Requirement for CD8+ T cells in the development of airway hyperresponsiveness in a marine model of airway sensitization.

To study the role of CD8+ T cells in allergic sensitization, we examined the effects of in vivo depletion of CD8+ T cells prior to sensitization on IgE production, immediate type cutaneous hypersensitivity and development of altered airway responsiveness. BALB/c mice were thymectomized and treated with anti-CD8 antibody resulting in depletion of CD8+ T cells (<1%) in spleen and lymphoid tissues. In these mice, sensitization to ovalbumin (OVA) via the airways still resulted in IgE anti-OVA responses and immediate cutaneous reactions to OVA, but the animals were unable to develop airway hyperresponsiveness, eosinophil infiltration of the lung parenchyma, or IL-5 production in the local lymph nodes of the airway. Transfer of CD8+ T cells from naive animals during sensitization (on day 8 of the 10-d protocol) fully restored the ability to develop airway hyperresponsiveness and this was accompanied by IL-5 production and eosinophil accumulation in the lung. These data indicate a critical role for CD8+ T cells in the production of IL-5 and the development of altered airway responsiveness after antigen sensitization through the airways.

Evaluation of norovirus removal performance in a coagulation-ceramic microfiltration process by using recombinant norovirus virus-like particles.

Norovirus (NV) is a prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis worldwide. Because of the lack of a cell culture system or an animal model for this virus, studies on drinking water treatment such as separation and disinfection processes are still hampered. In the present study, we investigated NV removal performance as particles during a coagulation-ceramic microfiltration (MF) process by using recombinant NV virus-like particles (rNV-VLPs), which are morphologically and antigenically similar to native NV. We also experimentally investigated the behaviors of two widely accepted surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, for comparison with the behavior of rNV-VLPs. More than 4-log removal was observed for rNV-VLPs with a 1.08 mg-Al/L dose of polyaluminium chloride in the coagulation-ceramic MF process. This high removal ratio of rNV-VLPs satisfies the U.S. Environmental Protection Agency requirement of 4-log removal or inactivation. In addition, the removal ratios of Qbeta and MS2 were approximately 2-log and 1-log, smaller than the ratio of rNV-VLPs. Accordingly, both bacteriophages have the potential to become appropriate surrogates for native NV in the coagulation-ceramic MF process, and, of the two, Qbeta is the more conservative surrogate.

Passive transfer of immediate hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin (Ig) E and IgG1 in mice.

In a proportion of atopic asthmatics, exposure to a relevant antigen is followed by chronic inflammation in the airways leading to altered airway responsiveness (AR). However, the mechanisms underlying the development of airway hyperresponsiveness still remain unclear. To elucidate the relationship between IgE-mediated reactions and airway hyperresponsiveness, a murine model of passive sensitization and airway challenge with ovalbumin (OVA) was developed using anti-OVA IgE and IgG antibodies from murine B cell hybridomas. Passive sensitization by intravenous injection of anti-OVA IgE resulted in immediate cutaneous hypersensitivity and, after airway challenge with OVA on two consecutive days, increased AR in BALB/c and SJL mice. Increased numbers of eosinophils were observed in bronchoalveolar lavage fluid, in cells extracted from the lungs, and in the peribronchial areas of BALB/c mice passively sensitized with IgE and challenged through the airways compared with nonsensitized mice. Eosinophil peroxidase activity was also elevated in lung tissue from these mice. Passive sensitization with anti-OVA IgG1 but not IgG2a or IgG3 was similarly associated with development of skin test reactivity and increased AR after airway challenge, accompanied by an increase in eosinophils in bronchoalveolar lavage fluid. These data suggest that IgE/IgG1-mediated reactions together with local challenge with antigen can result in allergic inflammation resulting in altered airway function.

Improved virus removal by high-basicity polyaluminum coagulants compared to commercially available aluminum-based coagulants.

We investigated the effects of basicity, sulfate content, and aluminum hydrolyte species on the ability of polyaluminum chloride (PACl) coagulants to remove F-specific RNA bacteriophages from river water at a pH range of 6-8. An increase in PACl basicity from 1.5 to 2.1 and the absence of sulfate led to a reduction of the amount of monomeric aluminum species (i.e., an increase of the total amount of polymeric aluminum and colloidal aluminum species) in the PACl, to an increase in the colloid charge density of the PACl, or to both and, as a result, to high virus removal efficiency. The efficiency of virus removal at around pH 8 observed with PACl-2.1c, a nonsulfated high-basicity PACl (basicity 2.1-2.2) with a high colloidal aluminum content, was larger than that observed with PACl-2.1b, a nonsulfated high-basicity PACl (basicity 2.1-2.2) with a high polymeric aluminum content. In contrast, although extremely high basicity PACls (e.g., PACl-2.7ns, basicity 2.7) effectively removed turbidity and UV260-absorbing natural organic matter and resulted in a very low residual aluminum concentration, the virus removal ratio with PACl-2.7ns was smaller than the ratio with PACl-2.1c at around pH 8, possibly as a result of a reduction of the colloid charge density of the PACl as the basicity was increased from 2.1 to 2.7. Liquid (27)Al NMR analysis revealed that PACl-2.1c contained Al30 species, which was not the case for PACl-2.1b or PACl-2.7ns. This result suggests that Al30 species probably played a major role in virus removal during the coagulation process. In summary, PACl-2.1c, which has high colloidal aluminum content, contains Al30 species, and has a high colloid charge density, removed viruses more efficiently (>4 log10 for infectious viruses) than the other aluminum-based coagulants-including commercially available PACls (basicity 1.5-1.8), alum, and PACl-2.7ns-over the entire tested pH (6-8) and coagulant dosage (0.54-5.4 mg-Al/L) ranges.

Estimation of norovirus removal performance in a coagulation-rapid sand filtration process by using recombinant norovirus VLPs.

Norovirus (NV) is an important human pathogen that causes epidemic acute nonbacterial gastroenteritis worldwide. Because of the lack of a cell culture system or an animal model for this virus, studies of drinking water treatment such as separation and disinfection processes are still hampered. We successfully estimated NV removal performance during a coagulation-rapid sand filtration process by using recombinant NV virus-like particles (rNV-VLPs) morphologically and antigenically similar to native NV. The behaviors of two widely accepted surrogates for pathogenic waterborne viruses, bacteriophages Qbeta and MS2, were also investigated for comparison with that of rNV-VLPs. Approximately 3-log(10) removals were observed for rNV-VLPs with a dose of 40 muM-Al or -Fe, as polyaluminum chloride at pH 6.8 or ferric chloride at pH 5.8, respectively. Smaller removal ratios were obtained with alum and ferric chloride at pH 6.8. The removal performance for MS2 was somewhat larger than that for rNV-VLPs, meaning that MS2 is not recommended as an appropriate surrogate for native NV. By comparison, the removal performance for Qbeta was similar to, or smaller than, that for rNV-VLPs. However, the removal performances for rNV-VLPs and Qbeta differed between the coagulation process and the following rapid sand filtration process. Therefore, Qbeta also is not recommended as an appropriate surrogate for native NV.

Antigen-dependent regulation of IgE antibody production by human antigen-specific B cells.

Ag-specific B cells from the peripheral blood of immunized donors were separated following rosette formation with tetanus toxin (TT)-coupled immunobeads to study the regulation of T cell-dependent Ab production. Surface IgM+ (sIgM+) B cells rosetting with TT-coated beads produced anti-TT IgM, IgG, and IgE Abs in cultures stimulated with EBV or anti-CD40 Ab in the presence of IL-4; this was associated with increased numbers of B cells positive for sIgG and sIgE. TT-binding sIgM+ B cells also produced significant amounts of anti-TT IgM, IgG, and IgE Abs when they were cultured with autologous T cells in the presence of IL-4. Addition of TT to the IL-4-stimulated, T cell-dependent cultures resulted in higher levels of TT-specific IgM and IgG Abs than observed with T cells alone, but the inclusion of TT with T cells suppressed anti-TT IgE production. Addition of TT Ag to cultures with allogeneic T cells failed to enhance IgM and IgG responses, and the inhibition of IgE production was reduced compared with cultures containing autologous T cells in the presence of Ag. These results demonstrate that the presence of Ag contributes to the regulation of Ab production, resulting in enhanced IgM and IgG responses, but down-regulation of IgE Ab production.

Engagement of the B-cell antigen receptor by antigen negatively regulates IgE production by antigen-specific B cells.

BACKGROUND: Functional differentiation of B lymphocytes can be regulated at different levels. Such B-cell responses, to a large extent, are under the control of T cells and the release of soluble factors from these cells. Crosslinking of B-cell antigen receptors also can direct B-cell differentiation, although the mechanisms are not well defined and the effects on specific antibody production have not been examined. OBJECTIVE: This study was conducted to determine whether crosslinking of the B-cell antigen receptor by antigen or antibody to the B-cell receptor could modulate specific antibody production, especially IgE production. METHODS: Antigen-specific human B cells were isolated and incubated with antigen or antibody to the B-cell antigen receptor. Specific antibody production was examined, as were surface isotype expression and levels of Ig heavy chain mRNA in T cell-independent cultures. RESULTS: The addition of antigen or anti-IgM antibody enhanced IgM and IgG antibody production in the presence of anti-CD40 and IL-4 but downregulated IgE synthesis. These changes in isotope production paralleled changes in the frequencies of surface Ig+ B cells and Ig mRNA levels. Exogenous IL-6 abolished the antigen-mediated downregulation of IgE production. CONCLUSIONS: These studies indicate that signals delivered by means of the B-cell antigen receptor regulate antibody production in a distinct manner, resulting in enhanced IgM and IgG responses but leading to the inhibition of IgE production. Endogenous IL-6 production by B cells may counter the negative regulatory effects of antigen on IgE synthesis. We suggest that although antigen-mediated T/B-cell interactions play a major role in IgM- and IgG-specific antibody production, antigen-independent interactions may be more effective for T-cell dependent, IL-4-induced IgE production by antigen-specific B cells. Furthermore, these findings provide a basis for further understanding of allergic responses and modulation by immunotherapy, where repeated exposure to large concentrations of allergen, administered by means of injection, enhance IgG responses and downregulate IgE synthesis.

Isolation and characterization of human antigen-specific B lymphocytes.

Antigen-specific B cells from the peripheral blood of immunized donors were isolated following rosette formation with antigen-coated immunomagnetic beads and were phenotypically and functionally characterized. B cells separated with tetanus toxin (TT)- or keyhole limpet hemocyanin (KLH)-coated beads produced significant amounts of specific IgM and IgG antibodies when they were transformed with Epstein-Barr virus or cultured with autologous T cells in the presence of a specific antigen. Cells isolated from peripheral blood mononuclear cells (PBMC) with TT-coated beads consisted mostly of B cells (88.7% CD20+). B cells detected among the rosetting cells were predominantly sIgM+ with low percentages of the other isotype-expressing B cells. The mean percentage of TT-specific B cells among adult donors was 0.34% of PBMC, and increased to up to 2% following a booster immunization with antigen. A similar increase in the number of rosetting B cells was also observed following in vitro culture of PBMC with antigen. These findings demonstrate that immunomagnetic bead selection serves as a specific and reliable approach in identifying and isolating antigen-specific B cells from human PBMC and provides a valuable method for studying T-B interactions in antigen-specific immune responses.

Role of IgE in the development of allergic airway inflammation and airway hyperresponsiveness--a murine model.

The importance of IgE in airway inflammation and development of AHR in allergen-sensitized mice has been compared and contrasted in different models of sensitization and challenge. Using different modes of sensitization in normal and genetically manipulated mice after anti-IgE treatment, we have been able to distinguish the role of IgE under these different conditions. Striking differences in the three sensitization protocols were delineated in terms of the role of allergen-specific IgE, extent of eosinophilic airway inflammation, and development of AHR (Table 1). The highest levels of IgE and eosinophil infiltration (approximately 20-fold increases) were achieved after systemic sensitization with allergen (plus adjuvant) followed by repeated airway challenge. Passive sensitization with allergen-specific IgE followed by limited airway challenge induced a modest eosinophilic inflammatory response in the airways despite high levels of serum IgE. Exposure to allergen exclusively via the airways also resulted in a modest serum IgE response and a limited eosinophilic inflammatory response (approximately fourfold increases). Under all of these conditions, inhibition of IL-5-mediated eosinophilic airway inflammation was associated with attenuation of AHR. In contrast, the differences in the responses to the different modes of allergen exposure were associated with differences in the requirements for IgE in the development of AHR (Table 1). In the two models associated with mild eosinophil infiltration (passive sensitization and exclusive airway exposure), IgE was required for the development of AHR but did not substantially enhance airway inflammation on its own. However, IgE-allergen interaction was able to enhance T-cell function in vitro and induce T-cell expansion in vivo. In mice systemically sensitized and challenged via the airways, IgE (or IgE-mediated mast-cell activation) was not required for T-cell activation, eosinophilic inflammation and activation in the airways, or development of AHR. This was most clearly seen in B-cell-deficient and mast-cell-deficient, low-IgE-responder mouse strains (B6, B10) and in anti-IgE-treated high-IgEresponder mice (BALB/c). At the same time, we confirmed the importance of IgE in the induction of immediate-type hypersensitivity (mast-cell activation, immediate cutaneous hypersensitivity, passive cutaneous and systemic anaphylaxis). These differences were also highlighted by the means used to detect altered airway function. Passive sensitization and limited airway challenge or exclusive airway exposure to allergen over 10 days elicited changes in airway function that could be detected only in tracheal smooth-muscle preparations exposed to EFS. In contrast, systemic sensitization followed by repeated airway challenge resulted not only in changes in the contractile response to EFS but also in increased responsiveness to inhaled MCh. Thus, these results distinguish not only the differential involvement of IgE and eosinophil numbers but also their contribution to the readouts used to monitor airway function. Based on these studies, we conclude that IgE plays an important role in the development of airway inflammation and AHR under conditions in which limited IL-5-mediated eosinophilic airway infiltration is induced. In conditions where a robust eosinophilic inflammation of the airways is elicited, IgE (and IgE-mediated mast-cell activation) does not appear to be essential for airway inflammation and the development of AHR, detected as increased responsiveness to inhaled MCh. These findings reveal the potential importance of differential targeting in the treatment of allergic diseases with a predominance of IgE-mediated symptoms, e.g., allergic rhinitis and conjunctivitis, where anti-IgE may be an effective therapy, compared to those diseases with a predominant inflammatory component, e.g., AHR in atopic bronchial asthma, where anti-inflammatory or anti-IL-5 therapy may be more beneficial.

Aggregation of the FcepsilonRI on mast cells stimulates c-Jun amino-terminal kinase activity. A response inhibited by wortmannin.

Aggregation of the high-affinity Fc receptors for immunoglobulin E (IgE) (FcepsilonRI) on the surface of mast cells initiates intracellular signal transduction pathways including the tyrosine phosphorylation of cellular proteins, phosphoinositide hydrolysis, an increase in intracellular calcium, and protein kinase C activation. These signals are believed to be involved in the exocytic release of inflammatory mediators such as vasoactive amines, cytokines, and lipid metabolites. However, the downstream consequences of these early activation events are not well defined. One exception is the activation of the extracellular signal-regulated kinases/mitogen-activated protein kinases. One member of the mitogen-activated protein kinase superfamily, designated c-Jun amino-terminal kinase (JNK), has been recently identified. JNK is activated following dual phosphorylation at a Thr-Pro-Tyr motif in response to diverse stimuli including tumor necrosis factor-alpha, heat shock, or ultraviolet irradiation. We found that JNK was strongly activated by antigen cross-linking in a mouse mast cell line passively sensitized with ovalbumin-specific IgE. Anti-mouse IgE antibody also activated JNK. MEK kinase 1 (MEKK1) which activates the JNK activator, JNK kinase (JNKK), was similarly activated by antigen stimulation. JNK but not p42(erk2) activation induced by antigen was significantly inhibited in the presence of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase. These results indicate that in response to the aggregation of FcepsilonRI on mast cells, phosphatidylinositol 3-kinase activation is involved in the stimulation of the MEKK1, JNKK, JNK pathway.

Differential expression of CD40 ligand on T cell subsets. Implications for different roles of CD45RA+ and CD45RO+ cells in IgE production.

Physical contact between human T lymphocytes and B lymphocytes is required for the induction of IgE production. In the present study, we examined the abilities of CD45RA+ and CD45RO+ human T cell subsets to provide help for IgE production by human peripheral blood B cells in the presence of IL-4. Purified peripheral CD45RA+ T cells are much better inducers of IgE synthesis than are CD45RO+ T cells. Activation of CD45RA+ T cells, but not CD45RO+ T cells, via the TCR/CD3 complex is sufficient to confer the ability to provide IgE help, suggesting that an inducible T cell surface molecule plays an important role in this system. The CD40 ligand, an inducible T cell surface molecule, is expressed at higher levels on CD45RA+ T cells as compared with CD45RO+ T cells following CD3-stimulation. Blocking of the CD40-CD40 ligand interaction in vitro by the addition of a soluble form of B cell CD40 Ag completely blocks IgE production induced by CD45RA+ T cells. Finally, the in vitro conversion of CD45RA+ T cells to the CD45RO+ phenotype is accompanied by a loss in the ability of these cells to express the CD40 ligand in response to anti-CD3 stimulation as well as a loss in their ability to provide IgE help. These results suggest that both CD45 subsets may play significant and distinct roles in the induction of IgE production under physiologic conditions: CD45RO+ T cells provide IL-4 and the CD45RA+ subset provides the second signal via the CD40 ligand.

Nerve growth-factor and anti-CD40 provide opposite signals for the production of IgE in interleukin-4-treated lymphocytes.

Nerve growth factor (NGF) is a well-known neurotrophic factor acting on both the peripheral and the central nervous systems. In addition, it has been shown to play a role in the function of the immune system through specific receptors. Both high-affinity and low-affinity NGF receptors (NGFR) are expressed on human B lymphocytes. The low-affinity NGFR has been shown to have structural homology with another specific B cell surface molecule, CD40, which plays an important role in IgE production. In view of the structural similarities of the p75 NGFR and CD40 we examined whether NGF may also be involved in the regulation of IgE production. We found that NGF and anti-CD40 exerted opposite effects on the induction of IgE by IL-4 in peripheral blood mononuclear cells. NGF inhibited the induction of IgE by IL-4 and this inhibition was not mediated through blocking of the induction of CD23 nor through inhibition of IL-4R expression. The inhibition of IL-4-dependent IgE production was observed on surface (s)IgE+ and sIgE-/sIgM+ B lymphocytes. Anti-CD40 on the other hand, exerted an enhancing effect on IgE production and its addition to IL-4 provided a signal that was resistant to the inhibitory effect of NGF. Antagonistic effects of NGF and IL-4 were also observed for other Ig isotypes since IL-4 prevented the increase in IgA and IgM production induced by NGF. These data indicate that although NGFR and CD40 belong to the same receptor superfamily and exert similar proliferative effects on B lymphocytes, they interact differently with IL-4 in the regulation of IgE production.

Transfer of immediate hypersensitivity and airway hyperresponsiveness by IgE-positive B cells.

The role of allergen-specific sIgE+ B cells in the development of airway hyperresponsiveness to electrical field stimulation was examined in a murine model of allergic sensitization. Ovalbumin (OVA)-specific B cells (OVA+) were isolated from mice that were sensitized to aerosolized OVA. The OVA+ B cell population was shown to be distinct from the remaining, non-OVA-responsive B cells (OVA-). There was a high frequency of sIgE+ B cells and a low frequency of sIgG+ B cells in the OVA+ population compared with the OVA- population, where the ratio was reversed. Although both populations produced immunoglobulin in vitro, only the OVA+ cells secreted anti-OVA antibodies. Transfer of 10(6) OVA+ B cells or as few as 5 x 10(4) OVA+/sIgE+ B cells was able to transfer the capability for anti-OVA IgE synthesis and cutaneous reactivity to OVA in naive recipients. Exposure to OVA via the airways in addition to transfer of OVA+ B cells was necessary for development of airway hyperresponsiveness, whereas recipients challenged with an irrelevant allergen, ragweed, had normal airway function. Transfer of up to 10(7) OVA- B cells failed to induce production of anti-OVA IgE. Despite production of polyclonal IgE, recipients of OVA- B cells did not develop airway hyperresponsiveness after OVA challenge. We conclude that both allergen-specific IgE production and local challenge via the airways with specific allergen are necessary to change airway function in this model.

Pretreatment with allergen prevents immediate hypersensitivity and airway hyperresponsiveness.

The ability of subcutaneous pretreatment with an immunogenic peptide derived from Fel d I, the major cat protein, to suppress the development of allergic responses was examined in a mouse model of antigen-induced sensitization. BALB/c mice exposed to aerosolized Fel d I chain 1 peptide developed antigen-specific IgE responses, immediate cutaneous reactivity to the peptide, and increased airway responsiveness (AR). Both subcutaneous and intraperitoneal administration of the peptide prior to sensitization caused a 50% reduction in cutaneous reactivity which was associated with a decrease in serum anti-Fel d I chain 1 IgE and IgG1 antibody responses and an increase in specific IgG. Pretreatment with the peptide also suppressed spleen and lymph node proliferative responses to the peptide. However, only subcutaneous peptide injections could prevent the development of increased AR. Transfer of spleen cells from subcutaneously peptide-treated mice to sensitized recipients reduced serum antigen-specific IgE and IgG1 antibody responses and skin test reactivity, and prevented alterations in AR. These data suggest that IgE (and IgG1) responses and airway hyperresponsiveness induced by allergen sensitization via the airways can be modulated by subcutaneous administration of peptide. Further, the results define a model for investigating the modulatory effects of subcutaneous administration of immunogenic peptides or protein on an ongoing allergic response.

Allergen-specific IgE and IL-5 are essential for the development of airway hyperresponsiveness.

The mechanisms underlying the development of airway hyperresponsiveness are not fully delineated. We addressed this question by studying the effects of passive sensitization with anti-OVA IgE on the development of altered airway responsiveness (AR) following local challenge with OVA in normal and athymic mice. Both normal and athymic BALB/c mice developed allergen-specific immediate cutaneous hypersensitivity after passive sensitization with anti-OVA IgE. In contrast, the combination of local challenge with allergen via the airways and passive sensitization triggered the development of airway hyperresponsiveness only in normal but not in athymic mice. Treatment of athymic mice with IL-5 significantly increased eosinophil accumulation in the lungs after local challenge with OVA; increased airway reactivity was only observed in athymic mice which received anti-OVA IgE, not an unrelated IgE, plus IL-5 treatment and airway challenge with OVA. These findings identify the requirement for allergen-specific IgE and IL-5 for the development of airway hyperresponsiveness following allergen challenge via the airways.

Allergic airway sensitization induces T cell activation but not airway hyperresponsiveness in B cell-deficient mice.

B cells play an important role in the allergic response by producing allergen-specific Igs as well as by serving as antigen-presenting cells. We studied the involvement of B cells in the development of responses in a murine model of allergic airway sensitization. Normal and B cell-deficient (muMt-/-) B10.BR mice were sensitized via the airways to ovalbumin; Ig production, cytokine elaboration from local lymph node cells, development of airway hyperresponsiveness, and histological changes in the airways were evaluated. Both strains of mice had increased production of T helper 2-like cytokines and developed an accumulation of eosinophils in the bronchial tissue after airway sensitization. However, only wild-type mice produced allergen-specific antibodies and exhibited altered airway function. B cell-deficient mice reconstituted with anti-ovalbumin IgE during the course of sensitization developed increases in airway responsiveness. These results indicated that neither B cells nor IgE were necessary for the induction of a T helper 2-type cytokine response or eosinophil infiltration of the airways after allergic sensitization but that IgE was required as a second signal for the development of airway hyperresponsiveness in this model of airway sensitization.

Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography.

To study the mechanisms and kinetics underlying the development of increased airway responsiveness (AR) after allergic sensitization, animal models have been invaluable. Using barometric whole-body plethysmography and increases in enhanced pause (Penh) as an index of airway obstruction, we measured responses to inhaled methacholine in conscious, unrestrained mice after sensitization and airway challenge with ovalbumin (OVA). Sensitized and challenged animals had significantly increased AR to aerosolized methacholine compared with control animals. AR measured as Penh was associated with increased IgE production and eosinophil lung infiltration. In a separate approach we confirmed the involvement of the lower airways in the response to aerosolized methacholine using tracheotomized mice. Increases in Penh values after methacholine challenge were also correlated with increased intrapleural pressure, measured via an esophageal tube. Lastly, mice demonstrating AR using a noninvasive technique also demonstrated increased pulmonary resistance responses to aerosolized methacholine when measured using an invasive technique the following day in the same animals. The increases in Penh values were inhibited by pretreatment of the mice with a beta 2-agonist. These data indicate that measurement of AR to inhaled methacholine by barometric whole-body plethysmography is a valid indicator of airway hyperresponsiveness after allergic sensitization in mice. The measurement of AR in unrestrained, conscious animals provides new opportunities to evaluate the mechanisms and kinetics underlying the development and maintenance of airway hyperresponsiveness and to assess various therapeutic interventions.

Modulation of antigen-induced B and T cell responses by antigen-specific IgE antibodies.

Ag-specific IgE Abs not only mediate immediate hypersensitivity through mast cell activation, but also enhance in vitro Ag presentation and in vivo specific Ab responses in mice. To delineate the role of IgE Ab in the modulation of Ag-specific responses, spleen cells from OVA-sensitized BALB/C mice were cultured together with OVA-specific IgE (or IgG isotypes). OVA-dependent proliferative responses and anti-OVA IgE production were enhanced in the presence of anti-OVA IgE. A significant decrease in IFN-gamma secretion in OVA-stimulated cultures was observed in the presence of anti-OVA IgE, but no changes in IL-4 production were detected. Anti-OVA IgG isotypes or anti-TNP IgE showed no significant effect on any of these Ag-dependent responses. Addition of anti-CD23 Ab abolished these effects of anti-OVA IgE. Further, OVA-sensitized spleen cells from CD23-deficient mice responded to in vitro stimulation with OVA, but demonstrated no modulation by anti-OVA IgE. These results demonstrate that Ag-specific IgE not only augments Ag presentation and T cell proliferation, but also alters the pattern of cytokine production and increases specific IgE synthesis. These modulatory effects of Ag-specific IgE appear to be mediated by binding to Fc epsilon RII/CD23.

Antiinterleukin-5 antibody prevents airway hyperresponsiveness in a murine model of airway sensitization.

Eosinophils play a central role in the inflammatory response associated with bronchial asthma. We studied the involvement of eosinophils in the development of airway hyperresponsiveness (AHR) in a mouse model of allergic airway sensitization. Sensitization of BALB/c mice to OVA via the airways induced allergen-specific T-cell responses, IgE production, immediate cutaneous hypersensitivity (ICH), and increased airway reactivity. Airway sensitization was associated with eosinophil infiltration of the airways and increased production of interleukin-5 (IL-5) in cultures of peribronchial lymph node cells. Treatment of OVA-challenged animals with anti-IL-5 antibody during the sensitization protocol completely abolished the infiltration of eosinophils into the lung tissue and prevented the development of AHR without affecting levels of allergen-specific IgE, cutaneous hypersensitivity and allergen-specific T cell responses. These findings demonstrate that infiltration of lung tissue by eosinophils, triggered by increased IL-5 production, is a major factor in the development of AHR in this mouse model of airway sensitization.

Mast cell tumor necrosis factor alpha production is regulated by MEK kinases.

Mast cells synthesize and secrete specific cytokines and chemokines which play an important role in allergic inflammation. Aggregation of the high-affinity Fc receptor (FcepsilonRI) for immunoglobulin E (IgE) in MC/9 mouse mast cells stimulates the synthesis and secretion of tumor necrosis factor alpha (TNF-alpha). FcepsilonRI aggregation activates several sequential protein kinase pathways, leading to increased activity of extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and the p38 mitogen-activated protein (MAP) kinase. Inhibition of ERKs with the compound PD 098059 had little effect on FcepsilonRI-stimulated TNF-alpha production. Aggregation of FcepsilonRI stimulated MEK kinase 1 (MEKK1) activity, which activates JNK kinase (JNKK), the kinase that phosphorylates and activates JNKs. Expression of activated MEKK1 (DeltaMEKK1) in MC/9 cells strongly stimulated JNK activity but only weakly stimulated p38 activity, and it induced a large activation of TNF-alpha promoter-regulated luciferase gene expression. Inhibitory mutant JNK2 expressed in MC/9 cells significantly blunted FcepsilonRI stimulation of TNF-alpha promoter-driven luciferase expression. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, diminished FcepsilonRI-mediated TNF-alpha synthesis, significantly blunted JNK activation and TNF-alpha promoter-driven luciferase expression, and only weakly inhibited p38 kinase activation. Inhibition of NFkappaB activation resulting from DeltaMEKK1 expression or FcepsilonRI stimulation did not affect TNF-alpha promoter-driven luciferase expression. Our findings define a MEKK-regulated JNK pathway activated by FcepsilonRI that regulates TNF-alpha production in mast cells.