Differential effects of phosphodiesterase type 4-specific inhibition on human autoreactive myelin-specific T cell clones

Journal of Neuroimmunology 98 Ž1999. 147–156

Differential effects of phosphodiesterase type 4-specific inhibition on human autoreactive myelin-specific T cell clones Martin Pette

a,b,)

, Paolo A. Muraro

a,c

, Dagmar F. Pette a , H. Dinter d , Henry F. McFarland a , Roland Martin a

a

b

Neuroimmunology Branch, National Institute for Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA Neurologische Klinik, UniÕersitatsklinikum Carl-GustaÕ-Carus der Technischen UniÕersitat ¨ ¨ Dresden, Fetscherstra b e 74, D-01307, Dresden, Germany c Neurological Clinic, Department of Oncology and Neuroscience, Medical School, UniÕersity ‘G. D’Annunzio’, I-66100 Chieti, Italy d Department of Immunology, Berlex Biosciences, Richmond, CA 94804, USA Received 13 January 1999; received in revised form 19 April 1999; accepted 19 April 1999

Abstract Proinflammatory cytokines, secreted by autoreactive CD4 q T lymphocytes may contribute to the pathogenesis of several human autoimmune diseases, including multiple sclerosis ŽMS.. Since the antigen specificities of these T cells are not known at present, therapeutic strategies aiming at common effector pathways, in particular cytokine secretion, may be more feasible in the near future. We have studied the influence of the isoenzyme-specific phosphodiesterase inhibitor rolipram on the proliferation and cytokine secretion of human myelin basic protein-specific T cell clones. The inhibition of proliferation correlated with interference with the IL-2rIL-2 receptor system, while the effects of rolipram on several T helper 1-ŽTNF-a, TNF-b, IFN-g . and T helper 2-like cytokines ŽIL-4, IL-13. as well as IL-10 revealed an interesting drug profile, with preferential inhibition of TNF-b, TNF-a and IL-10. This profile suggest that rolipram differs from other currently used immunomodulatory drugs. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Rolipram; Phosphodiesterase inhibition; Multiple sclerosis; T lymphocytes

1. Introduction Strategies to screen new drugs for their potential in multiple sclerosis ŽMS. include trials in experimental autoimmune encephalomyelitis ŽEAE. and in vitro studies on T lymphocytes reactive with proteins of central nervous system myelin. These approaches may provide opportunities to Ža. extend our understanding of the disease process by targeting individual steps of the postulated pathogenic cascade Žb. study the mechanism of action of immunomodulatory agents, and Žc. rationally design clinical trials for the evaluation of experimental treatments. The isoenzyme-specific phosphodiesterase ŽPDE.-inhibitors have recently gained interest as a potential treatment for MS. Given the relative overexpression of the type 4 isoenzyme ŽcAMP-specific. PDEs in cells of the acquired immune system, there is a focus on selective in-

)

Corresponding author. Tel.: q49-351-4582483; Fax: q49-3514584365; E-mail: [email protected]

hibitors of this subtype ŽEngels et al., 1994; Tenor et al., 1995; Giembycz et al., 1996.. Two studies on the acute model of EAE in the Lewis rat have described preventive effects of the PDE4 inhibitor rolipram Ž4-w3X-cyclopentyloxy-4X -methoxyphenylx-2-pyrrolidinone. ŽSommer et al., 1995; Jung et al., 1996., and in one of these two studies, a reduction in disease severity was observed when treatment was initiated following the onset of disease ŽSommer et al., 1995.. Reportedly, rolipram has also been successful in chronic forms of EAE, induced in non-human primates as well as in SJL mice ŽGenain et al., 1995; Sommer et al., 1997.. Furthermore, EAE could be treated with the unspecific PDE inhibitor pentoxifylline ŽRott et al., 1993.. As a possible mode of action, the investigators observed a variable inhibition of T cell proliferation and partial suppression of TNF-a secretion by T cells or, preferentially, macrophages ŽRott et al., 1993; Genain et al., 1995; Sommer et al., 1995; Jung et al., 1996.. A most recent study on myelin basic protein ŽMBP.specific T cell clones has focused on the PDE expression patterns of these cells ŽEkholm et al., 1997.. Total PDE

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 0 8 8 - 0

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activities as well as PDE3 to PDE4 ratios were highly variable and dependent on the state of activation. There was no obvious correlation between the relative proportions of IL-4 and IFN-g secretion and the expression of either PDE isoform activity. While the PDE3-specific inhibitor cilostamide, even in the presence of exogenous IL-2, caused significant inhibition of proliferation in some instances, the rolipram-mediated inhibition did not exceed 50% of the maximum. Since elevated levels of intracellular cAMP, as caused by rolipram, are known to interfere with the IL-2 gene expression ŽFarrar et al., 1987; Mary et al., 1987., we decided to evaluate a set of autoreactive T cell clones in the absence of exogenous IL-2. In order to examine more extensively the effect of PDE type 4 inhibition on cytokine expression, we used a panel of well characterized TCC specific for the human MBP peptide 111–129 in the context of HLA-DRB1)0401 ŽMuraro et al., 1997.. Using this approach, we demonstrate complex immunomodulatory effects of rolipram on proliferation and cytokine production in human antigen-specific T cells with a preferential inhibition of TNF-b, TNF-a and IL-10. With respect to differential inhibition of T cell proliferation our data indicate that rolipram variably interferes with the IL-2rIL-2R system.

taplate, Wallac.. All proliferation assays were performed in triplicate wells in a total volume of 200 mlrwell, and repeated at least twice. To obtain cell culture supernatants, 50 to 100 ml per well were harvested at 20 h Žfor IL-2 determination. or 40 h Žall other cytokines. and immediately analyzed ŽIL-2. or frozen and stored at y708C until analysis Žall other cytokines.. 2.2. FACS analysis of IL-2R a-chain (CD25) expression To study the effects of rolipram on the expression of CD25, T cells were activated with antigen as described above and stained with fluorochrome-labeled antibodies according to the manufacturer recommendations ŽCD25FITC, Immunotech, Westbrook, ME, CD2-PE, Becton Dickinson, Mountain View, CA.. Using a FACS w -Scan flow cytometer and CellQueste software ŽBecton Dickinson., T lymphocytes were identified and acquired Žat least 5000 events per sample. according to scatter parameters and CD2 expression. CD25 expression was quantified via

2. Materials and methods 2.1. Materials, establishment of T cell clones, proliferation assays Rolipram, 8-Bromo-cAMP and dibutyryl-cAMP were obtained from Calbiochem ŽLa Jolla, CA., dissolved and stored according to the manufacturers’ recommendations. Before adding these compounds to T cell cultures, we prepared appropriate dilutions in culture media. DMSO ŽSigma, St. Louis, MO., the solvent used for rolipram, when included as a control at the concentrations of 10 and 100 mM, did not affect the antigen-induced proliferation of T cells Žnot shown.. T cell clones ŽTCC. were established from cryopreserved PBMC, obtained by leukapheresis from MS patients and healthy donors. The details of the isolation method have been described elsewhere ŽMartin et al., 1992; Muraro et al., 1997.. Proliferation assays were performed by culturing 5 = 10 4 T cells together with 1 = 10 5 autologous or MHC-compatible PBMC as antigen presenting cells ŽAPC. and antigenic peptide at a final concentration of 10 mgrml in microtiter wells for 48 h, adding 0.5 mCi3 H-thymidine ŽDuPont NEN, Boston, MA. during the final 6 h. The antigen concentration chosen, as tested in dose-response proliferation assays, induced maximal proliferative responses in all TCC included in this study Ždata not shown.. Cells were harvested automatically ŽTomtec Harvester, Orange, CA. on glass fiber filter mats ŽWallac, Turku, Finland. and counted by liquid scintillation ŽBe-

Fig. 1. Modulation of antigen-induced proliferation of T cells clones by the PDE4-specific inhibitor rolipram. Individual clones were stimulated with MBP peptide 111–129 at 10 mgrml Žmaximal antigenic stimulation for all TCC. in the context of the DRB1)0401 ŽHLA-DR4. restriction element on PBMCs in the absence and presence of different concentrations of rolipram. Proliferation was measured after 48 h by w3 Hx-thymidine incorporation. Percent inhibition of individual clones was calculated according to the formula: %I s100yŽŽcpm with drugrcpm without drug.=100.. The cpm data of individual clones Žwithout antigenrwith antigen, mean"S.D. of triplicates. were: MS1-5A1 533"110, 4888" 643; MS3-2 463"93, 42956"1351; MS2-1B3 469"199, 7380"259; MS2-3E5 390"71, 2204"149; MS2-3C8 362"138, 26711"772; HD114 372"37, 45115"3825; HD3-5H2 494"102, 12469"1125; HD4-1C2 472"115, 37492"4227. In all proliferation assays performed during this study, the clones MS3-2 and HD1-14 required high rolipram concentrations up to 100 mM for a partial inhibition, while a considerable suppression of the clones MS2-3C8 and HD4-1C2 was achieved at lower drug concentrations Žsee also Fig. 4..

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the geometric mean of log fluorescence channel distribution. 2.3. Antigen-induced proliferation of T cell clones in the presence of neutralizing anti-IL-4 antibodies The proliferation assays were performed as described above in the presence of a neutralizing anti-IL-4 antibody Žclone 34019.111, IgG2b, R & D Systems, Minneapolis, MN. or an appropriate isotype control ŽR & D Systems. at a final concentration of 10 mgrml. 2.4. Measurement of cytokines Cell culture supernatants were obtained as described above, and cytokine concentrations were measured according to the manufacturers’ recommendations. We used the following commercially available kits: IL-2, IL-4, IFN-g Biosource International ŽCamarillo, CA. and Genzyme Duoset ŽCambridge, MA.; IL-10, IL-13 and TNF-a Biosource International; TNF-b R & D Systems ŽMinneapolis, MN. and Bender MedSystems ŽVienna, Austria.. 2.5. Distribution of PDE type 4 Isoenzyme mRNA in MBP-specific T cell clones A pair of degenerate oligonucleotide primers Ž5X CGCTTGGATCCCrACrTTCrGTGGGAGACTrArCTGG3X

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and 5X CGCGAAGCTTGTCrTAGTrCTCAAACTGAr GAA3X . were designed to anneal to regions encoding the proposed catalytic domain of all PDE 4, i.e., to the nucleotides corresponding to the amino acid sequence PLWETW and FQFELT Ž5X- and 3X-primer, respectively., as previously described ŽDinter et al., 1997.. These two sequences flank a short stretch which differs in the amino acids sequence among the four isozymes of PDE type 4. The oligonucleotide primer do not amplify preferentially a single PDE4 isoform, as suggested by earlier studies ŽDinter et al., 1997.. PDE4 mRNA expression was induced by treating the TCC Ž6 = 10 6 cells. with 200 uM dBcAMP for 2 h. mRNA was extracted from 1 = 10 6 cells using RNAzol B ŽTel-Test, Friendswood, TX.., and reverse-transcribed using Oligo-dT and SuperScript II ŽGibco-BRL, Rockville, MD. according to the manufacturer’s recommendations. The cDNA Ž0.5 ul per reaction. was used for PCR amplification ŽGeneAmp polymerase wPerkin Elmer, Branchburg, NJx, 35 cycles of denaturation at 948C for 1 min, annealing at 568C for 1 min, and extension at 728C for 1 min. The resulting fragments were purified using QIAquick spin purification columns ŽQiagen, Valencia, CA., digested with BamHI and HindIII, and ligated into the BamHI and HindIII sites of pBluescript SK ŽStratagene, La Jolla, CA.. E. coli XL-1 ŽStratagene, La Jolla, CA. were transformed with ligated plasmids. Approximately 40 independent clones out of each transformation

Fig. 2. Differential response of individual TCC with respect to proliferation and IL-2 secretion to the membrane-permeable cAMP derivatives 8-Bromo-cAMP Žopen bars. and dibutyryl-cAMP Ždashed bars.. The proliferation assays were performed by stimulating T cells with MBP peptide 111–129 at 10 mgrml on irradiated PBMC expressing the DRB1)0401 restriction element. w3Hx-thymidine incorporation was measured 48 h after stimulation, while IL-2 concentrations in aliquots of the corresponding culture supernatants were measured after 20 h. The raw values Žmean cpm " S.D. without drug, IL-2 pgrml without drug. were: TCC HD4-1C2 76362" 1499 cpm, 723 pgrml; MS2-3C8 83923" 4809, 2605 pgrml; MS3-2 70576 " 1168, 4367 pgrml; HD1-14 84901" 3463, 2855 pgrml. The differential sensitivities of the clones to rolipram well correlated with the response to membrane-permeable cAMP derivatives.

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Fig. 3. Comparison of a rolipram-sensitive ŽMS2-3C8. and resistant ŽMS3-2. TCC with respect to proliferation, IL-2 secretion and IL-2R a-chain ŽCD25. expression, measured as described in Section 2. Reproducibly, inhibition of proliferation corresponded to inhibition of IL-2 secretion and CD25 expression on TCC MS2-3C8, while the proliferation and IL-2 secretion of TCC MS3-2 was only affected at very high rolipram concentrations. The expression of CD25 was even enhanced in the presence of the drug. One experiment including the drug-responding TCC HD4-1C2 and the relatively resistant TCC HD1-14 yielded almost identical patterns Ždata not shown.. In the case of TCC HD1-14, however, measurable IL-2 levels increased from 130 Žno drug. to 165 pgrml at 100 mM rolipram.

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were grown in an overnight culture, and plasmid DNA was prepared and sequenced on an ABI-377 sequencer using big-dye terminators.

3. Results 3.1. Inhibition of antigen-induced T cell proliferation by rolipram The effect of rolipram on T cell proliferation was examined using MBP-specific TCC, generated from the peripheral blood of MS patients ŽTCC coded MS-. and healthy donors ŽTCC coded HD-., all specific for the human MBP-peptide 111–129 in the context of HLADRB1)0401. In initial screening experiments, we assessed the modulatory effects of rolipram at different concentrations on antigen-specific proliferation ŽFig. 1.. The highest concentration of rolipram Ž100 mM. resulted in a variable degree of inhibition of the proliferation of all TCC. However, wide differences in the response of individual clones were consistently detectable at 10 and 1 mM rolipram. Since these concentrations or lower ones are more likely to occur under pharmacological treatment, we based our distinction of drug-sensitive Ž) 30% inhibition. or drug-resistant ŽF 30%. TCC on the effect on proliferation observed at 1 mM rolipram. The dose–response curves of two sensitive ŽMS2-3C8, HD4-1C2. and two resistant clones ŽHD1-14, MS3-2., obtained during an independent experiment, are also shown in Fig. 4. Whether the TCC had been generated from MS patients or healthy donors, did not influence their response to the drug.

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interfere with IL-2 gene expression. Therefore, we asked whether heterogeneous responses of TCC to rolipram might reflect differential sensitivities of their IL-2 gene expression to the drug. As shown in Fig. 3, rolipram reduced dose-dependently both the measurable IL-2 concentrations in tissue culture supernatants and the CD25 ŽIL-2Ra . surface expression of the drug-sensitive TCC MS2-3C8. In contrast, the resistant clone MS3-2 failed to respond likewise. Rather, we observed a slight increase of CD25 expression, while the IL-2 concentration rose above the control value at up to 1 mM rolipram. Two repetitions of this experiment yielded identical results. 3.4. The addition of neutralizing anti-IL-4 antibodies does not affect the response of indiÕidual T cell clones to rolipram Since IL-4 is known to promote the proliferation of T cells under certain conditions and since rolipram modulated the IL-4 secretion of individual TCC differentially, we restimulated IL-4 producing, rolipram-responsive and non-responsive clones in the presence of neutralizing antiIL-4 antibodies and different concentrations of the drug. As shown in Fig. 4, the dose–response curves of all clones remained largely unaltered. This indicates that a given clone’s sensitivity to rolipram is not mediated via the IL-4rIL-4 receptor system.

3.2. The differential responses of indiÕidual TCC to rolipram can be mimicked by membrane-permeable cAMP deriÕatiÕes To answer the question whether the inhibition of TCC by rolipram corresponded to effects of cAMP, we exposed four TCC, two rolipram-sensitive and two resistant, to 8-bromo-cAMP and dibutyrl-cAMP. As shown in Fig. 2, both the proliferation and the IL-2 concentrations measurable in tissue culture supernatants could be inhibited by these compounds. However, only those clones responsive to rolipram ŽMS2-3C8, HD4-1C2. were significantly suppressed by cAMP, suggesting that the effects of rolipram on T cell proliferation are in fact mediated by the regulation of cyclic nucleotide-dependent signal transduction pathways. 3.3. Differential effects of rolipram on the IL-2 secretion and IL-2R a-chain (CD25) expression The proliferative response of T cells predominantly depends on the IL-2rIL-2R system. cAMP is known to

Fig. 4. Proliferation of IL-4-secreting TCC Žtwo rolipram-sensitive, two rolipram-resistant. in the presence of different concentrations of the drug and a neutralizing anti-IL-4 Žblack symbols. or isotype-control antibody Žwhite symbols.. Both antibodies were added immediately after antigenspecific stimulation at a concentration of 10 mgrml. Neutralization of IL-4 did not significantly alter the dose–response curve of the clones under investigation. This finding makes it unlikely that IL-4 plays a causal role in mediating resistance to rolipram.

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Fig. 5. Effects of rolipram on the T cell secretion of pro- and anti-inflammatory cytokines. Individual cytokines were measured by ELISA in culture supernatants, obtained 40 h after antigen-specific stimulation of TCC. Rolipram was added at a final concentration of 10 nM Žwhite bars., 1 mM Žlight gray bars. or 100 mM Ždark gray bars.. Individual cytokines were not detectable in some TCC. For each TCC, the presence of a valid antigen-specific proliferation was monitored in parallel by 3 H-thymidine incorporation Žnot shown.. Some clones were tested repeatedly. These results are given as mean values " S.D. The absolute values of cytokines measured per clone were: IFN-g MS1-5A1 1245 pgrml, MS2-3C8 959 pgrml, MS3-2 1608 pgrml, HD1-14 595 pgrml, HD3-5H2 1317 pgrml, HD4-1C2 1256 pgrml; TNF-a MS1-5A1 135 pgrml, MS2-3C8 not detectable, MS3-2 1240 pgrml, HD1-14 421 pgrml, HD3-5H2 283 pgrml, HD4-1C2 907 pgrml; TNF-b MS1-5A1 not detectable, MS2-3C8 59 pgrml, MS3-2 326 pgrml, HD1-14 98 pgrml, HD3-5H2 not detectable, HD4-1C2 674 pgrml; IL-4 MS1-5A1 2797 pgrml, MS2-3C8 782 pgrml, MS3-2 891 pgrml, HD1-14 1232 pgrml, HD3-5H2 904 pgrml, HD4-1C2 not detectable; IL-10 MS1-5A1 868 pgrml, MS2-3C8 1694 pgrml, MS3-2 56 pgrml, HD1-14 464 pgrml, HD3-5H2 3155 pgrml, HD4-1C2 1142 pgrml; IL-13 MS1-5A1 2328 pgrml, MS2-3C8 1249 pgrml, MS3-2 2366 pgrml, HD1-14 3617 pgrml, HD3-5H2 830 pgrml, HD4-1C2 not detectable.

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3.5. The effects of rolipram on cytokines produced by antigen-actiÕated T cell clones The effects of rolipram on pro- or anti-inflammatory cytokine levels measurable in tissue culture supernatants of antigen-activated T cells were assessed in six TCC ŽFig. 5.. The most consistent effect of the drug was an inhibition of TNF-b and IL-10 secretion of all clones which produced these factors. With one exception ŽTCC MS3-2, Fig. 5., this was also true for TNF-a. IFN-g was significantly lowered in two rolipram-sensitive clones ŽMS1-5A1, MS23C8., slightly suppressed in two resistant TCC ŽHD1-14, HD3-5H2., and barely affected in one sensitive ŽHD4-1C2. and in one resistant clone ŽMS3-2. ŽFig. 5.. An interesting profile was observed with respect to IL-4. While its concentration in the supernatants of two drug-sensitive clones were moderately reduced ŽMS1-5A1, MS2-3C8., production was slightly enhanced in two resistant clones ŽMS3-2, HD1-14, respectively.. The most resistant TCC with respect to inhibition of proliferation ŽMS3-2. also showed slightly enhanced IL-13 levels in the presence of rolipram, while the production of this cytokine was reduced in four other clones. In summary, rolipram significantly suppressed measurable TNF-b, TNF-a and IL-10 levels, while its effects on IFN-g, IL-13 and, perhaps most notably, IL-4 were moderately inhibitory, neutral or even enhancing. 3.6. mRNA encoding PDE type 4b predominates in human MBP-specific, autoreactiÕe T cell clones As described in Section 2, RNA was isolated from four T cell clones, reverse transcribed and PCR-amplified. The latter was accomplished by using a pair of primers which allow a homogenous amplification of RNA-sequences encoding the catalytic domains of the four PDE type 4 isoenzymes, A, B, C and D. The PCR fragments were cloned into plasmid vectors and introduced into E. coli. After expansion, a representative sample of individual colonies was randomly selected and sequenced. As shown in Table 1, all of the four tested clones abundantly expressed mRNA corresponding to the PDE type 4b isoenzyme. The expression patterns of rolipram-sensitive clones Table 1 Expression of PDE type 4 isoenzyme ŽA, B, C, D. mRNA in human MBP specific T cell clones. cDNA of each clone was amplified by PCR. After purification, PCR products were cloned into plasmids and, after transformation, expanded in E. coli. Approximately 40 colonies derived from each T cell clone were analyzed for their insert. Numbers indicate the frequency of colonies containing the respective isoenzyme sequence T cell clone

HD1-14 HD4-1C2 MS3-2 MS2-3C8

PDE type 4 isoenzyme

Total

A

B

C

D

6 0 1 0

29 29 34 31

2 0 0 0

2 8 5 4

39 37 40 35

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ŽMS2-3C8, HD4-1C2. did not differ from those of resistant clones. Thus, PDE 4b is the predominant isoenzyme in human TCC and mediates the effect of rolipram. 4. Discussion To further elucidate the immunomodulatory profile of the PDE Typ 4 specific inhibitor rolipram, we examined the responses of selected human MBP-specific TCC to rolipram. While the proliferation of some TCC was almost completely suppressible by the drug, others were only marginally affected even at high concentrations of rolipram ŽFig. 1.. The differential effects on individual TCC could be mimicked by exogenously adding membrane-permeable cAMP derivatives. These compounds inhibited the proliferation and IL-2 secretion of TCC sensitive to rolipram, but not of those resistant to the drug ŽFig. 2.. We were able to link the differential response of individual clones to the expression of IL-2 and its high affinity receptor CD25. Only sensitive cells expressed significantly lowered levels of IL-2 and CD25 in the presence of rolipram ŽFig. 3.. These observations strongly suggest that rolipram mediates T cell inhibition via elevation of intracellular cAMP. The latter compound modulates the protein kinase A signaling pathway ŽLaxminarayana and Kammer, 1996. and interferes with the transcription of the IL-2- and CD25-genes at different levels ŽAnastassiou et al., 1992; Paliogianni et al., 1993; Tamir et al., 1996.. To our knowledge, resistance of IL-2 secretion to elevated intracellular cAMP levels has not been reported previously ŽBetz and Fox, 1991.. T helper 1 ŽTh1. lymphocytes producing IL-2 were sensitive to intracellular cAMP induction by forskolin, while T helper 2 ŽTh2. cells were resistant ŽMunoz et al., 1990.. In a recent study, both human Th1 and Th2 clones, specific for ragweed, were inhibited by rolipram, with a slightly more pronounced response of Th2 clones ŽEssayan et al., 1997.. However, in this study the roles of IL-2 or IL-2 receptor expression, as well as the growth dependence on IL-4 were not addressed. Pentoxifylline, an unspecific PDE inhibitor, suppressed a rat-derived, encephalitogenic T cell clone with respect to proliferation and moderately interfered with IL-2 secretion ŽRott et al., 1993.. The complex regulation of IL-2 gene expression has been reviewed recently ŽJain et al., 1995.. Transcription of the gene results from binding of transcription factors belonging to at least four different families. These transcription factors are the nuclear factor of activated T cells ŽNF-AT., nuclear factor k B ŽNFk B., octamer binding protein ŽOct. and activating protein 1 ŽAP-1.. They are activated via different signal transduction pathways ŽCantrell, 1996.. Further complexity is introduced by the fact that different members of a given transcription factor family may bind at different time points after activation, e.g., p50rp65 complexes early after stimulation and complexes containing c-Rel later ŽJain et al., 1995.. Both transcription factors belong to the RelrNFk B family

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ŽBaeuerle and Henkel, 1994.. Several studies employing different experimental systems suggest that elevated intracellular levels of cAMP may variably interfere with the activation of NF-AT, AP-1 and members of the RelrNFk B family ŽAnastassiou et al., 1992; Paliogianni et al., 1993; Neumann et al., 1995; Lederer et al., 1996; Tamir et al., 1996.. It is likely that individual T cells differentially express these transcription factors upon stimulation, as already shown for NFk B ŽLederer et al., 1996.. Both the relative importance of a transcription factor for the proliferation of a given TCC and the sensitivity of the activating signal transduction pathway to elevated intracellular cAMP levels may finally determine, how a TCC responds to rolipram. We considered the possibility that differential responses of individual TCC to rolipram might correlate to the autocrine secretion of IL-4, a known T cell growth factor ŽSpits et al., 1987.. However, the addition of neutralizing anti-IL-4 antibodies did not significantly influence a given TCC’s proliferation in the presence of the drug ŽFig. 4., thus making this possibility very unlikely. Another explanation for the variable sensitivity of TCC to rolipram might lie in different PDE activities expressed by individual clones. A recent study from our laboratory, however, failed to reveal a simple relationship between effects of both PDE3 Žcilostamide. and PDE4 Žrolipram. inhibitors on proliferation and the ratios of PDE3 and PDE4 activities ŽEkholm et al., 1997.. Two pilot experiments including some of the clones examined in the present study were in line with these previous data Ždata not shown.. Similarly, the analysis of two resistant and two sensitive TCC with respect to their PDE type 4 isoenzyme expression did not explain the differential responses ŽTable 1.. In each instance, PDE type 4b mRNA prevailed. The effects of rolipram on cytokines secreted by TCC were also heterogeneous, but consisted preferentially in an inhibition of TNF-b, TNF-a and IL-10, the latter being secreted both by human Th1 and Th2 lymphocytes ŽDel Prete et al., 1993.. TNF-a was suppressed in five out of six secreting clones tested ŽFig. 5.. Notably, this inhibition also occurred in clones resistant to the anti-proliferative effect of rolipram. Also with respect to measurable IFN-g concentrations, there was no obvious correlation between the suppression of proliferation and the inhibition of cytokine release. One TCC ŽHD4-1C2., partially susceptible to the drug’s antiproliferative effects, released almost unaltered amounts of the cytokine. The secretion of IL-4 was suppressed in two and unaffected or slightly enhanced in three TCC. IL-13 was slightly enhanced in one TCC and slightly to moderately inhibited in four TCC ŽFig. 5.. These results, generated in a well-defined experimental system using a set of human CD4 q autoreactive TCC, reflect the complexity of the effects of cAMP modulation on cytokine expression and release. As summarized in

recent reviews ŽDent and Giembycz, 1996; Dent and Rabe, 1996., almost none of the presently examined cytokines responds uniformly to pharmacological cAMP elevation in T lymphocytes. A notable exception may be TNF-b Ž32. Žand present study., although the number of available studies is small. TNF-a andror IFN-g were inhibited ŽRott et al., 1993; Sommer et al., 1995; Genain et al., 1995; Dent and Giembycz, 1996; Nyman et al., 1997. Žand present study. or only marginally affected ŽJung et al., 1996. Žand present study.. A relative resistance of the IL-4 gene transcription to elevated intracellular cAMP levels induced by prostaglandin E2 has been reported ŽPaliogianni and Boumpas, 1996.. This contrasts to the marked rolipram-induced inhibition of IL-4 expression and secretion of ragweed-specific Th2-TCC, exceeding the drug’s effect on IFN-g secretion by ragweed-reactive Th1-TCC ŽEssayan et al., 1997.. Differences in PDE4 isoenzyme expression between the Th2 and Th1 clones were offered as a possible explanation, with the isoenzymes PDE 4c and 4d being exclusively expressed in Th2 clones ŽEssayan et al., 1997.. In whole blood cultures stimulated with lipopolysaccharides, prostaglandin E2 doubled the production of IL-10 Žvan der Pouw Kraan et al., 1995.. A significant enhancement was also observed upon stimulation of PBMCs in the presence of pentoxifylline ŽRieckmann et al., 1996.. On the other hand, all TCC included in our study were significantly inhibited with respect to IL-10 secretion, suggesting that increases in cAMP levels may determine divergent effects on distinct cell populations. Taken together, available data generated in different in vitro systems do not support the simplistic view that PDE inhibitors exclusively suppress Th1-derived cytokines while not affecting Th2-derived cytokines, as has been suggested ŽRott et al., 1993; Rieckmann et al., 1996.. We are well aware of current controversies on the importance of tumor necrosis factors and IL-10 for the development and attenuation of organ-specific autoimmunity ŽRott et al., 1994; Canella et al., 1996; van Oosten et al., 1996; Frei et al., 1997.. Likewise, we know about the difficulties in extrapolating our in vitro data on the complex situation in vivo. We believe, however, that there is by now enough evidence to suggest pharmacological inhibition of PDE 4 as a potential approach to modulate inflammatory responses during the course of autoimmune diseases in which Th1-mediated mechanisms are likely to play a role. However, a final evaluation of this approach will rely on the conduction of carefully designed clinical trials including further studies on the immunological and pharmacological characteristics of this interesting group of drugs. Acknowledgements M. Pette was supported by the Deutsche Forschungsgemeinschaft ŽPe 363r7-1.. We would like to thank Dr. D.

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Ekholm for measuring the activities of PDE isoenzymes in selected T cell clones. This study has been presented in part as a scientific poster during the 70s. Jahrestagung der DGN, Dresden, Germany, 1997.

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