UNLABELLED:TNF-alpha is a major inflammatory factor that is induced in response to injury, and it contributes to the normal regulatory processes of bone resorption. The role of TNF-alpha during fracture healing was examined in wild-type and TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice. The results show that TNF-alpha plays an important regulatory role in postnatal endochondral bone formation. INTRODUCTION:TNF-alpha is a major inflammatory factor that is induced as part of the innate immune response to injury, and it contributes to the normal regulatory processes of bone resorption. METHODS:The role of TNF-alpha was examined in a model of simple closed fracture repair in wild-type and TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice. Histomorphometric measurements of the cartilage and bone and apoptotic cell counts in hypertrophic cartilage were carried out at multiple time points over 28 days of fracture healing (n = 5 animals per time point). The expression of multiple mRNAs for various cellular functions including extracellular matrix formation, bone resorption, and apoptosis were assessed (triplicate polls of mRNAs). RESULTS AND CONCLUSIONS:In the absence of TNF-alpha signaling, chondrogenic differentiation was delayed by 2-4 days but subsequently proceeded at an elevated rate. Endochondral tissue resorption was delayed 2-3 weeks in the TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice compared with the wild-type animals. Functional studies of the mechanisms underlying the delay in endochondral resorption indicated that TNF-alpha mediated both chondrocyte apoptosis and the expression of proresorptive cytokines that control endochondral tissue remodeling by osteoclasts. While the TNF-alpha receptor ablated animals show no overt developmental alterations of their skeletons, the results illustrate the primary roles that TNF-alpha function contributes to in promoting postnatal fracture repair as well as suggest that processes of skeletal tissue development and postnatal repair are controlled in part by differing mechanisms. In summary, these results show that TNF-alpha participates at several functional levels, including the recruitment of mesenchymal stem, apoptosis of hypertrophic chondrocytes, and the recruitment of osteoclasts function during the postnatal endochondral repair of fracture healing.

OBJECTIVES:To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis. METHODS:Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha. RESULTS:Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha. CONCLUSIONS:Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.

OBJECTIVE:Osteoclast differentiation factor (ODF; also known as osteoprotegerin ligand, receptor activator of nuclear factor kappaB ligand, and tumor necrosis factor-related activation-induced cytokine) is a recently described cytokine known to be critical in inducing the differentiation of cells of the monocyte/macrophage lineage into osteoclasts. The role of osteoclasts in bone erosion in rheumatoid arthritis (RA) has been demonstrated, but the exact mechanisms involved in the formation and activation of osteoclasts in RA are not known. These studies address the potential role of ODF and the bone and marrow microenvironment in the pathogenesis of osteoclast-mediated bone erosion in RA.METHODS:Tissue sections from the bone-pannus interface at sites of bone erosion were examined for the presence of osteoclast precursors by the colocalization of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K in mononuclear cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify mRNA for ODF in synovial tissues, adherent synovial fibroblasts, and activated T lymphocytes derived from patients with RA.RESULTS:Multinucleated cells expressing both TRAP and cathepsin K mRNA were identified in bone resorption lacunae in areas of pannus invasion into bone in RA patients. In addition, mononuclear cells expressing both TRAP and cathepsin K mRNA (preosteoclasts) were identified in bone marrow in and adjacent to areas of pannus invasion in RA erosions. ODF mRNA was detected by RT-PCR in whole synovial tissues from patients with RA but not in normal synovial tissues. In addition, ODF mRNA was detected in cultured adherent synovial fibroblasts and in activated T lymphocytes derived from RA synovial tissue, which were expanded by exposure to anti-CD3.CONCLUSION:TRAP-positive, cathepsin K-positive osteoclast precursor cells are identified in areas of pannus invasion into bone in RA. ODF is expressed by both synovial fibroblasts and by activated T lymphocytes derived from synovial tissues from patients with RA. These synovial cells may contribute directly to the expansion of osteoclast precursors and to the formation and activation of osteoclasts at sites of bone erosion in RA.

Chemokines, including interleukin-8 (IL-8), function as key mediators in diverse inflammatory disorders via promoting the recruitment, proliferation, and activation of vascular and immune cells. IL-8 levels are elevated in inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, osteomyelitis, and periodontal disease, that also exhibit progressive bone loss. Therefore, it is possible that IL-8 contributes to the osteopenia associated with these pathological conditions. Although macrophages, neutrophils, and endothelial cells are considered the primary sources of inflammation-induced IL-8 increases, we report here for the first time that human bone marrow-derived osteoclast-like cells (hOCL) as well as authentic bone-resorbing human osteoclasts (hOC) isolated from osteoporotic femoral heads express messenger RNA (mRNA) for IL-8 and secrete high levels of IL-8 during culture. Basal IL-8 release by cultured hOC or hOCL was orders of magnitude greater than the release of the proinflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor-alpha. At a cellular level, in situ hybridization analysis revealed that IL-8 mRNA was expressed in resorbing hOC of rheumatoid arthritic pannus and was substantially greater than that expressed in hOC of noninflammatory giant cell tumor of bone tissue. Therefore, the potential inflammation-mediated induction of IL-8 was directly assessed using cultured hOCL. IL-8 release was stimulated by proinflammatory signals (IL-1alpha, tumor necrosis factor-alpha, lipopolysaccharide, or phorbol 12-myristate 13-acetate), unaffected by various other osteotropic modulators (transforming growth factor-beta1 and -beta3, IL-6, 17beta-estradiol, or calcitonin) and was decreased by interferon-gamma, vitamin D3, and the antiinflammatory glucocorticoid dexamethasone. Changes in IL-8 secretion were paralleled by corresponding changes in IL-8 mRNA steady state levels. We conclude that hOC and hOCL synthesize and secrete high constitutive and inflammation-stimulated levels of the chemokine IL-8. Consequently, hOC-derived IL-8 could act as an important regulatory signal for bone, vascular, and immune cell recruitment and activation during normal and pathological bone remodeling.

We used clinical criteria to study skin biopsy specimens with mild to moderate photoaging taken from the face and dorsal forearms of 74 Caucasian volunteers between the ages of 30 and 50. Facial skin had a greater number of granular cell layers, a higher degree of keratinocytic atypia, and more often showed a compact stratum corneum than arm skin. Furthermore, the dermis of facial skin had a more extensive perivascular and perifollicular lymphocytic infiltrate, more perifollicular fibrosis, a greater number of mast cells and melanophages, and thinner vascular walls than forearm skin. This study demonstrated that the photoaging process is different for face and arm skin. Appreciation of these differences should permit more refined studies of photoaging and the development of more efficient therapies.