Apoptosis in human germinal centre B cells by means of CC chemokine receptor 3 expression induced by interleukin-2 and interleukin-4

Background CC chemokine receptor 3 (CCR3), expressed on some inflammatory cells, is a member of the chemokine receptor family. Its ligand is eotaxin/CCL11. In this research, we studied the expression and function of CCR3 induced by interleukin-2 (IL-2) and interleukin-4 (IL-4) on human germinal centre (GC) B cells.Methods Cells isolated from human tonsils were stimulated with IL-2 or/and IL-4 followed by bonding with eotaxin/CCL11. Flow cytometry was used to detect expression of CCR3 on GC B cells and apoptosis of GC B cells. Real time quantitative reverse transcription polymerase chain reaction and Northern blot assays were used to analyse the CCR3 mRNA expressed in the GC B cells. Chemotaxis and adhesion assays were used to determine the effect of eotaxin/CCL11 ligand bonded to CCR3 on GC B cells.Results There was no CCR3 expression on human freshly isolated GC B cells. The combination IL-2 and IL-4 could upregulate CCR3 mRNA and protein expression on GC B cells. Eotaxin could not induce GC B cell chemotaxis and adhesion but triggered apoptosis of GC B cells.Conclusion IL-2 and IL-4 together induced expression of CCR3 on GC B cells, and the receptor acted as a death receptor.

CX3 chemokine receptor 1 deficiency leads to reduced dendritic complexity and delayed maturation of newborn neurons in the adult mouse hippocampus

Previous studies have shown that microglia impact the proliferation and differentiation of neu- rons during hippocampal neurogenesis via the fractalkine/CX3 chemokine receptor i （CX3CRI） signaling pathway. However, whether microglia can influence the maturation and dendritic growth of newborn neurons during hippocampal neurogenesis remains unclear. In the present study, we found that the number of doublecortin-positive cells in the hippocampus was decreased, and the dendritic length and number of intersections in newborn neurons in the hippocampus were reduced in transgenic adult mice with CX3CR1 deficiency （CX3CRl^GFP/GFe）. Furthermore, after experimental seizures were induced with kainic acid in these CX3CRl-deficient mice, the expression of c-fos, a marker of neuronal activity, was reduced compared with wild-type mice. Collectively, the experimental findings indicate that the functional maturation of newborn neu- rons during hippocampal neurogenesis in adult mice is delayed by CX3CR1 deficiency.

C-X3-C motif chemokine ligand 1/receptor 1 regulates the M1 polarization and chemotaxis of macrophages after hypoxia/reoxygenation injury

Background:Macrophages play an important role in renal ischemia reperfusion injury,but the functional changes of macrophages under hypoxia/reoxygenation and the related mechanism are unclear and need to be further clarified.Methods:The effects of hypoxia/reoxygenation on functional characteristics of RAW264.7 macrophages were analyzed through the protein expression detection of pro-inflammatory factors TNF-αand CD80,anti-inflammatory factors ARG-1 and CD206.The functional implications of C-X3-C motif chemokine receptor 1(CX3CR1)down-regulation in hypoxic macrophages were explored using small interfering RNA technology.Significance was assessed by the parametrict-test or nonparametric Mann-Whitney test for two group comparisons,and a one-way ANOVA or the Kruskal-Wallis test for multiple group comparisons.Results:Hypoxia/reoxygenation significantly increased the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and chemokine C-X3-C motif chemokine ligand 1(CX3CL1)/CX3CR1 and inhibited the protein expression of M2-related anti-inflammatory factors ARG-1 and CD206 in a time-dependent manner in RAW264.7 cells.However,the silencing of CX3CR1 in RAW264.7 cells using specific CX3CR1-siRNA,significantly attenuated the increase in protein expression of TNF-α(P<0.05)and CD80(P<0.01)and the inhibition of ARG-1(P<0.01)and CD206(P<0.01)induced by hypoxia/reoxygenation.In addition,we also found that hypoxia/reoxygenation could significantly enhance the migration(2.2-fold,P<0.01)and adhesion capacity(1.5-fold,P<0.01)of RAW264.7 macrophages compared with the control group,and CX3CR1-siRNA had an inhibitory role(40%and 20%reduction,respectively).For elucidating the mechanism,we showed that the phosphorylation levels of ERK(P<0.01)and the p65 subunit of NF-κB(P<0.01)of the RAW264.7 cells in the hypoxic/reoxygenation group were significantly increased,which could be attenuated by down-regulation of CX3CR1 expression(P<0.01,both).ERK inhibitors also significantly blocked the effects of hypoxic/reoxygenation on the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and M2-related anti-inflammatory factors ARG-1 and CD206.Moreover,we found that conditioned medium from polarized M1 macrophages induced by hypoxia/reoxygenation,notably increased the degree of apoptosis of hypoxia/reoxygenation-induced TCMK-1 cells,and promoted the protein expression of pro-apoptotic proteins bax(P<0.01)and cleaved-caspase 3(P<0.01)and inhibited the expression of anti-apoptotic protein bcl-2(P<0.01),but silencing CX3CR1 in macrophages had a protective role.Finally,we also found that the secretion of soluble CX3CL1 in RAW264.7 macrophages under hypoxia/reoxygenation was significantly increased.Conclusions:The findings suggest that hypoxia/reoxygenation could promote M1 polarization,cell migration,and adhesion of macrophages,and that polarized macrophages induce further apoptosis of hypoxic renal tubular epithelial cells by regulating of CX3CL1/CX3CR1 signaling pathway.

Effects of Curcumin on Pain Threshold and on the Expression of Nuclear Factor κ B and CX3C Receptor 1 after Sciatic Nerve Chronic Constrictive Injury in Rats

Objective：To investigate the effects of curcumin on pain threshold and the expressions of nuclear factorκB（NF-κB）and CX3C chemokine receptor 1（CX3CR1）in spinal cord and dorsal root ganglion（DRG）of the rats with sciatic nerve chronic constrictive injury.Methods：One hundred and twenty male Sprague Dawley rats,weighing 220-250 g,were randomly divided into 4 groups.Sham surgery（sham）group：the sciatic nerves of rats were only made apart but not ligated;chronic constrictive injury（CCI）group：the sciatic nerves of rats were only ligated without any drug treatment;curcumin treated injury（Cur）model group：the rats were administrated with curcumin 100 mg/（kg·d）by intraperitoneal injection for 14 days after CCI;solvent control（SC）group：the rats were administrated with the solvent at the same dose for 14 days after CCI.Thermal withdrawal latency（TWL）and mechanical withdrawal threshold（MWT）of rats were respectively measured on pre-operative day 2 and postoperative day 1,3,5,7,10 and 14.The lumbar segment L4-5 of the spinal cord and the L4,L5 DRG was removed at post-operative day 3,7 and 14.The change of nuclear factorκB（NF-κB）p65 expression was detected by Western blotting while the expression of CX3CR1 was determined by immunohistochemical staining.Results：Compared with the sham group,the TWL and MWT of rats in the CCI group were significantly decreased on each post-operative day（P〈0.01）,which reached a nadir on the 3rd day after CCI,and the expressions of NF-κB p65and CX3CR1 were markedly increased in spinal cord dorsal horn and DRG.In the Cur group,the TWL of rats were significantly increased than those in the CCI group on post-operative day 7,10 and 14（P〈0.05）and MWT increased than those in the CCI group on post-operative day 10 and 14（P〈0.05）.In addition,the administration of curcumin significantly decreased the positive expressions of NF-κB p65 and CX3CR1 in spinal cord and DRG（P〈0.05）.Conclusion：Our study suggests that curcumin could ameliorate the CCI-induced neuropathic pain,probably through inhibiting CX3CR1 expression by the activation of NF-k B p65 in spinal cord and DRG.