Effects And Mechanisms Of MRP8/14 In The Regulation Of Phagocytosis In Macrophages And Apoptosis In Alveolar Epithelial Cells

Lung infection has become a great health problem on a global scale which imposes huge socioeconomic burdens on the affected individuals,families and societies.Meanwhile,effective treatment is more challenging due to the emergence of microbial resistance to antibiotics.The two major components of lung tissue,macrophage and alveolar epithelial cells,play an important role in the lung infection.They are closely associated with the outcome and prognosis of lung infection.Macrophage cells are not only involved in the phagocytosis of pathogenic microorganisms,but also participate in the inflammatory injury and resolving of inflammation.Alveolar type Ⅱ epithelial cells(AT-Ⅱ)can prevent pneumonedema and repair the injured lung epithelial barrier.AIso they participate in the formation of first defending line.Apoptosis of alveolar epithelial cells can increase the permeability of lung epithelial barrier,which is definitely an important physiopathologic mechanism in the occurrence and development of acute lung injury(ALI)induced by lung infection.Therefore.further research about the functional changes and the mechanisms in macrophage and AT-Ⅱ will not only help us to understand the mechanisms of resisting lung infection,but also shed new light on the physiopathologic mechanisms of ALI induced by infection.All of these work will provide a new idea of treating lung infection for us.Myeloid-related protein 8/14(MRP8/14)is a heterodimer formed by MRP8 and MRP 14 and an important member of S100 calcium-binding protein family.MRP8 and MRP 14 are highly expressed and accounting for up to 40%total cytosolic proteins in neutrophil.Meanwhile these proteins can also be induced by special stimulation in other cell types such as keratinocytes.mature macrophages,fibroblasts and vascular endothelial cells.MRP8 and MRP 14 have two sites to bind calcium respectively and their biological functions can be regulated by calcium.MRP8 and MRP 14 can form homodimers,heterodimers or even more complex polymer.Heterodimers are different from homodimers in certain biological functions.Both of MRP8 and MRP 14 have many amino acid sites which can be oxidized to influence their functions.All the properties of the proteins mentioned above make sure that MRP8 and MRP 14 have complicated and extensive biological functions.MRP8 and MRP 14 are involved in the phosphorylation of proteins,cellular cytoskeletal rearrangement,metabolism of arachidonic acid and regulation of neutrophilic NADPH oxidase.MRP8/14 can be released from activated or necrotic neutrophils and monocytes to regulate inflammatory reaction and immunoreaction and becomes danger-associated molecular patterns(DAMPs).which exerts a great influence on the pathophysiology of infectious and inflammatory diseases.The levels of MRP8 and MRP 14 from serum and bronchoalveolar lavage fluid may increase obviously if patients were suffered from pneumonia induced by diverse pathogenic microorganisms.Some studies have showed that MRP8/14 contributes to protective immunity in pneumonia induced by Klebsiella pneumoniae.Acinetobacter baumannii,Candida albicans.However some other researchers have identified that MRP8/14 can exacerbate pathological damage of the sepsis and increase the mortality of mouse suffered from pneumococcal pneumonia.These two contradictory ideas strongly suggest that MRP8/14 may both have the positive and negative biological effects on the lung infection at the same time.The positive effects of MRP8/14 on lung infection may be associated with its various antibacterial actions.MRP8/14 can chelate Zn2+and Mn2+to disturb the metabolism of microorganism,which inhibits the growth of microorganism.It can also increase bactericidal activity of human neutrophils by enhancing phagocytosis in a signal transduction pathway.Besides,MRP8/14 can regulate the chemotactic movement of neutrophils,B cells and T cells,which can promote the accumulation of immunocytes toward infection sites.Phagocytosis of bacterial in macrophage is an important defence mechanism for lung infection.But,until now,the regulation of phagocytosis and its mechanism in macrophage induced by MRP8/14 still need further investigation.Exploration about this question will help us to understand the positive effects of MRP8/14 on lung infection better.The negative effects of MRP8/14 on lung infection may be acssociated with its apoptosis-promoting actions.It has been identified that MRP8/14 can lead to apoptosis of human microvascular endothelial cells(HMECs)via caspase-dependent or-independent pathways.What’s more,apoptosis of AT-II plays an important role in the occurrence and development of ALI induced by lung infection.Recent studies have shown that,MRP8/14 can regulate diverse functions of air way epithelium and alveolar epithelial cells.For instance,MRP8/14 can induce mucin secretion of airway epithelium or increase expression and release of interleukin-8(IL-8)in airway epithelium and alveolar epithelial cells.However,the effect of MRP8/14 on the apoptosis of AT-II is still not clear.So,further research about this issue will help us to understand the negative effects of MRP8/14 on the lung infection more deeply.MRP8/14 can active several intracellular signal transduction pathways as DAMPs,including nuclear transcription factor-κB(NF-κB),mitogen-activated protein kinases(MAPKs),Janus protein tyrosine kinase(JAK)/signal transducer and activator transcription(STAT).MAPKs signal pathways have closely correlations with phagocytosis of immunocyte.Extracellular signal-regulated kinase(ERK)participates in the enhanced phagocytosis in neutrophil induced by MRP 14.And in macrophage,lipopolysaccharide(LPS)increased the phagocyosis of E.coli in a p38-dependent pathway.NF-κB is associated with apoptosis of cells and its activation plays a key role in the apoptosis of AT-Ⅱ induced by LPS.All these studies have shown that MAPKs and NF-κB pathways play an important role in regulating the cell functions of macrophage and alveolar epithelium.However,the role of MAPKs and NF-κB pathway in the regulation of cell functions induced by MRP8/14 in macrophage and AT-II still need to be further studied.In conclusion,further research about effects of MRP8/14 on the regulation of phagocytosis in macrophage and apoptosis in alveolar epithelial cells,as well as explanation about the mechanism in these processes,will help us to understand the positive and negative effects of MRP8/14 on lung infection more comprehensively.All of these work may contribute to develop a new way of preventing and treating lung infection.Based on the above knowledge,the purposes of this study are as follows:1.To observe the effect of MRP8/14 on engulfment in macrophage.Raw264.7 macrophage cells were stimulated with MRP8/14 for different time and then infected with EGFP-E.coli at 37℃.The bacterial phagocytosis was observed under Zeiss fluorescence microscope,or quantitatively the mean fluorescence of cell lysate was detected by Spectra Max M5.Macrophage with or without stimulation were also infected by EGFP-E.coli at 4℃ to assess the adhesion between bacterial and macrophage.Raw264.7 cells were treated with heat-inactivated MRP8/14 and then internalization of EGFP-E.coli was analyzed as previous to rule out the influence of contaminating LPS on phagocytosis.2.To determine the effects of MRP8/14 on the MAPKs signal pathways.Phosphorylation of MAPK subtypes,p38,ERK1/2 and c-Jun N-terminal kinase(JNK)were detected by western blot.3.To verify the role of MAPKs in the phagocytosis of macrophage induced by MRP8/14.Raw264.7 cells administered with MRP8/14 were pretreated with p38 inhibitor SB203580,ERK inhibitor PD98059 or JNK inhibitor SP600125,and then the phagocytic capacity of every group was compared.4.To explore effects of calcium in the medium on the phagocytosis and MAPKs of macrophage treated with MRP8/14.Raw264.7 cells were treated with MRP8/14 in the presence of calcium with or without EGTA and then the phagocytic capacity and phosphorylation of p38 subtypes of all groups were compared.5.To investigate the role of toll-like receptor 4(TLR4)and receptor for advanced glycation endproducts(RAGE)in MRP8/14 modifying phagocytosis and MAPKs of macrophage.Bone marrow-derived macrophages(BMDMs)from WT,TLR4-/-,and RAGE-/-mice were stimulated with MRP8/14,and then phagocytic capacity and phosphorylation of MAPKs subtypes of all groups were compared.6.To investigate the effects of MRP8/14 on the survival and apoptosis of A549 cells.A549 cells were stimulated with different doses of MRP8/14 or treated with MRP8/14 for different times.The effect of MRP8/14 on the viability of A549 cell was determined by CCK-8 assay.The apoptotic rates were tested by flow cytometry with Annexin V/PI staining.7.To uncover the activation of NF-κB/p65 induced by MRP8/14 in A549 cells.The nuclear translocation of NF-κB/p65 was detected by Western blot and indirect immunofluorescence.Besides,the phosphorylation levels of NF-κB/p65 were determined by Western blot.8.To illuminate the role of p65 in the apoptosis of A549 cells induced by MRP8/14.A549 cells administered with MRP8/14 were pretreated with or without Bay11-7082,a specific inhibitor of NF-κB,and then differences of apoptotic rates in all groups were detected.The results were as follows:1.Stronger green fluorescence was observed by Zeiss microscope and higher mean fluorescence was determined by Spectra Max M5 in the RAW264.7 cells treated with MRP8/14 for 12 and 24 hours compared with the cells without stimulation.Comparing the macrophage treated with and without MRP8/14,no differences in intracellular green fluorescence and mean fluorescence were observed when macrophages were infected with EGFP-E.coli at 4℃.Mean fluorescence of macrophage stimulated with heat-inactivated MRP8/14 was no different from the mean fluorescence of no-stimulating group.2.MRP8/14 induced the phosphorylation and activation of MAPKs subtypes.p38.ERK1/2 and JNK.3.Specific inhibition of p38 activation with SB203580 completely abrogated MRP8/14-induced phagocytosis of EGFP-E.coli.However.neither inhibition of JNK nor ERK altered the phagocytosis of EGFP-E.coli.4.The presence of escalating dose of EGTA abrogated the increased phagocytosis and phosphorylation of p38 in MRP8/14 treated Raw264.7 cells.5.After stimulation with MRP8/14.BMDMs from WT and RAGE-deficient mice have significant increased phagocytosis of EGFP-E.coli and phosphorylation of p38.However,there was no significant difference in phagocytosis and phosphorylation of p38 in BMDMs from TLR4-deficient mice after stimulation.6.The viability of the A549 cell was affected by MRP8/14 in a dose-and time-dependent manner.Meanwhile the apoptotic rates were increased in the cells administered with MRP8/14.7.In A549 cells treated with MRP8/14,NF-κB/p65 was significantly phosphorylated and translocated into the nuclei.8.NF-κB-specific inhibitor Bay11-7082 significantly attenuated the cell apoptosis induced by MRP8/14.According to the above results,we could get conclusions as follows:1.MRP8/14 could increase the phagocytosis in macrophage via activation of p38 signal pathway through receptor TLR4.The evoked phagocytosis was dependent on calcium and might increase the bacteria elimination in lung infection.2.MRP8/14 at high concentration could induce apoptosis of alveolar type II epithelial cells via activation of NF-κB signal pathway.This may aggravate the occurrence and development of ALI induced by lung infection.