Differentiation of M1 myeloid precursor cells into macrophages results in binding and infection by Theiler's murine encephalomyelitis virus and apoptosis.
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Infection of susceptible mouse strains with BeAn, a less virulent strain of Theiler's murine encephalomyelitis virus (TMEV), results in immune system-mediated demyelinating lesions in the central nervous system (CNS) similar to those in multiple sclerosis. Since macrophages appear to carry the major detectable antigen burden in vivo, and purification of sufficient cell numbers from the CNS for detailed analysis is difficult, macrophage-like cell lines provide an accessible system with which to study virus-macrophage interactions. The myeloid precursor cell line M1 differentiates in response to cytokines and expresses many characteristics of tissue macrophages. Incubation of TMEV with undifferentiated M1 cells produced neither infection nor apoptosis, whereas differentiated M1 (M1-D) cells developed a restricted virus infection and changes indicative of apoptosis. Virus binding and RNA replication as well as cellular production of alpha/beta interferons increased with differentiation. Although the amount of infectious virus was highly restricted, BeAn-infected M1-D cells synthesized and appropriately processed virus capsid proteins at levels comparable to those for permissive BHK-21 cells. Analysis of Bcl-2 protein family expression in undifferentiated and differentiated cells suggests that susceptibility of M1-D cells to apoptosis may be controlled, in part, by expression of the proapoptotic alpha isoform of Bax and Bak. These data suggest that macrophage differentiation plays a role in susceptibility to TMEV infection and apoptosis. (+info)
The Salmonella typhi melittin resistance gene pqaB affects intracellular growth in PMA-differentiated U937 cells, polymyxin B resistance and lipopolysaccharide.
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Salmonella typhi is the causative agent of typhoid fever in humans. A cell-culture based assay involving the human monocyte macrophage cell line U937 has been developed to examine S. typhi invasion and survival. An S. typhi PhoP- (null) mutant was shown to be restricted in net growth in phorbol myristate acetate (PMA) differentiated U937 (PMA-U937) cells, and an S. typhi PhoPc (constitutive) mutant showed a defect in invasion. Neither of the phoP/Q mutants were growth impaired in HeLa cells, however the PhoPc mutant was impaired in invasion. As opposed to what was found for S. typhi, Salmonella typhimurium wild-type, PhoP- and PhoPc mutants grew equally well in PMA-U937 cells, indicating that the PhoP(-)-mediated net growth restriction in the PMA-U937 cells was S. typhi specific. An S. typhi mutation, pqaB::MudJ, recently shown to be a PhoP-activated locus, was shown to have a net growth defect in PMA-U937 cells. Sequencing of the S. typhipqaB gene revealed it had 98% identity to the fifth gene in a S. typhimurium PmrA/B regulated operon necessary for 4-aminoarabinose lipid A modification and polymyxin B resistance. The pqaB locus was regulated by PmrA/B (whose activity is modulated by PhoP-PhoQ) and the pqaB transposon mutant was sensitive to polymyxin B. The lipopolysaccharides (LPS) of S. typhi and S. typhimurium wild-type, PhoP- and PhoPc mutants, were compared by SDS-PAGE and silver staining. Differences in the LPS profile between the two Salmonella species were observed, and shown to be affected differently by the PhoPc mutation. Additionally, the pqaB::MudJ mutation affected S. typhi LPS. The effects on LPS may have ramifications for the difference between S. typhi and S. typhimurium infection of hosts. (+info)
Retinoids inhibit interleukin-12 production in macrophages through physical associations of retinoid X receptor and NFkappaB.
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Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that retinoids inhibit this LPS-stimulated production of IL-12 in a dose-dependent manner. The NFkappaB components p50 and p65 bound retinoid X receptor (RXR) in a ligand-independent manner in vitro, and the interaction interfaces involved the p50 residues 1-245, the p65 residues 194-441, and the N-terminal A/B/C domains of RXR. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the kappaB site, which significantly decreased upon addition of retinoids, as demonstrated by the electrophoretic mobility shift assays. In cotransfections of CV-1 and HeLa cells, RXR also inhibited the NFkappaB transactivation in a ligand-dependent manner, whereas a mutant RXR lacking the AF2 transactivation domain, which serves as ligand-dependent binding sites for transcription integrators SRC-1 and p300, was without any effect. In addition, coexpression of increasing amounts of SRC-1 or p300 relieved the retinoid-mediated inhibition of the NFkappaB transactivation. From these results, we propose that retinoid-mediated suppression of the IL-12 production from LPS-activated macrophages may involve both inhibition of the NFkappaB-DNA interactions and competitive recruitment of transcription integrators between NFkappaB and RXR. (+info)
Essential roles of retinoic acid signaling in interdigital apoptosis and control of BMP-7 expression in mouse autopods.
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We previously reported that mice lacking the RARgamma gene and one or both alleles of the RARbeta gene (i.e., RARbeta+/-/RARgamma-/- and RARbeta-/-/RARgamma-/- mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme (Ghyselinck et al., 1997, Int. J. Dev. Biol. 41, 425-447). In the present study, these compound mutants were used to investigate the cellular and molecular mechanisms involved in retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease was not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues which normally attract these cells into the INZs were not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis was also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bax, and p53. In contrast, the mutant INZs displayed a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RARbeta gene from the RARgamma null genetic background. As retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes, we propose that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression in the INZs. Approximately 10% of the RARbeta-/- /RARgamma-/- mutants displayed a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype (Dudley et al., 1995, Genes Dev. 9, 2795-2807; Luo et al., 1995, Genes Dev. 9, 2808-2820). BMP-7 was globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression. (+info)
Tenascin-C is expressed in macrophage-rich human coronary atherosclerotic plaque.
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BACKGROUND: Tenascin is a large extracellular matrix glycoprotein generally found in adult tissues undergoing active remodeling such as healing wounds and tumors. To determine the potential role of tenascin-C (TN-C) in the pathophysiology of atherosclerosis, we investigated the pattern of expression of TN-C in human coronary atherosclerotic plaques. METHODS AND RESULTS: Immunohistochemical staining and in situ hybridization demonstrated minimal and random expression of TN-C in fibrotic but lipid-poor atherosclerotic plaques. In contrast, all plaques with an organized lipid core or ruptured intimal surface strongly expressed TN-C, which was preferentially concentrated around the lipid core, shoulder regions, and ruptured area of the plaques but not in the fibrous cap. TN-C was not detected in normal arterial tissue. To identify the cellular source of TN-C, the plaques were stained with smooth muscle cell- and macrophage-specific antibodies. TN-C expression correlated with the infiltration of macrophages. Northern blot and immunoprecipitation analysis showed that macrophages expressed 7. 0-kb TN-C mRNA and 220-kDa protein. Reverse transcription-polymerase chain reaction of total RNA derived from macrophages showed that they express the small isoform of TN-C. Zymogram analysis revealed that macrophages markedly increased MMP-9 expression. CONCLUSIONS: This study demonstrates that the level of TN-C expression correlates with the degree of inflammation present, not with plaque size. In addition, cultured macrophages have the capacity to express the TN-C gene. These findings suggest the significance of macrophages in the remodeling of atherosclerotic plaque matrix composition. (+info)
Reduced tumor necrosis factor-alpha and transforming growth factor-beta1 expression in the lungs of inbred mice that fail to develop fibroproliferative lesions consequent to asbestos exposure.
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Tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta mRNA and protein expression and the degree of fibroproliferative response to inhaled asbestos fibers are clearly reduced in the 129 inbred mouse strain as compared with typical fibrogenesis observed in the C57BL/6 inbred strain. The C57BL/6 mice showed prominent lesions at bronchiolar-alveolar duct (BAD) junctions where asbestos fibers deposit and responding macrophages accumulate. The 129 mice, however, were generally indistinguishable from controls even though the numbers of asbestos fibers deposited in the lungs of all exposed animals were the same. Quantitative morphometry of H&E-stained lung sections comparing the C57BL/6 and 129 mice showed significantly less mean cross-sectional area of the BAD junctions in the 129 animals, apparent at both 48 hours and 4 weeks after exposure. In addition, fewer macrophages had accumulated at these sites in the 129 mice. Nuclear bromodeoxyuridine immunostaining demonstrated that the number of proliferating cells at first alveolar duct bifurcations and in adjacent terminal bronchioles was significantly reduced in the 129 strain compared with C57BL/6 mice at 48 hours after exposure (P < 0.01). TNF-alpha and TGF-beta1 gene expression, as measured by in situ hybridization, was reduced in the 129 mice at 48 hours after exposure, and expression of TNF-alpha and TGF-beta1 protein, as measured by immunohistochemistry, was similarly reduced or absent in the 129 animals. We postulate that the protection afforded the 129 mice is related to reduction of growth factor expression by the bronchiolar-alveolar epithelium and lung macrophages. (+info)
Echocardiography-derived left ventricular end-systolic regional wall stress and matrix remodeling after experimental myocardial infarction.
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OBJECTIVES: We tested the hypothesis that regional end-systolic left ventricular (ESLV) wall stress is associated with extracellular matrix remodeling activity after myocardial infarction (MI). BACKGROUND: Increased left ventricular (LV) wall stress is a stimulus for LV enlargement, and echocardiography can be used to estimate regional wall stress. A powerful validation of a noninvasive method of estimating wall stress would be predicting cellular responses after a MI. METHODS: Echocardiographic images were obtained in rats 1, 7, 14 or 21 days after coronary ligation (n = 11) or sham surgery (n = 5). End-systolic left ventricular wall stress was calculated by finite element analysis in three regions (infarcted, noninfarcted and border) from short-axis images. Matrix metalloproteinase-9 (MMP-9) and macrophage density were determined by immunohistochemistry, and positive cells were counted in high power fields (hpf). RESULTS: Average ESLV wall stress was higher in rats with MI when compared to shams irrespective of time point (p < 0.01), and ESLV wall stress in the infarcted regions increased with time (25.1 +/- 5.9 vs. 69.9 +/- 4.4 kdyn/cm2, day 1 vs. 21; p < 0.01). Matrix metalloproteinase-9 expression was higher in infarcted and border regions when compared to noninfarcted regions (22.1 vs. 25.7 vs. 0.10 cells/hpf, respectively; p < 0.01). Over all regions, ESLV wall stress was associated with MMP-9 (r = 0.76; p < 0.001), macrophage density (r = 0.72; p < 0.001) and collagen content (r = 0.67; p < 0.001). End-systolic left ventricular wall stress was significantly higher when MMP-9 positive cell density was greater than 10 cells/hpf (45+/-20 vs. 14+/-10 kdyn/cm2; p < 0.001). CONCLUSIONS: Regional increases in ESLV wall stress determined by echocardiography-based structural analysis are associated with extracellular matrix degradation activity. (+info)
Regulation of arachidonic acid release and cytosolic phospholipase A2 activation.
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The 85-kDa cytosolic PLA2 (cPLA2) mediates agonist-induced arachidonic acid release in many cell models, including mouse peritoneal macrophages. cPLA2 is regulated by an increase in intracellular calcium, which binds to an amino-terminal C2 domain and induces its translocation to the nuclear envelope and endoplasmic reticulum. Phosphorylation of cPLA2 on S505 by mitogen-activated protein kinases (MAPK) also contributes to activation. In macrophages, zymosan induces a transient increase in intracellular calcium and activation of MAPK, which together fully activate cPLA2 and synergistically promote arachidonic acid release. There are alternative pathways for regulating cPLA2 in macrophages because PMA and okadaic acid induce arachidonic acid release without increasing calcium. The baculovirus expression system is a useful model to study cPLA2 activation. Sf9 cells expressing cPLA2 release arachidonic acid to either A23187 or okadaic acid. cPLA2 is phosphorylated on multiple sites in Sf9 cells, and phosphorylation of S727 is preferentially induced by okadaic acid. However, the phosphorylation sites are non-essential and only S505 phosphorylation partially contributes to cPLA2 activation in this model. Although okadaic acid does not increase intracellular calcium in Sf9 cells, calcium binding by the C2 domain is necessary for arachidonic acid release. A23187 and okadaic acid activate cPLA2 by different mechanisms, yet both induce translocation to the nuclear envelope in Sf9 cells. The results demonstrate that alternative regulatory pathways can lead to cPLA2 activation and arachidonic acid release. (+info)