Volume 307, number I, 97-101 FEW I I242 July I9992 0 1992 Federation of European Biochemical Societies ;0145793/92/W_Kl

Minireview -8, a chemotactic and inflammatory

Marco Baggiolini” and Ian Clark-Lcwisb

“Tltcodor-Koclrer Iturirtrrr. ilttiversiryof Bern, 5ern, Snir:eriuttd and “7%~Biorttedicul Research Cotwe artdDeparmrmtr of Biocltern- istry, Uhversiry qf Rririslt Colttntbia, Vancouver, Cunuda

Received 12 May 1992 lntcrlcukind (IL-Q belongs to a family of small, structurally related cytokincs similar to platclct factor 4. It is produced by phagocytes and mcsenchymal cells exposed to inflammatory stimuli (e.g. interlcukin-I or tumor necrosis her) and activales ncutrophils inducing chcmotaxis, cxocytosis and the respiratory burst. In vivo, IL-8 elicits a massive neutropbil accumulation at thz site or injection. Five nuutrophil-activating similar to IL-8 in structure and function have been identified recently. IL-8 and the related cytokincs are produced in several tissues upon infection, inflammation, ischcmia, trauma etc., and are thought to bc the main cause of local ncutrophil accumul;ition.

Inurleukin 5; Rcccplor; Biological function; activation; Pathophysiology

1. INTRODUCTION fide bridges. It is resistant to plasma peptidases, heat, pM extremes and other denaturing treatments, but is The accumulation of neutrophil leukocytes in a tissue rapidly inactivated when the disulfide bonds are re- is the hallmark of inflammation, a defence reaction to duced [l]. Nuclear magnetic resonance spectroscopy infection or other injury, with participation of blood, and X-ray crystallography studies indicate that IL-8 blood vessel and tissue elements. The are forms a dimer [2]. The monomer has a short, confor- recruited from the blood by chemotactic stimuli that mationally flexible N-terminal domain that is anchored direct their migration through the vessel wall to the by the two disulfide bonds to a core structure consisting affected site. of three antiparallel b-strands followed by a terminal Interleukin-8 (lL-8) was identified in 1987 as a novel a-helix (Fig. 2). type of neutrophil-activating cytokine [l]. Today we know its sequence and 3D structure, the sequence and binding properties of its receptors, and the profile of IL-8 was originally recognized as a neutrophil-acti- biological activity. Several additional cytokines related vating protein on the basis of two in-vitro effects, che- to IL-8, but arising from different have also been motaxis and the release of granule enzymes [I]. These characterized. IL-8 and related cytokines are released effects suggested that IL-8 ~8s a novel type of chemoat- by phagocytes and a wide variety of tissue cells upon tractant, and for this reason it was compared exten- exposure to inflammatory stimuli. They are the main sively with well established chemotactic agonists, such tissue-derived chemoattractants for neutrophils. as CSa, filet-Leu-Phe, platelet-activating factor (PAF) and leukotriene BA(LTB,). These stimuli induce three main responses in neutrophils: (i) shape change and 2. PROPERTIES directional migration, (ii) exocytosis of storage proteins, 2.1. SrnKrure and (iii) the respiratory burst. lL-8 is generated as a precursor of 99 amino acids and The shape cltmge reflects the activation of the con- is secreted after cleavage of a signal sequence of 20 tractile system and enables the neutrophils to adhere to residues. N-terminal extracellular processing of the ma- cndothelial cells and to migrate. In ncutrophil suspen- ture form yields several biologically active variants (Fig. sions, the shape change can be monitored in a laser 1). The predominant variant consists of 72 amino acids nephclome~er as a transient rise in light transmission, and has a molecular weight of 8,383. It is a basic protein which reflects the decrease in cell body volume conse- (PI 8.3) and contains four cysteines that form two disul- quent to the formation of large and thin lamellipodia [3]. The shape-dependent transmission increase in re- Cor!rsponchw address: M. Baggiolini, Thcodor-Kochcr Institute, sponse to IL-8 is virtually instantaneous and is similar Cl-l-3000 Bern 9, Switzerland. Fax: (41) (31) 653 799. in kinetics and extent to that observed with CSa OF

Published by Elselscvbr Scietm Publislwrs R. V. 97 Volume 307, number I FEDS LETTERS July IX! fMet-Leu-Phe. When the response of the cells is syn- 2.4. Efjkcts itt viva chronized by prestimulation, the transmission changes Studies in rabbits showed massive neutrophil accu- induced by chemoattractants including IL-S show regu- mulation and plasma exudation upon intradermal injec- lar oscillations that correlate with the assembly and tion of human IL-8. The effect was rapid and long- disassembly of fllamentous actin, and are thought to lasting. The cellular infiltration was already significant reflect the protrusion and retraction of lamellipodia [3]. after I5 min, and the immigration of neutrophils re- E_socylosisleads to the release of soluble storage pro- mained detectable for up to 10 h [9,10]. Resistance to teins and the remodeling of the neutrophil plasma mem- inactivation and slow clearance from the injection site, brane by fusion with the storage organelles. IL-8 trig- possibly due to charge interaction with acidic tissue gers the exocytosis of specific granules and secretory matrix components, could explain the long duration of vesicles. Upon pretreatment with cytochalasin B, these action. Comparable responses to human IL-8 were ob- responses are markedly enhanced, and the release of served in mice, rats, guinea pigs and dogs. Studies in enzymes from the azurophil granules, e.g. elastase, is human skin showed the same kind of inflammatory observed in addition. IL-8 dependent SM$K~ntmbrme reaction with exclusive infiltration of neutrophils in par- rmrodehg during exocytosis leads to the expression of ticular around venules. numbers were not adhesion molecules, CD I I b/CD I S (complement recep- increased, and no basophils, eosinophils or tor type 3), CD1 It/CD 18 (pl50,95) and complement were found. IL-8 caused no wheal and Rare, itching or type I [4,5]. This upregulation markedly en- pain, suggesting that it does not induce histamine re- hances the ability of neutrophils to adhere to endothe- lease from skin mast cells [ 11,121. lial cells and to the extracellular matrix. The respircrtorybum is characteristic for stimulated 3. UBIQUITOUS PRODUCTlON phagocytes, Like other chemoattractants IL-S elicits the rapid and transient activation of the NADPH-oxidase, IL-8 was originally identified in the medium of leading to superoxide and HIOz formation. In terms of human blood monoq :es cultured in the presence of duration and intensity the respiratory burst induced by endotoxin, phorbol esters or lectins [I]. It was soon IL-8 is considerably weaker than that observed with realized, however, that many different cells have the flvIet-Leu-Phe or C5a, but more pronounced than that induced by PAF or LTB, [ 1, 61.

IL-8 receptors are coupled to BorrkteCla pcrrtrssis toxin-sensitive GTP-binding proteins. Signal transduc- tion depends on the activation of a phospholipase C specific for phosphatidylinositol 4,5-bisphosphate, which delivers two second messengers, IP3 and dia- cylglycerol. 1P3induces a rise in cytosolic free calcium. and diacylglycerol activates protein kinase C. ADP-ri- bosylation of GTP-binding proteins by pertussis toxin, depletion of mobilizable calcium, and exposure to pro- tein kinase inhibitors or to wortmannin can be used to modulate signal transduction and to influence shape change, exocytosis and the respiratory burst [7]. The responses of neutrophils to TL-8 and fMet-Leu- Phe are modified in a similar way by such treatments, indicating that they are controlled by a comparable mechanism [8].

EGAVPLRSARELREQ~... 79 aa

AVPLRSAKRLRSQ~... 77 aa

SAKELRiZQQ... 72 aa Fig. 2. Ribbon rcprescntation of the polypeptidc backbone of the 69 RELREQC... 70 aa amino acid form of IL-8 as a dimer. The N-terminal region (bottom right, top left) is anchored to the rest of the molecule by two disulfide ELRCQE.. . 69 aa bridges, An N-terminal loop follows the frst two cystcines and leads into the central/Sstrand thut interfaces with the other monomer. After Fig. l I N tcrmini of the naturally occurring, biologically active forms two additionalp-strands, the structure ends with a C-terminal a-helix. or IL-S. Diffcrcnt shading was used for the monomers.

98 Volume 307, number I FEBS LETTERS July 1992

l3TL-8 SAKELRCQCIKTISKPPHPKFZKELRV~ESGPSC NAP-Z A. . ..Mm...T.G-f...N.QS.E..GK.T.. GROa ASV.T...*.*LQ.LQG-I...N.QSYbl.KSP..,. GROR APL.'l?...*.*LQ.LQG-I.L.N.QSVK.K3P.... GROy ASVVT...a..LQ.LQG-T.L.N.QSVN.RSP.... ENA- AGBAAAVLR...*V*LQ.TQG-V...M.SN.Q.FAI..Q* PF-4 EAEEDGD.Q*L*V..T.Q-VR.RH.TS.E..KA...* IPICI VPLSRTV.rT..SfSNQ.VN.RSLEK.EiX.PASQFm IL-8 SAKELRCQCIKTISKPPHPKFIKELRYIBSGPnC

Fig, 3. N-tcrmiaal sequences of proteins related to lt-8 (72 amino acid form) aligned according IO the cystcincs. Note that the ELR (Glu-Lcu-Arg) sequence preceding the first cysuinc is shared by all proteins with ncutrophil-activating proprtim.

ability to produce IL-8 when appropriately stimulated. wald and Clark-Lewis, unpublished results), Finally, an The expression of IL-8 mRNA and the release of the epithelial cell derived neutrophil-activating peptide, biologically active cytokine was observed in endothelial EN’A-78,was discovered in the cultures of type-11 alve- cells, fibroblasts from different tissues, keratinocytes, olar cells [21]. synovial cells, chondrocytes, several types of epithelial IL-8 and its five related chemotactic cytokines arise cells as well as some tumor cells [ 1,131. Interestingly, from different genes clustered on 4 [22]. In even neutrophils can synthesize IL-8 [14], and may thus pathological conditions, it is likely that several of them intensify their own recruitment to sites of inflammation. are generated concomitantly as suggested by experi- Interleukin-1 (IL-l) and (TNF) ments with type-11 lung epithelial cells which release are clearly the most important stimuli, since they were IL-8, GROa and GROy in addition to ENA- [21]. It found to induce IL-8 expression and secretion in all cells has been shown in several laboratories that the expres- studied so far [1 51. Endotoxin is very effective on phag- sion of IL-8 and GROa is regulated in a similar way in ocytes and endothelial cells, but inactive on mesenchy- a variety of different cells [IS]. On the other hand, differ- mal cells, klonocytes and generate IL-8 ential expression in dependence of the tissue and the upon stimulation with IL-la, IL-l,& TNFa, IL-3, GM- stimulus has been reported for the three gro genes [20]. CSF, endotoxin, lectins, phorbol esters, immune com- The existence of so many structurally related proteins plexes and phagocytosis [16]. Opsonized particles are with comparable neutrophil-activating effects is aston- also a prime stimulus for IL-8 production by neutro- ishing. It may be taken to suggest that the function of phils [ 141. IL-8 is so important that several back-up genes have evolved. It is conceivable, however, that these different cytokines may have distinguishing biological properties 4. REDUNDANCY apart from their common effects on neutrophils. lL-8 belongs to a family of sequence-related 8-10 kDa proteins characterized by the conserved position of 5. IL-8 RECEPTORS their four cysteine residues. Platelet basic protein, con- nective tissue-activating peptide III and platelet factor Homologous desensitization was used initially to 4, which are components of the a-granules of blood demonstrate that IL-S acts via a selective receptor. It platelets, and y- inducible protein (IPlO) were was shown that neutrophils remain responsive to IL-8 known before IL-S. They differ from IL-8 by their lack after stimulation with fMet-Leu-Phe, CSa, PAF or of neutrophil-activating properties [l]. Several proteins LTB,, and that IL-8 does not desensitize the cells that share a!ructural and biological similarity to IL-8 against these other chemoattractants [23]. IL-8 recep- were discovered more recently, Neutrophil-activatitzg tors on human neutrophils and myeloid cell lines were peptide- 2 (NAP-2) derives from the N-terminal proc- then demonstrated in several independent studies. In essing of platelet basic protein or connective tissue-acti- general agreement with their results, we have shown vating peptide-III, which are released from stimulated that human neutrophils possess on average G4,KK.lf platelets [17]. GROa designates a protein that was orig- 14,000 receptors with an apparent Kd of 0. I8 10.07 nM inally described as melanoma growth stimulatory activ- [24]. Competition for the binding of radiolabelled IL-8 ity (MGSA) on the basis of its effects on certain mela- was obtained not only with unlabelled IL-S, but also noma cell lines [18], and was later shown to be a power- with NAP-2 and GROa. These studies revealed the exis- ful neutrophil chemoattractant [19]. Two related mole- tence of two types of receptors: one with high affmity cules, GROP and GROy, were found subsequently [20]. for all three ligands (& 0.1-0.3 nM). and one with the The three GRO proteins have about 90% sequence iden- same afhnity for IL-S, but lower affinity for NAP-2 and tity and similar neutrophil-activating properties (De- GROa (& 100-l 30 nM) f24]. Two membrane proteins

99 Volume 307, number I FEBS LETTERS July 1992 that bind lL-8, NAP-2 and GROa were found in receptor are required. Since nuclear magnetic resonance crosslinking experiments in agreement with Samanta et spectroscopy, shows that the N-terminus has a high de- al. [2S]. Furthermore cross-desensitization indicated gree of flexibility, the conformation of the entire protein that NAP-:! and GROa [24] as well as ENA- [21], appears to be important. This is in agreement with the GRO,8 and GROy (Dewald and Clark-Lewis, unpub- observation that IL-8 is inactivated when the disulfide lished results) share the receptors with IL-S. bonds are eliminated either by reduction or by substitu- tion of the cysteines. 5.1, Receptor structrrre A major advance in the understanding of neutrophil 6. PATHOPHYSIOLOGY activation was the demonstration that chemotactic ag onist receptors belong to the seven-transmembrane-do- The ability to attract and activate neutrophils quali- main type, Soon after the pioneering work of Boulay et fied IL-8 from the beginning as an inflammatory medi- al. [26] on the fMet-Leu-Phe receptor, two IL-8 receptor ator [I]. Meanwhile thi.s hypothesis has been amply ver- cDNAs were cloned and shown to code for similar in- ified, in particular by the study of inflammatory skin tramembrane molecules [27,28]. These observations and joint diseases. Considerable IL-8 immunoreactivity provided the last piece of evidence that IL-S, despite its is found in the skin of patients with psoriasis and palmo- name, must be regarded as a chemotactic agonist. Ex- plantar pustulosis [32-341, where expression of IL-8 tensive biological experimentation had already strongly mRNA is prominent in keratinocytes [35]. IL-8 accumu- indicated that the effects of IL-8 on neutrophils were lates in the synovial fluid of arthritic joints [16,36] and typical for chemoattractants [I], and the recent informa- is released by stimulated synovial cells [37] and chon- tion on the receptor sequence showed that this inrerpre- drocytes [38]. Mononuclear cells from the blood or syn- tation was correct. An important consequence of the ovial fluid of patients with rheumatoid arthritis release finding that IL-8 acts through a chemoattractant, rather much higher amounts of IL-8 than cells from healthy than a cytokine, or -type receptor is that controls after stimulation with LPS, IL-l, TNF and natural antagonists or soluble receptors, analogous to immune complexes [ 161. those for IL-I, TNF and some other cytokines [29], are IL-8 appears to be particularly suited for a role in unlikely to be found for IL-S. The cloning of two dis- inflammation and host defence. As a product of differ- tinct cDNAs is in agreement with the biochemical evi- ent types of cells it can arise irl any tissue when the levels dence for two IL-8 receptors. Information on the se- of IL-I and/or TNF (which are powerful inducers) are quence of the receptors identified by crosslinking must enhanced. Generation of IL-8 can be expected upon now be obtained. infection, ischemia, trauma, and other disturbances of tissue homeostasis, since IL-1 and TNF levels are ele- 5.2. Structurul determinants of receptor binding vated. In all these conditions IL-8 is likely to be the The 3D structure of the IL-8 dimer bears some resem- main cause of the local accumulation of neutrophils [ 1, blance with the antigen-binding domain of the HLA 133. Resisrmce to irzuctivationarzd slow clearance, as molecule. Since the groove that binds the antigen is shown by the long-lasting chemoattractant effect after formed by two a-helices it was thought that the C- intradermal injection [lo], suggests that IL-8 persists in terminal helices of the IL-8 dimer constitute the recep- active form within the immediate environment of the tor binding site. Recent structure-,activity relation stud- cells from which it is released. Other chemoattractants ies, however, indicate that IL-8 binds at the N-terminus like fMet-Leu-Phe, CSa, LTB4 and PAF, by contrast, [30]. Using chemically synthesized analogs, we have act more transiently because they are inactivated rap- found that removal of the entire C-terminal domain idly by oxidation or hydrolysis [lo]. decreased, but did not suppress biological activity. In Most remarkable is the high number of related cyto- contrast, no receptor binding or neutrophil activation kirzes. Five distinct chemotactic proteins have been was observed when the N-terminal sequence Glu-Leu- identified that share structural and biological similarity Arg that precedes the first cysteine was deleted. All with IL-S, and bind to the IL-8 receptors. Redundancy three residues, Arg in particular, are highly sensitive to strongly suggests that these cytokines are relevant in modification. The role of Glu-Leu-Arg is also empha- physiology and pathology. IL-8 and its related cytoki- sized by a recent mutagenesis study showing that re- nes can be expressed concomitantly by cells that are placement of these residues by alanine results in loss of stimulated with lL-l or TNF. They are likely to concur activity [31]. In this context it is interesting to note that in recruiting neutrophils, but may also fulfill additional, the Glu-Leu-Arg motif is common to all IL-8 related still unknown functions. chemotactic cytokines, but is not found in platelet fac- tor 4 and IPIO (Fig. 3). Short peptides containing the Acknosll~~~e/,~er~rs:This work was supported by the Swiss National Science Foundation. Grant 31-25700.88, and the Protein Engineering Glu- Leu-Arg motif are inactive, suggesting that either Network Ccntrcs of Excellence (PENCE). K.-L. is recipient of a a particular conformation of the tripeptide, or interac- scholarship from the Medical Research Council ofcanada. WC thank tions of other domains of the IL-8 molecule with the Dr. l3eatricc Dewald for her comments to the manuscript.

100 Volume 307, number 1 FEW LETTERS July 1992

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