Release of Interleukins 6 and 8 Induced by Zymosan and Mediated by MAP Kinase and NF-κB Signaling Pathways in Human Corneal Fibroblasts

Abstract

Purpose.: Zymosan is derived from the cell wall of yeast and induces immune responses associated with fungal infection. The effects of zymosan on the expression of proinflammatory cytokines, chemokines, and adhesion molecules and on the activity of signaling pathways were examined in cultured human corneal fibroblasts.

Methods.: Release of the proinflammatory cytokines interleukin (IL)-6, IL-1β, and IL-12 and of the chemokines IL-8, IP-10, and RANTES was measured with enzyme-linked immunosorbent assays. Expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) was evaluated by immunoblot and immunofluorescence analyses. Phosphorylation of mitogen-activated protein kinases (MAPKs) and the NF-κB inhibitory protein IκB-α was assessed by immunoblot analysis. Subcellular localization of the p65 subunit of the transcription factor NF-κB was determined by immunofluorescence analysis.

Results.: Zymosan induced the release of IL-6 and IL-8 from corneal fibroblasts without affecting either the release of IL-1β, IL-12, IP-10, or RANTES or the expression of ICAM-1 or VCAM-1. Zymosan also activated the MAPKs ERK, p38, and JNK and induced the phosphorylation of IκB-α and the nuclear translocation of p65 in these cells. The zymosan-induced release of IL-6 and IL-8 was attenuated by inhibitors of ERK, p38, JNK, and NF-κB signaling.

Conclusions.: Zymosan induces the release of IL-6 and IL-8 from human corneal fibroblasts in a manner dependent on MAPK and NF-κB signaling pathways. Corneal fibroblasts may thus modulate the local immune response to fungal infection in the corneal stroma.

Fungal keratitis refers to inflammation of the cornea as a result of ocular infection with a fungus. ^1,2 Such infection can occur as a result of trauma associated with plant material, the use of antibiotic or immunosuppressive drugs, or an otherwise immunocompromised state. The fungus grows and proliferates in the cornea and sometimes breaks through the Descemet membrane and passes into the anterior chamber. Inflammation caused by interactions among infiltrated cells, resident cells, and the fungus leads to edema, scarring, and neovascularization in the cornea. The factors responsible for such inflammatory events in the corneal stroma after fungal infection, however, remain incompletely characterized. 

Keratocytes are the resident cells of the corneal stroma and produce the extracellular matrix of this tissue that is responsible for corneal transparency. ^3,4 Keratocytes transform into corneal fibroblasts in response to injury or damage to the cornea. ^5,6 Corneal fibroblasts remodel the collagen structure of the stroma and contribute to the modulation of stromal inflammation through the production of cytokines, chemokines, and adhesion molecules. ^7,8 Proinflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-6, as well as chemokines such as IL-8, granulocyte colony-stimulating factor, and macrophage inflammatory protein-1β, are secreted by corneal fibroblasts in response to external inflammatory stimuli. ^9,10 Adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 are also expressed by corneal fibroblasts during corneal inflammation. ^10,11  

Zymosan is derived from the cell wall of yeast and consists of protein-carbohydrate complexes. ^12,13 It binds to Toll-like receptor 2 at the surface of immune cells and thereby induces the expression of proinflammatory cytokines. ^14,15 Exposure of corneal epithelial cells to zymosan has also been found to upregulate production of the proinflammatory cytokines IL-1β and IL-6. ¹⁶ To investigate the role of corneal fibroblasts in fungal keratitis, we have now examined the effects of zymosan on the production of proinflammatory cytokines (IL-1β, IL-6, IL-12) and chemokines (IL-8, interferon-inducible protein [IP]-10, regulated on activation normal T cell expressed and secreted [RANTES]) and on the expression of ICAM-1 and VCAM-1 in cultured human cells. The intracellular signaling pathways responsible for such effects were also examined. 

Materials and Methods

Materials

Eagle minimum essential medium (MEM), fetal bovine serum, and trypsin-EDTA were obtained from Invitrogen-Gibco (Carlsbad, CA), and 24-well culture plates and 60-mm culture dishes were from Corning-Costar (Corning, NY). Zymosan A was obtained from Sigma-Aldrich (St. Louis, MO), boiled for 1 hour, and washed three times with sterile phosphate-buffered saline (PBS). Antibodies to ICAM-1, VCAM-1, and the p65 subunit of nuclear factor (NF)-κB and normal rabbit or mouse immunoglobulin G (IgG) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to total or phosphorylated forms of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun NH₂-terminal kinase (JNK), and IκB-α were from Cell Signaling (Beverly, MA). PD98059 (tested at 10 μM), SB203580 (10 μM), JNK inhibitor II (10 μM), and IKK2 inhibitor IV (1 μM) were obtained from Calbiochem (La Jolla, CA), and a protease inhibitor cocktail and mouse monoclonal antibodies to β-actin were from Sigma-Aldrich. Nitrocellulose membranes and an enhanced chemiluminescence (ECL) kit were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden), and Alexa Fluor 488–labeled goat antibodies to mouse or rabbit IgG were from Molecular Probes (Eugene, OR). All media and reagents for cell culture were endotoxin minimized. 

Isolation and Culture of Human Corneal Fibroblasts

Human corneas were obtained for corneal transplantation surgery from NorthWest Lions Eye Bank (Seattle, WA). Human tissue was used in strict accordance with the tenets of the Declaration of Helsinki. Corneal fibroblasts were isolated and cultured as described previously. ¹¹ In brief, the endothelial layer of the rim of the cornea remaining after transplantation surgery was removed mechanically, and the tissue was incubated with dispase (2 mg/mL in MEM) for 1 hour at 37°C. After mechanical removal of the epithelial sheet, the tissue was treated with collagenase (2 mg/mL in MEM) at 37°C until a single-cell suspension of corneal fibroblasts was obtained. Isolated corneal fibroblasts were maintained under a humidified atmosphere of 5% CO₂ at 37°C in MEM supplemented with 10% fetal bovine serum. For experiments, cells were plated at a density of 1 × 10⁵ cells per well in 24-well plates or 2 × 10⁵ cells per 60-mm dish and were cultured for 24 hours in unsupplemented DMEM-F12 before exposure to test reagents in the same medium. 

Assay for IL-1β, IL-6, IL-8, IL-12, IP-10, and RANTES

Assays were performed as described previously. ¹⁷ Culture medium of cells incubated in 24-well plates was collected and centrifuged at 120g for 5 minutes, and the resultant supernatant was frozen at −80°C for subsequent assay of proinflammatory cytokines and chemokines. Cells were detached from the culture plate by exposure to trypsin-EDTA, and their number was determined with a particle counter. The morphology and number of cells were not affected by incubation with zymosan in the absence or presence of various inhibitors for 24 hours (data not shown). The concentration of each cytokine or chemokine in culture supernatants was measured with enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN). 

Immunoblot Analysis

Cells cultured in 60-mm plates were lysed in 300 μL solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na₃VO₄, and 1% protease inhibitor cocktail. Cell lysates were centrifuged at 15,000g for 10 minutes at 4°C, and the resultant supernatants were subjected to SDS-PAGE on a 10% gel. The separated proteins were transferred electrophoretically to a nitrocellulose membrane that was incubated at 4°C for 16 hours with blocking solution (20 mM Tris-HCl [pH 7.4], 5% dried skim milk, 0.1% Tween 20) and then for 16 hours with primary antibodies at a 1:1000 dilution in blocking solution. The membrane was washed with a solution containing 20 mM Tris-HCl (pH 7.4) and 0.1% Tween 20, incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies at a 1:1000 dilution in the same solution, washed again, and incubated with ECL detection reagents before exposure to film. 

Immunofluorescence Staining

Cells cultured in 24-well plates were fixed with 3.7% formaldehyde in PBS, washed with PBS, and permeabilized with 100% methanol for 5 minutes at −20°C. They were then washed with PBS, incubated at room temperature for 1 hour with 1% bovine serum albumin (BSA) in PBS, incubated again for 1 hour with primary antibodies (or with normal rabbit or mouse IgG as a control) at a 1:100 dilution in PBS containing 1% BSA, and then washed with PBS before incubation at room temperature for 1 hour with Alexa Fluor 488–labeled secondary antibodies (1:1000 dilution) in PBS containing 1% BSA. Finally, they were washed again with PBS and examined with a laser confocal microscope (LSM5; Carl Zeiss, Jena, Germany). 

Statistical Analysis

Data are presented as mean ± SD. Statistical analysis was performed with the Dunnett's multiple comparison test or the Tukey-Kramer test. P < 0.05 was considered statistically significant. 

Results

We first examined the effects of zymosan on the release of proinflammatory cytokines and chemokines and on the expression of adhesion molecules by human corneal fibroblasts. ELISA revealed that zymosan induced the release of IL-6 and IL-8 from corneal fibroblasts in a concentration-dependent manner (Fig. 1). In contrast, zymosan had no effect on the release of IL-1β, IL-12, IP-10, or RANTES by these cells (Fig. 1). Immunoblot and immunofluorescence analyses also revealed that zymosan had no effect on the expression or subcellular distribution of ICAM-1 or VCAM-1 (Fig. 2). 

Figure 1.

 

Effects of zymosan on the release of proinflammatory cytokines and chemokines from corneal fibroblasts. Cells were incubated for 24 hours in the presence of the indicated concentrations of zymosan, after which the amounts of the indicated proteins in culture supernatants were determined with ELISA. Data are mean ± SD of triplicates from an experiment repeated three times with similar results. *P < 0.05 (Dunnett's test) compared with the corresponding value for cells incubated without zymosan.

Figure 2.

 

Effects of zymosan on VCAM-1 and ICAM-1 expression in corneal fibroblasts. (A) Cells were incubated for 24 hours in the presence of the indicated concentrations of zymosan, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to VCAM-1, ICAM-1, or β-actin (loading control). (B) Cells incubated for 24 hours in the absence (a, c) or presence (b, d) of zymosan (600 μg/mL) were subjected to immunofluorescence analysis with antibodies to VCAM-1 (a, b) or to ICAM-1 (c, d) and with Alexa Fluor 488–conjugated secondary antibodies (gray). Scale bar, 50 μm. All data are representative of three independent experiments.

We next examined whether zymosan activated intracellular signaling by the MAPK ERK, p38, or JNK in corneal fibroblasts. Immunoblot analysis showed that zymosan induced the phosphorylation of ERK, p38, and JNK in a time-dependent manner (Fig. 3), with the phosphorylation level of each MAPK increased as early as 10 minutes after exposure to zymosan. We also examined whether zymosan affected the phosphorylation of the endogenous NF-κB inhibitor IκB-α in corneal fibroblasts. Immunoblot analysis revealed that exposure of the cells to zymosan for 15 minutes resulted in a concentration-dependent increase in the phosphorylation of IκB-α (Fig. 4A). Immunofluorescence analysis also showed that incubation of corneal fibroblasts with zymosan for 15 minutes induced translocation of the p65 subunit of NF-κB from the cytosol to the nucleus (Fig. 4B). 

Figure 3.

 

Time-dependent effects of zymosan on MAPK phosphorylation in corneal fibroblasts. Cells were incubated in the absence (Control) or presence of zymosan (600 μg/mL) for the indicated times, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p-) or total forms of ERK, p38 MAPK, or JNK. Data are representative of three independent experiments.

Figure 4.

 

Effect of zymosan on the activation status of NF-κB in corneal fibroblasts. (A) Cells were incubated with various concentrations of zymosan for 15 minutes, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated IκB-α. (B) Cells were incubated for 15 minutes in the absence (a) or presence (b) of zymosan (600 μg/mL) and then subjected to immunofluorescence analysis with antibodies to the p65 subunit of NF-κB and with Alexa Fluor 488–conjugated secondary antibodies (gray). Scale bar, 50 μm. All data are representative of three independent experiments.

Finally, we examined the effects of inhibitors of MAPK and NF-κB signaling on the zymosan-induced release of IL-6 and IL-8 from corneal fibroblasts. Cells were incubated with PD98059, an inhibitor of ERK signaling, or with the p38 MAPK inhibitor SB203580, JNK inhibitor II, or IκB kinase 2 (IKK2) inhibitor IV for 1 hour before incubation for 24 hours in the additional presence of zymosan. Zymosan-induced release of IL-6 and IL-8 was inhibited by inhibitors of MAPK and NF-κB signaling, respectively (Fig. 5). Percentage inhibition was calculated as 100% of inhibition of zymosan-induced IL-6 and IL-8 release by the IKK2 inhibitor. Zymosan-induced release of IL-6 and IL-8 was inhibited by 50% and 55%, respectively, in the presence of PD98059, by 36% and 49% in the presence of SB203580, and by 58% and 92% in the presence of the JNK inhibitor II. 

Figure 5.

 

Effects of inhibitors of MAPK or NF-κB signaling on zymosan-induced release of IL-6 and IL-8 from corneal fibroblasts. Cells were incubated first for 1 hour in the absence or presence of PD98059, SB203580, or JNK inhibitor II (each at 10 μM) or of IKK2 inhibitor IV (1 μM) and then for 24 hours in the additional absence or presence of zymosan (600 μg/mL), after which the amounts of IL-6 and IL-8 in culture supernatants were determined. Data are mean ± SD of four independent experiments. *P < 0.01 (Tukey-Kramer test).

Discussion

We have shown that zymosan induced the release of IL-6 and IL-8 without affecting either the release of IL-1β, IL-12, IP-10, or RANTES or the expression of ICAM-1 or VCAM-1 in human corneal fibroblasts. Zymosan also induced activation of the MAPKs ERK, p38, and JNK and the phosphorylation of IκB-α and nuclear translocation of the p65 subunit of NF-κB in these cells. The release of IL-6 and IL-8 induced by zymosan was inhibited by the MAPK inhibitors PD98059, SB203580, and JNK inhibitor II and by IKK2 inhibitor IV. 

Polymorphonuclear leukocytes (PMNs) are recruited to the site of fungal infection in the cornea as an early defense mechanism. ^18,19 Infiltration of PMNs contributes to the inflammatory response and is regulated by cytokines and chemokines. We have now shown that exposure of corneal fibroblasts to zymosan triggered the release of IL-6 and IL-8, both of which contribute to the recruitment of PMNs to the cornea. ^20,21 Keratocytes are the resident cells of the corneal stroma and differentiate into corneal fibroblasts in response to stromal injury. ^7,22 Our results now suggest that corneal fibroblasts respond to fungal infection by producing cytokines and chemokines that regulate the local infiltration and activation of PMNs. Stimulation of cells that express TLRs with the corresponding ligands regulates various cell activities, including proliferation, differentiation, and phagocytosis. ^23–25 Whether zymosan exhibits such actions in corneal fibroblasts remains to be determined. 

Corneal ulcers can result from ocular infection with bacteria, viruses, fungi, or parasites. ^26–28 Exposure of corneal fibroblasts to microbial components such as lipopolysaccharide, a component of Gram-negative bacteria, or to poly(I:C), an analog of viral double-stranded RNA, upregulates the expression of cytokines (IL-6, IL-1β), chemokines (IL-8, macrophage inflammatory protein-1β, IP-10, RANTES), and adhesion molecules (ICAM-1, VCAM-1). ^10,11 We have now shown that zymosan, derived from the cell wall of yeast, induced the release of IL-6 and IL-8 without affecting either the release of IL-1β, IL-12, IP-10, or RANTES or the expression of ICAM-1 or VCAM-1 in corneal fibroblasts. Expression patterns of cytokines, chemokines, and adhesion molecules in corneal fibroblasts exposed to zymosan differ from those in the same cells exposed to lipopolysaccharide or poly(I:C). The pathogenesis of corneal ulcer secondary to fungal infection may thus differ from that associated with bacterial or viral infection. 

TLRs serve as the pattern recognition receptors for microbial components and activate various signaling molecules, including NF-κB and the MAPKs ERK, p38, and JNK. ^29–31 We have previously shown that stimulation of TLR3 by poly(I:C) results in the activation of MAPK and NF-κB signaling pathways in corneal fibroblasts. ¹⁰ In the present study, zymosan induced the activation of ERK, p38 MAPK, and JNK signaling pathways and that of the NF-κB signaling pathway in corneal fibroblasts, and specific inhibitors of each of these pathways attenuated the stimulatory effects of zymosan on IL-6 and IL-8 release. The pattern of inhibition of zymosan-induced release of IL-6 and IL-8 by these various inhibitors differed from that observed for the effects of poly(I:C), suggesting that the relative contributions of the various signaling pathways differ correspondingly among pathogens. 

In summary, we have shown that zymosan induced the release of the proinflammatory cytokine IL-6 and the chemokine IL-8 in a manner dependent on the activation of MAPK and NF-κB signaling pathways in corneal fibroblasts. Our results therefore suggest that corneal fibroblasts play an important modulatory role in local immune and inflammatory responses associated with fungal infection in the corneal stroma. 

Footnotes

 Supported in part by Ministry of Education, Culture, Sports, Science, and Technology of Japan Grant 19791271.

Footnotes

 Disclosure: N. Nomi, None; K. Kimura, None; T. Nishida, None

The authors thank Yasumiko Akamatsu and the staff of Yamaguchi University Center for Gene Research for technical assistance.