Sarcoidosis involves the lung in more than 90% of patients. The disease is characterized by Th1 alveolitis, accompanied by a CD4+
T-cell predominance, leading to the formation of granulomas. No animal model, to the best of our knowledge, uses peptides that are unique to sarcoidosis. The mounting evidence for the involvement of mycobacterial antigens in the pathogenesis of sarcoidosis, and the specific identification of mycobacterial virulence factors in the BAL and granulomas of patients with sarcoidosis, led us to develop a murine model of pulmonary sarcoidosis. The administration of antigen-coated beads to elicit a delayed-type hypersensitivity reaction in mice has been widely used to study the cellular and molecular mechanisms for the selective recruitment of leukocytes to the airways (13
). After the embolization (via tail-vein injection) of antigen-coated beads, mice presensitized with the same antigen develop many features similar to human granuloma formation. For example, Mycobacterium bovis
purified protein derivative (PPD)–sensitized and challenged mice develop lung granulomas that are largest 4 days after challenge, and that resolve 8 days after challenge (13
). Similar to the situation in human pulmonary granulomas, sodA induced the formation of large granulomas throughout murine lungs. The murine model of sarcoidosis possesses the strongest pathologic hallmarks of sarcoidosis: hilar lymphadenopathy, lymphocytic infiltrates, the formation of giant cells, and a Th1 immunophenotype ( and ). This model demonstrates an additional strength, in that peptides isolated only from sarcoidosis granulomas are used to incite granulomatous inflammation. This unique Mycobacterium
sodA peptide sequence demonstrates the specificity of this peptide for eliciting the histologic features observed in human sarcoidosis.
In contrast, no sensitization (ConNS) or sensitization with IFA alone (ConIFA), followed by challenge with naked beads, led to the formation of small, foreign-body granulomas. To contrast the granuloma formation elicited by sodA to that elicited by a sarcoidosis-unrelated antigen, we used the SEA model developed by Chensue and colleagues (13
). As was the case in sodA-treated mice, SEA elicited granulomas throughout the lungs, with the formation of giant cells. In contrast to sodA-elicited granulomas, SEA granulomas were larger, they developed fibrosis, and they were associated with a Th2 immunophenotype ( and ) (13
). Taken together, these studies show that sodA elicits lung granulomas in mice, with the histologic features of human sarcoidosis.
Next, we determined the leukocyte components of sodA murine lung granulomas by immunohistochemistry, and compared them with those in human sarcoid pulmonary granulomas. Macrophages were the most abundant cell type in the granulomas of both sodA-treated mice and human sarcoidosis. Macrophages are known to be key players in sarcoidosis pathogenesis, because of their ability to present foreign antigens to innate and adaptive cellular components. Other cell types that have been the focus of intense investigation for their possible role in granuloma formation include T lymphocytes. We used CD3, a general marker for both CD4+
T cells. T cells were found in the middle layers of granulomas in both murine and human lungs. In addition, we localized abundant CD4+
T cells to the same region as the CD3+
cells in the sodA-elicited murine lung granulomas. A recent study of sarcoidosis BAL reported on CD4+
T-cell responses to mycobacterial antigens. We found similar responses in the murine model (). Although the formation of granulomas was evident in SEA-sensitized mice, T cells isolated from their BAL did not recognize sodA peptides (). Finally, B cells were also found sporadically in human and murine sarcoid lung granulomas. These observations are consistent with those of Minami and colleagues (19
), Nishiwaki and colleagues (20
), and McCaskill and colleagues (21
), who studied murine immune pulmonary responses to Propionibacterium acnes
. They found peribronchovascular granulomatous inflammation, composed of T and B cells and histiocytes, in response to P. acnes
. Although P. acnes
was implicated as a putative agent in Japanese and European patients with sarcoidosis, to the best of our knowledge, the molecular and immunologic evidence of its involvement in American patients with sarcoidosis was not previously reported.
Increasing evidence suggests that the degree of inflammation and fibroblast action/proliferation may be dependent on a balance of Th1-like and Th2-like cytokines expressed during the evolution of sarcoidosis (22
). A study by Moller and colleagues (23
) of BAL cells and fluid from sarcoid patients showed a predominant Th1 cytokine response, with an elevated gene and protein expression of IFN-γ and IL-12, but not IL-4 or IL-10. Moller and colleagues (23
) suggested that a chronic deregulation of IL-12 (which induces the production of IFN-γ, and directs T-cell expansion down the Th1 pathway) is the driving force of granuloma formation in sarcoidosis. The importance of these cytokines in the formation of granulomas was shown when IL-12 and IFN-γ knockout mice failed to develop granulomas after being challenged with granulomatous agents (24
). The lymphocytes from sodA-treated mice also demonstrated the Th1 immunophenotype seen in patients with sarcoidosis, compared with the Th2 granulomatous inflammation in SEA-treated mice (). Other early mediators include TNF-α, IL-1β, and IL-6, which both amplify and maintain the formation of granulomas (26
). The flow cytometry of murine BAL fluid demonstrates that no immune responses to microbial antigens occur at baseline, with an appropriate positive response to the positive control, SEB. BAL CD4+
T cells demonstrate antigen-specific responses to sodA-sensitized mice. A Th1 immunophenotype was demonstrated by the secretion of IL-2 and IFN-γ. A lack of recognition of sodA was evident in SEA-sensitized, ConNS, and ConIFA mice, as expected ( and ).
The role of genetics in sarcoidosis pathogenesis demonstrates that MHC Class II alleles are associated with susceptibility to, or protection from, sarcoidosis (5
). We were interested in the role of MHC Class II in the antigen presentation of sodA peptides in the murine model. A blockade of MHC Class II inhibited recognition of the sodA peptide, a result similar to what was described the recognition of mycobacterial antigens in patients with sarcoidosis (9
). This particular feature of the model will allow us to identify the molecular basis for genetic associations with sarcoidosis outcomes.
Experimental models of granulomatous inflammation, characterized by either a Th1 or Th2 response (22
), are also useful in delineating the mechanisms that maintain and resolve chronic granulomatous lung disease (29
). For example, in vivo
studies of the granuloma formation induced by Mycobacterium
infection showed that IFN-γ and TNF-α are necessary for the progression of lesions (13
). Moreover, McCaskill and colleagues (21
) revealed a Th1 cytokine profile in the BAL fluid and lungs associated with P. acnes
–induced granulomatous inflammation in murine lungs. In the present study, both IFN-γ and IL-2 were present in the BAL fluid of sodA-treated mice, consistent with the Th1 cytokine pattern observed in sarcoidosis BAL. On the other hand, the formation of granulomas induced by Schistosoma mansoni
was dependent on IL-4 (17
). In the SEA model, a shift in the cytokine profile occurred from Th1 to Th2 (i.e., IL-4, IL-5, and IL-10), which eventually resulted in fibrosis (13
). The activation of tissue remodeling, including the increased deposition and degradation of extracellular matrix, occurs simultaneously with the inflammatory response.
Thus, this murine model of sarcoidosis contains the gross, histologic, and immunologic features seen in active sarcoidosis. The work to date demonstrates that (1) mice can develop noncaseating granulomas from peptides unique to sarcoidosis; (2) the gross pathology and histology closely parallel those seen in human patients with sarcoidosis; (3) according to cell type and cytokine pattern, the immunology reflects observations in patients with sarcoidosis at presentation; and (4) MHC Class II alleles are also shown to be important in generating immune responses in this model. This model will serve as an important vehicle to facilitate the identification of mechanistic and immunologic contributors to the resolution of sarcoidosis or progression to fibrosis.