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Correspondence to: Lisa A. Miller, Dept. of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, U. of California, Davis, Davis, CA 95616. E-mail: lmiller@ucdavis.edu
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Summary |
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Inhalation of ozone by Rhesus monkeys results in epithelial injury and granulocyte influx in both conducting airways and respiratory bronchioles. We have reported that ozone-induced neutrophil recruitment and subsequent epithelial repair can be inhibited in vivo with a CD18 antibody. The antibody-mediated effect is abrogated by local instillation of C5a (a CD18-independent neutrophil chemoattractant), thereby demonstrating a role for neutrophils in lung epithelial repair processes. As an extension of this study, we examined the effect of ozone and neutrophil influx on epithelial expression of the ß6 integrin, an adhesion molecule associated with proliferation and repair. Expression of ß6 integrin was determined by immunohistochemistry for ozone-exposed monkeys treated with either control immunoglobulins or a CD18 antibody. The tracheal epithelium of ozone-exposed monkeys treated with control immunglobulins expressed the ß6 integrin. In contrast, the tracheal epithelium of ozone-exposed monkeys treated with CD18 antibody exhibited very low to undetectable expression of ß6 integrin. In association with C5a instillation and neutrophil influx, ß6 integrin was also observed in respiratory bronchiolar epithelium from both control and ozone-exposed animals. These findings cumulatively suggest that lung epithelial cell expression of ß6 integrin is associated with sites of neutrophil recruitment. (J Histochem Cytochem 49:4147, 2001)
Key Words: neutrophil, lung, epithelium, integrin
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Introduction |
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ELEVATED NUMBERS of neutrophils are typically observed within the airway lumen at the onset of an acute inflammatory response, implying an important role for this leukocyte as a host defense mechanism of the lung. Many pulmonary disease states can induce such a response, in addition to environmental exposure to pollutants. Short-term (4-hr) exposure of humans to ambient levels of ozone, a major constituent of air pollution, leads to enhanced accumulation of neutrophils in airways (
To further study the role of neutrophils in repair of ozone-injured epithelial cells, Rhesus monkey airway epithelium was evaluated for expression of ß6 integrin. The ß6 integrin subunit forms a heterodimer with the v integrin subunit. This adhesion molecule appears to be unique to epithelia and can mediate attachment to matrix proteins (fibronectin, tenascin, vitronectin), as well as TGFß1 (
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Materials and Methods |
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Antibodies
Mouse anti-human CD18 monoclonal antibody R15.7 (IgG1) was a generous gift from Dr. Robert Rothelin (Boehringer Ingelheim Pharmaceuticals; Ingelheim, Germany). IgG1 isotype control immunoglobulins were purified from the mouse plasmacytoma cell line MOPC-21 (ATCC; Rockville, MD). Mouse anti-human ß6 integrin monoclonal antibody Csbeta6 (IgG1) was generated by immunizing ß6 subunit knockout mice with secreted vß6 heterodimer as described (
Animals/Ozone Exposure
Young male Rhesus monkeys weighing 5.17.6 kg and aged from 3 years and 8 months to 3 years and 10 months were randomly assigned to either ozone or control (filtered air) experimental groups as follows: (a) filtered air + CD18 antibody (n = 2); (b) filtered air + isotype control (n = 2); (c) ozone + CD18 antibody (n = 6); and (d) ozone + isotype control (n = 6). All monkeys were given a comprehensive physical examination, including a chest radiograph and complete blood count, before exposures. Before initiation of experiments, monkeys were transferred to exposure chambers for a 1-week acclimatization in filtered air.
Eight hours before ozone/filtered air exposures, monkeys were given either anti-CD18 or isotype control immunoglobulins via the cephalic vein at 1 mg/kg body weight. It has previously been determined by flow cytometry that this concentration of CD18 antibody is saturating for circulating and lavage neutrophils for the duration of a 12-hr ozone exposure/postexposure period (
Immunohistochemistry
For immunoperoxidase staining, 5-µm cryosections of the accessory lobe and trachea were fixed in ice-cold acetone for 10 min and allowed to dry. To reduce endogenous peroxidase, cryosections were treated with 3% H2O2 for 5 min at room temperature (RT). Neutrophils in cryosections could not be completely blocked for endogenous peroxidase without destroying the integrity of the tissue. To block nonspecific binding of antibodies, cryosections were incubated overnight in goat serum at 4C before immunostaining. All monkeys in this study were treated with purified mouse IgG1 (either MOPC-21 or R15.7) as a part of the experimental/exposure design. Therefore, all tissue sections contained saturating quantities of mouse immunoglobulins as a control for immunohistochemical staining. For detection of ß6 integrin, cryosections were incubated at RT with mouse anti-human ß6 integrin monoclonal antibody (CSbeta6) for 1 hr. After a PBS wash, cryosections were incubated with biotinylated goat anti-mouse IgG (Vector Laboratories; Burlingame, CA) for 1 hr at 1:100 dilution. Sections were then incubated with a horseradish peroxidaseavidinbiotin complex (Vector Laboratories) for 30 min and developed using 0.5% 3,3'-diaminobenzidine (DAB)/0.01% H2O2 as a chromogen. For detection of ß6 integrin in monkeys previously treated with anti-CD18 antibody, CSbeta6 was directly biotinylated using a Sulfo-NHS-LC Biotinylation kit (Pierce; Rockford, IL). After a 1-hr incubation at RT with biotinylated Csbeta6, sections were incubated with the horseradish peroxidaseavidinbiotin complex for 30 min and developed using an enhancer kit for DAB (DAB-Black; Zymed Laboratories, South San Francisco, CA) as a chromogen.
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Results |
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We used an immunohistochemical approach to evaluate protein expression of the ß6 integrin subunit by conducting airway epithelium from Rhesus monkeys treated with isotype control immunoglobulins, comparing the effects of 8-hr exposure to 0.8 ppm ozone (n = 6) vs filtered air (n = 2). As expected, tracheal and bronchial epithelium of filtered air control monkeys had very low to undetectable ß6 integrin expression (Fig 1A). At the level of the trachea (Fig 1C) and bronchi (not shown), epithelial cell expression for ß6 integrin was observed in all ozone-exposed monkeys treated with isotype control immunoglobulins. In the trachea, immunohistochemical staining for ß6 integrin was detected in both apical and basolateral regions in individual epithelial cells.
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We have previously shown that ozone-induced neutrophil influx into Rhesus monkey airways is CD18-dependent, but instillation of C5a into distal airways results in CD18-independent neutrophil recruitment, most likely from the pulmonary circulation (
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On the basis of our findings of ß6 integrin expression in respiratory bronchioles of both filtered air- and ozone-exposed monkeys, we next evaluated if the expression of ß6 integrin was the direct result of neutrophil emigration into airways (as opposed to ozone exposure). Although C5a instillation in distal airways resulted in CD18-independent neutrophil emigration in respiratory bronchiolar epithelium, tracheal epithelium was not affected by this treatment. Therefore, we compared the expression of ß6 integrin on tracheal epithelium from ozone-exposed monkeys treated with isotype control immunoglobulins vs ozone-exposed monkeys treated with anti-CD18 immunoglobulins (n = 6). CD18 has been shown to play a significant role in the transepithelial migration of neutrophils across large airway epithelium in vitro (
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Discussion |
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Depending on the inflammatory stimuli, neutrophil recruitment in the lung can occur via CD18-dependent or CD18-independent mechanisms (
The vß6 integrin complex can mediate adhesion and spreading of epithelial cells on fibronectin in vitro (
vß6 may play a role in the migration of airway epithelial cells during reparative processes. In an animal model of epidermal injury,
vß6 is not essential for resolution of epidermal injury (
The regulation of vß6 integrin expression appears to be linked to epithelial cell proliferation. Although immunostaining of normal human proximal airways does not indicate expression of the ß6 subunit, proliferating cultures of primary tracheobronchial epithelial cells isolated from normal human subjects do express high levels of this integrin (
, and transforming growth factor ß can increase surface expression of
vß6 on primary cultures of human airway epithelial cells (
Mice that are homozygous for a null mutation of the ß6 subunit gene phenotypically exhibit pronounced lymphocyte accumulation in conducting airways, suggesting that the vß6 integrin complex may play a role in downregulating inflammation in the lung (
vß6 can bind and activate the latent form of TGFß1 (
) (
vß6 during inflammatory events in the airways. Given that localized expression of ß6 integrin in the Rhesus monkey appears to be associated with conditions of acute inflammation, these findings cumulatively suggest that expression of ß6 integrin by lung epithelium correlates with sites of leukocyte recruitment and may be related to the resolution of inflammatory events in the airways.
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Acknowledgments |
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Supported in part by Tobacco Related Disease Research Program Grants 6KT-0411 and 8IT-0054 (to LAM) and NIEHS ES-00628 (to DMH).
Received for publication August 31, 2000; accepted September 6, 2000.
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