Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA–/–) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA–/– mice. T cell apoptosis was abundant in thymi of ADA–/– mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA–/– mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA–/– T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA–/– mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.

J. Clin. Invest. 108:131–141 (2001). DOI:10.1172/JCI200110360.

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The hexahistidine-tagged mouse P2X1 receptor (H-mP2X1R), an ATP-gated ion channel receptor, was expressed in a baculovirus system using the pAcHLT-B transfer vector containing a hexahistidine tag. Both widely used denaturing (8M urea) and nondenaturing (such as 1% Triton X-100) solubilization conditions were compared, resulting in about 30% of the P2X1 receptors being solubilized (S1). However, at pH 13 most of the H-mP2X1R from the initially insoluble pellet fraction was solubilized (S2) and remained in the soluble fraction (S3) after dialyzing against a nondenaturing buffer. H-mP2X1Rs were purified sequentially through cobalt and ATP affinity columns. Receptors purified from S3 had higher purity than those from S1 (i.e., ~90% vs. ~75%). Circular dichroism spectra indicated identical protein secondary structures of the receptors from both sources. Autoradiographic data showed that the purified receptors from S3 had higher affinity for 8-azido-ATP-γ-32P than the receptors from S1. The binding of 8-azido-ATP-γ-32P to H-mP2X1R was inhibited by ATP-γ-S, α,β-me-ATP, and PPADS, but not by a nucleoside analog (N6-methyl-2′-deoxy-adenosine). In the presence of 2 mM Ca2+ or Mg2+ the binding was increased, but not when using a partially purified receptor fraction, in which unidentified proteins bound 8-azido-ATP-γ-32P or were phosphorylated at 4°C in the presence of 2 mM Mg2+. These data suggest that the decrease in potency of ATP in the presence of Ca2+ and Mg2+, as observed in functional studies, is not due to a direct effect of the cations on the binding of ATP to the receptor. Both cyanogen bromide and hydroxylamine cleavage further confirmed the peptide structure of the purified H-mP2X1R. Autoradiographic analysis of the cleavage products showed that 8-azido-ATP-γ-32P was crosslinked to the carboxyl side of the extracellular domain of the receptor.

Hypoxia inducible factor –1α (HIF-1α) plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that HIF-1α gene deficiency caused abnormal B cell development and autoimmunity. Here we show that HIF-1α-enabled glycolysis during B cell development is required in a developmental stage-specific manner. Supporting this conclusion are observations that the glycolytic pathway in HIF-1α deficient B220+ bone marrow cells is much less functionally effective than in wild-type control cells. The expression of genes encoding the glucose transporters and the key glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3 (pfkfb3), was greatly reduced in HIF-1α deficient cells. The compensatory adaptation to the defect of glycolysis was reflected in higher levels of expression of respiratory-chain related genes and TCA-cycle related genes in HIF-1α deficient cells than in wild-type cells. In agreement with these findings, HIF-1α deficient cells utilized pyruvate more efficiently than wild-type cells. The key role of HIF-1α-enabled glycolysis in bone marrow B cells was also demonstrated by glucose deprivation during in vitro bone marrow cell culture and by using a glycolysis inhibitor in the bone marrow cell culture. Taken together, these findings indicate that glucose dependency differs at different B cell developmental stages and that HIF-1α plays important role in the B cell development.

Toll-like receptors (TLRs) recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C) induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-inducible factor 1 (HIF-1) regulates several cellular processes, including apoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific I.3 isoform of HIF-1α and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF). Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1α. However, the transfection of I.3 isoform of hif-1α in LNCaP cells allows poly(I:C)-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1α expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.

Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene deleted mice to low oxygen containing inspired gas mixture allowed the investigation of the mechanism that controls inflammatory liver damage and protects the liver using a mouse model of T cell-mediated viral and autoimmune hepatitis. We tested the hypothesis that the inflammatory tissue damage-associated hypoxia and extracellular adenosine → A2AR signaling plays an important role in the physiological anti-inflammatory mechanism that limits liver damage during fulminant hepatitis. After induction of T cell-mediated hepatitis, mice were kept in modular chambers either under normoxic (21% oxygen) or hypoxic (10% oxygen) conditions for 8 h. It was shown that the whole body exposure to hypoxic atmosphere caused tissue hypoxia in healthy animals as evidenced by a decrease in the arterial blood oxygen tension and increase of the plasma adenosine concentration (P < 0.05). This “hypoxic” treatment resulted in significantly reduced hepatocellular damage and attenuated levels of serum cytokines in mice with acute liver inflammation. The anti-inflammatory effects of hypoxia were not observed in the absence of A2AR in studies of A2AR gene-deficient mice or when A2AR have been pharmacologically antagonized with synthetic antagonist. The presented data demonstrate that total body hypoxia-triggered pathway provides protection in acute hepatitis and that hypoxia (upstream) and A2AR (downstream) function in the same immunosuppressive and liver tissue-protecting pathway.

Background

Sepsis patients may die either from an overwhelming systemic immune response and/or from an immunoparalysis-associated lack of anti-bacterial immune defence. We hypothesized that bacterial superantigen-activated T cells may be prevented from contribution into anti-bacterial response due to the inhibition of their effector functions by the hypoxia inducible transcription factor (HIF-1α) in inflamed and hypoxic areas.

Methodology/Principal Findings

Using the Cre-lox-P-system we generated mice with a T–cell targeted deletion of the HIF-1α gene and analysed them in an in vivo model of bacterial sepsis. We show that deletion of the HIF-1α gene leads to higher levels of pro-inflammatory cytokines, stronger anti-bacterial effects and much better survival of mice. These effects can be at least partially explained by significantly increased NF-κB activation in TCR activated HIF-1 α deficient T cells.

Conclusions/Significance

T cells can be recruited to powerfully contribute to anti-bacterial response if they are relieved from inhibition by HIF-1α in inflamed and hypoxic areas. Our experiments uncovered the before unappreciated reserve of anti-bacterial capacity of T cells and suggest novel therapeutic anti-pathogen strategies based on targeted deletion or inhibition of HIF-1 α in T cells.

Acute respiratory distress syndrome (ARDS) usually requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the recent discovery of a critical tissue-protecting mechanism predicts that administration of oxygen to ARDS patients with uncontrolled pulmonary inflammation also may have dangerous side effects. Oxygenation may weaken the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism and thereby further exacerbate lung injury. Here we report experiments with wild-type and adenosine A2AR-deficient mice that confirm the predicted effects of oxygen. These results also suggest the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2AR-mediated lung tissue-protecting pathway. We show that this potential complication of clinically widely used oxygenation procedures could be completely prevented by intratracheal injection of a selective A2AR agonist to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous adenosine. The identification of a major iatrogenic complication of oxygen therapy in conditions of acute lung inflammation attracts attention to the need for clinical and epidemiological studies of ARDS patients who require oxygen therapy. It is proposed that oxygen therapy in patients with ARDS and other causes of lung inflammation should be combined with anti-inflammatory measures, e.g., with inhalative application of A2AR agonists. The reported observations may also answer the long-standing question as to why the lungs are the most susceptible to inflammatory injury and why lung failure usually precedes multiple organ failure.

A mouse model suggests that oxygen therapy may exacerbate lung injury by weakening the anti-inflammatory mechanisms driven by hypoxia.