Introduction
The pathogenesis of reactive arthritis triggered by a
Chlamydia trachomatis infection has remained difficult to define in the clinical setting [
1]. We have established a model of
C. trachomatis-induced arthritis (CtIA) in rats in which live
Chlamydia was grown up in carrier synovial fibroblasts and then injected directly into the knee joints of rats [
2]. We have defined the importance of the cytokine profile in the development of CtIA [
3] and have also addressed the role of complement in the resulting joint inflammatory process. To this end we attempted to modify joint inflammation by administrating cobra venom factor to decomplement the animals but no significant effect was seen. In our search for other complement-modifying agents, we have examined a synthetic compound, nafamostat mesylate (NM) for its effect on CtIA. NM, formerly known by the name FUT-175, is a serine protease inhibitor that has been used clinically in Asia, mainly Japan, as an anti-inflammatory agent [
4,
5]. It is generally well tolerated although rare occurrences of allergic reactions, hyperkalemia and hemolysis have been reported. It has been used for its complement-modifying effect and anticoagulant properties in the treatment of pancreatitis and disseminated intravascular coagulation. To date there are no data addressing the antimicrobial effects of NM, nor its effect on reactive arthritis.
Materials and methods
Rats
Eight-week old male Lewis rats were purchased from Harlan Laboratories (Indianapolis, IN, USA). They were maintained in microisolators under specific pathogen-free conditions in the animal care facility of the Toronto Western Hospital, University Health Network. All studies were conducted with the approval of the Animal Care Committee of the University Health Network.
Induction of arthritis
Arthritis was induced in the rats by the intra-articular injection of live
Chlamydia packaged in Lewis rat synovial fibroblasts as previously described [
2,
3]. Briefly,
C. trachomatis serotype L2 was inoculated into monolayers of the fibroblast lines in culture. After overnight incubation, the cells containing chlamydia were harvested and injected into the knee joint of each rat at 5 × 10
5 colony forming units (CFU)/joint. Rats were assessed on a daily basis and then sacrificed four days after injection. At necropsy, their knee joints were either processed for histopathology or for quantitation of intra-articular
Chlamydia.
Histopathology evaluation
For histology evaluations, joints were fixed in formalin. They were measured with a caliper and then decalcified as described previously [
2,
3]. They were sectioned and stained with H & E. Sections were evaluated and scored according to the system mentioned by us before.
Quantitation of Chlamydia in synovial tissues
We have adapted a clinical use ELISA kit, the IDEIA
PCE Chlamydia from OXOID-Dako (Basingstoke,, UK) for the quantitation of
Chlamydia in tissues [
6]. Synovial tissues from the joints were carefully dissected out and homogenized. They were suspended in the transportation medium provided by the ELISA kit in a fashion as to normalize samples to the same wet weight per volume. All samples were frozen and stored at -70°C until assayed.
Treatment of rats
NM was purchased from Sigma (St. Louis, MO, USA). The treatment scheme of Li
et al. [
7] for mice was followed. Daily intraperitoneal injections at a dose of 10 mg/kg body weight were made. NM was dissolved fresh in water each day just before use and then filtered sterilized before injection. The animals were briefly anesthetized with Isofurane (Zenoca Pharma, Missisauga, Ont., Canada), weighed and then the precise amount injected intraperitoneally. The injection schedule began on the day before
Chlamydia infection, and then on a daily basis until the rats were sacrificed. Sterile water was injected into the untreated control rats.
In vitro inhibition of Chlamydia proliferation
Lewis rat synovial fibroblast monolayers were set up on six well tissue culture plates. One hour prior to inoculation with Chlamydia, NM was added to the wells to give a range of concentrations from 0 to 200 μg/mL in duplicate wells. One hour after exposure to NM, the monolayers were infected at 105 CFU/well. The plates were spun down at 2 000 × G for 20 minutes to impact the bacteria onto the cells. Thereafter, plates were incubated at 37°C in a 5% CO2 incubator for 24 hours.
At the end of the incubation period, the monolayers were examined under phase contrast microscopy for Chlamydia inclusion bodies. Individual wells were harvested using cell scrapers and the contents frozen at -70°C until assayed.
The above IDEIA PCE Chlamydia kit from OXOID-Dako was also used for the quantitation of Chlamydia growth. Since the read out for this ELISA was in O.D., in order to combine different experimental runs, percent inhibition was calculated. The O.D. values from wells without NM was set at 0% inhibition.
The percentage difference from test wells with decreased O.D. values was calculated as percent inhibition.
Discussion
Among the spondyloarthropathies, the role of infection as a triggering factor is best established in reactive arthritis (ReA) in which a sterile synovitis follows an extraarticular infection. ReA occupies the conceptual ground somewhere between septic arthritis and the classic autoimmune joint diseases such as RA [
8]. Studies in ReA indicate that about 50% of such cases can be attributed to a specific pathogen by a combination of culture and serology, the predominant organisms being
Salmonella,
Yersinia and
Chlamydia. It has increasingly been recognized that post-viral arthritis also constitutes an important subset of ReA.
C. trachomatis has the strongest and most direct evidence for induction of ReA, with ReA occurring in 4% to 15% of those with chlamydial infections. Recent studies have demonstrated that current estimates for prevalence are likely underestimated as more than 60% of undifferentiated Spondyloarthritis represents post-
Chlamydia ReA [
9]. ReA is the paradigm of a rheumatic disease which reflects a dynamic interface between environmental triggers and genetic susceptibility. The fundamental distinctions between inflammatory joint disease as being autoimmune, autoinflammatory, or septic in nature are highlighted by the example of ReA [
10].
NM is a serine protease inhibitor used clinically as an anti-inflammatory. As a protease inhibitor, it is effective in neutralizing the enzymatic activities of activated complement components such as C1r, C1s, C3 and C5 convertases [
4,
7,
11‐
15]. It is also effective in inhibiting elements in the alternate pathway such as Factors B and D [
11,
13,
14]. Similarly it is able to counter the activation of key molecules in the coagulation cascade such as thrombin and plasmin [
14,
16,
17].
As an anti-inflammatory agent NM appears to prevent granulocyte and phagocytic cell accumulations into damaged tissues [
11,
12,
14]. Immunologically, NM is also known to suppress the production of cytokines IFNγ, TNF, IL17, IL4, IL5, IL6 and IL13 [
7,
16,
17]. NM has also been suggested as an anti-tumor agent since it interferes with NF-κB activation in cancer cell lines [
18] and in experimental rodent models [
19].
Because of its broad range of actions as a protease inhibitor, NM has been used experimentally in diverse settings. Ishizak
et al. [
12] used NM on a model of murine allergy while Hagiwara
et al. [
17] used it to prevent lung injury induced by LPS. Bonte
et al. [
11] and Schwertz
et al. [
14] used NM to reduce cardiac injury following experimental ischemia. Miyagi
et al. [
16] reported a good outcome in experimental liver transplant. Promising results were also reported in a rodent experimental autoimmune encephalomyelitis model [
7] as well as in a rabbit model of autoimmune Guillain-Barre syndrome [
13]. NM has also been used to treat adjuvant arthritis in rats to reduce the severity of the arthritis [
15]. Other than this early report in adjuvant arthritis, the effect of NM in other types of arthritis has not been evaluated.
Our data provide the first evidence that NM has important antimicrobial properties. This could have important implications for treatment options for clinical
Chlamydia-induced arthritis. At present the treatment options are few [
1]. There has recently been completed a randomized, placebo-controlled study of combination antibiotics for chronic
Chlamydia-induced arthritis [
20]. This has created renewed interest in examining agents that might have both anti-inflammatory and antimicrobial effects. Future studies will address time kinetics of different schedules of NM in the experimental model. Our findings in the experimental model indicate that NM would be an appealing candidate for such a therapeutic indication, and would be worthy of further investigation in this regard.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
BC carried out the in vitro and in vivo experiments. RI and BC jointly developed the first draft and the final revisions to the manuscript. Both authors have read and approved the final manuscript.