Ganoderma extract prevents albumin-induced oxidative damage and chemokines synthesis in cultured human proximal tubular epithelial cells.

BACKGROUND: Ganoderma lucidum (Ganoderma or lingzhi) is widely used as an alternative medicine remedy to promote health and longevity. Recent studies have indicated that components extracted from Ganoderma have a wide range of pharmacological actions including suppressing inflammation and scavenging free radicals. We recently reported that tubular secretion of interleukin-8 (IL-8) induced by albumin is important in the pathogenesis of tubulointerstitial injury in the proteinuric state. In this study, we explored the protective effect of Ganoderma extract (LZ) on albumin-induced kidney epithelial injury. METHODS: Growth arrested human proximal tubular epithelial cells (PTECs) were incubated with 0.625 to 10 mg/ml human serum albumin (HSA) for up to 72 h. HSA induced DNA damage and apoptosis in PTEC in a dose- and time-dependent manner. Co-incubation of PTEC with 4-64 microg/ml LZ significantly reduced the oxidative damage and cytotoxic effect of HSA in a dose-dependent manner (P<0.001). Increased release of IL-8 and soluble intercellular adhesion molecules-1 (sICAM-1) in PTEC induced by HSA was ameliorated by co-incubation with Ganoderma (16 microg/ml). To explore the components of LZ that exhibited most protective effect in HSA-induced PTEC damages, LZ was further separated into two sub-fractions, LZF1 (MW <30 kDa) and LZF2 (MW <3 kDa), by molecular sieving using millipore membrane. PTEC were incubated with 5 mg/ml HSA in the presence of different doses of LZF1, LZF2 or unfractionated LZ. RESULTS: There was no difference in the degree of protection from HSA-induced cytotoxicity or oxidative DNA damage between different fractions of LZ. However, low molecular weight LZ (<3 kDa) was most effective in reducing sICAM-1 released from HSA-activated PTEC whereas the high molecular weight LZ (unfractionated LZ) was more effective in diminishing IL-8 production. CONCLUSIONS: Our results suggest that Ganoderma significantly reduces oxidative damages and apoptosis in PTEC induced by HSA. The differential reduction of IL-8 or sICAM-1 released from HSA-activated PTEC by different components of the LZ implicates that components of Ganoderma with different molecular weights could play different roles and operate different mechanisms in preventing HSA-induced PTEC damage.     

Chemoattraction of T cells expressing CCR5, CXCR3 and CX3CR1 by proximal tubular epithelial cell chemokines.

BACKGROUND: Chemokines produced by resident renal cells promote the infiltration of leukocyte subsets. We have analysed the chemotactic responses of CD3+ peripheral blood lymphocytes (PBLs) to factors secreted by proximal tubular epithelial cells (PTEC), assessing the role of chemokines and chemokine receptors in this process. METHODS: By FACS we analysed expression of the chemokine receptors CCR5, CXCR3, CX3CR1, CCR2, CXCR1 and CXCR2 on both freshly isolated and activated PBLs. Using Boyden chambers we studied the chemotactic activity of supernatant from resting and cytokine-stimulated (TNF-alpha and IFN-gamma) PTEC towards PBLs. Soluble recombinant chemokines and blocking antibodies were used to study the role of individual chemokine receptors. Chemokine secretion by PTEC was analysed by ELISA. RESULTS: Only a small proportion of freshly isolated cells expressed the chemokine receptors and there was low grade chemotaxis of these cells towards cytokine-stimulated PTEC supernatant compared with unstimulated PTEC supernatant. After activation, 84% of PBLs expressed CCR5, 90% expressed CXCR3 and 19% expressed CX3CR1. There remained low expression levels of CXCR1, CXCR2 and CCR2. Activated PBLs showed strong chemotactic responses to supernatant from cytokine-stimulated PTEC compared with unstimulated PTEC (P<0.001). Chemotaxis of these cells was inhibited by blocking CCR5, CXCR3 and CX3CR1 by 69%, 71% and 29% respectively, with complete inhibition following combined blockade. ELISA showed high levels of the chemokine RANTES/CCL5 (for CCR5) and IP-10/CXCL10 (for CXCR3) in cytokine-stimulated PTEC supernatant. CONCLUSIONS: Chemokines produced by cytokine activated PTEC promote the selective recruitment of activated T cells via the receptors, CCR5, CXCR3 and CX3CR1. These receptors may be amenable to therapeutic manipulation in renal inflammation.     

趋化因子受体5促进乳腺癌干细胞的趋化与侵袭

目的 观察趋化因子受体5(CCR5)对乳腺癌干细胞(CD44+CD24-/low)趋化和侵袭作用.方法 应用流式细胞仪分选获得乳腺癌干细胞,逆转录-聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测乳腺癌MCF-72个SP侧群细胞(side population)6例样本CCR5 mRNA和蛋白质,在趋化因子5(CCL5)作用下通过趋化小室法检测2个SP侧群细胞趋化活性和侵袭活性,并与CCR5的封闭进行对照研究.结果 CCL5对MCF-7肿瘤干细胞有明显趋化[乳腺癌干细胞(86.0±14.8)个/HP与CD44+CD24+(72.0±13.5)个/HP,t=7.461,P<0.05]和侵袭作用[乳腺癌干细胞(25.0±8.3)个/HP与CD44+CD24+(16.0±5.4)个/HP,t=6.665,P<0.05].结论 CCB5表达能够促进乳腺癌干细胞趋化和侵袭.     

Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes

The infiltration of leukocytes plays a major role in mediating tubulointerstitial inflammation and fibrosis in chronic renal disease. CC chemokines participate in leukocyte migration and infiltration into inflamed renal tissue. Because CC chemokine-directed leukocyte migration is mediated by target cell expression of a group of CC chemokine receptors, this study examined the expression of CC chemokines and their receptors during initiation of tubulointerstitial fibrosis after unilateral ureteral obstruction in C57BL/6 mice. Obstructed kidneys developed hydronephrosis, tubular cell damage, interstitial inflammation, and fibrosis. From days 2 to 10, a progressive interstitial influx of F4/80+ macrophages and CD3+ lymphocytes occurred (macrophages, 4-fold; lymphocytes, 20-fold at day 10, compared with contralateral control kidneys). In parallel, the number of activated fibroblast-specific protein 1+ fibroblasts and interstitial collagen IV accumulation increased from days 2 to 10. The mRNA expression of CC chemokines (predominantly monocyte chemoattractant protein-1 [MCP-1]/CCL2, RANTES/CCL5) and their receptors CCR1, CCR2, CCR5 increased progressively from days 2 to 10. By in situ hybridization, a prominent interstitial mRNA expression of MCP-1 and RANTES and their receptors CCR2 and CCR5 localized to interstitial mononuclear cell infiltrates. MCP-1 and RANTES expression was also seen in tubular epithelial cells. Fluorescence-activated cell sorter analysis of single-cell suspensions from obstructed kidneys revealed a prominent expression of CCR2 and CCR5 by infiltrating macrophages, whereas most lymphocytes expressed CCR5 only. These data demonstrate an increased expression of MCP-1/CCL2 and RANTES/CCL5 at sites of tubulointerstitial damage and progressive fibrosis during unilateral ureteral obstruction that correlates with simultaneous accumulation of interstitial macrophages and T lymphocytes expressing the respective surface receptors CCR2 and CCR5. The chemokine receptor-mediated leukocyte influx into the tubulointerstitium could offer a new potential target for therapeutic intervention in progressive renal tubulointerstitial fibrosis.     

Activation of tubular epithelial cells by mesangial-derived TNF-alpha: glomerulotubular communication in IgA nephropathy

BACKGROUND: IgA nephropathy (IgAN), characterized by mesangial IgA deposition, runs a variable clinical course with tubulointerstitial damage and renal failure in no less than 30% of patients. Histologically, IgA is rarely detected in renal tubules. The direct toxicity by IgA on renal tubules remains uncertain. We hypothesize that mediators released from human mesangial cells (HMC) triggered by IgA deposition may lead to activation of proximal tubular epithelial cells (PTEC). METHODS: The binding of IgA to PTEC or HMC was assessed by flow cytometry. IgA-HMC medium was prepared by collecting the spent medium in which growth arrested HMC were incubated with IgA isolated from patients with IgAN, healthy control subjects, or other nephritic control patients. PTEC was cultured with the IgA-HMC medium in the presence or absence of neutralizing antibodies to TNF-alpha, IL-1beta, TGF-beta, or PDGF. Gene expression and protein synthesis of TNF-alpha, MIF, or ICAM-1 by PTEC were determined by RT-PCR and ELISA, respectively. RESULTS: The binding of IgA isolated from patients with IgAN to PTEC was increased when compared to binding of IgA from healthy control subjects (P < 0.005). However, the binding to PTEC was less than one tenth that of HMC in IgAN. The binding to PTEC was not mediated through known IgA receptors, as shown by competitive binding assays and gene expression of the receptors. Despite the in vitro binding, PTEC cultured with isolated IgA exhibited no increased cell proliferation or enhanced synthesis of TNF-alpha, MIF, or sICAM-1. However, when PTEC were cultured with IgA-HMC medium prepared from IgAN patients, there was enhanced proliferation of PTEC (P < 0.001) and increased synthesis of TNF-alpha, MIF, and sICAM-1 when compared with PTEC cultured with IgA-HMC medium from control subjects (P < 0.001). The synthesis of MIF and sICAM-1 by PTEC cultured with IgA-HMC medium was reduced by neutralizing antibodies to TNF-alpha (P < 0.001) but not by neutralizing antibodies to IL-1beta, TGF-beta, or PDGF. CONCLUSION: Our finding implicates that TNF-alpha released from the mesangium after IgA deposition activates renal tubular cells. The glomerulotubular communication could play an important role in the pathogenesis of tubulointerstitial damage in IgAN.     

Differential expression of beta-chemokines MCP-1 and RANTES and their receptors CCR1, CCR2, CCR5 in acute rejection and chronic allograft nephropathy of human renal allografts

BACKGROUND: The beta-chemokines MCP-1 (CCL2) and RANTES (CCL5) have been shown to play important roles in acute renal transplant rejection (AR) and chronic allograft nephropathy (CAN). The potential relationship of expression of these chemokines, their chemokine receptors CCR1, CCR2, CCR5, and the cell populations of inflammatory infiltrate, histological and clinical diagnoses were investigated in biopsies at the time of AR and compared with biopsies of CAN. METHODS: In 24 renal transplant biopsies with AR (n = 15) and CAN (n = 9), the expression of MCP-1 and RANTES, their receptors CCR1, CCR2, and CCR5 and the infiltration with monocytes/macrophages and T cells were studied. RESULTS: As previously described, chemokine and chemokine receptor expression was found mainly in mononuclear cells infiltrating the interstitium and glomeruli. In the tubulointerstitial area and glomeruli the expression of MCP-1, RANTES, and their receptors correlated with an infiltration by monocytes/macrophages. Biopsies with CAN revealed a lower expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in tubulointerstitial cells, and a significantly lower infiltration with MRP14-positive monocytes/macrophages than biopsies with AR. In AR, MCP-1 and CCR1 showed a lower expression compared to RANTES, CCR2, and CCR5. CONCLUSIONS: The positive correlation between chemokines and chemokine receptors and infiltrating leukocytes during acute rejection, the lower but detectable expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in CAN, and the differences in the quantity of expression between the different chemokines and chemokine receptors point to a complex regulation of chemokine expression in renal allografts. Since chemokines are not only involved in inflammation but also in tissue regeneration, this could have impact on the development of CAN.     

Expression of the chemokine receptor CCR1 in human renal allografts.

BACKGROUND: Chemokines are involved in the recruitment of leukocytes to vascularized allografts. CCR1 is a receptor for various proinflammatory chemokines and CCR1 blockade reduces renal allograft injury in rabbits. The purpose of the study was to characterize CCR1-positive cells in human renal allografts. METHODS: Formalin-fixed, paraffin-embedded allograft nephrectomies (n = 9) and non-involved parts of tumour nephrectomies (n = 10) were studied. Immunohistochemistry for CCR1, CD3 and CD68 was performed on consecutive sections. Double immunofluorescence for CCR1 and CD3, CD20, CD68, DC-SIGN and S100 was used on selected cases. Expression of CCR1 mRNA and the ligands CCL3 and CCL5 was studied in renal allograft biopsies with acute rejection (n = 10), with chronic allograft nephropathy (n = 8) and controls (n = 8). RESULTS: CCR1 protein was expressed by circulating cells in glomerular and peritubular capillaries, colocalizing with CD68. In renal allografts CCR1-positive cells were present within glomerular tufts, but only scattered CCR1-positive cells were found in tubulointerstitial infiltrates. CCR1 did not colocalize with the majority of CD68-positive cells in the interstitium. The small number of CCR1-positive interstitial cells were identified as CD20- or DC-SIGN-positive by double immunofluorescence. CCR1 mRNA was significantly increased in renal biopsies with acute allograft rejection (P < 0.001), and with chronic allograft nephropathy (P < 0.05), it correlated with the expression of CCL3 and CCL5, and with serum-creatinine. CONCLUSIONS: CCR1 mRNA expression was associated with renal function in allografts. CCR1 protein expression was restricted to monocytes, CD20-positive B cells and DC-SIGN-positive dendritic cells. Thus most interstitial macrophages were CCR1 negative, which may relate to down-regulation after migration into the interstitium in human renal allografts.     

Monocyte chemoattractant chemokines in cystic fibrosis

BackgroundNeutrophilic inflammation causes lung damage in cystic fibrosis (CF). Recent data from animal models suggest that the migration of blood monocytes into the airway supports neutrophil-mediated tissue injury. CF may therefore be associated with increased airway levels of chemoattractants for pro-inflammatory monocytes. In this study, we sought to assess the levels of monocyte chemoattractants CCL2 and CX3CL1 in the blood and airways of patients with CF, and expression of their respective receptors CCR2 and CX3CR1 on blood monocytes.MethodsBlood was obtained from 32 CF patients and 25 healthy controls. Induced sputum was obtained from a further 24 CF patients and 17 healthy controls. Expression of CCR2 and CX3CR1 on CD14++CD16− and CD14+CD16+ blood monocytes was determined by flow cytometry. CCL2 and CX3CL1 levels in blood and induced sputum were determined by ELISA.ResultsTotal blood monocyte concentration was not different between CF and controls. CCR2 was absent, and CX3CR1 higher on CD14+CD16+ monocytes from both CF and controls when compared with expression on CD14++CD16− cells. There was no difference in expression of chemokine receptors by either monocyte subpopulation between CF and controls. Blood CCL2, but not CX3CL1, was increased in CF patients (p = 0.006). Similarly, CF was associated with increased induced sputum CCL2, but not CX3CL1 (190.6 vs. 77.3pg/mL; p = 0.029).ConclusionCCL2, but not CX3CL1 is increased in the airway and blood of CF patients. Blood monocytes from CF patients are phenotypically competent to respond to CCL2, since they express normal levels of CCR2.     

Infiltration of activated dendritic cells and T cells in renal cell carcinoma following combined cytokine immunotherapy

OBJECTIVES: In a phase I study the feasibility, toxicity and immunological effects of peri-operative cytokine immunotherapy of renal cell carcinoma were studied. Main goals were to determine the maximal tolerable dose and detailed in situ analysis of tumor infiltrates. METHODS: Fifteen patients with renal cell carcinoma, undergoing nephrectomy, received subcutaneous immunotherapy, consisting of low-dose IL-2, IFNalpha and GM-CSF, from day -3 prior, until day +5 following surgery in a dose escalation study. Infiltrates from resected tumor tissues from patients undergoing immunotherapy or control patients that underwent nephrectomy only, were examined using quantitative immunohistological analysis and 3-color immunofluorescence staining and confocal laser scanning microscope analysis. RESULTS: Toxicity was limited and the maximal tolerable dose was established. In peripheral blood an increase was found in total lymphocytes, (activated) T cells, NK cells and monocytes. Quantitative immunohistological analysis of tumor infiltrates showed enhanced numbers of CD3+ T cells, S100+ DC, CD83+ DC and IL-2 receptor positive cells (4-fold, 2-fold, 10-fold and 20-fold, respectively, compared to controls). In treated patients preferential invasion was observed of TNFalpha positive CD8+ T cells and DC, positive for DC-SIGN (CD209), CD83, CD80, IL-12 and the DC specific chemokine, DC-CK1 (CCL18). CONCLUSIONS: These findings show increased infiltration of activated, mature DC and functionally active CD8+ T cells in renal tumors, which may suggest clinical potential of cytokine immunotherapy.     

Lupus nephritis: correlation of interstitial cells with glomerular function

Mononuclear inflammatory cells were studied using monoclonal antibodies in the interstitium and glomeruli of 35 renal biopsy specimens from patients with lupus nephritis already taking immunosuppressants. The aims of this study were to assess the composition and significance of the infiltrate, and to assess correlations with immediate glomerular function and ability to predict the future course of the disease. The majority of interstitial cells were T lymphocytes and monocytes/macrophages. The number of interstitial CD4 + ve T helper/inducer lymphocytes was greater than that of CD8 + ve T cytotoxic/suppressor cells in only 19 out of 35 biopsies, the mean CD4:CD8 ratio being only 1.5 +/- 1.2. NK cells and B lymphocytes were a minor component only. Some expression of IL-2, transferrin and C3b receptors was seen on interstitial cells, but HLA-DR expressing cells were much in excess of controls and the numbers of tubular cells expressing HLA-DR was also increased. The number of interstitial T cells, CD4 + ve cells and monocytes/macrophages was highly correlated with the extent of chronic damage judged by optical microscopy. There was also an association between glomerular function at biopsy and numbers of interstitial T cells, CD8 + ve cells, monocytes/macrophages and DR expressing cells. Subsequent decline in renal function, however, was associated only with numbers of monocytes/macrophages and the rather small number of C3b receptor-positive cells. The presence of tubulointerstitial immune aggregates of Ig and/or C in 63% of patients was associated with greater numbers of NK cells. As previously described, the degree of renal function at biopsy correlated with a chronicity index based on optical microscopy. No correlations were found between numbers or types (mostly monocyte/macrophages) of intraglomerular leukocytes and clinical or biopsy features, except that more proliferative types showed greater leukocyte numbers. One hypothesis consistent with our findings is that interstitial T cells and monocytes may be important determinants of pathogenesis and progression of lupus nephritis. While several mechanisms may play an initial role, interstitial monocytes may be the major factor in chronic injury.     

Compartment-specific expression and function of the chemokine IP-10/CXCL10 in a model of renal endothelial microvascular injury

The recruitment of inflammatory cells into renal tissue, mainly T cells and monocytes, is a typical feature of various renal diseases such as glomerulonephritis, thrombotic angiopathies, allograft rejection, and vasculitis. T cells predominantly infiltrate the tubulointerstitium, whereas monocytes are present in the tubulointerstitial and glomerular compartment. Because chemokines play a pivotal role in leukocyte trafficking under inflammatory conditions, this study investigated whether a differential expression of chemokines contributes to the precise coordination of leukocyte subtype trafficking in a rat model of renal microvascular endothelial injury. Renal microvascular endothelial injury was induced in rats by selective renal artery perfusion with an anti-endothelial antibody. Induction of the disease led to severe glomerular and tubulointerstitial endothelial injury with subsequent upregulation of chemokines followed by inflammatory cell recruitment. Among the analyzed chemokine mRNA, IP-10/CXCL10 (119-fold), acting via CXCR3 on activated T cells, and MCP-1/CCL2 (65-fold), acting via CCR2 on monocytes, were by far the most strongly upregulated chemokines. In situ hybridization revealed that IP-10/CXCL10 mRNA was selectively expressed by endothelial cells in the tubulointerstitial area, co-localizing with infiltrating T cells. Despite extensive damage of glomerular vasculature, no IP-10/CXCL10 expression by glomerular endothelial cells was detected. MCP-1/CCL2 mRNA in contrast was detectable in the glomerulus and the tubulointerstitium. Treatment with a neutralizing anti-IP-10/CXCL10 antibody significantly reduced the number of infiltrating tubulointerstitial T cells without affecting monocyte migration and led to an improved renal function. Our study demonstrates a role of IP-10/CXCL10 on T cell recruitment in a rat model of renal endothelial microvascular injury. Furthermore, a differential chemokine expression profile by endothelial cells in different renal compartments was found. These findings are consistent with the hypothesis that functional heterogeneity of endothelial cells from different vascular sites exists and provide an insight into the molecular mechanisms that may mediate compartment-specific T cell and monocyte recruitment in inflammatory renal disease.     

高迁移率族蛋白B1对小鼠调节性T细胞与CD4+CD25-T细胞相互作用的影响

目的 观察高迁移率族蛋白B1(HMGB1)对调节性T细胞(Treg)与CD4+CD25-T细胞相互作用的影响,并初步探讨其影响Treg抑制功能的机制.方法 免疫磁珠法分离正常BALB/c小鼠脾脏CD4+CD25+Treg及CD4+CD25-T细胞.采用固相包被抗CD3/可溶性抗CD28进行辅助活化,以不同时间及浓度HMGB1刺激Treg,ELISA法分析HMGB1刺激对Treg分泌IL-10的影响.将HMGB1(1000μg/L)刺激后的Treg与CD4+CD25-T细胞共培养,MTT法观察其对Treg抑制CD4+CD25-T细胞反应的作用,并分析HMGB1刺激后的Treg对CD4+CD25-T细胞IL-2生成及细胞功能极化的影响.结果 经抗CD3/CD28辅助活化的CD4+CD25+Treg在HMGB1作用下IL-2生成无显著差异(P＞0.05),但随时间延长及剂量增加,IL-10生成明显减少(P＜0.05).经HMGB1刺激的Treg对CD4+CD25-T细胞增殖的抑制反应减弱,同时诱导CD4+CD25-T细胞IL-2产生及细胞功能极化的能力均下降(P＜0.05).结论 HMGB1可通过诱导Treg抑制功能的下调,从而影响CD4+CD25-T细胞功能,进而调节炎症反应过程.     

CCR and CC chemokine expression in relation to Flt3 ligand-induced renal dendritic cell mobilization

BACKGROUND: We investigated the expression and function of CC chemokine receptors (CCR) on highly-purified kidney and blood dendritic cells isolated from mice in which dendritic cells were mobilized with fms-like tyrosine 3 kinase ligand (Flt3L). METHODS: CCR and CC chemokine expression were determined by RNase protection assay or flow cytometry, and dendritic cell migratory responses assayed using Transwell chambers. Chemokine production in renal tissue was detected by immunofluorescence staining. Trafficking of fluorochrome-labeled dendritic cells was monitored in vivo. RESULTS: Freshly-isolated renal dendritic cells expressed mRNA for CCR1, 2, 5, and 7 and CCR1 and 5 protein. They did not migrate to inducible chemokines--CCL3 [macrophage inflammatory protein (MIP)-1alpha], CCL5 [regulated upon activation, normal T cell expressed and secreted (RANTES)], or CCL20 (MIP-3alpha). Following lipopolysaccharide (LPS) stimulation, the dendritic cells down-regulated CCR1, 2, and 5 expression, up-regulated or sustained signals for CCR7, and migrated to the constitutively expressed ligands CCL19 (MIP-3beta) and CCL21 (secondary lymphoid tissue chemokine). Normal kidneys expressed weak message for CCL2, 3, and 4, with stronger signals for CCL5 and 19. Intrarenal CCL5 production was enhanced by Flt3L administration, in association with marked increases in interstitial CD45+ mononuclear cells. Mobilized blood dendritic cells migrated to CCR2 and CCR5 ligands and trafficked to renal intertubular sites following adoptive (intravenous) transfer. Their migration to the CCR5 ligand MIP-1beta (CCL4) and homing to kidneys of Flt3L-treated recipients were inhibited by CCR5 antagonism. CONCLUSION: These data implicate specific CCR and their ligands in regulation of the dendritic cell constituency of the kidney. CCR5 antagonism inhibits their directed migration and intrarenal accumulation.     

前列腺素E2对小鼠树突状细胞迁移能力及抗乳腺癌免疫作用的影响

目的观察前列腺素E2（PGE2）对体外培养的乳腺癌抗原负载小鼠树突状细胞（DC）迁移能力及抗乳腺癌免疫作用的影响.方法用重组小鼠粒细胞巨噬细胞集落刺激因子和重组小鼠白细胞介素-4培养BALB/c小鼠骨髓来源DC,负载乳腺癌抗原后加入PGE2,进行表型、CCR7mRNA及蛋白、同种异体混合淋巴细胞反应、特异性淋巴细胞（CTL）杀伤活性测定.将TM40D接种于小鼠左侧胸壁皮下制作乳腺癌动物模型,1周后皮下接种PGE2组及对照组DC,观察肿瘤抑制状况.结果体外实验显示,PGE2不影响DC的刺激淋巴细胞增殖能力和同种异体特异性杀伤活性.与对照组DC相比,PGE2培养组DC的CD80、CD86阳性细胞数增多,CCR7mRNA和蛋白表达上调（P＜0.05）.体外趋化试验显示,PGE2使DC对其配体CCL19和CCL21反应性增强（P＜0.05）.在乳腺癌动物模型中,PGE2培养组DC抑制肿瘤生长作用优于对照组.结论PGE2可以通过促进DC成熟并促进其体内迁移能力,提高抗乳腺癌DC疫苗的功效.     

FTY720 treatment of kidney transplant patients: a differential effect on B cells, naïve T cells, memory T cells and NK cells

FTY720 alters lymphocyte recirculation and homing by interfering with S1P receptors on lymphocytes, possibly in combination with chemokine receptors, and induces a decrease in PBL counts. In fresh, whole blood samples of 14 kidney transplant patients, we analyzed by flow cytometry the effect of FTY on the number of NK cells, monocytes, na?ve (CCR7+) T cells, memory (CCR5+) T cells and B cells. Patients treated with 0.5, 2.5 or 5mg FTY/day showed a strong decrease in T and B cell numbers. NK cells and monocytes were not affected. FTY reduced primarily na?ve T cells. From the memory T cells (CCR5+), predominantly CD8 cells, 40-60% remained in the circulation. The majority of the CCR7+ cells disappeared from the circulation within 3-6h, while a further reduction was achieved later. The more slowly decrease in na?ve CCR7+ T cell numbers was also observed in the group treated with 0.25mg FTY/day. Elispot assays revealed no IL-4 producing cells and a low frequency of IFN-gamma producing cells. We suggest that both CCR7 dependent and independent mechanisms are involved in the depletion of T cells from peripheral blood.     

IgA肾病伴扁桃体炎患者Th22细胞及CCL27/CCR10轴的表达

目的分析IgA肾病(IgAN)伴扁桃体炎患者辅助T细胞22(Th22)细胞及相关细胞因子、趋化因子轴的表达变化,探讨其与临床病理改变的关系。方法纳入2015年6月至2016年6月在中南大学湘雅医院经肾活检确诊为IgAN的患者为研究对象。按照肾脏病理类型及是否合并扁桃体炎分为IgAN合并扁桃体炎(IgAN+扁桃体炎)组、单纯IgAN(IgAN)组、系膜增生性肾小球肾炎(MsPGN)组和对照组(HC)组。采用流式细胞术检测外周血Th17、Th22细胞及Th22细胞CC型趋化因子受体(CCR)4、CCR6、CCR10表达阳性细胞占比;酶联免疫吸附法(ELISA)检测Th22细胞效应因子白细胞介素(IL)-22,促分化因子IL-1β、IL-6、TNF-α、CC型趋化因子配体(CCL)22、CCL20、CCL27水平。免疫组织化学方法(IHC)检测肾组织中CCL22、CCL20和CCL27的表达。比较分析各组临床病理指标、Th22细胞占比及相关趋化因子表达的差异。结果本研究共纳入44例IgAN患者,其中14例合并扁桃体炎,另纳入10例MsPGN患者及16例健康人作为对照。各组性别、年龄匹配,血压、肾功能、血脂等生化指标的差异无统计学意义(均P>0.05)。IgAN组、MsPGN组及IgAN+扁桃体炎组外周血Th22细胞、CCR10表达阳性细胞占比明显高于对照组,血清IL-22、IL-1β、IL-6、TNF-α、CCL20、CCL22、CCL27水平亦较高,且IgAN+扁桃体炎组更为显著(均P<0.05)。各组肾脏组织CCL20、CCL22、CCL27表达改变与其外周血变化一致。合并血尿的IgAN患者外周血Th22细胞占比显著高于无血尿组患者。病理分型(E1、S1或T1-2)较重的IgAN患者外周血Th22细胞占比显著高于病理分型较轻者(E0、S0或T0)。结论Th22细胞及CCL27/CCR10轴参与了IgAN发病过程,扁桃体炎可加重IgAN的临床与病理损伤。     

Direct anti-proliferative effect of adipose-derived mesenchymal stem cells of ankylosing spondylitis patients on allogenic CD4+ cells

ObjectivesT-cell-mediated adaptive immunity contributes to the development and persistence of ankylosing spondylitis (AS). Mesenchymal stromal/stem cells (MSCs) have immunomodulatory potential and are able to inhibit T-cell proliferation, but their functionality in AS patients is relatively unknown. The aim of the study was to assess the direct anti-proliferative effects of MSCs isolated from subcutaneous abdominal adipose tissue of AS patients (AS/ASCs) on allogeneic T lymphocytes, using commercially available ASC lines from healthy donors (HD/ASCs) as a control.Material and methodsCD3+CD4+ T-cells were isolated from peripheral blood of healthy blood donors, activated with anti-CD3/CD28 beads, and co-cultured for 5 days with untreated and TNF+IFN-γ pre-stimulated HD/ASCs (5 cell lines) and AS/ASCs, obtained from 11 patients (6F/5M). The proliferative response of T-cells was analysed by flow cytometry, while the concentrations of kynurenines, prostaglandin E2 (PGE-2), interleukin 10 (IL-10), and interleukin 1 receptor antagonist (IL-1Ra) were measured spectrophotometrically or using a specific enzyme-linked immunosorbent assay (ELISA).ResultsHD/ASCs and AS/ASCs similarly reduced the T-cell proliferation response, i.e. the percentage of proliferating cells, the proliferation, and replication indices, and these effects were dependent mostly on soluble factors. In the co-cultures of activated CD4+ T-cells with HD/ASCs and AS/ASCs significant increases of kynurenines, PGE-2, and IL-1Ra, but not IL-10, production were observed. The release of these factors was dependent either on cell-to-cell contact (IL-10, IL-1Ra) or soluble factors (kynurenines, PGE-2). There was a moderate to strong negative correlation between T-cell proliferative response, and the concentrations of kynurenines, PGE-2, and IL-10, but not IL-1Ra. This association was more evident in the case of TI-treated AS/ASCs than HD/ASCs.ConclusionsAS/ASCs, similar to HD/ASCs, exert a direct effective anti-proliferative impact on CD4+ T cells, acting via soluble factors that are released in cell contact-dependent (IL-10) and independent (kynurenines, PGE-2) pathways. Thus, our results suggest that AS/ASCs are potentially useful for therapeutic application.     

Differential impact of CD154 costimulation blockade on alloreactive effector and regulatory T cells in murine renal transplant recipients

BACKGROUND: Although CD154 costimulation blockade prolongs allograft survival in multiple transplantation models, the underlying immunological mechanisms remain to be elucidated. METHODS AND RESULTS: We used a murine orthotopic kidney allograft (KTx) model to analyze the impact of CD154 blockade on trafficking and function of alloreactive T effector versus T regulatory cells. A single dose of MR1 Ab treatment at the time of KTx significantly improved the survival of Balb/c KTx in na?ve C57BL/6 recipients (mean survival time >100 days vs. 52 days in controls; P<0.005), and improved graft histology, as evidenced by decreased lymphocyte infiltration and preservation of tissue architecture (days 6-8). In the early posttransplant phase, fluorescence-activated cell sorting analysis revealed preferential depression of T effector (CD8+CD25+) and relative enrichment of T-regulatory (CD4+ CD25+ CD152+) cells selectively in KTx. This pattern was further supported by intragraft gene expression analysis, which showed increased FoxP3/Tbet ratio and simultaneously decreased granzyme B/IFN-gamma levels in Ab-treated recipients. Additionally, MR1 Ab selectively up-regulated intragraft CCL17, but suppressed CXCL9/CCL5, in parallel with increased CCR4/CCR8 but unaltered CXCR3 expression. CONCLUSION: These results provide evidence, at both cellular and molecular levels, that CD154 blockade in murine KTx recipients differentially targeted T-effector and T-regulatory cell subsets by regulating intragraft induction of chemokines targeting distinct T-cell subsets.     

Back signaling of HLA class I molecules and T/NK cell receptor ligands in epithelial cells reflects the rejection‐specific microenvironment in renal allograft biopsies

The role of endothelial cells in the pathophysiology of antibody‐mediated rejection after renal transplantation has been widely investigated. We expand this scenario to the impact of epithelial cells on the microenvironment during rejection. Primary proximal tubular epithelial cells were stimulated via HLA class I, CD155 and CD166 based on their potential signal‐transducing capacity to mediate back signaling after encounter with either T/NK cells or donor‐specific antibodies. Upon crosslinking of these ligands with mAbs, PTEC secreted IL‐6, CXCL1,8,10, CCL2, and sICAM‐1. These proteins were also released by PTEC as consequence of a direct interaction with T/NK cells. Downmodulation of the receptor CD226 on effector cells confirmed the involvement of this receptor/ligand pair in back signaling. In vivo, CD155 and CD166 expression was detectable in proximal and distal tubuli of renal transplant biopsies, respectively. The composition of the protein microenvironment in these biopsies showed a substantial overlap with the PTEC response. Cluster and principal component analyses of the microenvironment separated unsuspicious from rejection biopsies and, furthermore, ABMR, TCMR, and borderline rejection. In conclusion, our results provide evidence that epithelial cells may contribute to the rejection process and pave the way to a better understanding of the pathomechanisms of kidney allograft rejection.