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Originally published online as doi:10.1189/jlb.1003479 on April 9, 2004

Published online before print April 9, 2004
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(Journal of Leukocyte Biology. 2004;76:185-194.)
© 2004 by Society for Leukocyte Biology

Synergy between proinflammatory ligands of G protein-coupled receptors in neutrophil activation and migration

Mieke Gouwy, Sofie Struyf, Julie Catusse, Paul Proost and Jo Van Damme1

Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Belgium

1Correspondence: Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: Jozef.Vandamme{at}rega.kuleuven.ac.be


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ABSTRACT
 
The chemokine dose and the time period during which the chemotactic gradient is established determine the number of leukocytes that infiltrate inflamed tissues. At suboptimal chemokine concentrations, neutrophils may require a priming agent or a second stimulus for full activation. An interesting mode of cooperative action to reach maximal migration is synergy between chemokines. This was first observed between the plasma CC chemokine regakine-1 and the tissue CXC chemokine ligand interleukin-8 (IL-8/CXCL8) in neutrophil chemotaxis. Addition of antibodies against IL-8 or regakine-1 in the Boyden microchamber assay abrogated this synergy. Other CC chemokines, such as CC chemokine ligand-2 monocyte chemotactic protein-1 (MCP-1/CCL2), MCP-2 (CCL8), and MCP-3 (CCL7) as well as the CXC chemokine receptor-4 (CXCR4) agonist stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12), also dose-dependently enhanced neutrophil chemotaxis toward a suboptimal concentration of IL-8. These chemokines synergized equally well with the anaphylatoxin C5a in neutrophil chemotaxis. Alternatively, IL-8 and C5a did not synergize with an inactive precursor form of CXCL7, connective tissue-activating peptide-III/CXCL7, or the chemoattractant neutrophil-activating peptide-2/CXCL7. In the chemotaxis assay under agarose, MCP-3 dose-dependently increased the migration distance of neutrophils toward IL-8. In addition, the combination of IL-8 and MCP-3 resulted in enhanced neutrophil shape change. AMD3100, a specific CXCR4 inhibitor, reduced the synergistic effect between SDF-1{alpha} and IL-8 significantly. SDF-1{alpha}, but not MCP-1, synergized with IL-8 in chemotaxis with CXCR1-transfected, CXCR4-positive Jurkat cells. Thus, proinflammatory chemokines (IL-8, MCP-1), coinduced during infection in the tissue, synergize with each other or with constitutive chemokines (regakine-1, SDF-1{alpha}) to enhance the inflammatory response.

Key Words: chemokines • chemotaxis • shape change • C5a • receptor


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INTRODUCTION
 
Among the cytokines released in the earliest phases of infection are members of the family of chemoattractant cytokines known as chemokines, which are related in amino acid sequence, with four conserved cysteines as a hallmark. Members of the chemokine family fall into two major groups, CC chemokine ligands (CCL) with two adjacent cysteines near the N terminus [e.g., monocyte chemotactic protein-3 (MCP-3)/CCL7] and CXC chemokine ligands (CXCL), for which the equivalent two cysteine residues are separated by another amino acid [e.g., interleukin-8 (IL-8)/CXCL8]. All chemokine receptors are integral membrane proteins containing seven membrane-spanning helices and belong to the family of G protein-coupled receptors (GPCR). Infection or physical damage to tissues initiates the production of chemokines by a wide variety of cell types. Chemokine receptor recognition and signaling via G proteins results in leukocyte migration, e.g., from the blood into the inflamed tissue, in response to the locally generated chemotactic gradient.

The recruitment of leukocytes under normal homeostasis and inflammatory conditions involves a multistep process [1 ]. In an initial step, leukocytes weakly adhere to the endothelial cell layer but are still allowed to roll along the venular wall. This rolling is promoted by vasodilation and by expression of selectins on leukocytes (L-selectin) and endothelial cells (E- and P-selectins). In the second step of extravasation, chemokine-stimulated leukocytes firmly attach to the endothelial layer and change in shape through interaction of integrins on the leukocyte with adhesion molecules on the endothelial cell. Integrin adhesiveness is enhanced by chemokines produced by leukocytes (e.g., IL-8) as well as by endothelial and tissue cells. Chemokines accumulate on the endothelial cell layer through glycosaminoglycan binding and activate leukocytes through specific recognition of their corresponding GPCR. In the third step, leukocytes migrate through the endothelial cell layer into the underlying tissue to the chemokine source. Activation of leukocytes by chemokines provokes the secretion of proteases, which can degrade the subendothelial extracellular matrix and facilitate the migration of leukocytes.

Many chemokines bind more than one receptor, and chemokine receptor generally bind more than one chemokine [2 ]. However, stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) is a CXC chemokine interacting with a single and unique receptor, CXCR4. This is supported by the identical phenotypes of SDF-1{alpha}–/– and CXCR4–/– mice, showing impaired hematopoiesis and embryogenesis [3 4 5 6 ]. The CXCR4 is not shared with any other chemokine but is a coreceptor for T-tropic human immunodeficiency virus type 1 (HIV-1) strains [7 ]. In contrast with SDF-1{alpha}, which is constitutively expressed in specific lymphoid or nonlymphoid tissues, other chemokines, such as platelet factor-4 (PF-4/CXCL4), connective tissue-activating peptide-III (CTAP-III/CXCL7), and regakine-1, are found at high concentrations in normal serum [8 9 10 ]. Recently, a high-affinity receptor for PF-4 was discovered, namely CXCR3-B, derived from an alternative splicing of the CXCR3 gene [11 ]. NH2-terminal processing is necessary for CTAP-III to recognize CXCR2. Indeed, after processing by proteases, CTAP-III becomes a functionally active chemokine known as the CXCR2 ligand neutrophil-activating peptide-2 (NAP-2/CXCL7) [12 ]. In contrast to constitutive chemokines, many chemokines, such as MCP-1 (CCL2), play an important role in inflammation [13 ]. Although MCP-1 also binds to a single receptor, CCR2 [14 ], the structurally related MCP-2 (CCL8) and MCP-3 (CCL7) each attract a different set of leukocyte cell types, which is a consequence of differential binding to shared receptors (CCR1, -2, -3, and -5) expressed on the target cells [15 16 17 18 ].

The chemokine dose and the time period during which the chemotactic gradient is established determine the number of leukocytes that will infiltrate the inflamed tissue. In addition, neutrophils may benefit from a priming agent or a second stimulus for full activation when only a low dose of chemokine is present, which would be insufficient to cause cell migration without priming. Some chemokines (e.g., MCP-1) are abundantly expressed, and the expression level of other chemokines, such as MCP-2 and MCP-3, is quantitatively more restricted [19 ]. For optimal chemokine production, synergy between several stimuli, e.g., cytokines, is often required. For example, costimulation of fibroblasts by IL-1ß and interferon-{gamma} resulted in a synergistic induction of MCP-2 in vitro [20 ]. Very recently, it was found that the anaphylatoxin C3a plays an essential role in promoting the homing of hematopoietic progenitor cells to the bone marrow through its synergistic interaction with the SDF-1{alpha}–CXCR4 signaling axis [21 ]. Human hematopoietic stem and progenitor cells express functional C3a receptors. After binding to its receptor, C3a sensitizes the responses of these cells to SDF-1{alpha} and thus, may be involved in promoting their homing to the bone marrow [21 ]. Furthermore, MCP-1 induces the release of arachidonic acid in monocytes, and this arachidonate release was potentiated by addition of platelet-activating factor [22 ]. These findings are in agreement with the observation that the constitutive plasma CC chemokine regakine-1 synergizes with C5a and the bacterial peptide formyl-Met-Leu-Phe (fMLP) in neutrophil chemotaxis [10 , 23 , 24 ]. Another interesting mode of cooperation in the inflammatory system is the synergy between chemokines. Indeed, regakine-1 also synergizes with the CXC chemokines IL-8, NAP-2, and granulocyte chemotactic protein-2 (GCP-2/CXCL6) [10 , 23 ]. In this study, we verified whether this cooperation between regakine-1 and other mediators of inflammation was restricted to constitutive chemokines present at high concentrations in the plasma. Here, we describe the synergistic effect between different CC or CXC chemokines and IL-8 or C5a in various neutrophil activation and migration tests.


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MATERIALS AND METHODS
 
Cells
Human neutrophilic granulocytes were freshly isolated from venous blood obtained from healthy donors (Blood Transfusion Center of Antwerp and Laboratory of Experimental Immunology, University of Leuven, Belgium). Erythrocytes were removed by sedimentation for 30 min at 37°C with hydroxyethyl starch solution (Plasmasteril, Fresenius AG, Bad Homburg, Germany). Mononuclear cells and granulocytes were separated by density gradient centrifugation for 30 min at 400 g on Ficoll-sodium diatrizoate (Lymphoprep, Invitrogen, Groningen, The Netherlands). Remaining erythrocytes were lysed by hypotonic shock for 30 s in bidistilled water. The human T cell line Jurkat, clone J45.01 was obtained from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK). Jurkat transfectants expressing the human CXCR1 (Jurkat/CXCR1) were generated using the pcDNA-3 expression vector (Invitrogen), which contains a neomycin (G418)-resistance gene [25 ]. Jurkat cells were grown at 37°C, 5% CO2, in RPMI 1640 (Invitrogen), supplemented with 10% (v/v) fetal calf serum (FCS; Invitrogen) and 0.5 mg/ml G418 (Invitrogen).

Chemoattractants, antibodies, and inhibitors
Natural human IL-8, NAP-2, and MCP-1 were purified to homogeneity from monocyte-derived, conditioned medium [26 , 27 ]. A fraction containing CTAP-III was purified from blood platelets [26 ]. Regakine-1 was isolated from FCS (Invitrogen) derived from Bos taurus [10 ]. Natural chemokines were purified by subsequent adsorption to silicic acid, heparin-Sepharose affinity chromatography (Amersham Biosciences, Uppsala, Sweden), mono S cation-exchange chromatography, and reversed-phase, high-performance liquid chromatography (RP-HPLC; Perkin-Elmer, Norwalk, CT), as described previously [10 ]. The CC chemokines MCP-2 and MCP-3 were synthesized by solid-phase peptide synthesis using fluorenylmethoxycarbonyl chemistry and were purified as described [28 ].

The anaphylatoxin C5a was purified to homogeneity from human plasma by heparin-Sepharose affinity chromatography, resource S cation-exchange chromatography (Amersham Biosciences), and RP-HPLC on a resource reversed-phase chromatography column (Amersham Biosciences) [24 ]. Recombinant PF-4 was purchased from Peprotech (Rocky Hill, NJ), and recombinant human SDF-1{alpha} was obtained from R&D Systems (Abingdon, UK). Polyclonal antibodies against regakine-1 (in rabbit) and IL-8 (in goat) were purified by protein A or protein G affinity chromatography, respectively (Amersham Biosciences), and dialyzed to pH 7.5. The CXCR4 antagonist AMD3100 was obtained from Sigma-Aldrich (St. Louis, MO) [29 ].

Chemotaxis through micropore filters
The capacity of purified chemokines to chemoattract human neutrophils or Jurkat cells was determined in a 48-well chemotaxis microchamber (NeuroProbe, Gaithersburg, MD). Cell fractions and samples were diluted in Hank’s balanced salt solution (HBSS; Invitrogen), supplemented with 1 mg/ml human serum albumin (HSA; Belgian Red Cross; dilution buffer), and tested in triplicate. The lower part of the Boyden chamber, containing the test sample or control dilution buffer, was separated from the upper plate containing neutrophils (1x106 cells/ml) or CXCR1-transfected Jurkat cells (2x106 cells/ml) by a polyvinyl pyrrolidone-free polycarbonate filter with a 5-µm pore size (Nuclepore, Corning Costar, Acton, MA). To test the synergistic effect of two chemoattractants, both molecules were added to the lower wells of the microchamber. After incubation of the chemotaxis chamber in a 5% CO2 incubator at 37°C for 45 min, cells were fixed and stained with Hemacolor solutions (Merck, Darmstadt, Germany). The migrated cells adherent to the lower surface of the membranes were counted microscopically in 10 oil immersion fields at 500x magnification. The chemotactic activity of the sample was expressed as a chemotactic index, calculated as the number of cells that migrated in response to the sample, divided by the number of cells that migrated toward the negative control (dilution buffer). In neutralization experiments, chemokine antibodies (rabbit polyclonal antiregakine-1 and goat polyclonal anti-IL-8) were incubated with the chemokines regakine-1 and/or IL-8 for 1 h at 37°C before addition of the mixture to the lower compartment of the microchamber. The stimulation index obtained with chemokines in the presence of antibody was calculated by dividing the number of migrated cells in response to chemokine plus antibody by the number of migrated cells in the presence of antibody alone. For inhibition of the chemokine activity by AMD3100, the CXCR4 inhibitor was added to the cells just before transfer to the upper compartment of the microchamber. The chemotactic indexes were calculated using the appropriate controls (AMD3100 treated vs. untreated control cells).

Chemotaxis under agarose
The assay under agarose was performed as described previously with minor modifications [30 ]. This method uses the migration distance of cells under agarose as a parameter to measure the chemotactic effect of chemoattractants. For preparation of the agarose plates, equal volumes of solution A and solution B were mixed. Solution A contained 10 ml prewarmed (48°C) medium, consisting of 2 ml FCS (Invitrogen), 2 ml 10x concentrated Eagle’s minimum essential medium (EMEM; Invitrogen) with Earle’s salts, and 6 ml pure water. Solution B was prepared by boiling 0.18 g agarose (Indubiose A37, Biosepra Inc., Marlborough, MA) in 10 ml pure water until completely dissolved and cooled to 48°C. In each plastic tissue-culture dish (diameter, 50 mm), 6 ml solution A/B was poured and cooled for 30 min before transfer to the refrigerator (4°C overnight). The day that the agarose assay was performed, six series (per dish) of three wells (3 mm in diameter and 3 mm interspace) were cut in the gel in a straight line using a template and a stainless-steel punch with inside bevel. The agarose cores were removed with a pipette by using vacuum. The gels were allowed to equilibrate at 37°C in a 5% CO2 incubator until sample dilutions and cells were prepared. Neutrophils (3x107 cells/ml) and chemokines were diluted in HBSS solution supplemented with 1 mg/ml HSA. The center well of each series of three wells was loaded with human neutrophils (3x105 cells in 10 µl), and the inner and outer wells were loaded with control dilution buffer and varying concentrations of chemoattractant, respectively. When a combination of two chemokines or a combination of a chemokine with cells was tested, the mixture was prepared before transfer to the wells. Agarose plates with cells were incubated (2 h/37°C/5% CO2), allowing sufficient time for neutrophils to migrate toward the chemotactic gradient of the test sample (distance X) or the control medium (random migration distance Y). The assay was terminated by adding 3 ml absolute methanol to the agarose plates at room temperature. After 30 min incubation, the methanol was removed, and the cells were fixed with formaldehyde (final concentration, 37%) for 30 min. After decanting the formaldehyde, the agarose was carefully removed from the culture dish, and the cells were stained with Hemacolor solutions (Merck). The migration distance (X–Y) was estimated and expressed as the percentage of maximal migration (3 mm).

Shape change
Purified human neutrophils (0.6x106 cells/ml) were diluted in HBSS supplemented with 10 mM HEPES (Invitrogen) and incubated in a 96-well microtiter plate in the presence of dilution buffer or chemoattractant solutions. At different time intervals (0, 0.5, 1, 3, 6, and 9 min), the cells were fixed by adding an equal volume of HBSS/HEPES buffer containing 4% formaldehyde. Changes in the cellular shape of the neutrophils were microscopically evaluated (500x magnification). Differential cell counts (rounded, blebbed, or spread) for each treatment, were obtained from four microscopic fields (each ~100 cells).

Measurement of intracellular calcium concentration
Changes in the intracellular calcium concentration [Ca2+]i were monitored using the fluorescent indicator fura-2 [31 ]. In brief, purified neutrophils (107 cells/ml) were incubated for 30 min at 37°C in EMEM + 0.5% FCS, supplemented with 2.5 µM fura-2/AM (Molecular Probes Europe BV, Leiden, The Netherlands) and 0.01% Pluronic F-127 (Sigma-Aldrich). After incubation, the cell suspensions were washed to remove excess of indicator and diluted to 106 cells/ml in calcium buffer (HBSS containing 1 mM Ca2+ and 0.1% FCS, buffered at pH 7.4, with 10 mM HEPES/NaOH). Fura-2 fluorescence was measured in an LS50B luminescence spectrophotometer and fitted with a water-thermostatable, stirred, four-position cuvette holder (Perkin-Elmer). The second stimulus was added 100 s after the first stimulus.


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RESULTS
 
CC and CXC chemokines synergize with IL-8 in the Boyden chamber chemotaxis assay for neutrophils
Previous findings demonstrated that the plasma CC chemokine and weak neutrophil chemoattractant regakine-1 synergized at physiological concentrations (100 ng/ml) with the CXC chemokines IL-8, GCP-2, and NAP-2 to chemoattract neutrophils in the Boyden chamber assay [10 , 23 ]. This phenomenon was further investigated in-depth using other CXC and CC chemokines in combination with IL-8. Preliminary experiments using low concentrations (30 ng/ml) of the CC chemokine MCP-3 in combination with IL-8 (5 ng/ml) indicated that the synergy could be restricted to regakine-1 [24 ]. However, Figure 1 shows that higher concentrations of MCP-3 (100 and 300 ng/ml) significantly increased (above the additive effect of the two chemokines; P<0.018 and P<0.012, respectively) the chemotactic response of human neutrophils toward a suboptimal concentration of IL-8 (5 ng/ml) in the Boyden microchamber assay. In addition, similar concentrations of MCP-1 and MCP-2 (100 and 300 ng/ml) also yielded a statistically significant enhancement of the chemotactic index obtained with IL-8, confirming that the synergy with IL-8 is not unique for regakine-1 (Fig. 1) . At 100 ng/ml, MCP-1, MCP-2, and MCP-3 yielded maximal monocyte chemotactic responses in this assay (data not shown), whereas these CC chemokines had only marginal neutrophil chemotactic activity (Fig. 1) . In contrast, IL-8 was not found to cooperate in the neutrophil chemotaxis assay when combined with 300 ng/ml of the CXC chemokine CTAP-III. This precursor form of CXCL7 does not exert a neutrophil chemotactic activity as a result of the lack of affinity for CXCR1 and/or CXCR2 or any other chemokine receptor [32 ]. NAP-2, a truncated form of CTAP-III, which like IL-8, binds to CXCR2, showed significant neutrophil chemoattraction on its own at 100 ng/ml but failed to further enhance the chemotactic response to IL-8 (5 ng/ml). In contrast, the CXC chemokine and CXCR4 agonist SDF-1{alpha} (100 and 300 ng/ml) did synergize with IL-8 (5 ng/ml) to chemoattract neutrophils in a statistically significant manner (P<0.037 and P<0.002, respectively), yielding two- to threefold higher chemotactic indexes compared with the additive effect of IL-8 and SDF-1{alpha}. The fact that 300 ng/ml SDF-1{alpha} induced neutrophil migration confirms the expression of CXCR4 on these cells.



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  		Figure 1. Synergy between CC and CXC chemokines with IL-8 to induce neutrophil chemotaxis in the Boyden chamber. The CC chemokines MCP-1, MCP-2, and MCP-3 (100 and 300 ng/ml; A), the plasma chemokine regakine-1 (RK-1; 300 ng/ml), and the CXC chemokines SDF-1{alpha} (100 and 300 ng/ml), PF-4 (300 ng/ml), CTAP-III (300 ng/ml), and NAP-2 (100 ng/ml; B) were tested in the presence (+) or absence (–) of IL-8 (5 ng/ml) in the lower compartment of the microchamber to measure human neutrophil chemotaxis. The chemotactic response is expressed as the mean chemotactic index ± SEM, derived from several (n≥3) independent experiments. Statistically significant differences in chemotactic indexes between the combination of IL-8 with another chemokine and the sum of the indexes obtained for the chemoattractants alone, determined by the Mann-Whitney test, are indicated by asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).

Synergy between IL-8 and MCP-3 to chemoattract neutrophils in the agarose assay
To confirm specific interaction between CC and CXC chemokines in neutrophil migration, rather than an aspecific interference with the Boyden microchamber assay equipment, similar experiments were done in the agarose assay (Fig. 2 ). In this less-sensitive test system, the migration distance, rather than the number of migrated neutrophils, was used as a parameter for chemotactic potency. In addition, chemokine (MCP-3) was added to the test cells in the central well or together with IL-8 in the outer well. It was observed that MCP-3 dose-dependently enhanced the migration distance of neutrophils toward IL-8 (50 ng/ml), irrespective of whether MCP-3 was added to the cells or combined with IL-8. In contrast with the more sensitive Boyden chamber assay, the maximal effect was already observed when a relatively low concentration of MCP-3 (30 ng/ml) was combined with an equimolar concentration of IL-8. Furthermore, higher MCP-3 (300 ng/ml) levels, mixed with IL-8 or the cells, reduced the synergy in this neutrophil migration assay, indicative for a classical bell-shaped dose-response curve. Application of MCP-3 in the agarose assay in the absence of IL-8 did not trigger a chemotactic response at any concentration, whereas a low concentration of IL-8 (5 ng/ml) also failed to significantly chemoattract neutrophils in this assay. It can be concluded that the synergistic effect between IL-8 and MCP-3 is not dependent on the chemotactic test system and that at suboptimal (50 ng/ml) concentrations, both agonists do cooperate. However, when a higher concentration of IL-8 (150 ng/ml) was applied in the agarose assay, synergy could not be observed (Fig. 2) .



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  		Figure 2. Synergy between IL-8 and MCP-3 to chemoattract neutrophils in the agarose assay. MCP-3 (30, 100, and 300 ng/ml) was added with the neutrophils in the central well (A) or together with IL-8 (50 and 150 ng/ml) in the outer well (B). The effective migration distance was calculated by substraction of the random migration (Y) from the induced migration (X). Distance X is the distance of neutrophil migration from the edge of the central well outward toward the well containing IL-8 plus MCP-3 or IL-8 alone. Distance Y is the random neutrophil motility from the edge of the well outward toward the well containing control medium. The migration distance (X–Y) is expressed as the percentage of maximal distance (3 mm). Results represent averages ± SEM of four independent experiments.

CXC and CC chemokines cooperate with C5a in neutrophil chemotaxis
Complement factor C5a is a potent neutrophil chemoattractant and a principal mediator in eliciting local and systemic inflammatory responses. Earlier experiments demonstrated a synergistic effect between regakine-1 (30 or 100 ng/ml) and C5a (30 or 100 ng/ml) on neutrophil chemotaxis in the Boyden chamber assay [24 ]. To verify whether such synergistic effect existed between C5a and other GPCR agonists, additional Boyden chamber chemotaxis experiments were performed (Fig. 3 ). The CC chemokines MCP-3 (300 ng/ml) and regakine-1 (100 ng/ml) caused a statistically significant (P<0.003 and P<0.02, respectively) increase above the additive neutrophil chemotactic index of C5a (30 ng/ml) and the CC chemokine. However, high concentrations (300 ng/ml) of platelet-derived PF-4 and CTAP-III, lacking significant chemotactic activity on human neutrophils, yielded no enhanced chemotactic response in combination with C5a (30 ng/ml). In addition, combined application of C5a and the CXCR2 ligand NAP-2 (100 ng/ml) did not result in chemotactic indexes higher than the additive effect of the two agonists, whereas the CXCR4 ligand SDF-1{alpha} (P<0.007) enhanced the neutrophil chemotactic response toward C5a significantly. The data indicate that the synergy observed between C5a and chemokines shows a similar pattern to that seen for IL-8.



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  		Figure 3. CXC and CC chemokines synergize with C5a in neutrophil chemotaxis. Regakine-1 (RK-1; 100 ng/ml), MCP-3 (300 ng/ml), SDF-1{alpha} (300 ng/ml), PF-4 (300 ng/ml), CTAP-III (300 ng/ml), and NAP-2 (100 ng/ml) were combined with C5a (30 ng/ml) in the lower compartment of the microchamber to measure human neutrophil chemotaxis. The chemotactic response is expressed as the mean chemotactic index ± SEM, derived from several (n≥4) independent experiments. Statistically significant differences in chemotactic indexes between the combination of C5a with a chemokine and the sum of the indexes obtained for the chemoattractants alone, determined by the Mann-Whitney test, are indicated by asterisks (*, P<0.05; **, P<0.01).

Chemokines synergize to induce a fast change in cell shape
A rapid test for activation of neutrophils is the morphological change (within a few minutes) after exposing the cells to a chemotactic factor. Neutrophil shape change was evaluated with light microscopy after fixation of the cells in suspension at different time intervals. Three dose- and time-dependent effects on neutrophilic morphology were observed and used as score: rounding, blebbing, and spreading of cells. Figure 4 (upper panel) shows the change in cell shape of human neutrophils stimulated for 6 min with MCP-3 (300 ng/ml), IL-8 (5 ng/ml), or the combination of MCP-3 (300 ng/ml) with IL-8 (5 ng/ml). Neutrophils treated with buffer or MCP-3 alone remained spherical in shape. The IL-8-treated cells were mainly blebbing cells, whereas neutrophils treated simultaneously with the two agonists (IL-8 and MCP-3) were more elongated and spread out in comparison with the cells treated with IL-8 alone. To quantify this result, we counted the rounded, blebbed, and spread cells in each condition of a typical experiment. After treatment with MCP-3 (300 ng/ml) plus IL-8 (5 ng/ml), a significant increase of blebbed and spread cells (P<0.02) was observed compared with the condition where the cells were treated with IL-8 (5 ng/ml) alone (Fig. 4 , lower panel). Thus, MCP-3 enhanced the shape change of neutrophils in cooperation with IL-8.



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  		Figure 4. Chemokines synergize to induce a fast change in cell shape. (Upper panel) Changes in cell shape of human neutrophils stimulated for 6 min with buffer (A), MCP-3 (300 ng/ml; B), IL-8 (5 ng/ml; C), and the combination of MCP-3 (300 ng/ml) with IL-8 (5 ng/ml; D). IL-8 treatment resulted in increased neutrophil blebbing, whereas further addition of MCP-3 provoked more pronounced spreading of the cells. Arrows indicated some neutrophils showing change in cell shape. (Lower panel) The number of rounded, blebbed, or spread cells was counted after 6 min of treatment of the neutrophils with buffer, MCP-3 (300 ng/ml), IL-8 (5 ng/ml), or the combination of MCP-3 (300 ng/ml) with IL-8 (5 ng/ml). Cell numbers are derived from one typical experiment by counting four microscopic fields (~4x100 cells) for each treatment. Statistically significant differences in cell numbers between the combination of IL-8 with MCP-3 and IL-8 alone, determined by the Mann-Whitney test, are indicated by asterisks (*, P<0.05).

The synergistic effect between CC and CXC chemokines is inhibited by a specific receptor antagonist or chemokine antibody
To exclude that the synergy between chemokines is a result of contamination of the preparation with impurities, other than chemokines, neutralization assays were performed with specific antibodies. Figure 5A shows that if IL-8 and regakine-1 were preincubated for 1 h at 37°C with their corresponding specific antibody before transfer to the microchamber, their neutrophil chemotactic capacity was completely abolished. In contrast, the chemotactic capacity was not significantly impaired by incubation of IL-8 with antibodies against regakine-1 and vice versa (data not shown). More importantly, the synergistic effect between regakine-1 (100 ng/ml) and IL-8 (5 ng/ml) was reduced significantly (P<0.02) by antibodies to regakine-1, to the level of chemotactic response obtained with IL-8 alone (Fig. 5A) . Moreover, if antibodies against IL-8 were added, the synergy between IL-8 and regakine-1 was also inhibited (P<0.02).



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  		Figure 5. The synergistic effect between chemokines is inhibited by specific chemokine antibody or receptor antagonist. (A) Regakine-1 (RK-1; 100 and 300 ng/ml), IL-8 (5 ng/ml), and regakine-1 (100 ng/ml) plus IL-8 (5 ng/ml) were preincubated with rabbit antiregakine-1 (1/100), goat anti-IL-8 (1/100), or buffer for 1 h at 37°C before addition to the lower compartment of the microchamber to measure neutrophil chemotaxis. The chemotactic response is expressed as the mean chemotactic index ± SEM, derived from four to six independent experiments. The combination of IL-8 and regakine-1 resulted in a significant (P<0.01) increase in chemotactic index compared with the additive effect of IL-8 and regakine-1. Statistically significant reductions in synergy between IL-8 (5 ng/ml) and regakine-1 (100 ng/ml) as a result of antibody treatment are calculated by the Mann-Whitney test and are indicated by an asterisk (*, P<0.05). (B) SDF-1{alpha} (300 ng/ml), IL-8 (5 ng/ml), and SDF-1{alpha} (300 ng/ml) plus IL-8 (5 ng/ml) were added in the lower compartment of the microchamber to measure human neutrophil chemotaxis. AMD3100 (100, 10, and 1 µg/ml) or buffer was added to the cells just before loading the upper compartment of the microchamber. The combination of IL-8 and SDF-1{alpha} resulted in a significant (calculated from three independent experiments; P<0.049) increase in chemotactic index (19.9±2.3) compared with the sum of the chemotactic indexes of IL-8 (8.4±1.7) and SDF-1{alpha} (2.8±0.8) alone. The statistically significant reductions in synergy between IL-8 and SDF-1{alpha} in the presence of various concentrations of AMD3100 (1, 10, 100 µg/ml), expressed as the percentage of the response to IL-8 alone, are determined by the Mann-Whitney test and are indicated by an asterisk (*, P<0.05).

To demonstrate that the synergy between CC and CXC chemokines in neutrophil chemotaxis implies receptor-mediated events, the combination of SDF-1{alpha} and IL-8 was evaluated in the microchamber assay in the presence of the specific CXCR4 inhibitor AMD3100 (Fig. 5B) , which at 100 µg/ml and 10 µg/ml, significantly (P<0.049) reduced the synergistic effect between SDF-1{alpha} (300 ng/ml) and IL-8 (5 ng/ml), observed in the absence of AMD3100. A lower dose of AMD3100 (1 µg/ml) was less efficient in blocking the synergy. The chemotactic effect of SDF-1{alpha} (300 ng/ml) alone was also blocked completely in the presence of AMD3100 (100, 10, and 1 µg/ml). We can conclude that the synergistic, chemotactic effect between SDF-1{alpha} and IL-8 implies receptor-mediated events.

Synergy between chemokines requires specific receptor-mediated events but not calcium signaling
Measurement of increased intracellular calcium concentrations is a standard test for cellular signaling through GPCR. Figure 6 illustrates that IL-8, but not MCP-3, was capable of triggering a detectable calcium signal in neutrophils. Also, lower concentrations of MCP-3 (30 and 100 ng/ml) did not induce a calcium response. Moreover, pretreatment of neutrophils for 100 s with 300 ng/ml MCP-3 did not desensitize the cells from responding to 3 ng/ml IL-8, indicating that the two ligands interact with their own receptor(s). However, simultaneous stimulation of neutrophils with MCP-3 (300 ng/ml) and a suboptimal concentration of IL-8 (1.5 ng/ml) did not provide an enhanced intracellular calcium concentration compared with addition of IL-8 alone, indicating that this signaling pathway is not involved in the synergy between CC and CXC chemokines. Similar results were obtained in the calcium assay when IL-8 was added together with CXC chemokines (SDF-1{alpha}) or plasma chemokines (regakine-1) in combinations, which yield synergy in the chemotaxis assay (data not shown).



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  		Figure 6. Synergy between chemokines does not affect calcium signaling. IL-8 but not MCP-3 was capable of providing a detectable calcium signal in neutrophils. Pretreatment of neutrophils with 300 ng/ml MCP-3 did not desensitize the cells from responding to 3 ng/ml IL-8. Simultaneous stimulation of neutrophils with MCP-3 (300 ng/ml) and IL-8 (1.5 ng/ml) did not enhance the intracellular calcium concentration compared with IL-8 (1.5 ng/ml) alone. The second stimulus was added 100 s after the first stimulus. One representative experiment is shown out of four independent experiments.

Synergy between IL-8 and SDF-1{alpha} to chemoattract Jurkat cells transfected with CXCR1
To investigate the mechanisms via which synergy among chemokines occurs, chemotaxis assays with Jurkat cells transfected with one of the human IL-8 receptors, i.e., CXCR1, were performed. Figure 7 shows that SDF-1{alpha} (30 and 100 ng/ml) induced significant chemotaxis of Jurkat/CXCR1-transfected cells, which reflects the expression of CXCR4 on these Jurkat cells. IL-8 significantly (P<0.028) attracted Jurkat/CXCR1-transfected cells, in contrast with Jurkat control cells (data not shown). Moreover, IL-8 significantly increased the chemotactic response of CXCR1 transfectants to SDF-1{alpha} (100 ng/ml). Such a cooperative, chemotactic response was not observed with a lower concentration of SDF-1{alpha}. Furthermore, MCP-1 (100 and 300 ng/ml) did not synergize with IL-8 (100 ng/ml) to chemoattract Jurkat/CXCR1 cells. MCP-1 alone had no chemotactic activity on Jurkat/CXCR1 cells, which reflects the absence of CCR2 on Jurkat cells. Thus, the synergy between IL-8 and SDF-1{alpha} occurs via their specific receptors, CXCR1 and CXCR4, and both receptors are necessary to obtain a synergistic effect.



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  		Figure 7. Synergy between IL-8 and SDF-1{alpha} to chemoattract Jurkat cells transfected with CXCR1. SDF-1{alpha} (30 and 100 ng/ml) and MCP-1 (100 and 300 ng/ml) were tested in the presence or absence of IL-8 (100 ng/ml) in the lower compartment of the microchamber to measure Jurkat/CXCR1 chemotaxis. The chemotactic response is expressed as the mean chemotactic index ± SEM, derived from five independent experiments. Statistically significant differences in chemotactic indexes between the combination of IL-8 with another chemokine and the sum of the indexes obtained for the chemoattractants alone, determined by the Mann-Whitney test, are indicated by an asterisk (*, P<0.05).


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DISCUSSION
 
Chemokines produced in response to infection recruit leukocytes to the site of inflammation and act in a complex network as stimulators or inhibitors via selective GPCR recognition. In vivo, the chemokine system is well controlled by ligand and receptor availability, chemokine receptor interaction, and (un)coupling of the G protein signal-transduction mechanism. Although chemokines can be proteolytically degraded [32 , 33 ] or scavenged by decoy receptors [34 ], an alternative set of chemokines and their receptors can continue to mediate the inflammatory response, as most leukocyte types express multiple, functional chemokine receptor, and most chemokines bind to more than one receptor [35 , 36 ]. Synergistic interaction between chemokines or between chemokines and other inflammatory mediators provides an additional mechanism to sustain the inflammatory response in the presence of suboptimal chemokine concentrations [10 , 21 22 23 24 ]. For example, the constitutive plasma CC chemokine regakine-1 synergizes with the CXC chemokine IL-8 or the complement factor C5a to chemoattract neutrophils [10 , 24 ]. As a consequence, constitutive plasma chemokines could help in recruiting neutrophils from the bone marrow during the inflammatory response. In this study, we have investigated in detail the synergy between IL-8 or C5a and chemokines, other than regakine-1, in three different neutrophil activation/migration assays, e.g., chemotaxis through micropore filters, migration under agarose, and change in cell shape. The CC chemokine MCP-3, which is a weak neutrophil chemoattractant [37 ], dose-dependently synergized with a suboptimal concentration of IL-8 in all three assays. Also, other CC chemokines, MCP-1 and MCP-2, yielded a statistically significant enhancement of the chemotactic response to low doses of IL-8 by binding to CCR1 and/or CCR2, two receptors that are expressed on neutrophils [38 ]. To point out that the synergy with IL-8 cannot be elicited by CC chemokines only, the CXCR4 ligand SDF-1{alpha} was also shown to synergize with IL-8. CXCR4 is constitutively expressed at low levels on various cell types including neutrophils [38 39 40 ]. SDF-1{alpha} released by bone marrow stromal cells influences the homing of myeloid progenitor cells and stem cells [41 , 42 ]. Hematopoietic stem-cell homing is not only regulated by adhesion molecules and cytokines but also by chemotactic factors that support migration across the bone marrow endothelium [43 , 44 ]. Conversely, the CXC chemokines CTAP-III and NAP-2 did not synergize with IL-8 in neutrophil chemotaxis. CTAP-III has no neutrophil chemotactic activity as a result of the lack of affinity for CXCR1 and/or CXCR2. The CXCR2 agonist NAP-2, which is the truncated and active form of CTAP-III, showed neutrophil chemotactic activity but failed to enhance the response to IL-8 above the cumulative effect of the agonists alone. Taken together, these data indicate that predominantly CXC chemokines, which bind to chemokine receptor other than those of IL-8, can synergize with IL-8 in chemotaxis assays. The fact that also CC chemokines, which do not recognize the IL-8 receptors, synergize with IL-8 can be explained by the presence of their receptors on neutrophils [38 39 40 , 45 ]. It is likely that receptor-mediated events are implicated in the synergy with CXCR2 ligands.

To further explore interaction between chemokines and other inflammatory mediators, parallel Boyden chamber experiments were performed with C5a. Similar to IL-8, CC (MCP-3, regakine-1) and CXC chemokines (SDF-1{alpha}) provoked a statistically significant increase in chemotactic response in combination with C5a (30 ng/ml). Alternatively, PF-4, CTAP-III, and NAP-2 did not significantly increase the chemotactic activity of C5a. The lack of synergy between NAP-2 and C5a is probably a result of their individual, potent chemotactic effect. To exclude that the synergy was associated with the Boyden microchamber device used for this unidirectional fluid-phase assay, our findings were confirmed in the agarose plate assay measuring the migration distance rather than the number of migrated cells. It was observed that MCP-3 dose-dependently enhanced the migration distance of neutrophils toward IL-8 (50 ng/ml), irrespective of whether MCP-3 was added together with the cells or with IL-8 but that the optimal chemokine concentrations were different from those in the microchamber assay. Indeed, the less-sensitive agarose assay required tenfold more IL-8 to induce neutrophil migration [26 ], whereas compared with the Boyden chamber assay, a tenfold-lower MCP-3 (30 ng/ml) concentration was required to reach maximal synergy with IL-8. As a consequence, the chemokines cooperate in this assay at an equimolar, physiological concentration. Moreover, the cooperative effect between chemokines showed a bell-shaped, dose-response curve in that at higher concentrations of MCP-3, the synergy with IL-8 was reduced.

To find out whether the synergy between chemokines is a direct phenomenon occurring at an early stage of neutrophil activation, shape-change assays were performed. In this assay, morphological changes of neutrophils are monitored that occur within minutes rather than an hour, the time period required for in vitro neutrophil migration. The number of neutrophils with a blebbing shape was increased after IL-8 (5 ng/ml) treatment, whereas with the combination of MCP-3 and IL-8 neutrophils, changed to a more spread-cell morphology. Taken together, it can be concluded that the synergistic effect between IL-8 and MCP-3 is not dependent on the test system used to measure neutrophil chemotaxis. Finally, to demonstrate that the synergy between chemokines is not the result of a contaminant present in the chemokine preparation, neutralization assays were performed with specific chemokine antibodies. The synergistic effect between regakine-1 and IL-8 was reduced by antibodies against regakine-1 or IL-8 to the chemotaxis level of IL-8 or regakine-1 alone, respectively. Furthermore, the CXCR4-specific inhibitor, AMD3100, was used to demonstrate that the synergistic effect between IL-8 and SDF-1{alpha} implies receptor-mediated events. The bicyclam AMD3100 was found to inhibit the replication of T-tropic HIV-1 strains, which use CXCR4 to enter the cells [29 ]. AMD3100 prevents the binding of a monoclonal antibody to CXCR4 and specifically inhibits the signal transduction initiated by the binding of SDF-1{alpha} with CXCR4 but does not act as a CXCR4 agonist [29 , 46 , 47 ]. Here, AMD3100 dose-dependently reduced the synergistic effect between IL-8 and SDF-1{alpha} in chemotaxis to the level of IL-8 alone, which points to the need of CXCR4 in this phenomenon.

The precise molecular mechanisms of this synergistic cooperation between chemokines remain unknown. Previous studies showing that regakine-1 did not compete for binding to the IL-8 or C5a receptors and that regakine-1 could not desensitize the IL-8 or C5a receptors were indicative of a separate receptor for this chemokine [23 , 24 ]. As chemokines (e.g., CTAP-III) not recognizing a GPCR and those competing for the same receptor (e.g., NAP-2 for CXCR2) do not synergize with IL-8, it is postulated that the above-described synergy between chemokines is dependent on binding of the chemokines to their own GPCR [48 ]. This hypothesis is supported by chemotaxis experiments with Jurkat cells transfected with CXCR1. Indeed, IL-8 but not MCP-1 significantly increased the chemotactic response to SDF-1{alpha} of these Jurkat cells, which constitutively express CXCR4 but not CCR2 [49 ]. We conclude that the expression of the two chemokine receptors is necessary to give a cooperation between the chemokines in cell migration. Once activated, these receptors transmit their signal to heterotrimeric G proteins, which activate downstream pathways leading to rapid cytoskeletal rearrangements and chemotaxis [50 51 52 ]. We speculate that the synergy of IL-8 with other chemokines occurs at the postreceptor-binding level, e.g., during intracellular signal transduction (enhanced protein kinase activity and/or enhanced production of second messengers). However, we could not observe enhanced intracellular calcium mobilization by combining IL-8 with chemokines that synergize in the chemotaxis assay. Although the synergy seems to be a rapid process, as observed in the shape-change assay, the impact on chemokine receptor recycling cannot be excluded [53 ]. Furthermore, the synergistic effect between C3a and SDF-1{alpha} to promote the homing of hematopoietic progenitor cells to the bone marrow was not mediated by the SDF-1{alpha}-dependent activation of the mitogen-activated protein kinase p42/44 or phosphatidylinositol-3 kinase–AKT signal-transduction pathway [21 ]. Other possibilities such as ligand or receptor heterodimerization are less likely but can at present not be excluded. Mellado et al. [54] provided evidence for functional CCR2 and CCR5 heterodimerization. Indeed, simultaneous stimulation with the two respective ligands MCP-1 and RANTES induced the formation of CCR2–CCR5 heterodimers and increased the efficiency of the Ca2+ signal and chemotactic response. Conversely, we have previously reported that the CC chemokine regakine-1 can cooperate with the bacterial tripeptide fMLP or the complement factor C5a [24 ], indicative that heterodimerization of ligands or receptors should be an unexpectedly broad phenomenon. Further research is needed for a better understanding of the molecular mechanisms implicated in the synergy between chemokines. However, it can be anticipated that the synergy between proinflammatory and constitutive ligands of GPCR observed in vitro is also biologically relevant in vivo. First, several proinflammatory chemokines are coinduced by viral or bacterial products in the infected tissue, even by a single-cell type, and can cooperate directly in situ [19 ]. The neutrophil by itself is a rich source of chemokines and can thereby provide synergy through autocrine release of inflammatory mediators, cooperating in the chemotactic response [55 ]. Second, some of the chemokines (SDF-1{alpha} and regakine-1) are constitutively produced and might play a central role in the distribution of immature and mature hematopoietic cells, including their release into the circulation or homing to the bone marrow [41 42 43 ]. The synergy occurs at physiological concentrations of the constitutive or inflammatory mediators. This allows us to conclude that the observed synergistic effect can occur under normal and disease conditions and that selective blockage of a single chemokine may have more pronounced consequences on the inflammatory response than currently considered.


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ACKNOWLEDGEMENTS
 
The Fund for Scientific Research of Flanders (F.W.O.-Vlaanderen), the Concerted Research Actions (G.O.A.) of the Regional Government of Flanders, the InterUniversity Attraction Poles Program-Belgian Science Policy (I.U.A.P.), the Cancer Research Foundation of Fortis AB, Belgium, and The Belgian Federation against Cancer and the Quality of Life Program of the European Community supported this work. P. P. and S. S. are senior research assistants of F.W.O.-Vlaanderen. We thank the members of the Laboratory of Clinical Immunology of the University of Leuven and the members of the Blood Transfusion Center of Antwerp for providing buffy coats. The technical assistance of R. Conings and J. P. Lenaerts is greatly appreciated.

Received October 15, 2003; revised February 18, 2004; accepted March 8, 2004.


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