Correlation of disease evolution with progressive inflammatory cell activation and migration in the IL-4 transgenic mouse model of atopic dermatitis

Atopic dermatitis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration in the skin. In order to assess the roles of inflammatory cells in this disease, we analysed the activation status and surface markers of various leucocytes in the IL-4 transgenic mouse model of atopic dermatitis, by flow cytometry, immuofluorescence microscopy, and T cell proliferation assays. The studies were performed with a nontransgenic mouse control and transgenic mice at three disease stages: before disease onset, early skin disease, and late skin disease, so that we can delineate the immunological sequence of events. As the skin disease evolves, the skin draining lymph node cells from IL-4-Tg mice show a spontaneous proliferation and a progressively enhanced proliferative response to stimulants including anti-CD3, Con A, PHA, and Staphylococcus enterotoxins A and B. As the disease evolves, the percent of lymphoid organ T cells expressing activation molecules (CD44 and CD69) and costimulatory molecules (ICOS and PD-1) are progressively increased; the percent and total number of T cells are reduced in an incremental manner in the secondary lymphoid organs while the number of T cells infiltrating the skin increases in an incremental fashion; the total number of dendritic antigen presenting cells, macrophages, and NK cells gradually increases in the lymphoid organs. Collectively, our results suggest that there is a continued and progressive migration of activated inflammatory cells from the secondary lymphoid organs into the skin where they participate in immune responses resulting in the pathology associated with inflammation.     

Interleukin-2 inhibits the interleukin-4-induced human IgE and IgG4 secretion in vivo.

The effect of interleukin-2 (IL-2) on IL-4-induced IgE and IgG4 secretion by B cells in peripheral blood mononuclear cell (PBMC) preparations from non-atopic healthy humans and atopic dermatitis patients was investigated. PBMC were cultured at an optimal concentration of recombinant IL-4 with or without addition of IL-2 for 10 days. Native and recombinant IL-2 inhibited the IL-4-induced IgE and IgG4 secretion in a dose-dependent manner by cells from both normal and atopic donors. Rabbit antibodies to IL-2 or to the monoclonal anti-IL-2 receptor antibody anti-TAC reversed the IL-2 effect. Culturing cells with IL-4 and IL-2 for 1 or 2 days only slightly suppressed the IgE and IgG4 secretion whereas addition of IL-2 to IL-4 containing cultures on day 4 or 5 inhibited the IgE and IgG4 secretion more effectively. This is in contrast to interferon-gamma (IFN-gamma) which inhibited the IL-4 induced IgE and IgG4 secretion when added for the first 24 or 48 h but had no effect when added on days 4 or 5. The data demonstrate that both IL-2 and IFN-gamma act as antagonists in the IL-4-induced IgE and IgG4 secretion by human B cells; while IL-2 appears to inhibit relatively late in culture, IFN-gamma has an early inhibitory effect, suggesting that the two lymphokines inhibit the IL-4 effect by different mechanisms.     

Different growth factor requirements for human Th2 cells may reflect in vivo induced anergy.

We previously reported the isolation of allergen-specific Th2 lines and clones from atopy patch test (APT) sites of atopic dermatitis (AD) patients. Upon stimulation with allergen or anti-CD3+ phorbol myristate acetate (PMA) IL-4 was released with or without IL-5, while no (or extremely low concentrations of) IL-2 and interferon-gamma (IFN-gamma) were detectable. A high IL-4/IFN-gamma ratio facilitates production of allergen-specific IgE, of which high levels are observed in AD patients. Here we show that the above mentioned Th2 cells are notably different from murine Th2 cells. Not IL-4, which is the autocrine acting growth factor for murine Th2 cells, but IL-2 was needed for proliferation of these human APT-derived Th2 lines and clones. Of significance, unless exogenous IL-2 was added, no proliferative response to allergen, presented by Epstein-Barr virus-transformed B (EBV-B) cells, non-T cells or IgE-bearing Langerhans cells (LC), occurred. Lack of proliferation and IL-2 production after full T cell receptor (TCR) triggering is a characteristic first described for in vitro anergized T cells. However, like the clones we describe in this study, anergic T cells may retain production of cytokines other than IL-2. A further resemblance between anergic T cells and the human Th2 clones reported here is that IL-4 can enhance IL-2-driven proliferation, but is not capable of inducing T cell growth by itself. The absence of IL-4-driven proliferation differentiates human Th2 cells from murine Th2 cells. Both produce IL-4 when stimulated in a cognate fashion, but only murine Th2 cells will proliferate. We conclude that the presently reported human Th2 cells are different from murine Th2 cells, in that they need other T cells to produce IL-2 required for their expansion. Moreover, the Th2 cells phenotypically resemble anergic T cells. As yet, however, we have no clue as to whether these features account for the current Th2 cells only or for human Th2 cells in general. We hypothesize that the Th2 phenotype of AD skin-derived, allergen-specific T cells may be induced in vivo by LC, which lack CD80, and therefore do not provide secondary signals through CD28-CD80 interaction.     

Involvement of interleukin (IL)-13, but not IL-4, in spontaneous IgE and IgG4 production in nephrotic syndrome

Nephrotic syndrome (NS) is a renal disease characterized by proteinuria and hypoalbuminemia. In NS patients without any allergic disease, serum IgE and IgG4 levels were selectively increased, and peripheral blood mononuclear cells (MNC) spontaneously produced IgE and IgG4. T cells produced interleukin (IL)-13 spontaneously, and B cells constitutively expressed IL-13 receptors (IL-13R). In addition, T cells stimulated surface IgE-negative (sIgE^?) and sIgG4^? B cells to produce IgE and IgG4, respectively, and IgE and IgG4 production was specifically blocked by anti-IL-13 antibody (Ab). MNC from atopic dermatitis (AD) patients also produced IgE and IgG4 spontaneously. However, in AD patients, T cells spontaneously produced IL-4, but not IL-13, and B cells constitutively expressed IL-4R, but not IL-13R. T cells stimulated sIgE^? and sIgG4^? B cells to produce IgE and IgG4, respectively, and the production was specifically blocked by anti-IL-4 Ab. On the other hand, sIgE⁺ and sIgG4⁺ B cells from both NS and AD patients spontaneously produced IgE and IgG4, respectively, and this production was not affected by T cells, anti-IL-4 Ab, or anti-IL-13 Ab. These results indicate that IL-13 is involved in the enhanced production of IgE and IgG4 in NS, while IL-4 is involved in these responses in AD.     

Enhanced expression of B7.2 (CD86) by percutaneous sensitization with house dust mite antigen

House dust mite antigen is a well-known allergen in the pathogenesis of atopic dermatitis (AD), a chronic relapsing inflammatory skin disease. We evaluated the AD model mice sensitized with house dust mite antigen and observed a Th2-dominant immune response. In this experiment, BALB/c mice were sensitized percutaneously with house dust mite antigen three times with 7 days interval after skin barrier disruption. A remarkable infiltration of polymorphonuclear granulocytes and monocytes in the cutis was observed in mice treated with this antigen, high serum IgE levels and IL-4 mRNA expression in local lymph node cells was also observed. CD19(+) B cell numbers overturned to CD4(+) helper T cells. In these mice, there was significant increase of B7.2 (CD86) expression on CD19(+) B cells. These results indicate that house dust mite antigen sensitizes BALB/c mice and skews their Th1/Th2 balance toward Th2.     

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.     

IgE‐expressing memory B cells and plasmablasts are increased in blood of children with asthma,food allergy,and atopic dermatitis

Despite the critical role of soluble IgE in the pathology of IgE‐mediated allergic disease, little is known about abnormalities in the memory B cells and plasma cells that produce IgE in allergic patients. We here applied a flow cytometric approach to cross‐sectionally study blood IgE+ memory B cells and plasmablasts in 149 children with atopic dermatitis, food allergy, and/or asthma and correlated these to helper T(h)2 cells and eosinophils. Children with allergic disease had increased numbers of IgE+CD27‐ and IgE+CD27+ memory B cells and IgE+ plasmablasts, as well as increased numbers of eosinophils and Th2 cells. IgE+ plasmablast numbers correlated positively with Th2 cell numbers. These findings open new possibilities for diagnosis and monitoring of treatment in patients with allergic diseases.     

Comparative analysis of mononuclear cell surface markers in atopic processes--a preliminary study

Atopic disorders are driven by the Th2 cell subset. We have determined the expression of costimulatory molecules and cell surface markers on peripheral CD4+ T cells and antigen presenting cells, in different atopic diseases, and we have also tried to correlate the expression of these markers with the severity of the disease. Cells from patients with atopic and contact dermatitis, mild or severe asthma, and symptomatic and non-symptomatic atopic rhinitis were analyzed by flow cytometry. Our results showed that CD30, CD124, and CD152 expression on CD4+ T cells was significantly higher in atopic dermatitis than in contact dermatitis patients (p < 0.05). It was interesting to observe that the cell surface expression of CD80 in T and B cells from atopic dermatitis patients was not enhanced as opposed to the other atopic diseases we analyzed. Our results suggest that there are differences in the immune mechanisms involved in the different atopic diseases, and that expression of CD30 in CD4+ T cells might be a marker of disease activity in atopic dermatitis.     

Regulation of allergic inflammation by skin-homing T cells in allergic eczema

BACKGROUND: In allergic inflammations of the skin, the pivotal role of CD45RO+ (memory/effector) T cells expressing the cutaneous lymphocyte-associated antigen (CLA) was demonstrated. In both atopic dermatitis (AD) and contact dermatitis (CD), T cells specific to skin-related allergens were confined to the CLA+ T cell population. Our research was aimed to further characterize these T cells in AD. METHODS: CD4+ and CD8+ subsets of CLA+ CD45RO+ T cells were purified from peripheral blood of AD patients and healthy control individuals. We studied, in vivo activation patterns, cytokine profiles, immunoglobulin isotype regulation and the influence of these cells on eosinophil survival and apoptosis. RESULTS: The CLA+ CD45RO+ T cells represent an in- vivo-activated memory/effector T cell subset as shown by surface expression of activation markers, spontaneous proliferation and a lower activation threshold via TCR/CD3 triggering. These cells contain and spontaneously release high amounts of preformed IL-5 and IL-13 but only very little IL-4 and IFN-gamma in their cytoplasm, as demonstrated by intracellular cytokine staining immediately after purification. Moreover, CLA+ memory/ effector T cells induce IgE production in B cells and enhance eosinophil survival by inhibiting eosinophil apoptosis in AD. In comparison, the CLA- population represents a resting memory T cell fraction, induces rather IgG4 in B cells and does not show any effect on eosinophil survival and apoptosis. CONCLUSION: Our results indicate that in-vivo-activated both CD4+ and CD8+ memory/effector T cells with skin-homing property play a specific and decisive role in the pathogenesis and exacerbation of AD. In contrast, resting memory T cells of atopic individuals retain normal, nonallergic immune functions.     

Comparative Analysis of Mononuclear Cell Surface Markers in Atopic Processes‐A Preliminary Study

Atopic disorders are driven by the Th2 cell subset. We have determined the expression of costimulatory molecules and cell surface markers on peripheral CD4 + T cells and antigen presenting cells, in different atopic diseases, and we have also tried to correlate the expression of these markers with the severity of the disease. Cells from patients with atopic and contact dermatitis, mild or severe asthma, and symptomatic and non‐symptomatic atopic rhinitis were analyzed by flow cytometry. Our results showed that CD30, CD124, and CD152 expression on CD4 + T cells was significantly higher in atopic dermatitis than in contact dermatitis patients (p < 0.05). It was interesting to observe that the cell surface expression of CD80 in T and B cells from atopic dermatitis patients was not enhanced as opposed to the other atopic diseases we analyzed. Our results suggest that there are differences in the immune mechanisms involved in the different atopic diseases, and that expression of CD30 in CD4 + T cells might be a marker of disease activity in atopic dermatitis.     

House dust mite-specific T cells in the skin of subjects with atopic dermatitis: frequency and lymphokine profile in the allergen patch test.

The mechanism underlying positive patch tests with house dust mite-allergen, Dermatophagoides pteronyssinus (Der p), in patients with atopic dermatitis was investigated by isolating T cells from the test sites of two patients. Eighty-five T cell clones (TCC) were established from the epidermis and dermis of lesional skin by the limiting-dilution method with Der p and interleukin (IL)-2. With restimulation assays, 29 of 60 TCCs tested demonstrated specific proliferation; 85% were of the CD3+, CD2+, and CD4+ phenotype. Der p-specific T cells constituted 0.4% to 2.7% of lesional T cells, and they were more frequent in the skin than in the blood of the patients by one order of magnitude. The mitogen-stimulated lymphokine profile of 55 TCCs was assessed; 42% (11/26) of the allergen-specific TCCs secreted IL-4 but almost no interferon-gamma, as described for the Th2 subset of the mouse. Also, six selected TCCs supported IgE secretion by autologous lymphocytes. Only three of 26 allergen-specific, skin-derived TCCs demonstrated a Th1-like lymphokine profile. These results support the specific nature of Der p-induced patch test lesions in patients with atopic dermatitis, and the results demonstrate also that a considerable proportion of lesional T cells are allergen-specific, IL-4-producing T cells that are capable of enhancing IgE production.     

Increased sensitivity to IL-4 in patients with allergic bronchopulmonary aspergillosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a heightened Th2 CD4+ cell response to Aspergillus fumigatus allergens and a hyper-IgE state compared to atopic asthmatic and cystic fibrosis patients without ABPA. We hypothesized that one reason for this response is increased sensitivity to IL-4 in ABPA, resulting in increased expression of CD23 and CD86, leading to a positive amplification mechanism which increases Th2 CD4+ T cell responses. METHODS: Peripheral blood mononuclear cells isolated from 10 ABPA, 9 atopic, and 8 nonatopic subjects and stimulated for 48 h with varying concentrations of rIL-4 ranging from 0.1 to 50 ng/ml. The percentages of CD23+ and CD86+ B cells and the number of CD23+ molecules on CD20+ and CD86+CD20+ B cells were quantified by flow cytometry. RESULTS: Total serum IgE levels were elevated in ABPA patients compared to atopic and nonatopic controls. At day 0 prior to culture, CD23 molecules per CD20+ B cell were significantly elevated in ABPA patients compared to atopic and to nonatopic patients. CD23 molecules per CD20+ B cell in ABPA and atopic patients decreased after 48 h in culture without IL-4 added and were similar. With IL-4 stimulation, ABPA patients had significantly increased rates of CD23 expression per B cell compared to atopic and nonatopic subjects (p < 0.001). Furthermore, ABPA had significantly increased numbers of CD23+ molecules per B cell and CD86+ B cell following IL-4 stimulation compared to atopic and nonatopic patients. Both ABPA and atopic patients at day 0 prior to culture had increased expression of CD86+ and CD23+CD86+ B cells compared to nonatopic patients. After 48 h in culture without IL-4, the percentages of CD86+ and CD23+CD86+ B cells decreased in ABPA and atopic patients. After stimulation with IL-4, ABPA patients had significant upregulation of CD23+CD86+ B cells compared to atopic and nonatopic patients. Similarly, the number of CD23 molecules per CD86+CD20+ B cell was significantly upregulated following IL-4 stimulation in ABPA patients compared to atopic and to nonatopic subjects. CONCLUSIONS: This is the first study to demonstrate that ABPA patients have increased sensitivity to IL-4 stimulation compared to other atopic individuals, such that ABPA > atopic > nonatopic patients. The B cells from ABPA patients were significantly more sensitive to IL-4 stimulation compared to atopic and nonatopic patients with upregulation of CD23 and CD86 expression. ABPA subjects had increased CD86+ and CD23+CD86+ B cell expression on day 0 prior to culture and with upregulation of CD23+ molecules on CD86+CD20+ B cells. IL-4 also stimulated upregulated CD86+ expression on B cells in atopic patients with little effect on nonatopic patients. This study supports the premise that IL-4, IL-4R alpha and CD86 are central targets in the treatment of ABPA and atopic disease.     

TLR7/8 ligand, R-848, inhibits IgE synthesis by acting directly on B lymphocytes

TLRs are involved in the regulation of immune responses. R-848, a TLR7/8 ligand, has potent anti-viral and anti-tumour properties and has been used as a new immune response modifier for enhancing Th1 immune response. In this study, we found that R-848 significantly inhibited IgE synthesis from murine B cells at the single cell levels by anti-CD40 plus IL-4-stimulated splenocytes, in which R-848 acted on the early stage of B cell differentiation to modulate IgE synthesis. This inhibitory effect of R-848 on IgE synthesis was not isotype specific as it also inhibited IgG1 synthesis. Moreover, R-848 had no significant effect on the production of IgG2a by anti-CD40 plus IL-4-stimulated splenocytes. Further studies showed that R-848 markedly promoted murine B cell activation induced by anti-CD40 plus IL-4 by up-regulating the expression of B cell activation markers CD25, CD69 and co-stimulatory molecule CD80. In contrast, R-848 inhibited the proliferation and division of murine B cells in anti-CD40 plus IL-4-stimulated splenocytes. R-848 promoted the production of IFN-γ and IL-12 that were partially responsible for its inhibitory effect on IgE production by anti-CD40 plus IL-4 because the addition of anti-IFN-γ or anti-IL-12 mAbs to the cultures could significantly restore IgE production by splenocytes. Importantly, R-848 had a direct effect on purified B cells to inhibit IgE production induced by anti-CD40 plus IL-4. Taken together, these results demonstrate that R-848 markedly inhibits IgE synthesis, and suggest that R-848 could be used to treat allergic diseases.     

Detection of IgE anti-parvovirus B19 and increased CD23+ B cells in parvovirus B19 infection: relation to Th2 cytokines

The immune profile of a parvovirus B19-infected patient (male, 8 years old) was studied on day 0 (initial presentation) and on days 14 and 210 post symptom presentation (psp). Before infection, the patient was skin test positive to various allergens, including ragweed and tree and grass pollens, and had a serum IgE level of 150 IU/mL. On day 0, the patient was diagnosed as parvovirus B19 infected, as judged by the presence of IgG anti-parvovirus Abs in serum (EIA) and presentation of "slap cheek" rash. The patient's serum IgE level increased from 150 IU/mL before infection to 256 IU/mL on day 0, was 233 IU/mL on day 14, and returned to preinfection levels on day 210. In contrast, there was little change in the levels of serum IgM, IgG, or IgA (nephelometry). IgE anti-parvovirus B19 protein (VP-N) was detected in serum (Western blot) on days 0, 14, and 210, despite the decrease in total IgE on day 210. Although there was no increase in total numbers of blood CD23+ B cells on day 0, by day 14 the numbers of these cells increased dramatically (93%), remaining high on day 210. In contrast, there were virtually no changes in total numbers of CD4+ and CD8+ T cells or CD16/56+ NK precursor cells on days 0-210. On day 0, when IgG and IgE anti-parvovirus were detected in serum, patient's peripheral blood mononuclear cells (PBMC) expressed mRNA for the Th2 cytokines IL-4 and IL-10, but not for the Th1 cytokines IFN-gamma or IL-2. However, by day 14 psp, PBMC expressed mRNA for the Th1 cytokines IFN-gamma and IL-2, as well as for IL-4 and IL-10. This is the first demonstration of the existence of IgE anti-parvovirus B19 Ab. The presence of IgE anti-parvovirus B19 Ab in serum on day 0 and its persistence in serum 7 months psp suggests that IgE anti-parvovirus may be useful in prognosis of parvovirus B19 infection. Our results reinforce the idea that IgE, in general, may play a major role in anti-viral immunity, perhaps in conjunction with CD23+ cells. The results further suggest that clearance of this infection is accompanied by a switch to Th1 cytokines.     

Effects of Staphylococcus aureus cell wall products (teichoic acid, peptidoglycan) and enterotoxin B on immunoglobulin (IgE, IgA, IgG) synthesis and CD23 expression in patients with atopic dermatitis.

The influence of staphylococcal cell wall products (teichoic acid, peptidoglycan) and enterotoxin B on peripheral blood lymphocytes (PBL) from patients with atopic dermatitis (AD) was investigated. The parameters studied were spontaneous and interleukin-inducible immunoglobulin (IgA, IgE, IgG) synthesis and CD23 expression. PBL from non-atopic donors served as controls. Teichoic acid and peptidoglycan induced an enhanced synthesis of IgA and IgG in normal donors. However, IgA and IgG synthesis in PBL from patients with AD was significantly suppressed by teichoic acid and enterotoxin B. The incubation of PBL from normal donors with enterotoxin B and interleukin-4 (IL-4) or IL-5 led to a significant suppression of IgA and IgG synthesis. Co-stimulation of PBL with teichoic acid or peptidoglycan and IL-4 led to a pronounced increase in IgE synthesis and CD23 expression in patients with AD. Our data indicate that cell wall products and toxins of staphylococci modulate the cytokine-dependent humoral immunity in patients with AD and may be responsible for allergic skin reactions in AD.     

CD19-deficient mice exhibit poor responsiveness to oral immunization despite evidence of unaltered total IgA levels, germinal centers and IgA-isotype switching in Peyer's patches

CD19 exhibits a critical role as a response regulator in B cells, influencing activation, differentiation and survival. Accordingly, CD19-deficient mice largely lack B-1 cells, and their conventional B-2 cells are poor responders to thymus-dependent antigen. Since both B-1 and B-2 cells may contribute to the total intestinal IgA production, we investigated whether lack of CD19 negatively affected mucosal immunity. We found that CD19(-/-) mice have near normal total IgA levels in serum and gut mucosa and, contrary to systemic lymphoid tissues, Peyer's patches (PP) had germinal centers to which also IgA+ B cells localized. However, the mice demonstrated severely impaired responses to oral immunization with keyhole limpet hemocyanin plus cholera toxin adjuvant. Mucosal responses to oral immunization were significantly more impaired than systemic responses. Despite normal specific IL-4 production, a selective defect in Th2-regulated B cell isotypes was observed, with poor or no mucosal IgA, low serum IgG1 and no IgE, but intact serum IgA and IgG2a production. Ex vivo experiments revealed strongly inhibited CD40-stimulated proliferation and IgA differentiation in CD19-deficient PP B cells. Taken together, an impaired CD40 responsiveness selectively affected Th2, but not Th1, coordinated B cell responses. The CD19(-/-) mice provide compelling evidence for the differential regulation of serum and mucosal IgA immunity.     

Roles of CD4+ and CD8+ T cells in adjuvant activity of diesel exhaust particles in mice

Through an imbalance in Th1 and Th2 cytokine profiles, diesel exhaust particles (DEP) are thought to induce Th2-dominated IgE and IgG1 production. However, the roles of CD4+ and CD8+ T-cell subtypes in the increased immune responses to antigen in mice exposed to DEP are unclear. In the present study, we investigated whether treatment with anti-CD4 or anti-CD8 mAb abrogated the adjuvant activity of DEP. On day -1 and day 1, each group of mice was injected intraperitoneally with anti-CD4, anti-CD8, or rat IgG (vehicle). On day 0, the mice were immunized with ovalbumin (OVA) or OVA plus DEP. After 3 weeks, each mouse was boosted with 10 microg of OVA alone. On day 7 after the first injection with OVA+DEP or OVA alone, the numbers of total, IA+, CD80+/IA+ and CD86+/IA+ cells in peritoneal exudate cells (PEC) were higher in OVA+DEP-immunized mice than in OVA-immunized mice. Depletion of CD8+ cells resulted in a modulation of the production of granulocyte-macrophage colony-stimulating factor, IL-12 and PGE(2) in peritoneal exudate fluid from OVA+DEP-immunized mice. On day 28, DEP injection markedly increased IL-4 production in the culture supernatants of spleen cells from CD4+ or CD8+-depleted mice. Depletion of CD8+ cells in OVA+DEP-immunized mice resulted in a decrease in IFN-gamma production compared with that in OVA-immunized mice. Adjuvant activity of DEP was observed in anti-OVA IgE, anti-OVA IgG1, anti-OVA IgG3, and total IgE production. Depletion of CD4+ T cells abrogated the adjuvant effect of DEP on anti-OVA IgE, and anti-OVA IgG1 production in plasma. However, depletion of CD8+ T cell inhibited the upregulated anti-OVA IgG3 production. These findings suggest that DEP injection may affect not only the function of CD4+ cells but also that of CD8+ T-cell subsets to modulate the synthesis of proinflammatory cytokine in PEC and type-1 and type-2 cytokine production in spleens.     

Do T helpers 1 and 2 recognize different class II MHC molecules? Humoral and cellular immune responses to soluble allergen from Aspergillus fumigatus Asp f2

Cellular signals leading to T helper (Th)1/Th2 shift are not well known. Here we demonstrate that Th1 possibly recognizes peptides presented by the IE molecule of MHC class II while Th2 is activated by the recognition of peptides presented by the IA molecule. BALB/c mice immunized with Asp f2 developed stable IA-restricted Th2 immune response to the 12th day after immunization, as analyzed by IL-2 production. On the contrary, early Th0 cells did not secrete IL-2 upon Asp f2 stimulation but did produce a high level of IL-2 if stimulated in the presence of anti-IA Abs. This effect of anti-IA Abs on early Th0 cells was both MHC IE and CD4(+) cell restricted. In vivo blocking of Asp f2 peptide presentation by the IA molecule led to the formation of antigen-specific cytotoxicity as demonstrated using immune splenocytes as effector cells and Asp f2 loaded P815 cells as targets.     

PI3K is a negative regulator of IgE production

The production of IgE, a main player in allergic disorders such as asthma and atopic dermatitis, is strictly regulated and the serum concentrations of IgE are normally kept at a much lower level than other isotypes. We found that mice deficient for the p85alpha regulatory subunit of class IA phosphoinositide 3-kinase (PI3K) produced increasing amounts of serum IgE. Purified p85alpha-/- B cells produced more IgE than wild-type B cells in vitro in response to anti-CD40 mAb and IL-4. PI3K inhibitors wortmannin and IC87114 enhanced IgE production by wild-type B cells stimulated with anti-CD40 mAb and IL-4. Under the same condition, antigen receptor cross-linking induced the expression of inhibitor of differentiation-2 and suppressed the expression of activation-induced cytidine deaminase and class switch recombination (CSR) in a PI3K-dependent manner. IgE production was also suppressed in a concentrated cell culture condition, which was completely reversed by PI3K inhibition. The selective suppression of IgE production by PI3K was also observed at a protein level after CSR. Our results indicate that PI3K negatively regulates IgE production at both CSR and protein levels.