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Induction of CD13 on T-lymphocytes by Adhesive Interaction with Gingival Fibroblasts

Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka Suita, Osaka 565-0871, Japan;

^* corresponding author, ipshinya{at}dent.osaka-u.ac.jp

	ABSTRACT

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Lymphocytes in peripheral blood do not express CD13 (aminopeptidase N), a membrane alanyl metallopeptidase. However, it has been demonstrated that locally infiltrated lymphocytes in chronic inflammatory sites can be CD13-positive, and possible involvement of stromal cell adherence in the induction of CD13 has been suggested. In this study, we examined whether T-lymphocyte/gingival-fibroblast interaction can activate T-lymphocytes to express CD13. CD13 expression was induced on PMA-activated T-lymphocytes only when they adhered directly to human gingival fibroblasts (HGF) at 2 hrs after the co-culture began, while an increase in the enzyme activity of CD13 was also confirmed in activated T-lymphocytes that had been co-cultured with HGF. Furthermore, CD13-positive T-lymphocytes were detected in inflamed gingival tissues in vivo. Analysis of these results indicates that direct interaction with HGF is essential for the induction of CD13 expression on T-lymphocytes that was also observed in periodontitis lesions.

KEY WORDS: T-lymphocyte • gingival fibroblast • adhesive interaction • CD13

	INTRODUCTION

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Chronic inflammatory reactions are usually characterized by inflammatory cell accumulation in extravascular connective tissue. In inflamed periodontal tissues, inflammatory cell accumulation can be observed histopathologically (Okada et al., 1983), and locally infiltrated cells are located adjacent to periodontal fibroblasts. Thus, it is reasonable to speculate that these immunocompetent cells have an opportunity to interact directly with each other and are mutually affected through heterotypic cell-cell interactions. Our previous studies demonstrated that peripheral blood T-lymphocytes (PBT) activated with phorbol 12-myristate 13-acetate (PMA) acquired an ability to bind human gingival fibroblasts (HGF), and this adherence was mediated by CD44/hyaluronate and LFA-1/ICAM-1, VLA integrins (Murakami et al., 1993a, b, 1994, 1996, 1997). Furthermore, we found that these adhesive interactions stimulated HGF to express cytokine mRNA (Murakami et al., 1999). Analysis of those results suggested that locally infiltrated lymphocytes may also be influenced by cognate interactions with HGF.

CD13 (aminopeptidase N) is a 150-kDa membrane alanyl metallopeptidase found on a variety of cells that is able to split amino acids from the N-terminus, with the exception of proline in the penultimate position (Hanson et al., 1967). Thus, CD13 on leukocytes is supposed to be involved in the degradation of neuropeptides (Giros et al., 1986; Furuhashi et al., 1988; Shimamura et al., 1991; Ahmad et al., 1992) and cytokines (Hoffmann et al., 1993; Kanayama et al., 1995), though its functions remain to be fully elucidated. CD13 has also been implicated in the Ag processing of peptides bound in the groove of MHC class II molecules (Hansen et al., 1993; Larsen et al., 1996), and is known to be involved in the regulation of lymphocyte activation and immune response (Ansorge et al., 1991). Although lymphocytes in peripheral blood do not express CD13, CD13-positive T-lymphocytes have been observed in chronic inflammatory sites (Riemann et al., 1993, 1994), and the possible involvement of stromal cell adherence in the induction of CD13 expression on T-lymphocytes has been suggested (Riemann et al., 1997). In the present study, we examined whether lymphocyte-HGF interaction can activate not only HGF but also T-lymphocytes to express CD13. Furthermore, we confirmed the presence of CD13-positive T-lymphocytes in periodontal lesions in vivo.

	MATERIALS & METHODS

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Human Gingival Fibroblasts
All human subjects participated in this study after providing informed consent to a protocol that was reviewed and approved by the institutional review board of the Osaka University Graduate School of Dentistry. Human gingival fibroblasts (HGF) were obtained from biopsies of healthy gingiva taken from healthy volunteers as previously described (Murakami et al., 1993a). HGF were passed by trypsinization and used for experiments at passages 4-10.

Purification of Peripheral Blood T-lymphocytes
Peripheral blood T-lymphocytes (PBT) were isolated from healthy donors as previously described (Murakami et al., 1993a). The purified PBT utilized in this study contained more than 95% CD3-positive cells.

Antibodies
Mouse monoclonal antibody (mAb) 22A5 (anti-CD13)-biotin conjugate was purchased from Leinco Technologies (Ballwin, MO, USA). Mouse mAb UCHT1 (anti-CD3) labeled with fluorescein isothiocyanate (FITC) was purchased from Pharmingen (San Diego, CA, USA).

Co-culture of PBT with HGF
PBT (1.5 x 10⁶/dish) were cultured in the presence or absence of 0.5 µg/mL phorbol 12-myristate 13-acetate (PMA) for 24 hrs and then added to six-well plates (no. 3516, Corning Inc., Corning, NY, USA) containing a confluent HGF monolayer. In some experiments, the requirement of direct interaction between PBT and HGF was examined by the use of cylindrical wells in which collagen-treated microporous membranes were assembled as previously described (Murakami et al., 1999). The plates were then incubated for the indicated period at 37°C. After the co-culture, PBT were harvested and utilized for the experiments.

RT-PCR Analysis
Total RNA and cDNA were prepared and PCR were performed according to the method of Yanagita et al.(2002). Oligonucleotide PCR primers specific for CD13 and hypoxanthine phosphoribosyl transferase (HPRT) were synthesized at Takara Shuzo Co. Ltd. (Kyoto, Japan). The primers for CD13 were: (sense) 5'-GTC TAC TGC AAC GCT ATC GC-3' and (antisense) 5'-GAT GGA CAC ATG TGG GCA CCT TG-3'. Those for HPRT were: (sense) 5'-CGA GAT GTG ATG AAG GAG ATG GG-3' and (antisense) 5'-GCC TGA CCA AGG AAA GCA AAG TC-3'.

Isolation of Mononuclear Cells from Gingival Tissues
Gingival tissues were obtained from periodontal biopsies taken from periodontitis patients. The gingival tissues were minced and washed extensively in phosphate-buffered saline (PBS), after which infiltrated cells were obtained by the use of an automated disaggregation machine (Medimachine, DAKO, Glostrup, Denmark) according to the manufacturer’s instructions. The harvested cells were washed with HBSS (Sigma Chemical Co., St. Louis, MO, USA) and transferred to RPMI 1640 (Sigma Chemical Co.) supplemented with 10% FCS (HyClone Sterile System, Logan, VT, USA). Mononuclear cells were then isolated by gradient centrifugation on a histopaque gradient (density 1.077 g/mL, Sigma Diagnostic, St. Louis, MO, USA). The whole isolation process described above was performed in 1.5 hrs at 4°C.

Surface Expression and Enzyme Activity of CD13
Cell suspensions were prepared in PBS without phenol red containing 1% BSA and 0.1% sodium azide, and 1 x 10⁶ cells were incubated for 30 min at 4°C with biotin-conjugated 22A5 mAb followed by phycoerythrin (PE)-conjugated streptavidin (SAv) (Pharmingen). In some experiments, single cell suspensions (1 x 10⁶) from gingival tissues were incubated for 30 min at 4°C with FITC-conjugated UCHT1 mAb and/or biotin-conjugated 22A5 mAb, followed by PE-conjugated SAv. After incubation, the cells were washed twice, re-suspended, and analyzed for fluorescence.

To examine CD13 enzyme activity, we prepared cell suspensions of PBT in 50 µL of PBS and incubated 1.5 x 10⁵ cells for 5–10 min at 37°C in a water bath. The enzyme substrate [non-fluorescent Di-(Alanyl)-Rhodamine 110 conjugate] (CellProbe Reagent; Coulter Co., Miami, FL, USA) was then added to each cell sample according to the manufacturer’s instructions. In some experiments, PBT were pre-treated for 1 hr with Bestatin (Sigma Chemical Co.), CD13-specific inhibitor. After incubation, samples were placed on ice for 3–20 min and suspended in 1 mL of PBS. Prepared samples were immediately placed on ice until analysis for fluorescence. Flow cytometric analyses were performed with the use of a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

Immunohistochemical Staining
Human gingival tissue specimens were obtained from patients during periodontal surgery. For fixation of gingival tissue specimens, the specimens were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 3 days, immersed overnight in 2% sucrose in PBS at 4°C, and embedded in OCT compound (Lab-TEK, Naperville, IL, USA), quickly frozen in liquid nitrogen. Serial sections (8 µm thick) were cut and put onto silanized glass slides (Nalge Nunc International Corp., Naperville, IL, USA). The tissue samples were incubated overnight at room temperature with FITC-conjugated UCHT1 mAb and biotin-conjugated 22A5 mAb, followed by PE-conjugated SAv. After incubation, the tissue samples were washed with distilled water, included with PermaFluor (Immunon, Pittsburgh, PA, USA), and analyzed for fluorescence with Axioskop 2 plus (Carl Zeiss, Göttingen, Germany).

Statistical Analysis
To test the statistical significance of the CD13-positive ratio between T-lymphocytes that had infiltrated gingival tissues and freshly isolated PBT, we compared data using Student’s t test.

	RESULTS

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Co-culture with HGF Induced CD13 Expression on PMA-stimulated PBT that were Strongly Bound to HGF.
CD13 expression on PBT was first analyzed by flow cytometry. As expected, both unstimulated and PMA-stimulated PBT did not express CD13 on the surfaces (Figs. 1A, 1B). We then cultured the PBT on HGF monolayers and examined whether CD13 expression was induced on the PBT. As shown in Fig. 1D, the cell-surface expression of CD13 was induced on PMA-stimulated PBT upon adhesion to HGF. In contrast, unstimulated PBT did not express CD13 on the surfaces, even when the cells were co-cultured on the HGF monolayer (Fig. 1C). As shown in Figs. 1E-1J, the expression of CD13 on PMA-stimulated PBT was first detected 2 hrs after the co-culture with HGF began, and it gradually increased over 24 hrs.

View larger version (32K): [in this window] [in a new window]   Figure 1. CD13 surface expression on PMA-stimulated PBT induced by co-culture with HGF. PBT were incubated in the absence (A,C) or presence (B,D) of 0.5 µg/mL PMA for 24 hrs. After being washed, PBT and HGF were cultured for 24 hrs in the same wells of a six-well culture plate without (A,B) or with (C,D) direct contact, as described in MATERIALS & METHODS. (E-J) In another experiment, PMA-stimulated PBT were co-cultured with HGF for the indicated times. PBT were stained with biotin-conjugated 22A5 (anti-CD13) followed by PE-conjugated SAv, and subjected to flow cytometric analysis. Dotted lines represent flow cytometric profiles of PBT with PE-conjugated SAv only as negative control. Shaded histograms represent flow cytometric profiles of PBT stained with 22A5.

View larger version (29K): [in this window] [in a new window]   Figure 2. CD13 expression by PMA-stimulated PBT cultured with HGF but separated by a microporous membrane. (A) Schematic illustration of the chamber system in which cylindrical wells with collagen-treated microporous membranes were assembled and introduced. In this culture system, PBT and HGF were cultured in the same wells of a six-well culture plate without direct contact, as described in MATERIALS & METHODS. PMA-stimulated PBT were cultured on HGF (B) or on porous membrane (C) for 24 hrs. PBT were stained with biotin-conjugated 22A5 (anti-CD13) followed by PE-conjugated SAv, and then subjected to flow cytometric analysis. Dotted lines represent flow cytometric profiles of PBT with PE-conjugated SAv only as negative control. Shaded histograms represent flow cytometric profiles of PBT stained with 22A5. (D) Gene expression of CD13. PMA-stimulated PBT were cultured on plate, or on either HGF monolayer or porous membrane, for 2 hrs in the same well as shown in (A). Total RNA was prepared from each cultured PBT, and cDNA syntheses by RT and PCR were performed as described in MATERIALS & METHODS. The results were a representative profile of three independent experiments.

View larger version (17K): [in this window] [in a new window]   Figure 3. Direct interaction with HGF increased enzyme activity of CD13 on PBT. PBT were incubated in the absence or presence of 0.5 µg/mL PMA for 24 hrs. After being washed, unstimulated PBT alone were incubated for 24 hrs, or PMA-stimulated PBT were incubated without (A) or with (B-D) HGF for 24 hrs. After incubation, PBT were harvested and cultured for 1 hr in the absence (A,B) or presence (C,D) of Bestatin (1 µg/mL or 10 µg/mL). The enzyme activity of CD13 on PBT was measured by means of CellProbe as described in MATERIALS & METHODS. Dotted lines, thin lines, and shaded histograms represent the flow cytometric profiles of CD13 enzyme activity on unstimulated PBT alone, PMA-stimulated PBT alone, and PMA-stimulated PBT co-cultured with HGF, respectively.

View larger version (79K): [in this window] [in a new window]   Figure 4. Two-color flow cytometric (A) and immunohistochemical (B-D) analyses of CD13+CD3+ cells that had infiltrated the gingival tissues of periodontitis patients. (A) Single-cell suspensions from gingival tissues and peripheral blood mononuclear cells of 10 periodontitis patients were stained with FITC-conjugated UCHT-1 (anti-CD3) and/or biotin-conjugated 22A5 (anti-CD13) followed by PE-conjugated SAv, and then subjected to flow cytometric analysis. Results are shown as Mean ± SD of percentage of CD13+CD3+/CD3+ cells in each sample. *Significantly different between gingival tissues and peripheral blood (p < 0.01 by Student’s t test). (B-D) Sections of gingival tissues from periodontitis patients were analyzed by immunohistochemistry as described in MATERIALS & METHODS. Sections were stained with FITC-conjugated UCHT-1 (anti-CD3) (B) and biotin-conjugated 22A5 (anti-CD13) followed by PE-conjugated SAv (C). The combined image of panels (B) and (C) is shown in panel (D). CD13+CD3+ cells are arrowed.

	DISCUSSION

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Interestingly, it was demonstrated that direct interaction between lymphocytes and HGF stimulated inflammatory cytokine mRNA expression in the HGF (Murakami et al., 1999) and that the lymphocytes were prevented from dying and leaving inflamed tissue by an inappropriate expression of pro-survival chemokines (IFN-ß) and pro-retention chemokines (SDF-1) by fibroblasts within the chronically inflamed tissue (Buckley et al., 2001). In the present study, we demonstrated that cognate-type interactions between HGF and PBT stimulated the PBT to induce CD13 expression on their surfaces. These results suggest that the adhesive interactions between lymphocytes and HGF have a mutual influence on the functional activities of each cell type (Murakami and Okada, 1997; Murakami et al., 1997).

In the present study, CD13 was detected on the cell surfaces of locally infiltrated T-lymphocytes in inflamed gingival tissues, but not on those of PBT isolated from the same donors (Fig. 4A). This strongly suggests that locally infiltrated T-lymphocytes have an opportunity to interact directly with neighboring HGF. Likewise, Riemann et al.(1993) examined CD13 surface expression on T-lymphocytes in synovial fluid and peripheral blood from patients suffering from juvenile chronic arthritis or rheumatoid arthritis, and reported that CD13 expression in T-lymphocytes was found only in synovial fluid from patients with chronic arthritis. Those and our results suggest that CD13-positive T-lymphocytes play some role in the formation of locally chronic inflammatory diseases.

With regard to the function of CD13, lymphocytic expression of CD13 represents a potentially increased cellular ability to inactivate inflammatory mediators. Furthermore, CD13 may be involved in degradation of the extracellular matrix during lymphocytic migration. As for T-lymphocytes, it was reported that the CD13 inhibitors probestin and actinonin reduced the proliferation of a CD13-positive T-cell line, KARPAS-299. Moreover, a study aimed at the identification of potential targets that mediate the anti-proliferative effects of CD13 expression and activity revealed a modulation of MAP kinase p42/Erk2 activity and mRNA levels in the same human T-cell line by probestin and actinonin (Lendeckel et al., 1998). This suggests that the enzyme activity of CD13 on T-lymphocytes may be related to proliferative responses of the T-lymphocytes. Recently, Mishima et al.(2002) indicated that the high level of CD13 expression on the surfaces of lymphoid cells could be important for their survival. Interestingly, we also found that T-lymphocytes survived over a long period of time when the cells were co-cultured with HGF (data not shown). Another important function of CD13 is to play a role in antigen processing by the trimming of peptides on the cell surface that protrude from MHC class II molecules. We found that the cell-surface expression of MHC class II molecules was also induced on PMA-stimulated PBT upon adhesion to HGF (data not shown). Taken together, these findings suggest that locally infiltrated T-lymphocytes express CD13 on the cell surface through direct adherence with HGF and survive over a long period of time, while locally infiltrated CD13-positive T-lymphocytes may act as effective antigen-presenting cells in inflamed gingival tissues.

The present findings—that direct interactions with HGF are essential for the induction of CD13 expression on PBT in vitro, and that CD13 is expressed on the cell surfaces of locally infiltrated T-lymphocytes in gingival tissues in vivo—strongly suggest that T-lymphocytes can be activated through direct adhesion with HGF in chronic periodontal lesions. Additional studies to examine the pathophysiological roles of CD13-positive T-lymphocytes will be necessary for a better understanding of the immunopathogenesis of periodontitis.

	ACKNOWLEDGMENTS

 
We thank Dr. K. Ueda for his technical assistance and helpful discussion. This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS) (Nos. 13557190, 13672185, and 15592186) and by the 21st Century COE Program from JSPS.

Received February 21, 2003; Last revision August 11, 2003; Accepted August 27, 2003

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