Lung transplantation remains the only effective therapy for patients with end-stage lung disease, but survival is limited by the development of obliterative bronchiolitis (OB). The chemokine receptor CXCR3 and two of its ligands, CXCL9 and CXCL10, have been identified as important mediators of OB. However, the relative contribution of CXCL9 and CXCL10 to the development of OB and the mechanism of regulation of these chemokines has not been well defined. In this study, we demonstrate that CXCL9 and CXCL10 are up-regulated in unique patterns following tracheal transplantation in mice. In these experiments, CXCL9 expression peaked 7 days posttransplant, while CXCL10 expression peaked at 1 day and then again 7 days posttransplant. Expression of CXCL10 was also up-regulated in a novel murine model of lung ischemia, and in bronchoalveolar lavage fluid taken from human lungs 24 h after lung transplantation. In further analysis, we found that 3 h after transplantation CXCL10 is donor tissue derived and not dependent on IFN-γ or STAT1, while 24 h after transplantation CXCL10 is from recipient tissue and regulated by IFN-γ and STAT1. Expression of both CXCL9 and CXCL10 7 days posttransplant is regulated by IFN-γ and STAT1. Finally, we demonstrate that deletion of CXCR3 in recipients reduces airway obliteration. However, deletion of either CXCL9 or CXCL10 did not affect airway obliteration. These data show that in this murine model of obliterative bronchiolitis, these chemokines are differentially regulated following transplantation, and that deletion of either chemokine alone does not affect the development of airway obliteration.

CARMA1 has been shown to be important for Ag-stimulated activation of NF-κB in lymphocytes in vitro and thus could be a novel therapeutic target in inflammatory diseases such as asthma. In the present study, we demonstrate that mice with deletion in the CARMA1 gene (CARMA1−/−) do not develop inflammation in a murine model of asthma. Compared with wild-type controls, CARMA1−/− mice did not develop airway eosinophilia, had no significant T cell recruitment into the airways, and had no evidence for T cell activation in the lung or draining lymph nodes. In addition, the CARMA1−/− mice had significantly decreased levels of IL-4, IL-5, and IL-13, did not produce IgE, and did not develop airway hyperresponsiveness or mucus cell hypertrophy. However, adoptive transfer of wild-type Th2 cells into CARMA1−/− mice restored eosinophilic airway inflammation, cytokine production, airway hyperresponsiveness, and mucus production. This is the first demonstration of an in vivo role for CARMA1 in a disease process. Furthermore, the data clearly show that CARMA1 is essential for the development of allergic airway inflammation through its role in T lymphocytes, and may provide a novel means to inhibit NF-κB for therapy in asthma.

body-antigen interactions in the airway initiate inflammation in acute asthma exacerbations. This inflammatory response is characterized by the recruitment of granulocytes into the airways. In murine models of asthma, granulocyte recruitment into the lung contributes to the development of airway hyperresponsiveness (AHR), mucus production, and airway remodeling. Leukotriene B4 is a mediator released following antigen challenge that has chemotactic activity for granulocytes, mediated through its receptor, BLT1. We investigated the role of BLT1 in granulocyte recruitment following antigen challenge. Wild-type mice and BLT1 Ϫ/Ϫ mice were sensitized and challenged with ovalbumin (OVA) to induce acute allergic airway inflammation. In addition, to explore the relevance to antibody-antigen interactions, we injected OVA bound to anti-OVA IgG1 or anti-OVA IgE intratracheally into naïve wild-type and BLT1 Ϫ/Ϫ mice. Cell composition of the lungs, cytokine levels, histology, and AHR were determined. After sensitization and challenge with ovalbumin, there was significantly reduced neutrophil and eosinophil recruitment into the airways of BLT1 Ϫ/Ϫ mice compared with wild-type animals after one or two daily antigen challenges, but this difference was not seen after three or four daily antigen challenges. Mucus production and AHR were not affected. Intratracheal injection of OVA bound to IgG1 or IgE induced neutrophil recruitment into the airways in wild-type mice but not in the BLT1 Ϫ/Ϫ mice. We conclude that BLT1 mediates early recruitment of granulocytes into the airway in response to antigen-antibody interactions in a murine model of acute asthma. lung inflammation; asthma; lipid mediator ACUTE ASTHMA EXACERBATIONS are life-threatening complications of allergic asthma that result in significant morbidity and health care costs (31). Inhalation of allergen initiates airway inflammation in part by binding of allergen to antigen-specific antibodies, which then bind to antibody receptors on innate immune cells, such as macrophages and mast cells. In response, these cells release numerous mediators, which initiate inflammation and induce the recruitment of neutrophils and eosinophils into the airways (5,7,13,25). Granulocytes are important sources of lipid mediators, cytokines, and cytotoxic proteins and thus could play an important role in the propagation of airway inflammation and airway remodeling. Consistent with this, recent studies in murine models of asthma have demonstrated that eosinophils are crucial for the development of airway hyperresponsiveness (AHR), mucus production, and airway remodeling (8,23,26,46). Furthermore, studies in humans have suggested that neutrophil and eosinophil recruitment into the airways may lead to airway fibrosis (9, 10, 14). Thus measures that inhibit the accumulation of granulocytes in the airway during asthma attacks could have profound effects on the development of airway inflammation and long-term complications of the disease.A commonly used murine model of asthma utilizes mice sensitize...

Site specific integration (SSI) expression systems offer robust means of generating highly productive and stable cell lines for traditional monoclonal antibodies. As complex modalities such as antibody-like molecules comprised of greater than two peptides become more prevalent, greater emphasis needs to be placed on the ability to produce appreciable quantities of the correct product of interest (POI). The ability to screen several transcript stoichiometries could play a large role in ensuring high amounts of the correct POI. Here we illustrate implementation of an SSI expression system with a single site of integration for development and production of a multichain, bi-specific molecule. A SSI vector with a single copy of all of the genes of interest was initially selected for stable Chinese hamster ovary transfection. While the resulting transfection pools generated low levels of the desired heterodimer, utilizing an intensive clone screen strategy, we were able to identify clones having significantly higher levels of POI. In-depth genotypic characterization of clones having the desirable phenotype revealed that a duplication of the light chain within the landing pad was responsible for producing the intended molecule. Retrospective transfection pool analysis using a vector configuration mimicking the transgene configuration found in the clones, as well as other vector configurations, yielded more favorable results with respect to % POI. Overall, the study demonstrated that despite the theoretical static nature of the SSI expression system, enough heterogeneity existed to yield clones having significantly different transgene phenotypes/genotypes and support production of a complex multi-chain molecule.