Journal of Clinical Immunology

, Volume 32, Issue 5, pp 1129–1140 | Cite as

Dual Analysis for Mycobacteria and Propionibacteria in Sarcoidosis BAL

  • Kyra A. Oswald-RichterEmail author
  • Dia C. Beachboard
  • Erin H. Seeley
  • Susamma Abraham
  • Bryan E. Shepherd
  • Cathy A. Jenkins
  • Daniel A. Culver
  • Richard M. Caprioli
  • Wonder P. Drake



Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious antigens continues to strengthen. Recent studies have reported molecular evidence of mycobacteria or propionibacteria. We assessed for immune responses against mycobacterial and propionibacterial antigens in sarcoidosis bronchoalveolar lavage (BAL) using flow cytometry, and localized signals consistent with microbial antigens with sarcoidosis specimens, using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS).


BAL cells from 27 sarcoidosis, 14 PPD- controls, and 9 subjects with nontuberculosis mycobacterial (NTM) infections were analyzed for production of IFN-γ after stimulation with mycobacterial ESAT-6 and Propionibacterium acnes proteins. To complement the immunological data, MALDI-IMS was performed to localize ESAT-6 and Propionibacterium acnes signals within sarcoidosis and control specimens.


CD4+ immunologic analysis for mycobacteria was positive in 17/27 sarcoidosis subjects, compared to 2/14 PPD- subjects, and 5/9 NTM subjects (p = 0.008 and p = 0.71 respectively, Fisher’s exact test). There was no significant difference for recognition of P. acnes, which occurred only in sarcoidosis subjects that also recognized ESAT-6. Similar results were also observed for the CD8+ immunologic analysis. MALDI-IMS localized signals consistent with ESAT-6 only within sites of granulomatous inflammation, whereas P. acnes signals were distributed throughout the specimen.


MALDI-IMS localizes signals consistent with ESAT-6 to sarcoidosis granulomas, whereas no specific localization of P. acnes signals is detected. Immune responses against both mycobacterial and P. acnes are present within sarcoidosis BAL, but only mycobacterial signals are distinct from disease controls. These immunologic and molecular investigations support further investigation of the microbial community within sarcoidosis granulomas.


Sarcoidosis mycobacteria propionibacteria bronchoalveolar lavage mass spectrometry MALDI-IMS 



We thank Joan Isom, L.P.N. for her assistance with data collection and coordination and Jamie Allen for sample preparation for MALDI analysis.

Conflict of interest statement

The authors declare that they have no conflict of interest.


  1. 1.
    M’Koma AE, Blum DL, Norris JL, Koyama T, Billheimer D, Motley S, et al. Detection of pre-neoplastic and neoplastic prostate disease by MALDI profiling of urine. Biochem Biophys Res Commun. 2007;353(3):829–34.PubMedCrossRefGoogle Scholar
  2. 2.
    Yanagisawa K, Shyr Y, Xu BJ, Massion PP, Larsen PH, White BC, et al. Proteomic patterns of tumour subsets in non-small-cell lung cancer. Lancet. 2003;362(9382):433–9.PubMedCrossRefGoogle Scholar
  3. 3.
    Demirkok SS, Basaranoglu M, Coker E, Karayel T. Seasonality of the onset of symptoms, tuberculin test anergy and Kveim positive reaction in a large cohort of patients with sarcoidosis. Respirology. 2007;12(4):591–3.PubMedCrossRefGoogle Scholar
  4. 4.
    Grunewald J, Wahlstrom J, Berlin M, Wigzell H, Eklund A, Olerup O. Lung restricted T cell receptor AV2S3+ CD4+ T cell expansions in sarcoidosis patients with a shared HLA-DRbeta chain conformation. Thorax. 2002;57(4):348–52.PubMedCrossRefGoogle Scholar
  5. 5.
    Kajdasz DK, Judson MA, Mohr Jr LC, Lackland DT. Geographic variation in sarcoidosis in South Carolina: its relation to socioeconomic status and health care indicators. Am J Epidemiol. 1999;150(3):271–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Morimoto T, Azuma A, Abe S, Usuki J, Kudoh S, Sugisaki K, et al. Epidemiology of sarcoidosis in Japan. Eur Respir J 2007 Oct 24.Google Scholar
  7. 7.
    Wilsher ML. Seasonal clustering of sarcoidosis presenting with erythema nodosum. Eur Respir J. 1998;12(5):1197–9.PubMedCrossRefGoogle Scholar
  8. 8.
    Sharma OP. Murray Kornfeld, American College Of Chest Physician, and sarcoidosis: a historical footnote: 2004 Murray Kornfeld Memorial Founders Lecture. Chest. 2005;128(3):1830–5.PubMedCrossRefGoogle Scholar
  9. 9.
    Drake WP, Pei Z, Pride DT, Collins RD, Cover TL, Blaser MJ. Molecular analysis of sarcoidosis tissues for mycobacterium species DNA. Emerg Infect Dis. 2002;8(11):1334–41.PubMedCrossRefGoogle Scholar
  10. 10.
    Dubaniewicz A, Dubaniewicz-Wybieralska M, Sternau A, Zwolska Z, Izycka-Swieszewska E, Augustynowicz-Kopec E, et al. Mycobacterium tuberculosis complex and mycobacterial heat shock proteins in lymph node tissue from patients with pulmonary sarcoidosis. J Clin Microbiol. 2006;44(9):3448–51.PubMedCrossRefGoogle Scholar
  11. 11.
    Song Z, Marzilli L, Greenlee BM, Chen ES, Silver RF, Askin FB, et al. Mycobacterial catalase-peroxidase is a tissue antigen and target of the adaptive immune response in systemic sarcoidosis. J Exp Med. 2005;201(5):755–67.PubMedCrossRefGoogle Scholar
  12. 12.
    Chen ES, Wahlstrom J, Song Z, Willett MH, Wiken M, Yung RC, et al. T cell responses to mycobacterial catalase-peroxidase profile a pathogenic antigen in systemic sarcoidosis. J Immunol. 2008;181(12):8784–96.PubMedGoogle Scholar
  13. 13.
    Allen SS, Evans W, Carlisle J, Hajizadeh R, Nadaf M, Shepherd BE, et al. Superoxide dismutase A antigens derived from molecular analysis of sarcoidosis granulomas elicit systemic Th-1 immune responses. Respir Res. 2008;9:36.PubMedCrossRefGoogle Scholar
  14. 14.
    Carlisle J, Evans W, Hajizadeh R, Nadaf M, Shepherd B, Ott RD, et al. Multiple Mycobacterium antigens induce interferon-gamma production from sarcoidosis peripheral blood mononuclear cells. Clin Exp Immunol. 2007;150(3):460–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Drake WP, Dhason MS, Nadaf M, Shepherd BE, Vadivelu S, Hajizadeh R, et al. Cellular recognition of Mycobacterium tuberculosis ESAT-6 and KatG peptides in systemic sarcoidosis. Infect Immun. 2007;75(1):527–30.PubMedCrossRefGoogle Scholar
  16. 16.
    Hajizadeh R, Sato H, Carlisle J, Nadaf MT, Evans W, Shepherd BE, et al. Mycobacterium tuberculosis Antigen 85A induces Th-1 immune responses in systemic sarcoidosis. J Clin Immunol. 2007;27(4):445–54.PubMedCrossRefGoogle Scholar
  17. 17.
    Oswald-Richter KA, Culver DA, Hawkins C, Hajizadeh R, Abraham S, Shepherd BE, et al. Cellular responses to mycobacterial antigens are present in sarcoidosis diagnostic bronchoalveolar lavage. Infect Immun. 2009;77(9):3740–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Dubaniewicz A, Trzonkowski P, Dubaniewicz-Wybieralska M, Dubaniewicz A, Singh M, Mysliwski A. Mycobacterial heat shock protein-induced blood T lymphocytes subsets and cytokine pattern: comparison of sarcoidosis with tuberculosis and healthy controls. Respirology. 2007;12(3):346–54.PubMedCrossRefGoogle Scholar
  19. 19.
    Ishige I, Usui Y, Takemura T, Eishi Y. Quantitative PCR of mycobacterial and propionibacterial DNA in lymph nodes of Japanese patients with sarcoidosis. Lancet. 1999;354(9173):120–3.PubMedCrossRefGoogle Scholar
  20. 20.
    Abe C, Iwai K, Mikami R, Hosoda Y. Frequent isolation of Propionibacterium acnes from sarcoidosis lymph nodes. Zentralbl Bakteriol Mikrobiol Hyg A. 1984;256(4):541–7.PubMedGoogle Scholar
  21. 21.
    Eishi Y, Suga M, Ishige I, Kobayashi D, Yamada T, Takemura T, et al. Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis. J Clin Microbiol. 2002;40(1):198–204.PubMedCrossRefGoogle Scholar
  22. 22.
    Yamada T, Eishi Y, Ikeda S, Ishige I, Suzuki T, Takemura T, et al. In situ localization of Propionibacterium acnes DNA in lymph nodes from sarcoidosis patients by signal amplification with catalysed reporter deposition. J Pathol. 2002;198(4):541–7.PubMedCrossRefGoogle Scholar
  23. 23.
    Ebe Y, Ikushima S, Yamaguchi T, Kohno K, Azuma A, Sato K, et al. Proliferative response of peripheral blood mononuclear cells and levels of antibody to recombinant protein from Propionibacterium acnes DNA expression library in Japanese patients with sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis. 2000;17(3):256–65.PubMedGoogle Scholar
  24. 24.
    Seeley EH, Caprioli RM. Molecular imaging of proteins in tissues by mass spectrometry. Proc Natl Acad Sci U S A. 2008;105(47):18126–31.PubMedCrossRefGoogle Scholar
  25. 25.
    Pathan AA, Wilkinson KA, Klenerman P, McShane H, Davidson RN, Pasvol G, et al. Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment. J Immunol. 2001;167(9):5217–25.PubMedGoogle Scholar
  26. 26.
    Oswald-Richter KA, Beachboard DC, Zhan X, Gaskill CF, Abraham S, Jenkins C, et al. Multiple mycobacterial antigens are targets of the adaptive immune response in pulmonary sarcoidosis. Respir Res. 2010;11:161.PubMedCrossRefGoogle Scholar
  27. 27.
    Breen RA, Hardy GA, Perrin FM, Lear S, Kinloch S, Smith CJ, et al. Rapid diagnosis of smear-negative tuberculosis using immunology and microbiology with induced sputum in HIV-infected and uninfected individuals. PLoS One. 2007;2(12):e1335.PubMedCrossRefGoogle Scholar
  28. 28.
    Fuhrmann S, Streitz M, Kern F. How flow cytometry is changing the study of TB immunology and clinical diagnosis. Cytometry A. 2008;73(11):1100–6.PubMedGoogle Scholar
  29. 29.
    Jafari C, Ernst M, Kalsdorf B, Greinert U, Diel R, Kirsten D, et al. Rapid diagnosis of smear-negative tuberculosis by bronchoalveolar lavage enzyme-linked immunospot. Am J Respir Crit Care Med. 2006;174(9):1048–54.PubMedCrossRefGoogle Scholar
  30. 30.
    Cornett DS, Mobley JA, Dias EC, Andersson M, Arteaga CL, Sanders ME, et al. A novel histology-directed strategy for MALDI-MS tissue profiling that improves throughput and cellular specificity in human breast cancer. Mol Cell Proteomics. 2006;5(10):1975–83.PubMedCrossRefGoogle Scholar
  31. 31.
    Hiramatsu J, Kataoka M, Nakata Y, Okazaki K, Tada S, Tanimoto M, et al. Propionibacterium acnes DNA detected in bronchoalveolar lavage cells from patients with sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis. 2003;20(3):197–203.PubMedGoogle Scholar
  32. 32.
    Smith J, Manoranjan J, Pan M, Bohsali A, Xu J, Liu J, et al. Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole. Infect Immun. 2008;76(12):5478–87.PubMedCrossRefGoogle Scholar
  33. 33.
    van Ingen J, De Zwaan R, Dekhuijzen R, Boeree M, van Soolingen D. Region of difference 1 in nontuberculous Mycobacterium species adds a phylogenetic and taxonomical character. J Bacteriol. 2009;191(18):5865–7.PubMedCrossRefGoogle Scholar
  34. 34.
    Barksdale L, Kim KS. Propionibacterium, Corynebacterium, Mycobacterium and Lepra bacilli. Acta Leprol. 1984;2(2–4):153–74.PubMedGoogle Scholar
  35. 35.
    Sahiratmadja E, Alisjahbana B, de Boer T, Adnan I, Maya A, Danusantoso H, et al. Dynamic changes in pro- and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment. Infect Immun. 2007;75(2):820–9.PubMedCrossRefGoogle Scholar
  36. 36.
    Furukawa A, Uchida K, Ishige Y, Ishige I, Kobayashi I, Takemura T, et al. Characterization of Propionibacterium acnes isolates from sarcoid and non-sarcoid tissues with special reference to cell invasiveness, serotype, and trigger factor gene polymorphism. Microb Pathog. 2009;46(2):80–7.PubMedCrossRefGoogle Scholar
  37. 37.
    Atarashi R, Moore RA, Sim VL, Hughson AG, Dorward DW, Onwubiko HA, et al. Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat Methods. 2007;4(8):645–50.PubMedCrossRefGoogle Scholar
  38. 38.
    Chaurand P, Schwartz SA, Reyzer ML, Caprioli RM. Imaging mass spectrometry: principles and potentials. Toxicol Pathol. 2005;33(1):92–101.PubMedCrossRefGoogle Scholar
  39. 39.
    Volkman HE, Pozos TC, Zheng J, Davis JM, Rawls JF, Ramakrishnan L. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science. 2010;327(5964):466–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Kyra A. Oswald-Richter
    • 1
    Email author
  • Dia C. Beachboard
    • 1
  • Erin H. Seeley
    • 2
  • Susamma Abraham
    • 3
  • Bryan E. Shepherd
    • 4
  • Cathy A. Jenkins
    • 4
  • Daniel A. Culver
    • 3
  • Richard M. Caprioli
    • 2
  • Wonder P. Drake
    • 1
    • 5
  1. 1.Department of Pathology, Microbiology and ImmunologyVanderbilt University School of MedicineNashvilleUSA
  2. 2.Mass Spectrometry Research Center and Department of BiochemistryVanderbilt University Medical CenterNashvilleUSA
  3. 3.Respiratory InstituteCleveland ClinicClevelandUSA
  4. 4.Department of BiostatisticsVanderbilt University Medical SchoolNashvilleUSA
  5. 5.Division of Infectious Diseases and Department of MedicineVanderbilt University Medical SchoolNashvilleUSA

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