Identification and Characterization of the Low-Affinity Receptor for Immunoglobulin E (FcεRII/CD23) on Murine Langerhans Cells
2002; Elsevier BV; Volume: 119; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.2002.01768.x
ISSN1523-1747
AutoresYuzuru Nagaoka, Koichiro Nakamura, Takahiro Watanabe, Akihiko Asahina, Kunihiko Tamaki, Nami Yasaka,
Tópico(s)Cell Adhesion Molecules Research
ResumoCD23 is a low-affinity receptor for immunoglobulin E and expressed on various hemopoietic cells. Although human epidermal cultured Langerhans cells express CD23, the study to identify CD23 on murine Langerhans cells has so far failed. In this study, using highly enriched (> 95%) Langerhans cells from murine epidermis obtained by the panning method, we investigated whether murine Langerhans cells express CD23. As the result of a series of experiments using fluorescence activated cell sorter analysis and the polymerase chain reaction method, it was revealed that CD23 is expressed on cultured Langerhans cells, but not on freshly isolated Langerhans cells. Comparison of the DNA sequence of polymerase chain reaction products of CD23 from cultured Langerhans cells with that from spleen leukocytes demonstrated that there were the same sequences between the two polymerase chain reaction products. The expression of CD23 on cultured Langerhans cells was downregulated when Langerhans cells were cultured with keratinocyte-derived cytokines: interleukin-1α, interleukin-18, macrophage colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Moreover, it was shown that murine IgE bound to cultured Langerhans cells and this binding was partially inhibited when Langerhans cells were cultured with monoclonal antibody against CD23 (B3B4). Thus this study revealed murine cultured Langerhans cells do express CD23 and the discrepancy from previous reports may be due to the influence of cytokines derived from keratinocytes. Furthermore, the finding that murine cultured Langerhans cells bind IgE through CD23 suggests that CD23 on murine Langerhans cells may be involved in IgE-mediated immune responses. CD23 is a low-affinity receptor for immunoglobulin E and expressed on various hemopoietic cells. Although human epidermal cultured Langerhans cells express CD23, the study to identify CD23 on murine Langerhans cells has so far failed. In this study, using highly enriched (> 95%) Langerhans cells from murine epidermis obtained by the panning method, we investigated whether murine Langerhans cells express CD23. As the result of a series of experiments using fluorescence activated cell sorter analysis and the polymerase chain reaction method, it was revealed that CD23 is expressed on cultured Langerhans cells, but not on freshly isolated Langerhans cells. Comparison of the DNA sequence of polymerase chain reaction products of CD23 from cultured Langerhans cells with that from spleen leukocytes demonstrated that there were the same sequences between the two polymerase chain reaction products. The expression of CD23 on cultured Langerhans cells was downregulated when Langerhans cells were cultured with keratinocyte-derived cytokines: interleukin-1α, interleukin-18, macrophage colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Moreover, it was shown that murine IgE bound to cultured Langerhans cells and this binding was partially inhibited when Langerhans cells were cultured with monoclonal antibody against CD23 (B3B4). Thus this study revealed murine cultured Langerhans cells do express CD23 and the discrepancy from previous reports may be due to the influence of cytokines derived from keratinocytes. Furthermore, the finding that murine cultured Langerhans cells bind IgE through CD23 suggests that CD23 on murine Langerhans cells may be involved in IgE-mediated immune responses. mean fluorescence intensity CD23/FcεRII, a low-affinity receptor for IgE, is expressed at increased levels in atopic individuals (Buckley et al., 1992Buckley C.C. Ivison C. Poulter L.W. Rustin M.H.A. FcεRII/CD23 receptor distribution in patch test reactions to aeroallergens in atopic dermatitis.J Invest Dermatol. 1992; 99: 184-188Crossref PubMed Scopus (30) Google Scholar) and has been associated with disorders characterized by chronic inflammation (Bonnefoy et al., 1996Bonnefoy J.Y. Plater-Zyberk C. Lecoanet-Henchoz S. Gauchat J.F. Aubry J.P. Graber P. A new role for CD23 in inflammation.Immunol Today. 1996; 17: 418-420Abstract Full Text PDF PubMed Scopus (56) Google Scholar). The expression of CD23 has been shown in multiple cell types such as lymphocytes, eosinophils, platelets, monocytes, and keratinocytes (Bonnefoy et al., 1996Bonnefoy J.Y. Plater-Zyberk C. Lecoanet-Henchoz S. Gauchat J.F. Aubry J.P. Graber P. A new role for CD23 in inflammation.Immunol Today. 1996; 17: 418-420Abstract Full Text PDF PubMed Scopus (56) Google Scholar;Becherel et al., 1997Becherel P.A. LeGoff L. Frances C. et al.Induction of IL-10 synthesis by human keratinocytes through CD23 ligation. A cyclic adenosine 3′,5′-monophosphate-dependent mechanism.J Immunol. 1997; 159: 5761-5765PubMed Google Scholar). Several functional roles of CD23 have been described, including T cell-dependent IgE production, augmentation of B cell proliferation, enhancement of antigen presentation, and mediation of T and B cell cognate interactions (Flores-Romo et al., 1993Flores-Romo L. Shields J. Humbert Y. et al.Inhibition of an in vivo antigen-specific IgE response by antibodies to CD23.Science. 1993; 261: 1038-1041Crossref PubMed Scopus (107) Google Scholar;Swendeman and Thorley-Lawson, 1987Swendeman S. Thorley-Lawson D.A. The activation antigen BLAST-2, when shed, is an autocrine BCGF for normal and transformed B cells.EMBO J. 1987; 6: 1637-1642PubMed Google Scholar;Kehry and Yamashita, 1989Kehry M.R. Yamashita L.C. Low-affinity IgE receptor (CD23) function on mouse B cells: role in IgE-dependent antigen focusing.Proc Natl Acad Sci USA. 1989; 86: 7556-7560Crossref PubMed Scopus (215) Google Scholar;Morris et al., 1994Morris S.C. Lees A. Holmes J.M. Jeffries R.D. Finkelman F.D. Induction of B cell and T cell tolerance in vivo by anti-CD23 MoAb.J Immunol. 1994; 152: 3768-3776PubMed Google Scholar). Furthermore, recent findings suggest a novel interaction between CD23 and the α chain of the β2 integrins, CD11b and CD11c, which leads to proinflammatory patterns of monocyte activation (Lecoanet-Henchoz et al., 1995Lecoanet-Henchoz S. Gauchat J.F. Aubry J.P. et al.CD23 regulates monocyte activation through a novel interaction with the adhesion molecules CD11b-CD18 and CD-11c and CD-18.Immunity. 1995; 3: 119-125Abstract Full Text PDF PubMed Scopus (133) Google Scholar,Lecoanet-Henchoz et al., 1997Lecoanet-Henchoz S. Plater-Zyberk C. Graber P. Gretener D. Aubry J.P. Conrad D.H. Bonnefoy J.Y. Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.Eur J Immunol. 1997; 27: 2290-2294Crossref PubMed Scopus (32) Google Scholar). Epidermis, the most superficial sheet of the skin, constitutes a heterogeneous cell population such as keratinocytes, Langerhans cells, melanocytes, and Merkel cells. Keratinocytes that constitute the major cell population of the epidermis are not only a physical barrier, but also are able to synthesize, under various stimuli, many different cytokines such as interleukin (IL)-1α, IL-6, IL-7, IL-10, IL-18, tumor necrosis factor (TNF)-α, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Matsue et al., 1992Matsue H. Cruz Jr P.D. Bergstresser P.R. Takashima A. Cytokine expression by EC subpopulations.J Invest Dermatol. 1992; 99: 42S-45SCrossref PubMed Scopus (77) Google Scholar), as well as to express major histocompatibility complex class II antigens upon interferon (IFN)-γ stimulation. Langerhans cells are bone-marrow derived major histocompatibility complex class II positive antigen-presenting cells (Katz et al., 1979Katz S.I. Tamaki K. Sachs D.H. Epidermal Langerhans cells are derived from cells originating in bone marrow.Nature. 1979; 282: 324-326Crossref PubMed Scopus (723) Google Scholar;Tamaki et al., 1979Tamaki K. Stingl G. Gullino M. Sachs D.H. Katz S.I. Ia antigens in mouse skin are predominantly expressed on Langerhans cells.J Immunol. 1979; 123: 784-787PubMed Google Scholar) in the epidermis and constitute only 1–3% of epidermal cells. They belong to a dendritic cell lineage, and are crucial for primary and secondary T cell dependent immune responses. In human epidermis, CD23 has been shown to be expressed on both cultured keratinocytes (Becherel et al., 1997Becherel P.A. LeGoff L. Frances C. et al.Induction of IL-10 synthesis by human keratinocytes through CD23 ligation. A cyclic adenosine 3′,5′-monophosphate-dependent mechanism.J Immunol. 1997; 159: 5761-5765PubMed Google Scholar) and cultured Langerhans cells (Bieber et al., 1989Bieber T. Rieger A. Neuchrist C. et al.Induction of FcεR2/CD23 on human epidermal Langerhans cells by human recombinant interleukin 4 and γ interferon.J Exp Med. 1989; 170: 309-314Crossref PubMed Scopus (166) Google Scholar); however, it is reported that neither murine cultured keratinocytes nor cultured Langerhans cells express CD23 (Hertl et al., 1996Hertl M. Asada H. Katz S.I. Murine epidermal Langerhans cells do not express the low-affinity receptor for immunoglobulin E, FcεRII (CD23).J Invest Dermatol. 1996; 106: 221-224Crossref PubMed Scopus (5) Google Scholar). Furthermore, although human freshly isolated Langerhans cells and cultured Langerhans cells express high-affinity receptor for IgE (FcεRI) (Bieber et al., 1992Bieber T. de la Salle H. Wollenberg A. et al.Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (FcεRI).J Exp Med. 1992; 175: 1285-1290Crossref PubMed Scopus (414) Google Scholar), it is reported that murine cultured Langerhans cells do not express FcεRI (Hayashi et al., 1999Hayashi S. Matsuda H. Okumura K. Ra C. Mouse Langerhans cells do not express the high-affinity receptor for IgE.Arch Dermatol Res. 1999; 291: 241-243Crossref PubMed Scopus (10) Google Scholar). With regard to Langerhans cells, we hypothesized that the failure to detect CD23 on murine Langerhans cells may be due to the effect of keratinocyte-derived cytokines because murine Langerhans cells were examined in relatively low purity with contaminating keratinocytes. As murine Langerhans cells of over 95% purity can be studied in each experiment (Salgado et al., 1999aSalgado C.G. Nakamura K. Sugaya M. et al.Differential effects of cytokines and immunosuppressive drugs on CD40, B7–1, and B7–2 expression on purified epidermal Langerhans cells.J Invest Dermatol. 1999; 113: 1021-1027Crossref PubMed Scopus (59) Google Scholar,Salgado et al., 1999bSalgado C.G. Nakamura K. Sugaya M. et al.Functional CD40 ligand is expressed on epidermal Langerhans cells.J Leukoc Biol. 1999; 66: 281-285PubMed Google Scholar), the CD23 expression on murine Langerhans cells was re-evaluated. Female BALB/c mice were purchased from Japan SLC Co. (Hamamatsu, Japan) and maintained in the Animal Center for Biomedical Research (Faculty of Medicine, University of Tokyo). BALB/c mice were used for experiments at the age of 8–12 wk old. The culture medium used was RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U penicillin per ml and 100 mg streptomycin per ml. Murine recombinant IL (rIL)-4, rIL-10, recombinant IFN-γ, and recombinant TNF-α were purchased from R&D Systems (Minneapolis, MN). Murine rIL-1α, rIL-12, recombinant GM-CSF, and rIL-18 were obtained from Pepro Tec Ec (London, U.K). Murine recombinant M-CSF was purchased from Update (New York, NY). Dispase was purchased from Godo Shusei (Tokyo, Japan) and diluted at a concentration of 20,000 U per ml in phosphate-buffered saline (PBS) and stocked at -20°C. Deoxyribonuclease I (DNase) was purchased from Sigma (St Louis, MO) and stored at -20°C. Red blood cell (RBC) lysing buffer was purchased from Serotec (London, U.K). Purified mouse monoclonal IgE anti-2,4,6-trinitrophenyl) was purchased from Pharmingen (San Diego, CA). The following monoclonal antibodies (MoAb) directed against murine antigens and isotype controls were used for immunostaining. Fluorescein isothiocyanate-conjugated (FITC-) anti-mouse CD23 (rat IgG2aκ, clone B3B4), FITC-anti-mouse CD11a, FITC-anti-mouse CD11b, FITC-anti-mouse CD11c, FITC-anti-mouse CD14, FITC-anti-mouse IgE (rat IgG1κ), phycoerythrin-conjugated (PE-) anti-mouse I-Ad (rat IgG2bκ), PE-anti-mouse CD23, purified anti-mouse CD23 (rat IgG2aκ, clone B3B4), purified anti-mouse FcγRII and FcγRIII (rat IgG2bκ, clone 2.4G2), FITC-mouse IgG2aκ, FITC-rat IgG1κ, FITC-rat IgG2aκ, PE-rat IgG2aκ and PE-rat IgG2bκ, were purchased from Pharmingen. FITC-goat anti-mouse IgG was obtained from Cappel (Durham, NC). FITC-anti-hamster IgG (Jackson Immunoresearch Laboratories, Baltimore, MA) and FITC-hamster IgG (Cedarlane, Ontario, Canada) were also used. To detect early or late apoptotic cells, FITC-Annexin V (R&D systems) was used. For enrichment of Langerhans cells, mouse anti-mouse I-Ad and goat anti-mouse IgG (Fc) were obtained from Meiji (Tokyo, Japan) and Cappel, respectively. The exact concentration of each antibody used in this study was 5 µg per ml. Epidermal cells suspensions were prepared by the method established previously with slight modification (Koyama et al., 1990Koyama Y. Kobayashi M. Ohashi K. Isolation and culture of panning method-enriched Langerhans cells from dispase-dissociated epidermal cells of the mouse.J Dermatol. 1990; 17: 211-217Crossref PubMed Scopus (21) Google Scholar;Salgado et al., 1999aSalgado C.G. Nakamura K. Sugaya M. et al.Differential effects of cytokines and immunosuppressive drugs on CD40, B7–1, and B7–2 expression on purified epidermal Langerhans cells.J Invest Dermatol. 1999; 113: 1021-1027Crossref PubMed Scopus (59) Google Scholar,Salgado et al., 1999bSalgado C.G. Nakamura K. Sugaya M. et al.Functional CD40 ligand is expressed on epidermal Langerhans cells.J Leukoc Biol. 1999; 66: 281-285PubMed Google Scholar). Briefly, mice were killed by cervical dislocation and shaved with a hair clipper. Then trunk skin was peeled and subcutaneous tissue was removed. These skin specimens were incubated with 3000 U per ml dispase in culture medium for 4 h at 37°C. Thereafter epidermal sheets were peeled gently with fine forceps from the underlying dermis and transferred to 0.025% DNase in RPMI 1640 containing 5% FBS. After incubation for 20 min at room temperature, epidermal sheets were vigorously stirred and epidermal cells suspensions were passed through the nylon filter to remove hair, stratum corneum, and undissociated fragments. Epidermal cells were harvested, washed twice in PBS containing 1% FBS by centrifugation and resuspended in culture medium. The number of epidermal cells was 1 × 107 per mouse and the viability of cells was always > 95% by trypan blue exclusion. Langerhans cell-enriched epidermal cells suspensions were obtained using a previously established method (Koyama et al., 1990Koyama Y. Kobayashi M. Ohashi K. Isolation and culture of panning method-enriched Langerhans cells from dispase-dissociated epidermal cells of the mouse.J Dermatol. 1990; 17: 211-217Crossref PubMed Scopus (21) Google Scholar;Salgado et al., 1999aSalgado C.G. Nakamura K. Sugaya M. et al.Differential effects of cytokines and immunosuppressive drugs on CD40, B7–1, and B7–2 expression on purified epidermal Langerhans cells.J Invest Dermatol. 1999; 113: 1021-1027Crossref PubMed Scopus (59) Google Scholar,Salgado et al., 1999bSalgado C.G. Nakamura K. Sugaya M. et al.Functional CD40 ligand is expressed on epidermal Langerhans cells.J Leukoc Biol. 1999; 66: 281-285PubMed Google Scholar). Briefly epidermal cells suspensions were treated with mouse anti-mouse I-Ad MoAb diluted 1:600 in RPMI containing 1% FBS for 45 min at 4°C. Cells were washed twice with RPMI containing 1% FBS by centrifugation and seeded on to panning dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ) for 45 min. The panning dishes had already been coated at room temperature for more than 4 h with goat anti-mouse IgG (Fc) diluted 1:100 in PBS, and then had been treated with RPMI containing 1% FBS for 60 min to avoid nonspecific adhesion of cells. Then in order to remove nonadhesion cells such as keratinocytes and melanocytes, the dishes were washed with PBS containing 1% FBS. Finally, adherent Langerhans cells were removed by vigorous pipetting with RPMI containing 1% FBS and enriched Langerhans cells were resuspended and cultured in 96-well tissue culture plates in culture medium with or without various cytokines (2 × 105 Langerhans cells per well). The spleen was removed and cell suspensions were prepared by mechanical vigorous agitation in PBS containing 1% FBS, and undissociated fragments were removed through the nylon filter. Cell suspensions were incubated in RBC lysing buffer for 10 min at room temperature to lyse spleen erythrocytes. The remaining spleen leukocytes were washed twice with PBS containing 1% FBS and resuspended and cultured in culture medium of 96-well tissue plates (2 × 105 spleen leukocytes per well). After isolation or after culture for 24 h or 48 h, spleen leukocytes were washed and resuspended and stained with FITC-MoAb diluted 1:100 in PBS containing 1% FBS for FACS analysis. For mRNA isolation with reverse transcription–polymerase chain reaction (reverse transcription–PCR), spleen leukocytes cultured for 36 h were used. After enrichment or after culture for 24 h or 48 h, cells were stained either with only the first MoAb for 30 min at 4°C or with the first MoAb followed by the second MoAb at 4°C for 30 min. Then, cells were washed twice and resuspended with PBS containing 1% FBS, and propidium iodide was added to exclude dead cells. After 10 min, cells were subject to analysis on FACSCalibur (Becton Dickinson). More than 5000 cells were used in each experiment and mean fluorescence intensity (MFI) for each surface molecule was analyzed according to CELLQuest Software User's Guide (Becton Dickinson). Percent inhibition was expressed as a percentage according to the following formula: MFI1−MFI2MFI1 where MFI1 is MFI of Langerhans cells cultured without cytokines and stained with anti-mouse CD23, and MFI2 is MFI of Langerhans cells cultured with cytokines and stained with anti-mouse CD23. mRNA was extracted from cultured Langerhans cells and cultured spleen leukocytes using Quickprep Micro mRNA Purification Kit (Pharmacia Biotech, Switzerland). About 100 ng of mRNA was routinely obtained from 5 × 106 cultured Langerhans cells and cultured spleen leukocytes. mRNA was denatured at 65°C for 10 min and cooled on ice for 2 min. Thereafter using First-Strand cDNA Synthesis Kit (Pharmacia Biotech), cDNA was synthesized in reaction mixture containing reverse transcriptase and random hexadeoxynucleotides at 37°C for 60 min. The synthesized cDNA was subject to 30 cycles of reverse transcription–PCR using Taq polymerase (Toyobo, Osaka, Japan) and the primers specific for CD23 and G3PDH with denaturation for 1 min at 94°C, annealing at 55°C for 1.5 min, and polymerization at 72°C for 2 min. The used primers for CD23 were purchased from Sawady Technology (Tokyo, Japan) and the sequences of the primers for CD23 were 5′-AGAAAGCGTTGCTGCTGTGCAAGAC-3′ (sense primer) and 5′-TCAAGCCCAGGGATTTG ATGTTGAC-3′ (anti-sense primer) (Hertl et al., 1996Hertl M. Asada H. Katz S.I. Murine epidermal Langerhans cells do not express the low-affinity receptor for immunoglobulin E, FcεRII (CD23).J Invest Dermatol. 1996; 106: 221-224Crossref PubMed Scopus (5) Google Scholar); the primers for G3PDH were purchased from Clontech Laboratories (Palo Alto, CA), and the sequences of the primers were 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ (sense primer) and 5′-CATGTAGGCCATGAGG; TCCACCAC-3′ (anti-sense primer). One milliliter of first PCR products was amplified using nested PCR primers. The used primers for nested PCR were also obtained from Sawady Technology and the sequences of the primers were 5′-GGATCCAGCACAGCAATGTGGGCTGGCC-3′ (nested sense primer) and 5′-GGATCCCTTAGATCCTCCTGGAGTCCGGT-3′ (nested anti-sense primer). Amplification was performed under the same conditions as reverse transcription–PCR except that the annealing temperature was 57°C. The nested PCR products were analyzed by electrophoresis on 1.5% agarose gels containing ethidium bromide. In preparation for sequencing, nested PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA) and sequenced by dideoxynucleotide termination method using ABI Prism BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Foster, CA). Briefly, purified nested PCR products were further amplified with AmpliTaq DNA Polymerase, FS (Applied Biosystems) using nested sense primer or nested anti-sense primer. Extension products were purified by ethanol precipitation method and electrophoresed on ABI Prism 377 DNA Sequencer (Applied Biosystems). Finally, sequence was analyzed using ABI Prism Sequencing Analysis (Applied Biosystems). After culture without cytokines, Langerhans cells and spleen leukocytes were harvested and resuspended in PBS containing 1% FBS. Langerhans cells (1 × 105) or spleen leukocytes (1 × 105) were incubated with mouse IgE for 50 min at 4°C in PBS containing 1% FBS, washed twice in PBS containing 1% FBS and then incubated with FITC-anti-mouse IgE for 50 min at 4°C in PBS containing 1% FBS. These cell suspensions were washed twice in PBS containing 1% FBS and subjected to FACS analysis. For blocking experiments, Langerhans cells (1 × 105) or spleen leukocytes (1 × 105) were incubated with purified anti-mouse CD23 (B3B4) for 30 min at 4°C. After washing, the cells were incubated with murine IgE, followed by FITC-anti-mouse IgE as above. Student's t test was used to compare the results. p < 0.05 was considered to be statistically significant. Freshly enriched preparations routinely contained > 95% Langerhans cells as determined by FACS analysis (Figure 1a). Freshly isolated Langerhans cells were NLDC145+/–, CD14–, CD11a+, CD11b+, and CD11c+. Cultured Langerhans cells were NLDC145+, CD14–, CD11a+, CD11b+, and CD11c+. These cells had intracytoplasmic Birbeck's granule. This is consistent with a previous observation (Salgado et al, 1999;Tada et al., 2000Tada Y. Asahina A. Nakamura K. Tomura M. Fujiwara H. Tamaki K. Granulocyte/macrophage colony-stimulating factor inhibits IL-12 production of mouse Langerhans cells.J Immunol. 2000; 164: 5113-5119Crossref PubMed Scopus (55) Google Scholar). The viability of freshly isolated Langerhans cells and cultured Langerhans cells was also similar to previous results (Salgado et al., 1999aSalgado C.G. Nakamura K. Sugaya M. et al.Differential effects of cytokines and immunosuppressive drugs on CD40, B7–1, and B7–2 expression on purified epidermal Langerhans cells.J Invest Dermatol. 1999; 113: 1021-1027Crossref PubMed Scopus (59) Google Scholar,Salgado et al., 1999bSalgado C.G. Nakamura K. Sugaya M. et al.Functional CD40 ligand is expressed on epidermal Langerhans cells.J Leukoc Biol. 1999; 66: 281-285PubMed Google Scholar). In the first set of experiments, freshly isolated Langerhans cells and spleen leukocytes were cultured without cytokines. Immediately after isolation and after culture for 24 h and 48 h, both of these cells were single-stained with FITC-anti-mouse CD23 or with FITC-rat IgG2a. Spleen leukocytes were used as positive controls because there are subpopulations that express CD23 in murine spleen leukocytes (Hertl et al., 1996Hertl M. Asada H. Katz S.I. Murine epidermal Langerhans cells do not express the low-affinity receptor for immunoglobulin E, FcεRII (CD23).J Invest Dermatol. 1996; 106: 221-224Crossref PubMed Scopus (5) Google Scholar). Although CD23 was not detectable in freshly isolated Langerhans cells, CD23 expression on cultured Langerhans cells was detectable in a time-dependent manner during culture (Figure 1b,c). After culture for 24 h and 48 h, a small number of cultured Langerhans cells (about 1%) was propidium iodide (PI) negative and annexin V positive, and part of these cells were CD23 positive (data not shown). As it was possible that the failure to detect CD23 on freshly isolated Langerhans cells was due to dispase used to obtain epidermal cell suspensions, the effect of dispase on the expression of CD23 was examined. For this, freshly prepared spleen leukocytes were stained with FITC-anti-mouse CD23 after incubation with 3000 U per ml dispase in culture medium for 10 min and compared with untreated spleen leukocytes. There was no change as for CD23 expression between these cells (data not shown). Thus, this study indicates that CD23 is dispase resistant and that freshly isolated Langerhans cells are virtually negative for CD23 at the protein level. Previous work has shown that human keratinocytes express CD23 (Becherel et al., 1997Becherel P.A. LeGoff L. Frances C. et al.Induction of IL-10 synthesis by human keratinocytes through CD23 ligation. A cyclic adenosine 3′,5′-monophosphate-dependent mechanism.J Immunol. 1997; 159: 5761-5765PubMed Google Scholar). As it would be possible that small numbers of contaminating keratinocytes existed even in our highly enriched cultured Langerhans cells, we next investigated whether keratinocytes expressed CD23. After culture for 48 h, the cells were double-stained with FITC-anti-mouse CD23 and PE-anti-mouse I-Ad or with FITC-rat IgG2a and PE rat IgG2b. As shown in Figure 2, CD23 expression was only detectable on I-Ad-positive cells (i.e., cultured Langerhans cells). There was a population of cultured Langerhans cells that do not express CD23. The percentage of CD23-positive cultured Langerhans cells from three different experiments was 34.7 ± 7.9 at 48 h. When Langerhans cell-depleted epidermal cells (i.e., keratinocytes) were stained for CD23, it was evident that these cells do not express CD23 at the protein level (Figure 3).Figure 3FACS analysis of CD23 expression on Langerhans cells and Langerhans cell-depleted epidermal cells. Immediately after preparation or after culture in the absence of exogenous cytokines, Langerhans cells and Langerhans cell-depleted epidermal cells were harvested and stained with FITC-anti-mouse CD23 or FITC-rat IgG2a (isotype control) for 30 min at 4°C, then washed and analyzed by FACSCalibur. Upper histogram: Langerhans cells; lower histogram: Langerhans cell-depleted epidermal cells; dotted line, FITC-rat IgG2a, solid line, FITC-anti-mouse CD23. The representative experiments are shown.View Large Image Figure ViewerDownload (PPT) Murine cultured Langerhans cells and cultured spleen leukocytes were harvested after culture for 36 h, and mRNA was extracted and cDNA was amplified and analyzed by electrophoresis. By reverse transcription–PCR and nested PCR, the band for CD23 mRNA was detected in cultured Langerhans cells and cultured spleen leukocytes. In freshly isolated Langerhans cells, however, the band for CD23 mRNA was not detected. Moreover, in freshly prepared keratinocytes or cultured keratinocytes, the band for CD23 mRNA was not detected either. Figure 4 shows the results of nested PCR. In order to determine whether nested PCR products obtained from cultured Langerhans cells and cultured spleen leukocytes were really CD23, we sequenced these two nested PCR products according to Materials and Methods and compared with the corresponding sequences from complete genomic sequences of murine CD23 (Richards et al., 1991Richards M.L. Katz D.H. Liu F.T. Complete genomic sequence of the murine low affinity Fc receptor for IgE.J Immunol. 1991; 147: 1067-1074PubMed Google Scholar). Our sequencing and comparison revealed the identity of nested PCR products of these cells with CD23 (data not shown). The effect of various cytokines derived from keratinocytes on the expression of CD23 on Langerhans cells was examined. When Langerhans cells were cultured with each cytokine (IL-1α, IL-10, IL-18, TNF-α, M-CSF, and GM-CSF) at a concentration of 0.1 ng per ml, IL-1α, IL-18, M-CSF, and GM-CSF, but not IL-10 or TNF-α, significantly inhibited CD23 expression on cultured Langerhans cells compared with that of Langerhans cells without exogenous cytokines (Figure 5). Also, these cytokines could not significantly induce apoptosis of cultured Langerhans cells (data not shown). When Langerhans cells were cultured for 24 h in a culture supernatant of keratinocytes, appreciable inhibition of CD23 expression was observed (about 40%). Although it is reported that membrane CD23 on human Langerhans cells was upregulated by IL-4 and/or IFN-γ (Bieber et al., 1989Bieber T. Rieger A. Neuchrist C. et al.Induction of FcεR2/CD23 on human epidermal Langerhans cells by human recombinant interleukin 4 and γ interferon.J Exp Med. 1989; 170: 309-314Crossref PubMed Scopus (166) Google Scholar), no changes were seen by coculture of murine Langerhans cells with IL-4 and/or IFN-γ (data not shown). In order to examine whether CD23 on murine cultured Langerhans cells were able to bind IgE, Langerhans cells cultured without cytokines for 36 h were successively incubated with mouse IgE and were stained with FITC-anti-mouse IgE. CD23 expression on both cultured Langerhans cells and spleen leukocytes were seen in Figure 6(b). As shown in Figure 6(e), cultured Langerhans cells and spleen leukocytes clearly bound IgE. Preincubation of cultured Langerhans cells with purified anti-CD23 (B3B4) partially inhibited the binding of IgE on cultured Langerhans cells (up to 52.3% at maximum, Figure 6(f). As B3B4 is known to interfere with the binding of IgE to CD23 on B cells (Rao et al., 1987Rao M. Lee W.T. Conrad D.H. Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE.J Immunol. 1987; 138: 1845-1851PubMed Google Scholar), these results indicate that murine cultured Langerhans cells bind IgE through CD23. Three IgE-binding structures have been reported in murine Langerhans cells: FcγRII, FcγRIII, and Mac-2 (Haines et al., 1983Haines K.A. Flotte T.J. Springer T.A. Gigli I. Thorbecke G.J. Staining of Langerhans cells with monoclonal antibodies to macrophages and lymphoid cells.Proc Natl Acad Sci USA. 1983; 80: 3448-3451Crossref PubMed Scopus (43) Google Scholar). It is known that the rat MoAb 2.4G2 (anti-mouse FcγRII and FcγRIII) inhibits the binding of IgE to FcγRII and FcγRIII (Takizawa et al., 1992Takizawa F. Adamczewski M. Kinet J.P. Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as FcγRII and FcγRIII.J Exp Med. 1992; 176: 469-476Crossref PubMed Scopus (125) Google Scholar) and cell surface Mac-2 can be detached from cells by lactose treatment (Frigeri and Liu, 1992Frigeri L.G. Liu F.-T. Surface expression of functional IgE binding protein, an endogenous lectin, on mast cells and macrophages.J Immunol. 1992; 148: 861-867PubMed Google Scholar). Preincubation of cultured Langerhans cells with 2.4G2, lactose, and B3B4 completely inhibited the binding of IgE on cultured Langerhans cells (Figure 6g). When we incubated cultured Langerhans cells with 2.4G2 or lactose, each inhibited the binding of IgE on cultured Langerhans cells to 30.0% or 27.9%, respectively. We have examined whether CD23 was expressed on murine Langerhans cells, and if so, whether Langerhans cells could bind IgE through CD23 molecule. In this study, it has been demonstrated that: (i) cultured Langerhans cells, but not freshly isolated Langerhans cells, express CD23; (ii) neither freshly isolated keratinocytes nor cultured keratinocytes express CD23; (iii) cultured Langerhans cells may be composed of a heterogeneous population in terms of the expression of CD23; (iv) cytokines derived from keratinocytes, such as IL-1α, IL-18, M-CSF, and GM-CSF, inhibit CD23 expression on cultured Langerhans cells; (v) murine IgE binds to CD23 on murine cultured Langerhans cells; and (vi) this binding is inhibited by the addition of anti-CD23 MoAb (B3B4) in culture medium. Previous study failed to detect CD23 on murine Langerhans cells. Hertl et al., 1996Hertl M. Asada H. Katz S.I. Murine epidermal Langerhans cells do not express the low-affinity receptor for immunoglobulin E, FcεRII (CD23).J Invest Dermatol. 1996; 106: 221-224Crossref PubMed Scopus (5) Google Scholar reported that neither freshly isolated Langerhans cells nor cultured Langerhans cells expressed CD23; however, our data clearly showed the expression of CD23 on cultured Langerhans cells. One possible explanation for this discrepancy is due to contaminating keratinocyte-derived cytokines in culture. It is known that murine keratinocytes are the major source of cytokines in the epidermis and they secrete various cytokines such as IL-1α, IL-6, IL-7, IL-10, IL-18, TNF-α, M-CSF, and GM-CSF (Matsue et al., 1992Matsue H. Cruz Jr P.D. Bergstresser P.R. Takashima A. Cytokine expression by EC subpopulations.J Invest Dermatol. 1992; 99: 42S-45SCrossref PubMed Scopus (77) Google Scholar). As revealed in this study, various keratinocyte-derived cytokines suppressed CD23 expression on cultured Langerhans cells. In this series of experiments, Langerhans cells of > 95% purity were used, which minimized the effect of contaminating keratinocytes. It would also be possible that freshly isolated Langerhans cells did not express CD23 because Langerhans cells resided among keratinocytes just before purification, thus keratinocyte-derived cytokines affected the expression of CD23 on freshly isolated Langerhans cells. During culture, Langerhans cells undergo various morphologic, functional, phenotypic changes. For example, morphologically, they increase in size, increase the number of the dendrites, and lose Birbeck granules. Functionally, they lose the capacity of processing naive peptides, but they activate allogeneic T cells more strongly than freshly isolated Langerhans cells. Phenotypically, they upregulate the expression of major histocompatibility complex class II antigens, ICAM-1, CD40, B7-1, and B7-2. In contrast, E-cadherin on cultured Langerhans cells is downregulated. Cultured Langerhans cells that exhibit these series of changes are similar to migrating Langerhans cells to draining lymph nodes in vivo where they present antigens to T cells (Schuler et al., 1985Schuler G. Romani N. Steinman R.M. A comparison of murine epidermal Langerhans cells with spleen dendritic cells.J Invest Dermatol. 1985; 85: 99s-106sCrossref PubMed Scopus (59) Google Scholar;Lappin et al., 1996Lappin M.B. Kimber I. Norval M. The role of dendritic cells in cutaneous immunity.Arch Dermatol Res. 1996; 288: 109-121Crossref PubMed Scopus (76) Google Scholar;Schwarzenberger and Udey, 1996Schwarzenberger K. Udey M.C. Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cells E-cadherin expression in situ.J Invest Dermatol. 1996; 106: 553-558Crossref PubMed Scopus (152) Google Scholar;Salgado et al., 1999aSalgado C.G. Nakamura K. Sugaya M. et al.Differential effects of cytokines and immunosuppressive drugs on CD40, B7–1, and B7–2 expression on purified epidermal Langerhans cells.J Invest Dermatol. 1999; 113: 1021-1027Crossref PubMed Scopus (59) Google Scholar,Salgado et al., 1999bSalgado C.G. Nakamura K. Sugaya M. et al.Functional CD40 ligand is expressed on epidermal Langerhans cells.J Leukoc Biol. 1999; 66: 281-285PubMed Google Scholar). Thus it is possible that CD23 expression is another phenotypical change seen in migrating Langerhans cells. Various functions have already been ascribed to CD23, including inhibition of apoptosis of germinal center cells, inhibition of IgE production, maturation of early thymocyte, B cell growth, and presentation of IgE immune complexes to T cells (Swendeman and Thorley-Lawson, 1987Swendeman S. Thorley-Lawson D.A. The activation antigen BLAST-2, when shed, is an autocrine BCGF for normal and transformed B cells.EMBO J. 1987; 6: 1637-1642PubMed Google Scholar;Kehry and Yamashita, 1989Kehry M.R. Yamashita L.C. Low-affinity IgE receptor (CD23) function on mouse B cells: role in IgE-dependent antigen focusing.Proc Natl Acad Sci USA. 1989; 86: 7556-7560Crossref PubMed Scopus (215) Google Scholar;Mossalayi et al., 1990Mossalayi M.D. Lecron J.C. Dalloul A.H. Soluble CD23 (FcεRII) and interleukin 1 synergistically induce early human thymocyte maturation.J Exp Med. 1990; 171: 959-964Crossref PubMed Scopus (90) Google Scholar;Bonnefoy et al., 1993Bonnefoy J.Y. Henchoz S. Hardie D. Holder M.J. Gordon J. A subset of anti-CD21 antibodies promote the rescue of germinal center B cells from apoptosis.Eur J Immunol. 1993; 23: 969-972Crossref PubMed Scopus (102) Google Scholar;Cho et al., 1997Cho S.W. Kilmon M.A. Studer E.J. van der Putten H. Conrad D.H. B cell activation and Ig, especially IgE, production is inhibited by high CD23 levels in vivo and in vitro.Cell Immunol. 1997; 180: 36-46Crossref PubMed Scopus (25) Google Scholar); in this study we have shown that murine cultured Langerhans cells can bind IgE through CD23 expressed on cultured Langerhans cells, which is consistent with that observed in human cultured Langerhans cells after culture with IL-4 and IFN-γ (Bieber et al., 1989Bieber T. Rieger A. Neuchrist C. et al.Induction of FcεR2/CD23 on human epidermal Langerhans cells by human recombinant interleukin 4 and γ interferon.J Exp Med. 1989; 170: 309-314Crossref PubMed Scopus (166) Google Scholar). Thus it would be possible that CD23 on Langerhans cells functions as a ligand for the IgE immune complex and enhances presentation of antigens to T cells as already reported in B cells (Kehry and Yamashita, 1989Kehry M.R. Yamashita L.C. Low-affinity IgE receptor (CD23) function on mouse B cells: role in IgE-dependent antigen focusing.Proc Natl Acad Sci USA. 1989; 86: 7556-7560Crossref PubMed Scopus (215) Google Scholar). As professional antigen presentation cells, antigen uptake, processing, and presentation are the main functions of Langerhans cells. In human Langerhans cells, it is reported that Langerhans cells use FcεRI and IgE for antigen internalization leading to subsequent efficient presentation and antigen focusing (Bieber et al., 1996Bieber T. Kraft S. Jurgens M. et al.New insights in the structure and biology of the high affinity receptor for IgE (FcεRI) on human epidermal Langerhans cells.J Dermatol Sci. 1996; 13: 71-75Abstract Full Text PDF PubMed Scopus (28) Google Scholar). We assume similar mechanisms are operating between CD23 and IgE. This study is under investigation in our laboratory. In humans two types of CD23, termed FcεRIIa and FcεRIIb, are known according to the differences of cytoplasmic amino acid sequences in the N terminus (Yokota et al., 1988Yokota A. Kikutani H. Tanaka T. Sato R. Barsumian E.L. Suemura M. Kishimoto T. Two species of human Fcε receptor II (FcεRII/CD23): Tissue-specific and IL-4-specific regulation of gene expression.Cell. 1988; 55: 611-618Abstract Full Text PDF PubMed Scopus (273) Google Scholar). The former is constitutively expressed on B cells and B cell lines, and is further upregulated by IL-4. The latter exists on various types of cells, such as monocytes and macrophages, and IL-4 is definitely required for its significant expression. These two molecules are functionally different: FcεRIIa is involved in FcεRII-mediated endocytosis, whereas FcεRIIb is associated with IgE-mediated phagocytosis (Yokota et al., 1992Yokota A. Yukawa K. Yamamoto A. Two forms of the low-affinity Fc receptor for IgE differentially mediate endocytosis and phagocytosis: Identification of the critical cytoplasmic domains.Proc Natl Acad Sci USA. 1992; 89: 5030-5034Crossref PubMed Scopus (86) Google Scholar). Although many investigators insisted that murine CD23 consists of a single isoform (Conrad et al., 1993Conrad D.H. Kozak C.A. Vernachio J. Squire C.M. Rao M. Eicher E.M. Chromosomal location and isoform analysis of mouse FcεRII/CD23.Mol Immunol. 1993; 30: 27-33Crossref PubMed Scopus (21) Google Scholar), some reports suggest the existence of murine FcεRIIb (Richards et al., 1991Richards M.L. Katz D.H. Liu F.T. Complete genomic sequence of the murine low affinity Fc receptor for IgE.J Immunol. 1991; 147: 1067-1074PubMed Google Scholar). So far, in the murine system two distinct chemical species have not been identified. The species of CD23 expressed on cultured Langerhans cells needs further investigation. It is interesting that a subpopulation of cultured Langerhans cells expressed CD23. Actually some authors insisted that Langerhans cells may consist of two functionally different populations. For example, Furue and Katz, 1989Furue M. Katz S.I. Direct effects of glucocorticosteroids on epidermal Langerhans cells.J Invest Dermatol. 1989; 92: 342-347Crossref PubMed Scopus (48) Google Scholar reported that cultured Langerhans cells may consist of a heterogeneous population in terms of sensitivity to dexamethasone, and that dexamethasone may completely abolish the expression of Ia antigens on a subpopulation of cultured Langerhans cells or may be cytolytic to this sensitive population. The experiments by Schwarzenberger and Udey, 1996Schwarzenberger K. Udey M.C. Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cells E-cadherin expression in situ.J Invest Dermatol. 1996; 106: 553-558Crossref PubMed Scopus (152) Google Scholar showed that cultured Langerhans cells may be divided into two subpopulations in terms of E-cadherin expression after application of contact allergens. Our results clearly showed that cultured Langerhans cells may be divided into two subpopulations in terms of CD23 expression during culture. With regard to subpopulations of Langerhans cells, Torresani et al., 1991Torresani C. Manara G.C. Ferrari C. De Panfilis G. Immunoelectron microscopic characterization of a subpopulation of freshly isolated epidermal Langerhans cells that reacts with anti-CD23 monoclonal antibody.Br J Dermatol. 1991; 124: 533-537Crossref PubMed Scopus (7) Google Scholar already reported that human freshly isolated Langerhans cells are composed of CD23-positive Langerhans cells (31.2%) and CD23-negative Langerhans cells (68.8%) by immunoelectron microscopic investigation, which is in accord with our results of murine cultured Langerhans cells. It would be possible that these subpopulations have distinct functions in terms of antigen presentation. This awaits further investigation. Thus, this study revealed that murine Langerhans cells express CD23 during culture, and the failure to detect CD23 on murine Langerhans cells may be due to contaminating keratinocyte-derived cytokines. Furthermore, the finding that murine cultured Langerhans cells bind IgE through CD23 suggests that CD23 on murine cultured Langerhans cells may be involved in IgE-mediated immune responses. The authors thank Dr. Stephen I. Katz for reviewing of this manuscript and R. Oh for technical advice in sequencing nested PCR products. This study was supported in part by the grants from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare, Healthy Science Research Grants, Japan.
Referência(s)