The sublingual locus has recently received great attention
as a delivery site for various immunotherapies, including
those that induce allergen-specific tolerance, and for vaccines
that generate protective immunity. To further understand
the immune functions of the human sublingual mucosa, we
characterized the distribution of various immunocytes
therein by immunohistochemistry. We identified professional
antigen presenting cells (APCs), including Langerhans cells
(LCs) and macrophages. CD1a+ and langerin+ LCs were
further found to be distributed in the basal and supra-basal
layers of the epithelium, and macrophages were identified in
the lamina propria. HLA-DR+ cells were observed in both
the epithelium and the lamina propria, which mirrors the
tissue distribution of LCs and macrophages within these
tissues. CD3+, CD4+, and CD8+ T cells were found to be
distributed along the basal layer of the epithelium and also
in the lamina propria. Although B cells, plasma cells, and
Foxp3+ regulatory T cells (Tregs) were only occasionally
observed in the human sublingual mucosa in the absence of
inflammation, they did show enrichment at inflammatory
sites. Hence, we have further elucidated the immune cell
component distribution in human sublingual mucosa

Choi, Youngnim

Cuburu, Nicolas

Czerkinsky, Cecil

Hong, Sung Doo

Lee, Jongho

Saletti, Giulietta

International Journal of Oral Biology

The sublingual locus has recently received great attention as a delivery site for various immunotherapies, including those that induce allergen-specific tolerance, and for vaccines that generate protective immunity. To further understand the immune functions of the human sublingual mucosa, we characterized the distribution of various immunocytes therein by immunohistochemistry. We identified professional antigen presenting cells (APCs), including Langerhans cells (LCs) and macrophages. CD1a+ and langerin+ LCs were further found to be distributed in the basal and supra-basal layers of the epithelium, and macrophages were identified in the lamina propria. HLA-DR+ cells were observed in both the epithelium and the lamina propria, which mirrors the tissue distribution of LCs and macrophages within these tissues. CD3+, CD4+, and CD8+ T cells were found to be distributed along the basal layer of the epithelium and also in the lamina propria. Although B cells, plasma cells, and Foxp3+ regulatory T cells (Tregs) were only occasionally observed in the human sublingual mucosa in the absence of inflammation, they did show enrichment at inflammatory sites. Hence, we have further elucidated the immune cell component distribution in human sublingual mucosa.N

Youngnim Choi

Sung Doo Hong

Jongho Lee

Nicolas Cuburu

Giulietta Saletti

Cecil Czerkinsky

SNU Open Repository and Archive

Immunohistochemical Characterization of the Human Sublingual Mucosa

International Journal of  Oral Biology, Vol. 34, No. 3,  September  30  2009, p. 131~135Copyright ⓒ 2009, The Korean Academy of Oral Biology 131InternationalJournal ofOral BiologyImmunohistochemical Characterization of the Human Sublingual MucosaYoungnim Choi*1, Sung Doo Hong2, Jongho Lee3, Nicolas Cuburu4, Giulietta Saletti4, and Cecil Czerkinsky4Departments of 1Oral Microbiology and Immunology, 2Oral Pathology,3Oromaxillofacial Surgery, Dental Research Institute and Seoul National University,4Laboratory Sciences Division, International Vaccine Institute (received August 24, 2009 ; revised August 28, 2009 ; accepted September 4, 2009)The sublingual locus has recently received great attentionas a delivery site for various immunotherapies, includingthose that induce allergen-specific tolerance, and for vaccinesthat generate protective immunity. To further understandthe immune functions of the human sublingual mucosa, wecharacterized the distribution of various immunocytestherein by immunohistochemistry. We identified professionalantigen presenting cells (APCs), including Langerhans cells(LCs) and macrophages. CD1a+ and langerin+ LCs werefurther found to be distributed in the basal and supra-basallayers of the epithelium, and macrophages were identified inthe lamina propria. HLA-DR+ cells were observed in boththe epithelium and the lamina propria, which mirrors thetissue distribution of LCs and macrophages within thesetissues. CD3+, CD4+, and CD8+ T cells were found to bedistributed along the basal layer of the epithelium and alsoin the lamina propria. Although B cells, plasma cells, andFoxp3+ regulatory T cells (Tregs) were only occasionallyobserved in the human sublingual mucosa in the absence ofinflammation, they did show enrichment at inflammatorysites. Hence, we have further elucidated the immune cellcomponent distribution in human sublingual mucosa.Key words: sublingual mucosa, immunohistochemistry,dendritic cells, macrophages, T cells, B cells, Treg cellsIntroductionThe sublingual mucosa has recently received great attentionfor its potential role as an attractive site for the delivery ofdrugs, immunotherapeutics, and vaccines (Holmgren et al.,2005). Due to its accessibility, high permeability, and highdegree of vascularization, the sublingual mucosa presents anon-invasive alternative to subcutaneous antigen delivery(Moingeon et al., 2006). The oral mucosal immune system,like mucosal immunity at other body sites, must continuouslymaintain tolerance to innocuous antigens, such as food andcommensal microbes, yet mount appropriate protectiveimmune responses against pathogens. Many published studiessupport the idea that sublingual delivery of allergens is asuitable treatment for allergic rhinitis in adult individualsand is safer than subcutaneous desensitization (Holmgren etal., 2005; Moingeon et al., 2006; Novak et al., 2008). Further-more, sublingual vaccination of mice with the influenzavirus induced systemic cytotoxic T lymphocyte (CTL)responses and antibody production, both systemically and inmucosal tissue-specific sites, including the oro-gastrointestinaland respiratory tracts, thus conferring protection againstinfection (Song et al., 2008). However, the regulatory me-chanisms that control sublingual mucosal immunity remainpoorly understood. In this study, we use immunohistochemicaltechniques to identify the subpopulations of immunocytesin specimens from human sublingual mucosa.Materials and methodsSpecimenThe use of human materials was approved by InstitutionalReview Board at Seoul National University Dental Hospital.Eight sublingual mucosal tissues were obtained from patients*Corresponding author: Youngnim Choi, Ph.D., Department ofOromaxillofacial Infection & Immunity, School of Dentistry andDental Research Institute, Seoul National University 28-2Yeongeon-dong, Jongno-gu, Seoul 110-749 Tel.: +82-2-740-8643;Fax.: +82-2-743-0311; E-mail: youngnim@snu.ac.kr132 Youngnim Choi, Sung Doo Hong, Jongho Lee, Nicolas Cuburu, Giulietta Saletti, and Cecil Czerkinskwith oral tumors during surgery under an informed consent.Small pieces (3 mm × 12 mm × 3 mm) of sublingual mucosawere excised avoiding tumor mass and fixed with formalin-free zinc fixative (BD Bioscience, San Diego, CA).AntibodiesPrimary antibodies and dilution used for immnohisto-chemistry are listed in table 1.ImmunohistochemistrySerial paraffin-embedded sections (4 µm) were deparaffi-nized in xylene, hydrated, and then incubated in Tris/EDTApH 9.0 buffer at 125oC for 3 min to expose antigens. Afterwashing with running water, sections were immersed in 3%hydrogen peroxide for 10 minutes to block endogenousperoxidase activity, and then subjected to immunohisto-chemistry using a Autoimmunostainer (LAB VISION 2D).Briefly, the sections were incubated with primary antibodiesat room temperature for 1 hour, washed with TBS (Tris-buffered saline and tween 20, pH 7.6) for five minutes threetimes, incubated with either anti-mouse EnVision+ system-HRP (DAKO, Denmark) or biotinylated anti-rat IgG (1:500,DAKO) followed by Streptavidin-HRP (DAKO) for 30minutes, washed with TBS again, and then visualized with3,3'-diaminobenzidine tetrahydrochloride (DAKO). Thesections were counterstained with Mayer’s hematoxylin andmounted. Primary antibodies and dilution used for immuno-histochemistry are listed in table 1. Immunolabeled cellswere counted in three high power fields (× 400) per sectionthat were chosen from the areas without inflammatoryinfiltrates, and the mean of three quantifications was obtainedfor each patient. The number of each immunolabeled cellsubpopulation in table 2 was expressed as the mean andrange of eight patients. ResultsDistribution of antigen-presenting cells (APCs)Transverse sections of human sublingual mucosal tissuereveal a non-keratinized epithelium with occasional regionsof para-keratinization (Fig. 1A and B). The distribution ofAPCs in the sublingual mucosa was evaluated by immuno-histochemistry. CD1a+ cells were distributed normally in theepithelium, but were absent from the lamina propria.Although most CD1a+ cells were found in the basal andsupra-basal layers of the epithelium, a few CD1a+ cells wereobserved in more superficial epithelial layers (Fig. 1C). Avery similar pattern of distribution was found for langerin+cells (Fig. 1E), which suggests that sublingual mucosa DCsare in fact Langerhans cells, an observation that has beenreported elsewhere (Allam et al., 2008). Furthermore, thepercentages of CD1a+ and langerin+ cells out of total kera-tinocytes were quite similar: 3.96% and 3.34%, respectively.Migrating CD1a+ and langerin+ cells were observed in boththe lamina propria and the epithelium at sites of inflammation(Fig. 1D and F). Macrophages, which are restricted to thelamina propria in the absence of inflammation, were alsofound in inflammatory foci within the epithelium (Fig. 1Gand H). HLA-DR+ cells were observed in both the epitheliumand in the lamina propria, a pattern that directly correlateswith the distribution of LCs and macrophages in these twoTable 1. Primary antibodies used for immunohistochemistryClone Isotype Dilution CompanyCD1a O10 Mouse IgG1 1:25 SerotecLangerin 306G9.01/HD24 Mouse IgG1 1:200 DendriticsMacrophage/histiocyte 3A5 Mouse IgG2b 1:50 SerotecHLA-DR YE2/36-HLK Rat IgG2a 1:100 SerotecCD20 7D1 Mouse IgG1 1:50 SerotecCD138 B-A38 Mouse IgG1 1:100 SerotecCD3 CD3-12 Rat IgG1 1:100 SerotecCD4 4B12 Mouse IgG1 1:50 MonosanCD8 4B11 Mouse IgG2b 1:50 SerotecFOXP3 221D/D3 Mouse IgG1 1:500 SerotecTable 2. The number of immune cell subpopulation in human sublingual mucosaaDC Macro-phageHLA-DRB cell T cellCD1a Langerin CD20 CD138 CD3 CD4 CD8 TregEp11.1         (4.7-20)9.3(5-16.3)5.9(2.7-11)12.8(5-20.3)7(1-17)10.3(3.3-16.7)0.7(0-2.7)LP13.7(4-26)7.5(1.7-14.3)0.7(0-2.3)3.6(0-13.3)20.6(6.3-40.7)12(2.3-29.3)12.3(2-27.3)0.2 (0-0.7)anumber per high power field (× 400) expressed as the mean and range of eight samples.Immunohistochemical Characterization of the Human Sublingual Mucosa 133loci (Fig. 1I). Based on the total counts of langerin+ cells,macrophages, and HLA-DR+ cells, we calculated that app-roximately 52% of macrophages and 63% of LCs appear toexpress HLA-DR (Table 2). These results indicate that LCsand macrophages are the most abundant APC subsets in thehuman sublingual mucosa. Distribution of lymphocyte subsetsWe also examined the distribution of lymphocytes in thesublingual mucosa. In the absence of inflammation, CD3+ Tcells were the most abundant lymphocyte populationpresent in the sublingual mucosa (Table 2), and were foundto be distributed equally both along the basal layer of theFig. 1. Paraffin embedded sections of sublingual biopsies were subjected to H&E staining (A, B) and immunohistochemistry using antibodiesfor CD1a (C, D), langerin (E, F), macrophage marker (G, H), and HLA-DR (I, J). Areas without inflammation (A, C, E, G, I) and those withinflammatory infiltrates (B, D, F, H, J) were photographed. Fig. 2. Paraffin embedded sections of sublingual biopsies were subjected to immunohistochemistry using antibodies for CD3 (A, B), CD4 (C, D),CD8 (E, F), FOXP3 (G, H), CD20 (I, J), and CD138 (K, L). Areas without inflammation (A, C, E, G, I, K) and those with inflammatory infil-trates (B, D, F, H, J, L) were photographed.134 Youngnim Choi, Sung Doo Hong, Jongho Lee, Nicolas Cuburu, Giulietta Saletti, and Cecil Czerkinskepithelium and within the lamina propria (Fig. 2A). Further-more, CD3+ T cells comprised a large proportion of the infil-trating immunocytes at inflammatory foci (Fig. 2B). Thepattern of distribution for CD4+ and CD8+ T cells was similarto that of CD3+ cells (Fig. 2C-F), and the ratio of CD4+ T cellsto CD8+ T cells was close to 1 (Table 2). Tregs were detectedusing an antibody against the transcription factor FOXP3(Nik et al., 2008). Whereas antibodies targeted against CDmarkers stained the plasma membrane of cells, the anti-FOXP3 antibody stained their nuclei specifically (Fig. 2Gand H). FOXP3+ cells were occasionally observed in theepithelium or lamina propria of healthy sublingual mucosaltissue (Fig. 2G), but were more frequently found within thepopulation of infiltrating inflammatory lymphocytes (Fig. 2H).In contrast to the pattern of distribution of T cells, CD20+B cells were rarely observed in either the epithelium or thelamina propria of non-inflamed tissue. However, many Bcells were found within the fraction of inflammatory infiltrates(Fig. 2I and J). The plasma cell marker CD138 (syndecan-1)was also strongly expressed on human oral epithelial cells,as reported by others (Sanderson et al., 1989; Soukka et al.,2000). Thus, this abundance of CD138 staining precludedour ability to determine an accurate assessment of thefrequency of plasma cells in the epithelium. Like B cells,plasma cells were rarely observed in the sublingual mucosallamina propria of non-inflamed tissue (Fig. 2K), but werereadily detected around salivary glands (data not shown)and at inflammatory loci (Fig. 2L).These results indicate that T cells are the predominantlymphocyte population in the immune compartment of non-inflamed sublingual mucosa. Regulatory T cells, B cells,and plasma cells, however, are detected in these tissues onlyin the context of inflammation.DiscussionWe evaluated the distribution of various immune cellsubsets within the human sublingual mucosa and found thatLCs, macrophages, and T cells, but not Tregs, B cells, orplasma cells, populate this tissue under normal conditions. Our data demonstrate that LCs and macrophages are theprinciple APCs present in the human sublingual mucosa.However, it is possible that intraepithelial LCs, not laminapropria macrophages, are the cell type responsible for theuptake of antigens that are administered to the sublingualmucosa. Although most LCs are found in the basal andsupra-basal layers of the epithelium, it has been reportedthat the dendrites of LCs residing in the gingival epitheliumare able to extend to the tissue surface (Ito et al., 1998). Inaddition, a thin and permeable epithelium in the sublingualmucosa might enable LCs to sample antigens more efficiently.The distribution of HLA-DR+ cells indicates that professio-nal APCs with antigen presenting ability reside in thehuman sublingual mucosa. Allam et al. reported that LCsisolated from the sublingual oral mucosa express high levelsof both MHC class II and the DC maturation marker CD83,and are able to stimulate allogeneic T cells to proliferate(2008). The presence of both LCs and T cells along the basallayer of the sublingual mucosal epithelium opens thepossibility that these cells are in direct contact with eachother for antigen presentation within this tissue in vivo.However, the lack of B cells within the sublingual mucosasuggests that the antigens are more likely to be delivered tothe draining lymph node for presentation to both B and Tcells. Whether antigen presentation occurs within thesublingual mucosa or in draining lymph nodes is aninteresting question that still needs to be answered.Regulatory T cells have been implicated in pro-tolerogenicmechanisms of the oral mucosa (Moingeon et al., 2006;Novak et al., 2008). Since Tregs are rarely observed in thesublingual mucosa in the absence of inflammation, theylikely migrate into this tissue in response to inflammatorycytokines and chemokines. Tregs might be recruited to sitesof inflammation to prevent excessive tissue destructionduring an inflammatory response. However, Tregs do notappear to be involved in the maintenance of tolerance tofood antigens and commensal microbes within the sublingualmucosa, at least at the peripheral site.Conflict of InterestAuthors declare there are no conflicts of interest.AcknowledgementThis work was supported by BK21 CLS to School ofDentistry, Seoul National University and grant F01-2007-000-10024-0 to Dr. Youngnim Choi from the Korea Scienceand Engineering Foundation.ReferencesAllam JP, Stojanovski G, Friedrichs N, Peng W, Bieber T,Wenzel J, Novak N. Distribution of Langerhans cells andmast cells within the human oral mucosa: new application sitesof allergens in sublingual immunotherapy? Allergy. 2008;63:720-7.Holmgren J, Czerkinsky C. Mucosal immunity and vaccines.Nat Med. 2005;11:S45-53. Ito H, Takekoshi T, Miyauchi M, Ogawa I, Takata T, Nikai H,Takemoto K. Three-dimensional appearance of Langerhanscells in human gingival epithelium as revealed by confocallaser scanning microscopy. Arch Oral Biol. 1998;43:741-4.Moingeon P, Batard T, Fadel R, Frati F, Sieber J, Van OvertveltL. Immune mechanisms of allergen-specific sublingual im-munotherapy. Allergy. 2006;61:151-65.Novak N, Haberstok J, Bieber T, Allam JP. The immuneImmunohistochemical Characterization of the Human Sublingual Mucosa 135privilege of the oral mucosa. Trends Mol Med. 2008;14:191-8.Sanderson RD, Lalor P, Bernfield M. B lymphocytes expressand lose syndecan at specific stages of differentiation. CellRegulation 1989;1:27-35. Song JH, Nguyen HH, Cuburu N, Horimoto T, Ko SY, ParkSH, Czerkinsky C, Kweon MN. Sublingual vaccination withinfluenza virus protects mice against lethal viral infection.Proc Natl Acad Sci U S A. 2008;105:1644-9.Soukka T, Pohjola J, Inki P, Happonen RP. Reduction ofsyndecan-1 expression is associated with dysplastic oralepithelium. J Oral Pathol Med 2000;29:308-13.Tavakoli N, Hambly BD, Sullivan DR, Bao S (2008). Forkheadbox protein 3: essential immune regulatory role. Int J BiochemCell Biol 2008;40:2369-73.

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Immunohistochemical Characterization of the Human Sublingual Mucosa

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