Przybilla, Bernhard

Sander, Christian

Summer, Burkhard

Thomas, Peter

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Poster PresentationCells and Cytokines in Allergic PatientsInt Arch Allergy Immunol 2001;124:292–295In vitro T Cell Reactivity in Nickel Allergy:Comparison of T Cell Clonality, CytokineExpression and Mediator ProductionPeter Thomas Burkhard Summer Christian Sander Bernhard PrzybillaKlinik für Dermatologie und Allergologie, Ludwig-Maximilians-Universität, München, DeutschlandCorrespondence to: PD Dr. P. ThomasKlinik und Poliklinik für Dermatologie und AllergologieLudwig-Maximilians-Universität München, Frauenlobstrasse 9–11D–80337 München (Germany), Tel. +49 89 5160 6314Fax +49 89 5160 6312, E-Mail Peter.Thomas@derma.med.uni-muenchen.deABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com© 2001 S. Karger AG, Basel1018–2438/01/1243–0292$17.50/0Accessible online at:www.karger.com/journals/iaaKey WordsLymphocytes W Nickel allergy W T cell receptor W IL-4 WIFN-Á W PCRIntroductionApart from typical symptoms of nickel contact allergy,there are also unusual manifestations, e.g. pseudolympho-ma formation, airborne contact dermatitis or implant-associated intolerance reactions [1]. Delayed-type hyper-sensitivity to nickel is reflected by eczematous reactionsupon patch test and by an antigen-specific T lymphocyteproliferation in vitro [2–4]. We now assessed to whatextent peripheral blood mononuclear cells (PBMC) fromnickel-allergic and from nonallergic individuals wouldproliferate in response to nickel stimulation in vitro. Inaddition, the production of IL-4 and IFN-Á in these cul-tures was monitored by RT-PCR of the respective mRNAand by immunoassay of the supernatants. In addition, weexamined with a PCR analysis of the several families ofthe T cell receptor gamma (TCR-Á) gene, if instead ofbeing randomly stimulated T cells would show a clonalexpansion pattern.Materials and MethodsSuspensions of PBMC were obtained by centrifugation from theperipheral blood of 10 nickel-allergic individuals and 5 controls notallergic to nickel. The subsequent cell culture was performed eitherwith complete 10% AB serum containing medium alone or with theaddition of phytohemagglutinin (PHA; 2.4 Ìg/ml), tetanus toxoid(TT; 5.0 Ìg/ml) or nickel sulfate (10–4M, 10–5M ). On day 5 a prolif-erative response was assessed by 3H-thymidine uptake and expressedas stimulation index.Released IL-4 [detection limit (d.l.): 1.5 pg/ml] and IFN-Á (d.l.:5.0 pg/ml) in the supernatants were measured by ELISA. Samplesbelow d.l. were set to 0.75 pg/ml (IL-4) and 2.5 pg/ml (IFN-Á). Toevaluate the respective actual mRNA levels extraction by the phenol/chloroform method was done. RT-PCR (IL-4, IFN-Á, ß-actin) wasapplied according to standard protocols. To cover the range of theTCR-Á chain we used a combination of primers for the main groupsof the variable region (VÁ1–8, VÁ9, VÁ10, VÁ11) and a primer mix,which contains several sequences of different joining regions. TheTCR region VÁ1–8 was detected by the consensus primer VÁ2. PCRproducts were separated on a 6% polyacrylamide gel and photo-graphed after staining with ethidium bromide. Clonal expansion offew specifically reacting T cell populations would result in definedbands in contrast to a ‘smear’ in the case of polyclonal TCR-Á rea-rangement.In vitro T Cell Reactivity Int Arch Allergy Immunol 2001;124:292–295 293Fig. 1. a Proliferative response (stimulation index) from PBMC of 10 nickel-allergic and 5 nonallergic individuals.b Production of IL-4 from PBMC of 10 nickel-allergic and 5 nonallergic individuals. d.l. was 1.5 pg/ml (samplesbelow d.l. are expressed as 0.75 pg/ml. c Production of IFN-Á from PBMC of 10 nickel-allergic and 5 nonallergicindividuals. d.l. was 5.0 pg/ml (samples below d.l. are expressed as 2.5 pg/ml). P = Nickel-allergic; [ = nonallergic.Fig. 2. RT-PCR products of mRNA from cell culture series (here nickel-allergic individual No. 10). 1 = Medium; 2 =PHA; 3 = TT; 4 = NiSO4 10–4M; 5 = NiSO4 10–5M; M = DNA marker.11294 Int Arch Allergy Immunol 2001;124:292–295 Thomas/Summer/Sander/PrzybillaTable 1. Analysis of TCR-Á specificity upon medium or allergenstimulation (clonality)AllergicindividualsMediumalonePHA TT NiSO410–4 MNiSO410–5 M– – VÁ11 – –2 – – VÁ11 VÁ10 –3 – – VÁ11 VÁ10 VÁ104 – – VÁ9 VÁ9 VÁ95 – – VÁ11 VÁ2, VÁ11 –6 VÁ9 VÁ9 VÁ9 VÁ9 VÁ97 – – – VÁ10 –8 – – VÁ11 VÁ10 VÁ109 – – – – –10 – – VÁ9 VÁ2 VÁ2Controls11 – – VÁ11 – VÁ1112 – – VÁ9 VÁ2, VÁ9 VÁ913 – – VÁ9 – –14 VÁ10 VÁ2 VÁ10 VÁ2 –15 – – – VÁ11 VÁ10– = No preferential TCR-Á expression; VÁ2, VÁ9, VÁ10, VÁ11 =predominant TCR-Á family.Table 2. Qualitative evaluation (band intensity) of RT-PCR products (IL-4/IFN-Á)AllergicindividualsMedium aloneIFN-Á IL-4PHAIFN-Á IL-4TTIFN-Á IL-4NiSO4 10–4 MIFN-Á IL-4NiSO4 10–5 MIFN-Á IL-4– – +++ – ++ – ++ – +++ –2 – – +++ ++ ++ – +++ – ++ +3 – – +++ + + – +++ – +++ –4 – – +++ – – – ++ – +++ –5 – – +++ – +++ – +++ – +++ –6 + – +++ – ++ – +++ – +++ +7 – – +++ – ++ – +++ – +++ –8 + – +++ + + – ++ – ++ –9 + – +++ – ++ + +++ – + –10 ++ – +++ – + – + – ++ –Controls11 – – +++ + – – + – – –12 – – +++ – + – + – + –13 – – +++ + ++ – – – + –14 – – +++ – – – + – – –15 – – +++ – – – – – + –mRNA was extracted from unstimulated (medium alone) or allergen stimulated cultures. – = Absent; + = visibleband; ++ = strong band; +++ = very strong expression. Evaluation was done with reference to ß-actin expression.Results and DiscussionPBMC of nickel-allergic patients proliferated upon theaddition of nickel sulfate to a varying degree. Cells of thedifferent blood donors – some of whom had actual eczema– showed a baseline IL-4 production. In contrast, IFN-Á inthe supernatants was enhanced in nickel-stimulated cul-tures of allergic individuals (fig. 1). This was reflected bythe predominant detection of IFN-Á-specific mRNA onday 5 (tables 1, 2, fig. 2). Despite most T cells showing sur-face expression of TCR-·ß, the analysis of the concomi-tant rearrangement of TCR-Á gene makes it possible toevaluate the array of T lymphocytes with regard to clonali-ty. Here the analysis of the TCR-Á spectrum in the specifi-cally stimulated cultures showed clonal expansion (ta-bles 1, 2) in contrast to the unspecific, broad stimulationby PHA. This would point to a rather specific response insensitized individuals in contrast to random T cell activa-tion. Thus, the combined analysis of proliferative responsein vitro, mRNA characterization and potential clonalTCR-Á rearrangement [5, 6] could further help to identifyspecific antigen/allergen-induced T cell activation.In vitro T Cell Reactivity Int Arch Allergy Immunol 2001;124:292–295 295References 1 Möller H: Nickel dermatitis: Problems solvedand unsolved. Contact Dermatitis 1990;23:217–220.2 Cederbrant K, Hultman P, Marcusson JA, Tib-bling L: In vitro lymphocyte proliferation ascompared to patch test using gold, palladiumand nickel. Int Arch Allergy Immunol 1997;112:212–217.3 Kapsenberg ML, Wierenga EA, Stiekema F,Tiggelman A, Bos JD: Th1 lymphokine pro-duction profile from nickel-specific CD4+ T-lymphocyte clones from nickel contact allergicand non-allergic individuals. J Invest Dermatol1992;98:59–63.4 Mitchison NA: Specialization, tolerance, mem-ory, competition, latency, and strife among Tcells. Annu Rev Immunol 1992;10:1–12.5 Thomas P, Thomas M, Summer B, NaumannT, Sander CA, Przybilla B: Intolerance of osteo-synthesis material: Evidence of dichromatecontact allergy with concomitant oligoclonal T-cell infiltrate and TH1-type cytokine expres-sion in the peri-implantar tissue. Allergy 2000;55:969–972.6 Werfel T, Hentschel M, Kapp A, Renz H:Dichotomy of blood- and skin-derived IL-4-producing allergen-specific T cells and re-stricted Vß repertoire in nickel-mediated con-tact dermatitis. J Immunol 1997;158:2500–2505.

In vitro T cell reactivity in nickel allergy: Comparison of T cell clonality, cytokine expression and mediator production

Peter Thomas

Burkhard Summer

Christian Sander

Bernhard Przybilla

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In vitro T Cell Reactivity in Nickel Allergy: Comparison of T Cell Clonality, Cytokine Expression and Mediator Production

http://epub.ub.uni-muenchen.de/16687/1/10_1159_000053736.pdf

In vitro T cell reactivity in nickel allergy: Comparison of T cell clonality, cytokine expression and mediator production

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