Articles de revues : « MCSF receptor »

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Azuma, C., F. Saji, T. Kimura, Y. Tokugawa, M. Takemura, Y. Samejima et O. Tanizawa. « Steroid hormones induce macrophage colony-stimulating factor (MCSF) and MCSF receptor mRNAs in the human endometrium ». Journal of Molecular Endocrinology 5, n^o 2 (octobre 1990) : 103–8. http://dx.doi.org/10.1677/jme.0.0050103.

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ABSTRACT We investigated the biological effects of sex-steroid hormones, secreted from the corpus luteum and placenta, on the induction of mRNAs encoding macrophage colony-stimulating factor (MCSF) and c-fms proto-oncogene (MCSF receptor) in human endometrium. RNA was extracted from the placenta and endometrium of both pregnant and non-pregnant women, and Northern blot analysis was performed on poly(A)+ RNA using MCSF or c-fms proto-oncogene cDNA as the probe. Results showed: (1) that MCSF mRNA was expressed in the placenta and endometrium of the pregnant uterus, (2) that c-fms proto-oncogene mRNA was also expressed in the placenta and endometrium of the pregnant uterus, and (3) that exogenous sex-steroid hormones could induce the expression of MCSF and c-fms proto-oncogene mRNAs in the endometrium of non-pregnant women. These results indicate that sex-steroid hormones secreted by the corpus luteum and/or placenta influence endometrial and placental growth and differentiation via a mechanism of action involving local production of MCSF and its receptor.

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Frangogiannis, Nikolaos G., Leonardo H. Mendoza, Guofeng Ren, Spyridon Akrivakis, Peggy L. Jackson, Lloyd H. Michael, C. Wayne Smith et Mark L. Entman. « MCSF expression is induced in healing myocardial infarcts and may regulate monocyte and endothelial cell phenotype ». American Journal of Physiology-Heart and Circulatory Physiology 285, n^o 2 (août 2003) : H483—H492. http://dx.doi.org/10.1152/ajpheart.01016.2002.

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Myocardial infarction is associated with the rapid induction of mononuclear cell chemoattractants that promote monocyte infiltration into the injured area. Monocyte-to-macrophage differentiation and macrophage proliferation allow a long survival of monocytic cells, critical for effective healing of the infarct. In a canine infarction-reperfusion model, newly recruited myeloid leukocytes were markedly augmented during early reperfusion (5–72 h). By 7 days, the number of newly recruited myeloid cells was reduced, and the majority of the inflammatory cells remaining in the infarct were mature macrophages. Macrophage colony-stimulating factor (MCSF) is known to facilitate monocyte survival, monocyte-to-macrophage conversion, and macrophage proliferation. We demonstrated marked induction of MCSF mRNA in ischemic segments persisting for at least 5 days after reperfusion. MCSF expression was predominantly localized to mature macrophages infiltrating the infarcted myocardium; the expression of the MCSF receptor, c-Fms, a protein with tyrosine kinase activity, was found in these macrophages but was also observed in a subset of microvessels within the infarct. Many infarct macrophages expressed proliferating cell nuclear antigen, a marker of proliferative activity. In vitro MCSF induced monocyte chemoattractant protein-1 synthesis in canine venous endothelial cells. MCSF-induced endothelial monocyte chemoattractant protein-1 upregulation was inhibited by herbimycin A, a tyrosine kinase inhibitor, and by LY-294002, a phosphatidylinositol 3′-kinase inhibitor. We suggest that upregulation of MCSF in the infarcted myocardium may have an active role in healing not only through its effects on cells of monocyte/macrophage lineage, but also by regulating endothelial cell chemokine expression.

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Wang, Tiehui, Tomoya Kono, Milena M. Monte, Haruka Kuse, Maria M. Costa, Hiroki Korenaga, Tanja Maehr, Mansourah Husain, Masahiro Sakai et Christopher J. Secombes. « Identification of IL-34 in teleost fish : Differential expression of rainbow trout IL-34, MCSF1 and MCSF2, ligands of the MCSF receptor ». Molecular Immunology 53, n^o 4 (avril 2013) : 398–409. http://dx.doi.org/10.1016/j.molimm.2012.09.008.

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Llewellyn, Heather P., Vishal S. Vaidya, Zhenyu Wang, Qinghai Peng, Craig Hyde, David Potter, Jianying Wang et al. « Evaluating the Sensitivity and Specificity of Promising Circulating Biomarkers to Diagnose Liver Injury in Humans ». Toxicological Sciences 181, n^o 1 (23 janvier 2021) : 23–34. http://dx.doi.org/10.1093/toxsci/kfab003.

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Abstract Early diagnosis of drug-induced liver injury (DILI) continues to be a major hurdle during drug development and postmarketing. The objective of this study was to evaluate the diagnostic performance of promising biomarkers of liver injury—glutamate dehydrogenase (GLDH), cytokeratin-18 (K18), caspase-cleaved K18 (ccK18), osteopontin (OPN), macrophage colony-stimulating factor (MCSF), MCSF receptor (MCSFR), and microRNA-122 (miR-122) in comparison to the traditional biomarker alanine aminotransferase (ALT). Biomarkers were evaluated individually and as a multivariate model in a cohort of acetaminophen overdose (n = 175) subjects and were further tested in cohorts of healthy adults (n = 135), patients with liver damage from various causes (n = 104), and patients with damage to the muscle (n = 74), kidney (n = 40), gastrointestinal tract (n = 37), and pancreas (n = 34). In the acetaminophen cohort, a multivariate model with GLDH, K18, and miR-122 was able to detect DILI more accurately than individual biomarkers alone. Furthermore, the three-biomarker model could accurately predict patients with liver injury compared with healthy volunteers or patients with damage to muscle, pancreas, gastrointestinal tract, and kidney. Expression of K18, GLDH, and miR-122 was evaluated using a database of transcriptomic profiles across multiple tissues/organs in humans and rats. K18 mRNA (Krt18) and MiR-122 were highly expressed in liver whereas GLDH mRNA (Glud1) was widely expressed. We performed a comprehensive, comparative performance assessment of 7 promising biomarkers and demonstrated that a 3-biomarker multivariate model can accurately detect liver injury.

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Gu, Hanjie, Bo Wang, Jiaojiao He et Yonghua Hu. « Macrophage colony stimulating factor (MCSF) of Japanese flounder (Paralichthys olivaceus) : Immunoregulatory property, anti-infectious function, and interaction with MCSF receptor ». Developmental & ; Comparative Immunology 116 (mars 2021) : 103920. http://dx.doi.org/10.1016/j.dci.2020.103920.

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JARRAR, Hala, Damla ÇETİN ALTINDAL et Menemşe GÜMÜŞDERELİOĞLU. « The inhibitory effect of melatonin on osteoclastogenesis of RAW 264.7 cells in low concentrations of RANKL and MCSF ». TURKISH JOURNAL OF BIOLOGY 44, n^o 6 (14 décembre 2020) : 427–36. http://dx.doi.org/10.3906/biy-2007-85.

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RAW 264.7 cells are one of the most recommended cell lines for investigating the activity and differentiation of osteoclasts. These cells differentiate into osteoclasts in the presence of two critical components: receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (MCSF). Melatonin (MEL) hormone has recently become one of the small molecules used in the field of bone regeneration and bone disease treatment, as it has the ability to inhibit the differentiation of osteoclasts directly by suppression of the NF-κB signaling pathway. The main aim of the current study is to determine sufficient RANKL/MCSF concentrations for differentiation of the cells to osteoclasts and to describe the repressive effect of MEL on the osteoclastogenesis of these cells. In this regard, it was found that 10 ng/mL of RANKL- and MCSF-containing medium is suitable for inducing osteoclastogenesis of the cells. In addition, melatonin at doses in the range of 100–1000 μM does not have a cytotoxic effect. Subsequently, results of tartrate resistant acid phosphatase (TRAP) activity, TRAP staining, and relative expressions of cathepsin K, nuclear factor of activated T cells one (NFATC1), and TRAP genes showed a suppressive effect of MEL —especially 800 μM— on RANKL-induced osteoclastogenesis of these cells.

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Langer, Thomas M., Suzanne E. Neumueller, Emma Crumley, Nicholas J. Burgraff, Sawan Talwar, Matthew R. Hodges, Lawrence Pan et Hubert V. Forster. « Ventilation and neurochemical changes during µ-opioid receptor activation or blockade of excitatory receptors in the hypoglossal motor nucleus of goats ». Journal of Applied Physiology 123, n^o 6 (1 décembre 2017) : 1532–44. http://dx.doi.org/10.1152/japplphysiol.00592.2017.

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Neuromodulator interdependence posits that changes in one or more neuromodulators are compensated by changes in other modulators to maintain stability in the respiratory control network. Herein, we studied compensatory neuromodulation in the hypoglossal motor nucleus (HMN) after chronic implantation of microtubules unilaterally ( n = 5) or bilaterally ( n = 5) into the HMN. After recovery, receptor agonists or antagonists in mock cerebrospinal fluid (mCSF) were dialyzed during the awake and non-rapid eye movement (NREM) sleep states. During day studies, dialysis of the µ-opioid inhibitory receptor agonist [d-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO; 100 µM) decreased pulmonary ventilation (V̇i), breathing frequency ( f), and genioglossus (GG) muscle activity but did not alter neuromodulators measured in the effluent mCSF. However, neither unilateral dialysis of a broad spectrum muscarinic receptor antagonist (atropine; 50 mM) nor unilateral or bilateral dialysis of a mixture of excitatory receptor antagonists altered V̇i or GG activity, but all of these did increase HMN serotonin (5-HT) levels. Finally, during night studies, DAMGO and excitatory receptor antagonist decreased ventilatory variables during NREM sleep but not during wakefulness. These findings contrast with previous dialysis studies in the ventral respiratory column (VRC) where unilateral DAMGO or atropine dialysis had no effects on breathing and bilateral DAMGO or unilateral atropine increased V̇i and f and decreased GABA or increased 5-HT, respectively. Thus we conclude that the mechanisms of compensatory neuromodulation are less robust in the HMN than in the VRC under physiological conditions in adult goats, possibly because of site differences in the underlying mechanisms governing neuromodulator release and consequently neuronal activity, and/or responsiveness of receptors to compensatory neuromodulators. NEW & NOTEWORTHY Activation of inhibitory µ-opioid receptors in the hypoglossal motor nucleus decreased ventilation under physiological conditions and did not affect neurochemicals in effluent dialyzed mock cerebral spinal fluid. These findings contrast with studies in the ventral respiratory column where unilateral [d-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO) had no effects on ventilation and bilateral DAMGO or unilateral atropine increased ventilation and decreased GABA or increased serotonin, respectively. Our data support the hypothesis that mechanisms that govern local compensatory neuromodulation within the brain stem are site specific under physiological conditions.

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Langer, Thomas M., Suzanne E. Neumueller, Emma Crumley, Nicholas J. Burgraff, Sawan Talwar, Matthew R. Hodges, Lawrence Pan et Hubert V. Forster. « State-dependent and -independent effects of dialyzing excitatory neuromodulator receptor antagonists into the ventral respiratory column ». Journal of Applied Physiology 122, n^o 2 (1 février 2017) : 327–38. http://dx.doi.org/10.1152/japplphysiol.00619.2016.

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Unilateral dialysis of the broad-spectrum muscarinic receptor antagonist atropine (50 mM) into the ventral respiratory column [(VRC) including the pre-Bötzinger complex region] of awake goats increased pulmonary ventilation (V̇i) and breathing frequency (f), conceivably due to local compensatory increases in serotonin (5-HT) and substance P (SP) measured in effluent mock cerebral spinal fluid (mCSF). In contrast, unilateral dialysis of a triple cocktail of antagonists to muscarinic (atropine; 5 mM), neurokinin-1, and 5-HT receptors does not alter V̇i or f, but increases local SP. Herein, we tested hypotheses that 1) local compensatory 5-HT and SP responses to 50 mM atropine dialyzed into the VRC of goats will not differ between anesthetized and awake states; and 2) bilateral dialysis of the triple cocktail of antagonists into the VRC of awake goats will not alter V̇i or f, but will increase local excitatory neuromodulators. Through microtubules implanted into the VRC of goats, probes were inserted to dialyze mCSF alone (time control), 50 mM atropine, or the triple cocktail of antagonists. We found 1) equivalent increases in local 5-HT and SP with 50 mM atropine dialysis during wakefulness compared with isoflurane anesthesia, but V̇i and f only increased while awake; and 2) dialyses of the triple cocktail of antagonists increased V̇i, f, 5-HT, and SP (<0.05) during both day and night studies. We conclude that the mechanisms governing local neuromodulator levels are state independent, and that bilateral excitatory receptor blockade elicits an increase in breathing, presumably due to a local, (over)compensatory neuromodulator response. NEW & NOTEWORTHY The two major findings are as follows: 1) during unilateral dialysis of 50 mM atropine into the ventral respiratory column to block excitatory muscarinic receptor activity, a compensatory increase in other neuromodulators was state independent, but the ventilatory response appears to be state dependent; and 2) the hypothesis that absence of decreased V̇i and f during unilateral dialysis of excitatory receptor antagonists was due to compensation by the contralateral VRC was not supported by findings herein.

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Chen, Haiming, Mingjie Li, Eric Sanchez, Abigail Gillespie, Cathy Wang, Tiffany Lee, Suzie Vardanyan et al. « Crosslinking of Fc Gamma-Rllb and Fc Epsilon-RI Binding Peptides Inhibits Osteoclast Formation in Multiple Myeloma through Inactivation of the ITAM Signaling Pathway ». Blood 126, n^o 23 (3 décembre 2015) : 2995. http://dx.doi.org/10.1182/blood.v126.23.2995.2995.

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Abstract Introduction: Overactivity of osteoclasts resulting in bone destruction is a hallmark of multiple myeloma (MM). Receptor for activation of NF-kB ligand (RANKL) and monocyte colony stimulating factor (MCSF) signaling pathways both promote proliferation and survival of the precursors of the osteoclast lineage, and have been widely investigated in MM. The third pathway involved in osteoclast differentiation is the immunoreceptor tyrosine-based activation motif (ITAM) with c-Fms signaling. ITAM and its inhibitor ITIM provide the basis for two opposed signaling modules that duel for control of osteoclast formation. Human monocyte/macrophage expresses the low-affinity FcγRIIb and high-affinity Fcε receptor 1 (FcεRI). Both receptors mediate Syk phosphorylation to activate or inactivate downstream ITAM or ITIM signaling molecules. In this study, we determined the effects of an IgG(CH2-CH3) and IgE(CH2-CH3-CH4) fusion protein that activates the ITIM inhibitory pathway on downstream signaling of Syk and osteoclast formation in monocytes from MM patients. Methods: We constructed IgG(CH2-CH3) with an IgE(CH2-CH3-CH4) fusion protein using standard cloning techniques. We evaluated the fusion protein on osteoclast formation using cells from either human monocytes isolated from MM patients' peripheral blood mononuclear cells (PBMCs) or bone marrow (BM) MCs with an anti-CD14 micro-bead affinity column and magnetic bead selection (Miltenyi Biotec, Auburn, CA). The monocytes were cultured on slide-culture dishes (2 X 105 cells/well). The cells were treated with the fusion protein or with IgE or IgG and subsequently treated with 50ng/ml RANKL (receptor for activation of nuclear factor kB and 10ng/ml MCSF (monocyte colony stimulating factor) in order to stimulate osteoclast formation at the beginning of the culture and during a medium change after 3 days with the same amount of growth factors added. The cells were fixed for tartrate resistant acid phosphatase (TRAP)-staining assay on day 21. To investigate ITIM signaling pathway we determined Syk phosphorylation of monocytes treated or without treated with fusion protein by Western blot analysis. Results: We found that in a concentration-dependent fashion, the fusion protein inhibited osteoclast cell formation from CD14+ MCs from PB or BM exposed to RANKL and MCSF. We further analyzed the effects on the FcγRIIb-SHIP signaling pathway in monocytes induced with 50ng/ml RANKL and 10ng/ml MCSF following exposure to fusion protein or control IgG or IgE. The results showed that the monocytes showed markedly lower Syk phosphorylation following exposure to the fusion protein (100-200ng/ml). There was no change of Syk phosphorylationl in monocytes treated with IgG or IgE or IgG with IgE. Conclusions: The results of our study show that intact human IgG or IgE does not affect the ITAM or ITIM signaling pathways. However, a fusion protein consisting of IgG(CH2-CH3) with IgE(CH2-CH3-CH4) showed the ability to activate the ITIM inhibition pathway through FcγRIIb to reduce osteoclast formation. Thus, blockage of ITAM may be treating novel treatment for preventing bone loss for MM patients. Disclosures No relevant conflicts of interest to declare.

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Azuma, C., F. Saji, T. Kimura, Y. Tokugawa, M. Takemura, M. Miki, M. Ono et O. Tanizawa. « The gene expressions of macrophage colony-stimulating factor (MCSF) and MCSF receptor in the human myometrium during pregnancy : Regulation by sex steroid hormones ». Journal of Steroid Biochemistry and Molecular Biology 39, n^o 6 (décembre 1991) : 883–88. http://dx.doi.org/10.1016/0960-0760(91)90345-6.

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Rovida, Elisabetta, Fabio Marra, Manuela Baccarini et Persio Dello Sbarba. « Constitutive activation of the MAPK pathway mediates v-fes–induced mitogenesis in murine macrophages ». Blood 95, n^o 12 (15 juin 2000) : 3959–63. http://dx.doi.org/10.1182/blood.v95.12.3959.

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Abstract Fes is a nonreceptor tyrosine kinase expressed at the highest level in macrophages. We previously showed that the overexpression of c-fes in murine macrophages of the BAC-1.2F5 cell line renders these cells independent of macrophage colony-stimulating factor (MCSF) for survival and proliferation, although no direct relationship could be established between tyrosine-phosphorylated substrates of Fes- and MCSF receptor–dependent signaling and mitogenesis. In this study, we investigated whether the mitogen-activated protein kinase (MAPK) pathway is involved in the growth factor–independent growth of v-fes–overexpressing macrophages. We found a constitutively increased phosphorylation of extracellularly regulated kinase (ERK) in v-fes–overexpressing macrophages as compared with mock-infected cells. This finding was associated with activation of mitogen/extracellular signal–regulated kinase (MEK) and with constitutive localization of ERK in the nucleus. Treatment of v-fes–overexpressing cells with the MEK-specific inhibitor PD98059 markedly reduced cell growth, hyperphosphorylation, and nuclear localization of ERK, indicating that the MAPK pathway mediates the mitogenic effect of v-fes.

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Rovida, Elisabetta, Fabio Marra, Manuela Baccarini et Persio Dello Sbarba. « Constitutive activation of the MAPK pathway mediates v-fes–induced mitogenesis in murine macrophages ». Blood 95, n^o 12 (15 juin 2000) : 3959–63. http://dx.doi.org/10.1182/blood.v95.12.3959.012k11_3959_3963.

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Fes is a nonreceptor tyrosine kinase expressed at the highest level in macrophages. We previously showed that the overexpression of c-fes in murine macrophages of the BAC-1.2F5 cell line renders these cells independent of macrophage colony-stimulating factor (MCSF) for survival and proliferation, although no direct relationship could be established between tyrosine-phosphorylated substrates of Fes- and MCSF receptor–dependent signaling and mitogenesis. In this study, we investigated whether the mitogen-activated protein kinase (MAPK) pathway is involved in the growth factor–independent growth of v-fes–overexpressing macrophages. We found a constitutively increased phosphorylation of extracellularly regulated kinase (ERK) in v-fes–overexpressing macrophages as compared with mock-infected cells. This finding was associated with activation of mitogen/extracellular signal–regulated kinase (MEK) and with constitutive localization of ERK in the nucleus. Treatment of v-fes–overexpressing cells with the MEK-specific inhibitor PD98059 markedly reduced cell growth, hyperphosphorylation, and nuclear localization of ERK, indicating that the MAPK pathway mediates the mitogenic effect of v-fes.

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Wiener, Doris, Xiaoming Gong, Raju Marisiddaiah et Lewis Rubin. « Effects of carotenoids on differential expression of scavenger receptors and cytokines in human monocyte-derived macrophage subpopulations (98.19) ». Journal of Immunology 184, n^o 1_Supplement (1 avril 2010) : 98.19. http://dx.doi.org/10.4049/jimmunol.184.supp.98.19.

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Abstract Circulating monocytes differentiate into macrophage (MΦ) subpopulations in response to environmental signals. Carotenoids may reduce MΦ oxidant stress, alter the inflammatory response, and lower risk of atherosclerosis. These observations prompted us to determine the effects of β-carotene, lycopene, astaxanthin and lutein on differentiation of human monocytes into MΦs (M1 and M2) phenotypes. Monocyte-to-MΦ differentiation was driven by treatment for 6 days with GMCSF or MCSF ± individual carotenoids. M1 MΦs (+GMCSF) were activated with LPS+IFNγ; M2 MΦs (+MCSF) were activated with IL-4. We validated that M1 and M2 cells showed distinct morphologies and expression patterns (qRT-PCR) of scavenger receptors (SR-A, LDLR, CD36, SR-B1) and cytokines (IL-10, IL-12). M1 and M2 markers (CD14, CD16, CD36, CD80, CD163) and cytokines (PGE2, IL-6, IL-8, IL-10, IL-12, TNFα, IFNγ) were confirmed by flow cytometry. Expression of CD14, CD16, CD36, CD163, and IL-10 was higher in M2 than M1 MΦs. Lycopene and astaxanthin decreased SRA, CD36 and LDLR mRNA levels in M1 cells but had no significant effect on M2 cells. Lutein also reduced SRA, CD36 and IL-10 expression in M2. Conversely, β-carotene increased M1 and decreased M2 expression of SRA, CD36, IL-10 and IL-12. Results show individual carotenoids can regulate MΦ scavenger receptor and cytokine expression. We speculate that carotenoids influence MΦ polarization, thereby inhibiting cholesterol accumulation in MΦ-derived atherogenic foam cells.

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Chen, Haiming, Richard A. Campbell, Melinda S. Gordon, Steven J. Manyak, Cathy Wang, Mingjie Li, Hee Jin Lee et al. « Circulating Tie2-Expressing Cells Are Increased in Multiple Myeloma Patients, Correlate with Serum Pleiotrophin Levels and May Develop from This Myeloma Angiogenic and Growth Factor. » Blood 106, n^o 11 (16 novembre 2005) : 2494. http://dx.doi.org/10.1182/blood.v106.11.2494.2494.

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Abstract Tie2, an endothelial cell-specific receptor kinase, plays an important role in tumor angiogenesis. This protein is essential to the development of embryonic vasculature as well as vascular growth and maintenance in adult tissues. Because of the increasing importance that angiogenesis has been shown to play in multiple myeloma (MM), we determined the number of Tie2-expressing cells in the peripheral blood (PB) of MM patients and its relationship to the serum levels and gene expression of a recently identified angiogenic factor, pleiotrophin (PTN). We have recently demonstrated that PTN is expressed and secreted by MM tumor cells, and serum levels of this protein are highly elevated in MM patients. We quantified the number of Tie2-positive cells in MM patients (n=15) and age-matched control subjects (n=10) using an immunohistochemical technique. Tie2-expressing cells were significantly elevated in the PB mononuclear cells (MCs) from MM patients compared to the normal controls (p<0.05). We also analyzed gene expression for Tie2 in these same samples using RT-PCR. The results showed that Tie2 mRNA was strongly expressed in the PBMCs from MM patients whereas control samples showed no or low expression of this gene. Serum levels of PTN were tested with ELISA, and PTN mRNA concentrations were quantified by RT-PCR in PBMCs from these same patients and control subjects. The results showed that serum levels of PTN correlated with the number of Tie2-expressing PBMCs in MM patients (R2=0.5778). PTN mRNA levels also correlated with Tie2 gene expression in PBMC samples. We further examined whether monocyte colony stimulating factor (mCSF), PTN and vascular endothelial growth factor (VEGF) may be capable of inducing Tie2 expression in highly purified human monocytes that lack Tie2 expression. Normal PB monocytes were purified using density centrifugation followed by anti-CD14 micro-bead affinity column selection. Although none of these three proteins alone or the combinations of either VEGF and mCSF or VEGF and PTN induced Tie2 gene expression in the monocytes following one week of incubation, the combination of PTN (100 nM) and mCSF (20 nM) led to expression of Tie2 in these cells. We quantified the proportion of cells expressing Tie2 in these samples with RT-PCR using serial dilutional analysis with B or T cells that lack Tie2 expression, and showed that approximately 0.1–1.0% of the monocytes expressed this gene following incubation with PTN and mCSF. Moreover, the addition of VEGF (20 ng/ml) to PTN and mCSF increased the proportion of cells expressing Tie2 (to >10%). Anti-PTN antibody blocked the induction of Tie2 gene expression in these monocytes by this cytokine combination. These results show that Tie2-expressing cells are elevated in the peripheral blood of MM patients, and correlate with PTN serum and PTN mRNA expression. PTN in combination with VEGF and mCSF induces Tie2 gene expression in a large proportion of circulating human monocytes. These results suggest that MM patients show increased numbers of vasculogenic progenitors in their circulation that may result from the presence of elevated levels of circulating angiogenic factors including PTN and VEGF.

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Lyros, Ioannis, Despoina Perrea, Konstantinos Tosios, Nikolaos Nikitakis, Ioannis A. Tsolakis, Efstratios Ferdianakis, Eleni Fora et al. « Histological and Biochemical Analysis after Posterior Mandibular Displacement in Rats ». Veterinary Sciences 9, n^o 11 (10 novembre 2022) : 625. http://dx.doi.org/10.3390/vetsci9110625.

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The present study aimed to investigate any biochemical and histological changes of the rat condyle and mandible in animals that had sustained mandibular growth restriction. Seventy-two male Wistar rats were divided into two equal groups, experimental and control. Each group consisted of three equal subgroups. The animals were sacrificed 30, 60, and 90 days after the start of the experiment. Blood samples were collected from the eye, and the osteoprotegerin (OPG), Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), and Macrophage Colony-Stimulating factor (MCSF)concentrations were measured by using enzyme-linked immunosorbent assay (ELISA) kits. A histological analysis was performed on the mandibular condyles. The blood serum values of OPG, RANKL, and MCSF did not exhibit any statistically significant difference between groups or subgroups. However, significant histological changes became evident after a histomorphometric condylar examination was performed. The Bone Surface/Total Surface ratio appeared reduced in the anterior and posterior regions of the condyle. In addition, the Posterior Condylar Cartilage Thickness was measured and determined to be significantly diminished. The present intervention that employed orthodontic/orthopedic devices did not prove to have any significant effect on the circulating proteins under study. Posterior displacement of the mandible may culminate only in local histological alterations in condylar cartilage thickness and its osseous microarchitecture.

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Koehler, Miriam I., Eliza S. Hartmann, Sabine Schluessel, Felicitas Beck, Julia I. Redeker, Baerbel Schmitt, Marina Unger et al. « Impact of Periprosthetic Fibroblast-Like Cells on Osteoclastogenesis in Co-Culture with Peripheral Blood Mononuclear Cells Varies Depending on Culture System ». International Journal of Molecular Sciences 20, n^o 10 (26 mai 2019) : 2583. http://dx.doi.org/10.3390/ijms20102583.

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Co-culture studies investigating the role of periprosthetic fibroblasts (PPFs) in inflammatory osteoclastogenesis reveal contrary results, partly showing an osteoprotective function of fibroblasts and high OPG expression in monolayer. These data disagree with molecular analyses of original periosteolytic tissues. In order to find a more reliable model, PPFs were co-cultivated with peripheral blood mononuclear cells (PBMCs) in a transwell system and compared to conventional monolayer cultures. The gene expression of key regulators of osteoclastogenesis (macrophage colony-stimulating factor (MCSF), receptor activator of NF-κB ligand (RANK-L), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)) as well as the ability of bone resorption were analyzed. In monolayer co-cultures, PPFs executed an osteoprotective function with high OPG-expression, low RANK-L/OPG ratios, and a resulting inhibition of osteolysis even in the presence of MCSF and RANK-L. For transwell co-cultures, profound changes in gene expression, with a more than hundredfold decrease of OPG and a significant upregulation of TNFα were observed. In conclusion, we were able to show that a change of culture conditions towards a transwell system resulted in a considerably more osteoclastogenic gene expression profile, being closer to findings in original periosteolytic tissues. This study therefore presents an interesting approach for a more reliable in vitro model to examine the role of fibroblasts in periprosthetic osteoclastogenesis in the future.

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Semnani, Roshanak Tolouei, Lily Mahapatra, Vanessa Moore, Vivornpun Sanprasert et Thomas B. Nutman. « Functional and Phenotypic Characteristics of Alternative Activation Induced in Human Monocytes by Interleukin-4 or the Parasitic Nematode Brugia malayi ». Infection and Immunity 79, n^o 10 (25 juillet 2011) : 3957–65. http://dx.doi.org/10.1128/iai.05191-11.

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ABSTRACTHuman monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) ofBrugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4+and CD8+T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly.

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Pakan, Prisca, Abeer Farhan Alanazi et Nicholas Andronicos. « Macrophage Colony-Stimulating Factor (MCSF) and Receptor Activator of NF₭B Ligand (RANKL) in Osteoclastogenesis ». International Journal of Current Research and Review 13, n^o 12 (2021) : 23–26. http://dx.doi.org/10.31782/ijcrr.2021.131234.

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Ma, Peilin, Holly Martin, Emily Sims, Ramdas Baskar, Joy Ghosh et Reuben Kapur. « Role of Intracellular Tyrosine Residues in Oncogenic KIT- Induced Transformamtion. » Blood 114, n^o 22 (20 novembre 2009) : 1435. http://dx.doi.org/10.1182/blood.v114.22.1435.1435.

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Abstract Abstract 1435 Poster Board I-458 Gain-of-function mutations of the receptor tyrosine kinase KIT have been associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myelogenous leukemia (AML). A mutation of aspartic acid to valine (KITD814V) in the activation loop of KIT results in altered substrate recognition and constitutive tyrosine autophosphorylation. However, the intracellular mechanisms that contribute to promiscuous signaling via this mutation are poorly understood. We have previously shown that KITD814V is sufficient to induce ligand independent growth in primary hematopoietic stem and progenitor cells (HSC/P) in vitro as well as transformation in vivo. However, it is not known whether ligand induced (i.e. KITL) signals in vivo contribute to transformation. To assess this, we generated a chimeric receptor (CHR) in which the extracellular domain of KIT was replaced with the human-macrophage colony stimulating factor receptor (h-MCSFR) to inhibit endogenous binding of murine SCF. In vitro thymidine incorporation assay confirmed that the WTCHR is only functionally responsive to human (h)-MCSF stimulation, but not to murine (m)SCF nor murine MCSF stimulation, and that CHRD814V receptor maintains ligand independent growth potential similar to the KITD814V receptor. To determine if the myeloproliferative disease (MPD) could occur in the absence of endogenous ligand binding, a murine transplantation model was utilized to compare CHRD814V and KITD814V-induced transformation. In vivo results demonstrated no significant difference in the onset of MPD in mice bearing KITD814V vs. CHRD814V (median survival 52 days vs. 49 days, n=10 to 20 mice), with similar disease manifestation in the two groups including splenomegaly, hepatomegaly, myeloid cell infiltration and elevated white blood cell counts (n=10 to 20). Transforming potential in the absence of KIT extracellular domain strongly suggested that self-association of KITD814V was sufficient to induce transformation rather than influences from endogenous ligand. To further assess the relative contribution of intracellular tyrosine residues in transformation, a CHRD814V mutant was generated in which seven critical tyrosine residues including the binding sites for Src family kinases, Grb2, p85α regulatory subunit of class IA PI3Kinase, PLC-g, Ras-GAP, and Grb7 were mutated to phenylalanines (i.e.CHRD814V-F7). Our results show that CHRD814V-F7 bearing primary bone marrow cells lost ligand independent growth potential in vitro, suggesting that seven tyrosine induced signaling may play a critical role in KITD814V-induced ligand independent transformation. Consistently, transplantation studies demonstrated that mice bearing bone marrow cells expressing CHRD814V-F7 exhibited a notable delay in MPD development (median survival= 95 days, n=4 to 8). These results suggest that intracellular tyrosines are crucial for ligand independent proliferation and transformation. Next, we individually restored each of the seven tyrosine residues back into CHRD814V-F7 to determine the importance of pathways essential for ligand independent growth and transformation. We found that among the single tyrosine add back receptors, only restoring p85α subunit binding site (Y719) alone was sufficient to induce promiscuous cellular growth, proliferation, and survival. Transplantation studies revealed that restoration of Y719 was sufficient to induce transformation in vivo (median survival= 55 days, n=5). Furthermore, restoration of other individual tyrosine residues demonstrated a notable delay in CHRD814V-induced transformation in vivo (median survival= 83∼120 days, n=5 to 12) and a minimal contribution to ligand independent growth in vitro. Taken together, our results demonstrate: 1) KITD814V induced transformation in vivo occurs in the absence of ligand stimulation; 2) intracellular tyrosine residues play an essential role in KITD814V induced transformation in vitro and in vivo and; 3) of all the critical tyrosine residues present in KIT, presence of tyrosine at position 719 is sufficient to completely restore ligand independent growth in vitro and transformation in vivo. Disclosures No relevant conflicts of interest to declare.

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McArthur, GA, LR Rohrschneider et GR Johnson. « Induced expression of c-fms in normal hematopoietic cells shows evidence for both conservation and lineage restriction of signal transduction in response to macrophage colony-stimulating factor ». Blood 83, n^o 4 (15 février 1994) : 972–81. http://dx.doi.org/10.1182/blood.v83.4.972.972.

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Abstract Retrovirus-mediated gene transfer was used to obtain expression of the macrophage colony-stimulating factor (MCSF) receptor, c-fms, on hematopoietic lineages that normally do not express this receptor. Cultures of murine fetal liver cells infected with the c-fms retrovirus developed erythroid colonies in cultures stimulated with M-CSF. However, these colonies were fewer and less hemoglobinized than colonies in parallel cultures stimulated by erythropoietin. Culture of isolated clones demonstrated a direct action of M-CSF on erythroid clones. Culture of c-fms retrovirus-infected adult murine bone marrow cells showed megakaryocyte and novel macrophage-megakaryocyte clones when stimulated by M-CSF. Culture of isolated clones again confirmed a direct action of M-CSF on megakaryocyte clones. In contrast, M-CSF stimulation of c-fms-infected granulocytes and granulocyte progenitor cells did not elicit proliferation, enhanced survival, or functional stimulation of granulocytes. These findings provide evidence for both conservation and lineage restriction of signal transduction in normal hematopoietic cells.

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McArthur, GA, LR Rohrschneider et GR Johnson. « Induced expression of c-fms in normal hematopoietic cells shows evidence for both conservation and lineage restriction of signal transduction in response to macrophage colony-stimulating factor ». Blood 83, n^o 4 (15 février 1994) : 972–81. http://dx.doi.org/10.1182/blood.v83.4.972.bloodjournal834972.

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Retrovirus-mediated gene transfer was used to obtain expression of the macrophage colony-stimulating factor (MCSF) receptor, c-fms, on hematopoietic lineages that normally do not express this receptor. Cultures of murine fetal liver cells infected with the c-fms retrovirus developed erythroid colonies in cultures stimulated with M-CSF. However, these colonies were fewer and less hemoglobinized than colonies in parallel cultures stimulated by erythropoietin. Culture of isolated clones demonstrated a direct action of M-CSF on erythroid clones. Culture of c-fms retrovirus-infected adult murine bone marrow cells showed megakaryocyte and novel macrophage-megakaryocyte clones when stimulated by M-CSF. Culture of isolated clones again confirmed a direct action of M-CSF on megakaryocyte clones. In contrast, M-CSF stimulation of c-fms-infected granulocytes and granulocyte progenitor cells did not elicit proliferation, enhanced survival, or functional stimulation of granulocytes. These findings provide evidence for both conservation and lineage restriction of signal transduction in normal hematopoietic cells.

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Wilkinson, Hannah, Daxin Chen et Anthony Dorling. « Thrombin fine tunes innate immune cell function ». Journal of Immunology 204, n^o 1_Supplement (1 mai 2020) : 74.2. http://dx.doi.org/10.4049/jimmunol.204.supp.74.2.

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Abstract Thrombin is the main effector protease in the coagulation cascade. It can also signal via protease activating receptors (PAR). The presence of these receptors on the surface of innate immune cells has been well reported but the functional consequence of activation has yet to be fully defined. This study aims to investigate the role thrombin has on innate immune cell function using murine bone marrow macrophages (BMM). In Vitro, stimulating mature MCSF-cultured BMM with thrombin did not affect gross markers of macrophage polarisation (iNOS or CD206). The stimulated cell supernatants contained increased amounts of Interferon gamma (IFNγ) but reduced IL10. Thrombin increased IFNγ receptor expression at the cell surface. Thrombin-treated cells had increased lipid rich microdomains by Cholera Toxin B staining and increased co-localisation of the LPS receptor within the lipid rafts. Compared to untreated cells, thrombin stimulated cells were highly sensitive to low dose M1 polarising stimuli, as evidenced by iNOS expression. The thrombin treated cells down regulated surface ABCA1 expression, but this was prevented by transfecting cells with siRNA against Cullin 3. This preserved ABCA1 expression and prevented the increase in lipid rich microdomains after thrombin stimulation and was associated with the loss of heightened sensitivity to low dose M1 stimuli. Taken together this shows a clear pro inflammatory signal on the thrombin treated cells and to date the first description of ABCA1’s key role in thrombin mediated inflammatory signalling.

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Fan, Xian, Diane M. Biskobing, Dongjie Fan, Willy Hofstetter et Janet Rubin. « Macrophage Colony Stimulating Factor Down-Regulates MCSF-Receptor Expression and Entry of Progenitors into the Osteoclast Lineage ». Journal of Bone and Mineral Research 12, n^o 9 (1 septembre 1997) : 1387–95. http://dx.doi.org/10.1359/jbmr.1997.12.9.1387.

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Bonis, J. M., S. E. Neumueller, K. L. Krause, T. Kiner, A. Smith, B. D. Marshall, B. Qian, L. G. Pan et H. V. Forster. « A role for the Kölliker-Fuse nucleus in cholinergic modulation of breathing at night during wakefulness and NREM sleep ». Journal of Applied Physiology 109, n^o 1 (juillet 2010) : 159–70. http://dx.doi.org/10.1152/japplphysiol.00933.2009.

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For many years, acetylcholine has been known to contribute to the control of breathing and sleep. To probe further the contributions of cholinergic rostral pontine systems in control of breathing, we designed this study to test the hypothesis that microdialysis (MD) of the muscarinic receptor antagonist atropine into the pontine respiratory group (PRG) would decrease breathing more in animals while awake than while in NREM sleep. In 16 goats, cannulas were bilaterally implanted into rostral pontine tegmental nuclei ( n = 3), the lateral ( n = 3) or medial ( n = 4) parabrachial nuclei, or the Kölliker-Fuse nucleus (KFN; n = 6). After >2 wk of recovery from surgery, the goats were studied during a 45-min period of MD with mock cerebrospinal fluid (mCSF), followed by at least 30 min of recovery and a second 45-min period of MD with atropine. Unilateral and bilateral MD studies were completed during the day and at night. MD of atropine into the KFN at night decreased pulmonary ventilation and breathing frequency and increased inspiratory and expiratory time by 12–14% during both wakefulness and NREM sleep. However, during daytime studies, MD of atropine into the KFN had no effect on these variables. Unilateral and bilateral nighttime MD of atropine into the KFN increased levels of NREM sleep by 63 and 365%, respectively. MD during the day or at night into the other three pontine sites had minimal effects on any variable studied. Finally, compared with MD of mCSF, bilateral MD of atropine decreased levels of acetylcholine and choline in the effluent dialysis fluid. Our data support the concept that the KFN is a significant contributor to cholinergically modulated control of breathing and sleep.

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Sarangi, Pranita P., Papiya Chakraborty, Larry Wahl et Yoshihiko Yamada. « Adhesion protein Fibulin-7 (Fbln7) regulate differentiation and polarisation of human monocytes and macrophages. » Journal of Immunology 198, n^o 1_Supplement (1 mai 2017) : 143.5. http://dx.doi.org/10.4049/jimmunol.198.supp.143.5.

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Abstract Fibulin-7 (Fbln7) is a member of the fibulin family of secreted glycoproteins, majorly found in developing teeth, bone and cartilage and shows interaction with other ECM proteins. Recently, it was shown that its C-terminal fragment (Fbln7C) and its peptides show anti-angiogenic effects. Fbln7 is also expressed in immune privileged tissues such as eye and placenta but to date very little is known about its functional significance. The results from this study show that human monocytes adhered to Fbln7 full-length (Fbln7-FL) and Fbln7C via integrin α5β1. Interestingly, actin stress fiber formation of monocytes was promoted by Fbln7-FL and not by Fbln7C. Morphological studies and surface expression analysis of CD14, mannose receptor (CD206), MHC-II and CD11b receptors revealed that both Fbln7-FL and Fbln7C inhibited MCSF induced monocyte differentiation with more significant effect with Fbln7C as compared to Fbln7-FL. Similarly, both protein fragments inhibited IFNγ and IL-4 induced M1 and M2 polarization of human blood monocyte derived macrophages. Collectively, these data suggests that Fbln7 and its fragments could negatively regulate immune cell in the expressed tissues.

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Vallet, Sonia, Noopur Raje, Kenji Ishitsuka, Teru Hideshima, Klaus Podar, Petter Veiby, Iris Breitkreutz et al. « MLN3897, a Novel CCR1 Antagonist, Inhibits Osteoclastogenesis by Blocking Early ERK Activation. » Blood 108, n^o 11 (16 novembre 2006) : 1636. http://dx.doi.org/10.1182/blood.v108.11.1636.1636.

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Abstract Osteolytic lesions are a hallmark of myeloma bone disease and other metastatic cancers, resulting in significant morbidity. In order to successfully target skeletal disease, it is critical to identify the mediators of osteoclastogenesis. Osteoclastogenesis is a multistep process regulated by receptor activator of nuclear factor κ B ligand (RANKL) and monocyte colony stimulating factor (mCSF). These cytokines recruit monocyte precursors, stimulate their fusion into multinucleated cells, and finally induce osteoclast (OC) formation and activation. Recent data suggest that several chemokines in combination with RANKL and/or vitamin D3 (Oba et al., Exp. Hematology 2005) modulate osteoclastogenesis. Moreover, the autocrine secretion of RANTES and MCP-1 mediate monocyte multinucleation (Kim et al., J. Biol. Chem. 2005), the initial step towards osteoclastogenesis. Since CCR1 is one of the main chemokine receptors expressed on monocytes and OC, we here studied the effects of CCR1 inhibition on osteoclastogenesis. MLN3897 is a small molecule, specific antagonist of the chemokine receptor CCR1. In order to analyze the effects of MLN3897 on osteoclastogenesis, we generated OC from peripheral blood mononuclear cells (PBMC) from healthy donors by stimulation with RANKL and M-CSF (50 ng/ml) for three weeks, in the absence or presence of MLN3897. Mature OC were multinucleated TRAP+ cells, and their functional activity was confirmed by a pit formation and a collagen release ELISA assay. Our data demonstrates that MLN3897 inhibits osteoclastogenesis in a dose and time-dependent fashion. MLN3897 at 10 nM concentration decreases OC number to 40% (range 20 to 70%) compared to untreated controls (p<0.05). Time course experiments demonstrate that MLN3897 inhibites OC formation if added at the beginning of culture, whereas no inhibition is noted after 7 or 14 days. The reduced OC number is associated with decreased OC activity as demonstrated by a decrease in collagen fragments (control 82 +/−8 vs treated 25+/− 19 nM). Because the effects of MLN3897 are noted at early time points, we hypothesized that MLN3897 interfers with the monocyte multinucleation process. Our data demonstrates that MLN3897 induces a 60% reduction in the multinucleated cell number at one week (control 61+/−14 vs treated 35+/−9), associated with decreased nuclei per cell at two weeks. This correlates with an early induction of CCR1 expression on monocytes by RANKL and mCSF. Flow cytometry confirms an increment of double-stained CD14/CCR1 monocyte population (from 20% to 50%) associated with increased ERK phosphorylation at 36 hours of stimulation, which is completely abrogated by MLN3897. Taken together, these data therefore suggest that CCR1 inhibition by MLN3897 blocks ERK pathway activation and impairs the monocyte multinucleation process, an early step in osteoclastogenesis. These studies delineate a novel mechanism of action of MLN3897 on osteoclastogenesis, and provide the rationale for its clinical evaluation to treat osteolytic bone disease.

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Fischer, P., E. Nacheva, DY Mason, PD Sherrington, C. Hoyle, FG Hayhoe et A. Karpas. « A Ki-1 (CD30)-positive human cell line (Karpas 299) established from a high-grade non-Hodgkin's lymphoma, showing a 2;5 translocation and rearrangement of the T-cell receptor beta-chain gene ». Blood 72, n^o 1 (1 juillet 1988) : 234–40. http://dx.doi.org/10.1182/blood.v72.1.234.234.

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Abstract We describe the characterization of a new human cell line, Karpas 299 (K299), established from blast cells in the peripheral blood of a 25- year-old white man. His illness, which began with enlarged occipital and axillary nodes and weight loss, ended after 7 months with generalized lymphadenopathy, pleural effusion, and bone marrow involvement. A lymph node biopsy showed a large cell lymphoma mainly sinusoidal in distribution. The blast cells with pleomorphic nuclei resembled primitive histiocytes. The cells, which expressed the T-cell- associated markers CD4 and CD5, were positive for HLA-DR, epithelial membrane antigen, and CD30 (Ki-1 antigen). The karyotype was aneuploid and included a translocation 2;5. The site of translocation on chromosome 5 (at 5q35.1) is in the region of the locus of the c-fms oncogene (receptor of the monocyte-macrophage colony-stimulating factor MCSF or CSF-1). The cell line Karpas 299 has the same karyotype and pattern of antigen expression as the patient's cells. Northern blot analysis of RNA showed an active rearrangement of the T-cell receptor beta-chain gene. This is to our knowledge the first Ki-1 antigen- positive line to be established from a case of non-Hodgkin's lymphoma.

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Fischer, P., E. Nacheva, DY Mason, PD Sherrington, C. Hoyle, FG Hayhoe et A. Karpas. « A Ki-1 (CD30)-positive human cell line (Karpas 299) established from a high-grade non-Hodgkin's lymphoma, showing a 2;5 translocation and rearrangement of the T-cell receptor beta-chain gene ». Blood 72, n^o 1 (1 juillet 1988) : 234–40. http://dx.doi.org/10.1182/blood.v72.1.234.bloodjournal721234.

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We describe the characterization of a new human cell line, Karpas 299 (K299), established from blast cells in the peripheral blood of a 25- year-old white man. His illness, which began with enlarged occipital and axillary nodes and weight loss, ended after 7 months with generalized lymphadenopathy, pleural effusion, and bone marrow involvement. A lymph node biopsy showed a large cell lymphoma mainly sinusoidal in distribution. The blast cells with pleomorphic nuclei resembled primitive histiocytes. The cells, which expressed the T-cell- associated markers CD4 and CD5, were positive for HLA-DR, epithelial membrane antigen, and CD30 (Ki-1 antigen). The karyotype was aneuploid and included a translocation 2;5. The site of translocation on chromosome 5 (at 5q35.1) is in the region of the locus of the c-fms oncogene (receptor of the monocyte-macrophage colony-stimulating factor MCSF or CSF-1). The cell line Karpas 299 has the same karyotype and pattern of antigen expression as the patient's cells. Northern blot analysis of RNA showed an active rearrangement of the T-cell receptor beta-chain gene. This is to our knowledge the first Ki-1 antigen- positive line to be established from a case of non-Hodgkin's lymphoma.

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Chen, Haiming, Mingjie Li, Richard A. Campbell, Melinda S. Gordon, Dror Shalitin, Cathy S. Wang, Ariana M. Berenson et al. « Arsenic Trioxide Affects Early Stage Angiogenesis through Inhibition of CD14 Monocyte Transdifferentiation into Endothelial Cells Induced by Pleiotrophin and mCSF. » Blood 108, n^o 11 (16 novembre 2006) : 1267. http://dx.doi.org/10.1182/blood.v108.11.1267.1267.

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Abstract We have discovered a novel mechanism leading to blood vessel formation involving transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (H. Chen et al, Blood, 2005; Yeh et al BJH, 2006). Arsenic trioxide (ATO) induces apoptosis of cancer cells directly through a number of mechanisms, and this drug has also been shown to inhibit angiogenesis. However, it remains unknown whether ATO affects the earliest stages of angiogenesis and vasculogenesis important in tumor development. We purified human monocytes (CD14+) and cultured these cells on collagen I-coated dishes. mCSF was added to the cells after 1 hour of culture. PTN was added twice to the culture, once after 24 hours and again after 5 days with or without ATO or bortezomib. FLK-1 expression (VEGFR-2) showed that the cells incubated on collagen I without drugs formed tube-like structures in the presence of PTN and mCSF. However, the tube-like structures disappeared after adding either the IC50 (5x10−6M) dose or low (5x10−7M) dose of ATO. FLK-1 staining remains in the tube-like structures with low doses (3x10−12M) of bortezomib. In order to examine whether ATO or bortezomib affects endothelial gene expression when monocytes are induced to transdifferentiate in the presence of these cytokines, we also examined expression using RT-PCR on endothelial cell genes (vascular endothelial growth factor receptor-2 (Flk-1), Tie-2 and von Willebrand factor (vWF)) and Western blot analysis for protein expression. The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at both the higher (5x10−6M) and lower (5x10−7M) doses of ATO. In contrast, the expression of endothelial markers was not reduced by adding low dose bortezomib (3x10−12M). We further examined the effects of ATO and bortezomib on early stage angiogenesis in vivo using the chorioallantoic membrane (CAM) assay. Fertilized chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. The tested micro-sponge with ATO (5x10−6M) or bortezomib (3x10−11M) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubator for 48 hours. The assay scored positive when two independent observers reported a significant reduction of vessels in the treated area. The results of the CAM assay showed that compared to saline, ATO significantly reduced new macroscopic and microscopic vessel formation. In contrast, bortezomib did not affect angiogenesis in the CAM assay. These experiments define a previously unrecognized novel mechanism by which ATO may have anti-angiogenetic effects in cancer patients-preventing the transdifferentiation of monocytes into endothelial cells by PTN. They also suggest ATO as a potential new specific agent to inhibit angiogenesis resulting from transdifferentiation of monocytes into vascular endothelial cells driven by pleiotrophin and mCSF. These results suggest a novel way by which anti-cancer agents may impact angiogenesis.

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Panda, Abir Kumar, Surajit Sinha, Kannan Natarajan, Jonathan M. Hernandez, David H. Margulies et Ethan M. Shevach. « Global inhibition of the interaction of NK inhibitory receptors with MHC-I augments coordinated innate and adaptive immunity against cancer metastasis. » Journal of Immunology 206, n^o 1_Supplement (1 mai 2021) : 57.11. http://dx.doi.org/10.4049/jimmunol.206.supp.57.11.

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Abstract Natural Killer (NK) cells are innate lymphocytes involved in the first line of immune defense and T cell adaptive immunity against viral infection and cancer, including metastasis. While on patrol, NK cells contact other cells and recognize MHC-I Ags via stochastically expressed MHC-I–specific inhibitory receptors (Ly49s in mice and KIRs in humans) that prevent NK cell activation via cytoplasmic ITIM. The binding site on MHC-Class-I for Ly49 inhibitory receptors is distinct from that for TCRs. The loss of MHC-I expression on tumor cells (“missing self”) abrogates inhibitory signals, resulting in NK activation. Global inhibition of the NK inhibitory receptor interactions in vivo by a pan-anti-MHC-I monoclonal antibody markedly activated IFNg-producing NK cells, independent of Fc receptors. NK cell-derived IFNg, along with other cytokines (MCSF & FLT3L), primed APC to induce IL-12/-15/-18/-21 cytokine cascades and enhanced levels of MHC-I and MHC-II expression that further drove the proliferation of NK cells and memory phenotype (MP) T cells. The global disruption of NK cell/MHC-I interactions significantly enhanced Th1 type signature transcription factors (Tbet & Eomes), cytokines (IFNg & Granzyme B), and chemokine receptors (CXCR3) on NK and MP T cells. Administration of pan-anti-MHC-I to unmanipulated mice profoundly augmented innate and adaptive immunity against viral infection, PD1-resistant transplanted tumors, and successfully constrained lung and liver metastasis, and protected the animals from tumor burden. Moreover, in vitro enhancement of human NK cell proliferation by a pan-anti-HLA Mab suggests that pan-anti-HLA could be a promising therapeutic strategy against chronic viral infection and cancer metastasis.

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Testa, U., C. Fossati, P. Samoggia, R. Masciulli, G. Mariani, HJ Hassan, NM Sposi et al. « Expression of growth factor receptors in unilineage differentiation culture of purified hematopoietic progenitors ». Blood 88, n^o 9 (1 novembre 1996) : 3391–406. http://dx.doi.org/10.1182/blood.v88.9.3391.bloodjournal8893391.

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We have evaluated the expression of growth factor receptors (GFRs) on early hematopoietic progenitor cells (HPCs) purified from human adult peripheral blood and induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), or monocytic (Mo) lineage. The receptors for basic fibroblast GF (bFGF), erythropoietin (Epo), thrombopoietin (Tpo), and macrophage colony-stimulating factor (MCSF) have been only assayed at mRNA level; the majority of GFRs have been evaluated by both mRNA and protein analyses: the expression patterns were consistent at both levels. In quiescent HPCs the receptors for early-acting [flt3 ligand (FL), c-kit ligand (KL), bFGF, interleukin-6 (IL-6)] and multilineage [IL-3, granulocyte-macrophage CSF (GM-CSF)] HGFs are expressed at significant levels but with different patterns, eg, kit and flt3 are detected on a majority and minority of HPCs, respectively, whereas IL-3Rs and GM-CSFRs are present on almost all HPCs. In the four differentiation pathways, expression of early-acting receptors shows a progressive decrease, more rapidly for bFGFR-1 and flt3 than for c-kit; furthermore, c-kit is more slowly downmodulated in the E and Mk than the G and Mo lineages. As a partial exception, IL-6Rs are still detected through the early or late stages of maturation in the Mk and Mo lineages, respectively. IL-3R expression is progressively and rapidly downmodulated in both E and Mk pathways, whereas it moderately decreases in the Mo lineage and is sustained in the G series. The expression of GM-CSFR is gradually downmodulated in all differentiation pathways, ie, the receptor density markedly decreases but late erythroblasts are still partially GM-CSFR+ and terminal G, Mk and Mo cells are essentially GM-CSFR+. Expression of receptors for late-acting cytokines is lineage-specific. Thus, EpoR, G-CSFR, TpoR, and M-CSFR exhibit a gradual induction followed by a sustained expression in the E, G, MK, and Mo lineages, respectively. In the other differentiation pathways the expression of these receptors is either absent or initially low and there-after suppressed. These observations are compatible with the following multi-step model. (1) The early-acting GFRs are expressed on quiescent HPCs with different patterns, whereas the multilineage GFRs are present on > or = 90% to 95% HPCs. (2) Multilineage GFs, potentiated by early-acting HGFs, trigger HPCs into cycling. HPC proliferation/differentiation is followed by declining expression of the early-acting GFRs and in part of multilineage GFRs (see above). (3) Multilineage GFs trigger the expression of the unilineage GFRs (see Testa U, et al: Blood 81:1442, 1993). Interaction of each unilineage GF with its receptor leads to sustained expression of the receptor (possibly via transcription factors activating the receptor promoter) and thus mediates differentiation/maturation through the pertinent lineage.

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Yu, Minjun, Jose Moreno et Achsah Keegan. « Regulation of the Receptor Activator of NF-κB Ligand (RANKL)-induced activation of the alternative NF-κB pathways by interleukin-4 (IL-4) (142.8) ». Journal of Immunology 184, n^o 1_Supplement (1 avril 2010) : 142.8. http://dx.doi.org/10.4049/jimmunol.184.supp.142.8.

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Abstract NF-κB signaling is essential for RANKL-induced osteoclast (OC) formation. IL-4 inhibited RANKL-induced OC differentiation, while at the same time promoted macrophage fusion to form multinucleated giant cells (MNG). Several groups have proposed that IL-4 acted by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not block canonical NF-κB signaling. Instead, we found that IL-4 inhibited alternative NF-κB signaling and induced p105/50 expression. Interestingly, in p105/50 deficient bone marrow macrophages (BMM), the formation of both multinucleated OC and MNG induced by RANKL or IL-4 respectively was impaired. This suggests that NF-κB signaling also plays an important role during macrophage fusion and MNG formation. To confirm this, the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) was used; PDTC blocked both RANKL-induced OC and IL-4-induced MNG formation. RT-PCR and western blot analyses showed that E-cadherin, and DC-STAMP, proteins important for macrophage fusion, were downregulated by PDTC. Furthermore, overexpression of p52 or RelB in p105/50 deficient BMM significantly enhanced both OC and MNG formation. DC-STAMP was upregulated in p52 and RelB transduced BMMs. These results suggest that the influence of IL-4 on NF-κB activation pathways is complex, and that NF-κB pathways positively regulate the IL-4-induced formation of MNG by MCSF-dependent macrophages.

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Chen, Haiming, Mingjie Li, Jennifer Li, Richard A. Campbell, Cathy S. Wang, Eric Sanchez, Jeffrey Steinberg et al. « Blockage of TRAF6 Signaling Inhibits Osteoclast Formation and Tumor Cell through Blocking the NF-kB and JNK Pathways in Multiple Myeloma. » Blood 110, n^o 11 (16 novembre 2007) : 2511. http://dx.doi.org/10.1182/blood.v110.11.2511.2511.

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Abstract We have recently shown that silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6) with a C-terminal siRNA inhibits proliferation and increases apoptosis of multiple myeloma (MM) tumor cells. In addition, TRAF6 ubiquitin ligase is also essential for receptor activator of nuclear factor kappa B ligand (RANKL) signaling and osteoclast differentiation. Based on TRAF6, CD40, and RANKL sequences and the TRAF6 interaction domain with CD40 or RANKL resides between residues 333 to 508, we cloned a sequence representing a 167 amino acid sequence from this domain in order to produce a TRAF6 dominant negative fragment (TRAF6dn) from the NIH gene bank (U78798) into the PCRII-TOPO vector. Subsequently, we re-cloned this fragment into an expression vector (pLenti6.2-hTRAF6dn). Expression of the TRAF6 dominant negative peptide was confirmed by Western blot analysis. We used human MM monocytes isolated by anti-CD14 micro-bead affinity column from MM patients’ peripheral blood (PB) or bone marrow (BM). In order to quantify osteoclast formation, the cells were fixed and stained for tartrate resistant acid phosphatase following seven days of culture. The BM and PB CD14+ cells were cultured on slide-culture dishes at a density of 2 × 105 cells per well. The cells infected with the pLenti6.2-hTRAF6dn or with the control vector, pLenti6.2/GW/EmGFP, were treated with 50ng/ml RANKL and 10ng/ml mCSF at the beginning of the culture period, and these factors were added again during a medium change after three days of incubation. We found that the TRAF6dn vector significantly inhibited osteoclast cell formation of CD14+ cells induced by RANKL and mCSF in a concentration dependent fashion compared with the control group. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The results showed that total endogenous c-Jun is reduced after TRAF6dn blocks TRAF6 signaling whereas cells infected with the control vector showed no changes in c-Jun. We further examined the effects of TRAF6dn on MM cell growth and apoptosis. Both tumor cells from fresh MM BM and the U266 and MM1s cell lines showed decreased cell proliferation and increased apoptosis in the presence of the TRAF6dn vector at 72 hours whereas the control vector had no effect on MM tumor cell growth or apoptosis. Furthermore, the TRAF6dn vector led to marked decreases in phospho-NF-kB protein levels compared to the control vector. Thus, we have demonstrated that inhibition of TRAF6 with a dominant negative construct both inhibits MM cell growth as well as osteoclast formation, and also reduces NF-kB activation and c-Jun levels which likely results in decreased activation of TRE/AP-1 elements. These studies suggest that the inhibition of TRAF6 may be an excellent therapeutic target for multiple myeloma since its inhibition results in suppression of tumor growth as well as osteoclast formation.

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Cunyat, Francesc, Jennifer N. Rainho, Brian West, Louise Swainson, Joseph M. McCune et Mario Stevenson. « Colony-Stimulating Factor 1 Receptor Antagonists Sensitize Human Immunodeficiency Virus Type 1-Infected Macrophages to TRAIL-Mediated Killing ». Journal of Virology 90, n^o 14 (27 avril 2016) : 6255–62. http://dx.doi.org/10.1128/jvi.00231-16.

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ABSTRACTStrategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4+T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophagesin vitro. PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs.IMPORTANCEAs the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4+T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4+T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4+T cells will likely dictate different approaches in order to achieve their elimination. For CD4+T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8+T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy.

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Michailov, Yulia, Ali AbuMadighem, Eitan Lunenfeld, Joseph Kapelushnik et Mahmoud Huleihel. « Granulocyte Colony-Stimulating Factor Restored Impaired Spermatogenesis and Fertility in an AML-Chemotherapy Mice Model ». International Journal of Molecular Sciences 22, n^o 20 (15 octobre 2021) : 11157. http://dx.doi.org/10.3390/ijms222011157.

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Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.

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Trojanowicz, Bogusz, Christof Ulrich et Matthias Girndt. « Uremic Apelin and Leucocytic Angiotensin-Converting Enzyme 2 in CKD Patients ». Toxins 12, n^o 12 (26 novembre 2020) : 742. http://dx.doi.org/10.3390/toxins12120742.

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Apelin peptides (APLN) serve as second substrates for angiotensin-converting enzyme 2 (ACE2) and, in contrast to angiotensin II (AngII), exert blood-pressure lowering and vasodilatation effects through binding to G-coupled APLN receptor (APLNR). ACE2-mediated cleavage of the APLN may reduce its vasodilatory effects, but decreased ACE2 may potentiate the hypotensive properties of APLN. The role of APLN in uremia is unclear. We investigated the correlations between serum-APLN, leucocytic APLNR, and ACE2 in 32 healthy controls (NP), 66 HD, and 24 CKD3–5 patients, and the impact of APLN peptides on monocytic behavior and ACE2 expression under uremic conditions in vitro. We observed that serum APLN and leucocytic APLNR or SLCO2B1 were significantly elevated in uremic patients and correlated with decreased ACE2 on uremic leucocytes. APLN-treated THP-1 monocytes revealed significantly increased APLNR and ACE2, and reduced TNFa, IL-6, and MCSF. Uremic toxins induced a dramatic increase of miR-421 followed by significant reduction of ACE2 transcripts, partially counteracted with APLN-13 and -36. APLN-36 triggered the most potent transmigration and reduction of endothelial adhesion. These results suggest that although APLN peptides may partly protect against the decay of monocytic ACE2 transcripts, uremic milieu is the most dominant modulator of local ACE2, and likely to contribute to the progression of atherosclerosis.

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Blanchfield, J. Lori, et Mark D. Mannie. « A GMCSF-neuroantigen (NAg) fusion protein targets NAg to dendritic cells (DC) and is an effective therapy for experimental autoimmune encephalomyelitis (EAE) (50.10) ». Journal of Immunology 182, n^o 1_Supplement (1 avril 2009) : 50.10. http://dx.doi.org/10.4049/jimmunol.182.supp.50.10.

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Abstract The GM-CSF and M-CSF cytokines were tested as domains in cytokine-NAg fusion proteins to assess targeting of NAg to different APC subsets. Fusion proteins were designed with a cytokine N-terminal domain and the encephalitogenic peptide 69-88 of guinea pig myelin basic protein (MBP) as the C-terminal domain. Studies measuring T-cell activation in vitro, prevention of EAE, and treatment of EAE showed that GMCSF-NAg was the most potent fusion protein, represented by the following rank order of activity (GMCSF-NAg > MCSF-NAg > GP69-88). GMCSF-NAg was 1000-fold more potent than GP69-88 in stimulating MBP-specific T-cell proliferation. The mechanism by which GMCSF-NAg promoted T-cell activation involved cytokine receptor-mediated uptake of NAg by antigen presenting cells (APC), because free GM-CSF inhibited the GMCSF-NAg potentiated response. GMCSF-NAg potently targeted NAg to dendritic cells and macrophages in vitro, but not to B or T cell APC. Covalent linkage between GM-CSF and NAg was required for enhanced potency of GMCSF-NAg in vitro and for the prevention and treatment of EAE in vivo. In conclusion, GMCSF-NAg potently targeted self-antigen to myeloid APC subsets and caused profound antigen-specific tolerance in EAE. This study was supported by research grants from the National Multiple Sclerosis Society and the Brody Brothers Endowment Fund.

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Gonzalez-Cotto, Marieli, Erika M. Palmieri, Walter A. Baseler, Christopher M. Rice et Daniel W. McVicar. « Trem2 Supports the Metabolic Program of Alternative Activated Macrophages ». Journal of Immunology 204, n^o 1_Supplement (1 mai 2020) : 73.19. http://dx.doi.org/10.4049/jimmunol.204.supp.73.19.

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Abstract Metabolism is a key modulator of macrophage differentiation, polarization and effector responses. While M1 favors a glycolytic configuration resembling the “Warburg effect”, M2 is fueled by oxidative metabolism. Here we describe a previously unappreciated metabolic phenotype controlled by The Triggering Receptor Expressed on Myeloid Cells (TREM)-2, a key player in inflammation and innate immunity. Trem2−/− macrophages show lower TCA cycle metabolites compared to wild type, but maintenance of glycolytic intermediates. Interestingly, while we show Trem2 abrogation is beneficial in M1 and protective against MCSF withdrawal, we find Trem2−/− cells to have disrupted lipid utilization as they accumulate both long chain free fatty acids (FFA) and carnitine-conjugated lipids. Because lipid metabolism is key for M2 responses, we hypothesized that Trem2 supports a metabolic program to maintain M2 activation. Indeed, in M2 cell, Trem2 sustains mitochondrial respiratory capacity, CD36 expression and lipoprotein and FFA uptake. Lipidomics confirmed Trem2−/− M2 cells had decreased FFA and enhanced triglycerides. Moreover, Trem2−/− M2 cells show a distinct metabolic profile with enhanced glutamine utilization, suggesting compensatory mechanisms in lieu of FFA for mitochondrial respiration. Transcriptome profiling decisively shows dysregulated genes related to defense response and glycerolipid metabolism, linking the metabolic findings to effector responses. Finally, in a model of in vivo Th2 response, Trem2−/− mice failed to control infection and exhibit metabolic dysregulation. Our findings suggest Trem2 is required for reprograming of macrophages by supporting the energetic requirements for M2 function.

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Mohamad, Safa F., Linlin Xu, Himes R. Evan, Hao Wu, Marta Alvarez, Paul Childress, Korbin Davis, Melissa Kacena et Edward F. Srour. « Calvariae-Resident Osteomacs That Are Phenotypically and Functionally Distinct from Marrow-Derived Macrophages Interact with Megakaryocytes to Regulate Hematopoietic Stem Cell Function ». Blood 128, n^o 22 (2 décembre 2016) : 28. http://dx.doi.org/10.1182/blood.v128.22.28.28.

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Abstract Maintenance of stem cell function is an orchestrated event requiring the participation of multiple cell types within the hematopoietic niche. Precise networking between hematopoietic stem cells (HSC) and these cell types is critical for the maintenance of the stem cell pool. Evidence is accumulating that multiple cell types cooperate to collectively maintain HSC function in the hematopoietic niche. We report here a detailed characterization of calvariae-resident osteomacs (OM) and outline how these cells require cooperation from megakaryocytes (MK) to sustain HSC function. We also describe in detail discriminating phenotypic and functional properties that clearly distinguish OM from marrow-derived macrophages (Mφ). Osteomacs, identified as CD45+F4/80+ cells, were easily detectable in calvarial cell (CC) preparations (3-5% of total CC) collected by the enzymatic digestion of calvariae from 2d-old pups. To assess the effect of MK, a known regulator of osteoblast (OB) proliferation and differentiation, on OM, we performed co-cultures using CC and MK prepared from fetal liver. In the absence of MK, OM did not increase in numbers over a period of 5 days in culture and remained approximately 5% of total cultured cells. However, in the presence of MK, OM significantly increased to become between 25% and 30% of total cells demonstrating that MK regulate OM proliferation. Clonogenic assays established that OM support hematopoiesis enhancing activity of OB and that this activity can be upregulated by MK. Interestingly, marrow-derived Mφ were unable to mediate the same hematopoiesis enhancing activity regardless of whether MK were present in the co-culture or not. These results were validated via primary and secondary transplantations in lethally irradiated hosts whereby the highest repopulating potential was observed among marrow-derived LSK cells co-cultured for 5 days with a mixture of OB, OM, and MK. Using eight surface markers and flow cytometric analysis, we established that although marrow-derived Mφ and OM share many phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and GR-1), only OM expressed MCSFR and CD166, thus providing a distinct and unique profile for these cells. To assess changes in pathway activation between resting and MK-activated OM, we performed single cell genomic analysis. This approach detected the upregulation of several canonical pathways important in HSC maintenance such as Ephrin receptor signaling, PDGF signaling, and leukocyte extravasation signaling in MK-stimulated OM. Single cell genomic analysis between CC-derived OM and marrow-derived Mφ (isolated from each tissue as CD45+F4/80+ cells) revealed 39 genes to be significantly different between the two cell types. Strikingly, many genes such as IGF1, KITL and NOTCH2 that have previously been implicated in HSC regulation were upregulated in OM. MCSFR1 a known regulator of proliferation, differentiation and survival of Mφ was also upregulated in OM corroborating the data previously collected from flow cytometric analyses. However, OM did not respond to exogenous MCSF stimulation suggesting that MCSF alone is not sufficient to induce OM proliferation or that direct contact with MK is required for induction of proliferation. To investigate changes at the protein translational level, we examined both cell types using CyTOF and a panel of 24 surface and intracellular antibodies. The surface marker CD169 which was previously associated with HSC retention when present on cellular components of the hematopoietic niche was expressed on OM but not on Mφ. Intriguingly, OM expressed both CD86 and CD206 which are known M1 and M2 Mφ markers, respectively. TNF-α, TIMP2, FGF2 and MCP1 which are known HSC regulators were also upregulated in OM. Finally, the majority of OM expressed embigin and IL-18, both of which have been implicated in the maintenance of HSC function. These data demonstrate that although bone-associated OM share many properties with marrow-derived Mφ, they are phenotypically and functionally distinct and are critical for the maintenance of HSC function. Furthermore, the function of OM, and consequently that of the two components of CC, namely OB and OM, is significantly augmented by interactions with MK demonstrating that the crosstalk between OM, OB and MK form a novel network in supporting HSC function. Disclosures No relevant conflicts of interest to declare.

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Li, Mingjie, Eric Sanchez, Cathy Wang, Megan Schultz, Jessica Wang, Emily Wong, Zhi-Wei Li et al. « Blockage of TRAF6 by Dominant Negative Peptides to Inhibit Multiple Myeloma (MM) Cell Proliferation and Osteoclast Formation through NF-κB, JNK and AKT Signal Transduction Pathways ». Blood 116, n^o 21 (19 novembre 2010) : 4068. http://dx.doi.org/10.1182/blood.v116.21.4068.4068.

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Abstract Abstract 4068 Several members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 have been implicated in regulating signal transduction from various TRAF family members. However, the unique biological function of TRAF6 is largely determined by its TRAF-C domain, which does not interact with peptide motifs that are recognized by TRAF1, -2, -3 or -5. We have reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-κB and JNK pathways through silencing TRAF6 C-domain mRNA and the dominant negative peptide expression vector (Chen H. et al, Oncogene, 2006; Li M. et al, Blood 2009). TRAF6 have been recently found as a ligase for Akt ubiquitination (Yang WL et al, Science, 2009). Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. In this study, we first investigated whether TRAF6 is over-expressed in MM tumor cells. Twelve MM fresh bone marrow (BM) aspirates derived from MM patients were assessed using Western blot analysis and immunohistochemical staining with anti-TRAF6 antibody. We found that TRAF6 protein was highly expressed in tumor cells from MM patients compared to normal human BM samples. Based on TRAF6, CD40, and RANKL sequences and crystal structures, we targeted the TRAF6 C-domain binding residues. We found that TRAF6 dominant negative binding peptide (TRAF6dn) significantly inhibited MM cell proliferation maximally at 72 hours using the MTS cell proliferation assay whereas effects on inducing MM cell apoptosis were most prominent at 48 hours as assessed with Annexin V staining with flow cytometric analysis. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration peptide-dependent fashion. Furthermore, phosphorylation of both AKT and NF-κB were also reduced using our human TRAF6dn or decoy peptides. We also examined the effect of the TRAF6dn peptide on the JNK pathway since this signaling pathway is also associated with cell cycle effects in MM. We measured JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. The results showed that the phosphorylation of JNKK is markedly reduced after treatment with the TRAF6dn peptide. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. We evaluated the effect of TRAF6dn peptide on osteoclast formation using cells from human monocytes isolated by anti-CD14 micro-bead affinity column from MM patients' BM or peripheral blood mononuclear cells. The monocytes were cultured on slide-culture dishes (2 × 105 cells/well).We found TRAF6dn markedly inhibited osteoclast cell formation from monocytes induced with RANKL and mCSF in a concentration- dependent fashion compared with a control group using tartrate resistant acid phosphatase staining. We further assessed whether TRAF6dn can reduce bone resorption using a dentin bone resorption assay. BM-derived monocytes were isolated as above and were cultured on dentin bone slides (4 × 105 cells/slide). The cells treated with a TRAF6dn peptide or the control peptide, were incubated with 50ng/ml RANKL and 10ng/ml MCSF. All cells were cultured for 21 days. It was found that TRAF6dn significantly inhibited lacunar resorption in a concentration-dependent fashion. These studies suggest that TRAF6 is over-expressed in MM and our TRAF6dn peptide inhibits many signaling pathways critical to the growth of MM and formation of osteoclasts resulting in marked anti-MM effects and reduction in osteoclast formation resulting in marked inhibition of bone resorption. Thus, this novel approach may offer a new therapeutic approach to both treat multiple myeloma and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures: No relevant conflicts of interest to declare.

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Valverde, P., T. Kawai et M. A. Taubman. « Potassium Channel-blockers as Therapeutic Agents to Interfere with Bone Resorption of Periodontal Disease ». Journal of Dental Research 84, n^o 6 (juin 2005) : 488–99. http://dx.doi.org/10.1177/154405910508400603.

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Inflammatory lesions of periodontal disease contain all the cellular components, including abundant activated/memory T- and B-cells, necessary to control immunological interactive networks and to accelerate bone resorption by RANKL-dependent and -independent mechanisms. Blockade of RANKL function has been shown to ameliorate periodontal bone resorption and other osteopenic disorders without affecting inflammation. Development of therapies aimed at decreasing the expression of RANKL and pro-inflammatory cytokines by T-cells constitutes a promising strategy to ameliorate not only bone resorption, but also inflammation. Several reports have demonstrated that the potassium channels Kv1.3 and IKCa1, through the use of selective blockers, play important roles in T-cell-mediated events, including T-cell proliferation and the production of pro-inflammatory cytokines. More recently, a potassium channel-blocker for Kv1.3 has been shown to down-regulate bone resorption by decreasing the ratio of RANKL-to-OPG expression by memory-activated T-cells. In this article, we first summarize the mechanisms by which chronically activated/memory T-cells, in concert with B-cells and macrophages, trigger inflammatory bone resorption. Then, we describe the main structural and functional characteristics of potassium channels Kv1.3 and IKCa1 in some of the cells implicated in periodontal disease progression. Finally, this review elucidates some recent advances in the use of potassium channel-blockers of Kv1.3 and IKCa1 to ameliorate the clinical signs or side-effects of several immunological disorders and to decrease inflammatory bone resorption in periodontal disease. ABBREVIATIONS: AICD, activation-induced cell death; APC, antigen-presenting cells; B(K), large conductance; CRAC, calcium release-activated calcium channels; DC, dendritic cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-γ, interferon-γ; IP3, inositol (1,4,5)-triphosphate; (K)ir, inward rectifier; JNK, c-Jun N-terminal kinase; I(K), intermediate conductance; LPS, lipopolysaccharide; L, ligand; MCSF, macrophage colony-stimulating factor; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T-cells; RANK, receptor activator of nuclear factor-κB; TCM, central memory T-cells; TEM, effector memory T-cells; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; OPG, osteoprotegerin; Omp29, 29-kDa outer membrane protein; PKC, protein kinase C; PLC, phospholipase C; RT-PCR, reverse-transcriptase polymerase chain-reaction; S(K), small conductance; TCR, T-cell receptor; and (K)v, voltage-gated.

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Karhunen, Ville, Dipender Gill, Jian Huang, Emmanouil Bouras, Rainer Malik, Mark J. Ponsford, Ari Ahola-Olli et al. « The interplay between inflammatory cytokines and cardiometabolic disease : bi-directional mendelian randomisation study ». BMJ Medicine 2, n^o 1 (février 2023) : e000157. http://dx.doi.org/10.1136/bmjmed-2022-000157.

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ObjectiveTo leverage large scale genetic association data to investigate the interplay between circulating cytokines and cardiometabolic traits, and thus identifying potential therapeutic targets.DesignBi-directional Mendelian randomisation study.SettingGenome-wide association studies from three Finnish cohorts (Northern Finland Birth Cohort 1966, Young Finns Study, or FINRISK study), and genetic association summary statistics pooled from observational studies for expression quantitative trait loci and cardiometabolic traits.ParticipantsData for 47 circulating cytokines in 13 365 individuals from genome-wide association studies, summary statistic data for up to 21 735 individuals on circulating cytokines, summary statistic gene expression data across 49 tissues in 838 individuals, and summary statistic data for up to 1 320 016 individuals on cardiometabolic traits.InterventionsRelations between circulating cytokines and cardiovascular, anthropometric, lipid, or glycaemic traits (coronary artery disease, stroke, type 2 diabetes mellitus, body mass index, waist circumference, waist to hip ratio, systolic blood pressure, glycated haemoglobin, high density lipoprotein cholesterol, low density lipoprotein cholesterol, total cholesterol, triglycerides, C reactive protein, glucose, fasting insulin, and lifetime smoking).Main outcome methodsGenetic instrumental variables that are biologically plausible for the circulating cytokines were generated. The effects of cardiometabolic risk factors on concentrations of circulating cytokines, circulating cytokines on other circulating cytokines, and circulating cytokines on cardiometabolic outcomes were investigated.ResultsGenetic evidence (mendelian randomisation P<0.0011) suggests that higher body mass index, waist circumference, smoking, higher concentrations of lipids, and systolic blood pressure increase circulating concentrations of several inflammatory cytokines and C reactive protein. Evidence for causal relations (mendelian randomisation P<0.0011) were noted between circulating cytokines, including a key role of vascular endothelial growth factor on influencing the concentrations of 10 other cytokines. Both mendelian randomisation (P<0.05) and colocalisation (posterior probability >0.5) suggested that coronary artery disease risk is increased by higher concentrations of circulating tumour necrosis factor related apoptosis-inducing ligand (TRAIL), interleukin-1 receptor antagonist (IL1RA), and macrophage colony-stimulating factor (MCSF).ConclusionThis study offers insight into inflammatory mediators of cardiometabolic risk factors, cytokine signalling cascades, and effects of circulating cytokines on different cardiometabolic outcomes.

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Uemura, N., K. Ozawa, K. Takahashi, A. Tojo, K. Tani, K. Harigaya, S. Suzu, K. Motoyoshi, H. Matsuda et H. Yagita. « Binding of membrane-anchored macrophage colony-stimulating factor (M- CSF) to its receptor mediates specific adhesion between stromal cells and M-CSF receptor-bearing hematopoietic cells ». Blood 82, n^o 9 (1 novembre 1993) : 2634–40. http://dx.doi.org/10.1182/blood.v82.9.2634.2634.

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Abstract To explore the biologic significance of the membrane-anchored form of macrophage colony-stimulating factor (M-CSF), we examined whether interaction between membrane-bound M-CSF and its receptor mediates intercellular adhesion as well as cell proliferation and differentiation. Human M-CSF receptors were expressed on a murine interleukin-2 (IL-3)-dependent cell line, FDC-P2, by DNA transfection with the c-fms gene (FDC-P2-MCSFR). A human bone marrow-derived stromal cell line, KM102, was used in the cell adhesion assay. The expression of membrane-bound M-CSF on KM102 cells was demonstrated by flow cytometry and immunoblot analysis. After the incubation of parent and transformed FDC-P2 cells on confluent KM102 cells, nonadherent cells were removed and the cells attached to KM102 cells were examined microscopically. Almost all parent FDC-P2 cells were nonadherent, whereas a significant number of FDC-P2-MCSFR cells bound to KM102 cells. The addition of anti-M-CSF or anti-M-CSF receptor neutralizing antibodies dose-dependently inhibited the binding of [3H]-thymidine- labeled FDC-P2-MCSFR cells to KM102 cells. An excess amount of M-CSF also inhibited the binding. On the other hand, the addition of antibodies against some representative adhesion molecules (vitronectin receptor, Pgp-1/CD44, and VLA-4) did not inhibit the adhesion between FDC-P2-MCSFR cells and KM102 cells. These results support the idea that membrane-anchored M-CSF and its receptor may function as mediators of cell-cell adhesion.

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Uemura, N., K. Ozawa, K. Takahashi, A. Tojo, K. Tani, K. Harigaya, S. Suzu, K. Motoyoshi, H. Matsuda et H. Yagita. « Binding of membrane-anchored macrophage colony-stimulating factor (M- CSF) to its receptor mediates specific adhesion between stromal cells and M-CSF receptor-bearing hematopoietic cells ». Blood 82, n^o 9 (1 novembre 1993) : 2634–40. http://dx.doi.org/10.1182/blood.v82.9.2634.bloodjournal8292634.

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To explore the biologic significance of the membrane-anchored form of macrophage colony-stimulating factor (M-CSF), we examined whether interaction between membrane-bound M-CSF and its receptor mediates intercellular adhesion as well as cell proliferation and differentiation. Human M-CSF receptors were expressed on a murine interleukin-2 (IL-3)-dependent cell line, FDC-P2, by DNA transfection with the c-fms gene (FDC-P2-MCSFR). A human bone marrow-derived stromal cell line, KM102, was used in the cell adhesion assay. The expression of membrane-bound M-CSF on KM102 cells was demonstrated by flow cytometry and immunoblot analysis. After the incubation of parent and transformed FDC-P2 cells on confluent KM102 cells, nonadherent cells were removed and the cells attached to KM102 cells were examined microscopically. Almost all parent FDC-P2 cells were nonadherent, whereas a significant number of FDC-P2-MCSFR cells bound to KM102 cells. The addition of anti-M-CSF or anti-M-CSF receptor neutralizing antibodies dose-dependently inhibited the binding of [3H]-thymidine- labeled FDC-P2-MCSFR cells to KM102 cells. An excess amount of M-CSF also inhibited the binding. On the other hand, the addition of antibodies against some representative adhesion molecules (vitronectin receptor, Pgp-1/CD44, and VLA-4) did not inhibit the adhesion between FDC-P2-MCSFR cells and KM102 cells. These results support the idea that membrane-anchored M-CSF and its receptor may function as mediators of cell-cell adhesion.

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Kolomansky, Albert, Naamit Deshet-Unger, Nathalie Ben-Califa, Zamzam Awida, Maria Ibrahim, Tamar Liron, Sahar Hiram-Bab et al. « B Cell Specific Knockdown of the Erythropoietin (EPO) Receptor Attenuates EPO-Induced Bone Loss in Mice ». Blood 134, Supplement_1 (13 novembre 2019) : 939. http://dx.doi.org/10.1182/blood-2019-127092.

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Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p<0.05, Figure 1). Remarkably, this effect was observed despite the fact that cKD mice attained higher hemoglobin levels following EPO treatment (21.1±0.1 mg/dL vs 20.4±0.2 mg/dL in the cKD vs control mice, p<0.05). An EPO-induced increase in CD115+ Pro-B cells was observed in EPO-treated control mice but was absent in the cKD mice. The latter finding correlates with the observed bone loss and indicates that the increased number of MCSF-R-expressing pro-B cells is dependent on B cell EPO-R. Supporting the osteoclastic potential of this specific B cell subpopulation is the fact that most of the CD115+ Pro-B cells also express β3 integrin (CD61) which is essential for osteoclast differentiation and function. Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.

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Li, Mingjie, Suzie Vardanyan, Jillian Gottlieb, Cathy Wang, Kevin Delijani, Ran Halleluyan, Joseph Ben-Zvi et al. « Higher TRAF6 and Fbxo 3 Expression Is With Progressive Multiple Myeloma and Blockage Of TRAF6 Inhibits Tumor Growth and Bone Resorption ». Blood 122, n^o 21 (15 novembre 2013) : 5362. http://dx.doi.org/10.1182/blood.v122.21.5362.5362.

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Abstract Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. F-box proteins such as the E3 ligase component Fbxo3 and another F-box family member Fbxl 2, regulate TRAF protein signaling. First, we investigated TRAF6 expression in bone marrow mononuclear cells (BMMCs) from multiple myeloma (MM) patients with progressive disease or in remission and healthy subjects. The results showed higher TRAF6 protein expression among MM patients with progressive disease than among those in remission or healthy subjects. Notably, changes in TRAF6 protein expression in MM BMMCs were found to correlate with response of individual patients to treatment with the proteasome inhibitors bortezomib or carfilzomib. We have previously reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-kB and JNK pathways using a silencing TRAF6 C-domain mRNA construct. In this study, we cloned a 167 amino acid (in residues 333 to 508) portion of the TRAF6 dominant negative (TRAF6dn) and synthesized decoy peptides of the TRAF6 interaction domain with CD40 and the TRAF6-RANK binding domain. Decreases in cell proliferation and increase in cell apoptosis in MM BMMCs treated with TRAF6dn occurred in a concentrationdependent fashion. We also found TRAF6dn markedly inhibited osteoclast cell formation of monocytes induced by RANKL and mCSF in a dose-dependent manner. Next, we examined the effect of TRAF6dn on NF-kB and JNK by determining phosphorylation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. MM BMMCs exposed to the TRAF6dn fragment or TRAF6 decoy peptide showed reduced phosphoNF-kB protein and phosphorylation of JNKK. These studies suggest that the TRAF6dn or combined TRAF6 decoy and CD40 decoy peptide may be excellent agents to block both MM cell growth and osteoclast formation in MM. We further investigated the protein expression of Fbxo 3, Fbxl 2 and TRAF6 in fresh BMMCs from MM patients with progressive disease or in remission. Results of Western blot analysis showed protein expression of Fbxo 3 and TRAF6 was increased and Fbxl 2 was decreased among patients with progressive disease compared to patients in remission. Thus, these results may offer a new mechanism through which MM tumor cells are regulated and provide a new therapeutic approach to treat MM and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures: No relevant conflicts of interest to declare.

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Zhang, Shu, Ping Lei, Xinyi Liu, Xiangrong Li, Kelcey Walker, Leela Kotha, Craig Rowlands et Stephen Safe. « The aryl hydrocarbon receptor as a target for estrogen receptor-negative breast cancer chemotherapy ». Endocrine-Related Cancer 16, n^o 3 (septembre 2009) : 835–44. http://dx.doi.org/10.1677/erc-09-0054.

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the relatively non-toxic selective aryl hydrocarbon receptor (AhR) modulator 6-methyl-1,3,8-trichlorodibenzo-furan (MCDF) induced CYP1A1-dependent ethoxyresorufin O-deethylase activity and inhibited proliferation of seven estrogen receptor (ER) negative breast cancer cell lines. MCDF, TCDD and structurally related 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 3,3′,4,4′,5-pentachlorobiphenyl induced CYP1A1 and inhibited proliferation of BT-474 and MDA-MB-468 cells. In BT474 and MDA-MB-468 cells transfected with a small inhibitory RNA for the AhR, the antiproliferative activity of the chlorinated aromatic compounds was reversed, whereas for MCDF, only partial reversal was observed, suggesting that this compound acts through both AhR-dependent and AhR-independent pathways in these two cell lines. MCDF also inhibited tumor growth in athymic nude mice in which MDA-MB-468 cells were injected directly into the mammary fat pad. These results suggest that the AhR is a potential drug target for treatment of ER-negative breast cancer.

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Wang, J. M., B. Sherry, M. J. Fivash, D. J. Kelvin et J. J. Oppenheim. « Human recombinant macrophage inflammatory protein-1 alpha and -beta and monocyte chemotactic and activating factor utilize common and unique receptors on human monocytes. » Journal of Immunology 150, n^o 7 (1 avril 1993) : 3022–29. http://dx.doi.org/10.4049/jimmunol.150.7.3022.

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Abstract The human macrophage inflammatory proteins-1 alpha and -beta (MIP-1 alpha and -beta), which are also known as LD78 and ACT2, respectively, are distinct but highly related members of the chemoattractant cytokine (chemokine) family. rMIP-1 alpha and -beta labeled with 125I specifically bind to human peripheral blood monocytes, the monocytic cell line THP-1, peripheral blood T cells, and the YT cell line. Steady state binding experiments revealed approximately 3000 high affinity binding sites/cell for MIP-1 alpha on human monocytes and on THP-1 cells, with Kd values of 383 pM and 450 pM, respectively. Human MIP-1 alpha and -beta had nearly identical affinities for the binding sites and each competed equally well for binding. Human monocyte chemotactic and activating factor (MCAF), a member of the same chemokine family, consistently displaced about 25% of human MIP-1 alpha and -beta binding on monocytes but not on YT cells, which did not bind MCAF. On the other hand, human rMIP-1 alpha and -beta partially inhibited binding of radiolabeled MCAF to monocytes. Both MIP-1 alpha and -beta were chemotactic for human monocytes. Preincubation of monocytes with human rMIP-1 alpha or -beta markedly reduced cell migration towards the other cytokine, whereas preincubation with human rMCAF only partially desensitized the monocyte chemotaxis response to human rMIP-1 alpha or -beta. These data suggest the existence of three subtypes of receptors, i.e., one unique receptor shared by MIP-1 alpha and -beta, a second unique receptor for MCAF, and a third species that recognizes both MCAF and MIP-1 peptides.

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Li, Ke, Vinit Karur, Bethany Vincent, Jennifer Curato, Reuben Kapur, Peter Besmer et Don M. Wojchowski. « Kit PY567- Directed Signals Are Important for Efficient Erythroid Progenitor Cell Proliferation and Survival, and Are Attenuated by Kit PY569. » Blood 104, n^o 11 (16 novembre 2004) : 2168. http://dx.doi.org/10.1182/blood.v104.11.2168.2168.

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Abstract Anemia incurred by mice with naturally occurring Kit mutations (eg, W41) clearly illustrates important contributions of SCF/Kit signaling to erythropoiesis. Specific cytoplasmic features of Kit and linked signaling events that support such contributions, however, are not well understood. To address this issue, the proliferative and survival signaling activities of a panel of Kit cytoplasmic PY mutants first were studied in SCF- and Epo- dependent G1E2 cells, and also in multi-potential SCF-dependent EML cells. Kit forms studied included PY567, PY569, PY567-plus-569, PY702, PY719, PY728 and PY745 phosphotyrosine add-back forms as well as a biologically inactive baseline mutant, Kit-F6 in which these six PY sites were mutated to phenylalanine. To by-pass endogenous Kit, cytoplasmic PY mutants were expressed from a MIEG3 vector as MCSF-R/Kit “MK” chimeras, and cells expressing matched and physiological levels of M/K chimeras were isolated by FACS. PY567 per se (ie, M/K PY567) selectively supported near wild-type proliferative and anti-apoptotic activities in both G1E2 and EML cells. By direct comparison, PY569 per se has diminished activity in G1E2 cells, and only baseline activity in EML cells. Moreover, the combined restoration of both PY567 and 569 uniformly attenuated the otherwise high proliferative and survival activities of PY567. These findings therefore indicate that Kit PY567 and PY569 do not necessarily exert overlapping roles, but that PY569 instead may preferentially link to negative effects and also act to attenuate key positive signals relayed via Kit PY567 (possibly in a lineage- restricted fashion). In keeping with recent studies in 32D cells, little if any bioactivities were conferred by PY702, 719, 728, or 745 per se. Mechanistically, PY567 also was observed to link with high efficiency to sustained Bcl-xl expression, and to be selectively efficient in Epo receptor co-signaling. While these findings predict that the mutation of Kit Y567 should perturb red cell development, erythropoiesis in Kit-F567 knock-in mice appears near-normal. However, primary erythroid progenitor cells isolated from the spleen of Kit F567 mice at 50hr post- phenylhydrazine injection proved to be markedly deficient in SCF responsiveness. In addition, a >5-fold decrease in SCF and Epo co-signaling was reproducibly observed for Kit-F567 progenitor cell preparations. These novel findings therefore lend new evidence to the case that PY567, in fact, contributes in important ways to Kit’s erythropoietic biofunction.

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Filipović, M., A. Šućur, D. Flegar, Z. Jajić, M. Ikić Matijašević, N. Lukač, N. Kovačić et al. « FRI0372 INCREASED EXPRESSION OF NOTCH RECEPTORS ON OSTEOCLAST PROGENITORS INDUCED BY RHEUMATOID ARTHRITIS ». Annals of the Rheumatic Diseases 79, Suppl 1 (juin 2020) : 783.1–783. http://dx.doi.org/10.1136/annrheumdis-2020-eular.1693.

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Background:Systemic and periarticular bone loss in rheumatoid arthritis (RA) is mediated by osteoclasts, multinucleated cells originating from the myeloid lineage. Recently, Notch signaling pathway has emerged as a potential regulator of osteoclast progenitor (OCP) differentiation and activation.Objectives:The exact role of Notch signaling in the context of arthritis is still unknown; however, its inhibition has beneficial effects in animal arthritis models. We aimed to determine the expression of Notch receptors and ligands on specific OCP subpopulations and define changes that occur in murine collagen-induced arthritis (CIA) and RA patients.Methods:Peripheral blood, synovial tissue and subchondral bone marrow were collected from RA patients, and periarticular bone marrow (PBM) and spleen (SPL) were harvested from male C57/Bl6 mice immunized with chicken type II collagen. Notch 1 to 4 receptor expression on OCPs was analyzed by flow cytometry. Gene expression of Notch receptors/ligands was determined by qPCR from tissues and sorted OCPs. Sorted OCPs were cultured, with addition of MCSF and RANKL, in control, IgG, Jagged (Jag) 1 or Delta (DLL) 1 coated wells. Immunohistochemistry (IHC) for Notch 1 and 2 was performed on sections of murine hind paws. Research was approved by Ethics committee.Results:We previously identified peripheral and periarticular subpopulations of murine and human OCPs, as CD45+CD3-B220-NK1.1-CD11blo/+CD115+CCR2+and CD45+CD3-CD19-CD56-CD11b+CD14+CCR2+respectively, specifically associated with arthritis. Flow cytometry revealed that majority of murine splenic and periarticular OCPs express Notch 2, whereas Notch 1 and 4 were expressed on approximately 10% of cells. In CIA, this highly osteoclastogenic population is expanded as is the expression of Notch 4 in PBM and Notch 3 in SPL. Majority of human peripheral-blood OCPs express Notch 2 and 4, with a specific increase in the expression of Notch 1 and 3 in RA. In contrast, RA synovial-derived OCPs mostly express Notch 1 to 3, whereas subchondral OCPs mostly express Notch 1 and 4. Notch ligands were analyzed at mRNA level and revealed expression of Jag1, Jag2 and DLL4 in murine sorted OCPs and Jag1 and DLL1 in human sorted OCPs. Expression of Notch 1 and 2 was confirmed by IHC on arthritic murine hind paws, with Notch 2 expressed by bone marrow, synovial tissue and chondrocytes and Notch 1 expressed by chondrocytes and synovial tissue. Increased expression of Notch 1, Notch 2 and Jag1 was also confirmed in murine arthritic periarticular tissue by qPCR. During osteoclastogenic culture, murine and human OCPs exhibit a similar gene expression pattern with higher initial expression of Notch 1 and 2, and increase in the expression of Notch 3 and 4 with differentiation. Osteoclasts were also differentiated under Notch-ligand stimulation. Coating with DLL1 results in a greater number of cells expressing osteoclast-specific TRAP, whereas Jag1 seemed to inhibit osteoclastogenesis.Conclusion:Our results indicate that murine and human OCPs express a distinct tissue-specific pattern of Notch receptors. Notch signaling in OCPs is increased in arthritis and may contribute to the osteoclastogenic potential and increased bone resorption. Our next aim would be to determine the effect of Notch inhibition on OCP activity and arthritis severity.Acknowledgments:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, IP-2014-09-7406 and DOK-2018-09-4276.Disclosure of Interests:None declared

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