β₂-Microglobulin participates in development of lung emphysema by inducing lung epithelial cell senescence

Abstract

β₂-Microglobulin (β₂M), the light chain of the major histocompatibility complex class I (MHC I), has been identified as a proaging factor and is involved in the pathogenesis of neurodegenerative disorders by driving cognitive and regenerative impairments. However, little attention has focused on the effect of β₂M in the development of lung emphysema. Here, we found that concentrations of β₂M in plasma were significantly elevated in patients with lung emphysema than those in normal control subjects (1.89 ± 0.12 vs. 1.42 ± 0.06 mg/l, P < 0.01). Moreover, the expression of β₂M was significantly higher in lung tissue of emphysema (39.90 ± 1.97 vs. 23.94 ± 2.11%, P < 0.01). Immunofluorescence showed that β₂M was mainly expressed in prosurfactant protein C-positive (pro-SPC⁺) alveolar epithelial cells and CD14⁺ macrophages. Exposure to recombinant human β₂M and cigarette smoke extract (CSE) in vitro enhanced cellular senescence and inhibited proliferation of A549 cells, which was partially reversed by the presence of anti-β₂M antibody. However, anti-β₂M antibody did not attenuate the elevated production of IL-1β, IL-6, and TNF-α in A549 cells that were exposed to CSE. Immunofluorescence showed that colocalization of β₂M, and the hemochromatosis gene (HFE) protein was observed on A549 cells. These data suggest β₂M might participate in the development of lung emphysema through induction of lung epithelial cell senescence and inhibition.

chronic obstructive pulmonary disease (COPD) is a major cause of mortality throughout the world and purports to be a significant global medical burden (3, 23). Aging and cigarette smoke remain the leading risk factors for COPD (13, 20, 26). COPD is aging-related disease, in which cellular senescence perhaps plays an important role in the pathogenesis of COPD (1, 5, 10, 16, 34). Moreover, cigarette smoke could accelerate the development of COPD and emphysema by inducing cellular senescence (24, 33, 34). However, the key factor causing the cigarette smoke extract (CSE)/aging-related lung emphysema is still unclear.

β₂-Microglobulin (β₂M) is the light chain of major histocompatibility complex class I (MHC I) molecules that form an important part of the adaptive immune system (2, 39). Recently, some studies have shown that β₂M also is a proaging factor in blood and increases susceptibility to chronic neurodegenerative diseases, which is closely related to age, through impairing hippocampal-dependent cognitive and regenerative faculties (7, 18, 30, 35, 36).

However, there is lack of evidence whether β₂M is associated with lung emphysema. To confirm this, β₂M expression in plasma and lung tissues from subjects with lung emphysema were detected. Besides, we explored the effect of β₂M on aging of human epithelial cells in vitro. We hypothesized that β₂M is able to accelerate CSE/age-related impairments in lung parenchyma and enlargement of alveolar spaces by inducing cellular senescence in alveolar epithelial cells in lung emphysema.

MATERIALS AND METHODS

Human subjects.

Thirty patients with COPD and lung emphysema and 21 age-matched subjects with normal lung function and nonemphysema were recruited from Chao-Yang Hospital, Capital Medical University, Beijing, China. The demographic characteristics of recruited subjects were shown in Table 1.

Table 1.

Subject demography

Subjects	n	Age, yr	Men/Women	Smoking History, pack/yr	FEV1, %pred
CN	21	59.0 ± 1.7	12/9	11.0 ± 4.2	109.1 ± 2.9
Emphysema	30	62.6 ± 1.3	27/3	44.8 ± 5.2	56.8 ± 3.5

Values are means ± SE; n, number of subjects. CN, control group; FEV₁, forced expiratory volume in 1 s; %pred, %predicted.

The inclusion criteria of lung emphysema were defined as low-attenuation areas in the lung as quantified by using a Hounsfield Unit (HU) threshold of −950 in CT (12, 37) and a postbronchodilator forced expiratory volume in 1 s/forced vital capacity ratio (post-FEV₁/FVC%) < 70%. The exclusion criteria included 1) patients who had hormone replacement therapy, 2) severe organ function failure, and 3) preexistent conditions, such as inflammatory disorders, including connective tissue diseases, diabetes, and tuberculosis.

In addition, a cohort of five subjects with COPD and lung emphysema (age: 55 ± 5, men (M)/women (W): 4/1, post-FEV₁/FVC% 56 ± 5) and four sex- and age-matched subjects (age: 52 ± 5, M/W: 3/1, post-FEV₁/FVC% 79 ± 2) with normal lung function and nonemphysema undergoing surgical resection for suspected or confirmed lung tumors were recruited from the same hospital. Lung tissues were collected from an area of the lung as far distal to surgical resection for suspected or confirmed lung tumor as possible and fixed by 10% formalin, embedded with paraffin.

The study was approved by the Ethics Committee of Beijing Chao-Yang Hospital (2016-ke-61). The subjects provided written informed consent.

CSE preparation.

CSE was prepared using a modification of the method previously described (22). A smoking machine (Beijing Beiyiminke Tech, Beijing, China) was used to direct main and side-stream smoke from one cigarette through 10-ml sterile culture medium. The generated CSE solution was filtered (0.22 μM) to remove large particles. The resulting solution was designated a 100% CSE solution. The CSE solution was used immediately after generation.

Culture human airways epithelial cells in the presence of β₂M, CSE, and an anti-β₂M antibody.

Alveolar epithelial cell line A549 (American Type Culture Collection, Manassas, VA) were cultured in RPMI-1640 (Life Technologies, Grand Island, NY). The starting cell density was 4 × 10⁴/ml in 24-well culture plates. Purified carrier-free human β₂M (Lee Biosolutions, Maryland Heights, MO) was dissolved in PBS and added to the cell cultures containing 2% FBS (Life Technologies) in the final concentrations of 0.1, 1, 10, 100, and 500 μg/ml for 48 h. In other experiments, the cells were incubated in 2% FBS medium with 0.2, 1, and 5% CSE solutions for 48 h. In the control groups, the cells were cultured in the serum-free media or 2% FBS medium for 48 h.

For detecting the effect of blockade of β₂M, the cells were cultured with a mouse monoclonal anti-β₂M antibody (ab181727; Abcam, Cambridge, MA) (2 or 4 μg/ml) for 2 h, then added 5% CSE, and 2% FBS medium for a further 48 h. The cells cultured with 2% FBS medium alone for 48 h were used as a control.

Immunohistochemistry.

Determining immunoreactivity for β₂M was performed by immunohistochemistry in the human lung. Briefly, slides were incubated with a mouse anti-β₂M (1:50; Abcam) overnight at 4°C, followed by horseradish peroxidase-conjugated second antibody (Abcam). The positive signals were detected with substrate 3, 3′-diaminobenzidine tetrahydrochloride (DAB; Abcam). The number of β₂M-immunoreactive cells within the lung was analyzed with a microscopy computed with an image system (Image-Pro Plus, Media Cybernetics, Bethesda, MD).

Immunofluorescence.

Colocalization of phenotypes of β₂M-immunoreactive cells was performed using immunofluorescence in the lung tissues. Briefly, slides were incubated with a mouse anti-β₂M (1:50; Abcam) and rabbit anti-pro-SPC (a marker for airway epithelial cells, 1:800, ab90716; Abcam), or a rabbit anti-CD14 (a marker for macrophage, 1:100, ab133335; Abcam) overnight at 4°C, followed by TRITC-conjugated goat anti-mouse (1:800, ab6786; Abcam) (red) and FITC-conjugated goat anti-rabbit IgG (1:800, ab6717; Abcam) (green). Nuclei were counterstained with DAPI (blue) (ab104139; Abcam).

To explore whether β₂M coexpressed with hemochromatosis gene (HFE) protein, the A549 cells, cultured in 2% FBS medium with 5% CSE solution for 48 h, were first stained with a mouse anti-β₂M (1:50; Abcam) and a rabbit anti-HFE (1:100; ab176123; Abcam), and then stained with fluorescence antibodies to detect the positive signals, as mentioned above. Images were captured by using a microscope (Olympus IX-51, Olympus Optical, Tokyo, Japan).

Measurements of β₂M concentration, cell senescence, and cellular proliferation.

The concentrations of β₂M in human blood and supernatants of the cultured cells were measured using an ELISA kit (ab108885; Abcam), according to the manufacturerʼs instructions. IL-1β, IL-6, and TNF-α also were measured using ELISA kits (eBioscience, Santiago, CA).

A commercial kit was employed to determine activity of senescence-associated β-galactosidase (SA-β-Gal), according to the manufacturerʼs protocol (4) (Beyotime, Beijing, China). 5-Ethynyl-2′-deoxyuridine (EdU) incorporation (9) (Click-iT Plus EdU Alexa Fluor 594 imaging kit; Life Technologies) was used to analyze cellular proliferation, according to the manufacturer’s instructions.

Statistical analysis.

Analyses were performed by using Prism 6.0 software (GraphPad, La Jolla, CA). Experimental data are expressed as means ± SE. Data were statistically tested by an unpaired two-tailed Student's t-test between two groups or one-way ANOVA in multiple groups. The relationship between β₂M and predicted FEV1% (FEV₁%pred) was tested by bivariate correlation analysis. Statistical significance was considered to be P < 0.05.

RESULTS

Increased expression of β₂M in lung tissues and plasma from patients with emphysema.

The expression of β₂M increased in lung tissues of emphysema compared with those in subjects with no emphysema and relative normal lung function (39.90 ± 1.97 vs. 23.94 ± 2.11%, P < 0.01) (Fig. 1, A and B). The SA β-Gal activity also increased in lung tissues of emphysema (Fig. 1C). When compared with that in healthy subjects, the concentration of β₂M was significantly elevated in plasma derived from patients with lung emphysema (1.89 ± 0.12 vs. 1.42 ± 0.06 mg/l, P < 0.01) (Fig. 2A). In addition, correlation analysis showed that β₂M has a good correlation with FEV₁%pred (r = −0.389, P = 0.005) (Fig. 2B).

Fig. 1.

β₂M immunoreactivity in lung tissue of emphysema. β₂-Microglobulin (β₂M)-immunoreactive cells (brown) increased in the lung tissue of patient with emphysema (A) (immunohistochemical staining, original magnification ×400) and (B) (a quantitative analysis of the percentage of β₂M⁺ cells in total cells of lung tissue). Increased SA β-Gal positive staining cells (blue, ×400) were shown in the lung tissue of patients with emphysema (C). Data are expressed as means ± SE. *P < 0.01.

Fig. 2.

Concentrations of β₂M in plasma of emphysema. A: concentrations of β₂M were elevated in plasma in patients with emphysema compared with normal control subjects (P-CN). Open circles (○) denote nonsmokers, while solid circles (●) denote smokers. Data are expressed as means ± SE. *P < 0.01. B: correlation of β₂M with FEV₁% pred (r = −0.389, P = 0.005).

Phenotypes of β₂M positive cells in lung.

Double-immunofluorescence staining displayed that β₂M immunoreactivity was mainly located on pro-SPC⁺ alveolar epithelial cells and CD14⁺ macrophages in lung of emphysema (Fig. 3).

Fig. 3.

Phenotypes of β₂M-immunoreactive cells. Double immunofluorescence showed that lung epithelial cells (pro-SPC-positive, green) and macrophages (CD14-positive, green) were the major source of β₂M-positive signals (red). DAPI was used for nuclear staining. Magnification: ×400.

β₂M induced cellular proliferation inhibition and senescence in A549 cells.

Exposure of A549 cells to human recombinant β₂M resulted in a concentration-dependent decline in proliferation (Fig. 4). SA-β-Gal-positive cells were elevated in the A549 cells cultured with β₂M. The absence of FBS (0%) and normal cultured A549 cells with 2% FBS were used as positive and negative controls, respectively (Fig. 5).

Fig. 4.

β₂M inhibited proliferation of A549 cells. A: images showed EdU staining of A549 cells exposed to different concentrations of β₂M (×200). B: quantification of percentages of EdU-positive cells. Data were expressed as the means ± SE. *P < 0.05.

Fig. 5.

β₂M induced cellular senescence in A549 cells. A: SA-β-Gal (blue, ×200)-positive cells increased after exposure of 100 μg/ml of β₂M, compared with the negative control. B: quantification of percentages of SA-β-Gal positive cells. Data were expressed as means ± SE. *P < 0.05.

CSE-mediated cellular proliferation inhibition and senescence in A549 cells.

CSE also induced a concentration-dependent proliferation inhibition of A549 cells (Fig. 6, A and B). With increasing concentration of CSE, the cell proliferation of A549 was decreased (Fig. 6, A and B), while 10%, CSE almost killed all A549 cells (data not shown). Accompanied by these findings, we also observed that exposure of CSE induced increasing expression of β₂M with a concentration-dependent manner in cultured A549 cells (Fig. 6C). In addition, cellular senescence manifested by SA-β-Gal-positive cells clearly increased in A549 cells after exposure of 5% CSE was also observed (Fig. 7).

Fig. 6.

CSE-mediated proliferation inhibition and expression of β₂M in A549 cells. A: images showed EdU staining (×200). B: quantification of percentages of EdU-positive cells. C: CSE exposure induced increased secretion of β₂M by A549 cells. Data were expressed as means ± SE. *P < 0.05.

Fig. 7.

CSE induced cellular senescence in A549 cells. A: SA-β-Gal-positive cells (blue, × 200) increased after 5% CSE stimulation, compared with the positive controls. B: quantification of percentages of SA-β-Gal positive cells. Data were expressed as means ± SE *P < 0.05.

Anti-β₂M antibody blocked CSE-induced cellular senescence and inhibition.

To detect whether β₂M participates in the CSE-induced cellular senescence and inhibition of alveolar epithelial cells, we attempted to test the effects of blocking endogenous β₂M in the above experiments. Adding an anti-β₂M antibody partially reversed CSE-induced proliferation inhibition (Fig. 8) and cell senescence (Fig. 9, A and B) in a concentration-dependent manner. However, adding an anti-β₂M antibody did not affect production of IL-1β, IL-6, and TNF-α induced by CSE (Fig. 9 C–E). In addition, β₂M and HFE protein were observed to coexpress in A549 cells (Fig. 9F).

Fig. 8.

Effect of anti-β₂M antibody on CSE-induced cell proliferation. A: images show EdU staining of A549 cells under the indicated condition (×200). B: quantification of percentages of EdU-positive cells. Data were expressed as the means ± SE. *P < 0.05.

Fig. 9.

Anti-β₂M antibody blocked CSE-induced cellular senescence. A and B: SA-β-Gal-positive-stained cells (blue, ×200) and quantification of percentages of SA-β-Gal-positive cells. Data were expressed as means ± SE. *P < 0.05. C–E: concentrations of IL-1β, IL-6, and TNF-α, respectively, after A549 cells exposed to CSE and anti-β₂M. F: colocalization of β₂M (red) and HFE protein (green) in A549 cells. DAPI was used for nuclear staining. Magnification: ×400.

DISCUSSION

β₂M comprises the light-chain MHC I molecules that form an active part of the adaptive immune system (38). It has been shown that the concentration of β₂M displayed an age-related increase in plasma from healthy individuals between 20 and 90 yr of age (30). Moreover, increased soluble β₂M have also been detected in the cerebral spinal fluid of patients with Alzheimer’s disease. These findings suggest β₂M to be potential proaging factors (30). COPD and emphysema are also acknowledged to be an aging-related disease. Furthermore, β₂M is known to be a protein that is used to evaluate glomerular filtration function and filtration load, while increased soluble β₂M has been found in many clinical situations, such as small-cell lung cancer, AIDS, rheumatoid arthritis, and acute tubular injury of renal allografts (21, 25, 27, 31, 32). Nobody, however, really knows why it happens and what the relationships between this molecule and pathogenesis of these diseases, either systemically or locally, including lungs. In our knowledge, the present study is the first to find that β₂M expression was elevated in plasma and lung tissue of patients with lung emphysema and that CSE exposure caused increasing β₂M expression in alveolar epithelial cells. Furthermore, our data showed that β₂M itself induced alveolar epithelial cell senescence and inhibition in vitro, but the effect of β₂M on alveolar epithelia cells did not seem to be explained simply by the inflammatory response.

Our studies and others' have shown that lung emphysema is an age-related disease, manifested as SA-β-Gal activity increased and telomere shortened in lung tissue with emphysema (5, 15, 16, 26). A major pathological feature of emphysema is the progressive loss of alveolar tissue, whereas alveolar epithelial cells are the major structural cell of lung tissue and the first gate defending the smoke's toxic particulates. A previous study has demonstrated that senescence of alveolar epithelial cells is accelerated in patients with emphysema (34), suggesting that epithelial cell senescence may be involved in the pathogenesis of emphysema by hampered tissue repair (1). Here, increased β₂M expression in lung tissue of patients with lung emphysema further supports that it could be a proaging factor, and β₂M might also contribute to the pathogenesis of lung emphysema.

In the present study, we observed that exposure of A549 to CSE caused a cellular senescence phenotype featured by increasing the expression of SA β-Gal activity; elevated levels of IL-1β, IL-6, and TNF-α; and cell proliferation inhibition. These findings are consistent with previous reports (24, 33). Meanwhile, our data showed that increasing expression of β₂M in lung tissue of patients with emphysema was mainly located in pro-SPC⁺ alveolar epithelial cells. Supporting this, the content of β₂M increased significantly in the culture supernatant after A549 cell exposure to CSE. These results implied that β₂M might contribute to the development of emphysema through inducing the senescence of alveolar epithelial cells.

Previous studies have indicated that epithelial cell senescence may be involved in the pathogenesis of emphysema by hampered tissue repair (1), whereas cigarette smoke can induce cellular senescence, which aggravates alveolar space destruction (24, 33). Our results showed that exposure of A549 cells to human recombinant β₂M resulted in decreased cell proliferation accompanying the cell senescence due to A549-manifested elevation of SA-β-Gal activity, whereas anti-β₂M antibody partially alleviated CSE-induced cell inhibition and the expression of SA-β-Gal activity. These data suggest that β₂M alone or combined with CSE, might participate, at least partially, in the pathogenesis of emphysema.

Although the causes of emphysema are still unclear, it has been shown that this disease is associated with a higher level of apoptosis in alveolar epithelial cells (38), while cigarette smoke and the senescence of alveolar epithelial cells might accelerate emphysema (34). Our data also showed that CSE caused senescence of alveolar type II–like epithelial cell line A549 cells and proliferation inhibition at 48 h after exposure. Interestingly, we found that secreted β₂M increased in A549 cells supernatant after 5% CSE exposure, which has not been reported yet. Therefore, it is reasonable to suppose that β₂M is likely to be involved in the process of CSE/aging-related lung pathological lesion in lung emphysema.

In addition to pro-SPC⁺ alveolar epithelial cells, immunoreactivity for β₂M was also observed in of CD14⁺ macrophages in lung tissues of emphysema. It has been known that pathological changes of epithelial cells and macrophages are closely related to the development of emphysema (11, 14, 34). Meanwhile, HFE protein was detected to coexpress with β₂M in A549 cells. The β₂M/HFE complex activates the iron metabolism, such as hypoxia-inducible factor-1α (HIF-1α) signaling, which induces epithelial cells to mesenchymal transition and causes reactive oxygen species (6, 17, 29), another two risk factors closely associated with pathogenesis in emphysema. Thus, it is necessary to demonstrate whether HFE could be a potential receptor for β₂M, causing senescence of alveolar epithelial cells in the future.

Compared with young people, airway inflammation is significantly higher in the elderly (28). It is also known that the inflammatory response also plays a key role in the process of lung aging (8, 19). Surprisingly, blockade of β₂M with a specific antibody did not affect production of proinflammatory cytokines IL-1β, IL-6, and TNF-α by A549 cells exposed to CSE, suggesting that β₂M as a proaging factor might induce cells senescence by the β₂M/HFE-HIF-1α pathways or others, rather than inflammatory pathways.

Although we found that β₂M increased in plasma and lung of emphysema and induced epithelial cell senescence to participate in CSE/aging-associated lung emphysema, there are some limitations in the present study. First, we did not investigate the relationship between β₂M and cigarette smoking, nor did we investigate the differences in β₂M in different COPD phenotypes because of the limited clinical sample size in the present study. Second, we could not directly confirm the relationship of smoking and β₂M in vitro. Finally, we still do not know why β₂M increases and whether this increase is associated with disease itself or with changes of immunity in lung emphysema. Therefore, these issues remain to be explored in further studies.

In summary, our study suggests the probability that targeting β₂M might provide potential benefits for CSE/aging-related impairments of lung parenchyma and enlargement of alveolar spaces observed in lung emphysema.

GRANTS

This study was supported by grants from the National Natural Science Foundation of China (81470238, 81670032) and the National Key Research and Development Program of China (2016YFC0901102).

DISCLOSURES

No conflicts of interest, financial or otherwise are declared by the authors.

AUTHOR CONTRIBUTIONS

N.G., Y.W., W.W., S.Y., and K.H. conception and design of research; N.G., Y.W., H.L., Y.-L.G and L.-L.X collected clinical data and samples; N.G., C.-M.Z., T.-T.F. performed experiments; N.G., Y.W., C.-M.Z., H.L., L.-L.X., Y.-L.G and Y.L. analyzed data; N.G., Y.W., C.-M.Z., Y.-L.G and Y.L. prepared figures; N.G., Y.W., W.W., S.Y., and K. H. interpreted results of experiments; N.G., S.Y., and K.H. drafted manuscript; S.Y., and K.H. edited and revised manuscript; S.Y., and K.H. approved final version of manuscript.