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Increased Platelet Binding to Circulating Monocytes in Idiopathic Pulmonary Fibrosis Ahmed Fahim • Michael G. Crooks Alyn H. Morice • Simon P. Hart
•
Received: 11 August 2013 / Accepted: 14 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Purpose Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia and its prognosis is poor. Epidemiological evidence suggests an association of IPF with vascular disease and thrombotic tendency, which may be related to platelet activation. Methods Platelet–monocyte adhesion in peripheral blood was examined by flow cytometry in patients with IPF (n = 19), interstitial lung disease (ILD) other than IPF (n = 9), and control subjects without pulmonary fibrosis (n = 14). Expression of platelet activation markers P-selectin (CD62P), PSGL-1 (CD162), and CD40 ligand (CD40L) on leukocytes and platelets were studied. Plasma concentrations of soluble P-selectin and CD40L were measured by ELISA. Results Significantly elevated levels of platelet–monocyte binding were found in patients with IPF (35.6 ± 4.34 % [mean ± SEM]) compared with patients with non-IPF ILD (23.5 ± 3.68 %) and non-ILD control subjects (16.5 ± 2.26 %; P \ 0.01). There was a trend towards increased divalent cation-independent platelet– monocyte binding in IPF (6.0 ± 0.77 % [mean ± SEM]) compared with non-IPF ILD (4.3 ± 1.38 %) and control subjects without ILD (3.1 ± 1.75 %; P = 0.058). There A. Fahim (&) M. G. Crooks A. H. Morice S. P. Hart Division of Cardiovascular and Respiratory Studies, Castle Hill Hospital, Castle Road, Cottingham HU16 5JQ, UK e-mail: [email protected] M. G. Crooks e-mail: [email protected] A. H. Morice e-mail: [email protected] S. P. Hart e-mail: [email protected]
was no differential surface expression of platelet activation markers on subsets of leukocytes or platelets. Plasma concentrations of CD40L and soluble P-selectin did not differ between IPF and control subjects. Platelet–monocyte binding had no significant correlation with percent predicted TLco or FVC. Conclusions Platelet–monocyte binding is increased in IPF, suggesting increased platelet activation. This conjugation is predominantly calcium-dependent, but there may be more calcium-independent adhesion in IPF. These findings support further research into the role of platelet activation in IPF. Keywords Idiopathic pulmonary fibrosis Interstitial lung disease Monocytes Platelets Platelet–monocyte complexes Abbreviations CD40L CD40 ligand FACS Fluorescent activated cell sorting IPF Idiopathic pulmonary fibrosis ILD Interstitial lung disease mAb Monoclonal antibodies PMN Polymorphonuclear leukocytes
Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease (ILD) associated with an insidious onset of dyspnoea, bi-basal inspiratory crackles, a restrictive ventilatory defect, and impaired gas exchange [1]. Radiologically, it is characterized by peripheral and basal reticular
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abnormalities with minimal ground glass change on highresolution computed tomography (HRCT) scans. Areas of fibrosis are interspersed with normal lung parenchyma in lung biopsy specimens consistent with geographical heterogeneity. IPF is the commonest of the idiopathic interstitial pneumonias (IIP) with a median survival of 2.5–3 years after diagnosis [2]. The cause is unknown, and there is no effective medical treatment. Progress in understanding the pathogenesis and potential treatment of IPF will depend on identifying the events that trigger and drive the disease. Epidemiological and autopsy studies have demonstrated a significant association of IPF with cardiovascular disease [3, 4]. Vascular remodeling in the form of luminal narrowing and progressive regression of vascularization has been observed in IPF [5–7]. Platelets are attractive candidates for linking vascular dysfunction with tissue fibrosis [8]. Mesenchymal growth factors, including platelet-derived growth factor (PDGF), insulinlike growth factor, and transforming growth factor-b (TGF-b) are upregulated in IPF [9–11]. PDGF along with other growth factors, including connective tissue growth factor (CTGF) and TGF-b, play a significant role in fibrogenesis as they are mitogens for fibroblasts [12, 13], the key effector cells in IPF. Platelets are activated following endothelial injury with upregulation of cell-surface receptors, such as CD40 ligand (CD40L) and P-selectin. The increased expression of these surface molecules results in platelet adhesion to the vascular wall. Activated platelets release certain chemokines with recruitment of inflammatory cells and formation of platelet– monocyte complexes [14]. Platelet adhesion to monocytes and polymorphonuclear leukocytes (PMN) may alter leukocyte activation and recruitment patterns [15–17]. Increased binding of platelets to circulating monocytes in peripheral blood of patients with myocardial infarction and unstable angina has been demonstrated [18]. Moreover, this increased adhesion has been linked to cardiovascular risk in patients with chronic obstructive pulmonary disease (COPD) [19]. We hypothesized that increased platelet activation in IPF would be associated with formation of circulating platelet–monocyte complexes. In the present study, we used flow cytometry to examine platelet–monocyte binding and markers of platelet activation in peripheral blood.
Methods Patient Selection and Blood Sampling Patients with IPF diagnosed according to ATS/ERS criteria were recruited from the interstitial lung disease clinic at our University Hospital [20]. At the time of recruitment,
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patients were in a clinically stable state and were excluded if there was evidence of acute infection, acute coronary syndrome, or acute exacerbation of IPF. The control group comprised patients with non-IPF ILD, including hypersensitivity pneumonitis, collagen vascular disease-associated ILD, and pneumoconiosis. All the patients in non-IPF ILD group were diagnosed following a multidisciplinary meeting with a respiratory clinician, thoracic radiologist, and histopathologist (in biopsied cases). The non-ILD control group comprised predominantly healthy volunteers and patients with COPD who had no evidence of interstitial lung disease. Patients’ age, sex, history of hypertension, ischemic heart disease, cerebrovascular disease, diabetes, smoking status, occupational dust exposure, and medication use were recorded. Blood was drawn by venipuncture via 19-gauge needles and collected into 6-mL tubes containing 102 IU of lithium heparin (BD Ltd, Plymouth, UK), which were gently inverted several times to avoid platelet activation. Because the choice of anticoagulant can have a significant effect on the extent of platelet monocyte adhesion and there is evidence of significantly reduced platelet monocyte binding with sodium citrate compared with heparin or hirudin, we used heparin as the anticoagulant for this investigation [21]. The study was approved by Hull and East Riding Ethics Committee (LREC No. 08/H1304/54), and written consent was obtained from all the participants before recruitment into the study. Antibodies, Immunolabeling, and Flow Cytometry Monoclonal antibodies (mAb) directly conjugated to fluorochromes were purchased from the following sources: FITC-conjugated CD42a and PE-conjugated CD14 from Serotec Ltd (Oxford, UK); PE-conjugated CD62P mAb AK-4, CD162 mAb KPL-1, CD40L mAb MR-1 and control IgG1 from BioLegend (San Diego, CA, USA). Blood (50 lL) was labeled with specific antibodies for 20 min at room temperature. CD42a-FITC and CD14-PE mAb were used at final concentrations of 2 lg/mL. PE-conjugated CD62P, CD162, CD40L, and control mAb had been optimized for flow cytometry by the manufacturer and were used at recommended dilutions. 300 lL of FACSLyse solution (Becton Dickinson, San Jose, CA, USA) was added after mAb incubation. Blood was labeled within 30 min and samples were analysed using a Becton Dickinson FACS Calibur flow cytometer, and data analyses were performed using CellQuest Pro (Becton Dickinson) software. Monocytes and neutrophils were distinguished by their distinct laser scatter properties (Fig. 1a, b). CD14 was used to identify monocytes. Platelets were identified using CD42a. Platelet–monocyte adhesion was determined by two-color flow cytometry using directly conjugated
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Fig. 1 Flow cytometric identification of monocytes and neutrophils. a Monocytes identified by distinct forward and side scatter properties (shown in grey). FSC forward scatter on x-axis; SSC side scatter on y-axis. b Neutrophils (shown in grey) with higher side scatter than monocytes
CD14-PE and CD42a-FITC mAb. The percentage of monocytes (CD14?) binding one or more platelets (CD42a?) was assessed by labeling blood with both CD42a and CD14 mAb with and without the addition of EDTA (Fig. 2a, b). Plasma concentrations of P-selectin and CD40L were measured in duplicate by ELISA according to the manufacturers’ protocols (Bender MedSystems, Vienna, Austria). Statistical Analysis Statistical analyses were performed using SPSS 13, Chicago, IL, USA). The Kolmogorov and method was used to assess the data distribution. isons between patient groups were made by
(Version Smirnov Comparone-way
Fig. 2 Flow cytometric identification of platelet–monocyte aggregates. a Platelet–monocyte complexes in the upper right quadrant (FL1 = CD42a; FL2 = CD14). In this example, 18.5 % of monocytes exhibit platelet binding. b Significant reduction in platelet– monocyte adhesion with the addition of EDTA to chelate calcium
ANOVA for normally distributed data. Data with skewed distributions were analyzed by nonparametric tests (Mann– Whitney U test and Kruskal–Wallis test). Comparisons of gender, smoking, and comorbidities were performed using chi-square tests. Correlations were analyzed by Pearson correlation. The level of statistical significance was set at 0.05. Results are presented as mean ± standard error of the mean (SEM).
Results The baseline characteristics and demographics of the study populations are shown in Table 1. IPF patients were slightly older than the control subjects. The only other significant difference between the study groups was the peripheral blood monocyte count, which was higher in nonILD control subjects. The other baseline parameters were similar between the groups.
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Non-IPF ILD
Non-ILD controls
Subjects
19
9
14
Age (years)
69 ± 7
58 ± 9
56 ± 21
Gender Male
P value
0.005* 0.064
15
6
4
3
8
Platelet count
246 ± 67
248 ± 72
265 ± 91
0.882
Monocyte count
0.73 ± 0.14
0.59 ± 0.2
0.95 ± 0.17
0.002*
White cell count
7.85 ± 2.2
7.43 ± 2.48
9.36 ± 0.7
0.290
1
1
0
Female
6
Smoking Current
0.052
Ex
16
5
6
Never smokers
2
3
8
Hypertension
8 (42)
1 (11)
3 (22)
0.285
Diabetes
1 (5)
1 (11)
4 (29)
0.168
IHD
1 (5)
0
1 (11)
0.642
CVA/TIA
2 (10)
1 (11)
0
0.168
Aspirin FVC % predicted
6 (32) 89 ± 18 (n = 17)
1 (11) 86 ± 11 (n = 7)
2 (14) NA
0.323 0.482
TLco % predicted
57 ± 19 (n = 17)
56 ± 15 (n = 7)
NA
0.356
Fig. 3 Platelet–monocyte complexes in the study groups showing a significantly higher proportion of complex formation in IPF compared with non-IPF ILD and non-ILD subjects (dark bars). There was a dramatic reduction in platelet–monocyte adhesion with the addition of EDTA (light grey bars), indicating the predominantly divalent cationdependent nature of platelet–monocyte adhesion
Data are presented as mean ± SD or numbers with percentages in parentheses unless stated otherwise CVA cerebrovascular event, TIA transient ischemic attack, FVC forced vital capacity, SD standard deviation, TLco total gas transfer coefficient for carbon monoxide, NA not available Non-IPF ILD group included following: rheumatoid lung n = 2; nonspecific interstitial pneumonia n = 2; pneumoconiosis n = 2; hypersensitivity pneumonitis n = 1; desquamative interstitial pneumonia n = 1; Crohn’s ILD n = 1 * Statistical significance at a-level of 0.05
Platelet–Monocyte Adhesion in Peripheral Blood Platelet–monocyte adhesion was significantly increased in IPF compared with non-IPF ILD and non-ILD controls (IPF 35.6 ± 4.34 %; non-IPF ILD 23.5 ± 3.68 %; nonILD controls 16.5 ± 2.26 %; P \ 0.01: one-way ANOVA; Fig. 3). A post-hoc analysis (Bonferroni) showed that the difference between platelet–monocyte binding between IPF and non-ILD controls was statistically significant (P = 0.004). When we added EDTA to study divalent cation-independent platelet–monocyte binding, there was a trend towards increased binding in patients with IPF (IPF 6.0 ± 0.77 %; non-IPF ILD 4.3 ± 1.38 %; non-ILD
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Fig. 4 No significant correlation between platelet–monocyte binding and forced vital capacity (a) or total gas transfer for carbon monoxide (b)
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Fig. 5 a PSGL-1 expression on leukocyte subsets in the study groups. There was no significant difference in the expression of PSGL-1 between groups. b Leukocyte CD40L expression data reflect a nonsignificant trend towards higher expression on neutrophils and monocytes in non-IPF ILD
controls 3.1 ± 1.75 %; Fig. 3), but this did not reach statistical significance (P = 0.058; one-way ANOVA). Platelet–Monocyte Complexes and Lung Function Parameters We also evaluated platelet–monocyte binding in terms of lung function impairment in IPF. Our data show that there was no significant correlation between platelet–monocyte binding and FVC (P = 0.15, R2 = 0.11) or TLco (P = 0.876, R2 = 0.01; Fig. 4). These data suggest that platelet–monocyte adhesion is independent of disease severity in IPF. PSGL-1 and CD40L Expression by Different Populations of Leukocytes PSGL-1 is a key molecule involved in platelet-leukocyteendothelial interactions and is constitutively expressed by leukocytes [22]. We studied the expression of PSGL-1 on different subsets of leukocytes and platelets. As shown in
Fig. 6 a Platelet expression of activation markers including P-selectin (P = 0.467), PSGL-1 (P = 0.779), and CD40L (P = 0.875) showed no differential expression between the three study groups. b Plasma concentrations of soluble P-selectin and CD40L. There were no significant differences between study groups (P = 0.532 for P-selectin and P = 0.621 for CD40L)
Fig. 5a, there was no differential expression of PSGL-1 on any of the leukocyte subpopulations. CD40L is a type 2 membrane glycoprotein that belongs to TNF family of cytokines and is expressed by CD4? T cells [23, 24], B cells [25], dendritic cells [26], and platelets [27]. There is evidence of significantly increased concentrations of this glycoprotein in blood and BAL fluid of patients with pulmonary fibrosis [28]. In this study, the expression of CD40L on subsets of leukocytes or platelets was not significantly different in study groups. However, there was a trend towards higher surface expression of CD40L on monocytes and neutrophils in non-IPF ILD compared with IPF or non-ILD control subjects (Fig. 5b). Platelet Expression of P-selectin, PSGL-1, and CD40L Platelet expression of P-selectin, PSGL-1, and CD40L was similar in IPF patients and controls (Fig. 6a).
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Plasma Concentrations of Soluble P-selectin and CD40L As an extension of our analysis of cell surface protein expression in IPF and control subjects in the flow cytometry study, concentrations of soluble CD40L and P-selectin were measured in plasma. The plasma concentrations of P-selectin and CD40L in IPF and control subjects are shown in Fig. 6b. There was no significant difference in the plasma levels of these molecules in study groups.
Discussion In view of the fact that there is increased risk of acute coronary syndrome and deep vein thrombosis before and after the diagnosis of IPF [3], it is plausible that these patients have a prothrombotic state that predisposes them to microvascular injury in the lungs. Indeed, individuals homozygous for the procoagulant factor V R506Q Leiden have more severe dyspnea as well as reduced lung function with a restrictive ventilatory defect [29]. There is evidence of activation of the clotting cascade and increased expression of the thrombin receptor protease activated receptor-1 in pulmonary fibrosis [30–32]. Furthermore, elevated levels of factor VIII and antiphospholipid antibodies have been reported in IPF [33]. Bargagli et al. [34] described a procoagulant state in IPF with elevated levels of factor VIIIc, D-dimer, fibrinogen, and homocysteine. Our findings of increased platelet–monocyte adhesion support the hypothesis of a prothrombotic state in IPF. A number of studies [18, 19, 35] have investigated platelet–monocyte aggregation in peripheral blood. Sarma et al. [18] investigated a cohort of patients with unstable angina (n = 12), acute myocardial infarction (n = 13), and noncardiac chest pain (n = 27). There was evidence of a significantly increased platelet–monocyte aggregation in acute myocardial infarction (70.1 ± 15.4 %) compared with noncardiac chest pain (45.4 ± 23.3 %; P \ 0.01). Similarly, Michelson et al. [35] reported that platelet– monocyte binding is significantly higher in patients with acute myocardial infarction AMI (n = 9) (34.2 ± 10.3 % [mean ± SEM]) than patients with no AMI (n = 84, 19.3 ± 1.4 %, P \ 0.05) and normal control subjects (n = 10, 11.5 ± 0.8%, P \ 0.001). More recently, Maclay et al. [19] studied 18 patients with COPD and 16 controls matched for age and cigarette-smoke exposure. Platelet–monocyte aggregation values were significantly increased in COPD (mean ± SD; 25.3 ± 8.3 %) compared with control subjects (19.5 ± 4.0 %, P = 0.01). Furthermore, this aggregation increased during an acute exacerbation of COPD. The proportion of platelet–
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monocyte aggregates in our study was comparable to the studies by Michelson et al. [35] and Maclay et al. [19]. However, the binding was lower than found by Sarma et al. [18]. This difference is likely to be related to the methodology, diversity of patient characteristics, and diseases evaluated in these studies. We did not demonstrate differences in levels of specific plasma or cell-surface markers of platelet activation in IPF, suggesting that increased platelet–monocyte interactions were mediated by other molecular mechanisms. Platelets release a number of mediators with profibrotic activity [12, 36], and platelet–monocyte complexes could conceivably be an inciting mechanism of alveolar injury culminating in fibrosis in IPF. Increased activity of metalloproteinases and vascular endothelial growth factor [37] could permit closer proximity of activated platelets to the intra-alveolar compartment, which could potentially initiate or contribute to alveolar injury at the vascular endothelial/alveolar epithelial interface. There are some similarities between the cell biology of atherosclerosis and the pathological lesions in IPF. In atherosclerosis, there is an exuberant fibro-proliferative response in the arterial wall with recruitment of leukocytes, extracellular matrix deposition [38], and smooth muscle cell proliferation [39]. Similarly, IPF is characterized by release of profibrotic mediators and myofibroblast stimulation culminating in an excessive fibroproliferative reaction in the lung interstitium, and smooth muscle hypertrophy in the pulmonary vasculature. Recruitment of monocytes to sites of vascular damage is a key initial step in atherosclerosis [40]. Adhesion of platelet–monocyte complexes to the vessel wall enhances monocyte clustering and is mediated by P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on platelets and monocytes respectively [41]. We cannot exclude that the trend towards higher rates of smoking, hypertension, and use of aspirin in the IPF cohort increased the likelihood of vascular dysfunction. However, only a small number of patients with these cardiovascular risk factors were present in our cohort and we did not demonstrate statistically significant differences in these potential confounders between patients and controls. Patients with IPF were slightly older than control subjects, suggesting that the increased platelet–monocyte adhesion observed in this study may be related to older age. However, a subgroup analysis of control subjects after stratification into younger (age \ 70 years) and older age groups (age [ 70 years) indicated that platelet–monocyte binding was not affected by age (older group 19.8 ± 10.6 %, younger group 18.5 ± 7.0 %; P = 0.79 unpaired t test). Hence, it is unlikely that age per se would have had an effect on platelet–monocyte adhesion.
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Limitations We acknowledge certain limitations of this study. First, the sample size is small and subgroup analysis is unlikely to yield significantly meaningful results. Second, we did not measure coagulation factors, such as factor VIII, Protein C and S, fibrinogen, and D-dimer. We measured platelet– monocyte aggregation in a cross-sectional manner, and it would be interesting to determine whether changes over time are associated with disease progression. Our study cohort had higher percent predicted FVC than many reported studies of IPF, but studies of patients with milder disease are important. It would be interesting to perform further studies looking at serial lung function in relation to platelet–monocyte binding to study the relationship with the natural history of IPF.
Conclusions This study is the first-reported investigation of platelet– monocyte interactions in IPF. Platelet–monocyte aggregation may be a potentially important mechanism of increased cardiovascular risk secondary to platelet activation. It will be important to further evaluate the pathogenesis of IPF at a microvascular level to better understand its etiology and pathogenesis. If confirmed, platelet inhibition may be a plausible therapeutic target on the basis of increased platelet activation in IPF. Acknowledgments The authors thank Dr. L. Sadofsky, Mr. C. Crow, Dr. V. Green, Dr. L. Madden, and Mr. W. Sheedy for their assistance in conducting the experiments during this study. We also acknowledge Dr. V. Allgar for her help with the statistical analyses. The study was funded by the University of Hull. Conflict of interest
None declared for any of the authors.
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Increased Platelet Binding to Circulating Monocytes in Idiopathic Pulmonary Fibrosis Ahmed Fahim • Michael G. Crooks Alyn H. Morice • Simon P. Hart
•
Received: 11 August 2013 / Accepted: 14 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Purpose Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia and its prognosis is poor. Epidemiological evidence suggests an association of IPF with vascular disease and thrombotic tendency, which may be related to platelet activation. Methods Platelet–monocyte adhesion in peripheral blood was examined by flow cytometry in patients with IPF (n = 19), interstitial lung disease (ILD) other than IPF (n = 9), and control subjects without pulmonary fibrosis (n = 14). Expression of platelet activation markers P-selectin (CD62P), PSGL-1 (CD162), and CD40 ligand (CD40L) on leukocytes and platelets were studied. Plasma concentrations of soluble P-selectin and CD40L were measured by ELISA. Results Significantly elevated levels of platelet–monocyte binding were found in patients with IPF (35.6 ± 4.34 % [mean ± SEM]) compared with patients with non-IPF ILD (23.5 ± 3.68 %) and non-ILD control subjects (16.5 ± 2.26 %; P \ 0.01). There was a trend towards increased divalent cation-independent platelet– monocyte binding in IPF (6.0 ± 0.77 % [mean ± SEM]) compared with non-IPF ILD (4.3 ± 1.38 %) and control subjects without ILD (3.1 ± 1.75 %; P = 0.058). There A. Fahim (&) M. G. Crooks A. H. Morice S. P. Hart Division of Cardiovascular and Respiratory Studies, Castle Hill Hospital, Castle Road, Cottingham HU16 5JQ, UK e-mail: [email protected] M. G. Crooks e-mail: [email protected] A. H. Morice e-mail: [email protected] S. P. Hart e-mail: [email protected]
was no differential surface expression of platelet activation markers on subsets of leukocytes or platelets. Plasma concentrations of CD40L and soluble P-selectin did not differ between IPF and control subjects. Platelet–monocyte binding had no significant correlation with percent predicted TLco or FVC. Conclusions Platelet–monocyte binding is increased in IPF, suggesting increased platelet activation. This conjugation is predominantly calcium-dependent, but there may be more calcium-independent adhesion in IPF. These findings support further research into the role of platelet activation in IPF. Keywords Idiopathic pulmonary fibrosis Interstitial lung disease Monocytes Platelets Platelet–monocyte complexes Abbreviations CD40L CD40 ligand FACS Fluorescent activated cell sorting IPF Idiopathic pulmonary fibrosis ILD Interstitial lung disease mAb Monoclonal antibodies PMN Polymorphonuclear leukocytes
Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease (ILD) associated with an insidious onset of dyspnoea, bi-basal inspiratory crackles, a restrictive ventilatory defect, and impaired gas exchange [1]. Radiologically, it is characterized by peripheral and basal reticular
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abnormalities with minimal ground glass change on highresolution computed tomography (HRCT) scans. Areas of fibrosis are interspersed with normal lung parenchyma in lung biopsy specimens consistent with geographical heterogeneity. IPF is the commonest of the idiopathic interstitial pneumonias (IIP) with a median survival of 2.5–3 years after diagnosis [2]. The cause is unknown, and there is no effective medical treatment. Progress in understanding the pathogenesis and potential treatment of IPF will depend on identifying the events that trigger and drive the disease. Epidemiological and autopsy studies have demonstrated a significant association of IPF with cardiovascular disease [3, 4]. Vascular remodeling in the form of luminal narrowing and progressive regression of vascularization has been observed in IPF [5–7]. Platelets are attractive candidates for linking vascular dysfunction with tissue fibrosis [8]. Mesenchymal growth factors, including platelet-derived growth factor (PDGF), insulinlike growth factor, and transforming growth factor-b (TGF-b) are upregulated in IPF [9–11]. PDGF along with other growth factors, including connective tissue growth factor (CTGF) and TGF-b, play a significant role in fibrogenesis as they are mitogens for fibroblasts [12, 13], the key effector cells in IPF. Platelets are activated following endothelial injury with upregulation of cell-surface receptors, such as CD40 ligand (CD40L) and P-selectin. The increased expression of these surface molecules results in platelet adhesion to the vascular wall. Activated platelets release certain chemokines with recruitment of inflammatory cells and formation of platelet– monocyte complexes [14]. Platelet adhesion to monocytes and polymorphonuclear leukocytes (PMN) may alter leukocyte activation and recruitment patterns [15–17]. Increased binding of platelets to circulating monocytes in peripheral blood of patients with myocardial infarction and unstable angina has been demonstrated [18]. Moreover, this increased adhesion has been linked to cardiovascular risk in patients with chronic obstructive pulmonary disease (COPD) [19]. We hypothesized that increased platelet activation in IPF would be associated with formation of circulating platelet–monocyte complexes. In the present study, we used flow cytometry to examine platelet–monocyte binding and markers of platelet activation in peripheral blood.
Methods Patient Selection and Blood Sampling Patients with IPF diagnosed according to ATS/ERS criteria were recruited from the interstitial lung disease clinic at our University Hospital [20]. At the time of recruitment,
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patients were in a clinically stable state and were excluded if there was evidence of acute infection, acute coronary syndrome, or acute exacerbation of IPF. The control group comprised patients with non-IPF ILD, including hypersensitivity pneumonitis, collagen vascular disease-associated ILD, and pneumoconiosis. All the patients in non-IPF ILD group were diagnosed following a multidisciplinary meeting with a respiratory clinician, thoracic radiologist, and histopathologist (in biopsied cases). The non-ILD control group comprised predominantly healthy volunteers and patients with COPD who had no evidence of interstitial lung disease. Patients’ age, sex, history of hypertension, ischemic heart disease, cerebrovascular disease, diabetes, smoking status, occupational dust exposure, and medication use were recorded. Blood was drawn by venipuncture via 19-gauge needles and collected into 6-mL tubes containing 102 IU of lithium heparin (BD Ltd, Plymouth, UK), which were gently inverted several times to avoid platelet activation. Because the choice of anticoagulant can have a significant effect on the extent of platelet monocyte adhesion and there is evidence of significantly reduced platelet monocyte binding with sodium citrate compared with heparin or hirudin, we used heparin as the anticoagulant for this investigation [21]. The study was approved by Hull and East Riding Ethics Committee (LREC No. 08/H1304/54), and written consent was obtained from all the participants before recruitment into the study. Antibodies, Immunolabeling, and Flow Cytometry Monoclonal antibodies (mAb) directly conjugated to fluorochromes were purchased from the following sources: FITC-conjugated CD42a and PE-conjugated CD14 from Serotec Ltd (Oxford, UK); PE-conjugated CD62P mAb AK-4, CD162 mAb KPL-1, CD40L mAb MR-1 and control IgG1 from BioLegend (San Diego, CA, USA). Blood (50 lL) was labeled with specific antibodies for 20 min at room temperature. CD42a-FITC and CD14-PE mAb were used at final concentrations of 2 lg/mL. PE-conjugated CD62P, CD162, CD40L, and control mAb had been optimized for flow cytometry by the manufacturer and were used at recommended dilutions. 300 lL of FACSLyse solution (Becton Dickinson, San Jose, CA, USA) was added after mAb incubation. Blood was labeled within 30 min and samples were analysed using a Becton Dickinson FACS Calibur flow cytometer, and data analyses were performed using CellQuest Pro (Becton Dickinson) software. Monocytes and neutrophils were distinguished by their distinct laser scatter properties (Fig. 1a, b). CD14 was used to identify monocytes. Platelets were identified using CD42a. Platelet–monocyte adhesion was determined by two-color flow cytometry using directly conjugated
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Fig. 1 Flow cytometric identification of monocytes and neutrophils. a Monocytes identified by distinct forward and side scatter properties (shown in grey). FSC forward scatter on x-axis; SSC side scatter on y-axis. b Neutrophils (shown in grey) with higher side scatter than monocytes
CD14-PE and CD42a-FITC mAb. The percentage of monocytes (CD14?) binding one or more platelets (CD42a?) was assessed by labeling blood with both CD42a and CD14 mAb with and without the addition of EDTA (Fig. 2a, b). Plasma concentrations of P-selectin and CD40L were measured in duplicate by ELISA according to the manufacturers’ protocols (Bender MedSystems, Vienna, Austria). Statistical Analysis Statistical analyses were performed using SPSS 13, Chicago, IL, USA). The Kolmogorov and method was used to assess the data distribution. isons between patient groups were made by
(Version Smirnov Comparone-way
Fig. 2 Flow cytometric identification of platelet–monocyte aggregates. a Platelet–monocyte complexes in the upper right quadrant (FL1 = CD42a; FL2 = CD14). In this example, 18.5 % of monocytes exhibit platelet binding. b Significant reduction in platelet– monocyte adhesion with the addition of EDTA to chelate calcium
ANOVA for normally distributed data. Data with skewed distributions were analyzed by nonparametric tests (Mann– Whitney U test and Kruskal–Wallis test). Comparisons of gender, smoking, and comorbidities were performed using chi-square tests. Correlations were analyzed by Pearson correlation. The level of statistical significance was set at 0.05. Results are presented as mean ± standard error of the mean (SEM).
Results The baseline characteristics and demographics of the study populations are shown in Table 1. IPF patients were slightly older than the control subjects. The only other significant difference between the study groups was the peripheral blood monocyte count, which was higher in nonILD control subjects. The other baseline parameters were similar between the groups.
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Non-IPF ILD
Non-ILD controls
Subjects
19
9
14
Age (years)
69 ± 7
58 ± 9
56 ± 21
Gender Male
P value
0.005* 0.064
15
6
4
3
8
Platelet count
246 ± 67
248 ± 72
265 ± 91
0.882
Monocyte count
0.73 ± 0.14
0.59 ± 0.2
0.95 ± 0.17
0.002*
White cell count
7.85 ± 2.2
7.43 ± 2.48
9.36 ± 0.7
0.290
1
1
0
Female
6
Smoking Current
0.052
Ex
16
5
6
Never smokers
2
3
8
Hypertension
8 (42)
1 (11)
3 (22)
0.285
Diabetes
1 (5)
1 (11)
4 (29)
0.168
IHD
1 (5)
0
1 (11)
0.642
CVA/TIA
2 (10)
1 (11)
0
0.168
Aspirin FVC % predicted
6 (32) 89 ± 18 (n = 17)
1 (11) 86 ± 11 (n = 7)
2 (14) NA
0.323 0.482
TLco % predicted
57 ± 19 (n = 17)
56 ± 15 (n = 7)
NA
0.356
Fig. 3 Platelet–monocyte complexes in the study groups showing a significantly higher proportion of complex formation in IPF compared with non-IPF ILD and non-ILD subjects (dark bars). There was a dramatic reduction in platelet–monocyte adhesion with the addition of EDTA (light grey bars), indicating the predominantly divalent cationdependent nature of platelet–monocyte adhesion
Data are presented as mean ± SD or numbers with percentages in parentheses unless stated otherwise CVA cerebrovascular event, TIA transient ischemic attack, FVC forced vital capacity, SD standard deviation, TLco total gas transfer coefficient for carbon monoxide, NA not available Non-IPF ILD group included following: rheumatoid lung n = 2; nonspecific interstitial pneumonia n = 2; pneumoconiosis n = 2; hypersensitivity pneumonitis n = 1; desquamative interstitial pneumonia n = 1; Crohn’s ILD n = 1 * Statistical significance at a-level of 0.05
Platelet–Monocyte Adhesion in Peripheral Blood Platelet–monocyte adhesion was significantly increased in IPF compared with non-IPF ILD and non-ILD controls (IPF 35.6 ± 4.34 %; non-IPF ILD 23.5 ± 3.68 %; nonILD controls 16.5 ± 2.26 %; P \ 0.01: one-way ANOVA; Fig. 3). A post-hoc analysis (Bonferroni) showed that the difference between platelet–monocyte binding between IPF and non-ILD controls was statistically significant (P = 0.004). When we added EDTA to study divalent cation-independent platelet–monocyte binding, there was a trend towards increased binding in patients with IPF (IPF 6.0 ± 0.77 %; non-IPF ILD 4.3 ± 1.38 %; non-ILD
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Fig. 4 No significant correlation between platelet–monocyte binding and forced vital capacity (a) or total gas transfer for carbon monoxide (b)
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Fig. 5 a PSGL-1 expression on leukocyte subsets in the study groups. There was no significant difference in the expression of PSGL-1 between groups. b Leukocyte CD40L expression data reflect a nonsignificant trend towards higher expression on neutrophils and monocytes in non-IPF ILD
controls 3.1 ± 1.75 %; Fig. 3), but this did not reach statistical significance (P = 0.058; one-way ANOVA). Platelet–Monocyte Complexes and Lung Function Parameters We also evaluated platelet–monocyte binding in terms of lung function impairment in IPF. Our data show that there was no significant correlation between platelet–monocyte binding and FVC (P = 0.15, R2 = 0.11) or TLco (P = 0.876, R2 = 0.01; Fig. 4). These data suggest that platelet–monocyte adhesion is independent of disease severity in IPF. PSGL-1 and CD40L Expression by Different Populations of Leukocytes PSGL-1 is a key molecule involved in platelet-leukocyteendothelial interactions and is constitutively expressed by leukocytes [22]. We studied the expression of PSGL-1 on different subsets of leukocytes and platelets. As shown in
Fig. 6 a Platelet expression of activation markers including P-selectin (P = 0.467), PSGL-1 (P = 0.779), and CD40L (P = 0.875) showed no differential expression between the three study groups. b Plasma concentrations of soluble P-selectin and CD40L. There were no significant differences between study groups (P = 0.532 for P-selectin and P = 0.621 for CD40L)
Fig. 5a, there was no differential expression of PSGL-1 on any of the leukocyte subpopulations. CD40L is a type 2 membrane glycoprotein that belongs to TNF family of cytokines and is expressed by CD4? T cells [23, 24], B cells [25], dendritic cells [26], and platelets [27]. There is evidence of significantly increased concentrations of this glycoprotein in blood and BAL fluid of patients with pulmonary fibrosis [28]. In this study, the expression of CD40L on subsets of leukocytes or platelets was not significantly different in study groups. However, there was a trend towards higher surface expression of CD40L on monocytes and neutrophils in non-IPF ILD compared with IPF or non-ILD control subjects (Fig. 5b). Platelet Expression of P-selectin, PSGL-1, and CD40L Platelet expression of P-selectin, PSGL-1, and CD40L was similar in IPF patients and controls (Fig. 6a).
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Plasma Concentrations of Soluble P-selectin and CD40L As an extension of our analysis of cell surface protein expression in IPF and control subjects in the flow cytometry study, concentrations of soluble CD40L and P-selectin were measured in plasma. The plasma concentrations of P-selectin and CD40L in IPF and control subjects are shown in Fig. 6b. There was no significant difference in the plasma levels of these molecules in study groups.
Discussion In view of the fact that there is increased risk of acute coronary syndrome and deep vein thrombosis before and after the diagnosis of IPF [3], it is plausible that these patients have a prothrombotic state that predisposes them to microvascular injury in the lungs. Indeed, individuals homozygous for the procoagulant factor V R506Q Leiden have more severe dyspnea as well as reduced lung function with a restrictive ventilatory defect [29]. There is evidence of activation of the clotting cascade and increased expression of the thrombin receptor protease activated receptor-1 in pulmonary fibrosis [30–32]. Furthermore, elevated levels of factor VIII and antiphospholipid antibodies have been reported in IPF [33]. Bargagli et al. [34] described a procoagulant state in IPF with elevated levels of factor VIIIc, D-dimer, fibrinogen, and homocysteine. Our findings of increased platelet–monocyte adhesion support the hypothesis of a prothrombotic state in IPF. A number of studies [18, 19, 35] have investigated platelet–monocyte aggregation in peripheral blood. Sarma et al. [18] investigated a cohort of patients with unstable angina (n = 12), acute myocardial infarction (n = 13), and noncardiac chest pain (n = 27). There was evidence of a significantly increased platelet–monocyte aggregation in acute myocardial infarction (70.1 ± 15.4 %) compared with noncardiac chest pain (45.4 ± 23.3 %; P \ 0.01). Similarly, Michelson et al. [35] reported that platelet– monocyte binding is significantly higher in patients with acute myocardial infarction AMI (n = 9) (34.2 ± 10.3 % [mean ± SEM]) than patients with no AMI (n = 84, 19.3 ± 1.4 %, P \ 0.05) and normal control subjects (n = 10, 11.5 ± 0.8%, P \ 0.001). More recently, Maclay et al. [19] studied 18 patients with COPD and 16 controls matched for age and cigarette-smoke exposure. Platelet–monocyte aggregation values were significantly increased in COPD (mean ± SD; 25.3 ± 8.3 %) compared with control subjects (19.5 ± 4.0 %, P = 0.01). Furthermore, this aggregation increased during an acute exacerbation of COPD. The proportion of platelet–
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monocyte aggregates in our study was comparable to the studies by Michelson et al. [35] and Maclay et al. [19]. However, the binding was lower than found by Sarma et al. [18]. This difference is likely to be related to the methodology, diversity of patient characteristics, and diseases evaluated in these studies. We did not demonstrate differences in levels of specific plasma or cell-surface markers of platelet activation in IPF, suggesting that increased platelet–monocyte interactions were mediated by other molecular mechanisms. Platelets release a number of mediators with profibrotic activity [12, 36], and platelet–monocyte complexes could conceivably be an inciting mechanism of alveolar injury culminating in fibrosis in IPF. Increased activity of metalloproteinases and vascular endothelial growth factor [37] could permit closer proximity of activated platelets to the intra-alveolar compartment, which could potentially initiate or contribute to alveolar injury at the vascular endothelial/alveolar epithelial interface. There are some similarities between the cell biology of atherosclerosis and the pathological lesions in IPF. In atherosclerosis, there is an exuberant fibro-proliferative response in the arterial wall with recruitment of leukocytes, extracellular matrix deposition [38], and smooth muscle cell proliferation [39]. Similarly, IPF is characterized by release of profibrotic mediators and myofibroblast stimulation culminating in an excessive fibroproliferative reaction in the lung interstitium, and smooth muscle hypertrophy in the pulmonary vasculature. Recruitment of monocytes to sites of vascular damage is a key initial step in atherosclerosis [40]. Adhesion of platelet–monocyte complexes to the vessel wall enhances monocyte clustering and is mediated by P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on platelets and monocytes respectively [41]. We cannot exclude that the trend towards higher rates of smoking, hypertension, and use of aspirin in the IPF cohort increased the likelihood of vascular dysfunction. However, only a small number of patients with these cardiovascular risk factors were present in our cohort and we did not demonstrate statistically significant differences in these potential confounders between patients and controls. Patients with IPF were slightly older than control subjects, suggesting that the increased platelet–monocyte adhesion observed in this study may be related to older age. However, a subgroup analysis of control subjects after stratification into younger (age \ 70 years) and older age groups (age [ 70 years) indicated that platelet–monocyte binding was not affected by age (older group 19.8 ± 10.6 %, younger group 18.5 ± 7.0 %; P = 0.79 unpaired t test). Hence, it is unlikely that age per se would have had an effect on platelet–monocyte adhesion.
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Limitations We acknowledge certain limitations of this study. First, the sample size is small and subgroup analysis is unlikely to yield significantly meaningful results. Second, we did not measure coagulation factors, such as factor VIII, Protein C and S, fibrinogen, and D-dimer. We measured platelet– monocyte aggregation in a cross-sectional manner, and it would be interesting to determine whether changes over time are associated with disease progression. Our study cohort had higher percent predicted FVC than many reported studies of IPF, but studies of patients with milder disease are important. It would be interesting to perform further studies looking at serial lung function in relation to platelet–monocyte binding to study the relationship with the natural history of IPF.
Conclusions This study is the first-reported investigation of platelet– monocyte interactions in IPF. Platelet–monocyte aggregation may be a potentially important mechanism of increased cardiovascular risk secondary to platelet activation. It will be important to further evaluate the pathogenesis of IPF at a microvascular level to better understand its etiology and pathogenesis. If confirmed, platelet inhibition may be a plausible therapeutic target on the basis of increased platelet activation in IPF. Acknowledgments The authors thank Dr. L. Sadofsky, Mr. C. Crow, Dr. V. Green, Dr. L. Madden, and Mr. W. Sheedy for their assistance in conducting the experiments during this study. We also acknowledge Dr. V. Allgar for her help with the statistical analyses. The study was funded by the University of Hull. Conflict of interest
None declared for any of the authors.
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