Role of IL-10 in the resolution of airway inflammation

IL-10 can be considered an important agent in the resolution of inflammation. Originally named "cytokine synthesis inhibitory factor" for its ability to inhibit IFN-gamma and IL-2 production in Th2 cells, it is secreted by monocytes, macrophages, mast cells, T and B lymphocytes, and dendritic cells (DCs). IL-10 production and release by monocytic cells in response to allergic challenge is upregulated by TNF-alpha, and by negative feedback regulation of itself. However, it is also secreted by T regulatory cells (Tregs), under the control of IL-2. Importantly in the context of asthma, IL-10 inhibits eosinophilia, by suppression of IL-5 and GM-CSF, by direct effects on eosinophil apoptosis, and effects on cell proliferation through down-regulation of IL-1. A number of its cytokine suppressive characteristics are now thought to occur through its upregulation of suppressor of cytokine signaling (SOCS)-3. IL-10 is also a suppressor of nitric oxide (NO) production, which may have ramifications for its role in airway inflammatory diseases. Initial clinical trials have demonstrated relative safety and few clinically adverse events at doses of recombinant human IL-10 below 50 microg/kg, with mixed success in treatment of patients with inflammatory bowel disease and psoriasis. However, both steroid therapy and allergen specific immunotherapy are known to elevate endogenous IL-10 levels, which may account for their efficacy, suggesting that further study of IL-10 as a target for treatment of airway inflammatory diseases such as asthma and COPD is warranted.     

Induction of prolonged asthma tolerance by IL-10-differentiated dendritic cells: differential impact on airway hyperresponsiveness and the Th2 immunoinflammatory response

IL-10-differentiated dendritic cells (DC10s) can prevent allergen sensitization and reverse the asthma phenotype in mice with established disease. However, little is known about the time-frames over which this tolerance is effective. We report that at 2 wk after i.p. or transtracheal delivery of 1 × 10(6) OVA-, but not house dust mite- presenting, DC10s to OVA-asthmatic mice, significant diminution of airway hyperresponsiveness (AHR) was first apparent, whereas AHR was abrogated between 3 and 10 wk posttreatment. At 13 wk, AHR returned to pretreatment levels but could again be reversed by DC10 retreatment. The impact of a single DC10 treatment on airway eosinophil and Th2 cytokine responses to recall OVA challenge, and on OVA-specific IgE/IgG1 responses, was substantial at 3 wk posttreatment, but progressively increased thereafter, such that at 8 mo, airway eosinophil and Th2 responses to recall allergen challenge remained ～85-95% suppressed relative to saline-treated asthmatic mice. Four biweekly DC10 treatments, whether transtracheal or i.p., reduced all asthma parameters to near background by 8 wk, whereas s.c. DC10 treatments did not affect AHR but did reduce the airway Th2 responses (i.v. DC10 had no discernible effects). Repeated challenge of the DC10-treated mice with aerosolized OVA (100 μg/ml) did not reverse tolerance, but treatment with the indoleamine-2,3-dioxygenase antagonist 1-methyltryptophan or neutralizing anti-IL-10R from days 12 to 21 after DC10 therapy partially reversed tolerance (Th2 cytokine responses, but not AHR). These findings indicate that DC10-induced Th2 tolerance in asthmatic animals is long lived, but that DC10s employ distinct mechanisms to affect AHR versus Th2 immunoinflammatory parameters.     

Dissociation by steroids of eosinophilic inflammation from airway hyperresponsiveness in murine airways

Background

The link between eosinophils and the development of airway hyperresponsiveness (AHR) in asthma is still controversial. This question was assessed in a murine model of asthma in which we performed a dose ranging study to establish whether the dose of steroid needed to inhibit the eosinophil infiltration correlated with that needed to block AHR.

Methods

The sensitised BALB/c mice were dosed with vehicle or dexamethasone (0.01–3 mg/kg) 2 hours before and 6 hours after each challenge (once daily for 6 days) and 2 hours before AHR determination by whole-body plethysmography. At 30 minutes after the AHR to aerosolised methacholine the mice were lavaged and differential white cell counts were determined. Challenging with antigen caused a significant increase in eosinophils in the bronchoalveolar lavage (BAL) fluid and lung tissue, and increased AHR.

Results

Dexamethasone reduced BAL and lung tissue eosinophilia (ED₅₀ values of 0.06 and 0.08 mg/kg, respectively), whereas a higher dose was needed to block AHR (ED₅₀ of 0.32 mg/kg at 3 mg/ml methacholine. Dissociation was observed between the dose of steroid needed to affect AHR in comparison with eosinophilia and suggests that AHR is not a direct consequence of eosinophilia.

Conclusion

This novel pharmacological approach has revealed a clear dissociation between eosinophilia and AHR by using steroids that are the mainstay of asthma therapy. These data suggest that eosinophilia is not associated with AHR and questions the rationale that many pharmaceutical companies are adopting in developing low-molecular-mass compounds that target eosinophil activation/recruitment for the treatment of asthma.     

Vaccination with heat-killed Listeria as adjuvant reverses established allergen-induced airway hyperreactivity and inflammation: role of CD8+ T cells and IL-18

Asthma is a respiratory disorder characterized by airway hyperreactivity (AHR) and inflammation and is associated with high serum IgE and overproduction of IL-4, IL-5, and IL-13 by allergen-specific Th2 cells. Our previous studies demonstrated that heat-killed Listeria monocytogenes (HKL) as an adjuvant in immunotherapy successfully reversed ongoing Ag-specific Th2-dominated responses toward Th1-dominated responses, but it was unclear if such immune modulation could reverse ongoing, established disease in target organs such as the lung. In this paper we show that a single dose of Ag plus HKL as adjuvant significantly reduced AHR in a murine model for asthma and reversed established AHR when given late after allergen sensitization. HKL as adjuvant also dramatically inhibited airway inflammation, eosinophilia, and mucus production, significantly reduced Ag-specific IgE and IL-4 production, and dramatically increased Ag-specific IFN-gamma synthesis. The inhibitory effect of HKL on AHR depended on the presence of IL-12 and CD8+ T cells and was associated with an increase of IL-18 mRNA expression. Thus, our results demonstrate that HKL as an adjuvant for immunotherapy mediates immune deviation from a pathological Th2-dominated response toward a protective immune response in peripheral lymphoid tissues and in the lungs and may be clinically effective in the treatment of patients with established asthma and allergic disease.     

Il-18 gene transfer by adenovirus prevents the development of and reverses established allergen-induced airway hyperreactivity

We examined the role of IL-18 in preventing the development of and in reversing established allergen-induced airway inflammation and airway hyperreactivity (AHR), the cardinal features of asthma. IL-18, which potently induces IFN-gamma, was administered into the respiratory tract as cDNA in a replication-deficient adenovirus (Adv). Treatment of OVA-sensitized mice with the IL-18-expressing Adv reduced allergen-specific IL-4 production, airway eosinophilia, and mucus production, increased IFN-gamma production, and prevented the development of AHR. The effects of the IL-18 Adv treatment were dependent on the presence of IFN-gamma and IL-12. Moreover, administration of the IL-18 Adv to mice with established AHR greatly reduced AHR and IL-4 production and increased IFN-gamma production. These results demonstrate that IL-18, when administered by Adv into the respiratory tract, effectively reduces AHR and replaces an established Th2-biased immune response with a Th1-biased response.     

Superoxide mediates acute renal vasoconstriction produced by angiotensin II and catecholamines by a mechanism independent of nitric oxide

NAD(P)H oxidases (NOX) and reactive oxygen species (ROS) are involved in vasoconstriction and vascular remodeling during hypertension produced by chronic angiotensin II (ANG II) infusion. These effects are thought to be mediated largely through superoxide anion (O(2)(-)) scavenging of nitric oxide (NO). Little is known about the role of ROS in acute vasoconstrictor responses to agonists. We investigated renal blood flow (RBF) reactivity to ANG II (4 ng), norepinephrine (NE, 20 ng), and alpha(1)-adrenergic agonist phenylephrine (PE, 200 ng) injected into the renal artery (ira) of anesthetized Sprague-Dawley rats. The NOX inhibitor apocynin (1-4 mg.kg(-1).min(-1) ira, 2 min) or the superoxide dismutase mimetic Tempol (1.5-5 mg.kg(-1).min(-1) ira, 2 min) rapidly increased resting RBF by 8 +/- 1% (P < 0.001) or 3 +/- 1% (P < 0.05), respectively. During NO synthase (NOS) inhibition (N(omega)-nitro-l-arginine methyl ester, 25 mg/kg iv), the vasodilation tended to increase (apocynin 13 +/- 4%, Tempol 10 +/- 1%). During control conditions, both ANG II and NE reduced RBF by 24 +/- 4%. Apocynin dose dependently reduced the constriction by up to 44% (P < 0.05). Similarly, Tempol blocked the acute actions of ANG II and NE by up to 48-49% (P < 0.05). In other animals, apocynin (4 mg.kg(-1).min(-1) ira) attenuated vasoconstriction to ANG II, NE, and PE by 46-62% (P < 0.01). During NOS inhibition, apocynin reduced the reactivity to ANG II and NE by 60-72% (P < 0.01), and Tempol reduced it by 58-66% (P < 0.001). We conclude that NOX-derived ROS substantially contribute to basal RBF as well as to signaling of acute renal vasoconstrictor responses to ANG II, NE, and PE in normal rats. These effects are due to O(2)(-) rather than H(2)O(2), occur rapidly, and are independent of scavenging of NO.     

Cutting edge: invariant V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity in an experimental asthma model

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jalpha18(-/-) mice, which are exclusively deficient in the invariant Valpha14(+) (iValpha14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iValpha14 NKT cells fully reconstitutes the capacity of Jalpha18(-/-) mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69(+)) iValpha14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iValpha14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iValpha14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.     

Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction

Recent data suggest that diabetes is a risk factor for pulmonary hypertension. The aim of the present study was to analyze whether diabetes induces endothelial dysfunction in pulmonary arteries and the mechanisms involved. Male Sprague-Dawley rats were randomly divided into a control (saline) and a diabetic group (70 mg/kg(-1) streptozotocin). After 6 wk, intrapulmonary arteries were mounted for isometric tension recording, and endothelial function was tested by the relaxant response to acetylcholine. Protein expression and localization were measured by Western blot and immunohistochemistry and superoxide production by dihydroethidium staining. Pulmonary arteries from diabetic rats showed impaired relaxant response to acetylcholine and reduced vasoconstrictor response to the nitric oxide (NO) synthase inhibitor L-NAME, whereas the response to nitroprusside and the expression of endothelial NO synthase remained unchanged. Endothelial dysfunction was reversed by addition of superoxide dismutase or the NADPH oxidase inhibitor apocynin. An increase in superoxide production and increased expression of the NADPH oxidase regulatory subunit p47(phox) were also found in pulmonary arteries from diabetic rats. In conclusion, the pulmonary circulation is a target for diabetes-induced endothelial dysfunction via enhanced NADPH oxidase-derived superoxide production.     

Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness

Increased arginase I activity is associated with allergic disorders such as asthma. How arginase I contributes to and is regulated by allergic inflammatory processes remains unknown. CD4+ Th2 lymphocytes (Th2 cells) and IL-13 are two crucial immune regulators that use STAT6-dependent pathways to induce allergic airways inflammation and enhanced airways responsiveness to spasmogens (airways hyperresponsiveness (AHR)). This pathway is also used to activate arginase I in isolated cells and in hepatic infection with helminths. In the present study, we show that arginase I expression is also regulated in the lung in a STAT6-dependent manner by Th2-induced allergic inflammation or by IL-13 alone. IL-13-induced expression of arginase I correlated directly with increased synthesis of urea and with reduced synthesis of NO. Expression of arginase I, but not eosinophilia or mucus hypersecretion, temporally correlated with the development, persistence, and resolution of IL-13-induced AHR. Pharmacological supplementation with l-arginine or with NO donors amplified or attenuated IL-13-induced AHR, respectively. Moreover, inducing loss of function of arginase I specifically in the lung by using RNA interference abrogated the development of IL-13-induced AHR. These data suggest an important role for metabolism of l-arginine by arginase I in the modulation of IL-13-induced AHR and identify a potential pathway distal to cytokine receptor interactions for the control of IL-13-mediated bronchoconstriction in asthma.     

Nitric oxide -- superoxide cooperation in the regulation of renal Na(+),K(+)-ATPase

The aim of this study was to investigate whether endogenous superoxide anion is involved in the regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities. The study was performed in male Wistar rats. Compounds modulating superoxide anion concentration were infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. We found that infusion of a superoxide anion-generating mixture, xanthine oxidase (1 mU/min per kg) + hypoxanthine (0.2 mumol/min per kg), increased the medullary Na(+),K(+)-ATPase activity by 49.5% but had no effect on cortical Na(+),K(+)-ATPase and either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase. This effect was reproduced by elevating endogenous superoxide anion with a superoxide dismutase inhibitor, diethylthiocarbamate. In contrast, a superoxide dismutase mimetic, TEMPOL, decreased the medullary Na(+),K(+)-ATPase activity. The inhibitory effect of TEMPOL was abolished by inhibitors of nitric oxide synthase (L-NAME), soluble guanylate cyclase (ODQ) and protein kinase G (KT5823). The stimulatory effect of diethylthiocarbamate was not observed in animals pretreated with a synthetic cGMP analogue, 8-bromo-cGMP. An inhibitor of NAD(P)H oxidase, apocynin (1 mumol/min per kg), decreased the Na(+),K(+)-ATPase activity in the renal medulla and its effect was prevented by L-NAME, ODQ or KT5823. In contrast, a xanthine oxidase inhibitor, oxypurinol, administered at the same dose was without effect. These data suggest that NAD(P)H oxidase-derived superoxide anion increases Na(+),K(+)-ATPase activity in the renal medulla by reducing the availability of NO. Excessive intrarenal generation of superoxide anion may upregulate medullary Na(+),K(+)-ATPase leading to sodium retention and blood pressure elevation.     

Further evidence for the role of free radicals in the limb teratogenicity of L-NAME

BACKGROUND: L-NAME (N(G)-nitro-(L)-arginine methyl ester), a nitric oxide synthase inhibitor, causes severe limb reduction malformations when gravid rats are treated intraperitoneally on gd-17. Hemorrhages, appearing within hours of L-NAME administration, and defects at term can be significantly reduced by co-treatment with PBN (alpha-phenyl-N-t-butylnitrone), a spin trap antioxidant. We have proposed that limb defects result from ischemia-reperfusion injury. We examine the role of xanthine oxidase and ROS formation in the limb effects of L-NAME. METHODS: Gravidas were treated with L-NAME (50 mg/kg) in the presence or absence of allopurinol, a xanthine oxidase inhibitor. Spatial patterns of limb hemorrhage were determined promptly and at term as was digit length at the latter interval. Xanthine oxidase activities were assayed in control and treated limbs with and without allopurinol co-treatment. RESULTS: Allopurinol significantly reduced hemorrhage severity in a dose-responsive fashion when fetuses were examined at term. Higher doses of allopurinol significantly preserved digit length. Xanthine oxidase activities in fetal limb were significantly increased by L-NAME treatment whereas co-treatment with allopurinol restored activities to near-control levels. CONCLUSIONS: These findings support the role of excess reactive oxygen species (ROS) formation in L-NAME-induced limb reduction. We propose that nitric oxide (NO) depletion by L-NAME interferes with vascular integrity, and causes vasoconstriction. Resultant hypoxia stimulates superoxide formation and nitric oxide formation catalyzed by the inducible isoform of nitric oxide synthase. The reduction products of superoxide or the products of its reaction with nitric oxide oxidize or nitrate endothelial components resulting in limb reduction defects.     

Critical role for IL-13 in the development of allergen-induced airway hyperreactivity

Airway hyperresponsiveness to a variety of specific and nonspecific stimuli is a cardinal feature of asthma, which affects nearly 10% of the population in industrialized countries. Eosinophilic pulmonary inflammation, eosinophil-derived products, as well as Th2 cytokines IL-13, IL-4, and IL-5, have been associated with the development of airway hyperreactivity (AHR), but the specific immunological basis underlying the development of AHR remains controversial. Herein we show that mice with targeted deletion of IL-13 failed to develop allergen-induced AHR, despite the presence of vigorous Th2-biased, eosinophilic pulmonary inflammation. However, AHR was restored in IL-13(-/-) mice by the administration of recombinant IL-13. Moreover, adoptive transfer of OVA-specific Th2 cells generated from TCR-transgenic IL-13(-/-) mice failed to induce AHR in recipient SCID mice, although such IL-13(-/-) Th2 cells produced high levels of IL-4 and IL-5 and induced significant airway inflammation. These studies definitively demonstrate that IL-13 is necessary and sufficient for the induction of AHR and that eosinophilic airway inflammation in the absence of IL-13 is inadequate for the induction of AHR. Therefore, treatment of human asthma with antagonists of IL-13 may be very effective.     

Ebselen suppresses late airway responses and airway inflammation in guinea pigs

Although ebselen, a seleno-organic compound, inhibits inflammation in various animal models, its efficacy as an anti-asthma drug remains to be clarified. In this study, we investigated the inhibitory effect of ebselen on a guinea pig asthma model. Ebselen was orally administered at dosages of 1-20 mg/kg 2 h before an ovalbumin (OA) challenge, and then airway responses, airway inflammation, the generation of superoxide, H(2)O(2), and nitrotyrosine, and the induction of inducible nitric oxide synthase (iNOS) were evaluated. Sensitized animals challenged with OA aerosol showed dual airflow limitations, i.e., immediate and late airway responses (IAR and LAR). Ebselen significantly inhibited LAR at dosages greater than 10 mg/kg, but did not inhibit IAR at any dosage. Bronchoalveolar lavage (BAL) examination showed that airway inflammation was significantly suppressed by ebselen at 10 mg/kg. The generation of superoxide and H(2)O(2) occurred on endothelial cells of LAR bronchi, and was inhibited by 10 mg/kg of ebselen. Superoxide generation was inhibited by diphenyleneiodonium chloride (DPI), a NAD(P)H oxidase inhibitor, but not by allopurinol, a xanthine oxidase inhibitor. Immunoreactivities for iNOS and nitrotyrosine were also observed on endothelial cells of LAR bronchi and were abolished in ebselen-treated animals. The present findings suggest that ebselen can be applied as a new therapeutic agent for asthma. The possible mechanisms by which ebselen inhibits LAR likely involve suppression of oxidant formation and iNOS induction in endothelial cells.     

Nitric oxide paradox in asthma

Asthma results from allergen-driven intrapulmonary Th2 response, and is characterized by intermittent airway obstruction, airway hyperreactivity (AHR), and airway inflammation. Accumulating evidence indicates that inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO). It has been shown that exhaled NO may be derived from constitutive NO synthase (NOS) such as endothelial (NOS 3) and neural (NOS 1) in normal airways, while increased levels of NO in asthma appear to be derived from inducible NOS2 expressed in the inflamed airways. Nevertheless, the functional role of NO and NOS isoforms in the regulation of AHR and airway inflammation in human or experimental models of asthma is still highly controversial. In the present commentary we will discuss the role of lipopolysaccharides contamination of allergens as key element in the controversy related to the regulation of NOS2 activity in experimental asthma.     

Inhibition of NADPH oxidase improves impaired reactivity of pial arterioles during chronic exposure to nicotine.

Our goals were to determine whether chronic exposure to nicotine alters nitric oxide synthase (NOS)-dependent reactivity of cerebral (pial) arterioles and to identify a potential role for NADPH oxidase in impaired NOS-dependent responses during chronic exposure to nicotine. We measured in vivo diameter of pial arterioles to NOS-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in saline-treated rats and rats chronically treated with nicotine (2 mg.kg(-1).day(-1) for 2 wk via an osmotic minipump). We found that NOS-dependent, but not -independent, vasodilatation was impaired in nicotine-treated compared with saline-treated rats. In addition, the production of superoxide anion (lucigenin chemiluminescence) was increased in rats treated with nicotine compared with saline-treated rats. Furthermore, using Western blot analysis, we found that chronic exposure to nicotine increased p47phox protein in the parietal cortex. Finally, we found that apocynin (40 mg.kg(-1).day(-1)) in the drinking water to inhibit NADPH oxidase alleviated impaired NOS-dependent cerebral vasodilatation in nicotine treated rats but did not alter NOS-dependent responses in saline treated rats and did not alter NOS-independent reactivity in saline- or nicotine-treated rats. These findings suggest that chronic exposure to nicotine impairs NOS-dependent dilatation of pial arterioles by a mechanism that appears to be related to the formation of superoxide anion via activation of NADPH oxidase.     

Invariant NKT cell defects in vitamin D receptor knockout mice prevents experimental lung inflammation

Vitamin D receptor (VDR) deficiency (knockout [KO]) results in a failure of mice to generate an airway hyperreactivity (AHR) response on both the BALB/c and C57BL/6 background. The cause of the failed AHR response is the defective population of invariant NKT (iNKT) cells in the VDR KO mice because wild-type (WT) iNKT cells rescued the AHR response. VDR KO mice had significantly fewer iNKT cells and normal numbers of T cells in the spleen compared with WT mice. In BALB/c VDR KO mice, the reduced frequencies of iNKT cells were not apparent in the liver or thymus. VDR KO and WT Th2 cells produced similar levels of IFN-γ and IL-5. On the BALB/c background, Th2 cells from VDR KO mice produced less IL-13, whereas on the C57BL/6 background, Th2 cells from VDR KO mice produced less IL-4. Conversely, VDR KO iNKT cells were defective for the production of multiple cytokines (BALB/c: IL-4, IL-5, and IL-13; C57BL/6: IL-4 and IL-17). Despite relatively normal Th2 responses, BALB/c and C57BL/6 VDR KO mice failed to develop AHR responses. The defect in iNKT cells as a result of the VDR KO was more important than the highly susceptible Th2 background of the BALB/c mice. Defective iNKT cell responses in the absence of the VDR result in the failure to generate AHR responses in the lung. The implication of these mechanistic findings for human asthma requires further investigation.     

Concomitant production of nitric oxide and superoxide in human macrophages

Many harmful effects of nitric oxide are caused by the reaction of NO with superoxide anion. The present study was carried out to find out the concomitant production of superoxide and to investigate a suitable inhibitor of NO, which is produced by iNOS. THP-1 cells were differentiated into macrophages by PMA and cytokine. Addition of L-NAME showed decrement in superoxide production. Addition of apocynin, aminoguanidine or ONO 1714 brought about a significant reduction in superoxide production. The expressions of p67 and p47(phox) were reduced by the addition of apocynin, aminoguanidine or ONO 1714 whereas xanthine oxidase and cyclooxygenase did not have a major role in superoxide production. The results of the present study show that iNOS and NADPH oxidase play an important role in superoxide release. It suggests that addition of iNOS inhibitor together with apocynin may be more effective in case of therapeutic application in disease conditions like atherosclerosis.     

Identification of TLR inducing Th1-responsive Leishmania donovani amastigote-specific antigens

Leishmania is known to elicit Th2 response that causes leishmaniasis progression; on the other hand, Th1 cytokines restricts amastigote growth and disease progression. In this study, we report the potential of two leishmanial antigens (65 and 98?kDa, in combination) which enhance strong macrophage effector functions, viz., production of respiratory burst enzymes, nitric oxide, and Th1 cytokines. The identification of antigens were done by resolving the crude soluble antigens on SDS-PAGE and eluted by reverse staining method. Further, RAW264.7 macrophages were challenged with eluted antigens, and the innate immune response was observed by detecting respiratory burst enzymes, nitric oxide (NOx), TNF-α, IFN-γ, IL-12, toll-like receptors (TLRs) gene expression, and TLR-signaling proteins. These antigens increased the production of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, NOx, TNF-α, IFN-γ, IL-12, TLR2, and p38 mitogen-activated protein kinase. These antigens also induced human peripheral blood mononuclear cells proliferation and Th1 cytokine production. This study concludes that these antigens induce innate immune response as well as have prophylactic efficacy.     

Distinct spatial requirement for eosinophil-induced airways hyperreactivity

T helper (Th)-2-derived cytokines and their involvement in the recruitment and activation of inflammatory cells crucially orchestrate asthma pathogenesis. A notable cellular component of this allergy-induced inflammation is the eosinophil. However, whether the eosinophil is an obligatory mediator for enhancing airways hyperreactivity (AHR) to cholinergic stimuli, a watershed of the asthmatic lung, is somewhat controversial. In this investigation we have endeavoured to define the spatial requirements for IL-4 and IL-13, and the downstream effector molecules, IL-5 and the CC chemokine eotaxin, for the recruitment of eosinophils and the development of AHR in a murine model of allergic pulmonary disease. These studies are of particular importance considering clinical trials, with either the soluble IL-4Ralpha subunit or a humanized anti-IL-5 antibody, are being conducted. Interestingly, our studies show that depletion of both IL-4 and IL-13 is necessary to ablate pulmonary eosinophilia and AHR, and that this may be attributed to the role these cytokines play in regulating the expression of the eosinophil- activating molecules, IL-5 and eotaxin. While it is clear that depletion of IL-5 diminishes pulmonary eosinophilia, we demonstrate in BALB/c mice that a deficiency in both IL-5 and eotaxin is necessary to abolish both the trafficking of eosinophils to the lung and AHR. However, in contrast to the neutrophil-rich inflammation observed in mice deficient in both IL-4 and IL-13, inflammation per se in mice deficient in both IL-5 and eotaxin is significantly attenuated. This suggests that asthma immunotherapy may be better directed towards the eosinophil- activating molecules IL-5 and eotaxin, rather than towards pleiotrophic molecules such IL-4 and IL-13, which are additionally important in modulating alternative inflammatory responses.