Cellular Recognition of Mycobacterium tuberculosis ESAT-6 and KatG Peptides in Systemic Sarcoidosis

Sarcoidosis is an enigmatic disease with a pathology similar to that of tuberculosis. We detected Th-1 immune responses to Mycobacterium tuberculosis ESAT-6 and KatG peptides from peripheral blood mononuclear cells from 15/26 sarcoidosis, 1/24 purified-protein-derivative-negative (PPD )( P < 0.0001, Fisher’s exact test), and 7/8 PPD-positive (PPD) subjects (P 0.21). This finding provides immunologic links between mycobacteria and systemic sarcoidosis. While the antigen(s) responsible for eliciting the sarcoidosis Th-1 immune response has not been identified (9, 13, 14), reviews of sarcoidosis immunology and pathology suggest that mycobacterial antigens could be important (6, 8). Previous studies have reported humoral responses to mycobacterial antigens among sarcoidosis subjects (4, 15) as well as the detection of mycobacterial nucleic acids and proteins in sarcoidosis granulomas (3, 5, 15). We performed enzyme-linked immunospot (ELISPOT) assays to assess for cellular recognition of two Mycobacterium sp. antigens (ESAT-6, an immunodominant Tcell antigen present in some members of the Mycobacterium tuberculosis complex but absent in Mycobacterium bovis BCG [2], and KatG, a catalase-peroxidase [7]) from peripheral blood mononuclear cells (PBMC) from 26 sarcoidosis, 24 purifiedprotein-derivative-negative (PPD), and 11 PPD-positive (PPD) subjects. This study was approved by the Vanderbilt University Institutional Review Board for human studies, and informed written consent was obtained from the study participants or their surrogates. All sarcoidosis subjects from the available patient database of the Vanderbilt University Pulmonary Clinic were invited to participate in the study. For inclusion in this study, the clinical, histological, and microbiologic criteria used to define sarcoidosis were as previously described (3). Healthy PPD volunteers were required to have undergone PPD testing by the Vanderbilt employee health services. PPD-positive subjects had written documentation of their PPD statuses and had no evidence of active disease at the time of study enrollment. The amino acid sequences for the 17 ESAT-6 peptides, 15-mers overlapping by 10 amino acids, were as previously described (11). KatG peptides, 15-mers overlapping by 10 amino acids, were derived from the amino acid sequence of M. tuberculosis (GenBank accession number NP 216424) and are listed in Table 1. Each ESAT-6 and KatG peptide was synthesized by solid-phase 9-fluorenylmethoxy carbonyl (Fmoc) chemistry (Genemed Synthesis, San Diego, CA), to a purity of 70%. Identity was confirmed by mass spectroscopy and purity by high-performance liquid chromatography. PBMC were isolated from blood samples drawn into tubes containing EDTA, separated by Ficoll-Hypaque density gradient separation (Amersham Biosciences), cryopreserved in fetal calf serum with 10% dimethyl sulfoxide, and stored in liquid nitrogen until the time of the analysis.