Monocytic Myeloid Cells Research Articles

NF-κB p50 Regulates C/EBPα Expression and Inflammatory Cytokine-Induced Neutrophil Production

NF-kappaB is a key transcriptional inducer of the inflammatory response in mature myeloid cells, and also stimulates cell survival, but its role in immature myeloid cell development has not been well characterized. C/EBPalpha is required for the development of monocytic and granulocytic myeloid cells from early progenitors, and NF-kappaB and C/EBPbeta cooperatively induce several inflammatory mediators. Having found that C/EBPalpha binds NF-kappaB p50 preferentially compared with NF-kappaB p65, we have now investigated myelopoiesis in nfkb1(-/-) mice lacking NF-kappaB p50. Absence of p50 leads to a significant reduction in the number of granulocytic progenitors, CFU-granulocyte, obtained with G-CSF or GM-CSF in vitro and reduces neutrophil production in vivo in response to G-CSF, with preservation of monopoiesis in vitro in response to cytokines or LPS. To gain insight into the mechanism underlying reduced granulopoiesis in the absence of NF-kappaB p50, we assessed the expression of several myeloid regulatory proteins in lineage-negative, immature myeloid cells. Although PU.1, C/EBPbeta, and STAT3 levels were unchanged, C/EBPalpha protein and RNA levels were reduced approximately 3-fold in the absence of NF-kappaB p50. In addition, NF-kappaB p50 and C/EBPalpha bound the endogenous C/EBPalpha promoter in a chromatin immunoprecipitation assay, and NF-kappaB p50 trans activated the C/EBPalpha promoter, alone or in cooperation with C/EBPalpha. Despite reduction of C/EBPalpha, G-CSFR and M-CSFR levels were maintained in total marrow and in lineage-negative cells. Together, these data indicate that acute inflammation not only activates mature myeloid cells, but also stimulates neutrophil production via NF-kappaB p50 induction of C/EBPalpha transcription.

Galactomutarotase and Other Galactose-Related Genes Are Rapidly Induced by Retinoic Acid in Human Myeloid Cells

Aldose-1-epimerase (mutarotase) catalyzes the interconversion of alpha and beta hexoses, which is essential for normal carbohydrate metabolism and the production of complex oligosaccharides. Galactose mutarotase (GALM) has been well characterized at the protein level, but information is lacking on the regulation of GALM gene expression. We report herein that all-trans-retinoic acid (RA), an active metabolite of vitamin A that is known to induce myeloid lineage cell differentiation into macrophage-like cells, induces a rapid and robust regulation of GALM mRNA expression in human myeloid cells. all-trans-RA at a physiological concentration (20 nM), or Am580, a ligand selective for the nuclear retinoid receptor RARalpha, increased GALM mRNA in THP-1 cells, with significantly increased expression in 2 h, increasing further to an approximately 8-fold elevation after 6-40 h (P < 0.005). In contrast, tumor necrosis factor-alpha did not increase GALM mRNA expression, although it is capable of inducing cell differentiation. RA also increased GALM mRNA in U937 and HL-60 cells. The increase in GALM mRNA by RA was blocked by pretreating THP-1 cells with actinomycin D but not by cycloheximide. GALM protein and mutarotase activity were also increased time dependently in RA-treated THP-1 cells. In addition to GALM, several other genes in the biosynthetic pathway of galactosyl-containing complex oligosaccharides were more highly expressed in RA-treated THP-1 cells, including B4GALT5, ST3GAL3, ST6GALNAC5, and GALNAC4S-6ST. Thus, the results of this study identify RA as a significant regulator of GALM and other galactose-related genes in myeloid-monocytic cells, which could affect energy utilization and synthesis of cell-surface glycoproteins or glycolipids involved in cell motility, adhesion, and/or functional properties.

Inflammatory cell infiltration of tumors: Jekyll or Hyde

Inflammatory cell infiltration of tumors contributes either positively or negatively to tumor invasion, growth, metastasis, and patient outcomes, creating a Dr. Jekyll or Mr. Hyde conundrum when examining mechanisms of action. This is due to tumor heterogeneity and the diversity of the inflammatory cell phenotypes that infiltrate primary and metastatic lesions. Tumor infiltration by macrophages is generally associated with neoangiogenesis and negative outcomes, whereas dendritic cell (DC) infiltration is typically associated with a positive clinical outcome in association with their ability to present tumor antigens (Ags) and induce Ag-specific T cell responses. Myeloid-derived suppressor cells (MDSCs) also infiltrate tumors, inhibiting immune responses and facilitating tumor growth and metastasis. In contrast, T cell infiltration of tumors provides a positive prognostic surrogate, although subset analyses suggest that not all infiltrating T cells predict a positive outcome. In general, infiltration by CD8(+) T cells predicts a positive outcome, while CD4(+) cells predict a negative outcome. Therefore, the analysis of cellular phenotypes and potentially spatial distribution of infiltrating cells are critical for an accurate assessment of outcome. Similarly, cellular infiltration of metastatic foci is also a critical parameter for inducing therapeutic responses, as well as establishing tumor dormancy. Current strategies for cellular, gene, and molecular therapies are focused on the manipulation of infiltrating cellular populations. Within this review, we discuss the role of tumor infiltrating, myeloid-monocytic cells, and T lymphocytes, as well as their potential for tumor control, immunosuppression, and facilitation of metastasis.

The Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder with variable clinical phenotypes that correlate with the type of mutations in the WAS protein (WASP) gene. WASP, a key regulator of actin polymerization in hematopoietic cells, has 5 well-defined domains that are involved in signaling, cell locomotion, and immune synapse formation. WASP facilitates the nuclear translocation of nuclear factor kappaB and was shown to play an important role in lymphoid development and in the maturation and function of myeloid monocytic cells. Mutations of WASP are located throughout the gene and either inhibit or dysregulate normal WASP function. Analysis of a large patient population demonstrates a phenotype-genotype correlation: classic WAS occurs when WASP is absent, X-linked thrombocytopenia when mutated WASP is expressed, and X-linked neutropenia when missense mutations occur in the Cdc42-binding site. The progress made in dissecting the function of WASP has provided new diagnostic possibilities and has propelled our therapeutic strategies from conservative symptomatic treatment to curative hematopoietic stem cell transplantation and toward gene therapy.

Is the CD200/CD200 receptor interaction more than just a myeloid cell inhibitory signal?

The membrane glycoprotein CD200, which has a widespread but defined distribution and a structurally similar receptor (CD200R) that transmits an inhibitory signal to cells of the hematopoetic lineage, especially myeloid cells, has been characterized. CD200R expression is restricted predominantly to cells of the myeloid lineage indicating that this ligand/receptor pair has a specific role in controlling myeloid cell function. In addition to CD200R, several related genes have been identified. Whether these gene products also regulate immune function is controversial. CD200R is also expressed by certain subsets of T cells and CD200 may be expressed by antigen-presenting cells, adding additional layers of complexity to the CD200/CD200R axis. Because monocytic myeloid cells provide a link between the innate and adaptive immune response, mechanisms to control their function through receptors such as CD200R will have therapeutic potential. Regulation of immune responses is accomplished by the concerted, but opposing, activity of kinases and phosphatases, fine control often being achieved through paired receptors. In this review, we will consider whether CD200R signaling functions within a framework of paired activating and inhibitory receptors and whether the inhibitory signal delivered has functional consequences beyond inhibition of myeloid cell proinflammatory activation.

Deregulation of homologous recombination DNA repair in alkylating agent-treated stem cell clones: a possible role in the aetiology of chemotherapy-induced leukaemia

Chemotherapeutic regimes involving alkylating agents, such as methylators and crosslinking nitrogen mustards, represent a major risk factor for acute myeloid leukaemia. A high frequency of microsatellite instability and evidence of MSH2 loss in alkylating chemotherapy-related acute myeloid leukaemia (t-AML) suggests that DNA mismatch repair (MMR) dysfunction may be an initiating event in disease evolution. Subsequent accumulation of secondary genetic changes as a result of DNA MMR loss may ultimately lead to the gross chromosomal abnormalities seen in t-AML. Homologous recombination repair (HRR) maintains chromosomal stability by the repair of DNA double-strand breaks, and is therefore a possible target for deregulation in MMR dysfunctional t-AML. In order to test this hypothesis Msh2- proficient and -deficient murine embryonic stem (ES) cells were used to examine the effects of MMR status and methylating agent treatment on cellular expression of DNA double-strand break repair genes. HRR gene expression was significantly deregulated in Msh2 null ES cell clones compared to wild-type clones. Furthermore, some Msh2 null clones expressed high levels of Rad51 specifically, a critical component of HRR. Such Rad51 superexpressing clones were also observed when expression was determined in monocytic myeloid cells differentiated from ES cells. A deregulated HRR phenotype could be partially recapitulated in MMR-competent wild-type cells by treatment with the methylating agent, N-methyl-N-nitrosourea. Furthermore, treatment with melphalan, a leukaemogenic DNA crosslinking chemotherapy nitrogen mustard predicted to elicit HRR, selected against cells with deregulated HRR. These data suggest a t-AML mechanism whereby DNA MMR loss promotes the emergence of HRR gene superexpressing clones, with concomitant chromosomal instability. However, melphalan selection against clones with deregulated HRR suggests that persistence and expansion of unstable clones may require additional genetic alterations that promote cell survival.

Melatonin influences interleukin-12 and nitric oxide production by primary cultures of rheumatoid synovial macrophages and THP-1 cells.

Because some of the clinical symptoms related to rheumatoid arthritis (RA) synovitis, such as joint morning stiffness and gelling, might be related to the effects exerted by the diurnal rhythmicity of the neurohormone melatonin (MLT) on synovial immune cell activation, we decided to evaluate the influence of MLT on the production of IL-12 and nitric oxide (NO) on primary cultures of RA synovial macrophages. Synovial macrophages were also prestimulated with lipopolysaccaride (LPS). Results were compared with those obtained on cultured human myeloid monocytic cells (THP-1). A significant increase in IL-12 (p = 0.01) was found in media of MLT-stimulated synovial macrophages versus RMPI-treated synovial macrophage controls. Interestingly, a significant decrease of IL-12 (p < 0.0001) was observed in media of synovial macrophages previously activated with LPS and then treated with MLT, when compared to synovial macrophages treated with LPS alone. A significant increase in NO levels (p = 0.01) was found in MLT-stimulated synovial macrophages versus RMPI-treated synovial macrophage controls. Interestingly, a nonsignificant increase of NO levels was observed in media of synovial macrophages previously activated with LPS and then treated with MLT, when compared to cynovial macrophages treated with LPS alone. Finally, a significant increase in IL-12 (p = 0.03) and NO (p = 0.002) concentrations was observed in media of MLT-stimulated THP-1 cells versus RMPI-treated controls. Our results therefore show that MLT induces IL-12 secretion and NO production by unstimulated cultured RA synovial macrophages and human monocytic myeloid THP-1 cells. The unexpected and opposite effects on IL-12 and NO production in RA synovial macrophages treated with LPS may be related to dose-dependent mechanisms exerted by MLT or to altered cell priming in RA macrophages; these are matters of our further research. This study strongly supports the role of MLT in immune response modulation and in particular suggests a close relationship between diurnal rhythmicity of neuroendocrine pathways, cytokine and reactive oxygen intermediate production by monocyte/macrophages, and synovial arthritis symptomatology, at least in RA.

NF-κB Protects HIV-1-Infected Myeloid Cells from Apoptosis

HIV-1 infection of primary monocytic cells and myeloid cell lines results in sustained NF-κB activation. Recently, NF-κB induction has been shown to play a role in protecting cells from programmed cell death. In the present study, we sought to investigate whether constitutive NF-κB activity in chronically HIV-1-infected promonocytic U937 (U9-IIIB) and myeloblastic PLB-985 (PLB-IIIB) cells affects apoptotic signaling. TNFα and cycloheximide caused infected cells to undergo apoptosis more rapidly than parental U937 and PLB-985 cells. Inhibition of TNFα-induced NF-κB activation using the antioxidantN-acetylcysteine (NAC) resulted in increased apoptosis in both U937 and U9-IIIB cells, while preactivation of NF-κB with the non-apoptotic inducer IL-1β caused a relative decrease in apoptosis. Inhibition of constitutive NF-κB activity in U9-IIIB and PLB-IIIB cells also induced apoptosis, suggesting that NF-κB protects cells from a persistent apoptotic signal. TNFα plus NAC treatment resulted in a marked decrease in Bcl-2 protein levels in HIV-1-infected cells, coupled with an increase in Bax protein compared to uninfected cells, suggesting that the difference in susceptibility to TNFα-induced apoptosis may relate to the differences in relative levels of Bcl-2 and Bax. The protective role of NF-κB in blocking TNFα- and HIV-1-induced apoptosis was supported by studies in Jurkat T cells engineered to express IκBα repressor mutants (TD-IκB) under the control of a tetracycline-responsive promoter. Cells underwent apoptosis in response to TNFα only when NF-κB activation was inhibited by TD-IκB expression. As was observed for the U9-IIIB cells, TNFα treatment also induced a marked decrease in Bcl-2 protein levels in TD-IκB expressing cells. These experiments demonstrate that apoptotic signaling is perturbed in HIV-1-infected U9-IIIB cells and indicate that NF-κB activation may play an additional protective role against HIV-1-induced apoptosis in myeloid cells.

Productive persistent infection of hematopoietic cells by human foamy virus.

Human foamy virus can establish persistent infections in human hematopoietic cell lines, such as H92.1.7 (erythroblastoid cells), Jurkat (CD4+ T cells), and U937 (myeloid-monocytic cells). The infection is characterized by constant production of infectious viruses (for > 2 1/2 years) with no cytopathic effects on the host cells. Electron microscopy of the infected cells showed a viral morphology similar to that observed for particles produced after acute infection. We have detected, in addition to the full-length form of bel1, a previously described deletion in the bel1 gene of the proviral DNA in these cells. RNA containing this 301-bp deletion, which mapped to the splice donor and acceptor sites of the intron of the bet gene, was also found in encapsidated virion RNA. However, the presence of this defective provirus harboring the deletion in bel1 does not prevent productive persistence in these chronically infected cells, since the virus titer does not decrease during cultivation.

Characterization of Cells of the Myeloid-Monocytic Lineage (ML-1, HL-60, THP-1, U-937) Chronically Infected with the Human Immunodeficiency Virus-1

The myeloid-monocytic cells ML-1, HL-60, THP-1 and U-937 were chronically infected (for more than 2 years) with the lymphotropic HIV-1 strain HTLV-IIIB. Reinfection experiments revealed that viruses obtained from chronically infected ML-1/HIV-1 and HL-60/HIV-1 cells show a low infectivity if tested with uninfected ML-1 and HL-60 cells in contrast to virus preparations from chronically infected THP-1/HIV-1 and U-937/HIV-1 with their corresponding uninfected cell lines. Analyses of selected cell surface markers showed a differential expression of CD4, CD8, CD11c, CD14, CD15, CD20, HLA-DR and HLA-DQ in non- or chronically infected cells. During chronical infection, the myeloid-monocytic cells lost their reactivity with peroxidase and esterase. In chronically infected cells, the steady-state levels for TNF-alpha mRNA remained unchanged while those for IL-6 decreased. The half-lives of transcripts of both TNF-alpha (t1/2: 70 min) and IL-6 (t1/2: 100 min) were nearly the same in uninfected and chronically infected HL-60 cells.

Leu-M1 immunoreactivity in nonhematopoietic neoplasms and myeloproliferative disorders. An immunoperoxidase study of paraffin sections.

Leu-M1 is a differentiation antigen present on human myelomonocytic cells, which also has been identified in Reed-Sternberg cells and variants of Hodgkin's disease. This study further defines the tissue distribution of Leu-M1, with immunoreactivity observed for neoplastic cells in 14 of 28 formalin-fixed, paraffin-embedded nonhematopoietic neoplasms. With the use of monoclonal antibodies and an indirect immunoperoxidase technic, Leu-M1 was detected in adenocarcinomas of various sites (breast, lung, colon, thyroid, pancreas, and stomach), in squamous cell and transitional cell carcinomas, and in a small-cell anaplastic carcinoma. Evaluation of a wide variety of myeloproliferative disorders indicated that Leu-M1 effectively characterized mature and immature monocytic cells and myeloid cells at late stages of granulopoiesis, but it was not a reliable marker for early myeloid cells including blasts. Leu-M1 monoclonal antibodies are a useful diagnostic reagent, particularly in the assessment of lymphoproliferative disorders, but must be used with extreme caution and full awareness of its staining profile.