Noriharu Shijubo

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.

Serum and BAL Clara cell 10 kDa protein (CC10) levels and CC10-positive bronchiolar cells are decreased in smokers

Cigarette smoking has diverse effects on the structure and function of the lung. Smoking appears to reduce the levels of Clara cell 10 kDa protein (CC10) in the alveolar lining fluid, but the influence of smoking serum on CC10 levels is still debated, and it has not been clear whether smoking reduces the number of CC10-producing lung cells. The aims of this study were to clarify the influence of smoking on CC10 levels in the alveolar lining fluid and bloodstream, and on the number of CC10-producing lung cells. CC10 concentrations were measured in sera and bronchoalveolar lavage (BAL) fluids, by means of enzyme-linked immunosorbent assay using monoclonal and polyclonal antibody, and the immunohistochemical expression of CC10 was examined in the lungs of nonsmokers and smokers using the monoclonal antibody, TY-5, against CC10/human urinary protein-1. CC10 concentrations in sera and in BAL fluids from healthy smokers were significantly lower than in healthy nonsmokers. Immunohistochemical expression of CC10 was found exclusively in nonciliated bronchiolar epithelial cells. As compared to that of nonsmokers, the mean percentage of CC10-positive bronchiolar epithelial cells was significantly decreased in lung tissue specimens obtained from smokers who had normal results in pulmonary function tests. It was concluded that smoking reduces the proportion of Clara cell 10 kDa protein-producing bronchiolar epithelial cells, resulting in decreased levels of Clara cell 10 kDa protein in the lower respiratory tract and in the bloodstream. The protein is a new blood biochemical and immunohistochemical marker, reflecting structural changes in peripheral airways induced by cigarette smoking.

Serum Levels of Clara Cell 10-kDa Protein Are Decreased in Patients with Asthma

Abstract. Clara cell 10-kDa protein (CC10), the predominant product from nonciliated cells in the epithelial lining of bronchioles (Clara cells), has been shown to have immunomodulatory and antiinflammatory activity and may play a role in controlling airway inflammation. This study was designed to measure serum CC10 concentrations in healthy and asthmatic nonsmokers. Serum CC10 concentrations in asthmatic nonsmokers were significantly lower than in healthy nonsmokers. Asthmatic patients with a long duration of the disease (≥10 years) had significantly lower serum CC10 levels than those with a short duration of the disease (<10 years). There was no significant difference in serum CC10 levels in asthmatic patients between the time of the asthmatic attack and the stable condition. Serum CC10 levels may reflect decreased production of CC10 caused by remodeling of the small airways in asthma.

Circulating intercellular adhesion molecule-1 (ICAM-1) antigen in sera of patients with idiopathic pulmonary fibrosis

Intercellular adhesion molecule‐1 (ICAM‐1), a member of immunoglobulin supergene family with a five‐domain structure, is known to play an important role in inflammatory diseases. An ELISA was developed using two MoAbs against human ICAM‐1 in order to detect the soluble shedding ICAM‐1 antigen in sera. We measured levels of circulating ICAM‐1 antigen in sera of patients with idiopathic pulmonary fibrosis (IPF), pulmonary sarcoidosis, hypersensitive pneumonitis, bacterial and mycoplasmal pneumonia, and inflammatory diseases of other organs. The results clearly demonstrated that IPF had significantly high levels of circulating ICAM‐1 in sera as compared with other disorders or normal controls. Moreover, immunohistochemical analysis with MoAb against human ICAM‐1 disclosed that in IPF, the expression of ICAM‐1 was intensively enhanced on alveolar epithelial cells. These results suggest that ICAM‐1 may contribute to the pathogenesis of IPF.

Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPNs functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence‐specific reagents, along with the previously described anti‐OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD‐dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit α9 integrin‐dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell‐binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration. J. Cell. Biochem. 84: 420–432, 2002.

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.

Aberrant Appearance of Lung Surfactant Protein A in Sera of Patients with Idiopathic Pulmonary Fibrosis and Its Clinical Significance

Pulmonary surfactant protein A (SP-A) is known to be a major phospholipid-associated glycoprotein in pulmonary surfactant, which is specific to the lung. In this study, the SP-A concentrations in sera of patients with various lung diseases were determined using an enzyme-linked immunosorbent assay. Patients with idiopathic pulmonary fibrosis (IPF) and pulmonary alveolar proteinosis (PAP) exhibited prominently high concentrations of serum SP-A compared to those of other lung diseases and healthy volunteers, although there were significant increases in serum SP-A concentrations in patients with pulmonary tuberculosis, chronic pulmonary emphysema, diffuse panbronchiolitis and bacterial pneumonia compared to those of healthy volunteers. Successive measurement in 2 patients with IPF showed that serum SP-A levels reflect the disease activity of IPF. In patients with IPF, serum SP-A concentrations were significantly correlated with those of serum lactate dehydrogenase, whereas there were no significant correlations of serum SP-A concentrations with erythrocyte sedimentation rate, arterial oxygen saturation, vital capacity and carbon monoxide diffusing capacity. Determination of serum SP-A will contribute to diagnosing IPF and PAP, and may reflect the disease activity of IPF.

Clinical significance of vascular endothelial growth factor-C and vascular endothelial growth factor receptor 3 in patients with T1 lung adenocarcinoma

Vascular endothelial growth factor‐C (VEGF‐C) plays an important role in lymphangiogenesis and activates VEGF receptor‐3 (VEGFR‐3). Lymphatic spread is an important prognostic factor in patients with lung adenocarcinoma. The aim of the current study was to determine whether the expression of VEGF‐C and VEGFR‐3 correlates with clinicopathologic factors and prognosis in patients with TNM classification T1 lung adenocarcinoma.

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.

Lobar distribution of emphysema in computed tomographic densitometric analysis.

RATIONALE AND OBJECTIVES The aims of this study were to determine the extent of emphysema in individual lobes and to investigate whether the lobar distribution of emphysema influences pulmonary function. METHODS Helical CT and pulmonary function tests were performed in 50 emphysema patients. Percentages of low attenuation volume (extent of emphysema) were calculated for each lobe by using CT densitometric analysis. RESULTS The extent of emphysema of the whole lung in these patients was 44%. Airflow limitation (r = -0.82, P<0.0001) and residual volume (r = -0.52, P<0.01) were closely correlated with the extent of emphysema in both lower lobes. Diffusing capacity (r = -0.61, P<0.0001) was closely correlated with the extent of emphysema in both upper lobes. On the basis of the lobar distribution of emphysema as determined by CT densitometry, we divided these emphysema patients into predominantly upper-lobe disease and predominantly lower-lobe disease groups. The predominantly lower-lobe disease group had significantly greater severe airflow limitation (P<0.0001), greater residual volume (P<0.01), and greater total lung capacity (P<0.05) than did the predominantly upper-lobe disease group. CONCLUSIONS CT densitometry showed a distinct lobar distribution of emphysema. Pulmonary function is significantly different between predominantly upper- and lower-lobe emphysema groups.