Distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5' -nucleotidase activity

Using a cytochemical assay we were able to show that the peritoneal macrophage population of normal nontreated mice (resident peritoneal macrophages) exhibits a heterogeneity with regard to the expression of the activity of the ecto-enzyme 5′-nucleotidase (5′-N). About 75% of the macrophages express high enzymic activity whereas the remaining 25% express low 5′-N activity. Macrophages accumulating in the peritoneum as a result of an inflammatory response are predominantly of the low activity type. In vitro activation of resident peritoneal macrophages by lymphokines does not result in a decrease in the number of macrophages expressing high enzymic activity though the level of the enzymic activity of these cells is reduced by about 36%. Bone marrow derived mononuclear phagocyte colonies developing in vitro, under liquid culture conditions, from bone marrows of normal mice can be divided into three types with respect to their expression of 5′-N activity: (1) high activity colonies–relatively small colonies in which all the cells express high 5′-N activity (about 20% of the colonies); (2) low activity colonies – relatively large colonies in which all the cells express low 5′-N activity (about 70% of the colonies); and (3) mixed colonies–relatively large colonies in which all the cells express low enzymic activity except for about 8% of cells located at the periphery of the colonies which express high enzymic activity (about 10% of the colonies). During an inflammatory response the frequency of the high activity colonies is significantly reduced. Our results provide evidence for distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5′-N activity and suggest that (1) the resident macrophages derive from a subpopulation of bone marrow precursor cells developing in vitro into high 5′-N activity mononuclear phagocytes, and (2) during an inflammatory response there is a preferential expansion of clones of the low enzymic activity phenotype.     

Infection of macrophages with Friend virus: relationship to the spontaneous regression of viral erythroleukemia.

Resident peritoneal macrophages from normal mice were refractory to infection with the RFV or conventional strains of Friend virus (FV). Stimulation of DNA synthesis in the macrophage population by induction of an exudate in vivo or treatment in vitro with macrophage colony-stimulating factor resulted in productive infection following exposure to virus. Similarly, normal resident macrophages did not become infected in vivo following transfer to leukemic mice, while exudate macrophages did become infected. Bone marrow macrophage stem cells were stimulated to replicate and mature in clonal agar cultures in the presence of colony-stimulating factor. These replicating stem cells could be infected with RFV, as shown by virus production in the resultant progeny macrophages. Transfer of normal resident peritoneal macrophages to leukemic progressor mice caused regression of the disease. In contrast, transfer of normal bone marrow cells was ineffective in causing leukemia regression. During erythroleukemogenesis induced by RFV, macrophage precursor cells in all of the mice became infected with virus. In mice with a progressive and lethal leukemia, mature end-stage macrophages were produced which were also infected with virus. In mice in which the leukemia would later spontaneously regress, the infected stem cells were eliminated and the marrow became repopulated with uninfected cells. The resultant progeny macrophages which appeared in the peritoneal cavity were uninfected and thus capable of participating in or causing leukemia regression.     

Detection of lymphoid leukemia colony-forming cells in abelson virus infected mice: Differences in inbred strains

BALB/c or DBA/2 mice were infected with Abelson murine leukemia virus (A-MuLV), pseudotype Molony murine leukemia virus (M-MuLV). Infection of these mice with 10⁴ focus-forming units of A-MuLV (M-MuLV) induced overt leukemia, detectable grossly or microscopically in 90% of the mice at 20–38 days. However, these methods did not detect leukemia at 17 days or before. Bone marrow cells from A-MuLV-infected leukemic or preleukemic mice were placed in tissue culture in a soft agarose gel. Cells from leukemic or preleukemic BALB/c mice grew to form colonies of 10³ cells or more, composed of lymphoblasts, whereas marrow cells from normal uninfected mice did not. Cells from these colonies grew to form ascitic tumors after intraperitoneal inoculation into pristane-primed BALB/c recipient. Colony-forming leukemia cells could be detected in the marrow of A-MuLV-infected mice as early as 8 days after virus incoluation. The number of colony-forming leukemia cells increased as a function of time after virus inoculation. Colony-forming leukemia cells require other cells in order to replicate in tissue culture. Normal bone marrow cells, untreated or after treatment with mitomycin-C, provide this “helper” function. Only in the presence of untreated or mitomycin-C treated helper cells was the number of colonies approximately proportional to the number of leukemia cells plated. Marrow cells from leukemic BALB/c mice form more colonies than those from leukemic DBA/2 mice. The number of colonies formed per 10³ microscopically identifiable leukemia cells plated was determined to be 2–3 for leukemic BALB/c mice and 0.3 for DBA/2 mice. Cocultivation of leukemic DBA/2 marrow cells with mitomycin-C treated normal BALB/c cells did not increase the number of colonies formed by the DBA/2 leukemic cells. Thus, the decreased ability of DBA/2 leukemia cells to form colonies appears to be a property of the leukemia cell population.     

Development of haematopoietic colonies on the macrophage layer formed in the peritoneal cavity of S1/S1d mice

The colony-forming ability of haematopoietic cells was examined on the macrophage layer formed in the peritoneal cavity of S1/S1d mice. The bone marrow cells of the congenic +/+ mice formed many macroscopic colonies on the macrophage layer of the S1/S1d mice although they did not form macroscopic colonies in the spleens of the same S1/S1d recipients. The size and the differentiation pattern of colonies on the macrophage layer of the S1/S1d mice were comparable to those of the colonies on the macrophage layer of the +/+ mice. There are two possible explanations for these results: (a) The microenvironmental defect of the S1/S1d mice has a more prominent effect on the development of spleen colonies than that of macrophage-layer colonies because 'Steel' locus may not be expressed significantly in the peritoneal macrophages or (b) because the cells that make colonies on the macrophage layer may be more differentiated cells than the multipotential stem cells that make colonies in the spleen.     

DEVELOPMENT OF HAEMATOPOIETIC COLONIES ON THE MACROPHAGE LAYER FORMED IN THE PERITONEAL CAVITY OF S1/S1D MICE

The colony-forming ability of haematopoietic cells was examined on the macrophage layer formed in the peritoneal cavity of S1/S1^d mice. the bone marrow cells of the congenic +/+ mice formed many macroscopic colonies on the macrophage layer of the S1/S1^d mice although they did not form macroscopic colonies in the spleens of the same S1/S1^d recipients. the size and the differentiation pattern of colonies on the macrophage layer of the S1/S1^d mice were comparable to those of the colonies on the macrophage layer of the +/+ mice. There are two possible explanations for these results: (a) the microenvironmental defect of the S1/S1^d mice has a more prominent effect on the development of spleen colonies than that of macrophage-layer colonies because ‘Steel’ locus may not be expressed significantly in the peritoneal macrophages or (b) because the cells that make colonies on the macrophage layer may be more differentiated cells than the multipotential stem cells that make colonies in the spleen.     

Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude-ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA-14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU-C) in short-term bone-marrow cultures. The two cell types that composed the MBA-14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF-1. In the parent MBA-14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen-primed lymph-node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony-stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.     

Plasmodium berghei: influence on granulopoiesis and macrophage production in BALB/c mice

Granulocyte and macrophage progenitor cells forming colonies in vitro (GM-CFC) from bone marrow, spleen, and peripheral blood of BALB/c mice infected with Plasmodium berghei were cultured at various times postinfection in a viscous, 0.8% methylcellulose system. The numbers of GM-CFCs from bone marrow increased gradually during the first week of infection, reaching a maximum around the tenth day of the disease. Subsequently, a rise of GM-CFCs in cultures of nucleated cells from the peripheral blood was observed and, with some delay, in spleen cell cultures also, with a maximum around the end of the second week. After the tenth day of malaria infection a fall of colony frequency in bone marrow-derived cells took place, leading to subnormal values of GM-CFCs during the third week of infection. Subsequently, a decrease in the spleen cell cultures followed, but colony numbers did not fall to normal values. The general increase in GM-CFCs in the different organs was preceded by a rise in serum levels of colony-stimulating activity (CSA), attaining a maximum 1 week after P. berghei inoculation. During the following period the CSA levels fell and reached normal values around the seventeenth day of the disease. Chemotherapy with chloroquine started on the fifteenth day of infection, when GM-CFCs in the bone marrow have dropped to normal values, stopped their further decrease. In the spleen a gradual normalization took more than 2 weeks. A challenge infection evoked an elevation of GM-CFC numbers in the bone marrow and in the spleen during the first 10 days in only about 50% of immune mice. The reaction was immediate in some animals, but generally lower and of shorter duration than during primary infection. The results have indicated that a lethal P. berghei infection in mice caused a transient increase in production of CSA followed by a general recruitment of GM-CFCs in all hemopoietic organs.     

Genetically mast cell-deficient W/Wv mice as a tool for studies of differentiation and function of mast cells

Genetically mast cell-deficient W/Wv mice are useful for the analysis of mast cell biology, especially as recipients of bone marrow cells and skin pieces. Inasmuch as suspension and clonal cultures of mast cells have been developed, we combined these in vivo and in vitro systems. Suspension-cultured mast cells had morphological and biochemical characteristics similar to those of mucosal mast cells (MMC). However, i.p. injection of such cultured mast cells gave rise to development of cells with characteristics similar to those of connective tissue mast cells (CTMC). When peritoneal cells of normal +/+ mice were cultured in methylcellulose, pure mast cell colonies appeared. Cells from individual mast cell colonies were divided and injected into the skin and stomach wall of W/Wv mice; CTMC developed in the skin and MMC in the stomach mucosa. This indicates the presence of a common precursor for CTMC and MMC. Morphology of such bipotent mast cell precursors was studied by using micromanipulation. About 4% of morphologically identifiable peritoneal mast cells may function as the bipotent precursors. Although W/Wv mice showed a defect in resistance against ixodid ticks, injection of suspension-cultured mast cells normalized the defect. The four examples mentioned above indicate that combinations of in vivo and in vitro systems increase the usefulness of W/Wv mice.     

Pre-CFU-f: young-type stromal stem cells in murine bone marrow following administration of DNA inhibitors

Occurrence of young-type stromal stem cells (defined here as "pre-CFU-f") in murine bone marrow is reported in this study. Two consecutive intraperitoneal (i.p.) cytosine arabinoside (ara-C) injections were administered to C57B1 mice (2 X 200 mg/kg at 6-h intervals). Two days later the bone marrow was collected and assayed for colony-forming units-fibroblastoid (defined here as "CFU-f"). In additional experiments, ara-C-treated marrow was exposed in vitro to hydroxyurea (HU; "hydroxyurea killing test"), prior to plating, to establish the cycling state of stromal stem cells. In separate cultures of ara-C-treated marrow, replating of adherent cells was carried out up to quaternary sub-cultures. The results indicate ara-C-treated marrow produces approximately 20% "huge" fibroblastoid colonies (approximately 5 mm diameter versus 0.5-2 mm normal size); most stromal stem cells producing huge colonies are cycling cells; and adherent cells from primary ara-C-treated marrow cultures replated to secondary cultures produce adherent layers with double the number of cells than in the control secondary cultures. We conclude that the ara-C-treated murine bone marrow contains certain young-type cycling stromal stem cells which we refer to as pre-CFU-f. These stem cells produce huge fibroblastoid colonies in culture, indicating that they probably go through more cell cycles than CFU-f during the culture period. Alternatively, pre-CFU-f may have a higher self-replicative capacity than CFU-f.     

COLONY-FORMING ABILITY OF BONE MARROW CELLS OF W ANAEMIC MICE ON MACROPHAGE LAYER FORMED IN PERITONEAL CAVITY OF MICE

The colony-forming ability of haematopoietic cells of W anaemic mice was examined on the macrophage layer formed in the peritoneal cavity of mice. Bone marrow cells of W anaemic mice formed a considerable number of colonies on the macrophage layer, notwithstanding they did not form any colonies in the spleen of the same recipients. As the colony-forming ability of the bone marrow cells was not reduced by the incubation with ³H-thymidine, most of the cells which formed colonies on the macrophage layer seemed to stay in G₀ state. The interrelationship between the spleen colony-forming cells, the macrophage-layer colony-forming cells, and in vitro colony-forming cells was discussed.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

锂对小鼠高增殖潜能集落形成细胞和粒巨噬系祖细胞体外增殖…

本文观察了锂对ＢＡＬＢ／Ｃ小鼠骨髓高增殖潜能集落形成细胞和粒巨噬系祖细胞ＣＦＵ－ＧＭ体外增殖的影响。ＨＰＰ－ＣＦＣ集落由ＩＬ－１,ＩＬ－６,ＷＥＨＩ３条件培养液及Ｌ９２９条件培养液所支持,而ＣＦＵ－ＧＭ由ＷＥＨＩ３－ＣＭ所支持。结果显示,ＬｉＣｌ浓度在０．４－２ｍｍｏｌ／Ｌ时呈现剂量依赖性抑制ＨＰＰ－ＣＦＣ增殖;而在０．４－１ｍｍｏｌ／Ｌ的浓度范围内,则对ＣＦＵ－ＧＭ的增殖起剂量依赖性促进作用。     

In vivo stimulatory effect of macrophage colony-stimulating factor on the number of stroma-initiating cells

The in vivo effect of human macrophage colony-stimulating factor (M-CSF) on the number of cells that formed stromal colonies in an in vitro culture system (stroma-initiating cells; SICs) was investigated. We found that the number of SICs in the femurs of C57BL/6 mice was significantly increased by the treatment with M-CSF. We also found that the SICs were resistant to at least three different chemotherapeutic reagents, 5-fluorouracil (5-FU), cytarabine, and cyclophosphamide, because the femoral cells of mice treated with these reagents contained higher numbers of SICs than those of untreated mice. M-CSF treatment also increased the number of SICs of the reagent-pretreated mice. The SICs detected in our culture system were present only in Mac-1(-)CD45(-) cells, and the M-CSF treatment of 5-FU-pretreated mice actually increased the number of Mac-1(-)CD45(-) SICs. The Mac-1(-)CD45(-) SICs collected from mice that were pretreated with 5-FU and then treated with M-CSF formed stromal colonies under in vitro culture conditions that did not contain M-CSF but did contain a high concentration of fetal calf serum. This result suggested that SICs collected following the treatment procedure did not necessarily require the presence of M-CSF for their in vitro proliferation. Our study indicated that M-CSF has the ability to increase the number of progenitor or precursor cells for bone marrow stromal cells in vivo system.     

Myeloproliferative sarcoma virus: its effects on erythropoiesis in adult DBA/2J mice

Adult susceptible mice (DBA/2J) infected with MPSV (myeloproliferative sarcoma virus), a defective RNA tumour virus, develop splenomegaly and progressive disruption of the haematologic system culminating in death. The present study was specifically directed toward determining the effects of the virus on erythroid differentiation. Early and late precursor cells (erythroid burst-forming units; BFU-E and colony-forming units; CFU-E, respectively) were evaluated by the ability of bone marrow and spleen cells to form colonies of fully differentiated erythroid cells in vitro. MPSV caused substantial modification of both the BFU-E and CFU-E populations in the bone marrow and spleen of infected animals. Changes were detected in the CFU-E population preceding any significant increase in spleen weight. In the bone marrow, the proportion of CFU-E cells increased almost twofold by days 5-10 after virus infection but decreased by day 15. In the spleen, CFU-E frequency rose 40-fold by days 10-15 and then declined steadily prior to death. At the peak of CFU-E expansion, a small proportion of the population appeared to be erythropoietin (Ep) independent, although there was no evidence of a complete switch to Ep-independence which occurs in Friend virus-induced erythroleukemia. Dose-response curves showed that none of these data could be explained in terms of a changing responsiveness to Ep. However, evidence is presented that indicates that BFU-E from MPSV-infected animals lose or have a reduced requirement for burst-promoting activity (BPA) relative to normal cells although their progeny still need Ep for terminal erythroid differentiation.     

Effect of internal contamination with tritiated water on the neoplastic colonies in the lungs,innate anti-tumour reactions,cytokine profile,and haematopoietic system in radioresistant and radiosensitive mice

Tritium is a potentially significant source of internal radiation exposure which, at high levels, can be carcinogenic. We evaluated whether single intraperitoneal injection of BALB/c and C57BL/6 mice with tritiated water (HTO) leading to exposure to low (0.01 or 0.1 Gy) and intermediate (1.0 Gy) cumulative whole-body doses of β radiation is immunosuppressive, as judged by enhancement of artificial tumour metastases, functioning of NK lymphocytes and macrophages, circulating cytokine’s levels, and numbers of bone marrow, spleen, and peripheral blood cells. We demonstrate that internal contamination of radiosensitive BALB/c and radioresistant C57BL/6 mice with HTO at all the absorbed doses tested did not affect the development of neoplastic colonies in the lungs caused by intravenous injection of syngeneic cancer cells. However, internal exposure of BALB/c and C57BL/6 mice to 0.1 and 0.01 Gy of β radiation, respectively, up-regulated cytotoxic activity of and IFN-γ synthesis in NK lymphocytes and boosted macrophage secretion of nitric oxide. Internal contamination with HTO did not affect the serum levels of pro- (IL-1β, IL-2, IL-6, TNF-α,) and anti-inflammatory (IL-1Ra, IL-4, IL-10) cytokines. In addition, exposure of mice of both strains to low and intermediate doses from the tritium-emitted β-particles did not result in any significant changes in the numbers of bone marrow, spleen, and peripheral blood cells. Overall, our data indicate that internal tritium contamination of both radiosensitive and radioresistant mice leading to low and intermediate absorbed β-radiation doses is not immunosuppressive but may enhance some but not all components of anticancer immunity.     

The effect of an antigenic stimulation on proliferative response in athymic mice

The proliferation activity of the main cellular categories of bone marrow after infusion of³H-thymidine was studied in nu/nu and +/+ 1-month- and 3-month-old BALB/c mice in comparison with lymphoid cells in the spleen and mesenteric lymph nodes. The stem cell defect in nu/nu mouse bone marrow is compensated by an, increased proliferation in myeloid series and in agranulocytes. The increase of proliferation activity among lymphoid cells in peripheral lymphoid organs was observed only in the 3-month-old mice with a delay in the nudes.     

Differences in antibody-dependent cellular cytotoxicity against tumor cells in in vitro differentiating mononuclear phagocytes from bone marrows of normal and inflamed mice

Mononuclear phagocytes, differentiated in vitro from bone marrow cells of mice inflamed in vivo with either Corynebacterium parvum or thioglycollate, expressed a higher activity in antibody-dependent cellular cytotoxicity (ADCC) against tumor cells, than those of normal mice. A good correlation between the cytolytic activity and chemiluminescence activity of the different mononuclear phagocyte populations was observed. The ADCC activity of BMDMP from normal mice was inhibited by exogenous prostaglandin E₂ (PGE₂) to a higher extent than that of BMDMP of inflamed mice. When the three BMDMP populations were cultured in the presence of aspirin (without exogenously added PGE₂), the ADCC was significantly increased. The three populations gave identical high values. This suggests that the differential ADCC activity of BMDMP from normal and inflamed mice is due to their differential response to endogenous prostaglandins. PGE₂ showed also a differential effect on the mononuclear phagocyte-forming capacity of bone marrow macrophage precursor cells from normal and inflamed mice. Bone marrow precursor cells from inflamed mice showed a higher resistance to the suppressive effect of PGE₂ (10^?5M) on mononuclear phagocyte-forming capacity than those of normal mice which were totally suppressed. It is concluded that the observed differential properties of the three bone marrow-derived mononuclear phagocyte populations originate at the level of bone marrow precursor cells and that, therefore, the similar functional differences observed in inflammation-induced peritoneal macrophage populations, observed by our and other groups, stem at least partly from differences at the level of bone marrow precursor cells.     

Inflammatory and Phagocytic Response to Experimental Campylobacter jejuni Infection in Mice

After intraperitoneal inoculation with Campylobacter jejuni BALB/c, Swiss and DBA mice show a peritoneal inflammatory response of different intensity. Only BALB/c mice have a strong peritoneal response. Simultaneous intraperitoneal inoculation of C. jejuni plus FeCl₃ increase both inflammatory response and phagocytic activity in Swiss mice, without production of diarrhea. Some thermostable compounds of C. jejuni have a very strong chemotactic activity against peritoneal cells of mice, whereas a diffusible, thermolabile and glutaraldehyde-resistant factor has an inhibitory effect over murine peritoneal cell phagocytosis. Bactericidal activity of peritoneal cells increased after in vitro re-challenge with C. jejuni. Bacteremia is present in all the mice strains tested, but the clearance is quick in DBA and slow in BALB/c and Swiss mice. These experiments confirm that in mice, peritoneal non-specific mechanisms of defense, such as macrophages, play an important role in order to control C. jejuni infection.