Signaling pathways regulating intercellular adhesion molecule 1 expression by endothelin 1: comparison with interleukin-1beta in normal and scleroderma dermal fibroblasts

OBJECTIVE: Endothelin 1 (ET-1) has been implicated in the pathogenesis of fibrotic and inflammatory diseases, including scleroderma. In addition to modulating vascular tone and extracellular matrix turnover, ET-1 up-regulates cell surface adhesion molecules including intercellular adhesion molecule 1 (ICAM-1), which is key to cell-cell and cell-matrix adhesion and leukocyte infiltration. This study was undertaken to delineate the signal transduction pathways utilized by ET-1 and compare them with those adopted by proinflammatory cytokine interleukin-1beta (IL-1beta) in normal and scleroderma dermal fibroblasts. METHODS: Protein expression induced by ET-1 and IL-1beta on normal dermal fibroblasts, with or without signaling inhibitors, was detected by enzyme-linked immunosorbent assay, while messenger RNA (mRNA) levels were analyzed by LightCycler polymerase chain reaction. Expression of protein kinase Cdelta (PKCdelta) and PKCepsilon protein in normal dermal fibroblasts and scleroderma dermal fibroblasts was determined by Western blotting, and PKCepsilon involvement in ET-1 signaling was confirmed through transfection of an ICAM-1 promoter construct into murine PKCepsilon-/- fibroblasts. NF-kappaB activation was confirmed via electrophoretic mobility supershift assay, and analysis of the ICAM-1 promoter region was achieved via transfection of deletion constructs into human dermal fibroblasts. RESULTS: In normal dermal fibroblasts, ET-1 induced ICAM-1 mRNA and surface protein expression in a dose- and time-dependent manner via both receptor subtypes, ET(A) and ET(B); antagonism of both abolished the ET-1 response. MEK was involved in the signaling cascade, but phosphatidylinositol 3-kinase and p38 MAPK were not. Key to the cascade was activation of NF-kappaB, achieved by ligation of either receptor subtype. PKCepsilon activation led to downstream activation of MEK and, in part, NF-kappaB. IL-1beta signaling required NF-kappaB and MEK activation, along with activation of PKCdelta. ET-1 and IL-1beta each utilized the same ICAM-1 promoter region and the same NF-kappaB site at -157 bp. Responses to ET-1 and IL-1beta differed in scleroderma dermal fibroblasts, with ET-1 sensitivity decreasing and IL-1beta responses remaining intact. Expression of PKCepsilon and PKCdelta in scleroderma dermal fibroblasts was also altered. CONCLUSION: The findings of this study indicate that differences in sensitivity to ET-1 and IL-1beta in scleroderma dermal fibroblasts may be explained by altered expression of the PKC isoforms and cytokine receptors.     

Increased expression of intercellular adhesion molecule 1 on the fibroblasts of scleroderma patients

The surface expression of intercellular adhesion molecule 1 (ICAM-1) and class I and class II major histocompatibility complex molecules on cultured dermal fibroblasts from 7 scleroderma patients and 6 control donors was compared. Scleroderma fibroblast lines contained 41.0 +/- 3.0% (mean +/- SEM) cells with high levels of ICAM-1 expression (ICAM-1-high), whereas 26.9 +/- 1.5% of control fibroblasts were ICAM-1-high (P = 0.0003). There were no differences in the expression of class I and class II molecules. ICAM-1-high and ICAM-1-low fibroblasts produced equal amounts of total protein and procollagen. The increase in the number of ICAM-1-high fibroblasts in scleroderma patients may facilitate T cell activation and lymphokine production, and thus indirectly contribute to the fibrotic process.     

Secretion and binding of transforming growth factor β by scleroderma and normal dermal fibroblasts

The production and binding of transforming growth factor β (TGF/β) were compared in dermal fibroblast lines derived from scleroderma patients and normal control donors. The mean ± SEM 24-hour level of secretion of TGFβ by fibroblast lines derived from the involved skin of scleroderma patients was 30.8 ± 5.5 pmoles/10⁶ cells, and it was 29.7 ± 5.8 pmoles/10⁶ cells for fibroblast lines derived from the normal skin of the healthy donors. Thus, we found that the fibroblasts themselves produced TGFβ. TGFβ production was equivalent in fibroblast lines derived from clinically involved and uninvolved skin of scleroderma patients. The mean ± SEM number of TGFβ receptors per cell on scleroderma fibroblasts was 9,736 ± 1,375, with a mean ± SEM K_d of 25.2 ± 3.9 pM. This value was similar in control fibroblasts, at 12,431 ± 2,425 TGFβ receptors per cell, with a mean ± SEM K_d of 27.1 ± 5.2 pM.     

Expression and shedding of intercellular adhesion molecule 1 and lymphocyte function–associated antigen 3 by normal and scleroderma fibroblasts.

Objective. To examine intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 3 (LFA-3) in cultures of normal and systemic sclerosis (SSc) dermal fibroblasts. Methods. The surface and soluble forms of ICAM-1 and LFA-3 were measured by flow cytometry and capture enzyme-linked immunosorbent assay, respectively. Results. Surface ICAM-1 was significantly higher on SSc fibroblasts compared with normal controls. β-estradiol did not directly enhance ICAM-1 or LFA-3 expression in either normal or SSc cells, but significantly augmented the cytokine-induced increase in ICAM-1. Soluble ICAM-1 (sICAM-1) and sLFA-3 were detected in fibroblast cultures. While no difference was found in the level of sLFA-3, the shedding of sICAM-1 was significantly increased (P < 0.001) in cells from SSc patients. Conclusion. SSc fibroblasts express intrinsically elevated levels of surface ICAM-1 and release higher levels of sICAM-1 in vitro. Increased expression of ICAM-1 by interferon-γ and tumor necrosis factor α alone, and the further induction in combination with β-estradiol may underlie an aspect of fibroblast dysfunction in SSc and the female predisposition to the disease.     

Up-regulation by tumor necrosis factor α of intercellular adhesion molecule 1 expression and function in synovial fibroblasts and its inhibition by glucocorticoids

Objective. To examine the regulation of the intercellular adhesion molecule 1 (ICAM-1) gene in cultured human synovial fibroblasts in response to tumor necrosis factor α (TNFα), and investigate its modulation by the synthetic glucocorticoid, dexamethasone. Methods. Cell surface expression of ICAM-1 was determined by flow cytometry, enzyme immunoassay, and immunoprecipitation. ICAM-1 messenger RNA (mRNA) levels were monitored by Northern blot. ICAM-1 function was determined by measuring the adhesion of monocytes to synovial fibroblasts. Results. ICAM-1 expression on unstimulated cells was weak but was rapidly enhanced in both a time- and dose-dependent manner following exposure to TNFα. Treatment of the cells with TNFα also resulted in both a time- and dose-dependent increase in steady-state ICAM-1 mRNA levels, as determined by Northern blot. The increased expression of ICAM-1 was inhibited by cycloheximide and actinomycin D. Cultured synovial fibroblasts from patients with rheumatoid and nonrheumatoid arthropathies responded similarly to TNFα. Adhesion studies demonstrated that ICAM-1 is involved in the adherence of peripheral blood monocytes to TNFα-activated synovial fibroblasts. In addition, dexamethasone inhibited TNFα-induced surface expression of ICAM-1, accumulation of ICAM-1 mRNA, and adhesion of monocytes to TNFα-activated synovial fibroblasts. Conclusion. These combined results provide further evidence of an important role of ICAM-1 in inflammatory synovitis, as well as a potentially novel site of antiinflammatory action of glucocorticoids.     

Elevated expression of β1 and β2 integrins,intercellular adhesion molecule 1, and endothelial leukocyte adhesion molecule 1 in the skin of patients with systemic sclerosis of recent onset

Objective. To investigate the possible role of integrins and cell adhesion molecules in the pathogenesis of the mononuclear cell infiltration and fibrosis of skin that occurs in systemic sclerosis (SSc). Methods. The presence and topographic distribution of β₁, β₂, and β₄ integrins, as well as of endothelial leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1), was examined immunohistochemically in affected skin from 8 patients with rapidly progressive SSc of recent onset. The expression of the β₁ integrin gene was also investigated by in situ hybridization with a human sequence-specific complementary DNA. Results. The presence of β₁ integrin epitopes and the corresponding messenger RNA within inflammatory cells surrounding small vessels was demonstrated in SSc skin but not in normal skin. Lymphocytes positive for β₂ integrin were also found only in SSc skin, and they appeared in close proximity to small blood vessels and collagen bundles. Immunostaining for β₄ integrin epitopes revealed no differences between normal and SSc skin. ELAM-1 and ICAM-1 monoclonal antibodies, which identify epitopes indicative of endothelial cell activation, stained endothelial cells in SSc skin but not normal skin. Conclusion. These observations suggest that the complex interactions of β₁ and β₂ integrins, as well as ELAM-1 and ICAM-1, may be intimately involved in the pathogenesis of SSc, perhaps by mediating the homing and targeting of pathogenetic lymphocytes to the affected tissues.     

Rosiglitazone induces interleukin‐1 receptor antagonist in interleukin‐1β–stimulated rat synovial fibroblasts via a peroxisome proliferator–activated receptor β/δ–dependent mechanism

Objective

To study the potency of 2 peroxisome proliferator–activated receptor γ (PPARγ) agonists, 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐deoxy‐PGJ₂) and rosiglitazone, to modulate the expression of interleukin‐1 receptor antagonist (IL‐1Ra) in rat synovial fibroblasts.

Methods

Levels of messenger RNA for IL‐1Ra and PPAR isotypes (α, β/δ, γ) were assessed by real‐time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL‐1β. PPAR levels were assessed by Western blotting and secreted IL‐1Ra levels by immunoassay. The potency of PPARγ agonists and the PPARβ/δ agonist GW‐501516 on IL‐1Ra levels was tested in the range of 1–10 μM and at 100 pM, respectively. The contribution of PPARγ to the effects of rosiglitazone on IL‐1Ra secretion was examined either by its overexpression or by inhibition using wild‐type or dominant‐negative constructs and the antagonist GW‐9662 (10 μM), respectively. The dominant‐negative strategy was also performed to investigate the possible contribution of PPARβ/δ and NF‐κB activation.

Results

IL‐1β–induced IL‐1Ra production was increased by 10 μM rosiglitazone but was reduced dose‐dependently by 15‐deoxy‐PGJ₂. Both agonists lowered IL‐1β secretion, but rosiglitazone alone reduced the imbalance of IL‐1β/IL‐1Ra toward basal levels. Enhancement of IL‐1β–induced IL‐1Ra production by rosiglitazone was not affected by PPARγ overexpression or by its inhibition with dominant‐negative PPARγ or GW‐9662. Inhibition of NF‐κB was also ineffective against rosiglitazone but abolished the stimulating effect of IL‐1β on IL‐1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARγ decreased dramatically upon IL‐1β exposure, whereas PPARβ/δ remained stable. Dominant‐negative PPARβ/δ abolished the enhancement of IL‐1Ra by rosiglitazone, whereas GW‐501516 reproduced the effect of rosiglitazone on IL‐1Ra secretion.

Conclusion

Rosiglitazone stimulates IL‐1Ra production by a PPARβ/δ mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.

     

Correlation of polymorphic variation in the promoter region of the interleukin‐1β gene with secretion of interleukin‐1β protein

Objective

Significant variation in interleukin‐1β (IL‐1β) protein secretion between subjects has been observed when using a lipopolysaccharide (LPS)/ATP–mediated ex vivo blood stimulation assay. To explore the potential relationships between genetic polymorphisms in the IL1B cytokine gene and cellular responses to inflammatory stimuli such as LPS, we investigated the hypothesis that polymorphisms within the promoter and exon 5 of the IL1B gene contribute to the observed differences in IL‐1β protein secretion.

Methods

The IL1B gene polymorphisms C−511T, T−31C, and C3954T were tested for association with LPS‐induced secretion of IL‐1β protein as measured by an ex vivo blood stimulation assay. Samples from 2 independent study populations (n = 31 and n = 25) were available for use in the ex vivo assay after consent was obtained to analyze the DNA.

Results

A specific haplotype, composed of the T allele at −511 and the C allele at −31, was significantly associated with a 2–3‐fold increase in LPS‐induced IL‐1β protein secretion. This association was observed in both of the independent study populations (P = 0.0084 and P = 0.0017).

Conclusion

These data suggest that polymorphisms within the promoter region of the IL1B gene contribute to observed differences in LPS‐induced IL‐1β protein secretion.

     

Estrogen receptor–related receptor α regulation by interleukin‐1β in prostaglandin E2– and cAMP‐dependent pathways in osteoarthritic chondrocytes

Objective

We reported previously that the orphan nuclear receptor, estrogen receptor–related receptor α (ERRα), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERRα is also dysregulated in patients with osteoarthritis (OA).

Methods

ERRα messenger RNA (mRNA) and protein were quantified in normal and OA cartilage samples and in OA chondrocytes in vitro, with and without short‐term treatment with a variety of OA‐associated factors and signaling pathway agonists and inhibitors.

Results

ERRα expression was lower in OA than in normal articular cartilage. Interleukin‐1β (IL‐1β) markedly up‐regulated ERRα expression in OA chondrocytes in vitro, and agonist or inhibitor treatment indicated that the up‐regulation was dependent on cyclooxygenase 2 (COX‐2; NS398), prostaglandin E₂, cAMP (8‐bromo‐cAMP), and protein kinase A (PKA; KT5720). Treatment with the ERRα inverse agonist XCT790 decreased the expression of SOX9 and the up‐regulation of ERRα by IL‐1β, suggesting autoregulation of ERRα in the IL‐1β pathway. Matrix metalloproteinase 13 (MMP‐13) expression was also decreased by treatment with XCT790 plus IL‐1β versus IL‐1β alone, and the down‐regulation of MMP‐13 mRNA and protein observed with XCT790 alone suggests that the up‐regulation of MMP‐13 by IL‐1β is ERRα‐dependent.

Conclusion

We report the first evidence that ERRα expression is regulated by IL‐1β in COX‐2–, cAMP‐, and PKA‐dependent pathways in OA chondrocytes. We confirmed that SOX9 is an ERRα target gene in human, as in rodent, chondrocytes and identified MMP‐13 as a potential new target gene, which suggests that ERRα may both respond to the healing signal and contribute to extracellular degradation in OA cartilage.

     

Association of interleukin‐18 expression with enhanced levels of both interleukin‐1β and tumor necrosis factor α in knee synovial tissue of patients with rheumatoid arthritis

Objective

To examine the expression patterns of interkeukin‐18 (IL‐18) in synovial biopsy tissue of patients with rheumatoid arthritis (RA), and to determine whether expression of this primary cytokine is related to the expression of other cytokines and adhesion molecules and related to the degree of joint inflammation.

Methods

Biopsy specimens of knee synovial tissue either without synovitis (n = 6) or with moderate or severe synovitis (n = 11 and n = 12, respectively) were obtained from 29 patients with active RA. Paraffin‐embedded, snap‐frozen sections were used for immunohistochemical detection of IL‐18, tumor necrosis factor α (TNFα), IL‐1β, IL‐12, and IL‐17. Furthermore, adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E‐selectin, and cell markers CD3, CD14, and CD68 were stained.

Results

IL‐18 staining was detectable in 80% of the RA patients, in both the lining and sublining of the knee synovial tissue. IL‐18 expression in the synovial tissue was strongly correlated with the expression of IL‐1β (in the sublining r = 0.72, in the lining r = 0.71; both P < 0.0001) and TNFα (in the sublining r = 0.59, P < 0.0007, and in the lining r = 0.68, P < 0.0001). In addition, IL‐18 expression in the sublining correlated with macrophage infiltration (r = 0.64, P < 0.0007) and microscopic inflammation scores (r = 0.78, P < 0.0001), and with the acute‐phase reaction as measured by the erythrocyte sedimentation rate (r = 0.61, P < 0.0004). Interestingly, RA synovial tissue that coexpressed IL‐18 and IL‐12 demonstrated enhanced levels of the Th1‐associated cytokine IL‐17.

Conclusion

Our results show that expression of IL‐18 is associated with that of IL‐1β and TNFα and with local inflammation in the synovial tissue of patients with RA. In addition, synovial IL‐18 expression correlates with the acute‐phase response. These data indicate that IL‐18 is a primary proinflammatory cytokine in RA that drives the local production of IL‐1β and TNFα.

     

A role for geranylgeranylation in interleukin‐1β secretion

Objective

Mevalonate kinase deficiency (MKD) is an autosomal‐recessive disorder characterized by recurring episodes of inflammation. MK catalyzes the phosphorylation of mevalonic acid, which is an early step in isoprenoid biosynthesis. The goal of our study was to determine whether a temporary shortage of certain isoprenoid end products and/or the accumulation of mevalonic acid is the cause of interleukin‐1β (IL‐1β) secretion in MKD.

Methods

We studied the effect of the addition of intermediate metabolites and inhibitors of the isoprenoid biosynthesis pathway on IL‐1β secretion by peripheral blood mononuclear cells (PBMCs) of patients with MKD and healthy controls.

Results

Inhibition of enzymes involved in geranylgeranyl pyrophosphate (GGPP) synthesis or geranylgeranylation of proteins led to a marked increase of lipopolysaccharide‐stimulated IL‐1β secretion in PBMCs of control subjects. Furthermore, the increased IL‐1β secretion by PBMCs of patients with MKD was reversed by supplementation with GGPP as well as with mevalonic acid. IL‐1β secretion was increased only when control PBMCs were incubated with excessive amounts of mevalonic acid. Finally, a reduction in IL‐1β secretion by MKD PBMCs was also observed when sterol biosynthesis was inhibited, favoring nonsterol isoprenoid biosynthesis.

Conclusion

Our results indicate that a shortage of geranylgeranylated proteins, rather than an excess of mevalonate, is likely to cause increased IL‐1β secretion by PBMCs of patients with MKD.

     

Regulation of interleukin‐1β–induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin‐3‐gallate in rheumatoid arthritis synovial fibroblasts

Objective

To evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin‐1β (IL‐1β)–induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP‐1/CCL2), epithelial neutrophil–activating peptide 78 (ENA‐78/CXCL5), growth‐regulated oncogene α (GROα/CXCL1), and matrix metalloproteinase 2 (MMP‐2) activity in rheumatoid arthritis (RA) synovial fibroblasts.

Methods

Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP‐1, ENA‐78, and GROα produced in culture supernatants were measured by enzyme‐linked immunosorbent assay. MMP‐2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF‐κB.

Results

EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 μM or 20 μM significantly inhibited IL‐1β–induced ENA‐78, RANTES, and GROα, but not MCP‐1 production in a concentration‐dependent manner. EGCG at 50 μM caused a complete block of IL‐1β–induced production of RANTES, ENA‐78, and GROα, and reduced production of MCP‐1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL‐1β–induced, and chemokine‐mediated MMP‐2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCδ and inhibited the activation and nuclear translocation of NF‐κB in IL‐1β–treated RA synovial fibroblasts.

Conclusion

These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.

     

Proteomic analysis by two‐dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor α and interleukin‐1β

Objective

To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) and to ascertain differences in the protein expression patterns of these 2 cytokines.

Methods

Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL‐1β (5 ng/ml) or with TNFα (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2‐dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix‐assisted laser desorption ionization−time‐of‐flight/time‐of‐flight technology.

Results

We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL‐1β–stimulated chondrocytes, IL‐1β was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up‐regulated. Proteins involved in cellular metabolism and energy (23%) that were up‐regulated or induced by IL‐1β included nicotinamide phosphoribosyltransferase, long‐chain fatty acid–coenzyme A ligase 4, δ‐aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde‐3‐phosphate dehydrogenase. In TNFα‐stimulated chondrocytes, TNFα was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3–like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up‐regulated. Eighteen proteins were differently modulated by IL‐1β and TNFα. Of these, 45% were related to metabolism.

Conclusion

IL‐1β and TNFα induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.

     

β1 integrin–mediated signaling induces intercellular adhesion molecule 1 and Fas on rheumatoid synovial cells and Fas‐mediated apoptosis

Objective

Rheumatoid arthritis (RA) synovial cells interact with inflammatory cells, as well as extracellular matrices, through integrins. However, the relevance of β1 integrin to inflammatory processes in RA remains unclear. We examined the role of β1 integrin–mediated signaling in RA.

Methods

Expression of cell‐surface molecules was assessed by FACScan. Engagement of β1 integrins was performed by crosslinking using a specific monoclonal antibody (mAb) and ligand matrices such as fibronectin or collagen. To determine the involvement of tyrosine kinases in β1 integrin–mediated signaling, the cells were pretreated with various inhibitors of intracytoplasmic signaling or were transfected with a wild‐type focal adhesion kinase (FAK) or a dominant negative truncation of the FAK expression plasmid via cationic liposome‐mediated transfection. Apoptosis of synovial cells was detected by double staining with propidium iodide and annexin V.

Results

β1 integrin was highly expressed on RA synovial cells. Engagement of β1 integrins by crosslinking as well as by ligand matrices markedly up‐regulated expression of intercellular adhesion molecule 1 (ICAM‐1) and Fas. Up‐regulation of ICAM‐1 and Fas induced by β1 integrin was mediated by the tyrosine kinase signaling pathway, especially involving FAK. Fas‐mediated early apoptotic change in the cells was amplified by β1 crosslinking.

Conclusion

Our results suggest that interaction of β1 integrins with extracellular matrix augments expression of ICAM‐1 and Fas on RA synovial cells, as well as Fas‐mediated apoptosis of synovial cells. This might lead to the spontaneous growth arrest through the Fas/Fas ligand pathway observed in RA synovitis.