Human Platelets Stimulate Mesangial Cells to Produce Monocyte Chemoattractant Protein-1 via the CD40/CD40 Ligand Pathway and May Amplify Glomerular Injury

Antibodies and Cytokines

The following Abs, recombinant cytokines, and proteins were used in this experiment: mouse monoclonal anti-human CD154 (CD40L) (Ancell, Baypor, MN); mouse monoclonal anti-human CD62P (P-selectin) (R&D systems, Minneapolis, MN); mouse monoclonal anti-β1-integrin (clone M13, kindly provided by Dr. Kenneth Yamada, NIDR, NIH, Bethesda, MD); mouse monoclonal anti-human CD40 (Biosource, Camarillo, CA); biotin-conjugated goat anti-mouse immunoglobulins; R-PE-conjugated goat anti-mouse immunoglobulins (Dako, Carpinteria, CA); recombinant human TNF-α (rTNF-α); rIFN-γ (both from R&D systems); and trimeric human CD40L/leucine-zipper fusion protein (rCD40L) (a gift from Immunex, Seattle, WA).

Preparation of Platelets

Platelets were carefully isolated as described elsewhere with slight modification (25). In brief, platelet-rich plasma (PRP) was isolated from freshly drawn citrated whole blood after centrifugation (200 × g for 20 min). PRP was recentrifuged (500 × g for 20 min) in the presence of acid-citrate-dextrose (ACD; 15% vol/vol). The supernatant was discarded, and Tris-EDTA saline (10 mM Tris, 1 mM EDTA, and 150 mM NaCl) was used to resuspend the platelet pellet. Platelet suspension was centrifuged (500 × g for 20 min), the supernatant was discarded, and the platelet pellet was resuspended in phosphate-buffered saline (PBS). For the experiment that used the pre-stimulated platelets (Figure 1B and 3), platelets were activated with 0.2 U/ml of human thrombin (Sigma, St. Louis, MO) or 1 μM of ADP (Sigma) for 10 min, washed twice with PBS, and then resuspended in PBS. In other experiments (Figure 3B), platelets were fixed for 10 min in 1% paraformaldehyde (Sigma) in PBS at 4°C with gentle agitation; this preserves membrane integrity but prevents secretory activity. To test the isolation-induced activation of platelets, P-selectin expression was examined by FACS analysis and found to be less than 5% (data not shown). In contrast, after the stimulation with thrombin or ADP, more than 90% of cells were positive for P-selectin. Contamination of leukocytes was assessed microscopically and found to be negligible.

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Figure 1. (A) Platelets induce monocyte chemoattractant protein-1 (MCP-1) upregulation by mesangial cells (MC) at the ratio of 1:100. (B) Prestimulation of platelets with either thrombin or ADP is not required. In panel A, platelets were isolated from normal human donors and cocultured with MC at various ratios for 24 h. Alternatively, MC were stimulated with 10 ng/ml of tumor necrosis factor–α (TNF-α) for 24 h (A). In panel B, platelets were pre-stimulated with 0.2 U/ml of thrombin or 1 μM ADP, washed, and then cocultured with MC. MCP-1 production in supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Data are means ± SE from three separate experiments.

Human Mesangial Cells (MC)

Human MC were established and characterized as reported previously (26). Cells were cultured in Waymouth medium (Life Technologies, Grand Island, NY) supplemented with 17% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 2 mM sodium pyruvate, 1% (vol/vol) nonessential amino acids, and 26 μg/ml of bovine insulin (all Life Technologies). Three independent cell lines were employed during passages 5 through 12.

Platelet/MC Coculture

Platelets were prepared to 1, 10, or 100 × 10⁵ /ml in Waymouth medium containing 1% FBS. MC were harvested at 80% confluence and cultured in 24-well plates at 6 × 10⁴ cells/0.6 ml per well in 1% FBS/Waymouth medium in duplicate for 16 h. Subsequently prepared 0.6 ml of platelets was added to 6 × 10⁴ of MC, resulting in 1:1, 1:10, or 1:100 ratio of coculture (MC:platelets). In the experiments examining the effect of cell contact, identical parallel cultures were established in which platelets were separated from MC by a 0.4-μm pore size semipermeable membrane (Biocoat, Becton Dickinson Labware, Bedford, MA) while sharing the same medium. In the experiments, which examined the effect of rTNF-α, rIFN-γ, and rCD40L, MC were thawed in a 24-well plate at the same concentration with coculture experiment for 16 h for the adherence. Then, MC were further incubated at final volume of 1.2 ml/well in the presence or absence of various cytokines for 24 h. After 24-h incubation at 37°C, MCP-1 synthesis in supernatants and MC CD40 expression were determined by enzyme-linked immunosorbent assay (ELISA, R&D systems) and FACS analysis, respectively.

Inhibition of Chemokine Synthesis by Antibodies or Chemical Inhibitors

In the blocking experiment with neutralizing Abs, coculture of MC and unstimulated platelets was carried out at the ratio of 1:100 (MC:platelets) in the presence or absence of 10 μg/ml Abs against P-selectin, β1-integrin, CD40L, or IgG₁ of irrelevant specificity (Sigma). All antibodies were added to MC simultaneously with platelets. In the experiments that used pyrolidine derivative of dithiocarbamate (PDTC, Sigma), SB203580, PD98059 (both from Calbiochem-Novabiochem, La Jolla, CA), and genistein (Sigma), MC were preincubated with those inhibitors for 2 h, and then washed four times with medium to remove added inhibitors completely. Coculture of MC and unstimulated platelets were subsequently performed at 1:100 ratio for 24 h. MCP-1 synthesis was determined by ELISA in the supernatants after 24 h.

Quantitative Real-Time PCR

MCP-1 mRNA expression in coculture was determined by real-time PCR, following the manufacture’s protocol. Briefly, total RNA was isolated from coculture of MC and unstimulated platelets (1:100 ratio) in 6-well plates after a variety time of incubation using RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was synthesized from 2 μg of total RNA by Superscript preamplification system for first strand cDNA system (Life Technologies), using oligo-dT primers. Subsequently, real-time PCR was performed in the ABI Prism 7700 Sequence Detection System (Perkin Elmer Applied Systems, Foster City, CA) using the SYBR Green I PCR kit (Perkin Elmer Applied Systems). Each reaction contained 25 μl of the 2× SYBR green Master Mix, 300 nM primers (MCP-1, forward: 5′-GAT CTC AGT GCA GAG GCT CG-3′, reverse: 5′-TGC TTG TCC AGG TGG TCC AT-3′; β-actin, forward: 5′-GAA CTT TGG GGG ATG CTC GC-3′, reverse: 5′-CGG GAA ATC GTG CGT GAC AT-3′) (27), 5 μl of a 1:10 dilution of the cDNA prepared above, and water to 50 μl. The reactions were incubated at 94°C for 10 min to activate the Amplitaq Gold polymerase (Perkin Elmer Applied Systems) followed by 40 cycles of 30 s at 94°C, 30 s at 60°C, and 60 s at 72°C. The ABI Prism 7700 Sequence Detection System software determined relative mRNA expression of MCP-1 and β-actin in each samples, based on the standard curve described below. MCP-1 mRNA expression in each sample were finally determined after correction with β-actin expression. Standard curve was generated as follows (28). In brief, cDNA was prepared from MC stimulated with 100 ng/ml of LPS (Sigma) for 8 h. Using this cDNA as template, PCR products for MCP-1 and β-actin were prepared with same primers. Each reaction contained 5 μl of the 10× PCR buffer; 500 nM forward and reverse primers; 0.5 μl of Taq Gold polymerase; 1 μl of the cDNA; and water to 50 μl. The reactions were incubated at 94°C for 10 min to activate the Amplitaq Gold polymerase followed by 40 cycles of 30 s at 94°C, 30 s at 60°C, and 60 s 72°C. After confirming specific single band on the agarose gels, serial dilutions (tenfold) of these PCR products (10⁻⁵∼10⁻⁹) were prepared and then amplified simultaneously with samples from coculture using SYBR green. A standard curve was determined by the ABI Prism 7700 Sequence Detection System software. The expression of MCP-1 and β-actin in each sample was quantitated in separate wells with the respective primers. No PCR products were detected in the real-time PCR procedure without reverse transcription, indicating that the contamination of genomic DNA was negligible. Gels of the PCR products after quantification of MCP-1 or β-actin by real-time PCR showed a single band (152 and 711 bp, respectively) with the expected size (data not shown).

Flow Cytometry

CD40 expression on MC was determined by FACS analysis. Briefly, MC were harvested with trypsin/EDTA and stained for CD40 expression by incubating the cells on ice with anti-human CD40 antibody or an isotype-matched control, followed by R-PE–conjugated goat anti-mouse immunoglobulins. CD40 expression was analyzed using CELLQUEST (Becton Dickinson).

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear proteins were isolated by the methods described previously with slight modification (29). Briefly, cocultures of MC and unstimulated platelets were performed at the ratio of 1:100 in 75-cm² flask for 1 h. Alternatively, MC were incubated for 1 h with 10 ng/ml of TNF-α or 100 ng/ml of LPS (Sigma) instead of platelets. After the treatment, cells were washed with ice-cold PBS, harvested by scraping, then spun-down by centrifugation. Pellets were resuspended in 1 ml of hypotonic buffer (10 mM Hepes, pH 7.9, containing 10 mM NaCl, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). After 15-min incubation on ice, 60 μl of 10% NP-40 was added. Cells were then vortexed vigorously for 15 s and then centrifuged for 1 min at 12,000 rpm at 4°C. After removing supernatants, the pellets were resuspended in 500 μl of extraction buffer (20 mM Hepes, pH 7.9, containing 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). After incubation for 15 min on ice, samples were vortexed vigorously for 15 s and then centrifuged at 12,000 rpm for 10 min. The supernatants containing nuclear protein were used for EMSA after determining protein concentration with the Brad-ford method (BioRad Protein Assay kit; BioRad Laboratories, Hercules, CA). EMSA of nuclear factor–κB (NF-κB) was carried out by use of Gel Shift Assay Systems (Promega, Madison, WI), following manufacture’s protocol. Briefly, double-stranded oligonucleotide containing the NF-κB binding element (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was end-labeled using [γ−32P]ATP (NEN Life Science Products, Boston, MA) and T4 polynucleotide kinase and then purified through G-25 spin columns (BioRad). Nuclear extracts (2 μg of protein) were incubated with radiolabeled probes for 20 min at room temperature in 10 μl of binding buffer (10 mM Tris-HCL, pH 7.5, containing 50 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA, 0.5 M DTT, 0.05 mg/ml poly (dl-dC)(dl-dC), 4% glycerol). Specific controls included unlabeled NF-κB or Sp-1 (5′-ATT CGA TCG GGG CGG GGC GAG C-3′) consensus oligonucleotides at 100-fold excess. The samples were separated on a nondenaturing 4% polyacrylamide gel in Tris-Borate buffer, dried, and then analyzed by autoradiography.

Statistical Analysis

All experiments were repeated at least three times. Results are presented as the mean ± SEM from three separate experiments where indicated. Statistical significance, where indicated, was determined by t test. A value of P < 0.05 was considered to represent a statistically significant difference between two groups.

Platelets Induce Upregulation of MCP-1 Production by MC

We first examined whether platelets induce MCP-1 production by MC. Platelets purified from normal human subjects were cocultured for 24 h with MC at various ratios. Platelets, at 1:100 ratio (MC:platelets), enhanced MCP-1 production by 18-fold above the basal level released by MC alone (Figure 1A). This was comparable to levels induced by recombinant TNF-α. We did not observe further increases in MCP-1 production by higher ratios up to 1:500 (data not shown).

We next examined the effect of prestimulation of platelets with ADP or thrombin. Platelets were isolated and stimulated with thrombin or ADP for 10 min. Activation of platelets was verified by the induction of P-selectin expression (data not shown). After washing, platelets were cocultured with MC at various ratios as before. Pretreatment of platelets with thrombin or ADP did not further enhance the expression of MCP-1 (Figure 1B), indicating that specific prior activation of platelets was not required. Stimulated platelets alone did not synthesize MCP-1 (data not shown).

We next determined whether the upregulation of MCP-1 production occurred at the transcriptional level. Platelets (without prior stimulation) were cocultured with MC at the ratio of 1:100. At various time points, cells were harvested and mRNA was extracted and quantified by real-time PCR. Unstimulated MC expressed low levels of basal MCP-1 mRNA. Platelets enhanced MCP-1 mRNA expression 22-fold at 6 h, which was sustained up to 12 h (Figure 2). Taken together, these data demonstrate that platelets induce MCP-1 upregulation by MC at both protein and mRNA levels. The magnitude of this response could not be further enhanced by specific prior in vitro platelet stimulation. This may have reflected activation of critical pathways during platelet isolation. This could have varied between donors, we therefore elected to stimulate platelets (judged by P-selectin expression) in subsequent mechanistic experiments to ensure maximal stimulation had been achieved in each experiment.

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Figure 2. Platelets induce upregulation of MCP-1 mRNA by MC. MC were cocultured with platelets at the ratio of 1:100 for various times, and then MCP-1 and β-actin mRNA expressions were analyzed by real-time PCR. Fold increase in MCP-1 expression at each time point, relative to 0 h, is shown after correction with β-actin expression. Data are means ± SE from three separate experiments.

Direct Cell-to-Cell Contact between Platelets and MC Is Essential for MCP-1 Production

Stimulation of MC by platelets could be mediated either by direct cell-to-cell contact or through soluble factors. We therefore determined whether platelets could induce MC MCP-1 release when separated by a semipermeable membrane that prevents direct cell-to-cell contact. Platelets were incubated with thrombin for 10 min and then cocultured with MC for 24 h at a 1:100 ratio in contact with or separated from MC. As shown in Figure 3A, separation of platelets from MC abrogated MCP-1 upregulation by MC, suggesting a role for cell-surface molecules. ADP-stimulated platelets also failed to induce MCP-1 when separated from MC (data not shown). These data implicate direct cell-to-cell contact in platelet-mediated MCP-1 upregulation in MC.

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Figure 3. (A) Direct cell-to-cell contact between platelets and MC is essential for MCP-1 upregulation. (B) Fixed activated platelets do not upregulate MCP-1 production by MC. In panel A, MC were cocultured in contact with or separated by a porous membrane insert from pre-stimulated platelets at 1:100 ratio for 24 h. In panel B, platelets were stimulated with thrombin and fixed with 1% PFA to preserve membrane integrity but prevent secretion of soluble factors. MC were subsequently cocultured with fixed or unfixed activated platelets at 1:100 ratio for 24 h. MCP-1 production in supernatants was determined by ELISA. Data are means ± SE from three separate experiments.

To address this further, platelets activated with thrombin were fixed with PFA to preserve membrane integrity but prevent secretion of soluble factors. Fixed, activated platelets were cocultured with MC at the ratio of 1:100 for 24 h. Figure 3B demonstrates that fixed activated platelets did not induce MCP-1 upregulation. Fixed activated platelets failed to secrete MCP-1 production, even at the ratio of 1:500 (data not shown). This suggests that cell-surface molecules alone are not sufficient and that soluble factors from activated platelets are also required for MCP-1 synthesis by MC.

CD40/CD40L Pathway is Involved in Platelet-Mediated MCP-1 Production by MC

We next sought to identify molecules involved in platelet/MC interactions. P-selectin plays a crucial role in leukocyte or endothelial cell activation by platelet binding (11,12), and β1-integrins are implicated in platelet interactions with extracellular matrix (30). Activated platelets also express surface CD40L through which platelets can induce proinflammatory responses by endothelial cells (13). We therefore examined the effect of neutralizing P-selectin, β1-integrins, and CD40L in coculture experiments. Platelets were cocultured with MC at the ratio of 1:100 in the presence or absence of antibodies for 24 h. Inhibition of CD40/CD40L reduced MCP-1 induction by approximately 60%, whereas blocking of P-selectin or β1-integrins did not significantly affect the results (Figure 4). Although immunohistochemical analysis has shown that CD40 is expressed on MC in renal biopsy from patients with a variety of glomerulonephritides (16), its expression on cultured MC has not been identified. Therefore we examined CD40 expression on MC by FACS analysis, and we found that MC constitutively expressed CD40 (Figure 5). In addition, inflammatory cytokines, such as TNF-α and interferon-γ (IFN-γ), or coculturing with platelets at the ratio of 1:100 increased CD40 expression (Table 1). To further examine the role of CD40 for MC activation, we stimulated MC with recombinant CD40L (rCD40L). MC were stimulated with 3 μg/ml of rCD40L for 24 h in the presence or absence of 500 U/ml of IFN-γ, which is known to function synergistically with CD40L in the monocyte/macrophage activation (15). In preliminary studies, the optimal concentrations of rCD40L and IFN-γ were determined. Although rCD40L alone did not upregulate MCP-1, rCD40L in combination with IFN-γ enhanced MCP-1 synthesis by twofold above basal levels in response to IFN-γ alone (Figure 6). These data suggest that CD40 is expressed on MC and is involved in their activation by platelets to release MCP-1.

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Figure 4. Inhibition of MCP-1 synthesis in MC/platelet coculture by neutralizing Abs. MC were cocultured with platelets at 1:100 ratio for 24 h in the presence or absence of neutralizing Abs for 24 h. Percent inhibition was calculated as follows: 100 − 100 × (MCP-1 synthesis with neutralizing Ab/MCP-1 synthesis without neutralizing Ab). Data are means ± SE from three separate experiments. * Satistically significant difference as compared with control (P < 0.01).

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Figure 5. MC constitutively express CD40. MC were stained with anti-human CD40 antibody or an isotype-matched control, followed by R-PE-conjugated goat anti-mouse immunoglobulins. CD40 expression was determined by FACS analysis. Representative data of three distinct experiments are shown. The left-hand histogram represents the staining with control Abs.

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Figure 6. rCD40L enhances MCP-1 production by MC in the presence of interferon-γ (IFN-γ). MC were cultured to adherence at 6 × 10⁴ cells/0.6 ml per well in 24-well plates in duplicate for 16 h. Cells were subsequently stimulated with rCD40L (3 μg/ml), IFN-γ (500 U/ml), or both. After 24 h, MCP-1 synthesis was determined by ELISA. Data are mean ± SE from three separate experiments.

Platelets Induce Mesangial MCP-1 Upregulation through NF-κB Activation

MCP-1 expression is regulated both in a stimulus-specific and a tissue-specific manner (31). NF-κB plays an essential role for MCP-1 upregulation (32). We performed gel-shift assays to examine the molecular mechanisms by which platelets activate MC. Platelets significantly enhanced NF-κB activation above basal levels detected in resting MC (Figure 7A). Binding was inhibited by adding cold probe against NF-κB, but not against SP-1, indicating specificity. To further investigate the involvement of NF-κB activation, we used PDTC, a specific inhibitor of NF-κB. PDTC reduced MCP-1 upregulation in a dose-dependent manner (Figure 7B). Collectively these data demonstrate that platelet contact induce NF-κB activation in MC, which in turn is essential for MCP-1 synthesis.

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Figure 7. (A) Platelet contact induces nuclear factor–κB (NF-κB) activation by MC. (B) PDTC, the specific inhibitor of NF-κB, reduced MCP-1 production in coculture in a dose-dependent manner. In panel A) MC were cocultured with platelets at 1:100 ratio for 1 h. Alternatively, MC were incubated in the presence or absence of LPS (100 ng/ml) or TNF-α (10 ng/ml) for 1 h. Nuclear extracts were subjected to electrophoretic mobility shift assay (EMSA) to examine NF-κB activation. Specificity was checked with an unlabeled probe (cold) against NF-κB or Sp-1. In panel B, MC pretreated with various concentrations of PDTC were cocultured with platelets at the ratio of 1:100 for 24 h. MCP-1 production was determined by ELISA. MCP-1 production in coculture without PDTC is shown as 100%. Data are means ± SE from three separate experiments.

Platelets Induce MCP-1 Production through p38 MAPK and PTK

Previous studies have shown that p38 MAPK activation is required in cytokine-induced MCP-1 expression (33,34). Similarly TNF-α or interleukin-1 (IL-1) induces MCP-1 production through protein tyrosine kinases (PTK), but not protein kinase C (PKC) or cAMP-protein kinase A (PKA) (35,36). To examine whether platelets induce MCP-1 synthesis by MC through activation of p38 MAPK and/or PTK, we used SB203580 or genistein, which are inhibitors of p38MAPK or PTK, respectively. As a control we used PD98059, an inhibitor of ERK1/2. MC were preincubated with these inhibitors for 2 h and then washed extensively with medium. In preliminary studies, the concentrations of inhibitors to achieve the maximal effects were established. Treatment with those inhibitors did not alter the basal expression of MCP-1 by MC. Platelets were added to inhibitor-treated MC at a final ratio of 1:100. SB203580 and genistein reduced MCP-1 production by 40 and 70%, respectively (Figure 8), whereas PD98059 had no effect. These data suggest that platelets induce MCP-1 synthesis by MC through activation of p38 MAPK and PTK.

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Figure 8. p38 MAPK and protein tyrosine kinases (PTK) are involved in mesangial MCP-1 upregulation induced by platelet binding. MC were pretreated with 50 μg/ml PD98059, SB203580, or genistein for 1 h. In preliminary studies, the concentrations of inhibitors to achieve the maximal effects were determined. Medium was changed four times to remove added inhibitors completely. Subsequently, coculture of MC and platelets were performed at the ratio of 1:100. ELISA determined MCP-1 synthesis after 24 h. MCP-1 production in coculture without inhibitors is shown as 100%. Data are means ± SE from three separate experiments.