Two human mast cell types were identified by immunohistochemical techniques in skin, lung, and small intestine. One type contains the neutral proteases, tryptase and chymotryptic proteinase, and is termed the TC mast cell. The second type contains only tryptase and is termed the T mast cell. Both types are fixed better by Carnoy's fluid than by formalin. (4,5,(9)(10)(11).The presence of distinct chymotryptic neutral proteases in rat mucosal and connective tissue mast cells is a useful criterion for designation of a rat mast cell to a particular type (12, 13). For example, the finding of rat mucosal mast cell chymotryptic protease in a tumor cell line previously considered to be a rat basophil leukemia tumor (RBL-1) indicated that this tumor may be a rat mucosal mast cell leukemia tumor (14). In dispersed human lung mast cells, the major neutral protease is tryptase (15-17). Tryptase is a specific marker for all mast cells in human lung, small bowel, and skin (17-19), including formalin-fixable and -nonfixable mast cells. A second neutral protease, termed human skin chymotryptic proteinase, has been purified from skin (20) and localized to mast cells in skin and lung (21,22). The present study uses murine monoclonal anti-tryptase and affinity-purified rabbit polyclonal anti-skin chymotryptic proteinase antibodies in a double indirect immunoenzymatic technique to demonstrate two mast cell types in human skin, lung, and small bowel, based on distinct protease compositions. MATERIALS AND METHODSMaterials. Peroxidase-conjugated goat anti-rabbit IgG (Cooper Biomedical, Malvern, PA), alkaline phosphataseconjugated goat anti-mouse IgG (Boehringer Mannheim), peroxidase-conjugated goat anti-mouse IgG (Bio-Rad), 3,3-diaminobenzidine tetrahydrochloride (Electron Microscopy Sciences, Fort Washington, PA), 30% H202 (Fisher), and naphthol AS-MX phosphoric acid and fast blue RR (Sigma) were obtained as indicated. Murine monoclonal IgG2b,K anti-tryptase, termed G5 (anti-T), and a negative control IgG2b,K, termed MPC-11 (17), as well as polyclonal rabbit IgG anti-human skin chymotryptic proteinase, termed C8 (anti-C), and IgG from nonimmunized rabbits (21) were purified as described previously. Fresh surgical or autopsy tissues were fixed in Carnoy's fluid (60% methanol/30% chloroform/10% glacial acetic acid) or in neutral buffered formalin (10% formalin in 0.08 M sodium phosphate, pH 7.4) and 5-,gm-thick sections were prepared. Macroscopically normal appearing areas of skin, lung, and small bowel were used. In addition, mast cells were enzymatically dispersed from human lung and cytocentrifuge preparations were obtained as described (17).Single Indirect Immunohistochemistry. Fixed sections were dewaxed in xylene over three 5-min periods. Endogenous peroxidase was blocked with 0.6% H202 in methanol for 30 min at 22°C. Rehydration was performed with graded ethanol solutions (100%, 95%, 80%, 70%, 50%) and H20, each for a 3-min period. Nonspecific staining was reduced by incubation with 10% heat-inactivated normal goat seru...

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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.

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.

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