Primary Bone Marrow Macrophages Research Articles

S100A4 Regulates Macrophage Chemotaxis

S100A4, a member of the S100 family of Ca(2+)-binding proteins, is directly involved in tumor metastasis. In addition to its expression in tumor cells, S100A4 is expressed in normal cells and tissues, including fibroblasts and cells of the immune system. To examine the contribution of S100A4 to normal physiology, we established S100A4-deficient mice by gene targeting. Homozygous S100A4(-/-) mice are fertile, grow normally and exhibit no overt abnormalities; however, the loss of S100A4 results in impaired recruitment of macrophages to sites of inflammation in vivo. Consistent with these observations, primary bone marrow macrophages (BMMs) derived from S100A4(-/-) mice display defects in chemotactic motility in vitro. S100A4(-/-) BMMs form unstable protrusions, overassemble myosin-IIA, and exhibit altered colony-stimulating factor-1 receptor signaling. These studies establish S100A4 as a regulator of physiological macrophage motility and demonstrate that S100A4 mediates macrophage recruitment and chemotaxis in vivo.

Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR Gαi proteins and matrix metalloproteinase-9

Anti-inflammatory activities of thymoquinone (TQ) have been demonstrated in in vitro and in vivo studies. However, the precise mechanism(s) of TQ in these anti-inflammatory activities is not well understood. Using a newly developed assay to detect sialidase activity in live macrophage cells (Glycoconj J doi: 10.1007/s10719-009-9239-8 ), here we show that TQ has no inhibitory effect on endotoxin lipopolysaccharide (LPS) induced sialidase activity in live BMC-2 macrophage cells. In contrast, the parent black seed oil (BSO) and another constituent of BSO para-cymene (p-CY) completely block LPS induced sialidase activity. All of these compounds had no effect on cell viability. On the other hand, TQ induces a vigorous sialidase activity in live BMC-2 macrophage cells in a dose dependent manner as well in live DC-2.4 dendritic cells, HEK-TLR4/MD2, HEK293, SP1 mammary adenocarcinoma cells, human WT and 1140F01 and WG0544 type I sialidosis fibroblast cells. Tamiflu (oseltamivir phosphate) inhibits TQ-induced sialidase activity in live BMC-2 cells with an IC(50) of 0.0194 microM compared to an IC(50) of 19.1 microM for neuraminidase inhibitor DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid). Anti-Neu1, -2 and -3 antibodies have no inhibition of TQ-induced sialidase activity in live BMC-2 and human THP-1 macrophage cells but anti-Neu4 antibodies completely block this activity. There is a vigorous sialidase activity associated with TQ treated live primary bone marrow (BM) macrophage cells derived from WT and hypomorphic cathepsin A mice with a secondary Neu1 deficiency (NeuI KD), but not from Neu4 knockout (Neu4 KO) mice. Pertussis toxin (PTX), a specific inhibitor of Galphai proteins of G-protein coupled receptor (GPCR) and the broad range inhibitors of matrix metalloproteinase (MMP) galardin and piperazine applied to live BMC-2, THP-1 and primary BM macrophage cells completely block TQ-induced sialidase activity. These same inhibitory effects are not observed with the GM1 ganglioside specific cholera toxin subunit B (CTXB) as well as with CTX, tyrosine kinase inhibitor K252a, and the broad range GPCR inhibitor suramin. The specific inhibitor of MMP-9, anti-MMP-9 antibody and anti-Neu4 antibody, but not the specific inhibitor of MMP-3 completely block TQ-induced sialidase activity in live THP-1 cells, which express Neu4 and MMP-9 on the cell surface. Neu4 sialidase activity in cell lysates from TQ-treated live THP-1 cells desialylates natural gangliosides and mucin substrates. RT-PCR and western blot analyses reveal no correlation between mRNA and protein values for Neu3 and Neu4 in human monocytic THP-1 cells, suggesting for the first time a varied post-transcriptional mechanism for these two mammalian sialidases independent of TQ activation. Our findings establish an unprecedented activation of Neu4 sialidase on the cell surface by thymoquinone, which is derived from the nutraceutical black cumin oil. The potentiation of GPCR-signaling by TQ via membrane targeting of Galphai subunit proteins and matrix metalloproteinase-9 activation may be involved in the activation process of Neu4 sialidase on the cell surface.

Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC(50) of 1.2 microM compared to an IC(50) of 1015 microM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFkappaB and the production of nitric oxide and pro-inflammatory IL-6 and TNFalpha cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.

Role and Regulation of CSF-1-Induced CSF-1 Receptor Interchain Disulfide Bonding in Receptor Activation in Macrophages

Colony stimulating factor-1 (CSF-1) is the major regulator of tissue macrophage development and function. The effects of CSF-1 are mediated by the CSF-1 receptor (CSF-1R), a class III receptor tyrosine kinase belonging to the PDGF receptor family. To study CSF-1R structure/function in macrophages using both genetic and proteomic approaches, we developed a novel, CSF-1R-deficient, mouse bone marrow macrophage (BMM) line (MacCsf1r−/−) (Yu et al., J. Leuk. Biol. 84: in press, 2008). MacCsf1r−/− macrophages are maintained in GM-CSF. Retroviral expression of the wild type CSF-1R fully rescued the CSF-1-induced survival, proliferation, differentiation and morphological characteristics, which resemble those of primary BMM. We have studied CSF-1-induced covalent modifications of the receptor in MacCsf1r−/− cells. Activation of the CSF-1R involves ligand-induced receptor dimerization and trans-phosphorylation of the cytoplasmic domains. Besides tyrosine phosphorylation, CSF-1 stimulation results in disulfide bonding of the CSF-1R dimers and cysteine mutagenesis experiments revealed that specific residues in the kinase domain are involved. Inhibition of the disulfide bond formation significantly compromised CSF-1-induced CSF-1R tyrosine phosphorylation, indicating its requirement for full receptor activation. Analysis of the regulation of CSF-1-induced CSF-1R interchain disulfide bonding revealed the participation of third party molecules with additional CSF-1R post-translational modifications. The importance of ligand-induced disulfide bonding for class III receptor tyrosine kinase activation may not be restricted to the CSF-1R as interchain disulfide bonding of PDGF receptor dimers is also ligand-induced.

IL‐23 promotes osteoclast formation by up‐regulation of receptor activator of NF‐κB (RANK) expression in myeloid precursor cells

Inflammation-mediated bone loss is a major feature of various bone diseases including rheumatoid arthritis, osteoarthritis and advanced periodontitis. Enhanced osteoclast development or activity at the inflammation site results in bone resorption. IL-23 is a heterodimeric cytokine belonging to the IL-6/IL-12 family that has been implicated in the pathogenesis of rheumatoid arthritis and demonstrated to play a role in osteoclastogenesis via stimulation of IL-17 production. In this study we investigated whether IL-23 contributes to the regulation of osteoclast differentiation independent of the IL-17 pathway. We show that IL-23 dose-dependently up-regulates receptor activator of NF-kappaB expression in primary murine bone marrow macrophages and RAW264.7 cells and thereby promotes commitment of myeloid precursor cells to receptor activator of NF-kappaB ligand-mediated osteoclastic differentiation. However, IL-23 by itself is insufficient to induce osteoclastogenesis. Increased osteoclastic differentiation of cells was associated with enhanced cathepsin K expression and dentine resorption indicating enhanced formation of functional osteoclasts. IL-17 was not detectable in culture supernatants and when added to cultures, did not promote differentiation of RAW264.7 cells. These results demonstrate that IL-23 can act directly on myeloid precursor cells in addition to indirectly stimulating receptor activator of NF-kappaB ligand production in osteoblasts and explains its potency in driving osteoclast development in inflammation-mediated bone pathology.

Protective Effect of Toll-like Receptor 4 in Pulmonary Vaccinia Infection

Innate immune responses are essential for controlling poxvirus infection. The threat of a bioterrorist attack using Variola major, the smallpox virus, or zoonotic transmission of other poxviruses has renewed interest in understanding interactions between these viruses and their hosts. We recently determined that TLR3 regulates a detrimental innate immune response that enhances replication, morbidity, and mortality in mice in response to vaccinia virus, a model pathogen for studies of poxviruses. To further investigate Toll-like receptor signaling in vaccinia infection, we first focused on TRIF, the only known adapter protein for TLR3. Unexpectedly, bioluminescence imaging showed that mice lacking TRIF are more susceptible to vaccinia infection than wild-type mice. We then focused on TLR4, the other Toll-like receptor that signals through TRIF. Following respiratory infection with vaccinia, mice lacking TLR4 signaling had greater viral replication, hypothermia, and mortality than control animals. The mechanism of TLR4-mediated protection was not due to increased release of proinflammatory cytokines or changes in total numbers of immune cells recruited to the lung. Challenge of primary bone marrow macrophages isolated from TLR4 mutant and control mice suggested that TLR4 recognizes a viral ligand rather than an endogenous ligand. These data establish that TLR4 mediates a protective innate immune response against vaccinia virus, which informs development of new vaccines and therapeutic agents targeted against poxviruses.

CSF-1 receptor structure/function in MacCsf1r–/– macrophages: regulation of proliferation, differentiation, and morphology

CSF-1 is the major regulator of tissue macrophage development and function. A GM-CSF-dependent, CSF-1 receptor (CSF-1R)-deficient F4/80(hi)Mac-1(+)Gr1(-)CD11c(+) bone marrow macrophage (BMM) line (MacCsf1r-/-) was developed to study the roles of the eight intracellular CSF-1R tyrosines phosphorylated upon receptor activation. Retroviral expression of the wild-type CSF-1R rescued the CSF-1-induced survival, proliferation, differentiation, and morphological characteristics of primary BMM. Mutation of all eight tyrosines failed to rescue, whereas the individual Y --> F mutants (544, 559, 697, 706, 721, 807, 921, 974) rescued these CSF-1-inducible phenotypes to varying degrees. The juxtamembrane domain Y559F and activation loop Y807F mutations severely compromised proliferation and differentiation, whereas Y706, Y721F, and Y974F mutations altered morphological responses, and Y706F increased differentiation. Despite their retention of significant in vitro tyrosine kinase activity, Y559F and Y807F mutants exhibited severely impaired in vivo receptor tyrosine phosphorylation, consistent with the existence of cellular mechanisms inhibiting CSF-1R tyrosine phosphorylation that are relieved by phosphorylation of these two sites. The MacCsf1r-/- macrophage line will facilitate genetic and proteomic approaches to CSF-1R structure/function studies in the major disease-related CSF-1R-expressing cell type.

The Ewing Sarcoma Protein (EWS) Binds Directly to the Proximal Elements of the Macrophage-Specific Promoter of the CSF-1 Receptor (csf1r) Gene

Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.

Adsorbed serum albumin is permissive to macrophage attachment to perfluorocarbon polymer surfaces in culture

Monocyte/macrophage adhesion to biomaterials, correlated with foreign body response, occurs through protein-mediated surface interactions. Albumin-selective perfluorocarbon (FC) biomaterials are generally poorly cell-conducive because of insufficient receptor-mediated surface interactions, but macrophages bind to albumin-coated substrates and also preferentially to highly hydrophobic fluorinated surfaces. Bone marrow macrophages (BMMO) and IC-21, RAW 264.7, and J774A.1 monocyte/macrophage cells were cultured on FC surfaces. Protein deposition onto two distinct FC surfaces from complex and single-component solutions was tracked using fluorescence and time-of-flight secondary ion mass spectrometry (ToF-SIMS) methods. Cell adhesion and growth on protein pretreated substrates were compared by light microscopy. Flow cytometry and integrin-directed antibody receptor blocking were used to assess integrins critical for monocyte/macrophage adhesion in vitro. Albumin predominantly adsorbs onto both FC surfaces from 10% serum. In cultures preadsorbed with albumin or serum-dilutions, BMMO responded similar to IC-21 at early time points. Compared with Teflon AF, plasma-polymerized FC was less permissive to extended cell proliferation. The beta(2) integrins play major roles in macrophage adhesion to FC surfaces: antibody blocking significantly disrupted cell adhesion. Albumin-mediated cell adhesion mechanisms to FC surfaces could not be clarified. Primary BMMO and secondary IC-21 macrophages behave similarly on FC surfaces, regardless of preadsorbed protein biasing, with respect to adhesion, cell morphology, motility, and proliferation.

Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase

Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

Co-regulated decrease of Neurokinin-1 receptor and Hemokinin-1 gene expression in monocytes and macrophages after activation with pro-inflammatory cytokines

Hemokinin-1 (HK-1), a potent ligand for the Neurokinin-1 receptor (NK-1) is thought to play a role in the immune system. To investigate the regulation of this receptor–ligand pair, we examined the effects of pro-inflammatory cytokines on their expression in the monocyte/macrophage cell lines Wehi-3 and RAW264.7. We demonstrate co-expression of NK-1 and HK-1 mRNA in both lines, as well as functional NK-1 receptor protein in Wehi-3 cells. Stimulation with IFN-γ, IL-1β and TNF-α markedly decreased NK-1 and HK-1 mRNA as well as NK-1 receptor protein, which coincided with monocytic differentiation. A co-regulated decrease could also be observed in differentiating primary bone marrow macrophages, suggesting that this receptor–ligand pair may be controlled by cytokine networks and may serve a developmental role in the immune system.

Negative Regulation of Osteoclastogenesis by Ectodomain Shedding of Receptor Activator of NF-κB Ligand

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.

FcγR-induced production of superoxide and inflammatory cytokines is differentially regulated by SHIP through its influence on PI3K and/or Ras/Erk pathways

Phagocytosis of IgG-coated particles via FcgammaR is accompanied by the generation of superoxide and inflammatory cytokines, which can cause collateral tissue damage in the absence of regulation. Molecular mechanisms regulating these phagocytosis-associated events are not known. SHIP is an inositol phosphatase that downregulates PI3K-mediated activation events. Here, we have examined the role of SHIP in FcgammaR-induced production of superoxide and inflammatory cytokines. We report that primary SHIP-deficient bone marrow macrophages produce elevated levels of superoxide upon FcgammaR clustering. Analysis of the molecular mechanism revealed that SHIP regulates upstream Rac-GTP binding, an obligatory event for superoxide production. Likewise, SHIP-deficient macrophages displayed enhanced IL-1beta and IL-6 production in response to FcgammaR clustering. Interestingly, whereas IL-6 production required activation of both PI3K and Ras/Erk pathways, IL-1beta production was dependent only on Ras/Erk activation, suggesting that SHIP may also regulate the Ras/Erk pathway in macrophages. Consistently, SHIP-deficient macrophages displayed enhanced activation of Erk upon FcgammaR clustering. Inhibition of Ras/Erk or PI3K suppressed the enhanced production of IL-6 in SHIP-deficient macrophages. In contrast, inhibition of Ras/Erk, but not PI3K, suppressed IL-1beta production in these cells. Together, these data demonstrate that SHIP regulates phagocytosis-associated events through the inhibition of PI3K and Ras/Erk pathways.

CpG DNA enhances macrophage cell spreading by promoting the Src-family kinase-mediated phosphorylation of paxillin

Macrophages are an important component of the innate immune response to infection by microbial pathogens. The activation of macrophages by pathogens is largely mediated by Toll-like receptors (TLRs). Bacterial DNA, which contains unmethylated CpG dinucleotide motifs, is specifically recognised by TLR9 and triggers the activation of a complex network of intracellular signalling pathways that orchestrates the ensuing inflammatory responses of macrophages to the pathogen. Here, we have established that CpG DNA promotes reorganisation of the actin cytoskeleton and enhances cell spreading by primary mouse bone marrow macrophages. CpG DNA stimulation resulted in an approximately 70% increase in cell size. Notably, CpG DNA-induced cell spreading was dependent on the activity of Src-family kinases. Tyrosine phosphorylation of several proteins was increased in a Src-family kinase-dependent manner following CpG DNA stimulation of bone marrow macrophages, including the cytoskeletal protein paxillin. Paxillin was phosphorylated both in vitro and in vivo by the Src-family kinase Hck. Significantly, paxillin from CpG DNA-stimulated bone marrow macrophages had a greater capacity to bind the SH2 domain of the adapter protein Crk than did paxillin from unstimulated bone marrow macrophages. Furthermore, phosphorylation of paxillin by Hck created a binding site for Crk. We propose that the formation of paxillin-Crk complexes may mediate the cytoskeletal changes that underlie the increased cell spreading of macrophages following their activation by CpG DNA.

Chlamydia pneumoniae augments the oxidized low-density lipoprotein-induced death of mouse macrophages by a caspase-independent pathway.

Chlamydia pneumoniae is a common respiratory pathogen that is associated with an increased risk of cardiovascular disease. However, the mechanisms by which C. pneumoniae contributes to cardiovascular disease have not been determined yet. C. pneumoniae infection may accelerate the death of cells within atherosclerotic lesions and contribute to the formation of unstable lesions. To test this hypothesis, the impact of C. pneumoniae infection on the death of lipid-loaded mouse macrophages was investigated. It was observed that RAW 264.7 cells are highly susceptible to the toxic effects of oxidized low-density lipoprotein (LDL) and exhibit markers of cell death within 24 h of treatment with as little as 5 microg/ml oxidized LDL. Subsequent infection with either live C. pneumoniae or heat-killed or UV-inactivated C. pneumoniae at a low multiplicity of infection for 24 to 72 h stimulated both additional binding of annexin V and the uptake of propidium iodide. Thus, C. pneumoniae augments the effects of oxidized LDL on cell death independent of a sustained infection. However, unlike oxidized LDL, C. pneumoniae infection does not activate caspase 3 or induce formation of the mitochondrial transition pore or the fragmentation of DNA, all of which are classical markers of apoptosis. Furthermore, primary bone marrow macrophages isolated from mice deficient in Toll-like receptor 2 (TLR-2) but not TLR-4 are resistant to C. pneumoniae-induced death. These data suggest that C. pneumoniae kills cells by a caspase-independent pathway and that the process is potentially mediated by activation of TLR-2.

Fc- and Complement-Receptor Activation Stimulates Cell Cycle Progression of Macrophage Cells from G1 to S

Phagocytosis of microorganisms by macrophages is an important host defense mechanism. While studying the phagocytosis of the human pathogenic fungus Cryptococcus neoformans, we noted that macrophage-like J774 cells with ingested fungal cells had frequent mitotic figures. By analyzing the relative proportion of phagocytic cells as a function of cell cycle phase, we observed an increase in S phase cells after Fc-mediated phagocytosis of polystyrene beads, live or heat-killed C. neoformans. This result was confirmed by increased nuclear BrdU incorporation after Fc-mediated phagocytosis. The induced progression to S phase was observed after both Fc- and complement-mediated phagocytosis of live yeasts. Fc-mediated stimulation of cell division did not require ingestion, because it could be triggered by incubating cells in IgG1-coated plates. Phagocytosis-mediated stimulation of replication was confirmed in vitro using primary bone marrow macrophages and in vivo for peritoneal macrophages. We conclude that phagocytosis of microbes or inert particles can stimulate macrophages to enter S phase and commence cell division. This observation suggests a potential mechanism for increasing the number of effector cells after microbial ingestion, but can also promote the spread of infection.

AZT enhances osteoclastogenesis and bone loss.

A variety of metabolic complications have been reported to be associated with highly active antiretroviral therapy (HAART), including osteopenia and osteoporosis. In this study, we determine the effects of zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, on osteoclastogenesis in a cultured mouse macrophage preosteoclast cell line (RAW264.7), in mouse primary bone marrow macrophage-monocyte precursors, and on bone mineral density in mice. The results indicate that AZT induces an increase in osteoclastogenesis in the mouse preosteoclast cell line and in mouse bone marrow osteoclast precursors in the presence of RANKL. This increased osteoclastogenesis is dependent upon the concentration of AZT. AZT increases the promoter activity of tartrate-resistant acid phosphatase (TRAP) and the binding and function of the nuclear transcription protein, NF-kappaB, in RAW264.7 cells. Therefore, the effect of AZT is mediated, at least in part, by enhancing RANKL-mediated osteoclastogenesis. Bone mineral density (BMD) in AZT-treated mice is decreased and histopathology shows marked osteopenia. These results support an important role of AZT-stimulated osteoclastogenesis in HAART-induced osteopenia.

Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers

Salmonella live vaccine strains harbouring mutations in htrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro. This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven. We report that the in vitro phenotype of increased susceptibility to oxidative stress of Salmonella typhimurium htrA mutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear. However, despite this change, htrA mutants are still attenuated in normal mice. In contrast, they were found to be lethal for gene targeted gp91 phox −/− mice deficient in NADPH oxidase, as was a S. typhimurium SPI-2 mutant known to be virulent in gp9l phox −/− mice. Infection with htrA mutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9l phox −/− mice, with more than 60% reduction in cell numbers 2.5 h after being infected. The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9l phox −/− mice, whereas an aroA live vaccine strain had no effect on either normal or gp9l phox −/− macrophages. Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.

Involvement of Upstream Stimulatory Factors 1 and 2 in RANKL-induced Transcription of Tartrate-resistant Acid Phosphatase Gene during Osteoclast Differentiation

Tartrate-resistant acid phosphatase (TRAP) plays an important role in bone resorption. TRAP expression in osteoclasts is regulated by receptor activator of NF-kappaB (RANKL), a potent activator of osteoclast differentiation. However, the molecular mechanism underlying the RANKL-induced TRAP expression remains unknown. Here we show that two regions in the mouse TRAP promoter (one at -1858 to -1239 and the other at -1239 to -1039, relative to the translation start site) are implicated in RANKL-induced TRAP transcription in RAW264.7 cells. A detailed characterization of the region at -1239 to -1039 identifies a 12-bp sequence, AGCCACGTGGTG, that specifically binds nuclear proteins from RAW264.7 cells and primary bone marrow macrophages (BMMs) in an electrophoretic mobility shift assay (EMSA). Moreover, the binding is significantly enhanced in EMSA with nuclear extracts from RANKL-treated RAW264.7 cells and BMMs, suggesting that the 12-bp sequence may be involved in RANKL-induced TRAP transcription. Various assays reveal that nuclear proteins binding to the 12-bp sequence are upstream stimulatory factors (USF) 1 and 2. Importantly, mutation of the USF-binding site partially blocks RANKL-induced TRAP transcription in RAW264.7 cells, confirming that USF1 and USF2 are functionally involved in RANKL-induced TRAP transcription. In summary, our data show that USF1 and USF2 play a functional role in RANKL-dependent TRAP expression during osteoclast differentiation.

Differential regulation of the mitogen-activated protein kinases by pathogenic and nonpathogenic mycobacteria.

Mycobacteria are the etiologic agents of numerous diseases which account for significant morbidity and mortality in humans and other animal species. Many mycobacteria are intramacrophage pathogens and therefore the macrophage response to infection, which includes synthesis of cytokines such as tumor necrosis factor alpha (TNF-alpha) and production of nitric oxide, has important consequences for host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection or how pathogenic mycobacteria may modulate the macrophage responses. Using primary murine bone marrow macrophages, we established that p38 and extracellular signal-regulated kinases 1 and 2 of the mitogen-activated protein kinase (MAPK) pathways are activated upon infection with different species of mycobacteria. However, we observed decreased MAPK activity over time in macrophages infected with pathogenic Mycobacterium avium strains relative to infections with nonpathogenic mycobacteria. Furthermore, macrophages infected with M. avium produced lower levels of TNF-alpha, interleukin 1beta, and inducible nitric oxide synthase 2 than macrophages infected with nonpathogenic species. Inhibitor studies indicate that the MAPKs are required for the Mycobacterium-mediated induction of these effector proteins. Our data indicate that MAPKs are activated in macrophages upon invasion by mycobacteria and that this activation is diminished in macrophages infected with pathogenic strains of M. avium, resulting in decreased production of important immune effector proteins. The decreased MAPK activation associated with M. avium infections suggests a novel point of immune intervention by this mycobacterial species.