Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis

Abstract

BackgroundKveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4–6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins.MethodsKv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay.ResultsWe observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4.ConclusionsStimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.

Imbalance in B cell and T Follicular Helper Cell Subsets in Pulmonary Sarcoidosis

“…Several studies revealed the increased levels of autoantibodies, including antinuclear 3 , anti-dsDNA 5 or anti-cyclic citrullinated peptide antibodies 4 in the sera of sarcoidosis patients. Moreover, B cells participate in the specific vimentin recognition that is also support the assumption about the role of humoral immune response in sarcoidosis 6,7 . Next, sarcoidosis is associated with a polyclonal hypergammaglobulinemia, where IgA−, IgG−, and IgM-secreting cells in bronchoalveolar lavage fluid are described 8 .…”

Etiology and Immunopathogenesis of Sarcoidosis: Novel Insights

“…Stimulation with vimentin could induce a specific proinflammatory cytokine response only in patients with sarcoidosis. 151 These data further strengthen the role of vimentin as a possible autoantigen in sarcoidosis pathogenesis. Given the lack of immune responses to vimentin in all patients, it is likely that other autoantigens may be involved in disease pathogenesis.…”

Key Players and Biomarkers of the Adaptive Immune System in the Pathogenesis of Sarcoidosis

“…Vimentin is a possible autoantigen proposed as the culprit of the granulomatous inflammation seen in sarcoidosis. IHC staining identified increased vimentin especially localized to granulomas in sarcoidosis patient spleens and stimulation of sarcoid peripheral blood mononuclear cells with vimentin induced higher IFN-γ and TNF-α secretion compared to those of tuberculosis patients or healthy controls [ 97 ].…”