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...

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-proteincoupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca 2؉. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca 2؉ . These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.

The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL-37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL-37 were identified in normal skin. Through the use of selective inhibitors, SELDI-TOF-MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5-deficient mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.

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.

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the high molecular weight serpin superfamily of serine proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in primary structure, the reactive site loop of each inhibitor suggests that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine proteinases. The purpose of this study was to determine whether SCCA2 inhibited a different family of proteolytic enzymes. Using recombinant DNA techniques, we prepared a fusion protein of glutathione S-transferase and full-length SCCA2 . The recombinant SCCA2 was most effective against two chymotrypsin-like proteinases from inflammatory cells, but was ineffective against papain-like cysteine proteinases. Serpin-like inhibition was observed for both human neutrophil cathepsin G and human mast cell chymase. The second order rate constants for these associations were on the order of ϳ1 ؋ 10 5 M ؊1 s ؊1 and ϳ3 ؋ 10 4 M ؊1 s ؊1 for cathepsin G and mast cell chymase, respectively. Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like serine proteinases.