Blood monocytes in rheumatoid arthritis are highly adherent to cultured endothelium.

Monocytes from 17 patients with rheumatoid arthritis (RA) were more adherent than monocytes from 17 control patients to monolayers of pig aortic endothelium irrespective of whether sera was included (median 27-34% increase; p = 0.002) or omitted (median 27% increase; p = 0.022) from the culture media. When human umbilical vein endothelial cells were used as the adherence substrate, rheumatoid monocytes from an additional 21 patients demonstrated a median 31% (p = 0.004) and 20% increase (p = 0.004) in adhesion when compared with monocytes from 21 normal healthy subjects in the absence and presence of autologous sera, respectively. Activation of control monocytes with muramyl dipeptide or treatment with RA sera increased their attachment to endothelium (mean 34 +/- 14% increase; p < 0.001). The expression of the adhesion molecules CD11b (p < 0.005), CD18 (p < 0.005), CD62L (p = 0.01) was enhanced on rheumatoid monocytes, but antibody-blocking studies suggested that CD18 and CD62L were not responsible for the augmented binding of the rheumatoid cells. A subpopulation of rheumatoid monocytes possessed a very low net negative surface charge, a property that favours binding to vessel walls. We propose that many rheumatoid monocytes are predisposed for sheer-resistant adhesion to vascular endothelium.

Persistent measles virus infection of the intestine: confirmation by immunogold electron microscopy.

This study sought to investigate persistent measles virus infection of the intestine: a novel protocol for immunogold electron microscopy was developed using a polyclonal anti-measles nucleoprotein antibody on reprocessed, formalin fixed paraffin wax embedded tissue sections. Antibody binding was detected using both immunoperoxidase and light microscopy on tissue sections, and 10 nm gold conjugated secondary antibody and electron microscopy on ultrathin sections. The techniques were validated using both measles infected vero cells and human tissues with established measles infection: these included brain affected by subacute sclerosing panencephalitis and acute measles appendicitis. The technique was applied subsequently to six untreated cases of granulomatous Crohn's disease, and two cases of ileocaecal tuberculosis, a granulomatous control. Mumps primary antibody--applied to both mumps infected vero cells, and measles infected vero cells and tissues studied by immunoperoxidase, and measles antibody on mumps infected cells studied by immunoperoxidase and immunogold--were used as specificity controls: the primary antibodies identified their respective target antigen and there was no antibody cross reactivity. Measles virus nucleocapsids labelled with gold conjugated antibody in both infected cells and tissues, including foci of granulomatous inflammation in five of six cases of Crohn's disease: in the fifth case, the granuloma could not be identified in ultrathin section. In one of the tuberculosis cases, a low level of signal was noted while the second case was negative. Labelling adopted a characteristic pattern in all infected tissues, strengthening the specificity of these findings. This study provides the first direct confirmation of persistent measles virus infection of the intestine.

Procoagulant and prothrombotic responses of human endothelium to indomethacin and endotoxin in vitro. Relevance to non-steroidal anti-inflammatory drug enteropathy.

BACKGROUND: In view of the association between non-steroidal anti-inflammatory drugs (NSAIDs) and microvascular injury in the intestine, this study investigated the procoagulant changes in cultured human umbilical vein endothelial cells (HUVEC) when exposed to indomethacin either alone or in the presence of bacterial lipopolysaccharide (LPS). METHODS: Confluent HUVEC cultures were cultured for 1, 6, 12, and 24 h in the presence of LPS (10 micrograms/ml) with or without indomethacin (1-100 microM). After incubation, supernatants were analysed for 6-keto-prostaglandin (PG) F1 alpha and PGE2 content, whereas cells were freeze-fractured and assayed in a one-stage clotting assay for the expression of procoagulant activity (PCA). RESULTS: LPS induced a significant expression of PCA at 6, 12, and 24 h, with a significantly increased production of 6-keto-PGF1 alpha, whereas the increased PGE2 production was much less pronounced. Indomethacin alone induced a time-dependent PCA response; when coincubated with LPS the PCA response was greater than that produced by either indomethacin or LPS alone. Indomethacin totally abolished the synthesis of antithrombotic eicosanoids. CONCLUSION: Indomethacin induces PCA in HUVEC and augments LPS-induced PCA, while it abolishes the antithrombotic prostanoid response in these cells. These observations may be relevant to the microvascular injury and thrombosis observed in NSAID enteropathy.

Measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro.

Measles virus infection of microvascular endothelium in vivo and ensuing endothelial cell activation may be important in the pathogenesis of subsequent inflammation in target organs. This study investigated the capacity of measles virus to induce procoagulant activity, in vitro, in endothelial cells isolated from human umbilical cord veins. Endothelial cells were infected with a clinical isolate of measles virus propagated in Vero cells. Cells were also incubated with bacterial lipopolysaccharide (10 micrograms/ml), herpes simplex virus type 1, cytomegalovirus or culture medium alone as positive and negative controls, respectively. Endothelial cell procoagulant activity was measured in a one-stage clotting assay. Measles virus stimulated both a time and dose-dependent endothelial cell procoagulant response by the induction of tissue factor synthesis, confirmed by both immunocytochemistry and its dependence on factor VII for activity. This activity was reduced by u.v.-irradiation of the virus. Infected cells were analysed by double immunofluorescent staining for both tissue factor and measles virus N-protein, and examined using confocal scanning laser microscopy. Cells expressing tissue factor were also positive for the measles virus N-protein. Low levels of interleukin-1 were detected in some viral inocula derived from measles virus-infected Vero cells, however neutralising antibody to interleukin-1 failed to inhibit the endothelial cell procoagulant response to measles virus, whereas it significantly reduced procoagulant activity induced in endothelial cells by recombinant interleukin-1. The capacity of measles virus to induce endothelial tissue factor in vitro, may be relevant to the thrombotic vasculopathy associated with measles virus infection in vivo.

Application of an immortalized human endothelial cell line to the leucocyte:endothelial adherence assay.

This report shows that an immortalized endothelial cell line (EA.hy 926) is able to substitute for secondary cultures of human umbilical vein endothelial cells (HUVEC) in the leucocyte:endothelial adherence assay. Enriched preparations of blood polymorphonuclear leucocytes, monocytes and resting and activated lymphocytes exhibited similar adherence characteristics to HUVEC and the EA.hy 926 cells. Cytokines such as tumour necrosis factor (TNF) act on endothelial cells to increase their adhesiveness for leucocytes and in this study there was no difference between TNF-treated HUVEC and EA.hy 926 cells in supporting the enhanced binding of leucocytes. The adherence promoting effect of TNF-treated EA.hy 926 cells appears to be dependent upon their endothelial properties since TNF treatment of A549 cells, the permanent human cell line used to generate the hybrid EA.hy 926 cells did not augment lymphocyte attachment. Monoclonal antibodies against CD11a and CD18 inhibited the binding of lymphocytes to untreated and TNF-treated HUVEC and EA.hy 926 cells and ICAM-1 expression was increased on both monolayers following treatment with TNF. The availability of a hybrid endothelial cell line whose adhesive properties are similar to those of recently isolated endothelial cells should benefit the study of factors that govern leucocyte-endothelial cell interactions and be advantageous to the longitudinal investigation of leucocyte adherence under static conditions.

Abnormal binding properties of blood monocytes in rheumatoid arthritis.

Blood monocytes from patients with RA exhibited a greater binding to monolayers of umbilical cord vein endothelium than monocytes from control subjects (mean 42% increase; p < 0.01). When control monocytes were added to TNF or IL-1 treated endothelium their adhesion was enhanced (mean 24% increase; p < 0.05), whereas the number of monocytes from RA patients binding to TNF or IL-1 treated monolayers was less than that adhering to untreated endothelial cells (mean 22% inhibition; p < 0.02). The surface expression of CD11b/CD18 on RA monocytes was increased and pretreatment of normal and RA cells with an anti-CD18 monoclonal antibody inhibited their attachment to untreated and cytokine-treated endothelial cells. Normal blood monocytes activated with LPS demonstrated an enhanced binding to untreated cultures (mean 23% increase; p < 0.05) and an inhibited attachment to cytokine-treated endothelial cells. This study suggests that blood monocytes in RA may be activated and that this property modifies the attachment of these cells to normal and "inflammatory" endothelium.