Research Paper Mediators of Inflammation, 9, 69–75 (2000)

The influence of anti-allergic drugs, epinastine hydrochloride (EP) and disodium cromoglycate (DSCG), on the co-stimulatory molecule expression was examined using in vitro cell culture technique. Spleen cells obtained from BALB/c mice 10 days after immunization with haemocyanin absorbed to aluminium hydroxide were cultured in the presence of 100.0 microg/ml haemocyanin and various concentrations of the agents. Low concentrations (<1.5 x 10(-4)M) of EP and DSCG did not influence spleen cell blastic activity induced by antigenic stimulation, whereas these agents caused significant inhibition of spleen cell activation when 2 x 10(-4) M of the agents were added to cell cultures. EP and DSCG also did not affect blastic activity of sensitized splenic T cells by anti-CD3 monoclonal antibody stimulation even when these cells were cultured in the presence of 2 x 10(-4) M of the agents. We next examined the influence of EP and DSCG on the expression of co-stimulatory molecules on spleen cells in response to antigenic stimulation. Sensitized spleen cells were cultured in the presence of 2 x 10(-4)M of the agents and the expression of molecules were examined by flow cytometer 24h later. EP and DSCG suppressed the expression of costimulatory molecules, CD40 and CD80, but not CD86, on splenic B cells which were enhanced by antigenic stimulation in vitro.


Introduction
Curre nt theories on the pathogene sis of allergic diseases such as asthma, atopic dermatitis and allergic rhinitis, clearly show that inflammatory responses are important determinants of the diseases. 1 Se veral inflammatory ce lls including eosinophils and mast ce lls, and IgE antibody have bee n postulated to participate in the inflammatory responses to antigenic stimulation. 2 ,3 It is gene rally ac ce pte d that the ce ntral ce lls in these re sponse s are the T lymphocytes: T lymphocyte s, espe cially helper T lymphocytes, upon contact w ith alle rgens, activate and re lease se veral types of lymphokines that control the e stablishment and re gulation of the inflammatory response s that underly many alle rgic disease s.
Antige n-spe cific T ce ll ac tivation requires triggering via the antigen-spec ific T ce ll rece ptor and a c ostimulatory signal usually provided by antigen prese nting c ells (APC) including B c ells and macrophage s. 4 -7 Interac tion of T ce lls w ith antige n in the abse nce of suffic ie nt c o-stimulation can result in T ce ll unresp onsive ness -te rme d anergy. 7,8 A number of APC surface de te rminants have be en show n to be capable of providing T c ell c o-stimulation. Ac tivated APC ex pre ss CD80, w hich c o-stimulate s T ce lls via binding to the c ounter re ce ptor CD28. 4 ,7 CD86, w hic h also binds to CD28, is constitutive ly ex pressed on professional and non-profe ssional APC e arlie r than CD80. 4 ,7 The inte rac tion betw e en CD40 mole cules and its ligand, CD40L, on T c ells has also be en identified as an important co-stimulatory signal required for sw itch re combination to IgE synthesis. 9,10 A number of anti-alle rgic drugs such as aze lastine, epinastine hydrochloride (EP), and disodium c romoglycate (DSCG) have been de velope d and are use d clinically in the treatme nt and the management of alle rgic disease s. The mechanisms of ac tion of these agents on alle rgic dise ases are ge nerally be lie ved to be ow ing, in part, to their inhibitory action on chemical me diator re le ase from effe ctor ce lls such as e osinophils and mast ce lls. 1 1,12 Although these age nts are also reporte d to display antagonistic e ffec ts on chemic al me diators, 13 ,1 4 there is little information about the influenc e of anti-allergic agents on co-stimulatory molecule ex pression. The re fore, in this study w e ex amined w hether anti-alle rgic age nts could modulate the ex p re ssion of co-stimulatory molec ule s on lymphocytes by an in v itro ce ll c ulture te chnique.

Mice
Spe cific pathogen free, male BALB/c mice , 5 w ee ks of age w e re purchase d from Charle s River Japan Inc. (Atsugi, Japan ). Each ex perimental and control group consis te d of five mice. Animal c are and handling w ere in ac cordance w ith the p rinc iples state d in the Guide for the Care and Use of Laboratory Animals (Prime Ministe r's Office of Japan, Public ation No. 85- 23,1985 ). The ex p erimental protocols of this study w ere approved by the Animal Care and Use Committe e of Show a Unive rsity.

Immunization
Mic e w e re immunize d by intraperitone al injec tion of 8.0 m g /ml haemoc yanin (Sigma Che mical Co., St Louis, MO) absorbed to 4.0 mg aluminium hydrox ide (Wako Che mical Co., Ltd, Osaka, Jap an ) in 0.5 ml saline .

Agents
EP w as kindly supplied by Boehringer Ingelhaim (Ingelhaim, Ge rmany ) as a w ater soluble pure powder. The pow der w as dissolve d in saline at a conc entration of 5 ´10 -2 M. This solution w as the n filtere d through a 0.22 m m filter (Nihon Millipore Co., Ltd, Yonezaw a, Japan ) and stored as a stock solution at 4°C until used. All dilutions used in this study w ere prepared from this stock solution by dilution w ith RPMI-1640 me dium (Wako Chemic al ) suppleme nte d w ith 10% foe tal c alf serum (Sigma chemic al; RPMI-FCS). DSCG w as p urchase d from Fujisaw a-Fison Co., Osaka, Japan as a solution in distilled w ater. This solution contained 5.0 M DSCG. All dilutions used in this study w e re prepared from this solution.

Cell preparation
Splee n w as remove d from five individual mice 10 days after the immunization. These organs w ere pre sse d through 60 gauge ste el sie ves into saline. The c ells w ere w ashed once w ith saline and then treate d w ith Tris-buffere d ammonium chloride (17 mM Tris-HCl-0.73% NH 4 Cl, pH 7.6 ) for 5 min to lyse red blood ce lls. After p assing through 200 gauge stee l sie ves, the residual c ells w ere w ashed thre e times, re suspende d in saline at a conce ntration of 1 ´10 6 ce lls /ml and used for splenocytes. In the case of ex pe riments using purified T ce lls, T c ells w ere se parated from splenocytes using a magne tic ce ll se parator (Milteny Biote c GmbH, Be rgis ch Gladbach, Ge rmany ) according to the manufac turer's instructions. Splenocyte s w ere labeled w ith anti-mouse CD19 monoclonal antibody (mAb )-c oate d magnetic microbeads (Milte ny Biote c GmbH). These ce lls w ere the n applied to the c olumn plac ed in the separator and the c olumn w as w ashe d w ith PBS. The entire effluate d c ells w ere w ashe d tw ice and re suspe nde d in saline at a c once ntration of 1 ´10 6 ce lls /ml. The purity of the ce lls w as che cke d by incubating separated c ells w ith fluore sce in is othioc yanate (FITC)-c onjugate d mAb to CD90 (Phar-Mingen, San Diego, CA ). Fluoresce nce mic roscopic ex amination revealed that more than 98% of the cells w ere CD90 positive, and the se c ells w e re used as sple nic T c ells.

Cell proliferation assay
Splenoc ytes pre pare d as above w ere w ashe d onc e and re suspende d in RPMI-1640 medium (Wako Chemical) supple mented w ith 10% foe tal c alf serum (Sigma

Flow cytometry
The mAbs use d for flow c ytometry w ere anti-mouse CD16 /32 mAb, FITC-c onjugated anti-mouse CD40, CD80 and CD86 mAbs. The y w ere p urchased from PharMinge n. Be fore staining CD40, CD80 and CD86 molecules w ith spec ific antibodies, the purifie d cultured B ce lls (1 ´10 6 c ells ) w e re treate d w ith 1.0 m g of anti-mouse CD16 /32 mAb for 5 min at 4°C to block non-spec ific Fc rec eptor me diate d binding of antibodie s. Pre-treate d c ells w ere the n staine d w ith either FITC-conjugated mAb to CD40, CD80 or CD86 for 25 min at 25°C. Afte r w ashing onc e, c ells w e re suspe nded in saline, and assayed for fluoresc enc e inte nsity on ce lls using Flow cytometer (Be cton Dickinson, Mountain Vie w, CA, USA).

Statistical analysis
The statistical significance of the diffe renc e in the me an value betw e en tw o groups w as ex amine d by the Mann -Whitne y U te st.

Influence of EP on proliferative response of lymphocytes in vitro
The first se t of ex p eriments w as de signed to ex amine the influenc e of EP on proliferative response of sple en c ells induce d by in v itro stimulation w ith antigens. Sple en cells prepare d from five individual mic e immunize d w ith haemoc yanin w ere c ultured in v itro in the presenc e of 100.0 m g /ml hae moc yanin and various c oncentrations of EP for 48 h. As show n in ex pre ssion on the cells, w hic h w ere e nhance d by in v itro stimulation w ith hae moc yanin. EP also suppresses the ability of the c ells to ex press CD80 molecules on the ir c ell surfac e induc ed by antige nic stimulation in vitro (Fig. 2 ).

Influence of DSCG on the response of splenocytes to antigenic stimulation
Sinc e many kinds of anti-allergic age nts are use d clinically, the re sults de sc ribe d above le ave open the questions of w he the r the se anti-alle rgic age nts have similar supp re ssive effe cts on c ell functions to those observe d in EP. There fore, w e chose DSCG, one of the best-know n anti-allergic age nts, and ex amine d w hether it also has inhibitory effects on c ell functions, such as prolife ration and c o-stimulatory molecule ex pression, induce d by in vitro antige nic stimulation. As in the c ase of EP, DSCG ex erted suppressive e ffec ts on antigen-induce d, but not anti-CD3-me diate d, p roliferative responses of splenocyte s such as snee zing, bronchosp asm and hive s are belie ve d to be largely the results of me diator re le ase from mast cells and basophils, 2,3 w hereas chronic symptoms, the re sult of allergic inflammation, can be ex plained on the basis of eosinophil-mediated tissue damage. 2,3 ,1 6 Base d on these e stablishe d ide as, the tre atment of alle rgic disease s still focuse s on the inhibition of mediator rele ase or on blocking the binding of soluble mediators to their rece ptors. How e ver, additional e videnc e cle arly show s that T ce lls play a c entral role in the driving and mainte nance of all these proc esses by elaboration of se veral type s of c ytokine s. 2,3,1 6,1 7 The re is now also circ umstantial e videnc e that the CD28 /B7 c o-stimulatory is c ritical in T ce ll activation, proliferation and cytokine production. 4 -7 ,18 Ye t the action of antialle rgic age nts on the co-stimulatory pathw ay is poorly understood. To ex amine the influenc e of antialle rgic age nts in the c o-stimulatory pathw ay, it w as first te ste d on the re sponse of lymp hocyte s to in v itro antigenic stimulation by ex amining DNA synthe tic activity. The prese nt results (Figs 1A and 3A) clearly show the inhibitory effect of EP and DSCG on DNA synthetic activity (proliferation ) of sensitize d sple en ce lls in response to in vitro stimulation w ith sp ecific antigen. How e ve r, EP and DSCG could not inhibit DNA synthe tic activity of sensitize d splenic T c ells by anti-CD3 stimulation (Figs 1B and 3B), suggesting that inhibition of the prolife rative response s of sensitize d sple en ce lls by specific antigen is not due to c ytotox ic action of the age nts to splenic T ce lls. It is reported that anti-alle rgic agents did not inte rfere w ith the ability of APC to proc ess antigens. 19 The prese nt results, therefore , may sugge st that EP and DSCG inhibit proliferation of se nsitized splee n ce lls induce d by antigenic stimulation through the suppression of signal transmission require d for DNA synthe sis.
T and B c ells play a c ritical role in the pathogene sis of allergic dise ases. Ac tivation of re sting lymphocytes requires the engage ment of the T c ell rec eptor w ith a pep tide /MHC as w ell as the engage ment of appropriate co-stimulatory molec ules. 4 -7 In particular, signals through the CD28 /CTLA-4 co-stimulatory pathw ay are essential for primary activation of antigen spe cific T ce lls. 20 It is reported that CTLA-4 immunoglobuline fusion protein (CTLA-4Ig ), a blocker of CD28 /B7 c o-stimulation, could block allergeninduc ed proliferation and cytokine production of peripheral blood mononucle ar c ells from atopic donors, w hen the cells w e re cultured in v itro in the presenc e of CTLA-4Ig. 18 In addition to the CD28 / CTLA-4 c o-stimulatory signals, CD40 ligand (CD40L) w as also re ported to play an important role in the inte rac tion betw ee n he lpe r T c ells and APC. 21 ,2 2 The CD40L-CD40 inte rac tion c an upre gulate the ex pression of B7 molec ule s on APC, w hich are essential for he lper T cell ac tivation, and enhances T c ell proliferative responses. 23,24 There fore, w e ex amine d w hether EP and DSCG suppress the ex pression of costimulatory mole cules, CD40, CD80 and CD86, on se nsitized B lymphocytes, induc ed by in vitro stimulation w ith specific antige n and re sulted in inhibition of in vitro T ce ll prolife rative resp onses. The prese nt results clearly show that EP and DSCG c ould suppre ss the ex pression of c o-stimulatory molec ule s, CD40 and CD80 but not CD86, on sensitized B c ells w hich w ere enhance d by in v itro stimulation w ith antigen (Figs. 2 and 4 ). Prophylactic treatme nt of the patients w ith anti-alle rgic age nts is rec ognized to pre vent the development of the diseases by inhibition of IgE hyper-produc tion, 9,25 but the mechanisms are not w ell defined. Induction of IgE synthesis from B ce lls requires tw o distinc t signals. The first signal is delive red by IL-4 and the se cond is provided by T-B ce ll contac t through the ligation of CD40 and CD40L. 9,1 0 Togethe r w ith the prese nt re sults, it may be spe culated that anti-allergic agents ex erte d inhibitory e ffec ts on the de velop ment of the dise ases through the suppression of CD40 ex pression, w hen agents w ere administe re d prophylactic ally.
Re ce nt re ports have ex amine d the co-stimulatory depende nce of allerge n-spe cific human T cells, and the role of the se CD28 and CD80 in animal models of alle rgic inflammation. De Boer e t a l. show ed that CD80 could ac t as a c o-stimulator molecule for Th2 type c ytokine , IL-4 and IL-5, p roduc tions from human p eriphe ral blood T c ells activated by mAb dire cted at T cell re ce ptor. 26 It is also reported that CD80 blockade pre vents antige n spec ific proliferative responses of T ce lls from atopic de rmatitis against De rm a to p ha g o ide s fa rin a e . 27 In murine mode ls for inflammatory disease s, blockade of CD80 is re ported to ameliorate the dise ases. 2 8 With re gard to the influe nce of CD80 on effe ctor cell (e.g. eosinophil) functions, Tsuyuki e t a l. show e d that intrana sal administration of anti-CD80 mAb inhibite d eosinophil accumulation in lungs and airw ay trac ts, but w as le ss effe ctive in inhibiting pe riphe ral blood eosinophilia induc ed by aerosol provocation of allergen. 2 9 CD80 blockade w as also reporte d to be able to prevent eosinophil influx into lungs and airw ay tracts w he n the mic e w ere injec te d w ith anti-CD80 mAb during the ae rosol challe nge. 3 0 These suppressive effects of CD80 may be ow ing to its inhibitory action on adhesion molec ule ex pre ssions, sinc e CD80 blockade is re ported to prevent ac quisition by lymphocytes including eosinophils of the adhesion molec ule s involved in re circulation through tissue, and upre gulation of their ligands such as ICAM-1 on endothelium. 31 Judging from these rep orts, the prese nt re sults show ing the suppressive e ffec t of anti-alle rgic agents on CD80, but not CD86, ex pre ssion sugge st that anti-allergic agents, espe cially EP and DSCG, are e ffec tive in the preve ntion of alle rgic inflammatory responses in lungs and airw ay trac ts.
Pharmacologic al studies re veale d that EP and DSCG pre ve nt c alcium cation influx into immune ce lls, 14 ,32 re sulting in reduced perme ability of ce ll me mbrane and inhibition of ex pression of proteins synthesized in the cytosol. It is also re ported that anti-alle rgic agents including EP and DSCG inc re ase intrace llular cyclic ade nosine monophosphate (cAMP) le vel by inhibiting adenylate c yclase ac tivity. 14 ,3 3 Ele vation of intrace llular cAMP le vel e laborates an important dow n-regulatory signal in the release of prote ins synthe size d in the c ytosol. 3 4,3 5 From these reports, it is possible that EP and DSCG cause p re vention of the ac cumulation of c alcium cation in the cytosol and enhanc e intracellular cAMP leve l re sulting in inhibition of CD mole cule ex pre ssions. Although the prese nt data add novel information about the be neficial effects of antialle rgic age nts on the diseases, the precis e mechanism(s ) by w hich the age nts inhibit co-stimulatory molecule ex pressions is not yet fully unde rstood. Furthe r ex p eriments are nee de d to clarify this point.