Evidence for oxidised low density lipoprotein in synovial fluid from rheumatoid arthritis patients.

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.

Activation of the transcription factor nuclear factor-kappaB in human inflamed synovial tissue.

OBJECTIVE: The transcription factor nuclear factor kappaB (NF-kappaB) has been implicated in the inflammatory response and is known to be activated by a process involving reactive oxygen intermediates. The purpose of the present study was to demonstrate the presence and distribution of activated NF-kappaB in synovium samples from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and from autopsy subjects with no known history of arthritis. METHODS: Immunohistochemical staining was performed using both polyclonal and monoclonal "activity-specific" antibodies to the Rel-A (p65) subunit of NF-kappaB (anti-Rel-A nuclear location sequences). Histologic features of inflammation were also scored. RESULTS: Both antibodies demonstrated positive staining of synovial tissue, with a cellular distribution that was nuclear. The staining was associated with specific cell types within the tissue, in particular, type A synoviocytes and vascular endothelium. Notably, lymphoid aggregates were unstained. Using the monoclonal antibody, a further study was carried out to investigate the distribution of staining in tissues from patients with different disease activities and clinical diagnoses, as well as in normal control tissue obtained at autopsy. Patients with acute RA more commonly showed vessel staining (P = 0.05) and, conversely, showed less frequent staining of the synovial lining (P < 0.005) compared with OA patients. Synovial tissue from controls exhibited either no staining or only weak staining in the synovial lining. CONCLUSION: The activation of NF-kappaB in vascular endothelium and type A synovial lining cells is a feature of synovial tissue from both RA and OA patients. The distribution of this staining appears to be related to the clinical diagnosis.

Copper-induced LDL peroxidation investigated by 1H-NMR spectroscopy.

Oxidatively modified LDL (oLDL) is thought to play a key role in the pathogenesis of atherosclerosis. We have studied Cu(2+)-induced peroxidation reactions of LDL and have elucidated the sequence of events which subsequently occur within LDL particles by 1H-NMR spectroscopy. Studies of chloroform/methanol extracts show that LDL arachidonate is oxidised by Cu2+ at a higher rate and to a greater extent than linoleate, giving isomeric hydroperoxides with predominantly trans,trans double-bonds, whilst only cis,trans isomers were detected as intrinsic hydroperoxides in control LDL samples. These intrinsic hydroperoxides were not degraded during peroxidation, suggesting that they are not involved in the initiation of Cu(2+)-induced peroxidation. Aldehydes arising from the decomposition of hydroperoxides were also detected, as well as saturated fatty acids which were released into the external aqueous medium. Decomposition pathways of the two major isomeric hydroperoxides are discussed. Cu(2+)-induced oxidation of LDL cholesterol appears to occur only after hydroperoxide breakdown, with esterified cholesterol being oxidised to a greater extent than free cholesterol. Phospholipid hydrolysis appeared to parallel the peroxidation of arachidonic acid, and the released lysophosphatidylcholine may become associated with apoB. These results suggest that hydroperoxide breakdown (probably in phospholipids) may be a key event in the peroxidation process, leading to the oxidation of cholesterol and propagation into the core of LDL.

Presence of foam cells containing oxidised low density lipoprotein in the synovial membrane from patients with rheumatoid arthritis.

OBJECTIVE: Increased concentrations of lipid peroxidation products have been described in the serum and synovial fluid from patients with rheumatoid arthritis. A large proportion of the unsaturated lipids in human extracellular fluids is a component of low density lipoprotein (LDL). The oxidative modification of LDL, and its subsequent uptake by macrophages, has been implicated in the pathogenesis of atherosclerosis, but not of rheumatoid arthritis. This study aimed to assess whether oxidatively modified LDL was present in the rheumatoid synovium. METHODS: A polyclonal antiserum raised in rabbits against oxidised LDL (o-LDL) was used to perform an immunohistochemical study of a series of synovial biopsy specimens from patients with rheumatoid arthritis. RESULTS: Collections of positively stained macrophages, arranged in a linear fashion and with the morphological characteristics of foam cells--that is, 'fatty streaks', were identified around blood vessels within the intimal connective tissue. In addition, scattered, positively stained foam cells were present in association with deposits of fibrin. These staining patterns were absent from control synovial membranes (traumatic knee injuries). CONCLUSIONS: The findings in all rheumatoid patients studied suggest that atherosclerosis and rheumatoid arthritis have analogous pathogenetic features.

Mechanical properties of HL60 cells: role of stimulation and differentiation in retention in capillary-sized pores.

Neutrophil sequestration in pulmonary capillaries occurs prior to the development of lung injury, but the mechanisms by which neutrophils are retained are unclear. We hypothesized that decreases in cell deformability, in the absence of an increase in cell surface adhesive properties, would be sufficient to cause cell retention in a filtration apparatus modeling the pulmonary microvasculature. The myelomonocytic cell line (HL60 cell line) was used to test the hypothesis since these cells were unable to increase adherence in response to n-formylmethionylleucylphenylalanine (FMLP) in either the undifferentiated state or when differentiated towards granulocytes. With differentiation, HL60 cell volume decreased, and f-actin organization changed from a thick cortical rim with focal areas of f-actin in undifferentiated cells to a thin rim in differentiated cells. Differentiated cells responded to FMLP by reorganizing f-actin and increasing stiffness. Undifferentiated cells did not exhibit changes in f-actin with stimulation, were stiffer than differentiated cells, and did not increase stiffness in response to FMLP. Cytochalasin D (CD), which disrupted the cytoarchitecture as assessed by confocal microscopy but did not affect cell volume or adherence, decreased the stiffness of undifferentiated and FMLP-stimulated differentiated cells, thus suggesting the importance of microfilament organization in the stiffness of these cells. Filtration of cells through 8-microns pores showed that undifferentiated cells were markedly retained and did not exhibit any further retention with FMLP. Differentiated cells exposed to FMLP exhibited a concentration-dependent increase in retention in 8-microns pores that was abolished by CD. In addition, CD reduced retention of undifferentiated cells, indicating that microfilament organization is an important factor in determining a cell's rheologic properties. In conclusion, FMLP-stimulated microfilament reorganization, which increased cell stiffness, was sufficient in the absence of adherence factors to cause cell retention in a filtration system. This lends support to the hypothesis that decreases in cell deformability contribute to neutrophil retention in the pulmonary microvasculature.