IL-1β Induces IL-6 Expression in Human Orbital Fibroblasts: Identification of an Anatomic-Site Specific Phenotypic Attribute Relevant to Thyroid-Associated Ophthalmopathy

Beiling Chen, Shanli Tsui and Terry J. Smith
J Immunol July 15, 2005, 175 (2) 1310-1319; DOI: https://doi.org/10.4049/jimmunol.175.2.1310

Abstract

Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we report that these fibroblasts, when treated with IL-1β, express high levels of IL-6, a cytokine involved in B cell activation and the regulation of adipocyte metabolism. The magnitude of this induction is considerably greater than that in dermal fibroblasts and involves up-regulation of IL-6 mRNA levels. IL-1β activates both p38 and ERK 1/2 components of the MAPK pathways. Disrupting these could attenuate the IL-6 induction. The up-regulation involves enhanced IL-6 gene promoter activity and retardation of IL-6 mRNA decay by IL-1β. Dexamethasone completely blocked the effect of IL-1β on IL-6 expression. Orbital fibroblasts also express higher levels of IL-6R than do skin-derived cells. When treated with rIL-6 (10 ng/ml), STAT3 is transiently phosphorylated. Thus, the exaggerated capacity of orbital fibroblasts to express high levels of both IL-6 and its receptor in an anatomic site-selective manner could represent an important basis for immune responses localized to the orbit in Graves’ disease.

The orbit is a site of intense inflammation and tissue remodeling associated with Graves’ disease (1, 2). That process, termed thyroid-associated ophthalmopathy (TAO)3, is connected in some as yet undefined manner with immune responses occurring in the thyroid gland. In TAO, connective tissue and extraocular muscles become infiltrated with T and B lymphocytes and mast cells (3, 4, 5). It is currently believed that these immunocompetent cells direct tissue-specific inflammatory responses and orbital fibroblast activation, culminating in hyaluronan accumulation, fibrosis, and eye motility dysfunction (6). The proximate causes for lymphocyte and tissue activation in TAO remain uncertain but are presumed to result from the complex interplay between highly specialized fibroblasts and bone marrow-derived cells recruited to the orbit. In fact, orbital fibroblasts display a number of surface receptor molecules and respond to several T cell-derived factors. This suggests potential cross-talk between lymphocytes and fibroblasts in situ (7, 8, 9, 10). Another characteristic of orbital fibroblasts is the diverse repertoire of cytokines they elaborate. When activated by IL-1β, leukoregulin, or CD154, they express high levels of IL-8, IL-16, RANTES, IL-1α, and IL-1β (9, 11, 12). Thus, they possess the potential to not only respond to cytokines but to generate these signals and, in so doing, influence the behavior of immunocompetent cells trafficked to the orbit.

A subset of orbital fibroblasts can differentiate into mature adipocytes when treated with cAMP-enhancing agents in concert with agonists of the peroxisome proliferator activator receptor γ (13, 14). This adipogenic potential has proximate relevance to the histopathology of TAO. In that condition, the orbital fat expands. At issue is whether orbital fibroblasts themselves generate high levels of factors, such as IL-6, which can influence terminal differentiation and metabolism of adipocytes.

Autoimmune thyroid disease involves the activation of multiple cytokine networks. Among these, IL-6 has generated considerable interest because it has been implicated in human autoimmune diseases and multiple myeloma (15, 16). IL-6 and soluble IL-6R, like several other important cytokines, have been found to be elevated in patients with Graves’ disease (17, 18, 19) and those with TAO (20, 21, 22, 23). IL-6 protein can be detected in s.c. fat in these patients (24). Hiromatsu et al. (23) found that extraocular eye muscle and orbital fat from patients with TAO express IL-6 mRNA and that orbital volumes correlated positively with transcript levels. Yet, the molecular basis for IL-6 expression in certain connective tissue depots is unclear.

IL-6 is a pleiotropic protein functionally and structurally related to several others, including oncostatin M, leukemia inhibitory factor, and IL-11 (25). The IL-6R belongs to the class I receptor family and functions as a complex, including the receptor and a ubiquitous 130-kDa signaling glycoprotein (gp130) (25, 26). The latter conveys high-affinity binding and is critical to the signal transduction occurring as a consequence of receptor occupancy using the Jak/STAT pathways (27).

In the present study, we report for the first time that orbital fibroblasts when activated by IL-1β express and release extremely high levels of IL-6. This results from a coordinated enhancement of IL-6 gene promoter activity and prolonged IL-6 mRNA stability. The aggregate of these actions culminates in substantially elevated steady-state IL-6 mRNA levels. Moreover, orbital fibroblasts express IL-6R and can respond to exogenous IL-6. High localized levels of IL-6 expression may result in enhanced adipogenesis and Ig production by orbital B cells in inflammatory diseases such as TAO.

Materials and Methods

Materials

Dexamethasone (1,4 pregnadien-9-fluoro-16α-methyl-11β,17α,21-triol-3,20-dione), 5,6-dichlorobenzimidazole (DRB) and cycloheximide were purchased from Sigma-Aldrich. IL-1β, IL-4, IL-5, IL-6, IL-12, IL-13, IFN γ, TNF-α, and TGF-β were purchased from BioSource International. CD154 (CD40L) was a kind gift of Dr. R. Phipps (University of Rochester, Rochester, NY) and was prepared by the method of Kehry and Castle (28). PD98059 and SB203580 were obtained from Calbiochem. A dominant negative (DN) mutant expression vector for p38 was generously provided by Dr. R. Davis (University of Massachusetts, Worcester, MA). The DN expression vector for ERK 1 was a gift from Dr. M. Cobb (University of Texas Southwestern, Dallas, TX). Abs against pan and phosphorylated p38 (detects p38α, p38β, and Mxi2) and ERK 1/2, IL-6R, and gp130 were obtained from Santa Cruz Biotechnology. Those against pan STAT3 and phosphorylated STAT3 were from Cell Signaling Technology. An ELISA for human IL-6 was purchased from Alpco Diagnostics.

Cell culture

Orbital fibroblast cultures were initiated from tissue explants obtained as surgical waste during decompression surgery for severe TAO or were from normal appearing orbital tissues in patients undergoing surgery for noninflammatory conditions. These activities have been approved by the Institutional Review Board of the Harbor-University of California at Los Angeles, Medical Center. Some of the fibroblast strains were kindly provided by Dr. R. Bahn (Mayo Clinic, Rochester, MN). Tissue fragments were generated by mechanical disruption of surgical explants, and fibroblasts were then allowed to outgrow the tissue and adhere to plastic culture plates (29). They were covered with Eagle’s medium to which 10% FBS, glutamine (435 μg/ml), and penicillin were added as described previously (29). Several dermal fibroblast strains were either generated from punch biopsies of normal appearing skin or were purchased from the American Type Culture Collection. Medium covering the cultures was changed every 3–4 days, and monolayers were maintained in a 5% CO2, humidified incubator at 37°C. Culture strains were used between the second and 12th passage from initiation. We have characterized these cultures extensively, established their purity, and found them to be essentially free from contamination by endothelial, epithelial, and smooth muscle cells (30).

RNA isolation and Northern hybridization

Fibroblasts were cultivated in 100-mm diameter plates to a confluent state. They were then treated with the test agents specified in the figure legends. Cellular RNA was extracted from monolayers by the method of Chomczynski and Sacchi (31) with an RNA isolating system purchased from Biotecx Laboratories. The nucleic acids were subjected to electrophoresis through denaturing, 1% agarose, formaldehyde gels. The RNA was transferred to Optitran membrane (Schleich and Schuell). Immobilized samples were hybridized with [32P]dCTP-labeled IL-6 and GAPDH cDNA probes generated by the random primer method. A 645-bp cDNA fragment encoding human IL-6 was cloned using the following primer sequences: (forward) 5′-CAGGAGCCCAGTATAACT-3′ and (reverse) 5′-GAATGCCCATGCTACATTT-3′. Hybridization was conducted in ExpressHyb solution (BD Clontech) for 1 h at 68°C. Membranes were washed under high stringency conditions, and then the RNA/DNA hybrids were visualized by autoradiography on X-Omat film (Kodak) following exposure at −80°C with intensifier screens. Bands resulting from radioactive hybrids were scanned by densitometry. Membranes were then stripped according to the instructions of the manufacturer and rehybridized with a GAPDH cDNA probe, and the band densities were normalized to this signal.

For IL-6 mRNA stability studies, cultures were treated with IL-1β for 6 h as a pretreatment. Cells were washed and incubated for an additional 7 h in growth medium. At time 0, DRB (20 μg/ml), an inhibitor of gene transcription, was added to the medium of all plates without or with IL-1β (10 ng/ml) for the intervals indicated in Fig. 4⇓B. Abundance of mRNAs was quantified by Northern blot hybridization and subjected to densitometry. IL-6 mRNA signals were normalized to their respective GAPDH levels.

Western blot analysis of fibroblast proteins

Cellular proteins were solubilized in ice-cold harvest buffer containing 0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), and 10 μM PMSF from rinsed fibroblast monolayers following the treatments indicated in the figure legends. Lysates were taken up in Laemmli buffer and subjected to SDS-PAGE, and the separated proteins were transferred to Immobilon membrane (Millipore). Primary mAbs were incubated with the membranes overnight at 4°C. Following washes, membranes were reincubated with secondary peroxidase-labeled Abs. The ECL (Amersham Biosciences) chemiluminescence detection system was used to generate signals.

Quantification of IL-6 protein released from IL-1β-activated fibroblasts

Confluent fibroblast monolayers in 24-well plates were shifted from growth conditions and treated with nothing or with IL-1β (10 ng/ml) without or with the other test compounds indicated. Aliquots of medium were collected and subjected to a specific ELISA for IL-6. Samples were assayed in triplicate using a standard curve, as suggested by the manufacturer.

Transient transfection of orbital fibroblasts with plasmids containing IL-6 promoter fragments and DN mutant p38 and ERK 1

With regard to promoter studies, an 1171-bp fragment of the human IL-6 promoter spanning −1168 to + 3 nt was cloned by PCR. Two primers used for the PCR include (forward) 5′-GGATCCTCCTGCAAGACAC-3′ and (reverse) 5′-GCCTCAGACATCTCCAGTCC-3′. The amplified fragment was sequenced and subcloned from the pCR2.1 TOPO vector (Invitrogen Life Technologies) into a promoterless pGL2-luciferase reporter vector (Promega). This was used to transiently transfect subconfluent fibroblast monolayers as described previously (32). Briefly, cultures were allowed to proliferate to 80–90% confluence in medium containing 10% FBS. The plasmid containing a fragment of the IL-6 gene promoter fused to a luciferase reporter gene was transiently transfected into fibroblasts using the LipofectAMINE PLUS system (Invitrogen Life Technologies). A total of 0.75 μg of pGL2 promoter DNA and 0.1 μg of pRL-TK vector DNA (Promega), serving as a transfection efficiency control, was mixed with PLUS reagent for 15 min before being combined with LipofectAMINE for another 15 min. The DNA-lipid mixture was added to culture medium of 80% confluent cells for 3 h at 37°C. DMEM containing 10% FBS replaced the transfection mixture overnight. Transfected cultures were then serum starved, and some received either IL-1β (10 ng/ml) for 2 h or nothing (control) as indicated in the figure legends. Cellular material was harvested in buffer provided by the manufacturer (Promega) and stored at −80°C until assayed. Luciferase activity was monitored with the Dual-Luciferase Reporter Assay System (Promega) in a FB12 tube luminometer (Zylux). Values were normalized to internal controls, and each experiment was performed at least three times.

To interrupt the expression of potentially relevant signaling pathway components, DN constructs of p38 and ERK 1 were ligated into pcDNA3.1 (Invitrogen Life Technologies). These were transiently transfected into cells as described above. Control cultures received a constant amount (2 μg) of empty vector DNA. The diminished levels of the kinases were documented by subjecting an aliquot of the lysate to Western blot analysis with relevant Abs.

Microscopy

Fibroblasts were cultured on glass coverslips (VWR Scientific) coated with collagen I (BD Biosciences). Cells were fixed with 4% paraformaldehyde and made permeable with Triton X-100 for 30 min. Following rinses with PBS, they were incubated with anti-IL-6R or anti-gp130 Abs (Santa Cruz Biotechnology) at a dilution of 1/200 in 10% goat serum containing PBS, alone or with their respective neutralizing peptides (Santa Cruz Biotechnology). Coverslips were rinsed and incubated with secondary Abs conjugated with Alexa fluorescent dye (Molecular Probes) at a dilution of 1/600. They were then incubated with 4′,6′-diamidino-2-phenylindole at a dilution of 1/300 (Molecular Probes). Images were acquired and analyzed using a Zeiss Axioskop40 microscope (Carl Zeiss Microimaging).

Results

IL-1β up-regulates the expression of IL-6 protein from orbital fibroblasts

The ability of orbital fibroblasts to express IL-6 was assessed by treating confluent cultures with IL-1β (10 ng/ml) for graded intervals, subjecting cell lysates to Western blot analysis and conditioned medium to a cytokine-specific ELISA. As the immunoblot in Fig. 1⇓A indicates, IL-6 levels in control (untreated) cell lysates are undetectable at time 0. The cytokine becomes detectable at 6 h and is maximal following 16 h of treatment with IL-1β. The protein migrates as a single 21-kDa band that begins to decline at 24 h when it is ∼50% lower. By 48 and 72 h, the duration of the study, the protein is barely detectable. When medium was subjected to an IL-6 ELISA, the cytokine became detectable after 6 h. It continued to accumulate for the duration of the study (48 h) (data not shown). The effects of IL-1β on IL-6 synthesis are concentration dependent. As Fig. 1⇓B indicates, the threshold of detectable induction occurs at an IL-1β concentration of 1 ng/ml. The effect was intermediate at a cytokine concentration of 5 ng/ml and was near-maximal at 10 ng/ml. Thus, the dose dependency is consistent with other established actions of IL-1β in orbital fibroblasts (32).

FIGURE 1.
FIGURE 1.

IL-6 production in orbital fibroblasts is induced in a time-dependent (A) and concentration-dependent (B) manner by IL-1β and released into the medium. The effects of IL-1β on IL-6 expression are mediated through a time-dependent up-regulation of steady-state levels of IL-6 mRNA (C). Confluent cultures, in this case from a patient with Graves’ disease, were treated with IL-1β (10 ng/ml) for graded intervals (A and C) or at varying concentrations of cytokine (B), as indicated. Cell layer material was harvested for Western (A) or RNA extracted for Northern blot analysis (C) of IL-6 protein and mRNA levels. Blots were then reprobed with anti-β-actin Ab in the Western blot or rehybridized with a GAPDH cDNA probe in the Northern blot. The columns represent the IL-6 signals normalized with their respective housekeeping signals. B, Media were collected and subjected to ELISA analysis as described in Materials and Methods. Data are shown as the mean ± SD of triplicate determinations.

Up-regulation of IL-6 by IL-1β in orbital fibroblasts involves the induction of its mRNA

The substantial time interval before a detectable increase in IL-6 protein provoked by IL-1β suggested that the action might be mediated at the pretranslational level. As the Northern blot analysis in Fig. 1⇑C indicates, this proved to be the case. Addition of IL-1β (10 ng/ml) to the culture medium resulted in the induction of the IL-6 transcript in a time-dependent manner. IL-6 mRNA was undetectable at time 0 but became apparent at 6 h. The single 1-kb band, consistent with the electrophoretic mobility reported previously in other cell types (33), was near-maximally induced at 16 h and then decreased in abundance at 24 h.

Induction of IL-6 in orbital fibroblasts is substantially greater than that in dermal cultures

In contrast to orbital fibroblasts, those derived from skin exhibited a substantially lower level of IL-6 induction by IL-1β (Fig. 2⇓). Several strains of orbital fibroblasts were examined for basal and cytokine-provoked IL-6 protein production, including those derived from patients with TAO and others from donors without orbital disease. As the data in Fig. 2⇓, A and C, indicate, all strains exhibited substantial increases following IL-1β treatment for 48 h. In contrast, IL-6 up-regulation in three dermal fibroblast strains treated with IL-1β was dramatically less robust (Fig. 2⇓, B and C). Of particular note is the blot shown in Fig. 2⇓C, where orbital and dermal fibroblast strains, derived from a single donor with severe TAO, were cultured and treated in parallel. IL-6 expression in the orbital strain is strongly induced while levels in the dermal strain remained undetectable. The cell type-specific differences in IL-6 expression were apparent at the level of IL-6 mRNA, as the Northern blot in Fig. 2⇓D illustrates.

FIGURE 2.
FIGURE 2.

Induction by IL-1β of IL-6 protein and mRNA is far more robust in orbital compared with dermal fibroblasts. Confluent orbital (A) or dermal (B) fibroblasts were treated with nothing or with IL-1β (10 ng/ml), and the cell material was solubilized and subjected to Western blot analysis with anti-IL-6 Abs (C). Orbital and dermal fibroblasts from a single patient with severe Graves’ disease were treated with nothing or the cytokine as described above and analyzed. Columns represent IL-6 signals normalized to those from their respective β-actin levels. D, Confluent fibroblasts were treated with IL-1β (10 ng/ml) for 16 h, and RNA was extracted and subjected to Northern blot hybridization with a probe generated with a fragment of the IL-6 cDNA. The membrane was stripped of radioactivity and reprobed with GAPDH.

IL-1β up-regulation of IL-6 is blocked by dexamethasone and represents a primary gene induction

Glucocorticoids exert powerful anti-inflammatory actions and influence the production and actions of cytokines, including IL-1β (34). Thus, we determined whether dexamethasone (10 nM), a powerful synthetic glucocorticoid used widely in clinical practice, could block the generation of IL-6 in these fibroblasts. As the data in Fig. 3⇓A indicate, the steroid, added to the medium of IL-1β-treated cultures, inhibited IL-6 release after 16 h. The concentration of dexamethasone used in these studies has been shown previously to result in a high fractional occupancy of the glucocorticoid receptor. Moreover, it has near-maximal inhibitory effects on cultured fibroblast metabolism (35). To determine whether this blockade of IL-6 induction was mediated at the pretranslational level, IL-1β-treated cultures were incubated without or with dexamethasone, and Northern blot analysis was conducted. The steroid completely attenuated the induction (Fig. 3⇓B). This may represent, at least in part, the basis for therapeutic benefit associated with glucocorticoid use in the inflammatory aspects of TAO.

FIGURE 3.
FIGURE 3.

A, Dexamethasone can block the induction of IL-6 protein expression by IL-1β in orbital fibroblasts. Confluent cultures were treated with IL-1β (10 ng/ml) or dexamethasone (10 nM) alone or in combination for 16 h. Media were collected and subjected to an IL-6 ELISA. B, Effects of dexamethasone and cycloheximide on the induction by IL-1β of IL-6 mRNA. Confluent cultures were treated with IL-1β without or with the steroid or cycloheximide (10 μg/ml) for 16 h. RNA was subjected to Northern blot hybridization with an IL-6 cDNA probe. C, Effects of IL-1β and several other proinflammatory cytokines on IL-6 protein expression. Cytokine concentrations were 10 ng/ml, except for IFN-γ (100 U/ml). Media samples were then subjected to an IL-6-specific ELISA. Data are presented as the mean ± SD of three individual determinations.

To determine whether the increased steady-state levels of IL-6 mRNA were a consequence of a primary IL-6 gene induction, cultures were treated with IL-1β alone or in combination with cycloheximide (10 μg/ml). The Northern blot in Fig. 3⇑B demonstrates that addition of cycloheximide to IL-1β enhances the cytokine’s induction of IL-6 mRNA, causing a “super induction” of the transcript. In contrast, the inhibitor failed to influence levels of IL-6 mRNA when added alone to the medium. It appears that the up-regulation of IL-6 mRNA by IL-1β represents a primary gene induction that does not require ongoing protein synthesis.

To address whether the induction of IL-6 is a generalized property of proinflammatory cytokines or represents a more specific action of IL-1β, a number of other cytokines were tested. In a survey including IL-1β, TNF-α, TGF-β, IL-4, IL-5, IL-12, IL-13 (all 10 ng/ml), and IFN γ (100 U/ml), only IL-1β-induced IL-6 protein following 16 h of treatment (Fig. 3⇑C). Importantly, both classical Th1 (IFN-γ) and Th2 (IL-4, IL-5, and IL-13) cytokines were included and all failed to up-regulate IL-6 expression in the orbital fibroblasts. In another study, CD154 could also induce IL-6 expression after 48 h of treatment (data not shown). Thus, it would appear that the induction of IL-6 in orbital fibroblasts exhibits considerable specificity with regard to the agents that provoke its up-regulation.

Induction of IL-6 mRNA by IL-1β is a consequence of enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability

A number of studies have demonstrated that the regulation of IL-6 expression by cytokines can be attributed to enhanced gene transcription (36, 37). Therefore, we cloned a 1171-bp fragment of the human IL-6 gene promoter spanning from −1168 to + 3 nt and fused it to a luciferase reporter gene. This construct was used to transiently transfect orbital fibroblasts from a patient with severe TAO and dermal fibroblasts. As data in Fig. 4⇓A demonstrate, the promoter exhibits some basal activity in untreated orbital fibroblasts. Addition of IL-1β (10 ng/ml) to the culture medium resulted in promoter activity that was substantially up-regulated at 2 h when it was 3-fold above basal levels. This effect was time dependent. In contrast, the IL-6 promoter was considerably less active in dermal fibroblast cultures under basal conditions, and the addition of IL-1β failed to influence its activity more than ∼50%. Thus, the substantial up-regulation of IL-6 mRNA in orbital fibroblasts appears to result, at least in part, from enhanced gene promoter activity.

FIGURE 4.
FIGURE 4.

A, Orbital fibroblasts exhibit substantial IL-6 gene promoter activity that is up-regulated by IL-1β in a time-dependent manner. Cultures of orbital and dermal fibroblasts were transiently transfected with an empty luciferase vector or one fused to an 1171-bp fragment spanning −1168 to + 3 nt of the human IL-6 gene promoter as described in Materials and Methods. Cultures were then treated with nothing or IL-1β (10 ng/ml) for 2 h. Data are presented and the mean ± SD of triplicate determinations. B, IL-1β enhances the stability of IL-6 mRNA in orbital fibroblasts. Cultures were treated as described in Materials and Methods. At time 0, fresh medium containing DRB (20 μg/ml) was added, and half of the plates received IL-1β or no cytokine (control) for the times indicated in the figure.

Stability of the IL-6 transcript could also play an important role as a determinant of steady-state mRNA levels. When cultures were preincubated in medium containing IL-1β (10 ng/ml) for 6 h and then treated with the inhibitor of gene transcription, DRB (20 μg/ml), without or with IL-1β, the cytokine could enhance IL-6 mRNA stability (Fig. 4⇑B). Transcript level decreased by ∼40% over a 7-h interval, the duration of the study, in cells not treated with the cytokine. In contrast, levels in cultures receiving IL-1β were sustained at levels similar to basal. Thus, it would appear that IL-1β exerts multiple effects on IL-6 expression in orbital fibroblasts. The increased IL-6 mRNA levels achieved in these cells are likely a consequence of both transcriptional and posttranscriptional actions of IL-1β.

Induction of IL-6 by IL-1β uses the p38 and ERK MAPK pathways

IL-1β induces a number of genes in orbital fibroblasts. Several of these inductions are mediated through the activation of p38 and ERK 1/2 MAPK pathways (32). Treatment with IL-1β (10 ng/ml) resulted in the very rapid phosphorylation of p38 (Fig. 5⇓A), which was strongly detectable after 10 and 30 min. Within 2 h, the signal was substantially reduced. Levels of phosphorylated ERK 1/2 were also strongly up-regulated within 10 min, and these too underwent rapid decline after 2 and 6 h (Fig. 5⇓B). Levels of p38 and ERK proteins, as assessed with pan anti-p38 and pan anti-ERK Abs, were unaffected by treatment with IL-1β over the duration of the study. We then tested the ability of specific inhibitors of both pathways to block the induction of IL-6 by IL-1β. SB203580 (10 μM) is a specific inhibitor of the p38 MAPK pathway (38), whereas PD98059 (10 μM) represents a highly specific inhibitor of MAPK kinase and therefore attenuates the activation of ERK 1/2 (39). As Fig. 6⇓A demonstrates, addition of either compound results in a substantial reduction (70–80%) in the up-regulation of IL-6 protein expression. The specificity of these compounds at the concentrations used in these studies has been well established (38, 39). The two signaling pathways appear relevant to the influence that IL-1β exerts on IL-6 mRNA stability because both compounds attenuate the cytokine’s effects on transcript survival (Fig. 6⇓C).

FIGURE 5.
FIGURE 5.

IL-1β treatment leads to a rapid phosphorylation of p38 and ERK components of the MAPK pathways in orbital fibroblasts. Confluent cultures of orbital fibroblasts, in this case from a patient with severe TAO, were treated with IL-1β (10 ng/ml) for the times indicated in the figure, and then protein from solubilized cell monolayers was subjected to Western blot analysis for pan- and phospho-p38 (A) or pan- and phopho-ERK1/2 (B). Blots were reprobed with an Ab against β-actin. Data are from a representative experiment.

FIGURE 6.
FIGURE 6.

Interrupting either p38 or ERK 1/2 pathway attenuates the induction of IL-6 by IL-1β in orbital fibroblasts. A, Confluent cultures were treated with IL-1β (10 ng/ml) alone or with either SB203580 (10 μM) or PD98059 (10 μM) for 16 h. Aliquots of medium were subjected to an IL-6-specific ELISA as described in Materials and Methods. Data are expressed as the mean ± SD of three replicates. B, Fibroblasts were transiently transfected with an empty vector or a plasmid containing either DN p38 or DN-ERK 1. The cultures were treated with nothing or IL-1β for 16 h, and then proteins were subjected to Western blot analysis for IL-6. C, Fibroblasts were pretreated with IL-1β (10 ng/ml) for 6 h, the monolayers were then treated with the cytokine alone or in combination with SB203580 or PD98059 for the time intervals indicated. RNA was harvested and subjected to Northern blot analysis for IL-6 and GAPDH mRNA levels. Data presented are from a representative experiment.

We then subjected orbital fibroblasts to transient transfection with DN mutant kinases. As Fig. 6⇑B indicates, introduction of either resulted in partial attenuation of the IL-6 induction. The fractional decline in IL-6 expression resulting from these transfections is entirely consistent with the 30–40% transfection efficiency we routinely achieve with these cells. The studies involving transfection of DN p38 and ERK were thus congruent with those using chemical inhibitors.

Orbital fibroblasts express IL-6R and gp130 and respond to rIL-6

We have begun to address the possibility that orbital fibroblasts might not only express high levels of IL-6 but could also respond to the cytokine. In initial studies, levels of IL-6R protein were assessed by Western blot analysis. As the results in Fig. 7⇓A indicate, IL-6R is readily detected in orbital fibroblasts obtained from three donors. The receptor appears as a single 80-kDa band. In contrast, IL-6R was barely detectable in three dermal fibroblast strains. Of importance is the common donor from whom the third orbital strain and first dermal strain were obtained. This patient had Graves’ disease and severe TAO. The signaling component associated with IL-6R, gp130, is shared with other receptors, including those for oncostatin M and leukemia inhibition factor (25). As Fig. 7⇓A also demonstrates, gp130 is expressed in high abundance in both orbital and dermal fibroblasts. Moreover, the level of this protein appears roughly equivalent in all strains tested. Thus, orbital fibroblasts specifically display considerably higher levels of IL-6R than do their dermal counterparts. The immunofluorescent pattern of IL-6R and gp130 protein expression was assessed using light microscopy. Although the IL-6R signal was strongly positive in orbital fibroblasts (Fig. 8⇓A), it was virtually absent in the cells derived from skin (Fig. 8⇓C). In contrast, the gp130 signal was easily detected in fibroblasts from both tissues (Fig. 8⇓, D and F). Neutralizing peptides for the proteins could completely quench the respective signals (Fig. 8⇓, B and E), documenting the specificity of these Abs. The images suggest membrane, cytoplasmic, and perinuclear expression patterns for these proteins.

FIGURE 7.
FIGURE 7.

A, Orbital fibroblasts express higher levels of IL-6R protein than do dermal cells. All strains expressed gp130 protein. Cell lysates from three orbital and three dermal fibroblast strains were subjected to Western blot analysis with Abs directed against IL-6R, gp130, or β-actin. B, IL-6 activates STAT3 in orbital fibroblasts. Confluent cultures were treated with rIL-6 (10 ng/ml) for the times indicated, and lysates were subjected to Western blot analysis with Abs against phospho-STAT3, pan-STAT3, or β-actin. The data presented are representative of three experiments performed.

FIGURE 8.
FIGURE 8.

Immunofluorescence signals for IL-6R and gp130 can be detected in orbital fibroblasts while only the latter protein is found in dermal cells. Orbital (A and B; D and E) and dermal (C and F) fibroblasts were cultured on collagen-coated glass coverslips, fixed, and stained with primary mAbs against IL-6R (A–C) or gp130 (D–F). B was incubated with neutralizing IL-6R peptide, whereas E received a gp130 peptide. Magnification, ×200.

We next determined whether abundant IL-6R protein would allow signaling through this protein and its associated complex in orbital fibroblasts. When cultures were treated with rIL-6 (10 ng/ml) for graded intervals, phosphorylated STAT3 could be detected (Fig. 7⇑B). As can be seen, the Western blot reveals a rapid and transient phosphorylation of the molecule, migrating as a 90-kDa protein. This was detected within 10 min of treatment and remained detectable for >30 min. After 2 h of treatment, the signal for phosphorylated STAT3 was undetectable. As the lower panel of the figure demonstrates, levels of STAT3 protein remain invariant with regard to cytokine treatment over the duration of the assay (6 h). Thus, it would appear that IL-6 might exert direct effects on orbital fibroblasts and thus could represent an autocrine (short-loop) factor in these cells.

Discussion

In the present study, we report for the first time that orbital fibroblasts, when treated with the proinflammatory cytokine IL-1β, express high levels of IL-6. This up-regulation appears to represent a primary gene induction because inhibiting ongoing protein synthesis fails to attenuate cytokine-dependent increases in IL-6 mRNA levels (Fig. 3⇑B). The induction is related in part to an increase in IL-6 gene promoter activity. IL-1β rapidly enhances promoter activity in orbital fibroblasts (3-fold at 2 h) but fails to do so in dermal fibroblasts (Fig. 4⇑A). A number of cell types have been shown previously to express high levels of IL-6 when treated with proinflammatory cytokines. Many of these effects are a consequence of up-regulated IL-6 gene transcription. For instance, the human intestine cell line, CaCo-2, when treated with IL-1β, exhibits an induction of IL-6 expression (40). This effect is enhanced by cAMP (40). Multiple response elements are critical to IL-6 promoter activation, including NF-κB, CREB, AP-1, and C/EBP. IL-1β can activate the IL-6 gene promoter in the rat osteoblastic cell line, UMR-106, an action that might be mediated through protein kinase C-β signaling (41).

In addition to the effects on gene promoter activity, IL-1β enhances the stability of the IL-6 transcript in orbital fibroblasts (Fig. 4⇑B). There was no appreciable mRNA decay observed in IL-1β-treated cultures over the duration of the stability studies (7 h). In contrast, mRNA levels in control cultures not receiving cytokine had declined by ∼40% over the same period. It would appear that the p38 MAPK pathway is especially important in the mediation of the IL-1β effect on IL-6 mRNA stability because SB203580 could inhibit the cytokine’s effects substantially (Fig. 6⇑C). Although of a smaller magnitude, the effect of PD98059, an inhibitor of MAPK kinase, suggests some involvement of the ERK pathway in IL-1β-dependent mRNA stability. Previously, p38 MAPK, but not ERK signaling, was implicated in the cAMP-dependent potentiation of IL-6 induction by IL-1β (40).

The high levels of IL-6 synthesis we find in fibroblasts from the orbit may underlie the particular susceptibility of these connective tissues to immune activation. Our current observations could directly influence at least two important aspects of orbital tissue dysfunction in the context of autoimmune disease. With regard to actions of IL-6 on connective tissue, a key pathological feature of TAO involves expansion of orbital fat content (6). This cytokine is an important determinant of fat metabolism, although its roles may vary from one adipose depot to another. IL-6 levels can become elevated in states of fat accumulation, such as obesity (42). Its expression can be enhanced substantially in differentiated 3T3F442A adipocytes by β-adrenergic activation, an effect also seen in cultures derived from s.c. breast tissue and abdominal wall (43, 44). The finding that IL-6 mRNA levels correlate positively with tissue volumes in TAO (23) suggests that a promotion by IL-6 of adipogenesis could underlie the expansion of orbital fat in TAO (1, 24).

With regard to the immunological consequences of localized, elevated IL-6 concentrations, the cytokine plays important roles in lymphocyte differentiation. In B cells, IL-6 drives the synthesis of Igs and is necessary for the normal development of plasma cells (36). Because the levels of IL-6 would be particularly high in the microenvironment surrounding activated orbital fibroblasts, it is plausible that B cells in the orbit might overproduce Igs, such as those associated with Graves’ disease. Concerning T lymphocytes, reports have appeared suggesting that IL-6 controls helper T cell differentiation. Specifically, it promotes Th2 differentiation while inhibiting that of Th1 cells (45). These actions appear to be mediated through separate pathways. The cytokine supports IL-4 synthesis through transcriptional activation of NFAT. In contrast, IL-6 interferes with IFN-γ signaling by up-regulating suppressor of cytokine signaling-1. The cytokine may also exert direct actions that promote T cell migration; these effects are mediated through MAPK, PI3K, and the Jak/STAT pathways (46). It also influences dendritic cell and macrophage differentiation in vitro (47, 48) and in vivo (49). It is noteworthy that STAT3 activation is required for the suppression by IL-6 of LPS-dependent cell maturation. IL-6 also induces through STAT3 the Ifi202 gene and p202 protein in mouse splenocytes (50). These findings are proximately relevant to those reported here because Ifi202 represents a candidate susceptibility gene for autoimmune diseases such as lupus erythematosus. Thus, the characteristic pattern of its induction coupled with potential actions of IL-6 in orbital fibroblasts suggests that this cytokine might locally bias inflammatory responses.

Besides exaggerated IL-6 production in IL-1β-activated orbital fibroblasts, we have also found that these cells express high levels of IL-6R (Fig. 7⇑). Coupled with the expression of gp130, our findings suggest that IL-6 acts in these cells (Fig. 7⇑). gp130 is a central determinant of signaling patterns associated with multiple IL-6-type cytokines (51). It directly interacts with principle components of the Jak/STAT pathway, including Jak 1, forming tight and enduring complexes (51, 52, 53). Multiple proline-rich regions of gp130, notably in the box1 region, are critical to Jak 1 binding. Our finding that STAT3 becomes phosphorylated transiently in orbital fibroblasts following IL-6 treatment is entirely congruent with previously established patterns of signaling in other cellular targets (54). STAT3 represents a key element mediating the cytokine’s biological impact (55). Its activation is mediated through multiple, nonequivalent gp130 motifs (56). Following binding to gp130, STAT3 becomes phosphorylated at Tyr705, leading to dimerization (57, 58). In addition to tyrosine, Ser727 can also be phosphorylated, albeit through the actions of an as yet unidentified kinase. It has been demonstrated recently that Rac1 mediates autocrine-dependent STAT3 activation through an indirect mechanism (54). In B cells, IL-6 induces a number of genes, and its actions are generally associated with antiapoptotic effects. It would appear that the activation of STAT3 is a requirement for the survival of INA-6 multiple myeloma cells and for the induction by IL-6 of several target genes in these cells (59). From our findings to date, the potential exists for IL-6 serving as an additional and potentially important autoregulatory molecule in orbital fibroblasts. Whether Rac1 or any of the other previously identified molecular mediators of IL-6 action play important roles in orbital fibroblasts will necessarily await further studies.

Abnormalities in IL-6 action and IL-6R activation have been associated previously with several human diseases, including multiple myeloma, rheumatoid arthritis, and inflammatory bowel disease (60, 61). Specifically relevant to rheumatoid arthritis, synovial fibroblasts have been shown previously to express IL-6, which is enhanced by IL-1β through a mechanism involving p38 MAPK-dependent stabilization of IL-6 mRNA (62, 63). Thus, our current findings are proximally relevant to other human autoimmune diseases. IL-6 has also been associated with the pathogenesis of Graves’ disease. For instance, cytokine levels are elevated in thyroid tissue from patients with active disease (64, 65). Wahrenberg et al. (24) have reported that adipose tissues from these individuals release several-fold higher levels of IL-6 than do similar tissues from control subjects. Circulating IL-6 levels have also been found to be elevated in Graves’ disease (17, 18, 19, 20, 21, 22). Moreover, treatment of thyrotoxicosis fails to normalize these levels, suggesting strongly that they are a consequence of the underlying autoimmune process.

The unexpected finding that IL-6 expression is considerably greater in cytokine-activated orbital fibroblasts compared with fibroblasts from skin suggests that a cytokine concentration gradient might underlie regional manifestations of this systemic disease. We have reported very recently that IgGs from patients with Graves’ disease (GD-IgG) can induce the expression of two important T cell chemoattractant molecules in fibroblasts (66). IL-16 and RANTES are substantially up-regulated by GD-IgG through a mechanism involving the insulin-like growth factor-1 receptor (IGF-1R) (7). Those earlier studies disclosed that fibroblasts from the orbit responded to GD-IgGs. Subsequently, synovial fibroblasts from patients with rheumatoid arthritis were also found to respond to IgGs directed against the IGF-1R (67). In contrast, fibroblasts from individuals without autoimmune diseases failed to respond to these Igs. From the current studies, it becomes clear that orbital fibroblasts, by virtue of their robust expression of IL-6, might activate B cells and support localized IgG production. It is the concentrated release of anti-IGF-1R Abs in TAO that may preferentially enhance T cell chemoattraction to the orbit. Given the direct actions now attributed to IL-6 as a T cell chemoattractant, it is possible that the site-restricted expression of these molecules by orbital fibroblasts might determine the profile of immunocompetent cells infiltrating the orbit in TAO. Thus, IL-6 and its receptor may represent an important pathogenic pathway, the interruption of which could result in effective therapeutic intervention.

Acknowledgments

We are grateful to Debbie Hanaya for expert assistance in preparing this manuscript.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported in part by National Institutes of Health Grants DK063121, EY008976, and EY011708 (to T.J.S.), RR00425 (to B.C.), and by the generous support provided by Dr. Steve and the late Dr. Milly Liu.

  • ↵2 Address correspondence and reprint requests to Dr. Terry J. Smith, Division of Molecular Medicine, Building C-2, Harbor-UCLA Medical Center, 1124 West Carson Street, Torrance, CA 90502. E-mail address: tjsmith{at}ucla.edu

  • ↵3 Abbreviations used in this paper: TAO, thyroid-associated ophthalmopathy; DRB, 5,6-dichlorobenzimidazole; DN, dominant negative; gp130, 130-kDa signaling glycoprotein; GD-IgG, IgG from patients with Graves’ disease; IGF-1R, insulin-like growth factor-1 receptor.

  • Received March 17, 2005.
  • Accepted May 6, 2005.

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