Discussion
We have shown that the basal generation of the proinflammatory LTB4 and the anti-inflammatory LXA4 were both lower in cultured AMs from severe asthmatics compared to those from non-asthmatics, while only LXA4 was lower in severe asthmatics compared to non-severe asthmatics. The LPS induced formation of LTB4 was higher in severe asthma compared to non-severe asthma and normal subjects, while the LPS induced production of LXA4 was significantly impaired in severe asthmatics compared to normal subjects, but to a similar extent as in non-severe asthma patients. The overall effect of LPS stimulation was a net pro-inflammatory balance in terms of enhanced generation of LTB4, and a decrease in LXA4 compared to AMs from normal subjects. In addition, while the LPS-induced LTB4 was largely suppressed by dexamethasone, it was only partly suppressed in AMs from severe asthma patients; by contrast, induced generation of LXA4 was suppressed in all three groups. Therefore, the overall balance of these 2 lipid mediators in severe asthma was in favour of an overall pro-inflammatory response through both increased production and relative corticosteroid insensitivity of LTB4 and decreased levels of LXA4 in severe asthmatics, as illustrated by the LTB4 to LXA4 ratios.
Human AMs can generate both 5-lipoxygenase and 15-lipoxygenase derived eicosanoids, including LTB
4 and LXA
4, from endogenous sources of arachidonic acid[
13]. LXA
4 generation from endogenous stores is low, but LX biosynthesis can be amplified by select TH2 cytokines, namely interleukin-4 and interleukin-13 [
14,
15]. In addition, exogenous LTA
4 can be converted by AMs to more substantial amounts of LXs [
16], as would occur during transcellular biosynthesis with LTA
4 donation from one cell to a second cell for enzymatic conversion by either 12- or 15-lipoxygenase to LXs. Our studies are the first to document both LTB
4 and LXA
4 generation from human AMs from asthmatic subjects. Although the levels of LXA
4 are low, they were detected reproducibly, validated with authentic material and picogram quantities of LXs are biologically active in resolving inflammation (reviewed in reference [
17]). Interestingly, when the data was expressed as a ratio of pro-inflammatory LTB
4 to anti-inflammatory LXA
4, there was an increased pro-inflammatory imbalance when the macrophages from both asthmatic groups were exposed to LPS, and this was not reversed by corticosteroids. Indeed, in macrophages from severe asthma, this pro-inflammatory ratio favouring LTB
4 over LXA
4 was further unbalanced by dexamethasone.
Our results indicate that the pulmonary macrophage can be an important source of lipid mediators, and that differences in LTB
4 and LXA
4 between the asthmatic groups are in general agreement with recent studies that have examined levels in whole blood[
9,
18] and BAL fluids [
10]. In the study of
Wenzel et al, levels of LTB
4 in BAL fluid from severe asthma were the highest compared to levels from moderate symptomatic asthma patients and normals [
19]. This indicates that the baseline contribution of LTB
4 from macrophages is unlikely to explain this increased levels found in BAL of severe asthma patients; however, following
ex vivo stimulation of macrophages from severe asthma patients, greater levels of LTB
4 were released compared to non-severe asthma patients. Using a similar method as our study to distinguish severe from non-severe asthma, a deficiency in both baseline and divalent cation ionophore-stimulated production of LXA
4 in whole blood of patients with severe asthma compared to moderate asthma was established, while the production of cysteinyl-leukotrienes and LTB
4 were increased[
9]. In addition, similar findings have been reported in airway fluids for the levels of these lipid mediators; thus, an increase in LTB
4 levels was found in the supernatant of induced sputum of severe asthma patients compared to non-severe asthma. In these same samples, LXA
4 levels were highest in the mild asthma group[
20,
21]. Moreover, LXA
4 levels in BAL fluids from patients with severe asthma recruited in the NHLBI Severe Asthma Research Program are decreased compared to non-severe asthma patients, and BAL cells from severe asthma patients had increased 5-LO and decreased 15-LO expression [
10]. These results are in line with the current observation of reduced basal and LPS-stimulated production of LXA
4 from alveolar macrophages of patients with severe asthma. In conjunction with the large number of alveolar macrophages in healthy and asthmatic lung, these observations provide support to the idea that the alveolar macrophage is a likely important source of LXA
4 in human airways.
There have been very few studies of the effect of LPS on human macrophages in terms of LT and LX generation. Brief exposure of murine macrophages to LPS can prime them to increase LT synthesis in response to an activating stimulus such as immune complexes or divalent cation ionophore A23187[
22], an observation that has been subsequently shown in human AMs [
23]. On the other hand, prolonged exposure to LPS impaired the capacity of rat macrophages to produce LTs in response to stimulating agents, a process that was due to the production of inhibitory substances such as nitric oxide [
24,
25]. LPS can induce human AM phagocytosis of apoptotic cells, but AMs from subjects with severe asthma display defective clearance mechanisms and lower levels of PGE
2 and 15-hydroxyeicosatetraenoic acid formation in response to LPS[
26]. Both PGE
2 and 15-HETE can play pivotal roles in establishing LX biosynthesis [
27,
28].
Differences in basal LTB
4 from AMs have not been previously observed between asthma and normal atopic or non-atopic control subjects[
29], or those with nocturnal asthma [
30]. However, asthmatic subjects in these studies would not have met the NHLBI Severe Asthma Research Program's criteria for severe asthma [
2]. Regarding calcium ionophore-induced LTB
4 biosynthesis by AMs, one study reported increased LTB
4 generation by cells in asthma compared to non-asthmatics [
31], while another study did not report any significant differences[
29]. Our study shows reduced baseline LTB
4 in non-severe asthma and no significant differences in stimulated production by LPS compared to non-asthmatics. Because LTB
4 biosynthesis by AMs
in vitro can be modulated by environmental factors
in vivo, such as cigarette smoking [
32], smokers were excluded from the study.
Glucocorticoids have been shown to inhibit LT generation through inhibition of phospholipase A
2 activity [
33,
34]. Chronic oral corticosteroid therapy may lead to a suppression of eicosanoid biosynthesis and could underlie the baseline reduction in LXA
4 and LTB
4 observed in the macrophages from patients with severe asthma. Both LTB
4 and LXA
4 stimulated by calcium ionophore in the circulating neutrophil was reduced in corticosteroid-dependent asthmatics who were on oral prednisolone [
35]. However, as far as the AM is concerned, there was no significant inhibition of LTB
4 from AMs of normal subjects treated with oral prednisolone despite the fact that direct incubation of these cells with dexamethasone leads to an inhibition of basal and calcium ionophore triggered formation of LTB
4 [
36]. Other work also indicate that oral short-term treatment with prednisone does not inhibit the levels of the eicosanoids, PGD
2, 5-HETE and LTE
4, in BAL from asthmatic subjects at baseline or after allergen challenge [
37]. However,
ex-vivo treatment of BAL cells with prednislone did cause inhibition of LTB
4 and thromboxane generation. Similarly, in the work of Wenzel
et al, a single dose of oral prednisone inhibited LTB
4 release from alveolar macrophages from patients with nocturnal asthma but not from those without nocturnal asthma [
30]. Only half of the patients with severe asthma in this study were on oral corticosteroid therapy and there was no significant differences in terms of LPS-induced LTB
4 or LXA
4 or of dexamethasone-induced suppression between macrophages of severe asthma patients who were on prednisolone versus those not on prednisolone. Similarly, in the non-severe asthma group, there was no difference in terms of LPS-induced LTB
4 or LXA
4 or of dexamethasone-induced suppression between macrophages of non-severe asthma patients who were on daily inhaled corticosteroids versus those not on inhaled corticosteroids. However, the influence of long-term oral or inhaled corticosteroid therapy, as contrasted to short-term, on this
ex-vivo production of arachidonic acid-derived mediators cannot be entirely excluded.
We have elected to group our asthmatic patients as non-severe and severe asthma patients on the basis of the definition of severe asthma proposed by the ATS [
12]. This definition of severe asthma is based on the lack of control of asthma despite taking maximal anti-inflammatory treatments with corticosteroids, while the non-severe asthma patients were those on no or only low-dose inhaled corticosteroids. We observed that there was relative CS insensitivity of LTB
4 generation but not of LXA
4 from AMs of patients with severe asthma. Previously, no differences in CS sensitivity of AMs in terms of calcium ionophore induced LTB
4 from asthmatics as compared to non-asthmatic macrophages have been reported[
29]. In a previous study, we have shown that AMs from patients with severe asthma demonstrate a reduced sensitivity to dexamethasone in terms of LPS-induced release of pro-inflammatory cytokines [
5].
Our data on LXA
4 is one of the first regarding its stimulated production by LPS, and its suppression by dexamethasone. Levels of anti-inflammatory LXs were low in severe asthmatics, and did not increase in response to LPS stimulation, further increasing the disparity between severe asthmatics and non-severe asthmatics in the levels of these mediators. Moreover, dexamethasone suppressed LPS-induced increases in LXA
4 in all groups. This differential response of AMs from severe asthmatics vis-a-vis LTB
4 and LXA
4 and the effect of corticosteroids (increased LTB
4 in response to LPS and impaired CS suppression of the rise in LTB
4 vs. little change in LXA
4 in response to LPS and unimpaired CS suppression of LXA
4 levels) may contribute to persistent airway neutrophilic inflammation since LXA
4 can inhibit LTB
4-induced chemotaxis, adhesion and transmigration[
17]. This potential role of LXA
4 in regulating neutrophil chemotaxis is supported by the inverse relationship between baseline LXA
4 and the percentage of neutrophils in bronchoalveolar lavage fluid.
Lipoxins are a distinct class of eicosanoids with anti-inflammatory properties at subnanomolar concentrations. Thus, although the basal and stimulated levels of LXA
4 from alveolar macrophages are in low picogram amounts, these levels would be predicted to have pro-resolving actions for airway inflammation (reviewed in [
17]. In support of the protective effect of LXA
4, we found a positive correlation between LPS-induced LXA
4 and lung function as represented by FEV
1. Indirectly, this protection in lung function may occur through an effect on neutrophilic inflammation, since there was an inverse correlation between BAL neutrophilia and FEV
1. LXA
4 can inhibit LTB
4-initiated chemotaxis, adhesion and transmigration. In addition, LXA
4 inhibits eosinophilic allergic inflammation [
38]. Thus, a possible imbalance in LTB
4 and LXA
4 in the airways would serve to increase airway neutrophil and eosinophil accumulation and activation. Interestingly, a similar imbalance between LT and LX generation is present in scleroderma lung disease [
39] and decreased lipoxin production has also been reported in inflammatory bowel disease [
40].
One of the potential limitations of our work regards the relative age of the healthy control group that were younger than the asthma groups. Generation of LXA
4 can decrease and LTB
4 increase with age [
41,
42], but there is no information available at present on the influence of age on the release of these eicosanoids from human alveolar macrophages. While there may be uncertainty about the effect of age, we are able to compare the non-severe with the severe group of asthmatic subjects as they were of comparable age group.
In summary, we demonstrate impaired corticosteroid modulation of the pro-inflammatory lipid mediator LTB4 but not of the anti-inflammatory lipoxin, LXA4, in AMs of severe asthma. Together with the augmented LPS induced formation of LTB4 and decreased generation of LXA4 in severe asthma, our observations indicate a net pro-inflammatory imbalance in severe asthma.
Authors' contributions
KFC and BDL conceived the study, PB and MH collected the samples, MP, SK and BDL did the measurements of lipoxins, PB, BDL, EI and KFC wrote the manuscript. All the authors have read the and approved the final manuscript.