In vitro immunosuppressive properties of cyclosporine metabolites

The in vitro biological activity of cyclosporine (CsA) and four of its metabolites (M1, M8, M17, and M21) was determined. M1, M17, and M21 are primary metabolites, while M8 is a secondary metabolite derived from either M1 or M17. The order of inhibitory activity in production assays was phytohemagglutinin (PHA), concanavalin A (ConA), mixed lymphocyte culture (MLC), and interleukin-2 (IL-2) CsA greater than M17 greater than M1 greater than M21 much greater than M8. In the PHA assay, CsA was significantly more inhibitory than M17, but in Con A and MLC assays, the inhibitory activity of M17 approached that of CsA. More importantly, M17 and M1 inhibited the production of IL-2 in the MLC to the same extent as CsA. M21 was significantly less inhibitory than either M17 or M1, and M8 appeared to be largely devoid of biological activity. These experiments demonstrate that single hydroxylations of amino acids 1 (M17) and 9 (M1) do not significantly affect the ability of the molecule to block IL-2 production, but hydroxylation of both amino acids renders the molecule virtually inactive. In addition, the presence of the N-methyl group on amino acid 4 appears to be very important, since removal of this group (M21) greatly diminishes the immunosuppressive activity.     

Dyslipidemia can reduce the immunosuppressive effects of cyclosporine

AIMS: Dyslipidemia is a significant risk factor for the development of atherosclerotic disease and of chronic allograft rejection. Few data are available on the effects of dyslipidemia on the immunosuppressive action of immunosuppressive agents. We investigate the in vitro effects of lipids solution on the immunosuppressive action of cyclosporine (CsA). METHODS: Peripheral blood mononuclear cells (PBMC) were PHA or OKT3 activated in vitro with/without different concentrations of Intralipid solution (INT, range 0.5% to 15%). CsA inhibition of activation was measured after a 3 day incubation, by adding H3-thimidine. The intracellular concentration of CsA was measured by radioimmunoassay and related to the CsA inhibitory effects. RESULTS: Increasing INT concentration in the medium, CsA inhibition of PBMC activation by PHA or OKT3 was reduced from 72+/-13% to 8+/-2% and from 80+/-10% to 18+/-3%, respectively. A significant reduction of the intracellular CsA concentration was also evident with increasing INT concentrations and was related to the inhibitory activity of CsA. CONCLUSIONS: These results suggest that dyslipidemia may reduce the availability of intracellular CsA concentration to inhibit the immune activation process and may explain the relationship between dyslipidemia and chronic allograft loss.     

A comparison of the inhibitory effects of immunosuppressive agents cyclosporine, tetranactin, and didemnin B on human T cell responses in vitro.

The agents cyclosporine, tetranactin (TN), and didemnin B (DB) were compared for their ability to inhibit proliferative human T cell responses in vitro, using anti-CD3, PHA, alloantigen, or tetanus toxoid as stimuli and using monocytes or Langerhans cells as antigen-presenting cells/accessory cells (APC/AC). We found that all three agents suppressed T cell activation in a dose-dependent fashion, irrespective of the stimulus of APC/AC type used. Both T cells and APC/AC were affected by the drugs. DB appeared to be the most potent suppressive drug (IC50 = 1-4 ng/ml), whereas CsA and TN exerted approximately similar potency (IC50 = 50-60 ng/ml). Remarkably however, DB was toxic at a concentration of 10 ng/ml, which is quite close to the inhibition-inducing dose. No toxicity was observed with CsA and TN at doses up to 5000 ng/ml. The agents TN and DB could interrupt ongoing T cell responses and could block responsiveness to exogenous recombinant IL-2. Expression of IL-2 receptors was slightly inhibited by all three drugs. Expression of MHC class II molecule HLA-D and of adhesion molecules LFA-1, LFA-3, and ICAM-1 was clearly reduced by DB, giving an explanation for the observed inhibition of cluster formation between T cells and APC/AC. Except for a slight reduction of LFA-3 by TN, CsA and TN did not affect the expression of any of these cell surface markers or the formation of clusters. Differences in the effects of CsA, TN, and DB on immune responses in vitro and on the phenotype of T cells and APC/AC suggest that these immunosuppressive drugs have different inhibitory mechanisms.     

Effect of interleukin 2 on the immunosuppressive action of cyclosporine

The influence of exogenous interleukin 2 (IL-2) on the immunosuppressive effect of cyclosporine in the mixed lymphocyte response (MLR) was examined. Results show that addition of exogenous IL-2 to a MLR containing graded doses of CsA (0.01-2.5 micrograms/ml) restored a normal proliferative response to alloantigens. In contrast, the effect of exogenous IL-2 on the induction of cytotoxic lymphocytes in primary MLR in the presence of CsA was variable. At the highest doses of CsA (0.5-2.5 micrograms/ml), no cytotoxic T cell activity could be detected, regardless of the presence of exogenous IL-2. However, at a lower dose of CsA (0.1 microgram/ml) that routinely resulted in the total inhibition of cytotoxic T cell induction, addition of exogenous IL-2 resulted in significant levels of detectable cytotoxic T cell activity. The effect of time-sequential addition of CsA or CsA-plus-exogenous-IL-2 on the proliferative and CML responses in MLR was also examined. Results show that addition of CsA to ongoing primary MLR cultures within the first 48-96 hr of culture results in the significant inhibition of the proliferative and CML response in MLR. Addition of CsA-plus-exogenous-IL-2 to ongoing cultures resulted in no significant inhibition of the proliferative response. In contrast, addition of CsA-plus-exogenous-IL-2 within the first 4 hr of culture did not overcome the immunosuppressive effect of CsA. At 18 hr of culture addition of CsA resulted in complete suppression of the CML response, whereas the addition of CsA-plus-IL-2 resulted in significant levels of cytotoxicity. Thereafter addition of CsA-plus-IL-2 resulted in enhanced levels of cytotoxic T cell activity compared with cultures receiving CsA alone. Taken together, our results suggest that: (1) exogenous IL-2 can overcome the immunosuppressive effect of CsA on the proliferative response in MLR to alloantigens; (2) at high levels of CsA, IL-2 cannot overcome the immunosuppressive effect of CsA on the induction of cytotoxic T-lymphocytes; (3) there are doses of CsA at least in vitro, that allow for the activation of the cytotoxic T cell, presumably with the acquisition of a receptor for IL-2 but without the clonal amplification due to inhibition of IL-2 production; and (4) time-sequential studies revealed that the development of responsiveness to IL-2 by the precursor cytotoxic T cell occurs 4-18 hr after exposure to the stimulating alloantigen with clonal expansion if IL-2 is present.     

Isolation and in vitro characterization of a novel immunosuppressive cyclosporine metabolite

A novel metabolite (M-E) was identified by high-performance liquid chromatography in the serum of cyclosporine-treated renal transplant recipients during a second wave of immunosuppressive activity after disappearance of the initial wave due to the direct effect of CsA. M-E was identified in human serum and porcine bile both by HPLC and by a preparative thin-layer chromatography (TLC). It demonstrated homogeneity with characteristic retention times on C8 and C18 column HPLC systems using a variety of elution systems, and distinctive TLC mobility (Rf 0.35). Metabolite E (M-E) was documented to be a CsA metabolite by radioactive tracer studies, by crossreactivity with a polyclonal sheep antibody in radioimmunoassay, and by the presence of a characteristic mass spectrum. Further, in vitro immunosuppressive assays documented effects of M-E similar to those of CsA. The relative activity of M-E versus CsA was quantitated by potency ratios: for inhibition of normal human mixed lymphocyte culture reactions, the ratio was 0.79 +/- 0.23. Interindividual differences were observed in patient susceptibility to MLR inhibition not only by CsA, as previously reported by others, but also by M-E. There was a lesser effect of M-E compared with CsA in inhibiting proliferation of, and IL-2 generation by, C3H murine splenocytes stimulated with concanavalin A: the potency ratios for both systems were about 0.5, possibly reflecting an interspecies variability in generation of or susceptibility to M-E. These studies suggest that heretofore unidentified metabolites--including, but not limited to, M-E--may play an important role in the immunosuppressive effect of CsA in man.     

Evidence that the immunosuppressive effects of FK506 and cyclosporine are identical.

The fungal metabolite FK506 was discovered because it shared an important property, the ability to inhibit production of IL-2, with another well-known immunosuppressive fungal metabolite, CsA. FK506 has, since its isolation, also been shown to share other immunosuppressive effects with CsA. This study was performed to further investigate the in vitro immunological properties of FK506, in comparison with and in combination with CsA, to evaluate the plausibility that their mechanisms of action were identical or similar, in spite of their different molecular structures. The ability to inhibit several responses of human peripheral blood lymphocytes to mitogenic and alloantigenic stimulation was explored. Synergistic effects of the two drugs were extensively studied, since this would provide additional information regarding their mechanisms of action. Also, similar immunosuppressive properties would enable the use of the two drugs in combination. We found that FK506 and CsA had very similar mechanisms of action and additive effects were recorded.     

The importance of the spleen for the immunosuppressive action of cyclosporine in transplantation

The spleen plays an important role in the response of the recipient's immune system to a primarily vascularized graft and cyclosporine treatment is known to alter this response. To investigate the interaction between the splenic immune response and CsA's immunosuppressive actions more thoroughly, Lewis recipients of Brown-Norway heterotopic heart grafts were treated i.p. daily with normal saline or with CsA doses of 0.75, 1.5, or 3.0 mg/kg/day from day 1 through day 50 or until rejection. Rats treated with 3 mg/kg were splenectomized intraoperatively (i.o.) or not splenectomized. Rats in subgroups of the other treatment groups were splenectomized i.o., on day 5, not splenectomized, or the recipient's spleen cells were reinfused after i.o. splenectomy. In non-CsA-treated rats, i.o. splenectomy (median survival time, [MST] = 11 days) and day 5 splenectomy (MST = 11 days) prolonged graft survival minimally in comparison with nonsplenectomized animals (MST = 7 days). Reinfusion of the spleen cells reversed this effect (MST = 7 days). Most interestingly, the immunosuppressive efficacy of 1.5 mg/kg of CsA (MST = 91 days) was reduced by day 5 splenectomy (MST = 24 days) and completely abolished by i.o. splenectomy (MST = 11 days). Spleen cell reinfusion partially restored the effect of CsA treatment (MST = 88 days). Since splenectomy resulted in a complete abrogation of the immunosuppressive efficacy of 1.5 mg/kg CsA, our results support the hypothesis that certain spleen cells augment immunosuppression by CsA. These findings provide additional evidence that the immune system's own regulation of its antigraft response can be an important component of the overall suppression of rejection that is associated with the use of certain immunosuppressive drugs.     

The immunosuppressive antagonism of low doses of FK506 and cyclosporine.

Clinical immunosuppression with potentially toxic agents may be optimized by combining drugs that act synergistically at low doses. The studies presented herein attempted to apply this strategy to the macrolide FK506 and the endecapeptide cyclosporine, which similarly inhibit T cell responses but display distinctive arrays of toxic side effects. The interaction between these agents was subjected to rigorous pharmacologic analysis using the median effect and combination index equations to determine synergistic, antagonistic, or additive drug interactions. FK506 and CsA showed pharmacologic antagonism in inhibiting in vitro proliferation upon phytohemagglutinin, anti-CD3 antibody, and mixed lymphocyte reaction (MLR) stimulation, and interleukin 2 generation by activated normal human peripheral blood lymphocytes. The antagonistic relationship was confirmed in vivo using low doses of FK506 in combination with CsA to treat Wistar-Furth recipients of heterotopic Buffalo rat cardiac allografts, a major plus minor histocompatibility barrier. This antagonistic relation suggests that FK506/CsA combination therapy does not permit dose reduction of the individual drugs to mitigate toxic complications.     

Studies on the multiple-drug induction immunosuppressive therapies with cyclosporine after renal transplantation

Our induction immunosuppressive therapies were carried out on patients split into three groups. The first group of 25 recipients were treated with regimen I [cyclosporin (CsA); 12 mg/kg/day and prednisolone (Pred)]. The second group of 16 recipients were treated with regimen II [CsA; 6 mg/kg/day, Pred and mizoribine (MIZ) or azathioprine (AZA)]. The third group of 14 recipients were treated with regimen III [CsA; 10 mg/kg/day, Pred and MIZ or AZA]. There was no significant difference among the three groups in renal function three months after renal transplantation. The frequency and grade of rejection were significantly higher in Group II than in the other groups. One of group I had CsA nephrotoxicity and none of group III had liver dysfunction three months after renal transplantation. Group I had a higher incidence of posttransplant hypertension. Hypertension of group I was very severe. We concluded that the triple-drug therapy on group III was the best induction immunosuppressive therapy after renal transplantation of the above three.     

Anti-inflammatory and immunosuppressive properties of corticosteroids

Glucocorticoids exhibit anti-inflammatory, anti-allergic and immunosuppressive properties, which contribute to their beneficial therapeutic effects. The biological effects of their interaction with their soluble receptor have been investigated as well as their inhibitory effects on several proteins implicated in the inflammatory process and on the tissular steps of inflammation. In addition, glucocorticoids exert multiple inhibitory effects both on immunocompetent cells (mainly T-lymphocytes) and on cytokines. Their role on Th17 lymphocytes and regulatory T cells has been recently studied and could represent new mechanisms of action of these drugs.     

Function of cryopreserved arterial allografts under immunosuppressive protection with cyclosporine A

Purpose: Cryopreserved arterial allografts may be used for arterial reconstructive procedures. In this experimental study cryopreserved arteries were used as autografts and as allografts with or without immunosuppression with cyclosporine A.Methods: In group A (three dogs, six bilateral grafts) cryopreserved carotid artery autografts were implanted. In groups B and C female mongrel dogs (three dogs and six bilateral grafts in each group) received cryopreserved male carotid artery allografts. Dogs in group C were treated with cyclosporine A (25 mg/kg/day). After 3 months of implantation patency was assessed by angiography. Contractile responses to KCl and phenylephrine (Phe) and the endothelium-dependent relaxation response to methacholine (Met) were examined in segments of the grafts after excision. Medial thickness was assessed semiquantitatively. The grafts were stained for sex chromatin analysis to determine the origin of cells in allografts.Results: Patency: group A, 100% (6 of 6), group B, 66.6% (4 of 6), and group C, 100% (6 of 6). Functional responses: before implantation, after thawing, 2.7 ± 0.5 mN (KCl), 4.8 ± 1.0 mN (Phe), and 0.0% ± 0.0% (Met), group A, 36.9 ± 10.6 mN (KCl), 31.5 ± 14.4 mN (Phe), and 59.3% ± 20.4% (Met), group B, 0 for all agents used, group C, 34.0 ± 7.5 mN (KCl), 28.8 ± 7.0 mN (Phe), and 46.2% ± 3.2% (Met). Morphologic characteristics: the media of grafts in group B showed significant thinning ( p < 0.05). Smooth-muscle cells in vessel walls of grafts in group C were of female origin.Conclusion: Arteries showed no function and loss of endothelial integrity after cryopreservation and thawing. After 3 months of implantation cryopreserved arterial autografts and allografts under immunosuppressive treatment with cyclosporine A showed 100% patency and return of functional responses resulting from repopulation of grafts by host cells. (J Vasc Surg 1996;24:876-82.)     

Analysis of the interactions of immunosuppressive drugs with cyclosporine in inhibiting DNA proliferation.

Though the high immunosuppressive efficacy of cyclosporine has revolutionized clinical transplantation, renal, hepatic, and neural toxicities limit its therapeutic potential. One approach to this problem is to combine CsA therapy with immunosuppressive agents that act synergistically with it, thereby permitting lower doses and mitigating drug-induced toxicity. The present study examines the in vitro interactions of various immunosuppressive agents--namely, 6-mercaptopurine (6MP), dexamethasone (Dexa), FK506, and Enisoprost (EP)--with CsA. These investigations dissect the impact of the drug combinations in inhibiting 3H-thymidine incorporation into DNA by normal human peripheral blood lymphocytes activated by mitogens or by alloantigens in mixed lymphocyte culture responses. The median-effect equation was used to analyze the dose-effect relationships of immunosuppressive drugs either alone or in combination. The interactions were quantified by calculation of combination indices (CI). CI values less than 1, greater than 1, and equal to 1 represent synergistic, antagonistic, and additive interactions, respectively. In combination with CsA, 6MP showed modest and Dexa profound synergism, while EP displayed modest and FK506 marked antagonism. These findings confirm the clinical impression of in vivo synergism between CsA and Dexa. They suggest that in vitro analysis of drug interactions may distinguish synergistic drug combinations potentially applicable to clinical practice.     

The effect of the CD28 activation pathway on the immunosuppressive action of cyclosporine.

The effect of the CD28 activation pathway on the immunosuppressive action of CsA was assessed. Human peripheral blood lymphocytes were stimulated with anti-CD3, bryostatin (Bryo) a novel activator of protein kinase C (PKC) and anti-CD28 singly or in combination, to which graded doses of CsA were added to determine relative sensitivity. Proliferation, IL-2 production, and IL-2 receptor expression were assessed and the IC50 determined. Lymphocytes stimulated with Bryo exhibited a marginal proliferative response but expressed the IL-2 receptor despite the presence of CsA. Addition of anti-CD3 or anti-CD28 to Bryo-stimulated lymphocytes promoted a vigorous proliferative response. CsA effectively inhibited the proliferative response and IL-2 production induced with anti-CD3 and Bryo but did not inhibit the response of cells stimulated with anti-CD28 and Bryo. However, II-2 receptor expression in both sets of cultures were comparable due to the induction of IL-2 receptor by Bryo and was not inhibited by CsA. Costimulation of lymphocytes with anti-CD3 plus anti-CD28 resulted in a 2-3-fold enhancement of proliferation compared with lymphocytes stimulated with anti-CD3 alone. Addition of CsA to lymphocytes stimulated with anti-CD3 resulted in the dose-dependent suppression of the proliferative response and IL-2 production (IC50 = 10-25 nM) but less so for IL-2 receptor expression (IC50 = 100-150 nM). In comparison, the proliferative response and IL-2 production elicited by anti-CD3 + anti-CD28 was more resistant to the effects of CsA (IC50 = 100-200 nM). However, IL-2 receptor expression exhibited comparable sensitivity to CsA (IC50 = 100-200 nM) in the presence of anti-CD28. Combination drug:drug studies revealed that CsA and the protein kinase C inhibitor H-7 were additive for both anti-CD3 and anti-CD3 plus anti-CD28 response. On the other hand, the cGMP-dependent protein kinase inhibitor H-8 was synergistic with CsA in inhibiting the response of lymphocytes to anti-CD3 plus anti-CD28 but only additive for responses to anti-CD3. Taken together, these data suggest that CsA inhibits T cell activation at two distinct levels, leading to inhibition of IL-2 production and inhibition of IL-2 receptor expression. Activation of the CD28 pathway partially overcomes the inhibitory activity of CsA on IL-2 production and may be mediated by indirect activation of a cGMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of diltiazem upon metabolism and immunosuppressive action of cyclosporine in kidney graft recipients

It is widely believed that calcium antagonists such as diltiazem exert immunosuppressive effects in kidney graft recipients--however, the mechanism is unclear. In a randomized controlled trial, kidney graft recipients who received diltiazem during transplantation and for an average of 12 months thereafter experienced significantly fewer rejection episodes than patients treated with cyclosporine and steroids alone. Furthermore, 1-year (97% vs. 85%) and 4-year (80% vs. 70%) graft survival rates were higher in diltiazem-treated patients, but the difference was not statistically significant. In vitro, diltiazem had little immunosuppressive activity. Concentrations of diltiazem which blocked the proliferation of PHA-stimulated human peripheral blood mononuclear cells, or prevented activation-associated accumulation of interleukin-2 mRNA, or p50- and p70-IL-2 receptor mRNA exceeded pharmacological concentrations by more than 100-fold. Both, CsA and high doses of diltiazem caused an increase of IL-6 mRNA. In contrast to these findings, the IL-6 plasma concentrations were comparable in both groups, whereas the serum concentration of soluble IL-2 receptors was decreased in patients treated with diltiazem. Administration of diltiazem caused an alteration of CsA metabolism. The whole-blood concentration of CsA metabolite 17 was significantly increased in diltiazem-treated patients, resulting in a five-times-higher concentration of this metabolite in the cellular blood compartment compared with the parent drug. Changes in metabolites 1, 8, and 18 levels were less pronounced. Although direct immunosuppressive properties of diltiazem are unlikely, diltiazem could support immunosuppression by altering CsA metabolism, and promoting accumulation of certain metabolites.     

Additive immunosuppressive effect of hydroxycamptothecin and cyclosporine on rejection of heart transplantation in rats

OBJECTIVE: The objective of this study was to study the inhibitory effects of hydroxycamptothecin (HCPT) and cyclosporine (CsA) on heart transplantation rejection in rats. MATERIALS AND METHODS: Inbred SD rats were used as donors and inbred Wistar rats as recipients. Cervical heterotopic heart transplantation was performed in 40 rats: group A received placebo; group B, HCPT; group C, CsA; group D, HCPT+CsA. RESULTS: The mean survival time was prolonged in group D with 5 grafts beating at more than 730 days. CONCLUSION: HCPT combined with CsA prevented acute rejection of allogeneic heart transplantations in rats, significantly prolonging graft mean survival time.