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Stroma Cell-Derived Factor 1 Mediates Desensitization of Human Neutrophil Respiratory Burst in Synovial Fluid from Rheumatoid Arthritic Patients¹

Département de Biologie Cellulaire, Institut Cochin, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Institut National de la Recherche Médicale Unité 567, and Université René Descartes, Paris, France

	Abstract

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Classical chemoattractants such as fMLP or the complement factor C5a use G protein (Gi)-coupled receptors to stimulate both chemotaxis and production of reactive oxygen species (respiratory burst, RB) by polymorphonuclear leukocytes (PMN). The chemokine stroma cell-derived factor 1 (SDF1) and its Gi-coupled receptor, CXCR4, regulate leukocyte trafficking and recruitment to the synovial fluid of rheumatoid arthritic patients (RA-SF). However, the role of SDF1 in the RB is unknown and was studied in this work in vitro with healthy PMN in the absence and presence of RA-SF. In healthy PMN, SDF1 failed to stimulate the RB, even though the p38 mitogen-activated protein kinase was activated to a similar level as in fMLP-stimulated PMN. In contrast, the SDF1-mediated calcium transients and activation of phosphatidylinositol 3-kinase/Akt were partially deficient, while p44/42 mitogen-activated protein kinases were not activated. SDF1 actually desensitized weakly the fMLP-mediated RB of healthy PMN. This cross-inhibitory effect was amplified in PMN treated with RA-SF, providing a protection against the exacerbation of RB induced by C5a or fMLP. This SDF1 beneficial effect, which was prevented by the CXCR4 antagonist AMD3100, was associated with impairment of C5a- and fMLP-mediated early signaling events. Thus, although SDF1 promotes leukocyte emigration into rheumatoid synovium, our data suggest it cross-desensitizes the production of oxidant by primed PMN, a property that may be beneficial in the context of arthritis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leukocyte chemoattractant receptors, which belong to the G protein-coupled receptor family, play an important role in nonspecific immunity and leukocyte migration to inflammatory sites. Chemoattractants may be grouped into classical chemotactic factors and chemokines. The former group is generally produced within inflammatory sites and comprises various types of inflammatory agents, such as N-formylated bacterial peptides, the C5a complement fraction, platelet-activating factor (PAF),⁴ and leukotriene B₄ (LTB₄). They all stimulate sustained chemotaxis of polymorphonuclear leukocyte (PMN) and variable levels of cytotoxic activity, such as the production of superoxide by the NADPH oxidase complex (respiratory burst, RB) and the release of lysosomal enzymes (exocytosis). Both activities can be activated and primed by various agents, such as fMLP, C5a, PAF, and LTB₄, and contribute to the bactericidal function of phagocytes and pathological processes (1). The biochemical pathways underlying these functions are initiated by the activation of pertussis toxin (PTX)-sensitive heterotrimeric G proteins and involve various signaling effectors, including phospholipases, calcium, lipid kinases, and protein kinases, such as tyrosine kinases, protein kinase C (PKC), and mitogen-activated protein (MAP) kinases (2).

Chemokines are a family of structurally related cytokines that have been classified on the basis of the spacing of the first two cysteines in CXC, CXXXC, C, and CC chemokines (3, 4, 5). Chemokines bind to the same class of receptors as classical chemoattractants (i.e., G protein-coupled receptor). Although many chemokines bind to more than one receptor (5), the stroma cell-derived factor 1 (SDF1), originally identified as a growth factor for murine pre-B cells (6, 7, 8), binds uniquely to CXCR4 (9, 10, 11, 12). SDF1 and its cognate receptor are expressed constitutively in various cell types and tissues and regulate hemopoietic progenitor cell trafficking and B cell proliferation (12). Targeted gene disruption of SDF1 or CXCR4 in mice is lethal because of cardiovascular and neurologic defects, defective B cell lymphopoiesis, and a severe impairment of bone marrow myelopoiesis (13, 14). SDF1 modulates cancer metastasis (15), and finally, CXCR4 acts as a coreceptor for T cell-tropic strains of the HIV (12).

CXCR4 is expressed by human PMN and mediates calcium mobilization and chemotaxis through the activation of a PTX-sensitive G protein (8, 11, 16). These observations and the phenotype of SDF1- and CXCR4-knockout mice (13, 14) have led to the notion that SDF1 regulates basal trafficking of hemopoietic cells and that, contrary to classical chemoattractants, SDF1 was not involved in acute inflammation (8). However, SDF1 is present at high levels in synovial fluid of arthritic patients (17) and contributes to accumulation of leukocytes (17, 18, 19). PMN play a major role in the progression of rheumatoid arthritis due to their ability to develop excessive oxygen-dependent cytotoxic functions (20). However, it is not known whether SDF1 regulates the RB of PMN in the synovial fluid of rheumatoid arthritis patients. Nevertheless, the fact that CXCR4 and receptors for classical chemoattractants couple to the same family of PTX-sensitive G proteins (21), and that these G proteins play a key role in oxygen-dependent cytotoxic functions of PMN (2, 22) suggests that SDF1 may directly or indirectly regulate reactive oxygen species (ROS) production by PMN.

In this study, we investigated the direct and indirect effects of SDF1 on ROS production and signaling events of human PMN, in comparison with those promoted by the chemotactic peptide fMLP. The effects of SDF1 were further studied using PMN modified in vitro following cell treatment in the presence of synovial fluid from rheumatoid arthritic patients (RA-SF). The results show that, unlike the classical chemoattractant fMLP, SDF1 failed to stimulate RB in healthy and RA-SF-primed PMN. In contrast, SDF1 desensitized the RB of healthy PMN induced by classical chemoattractants. This phenomenon was further amplified in the presence of RA-SF, providing protection against the exacerbation of ROS production. This novel property of SDF1 is of interest considering the damaging effects of the PMN RB in arthritis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and Abs

rSDF1 was from R&D Systems (Minneapolis, MN). fMLP, luminol, C5a, all-trans retinoid acid, fura 2-acetoxymethyl ester (fura 2-AM), and other reagents were from Sigma-Aldrich (St. Louis, MO). Stock solutions of fMLP, A23187, and fura 2-AM were prepared in DMSO and stored at –20°C. Abs against phosphorylated p44/42, p38 MAP kinase, and AKT or against p38 and AKT were from New England Biolabs (Beverly, MA). Anti-P44/42 MAP kinases and peroxidase- or alkaline phosphatase-conjugated Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Protease inhibitor mixture tablets were from Boehringer Mannheim (Meylan, France). PTX was from List Biological Laboratories (Campbell, CA). Synovial fluids from arthritic patients (RA-SF) were provided by X. Ayral (Hôpital Cochin, Paris, France) and L. Meccarely (Institut National de la Santé et de la Recherche Médicale Unité 507, Hôpital Necker, Paris, France). RA-SF were centrifuged at 4°C (3 min x 2000 x g), and the cell-free supernatants were stored at –80°C until use.

Preparation of human PMN, lymphocytes, and differentiated HL-60 cells

PMN from human venous blood, heparinized at 10 U/ml, were isolated by a first-step sedimentation of whole blood on 1% dextran T500, followed by centrifugation of the granulocyte-rich supernatant on a cushion of a mixture of Ficoll and Hypaque (Eurobio, Paris, France), as described (23). The purified PMN (95–97%) were subjected to hypotonic lysis, washed, and suspended in HBSS at pH 7.4 containing 1.2 mM calcium. Lymphocytes were purified by incubating the mononuclear cell population in petri dishes in PBS containing 5% decomplemented FCS for 2 h at 37°C. Nonadherent lymphocytes were purified by centrifugation (300 x g for 8 min).

HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FCS, as described (24). To induce myeloid differentiation, cells were seeded at a density of 0.5 x 10⁶/ml and treated with 1 µM all-trans retinoic acid for 5 days (25). The state of cell differentiation was checked by measuring the production of ROS using the luminol-ECL assay described below (26).

Production of ROS by PMN

RB was studied by continuously monitoring the reduction of cytochrome c (23) using a PerkinElmer (Wellesley, MA) 40 spectrophotometer equipped with thermostated (37°C) cuvette holder and magnetic stirrer. Suspensions of 2 x 10⁶ PMN were usually stimulated in 1 ml of HBSS containing 80 µM cytochrome c. In experiments with RA-SF, PMN were pretreated at 37°C for 15 min in 300 µl of HBSS containing 50% RA-SF, and the volume was adjusted to 1 ml for superoxide measurement.

ROS production was also measured by chemiluminescence (CL) assay (27) using cell suspensions of 10⁶ PMN in 300 µl of HBSS containing 10 µM luminol. CL was monitored every 5 s using a thermostated (37°C) bioluminometer (Packard Picolite luminometer; Packard Instrument, Meriden, CT), as described (26). In some experiments, the contribution of ROS production to the CL response of PMN was checked by measuring the inhibitory effect of a mixture of superoxide dismutase (4 U) and catalase (2 U). In experiments with RA-SF, suspensions of 3.5 x 10⁶ PMN in 300 µl of HBSS were pretreated in the absence (control) or presence of 50% RA-SF for 15 min at 37°C. Cells were centrifuged (1 min x 3500 x g) and suspended in HBSS for CL measurement. Results are expressed in cpm or as the percentage of maximal control values.

Chemotactic assay

Suspensions of 0.5 x 10⁶ PMN in 100 µl of HBSS containing 1% BSA were incubated in the upper compartment of the Boyden chamber (28), which was separated from the lower compartment by a cellulose filter with pores of 3 µm in diameter (Millipore, Bedford, MA). The lower compartment contained various concentrations of SDF1 or fMLP. Chambers were incubated at 37°C for 60 min in 95% humidified air and 5% CO₂; filters were treated with ethanol and stained with hemalum. Basal and directed migrations of PMN were measured as the front lead of migration (at least 10 PMN). Five fields were analyzed for each filter. Results are expressed in micrometer and represent the mean of four separate experiments.

Calcium flux

Changes in cytosolic-free calcium concentration ([Ca²⁺]_i) were measured in PMN loaded with fura 2, as described (29, 30). Briefly, PMN were loaded with 2 µM fura 2-AM in 20 mM HEPES/calcium-free HBSS for 45 min at 37°C, and the fluorescence assays were performed with suspensions of 4 x 10⁶ PMN in 2 ml of HBSS. In some experiments, PMN were treated with 1 µg/ml Bordetella PTX for 3 h at 37°C (31). In experiments involving RA-SF, PMN were treated in a 2-ml cuvette in the presence or absence of 50% RA-SF in 0.3 ml of HBSS for 15 min and the volume was adjusted to 2 ml. Changes in the fura 2 fluorescence (excitation and emission wavelengths of 340 and 510 nm, respectively) were monitored using a Jobin-Yvon 3D fluorometer (Longjumeau, France) equipped with a thermally controlled cuvette holder and a magnetic stirrer. The concentrations of cytosolic-free calcium were calculated using the equation [Ca²⁺]_i, nM = 224 (F – Fmin)/Fmax – F) (29). Tracings correspond to experiments performed with one population and are representative of at least three experiments.

Electrophoresis and Western blot analysis

Suspensions of 3 x 10⁶ cells in 300 µl of HBSS in Eppendorf tubes were pretreated at 37°C in the absence or presence of 50% RA-SF for 15 min. Cells were centrifuged, suspended in HBSS, and stimulated, as described in figure legends. To stop the reaction, tubes were filled with 0.5 ml of ice-cold PBS and immediately incubated in ice-cold methanol (–70°C) for 5 s to drop temperature. Cells were resuspended in 50 mM Tris-HCl, pH 7.5, containing 2.5 mM orthovanadate, 2.5 mM EDTA, and a mixture of antiproteases (Complete; Boehringer Mannheim), and lysed with 4x Laemmli sample buffer. Samples were boiled for 5 min at 95°C and stored at –85°C. Proteins were separated on a 10% SDS-PAGE and transferred to nitrocellulose membranes. Phosphorylated MAP kinases were analyzed in immunoblotting experiments using mAbs against the active form of p44/42 (Thr²⁰²/Tyr²⁰⁴), P38 (Thr¹⁸⁰/Tyr¹⁸²) MAP kinase and Akt(Ser⁴⁷³). ECL was used for detection of HRP-conjugated secondary Ab. Membranes were washed and subsequently reprobed with appropriate Abs to p44/42, p38, or Akt, and detection with alkaline phosphatase-conjugated secondary Abs was performed using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as substrate. The quantification of phosphorylated proteins was performed with the NIH Image 1.62 software.

Statistical analysis

Each experiment was performed in duplicate and repeated at least three times. Unless otherwise indicated, data represent means ± SEM. Statistically significant differences between means were identified by using Student’s paired t test with a threshold of p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Comparison of the effects of SDF1 and fMLP on PMN RB, chemotactic activity, and calcium transients

The RB of PMN was first studied using the conventional cytochrome c reduction assay for superoxide measurement under standard conditions (23). Unlike the classical chemoattractant fMLP, SDF1 (5–100 nM) failed to stimulate significant RB even in PMN primed with cytochalasin B (data not shown). With the highly sensitive CL assay, which measures the reactive oxygen species (ROS) generated by both the superoxide-generating NADPH oxidase (RB) and myeloperoxidases (32), SDF1 stimulated a very weak and transient CL response (p < 0.05; Fig. 1A), which reached 10–20% of that mediated by fMLP. This stimulation occurred at the saturating chemokine concentrations of 50–100 nM and may be mainly due to myeloperoxidase.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Effects of SDF1 and fMLP on ROS production, chemotactic activity of PMN, and intracellular calcium release. A, PMN were incubated in the presence of luminol and treated in the absence (buffer) or presence of 100 nM SFD1 or fMLP. Results represent the mean ± SEM of the maximal CL response obtained with SDF1 (n = 24) and fMLP (n = 12). B, Migration distances of PMN induced by various concentrations of SDF1 or fMLP. Results are expressed in micrometers (mean of four experiments ± SEM). C, Changes in the concentration of cytosolic-free calcium in fura 2-loaded PMN were induced by 10, 50, and 100 nM SDF1 or 50 nM fMLP. Each tracing is representative of at least three experiments obtained with different individuals.

The elevation of [Ca²⁺]_i is one of the earliest events that regulate both directed locomotion and ROS production induced by fMLP (2, 36). To identify the biochemical events responsible for the weak functional strength of CXCR4, we compared the peak of calcium elevation induced by both chemoattractants in fura 2-loaded PMN. SDF1-induced elevation of [Ca²⁺]_i was transient and proportional to the chemokine concentration from 10 to 100 nM (Fig. 1C). The maximal [Ca²⁺]_i did not exceed 300–400 nM, whereas fMLP-induced elevation of [Ca²⁺]_i routinely reaches micromolar levels (30), as confirmed in this study (Fig. 1C). SDF1-induced calcium transients did not require external calcium (data not shown) and were blocked in cells treated with PTX (Fig. 1C), confirming the coupling of activated CXCR4 to heterotrimeric G protein of the Gi group for stimulation of phospholipase C (2).

MAP kinases and Akt phosphorylation in SDF1- and fMLP-stimulated PMN

Two families of MAP kinases, the extracellular signal-regulated kinase (ERK or p42 ERK1)/p44 (ERK2) MAP kinase) and the p38 MAP kinases, are stimulated by fMLP in PMN and contribute to RB (37, 38, 39). SDF1 induced a rapid and substantial stimulation of the p38 MAP kinase, as shown by the time course study of phosphorylated p38 MAP kinases (Fig. 2A). This activation was proportional to the concentration of SDF1 (10–100 nM) (data not shown). FMLP-induced stimulation of p38 MAP kinase was also rapid and barely greater than that induced by SDF1. In contrast, SDF1 did not induce detectable amount of phosphorylated p44/42 MAP kinases in the same PMN extracts, unlike fMLP (Fig. 2B). This unexpected dysfunction of CXCR4 was selective to PMN because it was not observed with human lymphocytes and differentiated HL-60 cells (Fig. 2D). Stimulation of the p44/42 MAP kinase pathway by fMLP occurs downstream of activation of the phosphatidylinositol 3-kinase (PI3K) (40). We confirmed this in PMN treated with the PI3K antagonist dihydroxy-wortmannin (data not shown). Both fMLP and SDF1 induced a rapid stimulation of the PI3K pathway, as shown by the amount of phosphorylated form of Akt, a downstream target of PI3K (41). fMLP- and SDF1-induced phosphorylation of Akt was optimal at 30–60 s (Fig. 2C), although SDF1 was 2 times less potent than fMLP (Fig. 2E). Thus, deficiencies in CXCR4 signaling affected predominantly the ERK1/2 pathway (Fig. 2E) and to a lesser extent, the PI3K/Akt and phospholipase C/calcium pathways (Fig. 1C), which may contribute to the weak SDF1 functional effects (Fig. 1A).

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Effect of SDF1 and fMLP on MAP kinase and Akt phosphorylation in PMN. PMN were treated in the presence or absence of 100 nM SDF1 or fMLP for the indicated periods. PMN homogenates were submitted to SDS-PAGE, and Western blots were probed with Abs against the phosphorylated form of p38 (Phospho-p38) (A). The total amount of p38 MAP kinase was determined by reprobing the same membrane with Abs against p38 MAP kinase (p38). The same PMN homogenates were used to probe the phosphorylated form of Akt (Phospho-pAkt; C) and p44/42 MAP kinases (Phospho-p44 and p42; B). D, Shows the phosphorylated form of p44/42 MAP kinases in SDF1-stimulated (100 nM, 1 min) lymphocytes and in differentiated HL-60 cells stimulated with 100 nM SDF1 or fMLP for 1 min. Each blot is representative of three to four experiments obtained with different individuals. The maximal protein phosphorylation in experiments A–C was quantified and expressed as the percentage of that induced by fMLP (E).

SDF1-induced desensitization of RB in healthy and RA-SF-primed PMN

The RB of PMN can be potentiated upon cell pretreatment with certain cytokines (TNF-, GM-CSF) or low concentrations of chemoattractants (42). This priming phenomenon is believed to have physiopathological consequences (43). Because SDF1 was a weak activator of PMN (Fig. 1), we explored whether it primes the RB induced by fMLP. Treatment of PMN with low SDF1 concentrations (10 and 50 nM) for 5 min did not prime, but induced a concentration-dependent decrease in the fMLP-mediated RB (p < 0.05), with a maximal inhibition of 30%. In contrast, the RB induced by the direct PKC activator, PMA, was not altered.

The RB of PMN is greatly enhanced in various pathological situations and leads to tissue damages. In arthritis, for example, local production of ROS by PMN in synovial fluids from rheumatoid arthritis (RA-SF) patients contributes to cartilage degradation and the disease progression (20, 44). Because SDF1 induces recruitment of leukocytes into rheumatoid synovium (19), we investigated whether SDF1 regulates production of ROS by PMN under conditions close to those of rheumatoid synovium. For this purpose, we set up an in vitro model of arthritic-like PMN, which consists in pretreating healthy PMN in the presence of cell-free RA-SF in vitro before stimulation of RB by proinflammatory mediators such as C5a or fMLP. This pretreatment of PMN greatly potentiated the rate and total production of superoxide by PMN stimulated by fMLP (Fig. 3B), thus confirming the priming potency of RA-SF previously described with synovial PMN (45, 46). The RA-SF-priming property was confirmed by the measurement of the PMN CL response (Fig. 3, C and D), which further indicates that the RA-SF per se stimulated a substantial production of ROS.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Effect of SDF1 and RA-SF on the RB of PMN. A, PMN were treated in the absence or presence of various concentrations of SDF1 for 10 min, then with 100 nM fMLP or PMA. Results represent the mean value ± SEM of superoxide anion production and are expressed as the percentage of the respective controls (n = 5). A statistically significant difference is indicated by * (p < 0.05). B, Healthy PMN were preincubated without or with RA-SF for 15 min, then stimulated with 100 nM fMLP. The results show a representative time course production of superoxide induced by fMLP. The CL response of PMN treated or not with RA-SF was stimulated by 100 nM fMLP (C) or C5a (D). Data are expressed as the percentage of the stimulated control (fMLP or C5a) values (mean ± SEM of five experiments).

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Activated CXCR4 mediates inhibition of the RB and calcium transients in RA-SF-primed PMN. PMN pretreated with RA-SF for 15 min were incubated without (0) or with various concentrations of SDF1 for 5 min before stimulation with 100 nM fMLP or C5a (A). Results represent the maximal CL response and are expressed as the percentage of the control (mean ± SEM, n = 5–6 experiments). B, PMN pretreated with RA-SF were preincubated in the absence or presence of 3 µM AMD3100 (AMD) for 15 min. Cells were then treated in the presence or absence of 50 nM SDF1 for 5 min before stimulation with 100 nM fMLP. Results represent the maximal CL and are expressed as the percentage of initial basal values (mean ± SEM of five experiments). A statistically significant difference between groups of experiments is indicated by * (p < 0.05). C, PMN were treated in the absence or presence of RA-SF for 15 min, then with or without 50 nM SDF1 for 3 min before stimulation with 50 nM fMLP or C5a. Results are the mean of maximal calcium response and are expressed as the percentage of the stimulated (fMLP or C5a) control values (not treated with RA-SF). A statistically significant difference between groups of experiments is indicated by * (p < 0.05).

SDF1-induced desensitization of fMLP- and C5a-mediated signaling events in RA-SF-primed PMN

To further explore the mechanism by which SDF1 cross-inhibited the RB of PMN, we analyzed the effects of SDF1 on changes in [Ca²⁺]_i induced by fMLP and C5a. In healthy PMN, SDF1 alone induced weak calcium transients (Fig. 1C) without altering significantly the pattern of calcium elevation subsequently induced by 50 or 100 nM fMLP (data not shown). In contrast, the calcium transients induced by 100 nM SDF1 were completely inhibited in PMN pretreated with 100 nM fMLP for 3 min (data not shown), in agreement with previous reports (48). These data suggest that both receptors may be desensitized through different biochemical mechanisms. Treatment of PMN with RA-SF (Fig. 4C) induced a 2-fold increase in the maximal calcium response mediated by SDF1 (column 1 vs 2, p < 0.05), fMLP (column 3 vs 4), and C5a (column 6 vs 7), although SDF1 remained less potent than fMLP. However, in PMN treated with RA-SF, SDF1 greatly cross-inhibited the primed calcium response induced by fMLP (Fig. 4C, column 5 vs 4) or C5a (column 8 vs 7).

The treatment of PMN with RA-SF per se did not induce a detectable phosphorylation of the p44/42 MAP kinases (Fig. 5A), while the PI3K/Akt pathway was slightly increased (data not shown). In contrast, RA-SF primed the fMLP- or C5a-mediated stimulation of these two signaling pathways (Fig. 5, A and C). SDF1 was still unable to activate the p44/42 MAP kinase pathways in the presence of RA-SF (Fig. 5A), while the PI3K/Akt pathway was slightly increased (Fig. 5C). By contrast, SDF1 cross-inhibited the subsequent stimulation of the two signaling pathways by fMLP or C5a (Fig. 5, B and C).

View larger version (69K): [in this window] [in a new window]   FIGURE 5. SDF1-induced cross-inhibition ofMAP kinases and Akt phosphorylation in RA-SF-primed PMN. A, PMN were treated in the absence or presence of RA-SF for 15 min, then stimulated with 100 nM fMLP, C5a, or SDF1 for 1 min. B and C, PMN primed with RA-SF were treated in the absence or presence of 50 nM SDF1 for 5 min before stimulation with fMLP or C5a. Results represent the amount of phosphorylated p44/42 MAP kinases (B) and Akt (C) determined by Western blot experiments using specific Abs. Each blot is representative of three experiments obtained with different individuals.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The failure of SDF1 to induce significant RB was observed with healthy PMN and confirmed with PMN primed with RA-SF or cytochalasin B (data not shown). By contrast, SDF1 induced a weak PMN-directed migration in comparison with fMLP. These relative functional defects were associated with various defective signaling events. Among these, those dependent on the activation of Gi were markedly low, such as calcium transients (33), the activation of PI3K/Akt pathway, while the p44/42 MAP kinases were not activated. This latter defect, also reported in human platelets (49), may possibly be due to a deficient stimulation of upstream activators, such as the PI3K (50), as shown in this work. In contrast, the p38 MAP kinase was fully activated by SDF1 and fMLP, whereas in a murine pre-B cell line, the p38 MAP kinase was not activated by SDF1, unlike the p44/42 MAP kinases (51). These dissociated effects indicate that the activation of both MAP kinases by SDF1 may occur through distinct signaling cascade (50). The low stimulation of signaling events induced by SDF1 in comparison with fMLP may be due to impaired regulation of intracellular signaling partners by some selective domains of activated CXCR4 rather than differences in ligand-binding parameters. Indeed, both chemoattractants show similar affinity for their receptors (16, 34). CXCR4 was reported to be highly expressed on human PMN (52), although in some studies, CXCR4 expression was low and increased during PMN incubation (35, 53). In our PMN preparations, SDF1-binding parameters do not appear to be altered because SDF1 and fMLP stimulated similar level of phosphorylation of p38 MAP kinase. In addition, both chemoattractants also inhibited to a similar extent the forskolin-stimulated production of cAMP in PMN (data not shown), which further suggests that signaling through Gi may not be altered.

Unlike classical chemoattractants, SDF1 did not prime, but attenuated the subsequent fMLP-mediated RB. This unexpected observation is consistent with a recent finding that SDF1 impaired the PMN chemotactic migration induced by the chemokine growth-related oncogene- (35). The biochemical mechanism of this inhibitory effect remains unknown. However, the expression of growth-related oncogene- receptor at the PMN surface was not altered (35). Our observation that SDF1 did not inhibit the RB induced by PMA further suggests that PKC-dependent signaling may not be altered. Using an in vitro model of arthritic-like PMN, in which healthy PMN were primed with cell-free RA-SF before stimulation of RB by fMLP or C5a, we show that SDF1 greatly desensitized the RB induced by both C5a and fMLP. The range of SDF1-inhibitory concentrations (5–100 nM) was similar to that found in RA-SF (17), which strongly suggests that local productions of SDF1 in inflamed joints (17) may inhibit the RB of synovial PMN and possibly cartilage damage, although SDF1 induces leukocyte recruitment in RA-SF. This anti-PMN effect and potential anti-inflammatory property have been reported with some mediators, particularly with the lipid PMN chemoattractant lipoxin A4 (54, 55). Like SDF1, lipoxin A4 is a weak activator of PMN migration and calcium transients, but fails to stimulate RB, ERK1/2, and phospholipase A₂ (56). The detailed mechanism of the cross-inhibition of PMN RB and migration (35) by SDF1 is not known and appears to be mediated through heterologous desensitization of receptors, as indicated by the inhibition of C5a- and fMLP-mediated calcium transients, activation of the PI3K, and p44/42 MAP kinases. Chemoattractant-mediated heterologous desensitization of leukocytes is mediated by phosphorylation reactions at multiple levels, including membrane receptors, G proteins, and signaling effectors, such as phospholipase C (57, 58). This desensitization is mediated by both PKC and protein kinase A, whereas homologous desensitization, involves the phosphorylation of membrane receptors by G protein-coupled receptor kinases and PKC (57, 58). Although PKC and protein kinase A are potential candidates for SDF1-mediated cross-desensitization, their contribution may be different because the fMLP receptor is resistant to phosphorylation by PKC, unlike the C5a receptor (58). This is consistent with the fact that SDF1 was much more active in inhibiting the RB induced by C5a relative to fMLP. The relative contribution of PKC may likely be low because the main source of PKC activators, i.e., the inositol-specific phospholipase C/calcium pathway, was minimally activated by SDF1 even in PMN primed with RA-SF. Further studies of the signaling components involved in the anti-PMN action of SDF1 in healthy vs RA-SF-primed PMN may lead to the identification of appropriate targets to pharmacologically control the excessive RB of arthritic PMN and their pathological consequences.

In conclusion, this study shows that, unlike classical chemoattractants such as C5a or fMLP, SDF1 failed to activate the PMN RB due to defects affecting some CXCR4 signaling events. In contrast, SDF1 cross-inhibited the RB induced by a second agonist such as fMLP. This desensitization was amplified in the presence of RA-SF and provides protection against the exacerbation of RB and signaling events in PMN induced by the proinflammatory mediator C5a or fMLP. Taken together, the results suggest that SDF1/CXCR4 interactions may be potentially beneficial in reducing oxygen-dependent cytotoxic activities of PMN, particularly in arthritis.

	Acknowledgments

 
We thank the staff of the Blood Bank of St. Vincent de Paul’s Hospital (Paris, France) for providing blood samples, Dr. X. Ayral and L. Meccareli for providing us with RA-SF, and M. Alison for AMD3100. We also thank Drs. Y. O’Dowd for manuscript reading and S. Marullo for helpful suggestions.

	Footnotes

 
¹ This work was supported by grants from Agence Nationale de la Recherche sur le Sida.

² Current address: Faculté des Sciences Biologiques, El Alia, Alger, Algérie.

³ Address correspondence and reprint requests to Dr. Axel Périanin, Département de Biologie Cellulaire, Institut Cochin, Hôpital Cochin, Pavillon G Roussy, 27, rue du Faubourg St Jacques, 75014, Paris, France. E-mail address: perianin{at}cochin.inserm.fr

⁴ Abbreviations used in this paper: PAF, platelet-activating factor; [Ca²⁺]_i, cytosolic-free calcium concentration; CL, chemiluminescence; ERK, extracellular signal-regulated kinase; fura 2-AM, fura 2-acetoxymethyl ester; LTB₄, leukotriene B₄; MAP, mitogen-activated protein; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMN, polymorphonuclear leukocyte; PTX, pertussis toxin; RA-SF, synovial fluid from rheumatoid arthritic patients; RB, respiratory burst; ROS, reactive oxygen species; SDF1, stroma cell-derived factor 1.

Received for publication September 18, 2003. Accepted for publication March 8, 2004.

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