Patients, treatment and clinical assessments
Nine patients (seven females, two males) with RA (seven positive for rheumatoid factor, and two negative) according to the American College of Rheumatology (ACR) criteria, and arthritis of the knee joint, were recruited for this study. All patients were assessed for disease activity at baseline and at three months, with the Disease Activity Score counted on 28 joints (DAS28). ACR response criteria were also used to record the result of the therapy. Functional capacity was recorded with the health assessment questionnaire. The median DAS28 at inclusion was 5.95 (range 4.83 to 7.91), despite treatment with methotrexate (7.5 to 20 mg per week). The dose of methotrexate was stable during the study and at least one month before the first arthroscopy. Four patients received prednisolone in an equally stable dose of not more than 10 mg per day. The median age was 57 years (range 25 to 69) and the median disease duration was 6 years (range 0.5 to 18). The median duration of the current episode of arthritis in the knee was 17.5 days (range 3 to 365, lacking data for one patient).
Three intravenous infusions of infliximab (3 mg/kg; Centocor B.V, Leiden, The Netherlands) were given in accordance with the recommended standard treatment protocol, with the first infusion given 1 to 21 days after the first arthroscopy and the subsequent infusions given after 2 and 6 weeks.
Informed consent was obtained from all patients, and the study was approved by the local ethics committee at the Karolinska University Hospital.
Synovial biopsies, immunohistochemistry and computer-assisted image analysis
An arthroscopy with multiple biopsies of the knee joint was performed in all patients 1 to 21 days before the first infusion. A second arthroscopy was performed at 8 to 10 weeks (median 9 weeks) after the first infusion. Total knee joint replacement surgery was considered as a substitute for the second arthroscopy in patient no. 7. The same physician (EaK) performed all arthroscopies. At the first arthroscopy, biopsies were predominantly taken from areas of the synovial tissue with signs of maximal macroscopic inflammation, from the cartilage-pannus junction and from synovial villi. The biopsy site was documented photographically and at the second arthroscopy the biopsies were taken from the same area as the first biopsies. The biopsies were snap-frozen within 2 minutes in liquid isopentane and stored at -70°C until sectioned. Serial cryostat sections (7 µm) were fixed for 20 min with 2% (v/v) formaldehyde and stored at -70°C.
Several biopsies were taken to secure sufficient material. For each of the nine patients, the biopsy with the best morphology was selected for subsequent immunohistochemical staining. The staining was always performed in pairs before and after treatment infliximab, allowing a pairwise comparison.
Two anti-(human IL-15) mAbs were used, one neutralizing (B-E29) and one non-neutralizing (B-T15; both from Diaclone SAS, Besancon, France). The staining procedure has been described previously [
13]. Biopsy specimens were also analysed for the presence of the cytokines IL-1α, IL-1ß, IL-2, IL-4, IFN-γ, TNF mAb1 and mAb11, and for the presence of the cell surface markers CD3, CD19, CD20, CD68 and CD163, as described previously [
14]. In addition, for TNF a new neutralizing IgG1γ mAb, 2C8, was used (Biodesign, Maine, USA). Negative controls with isotype-matched IgG were included for each marker.
Two evaluators, blinded to the origin and order of the sections, performed a semi-quantitative analysis of the expression of cytokines and cell surface markers, considering the number of positive cells in the stained sections. TNF mAb1 and mAb11 and IFN-γ were scored with a semi-quantitative four-point scale as follows: 0 = no positive cells, 1 = 1 to 10 positive cells, 2 = 11 to 100 positive cells, and 3 = more than 100 positive cells [
15]. Cell surface markers (CD3, CD68 and CD163) were scored with another semi-quantitative four-point scale: 0 = no infiltration, 1 = minimal infiltration, 2 = moderate infiltration, and 3 = marked infiltration [
16]. In the blinded manner described, stained synovial biopsy sections were evaluated by computerized image analysis, in which the area of positive staining was expressed as a percentage of the total tissue area, for IL-15 neutralizing and non-neutralizing antibodies, IL-1α, IL-1ß and TNF mAb 2C8. Analysis of an entire tissue section typically involved 25 to 210 (median 70) microscope fields, corresponding to an area of 0.7 to 9.1 mm
2 (median 2.9 mm
2) at a magnification of × 250.
To evaluate which cells were predominantly expressing IL-15, double staining was performed on samples from two of the patients (nos 3 and 5) for IL-15 neutralizing antibody and IL-15 non-neutralizing antibody, together with CD markers CD3, CD19/20, CD68 and CD163.