Biomedical subjects

F Randow

Publications and source records attributed to F Randow.

16 recordsLinked to original sources

PubMed2001

The role of PPAR-gamma in macrophage differentiation and cholesterol uptake.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), the transcription factor target of the anti-diabetic thiazolidinedione (TZD) drugs, is reported to mediate macrophage differentiation and inflammatory responses. Using PPAR-gamma-deficient stem cells, we demonstrate that PPAR-gamma is neither essential for myeloid development, nor for such mature macrophage functions as phagocytosis and inflammatory cytokine production. PPAR-gamma is required for basal expression of CD36, but not for expression of the other major scavenger receptor responsible for uptake of modified lipoproteins, SR-A. In wild-type macrophages, TZD treatment divergently regulated CD36 and class A macrophage-scavenger receptor expression and failed to induce significant cellular cholesterol accumulation, indicating that TZDs may not exacerbate macrophage foam-cell formation.

Animals↗

PubMed2001

Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability.

Chaperone proteins are thought to promote the correct folding and assembly of newly synthesized proteins and to facilitate restoration of the folded state under environmental conditions that favour protein denaturation. They are among the most ubiquitous and highly conserved of all proteins. The eukaryotic endoplasmic reticulum (ER) chaperone gp96 in particular has long been thought to be indispensable for cell survival. Here we report that a screen for genes required for the immune response to bacterial endotoxins has identified a B-cell line deficient in gp96. Absence of gp96 is compatible with cellular survival even under stress conditions and causes a defect in the formation of only a small subset of cell surface receptors. Toll-like receptors are retained intracellularly in the absence of gp96, explaining the unresponsiveness of the mutant to microbial stimuli.

Animals↗

PubMed1997

Granulocyte-macrophage colony-stimulating factor and IFN-gamma restore the systemic TNF-alpha response to endotoxin in lipopolysaccharide-desensitized mice.

The influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-gamma on the restoration of impaired TNF-alpha release in LPS-desensitized mice or their refractory macrophages was investigated. Mice pretreated with GM-CSF or IFN-gamma (50 microg/kg i.v.) and injected with 3 mg/kg LPS i.p. displayed increased plasma TNF-alpha levels compared with LPS controls. IL-10 was marginally up-regulated by GM-CSF but abrogated by IFN-gamma pretreatment. LPS-tolerant mice (30 microg/kg LPS i.p., -24 h) showed an attenuated plasma TNF-alpha and IL-10 response to LPS and survived LPS shock. Pretreatment of such mice with GM-CSF or IFN-gamma restored the previously impaired TNF-alpha response. In cultures of murine monocyte/macrophage-containing cell populations, i.e., alveolar, peritoneal, spleen, bone marrow cells, or blood, the presence of GM-CSF or IFN-gamma (10 ng/ml) resulted in an enhanced release of TNF-alpha initiated by 1 microg/ml LPS. Cells from LPS-tolerant mice showed a diminished responsiveness to LPS. However, when exposed to GM-CSF or IFN-gamma ex vivo, their TNF-alpha response to LPS was partially restored. These findings characterize GM-CSF and IFN-gamma as potent enhancers of LPS-induced TNF-alpha production in normal as well as in experimentally immunocompromised mice and provide the rationale for further experiments to explore the pharmacologic use of these cytokines for restoration of immunocompetence in sepsis-associated immunosuppression.

Adjuvants, Immunologic↗

PubMed1997

In vitro prevention and reversal of lipopolysaccharide desensitization by IFN-gamma, IL-12, and granulocyte-macrophage colony-stimulating factor.

LPS tolerance is characterized by a diminished monocytic synthesis of TNF-alpha and, interestingly, IL-10 after LPS restimulation. We wondered whether granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, and IFN-gamma can prevent or reverse this down-regulation of TNF-alpha and IL-10 production. The LPS-induced TNF-alpha amounts in desensitized PBMC treated with GM-CSF, IFN-gamma, or IL-12 and in naive, non-cytokine-primed cultures were similar, while much more TNF-alpha was induced in cytokine-primed naive cells. The effect of IL-12 was dependent on the presence of nonmonocytic cells and could be completely blocked with an IFN-gamma antiserum. Treatment of LPS-desensitized pure monocytes with IFN-gamma or GM-CSF resulted in a very high TNF-alpha expression and no difference to cytokine-primed naive monocytes was evident any longer. While IFN-gamma and IL-12 decreased IL-10 expression in naive PBMC, it was increased by both and by GM-CSF in LPS-tolerant cultures. Again, only IL-12 was dependent on the presence of nonmonocytic cells. For prevention of LPS tolerance, similar results were obtained. Recently, we have shown that IL-10 and TGF-beta mediate LPS desensitization in vitro and can be used to establish LPS hyporesponsiveness in the absence of LPS. IFN-gamma and GM-CSF prevented and reversed down-regulation of TNF-alpha and IL-10 synthesis also in the model of IL-10/TGF-beta1-induced LPS hyporesponsiveness, while IL-12 was ineffective because of its obvious inability to induce IFN-gamma. In summary, after LPS desensitization/hyporesponsiveness, IFN-gamma and GM-CSF tended to normalize pro- and anti-inflammatory monocytic behavior. Our results suggest that during LPS desensitization/hyporesponsiveness, monocytes acquire a hitherto unknown functional state with an altered reaction to biologic response modifiers.

Desensitization, Immunologic↗

PubMed1997

Influence of monomethylfumarate on monocytic cytokine formation--explanation for adverse and therapeutic effects in psoriasis?

Although the effectiveness of systemic antipsoriatic treatment with fumaric acid esters has been proven, their mode of action is still not understood. Recent results indicate their potency in inducing cytokine production in stimulated T cells. Since monocytes and their cytokines are also considered to be of pathogenic importance in psoriasis, we investigated the effect of monomethylfumarate (MMF) on proinflammatory (TNF-alpha, IL-12) and antiinflammatory (IL-10, IL-1RA) cytokine production by peripheral blood mononuclear cells (PBMC) and separated monocytes. In 24-h PBMC cultures from both psoriatic patients (n = 6-13) and healthy volunteers (n = 7-9), MMF at 100 microM induced secretion of TNF-alpha, IL-10, and IL-1RA. Kinetics of IL-10 protein and mRNA expression indicated de novo production. Moreover, MMF significantly augmented endotoxin-induced synthesis of TNF-alpha, IL-10 and IL-1RA. In contrast, no influence on IL-12 secretion was found. Similar effects of MMF in purified monocytes indicated these cells to be responsible for aberrant cytokine formation. Furthermore, enhanced expression of costimulatory molecules after MMF stimulation confirmed monocyte activation. Multiple restimulation with fumaric acid esters in vitro, however, and immunomonitoring in a patient during Fumaderm initial therapy suggested that initial monocyte activation is followed by subsequent deactivation associated with an antiinflammatory response. Our results may explain the well-known effects of therapy with fumaric acid esters. Thus, initial treatment is often accompanied by adverse effects which may be caused by MMF-induced TNF-alpha formation. The change in the IL-10/IL-12 balance as a result of elective induction of IL-10, however, may have antipsoriatic activity by diminishing type-1/proinflammatory cytokine over-expression and the antigen-presenting capacity of monocytes/macrophages, and by upregulation of IL-1RA.

Cells, Cultured↗

PubMed1997

Monocyte deactivation in septic patients: restoration by IFN-gamma treatment.

Neutralization of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 (IL-1), decreases mortality in several animal models of sepsis. However, recent clinical trials did not show an unequivocal improvement in survival. In contrast to animals, which succumb to shock during the first 72 hours, we found that many patients die much later with signs of opportunistic infections accompanied by downregulation of their monocytic HLA-DR expression and reduced ability to produce lipopolysaccharide (LPS)-induced TNF-alpha in vitro. This phenomenon of monocyte deactivation in septic patients with fatal outcome shows similarities to experimental monocytic refractoriness induced by LPS desensitization or by pretreatment with its endogenous mediators IL-10 and transforming growth factor-beta (TGF-beta). In order to strengthen their antimicrobial defense, here we tested whether interferon-gamma (IFN-gamma) can improve monocytic functions in these patients and in experimental monocytic deactivation. The considerably lowered in vitro levels of LPS-induced TNF-alpha in these situations were significantly enhanced by IFN-gamma, but did not reach the extremely high levels of IFN-gamma primed naive cells from healthy donors. Moreover, IFN-gamma applied to septic patients with low monocytic HLA-DR expression restored the deficient HLA-DR expression and in vitro LPS-induced TNF-alpha secretion. Recovery of monocyte function resulted in clearance of sepsis in eight of nine patients. These data suggest that IFN-gamma treatment in carefully selected septic patients is a novel therapeutic strategy worth pursuing.

Adolescent↗

PubMed1996

Differential regulation of monocytic tumor necrosis factor-alpha and interleukin-10 expression.

Activation of human monocytes by bacterial endotoxin (LPS) results in an initial burst of inflammatory cytokines like tumor necrosis factor (TNF)-alpha which is followed by the secretion of anti-inflammatory mediators like interleukin (IL)-10. The signaling pathways in IL-10 induction are unknown. Here, we show that the regulation of IL-10 expression is more complex than that of TNF-alpha. LPS-induced TNF-alpha and IL-10 expression requires early activation of protein tyrosine kinases (PTK). Moreover, delayed addition of PTK inhibitors blocked IL-10, but not TNF-alpha, suggesting the impact of a late PTK activity. Two inducers of PTK activity are the downstream mediators of LPS activation, TNF-alpha and cyclic adenosine monophosphate (cAMP). Both mediators synergistically up-regulate IL-10 expression. Downstream of PTK activation, they use distinct pathways. TNF-alpha, but not cAMP-induced IL-10 gene expression was inhibited by pyrrolidine dithiocarbamate, suggesting the involvement of reactive oxygen species. Inhibition of protein kinase C (PKC) suppressed LPS-induced TNF-alpha and IL-10 expression as well, but, unlike TNF-alpha, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) did not induce IL-10 expression. Furthermore, PKC is not involved in late events of IL-10 activation, as delayed addition of PKC inhibitors did not suppress LPS-induced IL-10 expression and did not influence cAMP- or TNF-alpha-induced IL-10. The modulation of IL-10 expression by inflammatory mediators suggests a regulatory circuit of the inflammatory response.

Antioxidants↗

PubMed1995

Introduction of the octanucleotide restriction site SwaI into the bicistronic vector pTiSDT for high level synthesis of proteins.

The octanucleotide recognition site for the endonuclease SwaI was introduced into the Escherichia coli bicistronic expression vector pTiSDT by mutating a single position in the coupling SD sequence between a truncated form of the cro-gene and the multicloning site. This mutation does not influence the expression rate. The introduction of this restriction site allows high level production of proteins, that are modified only by an N-terminal methionine incorporated as the start codon.

Base Sequence↗

PubMed1995

Mechanism of endotoxin desensitization: involvement of interleukin 10 and transforming growth factor beta.

Tolerance of monocytes/macrophages to endotoxin (lipopolysaccharide [LPS]) can be induced both in vivo and in vitro by LPS itself. Exposure to LPS, even at a very low dose, induces a downregulation of cytokine response to a second high dose LPS challenge. To learn more about the unknown mechanisms of this phenomenon, we studied the role of antiinflammatory cytokines in this process. Preculture of human peripheral blood monocytes for 24 hours with low concentrations of LPS induced hyporesponsiveness to high-dose LPS rechallenge with respect to tumor necrosis factor (TNF) alpha and interleukin (IL) 10 but not IL-1RA production. These results suggest that LPS tolerance reflects a functional switch of monocytes rather than a general LPS hyporesponsiveness. IL-10 and transforming growth factor (TGF) beta 1 showed additive effects in replacing LPS for induction of LPS hyporesponsiveness in vitro. Additionally, neutralizing anti-IL-10 and anti-TGF-beta monoclonal antibodies prevented induction of LPS tolerance. In vitro induced LPS tolerance looks like the ex vivo LPS hyporesponsiveness of monocytes from septic patients with fatal outcome: downregulation of LPS-induced TNF-alpha and IL-10 production but not of IL-1RA secretion. LPS hyporesponsiveness in septic patients was preceded by expression of IL-10 at both the mRNA and protein level. In summary, our data suggests that IL-10 and TGF-beta mediate the phenomenon of LPS tolerance in vitro and perhaps in vivo (septic patients), too.

Cells, Cultured↗

PubMed1971