Neutrophil marrow release. A system analysis.

Neutrophil marrow egress is governed by several processes. The most important are cell maturation, functional behavior of marrow sinusoids and humoral or neuro-vascular factors. Neutrophil release cannot be observed directly but is reflected in the size, cellular composition and kinetics of the nonproliferating pool of granulocytopoiesis in bone marrow and of blood neutrophil pool. These experimentally determined parameters were used as the basis of a mathematical model study. The model describes two catenated compartments, the nonproliferating pool of granulocytopoiesis in marrow and the total blood granulocyte pool. Cell transit from one pool to the other was assumed to be age-dependent. It was expressed by a positive sloping sigmoidal function that defines the egress potential fo the cells that increases with cell maturation. During maturation granulocytopoietic cells develop intense motility which determines the morphology of the cells on smears. Relationship between cell motility and its morphology was defined by functions determining the age-dependent probabilities of cell fixation as metamyelocytes, band- and segmented forms, respectively. The parameters of this model could be so adjusted that all experimental data were matched within experimental errors. Thus, qualitative and quantitative information on neutrophil marrow egress was obtained for normal and pathological states of granulocytopoiesis.     

Granulocytopoiesis during the development of radiation injury from prolonged intake of tritium oxide

During chronic exposure to tritium oxide (a dose rate of 0.125 Gy/day-1, and a cumulative-absorbed dose of 22.1 Gy) different granulocytopoiesis compartments (i.e. polypotent CFUs, committed CFUc; proliferating, maturing, and functional pools) were differently damaged by radiation. In the course of the development of tritium oxide-induced affection granulocytopoiesis proceeds at an intense pace.     

Long-term overexpression of human granulocyte colony-stimulating factor in transgenic mice: persistent neutrophilia with no increased mortality for more than one year

To investigate possible adverse consequences of persistent neutrophil overproduction, mice transgenic for human granulocyte colony-stimulating factor (hG-CSF) were studied for more than 1 year. They showed marked granulocytopoiesis and neutrophilia. Continuous medullary and extramedullary granulocytopoiesis resulted in marked changes in bone and liver. In the liver, haemorrhage and focal necrosis and a few haemangiosarcomas were present, presumably caused by the destructive granulocytopoiesis. Despite the high incidence of lung infiltration by mature neutrophils, lung lesions rarely appeared. Although there was a persistent increase in neutrophils, mortality of the mice did not differ from that of non-transgenic littermates at least within 1 year after birth. Factors other than overproduction of G-CSF and extensive neutrophilia could be required for the development of neutrophil-mediated acute and chronic tissue damage.     

The functional condition of the immune system and the lymphocytes' hemopoiesis-modulating properties

Donor splenocytes and timocytes have an ability to stimulate erythropoesis after massive blood-letting. Changing of functional state of the immune system by means of immune modulators action affects the character and expressiveness of hemopoiesis regulation function of lymphocytes. Splinocytes and thymocytes depress granulocytopoiesis in the recipients' bone marrow after activation by T- and B-lymphocytes. The activation of B-lymphocytes determines the cells' capacity to increase concentration of thrombocytes in the blood. Donor thymocytes can activate erythropoiesis and granulocytopoiesis after macrophages stimulation.     

Changes in the activity of granulocytopoiesis during increasing and reducing the reactions of the lysosomal apparatus of circulating neutrophils in hypovolemic hypotension

An interrelation between the intensity of release of the lysosomal content of circulating neutrophils and the activation degree of granulocytopoiesis and neutrophilia was revealed by means of pharmacological influence on the lysosomal membrane stability in case of hypovolemic hypotension in rabbits. The activation of granulocytopoiesis and neutrophilia increased while intensifying the release of lysosomal factors from the circulating neutrophils and sharply decreased while restricting it. The intensity of hypotension decreased while intensifying the reaction of the lysosomal apparatus of circulating neutrophils and increased while depressing the reaction.     

The role of granulocytic chalone and antichalone in regulating granulocytopoiesis under stress

The experiments on rats have shown that immobilization stress induces a prolonged increase of the activity in the blood serum of tissue-specific granulocytopoiesis stimulator--granulocytic antichalone.     

The role of apoptosis in the pathophysiology of chronic neutropenias associated with bone marrow failure

Chronic neutropenia syndromes associated with bone marrow (BM) failure comprise distinct congenital and acquired hematologic disorders with varying degree of neutropenia due to decreased or ineffective BM neutrophil production. Recent evidence suggests that defective granulocytopoiesis in these neutropenia states is a consequence of accelerated apoptotic cell death of BM myeloid progenitor cells and/or their differentiated progeny. Inherited or spontaneously appearing mutations in the ELA2 gene encoding for neutrophil elastase have been implicated in the accelerated apoptotic process of the BM myeloid cells in patients with cyclic and severe congenital neutropenia. A disturbed balance between pro-apoptotic and anti-apoptotic intracellular or membrane molecules such as downregulation of the bcl-2 family members or upregulation of the death receptor Fas, have been implicated in neutropenia associated with myelokathexis, Shwachman-Diamond syndrome and acquired chronic idiopathic neutropenia of adult. In this review we summarize the available evidence suggesting that abnormally increased apoptosis and impaired proliferative and differentiating properties of neutrophil progenitor and precursor cells represent a common pathogenetic mechanism for impaired granulocytopoiesis in both acquired idiopathic and congenital neutropenia states. The underlying distinct cellular and molecular abnormalities and the role of the BM microenvironment are extensively analysed.     

Role of the thymus in regulating bone marrow hematopoiesis in the stress reaction

The role of thymus in the regulation of erythropoiesis during stress has been studied in experiments on immobilized mice. The development of pronounced leukopenia due to stimulation of erythro- and granulocytopoiesis was demonstrated. Thymectomy, performed a month before immobilization, abolished the phenomenon of erythropoietic cellularity, indicating an important role of thymus in the regulation of myelopoiesis during stress.     

The Role of Apoptosis in the Pathophysiology of Chronic Neutropenias Associated with Bone Marrow Failure

Chronic neutropenia syndromes associated with bone marrow (BM) failure comprise distinct congenital and acquired hematologic disorders with varying degree of neutropenia due to decreased or ineffective BM neutrophil production. Recent evidence suggests that defective granulocytopoiesis in these neutropenia states is a consequence of accelerated apoptotic cell death of BM myeloid progenitor cells and/or their differentiated progeny. Inherited or spontaneously appearing mutations in the ELA2 gene encoding for neutrophil elastase have been implicated in the accelerated apoptotic process of the BM myeloid cells in patients with cyclic and severe congenital neutropenia. A disturbed balance between pro-apoptotic and anti-apoptotic intracellular or membrane molecules such as down-regulation of the bcl-2 family members or up-regulation of the death receptor Fas, have been implicated in neutropenia associated with myelokathexis, Shwachman-Diamond syndrome and acquired chronic idiopathic neutropenia of adult. In this review we summarize the available evidence suggesting that abnormally increased apoptosis and impaired proliferative and differentiating properties of neutrophil progenitor and precursor cells represent a common pathogenetic mechanism for impaired granulocytopoiesis in both acquired idiopathic and congenital neutropenia states. The underlying distinct cellular and molecular abnormalities and the role of the BM microenvironment are extensively analysed.     

Action of "myelauxins", substances capable of accelerating granulocytic hematopoiesis, in vivo in rabbits]

Extracts from homologous blood serum had been shown by the author to stimulate the granulocytopoiesis of cells from chickens' embryonic spleen cultivated in vitro. The results presented here show that extracts obtained with the same method from rabbits' serum can induce the same activity in vivo in the adult mammals. Data relating to the nature of those active substances will be published later.     

Recovery of CFUc in rat bone marrow after prolonged fractionated irradiation with various total doses

The rate and the degree of recovery of committed precursors of granulocytes and monocytes (CFUc) following long-term fractionated irradiation were a function of a cumulative radiation dose. In rats exposed to doses of 9.7 and 19.4 Gy the number of CFUc of myelokaryocytes and granulocytes of blood reached the control values after 1-3 months. The increase in CFUc of animals exposed to a dose of 29.1 Gy was transient and did not provide the recovery of granulocytopoiesis.     

Cloning of canine hematopoietic cells in vitro]

A modified cloning method of the agar culture of canine bone marrow cells was described. A high efficiency of colony formation was seen only after addition to the agar medium of the colony-stimulating activity (CSA) from different sources. Dog serum in a 20% concentration was used in this case. With the optimal CSA concentration there was seen a linear relationship between the number of explanted cells and the number of produced colonies. This method is suitable for determination of committed granulocyte precursor cells, as well as for the study of potential humoral regulators of granulocytopoiesis in dogs.     

Morphological features of cellular responses to different rates of trematode Quinqueserialis quinqueserialis (Trematoda: Notocotilidae) invasion in muskrat (Ondatra zibethicus)

The results of investigation of leukocyte morphology and leukocyte contents of blood and caecum depending on the trematode Quinqueserialis quinqueserialis invasion rate in muskrats from natural population are given. At low trematode invasion rates, there was observed systemic activation of lymphopoiesis and neutrophil granulocytopoiesis with a decrease in the monocyte-macrophage response in caecum (trematode localization organ). At the same time, under high invasion rates, there was detected induction of T cell suppressor activity and the absence of a granulocyte response in the tissues under study. Intensification of B lymphocyte blast transformation in caecum tissues as well as the appearance of blast cells in the blood of infected muskrats was observed.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Effect of exposure to stress on the role of T- and B-lymphocytes in the response of the hematopoietic system

Cellular composition of the bone marrow, spleen and peripheral blood was studied after 6-hour immobilization on the back in experiments on 4 groups of (CBAxC57BL)F1 mice with varying degree of T lymphocyte deficiency (thymectomy, sham thymectomy, administration of antilymphocytic serum, B mice). The evidence obtained shows that the "lymphoid peak" recorded in the bone marrow during stress is likely to be formed at the expense of T and B lymphocyte migration from the peripheral lymphoid organs. The data have been also obtained, indicating that T lymphocytes migrating to the bone marrow during the first 6-9 hours after the exposure to stress may participate in granulocytopoiesis activation.     

Reutilization of DNA catabolites in granulocytopoiesis.

Different DNA labelling procedures for the proliferating granulocyte precursor compartments of rat bone marrow were evaluated by studying the appearance of labelled granulocytes in the blood stream by means of autoradiography. 3-H-thymidine was administered by single injection and continuous infusion. 125-I-Iododeoxyuridine, a DNA precursor showing a neglegible extent of reutilization, was studied in comparison. Labelling patterns of blood neurtrophils were identical after single injection and continuous infusion of 3-H-thymidine, while a different pattern was observed after use of a single injection of 125-I-Iododeoxyuridine. The results provide evidence that reutilization of label is an important event to be considered when kinetics of granulocytopoiesis are studied after application of 3-H-thymidine and indicate that reutilization occurs at the level of thymidinemonophosphate in this cell system.     

Quantitative determination of human bone marrow proliferation kinetics during three culture days]

In order to obtaon of human bone marrow cells, fresh bioptic material was homogenized and the cell suspensions were incubated for 72 hs in a fluid medium. After 24, 48 and 72 hs of incubation the total cell number of the culture was determined. At the same time differential counts of stained smears were performed. Both, erythrocytopoiesis and granulocytopoiesis showed regeneration, maturation, and an absolute increase of the number of precursors and of mature cells. The quantitative data obtained in vitro during 24 hs correspond with our data of kinetics obtained by observed mitotic duration and cell differential countings in vivo. However, after a longer cultivation time we found a diminution of divisible precursors, and an increase of mature erythroblasts as well as an excessibe survival of the PMNs.     

Hemopoietic precursor cells in the intima of the atheromatous human aorta

Granulocyte-macrophage progenitor cells (CFU-GM) were found in the intima of human atheromatous aorta. Granulocyte-macrophage colonies were recovered on the 14th day of culturing of intimal cells suspensions on agar. Medium conditioned by normal leukocytes in the presence of phytohaemagglutinin was used as a source of the colony-stimulating factor. Grossly normal and atheromatous intima contained different number of CFU-GM. No GM were recovered from fibrous plaques. By light and electron microscopies, the injured aortic intima contained the clusters of blood-born cells that were at various stages of granulocytopoiesis (including blasts and mature cells) and poorly differentiated lymphocyte-like cells. The results obtained suggest that in human aortic intima proliferation and differentiation of CFU-GM occur at early stages of atherogenesis, prior to fibrous plaque formation.     

Splenic monocyte-macrophage dependent suppression of autologous granulocyte-progenitors growth in Hodgkin's disease

The studies described compare the effect of spleen cell suspensions from 11 patients with Hodgkin's disease (HD) and 5 healthy subjects on the clonal growth of autologous marrow granulopoietic progenitors in diffusion chamber culture (CFU-G/D). Adherent monocyte/macrophage fraction of splenocytes from HD suppresses the proliferation of autologous CFU-G/D. This inhibition was mediated by an indomethacin-sensitive humoral factor(s). Non-adherent lymphoid cells stimulated myeloid colony formation. Dose response curves demonstrated a markedly increased inhibitory-activity production already by low numbers of splenic monocytes/macrophages from HD whereas a comparable counts of monocytes/macrophages from the spleens of healthy subjects stimulated the CFU-G/D growth. These results may suggest a possible activation of splenic monocytes/macrophages with an enhanced prostaglandin-mediated suppressor activity release for local granulocytopoiesis in the spleens of patients with HD.     

A histomorphometric analysis of trephine biopsies of bone marrow from 65 patients with chronic myeloid leukemia. Classification of patients into subgroups with different survival patterns

A histomorphometric (planimetric) study was performed on trephine biopsies of the bone marrow taken at presentation from 65 patients (31 males and 34 females, with a median age of 48 years) with chronic myeloid leukemia (CML). Specimens from 20 patients (9 males and 11 females, with a median age of 53 years) without any hematologic disorders served as controls. Of the various histologic variables tested, only the counts of neutrophilic granulocytes per 1 sq mm, the ratio of granulocytopoiesis to megakaryopoiesis and the density of reticulin (argyrophilic) fibers revealed a significant correlation with the prognosis. The CML patients were separated into two groups with different survival patterns. Group I (34 patients with a median survival of 24 months) mostly contained cases with the so-called "megakaryocytic subtype" of CML, which is accompanied by variable degrees of fibrosis; group II (31 patients with a median survival of 36 months) mainly contained cases with the "granulocytic subtype," which is not accompanied by myelofibrosis. Among the morphometric parameters, a positive correlation existed between the megakaryocyte count and the reticulin fiber density, which underlines the important role of that cell lineage in fibrillogenesis. There were multiple interrelationships between the histomorphometric variables and the laboratory data. Consequently, multivariate regression methods (using Cox's proportional hazards model) were applied to assess the relative predictive value of the patient characteristics for survival. The derived prognostic model divided the patients into two risk groups, with median survivals of 14 and 41 months, respectively. In order of their entry into the regression model, these variables were percentage of neutrophils in the differential blood count, amount of granulopoiesis, liver size, percentage of peripheral myeloblasts and density of reticulin fibers in the bone marrow. In comparing the two patient groups, based on bone marrow histomorphometric parameters, this model revealed that two of those factors (amount of granulopoiesis and density of reticulin fibers) had a significant correlation with the prognosis.