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Inflammatory Response in 7 Mg N02·M· a Larger Proportion of the AM Were 3 BALF After Exposure to 7 Mg N02·M· [16]

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Inflammatory Response in 7 Mg N02·M· a Larger Proportion of the AM Were 3 BALF After Exposure to 7 Mg N02·M· [16] Eur Reaplr J 1991, 3, 332-339 Inflammatory cell response in bronchoalveolar lavage fluid after nitrogen dioxide exposure of healthy subjects: a dose-response study T. Sandstrom•t, N. Stjernber~J", A. Eklund .. , M-C. Ledin·, L.. Bjermert, B. Kolmodin-Hedman·, K. Lmdstrom·, L. Rosenhallt, T. Angstromtt Inflammatory cell response in bronchoalveolar lavage fluid after nitrogen • National Institute of Occupational Health, dioxide exposure of healthy subjects: a dose response study. T. Sandstrom, Medical Division, Umel, Sweden. N. Stjernberg, A. Eklund, M-C. Ledin, L. Bjermer, B. Kolmodin-Hedman, K. Lindstrom, L. Rosenha/1, T. Angstr6m. ' Dept of Thoracic Medicine, Karolinska Hospital, ABSTRACT: The combinatJon of environmental chamber exposure and Stockhoim, Sweden. broncboalveolar lavage (BAL) was used to study the effects of the • • Dept of Thoracic Medicine, Karolinska Hospital, ). common air pollutant nitrogen dioxide (N01 Eighteen healthy Stockholm, Sweden. nonsmokers were exposed to N01 during 20 mln in an exposure chamber during llgbt bicycle ergometer work. All subjects were examined with "Cytology Laboratory, University Hospital, UmeA, BAL at least 3 wks before exposure, as a reference. The subjects were Sweden. re-examined wltb BAL, in groups of eight, 24 h after exposure to 4, 7 and 10 mg N0 ·m·' (2.25, 4.0 and S.S ppm), respectively. An Correspondence: T. SandstrOm, National Institute of 1 inflammatory cell response was found after exposure to all concentra­ Occupational Health, Medical Division, Box 6104, tions. An Increase In the number of lymphocytes in BAL fluid was S-900 04 Umel, Sweden. observed after 7 and 10 mg·m~ (p<0.05 and 0.02, respectively). An Keywords: Air pollution; bronchoalveolar lavage; Increase In the number of mast cells, that appears to be dose-dependent, lymphocyte; lysozyme; macrophage; mast cell. was found after exposure to all concentrations. The proportion of lysozyme posltJve alveolar macropbages was elevated after exposure to Received: February 8, 1990; accepted after revision 7 mg·m·'. The Inflammatory mediators fibronectln, hyaluronan, August 23, 1990. angiotensin converting enzyme (ACE) and beta -mfcroglobulln were 1 unchanged by exposure. Due to the findings of Inflammatory cell changes This study was supported by grants from the Swedish far below the peak exposure limits for work places In Industrialized Heart Lung Foundation, the University of UmeA and countries, 9-18 mg·m·', the safety of these limits Is questioned. the Norrlands Gas AB Fund. Studies are In progress In our laboratory using BAL to evaluate the effects of repeated N01 exposure. Eur Respir 1., 1991, 4, 332-339. Nitrogen dioxide (NOJ is a common air pollutant both occur after high N02 levels or long-term exposure [10, in community air in urban areas and in the indoor 11]. In animal studies increased numbers of neutrophils environment in industries using combustion processes. and macrophages [12-14], and in some investigations Due to its poor solubility it is to a large extent deposited also lymphocytes and mast cells [10, 15], have been in the peripheral air spaces [1, 2] where it acts on cells found in histological specimens and bronchoalveolar according to its oxidant and free radical properties [3]. lavage fluid (BALF). Inhalation of relatively low concentrations of N02 may In man bronchoalveolar lavage (BAL) has only increase airway resistance in healthy subjects and recently been employed in investigating the effects of repeated exposure may give enhanced susceptibility to N02 inhalation. Recently, we showed an altered airway infections [4, 5]. Reduced diffusion capacity composition of cells in BALF as the time-course was for carbon monoxide has been reported after a investigated for the inflammatory cell response induced 3 3 single exposure to 9 mg N02·m· [6]. Exposure to by 7 mg N02·m· healthy subjects [16]. N02 exposure high concentrations may result in chronic obstructive has also been shown to cause an altered antiprotease lung disease, emphysema or bronchiolitis obliterans activity, alpha2-macroglobulin increase in BALF and [7]. impaired virus inactivation by human alveolar Morphological studies in animals have shown that macrophages (AM) [17-19). the most sensitive part of the lung to N02 exposure is This investigation was undertaken to examine the the transitional zone between the terminal bronchiole dose-response relationship between N02 exposure in man and the alveolar duct [8, 9]. Alveolar effects mainly during light bicycle ergometer work and changes in the N0 EPFECI'S INVESTIGATED WITH BRONCHOALVEOLAR LAVAGE 2 333 BALF content of cells with regard to their concentra­ Bronchoalveolar lavage tion and function. This was measured as phagocytic ability and release of soluble components considered as All bronchoscopies were performed by the same a reflection of cellular activity. investigator. Atropine was given s.c. prior to the examination and lidocaine 200 mg was used for topical anaesthesia. No sedative or other complementary Subjects and methods medication was given. The flexible fibreoptic bronchoscope (Olympus BF 1T or BF 1T10, Japan) was Subjects inserted through the mouth with the subject in the supine position. After careful wedging of the broncho­ Eighteen healthy nonsmoking male volunteers with a scope tip in a middle lobe bronchus, sterile phosphate mean age of 26 yrs (range 22-32 yrs) participated. buffered saline pH 7.3 (PBS-A) at 37°C was infused in None of them had experience symptoms of airway four aliquots of 60 ml and gently suctioned back after infection for at least six weeks prior to the study or each infusion to a siliconized container placed in ice had a history of asthma. Informed consent was obtained water. and the study was approved by the local Ethics Committee. Lavage fluid analysis Design of the study Cell counts. The chilled BALF was filtered through a nylon filter (pore diameter 100 J.tm, Syntab Product AB, Malmo, Sweden) and centrifuged at 400 g for 15 min. Flexible fibreoptic bronchoscopy with BAL was The cell pellet was resuspended in balanced salt solu­ performed in all subjects in order to obtained reference 6 BALF. In the following exposure series, each subject tion to a concentration of 10 cells per ml. The total number of cells in the lavage fluid was counted in a was exposed to at least one N0 concentrations. Six of 2 Biirker chamber. Cytocentrifugal specimens with Sx104 the subjects were exposed to two N0 concentrations, 2 non-epithelial cells per slide were prepared using a with a time interval of at least 3 wks. The environmental Cytospin 2* (Shandon Southern Instruments Inc., chamber exposure was performed with 4, 7 and 10 Sewickly, PA, USA) 100 rpm (96 G) for 15 min. Slides mg·m·3 (2.25, 4.0 and 5.5 ppm) for 20 min. Eight were stained according to May-Griinwald-Giemsa for subjects were studied at each concentration, according standard cell differential counts and two hundred cells to a standardized protocol [20]. During the last 15 min per slide were counted. Mast cells were counted on 10 of the exposure the subjects were working on a bicycle visual fields at 16x magnification on slides stained with ergometer with a work load of 75 W. A second BAL acid toluidine blue and counterstained with Mayer's acid was performed 24 h after the end of N0 exposure, which 2 haematoxylin [21]. Lysozyme positive AM were was at least 4 wks after the first lavage. Immediately demonstrated with Lysozyme* antibody using an before and after exposure, and before the post-exposure immunoperoxidase technique (Dakopatts A/S, BAL, forced expiratory volume in one second (FEV ) 1 Copenhagen, Denmark). Two hundred alveolar and forced vital capacity (FVC) were recorded using a macrophages (AM) were counted. The ratio helper­ Vitalograph* spirometer (Vitalograph Ltd, Buckingham, inducer/cytotoxic-suppressor (CD8•/CD4•) T-cells was UK). counted on 200 lymphocytes using light microscopy using the Simultest T Helper/Suppressor Test11 (Becton Dickinsson AB, Stockholm, Sweden). Nitrogen dioxide exposure Non-cellular components. Albumin was measured with The exposure chamber measured 3.20 x 2.00 x 2.20 rocket electroimmunoassay according to LAuRELL [22]. 3 1 m with an air volume of 14.1 m • It was built with Concentrations were expressed in mg·l· • anodized aluminium, with windows in one wall. Fibronectin was analysed by a double-sandwich Ambient air was drawn continuously through the enzyme-linked immunosorbent assay (ELISA) developed 1 chamber at 150 m3·h· , resulting in one air exchange by Bu.sCHKE et al. [23]. The aliquots of BAL fluid were approximately every 6 min. During the exposures, the defrozen at 37°C. Briefly, microtitre plates (NUNC, chamber air temperature was kept at 21 °C and the Denmark) were coated with rabbit-antihuman fibronectin relative humidity at 45%. The desired N02 concen­ antibodies (Dakopatts, Denmark) in phosphate-saline trations in the exposure chamber were achieved by buffer, pH 7.2. After addition of unconcentrated BAL adding a gas stream from a gas tube containing 1% fluid samples the plates were incubated at room N02 gas into the chamber air inlet. The gas flow temperature for 2 h. Horse radish peroxidase-labelled was maintained evenly by a Mass Flowmeter, Brooks antihuman fibronectin (Dakopatts) was added as second 5850 Tr, Brooks Instruments, Vendeen, The Netherlands. antibody and the plates were incubated for 1 h. The The chamber air was continuously analysed with a amount of bound peroxidase, which is proportional direct indicating equipment (Nitrogen oxides analyzer to the amount of fibronectin in the sample, was 8440 B, Monitor Labs, San Diego, Ca, USA). measured by analysing the enzymatic activity on 334 T. SANDSTROM ET AL. orthophenylendiamine. Serum fibronectin of nephelom­ (Pharmacia Diagnostics AB, Sweden). The detection etric quality from Behringer-Hoechst (Frankfurt am limit was 40 f,.lg·J·1• Concentrations were expressed in Main, GFR) was used as standard.
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