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Originally published online as doi:10.1189/jlb.0107063 on April 18, 2007

Published online before print April 18, 2007
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(Journal of Leukocyte Biology. 2007;82:57-71.)
© 2007 by Society for Leukocyte Biology

Anti-CD44-mediated blockade of leukocyte migration in skin-associated immune diseases

Margot Zöller*,{dagger},1, Pooja Gupta*, Rachid Marhaba*, Mario Vitacolonna* and Pia Freyschmidt-Paul{ddagger}

* Department of Tumor Progression and Tumor Defense, German Cancer Research Center, Heidelberg, Germany;
{dagger} Department of Applied Genetics, University of Karlsruhe, Germany; and
{ddagger} Department of Dermatology, University Hospital Marburg, Germany

1 Correspondence: Department of Tumor Progression and Immune Defense, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: m.zoeller{at}dkfz.de

ABSTRACT

CD44 plays an important role in leukocyte extravasation, which is fortified in autoimmune diseases and delayed-type hypersensitivity (DTH) reactions. There is additional evidence that distinct CD44 isoforms interfere with the extravasation of selective leukocyte subsets. We wanted to explore this question in alopecia areata (AA), a hair-follicle centric autoimmune disease, and in a chronic eczema. The question became of interest because AA is treated efficiently by topical application of a contact sensitizer, such that a mild DTH reaction is maintained persistently. Aiming to support the therapeutic efficacy of a chronic eczema in AA by anti-CD44 treatment, it became essential to control whether a blockade of migration, preferentially of AA effector cells, could be achieved by CD44 isoform-specific antibodies. Anti-panCD44 and anti-CD44 variant 10 isoform (CD44v10) inhibited in vitro migration of leukocytes from untreated and allergen-treated, control and AA mice. In vivo, both antibodies interfered with T cell and monocyte extravasation into the skin; only anti-panCD44 prevented T cell homing into lymph nodes. Contributing factors are disease-dependent alterations in chemokine/chemokine receptor expression and a blockade of CD44 on endothelial cells and leukocytes. It is important that CD44 can associate with several integrins and ICAM-1. Associations depend on CD44 activation and vary with CD44 isoforms and leukocyte subpopulations. CD44 standard isoform preferentially associates with CD49d in T cells and CD44v10 with CD11b in monocytes. Accordingly, anti-panCD44 and anti-CD49d inhibit T cell, anti-CD11b, and anti-CD44v10 macrophage migration most efficiently. Thus, allergen treatment of AA likely can be supported by targeting AA T cells selectively via a panCD44-CD49d-bispecific antibody.

Key Words: rodent • autoimmunity • adhesion molecules • cell trafficking

INTRODUCTION

Alopecia areata (AA) is an autoimmune disease of the skin, which affects anagen-stage hair follicles [1 , 2 ]. Extensive AA is often treated by local application of a contact sensitizer, which induces a mild, chronic eczema [3 4 5 ]. Hair regrowth is observed in 50–70% of patients after 2–6 months [4 ]. Some C3H/HeJ mice spontaneously develop AA, which closely resembles the human disease. AA can also be transferred to nonaffected littermates using full-thickness skin grafts [6 , 7 ]. As in humans, AA of C3H/HeJ mice can be treated by repeated application of the contact sensitizer squaric acid dibutyl ester (SADBE), and hair regrowth is apparent after 6–10 weeks [8 , 9 ]. There is evidence that the curative effect of a chronic contact eczema in AA relies on impaired T cell activation as a result of hindrance in APC migration toward the draining lymph node (LN); however, T cell extravasation was not affected [10 ]. Because of the latter and also as allergen treatment is long-lasting and not always successful, we speculated that a blockade of leukocyte extravasation might provide an additive, therapeutic benefit. We focused on a blockade of CD44, as we demonstrated recently that AA induction can be prevented by treatment with a CD44 variant 10 isoform (CD44v10)-specific antibody [11 ]. However, anti-panCD44 and anti-CD44v10 have also been shown to interfere with the severity of delayed-type hypersensitivity (DTH) reactions [12 ]. Thus, the possibility arose that during AA treatment with a contact sensitizer, a blockade of CD44 might affect the (curative) DTH reaction, which would rather promote than inhibit AA progression. Therefore, it became important to explore the mechanisms, whereby anti-CD44 interferes with AA and DTH induction, whether these mechanisms are alike or distinct and whether a blockade of CD44/leukocyte extravasation could be directed selectively toward AA effector cells.

There is ample evidence that CD44, originally described as a leukocyte-homing receptor [13 ], plays an important role in the extravasation of T cells [14 , 15 ] as well as monocytes [16 , 17 ]. Earlier studies demonstrated that anti-CD44 inhibits induction of a DTH reaction during the first 24 h, and homing into LN is not affected, indicating that CD44 is required for extravasation [18 ]. The fact that leukocyte entry into inflamed tissue is impaired in mice with a targeted deletion of CD44 is in line with this assumption [19 ].

Several mechanisms have been discussed, whereby a blockade of CD44 could interfere with leukocyte extravasation. First, CD44 has been defined as the major hyaluronic acid (HA) receptor [20 ]. HA binding, which requires CD44 to be activated [21 22 23 24 ], is obviously of utmost importance for the involvement of CD44 in leukocyte migration [25 ]. In fact, in chronic inflammation, T cell rolling is up-regulated, and the percentage of T cells expressing activated CD44 is increased [26 ]. The inflammatory cytokines TNF-{alpha}, IL-1ß, as well as LPS induce HA and CD44 expression on endothelial cells [16 , 27 , 28 ], where HA is anchored via CD44. Thus, a positive loop is created, which supports leukocyte rolling further. Moreover, by a direct interaction between CD44 and CD49d, CD44 is involved in firm adhesion [29 ].

Second, CD44 can act as a chemokine receptor. It was shown that the hematopoietic [CD44 standard isoform (CD44s)] or CD44v acts as a receptor for osteopontin (OPN) [30 , 31 ]. The C-terminal region of OPN, in particular, interacts with CD44 and induces chemotaxis, and the N terminus interacts with ß3 integrins and induces spreading and activation [32 ]. Migration of Langerhans cells into the draining LN during initiation of a contact hypersensitivity reaction also depends essentially on OPN and its interaction with CD44 [33 ]. In addition, a motile phenotype can be acquired by an intracellular OPN-CD44 association [34 ], which is accompanied by colocalization of the complex with ezrin at the leading edge of the migrating cell [35 ]. Other chemokines, such as CCL5, also associate with CD44 [36 , 37 ]. The CCL5-CD44 complex supports formation of a signaling complex, which initiates p44/42 MAPK activation [36 ].

Third, CD44 can initiate signal transduction by itself or as an accessory molecule in concert with the TCR-CD3 complex [38 , 39 ]. It is notable too that the association of CD44 with CD49d does not only promote leukocyte adhesion [29 ] but also allows CD44 to make use of CD49d-associated, signal-transducing molecules and vice versa [40 ].

To evaluate whether a blockade of CD44 could provide an additive, therapeutic benefit in allergen-treated AA, it became essential to know which of the outlined, functional activities are dominating in AA and DTH reactions. Our data indicate that anti-CD44v10 interferes predominantly with macrophages (M{phi}) but also with T cell homing into the skin of AA and allergen-treated AA mice, and anti-panCD44 hampers, in addition, T cell homing into the draining LN. Furthermore, activated CD44/CD44v10 associates rather selectively with CD11b (M{phi}) and CD49d (T cells)—associations, which are of functional importance.

MATERIALS AND METHODS

Mice
C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, ME, USA) received autoclaved food pellets and acidified water ad libitum. AA was induced by grafting skin (1 cm2) from spontaneously AA-affected mice to normal-haired recipients [6 ]. Over 90% of grafted mice developed AA within 6–8 weeks after grafting. Only mice with extensive hair loss were included. Mice were sensitized on a 1-cm2 area of the back (outside the grafted area) with 2% SADBE in acetone, followed by weekly, topical applications of 0.5% or 1% SADBE in acetone on the entire back. The concentration and total volume of the contact allergen were chosen individually such that a moderately severe contact dermatitis, lasting for 2–3 days, was induced. Where indicated, mice received i.v. antibody injections (150 µg) two times weekly, starting at the first challenge with SADBE and concomitantly in control and AA mice. Previous studies revealed that SADBE-induced changes in leukocyte subpopulations, activation marker, and adhesion molecule expression had developed fully at 3 days after treatment—the effect being most pronounced after the third challenge. At this time, hair regrowth had not yet started (data not shown). Therefore, the impact of anti-CD44 treatment was evaluated 3 days after the third SADBE challenge. All ex vivo analyses were performed simultaneously in the four groups of diseased and control mice. The local government authorities approved all animal experiments.

Tissue preparation
Mice were killed by cervical dislocation. Skin, spleen, and draining LN were collected. Dorsal skin samples were embedded in OCT compound (Tissue Tek, Sakura, Zoeterwoude, Netherlands) and snap-frozen in liquid nitrogen. For the isolation of cutaneous leukocytes [skin-infiltrating leukocytes (SkIL)], fat and s.c. muscle tissue were removed. Skin was layered epidermis-uppermost on sterile gauze and incubated three times for 30 min with a 1-mg/ml trypsin/EDTA solution. After each incubation, the skin was pressed against the sterile gauze, and isolated cells were collected in RPMI 1640/10% FCS. After the final trypsin treatment, cells were pooled, washed, and resuspended in RPMI-1640/10% FCS and 10–3 M HEPES buffer and incubated at 37°C for 2 h for the recovery of cell surface molecule expression. For flow cytometry analysis, SkIL from three animals were pooled. Single cell suspensions from skin-draining LN and the spleen cells (SC) were prepared by pressing through fine gauze. Viability was determined by Trypan blue exclusion (SkIL: >70%; LN cells, SC: >95%). Where indicated, CD4+ or CD11b+ cells were enriched from LN or SC by magnetic bead sorting (Miltenyi, Mönchengladbach, Germany).

Cell lines and antibodies
The endothelial cell line C166 [41 ] was maintained in DMEM/10% FCS. For functional studies, the panCD44-specific antibody IM7 [42 ] and the CD44v10-specific antibody K926 [12 ] were used. Both antibodies do not interfere with HA binding [12 , 43 ]. The CD44v10-specific antibody K926 derives from a fusion of rat SC with Ag8 after vaccination with a CD44v4-v10-GST fusion protein. By peptide competition, the epitope could be restricted to AA 583-606, where only AA 594 differs between rat and mouse [12 ]. The following additional primary antibodies were used: antimouse CD4 (YTA3.2.1), CD8 (YTS169.4.2.1), CD11a (M17.5.2), CD11b (YBM6.6.10), CD54 (YN1/1.7.4; European Animal Cell Culture Collection, Porton Down, UK); antimouse CD25 (7D4), anti-panCD44 (KM81; American Type Culture Collection, Manassus, VA, USA); anti-CD49d (PS/2; kindly provided by Kaotu Miyazaki, Saga Medical School, Saga, Japan) [44 ]; antimouse CD11c (HL3), CD28 (37.51), CD40 (3/23), CD49f (GOH3; antihuman, cross-reacts with mouse), CD80 (1G10), CD86 (GL1), CD95 (Jo2), CD95L (MFL3), CD102 (3C4), CD152 (9H10), CD154 (MR1), CCL2/MCP-1 (2H5; BD PharMingen, Heidelberg, Germany); CCL1/TCA3 (polyclonal), CCL17/thymus and activation-regulated chemokine (monoclonal), CCL20/MIP-3{alpha} (monoclonal), CCR6 (monoclonal; R&D, Wiesbaden, Germany); CCL5/RANTES (polyclonal), CXCL10 (polyclonal; Biotrend, Köln, Germany); OPN (polyclonal; Assay Designs, Ann Arbor, MI, USA); CCR4 (polyclonal), CCR8 (polyclonal; Abcam Ltd., Cambridgeshire, UK). Secondary reagents were FITC-, PE-, or allophycocyanin-labeled antirat IgG, antirat IgM, antirabbit IgG, antihamster IgG, or streptavidin (Strep; Dianova, Hamburg, Germany; Becton Dickinson, Heidelberg, Germany; or Biotrend, Köln, Germany).

Flow cytometry
Flow cytometry followed routine procedures. For intracellular staining, cells were fixed and permeabilized in advance. Flow cytometry was evaluated using a FACSCalibur (Becton Dickinson). Contaminating keratinocytes in SkIL preparations were excluded by gating. Analysis was performed by the Cell Quest program. Experiments were repeated at least three times. Values represent the mean and SD. Significance of differences was evaluated using the two-tailed Student’s t test.

Immunohistology
Sections (5 µm) of snap-frozen skin were fixed (chloroform/acetone, 1:1, 4 min) and treated with levamisole to ablate tissue alkaline phosphatase activity. Nonspecific binding was blocked using an avidin-biotin-blocking kit (Vector Laboratories, Burlingame, CA, USA), and 2% normal serum was derived from the same species as the secondary antibodies. For chemokine staining, tissues were fixed in paraformaldehyde (4%) and permeabilized (0.1% Triton X-100, 4 min, 4°C). Tissues were incubated for 1 h with the primary antibody, washed, and exposed to biotinylated secondary antibodies (30 min) and an alkaline phosphatase-conjugated, avidin-biotin complex (5–20 min). Tissue sections were counterstained with Mayer’s hematoxylin. Primary antibodies were replaced with normal rat or rabbit IgG for negative controls.

ELISA
Standard ELISA procedures were used. Plates were coated with anti-CD44 (Clone KM81). Plates were washed, blocked (100 µg/ml BSA), and incubated overnight with 50 µl mouse serum (dilution 1:50–1:800). After washing, 50 µl biotinylated anti-CD44 (Clone IM7) was added (1 h, room temperature). After washing, alkaline phosphatase-coupled Strep and substrate (p-nitrophenyl phosphate) were added. Absorbance was read at 405 nm. Assays were run in triplicates.

Immunoprecipitation (IP) and Western blot (WB)
SC were lysed (30 min, 4°C) in HEPES buffer (25 mM HEPES, 150 mM NaCl, pH 7.4) containing 1% CHAPS, 1 mM PMSF, 1 mM NaVO4, 10 mM NaF, and a protease inhibitor mix (Boehringer Mannheim, Germany). Lysates were centrifuged (13,000 g, 10 min, 4°C) and used for IP (1 h, 4°C) followed by incubation with Protein G sepharose (1 h). Immune complexes were washed four times with lysis buffer, dissolved in Laemmli buffer, and resolved by SDS-PAGE. Samples were resolved on 8% SDS-PAGE, and proteins were transferred to nitrocellulose membrane (Amersham, UK; 30 V, 16 h, 4°C). Membranes were blocked (PBS/5% BSA/0.1% Tween-20, 1 h, room temperature) and blotted with primary antibodies, followed by HRP-conjugated secondary antibodies or Strep (1 h, room temperature). Blots were developed with the ECL detection system.

In vitro leukocyte migration
Draining LN cells (5x104) were seeded in the upper part of a Boyden chamber in 30 µl RPMI/0.1% BSA. Where indicated, cells had been loaded with antibody before seeding (10 µg/ml, 30 min, 4°C). Cells were washed and seeded as above. The lower chamber, separated by a 5-µm pore-size polycarbonate membrane (Neuroprobe, Gaithersburg, MD, USA) contained 30 µl RPMI/0.1% BSA plus 40 ng/ml OPN (R&D). The concentrations of OPN for optimal response were determined beforehand. After incubation (4 h), cells in the lower chamber were counted. Transendothelial cell migration was evaluated after seeding C166 cells on 3 µm polycarbonate tissue-culture inserts (Nunc, Roskilde, Denmark). After overnight culture to form a monolayer, OPN (40 ng/ml) was added to the lower chamber, and LN cells (2x105) were added to the upper chamber. Where indicated, C166 cells were activated by incubation with 10 ng/ml TNF-{alpha} for 4 h, or C166 cells were incubated with anti-CD44 (10 µg/ml, 30 min, 4°C). In some experiments, LN cells were preincubated with anti-CD44 as described above. Transendothelial cell migration was evaluated after 12 h. Assays were run in triplicates. Cell migration is presented as percentage of migrating cells taking the starting load as 100%.

Cell transfer and migration
Single LN cell suspensions from pooled, skin-draining LN were labeled with CFSE. Mice received an i.v. injection of 1 x 107 CFSE-labeled leukocytes concomitantly with 150 µg antibody. Mice were killed 3 days after treatment.

Statistics
Significance was evaluated by the two-tailed Student’s t test for unequal variance or by the two-tailed Wilcoxon rank sum test.

RESULTS

The experimental model
There is strong evidence that CD44 plays an important role in leukocyte extravasation. This is true for activated leukocytes in autoimmune disease as well as in allergic reactions [26 , 45 ]. It has not yet been explored whether a blockade of CD44 affects the same or different effector cells in autoimmune and allergic reactions, whether different CD44 isoforms are involved, and whether organ-related differences exist. AA can be treated by a chronic allergic eczema. Thus, allergen-treated AA mice in comparison with control, AA, or allergen-treated mice appeared to provide a suitable model to answer these questions. From a clinical point of view, we anticipated that an analysis of the three groups of diseased mice could provide additional information. We assumed that according to disease-related additive or selective effects of CD44 in leukocyte migration, a CD44 blockade might serve as a supporting therapeutic in allergen-treated AA. As previous work provided evidence that CD44v10 expression is particularly altered in skin-associated immune diseases [46 ], we concomitantly evaluated the effect of anti-CD44v10. Starting contemporarily with the first challenge in DTH and AA/DTH mice, mice received 5 x 150 µg antibody i.v.

Anti-CD44v10 and anti-panCD44 interfere distinctly with leukocyte migration
Skin-draining LN cells from AA mice display high migratory activity in vitro, even in the absence of any stimulus, whereas LN cells from control and allergen-treated mice migrated mostly in response to OPN or CXCL10 [10 ]. Thus, it became of interest to see whether anti-CD44 would hamper spontaneously migrating AA leukocytes similar to leukocytes from control and DTH mice and whether anti-CD44 would also be effective in the combined disease state. Indeed, irrespective of the above-mentioned, disease-associated differences, in vitro migration of draining LN cells of untreated and allergen-treated control and AA mice was inhibited significantly after LN cells had been preincubated with anti-panCD44 or anti-CD44v10. Although migration in the absence of an external stimulus was inhibited equally by anti-panCD44 and anti-CD44v10, migration of AA LN cells in response to OPN was inhibited more strongly by anti-CD44v10 than anti-panCD44 (Fig. 1A ).


Figure 1
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  		Figure 1. Impact of anti-panCD44 and anti-CD44v10 on migration of skin-draining LN cells (LNC), which (A) from untreated and SADBE-treated control and AA mice, killed 3 days after the third challenge, were incubated with control IgG, anti-panCD44, or anti-CD44v10 (10 µg/ml, 30 min, 4°C), washed, and seeded in the upper part of a Boyden chamber. The lower part contained RPMI/0.1% BSA or in addition, 40 ng/ml OPN. After 4 h (37°C, 5% CO2), cells in the lower chamber were counted. (B) Control, SADBE-treated, AA, and SADBE-treated AA mice received control IgG, anti-panCD44, or anti-CD44v10 for 2 weeks (150 µg per mouse, i.v., twice per week). Three days after the third challenge with SADBE, mice were killed, LN were excised, and LN cells were treated as described in A. (A and B) The mean percentage of migrating cells is shown (A, triplicates of five experiments; B, triplicates of three experiments with three mice per group in each experiment). (C) Mice were treated as described above. The number of draining LN cells and SkIL (mean values±SD of three experiments each with three mice per group) was evaluated. (D and E) Control, SADBE-treated, AA, and SADBE-treated AA mice received an i.v. injection of 1 x 107 CFSE-labeled LN cells from corresponding mice (i.e., LN cells from control mice into control mice) concomitantly with 150 µg control IgG, anti-panCD44, or anti-CD44v10. Mice were killed after 3 days. Skin and draining LN were collected. The percentage of CFSE-labeled LN cells and SkIL was evaluated by flow cytometry (exemplified in D). (E) Mean values ± SD (18 individual values per group) are shown. *, Significant differences (P<0.01) between control IgG and anti-CD44-treated cells/mouse; S, significant differences between anti-panCD44- and anti-CD44v10-treated LN cells (A) or between LN cells reloaded ex vivo with control IgG or anti-CD44 (B).

 
It is important that in vivo treatment with anti-panCD44 or anti-CD44v10 also sufficed for a significant reduction in draining LN cell migratory activity. Migration was inhibited to a comparable degree when mice had received anti-panCD44 or anti-CD44v10. Reloading the LN cells ex vivo with anti-panCD44 or anti-CD44v10 treatment strengthened the inhibitory effect of in vivo anti-CD44 treatment. With minor differences, this was true for all four groups of mice (Fig. 1B) .

To explore the impact of anti-CD44 on leukocyte migration in vivo, we evaluated the overall recovery of leukocytes first. As shown before [9 ], higher numbers of draining LN cells and SkIL were recovered from AA and allergen-treated mice. It is interesting that the recovery of draining LN cells was decreased only in anti-panCD44- but not in anti-CD44v10-treated mice and only in diseased animals. The overall recovery of SkIL was reduced slightly in all four groups of anti-panCD44- and anti-CD44v10-treated mice. The effect was slightly stronger in diseased rather than healthy mice (Fig. 1C) .

To confirm that only anti-panCD44 interferes with LN homing, and anti-panCD44 as well as anti-CD44v10 interfere with skin homing, mice received an i.v. injection of CFSE-labeled LN cells, which were derived from mice with corresponding health status; i.e., AA mice received LN cells from AA mice. Three days later, the recovery of dye-labeled cells was evaluated in skin-draining LN and the skin (an example is given in Fig. 1D ). LN cells from diseased mice homed more readily into the skin than LN cells of control mice. This was confirmed by a criss-cross transfer (control LN cells into AA or DTH mice and vice versa; data not shown). It is notable that in vivo migration toward the skin was inhibited by anti-panCD44 and anti-CD44v10. Only anti-panCD44, but not anti-CD44v10, inhibited the entry into LN of diseased mice. In the few dye-labeled cells recovered in LN of anti-panCD44-treated mice, CFSE intensity was high. Instead, in control IgG- or anti-CD44v10-treated mice, the intensity of the CFSE label ranged from 102 to 104 (Fig. 1E) . This could be indicative for anti-panCD44, inhibiting not only leukocyte migration but also leukocyte proliferation. A time-course analysis, where recovery of CFSE-labeled cells was recorded between 1 and 5 days after application (data not shown), supported this suggestion.

Taken together, anti-CD44 treatment exerted a strong effect on leukocyte migration in vitro and a distinct effect on leukocyte migration in vivo. As anti-CD44v10 inhibited skin but not LN homing, the question arose whether distinct CD44 isoforms are involved in LN versus skin homing and whether a panCD44/CD44v10 blockade might affect distinct leukocyte subpopulations.

Anti-panCD44 and anti-CD44v10 block skin homing of T cells and monocytes
Particularly in the skin, the composition of the major leukocyte subpopulations differs among control, AA, and DTH mice with a preponderance of T cells in AA and M{phi} in DTH (Fig. 2A and 2B ). Therefore, we compared the impact of anti-CD44 treatment on the total number of T cells and M{phi} rather than on the relative percentage. Anti-CD44 treatment had only a minor effect on the overall number of CD4+ and CD8+ cells in draining LN of all four groups of mice (data not shown). However, the number of freshly activated CD69+ and CD154+ T cells was reduced significantly in diseased mice (Fig. 2C) . In the skin, the effect of anti-CD44 treatment varied depending on the disease. In AA mice, CD4+ and CD8+ SkIL and in allergen-treated mice, CD11b+ SkIL were more strongly reduced. In AA/DTH mice, the number of CD4+, CD8+, and CD11b+ SkIL was reduced to a comparable degree (Fig. 2D) . Considering T cell extravasation, anti-panCD44 and anti-CD44v10 particularly inhibited extravasation of freshly activated T cells (Fig. 2E) . The finding was confirmed in vitro. Migration of CD4+ cells was inhibited most strongly by anti-panCD44 in AA and AA/DTH. Monocyte migration was inhibited by anti-panCD44 and slightly more efficiently by anti-CD44v10 (Fig. 2F) .


Figure 2
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  		Figure 2. Impact of anti-CD44 treatment on leukocyte subset distribution. (A and B) The number of CD4+, CD8+, and CD11b+ LN cells and SkIL was evaluated in untreated and SADBE-treated control and AA mice. (C–E) Mice as described in A received anti-panCD44 or anti-CD44v10 as described above. (C) The number of CD69+ and CD154+ LN cells, (D) CD4+, CD8+, and CD11b+ SkIL, and (E) CD69+ and CD154+ SkIL (mean±SD of three experiments; pooled SkIL from three mice) is shown. (F) CD4+ and CD11b+ LN cells of control and diseased mice were incubated with the indicated antibodies. Migration in response to OPN was evaluated as described in Figure 1A . *, Significant differences (P<0.01) in comparison with mice receiving a control IgG.

 
Taken together, anti-panCD44 and anti-CD44v10 interfere with T cell and M{phi} extravasation into the skin, such that depending on the disease, the dominating effector cell subpopulation—monocytes in contact allergy and CD4+ cells in AA—becomes affected most strongly. Inhibition of T cell extravasation preferentially affects activated T cells. Homing into the draining LN is only blocked by anti-panCD44 and mostly, affects T cells. According to these disease- and CD44 isoform-specific effects, it became of interest to elucidate the mechanism(s), whereby a blockade of CD44/CD44v10 interferes with leukocyte migration. We asked four questions: Will the treatment influence CD44 expression? Are chemokines of major importance? Are CD44-associating, adhesion molecules involved? What is the contribution of endothelial cells?

Anti-CD44 does not influence CD44 expression
CD44 is supposed to support leukocyte rolling by binding to HA on endothelial cells, a process assisted further by up-regulated CD44 expression on vessel endothelium, which retains HA [43 44 45 ]. However, neither K926 (anti-CD44v10) nor IM7 (anti-panCD44) occupies the HA-binding site. Accordingly, these antibodies did not compete with binding of FITC-labeled HA to activated T cells (data not shown). Alternatively, anti-CD44 could interfere with leukocyte egress by inducing internalization or shedding of CD44.

CD44 expression was not modulated in LN cells and SkIL of anti-CD44-treated AA or DTH mice. Solely, in control mice, anti-CD44v10 treatment was accompanied consistently by a slight reduction in CD44v10 expression (Fig. 3A 3B 3C ). There was also no evidence for an antibody-induced, pronounced shedding of CD44. Sera were collected 3 days after the last antibody treatment and were tested for the presence of CD44 by a sandwich ELISA. The relative amount of soluble CD44 in the sera was not increased in anti-CD44-treated healthy or diseased mice. These findings argue against a loss of CD44 being responsible for the reduced migratory activity (Fig. 3D) .


Figure 3
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  		Figure 3. Impact of anti-CD44 treatment on CD44 expression. Untreated and SADBE-treated control and AA mice had been treated with anti-panCD44 or anti-CD44v10 as described above. (A) Examples of CD44 expression in LN cells and SkIL of control and diseased mice, depending on anti-CD44v10 treatment. Single fluorescence overlays of the negative control (black line) and IM7 staining (gray area) are shown. The mean intensity (MI) of expression is indicated. rIgG, Rat IgG. (B and C) The percentage of CD44+- and CD44v10+-draining LN cells and SkIL is shown (mean±SD of three experiments, pooled LN cells, and SkIL from three mice in the individual experiment). *, Significant differences (P<0.01) in comparison with mice receiving control IgG. (D) Serum was collected and tested at the indicated dilutions in a sandwich ELISA for the presence of shedded CD44 (mean±SD of sera from three mice/group).

 
The impact of anti-CD44 on chemokine and chemokine receptor expression
CD44 is a receptor for several chemokines and can form complexes with chemokines and chemokine receptors [30 , 31 , 36 , 37 , 47 , 48 ]. In addition, chemokine expression is strongly up-regulated in the dermis of contact allergen-treated and although less pronounced, AA mice [10 ]. Thus, the anti-CD44-mediated blockade of leukocyte migration could be supported by hindrance in chemokine binding or by modulation of chemokine or chemokine receptor expression.

We first evaluated chemokine receptor expression and coexpression with CD44 in LN cells and SkIL of untreated and allergen-treated, control and AA mice. A representative example is shown in Figure 4A . CCR6 expression was high in LN cells and SkIL of C3H/HeJ mice and was increased further in diseased mice. CCR4 and CCR8 expression was also up-regulated in LN cells and SkIL of diseased mice. In all four groups of mice, CCR4+, CCR6+, and CCR8+ LN cells and SkIL coexpressed mostly CD44. Coexpression was increased further in SkIL of diseased mice (Fig. 4A) . It is important that anti-panCD44 treatment was accompanied consistently by up-regulated CCR6, CCR4, and CCR8 expression, exclusively in AA/DTH LN cells (Fig. 4B) but not in SkIL (data not shown).


Figure 4
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  		Figure 4. Chemokine receptor coexpression with CD44. (A) Draining LN cells and SkIL of untreated and allergen-treated, control and AA mice were double-stained with anti-CCR6, anti-CCR4, anti-CCR8, and anti-panCD44 or anti-CD44v10. A representative example is shown. (B) CCR4, CCR8, and CCR6 expression was evaluated in LN cells of control and diseased mice, which had received anti-CD44 or control IgG as described above. *, Significant differences (P<0.01) in comparison with mice receiving a control IgG.

 
Changes could have been expected in chemokine expression to correlate with those in chemokine receptor expression. However, this was not obvious in all instances. Thus, anti-panCD44 had no impact on chemokine expression in SkIL and LN cells (data not shown). Also, anti-CD44v10 induced only a slight reduction of CCL20+ [CCR6 ligand, dendritic cell (DC), and T cell recruitment] in AA SkIL and CCL17 (CCR4 ligand and T cell recruitment) and CCL1 (CCR8 ligand and monocyte recruitment) in allergen-treated SkIL. Expression of OPN, CCL2, CCL5, and CXCL10 was unaltered (data not shown). However, CCL2 (MCP-1 and leukocyte recruitment in general) expression was strongly and CCL17 expression distinctly reduced in the dermis of anti-CD44-treated DTH and AA/DTH mice (Fig. 5 ). Expression of the CD44 ligand OPN was reduced mainly in anti-CD44v10-treated AA mice (data not shown).


Figure 5
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  		Figure 5. Recovery of chemokines in the dermis of anti-CD44-treated mice. Shock-frozen sections of the skin of control and diseased mice, which were treated with control IgG, anti-panCD44, or anti-CD44v10, were stained with anti-CCL2 and anti-CCL17. Original scale bar, 50 µm.

 
Thus, anti-panCD44 affected the recovery of chemokine receptor expression only in AA/DTH LN cells. The increased percentage of chemokine receptor-expressing LN cells but not SkIL argues for an active retention in the draining node. Chemokine expression was reduced more strongly in the dermis rather than in SkIL and not in LN cells.

Does anti-CD44 hamper adhesion via CD44-associated molecules?
Activated CD44 can cooperate with CD49d to promote firm adhesion in inflammatory diseases [29 , 47 ]. We demonstrated recently that this also accounts for AA [40 ]. Thus, we first asked whether coexpression of CD44 and CD49d would also be strengthened in allergen-treated mice. Coexpression of CD44 with CD11b, CD49d, and CD54 was high in unseparated LN cells of all four groups of mice, and coexpression of CD44 and CD49d was strengthened in LN cells of diseased mice (data not shown). The same accounted for coexpression of CD44 with CD49d or CD54 in SkIL of the three groups of diseased mice. Coexpression with CD11b was only strengthened in unseparated SkIL of allergen-treated mice (Fig. 6A and 6B ). Coimmunoprecipitation of SC lysates revealed that the increase in coexpression correlates with associations of these molecules. CD44 coimmunoprecipitates with CD54 in SC from healthy and diseased mice, but coimmunoprecipitation is increased significantly in diseased mice. An association among CD44 and CD11a, CD11b, and CD49d was only observed in SC of diseased mice and was most pronounced in AA/DTH. It is notable that CD44v10 only associated with CD11b and only in diseased mice. An additional, weak association of CD44v10 with CD11a was observed consistently in AA/DTH (Fig. 6C) .


Figure 6
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  		Figure 6. Impact of AA and/or a DTH reaction on coexpression and association of CD44s and CD44v10 with additional adhesion molecules. (A) SkIL of control and diseased mice were double-stained with anti-CD44s or anti-CD44v10 and antibodies for the indicated adhesion molecules. The percentage of adhesion molecule+/CD44+ in comparison with adhesion molecule+ cells (taken as 100%) is shown (mean of three experiments; pooled SkIL from three mice in the individual experiment). The ratio of adhesion molecule+/CD44–:adhesion molecule+/CD44+ cells was calculated. Significant differences in the ratio between control and diseased mice were calculated by the Wilcoxon rank sum test. *, Differences reaching the minimal possible P value of 0.1. (B) Representative examples of SkIL double-stained with anti-panCD44 or anti-CD44v10 and anti-CD11b or anti-CD49d. (C) SC (5x107/precipitate) of mice as described above were lysed and precipitated with the indicated antibodies. After SDS-PAGE, lysates were blotted with anti-panCD44 and anti-CD44v10. (D and E) Unseparated LN cells, CD4+ LN cells, and CD11b+ SC of mice, as described above, were preincubated with anti-CD11a, -CD11b, -CD49d, and -CD54 (10 µg/ml, 30 min, 4°C), washed, and seeded in the upper part of a Boyden chamber. The lower part contained RPMI/0.1% BSA and 40 ng/ml OPN. After 4 h (37°C, 5% CO2), cells in the lower chamber were counted. *, Significant differences (P<0.01) in comparison with cells incubated with control IgG.

 
As revealed by antibody-blocking studies, these associations of CD44 with integrins and CAMs have bearing on leukocyte migration. Anti-CD11a, -CD11b, -CD49d, and -CD54 inhibited LN cell migration only in diseased mice, and the degree of inhibition correlated well with the degree of association between the respective molecules and CD44. (Fig. 6D) . To see whether the blockade by the integrin-specific antibodies is restricted to leukocyte subpopulations, CD4+ and CD11b+ cells were separated. In fact, anti-CD49d preferentially inhibited CD4+ T cell migration, and anti-CD11b inhibited M{phi} migration exclusively. Both antibodies inhibited only migration of CD4+/CD11b+ cells from diseased mice, where CD44/CD44v10 coimmunoprecipitated with CD49d and/or CD11b (Fig. 6E) .

Taken together, "activated" CD44 associates with additional adhesion molecules, such as CD49d, CD11b, and CD54. Accordingly, an anti-CD44 blockade could well-inhibit adhesion and migration of activated T cells and M{phi} via these associated molecules.

A blockade of CD44 on vascular endothelium can contribute to impaired leukocyte migration
Inflammation is accompanied by up-regulated CD44 expression on endothelial cells, which serves to harbor HA and chemokines to attract and capture leukocytes [49 , 50 ]. Thus, the effect of an in vivo anti-CD44 treatment on leukocyte migration in AA and DTH also could have been supported by a blockade of CD44 on endothelial cells.

We first evaluated panCD44 and CD44v10 expression on dermal vascular endothelium and on high endothelial venules of LN. CD44v10 expression was seen on dermal vascular endothelium, and expression was strong in diseased mice. panCD44 expression apparently was up-regulated in high endothelial venules of draining LN of diseased mice (Fig. 7A ). However, CD44v10 was hardly expressed (data not shown). To further evaluate a possible impact of anti-CD44 treatment on leukocyte migration via a blockade of CD44 on endothelial cells, we made use of an endothelial cell line [44 ], which expresses CD44 and CD44v10 at a high level (Fig. 7B) . Transmigration of leukocytes seeded on an endothelial cell monolayer varied depending on the leukocytes’ activation state. Endothelial cell activation by TNF-{alpha} treatment increased the transmigration rate of LN cells from healthy and diseased mice to a comparable degree (Fig. 7C) . When endothelial cells were preincubated with anti-panCD44 or anti-CD44v10 before addition of LN cells, transmigration was inhibited, although to a lesser degree than by preincubation of LN cells with anti-CD44. Furthermore, anti-CD44-mediated inhibition of transmigration was not strengthened by endothelial cell activation. Finally, transmigration of LN cells from AA and AA/DTH mice was even less impaired by a blockade of endothelial cell CD44 than transmigration of LN cells from healthy mice (Fig. 7D) .


Figure 7
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  		Figure 7. The impact of a blockade of CD44 on endothelial cells and leukocyte migration. (A) Shock-frozen sections of skin and LN of untreated and allergen-treated control and AA mice were stained with anti-panCD44, -CD44v10, and -Meca. Original scale bar, 50 µm. (B) The endothelial cell line C166 was stained with anti-panCD44 and anti-CD44v10. Single fluorescence overlays with the negative control are shown. (C and D) The upper part of a Boyden chamber was coated with a monolayer of unstimulated or stimulated (10 ng/ml TNF-{alpha}) C166 cells. (C) After washing, LN cells of control and diseased mice were seeded on top of the monolayer. (D) Before seeding the LN cells on top of the endothelial cell monolayer, the endothelial cells or the LN cells were incubated with control IgG or anti-CD44 or -CD44v10. (C and D) The lower chamber contained RPMI/0.1% BSA and 40 ng/ml OPN. After 12 h (37°C, 5% CO2), cells in the lower chamber were counted.

 
As dermal vascular endothelial cells strongly express CD44v10, the selective inhibition of skin homing could rely partly on a CD44v10 blockade on endothelial cells. Conversely, leukocyte migration was less inhibited by an endothelial cell than by a leukocyte CD44 blockade and was weakest by a CD44 blockade on activated endothelial cells. Although these findings require further analyses of the CD44-mediated crosstalk between endothelial cells and leukocytes, with respect to the effect of anti-CD44 on leukocyte migration in AA and DTH, leukocytes are obviously the major antibody target.

DISCUSSION

CD44 plays a major role in leukocyte extravasation in autoimmune disease and allergic reactions [25 , 51 ]. It has not yet been explored whether distinct CD44 isoforms are involved, whether there are organ-related differences, and whether an antibody-mediated blockade of CD44 affects distinct leukocyte subpopulations. AA is an autoimmune disease [1 , 2 , 52 ], which can be treated efficiently by induction of a mild, chronic allergic eczema [4 , 5 ]. It is interesting that induction of AA in C3H/HeJ mice can be prevented by repeated injections of anti-CD44v10 [11 ], and anti-CD44v10 suffices to delay the onset of Th1-based DTH reactions [12 ]. As autoimmune diseases require a persisting recruitment of effector T cells [53 ], and DTH reactions are characterized by an early influx of inflammatory cells, followed by Th1 cells, which sustain M{phi} activation [54 ], a comparative evaluation of the impact of anti-CD44 on leukocyte migration in a fully developed autoimmune disease and a superimposed, persisting allergic DTH reaction appeared well-suited to answer the questions of a potential organ and effector cell (T cells vs. monocytes) specificity of the CD44 involvement in leukocyte extravasation.

What are the targets of anti-CD44?
Endothelial cells as well as leukocytes express CD44, and a blockade at both levels could contribute to impaired leukocyte migration. This, apparently, is the case, but the endothelial cells’ contribution is minor.

Endothelial cells contribute to leukocyte adhesion via up-regulated CD44 expression, HA secretion, and chemokine binding [16 , 27 , 28 , 49 ]. In fact, in vitro studies provided evidence for a reduction in transendothelial lymphocyte migration by a CD44 blockade on endothelial cells. However, the blockade of transendothelial cell migration did not vary in dependence on the endothelial cells’ activation state and was much weaker than inhibition of migration by a blockade of CD44 on leukocytes. Thus, a blockade of CD44 on endothelial cells can contribute to the inhibition of leukocyte extravasation, but the different efficacy of an anti-CD44 blockade in diseased as compared with healthy mice apparently relies predominantly on the leukocyte.

CD44-mediated leukocyte rolling on hyaluronan secreted by endothelial cells [51 , 55 ] depends on the activation state of CD44 [21 22 23 24 ] and should be enhanced in inflammation [26 ]. Our results are in line with this observation, taking into account that anti-CD44-mediated inhibition was strengthened significantly in diseased as compared with healthy mice and that anti-CD44-mediated inhibition of T cell extravasation preferentially affected activated (CD69+, CD154+) T cells. Also, a CD154-CD40 interaction early during induction of response induces strong up-regulation of CD44 on T cells [56 ]. These strongly CD44-positive cells may be prone for targeting by anti-CD44, which interferes with their extravasation.

CD44 is also involved in M{phi} migration [16 , 17 , 28 ]. As AA is a T cell-mediated, autoimmune disease [52 , 57 ], and M{phi} are the primary effectors in DTH reactions [54 ], it became of special interest whether in allergen-treated AA mice, a blockade of CD44 affects T cell as well as M{phi} migration. As suggested by the reduction of CD11b+ SkIL in anti-CD44-treated mice and demonstrated by the in vitro inhibition of monocyte migration, anti-CD44, indeed, inhibits monocyte recruitment toward the skin in healthy and diseased mice. It is notable that although T cell migration was inhibited more strongly by anti-panCD44 than by anti-CD44v10, M{phi} recruitment was inhibited most strikingly by anti-CD44v10.

The finding that anti-panCD44/anti-CD44v10 inhibits skin homing of T cells and monocytes explains the efficacy of anti-CD44 in a skin-associated, T cell-mediated autoimmune disease and a chronic DTH reaction.

The organ selectivity of the anti-CD44-mediated blockade in leukocyte migration
Anti-panCD44 and anti-CD44v10 interfere with leukocyte extravasation into the skin. Anti-panCD44 but not anti-CD44v10 also interfered with T cell homing into LN. This organ selectivity of a CD44 isoform-specific blockade of leukocyte migration is by no means restricted to the model described; e.g., neither anti-CD44v10 nor anti-panCD44 interferes with leukocyte homing into inflamed gut mucosa (M. Zöller, unpublished finding).

The selective interference of anti-CD44v10 with skin homing corresponds to the predominant CD44v10 expression in malignant and reactive human skin lymphocytes [12 , 58 , 59 ] and as shown here, in dermal endothelial cells. CD44v10 expression mainly in M{phi} (and a subpopulation of CD8+ T cells) [12 ] may well contribute to the strong effect of anti-CD44v10 on leukocyte skin homing in mice suffering with a chronic DTH reaction. Finally, in vitro and in vivo, anti-CD44v10 inhibited migration in response to OPN more strongly than anti-panCD44. A lower in vivo efficacy of anti-panCD44 may be a consequence of the rather ubiquitous expression of CD44s [60 ] and accordingly, an insufficient amount of antibody. However, in in vitro studies, cells were incubated with an excess of antibody. Taking into account that OPN binds particularly CD44v [30 , 31 ], we hypothesize that OPN-binding CD44 isoforms likely include the v10 exon product. A blockade of CD44v10 as a selective chemokine ligand could add further to the organ selectivity of CD44-mediated leukocyte extravasation. Finally, in activated leukocytes, CD44v10 can associate with CD11b. The association with an integrin expressed mostly on M{phi} could well-strengthen the efficacy of anti-CD44v10 in mitigating DTH reactions.

The organ selectivity of distinct CD44 isoforms in leukocyte extravasation may provide a way out of severe side-effects encountered frequently in therapeutic settings using anti-pan CD44 [61 ].

The molecular mechanisms accounting for the anti-CD44-mediated blockade in leukocyte extravasation
The question about the mechanism(s), whereby anti-CD44 blocks leukocyte extravasation, could not yet be answered fully. However, our data provided hints toward potential molecular pathways: The anti-panCD44 (IM7) [43 ], and the anti-CD44v10 (K926) [12 ] antibodies do not interfere with HA binding. This excludes the most straight-forward explanation that leukocyte binding to endothelial cell-bound HA may be hampered [47 , 51 ].

Second, anti-CD44 does not modulate CD44 expression. A reduction in CD44 expression by antibody-initiated shedding has been demonstrated for IM7 in rheumatoid arthritis (RA) [62 , 63 ]. However, CD44 surface expression was hardly reduced in antibody-treated mice, and there was no evidence for antibody-induced, pronounced shedding. The different effect of IM7 on CD44 shedding in RA and our models could be a result of the chronic nature of AA, the repeated allergen treatment, and our long-lasting antibody treatment, such that a new balance between increased shedding and synthesis could have become established, which might hide an immediate effect of IM7 on CD44 shedding.

Third, anti-CD44 apparently has an impact on chemokine and chemokine receptor expression, which are important parameters in leukocyte migration [64 ]. Two features, which were modulated by anti-CD44, should be mentioned: First, CCR4 (T cells) [65 ], CCR6 (DC and T cells) [66 ], and CCR8 (monocytes) [67 ] expression was unaltered in SkIL but was increased in LN cells of anti-CD44-treated AA/DTH mice. Thus, it becomes likely that in AA/DTH mice, anti-CD44 hampered leukocyte egress. The mechanism and the selective circumstances, which promote retention only in allergen-treated AA mice, remain to be explored. Second, chemokine expression is particularly high in the dermis of allergen-treated mice and anti-CD44; mostly, anti-CD44v10 affected chemokine expression in the dermis rather than in LN cells or SkIL. Taking into account the quite selective interference of anti-CD44v10 with CCL1, CCL17, and OPN expression, we hypothesize that as outlined above, CD44v10 might function as a direct ligand for some chemokines or might bind to a ligand, which triggers selected chemokine expression. These alternative possibilities are currently explored for the CD44v10-OPN interaction.

Taken together, although functional activity of CD44 as a chemokine receptor may well-contribute to a blockade of leukocyte migration by anti-CD44, the observed effects were minor and do not support a decisive/exclusive role of altered chemokine and their receptor expression.

Finally, CD44-integrin associations are obviously of major importance in the CD44-mediated blockade of leukocyte migration. Depending on its activation state, CD44 associates with CD49d, which supports firm adhesion [29 ], lymphocyte migration, activation, and apoptosis resistance, which is a result of molecules gaining access to linker and signal-transducing molecules associated with the partner. These features were elaborated with freshly harvested lymphocytes of AA mice [40 ] and accordingly, are of in vivo relevance. Thus, we asked whether depending on the underlying disease and the preferentially activated leukocyte subpopulation, CD44 may associate with additional integrins and whether these associations are of functional relevance.

In fact, CD44 does not only associate with CD49d but also associates with CD11a, CD11b, and CD54. The integrin associations are only observed in diseased mice; the CD54 association is strengthened in diseased mice. It is notable too that CD44v10 only associated with CD11b and weakly with CD11a in AA/DTH mice. The CD44-integrin/ICAM associations have functional consequences: Anti-CD11a, CD11b, CD49d, and CD54 were only inhibitory when the molecules coimmunoprecipitated with CD44. Also, anti-CD11b inhibited mostly monocyte, and anti-CD49d inhibited mostly activated T cell migration. Migrating leukocytes develop a polarized phenotype, where CD44 relocalizes in the uropod of the cell [68 69 70 ], and Ets-related molecule proteins associate with [71 , 72 ] and link CD44 to the actin cytoskeleton [73 , 74 ], a process that involves the small GTPase Rac [75 ]. These studies did not take into account the association of CD44 with integrins. According to our studies, which demonstrated that by their association, CD44 and CD49d gain access to the partners, signal-transduction molecules [40 ], we consider it most likely that anti-CD44 prevents acquisition of the migratory phenotype by interrupting the association with and signal transduction by selected integrins and/or ICAMs.

We demonstrate for the first time that the involvement of CD44 in leukocyte extravasation varies depending on the CD44 isoform, the leukocyte subpopulation, and the organ environment. Anti-CD44, particularly anti-CD44v10, strikingly inhibits monocytes and T cells homing into the skin. Anti-panCD44 also interferes with LN homing of T cells. Mechanistically, our data argue against a direct blockade of HA binding by IM7 and K926 but point toward a distorted crosstalk between CD44 and integrins/CAMs, which could well-prohibit the acquisition of a migratory phenotype. In view of our intention to elaborate whether a CD44 blockade could provide an additive therapeutic in allergen-treated, AA-affected individuals, we consider a blockade of CD49d-associated CD44 on effector T cells via a bispecific antibody as most promising, as such a therapy should not interfere severely with the recruitment of DTH effector cells and their therapeutic potential. The feasibility of a selective CD44 targeting via bispecific antibodies has been demonstrated already for a B cell leukemia [61 ].

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft, grant Zo 40/9-1 (M. Z.) and FR 1509/1-2 (P. F-P.). We thank Dr. Kevin McElwee for great help with editing the manuscript and Dr. Annette Kopp-Schneider for advice in the statistical analysis.

Received January 24, 2007; revised February 24, 2007; accepted March 12, 2007.

REFERENCES

  1. McElwee, K. J., Tobin, D. J., Bystryn, J. C., King, L. E., Jr, Sundberg, J. P. (1999) Alopecia areata: an autoimmune disease? Exp. Dermatol. 8,371-379[Medline]
  2. Ito, T., Ito, N., Bettermann, A., Tokura, Y., Takigawa, M., Paus, R. (2004) Collapse and restoration of MHC class-I-dependent immune privilege: exploiting the human hair follicle as a model Am. J. Pathol. 164,623-634[Abstract/Free Full Text]
  3. Happle, R., Echternacht, K. (1977) Induction of hair growth in alopecia areata with D.N.C.B Lancet 2,1002-1003[Medline]
  4. Freyschmidt-Paul, P., Happle, R., McElwee, K. J., Hoffmann, R. (2003) Alopecia areata: treatment of today and tomorrow J. Investig. Dermatol. Symp. Proc. 8,12-17[CrossRef][Medline]
  5. Dall’oglio, F., Nasca, M. R., Musumeci, M. L., La Torre, G., Ricciardi, G., Potenza, C., Micali, G. (2005) Topical immunomodulator therapy with squaric acid dibutylester (SADBE) is effective treatment for severe alopecia areata (AA): results of an open-label, paired-comparison, clinical trial J. Dermatolog. Treat. 16,10-14[CrossRef][Medline]
  6. McElwee, K. J., Boggess, D., King, L. E., Jr, Sundberg, J. P. (1998) Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts J. Invest. Dermatol. 111,797-803[CrossRef][Medline]
  7. McElwee, K. J., Yu, M., Park, S. W., Ross, E. K., Finner, A., Shapiro, J. (2005) What can we learn from animal models of alopecia areata? Dermatology 211,47-53[CrossRef][Medline]
  8. Freyschmidt-Paul, P., Sundberg, J. P., Happle, R., McElwee, K. J., Metz, S., Boggess, D., Hoffmann, R. (1999) Successful treatment of alopecia areata-like hair loss with the contact sensitizer squaric acid dibutylester (SADBE) in C3H/HeJ mice J. Invest. Dermatol. 113,61-68[CrossRef][Medline]
  9. Zöller, M., Freyschmidt-Paul, P., Vitacolonna, M., McElwee, K. J., Hummel, S., Hoffmann, R. (2004) Chronic delayed-type hypersensitivity reaction as a means to treat alopecia areata Clin. Exp. Immunol. 135,398-408[CrossRef][Medline]
  10. Gupta, P., Freyschmidt-Paul, P., Vitacolonna, M., Kiessling, S., Hummel, S., Hildebrand, D., Marhaba, R., Zöller, M. (2006) A chronic contact eczema impedes migration of antigen presenting cells in alopecia areata J. Invest. Dermatol. 126,1559-1573[CrossRef][Medline]
  11. Freyschmidt-Paul, P., Seiter, S., Zöller, M., König, A., Ziegler, A., Sundberg, J. P., Happle, R., Hoffmann, R. (2000) Treatment with an anti-CD44v10-specific antibody inhibits the onset of alopecia areata in C3H/HeJ mice J. Invest. Dermatol. 115,653-657[CrossRef][Medline]
  12. Rösel, M., Seiter, S., Zöller, M. (1997) CD44v10 expression in the mouse and functional activity in delayed type hypersensitivity J. Cell. Physiol. 171,305-317[CrossRef][Medline]
  13. Goldstein, L. A., Zhou, D. F., Picker, L. J., Minty, C. N., Bargatze, R. F., Ding, J. F., Butcher, E. C. (1989) A human lymphocyte homing receptor, the hermes antigen, is related to cartilage proteoglycan core and link proteins Cell 56,1063-1072[CrossRef][Medline]
  14. Katakai, T., Hara, T., Sugai, M., Gonda, H., Nambu, Y., Matsuda, E., Agata, Y., Shimizu, A. (2002) Chemokine-independent preference for T-helper-1 cells in transendothelial migration J. Biol. Chem. 277,50948-50958[Abstract/Free Full Text]
  15. Grabbe, S., Varga, G., Beissert, S., Steinert, M., Pendl, G., Seeliger, S., Bloch, W., Peters, T., Schwarz, T., Sunderkotter, C., Scharffetter-Kochanek, K. (2002) ß2 integrins are required for skin homing of primed T cells but not for priming naive T cells J. Clin. Invest. 109,183-192[CrossRef][Medline]
  16. Gee, K., Kozlowski, M., Kumar, A. (2003) Tumor necrosis factor-{alpha} induces functionally active hyaluronan-adhesive CD44 by activating sialidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells J. Biol. Chem. 278,37275-37287[Abstract/Free Full Text]
  17. Leemans, J. C., Florquin, S., Heikens, M., Pals, S. T., van der Neut, R., Van Der Poll, T. (2003) CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis J. Clin. Invest. 111,681-689[CrossRef][Medline]
  18. Camp, R. L., Scheynius, A., Johansson, C., Pure, E. (1993) CD44 is necessary for optimal contact allergic responses but is not required for normal leukocyte extravasation J. Exp. Med. 178,497-507[Abstract/Free Full Text]
  19. Stoop, R., Gal, I., Glant, T. T., McNeish, J. D., Mikecz, K. (2002) Trafficking of CD44-deficient murine lymphocytes under normal and inflammatory conditions Eur. J. Immunol. 32,2532-2542[CrossRef][Medline]
  20. Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B., Seed, B. (1990) CD44 is the principal cell surface receptor for hyaluronate Cell 61,1303-1313[CrossRef][Medline]
  21. Lesley, J., Hyman, R. (1992) CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells Eur. J. Immunol. 22,2719-2723[Medline]
  22. DeGrendele, H. C., Estess, P., Picker, L. J., Siegelman, M. H. (1996) CD44 and its ligand hyaluronate mediate rolling under physiologic flow, a novel lymphocyte-endothelial cell primary adhesion pathway J. Exp. Med. 183,1119-1130[Abstract/Free Full Text]
  23. DeGrendele, H. C., Kosfiszer, M., Estess, P., Siegelman, M. H. (1997) CD44 activation and associated primary adhesion is inducible via T cell receptor stimulation J. Immunol. 159,2549-2553[Abstract]
  24. DeGrendele, H. C., Estess, P., Siegelman, M. H. (1997) Requirement for CD44 in activated T cell extravasation into an inflammatory site Science 278,672-675[Abstract/Free Full Text]
  25. Dunon, D., Piali, L., Imhof, B. A. (1996) To stick or not to stick: the new leukocyte homing paradigm Curr. Opin. Cell Biol. 8,714-723[CrossRef][Medline]
  26. Estess, P., DeGrendele, H. C., Pascual, V., Siegelman, M. H. (1998) Functional activation of lymphocyte CD44 in peripheral blood is a marker of autoimmune disease activity J. Clin. Invest. 102,1173-1182[Medline]
  27. Mohamadzadeh, M., DeGrendele, H., Arizpe, H., Estess, P., Siegelman, M. H. (1998) Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion J. Clin. Invest. 101,97-108[Medline]
  28. Gee, K., Lim, W., Ma, W., Nandan, D., Diaz-Mitoma, F., Kozlowski, M., Kumar, A. (2002) Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-{alpha} in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression J. Immunol. 169,5660-5672[Abstract/Free Full Text]
  29. Nandi, A., Estess, P., Siegelman, M. H. (2004) Bimolecular complex between rolling and firm adhesion receptors required for cell arrest; CD44 association with VLA-4 in T cell extravasation Immunity 20,455-465[CrossRef][Medline]
  30. Weber, G. F., Ashkar, S., Glimcher, M. J., Cantor, H. (1996) Receptor-ligand interaction between CD44 and osteopontin (Eta-1) Science 271,509-512[Abstract]
  31. Katagiri, Y. U., Sleeman, J., Fujii, H., Herrlich, P., Hotta, K., Tanaka, K., Chikuma, S., Yagita, H., Okumura, K., Murakami, M., Saiki, I., Chambers, A. F., Uede, T. (1999) CD44 variants but not CD44s cooperate with ß1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis Cancer Res. 59,219-226[Abstract/Free Full Text]
  32. Weber, G. F., Zawaideh, S., Hikita, S., Kumar, V. A., Cantor, H., Ashkar, S. (2002) Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation J. Leukoc. Biol. 72,752-761[Abstract/Free Full Text]
  33. Weiss, J. M., Renkl, A. C., Maier, C. S., Kimmig, M., Liaw, L., Ahrens, T., Kon, S., Maeda, M., Hotta, H., Uede, T., Simon, J. C. (2001) Osteopontin is involved in the initiation of cutaneous contact hypersensitivity by inducing Langerhans and dendritic cell migration to lymph nodes J. Exp. Med. 194,1219-1229[Abstract/Free Full Text]
  34. Suzuki, K., Zhu, B., Rittling, S. R., Denhardt, D. T., Goldberg, H. A., McCulloch, C. A., Sodek, J. (2002) Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts J. Bone Miner. Res. 17,1486-1497[CrossRef][Medline]
  35. Zohar, R., Suzuki, N., Suzuki, K., Arora, P., Glogauer, M., McCulloch, C. A., Sodek, J. (2000) Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration J. Cell. Physiol. 184,118-130[CrossRef][Medline]
  36. Roscic-Mrkic, B., Fischer, M., Leemann, C., Manrique, A., Gordon, C. J., Moore, J. P., Proudfoot, A. E., Trkola, A. (2003) RANTES (CCL5) uses the proteoglycan CD44 as an auxiliary receptor to mediate cellular activation signals and HIV-1 enhancement Blood 102,1169-1177[Abstract/Free Full Text]
  37. Charnaux, N., Brule, S., Chaigneau, T., Saffar, L., Sutton, A., Hamon, M., Prost, C., Lievre, N., Gattegno, L. (2005) RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44 Glycobiology 15,119-130[Abstract/Free Full Text]
  38. Marhaba, R., Bourouba, M., Zöller, M. (2005) CD44v6 promotes proliferation by persisting activation of MAP kinases Cell. Signal. 17,961-973[CrossRef][Medline]
  39. Föger, N., Marhaba, R., Zöller, M. (2000) CD44 supports T cell proliferation and apoptosis by apposition of protein kinases Eur. J. Immunol. 30,2888-2899[CrossRef][Medline]
  40. Marhaba, R., Freyschmidt-Paul, P., Zöller, M. (2006) In vivo CD44-CD49d complex formation in autoimmune disease has consequences on T cell activation and apoptosis resistance Eur. J. Immunol. 36,3017-3032[CrossRef][Medline]
  41. Wang, S. J., Greer, P., Auerbach, R. (1996) Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-function fps/fes proto-oncogene In Vitro Cell. Dev. Biol. Anim. 32,292-299[Medline]
  42. Picker, L. J., De los Toyos, J., Telen, M. J., Haynes, B. F., Butcher, E. C. (1989) Monoclonal antibodies against the CD44 [In(Lu)-related p80], and Pgp-1 antigens in man recognize the Hermes class of lymphocyte homing receptors J. Immunol. 142,2046-2051[Abstract]
  43. Zheng, Z., Katoh, S., He, Q., Oritani, K., Miyake, K., Lesley, J., Hyman, R., Hamik, A., Parkhouse, R. M., Farr, A. G. (1995) Monoclonal antibodies to CD44 and their influence on hyaluronan recognition J. Cell Biol. 130,485-495[Abstract/Free Full Text]
  44. Hession, C., Moy, P., Tizard, R., Chisholm, P., Williams, C., Wysk, M., Burkly, L., Miyake, K., Kincade, P., Lobb, R. (1992) Cloning of murine and rat vascular cell adhesion molecule-1 Biochem. Biophys. Res. Commun. 183,163-169[CrossRef][Medline]
  45. Gonda, A., Gal, I., Szanto, S., Sarraj, B., Glant, T. T., Hunyadi, J., Mikecz, K. (2005) CD44, but not l-selectin, is critically involved in leucocyte migration into the skin in a murine model of allergic dermatitis Exp. Dermatol. 14,700-708[CrossRef][Medline]
  46. Seiter, S., Schadendorf, D., Tilgen, W., Zöller, M. (1998) CD44 variant isoform expression in a variety of skin-associated autoimmune diseases Clin. Immunol. Immunopathol. 89,79-93[CrossRef][Medline]
  47. Steeber, D. A., Venturi, G. M., Tedder, T. F. (2005) A new twist to the leukocyte adhesion cascade: intimate cooperation is key Trends Immunol. 26,9-12[CrossRef][Medline]
  48. Freeman, C. M., Chiu, B. C., Stolberg, V. R., Hu, J., Zeibecoglou, K., Lukacs, S. A., Lira, N. W., Kunkel, S. L., Chensue, S. W. (2005) CCR8 is expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate Th2-mediated granuloma formation in mice J. Immunol. 174,1962-1970[Abstract/Free Full Text]
  49. Shimonaka, M., Katagiri, K., Nakayama, T., Fujita, N., Tsuruo, T., Yoshie, O., Kinashi, T. (2003) Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow J. Cell Biol. 161,417-427[Abstract/Free Full Text]
  50. Rouschop, K. M., Roelofs, J. J., Claessen, N., da Costa Martins, P., Zwaginga, J. J., Pals, S. T., Weening, J. J., Florquin, S. (2005) Protection against renal ischemia reperfusion injury by CD44 disruption J. Am. Soc. Nephrol. 16,2034-2043[Abstract/Free Full Text]
  51. Toole, B. P. (2004) Hyaluronan: from extracellular glue to pericellular cue Nat. Rev. Cancer 4,528-539[CrossRef][Medline]
  52. Gilhar, A., Kalish, R. R. (2006) Alopecia areata: a tissue specific autoimmune disease of the hair follicle Autoimmun. Rev. 5,64-69[CrossRef][Medline]
  53. Tesch, G. H., Maifert, S., Schwarting, A., Rollins, B. J., Kelley, V. R. (1999) Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice J. Exp. Med. 190,1813-1824[Abstract/Free Full Text]
  54. Kapp, A., Werfel, T. (1999) Allergic inflammation: skin Allergy 54(Suppl. 56),23-24
  55. Nandi, A., Estess, P., Siegelman, M. H. (2000) Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44 J. Biol. Chem. 275,14939-14948[Abstract/Free Full Text]
  56. Guo, Y., Wu, Y., Shinde, S., Sy, M. S., Aruffo, A., Liu, Y. (1996) Identification of a costimulatory molecule rapidly induced by CD40L as CD44H J. Exp. Med. 184,955-961[Abstract/Free Full Text]
  57. McElwee, K. J., Freyschmidt-Paul, P., Hoffmann, R., Kissling, S., Hummel, S., Vitacolonna, M., Zöller, M. (2005) Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(–) cells promote systemic alopecia areata and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model J. Invest. Dermatol. 124,947-957[CrossRef][Medline]
  58. Wagner, S. N., Wagner, C., Reinhold, U., Funk, R., Zöller, M., Goos, M. (1998) Predominant expression of CD44 splice variant v10 in malignant and reactive human skin lymphocytes J. Invest. Dermatol. 111,464-471[CrossRef][Medline]
  59. Yoshinari, C., Mizusawa, N., Byers, H. R., Akasaka, T. (1999) CD44 variant isoform CD44v10 expression of human melanoma cell lines is upregulated by hyaluronate and correlates with migration Melanoma Res. 9,223-231[Medline]
  60. Lesley, J., Hyman, R., Kincade, P. W. (1993) CD44 and its interaction with extracellular matrix Adv. Immunol. 54,271-335[Medline]
  61. Avin, E., Haimovich, J., Hollander, N. (2004) Anti-idiotype x anti-CD44 bispecific antibodies inhibit invasion of lymphoid organs by B cell lymphoma J. Immunol. 173,4736-4743[Abstract/Free Full Text]
  62. Shi, M., Dennis, K., Peschon, J. J., Chandrasekaran, R., Mikecz, K. (2001) Antibody-induced shedding of CD44 from adherent cells is linked to the assembly of the cytoskeleton J. Immunol. 167,123-131[Abstract/Free Full Text]
  63. Mikecz, K., Dennis, K., Shi, M., Kim, J. H. (1999) Modulation of hyaluronan receptor (CD44) function in vivo in a murine model of rheumatoid arthritis Arthritis Rheum. 42,659-668[CrossRef][Medline]
  64. Stein, J. V., Nombela-Arrieta, C. (2005) Chemokine control of lymphocyte trafficking: a general overview Immunology 116,1-12[Medline]
  65. Wenzel, J., Henze, S., Worenkamper, E., Basner-Tschakarjan, E., Sokolowska-Wojdylo, M., Steitz, J., Bieber, T., Tuting, T. (2005) Role of the chemokine receptor CCR4 and its ligand thymus- and activation-regulated chemokine/CCL17 for lymphocyte recruitment in cutaneous lupus erythematosus J. Invest. Dermatol. 124,1241-1248[CrossRef][Medline]
  66. Dieu-Nosjean, M. C., Massacrier, C., Homey, B., Vanbervliet, B., Pin, J. J., Vicari, A., Lebecque, S., Dezutter-Dambuyant, C., Schmitt, D., Zlotnik, A., Caux, C. (2000) Macrophage inflammatory protein 3{alpha} is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors J. Exp. Med. 192,705-718[Abstract/Free Full Text]
  67. Campbell, J. J., Hedrick, J., Zlotnik, A., Siani, M. A., Thompson, D. A., Butcher, E. C. (1998) Chemokines and the arrest of lymphocytes rolling under flow conditions Science 279,381-384[Abstract/Free Full Text]
  68. Millan, J., Montoya, M. C., Sancho, D., Sanchez-Madrid, F., Alonso, M. D. (2002) Lipid rafts mediate biosynthetic transport to the T lymphocyte uropod subdomain and are necessary for uropod integrity and function Blood 99,978-984[Abstract/Free Full Text]
  69. Shimonaka, M., Katagiri, K., Nakayama, T., Fujita, N., Tsuruo, T., Yoshie, O., Kinashi, T. (2003) Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow J. Cell Biol. 161,417-427[Abstract/Free Full Text]
  70. Lee, J. H., Katakai, T., Hara, T., Gonda, H., Sugai, M., Shimizu, A. (2004) Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation J. Cell Biol. 167,327-337[Abstract/Free Full Text]
  71. Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A., Tsukita, S. (1994) ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons J. Cell Biol. 126,391-401[Abstract/Free Full Text]
  72. Legg, J. W., Lewis, C. A., Parsons, M., Ng, T., Isacke, C. M. (2002) A novel PKC-regulated mechanism controls CD44 ezrin association and directional cell motility Nat. Cell Biol. 4,399-407[CrossRef][Medline]
  73. Del Pozo, M. A., Vicente-Manzanares, M., Tejedor, R., Serrador, J. M., Sanches-Madrid, F. (1999) Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes Eur. J. Immunol. 29,3609-3620[CrossRef][Medline]
  74. Seveau, S., Eddy, R. J., Maxfield, F. R., Pierini, L. M. (2001) Cytoskeleton-dependent membrane domain segregation during neutrophil polarization Mol. Biol. Cell 12,3550-3562[Abstract/Free Full Text]
  75. Föger, N., Marhaba, R., Zöller, M. (2001) Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells J. Cell Sci. 114,1169-1178[Abstract]