Pimecrolimus inhibits up-regulation of OX40 and synthesis of inflammatory cytokines upon secondary T cell activation by allogeneic dendritic cells

Abstract

Pimecrolimus is a new non-steroidal inhibitor of T cell and mast cell activation. In the present study, we compared the potency of pimecrolimus and cyclosporin A (CyA) to inhibit cytokine synthesis of alloantigen-primed T cells and the expression of CD134 (OX40), an inducible co-receptor molecule thought to be critical for the survival and expansion of inflammation-mediating T cells. To mimic the physiological situation of recurrent antigenic stimulation, we have used dendritic cells (DC) as stimulators of purified CD4+ T cells in the primary and secondary allogeneic mixed lymphocyte culture (allo-MLC). Pimecrolimus inhibited surface expression of OX40 and prevented the up-regulation of CD25 and CD54 with a 10-fold higher potency compared to CyA. Similarly, 50% inhibition of allo-DC-mediated T cell proliferation by pimecrolimus was obtained at 0.55 nm, compared to about 12 nm for CyA. Furthermore, pimecrolimus blocked the increase of OX40 on primed T cells restimulated on day 10 in secondary allo-MLC. Allo-DC-primed T cells showed a restricted cytokine profile characterized by the production of TNF-alpha, IFN-gamma and IL-2 but low to undetectable levels of IL-4 and IL-10. The synthesis of TNF-alpha and IFN-gamma and the up-regulation of OX40 on T cells after secondary allogeneic stimulation were almost entirely blocked by 10 nm pimecrolimus. Taken together, pimecrolimus inhibits T cell proliferation and Th1 cytokine synthesis and also prevents the up-regulation of the OX40 co-receptor on primed T cells indicating its potential in the therapy of chronic inflammation and autoimmunity.

Fig. 1

Chemical structure of pimecrolimus (SDZ ASM 981, Elidel®). Reproduced from Meingassner et al. Br J Dermatol 1997;137:568–76, with kind permission of the publisher.

Fig. 2

Inhibition of surface antigen expression on CD4⁺ T cells activated in primary MLC. (a) Purified CD4⁺ T cells were stained with antibodies specific for OX40 (CD134), CD25 and CD54 before stimulation (cross-hatched) and after stimulation for 5 days by allogeneic DC at a DC/T cell ratio of 1/40 in the absence of compounds (black bars) and in the presence of either pimecrolimus used at 100 nm (white), 10 nm (grey) or 1 nm (dark grey) or cyclosporin A used at 1000 nm, 100 nm or 10 nm (left-hatchted bars with increasing density). The percentage of positive T cells is shown as the mean (± s.d.) obtained in three independent experiments. (b) Mean percentage inhibition of surface marker induction by pimecrolimus or cyclosporin A was calculated according to the formula given in the legend to Table 1. The data displayed were obtained in one out of three similar and independently performed experiments.

Fig. 3

Compound-mediated inhibition of proliferation of CD4⁺ T cells in primary allogeneic MLC with DC as stimulator cells was determined by addition of [³H]-thymidine during the last 16 h of the 5-day MLR. Results are shown as mean cpm ± s.d. of quadruplicate culture wells and are representative of three independently performed MLC assays. Sigmoidal curve fitting of plots obtained for CyA (dotted line) and pimecrolimus (dashed line) allowed determination of IC₅₀ values using the Origin® software (version 6·1).., CyA; IC₅₀ = 11·8 nm; ▪, pimecrolimus; IC₅₀ = 0·55 nm.

Fig. 4

Pimecrolimus inhibits the increase of CD134 expression on primed T cells. Only few resting CD4⁺ T cells express CD134 or CD25 (filled black). DC-mediated T cell activation in MLC led to up-regulation of CD134 and CD25 as determined on day 10 (right-hatched). Thereafter, expression of both activation molecules dropped unless T cells were subjected to restimulation (RS) by DC on day 10. Relative to the level of surface expression on non-stimulated T cells (no RS control, set as 100% inhibition), CD134 and CD25 were inhibited to about 60% and 73% in the presence of 10 nm pimecrolimus (PiC) or 100 nm cyclosporin A (CyA) as indicated by downward arrows. Data are representative of three similar and independently conducted experiments. ▪, Day 1: start of culture; , day 10: time of RS; , day 14: no RS control; , day 14: RS control;, day 14: RS + CyA (100 nm); , day 14: RS + PiC (10 nm).

Fig. 5

Intracellular analysis of cytokine synthesis induced in MLC. Following priming in bulk MLC for 10 days, CD4⁺ T cells were restimulated by the addition of the same batch of allogeneic DC in the presence and absence of PiC or CyA and incubated for 22 h, the final 12 h thereof in the presence of Brefeldin A. Subsequently, appropriate aliquots of the secondary MLC were processed for intracellular cytokine staining as described in Materials and Methods. (a) Gates for analysis were set to exclude cellular debris based on forward and side scatter (R1) and CD40-FITC staining (R2) to exclude DC from evaluation. (b) Specific intracellular staining of T cell cytokines as determined by competition with unlabelled anti-cytokine MoAb (blocking control) in each staining reaction. Inserted numbers indicate percentage of cytokine-expressing T cells. The data shown were obtained in one of four independent experiments with similar results (see also Table 1).

Fig. 5

Intracellular analysis of cytokine synthesis induced in MLC. Following priming in bulk MLC for 10 days, CD4⁺ T cells were restimulated by the addition of the same batch of allogeneic DC in the presence and absence of PiC or CyA and incubated for 22 h, the final 12 h thereof in the presence of Brefeldin A. Subsequently, appropriate aliquots of the secondary MLC were processed for intracellular cytokine staining as described in Materials and Methods. (a) Gates for analysis were set to exclude cellular debris based on forward and side scatter (R1) and CD40-FITC staining (R2) to exclude DC from evaluation. (b) Specific intracellular staining of T cell cytokines as determined by competition with unlabelled anti-cytokine MoAb (blocking control) in each staining reaction. Inserted numbers indicate percentage of cytokine-expressing T cells. The data shown were obtained in one of four independent experiments with similar results (see also Table 1).

Fig. 6

Induction of intracellular cytokine synthesis by phorbol ester and Ca²⁺ ionophore. Allo-DC primed CD4⁺ T cells were restimulated on day 10 of MLC by addition of PMA and ionomycin for 6 h in the presence of Brefeldin A. T cell samples were fixed, permeabilized, stained with cytokine-specific or isotype-control MoAb and analysed after addition of 7-AAD to exclude dead cells from evaluation. Inserted numbers indicate percentage of cytokine-expressing T cells. The data shown were obtained in one of two independent experiments with similar results.