Background: Human mast cells are a rich and unique source of heparin, which is stored in cytoplasmic secretory granules and accounts for metachromasia, a staining property used to identify mast cells by light microscopy.
Objective: The sub-cellular locations of heparin in secretory human mast cells and human mast cells recovering from secretion are not known. Acquisition of this knowledge requires ultrastructural imaging of well-preserved cells with a visible probe which binds to heparin. We sought to develop this knowledge regarding human mast cell secretion by using a labelling method for heparin that depends on the well-known property of ribonuclease inhibition by heparin.
Methods: Human lung mast cells were isolated, partially purified, either stimulated or not stimulated to secrete with anti-IgE, and recovered 20 min or 6 h later for routine electron microscopy. Histamine secretion was also determined. A previously developed post-embedding, enzyme-affinity-gold electron microscopic technique to image ribonucleic acid (RNA) with ribonuclease-gold (R-G), which also binds to the enzyme inhibitor, heparin, was employed to determine the sub-cellular locations of heparin in non-secretory and secretory mast cells as well as in mast cells recovered from short-term cultures after secretion. Specificity controls for the novel use of this method and quantification of granule labelling in these controls were performed.
Results: Heparin was labelled by R-G in electron-dense granules within non-secretory human lung mast cells (HLMCs), in electron-dense granules that persisted in secretory HLMCs at the maximum histamine secretion time (20 min), and in electron-dense granules within recovering HLMCs. Specificity controls showed that gold alone did not label HLMCs and that absorption with heparin significantly reduced or abrogated HLMC granule staining with R-G, but that RNA absorption did not. Heparin stores were absent in newly formed, electron-lucent intracytoplasmic degranulation channels in secretory HLMCs. Electron-dense granule matrices in the process of extrusion to the cell exterior still retained heparin at the instant of cellular secretion. Non-granule heparin stores bound R-G in recovering HLMCs. These locations included resolving degranulation channels, as newly emergent granules partitioned and condensed within them, and electron-dense content-containing vesicles and progranules within synthetic mast cells. Ultimately, all known ultrastructural patterns of HLMC granules developed in recovering cells, and each of them contained heparin.
Conclusion: Heparin was secreted from HLMCs which were stimulated by anti-IgE, and heparin was recovered by a combination of conservative and synthetic mechanisms in HLMCs after a secretory event.