Objectives

1. Understand the importance of chronic inflammation in COPD.
2. Understand the significance of systemic manifestations in COPD.
3. Know the types of inflammatory cells that play a role in the systemic inflammatory component of COPD.
4. Become familiar with different types of inflammatory mediators involved in systemic inflammation in COPD.
5. Name the major hypotheses about the origin of systemic inflammation in COPD.

Abbreviations

AR = airway responsiveness; CD = cluster of differentiation; ICAM = intercellular adhesion molecule; IL = interleukin; NAD = nicotinamide adenine dinucleotide; PARP-1 = poly(ADP-ribose) polymerase-1; ROS = reactive oxygen species; sTNF-R = soluble TNF-receptor; TEAC = Trolox equivalent antioxidant capacity; TNF- a = tumor necrosis factor alpha

COPD as Chronic Inflammatory Disease

COPD is a complex heterogeneous respiratory disease characterized by the progressive development of airflow limitation that is largely irreversible.¹ The clinical syndrome of COPD encompasses different disease conditions ranging from chronic obstructive bronchitis with obstruction of respiratory bronchioles to emphysema characterized by enlargement of air spaces and destruction of lung parenchyma resulting in loss of lung elasticity. The association of an abnormal inflammatory response of the lungs to a variety of noxious inhaled particles or gases (mostly cigarette smoke) with airflow limitation in COPD, recently defined in a statement by the NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD),¹ indicates the critical role of the inflammatory process in the pathogenesis of this disease. A marked increase in activated neutrophils in induced sputum and BAL fluid, reflecting the airway compartment, was demonstrated in COPD patients vs smokers without airflow obstruction.^2-4 Resection and bronchial biopsy studies confirm these findings, but also show increased numbers of CD8+ lymphocytes throughout the whole lung.^5-7 The degree of airflow obstruction is correlated with the number of these types of inflammatory cells.^4,6 In addition, this cellular inflammatory response in the different compartments is associated with production of inflammatory mediators, including the chemokine interleukin (IL)-8, the proinflammatory cytokine tumor necrosis factor-alpha (TNF- a) and its soluble receptors (sTNF-R).^4,8 The intensity and characteristics (both cellular and molecular) of the inflammation vary as the disease progresses. The underlying mechanisms of inflammation leading to characteristic pathology observed in COPD are still incompletely understood. Recently, three main interacting processes have been postulated to be equally central in the process of tissue destruction and remodeling in COPD: (1) proteinase/antiproteinase imbalance; (2) oxidative stress; and (3) apoptosis.⁹ Additional studies both in vitro and in vivo clearly are required to elucidate the interaction between these processes in the origin and progression of COPD.

The chronic local inflammatory process in COPD is markedly different from that seen in asthma (reviewed by Jeffery¹⁰), in which clusters of (CD)4+ lymphocytes predominate; this may explain the differences in response to corticosteroid treatment in asthma vs COPD. Unlike chronic asthma, recent studies found no evidence that long-term treatment with high doses of inhaled corticosteroids reduces the progression of COPD, even when treatment was started before the disease became symptomatic, indicating that the inflammatory process leading to airflow limitation in COPD does not respond to steroids.^11-13 The generally accepted management goals for COPD patients are improvement in quality of life and in functional status in the absence of disease progression. Based on these goals and the limited outcomes that are possible with pharmacologic treatments directed to improve airflow limitation, there is at present a revival of research on the mechanisms of COPD, not limited to the local organ involvement but extending to the patient as a whole. The latter mechanisms are generally considered to be the systemic effects of the disease,¹⁴ and include systemic inflammation, skeletal muscle dysfunction, nutritional abnormalities, and weight loss. In addition to airflow limitation and loss of alveolar structure, systemic manifestations play an important role in dyspnea and exercise limitation in COPD, and thus the decline of health status.^14,15 This review focuses on several aspects of systemic inflammation in COPD-systemic oxidative stress, circulating inflammatory cells, and plasma proinflammatory mediators-and on their origins.

Systemic Oxidative Stress

The growing recognition of the role of oxidative stress in the pathogenesis of COPD has been assessed not only  in the airways and lung compartment, but also in the peripheral blood.¹⁶ As oxidative stress is difficult to assess in vivo owing to the very short half-life of reactive oxygen species (ROS), biological consequences of ROS are often used as indices of oxidative stress. ROS generated by neutrophils, whether circulating or sequestered in the pulmonary vasculature, are scavenged by blood antioxidants and antioxidant enzymes, indicating that an individual's ability to prevent the injurious effects of oxidative stress depends on the antioxidant capacity of the blood and the tissues. Rahman et al¹⁸ determined the Trolox (F. Hoffman-LaRoche, Ltd; Basel, Switzerland) equivalent antioxidant capacity (TEAC) of plasma and the levels of products of lipid peroxidation as indices of overall oxidative stress, and found that plasma TEAC was markedly reduced, with increased levels of lipid peroxidation products, in healthy smokers and COPD patients vs nonsmoking control subjects.^17,18 However, no relationship was found between spirometric data measured as FEV₁ predicted or FEV₁/FVC percent predicted and the plasma levels of TEAC in patients with COPD, healthy smokers, or healthy nonsmokers. These findings were recently confirmed and extended by Hageman et al,¹⁹ who also demonstrated a significant reduction of plasma uric acid as well as blood nicotinamide adenine dinucleotide (NAD)+ in stable COPD patients when compared with control subjects. Further evidence of persistent systemic oxidative stress in COPD patients was provided by the finding of higher urinary levels of isoprostane F₂a-II, a stable prostaglandin isomer formed by ROS-dependent peroxidation of arachidonic acid, which is excreted is urine, in COPD patients vs smoking control subjects.²⁰ Together, these studies indicate that both smoking and COPD are associated with significant systemic oxidative stress.

Circulating Inflammatory Cells

Many COPD-related articles have reported changes in various inflammatory cells in peripheral blood, including neutrophils and lymphocytes. Activation of peripheral blood neutrophils has also been shown, resulting in potentiation of cytotoxic and migratory responses. Noguera et al investigated ROS production and the expression of surface adhesion molecules in circulating neutrophils of COPD patients in a clinical stable condition.^21,22 Compared with control subjects, stable patients with disease showed an increased expression of Mac-1 (CD11b/CD18) in circulating neutrophils with lower levels of intercellular adhesion molecule (ICAM)-1. Increased plasma soluble ICAM-1, which is considered to be a surrogate of its expression on the endothelium, was reported by other investigators.²³ In addition, Noguera et al²² showed that blood neutrophils isolated from COPD patients produced more ROS under basal conditions and after stimulation in vitro as compared with smoking and nonsmoking control subjects, and this increased respiratory burst correlated with the elevated expression of adhesion molecules. In another in vitro study, Burnett et al^24,25 demonstrated that peripheral neutrophils isolated from COPD patients showed enhanced chemotaxis and extracellular proteolysis. In contrast, Cataldo et al²⁶ found no differences regarding matrix metalloproteinase-9 secretion by circulating granulocytes from COPD patients and control subjects. Even the expression of a G-protein subunit (stimulatory Ga, Gas ) in circulating neutrophils has been studied,²¹ which is a key protein in adhesion of human neutrophils to tissues as well as activation and respiratory burst. Gas was demonstrated to be down-regulated irrespective of the clinical condition of the patient. However, the pathogenic implications of most of these findings are still unclear, and need confirmation in well-characterized patient groups and in different phases of the disease process.

Although blood lymphocytes isolated from COPD patients have been less well studied, recent studies indicate abnormal lymphocyte function in COPD. Increased activity of cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, was reported in the lymphocytes of patients with COPD compared with healthy subjects,²⁷ and this was found to be significantly related to disease severity as reflected by the degree of airflow limitation. In a recent study, Hageman and colleagues¹⁹ investigated activation of nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1), which forms extensive poly(ADP-ribose) polymers from its substrate NAD+ after activation by ROS-induced DNA strand breaks. Activation of PARP-1 in peripheral blood lymphocytes of COPD patients was shown to be more prevalent than in lymphocytes of healthy, age-matched control individuals, supporting a contribution of PARP-1 activation to the pathophysiology of COPD.

In contrast to numerous studies investigating the lymphocyte CD4+/CD8+ ratio in the pulmonary compartment that have shown a decreased CD4+/CD8+ ratio in COPD in several compartments, this feature has not yet been well studied in the systemic compartment. Several studies suggest that cigarette smoke alone may trigger a shift in the numbers of CD4+ and CD8+ lymphocytes, which may be reversible after smoking cessation.^28-31 In this view, de Jong and coworkers³² investigated lymphocyte subsets in peripheral blood of COPD patients and healthy smokers, but no significant differences in lymphocyte subsets were found when either total groups or smokers from both groups were compared. However, these authors found that within the group of nonsmokers, the percentage of CD8+ lymphocytes was significantly higher in COPD patients than in control subjects, and the CD4:CD8 ratio was positively correlated with higher FEV₁ values (% predicted). Additional studies are necessary to understand the contribution of circulating lymphocytes to the pathogenesis of COPD.

Inflammatory Mediators in Plasma

During the last decade, studies investigating systemic inflammation in patients with COPD reported enhanced levels of circulating inflammatory mediators, such as acute-phase reactants and cytokines. Acute-phase reactants are key players in innate immunity and reduction of inflammatory reactions. Schols et al^33,34 demonstrated increased levels of C-reactive protein and lipopolysaccharide binding protein in stable COPD patients; these increases were most pronounced in a subset of COPD patients with an increased resting energy expenditure and decreased fat-free mass. The lack of a response to some intervention strategies, such as nutritional therapy, seems to be related to the level of this systemic inflammatory response.^34,35 A prospective epidemiologic study in a cohort of 8,955 subjects from a Danish general adult population study revealed that increased plasma levels of another acute-phase reactant, fibrinogen, are associated with reduced lung function and increased risk of COPD, independent of smoking status.³⁶ The rise in the systemic levels of acute-phase reactants suggest that the hepatocytes are activated to produce these reactants, although increasing evidence indicates that tissue-specific cells, such as lung epithelial cells, are able to produce acute-phase proteins.³⁷ The formation of acute-phase reactants is strongly induced by cytokines such as IL-6 or TNF- a . Indeed, enhanced circulating levels of IL-6 and TNF- a have been reported in COPD.^19,38-40 The detection of biologically active TNF- a can be hampered by its short half-life (approximately 6 to 7 min), the formation of complexes with both sTNF-R55 and sTNF-R75, and its renal clearance. Small but significant increases in circulating levels of both sTNF-R55 and sTNF-R75 have also been demonstrated in COPD.^4,33,41-43 Because inflammatory stimuli including TNF- a will induce shedding of membrane-bound TNF-R, 44 the enhanced levels of sTNF-R could reflect the enhanced inflammatory status of the patients. Yasuda et al⁴⁰ investigated the association between apoptosis-related factors and the progression of COPD, and demonstrated that plasma levels of soluble Fas (CD95), an inhibitor of apoptosis, were significantly increased in patients with severe COPD when compared with healthy control subjects and mild/moderate COPD patients. In contrast, another study suggested that the Fas-Fas ligand system does not independently play a role in the pathophysiology of COPD.⁴⁵ Future studies are needed to assess whether these systemic changes are continuously present as part of the stable state of the COPD process or reflect day-to-day variations in the inflammatory state.

Origin of Systemic Inflammation

The origin of the systemic inflammation present in COPD patients is still poorly understood, and several (independent) pathways may be involved. First, as smoking results in important extrapulmonary effects, such as cardiovascular diseases, tobacco smoke alone may significantly contribute to systemic inflammation in COPD. In this respect, both systemic oxidative stress and endothelial dysfunction of peripheral vessels were reported in passive smokers and smokers with only a few pack-years.^46,47 A second possible mechanism is the involvement of the local inflammatory response in the lung in systemic inflammation, which was recently investigated by Vernooy et al⁴ in patients with mild-to-moderate COPD. Comparison of levels of sTNF-R or IL-8 in sputum and plasma did not reveal direct correlations, suggesting that the systemic inflammatory response in mild-to-moderate COPD does not result from an overflow of inflammatory mediators from the pulmonary compartment, but rather that the inflammatory processes in the local and systemic compartment are differently regulated. Further evidence for the hypothesis that the inflammatory processes in the airways and the systemic circulation are independent processes comes from recent studies by Michel and colleagues.⁴⁸ Healthy subjects exposed to the proinflammatory compound lipopolysaccharide via inhalation were found to express differences with respect to changes in body temperature, airway responsiveness (AR), and FEV₁ values. Subjects with elevated body temperature showed an increase in systemic inflammatory response. Those with increased AR showed an increase in airway inflammation but not systemic inflammation. A lipopolysaccharide-induced increase in body temperature and change in AR were not necessarily associated in a given subject, suggesting that the underlying mechanisms are dissociated.

A third pathway was recently postulated by Takabatake et al.⁴¹ As in vitro studies have revealed that hypoxia will result in enhanced cytokine production by macrophages, the systemic hypoxemia observed in patients with COPD may contribute to the activation of the TNF system in blood.⁴¹ Indeed, significant inverse correlations between Pao₂ and circulating TNF and sTNF-R levels in patients were detected. These results suggest that changes in circulating cytokines resulting from deterioration of lung function will lead to enhanced systemic inflammation. In this respect, increased levels of inflammatory mediators in the blood of COPD patients originate from extrapulmonary cells. Circulating leukocytes, the endothelium, and muscle cells are possible candidate sources. Lastly, genetic factors may account for systemic abnormalities in patients with COPD, because only some smokers will eventually develop COPD.¹ In this respect, the expression of a certain genetic predisposition is the cause and systemic inflammation is then a consequence.

Summary

It is now recognized that COPD is characterized by a chronic inflammatory response that is not restricted to the respiratory compartment but also extends to the systemic compartment. The studies discussed in this review provide evidence that (1) both smoking and COPD are strongly associated with significant systemic oxidative stress; (2) numbers of neutrophils and lymphocytes are increased in COPD, and display an increased activation level resulting in potentiation of cytotoxic and migratory responses; and (3) levels of circulating inflammatory mediators, such as acute-phase reactants and cytokines, are enhanced in COPD. It is clear that we are only beginning to unravel the systemic inflammatory component in COPD. Studies in well-characterized patient groups are necessary to confirm and extend the present observations in the different phases of the disease process as well as unravel the origin of systemic inflammation in COPD.

References

1. Pauwels RA, Buist AS, Calverley PM, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med 2001; 163:1256-1276
2. Peleman RA, Rytila PH, Kips JC, et al. The cellular composition of induced sputum in chronic obstructive pulmonary disease. Eur Respir J 1999; 13:839-843
3. Pesci A, Balbi B, Majori M, et al. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur Respir J 1998; 12:380-386
4. Vernooy JH, Kucukaycan M, Jacobs JA, et al. Local and systemic inflammation in patients with chronic obstructive pulmonary disease: soluble tumor necrosis factor receptors are increased in sputum. Am J Respir Crit Care Med 2002; 166:1218-1224
5. Saetta M, Di Stefano A, Turato G, et al. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157:822-826
6. O'Shaughnessy TC, Ansari TW, Barnes NC, et al. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV₁. Am J Respir Crit Care Med 1997; 155:852-857
7. Saetta M, Baraldo S, Corbino L, et al. CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:711-717
8. Keatings VM, Collins PD, Scott DM, et al. Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996; 153:530-534
9. Tuder RM, Petrache I, Elias JA, et al. Apoptosis and emphysema: the missing link. Am J Respir Cell Mol Biol 2003; 28:551-554
10. Jeffery PK. Remodeling in asthma and chronic obstructive lung disease. Am J Respir Crit Care Med 2001; 164:S28-S38.
11. Burge PS, Calverley PM, Jones PW, et al. Randomised, double blind, placebo controlled study of fluticasone propionate in patients with moderate to severe chronic obstructive pulmonary disease: the ISOLDE trial. BMJ 2000; 320:1297-1303
12. Vestbo J, Sorensen T, Lange P, et al. Long-term effect of inhaled budesonide in mild and moderate chronic obstructive pulmonary disease: a randomised controlled trial. Lancet 1999; 353:1819-1823
13. Pauwels RA, Lofdahl CG, Laitinen LA, et al. Long-term treatment with inhaled budesonide in persons with mild chronic obstructive pulmonary disease who continue smoking: European Respiratory Society Study on Chronic Obstructive Pulmonary Disease. N Engl J Med 1999; 340:1948-1953
14. Wouters EF. Chronic obstructive pulmonary disease. 5: systemic effects of COPD. Thorax 2002; 57:1067-1070
15. Skeletal muscle dysfunction in chronic obstructive pulmonary disease: a statement of the American Thoracic Society and European Respiratory Society. Am J Respir Crit Care Med 1999; 159:S1-S40
16. Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease: Oxidative Stress Study Group. Am J Respir Crit Care Med 1997; 156:341-357
17. Rahman I, Morrison D, Donaldson K, et al. Systemic oxidative stress in asthma, COPD, and smokers. Am J Respir Crit Care Med 1996; 154:1055-1060
18. Rahman I, Swarska E, Henry M, et al. Is there any relationship between plasma antioxidant capacity and lung function in smokers and in patients with chronic obstructive pulmonary disease? Thorax 2000; 55:189-193
19. Hageman GJ, Larik I, Pennings HJ, et al. Systemic poly(ADP-ribose) polymerase-1 activation, chronic inflammation, and oxidative stress in COPD patients. Free Radic Biol Med 2003; 35:140-148
20. Pratico D, Basili S, Vieri M, et al. Chronic obstructive pulmonary disease is associated with an increase in urinary levels of isoprostane F 2 alpha-III, an index of oxidant stress. Am J Respir Crit Care Med 1998; 158:1709-1714
21. Noguera A, Busquets X, Sauleda J, et al. Expression of adhesion molecules and G proteins in circulating neutrophils in chronic obstructive pulmonary disease. 1998; 158:1664-1668
22. Noguera A, Batle S, Miralles C, et al. Enhanced neutrophil response in chronic obstructive pulmonary disease. Thorax 2001; 56:432-437
23. Riise GC, Larsson S, Lofdahl CG, et al. Circulating cell adhesion molecules in bronchial lavage and serum in COPD patients with chronic bronchitis. Eur Respir J 1994; 7:1673-1677
24. Burnett D, Chamba A, Hill SL, et al. Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis. Lancet 1987; 2:1043-1046
25. Burnett D, Chamba A, Hill SL, et al. Effects of plasma, tumour necrosis factor, endotoxin and dexamethasone on extracellular proteolysis by neutrophils from healthy subjects and patients with emphysema. Clin Sci (Lond) 1989; 77:35-41
26. Cataldo D, Munaut C, Noel A, et al. Matrix metalloproteinases and TIMP-1 production by peripheral blood granulocytes from COPD patients and asthmatics. Allergy 2001; 56:145-151
27. Sauleda J, Garcia-Palmer FJ, Gonzalez G, et al. The activity of cytochrome oxidase is increased in circulating lymphocytes of patients with chronic obstructive pulmonary disease, asthma, and chronic arthritis. Am J Respir Crit Care Med 2000; 161:32-35
28. Miller LG, Goldstein G, Murphy M, et al. Reversible alterations in immunoregulatory T cells in smoking: analysis by monoclonal antibodies and flow cytometry. Chest 1982; 82:526-529
29. Costabel U, Bross KJ, Reuter C, et al. Alterations in immunoregulatory T-cell subsets in cigarette smokers: a phenotypic analysis of bronchoalveolar and blood lymphocytes. Chest 1986; 90:39-44
30. Ekberg-Jansson A, Andersson B, Avra E, et al. The expression of lymphocyte surface antigens in bronchial biopsies, bronchoalveolar lavage cells and blood cells in healthy smoking and never-smoking men, 60 years old. Respir Med 2000; 94:264-272
31. Ekberg-Jansson A, Arva E, Nilsson O, et al. A comparison of the expression of lymphocyte activation markers in blood, bronchial biopsies and bronchoalveolar lavage: evidence for an enrichment of activated T lymphocytes in the bronchoalveolar space. Respir Med 1999; 93:563-570
32. de Jong JW, van der Belt-Gritter B, Koeter GH, et al. Peripheral blood lymphocyte cell subsets in subjects with chronic obstructive pulmonary disease: association with smoking, IgE and lung function. Respir Med 1997; 91:67-76
33. Schols AM, Buurman WA, Staal van den Brekel AJ, et al. Evidence for a relation between metabolic derangements and increased levels of inflammatory mediators in a subgroup of patients with chronic obstructive pulmonary disease. Thorax 1996; 51:819-824
34. Schols AM, Creutzberg EC, Buurman WA, et al. Plasma leptin is related to proinflammatory status and dietary intake in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160:1220-1226
35. Creutzberg EC, Schols AM, Weling-Scheepers CA, et al. Characterization of nonresponse to high caloric oral nutritional therapy in depleted patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000; 161:745-752
36. Dahl M, Tybjaerg-Hansen A, Vestbo J, et al. Elevated plasma fibrinogen associated with reduced pulmonary function and increased risk of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001; 164:1008-1011
37. Dentener MA, Vreugdenhil AC, Hoet PH, et al. Production of the acute-phase protein lipopolysaccharide-binding protein by respiratory type II epithelial cells: implications for local defense to bacterial endotoxins. Am J Respir Cell Mol Biol 2000; 23:146-153
38. Eid AA, Ionescu AA, Nixon LS, et al. Inflammatory response and body composition in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001; 164:1414-1418
39. Di Francia M, Barbier D, Mege JL, et al. Tumor necrosis factor-alpha levels and weight loss in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1994; 150:1453-1455
40. Yasuda N, Gotoh K, Minatoguchi S, et al. An increase of soluble Fas, an inhibitor of apoptosis, associated with progression of COPD. Respir Med 1998; 92:993-999
41. Takabatake N, Nakamura H, Abe S, et al. The relationship between chronic hypoxemia and activation of the tumor necrosis factor-alpha system in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000; 161:1179-1184
42. Dentener MA, Creutzberg EC, Schols AM, et al. Systemic anti-inflammatory mediators in COPD: increase in soluble interleukin 1 receptor II during treatment of exacerbations. Thorax 2001; 56:721-726
43. Takabatake N, Nakamura H, Abe S, et al. Circulating leptin in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 159:1215-1219
44. Porteu F, Nathan C. Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med 1990; 172:599-607
45. Takabatake N, Nakamura H, Inoue S, et al. Circulating levels of soluble Fas ligand and soluble Fas in patients with chronic obstructive pulmonary disease. Respir Med 2000; 94:1215-1220
46. Dietrich M, Block G, Hudes M, et al. Antioxidant supplementation decreases lipid peroxidation biomarker F(2)-isoprostanes in plasma of smokers. Cancer Epidemiol Biomarkers Prev 2002; 11:7-13
47. Dietrich M, Block G, Benowitz NL, et al. Vitamin C supplementation decreases oxidative stress biomarker f2-isoprostanes in plasma of nonsmokers exposed to environmental tobacco smoke. Nutr Cancer 2003; 45:176-184
48. Michel O, Dentener M, Corazza F, et al. Healthy subjects express differences in clinical responses to inhaled lipopolysaccharide that are related with inflammation and with atopy. J Allergy Clin Immunol 2001; 107:797-804