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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Clin Exp Allergy. 2021 Feb 2;51(4):564–573. doi: 10.1111/cea.13832

Increased IL-6 and Potential IL-6 Trans-signaling in the Airways after an Allergen Challenge

Stephane Esnault 1, Mehdi Khosravi 1,2, Elizabeth A Becky Kelly 1, Lin-Ying Liu 1,3, Yury A Bochkov 4, Matthew C Tattersall 5, Nizar N Jarjour 1
PMCID: PMC8020336  NIHMSID: NIHMS1686109  PMID: 33471392

Abstract

Background:

In asthma, IL-6 is a potential cause of enhanced inflammation, tissue damage and airway dysfunction. IL-6 signaling is regulated by its receptor, which is composed of two proteins, IL-6R and GP130. In addition to their membrane form, these two proteins may be found as extracellular soluble forms. The interaction of IL-6 with soluble IL-6R (sIL-6R) can trigger IL-6 trans-signaling in cells lacking IL-6R. Conversely, the soluble form of GP130 (sGP130) competes with its membrane form to inhibit IL-6 trans-signaling.

Objectives:

We aimed to analyze IL-6 trans-signaling proteins in the airways of subjects after an allergen challenge.

Methods:

We used a model of segmental bronchoprovocation with an allergen (SBP-Ag) in human subjects with allergy. Before and 48h after SBP-Ag, bronchoalveolar lavages (BAL) allowed for the analysis of proteins in BAL fluids (BALF) by ELISA, and membrane proteins on the surface of BAL cells by flow cytometry. In addition, we performed RNA-sequencing (RNA-seq) and used proteomics data to further inform on the expression of the IL-6R subunits by eosinophils, bronchial epithelial cells and lung fibroblasts. Finally, we measured the effect of IL-6 trans-signaling on bronchial fibroblasts, in vitro.

Results:

IL-6, sIL-6R, sGP130 and the molar ratio of sIL-6R/sGP130 increased in the airways after SBP-Ag, suggesting the potential for enhanced IL-6 trans-signaling activity. BAL lymphocytes, monocytes and eosinophils displayed IL-6R on their surface and were all possible providers of sIL-6R, whereas GP130 was highly expressed in bronchial epithelial cells and lung fibroblasts. Finally, bronchial fibroblasts activated by IL-6 trans-signaling produced enhanced amounts of the chemokine, MCP-1 (CCL2).

Conclusion and Clinical relevance:

After a bronchial allergen challenge, we found augmentation of the elements of IL-6 trans-signaling. Allergen-induced IL-6 trans-signaling activity can activate fibroblasts to produce chemokines that can further enhance inflammation and lung dysfunction.

Keywords: IL-6, IL-6 receptor, trans-signaling, asthma, allergy, bronchoalveolar lavage, CCL2, fibroblasts

Introduction

The pro-inflammatory cytokine IL-6 is elevated in the blood of subjects with asthma compared to healthy individuals [1] and IL-6 accumulates in bronchoalveolar fluid (BALF) of subjects with uncontrolled asthma [2]. Cross-sectional analyses in asthma have demonstrated that blood IL-6 levels correlate with older age, increased BMI, greater blood neutrophil count, lower lung function and more frequent asthma exacerbations [3–5]. Blood IL-6 levels are not correlated with cellular characteristics of type-2 asthma such as greater blood eosinophil counts, percentage sputum eosinophils, total serum IgE levels, or FENO values [5]. Conversely, airway IL-6 levels, correlate with a type-2 cytokine, IL-13, in induced-sputum samples from allergic asthma subjects compared to healthy controls [6]. In addition, enhanced levels of IL-6 along with type-2 cytokines, such as IL-4, IL-5 and IL-13 have been reported in the airways after allergen challenge (reviewed in [7]). These data suggest differences in IL-6 associations in the circulation versus the airways. While, macrophages are a predominant source of IL-6, both immune and non-immune cells produce and respond to IL-6 (reviewed in [8, 9]). For example, there is evidence implicating bronchial epithelial cells in the production of IL-6 in asthma [8, 10]. Eosinophils can produce IL-6 [11, 12] and we have shown that the content of degranulated eosinophils can induce fibroblast release of IL-6, which in turn recruits neutrophils [13, 14]. In addition to its role on neutrophils [15, 16], IL-6 enhances IL-17 production by naïve T cells and skews antigen-activated CD4 T lymphocytes into a type-2 phenotype and increases the production of IL-4 [17, 18]. In studies with allergen-challenged mice, the removal of IL-6 signaling indicates that IL-6 is involved in pulmonary fibrosis, mucus hypersecretion and airway hyperresponsiveness [8].

IL-6 signaling is controlled by different mechanisms involving its receptors. IL-6 signals after a first interaction with the IL-6 receptor (IL-6R; IL6R) followed by an interaction with two glycoprotein 130 (GP130; IL6ST) molecules [19]. In addition to their membrane forms, both IL-6R and GP130 can be found as soluble molecules. IL-6R is released as a soluble form (sIL-6R) due to either alternatively spliced transcript variants or shedding by the metalloproteinases, ADAM7 and ADAM10 [20, 21]. In a mechanism called “trans-signaling”, sIL-6R can recruit IL-6 to membrane GP130 (mGP130) to trigger intracellular signaling in cells lacking IL-6R, such as fibroblasts and epithelial cells [19]. The provenances of the sIL-6R are mainly monocytes, neutrophils and CD4 T lymphocytes [8, 22]. Soluble IL-6R is increased in the airways of asthma subjects [23] and is associated with severe asthma exacerbations in children and adults [24].

Unlike sIL-6R, the presence of soluble homodimer GP130 (sGP130) acts as an inhibitor of IL-6 trans-signaling by competing with membrane GP130 [25–27]. In addition to IL-6R, GP130 is a component of the receptors for IL-11, IL-27, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, cardiotrophin 1 and cardiotrophin-like cytokine. The making of sGP130 is the consequence of early stop codons due to alternatively spliced transcript variants [28]; however, the action of so far unidentified proteases and GP130 shedding cannot be ruled out [29]. The potential importance of GP130 in asthma is indicated by a study showing that among the 444 screened proteins in plasma of children with or without asthma, sGP130 is the only protein predicting childhood asthma and early life wheezing [30]. Yet, there is no report on the measurement of sGP130 in BALF or its association with allergen-induced airway inflammation. In this study, we aimed to measure airway sGP130, sIL-6R and IL-6 after a segmental allergen challenge (SBP-Ag) in allergic subjects, and evaluate the possible impact of IL-6 trans-signaling on bronchial fibroblasts.

Material and Methods

Subjects

Twenty-three allergic subjects (19 with asthma) were included in the segmental bronchoprovocation studies. These subjects’ characteristics are provided in Table I (A and B). All subjects had a positive skin prick test to one or more aeroallergens, were nonsmokers, and did not have a respiratory infection or asthma exacerbations within 30 days of study, and had not received long-acting ß-agonists within two days, antihistamines or leukotriene antagonists within 7 days, or corticosteroids within 30 days of study enrollment. An additional seven subjects (6 with allergic asthma and one without allergy and asthma) underwent bronchoscopy and endobronchial biopsies, which were used to culture fibroblasts. Blood from three additional subjects with allergic asthma was used to purify eosinophils for RNA-sequencing. These three subjects did not take topical or inhaled corticosteroids the day of blood draw. Informed consent was obtained from each subject prior to participation. The studies were approved by the University of Wisconsin-Madison Center for Health Sciences Human Subjects Committee (#1999-292; #1999-041 and #2013-1570).

Table I.

Subjects’ characteristics

A. Used for BALF ELISAs and subsequent correlation analyses with BAL cell counts
Number 18
Sex 9 F, 9 M
Age (yrs) 22 ± 1*
Baseline FEV1 (% Predicted) 96.9 ± 2.1*
Antigens Used for Challenge 8 Ragweed, 8 Dust mite, 2 Cat dander
B. Used for flow-cytometry analyses
Number 5
Sex 3 F, 2 M
Age (yrs) 22.4 ± 1.5*
Baseline FEV1 (% Predicted) 103 ± 2.1*
Antigen used for challenge 2 Ragweed, 1 Dust mite, 2 Cat dander
BAL EOS% 48 h after allergen challenge 46.3 ± 8.3*
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*

Mean ± SE

Segmental Bronchoprovocation with Allergen (SBP-Ag) and Bronchoalveolar Lavage (BAL)

Bronchoscopy, BAL, and SBP-Ag were performed as previously described [31]. In brief, the antigen dose leading to 20% FEV1 fall (antigen PD20) was calculated from a dose-response curve generated by a graded inhaled antigen challenge. A total dose of 30% of the antigen PD20 was administered for SBP-Ag; 10% in one segment and 20% in a second segment. BAL was performed in each segment before and 48 hours after SBP-Ag. BAL fluid from the two segments was pooled for fluid and cell analysis.

ELISA

As previously described [32], sandwich ELISA were used to measure IL-6, sIL-6Rα, sGP130 in BALF, and MCP-1 and fibronectin in fibroblast culture supernatants. All coating and biotinylated antibodies were purchased from R & D Systems (Minneapolis, MN). The assays for IL-6, sIL-6R, and sGP130 detect both free forms and complexes. The assay sensitivities were as follows: IL-6 (1 pg/ml); sIL-6R (8 pg/ml), sGP130 (80 pg/ml), MCP-1 (12 pg/ml), and fibronectin (12 pg/ml).

Determination of sIL-6R and dimer-sGP130 molecular weights

While the reported apparent molecular weight of sIL-6R may vary from 40 to 55 kDa, IL-6R cleavage by proteases gives a calculated molecular weight of ~40 kDa [33]. Therefore, for the calculation of molarity, we used 40 kDa as a molecular weight for sIL-6R. Macrophages, T cells and neutrophils mainly express an alternatively spliced transcript coding for a sGP130 protein with an apparent size around 60 kDa [34, 35], which is also close to the calculated size of the extracellular domain of GP130 (67 kDa). Therefore, although we did not analyze the size of sGP130 in BALF, we choose 60 kDa (120 kDa for the homodimer) as a molecular weight for calculation of molarity in BALF.

Flow cytometry analyses

Whole blood (100 μl), BAL cells (2×105 cells), or fibroblasts (1×105 cells) were stained with PE-conjugated monoclonal antibody (mAb) to IL-6Rα (CD126), GP130 (CD130), or an isotype control antibody. In blood and BALF, T cells were identified by FITC-conjugated mAb to CD3. Along with characteristic forward and side scatter, addition of the combination of FITC-CD16 plus FITC-CD14 allowed identification of neutrophils (CD16 positive/CD14 negative) and monocytes (CD16 negative/CD14 positive) (Supplemental Figure 1). In initial experiments, the identity of monocytes was confirmed by expression of CD163 (Supplemental Figure 2). Alveolar macrophages were identified by characteristic autofluorescence and forward and side scatter. Eosinophils were identified by characteristic forward and side scatter, autofluorescence, and negativity for CD16 and CD14 (Supplemental Figures 1 and 2) [31, 36]. All antibodies were from BD Biosciences, San Jose, CA. Before using the bronchial fibroblasts, CD90 staining (Immunotech-BeckmanCoulter, Fullerton, CA) was used to confirm the fibroblast phenotype [37]. For analysis, 10,000 events were collected using a Becton Dickinson FACScan II, and data were analyzed using the Cell Quest™ or FlowJo™ software package (BD Biosciences).

Eosinophil RNA-sequencing

Blood eosinophils were prepared from three subjects with allergy and mild asthma, and were cultured in RPMI and 10% serum with or without IL-3 (2 ng/ml) as previously described [38]. Eosinophils were activated with IL-3, as IL-3 is known to activate eosinophils after 20 h incubation in vitro, triggering a similar phenotype as airway eosinophils after in vivo activation by SBP-Ag [38–40]. As previously described [13], total RNA was extracted from eosinophils using the RNeasy Mini Kit (Qiagen, Valencia, CA) and treated with DNase (RNase-free DNase kit, Qiagen). RNA samples were submitted to the University of Wisconsin-Madison Biotechnology Center (Madison, WI) for RNA quality and integrity evaluation via Agilent 2100 Bioanalyzer platform (Agilent Technologies) and whole-transcriptome sequencing. The sequencing library from mRNA was prepared using TruSeq Stranded mRNA Library Preparation Kit (Illumina; San Diego, CA, USA) and RNA-seq (1 × 100 bases) was carried out using Illumina HiSeq 2500 platform. SeqMan Ngen (v.15) and ArrayStar with Qseq module (v.15) software packages (DNAStar, Madison, WI) were used to map sequence reads to human reference genome (GRCh38), apply statistical analyses and identify global gene expression changes. Raw data and normalized RPKM (Reads Per Kilobase per Million mapped reads) values have been deposited to the NCBI Gene Expression Omnibus (GEO) database and assigned accession number (GSE159782).

Fibroblast cultures

Fibroblasts were derived as previously described [32, 37] from endobronchial biopsies of seven subjects. Fifth passage primary bronchial fibroblasts (10,000 cells/well) were cultured in 96 well plates for 24 h, serum-starved overnight, and then treated for 5 days with human recombinant IL-6 (20 ng/ml), sIL-6R (50 ng/ml), sGP130 (500 ng/ml), IL-6/sIL-6R, or IL-6/sIL-6R/sGP130 (all recombinant proteins were from R & D Systems). For the condition IL-6/sIL-6R, IL-6 and sIL-6R were mixed and added together on fibroblasts. Fibroblasts were preincubated with sGP130 before addition of premixed IL-6/sIL-6R. Supernatant fluids were analyzed for MCP-1 and fibronectin.

Statistical analysis

A Wilcoxon signed rank test (or paired t-test, for normally distributed data) was used to compare data obtained at baseline and 48 hours after SBP-Ag. Correlations were made using Spearman rank order correlation. A p value of <0.05 was considered significant. Statistical analysis was performed using a SigmaStat software package (Jandel Scientific Software, San Rafael, CA).

Results

Concentrations of IL-6 and its soluble receptors increased in BALF 48 h after allergen challenge

At baseline (D0), IL-6 levels in BALF were low but detectable in 12 of 18 subjects; sIL-6R and sGP130 were both detectable in all subjects. As shown in Figure 1A, 48 hours (D2) after SBP-Ag, BALF concentrations of IL-6, sIL-6R and sGP130 were significantly elevated compared to baseline. The changes in the concentration of these factors were not due to differences in the return volume of BALF, which was 241±5 ml at baseline and 241±4 ml after challenge. Soluble GP130 was decreased or was not changed after challenge for eight of the eighteen subjects. Thus, to better evaluate the change of sIL-6R relative to that of the inhibitor of trans-signaling, sGP130, we calculated the molarity of sIL-6R and sGP130 homodimers (dimeric sGP130) in BALF, before (D0) and after SBP-Ag (D2). As an average, the molar ratio sIL-6R/dimer-sGP130 was increased by more than five-fold at D2 compared to D0 (Figure 1B). Taken together, the increase in IL-6, sIL-6R and the molar ratio of sIL-6R/sGP130 suggest the potential for enhanced IL-6 trans-signaling activity in BALF after allergen challenge.

Figure 1. Concentrations of IL-6 and its soluble receptor sIL-6R and sGP130 in BALF.

Figure 1.

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A/ BALF concentrations of IL-6, sIL-6R and sGP130 were determined by ELISA at baseline (D0) and 48 hours after SBP-AG (D2). B/ Molarities for sIL-6R and sGP130 in its homodimer form (dimer-sGP130) were calculated based on molecular weights of 40 kDa and 120 kDa, respectively, and ratio sIL-6R/dimer-sGP130 before (D0) and after challenge (D2) are shown. Boxes represent medians within 25th and 75th percentiles. Individual values for the 18 subjects are also shown using dots. For each subject, the dots at D0 and D2 are connected with straight lines.

Increase in lymphocyte number in BALF after allergen challenge positively correlated with changes in sIL-6R, sGP130 and the ratio sIL-6R/dimer-sGP130

Total numbers of BAL cells, eosinophils, lymphocytes, neutrophils and macrophages/monocytes significantly increased 48 h after SBP-Ag (Table II), with the percentage of BAL eosinophils increasing from 2 ± 1 % at baseline (D0) to 54 ± 4 % after challenge (D2). The change in IL-6 (pg/ml) in BALF after challenge compared to before challenge correlated with the change in number of BAL lymphocytes (r=0.585, p<0.01, Figure 2A). Changes in total cell and other cell-type numbers was not significantly associated with IL-6, although significance was almost reached for neutrophils (r=0.459, p-0.054; not shown). The change in the amount of sIL-6R in BALF after challenge correlated significantly with changes in total number of cells (r=0.752, p<0.0001), macrophage/monocytes (r=0.748, p<0.0002) and eosinophils (r=0.68, p<0.002) (Supplemental Figure 3). While the correlation of sIL-6R with the number of neutrophils was weak (r=0.50, p<0.035), the association with the number of lymphocytes was very high (r=0.851, p=0.0000002, Figure 2B, Supplemental Figure 3). Although sGP130 could be generated by non-inflammatory cells in the lung tissue, the change in sGP130 after challenge significantly correlated with changes in total cell number and macrophages/monocytes, with the highest correlations reached with the lymphocytes (r=0.728, p=0.0004, Figure 2C) and the eosinophils (r=0.676, p<0.002, not shown). Finally, the fold change in the ratio sIL-6R/dimer-sGP130 after allergen challenge correlated with most cell types except for the neutrophils (not shown). Notably, the lymphocyte number strikingly correlated with sIL-6R/dimer-sGP130 (r=0.849, p=0.0000002, Figure 2D). Altogether, these correlative analyses indicated that among the inflammatory cells, the lymphocytes are the most associated with IL-6 and potential IL-6 trans-signaling molecules in BALF after allergen challenge.

Table II.

BAL cell count before (D0) and after (D2) SBP-Ag

D0 D2 P values
Total cells (105) 356.2 ± 35 4026.0 ± 720 P< 0.001
Total eosinophils (105) 9.2 ± 5 2501.6 ± 540 P< 0.001
Total lymphocytes (105) 29.8 ± 5 301.3 ± 67 P< 0.001
Total neutrophils (105) 5.8 ± 2 130.9 ± 22 P< 0.001
Total macrophages/monocytes (105) 301.5 ± 34 1092.0 ± 146 P< 0.001
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All values at D0 and D2 are Mean ± SE, n=18 subjects described in Table I.A.

Figure 2. Correlation between BAL lymphocyte numbers with BAL concentrations of IL-6, sIL-6R, sGP130 and molar ratio of sIL-6R/dimer-sGP130.

Figure 2.

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For each subject, changes of IL-6, sIL-6R and sGP130 (D2-D0) between before (D0) and after SBP-Ag (D2), and fold change of the ratio sIL-6R/dimer-sGP130 (D2/D0) were calculated. Correlation between change in BAL lymphocyte numbers with change in BAL A/ IL-6 (pg/ml), B/ sIL-6R, C/ sGP130 and D/ molar ratio of sIL-6R/dimer-sGP130. Spearman rank order correlation coefficient r and p values are shown for n=18 subjects (subjects described in Table I.A.).

Expression of membrane IL-6 receptors by blood and BAL cells.

To determine whether BAL cells are a potential source of sIL-6R and sGP130, we used flow cytometry to compare the expression of these receptors on BAL compared to blood cells. IL-6R was present on blood neutrophils, monocytes, lymphocytes and eosinophils, and to a lesser degree on BAL monocytes, lymphocytes and eosinophils (Supplemental Figures 2 and 4). BAL neutrophils were not further evaluated due to their very low frequency in the majority of subjects. Compared to baseline (D0), there was no significant change in receptor-positive cells 48 h (D2) after SBP-Ag, (Figure 3). Blood monocytes and lymphocytes were also highly positive for surface GP130, which was reduced on BAL cells (Figure 3 and Supplemental Figures 2 and 4). Neither IL-6R nor GP130 were detected on alveolar macrophages (Supplemental Figures 2B and 4)

Figure 3. Expression of membrane IL-6R and GP130 on blood and BAL cells.

Figure 3.

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Paired samples of whole blood and unseparated BAL cells, collected at baseline (D0) and 48 hours (D2) after SBP-Ag, were analyzed by flow cytometry for cell surface expression of IL-6R (A) and GP130 (B). Percentage of positive cells for IL-6R and GP130 is shown. Boxes represent medians within 25th and 75th percentiles for n=5 subjects (Table I.B.). **p<0.001, *p=0.002, #p=0.005 compared to blood. At D0, the number of BAL eosinophils was too low to determine surface proteins (na).

The reduced expression of IL-6R and GP130 on BAL versus blood inflammatory cells (Figure 3) suggest that these cells are a source of soluble receptors that are shed from the cell surface. Because eosinophils are the predominant recruited cell type after airway allergen challenge (Table II) they were further evaluated as a potential source of IL-6R and GP130. We extracted data from previously published proteomic analyses [41] and performed new next-generation RNA-sequencing (RNA-seq) analysis (Table III). We found that blood eosinophils express IL-6R and ADAM10, a metalloproteinase that cleaves membrane IL-6R into its soluble form. In these same conditions however, eosinophils did not express GP130, ADAM7 or IL-6 (Table III). These data confirmed that eosinophils are probably not a source of sGP130 but they might play a role in IL-6 trans-signaling by shedding IL-6R from their surface.

Table III.

RNA-sequencing and proteomic analyses of IL-6R, GP130, ADAM7, ADAM10 and IL-6 expressions in human blood eosinophils

*RNA-sequencing & Proteomic
Rest-4h IL-3–4h IL-3–20h Rest-20h IL-3–20h
IL-6R (IL6R) 6.16 5.74 4.56 14.79 14.62
GP130 (IL6ST) −2.4 −2.32 −4.02 not detected not detected
ADAM10 2.99 3.01 3.29 21.25 21.33
ADAM7 −9.5 −9.5 −9.5 not detected not detected
IL-6 (IL6) −9.5 −9.5 −9.5 not detected not detected
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*

Eosinophils were prepared from three subjects with allergy and mild-asthma with no corticosteroids taken the day of blood draw. See Materials and Methods for eosinophil preparation and RNA-sequencing details. Mean values (n=3 subjects) are shown, and the range of expression for all values was from −9.5 to +15.1.

&

Eosinophils were prepared from five subjects with allergy including three with mild-asthma. No corticosteroids were taken the day of blood draw. The proteomic data have been previously published in Esnault et al. J Proteome Res. 2018 (Ref#47). Mean values (n=5) are shown, and the range of expression values was from 10.1 to 27.7.

Eosinophils were cultured in RPMI with 10% serum with either IL-3 (2 ng/ml) or remained in a resting state (without IL-3; Rest) for 4h and 20h before performing *RNA-sequencing or &Proteomic. In bold are the transcripts/proteins present in eosinophils.

To determine if airway stromal cells are potential sources of IL-6R and GP130, we examined data from our previously published RNA-seq analyses of both human bronchial epithelial cells and human lung parenchymal fibroblasts [13, 42] (Table IV). We found that these non-inflammatory cells express a high level of GP130 (Table IV), and expressed IL-6R, ADAM10 and some IL-6 (Table IV), suggesting that bronchial epithelial cells and lung parenchymal fibroblasts could both provide sGP130 in BALF, as well as enhance IL-6 trans-signaling in an autocrine way. However, we did not detect IL-6R protein on the surface of the bronchial fibroblasts (Supplemental Figure 5).

Table IV.

RNA-sequencing analyses of IL-6R, GP130, ADAM7, ADAM10 and IL-6 expressions in human bronchial epithelial cells and human lung fibroblasts

* Bronchial epithelial cells & Lung fibroblasts
IL-6R (IL6R) 4.92 2.19
GP130 (IL6ST) 7.37 6.22
ADAM10 7.25 6.34
ADAM7 not present −9.46
IL-6 (IL6) −0.38 0.76
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*

Epithelial cells were obtained from three healthy individuals and cultured at air-liquid interface (ALI) before RNA-sequencing analyses as published in Barretto et al Allergy 2020 (Ref #54). Mean values (n=3) are shown and the range of expression for all values was from −3.3 to +13.3.

&

Lung fibroblasts were obtained from healthy lung fragments from two subjects. Fibroblasts were cultured with 5% serum for 24h and RNA-sequencing was performed as published in Esnault et al. Respir Res. 2017 (Ref#15). Mean values (n=2) are shown and the range of expression for all values was from −9.46 to +13.5.

In bold are the transcripts present in bronchial epithelial cells and lung fibroblasts.

IL-6 trans-signaling increased MCP-1 production by fibroblasts

Several studies have proposed that IL-6 signaling triggers the production of MCP-1 (CCL2) that will further prolong recruitment of inflammatory cells, such as monocytes [19, 43]. Thus, we examined bronchial fibroblasts as a potential target of IL-6 trans-signaling to generate the chemokine MCP-1 following stimulation with the combination of IL-6 plus sIL-6R. We also analyzed in the same conditions, the extracellular matrix protein, fibronectin, which is implicated in the fibrotic process. Spontaneous release of MCP-1 was highly variable among fibroblast from different subjects (the median with range was 2412 (148–13552) pg/ml). Thus, data in Figure 4 are expressed as a fold change relative to medium used alone. When fibroblasts were stimulated with IL-6, MCP-1 increased in fibroblast from 5 of 7 subjects, but the change reached statistical significance only in the presence of a trans-signaling activity using both IL-6 and sIL-6R (Figure 4A). As expected, preincubation with GP130 prior to stimulation with IL-6 plus sIL-6R reduced the effect of trans-signaling on MCP-1 (Figure 4A). These changes in MCP-1 release due to presence of sIL-6R and sGP130 were confirmed using the raw data in pg/ml (Supplemental Figure 6). IL-6 trans-signaling had no effect on fibroblast generation of fibronectin (Figure 4B), which was spontaneously released in high amounts by bronchial fibroblasts (the median with range was 2503 (725–8021) pg/ml).

Figure 4. Bronchial fibroblast generation of MCP-1 by IL-6 trans-signaling.

Figure 4.

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Fibroblasts were prepared from endobronchial lung biopsies from seven human donors, and were cultured with medium alone, IL-6 (20 ng/ml), sIL-6R (50 ng/ml), sGP130 (200 ng/ml), or IL-6 plus sIL-6Rα. In another condition, cells were preincubated with sGP130 (200 ng/ml) prior to culture with IL-6 plus sIL-6Rα. After 5 days of culture, MCP-1 (A) and fibronectin (B) present in the culture supernatants were measured by ELISA. Spontaneous release of MCP-1 (pg/ml) was highly variable among fibroblast from different subjects. Thus, data were expressed as fold change relative to medium. Means with error bars are shown. * and # indicate statistical differences with IL-6 and with IL-6 plus sIL-6R plus sGP130, respectively.

Discussion

Using SBP-Ag to measure airway IL-6 and its receptors, we found that BAL IL-6, sIL-6R and the BAL molar ratio of sIL-6R/sGP130 were significantly augmented following allergen challenge. These findings demonstrate an increase of molecules important for both IL-6 signaling and trans-signaling in the airways after an allergen challenge. While sIL-6R is known to be elevated after an allergen challenge in asthma [23], we showed that potential IL-6 trans-signaling by sIL-6R and IL-6 is not likely to be significantly tampered by the inhibitory sGP130, which, compared to sIL-6R, was only modestly increased in BAL after challenge.

While sGP130 is known to occur via an alternatively spliced transcript variant [28]; the near absence of detectable cell surface GP130 on BAL cells and the striking reduction on BAL monocytes compared to blood raise the possibility that the receptor is cleaved from the surface by an unknown membrane-associated or soluble protease. Cell surface cleavage has been suggested in endometrial tissue; however, the proteinase has not been identified [29]. Additionally, the enhanced accumulation of sGP130 in the airways after SBP-Ag may originate from bronchial epithelial cells and lung fibroblasts, which both expressed a relatively high level of GP130 mRNA.

Conversely to GP130, and as expected [8, 19, 22, 44], IL-6R was clearly found on the surface of BAL inflammatory cells such as lymphocytes and monocytes. Although, IL-6R has been detected in tissue macrophages in other models [45, 46], it was not detectable by flow cytometry on alveolar macrophages in our study. Our data, however, are in agreement with an earlier study showing that IL-6R expression is reduced during differentiation of the monocytes into macrophages [47]. Whether IL-6R is shed from the surface of alveolar macrophages or its expression is decreased upon activation and differentiation remains to be determined. Due to the low frequency of neutrophils in the BAL it was not possible to fully characterize IL-6R expression on this cell population. However, the weak association between neutrophil number and sIL-6R and the lack of correlation with the molar ratio of sIL-6R/sGP130 may indicate that neutrophils are not an important source of sIL-6R in the SBP-Ag model. This discrepancy with other studies [19, 48] may be due to the more neutrophilic character of the diseases analyzed including chronic obstructive pulmonary disease and other asthma phenotypes where the neutrophil/eosinophil ratio is higher than in our SBP-Ag model.

Although eosinophils are known to express other GP130-family receptors including the IL-27 receptor [49], to our knowledge this is the first report showing the presence of IL-6R on the surface of blood and BAL eosinophils. We also showed that eosinophils produce a high amount of ADAM10, a protease that causes shedding of IL-6R [21]. Thus, eosinophils have the potential to be an important source of sIL-6R in the airway after an allergen challenge. This premise is supported by the significant correlations between eosinophil number after SBP-Ag and the changes in BAL sIL-6R and the molar ratio of sIL-6R/sGP130 after SBP-Ag, and it is in agreement with a study by Doganci et al. where sIL-6R correlates with eosinophil number in BAL after an allergen challenge in subjects with asthma [23].

Among the BAL cell types, the most striking associations with sIL-6R and sIL-6R/sGP130 occurred with the number of BAL lymphocytes. It is possible that BAL lymphocytes are a source of IL-6R; however, it is also possible that IL-6 function led to lymphocyte accumulation in BAL after challenge. It is known for instance that chemokines for T lymphocytes, such as CCL20, CCL22, CCL17, CXCL10 and CCL-2 (MCP-1) accumulate in BAL after SBP-Ag [50, 51]. Previous studies have reported that lymphocyte number peaked from 18 h to 48 h after SBP-Ag [52, 53] and remained elevated up to 7 days after challenge [53]. Therefore, we speculate that 48 h after SBP-Ag, IL-6 signaling could be involved in lymphocyte recruitment via production of chemokines by IL-6 trans-signaling-responding cells. In addition, direct effects of IL-6R and IL-6 trans-signaling on T lymphocytes have been demonstrated in allergic and pulmonary inflammatory mouse models where IL-6 signaling was responsible for decreased lymphocyte apoptosis and increased type-2 and type-17 lymphocytes [54, 55].

Because fibroblasts produce low levels of IL-6R and high amounts of GP130, they are an ideal target for IL-6 trans-signaling. The role of IL-6 trans-signaling has been studied using corneal and dermal fibroblasts [56–58]. In corneal fibroblasts, IL-6 trans-signaling results in the production of MCP-1 [56], which can lead to a transition from neutrophilic to monocytic inflammation [43]. In our present study, using bronchial fibroblasts obtained by endobronchoscopy, we also demonstrated that IL-6 trans-signaling led to enhanced release of MCP-1. Of note, MCP-1 is increased in the airway after SBP-Ag and it can recruit lymphocytes via CCR2 and CCR4 present on the BAL lymphocyte surface after SBP-Ag [59]. We did not observe an effect of IL-6 trans-signaling on fibronectin production. This was unexpected because IL-6 trans-signaling is critical for the production of fibronectin in a mouse model of idiopathic pulmonary fibrosis [60]. Whether the production of fibronectin in that model was a direct effect of IL-6 trans-signaling on fibroblasts or required a second signal such as TGF-β [58] is not known. The bronchial fibroblasts used in our study spontaneously released a high amount of fibronectin suggesting that alternative growth conditions are necessary to definitively show whether cells can or cannot release fibronectin in response to IL-6-trans-signaling.

A limitation of this study is that we did not directly measure IL-6 trans-signaling activity in the airways. Although MCP-1 is elevated in BALF [59] after SBP-Ag, MCP-1 cannot alone define the signature of IL-6 trans-signaling. Therefore, as a future direction, it would be important to first define the signature of IL-6 trans-signaling in vitro on bronchial fibroblasts and airway epithelial cells cultured at air-liquid interface using RNA-seq and protein multiplex analyses and then to utilize this signature to evaluate IL-6 trans-signaling activity in vivo. Additionally, despite increased sIL-6R/sGP130 ratio after SBP-Ag, it is unknown whether the amount of sGP130 after challenge remained sufficient to block IL-6 trans-signaling. Finally, future investigations to determine the conditions under which eosinophils release sIL-6R and the identification of the responsible proteinases would be of interest.

In conclusion, by measuring IL-6, sIL-6R and sGP130 in BAL, we showed that the elements of IL-6 trans-signaling were upregulated after bronchial allergen challenge. All inflammatory cells present in the airways after allergen challenge, except for the alveolar macrophages, were potential sources of sIL-6R, including eosinophils, which did not produce GP130. IL-6, sIL-6R and sIL-6R/sGP130 ratio were highly associated with lymphocyte number in the airways. The consequences of IL-6 trans-signaling include augmentation of chemokine production by fibroblasts. We may then speculate that after allergen challenge, enhanced IL-6 trans-signaling and chemokine production would prolong the inflammatory response and ultimately lead to lung function impairment. The beneficiaries of anti-IL-6 signaling therapies may include both patients with type-2 and non-type-2 asthma.

Supplementary Material

Supplemental Figure 1
Supplemental Figure 2A
Supplemental Figure 2B
Supplemental Figure 4
Supplemental Figure 5
Supplemental Figure 3
Supplemental Information
Supplemental Figure 6

ACKNOWLEDGMENTS

We thank our Research Nurses, Mary Jo Jackson and Erin Billmeyer for patient recruitment, screening, and assistance with bronchoscopies; Dr. Keith Meyer for assistance with bronchoscopies and Kristine Lee for directions on statistical analyses. We would like to acknowledge Drs. Mary Ellen Bates and Beatriz Quinchia-Rios for their input and support and the staff at University of Wisconsin pulmonary lab, Katie Clay, Rose de Grauw, Mollie Malaney and Ioana Oltean for technical assistance. This work was supported by HL64066, HL56396 and M01RR03186, Program Project grant P01 HL088594 and Clinical and Translational Research Center grant UL1 RR025011 from the National Institutes of Health.

Abbreviations:

BAL

bronchoalveolar lavage

BALF

bronchoalveolar fluid

mIL-6R

membrane IL-6 receptor

mGP130

membrane glycoprotein-130

RNA-seq

next generation RNA-sequencing

SBP-Ag

segmental bronchoprovocation with an allergen

sGP-130

soluble glycoprotein-130

sIL-6R

soluble IL-6 receptor

Footnotes

Conflict of Interest Statement: Dr Jarjour has received honoraria from Glaxo Pharmaceuticals, Astra Zeneca and Boehringer Ingelheim for consultations unrelated to the focus on this manuscript. None of the authors has relevant conflict of interest with this study.

Data Availability: All the data used in this manuscript are available to the public.

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Associated Data

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Supplementary Materials

Supplemental Figure 1
NIHMS1686109-supplement-Supplemental_Figure_1.pdf (171.8KB, pdf)
Supplemental Figure 2A
NIHMS1686109-supplement-Supplemental_Figure_2A.pdf (164.3KB, pdf)
Supplemental Figure 2B
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Supplemental Figure 4
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Supplemental Figure 5
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Supplemental Figure 3
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Supplemental Information
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Supplemental Figure 6
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