Katsunori Shigehara

IL-12 and IL-18 Are Increased and Stimulate IFN-γ Production in Sarcoid Lungs

Sarcoidosis is a systemic chronic granulomatous disease of unknown cause. Recent investigations revealed that the cytokine profile in inflamed lesions of sarcoidosis is Th1 dominant. To obtain better immunopathologic understanding of sarcoidosis, we examined the expression of IL-12 and IL-18 and their roles in IFN-γ production in pulmonary sarcoidosis. Sarcoid cases had significantly elevated levels of IL-12 (p40 and p70) and IL-18 in bronchoalveolar lavage (BAL) fluids compared with healthy subjects. IL-12 p70 and IL-18 were immunohistochemically expressed in the epithelioid cells and giant cells of sarcoid granulomas. Significant induction of IFN-γ, IL-12 p70, and IL-18 was observed from sarcoid BAL fluid cells with LPS stimulation, whereas LPS tended to induce only IL-12 p70 in BAL fluid cells from healthy subjects. Sarcoid cases had significantly greater IFN-γ induction with LPS stimulation than healthy subjects did. IL-18 mRNA expression was observed in freshly isolated sarcoid BAL fluid cells as well as in LPS-stimulated sarcoid BAL fluid cells, but IFN-γ and IL-12 mRNA expression was observed only in LPS-stimulated BAL fluid cells. Treatment with anti-IL-12- and anti-IL-18-neutralizing Abs significantly inhibited IFN-γ production from LPS-stimulated BAL fluid cells of sarcoid cases. Coadministration of rIL-12 or rIL-18 induced greater IFN-γ production in sarcoid BAL fluid cells than in normal BAL fluid cells. We concluded that bioactive IL-12 and IL-18 were produced in sarcoid BAL fluid cells and synergistically induced IFN-γ production, indicating important cytokines in the Th1 response of sarcoidosis.

Interferon γ assay for detecting latent tuberculosis infection in rheumatoid arthritis patients during infliximab administration

In rheumatoid arthritis (RA) patients treated with infliximab (IFX), QuantiFERON-TB Gold (QFT-G), an interferon γ assay for diagnosing tuberculosis infection, was performed to compare its effectiveness to conventional diagnostic procedures (tuberculin skin test, imaging and medical history) in diagnosing latent tuberculosis infection (LTBI). QFT-G was measured bimonthly in 14 rheumatoid arthritis patients during IFX treatment. Seven of 14 patients were confirmed as LTBI positive by at least one method. Of these, four were positive on QFT-G during the study period, and two were positive before the start of IFX administration. For two of the four QFT-G-positive patients, LTBI was diagnosed only by QFT-G. The rate of agreement between QFT-G and conventional procedures was 64.3%. A total of 5% of QFT-G tests were impossible to judge due to decreased reactions in the positive control. These results suggest that QFT-G is able to detect LTBI in RA patients overlooked by conventional methods. Conventional procedures and QFT-G should be employed in parallel, and LTBI should be assumed when one technique gives a positive result.

Increased circulating interleukin-12 (IL-12) p40 in pulmonary sarcoidosis.

In sarcoidosis, a T helper 1 (Th1) response is an essential event and the up‐regulation of interleukin‐12 (IL‐12) has been detected in affected disease sites. In order to investigate the clinical usefulness of circulating IL‐12, we measured the serum concentrations of IL‐12 by ELISA and performed immunohistochemistry using specific MoAbs for IL‐12 in the lungs and scalene lymph nodes of patients with sarcoidosis. The serum concentration of IL‐12 p40 was detectable in all 45 patients with pulmonary sarcoidosis and 18 normal controls, whereas that of IL‐12 p70 was undetectable. The serum concentrations of IL‐12 p40 in pulmonary sarcoidosis were significantly higher than those of the normal controls, especially in cases with abnormal intrathoracic findings detected by chest roentogenogram. The serum concentrations of interferon‐γ (IFN‐γ) also increased compared with those of normal controls and there was a significant positive correlation between the serum concentrations of IL‐12 p40 and IFN‐γ. Furthermore, serum angiotensin‐converting enzyme (ACE) and lysozyme, which are known to be useful markers for disease activity in sarcoidosis, correlated well with the serum concentrations of IL‐12 p40. The positive 67Ga scan group (for lung field) had significantly elevated serum IL‐12 p40 levels compared with those of the negative group. No bioactivity of IL‐12 p70 was detected in three sarcoid cases sera by using the IL‐12 responsive cell line. Finally, the immunohistochemical approach revealed that IL‐12 p40 was expressed in the epithelioid cells and macrophages of sarcoid lungs and lymph nodes. We concluded that the production of IL‐12 p40 was far greater in the sera and we have demonstrated this to be a useful clinical marker for disease activity and the Th1 response in pulmonary sarcoidosis.

Soluble intercellular adhesion molecule‐1 (ICAM‐1) in sera and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis

ICAM‐1 plays an important role in inflammatory diseases. To assess level of soluble ICAM‐1 in the circulation and inflamed lesions, we measured levels of soluble ICAM‐1 in the circulation and bronchial veolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF) and with pulmonary sarcoidosis (PS) and of healthy volunteers (HV), and we also analysed ICAM‐I expression of BALF cells in some patients and HV. IPF patients had significantly higher levels of circulating ICAM‐1 than HV, while PS patients did not. By contrast, significantly increased levels of BALF soluble ICAM‐1 were found in PS patients compared with those of HV, but not in IPF patients. There were no significant differences in the proportions of ICAM‐1+ BALF lymphocytes in IPF patients, PS patients and HV. whereas significantly increased proportions of ICAM‐1 pulmonary alveolar macrophages were found in PS patients compared with those of HV, but not in IPF patients. There was a significant positive correlation of BALF soluble ICAM‐1 levels to BALF lymphocyte proportions in PS patients. Although the source of BALF soluble ICAM‐1 is unclear. BALF soluble ICAM‐1 appears to reflect the grade of local activity of sarcoidosis. An interesting discrepancy between soluble ICAM‐1 levels in the circulation and BALF was found in IPF patients, and this might be an important clue to an understanding of this disorder.

Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma.

Allergic rhinitis (AR), the most common allergic disorder of the airway, is often accompanied by bronchial asthma. However, little is known about the mechanism by which AR advances to AR comorbid with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results showed polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, the %Breg cells in total B cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Moreover, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that relative decrease in Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.

Circulating γδ-T-Cell-Receptor-Positive Lymphocytes in Sarcoidosis

We investigated phenotypic surface markers of peripheral blood lymphocytes including expression of γδ T cell receptor (TCRγδ) in 185 patients with sarcoidosis and 42 normal subjects. The pr

Soluble intercellular adhesion molecule-1 (ICAM-1) in sera and bronchoalveolar lavage (BAL) fluids of extrinsic allergic alveolitis.

ICAM‐1 plays AN important role in inflammatory diseases. We analysed ICAM‐1 expression on BAL fluid cells and measured soluble ICAM‐1 (sICAM‐l) concentrations in sera and BAL fluids from patients with extrinsic allergic alveolitis (EAA). We found significantly increased cellular ICAM‐1 expression on BAL fluid lymphocytes and alveolar marcrophages, and significantly increased values of circulating and BAL fluid sICAM‐l in EAA patients compared with controls. Successive measurement showed prompt decrease of both sICAM‐1 values in EAA patients during periods when antigen exposure was prevented. In BAL fluids from EAA patients. sICAM‐l values significantly correlated to neutrophil and ICAM‐1+ lymphocyte counts. In EAA patients, circulating and BAL fluid sICAM‐l values has significant negative correlations to values of carbon monoxide diffusing capacity and to time intervals between last episode and sample collection. However, these values had significant positive correlation to values of alveolar‐arterial oxygen pressure difference. In EAA, antigen exposure appears to induce cellular ICAM‐1 expression on BAL fluid cells, and also appears to up‐regulate shedding of ICAM‐1 in the alveolar lining fluid and in the circulation. The sICAM values appear to reflect disease activity of EAA.

Cutting Edge: A Critical Role of Lesional T Follicular Helper Cells in the Pathogenesis of IgG4-Related Disease

IgG4-related disease (IgG4-RD) is a newly recognized systemic chronic fibroinflammatory disease. However, the pathogenesis of IgG4-RD remains unknown. To determine the pathophysiologic features of IgG4-RD, we examined T follicular helper (Tfh) cells in lesions and blood from patients with IgG4-RD. Patients with IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) showed increased infiltration of Tfh cells highly expressing programmed death 1 and ICOS in submandibular glands. Tfh cells from IgG4-DS submandibular glands had higher expression of B cell lymphoma 6 and a greater capacity to help B cells produce IgG4 than did tonsillar Tfh cells. We also found that the percentage of programmed death 1hi circulating Tfh cells in IgG4-DS patients was higher than that in healthy volunteers and was well correlated with clinical parameters. Our findings indicate that anomalous Tfh cells in tissue lesions of IgG4-RD have features distinct from those in lymphoid counterparts or blood and potentially regulate local IgG4 production in IgG4-RD.

Immunolocalization of extracellular matrix proteins and integrins in sarcoid lymph nodes.

Abstract To improve our understanding of the role of extracellular matrix (ECM) proteins and integrins during the processes of granuloma formation in sarcoidosis, we examined the distribution of ECM proteins and the expression of integrins in sarcoid lymph nodes by immunohistochemical methods. We also examined the expression of transforming growth factor-β1 (TGF-β1), which is one of major regulators for synthesis of ECM proteins. Most ECM proteins were detected in the periphery of the granulomas in a concentric pattern, and fibronectin was diffusely detected from an early to a regressive stage. Compared with normal lymph nodes, most β1-integrin subfamilies (α1, α4, α5 and α6) were more strongly expressed on lymphocytes around the granulomas. Epithelioid cells exhibited strong expression of the α5 molecule. Fibroblasts exhibited the expression of the α2 and α5 molecules surrounding ECM proteins. The α5β1 molecule had a distribution similar to that of fibronectin. TGF-β1 was detected in epithelioid cells throughout the various evolutional stages and its expression was especially marked in mature granulomas. Interaction of fibronectin and the α5β1 molecule may have an important role in the process of formation of sarcoid granuloma. The expression of TGF-β1 may be involved in the regression of sarcoid granuloma by initiating fibrosis and atrophy of epithelioid cells.

Bob1 limits cellular frequency of T‐follicular helper cells

T follicular helper (Tfh) cells are involved in specific humoral immunity at initial and recall phases. The fact that the transcription repressors B‐cell lymphoma‐6 and Blimp‐1 determine lineages of Tfh cells and other types of effector CD4+ T cells, respectively, suggests that there are unique mechanisms to establish Tfh‐cell identity. In this study, we found that Tfh cells preferentially express the transcriptional coactivator Bob1. Bob1 of Tfh cells was dispensable for the expression of B‐cell lymphoma‐6 and the functional property of the cells for B cell help. However, upon initial immunization of foreign antigens, the percentages of Tfh cells in Bob1−/− mice were much higher than those in wild‐type (WT) mice. In addition, expansion of Tfh cells within Bob1−/−CD4+ T cells transferred into WT mice revealed that the high frequency of Tfh cells was caused by a T‐cell‐intrinsic mechanism. These findings were further supported by the results of in vitro studies demonstrating that Bob1−/− Tfh cells had greater proliferative activity in response to stimuli by CD3/CD28 monoclonal antibody and were also refractory to CD3‐induced cell death in comparison to WT Tfh cells. These results suggest that Tfh cells harbor a Bob1‐related mechanism to restrict numerical frequency against stimulation of TCRs.