IL-8 is one of the major chemokines produced by monkey airway epithelium after ozone-induced injury

Mary Mann-Jong Chang1,2, Reen Wu1,2,3, Charles G. Plopper1,2, and Dallas M. Hyde1,2

1 Center for Comparative Respiratory Biology and Medicine, 2 Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, and 3 Pulmonary and Critical Care Medicine, School of Medicine, University of California, Davis, California 95616

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A rhesus monkey interleukin (IL)-8 cDNA clone with >94% homology to the human IL-8 gene was isolated by differential hybridization from a cDNA library of distal airways after ozone inhalation. In situ hybridization and immunohistochemistry showed increased IL-8 mRNA and protein levels in epithelial cells at 1 h but not at 24 h after inhalation of ozone. The appearance of IL-8 in airway epithelial cells correlated well with neutrophil influx into airway epithelia and lumens. Air-liquid interface cultures of tracheobronchial epithelial cells were exposed to ozone in vitro. We observed a transient increase in IL-8 secretion in culture medium immediately after ozone exposure and a dose-dependent increase in IL-8 secretion and mRNA production. In vitro neutrophil chemotaxis showed a parallel dose and time profile to epithelial cell secretion of IL-8. Treatment with anti-IL-8 neutralizing antibody blocked >80% of the neutrophil chemotaxis in vitro. These results suggest that IL-8 is a key chemokine in acute ozone-induced airway inflammation in primates.

chemotaxis; neutrophil; differential hybridization; in situ hybridization; immunohistochemistry; interleukin-8

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

OZONE IS A KEY air pollutant contaminating the atmosphere of most industrialized cities and is known to cause short-term pulmonary function impairment and chronic lung disease in humans (18). The respiratory system is the primary target of ozone toxicity (30). Acute exposure of humans or animals to ambient levels of ozone results in reversible changes in pulmonary function and pulmonary inflammation characterized by increased neutrophil influx (16). Although data have shown that ozone inhalation induces the production of different chemoattractants such as leukotreine B4, macrophage inflammatory protein-2, and interleukin (IL)-8 in various cases (13, 21), there is no direct in vivo evidence linking neutrophil influx and those chemoattractants. Therefore, the mechanism of neutrophil influx resulting from ozone inhalation is still not entirely clear.

To investigate the relationship between granulocyte migration and epithelial injury, we exposed rhesus monkeys to 0.96 part/million (ppm) ozone for 8 h and observed that epithelial necrosis and repair were associated with the presence of granulocytes in the epithelium and interstitium of the tracheobronchial airways during the week-long postexposure period (16). To understand the early cellular and molecular events that are important in the recruitment of neutrophils into the tracheobronchial airways, we hypothesized that one of the key target zones for ozone-induced injury, the respiratory bronchioles, upregulates gene expression and production of chemokines. To investigate the nature of the chemokine response to ozone, we used the microdissecting technique (23) to isolate different airway regions to establish a cDNA library for screening differentially expressed genes. We used the same ozone-exposed monkey lungs in which we have documented injured epithelial cells and increased neutrophil influx into respiratory bronchioles (16). This approach has allowed the isolation and characterization of several monkey ozone-responsive (MOR) genes (19). One of these differentially hybridized MOR cDNA clones, MOR6, had a DNA sequence with 94% homology to the human IL-8 gene. We used this clone in the present paper along with a human IL-8 antibody to investigate the presence of IL-8 mRNA and protein in respiratory bronchioles of monkeys after exposure to ozone. To evaluate the relative importance of IL-8 as a chemokine for neutrophil migration in human and monkey primary tracheobronchial epithelium after ozone exposure, we exposed primary human and monkey and human transformed (BEAS-2B) tracheobronchial epithelial cells to ozone and established that IL-8 is a key chemokine in neutrophil migration in response to ozone-induced epithelial cell injury.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In vivo ozone exposure of monkeys. Rhesus monkeys were exposed individually in exposure chambers of 4.2-m capacity that were ventilated at a rate of 30 changes per hour with chemical-biological and radiological-filtered air (FA) at 24 ± 2°C and 40-50% relative humidity. Ozone concentrations were measured using an ultraviolet ozone monitor and were reported with respect to the ultraviolet photometric standard. For additional details of the exposure and monitoring, please refer to the previous paper (16).

Isolation and characterization of monkey IL-8 cDNA clone. The airway microdissection technique described by Plopper et al. (23) was adapted in this study to isolate the distal airway region of monkey lungs after ozone or FA exposure. Poly(A)+ mRNAs were isolated from these tissues and used to establish cDNA libraries using the Lambda GEM-2 system (Promega, Madison, WI). The sizes of the cDNA libraries were 5 and 2 × 105 phages/library for FA and ozone-exposed tissues, respectively. With the use of differential hybridization (19), MOR genes were isolated. Among these MOR clones, MOR6 was sequenced using an automatic DNA sequencer (ABI sequencer), and we found a 94% homology to the human IL-8 gene in the 491-bp inserts of MOR6 (Fig. 1).


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Fig. 1.   Monkey ozone-responsive (MOR) 6 clone sequence. Gene sequence of monkey MOR6 clone (bottom) compared with human interleukin (IL)-8 gene (top). Common sequences appear in boxes. Note the high degree of homology between the monkey and human genes (GenBank search).

In situ hybridization. By subcloning the MOR6 into a pGEM4Z plasmid (Promega), sense and anti-sense IL-8 cRNAs were generated from MOR6 and labeled with 35S using the Maxiscript in vitro transcript kit (Ambion, Austin, TX). Paraffin sections from lung tissue of ozone- and FA-treated monkeys were used for hybridization, with conditions and washing following the procedure described by Angerer et al. (2). Autoradiography was carried out over a 7-day interval, and the developed slides were counterstained with methylene green.

Immunohistochemical staining with anti-human IL-8 antibody. Tissue sections were treated with methanol-acetone and then stained immunologically with a mouse anti-human IL-8 antibody (R&D Systems, Minneapolis, MI) using the Vectastain ABC Kit (Vector Laboratory, Burlingame, CA), an avidin-biotin peroxidase method to detect the reaction product. Controls used no primary antibody.

Cell culture. Tracheobronchial epithelial (TBE) cells were isolated from monkey or human as primary culture; cell isolation and medium conditions were carried out as previously described (31). Human tissues were obtained from organ donor patients or from autopsy through the University of California, Davis, Medical Center. Rhesus monkey tracheobronchial airways were obtained from the California Regional Primate Research Center, University of California, Davis. Two human bronchial epithelial cell lines were used in this study. BEAS-2B S-6 (S; serum-sensitive) clonal cell line, an immortalized cell line of normal human bronchial epithelium derived by transfection of primary cells with SV40 early-region genes, was obtained by courtesy of Dr. J. Lechner (Lovelace Inhalation Toxicology Research Institute, Albuquerque, NM). The HBE1 cell line, a papillomavirus immortalized human bronchial epithelial cell line, was generously provided by Dr. J. Yankaskas (University of North Carolina, Chapel Hill, NC). Cells were plated onto the membrane inserts (pore size 0.4 µm) of Costar six-well clustered Transwell chambers (Corning Costar, Cambridge, MA) at 2 × 105 cells/well and maintained in serum-free media with 1 ml on top of the cells (inside the insert, apical phase) and 2 ml medium beneath the cells (in the dish, basolateral phase). In the case of primary cultures, collagen was coated on top of the membrane inserts before the cells were plated, whereas in the case of BEAS-2B S cells and HBE1 cells, no collagen was added to the membrane inserts. After 7-10 days, when cells had reached confluency and maintained confluency for a couple of days, they were used for FA or ozone exposure. Immediately before exposure, the serum-free media were removed from both sides of the membrane inserts and washed two to three times with Hanks' balanced salt solution (HBSS), and then 1 ml of HBSS was added to the basolateral compartment so the cell could have direct contact with FA or ozone.

In vitro ozone exposure. The in vitro system for exposing airway epithelial cells to ozone has been previously described (28). To allow gases to interact with the cells, no medium was added to the apical compartment during exposure, and only 1 ml of HBSS was added to the basolateral phase.

Conditioned media. After ozone exposure, HBSS was removed, samples were further washed two times with HBSS, 1 ml of serum-free media was added to both the apical and basolateral compartments, cells were incubated for 2 or 4 h in a 37°C incubator, and then the media were collected as conditioned media. If the assays were not performed immediately, the supernatant was saved at -70°C until the assay was run, and the supernatant was never thawed more than one time. For the time-course study, at 0 or 16 h after ozone or FA exposure, cells were washed two times with HBSS and replaced with fresh media; conditioned media were collected after 4 h of incubation (4 and 20 h after ozone exposure).

Cell counting. Cells were trypsinized and stained with trypan blue, and then the viable, nonstained cells were counted using a microscope and a hemocytometer.

Quantitation of IL-8 by ELISA. The level of IL-8 in conditioned media was quantified by an ELISA kit obtained from Biosource International (Camarillo, CA).

In vitro neutrophil chemotaxis assay. Neutrophils were prepared from monkey or human blood according to the method of Haslett et al. (14) and then were labeled with 51Cr. We used a 24-well Transwell dish with inserts of 3-µm pore size membrane to evaluate chemotaxis according to the method of Casale and Abbas (5). To the outside of the filter insert, 450 µl of conditioned media (cell culture supernatant) were added to fill the well, whereas to the inside of the filter insert, 100 µl (1 × 106 cells) of freshly isolated and 51Cr-labeled neutrophils were added. The incubation was at 37°C for 3 h. The number of neutrophils that migrated to the bottom of the dish was measured by the 51Cr counts of the cell lysate. The chemotactic activity (CA) was calculated as follows
CA (%) = <FR><NU>sample counts − background</NU><DE>total counts − background</DE></FR> × 100
where background is defined as neutrophil migration in the well with fresh medium and total counts indicates the total count of the 200-µl neutrophils loaded onto the insert.

Northern blot hybridization. Total RNA was isolated by the single-step acid guanidium thiocyanate-phenol-chloroform extraction method described previously (8). Northern blot hybridization was done as previously described (15). 32P-labeled MOR6 and PG11-480 were used as probes, where MOR6 is the clone described above and PG11-480 is a clone using PGEM4Z (Promega) as the vector, and ligated with an insert of 469 bp of the human IL-8 sequence produced by the PCR method according to the published human IL-8 gene sequence.

Anti-IL-8 neutralizing antibody treatment. The conditioned media (collected 4 h after ozone exposure) were preincubated with increasing concentrations of 1) a mouse monoclonal anti-human IL-8 IgG (R&D Systems) or 2) a mouse anti-human mucin IgG (control antibody developed in our laboratory) for 1 h at 37°C. After this preincubation period, the pretreated (PCM) and untreated (UCM) conditioned media were evaluated in a chemotaxis assay. The inhibition of migration (IM) due to the pretreatment of antibody was calculated as follows
IM (%) = <FR><NU>chemotaxis of UCM − chemotaxis of PCM</NU><DE>chemotaxis of UCM</DE></FR> × 100
Statistics. All quantitative data were evaluated by group by ANOVA, and individual group comparisons were evaluated using Fisher's least significant difference test (SYSTAT 7.0, Chicago, IL). Differences were considered to be significant at P <=  0.05. Some data were evaluated using regression analysis.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Identification of a monkey IL-8 cDNA clone as one of the MOR genes. Only the cDNA library derived from ozone-exposed monkeys was used in this study to isolate the ozone-inducible cDNA clones. From a screening of ~5 × 105 phages, 16 MOR cDNA clones were isolated after several rounds of screening. Among these clones, MOR4 and MOR6 were identified as ozone-induced genes. DNA sequence information identified MOR4 as a monkey surfactant protein C cDNA clone, whereas the MOR6 clone had a DNA sequence with 94% homology to the human IL-8 gene (Fig. 1). In this study, we focused on the expression of the IL-8 gene after ozone exposure.

In situ hybridization and immunohistochemical identification of monkey IL-8. To further support the differential hybridization result, 35S-cRNA probes in both sense and antisense directions were generated from the MOR6 cDNA clone and used for in situ hybridization with paraffin lung sections derived from monkeys after the FA or ozone exposure. Similarly, a monoclonal antibody specific to the human IL-8 protein (R&D Systems), which has been shown to be monospecific to the monkey chemokine in a Western blot (unpublished data), was used for immunohistochemical evaluation on these monkey paraffin sections. Dark-field photomicrographs of in situ hybridization evaluation showed an elevated IL-8 mRNA steady-state level in monkey airway epithelium after acute exposure to ozone (Fig. 2B). In contrast, hybridization with sections derived from FA-exposed monkeys showed background staining (Fig. 2A) similar to the control hybridization with sense probe (Fig. 2C). Similar to in situ hybridization, immunohistochemical staining also showed enhanced IL-8 protein levels in monkey airway epithelium of sections after ozone exposure (Fig. 3). This increase in IL-8 protein level was found immediately after exposure (Fig. 3B) but declined 24 h after exposure to ozone (1 ppm for 8 h) (Fig. 3C). Interestingly, ozone-induced IL-8 expression appears to be somewhat cell-type and region specific. Immunostaining showed that ciliated cells are a primary target of ozone-induced IL-8 gene expression. Furthermore, IL-8 staining was patchy in the proximal airways, whereas it was more diffuse in the distal airways (Fig. 4). Most of the alveolar macrophages in distal airways stained positive for IL-8 in monkeys immediately after ozone exposure, but they were few in number compared with the positively stained epithelial cells. Few alveolar macrophages in more distal alveolar regions stained positive for IL-8. This staining pattern is consistent with elevated neutrophil influx in respiratory bronchioles compared with more proximal bronchi or more distal alveoli in these same monkeys immediately after ozone inhalation (16).


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Fig. 2.   Detection of IL-8 mRNA in monkey lung by in situ hybridization. Frozen lung sections from filtered air (FA)- or ozone (OZ)-exposed monkey were hybridized with 35S-labeled antisense cRNA of MOR6 clone; hybridization signals seen as white grains in the dark-field micrograph are concentrated on airway epithelium (B). No significant amount of signal was seen when sense cRNA was used as probe (C) or when antisense cRNA probe was used to hybridize the FA-exposed monkey frozen tissue (A). Dark-field photomicrograph. Scale bars, 20 µm.


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Fig. 3.   Comparison of IL-8 protein production in monkey respiratory bronchioles at different times after OZ or FA exposure as shown by immunohistochemical staining. Almost no detectable staining was shown on the tissue processed 1 h after FA exposure (A). Significant amount of IL-8 protein is shown on the tissue processed 1 h after OZ exposure (B), whereas, at 24 h after OZ exposure, the IL-8 production is hardly detectable (C). Bars, 20 µm.


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Fig. 4.   Comparison of proximal versus distal airway staining at 1 h after OZ exposure. A: typical negative staining for IL-8 in a proximal airway. B: positive focal staining for IL-8 in a proximal airway. C: typical positive staining for IL-8 in a respiratory bronchiole. D: negative control staining for IL-8. Bars, 20 µm.

In vitro studies of ozone-induced IL-8 gene expression in airway epithelial cells. To carry out in vitro ozone exposure, both established cell lines and primary cells were used. In most cases, monkey primary TBE cells were used; occasionally, human TBE cells were also used. For cell lines, we used BEAS-2B S cells and HBE1 cells. After cells reached confluency, these chambers were exposed to different ozone concentrations from 0.1 to 1.0 ppm for 60 min or to a constant ozone level (0.5 ppm) for various times. The control chambers were exposed to FA for the same period of time. The time- course study showed that ozone enhanced IL-8 secretion immediately after the exposure and that the enhancement was transient (Fig. 5). Also, ozone at 0.5 ppm caused substantial dose-dependent cell damage on the cultured BEAS-2B S (Fig. 6) and HBE1 (data not shown) cell lines. Concurrent to cell injury, there was a substantial increase in IL-8 in the media of the surviving cells over the 2 h after ozone exposure. By contrast, ozone exposure did not cause substantial damage to the primary culture of monkey TBE cells, even though the elevated IL-8 secretion persisted (Fig. 7). This result was also observed in primary cultures of human TBE cells (data not shown). To further elaborate the level of the change of IL-8 production by ozone, Northern blot hybridization was carried out to determine whether the change occurs at the mRNA level. As shown in Fig. 8, IL-8 message in both monkey and human primary TBE cells was increased by ozone exposure. These results show that ozone altered gene expression in the accumulation of IL-8 mRNA in culture.


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Fig. 5.   Comparison of IL-8 protein produced by primary monkey tracheobronchial epithelial cells at different times after FA or OZ exposure as measured by ELISA. Conditioned media with 4-h incubation period were collected at 4 or 20 h after cells were exposed to FA or various concentrations of OZ. Conditioned media at 4 h (containing the IL-8 production from 0 to 4 h postexposure) showed increased secretion of IL-8 in response to increased dosage of OZ. Conditioned media at 20 h (contained the IL-8 secretion between 16 and 20 h postexposure) showed no difference between FA or OZ exposure. To avoid collecting any IL-8 beyond the specific 4-h period, cells were washed 2 times with Hanks' balanced salt solution (HBSS) and replaced with fresh media for 4-h incubation and then the conditioned media were collected. ppm, Part/million. Values are means ± SE of n = 3 experiments per group. * Significantly greater than (P <=  0.05) 20-h value.


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Fig. 6.   Response of BEAS-2B cells to OZ exposure. BEAS-2B cells were exposed to FA or 0.5 ppm OZ for 30, 60, or 90 min. After the exposure, one-half of the cells were used for cell counting; the number of viable cells decreased in response to increased OZ dosage. The other half of the cells were washed 2 times with HBSS and replaced with fresh media. After 2 h of incubation, conditioned media were collected for ELISA. Data showed increased IL-8 production on a per-cell basis. Nos. on y-axis, ng IL-8 and cell number (millions). * Significantly decreased (P <=  0.05) compared with time 0 (FA). + Significantly increased (P <=  0.05) compared with time 0 (FA).


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Fig. 7.   Amount of IL-8 protein produced in conditioned media by primary monkey tracheobronchial epithelial cells as measured by ELISA. Conditioned media collected 4 h after exposure to FA or varying concentrations of OZ (0.2, 0.5, and 1 ppm) were quantified by ELISA. Values are means ± SE of n = 3 experiments per group. There was a significant (P = 0.0001) correlation between IL-8 protein and dose of OZ. * Significantly greater than (P <=  0.05) all other concentrations.


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Fig. 8.   Analysis of IL-8 mRNA expression in FA- or OZ (1 ppm)-exposed monkey and human primary tracheobronchial epithelial cells. Autoradiograph of the Northern blot is as shown. After scanning by a densitometer, IL-8 mRNA value is standardized by 18S, and the value is indicated below the autoradiograph.

Effects of ozone on epithelial cell-induced neutrophil chemotactic activity. Because epithelial cells are probably responsible for the first phase of inflammatory cell migration in vivo, we wondered whether in vitro ozone exposure also induces the secretion of neutrophil chemotactic activity. As shown in Fig. 9, ozone exposure enhanced neutrophil migration, and the activity appears to be dose dependent. To investigate the potential contribution of IL-8 in neutrophil chemotaxis in ozone-exposed culture, anti-IL-8 neutralizing antibody was used. As shown in Fig. 10, most neutrophil chemotactic activity in the conditioned medium from FA- and ozone-exposed cultures was inhibited by the IL-8 neutralizing antibody. This result supports the notion that IL-8 secretion by airway epithelium is a key chemokine in the initial phase of neutrophil chemotaxis in ozone-induced epithelial injury.


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Fig. 9.   Chemotactic activity. Migration of monkey neutrophils through 3-µm membranes to conditioned media of FA- or OZ-exposed monkey primary tracheobronchial epithelial cells was measured by chemotaxis assay. * Significantly greater than 0.2 ppm OZ (P = 0.032) and markedly increased compared with FA (P = 0.059).


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Fig. 10.   Neutralization of chemotactic activity by IL-8 neutralizing antibody. Monkey neutrophil chemotaxis to conditioned media from monkey primary tracheobronchial epithelial cells exposed to 0.5 ppm OZ for 1 h was inhibited in a dose-dependent fashion (%inhibition) by varying concentrations of IL-8 neutralizing antibody. There was a significant (P = 0.0001) correlation between the concentration of IL-8 neutralizing antibody and %inhibition of chemotaxis. Mouse anti-human mucin IgG antibody was used as the control. * Significantly greater inhibition than (P <=  0.05) all other concentrations.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

From the same monkeys in which neutrophil influx was associated with epithelial necrosis (16), we isolated an IL-8 cDNA clone from the cDNA library distal airway through differential hybridization. This clone was used for in situ hybridization, and an antibody to human IL-8 was used for immunohistochemical staining of monkey airways. Increased production of IL-8 in ozone-exposed monkey airways correlated well with our previous data showing an influx of neutrophils (16). When we used monkey/human primary cells or BEAS-2B S/HBE1 cells for in vitro evaluation, IL-8 production was also induced by ozone in a time- and dose-responsive manner. The transient nature of IL-8 induction was shown by a decrease in IL-8 production at 16 h after ozone exposure. Furthermore, the neutrophil chemotaxis assay of the cell culture supernatant indicated a direct relationship between the amount of IL-8 and neutrophil migration. When an IL-8 neutralization antibody was used, >85% of the chemotactic activity was blocked. From these studies, we concluded that ozone exposure of airway epithelial cells can induce the production of IL-8 in vivo and in vitro and that IL-8 is a key chemokine in neutrophil influx after ozone-induced epithelial injury.

Numerous in vivo experiments of ozone inhalation by humans have shown increased IL-8 secretion into bronchoalveolar lavage fluid. Aris et al. (3) monitored lavage fluid of healthy volunteers after ozone inhalation and showed a correlation between airway injury and increased IL-8 production. Additionally, McBride et al. (20) exposed subjects with asthma to an ambient concentration of ozone and found a strong correlation between IL-8 and white blood cell counts in nasal fluid. Together, their results suggest that IL-8 plays an important role in airway inflammation, although the specific lung site of IL-8 production cannot be deduced by bronchoalveolar lavage fluid analysis alone. Our investigations provide direct evidence of IL-8 production by airway epithelial cells in response to ozone inhalation in monkeys. First, the IL-8 monkey gene was isolated through differential hybridization from microdissected distal airways. This implies that ozone-exposed tissue is differentially regulated at the genetic level in reference to IL-8 gene expression. This concept is supported by the work of Jaspers et al. (17), which shows ozone exposure-induced DNA-binding activity of transcription factors at the prospective IL-8 gene regulation sites, nuclear factor-kappa B and nuclear factor IL-6 binding sites. Also, the use of distal airways facilitated the demonstration of this differentially expressed IL-8 gene, since previous pathological investigations into the effect of ozone exposure in monkeys revealed that the bronchiolar region showed the most consistent epithelial injury and neutrophil influx (16). Second, in vivo in situ hybridization and immunohistochemistry support this conclusion of direct IL-8 production by airway epithelial cells. These data show that induction of IL-8 gene expression after ozone exposure is greatest in airway epithelium, especially the distal airways. Epithelial cells are the first line of defense and the most predominant cells in the airway system, and in vitro studies have demonstrated that epithelial cells can produce a variety of cytokines and adhesion molecules (10). Our data suggest that epithelial cells not only serve as a line of defense but also play an important role in airway inflammation. Our data show that epithelial cells are a major source of IL-8 production in vivo, and its expression in epithelial cells can be modulated by ozone exposure.

In concert with our in vivo investigations, we also showed elevated IL-8 secretion and mRNA levels in cultures of immortalized human bronchial epithelial cell lines and primary monkey/human TBE cells by ozone using an in vitro exposure system. Similar in vitro observations have been demonstrated in other laboratories (9, 11). Devalia et al. (9) used an explant cultured system for human bronchial epithelial cells to show the enhancement of IL-8 of four to five times over background over a period of 24 h. However, the ozone exposure system was different, primarily because of the nature of explant culture; also, their exposure was at 100 parts per billion for 6 h. Our system is more comparable with that of Devlin et al. (11). However, our data yield several new observations as follows: 1) IL-8 production is dose dependent with ozone concentration; 2) although both cell lines and primary cell culture enhanced IL-8 production after ozone exposure, the toxicity of ozone on cell lines and primary cells is quite different; and 3) the enhancement of IL-8 is a transient phenomenon, with IL-8 production being increased immediately after ozone exposure but decreasing at 20 h after ozone exposure. Established cell lines, which were cultured on membranes without collagen, showed a significant amount of cell death. In contrast, primary cell culture of monkey or human cells cultured on collagen-coated membrane showed little cell death after ozone exposure. Immunohistochemical staining with anti-IL-8 antibody of ozone-exposed monkey distal airway sections also suggested a transient production of IL-8 in vivo. The nature of the transient production of IL-8 is unclear.

Hyde et al. (16) previously demonstrated in monkey lung that neutrophils were recruited to airway lumen, with a peak at 12 h after an 8-h ozone exposure and then a gradual decline to the baseline within 72 h postexposure. These times correlated well with the transient enhancement of IL-8 in vivo and in vitro. This observation suggests a possible role for airway epithelial cell-secreted IL-8 in the initial phase of neutrophilic recruitment in vivo. This notion is further supported by the in vitro chemotaxis assay of cultured medium from ozone-exposed cultures. We demonstrated that the neutrophilic chemotactic activity in culture medium of airway epithelial cells was elevated by ozone and that it could be blocked by a monoclonal anti-IL-8 neutralizing antibody. This inhibitory effect by anti-IL-8 neutralizing antibody strongly suggests that IL-8 is a key cytokine contributing to ozone-induced airway inflammation. Abdelaziz et al. (1) used conditioned media of primary human bronchial epithelial cells and showed that chemotactic activity for neutrophils could be blocked by IL-8 neutralizing antibody up to 50%. However, in our hands, the blocking activity can be as high as 85%. Further investigations with neutralizing anti-IL-8 antibody treatment in vivo with animals exposed to ozone are needed to assess how critical the chemokine IL-8 is in the initial phase of ozone-induced airway inflammation.

One interesting observation emerging from the immunohistochemical study is the demonstration that the ciliated cell type is the major contributor in vivo in ozone-induced IL-8 gene expression. We observed very little IL-8 gene expression in airway epithelium of control monkeys exposed to FA. However, immediately after acute ozone exposure, we saw increased staining for IL-8 in bronchiolar epithelium, especially in ciliated cells. The nature of this increase is not entirely clear. It has been demonstrated that ciliated cell type is a primary target of ozone toxicity (12, 22). Although there is no good explanation of why ciliated cell type is the major target of ozone toxicity, previous work has shown that the ozone effect is attributed to its ability to cause oxidation (6) or peroxidation (7) either directly or with the formation of free radicals (24). A sequence of the events may include lipid peroxidation, loss of functional groups of enzymes, and alteration of membrane permeability (4, 32). Because the membranes of ciliated cells project into the airway lumen via their cilia and have a much greater surface area compared with other airway epithelial cell types, they are more vulnerable to oxidant-induced injury. Therefore, it is reasonable to assume an association between elevated IL-8 gene expression and ciliated cell injury in vivo. However, we found it difficult to use "cell injury" to explain the current data. In immortalized human bronchial epithelial cell line, ozone exposure caused substantial damage in cells, although it also caused an increase in IL-8 secretion. In contrast, ozone caused little damage in primary human TBE cells, yet the enhanced IL-8 gene expression persisted. Thus cell injury by ozone is not critical for IL-8 gene expression. Our in vitro data show a strong correlation between cell viability and IL-8 production, whereas immunohistochemistry also showed staining of squamating epithelial cells (survivors) in monkey respiratory bronchioles. Nevertheless, the process of IL-8 gene activation may be part of the injury process. Recently, several reports have demonstrated that cell deformation (26) and cell detachment (29) in vitro can elevate IL-8 gene expression. Others (25, 27) have demonstrated that changes in the cellular redox mechanism can regulate IL-8 gene expression through the activation of transcriptional factors essential for IL-8 gene expression. These observations are consistent with the current view in which ozone may disrupt cell processes, and, as a consequence, IL-8 gene expression is activated.

Finally, we would like to comment on our general approach in this study. Using the molecular cloning technique, we have identified several MOR cDNA clones in which expression in monkey lung is both cell-type and regional specific and which is also altered by ozone. The success in identifying IL-8 as one of the MOR genes, despite the use of a genetic fishing expedition, demonstrates the utility of the airway microdissecting technique for isolating targeted tissues for various molecular, biochemical, and biological investigations. This type of approach is inevitable since the lung is a very complicated organ and not all conducting airways respond to environmental air pollutants in a similar manner.

    ACKNOWLEDGEMENTS

The technical support of Yu Hua Zhao on in situ hybridization and immunohistochemical staining is deeply appreciated, and we thank Brian Tarkington for assistance on ozone exposure.

    FOOTNOTES

This work is supported in part by National Institute of Environmental Health Sciences Grants ES-00628, ES-06553, and ES-06230.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. M.-J. Chang, Center for Comparative Respiratory Biology and Medicine, School of Medicine, Univ. of California, Davis, CA 95616.

Received 6 February 1998; accepted in final form 5 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abdelaziz, M. M., J. L. Devalia, Q. A. Khair, M. Calderon, R. J. Sapsford, and R. J. Davies. The effect of conditioned medium from cultured human bronchial epithelial cells on eosinophil and neutrophil chemotaxis and adherence in vitro. Am. J. Respir. Cell Mol. Biol. 13: 728-737, 1995[Abstract].

2.   Angerer, L. M., K. H. Cox, and R. C. Angerer. Demonstration of tissue-specific gene expression by in situ hybridization. Methods Enzymol. 152: 649-661, 1987[Medline].

3.   Aris, R. M., D. Christian, P. Q. Hearne, K. Kerr, W. E. Finkbeiner, and J. R. Balmes. Ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy. Am. Rev. Respir. Dis. 148: 1363-1372, 1993[Medline].

4.   Bhalla, D. K. Alteration of ozone-induced airway permeability by oxygen metabolites and antioxidants. Toxicol. Lett. 73: 91-101, 1994[Medline].

5.   Casale, T. B., and M. K. Abbas. Comparison of leukotriene B4-induced neutrophil migration through different cellular barriers. Am. J. Physiol. 258 (Cell Physiol. 27): C639-C647, 1990[Abstract/Free Full Text].

6.   Chen, L. C., and Q. Qu. Formation of intracellular free radicals in guinea pig airway epithelium during in vitro exposure to ozone. Toxicol. Appl. Pharmacol. 143: 96-101, 1997[Medline].

7.   Chitano, P., J. J. Hosselet, C. E. Mapp, and L. M. Fabbri. Effect of oxidant air pollutants on the respiratory system: insights from experimental animal research. Eur. Respir. J. 8: 1357-1371, 1995[Abstract/Free Full Text].

8.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

9.   Devalia, J. L., H. Bayram, C. Rusznak, M. Calderon, R. J. Sapsford, M. A. Abdelaziz, J. Wang, and R. J. Davies. Mechanisms of pollution-induced airway disease: in vitro studies in the upper and lower airways. Allergy 52: 45-51, 1997[Medline].

10.   Devalia, J. L., and R. J. Davies. Airway epithelial cells and mediators of inflammation. Respir. Med. 87: 405-408, 1993[Medline].

11.   Devlin, R. B., K. P. McKinnon, T. Noah, S. Becker, and H. S. Koren. Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L612-L619, 1994[Abstract/Free Full Text].

12.   Dimitriadis, V. K. Tracheal epithelium of bonnet monkey (Macaca radiaca) and its response to ambient levels of ozone. A cytochemical study. J. Submicrosc. Cytol. Pathol. 25: 53-61, 1993[Medline].

13.   Driscoll, K. E., L. Simpson, J. Carter, D. Hassenbein, and G. D. Leikauf. Ozone inhalation stimulates expression of a neutrophil chemotactic protein, macrophage inflammatory protein 2. Toxicol. Appl. Pharmacol. 119: 306-309, 1993[Medline].

14.   Haslett, C., L. A. Guthrie, M. M. Kopaniak, R. B. Johnston, Jr., and P. M. Henson. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am. J. Pathol. 119: 101-110, 1985[Abstract].

15.   Huang, T. H., D. Ann, Y. J. Zhang, A. T. Chang, J. W. Crab, and R. Wu. Control of keratin gene expression by vitamin A in tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 10: 192-201, 1994[Abstract].

16.   Hyde, D. M., W. C. Hubbard, V. Wong, R. Wu, K. Pinkerton, and C. G. Plopper. Ozone-induced acute tracheobronchial epithelial injury: relationship to granulocyte emigration in the lung. Am. J. Respir. Cell Mol. Biol. 6: 481-497, 1992[Medline].

17.   Jaspers, I., E. Flescher, and L. C. Chen. Ozone-induced IL-8 expression and transcription factor binding in respiratory epithelial cells. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L504-L511, 1997[Abstract/Free Full Text].

18.   Lippmann, M. Health effects of ozone. A critical review. J. Air Pollut. Control Assoc. 39: 672-695, 1989.

19.   Luo, G., G. An, and R. Wu. A PCR differential screening method for rapid isolation of clones from a cDNA library. Biotechniques 16: 673-675, 1994.

20.   McBride, D. E., J. Q. Koenig, D. L. Luchtel, P. V. Williams, and W. R. Henderson, Jr. Inflammatory effects of ozone in the upper airways of subjects with asthma. Am. J. Respir. Crit. Care Med. 149: 1192-1197, 1994[Abstract].

21.   McKinnon, K. P., M. C. Madden, T. L. Noah, and R. B. Devlin. In vitro ozone exposure increases release of arachidonic acid products from a human bronchial epithelial cell line. Toxicol. Appl. Pharmacol. 118: 215-223, 1993[Medline].

22.   Pino, M. V., J. R. Levin, M. Y. Stovall, and D. M. Hyde. Pulmonary inflammation and epithelial injury in response to acute ozone exposure in the rat. Toxicol. Appl. Pharmacol. 112: 64-72, 1992[Medline].

23.   Plopper, C. G., A. M. Chang, A. Pang, and A. R. Buckpitt. Use of microdissected airways to define metabolism and cytotoxicity in murine bronchiolar epithelium. Exp. Lung Res. 17: 197-212, 1991[Medline].

24.   Pryor, W. A. Mechanisms of radical formation from reactions of ozone with target molecules in the lung. Free Radic. Biol. Med. 17: 451-465, 1994[Medline].

25.   Schenk, H., M. Vogt, W. Droge, and K. Schulze-Osthoff. Thioredoxin as a potent costimulus of cytokine expression. J. Immunol. 156: 765-771, 1996[Abstract].

26.   Shibata, Y., H. Nakamura, S. Kato, and H. Tomoike. Cellular detachment and deformation induce IL-8 gene expression in human bronchial epithelial cells. J. Immunol. 156: 772-777, 1996[Abstract].

27.   Tanaka, C., H. Kamata, H. Takeshita, H. Yagisawa, and H. Hirata. Redox regulation of lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) gene expression mediated by NF kappa B and AP-1 in human astrocytoma U373 cells. Biochem. Biophys. Res. Commun. 232: 568-573, 1997[Medline].

28.   Tarkington, B. K., R. Wu, W. Sun, K. J. Nikula, D. W. Wilson, and J. A. Last. In vitro exposure of tracheobronchial epithelial cells and of tracheal explants to ozone. Toxicology 88: 51-68, 1994[Medline].

29.   Tomee, J. F., A. T. Wierenga, P. S. Hiemstra, and H. K. Kauffman. Proteases from Aspergillus fumigatus induce release of proinflammatory cytokines and cell detachment in airway epithelial cell lines. J. Infect. Dis. 176: 300-303, 1997[Medline].

30.   Wilson, D. W., C. G. Plopper, and D. L. Dungworth. The responses of the macaque tracheobronchial epithelium to acute ozone injury. Am. J. Pathol. 116: 193-206, 1984[Abstract].

31.   Wu, R., W. R. Martin, J. A. St. George, C. G. Plopper, G. Kurland, J. A. Last, C. E. Cross, R. J. McDonald, and R. Boucher. Expression of mucin synthesis and secretion in human tracheobronchial epithelial cells grown in culture. Am. J. Respir. Cell Mol. Biol. 3: 467-478, 1990[Medline].

32.   Yu, X. Y., N. Takahashi, T. L. Croxton, and E. W. Spannhake. Modulation of bronchial epithelial cell barrier function by in vitro ozone exposure. Environ. Health Perspect. 102: 1068-1072, 1994[Medline].


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