INTRODUCTION

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Smoking induces inflammation. It has effects on the bone marrow resulting in a peripheral leukocytosis (1) which, in heavy smokers, is associated with a shift in the balance of the CD4⁺ (T-helper)-to-CD8⁺ (T-suppressor) cell ratio in favor of the CD8⁺ population (2). In healthy smokers and those with chronic bronchitis (CB) and chronic obstructive pulmonary disease (COPD), this altered balance has also been found in the lungs in studies that have examined bronchoalveolar lavage fluid (BALF) (3), bronchial biopsies obtained during bronchoscopy (4), and airway tissue of lungs taken at resection from smokers with cancer (5). Accordingly, CB and COPD are now considered to be smoking-induced inflammatory conditions of the tracheobronchial tree, in which there is an alteration in the balance of T lymphocytes in favor of a CD8⁺ cell predominance, associated clinically with chronic productive cough and, in COPD, with fixed airflow obstruction. In contrast, asthma, at least in nonsmokers, is characterized by an "allergic profile" of inflammation that includes CD4⁺ T-helper type-2 cells associated with secretion of the regulatory cytokines interleukin (IL)-4 and IL-5, and with a tissue eosinophilia (6).

Studies of sputum obtained from smokers with bronchitis have shown that in contrast to the eosinophilia of asthma, the sputa of most bronchitic patients, at least in the stable phase of their condition, have relatively low numbers of eosinophils and high numbers of macrophages (7).

Subgroups of smokers, however, have been shown to have an eosinophilic component. For example, there is a subgroup of subjects with chronic productive cough without airway hyperresponsiveness or a history of asthma who have a marked sputum eosinophilia, which is reduced in response to inhaled corticosteroids (8). In other studies, investigation of BALF in subjects with bronchitis has shown variable numbers of both eosinophils and neutrophils in addition to macrophages, and measures of eosinophil cationic protein (ECP) are often increased (9, 10). More recently, there have been two reports of the result of bronchial biopsy of nonasthmatic bronchitic patients after an exacerbation of their condition in which the exacerbation was associated with a marked recruitment of tissue eosinophils (11, 12). Besides this, our recent study of airway wall tissue taken at resection from smokers demonstrated gene expression for both IL-4 and IL-5 in and around the hypertrophied bronchial mucus-secreting glands of patients with CB (13). Such an "allergic" profile of gene expression may partly explain how a tissue eosinophilia may develop in response to an exacerbation of CB caused by viral infection.

Our aim in the present study was to examine biopsy specimens of bronchitic patients who had had very recent exacerbations, in order to determine the cytokines and chemokines responsible for the tissue eosinophilia. Because antibodies applied to paraffin-embedded tissues often show a lack of sensitivity, we examined the patients' biopsied tissue sections for the regulatory cytokines' IL-4 and IL-5 messenger RNAs (mRNAs) as markers of these cytokines' gene expression. We wished to compare bronchial biopsies of well-characterized subjects with CB during a stable phase of their disease (i.e., at baseline) with those of subjects who had had a very recent exacerbation. This would determine whether there is an upregulation of IL-4 and IL-5 during exacerbation. However, IL-4 and IL-5 alone are insufficient to account for tissue recruitment of eosinophils; this also requires an eosinophil chemoattractant gradient to encourage the emigration of intravascular eosinophils into the surrounding mucosa, where they migrate toward, into and through the surface epithelium to enter the airway lumen. Thus, we set out to investigate which of three eosinophil chemoattractantseotaxin, monocyte chemoattractant protein (MCP)-4, or regulated on activation, normal T-cell expressed and secreted (RANTES) is upregulated, in order to explain more fully the increased tissue eosinophilia associated with such exacerbations of bronchitis. Our hypothesis was that both inflammatory cells and, more especially, the surface epithelium would express high levels of chemoattractant in order to initiate and maintain the required eosinophil chemoattractant gradient.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The study conformed to the declaration of Helsinki; all subjects were volunteers and gave their informed, written consent, and approval for the study was given by each of the participating institutions' local ethics committees. There were three subject groups studied in parallel: (1) normal, healthy, nonatopic nonsmokers without any history of bronchopulmonary disease and with an FEV₁ > 85% predicted (n = 7); (2) smokers with CB in its stable phase, examined at least 1 yr after the last clinical exacerbation and without a history or signs of asthma (stable CB) (n = 11); and (3) subjects with CB who sought medical advice for an exacerbation of their condition (CB exacerbation) (n = 9) (Table 1). The diagnosis of CB was based on a documented history of cough and sputum production on most days of the month occurring for at least 3 mo a year during the previous 2 yr (14). There was a lack of ₂-agonist-induced airflow reversibility, recorded as less than a 5% improvement of FEV₁ after salbutamol inhalation. No patient had a history of either asthma or allergic rhinitis, and all were nonatopic as defined by the absence of skin reactivity to a panel of common allergens including grass pollen sp., Parietaria sp., Artemisia sp., Cynodon sp., cat, molds, Dermatophagoides pteronyssinus, D. farinae, and Aspergillus sp. An exacerbation was defined as a worsening of symptoms (i.e., increased dyspnea associated with increased cough and sputum production), of sufficient severity to cause the subject to seek medical attention in the week preceding the bronchoscopy (11). All of the bronchitic subjects had a history of cigarette smoking. Those in a stable phase of their disease had smoked 43 ± 5 pack-years (mean ± SEM), whereas those with exacerbations had smoked 39 ± 7 pack-years. None of the subjects had received oral or inhaled glucocorticosteroids or antibiotics in the month preceding the study. All subjects underwent full clinical examination and had a chest radiograph, electrocardiogram, and routine blood tests. None had coin lesions on radiography, and there was no suspicion of cancer. Two subjects in the exacerbation group had a history of cardiac disease: Subject 8 in Table 1 had a history of atrial fibrillation and Subject 1 in Table 1 had a history of ischemic heart disease, but neither was receiving treatment during the week preceding the study. One subject in the stable-CB group (Subject 13 in Table 1) had a history of arterial disorder of the extremities, but he had not received treatment during the week preceding the study.

Bronchoscopy and Biopsies

Fiberoptic bronchoscopy was performed as previously described (11). Biopsies were taken with an Olympus BF type 1T 10- bronchoscope with sterile FB 15C forceps (Olympus Co., Tokyo, Japan) from the subcarina of the basal segmental bronchi of the right lower lobe. Biopsies were fixed immediately in 4% formaldehyde, and after dehydration were embedded in paraffin wax. Serial sections of 6-µm thickness were cut and stained with hematoxylin-eosin. Monoclonal antibodies (mAbs), that had been raised against eosinophil cationic protein (EG2) (activated eosinophils), a subset of CD4⁺ T-helper cells (henceforth referred to as CD4⁺ cells), CD4⁺ T-helper cells, and CD8⁺ T-cytotoxic/suppressor cells were applied to the sections. The technique of in situ hybridization was applied, using riboprobes that detected intracytoplasmic mRNA for IL-4, IL-5, human eotaxin, MCP-4, and RANTES.

Immunohistology

The identification of inflammatory cells within bronchial biopsies was done with the alkaline phosphatase-antialkaline phosphatase (APAAP) technique as previously described (15). Briefly, the sections were immunostained with mAbs to EG2-positive cells (Pharmacia & Upjohn Ltd, Milton Keynes, UK), CD4⁺ cells (M0834; DAKO Ltd., Cambridge, UK), and CD8⁺ cells (M7103; DAKO), appropriately diluted in Tris-buffered saline (TBS) and applied to biopsy sections in 50-µl aliquots for 30 min. Sections were washed and incubated for a further 30 min with unconjugated rabbit antimouse immunoglobulin (Z0259; DAKO) diluted 1 in 25 in TBS containing 20% normal human serum. Sections were then washed and incubated for 30 min with APAAP mouse mAb (D0651; DAKO) diluted 1 in 50 with TBS containing 20% normal human serum. After a further wash, bound alkaline phosphatase was detected as a red product after 20 min of incubation with Naphthol AS-MX phosphate and 1 mg/ml New Fuchsin. The slides were counterstained with hematoxylin to provide morphologic detail, and the specimens were then mounted in Aqua perm mounting medium (Lipshaw Immunon, Pittsburgh, PA, catolog no. 484990). We used an irrelevant mouse IgG1 kappa antibody (MOPC21; Sigma, Dorset, UK, catolog no. M7894) for the primary layer as a negative control procedure. We used the microwave antigen retrieval technique (1 mM ethylenediamine tetraactic acid [EDTA] at pH 8.0; 5 min) for detection of CD8⁺ cells.

Nonisotopic in situ Hybridization

Preparation of complementary RNA probes. Digoxigenin (Dig)-labelled antisense and sense complementary ribonucleic acid (cRNA) probes were generated from complementary deoxyribonucleic acid (cDNA) according to a well-tried and published method (16). A labelling mixture of 1 µg linearized DNA, 2 µl (of ×10 concentration) each of adenosine, guanosine, cytidine, and Dig-uridine triphosphates (ATP, GTP, CTP, and Dig-UTP, respectively) was added to 2 µl (of ×10 concentration) of transcription buffer, followed by 2 µl of SP6 or T7 RNA polymerase, with the final volume made up to a total of 20 µl with ribonuclease (RNAase)-free double-distilled water to produce either antisense or sense probes according to the orientation of each particular probe. After incubation for 2 h at 37° C, 20 U of RNAase-free deoxyribonucleasol (DNAase 1) was added and incubated for 15 min at 37° C. An aliquot of 2 µl EDTA (0.2 M, pH 8.0) was added to stop the reaction and precipitate the probe with cold ethanol. The sample was centrifuged and dried under vacuum. To check transcription efficiency and quantify the probe, 1 µl Dig-RNA was taken for electrophoresis in 1.2% agarose gel, after which it was stored at 80° C. The sizes (base pairs), vectors, and sources of the probes were as follows: IL-4 (318 bp; PGEM-1), IL-5 (412 bp; PGEM-IV), MCP-4 (306 bp; PBSIISK-), and RANTES (280 bp; PBSIISK-), all four of which were from Glaxo-Wellcome Biomedical, Geneva Switzerland, and eotaxin (300 bp; PCRscriptSK+; Leukosite Inc., Cambridge, MA).

Prehybridization procedures. The sections were dewaxed and washed in phosphate-buffered saline (PBS), and were then incubated with proteinase K to permeabilize the cells. After postfixation in 4% paraformaldehyde in PBS, hybridization buffer (×2 Denhardt's solution, 50 µg/ml salmon sperm DNA, 100 µg/ml yeast transfer RNA, 50% formamide) containing 50 to 100 ng/ml Dig-labeled cRNA probe was added and incubated at 42° C overnight. Sense probes were used as the most appropriate negative control. Slides were washed in different concentrations of sodium chloride/trisodium citrate (standard saline-citrate; SSC) and incubated in 20 µg/ml RNAase. Slides were washed twice in ×2 SSC at 37° C for 20 min each, and then in ×0.2 SSC at 37° C for 30 min, and the remaining probe was hybridized, with the mRNA of interest detected with anti-Dig antibody conjugated with alkaline phosphatase. Detection was done by adding 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate and incubating for 30 to 60 min to give a blue-black end-product. The nuclear counterstain was Nuclear Fast Red.

Quantification and Data Analysis

The slides were coded to avoid observer bias. Areas of subepithelium excluding muscle and gland were assessed with an Apple Macintosh computer and Image 1.5 software (Apple, Inc., Cupertino, CA). Cells positive for IL-4, IL-5, eotaxin, MCP-4, and RANTES mRNA, and EG2-positive CD8⁺ and CD4⁺ cells were counted under a Leitz Dialux 20 light microscope (Leitz, Wetzlaar, Germany), fitted with an eyepiece graticule divided into 100 squares, at ×200 magnification. A similar eyepiece graticule was used to "point count" and assess the percentage of epithelium expressing mRNA for the chemokine RANTES (17). To test the consistency of quantification and the inherent variation of repeated counts, one section was selected and counted five times over the period of the entire study. The coefficient of variation (CV) for repeated counts of cells positive for IL-4 mRNA by the same observer was 4%. The sections that had been hybridized with the sense (negative) probe were examined first in order to deduce background signals.

Statistical Analysis

The data for cell counts are expressed as number of positive cells per mm² subepithelial area. The CV (SD/mean × 100) was used to express the error of repeat counts. Mann-Whitney U tests were applied to test for differences between the three study groups. Spearman's rank correlation and Pearson's correlation (for data after log transformation) were used to determine the correlation between number of EG2-positive cells and cells positive for IL-4, IL-5, eotaxin, MCP-4, and RANTES mRNA, respectively. A value of p < 0.05 was accepted as indicating a significant difference in the Mann-Whitney U test. For Spearman's rank correlation, a value of p < 0.03 was used as the threshold for statistical significance following application of Bonferoni's correction for the multiple (i.e., 15) analyses we performed.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Clinical Findings

The characteristics of the healthy nonsmoker control subjects, those with CB in a stable phase of their disease, and those with exacerbations of bronchitis are shown in Table 1. The three groups were similar with regard to age. The two groups with CB were similar in their smoking history. Lung function data were similar in the two bronchitic groups: FEV₁ % predicted and FEV₁/VC% in each bronchitic group were significantly lower than in the normal nonsmoker controls (p < 0.01 and p < 0.05, respectively). All subjects were nonatopic (i.e., they had negative skin tests for a panel of common allergen extracts) (11). The numbers of blood eosinophils in the healthy nonsmokers and smokers with CB were similar, but in comparison with these two groups, their number in subjects with an exacerbation of CB was significantly increased (p < 0.01).

Inflammatory Cells

Biopsy tissue sections were immunostained to detect and quantify the CD4⁺ and CD8⁺ lymphocyte subsets and EG2-positive eosinophils (Figure 1). Table 2 shows that the numbers of CD4⁺ cells were significantly lower in both bronchitic groups than in the normal controls (p < 0.05). In comparison with the number of CD4⁺ cells present in the subjects with stable CB, exacerbations were associated with an increased number of these cells (p = 0.05). There were significantly increased ratios of CD8⁺ to CD4⁺ cells in the two bronchitic groups as compared with the healthy controls (p < 0.05). However, by comparison with the group with stable CB, those in exacerbation showed a relative decrease in the CD8⁺-to-CD4⁺ ratio, due primarily to the increase in the CD4⁺ cell subset.

View larger version (130K): [in this window] [in a new window]   Figure 1.   Light-microscopic image of immunostained bronchial mucosa from a biopsy of a patient with an exacerbation of bronchitis. There was an increase in the number of subepithelial EG2-positive eosinophils (shown stained red with the APAAP technique). (Original magnification: ×230.)

The numbers of activated (i.e., EG2-positive) tissue eosinophils were similar in the healthy nonsmoker and stable CB groups (Figure 2). By comparison, there was a significant (p < 0.05) sixfold increase in the number of eosinophils in the patients with an exacerbation (Figure 2).

View larger version (11K): [in this window] [in a new window]   Figure 2.   Results of counts for EG2-, IL-4-, and IL-5-positive cells in bronchial biopsies of subjects with CB associated with an exacerbation (E) and in a stable phase (S). The results are compared with respective counts for healthy nonsmoker controls (C). The results are expressed as the number of positive cells per mm2 of a subepithelial zone (Mann- Whitney U test: horizontal bar shows median values).

Gene Expression

Subepithelial lymphomononuclear cells showed mRNA positivity for IL-4 (Figures 3A and 3B) and IL-5 (Figure 4), and for eotaxin (Figure 5), MCP-4, and RANTES (Figures 6A through 6D). IL-4 was also expressed by surface epithelium, and eotaxin was also expressed in the endothelium of bronchial vessels and focally in the surface epithelium. RANTES was expressed focally and weakly in normal nonsmoking controls (Figure 6A); it was upregulated in smokers with CB (Figure 6B), and was particularly strongly expressed by both subepithelial cells and the surface epithelium in association with an exacerbation of CB (Figure 6C). All epithelial cell types showed mRNA positivity throughout the cytoplasm, albeit that in goblet cells it was particularly strong in the perinuclear region and missing in the areas of stored mucin. Where the epithelium was squamous and metaplastic, it also showed mRNA positive for RANTES.

View larger version (121K): [in this window] [in a new window]   View larger version (139K): [in this window] [in a new window]   Figure 3.   Nonisotopic in situ hybridization of a tissue section of a bronchial biopsy from a patient with bronchitis in a stable phase, showing focal cytoplamic localization of IL-4 mRNA in epithelial cells (arrowheads) and in many subepithelial lymphomononuclear cells (dark blue). (Original magnification; A: ×115; B: ×230.)

View larger version (140K): [in this window] [in a new window]   Figure 4.   In situ hybridization of a bronchial biopsy specimen from a patient with bronchitis in a stable phase, showing IL-5 mRNA in the cytoplasm of subepithelial lymphomononuclear cells (dark blue). (Original magnification: ×115.)

View larger version (131K): [in this window] [in a new window]   Figure 5.   In situ hybridization of a bronchial biopsy specimen from a patient with bronchitis associated with an exacerbation, showing eotaxin mRNA in epithelial cells (arrowhead ) and extensive expression of eotaxin in subepithelial lymphomononuclear cells (dark blue). (Original magnification: ×58.)

View larger version (125K): [in this window] [in a new window]   View larger version (122K): [in this window] [in a new window]   View larger version (122K): [in this window] [in a new window]   View larger version (145K): [in this window] [in a new window]   Figure 6.   In situ hybridization of a bronchial biopsy specimen for RANTES gene expression: (A) Specimen from a healthy nonsmoker, showing weak expression of RANTES in surface epithelial cells (arrowheads) and a relatively small number of RANTES-positive subepithelial lymphomononuclear cells (dark blue). (B) Specimen from a smoker with CB in a stable phase, showing moderate to intense staining of epithelial cells and an increase in the number of RANTES mRNA-positive inflammatory cells in the subepithelial zone. (C ) Specimen from smoker with an exacerbation of CB, showing strong expression of RANTES mRNA positivity in the cytoplasm of both epithelial and subepithelial lymphomononuclear cells. (D) Biopsy specimen from a patient with an exacerbation of CB shows an absence of staining with the sense control probe. Magnification of all: ×230.

The numbers of subepithelial cells expressing IL-4 and IL-5 mRNA were determined, and the results are shown in Figure 2. Cells expressing IL-4 and IL-5 were found in each of the three study groups: there were always significantly greater numbers of cells positive for IL-4 mRNA than for IL-5 mRNA (p < 0.05). There were, however, no significant differences between the three study groups. Cells expressing the genes for each of the three chemokines were identified and counted in each of the three study groups (Figure 7). As compared with that of the controls, there were significantly greater numbers of eotaxin-positive cells in the two groups of bronchitic subjects (p  0.01). The numbers of such cells in the two bronchitic groups were similar, and there was no difference associated with an exacerbation. There were no significant differences between the three groups with respect to the numbers of MCP-4-positive cells, albeit there was a tendency toward greater numbers in those with an exacerbation of CB. The only difference in chemokine expression associated with an exacerbation was the significant fivefold upregulation of RANTES by comparison with the level in the healthy controls (p < 0.01). The level of expression of RANTES in exacerbation was twofold greater than that seen in the bronchitic subjects with stable CB (p < 0.05). Compared with the controls, there was an increase in RANTES also seen in the stable CB group (p < 0.05). Epithelial expression of RANTES proved difficult to quantify because its expression was so intense. However, an attempt was made to quantify the area of the epithelium with RANTES mRNA positivity by use of an eyepiece graticule and application of a validated point-counting method (17). An average of 75.8% ± 10.5% (mean ± SEM) of the epithelium of the nonsmoking controls expressed RANTES, but it was very weakly expressed (a score of 1 on a scale of 1 to 3). By comparison, 100% of the epithelium in both of the smoker groups was positive for RANTES (p < 0.05), with 78% and 95% of the epithelium scoring for an intensity of 2 or 3 in the stable and exacerbation groups, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 7.   Counts of cells positive for eotaxin, MCP-4, and RANTES by in situ hybridization in bronchial biopsies of subjects with CB during an exacerbation (E) and in a stable phase (S), and in healthy nonsmoker controls (C). The results are expressed as the number of positive cells per mm2 subepithelial zone (Mann-Whitney U test: horizontal bar shows median values).

Correlations

There were no significant associations between the numbers of EG2-positive eosinophils and the numbers of cells expressing IL-4, IL-5, eotaxin, or MCP-4. However, there was a significant positive correlation between the numbers of EG2-positive cells and RANTES expression by subepithelial lymphomononuclear cells (Spearman's rank correlation: r = 0.51; p < 0.02). The correlation of log₂-transformed data for EG2-positive and RANTES-positive cells is shown in Figure 8 (Pearson's correlation: r = 0.69; p = 0.01). There were strong positive associations between IL-4 and IL-5 (r = 0.76; p < 0.001) and between IL-4 and eotaxin (r = 0.59; p < 0.01). IL-5 also showed statistically significant associations with eotaxin (r = 0.67; p < 0.001), MCP-4 (r = 0.61; p < 0.01), and RANTES (r = 0.53; p = 0.01). Eotaxin was significantly associated with both MCP-4 and RANTES, as were MCP-4 and RANTES with one another (r = 0.6; p < 0.01 for each correlation).

View larger version (11K): [in this window] [in a new window]   Figure 8.   Association between the numbers of EG2-positive eosinophils and subepithelial cells expressing RANTES per mm2 tissue (log2-transformed data and Pearson's correlation).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study shows that the mechanisms required to recruit eosinophils to the airway mucosa in exacerbations of bronchitis are present in subjects with CB. These bronchitic subjects had no prior history or pulmonary function findings suggestive of asthma. Eosinophils have occasionally been described previously in sputum (8, 18), BALF (9), and biopsy specimens (4) of subjects with CB in its stable phase, and infrequently in resection tissue obtained from smokers (19). In asthma, the infiltration of tissue by eosinophils, as an allergic reaction to allergen exposure, is part of a response in which memory T-helper cells regulate the specificity of the response, T-helper cells orchestrate a sequence of events via the production and secretion of interleukins, notably IL-4 and IL-5, and of eosinophil chemoattracants, which include eotaxin, MCP-4, and RANTES (20). These cytokines and chemokines are capable of multiple and interactive effects. IL-4 and IL-5 are required for tissue eosinophilia to occur. IL-5 encourages the terminal differentiation of eosinophils and stimulates their release from the bone marrow and their increase in peripheral blood (20). IL-4 induces upregulation of vascular cell adhesion molecule-1, whose ligand on eosinophils, very late antigen-4, encourages selective retention of eosinophils in subepithelial bronchial vessels (23); IL-4 may also have effects on lymphomononuclear cell recruitment (24). Importantly, the chemoattractant gradients that induce emigration and give direction to the movements of eosinophils within the tissues are provided by the secretion of chemokines produced by both inflammatory and structural cells, including airway surface epithelium and even bronchial smooth muscle (22, 25).

In the present study of nonasthmatic subjects with CB, we confirmed the previously reported increase of EG2-positive cells in exacerbations of bronchitis (11). In addition, we demonstrated with the molecular technique of in situ hybridization, that there is gene expression for IL-4 and also for IL-5 in CB, although the numbers of cells expressing these genes in stable bronchitis and in exacerbation are similar. The expression of IL-4 and IL-5 in smokers with mild CB is compatible with our recent demonstration of gene expression for these cytokines in the submucosal mucus-secreting glands of bronchitic smokers whose lungs had been resected for tumor (13) (Under review with American Journal of Respiratory and Critical Care Medicine; sent back to authors for revision). The novel identification in subjects with CB of gene expression for the eosinophil chemoattractants eotaxin, MCP-4, and RANTES emphasizes the similarities that can exist between the inflammation of a bronchitic exacerbation and asthma. As previously described in asthma, eotaxin mRNA is expressed by surface epithelium (22), and in the present study it was strongly expressed by subepithelial lymphomononuclear cells.

We also demonstrated for the first time the presence of gene expression for MCP-4 in CB: the distribution of MCP-4 mRNA was similar to that of mRNA for eotaxin. These chemokines have also been previously shown to be expressed constitutively in chronic sinusitis and rhinitis (26), and in experimental animal models of allergen challenge (25). Although eotaxin mRNA showed greater than normal expression in bronchitis, neither chemokine was upregulated significantly in association with an exacerbation, nor did the number of cells expressing either eotaxin or MCP-4 show a significant association with the increase in the number of tissue eosinophils. The most striking finding associated with an exacerbation was the upregulation of RANTES in both inflammatory and epithelial cells. We suggest that the significant positive relationship between RANTES and tissue eosinophilia found in our study best explains the mechanism for tissue eosinophilia in the population of bronchitic subjects that we studied.

It is probable that the factors initiating exacerbations were also responsible for the upregulation of RANTES. Although bacteria may also play a role (27), we speculate that viral infection is the most likely cause and inducer of epithelial RANTES expression during an exacerbation of bronchitis. Viruses such a rhinovirus (RV), respiratory syncytial virus (RSV), and influenza virus specifically target epithelial cells and have been shown to induce expression of intercellular adhesion molecule-1, via the induction of nuclear factor-kappa B (NF-B) (27, 28). With respect to chemoattractants, previous studies (31) have shown that RSV upregulates RANTES in vitro following RSV inoculation of both primary bronchial epithelial cells an the airway epithelial cell line BEAS 2B. During the logarithmic phase of infectious virus production, only RANTES, and not MCP-1, MCP-3, or MIP-1, was upregulated in an infection-dependent manner (31). Furthermore, the increase was found only in RSV-producing cells. To confirm these studies in vivo. RANTES has been measured in nasal lavage fluid obtained from children. RANTES was significantly increased in children with RSV infection as compared with a noninfected group and a group with infection in a stable phase. There is also evidence that both RANTES and IL-8 can be upregulated by inactive forms of RSV and can be detected along with MIP-1 in lower respiratory tract secretions in infants with RSV bronchiolitis (32). Additional evidence for the central role of RANTES production in response to viral infection comes from experimental studies of infection with influenza virus A (33). RANTES, IL-6, and IL-8 were released in significant amounts from the bronchial epithelial cell line NCI-H292, and RANTES mRNA and protein were detected in supernatants of cultured primary bronchial and nasal polyp epithelial cells at 24 to 72 h after influenza virus A infection. The supernatants of the virus-infected cells had potent chemotactic activity for eosinophils, which was attenuated after addition of anti-RANTES antibody (34). Sputum IL-6 has recently been shown to increase during exacerbations of patients with COPD, and increased sputum levels of IL-8, measured during the stable phase of disease, are associated with a relatively high frequency of exacerbation (35). It would also be instructive to measure RANTES in the sputum during such exacerbations.

We therefore propose that an exacerbation due to viral infection of airway surface epithelium in smokers with bronchitis induces a marked upregulation of epithelial RANTES. RANTES, acting through CCR3 receptors, is most responsible for the recruitment of tissue eosinophils in virally induced exacerbations of bronchitis, but also, via CCR3 and CCR4 receptors, recruits CD4⁺ memory cells (36), with consequent reduction of the normally high CD8⁺ to CD4⁺ cell ratio present in stable disease (Table 2). The prevailing balance between CD4⁺ and CD8⁺ cells at the time of an exacerbation may be critical. There is evidence that RANTES acts synergistically with CD8⁺ cytolytic cells to enhance FAS-ligand-dependent apoptosis of virally infected cells (37, 38). Thus, when CD8⁺ cells predominate, exacerbations and increased RANTES may promote CD8⁺ cell-mediated tissue damage. Increased frequency of viral exacerbations may thus destroy airway and alveolar tissue directly, encouraging the development of microscopic emphysema. In this way, repeated exacerbations due to viral infection may accelerate a decline in lung function in smokers whose CD8⁺ T-cell numbers are already increased (4). This would be particularly important in a subset of individuals with an already low genetically determined CD4⁺-to-CD8⁺ cell ratio (39) and whose smoking habit had elevated the numbers of their CD8⁺ cells even further (3). It would be reasonable to predict that CD8⁺ cytolytic activity in response to viral exposure would be more vigorous than normal in such individuals, and would result in enhanced tissue destruction (40, 41). This hypothesis requires further investigation both in vitro and in vivo.