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Referee comments: Referee 1 (Gerard Nash)

Posted by PLOS_ONE_Group on 25 Feb 2008 at 13:24 GMT

Referee 1's review (Gerard Nash):

General comments:
This paper reports parallel studies of neutrophil migration from microvessels and leakage of fluorescent albumin, in wild type or CD11b-deficient mice. The authors have reported previously that there is a delay in migration in the deficient mice, which is linked to the neutrophils migrating through the endothelial cells (transcellular route) rather than between them (paracellular route). Here it is shown that this there is also a delay in leakage of albumin, but that ultimately leakage (and total migration) are similar for the different mice. The authors originally hypothesised that there would be greater leakage for cells following the transcellular route, but they found that endothelial cells formed a dome over migrating neutrophils, which presumably limited loss of plasma protein during migration. In fact similar structures formed for neutrophils following the paracellular route, suggesting that such domes limit leakage for all migratory routes.

The findings are interesting and clearly described, and shed new light on the mechanisms controlling migration and vascular leakage in inflammation. A limitation is that the migration is directly induced by a chemotactic agent, MIP-2, acting on the neutrophils which were presumably otherwise rolling on the vessel wall. As the authors point out, recent interest in the transcellular route of migration started because of studies by Dvorak and colleagues, where neutrophil migration was induced by injection of chemotactic bacterial peptide into guinea pigs. Thus it is not clear that similar observations would apply for inflammation driven by tissue insult (such as ischaemia and reperfusion) or by cytokine(s) acting on endothelial cells.

Specific points:

The limitation linked with use of MIP-2 and the relevance to other inflammatory processes should at least be discussed.

The significance and meaning of the studies with low dose of MIP-2 need clarification. The data for low dose of MIP-2 don't show a delay in migration for the CD11b-/- mice, and the permeability changes are not so much subtle (as stated) but possibly non-existent. For the CD11b-/- mouse there seems to be a difference in permeability at 30min because permeability has decreased, and overall there is no increase in permeability compared to time zero in these animals.
With respect to the permeability analysis, it is not clear what the permeability increase is relative to, in any case. Clearly it is not relative to time zero in the same mouse, and permeability seems to be elevated at time zero in the treated mice (i.e., before treatment). The permeability analysis could be clarified.

In the abstract, it should be made more clear where the authors move from describing the previous studies to the new ones, and that migration here was induced by topical application of MIP-2.

Please state the numbers of mice compared in the various parts of the study.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.