Bernadeta Nowak

Taurine chloramine down-regulates the generation of murine neutrophil inflammatory mediators

We previously reported that taurine chloramine (TauCl), a product of activated neutrophils, inhibits the generation of macrophage inflammatory mediators such as nitric oxide (NO), TNF-alpha, and PGE2. Taurine, the most abundant free amino acid in the cytosol of neutrophils, is chlorinated to form TauCl by the halide-dependent myeloperoxidase (MPO) system. Under physiological conditions, TauCl reduces HOCl toxicity. In this study, we investigated the influence of TauCl on generation of oxygen free radicals, cytokines and eicosanoids by activated murine peritoneal neutrophils. We found that TauCl, but not taurine alone, inhibited the production of NO, prostaglandin E2, interleukin-6 and tumor necrosis factor-alpha, in a dose-dependent manner. In contrast, the products of the respiratory burst, as measured by luminol-dependent chemiluminescence (LCL), were reduced by both taurine and TauCl. However, taurine affected LCL at higher concentrations and to a lesser extent than TauCl. The results of these studies suggest that TauCl decreases production of tissue-damaging inflammatory mediators and may regulate the balance between protective, microbicidal and toxic effect of neutrophils.

A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse

The pathogenesis of diabetes in the nonobese diabetic (NOD) mouse is characterized by a selective destruction of the insulin‐producing β‐cells in the islets of Langerhans mediated by autoreactive T cells. The function of T cells is controlled by dendritic cells (DC), which are not only the most potent activators of naïve T cells, but also contribute significantly to the establishment of central and peripheral tolerance. In this study, we demonstrate that the NOD mouse (H2: Kd, Ag7, E°, Db) shows selective phenotypic and functional abnormalities in DC derived from bone marrow progeny cells in response to GM‐CSF (DCNOD). NOD DC, in contrast to CBA DC, have very low levels of intracellular I‐A molecules and cell surface expression of MHC class II, CD80, CD86 and CD40 but normal β2‐microglobulin expression. Incubation with the strong inflammatory stimulus of LPS and IFN‐γ does not increase class II MHC, CD80 or CD86, but upregulates the level of CD40. The genetic defect observed in the DCNOD does not map to the MHC, because the DC from the MHC congenic NOD.H2h4 mouse (H2: Kk, Ak, Ek, Dk) shares the cell surface phenotype of the DCNOD. DC from these NOD.H2h4 also fail to present HEL or the appropriate HEL‐peptide to an antigen‐specific T cell hybridoma. However all the DC irrespective of origin were able to produce TNF‐α, IL‐6, low levels of IL‐12(p70) and NO in response to LPS plus IFN‐γ. A gene or genes specific to the NOD strain, but outside the MHC region, therefore must regulate the differentiation of DC in response to GM‐CSF. This defect may contribute to the complex genetic aetiology of the multifactorial autoimmune phenotype of the NOD strain.

Regulation of murine dendritic cell functions in vitro by taurine chloramine, a major product of the neutrophil myeloperoxidase-halide system

Taurine chloramine (TauCl) is a major chloramine generated in activated neutrophils as a result of the reaction of highly toxic hypochlorous acid and taurine, the most abundant free amino acid in cytosol. In this study we have tested the influence of TauCl on the properties of murine dendritic cells (DC), the major cell population involved in the initiation of an adaptive immune response against pathogenic organisms. N418+, MHC II+, B7‐2+ dendritic cells, generated from the mouse bone marrow cells cultured in the presence of granulocyte–macrophage colony‐stimulating factor, were stimulated by interferon‐γ and lipopolysaccharide to produce nitric oxide, reactive oxygen species, interleukin‐6 (IL‐6), tumour necrosis factor‐α, and IL‐12, in the presence of different doses of TauCl. TauCl differently inhibited the generation of these inflammatory mediators in a dose‐dependent manner. Furthermore, TauCl selectively modulated the ability of DC to induce the release IL‐2 and IL‐10 from T cells. These results suggest that neutrophil‐derived mediators, such as TauCl, at a site of inflammation, may affect the functions of sentinel DC and macrophages, and play a role in maintaining the balance between the inflammatory response and the induction of an antigen‐specific immune response.

Antimicrobial and cytotoxic activity of hypochlorous acid: interactions with taurine and nitrite

Objective. HOCl, a major bactericidal product of neutrophil MPO - halide system reacts with taurine to form taurine chloramine (TauCl), a less toxic anti-inflammatory mediator. Recently, it has been reported that HOCl may also react with nitrite (NO2-), a major end-product of nitric oxide (NO) metabolism, to form very active oxidant, nitryl chloride (NO2Cl). The present study was conducted to elucidate the effect of nitrite on bactericidal and some immunoregulatory properties of HOCl and TauCl.¶Materials: TauCl was prepared from NaOCl and taurine. The reaction was carried out at pH 5.0 and pH 7.4, in the presence or absence of nitrite. All reactions were monitored by UV absorption spectra.¶Methods: Bactericidal activity of HOCl and TauCl in the presence of nitrite was tested by incubation of E. coli with the compounds and determined by the pour-plate method. To test the effect of the compounds on activity of inflammatory cells, murine peritoneal neutrophils (PMN) and macrophages were used. The cells were activated in vitro with either LPS, IFN-γ or zymosan and the production of following mediators was measured: reactive oxygen species using luminol-dependent chemiluminescence; nitric oxide by Griess reaction; TNF-α using capture ELISA. In addition, we tested the effect of HOCl and TauCl on activity of myeloperoxidase (MPO).¶Results: At physiological pH nitrite reacts with HOCl but not with TauCl. This reaction was abolished in the presence of taurine. Nitrite prevented HOCl-mediated bacterial killing, inhibition of MPO activity, cellular cytotoxicity and inhibition of TNF-α Production. Nitrite did not affect any activity of TauCl.¶Conclusion: We have shown that nitrite may react in vitro with HOCl but not with TauCl, to form new biologically active product(s). We did not confirm the hypothesis that a product of HOCl reaction with nitrite is more toxic than HOCl. To the contrary, we found that nitrite diminished bactericidal and immunoregulatory properties of HOCl. In vivo, nitrite will also compete with taurine for reaction with PMN-released HOCl. Nevertheless, due to high concentration of taurine in PMN cytosol, formation of TauCl will be a major regulatory mechanism of MPO-halide-system.

Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37. Effects on the production of inflammatory mediators by mouse macrophages

The ability to produce exopolysaccharides (EPS) is widespread among lactobacilli including Lactobacillus rhamnosus, the commonly used probiotic bacteria. Exopolysaccharides are a major component of the bacterial biofilm with a well‐documented impact on adherence of bacteria to host cells. However, their immunoregulatory properties are unknown. The aim of this study was to examine the immunostimulatory potential of EPS derived from L. rhamnosus KL37. We investigated the effect of EPS on the production of inflammatory mediators by mouse peritoneal macrophages and compared it with the effect of Lipopolysaccharide (LPS). Exopolysaccharides, at concentrations higher than those of LPS, stimulated production of both pro‐inflammatory (TNF‐α, IL‐6, IL‐12) and anti‐inflammatory (IL‐10) cytokines. Interestingly, analysis of the balance of TNF‐α/IL‐10 production showed a potential pro‐inflammatory effect of EPS. Furthermore, our data demonstrate that exposure of macrophages to LPS induced a state of hyporesponsiveness, as indicated by reduced production of TNF‐α after restimulation with either LPS or EPS (‘cross‐tolerance’). By contrast, EPS could make cells tolerant only to subsequent stimulation by the same stimulus. We also examined the relationship between TNF‐α production and activation of mitogen‐activated protein kinases (MAPKs) by EPS and LPS. Pretreatment of macrophages with specific inhibitors of p38 and ERK MAPKs reduced TNF‐α production induced by both stimuli to the same extent. In conclusion, these data demonstrate that EPS can effectively stimulate production of inflammatory mediators by macrophages in vitro. However, to predict whether EPS could be clinically useful as an immunomodulatory agent, further in vivo studies with highly purified EPS are necessary.

Lactobacillus rhamnosus exopolysaccharide ameliorates arthritis induced by the systemic injection of collagen and lipopolysaccharide in DBA/1 mice

Oral administration of some probiotic bacteria (e.g. Lactobacillus rhamnosus) attenuates various types of experimental arthritis, including collagen-induced arthritis (CIA) and inhibits arthritogenic autoantibodies. Much less is known about the possible anti-arthritogenic properties of exopolysaccharide (EPS), the major component of lactic bacteria biofilm. In this study, we asked the question whether systemic administration of EPS derived from L. rhamnosus KL37 depresses the production of anti-collagen IgG and affects the development of CIA in DBA/1 mice. Arthritis was induced employing two models of active CIA, in which mice were immunized with type II collagen (CII) either in the presence of lipopolysaccharide (LPS; mild arthritis with moderate CII-specific IgG production) or with Complete Freund’s Adjuvant and LPS (severe arthritis with massive CII-specific IgG production). Passive CIA was induced by intravenous injection of CII-specific monoclonal antibodies and LPS. Disease progression, the incidence and severity of arthritis, were determined. Serum concentration of CII-specific IgG was measured by enzyme-linked immunosorbent assay. Systemic administration of EPS markedly reduced CII-specific antibody production. Moreover, EPS significantly ameliorated arthritis in the active models of CIA, especially, when LPS alone was used as an adjuvant. In contrast, when arthritogenic antibodies were injected to mice in high amounts, the effect of EPS on the development of passive CIA was negligible and transient. These results show that EPS can suppress active CIA by the inhibition of arthritogenic antibodies production. Therefore, we suggest that EPS or EPS-producing probiotics may be promising agents for the supporting therapy of patients with rheumatoid arthritis.

The influence of opioids on the humoral and cell-mediated immune responses in mice. The role of macrophages

BACKGROUND Our experiments were aimed to test the influence of treatment with different opioids (morphine, fentanyl, methadone) on the humoral and cell-mediated immune responses. METHODS Mice were treated intraperitoneally (ip) with opioids for several days and next either immunized with sheep red blood cells (SRBC) to test the antibody production or skin-sensitized with hapten picryl chloride (PCL) to induce contact hypersensitivity (CHS). In addition, the effects of opioids on the production of reactive oxygen intermediates (ROIs) and cytokines by peritoneal macrophages (Mf) and on the expression of surface markers on these cells and blood leukocytes were estimated. RESULTS Opioids caused an enhancement of ROIs and cytokines production when macrophages were stimulated with zymosan or lipopolysaccharide (LPS) and reduced the expression of antigen presentation markers on Mf. Numbers of anti-SRBC plaque forming cells (PFC) and antibodies titres were lower in mice treated with all tested opioids. Depending on the use of particular opioid and the phase of allergic reaction, effects of the treatment on CHS were diverse. While morphine decreased the early and late phases of induction of CHS responses, methadone increased both reactions. In case of the effector phase of CHS, morphine and fentanyl increased both its early and late stages, while methadone decreased the late reaction. Treatment of recipients with opioids had diverse influence on the passive transfer of CHS in these animals. CONCLUSIONS Our experiments show that the action of opioids on the immune system is a complex phenomenon dependent on such variables as type of opioid, character of response (humoral versus cellular) and types of cells involved. Here Mf seem to play a significant role.

Changes in cell death of peripheral blood lymphocytes isolated from children with acute lymphoblastic leukemia upon stimulation with 7 Hz, 30 mT pulsed electromagnetic field.

Abstract Pulsed electromagnetic field (PEMF) influenced the viability of proliferating in vitro peripheral blood mononuclear cells (PBMCs) isolated from Crohn’s disease patients as well as acute myeloblastic leukemia (AML) patients by induction of cell death, but did not cause any vital changes in cells from healthy donors. Experiments with lymphoid U937 and monocytic MonoMac6 cell lines have shown a protective effect of PEMF on the death process in cells treated with death inducers.The aim of the current study was to investigate the influence of PEMF on native proliferating leukocytes originating from newly diagnosed acute lymphoblastic leukemia (ALL) patients. The effects of exposure to PEMF were studied in PBMCs from 20 children with ALL. PBMCs were stimulated with three doses of PEMF (7 Hz, 30 mT) for 4 h each with 24 h intervals. After the last stimulation, the cells were double stained with annexin V and propidium iodide dye to estimate viability by flow cytometric analysis.The results indicated an increase of annexin V positive as well as double stained annexin V and propidium iodide positive cells after exposure to threefold PEMF stimulation. A low-frequency pulsed electromagnetic field induces cell death in native proliferating cells isolated from ALL patients. The increased vulnerability of proliferating PBMCs to PEMF-induced interactions may be potentially applied in the therapy of ALL.The analysis of expression of apoptosis-related genes revealed changes in mRNA of some genes engaged in the intrinsic apoptotic pathway belonging to the Bcl-2 family and the pathway with apoptosis-inducing factor (AIF) abundance upon PEMF stimulation of PBMCs.

Air pollution, oxidative stress, and exacerbation of autoimmune diseases

A number of epidemiological studies have shown a strong association between exposure to ambient airborne particulate matter (PM 2.5, PM < 1.0) and lung or cardiovascular diseases characterised by high mortality and morbidity. However, much less is known about the role of air pollution in the pathogenesis of autoimmune diseases, which constitutes a significant problem in modern society. This paper summarises the state of current research regarding the influence of PM on the development and/or progression of autoimmune diseases. A brief review of the great body of research concerning pathogenesis of autoimmune disorders is presented. Then, the scope of our review is narrowed to the research related to the impact of particulate matter on oxidative and nitrosative stress, as well as exacerbation of chronic inflammation, because they can contribute to the development of autoimmune diseases. Moreover, we discuss the impact of various components of PM (metal, organic compounds) on PM toxicity and the ability to generate oxidants.

Distinct effects of Lactobacillus plantarum KL30B and Escherichia coli 3A1 on the induction and development of acute and chronic inflammation.

Objective Enteric bacteria are involved in the pathogenesis of ulcerative colitis. In experimental colitis, a breakdown of the intestinal epithelial barrier results in inflow of various gut bacteria, induction of acute inflammation and finally, progression to chronic colitis. Material and methods In the present study we compared pro-inflammatory properties of two bacterial strains isolated from human microbiome, Escherichia coli 3A1 and Lactobacillus plantarum KL30B. The study was performed using two experimental models of acute inflammation: peritonitis in mice and trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. Results Both bacterial strains induced massive neutrophil infiltration upon injection into sterile peritoneal cavity. However, peritoneal exudate cells stimulated in vitro with E. coli 3A1, produced far more nitric oxide, than those stimulated with L. plantarum KL30B. Interestingly, distinct effect on the development of TNBS-induced colitis was observed after oral administration of the tested bacteria. Lactobacillus plantarum KL30B evoked strong acute colitis. On the contrary, the administration of E. coli 3A1 resulted in a progression of colitis to chronicity. Conclusions Our results show that distinct effects of bacterial administration on the development of ongoing inflammation is strain specific and depends on the final effect of cross-talk between bacteria and cells of the innate immune system.