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20 Human basophils contain the eosinophil granule major basic protein
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D O-INDUCED HASOPBIL HISTAMINE HHLEASE IN A&HMATIC SUBJECTS: SECONDAKY TO C’ ACTIVATION PRODUCTS? W. Kazimierciak. H.L. Meier. M. Plaut, and L.M. Lichtenatein, Baltimore, MD. Basophile from a majority of asthmatic subjects spontaneously release histamine in the presence of D2C, suggesting that the cells are activated (J.I. 128:2067, 1982). In --in viva attempting to elucidate the nature of this phenomenon, we have found that : (1) The basophils of D20-responding subjects became sensitive only after they were removed from plasma. (2) Diluted plasma/serum (l:K)OO to 1: lo) inhibited D20-induced histamine release. (3) The inhibitory factor in plasma/serum was associated with a protein of MW 60,000 and was inactivated by heat (bloc for 2 hours), suggesting an enzymatic activity. (4) Inhibitors of carboxypeptidase N (CN) (anaphylatoxin inactivator) reversed the inhibitory action of diluted plasma/serum. (5) Plasma/serum treated with these CN inhibitors was able to cause greater than 50% histamine release from basophi15 of D20 responsive subjects even in the (6) Heating the plasma/serum absence of D20. (36’C for 30 minutes) completely abolished the releasing activity of plasma/serum caused by These data are compatible the CN inhibitors. with the hypothesis that D 0 release is due to its augmentation of the ef 1 ects of endogenously generated anaphylatoxins and that plasma/serum contains a C&like enzyme which can block this response.
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INTERACTION BETWEEN HYPEROSMOLAH AND I E INDUCED HISTAMINE RELEASE FROM BASOPHILS AND &ST CELLS &Q&g&&on MD. AK Sobotka PhD, R Schleimer PhD LM Lichtenstein MD PhD, Baltimore, MD Hyperosmolar solutions induce histamine release from basophils by uniquely Ca*independent mechanisms(JC1 67:1604). We have demonstrated that human lung mast cells(in suspension) also undergo noncytotoxic histamine release on exposure to hyperosmolar solutions. i!aximal release (6-28%) occurs over a broad range(640-101OmOsm) but the reaction in other aspects studied is similar to that in basophils. It is partially Ca* dependent, is enhanced by D20, and has a temperature optimum of 32-37. In Ca*, antiIgE enhanced hyperosmolar release from both cells (basophils:640 mOsm 21+3% release, anti&E .02-.2 ug/ml 22+4%, both 58+8%; mast cells: 640mOsm 7+2% release, antiIgE 12+4%, both 28+8%). In basophils. enhancement was seen in Ca * free buffers but not in 1 mM EDTA. In mast cells enhancement was not seen in Ca* free buffers. AntiIgE enhancement was lost when basophils were desensitized by preincubation with 1 ug/ml antiIgE in 1 mM EDTA for 1 hour. In Ca++ c AMP active drugs inhibited hyperosmolar releise and interaction with antiIgE only in the absence of Ca*. In Ca* 1mM dibutyryl CAMP inhibited antiIgE induced release 48% but did not affect enhancement; without Ca*, release to the combined stimulus was reduced 59%. These data demonstrate significant interaction between nonimmunologic and nonimmunologic stimuli, and confirm the model of a two stage release reaction in which one stage is Ca* dependent and unaffected by CAMP and the other is Ca* independent and cAM'P inhibitable.
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HLIMAN BASOPHILS CONTAIN THE BCSINOPHIL GRANULE MAJGR BASIC PROTEIN. S. J. Ackerman, Ph.D., G. M. Kephart, B.S., and G. J. Gleich, M.D., Mayo Clinic, Rochester, MM, In experiments using an immunofluorescent method to localize human eosinophil granule major basic protein (MBP) in cells and tissues, a small number of cells stained for MBP that subsequently could not be identified as eosinophils. Because Charwt-Leyden crystal protein, another eosinophil protein, was recently identified in basophils, we tested the hypothesis that MBP might also be a constituent of blood basophils. Highly purified, eosinophil free, basophil suspensions were prepared using the fluorescence activated cell sorter (FACS) to sort Ficoll-Hypaque interface cells stained with fluorescein-conjugated sheep antihuman IgE antibody. Utilizing these FACS-purified basophils, we found that: 1) enrichment for surface-IgE positive cells 090% basophils) by FACS also enriched for cells staining for MBP by immunofluorescence and MBP appeared to be localized in the histamine-, heparin-containing granules, 2) significant quantities of MBP were measurable by radioimmunoassay (RIA) in freezethaw, detergent extracts of purified basophils, and 3) RIA dose response curves for extracts of purified eosinophils and basophils had identical slopes. The MBP content of basophils from atopic individuals averaged 74 ng/106 cells, whereas purified eosinophils from atopics and patients with hypereosinophilic syndromes averaged 4,979 and 024 ng/106 cells, respectively. We conclude that hasophils contain a protein that is immunochemically indistinguishable from eosinophil MBP.
PASSIVE SENSITIZATION OF HUMAN BLOOD BASOPHILS. J. Pruzansky, Ph.D., R. Patterson, M.D., and L. Graavaer, M.D., Chicago, Illinois. The relative inability to passively sensi tize human basophils stems from the high affinity of their receptors for IgE. Since most individuals have serum IgE levels in the rig/ml range their basophils are largely occupied by endogenoue IgE which is not readily dissociated. Removal of this IgE consistent with functional integrity should greatly facilitate passive sensitization. Previous investigators (Ishizakas) succeeded in removing the IgE but reported spontaneous histamine release by those basophi Is. Leukocytes from a mold sensitive donor were treated at low pH which abolished reactivity to molds without causing unstimulated loss of histamine. Such cells were passively sensi tized with sera of ragweed sensitive donors, washed and challenged with ragweed extract. Histamine release occurred approaching the activity of untreated cells to mold allergens. Basophils of 6 of 6 atopic donors released significant histamine (4626%) as did 8 of 9 non atopic donors (36~7%) after removal of IgE. In contrast only 1 of the 9 non atopic donors could be sensitized without pretreatment. For 3 donors 7.4x104, zx105 and 2.4xlO5 molecules of IgE ware removed per basophil. Such cells will be useful in measuring meaningful kinetic and equilibrium parameters for human basophils and determining molecular requirements for sensitization for histamine release. Similar treatment of human and other mast cells should allow improved passive sensitization in those sys terns.
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D O-INDUCED HASOPBIL HISTAMINE HHLEASE IN A&HMATIC SUBJECTS: SECONDAKY TO C’ ACTIVATION PRODUCTS? W. Kazimierciak. H.L. Meier. M. Plaut, and L.M. Lichtenatein, Baltimore, MD. Basophile from a majority of asthmatic subjects spontaneously release histamine in the presence of D2C, suggesting that the cells are activated (J.I. 128:2067, 1982). In --in viva attempting to elucidate the nature of this phenomenon, we have found that : (1) The basophils of D20-responding subjects became sensitive only after they were removed from plasma. (2) Diluted plasma/serum (l:K)OO to 1: lo) inhibited D20-induced histamine release. (3) The inhibitory factor in plasma/serum was associated with a protein of MW 60,000 and was inactivated by heat (bloc for 2 hours), suggesting an enzymatic activity. (4) Inhibitors of carboxypeptidase N (CN) (anaphylatoxin inactivator) reversed the inhibitory action of diluted plasma/serum. (5) Plasma/serum treated with these CN inhibitors was able to cause greater than 50% histamine release from basophi15 of D20 responsive subjects even in the (6) Heating the plasma/serum absence of D20. (36’C for 30 minutes) completely abolished the releasing activity of plasma/serum caused by These data are compatible the CN inhibitors. with the hypothesis that D 0 release is due to its augmentation of the ef 1 ects of endogenously generated anaphylatoxins and that plasma/serum contains a C&like enzyme which can block this response.
19
INTERACTION BETWEEN HYPEROSMOLAH AND I E INDUCED HISTAMINE RELEASE FROM BASOPHILS AND &ST CELLS &Q&g&&on MD. AK Sobotka PhD, R Schleimer PhD LM Lichtenstein MD PhD, Baltimore, MD Hyperosmolar solutions induce histamine release from basophils by uniquely Ca*independent mechanisms(JC1 67:1604). We have demonstrated that human lung mast cells(in suspension) also undergo noncytotoxic histamine release on exposure to hyperosmolar solutions. i!aximal release (6-28%) occurs over a broad range(640-101OmOsm) but the reaction in other aspects studied is similar to that in basophils. It is partially Ca* dependent, is enhanced by D20, and has a temperature optimum of 32-37. In Ca*, antiIgE enhanced hyperosmolar release from both cells (basophils:640 mOsm 21+3% release, anti&E .02-.2 ug/ml 22+4%, both 58+8%; mast cells: 640mOsm 7+2% release, antiIgE 12+4%, both 28+8%). In basophils. enhancement was seen in Ca * free buffers but not in 1 mM EDTA. In mast cells enhancement was not seen in Ca* free buffers. AntiIgE enhancement was lost when basophils were desensitized by preincubation with 1 ug/ml antiIgE in 1 mM EDTA for 1 hour. In Ca++ c AMP active drugs inhibited hyperosmolar releise and interaction with antiIgE only in the absence of Ca*. In Ca* 1mM dibutyryl CAMP inhibited antiIgE induced release 48% but did not affect enhancement; without Ca*, release to the combined stimulus was reduced 59%. These data demonstrate significant interaction between nonimmunologic and nonimmunologic stimuli, and confirm the model of a two stage release reaction in which one stage is Ca* dependent and unaffected by CAMP and the other is Ca* independent and cAM'P inhibitable.
20
HLIMAN BASOPHILS CONTAIN THE BCSINOPHIL GRANULE MAJGR BASIC PROTEIN. S. J. Ackerman, Ph.D., G. M. Kephart, B.S., and G. J. Gleich, M.D., Mayo Clinic, Rochester, MM, In experiments using an immunofluorescent method to localize human eosinophil granule major basic protein (MBP) in cells and tissues, a small number of cells stained for MBP that subsequently could not be identified as eosinophils. Because Charwt-Leyden crystal protein, another eosinophil protein, was recently identified in basophils, we tested the hypothesis that MBP might also be a constituent of blood basophils. Highly purified, eosinophil free, basophil suspensions were prepared using the fluorescence activated cell sorter (FACS) to sort Ficoll-Hypaque interface cells stained with fluorescein-conjugated sheep antihuman IgE antibody. Utilizing these FACS-purified basophils, we found that: 1) enrichment for surface-IgE positive cells 090% basophils) by FACS also enriched for cells staining for MBP by immunofluorescence and MBP appeared to be localized in the histamine-, heparin-containing granules, 2) significant quantities of MBP were measurable by radioimmunoassay (RIA) in freezethaw, detergent extracts of purified basophils, and 3) RIA dose response curves for extracts of purified eosinophils and basophils had identical slopes. The MBP content of basophils from atopic individuals averaged 74 ng/106 cells, whereas purified eosinophils from atopics and patients with hypereosinophilic syndromes averaged 4,979 and 024 ng/106 cells, respectively. We conclude that hasophils contain a protein that is immunochemically indistinguishable from eosinophil MBP.
PASSIVE SENSITIZATION OF HUMAN BLOOD BASOPHILS. J. Pruzansky, Ph.D., R. Patterson, M.D., and L. Graavaer, M.D., Chicago, Illinois. The relative inability to passively sensi tize human basophils stems from the high affinity of their receptors for IgE. Since most individuals have serum IgE levels in the rig/ml range their basophils are largely occupied by endogenoue IgE which is not readily dissociated. Removal of this IgE consistent with functional integrity should greatly facilitate passive sensitization. Previous investigators (Ishizakas) succeeded in removing the IgE but reported spontaneous histamine release by those basophi Is. Leukocytes from a mold sensitive donor were treated at low pH which abolished reactivity to molds without causing unstimulated loss of histamine. Such cells were passively sensi tized with sera of ragweed sensitive donors, washed and challenged with ragweed extract. Histamine release occurred approaching the activity of untreated cells to mold allergens. Basophils of 6 of 6 atopic donors released significant histamine (4626%) as did 8 of 9 non atopic donors (36~7%) after removal of IgE. In contrast only 1 of the 9 non atopic donors could be sensitized without pretreatment. For 3 donors 7.4x104, zx105 and 2.4xlO5 molecules of IgE ware removed per basophil. Such cells will be useful in measuring meaningful kinetic and equilibrium parameters for human basophils and determining molecular requirements for sensitization for histamine release. Similar treatment of human and other mast cells should allow improved passive sensitization in those sys terns.
93