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Characterization of the CC Receptor 3 on Human Keratinocytes

Holger Petering, Christoph Kluthe, Yasmin Dulkys, Peter Kiehl, Paul D. Ponath,* Alexander Kapp, and JoÈrn Elsner Hannover Medical University, Department of Dermatology and Allergology, Hannover, Germany; *LeukoSite Inc., Cambridge, Massachusetts, U.S.A.

CC chemokine receptors are expressed on hemato- cytes than for eosinophils. In situ hybridization tech- poetic cells, and these may impart selective homing niques exhibited that basal cell layers of the of , leukocyte, and lymphocyte subsets to epidermis were stained homogeneously for CC sites of in¯ammation. CC 3 is chemokine receptor 3 mRNA with a decreasing the major receptor on eosinophils and is also signal to the upper epidermis showing that differenti- expressed on other in¯ammatory cells suggesting its ating and proliferating keratinocytes did express important role for allergic diseases such as atopic mRNA speci®c for CC chemokine receptor 3. dermatitis and bronchial asthma. Eotaxin, eotaxin-2 Immunohistochemical studies con®rmed low expres- and eotaxin-3 have been identi®ed as ligands that sion of CC chemokine receptor 3 protein on epider- only activate CC chemokine receptor 3. CC chemo- mal keratinocytes compared to the high level kine receptor 3 is also activated by other promiscu- observed on in®ltrating eosinophils. Furthermore, ous ligands, however, such as RANTES and stimulation of cultured keratinocytes with eotaxin monocyte chemotactic protein 4. To date, CC che- resulted in an increased [3H]thymidine incorporation mokine receptor 3 has not been reported to be indicating a role of CC chemokine receptor 3 in epi- expressed on nonhematopoetic cells. In this study, dermal proliferation and differentiation. These data we investigated whether keratinocytes possess auto- demonstrate that CC chemokine receptor 3 is crine and paracrine mechanisms for CC chemokine expressed not only on hematopoetic cells but also on secretion and receptor expression as reported for the keratinocytes as nonhematopoetic cells with ecto- expression of and its receptors. Reverse dermal origin. Therefore, the identi®cation of CC transcriptase polymerase chain reaction analysis chemokine receptor 3 on epidermal keratinocytes demonstrated that CC chemokine receptor 3 mRNA may indicate a role for CC chemokine receptor 3 and is expressed constitutively in cultured keratinocytes. its ligands in skin physiology and pathophysiology. The signal quantities of the CC chemokine receptor Key words: chemokine receptor/in¯ammation/keratinocytes. 3 amplicons showed lower intensities for keratino- J Invest Dermatol 116:549±555, 2001

hemokine receptors represent Gi-protein-coupled The CC eotaxin, eotaxin-2, and eotaxin-3 have seven transmembrane receptors that are divided into been identi®ed as ligands that exclusively activate the CCR3 and four subgroups (C, CC, CXC, CX3C) according to seem to be monospeci®c (Elsner et al, 1996a; 1998; Ponath et al, their ligands (Baggiolini, 1998; Luster, 1998). Each 1996b; Forssmann et al, 1997; White et al, 1997). Other Cmember is a receptor for a unique set of one or more chemokines recognize more than one chemokine receptor. CC chemokine agonists. Various CC chemokine receptors are CCR3 is also activated by other promiscuous ligands, however, expressed on hematopoetic cells, and these may impart selective such as RANTES, monocyte chemotactic protein 2 (MCP-2), homing of , leukocytes, and lymphocytes to sites of MCP-3, and MCP-4 (Alam et al, 1993; Dahinden et al, 1994; Kapp in¯ammation. CCR3 is the major receptor on eosinophils (Kitaura et al, 1994; Petering et al, 1998). et al, 1996; Ponath et al, 1996a) and is also expressed on basophils Recent studies have shown that eotaxin, eotaxin-2, and eotaxin- (Heath et al, 1997; Uguccioni et al, 1997) suggesting an important 3 not only are able to induce but also stimulate the role for allergic diseases such as atopic dermatitis and bronchial release of reactive oxygen species and induce changes in relative F- asthma. Furthermore, CCR3 expression was observed on macro- actin contents of human eosinophils as an indicator for locomotory- phages (Hariharan et al, 1999) and neural microglia cells (Albright et associated cellular responses (Elsner et al, 1996a, 1998). Therefore, al, 1999). eotaxin, eotaxin-2, and eotaxin-3 represent speci®c chemoattrac- tants for certain hematopoetic cells. Furthermore, CXC chemo- kines, e.g., interleukin-8 (IL-8), do not induce functional activity Manuscript received July 7, 1999; revised December 22, 2000; accepted even on activated human eosinophils indicating that CXCR1 and for publication December 29, 2000 CXCR2 are not expressed on the cell surface (Petering et al, 1999). Reprint requests to: Prof. JoÈrn Elsner M.D., Hannover Medical University, Department of Dermatology and Allergology, Ricklinger Str. These ®ndings may suggest an important role for CCR3 and its 5, D-30449 Hannover, Germany. Email: [email protected] ligands in chronic in¯ammatory disorders.

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 549 550 PETERING ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

The ®nding that CCR3 is the principal receptor for eosinophil (sense, 5¢-GAGCGGGAAATCGTGCGTGACATT-3¢; antisense, 5¢- responses to CC chemokines has led to the development of GAAGGTAGTTTCGTGGATGCC-3¢) were designed according to the monoclonal antibody (MoAb) 7B11 blocking CCR3 ligand published sequences (Accession No.: CCR3, AF026535; b-actin, binding (Daugherty et al, 1996a; Heath et al, 1997; Elsner et al, AB004047). PCR samples were run on a 1.8% agarose gel stained with 0.2 mg per ml ethidium bromide, and the PCR products were visualized 1998; Kitayama et al, 1998). In addition, CC chemokine receptor with ultraviolet light and photographed. directed antagonists such as Met-RANTES could be identi®ed. Met-RANTES is principally described as a CCR1 antagonist, but Automated sequencing of PCR products For automated sequencing has also weak inhibitory properties on CCR3 (Proudfoot et al, of PCR products, an Applied Biosystems Model 373 A (Forster City, 1996; Wells et al, 1996; Elsner et al, 1997). CA) was used. Nucleotide sequence data were analyzed with Lasergene To date, CCR3 has not been reported to be expressed on sequence analysis software (DNASTAR, Madison, WI). nonhematopoetic cells, whereas CXC chemokine receptors exhibit CCR3 riboprobes and in situ hybridization RNA probes for CCR3 a broad distribution not only on hematopoetic cells but also on were designed from a plasmid constructed by cloning a 578 bp RT- PCR fragment ampli®ed from human eosinophils into a polylinker site endothelial cells, ®broblasts, and melanocytes (Holmes et al, 1991; ± Murphy and Tiffany, 1991). Primary cultured human keratinocytes of transcription vector pBluescript II SK containing T3 and T7 polymerase recognition elements as described previously (Petering et al, are known to express mRNA speci®c for IL-8 as well as for the CC 1996). After blunt-end ligation, the orientation and DNA sequence of chemokine eotaxin (Hein et al, 1997). Human keratinocytes are both insert strains were determined by cycle sequencing as described known to synthesize and secret CXC chemokines, e.g., IL-8, and above. Antisense (T3) and sense (T7) riboprobes were transcribed can be stimulated by their own products. To investigate enzymatically and digoxygenin-labeled according to the manufacturer's whether human keratinocytes possess similar autocrine and instructions (Boehringer Mannheim, Germany). Probes were washed paracrine mechanisms for CC chemokine secretion and CC twice by ethanol precipitation to remove unincorporated nucleotides and chemokine receptor expression, reverse transcriptase polymerase stored in 10 nM dithiothreitol at ±80°C. chain reaction (RT-PCR) analysis, in situ hybridization, and For in situ hybridization, tissue sections were ®xed with 4% receptor protein expression analysis were performed with cultured paraformaldehyde and washed with 1% phosphate-buffered saline (PBS). Tissues were permeabilized with 50 mg per ml protease K (37°C, human keratinocytes and skin biopsies. Tissue sections were 30 min), washed once with 1% PBS, and incubated for 5 min with 2 3 obtained from healthy individuals, patients with chronic in¯am- standard saline citrate (SSC). The sections were dehydrated through matory diseases, and patients suffering from bullous pemphigoid. graded alcohol and air-dried. After prehybridization (55°C, 60 min) and This subepidermal blistering disease is characterized by a TH2-type hybridization (55°C, 12 h) with standard hybridization solution (10% cytokine pattern as well as peripheral and tissue eosinophilia. dextran sulfate, 40% formamide, 4 3 SSC, 1 3 Denhard's, 10 mM Therefore, in®ltrating eosinophils could be stained for CCR3 as a dithiothreitol, 100 mg per ml tRNA) slides were washed with SSC under positive control. In this study, we report that human keratinocytes stringent conditions. Staining for CCR3 was carried out with contain mRNA speci®c for the eotaxin receptor CCR3 and antidigoxygenin F(ab)¢ fragment conjugated to alkaline phosphatase at a express CCR3 protein. Functional data revealed that CCR3 may ®nal dilution of 1:1000 according to the manufacturer's instructions (Boehringer Mannheim). Slides stained with sense and antisense ribop- play a role in epidermal proliferation and differentiation. robes were read blindly by two dermatopathologists. Immunohistochemistry with human keratinocytes and MATERIALS AND METHODS eosinophils Tissue sections were cut to 5 mm and adhered to silanized slides. Immunostaining was performed (room temperature, 60 min) and Cell preparation was developed by the avidin±biotin-peroxidase complex technique with Human keratinocyte culture Normal human keratinocytes were obtained the chromogen fuchsin according to the manufacturer's from fresh trunk biopsies. Epidermal cells were disaggregated by recommendations (DAKO). Staining for CCR3 protein was performed trypsinization (4°C, 12 h) in Hank's balanced salt solution without Ca2+ with the anti-CCR3 MoAb 7b11 (750 mg per ml) at a ®nal dilution of and Mg2+ containing 0.25% trypsin. The cells were plated out at 107 1:100 in 1% bovine serum albumin/PBS. Staining for eosinophil matrix cells per 50 ml culture ¯ask in endotoxin-free keratinocyte growth granular proteins was performed with MoAb EG1 speci®c for ECP 2+ (Kiehl and Kapp, 1998). Non-reactive mouse antihuman antibodies of medium (KGM) containing 1.0 mM Ca and incubated with 10% CO2 (37°C). Normal keratinocytes were derived from the second to fourth IgG1 isotype were used as negative controls. passages grown as a monolayer to 80% con¯uence. Immuno¯uorescence of cultured keratinocytes Immuno- Isolation of human eosinophils Human eosinophils were isolated from ¯uorescence of cultured human trunk keratinocytes between the second heparin-anticoagulated venous blood from normal nonatopic healthy and fourth passages was performed using standard techniques. In brief, keratinocytes were adjusted to a density of 1 3 107 cells per ml. Aliquots donors. The isolation was performed using Ficoll (Pharmacia) density 5 gradient centrifugation and eosinophils were puri®ed by negative (20 ml) containing 2 3 10 cells were incubated at 4°C for 30 min with selection with anti-CD16 MoAb (clone 3G8, Immunotech, Hamburg, the indicated antibody or isotype control (IgG2). Thereafter, cells were Germany) coated Dynabeads M-450 (Dynal, Hamburg) as described washed twice with 4°C PBS. For indirect immuno¯uorescence, cells previously (Elsner et al, 1994). The resulting eosinophil purity were stained in a second step by a ¯uorescein isothiocyanate (FITC) was > 99.5% as determined by ¯ow cytometric analysis (FACScan, conjugated mouse antihuman antibody (Immunotech) and subsequently Becton Dickinson, Heidelberg, Germany) using phycoerythrin- washed twice. Thereafter, cells were analyzed by ¯ow cytometry conjugated anti-CD16 MoAb (clone 3G8, Immunotech). (FACScan). The samples were excited at 488 nm and emission was measured at 530 nm (FITC-labeled antibodies, ¯uorescence 1, green Chemokines, , and monoclonal antibodies Eotaxin was purchased ¯uorescence). The CCR3 receptor expression was analyzed after from Peprotech (London, U.K.). Bradykinin, epidermal growth factor preincubation of cultured keratinocytes for 30 min at 37°C with the (EGF), and C5a were obtained from Sigma (Deisenhofen, Germany). CCR3 ligand eotaxin or medium alone. The mean channel ¯uorescence The anti-CCR3 MoAb 7B11 was derived from C57BL6 mice of seven experiments was determined. Results were expressed as the immunized with CCR3 transfectants and prepared as described median ¯uorescence intensity after preincubation for 30 min versus the previously (Daugherty et al, 1996; Heath et al, 1997). MoAb EG1 against median ¯uorescence intensity after incubation with medium. Incubation eosinophilic cationic protein (ECP) was received from Pharmacia of cells for longer than 30 min did not change the results. (Uppsala). The human IgG1 isotype control was obtained from Sigma. Keratinocyte proliferation assay Keratinocytes were plated at CCR3 mRNA expression Total RNA was isolated from human 5 3 103 cells per well of a 96-well microtiter plate and cultured in keratinocytes and eosinophils using the TRIzol (Gibco Life KGM for 5 d. The medium in each well was then replaced with KGM Technologies, Karlsruhe, Germany) according to the manufacturer's supplemented with either EGF, IL-8, or eotaxin at the indicated instructions based on the guanidine isothiocyanate method. First-strand concentrations or KGM alone. Proliferation was measured by labeling six cDNA synthesis and PCR reaction were performed as described replicate cultures with 0.5 mCi ml [3H]thymidine (Amersham, previously (Elsner et al, 1996b; Petering et al, 1996, 1999). Primers for Braunschweig, Germany) during a further 48 h of incubation. After the ampli®cation of CCR3 (sense, 5¢-GCCATTTCGGACCTGCT- labeling, medium was removed, each well was washed with PBS, and CTT-3¢; antisense, 5¢-TCCGCTCACAGTCATTTCCA-3¢) and b-actin trichloroacetic acid (TCA) was added. The TCA-precipitated nucleic VOL. 116, NO. 4 APRIL 2001 CCR3 EXPRESSION ON HUMAN KERATINOCYTES 551

(Fig 2A). Immunohistochemical staining with MoAb EG1 recognizing matrix granular proteins identi®ed these cells as eosinophil granulocytes known to express CCR3 (Fig 2C). Like eosinophils, basal cell layers of the epidermis were stained homogeneously for CCR3 mRNA with a decreasing signal to the upper epidermis showing that differentiating and proliferating keratinocytes did express mRNA speci®c for CCR3 (Fig 2A). Negative controls hybridized with sense RNA probes only showed background signals (data not shown). To investigate whether the expression of CCR3 mRNA in keratinocytes is induced in the state of disease, specimens from healthy volunteers were analyzed by in situ hybridization. As seen in Figure 1. CCR3 mRNA is expressed in cultured human keratinocytes. mRNA from cultured human keratinocytes and puri®ed Fig 2(E), basal keratinocytes from normal individuals also showed eosinophils was isolated and ®rst-strand cDNA synthesis was performed. staining for CCR3 mRNA. A similar staining pattern could be PCR was carried out with primer pairs speci®c for CCR3 (expected size observed in tissue sections from chronic in¯ammatory diseases. 578 bp) and b-actin (expected size 221 bp) as an internal standard. The Figure 2(G) shows the expression of CCR3 mRNA in atopic amplicons were separated by electrophoresis in 1.8% agarose gel and dermatitis, a T-cell-mediated disease affecting the upper dermis and stained by ethidium bromide. Lane 1, 50 bp DNA size marker; lane 2, epidermis. Figure 2(H) demonstrates the expression of CCR3 human eosinophil mRNA; lane 3, human keratinocyte mRNA. One mRNA in psoriasis vulgaris, a chronic in¯ammatory disease representative experiment out of four is shown. characterized by epidermal hyperplasia and altered epidermal differentiation. These data reveal that CCR3 mRNA is expressed constitutively in human keratinocytes. The expression may not depend on the state of activation of basal keratinocytes, e.g., in acid was solubilized in 100 ml of 0.1 M sodium hydroxide, 1% sodium disease affecting the basement membrane zone. dodecyl sulfate, and 2% sodium carbonate for 30 min and the b decay was counted in a liquid scintillation counter. The stimulation index was de®ned by the ratio of mean counts per minute of stimulated to Immunohistochemical studies demonstrate that CCR3 is unstimulated cultures. expressed on epidermal keratinocytes To demonstrate that the transcription of CCR3 mRNA is followed by the expression of the receptor protein on the cell surface, bullous pemphigoid tissue RESULTS sections adjacent to those used for in situ hybridization were stained Cultured human keratinocytes express mRNA speci®c for immunohistochemically for CCR3. The colocalization of CCR3 RT-PCR analysis was performed to investigate whether keratinocytes and eosinophils allowed us again to compare the human keratinocytes express mRNA speci®c for CCR3. For this signal quantities. Expression of CCR3 protein was observed on purpose, human trunk keratinocytes were cultured and passaged epidermal keratinocytes with a marginally increased staining of the two to four times prior to total RNA isolation. Using the primer basal cell layers (Fig 2B). Immunohistochemical staining of pairs indicated, DNA fragments with an expected size of 578 bp specimens taken from healthy volunteers and patients suffering speci®c for human CCR3 could be detected in cultured human from atopic dermatitis and psoriasis vulgaris exhibited a similar keratinocytes (Fig 1, lane 3). To clarify whether the ampli®ed PCR staining pattern (data not shown). These results indicate that products were speci®c for CCR3 and did not represent other proliferating and differentiating keratinocytes of the epidermis are chemokine receptors, DNA sequence analysis was performed. PCR able to express CCR3 protein. products were sequenced automatically with vector-derived primer In comparison to human eosinophils in®ltrating the upper pairs. Computer-aided nucleotide sequence analysis revealed that dermis and epidermis, however, keratinocytes showed only a weak all PCR products represent DNA sequences speci®c for CCR3 CCR3 expression in terms of staining intensity indicating fewer mRNA. As expected, highly puri®ed human eosinophils did binding sites per cell (Fig 2B, D). Positive CCR3 staining of constitutively express CCR3 mRNA (Fig 1, lane 2). To eosinophils without immunohistochemical reaction on other demonstrate that all mRNA preparations were highly puri®ed, in®ltrating in¯ammatory cells, such as lymphocyte subpopulations, RT-PCR was performed simultaneously using primer pairs for b- demonstrated that anti-CCR3 MoAb 7B11 is also a speci®c marker actin as standard (221 bp) (Fig 1). The signal quantities of the for CCR3 in immunohistochemical studies (Fig 2D). CCR3 amplicons showed lower signal intensities for cultured keratinocytes than for isolated human eosinophils. The results Flow cytometry analysis reveals that CCR3 is expressed indicate that CCR3 is expressed at mRNA level in cultured human heterogeneously on cultured keratinocytes To con®rm the keratinocytes. immunohistochemical data, the expression of CCR3 on cultured In situ hybridization studies reveal that CCR3 mRNA is human trunk keratinocytes was investigated by ¯ow cytometry. expressed in epidermal keratinocytes To investigate whether Cells between the second and fourth passage were stained with the CCR3 mRNA is not only expressed in vitro in cultured anti-CCR3 MoAb 7B11 (Fig 3A). keratinocytes but can also be detected in vivo in epidermal Surprisingly, the expression of CCR3 on cultured keratinocytes keratinocytes, in situ hybridization studies were performed. Tissue was donor dependent and differed between the healthy volunteers. sections from ®ve patients suffering from bullous pemphigoid prior Cultured keratinocytes from two donors were negative or showed to immunosuppressive therapy were studied for the expression of only a weak staining for CCR3 in a small subpopulation of CCR3 mRNA. Bullous pemphigoid represents a chronic keratinocytes (Fig 3B). Preincubation of cultured keratinocytes for in¯ammatory autoimmune disease characterized by a local 30 min with TH2-type cytokines (IL-4, IL-5) did not alter CCR3 accumulation of eosinophils with subsequent subepidermal blister expression (data not shown). To demonstrate whether the anti- formation (Lever, 1979). Therefore, the colocalization of CCR3 MoAb 7B11 binds speci®cally to CCR3, cultured human keratinocytes and eosinophils in®ltrating the upper dermis and keratinocytes were incubated for 30 min with eotaxin at a epidermis allowed us to compare CCR3 signal speci®city and concentration of 125.0 nM or medium. As expected, preincuba- intensity between different cells. Specimens were obtained from tion of cultured keratinocytes with eotaxin induced downregula- perilesional and lesional skin and CCR3 mRNA expression was tion of CCR3 on the cell surface, indicating that the CCR3 ligand analyzed by in situ hybridization. Strong CCR3 mRNA expression eotaxin was able to bind speci®cally to its receptor expressed on was detected in in¯ammatory cells in®ltrating the upper dermis keratinocytes (Fig 3A). 552 PETERING ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 2. In situ hybridization and immunohistochemical analysis for CCR3 mRNA and protein expression on human keratinocytes. (A) In tissue sections from patients with bullous pemphigoid CCR3 mRNA expression was analyzed by in situ hybridization. Basal cell layers of the epidermis were homogeneously stained for CCR3 mRNA with a decreasing intensity to the upper epidermis. Strong CCR3 mRNA expression was detected in in¯ammatory cells in®ltrating the upper dermis. (B) Immunohistochemistry with anti-CCR3 MoAb 7B11 revealed that CCR3 protein is expressed on epidermal keratinocytes. (C) Immunohistochemical staining with MoAb EG1 recognizing ECP identi®ed in¯ammatory cells as human eosinophils. (D) Human eosinophils in®ltrating the upper dermis showed more intense immunohistochemical staining for CCR3 than epidermal keratinocytes indicating more binding sites per cell. (E) In situ hybridization revealed that basal keratinocytes from normal individuals also showed staining for CCR3 mRNA. A similar staining pattern could be observed in chronic in¯ammatory diseases: (F) expression of CCR3 mRNA in bullous pemphigoid; (G) expression of CCR3 mRNA in atopic dermatitis; (H) expression of CCR3 mRNA in psoriasis vulgaris. Scale bars:(A) 100 mm; (B±D) 50 mm.

Proliferation of cultured human keratinocytes after induced dose-dependently a proliferation of human keratinocytes. stimulation with eotaxin can be inhibited by anti- In this experiment, eotaxin was as effective as IL-8 with a CCR3 Previous studies have shown that eotaxin is expressed in maximum of cell proliferation at a concentration of 100 nM tissue specimens obtained from chronic in¯ammatory lesions (Fig 4). In addition, proliferation of cultured human keratinocytes (Rothenberg et al, 1995). These ®ndings prompted us to was also induced by other CCR3 ligands such as RANTES and investigate whether eotaxin may have any functional effects on MCP-4 representing two other potent activators of CCR3. At epidermal keratinocytes, e.g., cell proliferation and differentiation. equivalent concentrations eotaxin seemed to be more effective than Cultured human epidermal keratinocytes were stimulated with RANTES or MCP-4, however (Fig 4). EGF used as a positive eotaxin (10±1000 nM) and the incorporation of [3H]thymidine was control was more potent than eotaxin at concentrations above detected as a marker for cell proliferation. As seen in Fig 4, eotaxin 1 mM. Moreover, whereas EGF induced a dose-related curve all VOL. 116, NO. 4 APRIL 2001 CCR3 EXPRESSION ON HUMAN KERATINOCYTES 553

Figure 4. Proliferation of cultured human keratinocytes after stimulation with eotaxin can be inhibited by anti-CCR3 MoAb 7B11. Cultured human keratinocytes were stimulated with CC chemokines eotaxin, MCP-4, RANTES, and IL-8 as well as EGF in different concentrations (10±1000 nM). The incorporation of [3H]thymidine was detected as a marker for cell proliferation. Data are expressed as proliferation index (stimuli-induced [3H]thymidine incorporation/medium-induced [3H]thymidine incorporation). In one experiment keratinocytes were preincubated with the anti-CCR3 MoAb 7B11 and subsequently stimulated with eotaxin (10±7 M). One representative experiment out of four is shown indicating that eotaxin induces proliferation of human keratinocytes.

that the proliferation of keratinocytes after stimulation with eotaxin can be inhibited by antagonizing CCR3-mediated effects.

DISCUSSION Chemokines are proin¯ammatory cytokines known to be involved in chronic in¯ammatory diseases such as atopic dermatitis and allergic asthma (Baggiolini, 1998; Luster, 1998). Recent studies have shown that the CC chemokine eotaxin is elevated in the bronchial mucosa of asthmatic patients and is expressed by epithelial and endothelial cells (Ying et al, 1997). CCR3 was identi®ed Figure 3. Flow cytometry analysis reveals that CCR3 is expressed independently by two different groups as the eotaxin receptor on heterogeneously on cultured keratinocytes. Overlay histogram for human eosinophils (Daugherty et al, 1996; Ponath et al, 1996a) and the expression of CCR3 on cultured human trunk keratinocytes. Cells further studies showed the expression of CCR3 also on basophils between the second and fourth passage were stained for CCR3 with the suggesting that this chemokine receptor may play a central role in anti-CCR3 MoAb 7B11. (A) The bold line represents cultured chronic in¯ammatory disorders (Kitaura et al, 1996; Ponath et al, keratinocytes expressing CCR3. The dotted line indicates cultured 1996b; Uguccioni et al, 1997). So far, CCR3 has only been keratinocytes preincubated with 125.0 nM eotaxin for 30 min showing a downregulation of the CCR3. The thin line represents the IgG2a isotype identi®ed on hematopoetic cells whereas CXC chemokine control. These data demonstrate that eotaxin was able to bind speci®cally receptors are also expressed on nonhematopoetic cells such as to CCR3 expressed on human keratinocytes. (B) The expression of ®broblasts, melanocytes, and keratinocytes (Holmes et al, 1991; CCR3 was donor dependent and differed between healthy volunteers. In Murphy et al, 1991; Norgauer et al, 1996). In psoriatic skin, two donors cultured keratinocytes appeared negative or showed only a characterized by epidermal hyperplasia and altered epidermal weak staining for CCR3 in a small subpopulation of keratinocytes as differentiation, IL-8 receptors were found to be expressed at high demonstrated by the bold line. levels (Schulz et al, 1993; Kulke et al, 1998). In vitro studies con®rmed that keratinocyte migration and growth can be stimu- lated by its ligand IL-8 (Tuschil et al, 1992). As endothelial and epithelial cells of the lung and also epidermal keratinocytes are chemokines tested induced a bell-shaped curve with respect to known to produce CXC chemokines such as IL-8 (Sica et al, 1990; incorporation of [3H]thymidine in epidermal keratinocytes. Gillitzer et al, 1991; Huber et al, 1991), autocrine and paracrine To investigate whether the eotaxin-induced proliferation of mechanisms seem to be responsible for proliferation and differen- keratinocytes was mediated via CCR3, cultured keratinocytes were tiation of nonhematopoetic cells. preincubated with anti-CCR3 MoAb 7B11. As demonstrated Recently, increased levels of CC chemokine eotaxin have been previously, this antibody is able to block eotaxin binding to CCR3 found in respiratory epithelium and endothelium as well as in the completely and inhibits CCR3-speci®c cell activation as demon- intestinal mucosa (Garcia-Zepeda et al, 1996; Matthews et al, 1998). strated previously (Daugherty et al, 1996a; Heath et al, 1997; Elsner Hein et al detected eotaxin mRNA at low levels in stimulated et al, 1998). As seen in Fig 4 preincubation of keratinocytes with primary keratinocytes (Hein et al, 1997). Therefore, it is tempting the anti-CCR3 MoAb 7B11 inhibited the proliferation of to speculate that human keratinocytes also possess autocrine and keratinocytes induced by eotaxin. This experiment demonstrates paracrine mechanisms for CC chemokine secretion and CC 554 PETERING ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY chemokine receptor expression similar to their CXC counterpart. CCR3, which has recently been shown in eosinophils (Elsner et al, This study describes the functional analysis and expression of the 2000). Preincubation of keratinocytes with TH2-type cytokines predominant eosinophil CC chemokine receptor CCR3 on human did not alter CCR3 receptor expression. keratinocytes in vitro in comparison to isolated eosinophils and Recent studies have shown that the CXC chemokine IL-8 exerts in vivo to eosinophils in®ltrating the upper dermis and epidermis. a direct in¯uence on several keratinocyte functions including In the ®rst set of experiments, RT-PCR analysis was performed epidermal proliferation and differentiation (Kapp, 1993). demonstrating that cultured human keratinocytes constitutively Furthermore, in psoriatic skin, IL-8 and the growth-related express CCR3 mRNA. Signal quantities of CCR3 amplicons oncogen a, another ligand for CXCR2, are produced by lesional showed lower signal intensities for keratinocytes than for isolated keratinocytes especially from the upper epidermal cell layers (Larsen eosinophils. To rule out that the RT-PCR products were due to a et al, 1989) as part of an autocrine and paracrine cytokine network. false-positive ampli®cation of other chemokine receptors, e.g., The CC chemokine eotaxin can also be synthesized and released by CXCR1 or CXCR2, nucleotide sequence analysis of the keratinocytes and therefore not only may contribute to the local amplicons was performed and revealed a DNA sequence homolog accumulation of eosinophils and TH2-type lymphocytes but also to published CCR3 mRNA sequences. To con®rm that CCR3 may modulate the differentiation and proliferation of keratinocytes mRNA expression was also observed in vivo, skin biopsies were similar to IL-8 (Hein et al, 1997). To investigate whether such analyzed for CCR3 mRNA by in situ hybridization techniques. autocrine and paracrine mechanisms exist for CC chemokines, Again, CCR3 mRNA could be detected and was predominantly growth-promoting activities of eotaxin on cultured keratinocytes expressed in basal keratinocytes whereas in the upper cell layers the were analyzed. Our data suggest that eotaxin represents a weak receptor expression was decreased. To investigate whether the stimulus for proliferation of human keratinocytes. The observation expression of CCR3 mRNA is induced in the state of disease, skin that keratinocyte proliferation and differentiation was also induced biopsies from healthy volunteers and patients suffering from by RANTES and MCP-4, two other potent activators of CCR3, different in¯ammatory diseases (atopic dermatitis, psoriasis vulgaris, suggests that the effect of eotaxin may be due to CCR3 binding and bullous pemphigoid) were analyzed. Our data revealed that and signaling. Interestingly, all of the CCR3 ligands tested induced CCR3 mRNA is constitutively expressed on epidermal keratino- a bell-shaped curve with regard to keratinocyte proliferation. As we cytes and may not depend on the state of cell activation. The could clearly demonstrate that eotaxin was able to induce a ®nding that CCR3 mRNA is constitutively expressed on downregulation of CCR3 in CCR3+ keratinocytes it could be keratinocytes obtained from different in¯ammatory diseases affect- speculated that CCR3 ligands downregulate CCR3 at a concen- ing the epidermis (psoriasis vulgaris), basal membrane (bullous tration of 10±7 M. Thus, the reduced proliferation index of pemphigoid), and epidermis as well as the upper dermis (atopic keratinocytes in response to a higher concentration (10±6 M) of dermatitis) suggests that the low level of CCR3 mRNA expression CCR3 ligands could be explained by a downregulation of CCR3. may not have a pathophysiologic role in these disorders. Our results To further prove this hypothesis, cultured keratinocytes were indicate that in particular proliferating and differentiating kerati- preincubated with anti-CCR3 MoAb 7B11 known to speci®cally nocytes of the basal stratum express CCR3 mRNA. This study block the activation of human eosinophils by CCR3 ligands (Heath provides evidence that CCR3 mRNA is not only expressed on et al, 1997; Elsner et al, 1998; Kitayama et al, 1998). The anti- in¯ammatory cells derived from hematopoetic precursors but can CCR3 MoAb 7B11 was able to inhibit eotaxin-induced prolifer- also be expressed on human keratinocytes as nonhematopoetic cells ation of keratinocytes indicating that binding to CCR3 is the initial with ectodermal origin. step of the observed keratinocyte differentiation and proliferation. The expression of CCR3 protein on keratinocytes was inves- In summary, these data provide evidence that CCR3 is expressed tigated by immunohistochemical staining for CCR3 with tissue not only on hematopoetic cells but also on human keratinocytes as sections adjacent to those used for in situ hybridization. The nonhematopoetic cells with ectodermal origin. The level of experiments indicated that CCR3 protein is expressed in human receptor expression is donor dependent. In addition to its epidermis. Immunohistochemistry was performed with tissue involvement in chemotaxis and activation of the respiratory burst sections from patients with bullous pemphigoid, a blistering of human eosinophils eotaxin and other CCR3 ligands may autoimmune disease characterized by an excessive synthesis of contribute to local wound repair and healing mechanisms inte- proin¯ammatory cytokines. TH2-type cytokines such as IL-4 and grating different ways of tissue reconstitution. Therefore, the IL-5 but also CC chemokines, e.g., RANTES, have been identi®cation of CCR3 on epidermal keratinocytes suggests that identi®ed in the skin and the blister ¯uid of these patients and CCR3 and its ligands, especially eotaxin, may play an important contribute to the epidermal and dermal in®ltration of eosinophils role in skin physiology and pathophysiology. (D'Auria et al, 1998; Inaoki and Takehara, 1998). IL-4 and IL-5 may upregulate the expression of CCR3 on in¯ammatory cells and may also increase CCR3 protein synthesis in epidermal keratino- This work was supported by a grant from the Deutsche Forschungsgemeinschaft (EL cytes (Tiffany et al, 1998; Jinquan et al, 1999). Therefore, it was 160/6±1) and by the Hochschulinterne LeistungsfoÈrderung of the Medical obvious to speculate that cytokine priming of keratinocytes in vivo University Hannover. may contribute to the expression of CCR3 protein. 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