Dopamine D1-like receptor antagonist attenuates Th17-mediated immune response and ovalbumin antigen-induced neutrophilic airway inflammation

Allergic airway inflammation is generally considered a Th2-type immune response. Recent studies, however, demonstrated that Th17-type immune responses also play important roles in this process, especially in the pathogenesis of neutrophilic airway inflammation, a hallmark of severe asthma. We previously reported that dendritic cells release dopamine to naive CD4(+) T cells in Ag-specific cell-cell interaction, in turn inducing Th17 differentiation through dopamine D1-like receptor (D1-like-R). D1-like-R antagonist attenuates Th17-mediated diseases such as experimental autoimmune encephalomyelitis and autoimmune diabetes. However, the effect of antagonizing D1-like-R on Th17-mediated airway inflammation has yet to be studied. In this study, we examined whether D1-like-R antagonist suppresses OVA-induced neutrophilic airway inflammation in OVA TCR-transgenic DO11.10 mice and then elucidated the mechanism of action. DO11.10 mice were nebulized with OVA or PBS, and some mice received D1-like-R antagonist orally before OVA nebulization. D1-like-R antagonist significantly suppressed OVA-induced neutrophilic airway inflammation in DO11.10 mice. It also inhibited the production of IL-17 and infiltration of Th17 cells in the lung. Further, D1-like-R antagonist suppressed the production of IL-23 by lung CD11c(+) APCs. In contrast, D1-like-R antagonist did not increase Foxp3(+) regulatory T cells in the lung. D1-like-R antagonist neither suppressed nonspecific LPS-induced neutrophilic airway inflammation nor OVA-induced eosinophilic airway inflammation. These results indicate that D1-like-R antagonist could suppress Th17-mediated neutrophilic airway inflammation, raising the possibility that antagonizing D1-like-R serves as a promising new strategy for treating neutrophil-dominant severe asthma.     

Regulation of pulmonary T cell responses to inhaled antigen: role in Th1- and Th2-mediated inflammation.

DO11.10 transgenic mice, expressing an OVA-specific TCR, were used to study pulmonary T cell responses to inhaled Ags. Before OVA inhalation, the activation of lung parenchymal T cells elicited both strong proliferative responses and IL-2 production. However, following Ag inhalation the proliferative responses of the lung T cells, when restimulated in vitro with OVA323-339 peptide or immobilized anti-CD3, were severely attenuated and associated with a decrease in the level of production of IL-2 but not IFN-gamma. Such immune regulation was tissue-specific, because T cell responses in the lymph nodes and spleens were normal. This dramatic aerosol-induced attenuation of parenchymal T cell proliferation was also observed in BALB/c mice immunized with OVA and in BALB/c mice following adoptive transfer of DO11.10 T cells bearing either a Th1 or Th2 phenotype. In mice that had received Th2 cells, the reduced proliferative responses were associated with a decrease in IL-2 expression but augmented IL-4 and IL-5 production. Invariably, the inhibition of proliferation was a consequence of the action of F4/80+ interstitial macrophages and did not involve alveolar macrophages or their products. These observations demonstrate that clonal expansion of T cells in the lung compartment is prevented following the onset of either Th1- or Th2-mediated inflammation. This form of immune regulation, which appears as a selective defect in IL-2-driven proliferation, may serve to prevent the development of chronic pulmonary lymphoproliferative responses.     

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.     

IL-2 and autocrine IL-4 drive the in vivo development of antigen-specific Th2 T cells elicited by nematode parasites

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4-/- mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Ralpha-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4-/- recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.     

Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.     

Nippostrongylus brasiliensis can induce B7-independent antigen-specific development of IL-4-producing T cells from naive CD4 T cells in vivo

Th2 immune responses to a number of infectious pathogens are dependent on B7-1/B7-2 costimulatory molecule interactions. We have now examined the Th2 immune response to Nippostrongylus brasiliensis (Nb) in B7-1/B7-2(-/-) mice and show that Th2 effector cells develop that can mediate worm expulsion and produce substantial Th2 cytokines comparable with wild-type infected mice; however, in marked contrast, B cell Ag-specific Ab production is abrogated after B7 blockade. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of Nb plus OVA or either alone. Only the combination of Nb plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that Nb acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells. Furthermore, using the DO11.10 TCR-transgenic T cell adoptive transfer model, we show that blocking B7-1/B7-2 interactions does not impair nonparasite Ag-specific DO11.10 Th2 cell differentiation; however, DO11.10 T cell cycle progression and migration to the B cell zone are inhibited.     

The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.     

An optimized CD4 T-cell response can control productive and latent gammaherpesvirus infection

CD4 T cells are important for control of infection with murine gammaherpesvirus 68 (gamma HV68), but it is not known whether CD4 T cells function via provision of help to other lymphocyte subsets, such as B cells and CD8 T cells, or have an independent antiviral function. Moreover, under conditions of natural infection, the CD4 T-cell response is not sufficient to eliminate infection. To determine the functional capacities of CD4 T cells under optimal or near-optimal conditions and to determine whether CD4 T cells can control gamma HV68 infection in the absence of CD8 T cells or B cells, we studied the effect of ovalbumin (OVA)-specific CD4 T cells on infection with a recombinant gamma HV68 that expresses OVA. OVA-specific CD4 T cells limited acute gamma HV68 replication and prolonged the life of infected T-cell receptor-transgenic RAG (DO.11.10/RAG) mice, demonstrating CD4 T-cell antiviral activity, independent of CD8 T cells and B cells. Despite CD4 T-cell-mediated control of acute infection, latent infection was established in DO.11.10/RAG mice. However, OVA-specific CD4 T cells reduced the frequency of latently infected cells both early (16 days postinfection) and late (42 days postinfection) after infection of mice containing CD8 T cells and B cells (DO.11.10 mice). These results show that OVA-specific CD4 T cells have B-cell and CD8 T-cell-independent antiviral functions in the control of acute infection and can, in the absence of preexisting CD8 T-cell or B-cell immunity, inhibit the establishment of gammaherpesvirus latency.     

CD4+CD25+ T cells regulate airway eosinophilic inflammation by modulating the Th2 cell phenotype

We used a TCR-transgenic mouse to investigate whether Th2-mediated airway inflammation is influenced by Ag-specific CD4+CD25+ regulatory T cells. CD4+CD25+ T cells from DO11.10 mice expressed the transgenic TCR and mediated regulatory activity. Unexpectedly, depletion of CD4+CD25+ T cells before Th2 differentiation markedly reduced the expression of IL-4, IL-5, and IL-13 mRNA and protein when compared with unfractionated (total) CD4+ Th2 cells. The CD4+CD25--derived Th2 cells also expressed decreased levels of IL-10 but were clearly Th2 polarized since they did not produce any IFN-gamma. Paradoxically, adoptive transfer of CD4+CD25--derived Th2 cells into BALB/c mice induced an elevated airway eosinophilic inflammation in response to OVA inhalation compared with recipients of total CD4+ Th2 cells. The pronounced eosinophilia was associated with reduced levels of IL-10 and increased amounts of eotaxin in the bronchoalveolar lavage fluid. This Th2 phenotype characterized by reduced Th2 cytokine expression appeared to remain stable in vivo, even after repeated exposure of the animals to OVA aerosols. Our results demonstrate that the immunoregulatory properties of CD4+CD25+ T cells do extend to Th2 responses. Specifically, CD4+CD25+ T cells play a key role in modulating Th2-mediated pulmonary inflammation by suppressing the development of a Th2 phenotype that is highly effective in vivo at promoting airway eosinophilia. Conceivably, this is partly a consequence of regulatory T cells facilitating the production of IL-10.     

Establishment and characterization of a novel murine model for pollen allergy

Although there have been many studies revealing the mechanism and establishing the therapeutical method for allergic rhinitis, no suitable animal models for allergic rhinitis, especially for pollen allergy, are currently available. We therefore aimed in this study to develop a murine model producing IgE in response to an inhaled antigen without using any adjuvants. Ovalbumin (OVA)-specific T cell receptor transgenic mice (DO11.10) inhaled an OVA solution for one h, twice a week, for six weeks. The resulting increase of OVA-specific IgE in the serum was observed depending on the times of inhalation. Spleen cells from mice that had inhaled the antigen produced more IL-4 and less IFN-γ than those from the control mice in vitro. These results indicate that inhaled antigen enhanced the Th2-type responses and induced IgE production in a T cell-mediated manner. Our findings would contribute to studies on prevention and treatment of pollen allergy.     

TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice

Steroid-resistant asthma comprises an important source of morbidity in patient populations. T(H)17 cells represent a distinct population of CD4(+) Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of T(H)17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4(+) T cells from DO11.10 OVA-specific TCR-transgenic mice to a T(H)2 or T(H)17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of T(H)2 and T(H)17 cells. In vitro, T(H)17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of T(H)2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas T(H)17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of T(H)17 or T(H)2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the T(H)17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both T(H)2 and T(H)17 cells are able to induce AHR, whereas T(H)17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for T(H)17 cells in steroid-resistant asthma.     

Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease

Background

Th2 immune responses are linked primarily to mild and moderate asthma, while Th17 cells, Interleukin-17A (IL-17) and neutrophilia have been implicated in more severe forms of disease. How Th2-dependent allergic reactions are influenced by Th17 and IL-17-γδ T cells is poorly understood. In murine models, under some conditions, IL-17 promotes Th2-biased airway inflammatory responses. However, IL-17-γδ T cells have been implicated in the inhibition and resolution of allergic airway inflammation and hyperresponsiveness (AHR).

Methods

We compared airway responses in Balb/c mice sensitized to OVA with (and without) a Th2-skewing aluminum-based adjuvant and the IL-17 skewing, complete Freund’s adjuvant (CFA). AHR was measured invasively by flexiVent, while serum OVA-IgE was quantified by an enzyme immunoassay. Airway inflammatory and cytokine profiles, and cellular sources of IL-17 were assessed from bronchoalveolar lavage and/or lungs. The role of γδ T cells in these responses was addressed in OVA/CFA sensitized mice using a γδ T cell antibody.

Results

Following OVA challenge, all mice exhibited mixed eosinophilic/neutrophilic airway inflammatory profiles and elevated serum OVA-IgE. Whereas OVA/alum sensitized mice had moderate inflammation and AHR, OVA/CFA sensitized mice had significantly greater inflammation but lacked AHR. This correlated with a shift in IL-17 production from CD4⁺ to γδ T cells. Additionally, OVA/CFA sensitized mice, given a γδ TCR stimulatory antibody, showed increased frequencies of IL-17-γδ T cells and diminished airway reactivity and eosinophilia.

Conclusions

Thus, the conditions of antigen sensitization influence the profile of cells that produce IL-17, the balance of which may then modulate the airway inflammatory responses, including AHR. The possibility for IL-17-γδ T cells to reduce AHR and robust eosinophilic inflammation provides evidence that therapeutic approaches focused on stimulating and increasing airway IL-17-γδ T cells may be an effective alternative in treating steroid resistant, severe asthma.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0090-5) contains supplementary material, which is available to authorized users.     

Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4(+)CD25(+) T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4(+)CD25(+) regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA x DO11.10 mice had increased numbers of CD4(+)CD25(+) regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA x DO11.10 mice, T cells expressing endogenous TCR alpha beta chains were CD4(+)CD25(-) T cells, whereas T cells expressing autoreactive TCR were selected as CD4(+)CD25(+) T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA x DO11.10 mice. In contrast, in DO11.10 mice, CD4(+)CD25(+) T cells expressed endogenous TCR alpha beta chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4(+)CD25(+) T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4(+)CD25(+) T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4(+)CD25(-) T cells from autoreactive T cell repertoire.     

Suppression of oral tolerance by Lactococcus lactis in mice

Although oral ovabumin (OVA) administration suppressed the antibody (Ab) response in OVA-immunized mice, Lactococcus lactis increased OVA-specific IgG2a in these mice. L. lactis increased the casein-specific IgG level in NC/Nga mice fed on a casein diet. The percentage of CD4(+)CD25(+) cells was increased in DO11.10 mice orally given OVA, but this increase of CD4(+)CD25(+) cells were suppressed in L. lactis-fed DO11.10 mice.     

GIF inhibits Th effector generation by acting on antigen-presenting B cells

Glycosylation-inhibiting factor (GIF) is a 13-kDa cytokine secreted from T cells. Administration of bioactive recombinant GIF inhibits IgG1 and IgE Ab responses in vivo. Treatment of B cells with the cytokine reduces the secretion of IgG1 and IgE induced by LPS and IL-4. To examine the effect on cognate T-B interaction, GIF was added to low-density B cells from MD4 transgenic (Tg) mice, which express B cell receptor specific for hen egg lysozyme (HEL). The B cells were subsequently pulsed with HEL-OVA conjugate and cultured with OVA-specific naive CD4 T cells from DO11.10 Tg mice. Treatment of Ag-presenting B cells with GIF reduced expansion and IL-2 secretion of naive T cells and rendered them hyporesponsive to antigenic restimulation, resulting in 50--95% reduction of IL-4 and IFN-gamma secretion upon restimulation with Ag. GIF dramatically inhibited Th effector generation when it was added to B cells before pulsing with HEL-OVA, whereas it showed little to no effect when added after B cells were pulsed with Ag. GIF was more effective when B cells from MD4 Tg mice were pulsed with HEL-OVA than when they were pulsed with OVA. This cytokine did not affect Th effector generation when B cells or irradiated splenocytes pulsed with OVA(323--339) peptide stimulated naive DO11.10 T cells. Confocal microscopy revealed that GIF inhibited internalization of HEL by B cells from MD4 Tg mice. Therefore, the cytokine may regulate early steps of Ag presentation involving B cell receptors to diminish Th effector generation from naive CD4 T cells.     

Modulation of Th2 responses by peptide analogues in a murine model of allergic asthma: amelioration or deterioration of the disease process depends on the Th1 or Th2 skewing characteristics of the therapeutic peptide

Allergen-specific CD4+ Th2 cells play an important role in the immunological processes of allergic asthma. Previously we have shown that, by using the immunodominant epitope OVA323-339, peptide immunotherapy in a murine model of OVA induced allergic asthma, stimulated OVA-specific Th2 cells, and deteriorated airway hyperresponsiveness and eosinophilia. In the present study, we defined four modulatory peptide analogues of OVA323-339 with comparable MHC class II binding affinity. These peptide analogues were used for immunotherapy by s.c. injection in OVA-sensitized mice before OVA challenge. Compared with vehicle-treated mice, treatment with the Th2-skewing wild-type peptide and a Th2-skewing partial agonistic peptide (335N-A) dramatically increased airway eosinophilia upon OVA challenge. In contrast, treatment with a Th1-skewing peptide analogue (336E-A) resulted in a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production. Our data show for the first time that a Th1-skewing peptide analogue of a dominant allergen epitope can modulate allergen-specific Th2 effector cells in an allergic response in vivo. Furthermore, these data suggest that the use of Th1-skewing peptides instead of wild-type peptide may improve peptide immunotherapy and may contribute to the development of a successful and safe immunotherapy for allergic patients.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.