Elevated expression of the chemokine CCL18 in chronic rhinosinusitis with nasal polyps

Abstract

Background

Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with Th2-dominant inflammation including eosinophilia, in contrast to non-polypoid CRS (CRSsNP). Chemokine CCL18/PARC (pulmonary and activation regulated chemokine) is known to recruit naïve T cells, B cells, and immature dendritic cells, as well as activate fibroblasts. CCL18is thought to be involved in Th2-related inflammatory diseases including asthma and atopic dermatitis.

Objectives

The objective of this study was to investigate the expression of CCL18 in patients with CRS.

Methods

Using nasal polyp tissue (NP) and uncinate tissue (UT) from controls and patients with CRS, we examined the expression of CCL18 mRNA by real-time PCR and measured CCL18 protein by ELISA, western blot and immunofluorescence.

Results

Compared to UT tissue in control subjects, CCL18 mRNA was significantly increased in NP (p<0.001) and UT (p<0.05) from patients with CRSwNP but not in UT from patients with CRSsNP. Similarly, CCL18 protein was elevated in NP and UT from CRSwNP and levels were even higher in Samter’s triad patients. Immunohistochemical analysis revealed CCL18 expression in inflammatory cells and CCL18⁺ cells were significantly increased in NP. Immunofluorescence data showed co-localization of CCL18 in CD68⁺/CD163⁺/macrophage mannose receptor⁺ M2 macrophages and tryptase⁺ mast cells in NP. Levels of CCL18 correlated with markers of M2 macrophages but not with tryptase, suggesting that M2 macrophages are a major CCL18-producing cells in NP.

Conclusion

Overproduction of CCL18 might contribute to the pathogenesis of CRSwNP through its known activities, which include recruitment of lymphocytes and dendritic cells, activation of fibroblasts, and initiation of local inflammation.

INTRODUCTION

Chronic rhinosinusitis (CRS) is a chronic disease that afflicts roughly 10% of the U.S population.^1–3 CRS is characterized by inflammation of the sinonasal mucosa. Symptoms of CRS include anterior and/or posterior rhinorrhea, nasal obstruction, decreased sense of smell and nasal pressure, at least two of which persist for 12 weeks despite medical management.^1–3 The pathogenesis of CRS is not fully understood at this time, however allergy, bacterial and fungal infections as well as structural anomalies have all been theorized to play a role.^2–5 CRS is often divided into two groups based on histology and physical examination: CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). In general, CRSwNP is associated more closely with clinical complaints of nasal obstruction and olfactory loss and is characterized by eosinophilia and Th2-related inflammation especially in western countries.^{5, 6} It is also known that B cells, plasma cells, macrophages, neutrophils and dendritic cells (DCs) are present in high concentrations in patients with CRSwNP, making inflammatory cell recruitment a contributor to the pathogenesis of this disease.^5–13

Chemokines are a large group of proteins that participate in recruitment of inflammatory cells into tissue sites by binding G protein coupled receptors on their target cells.^{14, 15} They can be classified into several groups based on their molecular structure. While some chemokines are involved in housekeeping functions of the adaptive immune system, others are produced under pathological conditions in order to mount immune and inflammatory responses and initiate wound healing by selectively recruiting immune cells to the inflamed site.^14–16 Exaggerated expression of chemokines can result in tissue damage and inflammatory diseases via excessive recruitment of leukocytes. Therefore, chemokine receptors serve as potential targets for the treatment of many inflammatory diseases.^15–18

CCL18, also known as pulmonary and activation regulated chemokine (PARC), MIP-4, AMAC-1 and DC-CK1, is a 7.8 kDa protein composed of 69 amino acids.^19–22 CCL18 is thought to have been generated by the fusion of two CCL3-like genes likely occurring after the diversification of rodents and primates.^22–24 Therefore the ortholog of CCL18 in rodents has not been discovered. Although its receptor is also unknown at this time, CCL18 is known to be chemotactic for many cell types, including naïve T cells, skin homing memory T cells, Th2 cells and immature DCs.^{19, 23, 25} CCL18 is known to be expressed in lung, thymus, and lymph nodes and is secreted by monocytes, macrophages, DCs and mast cells.^{22, 26} Its expression is regulated by the Th2 cytokines IL-4 and IL-13, as well as by IL-10.^{19, 21, 22} CCL18 is highly expressed in a number of inflammatory diseases including atopic dermatitis, asthma, rheumatoid arthritis and pulmonary fibrosis.^{22, 25, 27–29} While several chemokines are known to be elevated in CRS, particularly in CRSwNP, expression of CCL18 has been minimally studied in this disease process. In this study, we found that CCL18 was elevated in patients with CRSwNP and examined the cell types that produce it.

METHODS

Patients and biopsies

CRS patients were recruited from the Allergy-Immunology clinic and the Otolaryngology clinic of the Northwestern Medical Faculty Foundation (NMFF) and the Northwestern Sinus Center at NMFF. Sinonasal and NP tissues were obtained from routine Functional Endoscopic Sinus Surgery in patients with CRS. All subjects met the criteria for CRS as defined by the American Academy of Otolaryngology-Head and Neck Surgery Chronic Rhinosinusitis Task Force.¹ Patients with an established immunodeficiency, pregnancy, coagulation disorder, Churg-Strauss syndrome, diagnosis of classic allergic fungal sinusitis or cystic fibrosis were excluded from the study. Details of subjects’ characteristics are included in Table I, Table E1–3 and in the Methods section in the Online Repository. All subjects signed informed consent forms and the protocol governing procedures for this study has been approved by the Institutional Review Board of Northwestern University Feinberg School of Medicine.

Table 1.

Subject characteristics

	Control			CRSsNP			CRSwNP			Samter’s
Total no. of subjects	n=28 (14M)			n=55 (26M)			n=92 (65M)			n=12 (6M)
Age (y), median (range)	48* (16–63)#			38 (18–64)			43 (22–74)			47 (26–61)
	Y	N	U	Y	N	U	Y	N	U	Y	N	U
Atopy	0	24	4	24	25	6	42	40	10	7	2	3
Asthma	0	27	2	12	33	10	40	51	1	12	0	0
Methodologies used	UT			UT			UT	NP		NP
Tissue RNA	n=9 (3M)			n=18 (8M)			n=17 (14M)	n=24 (18M)		-
Age	41 (16–59)			36 (23–64)			43 (26–61)	44 (26–74)		-
Tissue Extract	n=15 (9M)			n=32 (15M)			n=29 (20M)	n=39 (28M)		n=12 (6M)
Age	49 (19–63)			37 (18–64)			41 (18–67)	41 (22–72)		47 (26–61)
Immunofluorescence	n=11 (8M)			n=11 (6M)			n=11 (9M)	n=17 (11M)		-
Age	53 (19–62)			38 (21–61)			48 (34–72)	52 (22–70)		-

^*median,

^#(range). M; male, Y; yes, N; no, U; unknown.

Real-time PCR

Real-time RT-PCR was performed using the TaqMan method, as described previously.³⁰ Primer and probe sets for CCL18 (sense, 5′-CCAGCTCACTCTGACCACTTCTC-3′; antisense, 5′-GGTGCAGACGAGGACAAGGA-3′; probe, 5′-CTGCCCAGCATCATGAAGGGCCTT-3′) were purchased from Integrated DNA Technologies (Coralville, IA). The mRNA expression levels were normalized to the median expression of the housekeeping gene, β-glucuronidase. Details can be found in the Methods section in the Online Repository.

ELISA, Western blot and cell culture

The concentration of CCL18 in cell free supernatants was determined by specific ELISA kits. The minimal detection limit for these kits is 3.9 pg/ml (R&D systems, Minneapolis, MN). The concentration of CCL18 in tissue homogenate was normalized to the concentration of total protein. Western blot analysis was performed as described previously.³¹ Human mast cells were obtained as described previously.²⁶ Further details can be found in the Methods section in the Online Repository.

Immunofluorescence assay

Immunofluorescent localization of CCL18 was performed as described previously.¹³ Briefly, blocked sections were incubated with anti-CCL18 antibody (LifeSpan Biosciences, Seattle, WA) and either anti-CD68 mAb (Thermoscientific), anti-ECP mAb (Diagnostics Development, Sweden) or anti-tryptase mAb (Thermoscientific) at 4°C overnight. After washing, sections were incubated with Alexa Fluor-conjugated secondary antibodies for 1 hour in the dark. After a final washing, coverslips were mounted onto slides using SlowFade Gold with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Details can be found in the Methods section in the Online Repository.

Statistics

All data are reported as the mean ± SEM unless otherwise noted. Differences between groups were analyzed using the Mann-Whitney U-test and the one-way ANOVA Kruskal-Wallis test. Correlations were assessed using the Spearman’s rank correlation. A p value of less than 0.05 was considered significant.

RESULTS

CCL18 expression in CRS

We assessed the expression of CCL18 in UT from patients with CRSsNP, CRSwNP and controls, as well as in NPs from patients with CRSwNP. CCL18 mRNA was significantly increased in NPs from patients with CRSwNP (p<0.001) in comparison to UT from patients with either form of CRS or control subjects (p<0.001, Kruskal-Wallis test; Fig 1, A). CCL18 mRNA was also significantly increased in UT from patients with CRSwNP (p<0.001) in comparison to UT from control subjects (Fig 1, A). To confirm this observation at the protein level, we made detergent extracts from homogenates of UT and NPs and then measured the concentration of CCL18 using ELISA. CCL18 protein was significantly elevated in NPs from CRSwNP (4.75 ± 1.13 ng/mg (median; 1.58 ng/mg); n=39) compared to UT from CRSsNP (0.52 ± 0.06 ng/mg (median; 0.46 ng/mg); n=32), CRSwNP (1.52 ± 0.40 ng/mg (median; 0.59 ng/mg); n=29) and control subjects (0.52 ± 0.12 ng/mg (median; 0.42 ng/ml); n=15) (p<0.001, Kruskal-Wallis test; Fig 1, B). Although not statistically significant, CCL18 protein was also elevated in UT from CRSwNP (p=0.057) compared to UT from controls (Fig 1, B). We further confirmed this finding by western blot. The CCL18 protein was detected as a 7.8 KDa protein and found only in NPs but not in UT from patients with CRSsNP or control subjects (Fig 1, C and data not shown).

Figure 1.

Increased expression of CCL18 in nasal polyps. Total RNA was extracted from uncinate tissue (UT) and nasal polyps (Polyp) and expression of CCL18 RNA was analyzed using real-time PCR (A). Expression of CCL18 protein in tissue homogenates of UT and polyp from patients with aspirin-tolerant CRSwNP and Samter’s triad was measured using ELISA (B) and western blot (C). CCL18 concentration was normalized to the concentration of total protein (B). The results are representative of two separate experiments (C).* p < 0.05.

Nasal polyps in aspirin sensitive patients have generally more inflammation than in aspirin tolerant patients.³² Patients with clinical features including bronchial asthma, aspirin sensitivity and NPs are referred to as having Samter’s triad.^32–34 Interestingly, we found that the concentration of CCL18 in NPs was significantly higher in Samter’s triad patients (9.92 ± 1.93 ng/mg (median; 7.13 ng/mg); n=12, p=0.0015) compared to NPs from patients with aspirin tolerant CRSwNP (Fig 1, B).

Identification of CCL18 producing cells in nasal polyps

To examine the distribution of CCL18 in nasal mucosa, we used immunohistochemistry to detect CCL18⁺ cells in UT and NPs. As shown in Fig 2, CCL18 staining was observed mainly in submucosal inflammatory cells. Although CCL18 staining was also observed in some mucosal and glandular epithelium, levels of mRNA for CCL18 in epithelial cell scrapings from NPs were more than 500-fold lower than levels in whole NP tissue and we could not find elevation of CCL18 protein in nasal lavage from patients with CRSwNP (Fig 1, 2 and E1, and data not shown). We therefore excluded epithelial cells as major CCL18 producing cells and focused on inflammatory cells in this study. We found that CCL18⁺ inflammatory cells were highly elevated in NPs (Fig 2, D). We counted the number of CCL18⁺ inflammatory cells using a semi- quantitative method and found that CCL18⁺ inflammatory cells were significantly elevated in the NPs from CRSwNP (2.37 ± 0.43 CCL18⁺ cells/high power field (HPF); n=17) compared to UT from CRSsNP (1.16 ± 0.40 CCL18⁺ cells/HPF, p<0.05; n=11), CRSwNP (0.75 ± 0.18 CCL18⁺ cells/HPF, p<0.05; n=11) and controls (0.86 ± 0.19 CCL18⁺ cells/HPF, p<0.05; n=11) (p<0.01, Kruskal-Wallis test; Fig 2, F).

Figure 2.

Immunofluorescence of CCL18 was performed with an anti-human CCL18 antibody. Representative immunostaining (green) for CCL18 in uncinate tissue (UT) from a control subject (A), a patient with CRSsNP (B), a patient with CRSwNP (C), and in nasal polyp tissue (D). Negative control antibody staining in nasal polyp tissue from a patient with CRSwNP (E) is shown. Nuclei were counterstained with DAPI (blue). The number of CCL18 positive cells in UT from control (n=11), CRSsNP (n=11) and CRSwNP (n=11) and in nasal polyps (n=17) was counted using the NIH-issued Image J software (F). * p < 0.05.

We next attempted to identify the CCL18 producing cells in the nasal mucosa of NPs. We first assessed whether the expression of CCL18 correlated with the levels of mRNA expression for markers of inflammatory cells in NPs by real-time PCR. We found that the levels of expression of CCL18 mRNA in NPs were significantly and positively correlated with the expression of macrophage mannose receptor (MMR, r=0.796, p<0.0001) and Charcot-Leyden crystal protein (CLC, r=0.695, p=0.0002) but not with CD3, CD20, CD56, CD138, CD1c, tryptase or CXCR1 (Fig 3, A and data not shown), indicating that CCL18 might be expressed in macrophages and eosinophils.

Figure 3.

Detection of CCL18-producing cells in nasal polyps. Total RNA was extracted from nasal polyp tissue and the expression of CCL18 and cell specific markers was analyzed by real-time PCR (n=24) (A). The correlations were assessed using the Spearman rank correlation. An immunofluorescence assay was performed using anti-CCL18 (green), anti-CD68 mAb (red) for macrophages (B), anti-ECP mAb (red) for eosinophils (C), anti-tryptase mAb (red) for mast cells (D) and control IgG (E). Nuclei were counterstained with DAPI (blue). The results are representative of 5 to 8 separate patients.

We next performed dual immunofluorescence analysis using anti-CCL18 and antibodies against markers of macrophages (CD68) and eosinophils (ECP). We found CCL18 co- localization with CD68⁺ macrophages (Fig 3, B) but not with ECP⁺ eosinophils (Fig 3, C) in NPs. In addition to macrophages, we also found co-localization of CCL18 with tryptase⁺ mast cells (Fig 3, D). Although there was variability from patient to patient, 20–50% of CCL18⁺ cells were macrophages, 10–60% of CCL18⁺ cells were mast cells and another 10–50% of CCL18⁺ cells were unidentified inflammatory cells in NPs (n= 8; data not shown).

We have previously shown that mast cells are able to produce CCL18 during IgE- mediated reactions.²⁶ However, the effect of cytokines on the production of CCL18 is still unknown. Since we observed CCL18⁺ mast cells in NPs (above), we stimulated IgE-sensitized human peripheral blood-derived mast cells with cytokines TNF, IL-1β, IL-4, IL-13, IFN-γ and IL-17A alone or in combination with anti-IgE antibody in the presence of protease inhibitor cocktail (PIC) or DMSO (vehicle control) for 48 hours to assess production of CCL18. We found that the cytokines alone did not induce CCL18 production but IL-4, IL-13 and IFN-γ significantly enhanced IgE-mediated CCL18 production only in the presence of PIC (Fig E2 and data not shown). However, levels of CCL18 in mast cell culture supernatants were very low. In addition, levels of CCL18 mRNA did not correlate with levels of tryptase in NPs (Fig 3). This suggested to us that mast cells may not be a major CCL18 producing cell type in NPs and we therefore focused on macrophages.

Detection of CCL18 in M2 macrophages

Macrophages are now widely recognized to be polarized by their microenvironment, especially by Th cytokines and pathogens.^35–39 Classically activated macrophages (also known as M1 macrophages) are developed by the stimulation with Th1 cytokine IFN-γ and microbial products such as LPS. In contrast, alternatively activated macrophages are primed by Th2 cytokines IL-4 and IL-13, and therefore are called M2 macrophages. Importantly, CCL18 is known as a useful marker of M2 macrophages.^36–39 In vitro generated M2 macrophages or IL-4- stimulated monocytes/macrophages are known to release large amounts of CCL18. In addition, we found that expression of CCL18 was positively correlated in NPs with MMR, which is another well-known marker of M2 macrophages (Fig. 3, A). Therefore we next examined whether M2 macrophages were major CCL18 producing cells in NPs. We first determined levels of MMR and other M2 macrophages markers, CD163 and stabilin 1 (STAB1), in NPs by real- time PCR. Levels of mRNA for MMR, CD163 and STAB1 were significantly upregulated in NPs (p<0.05) in comparison with those seen in UT from either patients with CRS or control subjects (Fig 4, A). We also found that expression of CCL18 significantly and positively correlated with CD163 (r=0.630, p=0.001) and STAB1 (r=0.481, p=0.017) (Fig 4, B). Importantly, levels of MMR also significantly correlated with CD163 (r=0.704, p<0.001) and STAB1 (r=0.593, p=0.002) (n=24, Fig 4, B and data not shown).

Figure 4.

Correlation of CCL18 with markers of M2 macrophages in nasal polyps. Total RNA was extracted from UT from controls (n=9), CRSsNP (n=18) and CRSwNP (n=17) and nasal polyp tissue (n=24) (A). The expression of CCL18 and M2 macrophage markers, MMR, CD163 and STAB1 was analyzed by real-time PCR. The correlations in nasal polyps were assessed by using the Spearman rank correlation (B). * p < 0.05.

To further investigate whether M2 macrophages were CCL18 producing cells in NPs, we performed triple immunofluorescence analysis using anti-CCL18 and antibodies against markers of M2 macrophages including MMR and CD163. We detected CCL18 in CD68⁺, MMR⁺ cells and CD163⁺, MMR⁺ cells in NPs (Fig 5). These data suggest that M2 macrophages are major CCL18 producing cells in NPs.

Figure 5.

Detection of CCL18 in M2 macrophages in nasal polyps. An immunofluorescence assay was performed using anti-CCL18 (A and B; green), anti-CD68 mAb (A; orange), anti-CD163 mAb (B; orange), anti-MMR mAb (A and B; red), and control IgG (C). Nuclei were counterstained with DAPI (blue). The results are representative of four separate patients.

DISCUSSION

It has been reported that CCL18 is elevated in affected tissues in patients with Th2-related inflammatory diseases and that expression of CCL18 is regulated by Th2 cytokines.^{22, 28, 29} CRSwNP is well known to be characterized by Th2-mediated inflammation and eosinophilia.^{5, 6} In the current study we demonstrated that CCL18 was significantly increased in NPs (Fig 1). There are very few studies comparing identical tissue taken from patients with CRSsNP and CRSwNP and from controls, and most of those studies show elevation or reduction in NPs compared to non-polypoid tissue. In this study, we present rare evidence that up-regulation of mRNA and protein for CCL18 was also found in UT, which is one of the surrounding mucosal tissues in close proximity to NPs, from patients with CRSwNP compared to UT from control subjects (Fig 1). This suggests that CCL18 may be involved in the initiation of the inflammatory process in patients with CRSwNP. Interestingly CRSwNP in China has been reported to have less eosinophilia and Th2 inflammation than in Western countries.⁴⁰ It would be of interest to determine whether CCL18 elevation exists in this form of polypoid CRS.

Very recently Plager et al published a microarray study of asthmatic CRSwNP and showed that CCL18 mRNA was upregulated in NPs compared to control tissue.⁴¹ Our study unequivocally demonstrates elevation of CCL18 in CRSwNP. Plager et al focused on the expression of CCL18 only in asthmatic CRSwNP.⁴¹ When we evaluated whether overexpression of CCL18 in patients with CRSwNP was associated with asthmatic status, atopic status or glucocorticoid treatment, we found no significant difference in CCL18 levels between atopic and non atopic patients, asthmatic and non asthmatic patients or status of nasal steroid treatment (Fig E4). Since we might not have large enough subgroups of patients to make firm conclusions about the effect of oral steroid treatment, future studies will be required to test in larger cohort. In contrast, we found that levels of CCL18 in NPs were highest in patients with Samter’s triad compared to patients with aspirin tolerant CRSwNP (Fig 1). In general, tissues from aspirin sensitive patients are more inflamed than in aspirin tolerant patients.³² This suggests that levels of CCL18 in patients with CRSwNP may be related to severity of inflammation rather than asthmatic or atopic status.

It is well established that CCL18 is produced by monocytes and macrophages. Our immunofluorescence data clearly show that CCL18 was detected in CD68⁺ macrophages as well as tryptase⁺ mast cells. This suggests that macrophages and mast cells may be major CCL18 producing cells in NPs. However, the regulation of CCL18 in mast cells is poorly understood. We showed previously that mast cells could release CCL18 upon FcεRI cross-linking.²⁶ In our present study, we investigated the effect of cytokines on the production of CCL18 in mast cells. We found that the Th1 cytokine IFN-γ and Th2 cytokines IL-4 and IL-13 significantly enhanced IgE-mediated CCL18 production in mast cells. This suggests that the effect of IL-4 and IL-13 on the induction of CCL18 is well conserved across cell types. In contrast, IFN-γ is known to inhibit CCL18 expression in monocytes, macrophages and DCs, enhance it in keratinocytes and has no effect in PBMC.^{21, 25, 29} These data suggest that regulation of CCL18 by IFN-γ may be cell type specific. Although we detected CCL18 protein in mast cells (20–30 pg/10⁶ cells, Fig E2), these levels were more than 50-fold lower than those found to be produced by monocytes/macrophages (1–15 ng/10⁶ cells).^{25, 37, 42–44} These results suggest that mast cells may be a less important source of CCL18 in NPs.

CCL18 was detected in stimulated mast cells only in the presence of protease inhibitors. This demonstrates that CCL18 is highly sensitive to mast cell proteases. In a separate clinical study, we have found that mast cells are increased in NPs and some of them are degranulated (Takabayashi T, Kato A and Schleimer RP, manuscript in preparation). Although CCL18 mRNA was 500-fold higher in NP than control UT, up-regulation of CCL18 protein was only nine-fold. This suggests that local CCL18 production in NPs may be much more abundant but that a portion may be cleaved immediately by proteases from degranulated mast cells or other tissue cells. This may explain the variability of CCL18 protein levels in NPs. Future studies will be required to identify mast cells proteases that can cleave CCL18 and to investigate the effect of those proteases on the stability of CCL18 in NPs in vitro and in vivo.

Although CRSwNP is well known to be characterized by eosinophilia and Th2-related inflammation, the role of macrophages in the pathogenesis of CRS is poorly understood. Recently, Krysko et al showed that MMR⁺ M2 macrophages but not M1 macrophages were increased in NPs.¹² In our present study, we also found that M2 macrophage markers, MMR, CD163 and STAB1, were significantly upregulated in NPs (Fig 4). This suggests that upregulation of M2 macrophages in NPs should be considered as a feature of this disease. CCL18 is well known to be released from M2 macrophages.^37–39 We found that CCL18 was detected in MMR⁺ and CD163⁺ macrophages but not in MMR⁻ macrophages in NPs (Fig 5 and data not shown). In addition, we found that levels of M2 macrophage markers correlated with levels of CCL18 (Fig 4). As discussed above, mast cells were able to produce CCL18 but the levels were much weaker than what has been reported in macrophages. These results suggest that M2 macrophages are the major CCL18 producing cell type in NPs. We also found that CCL18 was increased in UT from patients with CRSwNP compared to UT in healthy subjects (Fig 1). In contrast to NPs, we could not find significant elevation of M2 macrophages or mast cells in UT from CRSwNP (Fig 4 and data not shown). Although CCL18 producing cells were not elevated in UT by immunofluorescence, the elevated protein level suggests that local cells produce it. Future studies will be required to identify the CCL18 producing cells in UT from patients with CRSwNP and whether they are distinct from the CCL18 producing cells in NPs.

Although we found that M2 macrophages were major CCL18 producing cells, little is known about the regulation of macrophage recruitment in NPs. Recently we showed that CCL23 was highly upregulated in eosinophilic CRSwNP and eosinophils were a major source of CCL23 in NPs.¹³ CCL23 is a known ligand of CCR1 and is able to recruit monocytes and macrophages.⁴⁵ Importantly, we showed that levels of CCL23 were significantly correlated with levels of CCR1 and MMR in NPs.¹³ Since NPs are usually characterized by eosinophilia and Th2-related inflammation, eosinophil-derived CCL23 might be involved in the recruitment of monocytes and macrophages to NPs, followed by polarization of the recruited cells to the M2 phenotype by Th2 cytokines. M2 macrophages are now known to be a rich source of eotaxins.^37–39 We found here that CCL18 was mainly produced by M2 macrophages in NPs. These findings suggest that accumulation and activation of M2 macrophages in NPs may further enhance tissue eosinophilia by the production of eotaxins and may induce local adaptive immunity by the production of CCL18 and its subsequent recruitment of naïve T cells and immature DCs. Importantly, both T cells and DCs were elevated in NPs.^{5, 6, 13} Future studies will be required to determine the relationship between these chemokines and inflammation in NPs.

We found that CLC, which is a marker of eosinophils, significantly and positively correlated with CCL18 in NPs. Since CCL18 has been described as an antagonist of CCR3, which is predominantly expressed on eosinophils,⁴⁶ we thought eosinophils may contribute to the production of CCL18. In this regard, Schraufstatter et al showed that peripheral blood eosinophils were able to produce CCL18.⁴² We also found that peripheral blood eosinophils were able to release small amounts of CCL18 (40–50 pg/10⁶ cells) but it was not enhanced by eosinophil activators or Th2 cytokines (Fig E3). Furthermore, our immunofluorescence data demonstrated that CCL18 was not detected in eosinophils in NPs (Fig 3). Another possibility is that CCL18 may be involved in the recruitment of eosinophils via a CCR3-independent manner. However, Nibbs et al showed that CCL18 inhibited CCL11-dependent peripheral blood eosinophil migration.⁴⁶ It is known that there are phenotypic differences between peripheral blood eosinophils and tissue eosinophils.⁴⁷ Therefore it is still possible that CCL18 is involved in the recruitment of NP eosinophils. Future studies will be required to investigate whether CCL18 contributes to the recruitment of eosinophils and whether eosinophils are a source of CCL18 in NPs.

In addition to its chemotactic activity, CCL18 is known to activate fibroblasts. CCL18 activated Sp1 and PKCα, and induced collagen production in lung fibroblasts.^{48, 49} Proliferation and activation of fibroblasts are considered to be important events leading to NP formation.³² Therefore the role of CCL18 in these processes is worthy of further investigation.

In summary, we report here that M2 macrophages and mast cells produce the chemokine CCL18 and that patients with CRSwNP have increased levels of CCL18 in polypoid tissue as well as in surrounding tissues. Our findings indicate that CCL18 is a novel marker in patients with CRSwNP and that the overproduction of CCL18 in NPs might contribute to the pathogenesis of CRSwNP.

Abbreviations

CRS

Chronic rhinosinusitis

NP

nasal polyp

CRSwNP

CRS with nasal polyps

CRSsNP

CRS without nasal polyps

CCL18

CC Chemokine Ligand 18

ECP

eosinophil cationic protein

UT

uncinate tissue

NP

nasal polyp

MMR

macrophage mannose receptor

CLC

Charcot-Leyden crystal protein

DAPI

4′,6-diamidino-2-phenylindole