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Originally published online as doi:10.1189/jlb.1103570 on June 3, 2004

Published online before print June 3, 2004
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(Journal of Leukocyte Biology. 2004;76:701-708.)
© 2004 by Society for Leukocyte Biology

I-TAC/CXCL11 is a natural antagonist for CCR5

Vibor Petkovic, Christian Moghini, Samantha Paoletti, Mariagrazia Uguccioni and Basil Gerber1

Institute for Research in Biomedicine, Bellinzona, Switzerland

1 Correspondence: Division of Allergology, Clinic for Rheumatology and Clinical Immunology/Allergology, Sahlihaus 1, Inselspital, CH-3010 Bern, Switzerland. E-mail: basil.gerber{at}dkf6.unibe.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The selective CXC chemokine receptor 3 (CXCR3) agonists, monokine induced by interferon-{gamma} (IFN- {gamma})/CXC chemokine ligand 9 (CXCL9), IFN-inducible protein 10/CXCL10, and IFN-inducible T cell {alpha} chemoattractant (I-TAC)/CXCL11, attract CXCR3+ cells such as CD45RO+ T lymphocytes, B cells, and natural killer cells. Further, all three chemokines are potent, natural antagonists for chemokine receptor 3 (CCR3) and feature defensin-like, antimicrobial activities. In this study, we show that I-TAC, in addition to these effects, acts as an antagonist for CCR5. I-TAC inhibited the binding of macrophage-inflammatory protein-1{alpha} (MIP-1{alpha})/CC chemokine ligand 3 (CCL3) to cells transfected with CCR5 and to monocytes. Furthermore, cell migration evoked by regulated on activation, normal T expressed and secreted (RANTES)/CCL5 and MIP-1ß/CCL4, the selective agonist of CCR5, was inhibited in transfected cells and monocytes, respectively. In two other functional assays, namely the release of free intracellular calcium and actin polymerization, I-TAC reduced CCR5 activities to minimal levels. Sequence and structure analyses indicate a potential role for K17, K49, and Q51 of I-TAC in CCR5 binding. Our results expand on the potential role of I-TAC as a negative modulator in leukocyte migration and activation, as I-TAC would specifically counteract the responses mediated by many "classical," inflammatory chemokines that act not only via CCR3 but via CCR5 as well.

Key Words: chemokine • receptor • competitive • leukocyte • migration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are small, secretory proteins produced by tissue cells and leukocytes, which play an important role in inflammatory and immune responses, primarily as a result of their chemotactic activities toward different subsets of leukocytes [1 2 3 ]. To date, all of the more than 40 known human chemokines act by activating heptahelical G protein-coupled receptors located on the leukocyte plasma membrane.

CXC chemokine receptor 3 (CXCR3) binds and is activated by CXC chemokine ligand 9 (CXCL9)/monokine induced by interferon-{gamma} (IFN-{gamma}; MIG), CXCL10/IFN-inducible protein 10 (IP-10), and CXCL11/IFN-inducible T cell {alpha} chemoattractant (I-TAC) [4 5 6 ]. These three chemokines attract and activate CXCR3-bearing cells such as peripheral T cells of the CD45RO+ memory phenotype, B cells, natural killer cells, and in vitro-generated T helper type 1 (Th1) cells [7 8 9 ]. The production of I-TAC, IP-10, and MIG is induced by IFN-{gamma}, the prototypical Th1 cytokine [5 , 10 , 11 ], and correlates with involvement of these chemokines, especially IP-10, in Th1-type driven inflammatory conditions such as multiple sclerosis [12 , 13 ], rheumatoid arthritis [9 , 14 ], and psoriasis [15 ]. I-TAC, IP-10, and MIG share 40% of sequence homology among each other, much more than to any other chemokine [5 ]. Of all three CXCR3 agonists, I-TAC is the most potent and efficacious, featuring the highest affinity for CXCR3 [5 ]. mRNA for I-TAC has been reported to be up-regulated in IFN-{gamma}-treated monocytes [5 ], bronchial epithelial cells [16 ], neutrophils [17 ], keratinocytes [18 ], and endothelial cells, hence implying a role in T lymphocyte recruitment to sites of inflammation [19 ]. It is interesting that two recent reports indicate that all three CXCR3 agonists are capable of inhibiting the activation of chemokine receptor 3 (CCR3) as well [20 , 21 ], suggesting that these chemokines feature regulatory properties beyond their agonistic and antimicrobial functions [22 ]. More recently, CXCL13/B cell-attracting chemokine-1 (BCA-1) has been reported as a CXCR3 agonist as well [23 ].

The chemokine receptor CCR5 is expressed on peripheral blood-derived dendritic cells (DCs) [24 , 25 ], CD34+ hematopoietic progenitor cells [26 ], activated T cells [14 ], and monocytes, which express low levels that can be increased by culture in vitro [27 ]. Expression of CCR5 on neurons, astrocytes, capillary endothelial cells, epithelium, and fibroblasts has also been reported (ref. [2 ] and references therein), but the functional role remains to be clarified.

Recently, several chemokines that act as natural antagonists have been described. Apart from being agonists for their corresponding receptors, these chemokines exert antagonism on one or several other receptor(s). The relevance of such dual-activity chemokines would lie in their potential as physiological regulators, as they would attract or activate a population of cells positive for one receptor and at the same time, prevent the recruitment or activation of a different cellular population bearing another receptor. So far, eight reports describe eight natural chemokine antagonists, namely chemokine ligand 18 (CCL18)/DC-derived chemokine (DC-CK1) for CCR3 [28 , 29 ], CCL7/monocyte chemoattractant protein-3 (MCP-3) for CCR5 [30 ], CCL11/eotaxin [31 , 32 ] and CCL26/eotaxin-3 for CCR2 [33 ], CCL4/macrophage inflammatory protein-1ß (MIP-1ß) for CCR1 [34 ], and as mentioned previously, all three CXCR3 agonists for CCR3 [20 , 21 ].

Early on, the use of chemokine receptor antagonists was recognized as a possible therapeutic approach to prevent inflammatory diseases by blocking the infiltration of leukocytes that bear the respective receptors. Consequently, a number of synthetically produced, truncated chemokine variants were tested and shown to be efficient antagonists in vitro. A truncated variant of CCL2/MCP-1 (9–76) prevented the onset of arthritis and reduced symptoms and cellular infilatrates in the MRL-lpr mouse model [35 ], and truncated forms of secondary lymphoid tissue chemokine inhibited the development of chronic graft-versus-host disease in mice [36 ], showing, most importantly, that such chemokine mutants with antagonistic properties can have antagonistic activity in vivo as well. The chemokines encoded by human viruses, especially viral MIP-II (vMIP-II), were also reported to act as antagonists on several human chemokine receptors [37 38 39 40 41 ]. vMIP-II inhibited leukocyte infiltration and attenuated glomerulonephritis in Wistar-Kyoto rats [42 ] and virus-induced, T cell-mediated inflammation in mice [43 ], demonstrating that the antagonistic properties of viral chemokines can have significance in vivo as well.

The existence of antagonistic properties of a number of natural chemokines and the proven antagonistic activities of mutant and viral chemokines in vivo suggest that chemokine antagonism might constitute an additional, regulatory mechanism for general leukocyte guidance. Thus, we have recently engaged in a large search for antagonistic activities of natural chemokines. In this study, we show I-TAC to have antagonistic activity on CCR5. By blocking this receptor, I-TAC would specifically counteract not just the action of MIP-1ß, the CCR5-specific, high-potency agonist, but also the activities of other promiscuous, inflammatory chemokines that act via CCR5.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Peripheral blood mononuclear cells were isolated from buffy coats of donor blood (Central Laboratory of the Swiss Red Cross, Basel) by Fycoll-Paque® density centrifugation. CD14+ monocytes were isolated by a positive immunoselection procedure (CD14 MicroBeads, Miltenyi Biotech, Germany), according to the manufacturer’s instructions. Monocytes were cultured overnight to up-regulate CCR5. Stable transfection of human CCR5 or CXCR3 into murine pre-B 300.19 cells was performed as described before [4 ].

Chemokine synthesis
All chemokines were chemically synthesized using tBoc solid-phase chemistry [44 ].

Chemotaxis assays
Chemotaxis was assayed in 48-well Boyden microchambers (Neuro Probe Inc., Gaithersburg, MD), using polyvinylpryrrolidone-free polycarbonate membranes with 5 µm pores (Nucleopore). Briefly, attracting chemokines diluted in chemotaxis buffer (RPMI 20 mM HEPES, pH 7.4) containing 0.05% pasteurized plasma protein (Swiss Red Cross Laboratory, Bern) were placed in the lower wells. In inhibition experiments, I-TAC was placed in the upper wells. Monocytes (5x104) or pre-B murine 300.19 cells transfected with human CCR5 were resuspended in chemotaxis buffer and added to the upper wells. After 60 min of incubation for monocytes and 120 min incubation for transfected cells, the membrane was removed, washed on the upper side with phosphate-buffered saline (PBS), fixed, and stained. Migrated cells were counted at 1000x magnification in five fields per well [migrated cells/5 h postfertilization (HPF)]. All experiments were performed in triplicates.

Chemokine receptor expression
CCR5 expression in CCR5-transfected cells and monocytes cultured overnight was analyzed by flow cytometry (FACScanTM, Becton Dickinson, San Jose, CA). Cells were incubated for 30 min in fluorescein-activated cell sorter (FACS) buffer [2% fetal calf serum (FCS), 0.1% sodium azide in PBS] with fluorescein isothiocyanate (FITC)- or phycoerythrin-coupled anti-CCR5 (MAB181, R&D Systems, Abington, UK), washed, and analyzed. Isotype-matched controls were treated accordingly.

Calcium transients
CCR5-transfected cells or monocytes were loaded in RPMI 1640 containing 20 mM HEPES and 5% FCS, with 0.16 nmol Fura-2/AM per million cells for 20 min. Intracellular Ca2+ mobilization ([Ca2+]i) was measured after a single or sequential stimulation with chemokines by recording [Ca2+]i-related fluorescence changes [45 ].

Actin polymerization
F-actin formation was determined with FITC-coupled phalloidin (Sigma-Aldrich Chemicals, St. Louis, MO) in CCR5-transfected cells, and human monocytes were cultured overnight. The reaction was stopped after 10 s, and cells were fixed with cold paraformaldehyde (4%) in PBS for 30 min on ice. For inhibition studies, transfected cells were pretreated with I-TAC for 3 min and stimulated with CCL5/regulated on activation, normal T cell expressed and secreted (RANTES) and monocytes, with CCL4/MIP-1ß. After paraformaldehyde fixation, cells were permeabilized with 0.1% Triton X-100, stained with FITC-conjugated phalloidin (6 µg/ml), and analyzed by FACS. All stimulations were performed in duplicate. Relative F-actin formation was calculated as fold increase over basal phalloidin staining (set to one for all experiments). Statistical analyses were performed using the ANOVA test to compare differences in actin polymerization.

Receptor binding
Binding assays were performed with CCR5-transfected cells and monocytes using 125I-CCL3/MIP-1{alpha} (Amersham Pharmacia Biotech, Uppsala, Sweden). Cells (2x106) in 120 µL RPMI 1640 containing 20 mM HEPES and 1% bovine serum albumin (BSA), pH 7.1, were incubated on ice for 90 min with 0.15 nM-labeled chemokine and increasing concentrations of unlabeled chemokines as indicated. Nonbound radioactivity was separated by centrifugation through 6% BSA in PBS, and cell-bound radioactivity was determined by {gamma}-counting. The difference between total and nonspecific binding, as determined in the absence of any unlabeled chemokine or the presence of 1 µM unlabeled MIP-1{alpha}, respectively, was taken as 100%. Data were analyzed with PRISM (GraphPad Software, San Diego, CA) using nonlinear regression in a one-site binding model.

Protein display and analysis
Multiple sequence alignments were performed using the standard settings of ClustalW at ch.EMBnet.org. I-TAC modeling of residues 6–73 and protein display was performed with Swiss Pdb Viewer [46 ], using residues 4–70 of interleukin (IL)-8 as reference structure [47 ]. Coordinates for IL-8 [Protein Data Bank (PDB) Code 1ICW] were obtained from PDB (www.rcsb.org/pdb).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
I-TAC is a natural antagonist of CCR5
The inhibitory effect of I-TAC on the rise of free [Ca2+]i was measured in a murine pre-B cell line, singly transfected with human CCR5 (Fig. 1A ), and in monocytes (Fig. 1B) . After isolation, monocytes were cultured overnight to up-regulate CCR5 and were shown to be CCR5+ by flow cytometry (data not shown). Cells were left unexposed or sequentially treated with increasing concentrations of I-TAC (transfected cells) or a single, 1µM concentration (monocytes) and with RANTES or MIP-1ß at concentrations sufficient to evoke cellular responses. In transfected cells, 30 nM RANTES induced a response that was partially inhibited by 300 nM I-TAC, and at a 1µM concentration, inhibition was complete. In monocytes, we observed partial inhibition after pretreatment with 1 µM I-TAC when cells were stimulated with 100 nM MIP-1ß.



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  		Figure 1. Inhibition of functional responses by I-TAC in CCR5-transfected cells and monocytes. Changes of [Ca2+]i-dependent fluorescence were recorded in Fura-2-loaded, CCR5-transfected cells (A) exposed to increasing concentrations of I-TAC and stimulated after 30 s with 30 nM RANTES and in monocytes (B) exposed to 1 µM I-TAC and sequentially stimulated with 100 nM MIP-1ß, respectively. Upper tracings show simultaneously performed controls with cells exposed to agonists only. Migration of CCR5-transfected cells (C) or of monocytes (D) in response to MIP-1ß alone (•) or in the presence of 1 µM I-TAC ({blacktriangleup}) and monocytes in response to 1 µM I-TAC alone ({triangleup}) or to buffer ({blacksquare}). Concentration-dependent inhibition of CCR5-transfected cells (E) in response to 10 nM RANTES and of monocytes (F) in response to 100 nM MIP-1ß by I-TAC. Actin polymerization induced by 10 nM RANTES in CCR5-transfected cells (G) and by 30 nM MIP-1ß in monocytes (H) and inhibition after pretreatment with 1 µM I-TAC following identical stimulation. Values are given as fold increase over stimulation with buffer. Assays were performed in triplicates (A–F) or duplicates (G and H). For all experiments, mean values (±SD) of three independent experiments are shown (monocytes from three different donors).

 
To more thoroughly characterize the antagonistic activity of I-TAC on other functional responses, we performed chemotaxis assays using increasing concentrations of RANTES as indicated and cells preincubated in the presence or absence (control migration) of 1 µM I-TAC. I-TAC-treated, transfected cells displayed a significant decrease in migration (Fig. 1C) . Similarly as with transfected cells, monocyte migration was markedly decreased when I-TAC was added together with MIP-1ß (Fig. 1D) . I-TAC at 1 µM did not induce any cell migration different from background movement (cell migration toward buffer).

Next, dose-response experiments were performed using optimal agonist concentrations for migration together with increasing concentrations of I-TAC as indicated. In CCR5-transfected cells (Fig. 1E) , I-TAC displayed almost complete inhibition of cell migration in response to 10 nM RANTES in a dose-dependent manner, with 50% inhibition occurring at approximately 300 nM. In monocytes (Fig. 1F) , I-TAC evoked the inhibition of cell migration in response to 10 nM MIP-1ß in a manner similar to the inhibition seen with CCR5-transfected cells.

When exposed to a chemotactic gradient, cells polymerize actin such that the leading edge, facing the highest concentration of the chemokine, becomes highly enriched in F-actin, allowing cells to move efficiently through the gradient. To test the capability of I-TAC to inhibit actin polymerization in CCR5-transfected cells (Fig. 1G) and in monocytes (Fig. 1H) , actin polymerization evoked by 10 nM RANTES (transfected cells) or by 30 nM MIP-1ß (monocytes) was taken as a positive control. In inhibition experiments, cells were exposed to 1 µM I-TAC prior to sequential stimulation with 10 nM RANTES (transfected cells) or to 30 nM MIP-1ß (monocytes). For transfected cells, we observed partial inhibition in the formation of F-actin after pre-exposure to 1 µM I-TAC, while for monocytes, inhibition was almost complete. Both cell types were stimulated with 1 µM I-TAC alone, which did not lead to any actin polymerization above background (stimulation with buffer).

Binding to a receptor without its triggering is considered to be the hallmark feature of antagonists. To characterize the ability of I-TAC to bind to CCR5 in transfected cells as well as in monocytes, we performed receptor-binding assays. In CCR5-transfected cells, I-TAC readily inhibited the binding of 125I-MIP-1{alpha} (Fig. 2A ). I-TAC had 33-fold and threefold lower inhibitory concentration 50% (IC50) values compared with RANTES and MIP-1{alpha}, with actual values of 2.4 nM for RANTES, 25 nM for MIP-1{alpha}, and 79 nM for I-TAC, respectively. In monocytes (Fig. 2B) , we observed a 26-fold lower IC50 of I-TAC compared with MIP-1ß, with actual values of 36 nM for MIP-1ß and 931 nM for I-TAC, respectively. MIP-1{alpha} displayed an IC50 of 23 nM, a value identical to the one measured for transfected cells.



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  		Figure 2. I-TAC inhibits 125I-MIP-1{alpha} binding on CCR5-transfected cells and monocytes. CCR5-transfected cells were incubated with 125I-MIP-1{alpha} (A) in the presence of increasing concentrations of unlabeled MIP-1{alpha} ({circ}), RANTES (•), and I-TAC ({blacktriangleup}). Monocytes (B) were incubated with 0.15 nM 125I-MIP-1{alpha} in the presence of increasing concentrations of unlabeled MIP-1{alpha} ({circ}), unlabeled MIP-1ß ({triangleup}), and I-TAC ({blacktriangleup}). Values were normalized by setting the specific binding of 0.15 nM 125I-MIP-1{alpha} to 100%, respectively. The curves show mean values (±SD) of two independent experiments performed in duplicates with CCR5-transfected cells or with monocytes from two different donors.

 
Absence of functional effects of the other CXCR3 agonists on CCR5 and of several CCR5 agonists on CXCR3
The antagonistic activity of I-TAC suggested that the other CXCR3 agonists, namely MIG, IP-10 [4 , 6 ], and CXCL13/BCA-1 [23 ], might inhibit CCR5 as well. We thus performed calcium release experiments with CCR5-transfected cells, where cells were stimulated with 30 nM RANTES in the presence or the absence of these three chemokines (Fig. 3A ). As opposed to I-TAC (see the uppermost and lowest tracings in Fig. 1A ), none of the three other CXCR3 agonists had any effect on CCR5-mediated calcium release, indicating that among all CXCR3 agonists, I-TAC is the only one featuring substantial CCR5 inhibition.



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  		Figure 3. (A) Changes of [Ca2+]i-dependent fluorescence in Fura-2-loaded, CCR5-transfected cells sequentially exposed to 1 µM MIG, IP-10, or BCA-1 and 30 nM RANTES. (B) Changes of [Ca2+]i-dependent fluorescence in Fura-2-loaded, CXCR3-transfected cells sequentially exposed to different CCR5 agonists at 1 µM concentration and 1 nM I-TAC at 30 s intervals. Results are representative of one out of two independent experiments. Upper tracings show simultaneously performed controls with cells exposed to agonists only.

 
To address whether any of the major CCR5 agonists such as RANTES, MIP-1{alpha}, MIP-1ß, and CCL8/MCP-2 exert antagonistic activity on CXCR3 (as an inverse control), a murine pre-B cell line singly transfected with human CXCR3 was tested using free [Ca2+]i changes as a functional readout. Cells were left untreated or exposed to 1 µM of different CCR5 agonists and were sequentially stimulated with 1 nM I-TAC. As shown in Figure 3B , none of the chemokines tested exerted any antagonistic activity on CXCR3.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that I-TAC, a highly potent, selective agonist for CXCR3, acts as an antagonist on CCR5. It inhibited the binding of 125I-MIP-1{alpha} in CCR5-transfected cells and monocytes and inhibited chemotaxis in transfected cells and monocytes. At 1 µM concentration, I-TAC inhibited actin polymerization partially in transfected cells and completely in monocytes. Free [Ca2+]i changes were completely inhibited in transfected cells and partially in monocytes. Further, I-TAC did not induce internalization of CCR5 in transfected cells (data not shown). Taken together, our results thus show the antagonistic activity of I-TAC on CCR5 to be sufficient to reduce chemotaxis, free [Ca2+]i changes and actin polymerization to minimum levels in transfected as well as primary cells.

I-TAC inhibited responses to both agonists tested: Although RANTES, being the most potent agonist of CCR5 [30 ], was used for transfected cells, MIP-1ß was used on monocytes to allow for specific stimulation of CCR5, as the presence of CCR1 prevented the application of MIP-1{alpha} or RANTES on these cells. I-TAC displayed a 12-fold lower IC50 value (931 nM) on monocytes as compared with the CCR5 transfectants (79 nM), and the homologous competition with MIP-1{alpha} featured identical values for both cell types (23 nM and 25 nM, respectively). Somewhat surprisingly, I-TAC showed similar inhibitory potencies (approximately 300 nM) for both cell types. For transfected cells, the binding and activity IC50 values are consistent with one another. For monocytes, the situation may be different: As determined in the binding experiments, receptor occupancy at 1 µM would be approximately 50%, which seems somewhat low to explain the almost complete functional inhibition (Fig. 1D and 1F) . In each single case, these differences are rather small. Collectively, however, they raise serious doubts as to if the inhibitory effects of I-TAC on CCR5 are a result of competitive receptor binding. We think it is likely that the low, apparent IC50 seen in the binding experiments on monocytes is a result of nondisplaced, MIP-1{alpha} binding to CCR1, although the values fit well to a one-site binding model. It is noteworthy that similar findings were reported independently by the two groups who found eotaxin to be a natural antagonist of CCR2 [32 , 33 ]: In both cases, the IC50 values were significantly lower in the binding experiments as compared with the activity assays. It is interesting that I-TAC has been shown to interact in an allotropic way with other chemokines at CXCR3 [48 ], and it cannot be excluded that I-TAC behaves accordingly at CCR5.

It should be mentioned that the antagonistic activity of I-TAC has briefly been touched upon before. To address the antagonistic specificity of I-TAC, Loetscher et al. [20 ] tested 14 murine pre-B cell lines singly transfected with different human chemokine receptors for antagonistic activity using [Ca2+]i changes as a functional readout. Apart from a potent inhibition of CCR3, they observed antagonistic activity of I-TAC on CCR5 but to a lesser extent (40% as opposed to substantial inhibition in our experiments) but did not expand or comment on their findings any further. I-TAC did not inhibit any of the other chemokine receptors tested. In particular, I-TAC did not affect the calcium response mediated by CCR1, suggesting that inhibition of monocyte responses, as demonstrated here, is a result of effects of I-TAC on CCR5 alone. Their and our own data together would thus indicate that I-TAC is a selective antagonist for CCR3 and CCR5.

Of the four agonists of CXCR3, I-TAC is the only one to inhibit CCR5. This fortuitous fact allows the identification of I-TAC residues that presumably mediate binding to CCR5 by sequence alignments, as such residues might consequently be conserved among all chemokines that bind to CCR5, including I-TAC, but not be conserved in the other three CXCR3 agonists. The alignments in Figure 4A and 4B , show the patterns of conservation among CCR5- and CXCR3-specific chemokines, respectively. Comparison of these two sets of residues reveals five amino acids that meet the two requirements: I43 and A61, which along with the corresponding residues of the other chemokines aligned, are light gray (Fig. 4A and 4B) and K17, K49, and Q51, which are similarly highlighted in dark gray or black (see figure legend and below for further explanations).



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  		Figure 4. Structural and sequence analysis of I-TAC binding to CCR5. (A) Alignment of all chemokines known to bind CCR5 with identical (*), highly conserved (:), and remotely conserved (.) residues indicated at the bottom of the alignment. Residues that are conserved among these chemokines but not among CXCR3 agonists are highlighted light and dark gray. The extended C terminus of human ß chemokine-4 (HCC-4) is shown below the alignment. (B) Multiple sequence alignment of all CXCR3 agonists, with patterns of conservation indicated (A) but residues chemically corresponding to those of CC chemokines, is in black. (C) Homology model of I-TAC in a ribbon representation with coloring of the amino acids shown with their van der Waals spheres as in B. The protein is shown with the N terminus in the upper-right quadrant. The ß1–ß2 loop (30 s loop) is at the top of the protein, partially hidden by the N terminus. The N-loop, the ß2–ß3 loop (40 s loop), and the ß3 strand, which compose a binding domain for the receptors’ N-termini in many chemokines, are facing the viewer. (D) I-TAC, as in C, where the surface representation of the protein is shown. K17, K49, and Q51 are colored gray, with their solvent-accessible side-chains clearly visible. I43 and A61 are buried within the chemokine and are not visible.

 
The structure of I-TAC has not been reported yet. To better understand the potential function of the residues identified, we developed a homology model of I-TAC, which was based on the solved structure of IL-8. According to this modeled structure, K17 is situated in the loop region proximal to the conserved cysteine pair (N-loop), and K49 and Q51 are found in the proximal portion of the ß3 strand (Fig. 4C) . Note that all three residues feature side-chains that are fully solvent-exposed (Fig. 4D) . At the positions corresponding to K17 of I-TAC (black in Fig. 4B ), all CCR5-binding chemokines feature basic amino acids as well. Although IP-10 displays a glutamine at this position, MIG and BCA-1 have completely divergent amino acids at the corresponding locations. R16, the corresponding amino acid of eotaxin, is involved in binding to the N-terminus of CCR3, as evidenced by 1H-15N correlation nuclear magnetic resonance spectroscopy [49 ]. R17, the respective residue of RANTES, is crucial for CCR-1 binding [50 ], and R18 of MIP-1{alpha} and MIP-1ß mediates chemokine interactions with glycosaminoglycans (GAG) [51 , 52 ].

A similar pattern is seen for K49 of I-TAC (black in Fig. 4B ). Again, all CCR5-binding chemokines display basic amino acids at this position, and a lysine is found in BCA-1 as well (black in Fig. 4B ). In MIG and IP-10, the respective residue is a glycine, and both chemokines would hence be devoid of this prominent basic protrusion. Of note, K49 of I-TAC corresponds to K48 of MIP-1ß, which has been reported to be a critical mediator for binding to CCR5 [53 ] as well as GAG [51 , 53 ]. R47, the corresponding residue of RANTES, mediates binding to CCR1 and CCR3 [54 ], and K47 in eotaxin is involved in CCR3 binding [49 ].

Further, the sequence alignments indicate Q51 of I-TAC to be of potential importance (black in Fig. 4B ). At this position, CCR5-binding chemokines feature a glutamine or an acidic residue. Although MIG displays a glutamine as well (black in Fig. 4B ), IP-10 and BCA-1 diverge at this position. We have not found any reports addressing the function of the corresponding residues in other chemokines, but its position in the third ß strand close to K49 and its chemical nature are certainly consistent with a role in receptor binding. Thus, I-TAC is the only CXCR3 agonist that features CC chemokine-like residues at all three positions, presumably crucial for CCR5 binding.

The two remaining hydrophobic residues, I43 and A61, differ in several respects from the three other hydrophilic residues that are conserved. They are located close to one another and both buried deep within the protein, which makes them unlikely mediators of receptor binding. Rather, their positions and vicinity would imply that via hydrophobic interactions, they serve to stabilize the protein structure. Consistent with this hypothesis, the corresponding positions in all CC chemokines are identical (featuring a phenylalanine and a valine) and are highly conserved between MIG and IP-10 as well (alanine and valine or isoleucine, respectively). In BCA-1, two hydrophobic residues are found, and I-TAC differs only in that the two residues have swapped their positions (isoleucine and alanine, respectively).

Our sequence analyses of all chemokines that bind to CCR5 and/or CXCR3 thus suggest that three amino acids of I-TAC, namely K17, K49, and Q51, may mediate its binding to CCR5. The corresponding residues of many CCR5 agonists are known to be involved in receptor and/or GAG binding, which may be taken as an indication for the validity of our approach. However, it is very much possible that chemokine antagonists might not bind to receptors in the same manner as chemokine agonists. It should also be noted that such an analysis, which essentially correlates receptor specificity with sequence conservation, will most likely not identify all residues that contribute to a particular function. Still, it is useful to furnish a starting point for understanding the structural requirements that govern receptor binding of antagonistic chemokines.

Although receptor antagonism by natural chemokines has been demonstrated in vitro in several cases, the concentrations required to obtain significant inhibition are most often high and exceed those necessary to activate the respective target receptors, and CCR5 inhibition by I-TAC "suffers" from the same problem. Thus, it seems likely that receptor inhibition would occur only in close proximity to the inflammatory sites where I-TAC is produced. As well, I-TAC antagonism of CCR5 would mainly be relevant for cells that do not coexpress CXCR3. Hence, T lymphocytes of the Th1 type, which express CXCR3 and CCR5 [7 8 9 ], are not the likely target. Immature DCs express CCR1 and CCR5 but no CXCR3 [55 , 56 ], and their response to CC chemokines activating CCR1 and CCR5 may thus at least partially become affected by CXCR3. The same conclusion may hold true for peripheral CD14++CD16– monocytes such as used here. It is interesting that CD14+CD16+ monocytes show lower and equal levels of CCR2 and CCR1 in comparison, respectively, CCR5 levels are increased, and functional responses reflect these changes [57 ]. As a result of this particular expression pattern, these cells may represent a specific target for CCR5 inhibition by I-TAC. Presumably, the homologous population of these cells has recently been identified in mice and is characterized by CX3CR1-dependent recruitment to noninflamed tissues [58 ].

The present study extends the already fairly extensive list of activities exerted by IFN-{gamma}-inducible agonists of CXCR3 even further. Initially considered to be specific agonists for CXCR3 only, they were later found to have antimicrobial activity [22 ] and antagonize CCR3 [20 , 21 ] as well, effects that have not been described so far for BCA-1, the fourth CXCR agonist. I-TAC now proves to be even more "versatile" in that with CCR5, it inhibits yet another receptor, thus counteracting in addition the activities of MIP-1{alpha} and MIP-1ß, the two inflammatory CC chemokines that act via CCR5 but not via CCR3.


   ACKNOWLEDGEMENTS
 
This work was supported by the Helmut Horten Foundation and the Roche Research Foundation.

Received November 18, 2003; revised March 11, 2004; accepted March 13, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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