Effect of immunization with polyvinylpyrrolidone on the counts of stromal precursor cells in bone marrow and spleen of CBA and CBA/N mice and cytokine gene expression in primary cultures of these cells.

Injection of polyvinylpyrrolidone (synthetic type 2 T-independent antigen) stimulated the efficiency of clone-forming efficiency and the content of stromal precursor cells in CBA mice in the femoral bone marrow (almost 3-fold) and in the spleen (by 1.7 times) with the peak within 24 h and normalization by day 3 after immunization. The expression of IL-6, IL-8, and TNF-α genes in bone marrow and spleen cultures from immunized animals appeared on day 1 and disappeared on day 3. Hence, stimulation of stromal tissue in response to polyvinylpyrrolidone immunization was significantly less pronounced in comparison with immunization with S. typhimurium antigens. The counts of stromal precursor cells in these organs did not increase in CBA/N mice not responding to polyvinylpyrrolidone because they had no xid-mutation in Brutton's tyrosine kinase (Btk) gene, and the proinflammatory cytokine genes expression in primary cultures derived from these animals did not increase either. These data indicated that the degree of stromal tissue stimulation in immunized mice correlated with the immune response intensity. This indicated a close relationship between the stromal tissue and immune system. Stromal tissue seemed to be stimulated not only and not so much through the stromal cell Toll-like receptors, but mainly through interactions of immunocompetent and stromal cells, the former presumably playing the leading role in this process.

Number of stromal precursors in mouse bone marrow and expression of cytokine genes in primary cultures of mouse bone marrow cells during various periods after immunization with S. typhimurium antigens.

Administration of S. typhimurium microbial mass to mice was followed by a significant increase (by 3-4 times) in the efficiency of cloning and number of stromal precursors in the femoral bone marrow. These parameters were maximum on days 1-3, but returned to normal by the 8th-15th day after immunization. As differentiated from intact animals, the expression of genes for proinflammatory cytokines IL-1ß (day 1 after immunization), IL-6 (days 1-3), TNF-α (days 1, 3, and 6), and IFN-α (days 1-3) was detected in bone marrow cultures from immunized mice. The expression of genes for IFN-γ, IL-18, and IFN-α was decreased on days 1, 3, and 6 after immunization of animals, respectively. Gene expression for the anti-inflammatory cytokine IL-4 was observed on day 6 after immunization. Therefore, this system was not characterized by a decrease in the immune response of stromal cells. The stromal component of hemopoietic and lymphoid organs has the vector of influences in response to bacterial antigens. This vector is directed to the stimulation and progression, but not to the suppression of immune reactions. Our results indicate that resident stromal cells play a role in the immune response of the body.

Counts of stromal precursor cells in the bone marrow and heterotopic bone marrow transplants from mice immunized with group A streptococcus antigens during different periods after immunization.

The counts of stromal precursor cells in bone marrow transplants obtained from animals 2 months after their immunization with killed type 5 group A streptococcus vaccine drop almost 3-fold in comparison with transplants from normal donors. Six months after donor immunization, the count of stromal precursor cells in the transplants reaches the normal level. The count of stromal precursor cells in bone marrow transplants from normal mice transplanted to recipients 6 months after their immunization with killed streptococcus vaccine also virtually did not change in comparison with the counts in bone marrow transplants from normal donors transplanted to normal recipients. The weight and size of bone capsules of 6.5-month bone marrow transplants in intact recipients after transplantation from donors immunized 2 months before with killed type 5 group A streptococcus vaccine was 3-fold lower than in bone marrow transplants collected from intact donors. The content of stromal precursor cells in the femoral bone marrow of animals immunized with killed streptococcus vaccine was 2.5 time higher in comparison with the parameter in the femoral bone marrow of normal mice even 8 months after immunization. The results indicate a significant long-acting effect of streptococcal antigens on the bone marrow stromal tissue, specifically, on its osteogenesis potential.

Effect of serum from mice immunized with group A streptococcus antigens on the efficiency of in vitro cloning of stromal precursor cells.

The efficiency of cloning of stromal precursor cell in mouse bone marrow culture increases significantly (2-3-fold) in the presence of serum from mice immunized with type 5 group A streptococcus antigens (5-20 microl serum/ml culture medium) in comparison with intact animal serum. The levels of TNF-alpha and IFN-gamma are significantly reduced (2.7 times and more than 6-fold, respectively) in the sera of immunized mice in comparison with normal serum. Serum levels of IL-2, -4, -5, -10, and -12 were about the same in both groups; no granulocyte-macrophage CSF was detected. These data attest to appreciable effect of immunization with streptococcal antigens on the bone marrow stromal tissue; this effect is presumably mediated through serum cytokines.

Effect of macrophage migration inhibition factor on the content of stromal precursor cells in mouse bone marrow and efficiency of bone marrow precursor cell cloning in vitro.

The content of stromal precursor cells in the bone marrow of mice decreased 2-5.7 times 24 h after injection of macrophage migration inhibition factor in doses of 0.1-50 ng/kg, this reduction depending on the dose of inhibition factor. The content of precursor cells in the bone marrow of mice increased 2-fold 24 h after injection of S. typhimurium bacterial mass. One day after injection of S. typhimurium bacterial mass, the count of precursor cells in mouse spleen was 7-fold higher than 24 h after injection of macrophage migration inhibition factor. The efficiency of cloning of mouse bone marrow stromal precursor cells in vitro was suppressed 1.7-2.8 times in the presence of macrophage migration inhibition factor in doses of 0.1 to 50 ng/ml culture medium. The effect of cloning inhibition was preserved, if macrophage migration inhibition factor was added to the culture medium after 2 days of bone marrow cell culturing. In general, macrophage migration inhibition factor inhibits stromal precursor cells in vivo and in vitro. The data also indicate that macrophage migration inhibition factor is not responsible for rapid and sharp increase in the count of stromal precursor cells after immunization of animals.

Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges.

Cells for study were obtained from mouse bone marrow by three methods: 1) Mechanical dissociation; 2) Trypsin release; and 3) Passage through a syringe and needle. The ratio of the fibroblast colony-forming cells in these suspensions was, respectively, 1:13:1. Of the colonies formed by cells obtained by trypsin release, 70% were alkaline phosphatase positive fibroblasts. Freshly isolated marrow cells and cells harvested, by trypsin treatment, from cultures established from the differently obtained bone marrow cell suspensions, were absorbed into porous sponges for transplantation under the renal capsule of mice. Ossicles containing bone marrow formed after transplantation of either freshly isolated cells, or cultured cells obtained from marrow by trypsin treatment, but not after the transplantation of cells cultured from marrow obtained by mechanical dissociation. The size of the bone marrow "organs" that formed was determined by both the number of cells grafted and the size of the sponge in which they had been absorbed for grafting.

Radiosensitivity and postirradiation changes of bone marrow clonogenic stromal mechanocytes.

The in vitro colony assay was used to determine radiosensitivity and post-irradiation changes of guinea pig clonogenic bone marrow stromal fibroblasts (CFU-F). D0 of the CFU-F survival curve proved to be about 200 rad, while n is 1.4. Immediately after total-body exposure to 400 rad and to local exposure of tibias to 400 rad, the number of CFU-F diminishes in accordance with D0; it sharply increases after 6 hours and then decreases again. In the period between the eighth and twelfth day following local irradiation, the number of CFU-F doubled in the course of 30-35 hours but later the rate of recovery slowed down. Practically no CFU-F were traced during 3 months after local exposure to 2 krad.

Origin of bone marrow stromal mechanocytes in radiochimeras and heterotopic transplants.

Strain histocompatibility antigens (detected by indirect immunofluorescence reaction) and sex chromosomes were used as markers of donor and recipient cells in monolayer cultures of bone marrow from radiochimeras and from heterotopic transplants. In contrast to macrophages and hemopoietic cells all bone marrow fibroblasts precursors in radiochimeras were of recipient origin; in heterotopic transplants they were of donor origin. These data point to the decisive role of stromal mechanocytes, and not macrophages or hemopoietic cells, in creating the bone marrow hematopoietic microenvironment.