Galioto GB (ed): Tonsils: A Clinically Oriented Update. Adv Otorhinolaryngol. Basel, Karger, 1992, vol 47, pp 16-20

Functional Morphology of Tonsillar Germinal Center I. Kawabata a, M . Nakamura b , H. Tabe a a Department

of Otolaryngology, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama, and bDepartment of Otolaryngology, Faculty of Medicine, Hongo Tokyo, University of Tokyo, Japan

The purpose of the present studies was to determine proliferation and migration of lymphocytes in tonsillar tissue using immunohistochemical methods. The germinal center and crypt epithelium are thought to be the morphological unit of tonsillar function. The outcoming antigens are captured in crypt epithelium, and immunoinformation is transferred to the germinal center, where antibodies are produced. The lymphocytes in the germinal center play an important role in the production of antibodies. Therefore, it is very important to know the cell proliferation and migration of lymphocytes in the germinal center for immunoactivity of the tonsil. Since Koburg [1] in 1963 reported the cell proliferation and migration oflymphocytes in the germinal center utilizing thymidine- 3R , several studies have been done. Recently the immunohistochemical method using anti-bromodeoxyuridine (BrdU, thymidine analogue) monoclonal antibodies has been tried extensively in various fields of medicine [2,3]. Using this method, we have attempted to detect the cell dynamics oflymphocytes in the germinal center. We report the results of both rabbit in vivo studies and human in vitro studies. Material and Methods

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In vivo Experiments on Rabbit Tonsil Healthy white rabbits weighing 2.0-2.5 kg were used in this study. The animals received BrdU (Sigma Chemical Co.), 60 mg/kg body weight, as a simple bolus injection administered intravenously. The animals were sacrificed and the tonsils were removed at

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15 min, 2 h, 14 h, and 24 h after injection respectively. Tonsils were fixed in Bouin's fluid, dehydrated, embedded in paraffin and sectioned into 5-llm thick slices. The specimens were incubated with the anti-BrdU monoclonal antibody for I h at room temperature, washed with PBS, and then incubated again with the anti-mouse IgG peroxidase conjugated for 30 min. The specimens were suspended in PB containing DAB to be stained at the site of BrdU incorporation.

In vitro Experiments on Human Tonsil Human tonsils taken at tonsillectomy were used for in vitro experiments. The small pieces of tonsillar tissue were incubated in artificial blood (FC-43) containing 0.5 mM BrdU at 37°C for 30 min under 3 atm pressure of mixed gases. The succeeding procedures were the same as those mentioned above.

Results Distribution of BrdU-Incorporated Cells in Tonsil The BrdU-incorporated cells were stained blue-black in the nucleus, and easily distinguished from the other ones. They were located in the basal layer of epithelium of the pharyngeal site, and were scattered mainly in the germinal center, however few in the crypt epithelium. The BrdUincorporated cells in the germinal center seemed to be the large or medium-sized lymphocytes in their appearance.

The Existence of Germinal Center without BrdU-Incorporated Cells It is well known that rabbit tonsil has 30-60 germinal centers. Whole germinal centers in one tonsil were examined by serial section to determine

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Localization of BrdU-Incorporated Cells in Germinal Center The distribution of BrdU-incorporated cells in the germinal center was changed with increasing time after administration ofBrdU. The nuclei of the large or medium-sized lymphocytes in the germinal center were already stained blue-black 15 min after injection of BrdU (fig. 1). The majority of those cells were found in the posterior portion (basal part) of the germinal center. With increasing time, BrdU-incorporated cells appeared in the central and anterior part (near the dark zone) (fig. 2). 14 h after injection, they reached the anterior portion (near the dark zone) (fig. 3). Most of them disappeared within 24 h after injection. Several cells were found in the parafollicular area and crypt epithelium (fig. 4). BrdU-incorporated cells were hardly found in the dark zone. Figure 5 shows the schematic drawing of migration of BrdU-incorporated cells in the germinal center.

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Fig. 1-4. Migration of BrdU-incorporated cells in the germinal center. la, 2a, 3a, and 4a show the localization of BrdU -incorporated cells at 15 min, 2 h, 14 h, and 24 h after BrdU injection respectively. 1b is an adjacent HE-stained section of la, as well as 2b, 3b, and 4b.

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6b Fig. 5. Schematic drawing of migration of BrdU-incorporated cells in the germinal Fig. 6. a Existence of the germinal center without BrdU-incorporated cells. b An adjacent HE-stained section. Fig. 7. a The distribution of BrdU-incorporated cells in human tonsil. b An adjacent HE-stained section.

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center.

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whether or not all germinal centers have a similar reaction to that mentioned above. We found that 20-30% of the germinal centers did not have any BrdU-incorporated cells (fig. 6).

In vitro Experiments on Human Tonsil In vitro experiments on human tonsil, BrdU-incorporated cells were found mainly in the germinal center and pharyngeal epithelium. In the germinal center they seemed to be the large or medium-sized lymphocytes and were distributed in the posterior portion as in the findings of in vivo experiments (fig. 7). This finding suggested that the posterior portion of the germinal center in human tonsil is the site of cell proliferation of lymphocytes.

Conclusion To conclude: (1) the immunohistochemical methods utilizing antiBrdU monoclonal antibodies were used to examine the cell dynamics of lymphocytes in the tonsillar tissue; (2) the germinal centers in the tonsil have a higher cell proliferation oflymphocytes, and a considerable number of lymphocytes migrate from the posterior to the anterior portion of the germinal center, and (3) the germinal centers without BrdU-incorporated cells are found.

References

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Koburg E: Zur Lymphocytopoese in den Tonsillen. Arch Ohren Nasen Kehlkopfheilkd 1965; 185:785. Gratzner HG: Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 1982;218:474. Shutte B, et al: Effect of tissue fixation on anti-bromodeoxyuridine immunohistochemistry. J Histochem Cytochem 1987;35: 1343.

Dr. I. Kawabata, Department of Otolaryngology, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama 350 (Japan)

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Functional morphology of tonsillar germinal center.

Galioto GB (ed): Tonsils: A Clinically Oriented Update. Adv Otorhinolaryngol. Basel, Karger, 1992, vol 47, pp 16-20 Functional Morphology of Tonsilla...
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