Delayed resolution of acute inflammation during zymosan-induced arthritis in leptin-deficient mice

Eight-week-old male, leptin deficient C57BL/6 ob/ob mice and their control +/+ or ob/+ (i.e. +/?) littermates, as well as OB-Rb leptin receptor deficient C57BL/KS db/db mice and their control db/+ littermates, were purchased from Elevage Janvier (Le Genest-St-Isle, France). Animals were housed under conventional conditions in the animal facility of the Geneva University School of Medicine. Water and standard laboratory chow were provided ad libitum. The experimental protocol received the approval from the Animal Ethics Committee of the Geneva University School of Medicine and of the Geneva Veterinarian Office.

Arthritis was induced by injection of 180 μg zymosan A via a small skin cut along the suprapatellar ligament directly into the knee joint cavity, as described previously [17]. Zymosan A (30 mg) from Saccharomyces cerevisiae (Sigma-Aldrich, Buchs, Switzerland) was suspended in 1 ml endotoxin-free saline (Laboratory Dr G Bichsel AG, Interlaken, Switzerland) by boiling and sonification. Mice were injected with 6 μl of this suspension into the right knee joint, under inhalation anaesthesia with 5% isofluran (Forene^®; Abbott AG, Baar, Switzerland). The left knee joint was simultaneously injected with an equal amount (6 μl) of saline and served as the control.

Ob/ob and +/? mice were killed at different time points after injection of zymosan A (i.e. on days 1 and 3 for evaluation of the acute phase of arthritis, and on days 14 or 21 for evaluation of the chronic phase). The knee joints were either processed for histology (days 3, 14 and 21) or used for RNA extraction (days 1 and 14). One experiment was performed using db/db and control db/+ mice to evaluate the effect of OB-Rb deficiency on ZIA. In this experiment, mice were killed on day 14 after injection of zymosan A and the knee joints were processed for histology. All animals were killed by exsanguination (cardiac puncture) followed by cervical dislocation, under intraperitoneal anaesthesia with 0.01 ml/g saline solution containing 12 mg/ml ketasol (Dr E Graub AG, Bern, Switzerland) and 0.16% rompun (Bayer, Provet AG, Lyssach, Switzerland).

Joint swelling was quantified at various time points after injection of zymosan A by measuring uptake of circulating ^99mTc-pertechnetate in the knee joint, as previously described [14, 17]. Animals were injected subcutaneously in the neck region with 10 μCi of ^99mTc-pertechnetate in 0.2 ml saline. After 30 min mice were sedated by inhalation anaesthesia with 5% isofluran, and accumulation of the isotope due to increased blood flow and oedema in the knee was determined in duplicate by external gamma counting. The ratio between ^99mTc-pertechnetate uptake in the inflamed and that in the contralateral knee joint was calculated. A ratio greater than 1.1 was taken to indicate joint swelling.

Knee joints were fixed in 10% formalin for a minimum of 24 hours and decalcified using 'd-calcifier' (Lerner Laboratories, Pittsburg, PA, USA), containing 14% HCl, over 6–7 hours. They were embedded in paraffin and serial sections of 4 μm were cut for histological analysis. Sections were stained with either haematoxylin–eosin or toluidine blue. Histological assessment was performed in a blinded manner, using an established scoring system for synovial hyperplasia (from 0 = no hyperplasia to 3 = most severe hyperplasia) and inflammatory cell infiltration in the synovium (from 0 = no inflammation to 3 = most severe inflammation). Cartilage damage was determined by toluidine blue staining (from 0 = no change and fully stained cartilage to 3 = total loss of toluidine blue staining and erosions in cartilage). These three scores were added together to obtain a total histological score, as previously described [18]. Only processes taking place inside the joint cavity were taken into account.

Blood samples (100 μl) were taken at baseline and different time points after injection of zymosan A from the tail vein and at the end of experiment by cardiac punction. Serum levels of IL-6 were measured using a commercial DuoSet ELISA Development System (R&D Systems, Abington, UK). Detection limit for this test is 39 pg/ml. Serum levels of serum amyloid A (SAA) were determined using a direct enzyme-linked immunosorbent assay, as previously described [19]. The detection limit for this test is 13 μg/ml. Serum corticosterone levels were determined by using a radioimmunoassay (Diagnostic Systems Laboratories, Webster, TX, USA), as previously described [20].

On days 1 and 14 after injection of zymosan A, knee joints from five ob/ob and five +/? mice were used for RNA isolation. Total RNA was prepared using the TRIzol reagent (Gibco – Life Technologies AG, Basel, Switzerland) according to the manufacturer's instructions. Expression levels of IL-12, IL-10, IL-1α, IL-1β, IL-1 receptor antagonist, IL-18, IL-6, interferon-γ, macrophage migration inhibitory factor (MIF), L32 ribosomal protein and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were analyzed by RiboQuant™ RNase protection assay, using the mCK-2b multiprobe template set from BD Biosciences (Heidelberg, Germany). Briefly, riboprobes were ³²P-labelled and hybridized overnight in solution with 10 μg total RNA. The hybridized RNA was digested with RNases A and T1, and the remaining RNase-protected probes were purified, resolved on denaturing polyacrylamide gels, and imaged by autoradiography according to the RiboQuant protocol. The protected bands representing cytokine mRNA expression were quantified by phosphor-imaging using a Cyclone Storage Phosphor System (PerkinElmer Life Sciences, Zaventem, Belgium) and normalized for GAPDH expression. Data represent mean ± SEM (n = 5) of values obtained for all detectable cytokines.

Arthritis was induced in ob/ob and control +/? C57BL/6 mice by injecting zymosan A into the right knee joint. To assess whether leptin deficiency had an effect on the development of ZIA, we quantified increased blood flow and oedema (which reflect the severity of acute arthritis) by measuring ^99mTc-pertechnetate uptake at various time points after zymosan A injection (Fig. 1a). The ratio between ^99mTc-pertechnetate uptake in the arthritic joint and that in the control knee increased within 6 hours after injection of zymosan A. They were maximal after 24 hours and declined thereafter. Joint swelling was similar at 6 and 24 hours after injection of zymosan A in ob/ob mice and in their +/? controls. However, on day 3 ^99mTc-pertechnetate uptake ratios remained elevated in ob/ob animals whereas they normalized in +/? controls. Resolution of the acute phase of ZIA thus appeared to be delayed in leptin deficient mice. Joint swelling was similar between groups at 7 days (Fig. 1a). In order to investigate whether the observed differences were mediated via OB-Rb, we investigated joint swelling in db/db mice and control db/+ C57BL/KS mice. As for ob/ob animals, acute arthritis was similar in db/db mice and in db/+ controls at 6 and 24 hours, but its resolution was delayed and swelling remained detectable on day 3 in db/db mice while subsiding in db/+ animals (Fig. 1b).

We then investigated the histological features of knee joints from ob/ob and +/? mice. In both groups overt signs of synovitis were observed in zymosan A injected joints at early (3 days) and late (14 and 21 days) time points after injection (Fig. 2a,2b). In the left control knees (i.e. saline injected) focal accumulation of some synovial cells was observed on day 3 in some of the mice, which was considered to be due to the mechanical injury during intra-articular injection, and no alterations were detected at later stages. On day 3 similar histological changes were observed in zymosan A injected knees of ob/ob and +/? mice. Both groups exhibited a moderate inflammatory cell infiltration associated with discrete synovial hyperplasia (Fig. 2a,2c). At this early time point cartilage was generally well preserved, as indicated by homogenous toluidine blue staining (data not shown). On day 3 histological scoring repeatedly revealed similar severity of articular lesions in ob/ob mice and controls (Fig. 2c). However, in later stages synovial hyperplasia, inflammatory infiltration and cartilage damage were generally greater in ob/ob mice than in controls, suggesting a delayed resolution of arthritis in leptin-deficient animals (Fig. 2b,2c). In fact, according to the total histological scores, arthritis always tended to be more severe in ob/ob than in +/? mice on days 14 and 21, although the differences did not achieve statistical significance (Fig. 2c). This was mostly due to large variation from animal to animal, which was particularly marked in the ob/ob group. It is important to note that, at later time points, some very severe cases of arthritis were observed exclusively in the groups of obese animals (Fig. 2b, right panel).

Similar histological features were observed on day 14 in db/db mice. Total histological scores tended to be somewhat higher in db/db than in db/+ animals, although again the differences were not statistically significant (total histological severity scores: 5.58 ± 0.80 for db/+ mice [n = 6] and 6.58 ± 1.11 for db/db mice [n = 6]). Leptin and OB-Rb deficiency thus appeared to similarly affect the course of ZIA.

The acute phase response was examined in ob/ob and control mice after injection of zymosan A by measuring circulating levels of IL-6 and of the acute phase protein SAA. In the sera of naïve mice from both groups, IL-6 was undetectable. Following intra-articular injection of zymosan A, serum IL-6 increased within 6 hours in all animals but IL-6 levels were significantly higher in ob/ob mice than in controls (Table 1). At 24 hours IL-6 levels were considerably reduced in both groups, and the differences were no longer significant. In db/db mice and their lean littermates a similar transient increase in IL-6 was observed, but differences in IL-6 levels between the two groups were not significant (data not shown).

Circulating levels of SAA increased in all animals during the first 3 days after intra-articular zymosan A injection (Table 1). Early after zymosan A injection the increase in circulating SAA was delayed in the ob/ob group as compared with +/? mice, but later, on days 1 and 3, SAA levels were significantly higher in ob/ob animals than in controls. Similar results were obtained in db/db and db/+ mice (data not shown).

Corticosterone secretion is known to be elevated in all forms of leptin deficiency and in leptin insensitivity [21, 22]. During ZIA corticosterone levels increased transiently in both +/? and ob/ob animals 6 hours after zymosan A injection; however, in the latter group they remained significantly higher than in +/? controls throughout the experiment (Table 1).

Expression of mRNAs encoding various cytokines was investigated in knee joints of ob/ob and +/? animals on days 1 and 14 after zymosan A injection. We observed increased expression of MIF, IL-1α, IL-1β, IL-1 receptor antagonist and IL-6 mRNA in zymosan A injected knees (Fig. 3). Furthermore, low levels of IL-18 were also detected on day 14 (data not shown). Expression levels of all of these cytokines were significantly lower or undetectable in the left, saline-injected knee joints in both groups (data not shown). IL-12, IL-10 and interferon-γ were undetectable in both zymosan A and saline injected joints. In contrast to the observed delayed resolution of arthritis and acute phase response in ob/ob animals, we could not detect any differences in the local expression of cytokine mRNA in knees injected with zymosan A between ob/ob animals and +/? controls.