- Email: [email protected]
Effects on cyclic AMP phosphodiesterase of drugs affecting mast cell degranulation
230
41
41a
P R O P H Y L A C T I C A N D T H E R A P E U T I C EFFECTS OF N A N D R O L O N E A N D ITS D E C A N O A T E E S T E R ( D E O A - D U R A B O L I N R ) IN M U R I N E L U P U S H. V e r h e u l l f W. S t i m s o n 2 ~ F. den H o l l a n d e r l ~ A . S c h u u r s l 1. B i o c h e m i c a l R e s e a r c h and D e v e l o p m e n t Labs. Organon, Oss ( N e t h e r l a n d s ) 2. D e p a r t m e n t of B i o c h e m i s t r y , S t r a t h c l y d e U n i v e r s i t y , G l a s g o w ( U . K . ) N Z B / W h y b r i d m i c e d e v e l o p an a u t o i m m u n e d i s e a s e , s t r o n g l y r e s e m b l i n g s y s t e m i c lupus e r y t h e m a t o s u s in h u m a n s . R o u b i n i a n et a l . d e m o n s t r a t e d t h a t c a s t r a t i o n of m a l e s , and c h r o n i c a d m i n i s t r a t i o n of o e s t r a d i o l a c c e l e r a t e , w h e r e a s t e s t o s t e r o n e and 5 ~ z - d i h y d r o t e s t o s t e r o n e d e l a y the o n s e t of the d i s e a s e . We d e m o n s t r a t e d that a l s o n a n d r o l o n e i m p l a n t s in s i l a s t i c t u b e s , and n a n d r o l o n e d e c a n o a t e ( D e c a - D u r a b o l i n R ) i n j e c t i o n s e v e r y 14 d a y s , f r o m 3 w e e k s after b i r t h o n w a r d s p r o d u c e a s i g n i f i c a n t r e d u c t i o n in m o r t a l i t y in f e m a l e and p r e p u b e r t a l l y c a s t r a t e d m a l e N Z B / W m i c e . P r o t e i n u r i a and a n t i - D N A IgG w e r e a l s o reduced in t h e s e m i c e . The c o r r e s p o n d i n g t e s t o s t e r o n e t r e a t m e n t s s h o w e d s i m i l a r e f f e c t s . M i c e s u r v i v i n g m o r e t h a n 65 w e e k s w e r e s a c r i f i c e d and s h o w e d s i g n i f i c a n t l y l e s s a n d r o g e n i c e f f e c t s ( a s m e a s u r e d by p r o s t a t e and s e m i n a l v e s i c l e w e i g h t ) after n a n d r o l o n e d e c a n o a t e t h a n after t e s t o s t e r o n e d e c a n o a t e t r e a t m e n t . When n a n d r o l o n e d e c a n o a t e t r e a t m e n t w a s s t a r t e d at the age of 27 - 29 w e e k s a s i m i l a r r e d u c t i o n in m o r t a l i t y w a s o b s e r v e d in p r e p u b e r t a l l y c a s t r a t e d m a l e m i c e . In f e m a l e m i c e the i m p r o v e m e n t was o n l y m a r g i n a l w h i c h m i g h t h a v e been due to the further a d v a n c e d s t a g e of the d i s e a s e at s t a r t of t r e a t m e n t ( f i r s t f e m a l e m i c e d i e d at 28 w e e k s ) . T e s t o s t e r o n e d e c a n o a t e was slightly less effective. The d a t a i n d i c a t e t h a t both n a n d r o l o n e ( - d e c a n o a t e ) and t e s t o s t e r o n e d e c a n o a t e t r e a t m e n t h a v e a b e n e f i c i a l p r o p h y l a c t i c and t h e r a p e u t i c e f f e c t in m u r i n e l u p u s . This e f f e c t d o e s not s e e m to be c o r r e l a t e d w i t h a n d r o g e n i c a c t i v i t y . In v i e w of its w e a k e r a n d r o g e n i c e f f e c t s and longer d u r a t i o n of a c t i o n , n a n d r o l o n e d e c a n o a t e ( D e c a - D u r a b o l i n R ) m i g h t p r o v e a b e t t e r c a n d i d a t e for the t r e a t m e n t of S L E , a d i s e a s e w h i c h is m o s t p r e v a l e n t in f e m a l e s . MODULATION OF MURINE LUPUS WITH SEX HORMONES N. Talal, J. Roubinian, and J. Greenspan Department of Medicine, University of California, and Veterans Administration Medical Center, San Francisco, California, USA. Systemic lupus erythematosus (SLE) is much more common in women than in men. The female: male ratio may be as high as 15:1 if one considers women during the child-bearing years, and declines to 2:1 for post-menopausal females. In NZB/NZW F I mice, an animal model for SLE, the disease occurs earlier and with greater severity in females. Prepubertal castration of male mice results in an accelerated disease so that the castrated males die at the same rate as females. Prepubertal castration of females does not improve survival. The administration of 5-e-dihydrotestosterone to prepubertal female mice suppresses autoantibody formation and immune complex glomerulonephritis. Mice so treated have an increased survival. By contrast, prepubertal mice treated with 17-B-estradiol have a decreased survival, and greater autoantibody formation and immune complex glomerulonephritis. If androgen administration is delayed until 3 or 6 months of age, B/W female mice again have an increased survival and less immune complex glomerulonephritis, but without a decline in antibodies to DNA. These results suggest that there may be several mechanisms by which androgens act to favorably influence the course of autoimmune disease in B/W mice.
MEDIATORS/DEGRANULATION
42
PHARMACOLOGICAL MODULATION OF HUMAN BASOPHIL DEGRANULATION H. L u c e , E. A l b e n q r e s , J.P. Tillement D4partement de Pharmacologie, Facult~ de M4decine de PARIS G4n~ral Sarrail, 94010 CRETEIL, France
XII,
8 rue
du
Anaphylactic reaction and its modulation by some pharmacological substances have been widely studied in a n i m a l s . However, results obtained were not directly extrapolated t o m a n , so w e d e v e l o p e d a technique (LEYNADIER F., L U C E H., D R Y J., 1977) t o c h e c k h u m a n b a s o p h i l degranulation and the effect
231
of v a r i o u s c o m p o u n d s . Subjects are selected regarding their reaginic hypersensitivity by intradermal skin testing. The most commonly implicated allergens were grass pollen and dermatophagoides pteronisinus. Corresponding allergens extracts (Institut Pasteur) a r e d i l u t e d in H e p ~ s b u f f e r t o t h e concentration of iOO/~g/ml. They are used, associated with solutions (in t h e s a m e b u f f e r ) of v a r i o u s c o m p o u n d s , EDTA, d i b u t y r y l A M P c , d i b u t y r y l GMPc, isoproterenol, disodium cromoglycate, colchicine and theophylline, or alone as c o n t r o l s . T h e t e c h n i q u e d e s c r i b e d involves the preparation of an e n r i c h i e d basophil cells suspension (E.C.B.S.) u s i n g an o r i g i n a l c o n c e n t r a t i o n gradient. T h e n 2 0 2 ~ i of E . C . B . S . is m i x e d w i t h I O ~ - i of b u f f e r s o l u t i o n c o n t a i n i n g allergens, either alone (controls) or with the tested compounds in p a r a f f i n circles on a slide. After incubation f o r 20 m i n u t e s at 37°C, s l i d e s a r e dried, fixed and coloured. B a s o p h i l s w e r e c o u n t e d on an e q u a l ~lumber of m i croscopic fields and degranulation i n d e x (D.I.) w a s d e t e r m i n e d . T h e D.I. of c o n t r o l s a l w a y s r e a c h e d m o r e t h a n 50 %. F o r E D T A (10 -2 M a n d 10 -4 M) a n d d i b u t y r y l A M P c (10 -3 M a n d 10 -4 M) D.I. = O : it a p p e a r s t h a t t h e o t h e r d r u g s h a v e n o t or o n l y a l i t t l e e f f e c t w h i c h w i l l be p r e c i s e d w i t h n e w p a t i e n t s .
EFFECTS ON CYCLIC AMP PHOSPHODIESTERASE
43
OF DRUGS AFFECTING MAST CELL DEGRANULATION
S.A. Saeed, N.M. Butt, D. Sheldon and W.A. Taylor Research Department, Miles Laboratories Limited, Stoke Poges, slough SL2 4LY, U.K. Cyclic AMP (cAMP) has been shown to modulate the release of histamine from mast cells and basophils in the allergic response. Sensitized mast cells degranulate and release histamine when challenged with appropriate antigen and it has been claimed that increase and decrease in the cAMP levels in these target cells would suppress and enhance, respectively, the release of histamine. We therefore tested the effects of a number of compounds that affect mast cell degranulation (MCD) on cAMP phosphodiesterase (PDE); the enzyme which controls intracellular levels of cAMP. Drugs were tested on (i) cAMP PDE from rat lung and peritoneal mast cells, and (2) MCD of isolated peritoneal mast cells harvested from rats passively sensitized with mouse anti-DNP reaginic serum and subsequently challenged with DNP26-bovine serum albumin conjugate. These experiments yielded the following conclusions. First, theophylline, isobutylmethylxanthine and several chromones including cromoglycate inhibited PDE and their effectiveness suggested some correlation with their ability to inhibit MCD. Second, indomethacin, which is an effective anti-inflammatory drug was a fairly potent inhibitor of PDE and also inhibited MCD, whereas diclofenac sodium which also inhibited PDE, caused a pronounced potentiation of MCD. It is concluded that the effect of PDE-inhibitors on MCD can be partly explained by decreased cAMP breakdown but it is necessary to take into account additional, presently unknown factors to explain fully the effects of these drugs on MCD.
MEDIATORS
44
A NOVEL C5 CLEAVING ACTIVITY IN NORMAL HAMSTER LUNG LAVAGE FLUID Usha Desai t D. L. Kreutzer and P. A. Ward Department of Pathology~ University of Connecticut Health Center 9 Farmington 9 CT USA
06032.
We have recently shown that intrapulmonary instillation of purified 5th component of human complement into hamster lungs induced acute inflammation (Am. J. Path. 97:61-73~ 1979). This acute alveolitis was also shown to be associated with in vivo fragmentation of C5, as seen on polyacrylamide gel electrophoresis of bronchoalveolar lavage fluid from hamster lungs instilled with C5-I 125 (Fed. Pro. 38:1343, 1979). Hence we undertook in vitro studies to determine whether C5 fragments thus produced were chemotactically active and to attempt characterization of this C5 fragment generating activity in normal hamster lungs. Normal hamster lungs were lavaged by repeated careful endobronchial washings with physiological saline. Vacuum dialysis was used to concentrate cell free supernates of
41
41a
P R O P H Y L A C T I C A N D T H E R A P E U T I C EFFECTS OF N A N D R O L O N E A N D ITS D E C A N O A T E E S T E R ( D E O A - D U R A B O L I N R ) IN M U R I N E L U P U S H. V e r h e u l l f W. S t i m s o n 2 ~ F. den H o l l a n d e r l ~ A . S c h u u r s l 1. B i o c h e m i c a l R e s e a r c h and D e v e l o p m e n t Labs. Organon, Oss ( N e t h e r l a n d s ) 2. D e p a r t m e n t of B i o c h e m i s t r y , S t r a t h c l y d e U n i v e r s i t y , G l a s g o w ( U . K . ) N Z B / W h y b r i d m i c e d e v e l o p an a u t o i m m u n e d i s e a s e , s t r o n g l y r e s e m b l i n g s y s t e m i c lupus e r y t h e m a t o s u s in h u m a n s . R o u b i n i a n et a l . d e m o n s t r a t e d t h a t c a s t r a t i o n of m a l e s , and c h r o n i c a d m i n i s t r a t i o n of o e s t r a d i o l a c c e l e r a t e , w h e r e a s t e s t o s t e r o n e and 5 ~ z - d i h y d r o t e s t o s t e r o n e d e l a y the o n s e t of the d i s e a s e . We d e m o n s t r a t e d that a l s o n a n d r o l o n e i m p l a n t s in s i l a s t i c t u b e s , and n a n d r o l o n e d e c a n o a t e ( D e c a - D u r a b o l i n R ) i n j e c t i o n s e v e r y 14 d a y s , f r o m 3 w e e k s after b i r t h o n w a r d s p r o d u c e a s i g n i f i c a n t r e d u c t i o n in m o r t a l i t y in f e m a l e and p r e p u b e r t a l l y c a s t r a t e d m a l e N Z B / W m i c e . P r o t e i n u r i a and a n t i - D N A IgG w e r e a l s o reduced in t h e s e m i c e . The c o r r e s p o n d i n g t e s t o s t e r o n e t r e a t m e n t s s h o w e d s i m i l a r e f f e c t s . M i c e s u r v i v i n g m o r e t h a n 65 w e e k s w e r e s a c r i f i c e d and s h o w e d s i g n i f i c a n t l y l e s s a n d r o g e n i c e f f e c t s ( a s m e a s u r e d by p r o s t a t e and s e m i n a l v e s i c l e w e i g h t ) after n a n d r o l o n e d e c a n o a t e t h a n after t e s t o s t e r o n e d e c a n o a t e t r e a t m e n t . When n a n d r o l o n e d e c a n o a t e t r e a t m e n t w a s s t a r t e d at the age of 27 - 29 w e e k s a s i m i l a r r e d u c t i o n in m o r t a l i t y w a s o b s e r v e d in p r e p u b e r t a l l y c a s t r a t e d m a l e m i c e . In f e m a l e m i c e the i m p r o v e m e n t was o n l y m a r g i n a l w h i c h m i g h t h a v e been due to the further a d v a n c e d s t a g e of the d i s e a s e at s t a r t of t r e a t m e n t ( f i r s t f e m a l e m i c e d i e d at 28 w e e k s ) . T e s t o s t e r o n e d e c a n o a t e was slightly less effective. The d a t a i n d i c a t e t h a t both n a n d r o l o n e ( - d e c a n o a t e ) and t e s t o s t e r o n e d e c a n o a t e t r e a t m e n t h a v e a b e n e f i c i a l p r o p h y l a c t i c and t h e r a p e u t i c e f f e c t in m u r i n e l u p u s . This e f f e c t d o e s not s e e m to be c o r r e l a t e d w i t h a n d r o g e n i c a c t i v i t y . In v i e w of its w e a k e r a n d r o g e n i c e f f e c t s and longer d u r a t i o n of a c t i o n , n a n d r o l o n e d e c a n o a t e ( D e c a - D u r a b o l i n R ) m i g h t p r o v e a b e t t e r c a n d i d a t e for the t r e a t m e n t of S L E , a d i s e a s e w h i c h is m o s t p r e v a l e n t in f e m a l e s . MODULATION OF MURINE LUPUS WITH SEX HORMONES N. Talal, J. Roubinian, and J. Greenspan Department of Medicine, University of California, and Veterans Administration Medical Center, San Francisco, California, USA. Systemic lupus erythematosus (SLE) is much more common in women than in men. The female: male ratio may be as high as 15:1 if one considers women during the child-bearing years, and declines to 2:1 for post-menopausal females. In NZB/NZW F I mice, an animal model for SLE, the disease occurs earlier and with greater severity in females. Prepubertal castration of male mice results in an accelerated disease so that the castrated males die at the same rate as females. Prepubertal castration of females does not improve survival. The administration of 5-e-dihydrotestosterone to prepubertal female mice suppresses autoantibody formation and immune complex glomerulonephritis. Mice so treated have an increased survival. By contrast, prepubertal mice treated with 17-B-estradiol have a decreased survival, and greater autoantibody formation and immune complex glomerulonephritis. If androgen administration is delayed until 3 or 6 months of age, B/W female mice again have an increased survival and less immune complex glomerulonephritis, but without a decline in antibodies to DNA. These results suggest that there may be several mechanisms by which androgens act to favorably influence the course of autoimmune disease in B/W mice.
MEDIATORS/DEGRANULATION
42
PHARMACOLOGICAL MODULATION OF HUMAN BASOPHIL DEGRANULATION H. L u c e , E. A l b e n q r e s , J.P. Tillement D4partement de Pharmacologie, Facult~ de M4decine de PARIS G4n~ral Sarrail, 94010 CRETEIL, France
XII,
8 rue
du
Anaphylactic reaction and its modulation by some pharmacological substances have been widely studied in a n i m a l s . However, results obtained were not directly extrapolated t o m a n , so w e d e v e l o p e d a technique (LEYNADIER F., L U C E H., D R Y J., 1977) t o c h e c k h u m a n b a s o p h i l degranulation and the effect
231
of v a r i o u s c o m p o u n d s . Subjects are selected regarding their reaginic hypersensitivity by intradermal skin testing. The most commonly implicated allergens were grass pollen and dermatophagoides pteronisinus. Corresponding allergens extracts (Institut Pasteur) a r e d i l u t e d in H e p ~ s b u f f e r t o t h e concentration of iOO/~g/ml. They are used, associated with solutions (in t h e s a m e b u f f e r ) of v a r i o u s c o m p o u n d s , EDTA, d i b u t y r y l A M P c , d i b u t y r y l GMPc, isoproterenol, disodium cromoglycate, colchicine and theophylline, or alone as c o n t r o l s . T h e t e c h n i q u e d e s c r i b e d involves the preparation of an e n r i c h i e d basophil cells suspension (E.C.B.S.) u s i n g an o r i g i n a l c o n c e n t r a t i o n gradient. T h e n 2 0 2 ~ i of E . C . B . S . is m i x e d w i t h I O ~ - i of b u f f e r s o l u t i o n c o n t a i n i n g allergens, either alone (controls) or with the tested compounds in p a r a f f i n circles on a slide. After incubation f o r 20 m i n u t e s at 37°C, s l i d e s a r e dried, fixed and coloured. B a s o p h i l s w e r e c o u n t e d on an e q u a l ~lumber of m i croscopic fields and degranulation i n d e x (D.I.) w a s d e t e r m i n e d . T h e D.I. of c o n t r o l s a l w a y s r e a c h e d m o r e t h a n 50 %. F o r E D T A (10 -2 M a n d 10 -4 M) a n d d i b u t y r y l A M P c (10 -3 M a n d 10 -4 M) D.I. = O : it a p p e a r s t h a t t h e o t h e r d r u g s h a v e n o t or o n l y a l i t t l e e f f e c t w h i c h w i l l be p r e c i s e d w i t h n e w p a t i e n t s .
EFFECTS ON CYCLIC AMP PHOSPHODIESTERASE
43
OF DRUGS AFFECTING MAST CELL DEGRANULATION
S.A. Saeed, N.M. Butt, D. Sheldon and W.A. Taylor Research Department, Miles Laboratories Limited, Stoke Poges, slough SL2 4LY, U.K. Cyclic AMP (cAMP) has been shown to modulate the release of histamine from mast cells and basophils in the allergic response. Sensitized mast cells degranulate and release histamine when challenged with appropriate antigen and it has been claimed that increase and decrease in the cAMP levels in these target cells would suppress and enhance, respectively, the release of histamine. We therefore tested the effects of a number of compounds that affect mast cell degranulation (MCD) on cAMP phosphodiesterase (PDE); the enzyme which controls intracellular levels of cAMP. Drugs were tested on (i) cAMP PDE from rat lung and peritoneal mast cells, and (2) MCD of isolated peritoneal mast cells harvested from rats passively sensitized with mouse anti-DNP reaginic serum and subsequently challenged with DNP26-bovine serum albumin conjugate. These experiments yielded the following conclusions. First, theophylline, isobutylmethylxanthine and several chromones including cromoglycate inhibited PDE and their effectiveness suggested some correlation with their ability to inhibit MCD. Second, indomethacin, which is an effective anti-inflammatory drug was a fairly potent inhibitor of PDE and also inhibited MCD, whereas diclofenac sodium which also inhibited PDE, caused a pronounced potentiation of MCD. It is concluded that the effect of PDE-inhibitors on MCD can be partly explained by decreased cAMP breakdown but it is necessary to take into account additional, presently unknown factors to explain fully the effects of these drugs on MCD.
MEDIATORS
44
A NOVEL C5 CLEAVING ACTIVITY IN NORMAL HAMSTER LUNG LAVAGE FLUID Usha Desai t D. L. Kreutzer and P. A. Ward Department of Pathology~ University of Connecticut Health Center 9 Farmington 9 CT USA
06032.
We have recently shown that intrapulmonary instillation of purified 5th component of human complement into hamster lungs induced acute inflammation (Am. J. Path. 97:61-73~ 1979). This acute alveolitis was also shown to be associated with in vivo fragmentation of C5, as seen on polyacrylamide gel electrophoresis of bronchoalveolar lavage fluid from hamster lungs instilled with C5-I 125 (Fed. Pro. 38:1343, 1979). Hence we undertook in vitro studies to determine whether C5 fragments thus produced were chemotactically active and to attempt characterization of this C5 fragment generating activity in normal hamster lungs. Normal hamster lungs were lavaged by repeated careful endobronchial washings with physiological saline. Vacuum dialysis was used to concentrate cell free supernates of