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S.R. Thurau, M. Diedrichs–Moehring, G. Wildner; Multiplex Analysis of Cytokine and Chemokine Responses of Peripheral Lymphocytes From Uveitis Patients in a Trial of Oral Tolerance Induction . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4513.
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© ARVO (1962-2015); The Authors (2016-present)
Multiplex technique offers the chance to investigate multiple cytokines and chemokines in small volumes of culture supernatant. We used this method to analyze autoantigen–specific in vitro responses of PBMC from uveitis patients orally treated with peptide B27PD, an S–antigen peptide mimotope.
Peripheral blood lymphocytes of eight uveitis patients who had received peptide B27PD for 12 weeks were collected before, during (4, 8, 12 weeks) and 24 weeks after starting peptide therapy. PBL were stimulated in vitro with retinal S–Ag, peptide PDSAg, therapeutic peptide B27PD, control peptide B7PD, IRBP peptides R14 and PI536, tetanus toxoid and PHA and were pulsed with 3H–thymidine after 5 days. Culture supernatants were collected daily and pooled for multiplex bead analysis of IL–1alpha, IL–2, IL–4, IL–6, IL–8, IL–10, IFN–gamma, TNF–alpha, GM–CSF, MCP–1 and MIP–1alpha. The study adhered to the Helsinki Declaration and was approved by the local ethical committee, patient informed consent was obtained.
PBMC from 4 of 8 patients showed proliferation to ocular antigens S–Ag or peptide PDSAg at least at one time point during the study. All patients' PBL proliferated in response to the recall antigen tetanus toxoid. We could not detect Th1 (IL–2, IFN–gamma) or Th2 (IL–4) cytokine secretion except in cultures stimulated with tetanus toxoid or PHA. Instead, we detected high levels of IL–8, IL–6, MCP–1 and MIP–1alpha. Cytokine and chemokine production in cultures did not correlate with proliferation, disease activity or treatment. During oral tolerance induction we could not detect a shift between Th1, Th2 or Tr1 cytokines.
This study shows, that culture supernatants of antigen– and/or mitogen–stimulated PBMC are not suitable to monitor treatment effects or reflect clinical activity in order to serve as surrogate marker.
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