Johan Grunewald

Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells

Objectives: A gene–environment interaction between HLA-DR shared epitope genes and smoking in anti-cyclic citrullinated peptide antibody-positive rheumatoid arthritis (RA) has been reported. Identification of citrullinated proteins in bronchoalveolar lavage (BAL) cells from smokers has led to the suggestion that citrullination induced by smoking might be the first step in the pathogenic chain of RA. Objective: To confirm and extend these findings. Methods: Immunohistochemistry was performed on BAL cells and bronchial mucosal biopsy sections obtained through bronchoscopy from 14 healthy smokers and 16 healthy non-smokers. Two antibodies recognising citrullinated proteins, two antibodies recognising peptidylarginine deiminase (PAD)2 enzyme and one recognising PAD4 enzyme were used. Results: Citrullinated proteins are upregulated in BAL cells of healthy smokers compared with healthy non-smokers. This was associated with higher expression of the PAD2 enzyme. The same level of citrullinated proteins was present in bronchial mucosal biopsy specimens of healthy smokers and non-smokers, despite higher expression of PAD2 in smokers. Conclusion: This study provides evidence that smoking enhances PAD2 expression in the bronchial mucosal and alveolar compartment, with consequent generation of citrullinated proteins in the latter. Smoking is an environmental factor that may lead to citrulline autoimmunity in genetically susceptible subjects.

Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid

Exosomes are 30–100 nm diameter vesicles formed by inward budding of endosomal compartments and are produced by several cell types, including T‐cells, B‐cells and dendritic cells (DC)s. Exosomes from DCs express major histocompatibility complexes (MHC) class I and II, and co-stimulatory molecules on their surface, and can induce antigen-specific activation of T‐cells. The aims of the present study were to investigate for the presence of exosomes in bronchoalveolar lavage fluid (BALF) from healthy individuals, and to establish if these exosomes bear MHC and co-stimulatory molecules. The authors analysed BALF taken from seven healthy volunteers and used exosomes from monocyte-derived DC (MDDC) cultures as a reference. After ultracentrifugation, exosomes were bound to anti‐MHC class II coated magnetic beads and analysed by flow cytometry and electron microscopy. The authors report for the first time that exosomes are present in BALF. These exosomes are similar to MDDC derived exosomes as they express MHC class I and II, CD54, CD63 and the co-stimulatory molecule CD86. The results demonstrate that exosomes are present in the lung, and since they contain both major histocompatibility complex and co-stimulatory molecules it is likely that they are derived from antigen presenting cells and might have a regulatory role in local immune defence.

Smoking, Use of Moist Snuff, and Risk of Chronic Inflammatory Diseases

RATIONALE Cigarette smoking is emerging as a strong risk factor in the otherwise unknown etiology of chronic inflammatory diseases. Whether the same applies also to smokeless tobacco remains unknown. Nicotine is a powerful modifier of the inflammatory response. By comparing risks associated with tobacco smoking and with smokeless tobacco, the role of nicotine in the development of chronic inflammation may be evaluated. OBJECTIVES To assess and compare the risks of rheumatoid arthritis (RA), ulcerative colitis (UC), Crohns disease (CD), sarcoidosis, and multiple sclerosis (MS) associated with cigarette smoking and with the use of Swedish moist snuff. METHODS We performed a cohort study of 277,777 males within a cohort of Swedish construction workers who had provided information about tobacco use in 1978-1993. Cross-linkage to the nationwide Swedish Hospital Discharge Register provided information about the occurrence of RA, UC, CD, sarcoidosis, and MS through 2004. MEASUREMENTS AND MAIN RESULTS Age-adjusted relative risks (RRs) associated with smoking and moist snuff, respectively, were estimated by Cox regression. Ever-smoking was associated with an increased risk for RA (RR, 2.1; 95% confidence interval [CI], 1.7-2.5), CD (RR, 1.5; 95% CI, 1.2-1.8), MS (RR, 1.9; 95% CI, 1.4-2.6), and UC (RR, 1.3; 95% CI, 1.1-1.5, confined to ex-smokers), and a decreased risk of sarcoidosis (RR, 0.5; 95% CI, 0.4-0.5). By contrast, ever-use of moist snuff, adjusted for smoking, was not associated with RA (RR, 1.0; 95% CI, 0.9-1.2), UC (RR, 1.1; 95% CI, 0.9-1.2), CD (RR, 0.9; 95% CI, 0.8-1.1), sarcoidosis (RR, 1.1; 95% CI, 0.8-1.5), or MS (RR, 1.0; 95% CI, 0.8-1.4). CONCLUSIONS Smokeless tobacco does not increase the risk of chronic inflammatory diseases, suggesting that inhaled nonnicotinic components of cigarette smoke are more important than nicotine itself in the etiology of these diseases.

Löfgren's Syndrome

RATIONALE Sarcoidosis may consist of a number of distinct disease entities, one of which could be Löfgrens syndrome. Patients with Löfgrens syndrome have an acute onset of erythema nodosum (EN) and/or periarticular inflammation or arthritis of the ankles, with bilateral hilar lymphadenopathy (and in some cases parenchymal infiltrates) and usually fever. There is a known association between HLA-DRB1*03 and Löfgrens syndrome. OBJECTIVES To investigate whether human leukocyte antigen type influences clinical manifestations, including the disease course in Löfgrens syndrome. METHODS We clinically characterized and HLA-DRB1 typed 301 patients with Löfgrens syndrome. A total of 275 of the patients were followed for more than 2 years and classified as having a nonresolving or a resolving disease. MEASUREMENTS AND MAIN RESULTS Almost every DRB1*03-positive patient had a resolving disease within 2 years, and 49% of the DRB1*03-negative patients developed a nonresolving disease. Mucosal granulomas were identified significantly more often in DRB1*03-negative patients. Among DRB1*03-negative patients who were treated with oral steroids at disease onset, 80% developed a nonresolving disease. CONCLUSIONS Patients with Löfgrens syndrome have a different disease course depending on whether they are DRB1*03 positive or not. This observation has clinical implications, and by comparing DRB1*03-positive and DRB1*03-negative patients with Löfgrens syndrome, we can search for additional markers of importance for developing a resolving or a nonresolving disease, respectively.

T Cell Responses to Mycobacterial Catalase-Peroxidase Profile a Pathogenic Antigen in Systemic Sarcoidosis

Sarcoidosis is a systemic granulomatous disease associated with local epithelioid granulomas, CD4+ T cells, and Th1 cytokines. The tissue Ags that drive this granulomatous inflammation are uncertain. In this study, we used IFN-γ-ELISPOT assays and flow cytometry to assess lung and blood T cell responses to the candidate pathogenic Ag, Mycobacterium tuberculosis catalase-peroxidase (mKatG) in patients with sarcoidosis from two centers. Despite differences in patient phenotypic, genetic, and prognostic characteristics, we report that T cell responses to mKatG were remarkably similar in these cohorts, with higher frequencies of mKatG-reactive, IFN-γ-expressing T cells in the blood of sarcoidosis patients compared with nontuberculosis sensitized healthy controls, and (in a subset) in greater numbers than T cells reactive to purified protein derivative. In sarcoidosis, mKatG-reactive CD4+ Th1 cells preferentially accumulated in the lung, indicating a compartmentalized response. Patients with or without Löfgren syndrome had similar frequencies of mKatG specific IFN-γ-expressing blood T cells. Circulating mKatG-reactive T cells were found in chronic active sarcoidosis but not in patients with inactive disease. Together, these results demonstrate that T cell responses to mKatG in sarcoidosis fit a profile expected for a pathogenic Ag, supporting an immunotherapeutic approach to this disease.

Structural Changes and Antibody Enrichment in the Lungs Are Early Features of Anti–Citrullinated Protein Antibody–Positive Rheumatoid Arthritis

It has been suggested that immunologic events in the lungs may be involved in triggering immunity, in particular production of anti–citrullinated protein antibodies (ACPAs) during early phases of rheumatoid arthritis (RA). The aim of this study was to investigate the structural and immunologic features of the lungs in incident cases of early RA in relation to ACPA presence and smoking status.

Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis

Background Sarcoidosis is a systemic disease of unknown aetiology characterised by granuloma formation and the presence of interferon γ (IFNγ)-producing T cells that cause inflammation and tissue damage in multiple organs, especially the lung. Exosomes are nano-sized immunomodulatory vesicles of endosomal origin released from a diverse range of cells and are also found in physiological fluids including bronchoalveolar lavage fluid (BALF) from healthy individuals. Objective To investigate whether exosomes are enriched in the lungs of patients with sarcoidosis compared with healthy individuals and whether they could contribute to pathogenesis. Design BALF exosomes from patients with sarcoidosis (n=36) and healthy controls (n=14) were compared by electron microscopy, flow cytometry, western blot analysis and mass spectrometry. BALF exosomes were incubated with autologous peripheral blood mononuclear cells (PBMCs) or the human bronchial epithelial cell line 16HBE14o-. Cytokines were measured by ELISPOT and ELISA. Results BALF from patients with sarcoidosis showed increased levels of exosomes compared with healthy individuals. Exosomes from patients showed significantly higher expression of MHC class I and II, tetraspanins CD9, CD63 and CD81 as well as neuregulin-1, known to be associated with cancer progression. Furthermore, BALF exosomes from patients induced significantly higher IFNγ and interleukin (IL)-13 production in autologous PBMCs compared with healthy individuals and could also stimulate IL-8 production from epithelial cells. Conclusion The results indicate for the first time a role for exosomes in human lung disease with possible contributions to the initiation and progression of inflammation in sarcoidosis. This suggests that exosomes may be a new potential target for the clinical treatment of lung diseases.

Phenotypic analysis of lymphocytes and monocytes/macrophages in peripheral blood and bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis

BACKGROUND The granulomatous inflammation in sarcoidosis is driven by the interplay between T cells and macrophages. To gain a better understanding of this process the expression by these cells of cell surface activation markers, co-stimulatory molecules, and adhesion molecules was analysed. METHODS CD4+ and CD8+ T lymphocytes from peripheral blood (PBL) or bronchoalveolar lavage (BAL) fluid, as well as paired peripheral blood monocytes and alveolar macrophages from 27 patients with sarcoidosis were analysed by flow cytometry. RESULTS CD26, CD54, CD69, CD95, and gp240 were all overexpressed in T cells from BAL fluid compared with those from PBL in both the CD4+ and CD8+ subsets, while CD57 was overexpressed only in BAL CD4+ cells. In contrast, CD28 tended to be underexpressed in the BAL T cells. Monocyte/macrophage markers included CD11a, CD11b, CD11c, CD14, CD16, CD54, CD71, CD80 and CD86 and HLA class II. CD11a expression in alveolar macrophages (and peripheral blood monocytes) was increased in patients with active disease and correlated positively with the percentage of BAL lymphocytes. Expression of CD80 in macrophages correlated with the BAL CD4/CD8 ratio. CONCLUSIONS Our data indicate substantial activation of both CD4+ and CD8+ lung T cells in sarcoidosis. There were also increased numbers of BAL lymphocytes whose phenotypic characteristics have earlier been associated with clonally expanded, replicatively senescent cells of the Th1 type.

Identification of HLA-DR–bound peptides presented by human bronchoalveolar lavage cells in sarcoidosis

Sarcoidosis is an inflammatory disease of unknown etiology, most commonly affecting the lungs. Activated CD4+ T cells accumulate in the lungs of individuals with sarcoidosis and are considered to be of central importance for inflammation. We have previously shown that Scandinavian sarcoidosis patients expressing the HLA-DR allele DRB1*0301 are characterized by large accumulations in the lungs of CD4+ T cells expressing the TCR AV2S3 gene segment. This association afforded us a unique opportunity to identify a sarcoidosis-specific antigen recognized by AV2S3+ T cells. To identify candidates for the postulated sarcoidosis-specific antigen, lung cells from 16 HLA-DRB1*0301pos patients were obtained by bronchoalveolar lavage. HLA-DR molecules were affinity purified and bound peptides acid eluted. Subsequently, peptides were separated by reversed-phase HPLC and analyzed by liquid chromatography-mass spectrometry. We identified 78 amino acid sequences from self proteins presented in the lungs of sarcoidosis patients, some of which were well-known autoantigens such as vimentin and ATP synthase. For the first time, to our knowledge, we have identified HLA-bound peptides presented in vivo during an inflammatory condition. This approach can be extended to characterize HLA-bound peptides in various autoimmune settings.

Changes in immune regulation in response to examination stress in atopic and healthy individuals

Background Stress can aggravate the allergic inflammation, but determinants of disturbed immune regulation are largely unknown.