INTRODUCTION

TOP ABSTRACT INTRODUCTION THE NEUTROPHIL IN COPD CHRONIC BRONCHIAL DISEASE REFERENCES

Chronic obstructive pulmonary disease (COPD) is a generic term covering several clinical syndromes with the common component of a degree of fixed airflow obstruction. However, our understanding of the pathogenic processes involved is largely based on studies confined to smoking-related chronic bronchitis and emphysema. Although these represent two separate components (at least anatomically), it is likely that both are predominantly the result of recruitment and activation of neutrophils in the lung. The strength of the association, and of the studies defining the mechanisms involved, date back to the original observation of ₁-antitrypsin deficiency in the early 1960s. Clinical studies (at least initially) indicated that most subjects with this deficiency developed both emphysema and bronchitis (1), particularly if they smoked.

After this association of an antiprotease deficiency with disease, there ensued extensive studies, both in vitro and in vivo, to determine the protease directly responsible for the lung damage. Eventually studies demonstrated that several human enzymes could directly produce many of the features of smoking-related COPD. For instance, neutrophil elastase (2), proteinase 3 (3), and cathepsin B (4) produce emphysematous lesions in experimental animals. In addition, neutrophil elastase (2), cathepsin G (2), and cathepsin B (5) produce bronchial disease in similar models. These observations were of importance since ₁-antitrypsin inhibits neutrophil elastase (NE), cathepsin G (CatG), and proteinase 3 (Pr3), all of which are stored in the same granule in the neutrophil and hence are released simultaneously. In addition, cathepsin B is released in the lung as a proenzyme that is activated by NE (6). Thus the neutrophil is the source of three of the enzymes shown directly to produce the features of COPD in vivo and can activate the fourth, hence potentially having the same effect by an indirect route. Thus there is considerable evidence to implicate the neutrophil in the pathogenesis of COPD.

	THE NEUTROPHIL IN COPD

TOP ABSTRACT INTRODUCTION THE NEUTROPHIL IN COPD CHRONIC BRONCHIAL DISEASE REFERENCES

As already indicated, the neutrophil (at least theoretically) is likely to play a major role in the pathogenesis of chronic bronchitis and emphysema. Studies in both health and disease have provided indirect evidence to support this role. Of importance is the observation that the lungs of smokers contain increased numbers of neutrophils (7). Thus the major known risk factor for COPD is associated with a greater lung burden of the cells thought to be responsible for the architectural damage seen in COPD. In addition, subjects with ₁-antitrypsin (₁AT) deficiency also have an increased neutrophil burden in the distal airways (8). Furthermore, bronchial secretions contain increased numbers of neutrophils in subjects with COPD (9), confirming the potential for a pathogenic role in bronchial disease (see below).

Serum studies have shown that soluble E-selectin and intercellular adhesion molecule 1 (ICAM-1) (adhesion molecules released as neutrophils migrate) are increased in COPD, suggesting cell activation and recruitment (10). Finally, histopathological studies have indicated that E-selectin (the receptor for neutrophil L-selectin) is upregulated on vascular endothelial cells and tissue neutrophils are increased in subjects with bronchial disease (11).

Neutrophil Recruitment

Neutrophils migrate into the lung as part of the secondary lung defenses in response to the presence or release of chemoattractants in the airways.

Leukotriene B₄ (LTB₄) has been implicated as the major chemoattractant responsible for neutrophil recruitment in ₁AT deficiency, as concentrations are increased in lavage fluids from such subjects (8). Studies indicated that the major source was probably the alveolar macrophage and it was proposed that recruitment of neutrophils could actually amplify the effect (8). Elastase release from the recruited neutrophils would be poorly inhibited because of the reduced ₁AT in the deficient subjects. The elastase could stimulate the macrophages to release more LTB₄, resulting in further neutrophil recruitment (8), and thereby perpetuating and amplifying the neutrophil-associated lung damage (Figure 1). However, it is likely that other chemoattractants also play a role, especially in subjects without ₁AT deficiency. Certainly interleukin 8 (IL-8) is present in the airways of patients with COPD (9) and is increased in the lavage of some smokers (12). Furthermore, cigarette smoke has been shown to increase IL-8 release by bronchial epithelial cells (13). In addition, nicotine itself may be a chemoattractant for neutrophils (14), providing further, potentially important, links between smoking and neutrophil recruitment leading to lung damage.

View larger version (53K): [in this window] [in a new window]   Figure 1.   Alveolar macrophage release of LTB4 results in recruitment of neutrophils to the airway. During this process degranulation causes connective tissue destruction that is enhanced by the low level of 1-antitrypsin. The free elastase being released by the neutrophil activates the macrophage to produce more LTB4, thereby continuing the cycle.

The Neutrophil and Connective Tissue

Destruction of interstitial lung elastin is thought to be central to the pathogenesis of emphysema. In health lung elastin is a long-lived connective tissue (15) and once destroyed by elastolytic enzymes, emphysema will develop even though elastin may reaccumulate (16). However, for an inflammatory cell to migrate from the circulation it must penetrate connective tissue and this requires proteolytic degradation as demonstrated for eosinophils (17). This process occurs in close proximity to the inflammatory cell and yet must be contained to prevent widespread indiscriminate connective tissue destruction.

Indeed, studies have shown that as neutrophils move in close proximity to connective tissue they degrade it; this can be reduced but not prevented by ₁AT (18). Previously it was believed that this inability to prevent completely the connective tissue degradation was due to restricted access of the inhibitor to the area of close contact between the cell and the connective tissue substrate (19). However, more recently it has been argued (20), and subsequently demonstrated (21), that the concentration of elastase released from the azurophil granule is supraphysiological and cannot, therefore, be totally controlled by the normal surrounding concentrations of inhibitors. However, as the enzyme diffuses away from the cell its concentration falls until it equals that of the surrounding inhibitors, whereupon its activity ceases owing to inactivation by complex formation. This overall process, therefore, enables cell penetration through the tight connective tissue matrix while limiting the area of damage. In this process, oxidation of ₁AT seems to play little or no role (19). It can, however, be predictably controlled by ₁AT alone, although the low concentration of ₁AT seen in genetic deficiency is associated with a much increased area of damage (21). Thus these studies explain the extensive disease seen in ₁AT deficiency while providing a mechanism to explain the development of emphysema in subjects with normal ₁AT.

In ₁AT deficiency a true protease/antiprotease imbalance exists whereas in subjects with normal ₁AT the development of disease depends largely on the normal process of connective tissue degradation as cells migrate, but it is influenced by the magnitude and persistence of neutrophil recruitment.

Neutrophil Responses

With a general understanding that the degree of neutrophil recruitment is a major determinant of the development of COPD it remains possible that crucial differences in neutrophil function may also prove critical in the development of disease.

For instance, studies have shown that neutrophils from patients with established COPD differ functionally from those of age- and smoking-matched healthy controls in two respects. First, the cells show an increased chemotactic response to a standard chemoattractant, and second, each cell has a greater ability to digest connective tissue both in the basal state and when upregulated (22). These changes were shown to be related to increased receptor expression (23). However, it has not been possible (so far) to reproduce these effects in vitro (24), suggesting that there may be a fundamental difference in the neutrophils produced by the patients with COPD.

These observations may be of major importance in the development of disease. In a smoker with more responsive neutrophils a greater number will be attracted to the lung in response to "normal" chemoattractant release. For each cell recruited the amount of connective tissue destroyed during cell migration will also be increased, leading (over time) to excess connective tissue damage and the development of clinical disease. This concept and the nature of the neutrophil changes await future elucidation.

The Influence of Connective Tissue on Proteinase Inhibition

As already indicated, the release of elastase from an activated neutrophil leads to an area of obligate connective tissue degradation until the enzyme diffuses far enough away from the cell, resulting in dilution to a concentration that equals the inhibitory function of surrounding antiproteases. However, even this process may be slightly more complicated in vivo since elastin will compete with the antiproteases and bind elastase, which then cannot be inactivated by ₁AT (25). Interestingly, however, this elastin-bound enzyme can be inhibited by secretary leukoprotease inhibitor (SLPI) (25). Nevertheless, again the concentration of elastase on the elastin will still depend on its distance from the released azurophil granule and hence even the effect of SLPI (and elastin-bound elastase) will be limited in close proximity to the cell.

In summary, in the absence of ₁AT deficiency the development and extent of lung damage leading to emphysema will be influenced by the nature and number of neutrophils recruited to the lung. This process has been argued on the understanding that NE is the major mediator of the necessary elastin degradation. However, both cathepsin G and Pr3 are present in concentrations similar to that of NE in the azurophil granule and similar arguments could therefore also apply to these two enzymes.

	CHRONIC BRONCHIAL DISEASE

TOP ABSTRACT INTRODUCTION THE NEUTROPHIL IN COPD CHRONIC BRONCHIAL DISEASE REFERENCES

There has been increasing interest in the role of neutrophils and particularly NE in the generation and perpetuation of chronic bronchial disease. Subjects with chronic bronchitis have excess production of mucus, patchy loss of ciliated epithelium, and reduced mucociliary clearance. Studies have confirmed that NE produces epithelial damage (26), mucous gland hyperplasia (2), reduced ciliary beating (27), and secretion of mucus (28), implying a major role for this enzyme in human disease.

In addition, NE has also been shown to compromise other critical components of the bronchial defenses including the C3Bi opsonophagocytic receptor (29) and immunoglobulin structure (30). Such major changes in the airway will reduce the ability to retain sterility and prevent infections. Indeed, the presence of bacteria in the airway may lead to further neutrophil recruitment and hence amplification and persistence of bronchial disease (31). These interrelationships are summarized in Figure 2.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Bacterial colonization in the lung leads to neutrophil recruitment by two mechanisms. First, bacterial products can act on airway epithelial cells to release interleukin 8; second, phagocytosis by resident macrophages leads to their activation and the release of LTB4 and IL-8. The release of these chemoattractants results in neutrophil recruitment and activation. These cells then phagocytose the colonizing bacteria and the processes of activation and phagocytosis themselves lead to further release of interleukin 8 and LTB4, amplifying the neutrophil recruitment. In addition, the release of elastase during activation of the neutrophil can damage host defenses, facilitating bacterial colonization and, at the same time, also stimulate macrophages to produce more LTB4, again resulting in amplification via a different route.

Studies in humans have confirmed that the necessary components of this perpetuating circle are in place. The secretions are chemotactic for neutrophils and both LTB₄ and IL-8 seem to contribute to this in vitro (32). The bronchial secretions contain neutrophils even in the stable clinical state (9) and NE is detectable immunologically, although activity of the enzyme is usually undetectable (33).

Mucociliary clearance is reduced (31) and bacterial colonization is often present (34). This whole process would be predicted to be self-perpetuating since the activated neutrophils will release both IL-8 (35) and LTB₄ (36), leading to their own recruitment.

As already indicated, enzyme activity is often undetectable by the time the secretions are collected and analyzed. However, elegant studies have demonstrated that the effect of neutrophils (at least on secretion of mucus is an elastase-mediated event that occurs in close proximity to the mucous glands (37). However, in this instance (unlike the degradation of connective tissue), the process can be prevented by antielastases. At present it is not clear why the effects are different, since presumably supraphysiological concentrations of enzyme will also be released in close proximity to the mucous glands and should be equally resistant to inactivation by inhibitors except at high concentrations. When airways inflammation is more intense, such as would occur in an acute airway infection, the neutrophil traffic is also increased and elastase activity becomes readily detectable (33) This represents a true protease/ antiprotease imbalance since airway inflammation also occurs, increasing leakage of ₁AT (33). Despite this adaptive response the NE released from the neutrophil remains partially active and capable of causing more widespread proteolytic damage in the airway. At the same time the enzyme has the potential to reduce concentrations of its own airway inhibitor, secretary leukoprotease inhibitor (38), while damaging epithelium, which will increase the leakage of proteins such as serum ₁AT (39). The reasons for this diametrically opposed physiological response are unclear although it results in persistent enzyme activity that may facilitate any antibacterial effect of elastase (40), or production of mucus, to assist bacterial clearance and hence removal of organisms from the airway epithelium. However, as already stated, the enzyme can also cause impairment of cilia function and epithelial damage, which may also facilitate bacterial colonization (41). The interactions are clearly complex and further studies will be necessary to dissect the beneficial effects from the detrimental effects, in order to determine the net effect of neutrophils on the bronchial tree in health and disease.