INTRODUCTION

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Keywords: chronic obstructive pulmonary disease; Haemophilus influenzae; immune response; cell mediated immunity; outer membrane protein

Chronic obstructive pulmonary disease (COPD) is characterized by periodic exacerbations in which the patient experiences worsening of symptoms, including increased cough and sputum production, increased sputum purulence, and shortness of breath (1). COPD is the fourth leading cause of death, and it is estimated that ~ 16 million persons in the United States have COPD (2). The cost of care for patients with COPD is estimated to be $15 billion annually (2).

Nontypeable Haemophilus influenzae (NTHI) is an important human respiratory tract pathogen in children and adults. NTHI is the most common bacterial cause of exacerbations of COPD (1). In addition, NTHI is a common cause of recurrent episodes of otitis media during childhood. Therefore, there is significant interest in developing a vaccine to prevent infections caused by NTHI in children and adults.

P6 is a 16-kD lipoprotein that makes up ~ 1 to 5% of the total outer membrane proteins and is highly conserved among strains of NTHI both at the protein level and at the DNA level (1, 3, 4). The results of human (5-8) and animal studies (3, 9- 12) indicate that antibody to P6 is bactericidal and suggests that antibody to P6 provides protection from infection or colonization. Therefore, this potentially protective antigen has been proposed as a possible candidate for vaccine formulation.

We speculate that patients with COPD who have exacerbations due to NTHI fail to make a robust immune response to P6 during infection. The aim of the present study was to investigate the in vitro lymphocyte proliferative response to P6 and quantitate serum antibody levels to P6 in adults with COPD.

	METHODS

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COPD Study Clinic

Peripheral blood samples were collected from adults enrolled in a prospective study of COPD at the Buffalo Veterans Affairs Medical Center (VAMC). The protocol was approved by the Human Studies Subcommittee of the VA Western New York Healthcare System, and informed written consent was obtained from all subjects. Inclusion and exclusion criteria in the study were: (1) the patient must have chronic bronchitis as defined by American Thoracic Society (13); (2) patients with asthma, bronchiectasis, malignancies, or other immunocompromising illnesses were excluded; and (3) patients receiving  20 mg prednisone daily were excluded. Patients were seen in the study clinic monthly and at the time of exacerbations. At each clinic visit, a clinical evaluation was done by a study nurse. In addition, a study physician (T.F.M., S.S.) evaluated each patient who was suspected of having an exacerbation. The examiner asked six questions regarding overall well being, dyspnea, cough, sputum production, sputum viscosity, and sputum purulence. Emphasis was placed on grading symptoms relative to baseline. The patient was evaluated with regard to overall appearance, respiratory rate, wheezes, and crackles. On the basis of this information, a clinical determination of whether the patient was having an exacerbation was recorded. Each patient brought an expectorated sputum sample produced the morning of the clinic visit.

Bacteriologic Methods

Quantitative sputum cultures were performed by previously described methods (14). NTHI and other bacteria were identified by standard techniques (15).

Pulmonary Function Tests

Pulmonary function tests were performed using a spirometer at the Buffalo Veterans Affairs Medical Center. Forced expiratory volume in 1 second (FEV₁) and FEV₁% predicted were used as indicators of airway obstruction (16).

Purification of P6

A modification of a previously described method was used to purify P6 from NTHI (3, 12, 17)

Lymphocyte Proliferation Assay

Peripheral blood mononuclear cells (PBMC) were prepared as described previously (18). PBMCs (2.5 × 10⁵) diluted in RPMI 1640, supplemented with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 5 × 10⁵ M 2-mercaptoethanol, 100 U/ml penicillin, 100-µg/ml streptomycin, and 10% heat-inactivated normal human male serum (Sigma, St. Louis, MO) were added to wells of 96-well plates (Costar; Corning Incorporated, Corning, NY). Appropriate concentrations of the stimuli were added to cells in triplicate wells (100 µl/well). The stimuli were: P6 (2, 10, and 50 µg/ml), tetanus toxoid (TT) (1:8 dilution), phytahemagglutinin (PHA), or medium alone. The cells were cultured for 5, 6, and 7 days in 5% CO₂ at 37° C. Three days before harvest, PHA (5 µg/ml) was added to triplicate wells as mitogen controls. One µCi of [³H]-thymidine (NEN, Boston, MA) was added to all wells 18 hours before the end of the culture period. The cells were harvested on glass-fiber filters, and the radioactivity incorporated was measured by liquid scintillation counting (1,450 Trilux Microbeta; Wallac Inc., Gaithersburg, MD). The proliferative response is expressed as the stimulation index (SI). SI is calculated by dividing the counts per minute (cpm) of [³H]-thymidine incorporated in triplicate experimental wells by the cpm of simultaneous triplicate control wells, which contained medium with no antigen stimulus.

Immunoglobulin G to P6

Immunoglobulin G (IgG) to P6 in human serum was measured by ELISA by a modification of a previously described method (19).

Data Analysis

Log transformed responses to P6 were compared among three groups (healthy control subjects, COPD patients with exacerbations due to NTHI in the past year, and COPD patients with no exacerbations due to NTHI in the past year) using ANOVA (20). In the case of different means, pairwise comparisons were performed using Tukey adjustment for multiple comparisons (20). p Values of < 0.05 were considered statistically significant. SAS 8.2 was used for all computations.

	RESULTS

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Patient Demographics

Thirty-six patients who fulfilled the inclusion criteria were enrolled. Table 1 shows the demographics and clinical information of the study population. The population was restricted entirely to the male sex with one exception as a result of the population who receives their medical care at the Buffalo VAMC. The results of pulmonary function tests represent baseline values in each patient because tests were performed during clinically stable periods. Of note is the fact that 32 of 36 patients (88.9%) had moderate or severe airway obstruction. All patients had received routine TT immunization in the past and although the precise timing was not available, none had received TT within 6 months before this study. Table 1 also shows the demographics of the 12 healthy volunteers who were enrolled.

In Vitro Lymphocyte Proliferative Response to P6

The in vitro lymphocyte proliferative response to P6 was investigated along with TT as an unrelated antigen. PHA was used as a T cell mitogen. Preliminary studies were performed to establish the optimal concentration of each stimulus and incubation period. Based on these preliminary assays, 6 days of incubation, 50 µg/ml of P6, 1:8 dilution of TT, and 5 µg/ml of PHA were selected for subsequent studies. The proliferative response is presented as SI. Figure 1 shows the results of a subject in which proliferation increased with P6 concentration (top panel) and the results of a subject in which there was no proliferative response to P6 (bottom panel).

View larger version (24K): [in this window] [in a new window]   Figure 1.   In vitro lymphocyte proliferative response to P6 in two patients with COPD. The x-axes show the in vitro stimuli. "Medium" indicates the proliferative response in antigen-free culture medium. Bars show counts per minute (cpm) noted on the y-axis. The values on the tops of the bars are stimulation index (SI). SI is calculated as follows: cpm observed with stimulus/cpm observed with antigen-free medium.

Correlation of In Vitro Lymphocyte Proliferative Response with Exacerbations due to NTHI

The 36 patients with COPD were divided into two groups based on their history of exacerbations due to NTHI in the one year before assaying the lymphocyte proliferative response to P6. An exacerbation due to NTHI is said to have occurred when the patient had the clinical features of an exacerbation as outlined in METHODS and NTHI was isolated from a sputum culture. Of the 36 patients with COPD studied, 10 had an exacerbation due to NTHI in the year before being studied and 26 did not. Therefore, three groups were compared: (1) COPD patients with exacerbations due to NTHI in the prior one year (group N); (2) COPD patients with no exacerbation due to NTHI in the prior one year (group C); and (3) age- and sex-matched healthy control subjects (group H).

The mean values of cpm and SI of TT and P6 were compared among the three groups. ANOVA was performed and initial analysis of residuals indicated that response measures were not normally distributed within different groups of individuals. Natural logarithmic transformation was applied, and ANOVA was repeated for mean responses on a logarithmic scale (20). Nonparametric ANOVA (Kruskal-Wallis) was also performed, but results were consistent with ANOVA results on a log scale, so only the latter are reported. Analyses were performed separately for P6 and TT. Mean responses were significantly different for P6 (p value = 0.013 for cpm; p value = 0.017 for SI). No difference in response to TT was observed among the groups. Observed means (log scale) and standard errors are shown in Figure 2. These data indicate that COPD patients with exacerbations due to NTHI in the prior year had significantly lower lymphocyte proliferative responses to P6 compared with COPD patients with no exacerbations due to NTHI in the prior year and compared with age- and sex-matched healthy control subjects.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Results of lymphocyte proliferative assays for three groups: H, healthy control subjects; C, COPD patients with no exacerbation due to NTHI in the prior year; N, COPD patients with exacerbation due to NTHI in the prior year. y-Axis: Log of stimulation index that is calculated by dividing the cpm of [3H]-thymidine incorporated in triplicate experimental wells by the cpm of simultaneous triplicate control wells, which contained medium with no antigen stimulus. Points are log transformed means, and error bars are standard errors. P6, outer membrane protein P6; TT, tetanus toxoid.

In addition, pairwise comparisons were performed to detect differences between groups. Tukey adjustment was used for these comparisons (20). Patients with exacerbations due to NTHI in the prior year showed significantly lower lymphocyte proliferative response to P6 compared with healthy controls (p = 0.010 for cpm; p = 0.018 for SI). The NTHI exacerbation group showed significantly decreased SI for P6 compared with COPD patients with no exacerbation due to NTHI (p = 0.044) and a borderline difference in cpm (p = 0.078). No difference was observed between COPD patients with no exacerbation due to NTHI in the prior year and healthy control subjects.

To test the possibility that a decreased proliferative response to P6 was related to pulmonary function, the relationship between FEV₁% predicted and proliferative response to P6 in COPD patients was investigated. There was no statistically significant correlation between FEV₁% predicted and proliferative response to P6 (cpm; Spearmann rank correlation = 0.215, n = 36, p = 0.2027, SI; Spearmann rank correlation = 0.078, n = 36, p = 0.6435).

Four patients were receiving chronic low-dose prednisone, and six patients received a short course of therapy with prednisone (40 mg daily tapering to zero over eight days) within three months of having the lymphocyte proliferative assays. The results of assays for TT in these patients are statistically not different from the rest of the patients studied. We conclude that chronic low-dose prednisone therapy in four patients and recent short courses of prednisone as administered to six patients did not suppress lymphocyte proliferative responses.

Correlation between Exacerbations due to NTHI and the Level of IgG to P6 in Serum

To test the hypothesis that the level of IgG to P6 in serum is associated with exacerbations due to NTHI, the concentration of IgG to P6 was measured in the serum of patients with COPD. The concentration of IgG to P6 in the group who had an exacerbation due to NTHI in the past year was 4,464.4 ± 2,982.7 ng/ml compared with 2,361.2 ± 1,215.5 ng/ml in the group with no exacerbation due to NTHI (p = NS). Therefore, the level of serum IgG to P6 was unrelated to history of exacerbations due to NTHI.

	DISCUSSION

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The present study demonstrated that patients with COPD who had exacerbations due to NTHI in the prior 12 months showed a lower lymphocyte proliferative response to P6 compared with patients with COPD who had no exacerbations due to NTHI in the prior 12 months and compared with healthy control subjects. This decreased lymphocyte proliferative response was specific for P6 because no difference in proliferative response to an unrelated antigen, TT, was observed. These results support the hypothesis that patients with COPD who experience exacerbations due to NTHI do not respond adequately to P6 and suggest that the ability to make a proliferative response to P6 is associated with protection from exacerbations due to NTHI.

Several limitations in this study should be considered. Sputum cultures do not reliably establish the etiology of individual exacerbations (1). It is possible that some of the exacerbations identified in this study as being caused by NTHI actually had different etiologies and NTHI was simply present as a colonizing organism. In addition, NTHI may have caused some of the exacerbations that were identified as having other etiologies if, for example, NTHI was present in numbers, which were below our level of detection, or if NTHI was outgrown by other organisms in culture. However, no other practical measures exist to establish unequivocally the etiology of exacerbations in a large-scale prospective study. In this study, the isolation of NTHI from the sputum of a patient with clinical features of an exacerbation was defined as an exacerbation due to NTHI, realizing the limitation of this approach. The observation that a statistically significant difference in lymphocyte proliferative response to P6 was observed in two groups defined by sputum culture, when overlap may be present in the two groups due to limitations of sputum cultures, adds even further credence to the result. One might speculate that if a method to reliably identify the etiology of exacerbations existed, the difference in response to P6 between the two groups would be even greater.

One must consider the possibility that the purified P6 used to stimulate lymphocytes in culture was contaminated with endotoxin. Purification of P6 by the method used in this study yields P6, which contains less than 0.025 units of endotoxin per microgram of P6, making contamination with significant amounts of endotoxin unlikely (17). Furthermore, the observation of a complete absence of a proliferative response to purified P6 in lymphocytes from many of the patients argues against nonspecific stimulation of proliferation by endotoxin. The present study demonstrated that a proliferative response to P6 is associated with relative protection from exacerbations due to NTHI. Other antigens of NTHI may also stimulate potentially protective immune responses, but one can draw conclusions only about P6 in the present study. These results do not exclude the possibility that other antigens of NTHI may stimulate proliferative responses as well.

Antibodies to P6 are bactericidal and are protective in animal model systems (3, 5, 9, 11). Furthermore, a decreased level of serum IgG to P6 is observed in otitis-prone children compared with non-otitis-prone children, suggesting that antibodies to P6 are associated with protection from otitis media due to NTHI (6, 21). To determine whether higher levels of antibodies to P6 were associated with protection from exacerbation, serum IgG was quantitated in the patients with COPD. No difference in serum level of IgG to P6 was seen in the patients with exacerbation due to NTHI compared with the other patients with COPD.

This is the first study to identify a potentially protective immune response to NTHI in patients with COPD. The observation in the present study that a decreased lymphocyte proliferative response to P6 is associated with exacerbation due to NTHI parallels the observations of Kodama and coworkers (22), who showed that impaired proliferation of adenoidal lymphocytes by P6 was associated with otitis media. The association of cellular immune responses with protection from NTHI infections of the respiratory tract has been in made in animal models of NTHI infection as well. Mucosal immunization of rats with P6 induced enhanced pulmonary clearance of NTHI (10). Analysis of the immune response revealed that Peyer's patch immunization with P6 induced serum antibodies to P6 and also induced proliferation of lymphocytes in mesenteric lymph nodes. More recent work with this animal model has shown that CD8+ T cells play an essential role in pulmonary clearance of NTHI (23). Interestingly, CD8+ T cells are present in the airways of patients with COPD. Elucidating the role of cellular immune responses in protection from bacterial infection in COPD is an important focus of research in the area.

In conclusion, this study establishes that patients with COPD who had exacerbations due to NTHI showed a decreased lymphocyte proliferative response to P6 compared with patients with COPD with no exacerbation due to NTHI, and compared with healthy control subjects. These results suggest that the failure to recognize P6 as an important antigen, as noted in otitis-prone children (6), may contribute to repeated exacerbations of COPD. The association of a lymphocyte proliferative response to P6 with relative protection from exacerbations of COPD provides rationale for studies to further evaluate P6 as a potential vaccine antigen.