Chronic inflammation plays a major role in the

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1 The Relation Between Peripheral Blood Leukocyte Counts and Respiratory Symptoms, Atopy, Lung Function, and Airway Responsiveness in Adults* Sarah A. Lewis, PhD; Ian D. Pavord, MD; John R. Stringer, FIBMS; Alan J. Knox, MD; Scott T. Weiss, MD, FCCP; John R. Britton, MD, MSc Study objectives: Eosinophils and neutrophils play major roles, respectively, in the pathogenesis of asthma and COPD, and it is well recognized that levels of these cells in peripheral blood are increased in relation to their pulmonary involvement. However, the relation between peripheral blood cell counts of the other major leukocyte groups and these lung diseases or markers of allergy or airflow obstruction is less clear. We have therefore investigated the association between peripheral blood levels of eosinophils, neutrophils, basophils, monocytes, and lymphocytes and the occurrence of chronic respiratory symptoms, atopy, lung function, and bronchial hyperresponsiveness, and the modifying effect of age, in adults. Design: A cross-sectional general population study. Setting: Data on > 2,000 British adults, who originally participated in a study of diet and lung health, were analyzed using multiple linear and logistic regression to adjust for potential confounders, including age, sex, and smoking history. Results: We found that, like eosinophils, the peripheral basophil count was increased in relation to asthma and associated symptoms, and to airway hyperreactivity and increased total IgE, but differed from eosinophils in that basophils were unrelated to atopy. Monocytes were predominantly associated with symptoms indicative of obstructive airway disease, in similar relation to neutrophils, but both of these leukocyte counts were also increased in asthma patients in older age groups. Lymphocyte counts were unrelated to any objective or subjective marker of disease. Conclusions: If peripheral blood cell counts reflect pulmonary involvement of these leukocyte groups, basophils and monocytes may play a distinct role in the pathogenesis of allergic and nonallergic respiratory disease. (CHEST 2001; 119: ) Key words: asthma; atopy; basophils; COPD; eosinophils; IgE; lung function; lymphocytes; monocytes; neutrophils *From the Division of Respiratory Medicine (Drs. Lewis, Knox, and Britton, and Mr. Stringer), City Hospital, University of Nottingham, Nottingham, UK; Department of Respiratory Medicine (Dr. Pavord), Glenfield Hospital, Leicester, UK; and Channing Laboratory (Dr. Weiss), Department of Medicine, Brigham and Women s Hospital, Boston, MA. Dr. Lewis is funded by the Medical Research Council. Manuscript received March 14, 2000; revision accepted July 17, Correspondence to: Sarah A. Lewis, PhD, Division of Respiratory Medicine, Clinical Sciences Building, City Hospital, Nottingham, UK NG5 1PB Chronic inflammation plays a major role in the pathogenesis of both asthma and COPD, and it has long been recognized that many of the inflammatory cells that are involved in mediating these processes reach the lung via the blood. In particular, the neutrophil has been implicated in the pathogenesis of COPD, being recruited into the lung in response to cigarette smoke inhalation and being responsible for the release of protease and oxidantproducing enzymes that cause bronchitis and emphysema. 1 4 It is also recognized that T lymphocytes and macrophages are involved in the pathogenesis of COPD. 5 7 In asthma, the dominant peripheral blood leukocyte found in the airway is the eosinophil, 8,9 and the degree of eosinophil involvement is correlated with various markers of severity including symptoms, 10 loss of lung function, 11,12 and with airway hyperresponsiveness. 10 In both COPD and asthma, the presence, respectively, of neutrophils and eosinophils in the airway is broadly reflected by increased numbers of these cells in peripheral blood However, the peripheral blood also contains cells from other major leukocyte groups potentially involved in these diseases, particularly basophils, monocytes, and lymphocytes, but the relation between these peripheral blood cell counts and either asthma or COPD is less clearly understood. We have therefore carried out a descrip- CHEST / 119 / 1/ JANUARY,

2 tive analysis of the relation between peripheral blood cell counts of neutrophils, eosinophils, basophils, monocytes, and lymphocytes and chronic respiratory symptoms, lung function, allergen skin sensitization, IgE, and airway hyperresponsiveness measured in a random adult population sample who participated in a study to investigate the relation between diet and lung disease. Materials and Methods This study is based on data from adults aged 18 to 70 years from a Local Authority Area of Nottingham who participated in a survey of diet and lung disease in 1991, and have been described in detail elsewhere. 15,16 In summary, 7,016 adults 18 years old were identified in a systematic sample from a random start point in local electoral registers and were invited to take part in the survey. Of these, 2,633 (representing between 48% and 59% of the initial sample subsequently ascertained to be alive and resident in the study area at the time of the study) provided data on respiratory symptoms and smoking history using a modified International Union against Tuberculosis and Lung Disease questionnaire. 17 These subjects also provided measurements of allergen skin sensitivity to Dermatophagoides pteronyssinus, grass pollen, and cat fur solutions (Bencard; Brentford, Middlesex, UK) by standard skin prick testing with histamine and saline solution controls, and of FEV 1 and FVC using a dry bellows spirometer (Vitalograph; Buckingham, UK). All subjects were invited to provide a venous blood sample, and those with a resting FEV 1 of 1.5 L and 60% of predicted were asked to undergo measurement of airway responsiveness to methacholine by the method of Yan et al. 18 A blood sample for peripheral blood cell counting was provided by 2,369 subjects (90%); of these, airway responsiveness was measured in 2,173 subjects (92%). Differential WBC counts were estimated in all samples within 24 h using a cell analyzer (Technicon H6000; Technicon; Basingstoke, UK). By this technique, total WBCs and differential neutrophils, lymphocytes, eosinophils, and combined monocytes/ basophils are counted optically on the peroxidase channel, hydrodynamically focused in a sheath stream flow cell. Basophils were counted optically after staining with Alcian blue dye, also hydrodynamically focused in a sheath stream flow cell. Nonsmokers were defined as those with a cumulative smoking history of less than one cigarette per day for 1 year, and ex-smokers were defined as those who had not smoked within 1 month of the study. Current smokers were categorized into three increasing levels of current cigarette consumption, using the ranges 0 to 10 cigarettes per day, 11 to 19 cigarettes per day, and 20 cigarettes per day; current and ex-smokers were categorized according to pack-years of consumption (1 pack-year 20 cigarettes per day for 1 year), from 1 to 10 pack-years, 11 to 20 pack-years, 21 to 30 pack-years, and 31 pack-years. Atopy was defined as a mean allergen skin-wheal diameter of at least 1 mm more than the saline solution control response. Airway responsiveness was computed for each subject as the two-point slope between FEV 1 after saline solution and after the maximum dose of methacholine given, using the method described by O Connor et al, 19 with augmentation of all values by 1.67 before log transformation to achieve normality. The presence of current respiratory symptoms was defined in terms of questionnairereported persistent cough (cough during the day or night on most days for at least 3 months each year), persistent phlegm (morning phlegm on most days for at least 3 months each year), and the occurrence within the past year of wheezing (wheezing or whistling in the chest), nocturnal dyspnea (woken by shortness of breath), and exercise-induced dyspnea (shortness of breath following strenuous exercise). A lifetime history of asthma (labeled ever asthma ) was defined as self-reported asthma that had been confirmed by a doctor. The subgroup of these subjects who reported having had an attack of asthma within the last 12 months were defined as having current asthma. Eosinophil, neutrophil, basophil, monocyte, and lymphocyte counts were log transformed to normality and then compared between sexes, smoking categories, and 10-year age bands by analysis of variance. Leukocyte counts were then divided into quintiles, and the odds of self-reported wheezing, nocturnal dyspnea, exercise-induced dyspnea, persistent cough and phlegm, asthma, and atopy were computed for each leukocyte quintile relative to the lowest, using multiple logistic regression, with adjustment for sex, age group, and smoking history. Statistical significance was assessed using a test for trend in odds across leukocyte quintiles. The modifying effects of age and atopic status on the association between leukocyte counts and current asthma were examined by augmenting the logistic regression model with the appropriate interaction terms, with age regrouped into three classes (18 to 35 years, 36 to 50 years, and 51 to 70 years) to improve statistical power. FEV 1 was compared between leukocyte quintiles in terms of standardized residuals after adjustment of FEV 1 for age, sex, height, and smoking history, and the two-point dose-response slope to methacholine (airway responsiveness) for age, sex, smoking history, and baseline FEV 1, FEV 1 percent predicted, and FEV 1 /FVC, since these three characteristics were previously shown to be independently related to a binary measure of airway responsiveness in this population. 15 All analyses were carried out using statistical software (SPSS version 8; SPSS; Chicago, IL) Statistical significance was inferred at a probability of 5% in all tests. Results The age, sex, and smoking characteristics of the 2,369 subjects who provided blood samples are shown in Table 1 and did not differ appreciably from those of the initial sample of 2,633 participants, described elsewhere. 16 All individual leukocyte counts were approximately log-normally distributed, with geometric means (interquartile ranges) for eosinophils of 151 cells/ L (range, 100 to 230); neutrophils, 3,637 cells/ L (range 3,000 to 4,450); basophils, 64 cells/ L (range, 50 to 90); monocytes, 438 cells/ L (range, 360 to 540); and lymphocytes, 2,104 cells/ L (range, 1,750 to 2,570). Each leukocyte count was increased in smokers in relation to current cigarette consumption, but with little difference between ex-smokers and nonsmokers for most leukocytes, and weaker relationships with the number of pack-years of smoking than with current cigarette consumption (Fig 1). Basophil and monocyte counts increased with age while lymphocyte counts decreased; there was no clear relation between age and neutrophil or eosinophil counts. Eosinophil, basophil, and monocyte counts were significantly higher and lymphocyte counts significantly lower in men. 106 Clinical Investigations

3 Table 1 Number, Sex, and Smoking Status by Age Group of Subjects Who Provided a Serum Sample Age Group, yr Variables Total Subjects, No. (%) 365 (15) 504 (21) 615 (26) 480 (20) 405 (17) 2,369 Women 184 (50) 283 (56) 303 (49) 237 (49) 184 (45) 1,191 (50) Smoking status Never smoked 252 (69) 277 (55) 270 (44) 225 (47) 137 (34) 1,161 (49) Ex-smoker 29 (8) 125 (25) 177 (29) 150 (31) 180 (44) 661 (28) Current smoker 84 (23) 102 (20) 168 (27) 105 (22) 88 (22) 547 (23) Eosinophils After adjustment for sex, age, and smoking history, there was a significant independent increase in the odds of wheezing, nocturnal dyspnea, exerciseinduced dyspnea, persistent cough, and current and lifetime history of asthma with eosinophil counts (Fig 2, 3), the relation with asthma being similar in atopic and nonatopic subjects (interaction p 0.2). The relation between eosinophil count and current asthma was also similar within all age groups (interaction p 0.5). The significance of the linear trend in each of these analyses was predominantly due to a more marked effect in the highest quintile. A high eosinophil count was also an independent predictor of atopy (Fig 3), reduced FEV 1, increased airway reactivity, and increased IgE (Fig 4) before and after exclusion of individuals with asthma from the analysis. Neutrophils Increasing neutrophil counts were independently associated with increased odds of nocturnal dyspnea, exercise-induced dyspnea, persistent phlegm, and current and lifetime history of asthma (Fig 2, 3), and with reduced FEV 1 (Fig 4). There was no evidence of a relation between neutrophil counts and either atopy or airway responsiveness (Fig 3, 4). The relation between neutrophil count and current asthma did not differ with atopic status, but was stronger in older individuals (interaction p 0.02; Fig 5) Basophils Increasing basophil counts were associated with increased odds of wheezing, nocturnal dyspnea, and with diagnosed asthma (Fig 2, 3), with impairment of FEV 1 and increased airway responsiveness and IgE (Fig 4). The relation with asthma was similar across age groups (interaction p 0.9) and within atopic and nonatopic asthmatics (interaction p 0.14). There was no evidence of an association between basophil counts and atopy (Fig 3). Monocytes Increasing monocyte counts were associated with increased odds of nocturnal dyspnea, persistent cough, and persistent phlegm (Fig 2), and with reduced FEV 1 (Fig 4). There was no evidence of association between monocyte counts and atopy or asthma, but there was a strong interaction such that monocyte counts were significantly negatively associated with asthma in the youngest age group but positively associated in the oldest age group (interaction p 0.01; Fig 5). There also tended to be a stronger positive association in nonatopic than in atopic individuals, but the interaction did not reach statistical significance (interaction p 0.1). Lymphocytes Lymphocyte counts in peripheral blood were not related to any respiratory symptom or diagnosis. Discussion In this analysis of peripheral blood leukocyte counts in a random and representative adult population, we have observed the previously well-documented associations between asthma, IgE, and blood eosinophil levels, and have shown that peripheral blood eosinophil counts were increased in relation to symptoms of wheeze, cough, and dyspnea, and to objective measures of allergen skin sensitization, reduced FEV 1, airway hyperresponsiveness, and IgE levels. These findings reiterate the central role of eosinophils in allergic asthma, 14,20,21 and also in the occurrence of chronic respiratory symptoms and airflow obstruction. 11,20,22 24 In our study, a relationship between eosinophil count and persistent cough persisted even after excluding asthmatics and those with a history of smoking, and after adjusting for airway reactivity and atopy (data not shown). These findings therefore suggest that eosinophilia may also be associated with nonspecific chronic cough. CHEST / 119 / 1/ JANUARY,

4 Figure 1. Geometric means ( 1 SE) of eosinophil, neutrophil, basophil, monocyte, and leukocyte counts by sex, age, group, and smoking status. *Implies significance at the 5% level. Our findings for neutrophils also confirm previous reports of the association between blood levels of these cells and the occurrence of chronic respiratory symptoms and reduction of FEV 1, 13 but without evidence of an independent association with atopy or airway hyperresponsiveness. This suggests that the major difference between the roles of eosinophils and neutrophils in these diseases is that neutrophils, although clearly involved in the pathogenesis of airflow obstruction, act through mechanisms relatively uninvolved with atopy. Neutrophils were significantly associated with diagnosed asthma, but we found an effect modification by age such that this association became apparent only with increasing age. This effect was not due to confounding by steroid therapy, since the relation of asthma and neutrophil counts in patients 50 years old (and the relation of eosinophils and basophils to asthma at all 108 Clinical Investigations

5 Figure 2. Odds ratios (95% confidence intervals) for wheeze, nocturnal dyspnea, exercise-induced dyspnea, persistent cough, and persistent phlegm with increasing quintiles of leukocyte counts relative to the lowest quintile, with adjustment for sex, age group, and smoking status. The lower cutoff points of eosinophil quintiles were 1, 100, 140, 190, and 260; basophils were 10, 60, 70, 80 and 110; neutrophils were 280, 2,870, 3,400, 3,930, and 4,630; monocytes were 40, 350, 420, 490, and 580; and lymphocytes were 340, 1,680, 2,000, 2,300, and 2,670. *Implies significance at the 5% level. OR odds ratio; CI confidence interval. CHEST / 119 / 1/ JANUARY,

6 Figure 3. Odds ratios (95% confidence intervals) for ever asthma, current asthma, and atopy with increasing quintiles of leukocyte counts relative to the lowest quintile, with adjustment for sex, age group. and smoking status. Quintile cutoffs are as in Figure 2. *Implies significance at the 5% level. See Figure 2 legend for abbreviations. ages) was apparent, if weaker, when analysis was restricted to the 61% who were currently receiving no form of oral or inhaled steroid. One likely explanation for the observed association between asthma and neutrophils is modification of the diagnostic labeling of asthma and asthma symptoms over time or between age groups; otherwise, this effect modification by age may reflect changes in the underlying disease pathology, with neutrophilic inflammation becoming a more important component in asthma 110 Clinical Investigations

7 Figure 4. Mean of the standardized residuals (Std Res) of FEV 1, airway reactivity, and logtransformed IgE by increasing quintiles of leukocyte counts. Airway reactivity is expressed as the log-transformed two-point slope. Quintile cutoffs are as in Figure 2. *Implies significance at the 5% level. See Figure 2 legend for abbreviations. pathogenesis in older age. It was also apparent that the nature of the relationship with asthma differed between the two leukocytes, such that asthma and hyperreactivity were largely associated with very high eosinophil counts while the risk of asthma increased with neutrophil counts in a more linear fashion. This suggestion of a difference in the doseresponse relationship may also reflect differences in the pathobiological importance of these two cells in the expression of asthma. Neutrophil counts would be expected to be associated with hyperresponsiveness independently from atopy because airflow obstruction is also associated with hyperresponsiveness, but we did not observe any such relation in this CHEST / 119 / 1/ JANUARY,

8 Figure 5. Odds ratios (95% confidence intervals) for current asthma with increasing quintiles of leukocyte counts relative to the lowest quintile by age group and with adjustment for sex and smoking status. Quintile cutoffs are as in Figure 2. *Implies significance at the 5% level. See Figure 2 legend for abbreviations. study, probably because our estimates of airway hyperresponsiveness were adjusted for baseline lung function. 15 Although the relationships between asthma and COPD and peripheral blood neutrophil and eosinophil counts are well documented, and the increase that we have observed in total and differential leukocyte counts in current smokers has been reported elsewhere, 25 there has been relatively little interest in the relation between other peripheral blood cells and markers of respiratory morbidity. We have therefore taken the opportunity to explore these relationships for peripheral blood basophil, monocyte, and lymphocyte counts. The method used to count different leukocytes in our blood samples is well validated and uses peroxidase and alcian blue stains to distinguish cell types on the grounds of both size and staining characteristics. To practical purposes, there is very little misclassification of cell types by this method, and particularly little between eosinophils and basophils, or between monocytes and neutrophils. We found that in contrast to the established involvement of lymphocyte subsets in airway pathology, 26 there was little evidence of any association of note between peripheral blood lymphocyte counts and any objective or subjective marker of disease, with the exception of smoking pattern. We were unable in this study to look at lymphocyte subsets, and so our data add nothing to previous evidence linking lymphocyte subsets with COPD. 27 However, in relation to basophils and monocytes, we obtained evidence of a differential pattern of relationships with disease markers. We found that monocytes were related to symptoms and to reduced FEV 1 in a similar pattern to neutrophils, consistent with a role similar to neutrophils in the pathogenesis of COPD. Peripheral blood monocytes are the precursors of tissue macrophages, 28 which are clearly implicated in the pathogenesis of COPD in conjunction with neutrophils. 5,7 Some degree of overlap in the relationships with disease markers is therefore consistent with these recognized roles in pathogenesis, though to our knowledge these associations have not previously been described in a general and representative population sample. We also found that basophils fol- 112 Clinical Investigations

9 lowed a similar, though less extensive, pattern of association with disease markers to eosinophils. Basophils were related to smoking, increasing age, wheeze, nocturnal dyspnea, and impairment of FEV 1, and as such were broadly associated with the characteristics of COPD, but were also associated with a diagnosis of asthma, hyperresponsiveness, and IgE level, though not with allergen skin sensitization. The role of the basophil in human lung disease is not clearly understood. The basophil is thought to differentiate from the same stem cell as the mast cell in response to interleukin 3, granulocyte macrophage colony-stimulating factor, and interleukin 5, and both cell lines are characterized by the expression of IgE receptors and the development of histaminecontaining granules that move to the cell surface and discharge their contents in response to crosslinking of IgE. 29 The releasability of basophil histamine appears to be closely correlated with the presence and severity of asthma, 30,31 consistent with a role of direct relevance to the pathogenesis of asthma. Basophils have been implicated in end-organ allergic responses in experimental models, and in the airway in exacerbation of asthma. 35 It is therefore reasonable to conclude that elevated peripheral blood levels of basophils in allergic disease reflect the involvement of these cells in the disease process, rather than a paraphenomenon driven by crossreaction to factors directed toward eosinophil stimulation and recruitment. Our study is a purely observational description of associations between markers of disease occurrence and severity and cell counts in peripheral blood, and it would be inappropriate to infer from these observations that any peripheral blood cell is necessarily actively involved in the pathogenesis of the diseases we have investigated. The degree of overlap with disease markers seen in our study also indicates that measuring peripheral differential WBC counts is unlikely to be useful clinically to diagnose or to distinguish between asthma and COPD. However, our studies suggest that in addition to well-documented roles for neutrophils and eosinophils in the etiology of COPD and asthma, monocytes and basophils may also be involved in these diseases; the roles of these cells in the pathogenesis and in the differential expression of allergic and nonallergic disease merit further investigation. References 1 MacNee W, Wiggs B, Belzberg AS, et al. The effect of cigarette smoking on neutrophil kinetics in human lungs. N Engl J Med 1989; 321: Sibille Y, Reynolds HY. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am Rev Respir Dis 1990; 141: McGowan SE, Hunninghake GW. Neutrophils and emphysema. N Engl J Med 1989; 321: Paakko P, Kirby M, Dubois RM, et al. Activated neutrophils secrete stored 1 -antitrypsin. Am J Respir Crit Care Med 1996; 154: Distefano A, Turato G, Maestrelli P, et al. Airflow limitation in chronic bronchitis is associated with T-lymphocyte and macrophage infiltration of the bronchial mucosa. 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10 22 Griffin E, Hakansson L, Formgren H, et al. Blood eosinophil number and activity in relation to lung function in patients with asthma and with eosinophilia. J Allergy Clin Immunol 1991; 87: McGarvey L, Forsythe P, Heaney L, et al. Bronchoalveolar lavage findings in patients with chronic nonproductive cough. Eur Respir J 1999; 13: Gibson P, Dolovich J, Denburg J, et al. Chronic cough: eosinophilic bronchitis without asthma. Lancet 1989; 2: Schwartz J, Weiss ST. Cigarette smoking and peripheral blood leukocyte differentials. Ann Epidemiol 1994; 4: Agostini C, Chilosi M, Zambello R, et al. Pulmonary immune cells in health and disease: lymphocytes. Eur Respir J 1993; 6: Dejong JW, VanderBeltGritter B, Koter G, et al. Peripheral blood lymphocyte cell subsets in subjects with chronic obstructive pulmonary disease: association with smoking, IgE, and lung function. Respir Med 1997; 91: Vignola A, Gjomarkaj M, Arnoux B, et al. Updates on cells and cytokines: monocytes. J Allergy Clin Immunol 1998; 101: Warner JA, Kroegel C. Pulmonary immune cells in health and disease: mast cells and basophils. Eur Respir J 1994; 7: Busse WW, Sedgwick J, Jarjour N, et al. Eosinophils and basophils in allergic airway inflammation. J Allergy Clin Immunol 1994: Casolaro V, Spadaro G, Marone G. Human basophil releasability: IV. Changes in basophil releasability in patients with allergic rhinitis or bronchial asthma. Am Rev Respir Dis 1990; 142: Liu MC, Hubbard WC, Proud D, et al. Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airways in allergic asthmatics: cellular, mediator, and permeability changes. Am Rev Respir Dis 1991; 144: Gaddy J, Busse WW. Enhanced IgE-dependent basophil histamine release and airway reactivity in asthma. Am Rev Respir Dis 1986; 134: Naclerio R, Proud D, Togia A. Inflammatory mediators in late antigen-induced rhinitis. N Engl J Med 1985; 313: Maruyama N, Tamura G, Aizawa T, et al. Accumulation of basophils and their chemotactic activity in the airways during natural airway narrowing in asthmatic individuals. Am J Respir Crit Care Med 1994; 150: Clinical Investigations