Spontaneous regression of Friend virus-induced erythroleukemia. III. The role of macrophages in regression.

The spontaneous regression of erythroleukemia induced by the RFV strain of Friend virus is a macrophage-dependent process. Functional suppression or elimination of the macrophage population in leukemic mice with silica, carrageenan, anti-macrophage serum, or trypan blue inhibited regression. Prior protection of the macrophages with PVNO allowed regression in silica or carrageenan-treated mice. Macrophage phagocytic activity was inhibited in about half the RFV-induced leukemic mice at 25 to 30 days post virus inoculation. Those animals with normal macrophages regressed, whereas whereas those with inhibited macrophages did not. Progressor mice could be induced to regress by inoculation with normal syngeneic macrophages; other cell types were ineffective. The inhibition of macrophage function in leukemic mice was the result of infection of the macrophages by virus. Removal of the infected cells by cytolysis with anti-virus antiserum and C restored the phagocytic activity of the population. Inhibited macrophages were less capable of responding to immobilized antigen-antibody complexes than normal macrophages, suggesting that the loss of function was due to a change in their Fc receptor.     

Infection of haematopoietic stem cells in mice with Friend virus induced erythroleukaemia

Animals infected with conventional anaemia (FVA) or polycythemia-inducing (FVP) strains of the Friend virus develop lethal erythroleukaemia. A variant strain, RFV, induces an initially identical disease except that it spontaneously regresses in 50% of infected mice. To determine whether pluripotent stem cells as measured by spleen colony forming units (CFU-s) in leukaemic mice are productively infected with virus and whether their infection influences the outcome of the disease, we tested CFU-s from leukaemic mice for susceptibility to cytotoxicity by monospecific antiviral gp70 antiserum. Spleen CFU-s from progressively leukaemic (FVP, FVA and RFV) mice were productively infected with virus. CFU-s in RFV progressors became infected by 40 days post-virus inoculation. FVA and FVP progressors became infected between 15 and 21 days post virus. Infection of CFU-s was accompanied by an increase in the proportion of replicating (S phase) CFU-s in these populations. Spleen CFU-s from fully regressed RFV regressor mice were uninfected and remained so throughout the course of their disease. Bone marrow CFU-s in both regressors and progressors remained uninfected and were not induced to increased cell cycling.     

Infection of Haematopoietic Stem Cells In Mice With Friend Virus Induced Erythroleukaemia

Abstract. Animals infected with conventional anaemia (FVA) or polycythemia-inducing (FVP) strains of the Friend virus develop lethal erythroleukaemia. A variant strain, RFV, induces an initially identical disease except that it spontaneously regresses in 50% of infected mice. to determine whether pluripotent stem cells as measured by spleen colony forming units (CFU-s) in leukaemic mice are productively infected with virus and whether their infection influences the outcome of the disease, we tested CFU-s from leukaemic mice for susceptibility to cytotoxicity by monospecific antiviral gp70 antiserum. Spleen CFU-s from progressively leukaemic (FVP, FVA and RFV) mice were productively infected with virus. CFU-s in RFV progressors became infected by 40 days post-virus inoculation. FVA and FVP progressors became infected between 15 and 21 days post virus. Infection of CFU-s was accompanied by an increase in the proportion of replicating (S phase) CFU-s in these populations. Spleen CFU-s from fully regressed RFV regressor mice were uninfected and remained so throughout the course of their disease. Bone marrow CFU-s in both regressors and progressors remained uninfected and were not induced to increased cell cycling.     

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.     

Cytotoxic T cells in the peritoneal cavity of mice infected with ectromelia virus.

Peritoneal exudate cells from mice infected with ectromelia virus were cytotoxic for virus-infected target cells as measured in a 51Cr release assay. Cytotoxic activity seemed to be T cell-dependent as it was largely abolished by treatment with anti-theta serum and complement but was not impaired by macrophage depletion. The kinetics of development of cytotoxicity in the peritoneal cavity lagged behind spleen cytotoxicity by 1-2 days. Peak activity in peritoneal cells was present about 6 days after intravenous infection with virus. These studies suggest that macrophages present in the free peritoneal cell populations of ectromelia-infected mice are not cytotoxic for virus-infected target cells. The effect of macrophages in virus clearance is therefore likely to be due to phagocytic rather than cytotoxic effects.     

Active immunotherapy of friend virus-induced erythroleukemia and its spontaneous regression

Summary We have tested the effects of specific and nonspecific immunostimulation on the spontaneous regression and recurrence of erythroleukemia induced by a strain of the Friend murine leukemia virus complex, RFV. Subcutaneous inoculation of mice with UV-irradiated allogeneic leukemic spleen cells (LSC) protected against subsequent virus challenge. RFV-leukemic mice injected with LSC into subcutaneous BCG-primed sites had a significantly increased incidence of leukemia regression. When leukemic mice received BCG or LSC alone or normal allogeneic spleen cells (NSC) in place of LSC the incidence of regression was not different from that recorded in untreated controls. A single treatment of recently regressed mice with LSC given into BCG-primed sites prolonged the disease-free period, while LSC alone, BCG alone, or NSC had no such effect. Our data support an immunological basis for spontaneous regression of erythroleukemia and for maintenance of the regressed state. This system provides a model for testing the efficacy of immunotherapeutic protocols for maintenance of leukemia remission and tumor dormancy.     

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.     

Identification of a macrophage-specific cell surface antigen.

A mouse-specific macrophage antigen (MSMA) was identified in NP-40 extracts of 125I-radiolabeled mouse preitoneal macrophages by using a rabbit anti-mouse macrophage serum (AMS) and SDS-polyacrylamide gel electrophoresis. The antigen was shown to have a m.w. of 83,000 daltons and was present on both normal and "activated" peritoneal macrophages. MSMA was also present on syngeneic adherent spleen cells, allogeneic peritoneal macrophages, a mouse macrophage cell line (P388D1), and exhibited some cross-reactivity with peritoneal macrophages from closely related species (rats and hamsters). MSMA was not present on nonadherent peritoneal exudate cells, spleen cells, erythrocytes, thymocytes, or bone marrow cells. Extensive absorptions of AMS with thymocytes and erytrocytes from mice were necessary to remove other antibodies that reacted with other mouse membrane antigens. An antiserum directed against a specific membrane antigen has great potential in elucidating structure-function relationships with regard to a number of macrophage activities.     

Selective induction of enhanced complement receptor 3 activity on murine macrophages

A high proportion of murine resident peritoneal macrophages bear complement receptors 1 and 3 (CR1, CR3) which bind C3b and iC3b components of complement, respectively. By contrast, macrophages derived from bone marrow, blood, and the elicited peritoneal exudate are predominantly CR1+3. To determine if the microenvironment of the normal peritoneal cavity influences CR3 phenotype, we studied the effects of lavage from the cavity on cultures of primary peritoneal exudate macrophages, and on macrophages derived from progenitors in the bone marrow, blood, and peritoneal exudate. The cell-free peritoneal lavage (CFPL), after 24 hr of culture, induced CR3 on primary and culture-derived populations of peritoneal exudate macrophages but had no effect on the CR3 phenotype of macrophages derived from bone marrow or blood. The CR3-inducing activity in CFPL was abolished by heating at 70 degrees C for 30 min and by trypsin, and was not affected by adsorption with EA(IgM)iC3b indicator cells, demonstrating that it is not soluble CR3. Finally, exudate macrophages exposed to CFPL required at least 24 hr before they expressed CR3; such macrophages regenerated CR3 after the receptors were removed by trypsin. The selective effect of the activity in CFPL for peritoneal exudate macrophages indicates that the local microenvironment of the peritoneal cavity can influence the expression of CR3.     

The cell content of peritoneal exudates following the injection of neutral thiol protease into guinea pigs.

The cell content of the peritoneal exudate was examined 4 days after an intraperitoneal injection of glycogen in uninfected and Paragonimus westermani-infected guinea pigs. In uninfected animals a reduction in macrophage count and an accumulation of granulocytes in the exudate were observed at 3 and 6 hr after an intraperitoneal injection of purified neutral thiol protease from P. westermani metacercariae. No such effect occurred after the enzyme was injected into infected animals. At 9 hr after enzyme injection, vacuoles were found in the cytoplasm of macrophages in uninfected animals.     

The regulation of bone marrow-derived mononuclear phagocyte differentiation and mobilization in syngeneic and allogeneic YAC-lymphoma-bearing mice

Abstract. YAC-lymphoma inoculation (ip) into syngeneic A/J mice results in a massive in situ proliferation of the lymphoma cells, with non-significant increases in the peritoneal macrophage population, and also in a remarkable and progressive increase in the bone marrow precursor cells forming both macrophage and fibroblastoid colonies in vitro . In the allogeneic situation (BALB/c mice), YAC-lymphoma cells proliferate to a limited extent before tumour regression. These processes (i.e. proliferation and regression) were accompanied by a progressive increase in the peritoneal macrophage population, which declined to normal after tumour regression. Macrophages accumulating at the regression stage exhibited in vitro cytolytic activity towards YAC-lymphoma cells and cytostatic activity towards unrelated cells. No correlation was observed between the limited asynchronous increase in the bone marrow level of precursor cells for in vitro differentiating macrophage and fibroblastoid colonies, on one hand, and macrophage accumulation at the tumour site in BALB/c mice on the other. Conditioned media from in vitro proliferating YAC-lymphoma cells exhibited high colony-stimulating activity for macrophage colony formation in vitro . Bone marrow cells taken from YAC-lymphoma inoculated A/J mice expressed potent tumorogenic potential at early stages after tumour inoculation. At day 2 after tumour inoculation the bone marrow cells were already tumorogenic and concomitantly expressed augmented levels of macrophage and fibroblastoid colony-forming capacity.
The results indicate an inter-relationship between tumour growth and mononuclear phagocyte differentiation and proliferation.     

Foreign body giant cell induction in the CSF-1-deficient osteopetrotic (op/op) mouse

The osteopetrosis (op) mutation in mice is characterized by generalized skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Macrophage polykaryons are known to be formed by fusion of mononuclear precursors and the presence of subcutaneous implants can elicit the formation of macrophage polykaryons. In order to determine if recruitment of foreign body giant cells is also impaired in osteopetrotic mice, tissue reactions to subcutaneously implanted polyvinyl sponges were studied and compared with normal mice. Our result showed that, in the op mouse, recruitment of macrophages and foreign body giant cells in response to the implants was quantitatively not different from that of normal mice. However, these cells were smaller and did not migrate as deeply into the implant as those seen in normal littermates. In contrast, resident macrophages obtained by peritoneal lavage were significantly reduced in op mice. These data indicate that there is a deficiency in the ability of op mice to mount a foreign body giant cell response to an implanted sponge characterized by a deficiency in the recruitment of precursor cells that are capable of either full development and spreading or migration into the implanted sponge. These data add to the emerging appreciation of the regional differences among macrophage populations in their dependence on CSF-1 for differentiation and survival.     

Regulation of macrophage populations. I. Preferential induction of Ia-rich peritoneal exudates by immunologic stimuli

The amounts of Ia-positive and -negative macrophages were studied in peritoneal exudates of normal mice or of mice injected with various inflammatory materials, infected with Listeria monocytogenes, or injected with hemocyanin. Ia-negative macrophages predominated in exudates from normal mice or from mice given mineral oil, peptone, thioglycollate, culture media, or endotoxin. Infection with Listeria caused a very marked increase in Ia-positive macrophages. The induction of Ia-positive macrophages by Listeria inoculation resulted in great part from an immune process. The Ia-positive exudates were more readily generated in immune mice given a secondary challenge with heat-killed organisms. Furthermore, immune T cells transplanted together with heat-killed organisms into normal mice resulted in Ia-rich exudates. Injection of hemocyanin also induced Ia-rich exudates involving an immune process. We conclude that an immune reaction involving T cells regulates the Ia phenotype of the exudate macrophage population. The Ia-positive macrophages were Fc and C3 receptor positive and phagocytized latex particles.     

The suppressive effect of peritoneal exudate macrophages on production of antibody to sheep erythrocytes in vitro

The effects of peritoneal exudate macrophages on antibody response to sheep erythrocytes (SRBC) were investigated in mice. Peritoneal exudate macrophages obtained from mice injected intraperitoneally with proteose peptone or Corynebacterium parvum 4 days earlier had stronger ability to phagocytize and degrade SRBC than normal resident macrophages. These macrophages suppressed antibody formation to SRBC in vitro as well as in vivo. This suppression was overridden by increasing the amount of SRBC and diminished completely by pretreatment of the macrophages with iodoacetate and partly by pretreatment with 2-deoxyglucose, both known to be inhibitors of phagocytosis, but not by addition of indomethacin to the in vitro culture. These results suggest that the suppression of antibody response by peritoneal exudate macrophages was due to the increased activity of these cells as scavenger cells, resulting in a reduced amount of effective antigenic stimulation, and that it was not mediated by a prostaglandin-dependent mechanism. The scavenger function of these macrophages may be due to Ia-negative macrophages.     

Aging impairs peritoneal but not bone marrow‐derived macrophage phagocytosis

Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age‐related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow‐derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow‐derived macrophages and bone marrow monocytes did not exhibit age‐related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age‐related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell‐derived IL‐10 was increased in resting and LPS‐activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue‐resident peritoneal macrophages, but not by bone marrow‐derived macrophages/monocytes, and suggest that age‐related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.     

Macrophages as effector cells of protective immunity in murine schistosomiasis

Mice infected with Schistosoma mansoni develop a dramatic (five- to eightfold) increase in numbers of peritoneal leukocytes, and approximately 65% of these cells are macrophages. By several biochemical and cytochemical criteria, these cells were comparable to resident peritoneal macrophages of normal mice. However, macrophages from schistosome-infected mice exhibited significant nonspecific tumoricidal activity in vitro, a function associated with immunologically activated cells. The time course for development of activated macrophages in the peritoneal cavity was dependent upon the route of infection. Cytotoxic cells were present in the peritoneal cavity by 3 weeks after intraperitoneal infection, but were not evident until several weeks later in animals infected percutaneously, subcutaneously, or intravenously. However, by 3 weeks after subcutaneous infection, tumoricidal macrophages appeared in the peritoneal cavity after intraperitoneal challenge with soluble schistosome antigens. Macrophage activation was independent of the development of egg granulomas, since tumoricidal cells could be found prior to the onset of egg production and were also present in mice infected with only male worms. Development of activated macrophages in these instances is thus consistent with previous observations on induction of T lymphocyte reactivity toward schistosomula. Since other manipulations known to activate macrophages have been shown to induce partial resistance to schistosome infection, the finding that macrophage activation results from primary S. mansoni infection itself suggests that these cells may play a major role in acquired immunity to this parasite.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.     

Aberrant expression of cytokine genes in peritoneal macrophages from mice infected with LP-BM5 MuLV, a murine model of AIDS.

Mice infected with LP-BM5 murine leukemia virus (MuLV) develop a syndrome denoted as murine AIDS. Macrophages harvested from the peritoneal cavities of these mice at 4 or 9 wk postinoculation with LP-BM5 MuLV were analyzed by Northern hybridization for the presence of the defective LP-BM5 virus and their ability to synthesize various cytokines upon induction with Newcastle disease virus (NDV) or (LPS). Neither IFN-alpha or IFN-beta was found to be constitutively expressed in LP-BM5-infected macrophages and in NDV induction studies, and the levels of biologically active IFN-alpha and its mRNA were found to be lower in LP-BM5 MuLV-infected macrophages than in the macrophages from uninfected controls. Similarly, after NDV or LPS induction, the levels of TNF mRNA and TNF protein were significantly lower in LP-BM5-infected macrophages than in macrophages from uninfected mice. The LP-BM5 MuLV-infected macrophages constitutively expressed low levels of IL-1 beta, and when induced with LPS, the relative levels of IL-1 beta were significantly higher in infected than in uninfected macrophages. Although no constitutive expression of IL-6 was detected, the levels of IL-6 mRNA induced with NDV were higher in LP-BM5 MuLV-infected macrophages than in controls. Thus, we found alterations in the expression of selected cytokines in macrophages from mice inoculated with LP-BM5 MuLV rather than a general deregulation of all cytokine expression. These results show that macrophages infected with the defective LP-BM5 virus respond differently to NDV- or LPS-stimulation and suggest that aberrant expression of certain cytokine genes may play a role in the immunopathologic condition in mice with murine AIDS.     

Impaired macrophage function in Friend virus leukemia: restoration by statolon.

Phagocytic and migratory functions of peritoneal macrophages from Friend virus (FV) leukemic mice are significantly depressed as compared with normal controls. Leukemic macrophages exposed in vivo and in vitro to statolon, an extract of the mold Penicillium stoloniferum, shown previously to suppress FV erythroleukemia, regain normal function and release reduced amounts of FV. Statolon's in vivo restoration of leukemic macrophage function is paralleled by restoration of humoral immune competence. Statolon induces interferon in vitro but its effects on leukemic macrophages are probably direct, since restoration of macrophage function occurs at dosage levels far below those that induce interferon. These studies suggest that macrophages play an integral role in both the pathogenesis and the statolon-induced suppression of FV disease.