Whereas molecular methods are quite sensitive, immune assays can be more powerful because trace amounts of antigen are sufficient to trigger an immune activation in a sensitized host. Moreover, the presence of preformed humoral or cellular mediators against an antigen supports prior exposure, even in the absence of active infection. Finally, a host immunologic response provides a direct mechanism by which mycobacterial antigens could drive disease development or progression in sarcoidosis.
Early observations of a host immune activation in sarcoidosis included an increase in the γ globulin fraction of sarcoidosis serum samples, which was remarkably similar to the spike found in tuberculosis blood samples (86
). This elevation in immunoglobulin probably reflects increased B-cell activation and antibody production in the early course of the disease (87
). Circulating antimycobacterial antibodies have been used to substantiate PCR findings detecting MAC genetic material in patients with sarcoidosis (56
). Sera from patients with sarcoidosis tested positive on immunoblots of recombinant mycobacterial antigens p36 and heat shock protein (hsp)65. Sera from normal patients did not react to these antigens, although 53% of patients with ulcerative colitis reacted to the p36 antigen (56
). Another study using ELISA showed high levels of anti-hsp70 antibodies in patients with sarcoidosis (88
). This antigen increases expression of costimulatory molecules, suggesting that MTB-hsp70 positivity could lead to a break in immune tolerance and subsequent autoimmune disease (89
). Consistent with these positive serology observations, mycobacterial hsp16, hsp65, and hsp70 have been detected in sarcoidal tissue by immunostaining (77
A robust step associating sarcoidosis and mycobacteria occurred with the identification of mKatG as an antigen and a target of the adaptive immune response in patients with sarcoidosis (81
). This work relied on identifying antigens in the Kveim reagent. In the Kveim reaction, lymph node or spleen homogenates from a subject with confirmed sarcoidosis injected into a patient with putative sarcoidosis resulted in the formation of granulomas at the inoculation site 80% of the time (90
). Song and coworkers reasoned that components of the Kveim reagent could be the antigen(s) responsible for triggering sarcoidal granuloma formation. Previous research on the Kveim reagent suggested that the antigen was protein in nature, given that its reactivity was abrogated by denaturants but was resistant to neutral detergents, acidity, and nucleases (91
). Dissociated sarcoidosis tissue produced poorly soluble aggregates that reacted with sera IgG of patients with sarcoidosis. When these aggregates were analyzed by mass spectrometry, the highest match was mKatG. The authors then found mKatG protein in sarcoidosis samples by immunoblotting in 55% of cases but not in control tissues. Furthermore, they discovered serum antibodies against recombinant mKatG in 48% of patients with sarcoidosis but not in control subjects (81
). These results demonstrated that a mycobacterial antigen was present in at least a subset of sarcoidosis tissues and that a humoral response against that antigen could be detected.
Subsequently it was shown that mKatG could trigger cellular immune responses in lymphocytes collected from patients with sarcoidosis. Drake and colleagues reported that peripheral blood mononuclear cells (PBMNs) from 15 of 26 patients with sarcoidosis responded to peptides from the mycobacterial antigens ESAT-6 and/or mKatG, compared with 1 of 24 purified protein derivative (PPD)-negative control subjects (92
). The same group observed higher rates of ESAT-6 and mKatG-dependent T-cell activation and proliferation in BAL samples from patients with sarcoidosis versus control subjects (93
). It was further shown that CD4+ T cells mediated a Th1
response in patients with sarcoidosis when these peptide antigens were presented by DRB1*1101-expressing antigen-presenting cells (94
). This finding links mycobacterial antigens to a typical sarcoidal Th1
response in the context of a well established sarcoidosis susceptibility MHC allele.
In similar studies, the group that originally identified mKatG in the Kveim reagent demonstrated that mKatG protein was able to activate PBMNs from patients with sarcoidosis (95
). Compared with PPD-negative control subjects, 67% of patients with sarcoidosis had significantly more IFN-γ–secreting, mKatG-reactive PBMNs. Moreover, 85% of sarcoidosis cases had high responses to mKatG or PPD components, suggesting prior exposure to mycobacteria. IFN-γ secretion from BAL cells was much higher than from PBMNs, demonstrating that mKatG-specific cells concentrate in the region of active disease. Finally, a higher percentage of BAL and PBMN CD4+ cells from patients with sarcoidosis proliferated in response to mKatG, and the level of mKatG responsiveness correlated to disease course. The authors concluded that mKatG behaves as a pathogenic antigen at least in a subset of sarcoidosis cases (95
If mKatG is driving an immune response in sarcoidosis, one would predict an antigenic difference between mycobacterial KatG protein and KatG from other bacterial species that can infect humans. To test if the antigenic KatG peptide associated with sarcoidosis was specific to mycobacteria, we performed a sequence analysis (F. Ramírez-Valle and S. Prystowsky, unpublished data). Whereas KatG protein is approximately 70% identical across several species of mycobacteria as well as other bacteria, including nocardia, the mKatG peptide 13 epitope tested by Drake and colleagues (92
) is approximately 90% identical among most mycobacteria (using the SIM protein alignment tool; http://ca.expasy.org/tools/sim-prot.html
). Restriction of this epitope sequence to mycobacterial species is evidenced by KatG peptide 13 having less than 60% homology between MTB and nocardia or other bacterial species. shows phylogenetic trees of the entire KatG and the KatG peptide 13 among several mycobacteria and other intracellular bacteria, demonstrating that the antigenic peptide 13 is relatively conserved among mycobacteria. Thus, mKatG peptide 13 appears to be an immunodominant peptide specific to mycobacteria that, in conjunction with HLA susceptibility, can induce a Th1 cellular immune response in patients with sarcoidosis.
Figure 1. The most antigenic epitope of mycobacterial catalase-peroxidase antigen (mKatG) in patients with sarcoidosis is conserved among mycobacterial species. (A) Phylogenetic diagram of full-length KatG protein showing high homology of KatG homologs across multiple (more ...)
Cellular responses to several additional mycobacterial antigens have also been reported. In one study, patients with sarcoidosis responded to the mycobacterial virulence factor antigen 85A (Ag85A), a mycolyl transferase (97
). The authors reported high IFN-γ production from PBMNs stimulated with whole recombinant Ag85A in 15 of 25 patients with sarcoidosis and in 2 of 22 PPD-negative healthy control subjects. The same group reported Th1
responses to antigens from mycobacterial ESAT-6, mKatG, and SodA (96
). In this study, BAL cells from pulmonary sarcoidosis responded more robustly to the mycobacterial antigens than did peripheral cells, adding to the evidence that antigen and antigen-responsive cells may cluster in areas of active disease. Dubaniewicz and colleagues found that PBMNs from patients with sarcoidosis and patients with tuberculosis were similarly activated by in vitro
exposure to recombinant MTB heat-shock proteins relative to cells from healthy control subjects (98
). Together these data imply that many different mycobacterial antigens may contribute to pathogenic responses in sarcoidosis. In contrast, PBMNs and BAL mononuclear cells collected from 17 German patients with sarcoidosis showed similar IFN-γ production compared with 35 PPD-negative control subjects with nongranulomatous lung disease when stimulated with mycobacterial antigens ESAT-6 or CFP-10 (78
). These findings suggest the possibility that immune cells isolated from diseased lungs possess a general hyperreactivity that is not specific to mycobacterial antigens. However, Drake and colleagues detected mycobacterial antigen–evoked cellular immune responses in cells from patients with sarcoidosis who did not react to Trypanosoma brucei
) or the neoantigen keyhole limpet hemocyanin (93
), demonstrating antigen specificity in the immune response.
Because mycobacterial genes and proteins can be found in sarcoidosis samples from patients with specific immune responses to the same proteins, one would expect that mycobacerial peptides might be bound to HLA on antigen-presenting cells in sarcoidal tissues. Yet when peptides bound to sarcoidosis-associated HLA protein DRB1*0301 were isolated in a Swedish population (99
), no mycobacterial antigens were observed. It is possible that these antigens are found in limited concentrations or are not amenable to elution and sequencing. Nonetheless, a follow-up study found that the cellular response of PBMNs and BAL cells to mKatG was stronger in Swedish patients with the DRB1*0301 HLA allele (95
), suggesting that it may be involved in initiating a pathogenic immune response to this mycobacterial antigen.