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(Journal of Leukocyte Biology. 2003;73:525-529.)
© 2003 by Society for Leukocyte Biology

NF-{kappa}B-dependent lymphocyte hyperadhesiveness to synovial fibroblasts by hypoxia and reoxygenation: potential role in rheumatoid arthritis

Myung-Kwan Han*, Jong-Suk Kim{dagger}, Byung-Hyun Park{dagger}, Jung-Ryul Kim{ddagger},§, Byung-Yun Hwang{ddagger},§, Hak-Yong Lee,§, Eun-Kyung Song* and Wan-Hee Yoo§,¶

Departments of
* Microbiology,
{dagger} Biochemistry,
{ddagger} Orthopedic Sugery, and
Internal Medicine, and
§ Research Institute of Clinical Medicine, Chonbuk National University Medical School, Chonju, Korea

Correspondence: Wan-Hee Yoo, M.D., Ph.D., Division of Rheumatology, Department of Internal Medicine, Chonbuk National University Medical School and Research Institute of Clinical Medicine, #634-18, Keum-Am Dong, Duck-Jin Gu, Chonju, Chonbuk, 561-712, Korea. E-mail: ywhim{at}moak.chonbuk.ac.kr


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ABSTRACT
 
Hypoxia/reoxygenation has been incriminated as a major factor in the pathogenesis of ischemia/reperfusion injury in various ischemic diseases such as rheumatoid arthritis (RA). In this study, we have investigated the effect of hypoxia/reoxygenation on the expression of intercellular adhesion molecule-1 (ICAM-1) in synovial fibroblasts and adherence of lymphocytes to synovial fibroblasts. Hypoxia/reoxygenation strongly activated nuclear factor-{kappa}B (NF-{kappa}B) in synovial fibroblasts to the levels produced by phorbol 12-myristate 13-acetate and caused lymphocyte hyperadhesiveness to synovial fibroblasts as well as up-regulation of ICAM-1, both of which were completely blocked by a NF-{kappa}B antagonist (pyrrolidine dithiocarbamate). These results indicate that hypoxia/reoxygenation has a major role in sequestration of inflammatory cells to synovium mediated by the activation of NF-{kappa}B. Our data suggest that hypoxia/reoxygenation could be an important target for the development of new, therapeutic strategies in RA.

Key Words: adhesion • synovial fibroblasts


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INTRODUCTION
 
Many studies have previously suggested that a rheumatoid synovial microenvironment is relatively ischemic and hypoxic [1 2 3 4 5 ]. The synovium of rheumatoid arthritis (RA) patients has some characteristic biochemical environments favorable for hypoxia-induced oxidative stress in the synovial joint because of increased pressure in the synovial cavity, reduced capillary density, vascular changes, and increased metabolic rate of synovial tissue [6 ]. This is supported by findings that mitochondrial reactive oxygen species (ROS) generation was enhanced, or levels of thioredoxin, an indicator of oxidative stress, were elevated in plasma of RA patients compared with healthy subjects or patients with nonrheumatic diseases [7 , 8 ]. Among the important stimuli in the production and release of cytokines in several cell types are hypoxia and/or reoxygenation [9 , 10 ]. It has been well known that the cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin-1 (IL-1) are important in mediating inflammation in RA [11 ]. These results strongly suggest that the hypoxic condition of synovial tissues might be involved in the pathogenesis of RA.

Fibroblasts are ubiquitous cells that have a major role as structural elements and as a resident sentinel cell with immunoregulatory function [12 ]. They are key sites of chemokine synthesis and have highly specialized roles in conditioning the cellular and cytokine environment in areas of inflammation by virtue of the complex array of factors they express. In rheumatoid synovium undergoing chronic and active inflammation, proliferating fibroblasts are considered to be responsible for the maintenance of chronic inflammation by supporting sequestration and activation of lymphocytes and monocytes. It is now generally accepted that inflammatory cells locally infiltrating the rheumatoid synovium play an important role in the pathogenesis of chronic inflammation of RA [13 , 14 ]. Specific cell–cell interactions, especially between synovial cells and invading mononuclear cells, play a pivotal role during the progression of disease. Various mediators, including inflammatory cytokines, chemokines, and adhesion molecules, have been implicated in the pathogenesis of RA [15 , 16 ]. Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), are thought to mediate these interactions.

Together, we have hypothesized that the leukocyte infiltrations may be mediated through effects of hypoxia/reoxygenation on synovial fibroblasts of RA synovium. In fact, hypoxic conditions can promote leukocyte-endothelial adherence via ROS [17 18 19 ]. However, there were no reports about the role of the effects of hypoxia/reoxygenation on fibroblasts in RA and fibroblasts in the recruitment of inflammatory cells.

Accordingly, we used an in vitro hypoxia/reoxygenation system to investigate the role of synovial fibroblasts on the infiltration of inflammatory cells to synovial tissues in RA. We also attempted to block the nuclear factor-{kappa}B (NF-{kappa}B) activation pathway using pyrrolidine derivative of dithiocarbamate (PDTC) and examined its effects on the induction of ICAM-1 and adhesive interaction between synovial fibroblasts and lymphocytes. Our result is the first evidence that adhesive capability of synovial fibroblasts for inflammatory cells is associated with the activation of NF-{kappa}B-dependent pathways caused by hypoxia/reoxygenation. Therefore, in this study, we provide new evidence showing that activation of synovial fibroblasts by synovial hypoxia/reoxygenation might be targets for therapeutic agents to reduce symptoms in RA.


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MATERIALS AND METHODS
 
Antibodies and reagents
Anti-CD3 monoclonal antibody (mAb), anti-CD68 mAb, anti-CD20 mAb, anti-vWF mAb, and anti-ICAM-1 mAb were purchased from R&D Systems (Minneapolis, MN). PDTC, phorbol 12-myristate 13-acetate (PMA), and 2,7-bis (carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) were obtained from Calbiochem (La Jolla, CA). M-Multilamellar vesicles RNase H–reverse transcriptase (RT), Trizol reagent, RPMI 1640, and fetal calf serum (FCS) were obtained from Gibco-BRL (Gaithersburg, MD). Hanks’ balanced salt solution (HBSS), formaldehyde, EDTA, and bovine serume albumin (BSA) were obtained from Sigma Chemical Co. (St. Louis, MO).

Isolation and cultures of human synovial fibroblasts
The synovial tissue specimens were isolated by the method of Matsuoka et al. [20 ] from RA patients. RA synovial fibroblasts were cultured in RPMI 1640 containing 10% FCS at a humid atmosphere (5% CO2, 95% air). The contamination of lymphocytes, macrophages, and endothelial cells into a synovial fibroblast cell batch was determined by flow cytometry (Beckton-Dickinson, Mountain View, CA) using anti-CD3 mAb, anti-CD68 mAb, anti-20 mAb, and anti-vWF, and these cells were less than l%. A specific staining for uridine diphosphoglucose dehydrogenase was determined to identify the synovial fibroblasts.

Hypoxia/reoxygenation treatments
Hypoxic conditions were obtained by placing the cells in a sealed hypoxic anaerobic chamber (Beckton-Dickinson). The oxygen levels in this chamber were maintained 1–2%, and the residual gas mixture contained 93–94% nitrogen gas and 5% carbon dioxide. Human synovial fibroblasts were cultured for 10 min under normoxia conditions for reoxygenation. The cell viability was determined by a trypan blue exclusion test [21 ].

Isolation of lymphocytes
Human lymphocytes were isolated from blood of normal, healthy donors. The blood was layered on 70% Percoll/55% Percoll and centrifuged at 500 g for 3 min. The pellet containing erythrocytes and lymphocytes was resuspended, and after hypotonic lysis of erythrocyte, lymphocytes were cultured in RPMI 1640 containing 10% FCS.

Flow cytometric analysis of ICAM-1 expression
Fluorescein-activated cell sorter analysis of synovial fibroblast was used to determine ICAM-1 expression. Synovial fibroblasts were washed two times with HBSS containing 1 mg/ml BSA, were suspended with staining buffer (0.01 M NaPO4, 0.15 M NaCl, pH 7.1, containing normal serum, 0.02% NaN3), and were incubated with fluorescein isothiocyanate (FITC)-conjugated human anti-ICAM-1 mAb or isotypic mAb (10 µg/ml). After washing with HBSS, synovial fibroblasts were fixed at 1% paraformaldehyde for flow cytometry.

RNA isolation and quantitative RT-polymerase chain reaction (PCR) of ICAM-1 expression
Total RNA was isolated from cells by using Trizol reagent following the manufacturer’s instructions. The first-strand cDNA was generated from RNA (1 µg) by using the random primer in the RT system (Promega, Madison, WI). PCR was performed in a reaction mixture containing 2 µl of the above first-strand cDNA, 10x PCR buffer, 3 mM MgCl2, Taq polymerase 1 unit, and 20 pmol each primer. After an initial denaturation for 2 min at 94°C, the conditions of PCR were 94°C for 40 s, 59°C for 30 s, and 72°C for 40 s in 30 cycles, and the final extension was performed at 72°C for 5 min (Perkin-Elmer thermal cycler, Perkin-Elmer, Foster City, CA). The ICAM sequence of the upstream primer was 5'-CACGCCTCCCTGAACCTA-3' and of the downstream primer, 5'-AGTTTACCCCGACCCTGA-3'. PCR reactions were performed with relative RT-PCR kit (Ambion, Austin, TX), each reaction containing the above ICAM primers, 18s rRNA primers, and 18s rRNA competimers. The competimers attenuated 18s rRNA amplification efficiently so that it could be multiplexed effectively with the much less abundant ICAM targets. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel. DNA was visualized by ethidium bromide staining at a concentration of 10 µg/ml. The intensities of the bands were evaluated using a UV–light box imaging system (Atto Co., Korea)

Western blot analysis of NF-{kappa}B p65
Nuclear extract was prepared as described previously [22 ]. Immunoblotting was performed with goat anti-human NF-{kappa}B p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was developed using alkaline phosphate-labeled anti-goat immunoglobulin G and a nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate kit (Sigma Chemical Co.).

Lymphocyte adherence assay
Human lymphocytes (2x106 cells/well) were at first stimulated with 5 ng/ml PMA at 37°C for 18 h and were then labeled with 50 µM BCECF-AM at 37°C for 30 min. Human synovial fibroblasts in six-well plates were cultured as the above-indicated hypoxic conditions. To measure lymphocyte adherence to synovial fibroblast, BCECF-AM-labeled human lymphocytes were added and incubated at 5% CO2 incubator. After incubation at 37°C for 10 min, each plate was washed three times with HBSS to remove unbound lymphocytes. Calculation of the fraction of adherent lymphocytes was done as follows: Fluorescence of each well was read by a fluorescent plate-reader (Perkin-Elmer, 485 nm exitation, 530 nm emission) to determine numbers of lymphocytes bound to fibroblasts compared with that of standard lymphocytes, and then, adherence was expressed as the ratio of bound lymphocytes and total lymphocytes added.

Statistical analysis
All experimental data are mean ± SD. Statistical analysis was performed using ANOVA test and Student’s test, and P values <0.05 were considered significant.


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RESULTS AND DISCUSSION
 
Hypoxia/reoxygenation induces activation of NF-{kappa}B in fibroblasts
PDTC, a potent inhibitor of NF-{kappa}B, prevents lung ischemia/reperfusion injury after transplantation, indicating that activation of NF-{kappa}B is associated with poor pulmonary graft function in transplant reperfusion injury [23 ]. The inhibition of NF-{kappa}B activity also blocks synovial joint inflammations in a rodent arthritis model [24 ]. Recent papers show that NF-{kappa}B activity of synovial fibroblasts is primarily involved in RA pathologies, including inflammation, synovial hyperplasia, and bone destruction [25 26 27 ]. Blockade of cytokine (TNF-{alpha} or IL-1ß)-induced activation of NF-{kappa}B gives rise to rapid and sustained decreases in symptoms and signs of disease [28 , 29 ]. Therefore, it has been suggested that NF-{kappa}B is a pivotal regulator in the pathogenesis of RA. However, there is no clear evidence concerned with NF-{kappa}B activation by hypoxia/reoxygenation in synovial fibroblasts. We designed an in vitro hypoxia/reoxygenation system to examine whether hypoxia induces activation of NF-{kappa}B in synovial fibroblasts. By Western blot analysis, a p65 subunit of NF-{kappa}B was detected in nuclear extracts of synovial fibroblasts (Fig. 1 ). A low amount of the p65 subunit was observed in untreated cells (Fig. 1 , lane 1). However, if synovial fibroblasts were cultured at hypoxia/reoxygenation for 3 h without a NF-{kappa}B inhibitor (Fig. 1 , lane 2), activated NF-{kappa}B was detected at high levels comparable with those induced by PMA (Fig. 1 , lane 4). The hypoxia-induced translocation of p65 into nucleus was completely blocked by the treatment of PDTC (Fig. 1 , lane 3). The significance of our result is supported by other observations, where activation of NF-{kappa}B of endothelial cells is in response to hypoxia or oxidative stress [30 31 32 ]. Thus, hypoxia/reoxygenation-induced activation of NF-{kappa}B of synovial fibroblasts may contribute to the intensive inflammations of RA synovium. These findings provide new evidence that the intra-articular hypoxic condition for activation of NF-{kappa}B in fibroblasts might be important targets for therapy and prevention of RA. The cell viability of fibroblasts was not changed during exposure to hypoxic conditions (data not shown).



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  		Figure 1. Effects of hypoxia/reoxygenation on translocation of NF-{kappa}B into nucleus. Human synovial fibroblasts from patients with RA were exposed to hypoxia for 3 h with (lane 3) or without (lane 2) PDTC (200 µM) and were treated with PMA as a positive control (lane 4, 100 nM). The nuclear extract of the cells was prepared, and Western blot analysis was performed.

Hypoxia/reoxygenation up-regulates ICAM-1 expression on fibroblasts
Next, we examined expression of an adhesion molecule in synovial fibroblasts after exposure to hypoxia/reoxygenation. Activation of NF-{kappa}B is involved in the induction of gene expressions, which encode for inflammatory cytokines and adhesion molecules of endothelial cells [30 31 32 ]. Furthermore, a variety of cytokines including TNF-{alpha}, IL-1ß, and IL-8 induce expression of ICAM-1 in synovial fibroblasts of RA [33 , 34 ]. Thus, we determined whether hypoxia induces the expression of ICAM-1 in synovial fibroblasts. The expression was determined by two distinctive methods: flow cytometric analysis for the cell-surface ICAM-1 molecule (Fig. 2 ) and relative quantitative RT-PCR for ICAM-1 mRNA (Fig. 3 ). As shown in Figure 2 , synovial fibroblasts exposed to hypoxic conditions exhibited higher expression patterns of ICAM-1 molecules of cell surfaces than control cells under normoxic conditions. The effects of hypoxia/reoxygenation on ICAM-1 expression were time-dependent, showing a maximal response of 84 ± 7% at 24 h compared with control cells (57±2.8%). Hypoxia/reoxygenation-induced up-expression of ICAM-1 in synovial fibroblasts was confirmed by relative quantitative RT-PCR for ICAM-1 mRNA. As shown in Figure 3 , ICAM-1 mRNA of the hypoxic fibroblast was increased twice compared with control cell when the cells were exposed to hypoxia for 6 h. The expressions of ICAM-1 mRNA started to increase at 6 h after hypoxia, and the increase was maintained for 24 h, whereas ICAM-1 protein started to express at 12 h after hypoxia, and the expression was maintained for 48 h. These responses were blocked by the NF-{kappa}B inhibitor, PDTC, indicating that up-regulation of ICAM-1 by hypoxia/reoxygenation is NF-{kappa}B-dependent.



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  		Figure 2. Surface expression of ICAM-1 on hypoxia/reoxygenation-induced human synovial fibroblasts. Synovial fibroblasts were exposed to hypoxia for 12, 24, or 48 h and were then reoxygenated. The cells were stained with FITC-conjugated human anti-ICAM-1 mAb (solid) or isotypic mAb (open), and then the cells were analyzed with a flow cytometer. Results were expressed as percentage of positive cells. *, P< 0.05 compared with normoxia (control); **, P < 0.05 compared with hypoxia.



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  		Figure 3. Changes in ICAM-1 mRNA levels on hypoxia/reoxygenation-induced human synovial fibroblasts. Synovial fibroblasts were exposed to hypoxia for 12, 24, or 48 h and were then reoxygenated. Samples were analyzed by relative quantitative RT-PCR analysis as described in Materials and Methods. The ICAM-1 mRNA/18 s rRNA ratio was presented as the amount of the induction of ICAM-1 mRNA transcription, and the results shown above are representative of three separate experiments.

Hypoxia/reoxygenation induces hyperadhesion of lymphocytes to synovial fibroblasts
Our experiments demonstrate for the first time that hypoxia/reoxygenation induces the hyperadhesion of lymphocytes to synovial fibroblasts. Light microscopic analysis of lymphocytes stained with colloidin blue or fluorophotometric analysis of BCECF-AM-labeled lymphocytes bound to fibroblasts showed hyperadhesion of lymphocytes to synovial fibroblasts. As shown at Figure 4 , synovial fibroblasts cultured under hypoxic condition have higher affinity for human lymphocytes than those under normoxic condition. The effect of hypoxia/reoxygenation on lymphocyte adhesion was time-dependent and reached a maximum at 24 h (Fig. 5 ). The increase of lymphocyte adhesion was blocked by the treatment of PDTC or anti-ICAM-1 antibody, whereas the equivalent concentration of isotype-control mAb did not (data not shown). The response of fibroblasts for lymphocyte adherence was also NF-{kappa}B-dependent and correlated with the expression of ICAM-1, indicating that hypoxia/reoxygenation induces the hyperadhesion of lymphocytes to synovial fibroblasts through NF-{kappa}B-dependent expression of ICAM-1.



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  		Figure 4. Effects of hypoxia/reoxygenation on the adhesion of lymphocytes to synovial fibroblasts. Light microscopic findings represent adherences of lymphocytes to synovial fibroblasts. Synovial fibroblasts were exposed to normoxia (A) or hypoxia for 24 h and were then reoxygenated (B). PMA-stimulated lymphocyte-enriched mononuclear cells were cocultured with a synovial fibroblast monolayer in culture plates for 10 min. Culture plates were washed three times with phosphate-buffered saline (PBS) for removal of nonadherent cells and were fixed with 10% formaldehyde and then stained with colloidin blue (x200). Black arrows indicate synovial fibroblasts, and white arrows indicate lymphocytes.



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  		Figure 5. Effect of NF-{kappa}B activity on the adhesion of lymphocytes to synovial fibroblasts induced by hypoxia/reoxygenation. Synovial fibroblasts were exposed to hypoxia for 6, 12, 24, or 48 h with vehicle (open bars), PDTC (200 µM; shaded bars), or anti-ICAM-1 antibody (10 µg/ml; solid bars) and were then reoxygenated. PMA-stimulated and BCECF-AM-labeled lymphocyte-enriched mononuclear cells were cocultured with a synovial fibroblast monolayer in culture plates for 10 min. Culture plates were washed three times with PBS for removal of nonadherent cells. Results were expressed as percentages of bounded lymphocytes to synovial fibroblasts. Each value denotes the mean ± SD of three separate experiments performed in triplicate. *, P < 0.01 compared with normoxia (control); **, P < 0.01 compared with hypoxia without PDTC.

In conclusion, although the detailed mechanisms are not fully understood, our data suggest that hypoxia/reoxygenation may be a common, inflammatory reaction mechanism by which RA synovium shows hyperplasia of synovial fibroblasts and infiltration of the inflammatory cells, predominantly T lymphocytes in vivo. Therefore, these findings point at synovial fibroblasts as a likely target for the anti-inflammatory effects of a variety of therapeutic agents on RA.


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ACKNOWLEDGEMENTS
 
The work was supported by a grant (6) from Kye-Nam, Kim Jae-Jung Memorial Fund. M-K. H. and J-S. K. contributed equally to this work.

Received May 28, 2002; revised December 16, 2002; accepted December 18, 2002.


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