Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation.

Enhanced generation of reactive oxygen intermediates by suppressor T cell-derived exosome-treated macrophages.

Macrophages (Mφ) as efficient phagocytes able to present the antigen and playing an effector role induce and orchestrate the immune response also through the release of soluble factors. Recently described T CD8+ cell-derived suppressive exosomes carrying miRNA-150, that act antigen-specifically, seem to inhibit murine contact sensitivity reaction indirectly by affecting antigen presenting cells, especially Mφ. Present studies investigated the influence of suppressive exosomes on secretory activity of Mφ assessed as their ability to generate reactive oxygen intermediates (ROIs), nitric oxide, cytokines as well as their viability and expression of antigen phagocytosis and presentation markers. Interestingly, in vivo and in vitro treatment of Mφ with assayed hapten-specific exosomes affected only ROIs generation, significantly enhancing their production. Current results suggest that ROIs may participate in antigen-specific tolerance mechanism mediated by suppressive T lymphocyte-derived exosome-influenced Mφ, by inhibition of effector T cell proliferation and induction of T regulatory lymphocytes.

Down-regulation of macrophage immune activity by natural CD8+ regulatory T cells.

To evaluate the influence of natural regulatory CD8+ T cells on macrophages we investigated in vitro production of cytokines, reactive oxygen intermediates (ROIs) and expression of CD80 surface costimulatory molecules by macrophages (MF) of wild type (WT) B10PL and syngeneic knock-out (KO) strains, TCRalpha-/-, beta2m-/- and CD1d-/- mice. MF of TCR alpha-/- (CD4- and CD8-) and beta2m-/- (CD8-) animals produced higher levels ofTNF-a, IL-6, IL-12 and ROIs and showed increased expression of CD80 costimulatory molecules in comparison to MF of WT or CD1d-/- (NKT-) mice. When MF of these strains were conjugated with TNP hapten and injected i.v. into WT mice to test either induction of contact sensitivity (CS) or tolerance, only TNP-MF of TCRalpha-/- and beta2m-/- animals induced a significant CS reaction, while cells of WT and CD1d-/- strains were tolerogenic. MF of the tested strains can be classified functionally as resembling either proinflammatory (TCRalpha-/- and beta2m-/-mice) or immunosuppressive (WT and CD1d-/-) phenotypes. We suggest the presence of an in vivo regulatory loop in which innate CD8+ Treg cells control the transition between MF phenotypes and thus adjust the magnitude of the inflammatory response to strictly local requirements.

Role of TLR ligands in epicutaneously induced contrasuppression.

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), 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.

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.

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

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-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.

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

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.

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

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 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.

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

BACKGROUND: 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.

Modulation of testicular macrophage activity by collagenase.

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 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.

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.

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 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.

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. 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.

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

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 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.

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-alphabeta lymphocytes, while Tgammadelta 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-beta 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.

In vitro response of macrophages to a new carbon-polylactide composite for the treatment of periodontal diseases.

The purpose of the study was to examine the response of macrophages and the concentration of selected released cytokines following contact with a new carbon-polylactide composite. The macrophages were grown on samples of the materials and on each of its components separately. Viability of the cells as well as concentrations of interleukins IL-6, IL-10, IL-12 and TNF-alpha were then determined. Some differences in the viability of the cells were demonstrated. They varied according to the kind of material used. After incubation with the serum, the composite and its components induced the release of IL-6, IL-12 and TNF-alpha which did not differ significantly from one another.