Structural and Immunological Characteristics of Chronically Inflamed Adenotonsillar Tissue in Childhood

Recurrent or chronic adenotonsillar infections mainly affectchildren and frequently involve otherwise healthy subjects.Therefore, having excluded systemic immunological deficiencies,this disease may be due to a local dysfunction of the epithelialstructures at either the rhino or oropharyngeal level. The aimof the present investigation was to analyze structural and immunologicalaspects of tonsils and adenoids in subjects who underwent adenotonsillectomybecause of recurrent inflammatory episodes with fever. Histologicalstudies and analyses of the cytokine patterns were carried outin palatine tonsils and adenoid samples from 105 patients whounderwent adenoidectomy and bilateral extracapsular tonsillectomyfor chronic inflammatory hypertrophy of these organs; 46 ofthe 105 cases examined presented hyperkeratosis of the cryptepithelium; in the remaining 59, the epithelium was hyperplasticwith no signs of keratosis. Scanning electron microscopy revealeda continuous epithelial surface of polygon-shaped flattenedcells with fissures towards the cryptic depressions. Titrationof interleukin-1ß and tumor necrosis factor alphain serum and tissues demonstrated higher concentrations in theadenotonsillar specimens, whereas the rise in interleukin-6was more modest.

INTRODUCTION

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Introduction

There are still some controversies among specialists in internalmedicine, pediatrics, and otorhinolaryngologists concerningthe diagnostic and therapeutic approach to recurrent and chronicinflammatory conditions of adenoids and tonsils in children.

Over the last few decades, immunobiological techniques haveallowed the identification of tonsillar cells responsible forinflammatory immune reactions (2, 5). Recurrent or chronic adenotonsillarinfections mainly affect children and frequently involve otherwisehealthy subjects. Therefore, having excluded systemic immunologicaldeficiencies, this disease may be due to a local dysfunctionof the epithelial structures, at either the rhino or oropharyngeallevel.

Several authors (4, 6, 8) have tried to explain why and howa modification in the balance between the local immunologicalfunction of the host and the infectious agents would lead toa clinical process characterized by recurrent inflammatory events.

The complex histological configuration of the parenchyma isfundamental for the uptake and presentation of antigens to thesubepithelial immunocompetent cells. This allows the whole organto act as a functional unit and hence to play an important rolein fighting microorganisms. It may therefore be hypothesizedthat persistent local inflammatory reactions in adenotonsillitismay, with time, lead to histomorphological changes and functionaldeficiencies in defense barriers (3).

The aim of the present investigation was to analyze structuraland immunological aspects of tonsils and adenoids in subjectswho underwent adenotonsillectomy because of recurrent inflammatoryepisodes with fever.

MATERIALS AND METHODS

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Materials and Methods

Patients. We selected 105 children affected by chronic inflammatory hypertrophyof palatine tonsils and adenoids which had not responded toprevious medical treatments; 54 males and 51 females, aged between4 and 18 years, were enrolled in the study.

All patients underwent adenoidectomy and bilateral extracapsulartonsillectomy, under general anesthesia. Signed written consentwas obtained from all patients.

Histological investigations. Each tonsil was divided into three parts. These were preparedfor study under the light microscope as well as the scanningelectron microscope and for immunohistochemical studies.

Light microscopy. Specimens were fixed in 10% buffered formalin (phosphate bufferaccording to Lillie), at room temperature, for 24 h, and thenfixed in paraffin and processed according to routine techniquesof light microscopy.

Slices 5 to 8 µm thick were stained with hematoxylin andeosin, Azan Mallory trichrome stain, and Gomori silver stain.

Scanning electron microscopy. Specimens were fixed in 2.5% aldehyde glutaraldehyde (in phosphatebuffer according to Sorensen at 4°C) for 48 h.

These were then rinsed in phosphate buffer for 30 min and inrunning water for 90 min, at room temperature. Postfixationwas carried out with OsO₄ (1.33%), for 2 h, with further rinsingin phosphate buffer for 20 min. This was followed by dehydrationin an increasing series of alcohol solutions and critical pointdrying by means of six cycles of 10 min each.

Once metallization in gold had been carried out by the EdwardsSputtering method, the samples were observed under the scanningelectron microscope (Cambridge 250 Stereoscan) at 20 kV.

Immunohistochemistry. The 5-µm sections were processed according to the peroxidase-antiperoxidasemethod for the detection of immunoglobulin E, immunoglobulinA, and J chains. Inhibition of endogenous peroxidase was performedprior to dehydration, for 5 min at room temperature, by meansof a hydrogen peroxide solution at 3% in methanol. Followingdehydration, trypsinization was carried out for 5 min in anaqueous solution of trypsin at 0.1% and calcium chloride at0.4%.

To detect J chains, the sections were subjected to preincubationin a 0.4 M urea solution, at pH 5.2, for 1 h at 4°C. Thiswas followed by immunohistochemical detection by means of theperoxidase-antiperoxidase method, with polyclonal antibodies(rabbit anti-human immunoglobulin A, Ortho code 00460; rabbitanti-human immunoglobulin G, Ortho code 00461; and J chain,Ortho code 00453).

Nuclear contrast staining was then carried out with Mayer hematoxylinfor 2 min. The samples were then mounted in an aqueous mediumand observed under a Zeiss FO-M12 photomicroscope.

Cytokine and immunoglobulin detection. In a randomly selected group of 10 children, 1 g of adenoidand 1 g of tonsil tissue, homogenized in 2 ml of physiologicalsolution, were centrifuged at 3,000 rpm, for 10 min.

Several kinds of immunoglobulins were assayed simultaneouslywith a simple radial immunodiffusion technique (LC-Partigen,Boehring, Germany).

Levels of interleukin-1ß, interleukin-6, and tumornecrosis factor alpha were determined in the supernatant, witha quantitative enzyme-linked immunosorbent assay (Quantikine;R and D Systems, Minneapolis, Minn.). The same method was alsoapplied to the serum. The lowest detectable values in the standardcurve, according to the manufacturer, were as follows: interleukin-1ß,4 pg/ml; tumor necrosis factor alpha, 12 pg/ml; and interleukin-6,3 pg/ml.

Detection of interleukin-2 and interleukin-4 was carried outby determining the mRNA with reverse transcription-PCR (RT-PCR)(7).

Total RNA was extracted from adenotonsillar cell homogenateswith organic solvents such as guanidine, isocyanate, and phenol-chloroform.Reverse transcription was then carried out with Moloney murineleukemia virus reverse transcriptase (8).

Clontech products (Advantage RT for PCR kit) were employed forthe reverse transcriptase reaction. The reverse transcriptasespecimens were incubated at 37°C for 30 min. The cDNA soobtained was subjected to amplification cycles, with the specificprimers for the PCR, in a DNA thermal cycler (Perkin-Elmer Cetus,Norwalk, Conn.). The amplification conditions were as follows:94°C for 1 min, 58°C for 45 s, and 72°C for 1 min,for a total of 35 cycles. Clontech and Promega products wereused for this reaction.

The amplification products for specific interleukin-2 and interleukin-4were subjected to electrophoresis on a 3% agarose gel stainedwith ethyl bromide and revealed by means of transilluminationunder UV rays.

RESULTS

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Results

Out of 105 patients examined, 46 presented hyperkeratosis ofthe epithelium in the bottom of the crypt (Fig. 1). This patternwas present throughout the tonsil tissue. In the other 59 cases,the epithelium presented a hyperplastic appearance, withoutany clear signs of keratosis.

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FIG. 1. (A) Normal tonsillar crypt. (B) Hematoxylin-eosin staining of chronically inflamed human tonsils. Hyperkeratosis of the epithelium is detectable in the bottom of the crypts (arrows). (C) Hematoxylin-eosin staining of chronically inflamed human tonsils. Hyperkeratosis of the cryptic epithelium (higher magnification).

Follicular hyperplasia was consistently present and was moreevident in the germinal centers; since the plasma cells arefound mainly in the cryptoreticular area, the hyperplasia appearedto be due to proliferation of B lymphocytes. A significant hyperplasiaof the mantle zone was observed only in 10 cases.

Furthermore, immunoglobulin G-positive B lymphocytes were presentin all specimens mainly in the germinal center, with only afew found in the follicular mantle.

Immunoglobulin A-positive B lymphocytes were observed in 43%of the samples and were mainly localized close to the cryptoreticularepithelium, whereas only a few were detected within the germinalcenters (Fig. 2).

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FIG. 2. Immunohistochemical staining for immunoglobulin A in chronically inflamed human tonsils. Immunoglobulin A-positive B lymphocytes are mainly localized close to the cryptoreticular epithelium (arrows).

In 29 of 105 cases examined (27.6%), B lymphocytes with intracytoplasmicstaining for the J chain were detected. T lymphocytes, on theother hand, were present in all the samples examined and werelocalized primarily in the mantle zone of the lymphatic folliclesbetween the crypt fundus and the germinal center. Only in afew samples were T lymphocytes found in the extrafolliculararea.

Scanning electron microscopy revealed a continuous epithelialsurface of polygon-shaped flattened cells, with fissures towardsthe cryptic depressions.

With higher magnification, the cell surfaces presented an irregularappearance due to the presence of numerous small folds (Fig.3).

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FIG. 3. Scanning electron microscopy of chronically inflamed human tonsils. Polygonal flattened cells presenting fissures tending towards the cryptic depressions are easily identifiable. Cell surfaces are irregular because of numerous small folds.

The cell edges within the crypt were raised, with small superficialfolds, and the bottom of the crypts showed a raised, dome-shapedepithelium, the diameter of which ranged between 10 and 30 µm.

In some crypts, apart from disseminated epithelial cells presentin various amounts, other cellular elements were also visibleon the fundus (plasma cells and lymphocytes) (Fig. 4). No Mcells were detected in the tonsils or on the surface of thecrypts.

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FIG. 4. Scanning electron microscopy of chronically inflamed human tonsils. Plasma cells, lymphocytes and disseminated epithelial cells were visible on the fundus of crypts (arrow).

The interleukin-1ß concentration was raised in allthe specimens studied (mean value ± standard deviation:tonsils, 258.5 ± 16.8 pg/ml; adenoids, 187.6 ±88.7 pg/ml) compared with the serum level (6.5 ± 3.6pg/ml) (P < 0.0001) (Table 1).

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TABLE 1. Concentrations of cytokines in tonsils, adenoids, and serum

The concentrations of interleukin-6 were also raised comparedwith the plasma level (2.1 ± 1.2 pg/ml), the mean values(± standard deviation) being 27.9 ± 15.3 pg/mlin tonsil tissue and 9.2 ± 3.7 pg/ml in adenoid tissue;the difference between the two tissues and plasma was statisticallysignificant (P < 0.0001) (Table 1).

The concentrations of tumor necrosis factor alpha were significantlyraised in both homogenates (serum level, 2.9 ± 2.0 pg/ml),the mean values (± standard deviation) being 138.7 ±49.7 pg/ml and 142.3 ± 42.9 pg/ml in tonsils and adenoids,respectively (P < 0.0001) (Table 1).

mRNA for interleukin-2 and interleukin-4 was detected only intwo cases.

DISCUSSION

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Discussion

Whereas chronic inflammation of palatine tonsils in childrenusually leads to tissue hyperplasia and hypertrophy, in adultsit is associated with sclerosis of the above-mentioned structures.

Histological analyses of the epithelial component demonstratedthe presence, in the base of the crypts, of a constant hyperplasiaand, in 44% of cases, of a hyperparakeratosis.

This observation gave rise to the hypothesis that epithelialmodification caused impaired antigen uptake and this, in turn,led to the recurrence of inflammatory processes with an increasinglysevere hyperparakeratosis, triggering a vicious circle for whichtonsillectomy was the only curative solution.

Immunohistochemical examination revealed a persistent hyperplasiaof B lymphocytes (often immunoglobulin G positive), which weremore evident in the germinal centers, and a rather constantdecrease in the T-cell compartment.

The observation of a negative J chain in 73% of the specimenswould appear to confirm that chronic inflammation of the Waldeyerring structures results in the inhibition or in a decrease inthe production of secretory immunoglobulin A. This is not unexpectedinasmuch as it is correlated with changes in the glandular epitheliumresponsible for the production of the secretory fragment. Themodifications observed under scanning electron microscopy maybe due to deregulation of the normal intercellular connectionsthat, in turn, may be caused by the intraepithelial infiltrationof the lymphocytes originating from the germinal centers below.

Our data do not support the hypothesis that M cells exist atthe level of the reticular crypt epithelium, which act eitheras an antigen-presenting cell or as a carrier of foreign particles(9). We did not find any differentiated M cells.

The identification of high tissue concentrations of tumor necrosisfactor alpha, interleukin-1ß, and interleukin-6 isan expression of local hyperproduction or overproduction, dueto monocyte-macrophage activation, caused by repeated stimulationby the pathogenic agents. The presence of high levels of thesecytokines, nevertheless, instead of inducing an increase inimmunological efficiency, may have a damaging effect on thesemechanisms in the long term. These mediators, in fact, inducethe activation and proliferation of endothelial cells and fibroblasts,which may result, with time, in the progressive replacementof immunologically active tissue with fibrotic tissue.

The first sign of a deficit in the activation of the immunesystem could be represented by the small quantity of mRNAs forinterleukin-2 and interleukin-4 detected in our population,suggesting a defective activation of Th1 and Th2 lymphocytes.

The results from this study offer the opportunity for a seriesof extremely promising investigations even if the mechanismsresponsible for chronic inflammation in some subjects and thepersistence, in others, of an immunological equilibrium remainto be clarified.

FOOTNOTES

* Corresponding author. Mailing address: E.N.T. Department, University of Siena Medical School Policlinico Le Scotte, Viale Bracci 53100, Siena, Italy. Phone: 0039/577/40035. Fax: 0039/577/47940. E-mail: passali{at}unisi.it.

REFERENCES

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