Suppression Of Granulopoiesis Research Articles

Mechanisms of glucocorticosteroid activity in patients with the preleukemic syndrome (hemopoietic dysplasia)

We have analyzed mechanisms of glucocorticosteroid activity in patients with the preleukemic syndrome by culturing in agar and methylcellulose bone marrow cells from 5 glucocorticosteroid-responsive patients with the preleukemic syndrome. We have found that methylprednisolone and cortisol are equally effective in enhancing colony growth in these patients, that enhancement occurs in either methylcellulose or agar matrix and in RPMI 1640, McCoy's 5a, and alpha medium. In 4 patients, glucocorticosteroids inhibited the activity of T-lymphocyte inhibitors of granulopoiesis in vitro. In one patient, these agents stimulated the immediate proliferation of CFU-C and in the presence of LCM enhanced CFU-C survival in suspension culture. These effects were blocked by progesterone and not due to enhancement of CSA production or inhibition of inhibitory T-cell function. Our results document the involvement of cortisol sensitive T-lymphocytes in the suppression of granulopoiesis in a small number of patients with the preleukemic syndrome and indicate that the mechanisms of glucocorticosteroid responses among patients with this syndrome are heterogeneous.

The role of colony-stimulating factor in granulopoiesis.

AbstractThe proliferation and maturation of granulocytic‐monocytic stem cells appears to be controlled by a series of closely related glycoproteins termed “colony‐stimulating factors” (CSFs). Recently, we devised a 6‐step scheme for the purification of murine fibroblast (L‐cell)‐derived CSF. Ten liter pools of conditioned media were concentrated by ultrafiltration, precipitated by ethanol, and separated on DEAE cellulose, Con‐A Sepharose, and Sephadex G 150. The CSF was separated from trace contaminants, including endotoxin, by density gradient centrifugation. The purified material was radioiodinated and used to define the serum half‐life and in vivo distribution. Following IV injection there was a biphasic serum clearance with a t½ of 24–40 min and 2–2½ hours in the first and second phases. Approximately 25% of the tracer was excreted in the urine at 6 h; however, urinary radioactivity was due to low molecular weight peptides. Simultaneous studies by radioimmunoassay showed a similar rapid serum clearance of unlabeled CSF but virtually no urinary CSF activity. Thus, assays for urinary CSF may not provide useful measures of in vivo CSF activity. Further in vitro studies have defined the interaction of CSF with responsive cells in the marrow. Varying doses of CSF were incubated with 107 marrow cells for intervals of 24–48 h. The major increment in cell‐associated radioactivity occurred between 6 and 16 h. The reaction was saturable with 1–2 ng/ml CSF. Binding was prevented by cold CSF, but not by other proteins. Irradiation yielded only a minimal reduction in CSF binding. The interaction of CSF with marrow cells appeared to require new protein synthesis, as binding was completely inhibited by cycloheximide and puromycin. Irradiated mice injected with antibodies to CSF showed an inhibition of granulopoiesis by marrow cells in peritoneal diffusion chambers; however, granulopoiesis in the intact bone marrow was unaffected. Granulpoiesis in long‐term marrow cultures was also unaffected by anti‐CSF. These different responses may be due to accelerated clearance of injected CSF in nonirradiated mice or to extensive stromal interactions that modulate and perhaps control granulocytic differentiation in the intact bone marrow microenvironment.

Myeloblastic leukemic cells causing suppression of granulopoiesis and excessive binding of colony-stimulating activity: The effect of antithymocyte globulin

The three and a half year course of an adult woman (65 years) with myeloblastic leukemia, the leukemic clone being trisomic for chromosome #8, was characterized by suppressed granulopoiesis despite a small mass of leukemic tissue relative to cytogenetically normal marrow precursors. The leukemic tissue was shown to be productive of mature neutrophilic granulocytes with hypersegmented nuclei and increased alkaline phosphatase content. These granulocytes showed active phagocytosis but diminished fungicidal capacity. The leukemic tissue inhibited granulocyte-monocyte colony formation in soft agar with colony-stimulating activity from human and murine sources when co-cultured with normal, chronic myelocytic leukemia, Philadelphia chromosome positive (CML Ph 1(+)) human and C57 black (BL) murine marrow. This inhibition was associated with binding of colony-stimulating activity at sites on the leukemic cells which failed to elicit colony formation by leukemia colony-forming units, culture (CFUc). Pretreatment of the leukemic tissue with antithymocyte globulin (ATG) before co-culture removed the inhibitory effect of the leukemic tissue and promoted colony formation by leukemic CFUc. This effect of ATG involved competitive binding of colony-stimulating activity and ATG by leukemic cells since the consumption of colony-stimulating activity by leukemic cells was blocked by prior treatment of leukemic cells with ATG. Infusion of autochthonous cytogenetically normal marrow after two years of cryopreservation was ineffective in altering the neutropenic state. Bacteremia (Klebsiella pneumoniae) at a time when the marrow contained mostly leukemic precursors was associated with transient neutrophilic granulocytosis involving mature leukemic progeny. ATG given to the patient in a preterminal state was associated with a simitar leukocytosis as well as colony formation in vitro by the leukemic marrow tissue.

Myeloblastic leukemic cells causing suppression of granulopoiesis and excessive binding of colony-stimulating activity. The effect of antithymocyte globulin : Craddock CG, Rodensky D, Forsen NR, Kaspersky CJ, Sparkes RS: Am J Med 64: 343–353, 1978

Myeloblastic leukemic cells causing suppression of granulopoiesis and excessive binding of colony-stimulating activity. The effect of antithymocyte globulin : Craddock CG, Rodensky D, Forsen NR, Kaspersky CJ, Sparkes RS: Am J Med 64: 343–353, 1978

Effect of Levamisole on Human Granulopoiesis in Vitro

SummaryThe effect of levamisole, a potent immunomodulator, was studied on granulopoiesis in vitro. Low concentrations of levamisole stimulated monocytes and macrophages and resulted in enhanced colony stimulating factor (CSF) release. Higher concentrations of levamisole were inhibitory to CSF release. Levamisole did not affect bone marrow directly to produce stimulation or inhibition of granulopoiesis. Levamisole did not degrade or potentiate preformed CSF. Improved survival of infected animals may in part be related to increased granulopoiesis. The dose-related effect of levamisole on CSF release by the monocyte-macrophage system may be responsible for the variable effect on the neu-trophil system noted in vivo.The authors gratefully acknowledge the expert technical assistance of Ms. Carla Drebing.

Inhibition of Growth of Normal Murine Granulocytes by Cocultured Acute Leukemic Cells

AbstractCoculture of equal numbers of C1498 murine acute myelogenous leukemia cells and normal mouse bone marrow cells in diffusion chambers (DC) led to complete suppression of normal granulocyte development within a few days. Suppression of granulopoiesis was not observed with irradiation-killed leukemic cells. Direct interaction between the leukemic and normal cells appears to be necessary since there was no inhibition when the two populations were separated by an intervening Millipore filter in double-DC cultures. When the ratio of leukemic to normal cells was reduced from 50/50 to 15/85, proliferation of granulocytic cells remained normal for a longer time but eventually ceased. With both the 15/85 and 50/50 cultures, the ratio of leukemic to normal granulocytic cells rose to about the same level, 6/1-7/1, just before granulopoiesis was suppressed. In both instances macrophage numbers were similar to those observed in control cultures of normal marrow cells alone. When mixtures of leukemic and normal bone marrow cells were assayed by the agar and spleen colony-forming techniques rather than by culture in DC, the formation of colony-forming cells in vitro was unaltered but there was a marked reduction in the formation of normal, differentiated spleen colony-forming cells. This finding suggests that the leukemic cells exert their inhibitory influence at the level of the pluripotential hemopoietic stem cell. The findings with mixed DC cultures of leukemic and normal marrow cells resemble those in the bone marrows of patients with acute myelogenous leukemia and provide an experimental model for elucidating the mechanisms by which leukemia subverts normal hemopoiesis.

Regulation of maturation rate of mouse granulocytes.

Regenerating mouse bone marrow cells were cultured i.p. in diffusion chambers (DC) to study factors affecting the maturation rate of granulocyte precursors. One day after exposing 3-day-old DC cultures to 3H-thymidine the cultures were harvested, and labelled proliferative and non-proliferative granulocytes were counted in radioautographs. The relative maturation rate--defined as the fraction of proliferative precursors maturing into the non-proliferative compartment per unit time--could be increased by different experimental procedures that inhibit cell production. Inhibition was obtained (a) by increasing culture cellularity; (b) by implanting DC into normal rats or rats with huge s.c. chloroma tumours rather than into mice; and (c) by treating the cells with leucocyte extracts (granulocyte chalone) during the last day of culture. Furthermore, a sudden inhibition of rapidly proliferating granulocytes by leucocyte extracts resulted in an increase (apparently transient) in the absolute number of labelled non-proliferative granulocytes. Such an increae was not detected in experiments involving a stronger or sustained inhibition of granulopoiesis, evidently because the size of the precursor population had been markedly reduced.

Regulation of bone marrow cell growth in diffusion chambers: the effect of adding normal and leukemic (CML) polymorphonuclear granulocytes

Inhibition of granulopoiesis was studied using the diffusion chamber (DC) technique. When mature granulocytes from human blood or syngeneic mouse peritoneal fluid were added to mouse bone marrow cells cultured in DC, a significant depression of granulopoiesis took place, and a stimulation of macrophage formation was observed in 7-day cultures. Human granulocytes had a stronger inhinitory capacity than mouse granulocytes. The inhibition appeared to be tissue-specific and caused by a diffusible factor. A time study showed that the added granulocytes had no observable effect on the growth of proliferative granulocytes and CFU-C during the first days of culture. A rapid decrease of proliferative granulocytes after day 5 was preceded by a similar reduction of CFU-C 1 day earlier. The effect of CFU-S was more variable. In one strain of mice, there was a consistent increase in the granulocyte co-culture group, whereas in another strain a significant increase was observed only on day 2. Hislotologic examination showed that mature granulocytes changed the colony distribution, so that a significant relative decrease of granuloid colonies occurred. The nature of this delayed suppression of granulopoiesis is not evident from these data. A possible explanation is that factors released by mature granulocytes prevent recruitment of CFU-C and granulocyte precursors from the CFU-S compartment by blocking the granulopoietic pathway. Leukemic (CML) granulocytes isolated from blood were less able to inhibit granulopoiesis than normal granulocytes with mouse bone marrow as well as human bone marrow as target cells.

Regulation of Bone Marrow Cell Growth in Diffusion Chambers: The Effect of Adding Normal and Leukemic (CML) Polymorphonuclear Granulocytes

Inhibition of granulopoiesis was studied using the diffusion chamber (DC) technique. When mature granulocytes from human blood or syngeneic mouse peritoneal fluid were added to mouse bone marrow cells cultured in DC, a significant depression of granulopoiesis took place, and a stimulation of macrophage formation was observed in 7-day cultures. Human granulocytes had a stronger inhinitory capacity than mouse granulocytes. The inhibition appeared to be tissue-specific and caused by a diffusible factor. A time study showed that the added granulocytes had no observable effect on the growth of proliferative granulocytes and CFU-C during the first days of culture. A rapid decrease of proliferative granulocytes after day 5 was preceded by a similar reduction of CFU-C 1 day earlier. The effect of CFU-S was more variable. In one strain of mice, there was a consistent increase in the granulocyte co-culture group, whereas in another strain a significant increase was observed only on day 2. Hislotologic examination showed that mature granulocytes changed the colony distribution, so that a significant relative decrease of granuloid colonies occurred. The nature of this delayed suppression of granulopoiesis is not evident from these data. A possible explanation is that factors released by mature granulocytes prevent recruitment of CFU-C and granulocyte precursors from the CFU-S compartment by blocking the granulopoietic pathway. Leukemic (CML) granulocytes isolated from blood were less able to inhibit granulopoiesis than normal granulocytes with mouse bone marrow as well as human bone marrow as target cells.

Stimulatory effect of staphylococcal leukocidin on granulopoiesis disturbed by cytostatic agents

Functional tests of granulopoiesis (leukocytosis, bone marrow picture, incubation in vitro of bone marrow with [3H]thymidine followed by radioautography and counting of labeled promyelocytes and myelocytes, serum muramidase level, liberation of granulocyte bone marrow reserve, Nitro-BT reduction in blood granulocytes, enzyme cytochemistry, and phagocytosis) were performed in rabbits given bubulphan (10 mg/kg) or 5-fluorouracil (200 mg/kg). Determinations were carried on serially during treatment with cytostatics. Some of the cytostatic-treated animals received intravenous (i.v.) injections of purified staphylococcal leukocidin in daily doses of 0.1 mg/kg. In control animals, theleukocidin resulted in stimulation of granulopoiesis (leukocytosis, increased number of [3H]thymidine-labeled myelocytes, elevated serum muramidase level). Animals receiving cytostatics suffered from marked inhibition of granulopoiesis accompanied by decrease of bone marrow granulocyte reserve. Injection of staphylococcal leukocidin during the period of myelosuppression evoked by cytostatics resulted in partial protection of granylopoiesis and faster regeneration of the leukocyte system.

Inhibition of granulopoiesis in diffusion chambers by a granulocyte chalone.

Abstract : Granulocyte chalone obtained from media conditioned by rat granulocytes was tested on normal and vinblastine treated regenerating mouse marrow placed in an in vivo diffusion chamber culture system. Raw granulocyte chalone when injected several times during the early phase of culture significantly altered the cellular growth pattern as well as the parent-progeny relationships of both normal and regenerating marrow established within the chamber milieu. The total number of chamber cells (granulocytes plus macrophages) produced per inoculated stem cell was reduced by approximately 30 percent due to the action of granulocyte chalone. The reduction of granulocyte progeny may be the result of chalone acting during the early phase of culture to reduce the active growth fraction of granulocyte progenitor cells and thereby diminishing the amplification potential inherent in the initial cell inoculum.

Inhibition of granulopoiesis by endogenous granulocyte chalone studied with the diffusion chamber technique

This study showed that growth of granulocytes, both normal and leukaemic, is inhibited in a specific manner in diffusion chamber cultures carried by chloroma-bearing host rats (→ elevated granulocyte chalone content in the body). This inhibition could be demonstrated directly by a reduced formation of granulocytes and by measuring the 3 H-thymidine uptake in the cells. Non-granulocytic cell lines (mastocytoma and HeLa cells) were not detectably inhibited when grown in identical conditions. The inhibition of granulocytic cells did not depend on a selective immunological reaction developed against granulocytic cells in the chloroma-bearing host rats.

PRIMARY SPLENIC NEUTROPENIA

Neutropenia can result from various causes, which include inhibition of granulopoiesis in the bone marrow, arrest of maturation and excessive destruction of neutrophils. It is also a common associated finding in other blood dyscrasias and in some infections and infectious diseases. Neutropenia that arises from depression of bone marrow often is associated with corresponding reduction of other elements formed in the marrow, for example, erythrocytes and platelets. This reduction occurs in the various types of aplastic anemia; in anemia secondary to roentgen and radium irradiation, to exposure to benzene and to the administration of various chemicals, such as derivatives of the benzene ring, arsphenamine, sulfonamide compounds and gold compounds, and in anemia in which the etiologic factor is unknown. The myelophthisic anemias that accompany myeloma, osteosclerosis, Hodgkin's disease, involvement of the marrow by metastatic carcinoma or tuberculosis, leukopenic forms of leukemia and Gaucher's disease often are associated with neutropenia. In