Spleen Cell Conditioned Medium Research Articles

Class II transactivator-mediated regulation of major histocompatibility complex class II antigen expression is important for hematopoietic progenitor cell suppression by chemokines and iron-binding proteins

Iron-binding proteins H-ferritin (HF) and lactoferrin (LF), as well as chemokines, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma suppress hematopoietic progenitor cell (HPC) proliferation. Major histocompatibility complex (MHC) class II antigens have been associated with suppressive effects of HF and LF. Because the transcription factor class II transactivator (CIITA) regulates expression of MHC class II antigens, we evaluated influences of CIITA and MHC class II antigens on suppression of colony formation by murine bone marrow HPC in response to HF, LF, CC, and CXC chemokines, TNF-alpha, and IFN-gamma. We also evaluated hematopoiesis in mice deficient in both CIITA and MHC class II antigens (CIITA -/-), in mice deficient in MHC class II antigens but not in CIITA (MHC class II -/-), and in mice deficient in CIITA but not in MHC class II antigens (CIITA-IE). HF, LF, CCL3/MIP-1alpha, CXCL5/ENA-78, CXCL8/IL-8, CCL5/RANTES, TNF-alpha, and IFN-gamma were assessed for effects on colony formation by bone marrow HPC (colony-forming unit granulocyte-macrophage, burst-forming unit erythroid, and colony-forming unit multipotential) stimulated in vitro by combinations of growth factors including erythropoietin, stem cell factor, pokeweed mitogen mouse spleen cell conditioned medium, and hemin. Bone marrow cells were from CIITA -/-, MHC class II antigen -/-, CIITA-IE, and littermate control mice. We also evaluated cycling status (percent cells in S-phase) and absolute numbers of marrow and spleen HPC in these mice. Multiple growth factor-stimulated colony formation by control bone marrow HPC was significantly suppressed by HF, LF, CCL3, CXCL5, CXCL8, TNF-alpha, and IFN-gamma, but not by CCL5. However, HPC from CIITA -/- and MHC class II antigen -/- mouse marrow was insensitive to inhibition by HF, LF, CCL3, CXCL5, CXCL8, and CCL5; these HPC were inhibited by TNF-alpha and IFN-gamma. Restoration of MHC class II expression in CIITA -/- (CIITA-IE) mice restored responsiveness of HPC to inhibition by HF, LF, CCL3, CXCL5, and CXCL8. Increased cycling of splenic HPC in CIITA -/- and MHC class II antigen -/-, compared to control and CIITA-IE, mice was noted. Myelosuppressive effects of iron-binding proteins HF and LF and chemokines CCL3, CXCL5, and CXCL8 on mouse bone marrow HPC require expression of MHC class II antigens, and CIITA is involved in this responsiveness through its regulation of expression of MHC class II antigens.

Requirement of Smad3 for mast cell growth

The involvement of the TGF-β family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-β and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-β pathway by anti-TGF-β neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-β and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-β-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion.

Mad2 (Mitotic Arrest Deficiency 2) Is Essential for the Synergistic Proliferation Induced by Stem Cell Factor Combined with GM-CSF in Hematopoietic Progenitor Cells, Effects Reflecting the Association of c-kit with Mad2.

Mitotic spindle checkpoint protein, Mad2, is required for proper functioning of the mitotic checkpoint which ensures correct chromosome segregation during cell division. Homozygous Mad2 gene deletion is embryonic-lethal. Mad2 interacts with mitosis-associated molecules such as Mad1 and anaphase promoting complex/cyclosome to ensure proper cell cycle progression. Recently, Mad2 was shown to physically associate with the common beta chain of the GM-CSF receptor which raises the possibility that Mad2 may also be involved in cytokine signaling and regulation of mitosis in hematopoietic progenitor cells. To investigate this, we studied hematopoiesis and cytokine signaling in Mad2-haploinsufficient (+/−) mutant mice (M2MT). Colony formation by granulocyte macrophage progenitor cells (CFU-GM) from bone marrow of wild type (WT) mice is synergistically stimulated in vitro by the combination of stem cell factor (SCF) and GM-CSF. We found that bone marrow CFU-GM from M2MT mice are deficient in the synergistic proliferative/colony formation response in vitro to stimulation with the combination of GM-CSF plus SCF. In contrast, there was no difference in stimulation of CFU-GM formation in response to the individual cytokines, GM-CSF or SCF alone, nor a difference in response to pokeweed mitogen mouse spleen cell conditioned medium between M2MT and WT mice. Because there was no difference in the frequency of c-kit+Sca-1+Lin- (KSL) cells nor a difference in the intensity of c-kit surface expression on KSL cells from wild type and M2MT mice, we considered whether the suppression of the SCF/GM-CSF synergy response was due to a difference in intracellular growth-factor receptor signaling pathways. We found that the kinetics of Erk1/2 phosphorylation signaling differ in M2MT Lin- cells compared to WT Lin- cells and that the duration of Erk1/2 phosphorylation in M2MT cells was at least one half of that in WT Lin- cells. On the other hand, we found no difference in the kinetics of Akt phosphorylation between WT and M2MT Lin- cells suggesting a specificity of involvement of the MAP-kinase pathways. To understand how Mad2 plays a role in SCF/GM-CSF synergy, we tested the physical interaction between Mad2 and c-kit in primary Lin- mouse bone marrow cells. Primary Lin- bone marrow cells from WT mice were expanded in liquid culture with SCF and thrombopoietin for 5 days. We found that Mad2 physically associated with c-kit as indicated by co-immunoprecipitation. These results suggest that Mad2 is required for the SCF/GM-CSF proliferative-synergy response in primary Lin- mouse bone marrow cells and that Mad2 is involved in growth-factor signaling pathways, such as the MAP-kinase cascade, in addition to spindle checkpoint function in primary hematopoietic cells. These effects are likely mediated through Mad2 interaction with c-kit and the beta chain of the GM-CSF receptor.

Effect of Spleen-Cell-Conditioned Medium on [3H]-Choline Uptake by Retinal Cells in vitro Is Mediated by IL-2

Cytokines are essential molecules throughout the development of the nervous system and also play an important role during the adult life span. In the present work, we analyzed in vitro the effect of spleen-cell-conditioned medium (SCM) on the survival and [³H]-choline uptake of neonatal rat retinal cells. SCM induced an increase in neuronal survival, glial cell proliferation and neurite outgrowth, as evaluated by biochemical and morphological criteria. These effects were time dependent; after 120 h, SCM induced a 6-fold increase in the total protein level. The effect of SCM was blocked both by the inhibition of protein tyrosine kinase activity (10 μM genistein) and by the inhibition of cell division (20 μM fluorodeoxyuridine). SCM also increased the uptake of [³H]-choline by retinal cells. The effect was time dependent. The maximum effect was obtained after 48 h and was maintained at a high level until 120 h. Treatment by 10 μM genistein and 15 μM bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) (an intracellular calcium chelator) completely blocked this effect. However, 20 μM fluorodeoxyuridine did not abolish it. Conditioned medium obtained from glial cells stimulated with SCM (S-GCM) induced an effect on [³H]-choline uptake earlier than that promoted by SCM. Anti-interleukin-2 (IL-2) antibodies blocked the effect of both SCM and S-GCM on [³H]-choline uptake after 48 and 72 h. IL-2 (50 U/ml) elicited the same effect as that observed when the cells were maintained in the presence of SCM. Taken together, our results suggest that IL-2 plays an important role in controlling the survival and differentiation of retinal cells in vitro.

Schistosoma mansoni infection in mice augments the capacity for interleukin 3 (IL-3) and IL-9 production and concurrently enlarges progenitor pools for mast cells and granulocytes-macrophages.

Mast cells and granulocytes-macrophages (GM) are components of the host defense system against worm infections, including schistosomiasis. Here we report the kinetics of changes in the number of colony-forming cells (CFC) for mast cells and GM during the course of a primary experimental infection of mice with Schistosoma mansoni cercariae over a period of 24 weeks postinfection (p.i.). Concurrently, we measured known myelopoietic and/or mast cell-stimulating cytokines (i.e., interleukin 3 [IL-3] and IL-9) in pokeweed mitogen-activated spleen cell-conditioned medium. Our results show that during the acute phase of the hepatic granulomatous reaction, the numbers of both mast-CFC and GM-CFC were significantly elevated in bone marrow. However, while femoral GM-CFC numbers had returned to normal control values at week 16 p.i., femoral and splenic mast-CFC numbers remained significantly elevated until week 20 p.i., which corresponds to the chronic fibrotic phase of hepatic granulomatous inflammation. Increased GM-CFC numbers correlated with elevated IL-3 levels, while increased mast-CFC numbers paralleled the increased IL-9 concentrations in spleen cell-conditioned medium. By the reverse transcription-PCR method, enhanced expression of IL-3 and IL-9 transcripts was found in RNA samples obtained from livers and spleens of infected mice. Our data demonstrate that during the course of infection of mice with S. mansoni, the coordinate need for mast cells and GM is at least partly regulated at the stage of progenitor cell commitment in the bone marrow and spleen. It appears that IL-3 and IL-9 help to promote at this stage the ultimate generation of mature effector cells.

Stem cell factor protects bone marrow-derived cultured mast cells (BMCMC) from cytocidal effect of nitric oxide secreted by fibroblasts in murine BMCMC-fibroblast coculture.

The survival of mast cells are dependent on two kinds of growth factors, one derived from T cells (IL-3) and another derived from fibroblasts (stem cell factor [SCF]). The 3T3 fibroblast cell line derived from WCB6F(1-)+/+ mouse embryos (+/+ 3T3 fibroblasts) supported the proliferation of bone marrow-derived cultured mast cells (BMCMC) in the PWM-stimulated spleen cell conditioned medium (PWM-SCM), whereas the 3T3 fibroblast cell line from WCB6F1-Sl/Sld mouse embryos (Sl/Sld 3T3 fibroblasts) did not. To study the role of nitric oxide (NO) on the growth of mast cells in BMCMC-fibroblast coculture, we used a NO synthase inhibitor, NG-monomethyl-L-arginine (NGMMA). NGMMA recovered survival and maintained proliferation of mast cells in BMCMC-Sl/Sld 3T3 fibroblasts coculture. Sl/Sld 3T3 fibroblasts as well as 3T3 fibroblasts from NIH(-)+/+, BALB(-)+/+ or Swiss(-)+/+ mouse embryos secreted NO in PWM-SCM, but not in alpha-MEM. SCF protected BMCMC from cytotoxicity of exogenous NO in IL-3-supplemented alpha-MEM. We concluded that SCF might protect BMCMC from cytocidal effect of NO in BMCMC-fibroblasts coculture.

Identification of a unique membrane-bound molecule on a hemopoietic stem cell line and on multipotent progenitor cells.

Hemopoietic stem cells are a distinct population of cells that can differentiate into multilineages of hemopoietic cells and have long-term repopulation capability. A few membrane-bound molecules have been found to be preferentially, but not uniquely, present on the surface of these primitive cells. We report here the identification of a unique 105-kDa glycoprotein on the surface of hemopoietic stem cell line BL3. This molecule, recognized by the absorbed antiserum, is not present on the surface of myeloid progenitors 32D and FDC-P1 cells, EL4 T cells, and NIH 3T3 fibroblasts. This antiserum can also be used to block the proliferation of BL3 cells even in the presence of mitogen-stimulated spleen cell conditioned medium, which is known to have a stimulating activity on BL3 cells. It can also inhibit development of in vitro, fetal liver cell-derived multilineage colonies, but not other types of colonies, and of in vivo bone marrow cell-derived colony-forming unit spleen foci. These data suggest that gp105 plays an important role in hemopoietic stem cell differentiation.

Infection With Nippostrongylus brasiliensis Induces Invasion of Mast Cell Precursors From Peripheral Blood to Small Intestine

Precursors of mast cells were defined as cells that formed mast-cell colonies in methylcellulose culture (CFU-mast). Mononuclear cells (MNC) were obtained from the bone marrow, peripheral blood, and small intestine of Ws/Ws rats with a small deletion at the tyrosine kinase domain of c-kit and of control normal (+/+) rats. In the culture containing concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM) alone, the numbers of mast-cell colonies produced by Ws/Ws MNC were comparable with those of +/+ MNC. In the culture containing both ConA-SCM and stem cell factor (a ligand of c-kit), however, the numbers of mast-cell colonies produced by +/+ blood MNC were 107 times as great as that of Ws/Ws blood MNC. Using this culture condition, we investigated changes in concentration of CFU-mast in the marrow, blood, and intestine of +/+ rats after infection with Nippostrongylus brasiliensis (NB), which induced marked mast-cell accumulation in the small intestine. The concentration of CFU-mast in blood dropped to 21% of preinfection levels 1 week after the NB infection. In contrast, a sevenfold increase of CFU-mast occurred in the small intestine. The proportion of CFU-mast in S phase of the cell cycle remained at low levels in the marrow and blood after NB infection, but it increased significantly in the small intestine. The present result suggests that NB infection induces the invasion of CFU-mast into the intestine from blood and their subsequent proliferation in the tissue site.

Immortalized hemopoietic cells with stem cell properties

We have established a hemopoietic cell line, BL3, that possesses a rearranged retroviral genome, which we used as a genetic tag for their engraftment into lethally irradiated mice. Analysis of recipients up to 7 months after engraftment indicates that the marker was present in differentiated cells of various hemopoietic organs, in colony-forming cells, pre-CFU-S-forming cells, and in organs of secondary recipients from bone marrow cells of primary recipients. BL3 cells are Thy-1 +, Sca-1 +, B220 −, Mac-1 -, and Gr-1 −. They express GATA-1 and are able to develop “cobblestones” with stromal cells. They do not express and respond to several hemopoietic growth factors known to facilitate marrow recovery. However, they are negatively regulated by TGFO and can be stimulated by mitogenstimulated spleen cell-conditioned medium. We conclude that BL3 cells possess properties of hemopoietic stem cells, including their capability to contribute to long-term repopulation.

Inhibition of tumor necrosis factor-mediated lysis by spleen cell-conditioned medium

A spleen cell-elaborated factor which inhibits both TNF and natural cytotoxic cell lytic activity has been described. Production of this factor is dependent on prostaglandins, and inhibitory activity is abrogated by protein synthesis inhibitors. Furthermore, it was demonstrated that the factor renders TNF sensitive cells refractory to TNF rather than acting on TNF directly. Here we show that the factor is produced by spleen cells from both BALB/c and C57bl/6 mice and inhibits TNF mediated lysis of various TNF sensitive cells. The inhibitory factor has a molecular weight greater than 30 kDal and is heat labile, suggesting it is a protein. Furthermore, the factor is produced by high density spleen cells (e.g. cells of the myeloid lineage). Although it is not clear which cell type(s) is responsible for the production of this factor, these findings suggest that high density spleen cells, which do not produce TNF, can modulate TNF lytic activity via this factor.

Characterization of Cultured Mast Cells Derived From Ws/Ws Mast Cell-Deficient Rats With a Small Deletion at Tyrosine Kinase Domain of c-kit

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP-I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I-/II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA-SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.

Characterization of cultured mast cells derived from Ws/Ws mast cell- deficient rats with a small deletion at tyrosine kinase domain of c-kit

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP- I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I- /II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA- SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.

A study of mast cells in autoimmune mice: the proliferative response of autoimmune mast cells to cytokines.

To investigate the mechanisms underlying the increased number of mast cells in autoimmune mice, the proliferative response of autoimmune mast cells to cytokines was examined. Bone marrow cells from autoimmune NZB mice produced scarcely any bone marrow derived mast cells (BMMCs) in the presence of interleukin 3 (IL-3), but were able to generate BMMCs when cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). In contrast, NZB BMMCs showed very little proliferation in the presence of PWM-SCM, but proliferated strongly when cultured with stem cell factor (SCF). Non-autoimmune NZW BMMCs showed a strong proliferative response to both IL-3 and PWM-SCM, but proliferated weakly in culture with SCF. Autoimmune NZB x NZW F1 (B/W) BMMCs shared the proliferative activities of both NZB and NZW BMMCs, showing strong proliferation in response to IL-3, PWM-SCM and SCF. All strains (including other non-autoimmune strains) except for NZW demonstrated synergism between PWM-SCM and SCF. This study suggests that the strong proliferative response of autoimmune mast cells to SCF plays a major role in, and that other cytokines are partially responsible for, increasing the number of mast cells in autoimmune mice. These mechanisms are discussed in relation to both constitutive and inducible hematopoiesis.

Clonal proliferation of murine lymphohemopoietic progenitors in culture.

We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.

A collection of mRNA species that are inducible in the RAW 264.7 mouse macrophage cell line by gamma interferon and other agents.

To identify genes induced during macrophage activation, a cDNA library was prepared from cultures of the RAW 264.7 mouse macrophage cell line that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the cDNA library was screened by differential plaque hybridization. Eleven cDNA clones, designated CRG-1 through CRG-11, corresponding to mRNA species inducible in RAW 264.7 cells by the spleen cell conditioned medium, were isolated. Inductions were not blocked by cycloheximide. All of the mRNAs were inducible by gamma interferon, and some were also inducible by alpha and beta interferons, by lipopolysaccharide, by phorbol 12-myristate 13-acetate, and by the calcium ionophore A23187. Sequencing of the cDNAs revealed that CRG-1, CRG-3, and CRG-5 are cDNAs of recently identified transcription factors IRF-1, zif/268, and LRF-1 respectively. As previously reported, CRG-2 and CRG-10 (MIG) encode new members of the platelet factor 4 family of cytokines. CRG-6 corresponds to a new member of a family of interferon-inducible genes clustered on mouse chromosome 1, CRG-9 corresponds to a prostaglandin synthase homolog, CRG-8 corresponds to beta 2-microglobulin, and CRG-4 corresponds to metallothionein II. CRG-11 contains sequences of a truncated L1Md repetitive element as well as nonrepetitive sequences. The nonrepetitive sequence of CRG-11 as well as the sequences of CRG-7 are not closely related to published sequences. The CRG genes and proteins are of interest because of their involvement in macrophage activation, because of their roles as mediators of the effects of gamma interferon and other pleiotropic agents, and because of their usefulness as tools for studying the signal pathways through which gamma interferon and other inducers exert their effects on gene and protein expression.

A collection of mRNA species that are inducible in the RAW 264.7 mouse macrophage cell line by gamma interferon and other agents.

To identify genes induced during macrophage activation, a cDNA library was prepared from cultures of the RAW 264.7 mouse macrophage cell line that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the cDNA library was screened by differential plaque hybridization. Eleven cDNA clones, designated CRG-1 through CRG-11, corresponding to mRNA species inducible in RAW 264.7 cells by the spleen cell conditioned medium, were isolated. Inductions were not blocked by cycloheximide. All of the mRNAs were inducible by gamma interferon, and some were also inducible by alpha and beta interferons, by lipopolysaccharide, by phorbol 12-myristate 13-acetate, and by the calcium ionophore A23187. Sequencing of the cDNAs revealed that CRG-1, CRG-3, and CRG-5 are cDNAs of recently identified transcription factors IRF-1, zif/268, and LRF-1 respectively. As previously reported, CRG-2 and CRG-10 (MIG) encode new members of the platelet factor 4 family of cytokines. CRG-6 corresponds to a new member of a family of interferon-inducible genes clustered on mouse chromosome 1, CRG-9 corresponds to a prostaglandin synthase homolog, CRG-8 corresponds to beta 2-microglobulin, and CRG-4 corresponds to metallothionein II. CRG-11 contains sequences of a truncated L1Md repetitive element as well as nonrepetitive sequences. The nonrepetitive sequence of CRG-11 as well as the sequences of CRG-7 are not closely related to published sequences. The CRG genes and proteins are of interest because of their involvement in macrophage activation, because of their roles as mediators of the effects of gamma interferon and other pleiotropic agents, and because of their usefulness as tools for studying the signal pathways through which gamma interferon and other inducers exert their effects on gene and protein expression.

Developmental kinetics of hemopoietic progenitors in the avian embryo spleen

By means of plasma clot clonal cultures, the content of the avian spleen in granulomonocytic progenitors was studied during ontogeny. Serum-free media were used that were supplemented with growth activities produced either by embryonic fibroblasts or adult spleen cells. These two conditioned media not only permitted the growth of M-CFC, G-CFC, and GM-CFC but also F-CFU (fibroblast colony-forming units) from quail or chick embryonic spleen cells. The presence of spleen cell-conditioned medium promoted the development of large colonies of immature granulocytes. In the chick the first hemopoietic progenitors appeared at E9 and their number displayed two peaks, one at E15 and a smaller one at E18. In the quail the first progenitors were detected as early as E7 and their number peaked at E10. In this species, hemopoietic progenitors disappeared definitively before hatching while in the chick some were still present at P3. The progenitor content of the chick embryo spleen was compared to that of the bone marrow. This content remained stable during all of embryonic life, while the bone marrow exhibited a very different profile, where a sharp peak at E16 was followed by an acute decline and a stabilization at a rather low level. The particular profile in the spleen speaks in favor of a special role of this organ in the development of the hemopoietic system.

In vitro cloning of murine megakaryocyte progenitors (CFU-Meg)

Convenient and reproducible culture systems for murine megakaryocyte progenitor cells (colony-forming unit-megakaryocytes, CFU-Meg) are described. Mouse bone marrow cells are cultured in fibrin clots supplemented with Iscove's modified Dulbecco's medium and fetal bovine serum or in fibrin clots supplemented with serum-free IMDM, bovine serum albumin, transferrin, cholesterol, and L-α-phosphatidylcholine. In the presence of murine interleukin-3 or pokeweed mitogen-stimulated murine spleen cell-conditioned medium, these cultures support CFU-Meg colony formation effectively. The cultivation and counting of colonies in these culture systems is considerably easier when compared with previously reported culture systems.

Identification of CRG-2. An interferon-inducible mRNA predicted to encode a murine monokine.

In order to identify novel proteins produced by activated macrophages, a cDNA library was made from cultures of the mouse macrophage-like cell line RAW 264.7 that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the library was screened by differential plaque hybridization. A cDNA clone was isolated that detected a 1.4-kilobase mRNA that accumulated dramatically in response to the spleen cell conditioned medium. The 1.4-kilobase mRNA encodes a predicted protein of 98 amino acids, designated CRG-2, molecular weight (Mr) 10,781, with a 21-residue signal peptide. The amino acid sequence indicates that CRG-2 is a member of the platelet factor 4 family (PF4) of cytokines. The CRG-2 mRNA was induced by alpha-, beta-, and gamma-interferons (IFNs) and by lipopolysaccharide. In response to IFN-gamma, the CRG-2 mRNA level reached a peak between 3 and 6 h. The accumulation of CRG-2 mRNA was not blocked by cycloheximide. Among the known members of the PF4 family, CRG-2 is most closely related to the interferon-inducible human protein IP-10. The 5'-flanking region of the crg-2 gene was isolated, and comparisons between crg-2 and IP-10 genes, mRNAs, and proteins reveal conserved features of possible functional importance. CRG-2 may play a role in host defense, particularly in the response to viral infection.

Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts

Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.