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The Journal of Immunology, 1998, 160: 4889-4895.
Copyright © 1998 by The American Association of Immunologists

Respiratory Syncytial Virus-Infected Pulmonary Epithelial Cells Induce Eosinophil Degranulation by a CD18-Mediated Mechanism1

Barbara Olszewska-Pazdrak*, Konrad Pazdrak{dagger}, Pearay L. Ogra* and Roberto P. Garofalo2,*

Departments of * Pediatrics and {dagger} Internal Medicine, The University of Texas Medical Branch, Galveston, TX 77555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the ß2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Since its initial isolation from infected humans 40 yr ago, respiratory syncytial virus (RSV)3 has emerged as the major cause of bronchiolitis in infants and young children throughout the world (1). Bronchiolitis is defined as a syndrome in young infants characterized clinically by wheezing, dyspnea, respiratory distress, and the radiologic evidence of hyperaeration of the lung (2). Given the ethical and procedural difficulties in performing bronchoscopic studies in children, little or no information is available about the histopathologic features of milder cases of viral bronchiolitis. However, postmortem studies have identified necrosis and, occasionally, proliferation of the bronchial epithelium and destruction of ciliated epithelial cells as the main lesions in fatal cases of RSV bronchiolitis (3, 4, 5). Moreover, the lumen of the small airways becomes obstructed from the sloughed epithelial cells and from increased mucus secretion, causing trapping of air peripheral to the sites of occlusion and formation of multiple areas of atelectasis and emphysema.

Although epithelial cells are the primary target of RSV and the infection proceeds by cell-to-cell spread of the virus (6), viral Ag has been found surprisingly scanty in amount and patchy in distribution in autoptic samples from patients with RSV bronchiolitis (5). These findings suggest that pathogenetic mechanisms other than those associated with direct viral infection are responsible to a great extent for the damage of airway mucosa seen in RSV bronchiolitis. Indeed, peribronchial infiltration of mononuclear cells with edema of surrounding tissue (4), and presence of basophil- and eosinophil-specific inflammatory and cytotoxic mediators (7, 8, 9, 10, 11) have been shown by histologic studies and by analysis of nasopharyngeal secretions of RSV-infected infants. Eosinophils, in particular, seem to play an important role in RSV bronchiolitis. Along this line, we and others have shown that eosinophil cationic protein (ECP), a toxic protein contained in the granules of eosinophils, is released in the respiratory tract of children with naturally acquired RSV infection (9, 10, 11). Significantly higher levels of ECP were recovered from subjects affected by bronchiolitis compared with those with localized upper respiratory tract illness (9, 10). ECP has been shown also in bronchial submucosa of subjects with asthma, especially in areas of epithelial cell sloughing (12), and its cytotoxicity for the epithelium has been confirmed by in vitro studies (13, 14).

Despite the amount of information generated by human, animal, and in vitro studies about the contribution of eosinophil granule proteins to the pathophysiology of allergic and nonallergic inflammation, the mechanisms that trigger eosinophil activation and degranulation remain unclear. Recent observations suggest that adhesion mechanisms most likely play a critical role in eosinophil degranulation (15, 16, 17). Furthermore, a growing body of experimental evidence indicates that the airway epithelium can function both as the target and the initiator of the inflammatory response in RSV infection (18). Thus, since little is known about the interaction between epithelial cells and eosinophils, we investigated the effect of RSV infection on ECP release using a coculture model of purified human eosinophils and lung type II alveolar epithelial cells. The effect of purified RSV virions as well as the direct and modulatory effect of soluble factors produced by infected epithelial cells upon eosinophil degranulation were also studied in this experimental model. Implications for the pathogenesis of viral-induced airway mucosa damage are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

The following Abs were used for flow cytometry: mAb anti-human CD11a (TS1.22, mouse IgG1) and anti-human CD11c (SHCL3, mouse IgG2b) from Dr. T. Springer (Dana-Farber Cancer Institute, Boston, MA), PE-conjugated anti-human CD11b (Becton Dickinson, San Jose, CA), anti-human ICAM-1 (HA58; PharMingen, San Diego, CA), isotype-matched PE-conjugated mouse IgG2a (Becton Dickinson), mouse IgG2a and mouse IgG1 (Dako, Carpenteria, CA), and anti-mouse F(ab')2 FITC-conjugated Ab (Tago, Camarillo, CA). The following Abs were used in neutralization studies: anti-human CD18 clones MHM23 (mouse IgG1; Dako), TS1.18 (mouse IgG1; from Dr. T. Springer), and R15.7 (mouse IgG1) and its F(ab')2 fragment (both a generous gift from Dr. Michele Mariscalco, Baylor College of Medicine, Houston, TX); anti-human ICAM-1 (RR1/1; first domain, mouse IgG1; Biosource, Camarillo, CA); and isotype-matched mouse IgG1 (Dako).

Eosinophil isolation

Heparinized (10 U/ml) venous blood was obtained from normal volunteers and sedimented with 6% dextran. The leukocyte-enriched buffy coats were harvested and overlaid onto Ficoll-Paque (Pharmacia, Piscataway, NY) and centrifuged at 400 x g for 20 min. The granulocyte-containing cell pellet was collected and washed with cold calcium- and magnesium-free HBSS. Erythrocytes were removed by hypotonic lysis. Eosinophils were negatively selected with anti-CD16 immunomagnetic beads to remove neutrophils using the MACS system (Miltenyi Biotec, Sunnyvale, CA) (19). The eosinophil purity was >99%, as determined by microscopic examination of Wright-stained cytospin preparations. Eosinophils were resuspended at 2 x 105 cells/ml in RPMI 1640 (Life Technologies, Grand Island, NY) containing 2% FCS.

Culture of airway epithelial cells

A549 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD). The cells were grown as monolayer in 24-well tissue culture plates (Becton Dickinson) in MEM supplemented with 10% FCS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all from Life Technologies) at 37°C with 5% CO2 in humidified air. All experiments were performed when the cells reached confluence.

RSV purification

The human long strain of RSV (A2) was grown in Hep-2 cells (ATCC) and purified by polyethylene glycol precipitation, followed by centrifugation on 35 to 65% discontinuous sucrose gradients, as described elsewhere (20). The virus was aliquoted, quick frozen on dry ice with ethanol, and stored at -70°C until needed. The virus titer of the purified RSV (pRSV) pools, as determined by a methylcellulose plaque assay (21), was 7 x 108 plaque-forming U/ml. No contaminating cytokines, including IL-1, IL-6, IL-8, TNF-{alpha}, granulocyte-macrophage CSF (GM-CSF), and IFNs, were found in these sucrose-purified viral preparations (22).

Preparation of supernatant from RSV-infected epithelial cells

Monolayers of A549 cells were infected with pRSV at multiplicity of infection (MOI) of 1 in MEM with 2% FCS. At 48 h, when the cells exhibited maximal cytopathic effect, the supernatant was collected, centrifuged at 300 x g, and finally exposed to a 245-nm UV light source for 5 min on ice to inactivate the virus. This RSV-conditioned medium (RSV-CM) was aliquoted and stored at -70°C until used. Control CM was collected from uninfected A549 cell cultures and treated as described for RSV-CM.

Eosinophil degranulation

To determine the role of epithelial cells in eosinophil degranulation, confluent monolayers of A549 cells were infected with pRSV at MOI of 1 or cultured in control medium (MEM with 2% FCS). At 24 h, medium was removed and 0.5 ml of eosinophil suspension (1 x 105 cells) was added to each well. In some experiments, anti-human CD18 mAb, anti-human ICAM-1 mAb, or mouse IgG isotype control, in concentration indicated in Results, was added to the wells at the beginning of the coculture. In other set of experiments, eosinophils were cocultured with epithelial cells in the presence of RSV-CM (50% v/v) with anti-CD18 mAb or mouse IgG isotype control (in concentration indicated in Results). Eosinophils and epithelial cells were then incubated for 16 h at 37°C in 5% CO2. At the end of the coculture period, supernatant was collected and stored at -70°C until ECP measurement was performed by a specific RIA (Pharmacia). Eosinophil viability at the end of culture period was assessed by the exclusion of trypan blue dye.

To determine the effect of purified RSV or supernatant from RSV-infected epithelial cells on eosinophil degranulation, equal number of eosinophils (1 x 105 cells/0.5 ml) was incubated with medium (RPMI 1640 with 2% FCS) or with pRSV (MOI = 10), or were treated with RSV-CM (50% v/v) for 16 h. For the measurement of the total content of ECP, eosinophils cultured in medium were lysed with 2% Triton X-100. Lysates were centrifuged at 300 x g, and supernatants were collected for ECP assay.

ICAM-1 expression by A549 cells

A549 cells were grown on 25-cm2 flasks in MEM containing 10% FCS. Confluent monolayers of epithelial cells were washed with PBS and were cultured with CM or with RSV-CM (50% v/v). After 16-h incubation, cells were harvested by 0.02% EDTA. Approximately 1 x 106 cells were stained with anti-human ICAM-1 mAb or irrelevant isotype control in 100 µl of FACS buffer (HBSS containing 1% BSA and 0.1% NaN3) for 40 min at 4°C. After two washes in FACS buffer, the cells were incubated with FITC-conjugated anti-mouse F(ab')2 Ab for 30 min at 4°C. The cells were finally washed and fixed in 1% paraformaldehyde, and 10,000 cells per condition were analyzed for fluorescence by single-color flow cytometry on a FACScan (Becton Dickinson).

Expression of CD11a, CD11b, and CD11c by eosinophils

Eosinophils (5 x 105 cells/ml) were incubated in control medium (RPMI with 2% FCS) or were exposed to CM, RSV-CM (50% v/v), or pRSV (MOI = 10). After 1.5 h of culture, eosinophils were stained with PE-conjugated anti-human CD11b or with an isotype control Ab in 100 µl of FACS buffer for 1 h at 4°C. To determine expression of CD11a and CD11c, eosinophils were stained with anti-CD11a, anti-CD11c, or irrelevant isotype Ab for 40 min at 4°C. After two washes, the cells were incubated with FITC-conjugated anti-mouse F(ab')2 Ab for 30 min at 4°C. The cells were washed in the FACS buffer and fixed with 1% paraformaldehyde. Expression of CD11a, CD11b, and CD11c was determined by flow cytometry using a FACScan analyzer (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of purified RSV or supernatant from RSV-infected epithelial cell on ECP release

We have shown previously that RSV, by binding to eosinophil cell membrane, directly stimulates superoxide production and primes them for enhanced leukotriene C4 release in response to subsequent challenge with calcium ionophore (23). Furthermore, RSV-infected airway epithelial cells release several cytokines, including TNF-{alpha} (24), GM-CSF (18, 25), RANTES (26, 27), and macrophage-inflammatory protein-1{alpha} (18), which have been shown to induce eosinophil degranulation (28). Thus, we initially examined whether pRSV or cytokines and other soluble mediators present in RSV-CM were able to induce eosinophil degranulation. As shown in Figure 1Go, exposure of purified blood eosinophils to pRSV or to RSV-CM for 16 h resulted in the release of small amounts of ECP (7.3 ± 2.7 ng/ml and 10.1 ± 7.2 ng/ml, respectively) that were comparable with those released spontaneously by nonstimulated eosinophils (11.8 ± 2.9 ng/ml). Eosinophils that had been lysed by treatment with Triton X-100 released 412 ± 39.5 ng/ml of ECP. Eosinophil viability after 16 h of culture was consistently >90%, regardless of the treatment conditions. These results indicate that eosinophils are not able to release ECP directly in response to pRSV or to soluble mediators present in the supernatant of RSV-infected epithelial cells.



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  		FIGURE 1. Effect of purified RSV or supernatant from RSV-infected epithelial cells on ECP release. Eosinophils were incubated with control medium (Eos), exposed to pRSV (Eos + pRSV), or treated with RSV-CM (Eos + RSV-CM) for 16 h. For the measurement of total content of ECP, eosinophils were lysed with Triton X-100 (Eos max). Each bar represents the mean ± SD obtained from four experiments performed in duplicate.

 
ECP release in coculture of eosinophils and RSV-infected lung type II cells

Adhesion mechanisms initiate and regulate several eosinophil effector functions, including superoxide production (17), leukotriene C4 generation (29), and cell degranulation (15, 16, 17). Therefore, we examined the role of RSV infection on ECP release in an in vitro coculture system of eosinophils and lung epithelial cells. In these experiments, we used the A549 cell line, which is derived from an alveolar cell carcinoma of the lung, displays features of type II epithelial cells (30), and is susceptible to RSV infection (31, 32). As shown in Figure 2Go, blood eosinophils cocultured with uninfected A549 cells for 16 h released ECP in concentrations similar to those released spontaneously by eosinophils cultured in absence of epithelial cells (14 ± 2 ng/ml) (see also Fig. 1Go). However, when eosinophils were cocultured with 24-h RSV-infected A549 cells, a significant fivefold enhancement in ECP release was observed (56.4 ± 16.6 ng/ml; p < 0.01).



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  		FIGURE 2. Effect of epithelial cells on ECP release by eosinophils. Confluent A549 cell monolayers were infected with RSV at MOI of 1, or were incubated with control medium for 24 h. After removing the medium, eosinophils (1 x 105) were added to uninfected (Ep + Eos) or RSV-infected (RSV-Ep + Eos) A549 cells and cocultured for 16 h. In other experiment, eosinophils (1 x 105) were added to uninfected A549 cells in the presence of RSV-CM (Ep + Eos + RSV-CM) and cocultured for 16 h. At the end of incubation, supernatants were collected and ECP was measured by RIA. The results are expressed as mean ± SD from separate experiments performed in duplicate; n = 7, *p < 0.01, compared with Ep + Eos; n = 5, **p < 0.01, compared with Ep + Eos.

 
During RSV or other mucosal viral infections, uninfected bystander epithelial cells are likely to become exposed to a number of soluble mediators produced by adjacent infected cells. Therefore, additional experiments were performed to determine whether eosinophil degranulation occurred in the presence of uninfected A549 cells and RSV-CM. As shown in Figure 2Go, eosinophils cocultured with uninfected A549 cells in the presence of RSV-CM released significantly higher amounts of ECP than eosinophils cocultured with A549 cells in the presence of CM, a supernatant obtained from control epithelial cells (25.8 ± 3.5 vs 9 ± 2 ng/ml, mean ± SD).

Expression of ICAM-1 on A549 cells and CD11b on eosinophils is up-regulated by the supernatant of RSV-infected epithelial cells

Integrins of the ß2 family (CD11b/CD18) play a critical role in eosinophil degranulation induced by platelet-activating factor or GM-CSF (17) and IgG (15). We and others have shown that RSV infection induces the expression of ICAM-1, the ligand for CD11b/CD18, on A549 cells via the autocrine effect of IL-1{alpha} (24). Since the expression of ICAM-1 by noninfected epithelial cells may also amplify eosinophil degranulation, we further examined by flow cytometry the expression of ICAM-1 on A549 cells following exposure to RSV-CM. As shown in Figure 3Go, exposure to RSV-CM induced ICAM-1 expression on A549 cells, while no effect was observed when the cells were exposed to supernatant of uninfected epithelial cells.



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  		FIGURE 3. Enhancement of ICAM-1 expression on A549 cells by supernatant from RSV-infected epithelial cells. Confluent monolayers of A549 cells were exposed to supernatant from uninfected (CM) or supernatant from RSV-infected (RSV-CM) epithelial cells for 16 h. At the end of the incubation period, the cells were stained for flow cytometry with anti-human ICAM-1 mAb or with an isotype control followed by FITC-conjugated anti-mouse F(ab')2 IgG Ab. The histogram shows overlays of logarithmic mean fluorescence frequency distribution of 10,000 cells. The bar graph shows the changes in mean fluorescence intensity values of ICAM-1 expression on A549 cells (n = 3; *p < 0.01, when compared with CM).

 
Additional experiments were performed to determine whether RSV-CM also had effect on the expression of CD11b by eosinophils. Freshly isolated blood eosinophils constitutively expressed CD11b (Fig. 4Go), as shown also in previous studies (33). Exposure to RSV-CM resulted in the up-regulation of CD11b expression on eosinophils, as indicated by a 92.6% increase in mean fluorescence intensity, compared with control. A smaller increase (12.2%) in CD11b expression was also observed when the eosinophils were exposed to the supernatant of uninfected epithelial cells. We did not observe significant changes in the expression (mean fluorescence intensity) of CD11a and CD11c on eosinophils exposed to control medium (22.4 ± 0.6 and 6.4 ± 1.2, respectively, mean ± SD, n = 3) or RSV-CM (32 ± 8.3 and 9.2 ± 0.14, respectively).



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  		FIGURE 4. Enhancement of CD11b expression on eosinophils by supernatant from RSV-infected epithelial cells. Eosinophils were incubated with control medium (Ctrl), or with supernatant from uninfected (CM) or RSV-infected (RSV-CM) A549 cells for 2 h. At the end of the incubation period, the cells were stained with anti-human CD11b PE-conjugated mAb or an isotype control. CD11b expression on eosinophils was determined by flow cytometry. The bar graph shows the changes in mean fluorescence intensity values of CD11b expression on eosinophils (n = 5; *p < 0.01, when compared with Ctrl or CM).

 
Effect of neutralizing Ab against adhesion molecules on ECP release

Since coculture of RSV-infected A549 cells with eosinophils resulted in release of ECP- and RSV-CM-induced expression of ICAM-1 on A549 cells and the up-regulation of CD11b on eosinophils, we examined the specific role of these adhesion molecules in eosinophil degranulation induced by RSV-infected epithelial cells. Neutralizing anti-ICAM-1 mAb (10 µg/ml) had no significant effect on eosinophil degranulation (data not shown). On the other hand, addition of the anti-CD18 mAb MHM23 to the coculture of RSV-infected A549 cells and eosinophils significantly inhibited ECP release by 64.4% (Fig. 5GoA). Similarly, the MHM23 mAb almost completely inhibited (84.7%) ECP release induced by the coculture of uninfected A549 cells and eosinophils in the presence of RSV-CM (Fig. 5GoB). In other experiments, the ability of various anti-CD18 mAbs to inhibit ECP release in coculture of RSV-infected A549 cells and eosinophils was compared. As shown in Figure 6Go, virtually complete inhibition of eosinophil degranulation was obtained with the mAbs TS1.18 and MHM23 (used at fivefold higher concentration than in the experiments presented in Fig. 5Go). F(ab')2 fragment of the R15.7 mAb had almost identical inhibitory activity than the parent Ab. Thus, these results suggest that a CD18-dependent mechanism is involved in eosinophil degranulation mediated by epithelial cells either directly infected by RSV or exposed to soluble mediators released by other infected cells. The neutralizing effect of anti-CD18, but not anti-ICAM-1 mAbs suggests that non-ICAM-1 epithelial ligand(s) may be recognized by eosinophil CD18 and is discussed further below.



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  		FIGURE 5. Inhibition of ECP release by anti-CD18 mAb. A, Eosinophils were cultured with uninfected A549 cells (Ep + Eos) or with RSV-infected A549 cells in the presence of mouse IgG isotype control (RSV-Ep + Eos + IgG) or anti-CD18 mAb (clone MHM23, 0.2 µg/ml) (RSV-Ep + E + anti-CD18). B, Eosinophils were cultured with uninfected A549 cells in the presence of RSV-CM with mouse IgG isotype control (Ep + Eos + RSV-CM + IgG) or with anti-CD18 mAb (clone MHM23, 0.2 µg/ml) (RSV-Ep + E + anti-CD18) (Ep + Eos + RSV-CM + anti-CD18). After 16 h, supernatants were collected and ECP was measured by RIA. Data are expressed as mean ± SD of n = 4 experiments performed in duplicate; *p < 0.01, when compared with Ep + Eos; **p < 0.01, when compared with RSV-Ep + Eos + IgG; #p < 0.01, when compared with Ep + Eos; ##p < 0.01, when compared with Ep + Eos + RSV-CM + IgG.

 


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  		FIGURE 6. Inhibitory effect of different mAbs anti-CD18 on ECP release. Eosinophils were cultured for 16 h with RSV-infected A549 cells in the presence of the anti-CD18 mAbs MHM23, TS1.18, R15.7, or R15.7 F(ab')2 fragment. mAbs were used at a final concentration indicated in parentheses. Data are expressed as mean ± SD of n = 4 experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Eosinophils are recognized as prominent effector cells in airway allergic inflammation by releasing basic granule proteins, including major basic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, and ECP (34). This conclusion is supported by immunohistologic studies in patients with asthma or chronic sinusitis that have identified deposition of eosinophil proteins on damaged respiratory epithelium (12, 35, 36), and by the demonstration of in vitro cytotoxicity of activated eosinophils and their proteins for nasal (37), bronchial (38), and lung epithelial cells (39). In addition, clinical and experimental observations suggest that eosinophils may play an important role in airway mucosa injury associated with naturally acquired RSV infection. In this regard, ECP and other inflammatory mediators produced by eosinophils have been demonstrated in the airways of infants with acute RSV infection (9, 10, 11). High levels of ECP were predictive of the development of bronchiolitis at the time of RSV infection and correlated with severity of disease (9). Despite the body of information generated about the role of eosinophil granule proteins in airway injury, the cellular and molecular mechanisms of eosinophil degranulation are incompletely understood.

In recent years, it has become clear that respiratory epithelial cells, which represent the main target of eosinophil cytotoxic proteins, are able themselves to initiate and modulate local inflammatory responses by releasing a variety of bioactive mediators and cytokines (40). Since respiratory epithelial cells are also the primary site of replication for RSV (6), our experimental approach was designed to mimic the in vivo condition of interaction between RSV-infected epithelium and eosinophils within the airway mucosa. For this purpose, ECP release was investigated using cocultures of human lung type II alveolar epithelial cells (A549) and purified blood eosinophils. We show in this work that eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. On the other hand, uninfected A549 cells, which failed to induce eosinophil degranulation, were able to trigger ECP release if cultured with eosinophils in the presence of supernatant obtained from RSV-infected cells (RSV-CM). The release of ECP, in cultures of eosinophils with RSV-infected or uninfected A549 cells in the presence of RSV-CM, was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb.

Based on these results, we asked the question concerning whether RSV, in the form of purified virions, and RSV-CM were able to trigger the process of eosinophil degranulation in the absence of epithelial cells. We have shown in previous studies that RSV, by binding to eosinophil cell membrane, is able to stimulate directly oxygen radical production and to prime eosinophils for enhanced superoxide generation and leukotriene C4 release (23). In addition, RSV-infected respiratory epithelial cells release several cytokines and chemokines with direct stimulatory activity on eosinophil degranulation (28), including TNF-{alpha} (24), GM-CSF (18, 25), RANTES (26, 27), and macrophage-inflammatory protein-1{alpha} (18). The results of these experiments clearly show that neither purified RSV nor RSV-CM was able to induce directly ECP release from eosinophils, indicating that the presence of RSV-infected epithelial cells is necessary to induce eosinophil degranulation. However, we demonstrate in this work that RSV-CM, by providing an appropriate milieu of inflammatory mediators, can activate eosinophils and uninfected epithelial cells and trigger efficient eosinophil degranulation.

With regard to eosinophil activation, RSV-CM induced the up-regulation of CD11b, the {alpha}-chain of the ß2 integrin Mac-1 (CD11b/CD18), on eosinophil membrane. Mac-1, which is involved in specific adhesion of leukocytes to endothelial cells and extracellular matrices, functions as a receptor for ICAM-1 (41) and is expressed in similar density by normodense and hypodense eosinophils (33). Increased adhesion avidity of ß2 integrins in eosinophils by the CC chemokines RANTES and monocyte chemotactic protein 3 has been recently reported (42). It is likely that RSV-CM, a rich source of CC chemokines, modulates adhesion and other functions of eosinophils by increasing both the number and the avidity of CD11b molecules on eosinophils. Several lines of evidence suggest that RSV-mediated up-regulation or activation of ß2 integrins on eosinophils is a crucial event for the induction of eosinophil degranulation. First, in the experimental model used in our studies, the release of ECP by eosinophils was inhibited significantly by three mAbs (with different epitope specificity) to CD18, the common ß-chain of the ß2 integrin family. Although adhesion studies were not performed, addition of neutralizing Ab anti-CD18 most likely resulted in inhibition of eosinophil adhesion to A549 cells. Previous studies have indeed shown that the process of eosinophil degranulation in the presence of bronchial cells (16) and immobilized IgG (15), or in response to GM-CSF and platelet-activating factor in albumin-coated polystyrene plates (17) is preceded consistently by cell adhesion. Second, neutrophil and eosinophil adhesion to respiratory epithelial cells has been shown to be greatly enhanced by RSV infection, to require prior activation of eosinophils, and to be reduced significantly by Ab to CD18 (43). Third, we have shown in these studies that uninfected A549 cells induced the release of significant amounts of ECP by eosinophils when RSV-CM was added to the coculture, resulting in the activation of epithelial cells and up-regulation of CD11b on eosinophils.

It has been shown recently that in cocultures of eosinophils and human bronchial epithelial cells, eosinophil degranulation occurred only if both cells were activated by IL-5 and TNF-{alpha}, respectively (16). We report in this work that A549 cells were induced to express high levels of ICAM-1 following exposure to RSV-CM. However, we were unable to block eosinophil degranulation using a well-characterized mAb to ICAM-1 (RR1/1), which has been shown previously, among other functions, to inhibit the adhesion of eosinophils to endothelial cells (44). In agreement with these findings, other investigators have shown that, although IL-1{alpha} or TNF-{alpha} treatment of respiratory epithelial cells induced enhanced expression of ICAM-1 and increased adherence of activated eosinophils, cell adherence was blocked by neutralizing mAb to ß2 integrins, but not to ICAM-1 (45). Moreover, IL-5-stimulated eosinophil degranulation in the presence of bronchial epithelial cells has been shown to be reduced significantly by mAbs to CD18, but not to ICAM-1 (16). Thus, while ß2 integrins seem to be key molecules for eosinophil adhesion to epithelial cells and degranulation, ICAM-1 appears to play a relatively minor role. One explanation for the different effect of anti-CD18 and anti-ICAM-1 on eosinophil adherence and degranulation in the presence of epithelial cells may be that ligands for the ß2 integrins, other than ICAM-1, are expressed on airway epithelial cells. Therefore, the induction of ICAM-1 that occurs as a result of direct viral infection or, in uninfected cells, by exposure to cytokines and soluble mediators released by neighbor-infected cells, may represent a paradigm of in vivo epithelial cell activation associated with the up-regulation of other adhesion molecules currently unknown.

Migration and/or activation of eosinophils in the airway mucosa are not unique to RSV among respiratory viruses. In this regard, it has been shown that rhinovirus infection in patients with allergic rhinitis is associated with the recruitment of eosinophils to the airways (46). Other investigators have extended these observations by showing a significant increase in the number of eosinophils in bronchial biopsies from normal volunteers experimentally infected with rhinovirus (47). Increased lung resistance and eosinophil infiltration have been reported in parainfluenza-infected guinea pigs (48). In vitro studies have also shown that influenza virus-infected respiratory epithelial cells are able to generate RANTES, a potent chemoattractant and activator of eosinophils (49), and to express ICAM-1 following infection with parainfluenza (50) or adenovirus (51). Therefore, the major respiratory viruses that cause bronchiolitis in infancy and trigger asthma attacks in children and older individuals (52) are able to induce the recruitment and perhaps to activate eosinophils by mechanisms still largely unknown. The results presented in this work demonstrate that eosinophil degranulation can be induced effectively by coculture of eosinophils with RSV-infected epithelial cells, and that this process is CD18 dependent. This observation has important implication for the pathogenesis of inflammation driven by the initial viral infection of epithelial cells. Indeed, the killing and eradication of infected cells by immunologic mechanisms mediated by Ag-specific CTLs or, as shown in this study, by cytotoxic eosinophil products may positively impact the outcome of RSV infection. On the other hand, eosinophil-mediated cytotoxicity against bystander uninfected epithelial cells may greatly contribute to the enhanced pathology observed in RSV-induced airway disease. Old and new therapeutic strategies for RSV airway disease should take into account the delicate balance between immunopathology and immunoprotection that is characteristic of RSV infection.


   Acknowledgments
 
We thank Dr. Michele Mariscalco for generously providing mAb R15.7, Dr. Frank Schmalstieg for helpful discussions, Todd Elliot for technical assistance, and Virginia Reimer for her help in the preparation of the manuscript.


   Footnotes
 
1 This work was supported by Grants AI 15939 and HD 27841 (P.L.O. and R.P.G.) from National Institutes of Health, and by a grant of John Sealy Memorial Endowment Fund for Biomedical Research (R.P.G.). K.P. was supported by McLaughlin Fellowship Fund. Back

2 Address correspondence and reprint requests to Dr. Roberto P. Garofalo, Department of Pediatrics, Division of Immunology/Allergy/Rheumatology, 301 University Blvd., Galveston, TX 77555-0369. E-mail address: Back

3 Abbreviations used in this paper: RSV, respiratory syncytial virus; CM, conditioned medium of epithelial cells; ECP, eosinophil cationic protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; MOI, multiplicity of infection; PE, phycoerythrin; pRSV, sucrose gradient-purified respiratory syncytial virus; RANTES, regulated upon activation, normal T cell expressed and secreted; RSV-CM, conditioned medium of respiratory syncytial virus-infected epithelial cells. Back

Received for publication September 29, 1997. Accepted for publication January 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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