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Modulation of Silica-Induced Pulmonary Toxicity by Dexamethasone-Containing Liposomes
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
142, 411–421 (1997)
TO968057
Modulation of Silica-Induced Pulmonary Toxicity by Dexamethasone-Containing Liposomes MICHAEL DIMATTEO
AND
MARK J. REASOR
Department of Pharmacology and Toxicology, Robert C. Byrd Health Sciences Center of West Virginia University, Morgantown, West Virginia 26506-9223 Received June 20, 1996; accepted October 28, 1996
Modulation of Silica-Induced Pulmonary Toxicity by Dexamethasone-Containing Liposomes. DIMATTEO, M., AND REASOR, M. J. (1997). Toxicol. Appl. Pharmacol. 142, 411–421. Human exposure to silica (SI) is of great occupational concern because it is marked by pulmonary inflammation and fibrosis. Our objective was to determine if early pharmacological intervention altered the inflammatory and fibrotic responses to silica in rats. Male Fisher-344 rats received intratracheal (IT) instillations of the anti-inflammatory steroid, dexamethasone (DEX), incorporated into a novel liposomal (LIP) delivery system (DEX–LIP), or buffer as control (HBSS) on Day 01 and every fourth day until euthanization. On Day 0, the DEX–LIP group received IT instillations of SI (10 mg/100g body wt, DEX–LIP–SI); half of the HBSS group received SI (10 mg/100g body wt, HBSS–SI) and the other half saline (HBSS–SAL). On Day 10 or 20, bronchoalveolar lavage (BAL) was performed for cellular, biochemical, and functional analyses of inflammation and damage. HBSS–SI rats had significant elevations in the neutrophil cell count over HBSS–SAL rats at both times. DEX–LIP treatment markedly reduced these values, indicating that DEX–LIP protected against SI-induced inflammation. In contrast, DEX–LIP did not protect against biochemical (albumin concentration, and b-glucuronidase and lactate dehydrogenase activities) and functional (luminol-dependent chemiluminescence) indices of SI-induced damage. At Day 20, the DEX– LIP treatment significantly reduced the SI-induced increase in right lung/total body weight ratio and right lung hydroxyproline content, a biochemical index of fibrosis. This attenuation of fibrosis was confirmed histopathologically on preserved left lungs from these same animals. These results show that administration of liposomes containing dexamethasone attenuated SI-induced pulmonary inflammation and fibrosis in rats, and that this protection is independent of some biochemical and functional parameters of damage. q 1997 Academic Press
As a known cytotoxic and fibrogenic dust, silica has been demonstrated to cause pulmonary inflammation (increases in numbers of one or more cell types, particularly macrophages, neutrophils, or lymphocytes; Khan and Gupta, 1991) and damage to the lung tissue. These processes have been shown to begin within 2 hr of exposure following intratracheal (IT)
administration of silica to rats (DiMatteo et al., 1995). This inflammation and damage can ultimately manifest itself as the fibrotic lung disease, pulmonary fibrosis (Adamson et al., 1992; Driscoll et al., 1991; Heppleston, 1982). Depending upon the dose, the appearance of fibrosis can occur as rapidly as 2 weeks after the introduction of silica (Reiser et al., 1982). Fibrosis development is characterized by a rapid increase in the rate of lung collagen synthesis followed by an increase in the deposition of the excess collagen. This collagen is abnormal with respect to normal lung collagen in that it has altered amino acid cross-links and has thus been termed ‘‘fibrotic collagen’’ (Last and Reiser, 1985). Lesions of fibrotic collagen appear as whorled, rounded nodules of concentric layers of tissue around an acellular center (Lapp and Castranova, 1993). The alveolar macrophage is the initial phagocyte exposed to foreign matter introduced into the airways, as is the case in silica exposure. In general, normal alveolar macrophages do not constitutively secrete significant levels of inflammatory and growth-associated cytokines; however, upon appropriate stimulation macrophages are capable of secreting a variety of cytokines including interleukin (IL)-1, IL-1 receptor antagonist (ra), IL-8, IL-10, platelet-derived growth factor (PDGF), and tumor necrosis factor-a (TNF-a) (Fiorentino et al., 1991; Mukaida et al., 1992; Piguet et al., 1993; Ross et al., 1986; Vilcek and Lee, 1991). Release of macrophage-derived inflammatory/fibrogenic factors represents a means of amplification of the overall reaction as it evolves within the structures of the lungs. In the disease process, it is felt that the secretion of IL-1 and TNF-a by the macrophage is most important since these two factors initiate the cascade of responses that lead to inflammation and fibrosis; resultantly, their secretion has been extensively examined in the silica-induced pulmonary toxicity model by several investigators (Driscoll et al., 1990; Driscoll and Maurer, 1991; DuBois et al., 1989). Because proinflammatory cytokines are believed to be central to the development of silica-induced pulmonary toxicities, inhibition of the production of these cytokines should
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result in protection. The anti-inflammatory, glucocorticoid steroid dexamethasone has been demonstrated to inhibit the production of proinflammatory mediators (Guyre et al., 1988; Kern et al., 1988; Ohtsuka et al., 1996; Van Furth et al., 1995). These inhibitory effects may occur through gene repression by activated glucocorticoid receptor binding to negative glucocorticoid response elements (GRE) in the 5*flanking region of the proinflammatory gene. Binding at the GRE down-regulates the activity of the nuclear transcription factor, NF-kB (Kleinert et al., 1996). NF-kB plays an essential role in the production of silica-induced proinflammatory mediators (Chen et al., 1995). In addition to its inhibition of proinflammatory mediator production, dexamethasone has the ability to enhance the production of anti-inflammatory cytokines, such as IL-10, by leukocytes (Van Furth et al., 1995). Despite these favorable properties, the dosage and duration of systemically administered dexamethasone is limited almost exclusively by the side effects of the steroid. These side effects include suppression of the hypothalamic– pituitary adrenal axis, shifts in water balance, weight loss, and diabetes (Kehrl and Fauci, 1983). Dexamethasone may affect fibrogenesis in ways other than through influencing the inflammatory process. This glucocorticoid can inhibit fibrogenesis by acting directly on the fibroblast to inhibit collagen biosynthesis (Robey, 1979; Siddiqui et al., 1986). It is believed that this occurs through down-regulation of procollagen gene expression (Cockayne et al., 1986; Meisler et al., 1995). Dexamethasone can inhibit the synthesis of growth factors produced by fibroblasts (Shull et al., 1995; Chedid et al., 1996). Additionally, dexamethasone can inhibit fibroblast proliferation (Frost et al., 1994; Torry et al., 1994), and in this way modulate fibrosis. Therefore, dexamethasone may exert antifibrotic actions at more than one site in the tissue and through more than one mechanism. In order to test our hypothesis that pharmacological intervention early in the inflammatory cascade will attenuate the late-phase toxic response of fibrosis, we utilized a novel liposomal drug delivery system. This encapsulation technique allows for: (1) ‘‘selective’’ delivery of drug to the cells of interest; liposomes are particulate and therefore their natural fate is phagocytosis (Gonzalez-Rothi et al., 1991), (2) the delivery of higher local drug concentrations (Debs et al., 1987), and (3) lower systemic drug levels and associated adverse effects (Shek et al., 1994).
silica was determined by automated X-ray diffractometer and was 99.5% a-quartz. Size fraction õ5 mm in diameter was made by a centrifugal airflow particle classifier (Accucut Particle Classifier, Donalson-Majal Division, St. Paul, MN). Ninety-eight percent of this fraction was õ5 mm in size with a median area equivalent diameter of 3.5 mm as estimated by scanning electron microscopic image analysis. Enzyme reagents, dexamethasone base, and cholesterol were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals used in the study were from Fisher Chemical Co. (Pittsburgh, PA) unless otherwise noted. Animal Treatment Male Fischer 344 rats (Hilltop Lab Animals, Scottdale, PA) weighing 200–250 g were housed in laminar flow hoods (2 per cage) and allowed at least 1 week for acclimation after arrival from the supplier. Rats were given a conventional laboratory diet (Purina Chow pellets) and tap water ad libitum. Dexamethasone–liposome (DEX–LIP) preparation. Phosphatidylcholine (100 mg, egg lecithin) in chloroform (20 mg/ml; Avanti Polar Lipids, Alabaster, AL), 8 mg of cholesterol, and 6 mg of dexamethasone base were combined in a 500-ml round-bottom flask in a final solvent system of chloroform:methanol (9:1). Glass beads were added to the flask to increase the surface area for film development. The solvent phase was removed by rotary-vacuum evaporation. A thin milky white film formed against the inner wall of the flask and around the glass beads. This film was rehydrated with 4 ml of dexamethasone phosphate (10 mg/ml, Steris Laboratories, Phoenix, AZ) for 1 hr. The milky white lipid suspension was kept at room temperature for 2 hr under nitrogen gas. After gentle shaking of the suspension, it was sonicated in a room temperature waterbath sonicator for 3 min. This suspension was kept overnight at 47C for swelling of the liposomes. Before using the liposomes, the nonencapsulated dexamethasone phosphate was removed by centrifugation (30,000g) of the liposomes. The liposome pellet was washed three times with 4 ml Ca2//Mg2/-free Hanks’ balanced salt solution (HBSS). The final liposome pellet was resuspended in 5 ml HBSS and used immediately or stored at 47C until it was used within 1 week of preparation. HPLC determination of dexamethasone. A 0.5-ml aliquot of each final liposome suspension was lyophilized. The dexamethasone was extracted from each liposome preparation with 1 ml of acetonitrile and sonicated in a water bath sonicator for 10 min and then left overnight at room temperature. The mixture was then sonicated for 40 min, transferred to a microfuge tube, and centrifuged for 3 min (Beckman Microfuge 11 at a speed setting of 7). Dexamethasone incorporation was determined with a Waters HPLC system equipped with a C18 mBondapak reverse-phase column and acetonitrile:water (1:1) as the elutant at a flow rate of 1 ml/min. The effluent was monitored at a wavelength of 254 nm and drug levels were quantified by comparison of sample peak heights to those of drug standards that were run above and below the range of experimental values to construct the standard curve. Testosterone was used as an internal standard.
MATERIALS AND METHODS
Intratracheal instillation. Rats were treated in a manner similar to that outlined by Brain et al. (1976). After being anesthetized with Brevital (sodium methohexital, Eli Lilly & Co., Indianapolis, IN) ip, rats were placed on their backs on a slanted board suspended by a wire under their maxillary incisors. The tongue was moved and a ball-tipped 20-gauge animal feeding needle fitted with an insulin syringe was placed into the trachea via the mouth.
Crystalline Min-U-Sil silica (U.S. Silica Corp., Berkeley Springs, WV) was a gift from Dr. Val Vallyathan (National Institute for Occupational Safety and Health, Morgantown, WV). The silica was cleaned by boiling in 1 M HCl for 60 min to remove contaminants such as iron. Purity of the
• Dexamethasone–liposomes—Rats were IT instilled with 0.5 ml DEX– LIP or an equal volume of HBSS as control on Day 01 and then every fourth day afterward (dosing regimen determined from preliminary experiments) until the time of euthanization. Rats received 0.25–0.34 mg of dexamethasone in each of the instillations. The animals remained in the slanted position for 1 min after the instillation to facilitate distribution in
Materials
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DEXAMETHASONE–LIPOSOMES MODULATE SILICA TOXICITY the lungs. Rats were observed until consciousness was regained. After recovery from anesthesia, the animals were returned to the animal quarters until the time for analysis. • Silica (SI)—On Day 0, the DEX–LIP group received instillations of SI (DEX–LIP–SI); half of the HBSS group received SI (HBSS–SI), and the other half received saline (HBSS–SAL). The silica particles were prepared at a concentration such that rats received 10 mg/100 g body wt in 0.5 ml SAL. Before instillation the silica suspension was sonicated for 10 min. A 0.5-ml aliquot of SI was instilled slowly into the trachea. In control groups the same volume of SAL was instilled. The animals remained in the slanted position for 1 min after the instillation to facilitate distribution in the lungs. Rats were observed until consciousness was regained. After recovery from anesthesia, the animals were returned to the animal quarters until the time for analysis. The HBSS–LIP–SI control group was omitted from these studies after it was determined from previous experiments that responses to these ‘‘empty’’ liposomes were no different from those of the HBSS–SI group in all performed analyses. Bronchoalveolar lavage (BAL). On Day 10 or 20, BAL was performed for cellular, biochemical, and functional analyses of inflammation and damage. Rats were anesthetized with sodium pentobarbital (The Butler Co., Columbus, OH) and exsanguinated by severing the abdominal aorta. The trachea was cannulated with PE160 tubing and the cells from the lungs were collected by BAL. While massaging the lungs, 2 ml/100 g body wt of warm HBSS was instilled into the lungs. For the first instillation HBSS remained in the lungs for 30 sec, was withdrawn, and then reinstilled for another 30 sec to maximize retrieval of biological markers (Lindenschmidt et al., 1990). After the second withdrawal the HBSS was saved for analysis in a separate tube. For additional lavages, 5-ml aliquots of HBSS were instilled sequentially, withdrawn, and placed in centrifuge tubes until É40 ml was obtained. All tubes from each rat were centrifuged at 800g for 7 min. The supernatant from the first lavage was transferred into a separate conical tube for further analysis. All other supernatants were discarded. Cell pellets were resuspended in 5 ml HBSS and the cells from each animal were pooled. The cells were recentrifuged, as above, and the pellets were resuspended in 1 ml HBSS for cell counting and further analysis. Cellular, Functional, and Biochemical Assays Cell counts. Cells harvested from lavage procedures were counted via a hemacytometer. Differential cell counts. For cellular differentiation, cells (1.5 1 105) were spun in a Shandon cytocentrifuge at 400 rpm for 4 min and allowed to affix to a microscope slide. Slides were stained with Wright–Giemsa Sure Stain. Cell types (macrophages, neutrophils, and lymphocytes) were differentiated under a light microscope by counting at least 200 cells per slide. Data are presented for neutrophils only because of their value in assessing the inflammatory response. Luminol-dependent chemiluminescence (LDCL). Chemiluminescence is a functional assay to assess release of oxidants from cells or tissue. Luminol was incorporated into the reaction cuvettes as an amplifying agent of chemiluminescence to study the oxidative activity of the lavagable cells. Luminol is first oxidized to an intermediate that subsequently converts to an aminophthalate product and the release of a photon (É425 nm). Luminol reacts with superoxide, nitric oxide, and their reaction product peroxynitrite (Radi et al., 1993). LDCL was followed for 20 min at 377C using a sixchambered Berthold LB 9505C luminometer. The integrated response was determined by an accompanying computer equipped with a KINB program that was supplied with the luminometer. • Cellular LDCL—Total cell concentrations obtained from BAL were adjusted to yield 1 1 107 cells/ml. Luminol was dissolved in DMSO and then diluted in physiological Hepes buffer (0.1 M, pH 7.4). The final concentration of the luminol in the cuvette reaction mixture was 1005 M. Phorbol
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myristate acetate (PMA) was used as a soluble stimulant at a final cuvette concentration of 2 1 1006 M. In each run an unstimulated baseline value was obtained by omitting the PMA from one cuvette. The final volume in each reaction cuvette was 500 ml (100 ml cells, 100 ml luminol, 100 ml PMA, and 200 ml Hepes). When PMA was excluded from the cuvettes, the volume was brought up to 500 ml with Hepes buffer. All cuvettes were incubated in a 377C waterbath for 10 min prior to PMA stimulation and being placed in the luminometer. • Total cellular LDCL—This was calculated by adjusting the values obtained on a per cell basis to the total number of cells recovered by BAL. BALF albumin. Albumin was assayed to assess damage/permeability of the alveolar–capillary barrier and is specific for assessing this damage since it is a blood-derived protein. The albumin content of the acellular fraction obtained by the first lavage was determined by using the Sigma Diagnostics albumin reagent kit. Absorbance was determined at 628 nm on a spectrophotometer. Quantification of albumin was accomplished by comparison to a bovine serum albumin standard curve and expressed in units of mg/100 ml. BALF b-glucuronidase (b-glu). b-Glu is a lysosomal enzyme and was assayed to assess phagocytic cell activation/damage. The activity of b-glu in the acellular fraction of the first lavage was analyzed according to the method of Lockard and Kennedy (1976). The absorbance was determined at 400 nm. The activity of the enzyme was calculated by using a known extinction coefficient (18.3 ml/mmol/cm) and was expressed in units of nmol/min/ml. BALF lactate dehydrogenase (LDH). LDH is a cytosolic enzyme and was assayed to assess general cell death. The activity of LDH in the acellular fraction of the first lavage was analyzed according to the procedure set forth by the Boehringer Mannheim LDH reagent kit and monitored at 340 nm in a dual-beam scanning spectrophotometer. The enzyme activity was calculated by using a known extinction coefficient (6.2 ml/mmol/cm) and was expressed in units of nmol/min/ml. Fibrosis. Animals designated for fibrosis assessment were given an overdose of sodium pentobarbital and exsanguinated. After the trachea was cannulated and removed along with the lungs, the right lung lobes were ligated, removed, weighed, and frozen immediately at 0807C for later determination of hydroxyproline, a biochemical index of fibrosis. The left lung was infused with 10% phosphate-buffered Formalin through the cannula. The trachea was ligated so the cannula could be cut away and the lung samples were stored in Formalin solution until processed for histopathology. • Hydroxyproline—Hydroxyproline is an amino acid that is found almost exclusively in the composition of collagen and was therefore used as an index of fibrosis. Frozen lung samples were thawed and minced then processed and analyzed according to the method of Witschi et al. (1985). Optical density was read at 560 nm on a spectrophotometer. Amount of hydroxyproline per right lung was calculated from a standard curve and expressed as mg OH-Pro/right lung. • Histopathology—Microscopic evaluation of disease development was accomplished by sectioning fixed lung lobes and then staining with hematoxylin and eosin or a trichrome stain. This evaluation of the samples was performed in collaboration with a veterinary pathologist.
Statistical Analysis All parameters were analyzed by the appropriate analysis of variance. Effects found to be significant were analyzed further by Tukey’s protected t test, a multiple comparison procedure used to determine significant differences between pairs of groups. The criterion for significance between groups was p õ 0.05. All statistical analyses were carried out using GB-STAT software.
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RESULTS
As a gross measure of lung damage, the ratio of right lung weight to total body weight was calculated. HBSS–SI animals had a significant increase (2.6-fold) in this parameter (Fig. 1). The DEX–LIP–SI animals had a 38% reduction of the SI-induced increase in this ratio when compared to the HBSS–SI group, but this reduction was not great enough to return the values to those of the HBSS–SAL control group. Silica instillation also induced a profound fibrosis as the HBSS–SI group displayed a significant increase (1.8-fold) in right lung hydroxyproline content on Day 20 after the IT instillation of silica (Fig. 2). The periodic administration of DEX–LIP to rats significantly decreased (73% reduction in the SI-induced increase) but did not eliminate the increase in lung hydroxyproline content. Histopathological evaluation confirmed the reduction of the response to silica associated with the DEX–LIP administration (Fig. 3). In the HBSS– SI group, the response was characterized by prominent foci of granulomatous pneumonitis within the lung structure and moderate, multifocal alveolar histiocytosis. Compared to controls, there was a moderate increase in the amount of collagen present as assessed by trichrome staining. In the DEX–LIP–SI group, the histiocytosis was present to a lesser degree, but the granulomatous lesioning was essentially absent; the trichrome staining was reduced compared to the HBSS–SI group but still more prominent than the HBSS– SAL group.
FIG. 1. Day 20 right lung/total body wt ratio of animals treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú DEX–LIP–SI and HBSS–SAL; b, DEX–LIP–SI ú HBSS–SAL.
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FIG. 2. Day 20 right lung hydroxyproline content of animals treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú DEX–LIP– SI and HBSS–SAL; b, DEX–LIP–SI ú HBSS–SAL.
The second aspect to this study involved analyzing the BAL fluid, on Days 10 and 20 after IT instillation of SI or HBSS, from animals of each treatment group to examine the parameters of inflammation and tissue injury. Silica produced a severe pulmonary inflammation which was reduced significantly by IT DEX–LIP treatment. Figure 4 depicts total neutrophil numbers recovered by BAL of rats from each of the three treatment groups. The HBSS–SI had a large increase in the number of neutrophils in the BAL fluid. Animals of the DEX–LIP–SI group also had neutrophils in the BAL fluid, but the level was significantly diminished (66% at Day 10 and 85% at Day 20) when compared to the HBSS–SI animals. It was also evident that the numbers of neutrophils increased with time in the HBSS-SI group, whereas the number remained unchanged in the DEX–LIP– SI group illustrating a sustained protective effect. At Day 10 there were marginal increases in the numbers of lavagable macrophages in both the HBSS–SI and DEX–LIP–SI groups; however, these increases returned to control levels by Day 20 (data not shown). Lymphocyte numbers were not affected by any instillation procedure at either time point (data not shown). The integrated response of LDCL on a per-cell-basis is presented in Fig. 5. At both times, unstimulated cellular LDCL from both treatment groups demonstrated no increases in the LDCL assay. However, with PMA stimulation, cells from both SI-exposed groups demonstrated tremendous increases in LDCL activity, with DEX–LIP treatment augmenting the generation of light (2.5-fold at Day 10 and
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3.7-fold at Day 20). The observed response at Day 20 was significantly less than that at Day 10, but the values were significantly greater than control. In contrast to these findings, when LDCL is expressed as total LDCL (Fig. 6), there is no significant difference between the light generated by the HBSS–SI and the DEX–LIP–SI groups at both times.
FIG. 4. Total number of neutrophils obtained by bronchoalveolar lavage of animals treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6) with the value for the Day 10 HBSS–SI control being 0.01 1 107. Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú DEX–LIP–SI and HBSS–SAL within an individual time; b, DEX–LIP– SI ú HBSS–SAL within an individual time. *Day 20 HBSS–SI ú Day 10 HBSS–SI.
Figure 7 shows a significant increase in albumin in the BAL fluid from the HBSS–SI animals. The DEX–LIP treatment had no effect on the SI-induced elevation of albumin in the acellular lavage at both times assayed. The HBSS –SI group caused a dramatic increase in bglucuronidase activity in the acellular lavage when compared to the HBSS– SAL control (Fig. 8). This enzymatic activity was enhanced by DEX– LIP treatment over that of the HBSS-SI group by 1.5-fold at Day 10 and 1.7-fold at Day 20. Similar results were noted for the analysis of LDH activity in the acellular BAL (Fig. 9). The HBSS–SI group had a significant increase in LDH activity that was further enhanced in DEX–LIP–SI group. At both times analyzed, the LDH activity of the HBSS–SI group was elevated by 1.4fold as a result of the DEX–LIP treatment. DISCUSSION
FIG. 3. Representative light photomicrographs of sections of lung tissue from the following groups at Day 20 after saline (SAL) or silica (SI) instillation: (A) HBSS–SAL, lung architecture is normal; (B) HBSS–SI, there is marked granulomatous pneumonitis and a moderate alveolar histiocytosis; (C) DEX–LIP–SI, there is an absence of the granulomatous response, and a reduction in the alveolar histiocytosis (original magnification, 401).
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Respirable particulate matter is an occupational concern because of the potential toxicity posed to the lungs. The ability of silica to cause the development of pulmonary inflammation and fibrosis in workers in various mining and sand-blasting occupations has been well established. However, most therapeutic attempts to treat the silica-induced lung damage have generally been unsuccessful. In an inter-
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esting and unusual clinical case (Goodman et al., 1992), a driller at a surface coal mine was exposed to and inhaled sufficient quantities of silica dust to demonstrate clinical symptomatology. His condition worsened and upon bronchoscopy and biopsy the patient was diagnosed with acute
FIG. 5. Cellular luminol-dependent chemiluminescence (LDCL) from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI) at Day 10 (A) and Day 20 (B). LDCL was monitored in the presence of luminol using a chemiluminometer. LDCL was determined at rest (unstimulated) or after stimulation with phorbol myristate acetate. Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú HBSS–SAL within an individual time; b, DEX–LIP–SI ú HBSS–SI and HBSS–SAL within an individual time. *Day 10 stimulated ú Day 20 stimulated within an individual group. 1, stimulated ú unstimulated within an individual group.
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FIG. 6. Total cellular luminol-dependent chemiluminescence (LDCL) from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI) at Day 10 (A) and Day 20 (B). Total LDCL was calculated by adjusting the values obtained on a per cell basis to the total number of cells recovered by BAL. Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI Å DEX–LIP–SI ú HBSS–SAL within an individual time. *Day 10 Stimulated ú Day 20 Stimulated within an individual group. 1, Stimulated ú Unstimulated within an individual group.
silicosis. The patient’s condition drastically improved when treated with a high-dose corticosteroids. Therapy was continued with oral corticosteroids at a reduced dosage which markedly reduced the severity of silica-induced lung nodules. Upon cessation of the steroid therapy (due to concerns of steroid-related toxicities) the patient’s condition
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FIG. 7. Albumin content in acellular bronchoalveolar lavage fluid from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotation represents significant rank order differences (p õ 0.05) as follows: a, HBSS–SI Å DEX–LIP–SI ú HBSS–SAL within an individual time.
worsened, his lungs became fibrotic, and he eventually died as a result of the silicosis. Thus, it appears that early and continual treatment with steroids could be life-saving in such conditions where silica causes major lung toxicities. However, due to the inherent
FIG. 8. b-Glucuronidase activity in acellular bronchoalveolar lavage fluid from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS– SI ú HBSS–SAL within an individual time; b, DEX–LIP–SI ú HBSS– SI and HBSS–SAL within an individual time.
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FIG. 9. Lactate dehydrogenase activity in acellular bronchoalveolar lavage fluid from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú HBSS–SAL within an individual time; b, DEX– LIP–SI ú HBSS–SI and HBSS–SAL within an individual time.
adverse effects associated with the long-term systemic administration of the steroids (suppression of the hypothalamic–pituitary adrenal axis, shifts in water balance, weight loss, and diabetes; Kehrl and Fauci, 1983), this route of delivery may be of little value in the treatment of silicainduced chronic lung disease. A novel and immunologically inert drug delivery system that can be administered locally may provide a new option in the treatment of such an inflammatory and fibrotic disease. Such a system exists in the liposomal encapsulation technique which allows for: (1) ‘‘selective’’ delivery of drug to the cells of interest (Gonzalez-Rothi et al., 1991). The usual fate of liposomes is ingestion and digestion by macrophages; therefore, liposomes have become a suitable tool to manipulate the function of the macrophage (Van Rooijen and Sanders, 1994); (2) the delivery of higher local drug concentrations (Debs et al., 1987); and (3) lower systemic drug levels and associated adverse effects (Shek et al., 1994). Encapsulation techniques have been used to deliver various drug entities in attempt to maximize the desired pharmacological effects and minimize the unwanted toxicological effects. The therapeutic efficacy of the antifibrotic agent cis-4hydroxy-L-proline (cHYP) in preventing bleomycin-induced pulmonary fibrosis was studied in both the free and liposomal formulation (Poiani et al., 1994). After once weekly intratracheal instillations of free cHYP or cHYP–liposomes, the investigators observed greater protection from bleomycin-induced increases in lung collagen as a result of liposo-
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mal encapsulation. The amount of lung collagen present in the free cHYP group was no different than that of the group treated with bleomycin alone. This study demonstrated that delivery of cHYP to the lung in carrier vehicles (i.e., liposomes) enhanced their efficacy in treating pulmonary fibrosis, over that of free unencapsulated cHYP alone. In a similar type of study, Tremblay and colleagues (1993) compared the effects of daily intranasal instillations of liposomal dexamethasone and free dexamethasone in a murine model of hypersensitivity pneumonitis. It was concluded from these studies that liposome-incorporated dexamethasone was a more effective treatment in reducing the lung inflammation and damage associated with hypersensitivity pneumonitis than was the free dexamethasone. In addition to the enhanced therapeutic value of liposomal encapsulation, this preparation did not inhibit adrenocorticotropic hormone secretion, therefore implying that the liposomal formulation was retained locally within the confines of the lungs and did not achieve blood concentrations sufficient to elicit a systemic effect. This protective effect is most likely mediated through glucocorticoid inhibition of inflammatory mediator production. Dexamethasone inhibits the production of proinflammatory mediators (Guyre et al., 1988, Kern et al., 1988, Ohtsuka et al., 1996, Van Furth et al., 1995) through gene repression by activated glucocorticoid receptor binding to negative glucocorticoid response elements (GRE) in the 5*-flanking region of proinflammatory cytokine genes (Kleinert et al., 1996). Binding at the GRE down-regulates the activity of the nuclear transcription factor, NF-kB, which plays an essential role in the production of silica-induced proinflammatory mediators (Chen et al., 1995). In addition to its inhibition of proinflammatory mediator production, dexamethasone enhances the production of anti-inflammatory cytokines, such as IL-10, by leukocytes (Van Furth et al., 1995). In the present study, we observed partial protection from silica-induced pulmonary inflammation and fibrosis by the administration of dexamethasone-containing liposomes. A reduction in the silica-induced increase in the right lung to total body weight ratio after DEX–LIP administration suggests that this formulation provided a general protection against silica toxicity. Additional protection by the DEX– LIP administration was evident when assessing the right lung hydroxyproline content, the biochemical index of fibrosis. Upon histopathological assessment, rats that received silica alone displayed prominent granulomatous lesions within the lung sections. However, a lesser degree of lesioning was present in the lungs of rats in the DEX–LIP– SI group. These observations confirm the biochemical findings in that the administration of dexamethasone-containing liposomes attenuated the development of silica-induced pulmonary fibrosis.
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Systemic dexamethasone administration has been shown to be effective in eliminating the acute silica-induced neutrophil accumulation 24 hr after instillation (Van Dyke et al., 1994). Inhibiting the production of proinflammatory and/or chemotactic factors may account for these effects. The IL8 family of cytokines is thought to be the primary signal for neutrophil recruitment, and dexamethasone has the ability to genetically suppress the production of the rat neutrophil chemoattractant CINC/gro, a member of the IL-8 family (Ohtsuka et al., 1996). In our study, the sustained reduction in lavagable neutrophil numbers that results from DEX–LIP treatment is most likely a direct response to this genetic down-regulation. While DEX–LIP administration afforded protection against silica-induced cellular inflammation and fibrosis, biochemical and functional indices of damage were unaffected or enhanced. It is possible that these augmented responses are a direct result of the ‘‘protective’’ effects observed by the dexamethasone administration and can be accounted for by the inherent functions of the steroid itself. Steroid therapy blunts the influx of blood-borne neutrophils into the airspaces. These recruited phagocytes have been shown to be beneficial in the clearance of silica particles from the lungs, and actually decrease the toxicity that normally results from silica (Adamson et al., 1992). As a result of decreased neutrophil influx and subsequent decreased particle clearance from the lungs, the burden of silica remains elevated at levels sufficient to allow for increased phagocyte–particle interactions and activation (increasing b-glucuronidase levels); in addition to increased cytotoxicity (increasing lactate dehydrogenase levels) produced by the silica itself. This cycling nature of silica (phagocytosis r cytotoxicity r ‘‘retoxification’’ r phagocytosis r . . .) may lead to the levels of BAL enzyme activity obtained in the present study. Thus, inhibiting the neutrophil influx with DEX–LIP administration may actually be potentiating the silica-induced damage observed in BAL fluid in our system. The lack of DEX–LIP protection afforded to the alveolar– capillary barrier, as assessed by the albumin analysis, is most likely due to the toxic surface properties of the silica itself. Since there is a lack of silica clearance by the inhibition of neutrophil recruitment, there is a greater chance that the silica can interact with the barrier and damage it, allowing the blood-derived protein to enter the airspaces. The DEX–LIP increase in silica-induced oxidant production, as assessed by chemiluminescence, may also be attributed to the inherent properties of the dexamethasone. It is known that silica as well as individual proinflammatory cytokines (IL-1 and IL-6) are able to increase antioxidant mRNA expression (Dougall and Nick, 1991; Janssen et al., 1991, 1994). Since inflammation is known to increase the oxidative status of the tissue (DiMatteo et al., 1995) as well as the cells
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(Antonini et al., 1994), the role of the increased antioxidant expression may be an attempt to protect against inflammation-induced oxidative damage. Thus: (1) dexamethasoneinduced inhibition of proinflammatory cytokine production results in decreased antioxidant mRNA expression (Chen et al., 1993; Dougall and Nick, 1991); (2) decreased antioxidant mRNA expression results in a decrease in the available antioxidant pool; and (3) the decreased antioxidant pool results in an enhanced oxidative status, as assessed by luminoldependent chemiluminescence. However, when expressed on a total cellular chemiluminescence basis, there is no difference in the oxidative state between the HBSS–SI and the DEX–LIP–SI groups since there are tremendously more cells in the HBSS–SI group than there are in the DEX– LIP–SI group. To validate this hypothesis, analysis of the antioxidant status of the BAL cells is required. It appears from our study that cellular events characteristic of silica exposures are critical for fibrogenesis, since protection from inflammation and fibrosis occurred independently of biochemical and functional indices. DEX–LIP administration suppressed inflammation and fibrosis by presumably acting through the NF-kB mechanism and the down-regulation of macrophage-derived proinflammatory/fibrogenic cytokine production. Direct action on fibroblast function may also have been involved. With their function in recycling surfactant, the alveolar type II cells could theoretically be a nonselective target for liposomal delivery and be acted upon by the encapsulated dexamethasone. Although this is possible, surfactant protein A (SP-A) is needed for alveolar type II cells to recycle phospholipids. It appears that SP-A regulates the uptake of phospholipids in some fashion (Bates et al., 1994; Kuroki et al., 1994; McCormack et al., 1994). Therefore, since there was no SP-A incorporated into the liposomes utilized in our study, the uptake of liposomes by alveolar type II cells would be negligible, and any anti-inflammatory/antifibrotic effects observed as a result of the administration of the dexamethasone-containing liposomes would not be mediated by the type II cells. From our study we can conclude that early pharmacological intervention, at the level of macrophage processing, signaling and cellular recruitment, and possibly effects directly on the fibroblast altered the inflammatory and fibrotic responses in rats. The administration of dexamethasone-containing liposomes to rats before and after the intratracheal instillation of silica results in the attenuation of the inflammation and fibrosis that is normally associated with exposure to this occupationally relevant dust. Additionally, this protection is independent of some biochemical and functional parameters of damage. Thus, overall we have reported a novel drug delivery system that can potentially be used in the treatment of silica-induced pulmonary inflammation and fibrosis.
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Direct administration of liposomes to the lungs can be achieved by intratracheal instillation or by aerosol inhalation. While the use of the instillation method serves as a means whereby a controlled dose can be delivered to the lung in an animal model, the instillation procedure is not practical for routine human use. For routine applications, aerosol inhalation would be considered the most convenient and practical approach to pulmonary liposome delivery. This delivery system would be advantageous over the current commercially available steroid inhalers since there would be enhanced targeting to the phagocytes, increased retention time within the lungs, and decreased extrapulmonary side/adverse effects. Invariably, these features would result in a greater therapeutic efficacy over that of conventional steroid inhalers. Although inhalational delivery of liposomes promises to be convenient and effective in many clinical application, delivery system engineering issues must be addressed prior to widespread human usage. ACKNOWLEDGMENTS This research was supported by the 1995 Hazleton/Society of Toxicology Graduate Student Fellowship (M.D.), a National Institute of Occupational Safety and Health Cooperative Agreement Grant for Occupational Respiratory Disease and Musculoskeletal Disorders, U60/CCU306149, and a National Institutes of Health Training Grant, 5 T32 GM07039-20. The authors thank Dr. Robert Garman and his staff for performing the histopathological analyses in this study.
REFERENCES Adamson, I. Y. R., Prieditis, H., and Bowden, D. H. (1992). Instillation of chemotactic factor to silica-injected lungs lowers interstitial particle content and reduces pulmonary fibrosis. Am. J. Pathol. 141, 319–326. Antonini, J. M., Van Dyke, K., Ye, Z., DiMatteo, M., and Reasor, M. J. (1994). Introduction of luminol-dependent chemiluminescence as a method to study silica inflammation in the tissue and phagocytic cells of rat lung. Environ. Health Perspect. 102, 37–42. Bates, S. R., Dodia, C., and Fisher, A. B. (1994). Surfactant protein A regulates uptake of pulmonary surfactant by lung type II cells on microporous membranes. Am. J. Physiol. 267, L753–L760. Brain, J. D., Knudson, D. E., Sorokin, S. P., and Davis, M. A. (1976). Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11, 13–33. Chedid, M., Hoyle, J. R., Csaky, K. G., and Rubin, J. S. (1996). Glucocorticoids inhibit keratinocyte growth factor production in primary dermal fibroblasts. Endocrinology 137, 2232–2237. Chen, F., Sun, S. C., Kuh, D. C., Gaydos, L. J., and Demers, L. M. (1995). Essential role of NF-kappa B activation in silica-induced inflammatory mediator production in macrophages. Biochem. Biophys. Res. Commun. 214, 985–992. Chen, Y., Whitney, P. L., and Frank, L. (1993). Negative regulation of antioxidant enzyme gene expression in the developing fetal rat lung by prenatal hormonal treatments. Pediatr. Res. 33, 171–176. Cockayne, D., Sterling, K. M., Jr., Shull, S., Mintz, K. P., Illeyne, S., and Cutroneo, K. R. (1986). Glucocorticoids decrease the synthesis of type I procollagen mRNAs. Biochemistry 25, 3203–3209.
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Debs, R. J., Straubinger, R. M., Brunette, E. N., Lin, J. M., Lin, E. J., Montgomery, A. B., Friend, D. S., and Papahadjopoulos, D. P. (1987). Selective enhancement of pentamidine uptake in the lung by aerosolization and delivery in liposomes. Am. Rev. Respir. Dis. 135, 731–737. DiMatteo, M., Antonini, J. M., Van Dyke, K., and Reasor, M. J. (1995). Characteristics of the acute-phase pulmonary response to silica in rats. J. Toxicol. Environ. Health. 46, 101–116. Dougall, W. C., and Nick, H. S. (1991). Manganese superoxide dismutase: A hepatic acute phase protein regulated by interleukin-6 and glucocorticoids. Endocrinology. 129, 2376–84. Driscoll, K. E., Lindenschmidt, R. C., Maurer, J. K., Higgins, J. M., and Ridder, G. (1990). Pulmonary response to silica or titanium dioxide: Inflammatory cells, alveolar macrophage-derived cytokines, and histopathology. Am. J. Respir. Cell Mol. Biol. 2, 381–390. Driscoll, K. E., Lindenschmidt, R. C., Maurer, J. K., Perkins, L., Perkins, M., and Higgins, J. (1991). Pulmonary response to inhaled silica or titanium dioxide. Toxicol. Appl. Pharmacol. 111, 201–210. Driscoll, K. E., and Maurer, J. K. (1991). Cytokine and growth factor release by alveolar macrophages: Potential biomarkers of pulmonary toxicity. Toxicol. Pathol. 19, 398–405. DuBois, C. M., Bissonnette, E., and Rola-Pleszczynski, M. (1989). Asbestos fibers and silica particles stimulate rat alveolar macrophages to release tumor necrosis factor. Am. Rev. Respir. Dis. 139, 1257–1264. Fiorentino, D. F., Zlotnik, A., Mosmann, T. R., Howard, M., and O’Garra, A. (1991). IL-10 inhibits cytokine production by activated macrophages. J. Immunol. 147, 3815–3822. Frost, G. H., Rhee, K., Ma, T., and Thompson, E. A. (1994). Expression of c-Myc in glucocorticoid-treated fibroblastic cells. J. Steroid Biochem. Mol. Biol. 50, 109–119. Gonzalez-Rothi, R. J., Straub, L., Cacace, J. L., and Schreier, H. (1991). Liposomes and pulmonary alveolar macrophages: Functional and morphologic interactions. Exp. Lung Res. 17, 687–705. Goodman, G. B., Kaplan, P. D., Stachura, I., Castranova, V., Pailes, W. H., and Lapp, N. L. (1992). Acute silicosis responding to corticosteroid therapy. Chest 101, 366–370. Guyre, P. M., Girard, M. T., Morganelli, P. M., and Manganiello, P. D. (1988). Glucocorticoid effects on the production and actions of immune cytokines. J. Steroid Biochem. 30, 89–93. Heppleston, A. G. (1982). Silicotic fibrogenesis: A concept of pulmonary fibrosis. Ann. Occup. Hyg. 26, 449–462. Janssen, Y. M., Marsh, J. P., Driscoll, K. E., Borm, P. J., Oberdorster, G., and Mossman, B. T. (1994). Increased expression of manganese-containing superoxide dismutase in rat lungs after inhalation of inflammatory and fibrogenic minerals. Free Radicals Biol. Med. 16, 315–22. Janssen, Y. M. W., Marsh, J. P., Absher, M. P., Hemenway, D., Vacek, P. M., Leslie, K. O., Borm, P. J. A., and Mossman, B. T. (1991). Expression of antioxidant enzymes in rat lungs after inhalation of asbestos or silica. J. Biol. Chem. 267, 10625–10630. Kehrl, J. H., and Fauci, A. S. (1983). The clinical use of glucocorticoids. Ann. Allergy. 50, 2–8. Kern, J. A., Lamb, R. J., Reed, J. C., Daniele, R. P., and Nowell, P. C. (1988). Dexamethasone inhibition of interleukin 1 beta production by human monocytes: Posttranscriptional mechanisms. J. Clin. Invest. 81, 237–244. Khan, M. F., and Gupta, G. S. D. (1991). Cellular and biochemical indices of bronchoalveolar lavage for detection of lung injury following insult by airborne toxicants. Toxicol. Lett. 58, 239–255. Kleinert, H., Euchenhofer, C., Ihrig-Biedert, I., and Forstermann, U. (1996). Glucocorticoids inhibit the induction of nitric oxide synthase II by down-
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regulating cytokine-induced activity of transcription factor nuclear factorkappa B. Mol. Pharmacol. 49, 15–21. Kuroki, Y., McCormack, F. X., Ogasawara, Y., Mason, R. J., and Voelker, D. R. (1994). Epitope mapping for monoclonal antibodies identifies functional domains of pulmonary surfactant protein A that interact with lipids. J. Biol. Chem. 269, 29793–29800. Lapp, N. L., and Castranova, V. (1993). How silicosis and coal workers’ pneumoconiosis develop—A cellular assessment. In Occupational Medicine: State of the Art Reviews (D. E. Banks, Ed.), pp. 35–56. Hanley & Belfus, Philadelphia. Last, J. A., and Reiser, K. M. (1985). Effects of silica on lung collagen. In Silicon Biochemistry (D. Evered and M. O’Conner, Eds.), pp. 180–187. Wiley, New York. Lindenschmidt, R. C., Driscoll, K. E., Perkins, M. A., Higgins, J. M., Maurer, J. K., and Belfiore, K. A. (1990). The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage. Toxicol. Appl. Pharmacol. 102, 268–281. Lockard, V. G., and Kennedy, R. E. (1976). Alterations in rabbit alveolar macrophages as a result of traumatic shock. Lab. Invest. 35, 501–506. McCormack, F. X., Kuroki, Y., Stewart, J. J., Mason, R. J., and Voelker, D. R. (1994). Surfactant protein A amino acids Glu195 and Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion, and the facilitated uptake of phospholipid by type II cells. J. Biol. Chem. 269, 29801–29807. Meisler, N., Shull, S., Xie, R., Long, G. L., Absher, M., Connolly, J. P., and Cutroneo, K. R. (1995). Glucocorticoids coordinately regulate type I collagen pro alpha 1 promoter activity through both the glucocorticoid transforming growth factor b response elements: A novel mechanism of glucocorticoid regulation of eukaryotic genes. J. Cell Biochem. 59, 376– 388. Mukaida, N., Harada, A., Yasumoto, K., and Matsushima, K. (1992). Properties of proinflammatory cell type-specific leukocyte chemotactic cytokines, interleukin 8 (IL-8) and monocyte chemotactic and activating factor (MCAF). Microbiol. Immunol. 36, 773–789. Ohtsuka, T., Kubota, A., Hirano, T., Watanabe, K., Yoshida, H., Tsurufuji, M., Iizuka, Y., Konishi, K., and Tsurufuji, S. (1996). Glucocorticoidmediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family, through impairment of NF-kappa B activation. J. Biol. Chem. 271, 1651–1659. Piguet, P. F., Vesin, C., Grau, G. E., and Thompson, R. C. (1993). Interleukin 1 receptor antagonist (IL-1ra) prevents or cures pulmonary fibrosis elicited in mice by bleomycin or silica. Cytokine. 5, 57–61. Poiani, G. J., Greco, M., Choe, J. K., Fox, J. D., and Riley, D. J. (1994). Liposome encapsulation improves the effect of antifibrotic agent in rat lung fibrosis. Am. J. Respir. Crit. Care Med. 150, 1623–1627. Radi, R., Cosgrove, T. P., Beckman, J. S., and Freeman, B. A. (1993). Peroxynitrite-induced luminol chemiluminescence. Biochem. J. 290, 51–57. Reiser, K. M., Hesterberg, T. W., Haschek, W. M., and Last, J. A. (1982). Experimental silicosis. Acute effects of intratracheally instilled quartz on collagen metabolism and morphologic characteristics of rat lungs. Am. J. Pathol. 107, 176–185. Robey, P. G. (1979). Effect of dexamethasone on collagen metabolism in two strains of mice. Biochem. Pharmacol. 28, 2261–2266. Ross, R., Raines, E. W., and Bowen-Pope, D. F. (1986). The biology of platelet-derived growth factor. Cell 46, 155–169. Shek, P. N., Suntres, Z. E., and Brooks, J. I. (1994). Liposomes in pulmonary applications: Physicochemical considerations, pulmonary distribution and antioxidant delivery. J. Drug Target. 2, 431–42. Shull, S., Meisler, N., Absher, M., Phan, S., and Cutroneo, K. (1995).
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Van Furth, A. M., Seijmonsbergen, E. M., Langermans, J. A., Van der Meide, P. H., and Van Furth, R. (1995). Effect of xanthine derivates and dexamethasone on Streptococcus pneumoniae-stimulated production of tumor necrosis factor alpha, interleukin-1 beta (IL-1 beta), and IL-10 by human leukocytes. Clin. Diag. Lab. Immunol. 2, 689–692. Van Rooijen, N., and Sanders, A. (1994). Liposome mediated depletion of macrophages: Mechanism of action, preparation of liposomes and applications. J. Immunol. Methods 174, 83–93. Vilcek, J., and Lee, T. H. (1991). Tumor necrosis factor: New insights into the molecular mechanisms of its multiple actions. J. Biol. Chem. 266, 7313–7316. Witschi, H. P., Tryka, A. F., and Lindenschmidt, R. C. (1985). The many faces of an increase in lung collagen. Fundam. Appl. Toxicol. 5, 240 – 250.
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TO968057
Modulation of Silica-Induced Pulmonary Toxicity by Dexamethasone-Containing Liposomes MICHAEL DIMATTEO
AND
MARK J. REASOR
Department of Pharmacology and Toxicology, Robert C. Byrd Health Sciences Center of West Virginia University, Morgantown, West Virginia 26506-9223 Received June 20, 1996; accepted October 28, 1996
Modulation of Silica-Induced Pulmonary Toxicity by Dexamethasone-Containing Liposomes. DIMATTEO, M., AND REASOR, M. J. (1997). Toxicol. Appl. Pharmacol. 142, 411–421. Human exposure to silica (SI) is of great occupational concern because it is marked by pulmonary inflammation and fibrosis. Our objective was to determine if early pharmacological intervention altered the inflammatory and fibrotic responses to silica in rats. Male Fisher-344 rats received intratracheal (IT) instillations of the anti-inflammatory steroid, dexamethasone (DEX), incorporated into a novel liposomal (LIP) delivery system (DEX–LIP), or buffer as control (HBSS) on Day 01 and every fourth day until euthanization. On Day 0, the DEX–LIP group received IT instillations of SI (10 mg/100g body wt, DEX–LIP–SI); half of the HBSS group received SI (10 mg/100g body wt, HBSS–SI) and the other half saline (HBSS–SAL). On Day 10 or 20, bronchoalveolar lavage (BAL) was performed for cellular, biochemical, and functional analyses of inflammation and damage. HBSS–SI rats had significant elevations in the neutrophil cell count over HBSS–SAL rats at both times. DEX–LIP treatment markedly reduced these values, indicating that DEX–LIP protected against SI-induced inflammation. In contrast, DEX–LIP did not protect against biochemical (albumin concentration, and b-glucuronidase and lactate dehydrogenase activities) and functional (luminol-dependent chemiluminescence) indices of SI-induced damage. At Day 20, the DEX– LIP treatment significantly reduced the SI-induced increase in right lung/total body weight ratio and right lung hydroxyproline content, a biochemical index of fibrosis. This attenuation of fibrosis was confirmed histopathologically on preserved left lungs from these same animals. These results show that administration of liposomes containing dexamethasone attenuated SI-induced pulmonary inflammation and fibrosis in rats, and that this protection is independent of some biochemical and functional parameters of damage. q 1997 Academic Press
As a known cytotoxic and fibrogenic dust, silica has been demonstrated to cause pulmonary inflammation (increases in numbers of one or more cell types, particularly macrophages, neutrophils, or lymphocytes; Khan and Gupta, 1991) and damage to the lung tissue. These processes have been shown to begin within 2 hr of exposure following intratracheal (IT)
administration of silica to rats (DiMatteo et al., 1995). This inflammation and damage can ultimately manifest itself as the fibrotic lung disease, pulmonary fibrosis (Adamson et al., 1992; Driscoll et al., 1991; Heppleston, 1982). Depending upon the dose, the appearance of fibrosis can occur as rapidly as 2 weeks after the introduction of silica (Reiser et al., 1982). Fibrosis development is characterized by a rapid increase in the rate of lung collagen synthesis followed by an increase in the deposition of the excess collagen. This collagen is abnormal with respect to normal lung collagen in that it has altered amino acid cross-links and has thus been termed ‘‘fibrotic collagen’’ (Last and Reiser, 1985). Lesions of fibrotic collagen appear as whorled, rounded nodules of concentric layers of tissue around an acellular center (Lapp and Castranova, 1993). The alveolar macrophage is the initial phagocyte exposed to foreign matter introduced into the airways, as is the case in silica exposure. In general, normal alveolar macrophages do not constitutively secrete significant levels of inflammatory and growth-associated cytokines; however, upon appropriate stimulation macrophages are capable of secreting a variety of cytokines including interleukin (IL)-1, IL-1 receptor antagonist (ra), IL-8, IL-10, platelet-derived growth factor (PDGF), and tumor necrosis factor-a (TNF-a) (Fiorentino et al., 1991; Mukaida et al., 1992; Piguet et al., 1993; Ross et al., 1986; Vilcek and Lee, 1991). Release of macrophage-derived inflammatory/fibrogenic factors represents a means of amplification of the overall reaction as it evolves within the structures of the lungs. In the disease process, it is felt that the secretion of IL-1 and TNF-a by the macrophage is most important since these two factors initiate the cascade of responses that lead to inflammation and fibrosis; resultantly, their secretion has been extensively examined in the silica-induced pulmonary toxicity model by several investigators (Driscoll et al., 1990; Driscoll and Maurer, 1991; DuBois et al., 1989). Because proinflammatory cytokines are believed to be central to the development of silica-induced pulmonary toxicities, inhibition of the production of these cytokines should
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result in protection. The anti-inflammatory, glucocorticoid steroid dexamethasone has been demonstrated to inhibit the production of proinflammatory mediators (Guyre et al., 1988; Kern et al., 1988; Ohtsuka et al., 1996; Van Furth et al., 1995). These inhibitory effects may occur through gene repression by activated glucocorticoid receptor binding to negative glucocorticoid response elements (GRE) in the 5*flanking region of the proinflammatory gene. Binding at the GRE down-regulates the activity of the nuclear transcription factor, NF-kB (Kleinert et al., 1996). NF-kB plays an essential role in the production of silica-induced proinflammatory mediators (Chen et al., 1995). In addition to its inhibition of proinflammatory mediator production, dexamethasone has the ability to enhance the production of anti-inflammatory cytokines, such as IL-10, by leukocytes (Van Furth et al., 1995). Despite these favorable properties, the dosage and duration of systemically administered dexamethasone is limited almost exclusively by the side effects of the steroid. These side effects include suppression of the hypothalamic– pituitary adrenal axis, shifts in water balance, weight loss, and diabetes (Kehrl and Fauci, 1983). Dexamethasone may affect fibrogenesis in ways other than through influencing the inflammatory process. This glucocorticoid can inhibit fibrogenesis by acting directly on the fibroblast to inhibit collagen biosynthesis (Robey, 1979; Siddiqui et al., 1986). It is believed that this occurs through down-regulation of procollagen gene expression (Cockayne et al., 1986; Meisler et al., 1995). Dexamethasone can inhibit the synthesis of growth factors produced by fibroblasts (Shull et al., 1995; Chedid et al., 1996). Additionally, dexamethasone can inhibit fibroblast proliferation (Frost et al., 1994; Torry et al., 1994), and in this way modulate fibrosis. Therefore, dexamethasone may exert antifibrotic actions at more than one site in the tissue and through more than one mechanism. In order to test our hypothesis that pharmacological intervention early in the inflammatory cascade will attenuate the late-phase toxic response of fibrosis, we utilized a novel liposomal drug delivery system. This encapsulation technique allows for: (1) ‘‘selective’’ delivery of drug to the cells of interest; liposomes are particulate and therefore their natural fate is phagocytosis (Gonzalez-Rothi et al., 1991), (2) the delivery of higher local drug concentrations (Debs et al., 1987), and (3) lower systemic drug levels and associated adverse effects (Shek et al., 1994).
silica was determined by automated X-ray diffractometer and was 99.5% a-quartz. Size fraction õ5 mm in diameter was made by a centrifugal airflow particle classifier (Accucut Particle Classifier, Donalson-Majal Division, St. Paul, MN). Ninety-eight percent of this fraction was õ5 mm in size with a median area equivalent diameter of 3.5 mm as estimated by scanning electron microscopic image analysis. Enzyme reagents, dexamethasone base, and cholesterol were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals used in the study were from Fisher Chemical Co. (Pittsburgh, PA) unless otherwise noted. Animal Treatment Male Fischer 344 rats (Hilltop Lab Animals, Scottdale, PA) weighing 200–250 g were housed in laminar flow hoods (2 per cage) and allowed at least 1 week for acclimation after arrival from the supplier. Rats were given a conventional laboratory diet (Purina Chow pellets) and tap water ad libitum. Dexamethasone–liposome (DEX–LIP) preparation. Phosphatidylcholine (100 mg, egg lecithin) in chloroform (20 mg/ml; Avanti Polar Lipids, Alabaster, AL), 8 mg of cholesterol, and 6 mg of dexamethasone base were combined in a 500-ml round-bottom flask in a final solvent system of chloroform:methanol (9:1). Glass beads were added to the flask to increase the surface area for film development. The solvent phase was removed by rotary-vacuum evaporation. A thin milky white film formed against the inner wall of the flask and around the glass beads. This film was rehydrated with 4 ml of dexamethasone phosphate (10 mg/ml, Steris Laboratories, Phoenix, AZ) for 1 hr. The milky white lipid suspension was kept at room temperature for 2 hr under nitrogen gas. After gentle shaking of the suspension, it was sonicated in a room temperature waterbath sonicator for 3 min. This suspension was kept overnight at 47C for swelling of the liposomes. Before using the liposomes, the nonencapsulated dexamethasone phosphate was removed by centrifugation (30,000g) of the liposomes. The liposome pellet was washed three times with 4 ml Ca2//Mg2/-free Hanks’ balanced salt solution (HBSS). The final liposome pellet was resuspended in 5 ml HBSS and used immediately or stored at 47C until it was used within 1 week of preparation. HPLC determination of dexamethasone. A 0.5-ml aliquot of each final liposome suspension was lyophilized. The dexamethasone was extracted from each liposome preparation with 1 ml of acetonitrile and sonicated in a water bath sonicator for 10 min and then left overnight at room temperature. The mixture was then sonicated for 40 min, transferred to a microfuge tube, and centrifuged for 3 min (Beckman Microfuge 11 at a speed setting of 7). Dexamethasone incorporation was determined with a Waters HPLC system equipped with a C18 mBondapak reverse-phase column and acetonitrile:water (1:1) as the elutant at a flow rate of 1 ml/min. The effluent was monitored at a wavelength of 254 nm and drug levels were quantified by comparison of sample peak heights to those of drug standards that were run above and below the range of experimental values to construct the standard curve. Testosterone was used as an internal standard.
MATERIALS AND METHODS
Intratracheal instillation. Rats were treated in a manner similar to that outlined by Brain et al. (1976). After being anesthetized with Brevital (sodium methohexital, Eli Lilly & Co., Indianapolis, IN) ip, rats were placed on their backs on a slanted board suspended by a wire under their maxillary incisors. The tongue was moved and a ball-tipped 20-gauge animal feeding needle fitted with an insulin syringe was placed into the trachea via the mouth.
Crystalline Min-U-Sil silica (U.S. Silica Corp., Berkeley Springs, WV) was a gift from Dr. Val Vallyathan (National Institute for Occupational Safety and Health, Morgantown, WV). The silica was cleaned by boiling in 1 M HCl for 60 min to remove contaminants such as iron. Purity of the
• Dexamethasone–liposomes—Rats were IT instilled with 0.5 ml DEX– LIP or an equal volume of HBSS as control on Day 01 and then every fourth day afterward (dosing regimen determined from preliminary experiments) until the time of euthanization. Rats received 0.25–0.34 mg of dexamethasone in each of the instillations. The animals remained in the slanted position for 1 min after the instillation to facilitate distribution in
Materials
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DEXAMETHASONE–LIPOSOMES MODULATE SILICA TOXICITY the lungs. Rats were observed until consciousness was regained. After recovery from anesthesia, the animals were returned to the animal quarters until the time for analysis. • Silica (SI)—On Day 0, the DEX–LIP group received instillations of SI (DEX–LIP–SI); half of the HBSS group received SI (HBSS–SI), and the other half received saline (HBSS–SAL). The silica particles were prepared at a concentration such that rats received 10 mg/100 g body wt in 0.5 ml SAL. Before instillation the silica suspension was sonicated for 10 min. A 0.5-ml aliquot of SI was instilled slowly into the trachea. In control groups the same volume of SAL was instilled. The animals remained in the slanted position for 1 min after the instillation to facilitate distribution in the lungs. Rats were observed until consciousness was regained. After recovery from anesthesia, the animals were returned to the animal quarters until the time for analysis. The HBSS–LIP–SI control group was omitted from these studies after it was determined from previous experiments that responses to these ‘‘empty’’ liposomes were no different from those of the HBSS–SI group in all performed analyses. Bronchoalveolar lavage (BAL). On Day 10 or 20, BAL was performed for cellular, biochemical, and functional analyses of inflammation and damage. Rats were anesthetized with sodium pentobarbital (The Butler Co., Columbus, OH) and exsanguinated by severing the abdominal aorta. The trachea was cannulated with PE160 tubing and the cells from the lungs were collected by BAL. While massaging the lungs, 2 ml/100 g body wt of warm HBSS was instilled into the lungs. For the first instillation HBSS remained in the lungs for 30 sec, was withdrawn, and then reinstilled for another 30 sec to maximize retrieval of biological markers (Lindenschmidt et al., 1990). After the second withdrawal the HBSS was saved for analysis in a separate tube. For additional lavages, 5-ml aliquots of HBSS were instilled sequentially, withdrawn, and placed in centrifuge tubes until É40 ml was obtained. All tubes from each rat were centrifuged at 800g for 7 min. The supernatant from the first lavage was transferred into a separate conical tube for further analysis. All other supernatants were discarded. Cell pellets were resuspended in 5 ml HBSS and the cells from each animal were pooled. The cells were recentrifuged, as above, and the pellets were resuspended in 1 ml HBSS for cell counting and further analysis. Cellular, Functional, and Biochemical Assays Cell counts. Cells harvested from lavage procedures were counted via a hemacytometer. Differential cell counts. For cellular differentiation, cells (1.5 1 105) were spun in a Shandon cytocentrifuge at 400 rpm for 4 min and allowed to affix to a microscope slide. Slides were stained with Wright–Giemsa Sure Stain. Cell types (macrophages, neutrophils, and lymphocytes) were differentiated under a light microscope by counting at least 200 cells per slide. Data are presented for neutrophils only because of their value in assessing the inflammatory response. Luminol-dependent chemiluminescence (LDCL). Chemiluminescence is a functional assay to assess release of oxidants from cells or tissue. Luminol was incorporated into the reaction cuvettes as an amplifying agent of chemiluminescence to study the oxidative activity of the lavagable cells. Luminol is first oxidized to an intermediate that subsequently converts to an aminophthalate product and the release of a photon (É425 nm). Luminol reacts with superoxide, nitric oxide, and their reaction product peroxynitrite (Radi et al., 1993). LDCL was followed for 20 min at 377C using a sixchambered Berthold LB 9505C luminometer. The integrated response was determined by an accompanying computer equipped with a KINB program that was supplied with the luminometer. • Cellular LDCL—Total cell concentrations obtained from BAL were adjusted to yield 1 1 107 cells/ml. Luminol was dissolved in DMSO and then diluted in physiological Hepes buffer (0.1 M, pH 7.4). The final concentration of the luminol in the cuvette reaction mixture was 1005 M. Phorbol
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myristate acetate (PMA) was used as a soluble stimulant at a final cuvette concentration of 2 1 1006 M. In each run an unstimulated baseline value was obtained by omitting the PMA from one cuvette. The final volume in each reaction cuvette was 500 ml (100 ml cells, 100 ml luminol, 100 ml PMA, and 200 ml Hepes). When PMA was excluded from the cuvettes, the volume was brought up to 500 ml with Hepes buffer. All cuvettes were incubated in a 377C waterbath for 10 min prior to PMA stimulation and being placed in the luminometer. • Total cellular LDCL—This was calculated by adjusting the values obtained on a per cell basis to the total number of cells recovered by BAL. BALF albumin. Albumin was assayed to assess damage/permeability of the alveolar–capillary barrier and is specific for assessing this damage since it is a blood-derived protein. The albumin content of the acellular fraction obtained by the first lavage was determined by using the Sigma Diagnostics albumin reagent kit. Absorbance was determined at 628 nm on a spectrophotometer. Quantification of albumin was accomplished by comparison to a bovine serum albumin standard curve and expressed in units of mg/100 ml. BALF b-glucuronidase (b-glu). b-Glu is a lysosomal enzyme and was assayed to assess phagocytic cell activation/damage. The activity of b-glu in the acellular fraction of the first lavage was analyzed according to the method of Lockard and Kennedy (1976). The absorbance was determined at 400 nm. The activity of the enzyme was calculated by using a known extinction coefficient (18.3 ml/mmol/cm) and was expressed in units of nmol/min/ml. BALF lactate dehydrogenase (LDH). LDH is a cytosolic enzyme and was assayed to assess general cell death. The activity of LDH in the acellular fraction of the first lavage was analyzed according to the procedure set forth by the Boehringer Mannheim LDH reagent kit and monitored at 340 nm in a dual-beam scanning spectrophotometer. The enzyme activity was calculated by using a known extinction coefficient (6.2 ml/mmol/cm) and was expressed in units of nmol/min/ml. Fibrosis. Animals designated for fibrosis assessment were given an overdose of sodium pentobarbital and exsanguinated. After the trachea was cannulated and removed along with the lungs, the right lung lobes were ligated, removed, weighed, and frozen immediately at 0807C for later determination of hydroxyproline, a biochemical index of fibrosis. The left lung was infused with 10% phosphate-buffered Formalin through the cannula. The trachea was ligated so the cannula could be cut away and the lung samples were stored in Formalin solution until processed for histopathology. • Hydroxyproline—Hydroxyproline is an amino acid that is found almost exclusively in the composition of collagen and was therefore used as an index of fibrosis. Frozen lung samples were thawed and minced then processed and analyzed according to the method of Witschi et al. (1985). Optical density was read at 560 nm on a spectrophotometer. Amount of hydroxyproline per right lung was calculated from a standard curve and expressed as mg OH-Pro/right lung. • Histopathology—Microscopic evaluation of disease development was accomplished by sectioning fixed lung lobes and then staining with hematoxylin and eosin or a trichrome stain. This evaluation of the samples was performed in collaboration with a veterinary pathologist.
Statistical Analysis All parameters were analyzed by the appropriate analysis of variance. Effects found to be significant were analyzed further by Tukey’s protected t test, a multiple comparison procedure used to determine significant differences between pairs of groups. The criterion for significance between groups was p õ 0.05. All statistical analyses were carried out using GB-STAT software.
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RESULTS
As a gross measure of lung damage, the ratio of right lung weight to total body weight was calculated. HBSS–SI animals had a significant increase (2.6-fold) in this parameter (Fig. 1). The DEX–LIP–SI animals had a 38% reduction of the SI-induced increase in this ratio when compared to the HBSS–SI group, but this reduction was not great enough to return the values to those of the HBSS–SAL control group. Silica instillation also induced a profound fibrosis as the HBSS–SI group displayed a significant increase (1.8-fold) in right lung hydroxyproline content on Day 20 after the IT instillation of silica (Fig. 2). The periodic administration of DEX–LIP to rats significantly decreased (73% reduction in the SI-induced increase) but did not eliminate the increase in lung hydroxyproline content. Histopathological evaluation confirmed the reduction of the response to silica associated with the DEX–LIP administration (Fig. 3). In the HBSS– SI group, the response was characterized by prominent foci of granulomatous pneumonitis within the lung structure and moderate, multifocal alveolar histiocytosis. Compared to controls, there was a moderate increase in the amount of collagen present as assessed by trichrome staining. In the DEX–LIP–SI group, the histiocytosis was present to a lesser degree, but the granulomatous lesioning was essentially absent; the trichrome staining was reduced compared to the HBSS–SI group but still more prominent than the HBSS– SAL group.
FIG. 1. Day 20 right lung/total body wt ratio of animals treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú DEX–LIP–SI and HBSS–SAL; b, DEX–LIP–SI ú HBSS–SAL.
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FIG. 2. Day 20 right lung hydroxyproline content of animals treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú DEX–LIP– SI and HBSS–SAL; b, DEX–LIP–SI ú HBSS–SAL.
The second aspect to this study involved analyzing the BAL fluid, on Days 10 and 20 after IT instillation of SI or HBSS, from animals of each treatment group to examine the parameters of inflammation and tissue injury. Silica produced a severe pulmonary inflammation which was reduced significantly by IT DEX–LIP treatment. Figure 4 depicts total neutrophil numbers recovered by BAL of rats from each of the three treatment groups. The HBSS–SI had a large increase in the number of neutrophils in the BAL fluid. Animals of the DEX–LIP–SI group also had neutrophils in the BAL fluid, but the level was significantly diminished (66% at Day 10 and 85% at Day 20) when compared to the HBSS–SI animals. It was also evident that the numbers of neutrophils increased with time in the HBSS-SI group, whereas the number remained unchanged in the DEX–LIP– SI group illustrating a sustained protective effect. At Day 10 there were marginal increases in the numbers of lavagable macrophages in both the HBSS–SI and DEX–LIP–SI groups; however, these increases returned to control levels by Day 20 (data not shown). Lymphocyte numbers were not affected by any instillation procedure at either time point (data not shown). The integrated response of LDCL on a per-cell-basis is presented in Fig. 5. At both times, unstimulated cellular LDCL from both treatment groups demonstrated no increases in the LDCL assay. However, with PMA stimulation, cells from both SI-exposed groups demonstrated tremendous increases in LDCL activity, with DEX–LIP treatment augmenting the generation of light (2.5-fold at Day 10 and
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3.7-fold at Day 20). The observed response at Day 20 was significantly less than that at Day 10, but the values were significantly greater than control. In contrast to these findings, when LDCL is expressed as total LDCL (Fig. 6), there is no significant difference between the light generated by the HBSS–SI and the DEX–LIP–SI groups at both times.
FIG. 4. Total number of neutrophils obtained by bronchoalveolar lavage of animals treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6) with the value for the Day 10 HBSS–SI control being 0.01 1 107. Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú DEX–LIP–SI and HBSS–SAL within an individual time; b, DEX–LIP– SI ú HBSS–SAL within an individual time. *Day 20 HBSS–SI ú Day 10 HBSS–SI.
Figure 7 shows a significant increase in albumin in the BAL fluid from the HBSS–SI animals. The DEX–LIP treatment had no effect on the SI-induced elevation of albumin in the acellular lavage at both times assayed. The HBSS –SI group caused a dramatic increase in bglucuronidase activity in the acellular lavage when compared to the HBSS– SAL control (Fig. 8). This enzymatic activity was enhanced by DEX– LIP treatment over that of the HBSS-SI group by 1.5-fold at Day 10 and 1.7-fold at Day 20. Similar results were noted for the analysis of LDH activity in the acellular BAL (Fig. 9). The HBSS–SI group had a significant increase in LDH activity that was further enhanced in DEX–LIP–SI group. At both times analyzed, the LDH activity of the HBSS–SI group was elevated by 1.4fold as a result of the DEX–LIP treatment. DISCUSSION
FIG. 3. Representative light photomicrographs of sections of lung tissue from the following groups at Day 20 after saline (SAL) or silica (SI) instillation: (A) HBSS–SAL, lung architecture is normal; (B) HBSS–SI, there is marked granulomatous pneumonitis and a moderate alveolar histiocytosis; (C) DEX–LIP–SI, there is an absence of the granulomatous response, and a reduction in the alveolar histiocytosis (original magnification, 401).
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Respirable particulate matter is an occupational concern because of the potential toxicity posed to the lungs. The ability of silica to cause the development of pulmonary inflammation and fibrosis in workers in various mining and sand-blasting occupations has been well established. However, most therapeutic attempts to treat the silica-induced lung damage have generally been unsuccessful. In an inter-
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esting and unusual clinical case (Goodman et al., 1992), a driller at a surface coal mine was exposed to and inhaled sufficient quantities of silica dust to demonstrate clinical symptomatology. His condition worsened and upon bronchoscopy and biopsy the patient was diagnosed with acute
FIG. 5. Cellular luminol-dependent chemiluminescence (LDCL) from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI) at Day 10 (A) and Day 20 (B). LDCL was monitored in the presence of luminol using a chemiluminometer. LDCL was determined at rest (unstimulated) or after stimulation with phorbol myristate acetate. Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú HBSS–SAL within an individual time; b, DEX–LIP–SI ú HBSS–SI and HBSS–SAL within an individual time. *Day 10 stimulated ú Day 20 stimulated within an individual group. 1, stimulated ú unstimulated within an individual group.
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FIG. 6. Total cellular luminol-dependent chemiluminescence (LDCL) from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI) at Day 10 (A) and Day 20 (B). Total LDCL was calculated by adjusting the values obtained on a per cell basis to the total number of cells recovered by BAL. Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI Å DEX–LIP–SI ú HBSS–SAL within an individual time. *Day 10 Stimulated ú Day 20 Stimulated within an individual group. 1, Stimulated ú Unstimulated within an individual group.
silicosis. The patient’s condition drastically improved when treated with a high-dose corticosteroids. Therapy was continued with oral corticosteroids at a reduced dosage which markedly reduced the severity of silica-induced lung nodules. Upon cessation of the steroid therapy (due to concerns of steroid-related toxicities) the patient’s condition
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FIG. 7. Albumin content in acellular bronchoalveolar lavage fluid from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotation represents significant rank order differences (p õ 0.05) as follows: a, HBSS–SI Å DEX–LIP–SI ú HBSS–SAL within an individual time.
worsened, his lungs became fibrotic, and he eventually died as a result of the silicosis. Thus, it appears that early and continual treatment with steroids could be life-saving in such conditions where silica causes major lung toxicities. However, due to the inherent
FIG. 8. b-Glucuronidase activity in acellular bronchoalveolar lavage fluid from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS– SI ú HBSS–SAL within an individual time; b, DEX–LIP–SI ú HBSS– SI and HBSS–SAL within an individual time.
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FIG. 9. Lactate dehydrogenase activity in acellular bronchoalveolar lavage fluid from rats treated intratracheally with HBSS or DEX–LIP and then with saline (SAL) or silica (SI). Values are means { SE (n Å 6). Denotations represent significant rank order differences (p õ 0.05) as follows: a, HBSS–SI ú HBSS–SAL within an individual time; b, DEX– LIP–SI ú HBSS–SI and HBSS–SAL within an individual time.
adverse effects associated with the long-term systemic administration of the steroids (suppression of the hypothalamic–pituitary adrenal axis, shifts in water balance, weight loss, and diabetes; Kehrl and Fauci, 1983), this route of delivery may be of little value in the treatment of silicainduced chronic lung disease. A novel and immunologically inert drug delivery system that can be administered locally may provide a new option in the treatment of such an inflammatory and fibrotic disease. Such a system exists in the liposomal encapsulation technique which allows for: (1) ‘‘selective’’ delivery of drug to the cells of interest (Gonzalez-Rothi et al., 1991). The usual fate of liposomes is ingestion and digestion by macrophages; therefore, liposomes have become a suitable tool to manipulate the function of the macrophage (Van Rooijen and Sanders, 1994); (2) the delivery of higher local drug concentrations (Debs et al., 1987); and (3) lower systemic drug levels and associated adverse effects (Shek et al., 1994). Encapsulation techniques have been used to deliver various drug entities in attempt to maximize the desired pharmacological effects and minimize the unwanted toxicological effects. The therapeutic efficacy of the antifibrotic agent cis-4hydroxy-L-proline (cHYP) in preventing bleomycin-induced pulmonary fibrosis was studied in both the free and liposomal formulation (Poiani et al., 1994). After once weekly intratracheal instillations of free cHYP or cHYP–liposomes, the investigators observed greater protection from bleomycin-induced increases in lung collagen as a result of liposo-
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mal encapsulation. The amount of lung collagen present in the free cHYP group was no different than that of the group treated with bleomycin alone. This study demonstrated that delivery of cHYP to the lung in carrier vehicles (i.e., liposomes) enhanced their efficacy in treating pulmonary fibrosis, over that of free unencapsulated cHYP alone. In a similar type of study, Tremblay and colleagues (1993) compared the effects of daily intranasal instillations of liposomal dexamethasone and free dexamethasone in a murine model of hypersensitivity pneumonitis. It was concluded from these studies that liposome-incorporated dexamethasone was a more effective treatment in reducing the lung inflammation and damage associated with hypersensitivity pneumonitis than was the free dexamethasone. In addition to the enhanced therapeutic value of liposomal encapsulation, this preparation did not inhibit adrenocorticotropic hormone secretion, therefore implying that the liposomal formulation was retained locally within the confines of the lungs and did not achieve blood concentrations sufficient to elicit a systemic effect. This protective effect is most likely mediated through glucocorticoid inhibition of inflammatory mediator production. Dexamethasone inhibits the production of proinflammatory mediators (Guyre et al., 1988, Kern et al., 1988, Ohtsuka et al., 1996, Van Furth et al., 1995) through gene repression by activated glucocorticoid receptor binding to negative glucocorticoid response elements (GRE) in the 5*-flanking region of proinflammatory cytokine genes (Kleinert et al., 1996). Binding at the GRE down-regulates the activity of the nuclear transcription factor, NF-kB, which plays an essential role in the production of silica-induced proinflammatory mediators (Chen et al., 1995). In addition to its inhibition of proinflammatory mediator production, dexamethasone enhances the production of anti-inflammatory cytokines, such as IL-10, by leukocytes (Van Furth et al., 1995). In the present study, we observed partial protection from silica-induced pulmonary inflammation and fibrosis by the administration of dexamethasone-containing liposomes. A reduction in the silica-induced increase in the right lung to total body weight ratio after DEX–LIP administration suggests that this formulation provided a general protection against silica toxicity. Additional protection by the DEX– LIP administration was evident when assessing the right lung hydroxyproline content, the biochemical index of fibrosis. Upon histopathological assessment, rats that received silica alone displayed prominent granulomatous lesions within the lung sections. However, a lesser degree of lesioning was present in the lungs of rats in the DEX–LIP– SI group. These observations confirm the biochemical findings in that the administration of dexamethasone-containing liposomes attenuated the development of silica-induced pulmonary fibrosis.
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Systemic dexamethasone administration has been shown to be effective in eliminating the acute silica-induced neutrophil accumulation 24 hr after instillation (Van Dyke et al., 1994). Inhibiting the production of proinflammatory and/or chemotactic factors may account for these effects. The IL8 family of cytokines is thought to be the primary signal for neutrophil recruitment, and dexamethasone has the ability to genetically suppress the production of the rat neutrophil chemoattractant CINC/gro, a member of the IL-8 family (Ohtsuka et al., 1996). In our study, the sustained reduction in lavagable neutrophil numbers that results from DEX–LIP treatment is most likely a direct response to this genetic down-regulation. While DEX–LIP administration afforded protection against silica-induced cellular inflammation and fibrosis, biochemical and functional indices of damage were unaffected or enhanced. It is possible that these augmented responses are a direct result of the ‘‘protective’’ effects observed by the dexamethasone administration and can be accounted for by the inherent functions of the steroid itself. Steroid therapy blunts the influx of blood-borne neutrophils into the airspaces. These recruited phagocytes have been shown to be beneficial in the clearance of silica particles from the lungs, and actually decrease the toxicity that normally results from silica (Adamson et al., 1992). As a result of decreased neutrophil influx and subsequent decreased particle clearance from the lungs, the burden of silica remains elevated at levels sufficient to allow for increased phagocyte–particle interactions and activation (increasing b-glucuronidase levels); in addition to increased cytotoxicity (increasing lactate dehydrogenase levels) produced by the silica itself. This cycling nature of silica (phagocytosis r cytotoxicity r ‘‘retoxification’’ r phagocytosis r . . .) may lead to the levels of BAL enzyme activity obtained in the present study. Thus, inhibiting the neutrophil influx with DEX–LIP administration may actually be potentiating the silica-induced damage observed in BAL fluid in our system. The lack of DEX–LIP protection afforded to the alveolar– capillary barrier, as assessed by the albumin analysis, is most likely due to the toxic surface properties of the silica itself. Since there is a lack of silica clearance by the inhibition of neutrophil recruitment, there is a greater chance that the silica can interact with the barrier and damage it, allowing the blood-derived protein to enter the airspaces. The DEX–LIP increase in silica-induced oxidant production, as assessed by chemiluminescence, may also be attributed to the inherent properties of the dexamethasone. It is known that silica as well as individual proinflammatory cytokines (IL-1 and IL-6) are able to increase antioxidant mRNA expression (Dougall and Nick, 1991; Janssen et al., 1991, 1994). Since inflammation is known to increase the oxidative status of the tissue (DiMatteo et al., 1995) as well as the cells
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(Antonini et al., 1994), the role of the increased antioxidant expression may be an attempt to protect against inflammation-induced oxidative damage. Thus: (1) dexamethasoneinduced inhibition of proinflammatory cytokine production results in decreased antioxidant mRNA expression (Chen et al., 1993; Dougall and Nick, 1991); (2) decreased antioxidant mRNA expression results in a decrease in the available antioxidant pool; and (3) the decreased antioxidant pool results in an enhanced oxidative status, as assessed by luminoldependent chemiluminescence. However, when expressed on a total cellular chemiluminescence basis, there is no difference in the oxidative state between the HBSS–SI and the DEX–LIP–SI groups since there are tremendously more cells in the HBSS–SI group than there are in the DEX– LIP–SI group. To validate this hypothesis, analysis of the antioxidant status of the BAL cells is required. It appears from our study that cellular events characteristic of silica exposures are critical for fibrogenesis, since protection from inflammation and fibrosis occurred independently of biochemical and functional indices. DEX–LIP administration suppressed inflammation and fibrosis by presumably acting through the NF-kB mechanism and the down-regulation of macrophage-derived proinflammatory/fibrogenic cytokine production. Direct action on fibroblast function may also have been involved. With their function in recycling surfactant, the alveolar type II cells could theoretically be a nonselective target for liposomal delivery and be acted upon by the encapsulated dexamethasone. Although this is possible, surfactant protein A (SP-A) is needed for alveolar type II cells to recycle phospholipids. It appears that SP-A regulates the uptake of phospholipids in some fashion (Bates et al., 1994; Kuroki et al., 1994; McCormack et al., 1994). Therefore, since there was no SP-A incorporated into the liposomes utilized in our study, the uptake of liposomes by alveolar type II cells would be negligible, and any anti-inflammatory/antifibrotic effects observed as a result of the administration of the dexamethasone-containing liposomes would not be mediated by the type II cells. From our study we can conclude that early pharmacological intervention, at the level of macrophage processing, signaling and cellular recruitment, and possibly effects directly on the fibroblast altered the inflammatory and fibrotic responses in rats. The administration of dexamethasone-containing liposomes to rats before and after the intratracheal instillation of silica results in the attenuation of the inflammation and fibrosis that is normally associated with exposure to this occupationally relevant dust. Additionally, this protection is independent of some biochemical and functional parameters of damage. Thus, overall we have reported a novel drug delivery system that can potentially be used in the treatment of silica-induced pulmonary inflammation and fibrosis.
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Direct administration of liposomes to the lungs can be achieved by intratracheal instillation or by aerosol inhalation. While the use of the instillation method serves as a means whereby a controlled dose can be delivered to the lung in an animal model, the instillation procedure is not practical for routine human use. For routine applications, aerosol inhalation would be considered the most convenient and practical approach to pulmonary liposome delivery. This delivery system would be advantageous over the current commercially available steroid inhalers since there would be enhanced targeting to the phagocytes, increased retention time within the lungs, and decreased extrapulmonary side/adverse effects. Invariably, these features would result in a greater therapeutic efficacy over that of conventional steroid inhalers. Although inhalational delivery of liposomes promises to be convenient and effective in many clinical application, delivery system engineering issues must be addressed prior to widespread human usage. ACKNOWLEDGMENTS This research was supported by the 1995 Hazleton/Society of Toxicology Graduate Student Fellowship (M.D.), a National Institute of Occupational Safety and Health Cooperative Agreement Grant for Occupational Respiratory Disease and Musculoskeletal Disorders, U60/CCU306149, and a National Institutes of Health Training Grant, 5 T32 GM07039-20. The authors thank Dr. Robert Garman and his staff for performing the histopathological analyses in this study.
REFERENCES Adamson, I. Y. R., Prieditis, H., and Bowden, D. H. (1992). Instillation of chemotactic factor to silica-injected lungs lowers interstitial particle content and reduces pulmonary fibrosis. Am. J. Pathol. 141, 319–326. Antonini, J. M., Van Dyke, K., Ye, Z., DiMatteo, M., and Reasor, M. J. (1994). Introduction of luminol-dependent chemiluminescence as a method to study silica inflammation in the tissue and phagocytic cells of rat lung. Environ. Health Perspect. 102, 37–42. Bates, S. R., Dodia, C., and Fisher, A. B. (1994). Surfactant protein A regulates uptake of pulmonary surfactant by lung type II cells on microporous membranes. Am. J. Physiol. 267, L753–L760. Brain, J. D., Knudson, D. E., Sorokin, S. P., and Davis, M. A. (1976). Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11, 13–33. Chedid, M., Hoyle, J. R., Csaky, K. G., and Rubin, J. S. (1996). Glucocorticoids inhibit keratinocyte growth factor production in primary dermal fibroblasts. Endocrinology 137, 2232–2237. Chen, F., Sun, S. C., Kuh, D. C., Gaydos, L. J., and Demers, L. M. (1995). Essential role of NF-kappa B activation in silica-induced inflammatory mediator production in macrophages. Biochem. Biophys. Res. Commun. 214, 985–992. Chen, Y., Whitney, P. L., and Frank, L. (1993). Negative regulation of antioxidant enzyme gene expression in the developing fetal rat lung by prenatal hormonal treatments. Pediatr. Res. 33, 171–176. Cockayne, D., Sterling, K. M., Jr., Shull, S., Mintz, K. P., Illeyne, S., and Cutroneo, K. R. (1986). Glucocorticoids decrease the synthesis of type I procollagen mRNAs. Biochemistry 25, 3203–3209.
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