Significance of increase in lipocortin-1 gene transcripts in the blood monocytes of patients with pulmonary sarcoidosis.

BACKGROUND AND AIM OF THE WORK: Lipocortin-1 (also known as annexin-1) is upregulated by corticosteroids and plays a prominent part in many of their anti-inflammatory actions. In our previou investigation, lipocortin-1 gene expression in blood monocytes was higher in sarcoidosis patients tha in healthy subjects or IPF patients. In this study, we examined the amounts of lipocortin-1 gene tran scripts [at baseline and after stimulation by synthetic adrenocorticotrophic hormone (ACTH)] in the blood monocytes of sarcoidosis patients, and then we analyzed the relationships between these amount and several clinical indexes. METHODS: Serum and blood monocyte samples were prepared from 21 sar coidosis patients before and after ACTH stimulation. The amounts of lipocortin-1 and beta-actin gene tran scripts obtained from monocytes were quantified by RT-PCR. The patients were prospectively followe for at least 2.5 years. RESULTS: 1. Serum cortisol was increased in all patients after synthetic ACTH in jection. 2. Lipocortin-1 gene transcripts tended to increase in parallel with increases in serum cortisol but the relationship was not significant. 3. The amounts of lipocortin-1 gene transcripts induced by syn thetic ACTH were related to two indexes of disease activity/extension: the presence of parenchymal lesions (p = 0.018), and the presence of extrathoracic lesions (p = 0.043). 4. After 3 years of follow-up the patients with higher basal amounts of lipocortin-1 gene transcripts in monocytes showed improve ment of sACE activity and FEV1/FVC%. CONCLUSION: The amounts of lipocortin-1 gene transcripts induced by synthetic ACTH were related to the presence of parenchymal lesions, while their basa amounts correlated with either improvement of sACE activity or stabilization of FEV1/FVC%.

Significance of the interleukin-1 receptor antagonist/interleukin-1 beta ratio as a prognostic factor in patients with pulmonary sarcoidosis.

BACKGROUND: Various factors such as serum angiotensin-converting enzyme (sACE) activity, bronchoalveolar lavage (BAL) fluid lymphocyte percent, CD4/CD8 ratio, and shadows on chest radiograph have been identified as indexes of disease activity in patients with sarcoidosis. However, it remains to be confirmed whether these factors can predict clinical outcomes. OBJECTIVE: To examine whether the interleukin-1 receptor antagonist (IL-1ra)/IL-1 beta ratio can predict the clinical course, we prospectively followed the clinical courses of 30 patients with pulmonary sarcoidosis 4 years after measurement of immunoreactive amounts of IL-1ra or IL-1 beta in the culture supernatants obtained from BAL fluid macrophages. METHODS: Immunoreactive amounts of IL-1ra or IL-1 beta were measured using ELISA. Changes in pulmonary function, sACE activity, and shadows on chest radiographs during observation periods were evaluated as markers of changes in disease activity. RESULTS: We found that the patients whose shadows on chest radiographs showed improvement had a higher molar IL-1ra/IL-1 beta ratio than the patients whose shadows persistently remained 4 years after BAL examination (p < 0.05). The molar ratio was found to be positively correlated with improvement of percent vital capacity (p < 0.05) and negatively correlated with the ratio of sACE activity at the time of the last observation to sACE activity at the time of BAL (sACE(LAST)/sACE(BAL), p < 0.01). The sACE(LAST)/sACE(BAL) ratio was significantly lower in patients whose shadows on chest radiographs decreased than in those whose shadows remained unchanged (p < 0.005). CONCLUSION: The IL-1ra/IL-1 beta ratio in the BAL fluid macrophage culture supernatants in patients with pulmonary sarcoidosis could be a useful marker in predicting the persistence of granulomatous lesions (chronicity).

Glucocorticoid-induced contractility and F-actin content of human lung fibroblasts in three-dimensional culture.

Fibroblast contractility plays a useful role in the wound healing process but contributes to architectural distortion in the lungs. Glucocorticoids (GCs) have been reported to reduce dermal fibroblast contractility, which may result in delaying wound healing, but the effects on lung fibroblasts are unknown. In this study, we examined how human lung fibroblast contractility is altered in the presence of GCs. Lung fibroblast cell lines (n = 5) were established from normal parts of surgically resected lung tissue. The effects of GCs on contractility were investigated with a type I collagen gel contraction assay. Filamentous actin (F-actin) content was detected by confocal microscopy and measured with a fluorescent phalloidin binding assay. GCs augmented fibroblast contraction in a concentration-dependent manner, with an approximate EC(50) of 1.8 x 10(-8) M, whereas other steroid derivatives had no effects. GC contractility needed de novo protein synthesis. The GC-induced increase in contractility was found to be consistent with an increase in F-actin content. In conclusion, lung fibroblast contractility was enhanced with GCs through an upregulation of lung fibroblast F-actin.

Proinflammatory or regulatory cytokines released from BALF macrophages of healthy smokers.

BACKGROUND: Chronic smoking influences bronchoalveolar lavage fluid (BALF) cell profiles in healthy subjects, which may alter profiles of inflammatory and regulatory cytokines. OBJECTIVE: We focused on the evaluation of smoking-related changes in the amounts of cytokines released from BALF macrophages. METHODS: We measured the amounts of immunoreactive culture supernatants by using ELISA. RESULTS: The amounts of IL-1 receptor antagonist (IL-1ra) were lower in smokers [n = 10, median 22.1 ng/ml, 25th and 75th percentiles (18.7-39.1)] than in nonsmokers [n = 10, 48.6 (39.2-66.1), p = 0.010]. In smokers, lipopolysaccharide stimulation revealed decreases in the amounts of interleukin-6 (IL-6) [nonsmokers: 2.1 ng/ml (0.68-5.4 vs. smokers: 0.5 (0.03-0.87), p = 0.049] as well as IL-1ra [nonsmokers: 69.2 ng/ml (48.3-83.8) vs. smokers: 27.3 (17.2-56.7), p = 0.028]. A delay in release from intracellular storage was not the cause of the reduced amounts of IL-1ra. In addition, interleukin-1beta (IL-1beta) was positively correlated with IL-6 and granulocyte macrophage colony-stimulating factor in nonsmokers, but not in smokers. Furthermore, the decreases in IL-1ra and interleukin-8 were correlated with the increase in the number of BALF macrophages in smokers, but not in nonsmokers. CONCLUSIONS: Chronic smoking caused changes in the profiles of cytokines released from BALF macrophages in healthy subjects. Decreases in the amounts of regulatory cytokines, but not prominent changes in the amounts of inflammatory ones, were characteristic.

Angiotensin II receptor on BALF macrophages from Japanese patients with active sarcoidosis.

BACKGROUND: Serum angiotensin converting enzyme (ACE) activity and its changes are important markers for disease activity in patients with sarcoidosis. We earlier reported that ACE and its enzymatic product, angiotensin II (A-II), might play a role in maintaining macrophage/T lymphocyte alveolitis by enhancing an accessory function of macrophages in chronic active cases with sarcoidosis. AIM OF THE WORK AND METHODS: We examined whether A-II receptor is present on BALF macrophages by a receptor binding assay using 125I-labeled A-II, and by amplification of A-II receptor gene transcripts using RT-PCR methods and quantification of the amounts of transcripts by HPLC. RESULTS: The receptor binding assay suggested that specific binding of A-II to BALF macrophages was somewhat more prevalent in active sarcoidosis than in controls. A specific band for A-II receptor was detected by RT-PCR. A-II receptor gene expression was standardized as the ratio to beta-actin. An increased ratio was shown in active sarcoidosis (0.70 +/- 0.19; n = 14) compared to inactive cases (0.14 +/- 0.06; n = 5; p = 0.023) and healthy subjects (0.15 +/- 0.09; n = 5, p = 0.029). The ratio correlated positively with the percentage of BALF T lymphocytes (r = 0.60, p = 0.0056), and negatively with BALF macrophages (r = 0.60, p = 0.0055). No difference was detected between nonsmokers and smokers. CONCLUSION: The amounts of A-II receptor gene expression of BALF macrophages correlated with disease activity in patients with pulmonary sarcoidosis.

Lipocortin I gene expression is higher in blood monocytes than in BAL fluid macrophages in patients with pulmonary sarcoidosis.

BACKGROUND AND AIM OF WORK: Lipocortin I, induced by corticosteroids in vivo is considered to be related to cell activation and differentiation in mononuclear phagocytes, key cells in the process of epithelioid cell granuloma formation in sarcoidosis. Therefore, we focused on the expression of lipocortin I gene in mononuclear phagocytes in patients with sarcoidosis. METHOD: The amounts of transcripts of lipocortin I gene obtained from both BAL fluid macrophages and blood monocytes were measured by RT-PCR and HPLC, and then standardized as the ratio of lipocortin I/beta-actin for comparison. RESULTS: 1. Smoking did not affect the expression of lipocortin I gene in BAL fluid macrophages and monocytes. 2. There were no differences in the amounts of expression in BAL fluid macrophages between patient groups (nonsmokers and smokers), or between patients and healthy subjects. 3. The gene expression in BAL fluid macrophages was higher than that in monocytes in patients with IPF (macrophages 1.23 +/- 0.48, vs monocytes 0.50 +/- 0.38, p < 0.05) and healthy subjects (nonsmokers: 1.66 +/- 1.17, vs 0.34 +/- 0.26, p < 0.05, smoker: 1.98 +/- 0.96, vs 0.34 +/- 0.48, p < 0.05, but sarcoidosis patients showed a higher expression of lipocortin I gene even in monocytes (2.62 +/- 1.87, vs 2.66 +/- 2.21). CONCLUSION: The increase in lipocortin I gene expression in monocytes was a disease-related finding in sarcoidosis.

Quantitative evaluation of the IL-1 beta and IL-1 receptor antagonist obtained from BALF macrophages in patients with interstitial lung diseases.

Our previous reports demonstrated the concomitant release of IL-1 beta and IL-1 inhibitory activity in the culture supernatants of BALF macrophages in both healthy subjects and patients with interstitial lung diseases. IL-1 inhibitory activities decreased in healthy smokers (HS), and patients with sarcoidosis (Sar), or idiopathic pulmonary fibrosis (IPF), compared with those in healthy nonsmokers (HNS), though an increase in IL-1 beta release was not detected. IL-1 inhibitory activity was mainly characterized as IL-1 receptor antagonist (IL-1ra). In this study, we confirmed a decrease in IL-1ra in terms of the amounts of protein (enzyme-linked immunoassay) and gene transcripts (reverse transcriptase polymerase chain reaction followed by high performance liquid chromatography). Imbalance between IL-1ra and IL-1 beta was expressed as a molar ratio of IL-1ra/IL-1 beta protein: (Sar; 4.20 +/- 2.06, IPF; 4.26 +/- 3.41, HS; 3.44 +/- 3.09 versus NS 8.33 +/- 2.77: P < 0.001). These results were similar in terms of the amounts of gene transcripts. In conclusion, the imbalance of IL-1 beta and IL-1ra production was confirmed at three levels: biological activity, amounts of protein, and gene transcript obtained from BALF macrophages in chronic inflammatory processes in the lungs.

Antifungal antibiotic benanomicin A increases susceptibility of Candida albicans to phagocytosis by murine macrophages.

Benanomicin A is an antifungal antibiotic produced by Actinomadura spadix. In the present study, we investigated the effect of benanomicin A on the phagocytosis of Candida albicans by murine peritoneal macrophages and on the cell-surface hydrophobicity (CSH) of C. albicans. Although pretreatment of macrophages with benanomicin A had no effect on the phagocytosis, addition of benanomicin A to the culture of macrophages and Candida cells increased the susceptibility of Candida cells to the phagocytosis by the macrophages. Pretreatment of Candida cells with benanomicin A also increased the susceptibility of Candida cells to the phagocytosis. When Candida cells were mixed with benanomicin A, the antibiotic bound irreversibly to Candida cells. These data suggest the possibility that the increased susceptibility of Candida cells to the phagocytosis is mediated by the binding of benanomicin A to Candida cells. Examination of physicochemical property of Candida cell surface showed that the CSH of Candida cells significantly decreased by the treatment with benanomicin A. Thus, binding of benanomicin A to Candida cells may induce biochemical/physicochemical alternation of the surfaces, so that they become more susceptible to phagocytosis by murine macrophages. These properties of benanomicin A, along with its antifungal activity, seem to be beneficial in the treatment of fungal infections.

T cell receptor (TCR) V gene segment use in HLA-typed Japanese healthy subjects.

The expression of 13 different alpha and beta V gene segments of the T cell receptor for antigen (TCR) was examined, using V gene-specific MoAbs, on human peripheral blood T lymphocytes from 32 healthy Japanese subjects. In addition, to examine associations between TCR V gene products and HLA alleles, the HLA class I and class II types of all subjects were serologically determined. The reactivities of the anti-TCR V-specific MoAbs were, with some significant exceptions, similar to those previously described in healthy Caucasian subjects. We found a non-random V gene usage as well as a statistically significant bias of the expression of eight V beta gene products towards the CD4+ subpopulation, and a significant skewness in the usage of V alpha 12 towards the CD8+ population. Some subjects showed increased reactivities (above 10%) of certain MoAbs, mainly in the CD8+ subpopulation. We found no distinct correlation between any certain HLA class I or II allele and TCR V gene usage in the CD8+ or CD4+ subpopulations, respectively. In conclusion, the pattern of anti-TCR V-specific MoAb reactivities found in CD4+ and CD8+ subsets of peripheral blood lymphocytes of healthy Japanese subjects was in general found to match that previously described in healthy Caucasian subjects.

T-cell receptor V gene expression in HLA-typed Japanese patients with pulmonary sarcoidosis.

The clinical features of sarcoidosis vary in different ethnic groups, suggesting that different genetic or environmental backgrounds influence the disease. In Scandinavian sarcoidosis patients, we have previously described a correlation between lung-accumulated CD4+ T cells expressing the T-cell receptor (TCR) V alpha 2.3 gene segment and a particular HLA type (DR3[17],DQ2). For purposes of comparison, we have in this study investigated TCR V gene usage and gamma delta TCR expression in CD4+ and CD8+ T cells in bronchoalveolar lavage (BAL) fluid and peripheral blood in an ethnically distinct and homogenous group of individuals consisting of Japanese sarcoidosis patients and healthy controls. We used a panel of 13 monoclonal antibodies (Mab) specific for different TCR V genes, which altogether stained approximately 50% of the T cells, and triple staining techniques with flow cytometry. The patients and controls were also HLA-typed. Our results show a high degree of expression of gamma delta TCR in peripheral blood T cells of close to half of the patients. Expansions of T-cell subsets were readily detected in the CD8+ T-cell population, while a more homogenous staining pattern was found in the CD4+ T-cell population. These findings show the importance of ethnic origin and environment in discussions of TCR V gene usage.

Streptomyces ATP nucleotide 3'-pyrophosphokinase-gene cloning and sequence analysis.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-beta, gamma-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.

TNF alpha mRNA, but not IL-1 beta, is differentially expressed in lung macrophages of patients with active pulmonary sarcoidosis.

In pulmonary sarcoidosis or experimental granuloma formation, interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) seem to play important roles during the inflammatory process. In order to examine whether IL-1 beta or TNF alpha mRNA expression in lung macrophages relates to disease activity or clinical course, ten cases with pulmonary sarcoidosis were divided into two groups: five cases with disease duration of more than 10 years (14.6 +/- 4.4 yrs; group A), and 5 cases with a duration of less than 3 years (1.7 +/- 1.1 yrs; group B). All cases showed both abnormal chest X-rays and elevated serum angiotensin converting enzyme activities. We compared these ten cases with 12 healthy subjects (6 nonsmokers: NS and 6 current smokers: S), and 5 cases with idiopathic pulmonary fibrosis (IPF) as disease control. Lavage macrophages were purified using the rosette forming method followed by plastic adhesion for one hour. Thereafter, RNA was extracted according to the AGPC method and amplified by the reverse transcription-polymerase chain reaction (RT-PCR). The results showed that IL-1 beta mRNA was detected in all samples studied, but TNF alpha mRNA expression was different among the groups: 5/5 (100%) in group A, 1/5 (20%) in group B, 5/5 (100%) in IPF, and 12/12 (100%) in healthy subjects. A constitutive expression was seen in healthy controls. On the other hand, no detection of TNF alpha mRNA was shown in pulmonary sarcoidosis. This fact may relate to a spontaneous regression of inflammation in sarcoidosis, as a substantial number of cases in group B may in time regress spontaneously.

Nucleotide 2',3'-cyclic monophosphokinase from actinomycetes.

Streptomyces nucleotide 3'-pyrophosphokinase does not only transfer the 5'-beta, gamma-pyrophosphoryl group of ATP, ATP 3'-pyrophosphate or dATP to a variety of nucleotides at the 3'-OH site, but also adds 2',3'-cyclic terminal monophosphate to some suitable nucleotides with the use of diadenosine 5',5'-polyphosphates (n = 3-5). Examples are pA greater than p, ppA greater than p, pG greater than p, CpG greater than p, etc.