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Background Persistent macrophage accumulation and alveolar enlargement are hallmark features of chronic obstructive pulmonary disease (COPD). A role for CD8+ lymphocytes in the development of COPD is suggested based on observations that this T cell subset is increased in the airways and parenchyma of smokers that develop COPD with airflow limitation. In this study, we utilize a mouse model of COPD to examine the contributions of CD8+ T cells in the persistent macrophage accumulation and airspace enlargement resulting from chronic irritant exposure. Methods We analyzed pulmonary inflammation and alveolar destruction in wild-type and Cd8-deficient mice chronically exposed to acrolein, a potent respiratory tract irritant. We further examined cytokine mRNA expression levels by RNase protection assay, matrix metalloproteinase (MMP) activity by gelatin zymography, and epithelial cell apoptosis by active caspase3 immunohistochemistry in wild-type and Cd8-deficient mice exposed chronically to acrolein. Results These studies demonstrate that CD8+ T cells are important mediators of macrophage accumulation in the lung and the progressive airspace enlargement in response to chronic acrolein exposures. The expression of several inflammatory cytokines (IP-10, IFN-γ, IL-12, RANTES, and MCP-1), MMP2 and MMP9 gelatinase activity, and caspase3 immunoreactivity in pulmonary epithelial cells were attenuated in the Cd8-deficient mice compared to wild-type. Conclusions These results indicate that CD8+ T cells actively contribute to macrophage accumulation and the development of irritant-induced airspace enlargement.

The NKG2D-activating receptor is expressed on cytotoxic lymphocytes and interacts with ligands expressed on the surface of cells stressed by pathogenic and nonpathogenic stimuli. In this study, we investigated the physiologic importance of NKG2D receptor-ligand interactions in response to acute pulmonaryPseudomonas aeruginosainfection.P. aeruginosainfection increased the expression of mouse NKG2D ligands (Rae1) in airway epithelial cells and alveolar macrophages in vivo and also increased the cell surface expression of human NKG2D ligands (ULBP2) on airway epithelial cells in vitro. NKG2D receptor blockade with a specific monoclonal antibody inhibited the pulmonary clearance ofP. aeruginosa.NKG2D receptor blockade also resulted in decreased production of Th1 cytokines and nitric oxide in the lungs ofP. aeruginosa-infected mice. Additionally, NKG2D receptor blockade reduced the epithelial cell sloughing that accompaniesP. aeruginosainfection. Macrophage phagocytosis and bronchoalveolar lavage cellularity were not different inP. aeruginosa-infected mice with and without NKG2D receptor blockade. These results demonstrate the importance of NKG2D-mediated immune activation in the clearance of acute bacterial infection and suggest that epithelial cell-lymphocyte interactions mediate pulmonary cytokine production, epithelial cell integrity, and bacterial clearance.

Complement is an ancient danger sensing system playing critical roles in host defense, immune surveillance and homeostasis1. C5a and its G-Protein-coupled receptor mediate many of the pro-inflammatory properties of complement2. Despite its critical role in allergic asthma3, autoimmune arthritis4, sepsis5 and cancer6, our knowledge about C5a regulation is limited. Here we demonstrate an unexpected link through which IgG1 immune complexes (IC), the inhibitory IgG receptor FcγRIIB and the C-type lectin-like receptor Dectin-1 suppress C5a receptor (C5aR) functions. Specifically, we found that IgG1 IC associate FcγRIIB with Dectin-1, resulting in phosphorylation of spleen tyrosine kinase (Syk) downstream of Dectin-1 and Src homology 2 domain containing inositol phosphatase (SHIP) downstream of FcγRIIB. This pathway blocks C5a receptor-mediated ERK1/2 phosphorylation and C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo including the development of skin blisters in experimental epidermolysis bullosa acquisita (EBA), an autoimmune skin disorder. Notably, high galactosylation of IgG N-glycan is critical for this inhibitory property of IgG1 IC as it promotes the association between FcγRIIB and Dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert immunoregulatory properties beyond their impact on activating FcγRs that may control allergy, autoimmunity and cancer.

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in dendritic cells (DC) affects the development of regulatory T cell (Treg) and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen-derived DC results in increased production of TGF-β leading to de novo differentiation of Foxp3+ Treg within 12h after co-incubation with CD4+ T cells from DO11.10/RAG2-/- mice. Stimulation of C5aR-/- DC with ovalbumin and TLR2 ligand Pam3CSK4 increased TGF-β production and induced high levels of IL-6 and IL-23 but only minor amounts of IL-12 leading to differentiation of Th cells producing IL-17A and IL-21. Th17 differentiation was also found in vivo after adoptive transfer of CD4+ Th cell into C5aR-/- mice immunized with OVA and Pam3CSK4. The altered cytokine production of C5aR-/- DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17 cell differentiation through regulation of DC function.

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 tran - script 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.

Immune surveillance of the airways is critical to maintain the integrity and health of the lung. We have identified a family of ligands expressed on the surface of stressed airway epithelial cells whose function is to bind the NKG2D-activating receptor found on several pulmonary lymphocytes, including natural killer cells, γδ+ T cells, and CD8+ T cells. We employed real-time PCR and flow cytometry in normal and transformed airway epithelial cell to demonstrate that major histocompatibility complex class I chain-related (MIC) B and the UL-16 binding protein (ULBP) ligands (ULBP1–4) are ubiquitously expressed at the mRNA level in all cell lines. MICA/B surface expression was present on 70% of transformed cell lines but was undetectable on primary cells. We demonstrate that MICA/B and ULBP 1, 2, 3, and 4 expression is rare or absent on the cell surface of unstimulated normal human bronchial epithelial cells although transcripts and intracellular proteins are present. Normal human bronchial epithelial cells exposed to 0.3 mM hydrogen peroxide exhibit an induction of all ligands examined on the cell surface. Surface expression is independent of changes in transcript level or total cellular protein and is mediated by the ERK family of mitogen-activated protein kinases. The induction of NKG2D ligands on stressed airway epithelial cells represents a potentially important mechanism of immune cell activation in regulation of pulmonary health and disease.

Regulatory T (T(reg)) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4(+) T cells in a cytokine dependent manner. T(reg) cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4(+)Foxp3(+) T(reg) cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient T(reg) cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2). In vivo, a small peripheral T(reg) pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral T(reg) cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.