Date Published: January 23, 2017
Publisher: Public Library of Science
Author(s): Christian Eberhardt, Muhunthan Thillai, Robert Parker, Nazneen Siddiqui, Lee Potiphar, Rob Goldin, John F. Timms, Athol U. Wells, Onn M. Kon, Melissa Wickremasinghe, Donald Mitchell, Mark E. Weeks, Ajit Lalvani, Francesco Dieli.
Kveim-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.
Kv 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.
We 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. Stimulation 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.
Sarcoidosis is a multi-organ granulomatous disease of unknown cause which occurs in genetically susceptible individuals  but primarily affects the lungs. The worldwide prevalence is 40 per 100,000 with highest incidence in North America, Scandinavia and Japan . Despite evidence for environmental triggers including clustered outbreaks and person-to-person transmission , there is no universally accepted cause of disease. The largest case controlled study to date comprised 705 patients and controls did not identify any common predominant triggers .
We identified a pro-inflammatory cytokine response in PBMCs from sarcoidosis patients when stimulated with Kveim reagent (Kv). This response was not observed in PBMCs from healthy volunteers stimulated with Kv nor in sarcoidosis PBMCs stimulated with control Kv made from healthy spleen tissue. Using two complementary proteomic approaches we identified 74 proteins specific to sarcoidosis tissue and found that one, vimentin, induced the similar pattern of cytokine secretion from sarcoidosis PBMCs. This response was not seen with vimentin stimulation of PBMCs from healthy volunteers or those with pulmonary tuberculosis.