title: Immunoferritin electron microscopic studies with antibasophil serum of guinea pig basophil degranulation and regranulation in vitro.
abstract: We have previously shown that degranulated guinea pig basophils in vitro do not die. Rather, they recover, and these mature cells rebuild granules in the absence of nuclear changes of immaturity or of mitoses. This ability to survive the explosive release of cytoplasmic granules and to rebuild granules was the first such demonstration for a mature granulocyte. We utilized this model of antigen-induced basophil degranulation and recovery in vitro, in conjunction with a specific antibasophil serum (ABS) and indirect immunoferritin electron microscopy, in order to localize the anatomic sites of expression of the antigen(s) recognizing ABS during the secretory and recovery events. Basophil surface membranes were labeled prior to degranulation, during degranulation, after degranulation, and as mature, polynuclear, granule-free cells began to rebuild new granules. In addition, the plasma membranes of non-granule-containing, glycogen positive, small mononuclear cells were labeled. These were few in number and were thought to be precursor cells in the basophil lineage. Controls, either nondegranulated basophils at a variety of culture intervals, or basophils following degranulation and during recovery, were done using normal rabbit serum (NRS) and an indirect immunoferritin electron microscopic technique. These were all negative. Moreover, contaminating eosinophils, neutrophils, and lymphocytes were also negative. Extruded granules were labeled with ferritin (ABS) on their membrane-free, finely granular exteriors, but not within the lamellar array of their matrixes. Extruded granules were not labeled in control experiments. When tested with ABS, membrane-bound granules in the cytoplasm prior to release and membrane-free, released granules contained within degranulation sacs, were negative, as was the membrane of sacs. Immature, membrane-bound granules were also negative. Ferritin binding to membranes was seen in narrow channels, vesicles, and larger vacuoles, as well as in multivesicular bodies, present in the peripheral cytoplasm. This distribution of labeled sites was not observed in neutrophils, eosinophils, and lymphocytes which were also routinely present in these preparations. These sites were not labeled in basophils or other cells in control studies using ferritin and NRS. This indicates the necessity for specific binding to basophils in order to visualize this process. Whether most such sites represent residual endocytosis at 4 degrees C or binding to open invaginations near surfaces of cells does not detract from their specificity and thus expression of specific antigen(s)-binding sites in basophils.
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001491:Basophils / Q000276:immunology* / Q000648:ultrastructure; D005293:Ferritins / Q000276:immunology*; D006168:Guinea Pigs; D008854:Microscopy, Electron; D009504:Neutrophils / Q000502:physiology / Q000648:ultrastructure; D014617:Vacuoles / Q000032:analysis / Q000648:ultrastructure
chemical_list: D000906:Antibodies; D005293:Ferritins