scholarly journals Knockout of the Murine Prostaglandin EP2 Receptor Impairs Osteoclastogenesis in Vitro*

Endocrinology ◽  
2000 ◽  
Vol 141 (6) ◽  
pp. 2054-2061 ◽  
Author(s):  
Xiaodong Li ◽  
Yosuke Okada ◽  
Carol C. Pilbeam ◽  
Joseph A. Lorenzo ◽  
Christopher R. J. Kennedy ◽  
...  
Keyword(s):  
Spleen Cell ◽  
Cell Cultures ◽  
Messenger Rna ◽  
Osteoblastic Cells ◽  
Spleen Cells ◽  
Dihydroxyvitamin D ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Ep2 Receptor

Abstract Prostaglandin E2 (PGE2) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE2 receptors, designated EP2 and EP4. We used cells from mice in which the EP2 receptor had been disrupted to test its role in the formation of TRAP + MNC. EP2 heterozygous (±) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP2 −/−) and wild-type (EP2 +/+) mice. PGE2, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP2 +/+ mice. In cultures from EP2 −/− animals, responses to PGE2, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP4 receptor antagonist (EP4RA) further inhibited TRAP+ MNC formation in both EP2 +/+ and EP2 −/− cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE2 or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP2− /− mice, by 88% or 68% when only osteoblastic cells were from EP2 −/− mice and by 58% or 35% when only spleen cells were from EP2 −/− mice. PGE2 increased receptor activator of nuclear factor (NF)-kB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP2 +/+ mice 2-fold but had little effect on cells from EP2 −/− mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE2 increased the number of TRAP+ MNC in spleen cell cultures from EP2 +/+ mice but not in cultures from EP2 −/− mice. EP4RA had no effect on the PGE2 response in spleen cell cultures. PGE2 decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP2+ /+ mice but had little effect on cells from EP2 −/− mice. These data demonstrate that the prostaglandin EP2 receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP2 −/− mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE2 in EP2 −/− mice.

scholarly journals H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins

1978 ◽  
Vol 147 (2) ◽  
pp. 352-368 ◽  
Author(s):  
A Schmitt-Verhulst ◽  
CB Pettinelli ◽  
PA Henkart ◽  
JK Lunney ◽  
GM Shearer
Keyword(s):  
T Cell ◽  
Spleen Cell ◽  
Cell Cultures ◽  
Sodium Dodecyl ◽  
Soluble Proteins ◽  
Immunofluorescent Staining ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Spleen Cells

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

Regulated control by granulocyte-macrophage colony-stimulating factor AU-rich element during mouse embryogenesis

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1281-1288 ◽  
Author(s):  
Laurent Houzet ◽  
Dominique Morello ◽  
Patrick Defrance ◽  
Pascale Mercier ◽  
Georges Huez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Regulatory Element ◽  
Constitutive Expression ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

In vitro studies have indicated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression is regulated at the posttranscriptional level by the AU-rich element (ARE) sequence present in its 3′ untranslated region (UTR). This study investigated the importance of the ARE in the control of GM-CSF gene expression in vivo. For this purpose, transgenic mice bearing GM-CSF gene constructs containing or lacking the ARE (GM-CSF AU+ or GM-CSF AU−, respectively) were generated. Both transgenes were under the transcriptional control of the immediate early promoter of the cytomegalovirus (CMV) to ensure their early, widespread, and constitutive expression. The regulation imposed by the ARE was revealed by comparing transgene expression at day 14 of embryonic development (E14); only the ARE-deleted but not the ARE-containing construct was expressed. Although GM-CSF AU+ embryos were phenotypically normal, overexpression of GM-CSF in E14 GM-CSF AU− embryos led to severe hematopoietic alterations such as abnormal proliferation of granulocytes and macrophages accompanied by an increased number of peroxidase-expressing cells, their putative progenitor cells. These abnormalities compromise development because no viable GM-CSF AU− transgenic pups could be obtained. Surprisingly, by E18, significant accumulation of transgene messenger RNA was also observed in GM-CSF AU+ embryos leading to similar phenotypic abnormalities. Altogether, these observations reveal that GM-CSF ARE is a developmentally controlled regulatory element and highlight the consequences of GM-CSF overexpression on myeloid cell proliferation and differentiation.

scholarly journals Hematopoietic precursors in disease induced by the myeloproliferative sarcoma virus

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 305-307 ◽  
Author(s):  
B Klein ◽  
JJ Akouala ◽  
C Le Bousse-Kerdiles ◽  
F Smadja-Joffe ◽  
C Jasmin
Keyword(s):  
Pokeweed Mitogen ◽  
Spleen Cells ◽  
In Vitro Proliferation ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Proliferation And Differentiation ◽  
Sarcoma Virus ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract Granulocyte and macrophage precursors (GM-CFU-C-), which differentiate in vitro without added granulocyte and macrophage colony stimulating factor (GM-CSF), can be detected in the hematopoietic organs of mice infected with myeloproliferative sarcoma virus (MPSV). Retransplantation experiments have shown that the GM-CFU-C- are incapable of autonomous growth and depend on a factor present in medium conditioned by MPSV spleen cells (MPSV-CM). This factor is not MPSV and is not produced by spleen cells of noninfected mice. Two classical sources of GM-CSF, lung GM-CSF and GM-CSF contained in the plasma of endotoxin-treated mice, cannot replace the MPSV factor. Inversely, MPSV- CM does not stimulate the growth of retransplanted clusters induced in normal bone marrow with lung GM-CSF, whereas lung GM-CSF does. Two conditioned media containing activity promoting the in vitro proliferation and differentiation of hematopoietic stem cells in the mixed colony assay stimulate the growth of MPSV clusters: one medium was conditioned by pokeweed-mitogen-stimulated spleen cells, the other by the WEHI 3 cell line. The implication of the results in the comprehension of MPSV disease is discussed.

scholarly journals THYMUS-INDEPENDENT (B) CELL PROLIFERATION IN SPLEEN CELL CULTURES OF MOUSE RADIATION CHIMERAS STIMULATED BY PHYTOHEMAGGLUTININ OR ALLOGENEIC CELLS

1972 ◽  
Vol 136 (4) ◽  
pp. 962-967 ◽  
Author(s):  
P.-F. Piguet ◽  
P. Vassalli
Keyword(s):  
T Cell ◽  
Spleen Cell ◽  
Cell Cultures ◽  
Bone Marrow Cells ◽  
Spleen Cells ◽  
Radiation Chimeras ◽  
Lymphocyte Cultures ◽  
Mixed Lymphocyte Cultures

Spleen cell cultures of radiation chimeras (thymectomized, lethally irradiated mice repopulated with bone marrow cells and thymocytes bearing different chromosomal markers) were stimulated by phytohemagglutinin (PHA) and F1 allogeneic spleen cells. Karyotypic analyses showed a marked predominance of T mitoses on the 2nd and 3rd days of culture followed by a strong predominance of B mitoses on the 4th and 5th days. Analysis of cells undergoing their first mitoses showed that the majority of T mitoses on day 3 resulted from continuous T cell division, and that most cells entering their first mitoses at that time were of B type. Mixed lymphocyte cultures (MLC) of chimeras immunized against allogeneic spleen cells showed sometimes, but not always, a response different from "primary" MLC, with an earlier and stronger predominance of BM mitoses. The role of stimulated T cells in the induction of B mitoses was shown by (a) the incapacity of T-depleted spleen cells to be stimulated by PHA or in primary or secondary MLC, and (b) the restoration of the mitotic response of B cells to PHA by adding to the T cell-depleted culture either a very small number of T cell (identified by their different karyotype: "in vitro chimeras") or the cell-free supernatant of a 24 hr MLC.

scholarly journals Hematopoietic precursors in disease induced by the myeloproliferative sarcoma virus

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 305-307
Author(s):  
B Klein ◽  
JJ Akouala ◽  
C Le Bousse-Kerdiles ◽  
F Smadja-Joffe ◽  
C Jasmin
Keyword(s):  
Pokeweed Mitogen ◽  
Spleen Cells ◽  
In Vitro Proliferation ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Proliferation And Differentiation ◽  
Sarcoma Virus ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Granulocyte and macrophage precursors (GM-CFU-C-), which differentiate in vitro without added granulocyte and macrophage colony stimulating factor (GM-CSF), can be detected in the hematopoietic organs of mice infected with myeloproliferative sarcoma virus (MPSV). Retransplantation experiments have shown that the GM-CFU-C- are incapable of autonomous growth and depend on a factor present in medium conditioned by MPSV spleen cells (MPSV-CM). This factor is not MPSV and is not produced by spleen cells of noninfected mice. Two classical sources of GM-CSF, lung GM-CSF and GM-CSF contained in the plasma of endotoxin-treated mice, cannot replace the MPSV factor. Inversely, MPSV- CM does not stimulate the growth of retransplanted clusters induced in normal bone marrow with lung GM-CSF, whereas lung GM-CSF does. Two conditioned media containing activity promoting the in vitro proliferation and differentiation of hematopoietic stem cells in the mixed colony assay stimulate the growth of MPSV clusters: one medium was conditioned by pokeweed-mitogen-stimulated spleen cells, the other by the WEHI 3 cell line. The implication of the results in the comprehension of MPSV disease is discussed.

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