Granuloma Formation In Sarcoidosis Research Articles

Aberrant Lipid Metabolism in Macrophages Is Associated with Granuloma Formation in Sarcoidosis.

Rationale: Chronic sarcoidosis is a complex granulomatous disease with limited treatment options that can progress over time. Understanding the molecular pathways contributing to disease would aid in new therapeutic development. Objectives: To understand whether macrophages from patients with nonresolving chronic sarcoidosis are predisposed to macrophage aggregation and granuloma formation and whether modulation of the underlying molecular pathways influence sarcoidosis granuloma formation. Methods: Macrophages were cultivated invitro from isolated peripheral blood CD14+ monocytes and evaluated for spontaneous aggregation. Transcriptomics analyses and phenotypic and drug inhibitory experiments were performed on these monocyte-derived macrophages. Human skin biopsies from patients with sarcoidosis and a myeloid Tsc2-specific sarcoidosis mouse model were analyzed for validatory experiments. Measurements and Main Results: Monocyte-derived macrophages from patients with chronic sarcoidosis spontaneously formed extensive granulomas invitro compared with healthy control participants. Transcriptomic analyses separated healthy and sarcoidosis macrophages and identified an enrichment in lipid metabolic processes. In vitro patient granulomas, sarcoidosis mouse model granulomas, and those directly analyzed from lesional patient skin expressed an aberrant lipid metabolism profile and contained increased neutral lipids. Conversely, a combination of statins and cholesterol-reducing agents reduced granuloma formation both invitro and invivo in a sarcoidosis mouse model. Conclusions: Together, our findings show that altered lipid metabolism in sarcoidosis macrophages is associated with its predisposition to granuloma formation and suggest cholesterol-reducing therapies as a treatment option in patients.

Immune mechanisms of granuloma formation in sarcoidosis and tuberculosis.

Sarcoidosis is a complex immune-mediated disease characterized by clusters of immune cells called granulomas. Despite major steps in understanding the cause of this disease, many questions remain. In this Review, we perform a mechanistic interrogation of the immune activities that contribute to granuloma formation in sarcoidosis and compare these processes with its closest mimic, tuberculosis, highlighting shared and divergent immune activities. We examine how Mycobacterium tuberculosis is sensed by the immune system; how the granuloma is initiated, formed, and perpetuated in tuberculosis compared with sarcoidosis; and the role of major innate and adaptive immune cells in shaping these processes. Finally, we draw these findings together around several recent high-resolution studies of the granuloma in situ that utilized the latest advances in single-cell technology combined with spatial methods to analyze plausible disease mechanisms. We conclude with an overall view of granuloma formation in sarcoidosis.

Activation of the pentose phosphate pathway in macrophages is crucial for granuloma formation in sarcoidosis.

Sarcoidosis is a disease of unknown etiology in which granulomas form throughout the body and is typically treated with glucocorticoids, but there are no approved steroid-sparing alternatives. Here, we investigated the mechanism of granuloma formation using single-cell RNA-Seq in sarcoidosis patients. We observed that the percentages of triggering receptor expressed on myeloid cells 2-positive (TREM2-positive) macrophages expressing angiotensin-converting enzyme (ACE) and lysozyme, diagnostic makers of sarcoidosis, were increased in cutaneous sarcoidosis granulomas. Macrophages in the sarcoidosis lesion were hypermetabolic, especially in the pentose phosphate pathway (PPP). Expression of the PPP enzymes, such as fructose-1,6-bisphosphatase 1 (FBP1), was elevated in both systemic granuloma lesions and serum of sarcoidosis patients. Granuloma formation was attenuated by the PPP inhibitors in in vitro giant cell and in vivo murine granuloma models. These results suggest that the PPP may be a promising target for developing therapeutics for sarcoidosis.

Dexamethasone alleviates pulmonary sarcoidosis by regulating the TGF-β/Smad3 signaling to promote Th17/Treg cell rebalance

Pulmonary sarcoidosis is an immune-mediated disorder closely related to Th17/Treg cell imbalance. Dexamethasone has been shown to regulate inflammation and immune responses in sarcoidosis patients. However, the underlying mechanisms of dexamethasone regulating Th17/Treg balance in sarcoidosis remain elusive. Herein, we elucidated the function role of TGF-β/Smad3 signaling in pulmonary sarcoidosis development and explored the underlying mechanism of dexamethasone in treating pulmonary sarcoidosis. We found that the TGF-β/Smad3 pathway was inactivated in pulmonary sarcoidosis patients. Propionibacterium acnes (PA) induced mouse model was generated to investigate the function of TGF-β/Smad3 signaling in vivo. Data indicated that IL17A inhibition with neutralizing antibody and activation of TGF-β/Smad3 signaling with SRI-011381 alleviated granuloma formation in the sarcoidosis mouse model. Moreover, we revealed that the Th17/Treg cell ratio was increased with PA treatment in mouse bronchoalveolar lavage fluid (BALF) and peripheral blood. The concentration of cytokines produced by Th17 cells (IL-17A, IL-23) was up-regulated in the BALF of PA-treated mice, while those produced by Tregs (IL-10, TGF-β1) presented significant reduction. The treatment of IL-17A neutralizing antibody or SRI-011381 was demonstrated to rescue the PA-induced changes in the concentration of IL-17A, IL-23, IL-10, and TGF-β1. Additionally, we demonstrated that dexamethasone treatment activated the TGF-β/Smad3 signaling in the lung tissues of pulmonary sarcoidosis mice. Dexamethasone was also revealed to promote the rebalancing of the Th17/Treg ratio and attenuated the granuloma formation in pulmonary sarcoidosis. In conclusion, dexamethasone activates the TGF-β/Smad3 signaling and induces Th17/Treg rebalance, alleviating pulmonary sarcoidosis, which suggests the potential of dexamethasone in treating pulmonary sarcoidosis.

916 Activation of the pentose phosphate pathway in macrophages is essential for granuloma formation in sarcoidosis

916 Activation of the pentose phosphate pathway in macrophages is essential for granuloma formation in sarcoidosis

Pentraxin 3 Inhibits Complement-driven Macrophage Activation to Restrain Granuloma Formation in Sarcoidosis.

Rationale: Sarcoidosis is a multisystemic inflammatory disease characterized by the formation of granulomas in response to persistent stimuli. The long pentraxin PTX3 (pentraxin 3) has emerged as a component of humoral innate immunity with essential functions in the resolution of inflammation, but its role during granuloma formation is unknown. Objectives: To evaluate PTX3 as a modulator of pathogenic signals involved in granuloma formation and inflammation in sarcoidosis. Methods: Peripheral blood mononuclear cells obtained from patients with sarcoidosis harboring loss-of-function genetic variants and gene-deleted mice were used to assess the role of PTX3 in experimental models of granuloma formation invitro and invivo. The identified mechanisms of granulomatous inflammation were further evaluated in tissue and BAL samples and correlated with the disease course. Measurements and Main Results: We have identified a molecular link between PTX3 deficiency and the pathogenic amplification of complement activation to promote granuloma formation. Mechanistically, PTX3 deficiency licensed the complement component C5a-mediated activation of the metabolic checkpoint kinase mTORC1 (mammalian target of rapamycin complex 1) and the reprogramming of macrophages toward increased glycolysis to foster their proliferation and aggregation. This process sustained the further recruitment of granuloma-promoting immune cells and the associated proinflammatory microenvironment and influenced the clinical course of the disease. Conclusions: Our results identify PTX3 as a pivotal molecule that regulates complement-mediated signaling cues in macrophages to restrain granulomatous inflammation and highlight the therapeutic potential of this signaling axis in targeting granuloma formation in sarcoidosis.

Transcriptome Analysis of Peripheral Blood Mononuclear Cells in Pulmonary Sarcoidosis.

BackgroundSarcoidosis is a granulomatous systemic disease of unknown etiology. Mononuclear cells such as macrophages or lymphocytes in lung tissue and hilar or mediastinal lymph nodes have been recognized to play an essential role in granuloma formation in pulmonary sarcoidosis. Peripheral blood mononuclear cells (PBMCs) consist of several immunocompetent cells and have been shown to play a mechanistic role in the pathogenesis of sarcoidosis. However, the genetic modifications that occur in bulk PBMCs of sarcoidosis remain to be elucidated.PurposeThis study aimed to explore the pathobiological markers of sarcoidosis in PBMCs by comparing the transcriptional signature of PBMCs from patients with pulmonary sarcoidosis with those of healthy controls by RNA sequencing.MethodsPBMC samples were collected from subjects with pulmonary sarcoidosis with no steroid/immunosuppressant drugs (n = 8) and healthy controls (n = 11) from August 2020 to April 2021, and RNA sequencing was performed with the PBMC samples.ResultsPrincipal component analysis using RNA sequencing datasets comparing pulmonary sarcoidosis with healthy controls revealed that the two groups appeared to be differentiated, in which 270 differentially expressed genes were found in PBMCs between sarcoidosis and healthy controls. Enrichment analysis for gene ontology suggested that some biological processes related to the pathobiology of sarcoidosis, such as cellular response to interleukin (IL)-1 and IFN-γ, regulation of IL-6 production, IL-8 secretion, regulation of mononuclear cell migration, and response to lipopolysaccharide, were involved. Enrichment analysis of the KEGG pathway indicated the involvement of tumor necrosis factor (TNF), toll-like receptor signaling, IL-17 signaling pathways, phagosomes, and ribosomes. Most of the genes involved in TNF and IL-17 signaling pathways and phagosomes were upregulated, while most of the ribosome-related genes were downregulated.ConclusionThe present study demonstrated that bulk gene expression patterns in PBMCs were different between patients with pulmonary sarcoidosis and healthy controls. The changes in the gene expression pattern of PBMCs could reflect the existence of sarcoidosis lesions and influence granuloma formation in sarcoidosis. These new findings are important to strengthen our understanding of the etiology and pathobiology of sarcoidosis and indicate a potential therapeutic target for sarcoidosis.

Current Sarcoidosis Models and the Importance of Focusing on the Granuloma.

The inability to effectively model sarcoidosis in the laboratory or in animals continues to hinder the discovery and translation of new, targeted treatments. The granuloma is the signature pathological hallmark of sarcoidosis, yet there are significant knowledge gaps that exist with regard to how granulomas form. Significant progress toward improved therapeutic and prognostic strategies in sarcoidosis hinges on tractable experimental models that recapitulate the process of granuloma formation in sarcoidosis and allow for mechanistic insights into the molecular events involved. Through its inherent representation of the complex genetics underpinning immune cell dysregulation in sarcoidosis, a recently developed in vitro human granuloma model holds promise in providing detailed mechanistic insight into sarcoidosis–specific disease regulating pathways at play during early stages of granuloma formation. The purpose of this review is to critically evaluate current sarcoidosis models and assess their potential to progress the field toward the goal of improved therapies in this disease. We conclude with the potential integrated use of preclinical models to accelerate progress toward identifying and testing new drugs and drug combinations that can be rapidly brought to clinical trials.

A Novel In Vitro Human Granuloma Model of Sarcoidosis and Latent Tuberculosis Infection.

Many aspects of pathogenic granuloma formation are poorly understood, requiring new relevant laboratory models that represent the complexity (genetics and diversity) of human disease. To address this need, we developed an in vitro model of granuloma formation using human peripheral blood mononuclear cells (PBMCs) derived from patients with active sarcoidosis, latent tuberculosis (TB) infection (LTBI), or normal healthy control subjects. PBMCs were incubated for 7 days with uncoated polystyrene beads or beads coated with purified protein derivative (PPD) or human serum albumin. In response to PPD-coated beads, PBMCs from donors with sarcoidosis and LTBI formed robust multicellular aggregates resembling granulomas, displaying a typical T-helper cell type 1 immune response, as assessed by cytokine analyses. In contrast, minimal PBMC aggregation occurred when control PBMCs were incubated with PPD-coated beads, whereas the response to uncoated beads was negligible in all groups. Sarcoidosis PBMCs responded to human serum albumin-coated beads with modest cellular aggregation and inflammatory cytokine release. Whereas the granuloma-like aggregates formed in response to PPD-coated beads were similar for sarcoidosis and LTBI, molecular profiles differed significantly. mRNA expression patterns revealed distinct pathways engaged in early granuloma formation in sarcoidosis and LTBI, and they resemble molecular patterns reported in diseased human tissues. This novel in vitro human granuloma model is proposed as a tool to investigate mechanisms of early granuloma formation and for preclinical drug discovery research of human granulomatous disorders. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

Innate immunity in sarcoidosis pathobiology.

The immunopathogenesis of sarcoidosis is considered to involve contributions from both adaptive and innate immune responses. Although the identification of adaptive responses to candidate pathogenic antigens will elucidate mechanisms that regulate inflammation in sarcoidosis, innate mechanisms likely represent the 'missing link' to the initiation, maintenance, and resolution of noncaseating granulomatous inflammation, the hallmark feature of sarcoidosis. Furthermore, environments that expose patients to candidate pathogenic antigens for sarcoidosis also provide opportunities for engagement with innate ligands. Several studies have identified enhanced responsiveness via pattern recognition receptor pathways, potentiating the local induction of cytokines relevant to granulomatous inflammation, such as through stimulation of nucleotide-binding oligomerization domain-like receptor and Toll-like receptor pathways. These pathways contribute to the inherent properties of granulomas including, in some cases, persistent localization of pathogens and antigen. Serum amyloid A has been identified to be abundant in sarcoidosis tissues, and this promiscuous host protein can serve as an innate ligand to regulate experimental granulomatous inflammation. Nascent evidence supports a potential role for alternatively activated macrophages to direct histopathological outcomes in sarcoidosis. Innate pathways deserve further investigation as potential therapeutic targets for inhibiting granuloma formation in sarcoidosis.

Exhaled breath markers of alveolar macrophage activity in sarcoidosis.

Granuloma formation in sarcoidosis is dependent upon the interaction between alveolar macrophages (AMs) and a CD4+-driven TH1 response. This study aimed to measure TNF-α and calcium ion concentrations as markers of AM activity, in addition to total protein as a non-specific inflammatory marker in the exhaled breath condensate (EBC) of patients with sarcoidosis as well as control subjects. EBC was collected from 17 sarcoidosis patients and 23 healthy volunteers. Protein was measured by the bicinchoninic acid assay, TNF-α concentration was measured by ELISA and Ca(2+) concentration was measured by inductively coupled plasma-mass spectrometry. Conductivity of EBC was assessed using a conductivity probe. Total protein concentration was significantly elevated in EBC from patients with sarcoidosis compared to control subjects (19.51±4.52 vs. 10.60±1.31µg/ml, p=0.020), as was TNF-α (3.37±0.38 vs. 2.59±0.40pg/ml, p=0.037) and conductivity (66.68±16.73 vs. 36.85±3.070µS/cm, p=0.044). EBC Ca(2+) concentration was significantly higher in healthy controls compared to patients with sarcoidosis (116.50±12.19 vs. 73.88±13.35µmol/l, p=0.018), although this was in the context of normal serum Ca(2+) in the sarcoidosis cohort. Total protein and TNF-α concentrations were elevated in EBC from patients with sarcoidosis and could indicate disease activity. The reduction in EBC Ca(2+) concentrations could represent granulomatous activity in the lung.

Gene expression profiles in granuloma tissue reveal novel diagnostic markers in sarcoidosis

Sarcoidosis is an immune-mediated multisystem disease characterized by the formation of non-caseating granulomas. The pathogenesis of sarcoidosis is unclear, with proposed infectious or environmental antigens triggering an aberrant immune response in susceptible hosts. Multiple pro-inflammatory signaling pathways have been implicated in mediating macrophage activation and granuloma formation in sarcoidosis, including IFN-γ/STAT-1, IL-6/STAT-3, and NF-κB. It is difficult to distinguish sarcoidosis from other granulomatous diseases or assess disease severity and treatment response with histopathology alone. Therefore, development of improved diagnostic tools is imperative. Herein, we describe an efficient and reliable technique to classify granulomatous disease through selected gene expression and identify novel genes and cytokine pathways contributing to the pathogenesis of sarcoidosis. We quantified the expression of twenty selected mRNAs extracted from formalin-fixed paraffin embedded (FFPE) tissue (n=38) of normal lung, suture granulomas, sarcoid granulomas, and fungal granulomas. Utilizing quantitative real-time RT-PCR we analyzed the expression of several genes, including IL-6, COX-2, MCP-1, IFN-γ, T-bet, IRF-1, Nox2, IL-33, and eotaxin-1 and revealed differential regulation between suture, sarcoidosis, and fungal granulomas. This is the first study demonstrating that quantification of target gene expression in FFPE tissue biopsies is a potentially effective diagnostic and research tool in sarcoidosis.

Fungal exposure and low levels of IL-10 in patients with sarcoidosis.

Background and Objectives. Sarcoidosis is an inflammatory disease with increased levels of inflammatory cytokines. Previous studies have shown a relation between the degree of granuloma infiltration and serum cytokine levels, except for interleukin- (IL-) 10. The aim of the study was to further investigate the serum levels of IL-10 in patients with sarcoidosis and relate them to fungal exposure in terms of the amount of fungi in the air of their homes and β-glucan in bronchoalveolar lavage (BAL) fluid. Methods. Patients with sarcoidosis (n = 71) and healthy controls (n = 27) were enrolled. IL-10 was determined in serum. BAL was performed and the amount of β-glucan was measured. Domestic exposure to fungi was determined by measuring airborne β-N-acetylhexosaminidase (NAHA) in the bedrooms. Results. At high levels of fungal exposure (domestic fungal exposure and β-glucan in BAL), serum IL-10 values were lower than at low and intermediate exposure levels. Conclusion. The low serum IL-10 values at high fungal exposure suggest that fungal cell wall agents play a role in granuloma formation in sarcoidosis by inhibiting the secretion of the anti-inflammatory cytokine IL-10.

The BTNL2 A allele variant is frequent in Danish patients with sarcoidosis

The butyrophilin-like 2 (BTNL2) gene is located on chromosome 6p21.3 close to the HLA-class II genes. An association has been reported between sarcoidosis and a single nucleotide polymorphism in BTNL2, rs2076530, also termed the A allele. To evaluate whether patients with sarcoidosis carry the A allele more frequently than healthy subjects. The series comprised 87 ethnic Danes with sarcoidosis and 113 healthy control subjects. Analysis of rs2076530 was performed by Taqman assay, polymerase chain reaction and sequencing of genomic DNA. Sarcoidosis patients had a higher frequency of the A allele than controls (73.9% vs 55.8%) (P < 0.025). The frequency of GG, GA and AA genotype was 5.7%, 40.2% and 54.0% in patients vs 16.0%, 56.6% and 27.4% in controls (P < 0.001). The AA genotype was associated with increased risk of sarcoidosis in both a dominant [odds ratio (OR) 3.1; 95% confidence interval (CI) 1.1-8.7; P < 0.03] and a recessive model (OR 3.1; 95% CI 1.72-5.61; P < 0.001). Population attributable fraction for disease was 50% in a dominant model and 25% in a recessive model. The BTNL2 A allele variant occurs with a high frequency in Danish patients with sarcoidosis and the AA genotype is associated with a ∼threefold higher risk of sarcoidosis than the GG genotype. Our results should encourage future studies on the interrelationship between the BTNL2 protein and granuloma formation in sarcoidosis.

Experimental epithelioid cell granuloma formation in Lewis rats induced by injection of cell wall fragments derived from alpha-streptococcus--etiologic relationship between bacterial cell wall and sarcoid granuloma

We observed the presence of epithelioid granulomas in follicles near lacunae in the tonsils from patients with active sarcoidosis. Bacteria isolated in these tissues were mostly alpha-streptococci. The present study was undertaken to determine whether alpha-streptococcus can induce granuloma formation. Streptococcal cell wall (SCW) fragments were injected into the foot pads of female Lewis rats. Epithelioid granulomas were abundantly formed in popliteal lymph nodes when SCW aqueous suspension was injected four times. SCW antigens were detected in macrophages in the granuloma but not in epithelioid cells, by immunoperoxidase method. These findings suggest that macrophages transform into epithelioid cells after phagocytosis and digestion of SCW, and that components of bacterial cell wall such as SCW may induce the granuloma formation in sarcoidosis.

NOD1 Engagement Leads to Increased IL12/IL23 p40 and decreased IL-10 Expressions in Sarcoidosis as Compared to Healthy Subjects (98.17)

Abstract Sarcoidosis is a systemic inflammatory disorder of unknown etiology affecting barrier organs like lung and skin. The presence of granuloma and its associated inflammation may lead to organ dysfunction. Two NLR family members, NOD1 and NOD2, trigger immune defenses in response to bacterial peptidoglycans (PGNs). Interaction between these sensors and microbial moieties leads to up-regulation of cytokines involved in innate and adaptive immunity. We hypothesize that granuloma formation in sarcoidosis is due to an abnormal sensing of specific microbial constituents through NOD1, leading to an aberrant induction of pro-inflammatory and/or a decreased anti-inflammatory cytokines. Methods: We assessed the functional responses of whole blood or isolated PBMC of patients with established diagnosis of sarcoidosis in response to NOD1 and TLR agonists and compared to those of healthy subjects. Pro- and anti-inflammatory cytokines were measured at the gene and protein levels. Our results demonstrate that subjects with sarcoidosis expressed several cytokines including IL-1β, IL-8 and IL-6 significantly higher at baseline as compared to healthy controls. Secondly we show for the first time that patients with sarcoidosis respond to NOD1 stimulation and to co-stimulation of NOD1 and TLR4 receptors with a significant increase of IL12/IL23 p40 and decrease of IL-10 expressions at both the gene and protein levels.

Absence of KIR2DS3, a receptor implicated in NK cell activation is associated with pulmonary sarcoidosis (136.23)

Abstract By interacting with certain HLA class I molecules, the killer cell immunoglobulin-like receptors (KIR) control the function of NK cells that trigger early immune response against infection and tumors. To determine whether certain KIR and HLA genes are associated with pulmonary sarcoidosis (PS), a chronic inflammatory disease characterized with pulmonary granuloma, we have characterized KIR and HLA genes in 149 PS patients and 186 healthy controls. There was a significant decrease in the frequencies of KIR2DL2 (56.4% vs. 71.5%, P = 0.0041; 95% CI, 0.33-0.81, OR = 0.52), 2DL5 (61.7% vs. 76.3%, P = 0.0041; 95% CI, 0.31-0.80, OR = 0.50) and 2DS3 (28.2% vs. 45.2%, P = 0.0015; 95% CI, 0.30-0.75, OR = 0.48) in PS compared to controls. Interestingly, these three KIR genes 2DL2, 2DL5 and 2DS3 are closely linked together in the cetromeric half of the KIR gene complex, and thus any of these KIRs or other linked loci may contribute to the risk for PS. Particularly, the decrease in activating receptor KIR2DS3 in PS is consistent with the clinical anergy and diminished cellular immunity evidenced in PS. The individuals missing KIR2DS3 may fail triggering early NK cell response to clear antigenic stimuli, which may contribute granulomas formation. This study offers new insights into the molecular mechanisms of granuloma formation in sarcoidosis and provides a framework for developing new therapeutic strategies. This work was supported by UCLA Department of Pathology and Laboratory Medicine to RR.

Prognosis of refractory neurosarcoidosis altered by thalidomide: a case report

IntroductionSarcoidosis is a multisystem disease characterized by noncaseating granulomas in the lungs, skin, lymph nodes, and, rarely, the nervous system. Granuloma formation in sarcoidosis is mediated by increased secretion of interferon-gamma, interleukin-2, and tumor necrosis factor-alpha. 25% of patients with neurosarcoidosis are steroid resistant and another 20–40% are resistant to any conventional immunosuppression, but the typical agents suppress the immune system in a non-specific fashion. Thalidomide has been shown to have activity specific to the inflammatory mediators of sarcoidosis, has been shown to be beneficial in cutaneous sarcoidosis, and provides an interesting observation in our patient with refractory neurosarcoidosis.Case presentationA 40 year old African-american female presented with refractory neurosarcoidosis. Over the course of several years, the patient was treated with high dose steroids, imuran, cytoxan, and cyclosporine without benefit. Then, the patient received thalidomide, slowly escalating to 650 mg. After 2 months radiologic improvement was noted and after 6 months clinical stabilization and improvement became apparent.ConclusionOur case report presents a difficult, refractory case of neurosarcoidosis that demonstrates an altered prognosis based on the addition of thalidomide.

Die Hyperkalzämie im Verlauf der Sarkoidose – Fallbeispiel, Prävalenz, Pathophysiologie und Therapiemöglichkeiten

Hypercalcemia is a highly prevalent complication of sarcoidosis. A medical history of a patient with sarcoidosis is shown as case report. Depending on the population studied about 2-63% of sarcoidosis patients show hypercalcemia. The major difference in the prevalence of hypercalcemia may be in part due to the undulating course of subacute sarcoidosis, so hypercalcemia may be missed when serum calcium is not frequently measured. Hypercalciuria appears to be twice as prevalent then hypercalcemia and should be looked for in every sarcoidosis patient. Hypercalcemia in sarcoidosis is due to the uncontrolled synthesis of 1,25-dihydroxyvitamin D3 by macrophages. 1,25-dihydroxyvitamin D3 leads to an increased absorption of calcium in the intestine and to an increased resorption of calcium in the bone. Immunoregulatory properties have been ascribed to 1,25-dihydroxyvitamin D3. It is an important inhibitor of interleukin-2 and of interferon-gamma-synthesis, two cytokines that are important in granuloma formation in sarcoidosis. It is thought that 1,25-dihydroxyvitamin D3 counterregulates uncontrolled granuloma formation. Treatment of hypercalcemia depends on the serum level of hypercalcemia and its persistence. Generally sarcoidotic patients should be advised to avoid sun exposition to reduce vitamin D3 synthesis in the skin, to omit fish oils that are rich of vitamin D and to produce more than two liters urine a day by adapting fluid intake. Although severe hypercalcemia seems to be rare, glucocorticosteroid treatment should be started if corrected total calcium level rises beyond 3 mmol/l. If hypercalcemia is symptomatic, treatment should be started even at lower levels. Glucocorticosteroids act by inhibition of the overly 1alpha-hydroxylase activity of macrophages. Alternatively, treatment with chloroquine or ketoconazole can be established. If isolated hypercalciuria without hypercalcemia is present with evidence for recurrent nephrolithiasis, patients can be treated with a thiazide diuretic.

ANALYSIS OF DIFFERENTIALLY REGULATED mRNAs IN MONOCYTIC CELLS INDUCED BY IN VITRO STIMULATION WITH KVEIM-SILTZBACH TEST REAGENT

Sarcoidosis is a granulomatous disorder of unknown origin. The sarcoid spleen-derived Kveim-Siltzbach test reagent (KSTR) elicits a sarcoid-specific, granulomatous response and was used to diagnose sarcoidosis. The active component and the pathomechanism by which KSTR induces granuloma are still unknown. This study investigated the KSTR-associated gene expression pattern in cells of patients with sarcoidosis or chronic beryllium disease (CBD). The monocytic-like cell line U937, alveolar macrophages of sarcoidosis patients, and peripheral blood monocytes of patients with CBD were stimulated with KSTR and other granuloma-associated stimuli. The KSTR-associated gene expression pattern was analyzed by means of differential display reverse transcription–polymerase chain reaction in combination with a multiple comparison of expressed sequence tags induced in response to KSTR and other granuloma-associated stimuli. Depending on the origin of cells tested, 3.7% to 14.6% of the analyzed sequence tags showed differential regulation induced by granuloma-associated stimuli. Alterations restricted to KSTR stimulation could be observed in 1.2% for the cell line U937, in 2.8% for blood monocytes of patients with CBD, and 1.3% for alveolar macrophages of sarcoidosis patients. These data are comparable to those achieved for the other granuloma-associated stimuli tested in this study. Therefore, it can be assumed that KSTR induces pathomechanisms for granuloma formation in sarcoidosis as beryllium in CBD.