T. A. Danilova

Effects of Combined Treatment with Complex S. typhimurium Antigens and Factors Stimulating Osteogenesis (Curettage, BMP-2) on Multipotent Bone Marrow Stromal Cells and Serum Concentration of Cytokines in CBA Mice

The content of multipotent stromal cells (MSC) in the bone marrow and efficiency of their cloning (ECF-MSC) increased by 3 times 1 day after administration of complex S. typhimurium antigens to CBA mice, while the relative content of alkaline phosphatase-positive MSC colonies (marker of osteogenesis; P+ colonies) decreased from 14% (control) to 3%. After administration of the complex S. typhimurium antigens to CBA mice 3 h after (or 3 h before) curettage or treatment with morphogenetic protein (BMP-2), the content of MSC and ECF-MSC decreased on the next day by ~3 times in comparison with animals receiving antigens alone and approached the control level. The relative content of P+ colonies increased to 20 and 35%, respectively, in comparison with animals receiving antigens (3%), but was signifi cantly lower than after curettage (34%) or BMP-2 (42%) administration. Expression of IL-1β, IL-6, IL-12, TNF-α, and IFN-γ genes in the primary cultures of stromal bone marrow cells induced by antigen administration was suppressed, while the concentrations of IL-12 and TNF-α in the culture medium sharply decreased after antigen treatment in combination with curettage or BMP-2 administration. Administration of complex S. typhimurium antigens after pretreatment with BMP-2 (3 h before) was associated with a decrease in serum levels of IL-2, IFN-γ, IL-12, and TNF-α in mice receiving BMP-2+S. typhimurium group 4 h after treatment in comparison with the animals receiving only S. typhimurium antigens alone by 1.9, 4.4, 1.5, and 6 times, respectively, i.e. to normal level or below it, while the concentration of IL-10 increased by almost 2 times, which probably reflected anti-inflammatory properties of BMP-2. These data probably attest to competitive relations between osteogenesis and immune response at the level of MSC.

Effect of BMP-2 protein on the count and osteogenic properties of multipotent stromal cells and expression of cytokine genes in primary cultures of bone marrow and spleen cells from CBA mice immunized with bacterial antigens

We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines.

Age-Associated Reduction of the Count and Functional Activity of Stromal Precursor Cells Can Be Caused by Both True Reduction (Exhaustion) of Cell Pool and Regulatory Effects of the Organism

The study was carried out on CBA mice using the method of heterotopic transplantation. A fragment of the femoral bone marrow (1/2) or spleen (1/5 of the organ) was transplanted under the renal capsule of a recipient. The following donor-recipient cross-transplantation variants were studied: young → young (Y → Y), young → old (Y → O), old → old (O → O), and old → young (O → Y). Cell suspensions were prepared from 2-month transplants inoculated in monolayer cultures and the cloning effi ciency (ECF-F) of stromal precursor cells (CFC-F) was evaluated. The bone marrow transplant ECF-F and the count of CFC-F in the O → O group were 8-fold lower than in the Y → Y group. In the O → Y group, ECF-F was 3-fold higher than in the O → O group, but by 2.5 times lower than in the Y → Y group. ECF-F in Y → O group was 2-fold lower than in Y → Y group. The ECF-F and CFC-F count in spleen transplants in the O → O group were 4- and 6-fold lower, respectively, than in Y → Y group. However, in O → Y group ECF-F was 7-fold higher than in O → O group and higher than even in Y → Y group. The weight of induced ectopic bone tissue after transplantation of the osteoinductor (fragments of the allogenic urinary bladder mucosa) was 2-fold lower in the O → O vs. Y → Y group. However, comparison of the ectopic bone tissue weights in different experimental groups showed that osteoinductor activity of the bladder epithelium did not decrease, but increased 3-fold with age (O → Y:Y → Y). A 5-fold reduction of this proportion in groups where the osteoinductor was transplanted from old donors to old and young recipients (O → Y:O → O) could be attributed to age-specifi c reduction of the count of inducible osteogenic precursor cells (IOPC). The data in general suggest that age-specifi c reduction of the stromal precursor count and functional activity could be caused by the true reduction (exhaustion) of cell pool (bone marrow CFC-F; presumably, IOPC) and by the regulatory effects of the organism (bone marrow and splenic CFC-F, IOPC). These data seem to be signifi cant for understanding of the role of osteogenic stromal precursor cells in the development of age-associated bone tissue defects, for example, senile osteoporosis.

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-1β (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 antiinflammatory 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 μl serum/ml culture medium) in comparison with intact animal serum. The levels of TNF-α and IFN-γ 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.

Successive Administration of Streptococcus Type 5 Group A Antigens and S. typhimurium Antigenic Complex Corrects Elevation of Serum Cytokine Concentration and Number of Bone Marrow Stromal Pluripotent Cells in CBA Mice Induced by Each Antigen Separately

Administration of bacterial antigens to CBA mice induced an increase in serum concentration of virtually all cytokines with a peak in 4 h after administration of S. typhimurium antigens and in 7 h after administration of streptococcus antigens. In 20 h, cytokine concentrations returned to the control level or were slightly below it. In 4 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, we observed a significant decrease in serum concentrations of IFN-γ, IL-10, GM-CSF, IL-12, and TNF-α, in comparison with injection S. typhimurium antigens alone and IL-5, IL-10, GM-CSF, and TNF-α in comparison with injection of streptococcus antigens alone; the concentrations of IL-2 and IFN-γ, in contrast, increased by 1.5 times in this case. In 20 h after administration of S. typhimurium antigens, the number of multipotential stromal cells (MSC) in the bone marrow and their cloning efficiency (ECF-MSC) increased by 4.8 and 4.4 times, respectively, in comparison with the control, while after administration of streptococcus antigens by 2.6 and 2.4 times, respectively. In 20 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, these parameters increased by 3.2 and 2.9 times, respectively, in comparison with the control, i.e. the observed increase in the level of MSC count and ECF-MSC is more consistent with the response of the stromal tissue to streptococcus antigens. Thus, successive administration of two bacterial antigens corrected both serum cytokine profiles and MSC response to administration of each antigen separately, which indicates changeability of the stromal tissue in response to changes in the immune response.

Counts of Stromal Precursor Cells in Heterotopic Bone Marrow Transplants in Mice Immunized with Group A Streptococcus Antigens

The count of stromal precursor cells in bone marrow transplants from CBA mice, transplanted to animals immunized with killed type 5 group A streptococcus vaccine, decreased 4.5–6.5 times (depending on the transplant age) in comparison with the grafts transplanted to normal recipients. The counts of stromal precursor cells in 1.5–3-month bone marrow transplants from animals immunized with killed streptococcal vaccine transplanted to normal mice were virtually the same, while in 7-month transplants they decreased 2-fold in comparison with their counts in bone marrow transplants from normal CBA mice transplanted to normal animals. The content of stromal precursor cells in the femoral bone marrow of animals immunized with killed streptococcal vaccine was appreciably (3.5 times) higher than in the bone marrow of normal mice. The results attest to an appreciable effect of streptococcal antigens on the bone marrow stromal tissue and suggest that not all stromal precursor cells, whose count increases after injection of antigens, are responsible for transplantability of the stromal tissue in case of its heterotopic transplantation.

Formation of colonies of clonogenic stromal precursors in bone marrow and splenic cell cultures in the presence of streptococcal antigens in the culture medium

The presence of streptococcal M protein and A polysaccharide in culture medium is shown to have an inhibitory effect on the growth of clonogenic stromal precursors in cultures of healthy murine bone marrow and of healthy guinea pig bone marrow and spleen. The efficacy of colony formation dropped 1.5- to 2-fold in the presence of antigens in a concentration of 25 μg/ml in the medium. The inhibitory effect was absent if antigens were added to adhesive cell cultures. The addition of antigens to cultures originating from animals immunized with streptococcus resulted in inhibition of the efficacy of colony formation in complete cultures and in cultures of adhesive cells. The presence of streptococcal antigens in guinea pig stromal fibroblast cultures of different strains did not affect their growth or colony formation. These data indicate that the effects of streptococcal antigens appear to be aimed at the stromal cells not directly, but rather via another cellular category in the bone marrow and splenic cell cultures, probably lymphocytes.

Specificity of monoclonal antibodies obtained by immunization of mice with trypsin-treated group a streptococcus culture

Hybridomas producing monoclonal antibodies intensively reacting with group A streptococcus antigens in enzyme immunoassay were obtained as a result of immunizing mice with pepsin-treated cultures of group A streptococcus. All antibodies were referred to class M immunoglobulins. The reactions of monoclonal antibodies were completely inhibited by the pepsin-treated culture of group A streptococcus. The degree of inhibition with A-polysaccharide was lower, being 17.5 to 50.0 in different monoclonal antibodies. All the monoclonal antibodies obtained cross-reacted with antigens of murine and human epithelial tissues of the thymus and skin.