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.

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.

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.

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.