Sarcoidosis is a systemic, granulomatous, and non-infectious disease, which most often affects the lungs. The key feature of sarcoidosis is a strong interaction of T-cells and macrophages leading to an exaggerated T
1 immune response, which is thought to be triggered by antigens in susceptible individuals. The obvious clinical heterogeneity of sarcoidosis supports the concept that several different sources of antigens such as bacteria, foreign particles, autoantigens or other endogenous factors may be involved. In the past decade, considerable progress has been made in defining potential antigens involved in sarcoidosis [
]. Especially antigens either derived from mycobacteria or from propionibacteria have been associated with sarcoidosis. For both pathogens a specific T-cell response was demonstrated in a high percentage of sarcoid patients [
Propionibacteria are gram-positive and aero-tolerant anaerobic bacteria. Particularly
Propionibacterium acnes (P. acnes
) is part of the skin, large intestine, mouth and conjunctiva flora and a common resident of the lung [
]. Mycobacteria and propionibacteria share many common features. Most importantly, both are intracellular pathogens and known for their persistence in macrophages due to the extraordinary high lipid content of their cell walls [
In the 80s of the last century
was identified as a putative causative agent of sarcoidosis [
]. The group of Abe and Homma cultivated
in homogenates of lymph nodes of patients with sarcoidosis [
]. Since then,
was detected by several, more sophisticated, methods: Hiramatsu et al. found elevated propionibacterial DNA in bronchoalveolar lavage cells from patients with sarcoidosis compared to healthy volunteers [
], Ishige and Ichikawa et al. confirmed and quantified the propionibacterial DNA in BAL cells, lymph nodes and lungs tissue [
] and Negi et al. detected
antigens with monoclonal antibodies in tissue sections [
] of sarcoid patients. Significantly higher levels of
genome were reported in sarcoid patients compared to patients with tuberculosis or healthy controls [
]. In addition, antibodies directed against RP35, a fragment of the
trigger factor, were found to be elevated in BAL fluid of sarcoidosis patients [
]. Most of these data are derived from studies by Japanese groups testing Japanese patients, while only very limited data exist for Caucasian patients. Furthermore,
is able to provoke a granulomatous T
1 inflammation in mice mimicking sarcoidosis [
]. Based on the Japanese data, Y. Eishi formulated the hypothesis that, in sarcoid patients, a latent
lung and/or lymph node infection can lead to a hypersensitivity granuloma formation directed against
and it’s derived proteins [
In sarcoidosis, elevation of several proinflammatory cytokines has been shown [
]. Tumour necrosis factor alpha (TNF-α) and granulocyte macrophage colony-stimulating factor (GM-CSF) are two key cytokines: TNF-α is required for granuloma formation, as shown in several studies in mice [
] and GM-CSF is able to induce transformation of alveolar macrophages into multinucleated giant cells [
]. Both, granulomas and multinucleated giant cells, are the hallmark histopathological changes in sarcoidosis.
Based on these data we investigated whether specific
P. acnes directed immunoglobulin production could be detected in Caucasian sarcoid patients, as specific antibodies reflect a previous,
in vivo, immune response to
P. acnes. As well, we attempted to prove an immunologic response of BAL cells to
P. acnes in vitro.
64 patients with sarcoidosis who underwent bronchoscopy for routine diagnostic evaluation and 21 healthy volunteers were included after obtaining their written informed consent. Diagnosis was made by clinical evaluation, HRCT, laboratory and histologic findings (non-caseating epitheloid granulomas on tissue biopsies) according to the American Thoracic Society/European Respiratory Society/World Association for Sarcoidosis and Other Granulomatous Disorders (ATS/ERS/WASOG) statement. Bronchoscopy and bronchoalveolar lavage (BAL) was performed using standard technique [
]. BAL cells were processed as previously described [
]. The study was approved by the local ethics committee of Albert-Ludwig University Freiburg (231/03).
IgG- and IgA-ELISA
We performed the following ELISA to quantify the total amount of IgG and IgA: 96 well plates were coated with 1:1000 diluted goat anti-human IgG + IgM + IgA antibodies (Dianova, Hamburg, Germany). We used 1:300 diluted goat anti-human IgG / IgA conjugated with alkaline phosphatase (Dianova, Hamburg, Germany) as detection antibodies and p-nitrophenylphosphate (Sigma-Aldrich, St. Louis, USA) as substrate. The detection limits were 0.8 ng/ml total IgG and total IgA, respectively.
Preparation of P. acnes or S. aureus antigen
(ATCC 12930) and
) were cultured, heat-killed and lyophilised as previously described [
]. The lyophilisate was resuspended in carbonate buffer (8.4 g NaHCO
& 3.56 g Na
in 1 l H
O, pH 9.5) by sonification (at 4 °C for 30 min). The bacterial suspension was centrifuged (at room temperature for 2 min, 20000 g) and concentration of the supernatant (antigen) was measured using BCA protein assay (Pierce, Bonn) and adjusted with carbonate buffer to 1 mg/ml.
antigens were used for specific ELISA and stimulation of BAL cells.
Anti-P. acnes-IgG/IgA- and Anti-S. aureus-IgG/IgA-ELISA
To measure the relative specific antibodies against
P. acnes and
S. aureus the following ELISA was utilized: 1 μg
P. acnes or 0.7 μg
S. aureus bacterial antigen was coated on 96-well ELISA maxisorb plates (Nunc, Roskilde, Denmark) and incubated at 37 °C for one hour followed by 4 °C over-night. The plates were washed twice with washing buffer (0.1 % Tween 20 in PBS), then blocked with 200 μl blocking buffer (2 % BSA in PBS) at room temperature for one hour. The BAL fluids were diluted with blocking buffer; one serum was used as standard in each experiment. After washing, the plates were loaded with standard or samples in triplicates, incubated at room temperature for two hours and then washed again. As detection antibodies we used 25 ng alkaline phosphatase linked anti-human IgA or IgA (Southern Biotechnology, USA) per well. 50 μg pNPP (Sigma, St. Louis, USA) per well served as substrate. Optical density (OD) was measured with an ELISA reader (Tecan, Crailsheim) at 405 nm against a reference value (492 nm). A non-linear regression standard curve was calculated (Prism software, GraphPad Inc.) using the ODs of the standard serum. After adjusting for the dilution factor relative antibody titres in relative arbitrary units (AU) were obtained.
Stimulation of BAL cells with P. acnes and cytokine measurement
BAL cells were suspended in RPMI medium (Gibco, Karlsruhe, Germany) supplemented with 10 % fetal calf serum and 1 % penicillin/streptomycin and put in 24-well plates (one million BAL cells/ml/well). If indicated, BAL cells were stimulated with
P. acnes lysate (1 μg/ml) in a 5 % CO
2 incubator at 37 °C for 24 h. The supernatant was harvested, frozen and stored at -80 °C until cytokine measurement. TNF-α and GM-CSF were quantified in duplicates using DuoSet ELISA Development System Kits (R&D Systems Europe, UK) according to the manufacturer’s instructions.
Values are expressed as means ± SD. Characteristics of patients with sarcoidosis and healthy volunteers were compared using a Mann-Whitney
U test for continuous measures and Fisher’s exact test for categorical measures. Wilcoxon signed-rank test was used to compare matched samples. Spearman rank correlation was utilized to establish possible correlations between numerical values. A significance level of p < 0.05 was used in all statistics.
is a gram-positive, low virulent bacterium and is thought to be, beside mycobacteria, a potential factor involved in the pathogenesis of sarcoidosis. A previous study reported the presence of
in BAL cells and lymph nodes of patients with sarcoidosis [
]. In this study we investigated the specific immune response to
in sarcoid patients.
In accordance with Vandenplas and colleagues, we showed increased local immunoglobulin production, particularly higher immunoglobulin G and A concentrations in the bronchoalveolar lavage of sarcoid patients [
]. The mechanisms of hypergammaglobulinemia in sarcoidosis are not fully understood, but three points seem to be clear: First, IFNg, a hallmark cytokine in sarcoidosis, is a potent stimulus of IgG secretion by B-cells [
]. Secondly, several studies have shown, that the IgG production in sarcoidosis is compartimentalized i.e. locally produced in the lung [
]. And thirdly, BAL T-cells from sarcoid patients stimulate B-cells to secret more immunoglobulins as well [
]. Furthermore, sarcoidosis is thought to be an antigen driven hypersensitivity reaction [
]. On the basis of these data, it was hypothesized that in the lungs in situ antigen presentation takes place leading to local immunoglobulin production.
Looking for specific antibodies against
, we used
as control since
belongs, similar as
, to commensals of the skin and the respiratory tract [
]. We found elevated total specific antibodies against
in sarcoid patients. The increased total amount of IgG and IgA in patients with sarcoidosis (Fig.
) probably accounts for the enhanced total
titre, since the relative
titres were not elevated. This differs completely from the anti-
titre which are relatively and absolutely elevated (Fig.
). Thus, the pulmonary immune system of sarcoid patients must have encountered
in the past. This is the first time an increased specific B-cell immune response to
could be proven in Caucasian sarcoid patients.
IgA and IgG prevent, amongst other things, bacteria and viruses from adhesion to the epithelium. Both, IgG and IgA are able to reach extravascular spaces; IgG by passive diffusion and IgA by an active transport across the epithelium [
-IgA was relatively elevated only in early disease, especially in Löfgren syndrome, while anti.-
-IgG was relatively increased in chronic disease as well. This finding might indicate that recent exposure to
is evident in Löfgren syndrome. The significance of all studies looking for a direct confirmation of
in sarcoid biopsies were limited to the fact that it is a commensal of the lung and also present in healthy individuals. Furthermore there is the possibility of contamination of the samples by
, as it is also a major commensal of the upper respiratory tract. With our approach in verifying a previously occurred exposure with
in vivo, we were able to abrogate this experimental limitation. Thus, our data suggest that Caucasian sarcoid patients had a recent immunologic response to
. Our study is limited by the fact, that our control group is significantly younger than the sarcoid patients. Aging leads to a decline of the immune system’s function, a phenomenon called “immunosenescence”; e.g., the reactivity to antigens is markedly reduced in older people [
]. We cannot exclude that part of the observed differences is due to aging, but we assume that the aging influence is marginal, because there was no correlation in sarcoid patients or healthy volunteers between total immunoglobulin levels and age. Because our study is biased regarding age, it may be possible that we underestimated the significance of relative IgG and IgA
titres rather than overestimated.
Next we explored, if there is an in vitro response in BAL cells stimulated with heat-killed
. Unstimulated BAL cells of patients with sarcoidosis already produced more TNF-α compared to control BAL cells, as shown by many groups [
]. The unstimulated GM-CSF increase was not significant, possibly due to a too small sample size, and in line with Prior et. al [
]. When stimulated with heat-killed
, BAL cells of healthy volunteers were anergic, yet in sarcoid patients, we saw a highly significant increase in proinflammatory cytokines such as TNF-α and GM-CSF, which has never been reported before. This results suggest that stimulation with
leads to an exaggerated local release of cytokines necessary for granuloma and multinucleated giant cell formation, the typical histopathological changes observed in sarcoidosis [
]. Of interest, increased IL-2 production and BAL T-cell proliferation following stimulation with
were reported by Mori and colleagues [
]. However, this phenomenon is not exclusively for sarcoidosis since also in skin acne an increase of TNF-α after
stimulation was reported [
]. Thus, our data indicate that contact of immune cells to
may boost the T
1 immune response in sarcoidosis.
The authors declare that they have no competing interests.
ST, NL and JS performed the experiments and the statistical analysis. AP designed of the study, performed the statistical analysis and wrote the manuscript. JS wrote the draft and designed the study. AP, JMQ and MF conceived of the study, participated in its design and coordination and helped to draft the manuscript. AP and JMQ supervised recruitment of patients and healthy volunteers. All authors read and approved the final manuscript.