Challenging the evidence for hepoxilin A₃ being a mediator of neutrophil epithelial transmigration

to the editor: A series of papers in the literature, beginning in 2004 and up to the present, implicate the eicosanoid hepoxilin A₃ (HxA₃) as the mediator of neutrophil chemotaxis produced by lung or gastrointestinal (GI) epithelial cells in response to bacterial infection (1, 2, 4–6, 8, 9, 11, 16–18). The production of HxA₃ and its biological activity as a chemotactic factor is thus cited as a key final step in the trans-epithelial polymorphonuclear leukocyte (PMN) recruitment to sites of mucosal inflammation and an important component of the inflammatory response. Biosynthesis of the chemotactic mediator is proposed to involve the release of arachidonic acid and its transformation by 12-lipoxygenase via the intermediate 12-hydroperoxy-eicosatetraenoic acid (12-HPETE), producing the epoxy-hydroxy derivative HxA₃. The following review of the published evidence challenges the contention that HxA₃ is correctly identified as the mediator of this chemotactic response in the lung and GI epithelium.

A critical review of the evidence finds shortcomings in that, 1) in the initial publication (9) the isolated chemotactic factor was not compared in analytical properties to HxA₃ (or to any authentic standard) nor was the isolated factor shown to possess biological activity; an early report of the same or similar analyses states that 210 pmol HxA₃ (71 ng) was detected in stimulated epithelial cell monolayers and <1 pmol in controls, but no experimental recordings are presented (5) and in subsequent papers using quantitative LC-MS assays the amounts of HxA₃ are reduced by approximately a thousand-fold (albeit from unspecified volumes or numbers of epithelial cells) (4, 17). HxA₃ is a mixture of two chromatographically separable hydroxy isomers (8R- and 8S-hydroxy) (10), whereas a single peak was identified as the PMN migration mediator (9). PLA₂ was implicated in the pathway although HxA₃ formation was not demonstrated (7). 2) In the original publication (9) the possibility that leukotriene B₄ (LTB₄) could be the natural factor was dismissed on the grounds that, in addition to its chemotactic activity, LTB₄ degranulates leukocytes whereas the natural factor does not; however, compared with its potent chemotactic activity, LTB₄ is a weak neutrophil secretagogue (3, 13, 14), and in the 2004 Proceedings of the National Academy of Sciences USA paper it was tested at 500 ng/mL (9), about three orders of magnitude higher than the concentration required to exhibit chemotaxis. 3) Authentic HxA₃ exhibits weak chemotactic activity and in comparable dose-response curves is about 100–1,000 times less potent than LTB₄ and lower in efficacy (6); 4) in line with this difference in potency, when authentic standards of the two were used in a neutrophil transmigration assay, the concentrations employed were 5 ng/mL (15 nM) LTB₄ and 16.7 μg/mL (50 μM) HxA₃ (8). 5) Evidence accrued using lipoxygenase inhibitors is of dubious value on account of the lack of specificity and general antioxidant activities of available inhibitors (e.g., Refs. 12, 15) and requires the use of “add back” experiments of the putative mediator, which has not been used to support the validity of this hypothesis. 6) When tested, the effect of mouse knockout on neutrophil migration via the putative hepoxilin synthesis pathway (Alox15^−/−) did not attain statistical significance (1).

The conclusion of this analysis is that the chemotactic factor(s) detected in bacterial-induced inflammatory epithelial exudates in the lung and GI tract is/are not identified, but on multiple grounds of evidence the factor is unlikely to be hepoxilin A₃.

GRANTS

A.R.B. is supported in this analysis by the National Institute of General Medical Sciences of the NIH under award number R01GM134548.

DISCLAIMERS

The content is solely the responsibility of the author and does not necessarily represent the official views of the NIH.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author.

AUTHOR CONTRIBUTIONS

A.R.B. drafted manuscript; A.R.B. edited and revised manuscript; A.R.B. approved final version of manuscript.