Janusz Marcinkiewicz

1-Methylnicotinamide and nicotinamide: two related anti-inflammatory agents that differentially affect the functions of activated macrophages

Introduction:1-Methylnicotinamide (MNA), a major metabolite of nicotinamide (NA), is known to exert anti-inflammatory effects in vivo. Treatment of inflammatory skin diseases by topical application of MNA provides certain advantages over the use of NA. However, in contrast to NA, the molecular mechanisms of the anti-inflammatory properties of MNA are not well known. In this study the influence of exogenous MNA and NA in vivo on the generation of inflammatory mediators by macrophages (Mϕ) was investigated.Materials and Methods:Peritoneal Mϕ of CBA/J mice were activated in vitro with lipopolysaccharide and incubated with MNA or NA. The effect of these compounds on biological functions of Mϕ was measured by evaluation of the production of reactive oxygen species (ROS) by luminol-dependent chemiluminescence, cytokines and prostaglandin E2 (PGE2) by ELISA, and nitric oxide (NO) by the Griess method. Moreover, the expressions of inducible NO synthase and cyclooxygenase-2 were measured by Western blotting.Results:It was shown that at non-cytotoxic concentrations, NA inhibits the production of a variety of pro-inflammatory agents, such as tumor necrosis factor α, interleukin 6, NO, PGE2, and the generation of ROS. In contrast to NA, exogenous MNA inhibited only the generation of ROS, while its effect on the synthesis of other mediators was negligible.Conclusions:These results indicate that the anti-inflammatory properties of MNA demonstrated previously in vivo do not depend on its capacity to suppress the functions of immune cells, but more likely may be related to its action on vascular endothelium. The authors suggest that the limited permeability for exogenous MNA, in contrast to that for NA, may be responsible for its lack of suppressor activity against Mϕ.

Effect of taurine chloramine, the product of activated neutrophils, on the development of collagen-induced arthritis in DBA 1/J mice.

Summary.u2002Taurine chloramine (TauCl), a product of neutrophil myeloperoxidase – halide system, formed by a reaction of taurine with HOCl, is known as an anti-microbial and anti-inflammatory long-lived oxidant. We previously reported that TauCl inhibits in vitro the production of proinflammatory cytokines (IL-6, IL-8) by RA synoviocytes. Therefore we performed this study to investigate the effect of TauCl treatment on the development of collagen-induced arthritis (CIA) in DBA1/J mice. Early administration of TauCl (after primary immunization) resulted in the delay of the onset of CIA, but had no effect on severity of arthritis. TauCl, given daily for 21 days after booster immunization, did not reduce the symptoms of arthritis in those mice, which already developed CIA, but significantly diminished incidence of the disease (55% vs. 90% of placebo mice). The mechanism of this effect is unknown. This is the first in vivo study suggesting that TauCl may be used for immune intervention in chronic inflammatory diseases.

Enhancement of immunogenic properties of ovalbumin as a result of its chlorination

1. Chlorination of ovalbumin results in its enhanced immunogenic properties. 2. This has been evaluated by the interleukin-2 production after incubation of the modified protein with antigen presenting cells and T helper cells.

Leukotrienes modulate cytokine release from dendritic cells

Leukotriene B4 (LTB4) and cysteinyl leukotrienes (CysLTs) are known as potent mediators of inflammation, whereas their role in the regulation of adaptive immunity remains poorly characterized. Dendritic cells (DCs) are specialized antigen‐presenting cells, uniquely capable to initiate primary immune responses. We have found that zymosan, but not lipopolysaccharide (LPS) stimulates murine bone marrow‐derived dendritic cells (BM‐DCs) to produce large amounts of CysLTs and LTB4 from endogenous substrates. A selective inhibitor of leukotriene synthesis MK886 as well as an antagonist of the high affinity LTB4 receptor (BLT1) U‐75302 slightly inhibited zymosan‐, but not LPS‐stimulated interleukin (IL)‐10 release from BM‐DCs. In contrast, U‐75302 increased zymosan‐stimulated release of IL‐12 p40 by ∼23%. Pre‐treatment with transforming growth factor‐β1 enhanced both stimulated leukotriene synthesis and the inhibitory effect of U‐75302 and MK886 on IL‐10 release from DCs. Consistent with the effects of leukotriene antagonists, exogenous LTB4 enhanced LPS‐stimulated IL‐10 release by ∼39% and inhibited IL‐12 p40 release by ∼22%. Both effects were mediated by the BLT1 receptor. Ligands of the high affinity CysLTs receptor (CysLT1), MK‐571 and LTD4 had little or no effect on cytokine release. Agonists of the nuclear LTB4 receptor peroxisome proliferator‐activated receptor‐α, 8(S)‐hydroxyeicosatetraenoic acid and 5,8,11,14‐eicosatetraynoic acid, inhibited release of both IL‐12 p40 and IL‐10. Our results indicate that both autocrine and paracrine leukotrienes may modulate cytokine release from DCs, in a manner that is consistent with previously reported T helper 2‐polarizing effects of leukotrienes.

Aggregates of denatured proteins stimulate nitric oxide and superoxide production in macrophages.

ObjectiveDenatured proteins are deposited in damaged tissues, around implanted biomaterials, during natural aging as well as in a heterogeneous group of amyloid diseases, such as Alzheimer’s disease. There is evidence that tissue damage observed in amyloidosis is mediated mainly by factors released from activated macrophages, such as superoxide and nitric oxide (NO), as opposed to direct interaction between amyloid fibrils and nonimmune cells.MethodsMouse resident peritoneal macrophages were stimulated in serum-free medium with different preparations of nonamyloidogenic proteins: alcohol dehydrogenase (AD), bovine serum albumin (BSA) or fibrinogen (FG). Intra- and extracellular superoxide production was measured by, respectively, nitro blue tetrazolium (NBT) reduction and lucigenin-enhanced chemiluminescence. Levels of nitrite (reflecting NO release) were measured in culture supernatants.ResultsAggregates of denatured, nonamyloidogenic proteins, but not their native or denatured but not aggregated counterparts, stimulated superoxide and/or NO production in macrophages. The NO production was mediated by β1 and β2 integrins, with a possible contribution of receptor for advanced glycation end products (RAGE). It was catalyzed by inducible NO synthase (iNOS), enhanced synergistically by interferon-γ (IFN-γ), and inhibited by covalently modified proteins—components of advanced glycation end products. Although intracellular superoxide production was stimulated significantly by denatured BSA and AD, but not by FG, both denatured BSA and FG strongly enhanced zymosan-stimulated extracellular release of reactive oxygen species.ConclusionOur results point at similarities in macrophage responses to denatured nonamyloidogenic proteins and to amyloid fibrils. Thus, the tissue injury observed in amyloidosis may result from overstimulation of mechanisms that, under physiological conditions, enable macrophages to recognize and remove denatured proteins.

Heme oxygenase-1 participates in the anti-inflammatory activity of taurine chloramine

Summary.Interleukin-6 (IL-6) and interleukin-8 (IL-8) are implicated in the pathogenesis of rheumatic diseases. In affected joints fibroblast-like synoviocytes (FLS) are the major source of these pro-inflammatory cytokines. We have previously found that production of both cytokines is inhibited in vitro by taurine chloramine (Tau-Cl). Heme oxygenase (HO-1) activity was also reported to restrict synthesis of various inflammatory mediators, including IL-6 and IL-8. The aim of present study was to investigate whether this enzyme activity is implicated in the mechanism of Tau-Cl suppressive effect. We have shown that in rheumatoid FLS both hemin (known HO-1 inducer) and Tau-Cl significantly up-regulate HO-1 expression at the mRNA and protein levels and simultaneously inhibit IL-1β-triggered production of pro-inflammatory cytokines. However, the inhibitory potency of these compounds differs, because hemin is more potent inhibitor of IL-8 than IL-6 production, while Tau-Cl exerts opposite effect. Importantly, pretreatment of the cells with HO-1 inhibitor completely reverses the inhibitory effect of hemin on both cytokines production. However, in Tau-Cl treated cells this inhibitor fully restores only IL-8 secretion but has weaker effect on IL-6 response. Thus, the present results: (i) support HO-1 activity to be relevant to negatively control production of pro-inflammatory cytokines, and (ii) underline implication of HO-1 in mediating Tau-Cl inhibitory action.

Prostanoids and MPO-halide system products as a link between innate and adaptive immunity.

and The crosstalk between innate adaptive immunity is regulated by cytokines and complex interactions between cells of the immune system. A variety of endogenous agents are involved in the regulation of the cytokine network. Especially, eicosanoids and ROIs have a great impact on the regulation of cytokine production. Eicosanoids (prostanoids, leukotrienes and lipoxins) are produced mainly by inflammatory cells while their receptors are distributed on the cells of both arms of the immune system. Depending on the predominant prostanoid produced and the profile of prostanoid receptors expression on immune cells, eicosanoids can selectively regulate the production of Th1 and Th2 driven cytokines. Inflammatory cells (neutrophils, macrophages), are also a rich source of large amounts of ROIs. In this paper we have focused on the role of taurine chloramine (TauCl), the physiological product of neutrophil MPO-halide system, in the regulation of immune system. It is well documented that TauCl has pleiotropic effects on the inductive phase of the immune response. TauCls immunoregulatory properties result from its ability to modulate the production of cytokines and eicosanoids. Finally, we conclude that eicosanoids and ROIs provide an important link between the afferent branches and the innate and adaptive immune response.

Further studies on the regulation of lymphokine biosynthesis in contact sensitivity

Contact sensitivity (CS) results from a series of cellular interactions involving CD4+ T cells and macrophages. We have studied the production of lymphokines by T cells from mice immunized by contact sensitization and by a variety of stimuli leading to CS or tolerance to CS. Primed T cells from mice immunized for CS are predominantly of the TH1 type. Conversely, a failure in the activation of these antigen-specific TH1 cells is a key step in the induction of tolerance to CS. Spleen cells from tolerant mice can suppress the production of lymphokines from primed cells, both in an adoptive transfer system and when mixed with effector cells in vitro. The relative importance of lymphokine competition, prostaglandin production by adherent cells, and other mechanisms underlying this suppression is discussed.

CYTOTOXICITY OF TAURINE METABOLITES DEPENDS ON THE CELL TYPE

We report that the effect of Tau-Cl on the cell fate strongly depends on the cellular context. In leukemic Jurkat cells Tau-Cl (> 200 microM) triggers mitochondrial, p53-independent apoptosis and amplifies PCD induced by anti-Fas treatment. In contrast, Tau-Cl affects RA FLS in a dose-dependent manner. At the noncytotoxic (200-400 microM) concentrations it induces: (i) p53-dependent growth arrest (Kontny et al., 2005), and (ii) Bax translocation and caspase 9 activity. Although the last events are characteristic for apoptotic state, there is not execution of RA FLS apoptosis, probably due to simultaneous inhibition of caspase 3 activity and prevention of PARP degradation. The last two events suggest an excessive ATP deprivation in Tau-Cl-treated RA FLS. At sufficiently high concentrations (> or = 500 microM) Tau-Cl causes therefore necrosis of these cells. Altogether our results suggest that Tau-Cl is able to eliminate the cells with both functional (RA FLS) and mutated (Jurkat) p53 tumor suppressor. This observation is clinically relevant because Tau-Cl is used in many animal inflammatory models and its sodium salt (used in this study) has been introduced to human therapy (Gottardi and Nagl, 2002; Teuchner et al., 2005).

Macrophage Function in Allergic and Autoimmune Responses

Macrophages are involved in various mechanisms of allergic and autoimmune response, especially in delayed-type hypersensitivity. Apart from antigen presentation, macrophages also act as effector cells in cellular hypersensitivity reaction induced by either pathogen during infection, hapten-antigen or self-antigen in pathological conditions. Their ability to secrete Th2-dependent cytokines as well as reactive oxygen and nitrogen intermediates exacerbates the severity of symptoms of allergy and asthma. Macrophage properties and activities could be modulated by numerous factors including drugs and biologically active compounds that may be used in future therapy. The present review is focused on the current knowledge about macrophage functions in allergy, autoimmunity and the mechanisms of their modulation.