Gregory T. Motz

Persistence of Lung CD8 T Cell Oligoclonal Expansions upon Smoking Cessation in a Mouse Model of Cigarette Smoke-Induced Emphysema

The role of adaptive immunity in the development or progression of chronic obstructive pulmonary disease (COPD) remains undefined. Recently, the presence of autoantibodies and autoreactive T cells has been demonstrated in COPD patients. In addition, oligoclonal expansions of lung T cells have been observed in COPD patients, but the overlapping incidence of infections, tumors, and cigarette smoke exposure obscures the antigenic stimulus. We analyzed the TCR Vβ repertoire of CD4 and CD8 T cells purified from the lungs and spleens of mice chronically exposed to cigarette smoke. In a mouse model of COPD, we demonstrate that chronic cigarette smoke exposure causes oligoclonal expansions of T cells isolated from the lungs, but not spleens. TCR Vβ repertoire analyses revealed oligoclonal expansions predominantly occurred in lung CD8 T cells, with preferential usage of Vβ7, Vβ9, Vβ13, and Vβ14. Using nucleotide sequence analysis based on Jβ analyses, we demonstrate selection of CDR3 amino acid motifs, which strongly suggests Ag-driven oligoclonal T cell expansion. Analysis of the lung TCR Vβ repertoire of mice with cigarette smoke-induced emphysema, which had undergone smoking cessation for 6 mo, revealed that oligoclonal expansions persisted. This study formally demonstrates that chronic cigarette smoke exposure, alone, causes a persistent adaptive T cell immune response. These findings have important implications for therapeutic approaches in the treatment of COPD, and provide insight into potential mechanisms involved in disease pathogenesis.

Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease

Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.

Chronic Cigarette Smoke Exposure Primes NK Cell Activation in a Mouse Model of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a debilitating, progressive lung disease punctuated by exacerbations of symptoms. COPD exacerbations are most often associated with viral infections, and exposure to cigarette smoke (CS) followed by viral infection has been shown experimentally to enhance lung inflammation, tissue destruction, and airway fibrosis. Despite this, however, the cellular mechanisms responsible for this effect are unknown. In this study, we examined NK cell function in a mouse model of COPD given the vital role of NK cells following viral infection. Ex vivo stimulation of lung leukocytes with poly(I:C), ssRNA40, or ODN1826 enhanced production of NK cell-derived IFN-γ in CS-exposed mice. NK cells from CS-exposed mice exhibited a novel form of priming; highly purified NK cells from CS-exposed mice, relative to NK cells from filtered air-exposed mice, produced more IFN-γ following stimulation with IL-12, IL-18, or both. Further, NK cell priming was lost following smoking cessation. NKG2D stimulation through overexpression of Raet1 on the lung epithelium primed NK cell responsiveness to poly(I:C), ssRNA40, or ODN1826 stimulation, but not cytokine stimulation. In addition, NK cells from CS-exposed mice expressed more cell surface CD107a upon stimulation, demonstrating that the NK cell degranulation response was also primed. Together, these results reveal a novel mechanism of activation of the innate immune system and highlight NK cells as important cellular targets in controlling COPD exacerbations.

NKG2D Is Critical for NK Cell Activation in Host Defense against Pseudomonas aeruginosa Respiratory Infection

Pseudomonas aeruginosa is a major cause of nosocomial respiratory infections. The eradication of P. aeruginosa from the lung involves the orchestrated actions of the pulmonary epithelium and both resident and recruited immune cells. The NKG2D receptor is constitutively expressed on the surface of circulating and tissue-resident NK cells (and other cytotoxic lymphocytes), and is capable of controlling NK cell activation and production of cytokines, such as IFN-γ via interactions with ligands expressed on the surface of stressed cells. Previously, we demonstrated that NKG2D mediates pulmonary clearance of P. aeruginosa. In the present study, we investigated the cellular and molecular mechanisms of NKG2D-mediated clearance of P. aeruginosa using a novel transgenic mouse model of doxycycline-inducible conditional expression of NKG2D ligands (retinoic acid early transcript 1, α) in pulmonary epithelial cells. NKG2D ligand expression in this model increased pulmonary clearance, cellular phagocytosis, and survival following P. aeruginosa respiratory infection. Additionally, NK cell sensitivity to ex vivo LPS stimulation was greater in lung cells isolated from naive transgenic mice administered doxycycline. We also showed that NK cells are the primary source of lymphocyte-derived IFN-γ in response to P. aeruginosa respiratory infection. Significantly, we demonstrated that NKG2D is critical to the nonredundant IFN-γ production by pulmonary NK cells following acute P. aeruginosa infection. These results represent the principal report of NKG2D-mediated activation of lung NK cells following respiratory infection with an opportunistic pathogen and further establish the importance of NKG2D in the host response against P. aeruginosa respiratory infection.

CCR7 deficiency leads to leukocyte activation and increased clearance in response to pulmonary Pseudomonas aeruginosa infection.

ABSTRACT CCR7 is a chemokine receptor expressed on the surfaces of T cells, B cells, and mature dendritic cells that controls cell migration in response to the cognate ligands CCL19 and CCL21. CCR7 is critical for the generation of an adaptive T cell response. However, the roles of CCR7 in the host defense against pulmonary infection and innate immunity are not well understood. We investigated the role of CCR7 in the host defense against acute pulmonary infection with Pseudomonas aeruginosa. We intranasally infected C57BL/6 mice with P. aeruginosa and characterized the expression of CCR7 ligands and the surface expression of CCR7 on pulmonary leukocytes. In response to infection, expression of CCL19 and expression of CCL21 were oppositely regulated, and myeloid dendritic cells upregulated CCR7 expression. We further examined the effects of CCR7 deficiency on the inflammatory response to P. aeruginosa infection. We infected Ccr7−/− and wild-type mice with P. aeruginosa and characterized the accumulation of pulmonary leukocytes, production of proinflammatory mediators, neutrophil activation, and bacterial clearance. CCR7 deficiency led to an accumulation of myeloid dendritic cells and T cells in the lung in response to infection. CCR7 deficiency resulted in higher expression of CD80 and CD86 on dendritic cells; increased production of interleukin-12/23p40 (IL-12/23p40), gamma interferon (IFN-γ), and IL-1α; increased neutrophil respiratory burst; and, ultimately, increased clearance of acute P. aeruginosa infection. In conclusion, our results suggest that CCR7 deficiency results in a heightened proinflammatory environment in response to acute pulmonary P. aeruginosa infection and contributes to more efficient clearance.

Nonredundant Functions of αβ and γδ T Cells in Acrolein-Induced Pulmonary Pathology

Acrolein exposure represents a significant human health hazard. Repeated acrolein exposure causes the accumulation of monocytes/macrophages and lymphocytes, mucous cell metaplasia, and epithelial injury. Currently, the mechanisms that control these events are unclear, and the relative contribution of T-cell subsets to pulmonary pathologies following repeated exposures to irritants is unknown. To examine whether lymphocyte subpopulations regulate inflammation and epithelial cell pathology, we utilized a mouse model of pulmonary pathology induced by repeated acrolein exposures. The role of lymphocyte subsets was examined by utilizing transgenic mice genetically deficient in either alphabeta T cells or gammadelta T cells, and changes in cellular, molecular, and pathologic outcomes associated with repeated inhalation exposure to 2.0 and 0.5 ppm acrolein were measured. To examine the potential functions of lymphocyte subsets, we purified these cells from the lungs of mice repeatedly exposed to 2.0 ppm acrolein, isolated and amplified messenger RNA, and performed microarray analysis. Our data demonstrate that alphabeta T cells are required for macrophage accumulation, whereas gammadelta T cells are critical regulators of epithelial cell homeostasis, as identified by epithelial cell injury and apoptosis, following repeated acrolein exposure. This is supported by microarray analyses that indicated the T-cell subsets are unique in their gene expression profiles following acrolein exposures. Microarray analyses identified several genes that may contribute to phenotypes mediated by T-cell subpopulations including those involved in cytokine receptor signaling, chemotaxis, growth factor production, lymphocyte activation, and apoptosis. These data provide strong evidence that T-cell subpopulations in the lung are major determinants of pulmonary pathology and highlight the advantages of dissecting their effector functions in response to toxicant exposures.

Alkaline pH-Induced Extracellular Regulated Protein Kinase Activation in Brain Microvascular Endothelial Cells: Differential Effects of Tris and Lowered CO2

As it was previously reported that Tris-elevated pH acutely activated extracellular regulated protein kinase (ERK) in rat aorta smooth muscle cells, this study tested whether this finding could be extended to endothelial cells and, moreover, the relevance of this finding in brain microvascular endothelial cells with respect to respiratory-induced hypocapnic alkalosis. Exposure of bovine brain microvascular endothelial cells to pH 7.90 due to Tris for 15 and 30 min activated ERK twofold. In contrast, pH elevated to 7.75 and 7.90 by lowered percent CO2 failed to activate ERK (15, 30, and 60 min). These results suggest that respiratory alkalosis due to hypocapnia does not activate ERK in brain microvascular endothelial cells. The ability of Tris to activate ERK suggests a novel pathway, possibly independent of pH elevation, whereby Tris activates ERK.