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JOURNAL OF AEROSOL MEDICINE Volume 3, Supplement I, 1990 Mary Ann Liebert, Inc., Publishers Overload of Lung Clearance Is Associated with Activation of Alveolar Macrophage Tumor Necrosis Factor and Fibronectin Release KEVIN E. DRISCOLL, JAMES K. MAURER, and LESLIE L. CROSBY Human Safety Department, The Procter & Gamble Company, P. O. Box 398707, Cincinnati, OH 45239 ABSTRACT This report summarizes recent findings on the relationships among overloaded lung clearance, activation of alveolar macrophages (AM) release of inflammatory mediators and the development of fibrosis using Ti02 as a model nuisance type dust. Briefly rats were intratracheally instilled with 2-100 mg Ti02/kg body weight and AM tumor necrosis factor or fibronectin release determined ex vivo 1, 7, 14 and 28 days after exposure. Lung dust burdens were determined 1 and 28 days after exposure. Histopathology was assessed 28 and 90 days after exposure. Intratracheal instillation of 50 mg/kg Ti02 resulted in overloaded lung clearance. Ti02 doses >50 mg/kg stimulated transient increases in AM TNF release and a persistent increase in AM fibronectin secretion. Histopathology demonstrated dose-related interstitial inflammation with fibrosis developing only after treatment with 250 mg/kg Ti02. Results from these studies suggest activation of AM secretory activity may play a key role in adverse pulmonary responses to high dust burdens of relatively innocuous materials. Studies investigating in vitro responses of AM to dust indicated that direct Ti02:AM interaction does not stimulate release of TNF or fibronectin, however, pre-exposure to y-interferon can render AM responsive to Ti02 with respect to increased TNF release. INTRODUCTION The secretory activity of alveolar macrophages (AM), while important to lung defense, is also thought to play a key role in the pathogenesis of interstitial lung disease. A variety of studies have demonstrated that pneumotoxic dusts such as crystalline silica and asbestos can activate AM to release factors which recruit and activate inflammatory cells, as well as, stimulate fibroblast proliferation and collagen synthesis (Davis, 1986). Two factors whose release is increased after Si02 or asbestos exposure are tumor necrosis factor (TNF) and fibronectin (Rom et al., 1986; Dubois et al., 1989; Driscoll et.al., 1990a) . TNF is a 17 kd peptide which possesses a number of pro-inflammatory activities. Fibronectin, a 440 kd glycoprotein, is a chemotactic and growth factor for mesenchymal cells. Thus, both these macrophage-derived mediators have the potential to play an important role in pulmonary inflammation and tissue repair processes. Key Words: alveolar macrophage, tumor necrosis factor, fibronectin, fibrosis, titanium dioxide, clearance overload S-83 In contrast to Si02 and asbestos, nuisance-type dusts, under most circumstances, are not associated with adverse pulmonary responses. However, exposure to relatively innocuous dusts can result in chronic inflammation and fibrosis when pulmonary dust burdens are reached which overload normal particle clearance mechanisms (Morrow, 1988; Oberdorster, 1988). Chronic inflammation and fibrosis have been observed at excessive lung burdens of titanium dioxide (Ti02), diesel exhaust and test toner; materials thought to possess a low degree of toxicity (Lee et al, 1986; Wolff et al., 1987; Muhle et al., 1988). At present, there is limited information on similarities and/or differences in mechanisms underlying the pulmonary responses to highly toxic dusts versus relatively innocuous dusts under conditions of clearance overload. This manuscript summarizes some of our recent findings on activation of alveolar macrophage secretory activity after exposure to excessive lung burdens of Ti02, a material generally regarded as a nuisance-type dust. Results presented demonstrate that at Ti02 lung burdens which overload clearance, AM become activated to release TNF and increased levels of fibronectin, responses associated with an infiltration of inflammatory cells and development of fibrosis, respectively. Additionally, results from in vitro exposure of AM to Ti02 or Si02 indicate the lung environment plays a key role in AM activation under conditions of overloaded clearance and suggest y-interferon may represent one factor which is important in rendering AM susceptible to activation by Ti02. MATERIALS AND METHODS Animals Specific pathogen-free male Fischer 344 rats weighing 180-200g (Charles River Breeding Laboratories, Kingston, NY) were housed in an air conditioned room (25C, 50% RH) on a 12 hour light/dark cycle. Animals were provided Purina Rodent Chow (5001) and tap water ad libitum throughout the study. D_ua.t_Exposures for Bronchoalveolar Lavage Studies The general experimental design and intratracheal instillation procedure have been described previously (Driscoll et al., 1990a). Briefly, animals were intratracheally instilled with sterile saline (control group) or saline suspensions of Ti02 (Anatase; Fisher Scientific, Fair Lawn, N J ) . Ti02 was heated to 200C for two hours for sterilization prior to use. The particle size (Feret's diameters) determined microscopically after 15 minutes of sonication was 2.1 1.5 (im and surface area determined by nitrogen adsorption was 8.8 m 2/ g . Instillations were performed at a dosing volume of 1.0 ml/kg body weight with Ti02 doses of 5, 10, 50, and 100 mg/kg body weight. Intratracheal instillations were performed on lightly anesthetized animals (Na pentabarbital, intraperitoneal injection; 3 mg/kg) using a pediatric laryngoscope and a #20 ball tipped dosing needle attached to a 1 ml syringe placed 1 cm into the trachea. Bronchoalveolar Lavage and Cell Culture At 1, 7, 14, and 28 days post-instillation 5 or 6 animals/treatment group were sacrificed by intraperitoneal injection of Na pentobarbital (50 mg/kg) and exsanguination via the abdominal aorta. Bronchoalveolar lavage (BAL) and AM culture were performed as described previously (Driscoll et al., 1990a). Briefly, the trachea was cannulated and the lung infused 6 times with Ca/Mg-free phosphate buffered saline solution (pH=7.2; PBS) at a volume of 8 ml/wash. The bronchoalveolar lavage fluid (BALF) was centrifuged at 300 x g for 10 minutes, and the cell pellet resuspended in RPMI 1640 (Gibco, Grand Island, NY) containing 25 mM HEPF.S buffer. BALF cell number and viability were determined by hemocytometer counting and trypan blue exclusion, respectively, and cell differentials were performed on cytocentrifuge preparations, fixed in methanol and stained with Diff QuikTM (American Scientific) . The BALF cell suspensions were a -35mm p a humi ri rusec incuba fracti filter until deterrr deterrr Histor anima ether inf us( sect i; Massor in .vit descr obtair media 10 6 1 steri. 100, suspe: maint, analy exper Addit pretr 24 hr Pulmo Ti02 sacri t rimir deter Stati are p bet we compa was c 100 n Ti02 / were adjusted to a concentration of 1 X 106 viable AM/ml and one ml seeded into 35mm plastic tissue culture dishes. AM were allowed to adhere for 60 minutes in a humidified incubator (37C and 5 % COj) after which adherent monolayers were rinsed vigorously 3 times with RPMI 1640 media. The rinsed monolayers were incubated (37C and 5% CO2 ) for 24 hr in 1 ml RPMI 1640 containing 2 mg/ml fraction V bovine serum albumin (BSA; Sigma). The AM conditioned media was filtered (0.45 (im pore size) to remove nonadherent cells and frozen at -70C until analysed for the presence of TNF or fibronectin. TNF bioactivity was determined using an L929 cell lysis assay and immunoreactive fibronectin was determined by ELISA as described previously (Driscoll et a l ., 1990a, b) . Histopathology On days 28 and 90 (at 100 mg/kg, day 60) after instillation, five animals/treatment group (at 100 mg/kg, 3 animals/treatment) were sacrificed by ether inhalation and exsanguination via the abdominal aorta. The lungs were infused to 25cm H20 with 10% buffered neutral formalin. Paraffin embedded sections from both lungs were stained with hematoxylin and eosin (H4E) or Masson's trichrome for light microscopic examinations. In Vitro Exposure of AM Methods used for in vitro exposure of F344 rats alveolar macrophages are described in detail elsewhere (Driscoll et al., 1990b and c ) . Briefly, AM were obtained by bronchoalveolar lavage of untreated F344 rats, suspended in RPMI 1640 media and 5 x 105 cells/well seeded for experiments examining TNF release and 2 x 106 AM/well seeded for experiments examining fibronectin release. Heat sterilized (200C x 2 hr) Si02 or Ti02 was suspended at concentrations of 10, 30, 100, or 300 )lg/ml in RPMI 1640 containing 2 mg/ml BSA and 1 ml of the dust suspension added to cultures of adherent AM for 48 hr. All cell cultures were maintained in a humidified incubator at 37C and 5% C02 . AM conditioned media was analysed for several consituents including LDH, TNF and fibronectin. The experiment was repeated 3 times, using cells pooled from at least 5 animals. Additional experiments were conducted to examine the effects of 24 hr pretreatment with 500 units/ml rat y-interferon on TNF release upon a subsequent 24 hr exposure to 100 |lg/ml Si02 or, 100 and 1000 Hg/ml Ti02. Pulmonary Dust Retention Groups of 15 rats were intratracheally instilled as described above with Ti02 at 2, 5, 10, 50, and 100 mg/kg body weight. Five animals/g roup were sacrificed at 1, 7, and 28 days after treatment, the lung3 removed enbloc, trimmed, weighed and lyophylized. Titanium levels in lung tissues were determined as described previously (Driscoll et al., 1990b). Statistical Analysis All data are expressed as actual values except dust retention data which are presented as a percentage of the day 1 group mean lung burdens. Differences between treatments were evaluated using a one-way ANOVA, followed by pairwise comparisons using the Newman-Keuls test (Zar, 1984) . Statistical significance was considered at p<0.05. RESULTS The lung burdens determined 24 hr after instillation of 2, 5, 10, 50 or 100 mg/kg Ti02 were 0.4 0.1, 1.1 0.2, 2.0 0.3, 8.8 0.5, 15.2 + 0.5 mg Ti02/g lung weight, respectively. Dust retention 28 days after instillation as a S-85 percentage of the day 1 lung burdens are shown in Figure 1. Instillation of 50 and 100 mg/kg Ti02 resulted in increased dust retention relative to that observed for instilled doses of 2, 5, or 10 mg/kg. I I 2 mg/kg V/A 5 mg/kg c o C 0> r- 75 ra O o 50 mg/kg 100 mg/kg Figure 1. Retention of Ti02 28 days after instillation. Results are presented as a percentage of lung burdens determined 24 hr post exposure, X+SE; N=5. (*) denotes a significant difference from 2, 5, and 10 mg/kg dose groups; p<0.05. Principal histological changes 28 days after Ti02 treatment consisted primarily of particle laden macrophages and interstitial inflammation with responses 90 days after exposure being of similar or decreased severity relative to those at day 28. Masson's trichrome stained sections were evaluated for increased prominence of collagen, which was interpreted to reflect fibrosis. At 28 days no fibrosis was observed for Ti02 exposed rats, however, at the later times fibrosis was present at Ti02 doses >50 mg/kg. The effects of dust exposure on AM release of TNF or fibronectin are described in detail elsewhere (Driscoll et al., 1990a and b) and summarized with histopathology (collagen staining) and lung burden data in Table 1. Ti02 at doses of 50 or 100 mg/kg stimulated transient increases in AM TNF release, with lower dose levels eliciting no significant TNF response. Instillation of 50 or 100 mg/kg Ti02 stimulated AM to release increased levels of fibronectin, in contrast, exposure to 10 mg/kg elicited only a transient (day 7) increase in fibronectin release and treatment with 5 mg/kg did not stimulate a fibronectin response. The effects of in vitro Si02 or Ti02 exposure on release of LDH, TNF and fibronectin are summarized for doses of 30 and 300 |ig/ml in Table 2. Si02, but not Ti02, resulted in increased LDH as well as TNF release. In contrast neither dust had a significant effect on fibronectin secretion. The effects of y-interferon pretreatment on dust-induced AM TNF release are shown in Figure 2. Treatment with y-interferon alone elicited small but significant increases in AM TNF release. Pretreatment with y-interferon followed by Si02 resulted in an increase in AM TNF release which was additive (equal to Si02 alone plus y-interferon alone). In contrast, combined treatment with 1000 (ig/ml Ti02 and y-interferon resulted in a stimulation of TNF release which was synergistic. Treatment of y-interferon primed cells with 100 |ig/ml Ti02 did not stimulate TNF release over that elicited by y-interferon alone. DISCUSSION A number of studies have demonstrated that lung burdens of low solubility dusts which overload normal lung clearance mechanisms are associated with development of chronic pulmonary inflammation, fibrosis and in some instances lung tumors (Morrow, 1988; 0berdor3ter, 1988) . Our laboratory has been investigating mechanisms underlying the pathogenesis of dust-induced interstitial lung disease. In the present report we summarize our recent observations on activation of AM secretory activity after exposure of rats to high and low lung burdens of Ti02, a material generally considered to be a nuisance-type dust. The results discussed demonstrate an association between overload of lung clearance, stimulation of AM TABLE 1. Pulmonary Responses to Intratracheally Instilled TiC>2 Relative to a Saline Instilled Control Group: Macrophage TNF and Fibronectin Release, Dust Retention, and Development of Pulmonary Fibrosis3 . Ti02 Dose 100 mg/kg 50 mg/kg 10 mg/kg 5 mg/kg AM TNF Release13 day 1 day 7 day 14 day 28 +- +- -+ + - -- - - - -- AM Fibronectin Release13 day 7 day 14 day 28 Dust Retention day 28 +++ increased +++ increased + - - normal "-- normal Fibrosis day 28 day 90c -+ -+ -~- 3 changes in dust retention are relative to Ti02 retentions observed for dose groups <10 mg/kg k (+) = statistically significant increase above control; (-) not statistically different from control c day 60 for 100 mg/kg groups S-87 TABLE 2. Response of Rat Alveolar Macrophages to In Vitro Treatment with Si02 and Ti02 (N=3; XSE) Treatment Lactate Dehydrogenase (units/106cells/24 hr) Tumor Necrosis Factor <units/106cells/24 hr) Fibronectin (ng/106cells/24 hr) Control 1.9 1.4 <2 Si02 : 30 |ig/ml 300 )ig/ml Ti02 : 30 |ig/ml 300 )ig/ml * 7.3 0.3 ,, 26.0 0.3 0.8 0.2 1.3 0.2 12.2 2.2 * 52.2 5.3 * <2 <2 fa significantly different from control; p<0.05 67.1 7.7 68.8 3.9 68.2 4.6 75.6 13.1 56.9 9.6 I I control 30 r H S I0 2 100 ng/ml E7] T i0 2 100 ug/ml Figure 2. Effect of a 24 hr pretreatment with 500 u/ml rat yinterferon on Ti02 and Si02induced AM TNF release. Data are presented as the XSD, N=3 . (*) denotes statistically significant difference from appropriate non dust exposed control; p<0.05. preincubation in media alone preincubation In Y-interferon to release TNF and fibronectin, and the eventual development of fibrosis. The in vitro data presented indicate that the pulmonary environment plays a key role in activation of AM after dust exposure and suggests y-interferon represents one factor with the potential to render AM susceptible to activation by Ti02. To further elucidate the mechanisms underlying the activation of inflammatory processes in the lung after mineral dust exposure, we have focused our attention on the AM. This cell, because of its ability to release mediators which can modulate the activities of inflammatory and immunocompetent cells, has the potential to play a key role in the initiation and perpetuation of inflammatory responses within the lung. TNF represents an AM-derived cytokine with pro-inflammatory activities. This cytokine can stimulate expression of adhesion molecules for inflammatory cells on capillary endothelium (Bevilacqua et al., 1989); activate release of neutrophil and monocyte chemotactic peptides by a variety of cells including macrophages, fibroblasts and endothelial cells (Larsen et al., 1989; Strieter et al., 1989); and stimulate phagocytic cells to release reactive oxygen species and lysosomal enzymes (Klebanoff et al., 1986; Tsujimoto et a l ., 1986). Thus, this cytokine has the potential to play a key role in the recruitment and activation of inflammatory cells to the lung after inhalation of noxious materials. More recently, the key role of TNF in lung fibrosis was demonstrated by Piguet et al. (1989, 1990) who reported that treatment of Si02 or bleomycin exposed mice with anti-TNF antibodies markedly attenuated collagen deposition. In the present report we summarize results suggesting a positive association between intratracheally instilled doses of Ti02 which overload lung clearance and activation of AM TNF release. Interestingly, statistical evaluation on an individual animal basis of the relationship between dust-induced increases in ex vivo AM TNF release and numbers of neutrophils in bronchoalveolar lavage fluid reveal a significant positive correlation (Driscoll et al, 1990a). This association strongly suggests a role for TNF in the recruitment of inflammatory cells under conditions of excessive dust exposure. Given the known pro inflammatory activities associated with this cytokine it is likely that TNF, at least in part, plays a role in the inflammation and fibrosis we observed at excessive lung burdens of Ti02. In addition to TNF, AM can release fibronectin, a mediator with the potential to influence development of pulmonary fibrosis. Fibronectin is chemotactic for fibroblasts (Postlethwaite et al., 1981); can mediate interactions between a variety of cell types and the extracellular matrix (Mosher, 1984; Macarak and Howard, 1983); and, acting in concert with other growth factors can stimulate fibroblasts to proliferate (Bitterman et al., 1983; Bitterman et al., 1986). Thus, fibronectin is thought to play a critical role in tissue repair; functioning to recruit cells to sites of tissue injury and inflammation, facilitating cell attachment to extracellular matrix proteins and promoting local cell proliferation. Excessive Ti02 lung burdens stimulated release of fibronectin, a response which persisted through the 28-day post instillation period. In contrast, dust burdens which did not overload lung clearance elicited only transient or no increase in fibronectin release. Imp eve precoa fib. s iti our supi of fib rel< a re puli dus und' lym; sub pre bal: lym; int7 -ir (10 obsi AM com app; of : fac ini' inf. to . lune may, fac insfor cel dev. pat': the fib suge resf Adair Rev Bevi turne ende and Bitt fibr Bitt Importantly, persistent increases in fibronectin release were associated with eventual increases in collagen deposition (i.e., fibrosis). In this respect, previous studies have demonstrated that individuals with silicosis, asbestosis or coal worker pneumoconiosis have AM populations releasing increased levels of fibronectin (Rom et al., 1986) as well as increased fibronectin deposition at sites of pneumoconiotic lesions (Wagner et al., 1982) . The prospective nature of our observations on increased fibronectin release and collagen deposition further support an important and early role for AM-derive fibronectin in the development of pulmonary fibrosis and suggest similarities in mechanisms of pulmonary fibrosis exist between highly toxic dusts and excessive lung burdens of relatively innocuous materials. The mechanisms by which Ti02 stimulates AM to release TNF and fibronectin are unknown, however, results from our in vitro experiments indicate the pulmonary environment plays a key role. In this respect, the response seen after du3t instillation could involve cells and or secondary mediators not present under in vitro exposure conditions. One cytokine, released by activated lymphocytes, which can "prime" macrophages to become more responsive to subsequent stimulation is y-interferon (Adams and Hamilton, 1984). Since we previously observed that high doses of Ti02 result in persistent increases in BALF lymphocyte numbers (Driscoll et a l ., 1990a), and thus, potentially increased lymphocyte secretory products, we investigated the effects of in vitro y- interferon pretreatment on responsiveness of AM to Ti02. These results indicated y-interferon can prime AM to release TNF upon in vitro exposure to high doses (1000 (ig/ml) of T i 0 2 . It is noteworthy that the priming response was only observed for the high dose of Ti02, suggesting that this effect may be unique to AM which become engorged with particulate materials such as occurs in vivo under conditions of overloaded clearance. Interestingly, a similar response was not apparent for Si02 exposure which suggests differences may exist in the mechanism of Si02 and Ti02-induced TNF release. These findings demonstrate that secondary factors can increase responsiveness of AM to dust exposure and suggest y- interferon has the potential, if released upon in vivo dust exposure, to influence AM responsiveness to Ti02. In conclusion, the findings summarized in this report suggest that similar to highly toxic dusts (e.g., Si02 and asbestos), the development of interstitial lung disease in animals exposed to excessive lung burdens of nuisance-type dusts may, at least in part, result from activation of AM to release pro-inflammatory factors. The correlation, observed between ex vivo AM TNF release after instillation of Ti02 and neutrophil numbers in BAL fluid clearly suggests a role for this cytokine in dust-induced recruitment and activation of inflammatory cells. Further, the association of persistent AM fibronectin release with the development of fibrosis provides additional support for this mediator in the pathogenesis of interstitial lung disease. 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