Evidence that SMS 201-995 enhances the immunosuppressive effect of FK506

\ PERGAMON

International Journal of Immunopharmacology 19 "0887# 368Ð389

Evidence that SMS 190!884 enhances the immunosuppressive e}ect of FK495 C[ Peregoa\ D[ Lattuadaa\ C[ Casnicia\ S[ Gattib\ R[ Orsenigob\ S[ Panagiotisb\ P[ Francoa\ O[ Marellia\ a

Department of Pharmacolo`y\ School of Medicine\ University of Milan\ Via Vanuitselli 21\ 19618 Milan\ Italy b Institute of Transplantation and Experimental Sur`ery\ School of Medicine University of Milan\ Ospedale Ma``iore I[R[C[ C[S[ Milan\ Italy Received 10 November 0886^ accepted 5 March 0887

Abstract Somatostatin "SS# was originally described as a growth hormone release inhibiting factor synthesised in the hypothalamus[ Recently\ SS and its receptor "SSTR# have been demonstrated in lymphoid tissues and seem to play a regulatory\ largely inhibitory\ role in immune responses[ The aim of the present study was to check the immunosuppressive e}ect of a SS derived peptide\ the octreotide "SMS 190!884# and to verify whether this molecule acted synergistically with FK495[ An immunosuppressive e}ect of SMS was observed on the proliferation of rat spleen cells induced in vitro\ either by polyclonal mitogens such as PHA or by alloantigens[ With PHA stimulation\ 09−03 M SMS signi_cantly enhanced the immunosuppressive action of 9[99990 mg:ml FK495[ The addition of SMS in MLR "09−00Ð09−8 M# increased the antiproliferative e}ect of both 9[9990 mg:ml and 9[99990 mg:ml FK495[ In consideration of the extremely low concentration of both drugs that was required to obtain a good immunosuppression in vitro\ we veri_ed the association of FK495 and SMS in vivo in an allogeneic skin graft model that used Lewis "Lew# rats as donors and Brown Norway "BN# rats as recipients[ BN treated with 9[0 mg:kg FK495 and 9[4Ð09 mg:kg SMS showed a signi_cant increase in mean skin allograft survival time when compared to either a monotherapy or control group[ None of the animals died or showed signs of drug!related toxicity[ In conclusion\ a combined therapy of SMS and FK495\ administered at lower dosages than those that are considered therapeutic\ led to an e}ective immunosuppression without any undesirable side e}ects[ Þ 0887 International Society for Immunopharmacology[ Published by Elsevier Science Ltd Key words] SMS 190!884^ FK495^ Rat^ Immunosuppression

0[ Introduction Recent evidence suggests that the nervous and the immune systems are closely linked and involved in bi!directional communication through soluble products such as neuropeptides and  Corresponding author[ E!mail] marellioÝimiucca[csi[unimi[it 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 3 8 Ð 5

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cytokines[ The concept that the nervous system can modulate immunological and in~ammatory responses has been supported by the identi_cation of neuropeptide receptors on leukocytes\ and the demonstration that these peptides can regulate leukocyte functions "Bellanti et al[\ 0883^ Blalock et al[\ 0883^ Madden + Felten et al[\ 0884#[ Furthermore\ the presence of SS m!RNA in lymphoid tissue "Aguila et al[\ 0880^ Elliot et al[\ 0883# indicates that lymphocytes are able to synthesise SS and this may then act as a paracrine factor and modulate immune responses[ Several reports indicate that SS has an inhibitory e}ect on mitogen!induced lamina propria and peripheral blood lymphocyte "PBL# proliferation "Fais et al[\ 0880#^ and\ _nally\ it has been reported that SS decreases human natural killer cell activity "Sirianni et al[\ 0883#[ Because of the very short plasma half!life of native SS of several minutes\ a number of long acting SS analogues\ such as SMS 190!884 "octreotide acetate# has been developed[ Recently\ _ve distinct SS receptors "SSTR0!4# have been cloned and sequence "Yamada et al[\ 0881a\ 0881b\ Yasuda et al[\ 0881^ Corness et al[\ 0882^ Bruno et al[\ 0881^ Rohrer et al[\ 0882^ Demchyshyn et al[\ 0882^ O|Carroll et al[\ 0881#[ These receptors have extensive similarities in amino acid sequence\ but di}er in their ligand a.nities\ mechanisms of signal transduction\ and capacities for desensi! tisation "Rens!Domiano et al[\ 0881^ Luthin et al[\ 0882^ Law et al[\ 0882^ Raynor et al[\ 0882#[ SMS 190!884 binds preferentially SSTR1 "Kaupmann et al[\ 0884# that has been localized on T lymphocytes isolated from the hepatic granulomas of Schistosome!infected mice "Elliott et al[\ 0883#[ The objectives of the present study were\ therefore\ to investigate the immunosuppressive action of SMS 190!884\ and to verify whether this molecule could act synergistically with FK495\ a novel immunosuppressant whose immunosuppressive activity on T lymphocytes has been widely known "Tocci et al[\ 0878^ Morris\ 0880^ Chang et al[\ 0880^ Thomson\ 0880^ Harding et al[\ 0878^ Bierer et al[\ 0882^ Dumont et al[\ 0889#[ Studies were carried out in vitro and in vivo in rats grafted with fully allogenic skin[

1[ Experimental procedures 1[0[ Cells isolation Rat spleen cells were aseptically removed and gently dissociated in HBSS "GIBCO Grand Island NY#[ The cell suspensions were centrifuged and the pellet was depleted of contaminating red cells by treatment with ACK!lysing bu}er "GIBCO Grand Island NY#[ After washing\ spleen cells were suspended in RPMI!0539 medium supplemented with 4) heat inactivated FCS\ 1 mM L!glutamine "all from GIBCO Grand Island NY#\ 099 U:ml penicillin and 099 mg:ml streptomycin "both from Squibb S[P[A\ Rome Italy#[ 1[1[ Proliferation assay 094 spleen cells were seeded in U!bottomed 85!well plates "Falcon\ NJ# in the presence of 3 mg:ml of phytohemagglutinin "PHA# "SIGMA\ St Louis\ Mo#\ FK495 "9[99990 or 9[9990 mg:ml# "Prograf\ Fujisawa GmbH\ Munich\ Germany# and octreotide acetate "SMS# "Sandostatin\Sandoz\ Italy# "from 09−03Ð09−6 M# either alone or in association[ Rat splenocytes were also stimulated\ in the

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presence of the above described concentrations of SMS\ with 4 mg:ml of anti!rat CD2 mAb or 4mg:ml of anti!rat CD17 mAb "both from Serotec Oxford\ U[K[# plus 4 ng:ml of tetra! decanoylphorbol 02!acetate "TPA# "SIGMA\ St Louis\ Mo#[ The cultures were incubated in a humidi_ed atmosphere with 4) CO1 at 26>C for 37 h^ SMS was added every 13 h[ 0 mCi of 2H!thymidine "Amersham Intl\ Amersham\ U[K[# was added into each well and after 07 h the cultures were sacri_ced[ Cell growth was measured by 2H!thymidine uptake[ The cells were harvested into glass _bre by a multiple cell!harvester "Titertek Flow Lab# and radioactivity was counted in an automatic b counter "Packard\ Meridien U[S[A[#[ The results were expressed as mean cpm of triplicate cultures[ 1[2[ MLR The mixed lymphocyte reaction was performed by mixing 094 responder spleen cells isolated from Brown Norway rats "Charles River Italy# with an equal number of spleen cells isolated from Lewis rats "Charles River Italy#\ previously incubated for 34 min at 26>C with 22 mg:ml mitomycin C "Kiowa\ Tokyo\ Japan#[ SMS was added every 13 h alone or in association with FK495 at the previously described concentration[ The cultures were carried out for 3 days in U!bottomed 85! well plates[ Cell growth was measured by 2H!thymidine uptake "see above#[ 1[3[ Animals Male Brown Norway "BN\ RT0n# and Lewis "LEW\RT0l# rats weighing 199Ð299 g were pur! chased as recipients and donors of the skin allografting\ respectively\ and maintained in con! ventional facilities housed 0:cage with food and water ad libitum[ 1[4[ Surgical procedures Skin grafting was performed using a modi_cation of the method of Helops "Helops\ 0855#[ A square of 0 cm skin was taken from the donor|s abdomen and placed on the recipient|s torso[ One live donor provide two skin grafts[ The transplanted skin was protected with bandages[ The graft assessments were made daily from the removal of bandages on 6th postoperative day "POD#[ Scoring of rejection was based on the degree of erythema and the area of the graft with intact epidermis versus scabbed or necrotic skin[ We considered day of rejection the day on which 099) graft scabbing was recorded "in all rats rejection started 2 days before the endpoint#[ 1[5[ Experimental groups Ten experimental groups were set up and each of them included 5 animals[ In group 0\ BN recipients received a LEW skin graft and no immunosuppression[ In group 1\ BN recipients were treated with FK495 at 9[0 mg:kg:day i[p[ The BN recipients in groups 2\ 3\ 4\ 5 were treated with SMS at 9[0Ð9[4Ð0Ð09 mg:kg:b[i[d[ i[p respectively[ Groups 6\ 7\ 8\ 09 received a combination of FK495 9[0 mg:kg:day i[p[ and SMS 9[0Ð9[4Ð0Ð09 mg:kg:b[i[d[ i[p\ respectively[

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1[6[ Statistical analysis Data were analysed using Student|s test for unpaired data and values given are means2standard error of the mean[

2[ Results The e}ects of SMS on the proliferation of rat spleen cells activated in vitro by polyclonal mitogens such as PHA or with alloantigens was assessed[ Results reported in Fig[ 0 show that SMS signi_cantly inhibited 2H!Thymidine incorporation by rat spleen cells activated by PHA or alloantigens although the peptide was more e}ective when the stimulus was an alloantigen rather then a polyclonal activator[ In this regard\ given that it is well known that activation and pro! liferation of T lymphocytes is mediated either via T cell antigen receptor complex "CD2:TCR# or via antigen independent pathways involving CD17 surface molecules "Linsley et al[\ 0882#\ the e}ect of SMS on proliferation of rat splenocytes stimulated by either anti!CD17 mAb plus TPA or via CD2 was investigated[ As shown in Fig[ 1\ SMS inhibited the proliferation induced by anti! CD17 mAb plus TPA\ while T cell proliferation induced by anti!CD2 mAb plus TPA\ was only marginally a}ected[ Since these _ndings support the hypothesis that SMS plays an inhibitory role on immune responses and at the same time indicate that SMS may act on CD17 rather than CD2 pathways\ it was of interest to associate SMS with FK495\ an immunosuppressive drug that has been shown to inhibit T cell proliferation via CD2 "Sigal et al[\ 0880#[ Thus\ rat spleen cells were stimulated by PHA in the presence of suboptimal concentrations of FK495 "9[9990 and 9[99990 mg:mL# and SMS[ Results reported in Fig[ 2 show that SMS at a low concentration\ 09−03 M\ signi_cantly enhanced the immunosuppressive e}ect of the lower FK495 dose that was only partially inhibitory when given alone\ with cpm 32022214 obtained by the association of the two compounds as compared with a cpm 82312710 of FK495 when given alone[ Higher doses of SMS "from 09−02Ð09−6 M# showed a good inhibitory e}ect\ while with an FK495 dose that caused good inhibition\ SMS was ine}ective[ Similar concentrations of SMS and FK495 were used to inhibit splenocyte proliferation induced by alloantigens and the results are reported in Fig[ 3[ In this case the SMS concentration that acted synergistically with FK495 was higher than the one reported above "from 09−00Ð09−8 M#^ furthermore\ SMS enhanced the antiproliferative e}ect of both of the FK495 doses used[ On the other hand\ while the inhibitory e}ect of FK495 was similar both with polyclonal or alloantigen stimulation at a dose of 9[99990 mg:ml\ at a dose one order of magnitude higher its action was more visible on mitogen stimulation rather than on alloantigen activation[ These _ndings support the concept that the addition of SMS signi_cantly enhances the immu! nosuppressive e}ect of suboptimal doses of FK495[ Furthermore\ this peptide was more e}ective when the stimulus was an oligoclonal activator such as an alloantigen and its synergic action with FK495 was more evident in this case[ The association of these two compounds was tested in vivo using an allogenic skin graft model[ Sixty BN rats were grafted with LEW rat skin and divided into ten experimental groups that either received no treatment^ SMS alone at di}erent con! centrations ranging between 9[0 mg:ml and 09 mg:ml\ or an association of SMS with FK495 at 9[0 mg:kg[ The FK495 regimen chosen was the highest dose that had previously been shown to either poorly or not protected at all the allografted animals[ SMS was administered twice and FK495

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Fig[ 0[ Spleen cell proliferation induced by PHA or alloantigens in the presence of di}erent concentrations of SMS[ Results are expressed as mean cpm2SE[ Data are representative of six independent experiments performed[ Unstimulated splenocytes "medium alone#] 456271[

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Fig[ 1[ E}ect of SMS on rat splenocytes stimulated by mAb a CD2 or a CD17 plus TPA[ The results are expressed as mean cpm2SE[ Unstimulated splenocytes "medium alone#] cpm 431276[ Data are representative of three independent experiments[

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Fig[ 2[ Inhibitory e}ect of the association of di}erent SMS doses with FK495 9[99990 and 9[9990 mg:ml compared with the e}ect on PHA stimulated rat splenocytes obtained by FK495 alone "broken line#[ The mean cpm2SE of PHA stimulated splenocytes are 085092189[ Data are representative of six independent experiments[

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Fig[ 3[ Inhibitory e}ect of the association of di}erent SMS doses with FK495 9[99990 and 9[9990 mg:ml compared with the e}ect on alloantigen stimulated rat splenocytes obtained by FK495 alone "broken line#[ The mean cpm2SE of alloantigen stimulated splenocytes are 071492234[ Data are representative of six independent experiments performed[

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Table 0 Mean survival time of LEW skin allografts "days# Exp[ Group

No

Therapy

MST2S[D[a

0 1 2 3 4 5 6 7 8 09

5 5 5 5 5 5 5 5 5 5

None FK495 9[0 mg:kg SMS 9[0 mg:kg SMS 9[4 mg:kg SMS 0 mg:kg SMS 09 mg:kg FK495 9[0 mg:kg¦SMS 9[0 mg:kg FK495 9[0 mg:kg¦SMS 9[4 mg:kg FK495 9[0 mg:kg¦SMS 0 mg:kg FK495 9[0 mg:kg¦SMS 09 mg:kg

6[529[4 7[429[4 7[429[4 829[5 829 829 8[429[4 0129[5 0029 0129[5

 P ¾ 9[995 vs 0 1 2[  P ¾ 9[9990 vs[ 0 1 2 3 4 5[ a Mean survival time of skin allograft2standard deviation[

once daily starting from the day of the transplant[ Results reported in Table 0 are expressed as mean survival time "MST# of the skin allograft2standard deviation[ Control animals\ that received saline solution\ rejected the skin graft with a MST of 6[4 days[ MST obtained in groups 1 "9[0 mg:kg FK495 once daily# and 2 "9[0 mg:kg SMS bid# were not signi_cantly higher when compared with the control group[ Animals receiving 9[4\ 0\ 09 mg:ml bid SMS alone showed a graft survival that was signi_cantly higher than the controls and when the same SMS concentrations were associated with 9[0 mg:ml FK495 the graft MST was signi_cantly higher when compared with each monotherapy group and\ of course\ with the control group[ None of the animals died or showed signs of drug!related toxicity[ 3[ Discussion Recent advances in understanding the cellular and molecular mechanisms that regulate immune responses\ including increasing evidence of interactions between neuropeptides and immu! nocompetent cells "Hagen et al[\ 0883#\ o}er the potential of new strategies in immunosuppression[ One of these peptides\ somatostatin and its receptor "SSTR#\ have been demonstrated to occur in normal and pathological lymphoid tissues and may play a regulatory\ largely inhibitory\ role in the immune response "Bhatena et al[\ 0870^ Himura et al[\ 0889#[ The results reported here were obtained using an SS derived peptide\ namely SMS 190!884\ that binds preferentially to the SSTR subtype 1 "SSTR1# "Reisine et al[\ 0884# that has been reported to be expressed on T lymphocytes "Elliot et al[\ 0883#[ SMS has the advantage that it is more stable than SS both in vitro and in vivo\ and we obtained evidence that it acts on the immune response via an inhibiting e}ect both on polyclonal and oligoclonal T cell induced proliferation\ with a more marked e}ect on the latter[ When SS rather than its analog was used\ we obtained the same inhibition pattern on lymphocyte

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proliferation "data not reported here#[ Furthermore\ we obtained evidence that SMS acts pref! erentially on the CD17 rather than the CD2 activation pathway[ On the other hand\ it is well known that immunosuppressive drugs like FK495 completely inhibit T cell activation via CD2 or CD1 pathways\ while the CD17 pathway is entirely una}ected "Sigal et al[\ 0880#[ These combined observations led to the hypothesis that SMS might act synergistically with FK495 by interfering with di}erent pathways of T cell activation[ Evidence that SMS enhanced the immunosuppressive e}ect of FK495 doses that were 099Ð0999 times lower than those that were e}ective on T lym! phocyte activation in vitro\ either by mitogens or by alloantigens\ demonstrate that the hypothesis is well!founded[ It is important to stress that the SMS concentrations that were e}ective in vitro are in the picomolar range\ close to physiological concentrations[ Moreover\ this peptide enhanced the antiproliferative e}ect of both of the FK495 doses used when the splenocyte were stimulated by alloantigens while it was e}ective only when tested together with the lower FK495 dose in the case of PHA stimulation[ This could be due to the fact that\ while at the dose of 9[99990 mg:ml FK495 was poorly inhibitory in both the system studied\ 9[9990 mg:ml of FK495\ by itself\ resulted in a good inhibition of PHA stimulation[ On the other hand\ it has recently been reported that the interaction of the CD17 molecule with its ligands "B6[0 and B6[1# during alloantigen stimulation is crucial to induce T cell proliferation "Linsley et al[\ 0882#\ hence\ the inhibition obtained in the MLR assay by the concomitant presence of SMS and FK 495 could be due to a down regulation of both CD2 and CD17 pathways[ The association of these two compounds was tested in vivo using an allogenic skin graft model[ The FK495 regimen chosen was the highest dose that was poorly:not e}ective in protecting the allograft and that showed no toxicity "Inamura et al[\ 0877#[ We selected SMS concentrations that were more e}ective in prolonging graft survival and were without side e}ects[ Animals receiving the two drugs in association showed a graft survival time that was signi_cantly improved when compared with each monotherapy group and\ of course\ with the control group[ In conclusion\ the results discussed in this paper indicate that a combined therapy based on FK495 and SMS could lead to a down regulation of the immune response without side e}ects and provides a promising beginning in the development of new strategies in immunosuppression[

Acknowledgments We thank Sandoz Italia and Fujisawa GmbH for providing the drugs[ This paper was partially supported by Associazione Italiana Ricerca sul Cancro "AIRC#[

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