Development of a sarcoidosis murine lung granuloma model using Mycobacterium superoxide dismutase A peptide

Abstract

Sarcoidosis is characterized by noncaseating granulomas containing CD4(+) T cells with a Th1 immunophenotype. Although the causative antigens remain unknown, independent studies noted molecular and immunologic evidence of mycobacterial virulence factors in sarcoidosis specimens. A major limiting factor in discovering new insights into the pathogenesis of sarcoidosis is the lack of an animal model. Using a distinct superoxide dismutase A peptide (sodA) associated with sarcoidosis granulomas, we developed a pulmonary model of sarcoidosis granulomatous inflammation. Mice were sensitized by a subcutaneous injection of sodA, incorporated in incomplete Freund's adjuvant (IFA). Control subjects consisted of mice with no sensitization (ConNS), sensitized with IFA only (ConIFA), or with Schistosoma mansoni eggs. Fourteen days later, sensitized mice were challenged by tail-vein injection of naked beads, covalently coupled to sodA peptides or to schistosome egg antigens (SEA). Histologic analysis revealed hilar lymphadenopathy and noncaseating granulomas in the lungs of sodA-treated or SEA-treated mice. Flow cytometry of bronchoalveolar lavage (BAL) demonstrated CD4(+) T-cell responses against sodA peptide in the sodA-sensitized mice only. Cytometric bead analysis revealed significant differences in IL-2 and IFN-γ secretion in the BAL fluid of sodA-treated mice, compared with mice that received SEA or naked beads (P = 0.008, Wilcoxon rank sum test). ConNS and ConIFA mice demonstrated no significant formation of granuloma, and no Th1 immunophenotype. The use of microbial peptides distinct for sarcoidosis reveals a histologic and immunologic profile in the murine model that correlates well with those profiles noted in human sarcoidosis, providing the framework to investigate the molecular basis for the progression or resolution of sarcoidosis.

Figure 1.

Timeline of murine model of lung sarcoidosis. C57Bl/6 mice were not sensitized (ConNS) or were sensitized subcutaneously on Day 1 with an emulsion of either incomplete Freund's adjuvant (IFA) + superoxide dismutase A (sodA) peptide, IFA + saline (ConIFA), or intraperitoneally with 3,000 Schistosoma mansoni eggs. Sensitized mice were challenged 14 days later via tail-vein injection of 6,000 Sepharose beads, covalently bonded to sodA or schistosome egg antigens (SEA), or by injection of naked beads (ConNS and ConIFA). Four days after challenge, BAL and lungs were collected and analyzed.

Figure 2.

Lung granuloma formation in mice 4 days after challenge. Lungs were paraffin-embedded, cut, and stained with hematoxylin and eosin. (A) Lung of a mouse sensitized and challenged with sodA (n = 16). Inset: Higher magnification of a granuloma in A reveals a multinucleated giant cell (arrow). (B) Lung granulomas of patient with sarcoidosis. Inset: High magnification of a lung granuloma from another patient with sarcoidosis. (C) Multinucleated giant cell (arrow) in mouse lung granuloma induced by sodA. (D) Lung granuloma from mice sensitized with Schistosoma mansoni eggs and challenged with SEA-coated beads (n = 4). (E) Lung from a nonsensitized (ConNS) mouse challenged with naked beads (n = 20). Inset: Higher magnification of granuloma in E. (F) Lung from a mouse sensitized with IFA only (ConIFA) and challenged with naked beads (n = 9). b, beads. Scale bars = 50 μm in A, C, D, E, and F; and 100 μm in B, inset in B, and inset in E.

Figure 3.

Morphometric analysis of lung granulomas elicited by different treatments. Granuloma area was determined for ConNS and ConIFA mice challenged with naked beads, sodA-sensitized and challenged mice, and SEA-sensitized and challenged mice, as described in Materials and Methods. Median and 25th and 75th percentile bars are shown; values were derived from analyses of 3–4 mice per treatment, totaling 24–125 granulomas. Statistical differences were determined using the Kruskal-Wallis test followed by Dunnett's multiple comparisons test. *P < 0.001. ^‡P = NS. ^§P < 0.001.

Figure 4.

Immunohistochemical analyses of leukocytes present in lung granulomas of sodA-treated mice (A, C, and E) and human patients with sarcoidosis (B, D, and F). (A and B) Macrophages. (C and D) CD3⁺ T cells. Inset in C: CD4⁺ T cells. (E and F) B cells. Scale bars = 25 μm in A, C, inset in C, and E, and 50 μm in B, D, and F.

Figure 5.

BAL fluid from sodA-treated mice demonstrates sodA-specific CD4⁺ Th1 immune response. Intracellular cytokine staining for IFN-γ was performed on BAL cells from baseline, SEA-treated, and sodA-treated mice, after stimulation with sodA peptide or staphylococcal enterotoxin B (SEB). Dot plots represent percentage of cells expressing IFN-γ within CD3⁺CD4⁺ gated subsets. These data are representative of three sets of mice (n = 9 per set).

Figure 6.

Cytokines present in BAL fluid from baseline, sodA- and SEA-treated mice. BAL fluid was collected and pooled from mice, and analyzed for (A) IL-2, (B) IFN-γ, (C) IL-4, and (D) IL-5. Statistical significance was determined using the Wilcoxon rank sum test. *P = 0.008. ^‡P = NS. ^§P = 0.02. These data are representative of three sets of mice (n = 9 per set).

Figure 7.

The sodA-specific CD4⁺ Th1 immune response is major histocompatibility complex (MHC) Class II–restricted. Intracellular cytokine staining for IFN-γ was performed on BAL cells from control and sodA-challenged mice, after stimulation with sodA peptide or SEB. Dot plots represent percentage of cells expressing IFN-γ within CD3⁺CD4⁺ gated subsets. These data are representative of three sets of mice (n = 9 per set).