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Platelet augmentation of basophil histamine release
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VIRUS IMPAIRMENTOF TIs CALMODULINANTAGONIST TRIFLUOPERAZINE (TFP) MODULATIONOF LEUKOCYTE HISTAMINE RELEASE (HR) W. Busse, M.D. and C. A. Swenson, B.S., Madison, Wisconsin During viral respiratory infections, many asthmatics have increased wheezing which may relate to increased mediator release. Previous work found enhanced basophilic-leukocyte HR with antigen and calcium ionophore following an in v i t r o virus incubation. Thoughassociated with interferon production, the mechanism of virus enhanced HR is unknown. Calmodulin (CM) is an i n t r a c e l l u l a r intermediary for calcium ions and is also linked to cyclic AMP metabolism through phosphodiesterase activation. Virus transformed cells may have increased CM (Biochem Biophys Res Commun 86:1169, 1979) which could affect calcium ion movement, reduce cAMP, and thus contribute to enhanced HR. CM a c t i v i t y is antagonized by TFP. This CM antagonist i n h i b i t s leukocyte.HR with maximal effects at IO-~M (ID~n=4.6xlO'bM). This TFP response provides a functional assay to measure virus incubation effects on basophilic CM a c t i v i t y in isolated leukocyte mixtures. Isolated leukocytes from 5 ragweed-sensitive patients were incubated (2 hr, 37~ with l i v e influenza A ( i n f e c t i v i t y of 1EIDsn:1 leukocyte) or medium control. Treated Cells were centrifuged, resuspended in fresh RPMI for an additional 22 hr incubation prior to measuring leukocyte HR with ragweed antigen E. TFP (10" M) inhibition of HR was s i g n i f i c a n t l y (p= 0.03) reduced in those cells incubated with virus. This suggests a virus-associated alteration in CM a c t i v i t y . These changes in basophil regulation can contribute to enhanced HR and airway i r r i t a b i l i t y in virus-induced asthma.
239
PLATELET AUGMENTATIONOF BASOPHIL HISTAMINE RELEASE. K. A. Knauer, M.D., A. Ka~e~-Sobotka, Ph.D., N. F. Adkinson, Jr., M.D. and L. M. Lichtenstein, M.D., Baltimore, MD. The effect of platelets (PLT) and PLT products on IgE mediated histamine release (HR) was studied by admixing the 2 cell types in ratios found in blood. In 18/18 experiments PLT enhanced antigen or a-lgE induced HR by 66 • ll% (control release 29 • 6%). The enhancement was positively correlated with PLT number over the range of 0.5 to 2xlO8/ml and appeared related to PLT secretion since: l ) augmentation of HR correlated with the release of the = granule marker PF4 (r = 0.75, p <.05), but not with PLT production of TxB2; 2) when PLTs were activated with thrombin, the effect was greater; enhancement of 90-230% in 5/5 experiments (control release 12-50%). PLTs increased both the rate and extent of HR equally, but neither stimulated nor non-stimulated PLTs consistently affected spontaneous HR. The factor(s) responsible are not yet identified, but l ) PLT-free supernatants are active, 2) the MWis >lO,O00 since they are not dialyzable, and 3) they are acid and heat stable. Highly purified basophils respond as well as mixed leukocytes, and PLTs also enhance release from isolated human lung mast cells. These results indicate that PLTs markedly influence IgE dependent mediator release, and presumably play an important role in the human allergic response.
240
SERUM INHIBITOR OF UROPOD FORMATION BY BASOPHILS.
MA Lett-Brown, Ph.D., WD Dickey, Ph.D., and FC Schmalstieg, M.D., Ph.D., Galveston, Texas Studies have shown that blood basophils can be recruited to enter the tissues in an immunologically specific manner. It is l i k e l y that uropod formation plays an important role in cell migration. The finding that serum stimulates uropod formation in basophils makes this hypothesis attractive. We have isolated a factor from human serum that inhibits uropod formation in guinea pig basophils (UIP=uropod inhibitory protein): TREATMENT % UROPODBEARING BASOPHILS RPMI-1640 59+9 RPMI-1640 +UIP 6~4 In addition, this factor inhlbits--in vitro human basophil chemotaxis to CSa. Phosphatidyl choline was a necessary cofactor for the action of the inhibitor. UIP is a trypsin sensitive protein that is associated with the low density lipoproteins (LDL). Isolation of UIP was accomplished by precipitation of LDL with heparin and was further purified by isopycnic ultracentrifugation. This material was used to prepare a rabbit-anti human UIP antibody. UIP was then purified by affinity chromatography followed by isoelectric focusing. Inhibitor activity appeared at PI's of 7.9 and 8.2. SDS-PAGE showed two bands of molecular weights 41,000 and 50,000 daltons. These proteins appear identical to the UIP previously described for T lymphocytes. Monospecific antisera to these proteins removes the inhibitory activity against both basophils and lymphocytes. The finding of factors in serum that both stimulate and inhibit uropod formation in basophils suggests that a control system for basophil movement may exist in serum.
THE EFFECT OF PRAZOSIN ON COLD AIR HYPERVENTILATION CHALLENGES (CAHC). A. Bewtra, M.D., N. Nair M.D., B. Alper~ M.D.~ R. Townley, M.D. Omaha~ PE This study was performed to evaluate the contribution of alpha adrenergic activity in cold air induced bronchospasm by blocking alpha~ adrenergic receptors with prazosin (P). Szx subjects with mild to moderate bronchial asthma were studied for effect of (P), (2 mg oral dose) in a randomized double blind, cross over trial. CAHC were conducted according to modification of the methodology described by Mcfadden et al, two hours post (P) or placebo. Subjects hyperventilated air cooled to -10 ~ to -20~ for five minute challenges at a fixed minute ventilation. The conditions of the challenges were kept constant for each test for a given subject. The results for the study are given in the table below: Treatment FEV 1 mean • S.E.M. baseline postdrug post CAHC Placebo 3.23• 3.21• 2.47• Prazosin 3.11• 3.16• 2.43• The data was analyzed by the paired t-test and shows that (P) does not provide protection against the bronchospasm caused by CAHC (p >.i). The results show that (P) had no effect on baseline pulmonary function (p >.i). This study demonstrates that adrenergic blockade induced by (P) under the conditions of this experiment provides no protection against cold air induced asthma and therefore we conclude that alpha-i adrenergic receptors do not have a significant role in the pathogenesis of cold air induced brenchospasm.
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238
VIRUS IMPAIRMENTOF TIs CALMODULINANTAGONIST TRIFLUOPERAZINE (TFP) MODULATIONOF LEUKOCYTE HISTAMINE RELEASE (HR) W. Busse, M.D. and C. A. Swenson, B.S., Madison, Wisconsin During viral respiratory infections, many asthmatics have increased wheezing which may relate to increased mediator release. Previous work found enhanced basophilic-leukocyte HR with antigen and calcium ionophore following an in v i t r o virus incubation. Thoughassociated with interferon production, the mechanism of virus enhanced HR is unknown. Calmodulin (CM) is an i n t r a c e l l u l a r intermediary for calcium ions and is also linked to cyclic AMP metabolism through phosphodiesterase activation. Virus transformed cells may have increased CM (Biochem Biophys Res Commun 86:1169, 1979) which could affect calcium ion movement, reduce cAMP, and thus contribute to enhanced HR. CM a c t i v i t y is antagonized by TFP. This CM antagonist i n h i b i t s leukocyte.HR with maximal effects at IO-~M (ID~n=4.6xlO'bM). This TFP response provides a functional assay to measure virus incubation effects on basophilic CM a c t i v i t y in isolated leukocyte mixtures. Isolated leukocytes from 5 ragweed-sensitive patients were incubated (2 hr, 37~ with l i v e influenza A ( i n f e c t i v i t y of 1EIDsn:1 leukocyte) or medium control. Treated Cells were centrifuged, resuspended in fresh RPMI for an additional 22 hr incubation prior to measuring leukocyte HR with ragweed antigen E. TFP (10" M) inhibition of HR was s i g n i f i c a n t l y (p= 0.03) reduced in those cells incubated with virus. This suggests a virus-associated alteration in CM a c t i v i t y . These changes in basophil regulation can contribute to enhanced HR and airway i r r i t a b i l i t y in virus-induced asthma.
239
PLATELET AUGMENTATIONOF BASOPHIL HISTAMINE RELEASE. K. A. Knauer, M.D., A. Ka~e~-Sobotka, Ph.D., N. F. Adkinson, Jr., M.D. and L. M. Lichtenstein, M.D., Baltimore, MD. The effect of platelets (PLT) and PLT products on IgE mediated histamine release (HR) was studied by admixing the 2 cell types in ratios found in blood. In 18/18 experiments PLT enhanced antigen or a-lgE induced HR by 66 • ll% (control release 29 • 6%). The enhancement was positively correlated with PLT number over the range of 0.5 to 2xlO8/ml and appeared related to PLT secretion since: l ) augmentation of HR correlated with the release of the = granule marker PF4 (r = 0.75, p <.05), but not with PLT production of TxB2; 2) when PLTs were activated with thrombin, the effect was greater; enhancement of 90-230% in 5/5 experiments (control release 12-50%). PLTs increased both the rate and extent of HR equally, but neither stimulated nor non-stimulated PLTs consistently affected spontaneous HR. The factor(s) responsible are not yet identified, but l ) PLT-free supernatants are active, 2) the MWis >lO,O00 since they are not dialyzable, and 3) they are acid and heat stable. Highly purified basophils respond as well as mixed leukocytes, and PLTs also enhance release from isolated human lung mast cells. These results indicate that PLTs markedly influence IgE dependent mediator release, and presumably play an important role in the human allergic response.
240
SERUM INHIBITOR OF UROPOD FORMATION BY BASOPHILS.
MA Lett-Brown, Ph.D., WD Dickey, Ph.D., and FC Schmalstieg, M.D., Ph.D., Galveston, Texas Studies have shown that blood basophils can be recruited to enter the tissues in an immunologically specific manner. It is l i k e l y that uropod formation plays an important role in cell migration. The finding that serum stimulates uropod formation in basophils makes this hypothesis attractive. We have isolated a factor from human serum that inhibits uropod formation in guinea pig basophils (UIP=uropod inhibitory protein): TREATMENT % UROPODBEARING BASOPHILS RPMI-1640 59+9 RPMI-1640 +UIP 6~4 In addition, this factor inhlbits--in vitro human basophil chemotaxis to CSa. Phosphatidyl choline was a necessary cofactor for the action of the inhibitor. UIP is a trypsin sensitive protein that is associated with the low density lipoproteins (LDL). Isolation of UIP was accomplished by precipitation of LDL with heparin and was further purified by isopycnic ultracentrifugation. This material was used to prepare a rabbit-anti human UIP antibody. UIP was then purified by affinity chromatography followed by isoelectric focusing. Inhibitor activity appeared at PI's of 7.9 and 8.2. SDS-PAGE showed two bands of molecular weights 41,000 and 50,000 daltons. These proteins appear identical to the UIP previously described for T lymphocytes. Monospecific antisera to these proteins removes the inhibitory activity against both basophils and lymphocytes. The finding of factors in serum that both stimulate and inhibit uropod formation in basophils suggests that a control system for basophil movement may exist in serum.
THE EFFECT OF PRAZOSIN ON COLD AIR HYPERVENTILATION CHALLENGES (CAHC). A. Bewtra, M.D., N. Nair M.D., B. Alper~ M.D.~ R. Townley, M.D. Omaha~ PE This study was performed to evaluate the contribution of alpha adrenergic activity in cold air induced bronchospasm by blocking alpha~ adrenergic receptors with prazosin (P). Szx subjects with mild to moderate bronchial asthma were studied for effect of (P), (2 mg oral dose) in a randomized double blind, cross over trial. CAHC were conducted according to modification of the methodology described by Mcfadden et al, two hours post (P) or placebo. Subjects hyperventilated air cooled to -10 ~ to -20~ for five minute challenges at a fixed minute ventilation. The conditions of the challenges were kept constant for each test for a given subject. The results for the study are given in the table below: Treatment FEV 1 mean • S.E.M. baseline postdrug post CAHC Placebo 3.23• 3.21• 2.47• Prazosin 3.11• 3.16• 2.43• The data was analyzed by the paired t-test and shows that (P) does not provide protection against the bronchospasm caused by CAHC (p >.i). The results show that (P) had no effect on baseline pulmonary function (p >.i). This study demonstrates that adrenergic blockade induced by (P) under the conditions of this experiment provides no protection against cold air induced asthma and therefore we conclude that alpha-i adrenergic receptors do not have a significant role in the pathogenesis of cold air induced brenchospasm.
152