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Published ahead of print on February 22, 2007, doi:10.1164/rccm.200602-161OC
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American Journal of Respiratory and Critical Care Medicine Vol 175. pp. 1125-1133, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200602-161OC

Original Article

Plasma Cells and IL-4 in Chronic Bronchitis and Chronic Obstructive Pulmonary Disease

Jie Zhu1, Yusheng Qiu1, Monica Valobra2, Shengyang Qiu1,3, Swati Majumdar1, Dean Matin1, Virginia De Rose2 and Peter K. Jeffery1

1 Lung Pathology Unit, Department of Gene Therapy, Imperial College London at the Royal Brompton Hospital, London, United Kingdom; 2 Department of Clinical and Biological Sciences, University of Torino, Torino, Italy; and 3 King's College, University of Cambridge, Cambridge, United Kingdom

Correspondence and requests for reprints should be addressed to Peter K. Jeffery, F.R.C.Path., D.Sc., Lung Pathology Unit, Department of Gene Therapy, Imperial College, London, at the Royal Brompton Hospital, Sydney St., London SW3 6NP, UK. E-mail: p.jeffery{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Airway wall inflammation, IL-4, and mucus hypersecretion are thought to be associated.

Objectives: To quantify bronchial inflammatory cells in smokers with chronic bronchitis (CB) with and without airflow obstruction (AO), determining the cells expressing IL-4 and IL-5 and their association with submucosal gland mucin.

Methods: We applied immunohistochemistry to identify, and double-labeling to colocalize, IL-4 and IL-5 to distinct inflammatory cells in resected bronchi from (1) 11 asymptomatic smokers (AS), (2) 11 smokers with CB, and (3) 10 smokers with CB and AO.

Measurements and Main Results: There were greater numbers of mucosal and gland CD45+ leukocytes in CB (epithelium, 673/mm2; subepithelium, 698/mm2; gland, 517/mm2) than in AS (331, 237, and 178/mm2, respectively; p < 0.01 for all) or CB + AO (375, 243, and 215/mm2, respectively; p < 0.05 for all). There were greater numbers of subepithelial and submucosal gland plasma cells in CB (subepithelium, 110/mm2; gland, 213/mm2) compared with AS (38 and 41/mm2, respectively; p < 0.01 for both), and more subepithelial mast cells in CB (204/mm2) than in AS (65/mm2; p < 0.01) or CB + AO (115/mm2; p < 0.01). In CB, the percentage of gland occupied by mucin was positively correlated with the numbers of interstitial CD45+ cells, plasma cells, and IL-4 protein+ cells. In CB, 69 and 62% of gland-associated plasma cells expressed IL-4 and IL-5, respectively.

Conclusions: Inflammatory cells are increased in bronchial submucosal glands and mucosa of large airways in smokers with CB. Gland-associated plasma cells express IL-4, and these likely promote mucus hypersecretion.

Key Words: chronic bronchitis • chronic obstructive pulmonary disease • IL-4 • inflammation • plasma cells



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Airway inflammation, hypertrophy of submucosal mucus-secreting glands, and goblet cell hyperplasia are abnormalities found in the airways of smokers with chronic bronchitis (CB) and chronic obstructive pulmonary disease. Airway wall inflammation and mucus hypersecretion are associated.

What This Study Adds to the Field
Inflammatory cells are increased in bronchial submucosal glands and mucosa of large airways in smokers with CB. Gland-associated plasma cells express IL-4 and these likely promote mucus hypersecretion.

 
Chronic bronchitis (CB) is defined on the basis of chronic cough and sputum due to mucus hypersecretion, a risk factor in chronic obstructive pulmonary disease (COPD) for accelerated lung function decline, increased hospitalization, and increased mortality (1–3). Histologically, airway inflammation, hypertrophy of submucosal mucus-secreting glands, and goblet cell hyperplasia are abnormalities found in the airways of smokers with CB (4, 5). Although an increase in gland size (i.e., hypertrophy) is one factor contributing to increased airway mucus (4), airway inflammation (including the presence of lymphocytes and plasma cells) rather than gland size per se is the feature most closely associated with expectoration of sputum (5–7). Hypersecretion of mucus-secreting glands may arise subsequent to chronic inflammation via the release of selected proinflammatory cytokines that include IL-4, a multifunctional cytokine that can facilitate the differentiation of both mucus-secreting tissue (8, 9) and B lymphocytes. B lymphocytes are the precursors of plasma cells. Recent studies of both small and large airways have highlighted the presence of B lymphocytes, the increasing number of which has been associated with increasing severity of COPD (10, 11).

We have already shown that there are increased numbers of inflammatory cells expressing IL-4 mRNA in the glands of smokers with CB, as compared with asymptomatic smokers (AS) and smokers with CB and coexisting airflow obstruction (AO) (12). However, it is not known which cell type(s) is mainly responsible for the production of IL-4 and other regulatory cytokines in CB.

Due to the known increase and predominance of CD8+ cells in COPD (13, 14), we had originally hypothesized that the type 2, cytotoxic subset of CD8 was the origin of IL-4. However, we found that no CD8+ cell colocalized with IL-4 (or IL-5) in the smokers with CB or CB + AO (12), thus leaving unresolved the question of the cellular origin of these two regulatory cytokines.

In the present study, we tested the hypothesis that airway wall inflammation, IL-4, and mucus hypersecretion are associated, with the rationale that IL-4 has a modulatory role on hypersecretion of mucus in CB. We considered, on the basis of prior information, that CD4+ lymphocytes or mast cells would be the most likely cell populations to express the genes for IL-4 (and IL-5). Accordingly, we counted inflammatory cells of distinct phenotype in defined tissue compartments of large airways (i.e., bronchi) and performed colocalization studies. Our focus here has been the cellular origin of the IL-4 associated with bronchial submucosal glands and, to be able to sample this tissue compartment, we have examined whole transverse sections of bronchi taken from grossly normal tissue in lungs surgically resected for tumor. Some of the findings of the present study have been published in abstract form (15).


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Three groups of smokers, 50–80 years of age (> 20 packs/yr, 31 current smokers and 1 ex-smoker), coming to surgery for resection of localized lung tumor were included. They were the same patients used in our previously reported study (12). Group I: AS without atopy or asthma (n = 11) with normal lung function (FEV1 > 80% of predicted; FEV1/FVC > 70%). Group II: smokers with CB (n = 11), as defined by the U.K. Medical Research Council and American Thoracic Society criteria for CB (1, 2), but with normal lung function (FEV1 > 80% of predicted; FEV1/FVC > 70%). Group III: smokers with CB, but additionally having persistent irreversible AO (i.e., CB + AO; n = 10) with FEV1 less than 80% of predicted (range, 43–77%) and FEV1/FVC less than 70% (range, 51–68%), satisfying the criteria for AO published by GOLD (Global Initiative for Chronic Obstructive Lung Disease) (16). For further details, see Table E1 and SUBJECTS in the online supplement.

Tissue Processing
Only grossly normal lobar and segmental bronchi were used for the present analyses (see the online supplement).

Histochemistry
A modified Unna-Pappenheim methyl green–pyronin stain (17) was used initially to demonstrate plasma cells present in airway tissue and to confirm the immunostaining specificity for plasma cells. Immunostaining was used for the counts presented herein (see the online supplement). The combined Alcian blue (AB)–periodic acid Schiff (PAS) stain, controlled for specificity with diastase digestion (17), was used to identify acidic and neutral mucopolysaccharides in both bronchial glandular and surface epithelium (see Figure E3).

Immunohistochemistry
Immunohistochemistry was used to label CD45+ inflammatory cells, plasma cells, mast cells, CD4+, CD8+, CD20+, and IL-4 protein+ cells (see the online supplement).

Preparation of cRNA Probes
Riboprobes for IL-4, IL-5, and tumor necrosis factor (TNF)-{alpha} cRNA were used for in situ hybridization procedures applied to tissue sections according to a well-tried and published method (12, 18) (see the online supplement).

Immunohistochemistry and Nonisotopic In Situ Hybridization Double Staining
The cells were immunostained for the purpose of identifying their phenotype (e.g., plasma cells, mast cells, CD4+ T lymphocytes), and hybridized to colocalize and identify their gene expression for IL-4 or -5, or TNF-{alpha}, the last included as a positive control (see the online supplement).

Immunofluorescence Double Staining
Indirect immunofluorescence double staining was used to detect whether plasma cells, CD20+ B lymphocytes, or CD68+ monocytes/macrophages also contained IL-4 protein. This technique was also used to confirm that mouse anti-human monoclonal antibodies (mAbs) to plasma cell–specific antigens did not cross react with CD20+ B lymphocytes (see the online supplement).

Quantification and Statistics
For details of the methods of quantification, variability, and how we expressed the data, see the online supplement. As the data of cell counts were nonnormally distributed, the Kruskal-Wallis test, and then the Mann-Whitney U test, where applicable, were applied to compare differences between the subject groups as well as to compare cell counts between different phenotypes in the same airway compartment. The data for percentage of AB-PAS+ epithelial areas were normally distributed and expressed as mean (± SEM). Analysis of variance and subsequent Student's t tests were applied to test for differences between the three groups. The Spearman rank correlation test was used to determine the associations between the numbers of plasma, mast, or CD4+ cells and IL-4 or IL-5 mRNA+ cells, and also correlations between percentages of AB-PAS+ epithelial areas and the numbers of CD45+, plasma, or IL-4 protein+ cells. A p value less than 0.05 was accepted as indicating a statistically significant difference, with a Bonferroni correction (p < 0.02) for multiple comparisons (i.e., 3). The Bland-Altman test was used to assess the level of agreement between counts of plasma cells identified either by immunohistochemistry or methyl green–pyronin stain.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient demography is shown in Table E1. The three groups of smokers were similar in respect to age and smoking history. By comparison with the AS and CB groups, the CB + AO group had a significantly lower value for FEV1% predicted (p < 0.0001 compared with either the AS or CB groups) and FEV1/FVC (p < 0.0001 vs. AS or CB).

Distribution of Inflammatory Cells
CD45+ leukocytes were identified throughout the depth of the airway wall, in the epithelium, subepithelium, and also in the interstitium (connective tissue) lying between the secretory acini and surrounding the gland ducts of submucosal mucus-secreting glands. Plasma cells, identified by immunohistochemistry, were present in the subepithelium (Figure 1A) and there were relatively large numbers found in association with submucosal glands (Figure 1B); this was especially apparent in the CB group. The plasma cells were mainly located in loose connective tissue nearby or within small blood vessels. Tryptase+ mast cells were located throughout the airway wall, but were most numerous in the subepithelium, with relatively few associated with submucosal glands. CD4+ cells were also located mainly in subepithelium and submucosal glands. We have observed that, unlike plasma cells, CD20+ B lymphocytes are mainly seen in lymphoid aggregates. In contrast, they are relatively spare in both subepithelial and glandular areas. The distributions of CD8+ cells, IL-4 mRNA+/protein+, and IL-5 mRNA+ cells in the bronchial wall have been reported previously (12, 19). Briefly, there was an abundance of strongly IL-4 protein+ mononuclear inflammatory cells located in the subepithelium, and this was particularly striking in the interacinar mucus-secreting gland connective tissue. An irrelevant antibody, mouse IgG1, did not stain these tissues (Figure 1C).


Figure 1
Figure 1
Figure 1
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  		Figure 1. (A–C) Light microscopic image of immunostained bronchial mucosa from a patient with chronic bronchitis (CB). Plasma cells (stained brown with the avidin–biotin complex [ABC] technique) are particularly abundant in (A) the subepithelium zone and (B) the interstitial tissue located between the submucosal gland acini. (C) There are no positive cells in negative control stained after using an irrelevant antibody. Scale bar = 20 µm.

 
Quantification
The results of counts for CD45+ cells (indicative of the total number of leukocytes), plasma cells, mast cells, and CD4+ T-cell subsets for the three patient groups are shown in Figures 2–4 and given in Table E2.


Figure 2
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  		Figure 2. Counts for CD45+ cells in distinct zones of the airway wall of asymptomatic (AS) smokers, smokers with chronic bronchitis (CB), and CB with airflow obstruction (AO) (CB + AO). The results are expressed as the number of positive cells/mm2 tissue: individual patient values and medians for the groups. The Mann-Whitney U test was used to compare the differences between subject groups. E = epithelium; G = gland; M = muscle; R = remainder of the airway wall; S = subepithelial zone.

 
CD45+ cells.
The CB-alone group had significantly more CD45+ cells in the epithelial, subepithelial, and glandular compartments than those of either the AS or CB + AO groups (p < 0.001 for each case) (Figure 2).

Plasma cells.
Compared with the AS group, the patients with CB alone had significantly greater numbers of plasma cells in both glandular and subepithelial compartments (p < 0.01 for both), whereas the patients with CB + AO had only a trend toward an increase in plasma cells in the glandular compartment (p = 0.06) and slightly higher numbers of subepithelial plasma cells (Figure 3). There were also significantly higher numbers of plasma cells in glandular compartment of CB than in the CB + AO group (p < 0.02), with a similar trend toward an increase in the subepithelial zone (p = 0.067) (Figure 3). There were greater numbers of plasma cells in the submucosal glands than in the subepithelium in the patients with CB (p < 0.02) and also in those with CB + AO (p < 0.05). This difference in tissue distribution was not apparent in the AS group (Figure 3).


Figure 3
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  		Figure 3. Counts for immunopositive plasma cells in distinct zones of the airway wall of asymptomatic smokers (AS) and smokers with chronic bronchitis (CB) and those smokers with CB + airflow obstruction (AO). The results are expressed as the number of positive cells/mm2 tissue: individual patient values and medians for the groups. The Mann-Whitney U test was used for the comparison of differences between distinct zones and groups. G = gland; R = remainder of the airway wall; S = subepithelial zone.

 
The specificity of the mouse anti-human plasma cell–specific antigen mAbs to detect plasma cells was confirmed by comparing counts of immunopositive plasma cells and methyl green–pyronin+ cells in both subepithelial and glandular areas in two serial sections from each of 11 patients with CB.

The Bland-Altman analysis showed that the means of the differences in subepithelial and glandular counts are close to zero: –0.91 and –0.64, respectively. The 95% limits of agreement were from –14.6 to 12.8 for subepithelium and from –11.5 to 10.2 for mucus-secreting gland. The differences lay entirely within 95% limits of agreement, indicating good agreement between the two staining methods. In addition, there was a strong positive correlation between numbers of immunopositive plasma cells and pyroninophilic plasma cells in both subepithelial and glandular compartments ({rho} = 0.93, p < 0.001; and {rho} = 0.91, p < 0.01, respectively), indicating good specificity of the antibody compared with the more traditional histochemical approach to identifying these cells.

Mast cells and CD4+ T cells.
There were significantly greater numbers of mast cells in CB patients than in the asymptomatic and CB + AO groups but only in the subepithelium (p < 0.05) (Figure 4). The number of mast cells in the subepithelium was significantly greater than that in epithelium, mucus-secreting glands, airway smooth muscle or the remaining compartment in each of three subject groups (p < 0.01). There were significantly more CD4+ cells in both subepithelial and glandular compartments than that in epithelium, muscle and remainder of the airway wall and this difference was so in each of the three subject groups (p < 0.01) (see Table E2). However, there were no significant differences between the groups in respect of CD4+ T cells (Table E2).


Figure 4
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  		Figure 4. Counts for tryptase+ mast cells in distinct zones of the airway wall of asymptomatic smokers (AS), smokers with chronic bronchitis (CB), and smokers with CB + airflow obstruction (AO). The results are expressed as the number of positive cells/mm2 tissue: individual patient values and medians for the groups. The Mann-Whitney U test was used to compare the differences between distinct tissue zones and subject groups. E = epithelium; G = gland; M = bronchial smooth muscle; S = subepithelial zone.

 
Compartmental Distribution
Comparisons for the number of distinct phenotypes of inflammatory cells infiltrating the different tissue compartments are shown in Table E2. The predominant epithelial population was of CD8+ cells in each of the three groups. The number of each type, in rank order expressed as median/mm2 epithelial area, was: CD8+ cells > mast cells > CD4+ cells > plasma cells. In the subepithelial area of the CB group, mast cells predominated relative to plasma, CD8+, and CD4+ cells. The rank order was: mast cells > plasma cells = CD8+ cells > CD4+ cells. The predominant population of inflammatory cells in bronchial smooth muscle was the mast cells in each of the three groups. The rank order was: mast cells > CD4 > CD8 > plasma cells. By contrast, plasma cells were the predominant cell type associated with submucosal glands as compared with mast cells, CD4+, and CD8+ cells in the CB and CB + AO groups. Gland-associated plasma cells were more than two- to six-times more numerous than mast cells, CD4+ and CD8+ cells (see Table E2).

The fold increases in number of CD45+ inflammatory cells, plasma cells, mast cells, CD4+, and CD8+ lymphocytes in CB as compared with those of AS and CB + AO are summarized in Table E3.

Association between Plasma Cells and Cells Expressing IL-4 and IL-5
The number of plasma cells showed a strong positive correlation with the number of cells expressing IL-4 mRNA, this apparent in both mucus-secreting glands and subepithelial compartments of patients with CB alone and, to a lesser extent, in those with CB + AO ({rho} = 0.82, p < 0.001; and {rho} = 0.66, p < 0.01, respectively) (Figure 5). The number of cells expressing IL-4 protein also showed a strong correlation with the number of plasma cells infiltrating both the gland and subepithelial areas in CB ({rho} = 0.72, p < 0.02; and {rho} = 0.74, p < 0.02, respectively) (Figure 6). There was also a significant association between the number of plasma cells and IL-5 mRNA+ cells in both the gland and subepithelial areas in CB and CB + AO groups ({rho} = 0.55, p = 0.01; and {rho} = 0.50, p < 0.03, respectively). In contrast, there were no significant correlations found between the number of mast or CD4+ cells and either IL-4 mRNA+ or IL-5 mRNA+ cells.


Figure 5
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  		Figure 5. Correlations between the numbers of plasma cells and numbers of IL-4 mRNA+ cells per mm2 of subepithelium and submucosal glands in the chronic bronchitis and chronic bronchitis + airflow obstruction groups (Spearman rank correlation; n = 21; subepithelium: {rho} = 0.66; p < 0.01; submucosal glands: {rho} = 0.82; p < 0.001).

 

Figure 6
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  		Figure 6. Correlations between the numbers of plasma cells and numbers of IL-4 protein+ cells per mm2 of subepithelium and submucosal glands in the chronic bronchitis group (Spearman rank correlation; n = 11; subepithelium: {rho} = 0.74; p < 0.02; submucosal glands: {rho} = 0.72; p < 0.02).

 
Colocalization of IL-4 or IL-5 mRNA to Inflammatory Cells
In smokers with CB, simultaneous double labeling was applied in order to phenotype and count the number of each inflammatory cell coexpressing IL-4 or IL-5 mRNA—this to identify the cellular source of these regulatory cytokines in CB. Double labeling of mast cells with TNF-{alpha} (i.e., mast cells/TNF-{alpha} mRNA) was used as a positive control. The colocalization and the counts of double-labeled IL-4 or IL-5 mRNA+ cells and plasma cells, tryptase+ mast cells, and CD4+ lymphocytes are shown in Figures 7A–7C and Table 1. Totals of 67 and 62% of plasma cells expressed IL-4 and IL-5 mRNA, respectively. A total of 6,694 IL-4 mRNA+ cells were counted, of which 40% were identified as plasma cells. Of 4,093 IL-5 mRNA+ cells, 34% were plasma cells; by comparison, only 2.3 and 1.2% of CD4+ cells expressed IL-4 and IL-5 mRNA, respectively, whereas only 2% of IL-4 and 3% of IL-5 mRNA+ cells were CD4+ cells. Similarly, only 1% of tryptase+ mast cells expressed IL-4 and IL-5 mRNA, respectively, and about 2% of IL-4 and IL-5 mRNA+ cells were tryptase+ mast cells, respectively (Table 1). In contrast, 13% of TNF-{alpha} mRNA+ cells were found to be mast cells, and 6% of mast cells expressed TNF-{alpha} mRNA.


Figure 7
Figure 7
Figure 7
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  		Figure 7. Double immunohistochemistry/nonisotopic in situ hybridization staining of bronchial mucosa from a patient with chronic bronchitis showing the plasma cells stained brown (with the avidin–biotin complex [ABC] technique) and mRNA+ cells as dark blue. The plasma cells coexpressing (A) IL-4 and (B) IL-5 mRNA are stained black-brown (arrows) located in a subepithelial zone. (C) There is no positivity for both immuno- and mRNA using irrelevant antibodies and IL-4 sense probes. Scale bar = 20 µm.

 

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  		TABLE 1. NUMBERS OF EACH INFLAMMATORY CELL TYPE IDENTIFIED BY THEIR IMMUNOPOSITIVITY AND THOSE OF EACH TYPE COEXPRESSING THE IL-4 OR IL-5 GENE IN AIRWAYS OF SMOKERS WITH CHRONIC BRONCHITIS

 
That plasma cells in CB produced and stored IL-4 protein was confirmed by double immunofluorescence staining with anti–plasma cell–specific antigen (red) and anti–IL-4 protein (green). Plasma cells located in the subepithelium and interstitium between mucus-secreting gland acini showed intense red tetramethylrhodamine isothiocyanate [TRITC] cytoplasmic fluorescence (Figure 8A). Infiltration of tissue by IL-4–producing cells was evident by their apple green cytoplasmic fluorescence: this was present in the same areas shown to be rich in plasma cells (Figure 8B). Cells double stained for both plasma cell–specific antigen (red) and IL-4 protein (green) were demonstrated clearly by their combined (yellow) cytoplasmic fluorescence (Figure 8C).


Figure 8
Figure 8
Figure 8
Figure 8
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  		Figure 8. Double immunofluorescence staining for identification of colocalization of plasma cells and IL-4 protein in the interstitium between submucosal gland acini of bronchi from a patient with chronic bronchitis. (A) Immunopositive plasma cells are illustrated with red fluorescence alone. (B) IL-4 protein positivity stained by anti-human IL-4 antibody and shown by the green fluorescein isothiocyanate fluorescence alone. (C) Coexpression of plasma cells with IL-4 protein are seen fluorescing yellow (i.e., a mixture of the two colors). (D) The negative control section stained with irrelevant antibody shows an absence of the red and green signals. Scale bar = 20 µm. Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI) blue.

 
Double immunofluorescence staining also identified some CD20+ B lymphocytes and CD68+ monocytes/macrophages that coexpressed IL-4 protein (see Figures E1A–E1F). However, CD20+ B lymphocytes did not stain with the anti–plasma cell–specific antigen mAb (see Figure E2), thus further supporting the specificity of the anti–plasma cell antigen mAb and also the immunohistochemical distinctiveness of these two inflammatory cell phenotypes. As a negative control for the immunostaining, there was an absence of either red or green signal using the irrelevant antibody instead of either the anti–plasma cell or anti–IL-4 primary antibody, respectively. Neither red nor green signal were seen when the two primary antibodies were replaced with an irrelevant antibody and the procedure continued with both the secondary antibodies (Figure 8D).

Glandular Mucin: Association with Inflammation and IL-4
To investigate the potential for a functional role in the production of airway mucus by infiltrating IL-4+ inflammatory cells, the relationships between their number and the percentage area of gland or surface epithelium containing AB-PAS+ mucin were analyzed. AS had 55.5 ± 4.7% (mean ± SEM) of their gland epithelium occupied by mucin, whereas smokers with CB had 74.6 ± 2.6%, and those with CB + AO had 70.6 ± 3.3% (p < 0.002 and 0.02, respectively) (Figure 9). The percentage of glandular epithelium occupied by mucin significantly correlated with the numbers of CD45+ inflammatory cells ({rho} = 0.81; p < 0.01; Figure 10A), interstitial plasma cells ({rho} = 0.81; p < 0.02; Figure 10B), and IL-4 protein+ (not mRNA+) inflammatory cells ({rho} = 0.61; p < 0.04; Figure 10C). By contrast, no association was found with mast, CD4+, or CD8+ cells. There were no significant differences in the percentages of surface epithelium occupied by mucin (see Figure 9), and no significant associations between surface epithelial mucin and either the numbers of subepithelial plasma cells or IL-4+ cells. Moreover, no such significant correlations were found in CB + AO groups.


Figure 9
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  		Figure 9. Results of point counts for percentages of surface and gland epithelium occupied by Alcian blue–periodic acid Schiff (AB-PAS)–positive mucin in bronchial mucosa of asymptomatic smokers (AS), smokers with chronic bronchitis (CB), and smokers with CB + airflow obstruction (AO). The results are expressed as individual patient values and means for the groups. The Student's t test was used to compare the differences between subject groups. GE = glandular epithelium; SE = surface epithelium.

 

Figure 10
Figure 10
Figure 10
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  		Figure 10. Correlations between percentages of gland epithelium occupied by Alcian blue–periodic acid Schiff–positive (AB-PAS+) mucin and (A) the numbers of CD45+ cells ({rho} = 0.81; p < 0.01), (B) plasma cells ({rho} = 0.64; p < 0.02), and (C) IL-4 protein+ cells in the smokers with chronic bronchitis–alone group ({rho} = 0.61; p < 0.04). Spearman rank correlation; n = 11.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrate a significantly greater number of airway wall inflammatory cells, glandular plasma cells, and subepithelial mast cells in smokers with nonobstructive CB (CB alone) as compared with AS and those with CB and AO (CB + AO). We show that a high percentage of gland-associated plasma cells express the genes for IL-4 and IL-5. The mucus-secreting glands of smokers with CB alone, and also those with CB + AO, have a significantly greater proportion of their mucus-producing glands occupied by mucin. However, only in CB alone does increased gland mucin correlate with the numbers of glandular inflammatory cells, plasma cells, and cells expressing IL-4 protein. In CB alone, CD8+ cells are the predominant cell type in surface epithelium, mast and plasma cells predominate in the subepithelium and submucosal glands, respectively, and mast cells predominate in bronchial smooth muscle.

Plasma Cells
The pioneering study of Soutar (20) reported the distribution and counts of plasma cells containing immunoglobulins in bronchi obtained post mortem. Compared with normal nonsmoking control subjects, plasma cells were plentiful among bronchial submucosal glands in the "incidental bronchitis" smokers, who had not suffered dyspnea and died of nonrespiratory causes. However, compared with the normal control subjects and those with "incidental" CB, the numbers of IgA plasma cells were significantly lower in patients with "fatal bronchitis" who had severe irreversible AO and had died as the result of a severe exacerbation. Soutar speculated that a preexisting bronchial IgA deficiency may have predisposed the patients with fatal bronchitis to infection, exacerbations, and the development of severe, eventually fatal, disease. We have advanced this early study by applying and validating, for the first time, an anti–plasma cell–specific antigen mAb, and, although our patient groups differed in several respects, our results are broadly supportive of these early findings.

As plasma cells are considered to be derived via maturation of B lymphocytes, it is reasonable to hypothesize that the numbers of plasma cells we report would mirror the reported increased numbers of B lymphocytes in COPD of increasing severity (11). In this recent report, the more severely obstructed (GOLD stage 3) patients had significantly greater numbers of B lymphocytes than those of GOLD stage 2 (11). Thus, at first appearance, our data and conclusions and those of Gosman and colleagues may appear discordant; however, our study designs and primary aims differ in two major respects: (1) we focused on plasma cells that, although related to B lymphocytes, are phenotypically distinct. Plasma cells are recognized by the acquisition of cytoplasmic immunoglobulins and loss of such common B-lymphocyte antigens as CD20 (21). The anti–plasma cell–specific antigen mAb used in the present study identifies a terminal B-lymphocyte–restricted antigen, termed HM1.24, selectively expressed on plasma cells (22). Using immunofluorescence double staining, we have here confirmed in resected bronchial tissues that the anti–plasma cell–specific mAb that we applied does not cross react with the CD20 antigen. The data of both studies show clear differences between our counts of subepithelial plasma cells, which were much higher (median, 55/mm2) than the counts of Gosman and colleagues' subepithelial CD20+ B lymphocytes (median, 8/mm2). Additionally, we have observed that the tissue distribution of plasma cells is quite different to that of CD20+ B lymphocytes. Whereas B lymphocytes are mainly seen in lymphoid aggregates, plasma cells are predominantly distributed in subepithelium and glandular areas around and within small blood vessels. These morphologic appearances imply that submucosal plasma cells were more likely to have originated, via the circulation, from bone marrow or lymphatic organs than to have been generated locally from resident CD20+ mature B lymphocytes in the inflamed airway. To support this possibility, previous studies in mice (23, 24) have demonstrated that plasma cells and plasmablasts were located in bone marrow and lymphatic organs from which they were recruited into inflamed tissues via the circulation. (2) Smokers with CB alone (GOLD stage 0) were not included for previous comparisons (11). If we omit the data for our CB-alone patients, comparison of the patients with GOLD stage 2 COPD of both studies with their respective control subjects shows both the increase of mucosal B lymphocytes in the study by Gosman and colleagues and also the trend toward increasing numbers of subepithelial and gland plasma cells in the present study. Thus, although the numbers of plasma cells and B lymphocytes do not match, the trends in the data are similar when clinically similar patients are compared.

Thus, the novelty of our data lies in the specific immunoidentification and quantification of distinct plasma cells in an airway wall compartment rich in mucus-secreting glands.

Mucin, Inflammation, Plasma Cells, and IL-4
We demonstrate both in CB alone and in CB + AO that there are significantly greater areas of their gland secretary acini occupied by mucin than in AS. In contrast, there are no differences between the smoking groups in the contents of epithelial mucin, a finding in keeping with a previous report (25). Thus, in agreement with early suggestions, it is most likely that the source of mucus and the reason that these patients have chronic expectoration lie in the glands rather than the surface epithelium.

The correlation between increases of gland mucin and the total numbers of inflammatory cells, plasma cells, and IL-4 protein+ inflammatory cells infiltrating gland interstitium in CB lends support to our initial hypothesis. IL-4 can induce glycoprotein synthesis, promote mucous cell hyperplasia, and, consequently, the secretion of airway mucus (8, 9). We show that over 60% of plasma cells express IL-4 mRNA. Our double immunostaining for IL-4 protein confirms the cytoplasmic presence of IL-4 in plasma cells. Although mycelia plasma cells have been reported to produce IL-6 (26), this is, to our knowledge, the first report of the localization of IL-4 and IL-5 to plasma cells, and indicates a novel role of plasma cells in the production of mucus. However, among our IL-4 mRNA+ cells, only about 40% were plasma cells, indicating that other cells also express IL-4. Consistent with others (27, 28), our double immunofluorescence staining confirms that bronchial mucosal CD20+ B lymphocytes and CD68+ monocytes/macrophages can also contribute to the production of IL-4.

We find interesting the absence of a correlation between numbers of CD45+ inflammatory cells, plasma cells or IL-4+ inflammatory cells and the glandular mucin that also increased in the group with CB + AO. This suggests that the evolution of COPD is not linear in terms of inflammation and there are likely to be other mechanism(s) that continue to drive mucin production as AO develops. It is known that transforming growth factor-beta1 and its receptor is can down-regulate mucin production after bacterial infection (29) whereas, in the bronchial glands of smokers with COPD, there is decreased expression of transforming growth factor-beta1 type II receptor. Thus, this alteration may also be associated with bronchial gland enlargement as AO worsens (30).

Mast Cells
There are conflicting reports as to alterations in numbers of mast cells in CB and COPD (7, 31). In the present study, we have demonstrated that, compared with AS or those with CB + AO, smokers with CB alone do have a statistically greater number of mast cells, but only in the subepithelial compartment. In respect to airway smooth muscle, our data are in agreement with previous findings in COPD (32): we found no greater numbers of mast cells in smokers with CB alone and CB + AO as compared with our asymptomatic control subjects.

Inflammation and AO
The increased total number of inflammatory cells that we found in patients with CB alone was greater than that in patients with CB + AO. However, several previous studies of the airway mucosa and parenchyma in COPD have shown that worsening of airflow is associated with increasing numbers of inflammatory cells. In general, these studies have shown these associations only for selected types of inflammatory cell (e.g., the CD8+ T cell, B lymphocyte, macrophage) (10, 11, 13, 14, 33–36), and not for the plasma cells that have formed the focus of the present study. In our early biopsy study of the bronchial mucosa (13), there was no difference between CB alone and CB + COPD. The apparent discrepancy between our present results in resected tissue and previous findings in biopsies may, in part, be explained by the opportunity we have had to count these cells in the entire depth of the subepithelium. Gamble and colleagues have demonstrated that, to achieve a representative count, when possible, greater volumes and depths of tissue need to be sampled than previously achieved, and surgically resected airway tissue favors this (37, 38). This aspect has been previously discussed in respect to the counts of CD8+ cells in resected bronchial tissue (12). Moreover, one biopsy study of very severe disease in COPD has also described a relative reduction of bronchial mucosal lymphocytes (39), and a recent study of airway secretions showed reduced spontaneous neutrophil migration and chemotaxis when COPD is in its most severe stages (40).

Thus these results draw attention not only to the florid nature of the inflammatory response in CB, but also to what appears to be a selective blunting of the inflammatory response as AO increases in severity. The underlying reasons for these reductions of inflammation in severe COPD are unclear, but we support the likely inclusion of genetic factors (16) and environmental influences, such as long-term oxidative stress with resultant damage to cellular components, including membrane lipids, proteins, and DNA (41).

Finally, we acknowledge that, to investigate more specifically the effect of airflow limitation alone on airway inflammation, the ideal design for our study would have to include a group of smokers with airflow limitation but without a current history of CB. However, this was not our primary goal and, in keeping with other published studies, such a group was not available to us from any source (6, 7, 11, 13, 14, 20, 35, 36).

In conclusion, we demonstrate that the numbers of CD45+ inflammatory cells, plasma cells, and mast cells are greater in the large airway walls of smokers with CB than in AS or smokers with CB + AO. The frequent presence of IL-4–expressing plasma cells associated with the increased mucin present in the bronchial submucosal glands of smokers with CB supports a modulator effect of plasma cells and associated IL-4 on hypersecretion of mucus in CB.


   Acknowledgments
 
The authors thank Professors R.A. Pauwels, and J.C. Kips from the University of Ghent, Belgium; Dr. A. Oliva, Torino, Italy, for obtaining lung tissue; and Mr. E. Inett for assistance with confocal microscopy.


   FOOTNOTES
 
Supported by the British Lung Foundation, London, United Kingdom, and the University of Ghent, Ghent, Belgium.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200602-161OC on February 22, 2007

Conflict of Interest Statement: J.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Y.Q. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.Q. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. V.D.R. has been reimbursed by pharmaceutical companies (Boehringer Ingelheim, Dompe, GlaxoSmithKline [GSK]) for attending conferences and received honoraria for speaking at scientific meetings sponsored by pharmaceutical companies (AstraZeneca, SIGMA-TAU); her institution has received an unrestricted educational grant from Boehringer Ingelheim. P.K.J. has been reimbursed by GSK, AstraZeneca and Merck, Sharpe & Dohme (Merck) for attending conferences, and has participated as a paid speaker in scientific meetings or courses organized and financed by various pharmaceutical companies (including GSK, AstraZeneca, Merck, and Boehringer Ingelheim); P.K.J. has served as a consultant to GSK and Novartis, and has recently or currently received research grants from GSK, Merck, and AstraZeneca, the first and last include grants for multicenter clinical trials.

Received in original form February 3, 2006; accepted in final form February 22, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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