Extracellular cyclophilins have been well described as chemotactic factors for various leukocyte subsets. This chemotactic capacity is dependent upon interaction of cyclophilins with the cell surface signaling receptor CD147. Elevated levels of extracellular cyclophilins have been documented in several inflammatory diseases. We propose that extracellular cyclophilins, via interaction with CD147, may contribute to the recruitment of leukocytes from the periphery into tissues during inflammatory responses. In this study, we examined whether extracellular cyclophilin-CD147 interactions might influence leukocyte recruitment in the inflammatory disease allergic asthma. Using a mouse model of asthmatic inflammation, we show that 1) extracellular cyclophilins are elevated in the airways of asthmatic mice; 2) mouse eosinophils and CD4+ T cells express CD147, which is up-regulated on CD4+ T cells upon activation; 3) cyclophilins induce CD147-dependent chemotaxis of activated CD4+ T cells in vitro; 4) in vivo treatment with anti-CD147 mAb significantly reduces (by up to 50%) the accumulation of eosinophils and effector/memory CD4+ T lymphocytes, as well as Ag-specific Th2 cytokine secretion, in lung tissues; and 5) anti-CD147 treatment significantly reduces airway epithelial mucin production and bronchial hyperreactivity to methacholine challenge. These findings provide a novel mechanism whereby asthmatic lung inflammation may be reduced by targeting cyclophilin-CD147 interactions.
Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2–/– mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.
Although the main regulators of leukocyte trafficking are chemokines, another family of chemotactic agents is cyclophilins. Intracellular cyclophilins function as peptidyl-protyl cis-trans isomerases and are targets of the immunosuppressive drug, cyclosporine A (CsA). Cyclophilins can also be secreted in response to stress factors, with elevated levels of extracellular cyclophilins detected in several inflammatory diseases. Extracellular cyclophilins are known to have potent chemotactic properties, suggesting they might contribute to inflammatory responses by recruiting leukocytes into tissues. The objective of the current study was to determine the impact of blocking cyclophilin activity using a cell-impermeable derivative of CsA, MM218, to specifically target extracellular pools of cyclophilins. We show that treatment with this compound in a mouse model of allergic lung inflammation: 1) demonstrates up to 80% reduction in inflammation, 2) directly inhibits the recruitment of antigen-specific CD4+ T cells, and 3) works equally well when delivered at 100-fold lower doses to the airways. Our findings suggest that cell-impermeable analogs of CsA can effectively reduce inflammatory responses by targeting leukocyte recruitment mediated by extracellular cyclophilins. Specifically blocking the extracellular function(s) of cyclophilins may provide a novel approach for inhibiting the recruitment of one of the principal immune regulators of allergic lung inflammation, antigen-specific CD4+ T cells, into inflamed airways and lungs.
The recruitment and trafficking of leukocytes are essential aspects of the inflammatory process. Although chemokines are thought to be the main regulators of cell trafficking, extracellular cyclophilins have been shown recently to have potent chemoattracting properties for human leukocytes. Cyclophilins are secreted by a variety of cell types and are detected at high levels in tissues with ongoing inflammation. CD147 has been identified as the main signaling receptor for cyclophilin A (CypA) on human leukocytes. It is interesting that the expression of CD147 is elevated on leukocytes from inflamed tissue, suggesting a correlation among the presence of extracellular cyclophilins, CD147 expression, and inflammatory responses. Thus, cyclophilin-CD147 interactions may contribute directly to the recruitment of leukocytes into inflamed tissues. In the current studies, we show that activated human T lymphocytes express elevated levels of CD147, compared with resting T cells and that these activated T cells migrate more readily to CypA than resting cells. Furthermore, we show that unlike resting CD4+ T cells, the cyclophilin-mediated migration of activated T cells does not require interaction with heparan sulfate receptors but instead, is dependent on CD147 interaction alone. Such findings suggest that cyclophilin-CD147 interactions will be most potent when leukocytes are in an activated state, for example, during inflammatory responses. Thus, targeting cyclophilin-CD147 interactions may provide a novel approach for alleviating tissue inflammation.
Chronic asthma is characterized by ongoing recruitment of inflammatory cells and airway hyperresponsiveness leading to structural airway remodeling. Although α4β1 and β2 integrins regulate leukocyte migration in inflammatory diseases and play decisive roles in acute asthma, their role has not been explored under the chronic asthma setting. To extend our earlier studies with α4Δ/Δ and β2−/− mice, which showed that both a4 and b2 integrins have nonredundant regulatory roles in acute ovalbumin (OVA)-induced asthma, we explored to what extent these molecular pathways control development of structural airway remodeling in chronic asthma.
Materials and Methods
Control, α4Δ/Δ, and β2−/−mouse groups, sensitized by intraperitoneal OVA as allergen, received intratracheal OVA periodically over days 8 to 55 to induce a chronic asthma phenotype. Post-OVA assessment of inflammation and pulmonary function (airway hyperresponsiveness), together with airway modeling measured by goblet cell metaplasia, collagen content of lung, and transforming growth factor β1 expression in lung homogenates, were evaluated.
In contrast to control and β2−/− mice, α4Δ/Δ mice failed to develop and maintain the composite chronic asthma phenotype evaluated as mentioned and subepithelial collagen content was comparable to baseline. These data indicate that β2 integrins, although required for inflammatory migration in acute asthma, are dispensable for structural remodeling in chronic asthma.
α4 integrins appear to have a regulatory role in directing transforming growth factor β-induced collagen deposition and structural alterations in lung architecture likely through interactions of Th2 cells, eosinophils, or mast cells with endothelium, resident airway cells, and/or extracellular matrix.
Selective leukocyte trafficking and recruitment is primarily regulated by a specific family of small proteins called “chemokines”. This extended family shepherds and guides leukocytes through their lives, facilitating their development, regulating their interactions with other leukocyte types, and guiding their recruitment to sites of inflammation.
Through the actions of chemokines, allergen sensitization is regulated in atopic asthma, through the controlled migration of dendritic cells, T- and B-lymphocytes, mast cells and basophils. Subsequently, atopic inflammation is driven by chemokine-directed recruitment of eosinophils, basophils and lymphocytes. Diseases from cancer to chronic obstructive pulmonary disease to interstitial fibrosis are all potential targets for chemokine receptor antagonism.
Innate immunity (the early pattern-recognition responses to stimuli such as lipopolysaccharide, viral proteins and bacterial DNA) needs to bridge the gap to specific immunity and antibody production and immunological memory. Again, chemokines are likely to be fundamental mediators of these responses.
Chemokines are fundamental regulators of leukocyte homeostasis and inflammation, and their antagonism by small molecule chemokine receptor antagonists may be of enormous importance in the future treatment of human respiratory disease.
Allergy; asthma; chemokines; immunoresponse; inflammation
Inflammatory infiltrates, airway hyper-responsiveness, goblet cell hyperplasia and subepithelial thickening are characteristic of chronic asthma. Current animal models of allergen-induced airway inflammation generally concentrate on the acute inflammation following allergen exposure and fail to mimic all of these features.
The aim of this study was to use a murine model of prolonged allergen-induced airway inflammation in order to characterize the cells and molecules involved in the ensuing airway remodelling. Moreover, we investigated whether remodelling persists in the absence of continued allergen challenge.
Acute pulmonary eosinophilia and airways hyper-reactivity were induced after six serial allergen challenges in sensitized mice (acute phase). Mice were subsequently challenged three times a week with ovalbumin (OVA) (chronic phase) up to day 55. To investigate the persistence of pathology, one group of mice were left for another 4 weeks without further allergen challenge (day 80).
The extended OVA challenge protocol caused significant airway remodelling, which was absent in the acute phase. Specifically, remodelling was characterized by deposition of collagen as well as airway smooth muscle and goblet cell hyperplasia. Importantly, these airway changes, together with tissue eosinophilia were sustained in the absence of further allergen challenge. Examination of cytokines revealed a dramatic up-regulation of IL-4 and tumour growth factor-β1 during the chronic phase. Interestingly, while IL-4 levels were significantly increased during the chronic phase, levels of IL-13 fell. Levels of the Th1-associated cytokine IFN-γ also increased during the chronic phase.
In conclusion, we have demonstrated that prolonged allergen challenge results in persistent airway wall remodelling.
airway remodelling; allergic airway inflammation; asthma; eosinophils; Th2 cytokines
Asthma is frequently caused and/or exacerbated by sensitization to fungal allergens, which are ubiquitous in many indoor and outdoor environments. Severe asthma with fungal sensitization is characterized by airway hyperresponsiveness and bronchial constriction in response to an inhaled allergen that is worsened by environmental exposure to airborne fungi and which leads to a disease course that is often very difficult to treat with standard asthma therapies. As a result of complex interactions among inflammatory cells, structural cells, and the intercellular matrix of the allergic lung, patients with sensitization to fungal allergens may experience a greater degree of airway wall remodeling and progressive, accumulated pulmonary dysfunction as part of the disease sequela. From their development in the bone marrow to their recruitment to the lung via chemokine and cytokine networks, eosinophils form an important component of the inflammatory milieu that is associated with this syndrome. Eosinophils are recognized as complex multi-factorial leukocytes with diverse functions in the context of allergic fungal asthma. In this review, we will consider recent advances in our understanding of the molecular mechanisms that are associated with eosinophil development and migration to the allergic lung in response to fungal inhalation, along with the eosinophil’s function in the immune response to and the immunopathology attributed to fungus-associated allergic pulmonary disease.
allergic asthma; inflammation; eosinophils; fungus
The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases.
Chemokines contribute to inflammatory responses by inducing leukocyte migration and extravasation. In addition, chemoattractants other than classical chemokines can also be present. Many chemokines have been demonstrated to cooperate, leading to an augmentation in leukocyte recruitment and providing a potential role for the presence of multiple chemoattractants. Extracellular cyclophilins are a group of alternative chemotactic factors, which can be highly elevated during various inflammatory responses and, as we have previously shown, can contribute significantly to neutrophil recruitment in an animal model of acute lung inflammation. In the current studies we investigated whether the most abundant extracellular cyclophilin, CypA, has the capacity to function in partnership with 2 classical chemokines known to be secreted in the same model, macrophage inflammatory protein (MIP)-2/CXCL2 and keratinocyte chemoattractant (KC)/CXCL1.
Neutrophil migration in response to combinations of CypA and MIP-2 or CypA and KC was measured by in vitro chemotaxis assays. Biochemical responses of neutrophils incubated with the combinations of chemoattractants were determined by changes in chemokine receptor internalization and actin polymerization measured by flow cytometry, and changes in intracellular calcium mobilization measured with a calcium sensitive fluorochrome.
A combination of CypA and MIP-2, but not KC, augmented neutrophil migration. Based on the level of augmentation, the cooperation between CypA and MIP-2 appeared to be synergistic. Evidence that CypA and MIP-2 cooperate at the biochemical level was demonstrated by increases in receptor internalization, calcium mobilization, and actin polymerization.
These findings provide evidence for the capacity of extracellular cyclophilins to interact with classical chemokines, resulting in greater and more efficient leukocyte recruitment.
chemokine; chemotaxis; inflammation
The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen-challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage-derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen-specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.
T lymphocytes; chemokines; allergy
The mechanisms underlying exacerbation of asthma induced by respiratory syncytial virus (RSV) infection have been extensively studied in human and animal models. However, most of these studies focused on acute inflammation and little is known of its long-term consequences on remodelling of the airway tissue.
The aim of the study was to use a murine model of prolonged allergen-induced airway inflammation to investigate the effect of RSV infection on allergic airway inflammation and tissue remodelling.
We subjected mice to RSV infection before or during the chronic phase of airway challenges with OVA and compared parameters of airway inflammation and remodelling at the end-point of the prolonged allergen-induced airway inflammation protocol.
RSV infection did not affect the severity of airway inflammation in any of the groups studied. However, RSV infection provoked airway remodelling in non-sensitized, allergen-challenged mice that did not otherwise develop any of the features of allergic airways disease. Increased collagen synthesis in the lung and thickening of the bronchial basal membrane was observed in non-sensitized allergen-challenged mice only after prior RSV infection. In addition, fibroblast growth factor (FGF)-2 but not TGF-β1 was increased in this group following RSV infection.
Our data show for the first time that RSV infection can prime the lung of mice that are not previously systemically sensitized, to develop airway remodelling in response to allergen upon sole exposure via the airways. Moreover, our results implicate RSV-induced FGF-2 in the remodelling process in vivo.
airway allergic inflammation; airway remodelling; asthma; RSV
Chemokines direct leukocyte trafficking and positioning within tissues, thus playing critical roles in regulating immune responses and inflammation. The chemokine system is complex, involving interactions between multiple chemokines and their receptors that operate in combinatorial cascades with adhesion molecules. The involvement of multiple chemokines and chemokine receptors in these processes brings flexibility and specificity to recruitment. The hepatic vascular bed is a unique low-flow environment through which leukocytes are recruited to the liver during homeostatic immune surveillance and in response to infection or injury. The rate of leukocyte recruitment and the nature of cells recruited through the sinusoids in response to inflammatory signals will shape the severity of disease. At one end of the spectrum, fulminant liver failure results from a rapid recruitment of leukocytes that leads to hepatocyte destruction and liver failure; at the other end, diseases such as chronic hepatitis C infection may progress over many years from hepatitis to fibrosis and cirrhosis. Chronic hepatitis is characterized by a T lymphocyte-rich infiltrate and the nature and outcome of hepatitis will depend on the T cell subsets recruited, their activation and function within the liver. Different subsets of effector T cells have been described based on their secretion of cytokines and specific functions. These include Th1 and Th2 cells, and more recently Th17 and Th9 cells, which are associated with different types of immune response and which express distinct patterns of chemokine receptors that promote their recruitment under particular conditions. The effector function of these cells is balanced by the recruitment of regulatory T cells that are able to suppress antigen-specific effectors to allow resolution of immune responses and restoration of immune homeostasis. Understanding the signals that are responsible for recruiting different lymphocyte subsets to the liver will elucidate disease pathogenesis and open up new therapeutic approaches to modulate recruitment in favor of resolution rather than injury.
Leukocytes; Chemokines; Hepatic inflammation
The development of chronic allergic dermatitis in early life has been associated with increased onset and severity of allergic asthma later in life. However, the mechanisms linking these two diseases are poorly understood. Here, we report that the development of oxazolone-induced chronic allergic dermatitis, in a mouse model, caused enhanced ovalbumin-induced allergic asthma after resolution of the former disease. Our findings show that oxazolone-induced dermatitis caused a marked increase in tissue mast cells, which persisted long after the resolution of this disease. Subsequent ovalbumin sensitization and airway challenge of mice that had recovered from dermatitis resulted in increased allergic airway hyperreactivity. The findings demonstrate that the accumulation of mast cells during dermatitis has the detrimental effect of increasing allergic airway hypersensitivity. Importantly, our findings also show that exposure to a given allergen can modify the immune response to an unrelated allergen.
Allergy; Fc receptors; IgE; Mast cells/basophils; Skin; Rodent
Cellular recruitment during inflammatory/immune responses is tightly regulated. The ability to dampen inflammation is imperative for prevention of chronic immune responses, as in asthma. Here we investigated the ability of lipoxin A4 (LXA4) stable analogs to regulate airway responses in two allergen-driven models of inflammation. A 15-epi-LXA4 analog (ATLa) and a 3-oxa-15-epi-LXA4 analog (ZK-994) prevented excessive eosinophil and T lymphocyte accumulation and activation after mice were sensitized and aerosol-challenged with ovalbumin. At <0.5 mg/kg, these LXA4 analogs reduced leukocyte trafficking into the lung by >50% and to a greater extent than equivalent doses of the CysLT1 receptor antagonist montelukast. Distinct from montelukast, ATLa treatment led to marked reductions in cysteinyl leukotrienes, interleukin-4 (IL-4), and IL-10, and both ATLa and ZK-994 inhibited levels of IL-13. In cockroach allergen-induced airway responses, both intraperitoneal and oral administration of ZK-994 significantly reduced parameters of airway inflammation and hyper-responsiveness in a dose-dependent manner. ZK-994 also significantly changed the balance of Th1/Th2-specific cytokine levels. Thus, the ATLa/LXA4 analog actions are distinct from CysLT1 antagonism and potently block both allergic airway inflammation and hyper-reactivity. Moreover, these results demonstrate these analogs’ therapeutic potential as new agonists for the resolution of inflammation.
resolution; lipid mediators; leukocytes
Allergic airway inflammation is characterized by elaboration of cytokines and chemokines leading to recruitment of inflammatory leukocytes, predominantly eosinophils, to the airways. Granulocyte macrophage colony stimulating factor (GM-CSF) is generated in the lungs of human subjects with asthma in response to allergen challenge and is necessary for the development of allergen-induced bronchial eosinophilia in mice. The effect of GM-CSF on human eosinophil and neutrophil interactions with the vascular endothelium under conditions of blood flow was investigated in post-capillary venules of the rabbit mesentery by intravital microscopy.
While GM-CSF significantly reduced the rolling fraction of neutrophils in vivo and induced consistent shedding of neutrophil L-selectin in vitro, its effect on eosinophil rolling was variable. Eosinophils from 57% of the donors demonstrated inhibition of rolling, while eosinophils from the remaining 43% of donors demonstrated no inhibition or increased rolling. The variable effect of GM-CSF on inhibition of eosinophil rolling was associated with variable shedding of L-selectin in vitro. In contrast to the differential effect of GM-CSF on neutrophils versus eosinophils, stimulation with phorbol myristate acetate demonstrated a similar degree of inhibition of rolling and L-selectin shedding by neutrophils and eosinophils suggesting that there was no defect in L-selectin shedding in the eosinophil donors who did not respond to GM-CSF.
Overall, these studies demonstrate that GM-CSF consistently inhibits interaction of neutrophils with endothelium in vivo, whereas its effect on eosinophil-endothelial interactions is variable. GM-CSF may thus be one factor accounting for the varying percentage of eosinophils and neutrophils recruited to sites of allergic inflammation in different individuals.
Allergic Inflammation; Cytokines; Leukocyte Rolling; L-selectin
The effects of chronic mild prenatal stress on leukocyte infiltration into the airways was investigated in rat offspring. The chronic prenatal stress consisted of transitory and variable changes in the rat's living conditions. Offspring at adult age were actively sensitized (day 0) and intratracheally challenged (day 14) with ovalbumin. Bronchoalveolar lavage was performed in the offspring at 48 h after intratracheal challenge with ovalbumin. A significant increase in total leukocyte infiltration was observed in the non-stressed offspring group and this was associated with a marked recruitment of eosinophils without a significant effect on the influx of neutrophils and mononuclear cells. In the prenatal stressed offspring, the counts of both total leukocyte and eosinophils, as well as mononuclear cells, was increased by 50% compared to the non-stressed offspring. We provide here the first experimental evidence that chronic mild unpredictable prenatal stress produces a marked increase in the allergen-induced airway inflammation in the rat offspring.
Rationale: The D6 chemokine receptor can bind and scavenge several chemokines, including the T-helper 2 (Th2)–associated chemokines CCL17 and CCL22. Although D6 is constitutively expressed in the lung, its pulmonary function is unknown.
Objectives: This study tested whether D6 regulates pulmonary chemokine levels, inflammation, or airway responsiveness during allergen-induced airway disease.
Methods: D6-deficient and genetically matched C57BL/6 mice were sensitized and challenged with ovalbumin. ELISA and flow cytometry were used to measure levels of cytokines and leukocytes, respectively. Mechanical ventilation was used to measure airway reactivity.
Results: The ability of D6 to diminish chemokine levels in the lung was chemokine concentration dependent. CCL17 and CCL22 were abundant in the airway, and their levels were attenuated by D6 when they were within a defined concentration range. By contrast, airway concentrations of CCL3, CCL5, and CCL11 were low and unaffected by D6. Allergen-challenged D6-deficient mice had more dendritic cells, T cells, and eosinophils in the lung parenchyma and more eosinophils in the airway than similarly challenged C57BL/6 mice. By contrast, D6-deficient mice had reduced airway responses to methacholine compared with C57BL/6 mice. Thus, D6 has opposing effects on inflammation and airway reactivity.
Conclusions: The ability of D6 to scavenge chemokines in the lung is dependent on chemokine concentration. The absence of D6 increases inflammation, but reduces airway reactivity. These findings suggest that inhibiting D6 function might be a novel means to attenuate airway responses in individuals with allergic asthma.
chemokines; lung; D6; allergic; transforming growth factor–; β
Allergic asthma is a chronic inflammatory disease of the upper airway. It is well appreciated that maladaptive Th2 immunity promotes the allergic phenotype, the underlying mechanisms of which remain elusive. The disease is associated with activation of complement, an ancient danger-sensing component of the innate immune system. Different models of experimental allergic asthma suggest that the small complement fragments of C3 and C5, the anaphylatoxins C3a and C5a, not only promote proallergic effector functions during the allergic effector phase but regulate the development of Th2 immunity during allergen sensitization. The available data support a concept in which C5a is dominant during allergen sensitization and protects against the development of maladaptive Th2 immunity. By contrast, C3a and C5a appear to act synergistically and drive allergic inflammation during the effector phase. In this article, we will review the recent findings in the field to judge the benefit of complement targeting in allergic asthma.
allergic asthma; anaphylatoxin; complement; dendritic cell; innate immunity; Th2 cytokine
Rationale: In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant.
Objectives: We investigated whether allergic sensitization through the airway elicits immune responses qualitatively different than those arising in the peritoneum.
Methods: Mice were sensitized to allergen through the airway using low-dose LPS as an adjuvant, or through the peritoneum using aluminum hydroxide as an adjuvant. After a single allergen challenge, ELISA and flow cytometry were used to measure cytokines and leukocyte subsets. Invasive measurements of airway resistance were used to measure allergen-induced airway hyperreactivity (AHR).
Measurements and Main Results: Sensitization through the peritoneum primed strong Th2 responses and eosinophilia, but not AHR, after a single allergen challenge. By contrast, allergic sensitization through the airway primed only modest Th2 responses, but strong Th17 responses. Th17 cells homed to the lung and released IL-17 into the airway on subsequent encounter with inhaled allergen. As a result, these mice developed IL-17–dependent airway neutrophilia and AHR. This AHR was neutrophil-dependent because it was abrogated in CXCR2-deficient mice and also in wild-type mice receiving a neutrophil-depleting antibody. Individually, neither IL-17 nor ongoing Th2 responses were sufficient to confer AHR, but together they acted synergistically to promote neutrophil recruitment, eosinophil recruitment and AHR.
Conclusions: Allergic sensitization through the airway primes modest Th2 responses but strong Th17 responses that promote airway neutrophilia and acute AHR. These findings support a causal role for neutrophils in severe asthma.
asthma; lung; immunity
L-selectin is a cell adhesion molecule, which mediates leukocyte rolling on bronchopulmonary endothelium. Previous studies in a murine model of allergic airways disease have shown that L-selectin plays a role in the regulation of airway hyperresponsiveness in asthma via mechanisms independent of inflammation. Airway remodeling has been shown to modulate airway hyperresponsiveness independently of inflammation.
Our aim was to determine if L-selectin influenced airway hyperresponsiveness via modulation of structural changes as a result of airway remodeling.
A chronic ovalbumin-induced allergic airways disease model was applied to L-selectin-deficient mice and wild-type control mice. The development of airway inflammation was assessed by examining leukocyte influx into bronchoalveolar lavage fluid. Airway remodeling changes were determined via histology and morphometric analysis of lung tissue sections, and the development of airway hyperresponsiveness was assessed by invasive plethysmography.
Total cell counts, but not individual differential cell counts, were reduced in the ovalbumin-treated L-selectin-deficient mice compared to wildtype ovalbumin-treated mice. L-selectin-deficient mice had significantly reduced epithelial thickness and smooth muscle thickness. Airway hyperresponsiveness was abrogated in ovalbumin treated L-selectin-deficient mice compared to wild-type controls.
L-selectin plays an important role in regulating airway remodeling in an animal model of chronic allergic airways disease. Abrogated airway hyperresponsiveness may be related to reduced remodeling changes in L-selectin-deficient mice. L-selectin represents a potential target for novel asthma treatment for airway remodeling and airway hyperresponsiveness.
asthma; L-selectin; airway hyperresponsiveness; airway remodeling
Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by polymerase chain reaction (PCR), but not by serology and culture, suggesting a low-level Mp involved in chronic asthma. However, the role of such a low-level Mp infection in regulation of allergic inflammation remains unknown.
To determine the impact of a low-level Mp infection in mice with established airway allergic inflammation on allergic responses such as eosinophilia and chemokine eotaxin-2, and the underlying mechanisms (i.e., prostaglandin E2 [PGE2] pathway) since PGE2 inhalation before allergen challenge suppressed eosinophil infiltration in human airways.
BALB/c mouse models of ovalbumin (OVA)-induced allergic asthma with an ensuing low-dose or high-dose Mp were used to assess IL-4 expression, BAL eosinophil, eotaxin-2 and PGE2 levels, and lung mRNA levels of microsomal prostaglandin E synthase-1 (mPGES-1). Primary alveolar macrophages (pAMs) from naïve BALB/c mice were cultured to determine if Mp-induced PGE2 or exogenous PGE2 down-regulates IL-4/IL-13-induced eotaxin-2.
Low-dose Mp in allergic mice significantly enhanced IL-4 and eotaxin-2, and moderately promoted lung eosinophilia, whereas high-dose Mp significantly reduced lung eosinophilia and tended to decrease IL-4 and eotaxin-2. Moreover, in both OVA-naïve and allergic mice, lung mPGES-1 mRNA and BAL PGE2 levels were elevated in mice infected with high-dose, but not low-dose Mp. In pAMs, IL-4/IL-13 significantly increased eotaxin-2, which was reduced by Mp infection accompanied by dose-dependent PGE2 induction. Exogenous PGE2 inhibited IL-4/IL-13-induced eotaxin-2 in a dose-dependent manner.
This study highlights a novel concept on how differing bacterial loads in the lung modify the established allergic airway inflammation, and thus interact with an allergen to further induce Th2 responses. That is: Unlike high-level Mp, low-level Mp fails to effectively induce PGE2 to down-regulate allergic responses (e.g., eotaxin-2), thus maintaining or even worsening allergic inflammation in asthmatic airways.
asthma; Mycoplasma pneumoniae; eotaxin-2; PGE2; alveolar macrophages
Asthma is a chronic inflammatory airway disease associated with increased generation of reactive oxidant species and disturbed antioxidant defenses. NRF2 is the master transcription factor that regulates the expression of Phase II antioxidant and detoxifying enzymes. Disruption of NRF2 augments oxidative stress and inflammation in a mouse model of asthma suggesting a protective role of NRF2 in the lungs in vivo. Yet, little is known about the regulation and function of NRF2 in human asthmatics. Using segmental allergen challenge, a well established experimental model of IgE-mediated asthma exacerbation in human atopic asthmatics, we investigated the effect of a specific allergen and the modulatory role of vitamin E on NRF2 and a NRF2-target gene, superoxide dismutase, in alveolar macrophages recovered from the airways at 24h after allergen instillation in vivo. Allergen-provoked airway inflammation in sensitive asthmatics caused a profound inhibition of macrophage NRF2 activity and superoxide dismutase, rendering them incapable of responding to the NRF2 inducers. Prolonged treatment with high doses of the antioxidant vitamin E lessened this allergen-induced drop in alveolar macrophage NRF2. These results are the first to demonstrate that NRF2 expression in human asthmatics is compromised upon allergen challenge but can be rescued by vitamin E in vivo.
Atopic asthma; human; alveolar macrophage; oxidative stress; NRF2; superoxide dismutase; antioxidant; vitamin E; segmental allergen challenge; BAL fluid; in vivo
Chronic inflammatory airway diseases including asthma are characterized by immune dysfunction to inhaled allergens. Our previous studies demonstrated that T cell priming to inhaled allergens requires LPS, which is ubiquitously present in household dust allergens. In this study, we evaluated the role of vascular endothelial growth factor (VEGF) in the development of T cell priming and its polarization to Th1 or Th17 cells when exposed to LPS-contaminated allergens. An asthma mouse model was induced by airway sensitization with LPS-contaminated allergens and then challenged with allergens alone. Therapeutic intervention was performed during allergen sensitization. The present study showed that lung inflammation induced by sensitization with LPS-contaminated allergens was decreased in mice with homozygous disruption of the IL-17 gene; in addition, allergen-specific Th17 immune response was abolished in IL-6 knockout mice. Meanwhile, in vivo production of VEGF was up-regulated by airway exposure of LPS. In addition, airway sensitization of allergen plus recombinant VEGF induced both type 1 and type 17 Th cell (Th1 and Th17) responses. Th1 and Th17 responses induced by airway sensitization with LPS-contaminated allergens were blocked by treatment with a pan-VEGF receptor (VEGFR; VEGFR-1 plus VEGFR-2) inhibitor during sensitization. These effects were accompanied by inhibition of the production of Th1 and Th17 polarizing cytokines, IL-12p70 and IL-6, respectively. These findings indicate that VEGF produced by LPS plays a key role in activation of naive T cells and subsequent polarization to Th1 and Th17 cells.
Allergic asthma, an inflammatory disease characterized by infiltration and activation of various leukocytes, production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious, and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine, and sensitized and challenged with allergens. Results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung, including eosinophilic/lymphocytic emigration; mRNA and/or protein expression of Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C4; and total as well as allergen-specific IgE. While nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, in spite of the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial stored mucosubstances, and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia, mucous production, and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.