Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A2B adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b+Gr1high cells in a mouse Lewis lung carcinoma (LLC) model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b+Gr1high subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b+Gr-1high subset had the highest levels of CD73 (ecto-5′-nucleotidase) expression (ΔMFI of 118.5±16.8), followed by CD11b+Gr-1int (ΔMFI of 57.9±6.8) and CD11b+Gr-1−/low (ΔMFI of 12.4±1.0). Even lower levels of CD73 expression were found on LLC tumor cells (ΔMFI of 3.2±0.2). The high levels of CD73 expression in granulocytic CD11b+Gr-1high cells correlated with high levels of ecto-5′-nucletidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation is significantly facilitated in the presence of the ecto-5′-nucletidase substrate 5′-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.
Osteopontin (OPN) is a matricellular protein with proinflammatory and profibrotic properties. Previous reports demonstrate a role for OPN in wound healing and pulmonary fibrosis. Herein, we determined if OPN levels are increased in a large cohort of systemic sclerosis (SSc) patients and if OPN contributes dermal fibrosis. Plasma OPN levels were increased in SSc patients, including patients with limited and diffuse disease, compared to healthy controls. Immunohistology demonstrated OPN on fibroblast-like and inflammatory cells in SSc skin and lesional skin from mice in the bleomycin-induced dermal fibrosis model. OPN deficient (OPN−/−) mice developed less dermal fibrosis compared to wild-type mice in the bleomycin-induced dermal fibrosis model. Additional in vivo studies demonstrated that lesional skin from OPN−/− mice had fewer Mac-3+ cells, fewer myofibroblasts, decreased TGF-beta (TGFβ) and genes in the TGFβ pathway and decreased numbers of cells expressing phosphorylated SMAD2 (pSMAD) and ERK. In vitro, OPN−/− dermal fibroblasts had decreased migratory capacity but similar phosphorylation of SMAD2 by TGFβ. Finally, TGFβ production by OPN deficient macrophages was reduced compared to wild type. These data demonstrate an important role for OPN in the development of dermal fibrosis and suggest that OPN may be a novel therapeutic target in SSc.
Gene expression can be regulated by chromatin modifiers, transcription factors and proteins that modulate DNA architecture. Among the latter, AT-hook transcription factors have emerged as multifaceted regulators that can activate or repress broad A/T-rich gene networks. Thus, alterations of AT-hook genes could affect the transcription of multiple genes causing global cell dysfunction. Here we report that targeted deletions of mouse AKNA, a hypothetical AT-hook-like transcription factor, sensitize mice to pathogen-induced inflammation and cause sudden neonatal death. Compared with wild-type littermates, AKNA KO mice appeared weak, failed to thrive and most died by postnatal day 10. Systemic inflammation, predominantly in the lungs, was accompanied by enhanced leukocyte infiltration and alveolar destruction. Cytologic, immunohistochemical and molecular analyses revealed CD11b+Gr1+ neutrophils as major tissue infiltrators, neutrophilic granule protein, cathelin-related antimicrobial peptide and S100A8/9 as neutrophil-specific chemoattracting factors, interleukin-1β and interferon-γ as proinflammatory mediators, and matrix metalloprotease 9 as a plausible proteolytic trigger of alveolar damage. AKNA KO bone marrow transplants in wild-type recipients reproduced the severe pathogen-induced reactions and confirmed the involvement of neutrophils in acute inflammation. Moreover, promoter/reporter experiments showed that AKNA could act as a gene repressor. Our results support the concept of coordinated pathway-specific gene regulation functions modulating the intensity of inflammatory responses, reveal neutrophils as prominent mediators of acute inflammation and suggest mechanisms underlying the triggering of acute and potentially fatal immune reactions.
in vivo; mouse genetics; gene network regulation; inflammation; pathogens; innate reactions
Adenosine has long been regarded as a crucial anti-inflammatory agent that protects the host from excessive damage. It has been reported to play an important role in suppressing immune activation, particularly that of T cells. However, it is a general observation that induction of T-cell activation is an efficient event despite the high adenosine levels that are often present in the affected host due to injury or stress. We report here that prior to antigenic stimulation via TCR/CD3, exposure of T cells to adenosine desensitizes adeno-sine receptors, so as to create a window of time where the T cells are insensitive to this ubiquitous suppressor. T cells from mice that were pre-exposed to this manipulation showed stronger responses to antigenic stimulation; therefore, the P1 adenosine receptor desensitization demonstrated an adjuvant-like effect. Our results suggest that adenosine receptor desensitization may be a mechanism for T cells to escape the general suppression during early points of T-cell activation and may emerge as a potential alternative for vaccine adjuvants.
Adjuvants; Immune regulation; T cells
Chronic lung diseases such as asthma, chronic obstructive pulmonary disease and interstitial lung disease are characterized by inflammation and tissue remodeling processes that compromise pulmonary function. Adenosine is produced in the inflamed and damaged lung where it plays numerous roles in the regulation of inflammation and tissue remodeling. Extracellular adenosine serves as an autocrine and paracrine signaling molecule by engaging cell surface adenosine receptors. Preclinical and cellular studies suggest that adenosine plays an anti-inflammatory role in processes associated with acute lung disease, where activation of the A2AR and A2BR have promising implications for the treatment of these disorders. In contrast, there is growing evidence that adenosine signaling through the A1R, A2BR and A3R may serve pro-inflammatory and tissue remodeling functions in chronic lung diseases. This review discusses the current progress of research efforts and clinical trials aimed at understanding the complexities of this signaling pathway as they pertain to the development of treatment strategies for chronic lung diseases.
asthma; fibrosis; emphysema; COPD; G-protein coupled receptors; adenosine receptors; inflammation
Granulomatous structures are highly dynamic during active mycobacterial infection, with accompanying responsive inflammation contributing to modulation of pathology throughout the course of disease. The heightened inflammatory response coinciding with initiation and maintenance of newly developing granulomatous structures must be limited to avoid excessive damage to bystander tissue. Modulating the cellular bioavailability of glucocorticoids by local regulation of 11βHSD enzymes within responding tissue and parenchyma would allow controlled inflammatory response during infection. Mycobacterial glycolipid trehalose 6,6′-dimycolate was used to induce strong pulmonary granulomatous inflammation immunopathology. Pulmonary corticosterone was significantly increased at days 3 and 5 after administration. An inverse relationship of 11βHSD1 and 11βHSD2 message correlated with pathology development. Immunohistochemical analysis also demonstrated that 11βHSD2 is expressed in proximity to granulomatous lesions. A role for pro-inflammatory IL-6 cytokine in regulation of converting enzymes to control the granulomatous response was confirmed using gene-disrupted IL-6–/– mice. A model is proposed linking IL-6 to endocrine-derived factors which allows modification of active corticosterone into inert 11-dehydrocorticosterone at the site of granuloma formation to limit excessive parenchymal damage.
Mycobacterial glycolipid; Granuloma; Inflammation; Lung; 11β-hydroxysteroid dehydrogenase
LEA (late embryogenesis abundant) proteins encode conserved N-terminal mitochondrial signal domains and C-terminal (A/TAEKAK) motif repeats, long-presumed to confer cell resistance to stress and death cues. This prompted the hypothesis that LEA proteins are central to mitochondria mechanisms that connect bioenergetics with cell responses to stress and death signaling. In support of this hypothesis, recent studies have demonstrated that mammalian LEA protein PRELI can act as a biochemical hub, which upholds mitochondria energy metabolism, while concomitantly promoting B cell resistance to stress and induced death. Hence, it is important to define in vivo the physiological relevance of PRELI expression.
Methods and Findings
Given the ubiquitous PRELI expression during mouse development, embryo lethality could be anticipated. Thus, conditional gene targeting was engineered by insertion of flanking loxP (flox)/Cre recognition sites on PRELI chromosome 13 (Chr 13) locus to abort its expression in a tissue-specific manner. After obtaining mouse lines with homozygous PRELI floxed alleles (PRELIf/f), the animals were crossed with CD19-driven Cre-recombinase transgenic mice to investigate whether PRELI inactivation could affect B-lymphocyte physiology and survival. Mice with homozygous B cell-specific PRELI deletion (CD19-Cre/Chr13 PRELI−/−) bred normally and did not show any signs of morbidity. Histopathology and flow cytometry analyses revealed that cell lineage identity, morphology, and viability were indistinguishable between wild type CD19-Cre/Chr13 PRELI+/+ and CD19-Cre/Chr13 PRELI−/− deficient mice. Furthermore, B cell PRELI gene expression seemed unaffected by Chr13 PRELI gene targeting. However, identification of additional PRELI loci in mouse Chr1 and Chr5 provided an explanation for the paradox between LEA-dependent cytoprotection and the seemingly futile consequences of Chr 13 PRELI gene inactivation. Importantly, PRELI expression from spare gene loci appeared ample to surmount Chr 13 PRELI gene deficiency.
These findings suggest that PRELI is a vital LEA B cell protein with failsafe genetics.
Asthma and chronic obstructive pulmonary disease (COPD) are pulmonary disorders characterized by various degrees of inflammation and tissue remodeling. Adenosine is a signaling molecule that is elevated in the lungs of patients with asthma and COPD. Adenosine elicits its actions by engaging cell surface adenosine receptors, and substantial preclinical evidence suggests that targeting these receptors will provide novel approaches for the treatment of asthma and COPD. Studies in animal models of airway disease suggest that there may be clinical benefit to the use of A1, A3 and A2B adenosine receptor antagonists in the treatment of features of asthma and/or COPD, while A2A agonists may also prove effective. Several adenosine receptor based pharmacologic agents have entered clinical development for the treatment of asthma and COPD; however, the studies have been limited and the efficacy of such approaches is not yet clear.
respiratory diseases; COPD; asthma; fibrosis; G-Protein coupled receptors; inflammation
Despite intensive research, efforts to reduce the mortality of septic patients have failed. Adenosine is a potent extracellular signaling molecule, and its levels are elevated in sepsis. Adenosine signals through G-protein–coupled receptors and can regulate the host’s response to sepsis. In this study, we studied the role of A2B adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A2B receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A2B knockout mice was associated with increased levels of inflammatory cytokines and chemokines and augmented NF-κB and p38 activation in the spleen, heart, and plasma in comparison with wild-type animals. In addition, A2B receptor knockout mice showed increased splenic apoptosis and phosphatase and tensin homolog activation and decreased Akt activation. Experiments using bone-marrow chimeras revealed that it is the lack of A2B receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A2B receptor-deficient mice. These results indicate that A2B receptor activation may offer a new therapeutic approach for the management of sepsis.
Mucin hypersecretion is a prominent feature of obstructive airway diseases such as asthma. Clara cells conditionally produce mucin in response to inflammatory signals in a process termed mucous metaplasia. This can be followed by mucin secretion stimulated by various signaling molecules. The cellular and molecular mechanisms that regulate mucin production and secretion are not well understood. Adenosine is a signaling nucleoside that has been implicated in airway diseases in which mucus obstruction is prominent. Furthermore, the A3 adenosine receptor (A3AR) is upregulated in mucin-producing goblet cells of the airway, thereby implicating it in processes involved in mucous cell biology. Here we use genetic approaches to investigate the contribution of A3AR signaling to mucus production and secretion in a mouse model of allergen-induced pulmonary disease. We found that the degree of mucin production in response to allergen is similar in wild-type and A3AR-deficient mice, and that overexpression of this receptor in Clara cells neither induces mucin production itself, nor enhances mucin production in response to allergen challenge. Collectively, these experiments demonstrate that the A3AR is neither necessary nor sufficient for mucous cell metaplasia. In contrast to the lack of effect on mucin production, agonist-induced mucin secretion was increased in goblet cells overexpressing the A3AR, and was absent in A3AR-deficient mice. Thus, the A3AR contributes to mucin secretion in allergen-induced metaplasia. Signaling through this receptor may contribute to mucus airway obstruction seen in pulmonary disorders in which adenosine levels are elevated.
mucin; mucous cell metaplasia; secretion; adenosine receptors; allergic lung disease
Pharmacologic evidence suggests that activation of A2B adenosine receptors results in proinflammatory effects relevant to the progression of asthma, a chronic lung disease associated with elevated interstitial adenosine concentrations in the lung. This concept has been challenged by the finding that genetic removal of A2B receptors leads to exaggerated responses in models of acute inflammation. Therefore, the goal of our study was to determine the effects of A2B receptor gene ablation in the context of ovalbumin-induced chronic pulmonary inflammation. We found that repetitive airway allergen challenge induced a significant increase in adenosine levels in fluid recovered by bronchoalveolar lavage. Genetic ablation of A2B receptors significantly attenuated allergen-induced chronic pulmonary inflammation, as evidenced by a reduction in the number of bronchoalveolar lavage eosinophils and in peribronchial eosinophilic infiltration. The most striking difference in the pulmonary inflammation induced in A2B receptor knockout (A2BKO) and wild-type mice was the lack of allergen-induced IL-4 release in the airways of A2BKO animals, in line with a significant reduction in IL-4 protein and mRNA levels in lung tissue. In addition, attenuation of allergen-induced transforming growth factor–β release in airways of A2BKO mice correlated with reduced airway smooth muscle and goblet cell hyperplasia/hypertrophy. In conclusion, genetic removal of A2B adenosine receptors in mice leads to inhibition of allergen-induced chronic pulmonary inflammation and airway remodeling. These findings are in agreement with previous pharmacologic studies suggesting a deleterious role for A2B receptor signaling in chronic lung inflammation.
adenosine; asthma; pulmonary inflammation; IL-4; transforming growth factor–β
Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase–deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883–treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy.
Adenosine is a signaling nucleoside that has been implicated in the regulation of asthma and chronic obstructive pulmonary disease. Adenosine signaling can serve both pro- and anti-inflammatory functions in tissues and cells. In this study we examined the contribution of A1 adenosine receptor (A1AR) signaling to the pulmonary inflammation and injury seen in adenosine deaminase–deficient (ADA-deficient) mice, which exhibit elevated adenosine levels. Experiments revealed that transcript levels for the A1AR were elevated in the lungs of ADA-deficient mice, in which expression was localized predominantly to alveolar macrophages. Genetic removal of the A1AR from ADA-deficient mice resulted in enhanced pulmonary inflammation along with increased mucus metaplasia and alveolar destruction. These changes were associated with the exaggerated expression of the Th2 cytokines IL-4 and IL-13 in the lungs, together with increased expression of chemokines and matrix metalloproteinases. These findings demonstrate that the A1AR plays an anti-inflammatory and/or protective role in the pulmonary phenotype seen in ADA-deficient mice, which suggests that A1AR signaling may serve to regulate the severity of pulmonary inflammation and remodeling seen in chronic lung diseases by controlling the levels of important mediators of pulmonary inflammation and damage.
Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility.
The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism.
Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol–modified ADA (PEG–ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG–ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation.
Main Outcome Measures
Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays.
We found that lowering adenosine levels in penile tissues by PEG–ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG–ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism.
Our studies have identified that PEG–ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation seen in two independent priapic animal models, and suggested its therapeutic possibility in men suffering from priapism.
Adenosine Signaling; Priapism; Novel Therapies; Pharmacologic Treatment of Priapism
We tested the impact of A1 adenosine receptor (AR) deletion on injury and oxidant damage in mouse hearts subjected to 25-min ischemia/45-min reperfusion (I/R). Wild-type hearts recovered ∼50% of contractile function and released 8.2 ± 0.7 IU/g of lactate dehydrogenase (LDH). A1AR deletion worsened dysfunction and LDH efflux (15.2 ± 2.6 IU/g). Tissue cholesterol and native cholesteryl esters were unchanged, whereas cholesteryl ester–derived lipid hydroperoxides and hydroxides (CE-O(O)H; a marker of lipid oxidation) increased threefold, and α-tocopherylquinone [α-TQ; oxidation product of α-tocopherol (α-TOH)] increased sixfold. Elevations in α-TQ were augmented by two- to threefold by A1AR deletion, whereas CE-O(O)H was unaltered. A1AR deletion also decreased glutathione redox status ([GSH]/[GSSG + GSH]) and enhanced expression of the antioxidant response element heme oxygenase-1 (HO-1) during I/R: fourfold elevations in HO-1 mRNA and activity were doubled by A1AR deletion. Broad-spectrum AR agonism (10 μM 2-chloroadenosine; 2-CAD) countered effects of A1AR deletion on oxidant damage, HO-1, and tissue injury, indicating that additional ARs (A2A, A2B, and/or A3) can mediate similar actions. These data reveal that local adenosine engages A1ARs during I/R to limit oxidant damage and enhance outcome selectively. Control of α-TOH/α-TQ levels may contribute to A1AR-dependent cardioprotection. Antioxid. Redox Signal. 11, 2641–2650.
Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1lAC) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1lAC vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1lAC kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5′ flanking region in Dot1lAC vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1lAC mice and in patients with diabetic nephropathy.
Adenosine deaminase (ADA) is a purine catabolic enzyme that manages levels of the biologically active purines adenosine and 2′-deoxyadenosine in tissues and cells. ADA-deficient mice die at 3 wk of age from severe respiratory distress. This phenotype is progressive and is linked to perturbations in pulmonary purine metabolism. The inflammatory changes found in the lungs of ADA-deficient mice included an accumulation of activated alveolar macrophages and eosinophils. These changes were accompanied by a pronounced enlargement of alveolar spaces and increases in mucus production in the bronchial airways. The alveolar enlargement was found to be due in part to abnormal alveogenesis. Lowering adenosine and 2′-deoxyadenosine levels using ADA enzyme therapy decreased the pulmonary eosinophilia and resolved many of the lung histopathologies. In addition, genetically restoring ADA to the forestomach of otherwise ADA-deficient mice prevented adenine metabolic disturbances as well as lung inflammation and damage. These data suggest that disturbances in purinergic signaling mediate the lung inflammation and damage seen in ADA-deficient mice.
eosinophil; asthma; emphysema; alveolar macrophage; adenosine deaminase
Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated.
The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A2BR are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A2BR in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A2BR, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A2BR on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators.
These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.
Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A3 adenosine receptor (A3R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A3R-deficient mice exhibit enhanced pulmonary inflammation that included an increase in eosinophils. Accordingly, there was a selective up-regulation of eosinophil-related chemokines and cytokines in the lungs of A3R-deficient mice exposed to bleomycin. This increase in eosinophil numbers was accompanied by a decrease in the amount of extracellular eosinophil peroxidase activity in lavage fluid from A3R-deficient mice exposed to bleomycin, an observation suggesting that the A3R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A3R-deficient mice treated with bleomycin, there was little difference in the degree of pulmonary fibrosis. Examination of fibrotic mediators demonstrated enhanced transforming growth factor (TGF)-β1 expression, but not a concomitant increase in TGF-β1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-β1, in alveolar macrophages and airway mast cells in the lungs of A3R-deficient mice. Together, these results suggest that the A3R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.
pulmonary fibrosis; adenosine receptors; inflammation; eosinophil; extracellular matrix
Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture (hu/moFTOC) to study ADA-deficient human thymocyte development in an “in vivo-like” environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular deoxyadenosine triphosphate (dATP). Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.
human; immunodeficiency diseases; apoptosis; thymus
Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.
ADA deficiency; human thymocyte development; deoxynucleoside kinases; apoptosis
After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naïve T cells with antigen-loaded dendritic cells. Here we show that CD73 (ecto-5′-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73-/- mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared to wild type mice 24 hours after LPS administration. Migration rates of cd73+/+ and cd73-/- lymphocytes into lymph nodes of wild type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A2B receptor is a likely target of CD73-generated adenosine, as it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up regulated by TNFα. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73-/- mice is largely normalized by pretreatment with the selective A2B receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.
Rodent; Cell Trafficking; Spleen and lymph nodes; Inflammation
Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.