Intraarticular glucocorticoid treatment reduces inflammation in synovial cell infiltrations more efficiently than in synovial blood vessels.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 12, December 2005, pp 3880–3889 DOI 10.1002/art.21488 © 2005, American College of Rheumatology Intraarticular Glucocorticoid Treatment Reduces Inflammation in Synovial Cell Infiltrations More Efficiently Than in Synovial Blood Vessels Erik af Klint, Cecilia Grundtman, Marianne Engström, Anca Irinel Catrina, Dimitrios Makrygiannakis, Lars Klareskog, Ulf Andersson, and Ann-Kristin Ulfgren Objective. To investigate whether intraarticular (IA) glucocorticoid (GC) therapy diminishes synovial cell infiltration, vascularity, expression of proinflammatory cytokines, and adhesion molecule levels in patients with chronic arthritides. Methods. Thirty-one patients with chronic arthritides received a single IA injection of triamcinolone hexacetonide to treat active large-joint inflammation. Synovial biopsy specimens were obtained with arthroscopic guidance before and 9–15 days after injection. The presence of T lymphocytes, macrophages, intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), the pan-endothelial marker CD31, and the proinflammatory cytokines interleukin1␣ (IL-1␣), IL-1␤, tumor necrosis factor (TNF), and high mobility group box chromosomal protein 1 (HMGB-1) was studied by immunohistochemistry and real-time reverse transcriptase–polymerase chain reaction. Results. IA GC treatment resulted in good clinical response in 29 of 31 joints. After therapeutic intervention, the number of synovial T lymphocytes declined, whereas the number of macrophages remained unchanged. Overall synovial protein expression of TNF, IL-1␤, extranuclear HMGB-1, VEGF, and ICAM-1 was reduced at followup tissue sampling, while no significant effects were observed regarding vascularity. In contrast, expression of IL-1␣, VEGF, and cytoplasmic HMGB-1 protein in vascular endothelial cells was not affected. GC therapy down-regulated levels of messenger RNA encoding IL-1␣ and IL-1␤, but not TNF or HMGB-1. Conclusion. Synovial cell infiltration and proinflammatory cytokine expression were affected in a multifaceted manner by IA GC treatment. Marked reduction of synovial T lymphocytes, TNF, IL-1␤, extranuclear HMGB-1, ICAM-1, and VEGF occurred in association with beneficial clinical effects. Unexpectedly, macrophage infiltration and proinflammatory endothelial cytokine expression remained unchanged. These findings may reflect mechanisms controlling the transiency of clinical improvement frequently observed after IA GC injection. Supported by The Swedish Rheumatism Association, King Gustaf V’s 80-year-Foundation, the Börje Dahlin’s Foundation, the Freemason Lodge in Stockholm, the Swedish Medical Research Council, and the Åke Wiberg Foundation. Erik af Klint, MD, Cecilia Grundtman, MSc, Marianne Engström, BSc, Anca Irinel Catrina, MD, Dimitrios Makrygiannakis, MD, Lars Klareskog, MD, Ulf Andersson, MD (current address: Astrid Lindgren Children’s Hospital, Stockholm, Sweden), AnnKristin Ulfgren, PhD: Karolinska Institutet, Stockholm, Sweden. Dr. af Klint and Ms Grundtman contributed equally to this work. Dr. Klareskog is a consultant for and/or has received research grants from Wyeth, Schering-Plough, Abbott, Astra-Zeneca, Bristol Myers-Squibb, and Centocor. Dr. Andersson has received research grants (more than $10,000 per year) from Critical Therapeutics and owns stock in Critical Therapeutics. Address correspondence and reprint requests to Ulf Andersson, MD, Department of Woman and Child Health, Astrid Lindgren Children’s Hospital, Q2:02, S-17176 Stockholm, Sweden. E-mail: firstname.lastname@example.org. Submitted for publication October 18, 2004; accepted in revised form August 31, 2005. The discovery more than 50 years ago of glucocorticoids (GCs) and their potent therapeutic effects in rheumatoid arthritis (RA) represents a major breakthrough for ameliorating inflammatory diseases. However, dose-related toxicity mediated by GCs constrains their systemic therapeutic usefulness and encourages local administration whenever feasible. Hence, intraarticular (IA) GC injections are widely used in the management of arthritic conditions. Their clinical efficacy is unpredictable, however, and varies from long-term remission to minimal benefit. The reasons for the diverse 3880 EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION clinical responses have not been fully elucidated, partly because of a paucity of published data on in vivo effects of IA GCs on chronic human synovitis. In vitro studies indicate that important antiinflammatory effects of GCs are mediated by suppression of transcription and release of multiple cytokines and chemokines, by inhibition of the influx of effector cells to sites of inflammation, and by redirection of T lymphocyte responses from a pro- to an antiinflammatory influence (for review, see refs. 1 and 2). In the present study we utilized sequential arthroscopic-guided biopsy and ex vivo techniques to examine synovial tissue changes in chronic arthritis reflecting cell migration and proinflammatory cellular functions after IA GC injection. A previous study demonstrated that IA GC therapy reduces the synovial membrane volume in inflamed joints (3). We analyzed whether this therapeutic effect could be linked to any decrease in T lymphocytes and/or macrophages, expression of intercellular adhesion molecule 1 (ICAM-1) in the synovial membrane, or the size of the vascular compartment. A recently reported study demonstrated a marked reduction of synovial macrophage numbers after 2-week systemic GC therapy for RA (4). We further assessed the expression of synovial proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin-1 (IL-1), since these cytokines are key mediators in chronic synovitis (5). The regulation of cytokine production and release is highly complex, and we consequently studied therapy-induced changes in both cytokine protein and cytokine messenger RNA (mRNA) levels. In addition, we investigated changes in the expression of high mobility group box chromosomal protein 1 (HMGB-1). HMGB-1 represents a proinflammatory molecule that is abundantly and aberrantly expressed in the synovial tissue in RA and other chronic arthritides (for review, see refs. 6 and 7). The intranuclear architectural protein HMGB-1 has recently been identified as a potent cytokine when present extracellularly (8). The molecule can be actively secreted by stimulated macrophages or monocytes in a process that requires HMGB-1 translocation from the nucleus to secretory lysosomes (9). Furthermore, HMGB-1 has been demonstrated to be a nuclear danger signal that is passively released by necrotic, as opposed to apoptotic, cells that will induce inflammation (10). Extracellular HMGB-1 acts as a cytokine by signaling via the receptor for advanced glycation end products and possibly via members of the Toll-like receptor family (11,12). 3881 PATIENTS AND METHODS Patients. Thirty-one patients with chronic arthritis (21 women and 10 men; mean age 48 years, range 20–83) who had active inflammation with effusion in at least 1 large joint (30 of 31 studied joints were knee joints), were included in this study. Clinical and demographic characteristics of the patients are presented in Table 1. Sixteen patients fulfilled the 1987 American College of Rheumatology criteria for RA (13) (10 patients were seropositive), 7 patients had unclassified oligoarthritis, 3 patients had unclassified polyarthritis, 2 patients had spondylarthritis, 2 patients had psoriatic arthritis, and 1 patient had juvenile idiopathic arthritis. Disease duration varied from 6 weeks to 20 years (mean 5.5 years). The investigated joints had exhibited active arthritis for 7 days to 3 years. Patients were recruited from the outpatient clinic of the rheumatology unit at Karolinska University Hospital, Solna, Sweden. All clinical examinations and sequential arthroscopic sampling were performed by the same physician (EaK). All patients had given informed consent, and the study was approved by the Karolinska University Hospital ethics committee. Arthroscopic biopsies. Synovial membrane samples were obtained by an arthroscopic technique as previously described (14) and were taken before and 9–15 days after IA injection of 40 mg triamcinolone hexacetonide (Lederspan; Wyeth Lederle, Solna, Sweden). Synovial biopsy sites were selected to be from areas exhibiting maximal signs of inflammation and were documented by photography and mapped in close proximity to the primary biopsy sites to avoid sampling variation at the second arthroscopy biopsy sampling. Tissue processing and immunohistochemical studies. The synovial biopsy specimens were snap frozen within 30 seconds in liquid isopentane, stored at ⫺70°C, and then embedded in OCT compound (TissueTek; Sakura Finetek, Zoeterwoude, The Netherlands) when sectioning was performed. Cryostat sections from the biopsy samples (6–8 m, cryostat setting 7 m) were placed on SuperFrost Plus slides (Menzel-Gläser, Braunschweig, Germany) and air dried for 30 minutes. Sections for cytokine staining were initially fixed for 20 minutes with freshly prepared 2% (volume/volume) formaldehyde (Sigma, St. Louis, MO), dissolved in phosphate buffered saline (PBS; pH 7.4) at 4°C, washed in PBS, and then left to air-dry before storage at ⫺70°C. Sections for phenotypic cellular characterization were fixed with acetone, then left to air-dry at room temperature before storage at ⫺70°C. The staining procedures have been described in detail previously (14). Primary antibodies. All primary antibodies used, apart from the anti–HMGB-1 and anti–vascular endothelial growth factor (anti-VEGF) antibodies, were mouse IgG1 monoclonal antibodies. Anti-CD3 antibody (clone SK7) was obtained from Becton Dickinson (San Jose, CA), anti-CD68 (clone PG-M1) and anti-CD163 (clone Ber-MAC3) from Dakopatts (Glostrup, Denmark), anti-CD54 (anti–ICAM-1) (clone 84H10) from Serotec Scandinavia (Oslo, Norway), anti-CD31 (human CD31) (clone EN4) from Sanbio Bio-Zac (Uden, The Netherlands), anti-TNF (clone 2C8) from Biodesign (Saco, ME), anti–IL-1␣ (clone 1277-89-7) and anti–IL-1␤ (clone 2D8 combined with 1437-96-5) from Immunokontakt (Bioggo, Switzerland), control mouse IgG1 from Dakopatts, anti– 3882 AF KLINT ET AL Table 1. Demographic and clinical characteristics of the patients at baseline* Patient Diagnosis RF Age/sex Duration of disease, months Duration of active arthritis, months Therapy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA RA Oligoarthritis Oligoarthritis Oligoarthritis Oligoarthritis Oligoarthritis Oligoarthritis Oligoarthritis Polyarthritis Polyarthritis Polyarthritis Spondylarthritis Spondylarthritis PsA PsA JIA ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ 32/F 40/F 42/F 46/F 53/M 57/F 58/F 63/M 65/F 68/F 35/F 38/M 44/F 60/M 70/F 83/M 21/M 28/M 39/F 53/M 56/F 61/F 66/F 20/F 43/F 73/F 20/M 36/F 53/F 52/F 33/M 3 180 3 3 84 72 23 3 36 12 240 36 60 3 60 18 4 48 168 120 8 12 1.5 8 144 24 6 72 12 36 204 0.75 0.75 3 3 0.75 6 0.75 1 2 1.75 0.5 36 1.5 2 3 0.5 4 2.5 7 2 3 6 1.5 8 1.5 0.5 1.5 2.5 3 0.25 0.25 NSAID Pred. 2.5 mg/day, NSAID NSAID 0 Pred. 7.5 mg/day, NSAID Etan. 50 mg/week, AZA 100 mg/day, NSAID 0 NSAID MTX 10 mg/week, NSAID NSAID MTX 15 mg/week, NSAID NSAID MTX 17.5 mg/week 0 MTX 20 mg/week MTX 15 mg/week, pred. 10 mg/day 0 HCQ 400 mg/day 0 0 NSAID 0 Pred. 5 mg/day, NSAID NSAID Pred. 2.5 mg/day, NSAID MTX 12.5 mg/week NSAID SSZ 2 gm/day, NSAID MTX 10 mg/week, NSAID MTX 20 mg/week, NSAID SSZ 3 gm/day, NSAID * RF ⫽ rheumatoid factor; RA ⫽ rheumatoid arthritis; NSAID ⫽ nonsteroidal antiinflammatory drug; pred. ⫽ prednisolone; etan. ⫽ etanercept; AZA ⫽ azathioprine; MTX ⫽ methotrexate; HCQ ⫽ hydroxychloroquine; SSZ ⫽ sulfasalazine; PsA ⫽ psoriatic arthritis; JIA ⫽ juvenile idiopathic arthritis. HMGB-1 peptide affinity-purified polyclonal rabbit IgG antibody from PharMingen (San Diego, CA), anti-VEGF polyclonal rabbit IgG antibody (A-20 [sc-152]) from Santa Cruz Biotechnology (Santa Cruz, CA), and control polyclonal rabbit IgG from Dakopatts. Secondary antibodies. Biotinylated F(ab⬘) 2 fragmented goat-anti-mouse IgG1 was purchased from Caltag (Burlingame, CA). Biotinylated horse anti-mouse IgG and biotinylated goat anti-rabbit IgG were from Vector (Burlingame, CA). Serum analysis. Serum levels of VEGF were assessed by standard sandwich enzyme-linked immunosorbent assay (ELISA) (Quantikine; R&D Systems Europe, Abingdon, UK). Recombinant human VEGF165 was used as a standard in the assay. All serologic analyses were performed in duplicate. Optical densities were determined using a microplate reader (Emax; Molecular Devices, Menlo Park, CA) at 450 nm with a connected software program (SoftMax version 2.01; SoftMax, Menlo Park, CA). Quantification of staining results. All stained sections were examined with a Polyvar II microscope (Reichert-Jung, Vienna, Austria) equipped with a Leica 300F digital color video camera that digitized the microscope images to be processed in a Quantimet 550S image analyzer (Leica Cambridge, Cambridge, UK) linked to a PC computer. The sections were assessed on 3 different occasions by 2 independent observers who were blinded to the identity of the specimens, with concordant results. The area of specific immunostaining was expressed as a percentage of the total counterstained tissue area evaluated. Analysis of an entire tissue section typically involved 22–253 microscopic fields at a magnification of ⫻ 250. RNA extraction and real-time reverse transcriptase– polymerase chain reaction (PCR). Total RNA was extracted from snap-frozen synovial biopsy samples from 12 patients before and after IA GC injection, using an RNeasy Minikit (Qiagen, Valencia, CA). Before RNA elution, residual genomic DNA was digested using RNase-Free DNase I (Qiagen). The integrity and quality of the total RNA were assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Total RNA (18–748 ng/l) was reverse-transcribed to complementary DNA (cDNA) using random hexamer (0.1 g/l; Gibco Life Technologies, Grand Island, NY) and SuperScript reverse transcriptase (200 units/ l; Gibco Life Technologies). Amplification was performed EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION Figure 1. Quantitative results of digital image studies of the immunostained tissue area occupied by T lymphocytes (CD3) or macrophages (CD68, CD163) before (white boxes and circles) and after (gray boxes and circles) intraarticular glucocorticoid therapy in 31 patients with chronic arthritis. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. Circles indicate outliers. Corticosteroid injection down-regulated the number of T lymphocytes (ⴱ ⫽ P ⬍ 0.05), but not the number of macrophages. with an ABI Prism 7700 Sequence Detection System (Perkin Elmer, Norwalk, CT) using the 5⬘ nuclease method (TaqMan) with a 2-step PCR protocol (95°C for 10 minutes, followed by 3883 40 cycles of 95°C for 15 seconds and 60°C for 1 minute) for IL-1␣. Amplification of IL-1␣ was performed using the human PCR cytokine kit (Applied Biosystems, Foster City, CA). GAPDH was used as a housekeeping gene control. For quantification of mRNA for IL-1␣, IL-1␤, HMGB-1, and TNF, a QuantiTec SYBR Green kit was used according to the instructions of the manufacturer (Qiagen). Amplification was performed using the same Sequence Detection System as above but with a 3-step PCR protocol (95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; then 95°C for 15 seconds and 60°C for 20 seconds and ramping for 20 minutes to 95°C for 15 seconds). Relative quantification of mRNA levels was performed using the standard curve method. The standard curve that consisted of a pool of all patient samples was created using 5 different dilutions (1, 1:5, 1:25, 1:125, and 1:625). The amount of mRNA in each sample and the amount of endogenous control GAPDH could be deduced from target and GAPDH mRNA standard curves, respectively. Standard curves were used for different targets; thus, the relative amounts cannot be compared between targets. The amount of each sample was calculated as the ratio between the relative amount of target and the relative amount of the corresponding endogenous control (GAPDH mRNA). The samples were run in duplicate. Samples without cDNA were used as negative controls. Primers and probe for IL-1␣ were obtained from Applied Biosystems and have been described previously (15). Primers for IL-1␤ and HMGB-1 (from CyberGene, Novum Research Park, Huddinge, Sweden) were as follows: IL-1␤ Figure 2. A, Cytokine protein expression before (white boxes and circles) and after (gray boxes and circles) intraarticular (IA) glucocorticoid (GC) therapy in biopsy specimens from 31 patients with chronic arthritis. Immunostaining for interleukin-1␣ (IL-1␣), IL-1␤, tumor necrosis factor (TNF), and high mobility group box chromosomal protein 1 (HMGB-1) was evaluated by digital image analysis. The overall protein expression of IL-1␤, TNF, and HMGB-1 was significantly reduced after therapy (ⴱ ⫽ P ⬍ 0.05). B, Expression of mRNA for IL-1-1␣, IL-1␤, TNF, and HMGB-1 before (white boxes and circles) and after (gray boxes and circles) IA GC therapy in paired biopsy specimens from 12 patients with chronic arthritis, analyzed by real-time reverse transcriptase–polymerase chain reaction. IL-1␣ and IL-1␤ mRNA levels were significantly reduced after a single injection (ⴱ ⫽ P ⬍ 0.05). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. Circles indicate outliers. 3884 AF KLINT ET AL Figure 3. IL-1␣ (A and B) and TNF (C and D) expression with brown (diaminobenzidine) immunoperoxidase staining in synovial sections from 1 GC-injected joint in a patient with rheumatoid arthritis. Cell nuclei were visualized with blue hematoxylin counterstaining. IL-1␣ was mainly observed in the vascular compartments, with no observable change of staining intensity after (B) versus before (A) IA GC injection. TNF was readily detectable before GC injection (C), intracellularly as well as extracellularly in inflammatory cell infiltrates (thick arrow in C), and its expression was substantially reduced 2 weeks after GC injection (D). Thin arrows indicate large vessels expressing IL-1␣ or TNF. See Figure 2 for definitions. (Original magnification ⫻ 250.) forward 5⬘-CTG-ATG-GCC-CTA-AAC-AGA-TGA-G, reverse 5⬘-GGT-CGG-AGA-TTC-GTA-GCT-GGA-T (16,17); HMGB-1 forward 5⬘-GCG-GAC-AAG-GCC-CGT-TA, reverse 5⬘-AGA-GGA-AGA-AGG-CCG-AAG-GA (18). Primers for TNF, designed with Primer Express software (Perkin Elmer) and obtained from CyberGene, were as follows: forward 5⬘-CCA-GGG-ACC-TCT-CTC-TAA-TCA-GC, reverse 5⬘-CTC-AGC-TTG-AGG-GTT-TGC-TAC-A (19). Primers and probe for GAPDH were designed with Primer Express software (primers obtained from CyberGene, probe obtained from Scandinavian Gene Synthesis [Köping, Sweden]) and were as follows: forward probe 5⬘-TAT-TGT-TGC-CATCAA-TGA-CCC-CTT-CAT-TGA, forward primer 5⬘-AGGGCT-GCT-TTT-AAC-TCT-GGT-AAA, reverse primer: 5⬘CAT-ATT-GGA-ACA-TGT-AAA-CCA-TGT-AGT-TG (19). Statistical analysis. Statistical analyses were performed using SPSS 11.5 for Windows (SPSS, Chicago, IL). Wilcoxon’s signed rank test was used to compare parameters before and after IA GC treatment. Adjustment for multiple comparisons was made by the Bonferroni correction method. P values less than or equal to 0.05 were considered significant. RESULTS Improvement in clinical signs of inflammation after IA GC injection. At the initial arthroscopy, all investigated joints had clinical signs of inflammation, with swelling and pain accompanied by IA effusion. At the time of followup arthroscopy 9–15 days after GC injection, the treated joints exhibited marked clinical improvement in the majority of the patients. In 2 injected joints, there were minimal or no clinical effects. In the remaining 29 joints, swelling and pain were reduced and effusion was undetectable. However, the postinjection arthroscopy still showed signs of persisting synovitis, including villus formation and increased vascularity in all patients. Reduced numbers of synovial tissue T lymphocytes, but not of macrophages, after IA GC injection. Before treatment, all joints expressed a considerable EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION 3885 Figure 4. HMGB-1 expression in inflammatory cells (A), lining layer (B), and endothelial cells (C and D) in biopsy specimens obtained before (A and C) and after (B and D) IA GC injection in the knee joint of a patient with rheumatoid arthritis. Massive HMGB-1 staining (brown) was evident both intracellularly and extracellularly in inflammatory infiltrates (thick arrow) before GC injection (A). This staining pattern was substantially changed after GC injection, when the extracellular HMGB-1 staining was almost abolished and the intracellular HMGB-1 was mainly confined to the lining layer cells (thick arrow) and was not seen in the sublining cell infiltrates (B). The prominent cytoplasmic HMGB-1 staining in vascular endothelial cells appeared unchanged after (D) versus before (C) GC injection (arrows). Thin arrow in A shows a large vessel with no HMGB-1 staining. Thin arrow in B shows HMGB-1 staining in a capillary. See Figure 2 for definitions. (Original magnification ⫻ 250 in A, C, and D; ⫻ 100 in B.) number of T lymphocytes in the synovium, mainly in the form of lymphoid aggregates and as scattered cells in the sublining tissue. The number of T lymphocytes was markedly reduced in the great majority of treated joints (Figure 1). Similarly, the number of lymphoid aggregates was diminished in almost every biopsy sample at followup. In contrast, no statistically significant effect on macrophage infiltration, as indicated by CD68 or CD163 phenotypic staining, was observed at the time of the second biopsy (Figure 1). Diverse modulation of synovial cytokine protein expression by IA GC. The expression of TNF and IL-1␤ protein detected by immunohistochemical staining in the synovial membrane was significantly diminished, but not abrogated, after IA GC injection (Figures 2A and 3C and D). TNF was mainly observed in sublining macrophage-like cells and in the lining layer, while IL-1␤ was demonstrated in macrophages and fibroblasts, as judged by morphology. In contrast, there were no differences in the appearance of IL-1␣ in tissues obtained before and after GC injections (Figures 2A and 3A and B). IL-1␣ was mainly observed in vascular endothelial cells present in the majority of blood vessels, and only occasionally in cells outside the vascular compartment. The proinflammatory mediator HMGB-1 was aberrantly expressed outside the cell nucleus in macrophage-like cells, in numerous synoviocytes, and in endothelial cells in a minority of blood vessels (Figure 4). Before GC injection, HMGB-1 was detectable extracellularly, with a staining pattern encompassing inflammatory cells that also expressed cytoplasmic HMGB-1 3886 AF KLINT ET AL Figure 5. A, Quantitative results of immunohistochemistry and digital image analysis of the expression of molecules associated with the vascular system, i.e., as the pan-endothelial marker CD31, intracellular adhesion molecule 1 (ICAM-1), and vascular endothelial growth factor (VEGF), before (white boxes and circles) and after (gray boxes and circles) intraarticular (IA) glucocorticoid (GC) therapy in 31 patients with chronic arthritis. Expression of ICAM-1 and VEGF was significantly reduced after therapy (ⴱ ⫽ P ⬍ 0.05). Vascularity did not change during the study period, as judged by CD31 staining. B, Serum VEGF levels were analyzed in blood samples obtained before (white box and circles) and 9–15 days after (gray box and circles) IA GC injection in 20 patients with chronic arthritis. The same analysis was also performed in 56 healthy age-matched blood donors, in a single blood test per person (stippled box and circles). VEGF levels were significantly higher in patients before versus after treatment, and both before and after treatment, patients exhibited higher serum VEGF levels than controls (ⴱ ⫽ P ⬍ 0.05). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. Circles indicate outliers. (Figure 4A). Overall HMGB-1 staining in the synovial tissue sampled postinjection was strongly diminished (Figures 2A and 4A and B), but in a selective manner. Extracellular HMGB-1 staining was almost eliminated at followup, and cytoplasmic HMGB-1 staining in synoviocytes and macrophage-like cells was considerably reduced. In contrast, the cytoplasmic HMGB-1 component in vascular endothelial cells and in macrophage-like cells in the lining layer did not decrease (Figures 4B and D). Nuclear HMGB-1 expression was not downregulated, and even appeared with increased intensity in many synovial cells (Figure 4B). Selective effects of IA GC injections on synovial cytokine mRNA levels. IA GC injection affected cytokine mRNA expression similarly among all 12 patients from whom paired synovial samples were technically suitable for mRNA extraction. However, results obtained in the cytokine mRNA analysis only partially paralleled those observed in cytokine protein assessments (Figure 2B). Levels of mRNA for both IL-1␣ and IL-1␤ were strongly down-regulated by IA GC. In contrast, TNF and HMGB-1 mRNA levels were not significantly altered. Effects of IA GC on the synovial vascular compartment. Immunohistologic studies did not reveal any reduction in the vascularity of the tissue as a conse- quence of GC therapy. We used the pan-endothelial marker CD31 for digital image analysis assessing tissue area (Figure 5A), and manual counting of blood vessels by microscopy (data not shown), and found no indications of significantly reduced vascularity. The adhesion molecule ICAM-1 was demonstrated by immunostaining to be present in blood vessels and in nonvascular tissue, particularly in the synovial lining layer. IA GC injection consistently and significantly down-regulated ICAM-1 expression in all studied tissue compartments in 30 of the 31 patients (Figure 5A). The expression of vascular growth factors, such as VEGF, was also significantly reduced in the synovial tissue sections after GC injection (Figure 5A). However, this decrease was seen only within the inflammatory cell infiltrates, and not within the vascular compartments. These immunohistochemical findings were further supported by results of ELISA study of peripheral blood samples (Figure 5B). In healthy controls, the mean ⫾ SD serum VEGF level was 331.4 ⫾ 33.3 pg/ml (range 25.2–1,315.1). In patients, the serum VEGF level was 923.7 ⫾ 105.1 pg/ml (range 366.8–2,198.8) before and 620.2 ⫾ 50.3 pg/ml (range 321.2–1,234.9) 9–15 days after GC injection. Although the serum VEGF levels in patients declined markedly (P ⬍ 0.05) after therapy, EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION they remained elevated when compared with those in healthy controls. DISCUSSION To our knowledge, this is the first study to examine therapeutic effects of IA GC injection on the target organ of chronic arthritides, the synovial membrane. However, arthroscopy and ex vivo studies, which form the basis for the observations described in this report, have inherent limitations. Although, it is desirable from a scientific standpoint to perform repeated arthroscopies during followup, ethical considerations preclude this strategy. Arthroscopy allows visualization mainly of the surface of synovium and not of pathologic processes in the adjacent bone tissue. Likewise, with synovial biopsy, only regions of superficial synovium of limited size, which may not necessarily reflect the global IA inflammatory activity, are sampled. However, one would expect that the strongest therapeutic effects would be on superficial surfaces, where injected GC reaches maximal levels. In the present study pretreatment biopsies were all obtained from areas expressing maximal signs of inflammation, and careful mapping was undertaken to direct the followup sampling to the same regions to enable evaluation of therapy-induced changes. Despite the fact that the IA GC injections led to good clinical recovery of almost all treated joints during the study period, both arthroscopy and microscopy examinations demonstrated that many signs of inflammation in the synovial tissue prevailed. IA GC injection markedly reduced the number of synovial T lymphocytes but not of macrophages, which differs from results observed after 2 weeks of treatment with systemically administered GC (4). One of the most striking findings of the latter study was that prednisolone therapy in RA was associated with a marked reduction in macrophage infiltration in the synovial tissue. Separate biologic effects exerted by local versus systemic GC administration possibly explain the discrepant results concerning macrophage accumulation. High-dose systemic, but not local, GC therapy will induce a profound monocytopenia (20), impeding the access of monocytes to the inflamed synovial tissue. Reduced numbers of macrophages as well as functionally pacified macrophages both correlate with good clinical efficacy of any therapy administered for synovitis. We cannot presently explain the mechanisms for the marked reduction of synovial T lymphocytes after IA GC injection in our study, which did not occur to the same extent after systemic GC administration (4). It has 3887 been demonstrated that activated human T lymphocytes are susceptible to induction of apoptosis in response to high doses of GC (21), which may be achieved only locally after IA GC injection. However, when we used the TUNEL assay staining technique in paired biopsy specimens from 20 treated joints, we could not demonstrate signs of increased apoptosis 9–15 days after GC injection (results not shown). We observed that IA GC injections affected the expression of cytokine protein and mRNA levels with, at first glance, contradictory outcomes. GC treatment did not diminish IL-1␣ protein expression during the study period, despite the fact that IL-1␣ mRNA was reduced. IL-1␤ protein as well as mRNA were down-regulated. Both TNF and HMGB-1 protein levels were decreased, while neither TNF mRNA nor HMGB-1 mRNA was significantly reduced. Transcription of IL-1␣, as well as IL-1␤, is under NF-B control (for review, see ref. 22), so the observed down-regulation of IL-1 mRNA levels by GC treatment is a predictable result. IL-1␣ protein lacks a known secretory pathway, precluding a hypothetical influence of GC on IL-1␣ release. Undiminished IL-1␣ protein expression, despite suppressed mRNA levels, could possibly be explained by a long half-life of IL-1␣ protein within the endothelial cells. IL-1␤ transcription is directly suppressed by a regulatory region containing a negative GC response element present in the IL-1␤ promoter, which would contribute to the IL-1␤ downregulation (23). TNF production is known to be negatively regulated by GCs, both transcriptionally by decreasing the steady-state level of TNF mRNA and posttranscriptionally by reducing the rate of secretion (24). Interferon-␥ (IFN␥) could possibly be a factor explaining the lack of effect of GCs on TNF mRNA in our study. IFN␥ strongly counteracts the effects of GCs on TNF at the mRNA level but not at posttranscriptional sites of action (24). HMGB-1 transcription is up-regulated by estrogen (25), but there are no reports of mediation of its regulation by GCs. The reduced extracellular HMGB-1 protein levels observed in our study are likely due to posttranscriptional therapeutic effects. GC-induced lipocortins suppress phospholipase A2 (PLA2) formation (26), a crucial factor for HMGB-1 protein secretion from activated macrophages (9,27). Several therapeutic antagonists of the NF-B pathway have been shown to inhibit extracellular HMGB-1 release (28), suggesting that this may also be a mechanism by which GCs may act to inhibit inflammatory HMGB-1 expression. 3888 There are few corroborating reports of studies investigating local effects of drug therapy on the synovial membrane in chronic arthritis. Two-week treatment of rheumatoid synovitis with high-dose oral prednisolone resulted in a clear trend toward reduction in levels of TNF and IL-1␤, but not IL-1␣ (4). A study of intravenous pulse methylprednisolone therapy with arthroscopically directed synovial biopsy before and 24 hours after infusion revealed a strong decrease of TNF and IL-8 levels in RA (29). Moreover, disease relapse was associated with recurrence of synovial TNF expression. However, no effects on IL-1␤ or IL-1 receptor antagonist levels were observed. Young and coworkers investigated therapeutic effects of IA GC injection in osteoarthritis, using serial arthroscopy sampling and immunostaining (30). They found a modest reduction of macrophage numbers in the lining layer, but no changes in the sublining tissue 1 month after injection. Cytokine assessment in a study of combination treatment with systemic GCs and gold showed no effect on synovial IL-1␤ expression 2 weeks after initiation of therapy, while a substantial reduction was noted 10 weeks later (31). To date, only 2 types of RA treatment have been shown to work within a time frame measured in days: GC and TNF-blocking therapy. We have previously published a study on anti-TNF (infliximab) treatment of patients with active RA, utilizing assessment methods similar to those in the present study (32). Synovial tissue TNF production was distinctly reduced 14 days after a single intravenous infliximab infusion, whereas no such changes in IL-1␣ and IL-1␤ expression in the synovium were recorded. This is the first report to address the therapeutic effects of GCs on the proinflammatory cytokine HMGB-1 in human arthritis. This protein is of major interest for several reasons: 1) it has been demonstrated to be present extracellularly in considerable amounts in inflamed, but not normal, joints; 2) it causes destructive arthritis when injected IA in animal studies; and 3) neutralizing anti–HMGB-1 therapy is beneficial in experimental arthritis (for review, see ref. 6). HMGB-1 is ubiquitously present as a nuclear factor in all mammalian cells, although we did not demonstrate the nuclear expression in all cells in our specimens, probably due to insensitivity of the staining technique. Nevertheless, it is the extranuclear expression of HMGB-1 that is of main interest for inflammation research. HMGB-1 may be passively released intracellularly by necrotic, but not apoptotic, cells, or it can be actively secreted by specialized cells including activated macrophages (9). It is thus biologically significant that in AF KLINT ET AL the present study, the extracellular HMGB-1 staining had decreased considerably in the postinjection specimens. Activation of macrophages results in an accumulation of HMGB-1 in cytoplasmic secretory lysosomes. The lysosomes then are exocytosed when triggered by lysophosphatidylcholine (9), a lipid generated by PLA2 at sites of inflammation. Since GCs are potent inhibitors of the synthesis of PLA2 (26), it is conceivable that IA GC injections caused reduced extracellular HMGB-1 secretion via a down-regulation of lysophosphatidylcholine formation required for HMGB-1 release. A recent study on systemic GC therapy in dermatomyositis and polymyositis likewise demonstrated a potent suppression of aberrant HMGB-1 expression in and adjacent to mononuclear inflammatory cells (33). A novel and thus unexpected result of this study was the finding of a relative insensitivity of the vascular compartment to the antiinflammatory effects of GCs. We did not observe a reduction in the area or numbers of blood vessels or a reduction of the proinflammatory cytokines IL-1␣ and HMGB-1 in the endothelial cells. The short followup period (maximum 15 days) complicates the interpretation of these findings. However, when patients with chronic myositis received systemic GC therapy for 3–6 months, inflammatory, extranuclear HMGB-1 expression prevailed selectively in the vascular endothelial cells (33), while vascular IL-1␣ expression declined but did not normalize (34). Furthermore, another study using cell cultures demonstrated strong inhibitory effects of dexamethasone on lipopolysaccharide-induced IL-1 formation in human monocytes, but not in endothelial cells (35). These findings together with the results of our study might have some relevance with regard to cardiovascular function. There are many inflammatory disorders that are successfully treated with systemic GCs, yet the same patients are at increased risk for developing cardiovascular disease. Other parameters related to vascular inflammation and activation, i.e., expression of ICAM-1 and production of VEGF, were susceptible to GC-induced suppression. The down-regulation of VEGF revealed by immunostaining of the synovial specimens was confirmed when serum VEGF levels were analyzed before and after the IA injections. In summary, the results of our study emphasize the central role of TNF and IL-1␤, and possibly also HMGB-1, in the pathogenesis of chronic arthritis. These 3 mediators are involved in a positive feedback system (5–8) and could thus be anticipated to be affected in the same direction by this therapy. The observed suppression of aberrant HMGB-1 expression after IA GC therapy represents a novel finding. Future studies are EFFECT OF IA GLUCOCORTICOIDS ON SYNOVIAL CELL INFLAMMATION needed to determine whether specific HMGB-1– blocking therapy will be beneficial for patients with chronic arthritis. REFERENCES 1. Almawi WY, Beyhum HN, Rahme AA, Rieder MJ. Regulation of cytokine and cytokine receptor expression by glucocorticoids. J Leukoc Biol 1996;60:563–72. 2. Moreland LW, O⬘Dell JR. Glucocorticoids and rheumatoid arthritis: back to the future? [review]. Arthritis Rheum 2002;46:2553–63. 3. Ostergaard M, Stoltenberg M, Gideon P, Henriksen O, Lorenzen I. Changes in synovial membrane and joint effusion volumes after intraarticular methylprednisolone: quantitative assessment of inflammatory and destructive changes in arthritis by MRI. J Rheumatol 1996;23:1151–61. 4. Gerlag DM, Haringman JJ, Smeets TJ, Zwinderman AH, Kraan MC, Laud PJ, et al. Effects of oral prednisolone on biomarkers in synovial tissue and clinical improvement in rheumatoid arthritis. Arthritis Rheum 2004;50:3783–91. 5. Feldmann M, Brennan FM, Foxwell BM, Maini RN. The role of TNF ␣ and IL-1 in rheumatoid arthritis. Curr Dir Autoimmun 2001;3:188–99. 6. Andersson U, Erlandsson-Harris H. HMGB1 is a potent trigger of arthritis. J Intern Med 2004;255:344–50. 7. Andersson U, Tracey KJ. HMGB1 as a mediator of necrosisinduced inflammation and a therapeutic target in arthritis. Rheum Dis Clin North Am 2004;30:627–37. 8. Andersson U, Wang H, Aveberger AC, Janson A, Palmblad K, Yang H, et al. High mobility group-1 (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 2000;192:565–70. 9. Gardella S, Andrei C, Ferrera D, Lotti LV, Torrisi MR, Bianchi ME, et al. The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep 2002;3:995–1001. 10. Scaffidi P, Misteli T, Bianchi ME. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 2002;418: 191–5. 11. Huttunen HJ, Kuja-Panula J, Sorci G, Agneletti AL, Donato R, Rauvala H. Coregulation of neurite outgrowth and cell survival by amphoterin and S100 proteins through receptor for advanced glycation end products (RAGE) activation. J Biol Chem 2000;275: 40096–105. 12. Park JS, Svetkauskaite D, He Q, Kim JY, Strassheim D, Ishizaka A, et al. Involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein. J Biol Chem 2004;279:7370–7. 13. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. 14. Ulfgren AK, Grondal L, Lindblad S, Johnell O, Klareskog L, Andersson U. Interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment. Ann Rheum Dis 2000;59: 439–47. 15. Seddighzadeh M, Larsson P, Ulfgren AC, Onelov E, Berggren P, Tribukait B, et al. Low IL-1␣ expression in bladder cancer tissue and survival. Eur Urol 2003;43:362–8. 16. Brink N, Szamel M, Young AR, Wittern KP, Bergemann J. Comparative quantification of IL-1␤, IL-10, IL-10r, TNF␣ and IL-7 mRNA levels in UV-irradiated human skin in vivo. Inflamm Res 2000;49:290–6. 17. Rad R, Dossumbekova A, Neu B, Lang R, Bauer S, Saur D, et al. Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and host specific colonisation during Helicobacter pylori infection. Gut 2004;53:1082–9. 3889 18. Pachot A, Monneret G, Voirin N, Leissner P, Venet F, Bohe J, et al. Longitudinal study of cytokine and transcription factor mRNA expression in septic shock. Clin Immunol 2005;114:61–9. 19. Khademi M, Illes Z, Gielen AW, Marta M, Takazawa N, BaecherAllan C, et al. T cell Ig- and mucin-domain-containing molecule-3 (TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis. J Immunol 2004;172:7169–76. 20. Fauci AS, Dale DC, Balow JE. Glucocorticosteroid therapy: mechanisms of action and clinical considerations. Ann Intern Med 1976;84:304–15. 21. Kirsch AH, Mahmood AA, Endres J, Bohra L, Bonish B, Weber K, et al. Apoptosis of human T-cells: induction by glucocorticoids or surface receptor ligation and ex vivo. J Biol Regul Homeost Agents 1999;13:80–9. 22. Auron PE, Webb AC. Interleukin-1: a gene expression system regulated at multiple levels. Eur Cytokine Netw 1994;5:573–92. 23. Zhang G, Zhang L, Duff GW. A negative regulatory region containing a glucocorticosteroid response element (nGRE) in the human IL-1␤ gene. DNA Cell Biol 1997;16:145–52. 24. Luedke CE, Cerami A. Interferon-␥ overcomes glucocorticoid suppression of cachectin/tumor necrosis factor biosynthesis by murine macrophages. J Clin Invest 1990;86:1234–40. 25. Borrman L, Kim I, Schultheiss D, Rogalla P, Bullerdiek J. Regulation of the expression of HMGB1, a co-activator of the estrogen receptor. Anticancer Res 2001;21:301–5. 26. Touqui L, Alaoui-El-Azher M. Mammalian secreted phospholipases A2 and their pathophysiological significance in inflammatory diseases. Curr Mol Med 2001;1:739–54. 27. Muller S, Ronfani L, Bianchi ME. Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function. J Intern Med 2004;255:332–43. 28. Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 2004;10:1216–21. 29. Youssef PP, Haynes DR, Triantafillou S, Parker A, Gamble JR, Roberts-Thomson PJ, et al. Effects of pulse methylprednisolone on inflammatory mediators in peripheral blood, synovial fluid, and synovial membrane in rheumatoid arthritis. Arthritis Rheum 1997; 40:1400–8. 30. Young L, Katrib A, Cuello C, Vollmer-Conna U, Bertouch JV, Roberts-Thomson PJ, et al. Effects of intraarticular glucocorticoids on macrophage infiltration and mediators of joint damage in osteoarthritis synovial membranes: findings in a double-blind, placebo-controlled study. Arthritis Rheum 2001;44:343–50. 31. Kirkham BW, Navarro FJ, Corkill MM, Panayi GS. In vivo analysis of disease modifying drug therapy activity in rheumatoid arthritis by sequential immunohistological analysis of synovial membrane interleukin 1␤. J Rheumatol 1994;21:1615–9. 32. Ulfgren AK, Andersson U, Engstrom M, Klareskog L, Maini RN, Taylor PC. Systemic anti–tumor necrosis factor ␣ therapy in rheumatoid arthritis down-regulates synovial tumor necrosis factor ␣ synthesis. Arthritis Rheum 2000;43:2391–6. 33. Ulfgren AK, Grundtman C, Borg K, Alexandersson H, Andersson U, Harris HE, et al. Down-regulation of the aberrant expression of the inflammation mediator high mobility group box chromosomal protein 1 in muscle tissue of patients with polymyositis and dermatomyositis treated with corticosteroids. Arthritis Rheum 2004;50:1586–94. 34. Lundberg I, Kratz AK, Alexandersson H, Patarroyo M. Decreased expression of interleukin-1␣, interleukin-1␤, and cell adhesion molecules in muscle tissue following corticosteroid treatment in patients with polymyositis and dermatomyositis. Arthritis Rheum 2000;43:336–48. 35. Zuckerman SH, Shelhaas JS, Butler LD. Differential regulation of lipopolysaccharide-induced interleukin 1 and tumor necrosis factor synthesis: effects of endogenous and exogenous glucocorticoids and the role of the pituitary-adrenal axis. Eur J Immunol 1989;19: 301–5.