ABSTRACT

Objective: Whether blood eosinophils (bEOS) in chronic obstructive pulmonary disease (COPD) are associated with disease progression is a topic of debate. We aimed to evaluate whether the differential white blood cell (WBC) count, symptoms and treatment may predict lung function decline and exacerbations in COPD patients. Methods: We retrospectively examined stable COPD patients with a minimum follow-up of 3 years at our outpatients' clinic. We collected information about lung volumes (FEV1, FVC), the total and differential WBC count, acute exacerbations of COPD (number in the 12 months before the beginning of the study=AE-COPD-B, and during the follow-up=AE-COPD-F), smoking status and treatment. FEV1 decline and AE-COPD-F were described by using a generalized linear model and a 2-level random intercept negative binomial regression, respectively. The models included eosinophil and neutrophil counts as potential predictors and were adjusted by sex, age, smoking status, AE-COPD-B, treatment with bronchodilators and inhaled corticosteroids (ICS). Results: Sixty-eight patients were considered, 36 bEOS- (<170 cells/µL, the median value) and 32 bEOS+ (=170 cells/µL). ?FEV1 was higher in bEOS+ than bEOS- (34.86 mL/yr vs 4.49 mL/yr, p=0.029). After adjusting for potential confounders, the eosinophil count was positively (ß=19.4; CI 95% 2.8, 36.1; p=0.022) and ICS negatively (ß=-57.7; CI 95% -91.5,-23.9; p=0.001) associated with lung function decline. bEOS were not found to be associated with the number of AE-COPD-F. Conclusion: In stable COPD patients, a higher level of blood eosinophils (albeit in the normal range) predicts a greater FEV1 decline, while ICS are associated with a slower progression of airflow obstruction.

Keywords: COPD; Blood eosinophils; FEV1 decline; Exacerbations; Biomarkers. Correspondence to:

RESUMO

Objetivo: Discute-se se eosinófilos no sangue (EOS) na doença pulmonar obstrutiva crônica (DPOC) são associados à evolução da doença. O objetivo deste estudo foi avaliar se a contagem diferencial de células brancas do sangue (CBS), os sintomas e o tratamento podem prever o declínio da função pulmonar e as exacerbações em pacientes com DPOC. Métodos: Foram retrospectivamente examinados pacientes com DPOC estável submetidos a um monitoramento mínimo de três anos em nossas clínicas ambulatoriais. Coletaram-se informações sobre volumes pulmonares (VEF1 e CVF), contagens total e diferencial de CBS, exacerbações agudas de DPOC (número nos 12 meses anteriores ao início do estudo = EA-DPOC-B; e durante o monitoramento = EA-DPOC-F), status tabagístico e tratamento. Os declínios de VEF1 e EA-DPOC-F foram descritos empregando modelo linear generalizado e regressão binomial negativa com interceptação aleatória de nível 2, respectivamente. Os modelos incluíram contagens de eosinófilo e neutrófilo como potenciais preditores e foram ajustados de acordo com sexo, idade, status tabagístico, EA-DPOC-B, tratamento com broncodilatadores e corticosteroides inalados (CSI). Resultados: 68 pacientes foram considerados, dos quais 36 para EOS- (< 170 células/μL, valor da mediana) e 32 para EOS+ (≥ 170 células/μL). ∆VEF1 foi maior em EOS+ do que em EOS- (34,86 mL/ano vs 4,49 mL/ano, p = 0,029). Após o ajuste em relação aos potenciais confundidores, as contagens de eosinófilos (β = 19,4; CI 95% 2,8,36,1; p = 0,022) e CSI (β = -57,7; CI 95% -91,5,-23,9; p = 0,001) foram positivamente e negativamente associadas ao declínio da função pulmonar, respectivamente. Os EOS não foram associados ao número de EA-DPOC-F. Conclusão: Em pacientes com DPOC estável, o maior nível de EOS (embora em um intervalo regular) prevê um maior declínio de VEF1, enquanto os CSIs são associados a uma evolução mais lenta da obstrução do fluxo aéreo.

Palavras-chave: DPOC; Eosinófilos no sangue; Declínio de VEF1; Exacerbações; Biomarcadores.

 
INTRODUCTION
 
Lung function decline is one of the most important features of the natural history in patients with Chronic Obstructive Pulmonary Disease (COPD). It is estimated that mean FEV1 decline is about 20-40 mL/year and that there are two subgroups of patients, called “faster decliners” and “slower decliners”(1).
 
Systemic and pulmonary inflammation may contribute to this decline. The primary involvement of neutrophils, whose levels are higher in the airways of COPD patients and are positively associated with airflow obstruction is common knowledge.(2) Moreover, even if eosinophils are the predominant granulocyte population in subjects with asthma, recent evidence suggests the presence of eosinophilic inflammation in a subgroup of COPD patients.(3) Eosinophils in the airways correlate with their blood levels and this correlation, albeit weak, is significant.(4)
 
COPD patients have more circulating eosinophils than the non-COPD smoker population,(5) even though it is not clear whether this result is clinically relevant, as well as statistically significant. The blood eosinophil count can predict the risk of having an acute exacerbation. In the Copenhagen Study,(6) a blood eosinophil count with more than 340 cells/μL was associated with an almost 2 times higher risk of incurring severe exacerbations. In addition, the greatest risk of having an increased concentration of circulating eosinophils during exacerbations is related to a greater eosinophil count during the stability phase.(7)
 
The literature supports the notion that blood eosinophils could be a predictor of response to corticosteroid therapy in patients with acute exacerbations of COPD (AE-COPD).(7,8) During an acute exacerbation, COPD patients with an eosinophil count greater than 2% of the total white blood cells (WBC) have a low probability of relapse if they are treated with systemic corticosteroid.(9) However, there are no prospective randomized studies that examine the role of the blood eosinophil count as a predictor of a response therapy during stable COPD.(10) Only few studies have investigated the association between the progression of airflow obstruction and blood eosinophils, with discordant results.(11-16) Thus, we conducted a longitudinal retrospective study aimed to investigate whether factors such as clinical characteristics, laboratory data and treatment were associated with FEV1 decline over time, focusing our attention on the differential count of circulating leukocytes.
 
METHODS
 
We conducted a retrospective observational study using the last 5-year data in the database of the outpatient clinic at the University Hospital of Verona. A total of 239 patients with COPD were considered. The diagnosis was based on the GOLD criteria.(17) The study was approved by the Ethic Committee of our Institution.
 
We selected patients with the following characteristics: 1) a stable phase of the disease, i.e., no AE-COPD and no change in the treatment in the previous 3 months; 2) a blood eosinophil count lower than 450 cells/μL (upper normal limit of our laboratory); 3) a minimum follow-up of 3 years. We excluded patients with other lung diseases, such as lung cancer, interstitial lung diseases, asthma, pulmonary resection, and pulmonary infections. We also excluded patients with diseases related to atopy (such as rhinitis) and those with unacceptable spirometry.(18)
 
Demographic information, including sex, age, height, weight, and smoking habit were collected from the patient’s medical record. Medical history and prescribed therapies for the respiratory disease (LABA, Long-acting β2-Agonists; LAMA, Long-acting Muscarinic Antagonists; Inhaled Corticosteroids, ICS) were also recorded. A patient was on therapy with LABA, LAMA or ICS when he/she took these drugs (alone or in combination) at the beginning of the observation period.
 
The values of spirometry parameters (Forced Expiratory Volume in 1 second, FEV1 L; Forced Vital Capacity, FVC L) were registered. Lung function decline (mL/year) was expressed as the FEV1 value at the beginning minus the FEV1 value at the end of the observational period, divided by the years of follow-up (∆FEV1, mL/yr).
 
Information on exacerbations, dyspnea (modified MRC scale(19)) were also available. An AE-COPD was defined as the worsening of respiratory symptoms requiring antibiotic or oral corticosteroid treatment.(17) AE-COPD-F was the number of exacerbations at the control visits performed during the follow-up period. AE-COPD-B was the number of exacerbations in the 12-months preceding the beginning of the period of observation.
 
Statistical analysis
 
The data are shown as means ± SD, or as median with interquartile range (IQR) as appropriate. The t-test or the Wilcoxon-Mann-Whitney test were used to assess the differences of continuous variables between groups of patients, accordingly. The chi-square test was used for the comparison of data expressed as percentages.
 
A multivariate generalized linear model was used to evaluate the association between the eosinophil and neutrophil blood count (independent variables) and ∆FEV1 (mL/yr) (dependent variable), controlling for sex, age, smoking status (active smoker vs former smoker) (Model I). At a later stage, baseline treatment with bronchodilators, ICS and number of AE-COPD-B were included in the model (Model II).
 
The association between the eosinophil and neutrophil blood count (independent variables) and AE-COPD-F (dependent variable) was assessed by using a 2-level random intercept negative binomial regression, with level 1 units (visits) nested into level 2 units (patients), adjusting for sex and age at baseline (Model III). Another model was fitted to the data adding to the previous model as independent variables treatment with bronchodilators, treatment with ICS at baseline and number of AE-COPD-B (Model IV). The estimated coefficients of the negative binomial regression were expressed as incidence rate ratios (IRR) and 95% confidence intervals were provided. Model III and IV were not adjusted for smoking habits, because the number of current smokers at baseline was very limited (n=5 out of 68 COPD patients) and none of them reported exacerbations in the follow-up.
 
The goodness-of-fit was assessed using the Akaike information criterion (AIC)(20) and the Bayesian information criterion (BIC):(21) the best performances in predicting FEV1 decline and the rate of exacerbations during follow-up were identified by the lowest AIC and BIC, when comparing model I with model II and model III with model IV.
 
The p value less than 0.05 was statistically significant.
 
The statistical analysis and graphs were processed using STATA/IC 16.1.
 
RESULTS
 
Two-hundred-and-thirty-nine medical records of COPD patients were analyzed. A total of 171 patients were excluded (Figure 1): 73 patients did not have a follow-up of at least 3 years (among them 11 had died); the blood eosinophil count of 75 patients was missing; 23 were not in a stable phase of the disease.

 

 
In total, 68 patients were included in the analysis (age 71.1±6.6 years; 18 (26.5%) females). The mean follow-up was 50.1±16.3 months. The median blood eosinophil count was 170 (IQR:115-260) cells per μL. Based on this value, the patients were divided into two groups, one with less than (36 patients, bEOS- group) and one with at least (32 patients, bEOS+) 170 blood eosinophils/μL.
 
The demographic and clinical characteristics of the COPD patients in the study are reported in the Table 1. Age, BMI, and male/female distribution were similar in the two groups. Active smokers were present in the bEOS- group only, whereas the median value of pack year was higher, although not significantly, in the bEOS+ group. The number of AE-COPD-B tended to be higher in EOS+, even though the difference between the groups was not statistically significant. The mMRC dyspnea scale and the use of LAMAs, LABAs and ICSs was comparable in bEOS+ and bEOS-. At the beginning of the study, the mean values of FVC, FEV1 as well as leucocyte, neutrophil and lymphocyte counts were similar in the two groups.
 

 
When considering the whole sample, the mean ∆FEV1 and the mean FVC annual decline resulted in 18.78 ± 58.25 mL/year and 9.5±125 mL/year, respectively. The ∆FEV1 was significantly higher in bEOS+ (34.86±50.33) than bEOS- (4.49±61.69) (p=0.029). On the contrary, no statistically significant difference was found in terms of the FVC annual change between the two groups (p=0.393). During the follow up, the median number of exacerbations was not significantly different between EOS+ and EOS- (median:0, IQR:0-2 in EOS- and median:0, IQR:0-1 in EOS+; p=0.868).
 
After adjusting for sex, age and smoking habits at baseline, the eosinophils turned out to be significantly associated with ∆FEV1 either when only neutrophils and eosinophils were considered as possible determinants (β=16.9; CI 95% 2.0,31.8; p=0.026) (Model I, Table 2) or in the complete model (β=19.4; CI 95% 2.8,36.1; p=0.022) (Model II, Table 2). Figure 2 shows the observed and adjusted mean of ∆FEV1 by eosinophil count in the complete model: in the case of an increase in the eosinophil count of 100 cells per μL the FEV1 decline was expected to increase by 19.4 mL/y. ICS treatment was found to be associated with a reduced lung function decline (β=-57.7; CI 95% -91.5,-23.9; p=0.001) (Model II, Table 2).
 

 


 
After adjusting for sex and age at baseline, the neutrophil count was found to be significantly associated with the number of AE-COPD-F when only neutrophils and eosinophils were considered as possible determinants: the estimated rate ratio of exacerbations for an increase in the neutrophil count of 100 cells/μL was 1.03 (CI 95% 1.00,1.07; p=0.032), i.e. holding all other variables in the model constant, the increase in the neutrophil count seems to increase the expected number of exacerbations by 3%. When baseline treatment with bronchodilators and with ICS and AE-COPD-B were included in the model (Model IV, Table 3), neither the eosinophil nor the neutrophil counts were significantly associated with the rate of exacerbations, whereas baseline treatment with bronchodilators resulted in a significant increase in the rate of exacerbations (estimated IRR=15.02, CI 95% 1.65,136.67).

 

 
The complete models (Model II and Model IV) had the best fitness, as shown by the lowest AIC and BIC (Table 2, Table 3).
 
DISCUSSION
 
The results of this retrospective, longitudinal study in COPD patients show that blood eosinophils are associated with a faster decline in FEV1. On the contrary, the blood eosinophil count does not predict a higher risk of exacerbations.
 
The association between eosinophilic inflammation and lung function decline has been investigated in few studies with contrasting results. One study, in agreement with our results, found that an eosinophil blood count greater than 2% was associated with a faster decline of FEV1.(11) Also, in a more recent paper,(15) it was shown that a blood eosinophil count of ≥300 cells/µL was an independent risk factor for accelerated lung function decline in a large Canadian cohort of older adults from the general population. On the contrary, in the ECLIPSE study,(13) COPD patients with a blood eosinophil count higher or lower than 2% had a similar FEV1 decline in the 3-year follow-up. The HOKKAIDO COPD cohort study(14) reported that COPD patients with higher levels of circulating eosinophils maintained FEV1 levels that were substantially stable over a period of 5 years, whereas a higher emphysema level and a greater circulating neutrophil count were predictors for a more rapid FEV1 decline. Ethnic and environmental differences between our and the Japanese study may account for the contrasting results. More recently, one study(22) demonstrated that in COPD patients with a high blood eosinophil count (≥350 cells/μL), the exacerbations were associated with the subsequent acceleration of FEV1 decline. In addition, the association between eosinophils in the sputum and lung function decline is uncertain. One study(23) found that both the neutrophil and eosinophil count in the sputum were related to FEV1 decline in COPD. Another study(24) reported the association between eotaxin-1 (a chemokine inducing eosinophil activation) in lung lavage and a faster progression of the disease in COPD patients.(11)
 
We failed to find an association between the blood eosinophil count and the risk of exacerbation, in agreement with two group of authors,(25,26) but in contrast to other studies reporting a role of circulating eosinophils in predicting the onset of AE-COPD.(27-30) In the present study, the circulating neutrophil count was associated with exacerbations during the follow-up, although this was true only in the model in which sex and age, but not treatments, were considered as confounders. This result partially supports a recent study suggesting that a high blood neutrophil count may provide a useful indicator of the risk of exacerbations in COPD patients.(31)
 
Our retrospective study does not reach reliable conclusions regarding the possible effect of inhaled drugs on the decline of respiratory function and exacerbations. However, it is worth noting the finding of a negative association between ICS and FEV1 decline, which may suggest a slowing-down effect of inhaled corticosteroids on the disease progression. The positive association between treatment with bronchodilators and the rate of exacerbations during the follow-up has no clear explanation. In our study, almost all patients were treated with a combination of drugs and only a minority of them were being treated with either LAMA or LABA alone, so that we are unable to evaluate the separate effect of the two types of bronchodilators. While keeping in mind this limit, we hypothesize that patients with more frequent exacerbations at baseline, and consequently with a higher risk of developing exacerbations in the follow-up, are more frequently treated. The use of medications may also explain the lack of an association between AE-COPD-B and AE-COPD-F. We are aware that only prospective, randomized studies are necessary to definitively clarify this point.
 
The blood eosinophil count is influenced by several factors, such as diurnal, seasonal, and hormonal variations.(32,33) Moreover, fluctuation in the disease and treatment may increase this variability.(32) Therefore, it has been pointed out that a single estimation of the eosinophil count, as in the case of our study, would be unlikely to reflect the overall pattern of blood eosinophilia.(34) However, a group of authors have shown that the proportion of COPD patients with a stable eosinophil count in a six-month time interval is high (93%) for subjects with a mean age of 70 years.(5) The variability is even more limited for an absolute eosinophil count lower than 340 cells/μL, which is similar to that found in our patients.(5) Finally, in the present study the eosinophil count was measured in a stable phase of the disease, at the same hour of the day, after an overnight fasting, all factors that may have further reduced the count variability.
 
Our study has some limitations. Other than the ones connected to its retrospective observational design, the choice of selecting patients in a stable phase of disease and the numerous exclusion criteria prevent from extending the findings to COPD patients with different characteristics. Another limitation is the small number of the patients, that may explain the lack of some associations, such as between eosinophils and exacerbations. However, the sample size was large enough to support the hypothesis of a relationship between eosinophils and FEV1 decline.
 
We considered an exacerbation to be ongoing when the patient reported the use antibiotics or corticosteroid after the worsening of symptoms, regardless of admission to an emergency room or hospitalization. Therefore, our results cannot be applied to the severe exacerbations (i.e., requiring hospitalization) or to the mild ones (i.e., those producing only a slight variation of the usual treatment).
 
We acknowledge that FEV1 decline was calculated on only two points at the beginning and at the end of the observational period. A higher number of measurements might have provided a more reliable value of functional change. However, lung function tests were performed during a stable phase of disease, adopting the criteria of the ATS technical statement,(16) a fact that enabled us to obtain high quality data.
 
In conclusion, our results suggest that in patients with stable COPD, a higher level of blood eosinophils (albeit in the normal range) predicts a greater decline in FEV1 over time, but not a higher number of exacerbations. Furthermore, ICS therapy seems to attenuate the progression of airflow obstruction, even though prospective randomized studies are needed to confirm these results.
 
AUTHOR CONTRIBUTIONS
 
MF, MP, LC: conception and design of the study. MF, MP, LC, VE, FS, SDM, LGDC, EC: acquisition, analysis, or interpretation of data. MF, MP, LC, LGDC, EC: drafting the work or revising it critically for important intellectual content. MF, LC, EC: approval of the final version of the manuscript.
 
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