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Topical exposure of mice to fluorescein isothiocyanate provokes a selective type 2 cvtokine secretion profile
204
Poster Session 4A. Immune Hypersensitivity
eters of placentas and newborns were collected. Placental samples were analyzed for 21 compounds of persistent organic pollutants (POP) by capillary gas chromatography. In umbilical blood samples the concentration of total IgE (as a biomarker of a sensitization of newborns) using CAP system was determined. Laboratory analyses have shown the statistically significantly higher concentrations of 17 compounds out of 21 POP investigated in the placental samples collected from the industrialized region if compared to the rural one. Paralelly, the increase in the total 19B level in the umbilical blood samples gathered from industrial region was found, evoked possibly by organic xenobiotics. External placental parameters differed too.
I P4A121
TOPICALEXPOSURE OF MICETO FLUORESCEIN ISOTHIOCYANATE PROVOKES A SELECTIVE TYPE 2 CVTOKINE SECRETION PROFILE
R.I. Dearman *, I. Kimber. Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire, UK It has been suggested previously that the sensitizing lIuorochrome lIuorescein isothiocyanate isomer I (FTIC) induces contact dermatitic reactions via a T helper (Th) 2 type mechanism. Antigenic restimulation in vitro of draining lymph node cells (LNC) isolated following topical exposure of BALB/c strain mice to FTIC was found to result in a higher frequency of LNC producing the type 2 cytokine interleukin 4 (lL-4) than the type I cell product interferon y (IFN-y). The converse Thl type pattern was observed when LNC were derived from animals which had received the contact allergen dinitrolIuorobenzene 1 • We have therefore examined the cytokine secretion profiles induced in mice following repeated topical treatment with FITC. BALB/c strain mice were exposed topically to 2% FITC in dibutylphthalate: acetone (I: 1) or to vehicle alone. Control animals were treated concurrently with the reference contact allergen 2,4-dinitrochlorobenzene (DNCB; I% in 4: 1 acetone:olive oil [AOO] vehicle) or with the reference respiratory chemical allergen trimellitic anhydride (TMA; 10% in AOO) Such treatment is known to result in the development of characteristic Thl and Th2 type cytokine production patterns, respectively. Thirteen days after the initiation of exposure, draining auricular lymph nodes were excised, LNC cultured for 12-120 hr and cytokine content of culture supernatants analyzed by enzyme-linked immunosorbent assays. DNCB-stimulated LNC produced high levels of IFN-y, but little of the Th2 type cytokines IL-4 or interleukin 10 (lL-IO). Exposure to TMA and FTIC, however, resulted in the converse Th2 type cytokine secretion pattern. LNC isolated from vehicle-treated animals failed to express significant levels of any cytokine. These data demonstrate that, in common with TMA, a Th2-type cytokine production profile is stimulated by FITC, providing confirmatory evidence that the skin lesions induced by this chemical may be associated with Th2 type cells. [I] Tang et al (1996). Journal of Immunology, 157: 117-125
§iiJ
EXTENDED COMPARISON OF THE LOCAL LYMPH NODEASSAYWITHGUINEAPIG TESTMETHODS
I. Kimber *1, R.I. Dearman", G.F. Gerberick'", L.J. Lea 2 , D.A. Basketter' . 1 Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire; 2 Unilever Environmental Safety Laboratory, Shambrook, Beds, UK; 3 Miami Valley Laboratories, Procter and Gamble Company, Cincinnati, OH, USA The murine local lymph node assay (LLNA) is an alternative method for the prospective identification of chemicals that have the f0tential to cause skin sensitization and allergic contact dermatitis . Sensitizing activity is measured as a function of proliferative responses induced in draining auricular lymph nodes following topical exposure of mice to the test chemical. As part of a programme of
continuing evaluation and validation of the LLNA we have extended previous comparisons between this method and the results of guinea pig predictive tests based upon either the guinea pig maximization test or the Buehler occluded patch test. In total, 124 chemicals were evaluated in the LLNA and in one or both guinea pig tests. Of these, 78 (62.9%) were positive in both the LLNA and a guinea pig test and 23 (18.5%) negative by both approaches. Of the chemicals tested, 9 (7.3%) that elicited positive responses in the LLNA were classified as negative in guinea pig tests, while 14 (11.3%) were positive in a guinea pig method, but negative in the LLNA. Taken together the results reveal an overall level of concordance of 81.4% between activity in the LLNA and the results of guinea pig test methods. These data provide additional evidence that the LLNA may be used for the accurate assessment of skin sensitizing potential. [1] Kimber, R.J. Dearman, E.W. Scholes and D.A. Basketter (1994) The local lymph node assay: developments and applications. Toxicology 93: 13-31.
I P4A141INTERLEUKIN-4 INDUCES SELECTION OF HUMAN PENICILLIN-5PECIFIC CD4+LYMPHOCYTES THROUGH REGULATION OF CELL CYCLEG1fS TRANSITION AND APOPTOSIS
I. Gaspard *1 , M. Pallardy 1 , A. vervisch'', Z. Mishal2 , H. Lebrec 1 . llmmunotoxicology group, INSERM U 461, Faculte de Pharmacie Paris Sud, 92296, Chdtenay-Malabry; 2 Lahoratoire de Cytometrie, CNRS, rue Guy Mocquet, 94800 Villejuij, France
,B-Iactam antibiotics are haptenic compounds known to induce hypersensitivity reactions. Clinically, penicillin-induced allergies include urticaria, angioedema, systemic life-threatening events such as anaphylactic shock, and bullous skin eruptions. These toxic effects involve activation of CD4+ helper T lymphocytes and/or CD8+ T lymphocytes, as well as production of specific antibodies. We previously showed that interleukin-4 (IL-4) plays a dominant role in controlling Thl- or Th2-like primary immune response to the hapten penicillin G (PNG) in the mouse. The present work focusses on the effects of IL-4 on differentiation of human penicillin-specific lymphocytes derived from allergic patients. Such lymphocytes proliferate in response to penicillin in the presence of irradiated autologous antigen-presenting cells (APCs: EBV-transformed B lymphocytes). This proliferation is MHC-restricted since no proliferation is induced with allogeneic APCs, and specific activation with PNG induces expression of IL-2, IL-4, IL-5, and IFN-y mRNAs. Long-term culture of this cell population in the presence of IL-4 and IL-2 induces selection of a pure CD4+ population, while culture with lL-2 alone induces selection of CD8+ cells. Propidium iodide staining of DNA in CD4+ and CD8+ penicillin-specific T lymphocytes showed that IL-4 increases IL-2-induced G liS cell cycle transition in CD4+ lymphocytes but not in CD8+ cells. In addition, activation-induced cell death of penicillin-specific CD4+ and CD8+ lymphocytes was assessed by measurement of reduced DNA stainability (sub Gl peak). When cultured with IL-2 alone, cell death was preferentially induced in CD4+ cells. Addition of IL-4 was able to reduce the sub G I peak as well as an anti-Fas blocking antibody. Together, these data indicate that lL-4 regulates penicillin-specific cellular response by regulating differently CD4+ and CD8+ cell growth and apoptosis.
Poster Session 4A. Immune Hypersensitivity
eters of placentas and newborns were collected. Placental samples were analyzed for 21 compounds of persistent organic pollutants (POP) by capillary gas chromatography. In umbilical blood samples the concentration of total IgE (as a biomarker of a sensitization of newborns) using CAP system was determined. Laboratory analyses have shown the statistically significantly higher concentrations of 17 compounds out of 21 POP investigated in the placental samples collected from the industrialized region if compared to the rural one. Paralelly, the increase in the total 19B level in the umbilical blood samples gathered from industrial region was found, evoked possibly by organic xenobiotics. External placental parameters differed too.
I P4A121
TOPICALEXPOSURE OF MICETO FLUORESCEIN ISOTHIOCYANATE PROVOKES A SELECTIVE TYPE 2 CVTOKINE SECRETION PROFILE
R.I. Dearman *, I. Kimber. Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire, UK It has been suggested previously that the sensitizing lIuorochrome lIuorescein isothiocyanate isomer I (FTIC) induces contact dermatitic reactions via a T helper (Th) 2 type mechanism. Antigenic restimulation in vitro of draining lymph node cells (LNC) isolated following topical exposure of BALB/c strain mice to FTIC was found to result in a higher frequency of LNC producing the type 2 cytokine interleukin 4 (lL-4) than the type I cell product interferon y (IFN-y). The converse Thl type pattern was observed when LNC were derived from animals which had received the contact allergen dinitrolIuorobenzene 1 • We have therefore examined the cytokine secretion profiles induced in mice following repeated topical treatment with FITC. BALB/c strain mice were exposed topically to 2% FITC in dibutylphthalate: acetone (I: 1) or to vehicle alone. Control animals were treated concurrently with the reference contact allergen 2,4-dinitrochlorobenzene (DNCB; I% in 4: 1 acetone:olive oil [AOO] vehicle) or with the reference respiratory chemical allergen trimellitic anhydride (TMA; 10% in AOO) Such treatment is known to result in the development of characteristic Thl and Th2 type cytokine production patterns, respectively. Thirteen days after the initiation of exposure, draining auricular lymph nodes were excised, LNC cultured for 12-120 hr and cytokine content of culture supernatants analyzed by enzyme-linked immunosorbent assays. DNCB-stimulated LNC produced high levels of IFN-y, but little of the Th2 type cytokines IL-4 or interleukin 10 (lL-IO). Exposure to TMA and FTIC, however, resulted in the converse Th2 type cytokine secretion pattern. LNC isolated from vehicle-treated animals failed to express significant levels of any cytokine. These data demonstrate that, in common with TMA, a Th2-type cytokine production profile is stimulated by FITC, providing confirmatory evidence that the skin lesions induced by this chemical may be associated with Th2 type cells. [I] Tang et al (1996). Journal of Immunology, 157: 117-125
§iiJ
EXTENDED COMPARISON OF THE LOCAL LYMPH NODEASSAYWITHGUINEAPIG TESTMETHODS
I. Kimber *1, R.I. Dearman", G.F. Gerberick'", L.J. Lea 2 , D.A. Basketter' . 1 Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire; 2 Unilever Environmental Safety Laboratory, Shambrook, Beds, UK; 3 Miami Valley Laboratories, Procter and Gamble Company, Cincinnati, OH, USA The murine local lymph node assay (LLNA) is an alternative method for the prospective identification of chemicals that have the f0tential to cause skin sensitization and allergic contact dermatitis . Sensitizing activity is measured as a function of proliferative responses induced in draining auricular lymph nodes following topical exposure of mice to the test chemical. As part of a programme of
continuing evaluation and validation of the LLNA we have extended previous comparisons between this method and the results of guinea pig predictive tests based upon either the guinea pig maximization test or the Buehler occluded patch test. In total, 124 chemicals were evaluated in the LLNA and in one or both guinea pig tests. Of these, 78 (62.9%) were positive in both the LLNA and a guinea pig test and 23 (18.5%) negative by both approaches. Of the chemicals tested, 9 (7.3%) that elicited positive responses in the LLNA were classified as negative in guinea pig tests, while 14 (11.3%) were positive in a guinea pig method, but negative in the LLNA. Taken together the results reveal an overall level of concordance of 81.4% between activity in the LLNA and the results of guinea pig test methods. These data provide additional evidence that the LLNA may be used for the accurate assessment of skin sensitizing potential. [1] Kimber, R.J. Dearman, E.W. Scholes and D.A. Basketter (1994) The local lymph node assay: developments and applications. Toxicology 93: 13-31.
I P4A141INTERLEUKIN-4 INDUCES SELECTION OF HUMAN PENICILLIN-5PECIFIC CD4+LYMPHOCYTES THROUGH REGULATION OF CELL CYCLEG1fS TRANSITION AND APOPTOSIS
I. Gaspard *1 , M. Pallardy 1 , A. vervisch'', Z. Mishal2 , H. Lebrec 1 . llmmunotoxicology group, INSERM U 461, Faculte de Pharmacie Paris Sud, 92296, Chdtenay-Malabry; 2 Lahoratoire de Cytometrie, CNRS, rue Guy Mocquet, 94800 Villejuij, France
,B-Iactam antibiotics are haptenic compounds known to induce hypersensitivity reactions. Clinically, penicillin-induced allergies include urticaria, angioedema, systemic life-threatening events such as anaphylactic shock, and bullous skin eruptions. These toxic effects involve activation of CD4+ helper T lymphocytes and/or CD8+ T lymphocytes, as well as production of specific antibodies. We previously showed that interleukin-4 (IL-4) plays a dominant role in controlling Thl- or Th2-like primary immune response to the hapten penicillin G (PNG) in the mouse. The present work focusses on the effects of IL-4 on differentiation of human penicillin-specific lymphocytes derived from allergic patients. Such lymphocytes proliferate in response to penicillin in the presence of irradiated autologous antigen-presenting cells (APCs: EBV-transformed B lymphocytes). This proliferation is MHC-restricted since no proliferation is induced with allogeneic APCs, and specific activation with PNG induces expression of IL-2, IL-4, IL-5, and IFN-y mRNAs. Long-term culture of this cell population in the presence of IL-4 and IL-2 induces selection of a pure CD4+ population, while culture with lL-2 alone induces selection of CD8+ cells. Propidium iodide staining of DNA in CD4+ and CD8+ penicillin-specific T lymphocytes showed that IL-4 increases IL-2-induced G liS cell cycle transition in CD4+ lymphocytes but not in CD8+ cells. In addition, activation-induced cell death of penicillin-specific CD4+ and CD8+ lymphocytes was assessed by measurement of reduced DNA stainability (sub Gl peak). When cultured with IL-2 alone, cell death was preferentially induced in CD4+ cells. Addition of IL-4 was able to reduce the sub G I peak as well as an anti-Fas blocking antibody. Together, these data indicate that lL-4 regulates penicillin-specific cellular response by regulating differently CD4+ and CD8+ cell growth and apoptosis.