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.     

The effects of immunosuppressive agents on in vitro production of human immunoglobulins.

We have evaluated the effects of CsA, methylprednisolone (MP), 6-mercaptopurine (6-MP), and FK506 on T cell-dependent and T cell-independent immunoglobulin production. FK506 and 6-MP were potent inhibitors of IgG and IgM production by PWM-stimulated peripheral blood mononuclear cells, which depend on the presence of T cells. CsA was less effective in this system and MP actually enhanced IgG and IgM production. In order to assess the direct effects of these various immunosuppressive agents on B cells, we utilized human B cell lines representing different stages of B cell differentiation. The B cell lines CESS and SKW6.4 exhibit increased production of IgG and IgM, respectively, in response to interleukin-6. These cells represent activated, but not fully differentiated, B cells. CsA inhibited IL-6-induced IgG production by CESS cells by 64% at 100 ng/ml and 6-MP inhibited this response by 82% at 250 ng/ml. Neither CsA nor 6-MP effectively inhibited IL-6-induced IgM production by SKW6.4 cells. MP at 250 ng/ml inhibited IL-6-induced IgG production by 89%, but enhanced IL-6-induced IgM production more than two-fold. FK506 did not inhibit IL-6-induced IgG or IgM production, suggesting that it has no direct effect on the ability of B cells to respond to this differentiation factor. These experiments clearly demonstrate that CsA, MP and 6-MP have direct inhibitory effects on the response of human B cells to IL-6. In contrast, FK506 has no direct effect on these B cells lines, but is more potent than the other agents at inhibiting T cell-dependent immunoglobulin production.     

The acute effect of FK506 and cyclosporine on endothelial cell function and renal vascular resistance.

We have previously documented that cyclosporine exerts a direct cytotoxic effect on endothelial cells and causes an increase in renal vascular resistance (RVR) in the rat. In the present study we investigated whether FK506, a novel immunosuppressive agent thought to be less nephrotoxic than CsA, impairs endothelial cell function in vitro and affects RVR in vivo. In vitro eicosanoid release and endothelin release were measured in bovine aortic endothelial cells in culture exposed for 1, 6, and 24 hr to increasing concentrations of FK506 (1 nM to 10 microM) or CsA (0.5, 10 microM). No significant changes in TxB2 and 6-keto-PGF1 alpha (the stable breakdown products of TxA2 and PGI2, respectively) and endothelin release were found after 1 and 6 hr of incubation with all the concentrations of FK506 and CsA considered. FK506 did not affect endothelin release even after 24 hr of incubation. In contrast, cell exposure to CsA was associated with a dose-dependent increase in TxB2, 6-keto-PGF1 alpha, and endothelin release that reached statistical significance after incubation with 10 microM CsA. FK506 did not induce cell detachment or lysis at any concentration and time considered, while 10 microM CsA induced a significant reduction in cell count accompanied by cell lysis. In vivo studies showed that a single i.v. injection of FK506 to rats within a broad range of doses (28 ng/kg to 2.8 micrograms/kg) did not modify RVR. This was true even for a dose as high as 20 mg/kg, while 20 mg/kg CsA caused a dramatic increase in RVR. We conclude that FK506, unlike CsA, does not induce endothelial cell injury in vitro. Whether this explains the differences in renovascular resistance observed in vivo after acute injection of FK506 and CsA is an attractive possibility that needs to be further explored.     

The effects of immunosuppressive drugs on the regulation of activation-induced apoptotic cell death in thymocytes.

This study investigated the effects of immunosuppressive drugs on the regulation of thymocyte sensitivity to clonal deletion via programmed cell death, or apoptosis. We have previously shown that TcR/CD3 cross-linking and intracellular stimuli that mimic TcR/CD3 cross-linking induce apoptosis in many immature thymocytes in the presence, but not in the absence, of cyclosporine (CsA). We have interpreted those results to suggest that TcR/CD3-associated signals induce a CsA-sensitive mechanism that protects the cells from activation-induced apoptosis. In the present study, we compared the effects of CsA, FK506, and rapamycin (RAP) on the regulation of thymocyte apoptosis. Optimal concentrations of CsA and FK506 augmented apoptosis to similar levels. However, FK506 was approximately 100-fold more potent than CsA in thymocytes, which parallels the relative potencies of these drugs in inhibiting mitogen-induced proliferation of mature T cells. In contrast to CsA and FK506, RAP did not exhibit substantial apoptosis-augmenting activity. However, RAP interfered with the activity of FK506. This pattern mirrors that of RAP in TcR/CD3-mediated signaling pathways in mature T cells. Together these results provide evidence (1) that CsA, FK506, and RAP can act on immature thymocytes, (2) that the mechanisms by which the drugs affect mature and immature T cell responses are similar, and (3) that immunosuppressive drug therapy may affect not only mature peripheral T cells but also developing immature thymic T cells.     

CD154 is expressed during treatment with calcineurin inhibitors after organ transplantation

BACKGROUND: CD154 (CD40 ligand) monoclonal antibody prevents allograft rejection in rodents and monkeys. Inasmuch as calcineurin inhibitors (CI) inhibit CD154 expression by pharmacologic agents in vitro, we investigated whether CD154 is also inhibited by CI in vivo and in vitro during allogeneic stimulation. METHODS: CD154 expression was determined by immunohistochemistry and flow cytometry in human lymph nodes and spleen sections from rhesus monkeys with or without CI treatment. The effect of CI on induction of CD154 expression was studied by stimulating lymphocytes with phorbol 12-myristate 13-acetate (PMA) and ionomycin or with allogeneic monocyte-derived mature dendritic cells. RESULTS: Lymph nodes from patients with or without CI cyclosporine (CsA) or FK506 (FK) treatment showed comparable CD154 expression, which was present on the cell surface of T cells. CD154-expressing cells were also present in spleens from monkeys treated with CsA in comparable numbers to those in the nontreated group. Moreover, in several liver transplant rejection biopsies taken during CI therapy CD154-expressing cells were observed. In vitro, CsA and FK strongly inhibited induction of CD154 expression on peripheral blood mononuclear cells by pharmacologic stimuli. Maximum inhibition was found at 50 ng/ml CsA and 20 ng/ml FK. CD154 expression induced by dendritic cells in peripheral blood mononuclear cells or spleen cells was also almost completely inhibited by CsA. CONCLUSION: Although CI strongly suppressed pharmacologic and allogeneic induction of CD154 expression on T cells in vitro at concentrations at approximately clinical trough levels, CD154 is prominently expressed during CI therapy in lymphoid tissue and (sporadically) in liver allografts. This suggests that the CD154-CD40 pathway remains functional during CI therapy, which may contribute to allograft rejections in the clinical setting.     

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-gamma)- and double-positive IL-2/IFN-gamma-producing whole T cells, CD4 + and CD8 + T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n = 7) or on FK506 (n = 7). Cytokine-expressing T-cell frequencies were assessed immediately pretransplantation (DO), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at DO the frequencies of IL-2-(22 +/- 2% vs. 22.2 +/- 2%), IFN-gamma-(26 +/- 3% vs. 29 + 3.4%) and IL-4-(0.8 +/- 0.2% vs. 1.4 +/- 0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-gamma double positive cells was higher in the latter (9.3 +/- 1.6%) than in the controls (5.6 +/- 0.8); p = 0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1%, in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (DO): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-gamma-expressing T cells, whereas those of IFN-gamma, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-gamma-expressing T cells affected CD4 + and CD8 + cells equally; (d) there was a marginal decrease in the frequency of IFN-gamma-expressing cells only in the CD4 + subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9+/-1.61%) than with CsA (16.3 +/- 1.8%; p = 0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-gamma in vivo but do not affect those expressing IFN-gamma. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.     

Tacrolimus reversibly reduces insulin secretion in paediatric renal transplant recipients.

BACKGROUND: Conflicting reports exist about the mechanism of tacrolimus-induced post-transplant diabetes mellitus. METHODS: We analysed intravenous glucose tolerance tests (IVGTT) of 14 paediatric renal transplant recipients on cyclosporin (CsA) microemulsion and 15 patients on tacrolimus (FK506). The groups were similar in age (13.2+/-4.2 vs 13.0+/-3.7 years), body mass index, serum creatinine concentrations (96+/-60 vs 97+/-44 micromol/l), time after renal transplantation, and cumulative steroid dose over 12 weeks prior to the test (3.4 vs 3.5 mg/m(2)/day, NS, Mann-Whitney). Parameters of glucose tolerance included glucose, insulin, C-peptide concentrations, and HbA1c. The mean concentrations of the primary immunosuppressant were similar to treatments employed in other centres (CsA 165+/-59 ng ml and FK506 7. 5+/-2.2 ng ml). RESULTS: Baseline glucose concentrations were significantly higher on FK506 therapy compared with CsA microemulsion therapy. Baseline insulin concentrations and C-peptide concentrations were identical in both treatment groups. FK506 trough levels correlated negatively with k values (glucose constant decay) in the FK506 group. There was a significant reduction of the insulin first-phase concentrations, both after 1 min and after 3 min in the FK506 group compared with the CsA group (112+/-17 vs 237+/-57 microU/ml, P=0.034). In patients with repetitive IVGTTs, glucose constant decay and insulin production improved after lowering FK506 whole-blood trough levels. CONCLUSIONS: We conclude that post-transplant glucose intolerance could be due to a dose-dependent, direct effect of FK506 on the pancreatic beta cell function, which can be controlled by dose reduction.     

Superior T-cell suppression by rapamycin and FK506 over rapamycin and cyclosporine A because of abrogated cytotoxic T-lymphocyte induction,impaired memory responses,and persistent apoptosis

Immunosuppressive therapy is best achieved with a combination of agents targeting multiple activation steps of T cells. In transplantation, cyclosporine A (CsA) or tacrolimus (FK506) are successfully combined with rapamycin (Rap). Rap and CsA were first considered for combination therapy because FK506 and Rap target the same intracellular protein and thus may act in an antagonistic way. However, in clinical studies, FK506+Rap proved to be effective. To date, there is no in vitro data supporting these in vivo findings, and it is unclear whether the observed effects are T-cell mediated. In a human polyclonal allogeneic in vitro model, we found that although combined drug treatment markedly reduced expansion of naive T cells, T-cell activation occurred irrespective of the drug combination used. The induction of cytotoxic effector T cells was reduced by CsA+Rap but completely abolished by FK506+Rap. Importantly, combined immunosuppression allowed generation of memory CD4+ and CD8+ T cells and hence did not result in T-cell anergy. However, FK506+Rap treatment resulted in a reduced number of allospecific memory T cells showing a decreased cell-cycle turnover and cytokine producing capacity. In contrast, CsA+Rap treatment led to increased memory T-cell numbers responding with elevated kinetics. The ability of Rap to promote apoptosis, which contributes to T-cell suppression, remained unaffected upon combination with FK506 or CsA. These data support the combined use of FK506+Rap over CsA+Rap for immunosuppressive therapy.     

The distinct effects of FK506 on the activation, proliferation, and differentiation of human B lymphocytes.

We examined the effect of FK506 on the activation, proliferation and differentiation of human B lymphocytes in vitro. FK506 inhibited the proliferative response of resting B cells induced by Staphylococcus aureus Cowan strain I (SAC) and phorbol myristate acetate (PMA) in a dose-dependent manner. Inhibition of cell proliferation by FK506 was caused by a selective block of G0 to G1 phase transition leading to cell arrest. In addition, the proliferative response of in vivo-activated B cells and lymphokine-driven B cell proliferation were also found to be sensitive to FK506. Interestingly, FK506 did not affect the expression of activation antigens such as CD23, IL-2 receptor (CD25), and transferrin receptor (CD71). Finally, FK506 had little effect on B cell antibody generation in a T cell-independent system. Conversely, FK506 suppressed neither proliferation nor immunoglobulin secretion in a human B lymphoblastoid cell line. These results indicate that FK506 has discrete effects on the different stages of the B cell maturation.     

FK506对许旺细胞体外增殖和分泌NGF的影响

目的 研究FK506促进许旺细胞体外增殖及对许旺细胞(SOs)分泌NGF的影响. 方法 将纯化的许旺细胞分6组:A组(空白对照组):含10%胎牛血清的DMEM/F12;B组:0.1 ng/mlFK506;C组:0.5 ng/ml FK506;D组:1.0 ng/ml FK506;E组:5.0 ng/mk FK506;F组:10 ng/ml FK506.将许旺细胞于倒置显微镜下观察并用S-100蛋白免疫组化染色鉴定;MTT法筛选FK506促SCs增殖的最佳作用浓度;流式细胞仪检测SCs周期;ELISA法检测培养72 h后SCs的NGF的分泌量. 结果 MTT法筛选:0.5 ng/ml FK506为促进SCs增殖的最佳作用浓度;当FK506浓度大于1.0 ng/ml时,SCs的生长活性逐渐下降并随着FK506浓度的逐渐增高,SCs的生长活性受抑制作用逐渐加强.流式细胞仪检测:含10%胎牛血清的DMEM/F12培养24 h、48 h、72 h,SCs S期百分比分别为:27.8%,39.3%和58.4%;0.5 ng/mlFK506培养24 h、48 h、72 h,SCs S期百分比分别为:54.2%、60.3%和94.6%.EUSA法检测FK506促SCs增殖后表达NGF的实验研究中发现:0.5 ng/ml FK506作用72 h后的SCs所分泌的NGF高达0.188 ng/ml. 结论 FK506应用于体外培养的SCs初期就能促进SCs增殖并使其保持良好的活性而高分泌NGF.     

Immunomodulatory properties of FK734, a humanized anti-CD28 monoclonal antibody with agonistic and antagonistic activities

BACKGROUND: We describe immunomodulatory effects of FK734, a humanized version of a mouse anti-human CD28 mAb (clone TN228), in vitro and in a chimeric human-mouse model of allograft rejection. METHODS: Cytokine production and proliferation were assessed in a mixed lymphocyte reaction containing FK734, human T cells, and endothelial cells or monocytes. FK734 was also administered to SCID mice engrafted with human skin and adoptively transferred with human peripheral blood mononuclear cells allogeneic to the skin graft. RESULTS: In vitro, FK734 enhanced secretion of interleukin-2 and interferon-gamma as well as proliferation of CD4+ and CD8+ T cells stimulated by allogeneic human leukocyte antigen (HLA)-DR+ human umbilical vein endothelial cells (which lack B7 molecules and FcgammaRs) or by blood monocytes (which express low levels of B7 molecules and FcgammaRs) compared with control mAb, but these effects were significantly smaller than those provided by mAb 28.2, a stimulatory mouse anti-human CD28 mAb, at comparable concentrations. However, FK734 generally inhibited cytokine secretion and T cell proliferation in cocultures with human umbilical vein endothelial cells transduced to express CD86. In vivo using SCID/beige mice bearing human skin with adoptively transferred peripheral blood mononuclear cells, administration of FK734 protected human endothelial cell-lined microvessels, significantly but incompletely reducing endothelial cell injury and T cell infiltration into the graft one or two weeks later. CONCLUSIONS: FK734 is a partial agonist of CD28 signaling that can reduce human T cell alloresponses in the presence of strong costimulation by B7 molecules in vitro and can reduce T cell-mediated skin allograft rejection in vivo.     

环孢素A与他克莫司对5-氟尿嘧啶治疗肝癌的影响及机制

目的　探讨环孢素A(CsA)与他克莫司 (FK5 0 6 )对 5 氟尿嘧啶 (5 FU)治疗肝癌的影响及机制。　方法　取DENA诱导的近交系wistar雄性原发性肝癌大鼠 ,随机分为A组 (肝癌鼠 )、B组(肝癌鼠加 5 FU)、C组 (肝癌鼠加 5 FU加CsA)、D组 (肝癌鼠加 5 FU加FK5 0 6 )处理 3d后取标本 ,光镜、流式细胞仪、激光共聚焦显微镜、透射电镜分别检测各组肝癌细胞周期 ,细胞凋亡、坏死定量及形态学改变 ,线粒体膜电位的变化。结果　D组G1/G0期肝癌细胞百分率明显减少 (P =0 0 0 ) ;S期肝癌细胞所占百分率各组之间差异均有显著意义 (P <0 0 5 )。凋亡细胞百分率加坏死细胞百分率各组之间差异均有显著意义 (P =0 0 0 0 )。透射电镜对照组呈正常肝癌细胞超微结构 ,治疗组可见大量不同时期的凋亡细胞和少量坏死细胞。肝癌细胞线粒体膜电位治疗组与对照组相比均有显著性下降 ,以B组下降最多 (P <0 0 5 )。　结论　CsA和FK5 0 6均对 5 FU治疗肝癌有协同作用 ,FK5 0 6在5 FU治疗时可促使G0 /G1期肝癌细胞向S期转化 ,CsA对肿瘤细胞周期无明显影响。     

The immunosuppressant rapamycin,alone or with transforming growth factor-beta,enhances osteoclast differentiation of RAW264.7 monocyte-macrophage cells in the presence of RANK-ligand

Immunosuppressant therapy is known to cause bone loss. Since this may partly result from direct effects on osteoclast development, we investigated whether cyclosporin A (CsA), rapamycin, or FK506 affect osteoclastic differentiation of RAW264.7 monocytic cells induced by RANK-ligand (RANKL). Furthermore, since the rapamycin receptor protein binds transforming growth factor beta (TGF-beta) receptors, and TGF-beta enhances osteoclastogenesis induced by RANKL, we also examined potential synergistic effects of rapamycin and TGF-beta1. Rapamycin inhibited cell proliferation and stimulated tartrate-resistant acid phosphatase (TRAP) activity of RAW cells in a dose-dependent manner. At the optimal concentration of 10 ng/ml, it increased the number of TRAP+ multinucleated cells (MNC) more than 20-fold and enhanced the expression of TRAP and calcitonin receptor (CTR) mRNAs 2.1- and 10-fold, respectively. CsA, at 125-2000 ng/ml, similarly inhibited proliferation, but at high doses (1000-2000 ng/ml) it decreased TRAP activity, TRAP+MNC formation, and the expression of TRAP and CTR mRNAs. FK506 had no effect on cell proliferation or TRAP activity at concentrations up to 2000 ng/ml; however, like CsA, 1000 ng/ml FK506 inhibited TRAP+MNC formation and the expression of TRAP and CTR mRNAs. The combination of rapamycin (10 ng/ml) and TGF-beta1 (1 ng/ml) increased TRAP+MNC 3.1- and 6.9-fold as compared with rapamycin or TGF-beta1 alone, respectively, and enhanced CTR mRNA expression induced by TGF-beta1 by 1.9-fold. Rapamycin also increased osteoclastic resorption activity by 6.5-fold compared with control, and this was enhanced further by the addition of TGF-beta by 3-fold, compared with rapamycin alone. These data thus indicate that rapamycin, alone or in synergy with TGF-beta, directly enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.     

A comparison of the sensitivity of pig and human peripheral blood mononuclear cells to the antiproliferative effects of traditional and newer immunosuppressive agents

Difficulty in preventing rejection of fetal pig islet-like cell clusters (ICCs) transplanted into pigs using traditional forms of immunotherapy has been reported. An in vitro study of the efficacy of seven different immunosuppressive agents to inhibit proliferation of pig peripheral blood mononuclear cells (PBMC) was performed, and a comparison was made between the human and pig to determine if the efficacy of these agents differed between species. The efficacy of cyclosporine (CsA), azathioprine (Aza), methylprednisolone (MP), FK506, rapamycin (RAP), mycophenolate mofetil (MMF) and deoxymethylspergualin (MeDSG) to inhibit pig and human PBMC proliferation in mitogenic experiments using phytohaemagglutinin (PHA) as a stimulus was performed. Further, allogeneic pig mixed lymphocyte reactions (MLR) were used to determine the activity of these agents in a model more comparable to the allograft rejection process. It was found that pig PBMC stimulated with PHA or in a MLR were inhibited by the agents tested, with the exception of MeDSG that was ineffective in mitogenic experiments. The inhibitory effects of these agents differed between PHA and MLR, the respective (50% inhibitory concentration) IC50 values for pig PBMC being 1.7 and 0.08 microg/ml for CsA, 1.4 and 4.4 microg/ml for Aza, 0.11 and 0.002 microg/ml for MP, 3.0 and 2.8 ng/ml for FK506, 2.1 and 0.3 ng/ml for RAP and 10.8 and 454 ng/ml for MME Pig PBMC were less sensitive than human PBMC to the antiproliferative effects of CsA, Aza, FK506, RAP and MMF, but not MP on PHA stimulation, the ratio of the pig to human IC50 values being 19, 11, 13, 2.3, 1.4, and 0.4, respectively. These data suggest that the doses of most immunosuppressive agents administered to prevent rejection in pigs need to be higher than those used to achieve therapeutic benefit in humans.     

Different effects of cyclosporine a and FK506 on potassium transport systems in MDCK cells

BACKGROUND: Hyperkalemia and metabolic acidosis are common manifestations in patients receiving the immunosuppressive agent cyclosporine A (CsA) and the recently introduced FK506. We compared the acute toxic and antiproliferative effects as well as the effects on the transport activity of Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter of CsA and FK506 in an established cell line of distal/collecting tubule origin (MDCK cells). METHODS: MDCK cells were exposed to various concentrations of CsA or FK506 and the effects on cell viability (MTT test and neutral red uptake), plasma membrane integrity (lactate dehydrogenase (LDH) release) and cell proliferation (bromodeoxyuridine (BrdU) incorporation) were compared. For transport studies, after confluence, MDCK cells were exposed to CsA or FK506 for 48 h in the presence and absence of aldosterone. Ouabain- and bumetanide-sensitive (86)Rubidium uptake measurements were used to study the activity of the Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter at the surface of intact cells. Results: After 24 h of exposure CsA reduced the number of viable cells to 50% at 30 microM, whereas for FK506 2-3 times higher concentrations had to be employed. Similarly, LDH release was stimulated tenfold by 30 microM CsA but only fourfold by 70 microM FK506. In contrast, DNA synthesis was affected at lower concentrations of FK506 than of CsA. In cells treated for 24 h BrdU incorporation was significantly inhibited by 3 microM FK506, whereas a similar inhibition required 10 microM CsA. The transport activity of Na(+)/K(+)-ATPase and of Na(+)/K(+)/2Cl(-) cotransporter were significantly decreased (37 and 63%, respectively) on CsA administration (8 microM). In CsA-treated cells the K(+) channel blockers barium (1 mM), TEA (10 mM) and quinine (1 mM) did not further inhibit the transport activities suggesting that CsA might also act via inhibition of K(+) channels. FK506 at 8 microM had no effect on Na(+)/K(+)-ATPase transport activity but stimulated Na(+)/K(+)/2Cl(-) cotransporter activity by 59%. The stimulatory effect was abolished by K(+) channel blockers indicating that recycling of K(+) might increase by FK506. The simultaneous presence of aldosterone (5 microM) protected the cells from the inhibitory effect of CsA on Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter activity. The stimulatory effect of FK506 on the Na(+)/K(+)/2Cl(-)cotransporter activity was completely abolished in the presence of aldosterone. Conclusions: Both CsA and FK506 showed acute toxicity in MDCK cells in vitro with the effects of FK506 being less pronounced. CsA and FK506 had different effects on the in vivo transport rates of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter; CsA inhibited the activity of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter whereas FK506 stimulated the activity of Na(+)/K(+)/2Cl(-) cotransporter. These effects were abolished by the application of aldosterone.     

Importance of CD49d- VCAM interactions in human monocyte adhesion to porcine endothelium

Abstract: By using a primate model of natural antibody depletion, we have previously shown that delayed rejection of porcine cardiac xenografts in unmodified primate recipients resulted from xenograft infiltration with monocyte/macrophage lineage cells. In the present study, we initially showed that human monocytes/macrophages demonstrated significantly greater adherence to unstimulated pig aortic endothelial cells (PAEC) than to human umbilical vein endothelial cells (HUVEC). Human TNF-alpha augmented monocyte adhesion to HUVEC by 5-fold higher levels than to PAEC. This effect could not be explained on the basis of incompatibility between human TNF-alpha and its receptor on PAEC since porcine VCAM expression increased by 75–85% after stimulation with TNF-alpha. TNF-augmented monocyte adherence was abrogated by either treatment of PAEC with an anti-VCAM Mab or monocytes with an anti-CD49d Mab. These results suggest that VCAM-CD49d interactions are important in adhesion of human monocytes to PAEC but may not be as effective as those between human monocytes and allogeneic endothelium, perhaps because of structural differences across species. Other interactions, as yet undefined, must explain the relative increase in adhesiveness of human monocytes for unstimulated PAEC versus HUVEC. In experiments investigating the functional consequences of this enhanced monocyte adherence, PAEC stimulation induced 10-fold higher levels of macrophage-derived IL-1 beta and 3-fold higher levels of T cell proliferation compared with HUVEC. Using an anti-DR Mab to interrupt antigen presentation by autologous macrophages markedly reduced the T cell proliferative response to PAEC. Together, these results indicate that the enhanced adherence of human monocytes to PAEC contributes to xenograft rejection beyond the hyperacute period by leading to tissue infiltration, elaboration of cytokines, and an augmented indirect pathway of T cell xenoantigen recognition.     

Effect of FK506 on donor T-cell functions that are responsible for graft-versus-host disease and graft-versus-leukemia effect

BACKGROUND: FK506 is a potent immunosuppressive agent that is used in human graft-versus-host disease (GvHD) prevention. However, the precise mechanisms for GvHD prevention and the effect on graft-versus-leukemia (GvL) activity are unknown. This study was undertaken to determine the effect of FK506, given at clinically relevant doses, on donor T-cell functions responsible for GvHD and GvL activity. METHODS: The effect of FK506 on GvHD prevention and GvL activity was investigated using a murine model of allogeneic bone-marrow transplantation in which mice were injected with a P815 leukemic cell line. The regulatory role of FK506 on donor T cells was tested by analysis of donor T-cell expansions in the spleen and donor anti-host T-cell proliferative and cytotoxic responses. mRNA expression of type 1 T helper (Th1), Fas ligand (L), and granzyme B were also evaluated in target organs of GvHD. RESULTS: FK506 significantly prolonged the survival of GvHD mice when given at the trough level of 17.6 ng/mL, whereas it also blocked GvL effect in P815-injected GvHD mice. FK506 reduced the expansion of donor CD8+ and, to a lesser extent, CD4+ T cells in the spleen and inhibited donor anti-host T-cell proliferative and cytotoxic responses. It also inhibited the induction of Th1, FasL, and granzyme B mRNA expression in target organs of GvHD. CONCLUSIONS: FK506 inhibits both GvHD and GvL activity when given at clinical doses by inhibiting donor T-cell expansion, donor anti-host T-cell reactivity, and Th1 immune responses.     

Immunosuppressive activity, lymphocyte subset analysis, and acute toxicity of FK-506 in the rat. A comparative and combination study with cyclosporine

The immunosuppressive and toxic properties of the recently discovered macrolide antibiotic FK506 were examined in comparison and in conjunction with cyclosporine administration in the rat. Male Sprague-Dawley rats were immunized systemically with sheep erythrocytes and received, from the same time, either FK506 (1 mg/kg/day) intramuscularly or CsA (25 mg/kg/day) by gavage, or both drugs in combination. Seven days after immunization, the splenic plaque-forming cell response and circulating antibody titers were reduced greater than 90% in animals receiving either FK506 or CsA and in the drug combination group. These immunosuppressive effects of FK506 and CsA were accompanied by significant increases in the incidences of splenic OX-8+ cells and by corresponding reductions in the W3/25+:OX-8+ ratio. No further changes in T cell populations were observed in animals given both drugs. A progressive monocytosis was found in response to CsA, but not in FK506-treated rats. Increases in plasma urea were observed in FK-506 and drug-combination or CsA-treated rats on day 7, whereas creatinine levels were raised only in the FK-506 groups. Elevated bilirubin levels and alterations in liver enzyme activities were observed in CsA-treated rats by day 4, whereas FK-506 alone produced no similar effects. CsA-treated rats also exhibited elevated blood and urinary glucose levels from day 4. No biochemical evidence of additive drug toxicity was detected. The only histological abnormalities observed were thymic medullary atrophy in all drug-treated animals, together with very minor reductions in bone marrow cellularity in a proportion of those rats given FK-506. These findings show that, at the dosage selected, the powerful immunosuppressive activities of FK-506 were associated with little evidence of acute toxicity and with no indications of additive toxicity with CsA.