Tryptase, a mediator of human mast cells

Transcription

1 Tryptase, a mediator of human mast cells Lawrence B. Schwartz, MD, PhD Richmond, Virginia Tryptase, a mediator secreted by human mast cells during immediate reactions, has demonstrated effects on several pathways in vitro. This enzyme can rapidly inactivate fibrinogen and, as a complex with heparin, may prevent coagulation that may otherwise occur when plasma enters tissues at sites of immediate reactions. Tryptase may also activate prostromelysin, which in turn activates latent collagenase. When canine pulmonary smooth muscle is incubated with canine tryptase, the contractile response to histamine is increased. Tryptase, quantifiable in complex biologic fluids by immunoassay, can serve as a specific indicator of mast cell involvement in certain clinical settings. For example, after bee sting-induced anaphylaxis, tryptase levels in the blood peak at approximately 1 hour, then decline with a half-life of approximately 2 hours. Additionally, elevated tryptase levels in bronchoalveolar lavage fluid of asymptomatic, atopic persons with asthma suggest ongoing mast cell activation, which may relate to adenosine hyperresponsiveness and a persistence of bronchial hyperreactivity. Tryptase levels in bronchial lavage fluid of atopic patients with asthma rise markedly after endobronchial allergen challenge but not after an exercise challenge, suggesting a lack of mast cell involvement in the latter condition. ( J ALLERGY CLIN IMMUNOL 1990;86.'594-8.) During immediate allergic reactions, human mast cells secrete a variety of mediators, ~ including several neutral proteases. These enzymes (e.g., tryptase, chymase, carboxypeptidase, and a cathepsin G-like protein) exist abundantly and selectively in the secretory granules of mast ceils. Tryptase, a tetrameric enzyme with trypsinlike substrate specificity, is present in all mast cells, whereas substantial amounts of the other proteases appear to be present in a mast cell subpopulation. The biologic function of tryptase in vivo has not yet been demonstrated convincingly, but it appears to have an effect on several pathways in vitro. POTENTIAL EFFECTS ON COAGULATION The potential effects of human mast cell tryptase on coagulation are shown in Fig. 1. Tryptase has been shown to destroy rapidly both high molecular weight kininogen (a procoagulant factor) 2 and fibrinogen, 3 after which the fibrinogen can no longer be clotted by thrombin in vitro. Tryptase, complexed with the proteoglycan heparin, may prevent coagulation, at least on the local level, and, indeed, examination of immediate reactions in the skin reveals an absence of fibrin deposition around the sites of activated mast From the Medical College of Virginia. Reprint requests: Lawrence B. Schwartz, MD, PhD, Professor of Medicine and Microbiology and Immunology, Medical College of Virginia, Box 263, Richmond, VA / 0 / Coagulation 1. HMWK Inactivation 2. Fibrinogen ~, Inactivation 3. Plasminogen ~ No Effect 4. Hageman Factor ~ ~ ) No Effect 5. ATIII ~ No Effect HMWK, high molecular weight kininogen. ATflI, antithrombin lli. FIG, 1, Potential effects of human mast cell tryptase on coagulation. cells. Because there is an abundance of mast cells in alveolar septa in the lung, this complex may also mediate the down-regulation of fibrin deposition at this sensitive site. On the other hand, the presence of fibrin deposition that is observed in late allergic reactions may occur because the activities of tryptase and heparin are overwhelmed by the influx of other cell types, and the enzyme may be inactive by this time. POTENTIAL EFFECTS ON CONNECTIVE TISSUE METABOLISM The potential effects of human mast cell tryptase on connective tissue metabolism are shown in Fig. 2. Gruber et al. 4 have demonstrated that when active tryptase is incubated with an extract of rheumatoid

2 VOLUME 86 NUMBER 41 PART 2 Connective Tissue Metabolism Prometalloproteinase III Metalloproteinase III (Prostromelysin) ~ (Stromelysin) Procollagenase Elastin; Collagens I - V; Fibronectin MMP Ill > MMP. matrix metalloproteinase I~1. Collagenase No Effect FIG. 2. Potential effects of human mast cell tryptase on connective tissue metabolism. synovial cells that contain latent collagenase or procollagenase, collagenase activity is expressed. The mechanism appears to involve tryptase activation of another metalloproteinase (known as prostromelysin or prometalloproteinase-3) to the active form of the enzyme, which in turn activates procollagenase to collagenase. ~ Tryptase seems to have minimal capacity to activate procollagenase directly. Similarly, tryptase has no effect on other connective tissue components (elastin, various collagen types, or fibronectin). These observations are notable because mast cells are often found at sites of connective tissue damage in rheumatoid arthritis 6' 7 and in other inflammatory diseases. Consequently, whether mast cells have a direct effect on either connective tissue injury or repair, through actions of the neutral proteases, is of great interest. POTENTIAL EFFECTS ON SMOOTH MUSCLE CONTRACTILITY Fig. 3 summarizes the potential effects of mast cell tryptase on smooth muscle cell contractility. Research has shown that when canine tryptase is incubated with canine tracheal smooth muscle, it increases the response of that smooth muscle to histamine. 8 Human tryptase also has been shown to cleave the complement protein C3 in vitro, generati.~ a.aphylatoxin C3a. 9 Because this process occurs very slowly in vitro, however, the physiologic relevance is uncertain. Nonetheless, both activities may play important roles in the pathogenesis of diseases such as asthma. TRYPTASE AS A MARKER OF MAST CELL INVOLVEMENT: IMMUNOASSAY DEVELOPMENT Tryptase is a tetramer; each of its four subunits has an active site.l~ Tryptase binds very tightly to negatively charged proteoglycans such as heparin; in fact, the active tetrameric configuration is stabilized by its Tryptase, a mediator of human mast cells 595 Smooth Muscle Contractility (Asthma) 1. Dog Tryptase ~ Increased Response to Histamine 2. Human Tryptase Generation of Complement 3a FIG. 3. Potential effects of human mast cell tryptase on smooth muscle contractility. binding to negatively charged macromolecules. 11, 12 When heparin is separated from tryptase in vitro, the enzyme can be stabilized in various ways, such as at acidic ph, with the addition of glycerol, at high salt concentrations, or at 4 ~ C. If the free molecule is placed at room temperature in a physiologic buffer, plasma, or serum, however, the subunits spontaneously dissociate from one another and undergo conformational changes that markedly alter the forms of the dominant epitopes that are available on the enzyme. 13 For example, monoclonal antibodies prepared against inactive tryptase will, in general, recognize only the inactive form of the enzyme, whereas those prepared against tetrameric tryptase will recognize only the active form of the enzyme. The immunoassay 14 we are currently using to measure tryptase detects only the inactive form; the assay conditions are such that all active enzyme is converted to the inactive enzyme during the measurement. 13, 14 Both tryptase and histamine are present in secretory granules of mast cells and are therefore released simultaneously.15 The different time course with which they each appear in the circulation 16 and in skin chamber fluid 17 after allergen-mediated stimulation of tissue mast cells probably reflects their unequal rates of diffusion from tissue to fluids. Mast cells of the connective tissue phenotype have 35 pg of tryptase and almost 2 pg of histamine18; mast cells in the mucosa have approximately 10 pg of tryptase and 1.6 pg of histamine. Thus similar amounts of histamine and somewhat similar amounts of tryptase, are present in the two types of mast cells. In the basophils, the only other cell type in which even trace amounts of tryptase have been identified, the levels of the enzyme are markedly lower than levels in mast cells (100 to 1000 times lower). 19 Histamine levels, on the other hand, are somewhat comparable in basophils and mast cells. This disparity provides a selective advantage when attempting to determine the degree of mast cell activation by measuring tryptase, because the effect of basophil activation will not be a confounding factor. Therefore either serum or plasma tryptase levels can be a specific

3 596 Schwartz J, ALLERGY CLIN. IMMUNOL. OCTOBER 1990 TABLE I. Tryptase as an indication of mast :cell activation 9 Selectively concentrated in mast cells 9 Prolonged presence in circulation 9 Immunoassay TABLE II. Mast cells and asthma 9 Mast cells in bronchial and alveolar walls and lumen 9 Increased luminal mast cells 9 Elevated tryptase and histamine 9 Hyperresponsive to adenosine 9 Increased ratio of histamine release activity to histamine release inhibitory activity indicator of mast cell involvement when the diagnosis of anaphylaxis is in question and the small amounts of basophil activation that may occur ex vivo during blood clotting or blood withdrawal are inconsequential. One of our early studies on the efficacy of tryptase levels as an indicator of mast cell activation was conducted in patients who were seen in the emergency room with clinically defined anaphylaxis within 1 to 4 hours of the onset of the event. 2~ All these patients had elevated levels of tryptase, whereas a variety of in-hospital control subjects with myocardial infarction. septic shock, and other ailments had essentially undetectable or normal levels of tryptase (<5 ng / ml). Thus tryptase levels appear to be clinically useful for the precise diagnosis of mast cell-mediated reactions (Table I). On the other hand, among a group of patients with systemic mastocytosis studied at the National Institutes of Health. approximately one third to half had elevated tryptase levels at baseline during asymptomatic periods. Therefore at the current level of sensitivity the tryptase immunoassay is not a sensitive indicator of mastocytosis in an asymptomatic person. On the other hand, tryptase levels may suggest mastocytosis (as well as anaphylaxis) in patients in whom flushing and hypotension develop. A normal tryptase level 1 to 3 hours after an attack begins strongly suggests an absence of mast cell involvement. The time course of tryptase appearance in the circulation during acute systemic anaphylaxis has been documented.16 Patients were subjected to a bee sting challenge, and blood was obtained at various times during the anaphylactic response. Three patients had anaphylactic reactions during their sting challenge. In each case histamine levels peaked at approximately 5 TABLE IlL Mast cell mediators in exercise-induced asthma Level (pg/ml) Mediator Before exercise After exercise Histamine Tryptase Prostaglandin D Leukotriene C4 < 1 < 1 Data from Broide et al. 26 minutes, then rapidly declined back to normal within approximately 30 minutes. In contrast, tryptase levels tended to remain within normal limits during the first 15 to 30 minutes, then increased, peaking at approximately 1 hour. Thereafter the levels declined, with a half-life of approximately 2 hours. These observations confirm the prevailing evidence that immediate reactions to allergen involve mast cell activation. A variety of data support the involvement of mast cells in asthma (Table II). First, high numbers of mast cells normally exist in both the bronchial and alveolar walls, some of which also appear in the lumen. 21 In asthma patients who shed their bronchial epithelium, the number of luminal mast cells often is modestly increased. Furthermore, in persons with asthma whose only abnormality is bronchial hyperreactivity (with otherwise normal airway geometry), both tryptase and histamine levels in bronchoalveolar lavage fluid are elevated. 22 Baseline tryptase levels of 2 ng/ml have been found in the bronchoalveolar lavage fluid of such patients, whereas levels in either atopic patients without asthma, asthma patients who are not atopic, or normal persons ranged from 0.5 to 0.6 ng/ml. The elevated tryptase levels suggest ongoing mast cell activation, which may correspond to persistent bronchial hyperreactivity in these persons. The hyperresponsiveness of mast cells to adenosine is also highly suggestive of ongoing mast cell activation in persons with hyperreactivity, z3 The adenosine-induced bronchospasm that occurs in such patients is probably as a result of the augmented release of preformed mediators from mast cells already undergoing a low degree of activation. 24 Consistent with this interpretation is the observation that adenosine-induced bronchospasm can be predominantly blocked by antihistamines. 25 Conversely, allergen-induced asthma is not substantially blocked by the same concentrations of antihistamines used to block the adenosine-induced hyperresponsiveness. Finally, it has been suggested that the ratio of

4 VOLUME 86 Tryptase, a mediator of human mast cells 597 NUMBER 4, part 2 histamine releasing activity to histamine releaseinhibiting activity may be increased in the asthmatic airway (Grant JA, Leh-Brown MA, Alam K, et al., unpublished observations). This is another possible mechanism to consider in explaining ongoing mast cell activation in persons with bronchial hyperreactivity. In a recently completed study, Broide et ai. 26 explored mast cell involvement in exercise-induced asthma. Before and after exercise, levels of a variety of mediators derived from mast cells were examined in the bronchoalveolar lavage fluid of atopic patients with exercise-induced asthma, as shown in Table III. Levels of histamine, tryptase, and prostaglandin D2 were not increased after exercise, and levels of leukotriene CA could not be detected either before or after exercise. It seems unlikely then that mast cells play a substantial role in exercise-induced asthma, even though the forced expiratory volume at 1 second is decreased by an average of 25% after exercise. CONCLUSION The presence of tryptase in the secretory granules of all mast cells has led us to address the possible biologic functions of this enzyme. Potential effects on coagulation during immediate reactions in vitro may reflect a beneficial down-regulation of fibrin deposition by mast cells in such tissues as those in the lung. The observation that tryptase activates latent collagenase may help clarify the role of mast cells at sites of connective tissue damage in rheumatoid arthritis and other collagen-vascular diseases. Tryptase may also increase the contractile response of pulmonary smooth muscle to histamine, which could be important in understanding the relationship of bronchial hyperreactivity to mast cell activation in asthma. Finally, assessments of tryptase levels in biologic fluids should advance our understanding of mast cell involvement in a variety of physiologic and pathophysiologic processes. REFERENCES 1. Schwartz LB, Austen KF. The mast cell and mediators of immediate hypersensitivity. In: Samter M, Talmage DW, Frank MM, Claman HN, eds. hnmunological diseases. Boston: Littie, Brown & Co, 1988: Maier M, Spragg J, Schwartz LB. Inactivation of human high molecular weight kininogen by human mast cell tryptase. J Immunol 1983;130: Schwartz LB, Bradford TR, Littman BH, Wintroub BU. The fibrinogenolytic activity of purified tryptase from human lung mast cells. J Immunol 1985;135: Gruber BL, Schwartz LB, Ramamurthy NS, Irani AM, Marchese MJ. Activation of latent rheumatoid synovial collagenase by human mast cell tryptase. J Immunol 1988;140: Gruber BL, Marchese MJ, Suzuki K, et al. Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation. J Clin Invest 1989;84: Crisp AJ, Chapman CM, Kirkham SE, Schiller AL, Krane SM. Articular mastocytosis in rheumatoid arthritis. Arthritis Rheum 1984;27: Godfrey HP, Ilardi C, Engber W, Graziano FM. Quantitation of human synovial mast cells in rheumatoid arthritis and other rheumatic diseases. Arthritis Rheum 1984;27: Sekizawa K, Caughey GH, Lazarus SC, Gold WM, Nadel JA. Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs. J Clin Invest 1989;83: Schwartz LB, Kawahara MS, Hugli TE, Vik D, Fearon DT, Austen KF. Generation of C3a anaphylatoxin from human C3 by human mast cell tryptase. J Immunol 1983;130: Schwartz LB, Lewis RA, Austen KF. Tryptase from human pulmonary mast cells. Purification and characterization. J Biol Chem 1981 ;256: Alter SC, Metcalfe DD, Bradford TR, Schwartz LB. Regulation of human mast cell tryptase. Effects of enzyme concentration, ionic strength and the structure and negative charge density of polysaccharides. Biochem J 1987;248: Schwartz LB, Bradford TR. Regulation of tryptase from human lung mast cells by heparin: stabilization of the active tetramer. J Biol Chem 1986;261: Schwartz LB, Bradford TR, Lee DG, Chlebowski JF. Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer: production of monoclonal antibodies recognizing active tryptase. J Immunol 1990;144: Wenzel S, Irani AA, Sanders JM, Bradford TR, Schwartz LB. Immunoassay of tryptase from human mast cells. J Immunol Methods 1986;86: Schwartz LB, Lewis RA, Seldine D, Austen KF. Acid hydrolases and tryptase from secretory granules of dispersed human lung mast ceils. J Immunol 1981;126: Schwartz LB, Yunginger JW, Miller JS, Bokhari R, Dull D. The time course of appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis. J Clin Invest 1989;83: Schwartz LB, Atkins PC, Bradford TR, Fleekop P, Shalit M, Zweiman B. Release of tryptase together with histamine during the immediate cutaneous response to allergen. J ALLERGY CLIN IMMUNOL 1987;80: Schwartz LB, Irani AMA, Roller K, Castells C, Schechter NM. Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells. J Immunol 1987:138: Castells MC, Irani AM. Schwartz LB. Evaluation of human peripheral blood leukocytes for mast cell tryptase. J Immunol 1987;138: Schwartz LB, Metcalfe DD. Miller JS. Earl H. Sullivan T. Tryptase levels as an indicator of mast-cell activation in systemic anaphylaxis and mastocytosis. N Engl J Med 1987: 316: Craig SS, Deblois G. Schwartz LB. Mast cells in human keloid, small intestine and lung by an immunoperoxidase technique using a murine monoclonal antibody against tryptase. Am J Pathol 1986;124: Wenzel SE, Fowler AA III, Schwartz LB. Activation of pulmonary mast cells by bronchoalveolar allergen challenge. In vivo release of histamine and tryptase in atopic subjects with and without asthma. Am Rev Respir Dis 1988;137: Cushley MJ, Tattersfield AE, Holgate ST. Adenosine-induced

5 598 Schwartz J. ALLERGY CLIN. IMMUNOL. OCTOBER 1990 bronchoconstriction in asthma: antagonism by inhaled theophylline. Am Rev Respir Dis 1984;129: Marquardt DL, Gruber HE, Wasserman SI. Adenosine release from stimulated mast cells. Proc Natl Acad Sci USA 1984; 81: Phillips GD, Holgate ST. The effect of oral terfenadine alone and in combination with flurbiprofen on the bronchoconstrictor response to inhaled adenosine 5'-monophosphate in nonatopic asthma. Am Rev Respir Dis 1989;139: Broide DH, Eisman S, Ramsdell JW, Ferguson P, Schwartz LB, Wasserman SI. A bronchoalveolar lavage study of mast cell derived mediators in exercise induced asthma. Am Rev Respir Dis 1990; 141: DISCUSSION Question. Are data available on the levels of tryptase in blood or lavage fluid after antigen challenge in patients with asthma? Dr. Schwartz. Yes, the tryptase levels increase dramatically following antigen challenge. In atopic patients with asthma, the levels increase approximately fivefold. In atopic persons without asthma the levels increase approximately threefold. Thus even in this nonasthmatic subpopulation, mast cells are still responsive to allergen. Question. Does this imply that one can differentiate socalled allergic and nonallergic asthmatic persons on the basis of their tryptase levels? If so, what implications would this have in terms of the involvement of mast ceils in different forms of asthma? Dr. Schwartz. From our limited work, we would conclude that mast cells are not involved in the nonatopic asthmatic subpopulation. The baseline levels have been normal in this group, and levels do not rise in response to allergen challenge. However, nonimmunologic bronchoconstrictive stimuli have not been assessed as to mast cell involvement in this nonatopic asthmatic population. Among atopic patients with exercise-induced asthma, we have not observed mast cell involvement. In light of these findings; I believe that mast cells play an important role in atopic asthma, but their activation is not necessary for bronchoconstriction to occur. Question. Does the measurement of tryptase offer a practical method by which to make a clinical diagnosis of certain diseases? Are elevated tryptase levels seen only when patients are hypotensive, or are they also evident in other generalized, nonhypotensive reactions? Dr. Schwartz. Among normal persons 95% or more have tryptase levels less than the current level of sensitivity of the assay. At this level of sensitivity, tryptase is a good marker for moderate to severe systemic reactions. In a patient with hypotension, tryptase shou/d be elevated if mast cells are substantially involved. The precise diagnosis of systemic anaphylaxis in antemortem as well as postmortem samples thereby can be determined. A negative tryptase level in a patient with hypotension, therefore, should provide a fairly good indication that mast ceils were not involved. In a person with a mild, local reaction (such as that occurring after a pulmonary allergen challenge that decreases the forced expiratory volume at 1 second by 25% to 50%), an elevation of circulating tryptase levels is not likely to be detected at the current sensitivity of the assay. We are currently working to increase the level of sensitivity by approximately tenfold. If we or other workers are successful in accomplishing this goal, the applicability of tryptase levels to milder reactions may be extensive.