DNA Synthesis In Macrophages Research Articles

Aberrant mitochondrial DNA synthesis in macrophages exacerbates inflammation and atherosclerosis

There is a large body of evidence that cellular metabolism governs inflammation, and that inflammation contributes to the progression of atherosclerosis. However, whether mitochondrial DNA synthesis affects macrophage function and atherosclerosis pathology is not fully understood. Here we show, by transcriptomic analyzes of plaque macrophages, spatial single cell transcriptomics of atherosclerotic plaques, and functional experiments, that mitochondrial DNA (mtDNA) synthesis in atherosclerotic plaque macrophages are triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice. Mechanistically, VCAM-1 activates C/EBPα, which binds to the promoters of key mitochondrial biogenesis genes - Cmpk2 and Pgc1a. Increased CMPK2 and PGC-1α expression triggers mtDNA synthesis, which activates STING-mediated inflammation. Consistently, atherosclerosis and inflammation are less severe in Apoe−/− mice lacking Vcam1 in macrophages. Downregulation of macrophage-specific VCAM-1 in vivo leads to decreased expression of LYZ1 and FCOR, involved in STING signalling. Finally, VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Collectively, our study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis pathology and proposes a self-acerbating pathway involving increased mtDNA synthesis.

African Swine Fever Virus Manipulates the Cell Cycle of G0-Infected Cells to Access Cellular Nucleotides.

African swine fever virus manipulates the cell cycle of infected G0 cells by inducing its progression via unblocking cells from the G0 to S phase and then arresting them in the G2 phase. DNA synthesis in infected alveolar macrophages starts at 10–12 h post infection. DNA synthesis in the nuclei of G0 cells is preceded by the activation of the viral genes K196R, A240L, E165R, F334L, F778R, and R298L involved in the synthesis of nucleotides and the regulation of the cell cycle. The activation of these genes in actively replicating cells begins later and is less pronounced. The subsequent cell cycle arrest at the G2 phase is also due to the cessation of the synthesis of cellular factors that control the progression of the cell cycle–cyclins. This data describes the manipulation of the cell cycle by the virus to gain access to the nucleotides synthesized by the cell. The genes affecting the cell cycle simply remain disabled until the beginning of cellular DNA synthesis (8–9 hpi). The genes responsible for the synthesis of nucleotides are turned on later in the presence of nucleotides and their transcriptional activity is lower than that during virus replication in an environment without nucleotides.

DNTP pool modulation dynamics by SAMHD1 protein in monocyte-derived macrophages.

BackgroundSAMHD1 degrades deoxyribonucleotides (dNTPs), suppressing viral DNA synthesis in macrophages. Recently, viral protein X (Vpx) of HIV-2/SIVsm was shown to target SAMHD1 for proteosomal degradation and led to elevation of dNTP levels, which in turn accelerated proviral DNA synthesis of lentiviruses in macrophages.ResultsWe investigated both time-dependent and quantitative interplays between SAMHD1 level and dNTP concentrations during multiple exposures of Vpx in macrophages. The following were observed. First, SAMHD1 level was rapidly reduced by Vpx + VLP to undetectable levels by Western blot analysis. Recovery of SAMHD1 was very slow with less than 3% of the normal macrophage level detected at day 6 post Vpx treatment and only ~30% recovered at day 14. Second, dGTP, dCTP and dTTP levels peaked at day 1 post Vpx treatment, whereas dATP peaked at day 2. However, all dNTPs rapidly decreased starting at day 3, while SAMHD1 level was below the level of detection. Third, when Vpx pretreated macrophages were re-exposed to a second Vpx treatment at day 7, we observed dNTP elevation that had faster kinetics than the first Vpx + VLP treatment. Moreover, we performed a short kinetic analysis of the second Vpx treatment to find that dATP and dGTP levels peaked at 8 hours post secondary VLP treatment. dGTP peak was consistently higher than the primary, whereas peak dATP concentration was basically equivalent to the first Vpx + VLP treatment. Lastly, HIV-1 replication kinetics were faster in macrophages treated after the secondary Vpx treatments when compared to the initial single Vpx treatment.ConclusionThis study reveals that a very low level of SAMHD1 sufficiently modulates the normally low dNTP levels in macrophages and proposes potential diverse mechanisms of Vpx-mediated dNTP regulation in macrophages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0063-2) contains supplementary material, which is available to authorized users.

DNTP pool modulation dynamics by SAMHD1 protein in monocyte-derived macrophages

SAMHD1 degrades deoxyribonucleotides (dNTPs), suppressing viral DNA synthesis in macrophages. Recently, viral protein X (Vpx) of HIV-2/SIVsm was shown to target SAMHD1 for proteosomal degradation and led to elevation of dNTP levels, which in turn accelerated proviral DNA synthesis of lentiviruses in macrophages. We investigated both time-dependent and quantitative interplays between SAMHD1 level and dNTP concentrations during multiple exposures of Vpx in macrophages. The following were observed. First, SAMHD1 level was rapidly reduced by Vpx + VLP to undetectable levels by Western blot analysis. Recovery of SAMHD1 was very slow with less than 3% of the normal macrophage level detected at day 6 post Vpx treatment and only ~30% recovered at day 14. Second, dGTP, dCTP and dTTP levels peaked at day 1 post Vpx treatment, whereas dATP peaked at day 2. However, all dNTPs rapidly decreased starting at day 3, while SAMHD1 level was below the level of detection. Third, when Vpx pretreated macrophages were re-exposed to a second Vpx treatment at day 7, we observed dNTP elevation that had faster kinetics than the first Vpx + VLP treatment. Moreover, we performed a short kinetic analysis of the second Vpx treatment to find that dATP and dGTP levels peaked at 8 hours post secondary VLP treatment. dGTP peak was consistently higher than the primary, whereas peak dATP concentration was basically equivalent to the first Vpx + VLP treatment. Lastly, HIV-1 replication kinetics were faster in macrophages treated after the secondary Vpx treatments when compared to the initial single Vpx treatment. This study reveals that a very low level of SAMHD1 sufficiently modulates the normally low dNTP levels in macrophages and proposes potential diverse mechanisms of Vpx-mediated dNTP regulation in macrophages.

Abundant Non-canonical dUTP Found in Primary Human Macrophages Drives Its Frequent Incorporation by HIV-1 Reverse Transcriptase

Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20-40 nm), compared with activated CD4(+) T cells (2-5 μm). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 μm) is ∼60 times higher than TTP in macrophages, whereas the concentrations of dUTP and TTP in dividing human primary lymphocytes are very similar. Specifically, we evaluated the contribution of HIV-1 reverse transcriptase to proviral DNA uracilation under the physiological conditions found in HIV-1 target cells. Indeed, biochemical simulation of HIV-1 reverse transcription demonstrates that HIV-1 RT efficiently incorporates dUTP in the macrophage nucleotide pools but not in the T cell nucleotide pools. Measurement of both pre-steady state and steady state kinetic parameters of dUTP incorporation reveals minimal selectivity of HIV-1 RT for TTP over dUTP, implying that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation. The RT of another lentivirus, simian immunodeficiency virus, also displays efficient dUTP incorporation in the dNTP/dUTP pools found in macrophages but not in T cells. Finally, 2',3'-dideoxyuridine was inhibitory to HIV-1 proviral DNA synthesis in macrophages but not in T cells. The data presented demonstrates that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.

Ribonucleoside Triphosphates as Substrate of Human Immunodeficiency Virus Type 1 Reverse Transcriptase in Human Macrophages

We biochemically simulated HIV-1 DNA polymerization in physiological nucleotide pools found in two HIV-1 target cell types: terminally differentiated/non-dividing macrophages and activated/dividing CD4+ T cells. Quantitative tandem mass spectrometry shows that macrophages harbor 22–320-fold lower dNTP concentrations and a greater disparity between ribonucleoside triphosphate (rNTP) and dNTP concentrations than dividing target cells. A biochemical simulation of HIV-1 reverse transcription revealed that rNTPs are efficiently incorporated into DNA in the macrophage but not in the T cell environment. This implies that HIV-1 incorporates rNTPs during viral replication in macrophages and also predicts that rNTP chain terminators lacking a 3′-OH should inhibit HIV-1 reverse transcription in macrophages. Indeed, 3′-deoxyadenosine inhibits HIV-1 proviral DNA synthesis in human macrophages more efficiently than in CD4+ T cells. This study reveals that the biochemical landscape of HIV-1 replication in macrophages is unique and that ribonucleoside chain terminators may be a new class of anti-HIV-1 agents specifically targeting viral macrophage infection.

MHV68 complement regulatory protein facilitates MHV68 replication in primary macrophages in a complement independent manner

Murine gammaherpesvirus-68 (MHV68) is genetically related to human Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and provides a tractable model to study gammaherpesvirus-host interactions in vivo and in vitro. The MHV68-encoded v-RCA product inhibits murine complement activation and shares sequence homology with other virus and host regulators of complement activation. Here we show that v-RCA is required for efficient MHV68 replication in primary murine macrophages, but not in murine embryonic fibroblasts. v-RCA-deficient MHV68 mutant viruses display defects in viral DNA synthesis in infected macrophages. Importantly, attenuated growth of v-RCA mutant viruses is not rescued in macrophages lacking critical components of the complement system including C3, indicating that the macrophage-specific role of v-RCA in MHV68 replication is complement-independent. This contrasts with the situation in vivo in which attenuated neurovirulence of v-RCA mutant viruses is rescued in C3-deficient mice. This study shows a novel, complement independent cell-type-specific function of a gammaherpesvirus RCA protein.

Ceramide 1-phosphate stimulates macrophage proliferation through activation of the PI3-kinase/PKB, JNK and ERK1/2 pathways

Ceramide 1-phosphate (C1P) was first shown to be mitogenic for fibroblasts, but the mechanisms whereby it stimulated cell proliferation have remained largely unknown. Here we demonstrate that C1P stimulates DNA synthesis and cell division in murine bone marrow-derived macrophages. C1P caused rapid phosphorylation of protein kinase B (PKB, also known as Akt), a downstream target of phosphatidylinositol 3-kinase (PI3-K). Selective inhibition of PI3-K blocked both DNA synthesis and cell growth. C1P induced phosphorylation of GSK-3β, which is a major target of PKB, and this effect was also abolished by inhibition of PI3-K. In addition, C1P upregulated the expression of cyclin D1 and c-Myc, two major targets of GSK-3β, which are important regulators of cell proliferation. C1P stimulated the activity of NF-κB, and inhibitors of this transcription factor completely blocked macrophage proliferation. Lastly, C1P induced phosphorylation of the mitogen activated protein kinases (MAPK) extracellularly regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). Inhibition of ERK1/2 and JNK also blocked C1P-induced macrophage proliferation. It can be concluded that C1P stimulates macrophage proliferation through activation of the PI3-K/PKB, ERK and JNK pathways, and that GSK-3β, c-Myc, cyclin D1, and NF-κB are important downstream effectors in this action.

Alzheimer’s disease amyloid beta and prion protein amyloidogenic peptides promote macrophage survival, DNA synthesis and enhanced proliferative response to CSF-1 (M-CSF)

Microglial cells, macrophage-lineage cells in the brain, are increased in amyloid-containing plaques in Alzheimer’s disease (AD) and in the lesions of prion diseases. Recent studies suggest that microglia have a central role in turnover of amyloid in these diseases. We report here that synthetic amyloid beta (Aβ) 1-42 and prion protein (PrP) 106-126 peptides promote macrophage survival; they also induce macrophage DNA synthesis, particularly in the presence of sub-optimal concentrations of the growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1). These responses are proposed to provide a means to increase brain microglia/macrophage numbers thereby enhancing subsequent inflammatory/immune responses. These fibrillogenic peptides join the list of aggregates having these effects on macrophages, indicating the generality of this type of response.

Cadmium-induced DNA synthesis and cell proliferation in macrophages: The role of intracellular calcium and signal transduction mechanisms

Cd 2+ exposure increases the risk of cancer in humans and animals. In this report, we have studied the effect of Cd 2+ on signal transduction and Ca 2+ mobilization in murine macrophages. At micromolar concentrations, Cd 2+ significantly increased cell division as judged by [ 3H]thymidine uptake and cell counts. Cd 2+-treated cells continued to proliferate even after more than 4 weeks in culture. Cd 2+ (1 μM) treatment induced a 1.5- to 2-fold increase in cytosolic free Ca 2+, [Ca 2+] i, which was transitory and/or oscillatory. The sources of this Ca 2+ included both inositol 1,4,5-trisphosphate (IP 3)-sensitive and -insensitive stores. Macrophage treatment with 1-(6-((17β-3-methoxyestra-1,2,5(10)-triene-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione (U73122), an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), decreased Cd 2+-induced formation of IP 3 in a concentration-dependent manner ( K d∼2 μM). This caused a concomitant, partial decrease in the effect of Cd 2+ on [Ca 2+] i. Cd 2+ itself crosses the macrophage membrane in part via L-type Ca 2+ channels, but it also interacts with a cell surface membrane protein(s) coupled to a pertussis toxin-sensitive G protein. Use of selective inhibitors of signal transduction and the quantitation of the levels of phosphorylated MAPK/ERK-activating kinase-1 (MEK1), extracellular signal-regulated kinase-1 (ERK1), and p38 mitogen-activated protein kinase (MAPK) suggests that the effects of Cd 2+ are mediated by the p21 ras -dependent MAPK, but not the phosphoinositide 3 (PI 3)-kinase signalling pathway. The effect of activating these pathways includes increased availability of the transcription factor NFκB as well as activation of the early genes c- fos and c- myc.

Lipopolysaccharide-induced Apoptosis of Macrophages Determines the Up-regulation of Concentrative Nucleoside Transporters Cnt1 and Cnt2 through Tumor Necrosis Factor-α-dependent and -independent Mechanisms

In murine bone marrow macrophages, lipopolysaccharide (LPS) induces apoptosis through the autocrine production of tumor necrosis factor-alpha (TNF-alpha), as demonstrated by the fact that macrophages from TNF-alpha receptor I knock-out mice did not undergo early apoptosis. In these conditions LPS up-regulated the two concentrative high affinity nucleoside transporters here shown to be expressed in murine bone marrow macrophages, concentrative nucleoside transporter (CNT) 1 and 2, in a rapid manner that is nevertheless consistent with the de novo synthesis of carrier proteins. This effect was not dependent on the presence of macrophage colony-stimulating factor, although LPS blocked the macrophage colony-stimulating factor-mediated up-regulation of the equilibrative nucleoside transport system es. TNF-alpha mimicked the regulatory response of nucleoside transporters triggered by LPS, but macrophages isolated from TNF-alpha receptor I knock-out mice similarly up-regulated nucleoside transport after LPS treatment. Although NO is produced by macrophages after LPS treatment, NO is not involved in these regulatory responses because LPS up-regulated CNT1 and CNT2 transport activity and expression in macrophages from inducible nitric oxide synthase and cationic amino acid transporter (CAT) 2 knock-out mice, both of which lack inducible nitric oxide synthesis. These data indicate that the early proapoptotic responses of macrophages, involving the up-regulation of CNT transporters, follow redundant regulatory pathways in which TNF-alpha-dependent- and -independent mechanisms are involved. These observations also support a role for CNT transporters in determining extracellular nucleoside availability and modulating macrophage apoptosis.

Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL

Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived ‘foam’ cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo.

Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL.

Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived "foam" cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo.

Comparison of macrophage responses to oxidized low-density lipoprotein and macrophage colony-stimulating factor (M-CSF or CSF-1)

Modification of low-density lipoprotein (LDL), for example by oxidation, could be involved in foam cell formation and proliferation observed in atherosclerotic lesions. Macrophage colony-stimulating factor (CSF-1 or M-CSF) has been implicated in foam cell development. It has been reported previously that oxidized LDL (ox.LDL) and CSF-1 synergistically stimulate DNA synthesis in murine bone-marrow-derived macrophages (BMM). The critical signal-transduction cascades responsible for the proliferative response to ox.LDL, as well as their relationship to those mediating CSF-1 action, are unknown. We report here that ox.LDL stimulated extracellular signal-regulated protein kinase (ERK)-1, ERK-2 and phosphoinositide 3-kinase activities in BMM but to a weaker extent than optimal CSF-1 concentrations at the time points examined. Inhibitor studies suggested at least a partial role for these kinases, as well as p70 S6-kinase, in ox.LDL-induced macrophage survival and DNA synthesis. For the DNA synthesis response to CSF-1, the degree of inhibition by PD98059, wortmannin and rapamycin was significant at low CSF-1 concentrations but was reduced as the CSF-1 dose increased. Using BMM from CSF-1-deficient mice (op/op) and a neutralizing antibody approach, we found no evidence for an essential role for endogenous CSF-1 in ox.LDL-mediated survival or DNA synthesis; likewise, with the same approaches, no evidence was obtained for an essential role for endogenous granulocyte/macrophage-CSF in ox.LDL-mediated macrophage survival and, in contrast with the literature, ox.LDL-induced macrophage DNA synthesis.

Inflammatory microcrystals induce murine macrophage survival and DNA synthesis.

The interaction of particulates with resident macrophages is a consistent feature in certain forms of crystal-induced inflammation, for example, in synovial tissues, lung, and the peritoneum. The mitogenic activity of basic calcium phosphate (BCP) crystals and calcium pyrophosphate dihydrate (CPPD) crystals on synovial fibroblasts has been considered relevant to the synovial hyperplasia observed in crystal-induced arthritis. The aim of the study was to determine whether microcrystals such as these could enhance macrophage survival and induce DNA synthesis, thus indicating that they may contribute to the tissue hyperplasia. Murine bone-marrow-derived macrophages were treated in vitro with microcrystals, the cell numbers were monitored over time, and DNA synthesis was measured as the incorporation of [methyl-(3)H]thymidine (TdR). We report here that BCP, monosodium urate, talc, and, to a lesser extent, CPPD crystals promote macrophage survival and DNA synthesis; the latter response is particularly striking in the presence of low concentrations of macrophage-colony stimulating factor (M-CSF, CSF-1). Enhanced macrophage survival or proliferation may contribute to the synovial hyperplasia noted in crystal-associated arthropathies, as well as to talc-induced inflammation and granuloma formation. The crystals studied join the list of particulates having these effects on macrophages, indicating the generality of this type of response.

MRL/lprand MRL+/+ Macrophage DNA Synthesis in the Absence and the Presence of Colony-Stimulating Factor-1 and Granulocyte-Macrophage Colony-Stimulating Factor

AbstractMacrophage accumulation and proliferation as well as altered macrophage properties have been observed in autoimmune MRL mice. To determine whether there might be innate differences in the proliferative responses, we examined the DNA synthesis responses of peritoneal macrophages and macrophages derived in vitro from bone marrow precursors (bone marrow-derived macrophages (BMM)). Murine peritoneal exudate macrophages normally require the addition of macrophage CSF (CSF-1) to enter cell cycle in vitro. In contrast, we have found that many thioglycollate-induced adherent peritoneal macrophages, but not resident peritoneal macrophages, from both MRL/lpr and MRL+/+ mice atypically underwent DNA synthesis even in the absence of added CSF-1. They also responded very well to granulocyte-macrophage CSF. These findings may help to explain the appearance of increased macrophage numbers in MRL lesions. In contrast to a previous report, it was found that MRL/lpr and MRL+/+ BMM did not have an enhanced response to CSF-1 and that modulation of CSF-1 receptor expression was not more rapid in MRL BMM. We also found no evidence for abnormal CSF-1 internalization and degradation or for the lpr mutation to have any enhanced effect on BMM survival in the absence of CSF-1. TNF-α lowered the DNA synthesis response to CSF-1 of MRL/lpr BMM rather than enhanced it, as has been reported. Our data suggest that the enhanced accumulation of macrophages in the MRL/lpr kidney cannot be explained by a proposed model of enhanced responsiveness of MRL/lpr BMM to CSF-1, including a contribution by TNF-α.

Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages

There are differing views regarding the roles of phosphatidylinositol 3-kinases (PI3-kinases) and p70 S6 kinase (p70 s6k) in growth factor-induced cellular responses. One approach that is widely employed to investigate these roles is to use the inhibitors, wortmannin and rapamycin, respectively. This approach is used here to study the responses in macrophages to colony stimulating factor-1 (CSF-1). Wortmannin (≥30 nM) and rapamycin (≥3 nM) both weakly inhibited CSF-1-stimulated DNA synthesis in murine bone marrow-derived macrophages (BMM), suggesting that there are PI3-kinase- and p70 s6k-independent pathways required for the onset of S phase; interestingly the combination of the drugs gave dramatic suppression. Inhibition of DNA synthesis by rapamycin on the BMM was much less than that observed with the CSF-1-dependent cell line, BAC1.2F5. In BMM, wortmannin suppressed CSF-1-stimulated increase in p70 s6k activity indicating that PI3-kinase activity may lie upstream. In contrast to some other growth factor/cell systems, no evidence was obtained using the inhibitors for the involvement of PI3-kinase or p70 s6k in CSF-1-mediated induction of c- fos mRNA expression or Erk-1 activity; in addition, no evidence was found for an involvement in the CSF-1-mediated increase in cyclin D1 expression or STAT activation. The findings reinforce the need to study the signal transduction cascades relevant to each individual growth factor and preferably not in cell lines.

Ligation of the α 2M signaling receptor with receptor-recognized forms of α 2-macroglobulin initiates protein and DNA synthesis in macrophages : The effect of intracellular calcium

We have previously reported that receptor-recognized forms of the proteinase inhibitor α 2-macroglobulin (α 2M *) bind to a distinct receptor (α 2MSR), K d∼50—100 pM, activating a signaling cascade, triggering tyrosine phosphorylation of phospholipase Cγ1, and raising cytosolic pH. We have now studied the effects of α 2M * or a cloned and expressed receptor binding fragment (RBF) on protein and DNA synthesis by macrophages. A nearly linear increase in total protein and DNA synthesis was noted at ligand concentrations up to 100 pM; thereafter, synthesis plateaued. The increase (1.5–2-fold) in protein and DNA synthesis was similar to that observed with known growth factors such as epidermal growth factor and platelet derived growth factor. Mutants of RBF which bind well to α 2MSR, also caused a similar increase in DNA synthesis. By contrast, mutant K1374R which binds poorly to α 2MSR demonstrated much less of an effect on DNA synthesis. Chelation of intracellular Ca 2+ drastically reduced protein and DNA synthesis induced by RBF or the human growth factors. These studies suggest that activation of native α 2M, such as would occur during tissue injury, produces a molecule with properties which are similar to growth factors.

Macrophages from rat livers with micronodular and macronodular cirrhosis differ with respect to mediator release and DNA-synthesis

Background/Aims: Liver macrophages play an essential role in necro-inflammatory liver damage which leads to fibrosis and cirrhosis. The aim of the present study was to compare the mediator release and the DNA synthesis of macrophages at an early and at a later stage of liver cirrhosis induced by thioacetamide. Methods: Liver macrophages were isolated by an enzymic digestion method, followed by elutriation. The release of reactive oxygen species and cytokines, and the synthesis of DNA were measured in cultivated cells. Results: The vitality of isolated macrophages from cirrhotic livers was always higher than 98%. The total yield of macrophages was less in micronodular cirrhotic livers and was markedly higher in macronodular cirrhotic livers when compared with age-matched controls. The cellular granules measured by sideward light scattering showed a shift to larger sizes in macrophages from micronodular cirrhotic livers when compared with the controls and the other experimental group. Macrophages from both cirrhosis groups exhibited a markedly higher unstimulated and lipopolysaccharide-stimulated IL-6 production than the controls. The release of TNF- a did not differ between controls and the experimental groups. Macrophages from macronodular cirrhotic livers produced higher amounts of nitric oxide but less superoxide anion radicals than the controls. DNA synthesis was 10–12-fold and 3–10-fold higher in macrophages from micronodular and macronodular cirrhotic livers, respectively, when compared with the age-matched controls. Conclusions: The data presented provide evidence that it is possible to isolate and to cultivate macrophages from livers with high yield and vitality at different stages of cirrhogenesis. Our results clearly demonstrate functional differences between macrophages from livers with micro- or macronodular cirrhosis; this finding may be important for the pathogenesis or perpetuation of the cirrhogenic process.

Endogenous IFN-alpha beta suppresses colony-stimulating factor (CSF)-1-stimulated macrophage DNA synthesis and mediates inhibitory effects of lipopolysaccharide and TNF-alpha.

Murine bone marrow-derived macrophages (BMM) are widely used as a suitable model to study the proliferative response to macrophage-CSF or CSF-1. We report here that the amount of DNA synthesis observed in BMM cultures in response to CSF-1 can be masked quite significantly by low levels of IFN-alpha beta produced in the cultures. It was found that Ab to IFN-alpha beta could enhance the proliferative response in CSF-treated BMM that were able to respond to endogenous IFN-alpha beta; however, BMM from mice lacking a component of the type I IFN receptor did not show any enhancement of CSF-1-dependent DNA synthesis on addition of the Ab. While DNA synthesis in CSF-1-stimulated BMM from normal mice was also very sensitive to the inhibitory actions of very low concentrations of added IFN-alpha beta, DNA synthesis in BMM from the "knockout" mice was not, indicating that the type I IFN receptor component containing the null mutation was essential for signal transduction. Previously it was shown that bacterial LPS, TNF-alpha, IFN-gamma, and cAMP could all inhibit CSF-1-stimulated BMM DNA synthesis and proliferation. Using the combined approach of blocking IFN-alpha beta Ab and the IFN receptor "knockout" mice, it was found here that the growth-inhibitory effects of LPS and TNF-alpha are due, to a significant extent, to endogenous IFN-alpha beta, whereas those of IFN-gamma and cAMP occur by a different mechanism. it is proposed that the type I IFN receptor (IFNAR 1) "knockout" mice may be useful in delineating some of the in vivo actions of CSF-1, LPS, TNF-alpha, and possibly other agents.