In the course of an ultrastructural cytochemical study of intracellular sulphated proteoglycans involving the addition of cetylpyridinium chloride in the primary aldehyde fixative, a remarkable ultrastructural preservation of the collagen-associated sulphated proteoglycans was observed. Together with the preservation of their localization among the collagen fibrils (with, for some of them, a 50 nm periodic association with d-bands) and of their native elongated shape, previously observed under similar technical conditions, these stick-shaped and chondroitinase ABC-sensitive proteoglycans exhibited a typical pattern with several dense longitudinal parallel tracks (periodicity: 3-4 nm) not described as yet. Readily observable without high iron diamine-staining, the morphology of these cetylpyridinium chloride-precipitated and collagen-associated polyanions was particularly enhanced after incubation in the diamine solution which ascertained their sulphate content. Such a common ultrastructural organization with parallel tracks for both intracellular (i.e., in eosinophilic polymorphonuclear cells and Kurloff cells) and extracellular CPC-precipitated sulphated proteoglycans could correspond to intrinsic properties of the complexed molecules and could be related to 'double track' proteoglycans observed under other technical conditions in basement membranes.

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Large populations of splenic Kurloff (150 - 200 X 10(6) Kurloff cells) were obtained from estrogenized guinea pigs by isopycnic centrifugation in a Percoll solution of 1.085 g/ml starting density. The Kurloff cells settled at a buoyant density of about 1.100 g/ml. The purity of these cell suspensions reached 95%, as assessed by phase contrast microscopy and by specific staining. The viability assessed by Trypan blue exclusion test was also about 95%. Moreover, the good transmission electron microscopic appearance of these Kurloff cells and their ability to take up 35S-methionine in culture confirmed their physiological integrity. By autohistoradiography, this protein labeling was localized between the nucleus and the Kurloff body, and also on the Kurloff body itself. This data reinforces the hypothesis of de novo synthesis of the Kurloff body.

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.