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The effect of blood storage on natural killer cell cytotoxicity and mitogen-induced proliferation
Abstracts
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78. PROSTAGLANDIN E2 AND THROM~3XANE A2 SECRETIOW FROM CULTUREO HUMANMONOCYTE$AND TNE MACmOPHAGESUBTYPES RM 3/1 AND 27E10. O. zl~dto-I(tarwsssor, D. Hitgere, S. Bent arm W. Schmutzter: I n s t i t u t e of Phammacotogy0 I~TH Aachen, F.R.G. #4acrophages (#40) have beGm shoran to be a major source of cyctooxygenase products. I t has also become clear that S~--~'~'~-~'r~r'-r-'LettonSof MO exist ~h|ch d t f f e r fn t h e t r a b i l i t y to generate prostagtandins. We therefore inwesttgated the proetglsnd|n E2 (PGE2) amndthromboxane A2 (TXA2) eecret|en of the fottoulng #40 subpoputatLono in romper|con to unoeperated c e l l s treated wtth Lipopotysecchamr|de (LPS), the gtucocort|co|d prednyL|dane (pred) or |ndomethacine (|ado): 27E10, an activated NO subtype found in acut Lnftamwtiorm end RM 3/1, • gtucocortico|d inducible type of I 0 associated w|th the do~n-reguLetory phase of the imlul!e response. Unseparated blood monocytes, 3/1 +, R#4 3/1", 27E10+ and 27£10" #40 subsete w~re cuttered for 1 or 2 days w|th the agents. Subpopuletiens were separeted from cultured NO with the mutational antibodies 27E10 and RM 3 / I I=xxrd to magnetobseda. The purity of either |x)s|tive subset was >98"A. TXB2 the stable product of I"XA2 and PGE2 were quantified in the culture amupernatent by redioimmunoessey. Un6epareted NO secreted three tLmee more TXA2 and 10 times sore PGE2 a f t e r st|mutet|en ~ith LPS. Pred and indo inhibited the TXA2 and PGE2 secretion in am dose dependent manner. NO etimotamted with LPS for 1 day generated POE2 for st least two days k~n|le TXA2 production was onLy Limited to amfew hours. R!q 3/1 + NO generated no PC~2 even when atimJLated with LPS. In the RM 3/1" poputatior~ LPS caused enty a weak increase ÷ . . of PGE2. RM 3/1 NO secreted even tess TXA~ then the RM 3/1 celLs. In both popuLar|one LPS s l i g h t l y increased the TXA~ secretion. 27E10+ NO secreted Large enounts of PGE2 e~d eteo elevated LeveLs of TXA2 compared to the 27E10", RN 3 / I + and RM 3/1" ~ J l e t i o n s . LPS d|d not enhance this secretion ~hile Pred reduced i t . These results indicete that PGE2 |s the major secretory product of LPS stimulated NO. The fact that LPS iam able to |nduce the 27EI0 + NO end to inhibit the appearance auf RM 3/1 + NO correlates with these findings. These data cLeeriy demor~;trate that various #4o subpop(JLetions d i f f e r in t h e | r potency to generate cyclooxygenaameproducts.
79.
REGULATION OF DELAYED-TYPE HYPERSENSITIVITY BY SPLEEN DENDRITIC CELLS Yoshihiro MORIKAWA, Masahiko FUROTANI, Yoshizo YANASE and Kenichi KAKUDO Department of Pathology, Wakayama Medical School, Wakayama City, Wakayama, 640 Japan We i n v e s t i g a t e d immunoregulatory effects o f mouse s p l e e n d e n d r i t i c cells (DC) on d e l a y e d type hypersensitivity (DTH) a n d a n t i b o d y r e s p o n s e . When k e y h o l e l i m p e t h e m o c y a n i n (KLH) - p u l s e d IX: w e r e t r a n s f e r r e d intravenously i n t , , n a i v e s y n g e n e i c m i c e , DTH was weak i n s p i t e of the elevation of anti-KLH antibody titer. Subcutaneous transfer o f K L H - p u l s e d DC i n d u c e d DTH, b u t a n t i - K L H a n t i b o d y t i t e r did not elevate. Furthermore, Intravenous transfer o f K L H - p u l s e d DC i n t o t h e s y n g e n e i c m i c e w h i c h w e r e i m m u n i z e d w i t h KLH-CFA in induction phase induced the suppression o f DTH a n d t h e a u g m e n t a t i o n of anti-KLH antibody production. When S ~ C r - l a b e l e d DC w e r e i n j e c t e d intravenously or subcutaneously into s y n g e n e i c m i c e , S~Cr l a b e l was d e t e c t e d a l m o s t i n t h e lymph n o d e in m i c e i n j e c t e d subcutaneously a n d in t h e s p l e e n i n i n j e c t e d intravenously. In a d d i t i o n , when g L H - p u ] s e d DC w e r e t r a n s f e r r e d intravenously into the splenectomized m i c e in i n d u c t i o n p h a s e , DTH c o u l d n o t be s u p p e r s s e d a n d a n t i b o d y p r o d u c t i o n could not be induce. Peritoneal exudate macrophages did not have these immunoregulatory effects. These findings suggested that the effects o f DC on DTH a n d a n t i b o d y p r o d u c t i o n may b e a t t r i b u t e d to t h e d i f f e r e n c e of the lymphoid tissue to w h i c h DC m i g r a t e , b u t n o t to t h e s p e c i f i c f u n c t i o n o f DC.
80. THE EFFECT OF BLOOD STORAGE ON NATURAL KILLER CELL CYTOTOXICITY AND MITOGEN-INDUCED PROLIFERATION. D.I. Cowden, D.G. Gilbert, R.A. Jensen, C.J. Meliska, M.E. Stunkard, J.M. Martinko, Southern Illinois University, Carbondale, IL 62901. In this study, an assessment was made of the effect of 24 hour blood storage on two parameters of lymphocyte function: natural killer cytotoxic activity (NKCA) and mitogen-induced proliferation. One hundred twenty whole blood samples were drawn from 20 subjects involved in a study of the effect of smoking cessation on immune function. Two aliquots of acid-citrate-dextrose anticoagulated blood were obtained from each subject. One specimen was separated and tested immediately for NKCA against the target K562 cell line by a 5lCr release assay and for mitogenic response to concanavalin A (ConA) and phythemaglutinin (PHA) by a 3H-thymidine uptake assay. A second, unseparated, specimen was stored at room temperature for 24 hours before separation and assay. Following storage, NKCA was reduced 34-46% at effector:target ratios of 40:1, 20:1, 10:1 and 5:1 (all p's, 0.001) as compared to NKCA assayed immediately. However, correlations between freshly assayed and stored blood NKCA values were high at all effector: target ratios (all r's > 0.8, p < 0.001). ConA proliferative response was depressed 24% following storage (p < .03). PHA response was not significantly depressed following storage (p > 0.10). While ConA and PHA responses were moderately correlated for fresh samples (r=0.493, p < 0.03), and highly correlated for stored samples (r=0.767, p < 0.001), lymphocyte proliferation in response to mitogens was not correlated with measures of NKCA at any effector:target ratios. Thus, storing blood at room temperature for 24 hours significantly decreases NKCA activity and lymphocyte proliferation in response to ConA, but not PHA. Furthermore, NKCA activity appears to vary independently of alLeges-induced lymphocyte proliferation.
743
78. PROSTAGLANDIN E2 AND THROM~3XANE A2 SECRETIOW FROM CULTUREO HUMANMONOCYTE$AND TNE MACmOPHAGESUBTYPES RM 3/1 AND 27E10. O. zl~dto-I(tarwsssor, D. Hitgere, S. Bent arm W. Schmutzter: I n s t i t u t e of Phammacotogy0 I~TH Aachen, F.R.G. #4acrophages (#40) have beGm shoran to be a major source of cyctooxygenase products. I t has also become clear that S~--~'~'~-~'r~r'-r-'LettonSof MO exist ~h|ch d t f f e r fn t h e t r a b i l i t y to generate prostagtandins. We therefore inwesttgated the proetglsnd|n E2 (PGE2) amndthromboxane A2 (TXA2) eecret|en of the fottoulng #40 subpoputatLono in romper|con to unoeperated c e l l s treated wtth Lipopotysecchamr|de (LPS), the gtucocort|co|d prednyL|dane (pred) or |ndomethacine (|ado): 27E10, an activated NO subtype found in acut Lnftamwtiorm end RM 3/1, • gtucocortico|d inducible type of I 0 associated w|th the do~n-reguLetory phase of the imlul!e response. Unseparated blood monocytes, 3/1 +, R#4 3/1", 27E10+ and 27£10" #40 subsete w~re cuttered for 1 or 2 days w|th the agents. Subpopuletiens were separeted from cultured NO with the mutational antibodies 27E10 and RM 3 / I I=xxrd to magnetobseda. The purity of either |x)s|tive subset was >98"A. TXB2 the stable product of I"XA2 and PGE2 were quantified in the culture amupernatent by redioimmunoessey. Un6epareted NO secreted three tLmee more TXA2 and 10 times sore PGE2 a f t e r st|mutet|en ~ith LPS. Pred and indo inhibited the TXA2 and PGE2 secretion in am dose dependent manner. NO etimotamted with LPS for 1 day generated POE2 for st least two days k~n|le TXA2 production was onLy Limited to amfew hours. R!q 3/1 + NO generated no PC~2 even when atimJLated with LPS. In the RM 3/1" poputatior~ LPS caused enty a weak increase ÷ . . of PGE2. RM 3/1 NO secreted even tess TXA~ then the RM 3/1 celLs. In both popuLar|one LPS s l i g h t l y increased the TXA~ secretion. 27E10+ NO secreted Large enounts of PGE2 e~d eteo elevated LeveLs of TXA2 compared to the 27E10", RN 3 / I + and RM 3/1" ~ J l e t i o n s . LPS d|d not enhance this secretion ~hile Pred reduced i t . These results indicete that PGE2 |s the major secretory product of LPS stimulated NO. The fact that LPS iam able to |nduce the 27EI0 + NO end to inhibit the appearance auf RM 3/1 + NO correlates with these findings. These data cLeeriy demor~;trate that various #4o subpop(JLetions d i f f e r in t h e | r potency to generate cyclooxygenaameproducts.
79.
REGULATION OF DELAYED-TYPE HYPERSENSITIVITY BY SPLEEN DENDRITIC CELLS Yoshihiro MORIKAWA, Masahiko FUROTANI, Yoshizo YANASE and Kenichi KAKUDO Department of Pathology, Wakayama Medical School, Wakayama City, Wakayama, 640 Japan We i n v e s t i g a t e d immunoregulatory effects o f mouse s p l e e n d e n d r i t i c cells (DC) on d e l a y e d type hypersensitivity (DTH) a n d a n t i b o d y r e s p o n s e . When k e y h o l e l i m p e t h e m o c y a n i n (KLH) - p u l s e d IX: w e r e t r a n s f e r r e d intravenously i n t , , n a i v e s y n g e n e i c m i c e , DTH was weak i n s p i t e of the elevation of anti-KLH antibody titer. Subcutaneous transfer o f K L H - p u l s e d DC i n d u c e d DTH, b u t a n t i - K L H a n t i b o d y t i t e r did not elevate. Furthermore, Intravenous transfer o f K L H - p u l s e d DC i n t o t h e s y n g e n e i c m i c e w h i c h w e r e i m m u n i z e d w i t h KLH-CFA in induction phase induced the suppression o f DTH a n d t h e a u g m e n t a t i o n of anti-KLH antibody production. When S ~ C r - l a b e l e d DC w e r e i n j e c t e d intravenously or subcutaneously into s y n g e n e i c m i c e , S~Cr l a b e l was d e t e c t e d a l m o s t i n t h e lymph n o d e in m i c e i n j e c t e d subcutaneously a n d in t h e s p l e e n i n i n j e c t e d intravenously. In a d d i t i o n , when g L H - p u ] s e d DC w e r e t r a n s f e r r e d intravenously into the splenectomized m i c e in i n d u c t i o n p h a s e , DTH c o u l d n o t be s u p p e r s s e d a n d a n t i b o d y p r o d u c t i o n could not be induce. Peritoneal exudate macrophages did not have these immunoregulatory effects. These findings suggested that the effects o f DC on DTH a n d a n t i b o d y p r o d u c t i o n may b e a t t r i b u t e d to t h e d i f f e r e n c e of the lymphoid tissue to w h i c h DC m i g r a t e , b u t n o t to t h e s p e c i f i c f u n c t i o n o f DC.
80. THE EFFECT OF BLOOD STORAGE ON NATURAL KILLER CELL CYTOTOXICITY AND MITOGEN-INDUCED PROLIFERATION. D.I. Cowden, D.G. Gilbert, R.A. Jensen, C.J. Meliska, M.E. Stunkard, J.M. Martinko, Southern Illinois University, Carbondale, IL 62901. In this study, an assessment was made of the effect of 24 hour blood storage on two parameters of lymphocyte function: natural killer cytotoxic activity (NKCA) and mitogen-induced proliferation. One hundred twenty whole blood samples were drawn from 20 subjects involved in a study of the effect of smoking cessation on immune function. Two aliquots of acid-citrate-dextrose anticoagulated blood were obtained from each subject. One specimen was separated and tested immediately for NKCA against the target K562 cell line by a 5lCr release assay and for mitogenic response to concanavalin A (ConA) and phythemaglutinin (PHA) by a 3H-thymidine uptake assay. A second, unseparated, specimen was stored at room temperature for 24 hours before separation and assay. Following storage, NKCA was reduced 34-46% at effector:target ratios of 40:1, 20:1, 10:1 and 5:1 (all p's, 0.001) as compared to NKCA assayed immediately. However, correlations between freshly assayed and stored blood NKCA values were high at all effector: target ratios (all r's > 0.8, p < 0.001). ConA proliferative response was depressed 24% following storage (p < .03). PHA response was not significantly depressed following storage (p > 0.10). While ConA and PHA responses were moderately correlated for fresh samples (r=0.493, p < 0.03), and highly correlated for stored samples (r=0.767, p < 0.001), lymphocyte proliferation in response to mitogens was not correlated with measures of NKCA at any effector:target ratios. Thus, storing blood at room temperature for 24 hours significantly decreases NKCA activity and lymphocyte proliferation in response to ConA, but not PHA. Furthermore, NKCA activity appears to vary independently of alLeges-induced lymphocyte proliferation.