Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

Abstract

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO(4) and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.

Fig. 1

Araldite sections of ear mast cells of (a) wild-type (C57BL/6JxC3HeB/FeJ) and (b) bm/bm (C57BL/6JxC3HeB/FeJ bm/bm) mice. Tissues were fixed with aldehydes and OsO4, and sections were post- labeled with avidin gold (AvG) and counterstained with lead citrate. Note the differences in granules size and labeling densities between control and bm/bm mast cells (arrows granules labeled predominantly in the periphery). Gold particle density within all ear mast cell granule profiles was 334±148 particles/μm2 for wild-type mice versus 180±78 particles/μm2 for bm/bm mice; P<0.001. Some AvG binding to nuclear heterochromatin was seen. For wild-type mice, nuclear chromatin gold particle density (arrowheads left) was 0.9±0.4 particles/μm2 and extracellular matrix (arrowheads top) density was 0.4±0.3 particles/μm2. Bar 1 μm

Fig. 2

Araldite sections of tongue mast cells of wild-type mice. Tissues were fixed with aldehydes and OsO4, sectioned, post-labeled with AvG, and counterstained with lead citrate. Note the differences in labeling densities between controls (a) and heparinase-Itreated mast cells (b). Note the granules labeled predominantly in the periphery (arrows). Bar 1 μm

Fig. 3

Densitometry histograms showing the characteristics of all mast cell granules as measured by optical density (a) or by AvG (b). Each graph represents data summed from all granules in all cell profiles analyzed (derived from a total of 22 wild-type and 17 bm/bm mast cells). Granules of control wild-type mice (continuous line) are darker than those of the bm/bm mutant mice (dashed line); P<0.001 (Kolmogorov-Smirnov test). The frequency histogram shows that granule anionic site density (b), as measured by AvG particles, is also higher in the mast cells of control wild-type mice than in the bm/bm mice; P<0.001 (Kolmogorov-Smirnov test)

Fig. 4

Densitometry characteristics of control wild-type (a-c) and bm/bm (d-f) mast cell granules. Each graph represents data summed from all granules within a single mast cell profile (arrowheads peaks of the gold particles located at about the same density in all six examined cells). The granule “content density” mode (arrows), which is a measure of the density excluding the gold particles, is roughly the same for the three wild-type mast cells but is lower for the three bm/bm mast cells

Fig. 5

Histograms of moving-bin analyses demonstrating multimodal frequency of mast granule equivalent volumes derived from control wild-type (a) and bm/bm mutant mouse (b) mast cells. The values for granule modes (indicating the calculated volumes of granules representing the unit granules (V1) and integral multimers of the unit granules from V2 to V10, are indicated by arrowheads (mast cell unit granule volume for wild-type mice = 0.0116 μm3 and for bm/bm mice = 0.0094 μm3; P<0.05, Student's t-test, two-tailed). The granule profile area histograms for each cell type are presented in the insets

Fig. 6

Scattergram analysis of the relationship between AvG particle density and mean granule profile equivalent volume. The locations of the arrows, indicating the calculated volumes of granules representing the unit granules (V1) and integral multimers of the unit granules from V2 to V7, are based on our calculation of the unit granule volume, as estimated in Fig. 5. The mean data, based on Fig. 3b, are indicated by horizontal solid lines