Acid-sensing Ion Channels Contribute To The Effect Of Acidosis On The Function Of Dendritic Cells

pH value is an important physiological indicator of internal environment homeostasis. Under normal conditions, pH values in Peripheral blood, tissues and organs are usually 7.2-7.4. However, in some diseases, such as inflammation, autoimmune disease and tumors, the values of extracellular pH are much lower than that of normal tissues, and low pH values play an important role in developments of these diseases. Therefore, it is necessary to clarify the effects of extracellular pH on immune cells and their function.As the most potent antigen-presentation cells (APCs) of the immune system, dendritic cells (DCs) are specialized to capture, process and present antigens to T lymphocytes to initiate Ag-specific T cell response. Some surface molecules of DCs are involved in the process of antigen presentation, such as MHC, costimulatory molecules CD80 and CD86. Many reports have showed that DCs play an important role in the developments of inflammatory diseases, tumors and autoimmune diseases. Although it has been reported that acidosis improves the uptake of antigens and MHC I-restricted presentation by DCs, the molecular mechanism through which DCs are regulated by acidosis has remained unknown.Acid-sensing ion channels (ASICs) represent an H+-gated subgroup of the degenerin/epithelial Na+ channel (DEG/ENaC) family of cation channels and are activated by extracellular protons. They are widely expressed in peripheral sensory neurons and in the neurons of the central nervous system. ASICs are involved in nociception, mechanosensation, learning and memory and formation of sour taste. Recent studies have reported that ASICs are also expressed in non neuronal cells and have important effects on their physiological and pathological function.We proposed that ASICs served as sensors for extracellular acidosis and mediate the responses of DCs to acidosis. In the present study, we investigated the expression of ASICs in mouse DCs and the role of ASICs in the effect of acidosis on DCs function.1. ASICs are functional expressed in DCsWe first performed RT-PCR and western blot to detect the expression of ASIC1,ASIC2 and ASIC3 at mRNA and protein levels, respectively. As a result, mRNAs and proteins for all the three ASICs were detected in DCs. Furthermore, we used immunocytochemistry and electron microscope to determine cellular and subcellular distributions of ASICs in DCs. The results showed that ASIC1 and ASIC3 were expressed in endoplasmic reticulum and mitochondria, while ASIC2 existed in cell membrane.To determine whether the expression of the ASIC proteins in DCs has function, we performed whole-cell patch clamp recordings. In the 85%(n=36/42) of the recorded DCs, the rapid drop of extracellular pH elicited inward ASICs-like currents, indicating the presence of functional ASIC channels. Usually, this transient inward current contained two phases:early fast and late slow inactivation. The mean amplitude of ASICs-like currents induced by pH 6.0 was 329.63±52.36 pA. The ASICs-like currents in DCs were reversibly inhibited by amiloride (100μM), a blocker of ASICs (n=6, P<0.05). The response of DCs to the drop of pH was in a dose-dependent manner. The threshold extracellular pH to elicit the inward current was about 7.0 and the maximum response appeared at 5.0. The extracellular pH producing 50% effect (pHso) was 6.08±0.05, and the Hill coefficient was 1.03±0.07 (n=6). 2. Acidosis induced the currents in DCs via ASICsAs a non-selective cation channel, TRPV1 also can evoke membrane currents at low extracellular pH (<6.0). Thereby, we examined the effect of Capsaicin (Cap), a selective agonist of TRPV1, on DCs with whole cell patch clamp technique. As a result,10μM Cap significantly evoked an inward current in cortex neurons. However,10μM Cap, even 100μM Cap failed to induce any inward currents in DCs. Moreover, we did not detect the TRPV1 mRNA expressing in DCs by PT-PCR. These results suggest that, the inward currents induced by decrease of extracellular pH are mediated by ASICs and not TRPV1 channels in DCs.Previous studies have demonstrated that NSAIDs significantly inhibited ASICs currents in hippocampus and sensory neurons. To investigate the effect of NSAIDs on ASICs in DCs, we tested the responses of ASICs currents to NSAIDs, ibuprofen or diclofenac. Extracellular application of 200μM ibuprofen significantly reduced ASICs currents from 364.17±57.15 pA to 176.07±28.77 pA. The inhibitory percentage was 51.49±3.75% and the inhibition could be reversed by extensive washes with bath solution. Similarly, diclofenac (200μM) also significantly reduced ASICs currents from 353.97±51.58 pA to 166.7±18.57 pA. The inhibition was also reversible and the inhibitory percentage was 51.67±8.3%. These data suggested that ASICs currents in DCs are also sensitive to NSAIDs.3. ASICs are involved in the up-regulation of surface molecules CD11c, MHCⅡ, CD80 and CD86 induced by acidosis in DCsWe incubated DCs in pH7.3 for 30 min at 37℃in the presence of amiloride (100μM), a blocker of ASICs, before exposuring to pH6.5 for 4 h at 37℃, and examined the MFI of surface molecules CD11c, MHCⅡ, CD80 and CD86 by flow cytometry. The up-regulations of CD11c, MHC classⅡ, CD80 and CD86 expressing stimulated by acidosis were significantly inhibited by amiloride, suggesting that ASICs are involved in up-regulation of surface molecules CD11c, MHCⅡ, CD80 and CD86 expressings in DCs induced by acidosis.Since we have confirmed that NSAIDs can inhibit the function of ASICs in DCs, we next examined the effect of NASIDs on up-regulation of CD11c, MHCⅡ, CD80 and CD86 expressing in DCs induced by acidosis. DCs were pre-incubated with Ibuprofen (200μM) or Diclofenac (200μM) in pH7.3 for 30 min at 37℃, and then exposured to pH6.5 for 4 h. As a result, the expressings of CD11c, MHCⅡ, CD80 and CD86 were significantly lower than that of pH6.5 treated alone, indicating that NASIDs can inhibited the up-regulation of CD11c, MHCⅡ, CD80 and CD86 expressings induced by acidosis.Conclusions1. ASICs are functional expressed in DCs.2. ASICs mediated the up-regulation of cell surface molecules CD11c, MHC classⅡ, CD80 and CD86 expressings induced by acidosis.3. ASICs may be a new therapeutic target for regulating function of DCs in inflammation, tumors and autoimmune disease.