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V Kolb-Bachofen

Publications and source records attributed to V Kolb-Bachofen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes.

BACKGROUND: Nitric oxide (NO) has frequently been shown to display immunosuppressive activities. We describe here a molecular mechanism contributing to this effect. MATERIALS AND METHODS: Murine T cell lymphoma EL4-6.1 cells were activated with the physiological stimulus interleukin (IL)-1beta to express IL-2 mRNA in the presence or absence of subtoxic concentrations of the physiological spontaneous NO donor S-nitrosocysteine (SNOC). Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyze the effects of NO on IL-2 mRNA expression and on the activity of the dominant regulating transcription factors Sp1, EGR-1, and NFATc. RESULTS: NO inhibits IL-1beta-induced IL-2 mRNA expression in EL4-6.1 cells. The suppressive activity of NO was concentration dependent and found to be completely reversible. Importantly, NO at the concentrations used induced neither apoptosis nor necrosis. Dominant regulation of IL-2 gene expression is known to reside in the zinc finger transcription factors Sp1 or EGR-1 and in the non-zinc finger protein NFAT. NO abrogates the DNA binding activities of recombinant Sp1 and EGR-1. More importantly, gel shift assays also showed a lack of DNA binding of native Sp1 derived from NO-treated nuclear extracts and that from NO-treated viable lymphocytes. This effect is selective, as the DNA binding activity of recombinant NFATc was not affected by NO. CONCLUSION: Inactivation of zinc finger transcription factors by NO appears to be a molecular mechanism in the immunosuppressive activity of NO in mammals, thus contributing to NO-mediated inhibition of IL-2 gene expression after physiological stimuli. The exact understanding of the molecular mechanism leading to NO-mediated, fully reversible suppression of immune reactions may lead to use of this naturally occurring tool as an aid in inflammatory diseases.

Animals↗
PubMed1998

Nitric oxide and its implications in skin homeostasis and disease - a review.

The recent identification of the nitric oxide synthase (NOS) pathway in various cell types in the skin has provided important insight into the molecular mechanisms underlying regulatory and homeostatic functions of the skin. Many studies also point to perturbations or defects in the signaling cascade of nitric oxide (NO) and reactive nitrogen intermediates as key players in skin disease pathogenesis. A critical role for NO is now established for a subset of human skin diseases, and new mechanism-based therapies may be available in the near future. This remarkable progress and the implications it may have for common forms of skin disease are reviewed here.

Animals↗
PubMed1998

Nitric oxide in human skin: current status and future prospects.

The gaseous free radical nitric oxide is an important biologic mediator with physiologic and pathophysiologic roles in nearly every organ system. Because of its unique biologic activity, unusual chemical structure, and unprecedented mechanisms of action, nitric oxide, arguably more than any other natural product, has opened new avenues to investigate cellular processes. Nitric oxide is generated in biologic tissues by specific nitric oxide synthases that metabolize arginine and molecular oxygen to citrulline and nitric oxide. Besides its function as a diffusible messenger in the vasculature and in neurons, nitric oxide also plays a key role in innate immunity and inflammation. Recent progress has allowed the identification of the nitric oxide pathway in several cell types that reside in the skin, including keratinocytes, melanocytes, Langerhans cells, fibroblasts, and endothelial cells. Convincing evidence suggests that nitric oxide synthesis in these cells can be modulated by calcium-mobilizing agonists as well as diverse inflammatory and immune stimuli, and thereby contributes to the pathogenesis of several human skin diseases. Characterization of these intrinsic and extrinsic regulatory stimuli of nitric oxide synthesis has afforded substantial insights into the role of nitric oxide in inflammatory, hyperproliferative, and autoimmune skin diseases, as well as skin cancer, and may ultimately form the basis for future therapeutic intervention. The demonstrable and potential roles of nitric oxide in skin disease pathogenesis and treatment are the subjects of this review.

Humans↗
PubMed1998

Aberrant timing in epidermal expression of inducible nitric oxide synthase after UV irradiation in cutaneous lupus erythematosus.

Photosensitivity is a main criterion for the diagnosis of systemic lupus erythematosus (LE), and ultraviolet (UV) irradiation plays a key role in the pathogenesis of cutaneous LE. Patients with a tentative diagnosis of LE are routinely tested for skin lesion development after experimental UV irradiation, providing an ideal opportunity to evaluate early, preclinical events involved in the pathogenesis of LE. Several reports have shown expression of the cytokine-inducible nitric oxide synthase (iNOS) in autoimmune diseases. Therefore, we investigated the role of iNOS expression at mRNA and protein level in the pathogenesis of LE lesions. Skin biopsies from patients with different subtypes of LE were examined, and iNOS expression was found in six of 18 biopsies from cutaneous LE patients and two of three biopsies from systemic LE patients. In biopsies taken 4-20 d after UV irradiation, epidermal iNOS expression was seen in all patients (n = 10) after UVB and in four of 10 patients provoked by UVA. In healthy controls (n = 8) epidermal iNOS expression was detected 24 h after UV irradiation, persisting for another day before subsiding on day 3. In LE patients (n = 8) the exact reverse situation was seen: an iNOS-specific signal was undetectable in keratinocytes for 2 d after UV irradiation, but became positive on day 3 and persisted for up to 25 d in the evolving skin lesions. Our findings demonstrate a time-restricted, UV-induced iNOS expression in human skin; moreover, the results indicate that both the kinetics of iNOS induction as well as the time span of local iNOS expression may be critical to the development of cutaneous LE lesions.

Adult↗
PubMed1998

Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts.

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.

Cell Differentiation↗
PubMed1997

The dominant role of exogenous or endogenous interleukin-1 beta on expression and activity of inducible nitric oxide synthase in rat microvascular brain endothelial cells.

In the brain large amounts of nitric oxide are produced in response to various pathological stimuli such as infectious agents, ischemia and trauma. Although it is known that endothelial cells can express the inducible isoform of nitric oxide synthase (iNOS) upon activation, the impact of different cytokines on iNOS expression in rat microvascular endothelial cells remains unclear. We now investigated iNOS mRNA expression and enzyme activity in primary cell cultures of rat microvascular brain endothelial cells after treatment with the proinflammatory cytokines interleukin-1beta (IL-1beta), Tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) alone or in combination. Cells were characterized by immunocytochemistry staining for von-Willebrand-factor and the rat brain endothelial antigen recognized by monoclonal antibody Ox2. iNOS-enzyme activity was determined by measurement of nitrite in the supernatants of cell culture using the Griess-reaction. In addition mRNA expression was analysed by RT-PCR with iNOS and IL-1beta specific primers. All cells in the endothelial cell culture were found to express the antigenic phenotype vWF+/Ox2+/Ox43-, thus identifying the cells as rat brain endothelial cells of microvascular origin. IL-1beta was the only cytokine that as a single stimulus induced iNOS mRNA expression and iNOS-enzyme activity in these endothelial cells. All combinations of two cytokines, including that of TNF-alpha and IFN-gamma--or the triple combination led to expression of iNOS-mRNA and active protein. Cell activation by the combination of TNF-alpha + IFN-gamma led to an early expression of IL-1beta by the endothelial cells suggesting iNOS induction as a consequence of endogenous IL-1beta production under this challenge. The experiments prove that rat brain microvascular endothelial cells express iNOS and produce large amounts of NO under inflammatory conditions. Furthermore, our results indicate a decisive role of IL-1beta in iNOS expression and NO generation.

Animals↗
PubMed1997

Nitric oxide mediates intracytoplasmic and intranuclear zinc release.

We previously described that NO. leads to destruction of ZnS clusters and release of Zn2+ from various proteins including zinc finger transcription factors. To assess the relevance in living cells, we investigated, whether exogenous NO. leads to an increase of cytoplasmic and nuclear free Zn2+. L929 cells, mouse splenocytes, or rat aorta endothelial cells were labeled with Zinquin-E, a Zn2+-specific fluorophore, and were treated with two different spontaneous NO donors, S-nitrosocysteine or DETA/NO. Both NO donors strongly increased the Zn2+-dependent fluorescence in the cellular cytosol and also in nuclei as compared to controls. NO-dependent Zn2+ release in splenocytes was quantitated by flow cytometry. These results show for the first time, that nitrosative stress mediates intracellular and intranuclear Zn2+ release which may be relevant in altering gene expression patterns.

Animals↗
PubMed1997

Nitric oxide: cytotoxicity versus cytoprotection--how, why, when, and where?

Nitric oxide (NO) has been found to play an important role as a signal molecule in many parts of the organism as well as a cytotoxic effector molecule of the nonspecific immune response. It appears paradoxical that NO on one side acts as a physiological intercellular messenger and on the other side may display cytotoxic activity in vivo. To make things even more complicated, cytoprotective properties of NO are also described. We here review the current understanding of cytotoxic versus cytoprotective effects of NO in mammalian cells and try to highlight the janus-faced properties of this important small molecule.

Animals↗
PubMed1997

Nitric oxide-induced expression of C-reactive protein in islet cells as a very early marker for islet stress in the rat pancreas.

In searches for marker molecules specifically expressed in nitric oxide-treated islet cells as a means to recognize early events in islet destruction, we now establish the presence of neo-C-reactive protein (neoCRP) in rat islet cells as early as 2 hr after treatment. We detected this altered molecular form of the acute-phase-reactant C-reactive protein (CRP) using immunocytochemistry with an anti-neoCRP-specific monoclonal antibody as well as reverse transcription-polymerase chain reaction with CRP-specific primers and in situ hybridization to demonstrate the presence of CRP-specific mRNA. After induction of a generalized inflammatory reaction in rats with heat-inactivated Corynebacterium parvum in vivo, neoCRP expression in islets is also found and within the pancreas restricted to pancreatic islet cells only. Our findings suggest an early heat-shock-like expression of this molecule in response to local nitrite oxide production or to exogeneously added nitric oxide in islet cells.

Animals↗
PubMed1997

Endothelial cells as cytotoxic effector cells: cytokine-activated rat islet endothelial cells lyse syngeneic islet cells via nitric oxide.

In vivo, each beta cell is located in proximity to at least one capillary islet endothelial cell. Rat aorta and islet endothelial cells can be activated in vitro to express inducible nitric oxide synthase by a cytokine mixture of tumour necrosis factor-alpha, gamma-interferon, and interleukin-1 beta and to produce high concentrations of nitric oxide. We have performed co-culture experiments with rat islet endothelial cells together with isolated syngeneic islet cells at low target:effector ratios with or without previous cytokine challenge of endothelial cultures. Co-cultures were always free of exogenous cytokines, which were removed prior to addition of islet cells. We found that pre-activated, in contrast to resident islet endothelial cells, at a target:effector ratio as low as 1:1 almost completely lysed syngeneic beta and non-beta cells with 24 h of co-culture. Lysis by pre-activated islet endothelial cells was found to be preceded by DNA damage found in 46% of islet cells after 8 h of co-culture with pre-activated vs 7% with resting islet endothelial cells. Lysis was blocked to control levels in the presence of the nitric oxide synthase inhibitor NG-methyl-L-arginine. With the results presented here, we demonstrate for the first time, that activated endothelial lining cells can express effector cell activity and thus can contribute to local tissue destruction, especially in organs that are densely capillarized such as pancreatic islets.

Animals↗
PubMed1997

Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells.

1. Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants. 2. In each of the different endothelial cells Mg-Dobesilate incubation (0.25-1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor N(G)-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects. 3. iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT-PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT-PCR.

Animals↗
PubMed1996

A proinflammatory activity of interleukin 8 in human skin: expression of the inducible nitric oxide synthase in psoriatic lesions and cultured keratinocytes.

Psoriasis is a common chronic skin disease mediated by cellular immune mechanisms and characterized by an intense neutrophil cell infiltrate and proliferative activation of epidermal keratinocytes. We have previously described the expression of the inducible nitric oxide synthase (iNOS) in epidermal keratinocytes of psoriatic skin lesions. In this study, the role of iNOS in psoriatic inflammation was explored ex vivo in psoriatic skin biopsies and in vitro in primary cultures of human keratinocytes. Messenger RNA for the iNOS enzyme (iNOS mRNA) was detected by reverse transcriptase polymerase chain reaction in skin biopsies from patients with psoriasis, but not in skin specimens from patients with atopic eczema or from healthy volunteers. As demonstrated by in situ hybridization and immunohistochemistry, expression of iNOS mRNA and its gene product was localized to the epidermal keratinocytes of psoriatic skin lesions. In situ hybridization further revealed a complete colocalization of mRNA expression for iNOS with interleukin (IL) 8 receptor-specific mRNA either in the basal germinative cell layer or at focal sites of ongoing neutrophil inflammation in suprabasal cell layers. Because psoriatic keratinocytes have previously been shown to express mRNA transcripts for IL-8, it seemed reasonable to hypothesize that iNOS expression could be induced in an autocrine loop by IL-8. This hypothesis was substantiated by our in vitro experiments showing that a combination of IL-8 and interferon gamma induces the expression of iNOS-specific mRNA and of the functional enzyme in cultured human keratinocytes. These results suggest an important role for iNOS in concert with IL-8 and its receptor early during the formation of psoriatic lesions.

Biopsy↗