Regulatory cells in human bone marrow: Suppression of an in vitro primary antibody response

Human bone marrow (BMC) contains regulatory cells that can suppress the in vitro primary PFC response of normal allogeneic spleen or tonsillar cells and autologous peripheral blood cells. Suppression is dependent upon the dose of BMC added, but is not due to cell crowding nor to excessive cytotoxicity, and requires the presence of viable, metabolically active BMC. BMC are maximally inhibitory when added during the first 24 hr of culture and do not cause an induced shift in the kinetics of the response. Thus, suppression reflects inhibition of early inductive events in the antibody response. The target of suppression is the non-T cell, with either polyclonal activator or Ag being required for maximal suppression. DNA synthesis of normal tonsillar cells is not inhibited by BMC. Characterization of the human bone marrow-suppressor cell has shown it to be radiosensitive, E-rosette negative, Fc receptor positive, and to reside in the large, weakly adherent cell population after velocity sedimentation and in the lymphocyte-depleted fraction after sucrose density gradient separation. Pretreatment of the bone marrow-suppressor cell with anti-human thymocyte serum does not abrogate suppression. We speculate on a possible physiologic role for this cell.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

The immunoregulatory role of bone marrow. II. Characterization of a suppressor cell inhibiting the in vitro antibody response.

The addition of bone marrow cells (BMC) to spleen cell cultures suppressed the antibody response in a dose-dependent manner. This suppression required viable cells. Treatment of BMC with anti-thymocyte serum did not affect the suppressive activity and BMC, but not spleen cells, from nude mice inhibited the antibody response to the same degree as marrow from normal littermates. BMC which had been depleted of macrophages with antimacrophage serum or carbonyl iron showed increased suppressor activity. Furthermore, fractionation of BMC by velocity sedimentation and resetting revealed the suppressor cell to be a medium-to-large Fc receptor-positive lymphocyte. Absence of detectable B or T cell markers on the suppressor cell indicates this cell to be an Fc-positive null lymphocyte, possibly a precursor cell, which inhibits the response of mature lymphocytes     

Chemotaxis of large granular lymphocytes

The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet-derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK-1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide.     

Interleukin 2 inhibits in vitro granulocyte-macrophage colony formation

We have previously shown that murine bone marrow cells cultured with interleukin 2 (IL-2) produce interferon-alpha/beta (MuIFN-alpha/beta) and that IFN-alpha/beta can suppress in vitro granulocyte-macrophage colony-forming cell formation (GM-CFC). In this study, IL-2 was directly assessed for its ability to inhibit in vitro granulocyte and/or macrophage colony-forming cell formation (GM-CFC/M-CFC). C57BL/6 bone marrow cells were cultured with different colony-stimulating factors (CSF), i.e., partially purified macrophage-CSF (M-CSF) or recombinant granulocyte and macrophage CSF (GM-CSF) in the presence or absence of different IL-2 preparations. Partially purified mouse IL-2 or recombinant human or mouse IL-2 (rHuIL-2 and rMuIL-2) totally inhibit GM-CFC and M-CFC formation at 7 days of culture. The level of inhibition mediated by IL-2 was concentration-dependent, with as little as 1 U/ml giving total inhibition of colony formation. The ability of IL-2 to inhibit colony formation was completely abolished by treatment with antisera to IL-2. MuIFN-alpha/beta and MuIFN-gamma appeared to play no role in IL-2-induced myelo-suppression in that addition of antisera to these IFN failed to block IL-2-induced suppression. Myelo-suppression mediated by IL-2 was independent of the concentration of CSF used in the bone marrow cultures. Suppression was also not dependent upon the initial presence of T cells or natural killer (NK) cells. Bone marrow cells depleted of Thy-1+, Lyt-1+, Lyt-2+, NK-1.1+, Asialo GM1+, or Qa-5+ cells were as susceptible to IL-2 induced suppression as untreated or complement-treated bone marrow cells. These results suggest that IL-2 may play an important role in regulating different aspects of hematopoiesis.     

Suppressor cell function of human granular lymphocytes identified by the HNK-1 (Leu 7) monoclonal antibody

The HNK-1 (Leu 7) differentiation antigen defines a subpopulation of human granular lymphocytes with natural killer (NK) and K cell function. In this study, we investigated whether HNK-1+ cells, identified with the monoclonal antibody and purified with a fluorescence-activated cell sorter (FACS), could function as suppressor cells. The results demonstrated that purified HNK-1+ cells efficiently suppressed both PWM-induced IgG production by B cells and T cell proliferation in mixed lymphocyte reactions (MLR). Manifestation of this suppressor cell activity required immune complex activation and was partially sensitive to 2000 rad irradiation. This suppressor cell activity was predominantly mediated by a subset of HNK-1+ cells that have previously been shown to have maximum NK function and lack expression of the E rosette (ER) receptor and T cell antigens (e.g., T3 and T8). Thus, HNK-1+ER- cells suppressed a MLR by an average 52%; HNK-1+ER+ were one-half as efficient, causing an average 23% suppression. For comparison, we also examined the characteristics of Leu 2a+ suppressor T lymphocytes. In contrast to HNK-1+ cells, unactivated Leu 2a+ cells suppressed both B and T cell responses. This suppressor activity was not augmented by immune complex activation and was absolutely radio-sensitive in PWM assays. HNK-1+ cells, especially the HNK+ER- subset, can therefore mediate suppressor cell function in addition to their spontaneous cytotoxic function. Furthermore, some of their suppressor cell properties are distinct from those attributed to other types of suppressor lymphocytes.     

Inhibition of herpes simplex virus type 1 replication in fibroblast cultures by human blood mononuclear cells.

An assay was developed to test the effect of human blood mononuclear cells (MNCs) on herpes simplex virus (HSV) replication. In this assay, human fibroblast monolayers were inoculated with HSV and then cultured with or without blood MNCs. Fewer HSV-infected cells were recovered from human fibroblasts cultured in the presence than in the absence of blood MNCs. This inhibition of viral replication by MNCs was independent of HLA matching between the MNCs and fibroblasts and persisted even when T cells were depleted by antibody and complement. However, depletion of Leu11+ MNCs either by panning or with antibody and complement reduced the ability of the cells to suppress HSV infection, whereas enrichment of Leu11+ cells by fluorescence-activated cell sorting increased the viral suppression. Depletion of OKM1+ MNCs also reduced the viral suppression. After coculturing of MNCs and HSV-infected fibroblasts for 3 days, alpha interferon (IFN) and gamma IFN were detected in the supernatants. Predepletion of Leu11+ MNCs reduced the amount of gamma IFN produced in these cultures. Incubation of the MNCs and HSV-infected fibroblasts with antibody specific for either alpha or gamma IFN resulted in reduced viral suppression. Preincubation of MNCs for 18 h with either interleukin 2 or alpha IFN or for 7 days with antigen increased the suppression of HSV infection. These results suggest that natural killer cells with the Leu11+ phenotype participate in the recognition of HSV-infected cells at a point sufficiently early to interfere with the spread of infection in vitro and may inhibit viral replication by natural killer cell cytotoxicity, by generation of interferon, and by lymphokine-activated killing.     

The immunoregulatory role of bone marrow. IV. Role of an immunoenhancing glycoprotein derived from murine bone marrow

Bone marrow-enhancing factor (B-EF) is the spontaneous product of whole bone marrow cells cultured in serum-free medium for a short term (24-48 hr). The factor is prepared by ultrafiltration of BMC supernates to yield a preparation with a MW of greater than 10,000. Production of the factor is not dependent upon antigenic or mitogenic stimulation of BMC, but is inhibited by treatment of BMC with cycloheximide. B-EF augments the in vitro primary PFC response to SRBC, as well as in vitro secondary IgM and IgG PFC responses to SRBC. Enhancement by B-EF is antigen dependent, genetically nonrestricted, and maximal when present at the initiation of culture. B-EF cannot induce a polyclonal antibody response like the polyclonal activator LPS. B-EF is directly mitogenic for thymocytes, bone marrow, and whole spleen cells, but fails to act as a costimulator of thymocyte proliferation in the presence of Con A. B-EF cannot support the growth of the IL-2-dependent cell line CTLL-2. Since B-EF has not been purified, the supernatant may contain more than one activity. The factor is heat labile at 65 degrees C and is sensitive to enzymatic digestion with trypsin and neuraminidase; this implies that B-EF may be a glycoprotein.     

Phenotypic analysis of a large number of normal human bone marrow sample by flow cytometry

Bone marrow aspirates from 48 healthy donors (34 adults, 14 children) were analyzed by flow cytometry (FACS Analyzer) after purification of low-density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labelling with 23 anti-hematopoietic cell monoclonal antibodies. Based on physical properties, these Ld BMC could be divided into four different populations called E, My, Mo and L, which comprised 14% +/- 9%, 31% +/- 16%, 10% +/- 5% and 45% +/- 14% of these cells, respectively. The phenotypic analysis of these different populations enabled the identification in E, of erythrocytes (Glycophorin A+, Rhesus D+, but negative for early erythroid differentiation markers such as the transferrin receptor (Tf. R) and the FA6-152 antigen); in My of cells of the myeloid lineage (VIM2+, HLA DR-); in Mo of cells of the monocytic lineage (VIM2+, CD14+) plus some myeloblasts (VIM2+, CD14-, HLADR+) and finally in L of a heterogeneous population including: 1. T lymphocytes labelled to the same extent by CD2, CD3, CD5 and CD6 (28% +/- 10%), B lymphocytes assessed by CD19 and CD20 (12% +/- 8%), Pre-B cells (CD10+ = 8% +/- 7%), less than 5% of "natural killer" cells (CD16+ or Leu7+) and finally, less than 6% of myelomonocytes (CD14+ and/or VIM2+). 2. The erythroid lineage (rhesus D+ = 42% +/- 20%, Tf.R+ and FA6-152+ = 32% +/- 12%). 3. Undifferentiated cells or progenitor cells (CD34+ = 7% +/- 5%). 4. Cells unlabelled by any antibodies (approximately 6%). We observed no difference between bone marrow samples from adults or children, with respect to physical properties, and with all but four immunological markers. A significantly higher proportion of B cells (CD19+ and CD10+) (P less than 0.001) and undifferentiated cells (CD34+ and HLADR+) (P less than 0.02) was observed in children. These data, obtained from a large number of bone marrow samples, could be used to quantify the imbalance of some bone marrow disorders.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

Characterization of human large granular lymphocyte subpopulations: comparison of the phenotype of NK cells and of interleukin 2-dependent progenitors of cytolytic effector cells

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.     

Unusual T cell populations in adult murine bone marrow. Prevalence of CD3+CD4-CD8- and alpha beta TCR+NK1.1+ cells

The T cell populations present in normal murine bone marrow have not been previously analyzed in detail, mainly because of their relative rarity. In order to permit such analyses, bone marrow T cells were enriched by depleting Mac1-positive cells, which constitute 65 to 90% of bone marrow cells (BMC), and then studied by two-color flow cytometry. Analysis of the remaining cells revealed that the T cell profile of adult murine bone marrow is markedly different from that of other lymphoid organs. A very high proportion of bone marrow CD3+ cells (approximately one-third) are CD4-CD8-. CD3+CD4-CD8- cells are much more concentrated among BMC T cells than among thymocytes or splenic T cells, suggesting that bone marrow may be either a site of extrathymic TCR gene rearrangement, or a major site to which such cells home from the thymus. The expression of NK1.1 was also evaluated on Mac1-depleted BMC populations. Surprisingly, up to 39% of alpha beta TCR+ BMC were found to express NK1.1. Most alpha beta TCR+NK1.1+ BMC also expressed CD4 or CD8. NK1.1+ alpha beta TCR+ cells represented a much greater proportion of BMC T cells than of other lymphoid (splenocyte or thymocyte) T cell populations. Mac1-depleted BMC of nude mice contained very few cells with this phenotype. These results are consistent with the hypothesis that NK1.1+ alpha beta TCR+ cells are generated primarily in the thymus of normal animals and migrate preferentially to bone marrow, where they may function as regulatory elements in hematopoiesis.     

Natural killing and antibody-dependent cellular cytotoxicity: characterization of effector cells by E-rosetting and monoclonal antibodies

Recent investigations have indicated that the OKM1 hybridoma monoclonal antibody reactive with cells of the myelomonocytic series identifies a subpopulation of human peripheral blood mononuclear cells (PBMNC) which mediate natural and antibody-dependent cellular cytotoxicity (ADCC). However, it was not clear whether this OKM1+ group was heterogeneous with regard to cytotoxic function or the presence of receptors for sheep erythrocytes. Thus, the purpose of the present study was to further define the phenotype of the ADCC effector cell and natural killer (NK) cell using a combination of reactivity with hybridoma antibodies and separation of subsets by sheep erythrocyte rosette (E+) formation. Furthermore, the phenotypes of the NK population were assessed directly by performing two-color immunofluorescent staining on tumor cell conjugates. These studies led to the following conclusions: (1) that NK activity is mediated by both E+ OKM1+ and E- OKM1+ cells; the E+ OKT3+ cell possessed essentially no ADCC or NK activity; (2) that E+ OKM1+ cells mediated more NK activity on a per cell basis than E- OKM1+ cells; this was verified by separating OKM1+ cells on a cell sorter into E+ and E- with the OKT11 monoclonal antibody (anti-E-receptor antibody); (3) that E+ OKM1+ cells mediated both ADCC and NK activity; (4) that the phenotypes of PBMNC forming tumor cell conjugates were (a) OKM1+ (both E-receptor positive and negative) and (b) OKM1- E-receptor positive.     

Natural killer cells are deficient in the surface expression of the complement regulatory protein, decay accelerating factor (DAF)

Decay-accelerating factor (DAF) is a 75,000 m.w. membrane protein that inhibits autologous complement C3 activation at the cell surface. One-color direct immunofluorescence with anti-DAF antibody and cytofluorographic analysis indicates that normal human monocytes and granulocytes are uniform in expression of DAF, whereas 23% of peripheral blood lymphocytes are DAF deficient. A two-color indirect immunofluorescence method, used to define the phenotype(s) of the DAF-deficient lymphocytes, was less efficient in DAF detection and led to overestimation of the fraction of deficient cells. Nonetheless, the difference between DAF expression by natural killer cells, identified by the CD16 and HNK-1 antigens, was marked. DAF deficiency was intermediate in cells expressing the CD2, CD3, CD4, or CD8 markers. On the basis of the phenotypic definition of natural killer cells and their contribution to the lymphocyte population, it is concluded that a uniform deficiency of DAF on natural killer cells accounts for about one-half of the DAF-deficient lymphocytes in peripheral blood of normal donors. The finding of a complete DAF deficiency in the lymphocytes from a patient with a lymphoproliferative disorder with the predominant proliferation of CD2+, CD3+, CD8+, HNK-1+ large granular lymphocytes gives additional support for the association of DAF-deficiency with natural killer cells.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

The distribution of cells expressing a natural killer cell marker (HNK-1) in normal human lymphoid organs and malignant lymphomas

A murine monoclonal antibody, HNK-1, is known to react with some human leukocytes including all natural killer (NK) cells in peripheral blood. The distribution of cells reacting with this antibody (HNK-1+ cell) was studied in human peripheral lymphoid organs, consisting of five lymph nodes, two specimens of gastric mucosa with lymphoid tissue, two tonsils, one appendix, and two thymuses. Fourteen cases of malignant lymphoma (ML) were also examined. For the demonstration of HNK-1+ cells, the peroxidase-antiperoxidase (PAP) bridge method was applied to cryostat sections of these specimens. It was found that in normal lymphoid organs most HNK-1+ cells were located in lymph follicles, especially in germinal centers, and some were found in 'mixed' regions which indicate outsides of both the follicles and T-zones. Amongst the ML, large clusters of HNK-1+ cells were observed only in two cases of follicular lymphoma, although a few scattered HNK-1+ cells were noted in other ML, including five diffuse B-cell lymphomas, six T-cell lymphomas and one null cell lymphoma. The possible significance of these findings is discussed.     

Regulation of the secondary in vitro antibody response by endogenous natural killer cells: kinetics, isotype preference, and non-identity with T suppressor cells

Our previous studies had demonstrated that depletion of endogenous natural killer (NK) cells resulted in an augmented primary antibody response in vivo and in vitro. We have now examined the effect of NK cell depletion on the in vitro secondary response to antigen. Treatment of primed murine spleen cells with anti-NK-1.1 allo-antibody and complement before culture resulted in a significant increase in the magnitude of the antigen-specific plaque-forming cell (PFC) response. This treatment did not affect the proportions of Lyt-2+, L3T4+, or sIg+ cells in the population, however, indicating that the augmentation in PFC was not due to changes in the ratio of T to B cells. Removal of endogenous NK cells had a greater effect on the IgG (indirect) PFC response (100 to 200% increase) than on the IgM (direct) PFC response (25 to 50% increase). In contrast, removal of Lyt-2+ cells before culture affected the IgM and IgG responses similarly. Moreover, the kinetics of augmentation differed between cultures depleted of Lyt-2+ cells and those depleted of NK-1.1+ cells. NK cells appeared to act earlier in the response than did T suppressor cells. The NK-1.1+ cells involved in antibody regulation were not involved in the generation of the in vitro derived T suppressor cells. The conclusion that the regulation of the antibody response by NK-1.1+ cells is distinct from that involving T suppressor cells was confirmed in experiments in which removal of both regulatory cell populations resulted in an increase in PFC that was greater than in cultures depleted of either NK or T suppressor cells.     

Inhibition of mouse bone marrow granulocyte-macrophage colony formation by factors derived from natural killer cells: an in vitro model for hybrid resistance

Investigations were performed to study whether soluble factors produced by NK-cells could mediate "hybrid resistance" in vitro. NK-cells enriched from spleens of B6D2F1 hybrid mice were incubated with parental B6 bone marrow, and the effect of the derived supernatants on the development of granulocyte-macrophage colony forming cells (GM-CFC) was assessed. Cell free supernatants obtained from low density cells (LDC) of B6D2F1 hybrids stimulated with bone marrow cells (BMC) from B6 mice inhibited GM-CFC formation. The inhibition was similar using B6, D2 or B6D2F1 bone marrow cells as the targets for GM-CFC growth. Our findings suggest that NK cells from F1 hybrid mice when stimulated with BMC from B6 mice release inhibitory factors, different from IFN-gamma and that this production may represent a mechanism of natural resistance to parental H-2b bone marrow grafts.     

Cardiac pressure overload initiates a systemic stem cell response

Background aims. Acute cardiac injury results in the activation and recruitment of resident and non-cardiac stem cells. In this study we sought to define the pattern of peripheral stem cells and resident cardiac stem cell (CSC) activation induced acutely by cardiac pressure overload (PO). Methods. PO was induced in mice by transaortic constriction (TAC). CSC, endothelial progenitor cells (EPC), hematopoietic stem cells (HSC) and stage-specific embryonic antigen (SSEA)-1(+) cells were profiled in the heart, spleen and bone marrow after TAC by flow cytometry. Results. The combination of a systemic and local stem cell response resulted in increases in SSEA-1 (+) cells and EPC in the heart 7 and 14 days post-TAC, respectively. Locally, modest SSEA-1(+) proliferation at 4 days preceded the elevated myocardial stem cell number. We observed no significant proliferation of EPC and CSC in the heart. The systemic stem cell response was characterized by a biphasic loss of splenic SSEA-1(+) cells at 2 and 7 days post-TAC and loss of bone marrow and spleen EPC at 4 and 7 days, respectively. Spleen size changed dynamically after TAC. A negligible response of HSC to TAC was observed. Significant EPC and SSEA-1(+) proliferation in the bone marrow and spleen occurred only after their local levels were decreased. Conclusions. Our results demonstrate that an orchestrated systemic stem cell response (EPC and SSEA-1 (+) ) takes place in response to TAC. The increase of SSEA-1(+) cells and EPC in the heart in response to pressure is likely to be because of a combination of local proliferation and stem cell recruitment.