ULTRASTRUCTURAL-LOCALIZATION OF HEPARIN TO HUMAN MAST-CELLS OF THE MC(TC) AND MC(T) TYPES BY LABELING WITH ANTITHROMBIN III-GOLD

BACKGROUND: Mast cells derived from human skin and lung have been repo rted to produce heparin and chondroitin sulfate E proteoglycans. Howev er, no information about the proteoglycans distribution among the diff erent human mast cell types (MC(TC) and MC(T)) is available. Conjugate s of antithrombin III-gold were used to assess the presence of heparin in both human mast cell subsets. EXPERIMENTAL DESIGN: Thin sections o f human and rodent tissues and dispersed cell preparations were labele d with the conjugate in the presence of saline, heparin, and chondroit in sulfates A and E and particle densities were measured over granules , perigranular regions, and extracellular space. Control sections were preincubated with heparinase, chondroitinase ABC, or buffer. RESULTS: Labeling with antithrombin III-gold particles was detected in essenti ally all granules of human mast cells in skin (predominantly MC(TC) ty pe), lung alveolar wall, and bowel mucosa (predominantly MC(T) type), but was negligible over human eosinophils. Consistent with the known d istribution of heparin in rodent mast cells, strong labeling was obser ved over rat peritoneal connective tissue type mast cells, but not ove r mucosal mast cells in bowel mucosa of Nippostrongylus brasiliensis-i nfected rats (which contain chondroitin sulfate di-B) nor over mouse P T-18 mast cells (which contain chondroitin sulfate E). Mast cell label ing was preferentially blocked by exogenous heparin, and virtually abo lished by heparinase but not chondroitinase ABC preincubation. CONCLUS IONS: The data with rodent mast cells indicate that antithrombin III-g old labels cells that contain heparin, but not those that contain only over-sulfated chondroitin sulfates. Specificity of the procedure for detecting heparin is further demonstrated by inhibition of labeling af ter preincubation with heparinase and by competition with exogenous he parin. On this basis, we conclude that heparin is present in essential ly all mast cells in normal skin, lung alveolar wall, and bowel mucosa . The presence of heparin in all human mast cells is different than fo r rodent mast cells, and probably accounts for the inability to clearl y distinguish different human mast cell types from one another with hi stochemical stains based on proteoglycan content.