The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Development of a new, more sensitive immunoassay for human tryptase: use in systemic anaphylaxis.

Tryptase, a neutral protease, is selectively concentrated in the secretory granules of human mast cells, and its release into the circulation serves as a clinical marker of mast cell activation. The current study describes a new, more sensitive ELISA utilizing a newly developed, mouse monoclonal IgG1 antibody for capture called B12 and capable of detecting tryptase in normal plasma and serum. The greater sensitivity of the new immunoassay results in part from a greater portion of tryptase being detected. Mean levels of tryptase in serum from normal subjects from Richmond, Virginia (4.9 ng/ml; n = 56), Munich, Germany (3.8 ng/ml; n = 19), and Amersfoort, The Netherlands (1.9 ng/ml; n = 8) were as indicated. In 62 subjects with ongoing allergic rhinitis, tryptase levels were no different in serum than for 19 normal controls, indicating that local mast cell activation is not necessarily reflected in the circulation. In 61 subjects sensitive to honey bee or yellow jacket venom by history, the 17 destined to have a severe, hypotensive response to a sting challenge had higher levels of tryptase at baseline than mild reactors, nonreactors, and controls, suggesting that baseline levels of tryptase may predict the severity of the clinical response to allergen in sensitive subjects.

Localization of rat tryptase to a subset of the connective tissue type of mast cell.

We examined the cellular distribution of rat tryptase in rat skin, lung, small intestine, and peritoneal lavage cells by immunohistochemical techniques. Tryptase purified to apparent homogeneity from rat skin was used to generate a goat polyclonal anti-rat tryptase antibody. Tryptase-containing cells were detected in lung, skin, and peritoneal lavage cells. Small intestine mucosa, on the other hand, showed few if any tryptase-positive cells. Sequential staining with Alcian blue and anti-tryptase antibody showed that tryptase is located only in mast cells. Sequential staining with safranin to identify the connective tissue type of mast cell and anti-tryptase antibody showed that tryptase resides only in this mast cell type. However, only a subpopulation of the safranin-stained mast cells contained tryptase. In lung, 53% of the mast cells stained with safranin; 94% contained tryptase. In skin, 80% stained with safranin; only 6% contained tryptase. In peritoneal cells, more than 95% of the mast cells were stained with safranin; 20% contained tryptase. In the bowel mucosa, where few cells are stained by safranin, no cells with tryptase were detected. The percentages of cells with chymase I that also contained tryptase were 80% and 84% for lung, 4% and 7% for skin, and 15% and 13% for peritoneal cells by respective simultaneous and sequential double labeling with anti-tryptase and anti-chymase I antibodies. This study suggests that the rat connective tissue type of mast cell is subdivided into two forms on the basis of the presence or absence of tryptase, whereas rat mucosal mast cells lack this enzyme. These results contrast with those in humans, in which tryptase is present in all mast cells, but are similar to mice, in which tryptase mRNA has been detected only in the connective tissue type.

A new radioimmunoassay for human mast cell tryptase using monoclonal antibodies.

A solid phase immunoradiometric assay was developed for the quantitation of tryptase released from activated human mast cells. Tryptase exhibits a linear dose-response curve over the standard range of 2-50 micrograms/l in buffer, serum, and plasma. The dose-response curve approached a plateau at a tryptase concentration of 100 micrograms/l and exhibited partial inhibition at concentrations above 10,000 micrograms/l. The sensitivity of the assay was 0.2-0.4 micrograms/l, and the intra-assay and interassay coefficients of variation were below 4% at 2 micrograms/l or higher tryptase concentrations. The recovery of known amounts of purified tryptase added to serum ranged from 91 to 115%. Detection of tryptase was evaluated with several body fluids and was accurate in sera, plasma, bronchoalveolar lavage fluid, nasal lavage fluid, and saliva. The concentration of tryptase was examined in serum samples from 100 healthy controls; in each case the level was less than 2 micrograms/l. The immunoassay also was utilized to examine serum levels of tryptase after the onset of a hypotensive reaction in one patient receiving general anesthesia. A maximally elevated level of tryptase (25 micrograms/l) was detected at the first time point, 0.5 h, and elevated levels persisted to 6 h before a return to normal levels was documented at 24 h. Thus, the involvement of mast cell activation in hypotensive subjects can be ascertained by this new tryptase radioimmunoassay.

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.

The catalytic activity of human tryptase, a mast cell neutral endoprotease, is expressed when the enzyme is in its tetrameric form, but is lost under physiologic conditions concomitant with a quaternary structural alteration involving conversion to a monomeric form. The associated changes in the CD spectra noted in the current study indicate accompanying alterations in the secondary structure of the protein. In particular, the progressive disappearance of the negative minimum centered at 228 nm suggests an effect on beta-sheet structure, which may be important for monomer-monomer interaction and/or stabilization of catalytic activity. Dextran sulfate, like heparin, stabilizes the catalytic activity and quaternary structure of tryptase and also maintains the native secondary structure of the enzyme at and beyond a temperature of 40 degrees C. Dextran sulfate-stabilized tryptase therefore was used as an immunogen to which were produced three murine mAb (B2, C11, and G4) recognizing the catalytically active form of the enzyme. Inactive tryptase bound to plastic microtiter wells was not recognized by any of the newly made antibodies, whereas inactive tryptase in solution was recognized by G4, which when biotinylated, could be used as a detector antibody in a sandwich ELISA for tryptase. Each of the newly made mAb recognized the catalytically active form of tryptase. Thus, alterations in epitopes, perhaps reflecting tertiary structural alterations as well as changes in secondary and quaternary conformations, occur with tryptase inactivation. A pragmatic result of these newly generated antibodies is the affinity purification to homogeneity of active tryptase by sequential chromatography with B2 coupled to CH-Sepharose and heparin-agarose. Tryptase purified by this technique had a specific activity with p-tosyl-L-arginine methyl ester of 117 +/- 9 U/mg and had 3.9 +/- 0.3 active sites per molecule of active enzyme (134,000 m.w.) as titrated with p-nitrophenyl-p'-guanidinobenzoate. The spectral and immunologic data in the current study are consistent with concerted conformational alterations in the secondary and tertiary as well as quaternary structures of tryptase associated with loss of catalytic activity. Failure to reverse any of these alterations with dextran sulfate suggests that the pathway of tetramer assembly in vivo is more complicated than simple subunit association.

Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies.

We developed an improved immunohistochemical technique for distinguishing human mast cells of the MCT (tryptase-positive, chymase-negative) and MCTC (tryptase-positive, chymase-positive) types utilizing a biotinylated murine anti-chymase monoclonal antibody (MAb), termed B7, and an alkaline phosphatase-conjugated murine anti-tryptase MAb, termed G3. The B7 MAb also was used to show the selective presence of chymase in mast cells. The distribution of MCT and MCTC cells in Carnoy's fluid-fixed tissue sections of human lung, skin, small intestine, and tonsils was analyzed by the new technique and the results compared to those obtained with the older method using a rabbit polyclonal antichymase antibody and a mouse anti-tryptase MAb in indirect immunoperoxidase and indirect immunoalkaline phosphatase protocols, respectively. In tissues known to contain predominantly mature mast cells, there were no quantitative differences between the two techniques, although the staining intensity achieved with the anti-chymase MAb was greater and without development of high background, compared to results achieved with the polyclonal antibody. MCT cells were the predominant type seen in the alveoli of the lung (93%) and in the small intestinal mucosa (81%). MCTC cells predominanted in the skin (99%) and in the small intestinal submucosa (77%) and, to a lesser degree, in tonsils (60%). However, in newborn foreskin tissue which contains predominantly immature forms of mast cells, 75% of all mast cells were stained uniformly and intensely with B7, whereas only 43% were stained with the polyclonal anti-chymase antibody. Therefore, the use of MAb provides for better standardization of reagents and more accurate assessment of the distribution of human MCT and MCTC cells in tissues than previously available methods.

Regulation of human mast cell tryptase. Effects of enzyme concentration, ionic strength and the structure and negative charge density of polysaccharides.

Tryptase was previously shown to undergo rapid inactivation under physiological conditions unless stabilized by the presence of heparin. The current study shows that increasing the concentration of free tryptase enhances the preservation of enzymic activity, consistent with dissociation of the tetramer, rather than autodegradation, as the mechanism of inactivation. Heparin glycosaminoglycan fragments of Mr greater than 5700 are necessary for complete stabilization of tryptase activity. This stabilizing effect depends upon negative charge density rather than carbohydrate composition. Thus, keratan sulphate or hyaluronic acid were no better than physiological buffer alone; chondroitin monosulphates and heparan sulphate each prolonged the t1/2 about 20-fold over buffer alone; chondroitin sulphate E prolonged the t1/2 69-fold; and dextran sulphate and heparin provided complete stabilization of tryptase activity for 120 min. Poly-D-glutamic acid prolonged the t1/2 55-fold. In each case the loss of tryptase activity followed apparent first-order kinetics. Increasing the NaCl concentration from 0.01 M to 1.0 M increased the stability of free tryptase. In contrast, increasing the NaCl concentration in the presence of stabilizing polysaccharides decreased the stability of tryptase until dissociation of tryptase from each polysaccharide presumably occurred; thereafter tryptase stability increased as did that of free tryptase. The effect of salt concentration on heparin-stabilized tryptase activity (as opposed to stability) was also evaluated. The mast cell proteoglycans heparin and chondroitin sulphate E, by virtue of containing the naturally occurring glycosaminoglycans of highest negative charge density, may play a major role in the regulation of mast cell tryptase activity in vivo.

Release of tryptase together with histamine during the immediate cutaneous response to allergen.

Better in vivo techniques are needed for objective assessment of mast cell-dependent events. Tryptase, a neutral protease selectively concentrated in human mast cells, appears along with histamine in skin chamber fluid overlying sites of allergen challenge in sensitive human subjects. Maximal amounts of histamine were found 0 minutes to 30 minutes after challenge; maximal amounts of tryptase were found 30 minutes to 60 minutes after challenge. The later appearance of tryptase most likely reflects its slower diffusion through tissue after release of tryptase from cutaneous mast cells as a macromolecular complex with proteoglycan. The mean weight ratio of tryptase (134,000 molecular weight tetramer) to histamine (111 molecular weight) in chamber fluid after allergen challenge during a 1-hour time course was 4:1. Total amounts of tryptase and histamine recovered in the 0.3 ml chamber fluid samples after a 1-hour challenge averaged 95 ng and 26 ng, respectively. Tryptase levels in skin chamber fluid are an accurate indicator of mast cell activation.

Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer.

Tryptase was shown to be stabilized as an enzymatically active tetramer by association with heparin and dissociated to inactive monomers in the absence of heparin at 37 degrees C in physiologic buffer and in plasma. There was a 50% loss of tryptase activity at 37 degrees C by 6-8 min in both physiologic buffer and plasma. When heparin glycosaminoglycan was present, tryptase retained nearly full activity for 2 h in buffer and in plasma. Tryptase activity also decayed under standard assay conditions in the presence of synthetic ester and peptide substrates unless bound to heparin. That tryptase is bound to heparin at the pH and physiologic NaCl concentrations employed was shown by chromatography of tryptase on heparin-agarose, gel filtration, and velocity sedimentation. Elution of tryptase from heparin-agarose occurred at 0.8 M NaCl. Maximal stabilization of tryptase by heparin occurred at a weight ratio to tryptase that was equal to or greater than unity. Kcat/Km ratios for tryptase-heparin at 0.15 M NaCl and 37 degrees C were 0.9 X 10(6) s-1 M-1 for tosyl-L-Gly-Pro-Lys-p-nitroanilide and 1.7 X 10(6) s-1 M-1 for p-tosyl-L-arginine methyl ester and are among the highest reported for tryptic enzymes. The mechanism of heparin-dependent stabilization of tryptase was not due to indirect ion binding properties of heparin and was analyzed by Superose 12 high performance liquid chromatography. Active enzyme eluted with an apparent Mr of 132,000 +/- 10,000 (n = 3, +/- S.D.), whereas tryptase inactivated by incubation without heparin eluted with an apparent Mr of 34,000. The tetrameric structure of diisopropyl fluorophosphate-inhibited tryptase was also preserved after incubation with heparin at 37 degrees C but was reduced to monomeric subunits after incubation without heparin. That no appreciable degradation of tryptase occurs under conditions that cause dissociation of subunits was directly shown by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Two different subunits of 34,000 and 33,000 Mr (after reduction) present in the intact enzyme (calculated to be 134,000 Mr) were also detected unchanged after inactivation of tryptase by dissociation of its subunits. Thus, the selective localization and association of heparin and tryptase in the human mast cell secretory granule most likely plays a major role in the regulation of tryptase after secretion.

Immunoassay of tryptase from human mast cells.

A sandwich ELISA was developed for the measurement of tryptase. The assay utilizes the mouse monoclonal anti-tryptase antibody, termed G5 (IgG2b kappa) in the solid phase and monospecific goat IgG anti-tryptase antibody together with tryptase in the fluid phase. The immunoassay will quantify 0.1 ng-5.6 ng of tryptase per 100 microliters of sample solution to within 0.1 ng. Intra-assay coefficients of variation were determined at 0.3 ng, 1.0 ng and 3.0 ng of tryptase per assay, respectively, to be 19%, 7% and 4% with buffer and 10%, 4%, and 4% in the presence of 20% plasma. Inter-assay coefficients of variation at the same respective levels of tryptase were 22%, 18% and 15% with buffer and 18%, 11% and 14% with 20% plasma. Net absorbance values obtained with a standard amount of tryptase in buffer alone and up to 50% (v/v) normal human citrate-treated plasma were within 10% of one another, indicating nearly complete detection of tryptase added to plasma. This represents the first sensitive immunoassay for a preformed mediator specific for human mast cells.

The fibrinogenolytic activity of purified tryptase from human lung mast cells.

The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.