Changes of Tumor Necrosis Factor, Surfactant Protein A, and Phospholipids in Bronchoalveolar Lavage

BIOMEDICAL AND ENVIRONMENTAL SCIENCES 19, 124-129(2006)Changes of Tumor Necrosis Factor, Surfactant Protein A, and Phospholipids inBronchoalveolar Lavage Fluid in the Development and Progression ofCoal Workers'PneumoconiosisJING-CAI XING"A WEI-HONG CHEN".2, WEN-HUI HAN, MEI-FENG GUOSTEFFENI REHN AND JOACHIM BRUCHDepartment of Occupational Environmental Health, School of Public Health, Tongji Medical CollegeHuazhong University of Sciencechnology, Wuhan 430030, Hubei, China; Institute of HygienecTheo nal Medicine, Medical School, University of Essen, Essen, GermanyA The Central Hospital of Xishan Coal& Power Company, Shanxi, ChinaObjective To evaluate the alterations of biomarkers in the development and progression of coal workerspneumoconiosis(CWP). Methods The type and number of cells, and the levels of tumor necrosis factor-alpha(TNF-a),pulmonary surfactant protein, phospholipids and fibronectin in bronchoalveolar lavage fluid were assayed in 14 health activecoal miners, 21 coal miners without CWP and 13 miners with CWP of OI to 1/1. Results Compared to active coal minerswithout CWP(8. 23 Hg/mL), TNF-a concentration was gradually decreased when dust exposure was stopped(5.90 ug/mL)Elevated surfactant protein A(SP-A)level and phosphatidylglycerol(PG)to phosphatidylinositol(PD) ratio were found inminers actively exposed to coal dust(6528 ng/mL for SP-A and 10. for PG/PD), and both parameters decreased when CWPprogressed from CWP (O/1)(3419 ug/mL for SP-A and 5.9 for PG/PD)to CWP(1/1)(1654 Hg/mL for SP-A and 5.5 for PG/PD)Conclusion Biomarkers in bronchoalveolar lavage fluid can be used to screen coal miners at high risk of developing coalKey words: Pneumoconiosis; TNF-alpha; Pulmonary surfactant-associated protein A; PhospholipidsINTRODUCTIONPrevious studies have shown that tumor necrosisfactor alpha(TNF-a), pulmonary surfactant-associatedCoal workers' pneumoconiosis (CWP) is protein, and phospholipids playcharacterized by interstitial pulmonary fibrosis the pathologic process of pulmonary fibrosis. Exceptinduced by inhalation of coal dust. Pneumoconiosis is that TNF-a is directly associated with fibroblast celltraditionally diagnosed on the basis of a known proliferation and collagen accumulation, it inducesexposure history, altered chest radiographs, and infiltration of different inflammatory cells and releasepulmonary malfunction. However, the use of these of fibrogenic cytokines". It has been reported thatmethods is limited in the diagnosis of early the levels of pulmonary surfactant-associated proteinpneumoconiosis because fibrotic changes are usually A (SP-A)(the most abundant surfactant protein)andmanifested after a long latency process of phospholipids(PL) [including phosphatidylcholineinflammation and tissue impairment leading to (PC), phosphatidylglycerol (PG), phosphatidylinositolclinical presentation. Thus, alterations of biomarkers (PD), phosphatidyethanolamine, and sphingomyelin]in the development and progression ofre altered in experimental animals and workerspneumoconiosis are of great interest in the earlyexposure to crystalline silica and coal dustdiagnosis of CWP.alterations have also been found inThis research was supported by University of Essen in Germany and Xishan Coal Power CompanyCorrespondence should be addressed to Dr. Wei-Hong CHEN, Department of Occupational& Environmental Health, Tongji MedicalCollege, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, Hubei, China. Tel: 86-27-83691677. E-mailBiographical note of the first author to Jing-Cai XING, female, bom in August 1955, Ph. D, candidate in Tongji Medical college,Abbreviations: CWP: coal workers' pneumoconiosis; TNF-a: tumor necrosis factor-alpha; SP-A: surfactant protein A; PG:phosphatidylglycerol: PL: phosphatidylinositol: PL: phospholipids: PC: phosphatedne: BALF: bronchoalveolar lavage fluid; IPFiopathic pulmonary fibrosis; RDS: respiratory distress syndrome; SP: simple中国煤化工CNMHCNl1-2816QCopyright o 2006 by China CDCCHANGES OF BIO-PARAMETERS IN BAL FOR MINERS OF CWPinflammatory interstitial pulmonary diseases such as staining in plate reader(Anthos, Vienna)at 450 midiopathic pulmonary fibrosis(IPF))and respiratoryultraviolet light(Labsystems Multiskan, Germany)distress syndrome (RDS). In general, theaccumulation of surfactant PL and proteins and their Protein Assay and Surfactant Protein Acomplex interaction contribute to the defensiveProtein concentration in BALF supernatant wassystem and pulmonary repair process after exposure measured by a modified method of LowryPrevious studies have investigated the humanPulmonary SP-A concentrations were measured by aSP-a specifice linkedparameters derived from dust exposure. However, we immunoabsorbant assay (ELISA). Immunoassayare interested in the trends of parametric alterations inbronchoalveolar lavage fluid(BALF) of workers plates were coated overnight4℃ with awithout and with the initial development of CWP.monoclonal antibody against anti-human SP-ATherefore. we included coal miners without CWP(BYK-Gulden, Konstanz), and then incubated withand coal miners with eaDy CWP in this study. The the test samples and standard dilutions of humanpurpose of this study was to identify possible SP-A at 37C for 4 h, followed by incubation with abiomarkers and to investigate their role in screening peroxidase conjugated anti-human SP-A polyclonalcoal miners at high risk of developing pneumoconiosis antibody for 2 h. The peroxidase was quantified byand in predicting or diagnosing early CWPadding the substrate OPD and stopping the reactionwith IM sulphuric acid. The peroxidized OPd wasat 405 nm ultraviolet light LabsystemsMETHODSMultiskan)SubjectsPhospholipid(PL) and Fibronectin AnalysisForty-eight coal miners were included in thisTotal PL was extracted from thesupernatantstudy who worked in Xishan Coal and Power grusing the method of FolchrieflyLimited Company in Shanxi Province of China. 10 mL cell-free supernatant was added to 30 mLAmong them, 14 were healthy active workers, 21 chloroform and methanol (2: 1 ). The mixture waswere diagnosed as having simple CWP, and 13 as centrifuged to separate the methanol and chloroformCWP 1/1. Most of the patients were relocated layers. The chloroform layer was removed,driedaway from dust exposure when they were diagnosed under nitrogen, and redissolved in 200 uL chloroformas having CWP 1/1(12 among 13). Information on and methanol (2: 1)for high-performance liquiddemographics and personal exposure to smoking andromatography(HPLC) analysis. PL was separatedoccupational hazards was obtained from each subject by HPLC (Gynotek Co., Munich, FRG)according tothrough a questionnaire. This study was approved by the method of Pison and colleagues PhospholipidTongji Medical College and local ethic committeecomposition was calculated by comparison withBronchoalveolar Lavage(BAL) and Cell Isolationstandard PL mixture(PG, Pl, PE, PC, SPh) obtained(Munich, FRG). FibronectinGO. BaL was performed by instillation of 150 mL concentration was determined by a fibronectinmL for the first time, then 40 mL for three times) specific ELISAsterile physiologic saline at 37C into the medial Statistical Analysissegment bronchus of right lobe during fibreopticbronchoscopy. Recoveries(>40%)were obtained byThe difference in the means of all quantitative80 mmHg negative pressure. The later three measurements between two groups was tested by therecoveries were mixed in one polypropylene plastic Mann-Whitney U-test. The principal componenttube for the following tests. Cells were harvested byy analysis was used to identify representative variablescentrifugation after BALF was filtered through from the biomarkers included. SAS version 6.12 wasdouble sterile surgical gauzused for statistical analysisNF-a AssayRESULTSTNF-a in balF was determined by a cell lyticassaylb!. Briefly, mouse L929 fibroblast cells wereffects of Age, Smoking, and dust Exposureincubated with the balf test samples and samples中国煤化工inhibited by anti-TNF-a at 37C in 5%CO2. After 18 showCN M H Gloyment with dushours, the test samples were removed, the plate was exposure in the three groups were compwashed, and cell lysis was detected by crystal violet difference in smoking habit was found among all groupXING ET AL.TABLE ICharacteristics of Study PopulationGroupsCoal Miners Without CWPCWP O1CWP 1/ge( Years,x土s)327±7940.1+3.6447.9±9.1Age Range(Years)25-4934-44Years in Exposure90+6214.5±3.71867.1Smoking Pack-years69±7118±14.7183±13.4Note ' Values are expressed as x+s"P<0.05, compared with healthy controls. "Packs of cigarettes per day x years.controls and other groups are shown in Table 3. TheDifferential Cells in BALFprotein level in lung BALF was increased in minersTotal cells and the percentages of differential with CWP. The mean SSP-a concentration was greatlycells in BALF for the three groups are listed in Table increased in coal miners without CWP (6528 ng/mL)2. The predominating cells in BALF were alveolarand gradually decreased to 3419 ng/mL in patientsmacrophages (AMs). Lymphocyte percentage waswith CWP O/1. The mean SP-A in patients with CWPgnificantly lower in CWP O/I than in controls1/1 showed a great scattering with a high value of5455 ng/mL for two cases and a low value of 386.4TNf-a Levels in Silica and Mixed Coal Dust ng/mL for other 11 cases. a parallel pattern wasExposure Groupsshown between the ratio of SP-a to total protein andSP-A levels(Table 3).The TNF-a concentrations in baLF in differentgroups are presented in Fig. 1. No correlation was Total PL, PL Composition, and Fibronectin Level inobserved between TNF-a level and exposure duration BALFto coal dust. The TNF-a levels were significantlyincreased in coal miners without CWP(8.2+1.6Three methods were used to determine the levelsunit/mL). With the progression of CwP, the TNF-aof total PL in cell-free BaLF Table 3). a goodcorrelation was shown between the results detectedlevels decreased from 6.5+2.5 unit/mL in minersby these methods(r=0.94 for UV method and r-d0.97with CwP 0/1 to 5.9+1.7 unit/mL in miners with for md method when correlated with phosphor method)CWP 1/1. TNF-a concentration in both CWP groups The total PL was slightly decreased in miners with CWP,was significantly lower than that in coal minersbut elevated in active coal miners. A significantwithout CWP(P<0.05)decrease(P<0.01)in total PL content of baLF wasobserved in coal miners with CWP 1/1 as comparedwith active coal miners. PL seemed to be stimulated bydust exposure to accumulate in BALF, and it was cut of7000Coal minewith the development of pulmonary fibrosisAs shown in Table 3, PC was the predominant PI▲CWPI/lwhich accounted for 76% of total PL in healthy controbjects. The percentage of PC in dust-exposed groups3000was lower than that in healthy controls, indicating that1000other components of PL were elevated in dust exposuregroups. A significantly elevated PG percentage was20 25 30 shown in active coal miners. the highest ratio of PG toPhospholipids(ug/m, MD method)Pi was observed in active coal miners without CwP asFIG. 1. Pointof SP-A anspholipids incompared with other groups( Table 3)BALF aminers without and with CwPNo correlation was observed between SP-A andPL-APL was highly中国煤化工Total Protein and sP-A in bALFCNMHG was shown inAtween healthy controlThe total protein concentration and SP-A in subjects and dust exposed groups( Table 3)CHANGES OF BIO- PARAMETERS IN BAL FOR MINERS OF CWPTABLE 2Cell Recovery Characteristics of BALF From Healthy Controls, Coal Dust Exposed Workers, and Patients with PneumoconiosisGroupsCoal miners without CwPCoal Miners With CWP OvCoal Miners With CWP 1/1Bronchitis Index3.3±133.9±1.3BAL Recovery(mL)800±12.170.0+20.5729±179Total Cells(10/mL159±8618.3±10.0240±6.8Differential Cell Count(%)Macrophages84.2+14.387.746.878.39.1Neutrophilsl.0±1.02.7+2122±13410.7532166±72Others0.6+1.11.1±22Note.ValueTABLE 3Biomakers in BALF in Coal Dust Exposed Workers and Pneumoconiosis Patientspal Miners without CWP Coal Miners With CWP OICoal Miners With CWP 1/1TNF(ug/mL)8.231.616.50±245Total protein(Hg/mL)1253+46.7140.7±413SP-A(ng/mL)6528±187243419323981+34°108±5.2(UV)280±12216.5+4.714.8±778.7±3.645+39°1.2±040.29±0.l10.2410050.22+0.05Fibronectin278±198390±226PC/P681±74271,2+46°69+782PG/PL55±7517.7+4.116.9±2.8PUPL2.8±1.13.8+2.83.7±15PG/PI10.1±3.325.9±2.7SP-A/PL496.1±17484749±4179103±3264623±3928.9±26.116.0±24.9Note ' Values are expressed as x+s. p<0.01, compared with healthy controls P<0. 05, compared with healthy controls.DISCUSSIONIndependent Factors for CWPPrincipal component analysis was used to searchThis study confirmed the alterations offor independent factors contributing to the progresses pulmonary surfactants and TNF-a in miners after coalof CWP. We obtained three independent groups. d中国煤化工 rnative trends oGroup A included SP-A, PG/Pl, and phospholipidsgroup B included lymphocytes and macrophagesCNM HGnparison of thesegroup C included TNF-acellular and biochemical alterations between minersXING ET AIwith CWP and healthy miners can help us to dust damage thus also moves to the interstitialnderstand the processes of lung fibrosis and to and the level of SP-A in BALF is recovereddistinguish pathological processes of earlyme. the disease is manifested as apneumoconiosis in clinical practiceinflammation reaction, a different phase in theTNF-a is an important cytokine in initiating development of pneumoconiosis. Third, activation ofpulmonary fibrosis. TNF-a is released from alveolar dust in the lungs is reduced over time. Hence,macrophages after dust particles are engulfed and is removal of patients from dust exposure wouldassociated with fibroblast cell proliferation and decrease the levels of surfactant. It was reported thatenhanced collagen accumulation o. In this study, SP-A becomes normal in monkeys one year afterTNF-a levels increased in coal miners exposed to removal from dust exposure 9mixed coal dust and then gradually decreased whenNo significant correlation was found betweenCWP progressed. The TNF-a level in miners with alveolar proteins or SP-A and PL in this study,CWP 1/1 was close to that in healthy controls. suggesting that the regulation of each surfactantThese results are highly comparable to those of Borm constituent may be under separate controlet al. and Schins2-13 who found that the highest mechanisms. These findings are consistent with therelease of TnF-a is only observed in early CWP, and conclusion derived by other research groups"5.20)CWP progressed. Our results are also McCormack and colleagues reported that SP-A/PL asconsistent with those of Lassalle et al. 4 who found a biochemical marker in lavage can predict survivalthat TNF-a level in dust exposed coal miners, coal time of patients with idiopathic pulmonary fibrosisminers with simple pneumoconiosis (SP) and (IPF). Reduced SP-A/PL ratios are associated withprogressive fibrosis(PMF) are significantly elevated. clinical deterioration and short survival time. We alsocould be compared to CWP O/I and CWP 1/1found significantly decreased SP-A/PL in CWP 1/1study and PMF is an advanced stage of CWP. which was much lower than that in the controls. ItThe decreased trend of TNF-a for CWP 1/1 in our seems that SP-A/PL may reflect pathogenic phases orstudy is at least partly owing to the removal of miners deterioration degree of CWP.from active exposure. All miners except one withSlightly reduced concentrations of total PL inCWP I/l removed from coal dust exposure. All these BALF were observed in patients with CWP in thisresults indicate that TNF-a is also a biomarker for study. Generally, changes in PL are the results ofactive coal dust exposurecomplex multifaceted inflammation damage repairEnhanced accumulation and decreased content of processes. Such a different profile remains unclearSP-A after dust exposure in this study are very clear. and needs further investigation. Both PG and PI areThe initial value of elevated SP-A was shown among synthesized from the same precursor CDP-DG ofall coal miners without CWP and CWP 0/1(7/13), alveolar type I cells, stored in the lamellar bodieswhich agrees with previous reports from animal and secreted into the alveolar space". Changes inexperiments of silica-treated rats and sheep 56. This the PG content in BALF appear to reflect morechange may be explained by elevated synthesis and precisely the damage to type II cells. Normally thesecretion of SP-a by activating type I alveolar concentration of PG is much higher than that of PI inepithelial cells. It was reported that SP-A is normal in pulmonary surfactant. Thus a switchover in therats exposed to dust", suggesting that accumulationof SP-A is an acute inflammatory and active repair mean that the lung surfactant is in the pathologicalprocess in the lung after dust exposure. Several state. These reductions have also been reportedontribute to the decreased in patients with interstitial lung disease IPFconcentrations of SP-a due to the progression ofA limitation of this study is that the control/P from O/1 to 1/1. First, the toxicity of coal dust subjects were younger than CWP patients althoughmay damage the function of type II cells and induce no correlation between age and TNF-a, SP-A and PLIterations of synthesis, secretion and degradation of levels was found in any group of this study. TheSP-A. The magnitude of reduction of SP-A can study of Schins and Borm excluded the effect of agepredict the reduction of survival in patients with on release of TNF-a from monocytes The effect ofidiopathic pulmonary fibrosis 8. In this study, SP-A smoking was not assessed with TNF-a in this studylevel was very low in patients with CWP 1However, a recent study showed that smoking hasindicating that serious impairment of type I particInfluence on TNF-a in the BAL 261epithelial cells can predict prognosis for these中国煤化工 centration and PIpatients. Second, coal dust particles in the perCNMHstinguish acutebronchoalveolar milieu are translocated rapidly to the inflamixed dust frominterstitial space, and cause interstitial fibrosis. The interstitial fibrosis. TNF-a level can be used to reflectCHANGES OF BIO-PARAMETERS IN BAL FOR MINERS OF CWPactive dust exposure in coal miners.pneumoconiosis. Eur Respir J8(10), 1658-166313.Chins R P, Borm P J(1995). Epidemiological evaluation ofrelease of monocyte TNF-alpha as an exposure and effectACKNOWLEDGEMENTmarker in pneumoconiosis: a five year follow-up study of coalworkers. Occup Environ Med 52(7), 441-450.14.Lassalle P, Gosset P, Aerts C, et al.(1990). Abnormal secretionThe authors thank Mrs. S. REHN for determiningof interleukin-1 and tumor necrosis factorby alveolarTNF-a; Mrs. A GONO for determining PL; Dr Jirrophages in coal worker's pneumoconcompansonMei WANG for convening patients, and the referringbetween simple pneumoconiosis and progressive massivephysicians and participating patientsfibrosis. Exp Lung Res 16(1), 73-8015.Lesur O, Veldhuizen R A, Whitsett J A, et aL. (1993)REFERENCESproportionally to alveolar phospholipids in sheep silicosis. Lung171(2,63-74.1. Piguet P F Grau G E, Vassalli P(1990). Subcutaneous perfusionrats. Am J Physiol 273, 395-400fibroblasts, capillaries, and epidermal cells, or massive tissue 17Spech R W, Wisniowski P, Kachel D L, et al.(2000). Surfactantnecrosis. Am J Pathol 136(1), 103-110A prevents silica-mediated toxicit2. Gosset P, Lassalle P, Vanhee D, et aL. (1991). Production ofmacrophages, Am J Physiol Lung Cell Mol Physiol2tumor necrosis factor-alpha and interleukin-6 by humanL713-718Iveolar macrophages exposed in vitro to coal mine dust. Am J18. Yang S, Milla C, Panoskaltsis-Mortari A, et aL.(2001). HumanRespir Cell Mol Biol 5(5), 431-436surfactant protein A suppresses T cell-dependent3. Driscoll K E, Lindenschmidt R C, Maurer J K, ef aL.(1990and attenuates the manifestations of idiopathicPulmonary response to silica or titanium dioxide: inflammatorysyndrome in mice. Am J Respir Cell Mol Biol 24(5ells, alveolar macrophage-derivedokines, and 19.Schengrund C L, Chi X, Sabol J, et aL. (1995)effects of instilled mineral dusts on pulmonar4. Gunther A, Schmidt R, Nix F, et al.(1999). Surfactantisolated from monkeys. Lung 173(3), 197-208abnormalities in idiopathic pulmonary fibrosis, hypersensitivity20. Nogee L M, Wisp JR( 1988). Effects of pulmonary oxygenneuronitis and sarcoidosis. Eur Respir J 143), 565-573injury on airway content of surfactant-associated protein A5. Hallman M, Spragg R, Harrell J H, et aL.(1982). Evidence ofPediatr Res 24(5), 568-573ng surfactant abnormality in respiratory failure. Study of21. Mc Cormack F x, King T E, Jr, Bucher B L, et aL.(1995)bronchoalveolar lavage phospholipids, surface activity,Surfactant protein A predicts survival in idiopathic pulmonaryactivity, and plasma myoinositol. J Clin Investfibrosis. Am J Respir Crit Care Med 152(2), 751-7590(3),673-622 Batenburg JJ, Klazinga W, L M. vG(1985). Regulation and6. Aggarwal B B, Eessalu T E, Hass P E(1985). Characterizationphosphatidylglycerol and phosphatidylinositolf receptors for human tumour necrosis factor and theiynthesis in alveolar type I cells isolated from fetal rat lungregulation by gamma- interferon Nature 318(6047), 665-667Biochim Biophys Acta 833, 17-2U. Nathan C F(985). A semi-automated 23Adachi H, Hayashi H, Sato H, et al.(1989). Characterization ofmicro-assay for Hnog2 release by human blood monocytes andaspholipids accumulated in pulmonary-sumouse peritoneal macrophages. J Immunol Methods 78(2).compartments of rats intratracheally exposed to silica323-336.J262(3),7818. Folch J, Lees M, Stanley G HS(1957). A simple method for the 24.Robinson PC, Watters L C, King T E, et al.(1988). Idiopathication and purification of total lipids from animal tissue. Julmonary fibrosis. Abnormalities in bronchoalveolar lavageBiol Chem 226, 497-509fluid phospholipids, Am Rev Respir Dis 137(3),585-5919. Pison U, E. G, T J, et al.(1986). High-performance liquid 25Honda Y, Tsunematsu K, Suzuki A, et al. (1988). Changes inchromatography of adult human bronchoalveolar lavage: assayphospholipids in bronchoalveolar lavage fluid of patients withfor phospholipid lung profile. J Chromatogr 377, 79-89interstitial lung diseases. Lung 166(5), 293-30110. Donaldson K, Bom PJ(1998quartz hazard: a variable26 Popp w, Plappert U, Muller W U, et aL. (2000). Biomarkers ofentity. Ann Occup Hyg 42(5), 287-294genetic damage and inflammation in blood and bronchoalveolar11. Borm P J, Palmen N, Engelen J J, et aL.(1988). Spontaneoulavage fluid among former German uranium miners: a pilotand stimulated release of tumor necrosis factor-alpha (TNF)study Radiat Environ Biophys 39(4), 275-282.from blood monocytes of miners with coal workers'pneumoconiosis. Am Rev Respir Dis 138(6), 1589-1594(Received December 6, 2004 Accepted July 19 2005)12. Schins R P, Borm P J(1995). Plasma levels of soluble tumourecrosis factor receptors are increased in coal miners with中国煤化工CNMHG