ARTICLE
INTRODUCTION
The pro-inflammatory cytokines TNF-alpha, IL-1beta, IL-6 and IL-8 are
important mediators in immune regulation and are produced during immune
and host defence responses.
Tumor necrosis factor (TNF-alpha) is a pleiotropic cytokine, produced
primarily by macrophages in response to bacterial and viral pathogens.
The pro-inflammatory functions of TNF-alpha include up-regulation of endothelial
cells adhesion molecule expression, stimulation of cytokine expression,
activation of neutrophils, stimulation of fibroblast proliferation, and
antiviral effects against both DNA and RNA viruses [1, 2].
IL-1beta is recognized as a central mediator of inflammation, produced
mainly by activated macrophages [3, 4]. IL-1beta has a wide range of target
cells, including fibroblasts (causing proliferation and inducing secretion
of collagenases and IL-6); endothelial cells (induces TNF-alpha release
and adhesion molecule expression); osteoclasts (activates the proton pump,
leading to bone resorption); mature T cells (induces proliferation of
Th2 cells); mature B cells, (induces proliferation and immunoglobulin
secretion); monocytes and neutrophils (induces secretion of several cytokines,
including IL-8 and IL-1 itself) [5, 6].
IL-6 possesses pleiotropic activities, which play a central role in
host defence. These activities include maturation of, and immunoglobulin
secretion by B cells, osteoclast activation, induction of monocyte differentiation
into macrophages, activation of T cells, stimulation of hepatic acute-phase
protein synthesis, maturation of megakaryocytes [7, 8].
IL-8 is related to the chemokine family of cytokines. IL-8 primarily
attracts and activates neutrophils and also induces the directional migration
of monocytes, T lymphocytes and basophils; it releases intracellular enzymes
from neutrophils and histamine from basophils; it regulates the adhesion
of neutrophils and plays a key role in the accumulation of leukocytes
at sites of inflammation [9, 10].
Reflecting their biological activities, TNF-alpha, IL-1beta, IL-6 and
IL-8 are involved in different inflammatory reactions, accompanying the
host immune response to microbial and viral infections and participate
in the pathogenesis of related diseases [11-13]. Investigations over the
last two decades demonstrate the involvement of TNF-alpha, IL-1beta, IL-6
and also IL-8 in the pathogenesis of otitis media with effusion (OME).
OTITIS MEDIA WITH EFFUSION - HISTOPATHOLOGICAL,
CELLULAR AND MOLECULAR CHARACTERISTICS
Otitis media with effusion is the commonest cause of childhood deafness
in the developed world. The disease is an inflammatory condition of the
middle ear cleft and characterized in the early stage by inflammatory
cell infiltration in the submucosal layer, cell proliferation in the epithelial
and mucosal linings of the middle ear cavity, and accumulation of neutrophils,
macrophages and lymphocytes in the middle ear fluid [14, 15]. Later accumulation
of effusion in the middle ear cleft occurs as a result of mucociliary
transport system dysfunction [16, 17]. In the late stage of the disease,
severe and sometimes irreversible tissue pathology can develop that includes:
fibrosis [18], granulation tissue, osteitis, osteoneogenesis, cholesteatoma
[19], and finally histological degeneration of the tympanic membrane as
a result of tissue proliferation in subepithelial and submucosal layers
[20, 21], leading to hearing loss.
Otitis media (OM) can be subdivided into 4 main types: purulent (acute)
(POM), serous (SOM), mucoid (MOM) and chronic (COM) [19]. These subdivisions
have specific histological (Figure 1) and clinical characteristics. The
symptoms of purulent otitis media include pain, fever, hearing loss and
occasional perforation of the tympanic membrane.
Serous and mucoid OM are less severe, without the pain and fever of
POM. However, hearing loss is present and changes in the tympanic membrane
do occur. SOM and MOM frequently develop from eustachian tube dysfunction
and appear to occur in a continuum. In the early stage of otitis media,
serous transudate from vessels in the subepithelial space (SES) can pass
to the middle ear cleft, leads to and forms the serous otitis media [19].
There is no evidence of secretory cell proliferation in SOM [22].
Mucoid otitis media is characterized by differentiation of basal cells
into goblet and ciliated cells [19], proliferation of goblet cells [22,
23], and formation of mucus and secretory gland populations [24, 25] in
the middle ear mucosa. These changes lead to the increased secretory activity
of the middle ear mucosa and promote the appearance of a thick mucoid
effusion in the middle ear cleft. However, with time, degeneration of
the new glands and a decrease in basal cells in the middle ear mucosa
may occur, and the middle ear epithelium returns to normal.
Chronic otitis media can also be a painless process without fever. As
in all other types of OM, hearing loss and tympanic membrane changes are
the main clinical signs of COM [19]. However, at the histological level,
irreversible tissue pathology is observed in COM (Figure 1). COM frequently
is associated with the presence of increased numbers of mast cells in
the middle ear cleft tissues [26-28]. An overlap of the histopathological
findings between the different types of otitis media is characteristic
(Figure 1). In addition to the main types of otitis media, there are also
intermediate types such as mucopurulent and mucoserous.
Mucins are important glycoproteins in the mucociliary transport system
of the middle ear and are the main component of middle ear effusions,
responsible for the viscous properties of effusions [31, 32]. Middle ear
mucins are able to bind to proteins in the outer membrane of bacteria
[33], and play an essential role in evacuating pathogens from the middle
ear. However, under disease conditions, alterations that occur in the
middle ear and eustachian tube mucin metabolism [34], in the structure
of mucin glycoproteins [35, 36], and in glycoconjugate expression in cilia
and goblet cells [37], can contribute to dysfunction of the normal mucociliary
transport system and promote the formation of effusion in the middle ear
cleft.
Three mucins, namely membrane-bound MUC4 [34] and secreted MUC5AC [36]
and MUC5B [34, 36, 38, 39] are found in the middle ear mucosa and mucus
secretions from patients with chronic and mucoid otitis media. MUC5B is
considered to be a major mucin in the middle ear mucosa and effusions.
Expression of the MUC5B mucin gene in pseudostratified middle ear mucosal
epithelia correlates with the expression of inflammatory molecules ICAM-1
(intercellular adhesion molecule-1) and RANTES (regulated upon activation,
normal T cell expressed and secreted) [40], and with infiltration of inflammatory
cells in the submucosa of the middle ear cleft [39]. These correlations
suggest that inflammation may initiate and maintain the hypersecretory
state of the middle ear mucosa leading to the chronicity of OME.
It is important to note that acute inflammatory changes are usually
seeing in purulent and serous OM, and chronic inflammatory changes are
more severe in mucoid and chronic OM [29]. However, the overlaps in histopathological
findings between different types of otitis media [29, 30] suggest that
all known categories of otitis media (purulent, serous, mucopurulent,
mucoserous, mucoid and chronic) can represent different stages in a continuum
of events, accompanying one disease - the otitis media with effusion.
INFLAMMATORY MEDIATORS IN OTITIS MEDIA WITH EFFUSION
The molecular background explaining the pathology of OME is still unclear.
However, the crucial process in the middle ear leading to OME is local
inflammation. Bacteria [41-43], viruses [44-46] and allergic reactions
[47, 48] are all implicated as initial stimuli of middle ear inflammation.
Different groups of inflammatory mediators, regulating different stages
of the inflammatory response, are identified in the human middle ear mucosa,
fluids (MEF) and effusions (MEE).
The early mediators of inflammation, initiating acute inflammatory reactions,
are detected in OME and include arachidonic acid metabolites (prostaglandin
PGE2 and leukotrienes LT-B4, LT-C4) [49,
50], histamine [51, 52], platelet-activating factor (PAF) [53], surface
cell adhesion molecules (intercellular adhesion molecule-1 (ICAM-1), vascular
cell adhesion molecule-1 (VCAM-1), endothelial leukocyte adhesion molecule-1
(ELAM-1), platelet endothelial cell adhesion molecule (PECAM)) [54, 55]
and the pro-inflammatory cytokines TNF-alpha [56], IL-1beta [57], IL-8
[58].
The mediators of the ongoing inflammatory process are also identified
in OME and include soluble cell adhesion molecules (sICAM-1 and sVCAM-1)
[59, 60], chemokine RANTES [61, 62], complement C3a anaphylatoxin [63],
interferon-gamma [64, 65] and the pro-inflammatory cytokine IL-6 [66].
The immunoregulatory cytokines IL-2 [64, 67] and IL-5 [68] and cytokines
that downregulate the inflammatory response, such as transforming growth
factor-beta (TGF-beta) [69] and IL-10 [70], are also present in OME.
All the aforementioned inflammatory mediators are involved in the pathogenesis
of OME. However, the pro-inflammatory cytokines TNF-alpha, IL-1beta, IL-6
and IL-8 are very likely to play a central role in middle ear inflammation
and in the stimulation of the molecular-pathological background of OME.
These cytokines are detected at high rates in middle ear effusions and
are present in effusions at high concentrations. Rates and the highest
mean concentration values are as follows: (63%-91%) and 234.2 ± 109.1
pg/mg of total protein (TP) for TNF-alpha [64, 71-73]; (51%-97%) and 4076
± 1510 pg/mg of TP for IL-1beta [64, 71-73]; (36%-83%) and 173.9
± 74.7 pg/mg of TP for IL-6 [66, 72]; (92%-100%) and 4805 ±
913 pg/mg of TP for IL-8 [71, 73].
TNF-alpha IN OTITIS MEDIA WITH EFFUSION
TNF-alpha was one of the first pro-inflammatory cytokines identified
in middle ear effusions from children with chronic otitis media with effusion
(COME) [56, 64]. Concentrations of TNF-alpha in effusions correlate with
age such that the older the child, the higher the level of TNF-alpha,
and the highest concentrations of TNF-alpha (up to 234 pg/mg of TP) are
associated with a history of multiple placements of tympanostomy tubes
[64, 72]. Children undergoing tympanostomy on multiple occasions have
average levels of TNF-alpha, nearly 14 times higher than those in children
undergoing their first tympanostomy [64].
The presence of TNF-alpha in otitis media correlates with the presence
of bacteria [72, 74], viruses and cell adhesion molecules [54] in effusions.
TNF-alpha is detected in all types of otitis media: purulent [72, 74,
75], mucopurulent [76], serous [77], mucoid [56, 73, 78] and chronic [64,
71, 72]. High concentrations of TNF-alpha (up to 67 pg/mg of TP) in mucoid
effusions [73], suggest the involvement of TNF-alpha in middle ear mucin
hypersecretion, one of the important molecular-pathological processes
occurring in OME. This suggestion is supported by investigations of the
effects of TNF-alpha on mucin gene expression and mucus secretion in the
middle ear with in vitro and in vivo models [79, 80]. The
results of these investigations are discussed below.
It is important to note that TNF-alpha soluble receptor (TNF-alphasolR)
has been identified together with TNF-alpha in COME [81]. The correlation
between TNF-alpha and TNF-alphasolR concentrations in effusions suggests
that the chronic inflammation seen in COME may be the result of an imbalance
in the ratio between TNF-alpha and its natural inhibitor - TNF-alpha
soluble receptor.
Although TNF-alpha is detected in different types of otitis media and
effusions, the level of TNF-alpha is always higher in the chronic stage
of the disease and therefore, TNF-alpha is considered to be a marker for
OME chronicity [72] and related to the persistence of OME [78].
The role of TNF-alpha in the pathogenesis of OME has been studied with
in vitro and in vivo models.
In vitro studies show that TNF-alpha up-regulates the expression
of RANTES in cultured rabbit middle ear epithelium [62], and stimulates
the secretion of mucous glycoprotein (MGP) in cultured chinchilla middle
ear epithelial cells [79].
For in vivo studies, investigators have used two types of animal
models. In the first model, recombinant TNF-alpha was directly injected
into middle ears of experimental animals and subsequent immuno-histopathological
changes were analyzed [80, 82, 83]. In the second model, the TNF-alpha-related
processes in otitis media were analyzed after injection of bacteria or
bacterial products (endotoxin) into middle ears of experimental animals
[84-89].
Numerous in vivo studies have presented the following observations.
TNF-alpha inoculated into the middle ear cavity of specific pathogen-free
rats markedly increases Muc2 mucin mRNA expression in the middle ear epithelium
and MGP secretion in the middle ear fluid [80].
Transtympanical injection of TNF-alpha induces acute experimental OME
in the guinea pig [82] and in the rat models [83]. The inflammatory cellular
effusion, containing up to 67% lymphocytes, develops within 24 hours after
injection of TNF-alpha [82]. The TNF-alpha-induced middle ear effusion
is accompanied by histopathological changes such as subepithelial edema,
marked infiltration of neutrophils and increased microvascular permeability
in the middle ear cleft. These immuno-histopathological changes are significantly
reduced by the TNF-alpha soluble receptor type I [83].
Hypersecretion of TNF-alpha (up to 200 ng/ml) is detected in experimental
endotoxin-induced OME [84], and interestingly, the histhopathological
changes in the middle ear caused by endotoxin are reduced significantly
by the injection of TNF-alpha-binding protein [85].
TNF-alpha is present in the middle ear mucosa, fluids and effusions
in experimental otitis media induced by the heat-inactivated [86] or viable,
Gram-positive bacterium Streptococcus pneumoniae [87,88] and Gram-negative
bacterium Haemophilus influenzae [89]. Up-regulation of TNF-alpha
mRNA expression in the middle ear mucosa occurs at 6 hours after bacterial
inoculation [87]. However, secretion of TNF-alpha is detected in MEF earlier,
within the first 2 hours after inoculation of the pathogens and afterwards
shows peaks at 6 hours, 48 hours, 72 hours and 96 hours [88, 89]. Concentrations
of TNF-alpha in effusions correlate significantly with bacterial product
[86], and in MEF, with the number of lymphocytes, macrophages and neutrophils
[88], suggesting that TNF-alpha activates inflammatory cells in middle
ear inflammation.
Investigations of TNF-alpha in human and experimental OME lead to the
following conclusions:
1) TNF-alpha is produced in the early stage of inflammation by the middle
ear mucosa and in the late stage by accumulating inflammatory cells, and
can be considered as the primary cytokine in OME.
2) TNF-alpha correlates with the number of inflammatory cells and the
presence of cell adhesion molecules in otitis media. In the middle ear
mucosa TNF-alpha stimulates the expression of RANTES - one of the
markers of ongoing inflammation. Therefore, TNF-alpha is a mediator of
ongoing inflammation in the middle ear and is involved in the pathogenesis
of purulent (acute) otitis media.
3) TNF-alpha up-regulates mucin gene expression and mucus secretion
in the middle ear and probably plays an essential role in the pathogenesis
of mucoid otitis media.
4) TNF-alpha induces severe histopathological changes in the middle
ear tissues and can contribute to chronic OME.
5) TNF-alpha is one of the primary cytokines in otitis media induced
by bacterial pathogens, both Gram-positive and Gram-negative, and participates
in viral OME.
6) TNF-alpha soluble receptor may be a natural inhibitor of OME.
Thus the pro-inflammatory cytokine TNF-alpha is one of the primary mediators
in middle ear inflammation (Figure 2), it regulates many molecular-pathological
processes in the middle ear, and can be considered as the key cytokine
involved in the aetiology of OME.
IL-1beta IN OTITIS MEDIA WITH EFFUSION
IL-1beta, as well as TNF-alpha, is present in all main types of otitis
media: purulent (acute) [57, 75, 78, 90], serous [57, 77], mucoid [73,
78] and chronic [64, 72]. Purulent effusions (POM) contain the highest
levels of IL-1beta, while serous (SOM) and mucoid (MOM) have the lowest
[57].
In acute otitis media, the concentrations of IL-1beta in MEF with viable
bacterial pathogens (culture-positive) are 3 times higher, than in culture-negative
ones, and usually, the concentration of IL-1beta decreases with the eradication
of bacterial pathogens following antibiotic therapy [90]. IL-1beta is
also present in middle ear effusions infected with viruses [54]. These
observations show that IL-1beta is a mediator of both bacterial and viral
otitis media.
Investigations of IL-1beta in MEE have found the following correlations:
(1) IL-1beta correlates inversely with the age of children, such that
younger groups have higher levels of IL-1beta than older groups [57, 64,
75]. (2) High level of IL-1beta in the purulent effusions correlates with
high level of total collagenase [75]. (3) The immuno-cytological analysis
of effusions shows the highest level of IL-1beta in the neutrophil-rich
effusions [78]. (4) Effusions that contain IL-1beta also contain TNF-alpha,
IL-6 and IL-8 [64,70-73]. (5) Statistically significant correlations are
found between the concentrations of IL-1beta and TNF-alpha [63, 70, 72],
IL-1beta and IL-6 [72], IL-1beta and IL-8 [70, 71] in effusions.
These findings present indirect evidence for a wide range of molecular
processes, which IL-1beta can stimulate during inflammation in the middle
ear (Figure 2), namely, up-regulation of primary (TNF-alpha and IL-1beta
itself) and secondary (IL-6 and IL-8) pro-inflammatory cytokine secretion;
activation of neutrophils in the zone of inflammation; fibroblast activation
with subsequent up-regulation of collagenase secretion.
The role of IL-1beta in OME has been studied in vivo, applying
two approaches.
In the first approach, recombinant IL-1beta (rIL-1beta) was transtympanically
injected into experimental animals. Analysis of the pathological changes
in the middle ear showed the following results. rIL-1beta (100 U) does
not produce significant effusions in normal guinea pigs [82]. However,
in the murine model (specific pathogen-free male BALB/c mice) of OME,
rIL-1beta (100 ng) induces a middle ear effusion within three days of
injection [91].
In the second approach, IL-1beta -related processes were analyzed in
otitis media provoked by injection of bacterial pathogens into the middle
ear cleft of experimental animals, with the following results: (1) IL-1beta
is the earliest pro-inflammatory cytokine detected in MEF, less than 1
hour, after inoculation of viable pneumococci, in the chinchilla model
of OME [88]. The level of IL-1beta peaks twice, at 6 hours after inoculation
and before appreciable inflammatory cell accumulation, and at 24 hours,
when the level of inflammatory cells is high. (2) The concentration of
IL-1beta in MEF correlates significantly with the number of neutrophils,
suggesting that IL-1beta activates neutrophils and induces up-regulation
of its own secretion by activated neutrophils in middle ear inflammation.
Correlations between the concentration of IL-1beta and the number of macrophages
and lymphocytes are less significant as compared with neutrophils [88].
(3) The significant correlations between the level of IL-1beta and levels
of TNF-alpha, IL-6, IL-8 and the dynamics of the secretion of these cytokines
in experimental otitis media [88], suggest that IL-1beta can up-regulate
TNF-alpha, IL-6 and IL-8 secretion in middle ear inflammation. (4) High
levels of IL-1beta are detected in middle ear effusions in experimental
otitis media induced by both Gram-positive (heat-killed Streptococcus
pneumoniae) [86] and Gram-negative (viable Haemophilus influenzae)
bacteria [89]. (5) Up-regulation of IL-1beta mRNA expression occurs in
the middle ear mucosa in experimental OME induced by the endotoxin [91].
Pathological changes in the middle ear stimulated by endotoxin [91] are
inhibited by the anti- IL-1 receptor antibody, suggesting an important
role for IL-1beta in endotoxin-induced OME.
Study of IL-1beta in human and experimental OME have produced the following
conclusions:
1) IL-1beta is the earliest cytokine in middle ear inflammation, produced
in the early stages of inflammation by cells in middle ear mucosa, and
later by accumulated inflammatory cells, and can be recognized, together
with TNF-alpha, as the primary cytokine in OME.
2) IL-1beta induces secretion of pro-inflammatory cytokines (IL-1beta
itself, TNF-alpha, IL-6, IL-8) in middle ear tissues and thus stimulates
the pro-inflammatory cytokine network in middle ear inflammation (Figure
2).
3) IL-1beta activates neutrophils and lymphocytes in middle ear inflammation
and promotes inflammatory cells infiltration into middle ear.
4) IL-1beta is involved in otitis media induced by viral and both Gram-positive
and Gram-negative bacterial pathogens.
In general, IL-1beta can be considered to be a mediator of acute and
ongoing inflammation in the middle ear, associated with the early stages
of the disease and the purulent (acute) type of otitis media. However,
IL-1beta can induce fibroblast activation and proliferation, growth of
osteoclasts in the middle ear tissues, with subsequent pathological changes
such as fibrosis and bone erosion and therefore can contribute to the
pathogenesis of the chronic type of OME.
IL-6 IN OTITIS MEDIA WITH EFFUSION
IL-6 has been identified in chronic OME, together with the primary pro-inflammatory
cytokines IL-1beta and TNF-alpha [66, 72], and in purulent (acute) OM
[92, 93]. IL-6 is detected in both bacterial and non-bacterial, acute
otitis media [92], and mRNA for IL-6 is also present in middle ear effusions
and mucosal tissues, infected with respiratory syncytial virus [54].
Studies of IL-6 in middle ear effusions revealed the following. (1)
Like IL-1beta, levels of IL-6 are higher in young children [66]. (2) Level
of IL-6 in purulent otitis media is higher as compared to chronic otitis
media, and the presence of IL-6 in biopsy specimens correlates positively
with the presence of macrophages and B cells [93]. (3) In bacterial otitis
media, IL-6 concentrations are higher in the culture-positive samples
as compared to culture negative ones, and the concentration of IL-6 in
MEF decreases significantly after antibiotic therapy [92]. (4) Concentrations
of IL-6 in effusions, correlate positively with concentrations of IL-1beta
and TNF-alpha [72].
These observations suggest the participation of IL-6 in the regulation
of acute and ongoing stages of middle ear inflammation, and involvement
of IL-6 in bacterial otitis media. Investigations of IL-6 in experimental
pneumococcal otitis media [87, 88, 94] confirm this suggestion.
IL-6 is detected in MEF in pneumococcal otitis media as early as 2h
after bacterial middle ear challenge [88]. Then the level of IL-6 decreases
and increases a second time with two peaks, one at 48 and one at 72 hours.
These changes in the level of IL-6 correlate with the inflammatory cell
concentration, suggesting that IL-6 is produced by the accumulated inflammatory
cells [88]. Transcripts of IL-6 (mRNA) have been observed in purulent
effusions and in the middle ear mucosa only 24 hours after inoculation
of pathogen, however, the IL-6 cytokine (protein) persisted in middle
ear tissues for at least 5 days following bacterial injection [94]. IL-6
is localized mainly in the mucosal epithelium, but also in the deeper
layers of the tissues such as the bullular bone, deep in the submucosa
and around blood vessels [94].
The concentration of IL-6 in MEF correlates significantly with the number
of inflammatory cells (especially macrophages and lymphocytes), and primary
cytokines (IL-1beta and especially TNF-alpha) [88]. It is important to
note that up-regulation of IL-6 mRNA (peak at 12 to 24 hours), in experimental
pneumococcal otitis media, occurs after up-regulation of TNF-alpha expression
(within 6 hours after inoculation of pathogens) [87].
Investigations of IL-6 in human and experimental OME led to the following
conclusions:
1) IL-6 is a mediator of middle ear inflammation, and is produced by
cells in the middle ear mucosa and by the accumulated inflammatory cells
(mainly macrophages and possibly T cells).
2) IL-6 activates B cells in middle ear inflammation.
3) The secretion of IL-6 during middle ear inflammation is under control
of the primary pro-inflammatory cytokines IL-1beta and TNF-alpha, and
IL-6 can be considered as the secondary cytokine in middle ear inflammation
(Figure 2).
4) IL-6 is involved in otitis media stimulated by both bacterial and
viral pathogens.
In general, IL-6 is the regulator of the ongoing inflammatory processes
in the middle ear associated with the early stage of OME. However, IL-6
can initiate a differentiation of macrophages to osteoclasts and thereby
participate in a bone remodeling process leading to development of tympanosclerosis
and switching the disease to the chronic stage.
IL-8 IN OTITIS MEDIA WITH EFFUSION
IL-8, as well as TNF-alpha, has been identified in most types of otitis
media: purulent (acute) [95, 97, 98, 99], serous [95-97], mucoserous [96],
mucoid [73, 95, 97], and chronic [97]. Concentrations of IL-8 in acute
otitis are significantly higher than in chronic OME [98] and mucoid OM
shows significantly higher mean concentrations of IL-8 in comparison with
serous OM [96, 97].
IL-8 is detected in a high proportion of effusions analyzed (up to 100%)
[73], and usually shows the highest mean concentration value of an the
other pro-inflammatory cytokines measured; typically an IL-8 concentration
of 4,805 pg/mg is present, whereas concentrations of IL-1beta and TNF-alpha
in the same effusions are 4,075 pg/mg and 163 pg/mg, respectively [71].
However, there are significant and positive correlations between the concentration
of IL-8 and concentrations of IL-1beta and TNF-alpha in effusions [70,
71], suggesting that IL-1beta and TNF-alpha can induce the IL-8 hypersecretion
seen in OME.
The presence of IL-8 in effusions correlates with the total number of
neutrophils, and purulent effusions contain more neutrophils than mucoid
and serous effusions [96]. Although IL-8 is an important chemotactic factor
for leukocytes, the analysis of chemotactic activity in effusions shows
that IL-8 cannot be considered as the main chemotactic component in MEE
[98].
Investigations of IL-8 in MEE show that IL-8 is involved in the pathogenesis
of otitis media induced by bacterial and viral pathogens. The IL-8 concentration
in effusions with bacteria is higher than in effusions without bacteria
[98, 99]. The decrease of the IL-8 concentration in middle ear fluid correlates
with bacterial eradication from the middle ear following antibiotic treatment
[99]. IL-8 mRNA is identified in effusions, together with mRNA for respiratory
syncytial virus and parainfluenza virus type 3 [61].
The significance of IL-8 in the aetiology of OME has been shown with
in vivo and in vitro models. Transtympanical injection of
human IL-8 (25 mug/ml) induces middle ear inflammation in experimental
OME [100]. The inflammation develops within the first 8 hours after IL-8
injection, and is accompanied by thickening of the epithelial layer and
infiltration of the inflammatory cells into the subepithelial space.
Gram-positive and Gram-negative, middle ear bacterial pathogens, induce
the IL-8 network within different types of experimental otitis media [88,
89, 101].
The secretion of IL-8 in MEF is detected in otitis media provoked by
viable Streptococcus pneumoniae [88] and non-viable Haemophilus
influenzae [89]. It is important to note that IL-8 is not detected
in MEF before 12 hours, but is significantly increased at 48 hours, 72
hours and 96 hours after inoculation of pathogens [88, 89], suggesting
secretion of IL-8 by the increased population of inflammatory cells during
the later stages of middle ear inflammation. Positive and significant
correlations exist between the concentration of IL-8 and number of neutrophils,
but not with lymphocytes, in MEF [88], supporting the theory that IL-8
is primarily a neutrophil chemoattractant. The IL-8-related accumulation
of neutrophils in the middle ear is also detected in otitis media stimulated
by endotoxin [101].
The kinetics of IL-8, IL-1beta and TNF-alpha secretion during experimental
middle ear inflammation caused by bacterial pathogens, and correlations
between the concentrations of these cytokines in the middle ear fluid
[88, 89] provide indirect evidence that IL-1beta and TNF-alpha control
IL-8 expression in otitis media.
A new gram-positive and OME-related bacterium Alloiococcus otitidis,
and soluble extracts from the inactivated A. otitidis, show the
ability to stimulate IL-8 secretion in cultured monocytes and epithelial
cell lines [102]. Although monocytes produce significantly higher levels
of IL-8 than the epithelial cells, these results demonstrate that both
monocytes and epithelial cells may be the source of IL-8 in middle ear
inflammation, and contribute to the IL-8 hypersecretion seen in OME.
The results of investigations of IL-8 in OME within clinical materials
(middle ear effusions, fluids and mucosal biopsy samples), and in vivo
and in vitro models allow us the following conclusions:
1) IL-8 is a mediator of middle ear inflammation, produced later than
other pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha ) by the
accumulated inflammatory cells and possibly by cells in the middle ear
mucosa.
2) IL-8 is the main chemotactic factor for neutrophils in middle ear
inflammation and is responsible for the accumulation of neutrophils in
middle ear effusions.
3) The primary pro-inflammatory cytokines, IL-1beta and TNF-alpha, can
control the IL-8 expression and secretion during middle ear inflammation,
therefore IL-8 can be considered as the secondary cytokine in middle ear
inflammation (Figure 2).
4) IL-8 participates in the middle ear local immune response to viral
and both Gram-positive and Gram-negative, bacterial pathogens.
5) IL-8 is present in different types of otitis media and is involved
in both acute and chronic inflammatory reactions, in the middle ear.
In general, IL-8 is the mediator of the ongoing inflammatory process
in the middle ear and is responsible for inflammatory cell infiltration
into the middle ear tissues and accumulation of inflammatory cells in
middle ear effusions.
Investigations of the pro-inflammatory cytokines in human and experimental
OME demonstrate that all four cytokines TNF-alpha, IL-1beta, IL-6 and
IL-8 are produced in the middle ear tissues in response to bacterial and
viral pathogens and participate in stimulation and regulation of the molecular
processes accompanying the middle ear inflammation (Figure 2).
ENDOTOXINS, PRIMARY CYTOKINES TNF-alpha
AND IL-1beta AND INFLAMMATORY REACTIONS IN THE MIDDLE EAR
All four cytokines, TNF-alpha, IL-1beta, IL-6 and IL-8, are involved
in the pathogenesis of otitis media caused by both Gram-positive [88]
and Gram-negative bacterial infection [89]. However, in this chapter we
are focusing on endotoxin-induced otitis media, because endotoxins (lipopolysaccharides),
the major components of the outer membrane of Gram-negative bacteria,
play an important role in the manifestation of Gram-negative infection
[103], and endotoxins are detected in middle ear effusions together with
the primary cytokines TNF-alpha and IL-1beta [72, 104]. It is important
to note that endotoxins can be also released in a biologically active
form during death of the microorganisms resulting from host immune mechanisms
or antibiotic treatment [105].
Endotoxins are identified in middle ear effusions from patients with
chronic OME [106, 107], and in the persistent effusions following acute
otitis media [108, 109]. Endotoxin is not easily eradicated by local host
defense mechanisms and persists in the middle ear, even after effective
antibiotic treatment, for up to 3 months [104]. Endotoxins are identified
in effusions more frequently than the viable bacterial pathogens [110,
111], suggesting that endotoxins by themselves (in the absence of viable
bacteria), can induce inflammation and pathological changes in the middle
ear. This assumption is confirmed in the experimental models of OME, where
endotoxins, derived from different bacterial pathogens (Haemophilus
influenzae, Klebsiella pneumoniae), induce similar histopathological
changes in the middle ear. These changes include: mucosal and submucosal
inflammatory infiltrate containing T-lymphocytes, macrophages and neutrophils
[112, 113], interstitial edema, thickened epithelium with intracellular
edema [114, 115], hyperplasia of goblet cells [116, 117] and dysfunction
of the mucociliary transport system resulting in the accumulation of effusions
in the middle ear cavity [118, 119].
Analysis of molecular mediators of endotoxin-induced OME within in
vivo models shows the following: 1) high concentrations of TNF-alpha
(up to 200 pg/ml) in the middle ear fluid [84] and expression of IL-1beta
messenger RNA in the middle ear mucosa [91] are detected in experimental,
endotoxin-induced OME; 2) the endotoxin-induced pathological changes
in the middle ear, such as vascular extravasation and accumulation of
effusion are significantly attenuated by TNF-alpha binding protein [85]
and anti-IL-1 receptor antibodies [91]. These findings demonstrate that
endotoxin stimulates the local production of TNF-alpha and IL-1beta in
the middle ear, and suggest that TNF-alpha and IL-1beta are the mediators
of endotoxin-induced middle ear inflammation.
The statistically significant correlations between concentrations of
bacterial endotoxin and the primary cytokines in the effusion samples
[76, 104, 120] confirm this hypothesis.
Concentrations of endotoxin and both TNF-alpha and IL-1beta correlate
positively and significantly in the culture-positive (containing viable
bacteria) and in culture-negative effusions [104, 120]. Concentrations
of TNF-alpha and endotoxin also correlate with the type of effusion: in
effusions classified as mucopurulent, both TNF-alpha and endotoxin levels
are significantly higher as compared to mucoid or serous types [76].
The adhesion molecules, intercellular (ICAM-I) and vascular (VCAM-1)
are also present in effusion samples with endotoxin and primary cytokines
[120], but their concentrations do not directly correlate with neither
endotoxin or TNF-alpha and IL-1beta. However, both cytokines TNF-alpha
and IL-1beta and, to some extent, bacterial endotoxin, stimulate ICAM-I
expression in the middle ear epithelium in vitro [121]. It is important
to note that ICAM-I is a specific cell-surface molecule. ICAM-I is expressed
by epithelial, endothelial and antigen-presenting cells [122], and, being
adhesive for ligands on circulating leukocytes, accumulates leukocytes
at sites of inflammation [123]. The membrane-bound integrin receptors
LFA-1 (lymphocyte function associated molecule-1) and Mac-1 (membrane
attack complex-1) are the ligands for ICAM-I [122]. These three molecules,
ICAM-I, LFA-1 and Mac-1, are markers of ongoing inflammation.
Thus, a direct link exists between the external stimulus of the middle
ear inflammation - bacterial endotoxins, and the internal, primary
cytokines of inflammation, TNF-alpha and IL-1beta, and ICAM-1, which is
expressed on the cell surface during inflammation. This fact gives us
the opportunity to show the start of the endotoxin-induced inflammatory
cascade in the middle ear, mediated by the primary pro-inflammatory cytokines
(Figure 3).
Bacterial endotoxin provokes the sustained production of TNF-alpha and
IL-1beta by cells (in particular, macrophages), in the middle ear mucosa.
TNF-alpha and IL-1beta, in turn, induce two important molecular processes:
secretion of secondary cytokine IL-8 by goblet cells and endothelial cells,
and up-regulation of ICAM-1 expression on the epithelium and vascular
endothelium and probably on the antigen-presenting cells. Secreted IL-8
induces chemotaxis of polymorphonuclear neutrophils (PMN), already activated
by the earliest mediators of inflammation (histamine, platelet-activating
factor (PAF), and leukotrienes), from the bloodstream, to the surface
of endothelial cells. The interaction between ICAM-1 receptors on endothelial
cells and its ligand LFA-1 on activated neutrophils binds neutrophils
very firmly to the endothelial surface and facilitates the subsequent
migration of neutrophils from the bloodstream into infected mucosa (Figure
3). The interaction between ICAM-I and its ligands LFA-1 and Mac-1 strengthens
the binding of immunocompetent cells to antigen-presenting cells, which
is a prerequisite for antigen recognition and subsequent immunostimulation
in the middle ear.
CONCLUSION
Local inflammation in the middle ear mucosa is the crucial event in the
development of middle ear effusion (OME). The pro-inflammatory cytokines,
TNF-alpha, IL-1beta, IL-6 and IL-8, are the key mediators of middle ear
inflammation. These cytokines regulate the molecular processes, which
lead to the pathological changes in the middle ear in early stages of
the disease such as: tissue infiltration of inflammatory cells, mucin
hypersecretion, accumulation of effusion in the middle ear cleft. However,
the pro-inflammatory cytokine network, probably against the background
of additional predisposing factors such as eustachian tube dysfunction,
obstructive adenoids, allergy and reflux, can provoke irreversible changes
in the middle ear tissues and encourage chronic otitis media with effusion.
Acknoledgements. We gratefully acknowledge the Wellcome Trust
and the Hearing Research Trust for their financial support.
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