Ewa Paleolog

Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor alpha blockade in patients with rheumatoid arthritis.

OBJECTIVE To verify the hypothesis that in rheumatoid arthritis (RA), tumor necrosis factor alpha (TNFalpha) plays a critical role in regulating leukocyte trafficking and chemokine levels. METHODS Ten patients with longstanding RA received a single 10 mg/kg infusion of anti-TNFalpha monoclonal antibody (cA2). The articular localization of autologous granulocytes, separated in vitro and labeled with 111In, was studied by analysis of gamma-camera images both before and 2 weeks after treatment. At the same sequential time points, synovial biopsy samples were assessed for infiltrating CD3+ T cells, CD22+ B cells, and CD68+ macrophages. Synovial tissue expression of the chemokines interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, Groalpha, and RANTES was also determined. Serum IL-8 and MCP-1 concentrations were measured by enzyme-linked immunosorbent assay. RESULTS Anti-TNFalpha therapy in RA significantly reduced 111In-labeled granulocyte migration into affected joints. There was a simultaneous and significant reduction in the numbers of infiltrating synovial CD3+ T cells, CD22+ B cells, and CD68+ macrophages and in the expression of IL-8 and MCP-1, with a trend toward a reduction in serum concentrations of these chemokines. CONCLUSION TNFalpha blockade reduces synovial expression of the chemokines IL-8 and MCP-1 and diminishes inflammatory cell migration into RA joints.

Modulation of angiogenic vascular endothelial growth factor by tumor necrosis factor alpha and interleukin-1 in rheumatoid arthritis.

OBJECTIVE To investigate the regulation of expression of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA), in order to determine whether new blood vessel formation could be a potential therapeutic target in RA. METHODS Dissociated RA synovial membrane cells were cultured in the presence of cytokine inhibitors, or under hypoxic conditions. Serum VEGF levels were serially measured in RA patients enrolled in clinical trials of anti-tumor necrosis factor alpha (anti-TNFalpha) monoclonal antibody treatment. RESULTS Combined neutralization of TNFalpha and interleukin-1 (IL-1) in RA synovial membrane cultures reduced VEGF release by 45% (P < 0.05 versus control), although blockade of either TNFalpha or IL-1 activities alone resulted in only small inhibitory effects. In addition, release of VEGF from RA synovial membrane cells was selectively up-regulated by hypoxia. Serum VEGF levels were significantly elevated in RA patients relative to control subjects, and correlated with disease activity. Treatment of RA patients with anti-TNFalpha significantly decreased serum VEGF, and this effect was enhanced by cotreatment with methotrexate. CONCLUSION Inhibition of TNFalpha and IL-1 activity in vivo could reduce the drive to new blood vessel formation, and hence pannus mass, adding to other therapeutic effects of anti-TNFalpha therapy in RA.

Raised serum vascular endothelial growth factor levels are associated with destructive change in inflammatory arthritis

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.

Monoclonal anti-TNF-alpha antibody as a probe of pathogenesis and therapy of rheumatoid disease

Rheumatoid arthritis is a common cause of chronic disability for which current therapies are of limited value in controlling the disease process and outcome. Our initial approach to understanding the pathogenesis of RA and defining a novel therapeutic target was to investigate the role of cytokines by blocking their action with antibodies on cultured synovial-derived mononuclear cells in vitro. These investigations suggested that neutralization of TNF alpha with antibodies significantly inhibited the generation of other pro-inflammatory cytokines also over-produced, such as, IL-1, GM-CSF, IL-6 and IL-8. The implication that blockade of a single cytokine, TNF alpha might have far-reaching effects on multiple cytokines and thereby exert significant anti-inflammatory and protective effects on cartilage and bone of joints, was tested in arthritic DBA/1 mice immunized with collagen II. Impressive amelioration of joint swelling and joint erosions in this model encouraged clinical trials with a monoclonal anti-TNF alpha antibody. The cA2 chimeric anti-TNF alpha high-affinity antibody was initially tested in an open-label study at a dose of 20 mg/kg on 20 patients, with substantial and universal benefit. Subsequently, a randomized placebo-controlled double-blind trial was performed on 73 patients comparing a single intravenous injection of placebo (0.1% human serum albumin) with two doses of cA2. Using a composite disease activity index, at 4 weeks post infusion, 8% of patients receiving placebo improved compared with 44% receiving 1 mg/kg cA/2 and 79% receiving 10 mg/kg. Between 2 to 4 repeated cycles of cA2 were administered to 7 patients and all patients showed improvement of a similar magnitude with each cycle. These data support our proposition that TNF alpha is implicated in the pathogenesis of RA, and is thus a key therapeutic target. Monoclonal anti-TNF alpha antibodies control disease flares and are candidate agents for longer-term control of RA, although repeated therapy with cA2 is associated with anti-idiotypic responses in 50% of patients and a trend toward shortening of the duration of response. In the DBA/1 arthritic mice, synergy of action of anti-TNF and anti-CD4 is observed together with suppression of an anti-globulin response, indicating one way in which benefit might be augmented in the future.

Human neutrophils secrete vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil‐derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation. J. Leukoc. Biol. 62: 397–400; 1997.

VEGF expression in human macrophages is NF-kappaB-dependent: studies using adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the IkappaB kinase 2.

Vascular endothelial growth factor (VEGF) is the most endothelial cell-specific angiogenic factor characterised to date, and it is produced by a variety of cell types. In macrophages, VEGF has been shown to be upregulated by the inflammatory mediator lipopolysaccharide (LPS) and by engagement of CD40 by CD40 ligand (CD40L). Because LPS and CD40L activate nuclear factor-κB (NF-κB) in monocytes, we investigated in this study whether VEGF production in macrophages, when stimulated with either LPS or CD40L, is NF-κB-dependent. We used adenoviral constructs over-expressing either IκBα (AdvIκBα), the endogenous inhibitor of NF-κB, or a kinase-defective mutant of IKK-2 (AdvIKK-2dn), an upstream activator of IκBα, to infect normal human monocyte-derived macrophages. We observed that LPS-induced production of VEGF in human macrophages was almost completely inhibited (>90%) following adenoviral transfer of IκBα. In addition, we observed significant inhibition of the CD40L-induced VEGF production in macrophages following infection with AdvIκBα. Expression of IKK-2dn in macrophages decreased VEGF production in response to LPS or CD40L by approximately 50%, suggesting that in addition to IKK-2, other kinases might be involved in NF-κB activation. These results show for the first time that VEGF production in human macrophages is NF-κB dependent. NF-κB regulates many of the genes involved in immune and inflammatory responses, and our study adds the angiogenic cytokine VEGF to the list of NF-κB-dependent cytokines.

Hypoxia--a key regulator of angiogenesis and inflammation in rheumatoid arthritis.

The importance of inflammation in rheumatoid arthritis (RA) is well understood. This knowledge has resulted in the development of anti-inflammatory therapies—either broadly acting (such as steroids) or more specific approaches (such as antibodies against TNF)—with biologic therapies (including TNF inhibitors) revolutionizing the treatment of RA. However, what is less well appreciated in RA are the links between inflammation, blood-vessel formation (angiogenesis) and cellular responses to changes in oxygen tension. Inadequate oxygenation, termed hypoxia, is thought to drive the increase in synovial angiogenesis that occurs in RA, through expression of hypoxia-inducible molecules, including vascular endothelial growth factor (VEGF). This process promotes further infiltration of inflammatory cells and production of inflammatory mediators, perpetuating synovitis. This Review highlights the molecular pathways activated by hypoxia, and how these pathways might interact with inflammatory signaling to promote and maintain synovitis in RA, with a particular focus on the response of macrophages to hypoxia in the context of RA. Successful treatment of RA, for example with anti-TNF antibodies, reduces levels of proangiogenic factors, including VEGF, and leads to normalization of the vasculature. These processes emphasise the close links between hypoxia, angiogenesis and inflammation in this disease and supports the concept that angiogenesis blockade could be of therapeutic benefit in RA.

Treatment with soluble VEGF receptor reduces disease severity in murine collagen-induced arthritis.

Maintenance of the invasive pannus in rheumatoid arthritis is an integral part of disease progression. The synovial vasculature plays an important role in the delivery of nutrients, oxygen, and inflammatory cells to the synovium. Vascular endothelial growth factor (VEGF), an endothelial mitogen expressed by cells within the synovial membrane, is thought to contribute to the formation of synovial blood vessels. Our objective in this study was to measure the kinetics of VEGF production in a murine model of collagen-induced arthritis and to determine whether VEGF blockade reduces disease progression. Synovial cells isolated from the knee joints of naive or sham-immunized mice, or from mice immunized with collagen but without arthritis, released little or no detectable VEGF. Onset of arthritis was associated with expression of VEGF mRNA and protein. The levels of VEGF secreted by synovial cells isolated from the joints of mice with severe arthritis were significantly higher than from mice with mild disease. To block VEGF activity, animals were treated after arthritis onset with a soluble form of the Flt-1 VEGF receptor (sFlt), which was polyethylene glycol (PEG)-linked to increase its in vivo half-life. Treatment of arthritic mice with sFlt-PEG significantly reduced both clinical score and paw swelling, compared with untreated or control-treated (heat-denatured sFlt-PEG) animals. There was also significantly less joint inflammation and reduced bone and cartilage destruction in sFlt-PEG-treated animals, as assessed by histology. Our data demonstrate that, in collagen-induced arthritis, expression of the potent angiogenic cytokine VEGF correlates with disease severity. Furthermore, specific blockade of VEGF activity results in attenuation of arthritis in both macroscopic and microscopic parameters. These observations indicate that blood vessel formation is integral to the development of arthritis and that blockade of VEGF activity might be of therapeutic benefit in rheumatoid arthritis.

T-cell-mediated signalling in immune, inflammatory and angiogenic processes: the cascade of events leading to inflammatory diseases.

In the last decade, the understanding of the molecular mechanisms of regulation of the inflammatory process in chronic inflammatory diseases has moved remarkably forward. Recent evidence in various fields has consistently indicated that T-cells play a key role in initiating and perpetuating inflammation, not only via the production of soluble mediators but also via cell/cell contact interactions with a variety of cell types through membrane receptors and their ligands. Signalling through CD40 and CD40 ligand is a versatile pathway that is potently involved in all these processes. In this article, we review how T-cells become activated by dendritic cells or inflammatory cytokines, and how these T-cells activate, in turn, monocytes/macrophages, endothelial cells, smooth muscle cells and fibroblasts to produce pro-inflammatory cytokines (tumour necrosis factor alpha, interleukin-6), chemokines (interleukin-8, monocyte chemotactic protein-1), tissue factor, the main initiator of the coagulation cascade in vivo, and finally matrix metalloproteinases, responsible for tissue destruction. Moreover, we discuss how CD40 ligand at inflammatory sites stimulates fibroblasts and tissue monocyte/macrophage production of VEGF, leading to angiogenesis, which promotes and maintains the chronic inflammatory process. This cascade of events is discussed in the context of disease initiation/progression, with particular reference to atherosclerosis and rheumatoid arthritis, and to potential novel therapeutic targets for their treatment.

Cyclooxygenase-2 Induction and Prostacyclin Release by Protease-activated Receptors in Endothelial Cells Require Cooperation between Mitogen-activated Protein Kinase and NF-κB Pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1α, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1α formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-κB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1α synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IκBα. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1α generation were markedly attenuated by the NF-κB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-κB, and thrombin-induced but not PAR-2-induced p65-NF-κB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-κB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IκBα-dependent NF-κB activation.