The recruitment of eosinophils into the airways after allergen exposure is dependent on interleukin (IL) 5 secreted from antigen-specific CD4+ T cells of the T helper cell (Th) 2 subset. However, while it is established that costimulation through CD28 is required for TCR-mediated activation and IL-2 production, the importance of this mechanism for the induction of a Th2 immune response is less clear. In the present study, we administered the fusion protein CTLA-4 immunoglobulin (Ig) into the lungs before allergen provocation to determine whether CD28/CTLA-4 ligands are required for allergen-induced eosinophil accumulation and the production of Th2 cytokines. Administration of CTLA-4 Ig inhibited the recruitment of eosinophils into the lungs by 75% and suppressed IgE in the bronchoalveolar lavage fluid. CTLA-4 Ig also inhibited the production of IL-4, IL-5, and IL-10 by 70–80% and enhanced interferon-γ production from CD3–T cell receptor–activated lung Thy1.2+ cells. Allergen exposure upregulated expression of B7-2, but not B7-1, on B cells from the lung within 24 h. Moreover, airway administration of an anti-B7-2 monoclonal antibody (mAb) inhibited eosinophil infiltration, IgE production, and Th2 cytokine secretion comparable in magnitude to that observed with CTLA-4 Ig. Treatment with an anti-B7-1 mAb had a small, but significant effect on eosinophil accumulation, although was less effective in inhibiting Th2 cytokine production. The anti-B7-2, but not anti-B7-1, mAb also inhibited antigen-induced airway hyperresponsiveness in vivo. In all of the parameters assessed, the combination of both the anti-B7-1 and anti-B7-2 mAb was no more effective than anti-B7-2 mAb treatment alone. We propose that strategies aimed at inhibition of CD28 interactions with B7-2 molecules may represent a novel therapeutic target for the treatment of lung mucosal allergic inflammation.
Elevated levels of immunoglobulin (Ig) E are associated with bronchial asthma, a disease characterized by eosinophilic inflammation of the airways. Activation of antigen-specific T helper (Th) 2 cells in the lung with the subsequent release of interleukin (IL) 4 and IL-5 is believed to play an important role in the pathogenesis of this disease. In this study, we have used a non-anaphylactogenic anti-mouse-IgE antibody to investigate the relationship between IgE, airway eosinophil infiltration, and the production of Th2 cytokines. Immunization of mice with house dust mite antigen increased serum levels of IgE and IgG. Antigen challenge of immunized but not control mice induced an infiltration of eosinophils in the bronchoalveolar lavage associated with the production of IL-4 and IL-5 from lung purified Thy1.2+ cells activated through the CD3-T cell receptor complex. Administration of the anti-IgE monoclonal antibody (mAb) 6h before antigen challenge neutralized serum IgE but not IgG and inhibited the recruitment of eosinophils into the lungs and the production of IL-4 and IL-5 but not interferon gamma. Studies performed using an anti-CD23 mAb, CD23 deficient and mast cell deficient mice suggest that anti-IgE mAb suppresses eosinophil infiltration and Th2 cytokine production by inhibiting IgE-CD23-facilitated antigen presentation to T cells. Our results demonstrate that IgE-dependent mechanisms are important in the induction of a Th2 immune response and the subsequent infiltration of eosinophils into the airways. Neutralization of IgE, for example, non- anaphylactogenic anti-IgE mAbs may provide a novel therapeutic approach to the treatment of allergic airway disease.
Reversible airway hyperreactivity underlies the pathophysiology of asthma, yet the precise mediators of the response remain unclear. Human studies have correlated aberrant activation of T helper (Th) 2-like effector systems in the airways with disease. A murine model of airway hyperreactivity in response to acetylcholine was established using mice immunized with ovalbumin and challenged with aerosolized antigen. No airway hyperractivity occurred in severe combined immunodeficient mice. Identically immunized BALB/c mice developed an influx of cells, with a predominance of eosinophils and CD4+ T cells, into the lungs and bronchoalveolar lavage fluid at the time that substantial changes in airway pressure and resistance were quantitated. Challenged animals developed marked increases in Th2 cytokine production, eosinophil influx, and serum immunoglobulin E levels. Neutralization of interleukin (IL) 4 using monoclonal antibodies administered during the period of systemic immunization abrogated airway hyperractivity but had little effect on the influx of eosinophils. Administration of anti-IL-4 only during the period of the aerosol challenge did not affect the subsequent response to acetylcholine. Finally, administration of anti- IL-5 antibodies at levels that suppressed eosinophils to < 1% of recruited cells had no effect on the subsequent airway responses. BALB/c mice had significantly greater airway responses than C57BL/6 mice, consistent with enhanced IL-4 responses to antigen in BALB/c mice. Taken together, these data implicate IL-4 generated during the period of lymphocyte priming with antigen in establishing the cascade of responses required to generate airway hyperractivity to inhaled antigen. No role for IL-5 or eosinophils could be demonstrated.
Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 (Th2)-mediated allergic airway inflammation in mice. However, it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness. Here, we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma. We could suppress GATA-3 expression in interleukin (IL)-4–producing T cells in vitro and in vivo by an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3, whereas nonsense control oligonucleotides were virtually inactive. In a murine model of asthma associated with allergic pulmonary inflammation and hyperresponsiveness in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate–labeled GATA-3 antisense oligonucleotides led to DNA uptake in lung cells associated with a reduction of intracellular GATA-3 expression. Such intrapulmonary blockade of GATA-3 expression caused an abrogation of signs of lung inflammation including infiltration of eosinophils and Th2 cytokine production. Furthermore, treatment with antisense but not nonsense oligonucleotides induced a significant reduction of airway hyperresponsiveness in OVA-sensitized mice to levels comparable to saline-treated control mice, as assessed by both enhanced pause (PenH) responses and pulmonary resistance determined by body plethysmography. These data indicate a critical role for GATA-3 in the effector phase of a murine asthma model and suggest that local delivery of GATA-3 antisense oligonucleotides may be a novel approach for the treatment of airway hyperresponsiveness such as in asthma. This approach has the potential advantage of suppressing the expression of various proinflammatory Th2 cytokines simultaneously rather than suppressing the activity of a single cytokine.
GATA-3; antisense DNA; asthma; T cells; Th2 cytokines
Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity.
Eosinophil recruitment is a classic characteristic of both allergic and parasitic helminth diseases. Elucidation of the role of eosinophils in these diseases is of pivotal importance for understanding the mechanisms of protection and the development of pathology. In the last few years, the part played by eosinophils in helminth-defence has been dissected using in vivo models and their importance in protection has been shown to be highly specific to the host-parasite combination. This study dissects the role of eosinophils during infection with the human lymphatic filarial parasite, Brugia malayi, which causes the major neglected tropical disease, lymphatic filariasis. In particular, we study the role of the eosinophil as a double–edged sword in generating both protection and pathology. We definitively confirm the importance of eosinophils in protection against B. malayi microfilariae and show that protection is not mediated by release of the eosinophil granule proteins, major basic protein or eosinophil peroxidase alone. Overall, we reveal that during an infection with B. malayi microfilariae, eosinophils are critical for primary protective responses. However, eosinophils contribute to nematode-induced lung dysfunction, while additionally, eosinophil granules are important negative regulators of parasite-induced lung inflammatory and some adaptive immune responses.
A T helper cell type 1–mediated colitis develops in severe combined immunodeficient mice after transfer of CD45RBhigh CD4+ T cells and can be prevented by cotransfer of the CD45RBlow subset. The immune-suppressive activities of the CD45RBlow T cell population can be reversed in vivo by administration of an anti-transforming growth factor β antibody. Here we show that interleukin (IL)-10 is an essential mediator of the regulatory functions of the CD45RBlow population. This population isolated from IL-10–deficient (IL-10−/−) mice was unable to protect from colitis and when transferred alone to immune-deficient recipients induced colitis. Treatment with an anti–murine IL-10 receptor monoclonal antibody abrogated inhibition of colitis mediated by wild-type (WT) CD45RBlow CD4+ cells, suggesting that IL-10 was necessary for the effector function of the regulatory T cell population. Inhibition of colitis by WT regulatory T cells was not dependent on IL-10 production by progeny of the CD45RBhigh CD4+ cells, as CD45RBlow CD4+ cells from WT mice were able to inhibit colitis induced by IL-10−/− CD45RBhigh CD4+ cells. These findings provide the first clear evidence that IL-10 plays a nonredundant role in the functioning of regulatory T cells that control inflammatory responses towards intestinal antigens.
interleukin 10; inflammatory bowel disease; CD4+ T lymphocyte; regulatory T lymphocyte
The molecular determinants of the severity and persistence of allergic asthma remain poorly understood. Suppressor Of Cytokine Signaling 1 (SOCS1) is a negative regulator of interleukin (IL)-4-dependent pathways in vitro and might therefore, control T helper type 2 (Th2) immunity associated traits, such as IgE levels, mucin production, IL-5 and IL-13 induction, and eosinophilic mucosal inflammation, which are implicated in allergic asthma.
To investigate the role of SOCS1 in regulating Th2-associated disease traits in a murine sub-chronic aeroallergen-driven asthma model.
Following sensitization and challenge with ovalbumin (OVA), bronchoalveolar lavage (BAL) and serum were collected from mice lacking the Socs1 gene on an interferon γ (IFN-γ) null background (Socs1−/−Ifnγ−/−). The composition of infiltrating cells in the lung, serum IgE and IgG1 levels and cytokine levels were analysed.
Serum IgE levels and infiltrating eosinophils were greatly increased in the lungs of OVA-treated Socs1−/−Ifnγ−/− mice compared to Ifnγ−/− and C57BL/6 controls. Expression of the Th2 cytokines, IL-4, IL-5 and IL-13 was increased in CD4+ cells and lung tissue from OVA-treated Socs1−/−Ifnγ−/− mice. IgE, IL-5 levels and infiltrating eosinophils were also elevated in saline-treated Socs1−/−Ifnγ−/− mice, suggesting that in the absence of SOCS1, mice are already biased towards a Th2 response. It is at present unclear whether the elevated cytokine levels are sufficient to result in the exacerbated Th2-response to OVA challenge or whether enhanced intracellular signalling also contributes. Surprisingly, of the various IL-4/IL-13 responsive genes tested, only Arginase I appeared to be modestly up-regulated in the lungs of OVA-treated Socs1−/−Ifnγ−/− mice, suggesting that regulation by SOCS1 occurs primarily in hematopoietic cells and not in the airway epithelium.
Together these results indicate that SOCS1 is an important regulator of the Th2 response.
SOCS1; asthma; Th2 type cytokines; bronchoalveolar lavage; eosinophils
Protection against infectious pathogens at mucosal surfaces is dependent on local antibody responses, production of inflammatory mediators, and recruitment of immune effector cells to the site of infection. Since Th1 and Th2 cells produce cytokines with pro- and anti-inflammatory activities, immunization with vaccines that induce these T-cell subtypes may regulate the subsequent inflammatory response to infection. We have demonstrated that immunization of mice with pertussis whole-cell or acellular vaccines (Pw or Pa) selectively induces Th1 and Th2 cells, respectively. In this study we have used a murine respiratory-infection model to demonstrate that priming with a Th1- or Th2-inducing pertussis vaccine can influence the local inflammatory response and immune effector cells in the lung following aerosol challenge with Bordetella pertussis. Analysis of bronchoalveolar lavage (BAL) fluid taken during the course of B. pertussis infection of naïve mice or mice immunized with Pw revealed an early influx of neutrophils and local production of interleukin 1β (IL-1β) in the lungs. In contrast, neutrophil infiltration and IL-1β production were not observed following challenge of mice immunized with the Th2-inducing Pa. Conversely, during infection local production of IL-6 and IL-1ra was significantly greater in mice immunized with Pa than in those immunized with Pw. Studies of knockout mice revealed neutrophil and lymphocyte infiltration in the lungs following B. pertussis infection of IL-4-defective (IL-4−/−) mice but not in wild-type mice immunized with Pa. Furthermore, the levels of IL-1β, IL-6, and IL-1ra in Pa-immunized IL-4−/− mice were comparable to those in mice immunized with Pw. These results demonstrate distinct influences of Th1- and Th2-inducing vaccines on the protective inflammatory responses in the lungs following challenge with B. pertussis and implicate IL-4 as an important regulator of inflammatory-cell recruitment.
Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen- induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.
Asthma is a chronic inflammatory disease induced by Type 2 helper T cells and eosinophils. Vascular cell adhesion molecule-1 (VCAM-1) has been implicated in recruiting eosinophils and lymphocytes to pathological sites in asthma as a regulatory receptor. Accordingly, monoclonal antibody (mAb) against VCAM-1 may attenuate allergic inflammation and pathophysiological features of asthma. We attempted to evaluate whether a recently developed human anti-VCAM-1 mAb can inhibit the pathophysiological features of asthma in a murine asthma model induced by ovalbumin (OVA). Leucocyte adhesion inhibition assay was performed to evaluate the in vitro blocking activity of human anti-VCAM-1 mAb. OVA-sensitized BALB/c mice were treated with human anti-VCAM-1 mAb or isotype control Ab before intranasal OVA challenge. We evaluated airway hyperresponsiveness (AHR) and bronchoalveolar lavage fluid analysis, measured inflammatory cytokines and examined histopathological features. The human anti-VCAM-1 mAb bound to human and mouse VCAM-1 molecules and inhibited adhesion of human leucocytes in vitro. AHR and inflammatory cell counts in bronchoalveolar lavage fluid were reduced in mice treated with human anti-VCAM-1 mAb as compared with a control Ab. The levels of interleukin (IL)-5 and IL-13, as well as transforming growth factor-β, in lung tissue were decreased in treated mice. Human anti-VCAM-1 mAb reduced goblet cell hyperplasia and peribronchial fibrosis. In vivo VCAM-1 expression decreased in the treated group. In conclusion, human anti-VCAM-1 mAb attenuated allergic inflammation and the pathophysiological features of asthma in OVA-induced murine asthma model. The results suggested that human anti-VCAM-1 mAb could potentially be used as an additional anti-asthma therapeutic medicine.
VCAM-1; monoclonal antibody; allergic inflammation; asthma; cell adhesion molecule; anti-inflammatory effect
The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae (Df) through the generation of cysteinyl leukotrienes (cys-LTs) and pro-inflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. Here, we demonstrate that administration of the Dectin-2 mAb solely at the time of Df challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage (BAL) fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n−/−) sensitized with the adoptive transfer of Df-pulsed wild-type (WT) bone marrow-derived DCs (BMDCs) also had less Df-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n−/− mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production, and with no reduction in serum IgE. WT and Clec4n−/− mice recipients, sensitized with Df-pulsed WT BMDCs, had comparable levels of Df-elicited IL-6, IL-23, TNF-α, and cys-LTs in the lung. By contrast, Df-elicited CCL4 and CCL8 production from pulmonary CD11c+CD11b+Ly6C+ and CD11c+CD11b+Ly6C−CD64+ monocyte-derived DCs was reduced in Clec4n−/− recipients. Addition of CCL8 at the time of Df challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n−/− recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at Df challenge to promote the local inflammatory response.
Knockout mice; Dendritic Cells; Lipid mediators; Allergy; Inflammation
T lymphocytes play a pivotal role in the immune response during viral infections. In a murine model of experimental respiratory syncytial virus (RSV) infection, mice sensitized to either of the two major glycoproteins of RSV develop distinct patterns of cytokine secretion and lung inflammation upon subsequent RSV infection. Mice sensitized to RSV-G (attachment) glycoprotein exhibit a strong interleukin (IL)-4 and IL-5 response and develop pulmonary eosinophilia, whereas mice sensitized to RSV-F (fusion) glycoprotein develop a predominantly T helper cell (Th)1 response and pulmonary inflammation characterized by mononuclear cell infiltration. In this study, we examined the potential role of virus-specific CD8+ T cytolytic T cells on the differentiation and activation of functionally distinct CD4+ T cells specific to these viral glycoproteins. Mice primed with recombinant vaccinia virus expressing RSV-F glycoprotein mounted a strong RSV-specific, MHC class I–restricted cytolytic response, whereas priming with recombinant vaccinia virus expressing RSV-G glycoprotein failed to elicit any detectable cytolytic response. Priming for a RSV-specific CD8+ T cell response, either with a recombinant vaccinia virus expressing RSV-G glycoprotein in which a strong CD8+ T cell epitope from RSV-M2 (matrix) protein has been inserted or with a combination of vaccinia virus expressing the matrix protein and the RSV-G glycoprotein, suppressed the eosinophil recruitment into the lungs of these mice upon subsequent challenge with RSV. This reduction in pulmonary eosinophilia correlated with the suppression of Th2 type cytokine production. The importance of CD8+ T cells in this process was further supported by the results in CD8+ T cell deficient, β2 microglobulin KO mice. In these mice, priming to RSV-F glycoprotein (which in normal mice primed for a strong cytolytic response and a pulmonary infiltrate consisting primarily of mononuclear cells on RSV challenge) resulted in the development of marked pulmonary eosinophilia that was not seen in mice with an intact CD8+ T cell compartment. These results indicate that CD8+ T cells may play an important role in the regulation of the differentiation and activation of Th2 CD4+ T cells as well as the recruitment of eosinophils into the lungs during RSV infection.
Cigarette smoke (CS) causes considerable morbidity and mortality by inducing cancer, chronic lung and vascular diseases, and oral disease. Despite the well-recognized risks associated with smoking, the habit remains unacceptably prevalent. Several toxins present in CS have immunomodulatory effects. CS also contains trace amounts of microbial cell components, including bacterial lipopolysaccharide. These and other CS constituents induce chronic inflammation at mucosal surfaces and modify host responses to exogenous antigens. The effects of CS on immunity are far-reaching and complex; both pro-inflammatory and suppressive effects may be induced. The net effect of CS on immunity depends on many variables, including the dose and type of tobacco, the route and chronicity of exposure, and the presence of other factors at the time of immune cell stimulation, such as Toll receptor ligands or other inflammatory mediators. CS impairs innate defenses against pathogens, modulates antigen presentation, and promotes autoimmunity. CS also impairs immunity in the oral cavity and promotes gingival and periodontal disease and oral cancer. The recognition of specific mechanisms by which CS affects host immunity is an important step toward elucidating mechanisms of tobacco-induced disease and may identify novel therapeutic approaches for the management of diseases that afflict smokers.
Abbreviations: AP-1, activator protein-1; CD, cluster of differentiation; COPD, chronic obstructive pulmonary disease; HLA, human leukocyte antigen; IFNγ, interferon gamma; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa-B; RAGE, receptors for advanced glycation end-products; ROS, reactive oxidative species; RORγτ, retinoic acid receptor-related orphan receptor transcription factor; STAT, signal transducer and activator of transcription; T-bet, T-box transcription factor; Th, T-helper; TLR, Toll-like receptors; TNFα, Tumor necrosis factor alpha; and TSLP, thymic stromal lymphopoeitin.
tobacco; immunity; inflammation; autoimmunity; cigarette smoke; nicotine
The feasibility of using the highly purified native attachment (G) protein in a subunit vaccine against respiratory syncytial virus (RSV) was examined in a murine model with or without the fusion (F) protein of RSV and the adjuvant QS-21. The studies established that QS-21 was more potent than AIOH as an adjuvant for both F and G glycoproteins. Augmented antigen-dependent killer cell activity and complement-assisted serum neutralizing and anti-F and G protein immunoglobulin G2a antibody titers were observed. Immunization with G/QS-21 generated immune responses that were characterized by low levels of antigen-dependent killer cell activity, elevated levels of interleukin-5 (IL-5) and percentages of eosinophils in the bronchoalveolar lavage fluids after challenge, and splenic immunocytes that secreted IL-5 but not gamma interferon (IFN-gamma) after in vitro stimulation with purified whole virus antigens. The pulmonary eosinophilia was similar to that induced by a facsimile of a formalin-inactivated vaccine used in previous clinical trials and was prevented by prior in vivo treatment with anti-IL-5 but not with control immunoglobulin G or anti-IFN-gamma neutralizing monoclonal antibodies. Thus the data implied that vaccination with G/QS-21 generated helper T-cell immune responses that were type 2 in nature. Alternatively, the data suggested that the helper T-cell immune responses elicited by F/QS-21 were more type 1 in character. Neither eosinophilia nor elevated levels of IL-5 were observed in the lungs of mice after challenge. Noteworthy levels of antigen-dependent killer cell activity was observed, and splenic immunocytes secreted copious quantities of IFN-gamma. Immunization with a combination vaccine composed of highly purified native F and G proteins plus QS-21 (F+G/QS-21) resulted in augmented complement-assisted serum neutralizing antibody titers compared with vaccination with either F/QS-21 or G/QS-21 alone. However, following vaccination with F+G/QS-21, the bronchoalveolar lavage fluids contained significant increases in IL-5 and percentages of eosinophils after challenge, the spleen cells appeared to secrete less IFN-gamma after in vitro stimulation, and there was no evidence of increased numbers of antigen-dependent killer cell precursors. Taken together, the data imply that native G protein influences the nature of the immune responses elicited by F/QS-21. The results therefore suggest that G, not F, protein has more potential to bias the host for atypical pulmonary inflammatory responses.
The coinhibitory receptor Programmed Death-1 (PD-1) inhibits effector functions of
activated T cells and prevents autoimmunity, however, cancer hijack this pathway
to escape from immune attack. The costimulatory receptor glucocorticoid-induced
TNFR related protein (GITR) is up-regulated on activated T cells and increases
their proliferation, activation and cytokine production. We hypothesize that
concomitant PD-1 blockade and GITR triggering would synergistically improve the
effector functions of tumor-infiltrating T cells and increase the antitumor
immunity. In present study, we evaluated the antitumor effects and mechanisms of
combined PD-1 blockade and GITR triggering in a clinically highly relevant murine
ID8 ovarian cancer model.
Mice with 7 days-established peritoneal ID8 ovarian cancer were treated
intraperitoneally (i.p.) with either control, anti-PD-1, anti-GITR or
anti-PD-1/GITR monoclonal antibody (mAb) and their survival was evaluated; the
phenotype and function of tumor-associated immune cells in peritoneal cavity of
treated mice was analyzed by flow cytometry, and systemic antigen-specific immune
response was evaluated by ELISA and cytotoxicity assay.
Combined anti-PD-1/GITR mAb treatment remarkably inhibited peritoneal ID8 tumor
growth with 20% of mice tumor free 90 days after tumor challenge while treatment
with either anti-PD-1 or anti-GITR mAb alone exhibited little antitumor effect.
The durable antitumor effect was associated with a memory immune response and
conferred by CD4+ cells and CD8+ T cells. The treatment of
anti-PD-1/GITR mAb increased the frequencies of interferon-γ-producing
effector T cells and decreased immunosuppressive regulatory T cells and
myeloid-derived suppressor cells, shifting an immunosuppressive tumor milieu to an
immunostimulatory state in peritoneal cavity. In addition, combined treatment of
anti-PD-1/GITR mAb mounted an antigen-specific immune response as evidenced by
antigen-specific IFN-γ production and cytolytic activity of spleen cells from
treated mice. More importantly, combined treatment of anti-PD-1/GITR mAb and
chemotherapeutic drugs (cisplatin or paclitaxel) further increased the antitumor
efficacy with 80% of mice obtaining tumor-free long-term survival in murine ID8
ovarian cancer and 4 T1 breast cancer models.
Combined anti-PD-1/GITR mAb treatment induces a potent antitumor immunity, which
can be further promoted by chemotherapeutic drugs. A combined strategy of
anti-PD-1/GITR mAb plus cisplatin or paclitaxel should be considered translation
PD-1; GITR; Monoclonal antibody; Immunotherapy
Background & Aims
TL1A is a tumor necrosis factor-like molecule that mediates a strong costimulation of T helper (TH) 1 cells. Expression of TL1A is increased in the mucosa of Crohn’s disease patients and murine models of ileitis. The aim was to determine the possible role of TL1A in chronic intestinal inflammation.
We used dextran sodium sulphate (DSS)-induced chronic colitis to investigate the effects of TL1A on the development of colitis. The cytokine profile in the gut-associated lymphoid tissue (GALT) was measured. Neutralizing anti-TL1A monoclonal antibodies (mAb) were injected intraperitoneally into DSS-induced chronic colitis and G protein αi2−/− T cell transfer colitis models. Severity of colitis was evaluated by body weight, colon length, histology, and cytokine production.
DSS-induced chronic colitis was characterized by the infiltration of CD4+ T cells. TL1A, death receptor 3, interferon (IFN)-γ and interleukin (IL)-17 were significantly increased in GALT of DSS-treated mice. TL1A up-regulated both IFN-γ production from TH1 cells and IL-17 production from TH17 cells in GALT CD4+ T cells. Furthermore, IFN-γ and IL-17 production from CD4+ T cells, induced by IL-12 and IL-23 respectively, was synergistically enhanced by combination with TL1A. Anti-TL1A mAb prevented chronic colitis and attenuated established colitis by down-regulation of both TH1 and TH17 activation.
Our results reveal that TL1A is an important modulator in the development of chronic mucosal inflammation by enhancing TH1 and TH17 effector functions. The central role of TL1A represents an attractive, novel therapeutic target for the treatment of CD patients.
The current paradigm surrounding allergen-mediated T helper type 2 (Th2) immune responses in the lung suggests an almost hegemonic role for T cells. Our studies propose an alternative hypothesis implicating eosinophils in the regulation of pulmonary T cell responses. In particular, ovalbumin (OVA)-sensitized/challenged mice devoid of eosinophils (the transgenic line PHIL) have reduced airway levels of Th2 cytokines relative to the OVA-treated wild type that correlated with a reduced ability to recruit effector T cells to the lung. Adoptive transfer of Th2-polarized OVA-specific transgenic T cells (OT-II) alone into OVA-challenged PHIL recipient mice failed to restore Th2 cytokines, airway histopathologies, and, most importantly, the recruitment of pulmonary effector T cells. In contrast, the combined transfer of OT-II cells and eosinophils into PHIL mice resulted in the accumulation of effector T cells and a concomitant increase in both airway Th2 immune responses and histopathologies. Moreover, we show that eosinophils elicit the expression of the Th2 chemokines thymus- and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in the lung after allergen challenge, and blockade of these chemokines inhibited the recruitment of effector T cells. In summary, the data suggest that pulmonary eosinophils are required for the localized recruitment of effector T cells.
T-cell immunoglobulin and mucin domain 3 (TIM-3) is known as a negative immune regulator and emerging data have implicated TIM-3 a pivotal role in suppressing antitumor immunity. The co-stimulatory receptor CD137 is transiently upregulated on T-cells following activation and increases their proliferation and survival when engaged. Although antagonistic anti-TIM-3 or agonistic anti-CD137 antibodies can promote the rejection of several murine tumors, some poorly immunogenic tumors were refractory to this treatment. In this study, we sought to evaluate whether combined TIM-3 blockade and CD137 activation would significantly improve the immunotherapy in the murine ID8 ovarian cancer model.
Mice with established ID8 tumor were intraperitoneally injected with single or combined anti-TIM-3/CD137 monoclonal antibody (mAb); mice survival was recorded, the composition and gene expression of tumor-infiltrating immune cells in these mice was analyzed by flow cytometry and quantitative RT-PCR respectively, and the function of CD8+ cells was evaluated by ELISA and cytotoxicity assay.
Either anti-TIM-3 or CD137 mAb alone, although effective in 3 days established tumor, was unable to prevent tumor progression in mice bearing 10 days established tumor, however, combined anti-TIM-3/CD137 mAb significantly inhibited the growth of these tumors with 60% of mice tumor free 90 days after tumor inoculation. Therapeutic efficacy was associated with a systemic immune response with memory and antigen specificity, required CD4+ cells and CD8+ cells. The 2 mAb combination increased CD4+ and CD8+ cells and decreased immunosuppressive CD4+FoxP3+ regulatory T (Treg) cells and CD11b+Gr-1+ myeloid suppressor cells (MDSC) at tumor sites, giving rise to significantly elevated ratios of CD4+ and CD8+ cells to Treg and MDSC; This is consistent with biasing local immune response towards an immunostimulatory Th1 type and is further supported by quantitative RT-PCR data showing the increased Th1-associated genes by anti-TIM-3/CD137 treatment. The increased CD8+ T cells produced high level of IFN-γ upon tumor antigen stimulation and displayed antigen-specific cytotoxic activity.
To our knowledge, this is the first report investigating the effects of anti-TIM-3/CD137 combined mAb in a murine ovarian cancer model, and our results may aid the design of future trials for ovarian cancer immunotherapy.
Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.
BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.
Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund's adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.
These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.
In both skin and synovial tissues of psoriatic arthritis (PsA) patients, there are prominent lymphocytic infiltrates localized to the dermal papillae in the skin and the sublining layer stroma in the joint. T-cells, with a predominance of CD4+ lymphocytes, are the most significant lymphocytes in the tissues; in contrast, this ratio is reversed in the epidermis, synovial fluid compartment, and at the enthesis, where CD8+ T-cells are more common. This differential tropism of CD8+ T-cell suggests that the CD8+ T-cells may be driving the immune response in the joint and skin. This is supported by an association with MHC class I. The cytokine network in the psoriatic skin and synovium is dominated by monocyte and T-cell-derived cytokines: IL-1β, IL-2, IL-10, IFN-γ, and TNF-α. In PsA synovium, higher levels of IFN-γ, IL-2, and IL-10 have been detected than in psoriatic skin. An analysis of T-cell receptor beta-chain variable (TCRβV) gene repertoires revealed common expansions in both skin and synovial inflammatory sites, suggesting an important role for cognate T-cell responses in the pathogenesis of PsA and that the inciting antigen may be identical or homologous between the afflicted skin and synovium. Traditionally, T-cells have been classified as T helper 1 (Th1) or Th2 cells by production of defining cytokines, IFN-γ and IL-4, respectively. Recently, a new type of T-cell, Th17, has been linked to autoimmune inflammation. T-helper 17 (Th17) cells are a unique effector CD4+ T-cell subset characterized by the production of interleukin (IL)-17. Murine diseases that were previously considered to be pure Th1-mediated responses have been shown to contain mixed populations of Th1 and Th17 cells. Also, in humans, a critical immunoregulatory role of Th-17 cells in infectious and autoimmune diseases has been identified. It has been postulated that IL-17 may be important in psoriasis. Our initial observations demonstrate that IL-17 and its receptor system are important for PsA also. In in vivo and in vitro studies we have demonstrated that IL-17/IL-17R are enriched in skin, synovial tissue, and synovial fluid of psoriatic arthritis patients and Th17 cells are functionally significant in the pathogenesis of psoriasis and psoriatic arthritis. Here we will share our experience of the SCID mouse model of psoriasis in respect to its use in investigating psoriatic diseases and development of immune-based drugs for psoriasis, psoriatic arthritis, and other autoimmune diseases.
Novel therapy; psoriasis; psoriatic arthritis; SCID mouse; Th17 cell
Respiratory virus infections, such as influenza, typically induce a robust type I (pro-inflammatory cytokine) immune response, however, the production of type 2 cytokines has been observed. Type 2 cytokine production during respiratory virus infection is linked to asthma exacerbation; however, type 2 cytokines may also be tissue protective. Interleukin (IL)-5 is a prototypical type 2 cytokine that is essential for eosinophil maturation and egress out of the bone marrow. However, little is known about the cellular source and underlying cellular and molecular basis for the regulation of IL-5 production during respiratory virus infection. Using a mouse model of influenza virus infection, we found a robust transient release of IL-5 into infected airways along with a significant and progressive accumulation of eosinophils into the lungs, particularly during the recovery phase of infection, i.e. following virus clearance. The cellular source of the IL-5 was group 2 innate lymphoid cells (ILC2) infiltrating the infected lungs. Interestingly, the progressive accumulation of eosinophils following virus clearance is reflected in the rapid expansion of c-kit+ IL-5 producing ILC2. We further demonstrate that the enhanced capacity for IL-5 production by ILC2 during recovery is concomitant with the enhanced expression of the IL-33 receptor subunit, ST2, by ILC2. Lastly, we show that NKT cells, as well as alveolar macrophages (AM), are endogenous sources of IL-33 that enhance IL-5 production from ILC2. Collectively, these results reveal that c-kit+ ILC2 interaction with IL-33 producing NKT and AM leads to abundant production of IL-5 by ILC2 and accounts for the accumulation of eosinophils observed during the recovery phase of influenza infection.
IL-5 is a cytokine that is typically associated with parasitic infections and allergic reactions. The primary role of IL-5 is thought to be for the development and maturation of an innate immune cell type, the eosinophil, which is also a culprit in allergic diseases such as asthma. During respiratory virus infection, such as influenza infection, IL-5 and eosinophils are not thought to play a major role in host defense. Here we show that IL-5 is produced in response to influenza infection and results in the progressive accumulation of eosinophils in the lung. We show that a newly discovered cell type, the group 2 innate lymphoid cell (ILC2), is responsible for IL-5 production during influenza infection and that the capacity of ILC2 to make IL-5 is greatly increased following virus clearance, i.e. during the recovery phase. The production of IL-5 by ILC2 is in part regulated by NKT cells and IL-33 produced by this cell type during the recovery phase of influenza infection.
The cytokine interplay during the development of protective immunity to the radiation-attenuated (RA) schistosome vaccine has been extensively characterized over recent years, yet the role of costimulatory molecules in the development of cell-mediated immunity is much less well understood. Here we demonstrate the importance of CD40/CD154 in vaccine-induced immunity, as CD154−/− mice exposed to RA schistosomes develop no protection to challenge infection. We showed that vaccinated CD154−/− mice have defective Th1-associated immune responses in the skin-draining lymph nodes and the lungs, with reduced or absent levels of interleukin-12p40 (IL-12p40), gamma interferon, and nitric oxide, but elevated levels of lung IL-4 and IL-5. The expression of major histocompatibility complex II (MHC-II) on antigen-presenting cells recovered from the lungs of vaccinated CD154−/− mice was also severely compromised. The administration of anti-CD40 monoclonal antibody (MAb) to CD154−/− mice did not reconstitute sustained Th1 responses in the lymph nodes or the lungs, nor did the MAb restore anti-parasite immunoglobulin G production or protective immunity. On the other hand, the administration of recombinant IL-12 (rIL-12) to CD154−/− mice shortly after vaccination caused elevated and sustained levels of Th1-associated cytokines, rescued MHC-II expression by lung CD11c+ cells, and restored the appearance of inflammatory effector foci in the lungs. However, the treatment of CD154−/− mice with rIL-12 did not restore protection. We conclude that protective immunity to the RA schistosome vaccine is CD154 dependent but is independent of IL-12-orchestrated cellular immune mechanisms in the lungs.
The cytokine interleukin (IL) 12 stimulates T cell and natural killer cell production of interferon (IFN) gamma and inhibits T cell production of IL-4. We investigated the effects of IL-12 on cytokine gene expression, immunoglobulin (Ig)E, mucosal mast cell, and eosinophil responses, and the course of infection in mice inoculated with the nematode parasite Nippostrongylus brasiliensis, as well as the IFN-gamma dependence of these effects. IL-12 stimulated IFN-gamma and IL-10 gene expression during primary and secondary N. brasiliensis infections and inhibited IL-3, IL-4, IL-5, and IL-9 gene expression during primary infections but had little inhibitory effect during secondary infections. IL-12 inhibited IgE, mucosal mast cell, and blood and tissue eosinophil responses during primary infections, but only eosinophil responses during secondary infections. IL-12 enhanced adult worm survival and egg production during primary, but not secondary infections. IL-12 needed to be administered by day 4 of a primary infection to inhibit IgE and mucosal mast cell responses, and by day 6 to strongly inhibit eosinophil responses and to enhance worm survival and fecundity. Anti-IFN-gamma mAb inhibited the effects of IL-12 on IgE secretion, intestinal mucosal mastocytosis, and parasite survival and fecundity, but did not affect IL-12 inhibition of eosinophilia. These observations indicate that IL-12, if administered during the initiation of eosinophilia. These observations indicate that IL-12, if administered during the initiation of an immune response, can change the response from one that is characterized by the production of T helper (Th)2-associated cytokines to one characterized by the production of Th-1 associated cytokines. However, IL-12 treatment has less of an effect once the production of Th2-associated cytokines has become established. In addition, our results provide evidence that Th2- associated responses protect against, and/or Th1-associated responses exacerbate, nematode infections.
Eosinophils arise from hematopoietic CD34+ stem cells in the bone marrow. They acquire IL-5Rα on their surface at a very early stage during eosinophilopoiesis, and differentiate under the strong influence of interleukin (IL)-5. They then exit to the bloodstream, and enter the lung upon exposure to airway inflammatory signals, including eotaxins. In inflamed tissues, eosinophils act as key mediators of terminal effector functions and innate immunity and in linking to adaptive immune responses. Transcription factors GATA-1, CCAAT/enhancer-binding protein, and PU.1 play instructive roles in eosinophil specification from multipotent stem cells through a network of cooperative and antagonistic interactions. Not surprisingly, the interplay of these transcription factors is instrumental in forming the regulatory circuit of expression of eosinophil-specific genes, encoding eosinophil major basic protein and neurotoxin, CC chemokine receptor 3 eotaxin receptor, and IL-5 receptor alpha. Interestingly, a common feature is that the critical cis-acting elements for these transcription factors are clustered in exon 1 and intron 1 of these genes rather than their promoters. Elucidation of the mechanism of eosinophil development and activation may lead to selective elimination of eosinophils in animals and human subjects. Furthermore, availability of a range of genetically modified mice lacking or overproducing eosinophil-specific genes will facilitate evaluation of the roles of eosinophils in the pathogenesis of asthma. This review summarizes eosinophil biology, focusing on development and regulation of eosinophil-specific genes, with a heavy emphasis on the causative link between eosinophils and pathological development of asthma using genetically modified mice as models of asthma.
Asthma; CCR3; eosinophils; eotaxin; GATA-1; IL-5
Among patients with cystic fibrosis (CF), females have worse pulmonary function and survival than males, primarily due to chronic lung inflammation and infection with Pseudomonas aeruginosa (P. aeruginosa). A role for gender hormones in the causation of the CF "gender gap" has been proposed. The female gender hormone 17β-estradiol (E2) plays a complex immunomodulatory role in humans and in animal models of disease, suppressing inflammation in some situations while enhancing it in others. Helper T-cells were long thought to belong exclusively to either T helper type 1 (Th1) or type 2 (Th2) lineages. However, a distinct lineage named Th17 is now recognized that is induced by interleukin (IL)-23 to produce IL-17 and other pro-inflammatory Th17 effector molecules. Recent evidence suggests a central role for the IL-23/IL-17 pathway in the pathogenesis of CF lung inflammation. We used a mouse model to test the hypothesis that E2 aggravates the CF lung inflammation that occurs in response to airway infection with P. aeruginosa by a Th17-mediated mechanism.
Exogenous E2 caused adult male CF mice with pneumonia due to a mucoid CF clinical isolate, the P. aeruginosa strain PA508 (PA508), to develop more severe manifestations of inflammation in both lung tissue and in bronchial alveolar lavage (BAL) fluid, with increased total white blood cell counts and differential and absolute cell counts of polymorphonuclear leukocytes (neutrophils). Inflammatory infiltrates and mucin production were increased on histology. Increased lung tissue mRNA levels for IL-23 and IL-17 were accompanied by elevated protein levels of Th17-associated pro-inflammatory mediators in BAL fluid. The burden of PA508 bacteria was increased in lung tissue homogenate and in BAL fluid, and there was a virtual elimination in lung tissue of mRNA for lactoferrin, an antimicrobial peptide active against P. aeruginosa in vitro.
Our data show that E2 increases the severity of PA508 pneumonia in adult CF male mice, and suggest two potential mechanisms: enhancement of Th17-regulated inflammation and suppression of innate antibacterial defences. Although this animal model does not recapitulate all aspects of human CF lung disease, our present findings argue for further investigation of the effects of E2 on inflammation and infection with P. aeruginosa in the CF lung.