ARTICLE
Auteur(s) :, Cengiz
KirmazCengiz
Kirmaz1,2,*, Papatya Bayrak1, Ozge
Yilmaz3, Hasan Yuksel3
1Celal Bayar University Medical Faculty, Department
of Internal Medicine, Division of Immunology and Allergy, Manisa,
Turkey
2275/8 Sok. No: 16, K: 3, D: 9, Hazal Apt. Bornova,
Izmir, Turkey
3Celal Bayar University Medical Faculty, Department of
Paediatric Allergy, Manisa, Turkey
Allergic rhinitis (AR) is clinically defined as a symptomatic
disease of the nasal mucosa caused by an IgE-mediated allergic
inflammation [1]. Recent insights into the development of allergic
diseases such as AR, asthma and atopic eczema are based on the
functional diversity of T helper (Th) 1 and Th2 lymphocytes [2].
Th2 cells (secreting IL-4, IL-5, IL-9 and IL-13) are now considered
to be responsible for the induction, as well as for many of the
manifestations of atopic diseases [2, 3]. In these diseases, IL-4
plays a key role in driving the differentiation of CD4+
Th precursor into Th2, and B cells into IgE-producing cells [4].
IL-5 stimulates eosinophil generation and chemotaxis; it activates
mature eosinophils while prolonging their survival [5]. T
cell-mediated overproduction of Th2 cytokines (IL-4, IL-5 and
IL-13) locally at the site of allergic inflammation in atopic
individuals, has been reported by several authors [6, 7].
Additionally, Th1 and Th2 cells can inhibit each other’s function
when they are activated, through several cytokines such as IL-10,
IL-4 for Th2 and IFN-α IL-12 for Th1 cells [4, 8].The trend in
immunological modulatory treatment with biological agents has
recently been a topic of interest, however, it was initiated by
research performed 30 years ago [9-11]. Pillemer and Ecker [12]
applied the name zymosan to a yeast cell wall fraction of
Saccharomyces cerevisiae, which produced prominent hyperplasia and
hyperfunction of the reticuloendothelial system when administered
to experimental animals. Glucan, a β-1,3 linked glucopyronase
polysaccharide of approximately 6.5 kD, has been isolated from
zymosan and shown to be a potent activator of the
macrophage/monocyte cell series, and also, responsible for most of
the stimulatory effects on the reticuloendothelial system [13-15].
Results of many studies following this up have revealed antitumor
properties of glucans [16-19]. The Th1 type of immune response
causes an activation of macrophage functions and growth, an
increase in accessory cell functions and antigen presentation, as
well as strong chemotaxis in lymphocytes and leucocytes.
Additionally, it leads to a strong cellular immune response against
tumor cells and viruses by producing cytokines (IFN-α, IL-12) [8].
It is known that an efficient antitumor response requires a
Th1-mediated immune reaction. Glucan may be causing a skewing of
the immune system towards a Th1 response. Nevertheless, lentinan,
which is an important source of Glucan, has been reported to be a
possible immunomodulator therapy in patients with cancer of the
digestive tract by decreasing the Th2-mediated immune response and
converting it to a Th1-mediated response [19].In the light of all
this knowledge, we designed this study to investigate the effects
of Glucan on immunopathogenic processes in the microenvironment and
its ability to restore a balance between Th2- and Th1-mediated
immune response in AR.
Materials and methods
Subjects and study design
The study was carried out according to a randomized, doubled-blind,
parallel group, placebo-controlled design.
A total of 26 patients with seasonal AR participated in this
study. All of the patients selected for this study were sensitive
only to olive pollen. The study was performed in autumn and winter,
out of the pollen season, to avoid natural allergen provocation.
Before the study, informed consent for the described procedures was
obtained from all patients. Approval for the study was given by the
local ethics committee of our hospital. Diagnosis of seasonal AR
was based on history, physical examination and laboratory findings.
Allergic sensitization was demonstrated by the skin prick test.
Skin prick tests were performed according to the EAACI guidelines
[20] for the most common inhalant allergens in Turkey, including
house dust mites (Dermatophagoides pteronyssinus and
Dermatophagoides farinae), fungi (Aspergillus fumigatus, Alternaria
alternata, Cladosporium herbarum, Penicillium notatum), grasses
(Lolium perenne, Fectuca pratensis, Phleum pratense, Poa pratensis,
Dactylis glomerata), weeds (Plantago lanceolata, Artemisia
vulgaris, Rumex acetosa, Taraxacum vulgare, Parietaria officinalis)
and trees (Sambucus nigra, Populus alba, Ulmus scabra, Salix
caprea, Fagus silvatica, Carpinus betulus, Quercus robur, Fraxinus
excelsior, Olea europea) with histamine and diluent control
(Allergopharma Ltd, Reinbek, Germany). Two patients were excluded
because the nasal provocation test (NPT) with Olea europea
(ALK-Abello, Madrid, Spain) could not be performed due to the total
obstruction of at least one nostril, as shown by peak nasal
inspiratory flow (PNIF) measurements. The NPT with Olea europea was
performed on 24 patients who were included in the study after
having stopped their medications (oral anti-histamines and nasal
topical corticosteroids for 1 week and nasal topical
anti-histamines for at least 48h before NPT). After 30 min of
NPT, nasal lavage was performed on all subjects for measurement of
IL-4, IL-5, IFN-α, IL-12 and eosinophil count (%). Blood samples
were obtained to measure blood eosinophils (%). Subjects were then
randomized into two matched groups: Glucan group (n = 12) to be
treated with β-1,3-1,6-glucan (Imuneks® capsule, Mustafa
Nevzat Ilac San., Istanbul, Turkey), 10 mg, twice a day for 12
weeks, and the placebo group (n = 12) with the same posology and
duration. Following both treatments, all subjects underwent a
second NPT with Olea europea, together with the nasal lavage, and
blood sampling.
Nasal allergen provocation test
Subjects were allowed to rest for 15 min to ensure that
equilibration of the nasal mucosa with the environmental conditions
of the laboratory was achieved. Symptom scores and PNIF values
measured with a PNIF meter device (Clement Clarke Int Ltd, Essex,
UK), which were reported to reveal quite specific and sensitive
results in the NPT, were recorded initially as a baseline [21, 22].
Specific allergen extract was insufflated into each nostril using
spray bottles containing incremental concentrations of allergen (2,
4, and 8 BU/mL); these insufflations delivered 0.16, 0.32, and 0.64
μg of allergen, respectively. At each insufflation, the nasal
reaction obtained was recorded according to a symptom score
technique. Patients were challenged with incremental doses of the
allergen (0.016, 0.032, and 0.064 μg in each nostril) until a
composite symptom score of 6 or more accompanied by a 20% or more
decrease in PNIF was obtained. The clinical criteria for a positive
NPT result included symptom assessment through the use of a
semiquantitative method similar to that used by others [23, 24].
Briefly, 3 major nasal symptoms (sneezing, rhinorrea, and nasal
obstruction) were recorded 10 minutes after nasal challenge,
according to a scale ranging from 0 (no symptoms) to 3 (severe). A
total nasal symptom score was calculated by adding these scores;
the maximum possible score was 9 for each subject. A composite
symptom score of 6 or more was the clinical criterion for a
positive response to the NPT. In addition to this, a 20% or more
decrease in PNIF was the laboratory criterion for a positive
response to NPT. Cumulative doses of allergen required to induce a
nasal response varied from 0.016 to 0.112 μg for each nostril.
Nasal lavage
Nasal lavages were performed 30 min after NPT. The subject, in
the sitting position, was instructed to extend the neck
approximately 30 degrees from the horizontal and to refrain from
breathing or swallowing. The nasal cavity was washed with
10 mL of physiological saline, warmed to 37 °C, by
reciprocating the piston of the syringe 10 times on each side while
the subject did not breathe or swallow. The subject flexed the neck
and expelled the nasal lavage fluid (NLF) into a collection pot 10
seconds after each lavage. All NLF was filtered through a
52 μm nylon filter to remove mucin, and the filtrate was
centrifuged at 4 °C for 10 min at 1000g. The supernatant
was stored at -80 °C until assayed for cytokine levels. The
sediment was subjected to cell count measurement.
Measurements of cytokines and eosinophil count
Levels of cytokines in NLF supernatant were measured using
commercially available ELISA kits (IL-4, Catalog # KHC0041; IL-5,
Catalog # KHC0051; IFN-α Catalog # KHC4021; IL-12, Catalog #
KAC1561. BioSource International, Inc., Camarillo, CA, USA)
following the manufacturer’s instructions. Levels of these
mediators below the sensitivity of the assay were 2 pg/mL for IL-4,
4 pg/mL for IL-5, 4 pg/mL for IFN-α, 1.5 pg/mL for IL-12.
The sediment from the NLF was resuspended in 1 mL RPMI 1640
(Gibco BRL, Life Technologies Ltd, Paisley, UK) containing 10% FCS.
An equal volume of 16% N-acetylcysteine in calcium-free, Hank’s
balanced salt solution (Gibco BRL, Life Technologies Ltd, Paisley,
UK) was added to each sample and samples were then incubated for 45
minutes at 37 °C to disaggregate remnant mucus and clumps.
Differential counts were performed on cytospin slides stained with
DiffQuick (DADE S.p.A. Rome, Italy) and examined by means of light
microscopy. Pre-treatment and post-treatment percentage eosinophil
values were used for statistical evaluation. Additionally, the
peripheral blood eosinophil count (%) of each subject was recorded
by a hemocounter pre- and post-treatment.
Statistical assessment
The significance of differences between within-group and
inter-group comparisons were determined using the Wilcoxon signed
rank’s test and the Mann-Whitney U test, respectively. Correlations
were evaluated by the Pearson’s correlation test. Differences were
considered significant when the p-value was less than 0.05.
Results
The study was completed with 24 patients. The demographical,
disease and SPT characteristics of our patients are summarized in
table 1( Table 1 ). There were no
differences in these characteristics between the groups.
Mean allergen dosage ± SEM required for a positive nasal
response in the Glucan group increased minimally from 0.04 ± 0.008
μg at the pre-treatment period to 0.056 ± 0.011 μg at the
post-treatment period. However, the difference between the two
values was not statistically significant (p = 0.21) ( (figure 1) ). Similarly,
the mean allergen dosage required for a positive nasal response in
the placebo group did not display a significant change at the
pre-treatment and post-treatment periods (0.04 ± 0.011 μg and 0.037
± 0.005 μg; respectively) (p = 0.78) ( (figure 1) ).
Eosinophil count
There were no differences in the pre-treatment values for the mean
eosinophil ± SEM count in NLF between the Glucan and placebo groups
(15.33 ± 0.90%, 13.50 ± 1.18%; respectively) (p = 0.23) ( (figure 2) ). The
eosinophil count in the NLF did not differ between the two groups
post-treatment either (11.83 ± 1.06% for the Glucan group versus
14.17 ± 0.87% for the placebo group) (p = 0.12) ( (figure 2) ). However,
eosinophil count in NLF from the Glucan group decreased
significantly after treatment (p = 0.01) ( (figure 2) ). The
percentage of eosinophils in the NLF did not differ between the
pre-treatment and post-treatment period in the placebo group (p =
0.53) ( (figure
2) ). Decreases in IL-5 values were significantly
correlated with the decrease in the eosinophil count in the NLF
following treatment in the glucan group (r = 0.676; p = 0.016),
while there was no correlation with the changes in other cytokines.
The peripheral blood eosinophil count was very similar in the
two groups before treatment. (5.75 ± 0.63% for Glucan group versus
5.58 ± 0.5% for the placebo group) (p = 0.86). Similarly, the
peripheral eosinophil count was very similar for the groups after
treatment, (5.83 ± 0.56% for the Glucan group versus 5.33 ± 0.33%
for the placebo group) (p = 0.46). Comparison of peripheral
eosinophil counts within the groups did not demonstrate any
significant difference for either group (p = 0.8, p = 0.56;
respectively).
Table 1 Demographical, disease and SPT characteristics
of our patients. NS, p-value not significant
|
Glucan group
|
Placebo group
|
p-value
|
|
(n = 12)
|
(n = 12)
|
|
Age (year) (mean ± SEM)
|
33.5 ± 3.34
|
32.25 ± 3.75
|
NS
|
|
Gender (F/M)
|
5/7
|
6/6
|
NS
|
|
Disease type
|
Seasonal AR
|
Seasonal AR
|
|
|
Disease duration (year) (mean ± SEM)
|
8.08 ± 0.72
|
7.42 ± 0.79
|
NS
|
- SPT result
- Olea europea/Others
|
12/0
|
12/0
|
|
Results for the cytokines
Results for all cytokines are summarized in table 2( Table 2 ) as mean±SEM. All cytokine levels in NLF
before treatment and in the Glucan group did not differ from the
values found in the placebo group. However, the IL-12 level in the
Glucan group was significantly higher than that in the placebo
group (p = 0.014) ( (figure 3d) ).
Comparison of IL-4 and IL-5 levels within the Glucan group
before and after treatment revealed a significant decrease after
treatment (p = 0.027, p = 0.04; respectively) ( (figure 3a,b) ). We
observed that the IL-12 level increased significantly in the Glucan
group after treatment (p = 0.008). However, we found that IFN- α
levels in the Glucan group before and after treatment did not
change significantly (p = 0.1) ( (figure 3c) ).
Additionally, cytokine levels obtained before treatment were seen
not to change significantly after treatment in the placebo
group.
Changes in any cytokine level with treatment in the Glucan group
were found not to be correlated with changes in any other
cytokine.
Table 2 Mean±SEM cytokine levels in nasal lavage fluid
of the Glucan and placebo groups before and after treatment
|
|
|
p-value
|
|
Pre-treatment
|
Post-treatment
|
Within group
|
Intergroup
|
|
Glucan
|
Placebo
|
Glucan
|
Placebo
|
Glucan
|
Placebo
|
Pre-treatment
|
Post-treatment
|
|
IL-4 (pg/mL)
|
5.48 ± 0.92
|
4.63 ± 0.69
|
3.66 ± 0.64
|
4.45 ± 0.85
|
0.027
|
0.622
|
0.624
|
0.272
|
|
IL-5 (pg/mL)
|
8.58 ± 1.58
|
6.78 ± 0.69
|
5.81 ± 0.83
|
6.57 ± 0.68
|
0.04
|
0.753
|
0.707
|
0.214
|
|
IFN-α (pg/mL)
|
6.19 ± 1.18
|
6.13 ± 0.58
|
7.83 ± 1.22
|
6.85 ± 0.80
|
0.1
|
0.384
|
0.386
|
0.84
|
|
IL-12 (pg/mL)
|
11.08 ± 2.43
|
10.48 ± 1.51
|
17.31 ± 2.75
|
9.97 ± 1.54
|
0.008
|
0.8
|
0.773
|
0.014
|
Discussion
Allergic rhinitis is a disease characterized by an IgE-mediated
inflammation, which presents with a Th2-mediated immune response
[1]. Eosinophils especially, play an important role in the allergic
inflammatory process. Specific IgE synthesis is known to be
mediated by IL-4 and IL-13 in patients with AR [1]. Previous
studies have found that Th2-originated cytokines predominated in
the evaluation of the NLF and in the activated peripheral blood
mononuclear cell supernatant of patients with AR when compared to
the non-atopic subjects [25-29]. Evaluation of treatment of AR and
other diseases with a similar allergic nature requires the
determination of the reversal of this cytokine pattern to one which
originates from Th1 [30]. We also detected a decrease in the levels
of Th2-originated IL-4 and IL-5 in NLF with Glucan treatment in
this study. These results suggest that the Th2-mediated immune
response is inhibited, especially in the microenvironment. During
their extensive investigations of the Th1 and Th2 phenomena,
Murata’s group administered lentinan to mice [31]. Lentinan is a
β-1,3-D-glucan from the medicinal mushroom Lentinus edodes. This
molecule closely resembles other glucans typically liberated from
fungal cell walls during digestion or in the course of an immune
response. When they gave lentinan intraperitoneally to mice, the
macrophage glutathione status as well as their capacity to produce
IL-12 improved, thus orientating them toward Th1 immunity. This
effect could be turned off by depleting glutathione, and then
restoring it using alpha-lipoic acid, an antioxidant nutrient that
replenishes glutathione [31]. Murata and colleagues also collected
Th cell populations from the mice following lentinan exposure,
assessed their cytokine production capacity, and found that they
had shifted toward Th1 production instead of Th2 [31]. Similarly,
the decrease in eosinophils that play an essential role in the
immunopathogenesis of AR, in NLF following treatment, may be due to
the inhibitory role of Glucan in allergic immunopathogenesis.
Correlation of the decrease in IL-5 levels and eosinophil counts in
NLF indicates that the changes in immunological parameters that we
detected coexist with the changes in inflammatory parameters. It
was not surprising to find that, in the NLF, the treatment-mediated
decrease in IL-5, which is a stimulator of eosinophil generation
and chemotaxis and which prolongs the survival of eosinophils,
parallels the decrease in the level of eosinophils after treatment
[5]. Absence of a change in the eosinophil count in peripheral
blood indicates that the immunological change mediated by treatment
may not be systemic.
The increase in IL-12, which is secreted from Th1, in the NLF of
patients receiving Glucan treatment, indicates that the systemic
use of Glucan in patients with AR may convert the immune response
towards Th1. There are earlier, cytokine-based studies involving
allergen-specific immunotherapy, which demonstrated that a
Th1-mediated immune response predominated with this treatment [30].
Therefore, the increase in IL-12 levels that we have detected may
indicate the predominance of a Th1 immunological response. Saito et
al. have reported that Glucan increases IL-12 levels in vitro and
decreases Ig-E synthesis in vivo, inhibiting the Th2-mediated
immune response, similar to our results [32]. Authors in this
previous study have concluded that glucans can be used in the
prevention and treatment of allergic diseases. However, the absence
of a change in the levels of IFN-α, which is also secreted from
Th1, may be the result of the lack of an adequate time period
during which the pattern of this cytokine may alter. Nevertheless,
previous research on AR patients receiving allergen-specific
immunotherapy revealed that changes in levels of IFN-α manifested
only during the second year of treatment [33].
Although insignificant, increases in the provocative allergen
dose required to induce a positive nasal response in the group
receiving Glucan treatment suggest that Glucan may suppress the
allergen-mediated response, along with changes in
microenvironment.
In conclusion, Glucan treatment may be used as an adjunct to
treatment in patients with AR because of the immunological
modulation it causes.
Acknowledgements
We thank Dr Ahmet VAR (Celal Bayar University Medical Faculty,
Department of Biochemistry) for expert technical support for the
ELISA application.
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