Jpn. J. Oral Biol., 36 : 222-229, 1994 .

ORIGINAL

Incorporation of instilled Candida albicans and a lipopolysaccharide

into the palatine tonsil of rabbit

Takaki Fukuizumi, Hirornasa Inoue, Yuichi Anzai,

Kyoko Hase and Choji Uchiyama

Department of Oral Bacteriology, Kyushu Dental College,

(Cheif: Choji Uchiyama)

2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803, Japan

•kAccepted for publication : October 15, 1993•l

Key words: palatine tonsil/deep cervical lymph nodes/ Candida albicans/lipopolysaccharides

Abstract: The palatine tonsil is thought to be the organ which accepts antigens to initiate an immunological

response, but the incorporation of the antigens from the oral cavity was not yet known . To show thisincorporation, fluorescence-labeled Candida albicans and a lipopolysaccharide were instilled around the

rabbit palatine tonsil. The distribution of fluorescence was examined in frozen sections of the tonsil after 30 ,60 and 180 minutes of the instillation. Candida albicans and a lipopolysaccharide were incorporated into the

cryptoepitherial tissue of the palatine tonsil within 30 minutes. The antigens in the epithelium were transpor-

ted to the intratonsillar follicles and partly to the deep cervical lymph nodes after 60 minutes . Thelipopolysaccharide was found intrafollicular by in both the tonsil and the deep cervical lymph nodes earlier

than Candida albicans was. Because the normal cryptoepithelium easily passed through the external antigens

which were transported to the neighboring lymphoid follicles, it was suggested that the cryptoepithelium was

the entrance of the immunological response in the palatine tonsil and neighboring lymphoid tissues in the

normal condition.

抄録:健 全状態 の 口蓋扁 桃 にお け る抗 原曝露 直後 の その侵入動 態 を検 索す るた め,Candida albicans(C.al-

bicans)お よびlipopolysaccharide(LPS)を ウサ ギ 口蓋 扁桃周 囲 に滴下投 与 した。 これ らの 分布 を30,60,180

分 後 と経 時的 に検 索 した結 果,C.albicansとLPSは30分 以 内に 口蓋 扁桃 の陰 窩上皮 部 に取 り込 まれ た。さ らに,

上皮 内 のそ れ らの抗 原 は,60分 後 には扁桃 濾胞 内 に認 め られ,一 部 は頚 部 リンパ 節 に も移 動 した。また,LPSは,

口蓋 扁桃 ・深頚 リンパ節 の いず れに おい て も,C.albicansよ りも速や か に濾 胞間領 域 お よび濾胞 内 に認 め られ た。

したが って,元 来,陰 窩 上皮 は容 易 に外来 性抗 原 を通 過 させ,抗 原情 報 を収 集 しや す くな ってお り,口 腔 領域 に

お け る免疫 応答 の初 期段 階 に深 く関与 す る重要 な機能 を有 してい るこ とが 示唆 され た。

Introduction

The palatine tonsil is one of the infra-epithelial

lymphatic tissues which directly contacts external

antigens through the epithelium1•`3). The infra-epith-

elial lymphatic tissues include the appendix and the

Peyer's patches, which are called gut-associated

lymphoid tissues (GALT). It was believed thàt GALT

incorporated antigens and the lymphocytes in GALT

were transported to the intestinal mucosa and

produced antibodies which were secreted from the

mucosa and from remote exocrine glands4) . Similar toGALT, the tonsil may incorporate antigens to initiate

an immunological response.

Although it was speculated some information on

T. Fukuizumi, et al.: Incorporation of antigen into the tonsil 223

A

antigens in the intra-oral cavity was caught in the

palatine tonsil and then transported to the other sys-

temic lymph tissues through the lymph vessels, the

details of this were not yet clear5). So far, many

reports have showed that in the inflamed tonsil, sol-

uble antigens pass through the epithelium, which is

reticulated because of inflammation, in a short

time6•`13). For the granular antigens, such as pyogenic

bacteria, one day or more was required to invade the

tonsillar tissue14•`18). One day may be enough to cause

the inflammation in the epithelial tissue which became

reticulated. Thus, we do not know whether antigens in

the oral cavity may access freely the normal tonsillar

tissue and whether there are any differences in the

distribution of antigens differing in size.

In the present experiment, we chose Candida al-

bicans (C. albicans) as one of the biggest antigens

among the microbes existing in the oral cavity, and a

lipopolysaccharide (LPS) as a bacterial small compo-

nent. These were instilled around the palatine tonsil,

and we examined the distribution in the tissue section

B

Fig.1 Fluorescence microscopic observation of the

thin section of the tonsil after 30 minutes of

the application of C. albicans.

A. Cryptoepitherial layer (•~100)

Many yeast form of C. albicans were observed in the

lymph-epithelial symbiotic part and infra-epith-

eliocortical layer.•¬=fluorescence

B. Infra-epitheliocortical layer (•~400)

Many phagocytes which engulfed C. albicans were

observed in the infraepitheliocortical layer.

at the appropriate times. We found that C. albicans

and LPS passed through the tissue very easily and

some information of the antigens may be presenting in

the tonsil and in neighboring lymph nodes.

Materials and methods

1. Rabbits

The animals used in the experiments were 8 weeks

old male Japanese albino rabbits, weighing from 1.0 to

1.2kg.

2. Preparation of antigens

C. albicans ATCC 18804 successively preserved in

our laboratory were cultured in brain heart infusion

broth (Difco Lab.) at 37•Ž for 18 hours, washed two

times with sterilized saline solution and centrifuged

(1,200•~g, 20 min). The cells were labeled with PKH

26 (fluorescent staining kit, Zynaxis)19) and suspended

in sterilized saline (1•~107 cells/ml).

Fluorescein isothiocyanate (FITC) labeled LPS

224 Jpn. J. Oral Biol., 36: 222-229, 1994.

from Escherichia coli (serotype 0111: B4) (Sigma)

was added with sterilized saline to adjust to 10ƒÊg/ml,

and was sterilized by filtration.

3. Antigens application

FITC-labeled LPS and PKH 26-labeled C. albicans

(0.5 ml each) were instilled around the rabbit palatine

tonsil under intravenous anesthesia with sodium

pentobarbital. At 30, 60 and 180 minutes after the

instillation, each rabbit was killed painlessly by exces-

sive intravenous administration of sodium pentobar-

bital. The palatine tonsil and the deep cervical lymph

nodes were excised, embedded in CRYO-M-BED

(BRIGHT Instrument) and rapidly frozen. The frozen

tissues were sectioned at 8 and 30ƒÊm in thickness.

4. Observation with fluorescence

The frozen sections were fixed with 2.5% glutaral-

dehyde for 5 minutes, washed two times with distilled

water, and air-dried at room temperature in the dark.

The sections were mounted with 90% glycerol in

phosphate buffered saline (PBS) containing 1% p-

phenylendiamine. The distribution of fluorescence-

labeled antigens was examined with a fluorescent

microscopic apparatus (New Vanox AH2-FL,

Olympus) and a confocal type of scanning laser-bio-

logical microscopy system (LSM-GB 200, Olympus).

Results

1. Candida albicans application

Thirty minutes after the application of C. albicans,

labeled yeasts were observed at the lymph-epithelial

symbiotic part in contact with the cryptocavity and in

the deep area of the symbiotic part of the palatine

tonsil (Fig. 1-A). The result showed that the C. al-

bicans passed through the epithelial tissue within 30

minutes. Many labeled C. albicans were engulfed by

the cells at the same site (Fig. 1-B). It was suggested

that many phagocytes as macrophages were located

in the epithelial tissue and phagocytosed the micro-

organisms in the normal condition.

Sixty minutes after the application of C. albicans,

the distribution of the yeasts in a part of the lymph-

epithelial symbiotic part and the infraepitheliocortical

Fig.2 Fluorescence microscopic observation of the

thin section of the tonsil after 60 minutes of

the applicatiton of C. albicans (•~200).

Fluorescence were observed in the cells in the cap

area surrounding the lymphoid follicles. •¬=fluo-

rescence

layer was similar to that of 30 minutes after the

application of C. albicans. Labeled yeasts were also

found in the part of the cap area at the epithelial side

surrounding the follicles (Fig. 2).

One hundred and eighty minutes after the applica-

tion of C. albicans, a few yeasts of free C. albicans

was detected in the palatine tonsil, and most labeled

yeasts were detected in the cells widely distributed in

the deep cortical layer (Fig. 3-A). Some of the cells

engulfing the C. albicans were localized in the part of

cortex laying around the follicles, and were seen in the

area of the germinal center (Fig. 3-B). Furthermore,

the cells engulfing the C. albicans were seen in the

afferent lymphatics around the deep cervical lymph

nodes (Fig. 3-C).

T. Fukuizumi, et al.: Incorporation of antigen into the tonsil 225

A

B

2. LPS application

30 minutes after LPS application, most of the fluo-

rescence was found in the cells which were widely

distributed in the cortical layer including the lymphoid

follicles of the palatine tonsil. Some amorphous fluo-

rescence was seen in part of the area between the

connective tissue surrounding the capsule and the

efferent lymphatics (Fig. 4).

60 minutes after the application of LPS, the distri-

bution of LPS in the palatine tonsil was similar to that

after 30 minutes of the application of LPS. In the deep

cervical lymph nodes, the cells containing fluores-

cence were found in the afferent lymphatics and

marginal sinus (Fig. 5).

180 minutes after the application of LPS, the cells

containing fluorescence were found in and out of the

lymphoid follicles, and in the germinal center of the

palatine tonsil. Fluorescence in the connective tissue

C

Fig. 3 Fluorescence microscopic observation of the thin

section of the tonsil after 180 minutes of the applica-

tion of C. albicans.

A. Cortical layer (non-confocal view,•~40)

Fungal cells and phagocytes which engulfed fungal cells

were widely distributed in the cortical layer and surrounding

area of the lymphoid follicles.

B. Surrounding area of the lymphoid follicles (confocal view

of X-Y scanning,•~100)

Part of phagocytes which engulfed C. albicans surrounding

the lymphoid follicles were observed in the germinal center.

Bar=50ƒÊm.

C. Afferent lymphatics around the deep cervical lymph

nodes (confocal view of vertical projection [integrated view

of X-Y scanning sections],•~400)

layer was greater than that after 30 and 60 minutes of

the application of LPS (Fig. 6-A). In the deep cervical

lymph nodes, the cells including fluorescence were

widely distributed in the cortical layer including the

lymphoid follicles (Fig. 6-B).

Discussion

We showed here first that soluble antigens were

incorporated into the normal tonsillar tissue. In the

LPS application, the fluorencesce incorporated in to

cells which moved in the tonsile in a similar fashion to

that in the C. albicans application.

Although several kinds of proteins incorporated

into the palatine tonsil6,9•`11,20), these reports were

based on the data of the tonsil after sensitization in

rabbits or on the data of human with inflammation of

the tonsil7,8). In the cases of habitual tonsillitis, it was

226 Jpn. J. Oral Biol.,. 36: 222-229, 1994.

Fig. 4 Fluorescence microscopic observation of the

thin section of the tonsil after 30 minutes of

LPS application (confocal view of vertical

projection [integrated view of X-Y scanning

sections],•~100).

Phagocytes which engulfed LPS and free LPS were

widely distributed in the cortical layer containing

the lymphoid follicles, and LPS in the intercellular

space in the connective tissue around the capsule.

Bar=50ƒÊm. •¬=fluorescence

shown that antigens easily passed through the

cryptoepithelium because of the progress of the

reticular formation of the epithelium12).

We showed that C. albicans incorporated into the

tonsil within 30 minutes. After the instillation of

Staphylococcus aureus at the same site 2 or 3 times a

day14) or after the topical application of india ink by

brush to the palatine tonsil surface21), the granular

antigens passed through the epithelial tissue in one

day and reached to a wide area between the infrae-

pithelium and the capsule. Although Hatano et al. and

Haraguchi et al. did not report the incorporation

Fig. 5 Fluorescence microscopic observation of the

thin section of the tonsil after 60 minutes of

LPS application (•~100).

Phagocytes which engulfed LPS were detected in

the afferent lymphatics and the marginal sinus of

the deep cervical lymph nodes. •¬=fluorescence

within one day, the bacterial cells possibly did within

30 minutes. Because the diameter of C. albicans was

several times greater than that of Staphylococcus

aureus and india ink, the incorporation of the bacteria

seems to be casier than that of the yeasts. Applying

Streptococcus pyogenes by the spraying around the

palatine tonsil, the bacteria adhered, but did not

invade the cryptoepithelium15•`18). It seemed that the

incorporation ratio of bacterial cells to the tonsil

depended on cell density of bacterial cells at the crypt

of the tonsil, rather than on the species specificity.

Application by instillation or topical application by

brush will provide a higher cell density in small area

than that by spray. The present result shows that the

cryptoepithelium has a structure passing through a

large antigen nearly 3•`5ƒÊm in diameter like the

yeast form of C. albicans.

At the lymph-epithelial symbiotic part , a number of

microcrypts were founds and were classified into

T. Fukuizumi, et al.: Incorporation of antigen into the tonsil 227

A

three types18,22•`24). One type of microcrypt named MC-

3 has a similar structure to M-cells which incorporate

antigens into intestinal mucosa. The other two types

are called MC-1 and MC-2 and they have small pores.

It is likely that the antigens instilled around the

palatine tonsil passed through the cryptoepithelium

from one of three microcrypts to the tonsillar paren-

chyma.

We showed that the site of fluorescence shifted

from the epithelial side to the cortical area, the sur-

roundings of lymphoid follicles, and the germinal

center with the time after application of C. albicans. It

was suggested that the phagocytes moved to these

tissues after phagocytosing the yeasts. Since the

major phagocytes which appeared in the tonsillar

epithelium and the cortical layer were macropha-

ges25,26), some macrophages must engulf bacterial

cells.

There are two trasferring pathways of antigens

from the palatine tonsil to the deep cervical lymph

nodes. The first pathway is through lymphatics and

the second one is through blood vessels. It was shown

B

Fig.6 Fluorescence microscopic observation of the thin sec-

tion of the tonsil after 180 minutes of LPS application.

A. Arounds the capsule and efferent lymphatics (confocal

view of vertical projection [integrated view of X-Y scanning

sections],•~200)

LPS was noted in the connective tissue.•¬=fluorescence

B. Cortical layer of the deep cervical lymph nodes (•~100)

Phagocytes which engulfed LPS and free LPS were widely

observed in the cortical layer containing the follicles of the

deep cervical lymph nodes.

that some pigments which were injected into the

lymph vessels of the human palatine tonsil moved to

the middle-deep cervical lymph nodes through the

efferent lymphatics surrounding the capsule27•`31). On

the other hand, it was reported that horseradish per-

oxidase reached the deep cervical lymph nodes by the

stream of blood circulating through the high endoth-

elial venules9). In the present study, phagocytes were

first seen in lymph sinuses distributed in the interfol-

licular area13) of the tonsil and then in the afferent

lymphatics of the deep cervical lymph nodes in both C .

albicans and LPS application. The result showed that

the antigens incorporating into the tonsil were trans-

ferred through the efferent lymphatics to the deep

cervical lymph nodes by phagocytes.

In sensitized tonsil, antibody producing cells and

specific antibodies were detected in the mixed tonsil-

lar cell culture with splenic cells32) and in the tonsillar

cell culture. In the sensitized tonsils, by topical appli-

cation and intra-cryptocavitary infusion, specific anti-

bodies were produced in all cases6,9•`11,20,33).

We supposed that some antigen information should

228 Jpn. J. Oral Biol., 36: 222-229, 1994.

be presented to T cells which helped B cells to pro-

duce the antibodies. This proposal was made from the

following observations. First, the incorporated

antigens were phagocytosed efficiently. We suggest

the major phagocytes were macrophages which were

one of the antigen presenting cells. Second,

phagocytes moved around the follicles, especially

localized in the part of the cap area at the epithelial

side of the follicles. It is believed that antigen present-

ing cells transmit the antigen information at the cap

area where the dominant cells are T cells, which are

believed to be special cells which accept information

from antigen presenting cells. Third, a few antigen

bearing cells moved to the germinal center, where B

cells proliferate and become antibody producing cells.

We did not show the trasfer of the information and

the intensity of the stimuli to initate the im-

munological response from the present study. The

results show that many phagocytes were located at

the area surrounding the follicles, and supports the

idea that a single instillation is enough to sensitize the

tonsillar cells to become antibody producing cells.

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