Major involvement of CD40 in the regulation of chemokine secretion from human peritoneal mesothelial cells

Kidney International, Vol. 64 (2003), pp. 2064–2071

Major involvement of CD40 in the regulation of chemokine secretion from human peritoneal mesothelial cells LIMOR MAN, ELI LEWIS, TOM EINBINDER, BORIS ROGACHEV, CIDIO CHAIMOVITZ, and AMOS DOUVDEVANI Department of Nephrology, Soroka University Medical Center, Ben-Gurion University of the Negev, Faculty of Health Sciences, Beer-Sheva, Israel

Major involvement of CD40 in the regulation of chemokine secretion from human peritoneal mesothelial cells. Background. CD40 is a member of the tumor necrosis factor (TNF) family of receptors, whose ligand, CD154, is expressed by activated mononuclear cells. CD40 activation is a major immune regulatory pathway and is important for the regulation of chemokine and cytokine secretion. This study investigates the effect of CD40 ligation on the secretion of chemokines from human peritoneal mesothelial cells (HPMC). Methods. We activated CD40 in HPMC along with combinations of TNF-a, interleukin-1 (IL-1), and interferon gamma (IFN-c ), and evaluated the mRNA levels and protein secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and IL-8. Results. CD40 ligation had a small stimulatory effect on the secretion of all three chemokines, while TNF-a, IL-1 and IFN-c induced their secretion in a dose-dependent manner. The combination of CD40 ligation with either IL-1 or TNF-a increased chemokine secretion additively. IFN-c and CD40 ligation acted in synergy to induce the secretion of the mononuclear recruiting chemokines RANTES and MCP-1 (up to ∼36-fold and ∼ threefold, respectively), for which the combination of all three cytokines with CD40 ligation was extremely potent. In contrast, the secretion of the neutrophil chemoattractant IL-8, induced by CD40 ligation or by the combination of IL-1 and TNF-a, was reduced in the presence of IFN-c . Conclusion. In light of our data, it is reasonable to suggest that in the mononuclear phase of peritonitis, IFN-c and CD154, expressed by activated mononuclear cells, diminish IL-8 secretion from HPMC and thus inhibit neutrophil recruitment. At the same time, the two act in synergy to induce the secretion of RANTES and MCP-1 from HPMC. Hence, by regulating chemokine secretion, CD40 may be involved in peritonitis and in the development of late phase mononuclear predominance.

Key words: CD40, human peritoneal mesothelial cells, chemokines, peritonitis. Received for publication November 25, 2002 and in revised form March 23, 2003, April 13, 2003, and July 8, 2003 Accepted for publication August 5, 2003
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2003 by the International Society of Nephrology

During peritonitis, mesothelial cells gate leukocyte trafficking into the peritoneal compartment [1]. Consistent with this role, following inflammation in vivo or cytokine stimulation in vitro, human peritoneal mesothelial cells (HPMC) up-regulate the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) [2–5], and respond to bacterial and inflammatory mediators by secreting proinflammatory cytokines and chemokines [6–11]. The early leukocytic influx during peritonitis is part of the nonspecific innate immune response and consists predominantly of neutrophils [10]. It is followed shortly by a mononuclear infiltrate, which is part of the specific immune response [10]. “Switching” between infiltrate phenotypes is important for a successful immune response and is a tightly regulated process. It has been suggested that the regulation of the infiltrate phenotype is controlled by chemokine secretion from mesothelial cells [12]. Cytokine-activated HPMC have been shown to secrete both neutrophil-specific CXC [interleukin-8 (IL-8)] and mononuclear-specific CC [regulated upon activation, normal T-cell expressed and secreted (RANTES) and monocyte chemoattractant protein (MCP)-1] chemokines [7]. In addition, specific antibody blocking studies have demonstrated a direct role for HPMC-derived IL-8 and MCP-1 in mediating leukocyte migration of appropriate phenotypes across the mesothelial layer [12]. CD40 is a cell surface receptor that belongs to the tumor necrosis factor (TNF)-receptor family (reviewed in [13]). Its critical role in T-cell–dependent immune responses has been demonstrated in patients with hyperIgM syndrome, as well as by gene targeting in mice. The CD40 receptor is expressed on B cells and monocytes, and on dendritic, endothelial, and epithelial cells. We recently demonstrated that mesothelial cells express CD40 [14]. The ligand for the CD40 receptor, CD154, is a member of the TNF family and is mainly expressed on activated CD4+ T cells. Unlike soluble TNF-a, CD154

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acts as a membrane-bound ligand. Thus, using CD154 via direct cell-to-cell contact, lymphocytes activate CD40 on their target cells to induce the secretion of cytokines and chemokines and to up-regulate the expression of adhesion and accessory molecules. Based on the effect of CD40 activation on chemokine secretion from several tissues [15–21], and our finding of a functional CD40 receptor on HPMC [14], we hypothesized that the CD40 signaling may be involved in the regulation of the chemotactic activity of HPMC and thus affect the trafficking of immune cells into the peritoneal cavity. In support of this hypothesis, we recently reported an inducible expression of the CD40 receptor on the peritoneal membrane in a mouse model of grampositive and gram-negative bacterial peritonitis (submitted for publication), as well as a gradual increase in CD154 expression on infiltrating leukocytes in the same model. We therefore investigated whether CD40 ligation in HPMC is involved in the regulation of leukocyte-specific chemokine secretion from activated HPMC. We stimulated HPMC using CD40 ligation in the presence of IL-1, TNF-a, and interferon-gamma (IFN-c ), which are cytokines that are involved in peritonitis [22, 23]. The resulting changes in the production and secretion of RANTES, MCP-1, and IL-8, chemokines that are involved in leukocyte recruitment in peritonitis [24–27] were then examined.

METHODS Cell preparation HPMC. Omentum-derived HPMC were isolated and characterized for their specific morphology and markers, as previously reported [28]. Experiments were performed on cells from the second to fourth passages.

CD154-expressing L cells (CD40L cells) For CD40 activation, we used a cell line of mouse fibroblast L cells, transfected with the complete human CD154 coding sequence and a neomycin resistance gene for selection of stable transfectants. Both types of cells were cultured in RPMI and supplemented with 10% heatinactivated fetal calf serum (FCS), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 lg/mL streptomycin (Biological Industries, Bet Haemek, Israel). The parental nontransfected cells (L cells) were used as a negative control. Both parental and CD154-transfected cells were kindly supplied by Schering Plough Corporation and have been previously described [29]. CD40 activation using CD154 expressing cells is frequently used to activate CD40 on various cell types in several experimental models [29–32].

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HPMC activation protocols HPMC were seeded in 24-well plates (2 × 104 cells/ well) and cultured to confluence. HPMC were washed twice with medium and incubated with TNF-a (Peprotech, Rocky Hill, NJ, USA), IL-1a (kindly provided by Hoffman-La Roche, Nutley, NJ, USA), or IFN-c (Peprotech) in various concentrations, and with CD40L cells (expressing CD154) or control L cells (5 × 104 ). The incubation time for chemokine determination was 24 hours for enzyme-linked immunosorbent assay (ELISA) analysis, and 6 hours for mRNA analysis. Incubation time for RNA analysis was optimized in preliminary experiments. To prevent overgrowth of L cells and CD40L cells in the co-culture experiments, the cells were grown in M-199 medium, which inhibits fibroblast growth. Supernatant was collected and stored at −20◦ C. Cells from two wells were counted in each plate to determine consistency of cell number per well. All tissue culture reagents contained less than 0.025 ng/mL endotoxin. As a positive control for stimulation for IL-1b production, HPMC were stimulated with IL-1a (100 U/mL), TNF-a (10 ng/mL), and lipopolysaccharide (LPS) (10 lg/mL) (Sigma, Rehovot, Israel). mRNA analysis IL-8, MCP-1, and RANTES mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) of total RNA extracted from HPMC. HPMC were incubated as described above. At the end of each experiment, murine cells were discarded and their absence was confirmed under light microscopy. Total RNA was extracted from HPMC using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The extracted RNA was immediately converted to cDNA. For cDNA generation, 13 lL of RNA sample were added to 7 lL reverse transcriptase reaction mixture, containing 1 lL of Moloney murine leukemia virus-reverse transcriptase (MMLVRT) (200 U/lL) (Gibco BRL, Gaithersburg, MD, USA), 4 lL reverse transcriptase buffer (Gibco BRL), 0.5 lL dithiothreitol (DTT) (0.1 mol/L) (Gibco BRL), 0.5 lL RNase inhibitor (40 U/lL) (Sigma), 1 lL oligo-d(T) 12– 18 mer (40 pmol/lL) (Roche), and 1 lL desoxynucleoside triphosphate (dNTP) (10 nmol/lL) (Sigma). The reaction tube was incubated for 1 hour at 37◦ C, after which the volume of each sample was adjusted to 60 lL and the enzyme was inactivated by incubation for 10 minutes at 65◦ C. Chemokines and b-actin cDNA were amplified by PCR using specific primers. RANTES sense TCT CCA CAG GTA CCA TGA AGG, RANTES antisense GGT TCA AGG ACT CTC CAT CC; MCP-1 sense TTG TGT GCC TGC TGC TCA TA, MCP-1 antisense GGT TTG CTT GTC CAG GTG GT; IL-8 sense TGA CTT CAA AGC TGG CCG TG, IL-8 antisense CCA CGT CTC CAA CAC CTC T; and b-actin sense ATG GAT GAT GAT

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ATC GCC GCG, b-actin antisense CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC. The primers were designed to be specific for human cDNA. A total of 5 lL of reverse transcription product were added to 45 lL of PCR reaction mixture containing 32.75 lL H2 O, 2.5 lL 5 primer (20 lmol/L), 2.5 lL 3 primer (20 lmol/L), 2 lL dNTP (10 nmol/lL) (Sigma), 5 lL reaction buffer, and 0.25 lL Taq DNA polymerase (Sigma). A negative control consisting of the reaction mixture without cDNA was included in each run. PCR was run for 20 to 25 cycles with b-actin primers under the following conditions: 90 seconds at 95◦ C, then 5 to 10 cycles of 45 seconds each at 95◦ C, 90 seconds at 60◦ C, and 1 minute at 72◦ C. The last 15 cycles were prolonged by 5 seconds in each cycle. PCR with RANTES, MCP-1, or IL-8 primers was run with the same protocol except that the number of cycles was 30 to 35, 20 to 25, 25 to 30, respectively. Each experiment was amplified with at least two different numbers of cycles to ensure that amplification was in the exponential phase of PCR. Under these conditions, we also found a linear dose-response of the PCR product to increasing doses of cDNA. Eight microliters of each sample containing amplified cDNA were loaded on an agarose gel (1.5%) containing ethidium bromide (0.5 lg/mL). A DNA size marker was run on the same gel (100 bp ladder) (Gibco BRL). PCR products were quantified by video densitometry with the ImageMaster VDS-CL (Amersham Pharmacia Biotech, Freiburg, Germany). To correct for differences in loading, densitometric values of RANTES, MCP-1, and IL-8 amplification products were corrected with corresponding values of b-actin. ELISA IL-8, MCP-1, RANTES, and IL-1b were measured in supernatants in duplicates using ELISA for human IL-8, MCP-1, RANTES, and IL-1b made with commercially available antibodies and standards (R&D Systems, Minneapolis, MN, USA). ELISA was performed according to the manufacturer’s protocol. The ELISAs are specific for human products, have no cross-reactivity, and exhibit no interference with 50 ng/mL of equivalent murine products. For RANTES, we used monoclonal antihuman RANTES (4 lg/mL, type MAB678), rhRANTES (39 to 2500 pg/mL), and biotinylated monoclonal antihuman RANTES antibody (200 ng/mL) (type BAF278). For MCP-1, we used monoclonal antihuman MCP-1 (4 lg/mL) (type MAB679), rhMCP-1 (31.25 to 2000 pg/mL) and biotinylated monoclonal antihuman MCP-1 antibody (50 ng/mL) (type BAF279). For IL-8, we used monoclonal antihuman IL-8 (4 lg/mL) (890804 DuoSet), rhIL-8 (31.25 to 2000 pg/mL), and biotinylated monoclonal antihuman IL-8 antibody (20 ng/mL) (890805 DuoSet). For IL-1b, we used mouse antihuman

IL-1b (4 lg/mL) (840168 DuoSet), rhIL-1b (3.1 to 250 pg/ mL) and biotinylated goat antihuman IL-1b antibody (100 ng/mL) (840169 DuoSet). For detection we used streptavidin horseradish peroxidase (HRP) (type 434323) (Zymed, San Francisco, CA, USA) (1:20,000) and tetramethylbenzidine solution (TMBSingle Solution) (Zymed). ELISA reaction was stopped with H2 SO4 (2 N). Optical density was read with an ELISA reader at 450 nm and at 550 nm for reference. The sensitivity was 2 pg/mL. No cross-detection of other cytokines was observed. Statistics Results are expressed as mean ± SD. To compare levels between groups we used one-way analysis of variance (ANOVA). P values below 0.05 were considered significant.

RESULTS All of the experiments in this study were performed on primary cultures of HPMC. The CD40 pathway was activated in these cells by exposure to a murine cell line genetically modified to express human CD154 (CD40L cells). The use of these cells has been validated in previous reports [14, 30]. We previously demonstrated that the interaction between these two cell populations is dependent on cell-to-cell contact [14]. We used modified murine cells, rather than activated lymphocytes, in order to isolate the role of CD154 from other lymphocytic signals. We also examined a soluble form of CD154 and found a similar trend of HPMC activation, but with diminished response amplitude (data not shown). Since the activation via cell-to-cell contact more accurately reflects the physiologic manner of HPMC activation, we chose to use the modified murine cell line for CD40 ligation. We activated the CD40 pathway with CD154 expressing murine cells (CD40L cells) and different combinations of TNF-a (0.001 to 10 ng/mL), IL-1 (0.01 to 100 U/mL), and IFN-c (0.05 to 500 U/mL). Following stimulation, the secretion of chemokines was assayed by ELISA and their mRNA levels were evaluated by RT-PCR. RANTES secretion (Fig. 1A) was induced by IL-1 or TNF-a in a dose-dependent manner and was augmented with the addition of CD154 (up to ∼1.6- and ∼2.4-fold, respectively, at the maximal cytokine dose). IFN-c alone had a small effect on RANTES secretion, but exhibited an impressive synergistic increase when combined with CD40 ligation (up to ∼36-fold). This effect was also observed when examining mRNA levels. RANTES mRNA levels following CD40 ligation and IFN-c stimulation could be detected following 30 amplification cycles, while five additional amplification cycles were needed to

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Fig. 1. Effect of CD40 ligation on chemokine secretion from human peritoneal mesothelial cells (HPMC) in the presence of tumor necrosis factor-a (TNF-a ), interleukin-1 (IL-1), or interferon-gamma (IFN-c ). Primary cultures of HMPC (2 × 104 cells/well) were incubated for 24 hours in the presence of varying concentrations of IL-1, TNF-a, or IFN-c (left, middle, and right panels, respectively) and were added either CD40L-expressing murine fibroblasts (
) or control L cells (4 × 105 cells/well) (). Levels of regulated upon activation, normal T-cell expressed and secreted (RANTES) (A), monocyte chemoattractant protein-1 (MCP-1) (B), and interleukin-8 (IL-8) (C) were assayed by specific enzyme-linked immunosorbent assay (ELISA). Results are average values ± SD of three experiments performed in triplicate. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005 versus the same experimental conditions without CD40 ligation; ˆP < 0.05 versus the same experimental conditions without IFN-c .

detect RANTES levels in control and in single treatment, reflecting the extent of induction by CD40 ligation and IFN-c (Fig. 2). IFN-c induction of RANTES secretion was also synergistic with TNF-a (∼59-fold) (Fig. 3). Maximal RANTES secretion was obtained by the combina-

tion of CD154, TNF-a, and IFN-c (∼69-fold). For all combinations of TNFa, IL-1, and IFNc , CD40-ligation significantly increased RANTES secretion (Fig. 3). MCP-1 secretion (Fig. 1B) was achieved in the presence of each cytokine alone in a dose-dependent manner. IL-1

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Fig. 2. Effect of interferon-gamma (IFN-c ) and CD40 ligation on chemokine mRNA levels in human peritoneal mesothelial cells (HPMC). Primary cultures of HMPC (2 × 104 cells/well) were incubated for 6 hours in the presence of IFN-c (5 U/mL) or CD40Lexpressing murine fibroblasts (4 × 105 cells/well) or both. Reverse transcription-polymerase chain reaction (RT-PCR) was performed with specific primers for regulated upon activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP1), interleukin-8 (IL-8), and b-actin and the products were run on 1.5% agarose gels containing ethidium bromide. Gels are presented in the left panels. The PCR products shown here are 30 and 35 amplification cycles (Cy) for RANTES, 25 cycles for MCP-1, and 30 cycles for IL-8. The right panel shows the intensity of the PCR products evaluated by video densitometry and normalized according to their b-actin levels. RANTES densitometry was performed on products after 35 cycles of PCR. Unstimulated control was normalized to 1 and the densitometry of all other samples were adjusted accordingly. Results are average values ± SD from four independent experiments performed in triplicate. ∗ P < 0.05 versus untreated control (CT).

or TNF-a exhibited an additive increase in secreted levels when combined with CD154. IFN-c induction of MCP-1 secretion was synergistic with CD154 (∼3-fold). CD40 ligation had no additional effect on TNF-a and IL-1 in the presence of IFN-c (Fig. 3). IL-8 secretion (Fig. 1C) was achieved to a small extent by IL-1 and TNF-a and was increased in the presence of CD154 (IL-1 ∼2.0-fold and TNF-a ∼1.3-fold). IFN-c had a negative dose dependent effect on the levels of IL-8 and was able to significantly diminish the effect of CD40 at concentrations higher than 5 u/mL. The most effective combination to achieve IL-8 secretion was CD40 ligation in the presence of TNF-a and IL-1. Any combination containing IFN-c diminished IL-8 secretion levels (Fig. 3). CD40 ligation had no additional effect when TNF-a, IL-1, and IFN-c were combined (Fig. 3). Since we previously demonstrated that stimulation of HPMC with inflammatory agents resulted in induction

of IL-1 [6], we tested whether IL-1 secretion is affected by CD40 ligation. We found that in contrast to the positive control for IL-1b induction, namely, stimulation with TNF-a, IL-1a, and LPS, HPMC exposed to CD40 L cells for 24 hours did not produce significant levels of IL-1b (Fig. 4).

DISCUSSION The present study supports our contention that CD40 has a regulatory role in the secretion of chemokines from HPMC. We demonstrated that when stimulated by TNF-a, IL-1, or IFN-c , secretion of the mononuclear specific chemokine, RANTES, by HPMC is up-regulated in the presence of CD154, the CD40 ligand. This phenomenon was particularly evident when HPMC were stimulated by a combination of IFN-c and CD154, which magnified secretion of RANTES by up to 59-fold. As we suspected that mesothelial cells are exposed to both CD154 and IFN-c at the point of mononuclear infiltration, rather than pretreating them with IFN-c , we simultaneously administered CD154 and IFN-c throughout the study. A similar pattern of RANTES secretion in the presence of CD154 and IFN-c has been described in a cervical cancer cell line [32] and in myocytes [33]. We also demonstrated that RANTES secretion was induced by up to 69-fold when mesothelial cells were treated with a combination of TNF-a, IFN-c , and CD154. Similar to RANTES, we found that secretion of another mononuclear specific chemokine, MCP-1, was up-regulated when HPMC were treated with TNF-a, IL-1, or IFN-c in the presence of CD154. A possible mechanism by which IFN-c and TNF-a increases the effect of CD40 ligation is by up-regulating CD40 levels on HPMC, as we have previously described [14]. Another mechanism for the synergism of CD40 and IFN-c is inferred by the findings of Genin et al [34] who have shown that optimal RANTES expression is attained by the cooperative actions of the transcription factors nuclear factor-kappaB (NF-jB) and IFN-regulatory factor-1 (IRF-1) [34]. Since CD40 activates NF-jB [13] and IFN-c activates IRF-1 [35], these transcription factors may play a major role in the synergistic induction of RANTES and MCP-1. Similar to the up-regulatory effects on RANTES and MCP-1, the secretion of IL-8 by mesothelial cells was up-regulated by a combination of CD154 with TNF-a or IL-1. In contrast, a significant down-regulatory effect on IL-8 secretion was detected when the cells were treated with IFN-c . Our data demonstrate that the effects of CD40 ligation on chemokine secretion by mesothelial cells are not mediated by IL-1 induction. We have previously shown that HPMC produce IL-1 in response to various inflammatory signals [6]. However, in the present study, CD40 ligation

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did not induce IL-1 production, which suggests that the effect of CD40 is direct. CD40 has been associated with several conditions that depend largely on the influx of a mononuclear infiltrate. For example, in collagen-induced arthritis [36] and acute and chronic graft rejection [37], anti-CD154 treatment alleviated disease parameters. Furthermore, we previously reported that mesothelial cells express an inducible and functional CD40 molecule [14]. We also demonstrated, in a mouse model of bacterial peritonitis, that there is a considerable induction of CD40 expression on mesothelial cells during peritonitis (submitted for publication). The involvement of CD40 in peritonitis is further supported by the finding of increased CD154 expression in

Fig. 3. Effect of CD40 ligation on chemokine secretion from human peritoneal mesothelial cells (HPMC) in the presence of various combinations of tumor necrosis factor-a (TNFa ), interleukin-1 (IL-1), and interferongamma (IFN-c ). Primary cultures of HMPC were incubated for 24 hours in the presence of IL-1a (10 U/mL), TNF-a (1 ng/mL), and IFN-c (5 U/mL) and were added 4 × 105 cells/well of either CD40L-expressing murine fibroblasts (
) or control L cells (). Levels of regulated upon activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and IL-8 were assayed by specific enzyme-linked immunosorbent assay (ELISA). Results are average values ± SD of three experiments performed in triplicates. ∗ P < 0.05; ∗∗∗ P < 0.005 versus the same experimental condition without CD40 ligation; ˆP < 0.05 versus the same experimental condition without IFN-c ; # P < 0.05 versus untreated control; ∼ P < 0.05 combined IL-1 and TNF-a versus IL-1 or TNF-a alone.

the mononuclear infiltrate in the same model. The timing of CD154 elevation, 12 to 48 hours after initiation of peritonitis (submitted for publication), which coincides with CD40 up-regulation in mesothelial cells during peritonitis and with the peak of the mononuclear infiltrate, also supports the conjunction that CD40 is involved in cell recruitment. During peritonitis, the immunologic response is characterized by the tightly orchestrated appearance of leukocytes in the peritoneal compartment and a synchronized secretion of proinflammatory cytokines [10]. This process consists of two major phases of leukocyte infiltration: an early phase of neutrophilic infiltration and a subsequent phase in which mononuclear cells predominate [10]. Our

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Fig. 4. CD40 ligation does not induce interleukin-1 (IL-1) production by human peritoneal mesothelial cells (HPMC). Primary cultures of HMPC were incubated for 24 hours in the presence of either CD40L expressing murine fibroblasts or control L cells. As a positive control for interleukin-1b (IL-1b) induction, HPMC were stimulated with IL-1a (100 U/mL), tumor necrosis factor-a (TNF-a) (10 ng/mL), and lipopolysaccharide (LPS) (10 lg/mL). IL-1b was assayed by specific enzyme-linked immunosorbent assay (ELISA) (no cross-reactivity or interference was observed when 50 ng/mL IL-1a was added in this assay). The figure is a representative experiment of two experiments performed in triplicate. Results are presented as mean ± SD. ∗∗∗ P < 0.005 versus untreated control.

to decline, maintenance of mononuclear cells infiltration will be mainly dependent on the up-regulation of CD40 and IFN-c pathways. Thus, up-regulation of RANTES and MCP-1 by the combination of CD154 and IFN-c ensure the predominance of mononuclear cells at the inflamed site. Our results also indicate that high levels of IFN-c cause a decrease in IL-8 secretion from the mesothelium, leading to a continuing decrease in neutrophil infiltration. The regulatory role of IFN-c that we suggest here is consistent with the results of Topley, Mackenzie, and Williams [10] who showed that IL-8 secretion from mesothelial cells diminishes in the presence of IFN-c , as well as those of Robson et al [39] who provided evidence that IFN-c affects the migration of neutrophils through the peritoneal membrane in vitro and in vivo. The latter study, which focused on the effects of the Th1-type cytokine IFN-c on leukocyte migration into the peritoneum, also showed that stimulation of HPMC with IFN-c alone or in combination with IL-1 or TNF-a upregulates MCP-1 and RANTES and inhibits IL-1– and TNF-a–induced IL-8 secretion. Since both CD154 and IFN-c are products of the mononuclear infiltrate, their coordinated effect on CC and CXC chemokine secretion from mesothelial cells indicates that in the late phase of peritonitis mononuclear cells play a major role in the regulation of leukocyte recruitment. CONCLUSION

results suggest that at the beginning of an immune response in the peritoneum, mesothelial cells react to the early cytokines TNF-a and IL-1 by elevating secretion of chemokines that recruit granulocytes such as IL-8. In this early phase, mononuclear cells are the minority of the recruited cells. This could be the result of low IFN-c and CD40 signals, which induce low RANTES and MCP-1 production. We found that the effects of CD40 ligation on chemokine induction in the presence of TNF-a and IL-1 are negligible, which suggests that in the first phase of inflammation the CD40 signal has only a minor role. Hurst et al [38] have reported that in the early phase of peritonitis, IL-6 production by mesothelial cells is induced and soluble IL-6 receptors (sIL-6R) are released from infiltrating neutrophils. They showed that mesothelial cells have no IL-6 receptor, and therefore signaling of IL-6 in mesothelial cells depends on granulocyte-sIL-6R. Most significantly, they found that the IL-6 signal on mesothelial cells down-regulates CXC chemokines and promotes CC chemokines such as RANTES and MCP-1. As such, by shedding sIL-6R, the influx of neutrophils induces its own down-regulation and increases mononuclear cell infiltration [38]. The decline in granulocyte number at the end of the first phase of inflammation probably results in reduction of sIL-6R levels. Our data support the assumption that at this point, when the IL-6 signal begins

We suggest that in the late phase of inflammation the mononuclear signals IFN-c and CD40 suppress granulocyte recruitment and increases mononuclear migration via differential control of chemokine secretion by mesothelial cells. This study provides further evidence for the cellular crosstalk between mesothelial and immune cells during peritonitis. ACKNOWLEDGMENTS This work was supported by the “Dr. Montague Robin Fleisher Kidney Transplant Unit Fund.” We would like to acknowledge Schering Plough Corporation for providing us with the CD40L cells. We also thank Mrs. Valeria Frishman for her excellent technical assistance. Reprint requests to Dr. Amos Douvdevani, Nephrology Laboratory, Soroka Medical Center, P. O. Box 151 Beer-Sheva 84101, Israel. E-mail: [email protected]

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