Suppression of Th2 immune responses by mekabu fucoidan from Undaria pinnatifida sporophylls

BACKGROUND: We demonstrated that mekabu fucoidan obtained from Undaria pinnatifida (Up) sporophylls augments the type 1 T-helper (Th1) cell response in normal BALB/c mice. In this study, we examined the effects of the fucoidan of mekabu on the type 2 T-helper (Th2) response in bronchoalveolar lavage fluid (BALF) after ovalbumin (OVA) aerosol challenge. METHODS: Mekabu fucoidan (50 mg/kg) was injected intraperitoneally into BALB/c mice for 4 days, and then the mice were sensitized with 50 microg/mouse of OVA plus alum (1 mg/mouse) 1 and 8 days later. The mice were challenged with OVA delivered using a nebulizer 7, 8 and 9 days after the second challenge with OVA plus alum. After 24 h, we assessed T cell responses in BALF by measuring the amount of Th2 cytokines (IL-4, IL-5, IL-13) and gamma-interferon (IFN-gamma) produced by Th1 cells. RESULTS: The production of Th2 cytokines was suppressed (p < 0.05), and the amount of IFN-gamma was not increased in the mice treated with mekabu fucoidan. Anti-OVA immunoglobulin E (IgE) and IgE levels in serum determined after challenge with aerosolized OVA at the end of the experiment were lower (p < 0.05) in the treated than in the control mice. CONCLUSIONS: The pulmonary inflammation was relieved by mekabu fucoidan, which also downregulated Th2-dominated responses. These results indicate that mekabu fucoidan modulates Th2 responses and might be useful for treating allergic inflammation.     

Interleukin-18 plays a role in both the alum-induced T helper 2 response and the T helper 1 response induced by alum-adsorbed interleukin-12

Previous studies have shown that the antigen-specific T helper 2 (Th2) response induced by alum adjuvants is interleukin (IL)-4 independent. As a role for IL-18 in Th2 induction has recently been described, in addition to its role in enhancing Th1 responses, we have studied the Th2 response induced by ovalbumin (OVA) adsorbed to alum in wild-type and IL-18-deficient mice. Our results indicate that while endogenous IL-18 facilitates alum-induced IL-4 production, OVA-specific immunoglobulin G1 (IgG1) and IgE production remain unaffected. Furthermore, antigen-specific Th1 responses induced with alum/IL-12-adsorbed OVA were demonstrated to be highly IL-18 dependent. Despite these observations, injection of BALB/c mice with exogenous IL-18 adsorbed to alum/OVA did not alter IL-4 or interferon-gamma production by T cells and had little effect on the relative production of IgG1/IgG2a antibody subclasses compared with alum/OVA inoculated mice. However, the previously described synergism between IL-12 and IL-18 in Th1 induction was evident as the Th1-promoting activity of alum/IL-12 against adsorbed OVA was greatly augmented by the coadministration of IL-18. These results indicate that while alum-induced IL-18 can facilitate Th2 induction, the addition of exogenous IL-18 cannot further enhance the alum-induced Th2 response.     

Antigen dose defines T helper 1 and T helper 2 responses in the lungs of C57BL/6 and BALB/c mice independently of splenic responses

To investigate the effect of antigen dose on immune response, C57BL/6 and BALB/c mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) then challenged with aerosolized OVA. Low-dose sensitization (less than 8 microg of OVA) elicited T helper 2 (Th2)-type immunoglobulins (Igs) secretion from C57BL/6 mice, including high levels of serum IgE, IgG1 and low levels of IgG2a, while BALB/c mice secreted T helper 1 (Th1)-type Igs, including low levels of IgE, IgG1 and high levels of IgG2a. In contrast, high-dose sensitization (more than 50 microgram) elicited Th1-type Igs secretion in C57BL/6mice, while BALB/c mice exhibited Th2-type Igs secretion. Furthermore, the number of eosinophils infiltrating into the lungs of low-dose OVA-sensitized C57BL/6 mice was significantly greater than in BALB/c mice sensitized with the same amount of OVA. Only a very high dose of OVA (1 mg) could induce greater eosinophil infiltration into the lungs of BALB/c mice compared with C57BL/6 mice. Additionally, low-dose sensitization generated Th2-type cytokines, including high levels of interleukin (IL) -4, IL-5 and a low level of interferon-gamma (IFN-gamma) in the lungs of C57BL/6 mice, while BALB/c mice generated Th1-type cytokines in their lungs, including low levels of IL-4, IL-5 and a high level of IFN-gamma. In contrast, high-dose sensitization elicited Th1-type cytokines production in the lungs of C57BL/6 mice, while BALB/c mice generated Th2-type cytokines in their lungs. Interestingly, splenocyte cultures from C57BL/6 mice produced Th1-type cytokines, while cultures from BALB/c mice produced Th2-type cytokines regardless of OVA sensitization dose (100 ng-1 mg). These results indicate that C57BL/6 and BALB/c mice have different susceptibilities to OVA-sensitization and OVA-induced pulmonary eosinophilia regulated by Th1- and Th2-type cytokines, independent of splenic Th1- and Th2-type cytokines production.     

Comparison between Ovalbumin and Ovalbumin Peptide 323‐339 Responses in Allergic Mice: Humoral and Cellular Aspects

Ovalbumin (OVA) is widely used in allergy research. OVA peptide 323‐339 has been reported to be responsible for 25–35% of isolated BALB/c mouse T‐cell response to intact OVA. An investigation of whether OVA and OVA 323‐339 molecules can induce equivalent in vivo and in vitro immune responses was conducted. Eight‐week‐old BALB/c mice were randomly divided into three groups: OVA, OVA 323‐339 and saline. On days 0, 7, 14, mice were intraperitoneally injected with 25 μg OVA or OVA 323‐339 absorbed on 300 μg Alum, or saline; on days 21–23, all groups were challenged intranasally with either 20 μl of 1% OVA, 1% OVA 323‐339 or saline. On day 28, after killing, splenocytes were isolated and cultured under the stimulus of each allergen or medium. Evaluated by hematoxylin/eosin and major basic protein immunohistochemical stainings, OVA and OVA 323‐339 induced similar lung inflammation. Interestingly, significant serum total IgE and OVA‐specific IgE were observed in OVA mice when compared to saline control. OVA 323‐339 mice showed higher serum OVA‐specific IgE, OVA 323‐339‐specific IgE, IL‐4 and lower IFN‐γ similar to OVA mice. The proliferative response to OVA was found in cultured splenocytes of both OVA and OVA 323‐339 mice, while the similar proliferative response to OVA 323‐339 was only observed in the splenocytes of OVA 323‐339‐sensitized and challenged mice. Although OVA 323‐339 induced a Th2‐like response in the mouse model as did OVA, OVA 323‐339 has clearly limited immunogenic potency to activate OVA‐sensitized and challenged mice splenocytes, unlike OVA.     

Interleukin-10-treated dendritic cells do not inhibit Th2 immune responses in ovalbumin/alum-sensitized mice

BACKGROUND: It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). METHODS: OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. RESULTS: Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. CONCLUSIONS: Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.     

4-1BB stimulation inhibits allergen-specific immunoglobulin E production and airway hyper-reactivity but partially suppresses bronchial eosinophilic inflammation in a mouse asthma model

BACKGROUND: 4-1 BB, a member of the tumour necrosis factor receptor superfamily, functions as a co-stimulatory molecule. Recently, stimulation of the 4-1 BB pathway was shown to suppress antigen-specific CD4(+) T cell and subsequent T cell-dependent humoral immune responses. OBJECTIVE: We examined the effect of agonistic anti-4-1 BB monoclonal antibody (mAb) treatment on allergic asthma, in which allergen-specific type 2 helper T cells (Th2) have been shown to play an important role. METHODS: BALB/c mice were systemically sensitized with intraperitoneal injections of ovalbumin (OVA) and alum on days 0 and 14, and then challenged with inhaled OVA on days 28, 29 and 30. In test groups, the agonistic anti-4-1 BB mAb was administered at the time of initial systemic sensitization with OVA. On day 31, mice were challenged with inhaled methacholine, and enhanced pause was measured as an index of airway hyper-responsiveness (AHR). Levels of OVA-specific IgE in serum, and levels of various cytokines in bronchoalveolar lavage (BAL) fluids were measured. The severity of airway inflammation was determined by differential cell counts in BAL fluids and histopathologic lung analysis. To evaluate local immunity, we cultured lymphocytes from draining perihilar lymph nodes and evaluated the proliferative response to OVA and the levels of IL-5 in the culture supernatant. In addition, the functional mechanism of 4-1 BB stimulation was evaluated in splenocytes obtained at day 7 after systemic OVA sensitization. RESULTS: We found that treatment with the anti-4-1 BB mAb significantly decreased AHR and the production of allergen-specific IgE. Bronchial inflammation, however, had only partially improved and the levels of IL-4 and IL-5 in BAL fluids showed only a small degree of reduction compared with the control Ig-treated mice. Thoracic lymphocytes from anti-4-1 BB-treated mice showed significant suppression of OVA-induced proliferation and IL-5 production. In anti-4-1 BB-treated mice, splenocytes exhibited poor proliferation and marked apoptosis 7 days after systemic OVA challenge. CONCLUSION: These results suggest that stimulation of the 4-1 BB pathway effectively suppresses some features of allergic asthma, including allergen-specific IgE production and AHR, through deletion of allergen-specific Th2 cells. However, we found that bronchial allergic inflammation was not strictly mediated by suppression of the Th2 immune response in this murine model of asthma. Despite these somewhat contradictory effects, intervention in the 4-1 BB pathway might provide a potential novel immunotherapeutic approach for treatment of allergic asthma.     

Importance of inflammatory and immune components in a mouse model of airway reactivity to toluene diisocyanate (TDI)

BACKGROUND: Nearly 9 million individuals are exposed to agents in the workplace associated with asthma, and isocyanates represent the most common cause of occupationally induced asthma. OBJECTIVES: Nonetheless, the immunological mechanisms responsible for isocyanate-induced asthma are not clear. A murine model for toluene diisocyanate (TDI) asthma is described and employed to examine inflammatory and immune components that may be involved in the disease. METHODS: Groups (n = 6) of C57BL/6J and athymic mice were sensitized by subcutaneous injection (20 microl on day 1, 5 microl on days 4 and 11), and 7 days later challenged by inhalation (100 p.p.b., days 20, 22 and 24) with TDI. Twenty-four hours following the last challenge the tracheae and lungs were examined for histological changes as well as for the expression of Th1, Th2 and pro-inflammatory cytokines. Mice were also examined for airway reactivity to methacholine challenge and for specific and total IgE and IgG antibodies. RESULTS: TDI sensitization resulted in increased reactivity to methacholine challenge as well as a significant inflammatory response in the trachea and nares of wild-type mice, but not in the athymic mice nor in the lungs of the C57BL/6J mice. Airway inflammation was characterized by inflammatory cell influx, goblet cell metaplasia and epithelial damage. Histological changes in the trachea were accompanied by increased mRNA expression of interleukin (IL)-4, tumour necrosis factor alpha, lymphotoxin beta, lymphotactin and Rantes, as well as TDI-specific IgG antibodies and elevated levels of total IgE. IgE-specific antibodies were not detected with this exposure regimen but were produced when the TDI concentrations were increased. CONCLUSIONS: These studies provide a unique murine model for occupational asthma that generates both inflammatory and immune mediators similar to those occurring in TDI-induced asthma in humans.     

Characteristic features of allergic airway inflammation in a murine model of infantile asthma

BACKGROUND: The pathophysiology of infantile asthma may differ from that in older children or in adults, partly because of the different immune response depending upon maturation. In adult mice, the sensitizing dose of antigen is known to be critical to the polarized development of helper T cell subsets and allergic airway inflammation. We wanted to know the characteristics of allergic airway inflammation of infantile asthma by developing a murine model. METHODS: BALB/C mice at different stages of maturation (juvenile: 3 days after birth; adult: 8 weeks of age) were sensitized with 10 or 1,000 microg ovalbumin (OVA) or vehicle. The animals were then challenged with aerosolized OVA or saline once a day during 6 consecutive days. After the final challenge, bronchial hyperresponsiveness (BHR), bronchoalveolar lavage fluid (BALF), histological changes in the airways and immunological status were examined. RESULTS: In both juvenile and adult animals, sensitization with 10 microg OVA induced the T helper 2 response (elevated IL-4 and decreased IFN-gamma levels). BHR, airway eosinophilia, the inflammatory cell infiltration, goblet cell metaplasia (GCM), and IgE antibody production were more prominent in animals given this dose than 1,000 microg OVA. Among these responses, GCM as well as BALF IL-4, and BHR were comparable between juvenile and adult animals, whereas other parameters were lower in juvenile animals, especially in those given 1,000 microg OVA. CONCLUSION: GCM and, consequently, airway mucus hypersecretion may be an important component of allergic airway inflammation in infantile asthma.     

Deviation of the allergic IgE to an IgG response by gene immunotherapy

The Th1/Th2 type immune response to E. coli beta-galactosidase (beta-gal) was compared to that to gene vaccination with plasmid (p) DNA encoding beta-gal. BALB/c mice were immunized with beta-gal in alum or a pDNA construct consisting of a CMV-based promoter and the beta-gal gene (pCMV-LacZ). Beta-gal in alum induced IgG1 and IgE antibodies and the CD4+ T cells from these mice secreted interleukin 4 (IL-4) and IL-5 but no interferon-gamma (IFN-gamma) after in vitro antigen stimulation. In contrast, mice immunized with pCMV-LacZ formed predominantly IgG2a antibodies and their CD4+ T cells secreted IFN-gamma but no IL-4 and IL-5. These data indicate that beta-gal induced a Th2 and the pCMV-LacZ a Th1 response to beta-gal. The pDNA induced Th1 response dominated over the Th2 response. Mice primed with pCMV-LacZ failed to produce IgE antibodies after a booster injection of beta-gal in alum. Boosting of mice primed with beta-gal in alum with pCMV-LacZ resulted in a 75% decrease in the IgE antibody titer within 6 weeks and IgG2a antibody formation and CD4+ T cells that secreted IFN-gamma in amounts similar to T cells from pDNA primed mice. As shown by adoptive cell transfer, both CD4+ and CD8+ T cells from pDNA immunized mice inhibited an IgE response to beta-gal in alum in the recipient mice. pDNA immunization also inhibited the eosinophilic infiltration of the lung of ovalbumin (OVA) immunized mice after OVA inhalation challenge in an animal model of the late phase reaction. The mechanism of the pDNA induced Th1 immune response was shown to be the result of stimulation by distinct non-coding immunostimulatory DNA sequences (ISS) in the backbone of the pDNA. The ISS induced antigen presenting cells to secrete cytokines that cause naive T cells to differentiate into Th1 cells (e.g. IFN-alpha, IL-12). The data indicate that gene vaccination induces a Th1 immune response that is capable of down-regulating a preexisting Th2 response and IgE antibody formation. Thus, immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.     

Prolonged antigen exposure ameliorates airway inflammation but not remodeling in a mouse model of bronchial asthma.

BACKGROUND: In naive rodents, repeated exposure to aerosolized antigen induces suppression of the Th2 response to the antigen. We hypothesized that more prolonged exposure of established asthma model to antigen aerosols may downregulate asthmatic phenotype. METHODS: After establishing an ovalbumin (OVA)-induced asthma model, mice were further exposed to OVA (prolonged exposure group) or phosphate-buffered saline (positive controls) 3 days per week for 6 weeks. During week 7, the mice of both groups were finally challenged with OVA. RESULTS: Prolonged OVA exposure resulted in marked suppression of serum OVA-specific immunoglobulin E (IgE) antibody levels, eosinophilia of the airway, and airway hyperresponsiveness (AHR). However, airway remodeling characterized by goblet cell hyperplasia and airway fibrosis was observed to the same degree in both groups. These effects were accompanied by diminished production of Th2 cytokines such as interleukin-4 (IL-4), IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF) and cultured supernatant of splenocytes. Furthermore, prolonged exposure markedly increased IL-12 levels in BALF. CONCLUSIONS: Prolonged antigen exposure has inhibitory effects on eosinophilic inflammation, AHR and IgE response to antigen, but not on airway remodeling, presumably via inhibition of Th2 cytokines and increased IL-12 production in the lungs.     

Effect of neonatal sublingual vaccination with native or denatured ovalbumin and adjuvant CpG or cholera toxin on systemic and mucosal immunity in mice

Sublingual immunotherapy has been applied for allergic diseases, but whether sublingual immunization in neonates can prevent sensitization has not been studied. In this study, we evaluate the effect of neonatal sublingual vaccination with native or denatured allergens alone or plus adjuvant on allergy prevention. Newborn BALB/ c mice were sublingually vaccinated daily for the first 3 days with native or denatured ovalbumin (OVA) only, or combined adjuvant CpG or cholera toxin (CT). They were sensitized with OVA adsorbed onto alum 7 weeks after the last vaccination. Specific secretory IgA antibody responses were readily induced by neonatal vaccination with antigen plus CpG or CT, but not with antigen alone. Whereas vaccination with denatured OVA plus CpG markedly enhanced T helper 1 (Th1) responses and inhibited IgE production, vaccination with denatured OVA plus CT increased cervical lymph node cell production of interleukin-4 (IL-4), IL-5, IL-6, and serum IgG1 responses. These data demonstrate that neonatal sublingual vaccination with denatured OVA and CpG not only preferentially induces systemic Th1 responses and mucosal immunity, but also simultaneously abrogates IgE production. Neonatal sublingual vaccines may play a role for the strategy of allergy prevention.     

Dietary nucleotides can up-regulate antigen-specific Th1 immune responses and suppress antigen-specific IgE responses in mice

BACKGROUND: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific Th1 and Th2 responses and IgE responses. METHODS: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(-) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA. RESULTS: The level of production of antigen-specific interferon-gamma by spleen cells was significantly higher in the OVA-TCR Tg mice fed the NT(+) diet than in the control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(-) diet. CONCLUSION: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response.     

Status of Stat3 in an ovalbumin-induced mouse model of asthma: analysis of the role of Socs3 and IL-6

BACKGROUND: Stat3, Socs3 and cytokines play an integral role in the coordination and persistence of inflammation. However, a clear understanding of the role played by the Stat3/IL-6 and Socs3 pathway in airway inflammation is lacking. We report the alteration in the status of expression and activation of Stat3 by ovalbumin (OVA), and establish its relationship with Socs3 and IL-6 in the lungs of mice with eosinophilic pulmonary inflammation and airway hyperresponsiveness. METHODS: Alterations in the expression of Stat3, Socs3 and IL-6 were determined in a murine model of asthma, where Balb/c mice were sensitized and challenged with OVA (OVA/OVA) and compared with control mice sensitized and challenged with saline (SAL) (SAL/SAL) mice. The OVA/OVA mice were characterized by a moderate increase in methacholine-induced specific airway resistance, the presence of 150 microg/ml of OVA-specific IgG and 8.93 microg/ml OVA-specific IgE antibody and elevated levels of eosinophils and Th2 cytokines (IL-4 and IL-5) in the bronchoalveolar lavage fluid. In contrast SAL/SAL mice had low eosinophils, IL-4 and IL-5 and no OVA-specific IgG and IgE antibodies in the BALF. Stat3 and Socs3 expression profiles were monitored in OVA/OVA and Stat3- and Socs3-silenced OVA/OVA mice. Furthermore, expression of IL-6 in Stat3- and Socs3-silenced mice and the exogenous effect of IL-6 on Stat3 were studied. RESULTS: The results show that expression and activation of Stat3 mRNA and proteins are significantly low in lung of OVA/OVA mice in comparison to SAL/SAL mice following OVA challenge. An increased pool of Socs3 mRNA is observed in OVA/OVA mice with or without OVA challenge and in SAL/SAL mice 24 h after OVA challenge. Transient in vivo blocking of Socs3 gene by Socs3 siRNA restores the expression of IL-6 mRNA and protein in OVA/OVA mice, and nasal administration of recombinant IL-6 to OVA/OVA mice enhanced Stat3 mRNA expression. CONCLUSIONS: Our data suggest that airway inflammation is associated with low expression of Stat3 and IL-6 and overexpression of Socs3 genes in a mouse model of asthma. Furthermore, IL-6 is under the influence of the Socs3 gene and may contribute to the negative regulation of Stat3 via IL-6 following a challenge with an allergen during the development of asthma.     

Ginsenoside Rg1 and aluminum hydroxide synergistically promote immune responses to ovalbumin in BALB/c mice

The combined adjuvant effect of ginsenoside Rg1 and aluminum hydroxide (alum) on immune responses to ovalbumin (OVA) in mice was investigated. BALB/c mice were subcutaneously (s.c.) inoculated twice with OVA alone or in combination with Rg1, alum, or Rg1 plus alum. Samples were collected 2 weeks after the boosting for the measurement of anti-OVA immunoglobulin G (IgG) isotypes in sera and gamma interferon (IFN-γ) and interleukin-5 (IL-5) produced in singular splenocyte cultures. Delayed-type hypersensitivity (DTH) responses were measured in mice immunized as described above. After 10 days, the mice were injected s.c. with OVA at the footpads. Thereafter, the thickness of the footpads was measured once daily for 5 days. The results indicated that alum enhanced mainly Th2 (IgG1 and IL-5) responses (P < 0.05), while Rg1 enhanced both Th1 (IgG1 and IL-5) and Th2 (IgG2a, IFN-γ, and DTH) responses (P < 0.05). The highest immune responses were found in the mice injected with OVA solution containing both alum and Rg1. In addition, the hemolytic activity of Rg1 was much lower than that of Quil A. Therefore, Rg1 deserves further studies in order to tailor desired immune responses when a mixed Th1/Th2 immune response is needed.     

IL-10 controls Th2-type cytokine production and eosinophil infiltration in a mouse model of allergic airway inflammation

Interleukin-10 was originally described as a factor that inhibits cytokine production by murine Th1 clones. Recent studies have since shown that IL-10 can also downregulate Th2 clones and their production of IL-4 and IL-5. Because of its immuno-suppressive properties, IL-10 has been suggested as a potential therapy for allergic inflammation and asthma. However, the pathophysiological role of IL-10 in vivo has not been clearly elucidated. We investigated the effects of IL-10 administration on the production of IgE, cytokine and allergen-induced Th2 cytokine production as well as its effects on eosinophilic inflammation. We established GATA-3/TCR double transgenic (GATA-3/TCR-Tg) mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice; these mice were then sensitized using an intraperitoneal injection of OVA adsorbed to alum and challenged with the intratracheal instillation of an allergen. When GATA-3/TCR-Tg mice sensitized with OVA and alum were injected with C57-IL-10 cells before OVA inhalation, the levels of IL-5, IL-13, and IL-4 decreased by 40-85% and number of eosinophils decreased by 70% (P < 0.03) in the murine bronchoalveolar lavage fluid (BALF). These results suggest that IL-10 plays an important role downstream of the inflammatory cascade in the Th2 response to antigens and in the development of BALF eosinophilia and cytokine production in a murine model of asthma. These immunosuppressive properties in animal models indicate that IL-10 could be a potential clinical therapy for the treatment of allergic inflammation.     

Mycobacterium vaccae administration during allergen sensitization or challenge suppresses asthmatic features

BACKGROUND AND OBJECTIVE: The hygiene hypothesis suggests that a lack of bacterial infections would favour the development of allergic disease. For this reason, bacteria or their components can be used as potential treatment for allergic asthma. We investigated whether heat-killed Mycobacterium vaccae is either able to suppress the induction of allergic asthma or able to suppress already established allergic asthma. METHODS: Mice were sensitized with ovalbumin (OVA)/alum on days 0 and 14. Thereafter, mice were challenged on days 35, 39 and 42 by inhalation of either OVA or saline aerosols. M. vaccae-treated mice received an injection with 106, 107 or 108 CFU heat-killed M. vaccae on days 0 and 14 or 107 CFU on days 35 and 39. On day 43, the airway responsiveness of the mice to increasing concentrations of methacholine was assessed, blood was withdrawn to measure serum parameters, and lung lavage was performed to detect cytokines and inflammatory cell number. RESULTS: Treatment of OVA-sensitized mice with 107 CFU M. vaccae either during sensitization or challenge suppresses airway hyper-responsiveness, airway eosinophilia and IL-5 production after OVA challenge. The increases in OVA-specific serum IgE and in IL-4 by respiratory challenges with OVA were only diminished after M. vaccae treatment (107 CFU) during sensitization. CONCLUSIONS: Heat-killed M. vaccae prevents allergic and asthmatic manifestations in a mouse model and, more importantly, M. vaccae treatment during challenge suppresses features of asthma, which opens up possibilities for new therapeutic interventions.     

Heat Denaturation of Egg-White Proteins Abrogates the Induction of Oral Tolerance of Specific Th2 Immune Responses in Mice

Human foods are usually prepared by cooking. Boiling of chicken egg-white (EW) led to decreased allergenicity, and abrogated intestinal uptake of immunoreactive ovalbumin (OVA) when fed to mice. Therefore, the effects of oral administration of boiled EW were examined further in BALB/c mice. Specific IgE, IgG₁ and IgG antibody responses were suppressed by raw EW, but not by EW boiled for 5 or 60 min, fed prior to sensitization with 10  μ g OVA or 1  μ g DNP–OVA in alum. Similar results were obtained when mice were sensitized with 10  μ g conalbumin, ovomucoid or lysozyme in alum. BALB/c spleen cell proliferation and secretion of Th2 cytokines IL-4 and IL-5 during in vitro stimulation with OVA were also suppressed by feeding raw EW, but not by boiled EW. Although heat denaturation of proteins can minimize allergenicity, the present results suggest that over-cooking of proteins may affect their intestinal antigen processing and thus prevent the induction of oral tolerance.     

Vaccinations with T-helper type 1 directing adjuvants have different suppressive effects on the development of allergen-induced T-helper type 2 responses

BACKGROUND: Allergen-induced T-helper type 2 (Th2) responses can be inhibited with Th1 directing vaccines. However, studies comparing the efficacy of the different adjuvants have not been performed in detail. OBJECTIVE: For this reason we compare the effects of live Bacillus-Calmette-Guerin(BCG), heat-killed (hk)-BCG, CpG-ODN (oligodeoxynucleotide) or PPD on the development of allergen-induced Th2 responses in mice. METHODS: Ovalbumin (OVA)-specific allergic responses were induced in C57BL/6 mice by two intraperitoneally (i.p.) applications of OVA/alum followed by the intranasal challenge with OVA. The different Th1-inducing adjuvants were applied to the mice together with OVA/alum i.p. during the OVA-sensitization period and, subsequently, different parameters of allergic immune responses were evaluated. RESULTS: All the adjuvants were effective in inhibiting the development of allergen-induced airway eosinophilia, mucous production and, with the exception of PPD, also airway hyper-reactivity, when they were applied together with OVA/alum. However, allergen-specific IgG1 and IgE serum levels were only reduced in live BCG- and PPD-treated mice. Suppression of airway eosinophilia was not observed in IFN-gamma- or IL-12-deficient mice (hk-BCG, CpG-ODN and PPD). Interestingly, live BCG was still able to suppress allergen-induced Th2 responses in the absence of either IFN-gamma or IL-12. When mice vaccinated with the different adjuvants together with OVA/alum were subjected to a second period of OVA/alum immunization, only live and hk-BCG were able to efficiently suppress the development of airway inflammation. This effect could be adoptively transferred by splenic CD4(+) T cells. CONCLUSIONS: Taken together our data suggest that live BCG>hk-BCG>CpG-ODN >PPD are effective in suppressing allergen-induced Th2 responses. The degree of suppression and the component of the Th2 response affected (airway inflammation vs. the production of allergen-specific IgE and IgG1) were dependent upon the adjuvant used and how it was applied. Our results contribute to the design of novel vaccines protecting humans from developing allergic disorders.     

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Mouse Reaginic Antibody Responses

The effect of chemical denaturation of ovalbumin (OVA) on the induction of oral tolerance of reaginic antibody responses was studied. Both urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by centrifugation. When compared with OVA and UD-OVA, CM-OVA had the least sensitizing capacity and allergenicity in IgE responses to OVA. BALB/c IgE, IgG1 and IgG antibody responses were suppressed by OVA, but not by UD-OVA or CM-OVA, fed prior to sensitization with OVA, UD-OVA, or CM-OVA in alum, respectively. The priming effect of specific IgG and IgG1 antibody responses was induced by CM-OVA fed prior to sensitization with OVA or CM-OVA. The proliferation of BALB/c spleen cells and their secretion of T helper type 2 (Th2) cytokines interleukin-4 (IL-4) and IL-5 were also orally tolerized by OVA, but not by denatured OVA. Although denatured OVA is hypoallergenic, the present result indicates that denaturation of a soluble protein prevents the induction of oral tolerance of Th2 responses.     

Diesel exhaust particles exert acute effects on airway inflammation and function in murine allergen provocation models

BACKGROUND: Epidemiologic studies show that sudden surges in ambient particulate matter (PM) levels can trigger acute asthma exacerbations. Although diesel exhaust particles (DEPs) act as an adjuvant for allergic sensitization, this is a delayed response and does not explain acute PM effects on airway hyperreactivity (AHR). OBJECTIVE: Our aim was to determine the acute effects of DEPs on AHR using a mouse model. METHODS: Three protocols were developed, 2 of which require OVA sensitization, whereas the third was OVA independent. In the mild sensitization protocol BALB/c mice receive intraperitoneal OVA without alum and are then challenged with aerosolized OVA with or without DEPs. In the postchallenge model DEPs are delivered after OVA challenge to animals sensitized by intraperitoneal OVA plus alum. In the third protocol nebulizer DEPs were also delivered to IL-5-overexpressing mice that exhibit constitutive airway inflammation. Animals were subjected to whole-body plethysmography (WBP) and then killed for performance of bronchoalveolar lavage, histology, and serology. RESULTS: DEP delivery concomitant with OVA challenge or after the induction of airway inflammation with this allergen induced increased AHR in models 1 and 2, respectively. Although these animals showed DEP-induced inflammation and mucus production in the intermediary airways, there was no effect on OVA-specific IgE or T(H)2 cytokine production. In the IL-5 transgenic mice it was possible to induce similar effects with DEPs in the absence of an allergen. CONCLUSION: We demonstrate that DEPs induced AHR independent of their adjuvant effects, suggesting the use of these models to study the mechanism or mechanisms of acute asthma exacerbation by means of PM.