A novel type of cytoplasmic granule in bovine neutrophils

We obtained cell preparations containing greater than 95% neutrophils from freshly drawn bovine blood. The cells were suspended in sucrose and disrupted in a Dounce homogenizer, and the postnuclear supernate was fractionated by zonal differential sedimentation and by isopycnic equilibration. The subcellular fractions were characterized biochemically by testing for marker enzymes and other constituents known to occur in azurophil and specific granules of other species, and by electrophoretic analysis of extracts of the particulate material. In addition, each fraction was examined by random-sampling electron microscopy. We found that bovine neutrophils contain in addition to azurophil and specific granules a third type of granule, not known to occur in neutrophils of other species. These novel granules are larger, denser, and considerably more numerous than the two other types. Except for lactoferrin, they lack the characteristic constituents of azurophil granules (peroxidase, acid hydrolases, and neutral proteinases) and of specific granules (vitamin B12-binding protein). Instead, they contain a group of highly cationic proteins not found in the other granules, and they are the exclusive stores of powerful oxygen-independent bactericidal agents. We studied the fate of the large granules in bovine neutrophils exposed to opsonized particles, the ionophore A 23187, or phorbol myristate acetate. The appearance in the cell-free media of antibacterial activity and of the characteristic highly cationic proteins as revealed by electrophoresis was monitored and compared with the release of azurophil and specific granule markers. In addition, changes of the relative size of the large granule compartment induced by phagocytosis were assessed by morphometry. The results show that exocytosis of the large granules occurs following both phagocytosis and exposure to soluble stimuli. Like the specific granules, the large granules appear to be discharged by true secretion under conditions where the azurophil granules are fully retained.

[1]  R. Gennaro,et al.  Isolation and partial characterization of the plasma membrane of purified bovine neutrophils. , 1980, European journal of biochemistry.

[2]  P. A. Lanzetta,et al.  An improved assay for nanomole amounts of inorganic phosphate. , 1979, Analytical biochemistry.

[3]  R. Gennaro,et al.  Ca2+-dependent ATPase activity of alveolar macrophage plasma membrane. , 1979, Biochimica et biophysica acta.

[4]  B. Dewald,et al.  A volume adapter for use in a B-XIV zonal rotor. , 1978, Analytical biochemistry.

[5]  G. Weissmann,et al.  Leukocytes as secretory organs of inflammation. , 1978, Agents and actions.

[6]  R. Gennaro,et al.  Biochemical Properties of Bovine Granulocytes , 1978, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[7]  C. Penney.  A simple micro-assay for inorganic phosphate. , 1976, Analytical biochemistry.

[8]  P. Rausch,et al.  Granule enzymes of polymorphonuclear neutrophils: A phylogenetic comparison. , 1975, Blood.

[9]  T. Peters,et al.  Analytical subcellular fractionation of human granulocytes with reference to the localization of vitamin B12-binding proteins. , 1975, Clinical science and molecular medicine.

[10]  S. Klebanoff.  Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. , 1975, Seminars in hematology.

[11]  M. Baggiolini,et al.  Subcellular Localization and Heterogeneity of Neutral Proteases in Neutrophilic Polymorphonuclear Leukocytes , 1975, The Journal of experimental medicine.

[12]  M. Leffell,et al.  Character of azurophil and specific granules purified from human polymorphonuclear leukocytes. , 1974, Laboratory investigation; a journal of technical methods and pathology.

[13]  M. Baggiolini,et al.  ASSOCIATION OF THE ALKALINE PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES WITH THE MEMBRANE OF THE SPECIFIC GRANULES , 1973, Journal of Cell Biology.

[14]  S. Spicer,et al.  Morphologic and cytochemical studies of rabbit heterophilic leukocytes. Evidence for tertiary granules. , 1973, Laboratory investigation; a journal of technical methods and pathology.

[15]  G. Carlson,et al.  Isolation of leukocytes from bovine peripheral blood. , 1973, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[16]  M. Leffell,et al.  Association of Lactoferrin with Lysozyme in Granules of Human Polymorphonuclear Leukocytes , 1972, Infection and immunity.

[17]  M. Farquhar,et al.  THE DEVELOPMENT OF NEUTROPHILIC POLYMORPHONUCLEAR LEUKOCYTES IN HUMAN BONE MARROW , 1971, The Journal of experimental medicine.

[18]  D. Wallach,et al.  Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. , 1971, Biochemistry.

[19]  M. Baggiolini,et al.  FURTHER BIOCHEMICAL AND MORPHOLOGICAL STUDIES OF GRANULE FRACTIONS FROM RABBIT HETEROPHIL LEUKOCYTES , 1970, The Journal of cell biology.

[20]  M. Baggiolini,et al.  ASSOCIATION OF LACTOFERRIN WITH SPECIFIC GRANULES IN RABBIT HETEROPHIL LEUKOCYTES , 1970, The Journal of experimental medicine.

[21]  M. Baggiolini,et al.  RESOLUTION OF GRANULES FROM RABBIT HETEROPHIL LEUKOCYTES INTO DISTINCT POPULATIONS BY ZONAL SEDIMENTATION , 1969, The Journal of cell biology.

[22]  H. I. Zeya,et al.  ARGININE-RICH PROTEINS OF POLYMORPHONUCLEAR LEUKOCYTE LYSOSOMES , 1968, The Journal of experimental medicine.

[23]  S. Spicer,et al.  Fine structural studies on the development of heterophil, eosinophil, and basophil granulocytes in rabbits. , 1967, Laboratory investigation; a journal of technical methods and pathology.

[24]  P. Baudhuin,et al.  ELECTRON MICROSCOPIC EXAMINATION OF SUBCELLULAR FRACTIONS , 1967, The Journal of cell biology.

[25]  E. Weibel,et al.  PRACTICAL STEREOLOGICAL METHODS FOR MORPHOMETRIC CYTOLOGY , 1966, The Journal of cell biology.

[26]  M. Karnovsky,et al.  THF EARLY STAGES OF ABSORPTION OF INJECTED HORSERADISH PEROXIDASE IN THE PROXIMAL TUBULES OF MOUSE KIDNEY: ULTRASTRUCTURAL CYTOCHEMISTRY BY A NEW TECHNIQUE , 1966, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[27]  J. Heremans,et al.  Immunochemical quantitation of antigens by single radial immunodiffusion. , 1965, Immunochemistry.

[28]  U. Lewis,et al.  Disk Electrophoresis of Basic Proteins and Peptides on Polyacrylamide Gels , 1962, Nature.

[29]  John H. Luft,et al.  IMPROVEMENTS IN EPOXY RESIN EMBEDDING METHODS , 1961, The Journal of biophysical and biochemical cytology.

[30]  G. L. Miller,et al.  Protein determination for large numbers of samples. , 1959 .

[31]  B. Dewald,et al.  Subcellular localization of the superoxide-forming enzyme in human neutrophils. , 1979, The Journal of clinical investigation.

[32]  M. Baggiolini,et al.  Capping of ricin-binding sites does not influence phagocytosis in human polymorphonuclear leukocytes. , 1978, Acta haematologica.

[33]  P. Henson.  Mechanisms of Mediator Release from Inflammatory Cells , 1974 .

[34]  H. Beaufay,et al.  Gradient Centrifugation of Cell Particles Theory and Applications , 1959 .