| Normally, the pH value of the organ and tissues of our body ranges from 7.2 to 7.4. In many pathological conditions, peripheral organizations typically present acidification, for example acute asthma, tumor, inflammation, autoimmune disease, etc. So, microenvironment acidification plays an important role in immune system.As one of the most important groups of immune cells, macrophages play a variety of roles in inflammation, tumor immunity, autoimmune disease, et al. When pathogens invade into the body, macrophages are one of the first troops migrating to the infected region to clear the pathogens. In addition, macrophages also collaborate with T and B cells, through both cell-to-cell interactions and fluid phase-mediated mechanisms, based on the release of cytokines, chemokines, enzymes, arachidonic acid metabolites, and reactive radicals.Acid-sensing ion channels (ASICs) belong to the degenerin/epithelial Na+ channel (DEG/ENaC) superfamily. ASICs are widely expressed in the central nervous system (CNS) and peripheral sensory neurons. Activation of these channels plays an important role in physiological and pathological processes such as nociception, mechanosensation, and acidosis-mediated neuronal injury. Recent studies have reported that ASICs are also expressed in T cells, B cells and DC and have important effects on their physiological and pathological functions. This study focus on:1). extracellular acidosis affect the function of bone marrow derived macrophages (BMM); 2). the role of ASICs in the effect of acidosis on BMM function.1. Extracellular acidosis affects the function of BMMs(1) Extracellular acidosis promotes endocytosis of BMMs(1) Extracellular acidosis promotes BMM s'pinocytosis of FITC-dextran: FITC-dextran was used to detect the endocytosis of BMM at acidic environment (pH 6.5,7% CO2) or normal environment (pH 7.3,7% CO2) separately. First, experiments were carried out using different concentrations of FITC-dextran and then for different time. The result showed that extracellular acidosis increased the uptake of FITC-dextran by BMM. And the accumulation of FITC-dextran in BMM was in a time dependent pattern either at pH 7.3 or at pH 6.5. The uptake of FITC-dextran was not enhanced at 5 or 15 min, but was markedly increased after 30 min of incubation.(2) Extracellular acidosis promotes BMM s'phygocytosis of IgG-coated latex: Then, endocytosis of IgG-coated latex was detected. Within 5 min of exposure to IgG-coated latex beads, BMM cultured in pH 6.5 showed substantial phagocytosis with most cells engulfed over 6 beads, while most BMMs cultured in pH 7.3 engulfed 0-2 beads. The phagocytic index of BMM in pH 7.3 was 55.8% lower than in pH 6.5 (n= 6, P<0.05). These results indicated that extracellular acidosis promotes endocytosis of BMMs(2) Extracellular acidosis up-regulates the expression of CD80, CD86 and MHC-Ⅱof BMMsIn our study, BMM were cultured for 3 h in pH 7.3,5% CO2, or pH 6.5,7% CO2 respectively, and the expression of CD80, CD86 and MHC-II were detected. The results certified that CD80, CD86 and MHC-Ⅱon BMM were up-regulated after exposure to pH 6.5 for 3 h. These results suggested that Extracellular acidosis up-regulates the expression of CD80, CD86 and MHC-Ⅱof BMM.(3) Extracellular acidosis promotes the secretion of IL-10 by BMMs, but does not affect the secretion of TNF-aAfter BMM were cultured in pH 7.3 or pH 6.5 for 3 h, the supernant was collected to detect the cytokine secretion. As a result, IL-10 secretion was higher in pH 6.5 than in pH 7.3. However the secretion of TNF-αwas not affected.2. Extracellular acidosis affects the function of BMMs via ASICs(1) ASICs are expressed in BMMASICs mRNA and protein expression was detected by way of RT-PCR and western blot. And the distribution of ASICs was detcted through immunocytofluorescence. The results showed that ASIC1 and ASIC3 were mainly expressed in the cytoplasm of BMM, while ASIC2 was not expressed.(2) Extracellular acidosis promotes pinocytosis of BMMs via ASICsTo confirm weather ASICs participated in the up-regulation of endocytosis of BMM, amiloride was used which was a non-specific blocker for ASICs. Before the exposure to pH 6.5 for 3 h at 37℃, BMMs were incubated in pH 7.3 for 30 min at 37℃in the presence of amiloride (100μM), and then collected the cells for further detection. As expected, the up-regulation of endocytosis of FITC-dextran, stimulated by acidosis was significantly inhibited by amiloride. These results suggested that ASICs are involved in the upregulation of endocytosis of BMM induced by acidosis.(3) Extracellular acidosis up-regulates the expression of CD80, CD86 and MHC-II of BMMs via ASICsTo confirm weather ASICs participated in the up-regulation of surface molecule expression of BMMs, we used. Before the exposure to pH 6.5 for 3 h at 37℃, BMMs were incubated in pH 7.3 for 30 min at 37℃in the presence of amiloride (100μM), and then collected the cells for further detection. As expected, the up-regulation of CD80, CD86 and MHC-II stimulated by acidosis was significantly inhibited by amiloride. These results suggested that ASICs were involved in the maturation of BMMs induced by acidosis. (4) NSAID inhibit BMMs maturation induced by acidosisSome researches have certified that NSAIDs could suppress ASICs current on neurons. So we investigated the effect of NSAIDs on BMMs maturation induced by acidosis. Before exposuring to pH 6.5 for 3 h, BMMs were pre-incubated with ibuprofen (200μM) or diclofenac (200μM) in pH 7.3 for 30 min at 37℃. As a result, ibuprofen and diclofenac significantly abrogated the up-regulation of CD80, CD86 and MHC-II by acidosis. This result indicated that NSAID can inhibit the maturation of BMM stimulated by acidosis.Conclusions1. Extracellular acidosis promotes endocytosis and surface molecular expression of BMMs and increases the secretion of cytokine IL-10;2. BMMs express ASIC1 and ASIC3;3. ASICs participate in the up-regulation of endocytosis and the cell surface molecules induced by extracellular acidosis;... |
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