In vivo stimulation and inhibition of granulopoiesis: the effect of an inflammatory reaction on murine diffusion chamber granulopoiesis

Humoral factors influencing granulopoiesis have been evaluated using diffusion chambers (DC) implanted in the peritoneal cavity of mice challenged by an aseptic abcess produced by the subcutaneous implantation of copper rods. This resulted in an increase in peripheral blood neutrophils and an increase in tibial granulocytic elements. When DC loaded with bone-marrow cells were implanted into mice stimulated the day before by an aseptic abcess significantly more CFU-s, CFU-c, proliferative and non-proliferative granulocytes were produced, as compared to DC implanted into control hosts. When DC were implanted 4-6 d after the induction of inflammation in mice a significant depression of DC granulopoiesis was observed. Levels of serum and DC fluid CSF and serum inhibitors of in vitro colony growth showed no correlation with DC myelopoiesis. The data show that mice undergoing an inflammatory reaction elaborate first humoral substance(s) enhancing CFU-s and granulocytic growth in DC and next inhibitory factor(s) of DC granulopoiesis.     

Effect of a congenital defect in hemopoiesis on myeloid growth and the stem cell (CFU) in an in vivo culture system.

W/Wv mice with congenitally defective CFU proliferation and their normal, congenic littermates were used as hosts for diffusion chamber (DC) implants. CFU growth in implanted allogenic CF1, or congenic +/+ marrow was significantly greater in W/Wv than in control hosts. When W/Wv mice were "cured" of their hemopoietic defect, CFU proliferation in the DCs decreased, but not to the control level. These observations have provided evidence for humoral control of CFU growth related to a genetic stem cell defect. Diffusion chamber myelopoiesis was also enhanced in W/Wv hosts. In comparison with their congenic controls, W/Wv mice were neutropenic and had decreased numbers of marrow myeloid elements. Thus, a humorally mediated feedback related to a defective myelopoiesis in the hosts might have accounted for increased DC myelopoiesis. However, a "spillover" effect from increased stem cell growth has not been excluded.     

The Effect of Neutropenia on Myeloid Growth and the Stem Cell in an In Vivo Culture System

An in vivo diffusion chamber (DC)culture technique was used to evaluate the role of circulating humoral factors in the control of myelopoiesis.Normal mouse bone marrow cell suspensions sealed in DC’s were implanted intraperitoneally into mice orrats rendered neutropenic by pretreatment with cyclophosphamide, and thegrowth of cells in the DC’s was evaluated at intervals thereafter. When marrow was cultured in hosts with gradedneutropenia, a dose-related augmentation of myelopoiesis was demonstrated. The growth of macrophagesand of pluripotent stem cells,measured by the spleen colony-forming technique, was also enhanced byculture in neutropenic hosts. Thesedata provide further evidence that therate of myelopoiesis is influenced bya circulating humoral factor. Theyfurther suggest that a humoral or hormonal factor is important in controlof the pluripotent stem cell population.

Submitted on March 30, 1972 Revised on May 27, 1972 Accepted on June 2, 1972     

Marrow culture in diffusion chambers in rabbits. I. Effect of mature granulocytes on cell production

Factors influencing granulopoiesis have been evaluated using diffusion chambers implanted in the peritoneal cavity of rabbits. An increase in granulopoiesis in chambers implanted in hosts made neutropenic by nitrogen mustard occurs in mice made neutropenic by x-ray or drug. The intraperitoneal injection of leukocytes inhibited the growth of cells in chambers implanted in rabbits. Removal of mature granulocytes from marrow prior to chamber inoculation produced a marked in-increase in cell growth, especially of granulocytes. Mature granulocytes clearly inhibited cell replication and this inhibition involved both myeloid and erythroid elements, although the data suggest a greater effect on myelopoiesis. In contrast to the mouse, erythropoiesis in chambers in rabbits remained prominent for over 1 wk.     

Culture of autologous bone marrow in diffusion chambers: effect of host irradiation

Autologous bone marrow (BM) cells were cultured in diffusion chambers (DC) implanted into whole-body irradiated, non-irradiated, or sham- irradiated goats. Proliferation was apparent in DC implanted in both irradiated and nonirradiated goats. However, cells in DC cultured in irradiated hosts increased in number beginning earlier, proceeded at a faster rate, and reached higher numbers than in DC in nonirradiated hosts. Growth enhancement could not have occurred as a result of radiation-induced immunosuppression in autologous hosts. The nonirradiated "target cells" in the DC therefore constituted an indicator system for stimulatory or inhibitory substances in the host. The simultaneous increase in the number of granulocytes in peripheral blood and in DC of irradiated hosts was paralleled by an initial rise in serum colony-stimulating factor (CSF). A second, prolonged period of severe granulocytopenia following irradiation of the host correlated with high levels of serum CSF. Increased numbers of megakaryocytes were seen in DC as thrombocytopenia developed in the irradiated host. DC erythropoiesis disappeared rapidly in nonirradiated goats; however, in DC of irradiated goats, the number of erythrocytic precursors increased exponentially during ablation of host erythroid marrow. Anemia did not develop in the host during the culture period. Proliferation of mononuclear cells in DC was markedly stimulated by irradiation of the host. Proliferation of macrophages appeared independent of host treatment. These observations provide strong evidence for diffusion of specific and/or nonspecific humoral hematopoietic stimulators from the host into the DC. This stimulation appears to be elicited and/or intensified by irradiation of the host.     

Leucopoiesis versus concentration of cytokines in diffusion chamber cultures of mouse bone marrow cells: clues to the physiological roles of growth factors

Physiological mechanisms that regulate formation of neutrophil granulocytes, macrophages and their precursor cells were studied with the diffusion chamber (DC) technique. DC inoculated with mouse bone marrow cells were implanted intraperitoneally into host mice. When these in vivo cultures had been established and their marrow populations were expanding (2-day cultures), the DC were transferred to different environments: new, normal mice, lethally irradiated mice, or incubation flasks with optimal concentrations of growth factors. Culture development during the following final culture period was correlated to the concentration of some select candidate growth regulators in DC. After 3 d the cellularity of DC in irradiated hosts had increased significantly more than in the normal hosts. A difference was detectable already after 1-2 d when preculturing was omitted. The increased growth appeared to take place at several stages of cell maturation and not only at the progenitor cell level. Colony stimulating factor(s) for granulocyte and macrophage progenitors, as well as cytokines stimulating the bone marrow-derived cell line, 14 M.1, were present in DC fluid (DCF) at higher concentrations in irradiated than in normal mice throughout the final culture period. On the other hand, DCF concentrations of tumour necrosis-like factor (that may either induce CSF secretion or directly inhibit myelopoiesis), were not significantly different in irradiated compared with normal DC hosts. The cytokines detected in the DC may at least in part stem from inflammatory cells accumulating around the chambers. This animal model should be useful in further investigation of the highly complex regulatory network governing formation of white blood cells in the intact organism.     

Regulation of diffusion-chamber granulopoiesis by colony-stimulating factor

Studies were undertaken to examine the role of colony-stimulating factor (CSF) in controlling murine peritoneal diffusion-chamber (DC) granulopoiesis. Serum CSF was unchanged for 1-3 days after 700-750 rad total body irradiation. Variable increments were noted on the fourth and seventh days after irradiation. DC CSF increased four- to fivefold over serum activity within 24 h of implantation in control mice. An eight- to tenfold increment over serum levels was noted in DC CSF from irradiated mice. Both serum and DC CSF were neutralized completely by repeated injections of anti-CSF serum. DC granulopoiesis was studied by immobilizing marrow cells in plasma clots in peritoneal DC. Repetitive administration of anti-CSF serum or a purified monospecific anti-CSF antibody caused a marked reduction in DC granulopoiesis. This was characterized by a decrease in both the number and size of granulocyte colonies, with a 40%-80% decrease in total granulocytes per chamber. These findings indicate that the granulopoiesis observed in the DC of irradiated hosts is associated with an increase in local rather than systemic CSF production. Neutralization of the CSF reduces proliferation of the granulocyte progenitor cell and maturation of more differentiated granulocytic cells.     

Alteration of colony-stimulating factor output, endotoxemia, and granulopoiesis in cyclic neutropenia.

Cellular and humoral factors involved in the regulation of granulopoiesis were evaluated in two patients with cyclic neutropenia by utilizing the agar-gel marrow culture technique to serially study marrow granulocytic colony-forming capacity (CFC) and the urinary output of colony-stimulating factor (CSF). CSF output varied inversely with peripheral neutrophil counts and directly with monocyte counts and evidence for infection (endotoxemia and/or staphylococcal abscesses). Following autologous infusion of one patient's plasma obtained during a period of neutropenia, increased urinary excretion of CSF occurred concomitant with increments in both marrow CFC and the proportion of granulocytic progenitor cells in DNA synthesis. Neutrophil periodicity was not altered by the administration of the neutropenic plasma. These findings are consistent with the hypothesis that cyclic neutropenia is caused by a quantitatively decreased entry of stem cells or granulocytic progenitor cells into granulopoiesis.     

Inhibition of Myeloid Differentiation by Mature Granulocytes can be Reversed by Humoral Factors

Human mature granulocytes added to in vivo diffusion chamber (DC) cultures of murine bone marrow cells (BMC) led to a late reduction of CFU-C, day 5, and proliferative granulocytes, day 6. Cell free DC fluid harvested on day 4 from chambers with BMC and mature granulocytes slightly reduced granulocyte formation in new DC cultures, but not more than DC fluid from control cultures (BMC). Mice which had carried chambers of either group for 4 d, were not different when used as hosts for new DC cultures. However, they were less able to stimulate cell growth in DC than untreated mice. Transfer of DC to new host mice on day 2 completely reversed the inhibition by mature granulocytes which otherwise could be detected in numbers of CFU-C on day 5 and proliferative granulocytes on day 7. However, CFU-S numbers were reduced on day 5 following reimplantation indicating an increased differentiation of CFU-S. Transfer of DC as late as day 4, rapidly restored the granulocyte numbers in the granulocyte co-culture group suggesting that also more differentiated granulocytes were stimulated to further growth and differentiation. It is proposed that factors from mature granulocytes reversibly inhibit myeloid differentiation. Differentiation can again be induced by factors which reach the DC shortly after intraperitoneal implantation in a mouse.     

Effect of anti-CSF on in vivo hemopoiesis

Studies were undertaken to determine whether colony-stimulating factor in the serum is important in the control of granulopoiesis and monocytopoiesis. Groups of mice were injected with either antiserum to colony-stimulating factor (CSF) or normal rabbit serum every 12 h for intervals of 6-7 days. Antibody treatment did not lead to a reduction in circulating granulocytes or monocytes nor a decrease in marrow cellularity. In further studies, two dose levels of purified anti-CSF were employed; rabbit IgG served as a control. Virtually no effect was observed on blood monocytes, blood granulocytes, or marrow granulocytes; however, antibody treatment did cause a decline in marrow colony-forming cells and a reciprocal increase in erythroid progenitor cells. Immunoassays showed a virtual absence of serum anti-CSF after five days, despite continued administration of the antibody. Modified immunoassays indicated that this was due to development of murine antibodies directed against anti-CSF. In further experiments, mice received the purified antibody or whole antiserum in conjunction with cortisone to reduce murine antibody formation. Despite persistent anti-CSF levels in the serum, blood granulocytes, blood monocytes, and marrow precursor cells were unaffected. These findings suggest that serum levels of CSF do not appear to be responsible for in vivo granulopoiesis or monocytopoiesis.     

Unique differentiation programs of human fetal liver stem cells shown both in vitro and in vivo in NOD/SCID mice.

Comparative measurements of different types of hematopoietic progenitors present in human fetal liver, cord blood, and adult marrow showed a large (up to 250-fold), stage-specific, but lineage-unrestricted, amplification of the colony-forming cell (CFC) compartment in the fetal liver, with a higher ratio of all types of CFC to long-term culture-initiating cells (LTC-IC) and a lower ratio of total (mature) cells to CFC. Human fetal liver LTC-IC were also found to produce more CFC in LTC than cord blood or adult marrow LTC-IC, and more of the fetal liver LTC-IC-derived CFC were erythroid. Human fetal liver cells regenerated human multilineage hematopoiesis in NOD/SCID mice with the same kinetics as human cord blood and adult marrow cells, but sustained a high level of terminal erythropoiesis not seen in adult marrow-engrafted mice unless exogenous human erythropoietin (Epo) was injected. This may be due to a demonstrated 10-fold lower activity of murine versus human Epo on human cells, sufficient to distinguish between a differential Epo sensitivity of fetal and adult erythroid precursors. Examination of human LTC-IC, CFC, and erythroblasts generated either in NOD/SCID mice and/or in LTC showed the types of cells and hemoglobins produced also to reflect their ontological origin, regardless of the environment in which the erythroid precursors were generated. We suggest that ontogeny may affect the behavior of cells at many stages of hematopoietic cell differentiation through key changes in shared signaling pathways.     

Control mechanisms of endotoxin and particulate material stimulation of hemopoietic colony forming cell differentiation.

Through injection of particulate materials and endotoxins (ET), possible control mechanisms of murine hemopoietic colony forming cells (CFC) were investigated. The experimental system employed two inbred strains of mice that responded with a similar increase in splenic CFC after injection of particulate materials, sheep red blood cells (SRBC) and colloidal carbon (cc), but differed in their response to ET. The strains similarly phagocytosed radiolabelled SRBC and ET, suggesting that the CFC changes were not dependent solely on phagocytosis of ET. The CFC from low ET responder strain responded in vitro to colony stimulating factor (CSF) elicited by ET in serum of the high responder strain, demonstrating that lack of response to ET is due, at least in part, to failure to produce CSF. Subjection of serum from either strain to chloroform extraction did not significantly increase its stimulating capacity, suggesting that the increased stimulatory ability in response to ET was due to increase in the CSF level rather than to a decrease in the level of inhibition.     

The marrow colony forming cell and serum colony stimulating factor of the chicken.

Marrow cells of the chicken produced colonies in semisolid media. Developing colonies consisted of granulocytes, macrophages or a mixture of these two cell types. The granulocyte-macrophage CFC was nonadherent. An adherent 'CFC' was also present and it differed in several ways from the nonadherent CFC: (a) clones contained only macrophages, (b) they contained a core of nonrefractile cells, (c) their appearance was delayed 1-2 weeks, (d) they were unaffected by the presence of erythrocytes and (e) the efficiency of cloning was increased but the percentage of clones able to produce 50 or more cells was markedly decreased, i.e., the cluster/colony ratio was increased. The growth of both colony types was strictly dependent on the presence of CSF. Data obtained from dose-response studies on unfractionated marrow indicated that clusters and colonies were derived from single cells. The CSF of chicken serum yielded sigmoid dose-response curves when tested on marrow cells. Calf serum could not support cluster or colony formation when tested alone but it did have an enhancing effect on the CSF of chicken serum. Levels of serum CSF were increased by injecting chickens with bacterial endotoxin. This phenomenon occurred with five chicken lines tested, but certain chickens of the Kimber line did not respond to endotoxin with elevated levels of CSF.     

Marrow culture in diffusion chambers in rabbits. II. Effect of competing demands for red cell and white cell production on cell growth

Studies were done of cell production by marrow in diffusion chambers implanted in the peritoneal cavity of rabbits subjected to various stimuli to hematopoiesis. In chambers in neutropenic hosts and in hosts injected with endotoxin, animals presumed to have an increased stimulus to granulopoiesis, there was increased production of granulocytes but there was also increased production of red cells. Although red cell production was decreased in chambers in polycythemic hosts, granulocyte production was not different from that in controls. Stimulation of erythropoiesis by erythropoietin injections or by exposure to hypoxia increased red cell production by marrow in the implanted diffusion chambers without diminishing granulopoiesis. Only in chambers in hosts made anemic by bleeding was there an increase in red cell production accompanied by a decrease in granulocyte production. In these anemic hosts induction of neutropenia led to an increase in granulopoiesis without any depression of erythropoiesis.     

Erythropoietin, thrombopoietin and colony stimulating factor in fetal mouse liver culture media.

Fetal mouse livers, days 13 to 19 of gestation, were cultured for 21 days and the culture media tested for erythropoietin (Ep), thrombopoietin (TSF) and colony stimulating factor (CSF). High levels of both Ep and CSF were released into the culture media. However, no detectable TSF was found. Maximum Ep culture activity was detected in day 14 and day 15 fetal liver cultures while maximum CSF was found in the day 16 fetal liver culture. These studies indicate that fetal liver cells in culture are capable of producing and/or releasing both Ep and CSF but not TSF.     

Antimyeloma efficacy of thalidomide in the SCID-hu model

To determine the mechanism of thalidomide's antimyeloma efficacy, we studied the drug's activity in our severe combined immunodeficiency-human (SCID-hu) host system for primary human myeloma. In this model, tumor cells interact with the human microenvironment to produce typical myeloma manifestations in the hosts, including stimulation of neoangiogenesis. Because mice are not able to metabolize thalidomide efficiently, SCID-hu mice received implants of fetal human liver fragments under the renal capsule in addition to subcutaneous implants of the fetal human bone. Myeloma cell growth in these mice was similar to their growth in hosts without liver implant, as assessed by change in levels of circulating human immunoglobulins and by histologic examinations. Thalidomide given daily by peritoneal injection significantly inhibited myeloma growth in 7 of 8 experiments, each with myeloma cells from a different patient, in hosts implanted with human liver. In contrast, thalidomide exerted an antimyeloma effect only in 1 of 10 mice without liver implants. Microvessel density in the untreated controls was higher than in thalidomide-responsive hosts but not different from nonresponsive ones. Expression of vascular endothelial growth factor by myeloma cells and by other cells in the human bone, determined immunohistochemically, was not affected by thalidomide treatment in any experiment. Our study suggests that thalidomide metabolism is required for its antimyeloma efficacy. Although response to thalidomide was strongly associated with decreased microvessel density, we were unable to conclude whether reduced microvessel density is a primary result of thalidomide's antiangiogenic activity or is secondary to a lessened tumor burden.     

Colony-stimulating factor in patients with chronic neutropenia.

Urinary and serum colony-stimulating factor (CSF) levels were measured in 11 patients with chronic idiopathic neutropenia without infections and in 10 normal individuals. Urinary CSF output was determined using mouse marrow target cells, and serum CSF activity was assayed with human marrow target cells by the double agar layer technique. Using these methods, there was no significant difference between CSF levels of neutropenic and normal subjects. These data indicate that CSF levels are not inversely related to the blood neutrophil count in chronic idiopathic neutropenia and suggest that CSF is not a hormone regulating the blood neutrophil count in a manner analogous to the erythropoietin regulation of circulating erythrocyte levels.     

Hemopoietic stem cells in murine embryonic yolk sac and peripheral blood.

Disaggregated embryonic yolk sac cells and circulating peripheral blood cells were obtained from normal murine day 9 embryos, prior to the formation of the fetal liver. These cells were microinjected transplacentally into days 11-15 W mutant anemic fetuses, when the fetal liver was the major hemopoietic organ. In a small proportion of the recipient animals examined after birth, long-term repopulation by the embryonic donor hemopoietic cells was observed. The donor hemopoietic stem cells proliferated and differentiated in the hosts as evidenced by the presence of donor hemoglobins in the growing recipient host animals. Some mothers of the pups were also repopulated by the donor stem cells. These results provide direct evidence that, during early murine embryogenesis, there are functional hemopoietic stem cells which are capable of colonizing the adult hemopoietic organs and probably the fetal liver and spleen to initiate hemopoiesis in these tissues.     

Erythroid differentiation of fetal, newborn and adult haemopoietic stem cells.

Erythroid regeneration was studied in lethally irradiated mice given transplants containing equivalent numbers of haemopoietic stem cells (i.e. CFU) from fetal liver, neonatal marrow or adult marrow. Adult marrow was taken from normal control mice, whose CFU for the most part were not in active cell cycle, as well as from phenylhydrazine-treated groups whose CFU were in similar state of proliferation (i.e. approximately 40-50% in DNA SYNTHESIS) AS THOSE DERIVED FROM FETAL LIVER AND NEONATAL MARROW. Splenic and femoral radioiron (59Fe) incorporation were measured at intervals after transplantation and were found to begin earliest in mice given fetal liver, then in animals given neonatal marrow and latest in recipients of adult marrow. Peripheral reticulocytes showed a similar pattern of recovery. The data reported herein suggest that the differences in erythroid regeneration evoked by transplants of fetal liver, neonatal marrow or adult marrow, are not solely attributed to the degree of proliferation in the pluripotential stem cell compartment. These data may, however, suggest a shorter doubling time for cells comprising the fetal and newborn committed erythroid compartments.     

Congenital neutropenia: Studies of pathogenesis

Congenital neutropenia (CN) was diagnosed in a five-month-old boy. A variety of studies was performed to define the pathogenesis of his disease. Opsonic antineutrophil antibodies were present in his serum. Transfused normal granulocytes circulated poorly. Incubation of the patient's serum with normal granulocytes failed to alter their metabolic or functional activity. The patient's marrow demonstrated increased numbers of colony-forming units (CFUs) in vitro compared with control marrow. The patient's parents had low marrow CFU activity. The patient's serum and peripheral lymphocytes failed to inhibit normal marrow CFU activity. The patient's marrow did inhibit CFU growth of an HLA-identical-sibling's marrow in coculture. Histocompatibility studies demonstrated the HLA-B12 antigen in this patient, a histocompatibility marker previously associated with CN. These studies suggest some cases of CN are associated with a genetically transmitted marrow factor capable of suppressing myelopoiesis in normal marrow.