184. Examination of an in vitro technique for assessing the relative immunosuppressive activity of synthetic and naturally occurring steroids related to hydrocortisone

lxii

Abstracts

l&I. EXAMINATION OF AN IN VITRO TECHNIQUE FOR ASSESSING THE RELATIVE ACTIVITY

OF SYNTHETIC

AND NATU~LLY

OCCURRING

STEROIDS

RELATED

IMMUNOSUPPRESSIVE TO HYDROCORTISONE

Browning, M.C.K., Roberts, C., Potts, R., Brown, R. and Beck, J.S. Ninewells

Hospital,

Hydrocortisone

University

of Dundee,

Scotland.

and chemically related steroids were tested for their depressive of lymphocytes from many normal subjects.

effecton PHA stimulation

In each experiment two or more compounds were tested on cells from one donor. The potencies of compounds were related to hydrocortisone (the reference compound). Reference steroids known to have no immunosuppressive action in viva had no action in vitro, whilst synthetic active glucocorticoids were shown to be immunosuppressive in vitro.

This in vitro technique appears to offer a simple screening method for determining the possible immunosuppressiveactivity of pharmacological products.

185.GLUCOCORTICOID ACTION ON HUMAN THYMOCYTE AND PERIPHERAL T-LYMPHOCYTE MITOGENESIS.Ranelletti,F.O,,Musiani,P., Lauriola,L.,Maggiano,N.and Piantelli M. Depts.of Histology-and Pathology,Univ.Cattolica S.Cuore.Rome,Italy. -7 The capacity of dexamethasone(DEX)(lO M)of inhibiting the peripheral blood lymphocyte(P8L)mitogenesi.s was inversely correlated with the phytohemagglutinin(PHA)concentration used(from40% to158 at suboptimal and optimal PHA concentrations,respectively).Conversely,DEX completely(>90%) inhibited the thymocyte(TY)mitogenesis,irrespective of PHA concentrations. T-cells purified from PBL(TC)(adherent cellsCl%)behaved as TY regarding the DEX inhibitory pattern.The addition of adherent cells(M interleukin 1fILl)was effective in removing the DEX inhibitory effect on TC but not on TY,in a PHA concentration dependent manner.However,a delayed addition(24 hr)of DEX to TY cultures was sufficient in giving an inhibitory pattern quite similar to that observed for TC.As on a wide range of IL1 concentrations,the DEX inhibitory capacity was only dependent on PHAdoses it seems likely that the main glucocorticoid inhibitory action on T-lymph2 cytmitogenesis is directed on the PHA blastic activation.

186.~-~,~~~~~ ~~~-~E~~~~ pT794 LACKS ~TI-GLUCOCORT~COID RECEPTOR-I~NOA~TIVITY. Evidence for the Absence of a Receptor

Domain Responsible

for,Biological

Effects.

Okret S, Stevens Y:W, Carlstedt-Duke .I,Wrange 6, Gustafsson.J-A,.and"stevensJ. Dept. Medical Nutrltlon. Karolinska Inst. Stoc_k~~_~,.,.,&&d~&~r~c C;mcarsnr;uu Glucocorticoid-resistant(CR), in contrast to glucocorticoid-sensitive(CS) mouse lymphoma P1798,was shown to lack anti-glucocorticoidreceptor-i~unoactivity. Antibodies raised against the purified rat liver glucocorticoid receptor (GR), crossreacted with the GR from CS but not with the GR from CR P1798 lymphoma. Using a highly specific antisera against the GR inan indirect competitive enzyme-linked immunosorbent assay (Okret, et.a1.(1981) Biochem Biophys Acta f&!, 205f, shows that%-chymotrypsin digestion of the GR from CS FL798 lymphomacause a separationof a "resistant-like"

non-immunogenic steroid-DNA-binding domain (Stokesradius3.3nm) from the immunoactive domain (Stokesradius 2.6nm).In contrastto CS P1798 lymphoma,both before and after at all could be found in the cytosol frsm *r.-chymotrypsin digestion,no inrmunoactivity CR Pl798 lymphoma. This was assayed both in whole cytosol and after chromatography on DNA-cellulose or gel filtration on Agarose A-0.5m. These results suggest that the domain of the CS GR conraining the immunoactive determinant(s), normally removed by limited proteolysis by ti-chymotrypsin,appears to be missing in CR P1798 lymphoma cytosol. It would seem, therefore, chat this domain plays an important role in the mechanism of action of glucocorticoids. It might suggest that a mutation has occured affecting the genome,resu-ltingin a defect transcrigti-on or the receptor gene.