2. Cellular Mechanisms Cellular Activation and Releasability in Asthma and in Chronic Obstructive Pulmonary Disease 1 , 2 TAK H. LEE
Introduction
Increased basophil releasability is a feature of asthma, and this refers to the increased capacity of this cell type to release histamine, both spontaneously and in response to nonspecific agents. The enhanced basophil releasability was shown to correlate with nonspecific bronchial hyperresponsiveness. Wenow know that in bronchial asthma, increased releasability is not restricted to the basophil and is not limited to histamine release. For instance, in a recent study investigating leukocyte activation and releasability in experimentially provoked asthma, Arm and coworkers (1) demonstrated that circulating peripheral blood neutrophils have an augmented capacity to produce LTB4 when stimulated by unopsonized zymosan or the calcium ionophore (A23187). The generation of LTB4 by peripheral blood neutrophils (PMN) isolated before and for as long as 6 h after exercise-induced asthma (EIA) was analyzed. At 3 and at 6 h after the development of EIA, PMN isolated from 10asthmatic subjects and stimulated in vitro by 2 x 108 and 4 x 108 zymosan particles per 2 x 106 PMN demonstrated a 12-and 4-fold enhancement, respectively, in the production of immunoreactive LTB4 as compared with PMN isolated before exercise. At 6 h after EIA, there was redistribution of generated LTB4 such that 30to 40070 of LTB4 produced by zymosan-activated PMN was released extracellularly compared with 10% before exercise. There was no significant enhancement in the generation of LTB4 by unstimulated PMN at any time point after exercise. Resolution by reverse-phase high performance liquid chromatography (RP-HPLC) of products from 3H arachidonic-labeled and zymosan-activated PMN demonstrated that, in addition to LTB4 , there was enhanced metabolism of 6-transLTB4 , omega oxidation products of LTB4 and 5-HETE. Stimulation of PMN with 10 JlM ionophore revealed a 2-, 6-, and 5-fold enhancement in the production of LTB4 , 6-transLTB4 , and 5-HETE, respectively, at 6 h after EIA, as measured by integrated ultraviolet absorbance after RP-HPLC. There was no significant enhancement in LTB4 generation by PMN in six asthmatic subjects after methacholine-induced bronchospasm, and after exercise in six subjects who did not develop asthma. The augmentation of PMN LTB4 generation in EIA correlated with the extent of the early decrease in specific conductance. These results suggested that the events associated with the acute bronchoconstriction 1162
in EIA increases the proinflammatory potential of PMN by priming them for enhanced LTB4 generation. The only previous comparison of leukocyte 5-lipoxygenase pathway activity between asthmatics and normal control subjects has shown that mixed leukocyte suspensions obtained from the asthmatic subjects at rest and stimulated by ionophore demonstrated an enhanced generation of slow-reacting substance of anaphylaxis, as measured by bioassay (2). Recent Studies on Monocyte and Macrophage Interactions
With this scenario, it becomes critical to understand the mechanisms that lead to priming and augmented proinflammatory potential of leukocytes in bronchial asthma. Our studies were prompted by several observations. Supernatants from activated peripheral blood mononuclear cells (PBMC) modulate arachidonic acid metabolism by the cyclooxygenase pathway in macrophages and fibroblasts and activate granulocytes for certain cytotoxic functions (3-10). We have shown that PBMC-derived supernatants prime human blood eosinophils and neutrophils for enhanced abilities to produce immunoreactive LTC4 and LTB4 after activation of the cells by the calcium ionophore (11). Maximal enhancement ranged from 20 to 4,500% for LTC4 generation by eosinophils (geometric mean, 87%) and from 30to 1,600% for LTB4 generation by neutrophils (geometric mean, 105%). The enhancing activity was produced by immunoglobulin-negative, leu-1 negative, adherent PBMC, which were presumed to be monocytes. The activity of the monocytederived enhancing material on each type of granulocyte was relatively heat-stable. It was associated with an acidic group of monocytederived molecules having isoelectric points of 4.2 to 4.3, 4.5 to 4.6, and 4.9. A major action of the monokines was permissive for the activation or catalytic expression of fatty acid hydrolases. In addition to augmenting the generation of 5-lipoxygenasepathway metabolites from granulocytes, lipopolysaccharide-stimulated monocyte supernatants also were capable of augmenting the antibodydependent cytotoxic killing of Schistosoma mansoni larvae by human eosinophils (12). At low antibody concentrations, control medium-treated eosinophils only adhered in small numbers to the larvae and killed 2 to 10% of the schistosomula. In contrast, eosinophils treated with condition media adhered in large numbers to schistosomula and
demonstrated high helminthotoxicity. Biochemical analysis of the enhancing activity for lipoxygenase pathway product generation and for helminthotoxicity suggested that they were carried by a unique molecule, or by a unique set of molecules, whose biochemical and functional properties weredifferent from those of previously described monokines such as interleukin-1 (IL-1), interferon-a and -P, colony-stimulating factor (CSF), and tumor necrosis factor (TNF). The enhancing activity had similar properties to a molecule described by Veith and Butterworth (10)termed eosinophil-activating factor (EAF). Most recently, the molecule has been purified to substantial homogeneity, and the partial NH r terminal amino acid sequence has been reported (13). The regulatory effects exerted by monokines on the 5-lipoxygenasepathway in human granulocytes and in eosinophil cytotoxicity support the view that positive interactions between monocytes and granulocytes may occur in pathologic processes and may contribute to the development of the inflammatory reaction in the airwaysin asthma. This hypothesis was supported by several pieces of work. Pennington and colleagues (14)demonstrated that alveolar macrophages placed in monolayer tissue cultures released a substance that could enhance the bactericidal capacity of human neutrophils. Neutrophils preexposed to supernatants killed Pseudomonas aeruginosa from 70 to 90% more efficiently than did control cells. The activity was present in macrophage supernatants within 4 h of incubation, and in vitro stimulation of the macrophage cultures with heatinactivated bacteria further increased its production. Physicochemical isolation demonstrated that the activity resided with a 5,OOO-D glycoprotein with an isoelectricpoint of 7.6. The secretion of the 5,OOO-D glycoprotein wasstimulated by the presenceof heatinactivated bacteria in the macrophage cultures, and culturing the macrophages in suspension did not lead to the release of this material, indicating that adherence was an important stimulus for its secretion. The macrophages obtained from patients with chronic obstructive bronchitis who were smokers did 1 From the Department of Allergy and Allied Respiratory Disorders, Guy's Hospital, London, United Kingdom. 2 Correspondence and requests for reprints should be addressed to Professor T. H. Lee, Department of Allergy and Allied Respiratory Disorders, 4th Floor, Hunt's House, Guy's Hospital, London SEl 9RT, UK.
AM REV RESPIR DIS 1991; 143:1162-1164
CELLULAR ACTIVATION AND RELEASABILITY IN ASTHMA AND COPD
not affect the secretion of this material. Thus, we reasoned that monocyte- and macrophagederived products could playa role in priming human inflammatory granulocytes for augmented proinflammatory functions (15). This view is further supported by the findings that certain cytokines can prime eosinophils for augmented pro inflammatory functions, enhanced survivalin culture, and to change from a normodense to a hypodense phenotype (16, 17). Immunology of Airways in Asthma and Chronic Bronchitis The immunology of the airways in asthma is probably different from that in chronic bronchitis. For instance, the numbers of eosinophils and IgE, but not IgG, IgA, or IgM concentrations, in the sputum of asthmatic subjects were substantially greater than those in patients with chronic obstructive bronchitis caused by smoking (18). Thus, if macrophages in asthma had a role to play in priming inflammatory granulocytes, they should provide molecules that could modulate eosinophil function.
Hypothesis We reasoned that if our hypothesis had any credence, it was necessary to demonstrate at least two things. These included evidence for monocyte and macrophage activation in asthma and, in addition, it was necessary to demonstrate the release of molecules from lung macrophages derived from asthmatic subjects that could influence eosinophil function. Evidence for Monocyte and Macrophage Activation in Asthma There is substantial evidence for monocyte and macrophage activation in asthma. This evidence includes the enhanced expression of FCsnR2 and complement receptors in peripheral blood monocytes of asthmatic patients and the increased expression of complement receptors, as measured by rosetting technique, in PBMC of asthmatic patients after allergen bronchoprovocation (19-21). The alveolar macrophages of patients with asthma demonstrate increased capacity to produce eicosanoid mediators and superoxide anion (5, 22). Furthermore, these cells bear CD23 on their surface and can be stimulated to release mediators for IgE-dependent events (5, 23, 24). Analysis of bronchoalveolar lavage fluid of patients with asthma after antigen challenge revealed increased amounts of p-glucuronidase, whereas macrophage intracellular levels were decreased (25,26). This suggests that macrophage secretory processes were activated by allergen. Finally, Metzger and coworkers (26) have shown that the numbers of monocytes in the airways increase at 48 h after antigen provocation in asthmatic patients.
1163
Effects of Lung Macrophage-derived Product(s) on Eosinophil Function Eosinophils incubated with supernatants derived from lung macrophages obtained from asthmatic patients by bronchoalveolar lavage, followed by stimulation with ionophore, resulted in enhancement of the capacity of eosinophils to secrete LTC4 (mean enhancement, 169070) (27). Macrophage supernatants derived from normal subjects had no enhancing effect when compared with culture medium. Partial purification of the enhancing activity by HPLC using TSK-G 3,000 SW column revealed that it had a molecular size of approximately 30,000D eluted in two consistent peaks at 0.17and 0.20 M salt after anion exchange HPLC using TSK-DE AE 5PW column (pH, 7.4). Activitiesweredistinct from IL-l and TNF. The major activity eluting at 0.2 M NaCI was further resolved by reversephase HPLC using a C18 spherisorb ODS column and a slope gradient of zero to 100070 acetonitrile/OJ 070 trifluoroacetic acid. This demonstrated a single peak activity, which eluted at 41070 acetonitrile. The activity could be inhibited by trypsin digestion and heat, but it was neutralized by incubation with specific antibodies to human granulocytemacrophage CSF (GM-CSF). This indicates that the major active component is identical or closely related to GM-CSF. This was supported by the observation that pretreatment of eosinophils with the recombinant GM-CSF primed the cells for enhanced LTC4 generation after stimulation with ionophore. GM-CSF is an acidicglycoprotein with a pi of 4.5 and a molecular weight of 22,000 D (28, 29). It elutes from sizeexclusion columns with an apparent molecular weight between 15,000and 40,000 D because of variations in its glycosylation. It stimulates the proliferation and differentiation of normal granulocytes and monocytic stem cells. It induces histamine release from basophils (30) and enhances eosinophil survival in culture (16, 17, 31). It primes eosinophils for augmented proinflammatory functions and changes the cellsinto hypodense phenotype (32).The presence of GM-CSF in the lung may precondition the eosinophils for enhanced inflammatory functions upon subsequent stimulation and, either alone or in concert with other cytokines, leads to eosinophil colony formation from bone marrow progenitors. This cytokine may play an important role in the amplification of the eosinophilic inflammation that is characteristic of asthmatic airways. References 1. Arm JP, Horton CE, House F, Clark TJH, Spur BW, Lee TH. Enhanced generation of leukotriene B4 by neutrophils stimulated by unopsonized zymosan and by calcium ionophore after exercise-induced asthma. Am Rev Respir Dis 1988; 138:47-53. 2. Wang SR, Yang CM, Wang SSM, Han SH, Chiang BN. Enhancement of A23187-induced production of the slow-reacting substance on peripheralleukocytes from subjects with asthma. J Allergy Clin Immunol 1986; 77:465-71. 3. Dessein AJ, Lenzi HL, David JR. Modulation of the cytotoxicity of human blood eosinophils by
factors secreted by monocytes and Tdymphocytes. Monogr Allergy 1983; 18:45-51. 4. Dore-Duffy P, Perry W, and Kuo HH. Interferon-mediated inhibition of prostaglandin synthesis in human mononuclear leukocytes. Cell Immunol 1983; 79:232-9. 5. Joseph M, Tonnel AB, Torpier G, et af. Involvement of immunoglobulin E in the secretory processes of alveolar macrophages from asthmatic patients. J Clin Invest 1983; 71: 221-30. 6. Kurland Jl, Bockman RS, Boxmeyer HE, et al. Limitation of excessivemyelopoiesis by the intrinsic modulation of macrophage-derived prostaglandin E. Science 1978; 199:552-5. 7. Kurland JI, Plus LM, Ralph P, et al. Induction of prostaglandin E synthesis in normal and neoplastic macrophages: role for colony-stimulating factor(s) distinct from effects on myeloid progenitor cell proliferation. Proc Nat! Acad Sci USA 1984;76:2326-30. 8. Dessein AJ, Lenzi HL, Bina JC, et al. Modulation of eosinophil cytotoxicity by blood mononuclear cellsfrom healthy subjects and patients with chronic Schistosomiasis mansoni. Cell Immunol 1984; 85:100-13. 9. Vadas MA, Nicola N, Lopez AF, et al. Mononuclear cell-mediated enhancement of granulocyte function in man. J Immunol 1984; 133: 202-7. 10. Veith MC, Butterworth AE. Enhancement of human eosinophil mediated killing of Schistosoma mansoni larvae by mononuclear cell products in vitro. J Exp Med 1983; 57:1828-43. 11. Dessein AJ, Lee TH, Elsas P, et al. Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated with calcium ionophore A23187. J Immunol1986; 136: 3829-38. 12. Elsas P, Lee TH, Lenzi HL, et al. Monocytes activate eosinophils for enhanced helminthotoxicity and increased generation of leukotriene C 4 • Arch Inst Pasteur Immunol 1987; 138: 97-116. 13. Silberstein DS, Mohammed H Ali, Baker SL, David JR. Human eosinophil cytotoxicityenhancing factor: purification, physical characteristics and partial amino acid sequence of an active polypeptide. J Immunol 1989; 143:979-83. 14. Pennington JE, Rossing TH, Boerth LW, et al. Isolation and partial characterization of a human alveolar macrophage-derived neutrophil activating factor. J Clin Invest 1985; 75:1230-7. 15. Lee TH. Interactions between alveolar macrophages, monocytes and granulocytes: implications for airway inflammation. Am Rev Respir Dis 1987; 135 (Suppl:14-7). 16. Dahinden CA, Zingg J, Maly FE, de Weck AJ. Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony stimulating factor and stimulated with the complement component C5a and FMLP as second signals. J Exp Med 1988; 167:1281-95. 17. Owen WF, Rothenburg ME, Silberstein DR, et al. Regulation of human eosinophil viability,density and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts. J Exp Med 1987; 166:129-41. 18. Kay AB. The sputum in bronchial asthma. In: Clark TJH, Godfrey S, eds. Asthma. 2nd ed. London: Chapman & Hall Medical, 1983; 99-110. 19. Melewicz FM, Zeiger RS, Mellon MH, et al. Increased peripheral blood monocytes with Fe receptors for IgE in patients with severe allergic disorders. J Immunol 1981; 126:1592-5. 20. Anderson CL, Spiegelberg HL. Macrophage receptors for IgE: binding of IgE to specific 19B receptors on a human macrophage cell line U937. J Immunol 1981; 126:2470-3. 21. Carroll MP, Durham SR, Walsh G, et al. Ac-
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tivation of neutrophils and monocytes after allergen- and histamine-induced bronchoconstriction. J Allergy Clin Immunol 1985; 75:290-6. 22. Cluzel M, Damon M, Chanez P, et at. Enhanced alveolar cellluminil-dependent chemiluminescence in asthma. J Allergy Clin Immunol1987; 80:195-201. 23. Rankin JA, Hitchcock M, Merrill WW, et at. IgE-dependent release of leukotriene C4 from alveolar macrophages. Nature 1982; 297:329-31. 24. Murray JJ, Tonnel AB, Brash AR, et at. Release of prostaglandin D 2 into human airways during acute allergen challenge. N Engl J Med 1986; 315:800-4. 25. Tonnel AB, Gosset PH, Joseph M, et at. Stimulation of alveolar macrophages in asthmatic patients after local provocation test. Lancet 1983; 1:1406-8. 26. Metzger WJ, Zavala D, Richerson HB, et al. Local allergen challenge and bronchoalveolar lavage of allergic asthmatic lungs. Am Rev Respir Dis 1987; 135:433-40. 27. Howell CJ, Pujol JL, Crea AEG, et al. Identification of an alveolarmacrophage derivedactivity in bronchial asthma which enhances leukotriene C 4 generation by human eosinophils stimulated by ionophore (A23187) as granulocyte-macrophage colony-stimulating factor (GM-CSF). Am Rev Respir Dis 1989; 140:1340-7. 28. Clark SC, Kamen R. The human hematopoietic colony-stimulating factors. Science 1987; 236:1229-37. 29. Nicola NA, Metcalf M, Johnson GR, et at. Separation of functionally distinct human granulocyte-macrophagecolony-stimulating factor. Blood 1979; 54:614-27. 30. Haak-Frendscho M, Arai N, Arai K-I,'et al.
TAK H. LEE
Human recombinant granulocyte-macrophage colony stimulating factor and interleukin-3 cause basophil histamine release. J Clin Invest 1988; 82:17-9. 31. Lopez AF, Williamson DJ, Gamble JR, et al. Recombi~ant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression and survival. J Clin Invest 1986; 78:1220-8. 32. Di Persio JF, Billing P, Williams R, Gasson JC. Human granulocyte-macrophage colony-stimulating factor and other cytokinesprime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis. J Immunol 1988; 140: 4315-22.
Comments Dr. Holgate: What is known of the intracellular physiologic and biochemical mechanisms of leukocyte "priming" by cytokines such as GM-CSF, IL-3, and IL-8? Dr. Lee: Most of the information on leukocyte priming by GM-CSF has been from studies on the neutrophil. In this cell type, GM-CSF appears to increasearachidonic acid release, which may be mediated by G-proteinrelated mechanisms. Dr. Koeter: Did you observe subjects with asthma who had primed cells after allergen
exposure or after an upper respiratory tract infection? Dr. Lee: We have not studied upper respiratory tract infections, but after both exerciseand antigen-induced bronchoconstriction there is evidence for cellular priming.
Dr. Schleimer: Does eosinophil priming with GM-CSF allow the cells to release LTC4 in response to C5a, as occurs with IL-3 in the basophil? Dr. Lee: We have not studied activation of eosinophils with C5a. Dr. Laitinen: What would be the defense mechanisms in vivo in asthmatics against the production of GM-CSF from macrophages? Dr. Lee: The mechanism(s) for the regulation of GM-CSF production is critically important and is under study at present. Dr. Pare: Are the macrophages that produce GM-CSF from the alveoli or from the airways? Dr. Lee: Wehave no evidence that "alveolar" and "airway" macrophages obtained from BAL have different capacities to produce GM-CSF. Dr. Pare: What stimulus is causing the macrophages to express GM-CSF in asthma? Dr. Lee: This is a critical issue for which we have no data. Studies on the regulation of cytokine generation are in progress.
Introduction
Increased basophil releasability is a feature of asthma, and this refers to the increased capacity of this cell type to release histamine, both spontaneously and in response to nonspecific agents. The enhanced basophil releasability was shown to correlate with nonspecific bronchial hyperresponsiveness. Wenow know that in bronchial asthma, increased releasability is not restricted to the basophil and is not limited to histamine release. For instance, in a recent study investigating leukocyte activation and releasability in experimentially provoked asthma, Arm and coworkers (1) demonstrated that circulating peripheral blood neutrophils have an augmented capacity to produce LTB4 when stimulated by unopsonized zymosan or the calcium ionophore (A23187). The generation of LTB4 by peripheral blood neutrophils (PMN) isolated before and for as long as 6 h after exercise-induced asthma (EIA) was analyzed. At 3 and at 6 h after the development of EIA, PMN isolated from 10asthmatic subjects and stimulated in vitro by 2 x 108 and 4 x 108 zymosan particles per 2 x 106 PMN demonstrated a 12-and 4-fold enhancement, respectively, in the production of immunoreactive LTB4 as compared with PMN isolated before exercise. At 6 h after EIA, there was redistribution of generated LTB4 such that 30to 40070 of LTB4 produced by zymosan-activated PMN was released extracellularly compared with 10% before exercise. There was no significant enhancement in the generation of LTB4 by unstimulated PMN at any time point after exercise. Resolution by reverse-phase high performance liquid chromatography (RP-HPLC) of products from 3H arachidonic-labeled and zymosan-activated PMN demonstrated that, in addition to LTB4 , there was enhanced metabolism of 6-transLTB4 , omega oxidation products of LTB4 and 5-HETE. Stimulation of PMN with 10 JlM ionophore revealed a 2-, 6-, and 5-fold enhancement in the production of LTB4 , 6-transLTB4 , and 5-HETE, respectively, at 6 h after EIA, as measured by integrated ultraviolet absorbance after RP-HPLC. There was no significant enhancement in LTB4 generation by PMN in six asthmatic subjects after methacholine-induced bronchospasm, and after exercise in six subjects who did not develop asthma. The augmentation of PMN LTB4 generation in EIA correlated with the extent of the early decrease in specific conductance. These results suggested that the events associated with the acute bronchoconstriction 1162
in EIA increases the proinflammatory potential of PMN by priming them for enhanced LTB4 generation. The only previous comparison of leukocyte 5-lipoxygenase pathway activity between asthmatics and normal control subjects has shown that mixed leukocyte suspensions obtained from the asthmatic subjects at rest and stimulated by ionophore demonstrated an enhanced generation of slow-reacting substance of anaphylaxis, as measured by bioassay (2). Recent Studies on Monocyte and Macrophage Interactions
With this scenario, it becomes critical to understand the mechanisms that lead to priming and augmented proinflammatory potential of leukocytes in bronchial asthma. Our studies were prompted by several observations. Supernatants from activated peripheral blood mononuclear cells (PBMC) modulate arachidonic acid metabolism by the cyclooxygenase pathway in macrophages and fibroblasts and activate granulocytes for certain cytotoxic functions (3-10). We have shown that PBMC-derived supernatants prime human blood eosinophils and neutrophils for enhanced abilities to produce immunoreactive LTC4 and LTB4 after activation of the cells by the calcium ionophore (11). Maximal enhancement ranged from 20 to 4,500% for LTC4 generation by eosinophils (geometric mean, 87%) and from 30to 1,600% for LTB4 generation by neutrophils (geometric mean, 105%). The enhancing activity was produced by immunoglobulin-negative, leu-1 negative, adherent PBMC, which were presumed to be monocytes. The activity of the monocytederived enhancing material on each type of granulocyte was relatively heat-stable. It was associated with an acidic group of monocytederived molecules having isoelectric points of 4.2 to 4.3, 4.5 to 4.6, and 4.9. A major action of the monokines was permissive for the activation or catalytic expression of fatty acid hydrolases. In addition to augmenting the generation of 5-lipoxygenasepathway metabolites from granulocytes, lipopolysaccharide-stimulated monocyte supernatants also were capable of augmenting the antibodydependent cytotoxic killing of Schistosoma mansoni larvae by human eosinophils (12). At low antibody concentrations, control medium-treated eosinophils only adhered in small numbers to the larvae and killed 2 to 10% of the schistosomula. In contrast, eosinophils treated with condition media adhered in large numbers to schistosomula and
demonstrated high helminthotoxicity. Biochemical analysis of the enhancing activity for lipoxygenase pathway product generation and for helminthotoxicity suggested that they were carried by a unique molecule, or by a unique set of molecules, whose biochemical and functional properties weredifferent from those of previously described monokines such as interleukin-1 (IL-1), interferon-a and -P, colony-stimulating factor (CSF), and tumor necrosis factor (TNF). The enhancing activity had similar properties to a molecule described by Veith and Butterworth (10)termed eosinophil-activating factor (EAF). Most recently, the molecule has been purified to substantial homogeneity, and the partial NH r terminal amino acid sequence has been reported (13). The regulatory effects exerted by monokines on the 5-lipoxygenasepathway in human granulocytes and in eosinophil cytotoxicity support the view that positive interactions between monocytes and granulocytes may occur in pathologic processes and may contribute to the development of the inflammatory reaction in the airwaysin asthma. This hypothesis was supported by several pieces of work. Pennington and colleagues (14)demonstrated that alveolar macrophages placed in monolayer tissue cultures released a substance that could enhance the bactericidal capacity of human neutrophils. Neutrophils preexposed to supernatants killed Pseudomonas aeruginosa from 70 to 90% more efficiently than did control cells. The activity was present in macrophage supernatants within 4 h of incubation, and in vitro stimulation of the macrophage cultures with heatinactivated bacteria further increased its production. Physicochemical isolation demonstrated that the activity resided with a 5,OOO-D glycoprotein with an isoelectricpoint of 7.6. The secretion of the 5,OOO-D glycoprotein wasstimulated by the presenceof heatinactivated bacteria in the macrophage cultures, and culturing the macrophages in suspension did not lead to the release of this material, indicating that adherence was an important stimulus for its secretion. The macrophages obtained from patients with chronic obstructive bronchitis who were smokers did 1 From the Department of Allergy and Allied Respiratory Disorders, Guy's Hospital, London, United Kingdom. 2 Correspondence and requests for reprints should be addressed to Professor T. H. Lee, Department of Allergy and Allied Respiratory Disorders, 4th Floor, Hunt's House, Guy's Hospital, London SEl 9RT, UK.
AM REV RESPIR DIS 1991; 143:1162-1164
CELLULAR ACTIVATION AND RELEASABILITY IN ASTHMA AND COPD
not affect the secretion of this material. Thus, we reasoned that monocyte- and macrophagederived products could playa role in priming human inflammatory granulocytes for augmented proinflammatory functions (15). This view is further supported by the findings that certain cytokines can prime eosinophils for augmented pro inflammatory functions, enhanced survivalin culture, and to change from a normodense to a hypodense phenotype (16, 17). Immunology of Airways in Asthma and Chronic Bronchitis The immunology of the airways in asthma is probably different from that in chronic bronchitis. For instance, the numbers of eosinophils and IgE, but not IgG, IgA, or IgM concentrations, in the sputum of asthmatic subjects were substantially greater than those in patients with chronic obstructive bronchitis caused by smoking (18). Thus, if macrophages in asthma had a role to play in priming inflammatory granulocytes, they should provide molecules that could modulate eosinophil function.
Hypothesis We reasoned that if our hypothesis had any credence, it was necessary to demonstrate at least two things. These included evidence for monocyte and macrophage activation in asthma and, in addition, it was necessary to demonstrate the release of molecules from lung macrophages derived from asthmatic subjects that could influence eosinophil function. Evidence for Monocyte and Macrophage Activation in Asthma There is substantial evidence for monocyte and macrophage activation in asthma. This evidence includes the enhanced expression of FCsnR2 and complement receptors in peripheral blood monocytes of asthmatic patients and the increased expression of complement receptors, as measured by rosetting technique, in PBMC of asthmatic patients after allergen bronchoprovocation (19-21). The alveolar macrophages of patients with asthma demonstrate increased capacity to produce eicosanoid mediators and superoxide anion (5, 22). Furthermore, these cells bear CD23 on their surface and can be stimulated to release mediators for IgE-dependent events (5, 23, 24). Analysis of bronchoalveolar lavage fluid of patients with asthma after antigen challenge revealed increased amounts of p-glucuronidase, whereas macrophage intracellular levels were decreased (25,26). This suggests that macrophage secretory processes were activated by allergen. Finally, Metzger and coworkers (26) have shown that the numbers of monocytes in the airways increase at 48 h after antigen provocation in asthmatic patients.
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Effects of Lung Macrophage-derived Product(s) on Eosinophil Function Eosinophils incubated with supernatants derived from lung macrophages obtained from asthmatic patients by bronchoalveolar lavage, followed by stimulation with ionophore, resulted in enhancement of the capacity of eosinophils to secrete LTC4 (mean enhancement, 169070) (27). Macrophage supernatants derived from normal subjects had no enhancing effect when compared with culture medium. Partial purification of the enhancing activity by HPLC using TSK-G 3,000 SW column revealed that it had a molecular size of approximately 30,000D eluted in two consistent peaks at 0.17and 0.20 M salt after anion exchange HPLC using TSK-DE AE 5PW column (pH, 7.4). Activitiesweredistinct from IL-l and TNF. The major activity eluting at 0.2 M NaCI was further resolved by reversephase HPLC using a C18 spherisorb ODS column and a slope gradient of zero to 100070 acetonitrile/OJ 070 trifluoroacetic acid. This demonstrated a single peak activity, which eluted at 41070 acetonitrile. The activity could be inhibited by trypsin digestion and heat, but it was neutralized by incubation with specific antibodies to human granulocytemacrophage CSF (GM-CSF). This indicates that the major active component is identical or closely related to GM-CSF. This was supported by the observation that pretreatment of eosinophils with the recombinant GM-CSF primed the cells for enhanced LTC4 generation after stimulation with ionophore. GM-CSF is an acidicglycoprotein with a pi of 4.5 and a molecular weight of 22,000 D (28, 29). It elutes from sizeexclusion columns with an apparent molecular weight between 15,000and 40,000 D because of variations in its glycosylation. It stimulates the proliferation and differentiation of normal granulocytes and monocytic stem cells. It induces histamine release from basophils (30) and enhances eosinophil survival in culture (16, 17, 31). It primes eosinophils for augmented proinflammatory functions and changes the cellsinto hypodense phenotype (32).The presence of GM-CSF in the lung may precondition the eosinophils for enhanced inflammatory functions upon subsequent stimulation and, either alone or in concert with other cytokines, leads to eosinophil colony formation from bone marrow progenitors. This cytokine may play an important role in the amplification of the eosinophilic inflammation that is characteristic of asthmatic airways. References 1. Arm JP, Horton CE, House F, Clark TJH, Spur BW, Lee TH. Enhanced generation of leukotriene B4 by neutrophils stimulated by unopsonized zymosan and by calcium ionophore after exercise-induced asthma. Am Rev Respir Dis 1988; 138:47-53. 2. Wang SR, Yang CM, Wang SSM, Han SH, Chiang BN. Enhancement of A23187-induced production of the slow-reacting substance on peripheralleukocytes from subjects with asthma. J Allergy Clin Immunol 1986; 77:465-71. 3. Dessein AJ, Lenzi HL, David JR. Modulation of the cytotoxicity of human blood eosinophils by
factors secreted by monocytes and Tdymphocytes. Monogr Allergy 1983; 18:45-51. 4. Dore-Duffy P, Perry W, and Kuo HH. Interferon-mediated inhibition of prostaglandin synthesis in human mononuclear leukocytes. Cell Immunol 1983; 79:232-9. 5. Joseph M, Tonnel AB, Torpier G, et af. Involvement of immunoglobulin E in the secretory processes of alveolar macrophages from asthmatic patients. J Clin Invest 1983; 71: 221-30. 6. Kurland Jl, Bockman RS, Boxmeyer HE, et al. Limitation of excessivemyelopoiesis by the intrinsic modulation of macrophage-derived prostaglandin E. Science 1978; 199:552-5. 7. Kurland JI, Plus LM, Ralph P, et al. Induction of prostaglandin E synthesis in normal and neoplastic macrophages: role for colony-stimulating factor(s) distinct from effects on myeloid progenitor cell proliferation. Proc Nat! Acad Sci USA 1984;76:2326-30. 8. Dessein AJ, Lenzi HL, Bina JC, et al. Modulation of eosinophil cytotoxicity by blood mononuclear cellsfrom healthy subjects and patients with chronic Schistosomiasis mansoni. Cell Immunol 1984; 85:100-13. 9. Vadas MA, Nicola N, Lopez AF, et al. Mononuclear cell-mediated enhancement of granulocyte function in man. J Immunol 1984; 133: 202-7. 10. Veith MC, Butterworth AE. Enhancement of human eosinophil mediated killing of Schistosoma mansoni larvae by mononuclear cell products in vitro. J Exp Med 1983; 57:1828-43. 11. Dessein AJ, Lee TH, Elsas P, et al. Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated with calcium ionophore A23187. J Immunol1986; 136: 3829-38. 12. Elsas P, Lee TH, Lenzi HL, et al. Monocytes activate eosinophils for enhanced helminthotoxicity and increased generation of leukotriene C 4 • Arch Inst Pasteur Immunol 1987; 138: 97-116. 13. Silberstein DS, Mohammed H Ali, Baker SL, David JR. Human eosinophil cytotoxicityenhancing factor: purification, physical characteristics and partial amino acid sequence of an active polypeptide. J Immunol 1989; 143:979-83. 14. Pennington JE, Rossing TH, Boerth LW, et al. Isolation and partial characterization of a human alveolar macrophage-derived neutrophil activating factor. J Clin Invest 1985; 75:1230-7. 15. Lee TH. Interactions between alveolar macrophages, monocytes and granulocytes: implications for airway inflammation. Am Rev Respir Dis 1987; 135 (Suppl:14-7). 16. Dahinden CA, Zingg J, Maly FE, de Weck AJ. Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony stimulating factor and stimulated with the complement component C5a and FMLP as second signals. J Exp Med 1988; 167:1281-95. 17. Owen WF, Rothenburg ME, Silberstein DR, et al. Regulation of human eosinophil viability,density and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts. J Exp Med 1987; 166:129-41. 18. Kay AB. The sputum in bronchial asthma. In: Clark TJH, Godfrey S, eds. Asthma. 2nd ed. London: Chapman & Hall Medical, 1983; 99-110. 19. Melewicz FM, Zeiger RS, Mellon MH, et al. Increased peripheral blood monocytes with Fe receptors for IgE in patients with severe allergic disorders. J Immunol 1981; 126:1592-5. 20. Anderson CL, Spiegelberg HL. Macrophage receptors for IgE: binding of IgE to specific 19B receptors on a human macrophage cell line U937. J Immunol 1981; 126:2470-3. 21. Carroll MP, Durham SR, Walsh G, et al. Ac-
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tivation of neutrophils and monocytes after allergen- and histamine-induced bronchoconstriction. J Allergy Clin Immunol 1985; 75:290-6. 22. Cluzel M, Damon M, Chanez P, et at. Enhanced alveolar cellluminil-dependent chemiluminescence in asthma. J Allergy Clin Immunol1987; 80:195-201. 23. Rankin JA, Hitchcock M, Merrill WW, et at. IgE-dependent release of leukotriene C4 from alveolar macrophages. Nature 1982; 297:329-31. 24. Murray JJ, Tonnel AB, Brash AR, et at. Release of prostaglandin D 2 into human airways during acute allergen challenge. N Engl J Med 1986; 315:800-4. 25. Tonnel AB, Gosset PH, Joseph M, et at. Stimulation of alveolar macrophages in asthmatic patients after local provocation test. Lancet 1983; 1:1406-8. 26. Metzger WJ, Zavala D, Richerson HB, et al. Local allergen challenge and bronchoalveolar lavage of allergic asthmatic lungs. Am Rev Respir Dis 1987; 135:433-40. 27. Howell CJ, Pujol JL, Crea AEG, et al. Identification of an alveolarmacrophage derivedactivity in bronchial asthma which enhances leukotriene C 4 generation by human eosinophils stimulated by ionophore (A23187) as granulocyte-macrophage colony-stimulating factor (GM-CSF). Am Rev Respir Dis 1989; 140:1340-7. 28. Clark SC, Kamen R. The human hematopoietic colony-stimulating factors. Science 1987; 236:1229-37. 29. Nicola NA, Metcalf M, Johnson GR, et at. Separation of functionally distinct human granulocyte-macrophagecolony-stimulating factor. Blood 1979; 54:614-27. 30. Haak-Frendscho M, Arai N, Arai K-I,'et al.
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Human recombinant granulocyte-macrophage colony stimulating factor and interleukin-3 cause basophil histamine release. J Clin Invest 1988; 82:17-9. 31. Lopez AF, Williamson DJ, Gamble JR, et al. Recombi~ant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression and survival. J Clin Invest 1986; 78:1220-8. 32. Di Persio JF, Billing P, Williams R, Gasson JC. Human granulocyte-macrophage colony-stimulating factor and other cytokinesprime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis. J Immunol 1988; 140: 4315-22.
Comments Dr. Holgate: What is known of the intracellular physiologic and biochemical mechanisms of leukocyte "priming" by cytokines such as GM-CSF, IL-3, and IL-8? Dr. Lee: Most of the information on leukocyte priming by GM-CSF has been from studies on the neutrophil. In this cell type, GM-CSF appears to increasearachidonic acid release, which may be mediated by G-proteinrelated mechanisms. Dr. Koeter: Did you observe subjects with asthma who had primed cells after allergen
exposure or after an upper respiratory tract infection? Dr. Lee: We have not studied upper respiratory tract infections, but after both exerciseand antigen-induced bronchoconstriction there is evidence for cellular priming.
Dr. Schleimer: Does eosinophil priming with GM-CSF allow the cells to release LTC4 in response to C5a, as occurs with IL-3 in the basophil? Dr. Lee: We have not studied activation of eosinophils with C5a. Dr. Laitinen: What would be the defense mechanisms in vivo in asthmatics against the production of GM-CSF from macrophages? Dr. Lee: The mechanism(s) for the regulation of GM-CSF production is critically important and is under study at present. Dr. Pare: Are the macrophages that produce GM-CSF from the alveoli or from the airways? Dr. Lee: Wehave no evidence that "alveolar" and "airway" macrophages obtained from BAL have different capacities to produce GM-CSF. Dr. Pare: What stimulus is causing the macrophages to express GM-CSF in asthma? Dr. Lee: This is a critical issue for which we have no data. Studies on the regulation of cytokine generation are in progress.
