INTRODUCTION

TOP ABSTRACT INTRODUCTION CONCLUSION DISCUSSION REFERENCES

The leukotrienes (LTs) can have effects on many parameters of airway function in asthma, including the development and regulation of inflammation. These features have prompted an extensive research effort to define the biological effects of the LTs, and, simultaneously, numerous laboratories began the search for drugs that would either block LT biosynthesis or antagonize LT actionswith the expectation that such agents would be effective and safe in asthma management.

	THE LEUKOTRIENES

The LTs are a family of lipid mediators derived from arachidonic acid via the 5-lipoxygenase pathway. The cysteinyl leukotrienes (cysLTs), LTC₄, LTD₄, and LTE₄, account for the bioactivity originally termed slow-reacting substance of anaphylaxis (SRS-A) (1), whereas LTB₄ was identified by its potent chemotactic activity for neutrophils (2).

Leukotriene B₄

LTB₄ stimulates neutrophil chemotaxis (3), enhances neutrophil-endothelial interactions (4), and stimulates neutrophil activation, leading to degranulation and the release of mediators, enzymes, and superoxides (5). LTB₄ affects cells other than neutrophils as well. For example, LTB₄ increases interleukin (IL)-6 production by human monocytes (6), and may affect the production of other cytokines by stimulating early gene transcription in mononuclear cells (7). Via a neutrophil-dependent process, LTB₄ may also be involved in inflammatory pain by reducing the nociceptive threshold (8).

Intratracheal instillation of LTB₄ in humans increased the total number of cells recovered by bronchoalveolar lavage (9) (Table 1). In comparison with a saline control, LTB₄ selectively increased the number and percentage of neutrophils. When expressed as a percentage of total cells obtained in bronchoalveolar lavage (BAL) fluid, there was a corresponding decrease in macrophages, although the absolute number of these cells was slightly elevated. Thus, LTB₄ has apparent specificity for neutrophil recruitment into the lung, where it has the capacity to activate these cells, presumably resulting in inflammation.

A number of factors can influence LTB₄ generation as well as the production of other inflammatory mediators. Preincubation of human neutrophils with granulocyte macrophage colony-stimulating factor (GM-CSF) resulted in a modest increase in LTB₄ production in response to the chemotactic peptide, f-met-leu-phe (fMLP), but a 10-fold increase after serum-treated zymosan stimulation. This illustrates a significant point: the quantity of LT produced will likely be affected by a number of factors, including exposure to cytokines and other pro-inflammatory substances that are generated during an inflammatory response and the activating factor that ultimately stimulates this cell to release its granular content.

Cysteinyl Leukotrienes

The cysLTs have profound effects on airway function. While many of these actions may not be labeled as proinflammatory, they are nevertheless important in the changes inherent to asthma. The cysLTs contract airway smooth muscle and are 100- to 1,000-fold more potent than histamine in this respect (10). Second, the cysLTs act on the vasculature to produce vasodilation and increase vascular permeability (11), processes that are likely relevant to the movement and recruitment of leukocytes to the site of an inflammatory response and that result in airway tissue edema; these changes can decrease airway caliber. Third, the cysLTs potently stimulate mucous secretion and interfere with mucociliary clearance, which further alter airway patency (12).

CysLTs are generated by a number of different cell types. The mast cell is a source of several preformed mediators, including histamine and tryptase, and synthesizes lipid mediators, including the cysLTs (13). Macrophages generate LTB₄ and the cysLTs as well (14). Both mast cells and alveolar macrophages are found in the airway and can therefore be sources of cysLTs when activated by any of a variety of factors, including IgE-dependent mechanisms.

Eosinophils appear to be important in asthma pathophysiology. There is also evidence that their presence in the lung is influenced by cysLTs (15). Eosinophil localization into the airway is often associated with increased LTC₄ production, which occurs in direct proportion to the number of recruited eosinophils (16). Furthermore, eosinophils are capable of producing significant quantities of LTC₄ (17); the amount produced is determined by the eosinophil activation state, which, in turn, depends on exposure to cytokines or other upregulating mediators.

	ROLE OF CYSTEINYL LEUKOTRIENES IN AIRWAY INFLAMMATION

Segmental bronchoprovocation with antigen has been used to study the allergic airway response. The immediate response to antigen is associated with mast cell activation, causing them to release preformed mediators, including histamine and tryptase. Bronchoprovocation studies can be performed using low, medium, or high antigen challenge doses, with saline challenge as a control. Shortly after saline or low-dose antigen challenge, only low concentrations of LTC₄ can be recovered by BAL (18). With high-dose antigen challenge, there was an immediate increase in LTC₄ levels, presumably as a result of mast cell release following the antigen trigger (18). However, 48 h after antigen challenge, the picture was quite different; antigen challenge markedly and dose-dependently increased the amount of LTC₄ in BAL fluid. LTC₄ concentrations strongly correlated with the number of eosinophils recruited into the airway (Figure 1), which suggests that antigen challenge causes recruitment and activation of eosinophils.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Correlation of eosinophils and LTC4 48 h after segmental antigen bronchoprovocation. The regression line (solid line) and 95% confidence levels (dotted lines) are shown. Reprinted from Reference 18 by permission.

The mechanisms of eosinophil recruitment and activation are not fully established; however, it is likely that eosinophils recruited to the airways release LTC₄, where it can affect airway smooth muscle tone and vascular permeability. Associated with the late response to antigen, there is generation of pro-inflammatory cytokines, including IL-5. This cytokine is important in the stimulation of bone marrow production of eosinophils, the recruitment (or availability for recruitment) of eosinophils to the airway, and possibly, the function of these cells. Whether IL-5 or other cytokines also determine the release of LTC₄ from eosinophils has yet to be fully established.

In a preliminary study involving four patients with asthma, inhalation of LTE₄ increased the number of eosinophils and neutrophils found in mucosal biopsies 4 h later (19). The average number of eosinophils was 10-fold higher than the number of neutrophils. Thus, LTE₄ inhalation not only can produce airways obstruction, but also appears capable of directing eosinophil recruitment.

Some patients with asthma are intolerant of aspirin, a response that appears to be dependent on the generation of cysLTs. This condition is usually found in individuals who have nonallergic asthma with coexisting nasal polyps and sinusitis, and more severe asthma. Following aspirin administration to these patients, a profound drop in FEV₁ occurs, with an associated increase in naso-ocular signs and symptoms (20). These individuals often have profound rhinorrhea that occurs in association with the development of airflow obstruction. Since tryptase and histamine are detectable in nasal secretions from these patients, this response to aspirin likely involves mast cell activation. Following aspirin exposure, there is a marked increase in urinary excretion of LTE₄ in asthmatic patients with aspirin sensitivity. In asthma patients who are not aspirin-sensitive, this increase in urinary LTE₄ excretion is not observed. Thus, aspirin-induced asthma is another situation in which the development of airflow obstruction is associated with increased cysLT production. Because aspirin-induced asthma is not always associated with increased airway hyperresponsiveness, this syndrome appears to be primarily an acute airway smooth muscle contractile response, for which the cysLTs are the major mediator.

There are observations that suggest that cysLTs appear to be involved in the inflammatory components of asthma. For example, antigen challenge in some patients with asthma produces both early- and late-phase airway obstruction. The FEV₁ decreases immediately after antigen, and then a second phase of airflow obstruction occurs about 6 h later. In patients who exhibit both early- and late-phase responses, there is a significant increase in urinary LTE₄ excretion in relation to antigen challenge, with higher LTE₄ levels detected 6 to 7 h after antigen challenge (21) (Figure 2). The late-phase response is associated with increased airway hyperresponsiveness and airway inflammation, as can be observed when BAL or mucosal biopsies are performed. Thus, one could postulate the following scenario: antigen challenge leads to cysLT generation, which, in turn, signals eosinophil recruitment. Recruitment of these cells to the airways can cause bronchial sensitivity to inflammatory effects and hyperresponsiveness. While this is obviously an oversimplification of actual events, it nonetheless illustrates the relationship of these processes to the pathophysiology of the late-phase reaction, a model of airway inflammation.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Change in FEV1 (%) in five subjects who developed an isolate late asthmatic response after inhaling occupational sensitizing agents (closed circle) and after inhalation of the appropriate diluent (open circle). Also illustrated are the levels of urinary LTE4 (mean and SEM) measured before inhalation of the diluent (open bars) or the sensitizing agent (solid bars) between 2 to 3 h and between 6 to 7 h after inhalation. Reprinted from Reference 21.

In studies evaluating epithelial cells and airway biopsies from patients with asthma, distinct markers for increased leukotriene generation have been found. The number of 15-lipoxygenase-positive cells in bronchial mucosal biopsies from patients with asthma were found to be significantly higher than in healthy subjects (22). This raises the possibility that there is a greater overall capacity for lipoxygenase product generation in patients with asthma.

Some evidence also points to the possibility that cysLTs play a direct role in the development of airway hyperresponsiveness. Histamine responsiveness has been shown to increase after inhalation of LTE₄an effect that was maximal 7 h later, but still evident after 4 d (23). In contrast, methacholine does not affect histamine responsiveness. In another study, histamine responsiveness was not altered over a 7-h period following exposure to saline or methacholine. However, histamine responsiveness was increased after inhalation of LTC₄, LTD₄, or LTE₄with maximal effects noted after 4 h (24). The mechanism by which cysLTs affected the airway response to histamine has not been described. However, it is tempting to speculate that it involves inflammatory events in the airways, and could involve eosinophil recruitment, as has previously been reported after LTE₄ inhalation (19).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION CONCLUSION DISCUSSION REFERENCES

The cysLTs have profound effects on features of asthma and, possibly, some inflammatory processes. First, the cysLTs directly contract airway smooth muscle. Second, the cysLTs increase vascular permeability, an effect that leads to changes in airway caliber. The altered geometry of the airway has been associated with increased airway reactivity. Third, the cysLTs can stimulate mucous secretion, which further compromises the airways. Finally, the cysLTs appear capable of recruiting eosinophils into the lung, which in turn leads to the production of additional cysLTs as well as other tissue-damaging mediators. The fact that 5-lipoxygenase inhibitors and cysLT receptor antagonists have efficacy in asthma confirms the importance of these lipid mediators in the disease process.

	DISCUSSION

TOP ABSTRACT INTRODUCTION CONCLUSION DISCUSSION REFERENCES

Drazen: We have looked at leukotriene inhalation in nonaspirin-sensitive and aspirin-sensitive asthma patients and have been unable to confirm that selective airway hyperresponsiveness to LTE₄ is associated with aspirin sensitivity. I was wondering if there is an explanation for this?

Busse: One explanation would be as follows. If the leukotrienes affect vascular permeability, they could cause a very transient thickening of the airway. Based on the observations of Dr. Peter Paré, a "thickening" of the airway changes its geometry. This may be the effect that is directly related to increases in airway responsiveness, and not the recruitment of inflammatory cells into the airway.

Dahlén: I think that the relative hyperresponsiveness to LTE₄ is an open question that needs confirmation. But an important report at the European Respiratory Society showed that LTD₄ inhalation caused an increased number of eosinophils in the induced sputum. That would support Dr. Tak Lee's LTE₄ observations.

Peters-Golden: I have a comment on another action of the cysteinyl leukotrienes relevant to asthma. They have effects that would promote airway remodelingsmooth muscle and fibroblast proliferation. In terms of chronic asthma, these effects may be relevant.

Busse: I think they are very relevant.

Calhoun: What is known about the direct effects of cysteinyl leukotrienes on the cell physiology of airway smooth muscle, as opposed to the intact airway, where hyperresponsiveness develops? Is the length-tension relationship of isolated airway smooth muscle actually altered?

Aharony: Based on studies by Dr. Carl Buckner, that is not the case. We have been unable to show any evidence for alteration of leukotriene receptors or the basic physiology of smooth muscle.

Drazen: The experiment that needs to be done would be to culture airway smooth muscle with leukotrienes for a prolonged period of time. The kinds of studies that you did with Dr. Buckner were short-term incubations in tissue baths.

Peters-Golden: You touched on a large body of work showing that various cytokines affect leukotriene production. However, I have seen very little work on the effect of cytokines on leukotriene receptors. Has this been studied?

Busse: I don't think that this has been looked at. Some of the effects on cysteinyl leukotriene generation appear to be via induction of the 5-LO enzyme, itself, intracellularly.

Drazen: That area has really been held up by the lack of a molecular definition for the leukotriene receptor.