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The immunosuppressive potency in vitro of physiological and synthetic steroids on lymphocyte cultures
Int. J. Immunopharmac., Vol. 9, No. 4, pp. 469-473, 1987. Printed in Great Britain.
0192-0561/87 $3.00+ .00 International Society for Immunopharmacology.
THE I M M U N O S U P P R E S S I V E P O T E N C Y I N VITRO OF P H Y S I O L O G I C A L A N D SYNTHETIC STEROIDS ON L Y M P H O C Y T E CULTURES ERIK LANGHOFF, KLAUSOLGAARD and JORGEN LADEFOGED Medical Department P, Rigshospitalet, Copenhagen, Denmark (Received 17 July 1986 and in final form 29 December 1986)
Abstract - - The immunosuppressive potency of five natural and seven synthetic steroids were tested in vitro
on phytohemagglutinin (PHA) stimulated peripheral lymphocytes (PBL) and T-lymphocytes and compared to their anti-inflammatory potencies. The physiological glucocorticoid, hydrocortisone, was of intermediate immunosuppressive potency in vitro, whereas the metabolites of hydrocortisone (cortisone, dihydrocortisol, and tetrahydrocortisol) and aldosterone were without effect. The synthetic steroids, methylprednisolone and fluorohydrocortisone were both highly potent in suppressing the PHA responses of both lymphocyte subsets. Prednisolone and dexamethasone were of intermediate potency and ranked similar to hydrocortisone which is in contrast to their anti-inflammatory properties. Prednisone, the biologically inactive metabolite of prednisolone, was without immunosuppressive properties. Deoxydeflazacort, the biologically active metabolite of deflazacort (a new oxazoline derivative of prednisolone) was comparable to prednisolone and hydrocortisone in suppressing lymphocyte proliferation but again there was a large discrepancy between the in vitro immunosuppressive effect and the anti-inflammatory potency. In conclusion, the present assay may therefore separate the immunosuppressive properties from the antiinflammatory properties of glucocorticoids. These findings may be useful for comparison of new synthetic steroids.
Steroid therapy alone or in combination with other Isolation o f mononuclear cells (PBL) immunosuppressive agents is the most frequent The peripheral blood lymphocytes were isolated as immunosuppressive regimen of a large number of previously described (Langhoff, Ladefoged & auto-immune diseases. However, steroids are Dickmeiss, 1985). Briefly, venous blood was added administered on the basis of their in vivo anti- to equal amounts of Hanks' balanced salt solution. inflammatory properties which may not be directly The PBLs were then isolated by lymphoprep (Nyco, correlated to their immunosuppressive properties, • Norway). The isolated cells were washed three times and adverse effects often occur such as osteopenia, with Hanks' solution and resuspended in RPMI-1640 and other metabolic disorders. (Gibco, Europe) containing 10070 of fetal calf serum, The immunosuppressive properties in human 10/ag-ml of gentamycine (Essex Chemie, lymphocyte subpopulations in vitro of natural Switzerland), and 2 mM of L-glutamine (Gibco, steroids, their metabolites, synthetic steroids, and Europe). synthetic steroids with bone-sparing properties were investigated. The in vitro immunosuppressive properties were compared to the anti-inflammatory Isolation o f nylon wool non-adherent T-lymphocytes potencies, the metabolic potencies, and the sodium Suspensions containing 15 x 106 PBL in 0.5 ml of retaining properties. growth medium were added to a 10 cm plastic drinking straw packed with 0.1 g of nylon wool EXPERIMENTAL PROCEDURES (Fenwal Laboratories, U.S.A.) preheated at 37°C (Langhoff et al., 1985). The cell suspension was Subjects allowed to absorb into the nylon wool and an Blood samples, 25 ml, were drawn by venepunc- additional 0.5 ml of growth medium (37°C) was ture from healthy donors aged 2 0 - 6 0 yr. added to the column. The column was then 469
470
E. LANGHOFF et al.
incubated for 30 min at 37°C in a humidified amosphere with 5070 carbon dioxide. The nonadherent T-cells (B-cell depleted lymphocytes) were then eluted by dropwise addition of 10 ml of growth medium (37°C) to the column.
PBL and T-cell proliferation assay Triplicates of PBL and T-cells were cultured in microtiter plates (Nunc, Denmark) containing 5 × 104 cells/well. Phytohemagglutinin (PHA; Difco, U.S.A.) 8/ag/well (optimal concentration) was used as mitogen. Glucocorticoids, in parallel experiments, were added to part of the triplicates in equimolar amounts. The following concentrations were used: 0, 1.2, 6.0, 24, 60, 240, 600 × 10 7 M, i.e. within in vivo physiological, pharmacological, and suprapharmacological concentrations of commonly used steroids. Two reference steroids, methylprednisolone, and hydrocortisone, were always included in each experiment. The total volume of incubation was 170/A/well and all cultures were incubated for 72 h in a humidified atmosphere with 5070 carbon dioxide at 37°C before the addition of the radioisotope for 20 h ('4Cthymidine, 20 nCi/well, Amersham, U.K.). The cultures then were harvested and counted (Packard, Tri-Carb, Liquid Scintillation Spectrometer). The suppressive effect of each steroid was expressed at each concentration level as the percent response of the maximal mitogen response without steroid. The relative potency of each steroid was defined (Langhoff et al., 1985) as the calculated concentration of the compound with the same 070 suppressive effect on cell proliferation in vitro as compared to that of methylprednisolone at 6 × 10 -7 M , i.e. within in vivo pharmacological concentrations of steroid.
Statistical analysis A paired t-test was used to compare the differences of the in vitro immunosuppressive potencies of the glucocorticoids tested in parallel cultures at 6 × 10-Tm. Student's t-test was used to compare PBL and T-cell cultures. Values of P <0.05 were considered significant.
RESULTS
Physiological glucocorticoid and aldosterone The physiological glucocorticoid, hydrocortisone, is shown as a reference glucocorticoid in the PBL cultures (Figs 1 - 3) and in the T-cell cultures (Fig. 4). The in vitro potency of the reference glucocorticoid, hydrocortisone, was significantly different from the high potency group of the synthetic steroid, methylprednisolone, (Fig. 1, P <0.0005) and the mineralocorticoid, aldosterone (P <0.01). Thus, the calculated relative potency of hydrocortisone to methylprednisolone (Table 1) was 1:11. The potency of hydrocortisone was not significantly different in PBL and T-cell cultures.
% RESPONSE 90
70
50
30
10-
Compounds Methylprednisolone hemisuccinate and hydrocortisone hemisuccinate (Upjohn, U.S.A.); deflazacort and 21-deacetyl-deflazacort (LePetit, Italy); prednisolone 21-phosphate and prednisone 21-hemisuccinate (Steroids, P730 and P320, U.S.A.); cortisone (Sigma C2755, U.S.A.). 5/3dihydrocortisol (Sigma P9771, U.S.A.); tetrahydrocortisol (Markor Chemicals 2495, Israel); dexamethasone phosphate (Merck Sharp & Dome, the Netherlands); 9-a-flurohydrocortisone (Florinef, Squibb, U.K.); aldosterone (Aldocorten, CibaGeigy, Switzerland).
t'2
~
2~.
6'0
2Zo 6o6.~o-7 M
STEROID CONCENTRATION
Fig. 1. The mean effect (-+ S.E.M.) of increasing concentrations of five tested steroids on the PHA response of PBL cultures from healthy donors. O Methylprednisolone succinate (r = 0.99, n = 55). VDeflazacort (r = 0.97, n = 11). [] 21-deacetyl-deflazacort (r = 0.95, n = 11). • Hydrocortisone hemisuccinate (r = 0.95, n = 54). • Aldosterone (r = 0.93, n = 13). Statistical comparison between groups of steroids are: hydrocortisone hemisuccinate and methylprednisolone succinate, P <0.0005; hydrocortisone hemisuccinate and aldosterone, P <0.01.
The Immunosuppressive Potency of Steroids
471
Table 1. Relative potency of genuine and synthetic steroids in PBL cultures Immunosuppressive potency in vitro
Anti-inflammatory potency*
Metabolic potency t
Sodium retention*
1 0 0.01 0 0 0.6 0 2.2 0.12 1.0 11 135
1 0.8 -0 0.2 4.0 4.0 25 40* 40* 5.0 10
1 0.8 --0 4.0 4.0 17 0.6 ~ 0.6 ~ 5.0 1 0 - 25
1 0.8 -0 250 0.8 0.8 0 --0.5 125
Hydrocortisone Cortisone Dihydrocortisol Tetrahydrocortisol Aldosterone Prednisolone Prednisone Dexamethasone Deflazacort Deoxy-deflazacort Methylprednisolone Fluorohydrocortisone * Haynes & Murad (1980).
t Documenta Geigy (1962). * Schiatti, Selva, Barone, Restelli & Glasser (1980). Buniva, Dubini & Sasella (1979).
S y n t h e t i c glucocorticoids P r e d n i s o l o n e a n d d e x a m e t h a s o n e as well as t h e n e w o x a z o l i n e derivative o f p r e d n i s o l o n e , d e f l a z a c o r t a n d its m e t a b o l i t e , d e o x y d e f l a z a c o r t , w e r e o f i n t e r m e d i a t e p o t e n c y (Figs 1 a n d 2). A c c o r d i n g l y , t h e
c a l c u l a t e d i m m u n o s u p p r e s s i v e p o t e n c i e s in vitro ( T a b l e 1) o f d e o x y d e f l a z a c o r t , p r e d n i s o l o n e , a n d d e x a m e t h a s o n e w e r e c o m p a r a b l e w h i c h is in c o n t r a s t to their anti-inflammatory properties. Methylprednisolone and fluorohydrocortisone were both highly
% RESPONSE
% RESPONSE
9oi 701 5oi
• •
• TETRAHYDROCORTISOL
70
DIHYDROCORTfSOL .
•
PREDNISOLONE HYOROCORTI~NE ~ DEXAMETHASONE
FLUOROHYDROCORTISONE
CORTISONE ~
HYDROCORTISONE
5O
30-
10-
1;2
~
2~
6b
zZo 6o6.1o .7 M
STEROID CONCENTRATION
Fig. 2. The m e a n effect (+ S.E.M.) of increasing concentrations o f five tested steroids on the P H A response of PBL cultures from healthy donors. • Fluorohydrocortisone (r = 0.93, n = 9). [] Prednisone 21-phosphate (r = 0.99, n = 14)./x Dexamethasone phosphate (r = 0.99, n = 12). • Hydrocortisone hemisuccinate (r = 0.95, n = 54). • Prednisone 21-hemisuccinate (r = 0.44, n = 13). Statistical comparison between groups o f steroids are: hydrocortisone hemisuccinate and fluorohydrocortisone, P <0.05; hydrocortisone hemisuccinate and prednisone 21 hemisuccinate, P <0.05.
t'2
6
2Z
6b
2Zo 606.10 .7 M
STEROID CONCENTRATION
Fig. 3. The m e a n effect (_+ S.E.M.) of increasing concentrations of three tested metabolites of hydrocortisone on the P H A response of PBL cultures from healthy donors. • Tetrahydrocortisol (r = 0.90, n = 38). [] dihydrocortisol (r = 78, n = 42). • Cortisone (r = 0.67, n = 27). • Hydrocortisone hemisuccinate (r = 0.95, n = 54). Statistical comparison between groups o f steroids are: hydrocortisone hemisuccinate and tetrahydrocortisol, P <0.005; hydrocortisone hemisuccinate and cortisone hemisuccinate, P <0.05; hydrocortisone hemisuccinate and dihydrocortisol P <0.001.
472
E. LANGHOFF et al.
potent lymphocyte suppressive steroids in the PBL cultures compared to hydrocortisone. Thus, the relative potencies of methylprednisolone and fluorohydrocortisone to hydrocortisone were 11:1 and 135:1. Prednisone, the inactive metabolite of prednisolone was without in vitro immunosuppressive effect. The T-cell cultures were significantly (P <0.001) more sensitive to these highly potent steroids than the PBL cultures (Figs 3 and 4).
Metabofites o f hydrocortisone
The potency of the physiological metabolites of hydrocortisone (cortisone, dihydrocortisol, and tetrahydrocortisol) were all significantly lower (P <0.05, P <0.001 and P <0.005, respectively) than that of hydrocortisone (Fig. 3) even at suprapharmacological (6 x 10 5 M) concentrations. The calculated relative potencies (Table 1) of the metabolites of hydrocortisone were below 1:100 compared to that of hydrocortisone. In the T-cell cultures the metabolites of hydrocortisone ranked similarly (Fig. 4).
°/. RESPONSE
j.
1.2
;i;;ii i2 (~
2~ 60 2~ 600"10"7/,4 STEROID CONCENTRATION
Fig. 4. The mean effect (+_ S.E.M.) of increasing concentrations of five tested steroids on the PHA response of T-cell cultures from healthy donors. C Methylprednisolone succinate (r = 0.98, n = 11). • Fluorohydrocortisone (r = 0.92, n = 10). • Hydrocortisone hemisuccinate (r = 0.93, n = 11). • Tetrahydrocortisol (r = 0.92, n = 8). [] Dihydrocortisol (r = 0.89, n = 11). Statistical comparison between groups of steroids are: hydrocortisone hemisuccinate vs fluorohydrocortisone and methylprednisolone succinate, P <0.005 and P <0.001, respectively; hydrocortisone hemisuccinate vs dihydrocortisol and tetrahydrocortisol, P <0.01 and P <0.005, respectively).
DISCUSSION Therapy with glucocorticoids is a widely used immunosuppressive regimen in the treatment of various autoimmune diseases such as systemic lupus erythematosis or rheumatoid arthritis. However, when using glucocorticoids several considerations must be taken into account such as (1) the immunosuppressive potencies, (2)the anti-inflammatory properties which may not be ranked similar to the immunosuppressive potencies, (3) the metabolic adverse effects such as demineralization of the skeleton. The present results clearly demonstrate large differences between the immunosuppressive properties in vitro and the in vivo anti-inflammatory properties which are based on assays such as inhibition of granuloma formation around cotton pellets implanted in rats (Lerner, Bianchi, Turkheimer, Singer & Borman, 1964). Thus, this concept of anti-inflammatory potency may not be extrapolated to the immunosuppressive potency of the compounds found in the present study. Furthermore, part of this inconsistency may be due to species variations in the sensitivity to the steroids in question. Accordingly, lymphocytes from guinea pigs and man are resistant to glucocorticoids whereas, murine lymphocytes are easily lysed upon exposure to glucocorticoids (Fauci, Muramami, Brandon, Loriaux & Lipsett, 1980; Cupps & Fauci, 1982). The lack of suppressive effects of the physiologically inactive metabolites of hydrocortisone (cortisone, dihydrocortisol, and tetrahydrocortisol), prednisone, and aldosterone support the reliability of the present assay. The present study showed that T-lymphocyte cultures were considerably more sensitive to steroids with high immunosuppressive potencies in vitro than the PBL cultures, but in both sets of cultures the immunosuppressive potencies of the tested glucocorticoids ranked similarly. The immunosuppressive mechanisms of glucocorticoids are at present unclear, but the high T-cell sensitivity may indicate that glucocorticoids interact with the proliferation of activated T-lymphocytes. This is in accordance with our previous results (Langhoff et al., 1985) of a high steroid resistance of NK and K cell functions which are not dependent of proliferation. Furthermore, pokeweed (PWM) responses which are partly B-cell responses (Stobo, 1980) have been shown to be more resistant to steroid than the concanavalin A (Con A) responses which are primarily of T-cell origin (Schmidke, Hatfield & Ferguson, 1979; Stobo, 1980).
The Immunosuppressive Potency of Steroids In c o n s i d e r a t i o n o f the adverse m e t a b o l i c effects o f the m o s t c o m m o n l y used steroids, deflazacort, a n oxazoline derivative o f prednisolone, has been r e p o r t e d to cause less b o n e d e m i n e r a l i z a t i o n ( G e n n a r i , I m b i b o , M o n t a g n a n a , Bernini, N a r d i & Avioli 1984; H a h n , Halstead, Strates, I m b i b o & Baran, 1980a). In the present assay the i m m u n o s u p p r e s s i v e effect o f the active m e t a b o l i t e o f deflazacort, 21-deacetyl-deflazacort, c o m p a r e s f a v o r a b l y to p r e d n i s o l o n e a n d h y d r o c o r t i s o n e . A g a i n , the a n t i - i n f l a m m a t o r y properties r a n k differently f r o m the i m m u n o s u p p r e s s i v e potencies in vitro (Table l). H a h n , Pletscher & M u n i a i n (1980b) have e x a m i n e d the i m m u n o s u p p r e s s i v e potencies in vitro o f deflazacort a n d its metabolite in mixed
473
l y m p h o c y t e cultures (MLC). Their studies o f the i m m u n o s u p p r e s s i v e properties in vitro o f deflazacort are in good a g r e e m e n t with o u r results. T h u s , based u p o n the b o n e - s p a r i n g p r o p e r t y o f deflazacort a n d the present d a t a which c o m p a r e the i m m u n o suppressive potency in vitro favorable to prednisolone, it m a y be considered t h a t this new steroid offers a n alternative to the currently available glucocorticoids. Acknowledgements - - This work has been supported by grants from the Danish Medical Research Council, Dagmar Marshall Foundation, 1870 Foundation, and NOVO's Foundation. We are grateful to Mrs V. Pless and Mrs K. Meibom for their invaluable technical assistance.
REFERENCES
BUNIVA, G., DUBINI, A. & SASELLA, D. (1979). Human bioassay of corticotropin-suppressing, eosinopenic and hyperglycaemic potencies of deflazacort and prednisone. Curr. ther. Res., 26, 6 9 - 8 1 .
CUPPS, T. R. • FAUCI,A. S. (1982). Corticoid-mediated immunoregulation in man. Immun. Rev., 65, 133 - 155. Documenta Geigy (1962). Scientific Tables (ed. Diem, K.) p. 491. J. R. Geigy, Basle, Switzerland. FAUCI, A. S., MURAMAMI,T., BRANDON, D. D., LORIAUX, D. L. & LIPSETT, M. B. 0980). Mechanisms of corticosteroid action on lymphocyte subpopulations. Cell Immun., 49, 43 - 50. GENNARI, C., IMaIMBO, B., MONTAGNANA,M., BERNINI, M., NARDI, P. & AVIOLI, L. V. (1984). Effects of prednisone and deflazacort on mineral metabolism and parathyroid hormone activity in humans. Calcif. Tissue Int., 36, 245 - 252. HAHN, T. J., HAESTEAD, L. R., STRATES, B., IMBIMBO, B. & BARAN, D. T. (1980a). Comparison of subacute effects of oxazacort and prednisone on mineral metabolism in man. Calcif. Tissue Int., 31, 109- 115. HAHN, B., PLETSCHER, L. S. & MUNIAIN, M. (1980b). Immunosuppressive effect of deflazacort. A new glucocorticoid with bones-sparing and carbohydrate-sparing properties: Comparison with prednisone. J. Rheumat., 8, 7 8 3 - 790. HAYNES, R. C. & MURAO, F. (1980). Hormones and hormone antagonists. In The Pharmacological Basis o f Therapeutics (eds Gilman, A. G., Goodman, L. S. and Gilman, A.) p. 1482. Macmillan, New York. LANGHOFE,E., LADEFOGED,J. & DICKMEISS, E. (1985). The immunosuppressive potency of various steroids on peripheral blood lymphocytes, T-cells, NK and K cells. Int. J. Immunopharmac., 7, 4 8 3 - 489. LERNER, L. J., BIANCHI, A., TURKHEIMER, A. R., SINGER, F. M. & BORMAN, A. (1964). Anti-inflammatory steroids: Potency, duration and modification of activities. Ann. N. Y. Acad. Sci., 116, 1071 - 1077. SCHIATTI, P., SELVA, D., BARONE, D., RESTELEI, A. & GLASSER, A. (1980). Antiinflammatory activity and other pharmacological properties of I 1,21-dihydroxy-2 ' -methyl-5 ' H-pregna- 1,4-dieno ( 17, 16-d) oxazole-3,20-dieone-2 lacetat (Deflazacort). Arzneimittel-Forsch., 30, 1543- 1549. SCHM1DKE, J. R., HATFIELD, S. M. & FERGUSON, R. M. (1979). Differential susceptibility of human peripheral blood lymphocyte subpopulations to mitogenic activation. Transplantation, 27, 319-323. STOBO, J. D. (1980). Mitogens. In Clinicallmmunology, Vol. 4 (eds Bach, F. H. and Good, R. A.) p. 55. Academic Press, New York.
0192-0561/87 $3.00+ .00 International Society for Immunopharmacology.
THE I M M U N O S U P P R E S S I V E P O T E N C Y I N VITRO OF P H Y S I O L O G I C A L A N D SYNTHETIC STEROIDS ON L Y M P H O C Y T E CULTURES ERIK LANGHOFF, KLAUSOLGAARD and JORGEN LADEFOGED Medical Department P, Rigshospitalet, Copenhagen, Denmark (Received 17 July 1986 and in final form 29 December 1986)
Abstract - - The immunosuppressive potency of five natural and seven synthetic steroids were tested in vitro
on phytohemagglutinin (PHA) stimulated peripheral lymphocytes (PBL) and T-lymphocytes and compared to their anti-inflammatory potencies. The physiological glucocorticoid, hydrocortisone, was of intermediate immunosuppressive potency in vitro, whereas the metabolites of hydrocortisone (cortisone, dihydrocortisol, and tetrahydrocortisol) and aldosterone were without effect. The synthetic steroids, methylprednisolone and fluorohydrocortisone were both highly potent in suppressing the PHA responses of both lymphocyte subsets. Prednisolone and dexamethasone were of intermediate potency and ranked similar to hydrocortisone which is in contrast to their anti-inflammatory properties. Prednisone, the biologically inactive metabolite of prednisolone, was without immunosuppressive properties. Deoxydeflazacort, the biologically active metabolite of deflazacort (a new oxazoline derivative of prednisolone) was comparable to prednisolone and hydrocortisone in suppressing lymphocyte proliferation but again there was a large discrepancy between the in vitro immunosuppressive effect and the anti-inflammatory potency. In conclusion, the present assay may therefore separate the immunosuppressive properties from the antiinflammatory properties of glucocorticoids. These findings may be useful for comparison of new synthetic steroids.
Steroid therapy alone or in combination with other Isolation o f mononuclear cells (PBL) immunosuppressive agents is the most frequent The peripheral blood lymphocytes were isolated as immunosuppressive regimen of a large number of previously described (Langhoff, Ladefoged & auto-immune diseases. However, steroids are Dickmeiss, 1985). Briefly, venous blood was added administered on the basis of their in vivo anti- to equal amounts of Hanks' balanced salt solution. inflammatory properties which may not be directly The PBLs were then isolated by lymphoprep (Nyco, correlated to their immunosuppressive properties, • Norway). The isolated cells were washed three times and adverse effects often occur such as osteopenia, with Hanks' solution and resuspended in RPMI-1640 and other metabolic disorders. (Gibco, Europe) containing 10070 of fetal calf serum, The immunosuppressive properties in human 10/ag-ml of gentamycine (Essex Chemie, lymphocyte subpopulations in vitro of natural Switzerland), and 2 mM of L-glutamine (Gibco, steroids, their metabolites, synthetic steroids, and Europe). synthetic steroids with bone-sparing properties were investigated. The in vitro immunosuppressive properties were compared to the anti-inflammatory Isolation o f nylon wool non-adherent T-lymphocytes potencies, the metabolic potencies, and the sodium Suspensions containing 15 x 106 PBL in 0.5 ml of retaining properties. growth medium were added to a 10 cm plastic drinking straw packed with 0.1 g of nylon wool EXPERIMENTAL PROCEDURES (Fenwal Laboratories, U.S.A.) preheated at 37°C (Langhoff et al., 1985). The cell suspension was Subjects allowed to absorb into the nylon wool and an Blood samples, 25 ml, were drawn by venepunc- additional 0.5 ml of growth medium (37°C) was ture from healthy donors aged 2 0 - 6 0 yr. added to the column. The column was then 469
470
E. LANGHOFF et al.
incubated for 30 min at 37°C in a humidified amosphere with 5070 carbon dioxide. The nonadherent T-cells (B-cell depleted lymphocytes) were then eluted by dropwise addition of 10 ml of growth medium (37°C) to the column.
PBL and T-cell proliferation assay Triplicates of PBL and T-cells were cultured in microtiter plates (Nunc, Denmark) containing 5 × 104 cells/well. Phytohemagglutinin (PHA; Difco, U.S.A.) 8/ag/well (optimal concentration) was used as mitogen. Glucocorticoids, in parallel experiments, were added to part of the triplicates in equimolar amounts. The following concentrations were used: 0, 1.2, 6.0, 24, 60, 240, 600 × 10 7 M, i.e. within in vivo physiological, pharmacological, and suprapharmacological concentrations of commonly used steroids. Two reference steroids, methylprednisolone, and hydrocortisone, were always included in each experiment. The total volume of incubation was 170/A/well and all cultures were incubated for 72 h in a humidified atmosphere with 5070 carbon dioxide at 37°C before the addition of the radioisotope for 20 h ('4Cthymidine, 20 nCi/well, Amersham, U.K.). The cultures then were harvested and counted (Packard, Tri-Carb, Liquid Scintillation Spectrometer). The suppressive effect of each steroid was expressed at each concentration level as the percent response of the maximal mitogen response without steroid. The relative potency of each steroid was defined (Langhoff et al., 1985) as the calculated concentration of the compound with the same 070 suppressive effect on cell proliferation in vitro as compared to that of methylprednisolone at 6 × 10 -7 M , i.e. within in vivo pharmacological concentrations of steroid.
Statistical analysis A paired t-test was used to compare the differences of the in vitro immunosuppressive potencies of the glucocorticoids tested in parallel cultures at 6 × 10-Tm. Student's t-test was used to compare PBL and T-cell cultures. Values of P <0.05 were considered significant.
RESULTS
Physiological glucocorticoid and aldosterone The physiological glucocorticoid, hydrocortisone, is shown as a reference glucocorticoid in the PBL cultures (Figs 1 - 3) and in the T-cell cultures (Fig. 4). The in vitro potency of the reference glucocorticoid, hydrocortisone, was significantly different from the high potency group of the synthetic steroid, methylprednisolone, (Fig. 1, P <0.0005) and the mineralocorticoid, aldosterone (P <0.01). Thus, the calculated relative potency of hydrocortisone to methylprednisolone (Table 1) was 1:11. The potency of hydrocortisone was not significantly different in PBL and T-cell cultures.
% RESPONSE 90
70
50
30
10-
Compounds Methylprednisolone hemisuccinate and hydrocortisone hemisuccinate (Upjohn, U.S.A.); deflazacort and 21-deacetyl-deflazacort (LePetit, Italy); prednisolone 21-phosphate and prednisone 21-hemisuccinate (Steroids, P730 and P320, U.S.A.); cortisone (Sigma C2755, U.S.A.). 5/3dihydrocortisol (Sigma P9771, U.S.A.); tetrahydrocortisol (Markor Chemicals 2495, Israel); dexamethasone phosphate (Merck Sharp & Dome, the Netherlands); 9-a-flurohydrocortisone (Florinef, Squibb, U.K.); aldosterone (Aldocorten, CibaGeigy, Switzerland).
t'2
~
2~.
6'0
2Zo 6o6.~o-7 M
STEROID CONCENTRATION
Fig. 1. The mean effect (-+ S.E.M.) of increasing concentrations of five tested steroids on the PHA response of PBL cultures from healthy donors. O Methylprednisolone succinate (r = 0.99, n = 55). VDeflazacort (r = 0.97, n = 11). [] 21-deacetyl-deflazacort (r = 0.95, n = 11). • Hydrocortisone hemisuccinate (r = 0.95, n = 54). • Aldosterone (r = 0.93, n = 13). Statistical comparison between groups of steroids are: hydrocortisone hemisuccinate and methylprednisolone succinate, P <0.0005; hydrocortisone hemisuccinate and aldosterone, P <0.01.
The Immunosuppressive Potency of Steroids
471
Table 1. Relative potency of genuine and synthetic steroids in PBL cultures Immunosuppressive potency in vitro
Anti-inflammatory potency*
Metabolic potency t
Sodium retention*
1 0 0.01 0 0 0.6 0 2.2 0.12 1.0 11 135
1 0.8 -0 0.2 4.0 4.0 25 40* 40* 5.0 10
1 0.8 --0 4.0 4.0 17 0.6 ~ 0.6 ~ 5.0 1 0 - 25
1 0.8 -0 250 0.8 0.8 0 --0.5 125
Hydrocortisone Cortisone Dihydrocortisol Tetrahydrocortisol Aldosterone Prednisolone Prednisone Dexamethasone Deflazacort Deoxy-deflazacort Methylprednisolone Fluorohydrocortisone * Haynes & Murad (1980).
t Documenta Geigy (1962). * Schiatti, Selva, Barone, Restelli & Glasser (1980). Buniva, Dubini & Sasella (1979).
S y n t h e t i c glucocorticoids P r e d n i s o l o n e a n d d e x a m e t h a s o n e as well as t h e n e w o x a z o l i n e derivative o f p r e d n i s o l o n e , d e f l a z a c o r t a n d its m e t a b o l i t e , d e o x y d e f l a z a c o r t , w e r e o f i n t e r m e d i a t e p o t e n c y (Figs 1 a n d 2). A c c o r d i n g l y , t h e
c a l c u l a t e d i m m u n o s u p p r e s s i v e p o t e n c i e s in vitro ( T a b l e 1) o f d e o x y d e f l a z a c o r t , p r e d n i s o l o n e , a n d d e x a m e t h a s o n e w e r e c o m p a r a b l e w h i c h is in c o n t r a s t to their anti-inflammatory properties. Methylprednisolone and fluorohydrocortisone were both highly
% RESPONSE
% RESPONSE
9oi 701 5oi
• •
• TETRAHYDROCORTISOL
70
DIHYDROCORTfSOL .
•
PREDNISOLONE HYOROCORTI~NE ~ DEXAMETHASONE
FLUOROHYDROCORTISONE
CORTISONE ~
HYDROCORTISONE
5O
30-
10-
1;2
~
2~
6b
zZo 6o6.1o .7 M
STEROID CONCENTRATION
Fig. 2. The m e a n effect (+ S.E.M.) of increasing concentrations o f five tested steroids on the P H A response of PBL cultures from healthy donors. • Fluorohydrocortisone (r = 0.93, n = 9). [] Prednisone 21-phosphate (r = 0.99, n = 14)./x Dexamethasone phosphate (r = 0.99, n = 12). • Hydrocortisone hemisuccinate (r = 0.95, n = 54). • Prednisone 21-hemisuccinate (r = 0.44, n = 13). Statistical comparison between groups o f steroids are: hydrocortisone hemisuccinate and fluorohydrocortisone, P <0.05; hydrocortisone hemisuccinate and prednisone 21 hemisuccinate, P <0.05.
t'2
6
2Z
6b
2Zo 606.10 .7 M
STEROID CONCENTRATION
Fig. 3. The m e a n effect (_+ S.E.M.) of increasing concentrations of three tested metabolites of hydrocortisone on the P H A response of PBL cultures from healthy donors. • Tetrahydrocortisol (r = 0.90, n = 38). [] dihydrocortisol (r = 78, n = 42). • Cortisone (r = 0.67, n = 27). • Hydrocortisone hemisuccinate (r = 0.95, n = 54). Statistical comparison between groups o f steroids are: hydrocortisone hemisuccinate and tetrahydrocortisol, P <0.005; hydrocortisone hemisuccinate and cortisone hemisuccinate, P <0.05; hydrocortisone hemisuccinate and dihydrocortisol P <0.001.
472
E. LANGHOFF et al.
potent lymphocyte suppressive steroids in the PBL cultures compared to hydrocortisone. Thus, the relative potencies of methylprednisolone and fluorohydrocortisone to hydrocortisone were 11:1 and 135:1. Prednisone, the inactive metabolite of prednisolone was without in vitro immunosuppressive effect. The T-cell cultures were significantly (P <0.001) more sensitive to these highly potent steroids than the PBL cultures (Figs 3 and 4).
Metabofites o f hydrocortisone
The potency of the physiological metabolites of hydrocortisone (cortisone, dihydrocortisol, and tetrahydrocortisol) were all significantly lower (P <0.05, P <0.001 and P <0.005, respectively) than that of hydrocortisone (Fig. 3) even at suprapharmacological (6 x 10 5 M) concentrations. The calculated relative potencies (Table 1) of the metabolites of hydrocortisone were below 1:100 compared to that of hydrocortisone. In the T-cell cultures the metabolites of hydrocortisone ranked similarly (Fig. 4).
°/. RESPONSE
j.
1.2
;i;;ii i2 (~
2~ 60 2~ 600"10"7/,4 STEROID CONCENTRATION
Fig. 4. The mean effect (+_ S.E.M.) of increasing concentrations of five tested steroids on the PHA response of T-cell cultures from healthy donors. C Methylprednisolone succinate (r = 0.98, n = 11). • Fluorohydrocortisone (r = 0.92, n = 10). • Hydrocortisone hemisuccinate (r = 0.93, n = 11). • Tetrahydrocortisol (r = 0.92, n = 8). [] Dihydrocortisol (r = 0.89, n = 11). Statistical comparison between groups of steroids are: hydrocortisone hemisuccinate vs fluorohydrocortisone and methylprednisolone succinate, P <0.005 and P <0.001, respectively; hydrocortisone hemisuccinate vs dihydrocortisol and tetrahydrocortisol, P <0.01 and P <0.005, respectively).
DISCUSSION Therapy with glucocorticoids is a widely used immunosuppressive regimen in the treatment of various autoimmune diseases such as systemic lupus erythematosis or rheumatoid arthritis. However, when using glucocorticoids several considerations must be taken into account such as (1) the immunosuppressive potencies, (2)the anti-inflammatory properties which may not be ranked similar to the immunosuppressive potencies, (3) the metabolic adverse effects such as demineralization of the skeleton. The present results clearly demonstrate large differences between the immunosuppressive properties in vitro and the in vivo anti-inflammatory properties which are based on assays such as inhibition of granuloma formation around cotton pellets implanted in rats (Lerner, Bianchi, Turkheimer, Singer & Borman, 1964). Thus, this concept of anti-inflammatory potency may not be extrapolated to the immunosuppressive potency of the compounds found in the present study. Furthermore, part of this inconsistency may be due to species variations in the sensitivity to the steroids in question. Accordingly, lymphocytes from guinea pigs and man are resistant to glucocorticoids whereas, murine lymphocytes are easily lysed upon exposure to glucocorticoids (Fauci, Muramami, Brandon, Loriaux & Lipsett, 1980; Cupps & Fauci, 1982). The lack of suppressive effects of the physiologically inactive metabolites of hydrocortisone (cortisone, dihydrocortisol, and tetrahydrocortisol), prednisone, and aldosterone support the reliability of the present assay. The present study showed that T-lymphocyte cultures were considerably more sensitive to steroids with high immunosuppressive potencies in vitro than the PBL cultures, but in both sets of cultures the immunosuppressive potencies of the tested glucocorticoids ranked similarly. The immunosuppressive mechanisms of glucocorticoids are at present unclear, but the high T-cell sensitivity may indicate that glucocorticoids interact with the proliferation of activated T-lymphocytes. This is in accordance with our previous results (Langhoff et al., 1985) of a high steroid resistance of NK and K cell functions which are not dependent of proliferation. Furthermore, pokeweed (PWM) responses which are partly B-cell responses (Stobo, 1980) have been shown to be more resistant to steroid than the concanavalin A (Con A) responses which are primarily of T-cell origin (Schmidke, Hatfield & Ferguson, 1979; Stobo, 1980).
The Immunosuppressive Potency of Steroids In c o n s i d e r a t i o n o f the adverse m e t a b o l i c effects o f the m o s t c o m m o n l y used steroids, deflazacort, a n oxazoline derivative o f prednisolone, has been r e p o r t e d to cause less b o n e d e m i n e r a l i z a t i o n ( G e n n a r i , I m b i b o , M o n t a g n a n a , Bernini, N a r d i & Avioli 1984; H a h n , Halstead, Strates, I m b i b o & Baran, 1980a). In the present assay the i m m u n o s u p p r e s s i v e effect o f the active m e t a b o l i t e o f deflazacort, 21-deacetyl-deflazacort, c o m p a r e s f a v o r a b l y to p r e d n i s o l o n e a n d h y d r o c o r t i s o n e . A g a i n , the a n t i - i n f l a m m a t o r y properties r a n k differently f r o m the i m m u n o s u p p r e s s i v e potencies in vitro (Table l). H a h n , Pletscher & M u n i a i n (1980b) have e x a m i n e d the i m m u n o s u p p r e s s i v e potencies in vitro o f deflazacort a n d its metabolite in mixed
473
l y m p h o c y t e cultures (MLC). Their studies o f the i m m u n o s u p p r e s s i v e properties in vitro o f deflazacort are in good a g r e e m e n t with o u r results. T h u s , based u p o n the b o n e - s p a r i n g p r o p e r t y o f deflazacort a n d the present d a t a which c o m p a r e the i m m u n o suppressive potency in vitro favorable to prednisolone, it m a y be considered t h a t this new steroid offers a n alternative to the currently available glucocorticoids. Acknowledgements - - This work has been supported by grants from the Danish Medical Research Council, Dagmar Marshall Foundation, 1870 Foundation, and NOVO's Foundation. We are grateful to Mrs V. Pless and Mrs K. Meibom for their invaluable technical assistance.
REFERENCES
BUNIVA, G., DUBINI, A. & SASELLA, D. (1979). Human bioassay of corticotropin-suppressing, eosinopenic and hyperglycaemic potencies of deflazacort and prednisone. Curr. ther. Res., 26, 6 9 - 8 1 .
CUPPS, T. R. • FAUCI,A. S. (1982). Corticoid-mediated immunoregulation in man. Immun. Rev., 65, 133 - 155. Documenta Geigy (1962). Scientific Tables (ed. Diem, K.) p. 491. J. R. Geigy, Basle, Switzerland. FAUCI, A. S., MURAMAMI,T., BRANDON, D. D., LORIAUX, D. L. & LIPSETT, M. B. 0980). Mechanisms of corticosteroid action on lymphocyte subpopulations. Cell Immun., 49, 43 - 50. GENNARI, C., IMaIMBO, B., MONTAGNANA,M., BERNINI, M., NARDI, P. & AVIOLI, L. V. (1984). Effects of prednisone and deflazacort on mineral metabolism and parathyroid hormone activity in humans. Calcif. Tissue Int., 36, 245 - 252. HAHN, T. J., HAESTEAD, L. R., STRATES, B., IMBIMBO, B. & BARAN, D. T. (1980a). Comparison of subacute effects of oxazacort and prednisone on mineral metabolism in man. Calcif. Tissue Int., 31, 109- 115. HAHN, B., PLETSCHER, L. S. & MUNIAIN, M. (1980b). Immunosuppressive effect of deflazacort. A new glucocorticoid with bones-sparing and carbohydrate-sparing properties: Comparison with prednisone. J. Rheumat., 8, 7 8 3 - 790. HAYNES, R. C. & MURAO, F. (1980). Hormones and hormone antagonists. In The Pharmacological Basis o f Therapeutics (eds Gilman, A. G., Goodman, L. S. and Gilman, A.) p. 1482. Macmillan, New York. LANGHOFE,E., LADEFOGED,J. & DICKMEISS, E. (1985). The immunosuppressive potency of various steroids on peripheral blood lymphocytes, T-cells, NK and K cells. Int. J. Immunopharmac., 7, 4 8 3 - 489. LERNER, L. J., BIANCHI, A., TURKHEIMER, A. R., SINGER, F. M. & BORMAN, A. (1964). Anti-inflammatory steroids: Potency, duration and modification of activities. Ann. N. Y. Acad. Sci., 116, 1071 - 1077. SCHIATTI, P., SELVA, D., BARONE, D., RESTELEI, A. & GLASSER, A. (1980). Antiinflammatory activity and other pharmacological properties of I 1,21-dihydroxy-2 ' -methyl-5 ' H-pregna- 1,4-dieno ( 17, 16-d) oxazole-3,20-dieone-2 lacetat (Deflazacort). Arzneimittel-Forsch., 30, 1543- 1549. SCHM1DKE, J. R., HATFIELD, S. M. & FERGUSON, R. M. (1979). Differential susceptibility of human peripheral blood lymphocyte subpopulations to mitogenic activation. Transplantation, 27, 319-323. STOBO, J. D. (1980). Mitogens. In Clinicallmmunology, Vol. 4 (eds Bach, F. H. and Good, R. A.) p. 55. Academic Press, New York.