| Objective Astrocytes play important physiological roles related to neuronal function.Upon injury of central nervous system(CNS),they are activated to participate in the neuropathological process by morphological and functional alterations,such as reactive gliosis.Astrocytes can also act as immune effector cells with the ability to produce a wide array of chemokines and cytokines following injury stimuli,which potentially exacerbate the extent of tissue damage.Macrophage migration inhibitory factor(MIF),a proinflammatory cytokine,can facilitate production of inflammatory cytokines in astrocytes of injured spinal cord.Whether it is able to promote release of chemokines from astrocytes remains elusive.Here,we are going to investigate the relationship between MIF,one of the proinflammatory cytokines,and chemokines release from astrocytes after spinal cord injury,as well as the underlying mechanisms.Methods For contusion SCI rat model,the spinal cord of adult male SD rats was injuried at T8-T10.The injured segments were perfused and collected at 0d,1d,4d,7d,respectively.Western blot was performed to examine the expression of MIF.ELISA and immunofluorescence were performed to examine the expression of CCL5.By using siRNA interference MIF receptor CD74,transcriptome sequencing and bioinformatics analysis were performed to analyze the differentially expressed genes.Primary cultured astrocytes were prepared from spinal cord of newborn SD rats,1–3 days after birth.Cells were treated with recombinant MIF,with or without MIF specific inhibitor 4-IPP,ELISA was performed to examine the expression of CCL5 to verify the transcriptome sequencing results.By using siRNA interference CD74 receptor,adding recombinant MIF,Western blot was performed to examine phosphorylation of MAPKs and NFκB expression.By using MAPKs inhibitors combined with recombinant MIF,ELISA detects the expression of CCL5.A Transwell assay was performed to determine the effect of CCL5 on astrocytes-stimulated macrophages migration.After contusion SCI rat model,4-IPP and recombinant CCL5 were injected into the injury site.After perfusion at different time points,sections were allowed to incubate with IBA-1 antibody to examine the effects of the migration of macrophages in vivo.Results1.The protein levels of chemokine CCL5/RANTES were remarkably increased in the astrocytes of rat injured spinal cord,in parallel with the expression of MIF.Treatment of MIF inhibitor 4-IPP in the lesion sites resulted in a significant decrease of CCL5 protein levels.2.In vitro study revealed MIF was capable of facilitating CCL5 production of astrocytes through interaction with CD74 membrane receptor,and knockdown of this receptor attenuated such effects.3.Production of CCL5 in astrocytes was significantly blocked by inhibitor of JNK,rather than by those of ERK and P38,suggesting an important role of JNK signaling in regulation of CCL5 expression.4.Recombinant CCL5 protein was found to be more effective in promoting migration of M2-,compared to M1-type macrophages.Our result has revealed a novel function of MIF in regulation of CCL5 release from astrocytes,which provides some clues for therapy of spinal cord inflammation.Conclusions1.MIF/CD74 axis regulates the expression of CCL5 in astrocytes after spinal cord injury in Rats2.MIF/CD74 axis regulates the production of CCL5 through the JNK signaling.3.CCL5 promotes the migration of M2 macrophages. |
