Ultrastructural localization of heparin to human mast cells of the MCTC and MCT types by labeling with antithrombin III-gold.

BACKGROUND

Mast cells derived from human skin and lung have been reported to produce heparin and chondroitin sulfate E proteoglycans. However, no information about the proteoglycans distribution among the different human mast cell types (MCTC and MCT) is available. Conjugates of antithrombin III-gold were used to assess the presence of heparin in both human mast cell subsets.

EXPERIMENTAL DESIGN

Thin sections of human and rodent tissues and dispersed cell preparations were labeled with the conjugate in the presence of saline, heparin, and chondroitin 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 essentially all granules of human mast cells in skin (predominantly MCTC type), lung alveolar wall, and bowel mucosa (predominantly MCT type), but was negligible over human eosinophils. Consistent with the known distribution of heparin in rodent mast cells, strong labeling was observed over rat peritoneal connective tissue type mast cells, but not over mucosal mast cells in bowel mucosa of Nippostrongylus brasiliensis-infected rats (which contain chondroitin sulfate di-B) nor over mouse PT-18 mast cells (which contain chondroitin sulfate E). Mast cell labeling was preferentially blocked by exogenous heparin, and virtually abolished by heparinase but not chondroitinase ABC preincubation.

CONCLUSIONS

The data with rodent mast cells indicate that antithrombin III-gold 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 after preincubation with heparinase and by competition with exogenous heparin. On this basis, we conclude that heparin is present in essentially all mast cells in normal skin, lung alveolar wall, and bowel mucosa. The presence of heparin in all human mast cells is different than for rodent mast cells, and probably accounts for the inability to clearly distinguish different human mast cell types from one another with histochemical stains based on proteoglycan content.