Krzysztof Bryniarski

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.

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

BACKGROUND T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. OBJECTIVE We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. METHODS T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. RESULTS Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8(+) suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH(-/-) or miRNA-150(-/-) mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. CONCLUSIONS This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity.

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5+, CD19+, CD22+, B220+, Thy1+, and Mac1+, suggesting that they are B-1 B cells. DTH initiation is absent in μMT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.

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.

Epicutaneous immunization induces αβ T-cell receptor CD4 CD8 double-positive non-specific suppressor T cells that inhibit contact sensitivity via transforming growth factor-β

Since it was previously shown that protein antigens applied epicutaneously in mice induce allergic dermatitis mediated by production of T helper 2 (Th2) cytokines we postulated that this might induce suppression of Th1 immunity. Here we show that epicutaneous immunization of normal mice with a different protein antigen applied on the skin in the form of a patch induces a state of subsequent antigen‐non‐specific unresponsiveness caused by suppressor T cells (Ts) that inhibit sensitization and elicitation of effector T‐cell responses. Suppression is transferable in vivo by αβ‐T‐cell receptor CD4+ CD8+ double positive lymphocytes harvested from lymphoid organs of skin patched animals and are not major histocompatibility complex‐restricted nor antigen specific. Both CD25+ and CD25– CD4+ CD8+ T cells are able to suppress adoptive transfer of Th1 effector cells mediating cutaneous contact sensitivity. In vivo treatment with monoclonal antibodies showed that the cytokines interleukin (IL)‐4, IL‐10 and transforming growth factor‐β (TGF‐β) are involved in the induction of the Ts cells. Additionally, using IL‐10–/– mice we found that IL‐10 is involved in skin induced tolerance. Further in vitro experiments showed that lymph node cells of skin tolerized mice non‐specifically suppress [3H]thymidine incorporation by antigen‐stimulated immune cells and this effect can be abolished by adding anti‐TGF‐β, but not anti‐IL‐4 nor anti‐IL‐10 antibodies. These studies indicate the crucial role of TGF‐β in skin induced tolerance due to non‐antigen‐specific Ts cells and also show that IL‐4, IL‐10 and TGF‐β play an important role in the induction of epicutaneously induced Ts cell suppression.

Subpopulations of mouse Testicular macrophages and their immunoregulatory function

Problem:  Testicular macrophages (TMf) participate together with Sertoli cells in formation of blood–testis barrier. The present experiments were aimed to test their immunoregulatory functions in vivo and in vitro.

Epicutaneously induced TGF-β-dependent tolerance inhibits experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a devastating autoimmune disorder of the central nervous system (CNS) with limited treatment modalities. To evaluate the efficacy of epicutaneous (e.c.) tolerance induction in the prevention of CNS autoimmunity, we utilized an animal model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE). We show that application of myelin basic protein (MBP) to the skin prior to the induction of EAE by immunization with MBP protected mice from developing disease. In addition, e.c. application of MBP at the first clinical signs of EAE, also resulted in suppression of disease. This therapeutic effect was transferable to naïve recipients with lymph node cells from MBP-treated mice. These regulatory cells were found to be antigen non-specific, as suppression of EAE also occurred when the foreign antigens OVA or TNP were e.c. applied. The mechanistic basis for the tolerance was found to be the production of TGF-beta by the antigen induced toleragenic regulatory T cells. These data demonstrate that e.c.-induced regulatory T cells are potent inhibitors of antigen-specific T cell responses, and suggest that e.c. tolerization may have potential effectiveness in the treatment of autoimmune disorders.

Epicutaneous Application of Protein Antigens Incorporated into Cosmetic Cream Induces Antigen-Nonspecific Unresponsiveness in Mice and Affects the Cell-Mediated Immune Response

Background: Protein antigens applied epicutaneously by the patch method induce allergic dermatitis mediated by IgE antibodies in mice and simultaneously significant suppression of Th1-mediated delayed-type hypersensitivity (DTH) reactions. Methods: We developed a method in which protein antigens (calf collagen, elastin, keratin, TNP-substituted mouse Ig) were homogenized with neutral cream. Animals treated epicutaneously with such preparations were tested for contact sensitivity to TNP hapten or DTH hypersensitivity to hemocyanin. Antigen specificity of induced unresponsiveness was tested in vivo in ‘transfer-in’ and ‘transfer-out’ experiments. The influence of skin-induced regulatory cells on in vitro [3H]thymidine uptake by immune cells as well as the possible mode of their action using anticytokine antibodies were tested. Results: Our procedure in which different protein antigens are applied on the skin in the form of cream induces a state of antigen-nonspecific unresponsiveness affecting cell-mediated immune responses in mice. Cream alone has no such effect. Suppression is transferable in vivo by TCR-αβ lymphocytes, while Tγδ cells show no activity. In vitro, lymphoid cells of skin-tolerized mice suppress [3H]-thymidine incorporation by immune cells and this effect can be abolished by adding anti-TGF-β but neither anti-IL-4 nor anti-IL-10 antibodies. Conclusions: Lack of antigen-specifity of unresponsiveness induced by epicutaneous deposition of cream containing protein antigens resembles ‘determinant spreading’ in oral tolerance induced by antigen feeding. This may suggest that similar immunoregulatory mechanisms operate on these two bodily surfaces.

Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice

2,4,6-Trinitrophenyl (TNP) hapten-labeled peritoneal macrophages (Mf) given intravenously (iv) to recipients are poor inducers of contact sensitivity (CS) reactions unless Mf donors are pretreated with low doses of cyclophosphamide (CY). In vivo CY is converted into active alkylating metabolites, phosphoramide mustard (PM) and acrolein (ACR). Our experiments aimed to test how in vitro treatment of non-immunogenic Mf with different concentrations (10(-5) to 10(-7) M) of CY metabolites will influence their immunogenicity and other biological functions. Instead of chemically unstable PM, we used structurally and functionally similar nitrogen mustard (NM). Our experiments show that treatment of Mf with ACR or NM stimulates the in vitro production of pro-inflammatory IL-6 and IL-12 and down-regulates anti-inflammatory IL-10 and TGF-beta cytokines. In vivo non-immunogenic TNP-Mf become capable of inducing CS reactions in two situations: first, after treatment with NM or ACR and second, when cell recipients are received iv before Mf transfer of monoclonal antibodies against IL-10 and/or TGF-beta (500 mug per animal). Treatment with NM, but not with ACR, was also an efficient stimulus for production by Mf of significantly increased levels of reactive oxygen intermediates (ROIs). In summary, our experiments show that CY metabolites can significantly increase the specific immune response as well as nonspecific innate reaction (ROIs production) and support the notion that CY and its metabolites can be a promising accessory tool when upregulation of the immune response is desired.

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases.