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. 2015 Jun 29;146(1):50–58. doi: 10.1111/imm.12478

Leukotriene B4—leukotriene B4 receptor axis promotes oxazolone-induced contact dermatitis by directing skin homing of neutrophils and CD8+ T cells

Jiaoyan Lv 1, Linlin Zou 1, Lina Zhao 1, Wei Yang 1, Yingluo Xiong 1, Bingji Li 1, Rui He 1,2,✉
PMCID: PMC4552500  PMID: 25959240

Abstract

Leukotriene B4 (LTB4) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8+ T cells. The role of the LTB4–BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8+ T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1β. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8+ T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1β. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8+ T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4–BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8+ T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis.

Keywords: CD8 T cells, elicitation phase of contact dermatitis, leukotriene B4–BLT1 axis, neutrophils, skin homing

Introduction

Contact dermatitis is a common inflammatory skin disease. Murine contact hypersensitivity induced by chemicals as the hapten is a T-cell-mediated inflammatory response and widely used as a model of contact dermatitis to investigate the underlying mechanism of the aberrant inflammatory response.1 The development of contact hypersensitivity includes the sensitization phase and the elicitation phase.2 During the sensitization phase, skin dendritic cells take up the exposed antigen, migrate to the draining lymph nodes where they prime the naive T cells to antigen-specific effector T cells. During the elicitation phase, hapten challenge of sensitized mice induces the rapid recruitment of the inflammatory immune cells to the challenged skin.2

Hapten-primed CD8+ T cells and neutrophils are two major types of inflammatory cells infiltrating in hapten-challenged skin,1,3 contributing to the inflammatory response. A variety of inflammatory cytokines have been suggested to temporally regulate the recruitment of CD8+ T cells and neutrophils to hapten-challenged skin during the elicitation phase. For example, interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α) have been reported to be required for the skin recruitment of hapten-specific effector CD8+ T cells, which, in turn, produce interferon-γ (IFN-γ) and IL-17A to stimulate the production of the neutrophil chemokines CXCL1 and CXCL2.4 CXCR3 and its ligands, CXCL9 and CXCL10, are also documented as being crucial for mediating the skin homing of hapten-primed CD8+ T cells.5,6 Interestingly, the intensity of neutrophil infiltration was reported to control the number of hapten-primed CD8+ T cells infiltrating in hapten-challenged skin.3 Recent study further suggested that neutrophil expression of Fas ligand and perforin could promote CD8+ T-cell infiltration by inducing T-cell chemokine production in hapten-challenged skin.7 These results together suggest a complex and dynamic cytokine network in the regulation of the skin recruitment of hapten-primed CD8+ T cells and neutrophils following hapten challenge during the elicitation phase.

Leukotriene B4 (LTB4) is an important lipid mediator rapidly generated from arachidonic acid through the sequential enzymatic actions of 5-lipoxygenase (5-LO) and leukotriene A4 hydrolase (LTA4H) at sites of acute inflammation.8 Leukotriene B4 is primarily produced by innate immune cells such as neutrophils, macrophages and mast cells in response to a variety of stimuli.9 BLT1 is the functional receptor of LTB4 with high affinity, and is predominantly expressed on leucocytes.10 The LTB4–BLT1 axis has been reported to be involved in the progression of several inflammatory diseases, such as rheumatoid arthritis, asthma and atopic dermatitis by controlling tissue recruitment of different inflammatory cells.11–13 Our previous study revealed a dual role of the LTB4–BLT1 axis in the different phases of acute lung injury, either pro-inflammatory by mediating airway recruitment of neutrophils in the initiation phase or anti-inflammatory by mediating airway recruitment of regulatory T cells in the resolution phase.14 However, little is known about the role of the LTB4–BLT1 axis in contact dermatitis and the underlying mechanism.

We here used BLT1−/− mice and the specific antagonist to block the BLT1 or LTB4 during the elicitation phase to investigate the influence of the LTB4–BLT1 axis on an oxazolone (OXA)-induced mouse model of contact dermatitis.

Materials and methods

Mice

Breeding pairs of BLT1−/− mice on the C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in the animal facility of Fudan University (Shanghai, China). Wild-type (WT) C57BL/6 mice that were purchased from the Chinese Academy of Science (Shanghai, China) and the WT littermates were used. All mice used were female, 6–8 weeks of age (18–20 g) and housed under specific pathogen-free barrier conditions. All experiments were performed according to the Animal Care and Use Committee at Fudan University.

Mouse model of OXA-induced contact dermatitis

A mouse model of contact dermatitis was induced with OXA (Sigma, St Louis, MO) as previously described.15 On day 0, mice were topically sensitized with 50 μl 2% OXA in acetone/alcohol (1 : 3) on the shaved abdomen. Five days later, mice were topically challenged with 25 μl 1% OXA on the right ear and a comparable volume of vehicle on the left ear. Measurement of ear thickness was performed with an engineer’s micrometer (Mitutoyo, Kawasaki, Japan) both before and at the indicated time-points after OXA challenge. Mice were killed with sodium pentobarbital via intraperitoneal injection at the indicated time-points.

BLT1 and LTB4 blockade by U-75302 and bestatin treatment

U-75302, a selective BLT1 antagonist, (10 μg/mouse) (Cayman, Ann Arbor, MI) or vehicle (ethanol) was topically applied to ear skin 3 and 8 hr after OXA challenge. In some experiments, bestatin (0·5 mg/mouse) (Cayman) or vehicle (DMSO) (Sigma, St Louis, MO) were similarly applied to both sides of the OXA-challenged ear.

Neutrophil depletion and subcutaneous injection of LTB4

Neutrophils were depleted by 75 μg anti-Gr-1 monoclonal antibody (clone RB6-8C5) (eBioscience, San Diego, CA) via intravenous injection 24 hr before OXA challenge. Mice treated with isotype antibody anti-mouse IgG2b served as control. About 97% of neutrophils were depleted from blood 24 hr later, and this high depletion efficiency was maintained for at least 48 hr. In some experiments, LTB4 (1 μg) (Cayman) in 15 μl PBS was subcutaneously injected into OXA-challenged skin of neutrophil-depleted mice. Ears were similarly treated with PBS served as controls.

Quantification of LTB4 in ear tissue

Total ear tissue was removed from the mice and stored at −80° until use. LTB4 was extracted from weighed ear tissue and measured using the LTB4 enzyme immunoassay kit (Cayman).

Histopathology

Ear tissues were harvested 24 hr after OXA challenge, fixed in 10% formalin and embedded in paraffin. Multiple 4-μm sections were stained with haematoxylin & eosin (H&E) for histopathological analysis.

Flow cytometry assay and ELISA

Mouse ears were separated into two parts and immersed in 0·25% Trypsin for 30 min at 37°C. Then the ears were cut into small pieces in RPMI-1640 medium with 10% fetal calf serum and shaken for 1 hr. Then, the digested skin cells were resuspended into PBS. The single cells were stained with anti-Gr-1 phycoerythrin, anti-CD11b FITC, anti-CD4 phycoerythrin/FITC, anti-CD8 allophycocyanin (all from eBioscience).

Draining lymph nodes were culture with the stimulation of anti-CD3 (5 μg/ml) for 4 days in vitro. Then the cells were stained with anti-CD4 FITC, anti-CD8 allophycocyanin and anti-IFN-γ phycoerythrin. The protein level of IFN-γ in the supernatant of the culture medium was determined by ELISA (eBioscience).

Real-time PCR

Total RNA was extracted from homogenized skin tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the provided instructions. Complementary DNA was generated using a PrimeScript RT reagent kit (TaKaRa Bio, Otsu shiga, Japan). Real-time RT-PCR was performed with SYBR green Gene Expression assay (Applied Biosystems, Grand Island, NY). Fold change of target gene expression was calculated using the comparative method for relative quantification by normalization to the internal control β-actin. The following primers were used: β-actin, forward: 5′-CCAGCCTTCCTTCTTGGGTATG-3′, reverse: 5′-TGTGTTGGCATAGAGGTCTTTACG-3′; IL-1β, forward: 5′-ACCTGTCCTGTGTAATGAAAGACG-3′, reverse: 5′-TGGGTATTGCTTGGGATCCA-3′; CXCL1, forward: 5′-GATTCACCTCAAGAACATCCAG-3′, reverse: 5′-TGGGGACACCTTTTAGCATC-3′; CXCL2, forward: 5′-TTCCAGGTCAGTTAGCCTTG-3′, reverse: 5′-CAGACAGAAGTCATAGCCAC-3′; CXCL9, forward: 5′-TCCTTTTGGGCATCATCTTCC-3′, reverse: 5′-TTTGTAGTGGATCGTGCCTCG-3′; CXCL10, forward: 5′-CGTCATTTTCTGCCTCATCC-3′, reverse: 5′-CAGACATCTCTGCTCATCATTC-3′; IFN-γ, forward: 5′-GGATGGTGACATGAAAATCCTGC-3′, reverse: 5′-TGCTGATGGCCTGATTGTCTT-3′; 5-LO, forward: 5′-TCATTGAGAAGCCAGTGAAGG-3′, reverse: 5′-GTTGGGAATCCTGTCTGGTGA-3′; LTA4H, forward: 5′-GAGGTCGCGGATACTTGCTC-3′, reverse: 5′-CTCCTGTGACTGGACCGTG-3′; BLT1, forward: 5′-ATGGCTGCAAACACTACATCTCCT-3′, reverse: 5′-CACTGGCATACATGCTTATTCCAC-3′.

Statistical analysis

Two-tailed unpaired Student’s t-test was used for the comparison of two groups either with genotype difference or with treatment difference. A P value < 0·05 was considered statistically significant.

Results

Elevated LTB4 synthesis and skin gene expression of BLT1 in OXA-induced contact dermatitis

We first determined whether the LTB4–BLT1 axis was involved in OXA-induced contact dermatitis. Wild-type mice were sensitized with OXA and challenged with OXA or vehicle as control. Twenty-four hours after challenge, markedly elevated gene expression of 5-LO and LTA4H and LTB4 production were detected in OXA-challenged ear skin compared with that of vehicle-challenged mice (Fig.1a,b), suggesting locally elevated LTB4 synthesis in the OXA-induced contact dermatitis model. Furthermore, elevated BLT1 expression was also found in the OXA-challenged ear skin of OXA-sensitized mice, which is indicative of skin accumulation of BLT1-expressing inflammatory leucocytes (Fig.1c).

Figure 1.

Figure 1

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Elevated leukotriene B4 (LTB4) synthesis and expression of the LTB4 receptor BLT1 in oxazolone (OXA) -challenged ear skin of OXA-sensitized mice. Ears were harvested 24 hr after OXA challenge from OXA-sensitized mice. (a) The skin expression of 5-lipoxygenase (5-LO), leukotriene A4 hydrolase (LTA4H) was examined by quantitative real-time PCR. (b) LTB4 level in ear skin was examined by LTB4 enzyme immunoassay kit. (c) BLT1 expression was examined by quantitative real-time PCR. Data are representative from three independent experiments and expressed as mean ± SEM (n = 3 or n = 4 per group). *P < 0·05, ***P < 0·001.

BLT1−/− mice exhibit attenuated skin inflammation in OXA-induced contact dermatitis

We next attempted to explore the role of the LTB4–BLT1 axis in the development of OXA-induced contact dermatitis. BLT1−/− mice and genetically matched WT C57BL/6 control mice were sensitized with OXA and challenged with OXA or vehicle as control. Neutrophil infiltration and ear swelling peaked at 24 hr and then declined over time after OXA challenge, whereas the infiltration of CD4+ T cells and CD8+ T cells significantly increased at 24 hr and peaked at 48 hr (data not shown). We therefore chose to evaluate the skin inflammatory response in WT and BLT1−/− mice at 24 hr after OXA challenge. We found that BLT1−/− mice exhibited a significant reduction in ear swelling and inflammatory infiltrates in both epidermis and dermis of the OXA-challenged ear compared with WT controls (Fig.2a,b). Furthermore, we examined the proportion of infiltrating CD8+ T cells and neutrophils by flow cytometric analysis of enzymatically digested skin tissues. Significantly decreased percentages of neutrophils and CD8+ T cells were observed in OXA-challenged skin of BLT1−/− mice compared with that of WT controls (Fig.2c,d). CD4+ T cells and macrophages have also been implicated in the development of contact dermatitis,16,17 however, comparable percentages of F4/80+ CD11b+ macrophages and CD4+ T cells were found between OXA-challenged skin of WT and BLT1−/− mice (Fig.2c,d). Consistently, there was a significant decrease in gene expression of CXCL1 and CXCL2, two major chemokines for neutrophil recruitment, as well as CXCL9 and CXCL10, two major chemokines for T-cell recruitment, in OXA-challenged skin of BLT1−/− mice compared with that of WT controls (Fig.2e). Furthermore, the gene expression of IL-1β and IFN-γ, two important pro-inflammatory cytokines that are primarily produced by neutrophils and T cells, respectively, were also significantly reduced in the OXA-challenged skin of BLT1−/− mice compared with that of WT mice (Fig.2e).

Figure 2.

Figure 2

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Attenuated skin inflammation following oxazolone (OXA) challenge in OXA sensitized mice deficient for the LTB4 receptor (BLT1−/− mice). Groups of OXA-sensitized BLT1−/− and wild-type (WT) mice were challenged with 1% OXA or vehicle on day 5, and 24 hr later OXA-challenged ears were harvested. (a) Ear swelling was measured with an engineer’s micrometer. (b) Representative photomicrographs of skin sections stained with haematoxylin & eosin and examined at 200× magnification. (c, d) The percentage of Ly6G+ CD11b+ neutrophils, F4/80+ CD11b+ macrophages (c), CD8+ T cells and CD4+ T cells (d) were examined by flow cytometric assay. (e) The skin expression of CXCL1, CXCL2, CXCL9, CXCL10, interferon-γ (IFN-γ) and interleukin-1β (IL-1β) was examined by quantitative real-time PCR. Data are representative from three independent experiments and expressed as mean ± SEM (n = 6 or n = 7 per group). NS: not significant; *P < 0·05, ***P < 0·001.

Previous study demonstrated that BLT1−/− dendritic cells exhibited decreased lymphoid homing ability, which impaired T-cell priming during the sensitization phase of dinitrofluorobenzene-induced contact dermatitis.18 We also found significantly impaired T-cell priming in OXA-sensitized BLT1−/− mice, as evidenced by great decreases in percentages of IFN-γ+ CD8+ T cells (see Supplementary material, Fig. S1a) and the concentrations of IFN-γ in culture of anti-CD3 stimulated cells that were collected from skin-draining lymph nodes (see Supplementary material, Fig. S1b). Collectively, these results suggested that the attenuated skin inflammation observed in BLT1−/− mice sensitized and challenged with OXA could be the result of the impairment in T-cell priming or skin recruitment of neutrophils and CD8+ T cells during the elicitation.

BLT1 blockade during the elicitation phase attenuates skin inflammation

We next elucidated the role of the LTB4–BLT1 axis during the elicitation phase of contact dermatitis by topically applying U-75302, a selective BLT1 antagonist, or vehicle as control, following OXA challenge. U-75302 treatment significantly attenuated OXA-induced skin inflammation, as evidenced by decreased ear swelling and inflammatory infiltrates both in epidermis and dermis compared with vehicle controls (Fig.3a,b). Furthermore, significantly decreased infiltrating neutrophils and CD8+ T cells, but slightly decreased CD4+ T cells, were found in OXA-challenged ears following U-75302 treatment (Fig.3c,d). Consistently, significantly decreased expression of CXCL1 and CXCL2 was detected in OXA-challenged ears following U-75302 treatment (Fig.3e). However, the gene expression of CXCL9 and CXCL10 was comparable between U-75302 treated mice and vehicle controls (Fig.3e). U-75302 treatment also led to significantly decreased gene expression of IFN-γ and IL-1β (Fig.3e). Furthermore, we confirmed the importance of the LTB4–BLT1 axis by topically applying bestatin, the inhibitor of LTA4H, to OXA-challenged ear. Significant decreases in ear swelling, infiltrating neutrophils and the expression of cytokines were also found in bestatin-treated mice compared with vehicle controls (see Supplementary material, Fig. S2). We noted that the percentages of neutrophils varied between mice that were treated with and without vehicle. Similar proportions of infiltrating neutrophils were observed in WT mice receiving ethanol or DMSO (Fig.3c and see Supplementary material, Fig. S2b), which, however, was much lower compared with those without vehicle treatment (Fig.2c), suggesting that vehicle itself could influence skin infiltration of neutrophils following OXA challenge. Collectively, these results demonstrated that the LTB4–BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils and CD8+ T cells during the elicitation phase.

Figure 3.

Figure 3

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Treatment of U-75302, the specific LTB4 receptor (BLT1) antagonist, impairs oxazolone (OXA) -induced contact dermatitis. Groups of mice were sensitized and challenged with OXA. U-75302 or vehicle was topically applied on OXA-challenged ear 3 and 8 hr later. Ears were harvested 24 hr following OXA challenge. (a) Ear swelling was measured with an engineer’s micrometer. (b) Representative photomicrographs of skin sections stained with haematoxylin & eosin and examined at 200× magnification. (c, d) The percentage of Ly6G+ CD11b+ neutrophils (c), CD8+ T cells and CD4+ T cells (d) were examined by flow cytometric assay. (e) The skin expression of CXCL1, CXCL2, CXCL9, CXCL10, interferon-γ (IFN-γ) and interleukin-1β (IL-1β) was examined by quantitative real-time PCR. Data are representative from three independent experiments and expressed as mean ± SEM (n = 3 to n = 5 per group). *P < 0·05, **P < 0·01, ***P < 0·001.

LTB4 is primarily produced by neutrophils and directs the skin homing of CD8+ T cells in the elicitation phase of OXA-induced contact dermatitis

Neutrophils are the most abundant infiltrating cells in OXA-induced contact dermatitis, and the LTB4–BLT1 axis was originally identified as being responsible for neutrophil migration.19 We next investigated whether depletion of neutrophils during the elicitation phase was able to mimic the attenuated skin inflammation observed in U-75302-treated mice following OXA challenge. Anti-Gr-1 monoclonal antibody (clone RB6-8C5) or isotype antibody as control was intravenously injected into the mice 1 day before OXA challenge. Neutrophils were efficiently depleted, as < 1% Gr-1+ cells were detected in the blood 48 hr after the treatment with anti-Gr-1 monoclonal antibody (Fig.4a). Neutrophil depletion caused significantly decreased ear swelling and inflammatory infiltrates both in epidermis and dermis in OXA-challenged skin compared with that of control mice (Fig.4b,c). Significantly decreased percentages of CD8+ T cells, but not CD4+ T cells, were found in OXA-challenged skin following neutrophil depletion (Fig.4d). Significantly decreased gene expression of CXCL2, IFN-γ and IL-1β, but comparable expression of CXCL9 and CXCL10 was detected in neutrophil-depleted mice compared with control mice following OXA challenge (Fig.4e). However, in contrast to U-75302 treatment, CXCL1 gene expression increased in neutrophil-depleted mice (Fig.4e). A previous study showed that neutrophil is a prominent source of LTB4.20 We found that neutrophil depletion caused significantly decreased gene expression of 5-LO and LTA4H and LTB4 production in OXA-challenged skin (Fig.5a), suggesting reduced LTB4 synthesis in neutrophil-depleted mice following OXA challenge. Previous study showed that LTB4 is responsible for the recruitment of CD8+ T cells into inflammatory tissue site.21 We therefore hypothesized that decreased skin-infiltrating CD8+ T cells could be due to reduced LTB4 synthesis in neutrophil-depleted mice. To test the possibility, we subcutaneously injected exogenous LTB4 into neutrophil-depleted mice following OXA challenge. We found that administration of exogenous LTB4 completely restored skin-infiltrating CD8+ T cells, but had no effect on CD4+ T cells, in neutrophil-depleted mice following OXA challenge (Fig.5b). However, LTB4 administration failed to restore the ear swelling in those mice (Fig.5c). These results demonstrated that neutrophil is critical for OXA-induced contact dermatitis, and neutrophil-derived LTB4 mediates the skin recruitment of CD8+ T cells following OXA challenge.

Figure 4.

Figure 4

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Neutrophil depletion reduces skin infiltrating CD8+ T cells and gene expression of CXCL2 and interleukin-1β (IL-1β) during the elicitation phase. Groups of oxazolone (OXA) -sensitized mice were treated with anti-Gr-1 monoclonal antibody (mAb) or ISO-IgG2b antibody (Ab) through intravenous (i.v.) injection 1 day before OXA challenge. Mice were killed 24 hr following OXA challenge. (a) The percentage of neutrophils in the blood was measured by flow cytometry assay 48 hr after i.v. anti-Gr-1 mAb. (b) Ear swelling was measured with an engineer’s micrometer. (c) Representative photomicrographs of skin sections stained with haematoxylin & eosin and examined at 200× magnification. (d) The percentage of CD8+ T cells and CD4+ T cells in OXA-challenged ear was examined by flow cytometric assay. (e) The skin expression of CXCL1, CXCL2, CXCL9, CXCL10, interferon-γ (IFN-γ) and IL-1β was examined by quantitative real-time PCR. Data are representative from three independent experiments and expressed as mean ± SEM (n = 3 to n = 5 per group). NS: not significant; *P < 0·05, **P < 0·01.

Figure 5.

Figure 5

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Neutrophil depletion causes decreased skin infiltration of CD8+ T cells, which is completely restored by local administration of exogenous leukotriene B4 (LTB4). LTB4 or PBS was subcutaneously injected into oxazolone (OXA) -challenged ear skin immediately after OXA challenge in neutrophil-depleted mice. Ears were harvested 24 hr later. (a) The skin expression of 5-lipoxygenase (5-LO) and leukotriene A4 hydrolase (LTA4H) was examined by quantitative real-time PCR. (b) LTB4 level in ear skin was examined by an LTB4 enzyme immunoassay kit. (c) The percentage of CD8+ T cells and CD4+ T cells in OXA-challenged ear was examined by flow cytometric assay. (d) Ear swelling was measured with an engineer’s micrometer. Data are representative from three independent experiments and expressed as mean ± SEM (n = 3 per group). NS: not significant; *P < 0·05, **P < 0.01.

Discussion

In this study, we have demonstrated that the LTB4–BLT1 axis contributes to both the sensitization and the elicitation phases of OXA-induced contact dermatitis, and is important for the skin recruitment of neutrophils, which act as a major cell source of LTB4, leading to subsequent skin homing of hapten-primed CD8+ T cells following OXA challenge.

Prete et al.18 have shown that the LTB4–BLT1 axis is important for the sensitization phase of dinitrofluorobenzene-induced contact dermatitis by up-regulating CCR7 expression on dendritic cells to promote their lymphoid homing to prime T-cell response. We confirmed their findings by demonstrating that BLT1−/− mice exhibited a significantly attenuated T-cell response characterized by greatly reduced IFN-γ+ CD8+ T cells in skin-draining lymph nodes of OXA-induced contact dermatitis. We further found that BLT1−/− mice exhibited attenuated skin inflammation characterized by greatly decreased ear swelling and infiltration of neutrophils and CD8+ T cells. Decreased skin inflammation in BLT1−/− mice sensitized and challenged with OXA could be the result of impaired T-cell priming in the sensitization phase or impaired inflammatory cell recruitment in the elicitation phase. We next demonstrated that the LTB4–BLT1 axis is critical for the elicitation phase of OXA-induced contact dermatitis, as blockade of LTB4 or BLT1 following OXA challenge greatly reduced ear swelling and infiltration of neutrophils and CD8+ T cells. Although BLT1-dependent tissue recruitment of CD4+ T cells has been implicated in several inflammatory conditions,14,15,22 interestingly, we found that skin infiltration of CD4+ T cells remained unchanged in BLT1−/− mice and antagonist-treated mice following OXA challenge, suggesting that the skin homing of CD4+ T cells could be BLT1-independent at least in OXA-induced contact dermatitis. We also found that CD8+ T cells are the primary cell source for IFN-γ, as the average percentage of IFN-γ+ CD8+ T cells was about 10-fold more than IFN-γ+ CD4+ T cells in the culture of stimulated cells from skin-draining lymph nodes (data not shown). A previous study demonstrated that CD8+ T-cell depletion caused decreased ear swelling to a greater extent than CD4+ T-cell depletion.16 Moreover, several studies suggested that CD4+ T cells could suppress skin inflammation as a negative regulatory cell in mouse models of contact dermatitis.23,24 These results together suggest that CD8+ T cells, rather than CD4+ T cells, are important effector T cells in hapten-induced contact dermatitis. Hence, our results indicated that the LTB4–BLT1 axis is important for skin homing of CD8+ T cells in OXA-induced contact dermatitis.

Neutrophil is crucial for the development of contact hypersensitivity and is implicated as an important target cell for glucocorticoid treatment of contact dermatitis.17,25 Recent studies suggested that BLT1 mediates early recruitment of neutrophils, which act as the primary cellular source of IL-1β, which in turn up-regulates the expression of neutrophil-attracting chemokines, such as CXCL1 and CXCL2, to further amplify neutrophil recruitment in a CXCR2-dependent way during the later phase in a mouse model of inflammatory arthritis.11,26 Similarly, we found that the blockade of the LTB4–BLT1 axis significantly reduced skin-infiltrating neutrophils, accompanied by a significant decrease in the skin expression of IL-1β and CXCL2 in our model. Consistently, neutrophil depletion also caused greatly reduced skin expression of IL-1β and CXCL2. Unexpectedly, in contrast to decreased CXCL2 gene expression, greatly increased CXCL1 gene expression was found in neutrophil-depleted mice following OXA challenge. Similarly elevated CXCL1 expression was reported in neutrophil-depleted mice by other investigators.4 We speculated that this might be a compensatory mechanism used by the host trying to recruit more neutrophils to inflammatory sites. Our results suggest that the LTB4–BLT1 axis directly mediates skin recruitment of neutrophils that promote the production of other inflammatory cytokines to amplify their recruitment and further promote the skin inflammation in OXA-induced contact dermatitis.

Previous studies demonstrated that the intensity of neutrophils in hapten-challenged skin could inversely control CD8+ T-cell infiltration.3,7 We confirmed these findings by showing that neutrophil depletion greatly blocked the skin infiltration of CD8+ T cells following OXA challenge. Several T-cell chemokines including CXCL9 and CXCL10 have been shown to be important for the skin homing of T cells;5,6,27 however, there was no change in gene expression of CXCL9 and CXCL10 in OXA-challenged ears following blockade of the LTB4–BLT1 axis or neutrophil depletion, suggesting that other important mediators could be more important for the skin homing of CD8+ T cells in OXA-induced contact dermatitis. Interestingly, significantly decreased gene expression of 5-LO and LTA4H as well as LTB4 production were detected in neutrophil-depleted mice, suggesting neutrophils as the major cell source for LTB4. BLT1 was reported to be expressed on effector CD8+ T cells and mediates tissue inflammation.21 Hence, it is possible that skin-infiltrating neutrophil-derived LTB4 mediates the subsequent recruitment of hapten-primed effector CD8+ T cells. In support of this, we found that administration of exogenous LTB4 completely restored skin infiltrating CD8+ T cells in neutrophil-depleted mice following OXA challenge. However, the complete restoration of skin infiltrating CD8+ T cells failed to restore the ear swelling. The possible explanation could be that CD8+ T cells are not able to cause ear swelling in the absence of infiltrating neutrophils in hapten-challenged skin. This is also consistent with the previous study showing that decreased ear swelling in CD8+ T-cell-depleted mice was accompanied by greatly decreased skin-infiltrating neutrophils.4 Hapten-primed CD8+ T cells have been shown to quickly migrate to the vasculature of hapten-challenged skin and direct early recruitment of neutrophils, which in turn mediates subsequent skin recruitment of CD8+ T cells through various mechanisms.3,7 Our data suggest a novel mechanism by which neutrophils amplify the skin recruitment of CD8+ T cells by producing LTB4.

In summary, our study reveals the important pro-inflammatory role of the LTB4–BLT1 axis in the development of contact dermatitis by attracting neutrophil and CD8+ effector T cells to hapten-challenged skin. Our study also identifies LTB4 as one of the important contributors to neutrophil-dependent skin recruitment of CD8+ T cells in contact dermatitis. Corticosteroids are widely used in clinical treatment of contact dermatitis, and neutrophils are revealed to be one of the important target cells of their action. However, long-term use of corticosteroids can cause side effects because of their non-specific anti-inflammatory effects. Our results suggest that the LTB4–BLT1 axis could be a potential therapeutic target in contact dermatitis.

Acknowledgments

This work is supported by National Natural Science Foundation of China Grant 813220437 (to R.H.).

Glossary

5-LO

5-lipoxygenase

BLT1

leukotriene B4 receptor

IFN-γ

interferon-γ

IL-1

interleukin 1

LTA4H

leukotriene A4 hydrolase

LTB4

leukotriene B4

OXA

oxazolone

U-75302

specific BLT1 antagonist

WT

wild-type

Author contributions

R.H. designed the research; J.L., L.Z., L.Z., W.Y. and Y.X. performed the research; J.L., L.Z. and B.L. analysed the data; and R.H. and J.L. wrote the paper.

Disclosures

The authors declare no financial and commercial conflict of interest.

Supporting Information

Figure S1. Attenuated T cell priming in OXA-sensitized BLT1−/− mice.

imm0146-0050-sd1.tif (660.7KB, tif)

Figure S2. Attenuated skin inflammation in OXA-challenged skin with beastatin treatment.

imm0146-0050-sd2.tif (1.3MB, tif)

 

imm0146-0050-sd3.doc (28KB, doc)

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Attenuated T cell priming in OXA-sensitized BLT1−/− mice.

imm0146-0050-sd1.tif (660.7KB, tif)

Figure S2. Attenuated skin inflammation in OXA-challenged skin with beastatin treatment.

imm0146-0050-sd2.tif (1.3MB, tif)

 

imm0146-0050-sd3.doc (28KB, doc)

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