A 61-year-old Japanese woman was admitted to our hospital complaining of fever and polyarthralgia. She had had arthralgia of her proximal interphalangeal joints and ankles for the past 3 years. Two years before admission, she was diagnosed with gouty arthritis because her excided left elbow node showed tophus with sodium urate crystals. She had suffered from polyarthritis, and serum creatinine concentration was already elevated due to renal dysfunction.
She was found to have symmetric polyarthritis of the elbows, knees, ankles, and metatarsophalangeal (MTP) joints of both feet. In addition, subcutaneous granulomatous nodules were detectable in her hands, ankles, and feet. Her serum laboratory data showed C-reactive protein (CRP), 29.8 mg/dL; ferritin, 2660 mg/L; creatinine, 2.46 mg/dL; blood urea nitrogen (BUN), 23.6 mg/dL; uric acid (UA), 8.7 mg/dL; aspartate aminotransferase (AST), 152 IU/L; and alanine aminotransferase (ALT), 245 IU/L. Her serum concentrations of cytokines measured by enzyme-linked immunosorbent assay (ELISA) were as follows: IL-1beta, under the detectable level; IL-6, 371 pg/mL; and tumor-necrosis factor (TNF)-alpha, 3.6 pg/mL (Table 1). Her synovial fluid IL-6 concentration was 62,800 pg/mL. An X-ray image of her feet showed subcutaneous crystals around the first MTP joints and the overhanging margin in her right first metatarsal bone (Figure 1(a), Before, arrow).
After admission, medication of prednisolone at 20 mg/day and colchicine at 0.5 mg/day improved her fever, joint swelling and joint tenderness. Colchicine had to be stopped because of hair loss after six weeks of medication. We reduced the dose of prednisolone gradually, and then her arthritis flared up when the dose of prednisolone was 5 mg/day. Treatment of tocilizumab (8 mg/kg, every 4 weeks) was started 4 weeks after admission with two intraarticular injections of 20 mg of methylprednisolone in the 8 weeks after tocilizumab treatment. As a result, her arthritis gradually improved, subcutaneous nodules were reduced in size as determined by an X-ray image (Figure 1(a), After) and the patient's laboratory data were also improved 24 weeks after starting the treatment of tocilizumab (Table 1) (Figure 1(b)). There was no fever or acute attack during the tocilizumab treatment (Figure 1(b)). Time course of medication and plotted body temperature, serum UA (mg/dL), serum CRP (mg/dL) and serum IL-6 (pg/mL) concentrations are indicated (Figure 1(b)).
The analyses of patient materials were approved by the Human Research Ethical Committees of Ehime University and Dogo Spa Hospital. We measured the concentrations of IL-1beta, IL-6, and TNF-alpha in the patient's serum before and after tocilizumab treatment (Table 1), and synovial fluid before tocilizumab treatment by ELISA.
Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-gradient centrifugation (GE Healthcare Bio-Sciences AB, Piscataway, NJ). Synthetic N-acetylmuramyl-l-alanyl-d-isoglutamine, muramyl dipeptide (MDP) and lipopolysaccharide (LPS) from Escherichia coli O55:B5, cell culture tested, purified by trichloroacetic acid extraction and gel filtration chromatography, were purchased from Sigma-Aldrich (St Louis, MO). The cells were cultured in 24-well flat-bottom plates (BD Biosciences, San Jose, CA) with a final cell density of 1 x 106/mL in a volume of 1 mL of RPMI1640, including 10% fetal bovine serum (FBS) with MDP, LPS or left untreated for 8 h at 37 C in a humidified atmosphere with 5% CO2. Concentrations of IL-1beta, IL-6, IL-8, and TNF-alpha in the culture supernatant were measured by ELISA using IL-1beta-, IL-6-, IL-8-, and TNF-alpha-specific ELISA sets according to the manufacturer's instructions (BD Biosciences). PBMCs isolated from the patient did not spontaneously secrete IL-1beta, IL-6, IL-8 or TNF-alpha without any stimuli, and secreted these cytokines due to MDP (10 ng/mL or 1 microg/mL), LPS (0.1 ng/mL or 10 ng/mL) or a combination of MDP (10 ng/mL or 1 microg/mL) + LPS (0.1 ng/mL) in a dose-dependent manner (Figure 1(c)). The cytokine secretion patterns of PBMCs isolated from the patient were almost the same as those from healthy volunteers (Figure 1(c)).
The biopsy specimen, previously diagnosed by pathologists, embedded in a paraffin block, was subjected to immunohistochemical analysis. A 3-microm-thick serial sections of tissue were cut, deparaffinized in xylene, and hydrated in ethanol, and endogenous peroxidase activity was blocked by soaking in absolute methanol containing 0.3% H2O2 for 30 min. Before immunostaining, antigen retrieval was carried out by incubating tissue sections in a microwave in 10 mmol/L Tris-HCl buffer (pH 8.0) containing 1 mmol/L ethylenediaminetetraacetic acid (EDTA). Sections were blocked with 1% normal goat serum in 50 mmol/L Tris-buffered saline (TBS) (pH 7.6) and incubated with primary antibodies specific for cryopyrin (mouse mAb, clone: Nalpy3-b Alexis, San Diego, CA), ASC (mouse mAb, clone: 23-4, MBL, Nagoya, Japan), caspase-1 (rabbit pAb, No. #2225, Cell Signaling, Danvers, MA), and IL-1beta (rabbit pAb, No. ab2105, abcam, Cambridge, MA) diluted 1:200, 1:100, 1:100 and 1:100, respectively, at 4 C overnight. After washing in TBS, slides were incubated with EnVision+ (DakoCytomation) for 60 min and 3,3-diaminobenzidine was used as the chromogen. These sections were counterstained with hematoxylin. In her excided tophus, expression signals of cryopyrin, ASC, caspase-1, and IL-1beta were all detected in the monocytic cells and multinuclear giant cells around the MSU crystal foreign body granuloma (Figure 1(d), arrowheads).