Hemopoietic colony studies. VI. Increased eosinophil-containing colonies obtained by antigen pre-treatment of irradiated mice reconstituted with bone marrow cells

The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.     

Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation.

Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.     

Evaluation of the marrow culture on the macrophage layer covering intraperitoneally implanted membrane as an assay for hemopoietic progenitor cells

When cellulose acetate membranes are implanted into abdominal cavity of mice they turn into a foreign body overgrown with macrophages. Such macrophage layer has been shown by other authors to be able to support the growth of hemopoietic colonies formed by intraperitoneally injected hemopoietic cells. This study confirms and extends this observation by showing that both granulopoietic and erythropoietic colonies may be observed. The number of colonies grown is in linear correlation with that of injected hemopoietic cells. The frequency of erythropoietic colonies was greatly enhanced by blood letting of the host mice. Colony forming cells were most numerous in the bone marrow then in the spleen and peripheral blood and hardly in the thymus. Prior irradiation of the host mice was essential for obtaining colony growth and the optimal dose was determined to be 6.0 Gy. This technique opens the way to studies into hemopoietic progenitor cells for laboratories having no sophisticated tissue culture equipment and where necessary reagents are easily available.     

Hemopoietic origin of certain decidual cell precursors in murine pregnancy

The possible hemopoietic origin of certain precursors of uterine decidual cells appearing during normal murine pregnancy was investigated in semiallogeneic hemopoietic chimeras with retained or regained fertility. Chimeras were produced by three different methods in two donor-host combinations: F1 [BALB/c female x C3H/HeJ male] cells introduced into the parental strain BALB/c female hosts or F1 [CBA/J female x C57BL/6 male] cells introduced into CBA/J female hosts. Prenatal chimeras (PN) were made by reconstituting mouse fetuses (day 13-17) with 10(6)-10(7) adult bone marrow or fetal liver cells through the yolk sac and they were allowed to be delivered naturally. Neonatal chimeras (NN) were made by injecting 1-2 x 10(7) adult bone marrow cells into the anterior facial vein of neonatal mice (less than 24 hr old). In both cases, experimental animals were raised to maturity. Ovary-transplanted chimeras (OT) were made by injecting 10(7) bone marrow cells into lethally irradiated (9.5 Gy) young adult female mice, followed 6 weeks later with bilateral orthotopic transplants of syngeneic ovary grafts to restore fertility. All female chimeras produced by the three different methods were mated with syngeneic male partners to produce normal pregnancy. The extent of chimerism at the cellular level was determined in all cases by a radioautographic identification of the H-2 phenotype of splenic lymphocytes and decidual cells and macrophages in the collagenase-dispersed decidua at day 11-16 of normal pregnancy, following a sandwich labelling with monospecific anti-H-2 antibodies and 125I-protein A. Morphological discrimination of typical decidual cells from macrophages in the collagenase-dispersed decidua was carried out on the basis of several distinctive markers: presence of surface Dec-1 and Thy-1 and absence of surface F4/80 or latex phagocytosis for decidual cells, in contrast to macrophages which were phagocytic and expressed F4/80 but not Dec-1 or Thy-1. While the degree of hemopoietic chimerism (judged by the incidence of donor-derived lymphocytes in the spleen) varied from animal to animal, in all three groups (PN, NN, and OT) comprising a total of 26 chimeras, the percentage of typical decidual cells expressing donor H-2 phenotype showed an excellent correlation with that for small lymphocytes in the spleen. These results reveal that at least a subpopulation of typical decidual cells of the pregnant uterus has a hemopoietic genealogy. A possible familial relationship of these cells to granulated metrial gland cells remains unclear.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. III. Significance of hemopoietic stem cells for induction and maintenance of Mls tolerance by continuous supply of tolerance-inducing nonlymphocytes

The role of hemopoietic stem cells and other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semi-allogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-1-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-10-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes.     

Protein-energy malnutrition decreases the expression of TLR-4/MD-2 and CD14 receptors in peritoneal macrophages and reduces the synthesis of TNF-alpha in response to lipopolysaccharide (LPS) in mice

Protein-energy malnutrition (PEM) modifies resistance to infection, impairing a number of physiological processes, including hematopoiesis. In this study, we examined a few aspects of the inflammatory response to LPS in a model of PEM. We evaluated the cellularity of the blood, bone marrow and spleen, as well as phagocytic, fungicidal and spreading activity, the production in vivo and in vitro of TNF-alpha, IL-1alpha and IL-6, and the expression of CD14 and TLR-4/MD-2 receptors in macrophages. Two-month-old male Swiss mice were submitted to PEM with a low-protein diet containing 4% protein as compared to 20% protein in the control diet. When the experimental group had attained about 20% loss of their original body weight, they were used in the experiments. Malnourished animals presented anemia, leucopenia and severe reduction in bone marrow, spleen and peritoneal cavity cellularity. The production of TNF-alpha, IL-1alpha and IL-6 stimulated in vivo with LPS and the production of IL-6 in bone marrow cells cultured with LPS and the production of TNF-alpha in bone marrow, spleen and peritoneal cells cultured with LPS were significantly lower in malnourished animals. The expression of CD14 and TLR-4/MD-2 receptors was found to be significantly lower in macrophages of malnourished animals. These findings suggest that malnourished animals present a deficient response to LPS. The lower expression of the CD14 and TLR-4/MD-2 receptors may be partly responsible for the immunodeficiency observed in the malnourished mice. These data lead us to infer that the nutritional state interferes with the activation of macrophages and with the capacity to mount an immune response.     

Effect of mononuclear phagocytes on the differentiation of syngeneic, allogeneic and xenogeneic hematopoietic stem cells

The colony formation in spleen of lethally irradiated syngeneic or hybrid recipients was studied after transplantation of bone marrow cells, with or without macrophages from lymph nodules or from peritoneal cavity of mice, cells of macrophage-like cell line J-774, and monocytes from peripheral blood of healthy donors. The direction of stem cell differentiations in the presence of all the types of mononuclear phagocytes was seen to change from mainly erythroid to mainly myeloid one. The ratio of erythroid to myeloid colonies became equal to 0.5-0.9 instead of 2.0, when bone marrow cells were injected with equivalent quantity of mononuclear phagocytes. This new regulatory function of mononuclear phagocytes is discussed.     

Il-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice

Autologous and allogeneic bone marrow transplantations have evolved as important cancer therapy modalities. For both indications, peripheral blood has been shown to have distinct advantages over bone marrow as the stem cell source. Cytokine combinations for mobilization have enhanced stem cell yield and accelerated engraftment. However, novel mobilizing agents and strategies are needed to further improve clinical outcomes. Within the donor graft, the dynamic equilibrium between T cells and stem cells critically influences engraftment and transplantation results. IL-17 is a cytokine produced almost exclusively from activated T cells. IL-17 was expressed in vivo with adenovirus technology. Here, proof-of-principle studies demonstrate that IL-17 effectively mobilizes hemopoietic precursor cells (CFU-granulocyte-erythrocyte-macrophage-monocyte, CFU-high proliferative potential) and primitive hemopoietic stem cells (Lin(-/low)c-kit(+)Sca1(+)). Moreover, mouse IL-17 adenovirus-mobilized peripheral blood stem cells rescued lethally irradiated mice. Bone marrow was found to be 45-75% of donor origin at 1 year. In secondary recipients, donor-derived bone marrow cells ranged from 45 to 95%. These data show that IL-17 mobilizes stem cells in mice with short- and long-term reconstituting capacity. Additional comparative studies are needed as well as studies in tumor models to refine distinct potential clinical applications for IL-17-mobilized peripheral blood stem cells.     

Immunofluorescent study of the hemopoietic organs of xenogeneic mouse radiation chimeras]

An immunofluorescent study of hemopoietic organs in xenogenic (mouse-rat) radiation chimaeras has been carried out by means of specific antiserum against hemopoietic cells of the rat bone marrow. The presence of donor cells was tested at different times after the transplantation in the bone marrow, spleen, lymph nodes, thymus and liver of radiochimaeras. The transplanted cells were shown to populate all hemopoietic organs of the recipient, first of all tissues of the bone marrow type and, then, lymphoid organs. The donor (bone marrow) origin of the extramedullar foci of hemopoiesis in the liver was established.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

Prevention of chronic rejection in murine cardiac allografts: a comparison of chimerism- and nonchimerism-inducing costimulation blockade-based tolerance induction regimens

We have previously described a nonirradiation-based regimen combining costimulation blockade, busulfan, and donor bone marrow cells that promotes stable, high level chimerism, deletion of donor-reactive T cells, and indefinite survival of skin allografts in mice. The purpose of the current study is to determine the efficacy of this tolerance regimen in preventing acute and chronic rejection in a vascularized heart graft model and to compare this regimen with other putative tolerance protocols. Mice receiving costimulation blockade (CTLA4-Ig and anti-CD40 ligand) alone or in combination with donor cells enjoyed markedly prolonged heart graft survival and initially preserved histological structure. However, tolerance was not achieved, as evidenced by the eventual onset of chronic rejection characterized by obliterative vasculopathy and the rejection of secondary skin grafts. In contrast, following treatment with costimulation blockade, busulfan, and bone marrow, heart grafts survived indefinitely without detectable signs of chronic rejection or structural damage, even 100 days after placement of a secondary donor skin graft. We detected multilineage chimerism in peripheral blood, spleen, lymph nodes, and thymus, and peripheral deletion of donor-reactive cells was complete by day 90. These findings indicate that only the CD40/CD28 blockade chimerism induction regimen prevents both acute and chronic rejection of vascularized organ transplants. Further testing of these strategies in a preclinical large animal model is warranted.     

Precursor cells of newly formed connective tissue in inflammatory foci in various organs in mice

The origin of fibroblast-like cells of the capsule around a foreign body in the spleen, liver, peritoneal cavity, subcutaneous connective tissue of mice, the localization of cells-precursors, their proliferative potencies and the ability to migrate through blood were studied using 3H-thymidine autoradiography. Precursors of the inflammation focus cells (irrespective of localization) reproduce intensively outside the limits of intraorganic connective tissue, supposedly, in hemopoietic organs of the bone marrow type and migrate, through the blood channel, into tissues (inflammation foci), where they terminate their differentiation.     

Production of donor T cells is critical for induction of donor-specific tolerance and maintenance of chimerism

Nonmyeloablative conditioning has significantly reduced the morbidity associated with bone marrow transplantation. The donor hemopoietic cell lineage(s) responsible for the induction and maintenance of tolerance in nonmyeloablatively conditioned recipients is not defined. In the present studies we evaluated which hemopoietic stem cell-derived components are critical to the induction of tolerance in a total body irradiation-based model. Recipient B10 mice were pretreated with mAbs and transplanted with allogeneic B10.BR bone marrow after conditioning with 100-300 cGy total body irradiation. The proportion of recipients engrafting increased in a dose-dependent fashion. All chimeric recipients exhibited multilineage donor cell production. However, induction of tolerance correlated strictly with early production of donor T cells. The chimeras without donor T cells rejected donor skin grafts and demonstrated strong antidonor reactivity in vitro, while possessing high levels of donor chimerism. These animals lost chimerism within 8 mo. Differentiation into T cells was aborted at a prethymic stage in recipients that did not produce donor T cells. Moreover, donor Ag-driven clonal deletion of recipient T cells occurred only in chimeras with donor T cells. These results demonstrate that donor T cell production is critical in the induction of transplantation tolerance and the maintenance of durable chimerism. In addition, donor T cell production directly correlates with the deletion of potentially alloreactive cells.     

Allogeneic chimerism in scid mice after neonatal transfer of bone marrow.

Allogeneic chimeras are valuable tools for studies of complex immune cell interactions in vivo. Mice with severe combined immune deficiency (scid) should be ideal hosts for chimerism with allogeneic bone marrow cells as these animals lack mature T and B lymphocytes capable of reacting against donor alloantigens. However, it has been difficult to achieve full reconstitution of adult scid mice even using coisogenic bone marrow grafts without prior irradiation of the recipient. We explored ways to generate complete reconstitution of scid mice with allogeneic bone marrow. Unirradiated adult scid recipients of allogeneic bone marrow were only marginally reconstituted. Adult scid mice pretreated with 250 R were reconstituted with allogeneic bone marrow as measured by serum IgM concentration, peripheral lymphoid cellularity, and mitogen responses, but a potentially important immunologic deficit was found in these mice: 250 R caused a 70% loss of scid macrophages and dendritic cells which persisted at least 5 months. By contrast, when scid mice were injected i.p. with allogeneic bone marrow within the first 24 h after birth, rapid and complete reconstitution of both T and B cell lineages was achieved, and the animals had APC that were both donor and host in origin. Considering the extent and duration of engraftment (43 wk) by allogeneic cells in neonatally transplanted scid mice, it was anticipated that their bone marrow would be chimeric. However, the bone marrow contained very few donor-derived cells, suggesting that lymphopoiesis may be taking place in other organs in these chimeras.     

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.     

The role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. I. Induction of regulatory macrophages in normal mice by the in vivo administration of rGM-CSF

Using a dimethylbenzanthracene-induced immunogenic nonmetastatic murine mammary adenocarcinoma in BALB/c mice, our previous work has shown that splenocytes from tumor bearers have reduced responses to both mitogens and Ag including tumor-associated Ag. NK and cytotoxic T cell activities are also reduced in splenocytes of tumor bearers. Mac-1+2+ macrophages induced in mammary tumor bearers are capable of down-regulating lymphocyte responses to mitogens and tumor-associated Ag by cell to cell contact interaction and increased PGE2 production. We have found that the tumor constitutively releases a granulocyte-macrophage (GM)-CSF-like factor in vivo and in vitro, which may be responsible for the systemic increase in cells of the macrophage lineage in tumor-bearing mice. A tumor cell line established from the in vivo tumor expresses and releases GM-CSF as shown by Northern and Western blot analyses. Daily i.p. injections for 3 wk of 10,000 U of rGM-CSF into normal mice induces hemopoietic and immunologic alterations similar to those observed in tumor bearers. Mac-1+ and/or Mac-2+ macrophages can also be detected in the spleens and bone marrow of the mice treated with rGM-CSF. Additionally, splenocytes from rGM-CSF-treated mice have reduced responses to mitogens and their peritoneal exudate cells can cause in vitro down-regulation of proliferative responses of lymphocytes from normal mice. The suppression can be partially reversed by the addition of indomethacin to the cultures suggesting that PGE2 may contribute to the effect. rGM-CSF enhances the in vitro release of PGE2 by the spleen, bone marrow, and peritoneal cells of normal mice. These data indicate that the high levels of GM-CSF constitutively produced by the tumor may be responsible for the hemopoietic changes and immunologic alterations observed in tumor-bearing mice.     

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.     

Host natural suppressor activity regulates hemopoietic engraftment kinetics in antibody-conditioned recipient mice

Resistance to semi-allogeneic or syngeneic hemopoietic stem cell engraftment can be reduced by treating the unirradiated host with anti-class I MHC antibody. In our previous studies we showed a direct correlation between such resistance and the level of natural suppressor (NS) activity in the host. Thus newborn mice that have high NS activity are very resistant to marrow engraftment, as are adults pretreated with CFA that increases NS activity in the bone marrow. We have now devised a method that allows us to follow hemopoietic engraftment kinetics within the marrow cavity itself by assaying individual CFU-granulocyte/macrophage progenitor cells for their host or donor origin over the immediate post-transplant period. By using this method, we find a close correlation between the rate of marrow engraftment and reduction in host NS activity. Marrow engraftment does not correlate with the reduction of either total host bone marrow cellular content or CFU-granulocyte/macrophage progenitor cell levels. NS activity is mediated by Thy-1-, partially radiosensitive, nylon wool nonadherent cells without NK activity. Adoptively transferred Thy-1-, irradiated spleen cells containing NS activity induced by pretreatment with CFA delayed engraftment kinetics in the marrow cavity. Thus hemopoietic engraftment in the marrow cavity appears to be controlled by an inhibitory regulatory activity that is reflected in the in vitro NS assay. These studies suggest new regulatory targets for selective host conditioning to eliminate resistance to marrow transplantation.     

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.     

Efficient natural defense mechanisms against Listeria monocytogenes in T and B cell-deficient allogeneic bone marrow radiation chimeras. Preactivated macrophages are the main effector cells in an early phase after bone marrow transfer

Radiation chimeras in the early phase after bone marrow transplantation are a good model to study the efficiency of the body's nonspecific defense system represented by macrophages (M phi), polymorphonuclear cells (PMN), and NK cells. These cell types are present in large numbers in spleen and liver at that time, whereas the specific immune system represented by T and B cells is functionally deficient. We previously reported enhanced activities in vitro of M phi (and PMN) from recipient animals in an early phase after allogeneic bone marrow transfer. We here demonstrate that these activities result in enhanced spontaneous resistance against Listeria monocytogenes in vivo: CFU of L. monocytogenes in spleen and liver 48 h after infection were about 1 or 2 to 4 log steps less than in untreated control mice of donor or host haplotype. This enhanced resistance decreased over the 4-mo period after marrow transfer. Preactivated M phi were identified as the most important effector cells. Isolated from spleen and peritoneal cavity, they performed enhanced killing of phagocytosed Listeria. Such preactivated M phi occurred in recipient animals after transfer of allogeneic but not of syngeneic bone marrow. The precise mechanism of M phi activation in the allogeneic radiation chimera in the complete absence of any detectable T cell function is not clear at present. However, these preactivated M phi display an important protective effect against L. monocytogenes: chimeras could eliminate Listeria without acquisition of positive delayed-type sensitivity when infected with 10(3) bacteria. An inoculum of 5 . 10(3) L. monocytogenes resulted either in prolonged survival compared with normal mice of the recipient haplotype or in definitive survival accompanied by a positive delayed-type sensitivity. We concluded that enhanced nonspecific immune functions can in part compensate for the defective specific immune system after bone marrow transfer.