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The Journal of Immunology, 1998, 161: 4944-4949.
Copyright © 1998 by The American Association of Immunologists

A His19 to Ala Mutant of Melanoma Growth-Stimulating Activity Is a Partial Antagonist of the CXCR2 Receptor

Deborah L. Baly1, Richard Horuk2, Daniel G. Yansura, Laura C. Simmons, Wayne J. Fairbrother, Claire Kotts, Cindy M. Wirth, Beth L. Gillece-Castro, Karen Toy, Joseph Hesselgesser2 and David E. Allison

Genentech, Inc., South San Francisco, CA 94080


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Melanoma growth-stimulating activity (MGSA)3 is a member of a family of proinflammatory cytokines collectively known as the chemokines (1). Members of this growing family of proteins are potent chemoattractants for different subsets of blood leukocytes and as such play an important role in modulating immune and inflammatory responses (2). The chemokines have been divided into two classes depending on whether the first two invariant cysteine residues are separated by an intervening amino acid (C-X-C) or whether they are adjacent (C-C) (1). The C-X-C chemokines include MGSA, IL-8, and platelet factor 4, while the C-C class includes RANTES, monocyte chemotactic peptide-1 (MCP-1), and the macrophage inflammatory protein (MIP)-1 proteins. In general, C-X-C chemokines, like IL-8 and MGSA, preferentially attract neutrophils and induce their activation by producing changes in neutrophil shape, transient increases in cellular calcium concentration and the up-regulation of surface adhesion proteins (3, 4). In contrast the C-C chemokines, for example monocyte chemotactic peptide-1 and RANTES, have no effects on neutrophils but are chemoattractant for monocytes and induce their degranulation (1).

The chemokines exert their biologic effects by interacting with specific receptors on the surface of their target cells (5). Two IL-8 receptors, CXCR1 and CXCR2, have been cloned and found to exhibit high affinity binding for IL-8 (6, 7). Given the important role of IL-8 in neutrophil attraction and activation and the involvement of neutrophils in inflammatory diseases of the lungs, such as adult respiratory distress syndrome, emphysema, and chronic bronchitis (8, 9, 10), considerable effort has been directed toward mapping out the residues of IL-8 that are involved in receptor binding and activation. Three independent studies have identified the E4L5R6 region of IL-8 and MGSA (which is conserved in all C-X-C chemokines that can chemoattract and activate neutrophils) as being important for IL-8 receptor binding (11, 12, 13). Recently, Moser et al. (14) have reported that a truncated form of IL-8 (IL-86–72) is an IL-8 receptor antagonist. However, since these studies were conducted in neutrophils that express both CXCR1 and CXCR2 and since IL-8 binds to both receptors with high affinity, presumably both receptors were antagonized by this mutant. Since it is not clear what the biologic significance of each of the IL-8 receptors is, it would be useful to have separate antagonists for each receptor to examine their role in the inflammatory response. In this context, antagonists based on the structure of MGSA would be useful because this chemokine binds to CXCR2 with high affinity (Kd = 2 nM) and to CXCR1 with low affinity (Kd = 450 nM) (15).

In an attempt to produce a CXCR2-selective antagonist, we generated a series of alanine scan mutants in which the charged side chains of the molecule were replaced by alanine (13). This approach has been successfully used in the past to examine the structure/function relationships for human growth hormone (hGH) (16). The mutants were all assayed for receptor binding in cells expressing CXCR2, and biologic assays were conducted in neutrophils, which, since MGSA binds with low affinity to CXCR1, are reflective of activation of CXCR2. One of the mutants, H19A, showed a dissociation between receptor binding to CXCR2 and biologic activity. These studies suggest that the H19 residue of MGSA is involved in signaling through CXCR2. Thus, this mutant may prove to be useful in helping to design CXCR2 antagonists and in mapping out the receptor binding and signaling regions of IL-8 and MGSA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Materials

[125I]MGSA (sp. act. 2200 Ci/mmol) was from NEN (Boston, MA). Unlabeled MGSA was purified as previously described (17). Reagents for electrophoresis were from Novex (San Diego, CA). HEPES and all other reagent grade chemicals were from Sigma (St. Louis. MO).

Plasmid construction

The expression plasmid for the alanine mutant was derived from the MGSA Escherichia coli secretion vector pMG34 (17). The mutation was incorporated into the original pMG34 vector by replacing an EcoRI-BsaJI fragment in that vector with synthetic DNA containing the respective codon change. The mutant plasmid was then sequenced over the entire region replaced by synthetic DNA to verify that the planned change was correct.

Induction cultures

The plasmid containing the alanine mutation was used to transform the E. coli strain 27C7 (tonA{Delta}ptr3 phoA{Delta}E15{Delta}(argF-lac)169ompT{Delta}degP41) by the CaCl2/heat shock method (18). A freshly transformed culture was grown in Luria-Bertani medium (LB) (19) containing 50 µg/ml carbenicillin for 3 to 4 h at 37°C. The LB culture was then diluted 100-fold into 1 L of a low phosphate minimal media (20) containing 50 µg/ml carbenicillin to induce the alkaline phosphatase promoter. After shaking for 21 h at 30°C, the cells were harvested by centrifugation, washed once with 30 ml of 50 mM Tris-HCl (pH 8), 5 mM EDTA, and 50 mM NaCl, and frozen until the MGSA protein could be extracted and purified. Small aliquots of the cells from induced cultures were analyzed by SDS-PAGE (18% Tris glycine; Novex) to verify the expression of the mutant MGSA protein. A significant fraction of the expressed MGSA can be found in the culture supernatant, particularly when the cells are grown in a fermenter (17). However, under the shake flask conditions described here, most of the expressed protein remained cell bound, and thus the remaining culture fluid was discarded.

Purification

The H19A MGSA mutant was extracted from E. coli cell pastes by treatment in 8 M urea/10 mM Tris (pH 7.8) containing 1% polyethyleneimine. The cell pastes were extracted in an end-over-end rotator for 10 min at room temperature and centrifuged at 20,000 x g for 10 min. The supernatant containing the MGSA was removed and purified by a combination of S-Sepharose chromatography and reverse-phase C4 HPLC as previously described (17). The purified mutant was analyzed by SDS-PAGE on 18% Tris glycine gels (Novex) followed by silver staining. The concentration of the MGSA and MGSA mutant was established by amino acid analysis.

Cell culture

Human embryonic kidney (HEK) cells stably expressing the CXCR1 and CXCR2 receptors were obtained as previously described (15) and maintained in F12/low glucose DMEM (Life Technologies, Grand Island, NY) containing 10% FCS. The cells were passaged weekly, and the medium was changed two additional times weekly. Cell number was determined by counting the cells in a hemacytometer.

Iodination of the H19A mutant

The H19A mutant was iodinated with the Bolton Hunter reagent (21) as previously described (22).

Receptor binding assays

HEK cells stably expressing CXCR2 receptors (2 x 106 cells/ml) and human neutrophils (4 x 106 cells/ml) were incubated with [125I]MGSA or 125I-labeled H19A mutant and varying concentrations of unlabeled ligands at 4°C for 1 h. The incubation was stopped by removing aliquots from the cell suspension and separating cells from buffer by centrifugation through a silicone/paraffin oil mixture as described previously (23). Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve fit with the computer program LIGAND (24) to determine the affinity (Kd), number of sites, and nonspecific binding.

Elastase release bioassay

Human neutrophils were isolated from healthy donors, and stimulus-dependent elastase release was determined after pretreatment with cytochalasin B, as previously described (25).

Flow cytometry

Neutrophils isolated as described previously (25) were resuspended (2.5 x 106 cells/ml) into FACS buffer (PBS containing 0.1% BSA) and incubated in the presence of increasing concentrations (0 to 1 µM) of MGSA, IL-8, and H19A for 2 h at 37°C. At the end of the incubation the cells were pelleted by centrifugation (300 x g) at 4°C and resuspended into FACS buffer. To determine CD18 up-regulation, the cells were incubated at 4°C for 60 min with a humanized anti CD18 Ab or no Ab as the control and then pelleted by centrifugation (300 x g) at 4°C and resuspended into FACS buffer. The cells were then incubated at 4°C for 30 min with anti-human F(ab')2 FITC and analyzed using a FACScan (Becton Dickinson, Mountain View, CA) flow cytometer.

Chemotaxis

Neutrophil migration was examined as previously described (26). The results were expressed as the percentage increase in migration of neutrophils by increasing chemokine concentrations. Background migration in the absence of added chemokine has been set as 0%.

Cytosolic Ca2+ measurements in HEK cells expressing the CXCR1 and CXCR2 receptors

Cells expressing the CXCR1 and CXCR2 receptors were loaded with 5 µM fura-2 AM (Molecular Probes, Eugene OR) for 1 h at 37°C in RPMI 1640 (Life Technologies) containing 10% serum, penicillin, and streptomycin. Cells were pelleted, washed with serum-free RPMI 1640 lacking phenol red, and resuspended in the same medium at a density of 2 x 106 cells/ml. This suspension (1.5 ml) or 3 x 106 cells were used for each experiment. Changes in the cytosolic free Ca2+ concentration were measured with an SLM/Aminco (Rochester, NY) model AB2 fluorescence spectrophotometer.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Nuclear magnetic resonance (NMR) studies of MGSA have shown that the overall structure of the molecule is very similar to IL-8 (27, 28), being a dimer in solution consisting of a six-stranded antiparallel ß-sheet packed against two C-terminal antiparallel {alpha}-helices. To examine the structure/function of MGSA for CXCR2 receptors, we constructed a series of alanine-scan mutants of MGSA (13) in which charged amino acids were replaced by alanine. One of the mutants generated, H19A, showed a dissociation between receptor binding and biologic activity and is the subject of this communication.

Under the conditions of growth described here, most of the expressed MGSA mutant remained cell bound, and the protein was extracted from the E. coli cell pastes and purified by a combination of S-Sepharose fast flow and reverse-phase chromatography, as described previously (17). The authenticity of the H19A mutant was determined by electrospray ionization mass spectrometry, which gave a mass of 7,796.6 Da, consistent with the predicted average mass of 7,796.2 Da (data not shown). Protein concentration was established by amino acid composition (17).

MGSA has a variety of biologic effects in neutrophils, including elastase release and chemotaxis (4). In addition, here we show that MGSA, like IL-8 (29), can up-regulate the expression of CD18 in neutrophils, which is important for extravasation. The relative potencies of the H19A mutant were compared with those of the wild-type MGSA in a number of assays (Figs. 1Go to 4). The most sensitive assay that we employed to measure the activity of the H19A mutant was a FACScan analysis of the expression of CD18. In this assay, the half-maximal effect for CD18 induction by MGSA was observed at a concentration of 0.41 nM, compared with a concentration of 115 nM for the H19A mutant, a 282-fold reduction in potency (Fig. 1Go).



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  		FIGURE 1. Expression of CD18 in neutrophils measured by FACScan analysis. Neutrophils (2.5 x 106 cells/ml) were incubated in the presence of increasing concentrations (0 to 1 µM) of MGSA and H19A for 2 h at 37°C. To determine CD18 up-regulation, the cells were incubated at 4°C for 60 min with anti-CD18 and then pelleted by centrifugation (300 x g) at 4°C and resuspended into FACS buffer. The cells were then incubated at 4°C for 30 min with anti-human F(ab')2 FITC and analyzed using a FACScan (Becton Dickinson) flow cytometer.

 
As shown in Figure 2GoA, the concentration of the H19A mutant required to induce a half-maximal biologic effect in the elastase release assay was 3595 nM, compared with 27 nM for wild-type MGSA, a 133-fold reduction in potency. The ability of the H19A mutant to antagonize MGSA action was assessed by preincubating neutrophils in the absence and presence of H19A (500 nM) and then measuring elastase release in the presence of increasing amounts of MGSA (Fig. 2GoB). Pretreatment of neutrophils with 500 nM H19A inhibited the release of elastase by MGSA and shifted the MGSA dose response curve to the right. The inhibition of elastase release by 500 nM H19A was most apparent at low concentrations of MGSA, 1 to 50 nM, and suggested that the H19A mutant was an antagonist of MGSA action. This inhibition was apparent, regardless of whether we preincubated with H19A or added the molecule at the same time as MGSA (data not shown), and this would tend to rule out receptor desensitization as a formal explanation for the inhibition that we observed here although it does still remain a formal possibility.



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  		FIGURE 2. Elastase release from cytochalasin B-treated human neutrophils stimulated with MGSA and mutant H19A. A, Dose-responsive induction of elastase release by MGSA ({blacksquare}) and the mutant H19A ({square}). Elastase activity was measured as the amount of substrate (methoxysuccinyl-Alanyl-Alanyl-Prolyl-Valyl-p-nitroanilide) converted to p-nitroanilide per min per 106 cells and has been normalized as percentage biologic activity. Data are mean values from four separate experiments ± SEM. B, Antagonism of MGSA-induced elastase release in neutrophils by the MGSA mutant H19A. Cytochalasin B-treated human neutrophils were incubated in the presence ({square}) or absence ({blacksquare}) of 500 nM H19A for 30 min at room temperature and were then stimulated with increasing concentrations of MGSA as indicated. Data are mean values from three separate experiments ± SEM.

 
Chemokines were originally defined and classified as potent leukocyte chemoattractants, mediating their effects through G-protein-coupled receptors (2). Thus, we measured the ability of MGSA and H19A to induce the cellular migration of neutrophils in a chemotaxis assay. As can be seen in Figure 3Go, the C-X-C chemokine MGSA induces the migration of neutrophils toward the chemokine gradient in a dose-responsive manner, with a typical bell-shaped curve at higher concentrations. Half-maximal stimulation was observed at an MGSA concentration of 10 nM. In contrast the H19A mutant, even at a concentration of 1250 nM, achieves only a modest 30% increase in neutrophil migration. Finally, addition of the H19A mutant at a concentration of 1 µM inhibited the migration of the cells in response to MGSA.



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  		FIGURE 3. The C-X-C chemokines MGSA and the MGSA mutant H19A induce neutrophil cell migration. Neutrophils were tested for their ability to migrate in response to various concentrations of chemokines as previously described (26). In addition, the effect of the addition of 1 µM H19A on the directed migration of neutrophils in response to MGSA is shown. The results are expressed as the percentage increase in migration of neutrophils by chemokine. Background migration in the absence of added chemokine has been set as 0%.

 
As an additional indication of the antagonist properties of the H19A mutant, we measured its ability to block the MGSA-induced transient rise in intracellular Ca2+. As shown in Figure 4GoA, pretreatment of cells expressing the CXCR2 receptor with increasing concentrations of the H19A mutant dose-responsively inhibited the transient rise in intracellular Ca2+ induced by MGSA. The IC50 of this inhibition was around 40 nM H19A (Fig. 4GoC). In addition the H19A mutant was also able to successfully antagonize the effects of IL-8 on the same receptor (Fig. 4GoB). In contrast to these data, however, the antagonist had no effects on the IL-8-induced transient rise in intracellular Ca2+ in cells expressing CXCR1 nor did MGSA produce any significant rise in intracellular Ca2+ in these cells (Fig. 4GoD). These studies are in line with previously published reports that demonstrate that IL-8 is a high affinity agonist for both CXCR1 and CXCR2 receptors but that MGSA is a high affinity agonist only for CXCR2 (15). These data clearly demonstrate that the effects of the H19A mutant observed in neutrophils ( Figs. 1–3GoGoGo) are manifested through the CXCR2 and not the CXCR1 receptor.



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  		FIGURE 4. Antagonism of MGSA and IL-8 induction of intracellular Ca2+ by the MGSA mutant H19A. A, The inhibition of MGSA-induction of Ca2+ transients in cells expressing CXCR2 by increasing concentrations of the MGSA mutant H19A. B, The inhibition of IL-8-induction of Ca2+ transients in cells expressing CXCR2 by the MGSA mutant H19A. C, Dose-response curve for the inhibition of MGSA-induction of Ca2+ transients by H19A. D, The failure of the MGSA mutant H19A to block the IL-8-induction of Ca2+ transients in cells expressing the CXCR1 receptor.

 
To examine the effect of the H19 to A replacement in MGSA on binding to CXCR2, we compared the abilities of wild-type MGSA and the H19A mutant to displace bound [125I]MGSA from cells stably expressing CXCR2 receptors and from neutrophils (Fig. 5Go). Scatchard analysis of competition binding studies revealed that wild-type MGSA had similar affinities for binding to the CXCR2-transfected cells and neutrophils, 3.6 ± 0.5 nM and 2.6 ± 1.1 nM, respectively (Fig. 5Go). These data agree well with the reported values (15). The H19A mutant was fully able to displace [125I]MGSA specifically bound to IL-8 receptors in cells transfected with CXCR2 receptors and in neutrophils, and the binding constants were 47 ± 5 nM and 31 ± 5 nM, respectively (Fig. 5Go). These data represent a 13-fold decrease in binding affinity over the binding affinity of wild-type MGSA.



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  		FIGURE 5. Scatchard analysis of the inhibition of binding by MGSA and H19A to HEK cells transfected with CXCR2 and neutrophils. Cells were incubated for 1 h at 4°C with [125I]MGSA in the presence of increasing amounts of unlabeled MGSA or H19A. The binding reactions were terminated by centrifugation of cells through a paraffin-oil mixture as described (23). Nonspecific binding was determined in the presence of 100 nM unlabeled MGSA and was less than 10% of total binding. Data are mean values from three separate experiments ± SEM. After adjustment of cell numbers, the IL-8 receptor site numbers were similar in all experiments.

 
Although we have demonstrated that the H19A mutant was able to displace MGSA specifically bound to CXCR2 receptors, it was important to show that the mutant was able to bind directly to CXCR2 with a similar affinity. Thus, we labeled the H19A mutant with Bolton-Hunter reagent (21) and conducted competition binding studies with unlabeled H19A and MGSA. The competition curves show that the H19A mutant is able to bind directly to cells transfected with the CXCR2 receptor and had a Kd of 95 ± 18 nM (Fig. 6Go). The dose response curve for MGSA was leftward shifted, consistent with a ligand with higher affinity, and gave a Kd of 8.1 ± 1.2 nM. These data represent a 12-fold decrease in binding affinity for the H19A mutant over the binding affinity of wild-type MGSA and are fully consistent with the binding data observed from the competition studies with radiolabeled MGSA.



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  		FIGURE 6. Scatchard analysis of the inhibition of 125I-labeled H19A binding by MGSA and H19A to HEK cells transfected with CXCR2.

 
This modest reduction in receptor binding affinity of the H19A mutant contrasts sharply with the greater than 100-fold reduction in biologic activity measured in three separate bioassays and suggests that the H19 residue is important for the biologic activity of MGSA. Interestingly, of the 11 human C-X-C chemokines cloned so far, this histidine residue is conserved in MGSA, IL-8, NAP-2, and ENA-78, C-X-C chemokines that can bind to CXCR2 and activate neutrophils (2), but not in other C-X-C chemokines like PF4, SDF-1, IP-10, BCA-1, or betaR1 that do not bind to the CXCR2 receptor. Conservation of the H19 residue in these chemokines is consistent with our proposal that it plays a role in the activation of CXCR2 by MGSA.

An exception to the above observation is a mutant form of human PF4, in which the N-terminal region is substituted with ELR (ELR-PF4) (30); this mutant binds to the IL-8 receptors and activates neutrophils with a potency 5-fold greater than MGSA, despite containing an R residue in the position corresponding to H19. A similar mutant form of IP10, (ELR-IP10), which has an N residue in the position corresponding to H19, does not bind the IL-8 receptor (30). These results suggest that a positive charge may be required in this position for activation of the receptors. At neutral pH, however, the H19 residues in MGSA and IL-8 are expected to be uncharged since the pKa values for H19 are 5.2 and 3.7 in MGSA and IL-8, respectively (28, 31). Interaction of the H19 side chain with a negatively charged region on the receptor may result in an increase in these pKa values and subsequent protonation of the imidazole ring, which "triggers" activation. Mutagenesis studies to replace H19 with an R or K residue can be designed to test this hypothesis. Interestingly, the C-X-C chemokine MIG, which, like IP10, is a high affinity ligand for CXCR3 (32), has an H residue at position 19, and it would be interesting to determine whether substitution of an ELR motif into this chemokine would enable it to bind to CXCR2. Other recently cloned chemokines, including SDF-1, which is a ligand for CXCR4 (33), and BCA-1, which is a high affinity ligand for CXCR5 (34), do not have a conserved ELR motif and have A and P residues, respectively, at position 19.

We have recently determined that the ELR region in MGSA is important for binding and activation of CXCR2 (13). We note that H19 is located more than 26 Å from the important ELR region in both the IL-8 and MGSA structures (28, 35). This suggests that the H19 forms a second MGSA binding site. The H18 residue in IL-8 has also been implicated recently as part of a region of the molecule that interacts with a highly acidic N-terminal fragment of CXCR1 (residues 1–40) (36). This binding event could represent nonspecific interaction between basic regions of IL-8 and the acidic peptide or may represent a secondary binding epitope of IL-8 (in addition to the previously identified N-terminal ELR motif). A second binding region on IL-8 and MGSA was also expected from recent results that suggest that a single subunit of IL-8 activates both CXCR1 and CXCR2 receptors and that receptor dimerization is not linked to signal transduction (37). In line with these experiments, a second binding region comprising the IL-8 residues 13–17 and the corresponding MGSA residues 15–18 was recently identified from loop swapping experiments (38). In these studies, replacement of the MGSA loop with the corresponding loop from IL-8, together with the substitution of residue 50 of MGSA from A to L, resulted in an MGSA variant that, like IL-8, had high affinity for both CXCR1 and CXCR2. Conversely, when the same loop from MGSA was swapped into IL-8 together with substitution of residue 49 of IL-8 from L to A, this produced an IL-8 variant that had low affinity for CXCR1, just like MGSA, and high affinity for CXCR2.

The ability of the H19A mutant to discriminate between the two IL-8 receptors could provide additional information into the biologic significance of these two receptors. Although neutrophils express both IL-8 receptors, their expression levels vary in different individuals (15). The relevance of this to the physiologic role of these two receptors is unknown. The H19A mutant, because it antagonizes only CXCR2, offers the opportunity to study the roles of these two receptors in neutrophil activation and in disease. Finally, the dissociation between receptor binding and activation for the H19A mutant suggests that it should be possible to design antagonists of MGSA that could have clinical utility.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Deborah Baly, 1 DNA Way, Genentech, Inc., South San Francisco, CA 94080. E-mail address: Back

2 Present address: Department of Immunology, Berlex Biosciences, Richmond, CA 94804. Back

3 Abbreviations used in this paper: MGSA, melanoma growth-stimulating activity; PF4, platelet factor 4; CXCR1, IL-8 receptor type A; CXCR2, IL-8 receptor type B; HEK, Human embryonic kidney; IP10, Interferon protein ENA-78 epithelial neutrophil activating protein-78; NAP-2, neutrophil activating protein-2; SDF, stromal derived factor. Back

Received for publication January 23, 1998. Accepted for publication June 25, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Schall, T.. 1994. The chemokines. A. Thompson, ed. The Cytokine Handbook 419.-460. Academic Press, San Diego.
  2. Baggiolini, M., B. Dewald, B. Moser. 1994. Interleukin-8 and related chemotactic cytokines: CXC and CC chemokines. Adv. Immunol. 55:97.[Medline]
  3. Baggiolini, M., I. Clark-Lewis. 1992. Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett. 307:97.[Medline]
  4. Moser, B., I. Clark-Lewis, R. Zwahlen, M. Baggiolini. 1990. Neutrophil-activating properties of the melanoma growth-stimulatory activity. J. Exp. Med. 171:1797.[Abstract/Free Full Text]
  5. Horuk, R.. 1994. Molecular properties of the chemokine receptor family. Trends Pharmacol. Sci. 15:159.[Medline]
  6. Holmes, W. E., J. Lee, W. J. Kuang, G. C. Rice, W. I. Wood. 1991. Structure and functional expression of a human interleukin-8 receptor. Science 253:1278.[Abstract/Free Full Text]
  7. Murphy, P. M., H. L. Tiffany. 1991. Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 253:1280.[Abstract/Free Full Text]
  8. Donnelly, S. C., R. M. Strieter, S. L. Kunkel, A. Walz, C. R. Robertson, D. C. Carter, I. A. Grant, A. J. Pollok, C. Haslett. 1993. Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups. Lancet 341:643.[Medline]
  9. Broaddus, V. C., C. A. Hebert, R. V. Vitangcol, J. M. Hoeffel, M. S. Bernstein, A. M. Boylan. 1992. Interleukin-8 is a major neutrophil chemotactic factor in pleural liquid of patients with empyema. Am. Rev. Respir. Dis. 146:825.[Medline]
  10. Mulligan, M. S., M. L. Jones, M. A. Bolanski, M. A. Baganoff, C. L. Deppeler, D. M. Meyers, U. S. Ryan, P. A. Ward. 1993. Inhibition of lung inflammatory reactions in rats by an anti-human IL-8 antibody. J. Immunol. 150:5585.[Abstract]
  11. Hébert, C. A., R. V. Vitangcol, J. B. Baker. 1991. Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J. Biol. Chem. 266:18989.[Abstract/Free Full Text]
  12. Clark-Lewis, I., C. Schumacher, M. Baggiolini, B. Moser. 1991. Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. J. Biol. Chem. 266:23128.[Abstract/Free Full Text]
  13. Hesselgesser, J., C. Chitnis, L. Miller, D. J. Yansura, L. Simmons, W. Fairbrother, C. Kotts, C. Wirth, B. Gillece-Castro, R. Horuk. 1995. A mutant of melanoma growth stimulating activity does not activate neutrophils but blocks erythrocyte invasion by malaria. J. Biol. Chem. 270:11472.[Abstract/Free Full Text]
  14. Moser, B., B. Dewald, L. Barella, C. Schumacher, M. Baggiolini, I. Clark-Lewis. 1993. Interleukin-8 antagonists generated by N-terminal modification. J. Biol. Chem. 268:7125.[Abstract/Free Full Text]
  15. Lee, J., R. Horuk, G. C. Rice, G. L. Bennett, T. Camerato, W. I. Wood. 1992. Characterization of two high affinity human interleukin-8 receptors. J. Biol. Chem. 267:16283.[Abstract/Free Full Text]
  16. Cunningham, B. C., J. A. Wells. 1989. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244:1081.[Abstract/Free Full Text]
  17. Horuk, R., D. G. Yansura., D. Reilly, S. Spencer, J. Bourell, W. Henzel, G. Rice, E. Unemori. 1993. Purification, receptor binding analysis, and biological characterization of human melanoma growth stimulating activity (MGSA): Evidence for a novel MGSA receptor. J. Biol. Chem. 268:541.[Abstract/Free Full Text]
  18. Mandel, M., A. Higa. 1970. Calcium dependent bacteriophage DNA infection. J. Mol. Biol. 53:159.[Medline]
  19. Miller, J. H.. 1972. Experiments in Molecular Genetics 37.-47. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  20. Chang, C. N., M. Rey, B. Bochner, H. Heyneker, G. Gray. 1987. High level secretion of human growth hormone by Escherichia coli. Gene 55:189.[Medline]
  21. Bolton, A. E., W. M. Hunter. 1973. The labeling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem. J. 133:529.[Medline]
  22. Bennett, G. L., R. Horuk. 1997. Iodination of chemokines for use in receptor binding analysis. Methods Enzymol. 288:134.[Medline]
  23. Robb, R. J., W. C. Greene, C. M. Rusk. 1984. Low and high affinity cellular receptors for interleukin 2: implications for the level of Tac antigen. J. Exp. Med. 160:1126.[Abstract/Free Full Text]
  24. Munson, P., D. Rodbard. 1980. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107:220.[Medline]
  25. Baly, D., U. Gibson, D. Allison, L. DeForge. 1997. Biological assays for C-X-C chemokines. Methods Enzymol. 287:69.[Medline]
  26. Hesselgesser, J., R. Horuk. 1997. Alanine scan mutagenesis of chemokines. Methods Enzymol. 287:59.[Medline]
  27. Fairbrother, W. J., D. Reilly, T. Colby, R. Horuk. 1993. 1H assignment and secondary structure determination of human melanoma growth stimulating activity (MGSA) by NMR spectroscopy. FEBS Lett. 330:302.[Medline]
  28. Fairbrother, W. J., D. Reilly, T. J. Colby, J. Hesselgesser, R. Horuk. 1994. The solution structure of melanoma growth stimulating activity (MGSA). J. Mol. Biol. 242:252.[Medline]
  29. Detmers, P. A., S. K. Lo, E. E. Olsen, A. Walz, M. Baggiolini, Z. A. Cohn. 1990. Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils. J. Exp. Med. 171:1155.[Abstract/Free Full Text]
  30. Clark-Lewis, I., B. Dewald, T. Geiser, B. Moser, M. Baggiolini. 1993. Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. Proc. Natl. Acad. Sci. USA 90:3574.[Abstract/Free Full Text]
  31. Clore, G. M., E. Appella, M. Yamada, K. Matsushima, A. M. Gronenborn. 1990. Three-dimensional structure of interleukin 8 in solution. Biochemistry 29:1689.[Medline]
  32. Loetscher, M., B. Gerber, P. Loetscher, S. A. Jones, L. Piali, I. Clark-Lewis, M. Baggiolini, B. Moser. 1996. Chemokine receptor specific for IP10 and Mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 184:963.[Abstract/Free Full Text]
  33. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J. Sodroski, T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382:829.[Medline]
  34. Legler, D. F., M. Loetscher, R. S. Roos, I. Clark-Lewis, M. Baggiolini, B. Moser. 1998. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5. J. Exp. Med. 187:655.[Abstract/Free Full Text]
  35. Baldwin, E. T., I. T. Weber, R. St. Charles, J.-C. Xuan, E. Appella, M. Yamada, K. Matsushima, B. F. P. Edwards, G. M. Clore, A. M. Gronenborn, A. Wlodawer. 1991. Crystal structure of interleukin-8: symbiosis of NMR and crystallography. Proc. Natl. Acad. Sci. USA 88:502.[Abstract/Free Full Text]
  36. Clubb, R. T., J. G. Omchinski, G. M. Clore, A. M. Gronenborn. 1994. Mapping the binding surface of interleukin-8 complexed with an N-terminal fragment of the type 1 interleukin-8 receptor. FEBS Lett. 338:93.[Medline]
  37. Rajarathnam, K., B. D. Sykes, C. M. Kay, B. Dewald, T. Geiser, M. Baggiolini, L. I. Clark. 1994. Neutrophil activation by monomeric interleukin-8. Science 264:90.[Abstract/Free Full Text]
  38. Lowman, H. B., P. H. Slagl, L. E. DeForge, C. M. Wirth, B. L. Gillece-Castro, J. H. Bourell, W. J. Fairbrother. 1996. Exchanging interleukin-8 and melanoma growth-stimulating activity receptor binding specificities. J. Biol. Chem. 271:14344.[Abstract/Free Full Text]



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