In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme-labeled antigen method

Abstract

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme-labeled antigen method was applied to detecting plasma cells producing P. gingivalis-specific antibodies in biopsied gingival tissue of periodontitis. N-terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro and Lys-hgp) were prepared by the cell-free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme-labeled antigen method on paraformaldehyde-fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme-labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.

Keywords: AlphaScreen method; antigen 53; gingipain; wheat germ cell free protein synthesis.

Figure 1

Schematic illustration of the AlphaScreen method for detecting a biotinylated protein–antibody interaction. The streptavidin-coated donor beads interact with the biotinylated target protein. The protein G-coated acceptor beads interact with the target protein via the specific antibodies. When the 680 nm excitation light is given, the donor beads generate singlet oxygens. The singlet oxygens promote the acceptor beads to emit luminescent light at 520–620 nm. This reaction occurs only in the presence of the specific antibodies in the solution, since the antigen–antibody reaction shortens the distance between both beads within 200 nm (top panel). No luminescence is seen without specific antibodies (bottom panel).

Figure 2

Electrophoretic analysis of biotinylated proteins without sugar moieties used in the present study. Protein bands showing appropriate molecular weights are visualized with the Western blot analysis using streptavidin-labeled Alexa Fluor 488. The estimated molecular weights of the proteins are: Ag53 = 53 kDa (lane 1), Arg-hgp = 103 kDa (lane 2), Lys-hgp = 103 kDa, (lane 3), Arg-pro = 44 kDa (lane 4), Lys-pro = 51 kDa (lane 5), and SpaP = 185 kDa (lane 6). The band of Arg-pro is relatively weak (arrowhead). Extra bands are also observed in each lane. M, molecular weight markers.

Figure 3

Plasma cells in consecutive frozen sections of the biopsied gingiva. Two representative lesions of gingivitis demonstrate dense (top panels: case 1) and moderate (bottom panels: case 9) infiltration of plasma cells beneath the squamous lining. Left panels: hematoxylin & eosin staining, right panels: immunostaining for CD138. Not only plasma cells located in the subepithelial layer but also covering squamous epithelial cells reveal membrane positivity for CD138. Bar indicates 50 μm.

Figure 4

The enzyme-labeled antigen method visualizing plasma cells producing antibodies reactive to Porphyromonas gingivalis proteins in proteinase K-pretreated, prefixed frozen sections of the biopsied gingiva. Arrowheads indicate plasma cells showing cytoplasmic labeling with the biotinylated proteins. The panels demonstrate anti-Ag53 antibodies, anti-Arg-hgp antibodies, anti-Lys-hgp antibodies, and anti-Arg-pro antibodies in case 2, and anti-Lys-pro antibodies in case 1. The plasma cells producing the pathogen-reactive antibodies are dispersed in the inflammatory stroma. Bar indicates 50 μm.

Figure 5

Absorption experiment using unlabeled Ag53, Arg-pro and Lys-pro. Anti-Ag53 reactivity in case 2, anti-Arg-pro reactivity in case 9, and anti-Lys-pro reactivity in case 1 are shown. Positive signals against the respective proteins (left panels) were abolished after absorption with an excess amount of the corresponding unlabeled proteins (right panels). Arrowheads indicate plasma cells producing the specific antibodies. Bar indicates 50 μm.

Figure 6

Absorption experiment using unlabeled Arg-hgp and Lys-hgp. The enzyme-labeled antigen method for Arg-hgp reactivity and Lys-hgp reactivity on consecutive frozen sections in case 1 is illustrated. The reactivities to Arg-hgp and Lys-hgp in the cytoplasm of plasma cells (left panels) are abolished with an excess amount of the corresponding unlabeled proteins (center top and right bottom panels), and partly eliminated with unlabeled Lys-hgp and Arg-hgp, respectively (center bottom and right top panels). Arrowheads indicate plasma cells producing antibodies reactive to the unique epitope on Arg-hgp or Lys-hgp. Bar indicates 50 μm.

Figure 7

Double immunofluorescence labeling with the biotinylated protein and a mouse monoclonal antibody to CD79a. Anti-Ag53 reactivity in case 2 and anti-Arg-pro reactivity in case 9 are shown. Cytoplasmic reactivities against the Porphyromonas gingivalis proteins are labeled green with Alexa Fluor 488 (left panels). CD79a along the plasma membrane and in the cytoplasm of B lymphocytes, including plasma cells, is stained red with Alexa Fluor 568 (center panels). Arrowheads indicate plasma cells producing P. gingivalis-reactive antibodies, fluoresced in yellow (right panels, the merged figure). Bar indicates 50 μm.