Tim-1 regulates Th2 responses in an airway hypersensitivity model.

T-cell immunoglobulin mucin-1 (Tim-1) is a transmembrane protein postulated to be a key regulator of Th2-type immune responses. This hypothesis is based in part upon genetic studies associating Tim-1 polymorphisms in mice with a bias toward airway hyperrespon-siveness (AHR) and the development of Th2-type CD4(+) T cells. Tim-1 expressed by Th2 CD4(+) T cells has been proposed to function as a co-stimulatory molecule. Tim-1 is also expressed by B cells, macrophages, and dendritic cells, but its role in responses by these cell types has not been firmly established. Here, we generated Tim-1-deficient mice to determine the role of Tim-1 in a murine model of allergic airway disease that depends on the development and function of Th2 effector cells and results in the generation of AHR. We found antigen-driven recruitment of inflammatory cells into airways is increased in Tim-1-deficient mice relative to WT mice. In addition, we observed increased antigen-specific cytokine production by splenocytes from antigen-sensitized Tim-1-deficient mice relative to those from controls. These data support the conclusion that Tim-1 functions in pathways that suppress recruitment of inflammatory cells into the airways and the generation or activity of CD4(+) T cells.

Immunohistochemical detection of MUC5AC and MUC5B mucins in ferrets.

OBJECTIVE: Cystic fibrosis (CF) is a genetic condition that causes abnormal mucus secretions in affected organs. MUC5AC and MUC5B are gel-forming mucins and frequent targets for investigations in CF tissues. Our objective was to qualify MUC5AC and MUC5B immunohistochemical techniques to provide a useful tool to identify, localize and interpret mucin expression in ferret tissues. RESULTS: MUC5AC and MUC5B mucins were detected most commonly in large airways and least in small airways, consistent with reported goblet cell density in airway surface epithelia. We evaluated whether staining method affected the detection of goblet cell mucins in serial sections of bronchial surface epithelia. Significant differences between stains were not observed suggesting common co-expression MUC5AC and MUC5B proteins in goblet cells of airway surface epithelia. Gallbladder and stomach tissues are reported to have differential mucin enrichment, so we tested these tissues in wildtype ferrets. Stomach tissues were enriched in MUC5AC and gallbladder tissues enriched in MUC5B, mucin enrichment similar to human tissues. Mucin immunostaining techniques were further qualified for specificity using lung tissue from recently generated MUC5AC-/- and MUC5B-/- ferrets. Qualified techniques for MUC5AC and MUC5B immunohistochemistry will be useful tools for mucin tissue studies in CF and other ferret models.

Anti-inflammatory effects of the neurotransmitter agonist Honokiol in a mouse model of allergic asthma.

Chronic airway inflammation is a hallmark of asthma, an immune-based disease with great societal impact. Honokiol (HNK), a phenolic neurotransmitter receptor (γ-aminobutyric acid type A) agonist purified from magnolia, has anti-inflammatory properties, including stabilization of inflammation in experimentally induced arthritis. The present study tested the prediction that HNK could inhibit the chronic inflammatory component of allergic asthma. C57BL/6 mice sensitized to and challenged with OVA had increased airway hyperresponsiveness to methacholine challenge and eosinophilia compared with naive controls. HNK-treated mice showed a reduction in airway hyperresponsiveness as well as a significant decrease in lung eosinophilia. Histopathology studies revealed a marked drop in lung inflammation, goblet cell hyperplasia, and collagen deposition with HNK treatment. Ag recall responses from HNK-treated mice showed decreased proinflammatory cytokines in response to OVA, including TNF-α-, IL-6-, Th1-, and Th17-type cytokines, despite an increase in Th2-type cytokines. Regulatory cytokines IL-10 and TGF-ß were also increased. Assessment of lung homogenates revealed a similar pattern of cytokines, with a noted increase in the number of FoxP3(+) cells in the lung. HNK was able to alter B and T lymphocyte cytokine secretion in a γ-aminobutyric acid type A-dependent manner. These results indicate that symptoms and pathology of asthma can be alleviated even in the presence of increased Th2 cytokines and that neurotransmitter agonists such as HNK have promise as a novel class of anti-inflammatory agents in the treatment of chronic asthma.

Utility of CD138/syndecan-1 immunohistochemistry for localization of plasmacytes is tissue-dependent in B6 mice.

OBJECTIVE: Inflammation is present in many diseases and identification of immune cell infiltration is a common assessment. CD138 (syndecan-1) is a recommended immunohistochemical marker for human plasmacytes although it is also expressed in various epithelia and tumors. Similarly, CD138 is a marker for murine plasmacytes, but its tissue immunostaining is not well-defined. Endogenous CD138 expression is an important confounding factor when evaluating plasmacyte infiltration. We studied two plasmacyte markers (CD138 and Kappa light chains) for endogenous immunostaining in five organs and one tumor from B6 mice. RESULTS: Plasmacytes in Peyer's patches were positive for CD138 and Kappa markers without endogenous immunostaining. Endogenous CD138 immunostaining was widespread in liver, kidney, lung and a malignant peripheral nerve sheath tumor (MPNST) versus regionalized immunostaining in skin and small intestine wall. Endogenous Kappa immunostaining was absent in all tissues except for plasmacytes. Tissues with widespread endogenous CD138 immunostaining were contrasted by absence of endogenous Kappa immunostaining. Here, plasmacytes would not be distinguished by CD138, but would be obvious by Kappa immunostaining. Our study suggests that utility of immunostaining for plasmacytes by CD138 is tissue dependent in mice. Additionally, Kappa immunostaining may be a useful alternative in mouse tissues with confounding endogenous CD138 immunostaining.

Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia.

Mutations in a group of genes that contribute to ciliary function cause Bardet-Biedl syndrome (BBS). Most studies of BBS have focused on primary, sensory cilia. Here, we asked whether loss of BBS proteins would also affect motile cilia lining the respiratory tract. We found that BBS genes were expressed in human airway epithelia, and BBS2 and BBS4 localized to cellular structures associated with motile cilia. Although BBS proteins were not required for ciliogenesis, their loss caused structural defects in a fraction of cilia covering mouse airway epithelia. The most common abnormality was bulges filled with vesicles near the tips of cilia. We discovered this same misshapen appearance in airway cilia from Bbs1, Bbs2, Bbs4, and Bbs6 mutant mice. The structural abnormalities were accompanied by functional defects; ciliary beat frequency was reduced in Bbs mutant mice. Previous reports suggested BBS might increase the incidence of asthma. However, compared with wild-type controls, neither airway hyperresponsiveness nor inflammation increased in Bbs2(-/-) or Bbs4(-/-) mice immunized with ovalbumin. Instead, these animals were partially protected from airway hyperresponsiveness. These results emphasize the role of BBS proteins in both the structure and function of motile cilia. They also invite additional scrutiny of motile cilia dysfunction in patients with this disease.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Glycogen depletion can increase the specificity of mucin detection in airway tissues.

OBJECTIVE: Mucin is an important parameter for detection and assessment in studies of airway disease including asthma and cystic fibrosis. Histochemical techniques are often used to evaluate mucin in tissues sections. Periodic acid Schiff (PAS) is a common technique to detect neutral mucins in tissue, but this technique also detects other tissue components including cellular glycogen. We tested whether depletion of glycogen, a common cellular constituent, could impact the detection of mucin in the surface epithelium of the trachea. RESULTS: Normal tissues stained by PAS had significantly more staining than serial sections of glycogen-depleted tissue with PAS staining (i.e. dPAS technique) based on both quantitative analysis and semiquantitative scores. Most of the excess stain by the PAS technique was detected in ciliated cells adjacent to goblet cells. We also compared normal tissues using the Alcian blue technique, which does not have reported glycogen staining, with the dPAS technique. These groups had similar amounts of staining consistent with a high degree of mucin specificity. Our results suggest that when using PAS techniques to stain airways, the dPAS approach is preferred as it enhances the specificity for airway mucin.

Effect of nitrogen dioxide exposure on allergic asthma in a murine model.

STUDY OBJECTIVES: The purpose of this study was to examine the effects of NO(2), a major component of air pollution, on airway eosinophilic inflammation and bronchial hyperreactivity, using a mouse model of asthma. SETTING AND SUBJECTS: BALB/c mice (eight mice per experimental group) were studied in a basic research laboratory at the University of Iowa. INTERVENTIONS: Using a standard murine model of asthma, BALB/c mice were sensitized to ovalbumin (OVA) by intraperitoneal (IP) injections (days 1 and 7) and were challenged with aerosolized OVA (days 13 and 14). Some mice were exposed to NO(2) (2 ppm) in an exposure chamber for 24 h before undergoing OVA aerosol challenge. A control group was exposed to OVA alone. MEASUREMENTS AND RESULTS: The outcomes assessed included airway inflammation, bronchial hyperreactivity to inhaled methacholine, and goblet cell hyperplasia. We found that NO(2) exposure modestly increased airway neutrophilia but not airway eosinophilia in OVA-exposed mice. These mice exhibited epithelial damage and loss of epithelial mucin. Surprisingly, nonspecific bronchial hyperreactivity (ie, enhanced pause index) was not increased, although baseline smooth muscle tone was increased (p < 0.05) in the mice exposed to NO(2). CONCLUSIONS: These data indicate that relatively short-term (24 h) exposure to NO(2) causes epithelial damage, reduced mucin expression, and increased tone of respiratory smooth muscle. Reduced mucin production may be a mechanism of injury following long-term exposure to inhaled NO(2). Despite enhancing epithelial damage in OVA-exposed mice, NO(2) exposure does not otherwise alter the expression of allergen-induced airway responses.

Safety assessment of inhaled xylitol in mice and healthy volunteers.

BACKGROUND: Xylitol is a 5-carbon sugar that can lower the airway surface salt concentration, thus enhancing innate immunity. We tested the safety and tolerability of aerosolized iso-osmotic xylitol in mice and human volunteers. METHODS: This was a prospective cohort study of C57Bl/6 mice in an animal laboratory and healthy human volunteers at the clinical research center of a university hospital. Mice underwent a baseline methacholine challenge, exposure to either aerosolized saline or xylitol (5% solution) for 150 minutes and then a follow-up methacholine challenge. The saline and xylitol exposures were repeated after eosinophilic airway inflammation was induced by sensitization and inhalational challenge to ovalbumin. Normal human volunteers underwent exposures to aerosolized saline (10 ml) and xylitol, with spirometry performed at baseline and after inhalation of 1, 5, and 10 ml. Serum osmolarity and electrolytes were measured at baseline and after the last exposure. A respiratory symptom questionnaire was administered at baseline, after the last exposure, and five days after exposure. In another group of normal volunteers, bronchoalveolar lavage (BAL) was done 20 minutes and 3 hours after aerosolized xylitol exposure for levels of inflammatory markers. RESULTS: In naive mice, methacholine responsiveness was unchanged after exposures to xylitol compared to inhaled saline (p = 0.49). There was no significant increase in Penh in antigen-challenged mice after xylitol exposure (p = 0.38). There was no change in airway cellular response after xylitol exposure in naive and antigen-challenged mice. In normal volunteers, there was no change in FEV1 after xylitol exposures compared with baseline as well as normal saline exposure (p = 0.19). Safety laboratory values were also unchanged. The only adverse effect reported was stuffy nose by half of the subjects during the 10 ml xylitol exposure, which promptly resolved after exposure completion. BAL cytokine levels were below the detection limits after xylitol exposure in normal volunteers. CONCLUSIONS: Inhalation of aerosolized iso-osmotic xylitol was well-tolerated by naive and atopic mice, and by healthy human volunteers.

Modulation of murine allergic rhinosinusitis by CpG oligodeoxynucleotides.

BACKGROUND: Allergic rhinosinusitis is characterized by eosinophilic inflammation of the upper airway, which is induced by TH-2 cytokines. CpG oligodeoxynucleotides (ODN) are known to induce TH-1 and to suppress TH-2 cytokines in a variety of settings, including murine models of asthma. OBJECTIVE: To examine the effect of CpG ODN in a murine model of upper airway allergic inflammation and to correlate with reduction of its manifestations of sneezing and nasal scratching. METHODS: BALB/c mice were sensitized using Ovalbumin (Ova) intraperitoneally and challenged with aerosolized Ova. CpG ODN were administered at the time of Ova sensitization. Outcomes measured included nasal symptoms, submucosal eosinophilia in the areas lined by respiratory or olfactory epithelium, and bone marrow eosinophilia. To delineate the mechanism of CpG ODN-induced suppression of eosinophilic inflammation, in vitro experiments were carried out to examine the effect of stimulation with Ova on splenocytes obtained from mice that were treated with CpG or control ODN (or no ODN) in vivo. Supernatant was collected after 72 hours of incubation and cytokines were measured by enzyme linked immunosorbent assay. RESULTS: CpG ODN administered at the time of Ova sensitization effectively abrogated nasal symptoms and eosinophilic upper airway inflammation compared with mice treated with control ODN or with no ODN. Cytokine data revealed that Ova sensitization suppressed IFN-gamma and induced IL-4 and IL-5 compared with non-sensitized mice. CpG ODN treatment reversed these effects. CONCLUSION: CpG ODN prevents the development of TH-2-mediated eosinophilic inflammation and symptoms in a murine model of allergic rhinosinusitis.

CaMKII is essential for the proasthmatic effects of oxidation.

Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.

Intestinal helminths protect in a murine model of asthma.

Underdeveloped nations are relatively protected from the worldwide asthma epidemic; the hygiene hypothesis suggests this is due to suppression of Th2-mediated inflammation by increased exposure to pathogens and their products. Although microbial exposures can promote Th2-suppressing Th1 responses, even Th2-skewing infections, such as helminths, appear to suppress atopy, suggesting an alternate explanation for these observations. To investigate whether induction of regulatory responses by helminths may counter allergic inflammation, we examined the effects of helminth infection in a murine model of atopic asthma. We chose Heligosomoides polygyrus, a gastrointestinal nematode, as the experimental helminth; this worm does not enter the lung in its life cycle. We found that H. polygyrus infection suppressed allergen-induced airway eosinophilia, bronchial hyperreactivity, and in vitro allergen-recall Th2 responses in an IL-10-dependent manner; total and OVA-specific IgE, however, were increased by worm infection. Finally, helminth-infected mice were protected against eosinophilic inflammation induced by adoptive transfer of OVA-stimulated CD4(+) cells, and transfer of cells from helminth-infected/OVA-exposed mice suppressed OVA-induced eosinophilic inflammation, suggesting a role for regulatory cells. Increased CD4(+)CD25(+)Foxp3(+) cells were found in thoracic lymph nodes of helminth-infected/OVA-exposed mice. Helminthic colonization appears to protect against asthma and atopic disorders; the regulatory cytokine, IL-10, may be a critical player.

Treatment of established asthma in a murine model using CpG oligodeoxynucleotides.

Allergen immunotherapy is an effective but underutilized treatment for atopic asthma. We have previously demonstrated that CpG oligodeoxynucleotides (CpG ODN) can prevent the development of a murine model of asthma. In the current study, we evaluated the role of CpG ODN in the treatment of established eosinophilic airway inflammation and bronchial hyperreactivity in a murine model of asthma. In this model, mice with established ovalbumin (OVA)-induced airway disease were given a course of immunotherapy (using low doses of OVA) in the presence or absence of CpG ODN. All mice then were rechallenged with experimental allergen. Untreated mice developed marked airway eosinophilia and bronchial hyperresponsiveness, which were significantly reduced by treatment with OVA and CpG. CpG ODN leads to induction of antigen-induced Th1 cytokine responses; successful therapy was associated with induction of the chemokines interferon-gamma-inducible protein-10 and RANTES and suppression of eotaxin. Unlike previous studies, these data demonstrate that the combination of CpG ODN and allergen can effectively reverse established atopic eosinophilic airway disease, at least partially through redirecting a Th2 to a Th1 response.

Immunomodulatory effects of CpG oligodeoxynucleotides on established th2 responses.

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-gamma) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-gamma, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8(+) T lymphocytes. Although IFN-gamma plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-gamma was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-gamma, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-gamma was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-gamma and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.

Mucosal immunotherapy with CpG oligodeoxynucleotides reverses a murine model of chronic asthma induced by repeated antigen exposure.

Murine models of acute atopic asthma may be inadequate to study the effects of recurrent exposure to inhaled allergens, such as the epithelial changes seen in asthmatic patients. We developed a murine model in which chronic airway inflammation is maintained by repeated allergen [ovalbumin (OVA)] inhalation; using this model, we examined the response to mucosal administration of CpG DNA (oligonucleotides) and specific antigen immunotherapy. Mice repeatedly exposed to OVA developed significantly greater airway hyperresponsiveness and goblet cell hyperplasia, but not airway eosinophilia, compared with those exposed only twice. CpG-based immunotherapy significantly reversed both acute and chronic markers of inflammation as well as airway hyperresponsiveness. We further examined the effect of mucosal immunotherapy on the response to a second, unrelated antigen. Mice sensitized to both OVA and schistosome eggs, challenged with inhaled OVA, and then treated with OVA-directed immunotherapy demonstrated significant reduction of airway hyperresponsiveness and a moderate reduction in eosinophilia, after inhalation challenge with schistosome egg antigens. In this model, immunotherapy treatment reduced bronchoalveolar lavage (BAL) levels of Th2 cytokines (IL-4, IL-5, IL-13, and IL-10) without changing BAL IFN-gamma. Antigen recall responses of splenocytes from these mice demonstrated an antigen-specific (OVA) enhanced release of IL-10 from splenocytes of treated mice. These results suggest that CpG DNA may provide the basis for a novel form of immunotherapy of allergic asthma. Both antigen-specific and, to a lesser extent, antigen-nonspecific responses to mucosal administration of CpG DNA are seen.

Vitamin A deficiency promotes bronchial hyperreactivity in rats by altering muscarinic M(2) receptor function.

Vitamin A deficiency (VAD) remains an important health problem among children in developing countries. Children living in these areas have a higher mortality from respiratory infections, which likely results in part from suboptimal nutrition, including VAD. Bronchial hyperreactivity can follow viral respiratory infections and may complicate the recovery. To investigate whether VAD promotes bronchial hyperreactivity, we have assessed methacholine-induced bronchoconstriction in VAD and vitamin A-sufficient rats. Bronchial constriction developed at lower concentrations of inhaled methacholine in VAD than in vitamin A-sufficient rats. This did not result from an increase in the bronchial wall thickness or the clearance of a small molecule (with a size similar to methacholine) from the air space. The function and abundance of the muscarinic M(2) receptors in bronchial tissue were reduced in VAD rats, suggesting that this receptor may contribute to these animals' diminished ability to limit cholinergic-mediated bronchoconstriction. A similar reduction in muscarinic M(2) receptor function has been observed in asthma. Vitamin A (retinol) and its congeners (retinoids) may be required to regulate bronchial responsiveness in addition to maintaining a normal bronchial epithelium.