Antibodies to mouse lung capillary endothelium.

We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.

Effects of ozone and endotoxin coexposure on rat airway epithelium: potentiation of toxicant-induced alterations.

Tropospheric ozone is the major oxidizing component in photochemical smog and is one of the most pervasive problems to human health of the criteria air pollutants for which the National Ambient Air Quality Standards have been designated by the Clean Air Act. Although many adverse health effects of ozone exposure have been documented in both humans and laboratory animals, controversy surrounds the establishment and implementation of ozone standards set forth by the U.S. Environmental Protection Agency. Because people are commonly exposed to more than one air pollutant at a time, studies that examine coexposures to airborne materials may be more relevant for assessing their risks to human health. Airborne biogenic substances such as pollens, spores, and bacterial products are ubiquitous in the environment, and when inhaled can cause adverse respiratory symptoms. One such biogenic agent, bacterial endotoxin, is a potent stimulus of airway inflammation and is a ubiquitous airborne contaminant commonly found in domestic, agricultural, and industrial settings. Little is known about the interaction of exposures to biogenic substances and criteria air pollutants such as ozone. In the last few years we have performed a series of studies in rodents that examined the biologic responses of the respiratory epithelium after airway exposures to both endotoxin and ozone. When exposed to ozone (0.5 ppm 8 hr/day for 3 days), Fischer rats develop lesions in the nasal transitional epithelium, whereas intranasal instillation of endotoxin (20 microg) elicits epithelial lesions in the respiratory epithelium of the nose and conducting airways. Our studies were designed to examine how exposure to one toxicant may affect the airway epithelial lesions induced by the other toxicant. We investigated the potential role of acute inflammation in the enhancement of airway epithelial lesions after exposure of these two toxicants in neutrophil-sufficient and neutrophil-deficient rodents. A summary of these results indicates that epithelial and inflammatory responses to coexposure of these two pollutants are greater than those elicited by either agent alone. Interestingly, each toxicant enhances the epithelial alterations induced by the other. Furthermore, the synergistic effects elicited by coexposure to ozone and endotoxin are mediated partly by neutrophils. These studies provided some new insights into how inhaled co-pollutants interact to initiate and promote alterations of airway epithelium. Further studies with these and other air pollutants will help define their true risk to human health.

In vivo effects of endotoxin on DNA synthesis in rat nasal epithelium.

Airway inflammation in bacterial infections is characterized by the presence of neutrophils and often epithelial injury and repair. Release of endotoxin from bacteria may contribute to these processes. The purpose of this study was to determine the in vivo effects of repeated endotoxin exposure on DNA synthesis in rat nasal epithelium in the presence and absence of neutrophilic influx. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillations, half of the saline and endotoxin-instilled animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum. Rats were sacrificed 6 or 24 h after the last instillation. Two hours prior to sacrifice, rats were intraperitoneally injected with bromodeoxyuridine (BrdU), an analog of thymidine that is incorporated in the nucleus of cells in the S-phase of the cell cycle. Nasal tissues were processed for light microscopy and immunohistochemical detection of BrdU in nasal epithelial cells. The numbers of nasal epithelial cells, BrdU-labeled epithelial nuclei, and neutrophils per millimeter of basal lamina in the epithelium lining the nasal turbinates in the proximal nasal passages were determined by morphometric analysis. We did not observe a neutrophilic influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, the numbers of nasal epithelial cells and the BrdU-labeling index were significantly increased compared to saline-instilled controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Responses of rat nasal epithelium to short- and long-term exposures of ozone: image analysis of epithelial injury, adaptation and repair.

This article reviews the use of computerized image analysis and standard morphologic techniques to characterize the responses of nasal epithelium in laboratory rats to single or repeated exposures to a common urban air-pollutant, ozone. Alterations in the number and composition of the epithelial cell populations after either short- or long-term exposures are described. The principal nasal epithelial alteration induced by repeated exposures to this irritating, oxidant pollutant is mucous cell metaplasia (i.e., transformation of airway epithelium, normally devoid of mucous cells, to a secretory epithelium containing numerous mucus-secreting cells). This metaplastic change, induced by acute or chronic ozone exposures, has been morphometrically examined at various times post-exposure. In this article, we describe our current understanding of the pathogenesis and persistence of ozone-induced mucous cell metaplasia in nasal epithelium based on the results of these morphometric studies.

Inflammatory and epithelial responses during the development of ozone-induced mucous cell metaplasia in the nasal epithelium of rats.

Rats repeatedly exposed to high ambient concentrations of ozone develop mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE). The present study was designed to determine the temporal relationships of ozone-induced inflammatory and epithelial responses and their correlation with subsequent MCM in the NTE of rats. Male F344/N rats were exposed to 0.5 ppm ozone, 8 h/day for 1, 2, or 3 days. Two h prior to sacrifice, all the rats were injected intraperitoneally with 5'-bromo-2-deoxyuridine (BrdU) to label epithelial cells undergoing DNA synthesis. Rats exposed to ozone for 1 or 2 days were killed 2 h after the exposure. Rats exposed to ozone for 3 days were killed 2 h or 1, 2, or 4 days after the exposure. Control rats were killed after a 7-day exposure to filtered air. One nasal passage from the anterior nasal cavity of each rat was fixed and processed for light microscopy to morphometrically determine the numeric densities of epithelial cells, neutrophils, and mucous cells, and the amount of intraepithelial mucosubstances in the NTE. The maxilloturbinate from the other nasal passage was processed for analysis of an airway mucin-specific gene (i.e., rMuc-5AC mRNA). Acute ozone exposure induced a rapid increase in rMuc-5AC mRNA levels prior to the onset of MCM, and the increased levels of rMuc-5AC mRNA persisted with MCM. Neutrophilic inflammation coincided with epithelial DNA synthesis and upregulation of rMuc-5AC, but was resolved when MCM first appeared in the NTE. The results of the present study suggest that upregulation of mucin mRNA by acute ozone exposure may be associated with the concurrent neutrophilic inflammation and epithelial hyperplasia in the NTE. Ozone-induced MCM may be dependent on these important pre-metaplastic responses (i.e., mucin mRNA upregulation, neutrophilic inflammation, and epithelial proliferation).

Endotoxin enhancement of ozone-induced mucous cell metaplasia is neutrophil-dependent in rat nasal epithelium.

Ozone, the primary oxidant gas in photochemical smog, causes neutrophilic inflammation and mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE) of rats and monkeys. Bacterial endotoxin is another common airborne agent that induces acute neutrophilic inflammation, but not MCM, in NTE. It does, however, enhance ozone-induced MCM in rat nasal airways (Fanucchi et al., 1998, Toxicol. Appl. Pharmacol. 152, 1-9). In the present study, F344 rats exposed to filtered air or 0.5 ppm ozone (8 h/day for 3 days) were intranasally instilled with sterile saline or 100 microg endotoxin 24 h and 48 h after the third ozone exposure. To determine the role of neutrophilic inflammation in endotoxin-induced potentiation of the MCM caused by ozone, half of the rats were depleted of circulating neutrophils prior to saline or endotoxin instillations. Rats were killed 6 h or 3 days after the last intranasal instillation, and nasal tissues were processed for (1) light microscopy and morphometric analysis to determine the number of infiltrating neutrophils and the volume amount (density) of stored mucosubstances in the NTE, and (2) quantitative RT-PCR analysis of steady-state mucin gene (rMuc-5AC) mRNA levels in the NTE. Endotoxin induced a transient influx of neutrophils in both air- and ozone-exposed rats that was completely blocked by neutrophil depletion. Endotoxin increased rMuc-5AC mRNA levels in the NTE of ozone-exposed rats. Neutrophil depletion, however, had no effect on endotoxin-induced upregulation of mucin gene mRNA levels. Endotoxin enhanced the ozone-induced increase in stored mucosubstances (4-fold increase), but only in neutrophil-sufficient rats. These data indicate that endotoxin enhancement of ozone-induced upregulation of rMuc-5AC mRNA levels is neutrophil-independent, while its effects on intraepithelial production and storage of mucus glycoproteins is dependent on the presence of neutrophils.

Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs.

Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity. LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins. Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure. The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis. Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone. Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice. Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p<0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone. When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p<0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone. The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

Ozone- and endotoxin-induced mucous cell metaplasias in rat airway epithelium: novel animal models to study toxicant-induced epithelial transformation in airways.

Mucous (goblet) cell proliferation and hypersecretion of airway mucus are important characteristics of human respiratory disorders, especially chronic bronchitis and cystic fibrosis. These changes in secretory patterns also occur in animals experimentally exposed to chemical irritants such as ozone (O3), sulfur dioxide (SO2), and cigarette smoke. The cellular and molecular mechanisms involved in irritant-induced mucous cell metaplasia (MCM; transformation of airway epithelium, normally devoid of mucous cells, to a secretory epithelium containing numerous mucous cells) are still unclear. We used two experimental models of toxicant-induced MCM in rat airways to study the cellular and molecular changes that occur during the development of this respiratory tract lesion. MCM can be induced in the nasal transitional epithelium of rats by repeated exposure to ambient levels of ozone. In addition, MCM can be induced in the tracheobronchial airways of rats repeatedly exposed to endotoxin, a lipopolysaccharide-protein molecule found in the outer walls of Gram-negative bacteria. The pathogenesis of ozone- or endotoxin-induced MCM has been partially characterized using a variety of morphometric and histochemical techniques. Toxicant-induced changes in the numbers and types of airway epithelial cells have been estimated using morphometric methods designed for estimating the abundance of cell populations. Nasal pulmonary airway tissues are also processed for light microscopy and stained with Alcian Blue (pH 2.5)/Periodic Acid Schiff (AB/PAS) for detection of acidic and neutral mucosubstances (the specific glycoprotein product of mucous cells), respectively, within the tissue. Computerized image analysis is used to quantitate the amount of the stained mucous product within the airway epithelium. To better characterize the molecular and cellular events in the pathogenesis of ozone- or endotoxin-induced MCM in the rat airway epithelium, we are conducting studies to determine when, and in which epithelial cells, the mucin gene is expressed after exposure to the toxicant. In these studies, rats undergo single or repeated exposures to ozone or endotoxin and are then sacrificed immediately or a few days after the end of the exposures. Airway tissues are microdissected from specific regions of the exposed respiratory tract, and changes in mucin core polypeptide mRNA are evaluated by Northern analysis using human and rat mucin cDNA. In future studies using in situ hybridization, we will establish when, and in which epithelial cells, the expression of high molecular weight airway mucin is initiated in response to ozone or endotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)

Pyridine induction of Sprague-Dawley rat renal cytochrome P4502E1: immunohistochemical localization and quantitation.

Previous research has shown that i.p. injection of rats with pyridine results in a significant increase in immunoreactive renal cytochrome P4502E1 (alcohol-inducible form) in a dose- and time-dependent manner. However, the cellular location of renal P4502E1 in rats was not reported. Thus, it was not known whether the pyridine-induced increase in renal P4502E1 resulted from increased production of the enzyme in cells which normally express P4502E1 or from de novo expression in cells normally devoid of the protein. To address these questions, rats were injected i.p. with either 200 mg pyridine/kg body wt./day for 1, 2, 3, or 4 days (n = 2/group) or injected once with an equal volume of sterile, pyrogen-free saline (control group; n = 2). Kidney tissue samples from saline- and pyridine-exposed rats were processed by light microscopy and were immunochemically stained to detect rat cytochrome P4502E1. Most of the immunoreactive P4502E1 was located within renal cortical epithelial cells lining proximal and distal tubules of the cortex with lesser--but consistent--amounts present in tubular epithelial cells within the inner and outer medulla. Pyridine exposure resulted in a 2-3-fold increase in P4502E1 immunoreactivity in proximal cortical tubules surrounding glomeruli and cortical blood vessels. The results of this study demonstrate a cell-specific distribution of cytochrome P4502E1 within the rat kidney and indicate that pyridine exposure results in a selective induction of immunoreactive P4502E1 in tubule epithelial cells which constitutively express this enzyme. The results of this study provide a morphologic basis for interpreting cell-specific nephrotoxicity due to xenobiotics that are biotransformed to toxic metabolites by renal P4502E1.

Chromosome damage in rat pulmonary alveolar macrophages following ozone inhalation.

To determine whether ozone is clastogenic at environmentally relevant exposure levels, rats were exposed for 6 h to 0.0, 0.12, 0.27, or 0.80 ppm ozone. The alveolar macrophages were isolated from animals sacrificed 28 h after the end of the exposure. The mitotic index and frequency of chromosome aberrations were determined. No change in the mitotic index was detected following 0.12 ppm ozone exposure. A significant decrease in mitotic index was observed after exposure to 0.27 ppm ozone; a significant (4-fold) increase in the frequency of dividing macrophages was detected following exposure to 0.8 ppm ozone. Only chromatid-type aberrations were observed. There was a significant increase in the frequency of cells with chromatid gaps and in the frequency of cells with chromatid deletions. Animals exposed to 0.27 ppm ozone had the highest proportion of cells with chromatid deletions (0.172) relative to background level (0.028). No exchanges or chromosome-type aberrations were detected in any of the animals. These data suggest that ozone, at relatively low levels, is clastogenic in macrophages from exposed rats.

Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part XII: Atrophy of bone in nasal turbinates.

As part of the National Toxicology Program/Health Effects Institute collaborative study of the health effects of prolonged ozone exposure, it was observed that rats chronically exposed to ozone had marked histopathologic changes in the upper respiratory tract, including atrophy of the nasal turbinates. The principal objective of the present study was to morphometrically assess the severity of the ozone-induced changes in the bony tissue of the maxilloturbinates in these chronically exposed rats. Male and female F344/N rats were exposed to 0, 0.12, 0.5, or 1.0 part per million (ppm) ozone, 6 hours/day, 5 days/week for 20 or 24 months. Rats were killed one week after the end of the exposure, and nasal tissues were processed for light and electron microscopy. Using image analysis and standard morphometric techniques, the amounts of bone, surface epithelium, and lamina propria comprising the maxilloturbinates were estimated by measuring the cross-sectional area of each tissue compartment at a defined location in the proximal nasal passage. Both male and female rats had significant morphologic and morphometric changes in the maxilloturbinates after prolonged exposures to 0.5 or 1.0 ppm ozone, but not to 0.12 ppm ozone. Ozone-exposed rats had significant reductions in the cross-sectional area of turbinate bone, reflecting the loss of bone in the maxilloturbinate after prolonged exposure. This ozone-induced bony atrophy was more severe in male than in female rats. Using electron microscopy, numerous bone-resorption sites were identified on the outer, periosteal, surface of the turbinate bone in ozone-exposed animals. Rats with bony atrophy also had a conspicuous influx and mixed inflammatory cells into the lamina propria surrounding the turbinate bone. In addition, ozone exposures caused reductions in the area of lamina propria, due to blood vessel constriction, and increases the in the area of the surface epithelium, due to hyperplasia and metaplasia. The results of the present tudy demonstrated that prolonged exposure of rats to ozone can cause marked loss of turbinate bone. The severity of this ozone-induced bony atrophy in rats is dependent on both concentration and gender.

A microspray nozzle for local administration of liquids or suspensions to lung airways via bronchoscopy.

A microspray nozzle has been developed for the localized administration of solutions or suspensions to discrete pulmonary airway regions via bronchoscopy. Toxicants or tracer particles can be administered to single or multiple, specific lung airway segments. This permits studies of local-airway, whole-lung, and systemic responses to inhaled materials. The nozzle is fabricated by ultra-precision machining, has an overall length of 1 mm, and a diameter small enough to fit in the end of a 1.1-mm inner diameter, 1.2-mm outer diameter catheter. In vitro studies demonstrated the delivery efficiency for 99mTc solutions (98 +/- 1% SD) and for suspensions of 99mTc sulfur colloid or 3-mum 85Sr-labeled microspheres (55 +/- 15%). Photographic and gamma camera images of material deposited in the airways of Beagle dogs demonstrated in vivo that the deposition patterns are compact and uniform. The technique may also have therapeutic medical applications.

Mucin genes in horse airways: MUC5AC, but not MUC2, may play a role in recurrent airway obstruction.

REASONS FOR PERFORMING STUDY: Increased mucin gene expression may be an important cause of mucus accumulation observed in recurrent airway obstruction (RAO)-affected horses. To date, however, no mucin gene sequences are available for the horse. OBJECTIVES: To identify equine homologues of gel-forming mucins and investigate their expression at different airway generations of healthy and RAO-affected horses. METHODS: Two equine homologues were identified by cloning and sequencing fragments of equine (eq)MUC5AC and eqMUC2. RESULTS: Semiquantitative RT-PCR on RNA from airways (generations 1, 5, 10, 15; small airways and parenchyma), stomach (glandular), and colon revealed that eqMUC5AC is expressed in equine stomach and in all of the airway samples. In contrast, eqMUC2 steady-state mRNA levels were detected in colon and very faintly in stomach, but not in airway tissue. EqMUC5AC expression was also compared to that of ZO-1, a tight junction protein, and eqMUC5AC/ZO-1 ratios were higher in RAO-affected compared to control horses at all airway generations. CONCLUSIONS: That eqMUC5AC is expressed in horse airways, but any expression of MUC2 is undetectable and unlikely to be of physiological consequence. POTENTIAL RELEVANCE: EqMUC5AC up-regulation may be a primary mechanism responsible for mucus hypersecretion and accumulation in RAO.

Assessment of the respiratory sensitization potential of proteins using an enhanced mouse intranasal test (MINT).

There remains a need for a simple and predictive animal model to identify potential respiratory sensitizers. The mouse intranasal test (MINT) was developed to assess the relative allergic potential of detergent enzymes, however, the experimental endpoints were limited to evaluation of antibody levels. The present study was designed to evaluate additional endpoints (serum and allergic antibody levels, pulmonary inflammation and airway hyperresponsiveness (AHR)) to determine their value in improving the predictive accuracy of the MINT. BDF1 mice were intranasally instilled on days 1, 3, 10, 17 and 24 with subtilisin, ovalbumin, betalactoglobulin, mouse serum albumin or keyhole limpet hemocyanin; challenged with aerosolized methacholine or the sensitizing protein on day 29 to assess AHR, and sacrificed on day 29 or 30. Under the conditions of this study, evaluation of AHR did not improve the predictive power of this experimental model. Allergic antibody responses and IgG isotype characterization proved to be the most sensitive and reliable indicators of the protein allergenic potential with BAL responses providing additional insight. These data highlight that the evaluation of the respiratory sensitization potential of proteins can be best informed when multiple parameters are evaluated and that further improvements and refinements of the assay are necessary.

Acute toxicologic and neurotoxic effects of inhaled 1,2-dichloroethane in adult Fischer 344 rats.

Acute toxicologic and neurotoxic effects were evaluated in Fischer 344 rats exposed to 0, 50, 200, 600, or 2000 ppm 1,2-dichloroethane (ethylene dichloride; EDC) for 4 h or 0, 50, 100 or 150 ppm for 8 h. Neurobehavioral and neuropathologic effects were assessed using a functional observational battery (FOB; baseline, days 1, 8, and 15), and by light microscopy, respectively. Acute toxicologic effects were assessed by bronchoalveolar lavage (BAL) and histopathology of the respiratory tract and selected target organs. Neurobehavioral effects consistent with central nervous system (CNS) depression were present at concentrations >200 ppm and were restricted to day 1. There were no neuropathologic changes in the CNS, however, olfactory epithelial regeneration 15 days after exposure to > or = 200 ppm was observed. The no-observed-effect concentration (NOEC) for behavioral neurotoxicity was 200 ppm EDC for 4 h. There were no effects on BAL parameters in any exposure group. Exposure to 2000 ppm EDC altered adrenal gland, kidney, and liver weights, and resulted in morphologic alterations in the kidney and liver. Degeneration/necrosis of the olfactory epithelium was observed at > or = 200 ppm for 4 h and > or = 100 ppm for 8 h. Based on olfactory epithelial degeneration/necrosis, the most sensitive indicator of toxicity in this study, the overall NOEC was 50 ppm EDC for up to 8 h in rats.

In vivo effects of endotoxin on intraepithelial mucosubstances in rat pulmonary airways. Quantitative histochemistry.

Bacteria-induced bronchopneumonias are often characterized by an influx of neutrophils and excess mucus in pulmonary airways. This study determined how endotoxin, a component of gram-negative bacteria and a potent inflammatory agent, affects the ultrastructure of the mucociliary apparatus and the amount of stored intraepithelial mucosubstances in the main axial airways within the lung. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Animals were sacrificed 1, 2, or 7 days after the last instillation. Microdissected intrapulmonary axial airways (generations 8-11) from the right caudal lobes of infusion-fixed lungs were processed for light and electron microscopy. Morphometric techniques were used to determine the volume densities (Vs) of histochemically stained intraepithelial mucosubstances and numerical densities of airway epithelial cells. There were marked increases, compared with controls, in the amount of intraepithelial mucosubstances in the intrapulmonary axial airways at generations 8 and 11 in the right caudal lobes from endotoxin-instilled rats sacrificed 1, 2, and 7 days after the last instillation. There were significantly greater numbers of surface epithelial cells per length of basal lamina (i.e., hyperplasia) in endotoxin-exposed airways compared with airways from controls. This endotoxin-induced hyperplasia was due primarily to an increase in the number of mucus-secretory cells, which in endotoxin-exposed epithelium were columnar and contained numerous, large confluent, electronlucent, secretory granules composed of acidic and neutral glycoproteins. In contrast, secretory cells in airway epithelium from controls were cuboidal and contained small discrete, electron-dense, granules composed of only neutral glycoproteins. The numbers of ciliated cells and basal cells were similar in both control and endotoxin-exposed epithelium. Only endotoxin-exposed epithelium, however, contained atypical epithelial cells with numerous basal bodies, few cilia, and few apical secretory granules. These results indicate that repeated airway instillations of endotoxin induce an increase in the amount of intraepithelial mucosubstances, secretory cell hyperplasia, and excess luminal mucus in pulmonary airways. Therefore, endotoxin released from gram-negative bacteria may be partially responsible for the structural alterations, in the airway surface epithelium, which result in the excess luminal mucus observed in bacteria-induced bronchopneumonias.

In vivo effects of endotoxin on nasal epithelial mucosubstances: quantitative histochemistry.

Airway inflammation induced by gram-negative bacteria is often characterized by an influx of neutrophils and hypersecretion of mucus. The purpose of this study was to determine how endotoxin, a component of gram-negative bacteria and a chemotaxinogen for neutrophils, affects the amount of stored intraepithelial mucosubstances in the rat nasal airway. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillation, half of the animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum. Rats were sacrificed 6 or 24 h after the last instillation. Nasal tissues were processed for light microscopy and histochemical detection of stored intraepithelial mucosubstances. The numbers of nasal epithelial cells and intraepithelial neutrophils per millimeter of basal lamina in the anterior nasal septum and the amounts of intraepithelial mucosubstances in the same nasal tissue were determined by image analysis. We did not observe a neutrophil influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, we did observe a significant increase in intraepithelial mucosubstances, compared to saline-instilled controls. In contrast, nonneutrophil-depleted animals had a marked neutrophilic influx and a concomitant decrease in stored mucosubstances, compared to saline-instilled controls. There was no significant difference in the number of nasal epithelial cells per millimeter of basal lamina among any of the experimental groups. These results indicate (1) that endotoxin induces an increase in the amount of intraepithelial mucosubstances only when intraepithelial neutrophils are absent, and (2) that the endotoxin-induced, neutrophil influx probably triggers mucous hypersecretion.

Endotoxin or cytokines attenuate ozone-induced DNA synthesis in rat nasal transitional epithelium.

Pretreatment of rats with endotoxin (E), a potent inducer of tumor necrosis factor alpha (TNF), and interleukin 1 beta (IL 1), or a combination of TNF and IL1, has been shown to increase levels of lung antioxidant enzymes and protect against pulmonary toxicity associated with hyperoxia. Inhalation of ozone (O3) induces cell injury, followed by increased DNA synthesis, cell proliferation, and secretory cell metaplasia in rat nasal transitional epithelium (NTE). This study was designed to test the effects of E, TNF, and IL1 pretreatment on acute O3-induced NTE cell injury as measured by changes in NTE cell DNA synthesis. Rats were exposed to either 0.8 ppm O3 or air for 6 hr in whole-body inhalation chambers. Immediately before exposure, rats in each group were injected intraperitoneally (ip) with either saline alone or saline containing E (1 microgram/g body wt), TNF (10 micrograms), IL1 (10 micrograms), or both TNF and IL1 (TNF/IL1; 10 micrograms each). Eighteen hours postexposure, rats were injected ip with bromodeoxyuridine (BrdU; 50 micrograms/g body wt) to label cells undergoing DNA synthesis and were euthanized 2 hr later. NTE was processed for light microscopy and immunochemically stained to identify cells that had incorporated BrdU into nuclear DNA. The number of BrdU-labeled NTE nuclei per millimeter of basal lamina was quantitated. There were no significant differences in the number of BrdU-labeled NTE nuclei in air-exposed rats that were injected with E, TNF, IL1, or TNF/IL1 compared with those in saline-injected, air-exposed controls. Rats that were injected with saline and exposed to O3 had approximately 10 times the number of BrdU-labeled NTE nuclei than saline-injected, air-exposed control rats. O3 exposure also induced a significant increase in labeled nuclei in rats that were pretreated with TNF alone. In contrast, pretreatment with E, IL1, or TNF/IL1 attenuated the O3-induced increase in NTE DNA synthesis. These results indicate that both E and the cytokines TNF and IL1 have physiologic effects that can attenuate O3-induced injury or modify the response to NTE cells to O3 exposure.

Effects of 0.12 and 0.80 ppm ozone on rat nasal and nasopharyngeal epithelial mucosubstances: quantitative histochemistry.

The present study was designed to characterize the quantity of mucosubstances in surface epithelia of the rat nasal cavity and nasopharynx after short-term ozone exposure. Rats were exposure. Nasal cavities were processed for morphometric analysis of intraepithelial mucosubstances. Compared to controls, rats exposed to 0.12 ppm ozone had increased amounts of stored mucosubstances within epithelium lining the medial aspect of the nasal turbinate, but no change within the epithelium of the nasopharynx. Rats exposed to 0.8 ppm ozone had increased quantities of stored mucosubstances within the transitional and respiratory epithelia lining turbinates and lateral walls of the anterior nasal airway, and significant decreases in stored mucosubstances within the epithelium of the nasal septum at the end of exposure. Seven days after the end of exposure, the amounts of intraepithelial mucosubstances returned to control levels along the septum, but remained greater than those of controls along the turbinates and nasopharynx. We conclude that exposures to ambient levels of ozone induce significant changes in the stored secretory product of nasal epithelium in the rat, and that these changes persist for at least 7 days after cessation of exposure.

A rat monoclonal antibody specific for murine type 1 pneumocytes.

A rat monoclonal antibody (MAb), 411-52, that binds specifically to murine pulmonary alveolar type 1 cells was developed. The cell-binding specificity of MAb 411-52 was assessed by light microscopy on immunoperoxidase-labeled tissue sections, electron microscopy on immunogold-labeled tissue blocks, and by flow cytometric analysis and fluorescence-activated cell sorting of immunofluorescently labeled cells enzymatically dissociated from murine lungs. The epitope recognized by MAb 411-52 was first detected in immunoperoxidase-stained sections of neonatal lungs of mice approximately 3 weeks after birth. In adult mice, the MAb 411-52-directed, immunoperoxidase-staining pattern was uniform throughout the lung parenchyma, was restricted to the luminal surfaces of alveoli, and was absent from type 2, endothelial, and interstitial cells, as well as from the epithelial cells of conducting airways. Electron microscopic analysis of immunogold-labeled lung tissue confirmed the type 1 cell binding specificity of MAb 411-52. Analysis by multiparameter, laser flow cytometry indicated that MAb 411-52 binds to 4.6 +/- 0.5% (mean +/- SD) of enzymatically dissociated cells from the lungs of normal adult mice. The absence of immunogold-labeling of type 2 cells suggested that the epitope recognized by MAb 411-52 might be a differentiation marker for the type 1 cell phenotype. With this MAb and standard immunohistochemical techniques, it is possible to visualize directly type 1 cells in paraffin sections.