In this study, we have identified a critical role for neutrophils in allergic skin inflammation in a mouse model with features of AD. We demonstrated that LTB4 production by neutrophils was essential for their accumulation in mechanically injured skin and for the recruitment of effector CD4+ T cells to cutaneous sites of antigen exposure and development of allergic skin inflammation.
Mechanical skin injury by scratching in humans, and its surrogate, tape stripping in mice, caused robust infiltration of neutrophils and accumulation of LTB4
and mRNA for its receptor, BLT1, in the dermis. The slight increase in epidermal thickness in injured human and mouse skin is possibly due to the release of cytokines that promote keratinocyte proliferation, which include IL-6 (Werner et al., 2007
). Accumulation of neutrophils and BLT1 mRNA was substantially greater in tape-stripped mouse skin than in scratched human skin, possibly because mouse skin is much thinner than human skin and might be more susceptible to mechanical skin injury. The relatively comparable elevation of LTB4
concentrations in tape-stripped mouse skin and scratched human skin suggests a more rapid washout of LTB4
from injured mouse skin. The baseline concentration of LTB4
may reflect a background signal intrinsic to the assay used and/or be derived from resident cells in the skin and may have not been biologically active, since neutrophils were not detected in unmanipulated skin. Studies in Lta4h−/−
mice demonstrated that LTB4
and BLT1 are both important for neutrophil accumulation into tape-stripped mouse skin. The role of LTB4
in neutrophil recruitment to the skin is supported by the observation that epicutaneous application of LTB4
in normal healthy volunteers results in local neutrophil infiltration (de Jong et al., 1992
induces itch-associated scratching response in mice (Kim et al., 2007
). Tape stripping induced significantly more scratching in WT mice than in Ltb4r1−/−
mice, suggesting that LTB4
is important in the scratching response to mechanical injury.
Neutrophils, but not mast cells, were the major source of the increased LTB4
in mouse skin 24 hrs following mechanical injury. LTB4
failed to accumulate in mice depleted of neutrophils using Gr-1 mAb or CTX, but accumulated normally in the mechanically injured skin of mast cell-deficient KitW/KitW-v
mice. Although mast cells could release LTB4
early after mechanical injury, mast cell-derived LTB4
was clearly not essential for neutrophil influx into mechanically injured skin. This is consistent with our previous observation that allergic skin inflammation elicited by EC sensitization of tape-stripped skin with OVA is intact in mast cell-deficient KitW/KitW-v
mice (Alenius et al., 2002
Adoptive transfer experiments demonstrated that neutrophil influx in tape-stripped mouse skin was largely dependent on BLT1 and on both paracrine and autocrine actions of neutrophil-derived LTB4
played a role in neutrophil recruitment to the skin in response to bacterial infection and sterile wounding. In addition, neutrophil-derived LTB4
is required for the recruitment of neutrophils to the joints and for the development of inflammatory arthritis (Chen et al., 2006
; Chou et al., 2010
; Kim et al., 2006
). These observations suggest that LTB4
generated by neutrophils is important for their recruitment to the skin in response to a variety of inflammatory stimuli, as well as for their recruitment to other tissues.
Additional factors, which include inflammatory chemokines and cytokines, produced by resident skin cells may contribute to the initial neutrophil influx in the tape-stripped skin, as suggested by the residual neutrophil infiltrate observed of Ltb4r1−/−
mice. Furthermore, production of IL-1b and IL-6 by skin infiltrating WT neutrophils may explain the finding that Ltb4r1−/−
neutrophils could be detected in tape-stripped skin when co-transferred with WT neutrophils, but not when transferred alone. Both the rapid induction (within 6 hrs) of CXCL2, IL-1b and IL-6 mRNA and neutrophil influx into tape-stripped skin, were blunted in Myd88−/−
mice. This observation suggests that signaling via TLR receptors, possibly by endogenous alarmins (Guiducci et al., 2010
) and/or signaling via IL-1 receptor (IL-1R) family members released from damaged keratinocytes, plays an important role in the initial neutrophil influx into injured skin.
A central finding of the present study is that CD4+ T effector cells from OVA-sensitized WT mice fail to transfer allergic inflammation to Ltb4r1−/− recipients. Taken together with the observation that Ltb4r1−/− CD4+ T cells fail to transfer allergic inflammation, this result indicates that the expression of BLT1 on both non-T cells and T cells is important for allergic skin inflammation. Co-transfer of WT neutrophils, but not Ltb4r1−/− neutrophils, rescued the ability of CD4+ effector T cells to accumulate in antigen-challenged skin of Ltb4r1−/− recipients and cause allergic skin inflammation. Intradermal administration of WT, and Ltb4r1−/−, but not Lta4h−/−, neutrophils rescued the ability of WT CD4+ effector T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. This finding demonstrates that neutrophil-derived LTB4 is critical for the ability of CD4+ effector T cells to cause allergic skin inflammation. These observations demonstrate that LTB4 production by neutrophils is critical both for the BLT1 dependent accumulation of neutrophils and the BLT1 dependent recruitment of antigen specific effector CD4+ T cells to cutaneous sits of antigen introduction.
The LTB4 synthesis inhibitor bestatin blocked neutrophil influx into tape-stripped skin and inhibited the development of allergic inflammation at sites of cutaneous antigen challenge in mice. This result suggests that blockade of LTB4-BLT1 interaction may be a useful strategy in preventing acute flares that are precipitated in quiescent skin in patients with AD by scratching and introduction of previously encountered antigen.
concentrations were elevated in OVA sensitized mouse skin sites, as in AD skin lesions, and mice EC-sensitized with OVA exhibited increased scratching. However, unlike AD skin lesions, which contain few, if any, neutrophils (Leiferman, 1994
), EC-sensitized mouse skin sites exhibit neutrophil infiltration (Spergel et al., 1998
). Neutrophils, like eosinophils, may not persist intact in chronic AD lesions. It thus remains possible that scratching established skin lesions induces a transient acute neutrophil influx, which triggers LTB4
-dependent recruitment of BLT1+
effector T cells, resulting in the exacerbation of allergic skin inflammation.
Scratching is a hallmark of AD and causes further disruption of an already abnormal skin barrier, allowing cutaneous introduction of antigen. Our finding that the LTB4-BLT1 axis is implicated in two sequential steps in allergic inflammation at sites of cutaneous antigen exposure, namely recruitment of neutrophils and neutrophil-dependent recruitment of effector T cells, make the LTB4-BLT1 axis a potential therapeutic target in AD.