Effect of stromal fibroblasts on antibody formation in cultures deficient in A-cells]

Stromal fibroblasts from the monolayer cultures of the rabbit thymus, spleen, and bone marrow when added to suspension cultures of non-adherent rabbit spleen cells had a significant effect on the response of the plaque-forming cells (PFC). Thymus-derived stromal fibroblasts caused an increase in the number of PFC, and bone marrow stromal fibroblasts suppressed the antibody formation in these cultures. Spleen-derived stromal fibroblasts influenced the PFC response similarly to adherent cells. Thymus-derived stromal fibroblasts irradiated by 5000 R influenced the PFC response in the same way as non-irradiated cells. The action of stromal fibroblasts on the antibody formation in the cultures depended on their attachment to the culture flask surface.     

Characteristics of heterologous antisera against stromal fibroblasts]

As shown with the use of heterologous rabbit antifibroblast sera (AFS) to stromal mechanocytes of guinea pig, the media from the cultured bone marrow, spleen, and thymus stromal fibroblasts contained specific trypsinoresistant fibroblast protein (AG-1), which was also present in the normal blood serum and on the surface of stromal fibroblasts. AG-1 was insensitive to collagenase effect and apparently differed from the chief surface protein of fibroblasts (CSP). AG-1 is referred to gamma1-globulin, and is probably a new specific surface fibroblast protein. AFS contained also antibodies to the nonspecific protein of fibroblasts (AG-2), alpha1,-globulin related to AG1.     

Effect of stromal mechanocytes of hematopoietic organs on in vitro formation of antibody producing cells

Cultured rabbit fibroblasts of bone marrow, thymus and spleen origin were added in spleen cell cultures in which the primary antibody response to SRBC was induced. Bone marrow fibroblasts caused strong inhibition of the response; thymus fibroblasts stimulated antibody formation; spleen fibroblasts inhibited the response when added in large amounts otherwise they produced no effect. The stimulation of antibody forming cell response by thymus fibroblasts proved independent of whether fibroblasts were irradiated or not. Bone marrow fibroblasts exhibited suppressive effect on the response predominantly during initial stages of antibody induction. All the 3 types of fibroblasts did not influence cell viability in spleen cells cultures, and were much more effective on addition to cultures of A-deficient spleen cells as compared to full spleen cells.     

Effect of hydroxyurea on the number of hematopoietic stem cells, stromal cell precursors and cell precursors of thymus lymphoid tissue in the bone marrow of mice during aging

The concentration of hemopoietic stem cells (colony-forming cells in the spleen - CFC-S) decreases in the bone marrow of CBA mice during ageing, whereas the concentration of precursors for stromal fibroblasts (colony-forming cells for fibroblasts-CFC-F) increases. The total numbers of nucleated cells, CFC-S and CFC-F in the bone marrow of old mice essentially increase. Hydroxyurea, administered in vivo, does not effect the concentration of CFC-S, but it increases CFC-F concentration in the bone marrow of mice. Hydroxyurea produces just the same suppressive effect on the numbers of nucleated cells, CFC-S and CFC-F in the mice of different ages, and stimulates the capacity of bone marrow donors to repopulate the thymus of the irradiated young recipients.     

Suppression of cytotoxic response to histoincompatible cells. I. Evidence for two types of T lymphocyte-derived suppressors acting at different stages in the induction of a cytotoxic response.

Spleen and thymus cell populations from normal or allograft tolerant mice have been cultured for 5 days with specific alloantigens and examined for their reactivity in three assay systems. No consistent correlation was observed between the production of cytotoxic T cells (CTL) in these cultures and the ability of such cultured cells to inhibit specifically a CML response from fresh normal spleen cells directed to the priming alloantigens. Furthermore, suppressor cells measured in this latter assay were apparently distinct from those able to inhibit the production of cytotoxic lymphocyte precursors (CTLp) from bone marrow stem cells in lethally irradiated bone marrow protected mice. Velocity sedimentation experiments confirmed that both the precursor and effector cells for the two suppressor systems were physically separable, and were distinct from CTLp or CTL, respectively. Precursor cells for the two suppressor systems investigated belong to the short-lived cortical thymus cell population.     

Organ interactions in the regulation of hematopoiesis: in vitro interactions of bone, thymus, and spleen with bone marrow stem cells in normal, Sl/Sld and W/Wv mice.

Hematopoietic cell differentiation is influenced by organ-dependent microenvironmental factors as well as humoral regulators. A technique is described for examining certain aspects of the hemopoietic inductive microenvironment in vitro. Suspension and agar cultures of mouse bone marrow were used to study the effects of organ stromal factors on cellular proliferation and differentiation. Bone, spleen, and thymus fragments from irradiated mice were placed in direct contact with or separated by a Nuclepore membrane from syngeneic marrow cells growing in suspension cultures. Normal adult mouse bone and spleen influenced granulocytic differentiation as well as cell proliferation. In this system, bone marrow and organ fragments from W/Wv and SlSld mice behaved like those of their non-anemic littermates. The most prominent difference between W/Wv and Sl/Sla mice and their normal counterparts was observed in the inductionof CFU-C from splenic precursors un-er the influence of CSA. In both types of anemic mice, in vitro generation of CFU-C from spleen was abnormal in young animals but was corrected by four months of age.     

Effect of opioid peptides on proliferation and differentiation of skeletogenic cells of rabbit bone marrow in vitro

The influence of opioid peptides DSLET and DAGO in doses 10(-5), 10(-7) or 10(-10) mg per 1 ml of the medium on colony formation in the culture of stromal bone marrow fibroblast precursors was investigated 5. 10(-6) bone marrow cells were placed in plastic containers (Costar). 12 day old cell cultures were fixed with ethanol and stained with hematoxyline-eosin. Effectiveness of fibroblast colony formation (EFFC) was detected. Grown fibroblast colonies were stained after Gomory for alkaline phosphatase. Opioid peptides DSLET and DAGO in the used doses exerted no influence on EFFC and percentage phosphatase-positive colonies, which casts doubt on a presumable direct action of opioid peptides on stromal bone marrow cell-precursors. But it does not seem unlikely that opioid peptides may affect stromal bone marrow precursors of fibroblasts through the cell environment, particularly, via macrophages.     

Effect of fibroblasts from monolayer cultures of hematopoietic and lymphoid tissue on the immune response in vitro]

Stromal fibroblasts from the monolayer cultures of human bone marrow, guinea pig bone marrow, spleen, thymus and peripheral blood suppressed the response of the plagueforming cells against sheep erythrocytes in the suspension cultures of mouse spleen cells. Combined cultivation of 20 X 10(6) fibroblasts from all the mentioned sources led to complete suppression of the immune response. This suppression was less in mice immunized three days before the spleen cell explantation into the suspension cultures and was absent entirely in case the pre-immunization of spleen cell donors was accomplished nine days before the explantation.     

Analysis of changes in natural cytostatic and cytotoxic activity of mouse spleen cells after treatment with cyclophosphamide and alpha-interferon

Cytostatic and cytotoxic activity of mouse spleen cells against normal and tumour target cells has been studied. The comparative analysis of mouse spleen cell cytostatic and cytotoxic activity after exposure to cyclophosphamide has shown that the effectors of natural cytotoxic activity are highly sensitive to cyclophosphamide, while cytostatic effectors are heterogeneous in their sensitivity to cyclophosphamide. Pretreatment of spleen cells with alpha-interferon produced an increase in cytotoxic and cytostatic activity against tumour target cells. The cells of lymphoid organs (spleen, bone marrow, thymus) had greater distinctions in cytotoxic than in cytostatic activity against tumour target cells.     

Adiponectin, a fat cell product, influences the earliest lymphocyte precursors in bone marrow cultures by activation of the cyclooxygenase-prostaglandin pathway in stromal cells

Adiponectin, an adipocyte-derived hormone, is attracting considerable interest as a potential drug for diabetes and obesity. Originally cloned from human s.c. fat, the protein is also found in bone marrow fat cells and has an inhibitory effect on adipocyte differentiation. The aim of the present study is to explore possible influences on lymphohematopoiesis. Recombinant adiponectin strongly inhibited B lymphopoiesis in long-term bone marrow cultures, but only when stromal cells were present and only when cultures were initiated with the earliest category of lymphocyte precursors. Cyclooxygenase inhibitors abrogated the response of early lymphoid progenitors to adiponectin in stromal cell-containing cultures. Furthermore, PGE(2), a major product of cyclooxygenase-2 activity, had a direct inhibitory influence on purified hematopoietic cells, suggesting a possible mechanism of adiponectin action in culture. In contrast to lymphopoiesis, myelopoiesis was slightly enhanced in adiponectin-treated bone marrow cultures, and even when cultures were initiated with single lymphomyeloid progenitors. Finally, human B lymphopoiesis was also sensitive to adiponectin in stromal cell cocultures. These results suggest that adiponectin can negatively and selectively influence lymphopoiesis through induction of PG synthesis. They also indicate ways that adipocytes in bone marrow can contribute to regulation of blood cell formation.     

A bicellular mechanism in the immune response to chemically defined antigens. 3. Interaction of thymus and bone marrow-derived cells

The role of thymus and bone marrow-derived cells in the in vitro response to the dinitrophenyl (DNP) determinant was studied using the millipore filter well technique for spleen organ cultures. Antibodies to DNP were assayed by the technique of inactivation of DNP-coupled T-4 bacteriophage. It was found that spleens of mice total-body irradiated at 750 R, treated with bone marrow and thymus cells after exposure and immunized against rabbit serum albumin (RSA) were able to produce antibodies to DNP when challenged in vitro with DNP-RSA. Such a response was not produced by spleen explants from x-irradiated mice treated with either thymus or bone marrow cells. Neither were antibodies to DNP produced by spleens of animals repopulated with thymus and bone marrow cells, but not immunized with the carrier. This carrier effect was manifested when the irradiated mice were treated with RSA and thymus cells 6–8 days before administration of the bone marrow cells. Yet, such an effect was not observed when the RSA and bone marrow cells were given 6–8 days before injection of the thymus cells. Thus, the thymus-derived cells appear to play the role of cells sensitive to the carrier (RSA), whereas the bone marrow seems to be involved in the production of antibodies.     

Increased bone mass is a part of the generalized lymphoproliferative disorder phenotype in the mouse

We investigated the bone phenotype of mice with generalized lymphoproliferative disorder (gld) due to a defect in the Fas ligand-mediated apoptotic pathway. C57BL/6-gld mice had greater whole body bone mineral density and greater trabecular bone volume than their wild-type controls. gld mice lost 5-fold less trabecular bone and had less osteoclasts on bone surfaces after ovariectomy-induced bone resorption. They also formed more bone in a model of osteogenic regeneration after bone marrow ablation, had less osteoclasts on bone surfaces and less apoptotic osteoblasts. gld and wild-type mice had similar numbers of osteoclasts in bone marrow cultures, but marrow stromal fibroblasts from gld mice formed more alkaline phosphatase-positive colonies. Bone diaphyseal shafts and bone marrow stromal fibroblasts produced more osteoprotegerin mRNA and protein than wild-type mice. These findings provide evidence that the disturbance of the bone system is a part of generalized lymphoproliferative syndrome and indicates the possible role of osteoprotegerin as a regulatory link between the bone and immune system.     

Origin of stromal bone marrow mechanocytes according to the results of typing them by isoantigens and chromosomal markers]

The bone marrow of radiochimaeras and heterotopic bone marrow transplants were used to study the origin of precursors of the fibroblasts growing in the monolayer cultures of hemopoietic tissue. In the bone marrow explants of the (C57BL/6 X CBA) F1 mice, in which the CBA bone marrow was transplanted following the lethal irradiation, the fibroblasts grown in the colonies were of recipient origin judging by isoantigens in the reaction of indirect immunofluorescence with the anti-C57BL/6-serum. At the same time in the bone marrow explants from heterotopic transplants (CBA leads to CBA X C57BL/6) the fibroblasts grown in colonies were of donor origin. The cultures of hemopoietic cells of the bone marrow of females heterotopically transplanted in the singenic male (guinea pigs Huston) contained only fibroblasts which were of donor origin judging by sex chromosomes in the metaphase plates of dividing cells. Hence, the bone marrow precursors of fibroblasts do not depend histogenetically on hemopoietic cells and are not replaced at the expense of repopulating cells of the second partner.     

SELF-MAINTENANCE ABILITY AND KINETICS OF HAEMOPOIETIC STROMA PRECURSORS

The use of repeated femoral curettages and repeated passages of ectopic haemopoietic foci has demonstrated the capacity of stromal precursor cells for repeated formation of haemopoietic microenvironment. During this process the stromal precursors undergo no less than ten to twelve mitoses. Precursors of bone marrow stroma proliferating slowly, if at all, in normal mice are triggered into cell cycle, as revealed by suicide methods, during formation of the ectopic haemopoietic focus.     

Alteration of allospecific t-cell receptors after differentiation from prethymic precursor cells in semiallogeneic environments

Murine bone marrow cells (strain A) have been allowed to differentiate in vivo in syngeneic (A) or semiallogeneic hosts (A × B) to produce mature splenic T lymphocytes. After stimulation of these cells with irradiated allogeneic (C) spleen cells in tissue cultures, the cytotoxic T-cell blasts (CTL) were purified by velocity sedimentation and used to immunize (A × C) F₁ hybrid mice, to produce antisera recognizing the receptor structure (for C) on the relevant A cytotoxic cells (and their precursors). Using these sera we have been able to show that the T-cell receptor for alloantigen C on strain A cytotoxic precursor lymphocytes (CTLp) seems to differ according to the host environment in which those T cells differentiate from immature bone marrow precursors.     

The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form

The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells. Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells. The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones. It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts. The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow). In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.     

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3–dimensional collagen sponges

Abstract. Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro , but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 × 10⁶ cells are loaded, mineralization as measured by ⁸⁵Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10⁷ cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phos-phatase positive cells. However, synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.     

Selective hybrid cell formation by human lymphoid cells

Subpopulations of human lymphoid cells are capable of spontaneously fusing with fibroblasts of human or murine origin to form human-human or human-mouse hybrid cells. These cells were present in thymus, spleen, and bone marrow. After fractionation on discontinuous bovine serum albumin gradients, the cells were found in the less dense layers of the gradient. Cells of fetal origin, except for bone marrow, fused spontaneously at a higher rate than those of adult origin. The highest rate of fusion was found with adult bone marrow cells. These lymphoid cells appear to be “thymus-derived” cells.     

Stromal precursor cell count in the bone marrow of young and old CBA mice

The content of stromal precursor cells in the bone marrow of young (1-4-month-old) and old (24-30-month-old) CBA mice was measured by cloning in primary monolayer cultures. It was found that both the concentration (fibroblast colony forming efficiency) and the total number of stromal precursors in femoral bone marrow markedly increased with aging.