VEGF release is associated with reduced oxygen tensions in experimental inflammatory arthritis.

OBJECTIVE: The contribution of local VEGF production and subsequent angiogenesis within the synovial membrane to the propagation of arthritis is unclear. The relationship between synovial oxygenation and blood flow in the development of arthritic disease is unknown. We have therefore measured oxygen levels and perfusion rates in the synovial space in a murine model of arthritis. METHODS: Arthritis was induced in DBA/1 mice by immunisation with type II collagen. Oxygen and perfusion levels were measured polarographically using silver needle microelectrodes within the knee joints prior to and 10 days after the onset of arthritis. In addition, synovial cells were isolated from knee joints of naive, pre-arthritic and arthritic mice. RESULTS: Onset of arthritis was associated with a marked reduction in synovial oxygen tensions (pO2). The perfusion rates in naive and arthritic animals were not significantly different: in naive mice, the rate was 0.58 +/- 0.11 ml/min/g and in arthritic joints, 0.64 +/- 0.17 ml/min/g. Furthermore, synovial cells isolated from the knee joints of naive animals did not express mRNA for VEGF, but significant levels were detected in cells from non-arthritic mice immunised with collagen. The onset of arthritis was associated with expression of VEGF mRNA and protein, and correlated negatively with pO2 levels. CONCLUSION: These data demonstrate that decreases in intra-articular pO2 occur in established arthritic conditions and may be the stimulus for local VEGF production. However, perfusion was not increased in arthritic animals and vascular density was unaltered, suggesting that the neovascularisation associated with inflammatory arthritis, is insufficient to restore oxygen homeostasis in the joint.

Dominant role of L- and P-selectin in mediating CXC chemokine-induced neutrophil migration in vivo.

The role of selectins in neutrophil emigration in response to the CXC chemokines KC and MIP-2 was investigated in wild type and P-selectin deficient mice. Intrapleural injection of KC or MIP-2 induced a rapid and specific neutrophil accumulation. Emigration 2 h after KC or MIP-2 was reduced 83 - 88% by anti-L-selectin mAb and 53 - 63% by anti-P-selectin mAb. Co-administration of anti-L- and P-selectin mAbs abolished neutrophil migration induced by either chemokine. An anti-E-selectin mAb tested alone did not affect KC-induced neutrophil migration after 2 or 4 h. Moreover, anti-E-selectin did not have an additive inhibitory effect on KC-induced neutrophil migration compared with P-selectin blockade alone. This was found when neutrophil migration was measured at 2 and 4 h after KC. Despite a blood neutrophilia, neutrophil migration at 2 and 4 h after KC was markedly smaller (by approximately 90%) in P-selectin deficient mice compared with wild type animals. Responses at both time points were not decreased further in animals given E-selectin mAb but were reduced to the PBS control level in the presence of anti-L-selectin. In vitro study of cultured murine endothelial cells demonstrated that KC can directly increase cell surface P-selectin expression. These data suggest that CXC chemokine-induced neutrophil accumulation is dependent on both neutrophil L-selectin and a rapid upregulation of endothelial P-selectin but there is no evidence for E-selectin induction.

New vessels, new approaches: angiogenesis as a therapeutic target in musculoskeletal disorders.

Musculoskeletal disorders such as rheumatoid arthritis (RA) and osteoarthritis are a common cause of pain and disability. The vasculature is an important component of the musculoskeletal system, and vascularization is a key event in the development of normal cartilage and bone. By promoting the delivery of nutrients, oxygen and cells, blood vessels help maintain the structural and functional integrity of joints and soft tissue and may facilitate tissue repair and healing. The identification of pro-angiogenic mediators such as vascular endothelial growth factor (VEGF) has led to the development of antiangiogenic therapies for the treatment of neoplastic diseases. The important role of angiogenesis, and especially VEGF, in the pathogenesis of joint disorders such as RA suggests that antiangiogenic therapy may be a useful adjunct to existing approaches in RA.

A comparison of the inhibitory activity of PDE4 inhibitors on leukocyte PDE4 activity in vitro and eosinophil trafficking in vivo.

1. Phosphodiesterase (PDE) 4 inhibitors have been shown to inhibit eosinophil PDE4 activity in vitro and accumulation of eosinophils in experimental airways inflammation. However, direct effects on eosinophil trafficking have not been studied in detail and it is not known if activity in vitro translates into efficacy in vivo. In the present study, we compared the activity of five PDE4 inhibitors in vitro and against trafficking of (111)In-eosinophils in cutaneous inflammation in the guinea-pig. 2. The rank order of potency for inhibition of PDE4 activity in guinea-pig eosinophil, neutrophil and macrophage, and human neutrophil lysates was RP73401 > SB207499 >CDP840 > rolipram > LAS31025. On TNFalpha production by human PBMC, all inhibitors with the exception of rolipram showed potency similar to their effect on neutrophil lysates. 3. In a brain cerebellum binding assay, the rank order of potency at displacing [3H]-rolipram was RP73401 > rolipram > SB207499 > CDP840 > LAS30125. 4. Trafficking of (111)In-eosinophils to skin sites injected with PAF, ZAP or antigen in sensitized sites was inhibited by oral administration of all PDE4 inhibitors. The rank order of potency was RP73401 = rolipram > LAS31025 > SB207499 > CDP840. 5. With the exception was RP73401, which was the most potent compound in all assays, there was no clear relationship between activity of PDE4 inhibitors in vitro and capacity to inhibit eosinophil trafficking in vivo. Thus, we conclude that in vitro activity of PDE4 inhibitors does not predict in vivo efficacy in an experimental model of eosinophil trafficking.

Suppression of acute lung injury in mice by an inhibitor of phosphodiesterase type 4.

The present study has investigated the therapeutic potential of a type 4 phosphodiesterase (PDE) inhibitor, rolipram, in experimental lung injury. Acute lung injury was induced in the mouse by combined treatment with lipopolysaccharide (LPS; 10 mg/kg, i.v.) and zymosan (3 mg/kg, i.v.), and assessed using extravascular albumin accumulation; neutrophil sequestration in pulmonary capillaries was also measured. The results show that pretreatment with rolipram (5 mg/kg, i.p.) was protective against the induction of lung injury by combined LPS and zymosan; extravascular albumin accumulation was reduced by 89% and neutrophil sequestration in lung tissue, as assessed by lung myeloperoxidase (MPO) activity was reduced by 75%. Pretreatment with rolipram also attenuated increases in serum tumor necrosis factor alpha (TNFalpha) levels induced by LPS and zymosan treatment, measured after 2.5 h. The role of endogenous TNFalpha in the induction of lung injury was therefore assessed. Blockade of endogenous TNFalpha by treatment with the soluble receptor p55-IgG fusion protein or an anti-murine TNFalpha monoclonal antibody, TN3. 19.12, had no protective effect against LPS and zymosan-induced lung injury. This suggests that there is a disassociation between TNFalpha production and the induction of injury in this model. Administration of rolipram after LPS and before zymosan treatment obliterated the increase in pulmonary vascular permeability, but its effect on sequestration of neutrophils in pulmonary microvessels, as measured by MPO, was less marked. The results of the present study suggest that use of agents such as rolipram that inhibit PDE4 may have a therapeutic role in treatment of acute lung injury, since we have shown that it is effective in attenuation of neutrophil activation even after sequestration. However, its effect appears to be independent of TNFalpha inhibition.

The role of lipocortin-1 in the inhibitory action of dexamethasone on eosinophil trafficking in cutaneous inflammatory reactions in the mouse.

1. The ability of glucocorticosteroids to inhibit tissue eosinophilia may be an important feature of their anti-inflammatory action in allergic diseases. Our previous work showed that an effect of dexamethasone on the release of eosinophils from the bone marrow could explain its inhibitory action on eosinophil accumulation in a mouse air-pouch model. Thus, it was unclear from that study whether dexamethasone could interfere with the process of eosinophil trafficking. In the present study, therefore, we used a newly developed mouse model to evaluate the effects of systemic treatment with dexamethasone on the recruitment of (111)In-labelled blood eosinophils to sites of cutaneous inflammation in the mouse and whether lipocortin-1 (LC-1) was involved. 2. The i.d. injection of ovalbumin (OVA) in sensitized mice induced a dose-dependent recruitment of (111)In-labelled blood eosinophils which peaked at 4 to 8 h after antigen challenge. Systemic treatment with dexamethasone (50 microg per mouse, 3 h after antigen) effectively inhibited (111)In-eosinophil recruitment in this reaction by 70 to 85%. Similarly, a 1 h pretreatment with dexamethasone significantly suppressed (111)In-eosinophil induced by platelet-activating factor (PAF), leukotriene B4(LTB4) and the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) by 40 to 70%. 3. Two experimental approaches were used to evaluate the role of LC-1: treatment with LC-1 fragment Ac2-26 and use of an anti-LC-1 antiserum. LC-1 fragment Ac2-26 (100 microg per mouse) failed to affect (111)In-eosinophil recruitment. Moreover, pretreatment of animals with an anti-LC-1 antiserum failed to reverse the inhibitory effects of dexamethasone on (111)In-eosinophil recruitment induced by MIP-1alpha and by antigen in sensitized mice. 4. In contrast, the LC-1 fragment significantly inhibited glycogen-induced neutrophil recruitment into the peritoneal cavity of mice. Furthermore, the anti-LC-1 antiserum reversed the inhibitory effects of dexamethasone on the glycogen-induced neutrophil recruitment. 5. Thus, our results suggest that dexamethasone can inhibit the recruitment of eosinophils in mouse skin independent of an action on the bone marrow. However, by use of two different approaches, we showed that LC-1 does not play a role in mediating the inhibitory action of dexamethasone on eosinophil migration into cutaneous inflammatory reactions in the mouse. These data add further support to a LC-1-independent action of dexamethasone on eosinophils in vivo.

Platelet-activating factor plays a pivotal role in the induction of experimental lung injury.

We have previously described a model of acute lung injury in the mouse in which intravenous administration of lipopolysaccharide (LPS) results in a marked sequestration of neutrophils in the pulmonary microvasculature, although this by itself was not sufficient to induce injury. If the sequestered neutrophils were exposed to zymosan, then a striking increase in pulmonary vascular permeability to albumin was found, suggesting that sequestered neutrophils may produce one or more mediators capable of acting directly on the capillary endothelium. Because activated neutrophils are known to release platelet-activating factor (PAF), we hypothesized that PAF produced locally within the pulmonary capillaries may be the mediator involved. Treatment of mice with the PAF antagonist UK-74,505 prior to administration of zymosan alone or combined LPS and zymosan resulted in a substantial attenuation of lung injury, as measured by the accumulation of extravascular 125I-labeled human serum albumin. UK-74,505 had no effect on neutrophil sequestration as measured by myeloperoxidase activity in whole lung tissue and as assessed by light microscopy. Administration of UK-74,505 after LPS, but before zymosan, was also effective at inhibiting lung injury but again, neutrophil sequestration was unaffected. In contrast, UK-74,505 had no effect on cobra venom factor-induced lung injury and neutrophil sequestration. These data suggest that PAF production is involved in the increases in pulmonary vascular permeability, but not in the sequestration of neutrophils, induced by zymosan alone or by combined LPS and zymosan treatment. Early treatment with PAF antagonists may be beneficial in preventing the development of acute lung injury in humans.

Angiogenesis in arthritis: role in disease pathogenesis and as a potential therapeutic target.

Rheumatoid arthritis (RA) is a chronic destructive musculo-skeletal disorder, associated with thickening of the synovial membrane lining the joints, inflammation and hyperproliferation of synovial cells, as well as a pro-inflammatory cytokine cascade, leukocyte infiltration, and tissue damage and bone resorption. An early event in RA is an alteration in blood vessel density and prominent neovascularisation. The hyperplasia of the synovium necessitates a compensatory increase in the number of blood vessels to nourish and oxygenate the tissue. However, angiogenesis may not keep pace with synovial proliferation, leading to regions of hypoperfusion and hypoxia. VEGF, a potent endothelial cell mitogen, is expressed in RA synovium and elevated in the serum of RA patients. We have reported that dissociated RA synovial membrane cells spontaneously secrete VEGF, and that release of VEGF by these cells is upregulated by cytokines and hypoxia. In a murine model of RA, VEGF is released from synovial cells isolated from the knees of arthritic but not healthy mice, and the extent of VEGF production correlates with the severity of arthritis. VEGF thus appears to play a key role in mediating alterations in synovial vessel density in arthritis. As a consequence, RA may be a potential target for anti-angiogenic therapy, and targeting VEGF may prove to be especially beneficial.

A comparison of the inhibitory activity of selective PDE4 inhibitors on eosinophil recruitment in guinea pig skin

The elevation of intercellular cyclic AMP by phosphodiesterase (PDE)4 inhibitors in eosinophils is associated with inhibition of the activation and recruitment of these cells. We have previously shown that systemic treatment with the PDE4 inhibitor rolipram effectively inhibit eosinophil migration in guinea pig skin. In the present study we compare the oral potency and efficacy of the PDE4 inhibitors rolipram, RP 73401 and CDP 840 on allergic and PAF-induced eosinophil recruitment. Rolipram and RP 73401 were equally effective and potent when given by the oral route and much more active than the PDE4 inhibitor CDP 840. We suggest that this guinea pig model of allergic and mediator-induced eosinophil recuitment is both a sensitive and simple tool to test the efficacy of PDE4 inhibitors in vivo.

A comparison of the inhibitory activity of selective PDE4 inhibitors on eosinophil recruitment in guinea pig skin.

The elevation of intracellular cyclic AMP by phosphodiesterase (PDE)4 inhibitors in eosinophils is associated with inhibition of the activation and recruitment of these cells. We have previously shown that systemic treatment with the PDE4 inhibitor rolipram effectively inhibit eosinophil migration in guinea pig skin. In the present study we compare the oral potency and efficacy of the PDE4 inhibitors rolipram, RP 73401 and CDP 840 on allergic and PAF-induced eosinophil recruitment. Rolipram and RP 73401 were equally effective and potent when given by the oral route and much more active than the PDE4 inhibitor CDP 840. We suggest that this guinea pig model of allergic and mediator-induced eosinophil recruitment is both a sensitive and simple tool to test the efficacy and potency of PDE4 inhibitors in vivo.

Selectins mediate eosinophil recruitment in vivo: a comparison with their role in neutrophil influx.

The role of selectins in mediating eosinophil recruitment in vivo was assessed in a model of lipopolysaccharide (LPS)-induced mouse pleurisy. LPS administration resulted in significant eosinophil influx at 24 hours, whereas neutrophil recruitment to the cavity peaked at 4 hours and persisted for 24 hours. The anti-L-selectin monoclonal antibody (MoAb) MEL-14 effectively inhibited (by 97%) eosinophil influx at 24 hours and also inhibited neutrophil recruitment at both times (75% to 95%). Eosinophil recruitment was partially reduced (54%) by the anti-P-selectin MoAb 5H1 but, in contrast, was unaffected by the anti-E-selectin MoAb 10E6. Neutrophil influx at 4 or 24 hours was not affected by the anti-P- or anti-E-selectin MoAbs. However, coadministration of anti-P-selectin and anti-E-selectin was very effective at inhibiting eosinophil influx at 24 hours (86%) and neutrophil influx at 4 (93%) and 24 hours (92%). These results show that all three selectins play a role in LPS-induced eosinophil and neutrophil recruitment in vivo, although P- and E-selectin show a degree of functional redundancy. The demonstration that P-selectin mediates eosinophil but not neutrophil influx suggests that suppressing the function of this adhesion molecule may be beneficial in blocking eosinophil accumulation in pleural inflammation.

A role for the beta2 integrin CD11b in mediating experimental lung injury in mice.

We have investigated the requirement of neutrophil emigration and the role for CD11b/CD18-mediated events in the experimental induction of acute lung injury. BALB/c mice received lipopolysaccharide (LPS) (3 mg/kg) intravenously (i.v.) 2 h prior to i.v. zymosan (10 mg/kg) and extravascular albumin accumulation was assessed after 30 min. Compared with saline-treated controls, zymosan alone caused a 6-fold increase in the accumulation of 125I-human serum albumin in whole lung tissue (P<0.05). Combined treatment with LPS and zymosan further increased extravascular albumin accumulation (P<0.05 compared with zymosan alone). The monoclonal antibody 5C6, directed against murine CD11b, was injected, 1 mg i.v. 15 min prior to LPS or 15 min before the zymosan, and compared with immunoglobulin G-injected controls. Albumin accumulation was significantly reduced by 5C6 when given prior to the LPS (P<0.01), but not when given before zymosan in the combined LPS and zymosan treatment. Interestingly, albumin accumulation induced by zymosan alone was not reduced by 5C6. The lungs of the mice treated with LPS and zymosan showed a marked, diffuse accumulation of inflammatory cells which, by light microscopy, appeared to be interstitial. Foci of neutrophil aggregates were seen in noncapillary microvessels, and pretreatment with 5C6 appeared to reduce their frequency. In the animals treated with zymosan alone, LPS alone, or LPS and zymosan in combination, electron microscopy established that approximately 25% of all nucleated cells were neutrophils: 99% of the neutrophils were restricted to the intravascular compartment. Pretreatment with 5C6 prior to LPS and zymosan treatment reduced the increase in percentage of neutrophils by half. These results indicate a disassociation between induction of permeability and neutrophil emigration in our murine model and suggest that the release of neutrophil-derived factors such as platelet-activating factor, proteases, or oxidants may be involved.