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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig 1

Total IFN-γ (Fig 1a) and TNF-α secretion (Fig 1c) after incubation of PBMCs from patients with confirmed Sarcoidosis (S) or healthy volunteers (H) with sarcoidosis (sKv) or control (cKv) Kveim reagent. Each dot represents mean cytokine concentration of one well stimulated in duplicate with antigen for 36 hrs. Fig 1b and 1d represent replicate experiments with the same PBMCs but using replicates of sKv and cKv. Statistical significance for each comparison was determined by Mann-Whitney test.

Fig 2

Images of colloidal Coomassie Blue stained 1D-SDS-PAGE gels loaded with biological replicates of historical diagnostically in vivo validated Kv (vKv) (Fig 2a), newly created Kv from sarcoidosis spleen (sKv) (Fig 2b) and newly created control Kv from healthy spleen tissue (cKv) (Fig 2c). Each lane was sliced into 10 equal sections. Proteins were subjected to in-gel tryptic digestion and peptides analysed by MS/MS and database searching for protein identification.

Fig 3. Example of a 2D-DIGE image (pI 6–9) depicting differences in protein abundance between sarcoidosis and healthy control spleen tissue.

Fig 4

Immunohistochemical staining of spleen tissue shows increased abundance of vimentin in sarcoidosis spleens at 200 x (Fig 4a) and 400 x magnification (Fig 4b) compared to healthy spleen tissue at the same magnifications (Fig 4c and 4d).

Fig 5

Total IFN-γ (Fig 5a) and TNF-α secretion (Fig 5b) after incubation of PBMCs from patients with sarcoidosis (S), tuberculosis (TB) or from healthy volunteers (H) with pooled recombinant human vimentin. Each dot represents the mean cytokine concentration of one PBMC sample stimulated in duplicate with protein for 36 hrs (after subtraction of the negative control well). Statistical significance between comparisons was determined by Mann-Whitney test.