PubMed Health⌕ Search

Biomedical subjects

K Nixdorff

Publications and source records attributed to K Nixdorff.

16 recordsLinked to original sources
PubMed2001

Negative regulation of IL-1beta production at the level of transcription in macrophages stimulated with LPS.

The IL-1beta gene is rapidly and transiently expressed in LPS-stimulated macrophages. While several studies have addressed the molecular basis of LPS-induced transcriptional activity, the mechanisms which underlie the subsequent decrease in IL-1beta gene expression have not been as extensively examined. In this regard, we found that the characteristic decrease in IL-1beta production after LPS stimulation could be abrogated by treatment of macrophages with the protein kinase inhibitor staurosporine. This inhibitor mediated an enhancement of IL-1beta production which was first evident 8-12 h after LPS stimulation and continued at peak levels for the rest of the incubation period (24 h). IL-1beta production was correlated with the level of mRNA specific for the cytokine. Staurosporine also mediated an enhancement of LPS-induced IL-1beta promoter activity measured in RAW 264.7 cells transiently transfected with an IL-1beta reporter plasmid. This increase paralleled the enhancement of IL-1beta mRNA by staurosporine both in intensity and time after LPS stimulation, suggesting that the negative regulation of IL-1beta is exerted primarily at the level of transcription. This regulation may be at least partially due to an observed inhibition of nitric oxide production by staurosporine in LPS-activated macrophages, which was correlated with enhanced IL-1beta production. However, the intensity of the observed effects suggested that additional staurosporine-sensitive regulatory mechanisms are in operation at the level of promoter activity.

Animals↗
PubMed1995

Enhancement of uptake of lipopolysaccharide in macrophages by the major outer membrane protein OmpA of gram-negative bacteria.

Monoclonal antibodies (MAb) to lipopolysaccharide (LPS) and to the major outer membrane protein OmpA from Proteus mirabilis were generated and used to monitor the kinetics of uptake in macrophages of LPS as well as LPS bound to OmpA. Uptake was measured by a modified enzyme-linked immunosorbent assay (ELISA) in a microtiter culture system. The MAb were of various immunoglobulin G subclasses and showed strong reactivities with their antigens. Four hybridoma clones recognizing LPS and three recognizing OmpA from P. mirabilis 19 were selected for the present study on the basis of reactions in ELISA and Western blot (immunoblot) analyses. In the uptake assay, it was possible to differentiate between antigen on the cell surface and antigen which had been internalized. Uptake of LPS by macrophages was relatively rapid during the first 4 h of culture and then progressed more slowly over the remaining 24-h observation period. The level of detection of LPS in this assay system was in the nanogram range. When macrophages were pulsed with LPS for 30 min and subsequently washed to remove antigen not bound to the cells, the amount of LPS detectable on the macrophage surface decreased progressively for 3 h after the pulse, which indicated internalization of the antigen. Thereafter, LPS rose to an increased level on the cell surface. The rate of uptake of LPS was more rapid when it was in complex with OmpA. When the fate of OmpA was monitored in the same LPS-protein complexes by use of MAb to OmpA in a pulse experiment, the level of protein measured on the cell surface decreased after an initial rise, which again indicated internalization, but the protein did not reappear on the cell surface in a form detectable with the MAb. Compared with the LPS monitoring system, detection of OmpA associated with macrophages was weak, although the MAb to OmpA reacted strongly with the protein in the ELISA and Western blot analyses.

Acute-Phase Proteins↗
PubMed1993

Differential modulation of the effects of lipopolysaccharide on macrophages by a major outer membrane protein of Proteus mirabilis.

We previously showed that a major protein isolated from purified cell walls of Proteus mirabilis (39-kDa protein) is a strong modulator of the specific immune responses to LPS from this bacterium in mice. When mixed with LPS before immunization, this protein enhances T cell-dependent, IgG antibody-producing cell responses specific for LPS. Furthermore, complexes of the 39-kDa protein with LPS drastically inhibit the production of oxygen radicals by murine macrophages activated with LPS, as measured in a chemiluminescence assay. In the present report, we have further investigated possible modulating effects of the protein at the level of LPS-macrophage interaction. When mixed with LPS, the 39-kDa protein inhibited IL-1 production by murine macrophages derived from bone marrow in a dose-dependent manner, as determined in an IL-2-dependent IL-1 assay. On the other hand, the protein had little effect on LPS-mediated suppression of MHC class II expression on the surface of macrophages induced with IFN-gamma. Some abrogation of suppression was observed, but the amounts of protein needed for this effect were quite large, in comparison with the amounts rendering inhibition of IL-1 production. In contrast, the 39-kDa protein enhanced the LPS-induced cytotoxicity of macrophages against L929 target cells, primarily as the result of production of TNF. These results show that the 39-kDa protein is a potent modulator of the interaction of LPS with macrophages, exerting its effects in a differential manner with respect to various parameters of LPS-induced activation of macrophages.

Animals↗
PubMed1993

The 39-kilodalton outer membrane protein of Proteus mirabilis is an OmpA protein and mitogen for murine B lymphocytes.

Partial amino acid sequence analysis of a major outer membrane protein of Proteus mirabilis (39-kDa protein) indicates that it is an OmpA protein. The mitogenic activities of the 39-kDa protein for murine lymphocytes were also investigated with T lymphocytes isolated by passing spleen cells over columns of nylon wool fiber and B lymphocytes obtained by treating spleen cells with monoclonal antibodies to Thy1 plus complement. The 39-kDa protein showed little activity in stimulating T cells to proliferate but was strongly mitogenic for B cells.

Amino Acid Sequence↗
PubMed1992

Modulation of effects of lipopolysaccharide on macrophages by a major outer membrane protein of Proteus mirabilis as measured in a chemiluminescence assay.

Our previous studies have shown that a major protein isolated from purified cell walls of Proteus mirabilis (39-kDa protein) is a strong modulator of the specific immune responses to lipopolysaccharide (LPS) from this bacterium. When the protein is mixed with LPS before immunization of mice, the responses of antibody-producing cells specific for LPS are greatly enhanced and converted predominantly to the immunoglobulin G isotype. In the present study, the immunomodulating effects of the 39-kDa protein were tested at the level of interaction of LPS with macrophages. Activation of macrophages was determined by measuring the production of oxygen radicals in a chemiluminescence assay with lucigenin as the amplifier. LPS from P. mirabilis induced strong oxidative metabolism in both peritoneal and bone marrow-derived murine macrophages. These responses were inhibited in a dose-dependent manner by mixing LPS with increasing amounts of the protein. In contrast, bovine serum albumin and methylated bovine serum albumin enhanced the response of macrophages dramatically when complexed with LPS. The inhibiting activity of the 39-kDa protein was also observed with LPS from Escherichia coli K-12.

Animals↗
PubMed1991

Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness.

A serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by beta-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.

Anti-Bacterial Agents↗
PubMed1983

Alteration of the immunoglobulin G subclass responses in mice to lipopolysaccharide: effects of nonbacterial proteins and bacterial membrane phospholipids or outer membrane proteins of Proteus mirabilis.

The immunoglobulin M (IgM) and the IgG1, IgG2ab, and IgG3 subclasses of plaque-forming cells (PFC) specific for lipopolysaccharide (LPS) were measured after immunization of mice with LPS alone and compared with the responses to LPS in combination with nonbacterial proteins and with bacterial membrane phospholipid vesicles or two major outer membrane proteins from Proteus mirabilis. The relative numbers of IgG PFC belonging to the IgG1, IgG2, or IgG3 subclasses induced by immunization with LPS alone depended upon the type of LPS administered. Phospholipids and the proteins effected characteristic alterations in not only the strength but also the subclass of the IgG responses to LPS. The results suggest that the hydrophobic-hydrophilic nature or state of aggregation of the preparations plays a role in the induction of IgG1 and IgG2 subclasses of PFC specific for LPS. Complex formation with LPS and adjuvant was apparently necessary to obtain these effects.

Adjuvants, Immunologic↗
PubMed1983

Modulation of the IgG subclass responses to lipopolysaccharide by bacterial membrane components: differential adjuvant effects produced by primary and secondary stimulation.

The characteristic modulating effects of bacterial membrane adjuvants on the IgG subclass responses to lipopolysaccharide (LPS) were further investigated. Previous studies indicated that more hydrophobic adjuvants preferentially augmented the number of LPS-specific IgG1 plaque-forming cells (PFC) whereas more hydrophilic adjuvants induced predominantly IgG2 PFC, and that complex formation between LPS and adjuvants was necessary for this modulation. In the present report, no carrier effect of adjuvants in the classic immunologic sense could be detected. Undiminished secondary IgG responses to LPS were obtained regardless of which adjuvant was used for the secondary injection. When IgG subclass responses were examined, however, differential effects of adjuvants produced by primary and secondary injections were observed. The adjuvant used in combination with LPS for the primary stimulus determined the number and subclass of LPS-specific IgG memory cells induced: The same or a different adjuvant used with LPS for the secondary stimulus effected the induction of these memory cells to antibody-producing cells. The pattern set by the primary stimulus was not altered by the secondary stimulus, regardless of the potential modulating effect of the second adjuvant.

Adjuvants, Immunologic↗
PubMed1981

Antibody-producing cell responses to an isolated outer membrane protein and to complexes of this antigen with lipopolysaccharide or with vesicles of phospholipids from Proteus mirabilis.

Antibody-producing cell responses of mice to a protein isolated from the outer membrane of Proteus mirabilis were typical of the responses to a thymus-dependent antigen. The immunoglobulin G antibody-producing cell responses to the protein were increased after administration of the antigen complexed with either lipopolysaccharide or with vesicles of phospholipids extracted from P. mirabilis. The protein in turn significantly increased the immune response to lipopolysaccharide and also converted this response from predominantly immunoglobulin M to predominantly immunoglobulin G.

Animals↗
PubMed1980

Immunological characterization of two major proteins isolated from the outer membrane of Proteus mirabilis.

Two proteins with apparent molecular weights of 39,000 and 36,000 (M(r) 39,000 and M(r) 36,000, respectively) were isolated from the outer membrane of Proteus mirabilis 19. M(r) 36,000 was shown to be free of detectable amounts of the M(r) 39,000 protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and free of lipopolysaccharide according to gas chromatographic analyses of 3-hydroxymyristic acid content. The M(r) 39,000 protein contained no detectable amount of lipopolysaccharide and only a trace of M(r) 36,000. Both isolated proteins gave strong reactions in antisera produced to purified P. mirabilis 19 cell walls (outer membrane proteins in the native state). This suggested that the proteins isolated by our methods essentially retained their native configuration upon resolubilization. Antisera produced in rabbits to the isolated proteins showed strongest reactions with the homologous antigen, but some cross-reactions with the heterologous protein and with P. mirabilis 19 lipopolysaccharide were observed. These cross-reactions could be attributed to specific responses to traces of the heterologous (contaminant) proteins present in the purified proteins used as immunizing antigens. The M(r) 39,000 and M(r) 36,000 proteins have no major antigenic determinants in common. Reactions with P. mirabilis 19 lipopolysaccharide in antisera to the outer membrane proteins could be completely removed by absorption of the antisera with the M(r) 36,000 protein.

Antigens, Bacterial↗
PubMed1980

Qualitative and quantitative changes in the antibody producing cell response to lipopolysaccharide induced after incorporation of the antigen into bacterial membrane phospholipid vesicles.

Lipopolysaccharide (LPS) isolated from Proteus mirabilis and administered to mice i.p. induced a primary immune response that consisted of the proliferation of only IgM antibody-producing cells. The response to a second stimulus 14 days later was also predominantly IgM, although a smaller number of IgG-producing cells was detected. The strength of the responses depended upon the dosage of LPS administered. When mice received a primary injection of the same amount of LPS incorporated into P. mirabilis phospholipid vesicles, the number of IgM-producing cells was significantly increased over that induced by LPS alone. In addition, IgG-producing cells appeared on day 5 and increased during the time course measured up to day 14. After a booster injection of the same amount of LPS-phospholipid vesicles on day 14, the numbers of IgM-producing cells increased approximately 3-fold and the numbers of IgG-producing cells approximately 16-fold over those of mice given LPS alone. These results demonstrate a pronounced adjuvant effect of bacterial membrane phospholipids that are able to alter not only the strength but also the type of response to LPS.

Animals↗
PubMed1978

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of Gram-negative bacteria.

In the presence of MgCl2, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl2 and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I MgCl2 and detergent were unable to either entrap or retain [14C]-sucrose, [3H=inulin or [3H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [3H]-inulin out of re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl2 to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occurring in their normal intestinal habitat.

Biological Transport↗
PubMed1977

Reconstitution of model membranes from phospholipid and outer membrane proteins of Proteus mirabilis. Role of proteins in the formation of hydrophilic pores and protection of membranes against detergents.

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine the cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes. Three major outer membrane proteins of apparent molecular weights 39000, 36000 and 17000 were isolated using acetic acid and sodium deoxycholate solution as solvents and avoiding the strongly denaturing sodium dodecyl sulfate. The isolated proteins were assayed for their ability to form hydrophilic pores in reconstituted membranes. The trypsin-sensitive 39000-Mr protein and the peptidoglycan-associated 36000-Mr protein were equally effective in this function whereas the 17000-Mr protein mediated little penetration of low molecular weight solute. The 39000-Mr and 36000-Mr proteins also protected reconstituted membrane vesicles from disruption by detergent while 17000-Mr protein was ineffective in this regard.

Cell Membrane↗