George T. De Sanctis

The IL-6R α chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo

The cytokine IL-6 acts via a specific receptor complex that consists of the membrane-bound IL-6 receptor (mIL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein 130 (gp130). In this study, we investigated the role of IL-6R components in asthma. We observed increased levels of sIL-6R in the airways of patients with allergic asthma as compared to those in controls. In addition, local blockade of the sIL-6R in a murine model of late-phase asthma after OVA sensitization by gp130-fraction constant led to suppression of Th2 cells in the lung. By contrast, blockade of mIL-6R induced local expansion of Foxp3-positive CD4+CD25+ Tregs with increased immunosuppressive capacities. CD4+CD25+ but not CD4+CD25- lung T cells selectively expressed the IL-6R alpha chain and showed IL-6-dependent STAT-3 phosphorylation. Finally, in an in vivo transfer model of asthma in immunodeficient Rag1 mice, CD4+CD25+ T cells isolated from anti-IL-6R antibody-treated mice exhibited marked immunosuppressive and antiinflammatory functions. IL-6 signaling therefore controls the balance between effector cells and Tregs in the lung by means of different receptor components. Furthermore, inhibition of IL-6 signaling emerges as a novel molecular approach for the treatment of allergic asthma.

Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A 4

The prevalence of asthma continues to increase and its optimal treatment remains a challenge. Here, we investigated the actions of lipoxin A4 (LXA4) and its leukocyte receptor in pulmonary inflammation using a murine model of asthma. Allergen challenge initiated airway biosynthesis of LXA4 and increased expression of its receptor. Administration of a stable analog of LXA4 blocked both airway hyper-responsiveness and pulmonary inflammation, as shown by decreased leukocytes and mediators, including interleukin-5, interleukin-13, eotaxin, prostanoids and cysteinyl leukotrienes. Moreover, transgenic expression of human LXA4 receptors in murine leukocytes led to significant inhibition of pulmonary inflammation and eicosanoid-initiated eosinophil tissue infiltration. Inhibition of airway hyper-responsiveness and allergic airway inflammation with a stable LXA4 analog highlights a unique counter-regulatory profile for the LXA4 system and its leukocyte receptor in airway responses. Moreover, our findings suggest that lipoxin and related pathways offer novel multi-pronged therapeutic approaches for human asthma.

Contribution of type I NOS to expired gas NO and bronchial responsiveness in mice

Nitric oxide (NO) can be measured in the expired gas of humans and animals, but the source of expired NO (FENO) and the functional contribution of the various known isoforms of NO synthase (NOS) to the NO measured in the expired air is not known. FENO was measured in the expired air of mice during mechanical ventilation via a tracheal cannula. FENO was significantly higher in wild-type B6SV129J +/+ mice than in mice with a targeted deletion of type I (neural) NOS (nNOS, -/-) (6.3 ± 0.9 vs. 3.9 ± 0.4 parts/billion, P = 0.0345, for +/+ and -/- mice, respectively), indicating that ∼40% of the NO in expired air in B6SV129 mice is derived from nNOS. Airway responsiveness to methacholine (MCh), assessed by the log of the effective dose of MCh for a doubling of pulmonary resistance from baseline (ED200 R L), was significantly lower in the -/- nNOS mice than in the wild-type mice (logED200 R L, 2.24 ± 0.07 vs. 2.51 ± 0.06 μg/kg, respectively; P = 0.003). These findings indicate that nNOS significantly contributes to baseline FENO and promotes airway hyperresponsiveness in the mouse.

IFN-γ, But Not Fas, Mediates Reduction of Allergen-Induced Mucous Cell Metaplasia by Inducing Apoptosis

Inflammatory responses induced by allergen exposure cause mucous cell metaplasia (MCM) by differentiation of existing and proliferating epithelial cells into mucus-storing cells. Airway epithelia have various mechanisms that resolve these changes to form normal airway epithelia. In this report, we first investigated the state of mucous cell metaplasia and the mechanisms by which MCM is reduced despite continued exposures to allergen. After 5 days of allergen exposure, extensive MCM had developed but was reduced when allergen challenge was continued for 15 days. During this exposure period, IL-13 levels decreased and IFN-γ levels increased in the bronchoalveolar lavage fluid. In contrast, IL-13 levels decreased but IFN-γ was not detected at any time point during the resolution of MCM following cessation of allergen exposure. Instillation of IFN-γ but not anti-Fas caused accelerated resolution of MCM and MCM was not resolved in Stat1-deficient mice exposed to allergen for 15 days, confirming that IFN-γ is crucial for reducing MCM during prolonged exposures to allergen. IFN-γ but not anti-Fas induced apoptotic cell death in proliferating normal human bronchial epithelial cells and in human bronchial epithelial cells from subjects with asthma. The apoptotic effect of IFN-γ was caspase dependent and was inhibited by IL-13, indicating that the Th2 milieu in asthmatics may maintain MCM by preventing cell death in metaplastic mucous cells. These studies could be useful in the understanding of deficiencies leading to chronicity in airway changes and designing novel therapies to reverse MCM and airway obstruction in asthmatics.

Interleukin-8 receptor modulates IgE production and B-cell expansion and trafficking in allergen-induced pulmonary inflammation

We examined the role of the interleukin-8 (IL-8) receptor in a murine model of allergen-induced pulmonary inflammation using mice with a targeted deletion of the murine IL-8 receptor homologue (IL-8r-/-). Wild-type (Wt) and IL-8r-/- mice were systemically immunized to ovalbumin (OVA) and were exposed with either single or multiple challenge of aerosolized phosphate-buffered saline (OVA/PBS) or OVA (OVA/OVA). Analysis of cells recovered from bronchoalveolar lavage (BAL) revealed a diminished recruitment of neutrophils to the airway lumen after single challenge in IL-8r-/- mice compared with Wt mice, whereas multiply challenged IL-8r-/- mice had increased B cells and fewer neutrophils compared with Wt mice. Both Wt and IL-8r-/- OVA/OVA mice recruited similar numbers of eosinophils to the BAL fluid and exhibited comparable degrees of pulmonary inflammation histologically. Both total and OVA-specific IgE levels were greater in multiply challenged IL-8r-/- OVA/OVA mice than in Wt mice. Both the IL-8r-/- OVA/OVA and OVA/PBS mice were significantly less responsive to methacholine than their respective Wt groups, but both Wt and IL-8r mice showed similar degrees of enhancement after multiple allergen challenge. The data demonstrate that the IL-8r modulates IgE production, airway responsiveness, and the composition of the cells (B cells and neutrophils) recruited to the airway lumen in response to antigen.

CC chemokine receptor-2 is not essential for the development of antigen-induced pulmonary eosinophilia and airway hyperresponsiveness.

Monocyte chemoattractant proteins-1 and -5 have been implicated as important mediators of allergic pulmonary inflammation in murine models of asthma. The only identified receptor for these two chemokines to date is the CCR2. To study the role of CCR2 in a murine model of Ag-induced asthma, we compared the pathologic and physiological responses of CCR2−/− mice with those of wild-type (WT) littermates following immunization and challenge with OVA. OVA-immunized/OVA-challenged (OVA/OVA) WT and CCR2−/− mice developed significant increases in total cells recovered by bronchoalveolar lavage (BAL) compared with their respective OVA-immunized/PBS-challenged (OVA/PBS) control groups. There were no significant differences in BAL cell counts and differentials (i.e., macrophages, PMNs, lymphocytes, and eosinophils) between OVA/OVA WT and CCR2−/− mice. Serologic evaluation revealed no significant difference in total IgE and OVA-specific IgE between OVA/OVA WT mice and CCR2−/− mice. Lung mRNA expression and BAL cytokine protein levels of IL-4, IL-5, and IFN-γ were also similar in WT and CCR2−/− mice. Finally, OVA/OVA CCR2−/− mice developed increased airway hyper-responsiveness to a degree similar to that in WT mice. We conclude that following repeated airway challenges with Ag in sensitized mice, the development of Th2 responses (elevated IgE, pulmonary eosinophilia, and lung cytokine levels of IL-4 and IL5) and the development of airway hyper-responsiveness are not diminished by a deficiency in CCR2.

Nitric oxide synthase-2 down-regulates surfactant protein-B expression and enhances endotoxin-induced lung injury in mice

Acute respiratory distress syndrome (ARDS) is a life‐threatening ailment characterized by severe lung injury involving inflammatory cell recruitment to the lung, cytokine production, surfactant dysfunction, and up‐regulation of nitric oxide synthase 2 (NOS2) resulting in nitric oxide (NO) production. We hypothesized that NO production from NOS2 expressed in lung parenchymal cells in a murine model of ARDS would correlate with abnormal surfactant function and reduced surfactant protein‐B (SP‐B) expression. Pulmonary responses to nebulized endotoxin (lipopolysaccharide, LPS) were evaluated in wild‐type (WT) mice, NOS2 null (‒/‒) mice, and NOS2‐chimeric animals derived from bone marrow transplantation. NOS2‒/‒ animals exhibited significantly less physiologic lung dysfunction and loss of SP‐B expression than did WT animals. However, lung neutrophil recruitment and bronchoalveolar lavage cytokine levels did not significantly differ between NOS2‒/‒ and WT animals. Chimeric animals for NOS2 exhibited the phenotype of the recipient and therefore demonstrated that parenchymal production of NOS2 is critical for the development of LPS‐induced lung injury. Furthermore, administration of NO donors, independent of cytokine stimulation, decreased SP‐B promoter activity and mRNA expression in mouse lung epithelial cells. This study demonstrates that expression of NOS2 in lung epithelial cells is critical for the development of lung injury and mediates surfactant dysfunction independent of NOS2 inflammatory cell expression and cytokine production.

Mutations in the human 5-lipoxygenase gene.

SummaryOur data demonstrate the presence of a naturally occurring family of alleles in the core promoter of the 5-LO gene, which is characterized by the deletion or addition of consensus Sp1 (—GGGCGG) and Egr-1 (—GCGGGGGCG—) binding motifs. Each of the variant alleles can bind SP1 and Egr-1 protein, as indicated by EMSA and supershift analysis with nuclear extracts. In addition, preliminary data from CAT reporter assays indicate that these alleles are less effective than the wild-type allele in initiating 5-LO gene expression. Whether patients harboring the various alleles identified herein have different capacities to transcribe the 5-LO gene and the importance of such potential regulation to the clinical expression of 5-LO have yet to be determined.

Lipoxins and Aspirin-Triggered Lipoxins in Airway Responses

Here, we have demonstrated potent inhibition of allergen-mediated pulmonary inflammation and airway hyper-responsiveness by a novel dual-pronged action of LXs. Moreover, these results indicate that LXs play pivotal and previously unappreciated roles in regulating allergy and pulmonary inflammation.

Ventilatory responses to acute hypoxia in neurokinin-1 receptor deficient mice

The regulatory effect of substance P on respiration is mediated via neurokinin (NK) receptors. While previous studies suggest that NK-1 receptors are involved, little is known about the role NK-2 receptors in ventilatory responses to hypoxia. Ventilatory responses to acute hypoxia (8% O2 in N2) were measured by indirect plethysmography in unanaesthetized, unrestrained NK-1 receptor gene deficient (NK-1-/-) and wild-type mice. In additional experiments mice were treated with an NK-2 receptor antagonist prior to hypoxic challenge. Resting ventilatory parameters were not different between groups. NK-1-/- mice displayed significantly greater shortening of expiratory time and higher increase of breathing frequency during hypoxia than wild-type mice. Treatment with the NK-2 receptor antagonist SR 48968 (1 mg/kg) resulted in a further shortening of inspiratory and expiratory time in NK-1-/- but not wild-type mice. These results demonstrate that both NK-1 and NK-2 receptors are involved in the modification of ventilation in response to acute hypoxia.