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Induction of CD13 on T-lymphocytes by Adhesive Interaction with Gingival Fibroblasts
T. Saho,
T. Kishida,
H. Hirano,
T. Hashikawa,
Y. Shimabukuro and
S. Murakami*
Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka Suita, Osaka 565-0871, Japan;
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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.
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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.
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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.
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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.
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Journal of Dental Research, Vol. 82, No. 11,
893-898 (2003)
DOI: 10.1177/154405910308201109
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