Results

PMNs express and release several cytokines among which interleukin (IL)-1β is a potent proinflammatory agent.16 We have recently investigated the actions of native LXA₄ and its stable analogues on TNFα induced IL-1β release.13 PMNs from healthy donors were exposed to increasing concentrations of 15R/S-methyl-LXA₄ or native LXA₄ in the presence of TNFα (25 000–50 000 activity units/ml) or vehicle alone. At a concentration of 100 nM, 15R/S-methyl-LXA₄inhibited approximately 60% of IL-1β release, a value which is comparable to that obtained with native LXA₄. Inhibition of IL-1β by LXA₄ and its stable analogue was, at least in part, the result of a downregulation in gene expression, since the IL-1β messenger RNA levels in cells treated with TNFα (10 ng/ml) plus 15R/S-methyl-LXA₄ (100 nM) were decreased by approximately 60% compared with cells treated with TNFα alone.13 Since IL-1β and TNFα are two cytokines considered to be important in inflammation, the inhibition of IL-1β suggested that ATL might exert an in vivo anti-cytokine action.

TNFα evokes leucocyte infiltration in a chemokine-dependent fashion in the murine six day air pouch.15 We therefore evaluated the impact of ATL stable analogues 15R/S-methyl-LXA₄ and 15-epi-16-(para-fluoro)-phenoxy-LXA₄ in this model to determine whether they also intersect the cytokine/chemokine axis in vivo and inhibit leucocyte infiltration.13 ,14 Murine TNFα gives a transient infiltration of leucocytes to the pouch cavity in a time-dependent fashion with maximal accumulation at four hours.13 15R/S-methyl-LXA₄ at a concentration of 25 nmol inhibited the TNFα stimulated recruitment of leucocytes by approximately 62%. Inhibition was evident at one hour and was maximal at 2–4 hours. At these intervals a reduction in leucocyte infiltration of more than 60% was noted that remained significantly reduced at eight hours. Inflammatory exudates were collected four hours after injections and trafficking of cell types into the cavity was measured. In the six day pouches given TNFα, PMNs constituted the major cell type present within the exudates at four hours with a range of 80–85%. Administration of both 15R/S-methyl-LXA₄ and TNFα into the six day air pouch cavity inhibited migration of PMNs, eosinophils, and basophils as well as mononuclear cells.13The fluorinated ATL analogue was compared with aspirin in this model and, when injected into the pouch with TNFα, more than 75% inhibition was achieved with 10 μg. This analogue also showed increased stability in whole blood (mouse and human) and intravenous administration was as effective as administration into the air pouch (topical). This stable ATL analogue was also about 100 times more potent than aspirin in blocking PMN influx to the pouch.14

Since macrophage inflammatory peptide (MIP)-2 is the major chemokine involved in recruiting PMNs to the pouch cavity following injection of TNFα,15 we determined the action of 15R/S-methyl-LXA₄ in this TNFα induced chemokine-cytokine axis. MIP-2 and IL-1β are important proinflammatory cytokines, and IL-4, IL-10, and IL-13 possess immunomodulatory properties. Exudates from selected time intervals were collected and cell-free supernatants assessed for the presence of these murine cytokines. 15R/S-methyl-LXA₄ (25 nmol) inhibited TNFα stimulated MIP-2 and IL-1β release by 48% and 30%, respectively (fig 2). 15R/S-methyl-LXA₄ alone in the air pouch did not stimulate MIP-2 or IL-1β release. In sharp contrast, 15R/S-methyl-LXA₄ stimulated the appearance of IL-4 within the exudates. This stimulation of IL-4 was observed both with and without TNFα. Neither IL-10 nor IL-13 was detected within the pouch exudates. These results show that administration of 15R/S-methyl-LXA₄ dramatically modified the cytokine/chemokine axis in TNFα initiated acute inflammation and, interestingly, this re-orientation of the cytokine/chemokine axis paralleled the reduction in leucocyte infiltration.

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Figure 2

15R/S-methyl-LXA₄ redirects the TNFα induced cytokine/chemokine profile in vivo. Sterile PBS (∼1 ml) containing either 0.1% ethanol, TNFα, 15R/S-methyl-LXA₄, or TNFα + 15R/S-methyl-LXA₄ was injected into the pouches and individual lavage exudates were collected at the indicated time periods. Measurements were performed with air pouch cell-free lavage exudates as described by Hachicha et al.13 The results are expressed as the % change compared with the quantities of cytokines found in TNFα injected air pouches in the absence of LXA₄(mean (SE) from three different mice for each time interval). Changes in IL-1β, MIP-2, and IL-4 secretion were statistically significant at all tested time intervals (p<0.01). At one hour the air pouches injected with TNFα alone generated 384 (12) pg MIP-2/pouch and 14.9 (2.3) pg IL-1β/pouch. 15R/S-methyl-LXA₄ alone generated 42.7 (0.7) pg IL-4/pouch.

Discussion

LXA₄ and ATL (generated by separate biosynthetic mechanisms in vivo; fig 1 and Serhan et al 10-12), as evidenced by the actions of the metabolically stable analogue 15R/S-methyl-LXA₄, are both potent cytokine regulating lipid mediators that can also have an impact on the course of inflammation initiated by TNFα. In this exudate and skin wound model,13 15R/S-methyl-LXA₄ not only inhibited TNFα elicited PMNs, monocytes, basophils, and eosinophils as well as the appearance of IL-1β and MIP-2, but also stimulated IL-4 appearance within the pouch. These results provide the first evidence to indicate that lipoxins and ATL induce upregulation of potential “anti-inflammatory” cytokines such as IL-4.17 It is likely that both the inhibition of IL-1β and MIP-2 within exudates and IL-4 levels in the surrounding tissues by metabolically stable LX analogues may represent, in part, some of the in vivo impact of LXA₄ and aspirin triggered 15-epi-LXA₄. These findings provide a new understanding of the relationship between “anti-inflammatory” cytokines and lipid mediators and also open new avenues to investigate protective lipid and protein mediators in host defence.

In summary, these results suggest that LXA₄ and aspirin triggered LXA₄ appear to be involved in controlling not only acute inflammatory responses, but also mechanisms that can influence chronic inflammatory responses. The recent results reviewed here also support the notion that aspirin may exert its beneficial action in part via the biosynthesis of endogenous ATLMs such as 15-epi-LXA₄ that can, in turn, act directly on PMNs as well as affect the appearance of chemokines and cytokines. Thus, LX-ATL can protect host tissues via multi-level regulation of proinflammatory signals and may be relevant new lipid mediators of interest in airway diseases.