IL-8

IL-8 Hisashi Iizasa1 and Kouji Matsushima2,* 1

Department of Pharmaceutics, Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan 2 Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan * corresponding author tel: 81-3-3812-2111, fax: 81-3-5800-6853, e-mail: [email protected] DOI: 10.1006/rwcy.2000.10003.

SUMMARY

Alternative names

Interleukin 8 (IL-8), a proinflammatory chemokine, is produced by various types of cells upon stimulation with inflammatory stimuli and exerts a variety of functions on leukocytes, particularly, neutrophils in vitro. Recent studies show that inhibition of IL-8 functions by either administration of specific antibody or disruption of the gene encoding the IL-8 receptor dramatically reduced neutrophils infiltration into acute inflamed tissues. IL-8 plays an pivotal role in acute inflammation by recruiting and activating neutrophils.

IL-8 has also been named monocyte-derived neutrophil chemotactic factor (MDNCF) (Matsushima et al., 1988), neutrophil-activating factor (NAF) (Walz et al., 1987), neutrophil-activating protein 1 (NAP-1) (Schroder et al., 1987), granulocyte chemotactic peptide (GCP) (Van Damme et al., 1988), leukocyte adhesion inhibitor (LAI) (Gimbrone et al., 1989).

BACKGROUND

Discovery In the early 1980s, Matsushima and coworkers purified IL-1 from LPS-stimulated human monocyte culture supernatants. Partially purified IL-1 or IL-1 had been claimed to be chemotactic for neutrophils at that time, although highly purified preparations of either form were not. This result indicated that contaminants in partially purified IL-1 had neutrophil chemotactic activity. The factor with this activity was then purified by Yoshimura et al. (1987) and molecularly cloned from the cDNA library of LPS-stimulated human monocytes (Matsushima et al., 1988). It was initially named monocyte-derived neutrophil chemotactic factor (MDNCF). MDNCF was found to have additional target cells including T lymphocytes (Larsen et al., 1989). Therefore, MDNCF was renamed as interleukin 8 (IL-8) (Balkwill and Burke, 1989).

Structure Four types of differentially processed forms of IL-8, consisting of 69, 72, 77, and 79 amino acids, are known. Among these variants, the 72 amino acid form is predominant and has greatest activity on neutrophils. In addition, IL-8 is a dimer at high concentrations. The dimer interface is formed by three antiparallel strands from each monomer and the  helices are formed in the C-terminal region.

Main activities and pathophysiological roles IL-8 has a chemotactic activity for neutrophils and also activates neutrophil functions. It also induces angiogenesis and inhibits the proliferation of myeloid progenitor cells. High levels of IL-8 have been detected in biofluids of various acute inflammatory diseases. IL-8 is an essential factor for neutrophil infiltration in most inflammatory reactions.

1062 Hisashi Iizasa and Kouji Matsushima

GENE AND GENE REGULATION

PROTEIN

Accession numbers

Accession numbers

See Table 1.

See Table 2.

Chromosome location

Sequence

4q12-4q21 (human).

See Figure 1.

Relevant linkages

Description of protein

Chr4. D4S392-D4S2947 (77.9±86 cM, human).

In human: pI 8.6 Amino acids size: precursor 99 mature 69±79 (main form is 72) Molecular weight of mature form 8 Disulfide bonds 2 N-linked glycosylation sites 0

Regulatory sites and corresponding transcription factors The 50 flanking region of the IL-8 gene contains potential binding sites for several transcription factors such as AP-1, NFB and NF-IL6 (Mukaida et al., 1989). NFB is essential in many cell types, but either NF-IL6 or AP-1 have to act together with NFB to activate the IL-8 gene (Matsusaka et al., 1983).

Cells and tissues that express the gene IL-8 is secreted from various cell types during inflammation. IL-8 has been detected in inflamed tissues and biofluids in many diseases. Table 1 Accession numbers for IL-8 mRNA and genes for various species Species

mRNA

Gene

Human

Y00787

M28130

Macaque

S78555

Sooty mangabey

U19839

Bovine

S82598

Sheep

X78306

Pig

M86923

Dog

U10308

Rabbit

M57439

Guinea pig

L04986

Chicken

X14971

AF061521

Discussion of crystal structure Structural analysis of recombinant IL-8 by NMR (Clore et al., 1990) and X-ray crystallography

Table 2 Accession numbers for IL-8 protein in various species Species

Accession numbers

Human

P10145

Macaques

P51495

Sooty mangabey

P46653

Bovine

P79255

Sheep

P36925

Pig

P26894

Dog

P41324

Rabbit

P19874

Guinea pig

P49113

Chicken

P08317

D14285 Figure 1 Amino acid sequence for human IL-8 precursor. M83361

Human (99 amino acids: precursor): MTSKLAVALL AAFLISAALC EGAVLPRSAK ELRCQCIKTY SKPFHPKFIK ELRVIESGPH CANTEIIVKL SDGRELCLDP KENWVQRVVE KFLKRAENS

IL-8 1063 (Baldwin et al., 1990, 1991) revealed that IL-8 is a dimer at a high concentration. This dimer consists of six strands and two  helices of the C-terminal in the antiparallel region. Interestingly, the IL-8 structure looks similar to class I major histocompatibility complex. However, it is not known whether dimer formation has any physiological significance, since the monomer of IL-8 is equally active to neutrophils in vitro (Rajarathnam et al., 1994).

Important homologies IL-8 is a prototype of the `chemokine superfamily' which consists of over 40 different molecules (Oppenheim et al., 1991). IL-8 belongs to the CXC chemokine subfamily, which consists of chemokines now known to regulate not only migration of various types of leukocytes but also movement of hematopoietic progenitor cells and homing of lymphocytes. The Glu-Leu-Arg (ELR) motif in the N-terminal region of IL-8 is essential for binding to its receptors (Herbert et al., 1991). This motif is also important for neutrophil chemotactic and angiogenic activities in other CXC chemokines such as GRO, NAP-2, and ENA-78, which bind to CXCR2 (Clark-Lewis et al., 1993). Nevertheless, some ELRÿ CXC chemokines, such as stromal derived factor 1 (SDF-1), inhibit angiogenesis without the ELR motif.

IL-8 secreted by inflamed tissues is internalized and transported through endothelial cells (Middleton et al., 1997). The C-terminal end of IL-8 is essential for internalization as well as heparin binding.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators LPS and inflammatory cytokines induce the production of IL-8. These inducers also activate NFB. In contrast, IL-4, IL-10, TGF , some interferons, and immunosuppressive drugs such as glucocorticoids, vitamin D3, and FK506 inhibit the expression of the IL-8 gene (Okamoto et al., 1994) through targeting NFB.

RECEPTOR UTILIZATION IL-8 binds to two types of receptors, CXCR1 and CXCR2, which belong to the G protein-coupled seven transmembrane receptor superfamily.

IN VITRO ACTIVITIES

Posttranslational modifications

In vitro findings

Secreted IL-8 is not glycosylated. IL-8 variants are derived by sequential cleavage from the N-terminal end of the molecule. In vitro, the most prominent active form of IL-8 is the 72 amino acid form. The 77 amino acid form of endothelial cell-derived IL-8 has been reported to only induce apoptosis against leukocytes (Terui et al., 1998). However, the pathophysiological role of these variants in vivo remains to be established.

IL-8 is a potent chemoattractant for neutrophils, but in vitro IL-8 also activates neutrophil function such as release of lysosomal enzymes, generation of superoxide/biolipids, and increases the expression of adhesion molecules on neutrophils as demonstrated by Peveri et al. (1988), Schroder (1989), and Paccaud et al. (1990), respectively. IL-8 also has been shown to chemoattract basophils, cytokine-stimulated eosinophils, human peripheral blood T lymphocytes by White et al. (1989), Warringa et al. (1991), and Larsen et al. (1989), respectively. Interestingly, IL-8 also increases the adhesion of neutrophils to unstimulated human umbilical cord vein cells but inhibits the adhesion of neutrophils to endothelial cells prestimulated by inflammatory cytokines such as IL-1 and TNF (Gimbrone et al., 1989). IL-8 enhances transendothelial migration of neutrophils (Huber et al., 1991), and induces angiogenesis in rat cornea without inducing leukocyte infiltration (Koch et al., 1992). These functions of IL-8 suggest important roles of IL-8 in inflammation as well as

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-8 is secreted from many cell types, including monocytes, lymphocytes, granulocytes, fibroblasts, endothelial cells, bronchial epithelial cells, keratinocytes, hepatocytes, mesangial cells, and chondrocytes.

1064 Hisashi Iizasa and Kouji Matsushima host defense (Table 3). On the other hand, IL-8 enhances viral replication including that of cytomegalovirus in human fibroblasts (Murayama et al., 1994) by inhibiting the antiviral activities of IFN (Khabar et al., 1997).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Bioassays used

In many diseases, IL-8 has been detected in inflamed tissues and biofluids. These include the skin lesions of psoriasis (Schroder and Christophers, 1986), and synovial fluids of rheumatoid arthritis (Brennan et al., 1990), osteoarthritis (Symons et al., 1992), and gouty arthritis (Terkeltaub et al., 1991). Elevated IL-8 levels are also detected in other biological fluids such as bronchoalveolar lavage (BAL) fluids (Carre et al., 1991), pleural fluids (Broaddus et al., 1992) and urine (Ko et al., 1993) (Table 4). Significant correlations between IL-8 levels and neutrophil infiltration in diseases has been reported. IL-8 is an essential factor for acute inflammation. However, recent studies have demonstrated that IL-8 is also important for noninflammatory reactions. In mice, a functional IL-8 homolog, MIP-2, induces migration of neutrophils into vagina in sexual cycle-dependent manner (Sonoda et al., 1998). In addition, systemic administration of IL-8 also rapidly induces migration of hematopoietic stem cells from bone marrow to peripheral blood (Laterveer et al., 1995). These result suggest that IL-8 also regulates noninflammatory physiological reaction in vivo.

IL-8 is measured by neutrophil chemotaxis and activation. However, this is not a specific assay since other CXC chemokines also induce migration of neutrophils in vitro. To distinguish between the effect of IL-8 and that of other chemokines, monoclonal anti-IL-8 antibody is useful. Table 3 Biological activities of IL-8 in vitro Target cells

Biological activities

Neutrophils

Chemotaxis Lysosomal enzyme release Respiratory burst Intracellular calcium influx Generation of superoxide anion Generation of biolipids (LTB4, 15-HETE, etc.) Induction of expression of adhesion molecules (CD11a, CD11b, CD11c, and CD18) Transendothelial migration

T cells

Chemotaxis

B cells

Inhibition of IL-4-induced IgE production

Basophils

Chemotaxis

Normal physiological roles

Table 4 Diseases with elevated expression of IL-8 Biological fluids

Diseases

BAL fluid

Acute respiratory distress syndrome (ARDS) Idiopathic pulmonary fibrosis

Inhibition of histamine release

Pulmonary edema (reperfusion injury)

Increased leukotriene release Monocytes

Intracellular calcium influx Respiratory burst Adhesion

Keratinocytes

Proliferation

Fibroblasts

Decrease of collagen mRNA expression

Pleural fluid

Empyema

Urine

Urinary tract infection (UTI) IgA nephropathy Acute glomerulonephritis (AGN) Purpuric nephritis Membranous proliferative glomerulonephritis (MGPN)

Induction of cytomegalovirus replication Endothelial cells

Proliferation

Smooth muscle

Chemotaxis

Stem cells

Inhibition of colony formation of myeloid progenitors

Lupus nepritis Synovial fluids

Rheumatoid arthritis Osteoarthritis Gout

IL-8 1065 IL-8 also induces the infiltration of T lymphocytes into inflamed tissue. Continuous injection of IL-8 caused massive migration of T lymphocytes into injected joints (Kudo et al., 1991). In a delayed type hypersensitivity reaction model, monoclonal antiIL-8 antibodies reduced the infiltration of both neutrophils and lymphocytes (Larsen et al., 1995). These results suggest that IL-8 also has a significant role in directly or indirectly regulating the migration of T lymphocytes in inflammatory reactions.

Species differences IL-8 homologs in other species have been cloned, including guinea pigs, sheep (Yoshimura and Johnson, 1993), rabbits (Yoshimura and Yuhki, 1991), pigs (Goodman et al., 1992), and dogs (Ishikawa et al., 1993). However, no clear-cut IL-8 homolog in rat or mouse has been identified so far, although MIP-2 and KC may be functional homologs of IL-8 in mice. Furthermore, IL-8 binds to two types of IL-8 receptors in human, but mouse has only one IL-8 receptor (homolog of human CXCR2).

Knockout mouse phenotypes Although mice lack an exact homolog of IL-8, the one IL-8 receptor that exists, MuCXCR2, is bound by MIP-2 and KC. Gene targeting of CXCR2 in mice exhibited inhibition of neutrophil infiltration into inflamed tissue although neutrophil function was normal (Cacalano et al., 1994). This phenotype is similar to blocking of IL-8 activity using anti-IL-8 antibodies. However, other neutrophil chemotactic factor-related gene knockout mice such as complement 5a receptor (Hopken et al., 1996) and the 5-lipoxygenase (Goulet et al., 1994) did not show any impairment of neutrophil influx into inflamed sites. These data strongly suggest that these classical chemotactic factors are not important for conventional neutrophil infiltration of inflammatory sites, and that IL-8 is essential for neutrophil infiltration of inflamed tissues.

Transgenic overexpression Mice transfected to overexpress IL-8 exhibit excessive accumulation of neutrophils in the microcirculation of the lung, liver, and spleen without neutrophil infiltration into any tissues, plasma exudation, or apparent tissue damage (Simonet et al., 1994). Lselectin expression is decreased on the surface of

circulating neutrophils in such IL-8 transgenic mice, whereas that of bone marrow neutrophils and neutrophil precursors was normal. In addition, no significant upregulation of the 2 integrins level was observed on peripheral blood neutrophils in this transgenic mouse. These results indicate that IL-8 modulates the surface expression of L-selectin without showing any effects on 2-integrin level.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Role in experiments of nature and disease states The in vitro biological activities of IL-8 and increased production of IL-8 in inflammatory diseases encouraged scientists to administer specific monoclonal antibodies (mAbs) against IL-8 in animal models of acute inflammatory diseases to establish the pathophysiological role of IL-8 in vivo. In the rabbit lung reperfusion injury model, administration of anti-IL-8 mAb dramatically blocked tissue injury by inhibiting neutrophil infiltration into lung tissue in spite of the presence of other neutrophil chemoattractants such as complement components (Sekido et al., 1993). Furthermore, treatment with anti-IL-8 mAb blocked neutrophil infiltration into inflamed tissue, and prevented tissue injury in many other animal models such as LPS-induced dermatitis (Harada et al., 1993), experimental acute immune complex-induced glomerulonephritis (Wada et al., 1994), acid aspirationinduced acute respiratory distress syndrome (ARDS) (Folkesson et al., 1995) and cerebral ischemiareperfusion injury (Matsumoto et al., 1997).

IN THERAPY

Clinical results Humanized monoclonal antibody against IL-8 is now in clinical trial by several companies. For example, Abgenix Inc. (Abgenix) reported an ongoing phase I/II clinical trial of anti-IL-8 antibody (they named ABX-IL8) in psoriasis (http://www.abgenix.com). ABX-IL8 is a completely human antibody derived from a transgenic mouse in which the mouse antibody was replaced by a human antibody gene. They plan to enroll 42 patients with moderate to severe psoriasis.

1066 Hisashi Iizasa and Kouji Matsushima They have already finished a phase I trial of ABX-IL8 in 33 patients with moderate to severe psoriasis. Next, they plan to test it in rheumatoid arthritis patients. Small chemical antagonists to IL-8 receptors have been also developed. Clinical development of such IL-8 antagonists will encourage the development of pharmaceuticals to be used in various other human inflammatory and immune diseases targeting other IL-8-related members of the chemotactic cytokine family.

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