G. Adolph Ackerman

HISTOCHEMICAL DIFFERENTIATION DURING NEUTROPHIL DEVELOPMENT AND MATURATION

Our studies concerning the hemopoietic system have been directed toward the morphological, histochemical, and submicroscopic aspects of cellular differentiation and maturation. This approach has proven most rewarding in being able to more clearly define and characterize changes taking place during cellular development as well as indicating cellular interrelationships, modulations, and the modes of origin of various cells of the hemopoietic system in both the definitive and embryonic conditions (G. A. Ackerman 1962a, 19626; G. A. Ackerman and R. A. Knouff 1959, 1960, 1962; J . A. Grasso et al., 1962; J. W. Houser et al., 1961; R. A. Knouff and G. A. Ackerman, 1963). The purpose of this paper is threefold: (1) to characterize histochemically the neutrophilic granulocyte during various phases of differentiation, (2) to indicate sequential patterns in the histochemical development and maturation of the neutrophilic granules as contrasted with the independent morphological stages of cellular differentiation and, (3) to discuss a working hypothesis concerning the possible origin and developmental pathways of granulocyte differentiation in normal human-bone marrow. Considerable work has been reported concerning the histochemical composition of the granulocytic elements of the blood and bone marrow, and the reader may gain familiarity with the present status of histochemistry of the cells of the blood and bone marrow by examining many of the excellent reports presented in the accompanying bibliography. Although changes in the histochemical reactivity of the granulocytic elements during their maturation and development have been observed, it would seem that these changes have received neither sufficient emphasis nor exploration. It has become increasingly essential to combine histochemical analysis with critical morphological study both at the microscopic and submicroscopic levels and whenever possible to relate changes in the histochemistry of these cells with alterations and modulations in cellular structure and composition. Ideally, correlated histochemical and biochemical analysis should be attempted, although it is beyond the scope of the present investigation. Histochemistry provides a major advantage over the biochemical method of studying the chemical composition of cells and tissues in that, by the use of selective histochemical “staining” techniques, it is possible to localize specific chemical substances and enzyme activities in individual cells and intracellular components.

Bursa of Fabricius in Chickens: Possible Humoral Factor

Chicks bursectomized by testosterone injection on the 5th day of incubation showed a marked inability to produce antibodies to Salmonella typhimurium. When portions of the bursa of Fabricius were enclosed in cell-impermeable Millipore diffusion chambers and implanted subcutaneously or intraperitoneally, the antibody-producing capacity of these animals was restored. Evidence strongly suggests that the bursa of Fabricius elaborates a non-cellular agent capable of restoring immunologic reactivity in bursectomized chicks.

Ultrastructural localization of peroxidase activity in normal human bone marrow cells

SummaryThe ultrastructural localization of peroxidase activity has been studied in the cells of normal human bone marrow using the diaminobenzidine peroxidase technique. Peroxidase activity has been localized within the primary (azurophil) granules of the neutrophilic series as well as in the cytoplasmic granules of eosinophils, basophils and monocytes. Peroxidase activity appears within the cisternal system (nuclear envelope, Golgi complex and rough endoplasmic reticulum) of these cells during the period of peroxidase-containing lysosome production. With the cessation of granulogenesis, peroxidase activity disappears from the cisternal system and does not reappear in subsequent developmental stages. In cells incubated in peroxide-free media, staining of granular components, but not of cisternae, is reduced. The inclusion of catalase in peroxide-free media eliminates all staining. This indicates that an endogenous peroxide is present within the cisternae and granules of these cell types.

A HISTOCHEMICAL EVALUATION OF THE PYROANTIMONATE-OSMIUM REACTION

The chemical nature of the pyroantimonate-osmium (PAO) reaction in normal human bone marrow cells has been evaluated by the application of a number of digestion and blocking procedures as well as the electron microprobe. Five foci of reactivity are localized in cells fixed directly in the PAO reagent, viz., nucleoli, heterochromatin, cytoplasmic granules, particulate glycogen and the outer plasmalemmal surface. Heterochromatin and nucleolar staining are attributable to calcium bound to nucleic acids as well as to reactive amino groups on histones. Granule staining is dissociable into acid-dialyzable, ribonuclease (RNase)-stable and RNase-labile components but sulfate groups of acid mucosubstances are probably not involved. Glycogen staining by the PAO reagent has been verified and shown to result from the formation of organometallic complexes between the pyroantimonate ion and the C2-C3-free hydroxy groups of the glucose residues. PAO reactivity has been definitely localized along the outer plasmalemmal surface, but this component of PAO staining is resistant to all control measures employed. These studies have illustrated the complexity and polyvalency of the PAO reaction and have shown that PAO staining is not exclusively associated with metallic cation localization.

Surface Differentiation of Hemopoietic Cells Demonstrated Ultrastructurally with Cationized Ferritin

SummaryThe ultrastructural cationized ferritin (CF) technique was employed as a probe of the surface binding characteristics of the various cell types present in normal human bone marrow. The number of CF particles per micron length of cell surface were counted and data subjected to statistical analysis. All cells of the bone marrow exhibited CF reactivity. The extent of labeling was cell specific and could be related to the stage of maturation of the cells in a given lineage. In the neutrophilic series, myeloblasts showed moderate labeling while promyelocytes and myelocytes revealed only minimal binding; CF binding increased sequentially in metamyelocytes, band and segmented neutrophils. Eosinophils and eosinophilic myelocytes showed similar membrane differentiation patterns while basophils exhibited stronger CF labeling than other granulocytic cells. Lymphocytes were strongly reactive while monocytes and their precursors were moderately labeled with CF. Surface reactivity of developing nucleated erythrocytic cells was similar to that of the lymphocytes. Surface labeling from the proerythroblast to early normoblast stage was identical, CP binding increased in the late normoblast stage and then decreased in the reticulocyte and mature erythrocyte stages. The extent of surface CP reactivity of the marrow cells was markedly different from that obtained with Thorotrast and colloidal iron. Thorotrast and colloidal iron stained the surface of all marrow cell intensely but failed to yield distinctive surface labeling patterns for the differing cell population in bone marrow.The ultrastructural cationized ferritin (CF) technique was employed as a probe of the surface binding characteristics of the various cell types present in normal human bone marrow. The number of CF particles per micron length of cell surface were counted and data subjected to statistical analysis. All cells of the bone marrow exhibited CF reactivity. The extent of labeling was cell specific and could be related to the stage of maturation of the cells in a given lineage. In the neutrophilic series, myeloblasts showed moderate labeling while promyelocytes and myelocytes revealed only minimal binding; CF binding increased sequentially in metamyelocytes, band and segmented neutrophils. Eosinophils and eosinophilic myelocytes showed similar membrane differentiation patterns while basophils exhibited stronger CF labeling than other granulocytic cells. Lymphocytes were strongly reactive while monocytes and their precursors were moderately labeled with CF. Surface reactivity of developing nucleated erythrocytic cells was similar to that of the lymphocytes. Surface labeling from the proerythroblast to early normoblast stage was identical, CP binding increased in the late normoblast stage and then decreased in the reticulocyte and mature erythrocyte stages. The extent of surface CP reactivity of the marrow cells was markedly different from that obtained with Thorotrast and colloidal iron. Thorotrast and colloidal iron stained the surface of all marrow cell intensely but failed to yield distinctive surface labeling patterns for the differing cell population in bone marrow.

Ultrastructural Localization of Concanavalin A Binding Sites on the Surface of Differentiating Hemopoietic Cells

SummaryThe surface characteristics of hemopoietic cells in normal human bone marrow have been explored by the use of the ultrastructural concanavalin A-peroxidase-diaminobenzidine (CAPD) procedure for the detection of specific carbohydrate residues (α-D-mannopyranoside, α-D-glucopyranoside, and α-N-acetyl-D-glycosaminide) associated with the cell surface. All cells present in the bone marrow were capable of binding Con A to their surfaces. The extent of binding proved cell specific and could be related to the stage of morphological development of each cell line. Maximum surface reactivity in the bone marrow cell population occurred with the most immature cells (myeloblast and erythroblast), lymphocytes, eosinophils, monocytes, macrophages and platelets, while mature neutrophils, basophils, and erythrocytes showed only minimum surface reactivity. These findings serve not only to expand our knowledge of the chemical nature of the surface of differentiating hemopoietic cells but provide striking evidence that modifications in the surface of hemopoietic cells occur during the process of normal cell differentiation in the bone marrow.

A CYTOCHEMICAL EVALUATION OF PYROANTIMONATE BINDING TO THE PLASMALEMMA OF BLOOD AND BONE MARROW CELLS AND ITS RELATIONSHIP TO CELLULAR MATURATION

Normal human blood and bone marrow cells exposed to the pyroantimonate reaction exhibit a selective binding of pyroantimonate to sites associated with the plasmalemma of neutrophilic leukocytes, erythrocytes and certain of their precursors. Chemical distinctions exist between the plasmalemma of different types of blood and bone marrow cells, and major changes in the cationic binding ability of neutrophilic and erythrocytic cells can be correlated with different phases in their differentiation. Plasmalemmal pyroantimonate-osmium-reactive cation is bound with glycoprotein in the erythrocytic elements and to both phospholipid and glycoprotein moieties in neutrophilic cells. The cellular distribution, membrane localization and degree of membrane reactivity obtained with the pyroantimonate-osmium reaction is distinct from that obtained with Thorotrast and ruthenium red.

Localization of pyroantimonate-precipitable cation and surface coat anionic binding sites in developing erythrocytic cells and macrophages in normal human bone marrow.

SummaryThe surfaces of developing erythrocytic cells and macrophages have been examined in normal human bone marrow by means of the pyroantimonate-osmium, ruthenium red and Thorotrast techniques for inorganic cations, surface glycoprotein-phospholipid complexes and surface anionic binding sites, respectively. No differences in the degree of surface coat reactivity were noted in the erythrocytic cells at different stages of maturation while pyroantimonate binding to the plasmalemma was not evident developmentally until the final stages of erythrocytic development. Rhopheocytotic invaginations proved to be chemically distinct from the remainder of the cell surface since they did not bind Thorotrast or pyroantimonate and gave more staining with ruthenium red. Pyroantimonate does not bind to the surface of macrophages and the binding of Thorotrast by these cells is less. Macrophage-erythrocytic cell contact zones did not stain with Thorotrast but stained with ruthenium red. The significance of these observations is discussed.

Influence of bursa implantation upon lymphocytic nodules and plasma cells in spleens of bursectomized chickens.

Summary Implantation of donor bursa in hormonally bursectomized chickens restored the immunological reactivity by means of a humoral substance capable of passing through cell-impermeable diffusion chambers. Lym-phocytic nodules, shown to develop under the influence of the humoral agent from the bursa, are felt, in part, to be the site of antibody production. Plasma cell development does not appear to be by means of the humoral substance alone.

Nuclear membrane contributions to the Golgi complex

SummaryElectron microscopic examination of a variety of rapidly growing or differentiating mammalian and avian cells suggests that many of the Golgi vesicles and saccules arise directly from the outer nuclear membrane. Evidence for this interpretation includes: (1) the presence of a continuum of vesicles which appears to originate from the outer nuclear membrane and to enlarge gradually into saccules in the region of the Golgi membrane complex; (2) the absence of ribosomes on the nuclear blebs and the vesicles formed in these regions along the nuclear envelope; (3) the presence of active nuclear vesiculation near the Golgi region in cells essentially devoid of rough and/or smooth endoplasmic reticulum, and (4) the demonstration of peroxidase activity in the cisternae of the nuclear envelope and in vesicles extending in rows from the nuclear envelope to the Golgi complex.