This study demonstrates that mycobacterial antigens induce a T-cell-specific response in the BAL fluid cells of sarcoidosis patients. The presence of these responses at the time of diagnosis is supportive of prior exposure to mycobacteria, and the confinement of responses to the lung at the time of diagnosis is congruent with prior epidemiologic studies suggesting that exposure to bioaerosols is a risk factor for the disease. These results are supported by a recent report showing responses to KatG whole protein in sarcoidosis patient BAL fluid from two research center study cohorts (10
It has been reported that superinfecting environmental mycobacteria preferentially home to tuberculous granulomas (11
). The significant absence of immune responses to mycobacterial virulence factors among granulomatous control subjects suggest that the immune responses detected in sarcoidosis subjects do not reflect superinfection but rather a role in pathogenesis. The presence of congruent recognition of two mycobacterial proteins, not just one, argues against the hypothesis that sarcoidosis is caused by retention of a single poorly cleared or poorly degraded antigen or protein (Fig. ). The specificity of the responses is evidenced by the lack of responses among the seven subjects with hypersensitivity pneumonitis or MycobacteriumY avium
infection. Furthermore, prior reports have identified the KatG protein by immunohistochemistry, mass spectrometry, and T-cell response assays with sarcoidosis subjects (9
). Moreover, a comparison of the KatG sequences of M. bovis
, M. avium
, M. smegmatis
, and M. tuberculosis
reveal a 99.9% identity in the 740-amino-acid overlap (GenBank accession no. NP_855594, NP_960602, YP_888029, and NP_216424, respectively). It has also been demonstrated that patients with pulmonary M. avium
do not react to ESAT-6, although rare responses have been reported (1
). Immune recognition of ESAT-6 has been reported in infections with other pathogenic mycobacteria, such as M. tuberculosis
, M. marinum
, and M. kansasii
). The significant difference in responses to mycobacterial antigens among sarcoidosis patient and control specimens supports the concept that ESAT-6-specific immune responses are not due to contamination of laboratory reagents with environmental mycobacteria (Fig. ). A lone control specimen (disease control 25) had strong reactions to KLH, ESAT-6, KatG, and propionibacterial supernatant. These responses may reflect infection with a microorganism that secretes these proteins, contamination, or possibly nonspecific reactivity.
Prior reports have found an association between sarcoidosis and propionibacteria in sarcoidosis patient specimens (25
). Although the assays were performed on limited numbers of sarcoidosis patient and control BAL fluid specimens, a preliminary investigation did not reveal immunologic evidence of P. acnes
in the study participants. Molecular analysis for P. acnes
was also performed and was negative for all five sarcoidosis patient specimens and four of the five control specimens tested (data not shown). Future studies will be performed to delineate the role of P. acnes
in American sarcoidosis subjects.
Another interesting observation was the presence of immune responses to mycobacterial antigens in BAL fluid cells and not in circulating cells. This has been reported in M. tuberculosis
infection also (6
). A recent study of tuberculosis patients found heterogeneity in recognition of mycobacterial antigens by BAL fluid cells (40
). In addition, the peripheral blood response to antigens did not always match the responses observed at the active site. In tuberculosis patients, the observed response to purified protein derivative of M. tuberculosis
in the lung was 100-fold greater than that in blood (4
). In prior reports, we demonstrated peripheral blood responses to mycobacterial antigens (9
). However, in these studies, the majority of the sarcoidosis subjects had been diagnosed for a year or longer, while in this study, we evaluated sarcoidosis subjects at the time of diagnosis. These data suggest that the immune response may be mainly localized to the site of active granulomatous inflammation.
The detection of CD8+
T-cell responses in sarcoidosis patient BAL fluid suggests important immunologic and genetic implications. The role of CD8+
T cells in sarcoidosis has not been well studied. It has been shown that CD8+
T cells secrete IFN-γ and tumor necrosis factor alpha in response to M. tuberculosis
antigen in M. tuberculosis
). There is mounting evidence that CD8+
T cells play a protective role in the host response to pathogenic mycobacteria (7
). Moreover, CD8+
T cells can kill infected cells via a granule-dependent mechanism involving perforin and granulysin, which also possess direct antimicrobial activities (45
). Therefore, CD8+
T cells may play a crucial role in the sarcoidosis clinical outcomes.
We are also interested in innate and adaptive signaling mechanisms important in the sarcoidosis patient immune response against microbial antigens. As cited earlier, direct interaction of mycobacterial ESAT-6 with TLR2 has been reported. It has also been demonstrated that TLR2 is recruited to the phagosomes of macrophages, thus allowing them to promptly respond to the invading pathogens (47
). A recent report suggests that ESAT-6 of M. tuberculosis
inhibits TLR2 signaling in macrophages (36
). While we were limited by cell availability, preliminary evidence suggests that inhibition of TLR2 signaling results in a reduced immune response. These preliminary results suggest that TLR2 signaling plays a role in the sarcoidosis patient immune response against ESAT-6. Due to limitations in the availability of BAL fluid cells, we were not able to investigate the role of other TLRs in this study. However, a more in-depth investigation including the role of other TLRs, such as TLR4 and TLR9, is warranted.
In conclusion, these findings demonstrate specific recognition of mycobacterial virulence factors at the time of diagnosis. It adds additional support to an association between mycobacteria and sarcoidosis but does not show causality. It remains feasible that the relationship of mycobacteria to sarcoidosis may represent an abnormal host response to nonviable organisms. Additional studies are also required to delineate the role of TLR2 signaling in the sarcoidosis patient immune response. An investigation to determine if pulmonary and extrapulmonary sarcoidosis involvements reflect the same pathogenesis is warranted. The current study cannot address extrapulmonary sarcoidosis involvement. Further investigation into the role of mycobacteria as an etiologic contributor to sarcoidosis pathogenesis is warranted.