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Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, JapanDepartment of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan
Serum neutrophil gelatinase-associated lipocalin (NGAL) was measured with ELISA.
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Sarcoidosis patients had significantly higher levels of NGAL than healthy subjects.
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Elevated NGAL levels at diagnosis were associated with subsequent steroid therapy.
Abstract
Background
Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein that is involved in the innate immune system and increased expression has been detected in diverse diseases. Sarcoidosis is a systemic granulomatous disorder and its clinical course and prognosis are changeable and highly divergent. This study aimed to examine the expression of NGAL in patients with sarcoidosis. In addition, we examined whether NGAL could serve as a marker of disease activity and prognosis.
Methods
Ninety-six sarcoidosis patients were studied. Serum samples collected at the time of diagnosis were examined for NGAL by cellular enzyme-linked immunosorbent assay. The level of NGAL was compared with clinical, radiological and laboratory data.
Results
Patients with sarcoidosis had significantly higher levels of NGAL (the median [interquartile range] was 35.1 ng/mL [23.5–60.8] in sarcoidosis patients versus 17.2 ng/mL [13.0–27.0] in the reference population, p < .0001). NGAL levels were not correlated with markers for disease activity. During the follow-up period, 26 patients (27.1%) deteriorated and received systemic corticosteroid therapy for organ dysfunction. In those patients, NGAL levels were significantly higher than in those who did not receive corticosteroid therapy (56.5 ng/mL [27.3–92.3] versus 34.3 ng/mL [23.0–53.0], p = .0201). Upon multivariate logistic regression analysis, elevated NGAL levels at diagnosis were associated with subsequent use of systemic corticosteroid therapy (hazard ratio, 1.20; 95% confidence interval, 1.09–1.31; p = .0004).
Conclusion
NGAL may be a useful marker to predict the disease course of sarcoidosis.
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
]. Although the exact disease etiology is unknown, currently, sarcoidosis is understood to be the consequence of a chronic immune response associated with genetic predisposition and unidentified antigens [
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
]. Immunologically, it is assumed that specific environmental factors like transmissible infectious origins are antigens and the pathogen-associated molecular patterns of the antigen can trigger or amplify inflammation in sarcoidosis [
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
Usefulness of plasma neutrophil gelatinase-associated lipocalin concentration for predicting the severity and mortality of patients with community-acquired pneumonia.
Usefulness of plasma neutrophil gelatinase-associated lipocalin concentration for predicting the severity and mortality of patients with community-acquired pneumonia.
Usefulness of plasma neutrophil gelatinase-associated lipocalin concentration for predicting the severity and mortality of patients with community-acquired pneumonia.
]. In 402 patients with chronic obstructive pulmonary disease, plasma levels of NGAL were elevated and the number of exacerbations was associated with higher NGAL expression [
]. The levels of NGAL in patients with community-acquired pneumonia presenting to the emergency department increased proportionally with the severity of pneumonia. Based on its relationship with cell growth, differentiation and apoptosis [
In this study, we aimed to examine the expression of NGAL in patients with sarcoidosis. In addition, we investigated the usefulness of NGAL as a marker of disease activity and prognosis.
2. Methods
2.1 Patient eligibility
A total of 96 consecutive patients who were diagnosed as having sarcoidosis and were observed for at least 2 years were included in this study (Table 1). They were ethnically homogeneous, Japanese patients. The diagnosis of sarcoidosis was based on the American Thoracic Society, the European Respiratory Society, and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) consensus statement [
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
]. This study was conducted in accordance with the amended Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of Hamamatsu University School of Medicine (Hamamatsu, Japan, approval number: 15-165). The study was registered with the University Hospital Medical Information Network Clinical Trial Registry (UMIN ID 000028108).
Lungs include pulmonary hilar and mediastinal lymphadenopathy; others include muscle, liver, kidney, hypercalcemia, parotid gland, and extramediastinal lymphadenopathy.
Lungs
96
Eyes
49
Skin
15
Others
51
Duration of follow-up, years
7.7 (4.8–13.0)
Smoker
39 (40.6)
Blood % neutrophil, %
64.7 (59.5–70.9)
Serum ACE, IU/L
18.5 (14.1–23.1)
Pulmonary function tests
FVC, % predicted
95.2 (84.4–107.4)
FEV1/FVC ratio, %
79.4 (74.4–84.3)
Radiologic stage 0/I/II/III
4/44/36/12
Bronchoalveolar lavage
Total cells, ×105/ml
1.40 (0.86–2.50)
Lymphocytes, %
9.6 (6.2–19.4)
Neutrophils, %
0.4 (0–1.0)
CD4/CD8 ratio
4.8 (2.6–8.5)
Data are expressed as number (%) or median (interquartile range) unless otherwise indicated.
ACE, angiotensin converting enzyme; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s. Radiographic stages 0, I, II, and III, represent a normal appearance, bilateral hilar lymphadenopathy (BHL) alone, BHL and lung parenchymal involvement, and lung parenchymal involvement without BHL, respectively.
a Lungs include pulmonary hilar and mediastinal lymphadenopathy; others include muscle, liver, kidney, hypercalcemia, parotid gland, and extramediastinal lymphadenopathy.
Clinical, radiological and laboratory data, including organs affected, pulmonary function, treatment, prognosis, bronchoalveolar lavage (BAL) and pathological information, were retrospectively obtained from medical records. Pulmonary function tests and laboratory parameters were assessed at the time of diagnosis.
2.3 ELISA for the detection of NGAL
Serum samples were collected at the time of diagnosis, when no patient was under systemic corticosteroid or immunosuppressive therapy, and stored at −30 °C until analysis. The enzyme-linked immunosorbent assay (ELISA) to detect serum NGAL was performed according to manufacturer's instructions (CircuLex Human NGAL kit; MBL, Nagoya, Japan). Forty-nine age- and gender-matched healthy subjects without any clinical, radiological, or serological evidence of infection, cardiovascular and renal disease, chronic obstructive pulmonary disease, tumor, sarcoidosis, or autoimmune disorders served as controls, which was approved by the ethical committee of Seirei Center for Health Promotion and Preventive Medicine (Hamamatsu, Japan, approval number; 26-05), and each subject directly provided informed consent.
2.4 Statistical analysis
The Wilcoxon signed rank test was used for continuous variables. Correlations between different parameters were undertaken using Spearman's rank correlation coefficient. Cox proportional hazard regression analysis was used to identify variables associated with the use of systemic corticosteroid therapy. p values of less than 0.05 were considered significant. Data are expressed as the median (interquartile range) unless indicated otherwise. All values were analyzed using JMP version 9.0.0 (SAS Institute Japan, Tokyo, Japan).
3. Results
3.1 Patient characteristics
Ninety-six patients with sarcoidosis were included in this study (Table 1). The median age at diagnosis was 55 years (range; 23–79 years) and 58 patients (60.4%) were female. Patients with sarcoid lesions in the lungs were divided into four groups based on the findings of chest radiography: four patients had a normal radiograph, 44 patients had bilateral hilar lymphadenopathy (BHL) alone, 36 patients had BHL and lung parenchymal involvement, and 12 patients had lung parenchymal involvement alone. The common extrapulmonary lesions were ocular and skin lesions, found in 51.0% and 15.6% of the patients, respectively. Of the 27 patients who had involvement of more than three organs, 85.2% had lung parenchymal lesions. About half of the patients had symptoms, three-fourths of which were ophthalmological manifestations. Serum angiotensin converting enzyme (ACE) levels were elevated above the upper normal range in 32 patients. In most patients, forced vital capacity (FVC), FVC% predicted, and forced expiratory volume in one second (FEV1)/FVC ratio were within the normal range.
3.2 Expression of NGAL and correlation with findings in sarcoidosis
There were no significant correlation of NGAL levels with sex (p = .11), age (r = 0.11, p = .31), absolute neutrophil counts (r = 0.15, p = .16), hemoglobin (r = 0.02, p = .84) or mean corpuscular volume (r = 0.002, p = .98), estimate glomerular filtration rates (r = −0.16, p = .83), body mass index (r = −0.03, p = .79), or smoking pack-year histories (r = −0.06, p = .57). The median NGAL was 35.1 ng/mL (23.5–60.8) in sarcoidosis patients, which was significantly higher than in the reference population (17.2 ng/mL, [13.0–27.0], Fig. 1; p < .0001). The levels of NGAL did not differ among the groups defined by chest radiographic findings. When we divided patients into two groups according to the number of organs involved: those with two or fewer affected organs and those with more than three organs affected, there was no difference in NGAL levels (35.0 ng/mL versus 45.0 ng/mL, p = .13). NGAL levels were not correlated with markers for disease activity, such as serum ACE levels (r = 0.10, p = .35), the percentage of alveolar lymphocytes (r = 0.14, p = .17) and the CD4/CD8 ratio in BAL (r = 0.02, p = .84).
Fig. 1Expression of serum NGAL in patients with sarcoidosis and the reference population. NGAL levels were significantly higher in sarcoidosis patients than in the reference population (*p < .0001). Horizontal lines, boxes, and error bars, represent the median, the 25th and 75th percentiles, and the 10th and 90th percentiles, respectively. Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin.
At the time of diagnosis, none of the patients had serious organ damage that needed systemic corticosteroid therapy. During the observation period (a median of 7.7 years), 26 patients deteriorated and received systemic corticosteroid therapy (median dose of 30 mg prednisolone per day, 0.5 mg/kg of body weight) for organ dysfunction; 16 patients for deterioration of pre-existing pulmonary disease with progressive loss of lung function, two for visual impairment due to deterioration of pre-existing ocular lesions, six for the appearance of cardiac sarcoidosis, and two for hypercalcemia and renal insufficiency. The NGAL levels in the 26 patients where the disease deteriorated and received systemic corticosteroid therapy during the follow-up period were significantly higher than in patients whose disease resolved spontaneously and did not require systemic corticosteroid therapy (56.5 ng/mL [27.3–92.3] versus 34.3 ng/mL [23.0–53.0], p = .0201, Fig. 2).
Fig. 2Serum NGAL levels in patients with sarcoidosis according to the use of systemic corticosteroid during follow-up period. When sarcoidosis patients were divided into two groups―those who received systemic corticosteroid therapy during follow-up period (Corticosteroid) and those who did not (No corticosteroid)―NGAL levels were higher in patients who received corticosteroid therapy (*p = .0201). Horizontal lines, boxes, and error bars represent the median, the 25th and 75th percentiles, and the 10th and 90th percentiles, respectively. Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin.
3.3 Univariate and multivariate logistic regression analyses for corticosteroid therapy
We evaluated the predictive factors associated with disease deterioration that needed systemic corticosteroid therapy (Table 2). In univariate logistic regression analysis, the number of affected organs, serum ACE level, reduced %FVC and serum NGAL level at the time of diagnosis were selected as candidate factors. Multivariate logistic regression analysis showed reduced %FVC and serum NGAL level at diagnosis were significantly associated with the use of systemic corticosteroids after adjusting for serum ACE and number of affected organs at baseline.
Table 2Logistic regression analyses for corticosteroid therapy.
Variables
Univariate
Multivariate
Hazard ratio
p-value
Hazard ratio
p-value
Age
0.99 (0.97–1.02)
.4388
Sex: male
2.17 (0.99–4.76)
.0501
Smoking habit: ever
1.11 (0.49–2.40)
.8008
Number of organs involved
1.62 (1.26–2.01)
.0005
1.15 (0.80–1.56)
.4405
Serum ACE
1.87 (1.26–2.65)
.0030
1.50 (0.90–2.36)
.1157
FVC %predicted
1.45 (1.20–1.75)
.0002
1.44 (1.17–1.80)
.0006
Serum NGAL
1.12 (1.04–1.19)
.0066
1.20 (1.09–1.31)
.0004
Data are expressed as mean (95% confidence interval). ACE, angiotensin converting enzyme; FVC, forced vital capacity; NGAL, neutrophil gelatinase-associated lipocalin.
In this study, the serum level of NGAL was elevated in patients with sarcoidosis. There was no significant relationship between the NGAL levels and clinical and radiological data and markers for disease activity in sarcoidosis. On the other hand, patients with sarcoidosis who received systemic corticosteroid therapy during the follow-up period had significantly higher NGAL expression at the time of diagnosis.
NGAL levels are considered to be as a useful biomarker in various diseases including autoimmune diseases. Chen et al. assessed the serum NGAL level in patients with anti-neutrophil cytoplasmic autoantibody-associated vasculitis [
]. The levels of serum NGAL was significantly higher in the active stage of disease compared with those in the remission stage, which suggested that serum NGAL is valuable to assess disease activity and identify a relapse of vasculitis. In sarcoidosis, there is still no general consensus regarding disease activity, and clinical symptoms, pulmonary function, chest radiographic findings, serum ACE level, BAL cell populations, and the ratio of CD4/CD8 in BAL are all used to assess disease activity [
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
]. In this study, serum NGAL level had no significant relationship with any of these parameters.
Clinical course and prognosis are variable in sarcoidosis, and the prediction of clinical course remains challenging. Currently, there is no accepted predictive factor in sarcoidosis [
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
]. Mode of onset, extent of disease, chronicity, serial functional changes and the severity of disease in involved organs are important predictors of the disease prognosis and clinical course [
]. In this study, we divided sarcoidosis patients into two groups, those who received systemic corticosteroid therapy during the follow-up period and those who did not receive corticosteroid therapy. Twenty-six patients received corticosteroid therapy during the follow-up period because they had exacerbated disease or developed new organ involvement, and those patients showed significantly elevated NGAL levels at the time of diagnosis than those who did not. When the clinical outcome status (COS) proposed by WASOG committee was used [
], persistent patients had higher NGAL levels than those with resolved or minimal disease five years after the initial diagnosis (43.5 ng/mL versus 31.6 ng/mL, p = .025). In the multivariate logistic regression analysis, the elevated NGAL at the time of diagnosis was an independent variable associated with systemic corticosteroid therapy. Thus, NGAL might be a useful marker to predict clinical course and prognosis in sarcoidosis.
The pathogenic implications of elevated NGAL expression in sarcoidosis remain unclear. As a function of NGAL in immunity, La Manna et al. showed NGAL upregulates human leukocyte antigen G expression in CD4+ T lymphocytes and amplifies T-regulatory cell anti-inflammatory and immune tolerance induction [
]. Using an immune complex-mediated inflammatory disorder model, Shashidharamurthy et al. showed that upregulated NGAL may regulate the migration of inflammatory cells to the site of inflammation and the resolution of inflammatory processes [
]. These studies showed that NGAL has immunomodulatory functions. On the other hand, NGAL may work as a microbicidal effector of the inflammatory response because NGAL is expressed in tissues and cells exposed to microorganisms and its synthesis is induced during inflammation [
]. To assess the role of NGAL, they generated NGAL-deficient mice. When infected intraperitoneally with Escherichia coli (E coli), the bacterial burden in blood, liver and spleen were increased and the administration of NGAL inhibited the growth of E. coli in a dose-dependent manner. Mori et al. demonstrated that the administration of recombinant NGAL protein inhibited the overgrowth of E. coli in gut tissues [
]. On the other hand, NGAL-deficient mice showed no difference in survival when intraperitoneally-infected with Staphylococcus aureus. NGAL confers a bacteriostatic effect by abrogating siderophore-dependent bacterial iron uptake, which was specific for iron-laden mechanisms. Currently, sarcoidosis is understood as a consequence of a chronic immune response to unidentified antigenic factors such as transmissible infectious origins in genetically susceptible individuals [
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
]. Otherwise, from the clinical and pathological similarities and the same predominance of Th1-type immunity, mycobacterial infection, particularly M. tuberculosis, has continued to be a candidate for an infectious cause of sarcoidosis. Guglani et al. showed that NGAL mRNA is induced in the lung parenchyma and epithelium in response to M. tuberculosis exposure [
]. They speculated that NGAL functions by regulating lymphocytic inflammation and promoting neutrophil accumulation, which in part participates in granuloma formation, during mycobacterial infection. If exposure or infection with mycobacteria is related to the pathogenic mechanism in sarcoidosis, NGAL might be induced in response to mycobacterial infection and function to inhibit the growth of mycobacteria by the innate immune system. There is currently no study that investigates the relationship between Propionebacterium infection and NGAL expression. The investigations of NGAL in sarcoidosis might be an additional process to identify specific environmental factors that are associated with the pathogenic mechanism of sarcoidosis.
There are several limitations in this study. First, this was a single-institution, uncontrolled, retrospective study. Second, the level of NGAL in BAL fluid was not investigated. Although the expression of NGAL has rarely been investigated in body fluids other than blood and urine [
], the level in BAL fluid might reflect pathology, particularly pulmonary sarcoidosis, more clearly. Third, all included patients in this study were Japanese. Racial factors can influence the clinical course of sarcoidosis [
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
]. Fourth, there is the possibility that other infectious or inflammatory conditions may have been present at the time of diagnosis, although we carefully excluded patients with other diseases and complications.
In conclusion, serum NGAL levels were significantly elevated in sarcoidosis. Patients who received systemic corticosteroids during the follow-up period had elevated levels of NGAL at the time of initial diagnosis. The measurement of NGAL might help in the management of sarcoidosis upon integration into the currently available clinical care of the patient. Further studies with a much larger series of patients of various races and from different areas and in comparison with other interstitial lung diseases or infections will be needed to confirm this.
Conflicts of interest
All authors declare no actual or potential conflicts of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Acknowledgments
The authors wish to thank S Kita and M Hashiguchi, Medical & Biological Laboratories Co., Ltd., Japan, for technical assistance with the experiments.
Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee.
Am. J. Respir. Crit. Care Med.February 1999; 160: 736-755
Usefulness of plasma neutrophil gelatinase-associated lipocalin concentration for predicting the severity and mortality of patients with community-acquired pneumonia.
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