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1.  Comparative analysis of circulating dendritic cell subsets in patients with atopic diseases and sarcoidosis 
Respiratory Research  2013;14(1):29.
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1186/1465-9921-14-29
PMCID: PMC3599330  PMID: 23497225
Dendritic cells; Peripheral blood; Sarcoidosis; Myeloid DC (mDC); CD1a; CD141
2.  The anergic state in sarcoidosis is associated with diminished dendritic cell function1 
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.
PMCID: PMC2593870  PMID: 18566441
Human; Dendritic Cells; Inflammation; Skin
3.  Correlation of CD4:CD8 ratio and tumour necrosis factor (TNF)α levels in induced sputum with bronchoalveolar lavage fluid in pulmonary sarcoidosis 
Thorax  2000;55(8):696-699.
BACKGROUND—An increased CD4:CD8 lymphocyte ratio and raised cytokine levels in bronchoalveolar lavage (BAL) fluid are characteristic of pulmonary sarcoidosis. Sputum induction has been used as a non-invasive tool for investigating the airways and may be useful in investigating inflammation in patients with sarcoidosis in whom endobronchial, peribronchial, and parenchymal inflammation is present. This study aimed to correlate the total and differential cell counts, CD4:CD8 ratio, and tumour necrosis factor (TNF)α levels between induced sputum and BAL fluid in patients with pulmonary sarcoidosis.
METHODS—Fourteen patients with newly diagnosed biopsy proven sarcoidosis and six healthy controls were investigated. Sputum induction and BAL was carried out at the initial visit and repeated following six months of treatment with oral prednisone.
RESULTS—There was no correlation of differential cell counts between induced sputum and BAL fluid. The CD4:CD8 ratio in induced sputum correlated strongly with that in BAL fluid (5.5 (0.4:1) versus 4.4 (0.2:1); r = 0.8, p<0.001) and the fall in the ratio following six months of treatment in sputum paralleled that in BAL fluid (3.4 (0.2:1) versus 2.4 (0.2:1)). The TNFα levels in sputum also correlated with levels in the BAL fluid (11.9 (1.5) pg/ml versus 17.6 (2.7) pg/ml; r = 0.8, p<0.001). The fall in sputum TNFα levels following six months of treatment paralleled the fall in BAL fluid levels (6.7 (0.9) pg/ml versus 11.6 (1.3) pg/ml).
CONCLUSIONS—The CD4:CD8 ratio and TNFα levels in induced sputum correlated with those in BAL fluid and paralleled changes with treatment. Induced sputum may therefore be a non-invasive surrogate for certain parameters in BAL fluid in patients with sarcoidosis.


doi:10.1136/thorax.55.8.696
PMCID: PMC1745837  PMID: 10899249
4.  Gamma/delta cells in tissue from patients with sarcoidosis. 
Thorax  1996;51(11):1123-1126.
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.
PMCID: PMC1090524  PMID: 8958896
5.  Clonal analysis of lung and blood T cells in patients with sarcoidosis. 
Thorax  1994;49(6):577-585.
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.
PMCID: PMC474948  PMID: 8016795
6.  Involvement of dendritic cells in sarcoidosis 
Thorax  2004;59(5):408-413.
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.
doi:10.1136/thx.2003.006049
PMCID: PMC1746987  PMID: 15115868
7.  Increased levels of Treg cells in bronchoalveolar lavage fluid and induced sputum of patients with active pulmonary sarcoidosis 
European Journal of Medical Research  2009;14(Suppl 4):165-169.
Objective
It has recently been described that circulatory and BAL regulatory T-cells (Tregs), defined as CD4+CD25highCD127low are increased in patients with active sarcoidosis compared with other interstitial lung diseases.
Materials and methods
We studied prospectively 17 patients (10 women, 7 men) of median age 39 years (range 27-65) with active granulomatous lung diseases (GLD) (10 patients with sarcoidosis (BBS), and 7 with hypersensitivity pneumonitis (HP), and 9 healthy controls. Bronchoalveolar lavage fluid (BAL) and induced sputum Treg counts, CD4+, CD8+, CD25+ cells were quantified by flow cytometry. Disease activity was measured by ACE serum level. Pulmonary function tests were performed using an Elite DL Medgraphics body box.
Results
We found Treg cells count significantly elevated in induced sputum from active GLD (38.3% vs. 7.1% and 5.3% in BBS, HP, and control, respectively). A significantly higher percentage of Treg cells characterized BAL cells from HP patients (2.27%; 9.5%; 2.1%, in BBS, HP and control, respectively). There was a strong correlation with ACE serum level and Treg cell count in BAL fluid of BBS patients, with no such correlation within HP patient group, nor Treg cell count and pulmonary function tests.
Conclusions
Our data suggest a potential role of CD4+CD25 high CD127 low induced sputum and BAL lymphocytes from patients with active granulomatous lung diseases and hypersensitivity pneumonitis. An increased number of Treg cells in active GLD may be involved in immune regulation in active granulomatous lung diseases. The results indicate that analysis of these cells could be useful as markers of disease activity in granulomatous lung diseases.
doi:10.1186/2047-783X-14-S4-165
PMCID: PMC3521330  PMID: 20156750
sarcoidosis; hypersensitivity pneumonitis; Treg cells; T cells
8.  Cell specific expression of peroxiredoxins in human lung and pulmonary sarcoidosis 
Thorax  2002;57(2):157-164.
Background: Six proteins of the peroxiredoxin (Prx) family have recently been characterised which have the capacity to decompose hydrogen peroxide in vivo and in vitro. These proteins may have an important role in the protection of human lung against endogenous and exogenous oxidant stress. However, the expression and distribution of these proteins in healthy human lung and diseased lung tissue is unknown.
Methods: The cell specific expression of Prxs in healthy lung tissue from four non-smokers and in parenchymal tissue from 10 subjects with pulmonary sarcoidosis was investigated by immunohistochemistry, and expression of these proteins in various cultured lung cells and cells of bronchoalveolar lavage (BAL) fluid of controls and patients with sarcoidosis was assessed by Western blot analysis.
Results: All six Prxs could be synthesised in cultured human lung cells. The bronchial epithelium showed moderate to high expression of Prxs I, III, V and VI, the alveolar epithelium expressed mainly Prxs V and VI, and alveolar macrophages expressed mainly Prxs I and III. Granulomas of subjects with sarcoidosis expressed mainly Prxs I and III. Samples of BAL fluid from controls and from subjects with sarcoidosis had very similar findings, except that Prxs II and III had a tendency for increased immunoreactivity in sarcoidosis tissue.
Conclusions: Prxs I, III, V, and VI, in particular, have prominent and cell specific expression in human lung tissue. High expression of Prxs I and III in granulomas and alveolar macrophages of sarcoidosis parenchyma may have a significant effect on the oxidant burden and the progression of lung injury in this disease.
doi:10.1136/thorax.57.2.157
PMCID: PMC1746258  PMID: 11828047
9.  Neurotrophins and neurotrophin receptors in pulmonary sarcoidosis - granulomas as a source of expression 
Respiratory Research  2010;11(1):156.
Background
Pulmonary sarcoidosis is an inflammatory disease, characterized by an accumulation of CD4+ lymphocytes and the formation of non-caseating epithelioid cell granulomas in the lungs. The disease either resolves spontaneously or develops into a chronic disease with fibrosis. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been suggested to be important mediators of inflammation and mediate tissue remodelling. In support of this, we have recently reported enhanced NGF levels in the airways of patients with pulmonary sarcoidosis. However, less is known about levels of BDNF and NT-3, and moreover, knowledge in the cellular sources of neurotrophins and the distribution of the corresponding neurotrophin receptors in airway tissue in sarcoidosis is lacking.
Methods
The concentrations of NGF, BDNF and NT-3 in bronchoalveolar lavage fluid (BALF) of 41 patients with newly diagnosed pulmonary sarcoidosis and 27 healthy controls were determined with ELISA. The localization of neurotrophins and neurotrophin receptors were examined by immunohistochemistry on transbronchial lung biopsies from sarcoidosis patients.
Results
The sarcoidosis patients showed significantly enhanced NT-3 and NGF levels in BALF, whereas BDNF was undetectable in both patients and controls. NT-3 levels in BALF were found higher in patients with non-Löfgren sarcoidosis as compared to patients with Löfgren's syndrome, and in more advanced disease stage. Epithelioid cells and multinucleated giant cells within the sarcoid granulomas showed marked immunoreactivity for NGF, BDNF and NT-3. Also, immunoreactivity for the neurotrophin receptor TrkA, TrkB and TrkC, was found within the granulomas. In addition, alveolar macrophages showed positive immunoreactivity for NGF, BDNF and NT-3 as well as for TrkA, TrkB and TrkC.
Conclusions
This study provides evidence of enhanced neurotrophin levels locally within the airways of patients with sarcoidosis. Findings suggest that sarcoid granuloma cells and alveolar macrophages are possible cellular sources of, as well as targets for, neurotrophins in the airways of these patients.
doi:10.1186/1465-9921-11-156
PMCID: PMC2994818  PMID: 21059230
10.  Apoptosis resistant bronchoalveolar lavage (BAL) fluid lymphocytes in sarcoidosis 
Thorax  2002;57(10):897-901.
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.
doi:10.1136/thorax.57.10.897
PMCID: PMC1746191  PMID: 12324678
11.  Expression of thioredoxin in granulomas of sarcoidosis: possible role in the development of T lymphocyte activation 
Thorax  2000;55(9):755-761.
BACKGROUND—Activated T lymphocytes are one of the characteristic features of sarcoidosis. The mechanism of T cell activation, expressing various activation markers including interleukin 2 receptor (IL-2R), has been extensively investigated but the precise mechanism remains unknown. Although thioredoxin (TRX) displays a number of biological activities including IL-2R inducing activity, its role in the induction of IL-2R expression on T cells in sarcoidosis has not been determined. The expression of TRX and IL-2R in granulomas of patients with sarcoidosis has been studied to clarify a possible role for TRX in the induction of IL-2R expression.
METHODS—Granulomas in specimens of lung tissue and lymph nodes from five patients with sarcoidosis were immunohistochemically stained with anti-TRX antibody and anti-IL-2Rα chain antibody and the concentration of TRX in the bronchoalveolar lavage (BAL) fluid from 20 patients with pulmonary sarcoidosis was measured.
RESULTS—Granulomas in lung and lymph node tissue from patients with sarcoidosis showed strong reactivity with anti-TRX antibody. Positive staining was present in the macrophages, epithelioid cells, and Langhans' type giant cells but not in lymphocytes. IL-2R was expressed on lymphocytes in the same granulomas. By contrast, positive immunoreactivity was not found in lung tissue specimens from 12 control subjects. Concentrations of TRX in BAL fluid were higher in patients with pulmonary sarcoidosis (median (range) 122.6 (20.9-303.3) ng/ml) than in control subjects (32.9 (16.8-52.8) ng/ml, p<0.05).
CONCLUSIONS—TRX is highly expressed and is locally produced by granulomas in patients with sarcoidosis. The coexistence of immunoreactive TRX and IL-2R in the same granulomas suggests that TRX might act as a local inducing factor for IL-2R expression on T cells.


doi:10.1136/thorax.55.9.755
PMCID: PMC1745843  PMID: 10950894
12.  Increased interleukin-13 expression in patients with sarcoidosis 
Thorax  2003;58(6):519-524.
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.
doi:10.1136/thorax.58.6.519
PMCID: PMC1746705  PMID: 12775866
13.  Analysis of T cell subsets and beta chemokines in patients with pulmonary sarcoidosis 
Thorax  1997;52(5):431-437.
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. 



PMCID: PMC1758558  PMID: 9176534
14.  Childhood sarcoidosis: A rare but fascinating disorder 
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.
doi:10.1186/1546-0096-6-16
PMCID: PMC2559831  PMID: 18811966
15.  Immunological features of lung lavage cells from patients with primary biliary cirrhosis may reflect those seen in pulmonary sarcoidosis. 
Gut  1990;31(2):208-212.
To investigate the basis of subclinical alveolitis in patients with primary biliary cirrhosis, 10 primary biliary cirrhosis patients were studied by bronchoalveolar lavage. Both bronchoalveolar lavage lymphoid and non-lymphoid cell populations were analysed using immunocytological methods to determine their proportions and phenotypic features in an attempt to gain information as to possible immune mechanisms active in the lung of these patients. Six of the 10 patients in our study showed evidence of an alveolitis (raised lymphocyte count: 27.6 (4.3)% of total count) on lavage. The results were compared with control groups of normal volunteers and patients with active pulmonary sarcoidosis. The six primary biliary cirrhosis patients with lymphocytosis had a raised CD4/CD8 T-cell ratio (4.13:1), similar to the sarcoid patients (5.60:1). A proportion of these T-lymphocytes expressed markers of activation (HLA-DR+ 7.5 (2.1)%); CD25 + 2.3 (0.9)%; CD7 + 5.8 (1.5)%. This increased T-cell activation was also seen in the sarcoid groups (HLA-DR+ 10.0 (1.9)%; CD25 + 3.0 (1.1)%; CD7 + 5.0 (0.2)%). This was not seen in the primary biliary cirrhosis patients without lymphocytosis and the normal volunteers. Within the non-lymphoid cell population, an increase in dendritic (RFD1+) cells was seen in primary biliary cirrhosis patients with lymphocytosis (31.2 (1.9)%) and sarcoid patients (46.3 (5.1)%) in contrast with the normal and primary biliary cirrhosis group without lymphocytosis. The primary biliary cirrhosis patients without lymphocytes had a relatively greater proportion of mature phagocytes (RFD7+). We postulate that these observations suggest the emergence in the lung of a granuloma producing mechanism similar to that occurring in the liver. By comparison, the alveolitis found in primary biliary cirrhosis is consistent with that observed in interstitial granulomatous lung disorders such as sarcoidosis.
PMCID: PMC1378382  PMID: 2311980
16.  Activation of peripheral blood mononuclear cells in bronchoalveolar lavage fluid from patients with sarcoidosis: visualisation of single cell activation products. 
Thorax  1994;49(11):1146-1151.
BACKGROUND--Interstitial lung diseases are characterised by the recruitment of mononuclear cells to disease sites where maturation occurs and activation products, including lysozyme (LZM), are released. Analysis of in vitro cell culture supernatants for activation products masks the functional heterogeneity of cell populations. It is therefore necessary to examine the secretion of activation products by single cells to assess whether the activation of newly recruited mononuclear phagocytes at the sites of disease in the lung is uniform and controlled by the local microenvironment. METHODS--The reverse haemolytic plaque assay was used to evaluate, at a single cell level, the ability of bronchoalveolar lavage (BAL) fluid from seven patients with sarcoidosis to activate Ficoll-Hypaque-separated peripheral blood mononuclear cells by comparison with BAL fluid from six normal volunteers and nine patients with systemic sclerosis. Monolayers of peripheral blood mononuclear cells and sheep red blood cells were cultured either alone or in the presence of 20% (v/v) BAL fluid with a polyclonal anti-LZM antibody. LZM/anti-LZM complexes bound to red blood cells surrounding the secreting cells were disclosed following complement lysis of red blood cells and quantification of plaque dimensions using microscopy and image analysis. RESULTS--Bronchoalveolar lavage fluid from all the patients with sarcoidosis increased LZM secretion by peripheral blood mononuclear cells compared with unstimulated mononuclear cells. By contrast, BAL fluid from the other individuals had no effect on LZM secretion. CONCLUSIONS--Single cells activated by BAL fluid can be evaluated by the reverse haemolytic plaque assay. BAL fluid from patients with sarcoidosis, but not from patients with systemic sclerosis or normal individuals, contains components capable of activating mononuclear phagocytes to secrete lysozyme.
Images
PMCID: PMC475278  PMID: 7831632
17.  Gamma/delta T lymphocytes in the blood of patients with sarcoidosis. 
Thorax  1995;50(8):858-862.
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.
PMCID: PMC474907  PMID: 7570437
18.  Imbalance between Th17 and Regulatory T-Cells in Sarcoidosis 
Sarcoidosis is a systemic granulomatous disease, which is thought to result from an aberrant immune response. CD4+ T lymphocytes play an important role in the development of granulomas. Previously, the immunopathogenesis of sarcoidosis was focused on Th1/Th2 disturbances. The aim of this study was to evaluate the balance between newer CD4+ T lymphocytes, i.e., Treg and Th17 cells. In our studies, a decrease in Treg cells and an increase in Th17 cells were observed in the peripheral blood and BALF of sarcoidosis patients. A significant increase in the Th17/Treg cell ratio was observed in sarcoidosis patients. After treatment with prednisone, the expression of Foxp3 mRNA was elevated in the peripheral blood, and expression of (ROR)γt mRNA showed a downward trend. These findings suggest that sarcoidosis is associated with an imbalance between Th17 and Treg cells in peripheral blood and BALF. Therefore, targeting the cytokines that affect the Th17/Treg ratio could provide a new promising therapy for pulmonary sarcoidosis.
doi:10.3390/ijms141121463
PMCID: PMC3856015  PMID: 24177566
granulomatous disease; sarcoidosis; regulatory T cells; Th17 cells; corticosteroids
19.  Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis 
Thorax  2001;56(6):487-493.
BACKGROUND—Pulmonary 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.
METHODS—Eighteen untreated patients with pulmonary sarcoidosis were evaluated and stratified according to whether they had acute or chronic disease.
RESULTS—Significantly 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.
CONCLUSIONS—Our data 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.


doi:10.1136/thorax.56.6.487
PMCID: PMC1746073  PMID: 11359967
20.  Inter-relationship between tumour necrosis factor-alpha (TNF-α) and TNF soluble receptors in pulmonary sarcoidosis 
Thorax  1999;54(6):524-530.
BACKGROUND—The importance of tumour necrosis factor-alpha (TNF-α) in the pathogenesis of pulmonary sarcoidosis has remained uncertain because of the paucity of clinical features associated with excessive levels of this cytokine. Increased levels of soluble TNF receptors (TNF-R), which are known to inhibit TNF-α activity, were recently described in the lungs of subjects with sarcoidosis. We hypothesised that TNF-α bioactivity may be inhibited in sarcoidosis by the presence of TNF-R. A study was therefore undertaken to investigate for the first time the relationship between soluble receptors and TNF-α bioactivity in the lungs of subjects with sarcoidosis.
METHODS—Alveolar macrophages (AMs) from 16 subjects with histologically proven sarcoidosis and 13 healthy controls were cultured in the presence and absence of lipopolysaccharide (LPS). The subjects with sarcoidosis were grouped by radiological assessment into stage I (n = 6) and stage II/III (n = 10). The cell culture supernatants and bronchoalveolar lavage (BAL) fluid were assayed for TNF bioactivity using the WEHI 164 clone 13 assay. Immunoreactive (bound and free) TNF-α and free TNF-Rs (p55 and p75) were determined by ELISA.
RESULTS—Bioactive TNF-α was undetectable in the BAL fluid of all the subjects with sarcoidosis and most of the healthy controls. However, there was significantly more immunoreactive TNF-α in the BAL fluid from subjects with sarcoidosis than from the controls (median values 0.304 ng/ml and 0.004 ng/ml, respectively, 95% CI 0.076to 0.455, p<0.001). The levels of both p55 and p75 in the BAL fluid were higher in both sarcoidosis groups than in the controls (p<0.0005 and p<0.001, respectively). In LPS stimulated AM supernatants reduced TNF-α bioactivity was seen in subjects with stage I sarcoidosis compared with those with stage II/III disease and healthy controls (median 0.333 ng/ml vs 1.362 ng/ml and 2.385 ng/ml, respectively, p<0.01). This contrasted with increased p55 levels in the AM supernatants derived from subjects with stage I sarcoidosis compared with those with stage II/III disease and healthy controls (median 0.449 ng/ml vs 0.058 ng/ml and 0.078 ng/ml, respectively, p<0.01). The levels of p75 were increased in unstimulated AM cultures in subjects with stage II/III disease compared with those with stage I disease and healthy controls (median 0.326 ng/ml vs 0.064 ng/ml and 0.102 ng/ml, p<0.05).
CONCLUSIONS—These results indicate that TNF-α bioactivity may be inhibited by increased soluble TNF-R in the lungs of subjects with sarcoidosis, and this inhibition may be greater in patients with stage I sarcoidosis than in those with stage II/III disease. This may represent a homeostatic mechanism which protects the lung from excessive TNF production characteristic of chronic inflammation.


PMCID: PMC1745499  PMID: 10335007
21.  Granuloma Formation in Pulmonary 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.
doi:10.3389/fimmu.2013.00437
PMCID: PMC3857538  PMID: 24339826
pulmonary sarcoidosis; granuloma; formation; integrity; dendritic cells; T helper 1 cells; T helper 17 cells; regulatory T cells
22.  Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis 
Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.
doi:10.1186/ar1864
PMCID: PMC1526567  PMID: 16507115
23.  Increased inflammatory cytokines and new collagen formation in cutaneous tuberculosis and sarcoidosis. 
Thorax  1996;51(12):1253-1261.
BACKGROUND: Interactions between mononuclear cells, vascular endothelium, fibroblasts, and cytokines during the inflammatory reaction within a granuloma have the potential to contribute to the progression to fibrosis. METHODS: Biopsy specimens of six tuberculous and eight sarcoidosis skin lesions were examined by immunohistochemistry to seek evidence for the presence of inflammatory and fibrotic reactions in human granulomatous disease. Additionally, to understand how a T cell mediated delayed type hypersensitivity reaction--a component of chronic granulomatous inflammation--could progress to fibrosis, the human in vivo model of the cutaneous tuberculin Heaf reaction to purified protein derivative (PPD) was studied in a group of 48 subjects. RESULTS: Granulomas from tuberculous and sarcoidosis skin biopsy specimens were seen to contain cells with marked staining by antibodies to fibronectin, transforming growth factor beta (pan TGF-beta), and type 1 procollagen (PCP-1). Accentuated staining of extracellular matrix was seen both in the granulomas and in the peri-granulomatous regions. Less prominent staining was observed using antibodies against interleukin 1 beta (IL-1 beta) and alpha-smooth muscle actin (alpha-SMA). Biopsies of Heaf reactions revealed cells staining for IL-1 beta, tumour necrosis factor alpha (TNF-alpha), platelet derived growth factor B (PDGF-B), and fibronectin which were detected as early as day 1 and persisted throughout the 14 day study period. Cells staining for PCP-1 increased to greatest abundance at day 14. All these cytokines were present in low abundance in biopsy specimens from sites inoculated with saline only. CONCLUSIONS: Evidence is provided that granulomas in tuberculosis and sarcoidosis behave as active centres of fibrogenesis. Using the Heaf model, the temporal relationship between the early appearance of cytokines and the later increase in the collagen precursor PCP-1 linked the immune mediated chronic inflammatory response with subsequent fibrosis and suggested that the tuberculin Heaf reaction will serve as a model for studying the early events of granuloma formation in patients with tuberculosis and sarcoidosis.
Images
PMCID: PMC472773  PMID: 8994525
24.  Immune response CC chemokines CCL2 and CCL5 are associated with pulmonary sarcoidosis 
Background
Pulmonary sarcoidosis involves an intense leukocyte infiltration of the lung with the formation of non-necrotizing granulomas. CC chemokines (chemokine (C-C motif) ligand 2 (CCL2)-CCL5) are chemoattractants of mononuclear cells and act through seven transmembrane G-coupled receptors. Previous studies have demonstrated conflicting results with regard to the associations of these chemokines with sarcoidosis. In an effort to clarify previous discrepancies, we performed the largest observational study to date of CC chemokines in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis.
Results
BALF chemokine levels from 72 patients affected by pulmonary sarcoidosis were analyzed by enzyme-linked immunosorbent assay (ELISA) and compared to 8 healthy volunteers. BALF CCL3 and CCL4 levels from pulmonary sarcoidosis patients were not increased compared to controls. However, CCL2 and CCL5 levels were elevated, and subgroup analysis showed higher levels of both chemokines in all stages of pulmonary sarcoidosis. CCL2, CCL5, CC chemokine receptor type 1 (CCR1), CCR2 and CCR3 were expressed from mononuclear cells forming the lung granulomas, while CCR5 was only found on mast cells.
Conclusions
These data suggest that CCL2 and CCL5 are important mediators in recruiting CCR1, CCR2, and CCR3 expressing mononuclear cells as well as CCR5-expressing mast cells during all stages of pulmonary sarcoidosis.
doi:10.1186/1755-1536-4-10
PMCID: PMC3080805  PMID: 21463523
25.  Etiologic Aspect of Sarcoidosis as an Allergic Endogenous Infection Caused by Propionibacterium acnes 
BioMed Research International  2013;2013:935289.
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.
doi:10.1155/2013/935289
PMCID: PMC3697284  PMID: 23844371

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