Macrophage expressed tartrate‐resistant acid phosphatase 5 promotes pulmonary fibrosis progression

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder involving scarring of pulmonary tissue and a subsequent decrease in respiratory capacity, ultimately resulting in death. Tartrate resistant acid phosphatase 5 (ACP5) plays a role in IPF but the exact mechanisms are yet to be elucidated. In this study, we have utilized various perturbations of the bleomycin mouse model of IPF including genetic knockout, RANKL inhibition, and macrophage adoptive transfer to further understand ACP5's role in pulmonary fibrosis. Genetic ablation of Acp5 decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix‐production) as well as histological damage. Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il‐1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions. Furthermore, antibody‐based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development. Adoptive transfer of Acp5−/− bone marrow‐derived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages. Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.

decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix-production) as well as histological damage.Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il-1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions.Furthermore, antibody-based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development.Adoptive transfer of Acp5 À/À bone marrowderived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages.Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.

INTRODUCTION
Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease, resulting in progressive decline of lung function and ultimately respiratory failure [1].Repeated alveolar epithelial microinjuries are considered paramount in pulmonary fibrosis development [2].Mechanistically, IPF is propelled by aberrant mesenchymal cell proliferation leading to the replacement of healthy alveolar lung tissues with excess extracellular matrix (ECM) [3,4].This highlights the requirement for tight regulation of wound healing responses, a role assigned to macrophages due to their programmable plasticity and involvement in critical regulatory phases of repair and fibrosis [5,6].In murine bleomycin models, which resemble the pathology of IPF, chemokine receptor type 2 (Ccr2) deletion mitigates fibrosis, with similar effects seen following deletion of CD11bexpressing monocyte-derived alveolar macrophages [7].Additionally, knockout of chemokine (C-C motif) ligand 3 (Ccl3), which is primarily produced by macrophages, protects against bleomycin-induced pulmonary fibrosis [8].Macrophages are rich sources of the phosphoglycoprotein osteopontin (OPN), that is established as a profibrotic mediator, partly through TGF-β 1 activation [9,10].
In this context, tartrate-resistant acid phosphatase 5 (ACP5, also designated TRAP5) is synthesized as a monomeric proenzyme (ACP5a) that undergoes posttranslational cleavage to reveal the enzymatically active phosphatase (ACP5b) [11].In bone matrix, ACP5 dephosphorylates OPN, which promotes osteoclast detachment and integrin-dependent migration on RGD-and RGD-like motifs of OPN [12].Additionally, OPN-rich macrophages have been reported in the airways and parenchyma of IPF patients [13].With the modulation of OPN tightly regulated by ACP5, it is hypothesized that a corresponding increase in ACP5 should be seen.ACP5 is abundantly expressed in several immune cells [14], colocalizing with OPN in alveolar macrophages where it serves to dephosphorylate OPN [15].Unlike other lysosomal hydrolases, ACP5 does not maintain housekeeping activities and can be actively secreted [16,17].ACP5's high presence in alveolar macrophages suggests it plays a vital role in pulmonary immunology; however, its precise function in this context is poorly understood.Interestingly, ACP5 is best characterized for its role in bone remodelling [18].Specifically, ACP5 is required for the function of osteoclasts, cells derived from macrophage progenitors, which are responsible for bone tissue homeostasis [19].Bone matrix turnover is a tightly regulated process that can give rise to numerous pathologies if unbalanced [20].Additional disease-states induced by deficiencies in ACP5 production include skeletal dysplasia disorders such as spondyloenchondrodysplasia, which are tightly linked with immune system dysregulation [21,22].Expression of the ACP5 gene is regulated by the transcription factor nuclear factor of activated T cells 1 (NFATC1), which is translocated to the nucleus upon RANKL stimulation, promoting transcription [23].
Despite the well-defined importance of macrophages in controlling fibrotic responses [24], no therapeutic macrophage-directed therapies have been introduced.Current IPF therapies focus on the inhibition of collagen deposition by blocking myofibroblast activation, with limited success in achieving overall IPF resolution necessitating the need for novel therapies [1].Therefore, therapeutic targeting of ACP5 in the context of pulmonary fibrosis could potentially decrease the rate of disease progression and allow patients to mediate disease symptoms, subsequently improving life expectancy associated with IPF.

MATERIALS AND METHODS
All materials and methods can be found in the Supporting Information.

RESULTS
ACP5 is expressed by human alveolar macrophages.
Previous studies have addressed the potential role of ACP5 in IPF [25]; however, numerous questions still surround the mechanism which drives these profibrotic changes.Therefore, we examined whether targeted ACP5 inhibition of alveolar macrophages serves as a novel clinical strategy to alleviate the onset and progression of pulmonary fibrosis.To assess the viability of this approach, we utilized the open access resource Human Protein Atlas to demonstrate the distribution of ACP5 within the human body [26].A predominant presence of ACP5 was seen in macrophages of the lung (Figure 1a).ACP5 gene expression was determined in various organs by single cell transcriptomics (Figure 1a), with read counts normalized to transcripts per million protein coding genes (nTPM).

Number of ACP5 expressing macrophages is increased in IPF.
As macrophages are the predominant cells in the lung expressing ACP5, 3,3 0 -diaminobenzinidine (DAB) chromogenic staining was conducted on human lung sections to investigate number of Acp5 positive macrophages in normal versus IPF lung tissue (details in Table 1).Staining of consecutive lung sections with antibodies directed at CD68 or ACP5 showed ACP5 positive macrophages in both IPF and healthy control tissue, with higher amounts of macrophages in the IPF samples (Figure 1b).Additionally, an increased clustering of ACP5 positive macrophages was found in the IPF tissue compared to the healthy control tissue (red arrows).Higher magnification revealed an increased staining of ACP5 in pneumocytes surrounding the macrophage clusters in the IPF tissue.Staining with isotype control antibodies showed no staining (Figure S1).Murine Acp5 À/À macrophages attenuate fibrotic fibroblast development Macrophage secreted cytokines and proteins, including ACP5 [27] has been shown to induce proliferation and differentiation in cells of mesenchymal origin.Therefore, we investigated if macrophage lysates affect fibroblast differentiation and thereby the progression of pulmonary fibrosis.F4/80-positive cells purified from BALF obtained from WT and Acp5 À/À mice exposed to bleomycin were utilized in a Transwell assay.F4/80+ lysates were prepared from both wild-type (WT) and Acp5 À/À knockout mice digests (Figure 2a) and added to naïve primary mouse lung fibroblasts (PMLF) generated from WT mice.Lysates from bleomycin treated Acp5-deficient macrophages lacked the ability to increase fibroblast proliferation (Figure 2b,c) as evidenced by a decrease in PMLF migration.Further analysis of fibrosis associated proteins, COL1A1, α-SMA and fibronectin, also shows that WT macrophages can stimulate production of these fibrosis associated proteins in fibroblasts, while Acp5 À/À macrophages cannot (Figure 2d).These data suggest that, not only endogenous fibroblastic ACP5 expression, but also macrophage derived ACP5 has a role in increased migration of fibrotic fibroblasts.The previous study by Hu et al [25] showed that TGF-β 1 induced ACP5 expression in primary lung fibroblast human cells (PHLF) and that ACP5 increased migration and differentiation of PHLF.This study confirms that knockdown of ACP5 (using ACP5-targeting siRNA) in TGF-β 1 stimulated primary lung fibroblasts (PHLF) results in reduced migration compared to non-transfected PHLF cells (Figure 2e,f).However, results from this study also shows that macrophage expressed ACP5 itself, directly or indirectly, seems to upregulate Acp5 expression in fibroblast leading to increased fibroblastic migration (Figure 2d).

ACP5 deficiency significantly attenuates mouse fibrotic lung progression
Global deletion of murine Acp5 has been shown to reduce fibrotic lung progression [25].To further dissect the requirement of ACP5 in the mouse lung, bleomycin was administered to WT and Acp5 À/À mice (Figure 3a).Acp5 À/À mice were significantly protected from both weight loss and death (Figure 3b,c).This was further supported by significant increases in total protein and hydroxyproline concentrations in the lungs of WT versus Acp5 À/À mice (Figure 3d,e).Confirmatory Western blots using murine lung homogenate showed decreases in key fibrotic proteins from Acp5 À/À mouse lung tissues including collagen 1A1 (Col 1a1), vimentin (Vim), fibronectin (Fn), and alpha-smooth muscle actin (α-SMA) compared to those from WT mice (Figure 3f).Additionally, significant increases in total Acp5 levels were seen in WT murine lung tissue and BALF compared to those from Acp5 À/À mice (Figure 3g).
Cell recruitment is attenuated following Acp5 deletion.
An important consideration for the progression of IPF is the involvement of immune cells, with macrophages providing large amounts of TGF-β 1 during fibrosis progression [28,29].Additionally, in a study on murine bacterial lung infection, ACP5 was shown to facilitate the migration of immune cells into the lumen of the lung [30].We sought to study the murine lung environment by performing flow cytometry on murine BALF samples.Quantitative analyses were used to determine neutrophils (Ly6G + /CD11b + ), inflammatory non-granulocytes (Ly6G À /CD11b + ), and alveolar macrophages (Ly6G À / CD11c + ).Significantly increased numbers of neutrophils and inflammatory non-granulocytes were seen in WT/bleomycin samples compared to those in the Acp5 À/À /bleomycin mice (Figure 4a).Confirmatory Giemsa-Wright-stained samples showed similarly increased immune cell concentrations within the WT/Bleomycin samples (Figure 4b).In addition, TGF-β 1 concentrations were decreased in Acp5 À/À animals compared to those in the WT control group (Figure 4c).

Histological damage is decreased by Acp5 knockout
Histological lung damage is a defining clinical feature of IPF.We assessed histological damage using H&E stain, with the bleomycin/WT samples displaying the greatest loss of lung structure and immune cell recruitment compared to the samples obtained from Acp5 À/À /bleomycin mice as denoted by positive pixel analysis (Figure 5a).
Additional collagen staining, using picrosirius red, revealed decreased collagen deposition within the lungs of the Acp5 À/À /bleomycin mice as compared to the lung tissue of WT/bleomycin mice (Figure 5b).

ACP5 deficiency results in altered chemokine expression in lung tissue
Given the decreased immune cell recruitment to the murine lung, we wanted to determine whether these changes are determined on a gene-level.We assessed fibrosis-specific gene regulation using a PCR array.Several genes were up-and down-regulated compared to a WT/bleomycin comparator.More specifically, gene fold regulation was increased when comparing Acp5 À/À and WT samples in decorin (Dcn), matrix metalloproteinase 9 (Mmp9), C-X-C motif chemokine receptor 4 (Cxcr4), TGF-β/Smad repressor TG-interacting factor 1 (Tgif1), and signal transducer and activator of transcription 6 (Stat6) (Figure 5c,d).Additionally, genes that displayed down-regulation included matrix metalloproteinase 13 (Mmp13), interleukin 1-alpha (Il1-α), and C-C motif chemokine ligand 3 (Ccl3).Differences in gene expression was analysed using Metascape, a program for system-level analysis of datasets [31], generating associated gene ontology (GO) terms.The top GO terms associated with the gene expression data were lung fibrosis, TGF-β signalling pathway, negative regulation of cell population proliferation and matrix metalloproteinases (Figure S3).

Inhibition of RANKL reduces immune cell recruitment but does not decrease lung injury or fibrosis
RANKL promotes cell migration and activates the transcription factor NFATC1, the latter inducing expression of ACP5.Consequently, it is possible that RANKL is involved in the development of fibrosis.Administration of a RANKL-inhibiting antibody, denosumab, significantly decreased immune cell recruitment and cytokine production but had very minor effects on lung weight, and total body weight (Figure 6a-e).More specifically, murine total bodyweight was unaffected by anti-RANKL treatment, while lung weights decreased in comparison to mice treated with the IgG2A control antibody (Figure 6b,c).Immune cell recruitment, including neutrophils, alveolar macrophages, and inflammatory non-granulocytes was significantly attenuated following anti-RANKL treatment converse to alveolar macrophage recruitment seen in the Acp5 À/À mice (Figure 6d).However, RANKL inhibition did not significantly decrease neither Acp5a nor Acp5b production, with only slight reductions seen for either protein in lung homogenates for RANKL antibody-treated mice compared to the mice treated with IgG2A control antibody (Figure 6e).Cytokine measurements in lung tissue and BALF of anti- RANKL/bleomycin mice reflected an altered chemokine/ cytokine profile, corresponding to the decreased immune cell recruitment when compared to the IgG2A/bleomycin control group.Particularly key cytokines associated with inflammation including IL-1α, IL-6, and KC (Figure 6f and Figure S4).However, cytokines associated with fibrotic changes were only marginally decreased including IL-4 and IL-13.The resultant pulmonary histology (Figure 6g) confirmed the lack of anti-fibrotic activity seen after anti-RANKL treatment, with soluble collagen (hydroxyproline) also being unaffected in comparison to the IgG2A/bleomycin control group (Figure 6h).
To confirm the inhibition of RANKL, immunofluorescence on murine lungs was conducted, with a reduction in RANKL intensity seen in the RANKL antibody-treated samples confirming the effect of RANKL inhibition in this context (Figure 6i).
Acp5 À/À macrophage add back results in improved lung function As macrophages present a large source of ACP5 [32], we wanted to determine the effects of adding back isolated bone marrow-derived monocyte (BMDM) derived macrophages from WT and Acp5 À/À mice.Macrophages were isolated and cultured according to Wang et al. [33] and adoptively transferred to murine lungs by intratracheal administration at Day 7 and Day 14 allowing for the temporal dissection of macrophage influence in the context of ACP5 (Figure 7a).Murine weight loss was not significantly attenuated by macrophage add back (Figure 7b).However, murine lung weight was subverted by the addition of Acp5 À/À macrophages at Day 7, with no other reported differences compared to the bleomycin/no treatment control (Figure 7c).Further changes in murine cytokine production were seen, with WT macrophage adoptive transfer at Day 14 resulting in significantly more cytokines than the bleomycin control in both the BALF and lung tissue, including IL-4, IL-6, and IL-13 (Figure 7d and Figure S5).Interestingly, add back of Acp5 À/À macrophages at Days 7 and 14 resulted in reduced production of cytokines compared to WT macrophage add back at Day 7.However, lung damage was only slightly attenuated by adding back Acp5 À/À macrophages at Day 14 versus WT macrophages added back at the same time point (Figure 7e,f).Hydroxyproline measurements in the murine lung revealed decreased hydroxyproline values for the mice receiving Acp5 À/À macrophages compared to those receiving WT macrophages at Day 14 and the bleomycin control (Figure 7g).Western blots revealed subsequent decreases in murine lung ACP5 content following Acp5 À/À macrophage add back, indicative of a potential downregulation of ACP5 production (Figure 7h).

DISCUSSION
Despite advances in IPF treatment, the understanding of molecular and physiological mechanisms that lead to disease progression remain poorly elucidated [34,35].Our study provides evidence for the involvement of ACP5 in IPF progression through the analysis of IPF patient samples and murine in vivo models of IPF.Importantly, we also delineate claims made by previous IPF studies regarding the immunological role of ACP5 in IPF.ACP5 is synthesized as a monomeric proenzyme (ACP5a), which undergoes post-translational cleavage and reveals the enzymatically active phosphatase (ACP5b) [11].ACP5 dephosphorylates the bone matrix protein osteopontin (OPN), which promotes osteoclast detachment and migration [12].Additionally, OPN-rich macrophages have been reported in the airways and parenchyma of IPF patients [13].With the modulation of OPN tightly regulated by ACP5, it is hypothesized that a corresponding increase in ACP5 should be seen.
Higher levels of ACP5 have indeed been shown in the sera of patients with IPF [25].However, little attention has been given to cell type assessment of ACP5 production.Herein, we show that macrophage levels of lungderived ACP5 were significantly higher than those of all other lung-based cell types.This is indicative of the role of macrophages, particularly those higher in ACP5, in IPF disease state.It is known that activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize ECM proteins such as collagen.Unfortunately, agents that suppress the release of these cytokines have hitherto been too toxic for administration [36].A benefit of our approach shows that by genetic ablation of Acp5, we were able to reduce the production of key profibrotic cytokines.This resulted in decreased immune cell recruitment to the lung, inhibiting further production of TGF-β 1 , which decreases fibroblast to myofibroblast transition [37].
Gene expression profiling of fibrosis related genes in lung tissue from Acp5 À/À and WT mice revealed a substantial reduction of Ccl3 expression in Acp5 À/À mice, and cytokine analysis of BALF and lung tissue showed a corresponding significant reduction of CCL3/MIP-1α.CCL3/MIP-1α plays a critical role in macrophage chemotaxis and collagen synthesis [38].Furthermore, regulation of CCL3/MIP-1α production in macrophages is mediated by IL-6 [39], a cytokine shown to have a diminished production in Acp5 À/À mice.Interestingly, expression of Ccl3 is induced by cell-to-cell interaction between macrophages and fibroblasts [40].The reduced recruitment of macrophages to the lumen of bronchi (as detected in BALF), seen in the Acp5 À/À mice, prevents the cell-to-cell interaction with fibroblasts, which could potentially hinder the induction of Ccl3 and further reduce macrophage recruitment in a negative feedback manner.
RANKL potently induces ACP5 expression in murine macrophages and lung slices making it an appropriate target for perturbation [14,41].Previous synthetic activators of RANKL have shown beneficial effects in the bleomycin model, inhibiting TGF-β 1 -induced Smad 2/3 phosphorylation [42].To further dissect the effects of ACP5 modulation within the lung environment, we employed a therapeutic antibody against RANKL.Interestingly, whilst the immune cell component and cytokines were significantly reduced by anti-RANKL treatment, little to no therapeutic effect was observed on pulmonary histology and collagen content, possibly indicating that RANKL is not involved in recruitment and/or differentiation of Acp5-expressing lung macrophages.
Macrophages provide perhaps the most plastic cell type in the haematopoietic system, allowing them to assume numerous roles during wound healing and tissue homeostasis [43].Additionally, macrophages, or subsets thereof, play a major role in IPF disease pathogenesis [44,45] and provide a source of ACP5.We further show that our adoptive transfer studies confirm the cell-specific nature of ACP5 in IPF progression.Furthermore, we illustrate through temporal add back of macrophages that the experimentally induced fibrotic response is influenced by the time of intervention.Despite differences in ACP5 protein levels in the macrophages, we cannot rule out other effectors including extracellular vesicles [46,47] and other macrophage-specific molecules [48] within this study.Further limitations to the study include the lack of dynamic ACP5 and cell-specific changes within the patient context.To fully understand the role of ACP5 in IPF, a larger patient cohort would need to be studied.Secondly, although we show a role for macrophage specific ACP5 in the experimental model of IPF, we have not further elucidated the role of other cell types within this context.Third, whilst cell recruitment is shown in this model it is demonstrated at a fixed time point (21 days).To fully elucidate the in vivo role of ACP5 in cell recruitment, precision cut lung slices or similar models would need to be utilized.
In conclusion, our study demonstrates the role of ACP5 in the context of IPF disease.Mice lacking Acp5 were protected from bleomycin-induced fibrotic lung damage.Furthermore, cell recruitment and cytokine production were significantly attenuated in Acp5-ablated animals.This was supported by genetic changes in murine lung tissue and anti-RANKL targeting antibody administration.Finally, the macrophage-specific role of ACP5 was suggested by the adoptive transfer of BMDMs into wildtype mice, whereas use of a knockout mouse baseline would further support these claims.Taken together, our results support ACP5 as a viable therapeutic target in the context of IPF.

F I G U R E 1
Distribution of ACP5 expression in human organs and cells during healthy conditions and in lungs of IPF patients.(a) Human Protein Atlas (v21.1.proteinatlas.org) was utilized with data generated from previous work [26].ACP5 gene expression was determined in various organs (liver, peripheral blood mononuclear cells [PBMCs], lung and kidney) by single cell transcriptomics shown as normalized transcripts per million protein coding genes (nTPM).(b) 3,3 0 -diaminobenzinidine (DAB) chromogenic staining showing ACP5-containing macrophages in lung sections from both IPF patients and healthy controls, with an increased amount of macrophages in the IPF patients.

F I G U R E 3
Acp5 À/À mice are protected against bleomycin induced pulmonary fibrosis.(a) Experimental design.Acp5 À/À and WT mice were intratracheally administered bleomycin (2.5 U/kg) and euthanized at Day 21.(b) Body weight of Acp5 À/À and WT mice ± bleomycin treatment displayed as percentage of starting weight.Data were analysed using one-way ANOVA with Dunnetts post hoc test (****p < 0.0001) (c) Survival curve of Acp5 À/À and WT mice ± bleomycin treatment with representative lung images alongside.Data were compared using a Mantel-Cox test.(d) Total protein amounts in lung homogenates from Acp5 À/À and WT mice ± bleomycin treatment.(e) Hydroxyproline production in the Acp5 À/À and WT mice ± bleomycin treatment.Data were analysed using one-way ANOVA with Dunnetts post hoc test (****p < 0.0001).(f, g) Production of fibrotic tissue, that is, Col1a1, fibronectin, α-SMA, and vimentin were assessed along with ACP5a and ACP5b in Acp5 À/À and WT mice ± bleomycin treatment.Statistical analysis was conducted using an unpaired Welch's t-test (*p < 0.05; **p < 0.01).

F
I G U R E 5 Acp5 knockout results in reduced histological damage and changes in gene-expression levels.(a, b) Murine lung sections were stained with haemotoxylin and eosin (H&E) and picrosirius red, with quantification reported alongside (one-way ANOVA; ***p < 0.001; ****p < 0.001).(c, d) Gene array data showing fold regulation of genes of proteins promoting fibrosis.A fold level change of 3 or higher was set as the cut-off value.

F
I G U R E 6 RANKL-targeting antibody decreases immune cell recruitment but fails to address lung damage.(a) Mice were administered bleomycin (2.5 U/kg) and allowed to progress to Day 14. Treatment using anti-RANKL antibody (ant-RL; clone IK22/5) or IgG control was administered on Days 14 and 18.(b) Murine weight was measured for all mice, with both bleomycin groups displaying similar levels of weight loss.(c) Murine lung weight was also measured, with statistical analysis conducted using one-way ANOVA (***p < 0.001; ****p < 0.0001).(d) Immune cell recruitment to the BALF was measured using flow cytometry with neutrophils (Ly6G+/CD11b+), inflammatory non-granulocytes (Ly6G À /CD11b + ), and alveolar macrophages (Ly6G À /CD11c + ).Statistical analysis was conducted using one-way ANOVA (**p < 0.01; ***p < 0.001; ****p < 0.0001).(e) Western blots were conducted to measure ACP5a and ACP5b in murine lung tissue.(f) Murine cytokines in the lung and BALF were measured and are presented in heatmaps (data normalized per cytokine).(g) Representative H&E-stained whole lung scans showing no changes in histological damage between the anti-RANKL and IgG2A-treated mice.(h) Murine lung hydroxyproline was measured, with statistical comparisons to the IgG2A/Bleomycin group conducted using one-way ANOVA (***p < 0.001).(i) Immunofluorescence measuring of RANKL is shown for murine lung samples.
Clinical data for patient samples included in this study.Control samples were obtained from organ donor lungs (F, female; M, male).Abbreviations: DLCO, diffusion capacity of the lungs to carbon monoxide (CO per unit time per mm of driving pressure of CO [cc of CO/s/mm of Hg]); FEV1, forced expiratory volume in 1 s; FEV1%, percentage forced expiratory volume % of expected FEV1 (in relation to age, height, and sex); FEV1/FVC, the proportion of a person's vital capacity expired in the first second of forced expiration (FEV1) divided by the forced vital capacity (FVC).
T A B L E 1Note: