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Rationale: The CD40 protein plays important roles in cell-mediated and humoral immune responses, especially in immunoglobulin class-switching to IgE. Objectives: We tested the association of CD40 polymorphisms with the risk of asthma and the level of serum IgE and investigated the functional effect of associated polymorphisms on the expression of CD40. Methods: We identified 17 CD40 single-nucleotide polymorphisms (SNPs) in the Korean population by direct sequencing, and we genotyped 7 of these in 487 subjects with asthma and in 161 normal subjects. Cell-surface expression of CD40 for B-cell lines of various SNP genotypes was measured using flow cytometry. The effects of SNPs in the promoter and 5'-untranslated regions (UTRs) of CD40 were assessed using pGL3 luciferase and enhanced green fluorescent protein (EGFP) reporter systems, respectively. Measurements and Results: None of the SNPs was associated with asthma risk, but total serum IgE levels were associated with the -580G>A and -1C>T polymorphisms in subjects with asthma (p = 0.007 and 0.005, respectively). The total amount of IgE was highest in the -580A or -1C homozygotes. More CD40 was expressed in B cells with the -1C allele than in those with the -1T allele (p < 0.001). EGFP expression from the CD40 5'-UTR-EGFP construct was higher for the -1C allele than the -1T allele. The -580G>A SNP did not affect promoter activity, even after IFN-gamma stimulation. Conclusions: CD40 gene polymorphisms exert a genetic effect on IgE production in patients with asthma through translational regulation of CD40 expression on B cells. [ABSTRACT FROM AUTHOR]

DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription.

Lung cancer is recently regarded as an overhealed inflammatory disease. Serum amyloid A (SAA) is known as an acute phase protein, but it is likely involved in the cancer pathogenesis. We identified both SAA1 and SAA2 in the pooled sera of lung cancer patients but not in the healthy control, by LC-MS/MS analysis. We found that about 14-fold higher levels of SAA in lung cancer patients' sera and plasma compared to healthy controls by ELISA using total 350 samples (13.89 ± 37.18 vs 190.49 ± 234.70 ug/mL). The SAA levels were also significantly higher than in other pulmonary disease or other cancers. An immunohistochemical study using tissue microarray showed that, unlike other cancer tissues, lung cancer tissues highly express SAA. Further in vitro experiments showed that SAA is induced from lung cancer cells by the interaction with THP-1 monocytes and this, in return, induces MMP-9 from THP-1. In in vivo animal models, overexpressed SAA promoted Lewis lung carcinoma (LLC) cells to metastasize and colonize in the lung. Our data suggest that a higher concentration of SAA can serve as an indicator of lung adenocarcinoma and represents a therapeutic target for the inhibition of lung cancer metastasis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is caused by alterations in expression of proteins involved in multiple pathways, including matrix deposition, inflammation, injury, and repair., Objectives: To understand the pathogenic changes in lung protein expression in IPF and to evaluate apolipoprotein (Apo) A-I as a candidate therapeutic molecule., Methods: Two-dimensional electrophoresis was adopted for differential display proteomics. Reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemical staining, and ELISA were performed for identification and quantitative measurement of Apo A-I in bronchoalveolar lavage fluids from subjects with IPF and experimental bleomycin-induced mice., Measurements and Main Results: Sixteen protein spots showed differences in relative intensity between IPF (n = 14) and healthy control subjects (n = 8). Nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed increase of haptoglobulin and decrease of alpha(1)-antitrypsin, alpha(1)-antichymotrypsin, macrophage capping protein, angiotensinogen, hemoglobin chain B, Apo A-I, clusterin, protein disulfide isomerase A3, immunoglobulin, and complement C4A in IPF compared with normal control subjects (P = 0.006-0.044). Apo A-I concentrations were lower in bronchoalveolar lavage fluids from subjects with IPF (n = 28) than in normal control subjects (n = 18; P < 0.01). In bleomycin-treated mice, Apo A-I protein in BALF was lower than that in sham-treated control animals. Immunohistochemical analysis showed positive staining on intraalveolar macrophages and epithelial cells of the lungs. Intranasal treatment with Apo A-I protein reduced the bleomycin-induced increases in number of inflammatory cells and collagen deposition in sham-treated mice in a dose-dependent manner., Conclusions: Alterations of several inflammatory and antiinflammatory proteins in the lungs may be related to the pathogenesis of IPF, and local treatment with Apo A-I is very effective against the development of experimental lung injury and fibrosis.

The gene induced in response to Cd exposure in microalga Nannochloropsis oculata was examined in this investigation. In order to isolate this gene by using nested PCR, degenerated primers were designed on the basis of highly conserved regions of the amino acid sequences of various phytochelatin synthase. The size of the nested PCR amplification product from Nannochloropsis oculata by using two degenerated primers was measured to be 266 bp. The 266 bp DNA fragment was sequenced, and it might be induced by Cd exposure in microalga Nannochloropsis oculata.

Background: Chitinase may play a role in regulating allergic diseases., Objective: We studied the role of chitinase in a mouse model exposed to diesel exhaust particles (DEP). Mice were exposed to intranasal DEP (0.6 mg/mL) for 5 days and challenged with aerosolized DEP (6 mg/m(3)) on days 6-8. Enhanced pause (Penh), as an airway obstruction marker, was measured on day 9, and bronchoalveolar lavage (BAL) fluid and lung tissues were collected on day 10. The expression of Ym1 and Ym2 mRNA was assessed in lung tissue extracts by reverse transcription-polymerase chain reaction., Results: DEP induced significant increases in methacholine-induced Penh and IL-4 levels in BAL fluid relative to the control group. Peribronchial and perivascular inflammatory cell infiltrates were prominent in the DEP group. DEP induced Ym1 and Ym2 mRNA expression in lung tissue extracts relative to the control group., Conclusion: These results demonstrate that DEP induced airway hyperresponsiveness and Ym mRNA expression via a Th2 cell-biased response, suggesting that chitinase may play an important role in airway inflammation and responsiveness upon exposure to DEP in a mouse model, and may therefore be involved in regulating allergic diseases.

Interleukin (IL)-5 and eotaxin families regulate the development of eosinophilic inflammation of asthma in a co-operative manner. The exposure to airborne lipopolysaccharide (LPS) induces varying degrees of airflow obstruction and neutrophilic airway inflammation. Production of IL-5 and eotaxin subfamily chemokines was analysed in response to Dermatophagoides pteronyssinus allergen (D.p.) according to the presence of specific IgE to D.p., and investigated the mechanism underlying their LPS-mediated regulation of these cytokines in response to the specific allergen. Peripheral blood cells (PBCs) from asthmatics with (group 1) or without (group 2) specific IgE to D.p. and from non-asthmatics with (group 3) or without (group 4) were stimulated with D.p. or LPS. For LPS-mediated inhibition of IL-5 and eotaxin-2 production, LPS-induced cytokines were added to the D.p.-stimulated PBCs. IL-5 and eotaxin-2, but not eotaxin-1 and 3, were significantly increased by D.p.-stimulated-PBCs from group 1, while only eotaxin-2 was elevated in group 3. Eotaxin-2 production was found in monocytes and correlated with the level of specific IgE to D.p. LPS treatment resulted in the decrease in eotaxin-2 and IL-5 production by the D.p.-stimulated PBCs. LPS-induced IL-10 completely inhibited D.p.-stimulated production of eotaxin-2 and IL-5. The differential responses of the eotaxin family to specific antigens suggest that the predominant role of eotaxin-2 and LPS may attenuate eosinophilic inflammation by inhibiting IL-5 and eotaxin-2 synthesis through IL-10 production.

Particle inhalation-induced lung inflammation acts as an adjuvant to allergens or respiratory viral infection in a process that is mediated by macrophages and epitheliums. The production of interleukin (IL)-4 and IL-13 by activated T cells is involved in the augmentation of Th2-type immune responses to particles, and IL-25 induces the synthesis of IL-4 and IL-13. However, whether IL-13 and IL-25 are directly regulated by particle instillation in the lung has not been studied. The aim of this study was to reveal particle induction of IL-13 and IL-25 in the lung. TiO(2) instillation potently induced the mRNA expression for IL-25 and IL-13 in lung tissue extracts 24 h after treatment, as compared with the sham group. Immunostaining for IL-25 and IL-13 showed strong positivity for macrophages in the inflammatory lung lesions of TiO(2)-treated rats. The alveolar macrophages expressed IL-25 and IL-13 24 h after in vitro stimulation with TiO(2) particles in dose- and time-dependent manners, with maximal induction at 24 and 48 h after stimulation, respectively. The sequence of the rat IL-25 gene is 95% homologous with the mouse IL-25 gene. These findings indicate that alveolar macrophages play an important role in particle-induced lung inflammation via direct induction of IL-13 and IL-25 production.

Background: Th2 cytokines play pivotal roles in allergic inflammation, including eosinophilia, and their actions are antagonized by Th1 cytokines, conferring them therapeutic potential., Methods: In this study, we examined the ability of a number of cytokines to suppress the activation of eosinophils that function as effector cells for allergic airway diseases., Results: Interleukin (IL)-5, IL-6, and tumor necrosis factor (TNF) induced an eosinophil shape change, whereas interferon (IFN)-gamma significantly inhibited the shape change. Other cytokines, including IL-1beta, IL-4, IL-10 and IL-13, had little or only slightly enhancing or reducing effects on the shape change. We further analyzed the IFN-gamma effect, showing that pretreatment with IFN-gamma strongly suppressed IL-5-induced eosinophil shape change, and cycloheximide (CHX) abrogated the suppression by IFN-gamma, suggesting that new protein synthesis is required for the inhibitory effect by this cytokine. In agreement with these results, IFN-gamma blocked the eosinophil migration and ERK phophorylation induced by IL-5, and the addition of CHX restored eosinophil chemotaxis., Conclusions: Collectively, IFN-gamma may attenuate eosinophilic inflammation by directly negating eosinophil mobilization., (Copyright (c) 2005 S. Karger AG, Basel.)

Eotaxin1 plays a pivotal role in eosinophil-associated inflammation. Previously, we demonstrated 14 single-nucleotide polymorphisms (SNPs) in the human eotaxin1 gene and the association between the EOT+67G>A allele and the level of IgE. In this study, we investigated the association between the SNPs and plasma eotaxin1 levels, peripheral blood eosinophil counts, and PC20 methacholine values in normal and asthmatic subjects, and the effects of SNPs on the process of eotaxin1 production. The EOT-576C>T and EOT-384A>G polymorphisms and haplotypes (ht1 and ht4) were significantly associated with plasma eotaxin1 levels in the asthmatics (p < 0.001-0.040). The log [plasma eotaxin1] values correlated with the log [serum total IgE] values in the asthmatics and the normal controls (p = 0.012 and p = 0.004, respectively), and with the log [PC20 methacholine] values in the asthmatics (p = 0.014). A DNA-protein complex was formed with EOT-384A>G, but not with the other SNPs of the promoter. The interaction was stronger with the minor allele than with the common allele, and was reduced upon TNF-alpha exposure. TNF-alpha-stimulated PBMCs from the asthmatics with the minor allele homozygote expressed significantly lower levels of eotaxin1 mRNA than those from individuals with the common allele. The EOT+67G>A polymorphism, which substitutes alanine with threonine, did not affect eotaxin1 production or activity. Our data suggest that the EOT-384A>G SNP participates in the regulation of eotaxin1 expression by providing a potential binding site for a repressor, and that the ANOVA of EOT-384A>G may predict asthma phenotypes.

Chemokine (C-C motif) ligand 24 (CCL24, eotaxin-2) is a CC chemokine that recruits and activates cells bearing the CC chemokine receptor 3, which play a major role in asthma. Previously, we observed a significant association between a single nucleotide polymorphism (SNP) in eotaxin-2 (CCL24+1272A--> G) and a lower risk of asthma. Consequently, this study has followed up on those genetic effects by evaluating the association between the SNP and plasma eotaxin-2 concentration in 172 asthmatics and 135 normal controls. Asthmatics had significantly higher plasma eotaxin-2 levels than did normal controls (P=0.02). The SNP (CCL24+1272A--> G) and two haplotypes (ht2 and ht6) were strongly associated with plasma eotaxin-2 levels in asthmatics (CCL24+1272A--> G: P=0.006, ht2: P=0.006, and ht6: P=0.002). The CCL24+1272A--> G allele and the ht2 and ht6 haplotypes showed a gene-dose effect on the plasma eotaxin-2 levels in asthmatics (P=0.005-0.032). In conclusion, the susceptibility of patients with asthma to high eotaxin-2 production may be due to genetic effects of the CCL24+1272A--> G polymorphism, ht2 and ht6 haplotypes.

Microglia, the major immune effector cells in the CNS, become activated when the brain suffers injury. In this study, we observed that prothrombin, a zymogen of thrombin, induced NO release and mRNA expression of inducible NO synthase, IL-1beta, and TNF-alpha in rat brain microglia. The effect of prothrombin was independent of the protease activity of thrombin since hirudin, a specific inhibitor of thrombin, did not inhibit prothrombin-induced NO release. Furthermore, factor Xa enhanced the effect of prothrombin on microglial NO release. Kringle-2, a domain of prothrombin distinct from thrombin, mimicked the effect of prothrombin in inducing NO release and mRNA expression of inducible NO synthase, IL-1beta, and TNF-alpha. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-kappaB in a similar pattern. NO release stimulated by either was similarly reduced by inhibitors of the extracellular signal-regulated kinase pathway (PD98059), p38 (SB203580), NF-kappaB (N-acetylcysteine), protein kinase C (Go6976, bisindolylmaleimide, and Ro31-8220), and phospholipase C (D609 and U73122). These results suggest that prothrombin can activate microglia, and that, in addition to thrombin, kringle-2 is a domain of prothrombin independently capable of activating microglia.

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Kringles are triple-disulfide-loop folding domains, which are found in several other blood proteins. In this study, we showed that recombinant human prothrombin kringle-1, -2. and -1-2 (rk-1, -2, -1-2) all have potent anti-angiogenic activities, which inhibit Lewis lung carcinoma (LLC) tumor growth and metastases. Recombinant human prothrombin kringles were expressed by an E. coli expression system and purified to apparent homogeneity from crude E. coli extracts. Purified rk-1, -2, -1-2 migrated with a molecular mass of 14, 19, and 31 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. rk-1, -2, -1-2 exhibited potent inhibitory effects on bFGF-stimulated bovine capillary endothelial cell growth with half-maximal concentrations (ED50) of approximately 41, 55, and 156 nM, respectively. All of the recombinant human prothrombin kringles also inhibited angiogenesis in the chorioallantoic membrane (CAM) of chick embryos at a dose of 20 microg. Systemic administration of rk-1, -2, -1-2 at a dose of 0.5 mg/kg/day suppressed the growth of primary LLC and at dose of 0.5 and 1.0 mg/kg/day inhibited LLC metastases in C57BL6/J mice lungs through their anti-angiogenic effects.

Previously, we reported that the rabbit prothrombin fragment 2 (kringle 2 domain) has an anti-endothelial cell proliferative effect (Lee et al., J. Biol. Chem., in press). In this report, we show that not only rabbit prothrombin fragment 2 but also human prothrombin fragment 1 and 2 have an inhibitory effect on bFGF-stimulated BCE cell growth. Human prothrombin fragment 1 and 2 obtained as proteolytic fragments of human prothrombin display potent inhibitory effects on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately 100 nM and 120 nM, respectively. As rabbit prothrombin fragment 2, the human prothrombin fragment 1 and 2 also inhibit angiogenesis in the chorioallantoic membrane (CAM) of chick embryos., (Copyright 1998 Academic Press.)

A TNF-like factor was purified from lipopolysaccharide (LPS) induced New Zealand white rabbit serum. The TNF-like factor was purified by DEAE-Sephacel, Sephacryl S-200, Mono-Q, CM-affi gel Blue, Superose 12 H/R preparative columns to the specific activity of 4 x 10(6) U/mg protein. The purified protein was 45 kDa in its oligomeric form and 22 kDa in its monomeric form. Rabbit TNF-like factor had a pI value of 5.0 and was resistant to trypsin digestion. The TNF-like factor reacted with polyclonal-Ab against human TNFalpha on immunoblot and immunoprecipitation analysis and interacted with human TNF receptors. Taken together, rabbit TNF-like factor might be a high molecular weight form of rabbit TNFalpha.