Experimental immunology
Tumor necrosis factor-α and matrix metalloproteinase-3 production in rheumatoid arthritis synovial tissue is inhibited by blocking gap junction communication

Introduction

The gap junctions are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (up to 1 kDa), such as calcium ions, inositol phosphatase and cyclic nucleotides, to pass from cells to cells. Each gap junction pore is formed by the juxtaposition of two hemichannels in adjacent cells. A gap junction hemichannel is composed of six gap junction protein subunits (connexin, Cx) that dock to a corresponding structure in neighboring cells [1].

More than a dozen connexins have been identified, including at least seven human isoforms. Cx43 is widely expressed in different tissues, including the brain, heart, kidney, smooth muscle, ovary, some epithelia and osteo­blasts [2]. It is the most abundant of the connexins. Immunologically, relevant peptides, normally between eight and ten amino acids in length, can be transferred through Cx43 gap junction [3]. Cross-presentation by intercellular peptide transfer through gap junction is also important for the function of the immune system and the infection system [4]. Gap-junctional intercellular communication also contributes to the regulation of osteoclastogenesis [1, 5-8]. The blocking of gap junction communication has been studied with, several inhibitors, such as heptanol [5], 182-glycyrrhetinic acid and deamide [9].

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease that is characterized by synovitis which leads to the destruction of articular cartilage and bone. Although the etiology of RA is not still fully understood, it is clear that both tumor necrosis factor- (TNF-) and matrix metalloproteinase 3 (MMP-3) play a pivotal role in inflammation and bone distraction.

In the current study, we investigated that the role of gap junctions in RA. We studied the effect of heptanol, an inhibitor of gap junctional communication, on the synovitis in RA. We showed that Cx43 was positive in RA synovial tissues. In addition heptanol decreased the production of both TNF- and MMP-3 from synovial tissues.

Material and methods

Tissue samples



Synovial tissues were obtained from patients with RA and OA at the time of TKA. Informed consent was obtained before the TKA. For the immunochemical studies, synovial tissues were mounted in OCT (Lab-Tek Products, Napervill, IL, USA) oriented on cork bases, immediately snap-frozen in isopentane/liquidN2 and stored at –80°C until use.



Immunochemistry



The sections were cut to a thickness of 6 m with a cryostat (Bright Instruments Huntington, UK) and air dried for overnight. The sections were immersed in cold acetone (4°C) for 5 min, followed by incubation with 0.03% hydrogen peroxide for 10 min to block endogenous peroxidase activities. The tissues were stained with Cx43 antibodies raised against Cx43 (antimouse Cx43 monoclonal antibody, DACO Co); the antibodies were used as a 1 : 100 dilution in phosphate-buffered saline (PBS) for 2 hours at 37°C. The tissues were fixed with 3% PFA and 2% sucrose in PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes on ice. The staining was carried out with rhodamineconjugated rabbit anti-mouse immunoglobulin secondary antibodies diluted to a strength of 1 : 100 in PBS for 30 minutes at 37°C.



Quantification of cell death



Cells death was quantified by trypan blue uptake.



Cell cultures



Synovial tissues were obtained at the time of TKA. A part of each tissue was dissected into small pieces (1 g) for organ culture and rinsed well with PBS (pH 7.4). Each piece of the synovial tissue was cultured in 1 ml of RPMI (GIBCO BRL, Gaithersburg, Maryland, USA) with various concentrations (0-3 mM) of a gap junction communication blocker (heptanol) for 48 hours in 5% CO2 in air at 37°C. Ethanol was used as a control. Culture supernatants were collected and kept frozen at –80°C until they were used for the measurement of the concentration of the MMP-3, TNF- and IL-1. The determination of MMP-3 used a one-step sandwich enzyme immunoassay system (Fuji Chemical Industries, Toyama, Japan) [10]. Two sets of monoclonal antibodies that recognize different epitopes on the human MMP-3 molecule were used in the assay system. The concentrations of TNF- were also measured by the sandwich enzyme assay (high sensitivity TNF- kit, Funakoshi Industries, Tokyo, Japan).



Statistical analysis



Correlation was estimated by Spearman’s rank correlation coefficient, and Wilcoxon test. All statistical analyses, including correlation coefficients and p values, were carried out using the Stat-view statistical package (Abacus Concepts, Berkeley, CA). Differences were considered significant if p was less than 0.05 by the Mann-Whitney U test.

Results

Immunochemistry



The Cx43 was positive in lining cells, sublining cells and along the lymphoid nodule area in synovial tissues from the RA patients (Fig. 1). Conversely, synovial tissues from the OA patients exhibited weak staining (Fig. 1).



Quantification of cell death



Cell death was quantified by trypan blue uptake. There was no trypan blue uptake by cells in either the heptanol treatment cultures or the ethanol treatment cultures (Fig. 2).



The concentrations of TNF-α, MMP-3 and IL-1β



The concentrations of TNF-, MMP-3 and IL-1 in supernatants decreased by adding heptanol dose-dependently (Fig. 3A-E), compared to the control. Heptanol, which is a gap-junction inhibitor, significantly reduced the amount of constitutive secretion of MMP-3 (p = 0.018) and TNF- (p = 0.04) (Fig. 3F, G). We performed the same study five times and obtained similar results each time.

Discussion

In this study, we demonstrated that the concentrations of TNF- and MMP-3 in supernatants in patients with RA decreased by adding a gap junction blocker (heptanol). In addition, Cx43 was positive in synovial tissues from the RA patients. Conversely, synovial tissues from the OA patients exhibited weak staining of Cx43. Thus, increased intercellular communication through gap junctions may contribute to the pathogenesis of synovitis in patients with RA.

We have reported that irsogladine (2,4-diamino-6-(2,5-dichlorophenly)-s-triazine maleate), a drug that reinforces gap-junctional intracellular communication and is used for the treatment of gastritis and peptic ulcers, reduces aphthous stomatitis/oral ulcers in patients with Behcet’s disease. The improvement of gap-junctional intercellular communication may contribute to the treatment of aphthous stomatitis in patients with BD [11]. Thus, controlling the gap junction communication may be a rational therapeutic target of some diseases.

Synovial cells are connected to each other by an extensive networks of gap junctions, and it may mediate some cellular processes [12, 13]. Kolomytkin et al. reported the existence of ICGP in synovial lining cells and in primary and passed cultures of human synovial cells. The formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of human synovial cells. Kolomytkin et al. also [14] reported that IL-1 produced a dose-dependent increase in MMP activity that was blocked by exposure to the gap junction inhibitors. The communication through gap junctions early in IL-1 signal transduction is critical to the process of cytokine-regulated secretion of MMPs by synovial cells in rabbit.

Marino et al. reported that increased ICJP may contribute to the progression of osteoarthritis [15]. The synovial lining cells from patients with osteoarthritis (OA) produced MMP constitutively and in response to stimulation by IL-1. ICJP is critical to the cell’s ability to secrete MMP.

Osteoclasts play an important role in the bone destruction of RA. Ilvesaro et al. [5] reported that bone-resorbing osteoclasts contained gap-junctional connexin 43, and a heptanol, a gap-junctional inhibitor, dramatically inhibited bone resorption, which caused a decrease in the number and activity of osteoclasts. Heptanol also decreased the actin rings and in the total resorbed area. In addition, RANKL-induced osteoclast fusion is shown to be associated with the upregulation of Cx43 expression in mouse bone marrow cultures [9]. When a gap junction inhibitor is added to the bone marrow-derived cultures, the number of TRAP-positive multinucleated cells is reduced. Thus, the gap junctions are needed for the fusion of precursors to mature osteoclasts; gap-junctional Cx43 plays a functional role in osteoclasts.

Recently, it was reported that cross-presentation by intercellular peptide transfer through gap junctions is also important for the function of the immune system and the infection system. In 2005, Neijssen et al. [4] presented a mechanism of the antigen presentation system of two adjacent cells and is lost in most tumors: The mechanism is that gap-junction-mediated intercellular peptides are coupled for the presentation by bystander major histocompatibility complex (MHC) class I molecules and transferred to professional antigen presenting cells for cross-priming. They found that peptides up to ten amino acids long derived from viral or cellular proteins may be transferred from one cell to its neighbor through gap junctions. These peptides follow the classical MHC I pathway and are displayed on the surface for recognition by killer T cells.

In summary, increased ICGJ may contribute to the pathogenesis of synovitis and bone destruction in patients with RA, because a gap junction blocker inhibited the production of TNF- and MMP-3. Thus, controlling the ICGJ may be a rational therapeutic target of synovitis and bone distraction of RA.

No financial support.

References

 1. Stains JP, Civitelli R (2005): Cell-to-cell interactions in bone. Biochem Biophys Res Commun 328: 721-727.

 2. Lecanda F, Warlow PM, Sheikh S, et al. (2000): Connexin 43 deficiency causes delyed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol; 151: 931-944.

 3. Eugenín EA, Bran~es MC, Berman JW, Sáez JC (2003): TNF-αlpha plus IFN-gamma induce connexin43 expression and formation of gap junctions between human monocytes/ macrophages that enhance physiological responses. J Immunology 170: 1320-1328.

 4. Neijssen J, Herberts C, Driijfhout JW, et al. (2005): Cross-pressentation by intercellular peptide transfer through gap junctions. Nature 434: 83-88.

 5. Ilvesaro J, Väänänen K, Tuukkanen J (2000): Bone-resorbing osteoclasts contain gap-junctional connexin-43. J Bone Miner Res 15: 919-926.

 6. Ilvesaro J, Tuukkanen J (2003): Gap-junctional regulation of osteoclast function. Crit Rev Eukaryot Gene Expr 13: 133-146.

 7. Vander Molen MA, Rubin CT, McLeod KJ, et al. (1996): Agap junctional intercellular communication contributes to hormonal responsivenessin osteoclastic networks. J Biol Chem 24: 12165-12171.

 8. Matemba SF, Lie A, Ransjö M (2006): Regulation of osteoclastogenesis by gap junction communication. J Cell Biochem 99: 528-537.

 9. Ransjö M, Sahli J, Lie A (2003): Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors. Biochem Biophys Res Commun 303: 1179-1185.

10. Obata K, Iwata K, Okada Y, et al. (1992): A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 3 (stromelysin-1) using monoclonal antibodies. Clin Chem Acta 212: 59-72.

11. Nanke Y, Kamatani N, Okamoto T, et al. (2008): Irsogladine is effective for recurrent oral ulcers in patients with Behcet’s disease. Drugs RD 9: 1.

12. Kolomytkin O, Marino A, Sadasivan K, et al. (2000): Gap Junctions in human synovial cells and tissue. J Cell Physiol 184: 110-117.

13. Marino AA, Kolomytkin OV, Frilot C (2003): Extracellular currents alter gap junction intercellular communication in synovial fibroblasts. Bioelectromagnetics 24: 199-205.

14. Kolomytkin OV, Marino AA, Waddell DD, et al. (2002):

IL-1beta-induced production of metalloproteinases by synovial cells depends on gap junction conductance. Am J Physiol Cell Physiol 282: 1254-1260.

15. Marino A, Waddell D, Kolomytkin O, et al. (2004): Increased intercellular communication through gap junctions may contribute to progression of osteoarthritis. Clin Orthop Relat Res 422: 224-232.