PENTOXIFYLLINE (PTX) SELECTIVELY POTENTIATES CYCLOSPORINE (CsA)-MEDIATED SUPPRESSION OF CELL-MEDIATED LYMPHOCYTOTOXICITY

Although cyclosporine (CsA*) is a widely used immunosuppressive agent, the side effects of CsA therapy constitute a major drawback in immunosuppression. Mainly, these effects are those associated with suppression of the immune system and organ toxicities such as renal dysfunction. Use of drugs that may be clinically safe and synergize with CsA may be a useful approach to minimize CsA dosage and alleviate CsA side effects. Pentoxifylline (PTX), a phosphodiesterase inhibitor initially characterized as a hemorheologic drug, was shown to inhibit tumor necrosis factor (TNF) production and has several in vivo benefits that may be attributed to PTX inhibition of TNF production (1-5). In this article, we addressed the possibility that PTX may potentiate CsA-mediated suppression of immunological functions. The results showed that PTX in a selective manner potentiates cell-mediated lymphocytotoxicity (CML) inhibition by CsA among several other in vitro functions. The median-effect analysis developed by Chou (6) was performed and demonstrated synergism and dose reductions in PTX and CsA combination treatment in CML inhibition. Peripheral blood mononuclear cells were separated by Ficoll-Hypaque density gradient, washed, and cultured in complete RPMI 1640 supplemented with 10% fetal bovine serum (ultralow endotoxin Hyclone, Inc., Logan, UT) or 10% human AB serum in case of mixed lymphocyte reaction (MLR). PTX (Sigma) was diluted in phosphate-buffered saline while CsA (Sandoz, Geneva, Switzerland) as a white powder was solubilized initially in ethanol and further diluted in the culture medium. Cells were seeded at 2×105 cell/well in 96-microwell plates and cultured in triplicate with 5 μg/ml phytohemagglutinin (PHA) or 10 ng/ml staphylococci enterotoxin A (SEA) for 3 days in the presence or absence of various concentrations of drugs. MLR was performed in triplicate by the coculture of 105 irradiated allogenic cells with 105 responder cells in the presence or absence of drugs for 6 days. Cells were pulsed with 1 μCi of 3[H]thymidine (Amersham, Bucking-hamshire, England) for an additional 18 hours and harvested to quantitate incorporated radioactivity. CML was generated by 4-hour incubation of effector cells, which were drug- or medium-treated MLR-generated responders, with 5×10451Cr-labeled Con A-blast target cells. Specific 51Cr release was quantitated as follows: 100 × (experimental - spontaneous)/(maximum - spontaneous). TNF activity (cytotoxin) was quantitated by the actinomycin-D-treated WEHI-13VAR cells as described in detail elsewhere (7); titers were taken at ED50 that gave 50% toxicity. TNF-α sandwich-type ELISA (monoclonal and polyclonal rabbit anti-TNF-α antibodies were obtained from Endogen, Boston, MA) was also performed. Samples, described in text, were analyzed by flow cytometry using FACScan (Becton Dickinson, BD, San Jose, CA) using fluorescein isothiocyanate-conjugated antibodies for interleukin (IL)-2R (CD25) and LFA-1α (CD11a) and phycoerythrin-conjugated antibodies for ICAM-1 (CD54). Mean channel fluorescence was obtained using LYSIS II software. The median-effect analysis was performed and assisted by computer software (6,8). The model analyzed dose-response parameters and quantitated synergism and dose-reduction indices. Details of the analysis are given in the text. PTX inhibited the proliferative responses of peripheral blood mononuclear cells due to the mitogen (PHA), antigen (SEA), and alloantigen (MLR) in dose-dependent manners; the ED50s were 400 μM, 700 μM, respectively. However, these concentrations exceed in many folds the physiological concentrations that can be attained in vivo (1,9). This is also true for the concentrations, though lower, required to inhibit TNF production in PHA, SEA, and MLR responses (ED50s were 50 μM, 35 μM, and 150 μM, respectively). Therefore, we addressed the possibility that the combination of CsA and PTX might result in synergistic inhibition of immunological functions. We chose therapeutically attained low concentrations of CsA (12.5 nM-100 nM) that would give 50% or less inhibition of immunological functions to facilitate comparison between the effect of CsA when alone and the effect of CsA in combination with PTX (also at therapeutic levels: 250 nM-2 μM). The combination of PTX (which alone was ineffective) and CsA results in isoeffective (addictive), rather than synergistic, inhibitions (Table 1). This is also the case with inhibition of TNF activity (Table 1). The adhesion molecules LFA-1α and ICAM-1 were also tested by flow cytometry in antigen and alloantigen cultures; there were no synergistic inhibitions (data not shown). However, PTX seems to selectively potentiate CsA-mediated suppression of CML (Table 1). Thus, the inclusion of therapeutic, yet ineffective, in vitro doses of PTX on CML may result in potentiation of CML inhibition by CsA. However, proper assessment of synergism and quantitation of dose reductions requires that the effects of drugs at their effective doses, alone or in combination, be carefully analyzed, such as by the use of the median-effect model. For reliable analysis (10), the drugs were combined in an equipotent combination (approximately in a ratio of 1:10,000) which reflects the relative difference in the activities of the drugs. Figure 1 shows the median-effect plots for the drugs alone and in combination. The plots were constructed to generate the median-effect parameters, median-effect dose (Dm), and slope (m), needed for the analysis. The linear correlation coefficients (r) indicate excellent correlation linearity (r>0.93) and applicability of the model. The slopes that were near 0.5-0.6 indicate semiflat dose-response curves for the effects of the drugs on CML. Table 2 shows detailed analysis of doses of each drug alone (Dx) and combination doses (Dc) that were calculated by the median-effect equation for the entire range of fractions affected (i.e. CML inhibitions). Dose-reduction indices (DRIs) indicated the degree of the fold decrease of the drug dose when in combination to give the same effect when the drug was alone. CsA dose reduction was almost constant (DRI = 5-6) over the entire range of fractions affected; approximately 80% decrease was achieved over the dose alone that gave the same effect. Interestingly, the PTX dose in combination required to inhibit CML at a given fraction was dramatically reduced and PTX reduction indices correlated with increasing doses of CsA, PTX, and higher CML inhibitions (r=0.87, 0.84, and 0.93, respectively, at P<0.01) (Table 2). Combination indices over the entire CML-inhibited fractions confirmed the synergistic effect. For example, at 0.5 CML fraction inhibited, combination index (CI) was calculated using the CI equation as follows: Equation which is less than 1 and thus is synergistic. The DRI for CsA and PTX at 0.5 fraction (i.e., 50% CML inhibition) was calculated using the formula:Equation with DRI defined as Dx/Dc. The results show that PTX potentiates CsA-mediated suppression of CML and this action is probably selective among other immune functions. This observation was confirmed by the use of the median-effect analysis for CML (Table 2). Median-effect analysis was also performed for MLR and showed no synergistic inhibitions with combination treatment (data not shown). The potency of PTX action in CML increased in combination treatment, as suggested by the increased dose-reduction indices of PTX (Table 2). This may suggest that most of the potentiation is on PTX-mediated pathways. The mechanism underlying this observation is not understood. The possible selective potentiation in CML among other functions suggested that unique pathways in the action of CML are the target of PTX. However, in vitro studies on CML lytic processes, such as granule exocytosis, perforin action, and DNA fragmentation, may be advisable to test whether they are subject to PTX regulation. PTX at therapeutic levels does not seem to directly influence cell-mediated immune functions and PTX inhibits CML at doses rather larger than those required to inhibit antigen and alloantigen responses. Thus, the direct benefits of PTX as a single drug in bone marrow transplantation are controversial (2,3,9). The in vivo mechanism of PTX in modulating immunological reactions is not known, but several lines of evidence suggest it is more potent in vivo than in vitro (1,2,11). It is more likely that PTX influences CML in vivo in synergism with CsA than alone. This may explain the reduction of organ toxicities seen in graft-versus-host disease patients in some clinical trials (1,2,5). Thus, the discrepancy of usefulness of PTX in clinical trials (1,2,5,9,12) may be explained in part by lack of controlling CsA synergism, which requires consideration of the two drug schedules. Also, in vivo discontinuous treatment with PTX yielded inhibition not only of TNF-α, but also of other proinflammatory cytokines (IL-1β, IL-6, and IL-8) (11), which may explain why oral but not continuous intravenous administration of PTX is beneficial. Although it is still to be confirmed in vivo, the results may be useful in two ways: (1) CsA dose reduction with combined treatment of PTX may reduce long-term CsA side effects, such as nephrotoxicity; and (2) prophylactic therapy for graft-versus-host disease and possibly acute rejection episodes may be achieved with the combination therapy. Careful assessment of PTX in conjunction with CsA therapy may be needed, such as in animal models or in randomized clinical trials. Acknowledgments. The authors thank Dr. Peter Ernst for his helpful comments. We also thank Ali El-Sofi for his technical assistance.Figure 1: Median-effect plot for CsA (•), PTX (▪), and their combination (▴) in inhibiting CML. Different doses (8 or 9 0.5 log data points) of CsA alone, PTX alone, or in combination (1 μM:10 mM ratio, i.e., 1:10,000) were tested for inhibition of CML. Dosage of drugs is expressed as log μM and log mM in case of CsA and PTX, respectively. Combination dosage is expressed as log sum of combined doses. At each dose, the fraction of CML inhibited (fa) is divided by the fraction of CML uninhibited (fu) and expressed as log (fa/fu). Data points were fitted by linear regression. Linear correlation coefficients (r), slope (m), and the median-effect dose, i.e., the dose that gives 50% inhibition in the median-effect plots (Dm), were generated from each plot. For CsA, r=0.99, m=0.64, and Dm=77 nM. For PTX, r=0.94, m=0.52, and Dm=2.5 mM. For combination, r=0.93, m=0.64, and Dm=0.15 (13.6 nM CsA/136 μM PTX). Drug doses were not toxic to cells as assessed by trypan blue exclusion dye. Data are mean of two independent experiments (of four performed) with approximate similar parameters.Footnotes Abbreviations: CI, combination index; CML, cell-mediated lymphocytotoxicity; CsA, cyclosporine; DIR, dose reduction index; IL, interleukin; MLR, mixed lymphocyte reaction; PHA, phytohemagglutinin; PTX, pentoxifylline; SEA, staphylococci enterotoxin A; TNF, tumor necrosis factor. REFERENCES 1. Bianco JA, Almgren J, Kern DL, et al. Evidence that oral pentoxifylline reverses acute renal dysfunction in bone marrow transplant recipients receiving amphotericin B and cyclosporine. 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