Localization of propionibacterium acnes in granulomas supports a possible etiologic link between sarcoidosis and the bacterium

Abstract

Sarcoidosis likely results from the exposure of a genetically susceptible subject to an environmental agent, possibly an infectious one. Mycobacterial and propionibacterial organisms are the most commonly implicated potential etiologic agents. Propionibacterium acnes is the only microorganism, however, found in sarcoid lesions by bacterial culture. To evaluate the pathogenic role of this indigenous bacterium, we screened for the bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger-factor protein (TIG antibody). We examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, and corresponding control samples from 275 patients with non-sarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients. Immunohistochemistry with PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. In nongranulomatous areas, small round bodies detected by PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some non-sarcoid patients. Large-spheroidal acid-fast bodies, Hamazaki-Wesenberg bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these Hamazaki-Wesenberg bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body. The high frequency and specificity of P. acnes, detected by PAB antibody within sarcoid granulomas, indicates that this indigenous bacterium might be the cause of granuloma formation in many sarcoid patients.

Figure 1

Western blot analysis for reactivity of the anti-P. acnes antibodies. Western blot analysis with PAB and TIG antibodies was performed using sonicated whole-cell lysate (lanes 1–10) with the appropriate positive control samples. Lane 1, serotype I P. acnes (ATCC 6919); lane 2, serotype I P. acnes (ATCC 11827); lane 3, serotype II P. acnes (ATCC 11828); lane 4, serotype I P. acnes (clinical isolate S4); lane 5, serotype II P. acnes (clinical isolate S7); lane 6, P. granulosum; lane 7, P. avidum; lane 8, M. tuberculosis; lane 9, M. avium; and lane 10, M. intracellulare. Positive control samples for the PAB antibody were lipoteichoic acid purified from serotype I (lane 11) and serotype II (lane 12) of P. acnes. Positive control samples for the TIG antibody were recombinant trigger-factor protein from the whole-genome sequence of P. acnes serotype I (lane 13) and serotype II (lane 14).

Figure 2

P. acnes within sarcoid granulomas of the lungs. Hematoxylin and eosin staining (left) and immunostaining with PAB antibody (right) are shown pairwise for three different video-assisted thoracic surgery samples of the lungs from sarcoid patients (original magnification, × 400). A higher magnification of the positive reaction products indicated by the red arrows is shown in each inset. In the granulomatous alveolitis lesion (a, b), some swollen macrophages of the immature granuloma were filled with many small round bodies detected by PAB antibody. In the granuloma lesion surrounded by prominent inflammatory cell infiltration (c, d), such small round bodies were detected not only in the granuloma cells but also in some of the inflammatory cells (black arrows). In a mature granuloma (e, f), a few granuloma cells with degraded forms of the small round bodies were sparsely distributed within the granulomas.

Figure 3

P. acnes within sarcoid granulomas of the lymph nodes. Hematoxylin and eosin staining (left) and immunostaining with PAB antibody (right) are shown pairwise for two Japanese samples (a, b, and c, d) and one German sample (e, f) of lymph nodes from patients with sarcoidosis (original magnification, × 1000). A higher magnification of the positive reaction products indicated by the red arrows is shown in each inset. Many small round bodies were detected within an immature granuloma (a, b) and a few small round bodies were detected within a mature granuloma (c, d). Granuloma cells filled with such small round bodies were observed at the rim of a granuloma (b, d). In many sarcoid granulomas, including in the German samples shown here (e, f), large spheroidal bodies detected by PAB antibody were sparsely distributed within a mature granuloma.

Figure 4

P. acnes in non-granulomatous areas of the sarcoid and non-sarcoid lungs and lymph nodes. Many macrophages with many small round bodies detected by PAB antibody were found in alveolar spaces adjacent to granulomas (^*) and areas of inflammation (indicated by the arrow heads) of many video-assisted thoracic surgery samples from sarcoid patients (a). A few such alveolar macrophages were also detected in some lung samples from non-sarcoid patients (b). Clusters of macrophages with many small round bodies detected by PAB antibody were found in granuloma-free paracortical areas of the lymph nodes with sarcoid granulomas (c). A few paracortical macrophages with many small round bodies detected by PAB antibody were also found in some samples from non-sarcoid patients (d). Higher magnification of the positive reaction products indicated by the red arrows is shown in each inset (a–d). A few large spheroidal bodies detected by PAB antibody, indicated by the black arrow, were occasionally observed in alveolar macrophages of the sarcoid lungs (e). Such a large spheroidal body also reacted with TIG antibody (f), as indicated by the black arrow and was acid-fast with Fite staining (f, inset). Original magnification: a–b, × 400; c–d, × 200; e–f, × 1000.

Figure 5

Immunofluorescence double-staining to phenotype the P. acnes-positive cells in non-granulomatous areas of sarcoid lungs and lymph nodes. Green signal (FITC): PAB antibody; red signals (TRITC): anti-CD68 antibody (a, c, e), anti-fascin antibody (b, d, f). Results of double-staining are merged in all pictures. In the sarcoid lung, all of the P. acnes signals overlapped (yellow) with CD68-positive alveolar macrophages (a), whereas the fascin-positive alveolar dendritic cells did not contain small round bodies detected by PAB antibody (b). In the paracortical area of the sarcoid lymph node, small round body detected by PAB antibody were found in the CD68-positive macrophages (c), but not in the fascin-positive dendritic cells (d). In the sinus of the sarcoid lymph node, Hamazaki-Wesenberg bodies detected by PAB antibody overlapped with CD68-positive macrophages (e), but not with the fascin-positive dendritic cells (f). Original magnification: a–b, × 400; c–f, × 1000.

Figure 6

Large-sized acid-fast bodies detected by PAB and TIG monoclonal antibodies in sarcoid lymph nodes. In this lymph node sample from a Japanese patient with sarcoidosis, many large-sized yellow-brown bodies, as indicated by the arrows, were observed with hematoxylin and eosin staining in hyperplastic sinuses (a). These bodies were strongly acid-fast with Fite staining (a, inset). Electron-microscopy of the same sample revealed typical features of Hamazaki-Wesenberg bodies (b). This sarcoid lymph node sample was examined by immunohistochemistry using PAB antibody (c, d) and TIG antibody (e, f) with light microscopy and electron microscopy. The Hamazaki-Wesenberg bodies reacted with PAB antibody (c) and abundant reaction products of the antibody, as indicated by the arrows, located along the outer margin of the body (d). The Hamazaki-Wesenberg bodies also reacted with TIG antibody (e) and spotty reaction products of the antibody, as indicated by the arrows, located throughout the inner areas of the body (f). Original magnification: a, c, and e, × 1000. Scale bars indicate 2 μm in b, d, and f.

Figure 7

Protrusions from Hamazaki-Wesenberg bodies in sarcoid lymph nodes. Electron-microscopic analysis revealed Hamazaki-Wesenberg bodies with protrusions in sinus macrophages of two different sarcoid lymph nodes. Multiple protrusions (a) are represented by fused bodies, with or without a central osmiophilic core, surrounded by a discontinuous limiting membrane. One-by-one protrusions (b) are represented by a large spheroidal body with two fused central cores (¶) and a separated core (^*) linked together with a small round body (indicated by the black arrow). Adjacent to and separated from this small round body, another similar-shaped small round body (indicated by the arrowhead) was observed, which might reveal how these small round bodies are derived from the Hamazaki-Wesenberg body. Scale bars indicate 2 μm.