Tumour necrosis factor α augments the inhibitory effects of CTLA-4-Ig on osteoclast generation from human monocytes via induction of CD80 expression.

Cytotoxic T lymphocyte antigen-4-immunoglobulin (CTLA-4-Ig) exerts anti-rheumatic action via negative regulation of the co-stimulation process between antigen-presenting cells and T cells. CTLA-4-Ig also binds to CD80/CD86 on monocytes of osteoclast precursors. However, little is known about the effect of CTLA-4-Ig on osteoclastogenesis in rheumatoid arthritis (RA). In this study we evaluated the effects of CTLA-4-Ig on osteoclast generation from human blood monocytes (PBM) and rheumatoid synovial fluid monocytes (RSFM). Highly purified monocytes were cultured with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of CTLA-4-Ig. CTLA-4-Ig inhibited RANKL-induced osteoclast generation in PBM and RSFM, as determined by tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay using osteo assay surface plates. In addition, CTLA-4-Ig reduced the gene and protein expressions of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and cathepsin K during osteoclastogenesis. Furthermore, CTLA-4-Ig significantly inhibited cell proliferation during osteoclastogenesis. Interestingly, the gene expression of indoleamine 2,3-dioxygenase-1, an inducer of apoptosis, was enhanced by CTLA-4-Ig. We next examined the effect of tumour necrosis factor (TNF)-α, a major inflammatory cytokine in rheumatoid synovium, on the expression of CD80 and CD86 by flow cytometric analysis. TNF-α potently induced the surface expression of CD80, which is known to have much higher affinity to CTLA-4-Ig than CD86, and this induction was observed at mRNA levels. Interestingly, freshly prepared rheumatoid synovial monocytes also expressed CD80 as much as TNF-α-treated PBM. Furthermore, TNF-α enhanced CTLA-4-Ig-induced inhibition of osteoclastogenesis and cell proliferation. Taken together, the TNF-α-induced CD80 may augment CTLA-4-Ig-induced inhibition of osteoclastogenesis, suggesting that CTLA-4-Ig potently inhibits osteoclast differentiation and protects bone destruction in rheumatoid inflamed joints.

[Experimental study on cryopreservation and intramuscular allografting of vertebral body and disc units].

This study investigated optimal conditions for preservation and intramuscular allografting of vertebral body and disc units. First, thirty-one vertebral body and disc units were removed from rabbits and divided into three groups: a slow freezing group, a quick freezing group, and a fresh non-freezing group. In the slow freezing group, disc units were slowly cryopreserved by decreasing temperatures from 4 to -80 degrees C using a program freezer. In the quick freezing group, they were rapidly cryopreserved by freezing to -80 degrees C without a program freezer. Second, to determine favorable duration of immersion in cryoprotectant (10% dimethyl sulfoxide), another 20 vertebral body and disc units were divided into four groups: immersed at 4 degrees C for 30, 90, 240 and 1440 minutes. After immersion, they were cryopreserved slowly using a program freezer. These disc units were thawed and examined histologically and biochemically. In the slow freezing group, both cell number in the anulus fibrosus and the value of 35SO4 incorporation in the disc were higher than those in the quick freezing group, and the disc viability of the 90-minute group was the highest. On the basis of these experiments, vertebral body and disc units were grafted into the back muscles of rabbits. To investigate the viability of the disc after allografting, another 38 specimens were grafted into the back muscles and examined after 4 or 12 weeks. The values for 35SO4 incorporation of each group at 4 weeks were about 80% of the pre-grafting level and those at 12 weeks were about 60%, and the viability of discs maintained. There were no significant differences between the slow freezing group, the quick freezing group, and the fresh non-freezing group after grafting. Further study is needed to determine a method in maintaining viability of the disc after grafting.