Presence Of Colony-stimulating Factor Research Articles

Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time.

Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.

The Presence of Colony-Stimulating Factor-1 and Its Receptor in Different Cells of the Testis; It Involved in the Development of Spermatogenesis In Vitro.

Spermatogenesis is a complex process, in which spermatogonial cells proliferate and differentiate in the seminiferous tubules of the testis to generate sperm. This process is under the regulation of endocrine and testicular paracrine/autocrine factors. In the present study, we demonstrated that colony stimulating factor-1 (CSF-1) is produced by mouse testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells (such as CDH1-positively stained cells; a marker of spermatogonial cells). In addition, we demonstrated the presence of CSF-1 and its receptor (CSF-1R) in testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells of human testis. We also show that the protein levels of CSF-1 were the highest in testis of 1-week-old mice and significantly decreased with age (2–12-week-old). However, the transcriptome levels of CSF-1 significantly increased in 2–3-week-old compared to 1-week-old, and thereafter significantly decreased with age. On the other hand, the transcriptome levels of CSF-1R was significantly higher in mouse testicular tissue of all examined ages (2–12-week-old) compared to 1-week-old. Our results demonstrate the involvement of CSF-1 in the induction the proliferation and differentiation of spermatogonial cells to meiotic and postmeiotic stages (BOULE- and ACROSIN-positive cells) under in vitro culture conditions, using methylcellulose culture system (MCS). Thus, it is possible to suggest that CSF-1 system, as a testicular paracrine/autocrine system, is involved in the development of different stages of spermatogenesis and may be used in the development of future therapeutic strategies for treatment of male infertility.

Abstract 2145: The role of MEN1611, a class I PI3-kinases (PI3Ks) inhibitor, in reprogramming the pro-tumoral inflammatory environment

Abstract MEN1611 (previously PA799) is a specific PI3Ks inhibitor with peculiar biochemical profile: it targets at the highest potency the mutated PI3Kα isoforms and the PI3Kγ isoform. PI3Kγ isoform is abundantly expressed in myeloid cells but not in cancer cells and promotes myeloid cell trafficking during inflammation and cancer. Previous studies have shown that selective inactivation of macrophage PI3Kγ is able to promote an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. Based on these evidences, we sought to investigate whether the anti-tumoral activity of MEN1611, that has been demonstrated especially in tumors harboring PI3Kα mutations, should be mediated also by inflammatory cells. To this aim we have set up an in vitro model in order to study macrophages polarization: in the presence of colony stimulating factor, mouse bone marrow-derived monocytes were directed into M0 macrophages differentiation, followed by M1 (by lipopolysaccharides and interferon γ) or M2 stimulation (by interleukin 4). Potential changes in macrophage phenotype induced by MEN1611 were measured by quantitative RT-qPCR and mouse cytokine antibody array. Moreover in order to evaluate in vivo the immune modulatory capacity of MEN1611 two syngeneic tumor models were established: 4T1 breast tumor model and CT26.wt colorectal tumor model. M1 or M2 polarized macrophages displayed different cell morphologies: M2 cells exhibited an higher degree of elongation compared with M1 or unstimulated M0 cells. Their activation status was tracked and confirmed by gene expression analysis, enzymatic assays (iNOS and arginase activity) and cytokine array. MEN1611 treatment was able to revert M2 cell morphology towards an M1-like round cell shape. Analysis of gene expression in MEN1611-treated macrophages showed a significant increase of immunostimulating factors mRNAs, such as IL-1b, IL-12b and iNOS, in M2 macrophages. MEN1611 also enhanced the secretion of pro-inflammatory cytokines both in M1 and M2 macrophages. In syngeneic mouse models, immune cell subsets were characterized via flow cytometry: both 4T1 and CT26.wt tumors had a similar baseline relative amount of M1 and M2 macrophages and were characterized by CD4+ and CD8+ T cells infiltrates. The impact of MEN1611 treatment on macrophages and T lymphocytes subsets infiltrating tumors is currently studied. In conclusion, we have demonstrated the ability of MEN1611 to modulate macrophage polarization towards an immune-activating phenotype at clinically relevant doses. These findings might suggest the involvement of PI3Kγ as part of MEN1611 mechanism of action. Citation Format: Stefania Capano, Alessio Fiascarelli, Andrea M Tomirotti, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Andrea Pellacani, Monica Binaschi. The role of MEN1611, a class I PI3-kinases (PI3Ks) inhibitor, in reprogramming the pro-tumoral inflammatory environment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2145.

Lithium attenuates lead induced toxicity on mouse non-adherent bone marrow cells

Lead is a poisonous heavy metal that occurs in all parts of environment and causes serious health problems in humans. The aim of the present study was to investigate the possible protective effect of lithium against lead nitrate induced toxicity in non-adherent bone marrow stem cells. Trypan blue and MTT assays represented that exposure of the cells to different concentrations of lead nitrate decreased viability in a dose dependent manner, whereas, pretreatment of the cells with lithium protected the cells against lead toxicity. Lead reduced the number and differentiation status of bone marrow-derived precursors when cultured in the presence of colony stimulating factor (CSF), while the effect was attenuated by lithium. The cells treated with lead nitrate exhibited cell shrinkage, DNA fragmentation, anion superoxide production, but lithium prevented lead action. Moreover, apoptotic indexes such as PARP cleavage and release of HMGB1 induced by lead, were protected by lithium, suggesting anti-apoptotic effect of lithium. Immunoblot analysis of histone H3K9 acetylation indicated that lithium overcame lead effect on acetylation. In conclusion, lithium efficiently reduces lead toxicity suggesting new insight into lithium action which may contribute to increased cell survival. It also provides a potentially new therapeutic strategy for lithium and a cost-effective approach to minimize destructive effects of lead on bone marrow stem cells.

Inflammatory response of TLR4 deficient spleen macrophages (CRL 2471) to Brucella abortus S19 and an isogenic ΔmglA deletion mutant

Brucellosis is a worldwide distributed zoonosis caused by members of the genus Brucella. One of them, Brucella abortus, is the etiological agent of bovine brucellosis. With the attenuated strain B. abortus S19 a vaccine is available. However, both, virulence (safety) and the ability to induce a protective B and T cell response (efficacy) have to be tested in suitable assays before successful use in the field. For this purpose, several macrophage cell lines of various origins have been used while splenic macrophages are the preferred host cells in vivo.We here characterized the in vitro response of the murine splenic macrophage cell line CRL 2471(I-13.35) to B. abortus. This cell line still depends on the presence of colony-stimulating factor 1 (CSF1) and is derived from LPS resistant (TLR4 deficient) C3H/HeJ mice. For infection the vaccine strain B. abortus S19A as well as the formerly described isogenic deletion mutant B. abortus S19A ΔmglA 3.14 were used.While numbers of viable bacteria did not differ significantly between the vaccine strain and the deletion mutant at 6h post infection, a higher bacterial load was measured in case of the mutant at 24h and 48h after infection. This was also true, when IFNγ was used for macrophage activation.A comprehensive gene expression profile of macrophages was analysed 6 and 24h after infection by means of an RT-PCR based gene expression array. The mutant strain B. abortus S19A ΔmglA 3.14 elicited a stronger cellular response of the splenic macrophages as compared to the parental vaccine strain. This was most prominent for the pro-inflammatory cytokines IL-1α, IL-1β, TNF-α and IL6 as well as for the chemokine ligands CXCL1, CXCL2, CXCL10, CCL2, CCL5, CCL7, CCL17 and the co-stimulatory molecules CD40 and ICAM1. While these differences were also present in IFNγ-stimulated macrophages, an addition of IFNγ after infection not only resulted in a dramatic increase of the translation of the afore mentioned genes but also resulted in the translation of IFNß1, IL12ß, MIP1α and β (CCL3, CCL4), NOS2 (and SOD2) and FAS. ConclusionThe TLR4 deficient murine splenic macrophage cell line CRL 2471 was used for the first time for the characterization of macrophage-Brucella interaction to investigate the pre-immune phase of brucellosis in vitro. Typical pro-inflammatory cytokines and certain surface receptors were differentially induced by B. abortus S19 A and an isogenic ΔmglA deletion mutant in vitro. This model may be useful for further studies to characterize the inflammatory response of splenic macrophages to intracellular gram-negative bacteria avoiding cell responses to soluble LPS.

Co-inhibition of colony stimulating factor-1 receptor and BRAF oncogene in mouse models of BRAFV600E melanoma

ABSTRACTThe presence of colony stimulating factor-1 (CSF1)/CSF1 receptor (CSF1R)-driven tumor-infiltrating macrophages and myeloid-derived suppressor cells (MDSCs) is shown to promote targeted therapy resistance. In this study, we demonstrate the superior effect of a combination of CSF1R inhibitor, PLX3397 and BRAF inhibitor, PLX4720, in suppressing primary and metastatic mouse BRAFV600E melanoma. Using flow cytometry to assess SM1WT1 melanoma-infiltrating leukocytes immediately post therapy, we found that PLX3397 reduced the recruitment of CD11b+ Gr1lo and CD11b+ Gr1int M2-like macrophages, but this was accompanied by an accumulation of CD11b+ Gr1hi cells. PDL1 expression on remaining myeloid cells potentially dampened the antitumor efficacy of PLX3397 and PLX4720 in combination, since PD1/PDL1 axis blockade improved outcome. We also reveal a role for PLX3397 in reducing tumor-infiltrating lymphocytes, and interestingly, this feature was rescued by the co-administration of PLX4720. Our findings, from three different mouse models of BRAF-mutated melanoma, support clinical approaches that co-target BRAF oncogene and CSF1R.

Isolation of bovine spermatogonial cells and co-culture with prepubertal sertoli cells in the presence of colony stimulating factor-1

BACKGROUND: Spermatogonial stem cells (SSCs) are infrequentself-renewing cells among the type A spermatogoniawithin the seminiferous tubules and are the basis of spermatogenesisin mammalian testis. An adequate number of SSCs is aprimary requirement for the study of their behavior, regulation, andfurther biomanipulation. OBJECTIVES: In this paper, we studiedthe development of the primary co-cultures of type A spermatogoniaand prepubertal bovine sertoli cells in the presence of ColonyStimulating Factor 1 (CSF1), a potential contributor in the SSCniche. METHODS: The effect of different concentrations of CSF1(0, 10, 50 and 100 ng/mL) on the colonization activity of spermatogonialcells was assessed 4, 7 and 11 days after the beginning of theculture by counting the total number of colonies and measuring theirarea in each group of the present experiment. Immunofluorescentstaining against OCT4 and vimentin led to the confirmation of thenature of both the SSCs and sertoli cells. RESULTS: Results showedthat the total number of colonies from day 4 to 11 increasedsignificantly in all groups, independent of CSF1 concentration. Inaddition, the total number and total area of colonies were higher (notsignificant) in 10 and 50 ng/mL CSF1 treatments than the controland 100 ng/mL CSF1 groups in all the three evaluations during theexperiment. However, this difference was only significant (p<0.05)between the total area of colonies in the control and 10 ng/mLCSF1groups at day 4 of co-culture. CONCLUSIONS:It was concluded thatCSF1 can be a suitable growth factor for improving SSCs colonizationin vitro, particularly during the first days of culture whereaccompanying sertoli cells still have not proliferated sufficiently tosupport the propagating spermatogonial cells.

A role for miR-142-3p in colony-stimulating factor 1-induced monocyte differentiation into macrophages

The differentiation of human peripheral blood monocytes into macrophages can be reproduced ex vivo by culturing the cells in the presence of colony-stimulating factor 1 (CSF1). Using microarray profiling to explore the role of microRNAs (miRNAs), we identified a dramatic decrease in the expression of the hematopoietic specific miR-142-3p. Up- and down-regulation of this miRNA in primary human monocytes altered CSF1-induced differentiation of monocytes, as demonstrated by changes in the expression of the cell surface markers CD16 and CD163. One of the genes whose expression is repressed by miR-142-3p encodes the transcription factor Early Growth Response 2 (Egr2). In turn, Egr2 associated with its co-repressor NGFI-A (Nerve Growth Factor-Induced gene-A) binding protein 2 (NAB2) binds to the pre-miR-142-3p promoter to negatively regulate its expression. Interestingly, the expression of miR-142-3p is abnormally low in monocytes from patients with the most proliferative forms of chronic myelomonocytic leukemia (CMML), and miR-142-3p re-expression in CMML dysplastic monocytes can improve their differentiation potential. Altogether, miR-142-3p which functions in a molecular circuitry with Egr2 is an actor of CSF1-induced differentiation of human monocytes whose expression could be altered in CMML.

Adenosine A1receptors (A1Rs) play a critical role in osteoclast formation and function

Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A(1)R, A(2A)R, A(2B)R, and A(3)R). Because A(1)R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A(1)R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A(1)R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the presence of colony stimulating factor-1 and receptor activator of NF-kappaB ligand in the presence or absence of the A(1)R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Osteoclast morphology was analyzed in tartrate-resistant acid phosphatase or F-actin-stained samples, and bone resorption was evaluated by toluidine blue staining of dentin. BMCs from A(1)R-knockout mice form fewer osteoclasts than BMCs from WT mice, and the A(1)R antagonist DPCPX inhibits osteoclast formation (IC(50)=1 nM), with altered morphology and reduced ability to resorb bone. A(1)R blockade increased ubiquitination and degradation of TRAF6 in RAW264.7 cells induced to differentiate into osteoclasts. These studies suggest a critical role for adenosine in bone homeostasis via interaction with adenosine A(1)R and further suggest that A(1)R may be a novel pharmacologic target to prevent the bone loss associated with inflammatory diseases and menopause.

Colony-Stimulating Factor-1 (CSF-1) Directly Inhibits Receptor Activator of Nuclear Factor-κB Ligand (RANKL) Expression by Osteoblasts

Colony-stimulating factor-1 (CSF-1), released by osteoblasts, stimulates the proliferation of osteoclast progenitors via the c-fms receptor (CSF-1R) and, in combination with receptor activator of nuclear factor-kappaB ligand (RANKL), leads to the formation of mature osteoclasts. Whether the CSF-1R is expressed by osteoblasts and mediates specific biological effects in osteoblasts has not been explored. Wild-type primary calvaria osteoblasts (OB) were analyzed for CSF-1R expression (RT-PCR and Western blot) and functionality (immunocomplex kinase assay). OB were serum starved for 24 h, and the effect of CSF-1 (0-100 ng/ml) on OB biological activities was determined at 48 h. In wild-type mouse bone marrow cultures, CSF-1 was tested for its effect on RANKL mRNA and osteoclast formation. Because ROS influence osteoblast RANKL expression, studies analyzed the effect of CSF-1 on reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and Nox1 and Nox4 proteins. Results indicate that OB express CSF-1R mRNA and protein and that CSF-1R could be phosphorylated in the presence of CSF-1. In osteoblasts, CSF-1 decreased RANKL mRNA in a dose- and time-dependent manner. Incubation of bone marrow cultures with CSF-1 resulted in a significant decline in tartrate-resistant acid phosphatase (TRACP) activity and CTR expression. RANKL-decreased expression by CSF-1 was correlated with a decrease of NADPH oxidase activity as well as Nox1 and Nox4 protein levels. These findings provide the first evidence that osteoblasts express CSF-1R and are a target for CSF-1 ligand. CSF-1-mediated inhibition of RANKL expression on osteoblasts may provide an important mechanism for coupling bone formation/resorption and preventing excessive osteoclastogenesis during normal skeletal growth.

Culture of Cells from Maternal Circulation, in Conditions Favoring Fetal Endothelial Cell Expansion, Does Not Facilitate the Preferential Expansion of Circulating Fetal Cells

Objectives: To establish optimal culture conditions for fetal endothelial cells, and determine whether these can be used for preferential expansion of fetal cells from maternal blood. Methods: Human adult microvascular and umbilical vein endothelial cells were cultured in the presence of colony-stimulating factor-1 (CSF-1), placental growth factor (PlGF), and transforming growth factor-β1 (TGF-β1). The effect of each cytokine was assessed. We expanded peripheral blood mononuclear cells (PBMCs) from 18 pregnant women using the conditions most favorable to fetal cells; in specimens from women carrying male fetuses (n = 9), cell origin was determined by PCR (SRY locus). Results: The optimal concentrations of CSF-1, PlGF and TGF-β1 were 10, 100, and 5 ng/ml, respectively. PBMCs from maternal blood expanded in the presence or absence of the cytokines; PCR analysis showed no Y sequences in cultured maternal samples. Conclusion: Optimal concentrations of CSF-1, PlGF and TGF-β1 for preferential expansion of fetal endothelial cells were determined in model cultures. However, when these conditions were applied to maternal blood samples, no fetal cells could be detected based on PCR for SRY in women carrying male fetuses.

Single Chain Antibody Vaccination in Mice Against Human Ovarian Cancer Enhanced by Microspheres and Cytokines

Experimental studies using monoclonal antibody have demonstrated that anti-idiotypic immunity can be enhanced by microspheres or cytokines. The underlying physiological principles behind these strategies involve sustained release of antigen and arousal of the immune system. In this study, a murine model was used to investigate the combination effect of microspheres and cytokines on anti-idiotypic response. A single chain antibody scFv-pDLl.O, which recognizes the human ovarian cancer antigen CA125 was formulated in microspheres and injected to mice alone or in the presence of colony stimulating factor (GM-CSF) or tumor necrosis factor (TNF-a). The immunization of mice with formulated single chain antibody and cytokines resulted in enhanced production of anti-idiotypic antibody, and which subsequently induced the production of anti-anti-idiotypic antibody. These results raise the possibility of cancer immunotherapy by administration of single chain antibody encapsulated in microspheres with GM-CSF or TNF-a.

Dendritic cell development from mobilized peripheral blood CD34+ cells.

Dendritic cells are not a homogenous population, but develop through different differentiation pathways, whose prominence is dependent on the cytokine milieu. Although dendritic cells can be readily derived from monocytes, dendritic cells that develop from CD34+progenitor cells have a more efficient antigen-presenting capability (1). However, even the development of dendritic cells from CD34+progenitor cells can occur through several different pathways (2,3). In one differentiation pathway, CD34+cells mature into committed CD14-CD1a+precursor cells that give rise to CD1a+dendritic cells. In a second differentiation pathway, CD34+cells first develop into bipotential CD14+CDla precursor cells, which mature into bipotential CD14+CD1a+ intermediate precursor cells. These intermediate cells can differentiate into monocytic cells in the presence of colony stimulating factor 1 (CSF-1), or into dendritic cells in the absence of CSF-1 (3,4). KeywordsDendritic CellPeripheral Blood CD34Isotype Control AntibodyResuspend CellConical Centrifuge TubeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Modulatory role of thymosin-alpha-1 in normal bone-marrow haematopoiesis and its effect on myelosuppression in T-cell lymphoma bearing mice

In continuation with the earlier and ongoing studies on Thymosin-alpha-1 (Tα1) exerting its immunomodulatory effects on various components of the immune system including T-cells, NK-cells, blood lymphocytes and macrophages, the role of Tα1 in normal bone-marrow haematopoiesis has been investigated in the present study. The haematopoietic alterations associated with the growth of murine T-cell lymphoma, Dalton's Lymphoma (DL) and subsequently its restoration by Tα1 was also investigated. It is observed that the non-adherent bone-marrow cells from normal mice (N-BMCs) exhibited enhanced proliferation on in vitro treatment with Tα1 (dose range of 1–100 ng/ml) with maximal response at 100 ng/ml of Tα1. In vitro stimulation with 100 ng/ml of Tα1 also resulted in increased myeloid colony formation, as manifested by the rise in total number of colonies, frequency of the individual colony types and their size. This response was further upregulated in the presence of various colony stimulating factors (CSFs) like MCSF, GMCSF, GCSF and IL-3. Similarly, in vivo administration of Tα1 (a single intraperitoneal injection of 10 μg per mouse) to normal mice also resulted in enhanced proliferation and colony formation by BMCs as compared with BMCs obtained from untreated mice. On the contrary, the progressive growth of T-cell lymphoma in mice led to suppressed myelogenesis, with marked reduction in the total colony numbers and their size. The BMCs from DL-bearing mice (DL-BMCs) displayed a preferential lineage-restricted differentiation towards the granulocytic-type colonies with maximum numbers of CFU-Gs and CFU-GMs, followed by CFU-Ms. However, incubation of DL-BMCs with 100 ng/ml of Tα1, in vitro restored their suppressed proliferation and colony forming ability (CFA) with significantly enhanced total number of colonies and individual colony types, which further increased in the presence of CSFs. In vivo studies with BMCs from DL-bearing mice treated with single intraperitoneal injection of 10 μg Tα1/mouse also resulted in significant enhancement in their proliferative as well as colony forming ability in comparison to that of untreated DL-mice. The present observations suggest that Tα1 can positively modulate the haematopoietic functions of normal murine BMCs, in addition to its myelorestorative role in tumour-bearing mice showing suppressed myelopoiesis.

Protein tyrosine phosphatase phi regulates paxillin tyrosine phosphorylation and mediates colony-stimulating factor 1-induced morphological changes in macrophages.

Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase phi (PTP phi). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTP phi and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTP phi induces cell rounding and ruffle formation, while C325S PTP phi has no effect. In contrast, in CSF-1-starved cells, C325S PTP phi behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTP phi increases adhesion in cycling cells, while WT PTP phi enhances motility. In WT PTP phi-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTP phi-expressing cells. Moreover, a complex containing PTP phi, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTP phi selectively binds paxillin and Pyk2 in vitro. Although PTP phi and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTP phi in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility.

Effect of angiotensin II on hematopoietic progenitor cell proliferation.

Angiotensin II (AII) induced the proliferation of hematopoietic progenitor cells (HPC) isolated from murine bone marrow or human cord blood. The formation of colonies with more than 50 cells increased approximately five-sevenfold in cultures of murine lineage-negative (Lin(-)) bone marrow cells both in the presence (day 10) and absence (day 13) of colony-stimulating factors (CSF). This could be blocked with addition of Losartan, an antagonist of AIITR1. The increase in proliferation of early hematopoietic progenitors (Lin(-)Sca l(+) cells) by AII was approximately threefold and occurred only in the presence of CSF, suggesting that AII may affect mesenchymal stromal cells to induce CSF production and might directly affect early HPC. These in vitro studies were replicated with human HPC isolated from cord blood. AII also accelerated the proliferation and formation of colony-forming units (CFU)-granulocyte/erythroid/macrophage/megakaryocyte and CFU-granulocyte/macrophage colonies by CD34(+)CD38(-) enriched progenitors but only in the presence of CSF. Additional studies also indicated that AII can act to increase proliferation in suspension culture. Exposure of CD34(+) cells to AII in suspension culture, prior to placement in a semisolid medium with erythropoietin, increased the formation of colonies with more than 50 cells and erythroid progenitors approximately five- and 20-fold, respectively. Further, mRNA for the AT1a receptor was expressed by human bone marrow CD34(+)CD38(-) cells, CD34(+)CD38(-) cells, and lymphocytes, but not mature myeloid cells. Similarly, mRNA for the AT1a receptor was expressed on human stromal cell clones, offering further support to the hypothesis that AII acts partially through the mesenchymal compartment of the bone marrow. These data suggest that AII may be a factor which stimulates the proliferation of hematopoietic progenitors.

Multinucleated giant cell formation by microglia: induction by interleukin (IL)-4 and IL-13

To investigate the cellular origin and the mechanisms of multinucleated giant cell (MGC) formation in the CNS, various cytokines were applied to isolated murine microglia. All the single cytokines failed to induce MGC. However, interleukin (IL)-13 and IL-4 induced microglial MGC formation in the presence of colony stimulating factors, which was inhibited by either anti-IL-13 or anti-fms antibody. T helper 2 (Th2)-derived cytokines can induce MGC formation even in the absence of infectious agents.

Separate precursor cells for macrophages and microglia in mouse brain: immunophenotypic and immunoregulatory properties of the progeny

The brain contains two populations of macrophages: the microglia of brain parenchyma, and the central nervous system (CNS) macrophages located in the perivascular spaces, the leptomeninges and the choroid plexus. The microglia are characterized, in part, by their paucity of major histocompatibility complex (MHC) molecules and lack of constitutive antigen (Ag)-presenting activity for naı̈ve CD4 + T-cells. Some CNS macrophages, on the other hand, constitutively express MHC molecules and present Ag to naı̈ve CD4 + T-cells. We have reported that mouse brain contains precursor cells that, in the presence of colony-stimulating factor-1, the macrophage growth factor, give rise to clones of cells that differ in their ability to constitutively present Ag to naı̈ve CD4 + T cells. Here we report that this population of precursor cells can be separated into two discrete subpopulations based on differences in cell density and that the two cell populations give rise to progeny that differ in their content of cells constitutively expressing MHC class II and CD86 molecules, and the ability to present Ag to naı̈ve CD4 + T-cells. A comparison of the level of CD45 staining of the progeny, an indication of a microglial or a CNS macrophage origin, suggests that one population of precursor cells yields immunologically immature microglia and the other CNS macrophages.

Synergistic Activation of Mitogen-Activated Protein Kinase by Cyclic AMP and Myeloid Growth Factors Opposes Cyclic AMP's Growth-Inhibitory Effects

AbstractColony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90rsk. cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1–mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.

Synergistic Activation of Mitogen-Activated Protein Kinase by Cyclic AMP and Myeloid Growth Factors Opposes Cyclic AMP’s Growth-Inhibitory Effects

Colony-stimulating factors (CSFs) promote the proliferation, differentiation, commitment, and survival of myeloid progenitors, whereas cyclic AMP (cAMP)-mediated signals frequently induce their growth arrest and apoptosis. The ERK/mitogen-activated protein kinase (MAPK) pathway is a target for both CSFs and cAMP. We investigated how costimulation by cAMP and colony-stimulating factor-1 (CSF-1) or interleukin-3 (IL-3) modulates MAPK in the myeloid progenitor cell line, 32D. cAMP dramatically increased ERK activity in the presence of CSF-1 or IL-3. IL-3 also synergized with cAMP to activate ERK in another myeloid cell line, FDC-P1. The increase in ERK activity was transmitted to a downstream target, p90(rsk). cAMP treatment of 32D cells transfected with oncogenic Ras was found to recapitulate the superactivation of ERK seen with cAMP and CSF-1 or IL-3. ERK activation in the presence of cAMP did not appear to involve any of the Raf isoforms and was blocked by expression of dominant-negative MEK1 or treatment with a MEK inhibitor, PD98059. Although cAMP had an overall inhibitory effect on CSF-1-mediated proliferation and survival, the inhibition was markedly increased if ERK activation was blocked by PD98059. These findings suggest that upregulation of the ERK pathway is one mechanism induced by CSF-1 and IL-3 to protect myeloid progenitors from the growth-suppressive and apoptosis-inducing effects of cAMP elevations.