Adenoids and Otitis Media With Effusion:

A Morphological Study

Mustafa Mete Kiro@u, MD, Kemal azbilgin, MD, Barlas Aydogan, MD,
Fikret Kir$lu, MD, Ozgiil Tap, MD, PhD, Mehmet Kaya, MD, PhD,
and Can Ozsahinoglu, MD

Purpose: Adenoidectomy, especially for the treatment of suppurative otitis media, has been
used for a very long time. In this study, the role of adenoids in the origin of otitis media with
effusion was investigated by using light microscopy, immunocytochemistry, enzymechemistry,
and electron microscopy.
Materials and Methods: A group of 28 children with otitis media with effusion (OME) was
identified. Ages ranged from 3 to 12 years. Acontrol group of 10 age-matched children without
any middle ear and upper respiratory tract infection served as the basis for comparison.
Specimens obtained at surgeries from both groups were divided into groups for light
microscopy, immunocytochemistry, enzyme cytochemistry, and electron microscopy and then
all were examined blindly. Also, quantitative analysis of antigen-presenting cells was
performed blindly on 10 patients and 10 controls.
Results: There was an increase in the number of lymphocytes, mast cells, plasma cells,
macrophages, dendritic cells, and M cells in the adenoids of patients with OME when
compared with the normal cases. Stratified squamous epithelial areas, collagenous fibers,
and fibrocytes were also increased in the patient group. Antigen-presenting functions of
epithelial cells are shown by major histocompatibilitycomplex (MHC) class II positivity of some
ciliated-columnar epithelial cells in the patient group.
Conclusion: Adenoid tissues of patients with OME in this study seem to be infectious foci,
aggravating immune reactions, which might attack the middle ear through an ascending route.
Copyright 0 1998 by W.B. Saunders Company.

Although the adenoids have been recog- cons of the adenoidectomy are still valid to-
nized as a factor in otitis media with effusion day.
(OME) since the definition of the disease, Morphological studies give clues about the
there is still controversy in its treatment, in- mucociliary transport system, secretory struc-
cluding adenoidectomy operations. tures, and the infectious and immunological
In fact adenoidectomy, especially for the events that take place in the etiopathogenesis
treatment of suppurative otitis media, has of OME. Adenoids are also investigated mor-
been performed since the beginning of the phologically, but the studies are concerned
century. Approximately one million tonsillec- with comparing the normal and hypertrophic
tomy and adenoidectomy procedures were adenoids, without taking OME into consider-
performed in the United States in the 197Os.l ation.2-6
In the last 20 years, the number of tonsillec- Previously, in our two consecutive studies,
tomy and adenoidectomy operations decreased
morphological changes in the middle ear and
dramatically. Discussions about the pros and
nasopharyngeal orifice of the eustachian tube
in patients with OME had been showm7J This
From the Departments of Otolaryngology, and Histol- study is a continuation of these previous stud-
ogy, University of Gukurova School of Medicine, Cuku- ies and investigates the ultrastructure of ad-
rova Universitesi Tip FakOltesi, KBB Anabilim Dali Balcali enoid tissues of patients with OME. The role
Adana, Turkey.
Address reprint requests to M. Mete Klroglu, MD, of adenoids in the origin of OME was investi-
Cukurova Universitesi TIP Fakiiltesi, KBB Anabilim gated by using light microscopy, immunocyto-
Dali Balcall Adana, Turkey.
Copyright 0 1998 by W.B. Saunders Company chemistry, enzymechemistry, and electron mi-
0196-0709/98/1904-0006$8.00/O croscopy.

244 American Journal of Otolaryngology, Vol 19, No 4 (July-August), 1998: pp 244-250

ADENOIDS AND OME 245

MATERIALS AND METHODS tion was carried out with a-naphtyl Aph (Sigma
7000, St Louis, MO) for 30 minutes at 37°C. After
Patients and Specimens precipitation of the final reaction product, prepara-
tions were rinsed and mounted in glycerol-jelly.
Specimens of adenoid tissue were obtained dur-
ing adenoidectomy operations on 28 children with
OME; their ages ranged from 3 to 12 years. Diagno- Quantitative Analysis of Antigen-Presenting
sis of OME was proven with the aspiration of Cells
effusion from the middle ear after myringotomy.
Also, adenoids were taken with cupped forceps The quantitative analysis of antigen-presenting
from 10 age-matched control cases who were oper- cells was evaluated on the HLA-DR MoAb and
ated on for foreign bodies in the airway. Informed u-naphtyl Aph-stained slides. From each group,
consent for biopsy was obtained before surgery slides of 10 patients and 10 controls (for a total of 40
from the patient’s parents. The normality of the ears slides) were selected for the analysis. On each slide
of the control group was tested with tympanometry of the series, an interfollicular subepithelial and a
and audiometry before or after the operation, de- follicular area (for a total of 80 areas) were ran-
pending on the urgency of the case. The adenoid domly chosen and photographed at 400x, original
tissue samples were divided into groups for light magnification. The antigen-presenting cells (inter-
microscopy, immunocytochemistry, enzyme cyto- digitating cell, follicular dendritic cell, and macro-
chemistry, and electron microscopy. Light micro- phage) and the other cells were counted on these
scopic and electron microscopic evaluations were photographs, and the ratio of the antigen-present-
performed on 28 patients and 10 controls, blindly. ing cells to the total counted cells was calculated.
In order to evaluate cytological changes more objec- Statistical analysis of the data was performed using
tively, quantitative analysis of antigen-presenting cells Mann-Whitney U test. (PI 85 was considered
was performed on 10 OME patients and 10 controls. significant.)
Light Microscopy
Electron Microscopy
Specimens were fixed with buffered neutral for-
malin and after dehydration were embedded in The tissue fragment from each subject was fixed
paraffin. Twenty semithin sections at 5-km thick- in glutaraldehyde for 4 hours at 4°C and post-fixed
ness were cut and stained with (1) hematoxylin- in osmic acid for 2 hours at 4°C. The specimens
eosin or (2) streptavidin-biotin immunoperoxidase. were dehydrated in graded ethanol series and were
Sections stained with hematoxylin-eosin were embedded in araldite. Twenty ultrathin sections
evaluated blindly. Also, some tissue samples were were stained with uranyl acetate and lead citrate.
frozen in - 70’ C for enzyme-cytochemistry. Sections were examined with electron microscope
blindly (Zeiss EM 900, Carl Zeiss, Oberkochen,
lmmunocytochemistry Germany).

Streptavidin-biotin immunoperoxidase staining

procedures were carried out on the paraffin blocks. RESULTS
After deparafinization, the sections were placed in
peroxidase-blocking reagent on each slide for 5 Light Microscopy
minutes, and the preparations were covered with
nonspecific blocking reagent for 28 minutes. After Control group. The surface of adenoids
taping off excess reagent, anti-human HLA-DR (Dako was covered with pseudostratified ciliated
775, Glostrup, Denmark) was incubated for 1 hour. columnar epithelium. The epithelium cover-
Linking reagents were added to each slide for 30
minutes; subsequently the slides were incubated ing the free surface was folded, but no deep
with streptavidin enzyme labels. Fresh working crypts were seen. There were several lymph
color reagent was prepared, and the slides were nodules in the subepithelial area. Three kinds
covered with this solution for 15 minutes. This of antigen-presenting cells were identified by
staining procedure was performed at room tempera- HLA-DR and Aph positivity: a few epithelial
ture, and preparations were washed with buffered
washing solution at each step. Finally, cells were cells; some dendritic cells, which were stained
stained with Mayer hematoxylin (Merck, Darm- only in perinuclear area; and some macro-
stadt, Germany) for 1 to 3 minutes, rinsed with tap phages, which were stained all through their
water, and mounted in glycerol-jelly. cytoplasm.
Enzyme Cytochemistry
Patient group. The surface epithelium was
On frozen preparations, acid phosphatase (Aph) similar to that of the control group, but strati-
staining was performed according to Barka.g Incuba- fied squamous epithelium was seen in some
246 KIRO6LU ET AL

areas. Epithelial and subepithelial areas were dendritic cells, reticular cells, and mast cells
infiltrated with lymphocytes (Fig lA), and were seen in some areas.
germinal centers of lymph nodules were promi-
nent. Cells positive for HLA-DR (Fig lB, 1C) Patient group. In addition to the findings
and Aph (Fig 1D) were increased in serial in the control group, some stratified squamous
sections and compared with the control group epithelial patches were seen. Also, there were
in the epithelial, subepithelial, interfollicular, some large spaces between epithelial cells,
and follicular areas: this increase was statisti- which were occupied with lymphocytes, cellu-
cally significant (P 5 .05) (Tables l-2). lar debris, and membrane-bounded body with
fine granular materials (Fig 2). Surface epithe-
Electron Microscopy lium was infiltrated with lymphocytes, and
Control group. The surface epithelium of degeneration was prominent. In the apical
adenoids consisted of ciliated columnar cells, surface of ciliated cells some bulb-like cyto-
columnar cells with microvilli, intermediate plasmic protrusions without organelles were
cells, and goblet cells. In the lymphoid tissue, seen between cilia, compound cilia, and micro-
small and large lymphocytes, plasma cells, villi (Fig 2). These protrusions contained elec-

Fig 1. (A) Lymphocyte infiltration is seen in pseudostratified ciliated epithelium and subepithelial area in the
patient group (hematoxylin & eosin; original magnification, x200). (B) lmmunocytochemical staining with HLA-DR.
MHC class II-positive dendritic cells having cytoplasmic extensions are seen (arrows) in the patient group (original
magnification; x200). (C) lmmunocytologi~al &ah-ring with HLA-DR. MHC class II-positive epithelial cells are seen
(arrow) in the patient group (original magnification, x200). (D) Frozen sections of adenoid tissue stained with
a-naphtyl Aph. Dendritic cells had Aph activity in a spot (arrows). In contrast, macrophages (arrowhead) had Aph
activity throughout the cytoplasm in the patient group (original magnification x 100).
ADENOIDS AND OME 247

TABLE 1. Percentages of Antigen-Presenting Ceils as

Determinated by HLA-DR MoAB

Control Patient P

lnterfollicular
subepithelial
area
IDC 1.180 2 0.103 1.810 5 0.168 <.05
M 0.615 ?I 0.133 0.875 k 0.146 <.05
Follicular area
FDC l.llO? 0.166 1.535 k 0.342 <.05
M 0.925 5 0.189 1.185 2 0.272 <.05

Abbreviations: IDC, interfollicular dendritic cells; M, macro-

phage; FDC, follicular dendritic cells.

tron-dense fine granular materials. Also, there

were structures very similar to these, envel-
oped with a membrane localized between
epithelial cells in close association with inter-
mediate and ciliated cells, leading to the con-
Fig 2. At the apical surface of the ciliated ceils
clusion that these were the eventually pinched-
bulb-like cytoplasmic bulgings (arrow) and compound
off epithelial protusions described previously. cilia (arrowhead) are seen. Lymphocytes (Ly) and mem-
It is interesting to note that these structures brane-bounded cytoplasmic bodies (asterisk) are ob-
served between epithelial cells in the patient group
were very similar to the cytoplasm of lympho- (original magnification, x6,300).
cytes (Fig 3). In some areas, goblet cells were
seen in the epithelium. M cells forming a cells and macrophages widespread in ad-
narrow barrier between the lumen and the enoids of the patients. The number of den-
lymphocyte were seen in some areas (Fig 4). dritic cells had increased in the subepithelial,
Lymphoid tissue consisted of different- interfollicular, and follicular areas. Dendritic
sized lymphocytes, mast cells, plasma cells, cells had an irregular shape, thin, long cyto-
macrophages, reticular cells, and dendritic plasmic extensions, and were in close contact
cells. In some lymphocytes there were cyto- with lymphocytes and plasma cells (Fig 6).
plasmic protusions with fine granular mate-
rial, in some areas these bulgings were con-
nected with a thin stalk to the lymphocyte,
and in some areas slits developed between
lymphocytes and their largely protuding cyto-
plasmic compartments (Fig 3). Both pycnotic
lymphocytes and remnants of degenerated cells
were observed in some areas. The number of
plasma cells had increased (Fig 5). Also, mast

TABLE 2. Percentages of Antigen-Presenting Cells as

Determined by a-naphtyl Acid Phosphate

Control Patient P

lnterfollicular
subepithelial
area
IDC 1.090 z 0.151 1.620 + 0.574 <.05
M 0.765 k 0.176 1.185 2 0.910 <.05
Follicular area
FDC 1.245 + 0.198 1.560 k 0.126 <.05
M 0.915 + 0.160 1.095 2 0.154 <.05 Fig 3. The large lymphocyte (Ly) had an indented
nucleus (N), scanty mitochondria (m), membranous
Abbreviations: IDC, interfollicular dendritic cells; M, macro- whorls (arrowhead), and cytoplasmic bulgings (arrow)
phage; FDC, follicular dendritic cells. in the patient group (original magnification, x8,000).
248 KlROrjLU ET AL

Fig 4. The thin cytoplasm of an M cell (M) on the Fig 6. The dendritic cells (DC) in the patient group
surface of the epithelium forming a single cell barrier were in close contact (arrow) with lymphocytes (Ly). The
between the lumen and intraepithelial channels. (Cc, dendritic cells had an indented nucleus(N), short granu-
ciliated cell; original magnification, x3,000). lar endoplasmic reticulum cisternae (GER), mitochon-
dria (m), smooth endoplasmic reticulum (SER), Golgi
complex (G), and dense bodies (db) in cytoplasm and a
Also, collagenous fibers had increased, and few lipid droplets (L) in their cytoplasmic extensions.
the fibroblasts were observed in the fibrocyte (PC, plasma cell; original magnification, x4,400).
form in many micrographs (Fig 7).
cells, and M cells in the adenoids of patients
DISCUSSION with OME when compared with the normal
cases. Although electron microscopic demon-
The current study shows that there is an stration of this increase is partly subjective,
increase in the number of lymphocytes, mast
cells, plasma cells, macrophages, dendritic

Fig 7. The fibroblasts (F) are mostly seen in inactive

fibrocyte form in patient group. The collagenous fibers
Fig 5. Increased plasma cells (PC) in patient group. A (Col) were increased. Between fibroblasts and collag-
nucleus (N), well-developed granular endoplasmic retic- enous fibers there are membrane-bound bodies with
ulum profiles (GER), and Golgi complex (G) in the finely granular material (asterisks) and cellular debris
cytoplasm (original magnification, x7,000). (d) (original magnification, x4,000).
ADENOIDS AND OME
objective measurements were performed on 10
patients and 10 controls for antigen-present-
ing cells. Also, there was epithelial destruc-
tion and a decrease in the number of ciliated
cells on the micrographs of the patient group.
We have also shown the immune function of
epithelial cells by HLA-DR positivity in serial
sections (Fig 1C). This finding (the antigen-
presenting activity of epithelial cells) is not
supported by all reports,4 but von-Nieuwkerk
et a15 showed the presence of major histocom-
patibility complex (MHC) class II-positive cili-
ated epithelial cells in all adenoid tissues that
they studied immunocytochemically. In our
opinion, the epithelial cells gain this function
in chronic infections; this is probably the
reason why we have seen extremely few HLA-
DR-positive epithelial cells in the control
cases. The similarity between the contents of
bulb-like cytoplasmic protusions in the apical
surface of the ciliated cells (Fig 2, arrow) and
the lymphocyte cytoplasm (Fig 3) seems to be
an electron microscopic support of the antigen-
presenting activity of epithelial cells. These
bulgings (which are very similar to the ones
branching from lymphocytes; Fig 3, arrow)
might be some useless parts of cytoplasm that
are lost in the way of maturation and specifica-
tion for antigen. This theory is supported by
the demonstration of vesicles that contain
horseradish peroxidase in the ciliated cells of
infected adenoids by Fujiyoshi et al3 who
suggested that immunocompetent M cells de-
velop from neighboring ciliated or nonciliated
cells under the influence of antigens or lym-
phocytes. Nevertheless, our assumption that
these cytoplasmic bulgings were related to
immune mechanisms needs to be proven with
immuno-electron microscopic methods.
It is obvious that persistent infection causes
epithelial destruction; this in turn can allow
penetration of foreign antigens through epithe-
lium that has lost its integrity. Formation
clefts down to the lymphoid tissue may lead
antigens to reach lymphocytes easily, augment-
ing the immunologic reactions. Also, some
large spaces between epithelial cells found in
some electron micrographs gave us the impres-
sion that these were structural units for a close
contact with the outside environment, similar
to intraepithelial lymphatic canals described
in 1994 by Winther and Innes.6
Our findings support the opinion that there
249
is no defect in the immune systems of patients
with OME. Moreover, increased amounts of
immune cells reflect increased antigenic load,
probably resulting from infection of the ad-
enoid tissue. The identification of the den-
dritic cells in contact with the lymphocytes
through their cytoplasmic elongations, espe-
cially in degenerative areas and in areas resem-
bling reparation where collagenous and reticu-
lar fibers are present, encourage us to think
that around these cells, charged to present
antigenic stimuli to lymphocytes, there would
be large numbers of antigens and antibodies,
which can activate the complement system.
The activation of the complement system
causes vasodilatation, increase in vascular per-
meability, and fluid exudation, all of which
precipitate OME.
An epithelial destruction resulting from cy-
totoxic activities of intraepithelial lympho-
cytes as proposed by Bani et al2 and Gallo et al4
also seems possible. Destruction of ciliated
epithelium can lead to OME by mucociliary
degeneration because of decreased middle ear
clearance. These mucociliary changes are seen
in some electron microscopic photographs of
the patient group in which compound cilia
formation and destruction of ciliated cells are
shown (Fig 2); changes in mucociliary trans-
port system in OME patients were shown in
our two previous electron microscopic stud-
ies.7s8The decrease of ciliated cells, a common
finding in enlarged adenoids,3v4 not only af-
fects mucociliary clearance adversely, but also
decreases local immunity and may lead to
secretory cell metaplasia.
In the areas of degenerated cells and cell
remnants, fibrosis develops, characterized by
the marked proliferation of fibroblasts and the
increase of collagen and reticular fibrils. The
injured parenchyme, resulting from degenera-
of tion of cells, was replaced by fibrotic material.
Increase of these areas will cause atrophy of
adenoids and may be the reason for the recov-
ery of the OME patients with or without any
therapy in the course of time.
In conclusion, adenoid tissues of patients
with OME seem to be infectious foci, aggravat-
ing immune reactions, which might attack the
middle ear through an ascending route. Future
studies are required to shed more light on the
multifactorial origins of OME.
250 KlROCiLU ET AL

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