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Are Products of Complement Activation C3a and C5a Relevant Factors in Bronchial Asthma?
RESUlIS
In the 10rabbits immunized ror an IgE response, only antiHRP antibodies of the IgE class were found. In the 11rabbits immunized for an IgG response, the homologous PCA test and the basophil degranulation test performed on whole blood were negative. However, in 3 rabbits a significant release of PAF from basophil-rich buffy coat cells was found, indicating that the rabbits had specifically sensitized basophils in the circulation. The plasma factor blocking degranulation of their basophils upon challenge with HRP was identified as anti-HRP IgG. Shortly after the start of HRP inhalation, rabbits immunized for an IgE response developed respiratory distress. The number of circulating basophils, WBCs, and platelets decreased, and in 5 animals PAF release in the circulation was documented (range: 2.5-3.9 nglml). These alterations were most pronounced about 30 minutes after the beginning of the HRP inhalation. When sacrificed, in 7 of the rabbits lung lesions were round. They consisted of interstitial edema and an influx of neutrophils and monocytes, resulting in thickening of the alveolar septa. In addition, degranulated platelets were round in alveolar capillary lumina. Immunofluorescence tests for tissue localization ofHRP and rabbit IgG and C3 were negative. In contrast, no change in any of the parameters studied was observed in rabbits immunized for an IgG response or in nonimmunized rabbits (n =8). DISCUSSION
Upon inhalation of HRP, rabbits with circulating IgE antibodies release PAF into the circulation. It is conceivable that cells other than those specifically sensitized participate in the in vivo generation ofthis mediator. 5 PAF may contribute to the lung lesions round in these animals. Rabbits with IgG antibodies do not develop any early abnormalities upon challenge with HRP, despite the fact that in some animals circulating specific IgE sensitized basophils can be demonstrated. REFERENCES
1 Halonen M, Palmer 10, Lohman IC, McManus LM, Pinckard RN. Respiratory and circulatory alterations induced by acetyl g1ycerylether phosphorylcholine, a mediator of IgE anaphylaxis in the rabbit. Am Rev Respir Dis 1980; 122:915-24 2 Camussi G, Pawlowski I, Tetta C, Roffinello C, Alberton M, Brentjens 1, et al. Acute lung inHammation induced in the rabbit by local instillation of l-O-octadecyl-2-acetyl-sn-g1yceryl-3-phosphorylcholine or of native platelet-activating factor. Lab Invest 1983; 112:78-88 3 Kravis TC. Henson PM. IgE-induced release of a plateletactivating factor from rabbit lung. 1 Immunoll975; 115:1677-81 4 Benveniste 1. Egido 1, Guttierrez Millet V, Camussi G. Detection of immediate hypersensitivity in rabbits by direct basophil degranulation. 1 Allergy Clin Immunoll977; 59:271-79 5 Camussi G. Pawlowski I. Bussolino F, Caldwell PRB. Brentjens 1. Andres G. Release of platelet activating factor in rabbits with antibody mediated injury of the lung: the role ofleukocytes and of pulmonary endothelial cells. 1 Immunoll983; 131:1802-7
Are Products of Complement Activation C3a and C5a Relevant Factors In Bronchial Asthma?* M. M. Glovsk". M.D.; S. Nagata. M.D.; R. R. ScheUenberg. M.D.;
J. Hogg, M.D.; S. Kunkel. Ph.D.; and P. A Ward. M.D.
Ativation of human complement after cardiopulmonary bypass in man results in cleavage of C3a from native C3, and sequestation of neutrophils in the lung. I Cobra venom factor activation of complement in the rat and dog is associated with C3 and C5 fixation, as well as endothelial damage to the lung. To explore the relative importance of generation of C3a and cSa in the induction of bronchial asthma, we have studied neutrophil aggregation, basophil histamine release, guinea pig trachea, and human pulmonary artery and vein constriction. Since the C-terminal octapeptide (ALA-ALAALA-LEU-GLY-LEU-ALA-ARG) has been shown to mimic the biologic responses to C3a, we have used it as well as C3a. I C5a was purified to homogeneity using an insolubilized anti C58t.. affinity column. Human PMN aggregation was induced with the octapeptide and cSah• with ED..,s of 3.2 X 10- aM and 3.5 X lO- 8M, respectively. As seen in 'Iable 1, f-Met-Leu-Phe had a similar ED.. to C5a for neutrophil aggregation. In contrast, neutrophil chemotaxis was 10 and 50 times more sensitive on a molar basis to the C3a octapeptide and cSa, respectively. Although increasing the magnitude of the aggregation response, eytocholasin B (5 mgIL) did not alter the ED..,s ror C3a peptide, C5a, or f-Met-Leu-Phe. Optimal basophil histamine release occurred with -100 ....glml octapeptide and -50 nglml C5a. C5~ or C5a added to serum were approximately 33%less active than cSa in the absence ofserum in histamine release, indicating that carboxypeptidase N only partially depresses cSa activity by cleavage of the C-terminal arginine. To demonstrate generation ofC3a and cSa in vitro. we used specific radioimmunoassays. After activation of human serum with house dust (1-64 ....glml). Escherichia coli endotoxin (1-64 ....glml), and zymosan (0.5-4.0 mglml), both C3a and cSa were generated in a dose- and time-dependent manner. Human pulmonary artery, pulmonary vein, and guinea pig trachea are constructed with l-loo-....glml octapeptide. Some extrinsic allergens such as house dust and molds show relatively poor IgE-specific correlations with atopic disease. Generation of C3a and C5a may in part be responsible for some of these dis·From the Southern Califurnia Permanente Medical Group, Los Angeles~ the Department of Medicine. University of British Columbia, Vancouver, B.C.; and the Department of Pathology • University of Michigan. Ann Arbor.
Table I-ED.for C3a Peptide, Coo, andf-MLP in Aggregation and Chemottuia Aggregation
C3a-Peptide
cSa
f-MLP
without cyt B
with cyt B (5 mg/L)
3.2x lO- a• 3.5x 10- 8 3.2x 10- 8
3.3x 10-· 2.4 x 10- 8 4.0x 10- 8
Chemotaxis
3.1 X 10- 1 1.6x 10- 11 2.5xl0- 1o
·Molar concentration. CHEST I 87 I 5 I MAY, 1985 I Supplement
1888
crepancies. We thus propose that C3a and C5a are potentially important agonists in bronchial asthma. Phannacologic control of C3a and cSa lysosomal and histamine-releasing activities and smooth muscle constriction should add new approaches to the control of human asthma. REFERENCES 1 Chenoweth OE, Cooper SW, Hugli TE, et al. Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylotoxms, N Engl J Med 1981; 304:497-503 2 GlovslcyMM, Hugli TE, lshizalca'I; Lichtenstein LM, Erickson BW Anaphylatoxin induced histaminee release with human leucocytes: studies of C3a leucocyte binding and histamine release. J Clio Invest 1979; 64:804-11
The Metabolism of Arachidonic Acid by Purified Human Lung Mast Cells* Stephen P. Peters, M.D.; Donold vv. MacGlashan, [r; M.D.; Robert P. Schleimer, Ph.D.; and Lawrence M. Lichtenstein, M.D.
T
he release of inflammatory substances from mast cells is thought to be of primary importance in many acute allergic and inflammatory disorders. Most closely associated with mast cell mediator release have been disorders of immediate hypersensitivity, including anaphylaxis, allergic rhinitis, and certain forms of asthma. Evidence is increasing that these cells may also play an important role in chronic inflammatory disorders such as inflammatory bowel disease and perhaps interstitial pulmonary fibrosis. EftOrts to study mast cell function have been greatly f8ciJjtated by the development of methods to obtain highly purified preparations of human lung mast cells, 1 allowing indepth exploration of several aspects of the cell biology of the human lung mast cell. Most recently we explored the metabolism of arachidonic acid by these cells. Purified human lung mast cells (>90% purity) were prelabeled with wC_ or 3H-arachidonic acid during overnight culture. After extensive washing, these cells showed label esterified into neutral lipids (73%)and phospholipids (21%). • In companion studies performed by Dvorak and associates,3 this label was localized, surprisingly, almost exclusively into osmiophilic, intracellular lipid bodies, with no detectable labeling (above background) in plasma membranes or other cellular organelles. It is not clear whether these lipid bodies represent an important site for arachidonic acid metabolism in these cells or are a relatively inactive storage pool of arachidonic acid. Mast cell activation with anti-IgE resulted in 16-30% histamine release and the release of 3-5% of cell-associated 3Has arachidonic acid metabolites and free arachidonic acid. In addition, a 15% decrease in 3H esterified into total phospholipids was observed, with significant decreases in 3H associated with phosphatidylcholine and phosphatidylinositol (to 75% and 84% of control levels, respectively). Characterization of the released metabolites by high-performance -From The Johns Hopkins University School of Medicine, Oe ment of Medicine, Divisions of Clinical Immunology and Bespiratory Medicine at The Good Samaritan Hospital, Baltimore.
Reprint requests: Dr: Peters, Good Samaritan Hospital, 5601 Loch Roven Blvd, Baltimare 21239
1708
liquid chromatography showed the presence ofprostaglandin D., leukotriene C., leukotriene B. isomers, and 5-hydroxyeicosatetraenoic acid in an approximate ratio of 25:10:8:1. The identities of PGD. and lJ'C. (VSO and V40 pmolllO" mast cells, respectively) was confirmed by specific radioimmunoassay. Although activation of these cells with anti-IgE and ionophore A23187 resulted in markedly different histamine release (16% vs 70% in one set of experiments), the quantity and distribution of arachidonic acid metabolites were the same.· Since the mast cell releases both preformed mediators (eg, histamine, lysosomalenzymes) and newly formed mediators, from both the cyclooxygenase (prostaglandin DJ and lipoxygenase (leukotriene C., B. isomers, and 5HETE) pathways after anti-IgE-mediated activation, we can explore in detail the effect ofselected pharmacologic agents on the production and release of these substances. The first agents chosen for study included indomethacin, a cyclooxygenase inhibitor; eicosatriynoic acid (ETI), a lipoxygenase inhibitor; and diethylcarbamazine (DEC), an inhibitor of leukotriene A synthesis.' Indomethacin (3 J.LM) markedly inhibited (>95%) PGD. release but did not inhibit or enhance the release of other arachidonic acid metabolites or histamine. No shunting was observed to products of the lipoxygenase pathway, particularly to leukotriene C. as has been observed in a number of different systems which employ lung fragments or contractile tissue. The lipoxygenase inhibitor ETI nonselectively inhibited the release of histamine and all arachidonic acid metabolites, including free arachidonic acid, with an IC50 of3-10 J.LM. No specificity was observed for products of the lipoxygenase pathway in these cells. DEC resulted in only slight inhibition of prostaglandin D. (V50% at 0.3 to 3.0 mM) and histamine (V50% at 3.0 mM) release at relatively high concentrations. Again, no specificity was observed fOr products of the lipoxygenase pathway. These results suggest that these pharmacologic agents can have profound effects which are species and tissue specific and that direct measurement of inflammatory mediators is needed to verify the expected effect in each system under investigation. Studies with purified preparations of human lung mast cells such as those described here, together with verification of these findings in in vivo model systems, promise to increase our understanding ofacute anaphylactic reactions as we attempt to improve treatment for diseases associated with the release of these inflammatory mediators. REFERENCES 1 Schulman ES, MacGlashan OW, Jr, Peters Sp' Schleimer RP, Newball HH, Lichtenstein LM. Human lung mast cells: purification and characterization. J Immunoll982; 129:2662-7 2 Peters Sp' MacGlashan OW, Jr, Schulman EE, Schleimer RP, Hayes EC, Rokach J, et al. Arachidonic acid metabolism in purified human lung mast cells. J Immunoll984; 132:1972-9 3 Dvorak AM, Dvorak HF, Peters Sp' Schulman ES, MacGlashan Ow, Jr, Pyne K, et al. Lipid bodies: cytoplasmic organelles important to arachidonic acid metabolism in macrophages and mast cells. J Immunoll983; 131: 2965-76 4 Peters Sp' MacGlashan OW, Jr, Schleimer RP, Hayes EC, Adkinson NF, Jr, Lichtenstein LM. The pharmacologic modulation of the release of arachidonic acid metabolites from purified human lung mast cells (submitted) 27lh Annual Aspen Lung ConlenInce
In the 10rabbits immunized ror an IgE response, only antiHRP antibodies of the IgE class were found. In the 11rabbits immunized for an IgG response, the homologous PCA test and the basophil degranulation test performed on whole blood were negative. However, in 3 rabbits a significant release of PAF from basophil-rich buffy coat cells was found, indicating that the rabbits had specifically sensitized basophils in the circulation. The plasma factor blocking degranulation of their basophils upon challenge with HRP was identified as anti-HRP IgG. Shortly after the start of HRP inhalation, rabbits immunized for an IgE response developed respiratory distress. The number of circulating basophils, WBCs, and platelets decreased, and in 5 animals PAF release in the circulation was documented (range: 2.5-3.9 nglml). These alterations were most pronounced about 30 minutes after the beginning of the HRP inhalation. When sacrificed, in 7 of the rabbits lung lesions were round. They consisted of interstitial edema and an influx of neutrophils and monocytes, resulting in thickening of the alveolar septa. In addition, degranulated platelets were round in alveolar capillary lumina. Immunofluorescence tests for tissue localization ofHRP and rabbit IgG and C3 were negative. In contrast, no change in any of the parameters studied was observed in rabbits immunized for an IgG response or in nonimmunized rabbits (n =8). DISCUSSION
Upon inhalation of HRP, rabbits with circulating IgE antibodies release PAF into the circulation. It is conceivable that cells other than those specifically sensitized participate in the in vivo generation ofthis mediator. 5 PAF may contribute to the lung lesions round in these animals. Rabbits with IgG antibodies do not develop any early abnormalities upon challenge with HRP, despite the fact that in some animals circulating specific IgE sensitized basophils can be demonstrated. REFERENCES
1 Halonen M, Palmer 10, Lohman IC, McManus LM, Pinckard RN. Respiratory and circulatory alterations induced by acetyl g1ycerylether phosphorylcholine, a mediator of IgE anaphylaxis in the rabbit. Am Rev Respir Dis 1980; 122:915-24 2 Camussi G, Pawlowski I, Tetta C, Roffinello C, Alberton M, Brentjens 1, et al. Acute lung inHammation induced in the rabbit by local instillation of l-O-octadecyl-2-acetyl-sn-g1yceryl-3-phosphorylcholine or of native platelet-activating factor. Lab Invest 1983; 112:78-88 3 Kravis TC. Henson PM. IgE-induced release of a plateletactivating factor from rabbit lung. 1 Immunoll975; 115:1677-81 4 Benveniste 1. Egido 1, Guttierrez Millet V, Camussi G. Detection of immediate hypersensitivity in rabbits by direct basophil degranulation. 1 Allergy Clin Immunoll977; 59:271-79 5 Camussi G. Pawlowski I. Bussolino F, Caldwell PRB. Brentjens 1. Andres G. Release of platelet activating factor in rabbits with antibody mediated injury of the lung: the role ofleukocytes and of pulmonary endothelial cells. 1 Immunoll983; 131:1802-7
Are Products of Complement Activation C3a and C5a Relevant Factors In Bronchial Asthma?* M. M. Glovsk". M.D.; S. Nagata. M.D.; R. R. ScheUenberg. M.D.;
J. Hogg, M.D.; S. Kunkel. Ph.D.; and P. A Ward. M.D.
Ativation of human complement after cardiopulmonary bypass in man results in cleavage of C3a from native C3, and sequestation of neutrophils in the lung. I Cobra venom factor activation of complement in the rat and dog is associated with C3 and C5 fixation, as well as endothelial damage to the lung. To explore the relative importance of generation of C3a and cSa in the induction of bronchial asthma, we have studied neutrophil aggregation, basophil histamine release, guinea pig trachea, and human pulmonary artery and vein constriction. Since the C-terminal octapeptide (ALA-ALAALA-LEU-GLY-LEU-ALA-ARG) has been shown to mimic the biologic responses to C3a, we have used it as well as C3a. I C5a was purified to homogeneity using an insolubilized anti C58t.. affinity column. Human PMN aggregation was induced with the octapeptide and cSah• with ED..,s of 3.2 X 10- aM and 3.5 X lO- 8M, respectively. As seen in 'Iable 1, f-Met-Leu-Phe had a similar ED.. to C5a for neutrophil aggregation. In contrast, neutrophil chemotaxis was 10 and 50 times more sensitive on a molar basis to the C3a octapeptide and cSa, respectively. Although increasing the magnitude of the aggregation response, eytocholasin B (5 mgIL) did not alter the ED..,s ror C3a peptide, C5a, or f-Met-Leu-Phe. Optimal basophil histamine release occurred with -100 ....glml octapeptide and -50 nglml C5a. C5~ or C5a added to serum were approximately 33%less active than cSa in the absence ofserum in histamine release, indicating that carboxypeptidase N only partially depresses cSa activity by cleavage of the C-terminal arginine. To demonstrate generation ofC3a and cSa in vitro. we used specific radioimmunoassays. After activation of human serum with house dust (1-64 ....glml). Escherichia coli endotoxin (1-64 ....glml), and zymosan (0.5-4.0 mglml), both C3a and cSa were generated in a dose- and time-dependent manner. Human pulmonary artery, pulmonary vein, and guinea pig trachea are constructed with l-loo-....glml octapeptide. Some extrinsic allergens such as house dust and molds show relatively poor IgE-specific correlations with atopic disease. Generation of C3a and C5a may in part be responsible for some of these dis·From the Southern Califurnia Permanente Medical Group, Los Angeles~ the Department of Medicine. University of British Columbia, Vancouver, B.C.; and the Department of Pathology • University of Michigan. Ann Arbor.
Table I-ED.for C3a Peptide, Coo, andf-MLP in Aggregation and Chemottuia Aggregation
C3a-Peptide
cSa
f-MLP
without cyt B
with cyt B (5 mg/L)
3.2x lO- a• 3.5x 10- 8 3.2x 10- 8
3.3x 10-· 2.4 x 10- 8 4.0x 10- 8
Chemotaxis
3.1 X 10- 1 1.6x 10- 11 2.5xl0- 1o
·Molar concentration. CHEST I 87 I 5 I MAY, 1985 I Supplement
1888
crepancies. We thus propose that C3a and C5a are potentially important agonists in bronchial asthma. Phannacologic control of C3a and cSa lysosomal and histamine-releasing activities and smooth muscle constriction should add new approaches to the control of human asthma. REFERENCES 1 Chenoweth OE, Cooper SW, Hugli TE, et al. Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a anaphylotoxms, N Engl J Med 1981; 304:497-503 2 GlovslcyMM, Hugli TE, lshizalca'I; Lichtenstein LM, Erickson BW Anaphylatoxin induced histaminee release with human leucocytes: studies of C3a leucocyte binding and histamine release. J Clio Invest 1979; 64:804-11
The Metabolism of Arachidonic Acid by Purified Human Lung Mast Cells* Stephen P. Peters, M.D.; Donold vv. MacGlashan, [r; M.D.; Robert P. Schleimer, Ph.D.; and Lawrence M. Lichtenstein, M.D.
T
he release of inflammatory substances from mast cells is thought to be of primary importance in many acute allergic and inflammatory disorders. Most closely associated with mast cell mediator release have been disorders of immediate hypersensitivity, including anaphylaxis, allergic rhinitis, and certain forms of asthma. Evidence is increasing that these cells may also play an important role in chronic inflammatory disorders such as inflammatory bowel disease and perhaps interstitial pulmonary fibrosis. EftOrts to study mast cell function have been greatly f8ciJjtated by the development of methods to obtain highly purified preparations of human lung mast cells, 1 allowing indepth exploration of several aspects of the cell biology of the human lung mast cell. Most recently we explored the metabolism of arachidonic acid by these cells. Purified human lung mast cells (>90% purity) were prelabeled with wC_ or 3H-arachidonic acid during overnight culture. After extensive washing, these cells showed label esterified into neutral lipids (73%)and phospholipids (21%). • In companion studies performed by Dvorak and associates,3 this label was localized, surprisingly, almost exclusively into osmiophilic, intracellular lipid bodies, with no detectable labeling (above background) in plasma membranes or other cellular organelles. It is not clear whether these lipid bodies represent an important site for arachidonic acid metabolism in these cells or are a relatively inactive storage pool of arachidonic acid. Mast cell activation with anti-IgE resulted in 16-30% histamine release and the release of 3-5% of cell-associated 3Has arachidonic acid metabolites and free arachidonic acid. In addition, a 15% decrease in 3H esterified into total phospholipids was observed, with significant decreases in 3H associated with phosphatidylcholine and phosphatidylinositol (to 75% and 84% of control levels, respectively). Characterization of the released metabolites by high-performance -From The Johns Hopkins University School of Medicine, Oe ment of Medicine, Divisions of Clinical Immunology and Bespiratory Medicine at The Good Samaritan Hospital, Baltimore.
Reprint requests: Dr: Peters, Good Samaritan Hospital, 5601 Loch Roven Blvd, Baltimare 21239
1708
liquid chromatography showed the presence ofprostaglandin D., leukotriene C., leukotriene B. isomers, and 5-hydroxyeicosatetraenoic acid in an approximate ratio of 25:10:8:1. The identities of PGD. and lJ'C. (VSO and V40 pmolllO" mast cells, respectively) was confirmed by specific radioimmunoassay. Although activation of these cells with anti-IgE and ionophore A23187 resulted in markedly different histamine release (16% vs 70% in one set of experiments), the quantity and distribution of arachidonic acid metabolites were the same.· Since the mast cell releases both preformed mediators (eg, histamine, lysosomalenzymes) and newly formed mediators, from both the cyclooxygenase (prostaglandin DJ and lipoxygenase (leukotriene C., B. isomers, and 5HETE) pathways after anti-IgE-mediated activation, we can explore in detail the effect ofselected pharmacologic agents on the production and release of these substances. The first agents chosen for study included indomethacin, a cyclooxygenase inhibitor; eicosatriynoic acid (ETI), a lipoxygenase inhibitor; and diethylcarbamazine (DEC), an inhibitor of leukotriene A synthesis.' Indomethacin (3 J.LM) markedly inhibited (>95%) PGD. release but did not inhibit or enhance the release of other arachidonic acid metabolites or histamine. No shunting was observed to products of the lipoxygenase pathway, particularly to leukotriene C. as has been observed in a number of different systems which employ lung fragments or contractile tissue. The lipoxygenase inhibitor ETI nonselectively inhibited the release of histamine and all arachidonic acid metabolites, including free arachidonic acid, with an IC50 of3-10 J.LM. No specificity was observed for products of the lipoxygenase pathway in these cells. DEC resulted in only slight inhibition of prostaglandin D. (V50% at 0.3 to 3.0 mM) and histamine (V50% at 3.0 mM) release at relatively high concentrations. Again, no specificity was observed fOr products of the lipoxygenase pathway. These results suggest that these pharmacologic agents can have profound effects which are species and tissue specific and that direct measurement of inflammatory mediators is needed to verify the expected effect in each system under investigation. Studies with purified preparations of human lung mast cells such as those described here, together with verification of these findings in in vivo model systems, promise to increase our understanding ofacute anaphylactic reactions as we attempt to improve treatment for diseases associated with the release of these inflammatory mediators. REFERENCES 1 Schulman ES, MacGlashan OW, Jr, Peters Sp' Schleimer RP, Newball HH, Lichtenstein LM. Human lung mast cells: purification and characterization. J Immunoll982; 129:2662-7 2 Peters Sp' MacGlashan OW, Jr, Schulman EE, Schleimer RP, Hayes EC, Rokach J, et al. Arachidonic acid metabolism in purified human lung mast cells. J Immunoll984; 132:1972-9 3 Dvorak AM, Dvorak HF, Peters Sp' Schulman ES, MacGlashan Ow, Jr, Pyne K, et al. Lipid bodies: cytoplasmic organelles important to arachidonic acid metabolism in macrophages and mast cells. J Immunoll983; 131: 2965-76 4 Peters Sp' MacGlashan OW, Jr, Schleimer RP, Hayes EC, Adkinson NF, Jr, Lichtenstein LM. The pharmacologic modulation of the release of arachidonic acid metabolites from purified human lung mast cells (submitted) 27lh Annual Aspen Lung ConlenInce