Interleukin-1β induced vascular permeability is dependent on induction of endothelial Tissue Factor (TF) activity
© Puhlmann et al; licensee BioMed Central Ltd. 2005
Received: 15 July 2005
Accepted: 30 September 2005
Published: 30 September 2005
IL-1β is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1β are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1β induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1β caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1β induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.
Keywordsvideomicroscopy inflammation neovasculature endothelial tissue
IL-1β is a pleotropic 17.5 kD cytokine, secreted primarily by monocytes and macrophages, that mediates the pathophysiology of various acute and chronic inflammatory conditions. High levels of circulating IL-1 have been identified in experimental models of endotoxic shock and acute bacterial infection and increased gene expression of IL-1 has been identified in tissues at sites of experimentally induced inflammation [1, 2]. Clinically, high levels of circulating IL-1 have been identified in patients with acute bacterial infections and elevated levels of IL-1 have been detected in the diseased articular tissues of patients with chronic rheumatoid arthritis [3, 4]. In experimental models of endotoxin or E. coli induced shock, immune complex colitis, cancer progression, cachexia, and non-specific inflammation, IL-1 blockade significantly ameliorates the pathophysiological host response in these conditions [5–9]. Of note, administration of recombinant IL-1ra is used clinically to ameliorate the symptoms of severe rheumatoid arthritis .
Although IL-1 receptors are widely expressed on different cell types, the protein appears to exert many of its physiological actions through effects on endothelial tissue. IL-1 induces endothelial cell (EC) production of cytokines such as IL-8, IL-6 and TNF, multiple cytokine receptors, adhesion molecules, growth factors, matrix metalloproteinases, and coagulation factors such as tissue factor, fibrinogen, urokinase plasminogen activator, type 1,2 plasminogen activator and protease nexin 1 [11–14].
Two sentinel homeostatic functions of vasculature are initiation of coagulation via the extrinsic clotting cascade and regulation of vascular permeability. Increased vascular permeability most likely represents the initial event in pathological or reparative inflammation or angiogenesis by allowing efflux of plasma proteins into interstitium to serve as a provisional matrix for circulating inflammatory cells or activated endothelium . IL-1 is known to induce a procoagulant phenotype on endothelial tissue primarily through induction of TF [16, 17] and has been shown to alter endothelial cell monolayer permeability in in vitro models [18, 19]. However, its effects on vascular permeability in vivo have not been consistent and the mechanisms for these effects are not known [20–22]. For example, in lapine models, systemic recombinant IL-1 has been shown to induce shock and significant pulmonary vascular injury manifested by marked perivascular pulmonary edema [20, 23]. However, in rodent or guinea pig models others have not demonstrated any independent effects of aerosolized or systemically administered IL-1 on pulmonary vascular permeability or injury [21, 22].
The present studies were performed to characterize the inflammatory properties of IL-1 as mediated by alterations in permeability across endothelial monolayers in vitro. The data indicate that IL-1 induces both procoagulant effects and permeability via a single TF dependent mechanism and suggest that TF may represent a novel target for acute or chronic inflammatory conditions mediated by IL-1.
Materials and methods
Human umbilical vein ECs (hUVECs) were obtained from Clonetics (Clonetics #CC 2519) and propagated at 37°C in a 5% CO2 incubator in EGM-2 media (Clonetics #CC-3156 and #CC-4176) according to the manufacturer's instructions but without addition of the VEGF-supplement. Cells in the experiments were passaged for a maximum of 5 generations. MC-38, a non-metastatic methylcholanthrene-induced murine adenocarcinoma was maintained in Dulbecco's modified Eagle's medium (DMEM) (Biofluids, Rockville, MD) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine and 50 U/mL penicillin/streptomycin.
In vitro permeability assay
In-vitro permeability was quantitated by spectrophotometric measurement of Evans Blue-bound albumin across functional hUVEC monolayers using a modified 2-compartment chamber model as previously described in detail. Briefly, hUVECs were plated (8 × 105/ well) in 1 μm PET Transwell filter inserts (Falcon #35 3102) using EGM-2 (as described above). On day 3, cells were washed and treated with EBM-2 (Clonetics #CC-3156) with 2% BSA with or without IL-1β with various concentrations as indicated. Except during time course experiments, cytokine exposure was kept constant at 2 hours (at 37°C, 5% CO2). Cells were briefly washed with PBS/ Ca2+ and Mg2+. Fresh EBM-2 including 1% Factor VIII-deficient plasma (George-King Biochemical) (where indicated) was added and incubated for an additional 1 hour at 37°C, 5% CO2. In experiments evaluating TF-blocking antibodies, designated wells were incubated first with a 1:100 dilution of antibody (American Diagnostica, #4509) and PBS/1% BSA. Inserts were washed with PBS/ Ca2+ and Mg2+ before adding 1.5 mL of Evans Blue (EB) bound to 0.1% BSA in PBS in the upper chamber. Two mL PBS/ Ca2+ and Mg2+ was added to the lower chamber in which absorbance of EB was determined after 1 hr using a spectrophotometer (620 nm). Experiments were performed in triplicates and repeated multiple times.
Tissue factor assay
Tissue factor activity was determined using a 1-stage coagulation assay. HUVECs were plated (8 × 105/ well) in a 6-well plate and incubated for 3 days. Cells were washed with PBS and treated with various concentrations of IL-1β (in EBM-2/ 2% BSA) for 2 hours at 37°C, 5% CO2. After this incubation, HUVECs were washed with sterile PBS followed by incubation for 10 min at 37°C in 1 ml TRIS (25 mM, pH 8). Plates were transferred to a freezer (-80°C) for at least 1 hour. After thawing the plates at 37°C, cells were harvested using a mechanical cell scraper and centrifuged in an Eppendorf table centrifuge at 14,000 rpm for 5 min. Cell pellets were suspended in 120 μL of assay buffer (25 mM TRIS/ PBS/0.1% BSA) and immediately assayed.
Tissue factor activity was measured using a 1-step coagulation assay (Amelung microcoagulation analyzer) by addition of 100 μL factor VIII-deficient human plasma (George-King Biomedical). Clotting time was determined at 37°C and measurement started after addition of 100 μL of 30 nM CaCl2 (Sigma). The reference curve for TF was obtained by reconstituting various concentrations of lipidated recombinant hTF (American Diagnostica, # 4500 L/B2) according to the manufacturer's recommendation and allowed for detection limits between 0.395 ng/mL to 25 ng/mL (7 – 470 pM/mL).
HUVECs were grown to confluence on fibronectin coated two well culture slides (BIOCOAT Fibronectin, Becton Dickinson, # 354628) and exposed to IL-1 as described for the permeability assays. After fixation with 4% formaldehyde (RT, 10 min), cells were permeabilized with 1% Triton X for 10 min. A 1:200 dilution of a polyclonal VE-cadherin antibody (Santa Cruz Biotechnology, #sc-6458) was used as primary antibody and incubated at RT for 1 hour. After several washes with PBS, cells were labeled with Alexa Fluor 488 donkey anti-goat IgG (Molecular Probes, #A-11055) for 1 hour at RT. After repeated washes, the F-actin cytoskeleton was stained with Texas Red labeled phalloidin (Molecular Probes, #T-7471) for 40 min at room temperature. Slides were mounted and sealed using Gel/Mount (Biomeda, #M01). Photomicrographs were obtained with a Zeiss Axiovert 200 M inverted fluorescent microscope (Zeiss, Germany) at 400 × magnification.
TF induction and permeability in endothelial cells by IL-1β in vitro
Activation of the extrinsic coagulation cascade by IL-1β leads to conformational changes of the EC cytoskeleton and loss of VE-cadherin complexes
We have previously shown that TNF shares these properties in an in vitro system highlighting the know widely overlapping biological activities of these two cytokines [24, 25] particularly on endothelial tissue . The acute loss of VE-cadherin complexes, contraction of F-actin cytoskeletal fibers, and the formation of large intercellular gaps due to TNF or IL-1 indicates that these morphological alterations are secondary to physiological EC responses rather than non-specific cell injury. EC monolayer integrity in this model is restored after the inflammatory stimuli is removed . The effects are only observed in the presence of plasma, and hence, with activation of the extrinsic coagulation cascade and thrombin production.
It is interesting that the selective procoagulant and permeability effects of TNF, a cytokine with broadly overlapping biological activities with IL-1, on tumor neovasculature have been applied in the clinical treatment of cancer. When high dose TNF is administered via an isolated organ perfusion there is increased local permeability followed by selective obliteration of the neovasculature . These selective effects on tumor neovasculature are thought to be important in augmenting delivery of chemotherapy, such as melphalan, to the tumor bed . Our data would suggest that IL-1 might be a suitable alternative to TNF in this capacity because of the similar vascular effects and its limited toxicity compared to the other.
In summary, these experiments demonstrate that IL-1 mediates potent permeability effects on endothelial tissue that is mediated through EC surface expression of TF. To that end, TF activity may represent a novel target to ameliorate pathological inflammatory conditions mediated by this cytokine.
- Zentella A, Manogue K, Cerami A: The role of cachectin/TNF and other cytokines in sepsis. Bacterial endotoxins: cytokine mediators and new therapies for sepsis. 1991, New York: Wiley-Liss, Inc, 9-24.
- Brandtzaeg P, Waage A, Mollnes TE, Oktedalen O, Kierulf P: Severe human septic shock involves more than tumor necrosis factor. Bacterial Endotoxins: cytokine mediators and new therapies for sepsis. 1991, New York: Wiley-Liss, Inc, 25-42.
- Ghivizzani SC, Kang R, Georgescu HI, Lechman ER, Jaffurs D, Engle JM, Watkins SC, Tindal MH, Suchanek MK, McKenzie LR, Evans CH, Robbins PD: Constitutive intra-articular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis. J Immunol. 1997, 159: 3604-12.PubMed
- Pettipher ER, Higgs GA, Henderson B: Interleukin 1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint. Proc Natl Acad Sci USA. 1986, 83: 8749-53.PubMed CentralView ArticlePubMed
- Mulcare RJ, Solis A, Fortner JG: Isolation and perfusion of the liver for cancer chemotherapy. J Surg Res. 1973, 15: 87-95. 10.1016/0022-4804(73)90147-9.View ArticlePubMed
- Gershenwald JE, Fong Y, Fahey TJI, Calvano SE, Chgizzonite R, Kilian PL, Lowry SF, Moldawer LL: Interleukin 1 receptor blockade attenuates the host inflammatory response. Proc Natl Acad Sci USA. 1990, 87: 4966-70.PubMed CentralView ArticlePubMed
- Fischer E, Marano MA, van Zee KJ, Rock CS, Hawes AS, Thompson WA, DeForge L, Kenney JS, Remick DG, Bloedow DC, Thompson RC, Lowry SF, Moldawer LL: Interleukin-1 receptor blockade improves survival and hemodynamic performance in Escherichia coli septic shock, but fails to alter host responses to sublethal endotoxemia. J Clin Invest. 1992, 89: 1551-7.PubMed CentralView ArticlePubMed
- Cominelli F, Nast CC, Clark BD, Schindler R, Lierena R, Eysselein VE, Thompson RC, Dinarello CA: Interleukin-1 (IL-1) gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitis. J Clin Invest. 1990, 86: 972-80.PubMed CentralView ArticlePubMed
- Alexander HR, Doherty GM, Venzon DJ, Merino MJ, Fraker DL, Norton JA: Recombinant interleukin-1 receptor antagonist (IL-1ra): Effective therapy against gram-negative sepsis in rats. Surgery. 1992, 112: 188-94.PubMed
- Jiang Y, Genant HK, Watt I, Cobby M, Bresnihan B, Aitchison R, McCabe D: A multicenter, double-blind, dose-ranging, randomized, placebo-controlled study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis: radiologic progression and correlation of Genant and Larsen scores. Arthritis Rheum. 2000, 43: 1001-9. 10.1002/1529-0131(200005)43:5<1001::AID-ANR7>3.0.CO;2-P.View ArticlePubMed
- Bevilacqua MP, Pober JS, Majeau GR, Cotran RS, Gimbrone MA: Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells. J Exp Med. 1984, 160: 618-23. 10.1084/jem.160.2.618.View ArticlePubMed
- Dias S, Choy M, Alitalo K, Rafii S: Vascular endothelial growth factor (VEGF)-C signaling through FLT-4 (VEGFR-3) mediates leukemic cell proliferation, survival, and resistance to chemotherapy. Blood. 2002, 99: 2179-84. 10.1182/blood.V99.6.2179.View ArticlePubMed
- Gloor SM, Weber A, Adachi N, Frei K: Interleukin-1 modulates protein tyrosine phosphatase activity and permeability of brain endothelial cells. Biochem Biophys Res Commun. 1997, 239: 804-9. 10.1006/bbrc.1997.7557.View ArticlePubMed
- Lebovic DI, Bentzien F, Chao VA, Garrett EN, Meng YG, Taylor RN: Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta. Mol Hum Reprod. 2000, 6: 269-75. 10.1093/molehr/6.3.269.View ArticlePubMed
- Carmeliet P: Mechanisms of angiogenesis and arteriogenesis. Nature Med. 2000, 6: 389-95. 10.1038/74651.View ArticlePubMed
- Osnes LT, Westvik AB, Joo GB, Okkenhaug C, Kierulf P: Inhibition of IL-1 induced tissue factor (TF) synthesis and procoagulant activity (PCA) in purified human monocytes by IL-4, IL-10 and IL-13. Cytokine. 1996, 8: 822-7. 10.1006/cyto.1996.0110.View ArticlePubMed
- Schwager I, Jungi TW: Effect of human recombinant cytokines on the induction of macrophage procoagulant activity. Blood. 1994, 83: 152-60.PubMed
- Didier N, Romero IA, Creminon C, Wijkhuisen A, Grassi J, Mabondzo A: Secretion of interleukin-1beta by astrocytes mediates endothelin-1 and tumour necrosis factor-alpha effects on human brain microvascular endothelial cell permeability. J Neurochem. 2003, 86: 246-54. 10.1046/j.1471-4159.2003.01829.x.View ArticlePubMed
- Nooteboom A, van der Linden CJ, Hendriks T: Tumor necrosis factor-alpha and interleukin-1beta mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood. Crit Care Med. 2002, 30: 2063-8. 10.1097/00003246-200209000-00019.View ArticlePubMed
- Goldblum SE, Yoneda K, Cohen DA, McClain CJ: Provocation of pulmonary vascular endothelial injury in rabbits by human recombinant interleukin-1 beta. Infect Immun. 1988, 56: 2255-63.PubMed CentralPubMed
- Schulman CI, Wright JK, Nwariaku F, Sarosi G, Turnage RH: The effect of tumor necrosis factor-alpha on microvascular permeability in an isolated, perfused lung. Shock. 2002, 18: 75-81. 10.1097/00024382-200207000-00014.View ArticlePubMed
- Reynolds AM, Holmes MD, Scicchitano R: Interleukin-1beta and tumour necrosis factor-alpha increase microvascular leakage in the guinea pig trachea. Respirology. 2002, 7: 23-8. 10.1046/j.1440-1843.2002.00362.x.View ArticlePubMed
- Okusawa S, Gelfand JA, Ikejima T, Connolly RJ, Dinarello CA: Interleukin-1 induces a shock like state in rabbits. J Clin Invest. 1988, 81: 1162-72.PubMed CentralView ArticlePubMed
- Dejana E, Bertocchi F, Bortolami MC, Regonesi A, Tonta A, Breviario F, Giavazzi R: Interleukin 1 promotes tumor cell adhesion to cultured human endothelial cells. J Clin Invest. 1988, 82: 1466-70.PubMed CentralView ArticlePubMed
- Sato N, Goto T, Haranka K, Satomi N, Nariuchi H, Mano-Hirano Y, Sawasaki Y: Action of Tumor Necrosis Factor on Cultered Vascular Endothelial Cells: Morphologic Modulation, Growth Inhibiton, and Cytoxicity. J Natl Cancer Inst. 1986, 76 (6): 1113-21.PubMed
- Zhao B, Stavchansky SA, Bowden RA, Bowman PD: Effect of interleukin-1beta and tumor necrosis factor-alpha on gene expression in human endothelial cells. Am J Physiol Cell Physiol. 2003, 284: C1577-C1583.View ArticlePubMed
- Friedl J, Puhlmann M, Bartlett DL, Libutti SK, Turner EN, Gnant MF, Alexander HR: Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF. Blood. 2002, 100: 1334-9.PubMed
- Alexander HR, Feldman AL: Tumor necrosis factor: Basic principles and clinical application in systemic and regional cancer treatment. Biologic Therapy of Cancer. Edited by: Steven A, Rosenberg MD. 2000, Philadelphia: Lippincott, 174-93.
- Olieman AFT, van Ginkel RJ, Hoekstra HJ, Mooyaart EL, Molenaar WM, Koops HS: Angiographic response of locally advanced soft-tissue sarcoma following hyperthermic isolated limb perfusion with tumor necrosis factor. Ann Surg Oncol. 1997, 4: 64-9.View ArticlePubMed
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