Modulation of inhaled antigen-induced IgE tolerance by ongoing Th2 responses in the lung.

The normal response to inhaled Ag is the absence of Ag-specific IgE and cytokine production to later Ag challenges. Although the mechanism of this aerosol-induced IgE tolerance is not completely understood, it may prevent sensitization to inhaled Ags, which could otherwise lead to allergy and asthma. We examined the consequences of ongoing Th1 and Th2 responses in the lungs of mice during OVA inhalation to mimic conditions that may subvert tolerance and lead to sensitization. We found that concurrent, secondary Th2 lung responses to keyhole limpet hemocyanin or primary responses to Nippostrongylus larvae or Asperigillus fumagatus extract prevented establishment of IgE tolerance to aerosolized OVA. Intranasal rIL-4 given before OVA aerosolization also prevented establishment of tolerance, whereas concurrent Th1 responses to influenza virus or Mycobacterium bovis bacillus Calmette-Guérin had no effect. However, once established, aerosol tolerance to OVA could not be completely broken by OVA rechallenge concurrent with a secondary Th2 response to keyhole limpet hemocyanin or A. fumagatus extract, or by intranasal rIL-4. These data suggest that the immune status of the lung of an individual may profoundly influence the initial response to inhaled Ag, and that aerosol-induced IgE tolerance may not be appropriately established in individuals undergoing concurrent, Th2-mediated responses to Ags or pathogens.

Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3).

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor, C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells, dendritic cells, or cells from the thymus, lymph node, or spleen of normal mice. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge, the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection, isolation, and in vivo depletion of eosinophils.

Aerosol-induced immunoglobulin (Ig)-E unresponsiveness to ovalbumin does not require CD8+ or T cell receptor (TCR)-gamma/delta+ T cells or interferon (IFN)-gamma in a murine model of allergen sensitization.

Mice exposed for 20 min daily to aerosolized ovalbumin (OVA) for 10 d at concentrations from 1 to 0.01% OVA made greatly reduced immunoglobulin (Ig)-E responses to subsequent immunogenic OVA challenges, given either intraperitoneally or by aerosol. This IgE-specific unresponsiveness lasted for at least four months. However, these aerosol-treated mice were primed for larger OVA-specific IgG1 and IgG2a responses. The specific reduction in IgE responses was not due to preferential induction of a T helper (Th)-1 response as aerosol OVA- primed mice made greatly reduced Th2 and no detectable Th1 response after rechallenge in vitro. Consistent with this, the increase in circulating eosinophils observed in control Th2-primed mice was absent in aerosol OVA-treated animals. Interferon (IFN)-gamma was not required for this unresponsiveness, as IFN-gamma knockout mice and anti-IFN-gamma antibody-treated wild-type mice had greatly reduced levels of IgE similar to wild-type controls. CD8+ T cells played a relatively small role as IgE responses were reduced to about the same extent in beta2 microglobulin-deficient, or in anti-CD8-treated wild-type mice as in normal mice after aerosol OVA treatment. Similarly, T cell receptor (TCR)-gamma/delta T cells were not required for maximal inhibition of the IgE response. These results demonstrate that exposure to inhaled protein antigens can induce a state of unresponsiveness of CD4+ T cells that results in a prolonged loss of IgE and eosinophil responses to subsequent challenges. This T cell unresponsiveness was shown not to require CD8+ or TCR-gamma/delta+ T cells or IFN-gamma.

Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis.

We have used interleukin-10 (IL-10) gene knockout mice (IL-10-/-) to examine the role of endogenous IL-10 in allergic lung responses to Aspergillus fumigatus Ag. In vitro restimulated lung cells from sensitized IL-10-/- mice produced exaggerated amounts of IL-4, IL-5, and interferon-gamma (IFN-gamma) compared with wild-type (WT) lung cells. In vivo, the significance of IL-10 in regulating responses to repeated A. fumigatus inhalation was strikingly revealed in IL-10-/- outbred mice that had a 50-60% mortality rate, while mortality was rare in similarly treated WT mice. Furthermore, IL-10-/- outbred mice exhibited exaggerated airway inflammation and heightened levels of IL-5 and IFN-gamma in bronchoalveolar lavage (BAL) fluids. In contrast, the magnitude of the allergic lung response was similar in intranasally (i.n.) sensitized IL-10-/- and wild-type mice from a different strain (C57BL/6). Using a different route of priming (intraperitoneal) followed by one i.n. challenge we found that IL-10-/- C57BL/6 mice had heightened eosinophilic airway inflammation, BAL-IL-5 levels, and numbers of alphabetaT cells in the lung tissues compared with WT mice. We conclude that IL-10 can suppress inflammatory Th2-like lung responses as well as Th1-like responses given the constraints of genetic background and route of priming.

Second-hand smoke is an adjuvant for T helper-2 responses in a murine model of allergy.

Epidemiologic studies have suggested that "second-hand smoke" increases the incidence and severity of allergies and asthma. To address this, we developed a murine model that included generation of and exposure to environmental tobacco smoke (ETS) followed by aerosolized Ag challenge. We report herein that exposure of OVA-sensitized BALB/c mice to ETS elicits a rapid and prolonged exaggerated response with respect to IgE, IgG1, eosinophils, and Th2 cytokines (particularly IL-4 and IL-10). There was a significant increase (p = 0.0159) in the total cells of the bronchoalveolar lavage in OVA-sensitized mice exposed to ETS when compared with those in ambient air. This is the first experimental data to demonstrate that "second-hand smoke" up-regulates the allergic response to inhaled Ag. The results have important health implications for children and allergic individuals residing in households with smokers.

Particulate Aspergillus fumigatus antigens elicit a TH2 response in BALB/c mice.

BACKGROUND: In order to understand the immunoregulation in allergic aspergillosis, a murine model was developed by exposing animals to particulate and soluble antigens of Aspergillus fumigatus. METHODS: BALB/c mice were exposed to soluble Aspergillus antigen intranasally. Animals showing moderate levels of IgE were subsequently exposed to soluble antigen or antigen conjugated to polystyrene beads intranasally. The IgE and IgG1 in the sera and the eosinophils in the blood and lungs were studied. The spleen and lung mononuclear cells were stimulated with both concanavalin A and antigen and evaluated for production of interleukin (IL)-4, IL-5, IL-10, and interferon-gamma. RESULTS: Animals exposed to particulate antigens showed more elevated serum IgE levels and increased numbers of eosinophils in the blood and lungs than those exposed to soluble antigen. Lung cell cultures from animals exposed to particulate antigens when stimulated with Aspergillus produced IL-4 and IL-5, indicating a TH2 type of response. Animals exposed to soluble antigens showed a weaker TH2 response, as evidence by low IgE levels in sera, fewer eosinophils in the blood, and low levels of cytokine production from lung and spleen cells. CONCLUSION: The results indicate that the physical nature of the antigen may have a major role in determining the type of immune response of the host.

A murine model of allergic bronchopulmonary aspergillosis with elevated eosinophils and IgE.

A model of allergic bronchopulmonary aspergillosis was developed by exposing BALB/c mice to Aspergillus fumigatus (AF) Ag. Animals immunized intranasally (i.n.) with soluble AF Ag produced low levels of serum IgE compared to animals given alum precipitated AF Ag i.p. The latter treatment also produced higher levels of serum IgG1 and AF-specific IgG1 than soluble AF given i.p. or i.n.. Blood and lung eosinophilia was detected in mice repeatedly exposed to AF by i.n. but not in the groups injected i.p. Particulate AF Ag-induced striking blood and lung eosinophilia and elevated levels of serum IgE in mice preexposed to AF Ag. The results indicate that route of inoculation and physical nature of Ag determine the immune response and can be manipulated to obtain enhanced IgE, eosinophils, or both in the animal model.

Effects of in vivo administration of interferon (IFN)-gamma, anti-IFN-gamma, or anti-interleukin-4 monoclonal antibodies in chronic autoimmune graft-versus-host disease.

These studies examined the role of cytokines in chronic autoimmune graft-versus-host disease (GVHD) in B6D2F1 mice injected with lymphoid cells from DBA/2 mice. Anti-interleukin (IL)-4 and anti-interferon (IFN)-gamma mAb, or IFN-gamma, were used in vivo to modulate B cell hyperactivity and disease. Kinetic experiments showed that, 2-3 weeks after induction, GVH mice had 100x elevated serum IgE, while IgG1 and IgG2a were 10x above normal. Early treatment with anti-IL-4 mAb or IFN-gamma decreased serum IgE and IgG1 and had no effect on IgG2a. Anti-IFN-gamma mAb treatment increased serum IgE and IgG1 while reducing IgG2a. This increase in serum immunoglobulins could be correlated with an increased spontaneous secretion of IL-4, IL-5, and IL-6 in spleen cell cultures from anti-IFN-gamma mAb-treated GVH mice. While neither anti-IFN-gamma nor IFN-gamma treatments altered the disease course, anti-IL-4 treatment delayed proteinuria and death in GVH mice. These observations suggest an important role for IL-4 in immune complex-mediated glomerulonephritis in chronic GVHD.

IL-4 can correct defective IgE production in SJA/9 mice.

SJA/9 mice are unable to produce significant serum IgE responses to either primary or secondary challenge with protein Ag or helminth parasites. Previous work has shown that this defect resides the T cell rather than with the B cells. These experiments test several possible explanations for the lack of IgE production in these mice. SJA/9 mice do not appear to overproduce IFN-gamma or any other antagonist of IL-4 function. Helminth parasite infection induces normal levels of IL-5-dependent eosinophilia, suggesting that there is not a complete absence of a Th2 response in these mice. The defect, however can be explained by a substantial reduction in IL-4 production in vivo and can be corrected by infusion with IL-4. The reason for this lack of IL-4 production is not yet clear and, paradoxically, SJA/9 T cells can be stimulated in vitro to produce IL-4 levels comparable to T cells from other strains.

In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice.

Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5-responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.

Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice.

When rodents are infected with the nematode Nippostrongylus brasiliensis, large numbers of eosinophils appear in their blood and lungs and their serum immunoglobulin E (IgE) is increased. Injection of a monoclonal antibody to interleukin-5 completely suppressed the blood eosinophilia and the infiltration of eosinophils in the lungs of parasitized mice but had no effect on serum IgE. In contrast, an antibody to interleukin-4 inhibited parasite-induced IgE but not the eosinophilia. These results show that interleukin-5 is important in eosinophil production in vivo and that IgE and eosinophil production are regulated by different cytokines produced by the TH2 subset of CD4-expressing T cells.

Similar chemotactic factor for monocytes predominates in different animal models of uveitis.

Anterior uveitis or iritis occurs in a variety of systemic diseases including sarcoid, Behcet's, and spondyloarthritis. Iritis is, therefore, presumed to result from a variety of pathogenetic mechanisms. We hypothesized that unique chemotactic factors should be associated with different etiologies for inflammation. We have tested this hypothesis using rabbit models of anterior uveitis. We have found that aqueous humor generally contained chemotactic activity for monocytes 24 h after an intravitreal injection of endotoxin, killed mycobacteria, or human serum albumin (in a rabbit previously immunized against human serum albumin). Anterior chamber paracentesis resulted in aqueous humor with a high protein content. However, in contrast to the other models of inflammation, paracentesis did not result in a cellular infiltrate in the anterior chamber, and aqueous humor after paracentesis was not chemotactic. For either immunologically mediated inflammation or for inflammation resulting from injection of a killed bacterial product, chemotactic activity could be digested by papain or trypsin and tended to coelute with albumin on either gel filtration or ion-exchange chromatography. These observations suggest that a similar chemotactic factor for monocytes appears to be associated with ocular inflammation that follows either an immune response or injection of a killed bacterial product.

The effect of corticosteroids or nitrogen mustard on aqueous humor chemotactic activity induced by intravitreal endotoxin.

In rabbits intravitreal injection of endotoxin induces potent chemotactic activity for monocytes in aqueous humor. We have assessed the effect of methylprednisolone (30 mg/kg intramuscularly) or nitrogen mustard (1.75 mg/kg intravenously) on the generation of this chemotactic activity. Histologic changes, anterior chamber protein extravasation, and aqueous humor cellular infiltration were reduced by corticosteroids to a variable degree. However, even in animals with a marked reduction in protein extravasation or histologic change, chemotactic activity was substantially preserved. Similarly, nitrogen mustard induced a leukopenia without affecting the ability of endotoxin to generate chemotactic activity in aqueous humor. In contrast, corticosteroids reduced both protein extravasation and the generation of chemotactic activity induced by intravenously injected endotoxin. The results suggest that the chemotactic activity induced in the eye by intravitreal endotoxin may be locally synthesized and may be present without a substantial leukocytic infiltrate.