Systemic TNF blockade does not modulate synovial expression of the pro-inflammatory mediator HMGB1 in rheumatoid arthritis patients--a prospective clinical study

Abstract

Introduction: High-mobility group box chromosomal protein 1 (HMGB1) has recently been identified as an endogenous mediator of arthritis. TNF and IL-1beta, pivotal cytokines in arthritis pathogenesis, both have the ability to induce the release of HMGB1 from myeloid and dendritic cells. It was, therefore, decided to investigate whether treatment based on TNF blockade in rheumatoid arthritis (RA) affects the expression of synovial HMGB1.

Methods: Repeated arthroscopy-guided sampling of synovial tissue was performed in nine patients with RA before and nine weeks after initiation of anti-TNF mAb (infliximab) therapy. Synovial biopsy specimens were analysed for HMGB1 protein by immunohistochemical staining and for HMGB1 mRNA expression by real-time reverse transcriptase PCR (RT-PCR). Statistical evaluations were based on Wilcoxon's signed rank tests or Spearman rank sum tests.

Results: Aberrant, extranuclear HMGB1 and constitutive nuclear HMGB1 expression, with histological signs of inflammation, were evident in all biopsies obtained before infliximab therapy. Signs of inflammation were still evident in the second biopsies obtained nine weeks after initiation of infliximab therapy. The cytoplasmic and extracellular expression of HMGB1 decreased in five patients, remained unchanged in one patient and increased in three patients, making the overall change in HMGB1 protein expression not significant. No correlation between the clinical response, as measured by disease activity score calculated for 28 joints (DAS28) or the American College of Rheumatology response criteria (ACR 20, 50, and 70), and the direction of change of HMGB1 expression in individual patients could be discerned. In addition, infliximab therapy did not alter HMGB1 mRNA synthesis.

Conclusion: Pro-inflammatory HMGB1 expression during rheumatoid synovitis was not consistently influenced by TNF-blocking therapy with infliximab. This suggests that TNF is not the main inducer of extranuclear HMGB1 during synovitis and that HMGB1 may represent a TNF-independent molecule that could be considered as a possible target for future therapeutic intervention in RA.

Figure 1

HMGB1 synovial protein expression before and during infliximab therapy as detected by immunohistochemistry. Nuclear, cytoplasmic and extracellular HMGB1 protein expression is evident in the RA synovia in the lining layer as well as in cellular infiltrates and endothelium. (a) Moderate aberrant synovial HMGB1 expression before the start of infliximab therapy from patient number 5. (b) The second biopsy in patient number 5 taken during infliximab treatment showing increased extranuclear HMGB1 staining. (c) A marked endothelial expression of HMGB1 is evident before infliximab therapy in synovitis obtained from patient number 7. (d) Infliximab therapy for nine weeks resulted in reduced endothelial HMGB1 expression in patient number 7. Original magnification ×250 for (a) and (b), ×100 for (c) and (d).

Figure 2

Change of HMGB1 synovial protein expression before and during infliximab therapy. Immunohistochemical scoring of HMGB1 protein expression was unchanged during the nine weeks of infliximab therapy. Values represent mean scores of highly concordant scoring recorded by the two independent investigators (ES and CG). The correlation was statistically confirmed by Spearman rank sum tests with a correlation coefficient (r_s)of 0.74 (significant at p < 0.002). Scoring for each section was evaluated using a 0 to 4 scale with increments of 0.5. Index 0 corresponds to no HMGB1 expression and 4 to highest degree of HMGB1 protein expression. Separate analyses were performed for lining layer, vessels and cellular infiltrates in each of the sections. (a) Change of overall HMGB1 protein expression for each patient. (b) Change of HMGB1 expression in cellular infiltrates. (c) Change of HMGB1 expression in lining layer. (d) Change of HMGB1 expression in endothelium.

Figure 3

Change of HMGB1 synovial mRNA expression before and after infliximab therapy. HMGB1 mRNA expression as determined by reverse-transcriptase PCR for six patients. Values are expressed as fold-change of up- or down-regulation using a log₂-scale. The last bar to the right represents the average value for the whole group and the standard deviation is indicated by whiskers.