Altered expression of IL-18, IL-18 binding protein and IL-18 receptor in blood monocytes of patients with allergic rhinitis

Background: Interleukin (IL)-18 is emerging as an attractive participant in allergic rhinitis (AR). However, correlation of IL-18 with IL-18 binding protein (BP) in plasma, and expression of IL-18, IL-18BP and IL-18 receptor (R) in AR blood monocytes remain obscure. Methods: We investigated IL-18, IL-18BP and IL-18R expression in monocytes by using ow cytometric analysis, murine AR model, and quantitative real-time PCR in the present study. Results: It was found that plasma IL-18 and IL-1β in AR patients was higher than those in healthy control subjects. Free (f)IL-18 had a high correlation with IL-18BP, IL-1β and TNF-α in AR plasma. Proportion of IL18 + monocytes was increased, whereas IL-18BP + monocytes were decreased in blood of patients with AR. It was found that Platanus pollen allergen extract provoked the elevated expression of IL-18 and IL-18R in AR blood monocytes. Dermatophagoides pteronyssinus , Artemisia sieversiana wild and Platanus pollen allergen extracts enhanced IL-18R protein and mRNA expression in primary monocytes from AR patients. Moreover, numbers of macrophages and IL-18R + macrophages in nasal lavage uid (NLF) were increased, and levels of IL-18 in both plasma and NLF were elevated in AR mice. Conclusions: IL-18 is likely to participate in the development of AR as a causative factor, therefore, it could be a therapeutic target for AR.

Background IL-18, initially discovered as interferon (IFN)-γ-inducing factor constitutively expressed by monocytes and macrophages, and plays regulatory roles in both innate and adaptive immunity. It is reported that polymorphisms of IL-18 gene was associated with AR [1][2][3][4][5], and up-regulated IL-18 is found in both nasal secretion and serum of AR patients [6,7]. It is also observed that elevated serum IL-18 during natural pollen exposure is closely associated with bronchial hyperresponsiveness in seasonal AR (sAR) patients [8]. These implicate that monocyte-and macrophage-derived IL-18 likely contributes to the pathogenesis of AR.
IL-18 binds initially to IL-18R then initiates MyD88-dependent signal pathway and exert immunomodulatory functions [9]. IL-18 binding protein (IL-18BP) is an endogenous soluble antagonist that speci cally inhibits IL-18 action by binding to IL-18 with high a nity [10]. Free serum IL-18BP is present at 20fold higher levels than free IL-18 in physiological status [11]; however, under allergic conditions IL-18 may be in excess [12]. These suggest an imbalance between IL-18 and IL-18BP expression may account for increased IL-18 activity in AR. Indeed, we reported recently that the role of IL-18 in atopic asthma is determined by the balance of IL-18/IL-18BP/IL-18R [13]. Since level of circulating IL-18BP in AR plasma/serum has not been reported, we examined level of IL-18BP in AR plasma in the present study.
It has been reported that pollen allergen speci c subcutaneous immunotherapy induced increased serum IL-18 in AR patients [14], pollen allergen extract could provoke IL-18 mRNA expression in PBMCs of patients with AR undergoing allergen immunotherapy [15][16][17], suggesting that allergens may contribute to AR through PBMCs and IL-18-related mechanisms. Since monocytes are one of the major cell types of PBMCs, and little is known about expression of IL-18, IL-18BP and IL-18R at protein and mRNA levels in monocytes of AR, particularly upon allergen challenge. Therefore, the aim of the present study is to investigate levels of IL-18 and IL-18BP in AR plasma, expression of IL-18, IL-18BP and IL-18R in monocytes of patients with AR, and in uence of allergens on their expression.

Subjects and animals
A total of 33 patients with perennial allergic rhinitis (pAR), 9 patients with sAR and 25 healthy control (HC) subjects were recruited in the study. Their general characteristics were summarized in Additional le 1 (Table S1). The diagnosing criteria of pAR and sAR were conformed to the Chinese Society of Allergy Immediately after con rmed diagnosis (acute stage), peripheral blood from each patient with allergic rhinitis was collected. Blood from HC were collected in the outpatient clinic. From each donor, 5 mL was taken into an EDTA containing tube before centrifugation at 450×g for 10 min. The cells were used for ow cytometric analysis, and plasma was collected and frozen at -80°C for analysis of cytokines. For CD14 + monocytes isolation study, 180 mL of peripheral blood was taken from each donor.
Five-week-old female BALB/c mice were obtained and maintained as described previously [12]. The animal experiment procedures were authorized by the Animal Care Committee at Jinzhou Medical University.
Isolation of CD14 + monocytes and allergen challenge test CD14 + cells were enriched by density gradient centrifugation and positive selection by using magnetic beads on magnetic cell sorting (MACS) according to the manufacturer's instructions. Final recovery of cells was determined with an improved Neubauer haemocytometer after being stained with trypan blue solution, and recovered cell purities were assessed by ow cytometry with an anti-human CD14 antibody.
To further investigate the direct action of allergen on the expression of IL-18, IL-18BP and IL-18R in monocytes, the isolated primary monocytes at a density of 1×10 6 per mL were cultured in RPMI 1640 medium containing 3% FBS and 100 U/ml penicillin/streptomycin in a 12-well cell culture plate (Nest, Wuxi, China) in the presence or absence of ASWAE, PPAE, DPAE (all at a concentration of 1.0 μg/mL) or IFNγ (as positive control) at 5 ng/mL for 10, 30 and 60 min, respectively at 37°C in a 5% (v/v) CO 2 , watersaturated atmosphere. Brefeldin A at 2 μg/mL was added in wells for detecting the intracellular expression of IL-18 and IL-18BP before stimulation. Cells were then harvested and centrifuged at 450×g for 10 min at 4°C. Cell pellets containing approximately 0.5×10 6 and 1×10 6 cells were resuspended in PBS for ow cytometric analysis, and in TRIzol reagent for RT-PCR, respectively. Cell culture supernatant was collected and frozen at -80°C for further use.
Flow cytometric analysis of IL-18, IL-18BP and IL-18R in human peripheral blood monocytes and isolated CD14 + monocytes The procedures for detecting IL-18, IL-18BP and IL-18R expression in human peripheral blood monocytes were mainly adopted from a previous study by Zhang et al [13]. Brie y, whole blood cells were challenged with or without ASWAE, PPAE, or DPAE (all at a concentration of 1.0 μg/mL) for 1 h, and isolated CD14 + monocytes were challenged for 10, 30 and 60 min at 37°C.
For cell surface molecules, whole blood cells were preincubated with human Fc receptor blocking solution and a live/dead cell dye (Zombie Green TM Fixable Viability Kit) [19] for 15 min, then stained with PE/Cy7 conjugated anti-human CD14 and APC conjugated anti-human IL-18Rα antibodies. Following red blood cell lysis, cells were analyzed with FACS Verse ow cytometer (BD Biosciences, San Jose, CA, USA). An irrelevant isotype-and concentration-matched antibody of anti-human IL-18Rα was used for uorescence minus one (FMO) control. Dead cells and doublets were discriminated by SSC-A-live/dead cell dye and FSC-H-FSC-A gating strategies. As for magnetic isolated monocytes, cells were processed as above.
For intracellular molecules, whole blood cells were preincubated with human Fc receptor blocking solution and a live/dead cell dye, subsequently stained with PE/Cy7 conjugated anti-human CD14 as described above. After lysing red blood cells, resuspended leukocytes were xed and permeabilized, and stained with PE conjugated anti-human IL-18 antibody, and anti-human IL-18BP primary antibody followed by the addition of BV510-conjugated donkey anti-rabbit polyclonal antibody. Finally, cells were processed and analyzed as above. As for isolated primary monocytes, cells were processed as above.
Establishment of mouse allergic rhinitis model OVA-induced allergic rhinitis mouse model was mainly adopted from a previous study by Mo JH et al [20]. Brie y, mice were sensitized on days 0, 7 and 14 by intraperitoneal injection of 25 μg OVA emulsi ed in 1 mg of alhydrogel. On days 21-27 mice were challenged by intranasal instillation with 500 μg of OVA dissolved in PBS (10 µL/nostril) once daily. For control experiments, healthy mice received vehicle only instead of OVA solution. At 24 h following the last OVA challenge, blood and nasal lavage uid (NLF) were collected from each mouse. Total cells were determined and collected after centrifugation as described above. The cells were used for ow cytometric analysis, plasma and NLF supernatant was collected and frozen at -80°C until use.
To evaluate allergic symptoms, numbers of sneezing and nasal-rubbing motions during the rst 15 min after each OVA challenge were recorded and compared with healthy control mice (HM) by observers blinded to the study. As presented in Additional le 2 ( Fig. S1a, b), the numbers of nasal rubbing and sneezing motion in AR mice were substantially higher than that in HM during a 7-day observation period.

Flow cytometric analysis of IL-18R in mouse blood monocytes and NLF macrophages
To detect IL-18R expression in mouse blood monocytes, whole blood cells were preincubated with antimouse CD16/32 antibody and a live/dead cell dye (Zombie NIR TM Fixable Viability Kit) [21] for 15 min.
Each labeled monoclonal antibody including BV510-conjugated anti-mouse Gr-1, PerCP-conjugated antimouse CD11b and APC-conjugated anti-mouse IL-18R was added into tubes for 15 min before red blood cells being lysed. Finally, cells were processed as for human blood samples and analyzed by using ow cytometry.
To detect IL-18R expression in NLF macrophages, cells were incubated with anti-mouse CD16/32 antibodies and a live/dead cell dye, followed by incubation with BV510-conjugated anti-mouse F4/80 and APC conjugated anti-mouse IL-18R antibodies, and analyzed as above.
Real-time PCR for IL-18, IL-18BP and IL-18R in isolated CD14 + monocytes Total RNA was extracted from magnetically sorted blood monocytes as described previously [22]. Brie y, after synthesizing cDNA from total RNA by using RT Master Mix Perfect Real Time, qPCR was performed with SYBR Premix Ex TaqII Kit on the Real-time Thermal Cycler (Thermo Fisher Scienti c Oy, Vantaa, Finland). Each reaction contains 12.5 μL of 2×SYBR green Master Mix, 300 nM oligonucleotide primers, and 10 μL of the cDNA. Untreated controls were chosen as the reference samples, and the ΔCt for all experimental samples were subtracted by the ΔCt for the control samples (ΔΔCt). The magnitude change of test gene mRNA was expressed as 2 −ΔΔCt . Each measurement of a sample was conducted in duplicate. The forward and reverse primers for human IL-18, IL-18BP and IL-18R were listed in Additional le 3 (Table S2).
Measurement of cytokine levels in plasma and NLF supernatant, and calculation of molar concentration ratio of plasma IL-18BP/IL-18 Levels of total IL-18 (tIL-18) and IL-18BP (tIL-18BP) in plasma or NLF supernatant were determined by using ELISA kit according to the manufacturer's instructions. Molar concentration of human IL-18 and IL-18BP was calculated using the equation of mass concentration divided by molecular weight, and free IL-18 (fIL-18) and IL-18BP (fIL-18BP) was calculated on the basis of a 1:1 stoichiometry in the complex of IL-18 and IL-18BP with a dissociation constant of 400 pM [23].
Human bio-plex panel (Bio-Rad Laboratories, California, USA) was employed to detect human plasma levels of IL-1β and TNF-α. The detection ranges for IL-1β and TNF-α were 0.24-3994 pg/mL and 0.57-9270 pg/mL, respectively.

Statistical analysis
Statistical analyses were performed by using SPSS software (version 21.0, IBM Corporation). Data are displayed as a boxplot, which indicates the median, interquartile range, the largest and smallest values for the number of experiments indicated. Where Kruskal-Wallis analysis indicated signi cant differences between groups, a pairwise test was used for multiple comparisons between the groups. Correlations were determined by using Pearson's correlation or Spearman rank correlation analysis. For all analyses, P < 0.05 was considered statistically signi cant.

Results
Elevated level of IL-18 in plasma of patients with pAR and sAR Using ELISA kits, it was observed that levels of tIL-18 (Fig. 1a) and fIL-18 (Fig. 1c) in plasma of patients with pAR and sAR were elevated in comparison with HC subjects. In contrast, there were no signi cant differences of plasma levels of tIL-18BP (Fig. 1b) and fIL-18BP (Fig. 1d) between patients with pAR or sAR and HC subjects being observed. It was found that the molar concentration ratio of fIL-18BP/fIL-18 for HC subjects (16.5) was markedly greater than that for patients with sAR (9.7) (Fig. 1e), indicating that IL-18 is likely to play a role in sAR. Moreover, signi cant correlations between fIL-18 and fIL-18BP were observed in patients with pAR, sAR and HC subjects (Fig. 1f).
Elevated levels of IL-1β and TNF-α in plasma of patients with sAR Since monocytes and macrophages are involved in the pathogenesis of AR possibly by overproducing IL-1β and TNF-α [24][25][26][27], we examined levels of IL-1β and TNF-α in plasma of patients with pAR, sAR and HC by using human bio-plex panel kit. As shown in Fig. 2a, plasma level of IL-1β was generally low, nevertheless it appeared that plasma level of IL-1β in pAR and sAR patients were higher than that in HC. The plasma level of TNF-α in sAR patient (8.7 pg/mL), but not the patients with pAR was signi cantly higher than that in HC subjects (7.2 pg/mL) (Fig. 2b). Moreover, fIL-18, IL-1β and TNF-α were shown to be correlated well between each other in plasma of patients with pAR and sAR (Fig. 2c).
Increased expression of IL-18 and IL-18R, and decreased expression of IL-18BP in monocytes of patients with pAR and sAR Since fIL-18, IL-1β and TNF-α were correlated well between each other in plasma of patients with pAR and sAR, and tIL-18 and fIL-18 in plasma of patients with pAR and sAR were elevated in comparison with HC subjects, we investigated the expressions of IL-18, its speci c receptor IL-18R, and its natural speci c neutralizer IL-18BP in peripheral blood monocytes in the present study. The results showed that the proportion of IL-18 + monocytes was increased by 4.2 and 9.1 fold, and IL-18BP + monocytes was decreased by 77.5% and 56.0% in pAR and sAR patients, respectively when compared with HC subjects (Fig. 3b, c). PPAE seemed to upregulate IL-18 and IL-18R expression in monocytes of pAR and sAR patients, respectively (Fig. 3b, c).
As for the expression intensity of a single positive cell (mean uorescence intensity, MFI), pAR and sAR patients appeared to have lower MFI of IL-18BP in monocytes than that in HC (Fig. 3d ii, e), and sAR patients had higher MFI of IL-18R on monocytes than that in HC and pAR patients (Fig. 3d iii, e).
Moreover, all allergens tested in this study including ASWAE, PPAE and DPAE enhanced the MFI of IL-18 + monocyte in HC, and ASWAE increased the MFI of IL-18 + monocyte in sAR patients (Fig. 3d i, e).

Allergens and IFNγ induced alteration of IL-18, IL-18BP and IL-18R expression in isolated monocytes
In order to evaluate the direct effects of allergens on the expression of IL-18, IL-18BP and IL-18R in puri ed monocytes (purity over 99.7%), we co-cultured DPAE, ASWAE, PPAE or IFNγ with puri ed monocytes in 12-well cell culture plate, and examined expression of IL-18, IL-18BP and IL-18R by ow cytometry. The results showed that proportion of IL-18BP + monocytes in AR patients was decreased compared with HC (Fig. 4a, c), and IL-18BP expression appeared to be up-regulated in HC following the stimulation of DPAE for 10 min (Fig. 4a, c). It was also shown that ASWAE, PPAE and IFNγ induced the elevated expression of IL-18R in monocytes of AR patients at 10 min following incubation. Moreover, DPAE increased the expression of IL-18R in AR patients at 30 min following stimulation (Fig. 4a, d).
However, number of IL-18 + monocytes in AR patients was decreased compared with that in HC (Fig. 4a,   b), which was in contrast with the result seen in Fig. 3b and 3c. In terms of MFI, DPAE and IFNγ seemed to down-regulate MFI of IL-18BP + monocyte in AR patients at 60 min following incubation (Fig. 4e i, g). While the enhanced MFI of IL-18R + monocyte in patients with AR was observed in comparison with HC (Fig. 4e ii, h), DPAE increased the MFI of IL-18R in HC at 10 min following incubation (Fig. 4e ii, h). However, allergens and IFNγ tested in the present study had little effect on IL-18 expression in monocytes (Fig. 4a, b, f).

Allergen induced IL-18R mRNA expression in primary monocytes
In order to further understand the effects of allergens on the expression of IL-18, IL-18BP and IL-18R in monocytes, we examined expression of IL-18, IL-18BP and IL-18R mRNAs in primary monocytes by using qPCR technique. As seen in Fig. 5, ASWAE, PPAE, DPAE upregulated expression of IL-18R mRNA in monocytes of AR patients by 2.5, 2.5 and 4.6 fold, respectively at 30 min following challenge. At 60 min following challenge, only DPAE-induced expression of IL-18R mRNA in monocytes of AR patients was observed. ASWAE-provoked expression of IL-18R mRNA in monocytes was also found in HC at 30 min following challenge. Allergens tested had little effect on expression of IL-18 and IL-18BP mRNAs in monocytes of AR and HC subjects following 10, 30 and 60 min challenge periods (data not shown).

Increased level of IL-18 in both plasma and NLF of AR mice
To understand further the role of IL-18 in AR, in uence of OVA challenge on IL-18 and IL-18BP production in AR mice was examined. The results showed that IL-18 (Fig. 6a, b), but not IL-18BP (data not shown) levels were elevated in both plasma and NLF of AR mice.

Down-regulated expression of IL-18R in blood monocytes and up-regulated expression of IL-18R in NLF macrophgaes of AR mice
To con rm the role of IL-18R in AR, we examined IL-18R expression in both blood monocytes and NLF macrophages of AR mice. Compared with HM group, while the number of monocytes (Fig. 7a, b) and MFI of IL-18R + monocyte (data not shown) in blood leukocytes had little change, the percentage of IL-18R + monocytes was reduced by 58.3% in AR mice (Fig. 7a, c). In contrast, the numbers of F4/80 + macrophages and IL-18R + macrophages were increased approximately 1.2 (Fig. 7d, e) and 1.4 fold (Fig.   7d, f), respectively in AR mice. Discussion IL-18 is a pro-in ammatory cytokine that induces IFN-γ production, which is closely related to the pathophysiologic mechanism of allergic respiratory disorders [28]. In the present study, we showed that free plasma IL-18 was elevated in pAR and sAR patients, sAR patients had a decreased IL-18BP/IL-18 ratio (9.7), and free IL-18 correlated well with free IL-18BP in the plasma of both pAR patients and sAR patients, indicating that the imbalance between IL-18 and IL-18BP is likely to be crucial to the development of AR as a molar excess of 10 of IL-18BP over IL-18 is required to decrease a pathological level of 400 pg/ml of IL-18 to a level of a HC subject (40 pg/ml) [11]. Since IL18BP has neutralizing capacity of IL-18 [10] and excessive free IL18 can cause in ammatory conditions [12,29], our data suggest that IL-18 may participate in the development of AR as a causative factor.
In the present study, we also observed that elevated levels fIL-18, IL-1β and TNF-α were correlated well between each other in plasma of patients with pAR and sAR. Given the fact that IL-1β and TNF-α are mainly produced by monocytes and macrophages [30,31], we anticipate that the elevated levels of fIL-18, IL-1β and TNF-α are at least partially originated from monocytes. Our observation that the proportion of IL-18 + monocytes was increased in pAR and sAR patients may support the above anticipation, we hence believe that monocyte-derived IL-18 is likely to play a role in AR. The decreased IL-18BP + monocytes were found in AR peripheral blood may help to understand excessive fIL-18 in AR plasma as reduced IL-18BP production can eliminate IL-18/IL-18BP complex, and consequently free more IL-18 in the plasma. The enhanced MFI of IL-18R on monocytes of sAR patients suggest that IL-18 may act on monocytes through its receptor, which could implicate a paracrine mechanism that monocytes secrete IL-18, and IL-18 act on adjacent monocytes via IL-18R.
The results in the present study that PPAE upregulates IL-18 and IL-18R expression in monocytes of pAR and sAR patients, and ASWAE enhances the MFI of IL-18 + monocyte in sAR patients suggest that airborne allergens can directly affect IL-18 and IL-18R expression in monocytes even though direct contact of allergens with blood monocytes hardly occurs in the body. Unexpectedly, the MFI of IL-18 + monocyte in HC can be enhanced by all allergens tested in this study including ASWAE, PPAE and DPAE. Since IL-18 plays regulatory roles in both innate and adaptive immunity [32], elevated serum IL-18 during natural pollen exposure is closely associated with bronchial hyperresponsiveness in seasonal AR patients [8], and monocytes are one of the major sources of IL-18, the enhanced expression of IL-18 on monocytes may help to promote sensitization of HC to airborne allergens. The nding that synergy of IL-5 and IL-18 in eosinophil mediated pathogenesis of allergic diseases [33] may also support the view that IL-18 promote allergy.
Using primary monocytes, the elevated expression of IL-18R in monocytes of AR patients induced by allergens ASWAE, PPAE and DPAE was con rmed at both protein and mRNA levels, suggesting that allergen-induced upregulation of expression of IL-18R is most likely a direct event. Although the proportion of IL-18BP + monocytes in AR patients was decreased compared with HC, and DPAE seemed to down-regulate MFI of IL-18BP + monocyte in AR patients, allergens tested had little effect on expression of IL-18BP mRNAs in monocytes of AR and HC subjects, suggesting that reduced IL-18BP expression in monocytes most likely occurred at protein synthesis process such as elongation, transport or modi cation stages.
On the other hand, compared with HC blood, less proportion of IL-18 + populations were found in primary monocytes from peripheral blood of AR patients. This is an unexpected result considering our previous observation that the proportion of IL-18 + monocytes was increased in peripheral blood of pAR and sAR patients. It is di cult to explain these con ict results without performing more detailed investigation, but the isolation procedure and individual difference between patients may take into account.
The results that IL-18 levels were elevated in both plasma and NLF of AR mice following OVA challenge support our observation that IL-18 level was increased in patients with AR. Since expression of IL-18R in blood monocytes appeared to be down-regulated and expression of IL-18R in NLF macrophages was upregulated in AR mice, increased IL-18 may contribute to AR through macrophages or IL-18R expressing cells other than monocytes.

Conclusions
In conclusion, we demonstrated for the rst time that enhanced fIL-18 in AR plasma, and upregulated expression of IL-18 and IL-18R expression in monocytes of AR patients, which implicate strongly that IL-18 may serve as a causative factor for AR. Regulation of expression of IL-18, IL-18BP and IL-18R in monocytes by speci c allergens suggests allergens can directly act on monocytes, thereafter modify IL-18, IL-18BP and IL-18R expression. These observations imply that monocyte-derived IL-18 is likely to contribute to the pathogenesis of AR, and therefore IL-18 could be therapeutic target for AR. Levels of total and free plasma IL-18 and IL-18BP in patients with AR. Scatter plots of levels of total IL-18 (tIL-18, a) and total IL-18BP (tIL-18, b), free IL-18 (fIL-18, c) and free IL-18BP (fIL-18, d) in plasma of patients with perennial allergic rhinitis (pAR) and seasonal allergic rhinitis (sAR), and healthy control (HC) subjects. (e) shows the molar concentration ratios of fIL-18BP/fIL-18. Each symbol represents the value from one subject. The median value of each de ned group of subjects is indicated as a horizontal line. The Pearson's correlation coe cient between plasma levels of fIL-18 and fIL-18BP is shown in (f). P < 0.05 was taken as statistically signi cant.

Figure 2
Plasma levels of IL-1β and TNF-α in AR patients and HC (Bio-plex). Scatter plots of levels of IL-1β (a) and TNF-α (b) in plasma of perennial allergic rhinitis (pAR) and seasonal allergic rhinitis (sAR) patients and healthy control (HC) subjects. Each symbol represents the value from one subject. The median value of each de ned group of subjects is indicated as a horizontal line. The Spearman's ρ correlation coe cient between the plasma levels of fIL-18, IL-1β and TNF-α in pAR and sAR patients is shown in (c). P < 0.05 was taken as statistically signi cant.   Induction of upregulated expression of IL-18R mRNA in sorted monocytes by allergens and IFNγ. Quantitative real-time PCR (qPCR) analysis of expression of IL-18 receptor (IL-18R) mRNA in isolated monocytes of patients with allergic rhinitis (AR) and healthy control (HC) volunteers. Cells were incubated in the presence or absence of Artemisia sieversiana wild allergen extract (ASWAE), Platanus pollen allergen extract (PPAE) and Dermatophagoides allergen extract (DPAE) or IFNγ. Expression of IL-18R mRNA was analyzed by qPCR. The data displayed as a boxplot for AR patients (n = 6) and HC volunteers (n = 6), which indicates the median, interquartile range, the largest and smallest values for the number of volunteers indicated. P < 0.05 was taken as statistically signi cant.

Figure 6
Increased level of IL-18 in plasma and nasal lavage uid of OVA induced AR mice. Levels of IL-18 in mouse plasma (a) and NLF (b). Following a seven-day OVA challergen or vehicle control treatment, plasma and NLF supernatant of OVA-induced allergic rhinitis (AR) mice and healthy mice (HM) were taken, and analyzed by using sandwich ELISA kits. Data are displayed as a boxplot for AR mice (n = 7) and HM (n = 7), which indicates the median, interquartile range, the largest and smallest values for the number of animals indicated. P < 0.05 was taken as signi cant. Expression of IL-18R in blood monocytes and NLF macrophages from OVA induced allergic rhinitis mice.
Expression of IL-18 receptor (R) in blood monocytes and nasal lavage uid (NLF) macrophages of OVAinduced allergic rhinitis (AR) mice or vehicle treated healthy mice (HM). (a) shows a gating strategy of CD11b+ Gr-1low monocyte expression in mouse leukocytes, and IL-18R expression in monocytes; (b, c) demonstrate percentages of monocytes in leukocytes, and proportions of IL-18R expressing monocytes, respectively; (d) represents a gating strategy of F4/80+ macrophage expression in mouse NLF, and IL-18R expression in macrophages of mouse NLF. (e, f) reveal percentages of macrophages in NLF, and proportions of IL-18R expressing NLF macrophages, respectively. Data are displayed as a boxplot for AR mice (n = 7) and HM (n = 7), which indicates the median, interquartile range, the largest and smallest values for the number of subjects indicated. P < 0.05 was taken as statistically signi cant. FMO = uorescence minus one control.

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