Phase-Dependent Roles of E-Selectin during Chronic Contact Hypersensitivity Responses

【摘要】  Chronic contact hypersensitivity (CH) models induced by repeated hapten exposure exhibit chronic dermatitis and immunological abnormalities resembling atopic dermatitis. To assess the contribution of endothelial selectins (P- and E-selectins) to cutaneous chronic inflammation, chronic CH responses were assessed in mice lacking P- or E-selectin. Elicitation with oxazolone on the ears of P-selectinC/C mice 7 days after the sensitization induced a typical delayed-type hypersensitivity response similar to that found in wild-type mice. By contrast, a significant increase in ear swelling was observed in E-selectinC/C mice 36 to 48 hours after first elicitation. E-selectinC/C mice showed augmented P-selectin up-regulation, and administration of anti-P-selectin monoclonal antibody significantly inhibited the enhanced ear response, suggesting that the enhanced ear-swelling response in E-selectinC/C mice resulted from compensatory increase in P-selectin expression. In the late phase of chronic CH, acceleration of ear swelling was significantly reduced in both E- and P-selectinC/C mice relative to wild-type littermates. Thus, the loss of P- or E-selectin suppressed inflammatory responses during the chronic phase of the chronic models, whereas early-phase inflammatory responses were exacerbated by E-selectin blockade. Collectively, P- and E-selectins cooperatively regulate CH response, although their roles may be different depending on the phase of the reaction.
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Leukocyte recruitment from the circulation into a site of inflammation involves adhesive interactions between the leukocyte and the vascular endothelium and has been implicated in the pathology of various inflammatory diseases.1-3 Leukocytes first tether and roll on vascular cells, before they are activated to adhere firmly and subsequently to immigrate into the extravascular space. The selectin family, which mediates tethering and rolling of leukocytes, consists of three cell-surface molecules expressed by leukocytes (L-selectin), vascular endothelium (E- and P-selectin), and platelets (P-selectin).1 E-selectin expression is induced within several hours after activation with inflammatory cytokines, whereas P-selectin is rapidly mobilized to the surface of activated endothelium or platelets.1
Interaction of adhesion molecules during the process of leukocyte migration into inflammatory sites is complex and highly regulated, where the three selectin members have partially overlapping functions.4,5 Specifically, E-selectin-deficient (E-selectinC/C) mice do not exhibit significant defects of leukocyte rolling and inflammation, which are virtually eliminated by the additional blockade of P-selectin.6-9 In addition, the relative contribution of each adhesion molecule to the inflammation varies according to the tissue site and the nature of the inflammatory stimuli.10 For example, in experimental models such as Arthus reaction and concanavalin A- or lipopolysaccharide-induced dermal inflammation, inhibition or loss of E-selectin expression does not induce a significant reduction of leukocyte rolling and recruitment, whereas inhibition or loss of P-selectin expression results in a significant reduction of leukocyte rolling and recruitment.11,12 By contrast, loss of P-selectin exacerbates certain inflammatory responses, such as glomerulonephritis and collagen-induced arthritis.13,14 Therefore, it is important to evaluate the differential contribution of each adhesion molecule to the diseases and their experimental models.
Contact hypersensitivity (CH) is a cutaneous immune reaction in sensitized individuals to subsequent contact with the sensitizing hapten, where local endothelial cell activation plays a critical role. Repeated application of a contact-sensitizing agent in mice induces chronic CH responses that are clinically relevant to human skin allergic diseases.15 Repeated epicutaneous application of antigen (Ag) results in a shift in the time course from a typical delayed-type hypersensitivity to an immediate-type hypersensitivity response followed by a late-phase reaction.15-17 This immediate-type hypersensitivity response is site-restricted and Ag-specific15,16 and is associated with a change from a local Th1-type to Th2-type cytokine pattern with elevated serum IgE levels.15,16,18 Because atopic dermatitis (AD) is often caused by repeated epicutaneous exposure to various environmental Ags, this is considered to serve as a potential animal model for AD. In acute CH models, L-selectinC/C mice and ICAM-1C/C mice exhibit reduced responses, with the combined loss of both molecules resulting in additional reductions.19 Furthermore, mice lacking both E-selectin and P-selectin expression have an impaired delayed-type hypersensitivity response, whereas no such impairment is seen in E- or P-selectinC/C mice.20
We previously reported that the induction of an immediate-type hypersensitivity response after chronic Ag exposure was completely eliminated by deficiency or blockade of L-selectin or ICAM-1, whereas development of the accompanying late-phase reaction was significantly inhibited. These findings suggest that adhesion molecules are potential therapeutic targets for regulating human allergic reactions.21 However, the relative contribution of E- and P-selectins to this CH model remains unknown. The current study demonstrates that loss of E- or P-selectin inhibits the chronic inflammatory response, whereas loss of E-selectin alone exacerbates inflammatory response in the early phase of this CH model, mainly attributable to excess expression of P-selectin.

【关键词】  phase-dependent e-selectin hypersensitivity responses

Materials and Methods

Mice

P-selectinC/C22 and E-selectinC/C mice8 and wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were healthy and fertile and did not display evidence of infection or disease. All mice were backcrossed between 10 generations onto the C57BL/6 genetic background. Mice used for experiments were 12 to 16 weeks old. All mice were housed in a pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science.

Sensitization and Elicitation Procedure

Mice were sensitized with 20 µl of a 1% oxazolone so-lution (4-ethyoxymethylene-2-phenyloxazolone; Sigma-Aldrich, St. Louis, MO) in acetone/sesame seed oil (4:1) applied to the right ear (10 µl on the dorsal side and 10 µl on the ventral side of the right ear) as described elsewhere.23 Starting 7 days after sensitization, 20 µl of 1% oxazolone was repeatedly applied to the original sensitized right ear as above at 2-day intervals until day 10. An identical amount of acetone/sesame seed oil (4:1) was administered to the left ear as control.

Ear thickness was measured using a dial thickness gauge (Ozaki Seisakusho Co., Tokyo, Japan) under light ether anesthesia at various time points during the course of the experiment. For detailed time-course analysis of ear-swelling reactions, ear thicknesses were measured before and 0.5, 1, 3, 6, 9, 12, 24, 36, and 48 hours after each elicitation on days 0, 4, and 8. Each ear lobe was measured three times at each time point, and the mean of those values was used for analysis. Six mice were used per each group in all experiments.

Histological Examination and Immunohistochemical Staining

A central strip of the ear was fixed in 3.5% paraformaldehyde and then paraffin-embedded. Sections (6 µm) were stained using hematoxylin and eosin (H&E) for general histological evaluation and toluidine blue for mast cell staining. Dermal leukocyte infiltration was evaluated by averaging the numbers of leukocytes present in 12 high-power fields (0.07 mm2). Each section was examined independently by two investigators in a blinded manner, and the mean was used for analysis. For immunohistochemistry, frozen tissue sections of skin biopsies were acetone-fixed and then incubated with 10% normal rabbit serum in phosphate-buffered saline (10 minutes, 37??C) to block nonspecific staining. Sections were then incubated with rat monoclonal antibodies (mAbs) specific for mouse CD4 (RM4-5; BD PharMingen, San Diego, CA), and CD8 (53-6.7; BD PharMingen). Rat IgG (Southern Biotechnology Associates Inc., Birmingham, AL) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 minutes, 37??C) with a biotinylated rabbit anti-rat IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) followed by horseradish peroxidase-conjugated avidin-biotin complexes (Vectastain ABC kit; Vector Laboratories). Sections were developed with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide and then counterstained with methyl green.

Blocking Study by mAbs

For a blocking study using mAbs to P-selectin and/or L-selectin and/or ICAM-1, mAbs were injected intravenously into E-selectinC/C mice simultaneously or 24 hours after the elicitation on day 0. Abs used in this blocking study included mAbs to murine L-selectin (MEL14, rat IgG2a, 100 µg per mouse; BD PharMingen),24 mAbs to murine P-selectin (RB40.34, rat IgG1, 30 µg per mouse; BD PharMingen),25 and mAbs to murine ICAM-1 (3E2, Armenian hamster IgG, 100 µg per mouse; BD PharMingen).26 These were the mAb concentrations required to inhibit L-selectin-, P-selectin-, and ICAM-1-dependent leukocyte recruitment in vivo as previously described.27,28 Irrelevant isotype-matched, purified rat IgG1 mAb (R3-34), rat IgG2a mAb (R35-95), and Armenian hamster IgG mAb (Ha4/8) served as controls (30 µg per mouse; BD PharMingen).

RNA Isolation and Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from the ear of mutant and wild-type mice using Qiagen RNeasy spin columns (Qiagen Ltd., Crawley, UK) and digested by DNase I (Qiagen Ltd.) to remove chromosomal DNA in accordance with the manufacturer??s protocol. Total RNA was reverse-transcribed to cDNA using a reverse transcription system with random hexamers (Promega, Madison, WI), and mRNA expression of ICAM-1 and P-selectin was analyzed by real-time quantitative RT-PCR using the TaqMan system (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize mRNA. We obtained all sequence-specific primers and probes from TaqMan gene expression assays (Applied Biosystems). Real-time PCR was performed on an ABI Prism 7000 sequence detector (Applied Biosystems) according to the manufacturer??s instructions. Relative expression of real-time PCR products was determined using the CT technique as previously described.29 In brief, we normalized each set of samples using the difference in threshold cycle (CT) between the target gene and GAPDH: CT = (CT target gene C CT GAPDH). Relative mRNA levels were calculated by the formula 2CCT where CT = CT sample (n) C CT calibrator (n). Each reaction was done in, at least, triplicate. One of the control samples was chosen as a calibrator sample.

Statistical Analysis

The Mann-Whitney U-test was used for determining the level of significance of differences in sample means, and Bonferroni??s test was used for multiple comparisons. All data are shown as mean ?? SEM.

Results

Ear-Swelling Response by Repeated Ag Elicitation

Mice sensitized on the right ears with oxazolone 7 days before the first elicitation were repeatedly exposed to oxazolone at 2-day intervals on the originally sensitized ear for 8 days. Acetone/sesame seed oil was applied to left ears as control. In addition, no swelling response was observed in the naïve mice treated on the ear with 1% oxazolone solution (data not shown). Total ear thickness was measured immediately before each elicitation. After repeated oxazolone elicitation, wild-type mice exhibited a dramatic increase in the total ear thickness, whereas repeated applications of carrier alone had no detectable effect (Figure 1) . In P-selectinC/C mice, increases in total ear thickness were almost identical to those found in wild-type mice. Initial velocity of ear-swelling response in wild-type mice and P-selectinC/C mice was relatively slow until day 4, when it was accelerated. By contrast, E-selectinC/C mice exhibited a more rapid onset of ear swelling on day 0, followed by an almost linear increase in thickness until day 6, resulting in a significant increase in total ear thickness in E-selectinC/C mice compared with wild-type mice from days 2 to 6 (1.5- to 5.1-fold, P < 0.05). There were no significant differences in total ear thickness between wild-type mice and E-selectinC/C mice on days 8 or 10 (Figure 1) . Thus, in the early-phase response, loss of E-selectin significantly enhanced the increase in total ear thickness by accelerating the velocity of ear swelling, whereas loss of P-selectin did not affect total ear thickness.

Figure 1. Total ear thickness after repeated 1% oxazolone solution elicitation in P-selectinC/C, E-selectinC/C, and wild-type mice. Mice sensitized on the ears with oxazolone 7 days before the first elicitation were repeatedly exposed for 8 days at 2-day intervals. Repeated administration with acetone/sesame seed oil alone served as control. Ear thickness was measured just before each elicitation until 10 days. All values represent the mean ?? SEM of results obtained from six mice in each group. Statistical analysis results are described in the Results section. Error bars are not shown when SEM values are smaller than their symbols. *P < 0.05 versus wild-type mice.

The Time Course of Ear-Swelling Response in P- and E-SelectinC/C Mice

A detailed time course of ear-swelling reaction by repeated oxazolone elicitation was assessed on days 0, 4, and 8. After the first challenge (day 0), wild-type mice exhibited typical delayed-type hypersensitivity response that peaked at 12 to 24 hours and then gradually decreased (Figure 2A) . Similar results were obtained in P-selectinC/C mice. In contrast, E-selectinC/C mice exhibited ear-swelling response that was similar to wild-type mice until 24 hours, whereas ear swelling did not decrease 24 to 48 hours after the first elicitation (Figure 2A) . By contrast to the day-0 response, the acceleration of ear-swelling reaction was significantly reduced in E-selectinC/C mice compared with wild-type mice at 3 to 48 hours after the elicitation on day 4 (>33% decrease, P < 0.05), although there were no significant differences between P-selectinC/C mice and wild-type mice (Figure 2B) . On day 8 in both P-selectinC/C mice and E-selectinC/C mice, the magnitudes of ear-swelling reaction peak were significantly suppressed relative to wild-type mice (33% decrease, P < 0.05, in P-selectinC/C mice and 50% decrease, P < 0.05, in E-selectinC/C mice; Figure 2C ). Thus, the loss of P- or E-selectin inhibited inflammatory response in the chronic phase, whereas acute inflammatory response was rather exacerbated by loss of E-selectin.

Figure 2. Time course of ear-swelling responses in P-selectinC/C, E-selectinC/C, and wild-type mice repeatedly elicited with oxazolone on days 0 (A), 4 (B), and 8 (C). Mice sensitized on the ears with oxazolone 7 days before the first elicitation were repeatedly exposed for 8 days at 2-day intervals. Ear thickness was measured before and at 0.5, 1, 3, 6, 9, 12, 24, 36, and 48 hours after each elicitation. The values indicate the difference of the ear thickness between these time points. All values represent the mean ?? SEM. These results were obtained from six mice in each group. Statistical analysis results are described in the Results section. Error bars are not shown when SEM values are smaller than their symbols. *P < 0.05 versus wild-type mice.

Histological Examination of the Ear-Swelling Reaction by Repeated Ag Exposure

To assess histological characteristics associated with the ear-swelling reaction, ear biopsies were examined on days 0 and 8. The ear biopsies from wild-type mice taken 24 hours after the Ag exposure revealed edema in the subcutaneous tissue (data not shown). Similar results were obtained by observation of tissues from P-selectinC/C and E-selectinC/C mice (data not shown). At 48 hours after the Ag challenge, subcutaneous edema was only modestly observed in wild-type mice and P-selectinC/C mice (Figure 3A) . By contrast, subcutaneous edema with leukocyte infiltration was more prominent in E-selectinC/C mice 48 hours after the elicitation (Figure 3A) . However, 24 hours after elicitation on day 8, decreases in area of edema and in infiltrated leukocytes were observed in the subcutaneous tissue from P-selectinC/C and E-selectinC/C mice relative to wild-type mice (Figure 3B) .

Figure 3. Histological sections of the ear pinnae from P-selectinC/C mice, E-selectinC/C mice, and wild-type mice 48 hours after oxazolone challenge on day 0 (A) and 24 hours after on day 8 (B). The repeated epicutaneous oxazolone elicitation was performed as described in Figure 2 . The sections were stained with H&E. Original magnifications: x100 (A); x40 (B).

Mast cell numbers were also assessed in the Ag-administrated ears from P- and E-selectinC/C mice and wild-type mice on days 0 and 8. Mast cell numbers 48 hours after the first challenge were comparable between wild-type mice and P-selectinC/C mice but were significantly higher in E-selectinC/C mice than wild-type mice (1.6-fold, P < 0.0001; Figures 4A and 5A ). However, after the repeated epicutaneous hapten application, mast cell numbers 24 hours after elicitation on day 8 were slightly but significantly reduced in P-selectinC/C mice (12% decrease, P < 0.05) and E-selectinC/C mice (17% decrease, P < 0.05) compared with wild-type mice (Figures 4B and 5C) . In addition, numbers of neutrophils, CD4+ T cells, CD8+ T cells, and macrophages were not significantly different between P- or E-selectinC/C mice and wild-type mice before or at any time points after elicitation (data not shown). Thus, although deficiency of E-selectin enhanced mast cell accumulation in the early phase, loss of E- or P-selectin expression led to significant inhibition of mast cell accumulation in the chronic phase.

Figure 4. Histological sections showing mast cell accumulation in the ear pinnae from P-selectinC/C mice, E-selectinC/C mice, and wild-type mice 48 hours after oxazolone challenge on day 0 (A) and 24 hours after on day 8 (B). The repeated epicutaneous oxazolone challenge was performed as described in Figure 2 . Mast cells were detected as cells with metachromatic staining of granules in toluidine blue-stained sections (arrows). Original magnifications, x100.

Contribution of Endothelial Selectins to the Enhanced Ear-Swelling Response in E-SelectinC/C Mice

A significant increase in ear swelling was observed in E-selectinC/C mice 24 to 48 hours after the first elicitation on day 0 (Figure 2A) . To assess the mechanisms of this enhanced ear-swelling response in E-selectinC/C mice, a blocking study using mAbs was performed. E-selectinC/C mice were intravenously treated with mAbs to L-selectin and/or P-selectin, or ICAM-1, or all simultaneously with the Ag administration (Figure 6A) or after 24 hours (Figure 6B) . E-selectinC/C mice that received injection of isotype-matched control mAbs exhibited kinetics and magnitudes of ear swelling similar to those of untreated E-selectinC/C mice (Figure 2A and data not shown). When E-selectinC/C mice were treated with mAbs to L-selectin and/or P-selectin, or ICAM-1, or all simultaneously with the Ag administration on day 0 (Figure 6A) , ear-swelling responses were significantly inhibited relative to wild-type mice and resulted in a significant further reduction of ear-swelling response compared with E-selectinC/C mice at 36 and 48 hours after elicitation (Figure 6A) . In particular, the blockade of P-selectin in E-selectinC/C mice remarkably resulted in 70 to 80% inhibition of the ear-swelling response in wild-type mice, and the inhibitory effect of P-selectin loss in E-selectinC/C mice on ear-swelling responses at 48 hours after elicitation was greater than that of L-selectin loss and ICAM-1 loss in E-selectinC/C mice.

Figure 6. The effect of adhesion molecule blockade by mAbs on ear-swelling responses. E-selectinC/C (E-selC/C) mice were treated intravenously with mAbs to L-selectin (L-sel), P-selectin (P-sel), and ICAM-1, simultaneously with the elicitation on day 0 (A) or after 24 hours (B). Ear thickness was measured at 36 hours (left) and 48 hours (right) after oxazolone challenge on day 0. The repeated epicutaneous oxazolone challenge was performed as described in Figure 2 . The values indicate the difference of the ear thickness between each time point. All values represent the mean ?? SEM. These results were obtained from four mice in each group. Statistical analysis results are described in the Results section.

Injection of mAbs to L-selectin, P-selectin, or ICAM-1 24 hours after the Ag administration also significantly inhibited the enhanced ear-swelling response in E-selectinC/C mice (Figure 6B) . Although the effect was modest compared with the 0-hour treatment, treatment with anti-P-selectin mAb significantly suppressed the exacerbated inflammatory response at 48 hours after elicitation, even compared with anti-L-selectin or anti-ICAM-1 mAb treatment (Figure 6B) . Thus, the enhanced ear-swelling response in E-selectinC/C mice may result from compensatory increase in P-selectin expression. Furthermore, the combination of P- and L-selectins with or without ICAM-1 blockade in E-selectinC/C mice significantly inhibited the enhanced ear-swelling response, which was equivalent to that found in wild-type mice (Figure 6B) .

Mast cell recruitment was also reduced in E-selectinC/C mice with cell adhesion molecule blockade. Mast cell numbers of E-selectinC/C mice treated with anti-P-selectin mAb were more reduced than anti-L-selectin or anti-ICAM-1 mAb treatment (Figure 7 and data not shown). These results were in parallel with the ear-swelling response, suggesting that the increase in mast cell numbers was mediated by adhesion molecules, especially P-selectin.

Figure 7. Histological sections at 48 hours after oxazolone challenge on day 0 in the ear pinnae from wild-type mice, E-selectinC/C mice and E-selectinC/C mice treated with mAb to P-selectin 24 hours after the first elicitation as was done in Figure 6B . The sections were stained with H&E and toluidine blue. Mast cells were detected as cells with metachromatic staining of granules in toluidine blue-stained sections (arrows). Original magnifications, x100.

P-Selectin Expression Was Markedly Up-Regulated in E-SelectinC/C Mice

Because the results indicated that P-selectin expression may compensate the loss of E-selectin, expression of P-selectin, as well as ICAM-1, was assessed in wild-type mice, P-selectinC/C mice, and E-selectinC/C mice at 12, 24, 36, and 48 hours after elicitation on day 0. ICAM-1 and P-selectin mRNA expression levels in the dermis were quantified by real-time RT-PCR. At 48 hours after the first elicitation, ICAM-1 mRNA level was decreased in P-selectinC/C mice compared with wild-type mice, although there was no significant difference (Figure 8A) . Furthermore, E-selectinC/C mice showed identical expression level of ICAM-1 as wild-type mice. Moreover, at different time points (12, 24, and 36 hours after the first challenge), no significant difference of ICAM-1 mRNA expression levels were observed in E- and P-selectinC/C mice relative to wild-type mice (data not shown). After the challenge, P-selectin expression was up-regulated in both wild-type and E-selectinC/C mice (Figure 8B) . P-selectin expression level in E-selectinC/C mice was slightly higher than that in wild-type mice after 12 and 24 hours, although there was no significant difference. In contrast to P-selectin mRNA expression in wild-type mice, which remained at similar levels until 48 hours, P-selectin mRNA expression levels in E-selectinC/C mice significantly increased after 36 hours (4.5-fold, P < 0.05) and 48 hours (5.6-fold, P < 0.05) compared with wild-type mice. Thus, E-selectin deficiency in early phase of CH resulted in the up-regulation of P-selectin expression.

Figure 8. mRNA expression of ICAM-1 (A) and P-selectin (B) in the dermis from P-selectinC/C mice, E-selectinC/C mice, and wild-type mice on day 0. mRNA expression was analyzed by real-time PCR. Relative expression of real-time PCR products was determined by using the Ct method to compare target gene and GAPDH mRNA expression. All values, which indicate mRNA expressions relative to those in wild-type mice at 48 hours, represent the mean ?? SEM. These results were obtained from five mice in each group.

Discussion

By assessing the contribution of selectins to chronic CH responses after repeated Ag exposure, the current study has demonstrated the critical role of P-selectin and E-selectin in chronic CH responses induced by repeated epicutaneous Ag elicitation, suggesting the cooperative regulation by all selectins. We previously showed that the loss of L-selectin, ICAM-1, or both significantly inhibited chronic skin inflammation.21 Although P-selectinC/C mice exhibited typical early-phase CH response that was similar to that found in wild-type mice, resolution of ear swelling was disturbed in E-selectinC/C mice 36 to 48 hours after the first elicitation (Figure 2A) . On the other hand, in the chronic phase of this CH model, acceleration of ear swelling was significantly reduced both in E- and P-selectinC/C mice compared with wild-type mice (Figure 2C) . Thus, the loss of E-selectin significantly altered the kinetics of ear swelling during the CH response; early-phase inflammatory responses were exacerbated by E-selectin blockade, whereas the loss of P- or E-selectin resulted in suppressed velocity of inflammatory responses during the chronic phase.

Loss of P- or E-selectin resulted in no inhibition of ear-swelling response 24 hours after the first elicitation (Figure 2A) , consistent with a previous report.20 At 36 and 48 hours after the first elicitation, ear-swelling response was not affected by loss of P-selectin, whereas loss of E-selectin resulted in impaired resolution of ear swelling (Figure 2A) . This enhanced ear-swelling response in E-selectinC/C mice was inhibited by administration of mAbs to cell adhesion molecules 24 hours after the first challenge, especially by blocking mAbs to P-selectin (Figure 6) . In addition, augmented P-selectin expression was observed in E-selectinC/C mice 36 and 48 hours after the first elicitation (Figure 8) . Taken together, these results suggest that the enhanced ear-swelling responses in E-selectinC/C mice are mainly attributable to a compensatory increase in P-selectin. Furthermore, the concomitant administration of anti-P- and anti-L-selectin mAbs completely abrogated the augmented swelling response in E-selectinC/C mice, suggesting that the expression of L-selectin ligands may be also up-regulated by the loss of E-selectin. Thus, although E-selectin-deficiency generally results in minimal phenotypic alteration in most inflammatory models, E-selectin plays a significant role in early-phase CH reaction by influencing the expression levels of other adhesion molecules.

By contrast, ear-swelling responses were significantly reduced in both E- and P-selectinC/C mice on day 8 compared with wild-type mice (Figure 2C) . Chronic Ag exposure leads to a shift in the time course of CH from a delayed-type hypersensitivity response that peaked at 24 hours to an immediate-type hypersensitivity response.15-17 Relative contribution of adhesion molecules may change during the shift, which may result in increasing the dependency on endothelial selectins. On the other hand, recruitment of Th2 cells to the skin may be more dependent on selectins than Th1 cells, although there have been no studies supporting this. Further investigation is required to clarify the mechanism.

Mast cell recruitment into tissues is considered to occur by release of immature mast cell precursors from the bone marrow into the peripheral blood, followed by migration of these precursors into tissue and their subsequent differentiation into mature mast cells.30 In addition, an increase in mast cell numbers at sites of inflammation have been reported.31 In the present study, chronic Ag exposure induced mast cell recruitment that was significantly inhibited by loss of P-selectin or E-selectin (Figures 4B and 5C) . These results suggest that chronic inflammatory response is dependent on the recruitment of mast cells, which is regulated by expression of adhesion molecules on the cells. In agreement with these results, mast cell numbers were significantly increased in E-selectinC/C mice 48 hours after the first elicitation (Figures 4A and 5A) . Moreover, mast cell recruitment was significantly reduced in E-selectinC/C mice treated with anti-P-selectin mAbs (Figure 7) . Several studies have shown that rolling of immature bone marrow-derived mast cell precursors is mediated by the interaction of P-selectin and P-selectin glycoprotein ligand-1.32,33 In addition, in the passive Arthus reaction, cutaneous and peritoneal mast cell recruitment is reduced in mice lacking P-selectin or E-selectin.12 Therefore, our results suggest that P-selectin and E-selectin regulate mast cell recruitment into inflammatory sites, presumably through the peripheral blood, and indicate that mast cells play a critical role in the progression of inflammatory responses.

AD is a chronic inflammatory skin disease with an allergic and genetic background.34,35 The prevalence of AD has increased in recent years, and it is now estimated to affect up to 20% of the general population.34 The present study demonstrates the differential contribution of P-selectin and E-selectin in a murine model of chronic CH responses that are clinically relevant to human skin allergic diseases such as AD.15 The results indicate that chronic inflammatory responses are inhibited by P-selectin or E-selectin blockade, whereas the loss of E-selectin exacerbated acute inflammatory response. Thus, although anti-adhesion therapy with mAb to these selectins is potentially beneficial for AD treatment, disrupting E-selectin expression alone may lead to the exacerbation of an acute inflammatory response, and simultaneous blockade of E- and P-selectins may be a preferable strategy.

Figure 5. Mast cell numbers at 48 hours after oxazolone elicitation on day 0 (A), 6 hours after on day 4 (B), and 24 hours after day 8 (C) in the dermis from P-selectinC/C mice, E-selectinC/C mice, and wild-type mice repeatedly elicited with oxazolone on the ears. The repeated epicutaneous oxazolone challenge was performed as described in Figure 2 . Mast cell numbers per dermis section in high-power microscopic field (0.07 mm2) were determined by counting in paraffin sections stained with toluidine blue. All values represent the mean ?? SEM of results from six mice in each group.

Acknowledgements

We thank Ms. M. Matsubara and Ms. Y. Yamada for technical assistance.

【参考文献】
  Tedder TF, Li X, Steeber DA: The selectins and their ligands: adhesion molecules of the vasculature. Adv Mol Cell Biol 1999, 28:65-111

Springer TA: Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 1995, 57:827-872

Butcher EC: Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 1991, 67:1033-1036

Ley K, Tedder TF: Leukocyte interactions with vascular endothelium: new insights into selectin-mediated attachment and rolling. J Immunol 1995, 155:525-528

Robinson SD, Frenette PS, Rayburn H, Cummiskey M, Ullman-Cullere M, Wagner DD, Hynes RO: Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment. Proc Natl Acad Sci USA 1999, 96:11452-11457

Labow MA, Norton CR, Rumberger JM, Lombard-Gillooly KM, Shuster DJ, Hubbard J, Bertko R, Knaack PA, Terry RW, Harbison ML, Kontgen F, Stewart CL, McIntyre KW, Will PC, Burns DK, Wolitzky BA: Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins. Immunity 1994, 1:709-720

Bullard DC, Kunkel EJ, Kubo H, Hicks MJ, Lorenzo I, Doyle NA, Koerschuk CM, Ley K, Beaudet AL: Infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in E-selectin and P-selectin double mutant mice. J Exp Med 1996, 183:2329-2336

Frenette PS, Mayadas TN, Rayburn H, Hynes RO, Wagner DD: Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins. Cell 1996, 84:563-574

Kanwar S, Bullard DC, Hickey MJ, Smith CW, Beaudet AL, Wolitzky BA, Kubes P: The association between alpha4-integrin, P-selectin, and E-selectin in an allergic model of inflammation. J Exp Med 1997, 185:1077-1087

Eppihimer MJ, Russell J, Anderson DC, Wolitzky BA, Granger DN: Endothelial cell adhesion molecule expression in gene-targeted mice. Am J Physiol 1997, 273:H1903-H1908

Issekutz AC, Issekutz TB: The role of E-selectin, P-selectin, and very late activation antigen-4 in T lymphocyte migration to dermal inflammation. J Immunol 2002, 168:1934-1939

Yanaba K, Kaburagi Y, Takehara K, Steeber DA, Tedder TF, Sato S: Relative contributions of selectins and intercellular adhesion molecule-1 to tissue injury induced by immune complex deposition. Am J Pathol 2003, 162:1463-1473

Bullard DC, Mobley JM, Justen JM, Sly LM, Chosay JG, Dunn CJ, Lindsey JR, Beaudet AL, Staite ND: Acceleration and increased severity of collagen-induced arthritis in P-selectin mutant mice. J Immunol 1999, 163:2844-2849

Rosenkranz AR, Mendrick DL, Cotran RS, Mayadas TN: P-selectin deficiency exacerbates experimental glomerulonephritis: a protective role for endothelial P-selectin in inflammation. J Clin Invest 1999, 103:649-659

Kitagaki H, Fujisawa S, Watanabe K, Hayakawa K, Shiohara T: Immediate-type hypersensitivity response followed by a late reaction is induced by repeated epicutaneous application of contact sensitizing agents in mice. J Invest Dermatol 1995, 105:749-755

Kitagaki H, Ono N, Hayakawa K, Kitazawa T, Watanabe K, Shiohara T: Repeated elicitation of contact hypersensitivity induces a shift in cutaneous cytokine milieu from a T helper cell type 1 to a T helper cell type 2 profile. J Immunol 1997, 159:2484-2491

Kitagaki H, Kimishima M, Teraki Y, Hayakawa J, Hayakawa K, Fujisawa S, Shiohara T: Distinct in vivo and in vitro cytokine profiles of draining lymph node cells in acute and chronic phases of contact hypersensitivity: importance of a type 2 cytokine-rich cutaneous milieu for the development of an early-type response in the chronic phase. J Immunol 1999, 163:1265-1273

Webb EF, Tzimas MN, Newsholme SJ, Griswold DE: Intralesional cytokines in chronic oxazolone-induced contact sensitivity suggest roles for tumor necrosis factor alpha and interleukin-4. J Invest Dermatol 1998, 111:86-92

Steeber DA, Campbell MA, Basit A, Ley K, Tedder TF: Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression. Proc Natl Acad Sci USA 1998, 95:7562-7567

Staite ND, Justen JM, Sly LM, Beaudet AL, Bullard DC: Inhibition of delayed-type contact hypersensitivity in mice deficient in both E-selectin and P-selectin. Blood 1996, 88:2973-2979

Shimada Y, Hasegawa M, Kaburagi Y, Hamaguchi Y, Komura K, Saito E, Takehara K, Steeber DA, Tedder TF, Sato S: L-selectin or ICAM-1 deficiency reduces an immediate-type hypersensitivity response by preventing mast cell recruitment in repeated elicitation of contact hypersensitivity. J Immunol 2003, 170:4325-4334

Bullard DC, Qin L, Lorenzo I, Quinlin WM, Doyle NA, Bosse R, Vestweber D, Doerschuk CM, Beaudet AL: P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli. J Clin Invest 1995, 95:1782-1788

Roberts LK, Spangrude GJ, Daynes RA, Krueger GG: Correlation between keratinocyte expression of Ia and the intensity and duration of contact hypersensitivity responses in mice. J Immunol 1985, 135:2929-2936

Gallatin WM, Weissman IL, Butcher EC: A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature 1983, 304:30-34

Bosse R, Vestweber D: Only simultaneous blocking of the L- and P-selectin completely inhibits neutrophil migration into mouse peritoneum. Eur J Immunol 1994, 24:3019-3024

Scheynius A, Camp RL, Pure E: Reduced contact sensitivity reactions in mice treated with monoclonal antibodies to leukocyte function-associated molecule-1 and intercellular adhesion molecule-1. J Immunol 1993, 150:655-663

Kumasaka T, Quinlan WM, Doyle NA, Condon TP, Sligh J, Takei F, Beaudet A, Bennett CF, Doerschuk CM: Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice. J Clin Invest 1996, 97:2362-2369

Kunkel EJ, Jung U, Bullard DC, Norman KE, Wolitzky BA, Vestweber D, Beaudet AL, Ley K: Absence of trauma-induced leukocyte rolling in mice deficient in both P-selectin and intercellular adhesion molecule 1. J Exp Med 1996, 183:57-65

Meijerink J, Mandigers C, van de Locht L, Tonnissen E, Goodsaid F, Raemaekers J: A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR. J Mol Diagn 2001, 3:55-61

Kirshenbaum AS, Kessler SW, Goff JP, Metcalfe DD: Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells. J Immunol 1991, 146:1410-1415

Ishizaka T, Ishizaka K: Biology of immunoglobulin E. Molecular basis of reaginic hypersensitivity. Prog Allergy 1975, 19:60-121

Sriramarao P, Anderson W, Wolitzky BA, Broide DH: Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo. Lab Invest 1996, 74:634-643

Steegmaier M, Blanks JE, Borges E, Vestweber D: P-selectin glycoprotein ligand-1 mediates rolling of mouse bone marrow-derived mast cells on P-selectin but not efficiently on E-selectin. Eur J Immunol 1997, 27:1339-1345

Leung DY, Soter NA: Cellular and immunologic mechanisms in atopic dermatitis. J Am Acad Dermatol 2001, 44:S1-S12

Beltrani VS: The clinical spectrum of atopic dermatitis. J Allergy Clin Immunol 1999, 104:S87-S98

作者单位：From the Department of Dermatology,* Kanazawa University Graduate School of Medical Science, Kanazawa; the Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo; and the Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan