Sarcoidosis is a chronic inflammatory disease of unknown cause, characterized by granuloma formation similar to tuberculosis, but without clear evidence of a microbial infection. Because sarcoidosis is linked with clinical anergy and other evidence of diminished cellular immunity, we hypothesized that decreased skin delayed-type hypersensitivity (DTH) responses to recall antigens in affected individuals would be associated with decreased function of their blood dendritic cells (DCs). Our study involved ex-vivo isolation, phenotyping and functional testing of myeloid DCs (mDCs), plasmacytoid DCs and T lymphocytes from blood of normal healthy volunteers and sarcoidosis subjects with active, untreated pulmonary disease. We found mDC function in the allogeneic mixed lymphocyte reaction directly corresponded to the magnitude of skin DTH reactions to recall antigens in both sarcoidosis subjects and normal volunteers. However, both of these outcomes were significantly decreased in the sarcoidosis group. Diminished mDC function occurred despite upregulated co-stimulatory and maturation markers. Clinical relevance is suggested by the inverse relationship between both mDC allogeneic responses and skin DTH responses with clinical disease severity as measured by chest radiograms. Since granulomas form when cellular immunity fails to clear antigenic stimuli, attenuated mDC function in sarcoidosis may contribute to susceptibility and persistence of the chronic inflammation characteristic of this disease.
Human; Dendritic Cells; Inflammation; Skin
Dendritic cells (DCs) are professional antigen-presenting cells that play a crucial role in the initiation and modulation of immune responses. Human circulating blood DCs are divided into two major subsets: myeloid DCs (mDCs); and plasmacytoid DCs (pDCs). Furthermore, mDCs are subdivided into two subsets: Th1-promoting mDCs (mDC1s); and Th2-promoting mDCs (mDC2s). Although CD1a, CD1c, and CD141 are generally used for classifying mDC subsets, their adequacy as a specific marker remains unclear. We performed this study to compare circulating mDC, pDC, mDC1, and mDC2 subsets between Th1- and Th2-mediated diseases using CD1a and CD141, and to analyze the adequacy of CD1a and CD141 as a marker for mDC1s and mDC2s, respectively.
Thirty patients with sarcoidosis, 23 patients with atopic diseases, such as atopic bronchial asthma, and 23 healthy subjects as controls were enrolled in this study. Peripheral blood DC subsets were analyzed with flow cytometry according to expressions of CD11c, CD123, CD1a, and CD141. For functional analysis, we measured interleukin (IL) 12p40 levels produced by the sorted mDC subsets.
The sarcoidosis group showed decreased total DC (P < 0.05) and mDC counts (P < 0.05) compared to controls. The atopy group showed decreased CD1a+mDC count (P < 0.05), and increased CD1a-mDC count (P < 0.05) compared to controls. CD141+mDC count in the atopy group was higher than controls (P < 0.05). Sorted CD1a+mDCs produced higher levels of IL-12p40 than CD1a-mDCs (P = 0.025) and CD141+mDCs (P = 0.018).
We conclude that decreased count of CD1a+mDC and increased count of CD141+mDC may reflect the Th2-skewed immunity in atopic diseases. The results of IL-12 levels produced by the sorted mDC subsets suggested the adequacy of CD1a and CD141 as a marker for mDC1 and mDC2, respectively, in vivo.
Dendritic cells; Peripheral blood; Sarcoidosis; Myeloid DC (mDC); CD1a; CD141
BACKGROUND: Because gamma/delta T lymphocytes (gamma delta cells) respond to myco-bacterial antigens in vitro and accumulate in the skin lesions of patients with certain granulomatous infections (leprosy, leishmaniasis), it was hypothesised that these cells might have a role in the pathogenesis of sarcoidosis, a disease also characterised by granuloma formation. Having failed to demonstrate an increase in gamma delta cells in the blood of patients with sarcoidosis, the aim of this study was to examine samples of bronchoalveolar lavage (BAL) fluid and biopsy tissue. METHODS: Samples from 23 patients (13 women) with newly diagnosed sarcoidosis, of mean age 31 years and median percentage of lymphocytes in the BAL fluid of 31%, were studied. Controls included normal subjects and patients with other interstitial lung diseases (ILD). Cytopreparations of BAL fluid (n = 13) and cryostat sections (five mediastinal nodes, 14 transbronchial biopsies) were stained with alkaline phosphatase-antialkaline phosphatase and monoclonal antibodies to CD3, CD4, CD8, CD25, and gamma delta T cell receptor (TCR). RESULTS: All patients had typical chest radiographs (16 stage I, four stage II, three stage III). All were Mantoux negative with negative tuberculosis cultures. Compared with normal controls and patients with other interstitial lung diseases there was no increase in gamma delta cells in the BAL fluid (sarcoidosis, 1% (range 0-4%) total cells; ILD, 1% (0-2%); controls, 0.5% (0-2%); p > 0.05, Kruskal-Wallis). Likewise, there was no increase in gamma delta cells in the transbronchial biopsy specimens (sarcoidosis, 1/high power field (hpf) (range 0-2); ILD, < 1/hpf (0-4); controls < 1/hpf (0-2); p > 0.05). gamma delta cells were rarely seen in the lymph nodes in spite of the presence of numerous granulomas. CONCLUSION: These results provide further evidence that gamma delta cells are not increased in most patients with sarcoidosis.
BACKGROUND: Sarcoidosis is a systemic granulomatous disorder of unknown origin characterised by accumulation of T lymphocytes and macrophages in multiple organs. Several cytokines and adhesion molecules may contribute to the accumulation of T lymphocytes in pulmonary sarcoidosis. The distribution of T lymphocyte subsets, T cell bearing CD11a and beta chemokines such as regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory peptide 1 alpha (MIP-1 alpha), and macrophage chemoattractant protein 1 (MCP-1) in bronchoalveolar lavage (BAL) fluid and peripheral blood were compared in untreated patients with sarcoidosis and normal subjects. METHODS: Flow cytometric analysis with monoclonal antibodies to cell surface antigens was used to identify T lymphocyte subsets in the BAL fluid of untreated patients with sarcoidosis (n = 40)--either without (group A, n = 12) or with (group B, n = 28) radiological evidence of pulmonary involvement--and in 22 normal subjects. The level of different beta chemokines was estimated by enzyme linked immunosorbent assay (ELISA). RESULTS: A high percentage of CD3+ cells, CD4+ cells expressing HLA-DR antigen, and a high CD4/CD8 ratio were detected in the BAL fluid of patients compared with normal subjects. In particular, CD4+ CD29+ memory T cells were significantly increased in patients with sarcoidosis. Furthermore, these cells were higher in those in group B than group A. The level of RANTES in the BAL fluid of patients was significantly higher than in normal subjects and correlated well with the percentage, number, and expression of CD29 on CD4 cells. The expression of CD11a (alpha chain of lymphocyte function associated antigen-1, LFA-1) on CD3+ cells in the BAL fluid of patients with sarcoidosis was not different from that of normal subjects. However, the expression of CD11a on CD3+ cells in the BAL fluid of patients in group A was significantly lower than that of patients in group B and normal subjects. CONCLUSIONS: These results suggest a possible interaction between activated memory T cells bearing CD11a and RANTES which may contribute to the pulmonary involvement in patients with sarcoidosis.
Sarcoidosis is a granulomatous disorder of unknown cause, affecting multiple organs, but mainly the lungs. The exact order of immunological events remains obscure. Reviewing current literature, combined with careful clinical observations, we propose a model for granuloma formation in pulmonary sarcoidosis. A tight collaboration between macrophages, dendritic cells, and lymphocyte subsets, initiates the first steps toward granuloma formation, orchestrated by cytokines and chemokines. In a substantial part of pulmonary sarcoidosis patients, granuloma formation becomes an on-going process, leading to debilitating disease, and sometimes death. The immunological response, determining granuloma sustainment is not well understood. An impaired immunosuppressive function of regulatory T cells has been suggested to contribute to the exaggerated response. Interestingly, therapeutical agents commonly used in sarcoidosis, such as glucocorticosteroids and anti-TNF agents, interfere with granuloma integrity and restore the immune homeostasis in autoimmune disorders. Increasing insight into their mechanisms of action may contribute to the search for new therapeutical targets in pulmonary sarcoidosis.
pulmonary sarcoidosis; granuloma; formation; integrity; dendritic cells; T helper 1 cells; T helper 17 cells; regulatory T cells
Considerable evidence supports the concept that CD4+ T cells are important in sarcoidosis pathogenesis, but the antigens responsible for the observed Th1 immunophenotype remain elusive. The epidemiologic association with bioaerosols and the presence of granulomatous inflammation support consideration of mycobacterial antigens. To explore the role of mycobacterial antigens in sarcoidosis immunopathogenesis, we assessed the immune recognition of mycobacterial antigens, the 6-kDa early secreted antigenic protein (ESAT-6) and catalase-peroxidase (KatG), by T cells derived from bronchoalveolar lavage (BAL) fluid obtained during diagnostic bronchoscopy. We report the presence of antigen-specific recognition of ESAT-6 and KatG in T cells from BAL fluid of 32/44 sarcoidosis subjects, compared to 1/27 controls (P < 0.0001). CD4+ T cells were primarily responsible for immune recognition (32/44 sarcoidosis subjects), although CD8+ T-cell responses were observed (25/41 sarcoidosis subjects). Recognition was significantly absent from BAL fluid cells of patients with other lung diseases, including infectious granulomatous diseases. Blocking of Toll-like receptor 2 reduced the strength of the observed immune response. The presence of immune responses to mycobacterial antigens in cells from BAL fluid used for sarcoidosis diagnosis suggests a strong association between mycobacteria and sarcoidosis pathogenesis. Inhibition of immune recognition with monoclonal antibody against Toll-like receptor 2 suggests that induction of innate immunity by mycobacteria contributes to the polarized Th1 immune response.
Background: Sarcoidosis is a chronic granulomatous lung disease of unknown origin. The accumulation of activated T cells at sites of inflammation represents an early stage in granuloma formation. Since mechanisms governing the normal resolution of inflammatory processes are poorly understood, this study aimed to investigate the apoptotic phenotype of peripheral blood and lung T lymphocytes from patients with sarcoidosis.
Methods: Bronchoalveolar lavage (BAL) was performed in 10 patients with active sarcoidosis and five healthy controls.
Results: Virtually no lymphocyte apoptosis, as determined by annexin V or Hoechst staining, was seen in either patients or controls. Sustained caspase-3 activity in non-apoptotic BAL fluid lymphocytes of the patients was detected, however, in agreement with in vitro studies demonstrating caspase activation after T cell receptor (TCR) triggering as a physiological response required for efficient T cell activation. Only 11.0% (range 7.7–17.6) of the BAL lymphocytes from sarcoidosis patients were annexin V positive after exposure to the apoptotic stimulus tributyltin compared with 55.0% (range 42.0–62.0) of BAL lymphocytes from healthy controls (p<0.001). After anti-Fas treatment only 8.5% (range 6–10) of BAL fluid lymphocytes from patients but 45.5% (range 38–62) from healthy controls were apoptotic.
Conclusion: BAL fluid lymphocytes from patients with sarcoidosis display a non-apoptotic morphology associated with endogenous caspase-3 activity. They seem to be resistant to apoptosis, which might contribute to the accumulation of inflammatory cells in the lungs, persistence of inflammation, and the development and maintenance of granuloma.
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=00.008 and p=00.71 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
Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells.
Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation.
Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p=0.03 and p=0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (p<0.001 and p=0.03, respectively). After polyclonal stimulation, Th-17 cells from sarcoidosis patients produced less IFN-γ than healthy controls.
Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis.
Sarcoidosis; Th-17; mycobacterial ESAT-6
Background: Sarcoidosis is a systemic granulomatous disease of unknown aetiology. It has been suggested that T helper type 1 (Th1) polarisation is associated with the pathophysiology of sarcoidosis, but the mechanism of skewing towards Th1 has not been elucidated. Dendritic cells (DCs) are known to regulate immune responses. This study was performed to determine whether DCs are involved in the aetiology of sarcoidosis.
Methods: The numbers of peripheral blood DCs in 24 patients with sarcoidosis were analysed and biopsy specimens from four patients were stained immunohistochemically using monoclonal antibodies.
Results: The numbers of both myeloid and lymphoid DC subsets were significantly decreased in the blood and mature DCs were found in the granulomas of patients with sarcoidosis. A number of interferon-γ (IFN-γ) producing T cells were also detected in the sarcoid granuloma, as well as many interleukin (IL)-4 producing T cells. Double staining of the biopsy specimen using anti-fascin and anti-CD3 antibodies showed an anatomical interaction between DCs and T cells.
Conclusions: These findings suggest that the blood DC subsets may migrate into the affected tissues, contributing to the formation of the granulomas in sarcoidosis. It is hypothesised that the migrating DCs may regulate the T cell response in sarcoidosis, at least in the granulomatous lesions.
Sarcoidosis is characterized by extensive local inflammation (granuloma, cytokine secretion) associated with anergy (poor response to antigens in vitro and in vivo). We postulated that this paradoxical situation would correspond to a disequilibrium between effector and regulatory T lymphocytes (T reg cells). We show that CD4+CD25brightFoxP3+ cells accumulate at the periphery of sarcoid granulomas, in bronchoalveolar lavage fluid, and in peripheral blood of patients with active disease. These cells exhibited powerful antiproliferative activity, yet did not completely inhibit TNF-α production. Sarcoidosis is therefore associated with a global T reg cell subset amplification whose activity would be insufficient to control local inflammation. At the same time, peripheral T reg cells exert powerful antiproliferative activity that may account for the state of anergy. Altogether, these findings advance our conceptual understanding of immune regulation in a way that resolves the immune paradox of sarcoidosis and permit us to envisage a profound clinical impact of T reg cell manipulation on immunity.
Activated pulmonary CD4+ T lymphocytes of the Th-1 type are essential for the inflammatory process in sarcoidosis, and IFN-γ production is crucial for the characteristic granuloma formation. Both the T cells and their inflammatory mediators may constitute possible targets for immunotherapy. A particular T-cell subset, the T-cell receptor (TCR) AV2S3+ bronchoalveolar lavage (BAL) CD4+ T cells, is found at dramatically increased levels in the BAL fluid of human leukocyte antigen (HLA)-DRB1*0301–positive and/or HLA-DRB3*0101–positive patients with sarcoidosis. The AV2S3+ BAL CD4+ T cells strongly associate with the sarcoid inflammation, and future studies on this particular T-cell subset to reveal their specificity may lead to the identification of sarcoidosis-specific antigen(s). T-cell subpopulations with regulatory functions (i.e., natural killer T cells and T regulatory cells) have recently been described as abnormal in sarcoidosis. Dysfunctional regulatory T cells may allow T effector cells to contribute to the formation of granulomas, and they may thus be relevant for the inflammatory process in this disease. These findings are exciting news and will be of help in designing new treatment strategies.
T cells; sarcoidosis; granuloma
granulomatous inflammation in sarcoidosis is driven by the interplay
between T cells and macrophages. To gain a better understanding of this
process the expression by these cells of cell surface activation
markers, co-stimulatory molecules, and adhesion molecules was analysed.
METHODS—CD4+ and CD8+
T lymphocytes from peripheral blood (PBL) or bronchoalveolar lavage
(BAL) fluid, as well as paired peripheral blood monocytes and alveolar
macrophages from 27 patients with sarcoidosis were analysed by flow cytometry.
CD69, CD95, and gp240 were all overexpressed in T cells from BAL fluid
compared with those from PBL in both the CD4+ and CD8+ subsets, while
CD57 was overexpressed only in BAL CD4+ cells. In contrast, CD28 tended
to be underexpressed in the BAL T cells. Monocyte/macrophage markers
included CD11a, CD11b, CD11c, CD14, CD16, CD54, CD71, CD80 and CD86 and
HLA class II. CD11a expression in alveolar macrophages (and peripheral
blood monocytes) was increased in patients with active disease and
correlated positively with the percentage of BAL lymphocytes.
Expression of CD80 in macrophages correlated with the BAL CD4/CD8 ratio.
indicate substantial activation of both CD4+ and CD8+ lung T cells in
sarcoidosis. There were also increased numbers of BAL lymphocytes whose
phenotypic characteristics have earlier been associated with clonally
expanded, replicatively senescent cells of the Th1 type.
BACKGROUND--Gamma/delta T lymphocytes are thought to have a role in granulomatous immune responses at peripheral sites of antigen contact such as the gut, skin and lung. The aim of this study was to determine if gamma/delta T lymphocytes are increased in the peripheral blood of patients with active sarcoidosis. METHODS--Peripheral blood from 21 untreated patients with a new presentation of sarcoidosis (12M, 9F), 20 normal volunteers (12M, 8F), and 12 patients with cavitary pulmonary tuberculosis were subjected to Ficoll Hypaque separation and flow cytometry analysis using monoclonal antibodies to CD3, 4, 8, 25, HLA-DR and gamma/delta T cell receptor. RESULTS--All patients with sarcoidosis had compatible chest radiographs and all were Mantoux negative in spite of previous BCG vaccination. In all but one patient histological examination showed non-caseating granuloma. There was no difference in the mean percentage or absolute numbers of gamma/delta positive peripheral blood lymphocytes between the three populations. Thirteen patients with sarcoidosis had an absolute lymphopenia and the mean percentage of CD3 positive peripheral blood lymphocytes in the group with sarcoidosis was lower than the other two groups. The percentage of CD25 and HLA-DR positive cells was higher in the group with sarcoidosis, supporting the fact that these patients had active disease. CONCLUSION--Gamma/delta T lymphocytes are not increased in the peripheral blood of patients with sarcoidosis and are unlikely to have a role in the pathogenesis of this disease.
sarcoidosis is characterised by a mononuclear alveolitis with a
predominance of CD4+ T cells and macrophages. We determined the
intracellular expression of interferon (IFN)γ, interleukin (IL)-2,
tumour necrosis factor (TNF)α, IL-4, IL-5 and IL-10 in CD4+ and CD8+,
naive and memory lymphocytes from blood and bronchoalveolar lavage
(BAL) fluid using three colour flow cytometry.
untreated patients with pulmonary sarcoidosis were evaluated and
stratified according to whether they had acute or chronic disease.
more T cells expressed Th1 than Th2 type cytokines in both BAL fluid
and peripheral blood samples, regardless of clinical presentation.
Significantly greater proportions of T cells secreted Th1 type
cytokines in BAL fluid than in peripheral blood. Th1 type cytokines
were more frequently expressed by peripheral and alveolar T cells in
acute disease than in chronic disease. There were no significant
differences between CD4+ and CD8+ T cells. Concerning naive and memory
lymphocytes, significantly higher CD45RO:CD45RA ratios were found in
BAL fluid than in blood, and increased expression of Th2 type
cytokines was found in peripheral compared with alveolar memory T cells.
support the immunopathogenetic concept of Th1/Th2 imbalance and
compartmentalisation in pulmonary sarcoidosis and suggest that the
cytokine patterns change during the course of disease. Expression of
Th2 type cytokines in memory lymphocytes is decreased in the alveolar
compartment compared with peripheral blood.
BACKGROUND--Sarcoidosis is a disease characterised by clinical "anergy" to delayed type hypersensitivity antigens and the formation of non-caseating granulomas, which frequently manifests in the lungs as a T lymphocyte/mononuclear cell alveolitis. Although there is an increased proportion of T cells in bronchoalveolar lavage (BAL) samples from these patients, and these T cells often show evidence of activation and spontaneous secretion of cytokines such as interleukin 2 (IL-2) and interferon gamma (IFN gamma)--a pattern similar to delayed type hypersensitivity reactions--it is unclear whether both cytokines are produced by the majority of T cells derived from the lungs of patients with sarcoidosis or whether unique subpopulations of T cells produce each cytokine. In this study the properties of T cells cloned from BAL fluid samples of patients with sarcoidosis have been analysed. METHODS--T cells were cloned by limiting dilution using IL-2, phytohaemagglutinin, and irradiated feeder cells. Cloning efficiencies were compared and phytohaemagglutinin induced clonal production of IL-2, IFN gamma, and IL-4 was determined by bioassay (IL-2 and IFN gamma) or ELISA (IL-4). RESULTS--T cells derived from the BAL fluid of patients with sarcoidosis cloned less efficiently than those from blood of the same individuals. Lung derived clones (CD4+ or CD8+) produced IFN gamma more frequently and to a higher titre than blood derived clones, whereas IL-2 production by CD4+ clones derived from BAL fluid was less than that from blood derived clones. Interestingly, IL-4 production by clones from both sites was similar. Analysis of the co-production of IL-2, IFN gamma, and IL-4 by these BAL fluid clones did not demonstrate a predominant "Th1"-like population which has been suggested to underlie delayed type hypersensitivity reactions. CONCLUSIONS--The reduced cloning efficiency of T cells from the lung compared with the blood in sarcoidosis is consistent with, although probably more pronounced than, previous observations in normal lungs and shows that T cell hyporesponsiveness is not overcome in the lungs of patients with sarcoidosis. Furthermore, major differences exist between the cytokine producing potential of T cells derived from the lung and the blood in sarcoidosis, and these parallel the differences in the properties of blood and lung T cells seen in healthy individuals.
Background: Sarcoidosis is a systemic granulomatous disorder of unknown origin. Lymphocytic inflammation is dominated by expression of Th1 type cytokines such as tumour necrosis factor α (TNFα). Interleukin 13 (IL-13) is a Th2 cytokine which is expressed by CD4+ T cells and has been shown to suppress TNFα in human blood monocytes. The role of IL-13 as a possible anti-inflammatory cytokine in sarcoidosis was investigated.
Methods: mRNA expression of IL-13, IL-4, IL-10, and TNFα in bronchoalveolar lavage (BAL) fluid cells and peripheral mononuclear blood cells (PBM) of 18 patients with sarcoidosis and nine healthy controls was assessed using RT-PCR. In addition, IL-13 protein levels in BAL cell culture supernatants from 12 patients and all controls were measured and immunocytochemistry of IL-13 protein was performed in BAL fluid cells of eight patients. TNFα concentrations were measured with and without stimulation with recombinant human (rh) IL-13, rhIL-10, and lipopolysaccharide (LPS).
Results: IL-13 mRNA expression was significantly increased in BAL cells and PBM of patients compared with controls (p<0.05). No significant difference was found in IL-4 mRNA or IL-10 mRNA expression in BAL fluid cells or PBM between the two groups. TNFα mRNA expression was significantly higher in BAL fluid cells of patients than controls (p<0.05). IL-13 protein levels in BAL cell culture supernatants were slightly raised in half the patients investigated but in only two controls. Immunocytochemistry detected IL-13 protein in alveolar macrophages of patients. IL-13 led to decreased TNFα concentrations (p<0.05).
Conclusions: IL-13 expression is increased in BAL cells and PBM in sarcoidosis and IL-13 is secreted from BAL cells. Alveolar macrophages may be the cellular source. These data suggest that IL-13 might have an anti-inflammatory effect by acting on TNFα.
BACKGROUND--Granulocyte-macrophage colony stimulating factor (GM-CSF) has several proinflammatory effects, some of which potentially favour granuloma formation. Its mRNA expression by the inflammatory cells recovered from lungs of patients with pulmonary sarcoidosis has been previously reported. In this study an association between GM-CSF expression and manifestations of the disease was explored. METHODS--GM-CSF mRNA was detected by reverse transcription polymerase chain reaction in the cells of bronchoalveolar lavage (BAL) fluid of 20 patients with pulmonary sarcoidosis. RESULTS--GM-CSF mRNA expression was positive in 15 of 20 patients with sarcoidosis. Fourteen of the 15 patients with positive mRNA expression had worsening or unchanged disease during the year preceding this study, on the basis of radiographic or physical findings, or both, whereas all five "negative" patients were judged to be improving. Similarly, serum levels of angiotensin converting enzyme, the proportion of lymphocytes in BAL fluid, and the CD4+/CD8+ ratio of lymphocytes in BAL fluid were significantly higher in the positive patients. CONCLUSIONS--There was an association between the presence of GM-CSF mRNA in the cells in BAL fluid and other indices of disease activity in sarcoidosis.
BACKGROUND: Mediators released by alveolar macrophages, as well as by T cells, play an important part in modulating local immune processes in sarcoidosis. Among alveolar macrophage secretory products, arachidonic acid metabolites are known to regulate inflammatory and immune reactions. It has been suggested that cyclo-oxygenase and lipoxygenase pathway metabolites of arachidonic acid modulate the evolution of the granulomatous inflammatory response in the lung differently. METHODS: Alveolar macrophages recovered from the bronchoalveolar lavage (BAL) fluid of 32 patients with sarcoidosis in different states of disease activity and 10 normal subjects were evaluated for their ability to release prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). Alveolar macrophages were cultured in the presence or absence of opsonised zymosan (500 micrograms/ml), and PGE2 and LTB4 levels in the culture supernatants were determined by enzyme immunoassay (EIA). RESULTS: Stimulated alveolar macrophages from patients with active sarcoidosis released higher LTB4 levels than those from normal subjects, but no differences in PGE2 release were observed between the two groups. The time course of LTB4 release by activated alveolar macrophages showed that normal cells produced similar levels of the hydroxyacid during the early and late times of culture while LTB4 release by activated cells from patients with sarcoidosis increased markedly after 60 minutes of culture, remaining elevated until 24 hours. Indomethacin (3 x 10(6) M) caused the expected inhibition of PGE2 formation without affecting LTB4 release. CONCLUSIONS: These results suggest that alveolar macrophages from the BAL fluid of patients with active sarcoidosis are primed to release LTB4, which may contribute to the locally heightened immune response.
Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis.
Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor.
When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed.
Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.
Sarcoidosis is a systemic granulomatous disease of unknown etiology. Propionibacterium acnes is the only microorganism that has been isolated from sarcoid lesions. Many P. acnes have been detected in sarcoid lymph nodes using quantitative PCR and in sarcoid granulomas by in situ hybridization. P. acnes trigger factor protein causes a cellular immune response only in sarcoid patients and induces pulmonary granulomas in mice sensitized with the protein and adjuvant, but only those with latent P. acnes infection in their lungs. Eradication of P. acnes by antibiotics prevents the development of granulomas in this experimental model. Although P. acnes is the most common commensal bacterium in the lungs and lymph nodes, P. acnes-specific antibody detected the bacterium within sarcoid granulomas of these organs. P. acnes can cause latent infection in the lung and lymph node and persist in a cell-wall-deficient form. The dormant form is activated endogenously under certain conditions and proliferates at the site of latent infection. In patients with P. acnes hypersensitivity, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Proliferating bacteria may escape granulomatous isolation, spreading to other organs. Latent P. acnes infection in systemic organs can be reactivated by another triggering event, leading to systemic sarcoidosis.
Rationale: The critical innate immune mechanisms that regulate granulomatous inflammation in sarcoidosis are unknown. Because the granuloma-inducing component of sarcoidosis tissues has physicochemical properties similar to those of amyloid fibrils, we hypothesized that host proteins capable of forming poorly soluble aggregates or amyloid regulate inflammation in sarcoidosis.
Objectives: To determine the role of the amyloid precursor protein, serum amyloid A, as an innate regulator of granulomatous inflammation in sarcoidosis.
Methods: Serum amyloid A expression was determined by immunohistochemistry in sarcoidosis and control tissues and by ELISA. The effect of serum amyloid A on nuclear factor (NF)-κB induction, cytokine expression, and Toll-like receptor-2 stimulation was determined with transformed human cell lines and bronchoalveolar lavage cells from patients with sarcoidosis. The effects of serum amyloid A on regulating helper T cell type 1 (Th1) granulomatous inflammation were determined in experimental models of sarcoidosis, using Mycobacterium tuberculosis catalase–peroxidase.
Measurements and Main Results: We found that the intensity of expression and distribution of serum amyloid A within sarcoidosis granulomas was unlike that in many other granulomatous diseases. Serum amyloid A localized to macrophages and giant cells within sarcoidosis granulomas but correlated with CD3+ lymphocytes, linking expression to local Th1 responses. Serum amyloid A activated NF-κB in Toll-like receptor-2–expressing human cell lines; regulated experimental Th1-mediated granulomatous inflammation through IFN-γ, tumor necrosis factor, IL-10, and Toll-like receptor-2; and stimulated production of tumor necrosis factor, IL-10, and IL-18 in lung cells from patients with sarcoidosis, effects inhibited by blocking Toll-like receptor-2.
Conclusions: Serum amyloid A is a constituent and innate regulator of granulomatous inflammation in sarcoidosis through Toll-like receptor-2, providing a mechanism for chronic disease and new therapeutic targets.
sarcoidosis; serum amyloid A; innate immunity; granuloma; cytokines
This study was designed to determine whether cell populations in bronchoalveolar lavage fluid represent a reflection of disease activity in sarcoidosis. Bronchoalveolar lavage fluid cells were obtained from 22 patients with sarcoidosis and from 10 normal control subjects and investigated by immunocytological methods. A panel of monoclonal antibodies was used to determine the relative proportions of phenotypically distinct subsets of macrophages and lymphocytes in the patients with sarcoidosis and to correlate them with clinical indices, such as disease duration, serum angiotensin converting enzyme, the chest radiograph, and results of pulmonary function tests. Patients with sarcoidosis had a higher percentage than the normal subjects of macrophage like cells expressing RFD1 (a class II associated antigen preferentially expressed by dendritic cells), an epithelioid cell antigen (RFD9), and a circulating monocyte antigen (UCHMI). The increase in RFD1+ cells appeared to be due to detection of antigen by this antibody on cells that were also expressing phenotypic markers of classical tissue macrophages (RFD7). The lymphocytes in lavage fluid from patients with sarcoidosis were characterised by increased expression of activation markers, such as interleukin-2 receptors (anti-Tac+), HLA-DR (RFDR+), and "blast" forms (expressing above normal concentrations of CD7 antigen). This was associated with increased proportions of the CD4+ (helper-inducer) T cell subset. Patients with sarcoidosis whose clinical indices suggested activity showed an increased number of macrophages coexpressing RFD1 and RFD7 antigens, of macrophages expressing UCHM1 and lymphocytes expressing activation markers. The expression of these markers was also increased on lavage cells from patients with radiographic evidence of widespread disease (chest radiographic stage II and III), but there was no relation with disease duration, pulmonary function, or serum angiotensin converting enzyme activity. Immunocytological analysis of lavage cells offers a probe for studying the pathogenesis of sarcoidosis and may be of value in monitoring disease activity.
Sarcoidosis is a disease of unknown etiology characterized by noncaseating epithelioid granulomas, oligoclonal CD4+ T cell infiltrates, and immune complex formation. To identify pathogenic antigens relevant to immune-mediated granulomatous inflammation in sarcoidosis, we used a limited proteomics approach to detect tissue antigens that were poorly soluble in neutral detergent and resistant to protease digestion, consistent with the known biochemical properties of granuloma-inducing sarcoidosis tissue extracts. Tissue antigens with these characteristics were detected with immunoglobulin (Ig)G or F(ab′)2 fragments from the sera of sarcoidosis patients in 9 of 12 (75%) sarcoidosis tissues (150–160, 80, or 60–64 kD) but only 3 of 22 (14%) control tissues (all 62–64 kD; P = 0.0006). Matrix-assisted laser desorption/ionization time of flight mass spectrometry identified Mycobacterium tuberculosis catalase–peroxidase (mKatG) as one of these tissue antigens. Protein immunoblotting using anti-mKatG monoclonal antibodies independently confirmed the presence of mKatG in 5 of 9 (55%) sarcoidosis tissues but in none of 14 control tissues (P = 0.0037). IgG antibodies to recombinant mKatG were detected in the sera of 12 of 25 (48%) sarcoidosis patients compared with 0 of 11 (0%) purified protein derivative (PPD)− (P = 0.0059) and 4 of 10 (40%) PPD+ (P = 0.7233) control subjects, suggesting that remnant mycobacterial catalase–peroxidase is one target of the adaptive immune response driving granulomatous inflammation in sarcoidosis.
Childhood sarcoidosis is a rare multisystemic granulomatous disorder of unknown etiology. In the pediatric series reported from the southeastern United States, sarcoidosis had a higher incidence among African Americans. Most reported childhood cases have occurred in patients aged 13–15 years. Macrophages bearing an increased expression of major histocompatibility class (MHC) II molecules most likely initiate the inflammatory response of sarcoidosis by presenting an unidentified antigen to CD4+ Th (helper-inducer) lymphocytes. A persistent, poorly degradable antigen driven cell-mediated immune response leads to a cytokine cascade, to granuloma formation, and eventually to fibrosis. Frequently observed immunologic features include depression of cutaneous delayed-type hypersensitivity and a heightened helper T cell type 1 (Th1) immune response at sites of disease. Circulating immune complexes, along with signs of B cell hyperactivity, may also be found. The clinical presentation can vary greatly depending upon the organs involved and age of the patient. Two distinct forms of sarcoidosis exist in children. Older children usually present with a multisystem disease similar to the adult manifestations, with frequent hilar lymphadenopathy and pulmonary infiltrations. Early-onset sarcoidosis is a unique form of the disease characterized by the triad of rash, uveitis, and arthritis in children presenting before four years of age. The diagnosis of sarcoidosis is confirmed by demonstrating a typical noncaseating granuloma on a biopsy specimen. Other granulmatous diseases should be reasonably excluded. The current therapy of choice for sarcoidosis in children with multisystem involvement is oral corticosteroids. Methotrexate given orally in low doses has been effective, safe and steroid sparing in some patients. Alternative immunosuppressive agents, such as azathioprine, cyclophosphamide, chlorambucil, and cyclosporine, have been tried in adult cases of sarcoidosis with questionable efficacy. The high toxicity profile of these agents, including an increased risk of lymphoproliferative disorders and carcinomas, has limited their use to patients with severe disease refractory to other agents. Successful steroid sparing treatment with mycophenolate mofetil was described in an adolescent with renal-limited sarcoidosis complicated by renal failure. Novel treatment strategies for sarcoidosis have been developed including the use of TNF-alpha inhibitors, such as infliximab. The long-term course and prognosis is not well established in childhood sarcoidosis, but it appears to be poorer in early-onset disease.