Inhibition of anti-CD3 antibody-induced mouse T cell activation by pentoxifylline in combination...

\PERGAMON International Journal of Immunopharmacology 19 "0887# 130Ð141

S9081Ð9450:87 ,08[99 Þ 0887 International Society for Immunopharmacology[ Published by Elsevier Science Ltd[PII] S 9 0 8 1 Ð 9 4 5 0 " 8 7 # 9 9 9 1 8 Ð 9

Inhibition of anti!CD2 antibody!induced mouse T cellactivation by pentoxifylline in combination with

rapamycin or A66 0615 "le~unomide#Martin Richard\ David W[ Hoskin�

Department of Microbiology and Immunology\ Faculty of Medicine\ Dalhousie University\ Halifax\Nova Scotia B2H 3H6\ Canada

Received 14 November 0886^ accepted 14 Februay 0887

Abstract

Pentoxifylline "PTX#\ rapamycin "RAP#\ and le~unomide are potent immunomodulatory drugs withdi}ering modes of action[ In order to develop new drug combinations for immunotherapy\ we tested thee}ects of PTX in combination with RAP or A66 0615 "the active metabolite of le~unomide# on in vitro Tcell activation in a mouse model system[ T lymphocytes in spleen cell preparations were stimulated withanti!CD2 monoclonal antibody alone\ or in the presence of PTX "14Ð199 mg:ml#\ RAP "9[4Ð4[9 ng:ml#\ A660615 "1[4Ð09[9 mM#\ PTX:RAP "14Ð199 mg:ml and 9[4Ð4[9 ng:ml\ respectively#\ or PTX:A66 0615 "14Ð199mg:ml and 1[4Ð09[9 mM\ respectively#[ Anti!CD2!induced T cell proliferation was inhibited in a dose!dependent fashion by the individual drugs[ An additive inhibitory e}ect was observed in cultures treatedwith PTX:RAP or PTX:A66 0615[ The e}ects of PTX\ RAP\ A66 0615\ PTX:RAP\ or PTX:A66 0615 "atconcentrations approximating the IC49 of individual drugs for inhibition of lymphoproliferation# on anti!CD2!activated killer "AK# cell induction\ CD14 expression\ and interleukin "IL#!1 synthesis in anti!CD2!activated spleen cell cultures were also determined[ Alone\ each drug was able to suppress AK cell inductionto varying degrees[ PTX plus RAP exhibited strong synergism\ while the combination of PTX and A66 0615had an additive inhibitory e}ect on AK cell induction[ CD14 expression was only weakly inhibited by A660615\ but the percentage of CD14!expressing cells was greatly reduced in cultures treated with PTX or RAP[The combination of PTX and RAP had an additive inhibitory e}ect on CD14 expression while PTX andA66 0615 together had an e}ect equivalent to PTX alone[ IL!1 synthesis was inhibited by PTX but wasuna}ected by RAP or A66 0615[ Treatment with PTX plus RAP led to a further reduction in IL!1 productionbut co!treatment with PTX and A66 0615 approximated the inhibitory e}ect of PTX alone[ We concludethat the combination of PTX and RAP is noteworthy for its potent immunomodulatory activity and maybe of use in clinical situations where it is desirable to prevent T cell activation[ Þ 0887 International Societyfor Immunopharmacology[ Published by Elsevier Science Ltd[

Key words] Pentoxifylline^ Rapamycin^ Le~unomide^ Immunosuppression^ T lymphocytes

� Corresponding author[ Tel[] 891!383!5498^ fax] 891!383!4014^ e!mail] dwhoskinÝis[dal[ca

M[ Richard\ D[W[ Hoskin:International Journal of Immunopharmacolo`y 19 "0887# 130Ð141131

0[ Introduction

There is growing interest in developing new strategies to use various combinations of immu!nosuppressive drugs with di}ering mechanisms of action to treat autoimmune diseases or preventallograft rejection in organ transplant recipients[ This led us to investigate the immunomodulatorypotential of pentoxifylline "PTX# in combination with rapamycin "RAP# or A66 0615\ the activemetabolite of le~unomide[ Although all three of these drugs are potent immunosuppressive agentsin their own right\ possible additive or synergistic activities which might be obtained by combiningPTX with RAP or A66 0615 have not yet been examined[

PTX is a xanthine!derived phosphodiesterase inhibitor which interferes with the synthesis ofproin~ammatory cytokines such as tumor necrosis factor a\ interferon g\ interleukin "IL#!1\ andIL!01 "Tilg et al[\ 0882^ Thanha�user et al[\ 0882^ Moller et al[\ 0886#[ Recently\ we have shown thatPTX is also able to suppress cytotoxic T lymphocyte "CTL# induction in a mouse model systemby inhibiting T cell expression of the cytolytic e}ector molecules granzyme B and perforin "Hoskinet al[\ 0885#[ In addition\ in vivo studies have revealed that PTX prevents the induction ofexperimental autoimmune encephalomyelitis in Lewis rats "Rott et al[\ 0882# and prolongs the lifeof MRL!lpr:lpr mice which spontaneously develop an autoimmune condition resembling systemiclupus erythematosus "Hecht et al[\ 0884#[ In the past\ PTX was used in clinical practice primarilyas a haemorrheological drug for the treatment of peripheral vascular disorders and cerebrovasculardisease "Ward and Clissold\ 0876#[ However\ PTX has recently been used in the treatment ofvarious immune!mediated disorders\ including rheumatoid arthritis and multiple schlerosis"Maksymowych et al[\ 0884^ Riekmann et al[\ 0885#[

RAP\ an immunosuppressive macrolide antibiotic obtained from Streptomyces hy`roscopicus\prolongs allograft survival in several animal models of acute transplant rejection "Chen et al[\0884^ Granger et al[\ 0884#[ RAP also protects against autoimmune disease in rodent models ofsystemic lupus erythematosus and insulin!dependent diabetes mellitus "Martel et al[\ 0866^ Kahanet al[\ 0880#[ RAP mediates immune suppression by preventing the activation of p69 S5 kinaseinvolved in signal transduction through cytokine receptors such as the IL!1 receptor "Kuo et al[\0881#[ As a result\ RAP inhibits lymphoproliferation induced by cytokines and\ in particular\ Tlymphocyte di}erentiation and progression through the cell cycle in response to IL!1 "Feuersteinet al[\ 0884#[ RAP also suppresses granzyme B and perforin gene expression following CTLactivation "Makrigiannis and Hoskin\ 0886#[

The isoxazol derivative le~unomide is a prodrug with potent immunosuppressive and anti!in~ammatory activities "Bartlett et al[\ 0880#[ Although the mechanism of action of le~unomide isnot entirely clear\ the active metabolite of the agent\ A66 0615\ is believed to inhibit T cellproliferation by "i# blocking pyrimidine biosynthesis and "ii# interfering with the enzymatic activityof protein tyrosine kinases involved in IL!1 receptor signal transduction "Elder et al[\ 0886#[ Incontrast\ A66 0615 inhibits B cell proliferation and and blocks cell cycle progression by actingprimarily at the level of pyrimidine biosynthetic pathways "Siemasko et al[\ 0885#[ Recently\le~unomide has been shown to prevent and reverse acute rejection of cardiac allografts in rats"Williams et al[\ 0883#\ as well as preventing the development of autoimmune pathology inMRL:MpJ!lpr:lpr mice "Xu et al[\ 0886# and experimentally induced myasthenia gravis in rats"Vidic!Dankovic et al[\ 0884#[

In this study we determined the e}ect of PTX in combination with RAP or A66 0615 on mouse

M[ Richard\ D[W[ Hoskin:International Journal of Immunopharmacolo`y 19 "0887# 130Ð141 132

T lymphocyte proliferation in spleen cell cultures activated with mitogenic anti!CD2 monoclonalantibody "mAb#[ We also tested the in~uence of co!treatment with PTX and RAP or PTX andA66 0615 on anti!CD2!activated killer "AK# cell development\ IL!1 production\ and expression ofthe a chain "CD14# of the high a.nity IL!1 receptor in anti!CD2!activated spleen cell cultures[

1[ Experimental procedures

1[0[ Mice

Female "5Ð7 weeks old# C46BL:5 "H!1b haplotype# mice were purchased from Charles RiverCanada "Lasalle\ PQ\ Canada# and were maintained in our animal care facilities on standardmouse chow and water supplied ad libitum[

1[1[ Materials

RPMI 0539 medium was supplemented with 099 mM L!glutamine\ 099 mg:ml streptomycin\ 099U:ml penicillin "all ICN Biomedicals Canada Ltd\ Mississauga\ ON\ Canada#\ 4 mM HEPESbu}er "Sigma Chemical Co[\ St Louis\ MO\ U[S[A[#\ and 4) heat!inactivated "at 45>C for 29 min#fetal calf serum "Gibco BRL\ Burlington\ ON\ Canada#[ Hereafter this will be referred to ascomplete medium[ The hamster anti!mouse CD2 mAb!secreting hybridoma 034!1C00 "Leo et al[\0876# was kindly provided by Dr J Bluestone\ University of Chicago\ IL\ U[S[A[ P704 mastocytomacells "H!1d haplotype# were obtained from the American Type Culture Collection\ Rockville\ MD\U[S[A[ All cell lines were maintained by in vitro passage in complete medium[ A66 0615 was agenerous gift from Hoechst AG\ Wiesbaden\ Germany[ PTX was purchased from Sigma ChemicalCo[\ St Louis\ MO\ U[S[A[ Both A66 0615 and PTX were dissolved in complete medium andstored as stock solutions "4 mM and 0 mg:ml\ respectively# at −19>C[ RAP from ResearchBiochemicals International "Natick\ MA\ U[S[A[# was dissolved in dimethyl sulfoxide and storedas a stock solution "149 mg:ml# at −69>C[ Fluorescein "FITC#!conjugated rat anti!mouse CD14"high a.nity IL!1 receptor# was purchased from Cedarlane Laboratories "Hornby\ ON\ Canada#[Rat anti!mouse IL!1 mAb and recombinant mouse IL!1 were from Genzyme Corp[\ Cambridge\MA\ U[S[A[ Rabbit anti!mouse IL!1 polyclonal antibody was purchased from CollaborativeResearch Inc[\ Bedford\ MA\ U[S[A[

1[2[ Spleen cell cultures

Spleens were removed aseptically from mice at sacri_ce and single cell suspensions were preparedby mechanical dissociation of spleens in cold phosphate bu}ered saline\ pH 6[1[ Erythrocytes weredepleted by osmotic shock and spleen cells were cultured in complete medium containing anti!CD2 mAb "0:19 dilution of hybridoma culture supernatant#\ with or without graded concentrationsof PTX\ RAP\ A66 0615\ PTX:RAP\ or PTX:A66 0615[ Spleen cell cultures were performed at0[14×095 cells:ml "9[1 ml volume# in 85!well round!bottom microtiter plates "proliferation assay#or at 3×095 cells:ml "1 ml volume# in 13!well ~at!bottom plates "all other assays# and maintainedat 26>C in a humidi_ed atmosphere containing 4) CO1[ Cell!free supernatants were collected


from 13!well plates at 37 h of culture and stored at −69>C for subsequent measurement of IL!1levels[

1[3[ T cell proliferation assay

T cell proliferation in response to anti!CD2 mAb stimulation was measured at 37 h of cultureby tritiated thymidine "ð2HŁTdR# incorporation as previously described "Fitzpatrick et al[\ 0885#[Brie~y\ 9[4 mCi ð2HŁTdR "speci_c activity 54 Ci:mmol^ ICN# was added to each well of themicrotiter plate during the last 5 h of culture[ Cultures were then harvested onto glass _ber matsand ð2HŁTdR incorporation was determined by scintillation counting[ Data from quadruplicatecultures are expressed as mean counts per minute "cpm# plus or minus the standard deviation "SD#[

1[4[ 40Cr!release cytotoxicity assay

AK cell!mediated cytotoxicity in 37 h cultures of anti!CD2!activated spleen cells was determinedby 40Cr!release assay as described earlier "Fitzpatrick et al[\ 0885#[ Brie~y\ P704 mastocytomatarget cells were labeled with 099 mCi ð40CrŁsodium chromate "speci_c activity 149Ð499 mCi:mgCr^ ICN# and combined in complete medium with anti!CD2!activated spleen cells in wells of a 85!well microtiter plate at various e}ector]target "E]T# cell ratios[ Following a 3 h incubation at 26>Cin a humidi_ed atmosphere containing 4) CO1\ 9[0 ml of cell!free supernatant was collected fromthe wells and the amount of 40Cr released from lysed P704 cells was determined by g counting[Data are presented as the mean ) lysis in triplicate samples "2SD#[

1[5[ Flow cytometry

The percentage of CD14¦ lymphocytes in 37 h cultures of anti!CD2!activated spleen cells wasdetermined by ~ow cytometry using a standard protocol "Fitzpatrick et al[\ 0885# and FITC!conjugated mAb to CD14[ Data are expressed as ) control "spleen cells activated with anti!CD2mAb in complete medium alone#[

1[6[ Enzyme!linked immunosorbent assay "ELISA#

IL!1 levels in supernatants from 37 h cultures of anti!CD2!activated T cells were measured bystandard sandwich ELISA using paired anti!IL!1 antibodies as described "Chen et al[\ 0885#[ IL!1levels in samples were determined using a standard curve constructed with recombinant mouse IL!1[ Data are presented as ) control "IL!1 produced by spleen cells activated with anti!CD2 mAbin complete medium alone#[

2[ Results

2[0[ Effect of PTX and RAP on T cell proliferation

Mitogenic anti!CD2 mAb was used to induce T cell proliferation in C46:BL5 mouse spleen cellcultures performed in the absence or presence of PTX "14Ð199 mg:ml# and RAP "9[4Ð4[9 ng:ml#\


Fig[ 0[ Additive inhibitory e}ect of PTX and RAP on anti!CD2!induced T cell proliferation[ Spleen cells were stimulatedwith anti!CD2 mAb in the absence or presence of PTX or RAP alone or in combination\ at the indicated concentrations[Following 37 h of culture\ DNA synthesis was measured by ð2HŁTdR incorporation[ Data are expressed as meancpm2S[D[ Results from one representative experiment out of six are shown[

alone or combination[ DNA synthesis was measured by ð2HŁTdR incorporation at 37 h of culturesince prior kinetic studies have determined that peak lymphoproliferation occurs at this time point[As shown in Fig[ 0\ both PTX and RAP inhibited DNA synthesis in a dose!dependent fashion[An additive inhibitory e}ect was observed in cultures co!treated with PTX and RAP[ Neither drugalone or in combination\ at the concentrations employed\ a}ected spleen cell viability over 37 h orculture\ as determined by trypan blue dye exclusion test[ The dimethyl sulfoxide vehicle for RAP\at the extremely low concentrations employed in our experiments\ had no e}ect on any of ourassay systems[

2[1[ Effect of PTX and A66 0615 on T cell proliferation

Anti!CD2!stimulated spleen cells were also treated with PTX "14Ð199 mg:ml# and A66 0615"1[4Ð09[9 mM#\ alone or in combination[ Figure 1 depicts the results of these experiments[ A dose!dependent inhibition of DNA synthesis in 37 h cultures was observed with both drugs[ PTX andA66 0615 together had an additive inhibitory e}ect[ Spleen cell viability over 37 h of culture wasnot a}ected by either drug\ alone or in combination[

2[2[ In~uence of co!treatment with PTX:RAP or PTX:A66 0615 on AK cell induction

We next compared the e}ect of PTX\ RAP\ A66 0615\ PTX:RAP or PTX:A66 0615 treatmenton AK cell induction in 37 h spleen cell cultures stimulated with anti!CD2 mAb[ We have previouslyshown that the major histocompatibility complex!unrestricted cytotoxic activity of AK cells againstP704 mastocytoma cells attains peak levels at 37 h of culture "Kaiser et al[\ 0882#[ PTX\ RAP\ andA66 0615 were used at concentrations "099 mg:ml\ 9[4 ng:ml\ and 4 mM\ respectively# which


Fig[ 1[ PTX and A66 0615 exert an additive inhibitory e}ect on T cell proliferation induced with anti!CD2 mAb[ Spleencells were stimulated with anti!CD2 mAb in the absence or presence of PTX or A66 0615 alone or in combination\ atthe indicated concentrations[ Following 37 h of culture\ DNA synthesis was measured by ð2HŁTdR incorporation[ Dataare expressed as mean cpm2S[D[ Results from one representative experiment out of six are shown[

approximated the IC49 of individual drugs for inhibition of T cell proliferation[ Generation of AKcells was inhibited to varying degrees by PTX\ RAP\ or A66 0615 "Fig[ 2#[ The relative order ofpotency was A66 0615 × RAP × PTX[ Co!treatment with PTX and A66 0615 had an additive

Fig[ 2[ Co!treatment with PTX and RAP or PTX and A66 0615 inhibits AK cell induction in anti!CD2!stimulatedspleen cell cultures[ To generate AK cells\ spleen cells were stimulated with anti!CD2 mAb in the absence or presenceof PTX\ RAP\ A66 0615\ PTX plus RAP\ or PTX plus A66 0615[ PTX was used at 099 mg:ml\ RAP at 9[4 ng:ml\ andA66 0615 at 4 mM[ Following 37 h of culture\ cytolytic activity against P704 mastocytoma cells at the indicated E]Tratios was measured in a 3 h 40Cr!release assay[ Data are expressed as mean percentage lysis2S[D[ Results from onerepresentative experiment out of three are shown[


Fig[ 3[ Flow cytometric analysis of changes in CD14 expression induced by PTX:RAP or PTX:A66 0615[ Spleen cellswere stimulated with anti!CD2 mAb in the absence or presence of PTX\ RAP\ A66 0615\ PTX plus RAP\ or PTX plusA66 0615[ PTX was used at 099 mg:ml\ RAP at 9[4 ng:ml\ and A66 0615 at 4 mM[ Following 37 h of culture\ the percentageof cells expressing CD14 was determined by ~ow cytometry[ Results are presented as ) control "mean2S[E[M[ offour identically performed experiments# where the control consisted of spleen cells exposed to anti!CD2 mAb alone[Mean CD14 expression in control cultures was 2324)[

inhibitory e}ect on AK cell induction while combined treatment with PTX and RAP was synergisticand nearly ablated the development of cytotoxicity in anti!CD2!activated spleen cell cultures[ AllAK cell populations used in the cytotoxicity assay were adjusted to contain the same number ofviable e}ector cells[

2[3[ Effect of PTX:RAP and PTX:A66 0615 on CD14 expression

Cell!surface expression of CD14 following T cell activation is critical for subsequent T cellproliferation and di}erentiation in response to IL!1 "Pimentel!Muin½os et al[\ 0883#[ Therefore\ weexamined the e}ect of treatment with PTX "099 mg:ml#\ RAP "9[4 ng:ml#\ A66 0615 "4 mM#\PTX:RAP "099 mg:ml and 9[4 ng:ml\ respectively#\ or PTX:A66 0615 "099 mg:ml and 4 mM\respectively# on CD14 expression in anti!CD2!activated spleen cell cultures[ As shown in Fig[ 3\the percentage of CD14!expressing cells was decreased markedly by both RAP and PTX "40)and 57) reduction\ respectively# while treatment with A66 0615 alone had only a weak inhibitorye}ect on CD14 expression[ Co!treatment with PTX and A66 0615 did not result in a furtherdecrease in CD14 expression beyond that achieved by treatment with PTX alone[ In contrast\ thecombination of PTX and RAP had an additive inhibitory e}ect\ reducing CD14 expression by72)[

2[4[ In~uence of PTX:RAP and PTX:A66 0615 on IL!1 synthesis

We also determined the e}ect of treatment with PTX "099 mg:ml#\ RAP "9[4 ng:ml#\ A66 0615"4 mM#\ PTX:RAP "099 mg:ml and 9[4 ng:ml\ respectively#\ or PTX:A66 0615 "099 mg:ml and 4mM\ respectively# on IL!1 production in 37 h anti!CD2!activated spleen cell cultures[ A previousstudy has shown that IL!1 levels are maximal at this time point in this particular culture system


Fig[ 4[ E}ect of PTX:RAP or PTX:A66 0615 on IL!1 production in anti!CD2!stimulated spleen cell cultures[ Spleencells were stimulated with anti!CD2 mAb in the absence or presence of PTX\ RAP\ A66 0615\ PTX plus RAP\ or PTXplus A66 0615[ PTX was used at 099 mg:ml\ RAP at 9[4 ng:ml\ and A66 0615 at 4 mM[ Following 37 h of culture\ IL!1levels in culture supernatants were determined by ELISA[ Results are presented as ) control "mean2S[E[M[ of fouridentically performed experiments# where the control consisted of spleen cells exposed to anti!CD2 mAb alone[ MeanIL!1 production in control cultures was 0321 units:ml[

"Kaiser et al[\ 0882#[ Figure 4 shows that RAP and A66 0615 alone had little\ if any e}ect on IL!1synthesis while treatment with PTX alone reduced IL!1 synthesis by 41)[ PTX and RAP togetherhad an additive inhibitory e}ect on IL!1 production\ reducing culture supernatant levels by 60)[Surprisingly\ the combination of PTX plus A66 0615 was less e}ective at inhibiting IL!1 synthesisthan treatment with PTX alone[

3[ Discussion

The immunomodulatory properties of PTX\ RAP\ and le~unomide have stimulated considerableinterest in the possible application of these drugs for the treatment of autoimmune disease\ as wellas for the prevention of allograft rejection in organ transplant recipients "Rott et al[\ 0882^ Chenet al[\ 0884^ Xu et al[\ 0886#[ Since each of these immunosuppressive agents has a unique mode ofaction\ combination therapies would be expected to exert additive or synergistic inhibitory e}ectson immune responses[ In this regard\ PTX has already been shown to synergize with dexamethasoneand cyclosporin A to produce profound inhibition of in vitro cytokine synthesis\ lympho!proliferation\ and adhesion molecule expression by mitogen!stimulated human peripheral bloodmononuclear cells "Funk et al[\ 0884#[ Here we demonstrate that PTX can be combined with RAPor A66 0615 to inhibit T cell activation with greater e.cacy than treatment with PTX\ RAP\ orA66 0615 alone[ The combination of PTX and RAP is shown to be an especially potent inhibitorof T cell activation[ In our studies we employed PTX at concentrations which approximate plasmaconcentrations of PTX\ including its metabolites\ observed in vivo "Funk et al[\ 0884#[ RAP andA66 0615 were used at concentrations substantially lower than those used in vivo "Chen et al[\0884^ Xu et al[\ 0886#[ The results which we obtained in vitro might therefore be relatively modestcompared to what one might expect in vivo using the maximum tolerated doses of RAP orle~unomide in combination with PTX[ It is also important to note that none of the drugs\ alone


or in combination\ at the concentrations used in our experimentation adversely a}ected lymphocyteviability over a 37 h interval\ thereby excluding non!selective drug toxicity as a possible explantationfor the inhibitory e}ects of these drugs on T cell function[

Our initial experiments revealed that PTX\ RAP\ or A66 0615 were able to inhibit T cellproliferation in a dose!dependent fashion with approximate IC49 values of 099 mg:ml\ 9[4 ng:ml\and 4 mM\ respectively[ Co!treatment of spleen cell cultures with PTX and RAP or PTX and A660615 resulted in an additive inhibition of T cell proliferation in response to stimulatory anti!CD2mAb[ Furthermore\ the proliferative response of T lymphocytes was nearly eliminated in thepresence of the highest concentrations of PTX:RAP or PTX:A66 0615 used in our studies\suggesting that these particular drug combinations may have clinical application in cases where itis desirable to prevent clonal expansion of autoreactive or alloreactive T cells[ Interestingly\ acomparison of the inhibitory activities of PTX\ RAP\ or A66 0615 "all used at the approximateIC49 for lymphoproliferation# on non!speci_c CTL "AK cell# induction revealed that the threedrugs are not equally e}ective inhibitors of cytotoxicity "inhibition by A66 0615 × RAP × PTX#[These data underscore the need to employ more than one assay system when evaluating theperformance of immunosuppressive agents in vitro[ Although PTX:A66 0615 was a more e}ectiveinhibitor of AK cell induction than either drug alone\ the e}ect was merely additive[ In contrast\the combination of PTX plus RAP was synergistic and virtually abrogated the developmentof cytotoxicity in anti!CD2!activated spleen cell cultures[ Since cell!mediated cytotoxicity is animportant aspect of allograft rejection "Sykes\ 0885#\ it is reasonable to speculate that co!treatmentwith PTX and RAP might signi_cantly prolong allograft survival in vivo[

To gain insight into the underlying mechanism"s# by which PTX:RAP and PTX:A66 0615interfere with anti!CD2!induced T cell proliferation and AK cell induction\ we examined the e}ectof these drug combinations on IL!1 production and CD14 expression in spleen cell culturesstimulated with anti!CD2 mAb[ Signalling through the high a.nity IL!1 receptor in response toendogenous IL!1 is a requirement for T cell proliferation following antigenic stimulation "Pimentel!Muin½os et al[\ 0883#[ Furthermore\ CTL development requires IL!1!induced signals "Maraskovskyet al[\ 0878# which upregulate perforin and granzyme B gene expression "Lui et al[\ 0889#[ Consistentwith previously published reports "Thanha�user et al[\ 0882^ Dumont et al[\ 0889^ Chong et al[\0882#\ treatment of anti!CD2!activated spleen cells with PTX inhibited IL!1 synthesis whereasRAP or A66 0615 had minimal e}ects on IL!1 production[ Reduced IL!1 production by Tlymphocytes in the presence of PTX has been attributed to an inhibitory e}ect of PTX on T cellactivation through the T cell receptor:CD2 and CD15 but not the CD17 signal pathway "Dong etal[\ 0886#[ Furthermore\ the accumulation of intracellular cyclic AMP has been implicated inthe PTX!mediated inhibition of IL!1 synthesis by T cells "Funk et al[\ 0884#[ Interestingly\ thecombination of PTX and RAP had a greater inhibitory e}ect on IL!1 synthesis than PTX alone[RAP interferes with T cell costimulation through CD17 "Bierer et al[\ 0880#\ as well as inhibitingIL!1 signal transduction "Feuerstein et al[\ 0884#[ One important function of the CD17 costimu!latory signal in T lymphocytes is to promote transcription of the IL!1 gene "Ghosh et al[\ 0882#[RAP may\ therefore\ enhance the inhibitory e}ect of PTX on IL!1 production by blocking CD17signal transduction in T lymphocytes concurrently with the PTX!induced blockade of T cellreceptor:CD2 and CD15 signalling[ Curiously\ the inhibitory e}ect of PTX on IL!1 productionwas partially reversed in the presence of A66 0615\ suggesting that A66 0615 either somehowinterferes with the ability of PTX to induce elevated levels of intracellular cyclic AMP in T


lymphocytes or modi_es the response of T cells to increased amounts of intracellular cyclic AMP"Funk et al[\ 0884#[

CD14 expression in anti!CD2!activated spleen cell cultures was strongly inhibited by treatmentwith PTX\ which is in line with the well established concept of IL!1 autoinduction of CD14expression by T lymphocytes "Depper et al[\ 0874#[ RAP also downregulated CD14 expressionfollowing mouse splenic T cell activation with anti!CD2 mAb[ A similar e}ect was observed byWoerly and colleagues "0885# when human peripheral blood lymphocytes were stimulated withanti!CD2 mAb in the presence of RAP[ Northern blot analysis revealed that the RAP!induceddefect in CD14 expression by T cells occurs at the level of CD14 gene transcription "Woerly et al[\0885#[ Presumably\ CD14 gene transcription is similarly depressed in mouse T lymphocytes acti!vated in the presence of RAP[ In contrast to PTX or RAP\ A66 0615 had only a marginal inhibitorye}ect on CD14 expression in anti!CD2!activated spleen cell cultures[ This _nding is consistentwith an earlier report that A66 0615 fails to signi_cantly inhibit CD14 expression by anti!CD2!activated human T cells "Chong et al[\ 0882#[ As expected\ co!treatment with PTX and A66 0615had an inhibitory e}ect on CD14 expression equivalent to that of treatment with PTX alone[However\ treatment with PTX in combination with RAP had an additive inhibitory e}ect onCD14 expression\ dramatically reducing the percentage of CD14¦ lymphocytes in anti!CD2!activated spleen cell cultures[ Failure of T lymphocytes to express adequate levels of the high a.nityIL!1 receptor in the presence of PTX:RAP\ combined with greatly diminished IL!1 production anda RAP!mediated blockade of IL!1 signalling\ likely accounts for the potent inhibitory e}ects ofPTX:RAP on in vitro T cell proliferation and subsequent AK cell induction[ Our _ndings in themouse system\ if they also hold true for humans\ suggest that PTX may be able to enhance theability of RAP to prevent T cell!mediated immune responses involved in acute allograft rejectionand the development of certain autoimmune diseases[

Acknowledgements

This work was supported by a grant to D[W[H[ from the Natural Sciences and EngineeringResearch Council of Canada[ We thank Dr L[ Best for assistance with the ~ow cytometric analysisand Dr A[ MacDonald for facilitating the gift of A66 0615 from Hoechst AG[

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Inhibition of anti-CD3 antibody-induced mouse T cell activation by pentoxifylline in combination...

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Transcript of Inhibition of anti-CD3 antibody-induced mouse T cell activation by pentoxifylline in combination...