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326 Induction of selective antigen-specific Th2 cell anergy
Abstracts
J ALLERGY CLIN IMMUNOL
S109
VOLUME 105, NUMBER 1. PART 2
gation, we determined the role of regulatory cytokines, including IL12, IFN-y, IL- 10, IL-4. and IL- 13 in the GC-mediated type- I/type-2 cytokine alterations in PBMC form healthy human volunteers stimulated with tetanus toxoid. Consistent with previous results, dexamethasone (DEX) decreased IFN-y production and increased IL-IO and IL-4 production by tetanus-stimulated human PBMC. The addition of either recombinant lL- 12~70 or IFN-y prevented the DEXmediated decrease in IFN-gamma and increase in IL-4 in a dosedependent fashion. The DEX-mediated increase in IL-10 production was abrogated by the addition of IFN-y only. Inhibition of type-2 cytokines with monoclonal antibodies specific for IL-IO, IL-4, or IL13 had no effect on the DEX-mediated suppression of IFN-yand only marginal effects on the DEX-mediated increase in IL-4 and IL-IO. Therefore, the DEX-mediated type-l/type-2 cytokine alterations are primarily dependent on modulation of type- I cytokines but not type-2 cytokines. These data suggest that interventions aimed at promoting the production of type-l cytokines may be sufficient to prevent or restore the type- I /type-2 cytokine balance during periods of stress. In contrast, inhibiting IL-4, IL-lo, and IL-13 may not be sufficient to alter the overall type-l/type-2 cytokine balance and may leave the individual at risk for immune-based diseases in which type-l cytokines are critical in the maintenance of health.
325 Lack of THl Cytokines and Atopic Disease P Wood*,
D Lammas*. J-L Casanovaf, S Holland& D Kumararatne* *Department of Immunology, Birmingham University, United Kingdom tUnite d’Immunologie, Hopital Necker-Enfants Malades. Paris, France SNIAID, NIH, Bethesda, MD, USA There is growing evidence that the allergic response is driven by cytokines released by TH2 cells. Although the mechanisms by which a TH2 response dominates the TH I reponse are not fully understood, the evidence that THl-type cytokines inhibit the progression of a TH2-type response has led to the suggestion that THl-type cytokines might be vital in inhibiting the development of allergy. This hypothesis is readily testable in a small number of families with defects in production of cytokines such as interleukin I2 and its receptor, or the interferon gamma receptor, which we have described previously. We have used simple clinical and laboratory assessment to establish that affected individuals in these families do not suffer from or have indicators of atopic disease. A questionnaire, based on the ISAAC study and concerning symptoms of allergic rhinitis, allergic asthma and dermatitis has been used in conjunction with laboratory measurements of total IgE and specific IgE to a number of common inhalant allergens (house dust mite; grass and tree pollen; cat and dog danders). Data from the questionnaires indicate that these patients do not suffer from significant clinical atopy and in addition the vast majority have normal total IgE and no specific IgE to common aeroallergens. This study suggests that unbalanced activity of TH2 cells caused by defective TH I cytokine responses does not underlie the development of atopy.
326 Induction of Selective Antigen-Specific Th2 Cell Anergy N Odu. K Minoguchi, A Tanaka, T Yokoe, M Adachi First Department of Internal Medicine, Showa University Interleukin (IL)-10 is regarded as an antiinflammatory cytokine to downregulate the functions of T helper I (Thl) cells. Recently, evidence is provided that IL- IO also inhibits ThZ-type responses. Here, we investigate the involvement of IL-IO on antigen-specific Th2 cell anergy. Peripheral blood mononuclear cells (PBMCs) were isolated from house dust mite (Dermatophagoides farinae. Der f)-sensitized asthmatic patients. PBMCs were primary stimulated with Der f anti-
gen or purified protein derivaties (PPD) immediately after purification or after48 hours of resting culture with medium alone for 7 days. These cells were then secondary stimulated with Der for PPD antigen with mitomysin C-treated PBMCs as antigen presenting cells, or phorbor my&ate acetate (PMA) plus calcium ionophore for 3 days. Proliferative responses were measured by incorporation of w3]methylthymidine for 7 days in primary response and for 3 days in secondary response. IL-5, IFN-gamma and IL-IO in the culture supernatant were measured by ELISA after stimulation for 3 days in each response. Moreover, we analyzed the surface expression of B7-1 and B7-2 and cytoplasmic expression of IL-IO by flow cytometry. Although PPD-mediated proliferative response and IFN-gamma production were not significant changed in each response, stimulation of PBMCs with Der f antigen after48 hours of resting culture with medium significantly decreased the proliferative response and IL-5 production, and markedly increased IL-10 production. Furthermore, no secondary proliferative response and IL-5 production were observed when the cells were stimulated with Der f antigen after 48 hours of resting culture. Decrease in Der f-mediated secondary proliferative response was not recovered by addition with recombinant IL-2, partially recovered by addition with anti-IL-10 antibodies, and fully recovered by the stimulation with PMA plus calcium ionophore. suggesting that Der f-specific Th2 cells rendered anergic state. Analysis by flow cytometry revealed that intracellular IL-IO was increased in monocytes after resting for 48 hours and CD4+ T cells after stimulation with Der f antigen. B7-I and B7-2 expression on B cells and monocytes were not significantly changed in these experimental condition. These results suggest that IL-IO produced by monocytes at antigen presentation may induce anergic state in antigen-specificTh2 cells and IL- 10 production by CD4+ T cells may maintain the anergy.
327 Antigen-Specific
Regulation
of CD1 Expression
in Man M
lJlanova*, A Tarkowskit, SA Porcellif, LA Hanson* *Department of Clinical Immunology, University of Gothenburg, Sweden tDepartment of Rheumatology, University of Gothenburg, Sweden During the last decade CD1 family of cell surface glycoproteins has been implicated in the presentation of nonpeptide antigens in man. Recent findings by our group indicate that CD1 molecules can be also involved in the presentation of certain bacterial proteins. However, CDla, b, and c (group 1 CD1 molecules) are not present at significant levels on circulating monocytes unless their expression is induced by cytokines such as GM-CSF. In this study we investigated the cell surface expression of CDI molecules following the antigenic stimulation in vivo via immunization of healthy volunteers with tetanus toxoid (TT) vaccine and in vitro cell cultures using the same antigen. Both in vivo and in vitro studies demonstrated clear up-regulation of the surface expression of CDla, b, and c on monocytes as a result of antigenic stimulation with ‘IT, supporting the idea that CD1 molecules participate in the presentation of this protein antigen in man. In vitro, antigen-triggered expression of these molecules was mediated by GM-CSF since neutralization of this cytokine with specific antibody totally abrogated CDla, b, and c expression. In contrast to the group 1 CD1 molecules, CDld was found to be. constitutively expressed on the majority of circulating monocytes and B lymphocytes prior to immunization. There. was no effect of antigenie stimulation with non the cell surface expression of CDld suggesting major differences in regulation of the expression and function of the different CDI molecules in man. Altogether our results point to antigen-driven up-regulation of CDI a, b, and c expression on human monocytes which is mediated by GM-CSF and no effect on CD Id expression.
J ALLERGY CLIN IMMUNOL
S109
VOLUME 105, NUMBER 1. PART 2
gation, we determined the role of regulatory cytokines, including IL12, IFN-y, IL- 10, IL-4. and IL- 13 in the GC-mediated type- I/type-2 cytokine alterations in PBMC form healthy human volunteers stimulated with tetanus toxoid. Consistent with previous results, dexamethasone (DEX) decreased IFN-y production and increased IL-IO and IL-4 production by tetanus-stimulated human PBMC. The addition of either recombinant lL- 12~70 or IFN-y prevented the DEXmediated decrease in IFN-gamma and increase in IL-4 in a dosedependent fashion. The DEX-mediated increase in IL-10 production was abrogated by the addition of IFN-y only. Inhibition of type-2 cytokines with monoclonal antibodies specific for IL-IO, IL-4, or IL13 had no effect on the DEX-mediated suppression of IFN-yand only marginal effects on the DEX-mediated increase in IL-4 and IL-IO. Therefore, the DEX-mediated type-l/type-2 cytokine alterations are primarily dependent on modulation of type- I cytokines but not type-2 cytokines. These data suggest that interventions aimed at promoting the production of type-l cytokines may be sufficient to prevent or restore the type- I /type-2 cytokine balance during periods of stress. In contrast, inhibiting IL-4, IL-lo, and IL-13 may not be sufficient to alter the overall type-l/type-2 cytokine balance and may leave the individual at risk for immune-based diseases in which type-l cytokines are critical in the maintenance of health.
325 Lack of THl Cytokines and Atopic Disease P Wood*,
D Lammas*. J-L Casanovaf, S Holland& D Kumararatne* *Department of Immunology, Birmingham University, United Kingdom tUnite d’Immunologie, Hopital Necker-Enfants Malades. Paris, France SNIAID, NIH, Bethesda, MD, USA There is growing evidence that the allergic response is driven by cytokines released by TH2 cells. Although the mechanisms by which a TH2 response dominates the TH I reponse are not fully understood, the evidence that THl-type cytokines inhibit the progression of a TH2-type response has led to the suggestion that THl-type cytokines might be vital in inhibiting the development of allergy. This hypothesis is readily testable in a small number of families with defects in production of cytokines such as interleukin I2 and its receptor, or the interferon gamma receptor, which we have described previously. We have used simple clinical and laboratory assessment to establish that affected individuals in these families do not suffer from or have indicators of atopic disease. A questionnaire, based on the ISAAC study and concerning symptoms of allergic rhinitis, allergic asthma and dermatitis has been used in conjunction with laboratory measurements of total IgE and specific IgE to a number of common inhalant allergens (house dust mite; grass and tree pollen; cat and dog danders). Data from the questionnaires indicate that these patients do not suffer from significant clinical atopy and in addition the vast majority have normal total IgE and no specific IgE to common aeroallergens. This study suggests that unbalanced activity of TH2 cells caused by defective TH I cytokine responses does not underlie the development of atopy.
326 Induction of Selective Antigen-Specific Th2 Cell Anergy N Odu. K Minoguchi, A Tanaka, T Yokoe, M Adachi First Department of Internal Medicine, Showa University Interleukin (IL)-10 is regarded as an antiinflammatory cytokine to downregulate the functions of T helper I (Thl) cells. Recently, evidence is provided that IL- IO also inhibits ThZ-type responses. Here, we investigate the involvement of IL-IO on antigen-specific Th2 cell anergy. Peripheral blood mononuclear cells (PBMCs) were isolated from house dust mite (Dermatophagoides farinae. Der f)-sensitized asthmatic patients. PBMCs were primary stimulated with Der f anti-
gen or purified protein derivaties (PPD) immediately after purification or after48 hours of resting culture with medium alone for 7 days. These cells were then secondary stimulated with Der for PPD antigen with mitomysin C-treated PBMCs as antigen presenting cells, or phorbor my&ate acetate (PMA) plus calcium ionophore for 3 days. Proliferative responses were measured by incorporation of w3]methylthymidine for 7 days in primary response and for 3 days in secondary response. IL-5, IFN-gamma and IL-IO in the culture supernatant were measured by ELISA after stimulation for 3 days in each response. Moreover, we analyzed the surface expression of B7-1 and B7-2 and cytoplasmic expression of IL-IO by flow cytometry. Although PPD-mediated proliferative response and IFN-gamma production were not significant changed in each response, stimulation of PBMCs with Der f antigen after48 hours of resting culture with medium significantly decreased the proliferative response and IL-5 production, and markedly increased IL-10 production. Furthermore, no secondary proliferative response and IL-5 production were observed when the cells were stimulated with Der f antigen after 48 hours of resting culture. Decrease in Der f-mediated secondary proliferative response was not recovered by addition with recombinant IL-2, partially recovered by addition with anti-IL-10 antibodies, and fully recovered by the stimulation with PMA plus calcium ionophore. suggesting that Der f-specific Th2 cells rendered anergic state. Analysis by flow cytometry revealed that intracellular IL-IO was increased in monocytes after resting for 48 hours and CD4+ T cells after stimulation with Der f antigen. B7-I and B7-2 expression on B cells and monocytes were not significantly changed in these experimental condition. These results suggest that IL-IO produced by monocytes at antigen presentation may induce anergic state in antigen-specificTh2 cells and IL- 10 production by CD4+ T cells may maintain the anergy.
327 Antigen-Specific
Regulation
of CD1 Expression
in Man M
lJlanova*, A Tarkowskit, SA Porcellif, LA Hanson* *Department of Clinical Immunology, University of Gothenburg, Sweden tDepartment of Rheumatology, University of Gothenburg, Sweden During the last decade CD1 family of cell surface glycoproteins has been implicated in the presentation of nonpeptide antigens in man. Recent findings by our group indicate that CD1 molecules can be also involved in the presentation of certain bacterial proteins. However, CDla, b, and c (group 1 CD1 molecules) are not present at significant levels on circulating monocytes unless their expression is induced by cytokines such as GM-CSF. In this study we investigated the cell surface expression of CDI molecules following the antigenic stimulation in vivo via immunization of healthy volunteers with tetanus toxoid (TT) vaccine and in vitro cell cultures using the same antigen. Both in vivo and in vitro studies demonstrated clear up-regulation of the surface expression of CDla, b, and c on monocytes as a result of antigenic stimulation with ‘IT, supporting the idea that CD1 molecules participate in the presentation of this protein antigen in man. In vitro, antigen-triggered expression of these molecules was mediated by GM-CSF since neutralization of this cytokine with specific antibody totally abrogated CDla, b, and c expression. In contrast to the group 1 CD1 molecules, CDld was found to be. constitutively expressed on the majority of circulating monocytes and B lymphocytes prior to immunization. There. was no effect of antigenie stimulation with non the cell surface expression of CDld suggesting major differences in regulation of the expression and function of the different CDI molecules in man. Altogether our results point to antigen-driven up-regulation of CDI a, b, and c expression on human monocytes which is mediated by GM-CSF and no effect on CD Id expression.