Boris Tartakovsky

Cytokines modulate preimplantation development and pregnancy.

Using two different models of pregnancy failure in mice, we show in this communication that preimplantation embryonic development can be controlled, in vivo, with cytokines. In one model, described by us previously, CSF-1 is shown to block gestations in plugged females due to its induction of defective early development. Pregnancies and preimplantation development are shown here to be restored to normal by TNF alpha or GM-CSF but not by TGF beta 1. In the second model, we reveal that CBA/J females plugged by various male strains produce spontaneously an extremely high proportion of abnormal precompaction embryos. Normal morulae and blastocysts, able to further develop and implant in vitro, are shown here to be induced by TNF alpha and to a lesser extent by GM-CSF and IL-1 alpha. These results suggest strongly that cytokines are potent modulators of early development. They may provide new and interesting insights into early events of mammalian embryonic development.

Methotrexate : mechanism of action in rheumatoid arthritis

Most studies of immune function in rheumatoid arthritis (RA) patients treated with methotrexate (MTX) show only marginal effects on humoral or cellular immune responses. These include measurements of lymphocyte subsets, proliferative responses to mitogens, immunoglobulin production, rheumatoid factor and immune complexes. The mechanism of action of MTX in RA might be more antiinflammatory than immunosuppressive. This is supported by the rapid clinical response to drug treatment and by data from in vitro and animal studies. The inhibition of interleukin-1 (IL-1) activity or other inflammatory cytokines by MTX may play an important role in the antiinflammatory effect of MTX. MTX effects in RA are not fully understood and further studies are needed to clarify its mechanism of action. MTX has crucial effects on the cascade of events initiated by some cytokines (IL-1, IL-6, tumor necrosis factor), which plays a major role in RA and other inflammatory diseases.

Monoclonal anticardiolipin antibodies derived from mice with experimental lupus erythematosus: Characterization and the induction of a secondary antiphospholipid syndrome

The primary antiphospholipid syndrome and the antiphospholipid syndrome in systemic lupus erythematosus (SLE) patients (defined as secondary antiphospholipid syndrome) are characterized by the presence of anticardiolipin antibodies, thrombosis, thrombocytopenia, and recurrent fetal loss. To determine the role of anticardiolipin antibodies in the pathogenesis of antiphospholipid syndrome, monoclonal anticardiolipin antibodies were derived from mice in which experimental lupus was induced by a murine monoclonal anti-16/6 Id antibody. Two murine monoclonal anticardiolipin antibodies (2C4C2, 2C4D1) were generated and characterized. The 2C4C2, but not the 2C4D1, monoclonal antibody demonstrated remarkable lupus anticoagulant activity. Furthermore, these murine anticardiolipin monoclonal antibodies appear to recognize antigenic epitopes similar to those recognized by anticardiolipin antibodies found in sera of SLE patients. The monoclonal anticardiolipin antibody 2C4C2 was injected into naive female mice. Following immunization, the mice developed high titers of autoantibodies reacting with cardiolipin, DNA, nuclear extract, 16/6 and anti-16/6 Id, and anticardiolipin antibodies. As early as 8 weeks after immunization these mice exhibited significant leukopenia, thrombocytopenia, and proteinuria with immune complex glomerulonephritis. Moreover, mating of 2C4C2-injected mice with allogenic males resulted in low pregnancy rates and a low number of fetuses with a high percentage of fetal loss. These studies provide a new experimental model for secondary antiphospholipid syndrome demonstrating the role of anticardiolipin antibodies in the pathogenesis of this syndrome.

CSF-1 induces resorption of embryos in mice

We have shown previously that C57BL/6J female mice inoculated with the syngeneic BL6-T2 regressor tumor resorb a high proportion of embryos sired by B6D2F1 or DBA/2 males but not embryos engendered by CBA/J or C57BL/6J males. In this present report, we provide a mechanism elucidating the nature of the fetal wastage observed. It was found that the BL6-T2 tumor secretes constitutively GM-CSF and CSF-1. Based on the hypothesis that those cytokines could interfere with normal fetal development, we injected semi-purified GM-CSF or CSF-1 into pregnant females, for five days. Results show that CSF-1 was able to induce a high rate of fetal resorption whereas GM-CSF had no effect.

Immunization with a syngeneic regressor tumor causes resorption in allo-pregnant mice

The purpose of our studies is to establish experimental systems in which one can deliberately disrupt the apparent maternal tolerance toward the semiallogeneic fetuses. Bases on the hypothesis that immunization against tumor-associated antigens may lead to a subsequent immune response directed against cross-reacting fetal antigens, we have immunized C57BL/6J female mice with a syngeneic regressor tumor. Mice were subsequently mated to B6D2F1, DBA/2, CBA/J or C57BL/6J males. We show that a high proportion of embryos sired by either B6D2F1 or DBA/2 males undergo resorption whereas those engendered by CBA/J or C57BL/6J males remain fully protected.

Serological detection of h-2k- and h-2d-gene products. I. Principal difference between t and b lymphocytes in expression of h-2d-encoded alloantigens.

Using the fluorescence-activated cell sorter (FACS II), we have analyzed the expression of H-2K- and H-2D-gene products on the membrane of various cellular components of the murine immune system. Using this serological technique we show a basic difference between T and B lymphocytes. Whereas all cellular components analyzed — hydrocortisone-resistant thymocytes, splenic T and B lymphocytes, macrophages and bone-marrow cells — expressed H-2K-subregion-encoded alloantigens at a high density, it seems that the high density expression of H-2D-encoded alloantigens is restricted mainly to B cells and to macrophages. Hydrocortisone-resistant thymocytes, splenic T lymphocytes and bone-marrow cells, on the other hand, showed significant expression of the H-2D alloantigens only at low membrane density. These results, then, provide evidence for the existence of an imbalance in serologically detectable expression of H-2K- and H-2D-region-gene products on the cell membrane of various cells comprising the murine immune system.

Production of colony-stimulating factor 1 by T cells: Possible involvement in their interaction with antigen-presenting cells

Colony-stimulating factor 1 (CSF-1) is required for the growth and differentiation of mononuclear phagocytes, and is also involved in modulating various activities in mature cells. We report herein that T-cell lines produce 4.6 and 1.5 kb mRNA species of CSF-1, and express the CSF-1 protein on their outer membranes, as determined by immunofluorescence staining with anti-CSF-1 antibodies. The CSF-1 protein is biologically active. Interested by the possible immunoregulatory function of CSF-1, we assessed its effect in an assay of antigen presentation to the T cell lines. We found that anti-CSF-1 antibodies inhibited T-cell stimulation. Moreover, soluble CSF-1 could not overcome this inhibition, but exerted a significant inhibitory activity on the interaction between T cells and antigen-presenting cells leading to T-cell activation and proliferation in vitro. Based on these observations we propose that T-cell CSF-1 may be involved in the interaction of these cells with CSF-1 receptor bearing antigen-presenting cells.

Macrophage Hybridomas: An Approach to the Analysis of the Functional Heterogeneity of Macrophages

Macrophages are wandering phagocytic cells known to participate in a wide variety of immunological processes, including phagocytosis, antigen presentation and cytostasis or killing of tumor cells. Despite their seemingly common histological origin, they do not consist of a homogeneous population. Macrophages seem to differ in morphology, expression of membrane markers (H-2I, Fc and C3 receptors), enzymatic contents and biological activities. Our previous studies concerning control and immunogenic properties of distinct subpopulations of macrophages revealed the existence of two major subpopulations, only one of which was highly efficient in presenting antigen to specific T lymphocytes (1). The other subpopulation, while highly phagocytic, was devoid of antigen-presenting capacity (1). The heterogeneity of cell types, the inability of macrophages to grow in culture, particularly as cloned populations, and the limitations of the methods for separation of distinct macrophage subpopulations do not enable a precise and detailed analysis of the molecular mechanisms and controlling signals which regulate the biological activity (i.e., phagocytosis, chemotaxis, secretion of interleukin 1, antigen presentation) in correlation to membrane molecules which characterize defined subsets of macrophages.

Fertility impairment and improved fetal survival induced by a tumor cell line in mice.

ABSTRACT: The BL6‐T2 tumor, a regressor melanoma line in C57BL/6J mice shown by us previously to be abortifacient, has been cloned in vitro. Clones obtained have been tested in vivo for tumorigenicity and effect on gestations, in order to correlate the immunogenicity of the tumor and its effect on fetal survival. Results demonstrate that high immunogenicity and impairment of fertility do not occur in the same tumor clones. We also show that this tumor produces and secretes GM‐CSF and CSF‐1 and that trophoblast cells express in situ mRNA encoding for the CSF‐1 receptor. We consequently hypothesize that this tumor exerts its abortifacient effect not via its strong immunogenicity but via cytokines it secretes.

Anticardiolipin, but not the 166 Id anti-DNA antibody induces pregnancy failure

The primary antiphospholipid syndrome or the antiphospholipid syndrome in systemic lupus erythematosus patients (defined as secondary antiphospholipid syndrome) are characterized by the presence of thrombosis, thrombocytopenia and recurrent fetal loss in association with anticardiolipin antibodies. To determine the causal role of these antibodies in the pathogenesis of pregnancy failure we studied the effects of immunization with monoclonal anti-DNA antibody (designated 16/6 Id; no cardiolipin reactivity) and anticardiolipin monoclonal antibody (designated 2C4C2; binds DNA as well) on the outcome of allogeneic pregnancies in BALB/c mice. Mating of BALB/c females 4 weeks after active immunization with the 16/6 Id, anti-DNA monoclonal antibody resulted in normal pregnancy outcome, similar to control mouse groups. In contrast to that, immunization with the 2C4C2 anticardiolipin antibodies resulted in severe gestational failure with low pregnancy rate, low numbers of fetuses and high rates of resorptions. The fertility index of those mice was extremely low as compared to the 16/6 Id-immunized mice or the control groups. Furthermore, a correlation was shown between the presence of anticardiolipin antibody levels in the sera of the mice at the time of gestation and the pregnancy fate. The 2C4C2-immunized mice which produced high levels of anticardiolipin antibodies demonstrated severe pregnancy failure, whereas normal gestations were observed in the 16/6 Id primed or the control mouse groups that did not produce measurable amounts of the latter antibodies. Thus, our studies demonstrate that anticardiolipin but not the 16/6 Id anti-DNA antibodies can induce severe gestational impairment.