IFNg

IFN Alfons Billiau* and Koen Vandenbroeck Rega Institute, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium * corresponding author tel: +32-16-337349, fax: +32-16-337340, e-mail: [email protected] DOI: 10.1006/rwcy.2000.07002.

SUMMARY Interferon (IFN ) is produced mainly by activated lymphocytes and has receptors on virtually all cell types of the body. It thus exerts a multitude of cellular biological effects. Among cytokines, IFN is the main activator of macrophages. In addition it also activates endothelial cells. Together, these two activities are the basis for assigning a proinflammatory role to IFN . The regulatory role of IFN on the antigen-specific phases of the immune response derive from its effects on antigen-presenting cells and on B and T lymphocytes. IFN augments expression of MHC molecules in professional as well as nonprofessional antigenpresenting cells; its effects on B and T lymphocyte proliferation and differentiation are complex. Production of IFN by T helper cells is a hallmark of the TH1-type phenotype. Thus, high-level production of IFN is typically associated with effective host defense against intracellular pathogens, and with immune and autoimmune pathology that depends upon delayed-type hypersensitivity. Genetic defects in the IFN system are rare; genetic polymorphism exists and its impact on disease susceptibility is under study. Despite the powerful immunoregulatory potential of IFN and the availability of preparations for administration to patients, clinical applications are limited. The reader is referred to previously published reviews on IFN by Young and Hardy (1995), Young (1996), Billiau (1996a, 1996b), Boehm et al. (1997), and Stark et al. (1998).

BACKGROUND

Discovery The protein which we now call interferon (IFN ) was discovered independently by two groups of

investigators and was originally given two different names after the biological activities studied: (1) type II or immune interferon, or IFN , and (2) macrophageactivating factor (MAF). Homonymy of IFN with IFN and IFN does not imply molecular relationship but merely reflects sharing of the biological property to be able to protect cells against virus infection. The corresponding bioassay is the so-called `antiviral assay', which consists in demonstrating that cultured cells exposed to IFN resist destruction by a standard challenge virus. Historically, the name `interferon' refers to the phenomenon of interference, i.e. the fact that cells infected with any one virus species are relatively resistant to infection with viruses of other species. However, whereas IFN and IFN (collectively called type I interferons) do play a role in such interference, IFN is not involved. In 1965 Wheelock and coworkers (Wheelock, 1965), demonstrated that an interferon-like antiviral activity, different from classical interferon by its lability in acid, appears in supernatants of mononuclear cells exposed to a mitogen. In fact, a 1964 report by Gresser and Nacify mentions the occurrence of acidlabile interferon in cerebrospinal fluids of patients with infectious and noninfectious neurological diseases (Gresser and Nacify, 1964). In the early 1970s the terms `type II interferon' (Youngner and Salvin, 1973) and `immune interferon' (Falcoff, 1972) were coined. The term immune interferon remained in use for some time in recognition of the awareness that the activity is associated with a protein physicochemically distinct from that responsible for the then classical interferon, that production of the factor is the prerogative of immune competent cell types, and that the factor possesses immune regulatory properties distinct from those of classical interferon. In 1980 an International Interferon Nomenclature Committee (Stewart et al., 1980) agreed on a new name for all interferons. Type I interferons, then known as leukocyte and fibroblast interferons, were renamed to

642 Alfons Billiau and Koen Vandenbroeck IFN and IFN , respectively; immune interferon was renamed to IFN . Availability of pure preparations of IFN and of monospecific antibodies made it possible to prove that MAF activity in biological fluids is largely if not entirely accounted for by IFN (Nathan et al., 1983). The term MAF appears in the literature around 1980, referring to the ability of supernatants of mitogenor antigen-challenged mononuclear cell cultures to augment various biological activities of macrophages. That stimulated lymphocytes' supernatants have these macrophage-stimulating abilities was first reported in 1966 by Bloom and Bennett. MAF bioassays have variably relied on intracellular killing of parasites or increased oxidative metabolism (Nathan et al., 1983), enhanced expression of class II antigens (Steeg et al., 1982) or enhanced tumor cell killing (Weinberg et al., 1978). Characterization of MAF with monoclonal antibodies soon revealed its identity with IFN (Nathan et al., 1983).

Alternative names For names used previously see section on Discovery.

Structure The size, amino acid sequence, and glycosylation of IFN are well conserved among animal species, and most studies on the structure of the glycoprotein have been done on either human or mouse IFN . In its biologically active form IFN is a 34 kDa homodimer stabilized by noncovalent forces. The peptide is N-glycosylated on two sites. Natural IFN is heterogeneous in size and charge, due to enzymatic trimming of the C-terminus and to variation in degree of glycosylation. This heterogeneity seems to be unimportant for biological activity on cells but might well influence the dynamics of tissue distribution. X-ray crystallographic analysis (Ealick et al., 1991) has revealed the subunits to consist of six  helices, accounting for 62% of the molecule, with no sheet domains. The two subunits, each of which is elongated in shape, are held together in an antiparallel configuration by intertwining of the helical domains. The N-terminus of each chain is juxtaposed to the C-terminus of the opposing chain. The symmetry in this structure allows IFN to bind a pair of identical receptor peptides, as has been demonstrated by analysis of the crystal structure of an IFN /IFN R complex. Peptide stretches at both the N- and the C-termini of IFN seem to have binding properties for the receptor, as demonstrated by competition-type experiments using the corresponding peptides (Griggs

et al., 1992). The N-terminal binding region has been identified (Van Volkenburg et al., 1993).

Main activities and pathophysiological roles IFN is a typical lymphokine, i.e. a product mainly but not exclusively of lymphocytes: T cells and NK cells. Under normal circumstances only minute but perhaps physiologically important quantities are produced. Larger quantities are produced only under pathologic circumstances such as trauma, infection, cancer, and autoimmunity. IFN acts on cells by inducing increased expression of several genes, the spectrum of which varies depending on the cell type concerned and on the presence of other cytokines, some of which (e.g. TNF) synergize with IFN , while others (e.g. IL-4) antagonize its actions. Receptors for IFN occur on virtually all cells of the body, so that many organs and systems undergo the action of IFN . IFN regulates cellular activities responsible for inflammation: the activation state of macrophages and endothelial cells. It also regulates the antigen-specific immune response by affecting both antigen-presenting cells and antigen-recognizing lymphocytes. Thus IFN is important in the initial phase of immune reactions (aspecific inflammatory and antigen-presenting), in the middle phase (expansion and differentiation of antigen-reactive lymphocyte clones), and in the end phase (sustained or final inflammation). Defects in the IFN mechanism are associated with severe impairment of resistance to infections caused by viruses and certain bacteria, in particular those which are normally killed by activated macrophages. Although IFN is often quoted to be a `proinflammatory' cytokine, the production of IFN during infections or as a result of immune reactions can under some circumstances enhance and in others inhibit inflammation and accompanying tissue damage. However, massive production of IFN , occurring as part of so-called acute cytokine release syndromes, is often associated with severe systemic manifestations, such as generalized bleedings and lethal shock.

GENE AND GENE REGULATION

Accession numbers Accession numbers for some mammalian and bird IFN cDNA (and genomic DNA) sequences are given in Table 1.

IFN Table 1 Accession numbers for IFN DNA of some mammalian and bird species (EMBL/ GenBank/DDBJ) Species

Genome

cDNA or CDS

V00536

X01992

Primates Human

J00219

X13274 a

M37265

X87308 M29383 V00543

Common marmoset (Callithrix jacchus)

X64659

±

Olive baboon (Papio hamadryas anubis)

±

To be added

Crab-eating macaque (Macaca fascicularis)

±

D89985

Rhesus monkey (Macaca mulatta)

±

L26024

Red-crowned mangabey (Cercocebus torquatus)

±

L26025

Pig-tailed macaque (Macaca nemestrina)

±

L26026

±

X86972

Carnivores Cat (Felis catus)

D30619 ±

Y11647b

Cow (Bos taurus)

Z54144

M29867

Goat (Capra hircus)

±

U34232

Eurasian badger (Meles meles) Artiodactyla

c

Z92887

X52640

Z73273a

A19173

Red deer (Cervus elaphus)

±

X63079

Pig (Sus scrofa)

X53085

S63967

A11777

U04050

Sheep (Ovis aries)

L07502 Perissodactyla Horse (Equus caballus)

D28520 Rodentia House mouse (Mus musculus)

±

K00083 M28621

Norway rat (Rattus norvegicus)

X02325

d

X02326d X02327

M29317 AF010466

d

Mongolian gerbil (Meriones unguiculatus)

±

L37782

Woodchuck (Marmota monax)

±

Y14138

±

AB010386

Common turkey (Meleagris gallopavo)

±

AJ000725

Helmeted guinea fowl (Numida meleagris)

±

AJ001263

Lagomorpha Rabbit (Oryctolagus cuniculus) Aves

643

644 Alfons Billiau and Koen Vandenbroeck Table I (Continued ) Species

Genome

cDNA or CDS

Chicken (Gallus gallus)

Y07922

U27465 X99774

Japanese quail (Coturnix japonica)

±

AJ001678

Ring-necked pheasant (Phasianus colchicus)

±

AJ001289

a

VNTR polymorphisms in intron 1 of human and ovine IFN genes.

b

Incomplete sequences.

c

Promoter fragment of the ovine IFN gene.

d

Unassembled exon sequences of the rat IFN gene.

In all species characterized to date, IFN is found to be coded for by a single-copy gene consisting of four exons. From 20 mammalian and five avian species, IFN -coding DNA sequences (CDS) are now available (Table 1). In addition, genomic regions spanning the structural gene of human, marmoset, cow, horse, pig, and chicken IFN have been sequenced. Comparison of these genes reveals a highly conserved overall structure with closely matching exon (and intron) sizes (Kaiser et al., 1998).

the IFN gene was physically localized to chromosome band 5p1.2-q1.1, close to the centromere (Johansson et al., 1993).

Chromosome location

Regulatory sites and corresponding transcription factors

Polymorphic sequences have been identified in the first intron of the human, bovine, and ovine, and in the third intron of the porcine IFN gene (Dijkmans et al., 1990; Ellegren et al., 1993). The polymorphism type and number of alleles, however, are different (Table 2). In the early 1980s, the human IFN gene was assigned to chromosome band 12q24.1 by in situ hybridization (Trent et al., 1982). This localization was only recently corrected by Bureau et al. (1995), who mapped the gene close to the D12S335 and D12S313 microsatellites on both the genetic and physical maps, and showed physical linkage with the MDM2 oncogene on chromosome band 12q15. This coincides with the assignment of the gene to 12q14 by fluorescence in situ hybridization (Zimonjic et al., 1995). In the mouse, the Mdm1, Ifg, and Myf6 loci were shown to be linked to each other in the telomeric part of chromosome 10. Other linkages in the same region include those between Ifg, Mdm1, Mdm2, and Mdm3. Thus, the similarity between the telomeric mouse chromosome 10 region and human chromosome band 12q15 is suggestive for a syntenic conservation during evolution of a large chromosome region that encompasses the IFN gene. In the pig,

Relevant linkages Relevant linkages are discussed under the section on Chromosome location. See also Table 2.

An overview of the most important regulatory sites is given in Table 3. The current state of evidence suggests a very complex mechanism of IFN gene transcription that takes place on three different levels of regulation: methylation, binding of activating transcription factors, and binding of repressors (for extensive review see Ye and Young, 1997, and Young, 1996). First, hypomethylation of CG islands in the proximal promoter region and the first intron correlates with expression of the gene. A significant difference in methylation status was observed in human CD4+ memory T cells (IFN producers) versus nonproducing thymocytes, neonatal cells, and adult naõÈ ve T cells (Melvin et al., 1995). The same holds true for mice where the promoter is not methylated in TH1 cells (IFN producers), but methylated in nonproducing TH2 cells (Young et al., 1994). Second, two distinct activation-specific regulatory elements are present in the human promoter region (pos. ÿ108 to ÿ40 bp), which are essential for induction by PMA and ionomycin (Phenix et al., 1993) (see Table 3 for a description). The first intron contains several important sites for control of transcriptional regulation.

IFN

645

Table 2 Chromosomal location, relevant linkages, and polymorphisms of the IFN gene Chromosomal location

Polymorphisms

Species

Chromosome

Relevant linkages

Types

Alleles

Location

Human

12q15 (centromeric)

MDM2, D12S335, D12S313

CA repeat

11

intron 1

Mouse

10 (telomeric)

Mdm1, Mdm2, Mdm3, Myf6

RFLP; SphI

2

±

Pig

5p1.2-q1.1 (centromeric)

Mucin, diacylglycerol kinase

[(GA)10(GAAG)8]n

n ˆ 1 or 2

intron 3

Sheep

±

±

(GTTT)n

n ˆ 5 or 6

intron 1

Table 3 Identification of important functional regulatory sites in the promoter and 1st intron of the IFN genea Core motif

Position

Regulatory site

Transcription or regulatory factors

Remarks

GGGGAGTTCC (r.o.)b

ÿ786 to ÿ776

IFN B site

NFB

Complex formed after T cell activation

AAAATATTCC (r.o.)

ÿ772 to ÿ762

C3-1P

NFB

Complex formed after T cell activation

AAAAATTTCC

ÿ278 to ÿ268

C3-3P

NF-ATc and NFB

Cyclosporin A-sensitive site; enhanced transcription through calcineurin-inducible and NFB transcription factors

ÿ251 to ÿ215

`Silencer' element

YY-1 and AP-2-like

Repression of basal and induced transcription repressor factors

CTATCT (r.o.)

ÿ95 to ÿ90

GATA site

GATA-3

Function not clear, but present in a complex from Jurkat nuclear extracts

TGTCACCA

ÿ90 to ÿ83

AP-1/CREB site

Dexamethasone inhibition of promoter activity

TACGTAA

ÿ57 to ÿ51

AP-1/CREB site

Dexamethasone inhibition of promoter activity

GTCACCAT

ÿ89 to ÿ82

GM-CSF/MIP-1 motif

Possibly related to expression in memory T cells

ACGTAATCC

ÿ56 to ÿ48

NFIL2A-site

Binding of transcription factors in Jurkat T cells; no binding of Oct-1

TAC*GTA (SnaB1)c

ÿ54

Methylation

Methyltransferase

TATAA

ÿ28 to ÿ23

TATA box

TFIIB/TBP

GAATTTTCC

460 to 468

C3 1st intron

c-Rel and NFB

Hypomethylation correlates to IFN expression Located in the first intron; complexes induced by PMA/ionomycin; works in concert with the promoter IFN B and C3-3P sites to enhance transcription

646 Alfons Billiau and Koen Vandenbroeck Table 3 (Continued ) Core motif

Position

Regulatory site

Transcription or regulatory factors

Remarks

CC*GG (HpaII)c

767

Methylation

Methyltransferase

Located in 1st intron

801±950

STAT sites

Located in 1st intron; adjacent and overlapping sites for STAT1, STAT4, STAT5, and STAT6

a

Regulatory sites represented in this table were collected from the papers referred to in the text.

b

r.o., reverse orientation.

c

Methylation-sensitive restriction site.

Not only does it contain a c-Rel and NFB p65/p50binding element (Brown et al., 1992; Sica et al., 1992) that enhances promoter activity, but also a pattern of adjacent sites for STAT1, STAT4, STAT5, and STAT6 has been identified in it (Xu et al., 1996). NFB and NF-ATc-binding sites are found in the promoter. NFB and calcineurin-inducible transcription factors form complexes with these regions upon T cell activation (Sica et al., 1997). Third, several lines of research have pointed to a crucial role for repressor nuclear factors in silencing IFN gene transcription. A silencer element (pos. ÿ251 to ÿ215 bp) has been identified to which both the nuclear repressor YY-1 (yin-yang-1) and an AP-2-like repressor can bind. This cooperative binding results in inhibition of IFN expression (Chrivia et al., 1990; Ye et al., 1994). Remarkably, a second YY-1-binding site has been identified that overlaps with an AP-1-binding enhancer site (Ye et al., 1996). While YY-1 activity is not modulated upon activation of T cells, that of AP-1 is upregulated. Thus, another mechanism for repression may consist of YY-1 blocking constitutive IFN gene transcription in resting T cells by competition for the overlapping binding site with the enhancer AP-1. Suppression of IFN transcription through glucocorticoids follows yet a different pathway. Dexamethasone-specific inhibition was localized to two enhancer CREB/AP-1 sites in the proximal promoter region (Cippitelli et al., 1995). As follows, the factors needed for glucocorticoid-mediated inhibition of IFN transcription utilize the same regulatory sites required for induction of the promoter. In conclusion, both enhancing and repressing transcription factors appear to regulate expression of the IFN gene through interaction with promoter and intronic cis-elements in a concerted manner.

PROTEIN

Accession numbers Accession numbers to IFN protein sequences (SwissProt) and crystal structures (Brookhaven Protein Database) are given in Table 4.

Sequence See Figure 1.

Description of protein The precursors of human and mouse IFN consist of 166 and 155 amino acids, respectively (Figure 1). Following removal of the signal peptide, mature forms with theoretical molecular weights of 17,100 (human IFN : 143 aa) and 15,900 (murine IFN : 133 aa) are generated. While mature MuIFN contains a single cysteine residue at its C-terminal position, HuIFN is devoid of cysteine. This excludes a role of disulfide bonds in tertiary structure formation. Most mammalian IFN species possess two N-glycosylation sequons, albeit at unconserved positions. Comparison of IFN sequences of various species shows that the most conserved sequences are found in helices C and F, which are the two most buried ones in the dimer. The most striking amino acid sequence conservation, however, is seen at the C-terminus. Here, a basic stretch of alternating lysine and arginine residues is found (K-128-RKRS) that is conserved in birds and mammals. During receptor binding, residues 130±132 of this cluster are probably targeted to an acidic stretch on the R C-terminal

IFN

647

Table 4 Accession numbers for IFN protein sequences and crystal structures for some mammalian and bird species Species

Protein sequence (SwissProt)

Crystal structure (PDB)

P05179

1HIG

Primates Human Common marmoset (Callithrix jacchus)

P28341

±

Crab-eating macaque (Macaca fascicularis)

P42163

±

Rhesus monkey (Macaca mulatta)

P42163

±

Red-crowned mangabey (Cercocebus torquatus)

P42162

±

Pig-tailed macaque (Macaca nemestrina)

P42163

±

P46402

±

Cow (Bos taurus)

P07353

1RFB

Goat (Capra hircus)

P79154

±

Sheep (Ovis aries)

P17773

±

Carnivores Cat (Felis catus) Artiodactyla

Red deer (Cervus elaphus)

P28333

±

Pig (Sus scrofa)

P17803

±

P42160

±

House mouse (Mus musculus)

P01580

±

Norway rat (Rattus norvegicus)

P01581

±

Mongolian gerbil (Meriones unguiculatus)

Q62574

±

P30123a

2RIG

P49708

±

Perissodactyla Horse (Equus caballus) Rodentia

Lagomorpha Rabbit (Oryctolagus cuniculus) Aves Chicken (Gallus gallus) a

Incomplete sequence.

domain, resulting in high-affinity binding of IFN on its receptor (Griggs et al., 1992). The secondary structure of the human IFN subunit contains six  helices, A±F, comprising about 60% of the overall structure, but no sheets (Figure 1). Apart from the AB loop, which is 13 residues long, all helices are connected by rather short loops of 3±5 amino acids (Ealick et al., 1991).

Discussion of crystal structure Information on crystal structures is available for human, bovine, and rabbit IFN (see Table 4 for

accession numbers to the Brookhaven Protein Database). Human IFN occurs in its natural conformation as a V-shaped globular dimer formed by association of two identical subunits that are both characterized by an extended flattened elliptical shape (Ealick et al., 1991); spatial dimensions of 60 AÊ  40 AÊ  30 AÊ). All 12 helices in the dimer are located parallel to the dimer 2-fold axis. Helices A, B, C, and D of each subunit form a cleft in which the C-terminal helix F of the other chain is packed. The latter helix is structurally different from the others because it shows a bend of about 125 at Glu112. This bend is necessary to make helix F fit into the cleft. Both N-glycosylation sites are on the surface of the dimer

648 Alfons Billiau and Koen Vandenbroeck Figure 1 Sequence and structure of human IFN . Boxed residues are conserved between human, baboon, bovine, ovine, porcine, murine, and rat species. Numbering starts with the first amino acid of the mature protein. The position of the six  helices in the crystal structure (Ealick et al., 1991) is indicated below the sequence. The C1 domain and a putative Grp78-binding site (Vandenbroeck and Billiau, 1998a) are indicated also. Asterisks indicate Asn25 and Asn97 N-glycosylation sites; filled circles indicate residues conserved in the dimer folds of all IFN and IL-10 amino acid sequences (Walter and Nagabhushan, 1995).

and thus exposed to the solvent. Receptor binding involves formation of a 2 : 1 R : IFN dimer complex. The receptor-binding interface of IFN consists of two discontinuous polypeptide chain segments, the first comprising helix A, the AB loop, and helix B, the second comprising helix F and part of the C-terminus. The long flexible loop that connects helices A and B is highly variable, and may, because of its essential role in receptor binding, explain some of the speciesspecificity of the molecule. While this loop exhibits no clear secondary structure in unbound IFN , receptor binding induces a conformational change including formation of a 310 minihelix (Walter et al., 1995).

Important homologies The IFN /IL-10 Fold and Topological Similarity The IFN dimer interface, centered at a pair of C±C0 helices, shows extensive interdigitation of both subunits. This type of intertwined interface is unique to a relatively rare subclass (7%) of symmetrical homodimers with high  helical content, in which both chains together fold into a single globular domain, i.e. the cytokines IL-5, IL-10, the mnt, arc, trp, and met repressors, and the fis, HU, and histone

B DNA-binding proteins (Larsen et al., 1998). In this subclass the contact surfaces at the interface are highly hydrophobic, thus providing stability reminiscent of monomeric proteins with an internally packed hydrophobic core. In contrast, the interfaces of the most common class of homodimers (65%) have a highly polar and hydrated nature, and both subunits form a separate compact domain. Tsai and Nussinov (1997) have postulated that the latter category folds by a three-state process, during which each subunit folds into a stable monomer that then combines into a dimer. IFN /IL-5/IL-10-type dimers are thought to form through a two-state process. Indeed, the highly intertwined structure of each subunit can be formed only upon dimerization, thus excluding the possibility of free monomers in solution. Similarly, any separation of the dimer must result in a pronounced disruption of the tertiary structure of the subunits. The dimer structure of IL-10, in particular, bears a striking resemblance to that of IFN , and five conserved residues are present in the dimer folds of all IFN and IL-10 sequences (Figure 1) (Zdanov et al., 1995; Walter and Nagabhushan, 1995). Although it is not yet known whether, and how, folding of this particular class of dimers is regulated in vivo, we have recently shown that both Hsp60type (GroEL) and Hsp70-type (DnaK) chaperon

IFN complexes can facilitate in vitro folding and dimerization of IFN at physiological temperature, a condition which is not permissive for spontaneous folding of the protein (Vandenbroeck and Billiau, 1998; Vandenbroeck et al., 1998a). A putative binding motif for the ER chaperon Grp78/BiP is located in helix C (Vandenbroeck and Billiau, 1998). Remarkably, IFN/ subtypes, though biologically related to IFN , are structurally different, in that they are not clearly dimeric in solution, but associate into 1 : 1 : 1 heterotrimeric complexes with R and R subunits. A Retroviral Homolog of IFN ? The p17 HIV matrix protein bears some structural similarity to IFN (Matthews et al., 1994): it has a similar helical/loop topology and also contains a flexible C-terminus. It is not known whether this resemblance has any functional significance.

Posttranslational modifications N-Glycosylation The primary sequences of murine, human, and porcine IFN contain two potential N-linked glycosylation sequons. Natural lymphocytic human IFN is heterogeneously glycosylated, and forms that are not glycosylated, or glycosylated either at a single site (20 kDa) or at both sites (25 kDa) exist (Yip et al., 1982; Kelker et al., 1984; Rinderknecht et al., 1984; Sareneva et al., 1996). Posttranslational modifications ± with an emphasis on glycosylation ± of recombinant human, mouse, and porcine IFN have been studied by using mammalian (CHO cells, transgenic mice) and baculovirus/insect cell expression systems (Scahill et al., 1983; Dijkmans et al., 1987; Sareneva et al., 1994; Vandenbroeck et al., 1994; James et al., 1995). Analysis of CHO cell-expressed recombinant MuIFN revealed that the highly heterogeneous nature of the secreted protein originates from three cumulative posttranslational modifications: Cterminal processing, N-glycosylation, and dimerization (Dijkmans et al., 1987). Similar observations were made later for human (Curling et al., 1990) and porcine IFN (Vandenbroeck et al., 1994). Since unglycosylated and N-glycosylated human IFN bind with practically identical affinities to the same receptors (Littman et al., 1985), glycosylation is unlikely to exert dramatic effects on the biological response. Neither does glycosylation alter or stabilize the conformation of the molecule, as shown by analysis of circular dichroic spectra (Arakawa et al., 1986). Thus, the relevance of IFN glycosylation should be sought 'upstream' from receptor binding. In this connection, it was demonstrated recently that

649

glycans at Asn25, but less at Asn97, are essential for protease resistance (Sareneva et al., 1995) and dimerization of IFN (Sareneva et al., 1994). Glycosylation at Asn25 occurs cotranslationally, while glycosylation at Asn97 occurs after dimerization. These observations are in line with the finding that in diverse expression systems, glycosylation occurs more efficiently at Asn25 than at Asn97 (James et al., 1995). In addition, the secretion level of a mutant IFN in which both N-glycosylation sites are abolished is much (10- to 100-fold) lower than that of the wildtype form (Sareneva et al., 1994). Taken together, the data indicate that glycosylation at Asn27 is critical for the formation of a secretion-competent form in the endoplasmic reticulum of producer cells that is resistant to proteolytic degradation. Detailed sugar chain analysis has been reported for CHO cell-derived (Mutsaers et al., 1986) and natural (Sareneva et al., 1996) human IFN , and revealed the presence of complex/sialylated and hybrid or high-mannose-type structures. Modulation of C-terminal Processing C-terminal truncation of IFN occurs at dibasic amino acid sites (James et al., 1996), and is responsible for the series of low molecular weight variants generally seen in the secreted pool. Proteolytic processing at the C-terminus results in a severe loss of receptor binding and biological activity (Leinikki et al., 1987), and may thus comprise a mechanism to modulate IFN activity. This can be understood in view of the observation that part of the C-terminal polybasic cluster K-125TGKRKR (C1 domain, Figure 1) is essential for binding to the  receptor chain (Griggs et al., 1992). The current state of evidence is suggestive of a more subtle regulation of IFN activity through its C-terminus. The C1 domain was recently found to bind with high affinity to heparin and heparan sulfate. Heparin-binding shifts the extent of proteolytic degradation from  18 to 10 C-terminal amino acids (Lortat-Jacob and Baltzer, 1996). While the molecule is inactivated in the former case, C-terminal removal of 10 amino acids increases IFN activity by 600%. In receptor-binding experiments, the C1 domain functions by enhancing the on rate of the IFN ±IFN R binding reaction (Sadir et al., 1998). IFN bound to heparin displays, however, a strongly reduced affinity for its receptor. It follows that the interaction of heparin with the C1 domain is regulatory on two levels: first, heparin controls C-terminal truncation and hence intrinsic activity of IFN , and second, it modulates receptor interaction by competitive binding to the C1 domain (Sadir et al., 1998).

650 Alfons Billiau and Koen Vandenbroeck

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IFN is a typical lymphokine, being produced almost exclusively by NK cells and certain subpopulations of T lymphocytes. Production takes place if these cells are properly activated, either in vivo or after in vitro cultivation. In the human system, T cells which express the activation-dependent CD30 membrane antigen have been identified as the principal IFN producing subset (Alzona et al., 1994). Both CD4+ and CD8+ lymphocytes can produce IFN . Cloned lines of T cells, especially those of murine origin, fall into categories that do (T helper 1 or TH1 lines) and others that do not (TH2 lines) produce IFN following appropriate stimulation. Sporadic literature reports describe production of IFN by cultured mononuclear phagocytes (Fultz et al., 1995; Gessani and Belardelli, 1998), neutrophil granulocytes ( Yeaman et al., 1998), and neurons (Neumann et al., 1997) or cell lines of neuronal origin (Watanabe et al., 1989). Tissue expression can be studied by various approaches, e.g. (1) immunofluorescent staining of producer cells tissue sections, (2) determination of the protein in tissue extracts, and (3) quantitation of IFN mRNA in tissue extracts. In vivo, IFN is produced (1) in tissues which are infiltrated by activated lymphocytes, in particular in foci of acute inflammation due to aspecific or antigen-specific immune responses (e.g. the central nervous system in experimental allergic encephalomyelitis (EAE), the joints in autoimmune arthritis), and (2) in peripheral lymphoid organs during local or generalized inflammation (e.g. as part of the acute phase response which follow exposure to endotoxin). Whenever IFN is produced somewhere in the body, it also enters the bloodstream but is quickly cleared, so that it becomes detectable in serum in usually small concentrations and for relatively short durations.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators NK and T cells do not produce IFN as long as they are in a resting state. Many agents can activate them to produce the cytokine. Although it has become customary to distinguish between those that can trigger production all by themselves (often referred to

as `inducers'), and those that can only enhance production that is being or has been triggered, this distinction is usually difficult to make. The main reason is that proper activation of NK or T cells by an exogenous agent requires cooperation of accessory cells, e.g. mononuclear phagocytes, which also need to be in some state of activation. These cells produce soluble factors, in fact cytokines (e.g. TNF, IL-12, IFN , and others), which act as endogenous inducers or enhancers of IFN production. In addition, these cells make physical contact with the lymphocytes through membrane-attached molecules, e.g. the CD40/CD40L system, and thereby provide so-called costimulatory signals which are needed for optimal activation. Exogenous Stimuli Exogenous stimuli which can elicit IFN production are very diverse: in principle they are ligands of receptor or receptor-like molecules on producer cells. Some arise in vivo in the cells' environment as a result of naturally occurring situations such as trauma, infection, cancer, allergy, or autoimmunity. Others are known in the literature because they have been used as artificial inducers in experiments. Any substance recognized by T lymphocytes as `non-self ' can activate these cells and, as a result, induce production of IFN , together with a number of other cytokines. A classical system consists of immunizing mice with a potent T cell antigen, e.g. killed mycobacteria, then culturing spleen or lymph node cells and stimulating these cultures with the same antigen. The in vivo preimmunization is necessary for clonal expansion of the reactive T cells to take place so that the cultures will contain sufficient numbers of memory T cells to generate measurable IFN yields. Some microorganisms that infect humans or animals produce superantigens, e.g. staphylococcal enterotoxins. Like antigens, these substances bind to the T cell receptor, but do so through a less variable part of the molecule, so that more clones are initially engaged in the reaction and so that clonal expansion is not required for sizeable IFN production. Hence superantigens can induce sizeable IFN production in fresh cultures of mononuclear leukocytes taken from nonimmunized animals or humans. As an exception to the general rule that IFN induction in NK cells requires help from accessory cells, it has been reported that the superantigen SEB can independently induce IFN production in human NK cells (D'Orazio et al., 1995). The lipopolysaccharide (LPS) of the outer membrane of gram-negative bacteria, also designated as

IFN endotoxin, is a potent inducer of IFN . Receptor systems for LPS are present on many cell types, ranging from leukocytes to fibroblasts. Injection of LPS in animals results in an IFN response which peaks at about 6 hours and wanes within 24 hours. IFN produced in response to injection of LPS apparently originates from NK cells as well as CD4+ and CD8+ T cells, since the mRNA is detectable in all three populations (Heinzel et al., 1994). A comparison of IFN production by cultured murine splenocytes of responsive and nonresponsive strains led to the conclusion that for gram-negative bacteria to induce IFN , the presence of mononuclear phagocytes and production of IFN is required (Yaegashi et al., 1995). Also, in endotoxin-injected mice, release into the circulation of IL-12, a prominent NK cell-stimulatory monokine, precedes that of IFN , and pretreatment with anti-IL-12 antibody inhibits such production of IFN (Heinzel et al., 1994). However, not only monocyte-derived cytokines, but also IL-2, produced by TH1 cells can stimulate IFN production by NK cells (Scharton and Scott, 1993). It is not excluded that bacterial components other than those mentioned can act as triggers of IFN production. In gram-positive-bacterial infections, e.g. in listeriosis, NK cells take a large part in IFN production, which accounts for resistance of the host to the infection (Dunn and North, 1991). Similarly, in parasitic infections, e.g. in leishmaniasis models, early production of IFN , which is of crucial importance for the further course of the infection, is accounted for by NK cells (Scharton and Scott, 1993). Vegetal lectins, in particular phytohemagglutinin A (PHA) and concanavalin A (Con A), by virtue of their affinity for lymphocyte membrane glycoproteins, are potent mitogens and cytokine inducers. Accordingly, they have been used as artificial inducers of IFN in leukocyte cultures. Similarly, antibodies directed at lymphocyte membrane components can be used experimentally as inducers of lymphokines, including IFN . Polyclonal antilymphocyte serum (ALS) as well as monoclonal antibodies, in particular those directed at the universal T lymphocyte antigen CD3, can be used, either in vitro or in vivo. These artificial inducers of IFN have been especially helpful in the elaboration of semi-large-scale production systems of the IFN protein and in analysing the role of different cell popuations, cytokines and other mediators as regulatory factors in IFN production. Endogenous Stimuli Two purely endogenous factors, IL-12 and IL-18 have so far been described as `IFN inducers',

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implying that they can trigger IFN production independently from the presence of exogenous stimuli, though perhaps not of other endogenous factors. IL-12, also known as a `natural killer cell stimulatory factor' or `cytotoxic lymphocyte maturation factor', is a heterodimeric cytokine made up of a 40 kDa (p40) and a 35 kDa (p35) subunit (reviews in references Bruna, 1994; Trinchieri, 1995). It is produced mainly by activated macrophages and B lymphocytes and exerts multiple effects on T cells and NK cells. It is a potent inducer of IFN production by these cells. In addition, IL-12 plays a major role in promoting the development and differentiation of IFN -producing TH1 lymphocytes (Manetti et al., 1993; Trinchieri, 1993). IL-18 (reviewed by Dinarello et al., 1998) is a monomeric protein (18±19 kDa), originally described as `IFN -inducing factor' (IGIF). Its folding pattern resembles that of IL-1. IL-18 is produced as a precursor by several cell types, including mononuclear phagocytes and keratinocytes. Pro-IL-18 is cleaved into active IL-18 by caspase 1; active IL-18 is deactivated by caspase 3. IL-18 induces or augments IFN production in the presence of other cytokines, namely IL-2 and/or IL-12. The role of IL-12 seems to consist in inducing expression of the IL-18 receptor complex in T cells. Numerous studies have demonstrated the crucial role of these two cytokines for (optimal) in vivo induction of IFN by various exogenous agents. Exogenous and Endogenous Modulators of Production Whereas certain cytokines enhance production of IFN , and may in fact be crucial for any IFN production to take place, one cytokine in particular, IL-10, is a strong inhibitor of IFN production. IL-10 (for review, see Moore et al., 1993) was originally described as `cytokine synthesis inhibitory factor' produced by TH2 cells. However, monocytes and B lymphocytes can also produce IL-10. Production of IL-10 by mononuclear phagocytes seems to be induced mainly in an autocrine fashion by TNF and may represent an important negative feedback pathway (Wanidworanun and Strober, 1993). The powerful inhibitory effect of IL-10 on IFN production is illustrated by the observation that IL-10 administration to mice can inhibit shock induced by SEB in mice (Florquin et al., 1994). The antagonistic effect of IL-10 on IFN is reciprocal. IL-10 production by monocytes is inhibited by IFN (Chomarat et al., 1993). It seems likely therefore that the balance between IL-10 and IFN in the initial stages of an immune response is of crucial

652 Alfons Billiau and Koen Vandenbroeck importance to determine the further course of the response. For instance, selective stimulation of IL-10 production has been proposed as a strategy used by certain microorganisms, in particular helminths and protozoans, to evade the IFN -mediated host defense. Production of IFN by T cells is also under regulatory control of prostaglandins. PGE2 inhibits production by TH1 cells of IFN and IL-2 but not production of IL-4 by TH2 cells. cAMP is involved in this control mechanism, which allows PGE2 to skew immune responses in the TH2 direction (Snijdewint et al., 1993).

RECEPTOR UTILIZATION The IFN receptor complex on human mononuclear leukocytes consists of at least three distinct proteins (Finbloom et al., 1991). One of these, the IFN receptor  chain (IFN R), is the membrane protein that primarily binds IFN with high affinity. It encompasses three domains (Aguet et al., 1988; Hemmi et al., 1989): the extracellular ligand-binding, the transmembrane, and the intracellular domain. For signals to be transmitted, the extracellular domain must interact with one or more species-matching proteins (Hibino et al., 1992). This protein, termed IFN R , has a structure resembling that of IFN R. Interaction of the  chain extracellular domain with this protein is sufficient for transmission of the signal to express MHC class II molecules, but insufficient for expression of the antiviral state. Therefore, interaction with a third protein is surmised. IFN binding to the receptor complex causes its dimerization, which in turn leads to intracellular signaling.

IN VITRO ACTIVITIES

In vitro findings Biochemical Changes in Cells One of the proteins induced by IFN is the enzyme indoleamine-2,3-dioxygenase (IDO). IDO is the first enzyme of the kynurenine pathway which links tryptophan to alanine and acetyl CoA. It catalyzes the oxidative cleavage of the pyrrole ring in tryptophan to yield N-formyl-kynurenine. Kynurenine is transformed by enzymes in liver and brain to alanine and metabolites, some of which, such as quinolinic acid, have neuroactive or toxic potential. Induction of IDO by IFN explains why exposure to endogenous or exogenous IFN is often associated with decreased

serum tryptophan levels and increased levels of kynurenine in serum and urine. Induction of IDO by IFN may indirectly function as a scavenger mechanism of the superoxide anion, as this anion is used in the IDO-catalyzed conversion of tryptophan to N-formyl-kynurenine. The cell- and tissuedamaging effect of IFN may thereby be dampened. IFN -induced depletion of tryptophan has also been speculated to contribute to the antiproliferative and immunosuppressive effects of IFN . IFN is well known to potentiate respiratory burst responsiveness of macrophages to stimulants, resulting in increased production of highly reactive oxidants such as H2O2 and the superoxide anion (O2ÿ) (Nathan et al., 1983). These effects of IFN are believed to be due to regulation of the transcription of genes coding for enzymes of the NADPH oxidase system (Cassatella et al., 1990). This membrane-associated system is dormant in resting cells, but becomes activated during phagocytosis or upon interaction with certain soluble stimuli. Besides, IFN also stimulates production of NO, which in turn may react with H2O2 to generate reactive oxygen. IFN plays a role in the synthesis of tetrahydrobiopterin, a limiting factor in the synthesis of NO (see below). Pteridins are synthesized from GTP, the first step being conversion to 7,8-dihydroneopterin 30 -triphosphate by the enzyme GTP cyclohydrolase I (Werner et al., 1993). The synthesis of this enzyme is upregulated by IFN . Further steps leading to production of tetrahydrobiopterin are catalyzed by enzymes that appear not to be under control of cytokines. The tetrahydrobiopterin concentration in cells is therefore mainly dependent on the level of GTP cyclohydrolase, although part of the primary product of this enzyme may also leave the cells after dephosphorylation. The latter escape pathway explains the increased levels of neopterin in urine and tissues of patients with inflammatory and infectious diseases. IFN has an important role in the generation of nitric oxide (NO). Various cells synthesize and release endogenous NO as a short-distance and short-lived messenger (Moncada and Higgs, 1993). NO is produced by NO synthases, which convert L-arginine to L-citrulline and NO. NO synthase occurs in at least three isoforms. Two forms are `constitutive', being produced mainly by endothelial cells and platelets on the one hand and by neurons on the other hand. The inducible NO synthase (iNOS) occurs, in vitro, in mononuclear phagocytes, in granulocytes, in fibroblasts, Kupffer cells, hepatocytes, endothelial cells, vascular smooth muscle cells and, probably, in many other cell types. The enzyme is not detectable in uninduced cells and differs from the constitutive type in that its induced release requires protein synthesis,

IFN that it can act in the absence of Ca2+ and that its activity is stimulated by flavin adenine dinucleotide (FAD) and reduced glutathione. In macrophages and fibroblasts, the known natural inducers of the enzyme are cytokines and endotoxin. Maximal activation of the cells to produce NO via this pathway is obtained by their exposure to a combination of IFN and endotoxin, IFN and TNF, or IFN and IL-1. As expected, macrophages of mice with a targeted disruption of the IFN receptor gene were found not to produce NO in response to IFN . Other cytokines, TNF and IFN/ , could only marginally substitute for IFN , indicating that IFN is indeed the major cytokine controlling the NOgenerating pathways (Kamijo et al., 1993b). The physiological receptor for NO is the intracellular soluble guanylyl cyclase, which generates cGMP. The enzyme is activated by binding of NO to its heme iron. Effects of NO mediated via this signaling pathway are: vasodilation, platelet inhibition, cell adhesion, and neurotransmission. By nitrosylating free thiols, NO can regulate the activity of certain enzymes and thereby exert physiological regulatory functions. Thus, the reaction of NO with cell surface thiols has been associated with antimicrobial effects, modulation of ligand-gated receptor (NMDA) activity, and alterations of smooth muscle function. The antimicrobial effect of NO may also be due to loss of iron from infected host cells (Weinberg, 1993). In vitro, cells producing NO, following exposure to IFN , can die a suicide-like death (Drapier and Hibbs, 1988; Dijkmans and Billiau, 1991), in particular when they have no access to glucose or when glycolysis is blocked, so that the respiratory chain is the only pathway for ATP generation. However, aside from being cytotoxic, NO can also exert cytostatic activity by causing arrest of DNA synthesis. This inhibition precedes inhibition of mitochondrial respiration and may be due to impairment of the enzyme ribonucleotide reductase which contains at least three targets for NO: a tyrosyl radical, cysteines, and an iron center. Macrophages in which NO production is induced by IFN have cytocidal activity towards other cells, e.g. tumor cells, in which no NO can be generated. Cell death in this case can be due to interruption of mitochondrial respiration (Henry et al., 1993), but generation of peroxynitrite may also be involved. Production of NO in phagocytic cells is associated with reduced survival of ingested microorganisms. The role of the IFN -induced NO synthase is therefore assumed to consist in augmented defense against infection with bacteria, molds, or protozoa (for review see Green et al., 1991). However, the cytotoxic effect of induced NO may also cause

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undesirable cell and tissue damage and may thus account for some of the deleterious in vivo effects of endogenous IFN . In view of the effects of NO on the vasculature, one may propose that NO produced by IFN -activated macrophages is in part responsible for local vasodilation in the inflammatory focus, and possibly for the hyperdynamic circulation response associated with inflammation. Also hypotension occurring as part of the septic shock syndrome seems in part to be due to excessive production, through the endotoxin/cytokine-induced pathway, of NO. Finally, NO production has also been found to contribute to the antiviral effects of IFN in macrophages infected with ectromelia virus, vaccinia virus, or herpes simplex 1 virus (Karupiah et al., 1993b). In the macrophage-like cell line, RAW 264.7, IFN induced NO was found to inhibit vaccinia virus DNA replication, late viral protein synthesis and particle assembly, but not to affect early protein synthesis. Virus replication was inhibited not only in the iNOSproducing macrophages themselves but also in bystander cells cocultured with the IFN -pretreated macrophages (Harris et al., 1995). Remarkably, this apparently paracrine effect was not seen when the cells were separated from each other by a semipermeable membrane, suggesting that, in addition to NO, cell±cell contact is also necessary for transfer of the antiviral state. Aside from the IFN -induced proteins and enzymes already mentioned, several others have been reported. Some of these proteins have been found serendipitously or by molecular screening strategies such as subtraction cloning. Often, the function of these proteins has remained unknown for some time. One example is the murine protein Mg21, identified by subtraction cloning of cDNA from peritoneal macrophages treated with IFN (Lafuse et al., 1995). This protein appears to belong to a family of GTP-binding proteins which also encompasses IRG-47 (Gilly and Wall, 1992), which is induced by IFN in B lymphocytes, and Mx which is an IFN/ -induced protein responsible for the antiviral effect against influenza virus. GBP-1 is a GTP-binding protein induced by IFN in human fibroblasts (Cheng et al., 1991) and in mouse macrophages (Wynn et al., 1991); however its sequence is unrelated to those of IRG-47 and Mg21. Expression of Membrane Proteins One of the best documented actions of IFN is the induction of MHC class II antigens on many but not all cell types in culture. IFN thus has the ability to enhance or to induce these cells' ability to present foreign antigens. MHC class I antigen expression can

654 Alfons Billiau and Koen Vandenbroeck also be enhanced under the influence of IFN . Cells in which this effect occurs may thus become better targets for cytotoxic T cells recognizing viral, tumor or autoantigens present in such cells. IFN regulates the expression on phagocytes of the high-affinity Fc receptor I (Fc RI). Resting monocytes express both Fc RI and the low-affinity receptors Fc RII and Fc RIII; resting polymorphonuclear cells express only the low-affinity receptors. IFN induces expression of Fc RI on neutrophils (Petroni et al., 1988) and augments its expression on mononuclear phagocytes (Guyre et al., 1983). Ligand binding to Fc Rs is widely recognized to stimulate effector functions of phagocytes, such as phagocytosis, tumor cell killing, and inflammatory mediator release. Therefore, augmented Fc R expression is one of the pathways by which IFN can act as a proinflammatory cytokine. Isolated rat brain microglia also display enhanced expression of Fc receptors after treatment with IFN , TNF, or IL-1. Remarkably, the combination of IFN and TNF has been shown to inhibit Fc receptor expression (Loughlin et al., 1992). IFN augments expression of Fc"R on the human mononuclear cell line U937 (Naray Fejes-Toth and Guyre, 1984) and on platelets (Pancre et al., 1988). In view of the important role of IgE in resistance to parasitic diseases and in type I allergic reactions, these effects of IFN need to be taken into account when considering the role of IFN in these diseases. IFN is among the cytokines that augment expression of the adhesion molecule ICAM-1 on various cell types, including cultured endothelial cells and epidermal keratinocytes (Dustin et al., 1988), resulting in increased adhesiveness for leukocytes expressing the integrin LFA-1. The significance of this effect may be that IFN produced early in an aspecific inflammatory focus (e.g. by NK cells) is co-responsible for firm adhesion of granulocytes to endothelial cells in postcapillary veins as a prelude to their spreading and diapedesis. Similarly, this mechanism is believed to promote proximity of dendritic cells, epidermal keratinocytes, and lymphocytes during antigen presentation in the skin (Dustin et al., 1988). Another important membrane protein induced by IFN is the B7 antigen, whose ligand on T cells is the CD28 molecule (Freedman et al., 1991). The presence of B7 on antigen-presenting cells is indispensable for them to avoid delivering an anergizing signal (Harding et al., 1992). IFN has also been reported to augment expression and/or shedding of tumor-associated antigens by tumor cells, thereby modulating their targetability for the corresponding antibodies or sensitized T cells (Marth et al., 1989; Leon et al., 1989; Greiner et al., 1990).

Bone marrow-derived macrophages express a protein with an epitope recognized by a monoclonal antibody specific for a peptide of the mycobacterial heat shock protein, hsp60. Exposure of the cells to IFN was found to result in increased expression of this crossreactive epitope. Antibodies recognizing heat shock proteins are believed to play a role in autoimmune diseases. Therefore, the augmenting effect of IFN on expression of hsp60-crossreactive proteins may be of relevance to the pathogenesis of such diseases (Wand-WuÈrttenberger et al., 1991). Whereas, as a general rule, IFN augments expression of membrane molecules involved in immune responses, there are some exceptions. Expression of gp39 (CD40 ligand) by activated murine TH1 cell and TH2 cell clones and by activated splenic CD4+ cells is inhibited by IFN (Roy et al., 1993). IFN was also reported to inhibit the ability of human dendritic cells to express CD1A, CD80, and CD4, whereas the expression of other membrane antigens is potentiated; concomitantly the dendritic cells were functionally depressed (Rungcun et al., 1998). Activation-induced expression of E- and P-selectin on human endothelial cells in culture was also found to be depressed (Melrose et al., 1998). These effects may account in part for the in vivo immunosuppressive role of IFN in some systems (see section on immunosuppression mediated by IFN ).

Effects on Mononuclear Phagocytes IFN has long since been recognized as the foremost important cytokine converting macrophages from a `resting' to an `activated' state. `Activation' is a rather indiscriminately used term, which has meaning only if placed in a context of a well-defined function or functional ability that is considered. The mononuclear phagocyte (MPC) population, to which macrophages belong, comprises cells in different stages of differentiation and maturation, i.e. bone marrow precursors, blood monocytes, and different types of tissue macrophages (e.g. connective tissue histiocytes, alveolar macrophages, Kupffer cells, exudate macrophages, microglial cells, osteoclasts, etc.). These stages are usually considered as steps in a process which, although regulated by environmental signals, is in essence irreversible. Activation states of tissue macrophages, as a contrast, are mostly seen as reversible changes determined by the temporally changing tissue microenvironment. IFN and other cytokines, being constituents of the cellular microenvironment, play an important role both in the differentiation and maturation of MPCs and in activation of tissue macrophages.

IFN Circulating monocytes, when placed in culture, undergo apoptosis unless provided with certain stimuli, e.g. LPS and/or certain cytokines. Typically, human blood monocytes will survive for more than 7 days in the presence of pure M-CSF, whereas in its absence, they will die within 24 hours by apoptosis and secondary necrosis. In the presence of M-CSF the 100% surviving monocytes become progressively resistant against withdrawal of the growth factor and can subsequently be activated by exposure to a stimulus (e.g. serum-activated zymosan) to become biologically active (as evident from adherence, phagocytic and respiratory activity). In the presence of not only M-CSF but also IFN , resistance to cytokine withdrawal develops more rapidly. Thus, although IFN cannot by itself replace M-CSF, it can be seen as a synergist for M-CSF to avoid apoptosis and promote maturation into a macrophage. However, remarkably, such monocytes which have avoided apoptotic death in the presence of both M-CSF and IFN , as opposed to those which have matured in the presence of only M-CSF, do undergo apoptosis when subsequently exposed to an activating stimulus (Munn et al., 1995). The authors speculate that this yin-yang type effect of IFN fulfils a useful function in host defense against infection in that it allows for rapid development of a microbicidal macrophage, but also for subsequent elimination of the macrophage which, by producing toxic metabolites, might otherwise inflict undue damage on the host's own tissues. Macrophages activated by IFN or other agents differ from resident ones by enhanced endocytic capacity, as manifested by increased pinocytosis and phagocytosis via receptors for complement and IgG2a. Such enhanced endocytic capacity does not, however, apply to all ligands. Expression of Fc receptors for other immunoglobulin classes, for instance, has been shown to be reduced in the activated state. Significantly, macrophages activated by IFN have been found to have reduced ability to ingest a variety of obligately intracellular microorganisms, e.g. rickettsiae, Trypanosoma cruzi, and Leishmania amastigotes. In the case of Leishmania in mouse macrophages, reduced binding was demonstrated to be due to reduced expression of lectin-like receptors for the organisms (Mosser and Handman, 1992). Reduced uptake of intracellular parasites by IFN -activated macrophages may represent one of the mechanisms by which IFN contributes to both specific and aspecific host resistance to these pathogens. IFN also affects accessory cell function of mononuclear phagocytes. In vivo, endogenous IFN seems to promote helper and to counteract suppressive circuitry, as evident from experiments in which anti-IFN antibody was injected in carrier-primed

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mice, whose spleen cells were subsequently immunized in vitro with carrier±haptene complex (Frasca et al., 1988). However, IFN may also activate suppression circuits in mixed mononuclear cell populations (Noma and Dorf, 1985; Ishikura et al., 1989). IFN regulates production of chemokines by macrophages (and some other cells) (Table 5). It is a potent inducer of IP-10 mRNA expression in human and murine macrophages (Luster et al., 1985; Hamilton et al., 1989). IP-10 belongs to the chemokine  family. Human IP-10 has been shown to exhibit chemoattractant activity for monocytes and activated T cells and to promote T cell adhesion to endothelial cells (Taub et al., 1993). On the other hand, IFN was found to suppress LPS-induced expression of certain other related chemokines, namely MCP-1 and KC (GRP/melanoma growth-stimulating activity) in mouse peritoneal macrophages (Ohmori and Hamilton, 1994). Similarly, induction of IL-8 by IL-2 or IL-1 in human monocytes was found to be inhibited by IFN (Gusella et al., 1993). However, IFN is not to be regarded as a general inhibitor of IL-8 expression, since it synergizes with TNF to induce IL-8 in other cells. Clearly, IFN has the ability to either stimulate or inhibit production of proinflammatory mediators by macrophages. Effects on Endothelial Cells Endothelial cells are of particular importance in the local inflammatory response. IFN , as well as IL-1 and TNF, augment expression of the adhesion molecule ICAM-1 on cultured endothelial cells of extracerebral (Dustin et al., 1993) as well as cerebral origin (McCarron et al., 1993), resulting in increased adhesiveness for leukocytes expressing the integrin LFA-1 (Yu et al., 1985). IFN also enhances the capacity of endothelial cells for producing IL-1 in response to LPS (Arenzana-Seisdedos et al., 1985; Haq et al., 1985; Miossec and Ziff, 1986). A similar effect has been noted in cultures of synovial cells (Johnson et al., 1990). IL-1, in turn, acts as an inducer of other inflammatory mediators, such as procoagulants and prostaglandins. Chemokine secretion by endothelial cells is under regulatory control by cytokines, in particular IFN and TNF: barely any RANTES was found to be produced by human vascular endothelial cell cultures treated with either IFN , TNF, or IL-1 . However, the combination TNF+IFN was highly effective in inducing RANTES production: pretreatment with IFN sensitized the cells to induction with TNF. This regulatory control was found to be exerted at the transcriptional level (Marfaing-Koka et al., 1995).

Table 5 Influence of IFN on chemokine production Cells tested

Inducer

Chemokine

Effect

Comment

References

Human fibroblastoid synoviocytes

IFN (+TNF)

MCP-1

Induction

Synergy with TNF

Hachicha et al., 1993

Human leukocytes

LPS + IFN

MIP-1, MIP-1 , (IL-8)

Early inhibition; later enhancement

IFN by itself inactive; anti-TNF abrogates enhancement

Kasama et al., 1995

Mouse peritoneal macrophages

LMW hyaluronan

MIP-1, MIP-1

Inhibition

Production of IL-12 enhanced

Hodge-Dufour et al., 1998

Human umbilical vein endothelial cells

TNF + IFN

RANTES

Enhancement

Single cytokines inactive; IFN pretreatment sensitizes; IL-4 and IL-13 inhibit; IL-10 has no effect

Marfaing-Koka et al., 1995

Human fibroblasts; Hep-2 and MG-63 tumor cells

IFN

MCP-1, MCP-2

Induction

MCP-2 more responsive to IFN ; synergy with IL-1

Van Damme et al., 1994; Van Coillie et al., 1997; Struyf et al., 1998

Human monocytes

-amyloid peptide+ IFN

MCP-1

Induction and enhancement

Human monocytes

IL-2+IFN

IL-8

Inhibition

IFN by itself inactive

Gusella et al., 1993

Murine peritoneal cells

LPS+IFN

KC/GRO/MGSA

Inhibition

Cell-specific effect (not in endothelial or 3T3 cells)

Ohmori and Hamilton, 1994

LPS+IFN

JE

Inhibition

Cell-specific effect (not in endothelial or 3T3 cells)

Ohmori and Hamilton, 1994

LPS+IFN

IP-10

No effect

Cell-specific effect (not in endothelial or 3T3 cells)

Ohmori and Hamilton, 1994

CC chemokines

Meda et al., 1996

CXC chemokines

IFN Effects on T Cell Proliferation and Apoptosis Whereas IFN acts as a mild inhibitor of proliferation for most cell types, it stimulates proliferation of mitogen-triggered primary T cells as well as a variety of T cell lines (see, for example, Landolfo et al., 1988). Remarkably, the antiviral effect of IFN is not expressed in these cells. It has been considered that this exceptional situation is due to modulation of signal transduction in T cells and that enhancement of IL-2 production and IL-2R expression are involved (Landolfo et al., 1988). In studies using murine T cell clones, it was found that IFN exerts a slight suppressive effect on IL-2- and IL-4-mediated proliferation of TH2 but not TH1 clones (Fernandez-Botran et al., 1988; Gajewski and Fitch, 1988). One aspect of the regulatory effect of IFN on lymphocytes is its ability to promote apoptosis under specific conditions. Thus, blockage of IFN inhibits cell death induced in effector T cells by TCR linkage in the absence of accessory cells (Liu and Janeway, 1990). Furthermore, both in normal and in cultured malignant lymphocytes, IFN has been shown to exert contrasting effects, i.e. apoptosis or proliferation, depending on the level of expression of IFN receptors: high-level expression is associated with an apoptotic response, low-level expression with a proliferative one (Novelli et al., 1994). In vivo apoptosis of thymocytes after treatment with anti-CD3 antibody is more pronounced in mice which are deficient in expression of the IFN R chain than in their wildtype counterparts, indicating that in this system IFN triggers an antiapoptotic pathway (Matthys et al., 1995a). IFN may play a certain role in the differentiation, either in vitro or in vivo, of T cells into populations with either a TH1 or TH2 cytokine production profile. The cytokines with the highest impact on this differentiation are IL-12 and IL-4. IL-12 is a strong inducer of IFN and a promotor of the TH1 track. IL-4, conversely, is an antagonist of IFN and a strong promoter of TH2 responses. Many effects of IL-12 have been shown to be mediated by IFN . Hence it is possible that IFN may act in this process as a mediator of the IL-12 effect. In fact, a basic difference between TH1 and TH2 lymphocytes appears to be the absence of the chain of the IFN receptor in the TH1 population (Pernis et al., 1995), implying that IFN should be inactive on these cells. IFN and the Generation of Cytotoxic Lymphocytes (CTLs) Exogenous IFN can augment the development of cytotoxic T lymphocyte activity in mixed lymphocyte cultures, and neutralizing antibodies to IFN can

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inhibit the development of CTL activity in antigen- or lectin-stimulated lymphocyte cultures (Siegel, 1988). Anti-IFN was also found to inhibit both proliferation and activation of CTLs in the primary in vitro mixed lymphocyte reaction (Landolfo et al., 1985; Simon et al., 1986), an observation not subsequently confirmed under apparently similar conditions (Bucy et al., 1988). A contribution of IFN to the generation of CTLs may, in principle, result from stimulatory effects on mononuclear phagocytes, on helper T cells, or on CTL precursors, or from inhibitory effects on suppressor T cells. In human mixed lymphocyte cultures the augmentation by IFN of CTL generation was found to result mainly from a direct effect on CD8+ T cells (Siegel, 1988). In a system suitable for the expansion of single murine CD4± xCD8+ T cells into clones, both IL-2 and IFN were found to be required (Maraskowsky et al., 1989). Effects on B Lymphocytes Reports on the effects of IFN on B cells are somewhat contradictory. In early studies recombinant murine IFN was found to possess B cell maturation factor activity for resting splenic B cells and the comparable B cell tumor line WEHI-279.1 (Sidman et al., 1984). However, on the other hand, IFN was found to inhibit LPS-induced IgM production in murine spleen-derived B cells, by reducing the number of IgM-forming cells and without affecting overall proliferation (Abed et al., 1994b). In fact, in B cells, as opposed to T cells, the stage of differentiation seems to codetermine the type of response to IFN . Resting B cells seem not to be affected by IFN , while preactivated B cells are inhibited from further differentiation. These cells also show increased expression of IFN receptors (Abed et al., 1994a, 1994b). Also, cultured normal murine pre-B cells (Grawunder et al., 1993; Garvy and Riley, 1994) or the human pre-B cell line (Trubiani et al., 1994) which are exposed to IFN undergo apoptosis, while human CD5+ chronic B lymphocytic leukemia cells, as a contrast, are protected from apoptosis by IFN (Buschle et al., 1993). IFN has been found to stimulate polyclonal immunoglobulin production by resting or activated human B cells (Sidman et al., 1984; Nakagawa et al., 1985). In cultured human PBMCs addition of IFN has been found to promote an anti-IFN antibody to inhibit spontaneous late production of IgG2; in PWM-stimulated cultures, exogenous IFN inhibited and anti-IFN stimulated early production of IgG1 (Kawano et al., 1994). In this system IFN did not seem to act as a IgG2 switch factor, since the IgG2promoting effect disappeared when the culture system had been depleted of sIgG2+ cells. However, in a

658 Alfons Billiau and Koen Vandenbroeck culture system in which T cells are eliminated, IFN seemed to possess IgG2-switching activity (Kitani and Strober, 1993).

IFN in Hematopoiesis Effects of IFN on hematopoiesis have been demonstrated in many studies, using different experimental settings, which invariably employ one or several hemopoietic factors and/or cytokines. The question as to whether the IFN effects depend on the presence of accessory cells (stroma cells, monocytes or lymphocytes) has been a matter of controversy in early studies (Broxmeyer et al., 1983; Mamus et al., 1985) and can as yet not be considered as being resolved. Also, IFN induces or enhances production of various other cytokines, and its effects on hematopoiesis may therefore be indirect. For instance, the IFN -inducible chemokine IP-10 has been shown to inhibit colony formation from early hematopoietic progenitors, apparently by counteracting r-steel factor (rSLF) (Sarris et al., 1993). In bone marrow cultures prepared from normal mice IFN was found to inhibit granulocyte-macrophage colony growth. However, in cultures from mice pretreated with post-5-fluorouracil it promoted colony formation. It was suggested that primitive progenitors require stem cell factor in the early stages and IFN for subsequent growth (Shiohara et al., 1993). In the human system IFN has similarly been shown by many studies to counteract the proliferative activity of colony-stimulating factors. However in cultures of pure CD34+ progenitor cells, IFN was found to synergize with IL-3 (Caux et al., 1992). IFN by itself did not affect proliferation, and in the presence of IL-3, while augmenting the number of colonies, failed to affect their size, indicating that it acted only on early progenitors. In fact, in long-term cultures, once mature cells appeared, IFN inhibited further proliferation. In a study on human bone marrow cultures, both IFN and NO were found to suppress colony formation, and pharmacological blockage of NO could partially prevent the suppressive effect of IFN (Maciejewski et al., 1995). IFN has been found to suppress erythropoiesis both in vitro and in vivo, an effect that may play a role in anemia which accompanies chronic infections or autoimmune diseases. In affecting erythropoiesis in vivo, IFN undoubtedly interacts with several other hematopoietic factors and cytokines. Studies have been aimed at revealing the most important of these interactions and at defining the most sensitive stages of erythropoiesis. In EPO-containing human CFU-E or BFU-E cultures addition of IFN was

found to inhibit colony formation, an effect that could be reversed by increasing EPO concentrations (Means and Krantz, 1991). Similarly, in murine macrophage-containing bone-marrow cultures supplemented with EPO, addition of IFN was found to suppress formation of both BFU-E and CFU-E colonies (Wang et al., 1995). However, the dose required was lower for suppression of BFU-E, and the overall effect was more pronounced as IFN was added earlier after culture initiation. The effect of IFN was not prevented by addition of single or combined antibodies against TNF, IL-1, or GMCSF. Accordingly, increased production of these cytokines in IFN -treated cultures, or synergy of these cytokines with IFN seemed not to play a significant role in these macrophage-containing cultures. Nevertheless, in macrophage-depleted cultures, IFN was shown to synergize with the antierythropoietic effect of TNF. Therefore, and also because cytokines other than those which were examined may be involved, it should not be concluded that IFN exerts a direct inhibitory effect on BFU-E colony formation independently of other cells and cytokines.

Effects on CNS Cells The two main types of CNS targets for IFN are microglia and astroglia. Microglial cells fulfil such functions as antigen presentation, phagocytosis, cytocidal activity, and production of tissue-degradative activity. Astrocytes act as regulators of the ionic balance in the CNS and neurotransmitter distribution. When cultured in vitro these cells spontaneously express MHC antigens. Exposure to IFN greatly enhances this expression. However, in astrocytes, as opposed to microglia, the effect of IFN can be counteracted by various factors including IFN , IL-1, TGF , glutamate and cAMP agonists, as well as contact with neurons. Normal human CNS astrocytes, when cultured, spontaneously express MHC antigens as well as ICAM-1. However this expression is greatly increased in the presence of IFN (Pulver et al., 1987; Mauerhoff et al., 1988; Frohman et al., 1989; Satoh et al., 1991; Yong et al., 1991; Aloisi et al., 1992). This enhancing effect of IFN on expression of class II MHC antigen, but not on expression of class I MHC antigen or ICAM-1, was found to be counteracted by IFN (Barna et al., 1989; Satoh et al., 1995). In a murine oligodendroglioma cell line (MOCH-1) IFN was found to induce a morphological change from a small round cell with thin branches to a large fibroblast-like cell. IFN also stimulated markedly enhanced expression of the astrocyte marker protein GFAP (Li et al., 1995).

IFN IFN and Connective Tissue Maintenance of intact and remodeling of traumatized or inflamed connective tissue is increasingly recognized to be controlled in part by the cytokine network. The contribution of IFN to this control mechanism derives at least in part from its ability to inhibit the synthesis of collagen and fibronectin by fibroblasts or chondrocytes in vitro (Jiminez et al., 1984; Amento et al., 1985). This inhibition is associated with reduction in mRNA levels (Rosenbloom et al., 1984; Goldring et al., 1986). Systemic administration of IFN was found to inhibit collagen synthesis in murine models: tissue reaction to a subcutaneous foreign body (Granstein et al., 1987) or to skin wounds (Granstein et al., 1989), and to alleviate pulmonary fibrosis induced by bleomycin (Giri et al., 1986). Effects on Endocrine Cells In cultured human thyrocytes IFN has been found to inhibit expression of thyrotropin receptors (Nishikawa et al., 1993), production of triiodothyronin (Kraiem et al., 1990) and transcription of the thyroglobulin gene (Kung and Lau, 1990). It also reduces basal thyroid peroxidase content and inhibits thyrotropin-induced increase of the enzyme (Asakawa et al., 1992). Similar effects have been noted to occur in a rat thyroid cell line (FRTL-5), although the effects were minor unless IFN was given in combination with TNF (Tang et al., 1995). These effects may in part explain the decrease in thyroid function in autoimmune thyroiditis. The effects of IFN on the function of the anterior pituitary have been studied in an in vitro system consisting of organotypic anterior pituitary cell aggregates (Vankelecom et al., 1990, 1992). In such aggregates secretion of prolactin and ACTH in response to hypothalamic stimulatory factor was found to be inhibited by IFN . This inhibitory effect was, however, codetermined by the composition of the aggregates. In particular it required the presence of the folliculo-stellate (FS) cell component. In in vitro systems, the FS cells have been shown to mitigate responses of secretory cells to stimulatory as well as inhibitory hypothalamic signals. One of their in vivo functions may therefore consist of avoiding overly brisk fluctuations in hormone levels. The FS cells also have properties and activities which resemble those of mononuclear phagocytes or glial cells: expression of typical macrophage and dendritic cell markers, phagocytosis and secretion of IL-6 (Vankelecom et al., 1989). The apparent kinship of FS cells with other typical IFN target cells, and the FS cell-dependent

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inhibitory effect of IFN on the anterior pituitary secretory activity invites speculation that IFN , produced during inflammatory or immune responses, uses the FS cell to act on the neuroendocrine axis (Vankelecom and Billiau, 1992). Effects on Adipocytes Dissipation of fat stores during cachexia may in part be due to direct effects of IFN (and other cytokines) on adipocytes. For instance, treatment of cultured adipocytes to IFN results in reduction of the amount of lipoprotein lipase activity releasable from the cells by incubation with heparin (Patton et al., 1986; Doerrler et al., 1994). This enzyme is responsible for hydrolyzing the triglycerides in circulating lipoproteins and is a major determinant of fat accumulation in adipocytes. IFN has also been shown to reduce the rate of fatty acid synthesis (Patton et al., 1986; Doerrler et al., 1994) and to cause increased hydrolysis of endogenous intracellular triglyceride in adipocytes (Feingold et al., 1992; Doerrler et al., 1994). In accord with these effects, IFN was found to reduce the level of lipoprotein lipase and fatty acid synthetase mRNAs. However, the levels of acetyl CoA carboxylase mRNA was unaffected and the level of hormone-dependent lipase mRNA was decreased rather than increased. Therefore posttranscriptional as well as transcriptional regulatory pathways seem to be involved (Doerrler et al., 1994). IFN and Epithelial Barriers Epithelia represent an example of tight but flexible permeability barriers within the body. In an in vitro model IFN was shown to modulate permeability of an epithelial layer. IFN was found to be rather unique in this respect, as the effect was not seen with IFN. Also, only the basolateral side and not the apical side of the epithelial layer was found responsive (Adams et al., 1993). Most epithelial cells contain cytokeratins, a family of proteins forming cytoskeletal filaments. The expression of at least one of these molecules, the acid cytokeratin K17, was shown to be enhanced in HeLa cells treated with IFN (Flohr et al., 1992), and the promoter of this protein was found to contain three putative GAS elements (Vogel et al., 1995). However, the exact mode of action of the promoter remains to be elucidated. Effects on Virus Replication Cells respond to IFN by entering a state of relative resistance to virus infection. The molecular and

660 Alfons Billiau and Koen Vandenbroeck cellular mechanisms underlying this resistance are similar to those initiated by IFN or IFN . Detailed information on these mechanisms comes almost exclusively from studies with the latter interferons and can be found in the relevant chapters. Only few comparative studies on antiviral action mechanisms of IFN and IFN/ are available. Both in the human and in the murine system, the specific antiviral activity (number of antiviral units per milligram of protein) is 10- to 100-fold lower for IFN than for IFN/ . Like IFN and IFN , IFN induces derepression of the genes for 20 ,50 -oligoadenylsynthase and dsRNA-dependent protein kinase, which account to a large degree for the broadspectrum antiviral effect in cells. However, IFN fails to induce the Mx protein, which accounts for an added antiviral effect against some virus families, e.g. influenza virus and Togotovirus. Protein synthesis inhibitors block induction of some genes by IFN , leaving the antiviral response to IFN unaffected (Kelly et al., 1985; Faltynek et al., 1985; Kusari and Sen, 1986). It is therefore possible that, in a first round of protein synthesis, IFN induces production of other proteins which in turn act as interferons. In particular, it has been shown that IFN induces production of IFN in the murine L929 (Hughes and Baron, 1987) and Friend erythroleukemia cell lines (Marziali et al., 1991) and in mouse macrophages (Hughes and Baron, 1989). In Friend cells the genes for IFN and IFN were found to be constitutively transcribed, and the mRNAs appeared to accumulate in response to IFN . Mutant Friend cell lines that are resistant to type I interferons retained responsiveness to IFN , indicating that induced production of IFN is only partially responsible for the antiviral effect of IFN . There are examples of IFN being more antivirally active in cells of heterologous animal species. For instance, this is the case for porcine IFN which is more active in bovine than in porcine cells, suggesting that in this species or in general, the direct antiviral effect of IFN is not a property among those that are most essential for its role in host defense against virus infections. A number of studies have been devoted to the possible effects of IFN on the replication of HIV in monocytic cells. The results have been confusing, as both inhibitory and stimulatory effects have been seen, each by several independent workers. IFN was found to inhibit HIV replication in U937 histiocytic lymphoma cells if added before and kept present continuously after virus inoculation; withdrawal of IFN was followed by resumption of virus replication and addition of IFN to U937 cells that were persistently infected with HIV had no effect (Hammer

et al., 1986). Similarly, in primary human monocytes IFN was reported to inhibit virus production in a dose-dependent fashion (Kornbluth et al., 1989; Fan et al., 1994; Dhawan et al., 1994). Time kinetic study indicates that virus production in these primaryinfection systems appears to result from multiple cycles. Virus replication per infected cell appears not to be inhibited by IFN . The antiviral action seems rather to be directed against virus in the fluid phase. Stimulatory effects of IFN on HIV replication in mononuclear phagocytic cells have also been reported (Koyanagi et al., 1988; Biswas et al., 1992). Although the mechanism of this effect remains obscure, it is speculated that activation of monocytes by IFN may go hand in hand with activation of the proviral genome. Support for this concept has come from a study on monuclear phagocytes of a transgenic mouse strain the genome of which contains the long terminal repeat sequence (LTR) of HIV linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). Because the LTR contains the transcription regulatory elements of HIV, alterations in HIV-LTRdirected expression of CAT can be surmised to be analogous to changes in HIV expression in cells harboring the provirus. In this study it was found that IFN by itself had little effect on CAT expression. However, combinations of IFN with LPS, IL-6, or TNF led to significant synergistically augmented levels of CAT expression. The synergic effect was also evident when the macrophages were first treated with IFN and then exposed to LPS, IL-6, or TNF (Warfel et al., 1995). Aside from exerting direct effects on HIV replication IFN may, in conjunction with other cytokines, play a role in AIDS by influencing immunopathogenesis. In particular, aberrant cytokine production due to retrovirus infection may promote the predominance of TH2- over TH1-type cytokines which is characteristic for AIDS. In accordance with this concept is the observation that, in a murine model for AIDS, administration of IFN together with anti-IL-4 antibody retards development of the disease manifestations (Wang et al., 1994). Poxviruses of different animal species, having coevolved with their host under intermittent pressure of the antiviral effect of IFN , have acquired the genetic information to instruct infected cells to produce soluble IFN receptor homologs. An example is the rabbit myxoma virus. Cells infected with this virus release a protein, M-T7, which can bind rabbit IFN and inhibit its biological activity. The reactivity was found to be animal species-specific (Upton et al., 1995). Poxviruses also express proteins which interfere with the biological activity of cytokines other than IFN , in particular IL-1 and TNF. Mutant

IFN viruses which lack the ability to produce these proteins replicate normally in cells in vitro, but tend to be attenuated in vivo (for review, see Gooding, 1992).

Regulatory molecules: Inhibitors and enhancers Synergy between IFN and TNF Many instances have been reported in which IFN synergizes with TNF either in vitro or in vivo: in vitro cytotoxicity for certain tumor cells, induction of microbicidal activity in macrophages, induction of NO release by various cell types, expression of cell surface adhesion molecules, in vivo antitumor effects, in vivo induction of other cytokines, systemic toxicity and lethality. The subcellular mechanism(s) underlying this synergy are poorly understood. IFN has been found to augment expression TNF receptors by certain cell lines (Aggarwal et al., 1985; Tsujimoto and Vilcek, 1986; Ruggiero et al., 1986). However, this is not the general rule and cannot explain all synergistic effects described. In explanted murine peritoneal macrophages IFN , when added at relatively high concentrations, was found to inhibit rather than enhance expression of TNF receptors: in freshly explanted macrophages it prevented the appearance of receptors, and in mature macrophages it downregulated receptors already expressed (Drapier and Wietzerbin, 1991). This inhibitory effect was not considered to be contradictory to the synergy between TNF and IFN , since such synergy was only seen at doses of IFN lower than those inhibiting TNF receptor expression. This dose-dependent difference in interaction between the two cytokines may be of particular importance for the interpretation of often contradictory effects of both cytokines seen in inflammatory models in vivo. It should also be mentioned that in these studies no distinction was made between effects of IFN on the low versus the high molecular weight receptor of TNF. Natural IFN Antagonists In several systems IL-4 and IFN have been found to have opposite effects and to antagonize each other when they are both present. Typical effects of IFN on monocytes and macrophages (induction of IFN responsive genes, H2O2 production, intracellular antimicrobial activity) are counteracted by IL-4 (Gaspari et al., 1988; Gautam et al., 1992), although

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synergy has also been reported (Belosevic et al., 1988). Also, the antiviral effect IFN on L929 cells has been found to be antagonized by IL-4 (Lohoff et al., 1990). IL-4 inhibits induction of chemokines by IFN (Larner et al., 1993; Marfaing-Koka et al., 1995). In reverse, typical IL-4 actions on B lymphocytes (isotype switch in favor of IgE) and T lymphocytes (inhibition of thymocyte proliferation) are counteracted by IFN (PeÁne et al., 1988a, 1988b; Plum et al., 1991). IL-4, finally, antagonizes IFN by inhibiting its production by peripheral blood mononuclear cells (Peleman et al., 1989; Wagner et al., 1989; Vercelli et al., 1990). The subcellular mechanism of antagonism between IFN and IL-4 in monocytic cells involves inhibition by IL-4 of transcriptional activation of the IFN inducible proteins, as was shown to be the case for the chemokine IP-10. The target for inhibition seems to be the ISRE (interferon-sensitive response element) in the promoter of the IP-10 gene (Larner et al., 1993). However, rather than inhibiting activation of the ISRE-binding transcription factor, ISFGF-3, IL-4 seems to induce a distinct ISRE-binding factor that functions to inhibit IFN -driven, ISRE-dependent transcription (Deng et al., 1994). The production of IFN by antigen-stimulated CD4+ T cells depends on the presence of IL-2 both during the priming and the expression phases. As a contrast, production of IL-4 requires IL-2 only during the priming phase. This difference in requirement of IL-4 and IFN production may be part of the mechanism directing the immune response towards TH1 or TH2 predominance. Type I interferons ( or ) counteract induction by IFN of MHC class II antigens in murine macrophages (Ling et al., 1985; Fertsch et al., 1987). The antagonistic effect has also been observed in cultured human astrocytes (Barna et al., 1989) and astrocytoma cells, but not in human monocytes (Ransohoff et al., 1991). As documented by nuclear run-on experiments, the antagonistic effect is exerted at the transcriptional level (Ransohoff et al., 1991). Although TGF 1 can by itself induce transcription of cytokine genes, it mostly inhibits production of cytokines, such as IL-1 , TNF, and IFN , induced by other agents, such as LPS or PHA. The mechanism of this inhibitory effect remains unclear; synthesis of PGE2 or cAMP, which have both been implicated in posttranscriptional control of cytokine expression, seem not to be involved. TGF 1 can also counteract IFN action as it was found to downregulate constitutive as well as IFN -induced expression of MHC class II antigens on a human melanoma cell line (Czarniecki et al., 1988). Another IFN -controlled

662 Alfons Billiau and Koen Vandenbroeck activity which is counteracted by TGF is induction of nitrogen oxide synthesis in macrophages (Ding et al., 1990). In reverse, IFN can antagonize actions of TGF . Thus, it has been reported that IFN reverses the stimulation by TGF of the collagen gene but not that of the fibronectin gene expression in normal human fibroblasts (Varga et al., 1990). Although IL-10, like all cytokines, possesses multiple biological activities, its main effect remains its capacity to inhibit the synthesis of IFN . In addition, however, IL-10 also counteracts the action of IFN , e.g. as it inhibits IFN -induced nitrogen oxide production (Gazinelli et al., 1992). The powerful inhibitory effect of IL-10 on IFN production is illustrated by the observation that IL-10 administration to mice can inhibit shock induced by staphylococcal enterotoxin B (SEB) (Florquin et al., 1994). The antagonistic effect of IL-10 on IFN is reciprocal. IL-10 production by monocytes is inhibited by IFN (Chomarat et al., 1993). It seems likely, therefore, that the balance between IL-10 and IFN in the initial stages of an immune response is of crucial importance to determine the further course of the response. For instance, selective stimulation of IL-10 production has been proposed as a strategy used by certain microorganisms, in particular helminths and protozoans, to evade the IFN -mediated host defense. TNF synergizes with IFN in many in vitro test systems. However, this is by no means a general rule. With regard to two important activities, enhancement of expression of MHC class II molecules and of Fc receptors, the combination of IFN and TNF has been reported to be less active than either cytokine alone, both in rat peritoneal macrophages and in microglia (Zimmer and Jones, 1990; Loughlin et al., 1992). In fact, whether IFN and TNF synergize or antagonize with each other may critically depend on the state of differentiation of the cells. Thus, TNF was found to enhance IFN -induced MHC class II molecule expression in undifferentiated macrophages and to inhibit such enhancement in mature macrophages (Watanabe and Jacob, 1991). IL-6 has been found to act as an antagonist for the toxoplasmacidal activity of IFN in murine peritoneal macrophages in vitro (Beaman et al., 1994). Combining TNF with IL-6 pretreatment resulted in restoration of the toxoplasmacidal activity, whereas addition of anti-TNF antibody to this combination resulted in enhancement of the IL-6-mediated impairment of IFN function. It would appear, therefore, that in this system IFN and IL-6 interact at the level of TNF triggering.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Nonviral Infections IFN is generally assumed to play a primordial role in defense against pathogens that reside in and use the intracellular environment as a shield against microbicidal antibodies. Specific host defense against these organisms is therefore mainly dependent on cellular immunity mechanisms. Many of these pathogens have their habitat in mononuclear phagocytes. The traditional view holds that CD4+ T helper cells which recognize microbial antigen on the class II antigenpositive infected phagocytes are activated to produce cytokines including IFN . This IFN then triggers several microbicidal mechanisms in the phagocyte, e.g. tryptophan oxidase and reactive oxygen and nitrogen. It should be noted, however, that this CD4+ T cell- and cytokine-dependent pathway is complemented by the cytotoxic T cell pathway, which kills phagocytes or other cells that harbor microbial pathogens (Ottenhof and Mutis, 1995). In a great variety of in vivo models for infectious diseases IFN has been demonstrated to influence the outcome of experimental infection. The two principal approaches used in these studies have been to demonstrate alterations in the course of infection under the influence of exogenous IFN , or conversely, an effect of blockage of endogenous IFN by administration of a neutralizing anti-IFN antibody. These data have been confirmed and extended by the use of mice which are engineered to be deficient in producing IFN or in expressing the IFN receptor. For instance, such mice were found to have lost the ability to mount a protective host response to Mycobacterium tuberculosis. Although granulomas and delayed-type hypersensitivity did develop, the mice succumbed to the infection (Cooper et al., 1993; Flynn et al., 1993). The cellular or physiological mechanisms by which IFN exerts its advantageous actions are diverse. Reduced binding of the parasite to the host cell has been described to be a possible mechanism in Leishmania infection (Mosser and Handman, 1992). Reduced availability of tryptophan, due to IFN induced enhanced production of indoleamine oxidase, has been pinpointed as a mechanism for reduced intracellular growth of Toxoplasma (Pfefferkorn, 1984; Byrne et al., 1986; Murray et al., 1989). Intracellular killing of organisms has been invoked as a

IFN mechanism of action against several agents. Oxygendependent killing by peroxide generation has been reported as a mechanism involved in killing of Listeria (Peck, 1989) and Salmonella (Kagaya et al., 1989) by IFN -activated macrophages. Production of NO reportedly contributes to IFN -mediated killing of Trypanosoma cruzi (Mayer et al., 1993), Mycobacterium tuberculosis (Flynn et al., 1993), and Histoplasma capsulatum (Nakamura et al., 1994). It should be noted, however, that at least some IFN activated macrophage lines can kill Listeria by a reactive nitrogen-independent mechanism (Leenen et al., 1994). Production of IFN by NK-like cell populations in the early stages of bacterial or protozoal infections is now generally accepted to constitute a crucial element in defense, because it generates a transient aspecific resistance. For instance, the IFN -dependent temporary resistance of mice to Toxoplasma infection was found to result from stimulation of CD4ÿCD8ÿ cells (Johnson et al., 1993). Not unexpectedly, IFN knockout mice succumb rapidly to infection with otherwise avirulent strains of Toxoplasma. In such mice neutralization of IL-12 activity by antibody blocked NK cell activity but did not further reduce resistance to infection, suggesting that all early resistance is mediated through IFN (Scharton et al., 1996). The IFN -producing cell population involved in this early resistance may not only be of the NK type but may also include CD4+ T cells, as evident from studies with cytokine receptor c chain-deficient mice (Scharton-Kersten et al., 1998). Another type of mechanism enabling early IFN production to determine the outcome of infections is the regulation of the balance between TH1 and TH2 lymphocyte development. A single administration of anti-IFN antibody before an experimental Leishmania infection in mice promotes susceptibility (Belosevic et al., 1989) and inhibits development of TH1 cells (Scott, 1991). Conversely, injection of IFN early in infection switches the early cytokine pattern in susceptible mice from a TH2 to a TH1 profile (Scott, 1991). The producers of early IFN in this model were found to be NK cells (Scharton and Scott, 1993). Mice from a genetically resistant background, lacking the IFN receptor gene, were found to be susceptible to infection but nevertheless to mount a TH1 type response (Swihart et al., 1995). Listeria infection is another example: treatment of inherently resistant mice with neutralizing anti-IFN antibodies resulted in delayed elimination of bacteria from spleens and livers, and in increased production of circulating IL-4 and IL-10 (TH2 cytokines) late in infection. However, additional treatment with antiIL-4 antibody restored resistance, suggesting that

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endogenous IFN is needed to avoid a TH2 response (Nakane et al., 1996). Remarkably, there are a few distinct exceptions to the general rule that endogenous IFN exerts a beneficial effect in infections. These exceptions can be divided in two categories: (1) adaptation of the microbial agent and (2) overproduction of the cytokine. Certain agents seem to have adapted so well to the immune system of the host that they have succeeded in perverting the cytokine network so that IFN acts to their advantage. Trypanosoma infections in rodents exemplify perversion of IFN by the parasite. These infections in general are associated with nonspecific immunosuppression, and various mechanisms have been shown to be involved. However, in the case of T. brucei and T. congolense infections in rats or mice, IFN production is a crucial element in bringing about immunosuppression. Lymphocytes from T. brucei-infected mice, while unable to express IL-2 receptor and to proliferate after in vitro stimulation with antigen, do produce normal quantities of IFN (Sileghem et al., 1987). The presence of macrophages was required for this state of immunosuppression to occur. In a culture system T. brucei was found to install a similar state of macrophage-dependent inhibited mitogen responsiveness with intact production of IFN (Darji et al., 1993). Remarkably, interference with endogenous IFN was found to relieve suppression. Similarly, in rat mononuclear cell cultures, T. brucei was found to induce a rapid, nonantigen-specific release of IFN which was dependent on the presence of CD8+ cells. A proliferative response was seen only if endogenous IFN was blocked by antibody. The parasites, conversely, proliferated more profusely in the presence than in the absence of IFN . Furthermore, by immunochemical staining, rat IFN was found to bind and possibly to be ingested by T. brucei (Olsson et al., 1991). Thus, IFN may act as a direct growth factor for the parasite. Remarkably, IFN of human origin did not bind to or affect growth of the organism. The immunosuppressive effect of IFN in T. congolense infection is illustrated by the observation that treatment with anti-IFN antibody protects highly susceptible Balb/c mice from infection (Uzonna et al., 1998). Two other agents that, at least in certain in vitro systems, thrive better in the presence than in the absence of IFN are Mycobacterium lepraemurium (Mor et al., 1989) and Candida albicans (Garner et al., 1989). Overproduction of IFN , in association with that of other cytokines, can act to the disadvantage of the host in two possible ways: (1) IFN may enhance the

664 Alfons Billiau and Koen Vandenbroeck production of other cytokines, e.g. TNF, which may cause tissue damage and death, or (2) IFN may act synergistically with such cytokines. Shock caused in mice by endotoxin is aggravated by endogenous as well as exogenous IFN . Thus, treatment with neutralizing antibody against IFN protects mice against induction of the lethal generalized Shwartzman reaction, and this protective effect is associated with lower TNF and IFN/ levels in serum (Billiau et al., 1987; Heremans et al., 1990; Billiau and Vandekerckhove, 1991). It can be inferred that in some instances of gram-negative sepsis endogenous IFN may contribute to, rather than antagonize fatal outcome (Silva and Cohen, 1994). Chronically infected mice develop a state of hypersensitivity to the toxic effects of endotoxin or TNF. Hypersensitivity to endotoxin does not, however, develop in BCG-infected mice which lack the receptor for IFN (Kamijo et al., 1993a), and endotoxin hypersensitivity in Propionibacterium acnes-infected mice can be blocked with neutralizing antibody against IFN (Katschinsky et al., 1992). Similarly, hypersensitivity of BCG-infected mice to the lethal effect of human TNF does not occur in mice which are deficient in expressing the IFN receptor; as a contrast, sensitization for TNF by tumors does occur in the IFN receptor-deficient mice (Cauwels et al., 1995). It should be noted that toxicity, as well as some other systemic effects of TNF are governed by multiple factors. In mice, whether toxicity occurs critically depends on which of the two TNF receptors becomes activated. Human TNF happens to trigger only the 55 kDa receptor (TNFRI, p55), whereas mouse TNF hits both this receptor and TNFRI (p75). This difference is correlated with a dramatically higher toxicity of homologous TNF than that of human TNF (Brouckaert et al., 1992). Chronic infections are often accompanied by cachexia. Overproduction of cytokines, in particular TNF (cachectin), has been incriminated as an important element in the pathogenesis of cachexia that develops in association with cancer (Tracey et al., 1988; Sherry et al., 1989). However, at least in tumor models in rats and mice, cachexia has been demonstrated to be due as much to IFN as to TNF (Langstein et al., 1991; Matthys et al., 1991a, 1991b; Billiau and Matthys, 1992). Viral infections IFN has the potential to protect the host against virus infection by virtue of its direct antiviral effect on most types of cells and by its regulatory activity on immunocytes. Not unexpectedly, exogenously administered IFN has been found to act prophylactically

against a variety of experimental virus infections, e.g. murine cytomegalovirus (CMV) infection in mice (Fennie et al., 1988) or rat CMV infection in rats (Haagmans et al., 1994). Of more fundamental importance are the studies analyzing production of IFN and the effects of its neutralization in model virus infections in mice and rats. Generally speaking these studies have indicated that endogenous IFN is essential for adequate host defense against virus infection, i.e. for elimination of the virus following primary infection and, in some instances, also for establishment of adequate immunity against reinfection. However, a question which remains largely unsolved is whether the in vivo antiviral effects of endogenous IFN are due to its direct antiviral effects on cells or to its immunomodulatory activities, such as activation of NK cells or maturation of cytotoxic T lymphocytes. Much relevant information has come from studies on lymphocytic choriomeningitis (LCM) virus infection in mice. LCM virus is indigenous to mice. Typically, the virus causes lethal choriomeningitis when inoculated intracerebrally in adult mice, but is poorly pathogenic when inoculated by other routes and/or at young age. Cell-mediated immunity is required for elimination of the virus in mice (LehmanGrube et al., 1985): in young or immunosuppressed mice, the infection tends to be inapparent and to become persistent. Cell-mediated antiviral immunity, when it arises, is also instrumental in bringing about tissue damage in mice infected under conditions that favor LCM pathology, e.g. in adult immunocompetent mice infected intracerebrally. In adult mice infected with LCM virus by inoculation in the footpad, the number of IFN -producing cells in the spleen increases significantly above low constititive levels without IFN becoming detectable in the circulation (Gessner et al., 1989, 1990). Pretreatment of the mice with anti-IFN antibodies delays virus elimination (Leist et al., 1989; Wille et al., 1989). Also, virus replicates to higher titers in organs of IFN receptor knockout mice than in those of wild-type counterparts (MuÈller et al., 1994). However, anti-IFN also converts an aggressive into an inapparent infection (Leist et al., 1989). The conclusion of these studies is that IFN contributes to cell-mediated immunity against the virus, the result of which may be favorable or unfavorable to the host depending on the circumstances. In mice infected with murine cytomegalovirus, clearance of the virus from the salivary glands was shown to depend on endogenous IFN (Lucin et al., 1992). SCID mice infected with murine CMV have activated macrophages with increased MHC gene product and ICAM-1 expression. Treatment with

IFN anti-IFN antibody prevented activation from occurring, caused virus replication in the spleen and liver to be increased, and pathology in spleen to be more pronounced (Heise and Virgin, 1995). Since SCID mice do not possess specific antigen-reactive T cells, it was concluded that IFN -dependent macrophage activation in this model of CMV infection occurred independently of T cells, implying that other IFN producing cells, conceivably NK cells, control macrophage activation in the infection. In rats and rat cells infected with a rat cytomegalovirus IFN affects virus replication in opposite ways depending on the dose and the circumstances (Haagmans et al., 1994). Rat CMV replicates in immunocompetent as well as immunosuppressed mice but is pathogenic only for the latter ones. High doses of IFN were found to act prophylactically both in vitro and in vivo in immunosuppressed rats. However, low doses of IFN caused enhanced replication in both fibroblast and macrophage cultures. Also, in immunocompetent rats, neutralization of endogenous IFN with antibody was associated with reduced replication of the virus in the spleen and helped to protect immunosuppressed rats partially reconstituted with sensitized T cells. The enhancing effect of IFN on rat CMV replication, especially in mononuclear phagocytes, is reminiscent of similar effects of IFN on replication of HIV in mononuclear leukocytes (Koyanagi et al., 1988; Biswas et al., 1992). The effect may be direct and virus-specific, e.g. if IFN action would activate promoter regions in the viral genome by the same pathways as it derepresses cellular IFN -inducible genes. A precedent for cytokines to act in this way is available: TNF and TGF have been shown to activate the immediate early genes of human CMV (Haagmans et al., 1992; Alcami et al., 1993). Alternatively, the effect may be indirect as is suggested by the fact that it is more pronounced in macrophages. IFN may alter production of other cytokines or may affect cellular receptiveness to such cytokines which have an influence on virus replication. Another mouse-indigenous virus is MHV (mouse hepatitis virus), a coronavirus of which there exist many strains. The virus is widespread among mouse colonies. Again the severity of experimental infection differs, depending on a number of defined variables. For instance, one colony of mice (A/J strain) was found to develop mild rather than severe disease after infection with MHV3, due to immunity resulting from inadvertent preceding infection with related coronaviruses. Interestingly, however, this partial immunity could be abrogated by pretreatment with neutralizing anti-IFN antibodies (Lucchiari et al., 1991, 1992). The findings imply that acquired

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resistance to virus infections can in some instances be infection-permissive and depend on early elimination of the challenge virus by cellular immunity. In mice infected in the footpad with the murine poxvirus (ectromeliavirus) treatment with anti-IFN antibody was found to result in enhanced spread of the virus to and efficient replication in the spleen, lungs, ovaries and, especially, liver (Karupiah et al., 1993a). Similarly, neutralization of endogenous IFN resulted in higher mortality in mice infected with human herpes virus (Stanton et al., 1995). Also, in IFN receptor gene knockout mice, a normally nonlethal vaccinia virus infection causes death (Huang et al., 1993; MuÈller et al., 1994). Vesicular stomatitis virus (VSV) infection (Huang et al., 1993) and Semliki Forest virus infection (MuÈller et al., 1994), however, were found to take a normal course. Mice infected with influenza virus via the respiratory route develop pneumonia. Recovery from this experimental infection depends on the development of virus-specific cytolytic T cell clones. Although IFN can in general terms affect development of cytolytic T cells, a study employing IFN gene knockout mice has indicated that, in mice infected with influenza virus, endogenous IFN plays little if any role in cellular immunity to the virus (Graham et al., 1993). As a contrast, the humoral immune response to the viral antigens was different in the knockout mice in that the proportion of IgG1 antibodies was increased. In mice lacking CD8+ T cells as a result of 2microglobulin gene disruption, neutralization of IFN was found to be associated with delayed virus clearance and increased proportions of lymph node B cells producing virus-specific antibodies of the IgG2a subclass (Sarawar et al., 1994). Cancer Rejection of some experimental tumors is associated with the presence of IFN in the tumor tissue. That this IFN contributes to this process is evident from studies with immunogenic autologous or isologous tumors, the rejection of which is abrogated by administration of neutralizing anti-IFN antibodies (Prat et al., 1987; Jarpe et al., 1989). Investigators have also inserted the IFN gene into nonimmunogenic murine tumor cells with high metastasizing potential and found that the IFN -secreting cells, when injected in syngeneic mice, had lesser ability than the parental cells to develop into tumors (Watanabe et al., 1989; Gansbacher et al., 1990); this suppression of tumorigenicity was reversed by the administration of anti-IFN or anti-CD8 antibodies. In a model of metastasis following operative removal of the primary tumor (Lewis lung), immunization

666 Alfons Billiau and Koen Vandenbroeck with an irradiated, highly productive IFN -transfected line of the tumor was found to be able to cure micrometastases (Porgador et al., 1993). Lowproducer lines and lines transfected with other IL-2 or IL-6 were ineffective. Dependence of tumor rejection on the presence of endogenous IFN and the generation of cytotoxic T cells was also demonstrated by the use of a transplantable mouse fibrosarcoma cell line whose IFN receptor was engineered so as to generate sublines that were either sensitive or insensitive to IFN (Dighe et al., 1994). The IFN -insensitive line was more tumorigenic and tumors induced by this line were more resistant to induction of rejection by LPS. Moreover, the IFN -insensitive line retained tumorigenic potential in vaccinated mice, and was itself unable to induce immunity to challenge with the standard line. More relevant for the natural occurrence of tumors was the approach in which primary tumors were induced by application of a carcinogen or by making use of an incapacitated anti-oncogene (Kaplan et al., 1998). IFN receptor-deficient as well as STAT1-deficient mice were found to develop methylcholanthrene-induced tumors more rapidly and with greater frequency than normal mice. Similarly, inactivation of the p53 anti-oncogene resulted in a larger number and a wider variety of tumors in these mice. From transplantation experiments with cells from carcinogen-induced tumors generated in IFN insensitive mice, it appeared that IFN acts at least in part by directly affecting the tumor cells, rendering them more immunogenic. Discordant with these observations are reports describing enhancement by IFN of tumor growth or metastatic potential of experimental tumors. A metastasizing murine mammary carcinoma (TS/A-pc), productively transfected with the IFN gene was found to metastasize more extensively than the untransfected tumor line (Ferrantini et al., 1994). Also, treatment of mice bearing Lewis lung tumors with anti-IFN antibodies was found to reduce tumor outgrowth (Matthys et al., 1991a). In vitro treatment of carcinoma cells with IFN prior to their inoculation in mice has been reported to enhance metastatic potential (Ramani and Balkwill, 1987); the mechanism involved in this model appeared to be augmentation by IFN of the tumor cells' resistance to the cytolytic effect of NK cells.

immunization systems testing for primary IgM antibody responses to sheep erythrocytes, blockage of endogenous IFN with antibodies that neutralize macrophage activation by IFN have indeed been found to reduce the amount of antibody produced (White-Helman and Wallace, 1989). IFN was also found to be a necessary component of T cell-derived helper factors for antibody induction in in vitro immunization systems (Brunswick and Lake, 1985). In contrast to this immunization-promoting effect of endogenous IFN , of which only minute amounts are actually detectable during the process, exogenous IFN suppresses early antibody formation (WhiteHelman and Wallace, 1989). IFN and IL-4 antagonize each other in a variety of systems, and it has become general knowledge that antibody responses depend in fact on the balance between two categories of cytokines, IFN and IL-2 belonging to the first one (the TH1 cassette) and IL-4, IL-5, IL-6, and IL-10 belonging to the second one (the TH2 cassette). In this setting the role of IFN consists in suppressing IgG1 and IgE antibody formation and stimulating IgG2a antibody formation (Finkelman et al., 1988; Snapper et al., 1993). Following infection with influenza virus (Graham et al., 1993) the virus-specific IgG1 response was found to be significantly higher in IFN -deficient than in normal mice, probably reflecting increased production of IL-4. Treatment with neutralizing antiIFN antibody in mice vaccinated with influenza virus antigens resulted in increased levels of antigenspecific IgG1 and IgE but reduced levels of IgG2 and IgG3 (Dobber et al., 1995). In IFN receptor knockout mice the IgG1 antibody response to ovalbumin was not different from that in normal mice, but the IgG2a isotype was reduced (Huang et al., 1993). When antigen in adjuvant (oil or alum) is injected, a very early change (day 3) is the appearance at the injection site of IFN -producing NK cells. Only later (day 7) do cytokine-producing T cells appear in draining lymph nodes. At this site IL-2 and IL-4 predominate over IFN (Bogen et al., 1993). The possibility is considered that early IFN production by NK cells is, in fact, secondary to IL-12 production by macrophages that respond to the antigen and/or the adjuvant. Early IL-12 and IFN may therefore play the crucial role in directing the immune response towards TH1 or TH2 predominance.

Antibody Formation

Immunosuppression Mediated by IFN

IFN augments expression of MHC class II antigen expression and may therefore be expected to facilitate antibody induction in systems in which antigen presentation is the limiting factor. In murine in vitro

The predominant responses of leukocytes and endothelial cells to IFN (enhanced expression of MHC molecules and of cell adhesion molecules, priming for production of other cytokines such as

IFN IL-1, TNF, and IL-12) are such that IFN is generally considered as an immunostimulant. Therefore, reference to immunosuppressive effects of IFN is counterintuitive. Yet, there are multiple reports of in vivo or ex vivo experimental systems in which higher production of IFN is associated with lesser responsiveness to antigens or mitogens. Graft-versus-host disease (GVHD), a major complication of bone marrow transplantation, is associated with suppression of cellular immune responses, as evident from reduced proliferative responses of lymphocytes to mitogens. Addition of monoclonal antibodies against IFN has been shown to relieve suppression, implying that endogenous IFN is involved (Wall et al., 1988). The target cell for IFN in this system is believed to be a so-called natural suppressor cell (Huchet et al., 1993). In fresh adherent mouse spleen macrophages as well as in macrophage hybridomas, IFN induces suppressive activity for proliferative responses of T cells. This induction is MHC class II antigenrestricted and specifically depends on expression of I-J region-coded antigens, which is stimulated by IFN (Noma and Dorf, 1985; Ishikura et al., 1989). Macrophage-like suppressor cells which inhibit mitogen-induced lymphocyte proliferation also occur in mice infected with Mycobacterium lepraemurium (Gosselin et al., 1995a) or Mycobacterium avium complex (Tomioka et al., 1995). Induction of these cells in vivo is independent of IFN but their suppressive action in vitro does require IFN . Acquisition of the suppressor activity requires cell-to-cell contact between the adherent-type and nonadherent-type leukocytes, the latter being different from classical T, B, and NK cells (Gosselin et al., 1995b). Another example in which IFN mediates generalized immunosuppression is in infection with Trypanosoma brucei in mice. A macrophage hybridoma, upon interacting with T. brucei was found to acquire the ability to suppress mitogenic responses of T lymphocytes (Darji et al., 1993). This suppressive activity was accompanied by decreased expression of IL-2 receptors but increased production of IFN . Addition of anti-IFN antibody to the system prevented suppression of the mitogenic responses and of IL-2 receptor expression, implying that IFN was instrumental in bringing about the suppressive activity. In chronic infection in mice, both spleen and lymph node cells acquire suppressive activity for mitogen-induced T cell proliferation (Sileghem et al., 1987). By the use of IFN knockout mice and inhibitors of iNOS, it could be demonstrated that, in this system, splenocytes acquire suppressive activity independently of IFN and of NO production (Beschin et al., 1998). In other studies using

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Trypanosoma brucei, NO production was found to indeed contribute to immunosuppressive effects of IFN -activated macrophages (Mabbott et al., 1995). In Trypanosoma congolense infections in highly susceptible Balb/c mice, immunosuppression by endogenous IFN may be mediated by IL-10: in vivo treatment with anti-IFN antibody was found to reduce plasma levels of IL-10 as well as secretion of IL-10 by splenocytes (Uzonna et al., 1998). Finally, IFN seems to be able to induce suppressor cells for delayed-type hypersensitivity (DTH) reactions. Splenic adherent cells incubated with haptene and then injected in mice were found to suppress DTH and induce appearance of haptene-specific suppressor cells, demonstrable by transfer into mice that were subsequently tested for DTH responsiveness. On prolonged culture the splenic cells lost their ability to induce suppression, but addition of IFN could restore this ability (Noma and Dorf, 1985). A possible mechanism of suppression by mononuclear phagocytes is generation of H2O2 and prostaglandins, since both catalase and indomethacin can alleviate suppression (Metzger et al., 1980). Boraschi et al. (1984), on the other hand, found that IFN reduces rather than stimulates murine macrophage suppressive activity by inhibiting PGE2 release and inducing IL-1 induction. Another pathway used by suppressor macrophages that can be activated by IFN , is the generation of nitric oxide (Mills, 1991; Albina et al., 1991). IFN also enhances release by mononuclear phagocytes of TGF (Twardzik et al., 1990), which is generally known as an anti-inflammatory cytokine. Another mechanism of immunosuppression by IFN may be inhibition of expression of CD40 ligand (Roy et al., 1993). Other mechanisms of immunosuppression by IFN include inhibitory effects on the expression of membrane-associated molecules and on production of chemokines. Acute Inflammation Application of various methods has documented production of IFN in lymphoid organs and in local sites of inflammation, e.g. in the CNS during experimental autoimmune encephalomyelitis (reviewed in Heremans and Billiau, 1997). However, most of our current understanding of the role of IFN in inflammation stems from experiments employing monoclonal antibodies to neutralize IFN in vivo, and from experiments involving the use of IFN or IFN receptor knockout mice, the former being unable to produce IFN , the latter being unable to to respond to it. A model of local inflammation is the local Shwartzman reaction elicited by a single injection of

668 Alfons Billiau and Koen Vandenbroeck endotoxin in the mouse footpad (Heremans et al., 1987). Pretreatment of the mice with monoclonal antibody to IFN resulted in a modification of the footpad swelling reaction: the early edema was reduced, whereas the later phase, consisting of cellular infiltration and intravascular thrombosis remained largely unchanged. This result indicates that IFN that is produced subsequent to the inflammatory stimulus exerts a local proinflammatory effect. However, it is unclear whether this IFN came from a local source (sporadic NK or T cells in the footpad) or had its origin in the spleen or lymph nodes. The local endotoxin injection most probably sufficed to induce a generalized cytokine response, as it was found to prime for a generalized Shwartzman reaction induced by a second systemic endotoxin injection (Heremans et al., 1990). Remarkably, pretreatment of the mice with exogenous IFN , instead of augmenting the local reaction, also inhibited it. A possible interpretation is that the effect of IFN differs depending on whether it hits the local site prior to or subsequent to application of endotoxin. Opposing effects of IFN depending on time of entry into the system is a recurrent theme in studies with animal models. This example illustrates the difficulty of distinguishing local from generalized pathogenetic inflammatory events. Model systems for generalized acute inflammation have been extremely popular in IFN studies. One of the first to be studied was the generalized lethal effect of endotoxin. Pretreatment of mice with anti-IFN antibody was found to make mice completely resistant to the generalized Shwartzman reaction. This resistance seemed to be related to a reduced systemic production of TNF (Heremans et al., 1990). As already mentioned, IFN and TNF synergize at the levels of both production and action. These early results from treatment with anti-IFN antibody have subsequently been reinforced by studies on knockout mice (Kamijo et al., 1993a; Car et al., 1994). Other models of acute inflammation in which the antibody approach led to evidence for a proinflammatory effect of IFN are the superantigen-induced shock syndrome (Matthys et al., 1995b), the anti-CD3 antibody-induced syndrome (Matthys et al., 1993), tumor-associated cachexia (Matthys et al., 1991a, 1991b) and the Con A-induced lethal hepatitis syndrome (Tagawa et al., 1997). In each of these instances, pretreatment with anti-IFN antibody protected the animals against the toxic manifestations of the inflammatory reaction. Of special note is the anti-CD3 syndrome, because the results and conclusions from antibody-mediated ablation of IFN were contradicted by the results from experiments with knockout mice. The CD3

membrane molecule is present on all T cells. Injection of the antibody initiates a response of all T cells, resulting in massive production of several cytokines including not only IFN , but also IL-2, IL-4, TNF, and several others. The result is a shock-like syndrome characterized by hypomotility and piloerection, hypothermia, and hypoglycemia. Depending on inherent sensitivity of the mice and on the dose of antibody, the syndrome may be self-contained or lethal. Mouse strains which are good IFN producers, e.g. Balb/c mice, were found to be more sensitive than others. Pretreatment of the mice with anti-IFN antibody prior to injection of the anti-CD3 antibody was found to provide near complete protection against the disease manifestations (Matthys et al., 1993), indicating that IFN produced as a result of the anti-CD3 challenge contributes to the severity of the syndrome. Studies with IFN receptor knockout mice, however, yielded data that led to a different conclusion (Matthys et al., 1995a). These studies were done with mice of the 129 background. Wild-type 129 mice are poorly sensitive to the anti-CD3 syndrome; the IFN R knockout mice, however, turned out to be more sensitive, indicating that, in this situation, an IFN -dependent protective pathway is operational. Obviously, IFN generates more than one pathway in the pathogenesis of the syndrome; some pathways make the syndrome worse, others rather provide protection. The question then is to know why the protective pathway is to prevail in one case and the disease-promoting pathway in the other. An obvious difference between blockage with anti-IFN antibody and blockage by knocking out the IFN receptor, is that the first one affects only IFN formed after antiCD3 challenge, while the second one affects all IFN , including any of it that is formed prior to the antiCD3 challenge. IFN produced in the animal's life time before exposure to the challenge with anti-CD3 antibody may be important for the protective pathway to be available; IFN produced as a result of the anti-CD3 challenge may be critical for full-blown disease to develop. Again, as was the case in the localized Shwartzman reaction, the effect of IFN seems to differ depending on the time of its entry into the system. In Vivo Modulation of Immune Pathology by IFN In various animal model systems IFN has been found to protect against immune pathology (Table 6). For instance, IFN seems to be able to induce suppressor cells for DTH reactions. Splenic adherent cells incubated with haptene and then injected in mice were found to suppress DTH and induce appearance of haptene-specific suppressor cells, demonstrable by

IFN

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Table 6 Animal model systems of immunopathology in which IFN acts as an immunosuppressant Model

Observation

References

DTH reaction

Haptene-specific suppressor cells lose in vivo suppressor activity upon prolonged culture

Addition of IFN restores suppressive activity

Noma and Dorf, 1985

Skin or heart allograft

Rejection prolonged by blockage of CD40±CD40L or B7±CD28 pathways

Rejection not prolonged in IFN knockout mice

Saleem et al., 1996; Konieczny et al., 1998

Experimental autoimmune encephalomyelitis (EAE)

Immunization with CNS antigens causes CNS inflammation and demyelination

Anti-IFN antibody enhances disease

Billiau et al., 1988; Duong et al., 1992, 1994; Lublin et al., 1993; Heremans et al., 1996b

Exogenous IFN alleviates disease

Billiau et al., 1998

IFN ligand and/or receptor knockout mice are more sensitive

Ferber et al., 1996

Anti-IFN antibody enhances disease

Caspi et al., 1994b

Exogenous IFN inhibits disease

Caspi et al., 1994a

Experimental autoimmune uveitis (EAU)

Immunization with bovine retinal antigen

transfer into mice that were subsequently tested for DTH responsiveness. On prolonged culture, the splenic cells lost their ability to induce suppression, but addition of IFN could restore this ability (Noma and Dorf, 1985). Treatment of skin allograft recipient mice with anti-IFN antibody has been found to prolong rejection if the graft is MHC class II antigen-incompatible, but not if it is only MHC class I-incompatible (Rosenberg et al., 1990), suggesting that, if endogenous IFN contributes to the rejection of a skin allograft, it does so because it induces class II expression on keratinocytes. More recent evidence from allograft rejection studies is rather indicative of a suppressive effect of endogenous IFN . The rejection rate of cardiac transplants was found to be similar in IFN gene knockout mice and in wild-types (Konieczny et al., 1998). However, in the knockout mice, and also in wild-type ones treated with neutralizing anti-IFN antibody, it proved impossible to prolong graft survival by blockage of the B7±CD28 or the CD40± CD40L pathways, as it could be done in the plain control wild-types (Saleem et al., 1996). Thus, it appears that endogenous IFN mediates a suppressive circuit when costimulation is inadequate. Experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU) in mice are two examples in which the net effect of endogenous IFN , produced in the course of the immune

process, is immunosuppression. In several variant models of the disease, treatment of the mice with neutralizing antibody against IFN was found to result in augmented symptoms and mortality (reviewed in Billiau, 1996b). Furthermore, both IFN and IFN receptor knockout mice were found to be more sensitive to induction of EAE than their wildtype counterparts (Ferber et al., 1996; Heremans et al., 1996a). Examples of models in which IFN accelerates or intensifies immune pathology are autoimmune diabetes and lupus-like syndrome in NZB/W mice, and inflammatory bowel disease (Table 7). Reports on the role of IFN in autoimmune diabetes models are controversial. Streptozotocin-induced diabetes, as assessed by hyperglycemia and body weight loss, was found to be more severe in mice that also received IFN injections (Campbell et al., 1988). Anti-IFN antibody pretreatment was found to reduce the incidence and severity of diabetes in nonobese diabetic (NOD/Wehi) mice in which occurrence of diabetes is boosted by cyclophosphamide (Debraye-Sachs et al., 1991; Campbell et al., 1991a). Counter to expectation, administration of IFN in this mouse model of diabetes did not affect blood glucose profiles. In fact, in combination with TNF, IFN treatment was associated with a reduction in severity of islet inflammation, although this treatment caused moderate to severe pancreatitis and several other pathologic

670 Alfons Billiau and Koen Vandenbroeck Table 7 Animal model systems of immunopathology in which IFN acts as an immunopotentiator Model

Observation

References

Streptozotocin-induced

Exogenous IFN aggravates disease

Campbell et al., 1988

Spontaneous or cyclophosphamideboosted diabetes in NOD mice

Anti-IFN antibody alleviates disease

Campbell et al., 1991a; Debraye-Sachs et al., 1991

IFN receptor knockout mice are protected

Wang et al., 1997

Anti-IFN antibody or soluble IFN receptor alleviate disease

Jacob et al., 1987; Ozmen et al., 1995

IFN receptor knockout mice are protected

Haas et al., 1998

MRL-lpr mice

IFN receptor knockout mice are protected

Balomenos et al., 1998

IL-10 knockout mice

Anti-IFN antibody prevents disease

Berg et al., 1996; Rennick et al., 1997

SCID mice reconstituted with CD45Rbhi CD4+ T cells

Anti-IFN antibody prevents disease

Powrie et al., 1994

IL-12-induced enteropathy

IFN knockout mice are insensitive; exogenous IFN induces enteropathy

Guy-Grand et al., 1998

Experimental autoimmune neuritis

Immunization of rats with peripheral nerve antigen in CFA

Anti-IFN antibody alleviates disease

Hartung et al., 1990; Strigard et al., 1989; Tsai et al., 1991

Experimental autoimmune orchitis

Immunization with syngeneic testicular germ cells (no adjuvant)

Late (day 20) treatment with anti-IFN antibody alleviates disease

Itoh et al., 1998

Autoimmune diabetes

Lupus-like disease

Inflammatory bowel disease

Female (NZB  NZW)F1 mice

changes (Campbell et al., 1991b). Breeding of a null mutation of the IFN receptor into the NOD mice resulted in a drastic reduction of insulitis and diabetes (Wang et al., 1997). Thus, the weight of the evidence favors the view that endogenous IFN stimulates autoimmune diabetes. The underlying mechanisms are considered to be (a) upregulation of MHC class I molecules, which could augment targeting of cytotoxic T cells to the islet cells, and (b) increased production of aspecific inflammatory mediators, e.g. NO. Female (NZB  NZW)F1 mice spontaneously develop a lupus-like syndrome, which has long been considered as a prototype autoantibody-mediated autoimmune disease. This view is reinforced by the cytokine production profile in these mice, which is of the TH2 type. Moreover, interventions which interfere with TH2-type cytokines, e.g. administration of anti-IL-10 antibodies, were found to alleviate disease. Endogenous or exogenous IFN would therefore be expected to also play a disease-alleviating role. However, the experiments turned out to yield the opposite result. Treatment with neutralizing anti-IFN

antibody (Jacob et al., 1987) or soluble IFN receptor (Ozmen et al., 1995) was found to prevent disease. These observations were recently reinforced by the report that IFN receptor-deficient (NZB  NZW)F1 mice are insensitive to the disease (Haas et al., 1998). The difference in sensitivity between the wild-type and mutant mice could entirely be explained by the different levels of autoantibody, indicating that IFN uses this pathway to accelerate disease. An obvious implication is that the TH1/TH2 concept fails to provide a suitable framework to explain the role of IFN in the NZB/NZW lupus model. Inflammatory bowel diseases (IBD) are now considered as disturbances in the delicate balance between immune reactivity and anergy towards microbial antigens and toxins present in the gut lumen. Few studies have been done on the role of IFN in models of IBD (see Table 7). The weight of the evidence is in favor of a disease-promoting role of endogenous IFN . Intestinal mucosal involvement in mice with graft-versus-host disease in SCID mice reconstituted with CD45Rbhi CD4+ T cells, and in

IFN IL-10 knockout mice was found to be alleviated by treatment with anti-IFN antibody (Powrie et al., 1994; Berg et al., 1996; Rennick et al., 1997). Also, IFN knockout mice were found to be resistant to IL-12-induced small bowel enteropathy, and exogenous IFN was found to be able by itself to cause mucosal epithelial damage (Guy-Grand et al., 1998), indicating that IFN produced by intraepithelial lymphocytes has direct cytotoxic effects on epithelial cells. Thus, in IBD, IFN seems to act more by its inflammatory than by its immunoregulatory potential. Table 7 reviews evidence for mixed immunosuppressive and immunostimulatory effects of IFN in a number of animal models. Graft-versus-host disease develops in irradiated mice reconstituted with allogeneic bone marrow. In several independent studies (Mowat, 1989; Brok et al., 1993) the contribution of this IFN to the disease manifestations has been assessed by the use of neutralizing anti-IFN antibodies. These studies are unanimous in observing that blockage of IFN inhibits disease development, in particular the lesions in the gut mucosa. On the other hand, as an apparent paradox, it has been reported that systemic administration of IFN inhibits disease development in much the same way as antiIFN antibody does. This inhibition was associated with reduced numbers of IFN -producing cells (Brok et al., 1993). Another model of graft-versus-host disease consists of inoculating semiallogeneic lymphocytes into neonatal mice, which allows for the persistence of the donor cells in the host. These cells differentiate into TH2-like cells as evident from predominance of IL-4 production over that of IL-2 and IFN . As an apparent consequence, donor B cells differentiate to produce large quantities of IgE and IgG1 autoantibodies, resulting in immune deposits and SLE-like pathology. In this model, exogenous IFN was found to prevent the disease, apparently by restoring the ability of the lymphocytes to produce IL-2 and IFN (Donckier et al., 1994). In collagen-induced arthritis treatment with antiIFN antibody has been reported to either inhibit or enhance disease, depending on the time of administration (Boissier et al., 1995). However, more recent studies have provided evidence for a uniform diseaseaggravating effect, as evident from accelerated occurrence of symptoms and a higher cumulative incidence (Williams et al., 1993; Vermeire et al., 1997; ManourySchwarz et al., 1997). Moreover, IFN receptor knockout mice were found to be more sensitive than corresponding wild types (Vermeire et al., 1997; Manoury-Schwarz et al., 1997). Remarkably, both experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis

671

(CIA) were found to be completely inhibited by treatment with anti-IL-12 antibody, indicating that, in these models, IL-12 acts independently from, and in opposite direction to IFN (Matthys et al., 1998; Heremans et al., 1999). On the basis of cytokine production profiles, EAE and CIA are both considered to result from TH1-type activity of autoreactive T cells. These observations are therefore seen to be at variance with the tenet, based on in vitro observations, that endogenous IFN participates in upregulating TH1-type reactivity. Recently, a possible explanation for the discrepancy has been proposed, based on the observation that the protective effect of endogenous IFN in CIA depends on the use of complete, as opposed to incomplete Freund's adjuvant. CIA induced by immunization with the aid of incomplete adjuvant, i.e. not containing heat-killed mycobacteria, was less pronounced in IFN receptor knockout mice or in anti-IFN antibody-treated mice than in corresponding controls. It was also found that the severity of the disease critically depends on the induction by the mycobacteria of an expansion of the Mac-1+ leukocyte population by intra- and extramedullary hematopoiesis, and that this myelopoiesis is much less pronounced in IFN -competent than in IFN -ablated mice (Matthys et al., 1999). In experimental autoimmune thyroiditis (EAT), a model which is operationally similar to EAE, EAU, or CIA, the role of endogenous IFN varies with genetic background and experimental conditions (Table 8). Deletion of the IFN receptor gene in H2k-haplotype mice has been reported to have little effect (Alimi et al., 1998), but deletion of the IFN ligand gene in H2q-haplotype mice resulted in more severe thyroiditis with granulomatous lesions and eosinophil infiltrations (Stull et al., 1992). On the other hand, treatment with anti-IFN antibody was found to inhibit actively induced disease (Tang et al., 1993), but to enhance disease if used to treat donor splenocytes in a transfer model (Stull et al., 1992).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Clinical evidence documenting the importance of IFN in human diseases is scarce. One reason for this is that determination of systemic or local IFN production in patients is impractical.

672 Alfons Billiau and Koen Vandenbroeck Table 8 Animal models of immunopathology in which IFN exerts mixed immunosuppressive and immunostimulatory effects Model Graft-versus-host disease

Collagen-induced arthritis (CIA)

Experimental autoimmune thyroiditis (EAT)

Observation

References

Reconstitution of irradiated mice with allogeneic bone marrow

Both IFN and anti-IFN antibody alleviate disease

Mowat, 1989; Brok et al., 1993

Allogeneic lymphocytes in neonatal mice cause TH2-associated SLE-like pathology

Exogenous IFN prevents disease, restores TH1 responsiveness

Donckier et al., 1994

Immunization with chicken collagen causes joint inflammation and deformity

Early anti-IFN antibody reduces severity; late antibody aggravates

Boissier et al., 1995

Anti-IFN antibody aggravates disease

Williams et al., 1993; Vermeire et al., 1997

IFN receptor knockout mice are more sensitive

Manoury-Schwarz et al., 1997; Vermeire et al., 1997

In vivo anti-IFN inhibits actively induced disease

Tang et al., 1993

Actively induced disease attenuated in IFN receptor knockout mice

Alimi et al., 1998

Anti-IFN -treated splenocytes are more pathogenic in the transfer model

Stull et al., 1992

IFN -deficient mice develop severe adoptive disease with eosinophilic infiltration

Tang et al., 1998

Immunization with thyroglobulin in CFA induces mononuclear infiltrate thyroiditis; transferable by spleen cells

Role in experiments of nature and disease states Genetic Defects Inherited deficiencies in the IFN receptor system have been described in children suffering from exceptional susceptibility to mycobacterial infections (for review, see Ottenhof et al., 1998). Complete IFN R1 deficiency, resulting from null mutations, is characterized by infections with low-virulent mycobacteria in early childhood. The granulomata are poorly differentiated and resemble those seen in lepromatous leprosy. Patients with partial IFN R1 deficiency, resulting from point mutations affecting the affinity of the receptor, have preserved the ability to form well-differentiated granulomata, but still are exceptionally susceptible to mycobacterial infections. A single case of complete IFN R2 deficiency, again suffering from early childhood mycobacterial infections, has also been described.

IFN Gene Polymorphisms and Disease A variety of animal experimental models have revealed a complex role, either disease-promoting or disease-limiting, for IFN in inflammatory diseases. Thus, the recent identification of a multiallelic microsatellite polymorphism in the human IFN gene has driven several research groups to investigate a potential genetic association with diverse autoimmune-type diseases, i.e. insulin-dependent diabetes mellitus, in Japanese (Awata et al., 1994) and Finnish and Danish individuals (Pociot et al., 1997); multiple sclerosis in German (Epplen et al., 1997), Finnish (Wansen et al., 1997), Sardinian (Vandenbroeck et al., 1998b) and Swedish (He et al., 1998) patients; and rheumatoid arthritis in British patients (John et al., 1998). The results of these analyses are summarized in Table 9. Though all these diseases show a certain degree of linkage to specific HLA class II alleles on chromosome 6, they are genetically highly heterogeneous and complex. In general, no global disease association with the IFN gene was seen in

IFN

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Table 9 Linkage and association analysis between a multiallelic CA repeat polymorphism in the first intron of the IFN gene and insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), and rheumatoid arthritis (RA) Disease type

Ethnicity (number of alleles detected)

Datasets (statistical tests used)

Statistically significant associations

References

Insulin-dependent diabetes mellitus (IDDM)

Japanese (8)

Case-control (chi square)

Global disease association; strong disease association in patients with onset  10 years of age

Awata et al., 1994

Danish/Finnish (5)

Case-control (chi square)

Global disease association in Finnish, but not Danish, case-control dataset

Pociot et al., 1997

Multiplex families (TDT)

No associations found

Pociot et al., 1997

German (11)

Case-control (chi square)

No associations found

Epplen et al., 1997

Finnish (5)

Case-control (chi square) Multiplex families (two point linkage analysis)

No associations found

Wansen et al., 1997

Sardinian (4)

Case-control (chi square) Simplex families (TDT)

Disease association in patients with low HLA-DR associated risk

Vandenbroeck et al., 1998b

Swedish (4)

Case-control (chi square)

No associations found

He et al., 1998

Multiplex families (two point linkage analysis)

Positive LOD score under assumption of recessive inheritance

Multiplex families (MLS IBD)

Increase in allele sharing in sibling pairs with an affected male

Multiple sclerosis (MS)

Rheumatoid arthritis (RA)

British

John et al., 1998

TDT, transmission disequilibrium test; MLS IBD, maximum likelihood score method for sharing of alleles by identity by descent.

any of the case-control data sets ± with the Japanese and Finnish insulin-dependent diabetes mellitus patients representing, however, a notable exception. By restricting the analysis to clinically (e.g. early disease onset) or genetically (e.g. family-based study designs) more homogeneous subsets of patients, significant associations were disclosed between specific IFNG CA-repeat alleles and insulin-dependent diabetes mellitus, multiple sclerosis, and rheumatoid arthritis. IgE Pathology Association of increased IgE levels with imbalance between IL-4 and IFN has been suggested to be involved in the pathogenesis of elevated IgE levels observed in patients with hyper-IgE recurrent infection syndrome (HIE) (King et al., 1989), atopic dermatitis,

or helminth infections. In HIE, increased IL-4 production has not been observed. Conversely, some but not all studies reported decreased production of IFN . Mitogen-driven IL-4 production by PBMCs of atopic subjects was found to be higher and production of IFN lower than that of PBMCs of normal subjects (Rousset et al., 1991). In patients with helminth infections, IgE levels were found to correlate with increased IL-4 and decreased IFN production by parasite antigen-stimulated lymphocytes (King et al., 1993). However, in a placebo-controlled trial, exogenous IFN failed to affect clinical parameters or IgE levels in patients with hayfever-type rhinitis due to ragweed allergy (Li et al., 1990). As a contrast, in a murine model for allergen sensitization, nebulized but not parenteral IFN was found to decrease IgE production and to normalize airway function (Lack et al., 1994).

674 Alfons Billiau and Koen Vandenbroeck Elevated levels of IgE are among the immunological characteristics of chronic atopic eczema. IFN responses have been reported to be defective in these patients (Reinhold et al., 1990). However, other changes are diminished delayed hypersensitivity reactions, diminished in vitro responsiveness to mitogens and recall antigens and a decreased proportion of CD8+ T-cells.

IN THERAPY

Preclinical ± How does it affect disease models in animals? See section on In vivo biological activities of ligand in animal models ± Normal physiological roles.

Clinical results Anti-infectious Potential Chronic granulomatous disease (CGD) is the name of a group of inherited deficiencies in the multicomponent enzyme NADPH oxidase, which is essential for the production by phagocytes of superoxide and related oxygen intermediates. Phagocytes of CGD patients ingest bacteria at a normal rate, but fail to effectively kill them. The patients suffer from recurrent and severe pyogenic infections that begin in early life and may lead to death. Pretreatment of normal phagocytes with IFN augments the respiratory burst triggered by other stimuli, such as LPS: IFN is believed to regulate the transcription of genes coding for the enzymes of the NADPH oxidase system. In a controlled clinical trial on CGD patients IFN was found to reduce the frequency of serious infections (International Chronic Granulomatous Disease Study Group, 1991). Although this study was initiated following reports (Ezekowitz et al., 1988; Sechler et al., 1988) of increased oxidative metabolism and bactericidal potential of CGD phagocytes, provoked by in vitro or in vivo exposure to IFN , there is controversy as to whether the clinical benefit is entirely or even partially accounted for by increased responsiveness of the NADPH oxidase system (International Chronic Granulomatous Disease Study Group, 1991; Muhlebach et al., 1992; Weening et al., 1996). The possibility is considered that oxygen-independent bactericidal mechanisms are involved. However, the clinical data have been confirmed (Weening et al., 1995) and extended to provide

evidence for a therapeutic effect in established hepatic abcess (Hague et al., 1993), which is a frequent type of infection in these patients. Obviously, in CGD patients spontaneous endogenous IFN production is insufficient to fully exploit what remains available of bactericidal potential. Optimal dosage regimens and differential responses of different categories of CGD patients are still under study (Ahlin et al., 1997). Visceral leishmaniasis (kala-azar), caused mainly by Leishmania donovani, is a severe disease characterized by fever, hepatosplenomegaly, anemia, and leukopenia. If left untreated, kala-azar mostly ends in progressive emaciation and death. The infection is endemic in most tropical and subtropical countries, with a focal distribution corresponding to the prevalence of specific sandfly vectors. The classical treatment consists of pentavalent antimony compounds or amphotericin B. Both types of treatment are frequently ineffective and mostly associated with toxic side-effects. Treatment with IFN in combination with antimony has been shown to provide a cure in some of the antimony-resistant (8 out of 10) or previously untreated (8 out of 9) cases (Badaro et al., 1990). In regions where the infection is highly resistant against antimony, the beneficial adjunctive effect of IFN seems to be limited (Sundar et al., 1997). Multidrug-resistant tuberculosis is likely to become an increasing concern in years to come. Preliminary evidence is available that treatment with IFN via aerosol may be part of alternative strategies (Condos et al., 1997). Prevention of infection in patients on cytostatic regimens represents another potential field of application. Aggressive therapy for certain forms of leukemia, while successful in children, fails in adults due to neutropenia resulting in uncontrollable infections (mostly pneumonia) with Pseudomonas, Staphylococci, and fungi. If IFN is to protect neutropenic patients it should do so mainly by acting on the mononuclear phagocyte system. A further proposed field of application of IFN is the control of infections occurring in patients with severe trauma, either accidental or resulting from aggressive surgery. Support for this possibility comes from results obtained in experimental wound infections with Klebsiella or Pseudomonas in mice (Hershman et al., 1988a, 1988b, 1988c). IFN might synergize with antibiotics and might thereby not only increase survival rates but also recovery rates. So far, placebo-controlled, double-blinded studies have failed to establish a clear-cut effect (Mock et al., 1996); one major difficulty being the large number and predominant impact of confounding variables

IFN affecting outcome. A European multicenter trial on the use of IFN to prevent burn-related infections also failed to reveal any beneficial effect (Wasserman et al., 1998). Anticancer Potential Ever since clinical-grade recombinant human IFN has become available, various regimens of treatment for malignant disease have been undertaken. It seems fair to state that, despite a tremendous input of effort, so far there have been no major breakthroughs in the field. Here, no attempt will be made to review these studies. Suffice it to state that the efforts are being continued along four main lines of investigation: 1. Systemic treatment of patients with recurrent or metastatic malignancy ± IFN alone or as adjunct to other drugs (IL-2, cytostatics) is being given to patients with melanoma (Kim et al., 1996; Kirkwood et al., 1997), colorectal cancer (Pavlidis et al., 1996), renal cell carcinoma (Lummen et al., 1996), small cell lung cancer (van Zandwijk et al., 1997), and non-Hodgkin lymphoma post stem cell transplantation (Nakao et al., 1997). These trials so far have borne out that IFN can be given without unsurmountable side-effects, for long periods of time, and at doses which affect immunological parameters. 2. IFN as an adjunct to TNF in isolated limb perfusion in primary or recurrent melanoma ± Isolated limb perfusion with TNF in conjunction with IFN has been shown to be effective in bringing about regression of melanoma skin lesions (Lienard et al., 1998). 3. Immunization with IFN gene-modified autologous melanoma cells ± Several strategies are currently being developed to combat cancer by immunization against cancer cell antigens. One approach consists in administering irradiated autologous tumor cells which have been engineered to express one or several immune-stimulating cytokines. In view of its immune-potentiating effects IFN is a prime candidate (Abdel et al., 1997). 4. Immunotherapy with autologous IFN -activated macrophages ± Activated macrophages, by virtue of their cytocidal capacities, are considered to be essential players in host defense against cancer. One strategy to put this anticancer potential to use consists in harvesting monocytes from the patient's blood and reinjecting them after they have been cultured in the presence of IFN (for review, see Andreesen et al., 1998).

675

Antiatopic Potential In a double-blind, placebo-controlled trial in patients with chronic atopic dermatitis (Hanifin et al., 1993) therapy with recombinant IFN has been found to result in improvement of the clinical parameters. However, therapeutic failures in children with severe refractory disease have also been reported (Horneff et al., 1994). Long-term IFN therapy for severe atopic dermatitis is considered a safe practise (Schneider et al., 1998).

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