Document EvZdLeN61oqQbyG0Qbj58zwLx

lYMPHOC ymphocyte Populations in Lung Tissue, Bronchoalveolar Lavage Fluid, and Peripheral Blood in Rats at Various Times at '! during the Development of Silicosis1-3 Bronch exsangi cutting cannul: and the Next, a nectedt DAVID STRUHAR, RONALD J. HARBECK, and ROBERT J. MASON salt soli (GIBC( msertec pulmor Introduction Silicosis is a chronic progressive granu- lomatotic-fibrotic lung disease caused by the inhalation of various forms of crys talline silica. In the naturally occurring disease in humans and in the experimen tally induced disease in animals, the ini tial histopathology of the silicotic nod ule is characterized by aggregates of mac rophages (1). This is followed by the infiltration of lymphocytes and a few neutrophils, along with alveolar type II cell hyperplasia. Later, focal fibrosis with brobiasts surrounding the zone of lym.. nocytes and macrophages appears. This may enlarge progressively. For a brief period of time after induc tion of silicosis, the bronchoalveolar la vage (BAL) fluid shows an inflammato ry cell accumulation (2). Later, there is an increase in the number of macro phages and lymphocytes, with the occur rence of occasional neutrophils. In early studies in which the interaction between silica and alveolar macrophages was ex amined in vitro, injury occurred to alve olar macrophages after the phagocyto sis of quartz particles (3). However, when alveolar macrophages from humans and animals were exposed to silica in vivo, injury was not apparent (2, 4, 5). The quartz particles were found to be coated with alveolar lining material which was thought to render them less toxic (6). Nevertheless, the ingestion of silica par ticles by the macrophages leads to the secretion of various factors, such as de rivatives of arachidonic acid, monokines, and growth factors (7-10). It is thought that one of the results of the secretion of these factors was an amplification of 'he initial response, i.e., lymphocyte re cruitment, and secretion of chemotactic and activating factors for macrophages. In animal models of bleomycin-in duced fibrosis, lymphocyte infiltration into the lung has been studied. There is a transient increase in the helper to sup SUMMARY Inflammatory celts and lymphocyte populations were examined in the bronchoalveolar lavage (BAL) fluid, lung tissues, and peripheral blood from rats at various times after the intratracheal instillation of silica. In lavage fluid, there was a rapid initial increase in the percentage and number of polymorphonuclear leukocytes (PMN), which slowly decreased during the course of the experi ment. In addition, compared to controls, there was an increased number and percentage of lympho cytes throughout the 75 days of the experiment. The lymphocytic populations, which were deter mined by an indirect immunofluorescence method with monoclonal antibodies to lymphocyte sur face markers, showed a predominance of the T-helper phenotype from Day 14 through the end of the experiment (Day 75). The number of PMNs obtained from collagenase digest of the lung was increased over control levels up to Oay 7 after silica administration and remained at a relatively constant level until Day 14, after which time they decreased slightly in number. The total number of lymphocytes peaked on Oay 14, with cells of the T-helper phenotype predominating after this time. In the peripheral blood, T-helper cells from silicotic rats were significantly increased over con trol rats on Days 7 and 14 but returned to normal control values after this time. The lymphocyte subsets in the BAL, but not in the peripheral blood, more closely reflect the lymphocyte patterns in the lung. The results of these experiments suggest that T-helper cells may play an important role in the inflammatory-fibrotic events in the lungs of rats with silicosis. AM REV RESPIR DIS 1989; 139:28-32 pressor ratio (11). Nevertheless, there was a prevalence of suppressor cells during the course of the disease, which correlat ed with both the decrease in collagen production and disease regression. The purpose of this study was to ex amine the inflammatory cells in the lungs during the development of silicosis in the rat. We compared the lymphocyte popu lations present in BAL fluid to those pres ent in lung tissue at various times during the development of the silicotic process. Our findings showed a predominance of helper T-cells relative to the suppressor T-cells in both the lavage and lung tissue during the development of silicosis. In addition, these changes in the BAL close ly reflect changes that occur in the lung, whereas the peripheral blood does not. mal care facility. Before killing, all animals were anesthetized with pentobarbital (2.5 mg/kg) and anticoagulated with 1,000 U of heparin. Before processing, the lungs of con trol and silicotic animals were examined macroscopically to exclude pulmonary disease caused by infection. Induction of Silicosis A tracheostomy was performed on all labo ratory animals, and a single intratracheal in jection of 10 mg silica (< 5 pm, Min-U-Sil; Pennsylvania Glass Sand Corp., Pittsburgh. PA) in 0.3 ml of sterile saline was given. Groups of four animals were killed at 3, ' 14, 30, and 75 days after silica instillation. (Received in originalform August 20, 1987 andin revised form June I, 1988) Methods Animats Adult, male, pathogen-free Sprague-Dawley rats weighing 200 to 225 g were purchased from Bantin and Kingman (Fremont, CA). These animals were maintained in a horizon tal laminar flow apparatus until the injection of silica, then housed in a conventional ani 1 From the Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado. * Supported by Grant No. HL-27353 from Ihe National Institutes of Health. 1 Requests for reprints should be addressed w Ronald J. Harbeck. Ph.D.. Department of Me1*1' cine. National Jewish Cemer for Immunology anJ Respiratory Medicine, 1400 Jackson Street, De"' ver, CO 80206. fusion i til the p cannul. inos ed. with 10 HBSS. proxim retrieve phosph aliquot determi prepare a cvtoc stained ing. In clear ce and de overlay um (LS After c the mo remove Lung inflated type I. ( phosph and inc ing wat and ma vortexe (PCS) ( and the asingie through Detroit and fin. Elmsfo used to their vi: a modi II cells Sreater "as sep Perig the aor saline, i tttonon identij Both B subpop Huores surface 28 LYHPH0CYTE populations in rat silicosis 29 a jls of ,>niCiSC -'0 :n Ml ;h ;n on ilK nil 't> ... itw | j :4 ;illjr.J >(- Control groups consisted ot ihree animals jeh from four groups killed 3, 7, 14. and 30 jjys after intratracheal saline administration. Cell Isolation gronchoa/veolar cells. After the animals were exsanguinated, the lungs were collapsed by cutting the diaphragm, and the trachea was cannulated. The thoracic cavity was opened, and the left and right ventricles were incised. Next, an 18-gauge polyethylene catheter con nected to a syringe filled with Hanks' balanced salt solution (without Ca" or MgJ`), (HBSS) (01BCO Laboratories, Grand Island, NY) was inserted through the right ventricle into the pulmonary artery. This was followed by per fusion of the lung circulation w ith HBSS un til the parenchyma appeared white. Next, the cannulated trachea, heart, and lung were re moved, and the lung was lavaged five times with 10 ml each time of 1% lidocaine in cold HBSS. In most experiments, we recovered ap proximately SO07)! of the instilled HBSS. The retrieved cells were washed twice with 0.1 M phosphate-buffered saline (PBS), pH 7.0. An aliquot was removed for counting and for the determination of viability. The viability of this preparation was between 80 and 90%. Also, a cytocentrifuge preparation was made and stained with Giemsa for differential count ing. In order to obtain an enriched mononulear cell preparation and to remove dead cells and debris, the remainder of the cells were overlayered on lymphocyte separation medi um (LSM) (Litton Bionetics, Charleston, SC). After centrifugation at 200 x g for 30 min. the mononuclear cells at the interface were removed and washed three times in PBS. Lung cells. After lavage, the lungs were inflated with 10 ml of 1 mg/ml collagenase, type 1, (Sigma, St. Louis, MO) in Dulbecco's phosphate-buffered saline (DBS) (G1BCO) and incubated at 37 C for 20 min in a shak ing water bath. After dissecting off the hila and major bronchi, the lungs were minced, vortexed for 1 min in 2 ml of fetal calf serum (ECS) (Hyclone Laboratories, Logan, UT), and then filtered through meshes to obtain a single cell suspension. The first passage was through a two-layer gauze filter (Parke-Davis, Detroit, MI), next a four-layer gauze filter, and finally a 20-pm mesh nylon filter (Tekto, Elmsford, NY). An aliquot of these cells was used to determine the total cells recovered, their viability and differential by Giemsa and a modified Papanicolaou for alveolar type II cells (12). The viability of these cells was *tester than 80%. The remainder of the cells was separated on LSM as described above. Peripheral blood. Blood was obtained via the aorta, diluted with an equal volume of saline, then separated on LSM to obtain the tononuclear fraction. identification of Lymphocyte Populations Both B- and T-lymphocytes and T-lymphocyte '^populations were identified by immuno fluorescence using specific antibodies to cell surface antigens, and thf percentage of posi- mc cells was determined bv flow cytometry lymphocyte jnaKsis. Each croup consisted with gating for lymphocytes by forward and ol at least four animals. right angle scatter (Ortho Cytofiuorograph). For the detection of B-!ymphocvtes or sur Statistics face immunoglobulin-bearing (Slg) cells, a Statistical analyses were carried out bv a one fluorescein-conjugated Flab"); preparation of way analysis of variance. goat IgG anti-rat IgG H and L chains (Cap- pel Laboratories, Downingtown, PA) was used. Total T-lymphocytes were identified by using indirect immunofluorescence and a W3/13 monoclonal antibody (Sera Labs. Westbury, NY), while cells expressing the T- Results Lung Injury The histologic evidence of lung injury was similar to what has been reported helper/inducer and T-suppressor/cvtotoxic previously after intratracheal administra phenotype were determined with W3/25 and tion of silica (13). The two major lesions OX-8 monoclonal antibodies (Sera Labs), respectively. To 1 x 106 of each cell prepara tion, 100 pi of an optimal saturating dilution of the appropriate monoclonal antibody was added and incubated at 4 C for 45 min. The optimal dilution of each antibody was deter mined previously on rat spleen cells. All cell suspensions were in PBS containing 0.1% so present in the lungs of silica-treated rats xveresilica-containine granulomas or sili cotic nodules and variable lipoproteinosis. These granulomas, some discrete and some coalescing, were scattered through out the lung lobes, with some localiza tion around airways. Within the granu dium azide and 1% FCS. After incubation, lomas, macrophages were seen surround the cells were washed with cold PBS and cen ed by lymphocytes. In addition, there trifuged at 200 x g for 10 min. To the pellet were scattered neutrophils and eosino ed cells, a fluoresceinated Flab'), goat anti mouse IgG (Cooper Biomedical Laboratories, Malvern, PA) was added, incubation was al lowed to proceed for an additional 45 min, and the cells were washed as previously. The cells were fixed with 2% formaldehyde in PBS, and the percentage of positive cells determined phils in the early disease; however, the eosinophils were not present in the ad vanced silicotic lung. Furthermore, there was focal interstitial fibrosis with alveo lar type II hyperplasia occurring first on Day 14. by flow cytometry. Controls for T-cells con sisted of the same cell populations stained BAL Cells with the secondary antibody only, while the There was a rapid increase in the num control for B-celi enumeration was fluoresce ber of inflammatory cells in the BAL flu inated anti-fibrinogen (Cappel Labs) of the id after instillation of silica (table 1). Dur same isotype as the B-cell marker (IgG,). The percentage of cells positive for each marker was determined by subtracting the percent age of positive cells in the control cells from the experimental cells. In order to obtain enough cells from BAL for analysis, the cells were pooled from the non-silica-treated con trol groups and in the 3-day post-silica instil ing the first 2 wk, the change in percent age of cells was attributable primarily to an increase in neutrophil percentage. The number of neutrophils was also relative ly high in those control animals killed 3 days after saline instillation; however, they never exceeded 6% of the total cell lation group. For the other groups, a suffi count (data not shown). The number of cient number of cells was recovered from in BAL lymphocytes increased with disease dividual animals in order to perform the progression, with an increase in both the TABLE 1 CELL TYPES RECOVERED FROM BAL FLUID Cell Type Controlstt 3* Days After Silica Administration* 7 14 30 75 Macrophages. % Lymphocytes, % Neutrophils. % Eosinophils. % Total 93 a 2.0< 2 0.5 4 2 0.5 1 2 0.5 8 a 1 oS 69 * 3.0 4 * 1.0 22 * 3.0 4 2 10 12 2.0 60 2 3.0 15 2 2.0 20 2 2.0 4 * 1.0 20 - 3.0 50 * 4 0 29 2 3.0 16 * 2.0 4 * 1.0 22 2 2.0 53 2 3.Q 35 2 4.0 11 2 1.0 1 2 0.5 20 2 2.0 54 2 3.0 35 r 3.0 9 2 10 1 2 0.5 19 2 2.0 An the results lor animals receiving silica are statistically significant (p < 0 OS) from control results exceoi for the oercentage of eosmoohils at Oays 30 and 75 after silica administration (p > 0.05) r The control group consisted of three animals eacn from four groups killed on Oays 3. 7. u. and 30 after mtratracneai saline administration * In order to ootam enough ceils lor analysis, the differential counts for the control group and the group of animals receiving silica and killed 3 days later represent tne number or each ceil fype/ammai Irom lour experiments witn the cells pooled from tnree ammais/expenmeni For the control group, the four expenments are trom animals killed on Oays 3. 7 u. or 30 arter saune ad ministration. ' Mean percentage of cells/animal SEM i Total numoer of cells x tC/ammal r SEM 30 STRUHAR MAR8ECK. ANO MASQs TABLE 2 TOTAL LYMPHOCYTE POPULATIONS IN 3AL FLUID (x 10* CELLS! Days After Silica Administration" Ceil Phenotype Lympnocytes Total T (W3/13) T-helpers (W3/25) T-suppressors (OX-8) 0-cells (Slg) Controls^* 0.16 0.01& 0 08 0.01 0.05 0.01 0.04 0.01 0.02 tOOl 3^ 0 4a 0.03 0.28 0 03 0.16 * 0.02 0.13 * 0.02 0.07 0.01 7 3.00 0.06 1 85 0 07 0.90 0 09 1.00 0 10 0.62 0.04 14 6.38 0.15 3.60 * 0 24 2.45 * 0.07 1.63 * 0 09 1 20 0 18 30 7 00 0 24 5 94 0 23 4.45 0 23 1 53 * 0 14 0 75 aOll 6 65 0.24 4 68 0 34 3 16 r 0.23 1 86 0 12 0 84 0 12 All th values tor the silica-treated rats are statistically different from the control values (p < 0 05) t The control group consisted of three animals each from tour groups killed on Oays 3. 7. 14. and 30 after intratracheal sa*<ne aom.mstrarion t in order to oOtam enough cells for analysis, the differential counts tor the control group and the group of animals receiving s<a ana miieo 3 aavs later represent the number of each cell type/animal from four experiments with the cells pooled from three animaisienperimen: For the control group the four experiments are from animals killed on Oays 3. 7. 14, or 30 after saline administration i Mean number o! positive cells/ammal SEM % Positive Lymphocytes total number of T- and B-cells (table 2). There was a nearly equal ratio of T-helper to T-suppressor cells through Day 7 af ter silica administration, with a slight in version of the ratio on Day 7 (figure la). However, on Days 30 and 75 there was a significant increase in this ratio (p < 0.05). Lung Tissue Cells There was an increase in the total num ber of inflammatory cells obtained from Fig. 1. Percentage of T-tymphocytes bearing the T-help er (circles) or T cytotoxic-suppressor (squares) pheno type obtained from 6AL fluid (a), coilagenase-digested lung tissue (b), and (he penpherat blood (c) from rats at vanous times after the administration of silica. The C in each panel refers to the control, non-siiica-treated rats. the dissociated lung tissue through the first 14 days after silica injection, although the increase in the total number of cells was proportionally less dramatic than that observed for the BAL cells (ta ble 3). On Day 75, a slight reduction in the total number of cells from Day 30 was observed, although the number of cells recovered at this time was still high er ihan the number of cells recovered from the control rats. The increased num ber of inflammatory cells infiltrating the lung tissue during the course of the dis ease vvas attributed to a generalized in crease in all types, including both lym phocytes and macrophages. The exami nation of neutrophil presence within the lungs showed a peak at Day 7, with a re turn to control levels by Day 75 after sili ca administration. A similar pattern was noted for the percentage of eosinophils infiltrating the lungs. The number of lymphocytes obtained from the digested lung tissue showed an increase through 14 days after silica administration, after which time there was a slight decrease in the total number recovered (table 3). However, animals killed at 75 days had levels of lymphocytes nearly three times that of the control animals. The numbers of both B- and T-lymphocytes showed a slight increase through Day 14 followed by a slight decrease through Day 75. The T-helper to T-suppressor cell ratio on Days 3 and 7 after silica administration was similar to the ratio obtained for the control animals, with an inversion of the ratio at 3 days followed by a further drop in the ratio at 7 days. Finally, there was a change to a higher ratio on Days 1530, and 75 (p < 0.05, figure lb). Days After Silica Instillation Peripheral Blood Lymphocytes Some degree of fluctuation occurs in the peripheral blood lymphocyte populations during the course of the disease (tig' ure 1c). The most dramatic change ov- VND MASOx iugh the tion, alnumber Jramatic cells (taiction in Day 30 mber of till highecovered sed numatine the .thf dizd<_ oth lyme examiithin the vith a reafter sili:tern was .inophils mber of digested through m, aftet crease in table 3). Jays had ree times numbers .howed a followed y 75. The ratio on listration d for the on of the her drop here was Days b). vies ars in the populaease(H?` ange oc- lymphocyte populations in rat silicosis 31 TABLE 3 INFLAMMATORY CELLS- RECOVERED FROM COLLAGEN A 5E-DIGES7ED LUNG TiSSUE Days After S/fica Administrationt Cell Type Controls^ 3* 7 14 30 75 Macrophages. % Lymphocytes. % Neutropfl'ls. % eosinophils. Total 20 3 Oil 10 2.0 2 0.2 1 * 0.2 80 2.21 25 * 2.8 15 1 5 4 .10 3 1.0 85 2 4 30 r 2 9 18 1 6 13 2.2 5 15 95 1.9 30 3 0 30 3 2 10 1 4 5 11 no 1.1 30 r 3.0 27 2 8 3 05 2 l0 110 1 2 30 3.2 25 3.0 2 05 0 95 r 1 1 * The remainder of the cells reoresenrs fiprooiasts eo'fheliai atveotar cells mast ceils, ana various other nonioentitieo lung -arenchymal cells f AH me results are statistically stgnilicani ip < 0 05) from control results e*ceot tor ine percentage of neutrconiis on Oay 75 jtter sdica administration Ip > 0.05) t fhe control group consisted of three animals each irom four groups `r'lied on Days 3 7, ta and 30 after mtrairacneal saline 3<jminstralion 11n order lo ooiain enough cells for analysis, the differential counts lor me control group and tne group ol animals receiving silica and Kilted 3 days later represent me numoer of each cell type/ammai from four experiments iih me cells pooled from three jmmais/expenment For tne control grouo. the four experiments are from animals <;iied on Oa>s 3 7. u. or 30 after saline ad- -cnisttaimn Mean percentage of cens/animal r 5EM 1 Total numoer of cells/animal x I04 : SEM. curs on Days 7 and 14 after silica adminis tration, where the percentage of T-helper cells increases from 35 to 54fo (p < 0.05, figure 1c). This increase in T-heiper cells precedes the increase noted in the BAL and lung tissue by 1 wk. There is no statistically significant difference at any of the other time points for the total num ber of peripheral blood T-cells, T-suppressor cells, or B-cells between control ani mals and animals receiving silica (p > 0.05). Discussion The results of this study demonstrate an increase in the total number of inflam matory cells including lymphocytes and macrophages in the lungs of rats during silica-induced disease. This increase oc curs both in the lung tissue and in the BAL fluid. Initially, PMNsare recruited to the lung, and later these are replaced by mononuclear cells. This sequence of events is in accordance with earlier re ports although there was no enumeration of the cells (13, 14). A previous study had suggested that the BAL cells may be rep resentative of the cell populations in the lung (15); however, our data show that there is an earlier and larger percent in crease of PMN in the BAL. Nevertheless, it may provide insight into changes oc curring in the lung tissue. The concomitant increase in the num ber of B-lymphocytes is not surprising because it is known that silicotic lung dis ease is accompanied by the presence of immunoglobulin in the lavage fluid of persons exposed to silica (16) as well as by the presence of autoantibodies and im mune complexes in the serum of patients with silicosis (17). However, the role of B-cell or B-cell products in the fibrotic response of the lung is unknow n. An in crease in the total number of T-cells in the lung tissue was observed 3 days after silica exposure and this increased progres sively until 14 days, when the number of cells recovered plateaued. Furthermore, there was a dramatic increase in the num ber ol'h mphocytes obtained in the BAL. The T-helper to T-suppressor cell ratio in the lung of rats was inverted on Dav 5 but then reverted back to a ratio on Day 7, nearly equivalent to that of the control rats. The ratio increased on Day 14 and was nearly 2: L on Days 30 and 75. A simi lar alteration in the T-helper to T-suppres sor ratio occurred in the BAL. but with out the inversion of the ratio on Day 3. In the peripheral blood, an increase in the T-helper to T-suppressor cell ratio oc curred 1 wk before the alterations in the BAL and lung tissue. This suggests the origin of the T-helper cells in the lung and BAL may be from the blood, perhaps in response to mediators released by the initial inflammatory ceils in the lung. One could speculate that the inversion of the T-helper to T-suppressor cell ratio in the lung on Day 7 may represent a migration of sensitized lung T-helper cells into the blood or lymph nodes where systemic amplification might result in a re-entry of an expanded population of these cells back to the lung at a later time. Lymphocytes and macrophages ap peared in close proximity to one another in developing silicotic nodules in both an imal models and human cases. Increased proportions of lymphocytes were found in BAL specimens from animals develop ing silicosis (18) and from humans with silica dust exposure (3, 19). However, the lymphocyte subpopulations and their traffic during the development of the dis ease were not studied. The cell-mediated immune responses have been reported to be normal in silicosis (20). In our study, the absence of significant change in the lymphocyte subpopulations in peripheral blood of rats with silicosis could support these findings. In contrast, in sarcoidosis, which is also characterized by lung lym phocytosis with helper preponderance, there is a decrease in cell-mediated im munity (21). TABLE 4 TOTAL LYMPHOCYTE POPULATIONS IN LUNG TISSUE OF RATS (x 10* CELLS) Oays After Silica Administration * Controls^* 3* 7 14 30 75 Lymphocytes Total T (W3/13) T-hetpers (W3/25) T-suppressors (OX-8) B<eits (Slg) 8.0 1.0 4 7 0.2 2.2 0.2 2 9 0.2 1Q Q1 12.8 2.0 7 2 0.3 4.3 02 3.4 0 2 2.5 0.2 17 1 2.0 9.9 0.2 4.3 0.1 5 4 0.1 2.7 * 0.5 33.0 3 0 19.7 1.6 13.4 0.9 8.1 0.8 5.6 1 0 29.7 3.0 18.3 1 9 13.6 1.6 6 2 * 1.0 4 0 1.4 23 8 r 3.0 15 2 1.7 11 2 1.6 5 9 1.0 3 1 1.1 * All the values for ihe siiica-trealed rats are statistically different from the control values (p < 0.05) t The control group consisted of three animats each from four groups Killed on Oays 3. 7 i. and 30 after intratracheal saline administration t,ln order to obtain enough cetts for analysis, the differential counts for the control grouo and the group of animals receiving silica and Killed 3 days later represent the numoer of each cell type/ammai from lour experiments with the cetts oooted from three ammals/expenment $ Mean number of positive ceils/ammal * SEM. 32 STRUHAH. HARBECK. ANO MASOi, The role of T-helper cell influx and per ils . in the lungs of silicone animals is u. -ar. In a previous investigation in which bleomycin was used to induce lung fibrosis (12), it was shown that at 14 days after bleomycin treatment, when there was a T-helper cell predominance in the lungs, the rate of collagen synthesis was significantly elevated, whereas at 30 days, when there was a predominance of sup pressor cells, the rate of collagen synthe sis was within normal limits. It has been suggested that the T-helper cells in the lung of bleomycin-treated animals may olay a role in enhancing collagen synthe sis (21). In silicosis, there is a progressive increase in lung collagen. Hence, the correlation between increased T-helper cells and collagen deposition may also oe true in silicotic lung as well. In summary, we were able to show that n the animals with silicotic lung disease he increase in the percentage of cells e.\iressing the T-helper phenotype preced 'd the increase of these cells in the BAL tnd lung tissue digest. Although the Mood T-helper and T-suppressor ratios .turned to levels comparable to levels biam-d in control animals, the T-helper red ance in the BAL and lung diest p--sists. Acknowledgment he writers thank Judith Pasternak for her xhnical assistance, and Suzanne Galt for her reparation of this manuscript. References I. Spencer H. Pathology of the lung. 3rd ed. Philadelphia: WB Saunders. 1977; 379-95. 3. Davis GS. Hemenwav DR. Evans JN. Lapenas DJ. Brody AR. Alveolar macrophages stimulation and population changes m silica-exposed rats. Chest 1981: S0<S):8-10. 3. Allison AC. Hartngton JS, Birbeck M. An ex amination of the cytotoxic effects of silica on mac rophages. i Exp Med 1966: 124:191-58. 4. Christman JW, Emerson RJ. Graham WGB, Davis GS. Mineral dust and ceil recovery from the bronchoalveoiar lavage of healthy Vermont gran ite workers. Am Rev Respir Dis 1985; 132:393-9. 5. Brody AR. Roe MW. Evans JN, Davis GS. Deposition and translocation of inhaled silica in rats. Quantification of particles distribution, mac rophage participation, and function. Lab Invest 1982: 47:533-42. 6. Emerson RJ. Davis GS. Effect of alveolar lin ing material-coated silica on rat alveolar macro phages. Environ Health Perspect 1983; 51:81-4. 7. Schmidt JA, Oliver CN, Lepe-Zuniga JL, Green I. Gerv I. Silica-stimulated monocytes release fibro blast proliferation factors identical to interleukin-1. A potential role for interleukin-l in the pathogen esis of silicosis. J Clin Invest 1984; 73:1462-72. 8. Lugano EM, Dauber JH, Daniele RP. Silica stimulation of ehemotactic factor release by guinea pig alveolar macrophages. J Reticuloendothel Six' 1981: 30:381-90. 9. Lugano EM. Dauber JH, Elias JA, Bashey Rl, Jimenez SA, Daniele RP. The regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis. Am Rev Respir Dis 1984; 129:767-71. 10. Martin TR. Altman LC, Albert RK. Hender son WR. Leukotriene B, production by the human alveolar macrophages: a potential mechanism for amplifying inflammation in the lung. Am Rev Respir Dis 1984; 129:106-11. 11. Thrall RS. Barton RW. A comparison of lym phocyte populations in lung tissue and in bronchoal- veolar lavage lluid ol rais ai various Mines dun- . I he development ol bleomycin-induced pulmona, fibrosis. Am Rev Respir Dis 1984; 129:279-si 12. Kikkawa Y. Yoneda K. The type II epnhel.j cell of ihe lung. I. Method of isolation. Lab lmc,, 1974; 30:76-S0. 13. Reiser KM, Haschek W M. Hesterberg TU Last JA. Experimental silicosis. II. Long-term cl. fects of intratracheally instilled quartz on collage metabolism and morphologic characteristics of ulungs. Am J Pathol 1983; 110:30-40. 14. Lugano EM. Dauber JH. Daniele RP. Acu1; experimental silicosis. Lung morphology, hivtctlo. gv. and macrophage chemotaxin secretion. Am i Pathol 1982; 109:27-36. 15. HunninghakeGW, KawanamiO. Ferrans V| Young RC. Roberts W'C. Crystal RG. Character, ization of the inflammatory and immune effector cells in the lung parenchyma of patients with inte: stitiai lung disease. Am Rev Respit Dis I9SI 123:407-12. 16. Calhoun VVJ. Christman JW, Ershier WB.Gra ham WGB. Davis GS. Raised immunoglobulin con centrauons in bronchoalveoiar lavage lluid of healthy granite workers. Thorax 1986; 41:266-7: 17. Doll NJ, Stankus RP, Hughes J, etai. Immune complexes and autoantibodtes in silicosis. J Aller gy Clin Immunol 1981; 68:281-5. 18. Callis AH, Sohnle PG. Mandel GS. Weissner J. Mande! NS. Kinetics of inflammatory and fibrotic pulmonary changes in tnuntic model of silico sis. J Lab Clin Med 1985; 105:547-53. 19. Schuyler M, Gauner HR, Stankus RP. Kai mal V, Hoffman E. Saivaggio J. Bronchoalveoiar lavage tn silicosis. Lung 1980; 157:95-102. 20. Schuyler M, Zisking M, Saivaggio J. Cellmediated immunity in silicosis. Am Rev Respir Dis 1977; 116:147-51. 21. Phan SH, Thrall RS, Williams C. Bleomycininduced pulmonary fibrosis. Effects of steroid on lung collagen metabolism. Am Rev Respir Dis 1981; 124:428-34. Bronch in Heal JEANNE MA and DENISE It Although the bestos exposure recognized sine turv, the pathc mg to asbestc related disease on animal moc icms (6-9) haw the acute and si liber depositioi ejaes, asbestos not detected ui posure (8). It 1 Juring this lag chemical and c ed with retentio lung, eventuall; A variety of s normalities rept populations ha immunologic p: development of (8.9.11-13). In ic findings, mor zested that abnc nary immune a present in asbesi and without ast tionship of the development of proved. ''e have pert lavage (BAL) or >ard workers v bestos exposure, her characteriz monary and svst may persist afte Potentially lead *e compared tl and lymphocyte :<1 and pecipht healthy, asbestos ars with that fro 'ithout known P ''bestos-exposed *ere recruited froi