David R. Moller

Mycobacterial catalase-peroxidase is a tissue antigen and target of the adaptive immune response in systemic sarcoidosis.

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.

Design of A Case Control Etiologic Study of Sarcoidosis (ACCESS)

Sarcoidosis is a chronic granulomatous disorder of unknown cause, characterized by activation of T-lymphocytes and macrophages. A Case Control Etiologic Study of Sarcoidosis (ACCESS) is a multicenter study designed to determine the etiology of sarcoidosis. The study organization includes 10 Clinical Centers, a Clinical Coordinating Center, specialized Core Laboratories, a Central Specimen Repository, and a Project Office at the National Heart, Lung, and Blood Institute. In addition to etiology, ACCESS will examine the socioeconomic status and clinical course of patients with sarcoidosis. We propose to enroll 720 newly diagnosed cases of sarcoidosis and compare them to 720 age, sex, and race matched controls and follow the first 240 cases for two years. Leads to the etiology of sarcoidosis have come from diverse sources: in clinical laboratory investigations, alveolitis has been found to precede granulomatous inflammation; in case control studies, familial aggregation has been identified; and in case reports, recurrence of granulomatous inflammation has been observed after lung transplantation. We describe the rationale for the study design based on genetic, environmental, infectious, and immune dysregulation hypotheses and the methods used for selecting controls. The cause may not prove to be a single, known exposure. Interactions of exposures with genetic predispositions would have important implications for our understanding of immune responses as well as the pathogenesis of sarcoidosis.

Serum Amyloid A Regulates Granulomatous Inflammation in Sarcoidosis through Toll-like Receptor-2

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)-kappaB 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-kappaB in Toll-like receptor-2-expressing human cell lines; regulated experimental Th1-mediated granulomatous inflammation through IFN-gamma, 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.

Selective activation and accumulation of oligoclonal V beta-specific T cells in active pulmonary sarcoidosis.

Sarcoidosis is a granulomatous disease in which activated T cells, responding to an unidentified stimulus, accumulate at sites of disease such as the lung. To evaluate the hypothesis that active sarcoidosis is characterized by a selective activation and expansion of a limited repertoire of T cell receptor (TCR) specific T cells, we analyzed TCR V beta gene expression in lung and blood T cells of patients with active sarcoidosis and, for comparison, normal individuals using polymerase chain reaction amplification of 20 V beta gene families. Analysis of normal bronchoalveolar lavage T cells revealed TCR V beta distributions similar to that of normal blood, providing evidence for a lack of generalized skewing of the T cell repertoire in the normal, noninfected lung. Compared to normal lung and blood, subgroups of individuals with sarcoidosis demonstrated biased expression of one or more V beta genes in either the lung or blood. Five V beta gene families (V beta 5, V beta 8, V beta 15, V beta 16, and V beta 18) were most frequently utilized in a biased fashion by sarcoid lung or blood T cells. Furthermore, dramatic skewing of the T cell repertoire was apparent when sarcoid lung and blood T cells were expanded by short-term culture with IL-2. Sequence analysis demonstrated a bias in V beta gene expression was usually due to expansion of select V beta-specific clones, some of which contained a similar V(D)J junctional region motif. These observations provide evidence for a selective activation and accumulation of antigen-specific V beta-expressing T cells in sarcoidosis.

Treatment of Sarcoidosis-Associated Pulmonary Hypertension: A Two-Center Experience

BACKGROUND Pulmonary hypertension (PH) is a common complication of sarcoidosis that is associated with increased mortality. The pathogenesis of PH in sarcoidosis is uncertain, and the role of pulmonary arterial hypertension (PAH)-specific therapies remains to be determined. METHODS We conducted a retrospective study of patients with sarcoidosis and PH at two referral centers. New York Heart Association (NYHA) functional class, exercise capacity, hemodynamic data, pulmonary function tests, and survival were collected and analyzed. RESULTS Twenty-two sarcoidosis patients treated with PAH-specific therapies were identified. After a median of 11 months of follow-up, NYHA class was improved in nine subjects. Mean 6-min walk distance (n = 18) increased by 59 m (p = 0.032). Patients with a higher FVC experienced a greater increment in exercise capacity. Among 12 patients with follow-up hemodynamic data, mean pulmonary artery pressure was reduced from 48.5 +/- 4.3 to 39.4 +/- 2.8 mm Hg (p = 0.008). The 1- and 3-year transplant-free survival rates were 90% and 74%, respectively. CONCLUSIONS PAH-specific therapy may improve functional class, exercise capacity, and hemodynamics in PH associated with sarcoidosis. Prospective, controlled trials of PAH therapies for sarcoidosis are warranted to verify this apparent benefit. Mortality among the study population was high, highlighting the need for urgent evaluation at a lung transplant center.

Inhibition of human interleukin-12 production by pentoxifylline

Pharmacological control of interleukin‐12 (IL‐12) production may be a key therapeutic strategy for modulating immunological diseases dominated by type‐1 cytokine responses. In this study, we investigated the effects of pentoxifylline on the production of IL‐12 by human blood mononuclear cells and primary human monocytes stimulated with heat‐killed Staphylococcus aureus Cowan strain I (SAC) or lipopolysaccharide (LPS). Pentoxifylline potently suppressed production of IL‐12 in a concentration‐dependent manner. In these same experiments, tumour necrosis factor‐α (TNF‐α) production was inhibited and IL‐10 and prostaglandin E2 (PGE2) production was enhanced by treatment with pentoxifylline. Suppression of IL‐12 production by pentoxifylline was found to be independent of several known endogenous inhibitors of IL‐12, such as IL‐10, transforming growth factor‐β (TGF‐β), IL‐4 and PGE2. RNase protection assays revealed that pentoxifylline inhibited accumulation of both IL‐12 p40 and p35 mRNA, suggesting a predominant mRNA locus for pentoxifylline‐induced IL‐12 inhibition. Low levels of pentoxifylline added to the suppression of IL‐12 production by suboptimal inhibiting doses of dexamethasone, suggesting that this drug combination may have therapeutic utility. These results provide a firm rationale for the use of pentoxifylline in clinical trials of immunological disorders characterized by inappropriate type‐1 immune responses.

Sarcoidosis—scientific progress and clinical challenges

Sarcoidosis is an uncommon systemic inflammatory disorder characterized by noncaseating granulomatous inflammation that most commonly affects the lungs, intrathoracic lymph nodes, eyes and skin. One-third or more of patients with sarcoidosis have chronic, unremitting inflammation with progressive organ impairment. Findings of family and genetic studies indicate a genetic susceptibility to sarcoidosis, with genes in the MHC region having a dominant role. Immunologic hallmarks of the disease include highly polarized expression of cytokines produced by type 1 T helper cells and tumor necrosis factor (TNF) at sites of inflammation. Increasing evidence obtained within the past decade suggests the etiology of sarcoidosis predominantly involves microbial triggers, with the most convincing data implicating mycobacterial or propionibacterial organisms. Innate immune mechanisms, possibly involving misfolding and aggregation of serum amyloid A, might have a critical role in the pathobiology of sarcoidosis. Despite these advances, there are no clinically useful biomarkers that can assist the clinician in diagnosis, prognosis or assessment of treatment effects. Corticosteroids remain the cornerstone of therapy when organ function is threatened or progressively impaired. The role of immunosuppressive drugs and anti-TNF agents in the treatment of sarcoidosis remains uncertain, and there are no FDA-approved therapies. Meaningful progress in developing clinically useful tools and new therapies will depend on further advances in understanding the pathogenesis of sarcoidosis and its disease-specific pathways.

Genome-wide search for sarcoidosis susceptibility genes in African Americans

Sarcoidosis, a systemic granulomatous disease of unknown etiology, likely results from an environmental insult in a genetically susceptible host. In the US, African Americans are more commonly affected with sarcoidosis and suffer greater morbidity than Caucasians. We searched for sarcoidosis susceptibility loci by conducting a genome-wide, sib pair multipoint linkage analysis in 229 African-American families ascertained through two or more sibs with a history of sarcoidosis. Using the Haseman–Elston regression technique, linkage peaks with P-values less than 0.05 were identified on chromosomes 1p22, 2p25, 5p15-13, 5q11, 5q35, 9q34, 11p15 and 20q13 with the most prominent peak at D5S2500 on chromosome 5q11 (P=0.0005). We found agreement for linkage with the previously reported genome scan of a German population at chromosomes 1p and 9q. Based on the multiple suggestive regions for linkage found in our study population, it is likely that more than one gene influences sarcoidosis susceptibility in African Americans. Fine mapping of the linked regions, particularly on chromosome 5q, should help to refine linkage signals and guide further sarcoidosis candidate gene investigation.

Original ResearchPulmonary HypertensionTreatment of Sarcoidosis-Associated Pulmonary Hypertension: A Two-Center Experience

BACKGROUND Pulmonary hypertension (PH) is a common complication of sarcoidosis that is associated with increased mortality. The pathogenesis of PH in sarcoidosis is uncertain, and the role of pulmonary arterial hypertension (PAH)-specific therapies remains to be determined. METHODS We conducted a retrospective study of patients with sarcoidosis and PH at two referral centers. New York Heart Association (NYHA) functional class, exercise capacity, hemodynamic data, pulmonary function tests, and survival were collected and analyzed. RESULTS Twenty-two sarcoidosis patients treated with PAH-specific therapies were identified. After a median of 11 months of follow-up, NYHA class was improved in nine subjects. Mean 6-min walk distance (n = 18) increased by 59 m (p = 0.032). Patients with a higher FVC experienced a greater increment in exercise capacity. Among 12 patients with follow-up hemodynamic data, mean pulmonary artery pressure was reduced from 48.5 +/- 4.3 to 39.4 +/- 2.8 mm Hg (p = 0.008). The 1- and 3-year transplant-free survival rates were 90% and 74%, respectively. CONCLUSIONS PAH-specific therapy may improve functional class, exercise capacity, and hemodynamics in PH associated with sarcoidosis. Prospective, controlled trials of PAH therapies for sarcoidosis are warranted to verify this apparent benefit. Mortality among the study population was high, highlighting the need for urgent evaluation at a lung transplant center.

Job and industry classifications associated with sarcoidosis in a case-control etiologic study of sarcoidosis (ACCESS)

Objectives: Objective: To determine whether specific occupations and industries may be associated with sarcoidosis. Methods: A Case Control Etiologic Study of Sarcoidosis (ACCESS) obtained occupational and environmental histories on 706 newly diagnosed sarcoidosis cases and matched controls. We used Standard Industrial Classification (SIC) and Standard Occupational Classification (SOC) to assess occupational contributions to sarcoidosis risk. Results: Univariable analysis identified elevated risk of sarcoidosis for workers with industrial organic dust exposures, especially in Caucasian workers. Workers for suppliers of building materials, hardware, and gardening materials were at an increased risk of sarcoidosis as were educators. Work providing childcare was negatively associated with sarcoidosis risk. Jobs with metal dust or metal fume exposures were negatively associated with sarcoidosis risk, especially in Caucasian workers. Conclusions: In this study, we found that exposures in particular occupational settings may contribute to sarcoidosis risk.