Departments of Internal Medicine, Urology and Biochemistry, Asan Medical Center, College of Medicine, University of Ulsan, Seoul, Korea
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (TNF-
) and interferon-
(IFN-
) 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-- (10 ng/ml) induced VCAM-1 expression (OD=1.14±0.15, 1.17±0.17 and 1.18±0.16 vs TNF-
=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-
-(10 ng/ml) induced expression of VCAM-1 (MFI: IL-1ß=90.8± 17.6, IL-1ß+TGF-ß1=37.8±14.9, TNF-
=113.6± 12.4, TNF-
+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-
.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-> (TNF-
) [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 cellcell recognition.
The most prominent mediators of endothelial cell activation are TNF- 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-
(IFN-
) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-, IFN-
, 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 510 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- (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 transcriptionpolymerase chain reaction (RTPCR), 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 MannWhitney U test. P<0.05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 38 well experiments) (Figure 2). However, VCAM-1 was induced rapidly after exposure to IL-1ß (5 ng/ml) or TNF-
(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-
(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-
(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-
(10 ng/ml) (figure 3
). 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-
(10 ng/ml) also increased the expression of VCAM-1 (MFI: TNF-
=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-
(10 ng/ml), but not IFN-
(10 ng/ml), increased VCAM-1 mRNA (Figure 4
).
|
|
|
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 5). Dexamethasone (10 µM) also reduced the TNF-
-(1 ng/ml) induced expression of VCAM-1 (1.50±0.30 vs TNF-
=2.26±0.39, n=6, P<0.05) (Figure 5
). 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 6
). The addition of TGF-ß1 (1, 10 and 25 ng/ml) also suppressed TNF-
- (10 ng/ml) induced VCAM-1 expression (OD=1.14±0.15, 1.17±0.17, 1.18±0.16 vs TNF-
=1.96±0.26, n=6, P<0.05) (Figure 7
). The same results were obtained by flow cytometry (Figure 8A
and B
). TGF-ß1 (10 ng/ml) inhibited both IL-1ß (5 ng/ml) and TNF-
- (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-
=113.6±12.4; TNF-
+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-
(Figure 9
).
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- and IL-1ß, the two key cytokines involved in endothelial cell activation. TNF-
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, HenochSchönlein 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. 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-
, IL-4, granulocytemacrophage colony-stimulating factor (GM-CSF), TGF-ß, or by TNF+IFN-
, 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-, but not by immune-regulatory cytokine, IFN-
.
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 (1015%) 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- but not IFN-
, 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 |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|