Biomedical subjects

H U Beuscher

Publications and source records attributed to H U Beuscher.

32 records · Page 2Linked to original sources

PubMed1990

IL-1 beta is secreted by activated murine macrophages as biologically inactive precursor.

IL-1 alpha and IL-beta are distinct cytokines, produced by activated macrophages. The temporal sequence in the processing and secretion as well as the mechanism(s) by which IL-1 is secreted from the cells remain undefined. Here we have studied the production of IL-1 from murine macrophages after stimulation with LPS or Listeria monocytogenes by two distinct methods: i) immunoprecipitation of radio-labeled IL-1 peptides from culture supernatants, and ii) determination of IL-1 activity by neutralization with monospecific antisera to either form of IL-1. We confirmed that precursor and mature forms of both IL-1 alpha and IL-1 beta can be detected in the culture supernatants after stimulation of the macrophages with 10 to 20 micrograms LPS/ml but, in addition, we report the novel finding that IL-1 beta is exclusively secreted in its unprocessed precursor form after stimulation of the cells with either 0.5 to 1 microgram LPS/ml or with L. monocytogenes. Exposure of the cells to increasing amounts of LPS led to the appearance of a 20-kDa IL-1 beta peptide in the culture supernatants concomitant with the release of a processing activity for the IL-1 beta precursor. These data therefore suggest that, in a first step, IL-1 beta is secreted as an unprocessed precursor protein that in a second, postsecretory step is cleaved by a LPS-inducible protease, thus generating the 20-kDa IL-1 beta peptide. The latter represents the biologically active IL-1 beta inasmuch as the generation of IL-1 beta activity in the culture supernatants strictly correlated with the appearance of the 20-kDa IL-1 beta peptide.

Animals↗

PubMed1990

Activation of bone marrow-derived mouse macrophages by bacterial lipopeptide: cytokine production, phagocytosis and Ia expression.

The lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl-a lanyl-glycine (Pam3Cys-Ala-Gly), a synthetic analogue of the N-terminal part of bacterial lipoprotein, induces the secretion of interleukin (IL) 1, IL 6 and tumor necrosis factor (TNF)-alpha in bone marrow-derived macrophages that have been cultured in vitro for up to 20 days. IL 6 and TNF-alpha secretion increased from day 6 to day 20 whereas IL 1 secretion increased until day 13 and decreased on day 20. In contrast to the enhancement of cytokine production, phagocytosis of IgG-coated sheep erythrocytes and Ia expression were found to be diminished after treatment with lipopeptide for 24 h. Morphological studies revealed lipopeptide-induced changes of macrophage cultures. The data presented here show the potential of the lipopeptide as a strong activator of bone marrow-derived macrophages.

Animals↗

PubMed1989

Proteolysis of the native murine IL 1 beta precursor is required to generate IL 1 beta bioactivity.

We utilized antisera specific for murine IL 1 alpha and IL 1 beta proteins to characterize cell-associated IL 1 according to two distinct criteria, immunochemical detection of radiolabeled IL 1 polypeptides and inhibition of IL 1 activity in cell lysates. Evidence is presented that IL 1 alpha and IL 1 beta are each synthesized by LPS-stimulated but not by unstimulated murine macrophages and accumulate within the cell as intracellular precursors in sizes of 33 kDa and 37 kDa, respectively. As judged by the respective rates of synthesis, IL 1 alpha was about 4-fold more abundant than IL 1 beta. Most (greater than or equal to 95%) of the cell-associated IL 1 activity was inhibited in the presence of the antiserum to IL 1 alpha, suggesting that the precursor of IL 1 beta is mostly biologically inactive. Incubation of cell lysates with papain but not with trypsin or plasmin markedly stimulates an increase in the level of cell-associated IL 1 beta activity and leads to the cleavage of 15-16 kDa carboxyl-terminal fragments from the IL 1 beta precursor. Together, these data indicate that proteolysis of the IL 1 beta precursor is required to generate bioactive IL 1 beta molecules and provide a basis for further investigations of the specific role of proteinases in processing the IL 1 beta precursor to a bioactive form.

Animals↗

PubMed1989

Reduction of murine interleukin-1 beta: a possible mechanism in the functional maturation of IL-1 beta.

Studies related to the functional activity of human endogenous pyrogen have emphasized its susceptibility to oxidative inactivation. Here we show that the specific activity of murine IL-1 beta polypeptides can be increased by reduction and subsequent alkylation. This treatment induces a change in the relative molecular weight from 20 kDa to 21 kDa of the molecule, suggesting that conformational changes may play a critical role in the functional maturation of IL-1 beta molecules.

Animals↗

PubMed1988

The precursor of interleukin-1 alpha is phosphorylated at residue serine 90.

Mononuclear phagocytes release interleukin-1 (IL-1), a 17-kDa polypeptide with diverse biological activities. IL-1 is synthesized as a precursor (31 kDa) which lacks a signal sequence or hydrophobic domains that could facilitate transmembrane translocation. Possible postsynthetic modifications of IL-1 that might account for its cellular transport were examined. We found that lipopolysaccharide stimulated, but not unstimulated, murine macrophages incorporated 32PO4 into the IL-1 alpha precursor (31 kDa) predominantly at residue serine 90. Released IL-1 alpha (17 kDa) is not phosphorylated in agreement with peptide sequence data that the site of 32P incorporation is in the amino-terminal one-third of the precursor. Approximately 10% of the phosphorylated IL-1 alpha precursor is membrane bound and associated with a fraction enriched in lysosomal vesicles. Together these data suggest mechanisms by which the postsynthetic proteolysis of the IL-1 alpha precursor may be modified and cellular transport of IL-1 alpha is accomplished.

Amino Acid Sequence↗

PubMed1988

Studies on the release of cell-associated interleukin 1 by paraformaldehyde-treated murine macrophages.

Experiments are presented demonstrating the release of interleukin 1 (IL1) by lipopolysaccharide-stimulated murine macrophages (M phi) after treatment of the cells with paraformaldehyde (PFA). The IL1 activity set free was completely blocked by a rabbit anti-mouse IL1 alpha antiserum. Immunoprecipitation studies revealed that the IL1 released was of Mr 33 kDa, 23 kDa and 17 kDa. Incubation of PFA-treated M phi in the presence of leupeptin reduced the amount of detectable low molecular mass (i.e. 23 kDa and 17 kDa) IL1 alpha. Addition of leupeptin to PFA-treated M phi also reduced the amount of released IL1 activity, suggesting that proteolytic modification of the IL1 alpha precursor is involved in the generation of biological active IL1 alpha. It is proposed that the cell-associated 33-kDa IL1 is released from the M phi by vesiculation of parts of the M phi cell membrane, followed by a proteolytic cleavage step.

Animals↗

PubMed1988

Structure and function of membrane IL-1.

Both interleukin-1 alpha (IL-1 alpha) and IL-1 beta are initially translated as approximately Mr 30,000 polypeptides, lacking hydrophobic or signal sequence that could facilitate transmembrane translocation and release of mature IL-1 (Mr 17,500). The current study utilizes an antiserum specific for murine IL-1 alpha in order to investigate membrane associated IL-1 alpha polypeptides and possible postsynthetic modifications of the IL-1 alpha precursor, that might account for its intracellular transport. Cell surface iodination of endotoxin stimulated murine macrophages allowed the detection of IL-1 molecules in size similar to the IL-1 alpha precursor (Mr 33,000). Membrane bound IL-1 alpha was sensitive to degradation by serine esterase activity to yield IL-1 peptides of Mr 16,000 to 18,000. Endotoxin stimulated macrophages, but not unstimulated cells, incorporated 32PO4 into the IL-1 alpha precursor. The phosphate label of the IL-1 alpha precursor is resistant to hydroxylamine and alkaline phosphatase treatment. Released IL-1 is not phosphorylated. Approximately 10% of the phosphorylated IL-1 alpha precursor is membrane bound and associated with fractions enriched in lysosomal vesicles. These data are consistent with a model for mIL-1 expression, in which pro IL-1 alpha is post-synthetically modified to achieve intracellular transport and further suggest that mIL-1 may be a prerequisite for the release of IL-1.

Animals↗

PubMed1988

Local extrahepatic expression of complement genes C3, factor B, C2, and C4 is increased in murine lupus nephritis.

Systemic lupus erythematosus (SLE) is associated with the presence of complement proteins and immune complexes in affected organs. Since complement proteins are synthesized in hepatic and extrahepatic sites, we studied a murine model of SLE to ascertain the relative importance of local and humoral (liver) synthesis of complement. C3, C4, and C2 mRNA increase in kidney coincident with the development of nephritis in the MRL lpr/lpr mouse, a strain that spontaneously develops SLE. Two factor B messenger RNA transcripts are expressed in kidney and intestine; SLE nephritis is associated with decrease in the long factor B mRNA and increase in the short form. Increased local synthesis of C3 and B protein and a concomitant glomerular and renal interstitial macrophage infiltrate paralleled the increase in mRNA content in the (lpr/lpr) mice. In addition to kidney, an increase in C3, C4, C2 and factor B mRNA was noted in the lung, heart and intestine and to a lesser extent in liver of (lpr/lpr) in comparison to the MRL (+/+) animals. These results suggest that in SLE local expression of complement genes plays a role in the pathogenesis of chronic glomerulonephritis and in the autoimmune arteritis of other organs.

Animals↗

PubMed1987

Macrophage membrane interleukin 1 regulates the expression of acute phase proteins in human hepatoma Hep 3B cells.

To assess the potential role of membrane interleukin 1 (mIL-1) in modulating expression of acute phase proteins, we studied the effect of fixed mouse peritoneal macrophages and isolated cell membranes on the synthesis of C3 and albumin in human hepatoma Hep 3B cells. An increase in C3 synthesis and decrease in albumin synthesis were detected after incubation of Hep 3B cells with fixed stimulated macrophages or with membrane preparations. Estimates of hepatocellular C3 and albumin mRNA indicated that the mIL-1 regulation was exerted at a pretranslational level. The changes in C3 and albumin expression induced by mIL-1 were inhibited by addition of anti-interleukin 1 (IL-1) antiserum to the macrophages, supporting the hypothesis that a form of IL-1 is present on the outer cell membrane of macrophages. Moreover, cell-surface iodination of macrophages allowed the detection of a 33-kDa IL-1 molecule, suggesting that an IL-1 species, similar to the intracellular precursor of IL-1 is present at the outer cell surface of macrophages. The expression of mIL-1 was temporally dissociated from IL-1 release, indicating that the cell-surface associated IL-1 activity is not the result of adsorbed soluble IL-1. These studies provide a basis for further investigation of the role of mIL-1 in modulating monocytes and macrophages function via cell-cell interactions.

Acute-Phase Proteins↗

PubMed1987

Constitutive and IL 1-regulated murine complement gene expression is strain and tissue specific.

To study the molecular mechanisms accounting for strain- and tissue-specific variations in the production of complement proteins, complementary DNA probes were used to assess qualitative and quantitative differences in specific mRNA content of complement proteins C2, factor B, and C3 in extracts of tissues (liver, lung, spleen, kidney, and peritoneal macrophages) isolated from various mouse strains. Northern blot analysis of total hepatic RNA revealed differences in C2, factor B, and C3 mRNA levels in strains that share B10 background but differ in the H-2 region (e.g., H-2k, H-2u, H-2d, H-2f). In each instance, hepatic mRNA specific for the individual gene product corresponded in amount to the serum levels. By contrast, specific mRNA content of C2 and factor B in macrophages differed significantly from those observed in liver for each strain. Modulation of C2, factor B, and C3 expression was studied after in vivo administration of recombinant IL 1 or endotoxin to H-2k (B10.AKM) or H-2u (B10.PL) strain mice. As assessed by Northern blot analysis, neither endotoxin nor IL 1 affected liver C2-specific mRNA but increased specific C2 mRNA levels in kidney and lung. For both strains, IL 1 increased specific factor B mRNA in all tissues examined except for the H-2u strain liver factor B mRNA content, which was not affected by IL 1, whereas that of H-2k mice was increased. The lack of factor B modulation by IL 1 in the H-2u lines was specific to that gene and not a reflection of a generalized IL 1 unresponsiveness. Differences in tissue and strain specific constitutive and IL 1-regulated expression of the C3 gene were also observed in the H-2u and H-2k strains.

Animals↗

PubMed1986

Lipopolysaccharides as complement inhibitors by complex formation with the purified third complement component (C3).

Lipopolysaccharides (LPS) from different bacteria in smooth or rough form (Y. enterocolitica, Y. pseudotuberculosis, E. coli, S. typhimurium, S. marcescens) strongly inhibited hemolytic C3 in incubation mixtures with purified C3. LPS from a core deficient mutant was still reactive, whereas lipid A no longer affected C3 activity. The physical state of LPS was critical for its effect on C3. Strand-like LPS structures formed by Ca++-induced aggregation of solubilized LPS, as shown by electron microscopy, demonstrated the highest reactivity with C3. Inhibition of hemolytic C3 was found to be due to complex formation between LPS and C3 by a hydrophobic reaction. The binding capacity of 1 microgram LPS-R and LPS-S was as high as 125 ng C3 and 56 ng C3, respectively. The C3b fragment required different reaction conditions for maximal binding. The strong binding capacity of LPS for the complement component C3 raises the possibility that LPS act as inhibitors of complement by interruption of the reaction cascade at local infectious sites with gram-negative bacteria.

Centrifugation, Density Gradient↗

PubMed1986

C3 activation by a new factor B-dependent enzyme detected in culture supernatant from guinea-pig peritoneal macrophages.

Culture supernatants (c.s.) collected from thioglycollate-elicited macrophages were concentrated and incubated with purified C3. In this reaction mixture loss of haemolytic C3 according to classical enzyme kinetics was observed. As revealed by SDS-PAGE, c.s.-catalysed fragmentation of the C3 alpha-chain occurred. The cleavage products were identified by size and function as C3a and C3b. The apparent molecular weight of this C3-activating enzyme in c.s. was approximately 220,000 according to ultracentrifugation studies. This large enzyme showed the following characteristics: it had no activity against C5; it was inhibited by EDTA; Mg2+ was required for its optimal function; its half-life at 37 degrees was approximately 35 min; it was completely inhibited by anti-B IgG. Thus, we were able to detect a C3-activating enzyme in c.s. containing B but differing otherwise from a preformed C3 convertase of the alternative pathway. The exact component composition of this new enzyme is under further investigation.

Animals↗

PubMed1984

Formation of EAC142 and EAC1423 with macrophage culture supernatant containing the secreted complement components C1 to C3.

Culture supernatants of thioglycollate-elicited guinea pig peritoneal macrophages contained hemolytic C1, C4, C2 and C3, whereas hemolytic C5, C6, C7, C8 or C9 were not detected. Activity of C1, C2 and C3 increased up to a 48 h culture period, whereas C4 activity already declined in 2 day old cultures. After secretion, the hemolytic activity of C1 was least stable in culture supernatant. Sensitized sheep erythrocytes (EA) when incubated with culture supernatant initiated activation and functional cooperation of secreted C1 to C3 as indicated by formation of EAC142 and EA1423 intermediates. Decay and regeneration with purified C2 was shown for EAC142 and deposition of C3 fragments on EAC1423 was demonstrated with anti-C3. On an average, supernatants of 2 day old macrophage cultures were most suitable for formation of EAC142 and EAC1423 . The rate of EAC142 and EAC1423 formation, and also of C2 and C3 inactivation, during incubation of EA with culture supernatant was slow; addition of purified C1 to culture supernatant, however, greatly enhanced the same reactions of EA with supernatant which indicated that C1 was the rate limiting factor. Local secretion of hemolytic C1, C4, C2 and C3 by macrophages may have an important role in antimicrobial defense mechanisms due to the well-known functional cooperation between macrophages and activated C3.

Animals↗

PubMed1983

[Results with the IgM-FTA-Abs and IgM-TPHA tests in the study of seropositive syphilitic sera].

The IgM fraction was separated by gel filtration chromatography from patient sera which were positive in the FTA-abs-test as well as in the TPHA-test. After absorption the isolated IgM fraction was tested for specific IgM antibodies against Treponema pallidum in the IgM-FTA-abs- and also in the IgM-TPHA-test. The following results were obtained: Both IgM tests often showed positive reactions even with negative results in the cardiolipin complement fixation assay (CFA) (Tables 1 and 2). In most positive cases specific IgM was detectable with both IgM tests in the same sample (Table 3). The CFA titers were low (less than 1:20) in the majority of cases which did not have detectable IgM (Tables 1 and 3). The CFA titers were prevailingly high (greater than 1:20) where the IgM tests gave positive results (Table 1 and 3). Clinical symptoms of active syphilis were only reported for cases with high CFA titer and positive IgM tests in the same sample. Therefore only these latter results of diagnostic tests can be clearly interpreted to indicate a requirement for specific therapy against syphilis.

Antibodies, Bacterial↗