Osteogenic potential of rat spleen stromal cells

Evidence is mounting that an increasing number of cell populations in the adult organism already committed and/or differentiated retain the ability to reprogram themselves and give rise to a different phenotype. Bone marrow stromal cells have long been recognized as early progenitor cells for osteoblasts, chondrocytes, hematopoietic-supportive fibroblasts and adipocytes. Recent reports though have demonstrated a potential of cell populations outside the bone marrow environment to sustain bone formation under specific circumstances. The formation of bone nodules in the spleen of IL-5 transgenic mice has been recently reported (Macias et al. (2001): J. Clin. Invest. 107, 949 - 959). We thus postulated that a cell population exists in the spleen that under particular microenvironmental conditions is able to reprogram itself and pursue a fate other than the tissue-specific one. Therefore we isolated and expanded in vitro spleen-derived stromal cells. After expansion, these cells were challenged with culture conditions designed to induce osteogenic differentiation. We hypothesized that the combination of a proliferating factor (fibroblast growth factor 2) and a differentiating hormone (dexamethasone) would allow us to induce spleen-derived stromal cells to proliferate and at the same time to express osteoblast-specific genes. Thus, spleen-derived stromal cells were isolated from rat spleen and expanded in the presence of fibroblast growth factor 2 and dexamethasone. Once primary cultures reached confluence they were either switched to an osteo-inductive medium or implanted in immunodeficient mice. Although no bone formation was observed in in vivo experiments, in vitro spleen-derived stromal cells were able to deposit a mineralized matrix. Gene expression, as revealed by RT-PCR analysis, evidenced that the deposition of a mineralized matrix was concomitant with the expression of CBFA1 and osteocalcin, along with alkaline phosphatase and bone sialoprotein. Our data suggest that rat spleen-derived stromal cells can undergo osteogenic differentiation in a permissive microenvironment.     

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.     

Combinations of two synthetic adjuvants: synergistic effects of a surfactant and a polyanion on the humoral immune response

Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.     

Ultrastructural characteristics of stromal mechanocytes and their interaction with hematopoietic cells in regenerating bone marrow transplants

Extramedullar grafts of the bone marrow have been studied electron microscopically 2-8 days after transplantation. The data on ultrastructural peculiarities of the stromal mechanocytes have been obtained at various stages of their differentiation, as well as topographic interrelationships between the mechanocytes and the hemopoietic cells. Digital junctions are described between the stromal mechanocytes (primitive) and the hemopoietic cells (in 3-day-old grafts) which are, probably, the first morphological signs demonstrating restoration of the hemopoietic microenvironment in the bone marrow grafts.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Antigen-specific suppression of the plaque-forming cell response induced polyclonally by lipopolysaccharides

Horse erythrocytes (HRBC) were added with LPS in mouse spleen cell cultures, and the effects of HRBC on the LPS-induced polyclonal PFC response were investigated by enumerating total IgM-secreting PFC, anti-HRBC PFC, and PFC against sheep erythrocytes (SRBC). The addition of HRBC influenced the frequencies of anti-HRBC PFC in the total IgM-secreting PFC, but did not influence those of anti-SRBC PFC. The augmentation of the frequencies of anti-HRBC PFC occurred only when an appropriate dose of HRBC was added in the cultures containing T cells. Higher doses of HRBC decreased the frequencies of anti-HRBC PFC whether T cells were present or absent. The degree of reduction of the frequencies of anti-HRBC PFC was dependent on the dose of HRBC, but independent of the dose of LPS. The addition of HRBC at 1 day after LPS stimulation also decreased the frequency of anti-HRBC PFC, though the addition of 2 or 3 days hardly suppressed it. These results suggest that the antigen-specific augmentation occurs via helper T cells, and the suppression is ascribed to the direct action of antigen on the antigen-specific B cells.     

Effect of polyinosinic-polycytidylic acid on the colony-forming ability of hematopoietic stem cells in conditions of allogeneic inhibition]

It was found that the colony-forming capacity of parental bone marrow transplant (C57BL/6) was partially restored in the (CBA X C57BL/6) hybrid recipient irradiated with 800 rad when poly I -- poly C preparation was injected. The effect of poly I -- poly C injection on the colony formation was equivalent to addition of the thymus cells syngeneic with the marrow. In either case the number of splenic colonies was more than double that in the control. On the other hand, it was found that in a completely syngeneic system the number of splenic colonies was not influenced by the thymus cells and poly I -- poly C preparation. Poly I -- poly C doses ranging from 50 to 100 mug and thymus cell doses ranging from 4-10(6) to 8-10(6) did not increase the efficiency of the colony formation with a stable bone marrow dose transplant.     

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.     

Dose-dependent induction of immunologic enhancement and suppression after oral administration of antigen

The effects of feeding various quantities of a particulate antigen, sheep red blood cells (SRBC), on plaque-forming cells (PFC) in the spleen were determined. Mice were given various numbers of SRBC orally daily for 14 days, then injected with SRBC intravenously. Splenic IgA PFC responses to SRBC were enhanced in the mice fed 5 X 10(8) SRBC and splenic IgG PFC responses to SRBC were depressed in the mice fed 5 X 10(9) SRBC. Adoptive transfer experiments showed that enhancement of splenic IgA PFC responses and suppression of splenic IgG PFC responses were induced by the T-cell rich fraction from Peyer's patches (PP) and the spleen in 5 X 10(8) SRBC- and 5 X 10(9) SRBC-fed mice, respectively. Kinetic studies revealed that IgA helper cells or IgG suppressor cells appeared in PP 2 days after oral administration and 4 days after it in the spleen.     

Differential regulation of activation, clonal expansion, and antibody secretion in human B cells

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.     

Inhibition of hemopoiesis in murine marrow cell cultures by recombinant murine tumor necrosis factor alpha: evidence for long-term effects on stromal cells

The addition of recombinant murine tumor necrosis factor alpha (rmTNF-alpha) to serum-free methylcellulose cultures inhibited macrophage colony formation stimulated by purified colony stimulating factor-1 (CSF-1), recombinant granulocyte-macrophage-CSF (rmGM-CSF), and recombinant interleukin 3 (rmIl-3). The concentration of rmTNF-alpha inhibiting colony formation by 50% (IC50) was between 2 and 20 ng/ml. Erythroid colony formation in cultures with erythropoietin (EPO) alone or EPO, rmIl-3, and rmGM-CSF in combination were reduced to a much lesser extent. In established long-term marrow cultures (LTMC), addition of 20 and 200 ng/ml of rmTNF-alpha resulted in release of cells from the adherent layer during the first week. Treatment of cultures with rmTNF-alpha for 4 consecutive weeks led to prolonged inhibition of cell production lasting up to 8 weeks after cessation of treatment. One day after addition of a low dose of TNF (2 ng/ml), "fat" cells were no longer observed in the adherent layer. Our results indicate that TNF inhibition of hemopoiesis occurs both at the progenitor cell and stromal cell levels.     

Heterologous antisera to stromal mechanocytes of the bone marrow]

Heterologous rabbit antifibroblast serum (AFS) to stromal fibroblasts of the guinea-pig bone marrow was obtained. AFS was shown to bind specifically stromal fibroblasts and their precursors (but not macrophages) in the monolayer primary cultures of the bone marrow, thymus and spleen of guinea pigs (in the complement-dependent cytotoxic reaction and in the indirect immunofluorescence test); AFS also bound precursors of blood fibroblasts and peritoneal exudate (in the cytotoxic reaction). Precursors of the thymus fibroblasts were found to be more sensitive to the AFS action than the spleen and bone marrow precursors, this suggesting that the thymic stromal mechanocytes had a greater concentration of common tissue-specific antigens on their surface. Precursors of the stromal fibroblasts in native cell suspensions were found to be essentially more sensitive to the cytotoxic action of AFS than the colony-forming fibroblasts on the passage cultures.     

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.     

Immunomodulatory activity of Sulforaphane, a naturally occurring isothiocyanate from broccoli (Brassica oleracea)

The effect of Sulforaphane on the immune system was studied using BALB/c mice. Intraperitoneal administration of five doses of Sulforaphane (500 microg/dose/animal/day) was found to enhance the total WBC count (12,950 cells/mm3) on 9th day. Bone marrow cellularity (23 x 10(6) cells/femur) and number of alpha-esterase positive cells (1346.66/4000 cells) were also increased by the administration of Sulforaphane. Treatment with Sulforaphane along with the antigen, sheep red blood cells (SRBC), produced an enhancement in the circulating antibody titre and the number of plaque forming cells (PFC) in the spleen. Maximum number of PFC (315.83 PFC/10(6) spleen cells) was obtained on the 6th day. Administration of Sulforaphane also showed an enhancement in the phagocytic activity of peritoneal macrophages. Moreover administration of Sulforaphane significantly reduced the elevated level of TNF-alpha production by LPS stimulated macrophages. These results indicate the immunomodulatory activity of Sulforaphane.     

Influence of the age of the recipient on the manifestation of the effect of allogeneic inhibition]

The expression of allogenic inhibition was studied when transplanting 10(5) cells of bone marrow of C57BL mice to the lethally irradiated recipients (CBA X C57BL) F1 of different age (2 to 11 months). In the control experiments the bone marrow cells at the same dose were introduced to the lethally irradiated syngenic (C57BL) mice. The most pronounced inhibition of the parental stem cells proliferation was registered in 2 months old recipients F1 (4.7 times), it was somewhat weakened in 3 months old and animal in 4--11 months old recipients (1.1 to 1.8 times). The thymectomy of adult recipients F1 did not eliminate the expression of allogenic inhibition.     

Use of the ELISA immunoenzyme method for the detection of the causative agent of tularemia

The conditions of the enzyme-linked immunosorbent assay (ELISA) for the detection of Francisella tularensis were worked out. In the study of 27 strains differing in their biological characteristics, the sensitivity of the assay was determined, varying within the range of 1 X 10(4)--5 X 10(4) million cells/ml and exceeding the sensitivity of the currently used methods for the immunodiagnosis of tularemia by 1-2 orders. ELISA also proved to be a highly effective technique for the detection of the specific antigen in the organs of infected animals. The antigen was regularly detected in the organs of white mice, beginning from day 3 after their infection with the minimal doses of F. tularensis. The method may be recommended both for the identification of isolated cultures and for the early diagnosis of tularemia infection.     

Free-radical inhibition of ATPase in hamster cheek pouch homogenates

The effects of free-radicals generated by either the oxidation of hypoxanthine by xanthine oxidase (HX/XO) or the lipoxidation of arachidonic acid (AA) on the ATPase of the hamster cheek pouch has been studied. Cheek pouches were removed from female golden syrian hamsters and homogenized. ATPase activity was measured by the production of Pi at 37 degrees. HX/XO and AA were added at a final concentration of 9.6 X 10(-5) M HX with 5 X 10(-2) units HX and 5 X 10(-5) M AA with and without 1 X 10(-4) M ouabain. HX/XO produced a 24.7% inhibition alone and 35.0% when combined with ouabain. Ouabain alone produced a 7.1% inhibition. AA produced a 23.6% inhibition alone and 24.3% inhibition when combined with ouabain. Ouabain alone produced a 5.4% inhibition in this series. When AA was added in doses ranging from 1 X 10(-5) to 2 X 10(-3) M, a plot of percent inhibition versus log dose followed a typical sigmoid type curve. The IC50 was 1.5 X 10(-4) M. These results suggest that free-radicals are capable of inhibiting the ATPase found in the hamster cheek pouch tissues. The possible modes of action of the free-radicals in producing this inhibition are discussed.     

Characterization of ultraviolet light-induced ouabain-resistant mutations in Chinese hamster cells.

Ouabain-resistant mutations in Chinese hamster cells have been quantitatively characterized. The mutation frequencies were found to be induced curvilinearly with treatments of increasing doses of ultraviolet light (UV). For the range of UV doses tested (5--20 J/m(2)), the observed frequency, Y, as a function of UV dose X, follows a curvilinear function, Y = (-28 + 13.37 X--1.52X(2) + 0.08X(3)) . 10(-6). The frequencies of UV-induced mutations were directly correlated with cell survival, indicating a similar causal relationship between cell killing and mutation induction. Under the same experimental conditions, X-rays induced 6--thioguanine-, but not ouabain-, resistant mutations. UV-induced ouabain-resistant (ouar) mutants exhibit a selection disadvantage. Their phenotypic expressions are modifiable by various agents. Wild type and 16 ouar mutants were compared with respect to their sensitivity to ouabain inhibition of 86Rb uptake by whole cells. All the ouar mutants assayed are less sensitive to the drug than are wild-type cells. In the absence of ouabain, the Na+--K+--ATPase activities can be significantly higher or lower than that of the wild-type cells.