Two types of human mast cells that have distinct neutral protease compositions.

Abstract

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...

Expression of a Functional High-Affinity IgG Receptor, FcγRI, on Human Mast Cells: Up-Regulation by IFN-γ

“…For extracellular/intracellular double staining to determine the mast cell phenotype, i.e., MC TC (tryptase/chymase-containing mast cells) and MC T (tryptase-containing mast cells) (16) and Fc␥RI expression on mast cell surfaces, cells were first incubated with FITC-conjugated human IgE (17) at 37°C for 1 h. Cells were then stained with PE-conjugated mouse antihuman Fc␥RI (clone 32.2) and PE cyanine 5-conjugated c-kit for 30 min at 4°C. After washing in PBS, cells were fixed in PBS plus 0.5% paraformaldehyde for 15 min at room temperature, followed by two washing steps in PBS.…”

The mouse mast cell–restricted tetramer‐forming tryptases mouse mast cell protease 6 and mouse mast cell protease 7 are critical mediators in inflammatory arthritis

“…Our group (1)(2)(3)(4) and many other investigators (5)(6)(7)(8)(9)(10)(11)(12)(13)(14) have obtained data implicating a prominent involvement of mast cells (MCs) and their mediators in RA and some animal models of this autoimmune disorder. On a weight basis, tetramer-forming ␤ tryptases (15)(16)(17)(18)(19) are the most abundant proteins present in the secretory granules of human MCs. Three ␤ tryptase complementary DNAs (designated hTryptase ␤1, ␤2, and ␤3) have been cloned.…”

Chymase Participates in Chronic Dermatitis by Inducing Eosinophil Infiltration

“…Chymase cleaves a variety of physiological substances, including metalloproteases (Fang et al, 1997), procollagen (Kofford et al, 1997), precursor of interleukin 1␤ (IL-1␤) (Mizutani et al, 1991b), and membrane-associated stem cell factor (Longley et al, 1997), while its precise role is not clear. Human mast cells are classified into two types, MC TC and MC T , based on their composition of serine protease (Irani et al, 1986). MC TC contains both chymase and tryptase, while MC T expresses tryptase but not chymase.…”