Objective To determine whether elevated levels of the angiogenic cytokine vascular endothelial growth factor (VEGF), detected on presentation to an early arthritis clinic, are associated with the development of chronic and erosive arthritis. Methods Concentrations of VEGF and its soluble receptor, soluble fms‐like tyrosine kinase 1 (sFlt‐1), were measured by enzyme‐linked immunosorbent assay in serum samples from patients with early (<2 years from onset) arthritic symptoms in the peripheral joints, namely early rheumatoid arthritis (RA), self‐limiting arthritis (viral, reactive, and idiopathic inflammatory arthritis), or psoriatic arthritis. In addition, measurements were made in random samples from patients with longstanding (>3 years from symptom onset) RA treated with disease‐modifying antirheumatic drugs, from patients with osteoarthritis (OA), and from patients with polyarthralgia without arthritis, as well as from nonarthritic controls. Results Serum VEGF levels at presentation were elevated in patients with inflammatory arthritis (RA, psoriatic, and self‐limiting arthritis) as well as in patients with OA, in comparison with nonarthritic controls. Moreover, serum VEGF concentrations were significantly higher in patients with early RA than in patients with self‐limiting arthritis. Serum VEGF levels at presentation in patients with early RA correlated significantly with the development of radiographic damage after 1 year. Improvement in the clinical symptoms of RA was associated with a reduction in serum VEGF levels. Serum sFlt‐1 levels were raised in patients with early and longstanding RA and in those with self‐limiting arthritis, and correlated positively with the serum VEGF concentrations in patients with inflammatory arthritis. Conclusion These findings implicate the proangiogenic cytokine VEGF in the persistence of inflammatory arthritis, and support the hypothesis that expansion of the synovial vasculature is important for the development of joint destruction in RA.

Human interleukin-4 possesses two distinct sites for receptor activation. A signalling site, comprising residues near the C-terminus on helix D, determines the efficacy of interleukin-4 signal transduction without affecting the binding to the interleukin-4 receptor a subunit. A complete antagonist and a series of low-efficacy agonist variants of human interleukin-4 could be generated by introducing combinations of two or three negatively charged aspartic acid residues in this site at positions 121, 124, and 125. One of the double variants, designated [R121D,Y124D]interleukin-4, with replacements of both Argl21 and Tyrl24 by aspartic acid residues was completely inactive in all analysed cellular responses. The loss of efficacy in [R121D,Y 124Dlinterleukin-4 is estimated to be larger than 2000-fold. Variant [R121D,Y124D]interleukin-4 was also a perfect antagonist for inhibition of interleukin-13-dependent responses in B-cells and the TF-1 cell line with a K, value of approximately 100 pM. In addition, inhibition of both interleukin-4-induced and interleulun-13-induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin-4Re,, the extracellular domain of the interleukin-4 receptor a subunit. These results indicate that efficient interleukin-4 antagonists can be designed on the basis of a sequential two-step activation model. In addition, the experiments indicate the functional participation of the interleukin-4 receptor a subunit in the interleukin-13 receptor system.Human interleukin-4 (IL-4) activates its cognate receptor system by sequential binding to two different receptor subunits. The first high-affinity binding step (& 100 pM) involves the interaction between IL-4 receptor a subunit (Beckmann et al., 1992;Galizzi et al., 1990;Idzerda et al., 1990) and IL-4 amino acid residues located on helices A and C (Kruse et al., 1993;Ramanathan et al., 1993). The subsequent second binding step involves a signalling site on helix D of IL-4 (Kruse et al., 1992) and a further receptor subunit, which according to recent evidence (Kondo et al., 1993;Russell et al., 1993) is probably the y chain of the IL-2 receptor system, now designated yc.Such a sequential two-step binding mechanism leading to receptor activation suggests a rationale for the design of antagonistic cytokine variants (Cunningham et al., 1991 ;De Vos et al., 1992). If the first high-affinity binding site is retained and the second signalling site is either destroyed by (Fuh et al., 1992;Kruse et al., 1992) or blocked by monoclonal antibodies (Reusch et al., 1994), the oligomerisation process stops at the level of the unproductive 1 : 1 complex between the cytokine and the first receptor subunit. The signalling site of human IL-4 comprises side chains of Argl21, Tyrl24 and Ser125. Previous results have shown that variants [R121D]IL-4 and [S225D]IL-4 have approximately 30% partial agonist activity and variant [Y124D]IL-4 has less than 1 % partial agonist activity for T-cell proliferation compared to wild-type IL-4. High-affinit...

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGFstimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

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The transmembrane tyrosine kinase TIE-2, the receptor for the angiopoietins-1 and -2, has been shown to be involved in angiogenic processes. Investigating the regulation of TIE-2 expression on endothelial cells, we found that stimulators such as PMA induce a decrease of TIE-2 protein from the cell surface without affecting TIE-2 mRNA. In conditioned media of PMA stimulated endothelial cells, a soluble form of this receptor comprising parts of the extracellular domain can be detected. Using a sandwich ELISA, we were able to detect and quantify TIE-2 receptors in cell lysates (representing the whole transmembrane receptor) and in cell culture supernatants (representing a soluble form of this receptor, sTIE-2). Several factors influencing the shedding process e.g. basic FGF could be identified. Finally, the soluble form of TIE-2 could also be detected in human biological fluids such as sera and plasma from healthy controls.