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Inflammatory Cells and Ferruginous Bodies in Bronchoalveolar Lavage in Urban Dogs

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Inflammatory Cells and Ferruginous Bodies in Bronchoalveolar Lavage in Urban Dogs
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  939 OBJECTIVE: To investigate whether high levels of at-mospheric pollution modify the inflammatory cell countof the canine lung and to de-tect the presence of ferrugi-nous bodies (FB) in the respiratory system.STUDY DESIGN: Bron-choalveolar lavage (BAL)was performed on dogs from four different areas of MexicoCity and a rural area. Withthe BAL fluid recovered,total and differential cell counts were made, and ferrugi-nous bodies were isolated by sodium hypochlorite diges-tion. They were counted by light microscopy and evaluatedas a marker of exposure to particulate pollutants.RESULTS: There was no signi cant difference in thetotal cell count or in the macrophage number between the ve groups. The neutrophil count was statistically high-er in dogs from the southwest area as compared to thenortheast and rural areas ( P <.05). The lymphocytecount was signi cantly greater in the southwest, also.The northeast part of the city showed the highest numberof FB in BAL fluids.CONCLUSION: The most polluted areas, in terms of  particulate matter, were the northeast and the northwest;those are the locations of heavy industry. In contrast,in the southwest, wheremore inflammation wasseen, the highest levels of ozone were registered dur-ing the year. (Acta Cytol1998;42:939–944) Keywords:  bronchoalve-olar lavage, air pollution, dogs, inflammatory cells,ferruginous bodies.Bronchoalveolar lavage (BAL) is an easy and safetechnique for studying cell populations 6,19 andevaluating whether atmospheric pollution can in-duce alterations in the inflammatory cell count of the lung. BAL allows the detection of inorganic pol-lutants in normal and diseased lungs. 5,13 Mexico City is a good place to observe some of the effects of pollution on the respiratory system be-cause it is in a valley surrounded by mountains, From the Mexican Institute of Respiratory Diseases, the Veterinary Faculty of the National Autonomous University of Mexico and theGeneral Hospital of Mexico, Mexico City, Mexico.Drs. Vanda, Jasso, Olmos, Arreola, Santillán and Vargas are Researchers, Mexican Institute of Respiratory Diseases. Dr. de Buen is Chief, Department of Veterinary Pathology, National Autonomous University of Mexico.Dr. Valero is Lecturer, Department of Veterinary Pathology, National Autonomous University of Mexico.Dr. Alonso is Professor of Pathology, General Hospital of Mexico.Address reprints requests to: Beatriz Vanda, D.V.M., M.Sc., Unidad de Investigación, Instituto Nacional de EnfermedadesRespiratorias, Calz. Tlalpan 4502, México D.F. 14080, México.This study was supported by Consejo Nacional de Ciencia y Tecnologia (grant no. 1343-M9206) and by the Mexican Institute of Respiratory Diseases.Received for publication June 30, 1997.Accepted for publication October 9, 1997. Inflammatory Cells and Ferruginous Bodies inBronchoalveolar Lavage in Urban Dogs Beatriz Vanda, D.V.M., M.Sc., Nuria de Buen, D.V.M., M.Sc., Rogelio Jasso, M.D., Germán Valero, D.V.M., M.Sc., Mario H. Vargas, M.D., M.Sc., Raúl Olmos, D.V.M.,  José Luis Arreola, D.V.M., Patricio Santillán, M.D.,and Patricia Alonso, M.D., M.Sc. Acta Cytologica 0001-5547/98/4204-0939/$06.00/0 © The International Academy of CytologyActa Cytologica BAL is a minimally invasivemethod that...can be used as a diagnostic tool in patients withradiologic findings suggestive of asbestosis and related diseases.  with 20 million inhabitants, 2.5 million vehicles andan industrial area on the north side of the city. Thisgenerates a complex mixture of pollutants, such asphotochemical gases, heavy metals and aromatichydrocarbons, and a broad spectrum of inorganicsuspended particles (asbestos fibers, silicate dusts,charcoal, etc.). 7 When particles <10 µm (PM10) areinhaled, they lodge in the alveolar spaces, wherethey are engulfed by macrophages but can be nei-ther digested nor dissolved and are coated withgranules of ferritinlike protein derived from the breakdown of hemoglobin, resulting in ferruginous bodies (FB) 8,17 (Figures 1 and 2).For many years FB have been observed in lungsfrom people with high exposure to asbestos dusts,such as mechanics and building, steel, textile andmining workers. Nevertheless, in recent decades,FB have been found in lung tissue and BAL fluidfrom patients without occupational exposure. 1,4,6 The aim of this study was to investigate, throughBAL, whether high levels of atmospheric pollutantscan alter the inflammatory cell count and to quanti-fy FB in BAL fluid in order to estimate the environ-mental exposure to inorganic particles in four areasof Mexico City and a rural area, using dogs as a nat-ural exposure model. These findings were correlat-ed with the records of air quality measured by theautomated atmospheric monitoring network of theValley of Mexico. Materials and Methods Bronchoalveolar Lavage Fluid Samples Forty-two mongrel dogs (21 males and 21 females)from different areas in Mexico City and a rural areawere studied. The dogs were between 2 and 7 yearsold, with 16 ±5.6 kg mean body weight; they wereclinically healthy, with normal thoracic radiogra-phy and without coughs or rhinorrhea. The animalswere divided into five groups according to theirplace of srcin, as follows (Figure 3): group 1, north-east (NE) (n=7); group 2, northwest (NW) (n=10);group 3, southeast (SE) (n=10); group 4, southwest(SW) (n=9); and group 5 (control), from a rural arealocated 70 km from Mexico City (n=6). All dogswere dewormed and lodged in individual, well-ventilated indoor kennels, receiving water andcommercial dog food ad libitum. Bronchoalveolar lavage was performed with thedogs positioned in dorsal recumbency, under gen-eral anesthesia with intravenous sodium pentobar- bital (28 mg/kg) and previous medication with at-ropine (0.04 mg/kg subcutaneously). Oxygen wasavailable in case of respiratory compromise; how-ever, it was not necessary in any of the procedures. A 6-mm flexible fiberoptic bronchoscope (Olym-pus BF, type 1T20D, Olympus, Lake Success, NewYork, U.S.A.) was passed through the larynx andtrachea and was wedged in the segmental bronchus 940 Acta Cytologica Vanda et al Figure 1  Typical ferruginous body (arrow) surrounded by macrophages (  1,500). Figure 2 Atypical ferruginous body (arrow) from BALF (  1,500).  from the right diaphragmatic lobe. Four sequential20-mL lavages were done on each dog, for a total in-stilled volume of 80 mL of warm (37 ° C), sterile 0.9%saline solution. 19 The bronchoalveolar lavage fluid(BALF) was recovered by a suction machine con-nected to a specimen trap. The first sample of re-covered fluid was discarded because it was consid-ered a “bronchial” specimen rather than “alveolar”and was not representative because of a higher per-centage of bronchial epithelial cells and neu-trophils. 5,19 The next three lavage aliquots werepooled and measured, designated an “alveolar”specimen. 10 The total recovered volume was always>77% of the introduced volume and did not differsignificantly between groups. Inflammatory cellcount, cell viability evaluation and search for ferrug-inous bodies were carried out with the alveolar fluid. Viability and Total Cell Count From the pooled, unfractionated BALF, 50 µL wastaken and mixed into a tube with 50 µL of Trypan blue supravital stain. This dilution was placed in ahemocytometer (Neubauer chamber) for a totalleukocyte cell count under a light microscope (40 × )and to evaluate the percentage of viability. All 42specimens had >80% of live cells, and none of thesamples was contaminated with erythrocytes. Differential Cell Count  A 10-mL aliquot was removed from the pooledalveolar fluid, placed in a tube, fixed in an equalvolume of carbowax 14 and centrifuged (BeckmanCS-6R, Beckman, Palo Alto, California, U.S.A.) at600 g for 10 minutes at 4 ° C. The supernatant wasdiscarded, and with the cell pellet three slides weremade and stained with Papanicolaou stain, hema-toxylin-eosin and Diff-Quik. At least 200 cells werecounted from each slide and identified as macro-phages, lymphocytes, neutrophils and eosinophils.Cell counts were expressed both as a percentage of each cell type and as cells per milliliter of BALF. Ferruginous Body Quantitation The remaining BALF (20–50 mL) was placed inpolyethylene tubes and centrifuged at 1,200 g for 15minutes. The supernatant was discarded, and 5 mLof a filtered 12% sodium hypochlorite solution wasadded, shaken and allowed to react with the pelletfor two hours at room temperature. After digestionwas complete, a second centrifugation and elimina-tion of sodium hypochlorite were done; the sedi-ment was resuspended in 2 mL of chloroform plus2 mL of 50% ethanol. This mixture was centrifugedand the supernatant carefully removed by aspira-tion with a micropipette, leaving the interface in the tube. 3 Then, 4 mL of 95% ethanol was added,and after 12 hours the samples were filtrated in a vacuum and recovered through nitrocellulosemembranes (Millipore, type HA, 0.45 µm, Milli-pore, Bedford, Massachusetts, U.S.A.), which were cleared in xylene and mounted on slides withsynthetic resin. The filters were observed underphase contrast microscopy at 100 × magnification,and the FB were counted and expressed per 5 mL of BALF. Statistical Analysis The results are presented as the mean ±SD for eacharea and are expressed as cells per milliliters of BALFand as FB/5 mL of BALF. For testing for differences between groups, ANOVA was used, followed byStudent’s t test. The Bonferroni method was appliedto correct for multiple comparisons. In order to es-tablish an association between such variables asweight, age, cell number and FB, linear regressionand Pearson correlation were done. A value of P <.05was considered statistically significant. Volume 42, Number 4/July–August 1998 941 Bronchoalveolar Lavage in Dogs Figure 3 Mexico City showing the four areas where dogs werecollected for the study.  Results Cell Counts in BALF  The percentage of cell viability in BALF was, on av-erage, >84% for all groups. There was no significantdifference in the mean total cell count (Table I) or inthe macrophage numbers between the five areas.There was no difference in the number of eosinophils, either, and they were absent from>50% of the dogs. Figure 4 shows that in lympho-cyte counts, differences were found only for the SW,which had a significantly higher number (143,093 ±32,204) than both the NW (81,741±12,340; P <.05)and SE (79,996±10,787; P <.05). The SW areashowed higher neutrophil counts in BALF (32,878 ±8,161) than did the NE (7,729 ±2,335; P <.05) and therural areas (8,855 ±3,663; P <.04) (Figure 5).Statistically significant differences were not seenwhen the average counts of each cell type of allurban dogs were compared against those from therural area; only the neutrophil count was signifi-cantly different (19,736 ±2,837 vs. 8,855 ±3,663; P <.05). The epithelial cells did not show any mor-phologic alteration. FB In Mexico City, an average of 4.37 FB/5 mL of BALFwas found, and these results were significantlyhigher than those in the rural area ( P <.05), where amean of 1.17 FB/5 mL was observed. The NE zonehad more FB/5 mL of BALF (9.9 ±3.2) than the SWzone (1.9 ±0.69; P <.02) and the rural area (1.17±0.33; P <.01). The NW area also had a higher numberof FB (4.04 ±1.5; P <.05) than the rural area (Figure6). The numbers of FB and inflammatory cells werenot related to age or body weight. The presence of FB was not related to inflammatory cell counts. Discussion When our results were compared to BALF reportson dogs in other countries, 11,19 Mexico City showeda low percentage of macrophages and the highestnumber of lymphocytes (Table II). Nevertheless, thecell percentages in BALF from dogs from this city 942 Acta Cytologica Vanda et al Figure 4 Histogram representing the mean values of inflammatory cell count from BALF in each area. There were nosignificant differences in the macrophage counts between the fiveareas. The SW area had a higher lymphocyte count (cross-hatched bars) than the NW and SE areas ( P <.05) and had moreneutrophils (black bars) than the NE and rural areas ( P <.05). M =macrophages, L = lymphocytes, N = neutrophils. Table I Total Inflammatory Cell Counts in BALF from Urbanand Rural Dogs AreaTotal cells ( × 10 3 )/mL of BALF NE339.4±68NW327.0±39SE346.3±33SW467.3±93Rural360.3±94 Values represent the mean ±SD. Figure 5 A large number of neutrophils were observed in bronchoalveolar lavage fluids from the SW area (hematoxylin-eosin,  1,500).  are very similar to those found in humans whowere asymptomatic contacts of patients with inter-stitial pulmonary diseases in Mexico, as reported bySansores et al 15 : 72% macrophages, 21% lympho-cytes and 6% neutrophils.The SW area of Mexico City had the highest per-centage of neutrophils in BALF from dogs; it corre-lated with the peaks in contaminant gases ( r =.45; P <.01) since that area often has the highest amountsof O 3 , with averages between 0.17 and 0.28 ppm(Table III), exceeding the air quality standard (0.11ppm O 3 per hour once a year) most of the time. Thisseems to be due mostly to the fact that the majority of the polluting gases, such as nitrogen dioxide, sulfurdioxide and hydrocarbons, are generated in thenorthern area of the city and are carried by the pre-vailing winds toward the southwest, where they re-main trapped by the mountains surrounding the Val-ley of Mexico. 2 These gases are known to be harmful by themselves and capable of eliciting neutrophil in-flammation; also, they are O 3 precursors. 9,16 The NW area was heavily polluted with O 3 andwas the second highest area in neutrophil counts.A report by the Metropolitan Council for the Pre-vention and Control of Environmental Pollution inthe Valley of Mexico states that the NE area has thehighest level of breathable suspended particles(PM10), with an average of 134 µg/m 3 , followed bythe NW, which also exceeds the standard. Thesedata match our findings: the animals with the high-est FB counts came from the NE and NW areas,where most of the city’s steel, smelting, rubber,glass and textile industries are found. The resultsalso seem to indicate that the number of FB found inthe respiratory system of dogs was associated withthe number of factories in the area ( P <.05). Theareas with the smallest amounts of FB, the SW andrural areas, have practically no industry.The harm caused by inhalation of particulate isplain. Their surface can condense nitrogen and sul-fur dioxide vapors and microdroplets, which turninto acids, thus increasing the harm done whenthey are in contact with body tissues. Particles canalso carry heavy metals (lead, cadmium, copper,cobalt and mercury) and carcinogens, such as ben-zene and other aromatic hydrocarbons, easing theirentrance into the respiratory system 12 and increas-ing their likelihood of permanence in it. 18 The ef-fects of pollutants should not be studied in isola-tion: in cities with high levels of air pollution, thesubjects breathe air with diverse pollutants, bothgases and particulate, which act synergistically toharm them. Conclusion The SW area, which usually has the highest ozonelevels in Mexico City, had the highest percentage of neutrophils in BALF, while the NE and NW areas,where most of the industry is located, had the high-est level of breathable suspended particles (PM10)and the greatest number of FB in dogs. Volume 42, Number 4/July–August 1998 943 Bronchoalveolar Lavage in Dogs Table II Comparative Cell Counts of BAL Performed on Dogs from Different Areas Total cells Macrophages Lymphocytes Neutrophils EosinophilisAuthor (location) ( × 10 3 )/mL (%) (%) (%) (%) Vanda (Mexico City)370±65682650.5Mayer (Vienna, Austria)1,425±190512220Vail (Madison, Wisconsin, U.S.A.)400±28779140.64Hawkins (West Lafayette, Indiana, U.S.A.)200±8678756  Total cell values represent the mean ±SD. Figure 6 Histogram representing the mean count of ferruginousbodies per 5 mL of BALF in each area. * P <.01. ** P <.05.
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