Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

Abstract

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3 M MgCl2 with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3 M MgCl2, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.

Sulfated glycosaminoglycans in guinea pig basophils studied by means of cationic colloidal gold

“…This explains the greatly reduced labelling of CCG after enzyme degradation. Our results also support those of Landemore et al (1995) and Skutelsky et al (1995) who reported that chondroitinase ABC abolished the staining of cuprolinic blue in basophils and heparinase I reduced CCG staining by 97.4% in mast cells.…”

“…Sections were incubated in either of the following enzymes: (1) 1 U/ml heparinase I (Sigma, USA) dissolved in TRIS-buffered saline, pH 7.0, containing 0.04 M calcium chloride (Goode et al 1991); or (2) 1 U/ml protease-free chondroitinase ABC (Proteus vulgaris, EC 4.2.2.4; Seikagakukogyo, Japan), dissolved in TRIS-HCl buffer, pH 8.0 (modification of Landemore et al 1995). Both incubations were carried out in a moisture chamber at 37°C overnight.…”

“…These studies did give useful information about the localization of sulfated glycoconjugates in basophils, but a more precise description about the ultrastructural arrangement of basophil granules was handicapped by the relatively low electron resolution. Landemore et al (1995) demonstrated the ultrastructure of proteoglycans in specific basophil granules of guinea pig using cuprolinic blue staining. However, there has been no study designed to examine the sites of sulfation of sulfated GAGs in basophil granules.…”