TGF-ß1 down-regulates inflammatory cytokine-induced VCAM-1 expression in cultured human glomerular endothelial cells

Su-Kil Park, Won Seok Yang, Sang Koo Lee, Hanjong Ahn, Jung Sik Park, Onyou Hwang and Jae Dam Lee

Departments of Internal Medicine, Urology and Biochemistry, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Korea



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Endothelial cells are active participants in the processes controlling coagulation, inflammation and the immune response. Variations are recognized between endothelia isolated from different vascular beds as well as from different species. Though transforming growth factor-ß1 (TGF-ß1) has been known to have an anti-inflammatory action, little is known about its effect on expression of cellular adhesion molecules during the inflammatory process in human glomerular endothelial cells. The aim of this study was to determine the effect of TGF-ß1 on the inflammatory cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human glomerular endothelial cells.

Methods. The culture of human glomerular endothelial cells was established using the normal portion of nephrectomized renal tissues and identified by factor VIII staining and cellular uptake of fluorescent-labelled acetylated low-density lipoprotein (LDL). The endothelial cells were stimulated by interleukin-1ß (IL-1ß), tumour necrosis factor-{alpha} (TNF-{alpha}) and interferon-{gamma} (IFN-{gamma}) with or without TGF-ß1. Cellular expression of VCAM-1 was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and VCAM-1 mRNA was measured by Northern blot analysis.

Results.TGF-ß1 (1, 10 and 25 ng/ml) blunted IL-1ß- (5 ng/ml) induced VCAM-1 expression significantly (OD=1.08±0.14, 1.10±1.16 and 1.05±0.14 vs IL-1ß=1.97±0.29, n=6, P<0.05) in ELISA. The addition of TGF-ß1 (1, 10 and 25 ng/ml) also suppressed TNF-{alpha}- (10 ng/ml) induced VCAM-1 expression (OD=1.14±0.15, 1.17±0.17 and 1.18±0.16 vs TNF-{alpha}=1.96±0.26, n=6, P<0.05). The same results were obtained by flow cytometry. TGF-ß1 (10 ng/ml) inhibited both IL-1ß- (5 ng/ml) and TNF-{alpha}-(10 ng/ml) induced expression of VCAM-1 (MFI: IL-1ß=90.8± 17.6, IL-1ß+TGF-ß1=37.8±14.9, TNF-{alpha}=113.6± 12.4, TNF-{alpha}+TGF-ß1=64.3±13.8, mean±SD, n=3, P<0.05). By Northern blot analysis, TGF-ß1 (10 ng/ml) significantly suppressed the stimulatory effect of IL-1ß and TNF-{alpha}.

Conclusions. These results show that TGF-ß1 down-regulates the inflammatory cytokine-induced expression of VCAM-1 in human glomerular endothelial cells, which could be a novel mechanism for the anti-inflammatory action of TGF-ß1 during the inflammatory processes in human glomerular diseases.

Keywords: cytokines; culture; glomerular endothelial cells; TGF-ß1; VCAM-1



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endothelial cells are active participants in the processes controlling coagulation, inflammation and immune responses [1]. In response to inflammatory stimuli, these cells secrete cytokines, including chemotactic factors [2,3]. Endothelial cells can also serve as targets that undergo cell injury [4]. The biological responses of endothelial cells can vary depending on their vascular origin. Differences are recognized between endothelia isolated from different vascular beds within the same species, as well as from different species [5]. Large vessel endothelium does not normally play a major role in the immune response, except when components of the vessel wall themselves come under immune attack, as observed during vasculitis. In contrast, capillary and post-capillary venular endothelial cells play a central role during immune responses because their activation serves to facilitate the recruitment of leukocytes to the locus of inflammation [6]. Glomerular endothelial cells belong to a specialized microvascular bed, and only recently has it been possible to isolate and propagate these cells [5,7]. For this reason, little has been known about their function in normal and pathological conditions [8–10].

Vascular cell adhesion molecule-1 (VCAM-1) was first identified as an endothelial cell surface adhesion receptor, the expression of which was induced by interleukin-1ß (IL-1ß) and tumour necrosis factor-{alpha}> (TNF-{alpha}) [11]. As a member of the immunoglobulin gene superfamily, this molecule is a ligand for the very late antigen-4 (VLA-4) integrin. It arrests circulating leukocytes and performs the first step in leukocyte recruitment to an inflamed tissue site [12]. In addition, VCAM-1 is involved in the initiation of T-cell activation and proliferation, and ultimately, T-cell death [13]. It may play important roles in a wide range of pathological states involving cell–cell recognition.

The most prominent mediators of endothelial cell activation are TNF-{alpha} and IL-1ß, though several components of the complement cascade, lipid mediators and other inflammatory cytokines also participate in eliciting the endothelial responses to immune injury [6]. Interferon-{gamma} (IFN-{gamma}) was the first identified cytokine shown to affect endothelial cells [4]. TGF-ß is a multifunctional cytokine with potent effects on development, cell growth, chemotaxis and immune function [14]. Evidence exists that TGF-ß down-regulates cytokine-stimulated leukocyte adhesion to the endothelium. Gamble et al. [15] reported an inhibitory effect of TGF-ß on neutrophil adherence to human umbilical vein endothelial cells by inhibition of E-selectin expression. Though TGF-ß1 has been known to have an anti-inflammatory action, little is known about its effects on expression of cellular adhesion molecules during inflammatory process in human glomerular diseases. In the present study, we investigated the effect of inflammatory cytokines on VCAM-1 expression in cultured human glomerular endothelial cells and the regulatory effect of TGF-ß1 on VCAM-1 expression.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Cell culture and characterization
Human glomerular endothelial cells were isolated from a normal portion of nephrectomized tissues from patients with renal cell carcinoma, as described previously [5]. The renal cortex was minced and passed through 250 µm mesh sieves. The cortical mash was collected in Hanks' balanced salt solution (HBSS) containing antibiotics. The glomerular suspension was washed and centrifuged four times at 800 g until free of blood. The suspension was then filtered sequentially through 200 and 150 µm mesh sieves. Glomeruli were collected from the surface of the 150 µm sieve. After centrifugation with HBSS, the glomerular suspension was resuspended in Waymouth media (Gibco Laboratories, Grand Island, NY) containing 20% fetal calf serum (FCS) and passed through a 25 gauge needle five times to rupture Bowman's capsule. The glomeruli were then centrifuged, washed and resuspended in Waymouth MB 752/1 supplemented with 20% FCS, fetuin (100 µg/ml, Gibco Laboratories), glutamine (2 mM, Gibco Laboratories) and antibiotics (basal culture medium). After washing and centrifugation, the pellet was incubated for 30 min at 37°C in sterile Waymouth medium containing 6 mg/ml collagenase type III (Worthington Biomedical, NJ). The glomerular pellet was then resuspended in basal culture medium, centrifuged and filtered sequentially by gravity through 100 and 50 µm sieves. Glomerular segments retained by the 50 µm filter were collected and plated onto two fibronectin-coated surfaces (60 mm dish) in basal culture medium containing heparin (100 µg/ml, Sigma Chemical, St Louis, MO) and endothelial cell growth factor (200 µg/ml, ECGF), which was semi-purified from bovine hypothalamus as described by Maciag et al. [16]. After 10–14 days of primary culture, usually 10–20 microcolonies of the cells could be obtained in each 60 mm dish. Most cells in the microcolonies had a cobblestone appearance. A few microcolonies with atypical appearance were removed mechanically by a rubber policeman. In 3–4 weeks, the cells were grown to confluence. The adherent cells were then trypsinized and transferred to a T 25 flask coated with fibronectin and cultured in the presence of puromycin (10 µg/ml, Sigma Chemical Co.) for 24 h to prevent epithelial cell contamination after 4–5 days of first subculture. The monolayers of endothelial cell were fed every 3–4 days, and experiments were performed on cells between passages 4 and 8.

Endothelial cells were identified by phase-contrast microscopy, immunofluorescence staining and immunohistochemical staining. Rabbit anti-human factor VIII antibody (Dako A0082; Dako, Glostrup, Denmark), mouse anti-human cytokeratin antibody (Dako M0821) and mouse anti-human smooth muscle actin (Dako M0851) were used for immunohistochemical staining. The ability to take up fluorescent-labelled (1,1-dioctadecyl-1-1-3,3,3,3-tetramethyl-indocarbo-cyanine perchlorate)-acetylated low-density lipoprotein (LDL) (DiI-Ac-LDL, Biomedical Technologies, Stoughton, MA) was used as a positive marker for endothelial cells [17].

Cellular ELISA
IL-1ß, TNF-{alpha}, IFN-{gamma}, TGF-ß1 and mouse anti-human VCAM-1 monoclonal antibody were purchased from R&D Systems (Abingdon, UK). Dexamethasone was purchased from Sigma. Total cellular expression of VCAM-1 was measured by enzyme-linked immunosorbent assay (ELISA) on fixed adherent cells as previously described [18]. In brief, 1x104 endothelial cells were cultured in fibronectin-coated 96-well plates with basal culture medium for 48 h, and washed with phosphate-buffered saline (PBS) twice. The medium subsequently was changed to serum-free medium, and the cytokines were applied for 6 h. The cells were washed and fixed in fresh 3.7% formaldehyde buffer for 5–10 min, and washed three times with PBS. After incubation in 1% bovine serum albumin (BSA) in PBS for 1 h at 37°C to block non-specific binding, the cells were incubated with VCAM-1mAb for 18 h at room temperature and washed three times with 1% BSA in PBS. Peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, Westgrove, PA) was applied and incubated at 37°C for 60 min. After washing, 100 µl of TMB/E substrate solution was added for the colour reaction, which was stopped with addition of 100 µl of 2 M sulfuric acid after 10 min. Optical density at 450 nm was determined spectrophotometrically using a Molecular Devices (Menlo Park, CA) microplate reader.

Flow cytometry
Single cell suspensions, obtained mechanically by a rubber policeman from a fibronectin-coated 100 mm dish, were centrifuged (200 g, 5 min) and resuspended in PBS. The cells (5x104) were incubated in microplates at 4°C with gentle stirring, with fluorescein isothiocyanate (FITC)-labelled antibody BBA22 anti-human VCAM-1 (R&D, Abingdon, UK). The final suspension was made in 200 µl of medium containing 1% paraformaldehyde. Cell samples were analysed by flow cytometry using FACScan (Becton Dickinson).

RNA isolation and Northern hybridization
Endothelial cells grown to confluence in 100 mm fibronectin-coated dishes (Corning) were treated with IL-1ß (5 ng/ml) and TNF-{alpha} (10 ng/ml) in serum-free media. At the indicated time points, total cellular RNA were isolated by using the Tri reagent kit® (Molecular Research Center Inc., Cincinnati, OH). The amount of RNA was measured by absorbance at 260 nm and its purity assessed by the absorbance ratio at 260/280 nm. Northern hybridization was performed as previously described [19]. Briefly, RNA (10 µg/lane) was electrophoresed through a gel containing 1% agarose and 2.2 M formaldehyde with MOPS buffer, followed by capillary transfer to a nylon membrane. After transfer, RNA integrity was assessed by methylene blue staining. After baking for 2 h at 80°C, the membrane was pre-hybridized at 65°C in 0.5 M sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, 7% SDS and 1% bovine serum albumin. The membrane was then hybridized overnight at 65°C with 100 µg/ml salmon sperm DNA and 32P-labelled cDNA probes, and labelled with 32P by the random priming method (MegaprimeTM DNA labelling system, Amersham, UK). After a series of washing, the filters were autoradiographed at -80°C and the intensity of the bands on the autoradiogram was measured by scanning laser densitometry (GS-670 imaging densitometer, Bio-Rad). The same filters were rehybridized with a cDNA specific for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) to correct for variation in RNA loading and transfer efficiency. The level of VCAM-1 mRNA was normalized against that of GAPDH mRNA from each sample.

Generation of cDNA probes
cDNA probes specific for VCAM-1 and GAPDH were made by reverse transcription–polymerase chain reaction (RT–PCR), as described previously [19]. Each PCR primer set was commercially synthesized (by Korea Biotech, Inc., Korea) based on the published cDNA sequences for human VCAM-1 [20] and GAPDH [21]. The primer sequences were as follows: VCAM-1: forward primer, 5'-TGGCCTCGTGAATGGGAGC-3'; reverse primer, 5'-CCGCATCCTTCAACTGGGC-3'; GAPDH: forward primer, 5'-TCACCATCTTCCAGGAGCG-3'; reverse primer, 5'-CTGCTTCACCACCTTCTTGA-3'. The identity of the PCR products was confirmed by detection of the expected size on an agarose gel. Each PCR product was purified using a Jetsorb gel extraction kit (Genomed Inc., USA).

Statistical analysis
Data are presented as the mean±SE, with n representing the number of different experiments. Comparisons of the values between groups were performed by Mann–Whitney U test. P<0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characterization of human glomerular endothelial cells
The cells were identified as endothelial cells by their characteristic morphology under phase-contrast microscopy. After removing morphologically uncharacteristic cells with a rubber policeman, homogenous microcolonies of endothelial cells could be obtained. They appeared polygonal with indistinct cell borders and, when grown to confluence in medium containing FCS, they exhibited a characteristic cobblestone appearance (Figure 1AGo). To confirm their endothelial cell origin, expression of factor VIII antigen by immunohistochemical staining and the uptake of acetylated LDL were evaluated (Figure 1BGo and CGo). Immunohistochemically, cytokeratin and smooth muscle actin were negative, and virtually all cells demonstrated homogenous perinuclear cytoplasmic staining of factor VIII. The cytoplasmic granular uptake of DiI-Ac-LDL was also observed in all cells with >70–80% cells demonstrating intense uptake of this functional marker. The isolation of glomerular endothelial cells was more successful with kidneys from younger patients, but unsuccessful with those from patients aged >60 years. Cells from 18 different patients were used for the experiments. After nine passages, the cellular morphology changed and the cell growth rate rapidly declined, thus the culture could not be maintained.



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Fig. 1. Characterization of human glomerular endothelial cells. (A) Phase-contrast photomicrograph of confluent glomerular endothelial cells (x100). (B) Immunohistochemical staining of glomerular endothelial cells. Virtually all of the cells demonstrated perinuclear cytoplasmic staining of factor VIII (passage 4, x200). (C) Immunofluorescent photomicrograph of glomerular endothelial cells (passage 6) labelled with DiI-Ac-LDL, indicating the presence of scavenger receptor on the cells (x200). After nine passages, the cellular morphology changed and the growth rate declined rapidly; therefore, the endothelial cell culture could not be maintained.

 

The effect of proinflammatory cytokine on the expression of VCAM-1
Expression of VCAM-1 on basal human glomerular endothelial cells was not detectable above background level (OD of control=0.36±0.02, mean±SE, n=24; each n is the mean of 3–8 well experiments) (Figure 2Go). However, VCAM-1 was induced rapidly after exposure to IL-1ß (5 ng/ml) or TNF-{alpha} (1 and 10 ng/ml). The OD, indicating the expression of VCAM-1, of the wells treated with IL-1ß (5 ng/ml) for 6 h (n=22, 1.76±0.15, P<0.05 compared with control) was significantly greater than that of control. TNF-{alpha} (1 and 10 ng/ml) also increased the expression of VCAM-1 (1.95±0.35, 1.88±0.17, n=8, 12, P<0.05 compared with control), but IFN-{gamma} (10 ng/ml) did not (0.36±0.08, n=4). TGF-ß1 alone had no effect on VCAM-1 expression (data not shown). Surface expression of VCAM-1 in flow cytometric analysis was also increased by IL-1ß (5 ng/ml) as well as by TNF-{alpha} (10 ng/ml) (figure 3Go). IL-1ß (5 ng/ml) increased the expression of VCAM-1 after 6 h incubation [mean fluorescence intensity (MFI): IL-1ß=51.0±38.8, control=5.6±2.7, mean±SD, n=7, P<0.05]. TNF-{alpha} (10 ng/ml) also increased the expression of VCAM-1 (MFI: TNF-{alpha}=113.6±12.4, control=5.2±2.1, mean±SD, n=3, P<0.05). By Northern blot analysis, IL-1ß (5 ng/ml) and TNF-{alpha} (10 ng/ml), but not IFN-{gamma} (10 ng/ml), increased VCAM-1 mRNA (Figure 4Go).



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Fig. 2. Expression of VCAM-1 on human glomerular endothelial cells. Expression of VCAM-1 was measured by cellular ELISA. Human glomerular endothelial cells were plated in fibronectin-coated 96-well plates. IL-1ß (5 ng/ml) and TNF-{alpha} (1 and 10 ng/ml) increased the expression of VCAM-1 after 6 h incubation (OD=1.76±0.15, 1.95±0.35, 1.88±0.17, control=0.36±0.03, mean±SE n=8–24; each n is the mean of 3–8 well experiments, *P<0.05). IFN-{gamma} (10 ng/ml) did not stimulate the expression of VCAM-1 (n=4).

 


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Fig. 3. Expression of VCAM-1 on human glomerular endothelial cells. The expression of VCAM-1 was measured by flow cytometry. The results shown are representative of 3–7 experiments. Human glomerular endothelial cells were plated in fibronectin-coated 100 mm dishes. IL-1ß (5 ng/ml) increased the expression of VCAM-1 after 6 h incubation (MFI: IL-1ß=51.0±14.6, control=5.6±1.0, mean±SE, n=7, P<0.05). TNF-{alpha} (10 ng/ml) also increased the expression of VCAM-1 after 6 h incubation (MFI: TNF-{alpha}=113.6±12.4, control=5.2±0.9, mean±SE, n=3, P<0.05). The vertical axis indicates the cell numbers in each channel and the horizontal axis the intensity of fluorescence on a log scale. MFI: mean fluorescent intensity.

 


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Fig. 4. Effect of proinflammatory cytokine on the induction of VCAM-1 mRNA. Human glomerular endothelial cells were stimulated with IL-1ß (5 ng/ml), TNF-{alpha} (10 ng/ml) and IFN-{gamma} (10 ng/ml) for the indicated time period. RNA extracts were examined by Northern blot analysis for VCAM-1 mRNA and GAPDH mRNA. IL-1ß and TNF-{alpha} increased the expression of mRNA of VCAM-1, but IFN-{gamma} did not. The results shown are representative of two experiments.

 

The effect of TGF-ß1 and dexamethasone on cytokine-induced VCAM-1 expression
Co-incubation of dexamethasone (10 µM) with IL-1ß (5 ng/ml) partially decreased the expression of VCAM-1 (OD=1.70±0.28 vs IL-1ß=2.32±0.32, n=6, P<0.05) (Figure 5Go). Dexamethasone (10 µM) also reduced the TNF-{alpha}-(1 ng/ml) induced expression of VCAM-1 (1.50±0.30 vs TNF-{alpha}=2.26±0.39, n=6, P<0.05) (Figure 5Go). Interestingly, TGF-ß1 (1, 10 and 25 ng/ml) blunted IL-1ß- (5 ng/ml) induced VCAM-1 expression significantly (OD=1.08±0.14, 1.10±1.16, 1.05±0.14 vs IL-1ß=1.97±0.29, n=6, P<0.05) (Figure 6Go). The addition of TGF-ß1 (1, 10 and 25 ng/ml) also suppressed TNF-{alpha}- (10 ng/ml) induced VCAM-1 expression (OD=1.14±0.15, 1.17±0.17, 1.18±0.16 vs TNF-{alpha}=1.96±0.26, n=6, P<0.05) (Figure 7Go). The same results were obtained by flow cytometry (Figure 8AGo and BGo). TGF-ß1 (10 ng/ml) inhibited both IL-1ß (5 ng/ml) and TNF-{alpha}- (10 ng/ml) induced expression of VCAM-1 (MFI: A, IL-1ß=90.8±17.6, IL-1ß+TGF-ß1=37.8±14.9; B, TNF-{alpha}=113.6±12.4; TNF-{alpha}+TGF-ß1=64.3±13.8, mean± SD, n=3, P<0.05). By Northern blot analysis, TGF-ß1 (10 ng/ml) significantly suppressed the stimulatory effect of IL-1ß and TNF-{alpha} (Figure 9Go).



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Fig. 5. Effect of dexamethasone on the expression of VCAM-1. The expression of VCAM-1 was measured by cellular ELISA. Co-incubation of dexamethasone (10 µM) with IL-1ß (5 ng/ml) decreased the expression of VCAM-1 (OD=1.70±0.28, IL-1ß=2.32±0.32, n=6, *P<0.05). Dexamethasone also reduced the TNF-{alpha}- (1 ng/ml) induced expression of VCAM-1 (1.50±0.30, TNF-{alpha}=2.26±0.39, n=6, *P<0.05). The results are given as mean±SE. Each experiment was done in 4–8 wells.

 


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Fig. 6. Effect of TGF-ß1 on the IL-1ß-induced expression of VCAM-1. The expression of VCAM-1 was measured by cellular ELISA. Human glomerular endothelial cells were grown in fibronectin-coated 96-well plates. Co-incubation of TGF-ß1 (1, 10 and 25 ng/ml) inhibited IL-1ß- (5 ng/ml) induced VCAM-1 expression (OD=1.08±0.14, 1.10±0.16, 1.05±0.14, IL-1ß=1.97±0.29, n=6, *P<0.05) in a 6 h incubation. Lower concentrations of TGF-ß1 (0.001, 0.01 and 0.1 ng/ml) did not inhibit the IL-1ß-induced expression of VCAM-1. The results are given as mean±SE. Each experiment was done in four wells.

 


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Fig. 7. Effect of TGF-ß1 on the TNF-{alpha}-induced expression of VCAM-1. TGF-ß1 (1, 10 and 25 ng/ml) suppressed TNF-{alpha}- (10 ng/ml) induced VCAM-1 expression (OD=1.14±0.15, 1.17±0.17, 1.18±0.16, TNF-{alpha}=1.96±0.26, n=6, *P<0.05). Lower concentrations of TGF-ß1 (0.001, 0.01 and 0.1 ng/ml) did not inhibit the TNF-{alpha}-induced expression of VCAM-1. The results are given as mean±SE. Each experiment was done in four wells.

 


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Fig. 8. The effect of TGF-ß1 on the proinflammatory cytokine-induced expression of VCAM-1. The expression of VCAM-1 was measured by flow cytometry. The results shown are representative of three experiments. Human glomerular endothelial cells were plated in fibronectin-coated 100 mm dishes. TGF-ß1 (10 ng/ml) inhibited both IL-1ß- (5 ng/ml) and TNF-{alpha}-(10 ng/ml) induced expression of VCAM-1 (MFI: A, IL-1ß=90.8±10.2, IL-1ß+TGF-ß1=37.8±8.5; B, TNF-{alpha}=113.6±12.4, TNF-{alpha}+TGF-ß1=64.3±8.0, mean±SE, n=3, P<0.05). The vertical axis indicates the cell numbers in each channel and the horizontal axis the intensity of fluorescence on a log scale. MFI: mean fluorescent intensity.

 


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Fig. 9. Effect of TGF-ß1 and dexamethasone on the proinflammatory cytokine-induced expression of VCAM-1 mRNA. Human glomerular endothelial cells were stimulated with IL-1ß (5 ng/ml) or TNF-{alpha} (10 ng/ml) with or without TGF-ß1 (10 ng/ml) or dexamethasone (10 µM) for the indicated time period. RNA extracts were examined by Northern blot analysis for VCAM-1 and GAPDH mRNA. TGF-ß1 as well as dexamethasone inhibited IL-1ß- or TNF-{alpha}-induced expression of VCAM-1 mRNA. The results shown are representative of two experiments.

 



   Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
Our results demonstrate that IL-1ß and TNF-{alpha}, but not IFN-{gamma}, stimulate expression of VCAM-1 in cultured human glomerular endothelial cells. We also found that TGF-ß1 suppressed the stimulatory effect of these cytokines on the expression of VCAM-1.

Regulation of the adhesion molecules has been well characterized in human umbilical vein endothelial cells, but not in human glomerular endothelial cells. Large vessel endothelium normally does not play a major role in the immune response. In contrast, capillary and post-capillary venular endothelial cells play a central role during immune responses because their activation serves to facilitate the recruitment of leukocytes to the locus of inflammation [6]. Glomerular endothelial cells belong to a specialized microvascular bed [5]. Infiltration of the renal parenchyma with inflammatory cells, including lymphocytes, monocytes and granulocytes, characterizes the histological picture of inflammatory kidney diseases, autoimmune disorders and renal allograft rejection [22]. In many forms of immune-mediated glomerular disease, there is evidence for local glomerular synthesis of TNF-{alpha} and IL-1ß, the two key cytokines involved in endothelial cell activation. TNF-{alpha} is produced primarily by inflammatory cells and non-endothelial glomerular cells, while IL-1ß is produced by endothelial cells as well [22].

Normal kidney glomerular endothelial cells express interstitial cell adhesion molecule (ICAM-1) constitutively, but do not express VCAM-1 or E-/P-selectin [23,24]. Although Seron et al. [25] could not identify VCAM-1 on vascular endothelium in human renal disease, the expression of VCAM-1 in glomerular capillary wall was increased histologically in vasculitis, Henoch–Schönlein purpura, lupus nephritis and membranoproliferative glomerulonephritis [26,27]. Hauser et al. [28] also reported that vascular and/or glomerular VCAM-1 and E-selectin expression was pronounced in severe acute allograft rejection and in primary renal diseases with or without autoimmune disorders. Nikolic-Paterson et al. [29] found that glomerular VCAM-1 mRNA was up-regulated following 6 h culture with IL-1{alpha}. In in vitro experiments using isolated glomeruli, Savage et al. [30] reported that VCAM-1 was not inducible in isolated glomeruli or in kidney pieces by IL-1ß, TNF, IFN-{gamma}, IL-4, granulocyte–macrophage colony-stimulating factor (GM-CSF), TGF-ß, or by TNF+IFN-{gamma}, but was weakly inducible by TNF+IL-4.

As VCAM-1 has a role in arresting circulatory leukocytes and recruiting leukocytes to the inflammatory site [12], it could be said that VCAM-1 is very important in the initiation of human glomerular disease.

In the present study using cultured human glomerular endothelial cells, we confirmed that VCAM-1 could be induced by proinflammatory cytokines, IL-1ß and TNF-{alpha}, but not by immune-regulatory cytokine, IFN-{gamma}.

TGF-ß is a multifunctional cytokine with potent effects on development, cell growth, chemotaxis and immune function [14]. There are five known isoforms of TGF-ß, three of which are expressed in human (TGF-ß1, TGF-ß2 and TGF-ß3). TGF-ß1 is the dominant TGF-ß isoform found in nephritic tissue [31]. Diffuse proliferative lupus nephritis and rapidly progressive glomerulonephritis showed more expression of TGF-ß1 than did focal proliferative lupus nephritis or IgA nephropathy [31]. TGF-ß1 synthesis and activation of latent TGF-ß are enhanced in activated endothelial cells [32] and mesangial or tubular cells [33].

Gamble et al. described an inhibitory effect of TGF-ß on neutrophil adherence in human umbilical vein endothelial cells by inhibition of E-selectin expression, but they could not find an inhibitory effect of TGF-ß on the expression of VCAM-1 [15]. Another study using human astroglioma cell lines and primary human fetal astrocytes showed that TGF-ß inhibited the proinflammatory cytokine-induced expression of VCAM-1 [34]. In the present study, we observed that TGF-ß1 as well as dexamethasone inhibited the cytokine-induced expression of VCAM-1; therefore, TGF-ß1 could down-regulate the inflammatory process in human glomerular disease. The difference between our results and those of Gamble et al. might be due to the difference of vascular beds.

TGF-ß, a well-known fibrogenic cytokine, acts as a biological rescue molecules to maintain homeostasis and initiate repair in tissue injury [35]. As a healing process, TGF-ß may well down-regulate the expression of inflammatory cytokine-induced VCAM-1. However, to identify the precise mechanisms and potential significance of TGF-ß in regulation of the adhesion molecule, further study will be needed.

Grandaliano et al. reported that in bovine glomerular endothelial cells, the factor VIII antigen disappeared with the passage of endothelial cells [36]. They found that only a small percentage (10–15%) of the cells demonstrated factor VIII staining, and they could maintain the cells up to passage 22. On the contrary, we could not maintain the endothelial cells beyond nine passages in our experiments, and when the age of patient was ~60 years, the cells did not survive after six passages. Only the cells from young patients could be maintained until eight passages (data not shown). To obtain glomerular endothelial cell culture from human kidney, the cells from a young patient would be better than those from older patients.

In summary, expression of VCAM-1 was induced by IL-1ß and TNF-{alpha} but not IFN-{gamma}, and TGF-ß1 as well as dexamethasone significantly inhibited the expression of VCAM-1. This study demonstrates an important biological effect of TGF-ß1 in human glomerular endothelial cells, which could be an important mechanism of anti-inflammatory action of TGF-ß1 during the inflammatory process in human glomerular disease.



   Acknowledgments
 
We thank Nam Jung Han for excellent technical help and acknowledge the financial support of the Korea Research Foundation 1998.



   Notes
 
Correspondence and offprint requests to: Su-Kil Park, MD, PhD, Department of Internal Medicine, Asan Medical Center, College of Medicine, University of Ulsan, Song-Pa, PO Box 145, Seoul 138-736, Korea. Back



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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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