Interleukin-33 amplifies IgE synthesis and triggers mast cell degranulation via interleukin-4 in naïve mice.

BACKGROUND: The regulation and function of IgE in healthy individuals and in antigen-naïve animals is not well understood. IL-33 administration increases serum IgE in mice with unknown mechanism. We tested the hypothesis that IL-33 provides an antigen-independent stimulus for IgE production and mast cell degranulation. METHODS: IL-33 was administered to naïve wild-type (WT), nude and ST2(-/-) , IL-4(-/-) , IL4Rα(-/-) and T-or B-cell-specific IL-4Rα(-/-) mice. IgE and cytokines were quantified by ELISA. T- and B-lymphocyte numbers and CD40L expression were determined by flow cytometry. Anaphylaxis was measured by temperature, mast cell degranulation and histamine release. RESULTS: IL-33 enhanced IgE production in naïve WT, T-IL-4Rα(-/-) but not in ST2(-/-) , IL-4(-/-) , IL-4Rα(-/-) or B-cell-specific IL-4Rα(-/-) mice, demonstrating IL-33 specificity and IL-4 dependency. Moreover, IL-4 was required for IL-33-induced B-cell proliferation and T-cell CD40L expression, which promotes IgE production. IL-33-induced IL-4 production was mainly from innate cells including mast cells and eosinophils. IL-33 increased mast cell surface IgE and triggered degranulation and systemic anaphylaxis in allergen-naïve WT but not in IL-4Rα(-/-) mice. CONCLUSION: IL-33 amplifies IgE synthesis and triggers anaphylaxis in naïve mice via IL-4, independent of allergen. IL-33 may play an important role in nonatopic allergy and idiopathic anaphylaxis.

Spread of malignant cells in the surgical margin with stapled excision of lung cancer: comparison of aggressive clump and less traumatic jaw closure type staplers.

BACKGROUND: During stapled excision of lung cancer tissue, malignant cells can spread in the surgical margin. Stapling methods can be classified as aggressive clumping (AC) and less traumatic jaw closing (LTJC) types, thus the ratio of malignant margins may differ between stapler types. METHODS: The malignant status of the stapled margin was retrospectively investigated in 112 cases using a cytology technique. Stapler type, maximum tumor diameter, distance from surgical margin, thoracotomy type, and tumor location were used as variables. In addition, clinical results of excision cases were assessed. RESULTS: The ratio of malignant margins was 22/54 (41 %) in the AC group and 11/58 (19 %) in the LTJC group ( P = 0.01). Multivariate analysis revealed that the stapling method and tumor location were an independently significant factor. Surgical margin recurrence occurred only in 4 (57 %) of 7 cases with malignant margin. CONCLUSIONS: The AC type method showed a greater potential to spread malignant cells, thus there seems to be a higher possibility of regional relapse with that technique.

Characterization of initial halogen adsorption on Si(111) surface by scanning tunnelling microscopy: correlation with optical measurements.

Initial adsorption processes of halogen atoms on a Si(111)-(7 × 7) surface were studied by means of scanning tunnelling microscopy (STM). The adsorption sites of halogen atoms were clarified directly with STM, and the results were compared with the partial coverage at each site, estimated previously from surface differential reflectance and thermal desorption spectroscopic analyses. The microscopic geometry of the atomic structure showed a good correspondence with the optical measurements, especially in terms of the density of the reacted sites. Bromine atoms were predominantly adsorbed near already adsorbed bromine, while chlorine atoms were almost randomly adsorbed. Polybromide formation occurred at coverage levels above 0.1 ML. Bromine atoms break the back-bonds of Si adatoms at lower levels of coverage than do chlorine atoms. The reason for the difference in adsorption behaviour between chlorine and bromine is discussed.

Interleukin-18 levels in induced sputum are reduced in asthmatic and normal smokers.

BACKGROUND: IL-18 is a cytokine which is known to have an important role in the development of a Th1 lymphocyte response. As such, it may have a regulatory role in asthma by modifying Th2 lymphocyte responses. Cigarette smoking may amplify the airway inflammation associated with asthma. OBJECTIVE: This study investigated if IL-18 could be detected in induced sputum from asthmatics and normal subjects and if smoking altered IL-18 levels. METHODS: Induced sputum was obtained from asthmatic (31 smokers, 35 non-smokers) and normal (20 smokers, 20 non-smokers) subjects. All smokers had a smoking history of > or =15 pack years. IL-18 levels in sputum supernatant were measured by ELISA. IL-18 mRNA expression and cellular localization were assessed by quantitative PCR and immunocytochemistry, respectively. RESULTS: Smoking was associated with a significant reduction in IL-18 levels (median (interquartile range) - smokers 20 (0-102) pg/mL vs. non-smokers 358 (50-876) pg/mL, P<0.001). This was more pronounced in asthmatics (smokers, 47 (40-64) pg/mL vs. non-smokers, 530 (30-1484) pg/mL; P<0.001) than in normal subjects (smokers, 25 (0-78) pg/mL vs. non-smokers, 247 (50-656) pg/mL; P<0.01). Within each of the smoking and non-smoking groups there was no significant difference in IL-18 levels between asthmatic and normal subjects. There was no correlation between sputum IL-18 levels and any specific cell type in the sputum samples nor serum IgE levels. IL-18 mRNA expression was reduced in asthmatic smokers compared with non-smokers. IL-18 production was localized to sputum macrophages by immunocytochemistry. CONCLUSIONS: IL-18 is detectable in induced sputum samples from both asthmatic and normal subjects. Cigarette smoking significantly reduces sputum IL-18 levels. This effect is more pronounced in asthmatics than in normal subjects.

A proinflammatory role of IL-18 in the development of spontaneous autoimmune disease.

Serum from patients with systemic lupus erythematosus (SLE) contained significantly higher concentrations of IL-18 than normal individuals. MRL/lpr mice, which develop spontaneous lupus-like autoimmune disease, also had higher serum levels of IL-18 than wild-type MRL/++ mice. Daily injections of IL-18 or IL-18 plus IL-12 resulted in accelerated proteinuria, glomerulonephritis, vasculitis, and raised levels of proinflammatory cytokines in MRL/lpr mice. IL-18-treated MRL/lpr mice also developed a "butterfly" facial rash resembling clinical SLE. In contrast, MRL/lpr mice treated with IL-18 plus IL-12 did not develop a facial rash. The facial lesion in the IL-18-treated mice showed epidermal thickening with intense chronic inflammation accompanied by increased apoptosis, Ig deposition, and early systemic Th2 response compared with control or IL-12 plus IL-18-treated mice. These data therefore show that IL-18 is an important mediator of lupus-like disease and may thus be a novel target for therapeutic intervention of spontaneous autoimmune diseases.

Immuno-regulatory cytokines in asthma: IL-15 and IL-13 in induced sputum.

BACKGROUND: The importance of Th2-type lymphocyte function in asthmatic airway inflammation is well recognized, but less is known about the factors which regulate the function of these lymphocytes in asthma. The macrophage-derived cytokine, interleukin (IL)-15 has a number of T cell regulatory properties which might be of relevance to asthma and its treatment. OBJECTIVE: The aims were to identify and quantify the T cell regulatory cytokine IL-15 in induced sputum samples from asthmatic patients, in comparison with IL-13, and to relate the levels of these cytokines to treatment with inhaled steroids. METHODS: Induced sputum was collected from 16 asthmatics (eight steroid and eight non-steroid treated) and eight normal controls. IL-15 and IL-13 levels were measured by enzyme-linked immunoassay (ELISA) in sputum. IL-15 levels were also measured in sputum cell culture supernatants and localized to specific sputum cells by immuno-cytochemistry. RESULTS: IL-15 levels were increased and IL-13 levels were decreased in sputum fluid from steroid-treated compared with non-steroid-treated asthmatics. IL-15 was localized specifically to macrophages and the proportion of these cells expressing IL-15 correlated with sputum fluid IL-15 and IL-15 levels in cell culture supernatants, and all were higher in the steroid-treated asthmatics. CONCLUSION: IL-15 and IL-13 production appears to be reciprocally regulated by steroid therapy in asthma patients. The steroid-associated increase in IL-15 may regulate a fundamental shift away from an inflammatory Th2-type environment in asthma and may be an essential component of the cytokine modulation underlying the therapeutic benefit of corticosteroids in this condition.

Remodeling of the major pig xenoantigen by N-acetylglucosaminyltransferase III in transgenic pig.

We have been successful in generating several lines of transgenic mice and pigs that contain the human beta-d-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galalpha1-3Galbeta1-4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.

Down-regulation of the alpha-Gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene. Modulation of the biosynthesis of terminal structures by a bisecting GlcNAc.

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases.

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.

ESkine, a novel beta-chemokine, is differentially spliced to produce secretable and nuclear targeted isoforms.

Using the murine embryonal stem cell system, we have identified a novel gene encoding a highly divergent member of the beta-chemokine family of proinflammatory mediators and have called this protein ESkine. Much of the coding sequence for ESkine overlaps with the 3'-end of a novel interleukin 11 receptor alpha-like sequence on murine chromosome 4. ESkine is produced as two splice variants. One of these variants encodes a classical chemokine with an associated signal peptide, while the other variant (PESKY) possesses the main body of the chemokine but has replaced the signal peptide with an alternative stretch of amino acids that allows for nuclear targeting of this isoform. This differential splicing arises as a result of alternative 5' exon usage. These differentially spliced forms are expressed at discrete tissue loci. Thus, while ESkine is highly expressed in the placenta, PESKY is mainly expressed in the Testes and brain and weakly in the developing embryo. Studies on the proinflammatory properties of ESkine reveal it to be active in inducing polarization of CD4(+) T cells but to be inactive on other hemopoietic cellular populations.

Masking and reduction of the Galactose-alpha1,3-Galactose (alpha-Gal) epitope, the major xenoantigen in swine, by the glycosyltransferase gene transfection.

The alpha-Gal epitope (Gal-alpha1-3Gal-beta1-4-GlcNAc-R), which is biosynthesized by the UDP-Gal:alpha1-3-galactosyltransferase (alpha1, 3GT), is highly associated with hyperacute rejection in swine to human xenotransplantation. A variety of strategies have been pursued to reduce or eliminate this epitope from swine tissues. Since swine ES cells are not available at present, the targeted knock out of the alpha1,3GT is restricted. Other strategies, such as enzyme competition of the alpha1,3GT with other glycosyltransferases and/or control of sugar processing by the glycosyltransferases, provide a new insight into the downregulation of the alpha-Gal epitope. This review will focus on this type of strategy, which involves a gene transfection of variety of glycosyltransferases as competitors against alpha1,3GT.

Reduction of the major swine xenoantigen, the alpha-galactosyl epitope by transfection of the alpha2,3-sialyltransferase gene.

alpha2,3-Sialyltransferase represents a putative enzyme that reduces the Galalpha1-3Gal beta1-4GlcNAc-R (the alpha-galactosyl epitope) by intracellular competition with alpha1,3-galactosyltransferase for a common acceptor substrate. This study demonstrates that the overexpression of the alpha2,3-sialyltransferase gene suppresses the antigenicity of swine endothelial cells to human natural antibodies by 77% relative to control cells and by 30% relative to cells transfected with alpha1,2-fucosyltransferase, and in addition, it reduces the complement-mediated cell lysis by 75% compared with control cells and by 22% compared with cells transfected with alpha1, 2-fucosyltransferase. The mechanism by which the alpha-galactosyl epitope was reduced was also studied. Suppression of alpha1, 3-galactosyltransferase activity by 30-63% was observed in the transfectants with alpha2,3-sialyltransferase, and mRNA expression of the alpha1,3-galactosyltransferase gene was reduced as well. The data suggest that the alpha2,3-sialyltransferase effectively reduced the alpha-galactosyl epitope as well as or better than the alpha1, 2-fucosyltransferase did and that the reduction of the alpha-galactosyl epitope is due not only to substrate competition but also to an overall reduction of endogenous alpha1, 3-galactosyltransferase enzyme activity.

Antigen-specific chemotaxis of B cells.

The chemoattractant effect of soluble protein antigens for B cells from immunized mice was examined. Mice were immunized either via the footpad with ovalbumin (OVA) in complete Freund's adjuvant (CFA) or with CFA alone; or intraperitoneally with OVA incorporated in immune-stimulating complexes (OVA-ISCOM) or with saline. After 8-14 days B cells were purified from the spleens or the draining popliteal nodes and tested in vitro for locomotor responses to antigen using polarization (shape-change) and filter assays. Cells obtained by both routes of immunization, but not cells from control mice, gave locomotor responses to OVA. Responses were seen in B cells directly after preparation (5-8% of cells responding) but were enhanced if the cells were cultured overnight in the presence of interleukin-4 (IL-4) before testing (10-12% of cells responding). Checkerboard filter assays suggested that the response to OVA was chemotactic. The response was antigen specific since cells from OVA-immunized mice did not respond to bovine serum albumin (BSA), and cells from BSA-immunized mice responded to BSA but not to OVA. The response to OVA was inhibited by preincubation of OVA with anti-OVA but not with anti-BSA. Many of the cells that polarized in response to antigen were larger than any B cells found in control populations suggesting that the responsive cells are those that had been stimulated to enter cell cycle following immunization.

TGF-beta stimulates but IFN-gamma inhibits growth-related activation of locomotion of human B cells.

Effects of TGF-beta and IFN-gamma on locomotion of high density B cells from human tonsils were studied using polarization and filter assays. Culture with TGF-beta (10 pg to 1 ng/ml) induced a gradual increase in locomotor morphologies in 40 to 50% of B cells during overnight culture. This was not immediate (<30 min) suggesting that TGF-beta is not a chemotactic factor. The time course suggests that B cells acquired a locomotor phenotype during the first few hours of culture. B cells cultured in TGF-beta increased in size, but to a lesser extent than those cultured in IL-4 used as a control. More cells were polarized in TGF-beta + IL-4 than in either alone. Following culture in TGF-beta, B cells showed vigorous responses to anti-IgD used as a chemoattractant in short-term assays. In contrast, addition of IFN-gamma (20-100 U/ml) to B cells in culture in IL-4, anti-CD40, or TGF-beta inhibited activation of locomotor capacity by the latter agents, and IFN-gamma-cultured B cells showed an even lower response to anti-IgD in a short-term polarization or filter assay than those cultured in medium alone. IFN-gamma also inhibited uridine incorporation by cultured B cells, and cells cultured in IFN-gamma showed no size increase. We suggest that IFN-gamma prevents locomotor activation by inhibiting progress of B cells into the G1 phase of growth.