Interleukin-8 Antagonists Generated by N-terminal Modification*

We have previously shown that the residues Glu4- Leu6-Arge (ELR) preceding the first cysteine at the N terminus of the 72-residue form of interleukin-8 (IL-8) are essential for receptor binding and neutrophil activation We have now synthesized a series of analogs of IL-8(4-72), the truncated form of IL-8 with the N-terminal sequence ELRC, as potential IL-8 antagonists. Among 26 analogs with deletions or amino acid replacements in the ELR region several inhibited IL-8 function. The most potent were IL-8(6-72), with Arg" at the N terminus, and IL-8,AAR(7-72) with N-terminal Ala4-Ala6 instead of Glu4-Leu'. They inhib- ited IL-8 receptor binding, exocytosis (ICso 0.3 p ~ ) , as well as chemotaxis and the respiratory burst. Inhibi- tion was restricted to responses elicited by IL-8, GROa, or NAP-2, and no effect was observed when the unre- lated agonists met-Leu-Phe or C5a were used as stim-uli.

Interleukin-8 (IL-8)' belongs to a family of small cytokines which are structurally related to platelet factor 4. It is produced by activated phagocytes and mesenchymal cells and activates neutrophils inducing chemotaxis, exocytosis and the respiratory burst (1,2). IL-8 is generated as a precursor of 99 amino acids and is secreted after cleavage of a leader sequence of 20 amino acids. Proteolytic processing at the N terminus yields several variants, the most prominent of which consists of 72 residues (3). The 4 conserved cysteines, which are characteristic for all members of the IL-8 family, form two disulfide bonds that are essential for biological activity (2, 4). The disulfide bonds link a short conformationally flexible Nterminal region to the core structure consisting of three antiparallel P-strands followed by a C-terminal a-helix.
As part of a study of the relationships between the structure *This work was supported in part by the Protein Engineering Network of Centres of Excellence, a grant from the Swiss Federal Commission for Rheumatic Diseases (to M. B.), and by Grant 31-25700-88 from the Swiss National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' T h e abbreviations used are: IL-8, interleukin-8 HPLC, highperformance liquid chromatography. and function of IL-8, we chemically synthesized analogs of the 72-residue form of IL-8 that were shortened at the Nor C-terminal ends. The results demonstrated that the integrity of each of the 3 residues, Glu4-Leu5-Arg' (ELR), that precede the first cysteine (Table I) is critical for receptor binding and neutrophil-stimulating activity (5). , the derivative with Glu4 at the N terminus, had maximal neutrophil-stimulating activity and binding affinity. Elimination of Glu4 (derivative IL-8(5-72)) led to a marked reduction in activity, and further truncation yielded two completely inactive derivatives,  and   (5). Evidence for the importance of the N terminus for IL-8 activity was also obtained in a mutagenesis study showing that replacement of Glu4, Leu', or Arg' with alanine leads to inactivation (6).
IL-8 is a potent neutrophil attractant in vivo (7)(8)(9), and its expression is enhanced in a variety of inflammatory conditions (2). The potential role of IL-8 as a mediator of inflammation has stimulated the search for inhibitors that could be used therapeutically. In view of the critical role of the Nterminal domain, we have attempted to produce such inhibitors by modification of the ELR sequence. In this paper, we describe several analogs that selectively bind to IL-8 receptors and inhibit IL-&mediated neutrophil responses, and thus qualify as IL-8 receptor antagonists.

EXPERIMENTAL PROCEDURES
Chemical Synthesis-IL-8, GROa, NAP-2, and the IL-8 analogs listed in Table I were synthesized by solid-phase methods using the tertiary butyloxycarbonyl and benzyl protection strategy (10). After deprotection with hydrogen fluoride, the material was folded by air oxidation and purified by reverse-phase HPLC. Purity was assessed by reverse-phase HPLC and isoelectric focusing. Amino acid incorporation was monitored during synthesis, and the final composition was determined by amino acid analysis. The exact procedure has been reported previously (10).
Biological Assays-Human neutrophils were isolated from buffycoats of donor blood (4). The final suspension containing IO8 cells/ ml in 0.15 M NaCl supplemented with 0.05 mM CaC12 was kept at 10 "C until use. I n vitro chemotaxis was measured in multiwell chemotaxis chambers (NeuroProbe, Cabin John, MD) equipped with polyvinylpyrrolidone-free polycarbonate membranes (Nucleopore) with pores of 5 pm (11). The chemotactic index is defined as the ratio of the numbers of cells migrated in the presence and absence of chemoattractant. Elastase release from cytochalasin B-pretreated neutrophils (4) and the respiratory burst (12) were determined as described previously.
Receptor Binding Assays-IL-8 iodination, binding assays, and the calculation of binding parameters were performed as described (5).

RESULTS
The effects of the truncated IL-8 analogs IL-8(5-72), IL-8 , and IL-8(7-72) on the binding of IL-8 to its receptor and on IL-&dependent neutrophil activation were compared. As shown in Fig. lA,  inhibited the release of elastase induced by IL-8. The effect was concentration-de- Analogs with substitutions of Gh4, Leu5, or Arg' were designated with one letter code for the N-terminal amino acids preceding the first  Leu Gln C y s Gln C y s . . .

Glu Leu
Orn C y s Gln C y s . . .

Ala Ala
& C y s Gln C y s . . .    its antagonistic effect is selective for the IL-8 receptor (Fig.  2 A ) . In addition to exocytosis, IL-8(6-72) inhibited in vitro chemotaxis (Fig. 2B) and the respiratory burst (Fig. 2C). Inhibition was again restricted to the responses induced by  (13) and further support the IL-8(6-72) binds to IL-8 receptors, its activity as an agonist was thoroughly tested. IL-8(6-72) did not elicit exocytosis p~, but showed weak chemotactic activity at 1 and 3 pM (Fig.  2B).
The ELR tripeptide itself and other oligopeptides containing the ELR sequence were not active as agonists or antagonists and did not bind to receptors, suggesting that additional structural features are required for IL-8 function.

DISCUSSION
It had been previously shown that elimination of the Nterminal ELR sequence abolishes IL-8 receptor binding and biological activity, indicating that this region is essential for IL-8 function (5,6). The present results show that modification of ELR can yield IL-8 derivatives that have lost neutrophil-stimulating activity but still bind to the receptors, thereby acting as selective IL-8 antagonists.
Two types of N-terminal modification were found to generate antagonists: the substitution or elimination of Glu4 and Leu' with conservation of Arg', or the substitution of ArgG with conservation of Glu4 and Leu'. The present results indicate that Arg' is the most critical residue of the ELR sequence for high-affinity binding to the IL-8 receptor. Analogs with substitution of Arg' showed comparable potency as those with conserved Arg' for inhibiting neutrophil activation, but, for reasons that could not be established, had lower affinity for the IL-8 receptor as assessed by competition assays.
Among the IL-8 derivatives with antagonistic properties IL-8  and IL-8,AAR  appear to be especially promising.  affected IL-&induced chemotaxis, exocytosis (elastase release), and the respiratory burst, and thus behaved as an overall inhibitor of receptor-dependent neutrophil activation. The effect on chemotaxis, however, differed somewhat from that on exocytosis and the respiratory burst. At concentrations between 1 and 10 PM IL-8(6-72) displayed weak chemotactic activity without inducing enzyme release or the respiratory burst. Since it has been shown that the various neutrophil responses depend on different levels of receptor occupancy (14) and signal transduction control (15), it is conceivable that a receptor-bound antagonist may transmit a signal for chemotaxis, but not for exocytosis or the respiratory burst.
IL-8(6-72) and IL-8,AAR(7-72) inhibited to similar extents A the elastase release induced in neutrophils by IL-8 and its homologs, GROa and NAP-2. This observation is of major practical significance, because it is known that in different types of stimulated cells IL-8 is often generated together with related chemotactic cytokines (16-18). These factors are believed to act in concert in pathological conditions by recruiting and activating neutrophils. It is, therefore, desirable to obtain inhibitors, like those described in this study, that diminish or prevent the action of all chemotactic cytokines of the IL-8 family.

IL-8 Receptor Antagonists
The present results further emphasize the importance of the ELR motif in IL-8 receptor binding and triggering. However, the ELR motif alone, as a peptide or in peptide constructs, is not sufficient for effective receptor interaction. The challenge will be to identify the additional structural requirements for the design of future generations of inhibitors with potential therapeutic application.