Maria Ptak

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.

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.

Modulation of Macrophage Activity by Proteolytic Enzymes. Differential Regulation of IL-6 and Reactive Oxygen Intermediates (ROIs) Synthesis as a Possible Homeostatic Mechanism in the Control of Inflammation

Inflammatory foci are rich in proteases released by neutrophils (serine proteases) and macrophages (metalloproteases). These enzymes can degrade extracellular matrix proteins and cell membrane bound proteins thus contributing to the development and progression of inflammatory reaction. In this study we have investigated the influence of collagenase (metalloprotease) and trypsin (serine protease) on murine resident and oil-induced peritoneal macrophages (Mf). Short in vitro treatment of Mf, not affecting cell viability, significantly reduced the release of reactive oxygen intermediates (ROIs) and at the same time triggered the increase of IL-6 production and to lesser extent of TNF-α production. Both these effects were dependent on enzyme concentration used and were particularly well pronounced in resident macrophages. In addition both enzymes cleaved a number of cell-membrane molecules, including CD23, CD14, CD95L, and Mac-3. We hypothesize that the enzymatic digestion of certain Mf surface receptor proteins in inflammatory foci may be responsible for modification of cell behaviour either by preventing the generation of specific signal or alternatively by delivering a mock substitute signal to the cell interior. In effect inhibition of ROIs production limits their destructive effects and the increase in the secretion of IL-6 stimulates the synthesis of acute phase proteins and triggers other anti-inflammatory mechanisms thus directing Mf present in inflammatory foci into regulatory pathway rather than allowing them to perform solely the effector function.

Macrophage function in alloxan diabetic mice : Expression of adhesion molecules, generation of monokines and oxygen and NO radicals

The increased incidence of bacterial and mycotic infections in poorly controlled diabetic patients or animals is frequently attributed to impaired activities of professional phagocytes (granulocytes, macrophages) in hypoinsulinaemic milieu. We measured production of monokines (IL‐6 and tumour necrosis factor‐alpha (TNF‐α)), active NO and reactive oxygen intermediates (ROIs), as well as expression of several cell surface adhesion molecules (Mac‐1, ‐2 and ‐3, intercellular adhesion molecule‐1 (ICAM‐1) and FcγRII), by thioglycollate medium‐induced peritoneal macrophages of normoglycaemic and alloxan diabetic CBA/J mice (blood glucose level in the range 300 or 500 mg/dl). Macrophages of animals with moderate diabetes (300 mg/dl) produced significantly more IL‐6 and TNF‐α and ROIs than cells of control mice and showed an increased expression of all cell surface molecules, except Mac‐3. NO/NO2 production was not affected. Administration of insulin restored enhanced values to normal levels, except for the production of ROIs which remained unusually high. We conclude that two separate mechanisms influence macrophage physiology in diabetes—lack of saturation of insulin receptors on macrophages and an indirect effect due to formation of advanced glycosylation endproducts (AGE) on their surfaces. The latter is possibly responsible for increased generation of ROIs, since it cannot be down‐regulated by prolonged insulin treatment. How the increased activity of macrophages of moderately diabetic mice (enhanced production of proinflammatory monokines and oxygen radicals as well as expression of molecules) is related to their ability to kill bacteria is now under investigation.

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.

Epicutaneous Immunization with Protein Antigen in the Presence of TLR4 Ligand Induces TCRαβ+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-β. 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 TCRαβ 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-γ, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-γ mAb showed that IFN-γ 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.

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.

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

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 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, TNF alpha 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

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.