A potential role for lymphocytes in COPD pathologies is based primarily on reports demonstrating that the number of lymphocytes in the lung correlates with the extent of COPD clinical endpoints (31
). However, the mechanism whereby lymphocytes become activated and contribute to COPD pathologies remains unclear. In the present study, we provide evidence, which we believe to be novel, that a lymphocyte activation pathway, NKG2D ligand expression, is associated with COPD pathologies in mouse models of disease and in human patients. Furthermore, we demonstrated that pulmonary expression of NKG2D ligands is sufficient for the development of emphysema in a mouse model.
Based on our experimental findings, we postulate that sustained expression of NKG2D ligands leads to the disruption of the alveolar architecture by CTL-mediated apoptosis of pulmonary epithelial cells. Given that NKG2D-bearing cells were abundant in the lung and that NKG2D lymphocyte populations did not considerably change in our mouse models of emphysema or COPD patients, we conclude that the mechanisms controlling this pathway are primarily dependent on the anomalous, sustained expression of NKG2D ligands on pulmonary epithelial cells and are independent of NKG2D receptor regulation. Several lines of evidence from in vitro and in vivo models demonstrate that the NKG2D pathway directly contributes to the pathogenesis of COPD. First, we showed that NKG2D ligands were not constitutively expressed in pulmonary epithelial cells of healthy subjects or naive mice, but cigarette smoke exposure associated with NKG2D ligand expression in both airway and peripheral tissues. Second, we showed that cell-mediated cytotoxicity against cigarette smoke–exposed alveolar epithelial cells was dependent on NKG2D receptor signaling. Third, transgenic expression of NKG2D ligands by pulmonary epithelial cells resulted in increased apoptosis of epithelial cells and emphysema, which was critically dependent on the function of NKG2D and NK cells. Fourth, we used multiple mouse models of emphysema to establish a causal relationship between NKG2D ligand expression and the typical histological changes of emphysema.
The mechanisms regulating NKG2D-mediated effector functions have been thoroughly characterized (reviewed in ref. 35
). NKG2D engagement is dependent on DAP10/DAP12 adaptor molecules that recruit Src family kinases and initiate multiple downstream signaling pathways that culminate in granule polarization, cytokine release, and cytotoxicity. In addition to ligand engagement, the expression and function of NKG2D can be influenced by other factors in the immune microenvironment, including IL-15 (36
), IL-21 (37
), and TGF-β1 (38
). However, the number of effector lymphocytes expressing the NKG2D receptor was not affected by increased NKG2D ligand expression. Our present study as well as previous studies by others (28
) demonstrate that CTLs, including NKG2D+
lymphocytes, are constitutively present in substantial numbers in the lung parenchyma. The differences in NKG2D receptor expression between mouse and human pulmonary lymphocytes represent a salient consideration in the interpretation of the data presented in the present study. In humans, NKG2D is present on the majority of CD8+
T cells and NK cells. In contrast, NKG2D is expressed primarily on NK cells, but not CD8+
T cells, in mice. Therefore, our data demonstrating that NK cell depletion, but not CD8+
T cells, ablated RAET1-mediated alveolar destruction in the transgenic mouse model may not represent a congruent response in human disease. It is more likely that the response to NKG2D ligands in humans is also mediated by the effector functions of CD8+
T cells and includes potentially confounding factors such as concurrent T cell receptor activation and the impact of pathogen-based disease exacerbations (39
). We conclude that induced NKG2D ligand expression, as opposed to receptor expression, is the major determinant of NKG2D-attributed pathologies in COPD. This conclusion is supported by observations that nonsmokers had no MICA expression, but expressed the same proportion of NKG2D+
CTLs, compared with COPD patients. Moreover, exogenous RAET1 expression in pulmonary epithelial cells caused an emphysematous phenotype without altering the CTL number in the parenchyma. Our conclusion is also bolstered by the lack of modulation of NKG2D+
cells observed in both cigarette smoke–exposed mouse lungs and Raet1a
Tg mice, and the lack of pulmonary inflammation or macrophage activation (as evidenced by MMP activation) observed in mice expressing exogenous RAET1.
The apparent lack of MICA expression in some smokers and COPD patients is intriguing. Current paradigms of complex disease pathogenesis, including COPD, incorporate multiple factors and pathways and include the possibility of interactions among them. The absence of MICA in a subgroup of smokers and COPD patients could be a reflection of several possibilities. These include an actual absence of MICA in some patients, attributable to interindividual variability in response to exposures inherent to any biological process; potential anatomical heterogeneity of the MICA expression (i.e., a negative biopsy obtained from an anatomical location does not exclude MICA expression in other airways); expression levels below our detection limits, and the potential involvement of other NKG2D ligands, such as ULBP proteins. The presence of aberrant polymorphic MICA alleles may circumvent detection with the antibody and also contributes to a failure to detect MICA expression (40
). For example, the MICA-A5.1 allele has 1 extra nucleotide in the transmembrane region compared with the MICA-A5, which leads to a frame shift. This results in a premature stop codon within the transmembrane region, and, consequently, no cytoplasmic tail is present in these MICA molecules, which can lead to aberrant expression on the cell surface. Furthermore, we cannot exclude the possibility that smokers without a COPD diagnosis that demonstrate MICA expression in the epithelium are absolutely free of pulmonary disease. According to present definitions, diagnosis of airflow obstruction and COPD itself is based on pulmonary function tests, mainly spirometry. Nevertheless, several studies demonstrate that high-resolution CT is more sensitive and specific than commonly used functional tests for the evaluation of initial emphysema in asymptomatic smokers, despite normal or minimal abnormal changes in spirometry, TLCO
, or other variables (41
). In other words, we speculate that it is probable that the definition of COPD according to current guidelines, and hence assignation of our volunteers to one group, could underestimate the presence of early phases or less obstructive phenotypes of the disease. Additionally, the potential abrogation of bronchial MICA expression in response to chronic inhaled treatment may confound the detection of ligand expression, as most COPD patients receive inhaled β-adrenergic and inhaled steroids that potentially regulate epithelial cell gene expression. Finally, interindividual variability in treatment compliance may also represent a variable explaining potential MICA downregulation in some COPD patients.
The results of the present study indicate that the progressive alveolar destruction observed in Raet1a
Tg mice is primarily a consequence of directed cytotoxicity against RAET1+
cells. This response was associated with an increase in perforin/granzyme activity that was accompanied by an increase in apoptotic pulmonary epithelial cells. These results were not surprising, because previous studies have demonstrated that exogenous expression of NKG2D ligands elicits perforin/granzyme-mediated cytolysis in vitro (43
). However, these findings represent an expansion of earlier studies investigating the role of CTLs in COPD. Along these lines, increased expression of perforin, a pore-forming protein that causes apoptosis of target cells via cytolytic granules, has previously been reported in CTLs in patients with COPD (45
), and increased alveolar epithelial cell apoptosis is correlated with the number of CTLs in patients with emphysema (34
). In addition, recent studies demonstrate that cytotoxic T cells are necessary for the development of toxicant-induced emphysema in mice (27
Our findings of no major alterations in the cellular composition of the BAL or whole lung compartments of Raet1a
Tg mice coincident with the development of alveolar destruction were somewhat unexpected. NKG2D receptor activation in vitro results in the elaboration of several cytokines (IFN-γ, TNF-α, IL-2, IL4, and GM-CSF; refs. 16
), and chemokines (macrophage inflammatory protein 1-β and I-309; refs. 16
) that are capable of inducing the accumulation and activation of multiple leukocyte populations. Therefore, the lack of BAL and lung inflammation in Raet1a
Tg mice suggests that the binding of mouse NKG2D by RAET1 in vivo elicits a fundamentally different response than that observed in vitro (i.e., inflammatory cytokine elaboration), or that these mediators are not produced in sufficient quantities in vivo to induce overt inflammation. The implication of these findings is that the effector function of RAET1 expression and/or NKG2D receptor activation is downstream of both the inflammation and excess proteolytic activity observed in the natural progression of pulmonary emphysema. It is important to emphasize that this is not necessarily the same expectation we would have for the effects of endogenous NKG2D ligand expression induced in a toxicant-induced model of airspace enlargement. In the instance of endogenous induction by cigarette smoke exposure, we expect that direct effects of the smoke, inflammation, and increased matrix degradation contribute to the stress imposed on the epithelium, which leads to the induction of RAET1 expression. Our present results highlight the difficulties of dissecting the cellular and molecular mechanisms of a complex disease, but demonstrate that the development of an experimental model of reduced complexity can help delineate the role of CTLs in COPD pathogenesis.
An important consideration in these studies is that the pathologies are dependent on sustained as opposed to transient expression of NKG2D ligands in the pulmonary microenvironment. Several acute experimental exposures initiate pulmonary NKG2D ligand expression in mice in a transient manner, including pulmonary bacterial infection (23
) and exposure to pulmonary irritants (e.g., ozone and acrolein; M.T. Borchers, unpublished observation). Similar to in vitro studies with human pulmonary epithelial cells (22
), acute exposures lead to maximal induction of NKG2D ligand expression at 24–48 h that rapidly returns to baseline. Under all the above-described conditions, acute expression of NKG2D ligands does not lead to irreversible changes in the pulmonary architecture. However, the present study demonstrated that inducible, sustained expression of NKG2D ligands in mice occurred following chronic exposure to cigarette smoke and that sustained pulmonary expression of NKG2D ligands in the Raet1a
Tg mouse model was sufficient to cause pulmonary emphysema. More importantly, we demonstrated that upregulation of NKG2D ligands in COPD patients was likewise persistent, as evidenced by the lack of association between MICA upregulation and either infection or smoking status. These data also demonstrate the importance of determining whether NKG2D pathway blockade is effective in attenuating experimental emphysema development induced by cigarette smoke.
The immune system can be activated by signals from cells exposed to pathogens, environmental stimuli, or mechanical damage. The presumed function of stress immunosurveillance is to contribute to tissue repair and maintenance by eliminating stressed or damaged cells and to facilitate the restoration of healthy cells (7
). In the context of COPD, the local pulmonary immune system appears to be chronically responding as if it recognizes a tumor cell or virus-infected cell. Although CTL activation in response to tumors and pathogens is highly beneficial to the host, the CTL response to chemically stressed cells (e.g., cigarette smoking) is ultimately detrimental to the susceptible host as it contributes to the imbalance in tissue injury/repair that is a pathognomonic feature of COPD. Previously, we reported that cultured human airway epithelial cells respond to acute oxidative stress by transiently expressing several known ligands for NKG2D (22
). Although the present study did not address the mechanisms of NKG2D ligand induction on pulmonary cells, we presume that it is likely a combination of direct effects of the many toxicants present in cigarette smoke and the indirect effects of products of tissue damage. Along these lines, NKG2D ligand induction has previously been reported in response to toll-like receptor stimulation (51
) and DNA damage (10
). In this context, NKG2D ligand induction represents a general mechanism of immune cell activation used by the lung under stress.
We provide the first evidence to our knowledge that the NKG2D pathway is associated with COPD, particularly the morphological and/or physiological endpoints of emphysema. We demonstrated that in multiple mouse models of emphysema, RAET1 expression was strongly associated with the development of airspace enlargement. In human COPD patients, bronchial biopsies demonstrated that MICA staining was associated with cigarette smoking and emphysema development. Similarly, MICA expression increased in peripheral lung tissue from a separate COPD patient population. Investigators have hypothesized that COPD represents an autoimmune disease and suggested that aberrant activation of the immune system accounts for the findings that inflammation persists and lung function continues to decline in former smokers (52
). Several findings support the concept that autoimmune reactions accompany COPD pathogenesis. Oligoclonal expansions of CD4+
T cells in emphysematous lung tissue of patients undergoing thoracic surgery have been reported (54
), but these findings may reflect the response to current infection or tumor. More recently, we have demonstrated clonal expansions in predominantly CD8+
T cells in a mouse model of COPD devoid of infections or tumors (55
). Direct evidence of autoimmunity is provided by the reports that circulating antibodies against elastin fragments correlate with emphysema severity (56
) and that autoantibodies against pulmonary epithelial cells are present in COPD patients (57
). Our present findings support the hypothesis that persistent NKG2D ligand expression on pulmonary epithelial cells, and the subsequent pathological consequences, is a form of autoimmunity because it reflects a breakdown in self tolerance. The emerging picture is that immunopathogenesis represents an important component of COPD progression.
Presently, it is unknown whether activation of the NKG2D pathway contributes to other chronic pulmonary diseases, or whether other exposures (e.g., passive exposure to cigarette smoke or other inhaled pollutants or viral infections), if any, are also associated with aberrant MICA expression in the lung. However, the potential pathogenic role of current or former active smoking is highlighted in the present study, as MICA was associated with smoking whereas it was not detected in any bronchial sample obtained from the limited number of patients with nonsmoking COPD or in former smokers without COPD. In addition, examination of a limited number of never smoker asthma patients failed to reveal any MICA immunoreactivity in bronchial biopsy material (data not shown). There was no a priori evidence that any immune functions are disease specific. Indistinguishable lymphocyte effector functions are triggered in response to pathogens, cancer, and autoimmune reactions. Therefore, the consequences of NKG2D ligand expression are likely dependent on temporal and spatial expression (airway versus alveoli) and the context of the unique pulmonary immune microenvironment.
In summary, our findings that NKG2D mediated CTL activation after exposure to cigarette smoke, that NKG2D ligand expression caused emphysema in transgenic mice, and that NKG2D ligands were aberrantly expressed in the pulmonary epithelium of both animal models and patients with COPD, which coincided with emphysema development, provide mechanistic insight into COPD pathogenesis. These data provide a more comprehensive picture of COPD and increase our understanding of the effects of current treatments and new therapeutics developed for COPD patients. Moreover, we demonstrated that anti-NKG2D blocking antibody abrogated emphysema development in the animal model, which suggests the NKG2D pathway maybe a molecular target for complementary treatment and/or prevention of emphysema. An increased understanding of how the pulmonary epithelium communicates with the immune system in order to maintain healthy tissue will provide the foundation for future investigations to determine the role of lymphocyte subpopulations in pulmonary diseases in which injury and repair are in disequilibrium.