MethodsMice. Mice deficient for IL-10 (IL-10 -/-mice), corresponding wild-type mice (IL-10 +/+ mice) on a C57BL/6 genetic background, genetically mast cell-deficient (MC-deficient) Kit W /Kit W-v mice, and The development and mechanisms of tolerance to allergens are poorly understood. Using the murine low zone tolerance (LZT) model, where contact hypersensitivity (CHS) is prevented by repeated topical low-dose applications of contact allergens, we show that LZT induction is IL-10 dependent. IL-10 is required for the generation of LZT effector cells, that is, CD8 + regulatory T cells. Only T cells from tolerized IL-10 +/+ mice or IL-10 -/-mice reconstituted with IL-10 during LZT induction adoptively transferred LZT to naive mice and prevented CHS, whereas T cells from IL-10 -/-mice failed to do so. The IL-10 required for normal LZT development is derived from lymph node CD4 + T cells, the only skin or lymph node cell population found to produce relevant amounts of IL-10 after tolerization. CD4 + T cells derived from IL-10 +/+ mice, but not from IL-10 -/-mice, allowed the induction of LZT in adoptively transferred T cell-deficient mice. Interestingly, IL-10 injections during tolerization greatly enhanced LZT responses in normal mice. Thus, the generation of CD8 + LZT effector T cells by CD4 + regulatory T cells via IL-10 may be a promising target of strategies aimed at preventing contact allergies and other harmful immune responses.This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

MethodsMice. Mice deficient for IL-10 (IL-10 -/-mice), corresponding wild-type mice (IL-10 +/+ mice) on a C57BL/6 genetic background, genetically mast cell-deficient (MC-deficient) Kit W /Kit W-v mice, and The development and mechanisms of tolerance to allergens are poorly understood. Using the murine low zone tolerance (LZT) model, where contact hypersensitivity (CHS) is prevented by repeated topical low-dose applications of contact allergens, we show that LZT induction is IL-10 dependent. IL-10 is required for the generation of LZT effector cells, that is, CD8 + regulatory T cells. Only T cells from tolerized IL-10 +/+ mice or IL-10 -/-mice reconstituted with IL-10 during LZT induction adoptively transferred LZT to naive mice and prevented CHS, whereas T cells from IL-10 -/-mice failed to do so. The IL-10 required for normal LZT development is derived from lymph node CD4 + T cells, the only skin or lymph node cell population found to produce relevant amounts of IL-10 after tolerization. CD4 + T cells derived from IL-10 +/+ mice, but not from IL-10 -/-mice, allowed the induction of LZT in adoptively transferred T cell-deficient mice. Interestingly, IL-10 injections during tolerization greatly enhanced LZT responses in normal mice. Thus, the generation of CD8 + LZT effector T cells by CD4 + regulatory T cells via IL-10 may be a promising target of strategies aimed at preventing contact allergies and other harmful immune responses.This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

No abstract

Low zone tolerance (LZT) to contact allergens is induced by epicutaneous exposure to haptens in subsensitizing doses resulting in an inhibition of contact hypersensitivity (CHS), which, in contrast, occurs after sensitization with immunogenic doses of allergens. Performing the protocol of tolerance induction resulted in robust LZT to allergens in B cell-deficient mice in vivo, indicating that B cells are not required for the induction and effector phase of LZT. However, CHS reactions in vivo were restricted in B cell-deficient mice as compared to wild-type (WT) mice. In contrast, analysis of hapten-specific T cell activation in vitro revealed a strong proliferative response of T cells derived from both WT and B celldeficient sensitized mice. Similar to WT animals, T cells obtained from tolerized B celldeficient mice produced a Tc2 cytokine pattern of LZT with high levels of IL-4 and IL-10, whereas sensitization of B cell-deficient mice resulted in the typical Tc1 cytokine profile of CHS. Adoptive transfer of CD8 + effector T cells from tolerized or sensitized B cell-deficient mice induced significant LZT or CHS reactions, respectively, in WT recipients, demonstrating that the development of hapten-specific effector CD8 + T cells of LZTand CHS is independent of B cells.

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q0, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K m-values for both pyridine nucleotides were in the μmolar range (K m[NADH]=9.8 μM, K m[NADPH]=3.2 μM calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5-8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP(+) reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP(+) reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.