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The Journal of Immunology, 2003, 170: 4325-4334.
Copyright © 2003 by The American Association of Immunologists

L-Selectin or ICAM-1 Deficiency Reduces an Immediate-Type Hypersensitivity Response by Preventing Mast Cell Recruitment in Repeated Elicitation of Contact Hypersensitivity1

Yuka Shimada*, Minoru Hasegawa*, Yuko Kaburagi*, Yasuhito Hamaguchi*, Kazuhiro Komura*, Eriko Saito*, Kazuhiko Takehara*, Douglas A. Steeber{dagger}, Thomas F. Tedder{dagger} and Shinichi Sato2,*

* Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; and {dagger} Department of Immunology, Duke University Medical Center, Durham, NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Repeated Ag exposure results in a shift in the time course of contact hypersensitivity (CH) from a typical delayed-type to an immediate-type response followed by a late phase reaction. Chronic CH responses are clinically relevant to human skin allergic diseases, such as atopic dermatitis, that are usually caused by repeated stimulation with environmental Ags. Chronic inflammatory responses result in part from infiltrating leukocytes. To determine the role of leukocyte adhesion molecules in chronic inflammation, chronic CH responses were assessed in mice lacking L-selectin, ICAM-1, or both adhesion molecules. Following repeated hapten sensitization for 24 days at 2-day intervals, wild-type littermates developed an immediate-type response at 30 min after elicitation, followed by a late phase reaction. By contrast, loss of ICAM-1, L-selectin, or both, eliminated the immediate-type response and inhibited the late phase reaction. Similar results were obtained when wild-type littermates repeatedly exposed to hapten for 22 days were treated with mAbs to L-selectin and/or ICAM-1 before the elicitation on day 24. The lack of an immediate-type response on day 24 paralleled a lack of mast cell accumulation after 30 min of elicitation and decreased serum IgE production. Repeated Ag exposure in wild-type littermates resulted in increased levels of serum L-selectin, a finding also observed in atopic dermatitis patients. The current study demonstrates that L-selectin and ICAM-1 cooperatively regulate the induction of the immediate-type response by mediating mast cell accumulation into inflammatory sites and suggests that L-selectin and ICAM-1 are potential therapeutic targets for regulating human allergic reactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukocyte recruitment from the circulation into a site of inflammation, a multistep process regulated by multiple adhesion molecules, is a critical component of both innate and acquired immunity, but has also been implicated in the pathology of various inflammatory diseases (1, 2, 3). Leukocytes first tether and roll on vascular endothelial cells before they are activated to adhere firmly and subsequently emigrate into the extravascular space. The selectin family (L-, E-, and P-selectins) mediates tethering and rolling of leukocytes while Ig superfamily members, including ICAM-1, and their integrin ligands are critical for the firm adhesion. Furthermore, ICAM-1 functions cooperatively with L-selectin to mediate optimal leukocyte rolling and recruit leukocytes into inflammatory sites (4, 5, 6).

The physiological process of leukocyte migration into inflammatory sites is complex. For example, functional redundancy exists between families of adhesion molecules: interactions between integrins and their Ig superfamily ligands can mediate rolling as well as firm adhesion in concert with selectins (4, 7, 8). In addition, the relative contribution of each adhesion molecule to the inflammatory process varies according to the tissue site of inflammation and the nature of the inflammatory stimuli (2). Furthermore, the frequency of the inflammatory stimuli encountered (single vs repeated exposure) may be an important factor in determining the relative usage of adhesion molecules. Indeed, recent studies have demonstrated that repeated Ag application induces inflammatory responses that are quantitatively and qualitatively different from those elicited with a single encounter with Ag. Specifically, chronic Ag exposure leads to a shift in the time course of contact hypersensitivity (CH)3 from a delayed-type hypersensitivity (DTH) response to an immediate-type hypersensitivity (ITH) response (9, 10, 11).

In acute CH models, inflammatory responses are elicited by a single epicutaneous application of a contact-sensitizing agent in animals previously sensitized with the same Ag. Under these conditions, L-selectin-deficient (L-selectin-/-) and ICAM-1-/-mice exhibit reduced CH responses, with the combined loss of both molecules resulting in additional CH response reductions (5). In contrast to acute CH models, repeated Ag stimulation results in chronic inflammation that is distinct from acute CH (9, 10, 11). Importantly, atopic dermatitis (AD) is usually caused by repeated epicutaneous exposure to various environmental Ags. Although L-selectin deficiency reduces airway hyper-responsiveness in a murine asthma model induced by repeated Ag exposure through the respiratory mucosa (12), the contribution of adhesion molecules to the development of chronic cutaneous inflammation remains unknown. However, Kitagaki et al. (9) have established a murine model of chronic CH responses that provides an appropriate animal model for chronic skin inflammation. Repeated epicutaneous application of a contact-sensitizing agent results in a shift in the time course from a typical DTH to an ITH response, followed by a late phase reaction (9). The development of the ITH response is site restricted and Ag specific (9, 11) and is associated with a change from a local Th1- type to Th2- type cytokine pattern with elevated serum IgE levels (9, 10, 11, 13). Since this model may be highly relevant to chronic allergic diseases in human, such as AD, it was assessed whether L-selectin or ICAM-1 contribute to chronic cutaneous inflammation induced by repeated epicutaneous application of oxazolone. The results of this study demonstrate that L-selectin and ICAM-1 cooperatively contribute to the induction of an ITH response by mediating mast cell accumulation into the inflammatory sites.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

L-selectin-/- mice were produced as described elsewhere (14). ICAM-1-/- mice (15), expressing residual amounts of ICAM-1 splice variants in the thymus and spleen but not in other organs including skin (16), were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice lacking both L-selectin and ICAM-1 were generated as described previously (4). All mice were healthy, fertile, and did not display evidence of infection or disease. All mice were backcrossed between 5 and 10 generations onto the C57BL/6 genetic background. Mice used for experiments were 12- to 16-wk old. Age-matched wild-type littermates and C57BL/6 mice (The Jackson Laboratory) were used as controls with equivalent results; therefore, all control results were pooled. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of the Kanazawa University School of Medicine.

Sensitization and elicitation procedure

Mice were sensitized with 20 µl (10 µl on the dorsal side and 10 µl on the ventral side) of a 1% oxazolone solution (4-ethyoxymethylene-2-phenyloxazolone; Sigma-Aldrich, St. Louis, MO) in acetone/sesame seed oil (4:1) applied to the right ear as described elsewhere (17). Starting 7 days following sensitization, 20 µl of 1% oxazolone was repeatedly applied to the original sensitized right ear as above at 2-day intervals until day 24 (9, 11). 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, 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 h after each elicitation on days 0, 4, 8, 16, and 24. Results were expressed as the difference between the ear thickness before and after each elicitation. Then 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. The ITH response was defined as the ear swelling within the first 30 min, the late phase reaction as that occurring between 3 and 6 h, and the DTH response as that occurring after 12 h.

Blocking study by mAbs

For a blocking study using mAbs to L-selectin and/or ICAM-1, mAbs were injected i.v. 1 h before the elicitation on day 24 into wild-type littermates repeatedly exposed to oxazolone at 2-day intervals on the sensitized ear for 22 days. Abs used in this blocking study included mAbs to murine L-selectin (MEL14, rat IgG2a, 5 mg/kg; BD PharMingen, San Diego, CA) (18) and mAbs to murine ICAM-1 (3E2, Armenian hamster IgG, 5 mg/kg; BD PharMingen) (19). These were the mAb concentrations required to inhibit L-selectin- and ICAM-1-dependent leukocyte recruitment in vivo as previously described (20, 21). Irrelevant isotype-matched, purified rat IgG2a mAb (R35-95) and Armenian hamster IgG mAb (Ha4/8) served as controls (5 mg/kg; BD PharMingen).

Histological examination and immunohistochemical staining

A central strip of the ear was fixed in 3.5% paraformaldehyde and then paraffin embedded. Six-micrometer sections were stained using 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 fashion. For immunohistochemistry, frozen tissue sections of skin biopsies were acetone fixed and then incubated with 10% normal rabbit serum in PBS (10 min, 37°C) to block nonspecific staining. Sections were then incubated with rat mAbs specific for mouse ICAM-1 (clone KAT1; Beckman Coulter, Miami, FL), macrophages (F4/80), CD4 (Clone RM4-5; BD PharMingen), and CD8 (clone 53-6.7; BD PharMingen). Rat IgG (Southern Biotechnology Associates, Birmingham, AL) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 min, 37°C) with a biotinylated rabbit anti-rat IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and then HRP-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.

Measurement of soluble L-selectin (sL-selectin)

Blood samples were obtained by retro-orbital venous plexus puncture of anesthetized mice on days 0, 7, 14, and 24 and were centrifuged shortly after clot formation. All serum samples were stored at -40°C until use. Serum sL-selectin levels were measured by ELISA as described previously (22). Briefly, 96-well microtiter plates (Costar, Cambridge, MA) were coated with an anti-L-selectin mAb (LAM1-102) (23) in 0.1 M borate buffer (pH 8.4) at 4°C for 18 h and were then blocked with 2% BSA and 1% gelatin in TBS for 1 h at 37°C. Serum samples diluted 1/200 were incubated in triplicate wells for 90 min at 20°C. Subsequently, the plates were incubated with a biotinylated anti-L-selectin mAb (LAM1-116) (23) for 60 min at 20°C and then with avidin-HRP (Sigma-Aldrich) for 30 min at 4°C. The plates were developed using o-phenylenediamine (Sigma-Aldrich) as a substrate in 0.1 M citrate buffer (pH 4.5) in the presence of H2O2 and were read at 450 nm. The relative concentration of sL-selectin in individual samples was calculated by comparing the mean OD to a semilog standard curve of a titrated standard serum sample of known sL-selectin concentration using linear regression analysis.

ELISA for serum IgE

Total serum IgE levels were quantified by sandwich ELISA according to the manufacturer’s protocol (BD PharMingen). Rat anti-mouse IgE mAb (clone R35-72) was used for coating the plates and rat anti-mouse IgE mAb (clone R35-92) was used for detection. The relative concentration of total IgE levels in individual samples was calculated by comparing the mean OD to a semilog standard curve of the titrated purified mouse IgE using linear regression analysis.

Statistical analysis

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Edema following repeated Ag elicitation was inhibited by adhesion molecule deficiency

Mice sensitized on the ears with oxazolone 7 days before the first elicitation were repeatedly exposed to oxazolone on the sensitized ears at 2-day intervals for 24 days. Ear thickness was measured immediately before each elicitation. Repeated Ag applications in wild-type littermates induced a dramatic increase in the total ear thickness, whereas repeated applications of carrier alone had no detectable effect (Fig. 1). Following repeated elicitation, L-selectin-/- and ICAM-1-/- mice exhibited increases in total ear thickness that were generally similar to those found in wild-type littermates. By contrast, total ear thickness in L-selectin/ICAM-1-/- mice was significantly reduced (by 15–28%, p < 0.05) from days 2 to 26 (except days 12 and 14) compared with wild-type littermates. Furthermore, L-selectin/ICAM-1-/- mice exhibited significantly decreased ear thickness on day 26 relative to either L-selectin-/- (p < 0.01) or ICAM-1-/- (p < 0.005) mice. Thus, although loss of either L-selectin or ICAM-1 alone did not reduce total ear thickness following repeated elicitation, the loss of both molecules significantly inhibited the increase in total ear thickness.



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  		FIGURE 1. Total ear thickness following repeated elicitation with oxazolone in adhesion molecule-deficient and wild-type littermates. Mice sensitized on the ears with oxazolone 7 days before the first elicitation were repeatedly exposed to oxazolone at 2-day intervals on the originally sensitized ear for 24 days. Repeated treatment with acetone/sesame seed oil alone served as control. Ear thickness was measured immediately before each elicitation until 26 days after the first elicitation. Data are expressed as mean ear thickness ± SEM. These results were obtained from six mice in each group. *, p < 0.05 vs wild-type littermates and #, p < 0.05 vs both L-selectin-/- mice and ICAM-1-/- mice.

 
The induction of an ITH response was eliminated by adhesion molecule deficiency

A detailed time course of ear swelling elicited by repeated oxazolone challenge was assessed at 2-day intervals for 24 days (Fig. 2). A typical DTH response that peaked after 12 h was elicited in wild-type littermates on day 0 (Fig. 2A). As reported previously (5, 24), a significant reduction in ear swelling was observed in ICAM-1-/- (21% decrease, p < 0.05), L-selectin-/- (37%, p < 0.05), and L-selectin/ICAM-1-/- (79%, p < 0.0001) mice relative to wild-type littermates 24 h after the first elicitation (Fig. 2A). As elicitation was repeated in wild-type littermates, kinetic profiles of the ear swelling response were remarkably altered. Specifically, while the DTH response was gradually declining, a late phase reaction that peaked at 3–6 h was appearing by day 8 (Fig. 2, B and C). In addition, the ITH response that reached its maximum within 30 min was developing by day 16 (Fig. 2D). On day 24, the DTH response was suppressed while the ITH response, followed by the accompanying late phase reaction, was fully developed (Fig. 2E).



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  		FIGURE 2. Time course of ear swelling responses in adhesion molecule-deficient and wild-type littermates repeatedly exposed to oxazolone on days 0 (A), 4 (B), 8 (C), 16 (D), and 24 (E). Mice sensitized on the ear with oxazolone 7 days before the first elicitation were repeatedly challenged on the original sensitized ear with oxazolone for 24 days at 2-day intervals. Ear thickness was measured at multiple time points (0, 0.5, 1, 3, 6, 9, 12, 24, 36, and 48 h) after each challenge. The ear swelling results were expressed as the difference between the ear thickness before and after each elicitation. All values represent the mean ± SEM. Note that early time points (0–12 h) on the horizontal axis were expanded to easily compare the ear swelling response at these time points. These results were obtained from six mice in each group. *, p < 0.05 vs wild-type littermates and #, p < 0.05 vs both L-selectin-/- mice and ICAM-1-/- mice.

 
Adhesion molecule deficiency significantly suppressed the induction of the late phase reaction that was defined as the ear swelling occurring between 3 and 6 h. On day 24, the late phase reaction peak in all adhesion molecule-deficient mice occurred at 6 h instead of at 3 h as in wild-type littermates (Fig. 2E). Furthermore, peak ear swelling of the late phase reaction was significantly reduced in L-selectin-/- (44% decrease, p < 0.05), ICAM-1-/- (39%, p < 0.05), and L-selectin/ICAM-1-/- (48%, p < 0.01) mice compared with wild-type littermates (Fig. 2E). Remarkably, the loss of L-selectin, ICAM-1, or both completely eliminated the induction of the ITH response even on day 24 when the ITH response was defined as the ear swelling within the first 30 min (Fig. 2E). On day 24, unlike day 0, all adhesion molecule-deficient mice exhibited comparable magnitudes of ear swelling with similar kinetics following Ag challenge (Fig. 2, A and E). Thus, after chronic exposure to Ag, development of the late phase reaction was significantly inhibited by loss of L-selectin and/or ICAM-1, whereas the induction of the ITH response was eliminated.

The induction of the ITH response was eliminated by mAbs to adhesion molecules

To confirm a role of L-selectin and/or ICAM-1 in the induction of the ITH response, a blocking study by mAbs was performed. Wild-type littermates repeatedly exposed to oxazolone at 2-day intervals on the sensitized ear for 22 days were treated with mAbs to L-selectin, ICAM-1, or both i.v. 1 h before the elicitation on day 24. Wild-type littermates injected with isotype-matched control mAbs exhibited kinetic profiles of the ear swelling similar to that of untreated wild-type littermates (Figs. 2E and 3). By contrast, injection with mAbs to L-selectin, ICAM-1, or both significantly suppressed the development of the late phase reaction relative to wild-type littermates treated with control mAbs (Fig. 3). Furthermore, treatment with mAbs to L-selectin, ICAM-1, or both completely abrogated the induction of the ITH response. Thus, the induction of the ITH response was also eliminated by treatment with mAbs to L-selectin and/or ICAM-1 before the elicitation on day 24.



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  		FIGURE 3. Effect of adhesion molecule blockade by mAbs on time course of ear swelling responses on day 24. Wild-type littermates repeatedly exposed to oxazolone at 2-day intervals on the sensitized ear for 22 days were treated with mAbs to L-selectin (rat IgG2a), ICAM-1 (Armenian hamster IgG), or both i.v. 1 h before the elicitation on day 24. Wild-type littermates injected with irrelevant isotype-matched control (CTL) mAbs (rat IgG2a mAb for anti-L-selectin mAb, Armenian hamster IgG mAb for anti-ICAM-1 mAb, and both for mAbs to L-selectin and ICAM-1) served as control. The results of adhesion molecule-deficient mice were also included. The repeated epicutaneous oxazolone challenge was performed as described in Fig. 2. All values represent the mean ± SEM. These results were obtained from six mice in each group. *, p < 0.05 vs wild-type littermates.

 
Early edema induced by the ITH response was abrogated by adhesion molecule deficiency

To assess histological characteristics associated with the ITH response and the late phase reaction, ear biopsies were examined before and 0.5 and 6 h after elicitation on day 24. Before elicitation on day 24, intense leukocyte infiltration into the treated ears of both adhesion molecule-deficient and wild-type littermates was observed (Fig. 4A and data not shown). The ear biopsies of wild-type littermates taken 30 min after the oxazolone challenge revealed extensive s.c. edema (Fig. 4B). By contrast, early edema formation was completely eliminated in tissues from L-selectin-/-, ICAM-1-/-, and L-selectin/ICAM-1-/- mice (Fig. 4B and data not shown). At 6 h after the Ag administration, edema spread throughout the entire dermis in wild-type littermates while only slight s.c. edema was observed in the adhesion molecule-deficient mice (Fig. 4C and data not shown). After 6 h, overall inflammatory cell infiltration appeared to be similar for adhesion molecule-deficient and wild-type littermates. After 24 h, there was no detectable difference between adhesion molecule-deficient and wild-type littermates in either edema or leukocytes infiltration (data not shown). Thus, early edema formation induced by the ITH response was completely abrogated in the absence of L-selectin and/or ICAM-1 expression.



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  		FIGURE 4. Representative histological sections of the ear pinnae from L-selectin/ICAM-1-/- and wild-type littermates before (A) and 30 min (B) and 6 h (C) after oxazolone challenge on day 24. The repeated epicutaneous oxazolone challenge was performed as described in Fig. 2. The sections were stained with H&E. Original magnification, x50.

 
Mast cell recruitment was prevented by deficiency or blockade of adhesion molecules

A shift in the time course of ear swelling during the ITH response is associated with a dramatic increase in the numbers of dermal mast cells and CD4+ T cells (9). Therefore, numbers of neutrophils, CD4+ T cells, CD8+ T cells, macrophages, and mast cells were assessed in the ear biopsies taken before and 0.5, 6, and 24 h after the elicitation on day 24. In wild-type littermates, CD4+ T cell numbers were significantly increased after 24 h relative to those before elicitation on day 24 (44.7 ± 3.31/0.07 mm2 vs 32.7 ± 2.8, respectively, p < 0.05) in immunohistochemistry sections, but numbers of neutrophils, CD8+ T cells, and macrophages did not significantly increase at any time point after elicitation (data not shown). Furthermore, numbers of neutrophils, CD4+ T cells, CD8+ T cells, and macrophages were not significantly different between adhesion molecule-deficient and wild-type littermates before or at any time point after elicitation on day 24 (data not shown).

Mast cell numbers in the challenged ears were similar for adhesion molecule-deficient and wild-type littermates at all time points on day 0 (Figs. 5A and 6A). Following the repeated epicutaneous hapten application, mast cell numbers before elicitation on day 24 were significantly increased in wild-type (6.1-fold increase, p < 0.001), L-selectin-/- (3.5-fold, p < 0.0005), ICAM-1-/- (5.1-fold, p < 0.0001), and L-selectin/ICAM-1-/- (3.3-fold, p < 0.001) littermates compared with those observed before elicitation on day 0 (Figs. 5, A and B, and 6, A and B). However, before elicitation on day 24, mast cell numbers in L-selectin/ICAM-1-/- mice were significantly lower than those found in L-selectin-/-, ICAM-1-/-, or wild-type littermates (p < 0.05, Figs. 5B and 6B). After elicitation on day 24, mast cell numbers peaked by 30 min in wild-type littermates, whereas the peak was delayed after 24 h in all adhesion molecule-deficient mice. Thus, 30 min after elicitation, mast cell numbers were significantly reduced in L-selectin-/- (38% decrease, p < 0.001), ICAM-1-/- (33%, p < 0.005), and L-selectin/ICAM-1-/- (49%, p < 0.0001) mice relative to wild-type littermates (Figs. 5C and 6B). The concurrent loss of L-selectin and ICAM-1 resulted in a greater reduction in mast cell numbers compared with loss of either L-selectin (p < 0.05) or ICAM-1 (p < 0.01) alone. After 24 h on day 24, mast cell numbers in L-selectin-/- and ICAM-1-/- mice reached levels comparable to those found in wild-type littermates; however, mast cell numbers remained significantly decreased in L-selectin/ICAM-1-/- mice compared with either L-selectin-/-, ICAM-1-/-, or wild-type littermates (p < 0.01, Fig. 6B). Thus, although deficiency of L-selectin or ICAM-1 inhibited mast cell accumulation similarly, a double deficiency led to more significant inhibition of mast cell accumulation.



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  		FIGURE 5. Representative histological sections of mast cell accumulation in the ear pinnae from L-selectin/ICAM-1-/- and wild-type littermates before the first oxazolone challenge on day 0 (A), before (B), and 30 min (C) after oxazolone challenge on day 24. The repeated epicutaneous oxazolone challenge was performed as described in Fig. 2 legend. Mast cells are detected by the metachromatic staining of cytoplasmic granules in toluidine blue-stained sections (arrows). Original magnification, x50.

 


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  		FIGURE 6. Mast cell numbers on day 0 (A) and day 24 (B) in the dermis from adhesion molecule-deficient and wild-type littermates repeatedly treated with oxazolone on the ears. The repeated epicutaneous oxazolone challenge was performed as described in Fig. 2 legend. The effect of adhesion molecule blockade by mAbs on mast cell numbers at day 24 is also shown (C). The blocking study by mAbs was performed as described in Fig. 3 legend. Mast cell numbers were similar in untreated wild-type littermates and those injected with isotype-matched control mAbs. Therefore, results of wild-type littermates treated with both control mAbs (rat IgG2a and hamster IgG) are only shown as control. Mast cell numbers per high-power microscopic field (0.07 mm2) were counted in paraffin sections stained with toluidine blue. All values represent the mean ± SEM. These results were obtained from six mice in each group. *, p < 0.05 vs wild-type littermates and #, p < 0.05 vs both L-selectin-/- mice and ICAM-1-/- mice.

 
To confirm that the increases in mast cell numbers after elicitation on day 24 were mediated by L-selectin and/or ICAM-1, wild-type littermates repeatedly exposed to oxazolone at 2-day intervals for 22 days were injected with mAbs to L-selectin, ICAM-1, or both i.v. 1 h before the elicitation on day 24. Mast cell numbers were similar in untreated wild-type littermates and those injected with isotype-matched control mAbs (Fig. 6, B and C and data not shown). Therefore, in Fig. 6C, the results of wild-type littermates treated with both control mAbs (rat IgG2a and Armenian hamster IgG) are only shown as control. Similar to adhesion molecule-deficient mice, mast cell numbers at 30 min after elicitation were significantly reduced by L-selectin blockade (26% decrease, p < 0.01), ICAM-1 blockade (19%, p < 0.01) and combined blockade of L-selectin and ICAM-1 (23%, p < 0.01) relative to control mAb treatment (Fig. 6C). Mast cell numbers remained significantly reduced by blockade of both L-selection and ICAM-1 6 h after elicitation (p < 0.05) compared with control mAb injection while such difference was no longer observed after 24 h. Unlike adhesion molecule-deficient mice, there was no significant difference between single blockade of L-selection or ICAM-1 and double blockade at any time points after the elicitation. Thus, blockade of L-selectin and/or ICAM-1 by mAbs eliminated rapid mast cell accumulation.

Adhesion molecule deficiency suppressed serum IgE levels

IgE-triggered release of mediators by dermal mast cells is responsible for onset of the edema response that occurs 30 min after elicitation (9, 13). Therefore, serum total IgE levels were assessed on days 0, 7, 14, and 24 of Ag challenge. Before the first elicitation, IgE levels were not significantly different between adhesion molecule-deficient and wild-type littermates (Fig. 7). In wild-type littermates, repeated elicitation for 24 days led to a 6-fold increase in serum IgE levels compared with those found before the first elicitation (p < 0.05). By contrast, IgE levels did not significantly increase in L-selectin-/-, ICAM-1-/-, or L-selectin/ICAM-1-/- mice. IgE levels on day 24 were significantly lower in L-selectin-/- (75% decrease, p < 0.05), ICAM-1-/- (80%, p < 0.01), and L-selectin/ICAM-1-/- (51%, p < 0.05) mice than those found in wild-type controls. Thus, the increase in serum total IgE levels following Ag administration was significantly suppressed by the loss of L-selectin, ICAM-1, or both.



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  		FIGURE 7. Serum total IgE levels on days 0, 7, 14, and 24 in adhesion molecule-deficient and wild-type littermates repeatedly treated with oxazolone on the ears. The repeated epicutaneous oxazolone challenge was performed as described in Fig. 2. Serum total IgE levels were determined by ELISA. All values represent the mean ± SEM. These results were obtained from six mice in each group. *, p < 0.05 vs wild-type littermates.

 
Repeated Ag challenge markedly altered the kinetics of ICAM-1 expression on the endothelium

To assess whether chronic exposure to Ag altered the kinetic profiles of ICAM-1 expression after elicitation, ICAM-1 expression was examined before and after 24 days of treatment using immunohistochemistry. In normal skin, weak ICAM-1 expression was detected exclusively on endothelial cells (Fig. 8A). ICAM-1 expression remained similarly weak at 30 min following the first elicitation, but was up-regulated after 24 h. Repeated elicitation resulted in slightly augmented ICAM-1 expression before elicitation on day 24 compared with that of normal skin (Fig. 8B). On day 24, ICAM-1 expression was rapidly up-regulated 30 min after elicitation and remained up-regulated after 24 h. By day 24, weak ICAM-1 expression was also detected on keratinocytes, but not on fibroblasts or infiltrating inflammatory cells (Fig. 8B and data not shown). In addition, on both days 0 and 24, loss of L-selectin expression did not affect ICAM-1 expression at any of the time points examined (data not shown). Furthermore, ICAM-1 expression was not detected before or after repeated Ag elicitation in the skin of ICAM-1-/- mice (data not shown). Thus, chronic administration of Ag resulted in a more rapid up-regulation of ICAM-1 expression following elicitation.



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  		FIGURE 8. ICAM-1 expression on day 0 (A) and day 24 (B) in the ear pinnae from wild-type mice repeatedly treated with oxazolone as described in Fig. 2 legend. ICAM-1 expression was assessed in tissue biopsies by immunohistochemistry using anti-ICAM-1 Abs. Arrows indicate ICAM-1 expression on endothelial cells. Original magnification, x400.

 
Serum L-selectin levels were elevated following repeated elicitation

L-selectin is rapidly released from the surface of leukocytes following cellular activation (25, 26). sL-selectin is present at relatively high levels in human plasma and remains functionally active (27). Interestingly, serum sL-selectin levels are elevated in patients with AD (28). Therefore, sL-selectin levels were measured by ELISA in serum samples from ICAM-1-/- and wild-type littermates on days 0, 7, 14, and 24 of Ag challenge. Although sL-selectin levels in wild-type littermates did not significantly increase on days 7 and 14 compared with day 0, sL-selectin levels on day 24 were elevated by 2.3-fold over those before the first elicitation (p < 0.005, Fig. 9A). ICAM-1-/- mice exhibited significantly increased sL-selectin levels on day 14 by 3.2-fold relative to those found on day 0 (p < 0.001), but significantly decreased sL-selectin levels on day 24 by 40% (p < 0.005, Fig. 8B). Serum sL-selectin levels were overall higher in ICAM-1-/- mice compared with wild-type littermates on day 0 (51% increase, p < 0.005), day 7 (45%, p < 0.01), and day 14 (180%, p < 0.01); by contrast, on day 24, sL-selectin levels were significantly 61% lower than those in wild-type littermates (p < 0.005). Thus, the repeated elicitation of CH resulted in the sL-selectin elevation that was generally enhanced by ICAM-1 deficiency.



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  		FIGURE 9. Serum sL-selectin levels in wild-type littermates (A) and ICAM-1-/- mice (B) repeatedly exposed with oxazolone on the ears as described in Fig. 2. Serum sL-selectin levels were determined by ELISA and represent the mean ± SEM of results obtained from six mice in each group. *, p < 0.005 vs day 0 and #, p < 0.01 vs wild-type littermates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, the induction of an ITH response following chronic Ag exposure was completely eliminated by deficiency or blockade of L-selectin, ICAM-1, or both, while development of the accompanying late phase reaction was significantly inhibited (Figs. 2 and 3). Consistently, early edema formation caused by the ITH response was not present in adhesion molecule-deficient mice (Fig. 4 and data not shown). Thus, this is the first report demonstrating the critical role of L-selectin and ICAM-1 in chronic CH responses induced by repeated epicutaneous Ag elicitation. In addition, total ear thickness following chronic Ag administration was significantly reduced by loss of both L-selectin and ICAM-1 (Fig. 1). Therefore, the present study demonstrates that L-selectin and ICAM-1 cooperatively regulate CH responses to chronic Ag stimulation both qualitatively and quantitatively.

The abrogated ability to mount an ITH response observed in adhesion molecule-deficient mice correlated with significantly decreased accumulation of mast cells, but not other types of immune cells, including CD4+ T cells, at 30 min after elicitation (Figs. 5C and 6B). Consistent with this finding, the ITH response is virtually eliminated in the ears of S1/S1d mice that lack dermal mast cells (9). Mast cell recruitment into tissues is thought to occur by release of mast cell precursors from the bone marrow into the peripheral blood, followed by migration of these precursors into tissues and their subsequent differentiation into mature mast cells (29). Increases in mast cell numbers at sites of inflammation has also been observed (30). Our present findings following chronic Ag exposure also demonstrate increased mast cell accumulation before elicitation on day 24 (Figs. 5B and 6B). However, despite this mast cell accumulation, the ITH response to oxazolone was not induced by deficiency or blockade of L-selectin, ICAM-1, or both (Figs. 2 and 3). At 30 min following elicitation on day 24, wild-type littermates exhibited rapid dermal mast cell recruitment that was virtually eliminated by loss or blockade of L-selectin, ICAM-1, or both (Figs. 5C and 6, B and C). This observation suggests that induction of the ITH response is dependent on the rapid recruitment of mast cells that is regulated by expression of adhesion molecules on these cells. Indeed, peritoneal murine mature mast cells express significant levels of L-selectin, CD18 ({beta}2 integrin), and CD11b (31, 32). Furthermore, 4 h following immune complex challenge in the passive reverse Arthus reaction, wild-type littermates exhibit rapid cutaneous and peritoneal mast cell recruitment that is markedly reduced in mice lacking L-selectin, ICAM-1, or both (32). Similar rapid mast cell migration into the CNS from the blood within 1–2 h in response to altered physiological conditions is also observed (33). The finding that ICAM-1 was expressed strongly on the endothelium and only weakly or not at all on epidermal cells, fibroblasts, or inflammatory infiltrating cells following chronic inflammation (Fig. 8) indicates that mast cell accumulation is primarily mediated by ICAM-1 expression on endothelial cells. Therefore, our results suggest that L-selectin and ICAM-1 regulate mature mast cell recruitment into inflammatory sites presumably through the peripheral blood.

Ag-specific IgE Abs are developed in parallel with the induction of the ITH responses (9). Therefore, IgE-triggered release of mast cell mediators in response to Ag may be responsible for onset of the ITH response at 30 min after elicitation (9). The current study confirms the presence of elevated serum total IgE in wild-type littermates following repeated elicitation (Fig. 7). In addition, serum total IgE was significantly reduced in adhesion molecule-deficient mice after 24 days of repeated elicitation (Fig. 7). Both humoral and cellular immune responses are generally normal in these adhesion molecule-deficient mice (5, 34, 35). In addition, loss of L-selectin or ICAM-1 does not affect total or Ag-specific IgE production in a murine model of asthma (12, 36). Therefore, it is unlikely that the observed reduction in IgE production is due to a defective immune response in the adhesion molecule-deficient mice. The development of the ITH responses following repeated elicitation is associated with preferential local production of Th2-type cytokines that induce IgE production (10, 11). Immunohistochemical analysis has shown that Th2-type cytokines, especially IL-4, are predominantly produced by mast cells and CD4+ T cells in this chronic CH model (11). Consistent with this, mast cells are one of the major sources of IL-4 in lesional skin from patients with AD (37). Therefore, the results of this study suggest that L-selectin and ICAM-1 regulate IgE production indirectly by controlling mast cell accumulation at sites of inflammation.

Chronic Ag exposure resulted in an altered kinetic profile of ICAM-1 expression. Specifically, the first elicitation up-regulated ICAM-1 expression at 24 h while the repeated elicitation for 24 days up-regulated ICAM-1 expression at 30 min and remained similarly high at 24 h (Fig. 8). This shift in expression may contribute to the rapid recruitment of mast cells into the inflammatory sites 30 min after elicitation on day 24 (Figs. 5C and 6B) and thereby may be associated with induction of the ITH response (Fig. 2). Although the ITH response did not develop in L-selectin/ICAM-1-/- mice even after 24 days of treatment, a weak late phase reaction was induced (Fig. 2E). This suggests that other adhesion pathways, which do not require expression of L-selectin and ICAM-1, are operable for induction of the late phase reaction by day 24. Consistent with this, a chronic inflammatory state is suggested to alter adhesion molecule requirements for acute neutrophil emigration (38). In addition, using a short-term chronic inflammation model, development of an L-selectin-independent pathway that is mainly mediated by the interaction between {alpha}4 integrin and VCAM-1 has been reported (39). Thus, ICAM-1- and L-selectin-independent adhesion pathways may also be involved in the regulation of chronic skin inflammation.

The repeated epicutaneous Ag elicitation for 24 days led to significantly increased sL-selectin levels compared with those before the first elicitation (Fig. 9A). Furthermore, ICAM-1 loss generally increased sL-selectin levels compared with wild-type littermates (Fig. 9B), as previously reported (22). This finding may be related to increased circulating leukocyte numbers by 2.9- to 4.1-fold in ICAM-1-/- mice (4, 15). Alternatively, since ICAM-1 regulates leukocyte rolling in concert with L-selectin (4), the increased sL-selectin levels in ICAM-1-/- mice may be interpreted as the increased leukocyte-endothelium interaction through L-selectin to compensate the rolling reduced by ICAM-1 deficiency at the sites of inflammation, resulting in the increased L-selectin shedding. However, sL-selectin levels on day 24 were decreased in ICAM-1-/- mice relative to wild-type littermates (Fig. 9B). Although the reasons for this remain unknown, it may be due to a decrease in the sL-selectin source since L-selectin expression levels on circulating leukocytes from ICAM-1-/- mice were significantly reduced on day 24 compared with those found on day 0 (data not shown) and the t1/2 of sL-selectin is estimated at ~25 h (22). Like the murine chronic CH model (Fig. 9A), sL-selectin levels are elevated in patients with AD and correlate with both the severity of AD and total IgE levels (28). Therefore, these results suggest that the murine chronic CH model reflects the chronic inflammation associated with AD with respect to regulation of L-selectin shedding.

AD is a chronic inflammatory skin disease with an allergic and genetic background (40, 41). The prevalence of AD has increased in recent years, and it is now estimated to affect up to 20% of the general population (40). Patients with AD are best recognized by their propensity to produce large amounts of IgE Abs and to mount skin ITH responses against various environmental allergens (41). The allergic skin manifestation in AD is also characterized by an IgE-dependent late phase reaction that may correspond to the late phase reaction in the murine chronic CH model (9, 40). The present study demonstrates that the loss of L-selectin, ICAM-1, or both significantly inhibited the late phase reaction, virtually abrogated induction of the ITH response, and suppressed IgE production, likely by reducing rapid mast cell recruitment into the inflammatory site (Figs. 2–7). Therefore, the results of this study indicate that L-selectin and ICAM-1 regulate induction of the ITH response during chronic skin inflammation and suggest that these cell adhesion molecules would be potential therapeutic targets for controlling human chronic allergic diseases, such as AD.


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


   Footnotes
 
1 This work was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan (to S.S.) and National Institutes of Health Grants CA54464 and CA81776 (to T.F.T.). Back

2 Address correspondence and reprint requests to Dr. Shinichi Sato, Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail address: s-sato{at}med.kanazawa-u.ac.jp Back

3 Abbreviations used in this paper: CH, contact hypersensitivity; DTH, delayed-type hypersensitivity; ITH, immediate-type hypersensitivity; AD, atopic dermatitis; sL-selectin, soluble L-selectin. Back

Received for publication May 30, 2002. Accepted for publication February 13, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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