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IgE immune complexes induce leukotriene B4 release from rat alveolar macrophages
364
12e F O R U M D ' I M M U N O L O G I E
IgE I M M U N E C O M P L E X E S I N D U C E L E U K O T R I E N E B4 R E L E A S E F R O M RAT A L V E O L A R M A C R O P H A G E S by J.A. Rankin
Pulmonary Section, Department o f Medicine, Yale University School of Medicine, P.O. Box 3333, 333 Cedar Street, New Haven, CT 06510 (USA) Introduction. The mast cell plays a central role as a source of pathogenic mediators in IgE-dependent reactions. Indeed, the mast cell is a good source of several mediators of putative importance in asthma syndromes such as prostaglandins, histamine and leukotrienes. However, inflammatory reactions generally are complex and involve many mediators and many different cell types. Two separate observations led us to study the potential role of an additional cell, the alveolar macrophage in IgE-dependent reactions. First, Capron and co-workers demonstrated that rat peritoneal macrophages could be activated to a cytotoxic state by the interaction of IgE immune complexes with IgE receptors (IgEFcR) on the surface of the macrophage [1]. These studies were the first to identify that macrophages possess IgEFcR and suggested that these cells have a role in IgE-dependent disease states. The second observation was made by Bach and co-workers, who demonstrated that rat peritoneal macrophages would release leukotrienes C4 and D4 when stimulated with a non-specific stimulus, calcium ionophore A23187 [2]. The work of both investigators led us to hypothesize that rat alveolar macrophages would release leukotriene C4 and D 4 when stimulated by IgE and specific antigen, and thereby portend a role for this cell in lung diseases mediated by IgE and antigen. In experiments performed previously, we demonstrated that alveolar macrophages from normal healthy rats release readily quantifiable amounts of LTC4, but not LTD4, when challenged with a mouse monoclonal hybridoma anti-dinitrophenyl (DNP)
IgE antibody and its specific DNP-HSA antigen [3, 4]. Experiments revealed that LTC4 release was initiated by the interaction of immune complexes containing IgE and antigen with the alveolar macrophages, and that complexes containing as little as 2 ng of antigen were effective in eliciting LTC4 release. Leukotriene B4 also is derived from arachidonic acid metabolism via the lipoxygenase pathway. This substance is a very potent chemotactic factor for polymorphonuclear leukocytes (PMNL) in vitro and causes PMNL adherence to vascular endothelium and migration into extravascular spaces in vivo [5]. Numerous cells such as macrophages, neutrophils and monocytes release LTB4, suggesting that a role exists for this mediator in inflammatory reactions [5]. The current study was performed to determine whether rat alveolar macrophages challenged with IgE immune complexes would release LTB4.
Materials and methods. Alveolar macrophages from healthy Sprague-Dawley rats were harvested and counted as described previously I3]. In experiments performed on two separate days, alveolar macrophages from 6-8 rats were pooled, suspended in Hanks' balanced salt solution (Grand Island Biological, Grand Island, NY) at 5 million viable macrophages/ml, and challenged with 10 ng/ml of IgE and 100 ~tg/ml of DNP-HSA at 37~ precisely the same conditions which result in maximal release of LTC 4 [3]. After a 20-min incubation, the supernatants were harvested, pooled, and an aliquot
IgE R E C E P T O R S stored at - 7 0 ~ for LTB 4 quantitation by radioimmunoassay [6]. The remainder of the pooled supernatants were extracted with methanol 1/4 vol/vol, and evaporated to dryness under a stream of nitrogen. The dried material was resuspended in methanol:water 1/1 vol/vol. An aliquot was subjected to reverse-phase high performance liquid chromatography (HPLC) (Waters NOVA-PAK C~8) and eluted isocratically with a mobile phase consisting of methanol/water/acetic acid/ ammonium hydroxide (67/33/0.8/0.4) pH 5.85 at a flow rate of lml/min. Results.
By RIA, rat alveolar macrophages released 72.2 and 59.1 ng LTB4/5 million viable macrophages in experiments performed on the two separate days. That the material recognized in the RIA was LTB 4 was confirmed by its coelution with pure synthetic standard on reverse-phase H P L C , figure 1. Retention times of synthetic LTB 4 and LTC 4 standards (J. Rokach Merck Frosst, Canada) were 6.84 min (range 6.7-7.0) and 13.7 min (range 13.4-14), respectively. In addition, collected eluates were dried under nitrogen, resuspended in RIA buffer, and LTB4 quantitated by RIA. Approximately 60-80 % of LTB 4 was recovered in fractions that co-eluted with synthetic standard. Results from previous experiments and results from one of the experiments above demonstrate that rat alveolar macrophages release 4-10 times more LTB 4 than LTC4 in response to an IgE and antigen stimulus.
365
ning rat alveolar macrophage release of LTC4 [3]. First, release occurs as early as two minutes after IgE and antigen challenge. Second, peak release occurs within ten to twenty minutes. Third, the mechanism of release involves the interaction of IgE immune complexes with the macrophages. Fourth, immune complexes containing as little as 2 ng of antigen will elicit LTC 4 release, and fifth, alveolar macrophages from individual rats differ markedly in the quantity of LTC4 they will release [3]. It is likely that these observations hold true for LTB 4 release as well. Oxidative metabolism of arachidonic acid via the lipoxygenase pathway results in the simultaneous appearance o f leukotriene products in supernatants o f cells capable of the synthesis and release of these substance [7]. In addition, in our current experiments, the conditions used to stimulate rat alveolar macrophages were identical to those used in previous expe-
Lr~
Discussion.
The results of this study demonstrate that rat alveolar macrophages release readily quantifiable amounts of LTB 4 in response to challenge with IgE and specific antigen. The presence of LTB 4 in supernatants was verified using a monospecific antibody and by reversephase H P L C . Our previous experiments have established several characteristics concer-
i 0
I 4
I
I 8
I
i 12
I
I 16
TIME(rain)
FIG. i. -- Reverse-phase HPLC of pooled supernatants obtained after challenging pooled aliquots of alveolar macrophages from 6-8 rats with lgE immune complexes. The arrows indicate the retention times of pure synthetic standards.
366
12e F O R U M D ' I M M U N O L O G I E
riments. In these previous studies, LTB4 was not detected because we did not assay for the presence of LTB4 either with functional assays or by HPLC. The results of animal studies recently concluded by Nadel and co-workers suggest that airway hyperreactivity is a consequence of airway inflammation in which PMNL may play a critical role [8]. Furthermore, recently reported analyses of bronchoalveolar lavage fluid from individuals with asthma reveal that PMNL and eosinophils are present in increased numbers after local airway challenge with an antigen to which the asthmatic individual is sensitive [9]. The mechanisms responsible for attracting these proinflammatory cells -- normally present as less than 1 % of all BAL cells in non-smoking healthy normal individuals -- to the airways is not yet clear. The lower respiratory tract of normal humans is coated by epithelial lining fluid that is rich in IgG [4]. In addition, IgE can be quantitated in BAL fluid from about 80 % of normal individuals [4]. Therefore, one plausible hypothesis proposes that antigen reaching the lower respiratory tract forms immune complexes with IgE or IgG present in epithelial lining fluid and these com-
plexes in turn activate macrophages via their respective receptors to release LTB4, which is a potent chemotactic agent for PMNL and eosinophils. Indeed, human alveolar macrophages from normal individuals are capable of producing as much as 400 ng of LTB4 per million cells after challenge with calcium ionophore A23187 [10]. Most recently, we have shown that normal human alveolar macrophages under appropriate conditions will also release LTB4 in response to an IgG stimulus [11]. Currently, we are evaluating the possibility that normal human alveolar macrophages also will release LTB4 in response to IgE stimuli. In summary, evidence continues to mount that the alveolar macrophage may play a crucial role in IgEmediated reactions in the lung. The observation that IgE complexes elicit the rapid release of significant quantities of LTB4 from rat alveolar macrophages suggests that human asthmatic alveolar macrophages may also respond in this manner. Studies of macrophages taken from the lower respiratory tract of normals and individuals with asthma hopefully will reveal new insights into the immunopathogenesis of asthma.
References. [1] CAPRON, A., DESSAINT, J.-P., ROUSSEAU, R., CAPRON, M. & BAZIN, H., Interaction between IgE complexes and macrophages in the rat: a new mechanism of macrophage activation. Europ. J. Immunol., 1977, 7, 315-322. [2] BACH, M.K. & BRASHLER,J.R., Ionophore A23187-induced production of slowreacting substance of anaphylaxis (SRS-A) by rat peritoneal cells in vitro: Evidence for production by mononuclear cells, d. Immunol., 1978, 120, 998-1005. [3] RANKIN,J.A., HITCHCOCK,M., MERRILL,W.W., HUANG, S.S., BRASHLER,J.R., BACH, M.K. & ASKENASE,P.W., IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages. J. Immunol., 1984, 132, 1993-1999. [4] RANKIN, J.A. & ASKENASE,P.W., The potential role of alveolar macrophages as a source of pathogenic mediators in allergic asthma, in <>(A.B. Kay, K.F. Austen & L.M. Licbenstein) (p. 157), Academic Press, London, New York, 1984. [5] BRAY, M.A., The Pharmacology and pathophysiology of leukotriene B4. Brit. reed. Bull., 1983, 39, 249-254.
IgE R E C E P T O R S
367
[6] ROKACH, J., HAYES, E.C., GIRARD, Y., LOMBARDO, D.L., MAYCOCK, A.L.,
[7] [8]
[9] [10]
[11]
ROSENTHAL,A.S., YOUNG,R.N., ZAMBONI,R. • ZWEERINK,H.J., The development of sensitive and specific radioimmunoassays for leukotrienes. PG Leuk. Med., 1984, 13, 21-25. LEwis, R.A. & AUSTEN, K.F., Mediation of local homeostasis and inflammation by leukotrienes and other mast-cell-dependent compounds. Nature (Lond.), 1981, 292, 103-108. NADEL, J.A. & HOLTZMAN, M.J., Regulation of airway responsiveness and secretion: role of inflammation, in ~>(A.B. Kay, K.F. Austen & L.M. Lichtenstein) (p. 129). Academic Press, London, New York, 1984. METZGER,W.J., Hunninghake, G.W. & RICHERSON, H.B., Late asthmatic responses: inquiry into mechanisms and significance. Clin. Rev. Allergy, 1985, 3, 146-165. GODARD, P., DAMON, M., MICHEL, F.B., COREY, E.J., AUSTEN, K.F. & LEWIS, R.A., Leukotriene B4 production from alveolar macrophages. Clin. Res., 1983, 31, 548 A. RANKIN, J.A., SCHRADER,C.E. & LEWIS, R.A., Leukotriene B4 release from human alveolar macrophages incubated with IgG: effect of -f-interferon. Fed. Proc., 1986 (abstract).
Supported by the Parker B. Francis Foundation and a Research Associate Grant from the Veterans Administration.
Fc~R-POSITIVE C E L L S A N D M O N I T O R I N G OF INFLAMMATORY REACTION by A . B . T o n n e l
Centre d'Immunologie et de Biologie Parasitaire, Unit~ Mixte I N S E R M 167-CNRS 624, Institut Pasteur, Lille 59019 (France)
Besides mast cells and basophils, receptors for the Fc fragment of immunoglobulin E have been successively demonstrated on different cell populations: mononuclear phagocytes, lymphocytes, eosinophils and more recently platelets [3]. One of the main consequences of these findings was to indicate that, following IgE-dependent stimulation, cells bearing FcE receptors participated in immune defence against metazoan parasites. They also have a particular significance in the context of human allergic diseases: Fc~R-positive cells could indeed be activated by antiIgE or allergen triggering and play a role in the initiation and the modulation of
the pro-inflammatory response in various hypersensitivity diseases such as allergic asthma or systemic anaphylactic reactions of hymenoptera venom hypersensitivity. Antiallergic drugs also appeared to be able to interfere with the inflammatory reaction by acting not only on mast cells but also on the other cells expressing the Fc~ receptor. I. - - Alveolar macrophage and allergic asthma.
With normal alveolar macrophages (AM) obtained by bronchoalveolar lavage, the successive incubation either
12e F O R U M D ' I M M U N O L O G I E
IgE I M M U N E C O M P L E X E S I N D U C E L E U K O T R I E N E B4 R E L E A S E F R O M RAT A L V E O L A R M A C R O P H A G E S by J.A. Rankin
Pulmonary Section, Department o f Medicine, Yale University School of Medicine, P.O. Box 3333, 333 Cedar Street, New Haven, CT 06510 (USA) Introduction. The mast cell plays a central role as a source of pathogenic mediators in IgE-dependent reactions. Indeed, the mast cell is a good source of several mediators of putative importance in asthma syndromes such as prostaglandins, histamine and leukotrienes. However, inflammatory reactions generally are complex and involve many mediators and many different cell types. Two separate observations led us to study the potential role of an additional cell, the alveolar macrophage in IgE-dependent reactions. First, Capron and co-workers demonstrated that rat peritoneal macrophages could be activated to a cytotoxic state by the interaction of IgE immune complexes with IgE receptors (IgEFcR) on the surface of the macrophage [1]. These studies were the first to identify that macrophages possess IgEFcR and suggested that these cells have a role in IgE-dependent disease states. The second observation was made by Bach and co-workers, who demonstrated that rat peritoneal macrophages would release leukotrienes C4 and D4 when stimulated with a non-specific stimulus, calcium ionophore A23187 [2]. The work of both investigators led us to hypothesize that rat alveolar macrophages would release leukotriene C4 and D 4 when stimulated by IgE and specific antigen, and thereby portend a role for this cell in lung diseases mediated by IgE and antigen. In experiments performed previously, we demonstrated that alveolar macrophages from normal healthy rats release readily quantifiable amounts of LTC4, but not LTD4, when challenged with a mouse monoclonal hybridoma anti-dinitrophenyl (DNP)
IgE antibody and its specific DNP-HSA antigen [3, 4]. Experiments revealed that LTC4 release was initiated by the interaction of immune complexes containing IgE and antigen with the alveolar macrophages, and that complexes containing as little as 2 ng of antigen were effective in eliciting LTC4 release. Leukotriene B4 also is derived from arachidonic acid metabolism via the lipoxygenase pathway. This substance is a very potent chemotactic factor for polymorphonuclear leukocytes (PMNL) in vitro and causes PMNL adherence to vascular endothelium and migration into extravascular spaces in vivo [5]. Numerous cells such as macrophages, neutrophils and monocytes release LTB4, suggesting that a role exists for this mediator in inflammatory reactions [5]. The current study was performed to determine whether rat alveolar macrophages challenged with IgE immune complexes would release LTB4.
Materials and methods. Alveolar macrophages from healthy Sprague-Dawley rats were harvested and counted as described previously I3]. In experiments performed on two separate days, alveolar macrophages from 6-8 rats were pooled, suspended in Hanks' balanced salt solution (Grand Island Biological, Grand Island, NY) at 5 million viable macrophages/ml, and challenged with 10 ng/ml of IgE and 100 ~tg/ml of DNP-HSA at 37~ precisely the same conditions which result in maximal release of LTC 4 [3]. After a 20-min incubation, the supernatants were harvested, pooled, and an aliquot
IgE R E C E P T O R S stored at - 7 0 ~ for LTB 4 quantitation by radioimmunoassay [6]. The remainder of the pooled supernatants were extracted with methanol 1/4 vol/vol, and evaporated to dryness under a stream of nitrogen. The dried material was resuspended in methanol:water 1/1 vol/vol. An aliquot was subjected to reverse-phase high performance liquid chromatography (HPLC) (Waters NOVA-PAK C~8) and eluted isocratically with a mobile phase consisting of methanol/water/acetic acid/ ammonium hydroxide (67/33/0.8/0.4) pH 5.85 at a flow rate of lml/min. Results.
By RIA, rat alveolar macrophages released 72.2 and 59.1 ng LTB4/5 million viable macrophages in experiments performed on the two separate days. That the material recognized in the RIA was LTB 4 was confirmed by its coelution with pure synthetic standard on reverse-phase H P L C , figure 1. Retention times of synthetic LTB 4 and LTC 4 standards (J. Rokach Merck Frosst, Canada) were 6.84 min (range 6.7-7.0) and 13.7 min (range 13.4-14), respectively. In addition, collected eluates were dried under nitrogen, resuspended in RIA buffer, and LTB4 quantitated by RIA. Approximately 60-80 % of LTB 4 was recovered in fractions that co-eluted with synthetic standard. Results from previous experiments and results from one of the experiments above demonstrate that rat alveolar macrophages release 4-10 times more LTB 4 than LTC4 in response to an IgE and antigen stimulus.
365
ning rat alveolar macrophage release of LTC4 [3]. First, release occurs as early as two minutes after IgE and antigen challenge. Second, peak release occurs within ten to twenty minutes. Third, the mechanism of release involves the interaction of IgE immune complexes with the macrophages. Fourth, immune complexes containing as little as 2 ng of antigen will elicit LTC 4 release, and fifth, alveolar macrophages from individual rats differ markedly in the quantity of LTC4 they will release [3]. It is likely that these observations hold true for LTB 4 release as well. Oxidative metabolism of arachidonic acid via the lipoxygenase pathway results in the simultaneous appearance o f leukotriene products in supernatants o f cells capable of the synthesis and release of these substance [7]. In addition, in our current experiments, the conditions used to stimulate rat alveolar macrophages were identical to those used in previous expe-
Lr~
Discussion.
The results of this study demonstrate that rat alveolar macrophages release readily quantifiable amounts of LTB 4 in response to challenge with IgE and specific antigen. The presence of LTB 4 in supernatants was verified using a monospecific antibody and by reversephase H P L C . Our previous experiments have established several characteristics concer-
i 0
I 4
I
I 8
I
i 12
I
I 16
TIME(rain)
FIG. i. -- Reverse-phase HPLC of pooled supernatants obtained after challenging pooled aliquots of alveolar macrophages from 6-8 rats with lgE immune complexes. The arrows indicate the retention times of pure synthetic standards.
366
12e F O R U M D ' I M M U N O L O G I E
riments. In these previous studies, LTB4 was not detected because we did not assay for the presence of LTB4 either with functional assays or by HPLC. The results of animal studies recently concluded by Nadel and co-workers suggest that airway hyperreactivity is a consequence of airway inflammation in which PMNL may play a critical role [8]. Furthermore, recently reported analyses of bronchoalveolar lavage fluid from individuals with asthma reveal that PMNL and eosinophils are present in increased numbers after local airway challenge with an antigen to which the asthmatic individual is sensitive [9]. The mechanisms responsible for attracting these proinflammatory cells -- normally present as less than 1 % of all BAL cells in non-smoking healthy normal individuals -- to the airways is not yet clear. The lower respiratory tract of normal humans is coated by epithelial lining fluid that is rich in IgG [4]. In addition, IgE can be quantitated in BAL fluid from about 80 % of normal individuals [4]. Therefore, one plausible hypothesis proposes that antigen reaching the lower respiratory tract forms immune complexes with IgE or IgG present in epithelial lining fluid and these com-
plexes in turn activate macrophages via their respective receptors to release LTB4, which is a potent chemotactic agent for PMNL and eosinophils. Indeed, human alveolar macrophages from normal individuals are capable of producing as much as 400 ng of LTB4 per million cells after challenge with calcium ionophore A23187 [10]. Most recently, we have shown that normal human alveolar macrophages under appropriate conditions will also release LTB4 in response to an IgG stimulus [11]. Currently, we are evaluating the possibility that normal human alveolar macrophages also will release LTB4 in response to IgE stimuli. In summary, evidence continues to mount that the alveolar macrophage may play a crucial role in IgEmediated reactions in the lung. The observation that IgE complexes elicit the rapid release of significant quantities of LTB4 from rat alveolar macrophages suggests that human asthmatic alveolar macrophages may also respond in this manner. Studies of macrophages taken from the lower respiratory tract of normals and individuals with asthma hopefully will reveal new insights into the immunopathogenesis of asthma.
References. [1] CAPRON, A., DESSAINT, J.-P., ROUSSEAU, R., CAPRON, M. & BAZIN, H., Interaction between IgE complexes and macrophages in the rat: a new mechanism of macrophage activation. Europ. J. Immunol., 1977, 7, 315-322. [2] BACH, M.K. & BRASHLER,J.R., Ionophore A23187-induced production of slowreacting substance of anaphylaxis (SRS-A) by rat peritoneal cells in vitro: Evidence for production by mononuclear cells, d. Immunol., 1978, 120, 998-1005. [3] RANKIN,J.A., HITCHCOCK,M., MERRILL,W.W., HUANG, S.S., BRASHLER,J.R., BACH, M.K. & ASKENASE,P.W., IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages. J. Immunol., 1984, 132, 1993-1999. [4] RANKIN, J.A. & ASKENASE,P.W., The potential role of alveolar macrophages as a source of pathogenic mediators in allergic asthma, in <
IgE R E C E P T O R S
367
[6] ROKACH, J., HAYES, E.C., GIRARD, Y., LOMBARDO, D.L., MAYCOCK, A.L.,
[7] [8]
[9] [10]
[11]
ROSENTHAL,A.S., YOUNG,R.N., ZAMBONI,R. • ZWEERINK,H.J., The development of sensitive and specific radioimmunoassays for leukotrienes. PG Leuk. Med., 1984, 13, 21-25. LEwis, R.A. & AUSTEN, K.F., Mediation of local homeostasis and inflammation by leukotrienes and other mast-cell-dependent compounds. Nature (Lond.), 1981, 292, 103-108. NADEL, J.A. & HOLTZMAN, M.J., Regulation of airway responsiveness and secretion: role of inflammation, in ~
Supported by the Parker B. Francis Foundation and a Research Associate Grant from the Veterans Administration.
Fc~R-POSITIVE C E L L S A N D M O N I T O R I N G OF INFLAMMATORY REACTION by A . B . T o n n e l
Centre d'Immunologie et de Biologie Parasitaire, Unit~ Mixte I N S E R M 167-CNRS 624, Institut Pasteur, Lille 59019 (France)
Besides mast cells and basophils, receptors for the Fc fragment of immunoglobulin E have been successively demonstrated on different cell populations: mononuclear phagocytes, lymphocytes, eosinophils and more recently platelets [3]. One of the main consequences of these findings was to indicate that, following IgE-dependent stimulation, cells bearing FcE receptors participated in immune defence against metazoan parasites. They also have a particular significance in the context of human allergic diseases: Fc~R-positive cells could indeed be activated by antiIgE or allergen triggering and play a role in the initiation and the modulation of
the pro-inflammatory response in various hypersensitivity diseases such as allergic asthma or systemic anaphylactic reactions of hymenoptera venom hypersensitivity. Antiallergic drugs also appeared to be able to interfere with the inflammatory reaction by acting not only on mast cells but also on the other cells expressing the Fc~ receptor. I. - - Alveolar macrophage and allergic asthma.
With normal alveolar macrophages (AM) obtained by bronchoalveolar lavage, the successive incubation either