Combined antibiotic and free radical trap treatment is effective at combating Staphylococcus-aureus-induced septic arthritis
© Sakiniene and Collins, licensee BioMed Central Ltd 2002
Received: 30 August 2001
Accepted: 29 November 2001
Published: 15 January 2002
Although early antibiotic treatment of patients with septic arthritis eradicates bacteria, joint destruction commonly results from the unregulated host inflammatory responses to infection. The spin trap compound phenyl-N-tert-butyl nitrone (PBN) has been shown to have both anti-inflammatory and antioxidant effects. The aim of this study was to assess the effect of combined systemic administration of PBN and cloxacillin on the development of Staphylococcus aureus arthritis.
Three days after Naval Medical Research Institute (NMRI) mice were infected intravenously with S. aureus LS-1, daily treatment was started with cloxacillin alone, PBN alone, or cloxacillin and PBN. Arthritis, weight loss and general condition were evaluated for each mouse, and joints were analyzed histopathologically. Systemic administration of PBN in conjunction with cloxacillin ameliorated the course of experimental S. aureus arthritis, as evidenced by an increased cure rate. Thus, combinatorial antioxidant plus antibiotic anti-inflammatory therapies represent a potentially efficacious approach to the management of septic arthritis.
Keywordsarthritis murine spin-trap Staphylococcus aureus treatment
Staphylococcus aureus is the most common causative agent of septic arthritis [1–3], a severe, rapidly progressing, erosive disease with high morbidity and mortality. Inflammatory processes during septic arthritis erode articular cartilage, destroy bone and promote joint destruction leading to irreversible loss of joint function in 25–50% of patients [4, 5]. Early administration of antibiotics eradicates the bacteria, but does not stop joint destruction. We have previously shown that antibacterial therapy combined with systemic corticosteroid administration ameliorated S. aureus arthritis in mice . The compound α-phenyl-N-tert-butyl nitrone (PBN) was originally developed as a means of trapping and detecting free radical intermediates . PBN and related nitrones have a variety of anti-inflammatory and antioxidant properties. Therefore we considered whether PBN might be an effective therapeutic in septic arthritis. In this study, we evaluated the efficacy of a combined PBN and antibiotic (cloxacillin) treatment in reducing joint destruction during staphylococcal arthritis in a murine model of hematogenously spread S. aureus sepsis and septic arthritis [8, 9].
Materials and methods
The arthritis model
Female 5–8 week-old Naval Medical Research Institute (NMRI) mice were injected intravenously in the tail vein with an arthritogenic dose of S. aureus LS-1 . Limbs were inspected (by a blinded observer) at various time-points after bacterial inoculation. A system of clinical scoring (arthritic index; 0–3 scale) was used to assess the severity of arthritis in each limb . The total index was calculated by adding the individual limb scores for each animal tested. The cure rate was estimated by subtracting the arthritic index at day 10 (i.e. seven days after treatment commencement) from that at day three (i.e. just prior to treatment commencement).
The animals were sacrificed ten days after inoculation of bacteria and the joints were examined histologically  for synovial hypertrophy (synovial membrane thickness of more than two cell layers ), pannus formation and destruction of cartilage and subchondral bone. To evaluate the severity of synovitis and cartilage and/or bone destruction, a histological scoring system (histological index) was employed . The total histological index was calculated by totaling all the scores for each animal tested.
After sacrifice, kidneys were aseptically removed, homogenized manually at 4°C, diluted in PBS, and inoculated on horse blood agar in serial dilutions to estimate the bacterial load in each organ.
Cloxacillin (Astra, Södertälje, Sweden) was dissolved in sterile PBS, and mice were injected intraperitoneally with 0.1 ml of the solution, corresponding to 500 mg/kg body weight, every 12 hours, starting on day three after inoculation of bacteria and continuing until the animals were sacrificed.
Phenyl-N-tert-butyl nitrone (PBN) (Sigma, St Louis, MO, USA) was diluted in 0.1 ml of sterile PBS and injected intraperitoneally (40 mg/kg body weight) every 12 hours, starting on day zero or day three after inoculation of bacteria and continuing until the animals were sacrificed. PBN is not bactericidal for S. aureus.
The differences between parametric and non-parametric values in all treatment groups were tested for significance by use of the two-tailed Student's t-test and the Mann-Whitney U-test, respectively. Differences between groups in the incidence of arthritis and mortality were analyzed by the Fisher's exact test. Results are presented as the mean ± SEM. A P value of less than 0.05 was considered statistically significant.
The effect of PBN-alone treatment on sepsis and septic arthritis
The treatment with PBN alone started on day zero, and had no effect either on the prevalence or severity of arthritis. Thus, 21 days after the treatment was started, six out of nine mice in the control group, and four out of seven in the PBN-treated group exhibited symptoms of arthritis The mean arthritic index was 1.7 ± 0.1 in both groups. However, we observed a moderate increase in the mortality rate in the PBN-treated animals; 30% of the PBN-treated animals died, compared to 10% of control animals (n = 10 per group) by day 21 post-infection (data not shown).
The effect of combined PBN-cloxacillin treatment on the clinical course of sepsis and septic arthritis
In order to evaluate the effect of combined PBN-cloxacillin treatment, forty 5–6-week-old female NMRI mice were injected intravenously with arthritogenic doses of S. aureus LS-1. The mice were subdivided into four groups and treatment began three days after infection. The first (control) group was given no treatment, the second group was treated with cloxacillin alone, the third group was given PBN plus cloxacillin, and the fourth group received PBN only. The experiment was performed three times. The fourth group was excluded in the last experiment. Since all three experiments displayed similar outcomes, the clinical, bacteriologic and histologic results were pooled.
We used a murine model of hematogenously acquired S. aureus arthritis to evaluate the effects of PBN treatment, given alone or in combination with cloxacillin. Treatment efficacy was expressed as the cure rate (i.e. arthritic index changes following treatment of infected mice). The combined PBN-cloxacillin treatment significantly (P < 0.05) increased the cure rate, compared to cloxacillin-alone treatment. Histological investigation confirmed these results. Joint destruction develops early (within 48 hours) in S. aureus infection [12, 13]. Therefore, the occurrence, even in the treated animals, of erosions is not surprising, bearing in mind that the treatment was started three days after inoculation of bacteria. This was done in an attempt to reflect the clinical situation when patients present with staphylococcal infections. It is important to note that despite the higher prevalence and severity of arthritis at the start of the treatment modalities, the joints of mice receiving combined treatment exhibited less severe histological changes. Therefore, PBN appears to exert an ameliorating effect on arthritis progression and joint destruction when given in combination with an antibiotic.
Treatment with PBN alone had no beneficial effect on disease. Indeed, there was a tendency towards increased mortality in animals receiving PBN from the first day of infection. Phagocytes manufacture large amounts of reactive oxidants that participate in the destruction of invading microorganisms . PBN scavenges radicals and probably decreases phagocyte bactericidal capacity, thereby increasing the bacterial burden and contributing to sepsis-induced mortality. On the other hand, the ability of PBN to spin trap free radicals could have directly diminished joint tissue damage. The role of oxygen-derived free radicals in inflammation and tissue damage is well established . Interestingly, another nitrone, tempol, has been shown to have beneficial effects on collagen-induced arthritis in rats .
PBN suppresses proinflammatory cytokine production (e.g. interleukin-1 and tumor necrosis factor-α) by monocytes in vitro. The involvement of proinflammatory cytokines in the pathogenesis of S. aureus infection has been previously established [18–22] Thus, both direct and indirect effects of PBN on immune cells and on the production of cytokines might have contributed to the observed amelioration of arthritis.
This is the first infectious model of inflammatory disease in which PBN has been tested as a potential therapeutic agent. We have shown that systemic administration of PBN concomitant to antibiotic therapy improves the cure rate in S. aureus-induced arthritis. The pleiotropic effects of PBN in modulating macrophage and neutrophil activities within the joint may reduce destructive arthritis. Therefore, PBN might have therapeutic applications to nonseptic as well as septic inflammatory disease.
Naval Medical Research Institute
We thank Ing-Marie Nilsson and Liu Zai-Qing for excellent technical assistance. This work was supported by grants from the Gothenburg Medical Society, Nanna Svartz Foundation, the Swedish Medical Research Council, the King Gustaf V 80 Years Foundation, the Swedish Association against Rheumatism, Börje Dahlins Foundation, and Rune and Ulla Amlövs Foundation for Neurological, Rheumatological and Audiological Research.
- Studahl M, Bergman B, Kälebo P, Lindberg J: Septic arthritis of the knee: a 10-year review and long-term follow-up using a new scoring system. Scand J Infect Dis. 1994, 26: 85-93.PubMedView ArticleGoogle Scholar
- Kaandorp CJ, Dinant HJ, van de Laar MA, Moens HJ, Prins AP, Dijkmans BA: Incidence and sources of native and prosthetic joint infection: a community based prospective survey. Ann Rheum Dis. 1997, 56: 470-475.PubMedPubMed CentralView ArticleGoogle Scholar
- Lundy DW, Kehl DK: Increasing prevalence of Kingella kingae in osteoarticular infections in young children. J Pediatr Orthop. 1998, 18: 262-267. 10.1097/00004694-199803000-00025.PubMedGoogle Scholar
- Goldenberg DL: Septic arthritis. Lancet. 1998, 351: 197-202. 10.1016/S0140-6736(97)09522-6.PubMedView ArticleGoogle Scholar
- Goldenberg DL, Reed JI: Bacterial arthritis. N Engl J Med. 1985, 312: 764-771.PubMedView ArticleGoogle Scholar
- Sakiniene E, Bremell T, Tarkowski A: Addition of corticosteroids ameliorate the course of experimental Staphylococcus aureus arthritis. Arthritis Rheum. 1996, 39: 1596-1605.PubMedView ArticleGoogle Scholar
- Janzen EG, Blackburn BJ: Detection and identification of short-lived free radicals by en electron spin resonance trapping technique. J Am Chem Soc. 1968, 90: 5909-5910.View ArticleGoogle Scholar
- Bremell T, Lange S, Yacoub A, Rydén C, Tarkowski A: Experimental Staphylococcus aureus arthritis in mice. Infect Immun. 1991, 59: 2615-2623.PubMedPubMed CentralGoogle Scholar
- Tarkowski A, Collins LV, Gjertsson I, Hultgren OH, Jonsson I, Sakiniene E, Verdrengh M: Model systems: Modeling human staphylococcal arthritis and sepsis in the mouse. Trends Microbiol. 2001, 9: 321-326. 10.1016/S0966-842X(01)02078-9.PubMedView ArticleGoogle Scholar
- Abdelnour A, Arvidson S, Bremell T, Rydén C, Tarkowski A: The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect Immun. 1993, 61: 3879-3885.PubMedPubMed CentralGoogle Scholar
- Goldenberg DL, Cohen AS: Synovial membrane histopathology in the different diagnosis of rheumatoid arthritis, gout, pseudogout, systemic lupus erythematosus, infectious arthritis and degenerative joint disease. Medicine. 1978, 57: 239-252.PubMedView ArticleGoogle Scholar
- Sakiniene E, Bremell T, Tarkowski A: Complement depletion aggravates Staphylococcus aureus septicemia and septic arthritis. Clin Exp Immunol. 1999, 115: 95-102. 10.1046/j.1365-2249.1999.00771.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Bremell T, Abdelnour A, Tarkowski A: Histopathological and serological progression of experimental Staphylococcus aureus arthritis. Infect Immun. 1992, 60: 2976-2985.PubMedPubMed CentralGoogle Scholar
- Babior BM: Phagocytes and oxidative stress. Am J Med. 2000, 109: 33-44. 10.1016/S0002-9343(00)00481-2.PubMedView ArticleGoogle Scholar
- McCord JM: Oxygen-derived free radicals. New Horiz. 1993, 1: 70-76.PubMedGoogle Scholar
- Cuzzocrea S, McDonald MC, Mota-Filipe H, Mazzon E, Costantino G, Britti D, Mazzullo G, Caputi AP, Thiemermann C: Beneficial effects of tempol, a membrane-permeable radical scavenger, in a rodent model of collagen-induced arthritis. Arthritis Rheum. 2000, 43: 320-328. 10.1002/1529-0131(200002)43:2<320::AID-ANR11>3.0.CO;2-9.PubMedView ArticleGoogle Scholar
- Sang H, Wallis GL, Stewart CA, Kotake Y: Expression of cytokines and activation of transcription factors in lipopolysaccharide-administered rats and their inhibition by phenyl N-tert-butylnitrone (PBN). Arch Biochem Biophys. 1999, 363: 341-348. 10.1006/abbi.1998.1086.PubMedView ArticleGoogle Scholar
- Hultgren O, Eugster HP, Sedgwick JD, Korner H, Tarkowski A: TNF/lymphotoxin-alpha double-mutant mice resist septic arthritis but display increased mortality in response to Staphylococcus aureus. J Immunol. 1998, 161: 5937-5942.PubMedGoogle Scholar
- Zhao YX, Nilsson IM, Tarkowski A: The dual role of interferon-gamma in experimental Staphylococcus aureus septicaemia versus arthritis. Immunology. 1998, 93: 80-85. 10.1046/j.1365-2567.1998.00407.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Dinarello CA: Interleukin-1 and interleukin-1 antagonism. Blood. 1991, 77: 1627-1652.PubMedGoogle Scholar
- Brennan FM: Role of cytokines in experimental arthritis. Clin Exp Immunol. 1994, 97: 1-3.PubMedPubMed CentralView ArticleGoogle Scholar
- Hultgren O, Stensson M, Tarkowski A: Role of IL-12 in Staphylococcus aureus-triggered arthritis and sepsis. Arthritis Res. 2001, 3: 41-47. 10.1186/ar138.PubMedPubMed CentralView ArticleGoogle Scholar