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Lipopolysaccharide (LPS) activation of human monocyte cytotoxicity: Augmentation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and formyl peptide (FMLP) and inhibition by cyclic AMP
Abst facts
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66 L I P O P O L Y S A C C H A R I D E (LPS) A C T I V A T I O N OF H U M A N M O N O C Y T E CYTOTOXICITY: P A R A D O X I C A L EFFECTS OF P R O T E I N K I N A S E C S T I M U L A N T S AND INHIBITORS. R o n a l d G. Coffew, L a u r a L. Weakland~ and V i c k i A. Alberts. D e p a r t m e n t of P h a r m a c o l o g y & Therapeutics, U n i v e r s i t y of South Florida C o l l e g e of Medicine, Tampa, FL 33612 H u m a n b l o o d m o n o c y t e s w e r e a c t i v a t e d by LPS (i0 ng/ml) for c y t o t o x i c i t y of W E H I - 1 6 4 mouse f i b r o s a r c o m a cells, d e t e r m i n e d by r e l e a s e of 51Cr from W E H I - 1 6 4 tumor cells incubated w i t h m o n o c y t e supernatants. A d d i t i o n of the tumor p r o m o t e r p h o r b o l - 1 2 - m y r i s t a t e - 1 3 - a c e t a t e (PMA) at c o n c e n t r a t i o n s or 3XIO "l° M to 3XlO 9 M r e s u l t e d in 2-4 fold a u g m e n t a t i o n of L P S - e v o k e d cytotoxicity. By contrast, higher c o n c e n t r a t i o n s of PMA c a u s e d inhibition. Phorbol d i b u t y r a t e had similar effects. Both the s t i m u l a t o r y and the i n h i b i t o r y e f f e c t s of PMA were e n h a n c e d by longer times of i n c u b a t i o n (i h) b e f o r e a d d i t i o n of LPS. The PKC i n h i b i t o r s H-7 (30 gM) and s t a u r o s p o r i n e (I0 DAM) did not p r e v e n t the s t i m u l a t o r y or the i n h i b i t o r y effects of PMA. In fact, in some e x p e r i m e n t s they s t i m u l a t e d cytotoxicity. These results suggest that if p r o t e i n kinase C (PKC) m e d i a t e s the effect of PMA it is an u n u s u a l isozyme. O k a d a i c acid (20 ng/ml), an inhibitor of p h o s p h a t a s e s I and IIa, a u g m e n t e d the e f f e c t s of LPS and of PMA. A c t i v a t i o n by LPS may involve a t y r o s i n e kinase since it was i n h i b i t e d by the t y r o s i n e k i n a s e inhibitor g e n i s t e i n (30300 gM). Curiously, lower c o n c e n t r a t i o n s w e r e stimulatory. L e u p e p t i n (50 gM), an inhibitor of Ca2+-dependent p r o t e a s e s w h i c h can d e g r a d e PKC, a u g m e n t e d cytotoxicity. Pertussis toxin (0.I 0.5 Gg/ml) also a u g m e n t e d LPS effects. The results, taken together, strongly suggest that LPS s t i m u l a t i o n of TNF r e l e a s e evokes a s i g n a l i n g system that involves kinases with some c h a r a c t e r i s t i c s of t y r o s i n e kinases and some c h a r a c t e r i s t i c s of PKC. In addition, an i n h i b i t o r y role of a P e r t u s s i s t o x i n - s e n s i t i v e G p r o t e i n is suggested. S u p p o r t e d by The A m e r i c a n C a n c e r Society grant #CH-444.
67 L I P O P O L Y S A C C H A R I D E (LPS) A C T I V A T I O N OF H U M A N M O N O C Y T E CYTOTOXICITY: A U G M E N T A T I O N BY G R A N U L O C Y T E M A C R O P H A G E C O L O N Y - S T I M U L A T I N G F A C T O R (GM-CSP) AND FORMYL P E P T I D E (FMLP) AND I N H I B I T I O N BY C Y C L I C AMP. V i c k i A. Albertsf Laura L. W e a k l a n d r and R o n a l d G. Coffey. D e p a r t m e n t of P h a r m a c o l o g y & Therapeutics, U n i v e r s i t y of South F l o r i d a C o l l e g e of Medicine, Tampa, FL 33612 Human b l o o d m o n o c y t e s were a c t i v a t e d by LPS (i0 ng/ml) for c y t o t o x i c i t y of W E H I - 1 6 4 m o u s e f i b r o s a r c o m a cells, d e t e r m i n e d by r e l e a s e of 51Cr d u r i n g a 6 h i n c u b a t i o n w i t h m o n o c y t e supernatants. K i l l i n g was m e d i a t e d by t u m o r n e c r o s i s factor (TNF) since it was c o m p l e t e l y p r e v e n t e d by 100-300 units of a n t i - h u m a n r e c o m b i n a n t TNF-a a n t i b o d y (Genentech). Monocyte a c t i v a t i o n by LPS was a u g m e n t e d by FMLP (3XI09-10"SM). FMLP s t i m u l a t e d inositol p h o s p h a t e production, i n d i c a t i n g the p a r t i c i p a t i o n of p h o s p h o l i p a s e C. P r e - t r e a t m e n t of m o n o c y t e s with d e x a m e t h a s o n e (10 .7 M, 24 h) i n h i b i t e d L P S - e v o k e d cytotoxicity, p r o v i d i n g further support for a role of a p h o s p h o l i p a s e . I n d o m e t h a c i n (i pM) was stimulatory, suggesting a n e g a t i v e role for cyclooxygenase. A d e n y l a t e c y c l a s e s t i m u l a n t s (histamine, 100 ~M, 5 ' - N - e t h y l c a r b o x a m i d o - a d e n o s i n e , i00 ~M, p r o s t a g l a n d i n E2, 0.I ~M, Rolipram, 50 pM) and l i p o p h i l i c analogues of cyclic AMP (monobutyryl, dibutyryl, and 8 - B r - c y c l i c AMP, but not 8 - B r - c y c l i c GMP) w e r e also inhibitory. R e c o m b i n a n t human G M - C S F (Immunex) a u g m e n t e d LPS a c t i v a t i o n of WEHI cell c y t o t o x i c i t y in m o n o c y t e s from over half of all blood donors. This a u g m e n t a t i o n ranged up to 3 fold (1.64 ± .ii, n = 21) and o c c u r r e d m o r e often w i t h cells that r e s p o n d e d p o o r l y to LPS. The effect of GM-CSF was m a x i m a l at i00 U/ml, and was p r e v e n t e d by a n t i - T N F antibody. The cyclic AMP analogues, a d e n y l a t e c y c l a s e stimulants, and d e x a m e t h a s o n e b l o c k e d the a u g m e n t i n g effects of GM-CSF. These results suggest that the a u g m e n t i n g effects of G M - C S F may be due in part to s t i m u l a t i o n of a r a c h i d o n a t e release. (Supported by ACS grant # CH-444.)
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THE E L U T I O N P R O F I L E OF THE A549 BETA ADRENERGIC (flAR) REGULATING ACTIVITY OF LYMPHOCYTE CONDITIONED MEDIUM (LCM) OF IM9 CELLS DEVELOPED BY DEAE ION EXCHANGE HPLC A. Szentivanvi. M.D.. D.Sc,, M. Schultze, M.D., O. Heim, M.D., S. Reiner, M.D., S. Robicsek, Ph.D., J. Zority, M.D., ICJ. Hurt, M.S., J.F. Hackney, Ph.D., E.G. Calderon, M.D., J.J. Dwornik, Ph.D. and R,F. Locke),, M.D., Depts. of Pharmacology and Internal Medicine, Univ. of South Florida College of Medicine, Tampa, Florida Stern and Kunos (J. Biol. Chem. 1988) by co-culturing A549 human lung cells with IM9 human lymphoid ceils showed a 3- to 5-fold increase in the density of BAR in A549 cells. We confirmed these findings (Szentivanyi et al., Cytokine 1989) except that a) HPLC yielded 4 distinct fractions (activity peaks) instead of the reported 3; b) Peaks II and III showed BAR upregulating activity; and c) Peak I yielded a significant BAR downregulating effect and Peak IV was inactive. The elution profile of the BAR regulating activity of LCM of IM9 cells by DEAE ion exchange HPLC was developed by incubating the LCM with A549 cells as adrenergic effector cells for 24 hrs. The activity peaks were distinguished by: a) retention time on ion exchange column; b) ability to significantly increase or decrease BAR density relative to that of untreated A549 cells; and c) immunoneutralization of the A549 BAR upregulating or downregulating effect of LCM peaks by antibodies to lymphokines. BAR densities were measured by [3H]dihydroalprenoloL Activity Peaks II and III showed a 311 and 196% increase, whereas Peak I showed a 59% decrease in BAR concentrations of untreated A549 cells.
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66 L I P O P O L Y S A C C H A R I D E (LPS) A C T I V A T I O N OF H U M A N M O N O C Y T E CYTOTOXICITY: P A R A D O X I C A L EFFECTS OF P R O T E I N K I N A S E C S T I M U L A N T S AND INHIBITORS. R o n a l d G. Coffew, L a u r a L. Weakland~ and V i c k i A. Alberts. D e p a r t m e n t of P h a r m a c o l o g y & Therapeutics, U n i v e r s i t y of South Florida C o l l e g e of Medicine, Tampa, FL 33612 H u m a n b l o o d m o n o c y t e s w e r e a c t i v a t e d by LPS (i0 ng/ml) for c y t o t o x i c i t y of W E H I - 1 6 4 mouse f i b r o s a r c o m a cells, d e t e r m i n e d by r e l e a s e of 51Cr from W E H I - 1 6 4 tumor cells incubated w i t h m o n o c y t e supernatants. A d d i t i o n of the tumor p r o m o t e r p h o r b o l - 1 2 - m y r i s t a t e - 1 3 - a c e t a t e (PMA) at c o n c e n t r a t i o n s or 3XIO "l° M to 3XlO 9 M r e s u l t e d in 2-4 fold a u g m e n t a t i o n of L P S - e v o k e d cytotoxicity. By contrast, higher c o n c e n t r a t i o n s of PMA c a u s e d inhibition. Phorbol d i b u t y r a t e had similar effects. Both the s t i m u l a t o r y and the i n h i b i t o r y e f f e c t s of PMA were e n h a n c e d by longer times of i n c u b a t i o n (i h) b e f o r e a d d i t i o n of LPS. The PKC i n h i b i t o r s H-7 (30 gM) and s t a u r o s p o r i n e (I0 DAM) did not p r e v e n t the s t i m u l a t o r y or the i n h i b i t o r y effects of PMA. In fact, in some e x p e r i m e n t s they s t i m u l a t e d cytotoxicity. These results suggest that if p r o t e i n kinase C (PKC) m e d i a t e s the effect of PMA it is an u n u s u a l isozyme. O k a d a i c acid (20 ng/ml), an inhibitor of p h o s p h a t a s e s I and IIa, a u g m e n t e d the e f f e c t s of LPS and of PMA. A c t i v a t i o n by LPS may involve a t y r o s i n e kinase since it was i n h i b i t e d by the t y r o s i n e k i n a s e inhibitor g e n i s t e i n (30300 gM). Curiously, lower c o n c e n t r a t i o n s w e r e stimulatory. L e u p e p t i n (50 gM), an inhibitor of Ca2+-dependent p r o t e a s e s w h i c h can d e g r a d e PKC, a u g m e n t e d cytotoxicity. Pertussis toxin (0.I 0.5 Gg/ml) also a u g m e n t e d LPS effects. The results, taken together, strongly suggest that LPS s t i m u l a t i o n of TNF r e l e a s e evokes a s i g n a l i n g system that involves kinases with some c h a r a c t e r i s t i c s of t y r o s i n e kinases and some c h a r a c t e r i s t i c s of PKC. In addition, an i n h i b i t o r y role of a P e r t u s s i s t o x i n - s e n s i t i v e G p r o t e i n is suggested. S u p p o r t e d by The A m e r i c a n C a n c e r Society grant #CH-444.
67 L I P O P O L Y S A C C H A R I D E (LPS) A C T I V A T I O N OF H U M A N M O N O C Y T E CYTOTOXICITY: A U G M E N T A T I O N BY G R A N U L O C Y T E M A C R O P H A G E C O L O N Y - S T I M U L A T I N G F A C T O R (GM-CSP) AND FORMYL P E P T I D E (FMLP) AND I N H I B I T I O N BY C Y C L I C AMP. V i c k i A. Albertsf Laura L. W e a k l a n d r and R o n a l d G. Coffey. D e p a r t m e n t of P h a r m a c o l o g y & Therapeutics, U n i v e r s i t y of South F l o r i d a C o l l e g e of Medicine, Tampa, FL 33612 Human b l o o d m o n o c y t e s were a c t i v a t e d by LPS (i0 ng/ml) for c y t o t o x i c i t y of W E H I - 1 6 4 m o u s e f i b r o s a r c o m a cells, d e t e r m i n e d by r e l e a s e of 51Cr d u r i n g a 6 h i n c u b a t i o n w i t h m o n o c y t e supernatants. K i l l i n g was m e d i a t e d by t u m o r n e c r o s i s factor (TNF) since it was c o m p l e t e l y p r e v e n t e d by 100-300 units of a n t i - h u m a n r e c o m b i n a n t TNF-a a n t i b o d y (Genentech). Monocyte a c t i v a t i o n by LPS was a u g m e n t e d by FMLP (3XI09-10"SM). FMLP s t i m u l a t e d inositol p h o s p h a t e production, i n d i c a t i n g the p a r t i c i p a t i o n of p h o s p h o l i p a s e C. P r e - t r e a t m e n t of m o n o c y t e s with d e x a m e t h a s o n e (10 .7 M, 24 h) i n h i b i t e d L P S - e v o k e d cytotoxicity, p r o v i d i n g further support for a role of a p h o s p h o l i p a s e . I n d o m e t h a c i n (i pM) was stimulatory, suggesting a n e g a t i v e role for cyclooxygenase. A d e n y l a t e c y c l a s e s t i m u l a n t s (histamine, 100 ~M, 5 ' - N - e t h y l c a r b o x a m i d o - a d e n o s i n e , i00 ~M, p r o s t a g l a n d i n E2, 0.I ~M, Rolipram, 50 pM) and l i p o p h i l i c analogues of cyclic AMP (monobutyryl, dibutyryl, and 8 - B r - c y c l i c AMP, but not 8 - B r - c y c l i c GMP) w e r e also inhibitory. R e c o m b i n a n t human G M - C S F (Immunex) a u g m e n t e d LPS a c t i v a t i o n of WEHI cell c y t o t o x i c i t y in m o n o c y t e s from over half of all blood donors. This a u g m e n t a t i o n ranged up to 3 fold (1.64 ± .ii, n = 21) and o c c u r r e d m o r e often w i t h cells that r e s p o n d e d p o o r l y to LPS. The effect of GM-CSF was m a x i m a l at i00 U/ml, and was p r e v e n t e d by a n t i - T N F antibody. The cyclic AMP analogues, a d e n y l a t e c y c l a s e stimulants, and d e x a m e t h a s o n e b l o c k e d the a u g m e n t i n g effects of GM-CSF. These results suggest that the a u g m e n t i n g effects of G M - C S F may be due in part to s t i m u l a t i o n of a r a c h i d o n a t e release. (Supported by ACS grant # CH-444.)
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THE E L U T I O N P R O F I L E OF THE A549 BETA ADRENERGIC (flAR) REGULATING ACTIVITY OF LYMPHOCYTE CONDITIONED MEDIUM (LCM) OF IM9 CELLS DEVELOPED BY DEAE ION EXCHANGE HPLC A. Szentivanvi. M.D.. D.Sc,, M. Schultze, M.D., O. Heim, M.D., S. Reiner, M.D., S. Robicsek, Ph.D., J. Zority, M.D., ICJ. Hurt, M.S., J.F. Hackney, Ph.D., E.G. Calderon, M.D., J.J. Dwornik, Ph.D. and R,F. Locke),, M.D., Depts. of Pharmacology and Internal Medicine, Univ. of South Florida College of Medicine, Tampa, Florida Stern and Kunos (J. Biol. Chem. 1988) by co-culturing A549 human lung cells with IM9 human lymphoid ceils showed a 3- to 5-fold increase in the density of BAR in A549 cells. We confirmed these findings (Szentivanyi et al., Cytokine 1989) except that a) HPLC yielded 4 distinct fractions (activity peaks) instead of the reported 3; b) Peaks II and III showed BAR upregulating activity; and c) Peak I yielded a significant BAR downregulating effect and Peak IV was inactive. The elution profile of the BAR regulating activity of LCM of IM9 cells by DEAE ion exchange HPLC was developed by incubating the LCM with A549 cells as adrenergic effector cells for 24 hrs. The activity peaks were distinguished by: a) retention time on ion exchange column; b) ability to significantly increase or decrease BAR density relative to that of untreated A549 cells; and c) immunoneutralization of the A549 BAR upregulating or downregulating effect of LCM peaks by antibodies to lymphokines. BAR densities were measured by [3H]dihydroalprenoloL Activity Peaks II and III showed a 311 and 196% increase, whereas Peak I showed a 59% decrease in BAR concentrations of untreated A549 cells.