Krzysztof Bryniarski

Publications of Krzysztof Bryniarski

• [Immunoregulation by interference RNA (iRNA) - mechanisms, role, perspective].

  Authors: Emilia Sikora, Włodzimierz Ptak, Krzysztof Bryniarski

  Postȩpy higieny i medycyny doświadczalnej (Online). 01/2011; 65:482-95.

  The functioning of an organism depends on the precise control mechanisms, constantly adjusted to the actual state. Therefore, there is a need for efficient communication between both adjacent andThe functioning of an organism depends on the precise control mechanisms, constantly adjusted to the actual state. Therefore, there is a need for efficient communication between both adjacent and distant cells, which may be executed by proteins such as hormones, neurotransmitters and cytokines. Recently another means of regulation has emerged - short regulatory RNAs (srRNAs). Although discovered only a couple of years ago, the mechanism of RNA interference has already become a topic of thousands of publications, defining its roles in both physiological and pathological processes, such as cancerogenesis and autoimmunization. RNAs regulating cell function may be coded in its genome (both exons and introns) or be introduced from the external environment. In mammals microRNAs (miRNAs) cooperate with proteins from the Ago/PIWI family to form effector ribonucleoprotein complexes, and owing to their complementarity to the target mRNA, control genes' expression at the posttranscriptional level, either through the suppression of mRNA translation or through mRNA degradation. SrRNAs are crucial regulators throughout the development of immune cells, starting from hematopoietic stem cells, up to the effector cells of the adaptive immune response. Moreover, some of the regulatory cells perform their function by releasing miRNAs, which are then transported to the target cells, possibly enclosed in the exosomes.

• Epicutaneous immunization with protein antigen in the presence of TLR4 ligand induces TCRalphabeta+CD4+ T contrasuppressor cells that reverse skin-induced suppression of Th1-mediated contact sensitivity.

  Authors: Wlodzimierz Ptak, Monika Majewska, Krzysztof Bryniarski, Maria Ptak, Francis M Lobo, Katarzyna Zajac, Philip W Askenase, Marian Szczepanik

  Journal of immunology (Baltimore, Md. : 1950). 02/2009; 182(2):837-50.

  Our previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecificOur previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecific unresponsiveness due to suppressor CD4+8+ T cells (Ts) that inhibit Th1-mediated contact sensitivity (CS) reactions via released TGF-beta. In the present work we show that EC immunization with Ag together with the TLR4 ligand LPS induced cells that could prevent suppression by the Ag-nonspecific Ts. These up-regulatory cells, called contrasuppressor T cells (Tcs), belong to a population of Ag-specific TCRalphabeta CD4+ lymphocytes and are different from Th1 CD4+ cells that mediate the CS reaction. Experiments using knockout mice showed that EC induced contrasuppression is MyD88, INF-gamma, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-gamma mAb showed that IFN-gamma is required for induction of Tcs cells but does not play a crucial role in the effector phase of contrasuppression. Additionally, treatment of CS effector cells with rIL-12 makes them resistant to EC induced suppression without affecting Ts cells, whereas IL-12 neutralization in vitro abrogates contrasuppression. These data show that IL-12 is indeed involved in the effector phase of EC induced contrasuppression and that this cytokine does not act directly on Ts cells. The mechanism of action of Tcs protects Th1 effector cells mediating CS from the nonspecific Ts, leaving suppression to other Ags intact. Ts and Tcs cells do not influence each other and can be induced simultaneously in the same animal.

• Anti-inflammatory effect of 1-methylnicotinamide in contact hypersensitivity to oxazolone in mice; involvement of prostacyclin.

  Authors: Krzysztof Bryniarski, Rafal Biedron, Andrzej Jakubowski, Stefan Chlopicki, Janusz Marcinkiewicz

  European journal of pharmacology. 02/2008; 578(2-3):332-8.

  1-methylnicotinamide (MNA) displays anti-inflammatory effects in patients with contact dermatitis, though the mechanisms involved remain unknown. Herein, we examined the anti-inflammatory effects of1-methylnicotinamide (MNA) displays anti-inflammatory effects in patients with contact dermatitis, though the mechanisms involved remain unknown. Herein, we examined the anti-inflammatory effects of MNA and its parent molecule, nicotinamide, in the contact hypersensitivity reaction to oxazolone in CBA/J inbred mice. Feeding mice with MNA or nicotinamide (100 mg/kg, 10 days) resulted in the inhibition of the development of contact hypersensitivity reaction by 37% and 35%, respectively, as assessed by the magnitude of ear swelling. This effect was not associated with changes in the expression of adhesion molecules (CD49d(+) and CD54(+)) on CD4(+) and CD8(+) oxazolone-specific T lymphocytes, the major cell component of an inflammatory infiltrate in contact hypersensitivity reaction. Furthermore, in the adoptive transfer model of contact hypersensitivity reaction, pretreatment of mice (recipients of oxazolone-specific T cells), with MNA, resulted in a remarkable anti-inflammatory effect (inhibition of contact hypersensitivity reaction by 66%). Interestingly, in the presence of prostanoid IP receptor antagonist R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO-3244794) (10 mg/kg) the MNA was inactive. In summary, pretreatment with MNA profoundly attenuated contact hypersensitivity reaction in vivo. In particular, the vessel dependent phase of contact hypersensitivity reaction was affected, in spite of the fact that MNA did not alter the expression of adhesive molecules on oxazolone-specific T lymphocytes. However, the anti-inflammatory action of MNA was completely reversed by the antagonist of prostanoid IP receptor. Accordingly, our results demonstrate for the first time that anti-inflammatory properties of MNA are linked to endothelial, PGI(2)-mediated mechanisms.

• Mesenteric lymph node Tgammadelta cells induced by gastrectomy in mice suppress cell-mediated immune response in vitro via released TGF-beta.

  Authors: Andrzej Gryglewski, Pawel Majcher, Krzysztof Bryniarski, Stanislaw Konturek, Maria Ptak, Wlodzimierz Ptak, Marian Szczepanik

  The Journal of surgical research. 10/2007; 142(1):66-71.

  We have shown previously that gastrectomy, but not laparotomy alone, severely impairs contact sensitivity responses in vivo and selectively alters cell trafficking in gut associated lymphatic tissue.We have shown previously that gastrectomy, but not laparotomy alone, severely impairs contact sensitivity responses in vivo and selectively alters cell trafficking in gut associated lymphatic tissue. Here, we investigate the immunological role of different subpopulations of mesenteric lymph node cells (MLNCs) in the inhibition of contact sensitivity as well as their suppressive mechanisms. Suppressive cells were isolated from the mesenteric lymph nodes of gastrectomized mice and were added to cultures of lymphocytes from mice immunized with trinitrophenyl-chloride. These MLNCs inhibited the proliferation of sensitized lymphocytes in response to antigen. Depletion experiments revealed that the suppressive MLNCs are Tgammadelta+ cells, but not Talphabeta+ cells. Neutralizing antibodies to IL-4, IL-10, and tissue growth factor-beta (TGF-beta) revealed that suppression was dependent on TGF-beta, but not the other cytokines. We conclude that surgical stress induced by gastrectomy causes accumulation of Tgammadelta+ lymphocytes in gut associated lymphatic tissue and that these cells suppress the cell-mediated response in vitro in an antigen-non specific manner via TGF-beta. This cytokine can possibly prevent in vivo the development of autoimmune responses following severe tissue trauma in the gastrointestinal tract.

• Toll-like receptor ligands reverse suppression of contact hypersensitivity reactions induced by epicutaneous immunization with protein antigen.

  Authors: Włodzimierz Ptak, Krzysztof Bryniarski, Maria Ptak, Monika Majewska, Andrzej Gamian, Francis M Lobo, Marian Szczepanik

  International archives of allergy and immunology. 02/2006; 139(3):188-200.

  BACKGROUND: Epicutaneous (EC) immunization with protein antigens has been shown to induce antigen nonspecific suppression of subsequent T cell-dependent contact hypersensitivity (CS) reactions afterBACKGROUND: Epicutaneous (EC) immunization with protein antigens has been shown to induce antigen nonspecific suppression of subsequent T cell-dependent contact hypersensitivity (CS) reactions after active immunization. The aim of this work was to test if EC application of Toll-like receptor (TLR) ligands together with protein antigen could reverse suppression of CS. METHODS: Mice were EC immunized by applying gauze patches soaked with a solution of protein antigen alone or in the presence of crude bacterial material (bacterial lysates or heat-killed bacteria) or purified TLR ligands and then tested for CS response. To test if reversal of EC-induced suppression is antigen-specific, mice were patched with TNP- or OX-substituted mouse Ig alone or together with LPS and then tested for CS with corresponding or non-cross-reacting hapten. Influence of EC immunization on cytokine production by lymph node cells was measured by ELISA. RESULTS: EC immunization with protein antigen induces antigen nonspecific suppression that can be reversed by crude bacterial material as well as purified TLR-2, TLR-3, TLR-4, and TLR-9 ligands. The effect of TLR-4 ligand LPS was not observed in the Tlr-4 mutant C3H/HeJ mouse, indicating that this effect was dependent upon intact TLR-4 signaling. Unlike the antigen nonspecific suppression of CS by EC immunization with antigen alone, the reversal of suppression by TLR ligands was specific for the protein antigen applied in the EC protocol. CONCLUSIONS: Our results strongly suggest that EC immunization with protein antigen together with TLR ligands induces a particular antigen-specific cell population, akin to previously described contrasuppressor cells, which protects immune cells against the action of suppressor cells but have no direct influence on antigen nonspecific suppressor cells induced by antigen alone.

• Epicutaneously induced TGF-beta-dependent tolerance inhibits experimental autoimmune encephalomyelitis.

  Authors: Marian Szczepanik, Monika Tutaj, Krzysztof Bryniarski, Bonnie N Dittel

  Journal of neuroimmunology. 08/2005; 164(1-2):105-14.

  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 inductionMultiple 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 immunization induces alphabeta T-cell receptor CD4 CD8 double-positive non-specific suppressor T cells that inhibit contact sensitivity via transforming growth factor-beta.

  Authors: Marian Szczepanik, Krzysztof Bryniarski, Monika Tutaj, Maria Ptak, Joanna Skrzeczynska, Philip W Askenase, Wlodzimierz Ptak

  Immunology. 06/2005; 115(1):42-54.

  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 induceSince 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 alphabeta-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-beta (TGF-beta) 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 [(3)H]thymidine incorporation by antigen-stimulated immune cells and this effect can be abolished by adding anti-TGF-beta, but not anti-IL-4 nor anti-IL-10 antibodies. These studies indicate the crucial role of TGF-beta in skin induced tolerance due to non-antigen-specific Ts cells and also show that IL-4, IL-10 and TGF-beta play an important role in the induction of epicutaneously induced Ts cell suppression.

• [Experimental methods in the treatment of multiple sclerosis--new possibilities and hopes]

  Authors: Marian Szczepanik, Monika Tutaj, Iwona Filipczak-Bryniarska, Krzysztof Bryniarski

  Przegla̧d lekarski. 02/2005; 62(2):115-8.

  Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelinization. Although our knowledge on the pathomechanism of MS has evolved dramaticallyMultiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelinization. Although our knowledge on the pathomechanism of MS has evolved dramatically in recent years; however there is still no effective cure for MS. So far, non-specific immune suppressive agents are used to slow down the disease. Recently, there are many efforts to find a new method that would allow to control specifically unwanted immune response. It might be achieved by affecting antigen recognition or induction of "immune deviation".

• Modulation of testicular macrophage activity by collagenase.

  Authors: Krzysztof Bryniarski, Marian Szczepanik, Maria Ptak, Włodzimierz Ptak

  Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society. 02/2005; 43(1):37-41.

  Testicular macrophages (TMs) are located in the interstitial tissue of male gonad. These phagocytic cells take part in forming the organ-specific functional blood-testis barrier and participate inTesticular macrophages (TMs) are located in the interstitial tissue of male gonad. These phagocytic cells take part in forming the organ-specific functional blood-testis barrier and participate in the regulation of the local hormonal balance. In the present study, we isolated TMs from testicular tissues using previously described methods--mechanical (M-TMs) or enzymatic, by treatment with collagenase (E-TMs) and then we studied production by these cells of several cytokines and reactive oxygen intermediates (ROI's). Similarly treated oil-induced peritoneal macrophages (PMs) were used as control cells. PMs had a higher baseline level of production of TNF-alpha, IL-6, IL-10 and IL-12 than M-TMs and collagenase treatment increased the production of these cytokines (except IL-12) by both cell populations. This effect was significantly more expressed in TMs. In contrast to PMs, TMs produced little ROI's when stimulated by zymosan. We conclude that in the case of local inflammation in the testis, ROI-negative TMs do not contribute to the tissue damage and instead may direct the local immune response into humoral pathway.

• Subpopulations of mouse testicular macrophages and their immunoregulatory function.

  Authors: Krzysztof Bryniarski, Marian Szczepanik, Katarzyna Maresz, Maria Ptak, Włodzimierz Ptak

  American journal of reproductive immunology (New York, N.Y. : 1989). 08/2004; 52(1):27-35.

  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 vivoPROBLEM: 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. METHOD OF STUDY: TMf were purified by glass adherence, rosetting with opsonized erythrocytes and fractionation on discontinuous Percoll gradient (over 95% purity). Their antigen-presenting capacity in humoral and cell-mediated responses was tested in vitro (Mishell-Dutton cultures, proliferation assay) and in vivo (induction of contact sensitivity reaction). RESULTS: TMf represent a heterogeneous cell population. Heavier Percoll fractions produce little transforming growth factor (TGF)-beta and are efficient antigen-presenting cells in humoral and cell-mediated immune responses. Lighter fractions produce high amounts of TGF-beta and are rather tolerogenic than immunogenic. Their immunosuppressive activity can be prevented by treatment of TMf donors with cyclophosphamide or in vitro by anti-TGF-beta monoclonal antibody. In non-separated TMf population the immunosuppressive activity prevails. CONCLUSIONS: Subpopulation of TMf able to trigger specific immune responses is present in the testis but remains under control of other TMf subpopulation which minimizes the risk of development of autoimmune reactions.

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

  Authors: Marian Szczepanik, Moe Akahira-Azuma, Krzysztof Bryniarski, Ryohei F Tsuji, Ivana Kawikova, Wlodzimierz Ptak, Claudia Kiener, Regis A Campos, Philip W Askenase

  Journal of immunology (Baltimore, Md. : 1950). 01/2004; 171(11):6225-35.

  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 sequentialWe 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 micro MT 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.

• A two step procedure to fractionate mouse testicular macrophages with different cytokine profiles.

  Authors: Krzysztof Bryniarski, Katarzyna Szewczyk, Maria Ptak, Małgorzata Bobek, Włodzimierz Ptak

  Archivum immunologiae et therapiae experimentalis. 02/2002; 50(3):225-9.

  Cells isolated enzymatically from the interstitial tissue of mouse male gonads are composed of macrophages, Leydig cells, and myofibroblasts. They can be separated on density gradients, either byCells isolated enzymatically from the interstitial tissue of mouse male gonads are composed of macrophages, Leydig cells, and myofibroblasts. They can be separated on density gradients, either by sedimentation (Ficoll) or flotation (Percoll) into several fractions according to the different buoyant densities of the different cells in the mixture. Macrophages (Fc gamma R+, esterase+) present in cell mixtures can by highly enriched (to 95% purity) in a single step by rosetting with opsonized erythrocytes followed by sedimentation on Lymphoprep. Separate fractions of highly purified (over 95%) macrophages obtained by the successive use of density gradients and rosetting differ significantly in the production of cytokines, e.g. cells from fractions at lower density produce little IL-6, cells from fractions at higher density are poor producers of TNF-alpha, while testicular macrophages (TMf) in intermediate fractions produce significant amounts of both cytokines. These differences may suggest that particular subpopulations of testicular macrophages play different biological roles in the testis.

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

  Authors: Janusz Marcinkiewicz, Agnieszka Grabowska, Joanna Bereta, Krzysztof Bryniarski, Bernadeta Nowak

  Immunopharmacology.

  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-α, and PGE2.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-α, 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-α, 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.

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

  Authors: Marian Szczepanik, Monika Tutaj, Krzysztof Bryniarski, Bonnie N. Dittel

  Journal of Neuroimmunology.

  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 inductionMultiple 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-β 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.

• The in vivo and in vitro effects of an alkylating agent, mechlorethamine, on IL-6 production in mice and the role of macrophages

  Authors: Krzysztof Bryniarski, Maria Ptak, Wtodzimierz Ptak

  Immunopharmacology.

  Alkylating agents, cyclophosphamide (CY) and the related compound mechlorethamine (NM), significantly increase in vivo the blood level of IL-6 but not of IL-1. Since in vitro CY is inactive we haveAlkylating agents, cyclophosphamide (CY) and the related compound mechlorethamine (NM), significantly increase in vivo the blood level of IL-6 but not of IL-1. Since in vitro CY is inactive we have used in our experiments NM, a compound structurally and functionally related to phosphoramide mustard, the natural biologically active metabolite of CY. Thioglycollate or oil-induced peritonal macrophages (Mf) of four different mouse strains treated with NM produce significantly more IL-6 than the non-treated cells. In contrast, under these conditions, the production of IL-1, TNFα and NO/NO2 radicals is not affected. The NM-induced elevated production of IL-6 by Mf could not be further increased by the treatment of cells with LPS, which may suggest that both agonists stimulate the same signalling pathway. The antineoplastic activity of the alkylating agents like CY or NM is usually attributed to the interstrand cross-linking of the DNA of the dividing cells. Our experiments can be tentatively interpreted as the induced overplus of IL-6 can also contribute to the stimulation of the cytotoxic NK-cells.

• Role of TLR ligands in epicutaneously induced contrasuppression.

  Authors: Monika Majewska, Krzysztof Bryniarski, Maria Ptak, Katarzyna Zajac, Magdalena Zemelka, Francis M Lobo, Włodzimierz Ptak, Marian Szczepanik

  Pharmacological reports : PR. 61(3):539-49.

  Our previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4(+)8(+) T cells (Ts), whichOur previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4(+)8(+) T cells (Ts), which inhibited contact hypersensitivity (CS). Simultaneous EC immunization with Ag and various Toll-like receptor (TLR) ligands reversed skin-induced suppression. Our present study shows that this process activates Ag-specific T contrasuppressor (Tcs) cells and leads to the protection of CS effector T cells from suppression. Epicutaneous immunization with Ag and the TLR4 ligand lipopolysaccharide (LPS) led to a significant increase in IFN-gamma production by lymph node and spleen cells. Ag and TLR ligands, like LPS, CpG or lipoteichoic acid did not need to be applied concomitantly to the skin. An identical contrasuppressive effect was observed when the Ag and TLR ligands were deposited on distant skin areas, suggesting that both the generation of Ts and Tcs are independent. To corroborate this finding, we used a model system that uses macrophages (Mf) as Ag-presenting cells. Mf labeled in vitro with Ag (Mf-Ag) induced, upon intravenous (iv) administration, an unresponsiveness reaction that was mediated by Ts cells. When treated simultaneously with LPS-treated Mf (Mf-Ag-LPS), a TLR-ligand could induce CS. Both the Ag and the LPS signal could be uncoupled i.e., Mf-Ag and Mf-LPS given at separate time points (with an 1 h interval between injections) induced immunity.We also found that LPS-treated Mf also produced significant amounts of IL-12, a cytokine that has well-known anti-tolerogenic properties. Our experiments suggest that reversal of EC-induced suppression by TLR-ligands may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.

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

  Authors: Krzysztof Bryniarski, Marian Szczepanik, Maria Ptak, Magdalena Zemelka, Włodzimierz Ptak

  Pharmacological reports : PR. 61(3):550-7.

  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 pretreated2,4,6-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.

• Effect of ovoalbumin on the survival of an H-Y incompatible skin graft in C57BL/6 mice.

  Authors: Minika Majewska, Katarzyna Zajac, Marta Kubera, Krzysztof Bryniarski, Maria Ptak, Agnieszka Basta-Kaim, Lucyna Ksiazek, Włodzimierz Ptak, Władysław Lasoń, Marian Szczepanik

  Pharmacological reports : PR. 58(3):439-42.

  Minor histocompatibility (H) antigens create a barrier to the transplantation of organs and tissues between individuals matched for strong antigens, encoded by the major histocompatibility complexMinor histocompatibility (H) antigens create a barrier to the transplantation of organs and tissues between individuals matched for strong antigens, encoded by the major histocompatibility complex (MHC) in humans and by H-2 in mice. The male-specific antigen H-Y, which belongs to H antigens, provides a well-characterized system for studying graft rejection. C57BL/6 female mice are able to generate a strong cellular response against syngeneic male grafts. This reaction can be alleviated by immunosuppressive drugs, but they are usually hepatotoxic and increase the risk of infection and tumor development. Our previous works showed that epicutaneous (ec) immunization with protein antigen led to a profound antigen-non-specific suppression of some aspects of cell-mediated immunity (e.g. contact hypersensitivity, delayed-type hypersensitivity). The present study was aimed at checking the effect of ec pretreatment of female C57BL/6 mice with ovoalbumin (OVA) on the survival of syngenic male skin transplants. Indeed, we found that ec immunization with a protein antigen prior to grafting significantly prolonged the survival of the graft. The ease of application makes ec tolerization an attractive treatment strategy for the suppression of graft rejection.