Safety, tolerability, pharmacokinetics, pharmacodynamics and efficacy of the monoclonal antibody ASK8007 blocking osteopontin in patients with rheumatoid arthritis: a randomised, placebo controlled, proof-of-concept study.

OBJECTIVES: Osteopontin is an extracellular matrix protein with diverse immunomodulatory functions. The authors assessed the safety, tolerability, pharmacokinetics, pharmacodynamics and initial efficacy of the humanised monoclonal antibody ASK8007, which blocks osteopontin. METHODS: In this double-blind, multicentre, combined first-in-man, single-dose escalation (phase I, part A) and proof-of-concept, multiple-dose (phase IIA, part B) study, rheumatoid arthritis (RA) patients with active disease were randomly assigned to receive ASK8007 or placebo intravenously. Safety monitoring, pharmacokinetic and pharmacodynamic analyses and clinical assessments were performed throughout the study. The expression of phenotypic cell markers was evaluated in synovial tissue biopsy samples obtained at baseline and 43 days after initiation of treatment (part B) by immunohistochemistry and digital image analysis. Two co-primary efficacy endpoints were the change from baseline in the disease activity score evaluated in 28 joints (DAS28) and the change from baseline in the number of CD68 synovial sublining macrophages, both assessed on day 43 (part B). RESULTS: ASK8007 was overall safe and well tolerated up to the highest studied dose (20 mg/kg). Quantifiable concentrations of ASK8007 were detected in synovial fluid. No differences were observed for changes from baseline in DAS28 and CD68 sublining macrophages between ASK8007 and placebo-treated patients. Within the ASK8007 treatment group, there were also no apparent clinical responses or changes in sublining macrophages. In addition, ASK8007 treatment did not change other assessed biomarkers. CONCLUSIONS: Osteopontin blockade is well tolerated and not related to safety concerns. These results consistently show that osteopontin blockade is unlikely to induce robust clinical improvement in RA patients.

Retinal pigment epithelium-immune system interactions: cytokine production and cytokine-induced changes.

Vision is dependent on proper function of several intraocular structures. Immune responses to eliminate invading pathogens from the eye may threat vision by causing damage to these structures. Therefore, immunological defence of the eye should be carefully balanced between efficacy and maintenance of functional integrity. The eye is equipped with several regulatory mechanisms to prevent certain immune and inflammatory responses and is, therefore, regarded as an immune privileged site. The retinal pigment epithelium (RPE) contributes to the immune privileged status of the eye as part of the blood-eye barrier and by the secretion of immunosuppressive factors inside the eye. RPE cells, however, may also play an important role in the development of immune and inflammatory responses in the posterior part of the eye. During the last decade it has become clear that RPE cells are highly sensitive to a variety of inflammatory cytokines. Under inflammatory conditions, RPE cells produce a myriad of cytokines that may activate the resident ocular cells or attract and activate leukocytes. Cytokine stimulation of RPE cells causes profound effects, including nitric oxide secretion, cell surface expression of MHC class II and adhesion molecules and abrogation of barrier function. This article provides a comprehensive review of the literature concerning RPE cells and cytokines.

Domains of rat interleukin 1 beta involved in type I receptor binding.

In the present study the inhibitory effects of a panel of 21 monoclonal antibodies (moabs) to rat interleukin 1 beta (rIL-1 beta) on the binding of 125I-labeled rIL-1 beta to murine type I IL-1 receptors on EL4 cells were investigated. Furthermore, the epitopes of these moabs were determined by the use of the pepscan technique, and these epitopes were visualized on a three-dimensional model of rIL-1 beta. Some moabs (SILK 3, 4, 5, 6, and 22) inhibited receptor binding of radioiodinated rIL-1 beta at concentrations that are similar to the dissociation constant values of antibody-rIL-1 beta binding. Another group of moabs (SILK 7, 11, 20, 21, and 23) also inhibited receptor binding but only at concentrations that are 10-150 times higher than their dissociation constants. A large group of moabs did not affect receptor binding in the concentration range tested, and two moabs enhanced the binding of rIL-1 beta to type I receptors. The result of pepscan analysis shows that the moabs bound to one or more of the amino acid sequences 35-49, 66-85, 78-97, 106-124, and 123-143 of mature rIL-1 beta. Modeling of rIL-1 beta shows that the binding domains of SILK 4, 5, 6, and 22 (sequence 123-143) is located at the closed end of the molecule, indicating that this part of rIL-1 beta harbors domains that are crucial for type I receptor binding. The binding domain of SILK 3 (sequence 66-85) is also located at this end of the molecule. In contrast, the binding domains of SILK 7, 11, 20, 21, and 23 (sequence 78-97) are located at the open end of the molecule, which is at the same face as the amino- and carboxy-terminals. The binding domain of SILK 16 (sequence 106-124) is positioned at the center of the molecule. It is concluded that the closed end of rIL-1 beta contains sequences that are crucial for its binding to type I receptors on murine EL4 cells. Because of the high concentrations of antibodies to residues 78-97 of rIL-1 beta that are needed to interfere with receptor binding, the importance of these domains in binding to type I receptors remains uncertain.

Circulating tumor necrosis factor-alpha does not mediate endotoxin-induced hypothermia in rats.

The present study was designed to investigate the role of macrophages and circulating tumor necrosis factor-alpha (TNF-alpha) in the endotoxin-induced hypothermic responses in rats. Intravenous as well as intraperitoneal administration of endotoxin to male Wistar rats (0.5 mg/kg) led to increased plasma TNF-alpha concentrations and a transient hypothermia, which reached its nadir after 90 min. The hypothermia and plasma TNF-alpha responses to endotoxin were abolished after elimination of peripheral macrophages. Seven days after the first challenge, tolerance of the hypothermic response was found if the same dose was administered intraperitoneally but not if it was administered intravenously. Tolerance of the TNF-alpha response was induced irrespective of the route of endotoxin administration. We hypothesize that, after intravenous administration of endotoxin, macrophage-dependent and -independent mechanisms are activated, whereas the hypothermic response to intraperitoneal endotoxin involves primarily macrophage-dependent mechanisms. These mechanisms may relate to the prime targets reached by endotoxin, such as macrophages and endothelial cells. Because the development of tolerance of the hypothermic response is dependent on the route of endotoxin administration, whereas that of the plasma TNF-alpha response is not, we conclude that circulating TNF-alpha is not the macrophage-derived cryogenic signal that triggers the hypothermic response.

Analysis of the secretion pattern of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta 2 (TGF-beta2) by human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells, situated between the neurosensory retina and the vascularized choroid, form part of the blood-eye barrier and are important for homeostasis of the outer retina. These cells are able to produce a variety of cytokines which may play a role in the maintenance of the immunosuppressive milieu inside the eye and in intraocular inflammatory responses. In the present study, we investigated whether RPE cells secreted the anti-inflammatory cytokine TGF-beta2 and the proinflammatory cytokine MCP-1 in a polarized manner. Monolayers of human donor RPE cells were cultured on transwell filters. Secretion of TGF-beta2 and MCP-1 at either the apical or basal side of the RPE cell monolayers, that were not treated or stimulated with IL-1beta (200 U/ml), was analysed by ELISA. All three cell lines examined had a different TGF-beta2 secretion pattern. In two of the three donor RPE cell lines tested, TGF-beta2 secretion was polarized, but not in the same direction. TGF-beta2 secretion was not up-regulated by stimulation with IL-1beta. In contrast, IL-1beta strongly induced MCP-1 secretion preferentially into the basal compartment of all RPE monolayers tested. These data indicate that human RPE cells are able to secrete TGF-beta2 and MCP-1 in a polarized fashion. Our results suggest that MCP-1 can be secreted by RPE cells in the direction of choroidal vessels during inflammatory responses in the posterior part of the eye, which may limit damage to the neurosensory retina.

Expression of multiple forms of IL-1 receptor antagonist (IL-1ra) by human retinal pigment epithelial cells: identification of a new IL-1ra exon.

The eye is considered an immunologically privileged organ and is separated from the rest of the body by blood-ocular barriers. Part of the blood-retina barrier consists of the retinal pigment epithelium (RPE). In addition to the physical barrier which the monolayer of RPE cells forms, these cells contribute to ocular immune privilege by producing anti-inflammatory molecules that down-regulate potential damaging immune reactions. In this study the mRNA expression of IL-1 receptor antagonist (IL-1ra) by RPE cells was studied in 15 donor-derived cell lines. Expression of both the intracellular and secreted IL-1ra was detected in unstimulated and IL-1beta- or phorbol 12-myristate 13-acetate-exposed RPE. Analysis of IL-1ra protein in RPE cell lysates and cell culture supernatants indicated that these cells produce mainly intracellular IL-1ra. No correlation between IL-1ra expression levels and the IL-1ra gene polymorphism could be detected. In addition to the two known intracellular IL-1ra variants (intracellular IL-1ra type I and type II) evidence is provided for the expression of a hitherto unknown splice variant of the IL-1ra mRNA by RPE cells. Expression was not confined to RPE cells and could also be detected in cultured human fibroblasts and macrophages. This variant, which we have tentatively named intracellular IL-1ra type III, encodes a C-terminally truncated protein of only 27 amino acids.

Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells.

A number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood-retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE-19 cell line) cultured on transwell filters were stimulated to produce IL-6 and IL-8 by adding IL-1beta (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL-6 and IL-8 towards the basal side, irrespective of the side of stimulation. The ARPE- 19 cell line also secreted IL-6 and IL-8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL-6 and IL-8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.

Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation.

The retinal pigment epithelium (RPE) maintains the choriocapillaris (CC) in the normal eye and is involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Vascular endothelial growth factor-A (VEGF) is produced by differentiated human RPE cells in vitro and in vivo and may be involved in paracrine signaling between the RPE and the CC. We investigated whether there is a polarized secretion of VEGF by RPE cells in vitro. Also, the localization of VEGF receptors in the human retina was investigated. We observed that highly differentiated human RPE cells, cultured on transwell filters in normoxic conditions, produced two- to sevenfold more VEGF toward their basolateral side as compared to the apical side. In hypoxic conditions, VEGF-A secretion increased to the basal side only, resulting in a three- to 10-fold higher basolateral secretion. By immunohistochemistry in 30 human eyes and in two cynomolgus monkey eyes, KDR (VEGFR-2) and flt-4 (VEGFR-3) were preferentially localized at the side of the CC endothelium facing the RPE cell layer, whereas flt-1 (VEGFR-1) was found on the inner CC and on other choroidal vessels. Our results indicate that RPE secretes VEGF toward its basal side where its receptor KDR is located on the adjacent CC endothelium, suggesting a role of VEGF in a paracrine relation, possibly in cooperation with flt-4 and its ligand. This can explain the known trophic function of the RPE in the maintenance of the CC and its fenestrated permeable phenotype and points to a role for VEGF in normal eye functioning. Up-regulated basolateral VEGF secretion by RPE in hypoxia or loss of polarity of VEGF production may play a role in the pathogenesis of choroidal neovascularization.