2-4-dinitrofluorobenzene-sulfonic-acid and Dermatitis--Contact

Soyasaponins (SSs) are abundant in soybeans and display inhibitory activity against contact hypersensitivity (CHS), which is often used as a mouse model for allergic contact dermatitis (ACD); however, their therapeutic mechanisms remain unknown. Here, we attempted to clarify the role of gut microbiota in the inhibition of CHS by dietary soyasaponins. For antibiotic treatment, mice were administered a mixture of ciprofloxacin and metronidazole or vancomycin. These antibiotics and SSs were given to mice via drinking water 3-weeks prior to CHS induction with 2,4-dinitrofluorobenzene, and the mice were analysed for ear swelling, tissue oedema, infiltration of Gr-1-positive immune cells, the composition of faecal microbiota and regulatory T (T

Topics: Animals; Anti-Bacterial Agents; Cells, Cultured; Dermatitis, Allergic Contact; Dermatitis, Contact; Diet; Dinitrofluorobenzene; Disease Models, Animal; Edema; Female; Gastrointestinal Microbiome; Glycine max; Humans; Mice; Mice, Inbred BALB C; Saponins; T-Lymphocytes, Regulatory

L-selectin, an adhesion molecule constitutively expressed on leukocytes, is important for primary adhesion and extravasation of lymphocytes at specialized high endothelial venules within lymph nodes and other leukocytes at sites of inflammation. We have generated L-selectin-deficient mice by targeted disruption, and have confirmed a previously reported phenotype which includes strikingly impaired contact hypersensitivity (CHS) responses to reactive haptens (Tedder, T.F., D.A. Steeber, and P. Pizcueta. 1995. J. Exp. Med. 181:2259-2264; Xu, J.C., I.S. Grewal, G.P. Geba, and R.A. Flavell. 1996. 183:589-598.). Since the mechanism of this impairment has not been clarified, we sought to define the stage(s) at which the CHS response is affected in L-selectin-deficient mice. We show that epidermal Langerhans cells in L-selectin-deficient mice are normal in number, migrate to peripheral lymph nodes appropriately, and are functional in presenting allogeneic and haptenic antigens. Moreover, T cells, as well as neutrophil and monocyte effector populations, are fully capable of entry into the inflamed skin sites in the absence of L-selectin. Thus, antigen presentation and effector mechanisms are intact in L-selectin deficient mice. In contrast, virtually no antigen-specific T cells can be found within draining peripheral nodes after a contact challenge, suggesting that the defect resides primarily in the inability of antigen-specific T cells to home to and be activated in these nodes. Indeed, L-selectin-deficient mice mount completely normal CHS responses when alternate routes of immunization are used. These studies pinpoint the lesion in CHS to a discrete stage of the afferent limb of the response, clarify the role of L-selectin on effector populations, and illustrate the critical importance of the route of antigen entry to the successful execution of an immune response.

Topics: Adoptive Transfer; Animals; Antigens; Dermatitis, Contact; Dinitrofluorobenzene; Hypersensitivity, Delayed; Inflammation; L-Selectin; Langerhans Cells; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Monocytes; Neutrophils; Skin; Spleen; T-Lymphocytes

In a rechallenge system examining murine contact hypersensitivity to DNFB in BALB/c mice, the reactivity to the specific antigen at the previously responded site and the persistence of an immunological memory were investigated. Flare-up reactions were induced 4 weeks after the first challenge only at the previously responded site by local or systemic administrations of minute quantities of a specific antigen. The intensity of ear swelling was dependent on the quantity of applied antigen at the time of the rechallenge. The local hypersensitivity to the specific antigen observed in the previously responded site and the regional lymph node persisted for at least 1 year. These results suggest that the sensitivity to a specific antigen at the previously responded site is intensified and the immunological memory persists for a long time. This may explain the mechanisms by which chronic contact dermatitis recurs readily at the limited site by exposure to minute quantities of causative antigens.

Topics: Administration, Cutaneous; Animals; Antigens; Cell Division; Dermatitis, Contact; Dinitrofluorobenzene; Dose-Response Relationship, Drug; Ear Diseases; Ear, External; Edema; Epitopes; Female; Immunization; Immunologic Memory; Injections, Intravenous; Lymph Nodes; Mice; Mice, Inbred BALB C; Mice, Inbred Strains

Dose-response relationships in contact sensitivity were evaluated in guinea pigs using an in vitro assay. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with DNCB and 2,4-dinitrobenzene sulfonic salt (DNBS). Lymph node cells from sensitized and control guinea pigs were cultured in the presence of different doses of DNCB and DNBS at 8 x 10(5) cells/well, respectively. The sensitivity was evaluated by the lymphocyte transformation test (LTT), which was assessed by uptake of 3H-thymidine. The results indicated that there were significant correlations between the doses of sensitizers and the values of LTT in both phases of induction and challenge. Thus, the presence of higher numbers of LTT-reactive lymphocytes in the circulation may well correlate with the sensitizing doses. The values examined by in vitro assay correlated well with patch test readings (r = 0.653), indicating that following the increment of degree of patch test reactions, the values of SI were also increased. The in vitro LTT may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. The in vitro assay reproduced the cross-reaction between DNCB and DNBS which was confirmed in vivo.

Topics: Animals; Cells, Cultured; Cross Reactions; Cytotoxicity Tests, Immunologic; Dermatitis, Contact; Dinitrochlorobenzene; Dinitrofluorobenzene; Dose-Response Relationship, Drug; Female; Guinea Pigs

Skin blood flow in allergic contact reactions and cross-sensitivity were evaluated using laser Doppler flowmetry (LDF) to study the dose-response relationships in phases of induction and challenge in guinea pigs. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with different doses of DNCB and 2,4-dinitrobenzene sulfonic sodium salt (DNBS). The skin reactions were evaluated by LDF and visual reading score. The results indicated that there were dose-response relationships between the doses of DNCB and LDF measurements in both phases of induction and challenge, that there was a cross-reaction between DNCB and DNBS, and that the reactions at 24 h were greater than that at 48 h after removal of the patches. LDF may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. This is because vascular dilatation and increase of flow already reaches a maximum in weak reactions. The more advanced phases are dominated by oedema formation. This is simply the nature of the inflammatory reaction, rather than a methodological error. The important point is that LDF can separate positive reactions from negative/uncertain reactions. The results indicated that LDF, as a non-invasive technique, may objectively and quantitatively evaluate the dose-response relationships of contact sensitivity of sensitizers.

Topics: Animals; Cross Reactions; Dermatitis, Contact; Dinitrochlorobenzene; Dinitrofluorobenzene; Dose-Response Relationship, Drug; Evaluation Studies as Topic; Female; Guinea Pigs; Lasers; Patch Tests; Skin

Two signals are required for the in vitro activation of Lyt2+ T suppressor cells (Ts) from mice tolerized with 2,4-dinitrobenzene sulfonate (DNBS) to produce soluble suppressor factors (SSF) which suppress the transfer of contact sensitivity to dinitrofluorobenzene (DNFB). Recognition of DNP/class I MHC (signal one) stimulates the Ts to synthesize SSF. Release of SSF requires a soluble mediator (signal two) produced by the interaction of L3T4+ T cells from tolerant mice with I-A on metabolically functional cells in the DNP-presenting cell population. The purpose of this study was to examine the nature of this second Ts activation signal. Coculture of tolerant spleen cells and glutaraldehyde-fixed (Glu-) DNP-labeled spleen cells (DNP-SC) resulted in the synthesis but not release of SSF. Addition of either IL-1 or IL-2 to these cultures induced SSF release. Treatment of such cultured cells with the anti-murine IL-2 receptor antibody PC 61.5.3 blocked the IL-2- and IL-1-stimulated release of SSF. Release of SSF was also blocked when tolerant cells were cultured with (unfixed) DNP-SC in the presence of a monoclonal anti-IL-2 antibody. IL-2 but not IL-1 was able to stimulate the Ts to release synthesized SSF in the absence of L3T4+ TH activity. First, addition of IL-2 to cocultures of tolerant cells and DNP-presenting I-A- cells induced release of the synthesized SSF, whereas addition of IL-1 did not. Second, IL-2 also stimulated SSF release in cocultures of L3T4+ T cell-depleted tolerant cells and Glu-DNP-SC, whereas IL-1 did not. Tolerant cells pretreated with IL-2 and then washed were able to synthesize and release SSF upon culture with Glu-DNP-SC. Pretreatment of tolerant cells with IL-1 did not stimulate SSF release upon subsequent culture with Glu-DNP-SC. These results indicate that the Lyt2+ Ts from DNBS-tolerant mice express IL-2 receptors and IL-2 is the lymphokine which induces the Ts to release synthesized SSF. Thus, IL-2 provides a differentiative signal during the functional activation of these regulatory T cells.

Topics: Animals; Cell Differentiation; Dermatitis, Contact; Dinitrofluorobenzene; Immune Tolerance; Interleukin-1; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Receptors, Interleukin-2; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory

We studied the effects of H1 and H2 histamine receptor antagonists on down regulation of contact sensitivity (CS) to dinitrofluorobenzene (DNFB). Two H2 receptor antagonists, cimetidine and ranitidine, reversed the nonspecific immunosuppression of CS induced by burns. On the other hand, these two drugs did not affect the antigen-specific suppressor T cell-mediated tolerance to DNFB induced by dinitrobenzene sulfonic acid. Two H1 antagonists did not affect the down regulation of CS induced by either tolerance or burning. The differential sensitivities to histamine 2-receptor antagonists indicate that the mechanisms for nonspecific burn-induced immunosuppression are different from those for hapten-specific tolerance to DNFB.

Topics: Animals; Burns; Cimetidine; Dermatitis, Contact; Dinitrofluorobenzene; Female; Histamine Antagonists; Immune Tolerance; Mice; Mice, Inbred BALB C; Ranitidine; T-Lymphocytes, Regulatory

Several mast cell-derived mediators, when tested individually, have actions that may be considered immunosuppressive or anti-inflammatory. Yet evidence concerning the importance of mast cells to the down regulation of T cell-dependent immune responses in vivo is scanty and contradictory. To test directly the net contribution of intact mast cells to the suppression of delayed hypersensitivity reactions in vivo, we attempted to elicit tolerance to contact sensitivity in W/Wv or Sl/Sld mast cell-deficient mice, and compared their responses with those of littermate control mice with normal numbers of mast cells. By using three different measures of delayed hypersensitivity (ear swelling, weight ratios of challenged and control ears, and 125I-IUDR-labeled leukocyte infiltration into challenged and control ears), we detected no deficiency in the 24 hr contact sensitivity reactions to DNFB in control (nontolerized) W/Wv or Sl/Sld mice. We thus confirmed previous work indicating that mast cells are not essential for the induction and elicitation of delayed hypersensitivity. Furthermore, mast cell-deficient and control mice developed equivalent levels of tolerance to contact sensitivity. This was true for tolerance induced by DNBS administered i.v. 7 days before epicutaneous sensitization with DNFB, and for tolerance induced by supraoptimal sensitization with DNFB. W/Wv and Sl/Sld mice also served as suitable donors and recipients for putative suppressor T cells (Ts) induced by i.v. DNBS. We conclude that mast cells make little or no contribution to the modulation of Ts activity in two different models of Ts-dependent tolerance to contact sensitivity.

Topics: Animals; Dermatitis, Contact; Dinitrofluorobenzene; Disease Models, Animal; Immune Tolerance; Immunization; Immunization, Passive; Mast Cells; Mice; Mice, Mutant Strains; T-Lymphocytes, Regulatory

Topics: Animals; Bone Marrow Transplantation; Dermatitis, Contact; Dinitrofluorobenzene; Female; H-2 Antigens; Histocompatibility; Immune Tolerance; Mice; Mice, Inbred Strains; Radiation Chimera; T-Lymphocytes, Regulatory