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(American Journal of Pathology. 2003;163:261-268.)
© 2003 American Society for Investigative Pathology

Infiltrating CD8⁺ T Cells in Oral Lichen Planus Predominantly Express CCR5 and CXCR3 and Carry Respective Chemokine Ligands RANTES/CCL5 and IP-10/CXCL10 in Their Cytolytic Granules

A Potential Self-Recruiting Mechanism

Wakana Iijima^*, Haruo Ohtani, Takashi Nakayama, Yumiko Sugawara^*, Eiichi Sato, Hiroshi Nagura, Osamu Yoshie and Takashi Sasano^*

From the Division of Oral Diagnosis and Radiology, ^* Tohoku University Graduate School of Dentistry, Sendai; the Department of Pathology, Tohoku University Graduate School of Medicine, Sendai; Mito National Hospital, Mito; and the Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, Japan

	Abstract

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Lichen planus is a chronic inflammatory disease of the skin and oral mucosa in which the cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. To understand its pathophysiology further, the present study examined the in situ expression of chemokines and chemokine receptors in oral lichen planus. Immunohistochemical analysis of 15 cases has consistently revealed that infiltrating CD4⁺ and CD8⁺ T cells in the submucosa predominantly expressed CCR5 and CXCR3. Furthermore, infiltrating T cells, particularly CD8⁺ T cells, were positive for RANTES/CCL5 and IP-10/CXCL10, the ligands of CCR5 and CXCR3, respectively. By immunoelectron microscopy, these chemokines were localized in the cytolytic granules of CD8⁺ T cells. Lesional keratinocytes also overexpressed the ligands of CXCR3, namely, MIG/CXCL9, CXCL10, and I-TAC/CXCL11. Our data suggest that the chemokines signaling via CCR5 and CXCR3, which are known to be selectively expressed by type 1 T cells, are predominantly involved in T-cell infiltration of oral lichen planus. Furthermore, the presence of CCL5 and CXCL10 in the cytolytic granules of tissue-infiltrating CD8⁺ T cells expressing CCR5 and CXCR3 reveals a potential self-recruiting mechanism involving activated effector cytotoxic T cells.

Chemokines are small cytokines that induce leukocyte migration into inflammation sites or regulate leukocyte trafficking through lymphoid tissues.⁶ Based on the spacing of two cysteines in the N-terminal region of the molecules, chemokines are grouped into four subfamilies: CXC, CC, C, and CX3C chemokines. To date, more than 45 chemokines and 18 functional chemokine receptors (CXCR1–6, CCR1–10, XCR1, and CX3CR1) have been identified in humans.⁶ Based on the classification of the subfamilies, a new systematic nomenclature system of the chemokine ligands has been proposed.⁶

After priming, T cells are differentiated into two functional subsets: type 1 T cells (Th1, Tc1) that produce tumor necrosis factor-ß/interferon (IFN)- and type 2 T cells (Th2, Tc2) that produce interleukin (IL)-4, IL-5, IL-6, IL-10, and IL-13.^7,8 It is now known that type 1 T cells selectively express CCR5/CXCR3, and type 2 T cells express CCR3, CCR4, and CCR8.⁶

One of the type 1-selective chemokine receptors, CXCR3, is primarily expressed on activated T lymphocytes, and its specific ligands are the trio of CXC chemokines commonly inducible by the type 1 cytokine IFN-, interferon-inducible protein-10 (CXCL10/IP-10), monokine induced by interferon (CXCL9/MIG) and interferon-inducible T-cell -chemoattractant (CXCL11/I-TAC).⁶ CCR5, another type 1-selective chemokine receptor, is expressed on activated T cells, particularly on exposure to IL-12.⁹ Regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), macrophage inflammatory protein-1 (MIP-1/CCL3) and MIP-1ß/CCL4 are the three CC chemokines that signal via CCR5.⁶

Previous reports on skin lichen planus and other inflammatory dermatoses revealed that mRNAs for MIG/CXCL9 and IP-10/CXCL10 were expressed by basal keratinocytes and by infiltrating cells in the dermal-epidermal junction, whereas I-TAC/CXCL11 mRNA was expressed exclusively by basal keratinocytes.¹⁰ Their shared receptor, CXCR3, was expressed by a vast majority of the dermal infiltrating T cells. Expression of RANTES/CCL5 in keratinocytes was rather sparse in skin lichen planus, while abundant in psoriasis.¹¹ On the other hand, T cells, mast cells, and epithelial cells were shown to express CCL5 and CCR1 in oral lichen planus.¹²

To extend these previous observations, we studied the involvement of chemokines and their receptors in T-cell infiltration of oral lichen planus by double-labeling immunohistochemistry and immunoelectron microscopy. We show here for the first time that the majority of infiltrating T cells, particularly CD8⁺ T cells, not only express CCR5 and CXCR3 but also contain their respective ligands, RANTES/CCL5 and IP-10/CXCL10, in their cytolytic granules. We have also demonstrated the prominent production of the ligands of CXCR3, IP-10/CXCL10, MIG/CXCL9, and I-TAC/CXCL11, by the lesional keratinocytes. Correctively, our findings support the dominant roles of type 1 chemokines and their receptors in the accumulation of T cells in oral lichen planus, and further suggest a potential self-recruiting mechanism involving type 1 chemokines in tissue infiltration of CD8⁺ T cells.

	Materials and Methods

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Specimens

Biopsy specimens were obtained from lesional buccal mucosa of patients with oral lichen planus (n = 15). Diagnosis was made by clinical features and histopathological findings. In all cases, the lesions showed the characteristic clinical features of the erosive form of oral lichen planus. Biopsies from the normal buccal mucosa of healthy volunteers (n = 3) were used as controls. This study was approved by the Ethical Committee of Tohoku University School of Dentistry. For reverse transcriptase-polymerase chain reaction (RT-PCR), samples were snap-frozen and stored at -80°C until the preparation of total RNA. For immunohistochemistry and immunoelectron microscopy, specimens were fixed in periodate-lysine-4% paraformaldehyde for 4 hours at 4°C. After washing in phosphate-buffered saline containing sucrose, fixed specimens were embedded in OCT compound (Miles, Elkhart, IN) and immediately frozen.

RT-PCR Analysis

A semiquantitative RT-PCR was performed as described previously.¹³ In brief, total RNA samples were prepared from buccal mucosa with lichen planus (n = 3) and normal buccal mucosa obtained from healthy volunteers (n = 3). The primers used were: +5-CAACGCCACCCACTGCCAATACAA-3 and -5-CAGGCGCAAGAGCAGCATCCACAT-3 for CXCR3; +5-CTGGCCATCTCTGACCTGTTTTTC-3 and -5-CAGCCCTGTGCCTCTTCTTCTCAT-3 for CCR5; +5-AAGAAGAACAAGGCGGTGAAGATG-3 and -5-AGGCCCCTGCAGGTTTTGAAG-3 for CCR4; +5-CCCCGTGCCCACATCAAGGAGTATTT-3 and -5-CGTCCAGCCTGGGGAAGGTTTTTGTA-3 for RANTES/CCL5; +5-GCCCGGTGTCATCTTCCTAACCAAGC-3 and -5-AGGGGACAGGGGAACTCTCAGAGCAA-3 for MIP-1/CCL3; +5-TGCTGCTTTTCTTACACCGCGAGGAA-3 and -5-AGAAGGGACAGGAACTGCGGAGAGGA-3 for MIP-1ß/CCL4; +5-TGCAAGCCAATTTTGTCCACGTGTTG-3 and -5-GCAGCTGATTTGGTGACCATCATTGG-3 for IP-10/CXCL10; +5-CATGCTGGTGAGCCAAGCAGTTTGAA-3 and -5-CACTTCTGTGGGGTGTTGGGGACAAG-3 for MIG/CXCL9; +5-CGATGCCTAAATCCCAAATCGAAGCA-3 and -5-AATTGCTGGACTCCTTTGGGCAGTGG-3 for I-TAC/CXCL11; +5-GAGCATCCAAAAGAGTGTGGAGA and -5-AGGACAACCATTACTGGGATGCT for IFN-; +5-CATTGTCACTGCAAATCGACACCTAT and -5-GTCCTTCTCATGGTGGCTGTAGAACT for IL-4; +5-GCCAAGGTCATCCATGACAACTTTGG-3 and -5-GCCTGCTTCACCACCTTCTTGATGTC-3 for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). PCR amplification was performed by denaturation at 94°C for 30 seconds (5 minutes at the first cycle), annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds (5 minutes at the last cycle) in 35 cycles for chemokines and chemokine receptors, in 37 cycles for IFN- and IL-4, and in 27 cycles for G3PDH.

Antibodies and Immunohistochemistry

We used the following murine monoclonal antibodies: anti-CCR4 (clone KM2160, IgG1, applied at 1.14 µg/ml),¹³ anti-CCR5 (clone 2D7, IgG2a, applied at 0.12 µg/ml; PharMingen, San Diego, CA), anti-CXCR3 (clone 49801.111, IgG1, applied at 0.1 µg/ml; PharMingen), anti-RANTES/CCL5 (clone VL1, IgG2b,applied at 2.5 µg/ml; Biosource International, Camarillo, CA), anti-MIP-1/CCL3 (clone 14215.41, IgG2a, applied at 20 µg/ml; DAKO Japan, Kyoto, Japan), anti-MIP-1ß/CCL4 (clone 24996.111, IgG2b, applied at 5 µg/ml; DAKO Japan), anti-IP-10/CXCL10 (clone 33036.21, IgG1, applied at 10 µg/ml; DAKO Japan), anti-MIG/CXCL9 (clone 49106.11, IgG1, applied at 0.55 µg/ml; DAKO Japan), and anti-perforin (clone delta G9, IgG2b, applied at 5 µg/ml; Ancell, Bayport, MN). Goat polyclonal anti-I-TAC/CXCL11 was purchased from DAKO Japan and applied at 10 µg/ml. For double immunohistochemistry, we also used murine monoclonal antibodies for CD4 (clone SK3, IgG1, applied at 1:100 or 1:50; BD, San Jose, CA) and CD8 [clone SK1, IgG1, applied at 1:50 (BD) and clone C8/144B, IgG1, applied at 0.75 µg/ml (DAKO A/S, Glostrup, Denmark)].

Six-µm frozen tissue sections were incubated with primary antibodies overnight at 4°C. Then peroxidase-labeled secondary antibodies such as the goat anti-mouse Envision plus system (DAKO Corp., Carpinteria, CA) and rabbit anti-goat simple stain MAX PO (Nichirei, Tokyo, Japan) were applied for 1 hour. The chromogen was 3',3-diaminobenzidine tetrahydrochloride (Dojin, Kumamoto, Japan). The sections were counterstained with methyl green. As negative controls, murine isotype-matched monoclonal IgG1, IgG2a, and IgG2b (DAKO Corp.) and normal goat serum (Zymed, San Francisco, CA) were used. To obtain a better labeling of the granular contents with anti-CCL5, CXCL10, or perforin antibodies, we modified our staining method as follows: the incubation time of Envision was set overnight and the specimens were immersed in 3',3-diaminobenzidine tetrahydrochloride solution without sodium azide or hydrogen peroxide for 5 minutes before the enzymatic reaction.

Double Immunohistochemistry

Double enzyme-linked immunohistochemistry was performed in three representative cases using periodate-lysine-4% paraformaldehyde-prefixed frozen sections as described previously.¹⁴ The combinations of antibodies included anti-CCR5 + anti-CD4, anti-CCR5 + anti-CD8, anti-CXCR3 + anti-CD4, and anti-CXCR3 + anti-CD8. For the following combinations of double staining, anti-RANTES/CCL5 + anti-CD4, anti-CCL5 + anti-CD8, anti-IP-10/CXCL10 + anti-CD4, and anti-CXCL10 + anti-CD8, we used the following method. First, anti-CCL5 or CXCL10 antibody was applied overnight with the Envision plus system. 3'-3-Diaminobenzidine tetrahydrochloride was used as chromogen. The sections were then treated with glycine-HCl buffer (pH 2.2) for 15 minutes, and anti-CD4 or anti-CD8 (clone C8/144B) was applied overnight followed by application of Envision plus system. The chromogen in this second system was TrueBlue (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). After drying, the specimens were mounted. For negative controls, isotype-matched control antibodies were used in each step of double immunohistochemistry.¹⁵ To get a better labeling of granular contents with anti-CCL5 or CXCL-10 antibodies, we treated the specimens with methanol for 3 minutes to increase the membrane permeability before the blocking process of the endogenous peroxidase activity.

Immunoelectron Microscopy for RANTES/CCL5 and IP-10/CXCL10

We adopted the pre-embedding immunoperoxidase method in two cases as described previously¹⁴ with one modification: the fixation condition before the enzymatic reaction was 0.25% glutaraldehyde for 20 seconds.

	Results

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RT-PCR Analysis

As shown in Figure 1 , RT-PCR analysis demonstrated marked and consistent increases in the transcripts of IFN-, CXCR3, MIG/CXCL9, and IP-10/CXCL10 (both CXCR3 ligands), and RANTES/CCL5, MIP-1/CCL3, and MIP-1ß/CCL4 (the CCR5 ligands) in lesional tissues of oral lichen planus (n = 3) in comparison with normal buccal tissues (n = 3). In some patients, signals were also noted in the transcripts of IL-4, CCR5, CCR4, I-TAC/CXCL11 (a CXCR3 ligand), and TARC/CCL17 and MDC/CCL22 (the CCR4 ligands). Collectively, in oral lichen planus, there were remarkable increases in the transcripts of type 1 cytokine IFN- and type 1 chemokines (CXCL9, CXCL10, CCL3, and CCL4) as well as their respective receptors CXCR3 and CCR5 over those of type 2 cytokine IL-4, type 2 chemokines (CCL17 and CCL22), and their shared receptor CCR4.

View larger version (54K): [in this window] [in a new window]   Figure 1. RT-PCR analysis for expression of cytokines, chemokines, and their receptors in oral lichen planus. RT-PCR analysis was performed with total RNA samples prepared from PHA-treated peripheral blood mononuclear cells (positive controls), three samples of normal oral mucosa, and three samples of oral lichen planus. Representative results from two separate experiments are shown.

We next performed immunohistochemical analysis of oral lichen planus to identify the cell types expressing individual chemokine receptors (Figure 2) . In all 15 cases, the majority (70 to 80%) of infiltrating mononuclear cells in the submucosa was positive for CCR5 (Figure 2A) and CXCR3 (Figure 2B) . Furthermore, 30 to 40% of infiltrating cells in 13 of 15 cases and 70% in 2 cases were positive for CCR4 (Figure 2C) . Staining with isotype-matched control IgG showed no reactivity (Figure 2D) . Because the sum of CCR5⁺ cells and CCR4⁺ cells or that of CXCR3⁺ cells and CCR4⁺ cells in infiltrating mononuclear cells exceed 100% in 14 of 15 cases, we presume that some fractions of T cells expressing CCR5 or CXCR3 (type 1 chemokine receptors) also co-express CCR4 (type 2 chemokine receptor). This, however, remains to be proven. Intraepithelial lymphocytes, which were far fewer than submucosal infiltrating T cells, expressed CCR5 (Figure 2E , arrows) and less frequently CXCR3 (data not shown). CCR4 expression in intraepithelial lymphocytes resembled that of CXCR3 (data not shown). Double staining revealed that both CD4⁺ and CD8⁺ T cells expressed CCR5 (Figure 2, F and G) and CXCR3 (Figure 2, H and I) . These findings in oral lichen planus were in sharp contrast to those in the normal buccal mucosa, where only sporadic cells expressing either CCR4, CCR5, or CXCR3 were observed in the submucosa and epithelium (data not shown).

View larger version (120K): [in this window] [in a new window]   Figure 2. Immunohistochemistry for chemokine receptors in oral lichen planus. CCR5 (A) and CXCR3 (B) are expressed in infiltrating mononuclear cells in the submucosa adjacent to epithelial cells (subepithelial). The positivity rate is 70% for both in this case. CCR4 (C) is expressed in the same areas by a minor fraction of mononuclear cells (40% in this case). Negative control (IgG1) (D). Intraepithelial cells (arrows) express CCR5 (E). Double immunohistochemistry for CCR5 (red) + CD4 (blue) (F) and CCR5 (red) + CD8 (blue) (G) in oral lichen planus. CCR5+ cells are double positive for either CD4 (F) or CD8 (G). Double immunohistochemistry for CXCR3 (red) + CD4 (blue) (H) and CXCR3 (red) + CD8 (blue) (I) in oral lichen planus, showing double positivity of CXCR3 and CD4 (H) or CD8 (I). Scale bars: 100 µm (A–D); 50 µm (E–I).

Ligands of CCR5 (RANTES/CCL5, MIP-1/CCL3, MIP-1ß/CCL4)

In all 15 cases, RANTES/CCL5 was expressed with a characteristic dotted pattern in mononuclear cells infiltrating the submucosa. Intraepithelial lymphocytes, although small in number, also expressed CCL5 in the same dotted pattern. This localization pattern, which was clearly discernible under oil-immersion observation (Figure 3, A and B) , was quite similar to that of perforin (Figure 3C) , one of the representative contents of cytolytic granules of CTLs and natural killer cells. Double staining revealed that CCL5-positive mononuclear cells were mostly CD8⁺ T cells and some were CD4⁺ T cells (Figure 3, D and E) . On the other hand, keratinocytes were negative for CCL5. No dendritic-shaped cells were positive for CCL5 in the submucosa. In the normal buccal mucosa, anti-CCL5 stained small numbers of mononuclear cells present in the submucosa (data not shown). In the present study, we were unable to demonstrate any clear-cut immunoreactivity for MIP-/CCL3 or MIP-1ß/CCL4 in oral lichen planus. This may be because of the limitation of antibodies used (data not shown).

View larger version (117K): [in this window] [in a new window]   Figure 3. Immunohistochemistry for chemokine ligands in oral lichen planus. RANTES/CCL5 is positive with a dotted pattern in infiltrating lymphocytes in the submucosa (A) and in the epithelium (B, arrows). This staining pattern is similar to that of perforin (C). Double staining for RANTES/CCL5 (brown) and CD8 (blue) (D), and RANTES/CCL5 (brown) and CD4 (blue) (E). The majority of RANTES/CCL5+ cells are double positive for CD8 (D) and a few are double positive for CD4 (E). IP-10/CXCL10 is not expressed in the normal tissue (F), while overexpressed in keratinocytes in oral lichen planus (G). IP-10/CXCL10 is also expressed in infiltrating lymphocytes with a dotted pattern (H). Double immunohistochemistry revealed that CD8+ cells (blue) express IP-10/CXCL10 (brown) (I, arrows), and a few CD4+ do (J). Diffuse expression of MIG/CXCL9 in keratinocytes in oral lichen planus (K). Scale bars: 20 µm (A–E, H–J); 100 µm (F, K); 50 µm (G).

The normal oral mucosal keratinocytes were essentially negative for IP-10/CXCL10, with only sporadic mononuclear cells positive for CXCL10 (Figure 3F) . In sharp contrast, keratinocytes near the basal layer in lichen planus were clearly positive for CXCL10 in all 15 cases, particularly in the areas where intraepithelial T cells were present (Figure 3G) . Mononuclear cells infiltrating the submucosa and epithelium were also positive for CXCL10 with the characteristic dotted pattern in all cases (Figure 3H) . This staining pattern was quite similar to those of CCL5 and perforin stated above, but the number of cells positive for CXCL10 were less than those positive for CCL5. Double staining revealed that the majority of CXCL10-positive cells were CD8⁺ T cells, as were those positive for CCL5 (Figure 3, I and J) . The lesional keratinocytes were also clearly positive for MIG/CXCL9 (Figure 3K) and I-TAC/CXCL11 (data not shown). Furthermore, some infiltrating mononuclear cells were positive for CXCL9 and CXCL11 (data not shown), but their identity was not determined. In the normal buccal mucosa, CXCL9 and CXCL11 were weakly expressed by keratinocytes and by a few submucosal mononuclear cells (data not shown). No reactivity was observed with isotype-matched murine IgG or normal goat serum (data not shown).

Immunoelectron Microscopy of RANTES/CCL5 and IP-10/CXCL10 (Performed in Two Representative Cases)

As described above, RANTES/CCL5 and IP-10/CXCL10 were positive in CD8⁺ T cells with the characteristic dotted pattern. Immunoelectron microscopy clearly revealed that CCL5 (Figure 4, A and B) and CXCL10 (Figure 4C) were localized in the membrane-bound granules (ie, cytolytic granules) within the lesional lymphocytes. The diameter of the granules ranged from 85 to 250 nm. The number of positive granules was one to two per cell. Furthermore, CXCL10 was localized in the lumen of rough endoplasmic reticulum of lesional keratinocytes (Figure 4D) . The presence of desmosomes helped to identify keratinocytes. No reactivity was observed with isotype-matched murine IgGs (data not shown).

View larger version (144K): [in this window] [in a new window]   Figure 4. Immunoelectron microscopy for RANTES/CCL5 and IP-10/CXCL10 in oral lichen planus. The immunoreactivity for RANTES/CCL5 is localized in membrane-bound granules in lymphocytes (A and B in higher magnification). IP-10/CXCL10 is also localized in membrane-bound granules in lymphocytes (C, arrows). IP-10/CXCL10 is expressed in the lumen of rough endoplasmic reticulum of keratinocytes as black deposits (D). Note the presence of desmosomes in these keratinocytes (arrows). Scale bars: 1 µm (A); 500 nm (B–D).

	Discussion

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CTLs are characterized by the presence of cytolytic granules, which contain perforin and granzymes.¹⁶ A previous report demonstrated co-localization of RANTES/CCL5 and granzyme A, a content of cytolytic granules, within in vitro cultured HIV-1-specific CD8⁺ CTL.¹⁷ A recent study further demonstrated that mouse T cells, particularly memory CD8⁺ T cells, produce CCL5 protein on TCR stimulation.¹⁸ Co-up-regulation of transcripts for CCR5, CCL5, and MIP-1ß/CCL4 in effector CD8⁺ T cells in comparison with naive CD8⁺ T cells was also reported.¹⁹ Other studies reported secretion of IP-10/CXCL10 by the myelin proteolipid protein-specific CD8⁺ CTL derived from multiple sclerosis.^20,21 These data fully concur with our in vivo findings that CCL5 and CXCL10 were localized in the cytolytic granules of CD8⁺ T cells in oral lichen planus. The localization of CCL5 and CXCL10 within the cytolytic granules of infiltrating CD8⁺ T cells and the expression of CCR5 and CXCR3 by the majority of infiltrating T cells strongly suggests a self-recruiting mechanism for accumulation of CCR5⁺ and/or CXCR3⁺ T cells into the lesion of oral lichen planus. Double staining of CCR5-CCL5 or CXCR3-CXCL10 would be useful to further support this concept. However, we were unable to obtain clear results because of technical difficulties (data not shown).

A previous report by our group demonstrated the expression of MDC/CCL22, the ligand of CCR4, in dendritic cells in inflamed skin tissue.¹⁵ In the present study, we did not observe the expression of RANTES/CCL5 in dendritic-shaped cells in oral lichen planus tissue either in the epithelium or in the submucosa. As for the CXCR3 ligands, a further study is needed to test their production by dendritic cells in inflamed tissues.

CXCR3 is expressed by the majority of infiltrating CD4⁺ and CD8⁺ T cells in skin lichen planus.¹⁰ Our study has further revealed that not only CXCR3 but also CCR5, both of which are known to be selective for type 1 T cells,⁶ are expressed by the majority of infiltrating T cells in oral lichen planus. Even though a mixed cytokine profile, not entirely shifted to either Th1 or Th2, was reported in oral lichen planus,²² our present results based on the expression of chemokines and their receptors together with the predominance of mRNA of IFN- over that of IL-4 indicated that type 1 T cells are predominant over type 2 T cells in oral lichen planus. In fact, cell-mediated cytotoxicity is considered to be the major pathological mechanism of the disease^3-5 and mRNA for IFN-, one of the representative cytokines to promote cell-mediated immune responses,²³ was detected in keratinocytes and infiltrating cells in oral lichen planus.²⁴ However, a detailed analysis of the cytokine profiles in oral lichen planus would be required to further clarify this issue. Compared to the frequencies of CD4⁺ T cells expressing CCR5 or CXCR3 in the peripheral blood,^25,26 the frequencies of CD4⁺ T cells expressing these receptors in skin tissues of delayed-type hypersensitivity were much higher.²⁵ This further underscores the importance of chemokines in selective recruitment of particular T-cell subsets into inflamed tissues.

IP-10/CXCL10, MIG/CXCL9, and I-TAC/CXCL11, the ligands of CXCR3 were all expressed by lesional keratinocytes. Immunoelectron microscopy further demonstrated that CXCL10 is localized in the rough endoplasmic reticulum, indicating de novo synthesis of CXCL10 by lesional keratinocytes. Human keratinocytes in culture were shown to up-regulate the expression of mRNAs for CXCL10 and CXCL9 on stimulation with tumor necrosis factor- and IFN-,²⁷ and they also expressed CXCL11 mRNA when stimulated by IFN-.²⁸ In oral lichen planus, IFN- mRNA and tumor necrosis factor- protein were demonstrated in keratinocytes and infiltrating cells.^24,29 Therefore, it is likely that IFN- and tumor necrosis factor- induce lesional keratinocytes to produce these chemokines in oral lichen planus. Up-regulation of mRNAs for CXCL9, CXCL10, and CXCL11 in our RT-PCR analysis also supports their de novo synthesis in lichen tissues (Figure 1) . Together with the demonstration of the expression of CXCR3 by the majority of infiltrating T cells, our data support the dominant pathological roles of these chemokines in oral lichen planus.

In conclusion, in oral lichen planus, the chemokines that attract type 1 effector T cells are released not only by target epithelial cells (CXCL9, CXCL10, and CXCL11) but also by infiltrating T cells themselves (CCL5 and CXCL10) that express CCR5 and CXCR3 (the receptors for CCL5 and CXCL10, respectively). The present histochemical findings support the predominant role of type 1 chemokines and their receptors in the recruitment of T cells including CTLs in oral lichen planus. Furthermore, CTLs may sustain their recruitment via a self-recruiting mechanism. These mechanisms of T cell recruitment into the lesions could be responsible for the prolonged clinical course of the disease.

	Acknowledgements

 
We thank Prof. Kouji Matsushima, Department of Molecular Preventive Medicine, University of Tokyo, for supplying anti-CCR4 antibody.

	Footnotes

 
Address reprint requests to Wakana Iijima, D.D., Division of Oral Diagnosis and Radiology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: wakana{at}mips.ne.jp

Supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Accepted for publication April 8, 2003.

	References

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1. Scully C, Beyli M, Ferreiro MC, Ficarra G, Gill Y, Griffiths M, Holmstrup P, Mutlu S, Porter S, Wray D: Update on oral lichen planus: etiopathogenesis and management. Crit Rev Oral Biol Med 1998, 9:86-122[Abstract/Free Full Text]
2. Takeuchi Y, Tohnai I, Kaneda T, Nagura H: Immunohistochemical analysis of cells in mucosal lesions of oral lichen planus. J Oral Pathol 1988, 17:367-373[Medline]
3. Sugerman PB, Satterwhite K, Bigby M: Autocytotoxic T-cell clones in lichen planus. Br J Dermatol 2000, 142:449-456[Medline]
4. Kilpi AM: Activation marker analysis of mononuclear cell infiltrates of oral lichen planus in situ. Scand J Dent Res 1987, 95:174-180[Medline]
5. Jungell P, Konttinen YT, Nortamo P, Malmstrom M: Immunoelectron microscopic study of distribution of T cell subsets in oral lichen planus. Scand J Dent Res 1989, 97:361-367[Medline]
6. Yoshie O, Imai T, Nomiyama H: Chemokines in immunity. Adv Immunol 2001, 78:57-110[Medline]
7. Carter LL, Dutton RW: Type 1 and type 2: a fundamental dichotomy for all T-cell subsets. Curr Opin Immunol 1996, 8:336-342[Medline]
8. Mosmann TR, Sad S: The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996, 17:138-146[Medline]
9. Iwasaki M, Mukai T, Gao P, Park WR, Nakajima C, Tomura M, Fujiwara H, Hamaoka T: A critical role for IL-12 in CCR5 induction on T cell receptor-triggered mouse CD4⁺ and CD8⁺ T cells. Eur J Immunol 2001, 31:2411-2420[Medline]
10. Flier J, Boorsma DM, van Beek PJ, Nieboer C, Stoof TJ, Willemze R, Tensen CP: Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J Pathol 2001, 194:398-405[Medline]
11. Raychaudhuri SP, Jiang WY, Farber EM, Schall TJ, Ruff MR, Pert CB: Upregulation of RANTES in psoriatic keratinocytes: a possible pathogenic mechanism for psoriasis. Acta Dermatol Venereol 1999, 79:9-11[Medline]
12. Zhao ZZ, Sugerman PB, Walsh LJ, Savage NW: Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration. J Oral Pathol Med 2002, 31:158-162[Medline]
13. Imai T, Nagira M, Takagi S, Kakizaki M, Nishimura M, Wang J, Gray PW, Matsushima K, Yoshie O: Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int Immunol 1999, 11:81-88[Abstract/Free Full Text]
14. Katou F, Ohtani H, Saaristo A, Nagura H, Motegi K: Immunological activation of dermal Langerhans cells in contact with lymphocytes in a model of human inflamed skin. Am J Pathol 2000, 156:519-527[Abstract/Free Full Text]
15. Katou F, Ohtani H, Nakayama T, Ono K, Matsushima K, Saaristo A, Nagura H, Yoshie O, Motegi K: Macrophage-derived chemokine (MDC/CCL22) and CCR4 are involved in the formation of T lymphocyte-dendritic cell clusters in human inflamed skin and secondary lymphoid tissue. Am J Pathol 2001, 158:1263-1270[Abstract/Free Full Text]
16. Peters PJ, Borst J, Oorschot V, Fukuda M, Krahenbuhl O, Tschopp J, Slot JW, Geuze HJ: Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med 1991, 173:1099-1109[Abstract/Free Full Text]
17. Wagner L, Yang OO, Garcia-Zepeda EA, Ge Y, Kalams SA, Walker BD, Pasternack MS, Luster AD: Beta-chemokines are released from HIV-1-specific cytolytic T-cell granules complexed to proteoglycans. Nature 1998, 391:908-911[Medline]
18. Swanson BJ, Murakami M, Mitchell TC, Kappler J, Marrack P: RANTES production by memory phenotype T cells is controlled by a posttranscriptional, TCR-dependent process. Immunity 2002, 17:605-615[Medline]
19. Kaech SM, Hemby S, Kersh E, Ahmed R: Molecular and functional profiling of memory CD8 T cell differentiation. Cell 2002, 111:837-851[Medline]
20. Biddison WE, Taub DD, Cruikshank WW, Center DM, Connor EW, Honma K: Chemokine and matrix metalloproteinase secretion by myelin proteolipid protein-specific CD8⁺ T cells: potential roles in inflammation. J Immunol 1997, 158:3046-3053[Abstract]
21. Biddison WE, Cruikshank WW, Center DM, Pelfrey CM, Taub DD, Turner RV: CD8⁺ myelin peptide-specific T cells can chemoattract CD4⁺ myelin peptide-specific T cells: importance of IFN-inducible protein 10. J Immunol 1998, 160:444-448[Abstract/Free Full Text]
22. Simark-Mattsson C, Bergenholtz G, Jontell M, Eklund C, Seymour GJ, Sugerman PB, Savage NW, Dahlgren UI: Distribution of interleukin-2, -4, -10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus. Arch Oral Biol 1999, 44:499-507[Medline]
23. Abbas AK, Murphy KM, Sher A: Functional diversity of helper T lymphocytes. Nature 1996, 383:787-793[Medline]
24. Fayyazi A, Schweyer S, Soruri A, Duong LQ, Radzun HJ, Peters J, Parwaresch R, Berger H: T lymphocytes and altered keratinocytes express interferon-gamma and interleukin 6 in lichen planus. Arch Dermatol Res 1999, 291:485-490[Medline]
25. Kunkel EJ, Boisvert J, Murphy K, Vierra MA, Genovese MC, Wardlaw AJ, Greenberg HB, Hodge MR, Wu L, Butcher EC, Campbell JJ: Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes. Am J Pathol 2002, 160:347-355[Abstract/Free Full Text]
26. Lohmann T, Laue S, Nietzschmann U, Kepellen TM, Lehmann I, Schroeder S, Paschke R: Reduced expression of Th1-associated chemokine receptors on peripheral blood lymphocytes at diagnosis of type 1 diabetes. Diabetes 2002, 51:2474-2480[Abstract/Free Full Text]
27. Rottman JB, Smith TL, Ganley KG, Kikuchi T, Krueger JG: Potential role of the chemokine receptors CXCR3, CCR4, and the integrin alphaEbeta7 in the pathogenesis of psoriasis vulgaris. Lab Invest 2001, 81:335-347[Medline]
28. Tensen CP, Flier J, Van Der Raaij-Helmer EM, Sampat-Sardjoepersad S, Van Der Schors RC, Leurs R, Scheper RJ, Boorsma DM, Willemze R: Human IP-9: a keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3). J Invest Dermatol 1999, 112:716-722[Medline]
29. Younes F, Quartey EL, Kiguwa S, Partridge M: Expression of TNF and the 55-kDa TNF receptor in epidermis, oral mucosa, lichen planus and squamous cell carcinoma. Oral Dis 1996, 2:25-31[Medline]

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