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.

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.

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.

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.

Locomotor properties of human germinal centre B cells: activation by anti-CD40 and IL-4 allows chemoattraction by anti-immunoglobulin.

The locomotor properties of B cells isolated from the germinal centres (GC) of human tonsils were studied using polarization, collagen gel invasion and micropore filter assays. The proportion of motile GC cells in the freshly isolated population was small. During culture in interleukin-4 (IL-4)+anti-CD40, but not in control medium, the proportion of polarized cells increased and these cells migrated actively into collagen gels. After 24 hr culture, most of the surviving population was in locomotor morphology. The locomotor population consisted mainly of centrocytes in the G1 phase of growth. More locomotor cells than spherical cells took up [3H]uridine, but locomotor cells did not take up [3H]thymidine. After culture for 6 hr in IL-4+anti-CD40, GC B cells were tested in short-term polarization assays and filter assays for their response to chemoattractants. In both assays, a proportion of the cells responded to anti-IgA and to anti-IgA F(ab')2 fragments at 1 ng/ml., or to anti-IgG, anti-IgM and F(ab')2 fragments of these antibodies at 100 ng-1 microgram/ml. A checkerboard filter assay showed a good chemokinetic response and a weaker chemotactic response of GC cells to anti-IgA. Expression of Fc gamma RII (CD32) was increased after culture in IL-4+anti-CD40, and these cultured cells responded in filter and polarization assays to anti-CD32. Thus culture in IL-4 and anti-CD40 not only rescues GC B cells, but also increases their locomotor capacity and allows them to respond in chemotaxis assays to anti-immunoglobulin.

Locomotion and chemotaxis of lymphocytes.

The behaviour of locomotor T and B lymphocytes and the chemoattractants to which they respond in vitro are reviewed. Following activation, T cells respond by locomotion and chemotaxis to cytokine attractants including IL-15 and IL-2 and several chemokines. In activated B cells chemotaxis may be signalled through the antigen receptor. Conversely resting lymphocytes respond poorly to the above signals though their locomotion is activated by contact with high endothelial venular cells. These differences in locomotion between resting and activated lymphocytes, together with differences in adhesion, may explain why activated lymphocytes migrate preferentially into inflammatory sites while resting cells recirculate.

Chemotaxis of human B lymphocytes to anti-IgD.

The resting population of small surface IgM+ and surface IgD+ B cells from the human tonsil can be preactivated by overnight culture in interleukin-4 (IL-4) to show locomotor responses to anti-IgM and anti-IgD at between 10 ng and 1 microgram/ml. Because this locomotion is activated through the antigen receptor and may simulate a response to antigen, we set out to establish whether this was a chemotactic response using a checkerboard filter assay with a range of concentrations and concentration gradients of anti-IgD. At high concentrations (100 ng/ml to 1 microgram/ml), a chemokinetic response, but no chemotaxis, to anti-IgD was seen. However, in concentration gradients set up at lower concentrations (0-50 ng/ml) a chemotactic response was demonstrable. During the period of culture in anti-IgD at 1 microgram/ml, a progressive loss of surface IgD from the cells was seen, but there was no loss at 10 ng/ml. This receptor loss from the cell surface may account for the lack of chemotactic effect of the anti-IgD at higher concentrations.

Interactions between IL-4, anti-CD40, and anti-immunoglobulin as activators of locomotion of human B cells.

The locomotor properties of small, surface IgM+ and surface IgD+ B cells from the human tonsil were studied using polarization assays and collagen gel invasion assays. These cells gave poor locomotor responses when freshly isolated from the tonsil; but 30 to 40% of the cells polarized and invaded collagen gels after overnight culture in IL-4 or anti-CD40. IL-13 had a similar but weaker effect. Culture with anti-CD40 and IL-4 together gave a higher proportion of polarized cells than either alone, and culture in anti-CD40, IL-4, and anti-IgM gave a still higher proportion (> 60% of B cells polarized). Polarization increased gradually, during hours of culture, unlike the typical rapid response to chemoattractants. We also studied the immediate (< 30 min) effects of chemoattractants on B cell polarization. B cells cultured overnight then washed, but not B cells fresh from the tonsil, polarized immediately in response to anti-CD40. Similar responses to anti-IgM and anti-IgD, both pre- and postculture were also observed, but the response of cultured cells was stronger. IL-4-cultured B cells invaded collagen gels incorporating anti-IgM, anti-IgD, or anti-CD40 in higher numbers than control gels. Most of the invading cells were surface IgM+. These results suggest that locomotor activation in B cells requires two steps. The capacity for locomotion is growth-related and is first activated by IL-4 or by anti-CD40, enhanced by the presence of anti-IgM. Following activation, the cells respond rapidly to chemoattractants such as anti-Ig or anti-CD40.