Interaction between proximal tubular epithelial cells and infiltrating monocytes/T cells in the proteinuric state

We hypothesize an interaction between T cells/monocytes and the tubules in the development of tubulointerstitial injury in chronic proteinuric nephropathy. We established in vitro co-culture systems of proximal tubular epithelial cells (PTEC) and T cells/monocytes to study the contribution of soluble factors and cell-to-cell contact in the development of tubulointerstitial injury. The release of monocyte chemoattractant protein-1 (MCP1 or CCL2), Regulated upon Activation, normal T cell Expressed and Secreted (RANTES or CCL5), soluble intracellular adhesion molecules-1 (sICAM-1), or interleukin-6 (IL-6) was increased in PTEC following apical exposure to human serum albumin (HSA). The release of CCL2, CCL5, or sICAM-1 from PTEC was enhanced by contact of monocytes/T cells on the basolateral surface. Tumor necrosis factor-alpha (TNF-alpha) and IL-1beta are important soluble factors as suggested by the blocking effect of antibodies (Abs) against TNF-alpha or IL-1beta but not against other cytokines. The percentage of CD4+ T cells expressing both chemokine receptors, CCR2 and CCR5, was increased after culturing with supernatant from the apical or basolateral surface of PTEC following apical exposure to HSA. However, only CCR2 was upregulated in CD8+ T cells, whereas CCR5 expression was increased in monocytes. The chemotaxis of CD4+ or CD8+ T cells to supernatant from PTEC upon apical exposure to HSA was reduced with neutralizing Abs against CCL5 and/or CCL2, whereas the chemotaxis of monocytes was only reduced by anti-CCL5 but not by anti-CCL2. In summary, chemokines released by HSA-activated PTEC are amplified by monocytes/T cells. Mediators released by HSA-activated PTEC can differentially modulate the expression of chemokine receptors in monocytes/T cells and hence, alter their chemotaxis towards activated PTEC. These interactions are pivotal in the development of tubulointerstitial injury.     

B7-1 (CD80) and B7-2 (CD 86) expression in human tubular epithelial cells in vivo and in vitro

BACKGROUND: Tubulointerstitial inflammation with infiltration of mononuclear cells plays an important role in acute allograft rejection and in the progression of renal diseases. We therefore investigated in vivo the expression of the costimulatory molecules B7-1 and B7-2 on proximal tubular epithelial cells (PTEC) under normal and pathologic conditions and analyzed the regulation and functional role of these molecules after cytokine and CD40 activation in vitro. METHODS: Immunohistological staining for B7-1 and B7-2 on cryostat sections of core needle biopsies from patients with different renal diseases was examined. Patients were divided into three groups: group A: diffuse interstitial inflammation; group B: minor interstitial inflammation; group C: no interstitial inflammation. In addition, the expression of B7-1 and B7-2 protein and mRNA of cultured PTEC that had been stimulated with cytokine-combinations in absence or presence of a stimulatory anti-CD40 antibody was investigated by means of FACS analysis and RT-PCR. The functional role was analyzed in MKLCs with cytokine and anti-CD40 prestimulated PTEC by measuring IFN-gamma and IL-2 expression in absence or presence of CTLA4-Ig by ELISA. RESULTS: Group A patients showed intense tubular staining for B7-1 and B7-2, group B patients showed mild staining, whereas in group C patients B7-1 and B7-2 staining was negative or only weakly positive. In vitro, the presence of B7-1 and B7-2 on PTEC was increased after stimulation with combinations of IL-1alpha, IL-4, IFN-gamma or IL-13 instead of IL-4 and CD40 activation. B7-1 and B7-2 mRNA could be detected in PTEC as well. In MKLCs only cytokine and anti-CD40 prestimulated PTEC were able to stimulate IFN-gamma and IL-2 production by purified T cells, which could be blocked dose-dependently by CTLA4-Ig. CONCLUSIONS: This study clearly shows that B7-1 and B7-2 can be induced on PTEC in vivo and in vitro. After B7-1 and B7-2 induction, PTEC costimulate CD28 on T lymphocytes resulting in cytokine production. This might be of relevance in allograft rejection and in various kidney diseases.     

Major involvement of CD40 in the regulation of chemokine secretion from human peritoneal mesothelial cells

BACKGROUND: CD40 is a member of the tumor necrosis factor (TNF) family of receptors, whose ligand, CD154, is expressed by activated mononuclear cells. CD40 activation is a major immune regulatory pathway and is important for the regulation of chemokine and cytokine secretion. This study investigates the effect of CD40 ligation on the secretion of chemokines from human peritoneal mesothelial cells (HPMC). METHODS: We activated CD40 in HPMC along with combinations of TNF-alpha, interleukin-1 (IL-1), and interferon gamma (IFN-gamma), and evaluated the mRNA levels and protein secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and IL-8. RESULTS: CD40 ligation had a small stimulatory effect on the secretion of all three chemokines, while TNF-alpha, IL-1 and IFN-gamma induced their secretion in a dose-dependent manner. The combination of CD40 ligation with either IL-1 or TNF-alpha increased chemokine secretion additively. IFN-gamma and CD40 ligation acted in synergy to induce the secretion of the mononuclear recruiting chemokines RANTES and MCP-1 (up to approximately 36-fold and approximately threefold, respectively), for which the combination of all three cytokines with CD40 ligation was extremely potent. In contrast, the secretion of the neutrophil chemoattractant IL-8, induced by CD40 ligation or by the combination of IL-1 and TNF-alpha, was reduced in the presence of IFN-gamma. CONCLUSION: In light of our data, it is reasonable to suggest that in the mononuclear phase of peritonitis, IFN-gamma and CD154, expressed by activated mononuclear cells, diminish IL-8 secretion from HPMC and thus inhibit neutrophil recruitment. At the same time, the two act in synergy to induce the secretion of RANTES and MCP-1 from HPMC. Hence, by regulating chemokine secretion, CD40 may be involved in peritonitis and in the development of late phase mononuclear predominance.     

Effect of 15d-PGJ2 on the expression of CD40 and RANTES induced by IFN-gamma and TNF-alpha on renal tubular epithelial cells (HK-2)

BACKGROUND/AIMS: Recent evidence shows that peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ameliorates a variety of inflammatory conditions. CD40 is a co-stimulatory molecule and its ligation induces production of different proinflammatory cytokines including RANTES (regulated upon activation, normal T cell expressed), which are considered as important factors in the initiation and maintenance of inflammatory response. The aim of this study was to investigate the effect of PPAR-gamma on CD40 and RANTES production on cultured human renal proximal tubular epithelial (HK-2) cells. METHODS: HK-2 cells were maintained under defined in vitro conditions and treated with either PPAR-gamma agonist 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) or 15d-PGJ2 + PPAR-gamma antagonist GW9662, and then stimulated with a combination of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The CD40 and RANTES levels were investigated. RESULTS: HK-2 cells expressed low levels of CD40 and RANTES. Activation of HK-2 cells by combined treatment of TNF-alpha and IFN-gamma results in strong synergistic effects on the expression of CD40 and the secretion of RANTES. 15d-PGJ2 significantly decreased CD40 and RANTES expression and GW9662 partly abrogated the inhibition of 15d-PGJ2 on CD40 and RANTES. CONCLUSION: 15d-PGJ2 significantly decreased CD40 and RANTES expression in HK-2 cells, which were partially mediated by PPAR-gamma-dependent pathways. These results point to PPAR-gamma as a remarkable new target in the prevention of tubular inflammatory injury associated with renal disease.     

Effect of human anti-DNA antibodies on proximal renal tubular epithelial cell cytokine expression: implications on tubulointerstitial inflammation in lupus nephritis

This study aimed to investigate the effects of human anti-DNA antibodies (Ab) from patients with lupus on renal proximal tubular epithelial cells (PTEC), focusing on alterations in cell morphology and proinflammatory cytokine synthesis. Immunohistochemistry showed increased tubulointerstitial IL-6 expression and IgG deposition in renal biopsies from patients with diffuse proliferative lupus nephritis, not observed in controls or membranous lupus nephritis, which correlated with the severity of inflammatory cell infiltration. Sera from patients with lupus nephritis contained IgG that bound to cultured PTEC. Such binding increased with disease activity and correlated with the level of anti-DNA Ab. Incubation of PTEC with anti-DNA Ab that were isolated during active (active Ab) or inactive (inactive Ab) disease induced IL-6 synthesis, both apically and from the basolateral aspect. This was accompanied by altered cell morphology, increased cell proliferation (P < 0.05), and lactate dehydrogenase release (P < 0.05). The binding of inactive Ab and active Ab to PTEC resulted in differential and sequential upregulation of TNF-alpha, IL-1beta, and IL-6 secretion (P < 0.05). Early induction of TNF-alpha was observed with active Ab; the two then acted synergistically to induce IL-6 secretion. Exposure of PTEC to inactive Ab was associated with modest induction of TNF-alpha, which was not involved in downstream induction of other proinflammatory peptides. These data suggest distinct immunopathogenetic mechanisms during disease flare or remission. Conditioned media from human mesangial cells acted synergistically with anti-DNA Ab to induce cytokine secretion in PTEC. Results from these studies underscore the pivotal role of PTEC in the pathogenesis of tubulointerstitial inflammation and fibrosis in lupus nephritis.     

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.     

High-avidity antitumor T-cell generation by toll receptor 8-primed, myeloid- derived dendritic cells is mediated by IL-12 production

BACKGROUND: High-level production of heterodimeric p70 interleukin (IL)-12 by myeloid-derived dendritic cells (DCs) requires 2 signals: interferon gamma (IFN-gamma) and a maturation signal provided by CD40 ligation (CD40L) or lipopolysaccharide (LPS). METHODS: In the current study we demonstrate that signaling through toll-like receptor (TLR) 8, but not TLR3, TLR2, or TLR4, provides a priming signal to myeloid-derived DC for high IL-12 p70 heterodimer production. RESULTS: All the TLR agonists induced maturation of DC as evidenced by increased expression of CD83, CD80, and CD86. Both IFN-gamma and TLR7/8 agonist R848 increased expression of TLR8 in immature monocyte-derived DCs. The combination of TLR7/8 agonist R848 and maturation signals LPS or CD40L induced high-level expression of IL-12p35 and p40 similar to that induced by IFN-gamma plus LPS. In contrast, receptor agonists specific for TLR7 did not prime for IL-12 production. The p70 IL-12 produced by the TLR8-primed DC polarized CD4+ T for Th1 cytokine production and induced CD8+ T cells, displaying high functional avidity with enhanced tumor cell recognition. CONCLUSIONS: The data suggest that toll 8 receptor agonists are useful for inducing type-1 polarized DCs for vaccine design in treating cancer and infectious disease.     

Sequential delivery of maturation stimuli increases human dendritic cell IL-12 production and enhances tumor antigen-specific immunogenicity

BACKGROUND: Despite the increasing use of dendritic cells (DCs) in clinical trials, questions regarding the optimal means of DC preparation, in particular how to achieve optimal maturation, remain unanswered. We hypothesized that delivering two separate sequential maturation signals to DC in vitro, mimicking the process of DC maturation that occurs in vivo, would enhance the ability of DCs to generate antigen-specific effector T cells in an experimental in vitro antimelanoma model. MATERIALS AND METHODS: Human monocyte-derived DCs were transfected with mRNA encoding melanoma-associated antigen Mart-1 (MART) or influenza M1 matrix protein (M1). After mRNA transfection, DCs were left untreated or exposed to different maturation stimuli either added simultaneously or delivered sequentially 18 h after first stimulation. Phenotypic DC cell-surface marker changes and IL-12 secretion were analyzed. Specific antigen presentation by DCs was measured by IFN-gamma release Elispot assay using a CD8(+) MART peptide-specific T cell clone. RNA-transfected and treated DCs were cultured with autologous naive T cells and the induction of antigen-specific effector T cells were assessed by IFN-gamma release Elispot assay. RESULTS: DCs transfected and matured had increased cell-surface expression of CD40 and costimulatory molecules CD80, and CD86. DCs matured and further treated by soluble CD40 ligand (sCD40L) had a 10- and 2-fold increase in MART antigen presentation compared to untreated (immature) DCs and DCs treated only with a first maturation signal, respectively (Elispot P = 0.02). Delivery of sequential maturation stimuli resulted in maximal DC IL-12 secretion compared to simultaneous stimuli. Last, generation of antigen-specific effector T cells more than doubled with the sequential addition of sCD40L to mature DC stimulators (Elispot P = 0.009). CONCLUSIONS: Maturation of DCs following mRNA transfection increases expression of cell-surface costimulatory molecules. Delivery of a second sequential maturation stimulus enhances antigen presentation, increases IL-12 secretion, and augments immunogenicity as evidenced by generation of tumor antigen-specific effector T cells. This strategy should be considered in the future development of RNA-based DC vaccine strategies for the treatment of cancer.     

CD40L proinflammatory and profibrotic effects on proximal tubular epithelial cells: role of NF-kappaB and lyn

Chronic allograft nephropathy (CAN) is the main cause of renal graft loss, but its pathogenic mechanisms are still unclear. Immune system activation has been suggested as a key event in the development of CAN. CD40 is a co-stimulatory protein whose expression is upregulated in proximal tubular epithelial cells (PTEC) in acute rejection. This receptor interacts with CD40L, expressed by activated T cells. CD40L induces the production by PTEC of different proinflammatory cytokines, but very little is known of its profibrotic effects. The aim of this study was to investigate the effect of CD40/CD40L interaction on PTEC expression of plasminogen activator inhibitor-1 (PAI-1), a powerful profibrotic mediator, and monocyte chemoattractant protein-1 (MCP-1), a proinflammatory cytokine, and to investigate the signaling pathways that lead to these effects. Soluble CD40L induced a time-dependent increase in both PAI-1 and MCP-1 gene expression and protein production in PTEC. CD40 cross-linking on PTEC caused TNF-R-associated factors 2 and 6 membrane translocation. This event led to NF-kappaB activation, through the NF-kappaB-inducing kinase, and to a significant increase in the phosphorylation of lyn, a src-related tyrosine kinase. Lyn, upon phosphorylation, became strictly associated with caveolin-1, a scaffolding protein enriched in caveolae. Lyn inhibition did not have any effect on CD40L-induced NF-kappaB activation and MCP-1 expression but abolished PAI-1 induction. On the contrary, NF-kappaB inhibition significantly reduced only MCP-1 expression. In conclusion, CD40L could play a key role in the pathogenesis of CAN through PAI-1 induction. CD40L profibrotic and proinflammatory effects are mediated by different signaling pathways, suggesting that drugs that inhibit inflammation may not be equally effective in reducing fibrosis.     

Influence of albumin on expression of NLRP3 inflammasome in renal tubular epithelial cells

Objective To investigate the effect of albumin on expression of NLRP3 inflammasome and its downstream cytokines IL-1β and IL-18 in tubular epithelial cells. Methods Thirty mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria were selected, and their renal biopsy samples were stained by PAS and Masson to observe tubular epithelial cells injury and inflammatory cells infiltration. NLRP3, caspase-1, IL-1β and IL-18, as well as different inflammatory cells, were detected by immunohistostaining. In vitro, Western blotting and real-time PCR were employed to detect NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA in HK-2 cells stimulated by bovine serum albumin (BSA) (20 g/L). Results In MsPGN patients with high levels of proteinuria, there were obvious renal tubular epithelial cell injury and inflammatory cells infiltration (all P＜0.05), and the expressions of NLRP3, caspase-1, IL-1β and IL-18 were up-regulated compared to patients with low levels of proteinuria (all P＜0.05). Furthermore, IL-1β and IL-18 expressions were positively correlated with the degree of proteinuria (r=0.836, P＜0.05; r=0.901, P＜0.05). NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA were significantly increased in HK-2 cells stimulated by BSA compared to the control group (all P＜0.05). Conclusions Albumin is able to induce NLRP3 inflammasome activation in tubular epithelial cells, which may be the mechanism of tubulointerstitial injury and inflammation caused by proteinuria.     

The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells.

BACKGROUND: Activation of urinary complement proteins in situ by proximal tubular epithelial cells (PTEC) may contribute to the mediation of tubulointerstitial injury in patients with significant proteinuria. However, the mechanism involved is unclear, and the role of changes in urinary pH and in the concentrations of urea or ammonia requires further clarification. METHODS: The protein fraction of urine samples from nine patients with proteinuria >1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.5-8.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition. RESULTS: Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components. CONCLUSIONS: These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a 'protected site' on their surface, rather than by the activation of C3 by convertase-like protease(s).     

Effect of beta2-adrenergic receptor agonists on intercellular adhesion molecule (ICAM)-1, B7, and CD40 expression in mixed lymphocyte reaction

BACKGROUND: The plasma interleukin (IL)-18 level is elevated in acute rejection after organ transplantation. Although beta2-adrenergic receptor (AR) agonists suppress the rejection of organ and tissue transplants, little is known about their action mechanisms. We examined the effects of endogenous catecholamines and beta2-AR agonists on the expression of intercellular adhesion molecule (ICAM)-1, B7.1, B7.2, CD40, and CD40 ligand (CD40L) in human mixed lymphocyte reaction (MLR) and in an in vitro model of acute rejection in the presence or absence of IL-18. METHODS: ICAM-1, B7.1 B7.2, CD40, and CD40L expression on monocytes was measured by flow cytometry, and the production of interferon (IFN)-gamma and IL-12 was determined by enzyme-linked immunosorbent assay. Lymphocytes proliferation in MLR was measured by [3H]-thymidine uptake. The relevant AR subtypes were characterized using subtype-selective agonists and antagonists. RESULTS: beta2-AR agonists inhibited the expression of ICAM-1 and CD40 during MLR in the absence of IL-18. Among IL-18-induced expression of ICAM-1, B7.1, B7.2, CD40, and CD40L, beta2-AR agonists inhibited ICAM-1 and CD40 expression. beta2-AR agonists prevented the production of IFN-gamma and IL-12 in the presence of IL-18 but had no effect in the absence of IL-18. beta2-AR agonists inhibited lymphocyte proliferation in IL-18-treated MLR. CONCLUSIONS: We found that beta2-AR agonists strongly inhibited the expression of ICAM-1 and CD40, irrespective of the presence or absence of IL-18, which is different from that of histamine and prostaglandin E2.     

经HBx转染后的肾小管上皮细胞对T细胞激活、分化的影响

目的 探讨乙型肝炎病毒x基因(hepatitis B virus x,HBx)转染人近端肾小管上皮细胞系(HK-2)后其对T细胞激活、分化的影响.方法 分4组:实验组(即转染HBx质粒的HK-2细胞+CD4+T细胞)、阴性对照组(即转染HBx空载质粒的HK-2细胞+CD4+T细胞)、单独培养组(即CD4+T细胞)和空白对照组(未处理HK-2细胞+CD4+T细胞).体外培养HK-2,用分子克隆的方法构建pcDNA3.1-myc-HBx质粒,采用脂质体转染法瞬时转染HK-2细胞,实时荧光定量PCR及Western印迹法验证HBx在HK-2细胞中的表达.免疫磁珠法分选健康志愿者外周血CD4+T细胞,分别与HK-2细胞共培养,流式细胞术检测HK-2细胞共刺激分子CD40的表达、CD4+T细胞CD40配体(CD40L)表达及细胞周期情况,ELISA检测各组细胞培养上清中Th1型细胞因子IFN-γ及Th2型细胞因子IL-4水平.结果 HK-2细胞经转染pcDNA3.1-myc-HBx质粒后,可高表达HBx.转染HBx基因后HK-2细胞CD40表达显著上调(P＜0.01);与对照组相比,实验组CD4+T细胞表面CD40L表达也显著增加(P＜0.01),S期与G2/M期细胞数之和增多(P＜0.01),培养上清中IFN-γ水平升高(P＜0.05),IL-4水平降低(P＜0.05).结论 肾小管上皮细胞经HBx转染后,其共刺激分子表达上调,并促进CD4+T细胞增殖,诱导CD4+T细胞向Th1方向分化.由此推测,乙肝相关性肾炎肾组织免疫损伤所导致病变的持续进展也可能与此因素的参与相关.     

Triptolide is a potent suppressant of C3, CD40 and B7h expression in activated human proximal tubular epithelial cells

BACKGROUND: Previous studies have shown that triptolide possesses potent immunosuppressive and anti-inflammatory properties. Increasing recognition of the importance of the proximal tubular epithelial cells (PTEC) in renal disease and renal transplantation raises the question of whether triptolide suppresses the pro-inflammatory activity of PTEC. METHODS: Cultured human PTEC were exposed to tumor necrosis factor-alpha (TNF-alpha) and immunosuppressant (triptolide or CsA or FK506) for 24 hours, followed by RT-PCR, ELISA, flow cytometry and Western blotting analysis for complement C3, CD40, B7h expression. RESULTS: TNF-alpha up-regulated C3, CD40 and B7h production by PTEC. This up-regulation was inhibited by all three immunosuppressants with different intensity. Firstly, triptolide (4 to 8 ng/mL), CsA (4000 to 6000 ng/mL) and FK506 (2000 ng/mL) inhibited up-regulation of C3 mRNA, but CsA and FK506 had less of an effect than triptolide. Secondly, triptolide (4 to 8 ng/mL) completely inhibited C3 expression at both mRNA and protein levels. In contrast, CsA and FK506 had only slight effects on C3 expression at the protein level. Thirdly, triptolide (4 to 8 ng/mL), CsA (500 to 2500 ng/mL) and FK506 (1250 ng/mL) inhibited up-regulation of CD40 and B7h mRNA, the effect on B7h and CD40 mRNA expression by CsA and FK506 being greater than that on C3 mRNA expression. CONCLUSION: Triptolide effectively inhibited up-regulation of C3, CD40 and B7h on PTEC. Triptolide was more effective than CsA and FK506 at inhibiting C3 expression. This suggests that triptolide, at non-cytotoxic concentrations, has the potential to reduce the inflammatory and immunostimulatory properties of PTEC, in addition to any of the previously reported actions on T cell or B cell function.