Inhibitory effect of TIMP influences the morphology of varicose veins.

OBJECTIVES: Imbalance of matrix metalloproteinase enzymes (MMP) and their inhibitors (TIMPs) may contribute to the development of varicose veins. We hypothesised that, histological changes in varicose vein wall correlate with alterations in expression of MMP/TIMP. METHODS: Varicose veins (n=26) were compared with great saphenous vein (GSV) segments (n=11) from arterial bypass, and with arm and neck veins from fistula and carotid operations (n=13). Varicose vein wall thickness was measured, enabling categorisation as atrophic and hypertrophic. MMP-2, MT1-MMP, TIMP-2, and TIMP-3 expression were quantitatively analysed by immunohistochemistry. RESULTS: There was significantly higher expression of TIMP-2 (immunopositive area 4.34% versus 0.26%), linked with connective tissue accumulation in the tunica media of varicose veins as compared with arm and neck vein controls. TIMP-2 and TIMP-3 expression was higher in hypertrophic than atrophic segments (3.2% versus 0.99% for TIMP-2, 1.7% versus 0.08% for TIMP-3). Similarly, TIMP-2 and TIMP-3 had elevated expression in the thicker proximal varicose vein segments compared to distal (4.3% versus 1.3% for TIMP-2 and 0.94% versus 0.41% for TIMP-3). CONCLUSIONS: This study linked morphological changes in varicose vein walls with MMP/TIMP balance. A higher TIMP expression favours deposition of connective tissue and thus thicker vein wall, reducing matrix turnover by suppression of protease activity.

In vitro target validation and in vivo efficacy of p38 MAP kinase inhibition in established chronic collagen-induced arthritis model: a pre-clinical study.

OBJECTIVES: The aim of the present study was to determine the in vivo efficacy of p38 mitogen-activated protein kinase (MAPK) inhibitors, namely GW856553X and GSK678361, in murine models of arthritis. METHODS: The effect of p38 MAPK inhibitors was tested in 2 variants of the collagen-induced arthritis model (CIA) in DBA/1 mice, acute arthritis induced by heterologous collagen and chronic relapsing arthritis induced by homologous collagen. Animals were treated after onset of arthritis. Furthermore, post-onset disease efficacy of GSK678361 was tested in the chronic model, so as to determine the effects on established arthritis. In vitro studies were carried out with GW856553X, using human umbilical vein endothelial cells, to determine potential effects of GW856553X on the vasculature. RESULTS: In both acute and chronic arthritis, GW856553X reduced signs and symptoms of disease, and protected joints from damage. The effect of GW856553X in chronic CIA was confirmed using an alternative compound, GSK678361. Importantly, treatment with GSK678361 from 14 days post-onset of chronic arthritis completely reversed signs of established disease and joint destruction. Mechanism of action studies demonstrated that GW856553X inhibited endothelial cell migration and angiogenesis in vitro, with reduced pro-inflammatory cytokine production. CONCLUSIONS: Suppression of murine CIA by the p38 MAPK inhibitors GW856553X and GSK678361 suggests that they may have therapeutic potential for future use in RA if safe clinical dosing achieves adequate compound exposure.

Adenoviral delivery of soluble VEGF receptor 1 (sFlt-1) abrogates disease activity in murine collagen-induced arthritis.

The angiogenic factor VEGF promotes synovitis and bone erosion in rheumatoid arthritis (RA). Previously, we have demonstrated that VEGF expression correlates with disease severity in RA patients and in murine collagen-induced arthritis (CIA). In this study, we adopted an adenoviral gene delivery system expressing soluble VEGF receptor 1 (sFlt-1) to further study the role of VEGF in CIA. Arthritis was induced in DBA/1 mice by injection of bovine collagen. Adenoviruses expressing human soluble VEGF receptor 1 (AdvsFlt-1), or without transgene (Adv0), were injected intravenously on the first day of arthritis. We found that disease severity and paw swelling were significantly suppressed in mice receiving AdvsFlt-1, when compared to untreated or Adv0-treated mice. Expression of sFlt-1 peaked 24 h after injection, with protein detectable in the liver, synovial issue and serum, but rapidly decreased by 72 h. The effect of sFlt-1 expression on signs of disease was paralleled by reduced joint destruction and decreased expression of the vascular marker von Willebrand factor. In summary, adenoviral delivery of human soluble VEGF receptor type 1 significantly suppressed disease activity in CIA. The actions of AdvsFlt-1 are likely to be mediated by reduced synovial neovascularization, and these results support the concept that VEGF blockade may be an effective therapeutic adjunct for the treatment of RA.

Diagnostic strategy for the assessment of rheumatoid vasculitis.

OBJECTIVE: To determine the clinical features associated with histologically proven rheumatoid vasculitis (HRV) and the additional diagnostic value of serological markers in an inception cohort of 81 patients with rheumatoid arthritis (RA) suspected of RV. METHODS: The presence and number of recently developed extra-articular manifestations (EAMs) and a weighted EAM score, as well as the levels of serological markers, were compared between 31 patients with RA with histologically proven vasculitis and 50 patients with RA in whom vasculitis could not be documented histologically. The following markers were evaluated: circulating immune complexes, complement components C3 and C4, class-specific rheumatoid factors (IgM RF, IgG RF, IgA RF), antineutrophil cytoplasmic antibodies, antinuclear antibodies, antiendothelial antibodies, circulating intercellular adhesion molecule-1 and -3, circulating vascular cell adhesion molecule and E-selectin, cellular fibronectin, von Willebrand factor antigen, and C reactive protein. The diagnostic value of these markers, in addition to the clinical features, was evaluated with logistic regression analysis. RESULTS: Peripheral neuropathy or purpura/petechiae, or both, were the most important clinical features to discriminate patients with RA with and without histologically proven RV. The presence of a high number of EAMs and a higher weighted EAM score in patients with RA suspected of vasculitis were also associated with an increased probability of histologically proven RV. After adjustment for EAMs, only the combination of an increased serum IgA RF level and a decreased serum C3 level appeared to make an additional contribution to the diagnosis histologically proven RV. Evidence of systemic vasculitis was found in a muscle biopsy of the rectus femoris in 9/14 (64%) patients with vasculitis with neuropathy and in 3/11 (27%) patients with purpura/petechiae and vasculitis of the skin. CONCLUSIONS: In the diagnostic process of RV the presence of peripheral neuropathy and/or purpura/petechiae or a high weighted EAM score will increase the probability of histologically proven RV. Of the circulating factors previously suggested to be markers for RV only IgA RF and C3 further increase the probability of histologically proven RV and may be useful to guide diagnostic decisions.

The role of the angiogenic molecule VEGF in the pathogenesis of rheumatoid arthritis.

The expansion of the synovial lining of joints in rheumatoid arthritis (RA), and the subsequent invasion by the pannus of underlying cartilage and bone, necessitates an increase in the vascular supply to the synovium, to cope with the increased requirement for oxygen and nutrients. New blood vessel formation - 'angiogenesis' - is now recognised as a key event in the formation and maintenance of the pannus in RA. Although many pro-angiogenic factors have been demonstrated to be expressed in RA synovium, the potent pro-angiogenic cytokine vascular endothelial growth factor (VEGF) has been demonstrated to have a central involvement in the angiogenic process in RA. The additional activity of VEGF as a vascular permeability factor may also increase oedema and hence joint swelling in RA. Several studies, including those from the Kennedy Institute of Rheumatology Division, have shown that targeting angiogenesis in animal models of arthritis ameliorates disease. Inhibition of angiogenesis, as an adjunct to existing therapy of RA, or even as a stand-alone treatment, would not only prevent delivery of nutrients to the synovium, but could also lead to vessel regression and possibly reversal of disease.

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.

Neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells.

Defensins, a family of small, cationic, antimicrobial peptides, are found in mammals, insects and plants. alpha-defensins are stored in granules of neutrophils and released upon activation by exocytosis. It was shown here that human neutrophil peptide (HNP), at concentrations of 10(-8) -10(-9) M, up-regulated the expression of TNF-alpha and IL-1 beta in monocytes activated with Staphylococcus aureus or PMA, while expression of IL-10 mRNA was down-regulated and production of IL-8 was not affected. HNP alone was unable to induce TNF-alpha or IL-1 beta expression in resting monocytes. At concentrations of 10(-4) -10(-5)M, HNP was cytotoxic for monocytes in serum-free medium. The cytotoxicity was abrogated in the presence of serum, while a cytokine-modulating effect of HNP was observed in the presence of serum and in whole blood, suggesting that this mechanism may function in vivo. Similarly, serum did not abrogate bactericidal activity of HNP. It was also demonstrated herein that HNP at 10 (-8) -10(-9) M, attenuated the inhibitory action of dexamethasone on TNF-alpha production. In parallel to monocyte studies, we have showed that HNP at concentrations ranging from 10(-9)M to 10(-6)M caused about 5-fold suppression of VCAM-1 expression in TNF-alpha-activated human umbilical vein endothelial cells, while the ICAM-1 expression was not affected. Our findings suggest that neutrophil defensins have the potential to modulate the inflammatory responses through regulation of cytokine production and adhesion molecule expression.

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.

Vascular endothelial growth factor in normal human salivary glands and saliva: a possible role in the maintenance of mucosal homeostasis.

Saliva is an enriched milieu containing biologically active proteins, including several different growth factors and cytokines. This study documents that vascular endothelial growth factor (VEGF), a potent, multifunctional, angiogenic cytokine, is a component of normal human saliva. VEGF was measured by ELISA in whole saliva (median concentration, 460 pg/ml) and in ductal secretions obtained from the parotid (277 pg/ml) and the submandibular-sublingual (80 pg/ml) salivary glands. VEGF seems to be synthesized endogenously by the salivary glands because both VEGF mRNA and protein (as revealed by in situ reverse transcriptase-PCR and by immunohistochemistry, respectively) colocalized to serous acinar cells and ductal epithelial cells within the parotid, submandibular, and minor salivary glands. These findings point to the existence of a "salivary VEGF system." It is possible that salivary VEGF plays a role in regulating physiologic and pathologic angiogenic and other vascular responses in salivary and mucosal tissues. And in particular, the presence of VEGF in saliva may contribute to the remarkable healing capacity of the oral mucosa as well as other regions of the digestive tract.

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.

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.

Selective enhancement of endothelial cell VCAM-1 expression by interleukin-10 in the presence of activated leucocytes.

Although initially described as an immunomodulatory cytokine, interleukin-10 (IL-10) has also been proposed to exert proinflammatory effects both in vivo and in vitro. In particular, studies in IL-10 transgenic mice have suggested that IL-10 may activate vascular endothelium to promote leucocyte adhesion and extravasation. In the present study we investigated whether IL-10 activates endothelial cells either directly or indirectly, via signals produced by leucocytes in the endothelial cell environment, using a co-culture of human umbilical vein endothelial cells and peripheral blood mononuclear cells (PBMC). No direct effects of IL-10 on endothelial cell responses were observed. However, in the presence of phytohaemagglutinin-activated PBMC, IL-10 increased the expression on endothelial cells of vascular cell adhesion molecule-1 (VCAM-1) but not of intercellular adhesion molecule-1, E-selectin or major histocompatibility complex (MHC) antigens, an effect mediated by PBMC-derived soluble factors. We also observed that interferon-gamma (IFN-gamma) antagonized VCAM-1 expression on endothelial cells mediated by IL-4 and IL-13. Since IL-10 has previously been documented to down-regulate release of IFN-gamma by PBMC, we propose that the IL-10-mediated reduction of IFN-gamma production by PBMC results in enhanced responsiveness of endothelial cells to PBMC-derived IL-4 and IL-13, and thus increased expression of VCAM-1. Our results suggest that the relative balance of cytokines produced by infiltrating cells in developing inflammatory lesions may differentially modulate endothelial responsiveness in vivo, and that IL-10 might indirectly stabilize VCAM-1 expression on endothelial cells by affecting the balance of leucocyte-derived cytokines in the endothelial environment.

The role of receptors for tumour necrosis factor-alpha in the induction of human polymorphonuclear neutrophil chemiluminescence.

Tumour necrosis factor-alpha (TNF-alpha) is a potent mediator of inflammation, which exerts profound effects on polymorphonuclear neutrophils (PMN). TNF-alpha binds to distinct cell surface receptors termed p55 and p75, expressed in approximately equal amounts on the PMN surface. We have studied the effects of TNF-alpha on the priming of F-Met-Leu-Phe (FMLP)-stimulated oxidative metabolism of PMN, using a luminol-enhanced chemiluminescence assay, and have examined the relative roles of PMN receptors for TNF-alpha in priming this oxidative metabolism, using antibodies with p55 and p75 receptor-specific agonistic and antagonistic activities. We have obtained the following results: (1) Antibody Htr-9 with agonistic activity at the p55 receptor mimicked the effect of TNF-alpha; however, a combination of Htr-9 and TNF-alpha did not results in any further increase in chemiluminescence relative to the response observed with TNF-alpha alone. The p75 agonistic antibody MR2-1 actually decreased basal and FMLP-enhanced chemiluminescence. Additionally, MR2-1 substantially inhibited the effects of both TNF-alpha itself and of the p55 agonist Htr-9. (2) Addition of antibodies with antagonistic activities at the p55 (antibody TBP-2) and p75 (antibody Utr-1) receptors resulted in a marked inhibition of the PMN response to TNF-alpha. A combination of both Utr-1 and TBP-2 was most effective at inhibiting the action of TNF. We have confirmed previously published observations that TNF-alpha alone effectively stimulates the oxidative metabolism of PMN in vitro, and that pre-incubation of PMN with TNF-alpha enhances subsequent generation of oxidative metabolites in response to FMLP. We conclude that both p55 and p75 receptors play a critical role in mediating the activation of PMN by TNF-alpha.

Deactivation of vascular endothelium by monoclonal anti-tumor necrosis factor alpha antibody in rheumatoid arthritis.

OBJECTIVE: To assess whether monoclonal antibody to tumor necrosis factor alpha (TNF alpha) reduces endothelial activation in rheumatoid arthritis (RA). METHODS: Levels of serum E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), and circulating leukocytes (differential counts) were measured in RA patients before and up to 4 weeks after infusion of either placebo or chimeric anti-TNF alpha antibody cA2 (1 or 10 mg/kg). RESULTS: Treatment with anti-TNF alpha decreased serum E-selectin and ICAM-1 levels, with the earliest detectable changes observed on days 1-3 after anti-TNF alpha infusion. No effect on VCAM-1 levels was detected. In parallel, there was a rapid and sustained increase in circulating lymphocytes. The extent of the decrease in serum E-selectin and ICAM-1 levels and the increase in lymphocyte counts was significantly higher (P < or = 0.05) in patients in whom a clinical benefit of anti-TNF alpha was observed ( > or = 20% response, by Paulus criteria, at week 4) compared with that in patients who failed to respond to anti-TNF alpha at this time point. CONCLUSION: We propose that decreased serum levels of adhesion molecules may reflect diminished activation of endothelial cells in the synovial microvasculature, leading to reduced migration of leukocytes into synovial joints, and thus prolonging the therapeutic effect of anti-TNF alpha in RA.

Functional activities of receptors for tumor necrosis factor-alpha on human vascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the control of endothelial cell function and hence in regulating traffic of circulating cells into tissues in vivo. Stimulation of endothelial cells in vitro by TNF-alpha increases the surface expression of leukocyte adhesion molecules, enhances cytokine production, and induces tissue factor procoagulant activity. In the present study, we have examined the relative roles of the two cell surface receptors for TNF-alpha (p55 and p75) on endothelial cells, using antibodies with both agonistic and antagonistic activities. We report that anti-p55 receptor agonistic antibody Htr-9 induces the expression of tissue factor antigen and the release of interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In contrast, there is very little or no activation of endothelial cell responses by an anti-p75 agonist. TNF-alpha-induced expression of tissue factor and adhesion molecules, and release of IL-8 and GM-CSF, are decreased by antibodies with antagonistic activities for either receptor, although the effect of anti-p55 antibodies is markedly greater than that of anti-p75 antibodies. The responses of endothelial cells to lymphotoxin/TNF-beta are significantly decreased by anti-p55 antagonists alone. Our data suggest that endothelial cell responses to TNF-alpha, such as expression of tissue factor and adhesion molecules for mononuclear cells, which may be important in the pathogenesis of atherosclerosis, are mediated predominantly, but not exclusively, by the p55 TNF receptor.

Antilipid A monoclonal antibody HA-1A decreases the capacity of bacterial lipopolysaccharide to activate human vascular endothelial cells by an immune adherence mechanism.

Human monoclonal IgM antibody HA-1A, which recognizes the lipid A component of bacterial lipopolysaccharide (LPS), has been shown to reduce mortality in Gram negative septicemia. The vascular endothelial lining of blood vessels, which controls leucocyte traffic and activation, as well as haemostatic balance, may be one of the primary targets of LPS action during sepsis. In earlier studies we have described HA-1A-induced immune adherence of LPS to complement receptors on erythrocytes, and showed that pre-incubation with HA-1A, in the presence of complement and red blood cells, markedly reduced LPS-induced cytokine production from peripheral blood mononuclear cells. In the present study, we measured the effect of immune adherence of LPS in the presence of HA-1A on the responses of cultured endothelial cells, and found that subsequent expression of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1, and secretion of the cytokines interleukin-6 and granulocyte-macrophage colony stimulating factor were markedly reduced. Moreover, the ability of LPS to increase levels of tissue factor procoagulant activity on endothelial cells was markedly diminished by LPS immune adherence to HA-1A. This decrease in endothelial activation in response to LPS following immune adherence to HA-1A may play a significant role in the protective effect of HA-1A in vivo during the course of Gram negative sepsis.

Comparison of the functional activities of two different preparations of human recombinant tumor necrosis factor alpha.

The role of tumor necrosis factor (TNF-alpha) in physiological and pathological reactions has resulted in a progressive increase of expensive TNF-alpha consumption for laboratory and clinical purposes. Following this trend, the first chemical synthesis of the gene for rHuTNF-alpha gene in Poland and its subsequent successful expression in E. coli was recently reported. In the present paper, we verify the in vitro biological activities of this TNF-alpha preparation (CMMS/TNF-alpha) in comparison with a commercially available preparation of TNF-alpha. We demonstrate that our TNF-alpha possesses strong cytotoxic activity against WEHI 164 (clone 13) cells, binds the p55 and p75 TNF receptors on cell lines, induces intercellular adhesion molecule-1 (ICAM-1) expression, and interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) release from human umbilical vein endothelial cells (HUVEC). We demonstrate its usefulness for further investigations as an effective reagent for in vitro assays.

Contrasting effects of interferon gamma and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor alpha.

Tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelial cells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-alpha-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from TNF-alpha-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-alpha-induced responses to those observed with endothelial cells of foetal origin. Additionally, we report here that TNF-alpha and interferon gamma (IFN-gamma) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-alpha-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-gamma plus TNF-alpha was markedly decreased when compared to the response induced by TNF-alpha alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.