Keywords
rhinosinusitis - airway - nasal polyposis - inflammation - allergy
Introduction
Recent studies show that nasal polyposis (NP) is more than a chronic inflammatory
disorder: it is a disease characterized by a mechanical dysfunction of the nasal mucosa,
namely, a tissue laxity that leads to loss of equilibrium between hydrostatic and
oncotic pressures, the mechanism responsible for limiting the formation of tissue
edema.[1]
[2]
[3]
This inability to properly elevate interstitial hydrostatic pressure in the polypoid
nasal mucosa is closely related to the unique makeup of its extracellular matrix,
which develops as the result of an anomalous tissue remodeling process.[4]
[5] In this context, transforming growth factor beta 1 (TGFβ-1) plays a pivotal role
in the remodeling process of abnormal tissue in cases of NP.[6]
[7]
[8]
[9]
In a previous study,[10] we demonstrated that fibroblasts isolated from patients with chronic rhinosinusitis
without NP exhibit increased matrix metalloproteinase 1 (MMP1) and cysteinyl leukotriene
receptor 1 (CysLTR1) after stimulation with leukotriene D4 (LTD4), with an additional
increase occurring in the presence of TGF-β1. In this previous study, the CysLT receptor
antagonist montelukast effectively reduced the MMP1 and CysLTR1 expression after stimulation
with LTD4 or TGF-β1.
In a recent study, we demonstrated that TGF-β1 is reduced not only in the nasal tissue,
but also in the plasma of aspirin-intolerant patients,[11] due to the chronic systemic inflammatory nature of this disease, which is not limited
to the airways. Patients with a history of aspirin intolerance exhibit an imbalance
in the production of eicosanoids characterized by an increase in proinflammatory mediators
(leukotrienes) at the expense of anti-inflammatory mediators (prostaglandin E2, PGE2).[12]
The aspirin-intolerant subgroup of patients with NP is characterized by a more intense
inflammatory process, which is reflected by higher rates of comorbidity and recurrence
after treatment. Thus, it is important that the relationship between the production
of leukotrienes and TGF-β1 be assessed.
In the present study, we evaluated the effects of the CysLT receptor antagonist montelukast
on the systemic production of TGF-β1 in patients with NP, with or without concomitant
aspirin intolerance.
Materials and Methods
The present study was conducted at the Department of Otolaryngology and Head and Neck
Surgery of our institution between 2015 and 2017. The project was approved by the
local Ethics in Research Committee under protocol number 70890617800005505. All individuals
willingly agreed to take part in the study and provided written informed consent.
The sample comprised 48 individuals with a European Position (EPOS) 2012[13] diagnosis of NP, and 15 healthy controls for comparison of the baseline TGF-β1 levels
in the peripheral blood.
The age of the subjects ranged from 18 to 65 years, and those who had used oral or
topical corticosteroids, immunosuppressants, or antihistamines in the 30 days preceding
the collection of peripheral blood collection and during the clinical assessment were
excluded from the study.
To assess the effects of the montelukast therapy on the production of TGF-β1, 20 of
the 48 patients diagnosed with NP were submitted to a treatment with montelukast for
30 days at a daily dose of 10 mg.
Of these 20 patients with NP, 9 patients with NP without asthma or aspirin intolerance,
6 also had a diagnosis of asthma, and 5 also had a diagnosis of asthma and aspirin
intolerance.
The measurement of the levels of TGF-β1 was performed in peripheral blood samples
collected in 4-mL ethylenediaminetetraacetic acid (EDTA) tubes (Becton Dickinson and
Company, Franklin Lakes, NJ, US) and centrifuged for 10 minutes at 1,000 G . The blood
plasma was collected and stored at -80°C.
The plasma concentrations of TGF-β1 were measured using TGF-β1 MILLIPLEX MAP (EMD
Millipore, St. Charles, MO, US) kits, in accordance with manufacturer instructions.
Briefly, 25 µl of plasma were incubated overnight with color-coded beads coated with
capture antibodies for TGF-β1 at 4°C, with constant agitation on a plate shaker. After
washing, the beads were incubated with biotinylated TGF-β1 secondary detection antibodies
for 1 hour at room temperature, followed by incubation with streptavidin-phycoerythrin
for 30 minutes at room temperature, all under constant agitation on a plate shaker.
Finally, the measurement was performed using a Luminex 100/200 System (Luminex Corporation,
Austin, TX, US). According to the standard curves, the concentration of the respective
cytokines was calculated and expressed in pg/ml.[14]
Statistical Analysis
The statistical analyses were performed using the Statistical Package for the Social
Sciences (SPSS, IBM Corp., Armonk, NY, US) software, version 22, and GraphPad Prism
(GraphPad Software Inc., San Diego, CA, US) software, version 7. The nonparametric
Wilcoxon test was used to assess the differences within the groups before and after
the montelukast treatment. The Mann-Whitney U test was used to assess the differences
in the baseline level of TGF-β1 between two independent samples (individuals with
and without NP). In all cases, values of p < 0.05 were deemed statistically significant.
Results
There were no gender differences in the levels of TGF-β1 in either group. The mean
age was significantly higher in the NP group (p < 0.05)
Baseline TGF-β1 Levels
The comparison of the plasma levels of TGF-β1 between the control and NP groups revealed
no significant difference. However, there was a trend toward lower levels of TGF-β1
in the NP group (p = 0.09, [Figure 1], [Table 1]).
Table 1
Levels of transforming growth factor beta 1 (TGF-β1; pg/ml)
|
Minimum
|
Maximum
|
Mean
|
Standard deviation
|
|
Control (baseline, healthy nasal mucosa)
|
304
|
7,299
|
3,150
|
2,045
|
|
Nasal polyposis without aspirin intolerance (before the treatment)
|
2.04
|
5,539
|
1,905
|
1,922
|
|
Nasal polyposis with aspirin intolerance (before the treatment)
|
2.04
|
4,289
|
1,914
|
1,310
|
|
Nasal polyposis without aspirin intolerance (after the treatment)
|
2.04
|
6,469
|
2,410
|
1,988
|
|
Nasal polyposis with aspirin intolerance (after the treatment)
|
3.73
|
5,239
|
1,794
|
1,610
|
Fig. 1 Trend toward lower baseline levels of transforming growth factor beta 1 (TGF-β1)
in the nasal polyposis group compared to the control group.
When the NP group was further subdivided into patients with NP (NP alone and NP + asthma
without aspirin intolerance) and NP + aspirin intolerance, there was no statistically
significant difference in the baseline levels of TGF-β1 among these subgroups.
Effect of the Leukotriene Receptor Blocker Montelukast on the production of TGF-β1
There was no statistically significant difference in the plasma levels of TGF-β1 in
the patients with NP who underwent the montelukast (p = 0.9) treatment. There was also no difference in the change in TGF-β1 levels after
the treatment with montelukast in the subgroup of patients with NP and asthma (p = 0.82) and in the subgroup with NP, asthma, and aspirin intolerance (p = 0.51), [Figure 2], [Table 1].
Fig. 2 Comparison of transforming growth factor beta 1 (TGF-β1) levels before and after
the montelukast treatment between different nasal polyposis subgroups, showing no
statistical significance.
Discussion
It is believed that NP is triggered by a chronic inflammatory process that leads to
a mechanical dysfunction of the nasal mucosa in predisposed patients.[1] This mechanical dysfunction is associated with a unique remodeling process, in which
TGF-β1 plays a pivotal role.[6]
Many studies[7]
[8]
[9] suggest that TGF-β1 is decreased in the polypoid nasal tissue, and systemic (plasma)
levels of this cytokine have also been shown to be decreased in patients with aspirin
intolerance.[11]
A research[12] has also shown that patients with aspirin intolerance have abnormal eicosanoid production,
with increased synthesis of leukotrienes. In NP, leukotriene receptors are overexpressed
in the polyp tissue.[15]
In cases of chronic rhinosinusitis, LTD4 and TGFβ-1 induce the expression of metalloproteinase
through CysLTR1,[10] and an increased expression of metalloproteinases is found in the nasal polyp tissue,
such as MMP7 and MMP9.[6] The close correlation between TGF-β1 and the production of leukotrienes in cases
of NP can also be highlighted by the fact that leukotrienes are found increased in
NP, especially in aspirin-intolerant patients,[12] who present a descreased systemic expression of TGF-β1 levels not only in the tissue,
as it is usually found in cases of NP without aspirin intolerance,[11] showing that the altered production of eicosanoids in aspirin-intolerant patients
provokes a systemic inflammation, unlike the local inflammation found in NP. On the
other hand, TGF-β1, a protein that plays a pivotal role in the balance of fibrinolysis
and fibrogenesis and, consequently, on the extracellular matrix production,[10] is found decreased in nasal polypoid tissue.[9]
Within this context, the present study sought to investigate whether there is a direct
relationship between the exacerbated effects of leukotrienes[16] and the decreased concentrations of TGF-β1 found in cases of NP.
In a previous study,[10] we demonstrated that the association of TGF-β1 with LTD4 in the nasal polyp tissue
stimulates the production of MM1 via the leukotriene receptor.
In the present study, although we found no significant difference, there was a trend
toward lower baseline levels of TGF-β1 in the NP group compared with the healthy controls,
which is consistent with the findings reported in the literature.[11]
It is noteworthy that, although aspirin intolerance is characterized by its systemic
nature,[11] the effects of the inflammatory process in cases of NP are often exclusively localized,
with no detection in the plasma mediators. Further studies are warranted, with a particular
focus on measuring tissue levels of TGF-β1, to elucidate the actual role of leukotriene
receptor blockers in modifying the nasal mucosa remodeling process. Studies designed
to assess quality of life and nasal symptoms after the administration of these drugs
would be particularly important to demonstrate the true dimension of their potential
for clinical use.
Conclusion
We found no impact of the therapy with a leukotriene receptor blocker on the production
of TGF-β1, making antileukotriene therapy a highly questionable choice for the treatment
of NP, particularly from the standpoint of seeking to modify the remodeling process
in this disease.
