TNF-alpha modulates angiopoietin-1 expression in rheumatoid synovial fibroblasts via the NF-kappa B signalling pathway.

Angiopoietin-1 (Ang-1) is one of a family of ligands for the Tie-2 receptor which has been demonstrated to be involved in angiogenesis. Little is known about the regulation of Ang-1 gene expression. We have previously demonstrated that TNF-alpha is able to up-regulate the expression of Ang-1 mRNA in synovial fibroblasts. This present study investigated the signal transduction pathways involved in the TNF-alpha induced expression of Ang-1. TNF-alpha signals primarily through the p38, JNK, MAP kinase, and IKK pathways resulting in the activation of the transcription factors AP-1 and NF-kappa B. Experiments with inhibitors and siRNA for these various signal transduction pathways revealed that TNF-alpha stimulation of Ang-1 expression occurs via the NF-kappa B signal transduction pathway.

Production and characterization of a Tie2 agonist monoclonal antibody.

The Tie2 receptor and its known ligands, the angiopoietins, play a critical role in endothelial cell differentiation during the process of angiogenesis. Recent experimental observations indicate that the agonistic ligand, angiopoietin-1, can stimulate endothelial cell sprouting and act as a chemo-attractant in vitro and induce increased and enhanced angiogenesis both alone and in conjunction with vascular endothelial growth factor (VEGF) in vivo. Here, we present a monoclonal antibody (MAb), which binds to the extracellular portion of the Tie2 receptor and elicits similar agonist effects. Upon MAb binding to the native Tie2 receptor of cultured human umblical vein endothelial cells (HUVEC), there is a rapid increase in receptor autophosphorylation with a concomitant enhancement in the recruitment and association of the signalling intermediates Grb2 and SH-PTP2. The antibody further demonstrates functional activity in vascular tissues. In vitro, the antibody promotes the survival of cultured HUVEC and elicits a dose dependent outgrowth and branching of microvessels from cultured explants of rat aorta. When administered in vivo, the antibody enhances the vascularization of subcutaneous Matrigel implants in mice. Together these data suggest that the antibody is capable of acting as a surrogate ligand for Tie2 and further confirms the role of Tie2 in the differentiation of endothelial cells during angiogenesis.

Inhibition of CD23 processing correlates with inhibition of IL-4-stimulated IgE production in human PBL and hu-PBL-reconstituted SCID mice.

BACKGROUND: CD23, the low affinity serum immunoglobulin E (IgE) receptor, is upregulated on B cells following interleukin (IL)-4 stimulation and is concomitantly cleaved to generate soluble CD23 (sCD23) fragments with cytokine-like activity. OBJECTIVE: Compounds that selectively inhibit the proteolytic release of CD23 to generate sCD23 were assessed for their ability to inhibit IgE production in order to evaluate the contribution of sCD23 in the production of human IgE as well as the ability of such compounds to block IgE production. METHODS: IgE production was measured in IL-4-stimulated human peripheral blood lymphocytes (PBL) and PBL-reconstituted SCID mice in the presence of a broad-spectrum matrix metalloprotease (MMP) inhibitor, a compound selective for inhibition of CD23 processing over MMPs and an anti-CD23 mAb, MHM6. RESULTS: The two compounds were equipotent in inhibiting IgE production without inhibition of IgG production by IL-4/anti-CD40-stimulated PBL. Soluble CD23 release was also shown to precede IgE accumulation in the cell-free medium. Addition of compound at later times other than day 0 in the 14 day assay resulted in progressively less inhibition of both IgE and sCD23, and exactly paralleled the effect of an anti-CD23 mAb, MHM6 on IgE levels. Both compounds also inhibited the release of CD23 from human RPMI 8866 cells adoptively transferred i. p. to mice. Doses required for inhibition of CD23 correlated well with the doses required for inhibition of IgE production in IL-4-challenged hu-PBL-SCID mice. IgE was selectively inhibited over total IgG in the SCID mice as well. CONCLUSIONS: Inhibition of CD23 processing alone is sufficient to inhibit IL-4-stimulated IgE production both in vitro and in vivo.

Inhibitors of the p38 mitogen-activated kinase modulate IL-4 induction of low affinity IgE receptor (CD23) in human monocytes.

CD23, the low affinity IgE receptor, is up-regulated on the surface of IL-4-treated B cells and monocytes and is immediately proteolytically processed, releasing soluble fragments of CD23. Here, we report that inhibitors of the p38 mitogen-activated kinase (p38 MAPK), SK&F 86002 or the more selective inhibitor, SB 203580, reduce the levels of soluble CD23 formed by IL-4-stimulated human monocytes or the human monocytic cell line, U937. In contrast to compounds such as the metalloprotease inhibitor batimastat ([4-(N-hydroxyamino)-2-(R)-isobutyl-3-(S)-(2-thiophenethiomethyl)s uccinyl]-(S)-phenylalanine-N-methylamide, sodium salt), p38 MAPK inhibitors do not directly inhibit proteolytic processing of CD23. Further, evaluation of surface intact CD23 (iCD23) by flow cytometry demonstrated that SK&F 86002 and SB 203580 reduced the surface expression of iCD23 in a concentration-dependent fashion, while batimastat increased the surface expression of iCD23. The decrease in surface iCD23 was accompanied by a decrease in total cell-associated CD23 protein levels but not CD23 mRNA. IL-4 induced a late (>4-h) increase in p38 MAPK activity and corresponding activation of its substrate MAPKAPK-2. This activation was blocked by addition of SB 203580 before IL-4 induction, in parallel with the inhibition of CD23 expression. Modulation of CD23 by antibodies has been shown to alleviate the symptoms of murine collagen-induced arthritis, implicating CD23 as an important proinflammatory agent. These data show that in addition to the known cytokine inhibitory actions of SK&F 86002 and SB 203580, they also confer an additional potential anti-inflammatory activity through modulation of CD23 expression.

IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (Fc epsilonRII).

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

Induction of the lutropin/choriogonadotropin receptor in rat ovary during luteinization.

Molecular analysis of the induction of the lutropin/choriogonadotropin receptor during the process of luteinization of the rat ovary was performed. The appearance of receptor binding activity and an immunological analysis of the receptor using Triton solubilized membrane proteins show little receptor present in luteal tissue through day 3 subsequent to hCG treatment, with some in day 4, and a marked increase by day 5. A similar pattern was found in the analysis of RNA hybridizing to several probes derived from the published cDNA sequence suggesting that receptor induction occurs primarily at the level of transcription.