A1 and A2A adenosine receptors play a protective role to reduce prevalence of autoimmunity following tissue damage.

Adenosine is a potent modulator that has a tremendous effect on the immune system. Adenosine affects T cell activity, and is necessary in maintaining the T helper/regulatory T cell (Treg ) ratio. Adenosine signalling is also involved in activating neutrophils and the formation of neutrophil extracellular traps (NETs), which has been linked to autoimmune disorders. Therefore, adenosine, through its receptors, is extremely important in maintaining homeostasis and involved in the development of autoimmune diseases. In this study, we aim to evaluate the role of adenosine A1 and A2A receptors in involvement of autoimmune diseases. We studied adenosine regulation by NETosis in vitro, and used two murine models of autoimmune diseases: type I diabetes mellitus (T1DM) induced by low-dose streptozotocin and pristane-induced systemic lupus erythematosus (SLE). We have found that A1 R enhances and A2A R suppresses NETosis. In addition, in both models, A1 R-knock-out (KO) mice were predisposed to the development of autoimmunity. In the SLE model in wild-type (WT) mice we observed a decline of A1 R mRNA levels 6 h after pristane injection that was parallel to lymphocyte reduction. Following pristane, 43% of A1 R-KO mice suffered from lupus-like disease while WT mice remained without any sign of disease at 36 weeks. In WT mice, at 10 days A2A R mRNA levels were significantly higher compared to A1R-KO mice. Similar to SLE, in the T1DM model the presence of A1 R and A2A R was protective. Our data suggest that, in autoimmune diseases, the acute elimination of lymphocytes and reduction of DNA release due to NETosis depends upon A1 R desensitization and long-term suppression of A2A R.

Pharmacological preconditioning with adenosine A1 receptor agonist induces immunosuppression and improves graft survival in novel allogeneic transplantation models.

Adenosine is widely known as a potent modulator of innate and acquired immunity. It is released during transplants, and acts on four subtype receptors. In previous studies, we demonstrated that pharmacological preconditioning (PPC), pre-administration of the selective A1 receptor (A1R) agonist led to A1R desensitization, is followed by upregulation of the adenosine A2A receptor. This immunosuppressive effect resulted in lymphopenia, and it reduced T-cell reactivity. The aim of the current study was to challenge the immunosuppressive effects of A1R-PPC in models of allogeneic grafts. PPC mice were treated by intraperitoneal injection using specific adenosine A1R agonist 24 h and 12 h before starting any procedure. We challenged our method in novel allogeneic muscle and skin grafts models. Mice and grafts were assessed by complete blood counts, MLR from PPC splenocytes, and pathological evaluation. We found a significant reduction in WBC and lymphocyte counts in PPC-treated mice. Two-way MLR with splenocytes from PPC grafted mice showed decreased proliferation and anergy. Histology of PPC allogeneic grafts revealed profoundly less infiltration and even less muscle necrosis compared to vehicle treated allografts. Similar results observed in PPC skin transplantation. To conclude, PPC moderated graft rejection in separate allogeneic challenges, and reduced lymphocytes infiltration and ischemic damage.

Systemic inflammatory response syndrome-related lymphopenia is associated with adenosine A1 receptor dysfunction.

SIRS is associated with lymphopenia, and prolonged lymphopenia of septic patients has been associated with increased mortality risk. We hypothesize that elevated adenosine during SIRS down-regulates Gi-coupled A1R, which signals an effect that sensitizes a cAMP-dependent lymphotoxic response. In this study, we evaluate the role of adenosine in SIRS-mediated lymphopenia and impaired IL-15 production. Cecal ligation and puncture was used to induce sepsis-associated SIRS in mice. BMDCs were cultured and used to measure the effect of adenosine on IL-15. We found that A1R mRNA levels were significantly down-regulated and A1R-dependent Gi activity was abolished in T cells of septic mice. In accordance, cAMP was elevated in isolated T cells from cecal ligation and puncture compared with sham-treated mice. Similar to septic mice, leukopenia was evident in sham A1R-KO mice, after treatment with the A1R antagonist (8-cyclopentyl-1,3-dipropylxanthine), or after A1R desensitization. In contrast, A2AR-KO mice were protected from leukopenia. In addition, we observed that septic A1R-KO mice exhibited low IL-15 levels. Cultured BMDC agonists of A2AR and A2BR inhibited IL-15 production and adenosine blocked IL-15-dependent proliferation of cytotoxic T cells that were cocultured with stimulated BMDCs. To conclude, we suggest that SIRS-associated lymphopenia is initiated by A1R desensitization and adenosine-mediated inhibition of IL-15 production is part of the mechanism that accounts for the delay in leukopenia recovery in patients with severe sepsis. Interference with adenosine signaling may thus be potentially beneficial for septic patients with leukopenia.

Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor.

Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in anti-tumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR(-/-)) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA-induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p=0.0286, n=15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR(-/-) mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNγ levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development.

Pharmacological preconditioning with adenosine A(1) receptor agonist suppresses cellular immune response by an A(2A) receptor dependent mechanism.

Under stressful conditions such as ischemia, sepsis, and severe trauma, adenosine levels are elevated and protect the tissue by interaction with G coupled receptors. In a model of peritonitis, we previously found that pharmacological preconditioning (PPC) of mice with a selective adenosine A1 receptor (A1R) agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), induced the A2AR which reduces cytokine secretion and leukocyte recruitment. In our present study we determined whether mice PPC will moderate cellular immune response by the same mechanism. Similar to the effect on inflammation, PPC reduced the response to lymphocyte mitogens and allogeneic MLR response. The inhibitory effect of PPC on the immune response was A1R and A2AR dependent as illustrated by experiments with antagonists of these receptors and mice with knock down (KO) receptors. In MLR with PPC splenocytes we found reduced levels of pro-inflammatory cytokines (IFN-γ, IL-15, TNF-α) and elevation of IL-10, as well as elevation of regulatory T-cell. Our data indicate that PPC is able to remarkably suppress cellular immune response due to the sensitization A2AR. This effect of PPC sheds light on the protective role of adenosine in ischemic preconditioning and makes this treatment candidate for the prevention of graft rejection.

Association between renal injury and reduced interleukin-15 and interleukin-15 receptor levels in acute kidney injury.

Interleukin (IL)-15 serves as a survival factor for a broad array of cells. Renal cells express both IL-15 and its receptor (IL-15R); however, the role of IL-15 in the kidney is yet to be determined. We examined IL-15 and IL-15R levels in sepsis-related renal injury, ischemia-reperfusion injury (IRI), and cisplatin-induced nephrotoxicity. To test the anti-apoptotic effect of IL-15, Bcl-2/Bax mRNA levels were assessed in kidneys of IL-15Ralpha(-/-) mice and in IL-15-stimulated renal epithelial cells (RECs). In addition, RECs were exposed to cisplatin and apoptosis was evaluated by TUNEL staining, caspase-3 activity, and cell cycle analysis. Intrarenal IL-15 levels decreased 24 h after initiation of all three examined pathologies by 5.8-fold (sepsis), 11-fold (IRI), and 23-fold (cisplatin-induced nephrotoxicity). Further experiments revealed that while addition of rIL-15 (1 ng/mL) to wild-type (WT) RECs increased Bcl-2/Bax ratio by 2-fold, kidneys of IL-15Ralpha(-/-) mice exhibited 4-fold lower Bcl-2/Bax ratio compared to WT mice. Accordingly, IL-15 lowered the apoptotic rate in cisplatin-treated cultured REC, and IL-15Ralpha(-/-) renal cells exhibited a higher rate of cisplatin-induced apoptosis. Furthermore, IL-15 levels negatively correlated with BUN of cisplatin-treated mice (R = -0.69, P = 0.003), suggesting that a decline in renal-derived IL-15 is detrimental to renal cell survival and kidney function during pathological stress.

Regulation of adenosine system at the onset of peritonitis.

BACKGROUND: Adenosine, a potent regulator of inflammation, is produced under stressful conditions due to degradation of ATP/ADP by the ectoenzymes CD39 and CD73. Adenosine is rapidly degraded by adenosine deaminase (ADA) or phosphorylated in the cell by adenosine kinase (AK). From four known receptors to adenosine, A(1) (A(1)R) promotes inflammation by a G(i)-coupled receptor. We have previously shown that A(1)R is up-regulated in the first hours following bacterial inoculation. The aim of the current study is to characterize the inflammatory mediators that regulate adenosine-metabolizing enzymes and A(1)R at the onset of peritonitis. METHODS: Peritonitis was induced in CD1 mice by intraperitoneal injection of Escherichia coli. TNFalpha and IL-6 levels were determined in peritoneal fluid by enzyme-linked immunosorbent assay. Adenosine-metabolizing enzymes and the A(1)R mRNA or protein levels were analyzed by quantitative PCR or by Western blot analysis, respectively. RESULTS: We found that CD39 and CD73 were up-regulated in response to bacterial stimuli (6-fold the basal levels), while AK and ADA mRNA levels were down-regulated. Cytokine production and leukocyte recruitment were enhanced (2.5-fold) by treatment with an A(1)R agonist (2-chloro-N(6)-cyclopentyladenosine, 0.1 mg/kg) and reduced (2.5-3-fold) by the A(1)R antagonist (8-cyclopentyl-1, 3-dipropylxanthine, 1 mg/kg). In contrast to lipopolysaccharide, IL-1, TNF and IFNgamma, only low IL-6 levels (0.01 ng/ml), in the presence of its soluble IL-6R (sIL-6R), were found to promote A(1)R expression on mesothelial cells. In mice, administration of neutralizing antibody to IL-6R or soluble gp130-Fc (sgp130-Fc) blocked peritoneal A(1)R up-regulation following inoculation. CONCLUSION: Bacterial products induce the production of adenosine by up-regulation of CD39 and CD73. Low IL-6-sIL-6R up-regulates the A(1)R to promote efficient inflammatory response against invading microorganisms.

Blocking adenosine A2A receptor reduces peritoneal fibrosis in two independent experimental models.

BACKGROUND: Long-term peritoneal dialysis (PD) is associated with peritoneal fibrosis and loss of function. It has been shown that activation of the adenosine A(2A) receptor (A(2A)R) promotes tissue repair, wound healing and extracellular matrix (ECM) production. We have previously shown that adenosine is a potent regulator of inflammation in the peritoneum. In the current study, we explored the role of adenosine and the A(2A)R in two experimental models. METHODS: Collagen deposition was evaluated in primary peritoneal fibroblasts following treatment with an A(2A)R agonist and antagonist. In addition, peritoneal fibrosis was induced by i.p. injection of either chlorhexidine gluconate for 2 weeks or 4.25% glucose peritoneal dialysis fluid (PDF) for 1 month. The development of fibrosis was compared between wild-type (WT) and WT mice treated with caffeine (an A(2A)R antagonist) in drinking water or between (A(2A)R(+/+)) mice and A(2A)R-deficient mice (A(2A)R(-/-)). RESULTS: Adenosine or the A(2A)R agonist CGS21680 stimulated collagen production by peritoneal fibroblasts in vitro and A(2A)R antagonists (ZM241385 and caffeine) blocked this effect. Consistent with these results, caffeine-treated WT or A(2A)R(-/-) mice had reduced submesothelial thickness, collagen deposition and mRNA levels of fibroblast-specific protein (FSP-1) and connective tissue growth factor (CTGF). In addition, treatment with caffeine in vitro and in vivo diminished A(2A)R and A(2B)R mRNA levels induced by CG or PDF while it upregulated A(1)R levels. CONCLUSION: Our data suggest that adenosine through its A(2A)R promotes peritoneal fibrosis and therefore should be considered as a target for pharmacological intervention.

Erythropoietin prevents dialysis fluid-induced apoptosis of mesothelial cells.

BACKGROUND: In peritoneal dialysis (PD)-treated patients, denudation of the mesothelium correlates with peritoneal fibrosis and vascular changes. Since recombinant human erythropoietin (rHuEPO) induces a range of cytoprotective cellular responses, rHuEPO treatment may reduce PD fluid (PDF)-induced damage. METHODS: To investigate the antiapoptotic effect and mechanism of rHuEPO in peritoneal mesothelial cells (PMCs), isolated mice PMCs were used for in vitro characterization of rHuEPO effects. To confirm the in vitro effects, active caspase-3 was analyzed in imprints of liver visceral peritoneum of mice pretreated overnight with rHuEPO (5000 U/kg intraperitoneally) and exposed to PDF (Dianeal 4.25%; Baxter Healthcare, Deerfield, Illinois, USA) for 4 hours. RESULTS: Mouse PMCs expressed EPO-receptor mRNA and protein. Short exposure to rHuEPO (5 U/mL) induced phosphorylation of JAK2, STAT5, and ERK1/2. PMCs pretreated for 1 hour with rHuEPO showed reduced PDF-induced caspase-3 activation (49.6%) and DNA fragmentation (38.4%) in comparison to cells treated by PDF alone (p < 0.05). rHuEPO treatment induced an increase in ERK1/2 phosphorylation and reduced levels of PDF-induced phospho-P38. PD98059, a specific inhibitor of ERK activation, fully blocked the protective effect of rHuEPO. In mice, rHuEPO reduced the apoptotic effect of PDF, as assessed by the level of active caspase-3. CONCLUSIONS: Our study presents new insights into clinical use of rHuEPO in the setting of PD. We found that rHuEPO provides ERK1/2-dependent protection to PMCs from PDF-induced apoptosis. The use of rHuEPO, or any of its new derivatives that do not stimulate erythropoiesis, should be considered for peritoneal preservation.

Anti-inflammatory preconditioning by agonists of adenosine A1 receptor.

BACKGROUND: Adenosine levels rise during inflammation and modulate inflammatory responses by engaging with four different G protein-coupled receptors. It is suggested that adenosine exhibits pro-inflammatory effects through its A(1) receptor (A(1)R), and anti-inflammatory effects through A(2A) receptor (A(2A)R). Therefore, understanding of the mechanisms that govern adenosine receptor regulation may advance treatment of various inflammatory disorders. We previously reported that peak A(1)R expression during leukocyte recruitment, is followed by a peak in A(2A)R during inflammation resolution. PRINCIPAL FINDINGS: Here, we examined whether A(1)R activation sequentially induces A(2A)R expression and by this reverses inflammation. The effect of adenosine on A(1)R mediated A(2A)R expression was examined in peritoneal macrophages (PMPhi) and primary peritoneal mesothelial cells (PMC) in vitro. Induction of A(2A)R was inhibited by pertussis toxin (PTX) and partly dependent on A(2A)R stimulation. Administration of A(1)R agonists to healthy mice reduced A(1)R expression and induced A(2A)R production in PMC. Mice that were preconditioned with A(1)R agonists 24 hours before E. coli inoculation exhibited decreased TNFalpha and IL-6 sera levels and reduced leukocytes recruitment. Preconditioning was blocked by pretreatment with A(1)R antagonist, as well as, or by late treatment with A(2A)R antagonist, and was absent in A(2A)R(-/-) mice. CONCLUSIONS: Our data suggest that preconditioning by an A(1)R-agonist promotes the resolution of inflammation by inducing the production of A(2A)R. Future implications may include early treatment during inflammatory disorders or pretreatment before anticipated high risk inflammatory events, such as invasive surgery and organ transplantation.

The anti-inflammatory activity of 1,25-dihydroxyvitamin D3 in macrophages.

In a previous study we demonstrated a down-regulatory effect of vitamin D active metabolite (1,25(OH)(2)D(3)) and its vitamin D(2) analog (1,24(OH)(2)D(2)) on TNFalpha expression in macrophages. We also found an inhibitory effect in the physiological concentration (10(-10)M) of 1,25(OH)(2)D(3) which was dose-dependent. This down-regulation, caused by the decrease in NFkappaB activity by 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2), was demonstrated in P388D1 cells transfected with NFkappaB reporter gene (p NFkappaB-Luc) and by EMSA. In our present study we investigated the processes leading to reduced NFkappaB activity on P388D1 cells. A decrease in nuclei NFkappaB-p65 and an increase in cytosolic NFkappaB-p65, were measured, while no changes in total NFkappaB-p65 mRNA and protein levels were observed. Simultaneously, a significant increase in both mRNA and protein levels of the NFkappaB-cytosolic inhibitor, IkappaBalpha, were determined. The half-life of IkappaBalpha-mRNA increased, with a parallel decrease in the phosphorylation of its protein, as the first step of ubiquitinization and degradation. The present results demonstrate that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) inhibit TNFalpha expression in macrophages, by increasing IkappaBalpha and decreasing NFkappaB activity. Since NFkappaB is a major transcription factor for TNFalpha and other inflammatory mediators, these findings suggest that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) may be used therapeutically as anti-inflammatory agents.

Involvement of graft-derived interleukin-15 in islet allograft rejection in mice.

The possibility that islets play a role in graft rejection during islet transplantation for type-1 diabetes patients holds promise for ex vivo islet manipulation and for specific anti-rejection therapy. Interleukin (IL)-15 is a T cell growth factor and chemoattractant that is expressed by non-T cells. Intragraft expression of IL-15 is elevated during acute rejection in patients and in mice, and systemic blockade of IL-15 in mice prolongs allograft survival. However, the source of IL-15 in these conditions is undetermined. Since epithelial cell-derived IL-15 promotes lymphocyte proliferation in culture, we sought to determine whether islet-derived IL-15 promotes rejection in mice. We designed antisense oligodeoxyribonucleotide molecules that target mouse IL-15. Uptake of FITC-labeled antisense molecules and efficacy of IL-15 inhibition in IFNgamma-stimulated islets were evaluated. Islets exhibited typical cytoplasmatic distribution of antisense molecules and produced IL-15 levels that were comparable to non-stimulated cells. Antisense-treated islet allografts, that were transplanted across multiple minor-histocompatibility-antigen mismatched strains of mice, were accepted at a higher rate than control-antisense treated islets or untreated islets (88.9% vs. 37.5% and 20%, respectively). Our results suggest that islet-derived IL-15 may be involved in acute islet allograft rejection.

Vitamin D decreases NFkappaB activity by increasing IkappaBalpha levels.

BACKGROUND: In a previous study we demonstrated the inhibitory effect of 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) and its less calcaemic analog 1,24(OH)(2)D(2) on the production of tumour necrosis factor alpha (TNFalpha) by human peritoneal macrophages. The aim of the present study is to examine whether this vitamin D inhibition of TNFalpha is mediated by its major transcription factor, nuclear factor-kappaB (NFkappaB). METHODS: Murine macrophage cells (P388D1) were incubated with 10(-7) M 1,25(OH)(2)D(3) or 1,24(OH)(2)D(2) and then stimulated with lipopolysaccharide. NFkappaB activity was assayed using a reporter gene and by electrophoretic mobility shift assay (EMSA). In addition, we evaluated mRNA and protein levels of NFkappaB-p65 and of IkappaBalpha, a potent NFkappaB inhibitor, and phosphorylated IkappaBalpha. RESULTS: Both 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) induced a 60% reduction of TNFalpha secretion. By using a reporter gene and EMSA we found that vitamin D markedly reduced NFkappaB activity. 1,25(OH)(2)D(3) or 1,24(OH)(2)D(2) decreased NFkappaB-p65 levels in the nucleus and increased NFkappaB-p65 levels in the cytosol; no changes were observed in the total levels of NFkappaB-p65 protein and mRNA. Concurrently, vitamin D induced a significant increase in mRNA and protein levels of IkappaBalpha (approximately 6.5- and 4.5-fold, respectively). Elevated levels of IkappaBalpha can be explained by the vitamin D-induced prolongation of IkappaBalpha-mRNA half-life from 110 to 190 min and by the decrease in IkappaBalpha phosphorylation. CONCLUSIONS: Vitamin D up-regulates IkappaBalpha levels by increasing mRNA stability and decreasing IkappaBalpha phosphorylation. The increase in IkappaBalpha levels reduces nuclear translocation of NFkappaB and thereby downgrades its activity. Since NFkappaB is a major transcription factor of inflammatory mediators, these findings suggest that the less-calcaemic analog, 1,24(OH)(2)D(2) may be effective as an anti-inflammatory therapeutic agent.

Involvement of adenosine in the antiinflammatory action of ketamine.

BACKGROUND: Ketamine is an anesthetic drug. Subanesthetic doses of ketamine have been shown to reduce interleukin-6 concentrations after surgery and to reduce mortality and the production of tumor necrosis factor alpha and interleukin 6 in septic animals. Similarly, adenosine was shown to reduce tumor necrosis factor alpha and mortality of septic animals. The aim of this study was to determine whether adenosine mediates the antiinflammatory effects of ketamine. METHODS: Sepsis was induced in mice by lipopolysaccharide or Escherichia coli inoculation. Leukocyte recruitment and cytokine concentrations were used as inflammation markers. Adenosine concentrations were assayed by high-performance liquid chromatography, and the involvement of adenosine in the effects of ketamine was demonstrated by adenosine receptor agonists and antagonists. RESULTS: Ketamine markedly reduced mortality from sepsis, leukocyte recruitment, and tumor necrosis factor-alpha and interleukin-6 concentrations. Ketamine administration in mice and rats was associated with a surge at 20-35 min of adenosine in serum (up to 5 microm) and peritoneal fluid. The adenosine A2A receptor agonist CGS-21680 mimicked the effect of ketamine in peritonitis, whereas the A2A receptor antagonists DMPX and ZM 241385 blocked its antiinflammatory effects. In contrast, A1 and A3 receptor antagonists had no effect. ZM 241385 reversed the beneficial effect of ketamine on survival from bacterial sepsis. CONCLUSIONS: The current data suggest that the sepsis-protective antiinflammatory effects of ketamine are mediated by the release of adenosine acting through the A2A receptor.

CD40 ligand (CD154) takes part in regulation of the transition to mononuclear cell dominance during peritonitis.

BACKGROUND: CD40 is a member of the tumor necrosis factor (TNF) family of receptors whose ligand (CD154) is found mainly on membranes of activated mononuclear cells. CD154-CD40 cross-linking is a central event in antigen presentation, B-cell activation, and regulation of cytokine and chemokine secretion from various types of cells. We have previously demonstrated in vitro the presence of CD40 on human peritoneal mesothelial cells (PMC) and have also shown that CD40 ligation synergizes with interferon-gamma (IFN-gamma) to up-regulate CC chemokine secretion from these cells. The aim of the present study was to investigate the role of CD40 ligation in leukocyte recruitment during peritonitis. METHODS: Peritonitis was induced in mice by bacterial inoculation, CD40 levels were analyzed on PMC by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. CD154 levels on leukocytes were analyzed by flow cytometry and RT-PCR. Chemokines mRNA levels were analyzed by RT-PCR. CD154 was blocked in vivo using monoclonal antibodies. Results. In mice inoculated by Staphylococcus epidermidis or Escherichia coli, CD40 in PMC increased twofold at 24 hours and CD154 was induced and reached a peak at 48 hours. In both Gram-positive and Gram-negative-peritonitis, peritoneal macrophages were the main peritoneal leukocyte population to express CD154. Similar results were observed in human subjects during peritonitis. Injection of CD154 blocking monoclonal antibody (MR1) reduced the mononuclear infiltrate by 50% and had no effect on granulocyte recruitment 48 hours after inoculation of S. epidermidis. CONCLUSION: Our data suggest that CD40 plays a significant role in the process of the mononuclear infiltration during peritonitis.

Renal cells express a functional interleukin-15 receptor.

BACKGROUND: Interleukin (IL)-15 is a pleiotropic cytokine known to be involved in graft rejection and to serve as a survival factor for leukocytes and epithelial cells, including renal cells. It utilizes a heterotrimeric receptor complex that consists of the IL-2 receptor betagammac subunits (IL-2/15Rbetagammac) and a unique high-affinity alpha-chain responsible for IL-15 specificity. METHODS: The cDNA of IL-15Ralpha main mRNA product was isolated from primary human tubular epithelial cells (TEC) and sequenced. IL-15R expression in TEC and in murine renal tissue was demonstrated using western blotting, ligand binding and flow cytometry. TEC were activated with combinations of IL-15, IL-2 and interferon-gamma (IFN-gamma), and mRNA and protein levels of IL-15R were determined. Jak-STAT tyrosine phosphorylation was assayed following IL-15 exposure. RESULTS: The full-length alpha-chain mRNA bearing exons 1-7 is expressed in TEC. IL-15Ralpha protein was detected on intact cells by flow cytometry and in extracts of human and mice renal cells using a specific anti-IL-15Ralpha antibody and by ligand-binding assay. The three subunits of the IL-15R were similarly expressed in cortex and medulla of mice kidney. Stimulation of TEC with IFN-gamma upregulated the alpha-chain while IL-2 and IL-15 had no effect on its expression. A short IL-15 stimulation of TEC induced tyrosine phosphorylation of the main IL-15 signalling molecules (Jak-1, Jak-3, STAT-3 and STAT-5). CONCLUSIONS: Our data demonstrate the presence of a functional IL-15 receptor in the kidney. Since renal cells produce IL-15, this cytokine may have an autocrine/paracrine regulatory role in the kidney.

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-alpha, interleukin-1 (IL-1), and interferon gamma (IFN-gamma), 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-alpha, IL-1 and IFN-gamma induced their secretion in a dose-dependent manner. The combination of CD40 ligation with either IL-1 or TNF-alpha increased chemokine secretion additively. IFN-gamma and CD40 ligation acted in synergy to induce the secretion of the mononuclear recruiting chemokines RANTES and MCP-1 (up to approximately 36-fold and approximately 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-alpha, was reduced in the presence of IFN-gamma. CONCLUSION: In light of our data, it is reasonable to suggest that in the mononuclear phase of peritonitis, IFN-gamma 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.

Correction of anemia in uremic mice by genetically modified peritoneal mesothelial cells.

BACKGROUND: During peritoneal dialysis, mesothelial cells become detached from the peritoneum and accumulate in the dialysate. Our aim was to evaluate the potential of peritoneal effluent (PF)-derived human peritoneal mesothelial cells (HPMC) as target for gene therapy. We used erythropoietin (EPO) as our target gene. METHODS: Various extracellular matrixes (ECM) were tested for optimal adhesion and growth of HPMC. The EPO gene was introduced to mouse peritoneal mesothelial cells (MPMC) and HPMC by transfection or retroviral transduction. EPO secretion from PMC was measured by enzyme-linked immunosorbent assay (ELISA) and by the TF-1 cell proliferation assay. We performed intraperitoneal or intramuscular transplantations of the genetically modified cells into regular or 5/6 nephrectomized Balb/c mice and nude mice. Finally, we measured serum EPO and hematocrit levels. RESULTS: ECM-coated plates provided up to sixfold increase in the efficiency of PMC isolation from PF. Gelatin coated dishes (20 microg/cm2) were found optimal for isolation of PF-HPMC. RPR-120535 liposome was found to be best for PMC transduction. In vitro studies showed EPO secretion from modified HPMC over 6 months. Intraperitoneal transplantation aided with collagen matrix was the most effective. EPO, in MPMC transplanted mice, was detected up to 3 weeks (peak at 13 +/- 1 mIU/mL), and anemia of uremic mice was corrected (35.3 +/- 0.9 mIU/mL to 41.9 +/- 1.1 mIU/mL). CONCLUSION: PF-HPMC can be considered as an appropriate target for gene therapy since these cells can be efficiently isolated, modified, and transplanted. Nevertheless, implantation techniques in the peritoneum should be directed at obtaining longer duration of transgene expression in vivo, and means should be developed for enabling regulated expression of the gene.

Induction of protein oxidation by intravenous iron in hemodialysis patients: role of inflammation.

BACKGROUND: Oxidative stress and inflammation contribute to the high prevalence and severity of atherosclerosis, infections, and beta2-microglobulin amyloidosis; and thus, to reduced survival rate and quality of life in hemodialysis (HD) patients. Inflammation induces oxidative stress by production of the oxidants: superoxide anion, hydrogen peroxide, and hypochlorite. Intravenous iron (IVIR), administered in HD patients to correct anemia, can release free iron, that may react with hydrogen peroxide to produce the strong oxidant hydroxyl radical. Inflammation-induced lipid and protein oxidation and IVIR-induced lipid oxidation were shown in HD patients. However, IVIR-induced protein oxidation and a relationship between inflammation and IVIR-induced oxidative stress have not been reported to date. METHODS: We examined the effect of IVIR administration on markers of protein oxidation in HD patients (advanced oxidation protein products [AOPPs], thiol, and dityrosine) in relation to such inflammatory markers as C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-alpha). Iron saccharate, 100 mg, was administered to 19 HD patients for 1 hour after 3.5 hours of high-flux dialysis. Blood samples were drawn pre-HD, pre-IVIR, and post-IVIR for iron, transferrin, TNF-alpha, AOPP, thiol, total antioxidant capacity (TEAC), and dityrosine levels and pre-HD for ferritin and CRP levels. RESULTS: IVIR administration induced a 37% increase in AOPP level (P < 0.001), which correlated positively with pre-HD CRP level (r = 0.72; P < 0.05) and was greater in patients with a greater pre-HD TNF-alpha level (P < 0.05). IVIR administration did not affect TEAC, thiol, dityrosine, or TNF-alpha levels. CONCLUSION: IVIR administration induced an increase in protein oxidation (AOPP levels) that was related to the degree of inflammation.

Novel role of 1,25(OH)(2)D(3) in induction of erythroid progenitor cell proliferation.

OBJECTIVE: Burst-forming unit erythroid and colony-forming unit erythroid growth in vitro is lower in studies of continuous ambulatory peritoneal dialysis patients than healthy controls. Burst-forming unit erythroid growth was potentiated by addition of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and normalized by erythropoietin (Epo) therapy, suggesting an interaction between Epo and 1,25(OH)(2)D(3) at the stem cell level. The objective of this study was to determine the mechanism by which 1,25(OH)(2)D(3) enhances the stimulatory effect of Epo on the growth of erythroid precursor cells. MATERIALS AND METHODS: We examined the effect of 1,25(OH)(2)D(3) and Epo on stem cell proliferation. Proliferation of TF1 cells of erythroid origin was measured by the XTT method, 3[H] thymidine incorporation, and cell counting by trypan blue exclusion; cord blood (CB) stem cells were counted. Epo receptor (EpoR) quantitation was evaluated by 125I-Epo binding and Scatchard analysis, immunoprecipitation, and Western blotting. Expression of EpoR mRNA was measured by reverse transcriptase polymerase chain reaction. RESULTS: The stem cell factor-dependent CB stem cells and the TF1 cells responded to Epo and 1,25(OH)(2)D(3) by increased proliferation, while their simultaneous addition potentiated cell proliferation in a synergistic manner (25.67% +/- 4.8% of Epo proliferation at day 10 for CB cells; p < 0.005). 1,25(OH)(2)D(3) produced an up-regulation of EpoR number in TF1 cells and increased the expression of EpoR mRNA (p < 0.01). CONCLUSIONS: The increase in EpoR expression induced by 1,25(OH)(2)D(3) might explain the synergistic interaction between Epo and 1,25(OH)(2)D(3) in stem cells.