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D Radzioch

Publications and source records attributed to D Radzioch.

At least 55 records · Page 3Linked to original sources
PubMed1994

Nitrite production by macrophages derived from BCG-resistant and -susceptible congenic mouse strains in response to IFN-gamma and infection with BCG.

Reactive nitrogen intermediates (RNI) have been implicated in the interferon-gamma (IFN-gamma)-induced anti-microbial action of macrophages against a wide variety of pathogens. We have been studying the production of NO2- by macrophage lines derived from the bone marrow of either B10.A (Bcgs) strain mice (B10S cell lines), or their congenic BCG-resistant partners of the B10A.Bcgr (Bcgr) strain (B10R cell lines). We have discovered that there is a significant difference in the production of NO2- of B10S compared with B10R macrophages in response to IFN-gamma. By 48 hr following treatment with 10 U/ml IFN-gamma, B10R macrophages had produced an approximately threefold higher level of NO2- than B10S macrophages. Similar results were obtained when experiments were performed with total splenic cells harvested from the spleens of B10.A.Bcgr and B10.A strain mice. The bacteriostatic activity, as assessed by the [3H]uracil incorporation by Mycobacterium bovis BCG, was higher in B10R macrophages compared to B10S macrophages. The bacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide produced by the macrophages. The anti-mycobacterial activity was inhibited by NgMMLA, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Addition of L-arginine to IFN-gamma-stimulated macrophages in the presence of NgMMLA restored nitrite production and bacteriostatic activity of macrophages. Northern blot analysis of macrophage nitric oxide synthase (iNOS) revealed that the difference in NO2- production by IFN-gamma-treated B10S and B10R lines was reflective of the difference in iNOS mRNA expression.

Animals↗
PubMed1994

Prostaglandins inhibit lipoprotein lipase gene expression in macrophages.

In the present investigation of the effects of prostaglandin E2 (PGE2) on lipoprotein lipase (LPL) gene expression in macrophages, we observed that treatment of macrophages with PGE2 increased the levels of adenosine 3',5'-cyclic monophosphate (cAMP), while the addition of exogenous 5-bromo-cAMP to macrophage cultures resulted in down-regulation of LPL expression. Using indomethacin (INDO), an inhibitor of cyclo-oxygenase and prostaglandins production, we determined that PGE2 acts as a feedback inhibitor of LPL expression. We found that inhibited secretion of LPL protein in lipopolysaccharide (LPS)-treated macrophages could be restored to control levels by the addition of INDO to the medium. In contrast, INDO did not reverse the inhibition of LPL mRNA induced by LPS. Overall, our results have demonstrated that PGE2 is a potent inhibitor of LPL gene expression and indicated that its action may play an important physiological role in the regulation of LPL gene expression during bacterial infections.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

T-helper 1-like subset selection in Mycobacterium bovis bacillus Calmette-Guérin-infected resistant and susceptible mice.

The Bcg gene has been shown to control natural resistance of mice to intravenous infection with low doses of Mycobacterium bovis (bacillus Calmette-Guérin; BCG). In the present study, we evaluated the impact of the Bcg gene on the development of T-cell reactivity during the early stages of infection. Congenic strains of mice, bearing 'r' and 's' alleles of the Bcg gene on B10.A and BALB/c backgrounds, were studied at different time-points for 2 weeks after infection. The in vitro proliferative response of spleen cells, induced by mycobacteria or concanavalin A, was depressed in the Bcgs mice compared to the Bcgr congenic mice 14 days after infection with 10(5) colony-forming units (CFU) of BCG. Polymerase chain reaction (PCR)-based methodology was used to compare the level of lymphokine gene expression in the spleens of infected congenic mice both ex vivo and after in vitro stimulation. In both cases, preferential expression of interferon-gamma (IFN-gamma), lymphotoxin, interleukin-2 (IL-2) and IL-2 receptor genes was observed. The lymphokine gene expression profiles indicated that T lymphocytes activated in the course of the BCG infection preferentially expressed the T-helper 1-specific pattern, irrespective of the allele of the Bcg gene. We showed that this bias in T-cell differentiation could not be attributed to either down-regulation of IL-4 gene expression or modulation of the macrophage co-stimulatory activity by live M. bovis BCG. We conclude that the mechanism of phenotypic expression of the Bcg gene resides in the differential ability of macrophages to be activated by lymphokines produced by protective T cells, rather than in the lack of these lymphokines in susceptible animals.

Animals↗
PubMed1994

Induction of tumor necrosis factor alpha gene expression by lipoprotein lipase.

In view of the suppressive effect of tumor necrosis factor alpha (TNF alpha) on lipoprotein lipase (LPL) and of the potential proatherogenic effects of these two macrophage secretory products, we have tested the possibility that LPL could modulate the production of TNF alpha. Treatment of macrophages with lipoprotein lipase induced tumor necrosis factor alpha gene expression and protein secretion. Maximal increase of TNF alpha mRNA levels occurred after a 3-h treatment with 200 ng/ml LPL. An additive effect of interferon gamma (IFN gamma) was observed on LPL-induced TNF alpha mRNA expression. De novo mRNA synthesis was required for induction of TNF alpha mRNA by LPL as no induction was observed when macrophages were pretreated with actinomycin D before adding LPL. We further established that LPL induced the transcription of the TNF alpha gene in macrophages. We also found that LPL caused the nuclear migration of one member of the NFkB family that appears to bind to a site in the murine TNF alpha gene promoter. Furthermore, we demonstrated that the treatment of macrophages with LPL increased the stability of TNF alpha mRNA. These results show that the TNF alpha gene induction in response to LPL involves both transcriptional activation and the enhancement of TNF alpha mRNA stability. Overall, our data demonstrate a new role for LPL, that of modulating macrophage TNF alpha gene expression. This effect may represent one of the mechanisms by which LPL may favor the development of atherosclerosis.

Animals↗
PubMed1993

Assessment of lymphokine profiles in activated lymphocytes by semiquantitative PCR.

Several methods are currently used to detect the expression of specific mRNAs in leukocytes. While Northern blot analysis and RNase protection assays are commonly chosen for quantitative assessment of mRNA levels, these methods require a significant quantity of RNA, making their use unfeasible when limiting numbers of cells are available. Alternatively, use of the reverse transcription-polymerase chain reaction (RT-PCR) technique allows detection of specific mRNAs even at low copy number. It is, however, difficult to establish the conditions which allow consistent semi-quantitative assessment of specific mRNA expression, using the RT-PCR method. We report here a modification of the RT-PCR technique, which has enabled us to compare lymphokine mRNA expression profiles in mixed cell populations activated either in vivo or in vitro. This modification is based on the use of standard RNAs generated by in vitro transcription of size-modified CD3 and IFN-gamma-specific PCR products subcloned into the pGEM3 plasmid. Equal amounts of standard RNAs are introduced into each sample, reverse transcribed and co-amplified with cellular mRNA to control the reproducibility and efficiency of the method. The template therefore follows the cellular RNA through all steps of the analysis, and the corresponding 32P-labelled PCR products are subsequently separated by PAGE procedure. The amount of radioactivity incorporated into lymphokine-specific bands is determined by densitometry and normalized against the density of standard bands. Under optimal PCR conditions this method is linear over a 50-fold range of dilutions. The technique is specific, reproducible and fast, allowing an analysis of lymphokine-specific mRNA profiles in samples containing 10(4)-10(6) cells.

Animals↗
PubMed1993

Picolinic acid, a catabolite of L-tryptophan, is a costimulus for the induction of reactive nitrogen intermediate production in murine macrophages.

In this study we investigated the effects of picolinic acid, a catabolite of L-tryptophan, on the production of L-arginine-derived reactive nitrogen intermediates in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce nitrite (NO2-) constitutively, but accumulated detectable levels of NO2- on exposure to IFN-gamma. Picolinic acid, although ineffective by itself, augmented IFN-gamma-induced NO2- production. The activity of picolinic acid was evident at 1 mM and reached its maximum at 4 mM. Picolinic acid also augmented the IFN-gamma-dependent expression of TNF-alpha mRNA, but did not appreciably affect the secretion of the TNF-alpha protein. Neutralizing concentrations of anti-TNF mAb completely abrogated IFN-gamma- and IFN-gamma plus rTNF-alpha-induced NO2- production in ANA-1 macrophages, but only decreased by approximately 50% the synergistic interaction between IFN-gamma and picolinic acid. Although IL-4 inhibited the expression of IFN-gamma plus picolinic acid-induced TNF-alpha mRNA and protein, it only partially suppressed picolinic acid-dependent NO2- production. Therefore, picolinic acid may affect NO2- production via both TNF-alpha-dependent and TNF-alpha-independent pathways. Overall, this study suggests that amino acid catabolites may be important for the activation and the expression of effector functions by murine macrophages, and provides the first evidence of a possible connection between tryptophan and arginine metabolism.

Animals↗
PubMed1993

Assessment of mycobacterial infection and multiplication in macrophages by polymerase chain reaction.

The amplification of mycobacterium-specific DNA sequences from samples obtained from infected patients by the polymerase chain reaction (PCR) has been useful in the clinical diagnosis of mycobacterial diseases. Using 20 bp oligonucleotide primers that recognize a 123 bp repeated sequence present in M. bovis and M. tuberculosis DNA, we describe in detail the conditions of the PCR reaction that allow an assessment of the mycobacterial content of infected macrophages. The results of the highly reproducible, time-efficient PCR technique show good correlation with the widely used colony forming unit (CFU) and [3H]uracil incorporation methods for the detection of Mycobacterium. Our method allows an assessment of the level of M. bovis BCG infection from a variety of sources, including peritoneal macrophages and macrophage lines, within a few hours, making it the assay of choice for rapid determination of the level of mycobacterial growth in infected cells, in experimental models of mycobacterial infection.

Animals↗
PubMed1993

Does a genetically determined aggressive locomotion favour the establishment of intracellular macrophage parasites?

A novel computerized methodology was used to quantify, for the first time, the 3-dimensional locomotory phenotypes of individual macrophages moving within an extracellular matrix-like hydrated collagen lattice/gel. Comparisons between two macrophage lines genetically susceptible to intracellular parasites (B10S4 and ANA-1) and two genetically resistant (B10R4 and CD2) demonstrated that Bcgs alleles consistently endowed macrophages with an outward searching and active locomotion, whereas Bcgr imparted a retrocessive phenotype. Several different macrophage pathogens may have exploited this genetically determined aggressive locomotory behavior of susceptible macrophages to allow them to rapidly enter a sanctuary.

Alleles↗
PubMed1993

Dietary n-3 polyunsaturated fatty acids prevent the development of atherosclerotic lesions in mice. Modulation of macrophage secretory activities.

We examined the effects of dietary n-3 polyunsaturated and saturated fatty acids on the development of the atherogenic process in mice and on the macrophage ability to secrete several effector molecules that may be involved in the atherogenic process. The secretion of inflammatory proteins such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) and the production of lipoprotein lipase (LPL), nitrogen oxide (NO2), and prostaglandin E2 (PGE2) were evaluated in peritoneal macrophages isolated from atherosclerosis-susceptible C57BL/6J mice. The mice were assigned at random to three experimental groups: the first group was fed a semi-defined control diet (control diet); the second group was maintained on the control diet supplemented with 10% menhaden oil (menhaden diet); and the third group received the control diet supplemented with 10% palm oil plus 2% cholesterol (saturated fat diet). Macrophages derived from mice fed the menhaden diet showed a suppression of their basal TNF-alpha mRNA expression and production. They also presented a dramatically decreased ability to express TNF-alpha and IL-1 beta mRNAs in response to exposure to lipopolysaccharide (LPS) compared with the macrophages from the control group. LPL mRNA and protein expression were downregulated after 6 and 15 weeks of menhaden-diet feeding. Significantly higher NO2 production in response to interferon gamma was found, both after 6 and 15 weeks of diet feeding, in the menhaden group compared with the control group. In addition, prostaglandin production and macrophage tumoricidal activity in response to LPS were decreased in this group compared with the control group. Macrophages derived from the saturated fat group did not show any significant alterations in TNF-alpha, LPL, NO2, or PGE2 secretion compared with controls. Interestingly, we observed a progressive increase of the LPS-induced IL-1 beta gene expression and secretion among macrophages harvested from mice receiving the dietary supplement of saturated fatty acids. At 6 and 15 weeks histologic examination of the atherosclerotic lesions did not reveal any important lesions in the control and menhaden groups, whereas a gradual development of fatty streaks was observed in the menhaden experimental diets for 10 additional weeks resulted in a major development of lesions in the control group, whereas only slight lesions were observed in the menhaden group.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

High macrophage lipoprotein lipase expression and secretion are associated in inbred murine strains with susceptibility to atherosclerosis.

To test the possibility that variations in macrophage lipoprotein lipase (LPL) secretion may constitute one of the hereditary components of atherosclerosis, we evaluated LPL gene expression and secretion in macrophages harvested from inbred mouse strains differing in their susceptibility to the diet-induced development of atherosclerosis. Inflammatory peritoneal macrophages harvested from atherosclerosis-susceptible C57BL/6J mice showed twofold to threefold higher basal LPL mass, activity, and mRNA levels than those isolated from atherosclerosis-resistant C3H/HeN mice. We determined LPL secretion and gene expression in the susceptible C57BL/6J (B), resistant A/J (A), and A x B/B x A recombinant inbred strains of mice typed as atherosclerosis resistant (A-like) or atherosclerosis susceptible (B-like). Macrophage LPL secretion and mRNA expression were twofold higher in the susceptible C57BL/6J (B) mice than in the resistant A/J (A) mice. Significantly higher LPL secretion, activity, and gene expression were found in recombinant inbred mouse strains that typed B-like than in those typed A-like. These results indicate that susceptibility to atherosclerosis is associated in inbred mouse strains with high LPL secretion and mRNA levels, whereas lower LPL secretion and mRNA expression are observed in atherosclerosis-resistant mice. These observations suggest a contributive role for LPL in the development of atherosclerosis.

Animals↗
PubMed1991

Genetic resistance/susceptibility to mycobacteria: phenotypic expression in bone marrow derived macrophage lines.

Congenic strains of mice susceptible (B10A.Bcgs) or resistant (B10A.Bcgr) to BCG were established. Here we describe the model system which has been established to analyze the functional activities of macrophages in the two strains. We have immortalized bone marrow macrophages from B10A.Bcgs and B10A.Bcgr congenic strains of mice and derived cloned macrophage lines designated B10S and B10R, respectively. B10R and B10S cell lines exhibited surface markers and morphology typical of macrophages. B10S and B10R were similar in their phagocytic activity, in their level of c-fms, in their transforming growth factor beta (TGF beta) mRNAs expression, and in their expression of tumoricidal activity in response to interferon-gamma (IFN gamma) plus lipopolysaccharides (LPS). However, B10R macrophages expressed a higher level of la mRNA when activated with IFN gamma compared with B10S macrophages. Analysis of the bacteriostatic activity of the two cell lines revealed that B10R macrophages were much more active in inhibiting Mycobacterium smegmatis replication than B10S. To measure the intracellular destruction of bacilli, a bactericidal assay based on hybridization with an oligonucleotide probe specific for mycobacterial ribosomal RNA was designed. The results demonstrated that B10R macrophages were endowed with enhanced constitutive bactericidal activity as compared with B10S. In conclusion we have obtained macrophage lines from bone marrow of B10A.Bcgs and B10A.Bcgr mice that express to a similar extent functional and phenotypic characteristics of macrophages. However, we demonstrate that relative to B10S macrophages, the B10R macrophages have higher expression of la mRNA and that they are constitutively more active in expressing mycobactericidal activity.

Animals↗
PubMed1991

Genetic control of innate resistance to mycobacterial infections.

The Mendelian segregation of resistance to infection in different strains of mice infected with mycobacteria, Salmonella and Leishmania spp, all of which live in macrophages, is currently under close scrutiny. Here, Erwin Schurr and colleagues review the nature and function of the Bcg gene in controlling innate resistance to mycobacterial infection in mice and speculate on the occurrence of a possible human equivalent.

Animals↗
PubMed1991

c-fos mRNA expression in macrophages is downregulated by interferon-gamma at the posttranscriptional level.

Treatment of macrophages with interferon-gamma (IFN gamma) strongly decreased the induction of c-fos mRNA by 12-O-tetradecanoylphorbol-13-acetate (TPA), lipopolysaccharide, or calcium ionophore A23187 in macrophages. Under the same experimental conditions, IFN gamma induced oligo(A) synthetase mRNA and did not affect the constitutive expression of transforming growth factor beta mRNA, indicating that IFN gamma did not induce general degradation of mRNAs. Run-on experiments indicated that c-fos was constitutively transcribed at low levels and that TPA augmented c-fos transcription. IFN gamma did not inhibit constitutive or TPA-induced c-fos transcription. However, IFN gamma decreased c-fos mRNA stability, as assessed by measuring the half-life of c-fos mRNA in actinomycin D-treated cells. These results indicated that IFN gamma inhibited c-fos mRNA induction by TPA at the posttranscriptional level.

Animals↗
PubMed1991

Molecular genetics of inherited susceptibility to intracellular parasites.

In the mouse, innate susceptibility to infection with mycobacteria is controlled by the Bcg host resistance locus. Phenotypically, the locus displays two alleles, a dominant resistance and a recessive susceptibility allele. The cell type expressing the resistance locus is the mature tissue macrophage. We have immortalized macrophages from Bcgr and Bcgs mice and found that macrophage cell lines of Bcgr origin, in comparison to those derived from Bcgs mice have an increased microbicidal activity and show increased expression of a number of molecules known to be associated with macrophage activation. This is in agreement with our earlier finding that Bcgr macrophages are at an advanced stage of macrophage priming for activation. The Bcg gene is located on the proximal region of mouse Chromosome 1. We have obtained a detailed genetic map in the vicinity of Bcg and identified three markers within 1 cM of the resistance locus. These three markers have been used for the generation of a physical map of this area of mouse Chromosome 1 and can be used as anchor points for the cloning of the Bcg host resistance gene. The chromosomal segment in the proximity of the Bcg locus in the mouse is precisely conserved on the telomeric region of the long arm of human Chromosome 2. Investigations of linkage of genetic markers present on human Chromosome 2q with susceptibility to tuberculosis have so far generated a LOD score of + 2.4. Linkage of Chromosome 2q markers with susceptibility to mycobacterial disease supports the existence of susceptibility genes in humans and suggests a human homologue of the mouse Bcg gene as a prime candidate for a human innate susceptibility locus.

Animals↗
PubMed1991

Human Harvey-ras is biochemically different from Kirsten- or N-ras.

The biochemical effects of the human H-, N- and K-ras oncogenes were studied. We analysed the induction of c-fos mRNA and protein by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in exponentially growing NIH3T3 fibroblasts transformed by transfection with ras oncogenes. We found that H-ras has the unique ability to inhibit c-fos induction by TPA. In contrast, normal c-fos expression was induced by TPA in fibroblasts transformed by N- or K-ras or by the ras-unrelated oncogenes dbl and trk. The inhibition of c-fos induction by H-ras was not due to alteration in the binding of TPA to the transformed cells or to the selection of idiosyncratic clones. These results provide clear evidence that H-ras is functionally different from K- or N-ras.

Animals↗
PubMed1990

Picolinic acid, a catabolite of tryptophan, as the second signal in the activation of IFN-gamma-primed macrophages.

We have studied the effects of picolinic acid, a product of tryptophan degradation, on the activation of mouse peritoneal macrophages (M phi). Picolinic acid acts synergistically with IFN-gamma in activating M phi from C57BL/6 mice. Moreover, M phi from C3H/HeJ mice and C3H/HeN that do not become cytotoxic in response to IFN-gamma alone could be fully activated by exposure to picolinate plus IFN-gamma. These results indicate that picolinic acid is a potent costimulator of M phi activation that functions as a second signal. Inasmuch as we have previously demonstrated that the activation of cytotoxic M phi correlates with specific changes in ribosomal RNA (rRNA), we investigated whether picolinic acid could modify M phi RNA metabolism. Picolinic acid inhibited the synthesis of total M phi RNA, the accumulation of newly synthesized 28S rRNA, and augmented the steady state levels of rRNA precursors (pre-rRNA). These changes in RNA metabolism were similar to those previously described in murine M phi activated in vitro or in vivo to express tumoricidal activity. These results demonstrate that picolinic acid is a potent, biologic M phi second signal, suggest that the changes in rRNA are causally connected with the expression of tumoricidal activity, and suggest the existance of an autocrine effect mediated by picolinic acid.

Animals↗
PubMed1989

Heterogeneity of hematopoietic cells immortalized by v-myc/v-raf recombinant retrovirus infection of bone marrow or fetal liver.

The J2 recombinant retrovirus expressing v-myc/v-raf (also known as MYC/RAF1) immortalized macrophages from the bone marrow of lipopolysaccharide-responsive mouse strains, producing the ANA-1 cell line from C57BL/6 mice and the INF-3A cell line from C3H/HeN mice. In contrast, J2 recombinant retrovirus infection of the fetal liver from C57BL/6-Ly-5a mice immortalized a cell line (GGD) that did not exhibit the characteristics of mature macrophages. The GGD cell line was classified as leukocytic on the basis of its expression of the Ly-6B.2, Fc gamma R, and Ly-5.2 antigens. Our results indicate that the J2 recombinant retrovirus selectively immortalizes macrophages from the bone marrow of C57BL/6 and C3H/HeN mice but immortalizes cells without definitive macrophage characteristics from murine fetal liver under the same culture conditions.

Animals↗