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Nickel allergy is a major health problem and shows clinical manifestation of contact eczema. The response of specific lymphocyte subpopulations in sensitized patients after new challenge to nickel has until now not been studied in detail. To evaluate if nickel-based elicitation reaction could be objectively identified by multi-parametric flow cytometry, immunophenotyping of specific T cells was applied. White blood cells from 7 patients (4 positive in patch test, 3 negative) were challenged by nickel and in vitro short-term culture. Standardized antibody-dye combinations, specific for T helper(h)1, Th17 and cytotoxic T cell activation, were selected according to the recommendations of Stanford Human Immune Monitoring Center. In cytotoxic CD8+CCR7+CD45RA+ T cells from patients suffering from nickel allergy, CD38 and HLA-DR were elevated comparing to healthy donors. After challenge to nickel in vitro both markers decreased in CD8+CCR7+CD45RA+ T cells but found up-regulated in CD4+CCR7+CD45RA+CCR6−CXCR3+Th1 cells. Intracellular expression of T-bet and RORγt further indicated Th1 and Th17 cells. Finally, CD4+CD25+CCR4− T cells increased after challenge with nickel in PBMCs of patients with nickel allergy. Flow cytometry based quantification of T cell markers might be used as a specific and reliable method to detect chemical induced skin sensitization and confirm diagnostic patch testing in the clinics. [ABSTRACT FROM AUTHOR]

The Vitamin-A-metabolite retinoic acid (RA) acts as a master regulator of cellular programs. Mast cells (MCs) are primary effector cells of type-I-allergic reactions. We recently uncovered that human cutaneous MCs are enriched with RA network components over other skin cells. Yet, direct experimental evidence on the significance of the RA-MC axis is limited. Here, skin-derived cultured MCs were exposed to RA for seven days and investigated by flow-cytometry (BrdU incorporation, Annexin/PI, Fc"RI), microscopy, RT-qPCR, histamine quantitation, protease activity, and degranulation assays. We found that whileMCsize and granularity remained unchanged, RA potently interfered with MC proliferation. Conversely, a modest survival-promoting effect from RA was noted. The granule constituents, histamine and tryptase, remained unaffected, while RA had a striking impact on MC chymase, whose expression dropped by gene and by peptidase activity. The newly uncovered MRGPRX2 performed similarly to chymase. Intriguingly, RA fostered allergic MC degranulation, in a way completely uncoupled from Fc"RI expression, but it simultaneously restricted MRGPRX2-triggered histamine release in agreement with the reduced receptor expression. Vitamin-A-derived hormones thus re-shape skin-derived MCs numerically, phenotypically, and functionally. A general theme emerges, implying RA to skew MCs towards processes associated with (allergic) inflammation, while driving them away from the skin-imprinted MCTC ("MCs containing tryptase and chymase") signature (chymase, MRGPRX2). Collectively, MCs are substantial targets of the skin retinoid network. [ABSTRACT FROM AUTHOR]

Nickel allergy is a major health problem and shows clinical manifestation of contact eczema. The response of specific lymphocyte subpopulations in sensitized patients after new challenge to nickel has until now not been studied in detail. To evaluate if nickel-based elicitation reaction could be objectively identified by multi-parametric flow cytometry, immunophenotyping of specific T cells was applied. White blood cells from 7 patients (4 positive in patch test, 3 negative) were challenged by nickel and in vitro short-term culture. Standardized antibody-dye combinations, specific for T helper(h)1, Th17 and cytotoxic T cell activation, were selected according to the recommendations of Stanford Human Immune Monitoring Center. In cytotoxic CD8 + CCR7 + CD45RA + T cells from patients suffering from nickel allergy, CD38 and HLA-DR were elevated comparing to healthy donors. After challenge to nickel in vitro both markers decreased in CD8 + CCR7 + CD45RA + T cells but found up-regulated in CD4 + CCR7 + CD45RA + CCR6 - CXCR3 + Th1 cells. Intracellular expression of T-bet and RORγt further indicated Th1 and Th17 cells. Finally, CD4 + CD25 + CCR4 - T cells increased after challenge with nickel in PBMCs of patients with nickel allergy. Flow cytometry based quantification of T cell markers might be used as a specific and reliable method to detect chemical induced skin sensitization and confirm diagnostic patch testing in the clinics.

The Vitamin-A-metabolite retinoic acid (RA) acts as a master regulator of cellular programs. Mast cells (MCs) are primary effector cells of type-I-allergic reactions. We recently uncovered that human cutaneous MCs are enriched with RA network components over other skin cells. Yet, direct experimental evidence on the significance of the RA-MC axis is limited. Here, skin-derived cultured MCs were exposed to RA for seven days and investigated by flow-cytometry (BrdU incorporation, Annexin/PI, FcεRI), microscopy, RT-qPCR, histamine quantitation, protease activity, and degranulation assays. We found that while MC size and granularity remained unchanged, RA potently interfered with MC proliferation. Conversely, a modest survival-promoting effect from RA was noted. The granule constituents, histamine and tryptase, remained unaffected, while RA had a striking impact on MC chymase, whose expression dropped by gene and by peptidase activity. The newly uncovered MRGPRX2 performed similarly to chymase. Intriguingly, RA fostered allergic MC degranulation, in a way completely uncoupled from FcεRI expression, but it simultaneously restricted MRGPRX2-triggered histamine release in agreement with the reduced receptor expression. Vitamin-A-derived hormones thus re-shape skin-derived MCs numerically, phenotypically, and functionally. A general theme emerges, implying RA to skew MCs towards processes associated with (allergic) inflammation, while driving them away from the skin-imprinted MC TC ("MCs containing tryptase and chymase") signature (chymase, MRGPRX2). Collectively, MCs are substantial targets of the skin retinoid network.