American Journal of Respiratory Cell and Molecular Biology

Allergic asthma is a heterogeneous disease with no curative therapies. T cells infiltrate the airway smooth muscle (ASM) layer and may be implicated in airway remodeling and the increase of ASM mass, a cardinal feature of asthma. The mechanism by which CD4+ T cells drive airway remodeling remains unknown. This study sought to determine the T cell–mediated mechanism of ASM cell proliferation. We hypothesized that CD4+ T cells adhere to ASM cells via CD44, and induce ASM cell proliferation through the activation of the epidermal growth factor receptor (EGFR). A coculture model showed that the contact of antigen-stimulated CD4+ T cells with ASM cells induced high levels of EGFR ligand expression in CD4+ T cells and the activation of matrix metalloproteinase (MMP)–9, required for the shedding of EGFR ligands. The inhibition of EGFR and MMP-9 prevented the increase of ASM cell proliferation after coculture. The hyaluronan receptor CD44 is the dominant mediator of the tight adherence of T cells to ASM and is colocalized with MMP-9 on the cell surface. Moreover, the neutralization of CD44 prevents ASM cell hyperplasia. These data provide a novel mechanism by which antigen-stimulated CD4+ T cells induce the remodeling indicative of a direct trophic role for CD4+ T cells.

Asthma is characterized functionally by airway hyperresponsiveness (AHR) and intermittent airway obstruction, and pathologically by inflammation and structural changes of the airways, which are termed airway remodeling. Structural changes include subepithelial fibrosis and increases in airway smooth muscle (ASM) mass. One of the main mechanisms contributing to the increase in ASM mass, which correlates with the severity of disease, is an increase in ASM cell proliferation (1). ASM cells are important structural cells that contribute to AHR and airway remodeling (2). Despite the presence of T cells within and adjacent to the ASM layer (3, 4), few studies have examined the role of T cells in ASM growth. In vitro studies have shown that T lymphocytes adhere to ASM cells via CD44, intercellular adhesion molecule–1 (ICAM-1), and vascular cell adhesion molecule–1 (VCAM-1). This direct contact is required for ASM cell proliferation (5). In an animal model of asthma, antigen-stimulated CD4+ T cells were found in contact with ASM cells in vivo, and in vitro these same cells triggered ASM cell hyperplasia through direct cell–cell contact (6). Furthermore, strong relationships are evident among asthma severity, airway smooth muscle mass, and the T cell infiltration of smooth muscle bundles in human subjects (4). CD4+ T cells secrete a panoply of cytokines, but many studies looking at the plausibility of Th1 and Th2 cytokines as mediators of ASM cell proliferation have failed to implicate these molecules. Indeed, two prominent Th2 cytokines, IL-4 and IL-13, which are strongly associated with asthma, have demonstrated antiproliferative properties, suggesting that proinflammatory Th2 cytokines could not mediate ASM cell proliferation (7, 8).

An up-regulation of the epidermal growth factor receptor (EGFR) and one of its ligands, heparin-binding epidermal growth factor (HB-EGF), has been observed in asthmatic airway tissues (9, 10). EGFR is a tyrosine kinase receptor involved in many processes such as cell differentiation, proliferation, and migration (11). Other ligands of EGFR include epidermal growth factor (EGF), amphiregulin (Areg), transforming growth factor–α (TGF-α), betacellulin, epiregulin, and epigen. EGFR ligands are synthesized as precursor ligands in a plasma membrane–anchored form, and require cleavage by proteolytic enzymes such as metalloproteinases (MMPs) and “a disintegrin and metalloproteinases” (ADAMs) (12). In particular, HB-EGF is a strong mitogen for ASM cells, and its expression in the airways is strongly associated with severe asthma (10, 13).

We reasoned that because CD4+ T cells induce ASM cell proliferation in vitro and that EGFR is involved in airway remodeling in vivo, perhaps the T cell mediates its direct effects through this receptor. Therefore, we hypothesized that CD4+ T cells in contact with ASM may be a significant source of EGFR ligands and induce ASM cell proliferation through the paracrine activation of the EGFR. We explored this hypothesis using a coculture model. Our results show that antigen-stimulated CD4+ T cells are a significant source of EGFR ligands that lead to ASM cell proliferation and require the participation of MMP-9 and CD44.

Cervical lymph nodes from sensitized Brown Norway rats (Harlan, London, UK) were cultured in complete Dulbecco’s Modified Eagle’s Medium (Gibco, Burlington, ON, Canada) containing 10% FBS (Gibco), 50 μM 2-mercaptoethanol, 1% nonessential amino acids (Gibco), 200 μg/ml ovalbumin (OVA), and 10 U/ml IL-2 (BD Biosciences, Mississauga, ON, Canada). After in vitro OVA stimulation, CD4+ T cells were enriched by immunomagnetic negative selection. Cell purity was assessed.

The tracheas of OVA-sensitized animals were digested in 0.05% elastase Type IV (Sigma-Aldrich, St. Louis, MO) and 0.2% collagenase Type IV (Sigma-Aldrich). Primary ASM cells were subcultured onto six-well plates, and at approximately 80% confluence, the cells were placed in serum-free medium containing 0.2% BSA (Sigma-Aldrich) for 72 hours. ASM cells were cultured under the specific conditions for 48 hours of 10% FBS, and 0.5% FBS containing 10 U/ml IL-2 in the presence or absence of 4 × 106 CD4+ T cells/well for 48 hours. Various inhibitors and neutralizing antibodies were added separately to the coculture medium, namely, 10 μM tyrphostin AG1478 (Cayman Chemical, Ann Arbor, MI), 25 μM GM6001 (3-(N-hydroxycarbamoyl)-2(R)-isobutylpropionyl-l-tryptophan methylamide; Calbiochem, Billerica, MA), 1 μM MMP-9 inhibitor (Calbiochem), 1 μg/ml CD44 (Abcam, Cambridge, MA), 10 μg/ml HB-EGF neutralizing antibodies (Abcam), 1 μg/ml of anti-CD11a and anti-CD49d (BD Biosciences), and 1 μM of TNF-α protease inhibitor-1 (TAPI1; Millipore, Billerica, MA). The concentrations used are the maximally effective doses. After coculture, the CD4+ T cells were detached by repeated washes with PBS, and ASM cell proliferation was assessed by flow cytometry, using an FITC–bromodeoxyuridine (BrdU) kit (BD Pharmingen, San Jose, CA).

Real-Time Quantitative PCR

The mRNA expression of EGFR ligands, CD44, and MMP-9 was measured by quantitative RT-PCR. The RNA of CD4+ T cells and ASM cells was extracted using the RNeasy Mini Kit, according to the manufacturer’s instructions (Qiagen, Toronto, ON, Canada). The RNA yield was measured using the NanoDrop system (Thermoscientific, Wilmington, DE), and RNA quality was assessed with an automated electrophoresis system (Bio-Rad, Hercules, CA). We prepared cDNA by using 100 μg/ml of total RNA, oligo(dT)12–18 primer, dNTP mix, RNaseOUT, and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. RT-PCR was performed using a LightCycler (Roche, Indianapolis, IN) and a QuantiTect SYBR Green PCR Kit (Qiagen).

Zymography

Further information on zymography is available in the online supplement.

Immunofluorescence microscopy

Cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature, and washed three times with PBS. Cells were stained for phosphorylated EGFR, using anti-rat phosphorylated EGFR (Abcam) and anti-mouse Alexa 488 (Invitrogen). Images were taken at ×40, using a fluorescent microscope. Confocal microscopy was used to assess CD44 and MMP-9 colocalization. Coverslips of cocultures were incubated with Dako blocking solution for 30 minutes (Dako, Burlington, ON, Canada). Cells were stained with FITC-conjugated anti-rat CD44 (1:100; Millipore), anti-rat MMP-9 (1:200), and Alexa 546 (1:2,000; Abcam). Images were collected on a Zeiss LSM-510 Meta laser scanning microscope (Zeiss, Jena, Germany).

Laser Tweezers

Further information on laser tweezers is available in the online supplement.

Statistical Analysis

Data are expressed as means ± SEs. For BrdU assays, data were normalized to the 10% FBS samples. When the data were not normally distributed, the data were log-transformed. Using GraphPad Prism (GraphPad Software, La Jolla, CA), one-way ANOVA with a Neuman-Keuls test was applied to assess significance. P ≤ 0.05 was considered statistically significant.

EGFR Is Involved in the CD4+ T Cell–Mediated Mechanism of ASM Cell Proliferation

We first explored the role of EGFR in the CD4+ T cell–mediated increase of ASM cell proliferation. Antigen-stimulated CD4+ T cells were obtained from the lymph nodes of OVA-sensitized Brown Norway rats and cocultured in the presence ASM cells for a period of 48 hours, which was previously shown to be the necessary time period to induce ASM cell proliferation (6). ASM cells were obtained from OVA-sensitized animals, grown to 80% confluence, and serum-deprived for 72 hours. At the end of the coculture period, nonadherent and adherent CD4+ T cells were removed by repeated washes. Remaining CD4+ T cells were distinguished from ASM cells by side-scattering on flow cytometry. Flow cytometry for BrdU incorporation revealed an increase in ASM cell proliferation after a 48-hour coculture period (Figure 1A). In the presence of AG1478 (10 μM), a specific EGFR inhibitor, at the time of coculture, we observed the complete prevention of ASM cell proliferation. AG1478 exerted no effect on the baseline proliferation of ASM cells. The concentration of AG1478 was determined to be optimal for inhibiting the exogenous HB-EGF–induced increase of ASM cell proliferation. We note that the percentages were normalized to 10% FBS (positive control) to reduce interexperiment variability. The average for 10% FBS using raw data was 31%, and 11% for 0.5% FBS.

ASM cells cultured with OVA-stimulated CD4+ T cells were assessed by immunofluorescence for evidence of EGFR phosphorylation, using a phosphospecific antibody for EGFR. After 48 hours of coculture, we observed strong evidence of EGFR phosphorylation in ASM cells (Figure 1B), compared with ASM before coculture. AG1478 added at the time of coculture completely prevented the phosphorylation of EGFR. We thus conclude from these findings that EGFR is involved in the CD4+ T cell–mediated increase of ASM cell proliferation.

Antigen-Stimulated CD4+ T Cells Are a Source of EGFR Ligands

EGFR ligands such as HB-EGF have been established to induce ASM cell proliferation in vivo and in vitro (10, 14). However, whether CD4+ T cells are a source of these ligands after antigen stimulation remains unknown. We hypothesized that antigen-stimulated CD4+ T cells express one or more of the EGFR ligands that contribute to ASM cell proliferation. To test this hypothesis, adherent OVA-stimulated CD4+ T cells were separated from ASM cells after the 48-hour coculture period by repeated washes. The cell purity of the CD4+ T cell population was verified by the negative expression of the myosin heavy chain (data not shown). We compared the expression of ligands of interest by these cells to that of OVA-stimulated CD4+ T cells from OVA-sensitized animals before coculture and nonstimulated CD4+ T cells from naive animals. After coculture, OVA-stimulated CD4+ T cells expressed significantly elevated concentrations of HB-EGF and TGF-α mRNA. HB-EGF expression was increased approximately 150-fold, compared with nonstimulated CD4+ T cells. TGF-α expression was increased approximately 70-fold, compared with nonstimulated cells. Although an increase in the expression of other ligands was evident, this was not as marked as that of the ligands already mentioned (Figures 2A–2E). The expression of EGFR ligands by ASM cells was studied to confirm that the major source of HB-EGF is CD4+ T cells. ASM cells after coculture expressed very little HB-EGF (Figure 2F). In vivo studies have shown that HB-EGF is the only EGFR ligand that induces ASM cell proliferation (13). ASM cell proliferation after coculture could be completely blocked by HB-EGF neutralization (10 μg/ml) without affecting baseline proliferation, indicating that HB-EGF is the major EGFR ligand involved in this process (Figure 3).

MMP-9 Is Involved in ASM Cell Proliferation, and Colocalizes with CD44 on the CD4+ T Cell Surface

EGFR ligands are synthesized as inactive proligands that are shed by MMPs and ADAMs to release the active form of the protein (15). Human ASM cells express active MMP-9, which has been linked with airway remodeling (16, 17). Therefore, we studied the involvement of MMP-9 in the cleavage of EGFR ligands and the increase of ASM cell proliferation. First, we looked at the expression of MMP-9 by ASM cells before and after coculture. A significant increase in MMP-9 expression was evident after coculture (Figure 4A). ADAM17 has also been involved in the shedding of EGFR ligands (18). Therefore, we looked at the expression of ADAM17 after coculture. No significant increase was evident in the expression of ADAM17 by ASM cells (Figure 4B). The supernatant was collected before and after coculture, and identification of the proteolytic enzyme was assessed using gelatin zymography. An increase in the release of MMP-9 was evident in the supernatant after coculture, although the exact source of the MMP-9 remains unknown (Figure 4F). Both a broad-spectrum MMP inhibitor (GM6001) and a specific MMP-9 inhibitor added at the time of coculture significantly attenuated ASM cell proliferation, supporting the idea that MMP-9 is upstream of EGFR activation by its ligands (Figure 4C). MMP-9 inhibition also inhibited EGFR phosphorylation (Figure IB). We then looked at the effect of blocking ADAM17, using a specific inhibitor, TAPI1 (1 μg). Blocking ADAM17 exerted no effect on ASM cell proliferation after coculture (Figure 4E).

To evaluate whether CD44 is necessary for the increase in ASM cell proliferation and the adhesion of CD4+ T cells to ASM cells, a neutralizing antibody to CD44 was used. CD44 neutralization prevented the increase in ASM cell proliferation, compared with coculture and the medium alone (Figure 4C). However, the neutralization of very late antigen–4 (VLA-4) and lymphocyte function–associated antigen–1 (LFA-1) exerted a mild effect on ASM cell proliferation (Figure 4D). Combining the neutralizing antibodies exerted no additive effect on ASM cell proliferation (data not shown). CD44 serves as a docking molecule for MMP-7 on neurite extensions of various tumor cell lines (19). Confocal microscopy was used to explore the possible association of CD44 with MMP-9 in our study. After the coculture of ASM cells and CD4+ T cells, an evident colocalization of CD44 and MMP-9 was observed on the surface of CD4+ T cells, as shown by the merged images suggesting that CD44 may also serve as a docking molecule for MMP-9, which leads to the shedding of EGFR ligands (Figure 5).

CD44 Is Sufficient to Mediate the Adhesion of CD4+ T Cells to ASM Cells

The next question involved whether CD44 was the predominant adhesion receptor mediating the attachment of CD4+ T cells to ASM cells. Using optical tweezer technology (laser trapping), we tested the strength of adhesion of T cells to the ASM cell. Cells were cocultured in the presence and absence of neutralizing antibody for CD44 (1 μg/ml) for 48 hours. In the absence of the neutralizing antibody, we observed a firm adhesion of CD4+ T cells to ASM cells, sufficient to prevent the detachment of T cells by the optical tweezers (Figures 6B and 6C). In the absence of anti-CD44, none of the CD4+ T cells was detachable from the ASM cells. After firm attachment had been established, to reverse it was still possible with anti-CD44 antibody added after the 48-hour coculture period. As early as 6 minutes after the addition of anti-CD44, we were able to detach a fraction of the CD4+ T cells that were previously firmly attached to the ASM cells (Figure 6C). After 1 hour of the incubation of anti-CD44 with the cells, we were able to detach approximately 50% of the cells that were targeted previously for their firm adhesion to ASM cells.

Many mediators may trigger ASM cell hyperplasia, and among the strongest mitogens are growth factors such as HB-EGF (20). Moreover, OVA-stimulated CD4+ T cells in contact with ASM cells induce proliferation in vitro (6). To our knowledge, this is the first study to show that antigen-stimulated CD4+ T cells express HB-EGF and other EGFR ligands, in turn inducing the paracrine activation of EGFR and proliferation of ASM cells in a contact-dependent manner. We have investigated the mechanism by which this occurs. An up-regulation of MMP-9 expression is observed in ASM cells after coculture. MMP-9 was also present in the coculture medium. We have shown that MMP-9 colocalizes with CD44, which is involved in the adhesion of CD4+ T cells to ASM cells. The inhibition of CD44 or MMP-9 is sufficient to prevent ASM cell proliferation.

EGFR has been strongly associated with airway remodeling. Studies targeting EGFR signaling have shown a decrease in features of airway remodeling, such as ASM cell hyperplasia and goblet cell proliferation in animal models (21). A coculture model of Th17 cells and epithelial cells has been shown to induce HB-EGF expression and release (14). However, the release of HB-EGF or other ligands of EGFR has not been documented in antigen-stimulated CD4+ T cells. For the first time, we show that OVA-stimulated CD4+ T cells from OVA-sensitized animals express high concentrations of HB-EGF and TGF-α, compared with unstimulated CD4+ T cells from naive animals (Figures 2A–2E). Moreover, this expression is further enhanced after coculture, suggesting that the direct contact of CD4+ T cells and ASM cells stimulates the expression of EGFR ligands.

In vivo, HB-EGF is the only EGFR ligand shown to date to induce ASM cell hyperplasia. For this reason, we decided to focus on the role of HB-EGF in the CD4+ T cell–mediated increase of ASM cell proliferation. Moreover, HB-EGF was the most increased EGFR ligand at the mRNA level in antigen-stimulated CD4+ T cells before and after coculture. Neutralization of HB-EGF completed prevented the in vitro increase of ASM cell proliferation. HB-EGF at the protein level was not measured because of the lack of ELISA kits available for rat HB-EGF. Interestingly, EGFR ligands have been noted to possess the ability to bind and activate the receptor located on a juxtacrine cell, which may contribute to our observation that the proximity of CD4+ T cells and ASM cells is important (22). Other studies have shown that HB-EGF expression is up-regulated in proliferating ASM cells in culture and in severely asthmatic tissue (10). However, RT-PCR showed little expression of HB-EGF in ASM cells after coculture, compared with ASM cells before coculture, confirming that antigen-stimulated CD4+ T cells were the major source of HB-EGF in a coculture of T cells and ASM cells. It is difficult to assess the relative importance of direct T cell–ASM interactions that are dependent on EGFR activation with potential EGFR ligand release elsewhere. Epithelial EGFR expression appears sufficient for ASM thickening in a murine model driven by house dust mites (12). Such an epithelial dependence of remodeling may be more important in the thin airways of a mouse, or else changes in epithelial properties that are associated with deficient EGFR signaling may exert other effects. Further studies will be required to address this issue.

EGFR ligands are synthesized as proligands that require cleavage by certain MMPs and ADAMs. ASM cells express certain MMPs, such as active MMP-3 and MMP-9 (16). MMP-9 is present in low amounts in healthy adult lungs, but is increased during asthma, chronic obstructive pulmonary disease, and fibrosis. Moreover, lung remodeling is strongly correlated with increased concentrations of MMP-9. MMP-9 is the predominant MMP found in bronchoalveolar lavage fluid, sputum, transbronchial biopsy tissue, and the blood of patients with asthma (23). Therefore, we investigated the expression of MMP-9 in ASM cells before and after coculture, and observed a significant increase in MMP-9 expression after coculture. EGFR ligands have long been suggested to require the shedding by MMPs to activate the receptor. MMP-9 is associated with the release of HB-EGF, whereas the anti–MMP-9 antibody blocks the purinergic receptor–triggered release of HB-EGF from Muller glial cells (24). We confirmed the presence of MMP-9 in the supernatant by zymography. In the presence of a specific inhibitor of MMP-9, we observed a prevention of ASM cell proliferation and EGFR phosphorylation, possibly via the failure to cleave pro–HB-EGF. Moreover, ADAM17 promotes the shedding of TGF-α, Areg, and HB-EGF, and may be associated with airway remodeling (25). However, we did not observe an increase in ADAM17 expression among ASM cells after coculture, and blocking ADAM17 exerted no effect on ASM cell proliferation, suggesting that MMP-9 is the main proteolytic enzyme involved in this process (Figures 4B and 4E).

Because pro-EGFR ligands are plasma-membrane–anchored proteins, we investigated whether CD44 is involved in the recruitment of MMP-9 to the cell surface of antigen-stimulated CD4+ T cells after coculture to facilitate the cleavage of EGFR ligands. In fact, CD44 and the proteolytic form of MMP-9 have been shown to associate on the surface of mouse mammary carcinoma and human melanoma cells. This interaction mediates the degradation of collagen IV, and allows for tumor invasion (26). In a later study, the same authors showed that this interaction promoted TGF-β activation (27). Our results using confocal microscopy confirm the colocalization of CD44 and MMP-9 on the cell surface of antigen-stimulated CD4+ T cells after the 48-hour coculture period. These results implicate CD44 not only as a receptor involved in the adhesion of cells, but also as a docking molecule for MMP-9, possibly facilitating the cleavage of EGFR ligands. Moreover, studies have shown that CD44 also acts as a surface binding site for many proinflammatory mediators such as macrophage inflammatory protein–1β (28), and growth factors such as basic fibroblast growth factor and HB-EGF (29). The antigen stimulation of splenic CD4+ T cells induced an increase in CD44 expression (30). We noted a trend toward an increase in CD44 expression in CD4+ T cells after OVA stimulation in vitro (data not shown). Using a neutralizing antibody for CD44, we were able to inhibit ASM cell proliferation, as assessed by BrdU incorporation after coculture. The administration of anti-CD44 to an Ascaris suum antigen–induced murine model of pulmonary eosinophilia has been shown to prevent airway inflammation and AHR (31). To our knowledge, the effect of CD44 neutralization on airway remodeling was not previously documented.

As stated previously, CD44 is involved in the adhesion of T cells to ASM cells, in conjunction with ICAM-1 and VCAM-1 (5). Whether this glycoprotein is sufficient for mediating antigen-stimulated CD4+ T cell adhesion to ASM cells is addressed in the present study. First, we showed that the neutralization of CD44 before the coculture prevented the CD4+ T cell–mediated increase in ASM cell proliferation. This result suggests that CD44 plays a role in ASM cell hyperplasia. Second, using laser tweezers, we confirmed that CD44 is necessary for CD4+ T cell adhesion to ASM cells. The neutralization of CD44 before the coculture prevented the adhesion of CD4+ T cells to ASM cells. Moreover, this adhesion was partly reversible when anti-CD44 was added after the coculture period. Interestingly, a study has shown that CD44 is important in the accumulation of Th2 cells, but not Th1 cells, after antigen challenge. Moreover, a lack of CD44 failed to induce AHR (32). However, neutralizing VLA-4 and LFA-1 did not completely block ASM cell proliferation after coculture.

In conclusion, we have provided evidence of a novel trophic role for CD+ T cells in ASM cell proliferation through direct interaction with the cells through CD44. We defined the pathway to the activation of EGFR via the MMP9-induced release of HB-EGF. This mechanism may be of particular relevance for pathological processes such as asthma, in which T cell infiltration is present in the smooth muscle of the airway, and our findings should prompt the further exploration of this pathway for its therapeutic potential. This study may contribute to a better understanding of the role of CD4+ T cells in airway remodeling, and more particularly in ASM cell hyperplasia. Moreover, this study provides many targets that can be further investigated. Among the most relevant targets in this study is the role of CD44 in airway remodeling. To our knowledge, few studies have investigated the role of CD44 in ASM cell proliferation. We believe this would be an interesting target for investigation in future studies.

The authors thank Venkatesan Narayanan and Muhannad Hassan for technical assistance.

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Correspondence and requests for reprints should be addressed to James G. Martin, M.D., Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St.-Urbain, Montreal, PQ, H2X 2P2 Canada. E-mail:

This work was supported by the Richard and Edith Strauss Canada Foundation, and by the Canadian Institutes of Health Research.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2012-0356OC on May 8, 2013

Author disclosures are available with the text of this article at www.atsjournals.org.

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