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* Institut National de la Santé et de la Recherche Médicale Unité 700, Physiopathology and Epidemiology of Respiratory Insufficiency, Paris, France;
Université Paris 7, Faculté de Médecine Denis Diderot, Site Bichat, Paris, France;
Department of Autoimmunity, Inflammation and Respiratory Disease, MedImmune, Gaithersburg, MD 20878;
Département de Pneumologie A, Centre Hospitalier Universitaire Bichat-Claude Bernard, Paris, France; and
¶ Assistance Publique Hôpitaux de Paris, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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0.027) and BAL (p 0.007), more YKL-40-positive cells in bronchial biopsies (p 0.03), and a greater proportion of alveolar macrophages expressing YKL-40 than smokers without COPD or never-smokers. YKL-40 levels in serum and BAL were associated with airflow obstruction (pre-β2 agonist forced expiratory volume in 1 s, rs = –0.3892, p = 0.0072 and rs = –0.5491, p < 0.0001, respectively) and impaired diffusion lung capacity (transfer factor of the lung for carbon monoxide, rs = –0.4667, p = 0.002 and rs = –0.3854, p = 0.0045, respectively). TNF-
stimulated YKL-40 synthesis in alveolar macrophages from smokers with COPD, and exposure of these cells to YKL-40 promoted the release of IL-8, MCP-1, MIP-1, and metalloproteinase-9. We conclude that YKL-40 is up-regulated in COPD, in which it may contribute to tissue inflammation and remodeling by sustaining the synthesis of proinflammatory and fibrogenic chemokines and of metalloproteinases by alveolar macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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YKL-40 is a highly phylogenetically conserved chitin-binding glycoprotein that belongs to the family of chitinase-like proteins, defined by their structural similarity to bacterial chitinases, although lacking their characteristic enzymatic activity (4). YKL-40 controls fundamental functions in mammalian cells, such as mitogenesis, differentiation, and extracellular matrix homeostasis (5, 6, 7). Prominent expression of YKL-40 has been associated with pathologic conditions characterized by tissue inflammation and remodeling and by aberrant cell growth, such as atherosclerosis, asthma, liver fibrosis, and several malignancies (8, 9, 10, 11, 12, 13, 14). For some of these diseases, the assessment of serum YKL-40 has proven to be of both diagnostic and prognostic value (9, 12).
In the current study, we hypothesized that YKL-40 is up-regulated in COPD patients and contributes to tissue inflammation and remodeling by activating alveolar macrophages, the main orchestrators of these responses (3, 15).
Therefore, we compared levels of YKL-40 in serum and in proximal and peripheral airways of healthy never-smokers and of heavy smokers with and without COPD, and tested whether these values correlate with clinical outcomes of disease severity and with smoking history. In addition, we examined the capacity of healthy and diseased alveolar macrophages to synthesize YKL-40 and to respond to this glycoprotein by releasing factors involved in chronic airway inflammation and remodeling, such as proinflammatory and fibrogenic chemokines and the matrix-degrading metalloproteinase (MMP), MMP-9.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Fifteen healthy never-smoker volunteers, 20 current heavy smokers without COPD, and 30 heavy smokers (14 current and 16 former smokers) with COPD were recruited (Table I). COPD severity was graded into stages II (n = 23), III (n = 4), and IV (n = 3) following the Guidelines of the Global Initiative for Obstructive Lung Disease (GOLD) (16). Heavy smokers without COPD reported chronic cough and sputum, but had normal measurements on spirometry. None of the patients were taking oral steroids or statins, and none of them had other inflammatory diseases. In parallel, 40 nonsmoker asthmatic subjects (15 mild, 10 moderate, and 15 severe), mainly atopics and fulfilling the criteria of the Guidelines for the Diagnosis and Management of Asthma of the National Heart, Lung, and Blood Institute/World Health Organization (17), were recruited. These asthmatic subjects have already been included in a recent study (14). Bronchoalveolar lavage (BAL) was collected by bronchoscopy by instilling and recovering 150 ml of 0.9% NaCl (18, 19), then centrifuged (400 x g, 5 min, 4°C) and stored at –80°C until use. Cytospin preparations from BAL cells were fixed in acetone for 10 min at room temperature and stored at –20°C until use. Differential cell counting was performed by microscopic examination using Diff-Quick staining (Baxter Dade).
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This protocol was approved by the Human Ethics Committee of the Bichat Hospital, and all subjects gave their written informed consent.
Ab and protein
The 293-F cell line (Invitrogen) was transfected with a plasmid-encoding human YKL-40, and His Tag was expressed at C terminus of this protein. rYKL-40 was purified from supernatant using Ni columns and was characterized by SDS PAGE, Western blot, protein assay, absorption at 280 nm, and mass spectrometry. Preparations were sterilized by passage through 0.2-µm filters. Endotoxin content was below 3 EU/mg protein, as tested by Lymulus Amoebocyte Lysate assay (Associates of Cape Cod).
Rabbits were immunized with rYKL-40 in CFA, and serum IgG was purified on protein G columns. Specificity and titers of the immune sera and the purified IgG were determined by Western blot and ELISA, using rYKL-40 and the unrelated His-tagged protein (14).
Immunohistochemistry and immunofluorescence
Serial 5-µm acetone-fixed sections from frozen bronchial tissue and cytospin preparations from BAL were sequentially incubated with 2 µg/ml rabbit polyclonal Ab directed against human YKL-40, biotin-conjugated goat anti-rabbit Ab, and streptavidin-alkaline phosphatase complex from the Vectastain kit (Vector Laboratories). Reaction was developed using Fast Red as a substrate (DakoCytomation), and tissues were counterstained using light nuclear Mayer’s hematoxylin (Sigma-Aldrich). Staining of formalin-fixed paraffin-embedded sections was performed using 4 µg/ml YKL-40 Ab. Immunostaining specificity was determined using the corresponding control isotype, i.e., rabbit IgG (DakoCytomation), instead of the primary Ab. Biopsy areas were determined by morphometry and computer-assisted image analysis (Microvision Instruments, Histolab), and results were expressed as numbers of YKL-40-positive cells/mm2 bronchial tissue (14, 19). Biopsy area was comparable in the three patient groups (median values ranging from 0.446 to 0.496 mm2; overall p value = 0.168). For some subjects, the tissues recovered during the fibroscopy were not of good enough quality to perform or to quantify immunostaining.
The proportion of YKL-40-positive BAL macrophages in cytospin preparations was determined after counting at least 200 cells in randomly selected fields, by moving the objective of the light microscope onto the cytospin spot in a cross fashion, from the top to the bottom and from the left to the right.
Immunofluorescence double staining was performed on paraffin-embedded lung tissue sections using a mouse anti-human CD68 mAb (clone PG-M1; DakoCytomation), or its control isotype, mouse IgG3
, in combination with the anti-YKL-40 Ab, followed by goat anti-mouse AlexaFluor 488 and anti-rabbit AlexaFluor 568 Abs (Invitrogen). Sections were examined using a Plan Apochromat 63 x 1.4 oil differential interference contrast objective on the LSM 510 confocal microscope, and fluorescence was analyzed with the LSM META Acquisition and Image Browser softwares (all from Carl Zeiss).
Alveolar macrophage isolation and stimulation
Alveolar macrophages were isolated by adhesion on plastic from BAL obtained by bronchoscopy (20). Because the number of alveolar macrophages in most of the BAL samples was not sufficient to perform the whole series of in vitro stimulations, final study groups were composed of cells obtained from 8 healthy never-smokers, 12 smokers without COPD (stage 0), and 12 smokers with COPD (GOLD stage II, n = 9, and GOLD stage III, n = 3). Smokers with COPD were former (n = 5) or current smokers (n = 7). BAL cells were resuspended in RPMI 1640 medium containing 25 mM HEPES, 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin (all from Invitrogen), and were seeded at 500,000 macrophages/1 ml in 12-well plates (Corning-Costar). After 24-h incubation at 37°C in a humidified 5% CO2 incubator, medium was removed and cells were either collected for RNA isolation or further stimulated for 24 h with 0.1, 1, and 10 µg/ml purified YKL-40, or with 10 ng/ml human rTNF- (R&D Systems), or with the medium alone. Cell-free supernatants were collected and stored at –80°C until use.
Quantitative real-time PCR
Total RNA was extracted from alveolar macrophages using the Nucleospin RNA II kit (Macherey-Nagel), according to the manufacturer’s instructions, and reverse transcribed using Moloney murine leukemia virus enzyme (Invitrogen), as described (21). The levels of the transcript encoding YKL-40 were assessed by quantitative real-time PCR, using an Mx 3000P apparatus (Stratagene Europe). The expression of the transcript was normalized to that of ubiquitin C, used as the most stable housekeeping gene. Calculation and normalization were performed using GeNorm software (21). Primers were designed using Primer Express 2 Software (Applied Biosystems) and were synthesized by Sigma-Genosys. Primer sequences were as follows: YKL-40 (GenBank Identifier M80927), sense 5'-TGCCCTTGACCGCTCCTCTGTACC-3', antisense 5'-GAGCGTCACATCATTCCACTC-3'; ubiquitin C (GenBank Identifier NM_004168), sense 5'-CACTTGGTCCTGCGCTTGA-3', antisense 5'-TTTTGGGAATGCAACAACTTT-3'. Their sequences were blasted against Basic Local Alignment Search Tool database (http://www.ncbi.nlm.nih.gov/BLAST).
Mediator assessment
Concentrations of YKL-40 in serum, BAL, and macrophage supernatants were measured by sandwich-type ELISA developed in-house. Anti-YKL-40 mAb (MedImmune) was used for capture, and biotinylated anti-YKL-40 polyclonal Ab, followed by HRP-labeled streptavidin (Amersham, GE Healthcare), was used for detection. This ELISA allowed to decrease the detection limit of human YKL-40 from 250 pg/ml, in the commercially available kit (14, http://www.quidel.com) down to 100 pg/ml. IL-8, MCP-1, and MIP-1 were assessed by commercially available ELISA (R&D Systems), and MMP-9 was measured by Biotrak activity assay (GE Healthcare). Sensitivities were of 250 pg/ml (for MMP-9), 6.5 pg/ml (for IL-8), and 12.5 pg/ml (for MCP-1 and MIP-1).
Statistical analysis
Data were analyzed statistically using the GraphPad Prism, version 4.0 for MacIntosh (GraphPad, eBizcuss). Results are expressed as median (interquartile range), except those obtained with purified alveolar macrophages, which represent means ± SEM. ANOVA was assessed with use of the nonparametric Kruskal-Wallis test. When an overall significant difference was detected (p 0.05), pairwise group tests were performed using the Mann-Whitney U test.
Univariate regression analyses were performed by the Spearman’s rank-order method, and correlation coefficients (rs) were calculated. Only smokers with and without COPD were included in the analysis. The ANOVA was assessed with the use of the nonparametric Kruskal-Wallis test. To account for multiple tests because of the eight comparisons across the three study groups, we used a Benjamini and Hochberg correction with a family-wise error rate of 0.05 (22). The p values were sorted in ascending order, and the smallest p value, P1, was compared with (0.05 x 1)/6 = 0.0063, and if P1 was less than 0.0063, P2 was compared with (0.05 x 2)/8 = 0.0125, etc.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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YKL-40 was detectable in serum and BAL samples from healthy never-smoker individuals (median (interquartile range), 13.5 ng/ml (10.1–40.3) and 2.8 ng/ml (1.4–5.1), respectively; Fig. 1). BAL, but not serum, levels of YKL-40 were slightly, but significantly increased in smokers without COPD (7.6 ng/ml (2.4–12.0), p = 0.0007; Fig. 1). Smokers with COPD had highly augmented levels of YKL-40 in serum (85.6 ng/ml (17.3–140.1)) and in BAL (19.0 ng/ml (10.0–34.1); Fig. 1).
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Univariate regression analyses in smokers with and without COPD (n = 50 subjects included) demonstrated that the levels of YKL-40 in serum and BAL were strongly associated with airflow obstruction, as determined by the assessment of percentage of predicted prebronchodilator, forced expiratory volume in 1 s (Table II). YKL-40 levels in serum and BAL also correlated significantly with transfer factor of the lung for carbon monoxide (DLCO) (Table II). Neither YKL-40 in serum, nor in BAL, correlated with total lung capacity or smoking history (Table II). In a further subanalysis performed to account for smoking exposure, we found similar serum and BAL levels of YKL-40 in patients with COPD, either former or current smokers. Indeed, levels of YKL-40 in the serum of former (n = 14) and current (n = 16) smokers were 86.7 (19.5–154.0) and 61.0 (19.9–132.0) ng/ml, respectively (p = 0.403), and those found in BAL were 30.0 (17.5–96.8) and 23.8 (9.2–41.3) ng/ml, respectively (p = 0.140).
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YKL-40 expression in bronchial biopsy and in lung tissue sections
Immunohistochemical detection of YKL-40 in bronchial biopsies from never-smokers showed only scarce, if any, positive cells (median (interquartile range), 3.1 cells/mm2 (2.1–5.9); Fig. 2, A and B). In smokers without COPD, the number of cells expressing YKL-40 tended to increase (4.1 cells/mm2 (0.0–6.3), p = 0.191; Fig. 2, A and C), and it was highly augmented in smokers with COPD as compared with never-smokers and to smokers without COPD (13.6 cells/mm2 (9.3–14.2), p = 0.011 and p = 0.03, respectively; Fig. 2, A and D). YKL-40 mainly localized to cells infiltrating the bronchial epithelium and the submucosa (Fig. 2, C and D), but was neither expressed by the bronchial epithelium (Fig. 2, C and D), nor by vascular endothelium, or airway smooth muscle (data not shown). No immunostaining was observed in control isotype rabbit IgG-treated tissue sections (Fig. 2E).
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Because alveolar macrophages were the major cell source of YKL-40, we next examined whether its expression was differentially regulated in relation with the disease status of the donors. The proportion of alveolar macrophages in BAL cells was higher in smokers with and without COPD (91.3 and 95.1%, respectively), as compared with never-smokers (81.3%). Immunostaining of cytospin preparations of BAL cells revealed weak or no expression of YKL-40 in the cytosol of alveolar macrophages of never-smokers (Fig. 4A), a consistent increment in smokers without COPD (Fig. 4B) and a strong immunostaining in smokers with COPD (Fig. 4C). Significantly higher proportion of YKL-40-positive alveolar macrophages was found in smokers without COPD (mean ± SEM, 26.7 ± 6.0%) in relation to never-smokers (3.3 ± 1.2%). This proportion was further increased in smokers with COPD (69.3 ± 5.8) (Fig. 4D).
To determine whether the increased expression of YKL-40 resulted from its elevated intrinsic baseline and stimulated synthesis, we examined the levels of YKL-40 transcript and protein in unstimulated and in TNF--exposed healthy and diseased alveolar macrophages. By quantitative real-time PCR, we found higher basal levels of YKL-40 mRNA in alveolar macrophages from smokers with and without COPD, in relation to never-smokers (Fig. 4E). This was accompanied with elevated amounts of the YKL-40 protein in the supernatants, as assessed by ELISA (Fig. 4F). The expression of both YKL-40 mRNA and protein did not differ significantly between never-smokers and smokers without COPD, whereas it was increased significantly in alveolar macrophages from smokers with COPD (Fig. 4, E and F). Stimulation of alveolar macrophages during 24 h with 10 ng/ml TNF- up-regulated the levels of YKL-40 mRNA and secreted protein in never-smokers and in smokers with and without COPD (Fig. 4, E and F). However, these levels were substantially increased in smokers with COPD in relation to the other patient groups (Fig. 4, E and F).
Effect of YKL-40 on alveolar macrophages
Finally, we examined the ability of YKL-40 to promote the release of proinflammatory and fibrogenic chemokines and of MMP-9 by alveolar macrophages. BAL alveolar macrophages were incubated during 24 h with 0.1, 1, and 10 µg/ml purified human YKL-40, or with 10 ng/ml TNF-, used as positive internal control. Higher levels of IL-8, MCP-1, and MIP-1 were found in the supernatant of unstimulated alveolar macrophages from smokers with and without COPD than in that from never-smokers (Fig. 5). YKL-40 dose-dependently promoted mediator release, with a maximal increase at the concentration of 10 µg/ml (Fig. 5).
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0.02). Similarly, YKL-40 promoted the release of higher quantities of MCP-1 in smokers with and without COPD (mean net levels of 39.9 and 56.0 pg/ml, respectively), as compared with never-smokers (15.6 pg/ml, p 0.04), as well as of MIP-1 (mean net levels of 100.2, 368.0, and 466.3 pg/ml in never-smokers, smokers with COPD, and smokers without COPD, respectively, p 0.03).
The ability of YKL-40 (10 µg/ml) to promote chemokine and MMP-9 release from alveolar macrophages was comparable to that observed with TNF- (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Recently, we have demonstrated that serum levels and number of cells expressing YKL-40 in bronchial biopsies were elevated in subjects with severe asthma (14), another debilitating respiratory disease characterized by airway inflammation and remodeling (23). Although in these subjects serum levels of YKL-40 were in the same range as those presently reported in subjects with COPD, this glycoprotein was almost undetectable in BAL fluid. This difference may simply reflect the airway infiltration by different cell types (e.g., neutrophils vs eosinophils) between these two diseases.
To determine YKL-40 localization in the airways, immunohistochemical detection was next performed on bronchial biopsy and lung tissue sections. In both types of specimens, very few YKL-40-positive cells were found in never-smokers, whereas these numbers were higher in smokers without COPD and further augmented in smokers with COPD. Study of the staining pattern of YKL-40 by double immunofluorescence coupled to confocal microscopy led to the identification of alveolar macrophages as the main cell source of YKL-40 in the lung of smokers with and without COPD. However, when present, neutrophils also stained positively for YKL-40. Together, these findings extend previous observations showing that phagocytes synthesize abundantly YKL-40, both in the systemic circulation and in normal and diseased tissues (8, 24, 25).
The number of macrophages in the lung tissue and the alveolar compartment is higher in COPD (26), and this may account, at least in part, for the increase in BAL levels of YKL-40 observed in smokers with COPD. However, we also found that the proportion of alveolar macrophages containing YKL-40 was substantially increased in smokers with COPD, when compared with smokers without COPD or to never-smokers, with 
70% of cells expressing this protein. In addition, alveolar macrophages from smokers with and without COPD showed increased baseline mRNA and higher spontaneous as well as TNF--induced secretion of YKL-40, as compared with never-smokers. These observations extend previous results showing strong up-regulation of YKL-40 expression in macrophages trapped in vessel atherosclerotic plaques (8) and suggest that elevated numbers of alveolar macrophages, associated with an intrinsically greater potential of synthesizing YKL-40, participate in the elevation of this glycoprotein in BAL from smokers with COPD.
Intriguingly, visual inspection also revealed that certain subsets of BAL alveolar macrophages failed to stain for YKL-40, suggesting that the expression of this glycoprotein reflects differences in phenotype within the whole macrophage population, or in the activation status, or in the degree of maturation, as previously proposed (27).
Recent studies conducted on articular chondrocytes demonstrated that IL-1β and TNF- efficiently promote the synthesis of YKL-40 (28). These proinflammatory cytokines are elevated in the lung of patients with COPD (11), suggesting that chronic exposure of alveolar macrophages to the inflammatory airway environment augments YKL-40 levels. Whether other factors that play a role in the pathogenesis of COPD regulate YKL-40 expression is an area for further investigation.
Once generated within the airways, YKL-40 may interact in an autocrine and/or paracrine manner to promote the generation of factors involved in COPD pathogenesis. Therefore, we tested the possibility that YKL-40 induced the release, by alveolar macrophages, of IL-8, MCP-1, and MIP-1, three chemokines that sustain tissue inflammation and fibrosis, and of MMP-9, which is primarily involved in matrix degradation and in the accompanying alveolar destruction seen in emphysema (11). Extending previous observations (19, 29), alveolar macrophages from smokers with and without COPD constitutively produced higher amounts of IL-8, MCP-1, and MIP-1 than those from never-smokers. Exposure of alveolar macrophages to YKL-40 promoted chemokine and MMP-9 release, and this stimulatory effect was comparable to that obtained upon cell activation with TNF-. It is noteworthy that YKL-40 was biologically active at 1 and, more so, at 10 µg/ml. These apparent high concentrations are, however, in the lower range of those routinely found in the serum of patients with chronic inflammatory diseases characterized by aberrant macrophage activation and tissue fibrosis (9, 10, 11).
Peribronchiolar fibrosis is another important histopathologic feature of tissue remodeling in COPD that is strongly associated with airflow obstruction and emphysema (2, 30). Recent studies have shown that YKL-40 is preferentially expressed in areas with active fibrogenesis in patients with hepatic fibrosis (10), where it may act synergistically with insulin-like growth factor I to stimulate the growth of fibroblasts (6). YKL-40 may also contribute to fibrosis by modulating the rate of type I collagen fibril formation (31). Whether YKL-40 participates in the onset of fibrosis of the small airways in patients with COPD remains to be determined. However, our present findings suggest that YKL-40 may influence extracellular matrix deposit and turnover by inducing MMP-9 production by alveolar macrophages (32, 33).
In conclusion, the present findings demonstrate that circulating and alveolar levels of YKL-40 are elevated in patients with COPD in association with disease severity. In addition, the in vitro data showing that alveolar macrophages are both source and target for YKL-40 make this glycoprotein a plausible candidate for playing a role in COPD pathogenesis. Overall, the current report brings a novel view on the pathophysiology of COPD and may lead to develop new therapeutic strategies for better management and benefit of the patients.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by the Fondation pour la Recherche Médicale (fellowship to S.L.) and by grants from the Agence Nationale de la Recherche (Santé et Environnement-Santé Travail) and from MedImmune. 
2 S.L. and A.K. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Marina Pretolani, Inserm Unité 700, Faculté de Médecine Denis Diderot, Site Bichat - 16, rue Henri Huchard, 75018 Paris, France. E-mail address: marina.pretolani{at}inserm.fr
4 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; BAL, bronchoalveolar lavage; DLCO, transfer factor of the lung for carbon monoxide; GOLD, Global Initiative for Obstructive Lung Disease; MMP, matrix metalloproteinase.
Received for publication January 10, 2008. Accepted for publication August 4, 2008.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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