Introduction
Leptin, the 16-kDa product of the obese (
In mice, leptin production is increased by stimuli such as TNF, IL-1 and lipopolysaccharide [16,17,18]. In rats, infection with
Septic arthritis is a highly and rapidly destructive hematogenously spread joint disease, which usually produces sequelae in the affected person and can at worst cause death [22]. The most common pathogen in bacterial arthritis is
While leptin levels do not correlate with disease activity in patients with rheumatoid arthritis [25], the impact of
Materials and methods
Mice, bacteria and infection
Male 5- to 8-week-old C57BL/6 and NMRI mice (B&K Universal, Uppsala, Sweden) were used throughout the study. Mice were housed, fed and intravenously inoculated as described elsewhere [26]. For administration of recombinant mouse leptin (rleptin) (R&D Systems, Abingdon, Oxon, UK), the mice were given intraperitoneal injections of 0.2 ml twice daily (50 μg/mouse per day [27]), the first dose being given 2 hours before inoculation of bacteria. Control animals received phosphate-buffered saline (PBS). Intra-articular injections of rleptin were given in a knee joint in a volume of 30 μl containing 40 μg/ml. PBS was used as control injection.
Experimental protocol
Experiment 1 was performed using 13 male C57BL/6 mice. Nine mice were each intravenously inoculated with 1.6 × 107
In experiment 2, 20 male NMRI mice received 8 × 106
Experiment 3 differed from experiment 2 in that the mice were bled only on day 1, to check for the presence of staphylococci in the blood, and that the joints were checked for the presence of staphylococci on day 8.
In experiment 4, the setup was the same as in experiments 2 and 3. The mice were killed on day 2 and the paws were kept for histopathological examination.
In experiment 5, four male C57BL/6 mice were used. They were given an intra-articular injection of leptin into one knee and of PBS into the other knee, as described above. The mice were killed 3 days after inoculation and the knees were kept for histopathological examination.
Evaluation of arthritis and weight
All mice were labeled and monitored individually. Their limbs were inspected by two observers at regular intervals. Clinical examinations for arthritis and histopathological examinations were performed as described elsewhere [26]. To evaluate the intensity of synovitis and of cartilage and bone destruction, we used a histological index based on microscopic inspection, assigning a score of 0-3 points for each joint (elbow, wrist, carpal joints, fingers, knee, ankle, tarsal joints and toes) with regard to synovial hypertrophy and to destruction of cartilage and bone. Synovitis was scored as follows: 0 = no synovial hypertrophy; 1 = mild synovial hypertrophy, consisting of up to five cell layers; 2 = moderate hypertrophy, up to 10 cell layers; 3 = marked hypertrophy, >10 cell layers. Cartilage and bone destruction were each scored separately, as follows: 0 = no destruction; 1 = mild destruction; 2 = moderate destruction; 3 = severe destruction; the scores for cartilage and for bone were summed to yield an overall score for cartilage and bone. The total index was calculated by adding all the scores within each animal tested.
Determination of bacterial load
Bacterial samples were obtained as described elsewhere [26]. Colonies were tested for reactivity to catalase (using 18% hydrogen peroxide) and to coagulase (using rabbit plasma with EDTA [Becton Dickinson Microbiology Systems, Sparks, MD, USA]).
Cytokine reagents and analyses
rleptin (R&D Systems) was reconstituted in accordance with the manufacturer's instructions, then further diluted in PBS to achieve the desired concentrations.
IFN-γ and IL-6 were measured as described elsewhere [28]. Leptin, TNF, IL-1β and IL-10 were measured using Quantikine mouse immunoassays (R&D Systems). Assays were performed in accordance with the manufacturer's instructions.
Statistics
The Mann–Whitney
Results
Decreased leptin production during septic arthritis
The serum leptin concentration before inoculation of staphylococci ranged from 1.0 to 8.0 ng/ml (median 2.5 ng/ml). Previous studies measuring leptin in C57BL/6 mice have found similar levels [16,29]. The serum concentration of leptin decreased during
Box-and-whisker plots of (a) serum concentration of leptin; (b) weight change in C57BL/6 mice (
Box-and-whisker plots of (a) serum concentration of leptin; (b) weight change in C57BL/6 mice (
Decreased severity of septic arthritis after treatment with leptin
The severity of
Box-and-whisker plots showing the severity of
Box-and-whisker plots showing the severity of
The destruction of bone and cartilage was not significantly affected by the addition of rleptin. At day 8, 89% in each group showed such destruction, and the severity score was 10 versus 16 (median, leptin-treated vs controls, n.s.). In each group, 89% displayed synovitis; the median severity score was 8 versus 12 (treated vs controls, n.s.).
Decreased interleukin-6 production in leptin-treated mice with septic arthritis
The leptin-treated mice had significantly lower IL-6 levels than the controls at day 1, before the differences in joint pathology appeared (1.1 ± 0.3 ng/ml vs 1.4 ± 0.1 ng/ml;
Administration of leptin does not influence mortality, weight loss or staphylococcal load in tissues
Ten percent (2/20) of the leptin-treated mice died during the two 8-day experiments, versus 5% (1/20) of controls. No effect of exogenous leptin on weight loss was recorded, either early or late. At the end of the experiment, the mean weight loss was 18% in both groups. The load of
Discussion
This is the first report of a study of leptin production in response to bacterial arthritis. The leptin production was clearly downregulated during
Leptin has been shown to be involved in inflammation, both as its initiator and as a downregulator. The administration of rleptin induces production of proinflammatory cytokines, e.g. TNF [30], and directs a T-cell response into a Th1 type of response [27]. Leptin is proinflammatory in case of initiation and progression of experimental autoimmune encephalomyelitis [31]. In this study, we evaluated if leptin itself could initiate joint inflammation, but no signs of synovitis were seen when knees were exposed to microgram doses of the molecule (results not shown).
Besides being an inducer of proinflammatory mediators, leptin may be induced by, for example, TNF and IL-1β. Since anorexia is one of the symptoms of murine septic arthritis, and the production of several proinflammatory cytokines is increased during the course of disease, we hypothesized that anorexia could be induced by increased leptin production during
Previous reports on the correlation between leptin and arthritis are rare [25]. Our data show that the administration of rleptin leads to a decreased severity of septic arthritis, indicating an anti-inflammatory effect. Leptin has previously been reported to exert a protective role against endotoxin-induced lethality, possibly mediated through increasing levels of IL-10 and IL-1-receptor antagonist [32] or through downregulation of TNF production [33]. Leptin also mediates the protection of TNF-induced lethality [15]. The proinflammatory importance of TNF/lymphotoxin-α and TNF itself for the development of septic arthritis has been described elsewhere [34]. Potentially, exogenous leptin may exert its anti-inflammatory effect on septic arthritis through protection against TNF-mediated inflammation rather than downregulation of TNF production. Indeed, our experiments did not show any significant differences with respect to circulating TNF levels in leptin-treated versus control mice. The decreased production of IL-6 in response to exogenous leptin might be a possible pathway. Decreased production of IL-6 could be due to a downregulation of TNF, or, alternatively, could be a direct effect of leptin. There are some indications that leptin directly influences the production of IL-6. Macrophages from
We did not find any differences with regard to clearance of staphylococci from the blood stream, joints or kidneys, though results
Conclusion
The production of leptin is decreased during the course of
Abbreviations
IFN = interferon; IL = interleukin; n.s. = not significant; PBS = phosphate-buffered saline [solution]; rleptin = recombinant mouse leptin; SEM = standard error of the mean; TNF = tumor necrosis factor.