CD40/CD154 ligation induces mononuclear cell adhesion to human renal proximal tubule cells via increased ICAM-1 expression
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《美国生理学杂志》
Division of Nephrology, Department of Medicine, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York
ABSTRACT
The role of CD40/CD154 ligation in the upregulation of genes of the proinflammatory nuclear factor-B (NF-B) signal transduction pathway was explored in primary cultures of human renal proximal tubule epithelial cells. Using a cDNA gene array specific for human NF-B signal pathway genes, 38 genes were upregulated at 1 h, and 7 of these genes remained upregulated at 3 h. Of these genes, intercellular adhesion molecule-1 (ICAM-1) was explored in further detail. Quantitative real-time PCR for ICAM-1 mRNA expression confirmed the gene array findings. Western blot analysis and quantitative sandwich-enzyme ELISA confirmed this observation at the protein level. A cell-surface ELISA assay showed that ICAM-1 expression doubled by 48 h of CD154 exposure, and fluorescence-activated cell sorter analysis suggested that both the number of cells expressing ICAM-1 and the expression of ICAM-1 on these cells had increased. A cell adhesion assay using fluorescein-labeled human peripheral mononuclear cells showed that ICAM-1 upregulation resulted in increased mononuclear cell adhesion to the monolayer, which was abrogated by pretreatment of the monolayer with a neutralizing ICAM-1 antibody. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB-203580 but not the extracellular signal-regulated kinase 1/2 inhibitor (PD-98059) nor the protein kinase C inhibitor (calphostin) blunted ICAM-1 expression and mononuclear cell adhesion to the monolayer. We conclude that, in human renal proximal tubule epithelial cells, CD40 activation upregulates ICAM-1 (and other NF-B pathway genes) expression with concomitant enhanced adhesion of mononuclear cells, which is mediated via the p38 MAPK signal transduction pathway.
nuclear factor-B signal pathway genes; cell adhesion; mitogen-activated protein kinases; interstitial inflammation; intercellular adhesion molecule-1
PREVIOUS STUDIES FROM THIS laboratory have identified the CD40 receptor on primary cultures of human renal proximal tubule epithelial cells (PTCs; see Ref. 16). Upon activation by its cognate ligand, CD154, CD40 engages tumor necrosis factor receptor-associated factor 6 (TRAF6). CD40 and TRAF6 translocate from separate membrane microdomains to associate with one another in the cytoplasmic compartment where TRAF6 in turn activates phosphorylation of the Jun kinase and p38 mitogen-activated protein kinase (MAPK) pathways. These in turn stimulate interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 production by these cells. Furthermore, CD40 and its downstream MAPK signaling proteins (but not TRAF6) are located in membrane rafts, and disruption of caveolae or dislodgment of signaling proteins from the caveolin-1 scaffolding domain diminishes MAPK activation and IL-8 and MCP-1 production in these cells (17).
Intercellular adhesion molecule-1 (ICAM-1), or CD54, is an inducible cell adhesion glycoprotein of the immunoglobulin supergene family expressed on the surface of a wide variety of cell types (23). ICAM-1 interacts with two integrins belonging to the 2-subfamily, namely CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), found on leukocytes, which plays a key role in transendothelial migration of leukocytes and activation of T lymphocytes (23). ICAM-1 is present constitutively on the surface of a wide variety of cell types and is upregulated by numerous inflammatory mediators and proinflammatory cytokines (23).
In the current study, we examined whether CD40/CD154 ligation upregulates genes of the proinflammatory nuclear factor-B (NF-B) signal transduction pathway. A total of 38 genes were upregulated, including ICAM-1, MCP-1, and IL-8. With regard to ICAM-1, the gene array data were confirmed by real-time PCR. ICAM-1 protein expression was upregulated in parallel. At the functional level, CD40 mediated ICAM-1 upregulation correlated with enhanced mononuclear cell (MC) adhesion to the epithelial cell, which was mediated via the p38 MAPK signal transduction pathway.
MATERIALS AND METHODS
Cell culture. All experiments were carried out on primary cultures of human renal PTCs. Normal renal tissue was obtained from nephrectomy specimens during the course of cancer surgery (2), and verification of proximal tubule origin was performed as previously described (21). Cultures were maintained in a defined medium composed of 1:1 (vol/vol) mixture of DMEM and Ham's F-12 medium supplemented with insulin (5 μg/ml), transferrin (5.5 μg/ml), hydrocortisone (50 nM), triiodothyronine (5 pM) and sodium selenate (10 nM), and to which 50 IU/ml penicillin and 50 μg/ml streptomycin were added. Cells were usually grown in 25-cm2 tissue culture flasks or in 24- or 96-well plates (ELISA) and perpetuated in a humidified incubator at 37°C in 95% air-5% CO2 (culture medium pH 7.3). Media were exchanged at 48- to 72-h intervals, and cells were propagated through 8–10 passages.
GEArray. The relative mRNA expression of 96 human NF-B signal pathway genes was analyzed by GEArray (SuperArray Bioscience, Frederick, MD) according to the manufacturer's instructions. Confluent monolayers were challenged with recombinant human soluble (rhs) CD154 (100 ng/ml + 1 μg/ml enhancer) for 1 or 3 h, and total RNA was extracted with Tri-Zol reagent (Life Technologies, Rockville, MD) and compared with the vehicle-treated condition. Briefly, 5 μg total RNA were added to 3 μl GEAprimer Mix and heated to 70°C for 3 min for annealing. Reverse transcription proceeded by incubating the annealing mix with labeling master mix containing 1 mM biotin-16-dUTP (Roche Diagnostic, Mannheim, Germany) and 50 U/μl Moloney murine leukemia virus RT (Promega, Madison, WI) at 42°C for 90 min. The biotin-labeled cDNA probe was denatured using denaturing solution at 68°C for 20 min and hybridized with prewetted GEArray Q Series membrane under conditions of continuous agitation at 7 rpm/min overnight in a hybridization cylinder. After three washes, the membrane was blocked with 1.5 ml GEA blocking Solution Q (room temperature, 40 min) and then incubated with alkaline phosphatase-conjugated streptavidin (1:5,000 in washing buffer) at room temperature for 10 min with gentle mixing. After further washes, the GEArray Q Series membrane was detected with CDP-Star R Solution and X-ray film. The images were analyzed with GEArray Analyzer software.
RNA isolation and cDNA synthesis. Confluent monolayers were challenged with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) for 2–24 h, or vehicle (control), and total RNA was extracted with Tri-Zol reagent (Life Technologies), as previously described (4, 5, 16). From each sample, 5 μg total RNA was reverse-transcribed to cDNA using oligo(dT) Primer and Superscript II RT (both from Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Reverse transcription was carried out at 42°C for 1 h in a 20-μl reaction volume.
Quantitative real-time PCR. In preparatory experiments, standard PCR was performed to test the following primer pairs for ICAM-1: sense, 5'-TATGGCAACGACTCCTTCT-3' and antisense, 5'-CATTCAGCGTCACCTTGG-3' (1, NM_000201 [GenBank] ; see Ref. 15). -Actin was incorporated as a control. The primer pairs for -actin were as follows: sense, 5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG GCG-3' and antisense, 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3' (20). The PCR protocol was as follows: denature at 94°C for 30 s, anneal at 55°C for 30 s, and elongate at 72°C for 1 min using an enzyme-dye mixture from QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA). The amplified products were size fractionated on a 1% Tris-borate-EDTA-agarose gel containing ethidium bromide for resolution. The predicted size for the ICAM-1 and -actin fragments was 238 and 838 bp, respectively. The PCR products were excised from the agarose gel, purified by GFX PCR DNA and GEL Band Purification Kit (Amersham Biosciences, Piscataway, NJ), and quantified by UV-Visible Recording Spectrophotometer (Shimadzu Scientific Instruments, Braintree, MA). A range of the products from 10–9 to 10–3 pmol was used as templates in the standard curve for quantitative real-time PCR.
Quantitative real-time PCR was performed on the cDNA using the QuantiTect SYBR Green PCR Kit (Qiagen) indicated above, according to the manufacturer's protocol. The PCR master mix was prepared by combining the following reagents to the final volume of 20 μl: 1 μl primer.up (6.25 pmol/μl), 1 μl primer.dn (6.25 pmol/μl), 10 μl of the enzyme-dye mixture, and 8 μl 1:16 cDNA. The PCR master mix was placed in a 96-well PCR plate and, after an initial denaturing step (95°C for 15 min), processed according to the following PCR protocol: denature 95°C for 30 s, anneal at 55°C for 30 s, and elongate at 72°C for 1 min for 39 cycles in a DNA Engine Option System (MJ Research, Alameda, CA). Plate read temperature was 80°C. The melting curve was performed from 65 to 95°C with reading every 0.2°C and holding for 5 s between reads. The final cooling temperature was set at 12°C. The data generated were analyzed by Opticon Monitor Software (MJ Research). Three replicates were performed on each sample, and four samples were taken for each time point. Gene expression was normalized to -actin expression levels (9).
Western blot analysis. Western blot analysis was performed using protocols previously described by this laboratory (1, 16). Briefly, monolayers were challenged with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle (control) for the time periods indicated and then washed extensively with PBS (4°C). Cells were next lysed in 100 μl SDS sample buffer (1) and scraped, and the extracts were transferred to microcentrifuge tubes for sonication (10–15 s) to reduce sample viscosity. Samples were heated to 95°C for 5 min, and matched aliquots were subjected to SDS-PAGE in 10% gels, transferred to membranes, and blocked with 5% nonfat dry milk suspended with 143 mM NaCl, 0.1% Tween 20, and 20 mM Tris base, pH 7.6 (TBST; room temperature, 1 h). Filters were probed with the antibody of interest (1:1,000, overnight at 4°C), washed (3 times) with TBST, and subsequently incubated with peroxidase-linked goat anti-rabbit IgG in blocking buffer (1:2,000, 45 min, room temperature). After additional washes (3 times), antibody-bound proteins were detected using an enhanced chemiluminescence (ECL) system according to the manufacturer's instructions (Pierce, Rockford IL), and membranes were exposed to Hyperfilm ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK). Molecular weight markers were used to determine the size of the detected band. Where required, relative densities of the protein bands were determined with a GS-670 imaging densitometer and Molecular Analyst PC software program (Bio-Rad, Richmond, CA). Antibodies were obtained from commercial sources and are detailed in MATERIALS AND METHODS.
ELISA and cell surface ELISA. Soluble ICAM-1 production by PTCs was measured by the quantitative sandwich enzyme immunoassay technique according to the manufacturer's instructions (R&D Systems, Minneapolis, MN) and as previously reported by this laboratory (16). PTCs were grown in 24-well culture plates, and the experimental maneuver was performed by adding rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle to the supernatant for the indicated time periods, which was assayed for adhesion molecule production. Optical density of each microtiter plate was read at 450 nm on a Biomed microplate reader. Recombinant ICAM-1 standards from 0 to 49.55 pg were used to generate standard curves. Quadruplicate determinations in three to four different series of experiments were performed. Data are presented as means ± SE as the index of dispersion. ANOVA was used to compare group means, and the null hypothesis was rejected at P < 0.05.
ICAM-1 expression on the surface of PTCs was detected by cell-surface ELISA according to Chen et al. (3) with modifications. PTCs grown in 96-well plates were stimulated with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle for the indicated time periods and then fixed with 1% paraformaldehyde (10 min). All subsequent steps were performed at room temperature. Cells were washed with PBS (2 times) and then incubated in a blocking solution comprised of 1% BSA suspended in TBST for 1 h. Fixed cells were probed with an anti-ICAM-1 rabbit anti-human polyclonal antibody (1:100 in 1% BSA TBST; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h or with 1% BSA in TBST as a control. After six vigorous washes with TBST, cells were incubated with anti-rabbit secondary antibody linked to horseradish peroxidase (1:500 in TBST) for 30 min. TMB Microwell Peroxidase Substrate System (Kirkegaard and Perry, Gaithersburg, MD) was applied to the cells according to the manufacturer's instructions, and 1 M phosphoric acid was used to terminate the reaction. The optical density of each microtiter plate was read at 450 nm on a Biomed microplate reader. Quadruplicate determinations in three to four different series of experiments were performed. Data are presented as detailed above.
Flow cytometry cell analysis. Monolayers incubated with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle for 48 h were detached with 0.5 mM EDTA for 5 min, and the cell suspension was then adjusted to 106 cells/ml with RPMI 1640. Suspended cells were incubated for 20 min at room temperature in a dark area with 40 μg/ml mouse anti-human monoclonal ICAM-1 antibodies conjugated to phycoerythrin (PE; Santa Cruz Biotechnology). Suspensions were fixed in a 0.5% formalin/PBS solution and then washed (2 times) with 1% sodium azide/PBS solution and analyzed with a FACScan (Becton-Dickinson, San Jose, CA). Data were analyzed with Simulset software. Erythrocyte-lysed whole blood from healthy individuals was labeled with Simultest LeucoGate (CD54-FITC/CD14-PE) and Simultest Control 1/2 (IgG1-FITC/IgG2a-PE; both from Becton-Dickinson) to set up the gates and act as a negative control, respectively.
Adhesion assay for peripheral MCs to PTCs. PTCs grown in monolayers in 96-well plates were treated with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) for 24 or 48 h at 37°C, pretreated with neutralized ICAM-1 antibody (10 μg/ml, 30 min) or mouse IgG1 isotype control (10 μg/ml, 30 min before CD154 stimulation), or left untreated (control). Human peripheral MCs were isolated from healthy human peripheral blood using Ficoll-Paque (Amersham Biosciences) according to the manufacturer's instructions. MCs were labeled with 10 ng/ml BCECF-AM (30 min at 37°C), adjusted to a concentration of 2.5 x 105/ml with RPMI 1640, and added to the monolayers of PTCs in a final volume of 100 μL. PTCs and MCs were cocultured in a CO2 incubator for 1 h. Nonadherent cells were removed from the plate by gentle washing with PBS. The adherent cells were manually counted under the fluorescence microscope at x20 magnification.
Materials. DMEM, Ham's F-12 medium, RPMI 1640, and the penicillin-streptomycin solution were purchased from GIBCO Laboratories (Grand Island, NY), and newborn calf serum was from Sigma Chemical (St. Louis, MO). The rhs CD40 ligand or CD154 and enhancer (an antibody that binds CD154 trimers and has the effect of multimerizing the CD40 receptor) were purchased from Alexis (San Diego, CA). For Western blot analysis, rabbit anti-human ICAM-1 polyclonal antibody and rabbit anti-p38-MAPK antibody were purchased from Santa Cruz Biotechnology. For the cell adhesion assays, the neutralizing ICAM-1 antibody and mouse IgG1 isotype control were purchased from R&D Systems. BCECF-AM was obtained from Molecular Probes (Eugene, OR). The selective mitogen/extracellular signal-regulated kinase inhibitor PD-98059 was purchased from Cell Signaling Technology (Beverly, MA); the selective p38 inhibitor SB-203580 and the protein kinase C inhibitor calphostin C were purchased from Calbiochem (San Diego, CA). Electrophoretic-grade reagents used for SDS-PAGE were obtained from Bio-Rad Laboratories (Melville, NY). All standard chemicals used were purchased at the highest commercial grade available.
Statistical analysis. ANOVA was used to compare group means (Tukey test), and the null hypothesis was rejected at P < 0.05.
RESULTS
CD154 induces mRNA expression of NF-B signal pathway intermediates in PTCs. A cDNA gene array containing 96 genes specific for human NF-B signal pathway genes identified several genes to be significantly upregulated in human renal PTCs after exposure to rhs CD154 (100 ng/ml + 1 μg/ml enhancer). With the use of a twofold upregulation cutoff point, 38 genes were upregulated at 1 h (Table 1) and 7 genes remained upregulated at 3 h of exposure (Table 2). Of these, ICAM-1 mRNA expression was increased 7.1-fold by 1 h (Fig. 1A and Table 1) and 4.9-fold by 3 h (Fig. 1B and Table 2) compared with the vehicle-stimulated condition. IL-8 mRNA expression increased 5.2-fold (Fig. 1A and Table 1) and 2.4-fold (Fig. 1B and Table 2) at these two time points. MCP-1 mRNA expression increased 2.0-fold (Fig. 1A) and 1.2-fold (Fig. 1B) at 1 and 3 h, respectively. The latter observations are in keeping with previous published data from this laboratory, namely, that CD154 stimulates IL-8 and MCP-1 production in human renal PTCs (16). Because we wished to further explore the role of CD154/CD40 engagement in the inflammatory process, we focused our efforts on ICAM-1 expression with the consideration that this adhesion molecule might amplify the inflammatory response.
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CD154 stimulates ICAM-1 mRNA expression and protein production. Quantitative real-time PCR was used to confirm enhanced ICAM-1 expression consequent to rhs CD154 stimulation. The data are summarized in Fig. 2 and are normalized for -actin expression levels (9). Compared with the unstimulated condition, ICAM-1 expression increased 15.2 ± 1.7 (2 h)-, 6.4 ± 0.6 (4 h)-, 3.7 ± 0.4 (6 h)-, 2.5 ± 0.4 (15 h)-, and 3.2 ± 0.3 (24 h)-fold (n = 4, P < 0.05 for the 2- and 4-h time points vs. 0 h). If ICAM-1 expression levels are not corrected for -actin expression levels in this experiment, actual mRNA values can be obtained. By 2 h, ICAM-1 mRNA had increased about eightfold from an unstimulated value of 3.9 ± 1.2 (10–7 pmol) to 31.8 ± 0.8 (10–7 pmol; n = 3, P < 0.001). By 4 h, ICAM-1 mRNA remained incremented by about fourfold above the control condition, i.e., 3.9 ± 1.2 (10–7 pmol) vs. 14.8 ± 1.2 (10–7 pmol; n = 3, P < 0.001). The 6-, 15-, and 24-h values were 6.3 ± 0.4, 6.0 ± 0.7, and 6.3 ± 0.2 (10–7 pmol), respectively, none of which were significantly different from the control value of 3.9 ± 1.2 (10–7 pmol).
In parallel experiments, CD154-induced ICAM-1 protein expression was explored by Western blot analysis (Fig. 3A). Compared with p38 MAPK (whose activity but not amount is stimulated by CD154 treatment), ICAM-1 expression increased by 1.2-fold at 24 h and 1.7-fold at 48 h (Fig. 3B). Later time points were not explored. Soluble ICAM-1 expression, as measured by quantitative sandwich-enzyme ELISA, was in keeping with these observations. As shown in Fig. 4A, soluble ICAM-1 expression peaked at 48 h of rhs CD154 (100 ng/ml + 1 μg/ml enhancer) exposure to a value of 49.4 ± 0.3 vs. 19.9 ± 0.3 ng/ml in the vehicle-treated group (n = 3, P < 0.001). Earlier and later time points showed lower levels of soluble ICAM-1 expression than at 48 h, but at each time point the CD154-stimulated value was significantly higher than the control value: 12 h, (ng/ml) 17.5 ± 0.3 (CD154) vs. 13.2 ± 0.3 (control); 24 h, 32.8 ± 0.4 (CD154) vs. 17.3 ± 0.4 (control); 72 h: 33.5 ± 0.7 (CD154) vs. 23.3 ± 0.4 (control).
A more physiologically relevant measurement of ICAM-1 expression might be the cell surface expression of the adhesion molecule, rather than a measurement of the soluble product outlined above. For this purpose, a cell-surface ELISA assay described in METHODS AND MATERIALS was used. As expected, early time points (up to 20 min) failed to detect any increase in cell-surface expression of ICAM-1 on PTCs vs. the control value (0 min; Fig. 4B). Similar to the soluble ICAM-1 data, cell surface expression of the adhesion molecule peaked at 48 h to about double the baseline value. Fluorescence-activated cell sorter (FACS) analysis further confirmed this observation (Fig. 5A). The number of cells labeled with ICAM-1-conjugated PE increased from 2,500 to 4,000 (Fig. 5B), and the expression of ICAM-1 on these cells, as measured by PE intensity, increased from 4,500 to 5,400 arbitrary units (Fig. 5C). In aggregate, these observations provide compelling evidence for CD154-mediated ICAM-1 upregulation in human PTCs.
CD154-mediated ICAM-1 expression promotes MC adhesion to PTCs. To establish the functional consequence of CD154-induced ICAM-1 expression by PTCs, we explored whether this maneuver might enhance MC adhesion to the monolayer. BCECF-AM-labeled human peripheral MCs were added to the monolayer under different conditions; after a 1-h coincubation period (and the appropriate washes), cells were visualized under phase and fluorescent microscopy, and MC cells were counted manually. As shown in Fig. 6, the untreated monolayer, visualized under phase microscopy (Fig. 6A and C, left), did not autofluoresce when examined under the fluorescent microscope (Fig. 6B), and only BCECF-labeled MCs were detected by fluorescent microscopy (Fig. 6C, right). After rhs CD154 (100 ng/ml + 1 μg/ml enhancer) treatment, the number of MCs that adhered to the PTC monolayer had increased at 24 h (Fig. 6E) and 48 h (Fig. 6F) compared with the untreated condition (Fig. 6D). MC adhesion to CD154-treated PTC monolayers was largely blunted in the presence of a neutralizing ICAM-1 antibody (10 μg/ml, 30 min) both at the 24-h (Fig. 6H) and 48-h (Fig. 6I) time points. The untreated condition with or without neutralizing antibody was comparable (Fig. 6, G and D). Finally, pretreatment with mouse IgG1 (isotype control, 10 μg/ml for 30 min) had no effect on MC adhesion to CD154-treated PTC monolayers at both the 24-h (Fig. 6K) and 48-h (Fig. 6L) time points compared with Fig. 6, E and F, respectively. The untreated condition with or without the isotype control was comparable (Fig. 6, J and D). The specificity of these observations suggests that CD154-mediated ICAM-1 expression by PTCs in monolayers enhances peripheral MC adhesion.
MC adhesion to PTCs is mediated through p38 MAPK. Because CD40 signals through MAPK pathways in PTCs (16), we explored whether CD154/CD40 activation of ICAM-1 proceeded in this manner. Confluent monolayers were challenged with vehicle or rhs CD154 (100 ng/ml + 1 μg/ml enhancer) in the absence and presence of the p38 inhibitor SB-203580 and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD-98059 and then subjected to Western blot analysis. The protein kinase C inhibitor calphostin C was included as a control. A representative experiment is depicted in Fig. 7 in the format of a Western blot (Fig. 7A) and the densitometric ratio of ICAM-1 to p38 MAPK (Fig. 7B). We have previously shown that the p38 inhibitor SB-203580 blunts p38 MAPK activity but not the level of protein expression (16). CD154 increased ICAM-1 expression at 24 h (lane 2) and 48 h (lane 6) vs. the control condition (lane 1). The ICAM-1-to-p38 ratio for these data was 0.88 at 24 h (bar 2) and 1.48 at 48 h (bar 6) vs. the control condition (0.66, bar 1). The ERK1/2 inhibitor PD-98059 somewhat decreased CD154-mediated ICAM-1 expression at 24 h (0.88 vs. 0.56; lane/bar 2 vs. 3) but was without effect at 48 h (1.48 vs. 1.33; lane/bar 6 vs. 7). In contrast, the p38 inhibitor SB-203580 markedly blunted CD154-evoked ICAM-1 expression at 24 h (0.88 vs. 0.20; lane/bar 2 vs. 4) and more impressively at 48 h (1.48 vs. 0.54; lane/bar 6 vs. 8). The protein kinase C inhibitor calphostin C was without effect at 24 h (0.88 vs. 0.78; lane/bar 2 vs. 5) but appeared to increase ICAM-1 expression at 48 h (1.48 vs. 2.03; lane/bar 6 vs. 9).
The functional relevance of this observation was explored using the cell attachment assay described earlier, using the 48-h data points. A representative experiment is shown in Fig. 8. Treatment of monolayers with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) increased MC adhesion (Fig. 8D) compared with the vehicle-treated condition (Fig. 8A). The p38 inhibitor SB-203580 prevented MC attachment to CD154-treated monolayers (Fig. 8E). Indeed, MC attachment was no different from the control condition, where monolayers were similarly treated with SB-203580 (Fig. 8B). In contrast, the ERK1/2 inhibitor PD-98059 had no effect on MC adhesion to the monolayer (Fig. 8F) and was clearly different from the control experiment similarly treated with PD-98059 (Fig. 8C). In aggregate, these data are in keeping with the hypothesis that CD154-mediated ICAM-1 expression and the attendant adhesion of MC to the PTC are mediated through p38 MAPK.
DISCUSSION
The results presented demonstrate that CD154/CD40 ligation of human renal PTCs in primary culture upregulates 38 NF-B signal pathway genes by twofold at 1 h and that seven of these genes remain upregulated at 3 h (Tables 1 and 2). Of these upregulated genes, we further explored ICAM-1 (Fig. 1). Quantitative real-time PCR for ICAM-1 mRNA expression confirmed the gene array findings (Fig. 2). ICAM-1 protein expression was enhanced in parallel (Fig. 3), both in the format of soluble (Fig. 4A) and cell surface (Figs. 4B and 5) ICAM-1. CD154-mediated ICAM-1 expression by PTCs promoted MC adhesion to the monolayer (Fig. 6), which was mediated via activation of the p38 MAPK signal transduction pathway (Figs. 7 and 8).
CD40 was initially described as a B cell-specific receptor that, upon engagement by its cognate ligand CD154, resulted in B lymphocyte activation and proliferation and immunoglobulin production (6). It is now well recognized that CD40 is expressed on a wide variety of other cells, including dendritic cells, fibroblasts, and epithelial and endothelial cells, and plays a pivotal role not only in the regulation of immune responses but also in aspects of the inflammatory response in nonlymphoid cells (8, 14, 22). Because the transcription factor NF-B has been recognized as an early mediator of immune and inflammatory responses (25), it seemed logical to screen other genes of this signal transduction pathway for CD40/CD154 activation. We focused our efforts on ICAM-1 because we reasoned that adhesion of circulating monocytes to the PTC could potentially amplify the inflammatory response. Future studies may elaborate on the potential role of CD40-mediated upregulation of the other genes identified herein.
Precedent exists for CD40/CD154 stimulation of ICAM-1 production by nonlymphoid cells. For example, endothelial cells have been shown to acquire a proinflammatory and proatherogenic phenotype and express ICAM-1 and other leukocyte adhesion molecules in response to CD40/CD154 activation (13, 18). A similar type of scenario has been proposed in the setting of lupus nephritis where tubular lesions have been shown to be a good predictor of the renal outcome (9). In a retrospective study with immunohistochemistry performed on renal biopsy material obtained from 152 patients, the authors observed a high incidence of CD40 and ICAM-1 expression on damaged proximal (and distal) tubule cells. Interestingly, ICAM-1 immunostaining correlated with CD40 immunostaining. These observations suggest but do not prove that the epithelial cells of the proximal (and distal) tubule play an important role in recruiting circulating monocytes. In keeping with this concept is our demonstration that CD154-mediated ICAM-1 upregulation resulted in increased adhesion of MCs to the PTC in culture (Figs. 6 and 8). In preparatory studies, we have also demonstrated that prolonged exposure of PTCs to CD154 results in their transdifferentiation into mesenchymal cells (19). Future studies in an in vivo model may establish how these events might be interrelated.
The major intracellular signal transduction pathways involved in the regulation of ICAM-1 expression include the MAPKs, NF-B, protein kinase C, and the JAK/STAT pathway (23). In addition, a number of nuclear transcription factors have been shown to activate ICAM-1 expression (23), which enhances the complexity of the cell type-specific and stimulus-specific regulation of the ICAM-1 gene. For example, in cultured human umbilical vein endothelial cells, tumor necrosis factor (TNF)--induced ICAM-1 expression was mediated via p38 MAPK (11), whereas, in HTB-94 chondrosarcoma cells, TNF--increased ICAM-1 expression is independent on both p38 and ERK1/2 MAPK pathways (12). On the other hand, different agonists can stimulate ICAM-1 production via different MAPK activation in the same cell type. In A549 cells, both p38 and ERK1/2 MAPK pathways are involved in adenovirus-induced ICAM-1 expression (24), but neither of these pathways is involved in TNF-- and IL-1-increased ICAM-1 production (10). The current study shows that, in cultured human renal PTCs, p38 MAPK but not ERK1/2 MAPK is involved in CD40/CD154-induced ICAM-1 production (Fig. 7). The cell adhesion assay (Fig. 8) convincingly confirms this observation at the functional level.
Previous studies from this laboratory have demonstrated that CD40/CD154 ligation in human PTCs stimulates MCP-1 and IL-8 production via TRAF6 recruitment and MAPK activation (16). Using a similar approach, we now show that this proinflammatory agonist upregulates ICAM-1 expression. It is interesting to speculate whether CD40 activation might not directly enhance ICAM-1 expression and whether CD40-mediated MCP-1 and/or IL-8 production by the PTC may be the stimulus for ICAM-1 upregulation. Alternatively, CD40-mediated ICAM-1 expression may be amplified in an autocrine manner by MCP-1 and/or IL-8. These possibilities were not explored during the course of this project but form the basis for future studies.
In summary, we show that the human renal PTC, in response to CD154/CD40 ligation, upregulates a number of NF-B pathway genes and that enhanced expression of ICAM-1 correlates with increased adhesion of MCs to the epithelial cell. These findings are in keeping with the concept that the PTC, under defined conditions, produces proinflammatory chemokines and attracts leukocytes that may amplify the proinflammatory effect. Hence, the PTC appears to be a contributor to, rather than a victim of, interstitial inflammation.
GRANTS
This study was supported by a grant from the Paul Teschan Research Fund (Dialysis Clinics, Inc.).
ACKNOWLEDGMENTS
The expert secretarial assistance of Sherryl Krulik is gratefully acknowledged.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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ABSTRACT
The role of CD40/CD154 ligation in the upregulation of genes of the proinflammatory nuclear factor-B (NF-B) signal transduction pathway was explored in primary cultures of human renal proximal tubule epithelial cells. Using a cDNA gene array specific for human NF-B signal pathway genes, 38 genes were upregulated at 1 h, and 7 of these genes remained upregulated at 3 h. Of these genes, intercellular adhesion molecule-1 (ICAM-1) was explored in further detail. Quantitative real-time PCR for ICAM-1 mRNA expression confirmed the gene array findings. Western blot analysis and quantitative sandwich-enzyme ELISA confirmed this observation at the protein level. A cell-surface ELISA assay showed that ICAM-1 expression doubled by 48 h of CD154 exposure, and fluorescence-activated cell sorter analysis suggested that both the number of cells expressing ICAM-1 and the expression of ICAM-1 on these cells had increased. A cell adhesion assay using fluorescein-labeled human peripheral mononuclear cells showed that ICAM-1 upregulation resulted in increased mononuclear cell adhesion to the monolayer, which was abrogated by pretreatment of the monolayer with a neutralizing ICAM-1 antibody. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB-203580 but not the extracellular signal-regulated kinase 1/2 inhibitor (PD-98059) nor the protein kinase C inhibitor (calphostin) blunted ICAM-1 expression and mononuclear cell adhesion to the monolayer. We conclude that, in human renal proximal tubule epithelial cells, CD40 activation upregulates ICAM-1 (and other NF-B pathway genes) expression with concomitant enhanced adhesion of mononuclear cells, which is mediated via the p38 MAPK signal transduction pathway.
nuclear factor-B signal pathway genes; cell adhesion; mitogen-activated protein kinases; interstitial inflammation; intercellular adhesion molecule-1
PREVIOUS STUDIES FROM THIS laboratory have identified the CD40 receptor on primary cultures of human renal proximal tubule epithelial cells (PTCs; see Ref. 16). Upon activation by its cognate ligand, CD154, CD40 engages tumor necrosis factor receptor-associated factor 6 (TRAF6). CD40 and TRAF6 translocate from separate membrane microdomains to associate with one another in the cytoplasmic compartment where TRAF6 in turn activates phosphorylation of the Jun kinase and p38 mitogen-activated protein kinase (MAPK) pathways. These in turn stimulate interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 production by these cells. Furthermore, CD40 and its downstream MAPK signaling proteins (but not TRAF6) are located in membrane rafts, and disruption of caveolae or dislodgment of signaling proteins from the caveolin-1 scaffolding domain diminishes MAPK activation and IL-8 and MCP-1 production in these cells (17).
Intercellular adhesion molecule-1 (ICAM-1), or CD54, is an inducible cell adhesion glycoprotein of the immunoglobulin supergene family expressed on the surface of a wide variety of cell types (23). ICAM-1 interacts with two integrins belonging to the 2-subfamily, namely CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), found on leukocytes, which plays a key role in transendothelial migration of leukocytes and activation of T lymphocytes (23). ICAM-1 is present constitutively on the surface of a wide variety of cell types and is upregulated by numerous inflammatory mediators and proinflammatory cytokines (23).
In the current study, we examined whether CD40/CD154 ligation upregulates genes of the proinflammatory nuclear factor-B (NF-B) signal transduction pathway. A total of 38 genes were upregulated, including ICAM-1, MCP-1, and IL-8. With regard to ICAM-1, the gene array data were confirmed by real-time PCR. ICAM-1 protein expression was upregulated in parallel. At the functional level, CD40 mediated ICAM-1 upregulation correlated with enhanced mononuclear cell (MC) adhesion to the epithelial cell, which was mediated via the p38 MAPK signal transduction pathway.
MATERIALS AND METHODS
Cell culture. All experiments were carried out on primary cultures of human renal PTCs. Normal renal tissue was obtained from nephrectomy specimens during the course of cancer surgery (2), and verification of proximal tubule origin was performed as previously described (21). Cultures were maintained in a defined medium composed of 1:1 (vol/vol) mixture of DMEM and Ham's F-12 medium supplemented with insulin (5 μg/ml), transferrin (5.5 μg/ml), hydrocortisone (50 nM), triiodothyronine (5 pM) and sodium selenate (10 nM), and to which 50 IU/ml penicillin and 50 μg/ml streptomycin were added. Cells were usually grown in 25-cm2 tissue culture flasks or in 24- or 96-well plates (ELISA) and perpetuated in a humidified incubator at 37°C in 95% air-5% CO2 (culture medium pH 7.3). Media were exchanged at 48- to 72-h intervals, and cells were propagated through 8–10 passages.
GEArray. The relative mRNA expression of 96 human NF-B signal pathway genes was analyzed by GEArray (SuperArray Bioscience, Frederick, MD) according to the manufacturer's instructions. Confluent monolayers were challenged with recombinant human soluble (rhs) CD154 (100 ng/ml + 1 μg/ml enhancer) for 1 or 3 h, and total RNA was extracted with Tri-Zol reagent (Life Technologies, Rockville, MD) and compared with the vehicle-treated condition. Briefly, 5 μg total RNA were added to 3 μl GEAprimer Mix and heated to 70°C for 3 min for annealing. Reverse transcription proceeded by incubating the annealing mix with labeling master mix containing 1 mM biotin-16-dUTP (Roche Diagnostic, Mannheim, Germany) and 50 U/μl Moloney murine leukemia virus RT (Promega, Madison, WI) at 42°C for 90 min. The biotin-labeled cDNA probe was denatured using denaturing solution at 68°C for 20 min and hybridized with prewetted GEArray Q Series membrane under conditions of continuous agitation at 7 rpm/min overnight in a hybridization cylinder. After three washes, the membrane was blocked with 1.5 ml GEA blocking Solution Q (room temperature, 40 min) and then incubated with alkaline phosphatase-conjugated streptavidin (1:5,000 in washing buffer) at room temperature for 10 min with gentle mixing. After further washes, the GEArray Q Series membrane was detected with CDP-Star R Solution and X-ray film. The images were analyzed with GEArray Analyzer software.
RNA isolation and cDNA synthesis. Confluent monolayers were challenged with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) for 2–24 h, or vehicle (control), and total RNA was extracted with Tri-Zol reagent (Life Technologies), as previously described (4, 5, 16). From each sample, 5 μg total RNA was reverse-transcribed to cDNA using oligo(dT) Primer and Superscript II RT (both from Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Reverse transcription was carried out at 42°C for 1 h in a 20-μl reaction volume.
Quantitative real-time PCR. In preparatory experiments, standard PCR was performed to test the following primer pairs for ICAM-1: sense, 5'-TATGGCAACGACTCCTTCT-3' and antisense, 5'-CATTCAGCGTCACCTTGG-3' (1, NM_000201 [GenBank] ; see Ref. 15). -Actin was incorporated as a control. The primer pairs for -actin were as follows: sense, 5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG GCG-3' and antisense, 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3' (20). The PCR protocol was as follows: denature at 94°C for 30 s, anneal at 55°C for 30 s, and elongate at 72°C for 1 min using an enzyme-dye mixture from QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA). The amplified products were size fractionated on a 1% Tris-borate-EDTA-agarose gel containing ethidium bromide for resolution. The predicted size for the ICAM-1 and -actin fragments was 238 and 838 bp, respectively. The PCR products were excised from the agarose gel, purified by GFX PCR DNA and GEL Band Purification Kit (Amersham Biosciences, Piscataway, NJ), and quantified by UV-Visible Recording Spectrophotometer (Shimadzu Scientific Instruments, Braintree, MA). A range of the products from 10–9 to 10–3 pmol was used as templates in the standard curve for quantitative real-time PCR.
Quantitative real-time PCR was performed on the cDNA using the QuantiTect SYBR Green PCR Kit (Qiagen) indicated above, according to the manufacturer's protocol. The PCR master mix was prepared by combining the following reagents to the final volume of 20 μl: 1 μl primer.up (6.25 pmol/μl), 1 μl primer.dn (6.25 pmol/μl), 10 μl of the enzyme-dye mixture, and 8 μl 1:16 cDNA. The PCR master mix was placed in a 96-well PCR plate and, after an initial denaturing step (95°C for 15 min), processed according to the following PCR protocol: denature 95°C for 30 s, anneal at 55°C for 30 s, and elongate at 72°C for 1 min for 39 cycles in a DNA Engine Option System (MJ Research, Alameda, CA). Plate read temperature was 80°C. The melting curve was performed from 65 to 95°C with reading every 0.2°C and holding for 5 s between reads. The final cooling temperature was set at 12°C. The data generated were analyzed by Opticon Monitor Software (MJ Research). Three replicates were performed on each sample, and four samples were taken for each time point. Gene expression was normalized to -actin expression levels (9).
Western blot analysis. Western blot analysis was performed using protocols previously described by this laboratory (1, 16). Briefly, monolayers were challenged with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle (control) for the time periods indicated and then washed extensively with PBS (4°C). Cells were next lysed in 100 μl SDS sample buffer (1) and scraped, and the extracts were transferred to microcentrifuge tubes for sonication (10–15 s) to reduce sample viscosity. Samples were heated to 95°C for 5 min, and matched aliquots were subjected to SDS-PAGE in 10% gels, transferred to membranes, and blocked with 5% nonfat dry milk suspended with 143 mM NaCl, 0.1% Tween 20, and 20 mM Tris base, pH 7.6 (TBST; room temperature, 1 h). Filters were probed with the antibody of interest (1:1,000, overnight at 4°C), washed (3 times) with TBST, and subsequently incubated with peroxidase-linked goat anti-rabbit IgG in blocking buffer (1:2,000, 45 min, room temperature). After additional washes (3 times), antibody-bound proteins were detected using an enhanced chemiluminescence (ECL) system according to the manufacturer's instructions (Pierce, Rockford IL), and membranes were exposed to Hyperfilm ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK). Molecular weight markers were used to determine the size of the detected band. Where required, relative densities of the protein bands were determined with a GS-670 imaging densitometer and Molecular Analyst PC software program (Bio-Rad, Richmond, CA). Antibodies were obtained from commercial sources and are detailed in MATERIALS AND METHODS.
ELISA and cell surface ELISA. Soluble ICAM-1 production by PTCs was measured by the quantitative sandwich enzyme immunoassay technique according to the manufacturer's instructions (R&D Systems, Minneapolis, MN) and as previously reported by this laboratory (16). PTCs were grown in 24-well culture plates, and the experimental maneuver was performed by adding rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle to the supernatant for the indicated time periods, which was assayed for adhesion molecule production. Optical density of each microtiter plate was read at 450 nm on a Biomed microplate reader. Recombinant ICAM-1 standards from 0 to 49.55 pg were used to generate standard curves. Quadruplicate determinations in three to four different series of experiments were performed. Data are presented as means ± SE as the index of dispersion. ANOVA was used to compare group means, and the null hypothesis was rejected at P < 0.05.
ICAM-1 expression on the surface of PTCs was detected by cell-surface ELISA according to Chen et al. (3) with modifications. PTCs grown in 96-well plates were stimulated with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle for the indicated time periods and then fixed with 1% paraformaldehyde (10 min). All subsequent steps were performed at room temperature. Cells were washed with PBS (2 times) and then incubated in a blocking solution comprised of 1% BSA suspended in TBST for 1 h. Fixed cells were probed with an anti-ICAM-1 rabbit anti-human polyclonal antibody (1:100 in 1% BSA TBST; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h or with 1% BSA in TBST as a control. After six vigorous washes with TBST, cells were incubated with anti-rabbit secondary antibody linked to horseradish peroxidase (1:500 in TBST) for 30 min. TMB Microwell Peroxidase Substrate System (Kirkegaard and Perry, Gaithersburg, MD) was applied to the cells according to the manufacturer's instructions, and 1 M phosphoric acid was used to terminate the reaction. The optical density of each microtiter plate was read at 450 nm on a Biomed microplate reader. Quadruplicate determinations in three to four different series of experiments were performed. Data are presented as detailed above.
Flow cytometry cell analysis. Monolayers incubated with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) or vehicle for 48 h were detached with 0.5 mM EDTA for 5 min, and the cell suspension was then adjusted to 106 cells/ml with RPMI 1640. Suspended cells were incubated for 20 min at room temperature in a dark area with 40 μg/ml mouse anti-human monoclonal ICAM-1 antibodies conjugated to phycoerythrin (PE; Santa Cruz Biotechnology). Suspensions were fixed in a 0.5% formalin/PBS solution and then washed (2 times) with 1% sodium azide/PBS solution and analyzed with a FACScan (Becton-Dickinson, San Jose, CA). Data were analyzed with Simulset software. Erythrocyte-lysed whole blood from healthy individuals was labeled with Simultest LeucoGate (CD54-FITC/CD14-PE) and Simultest Control 1/2 (IgG1-FITC/IgG2a-PE; both from Becton-Dickinson) to set up the gates and act as a negative control, respectively.
Adhesion assay for peripheral MCs to PTCs. PTCs grown in monolayers in 96-well plates were treated with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) for 24 or 48 h at 37°C, pretreated with neutralized ICAM-1 antibody (10 μg/ml, 30 min) or mouse IgG1 isotype control (10 μg/ml, 30 min before CD154 stimulation), or left untreated (control). Human peripheral MCs were isolated from healthy human peripheral blood using Ficoll-Paque (Amersham Biosciences) according to the manufacturer's instructions. MCs were labeled with 10 ng/ml BCECF-AM (30 min at 37°C), adjusted to a concentration of 2.5 x 105/ml with RPMI 1640, and added to the monolayers of PTCs in a final volume of 100 μL. PTCs and MCs were cocultured in a CO2 incubator for 1 h. Nonadherent cells were removed from the plate by gentle washing with PBS. The adherent cells were manually counted under the fluorescence microscope at x20 magnification.
Materials. DMEM, Ham's F-12 medium, RPMI 1640, and the penicillin-streptomycin solution were purchased from GIBCO Laboratories (Grand Island, NY), and newborn calf serum was from Sigma Chemical (St. Louis, MO). The rhs CD40 ligand or CD154 and enhancer (an antibody that binds CD154 trimers and has the effect of multimerizing the CD40 receptor) were purchased from Alexis (San Diego, CA). For Western blot analysis, rabbit anti-human ICAM-1 polyclonal antibody and rabbit anti-p38-MAPK antibody were purchased from Santa Cruz Biotechnology. For the cell adhesion assays, the neutralizing ICAM-1 antibody and mouse IgG1 isotype control were purchased from R&D Systems. BCECF-AM was obtained from Molecular Probes (Eugene, OR). The selective mitogen/extracellular signal-regulated kinase inhibitor PD-98059 was purchased from Cell Signaling Technology (Beverly, MA); the selective p38 inhibitor SB-203580 and the protein kinase C inhibitor calphostin C were purchased from Calbiochem (San Diego, CA). Electrophoretic-grade reagents used for SDS-PAGE were obtained from Bio-Rad Laboratories (Melville, NY). All standard chemicals used were purchased at the highest commercial grade available.
Statistical analysis. ANOVA was used to compare group means (Tukey test), and the null hypothesis was rejected at P < 0.05.
RESULTS
CD154 induces mRNA expression of NF-B signal pathway intermediates in PTCs. A cDNA gene array containing 96 genes specific for human NF-B signal pathway genes identified several genes to be significantly upregulated in human renal PTCs after exposure to rhs CD154 (100 ng/ml + 1 μg/ml enhancer). With the use of a twofold upregulation cutoff point, 38 genes were upregulated at 1 h (Table 1) and 7 genes remained upregulated at 3 h of exposure (Table 2). Of these, ICAM-1 mRNA expression was increased 7.1-fold by 1 h (Fig. 1A and Table 1) and 4.9-fold by 3 h (Fig. 1B and Table 2) compared with the vehicle-stimulated condition. IL-8 mRNA expression increased 5.2-fold (Fig. 1A and Table 1) and 2.4-fold (Fig. 1B and Table 2) at these two time points. MCP-1 mRNA expression increased 2.0-fold (Fig. 1A) and 1.2-fold (Fig. 1B) at 1 and 3 h, respectively. The latter observations are in keeping with previous published data from this laboratory, namely, that CD154 stimulates IL-8 and MCP-1 production in human renal PTCs (16). Because we wished to further explore the role of CD154/CD40 engagement in the inflammatory process, we focused our efforts on ICAM-1 expression with the consideration that this adhesion molecule might amplify the inflammatory response.
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CD154 stimulates ICAM-1 mRNA expression and protein production. Quantitative real-time PCR was used to confirm enhanced ICAM-1 expression consequent to rhs CD154 stimulation. The data are summarized in Fig. 2 and are normalized for -actin expression levels (9). Compared with the unstimulated condition, ICAM-1 expression increased 15.2 ± 1.7 (2 h)-, 6.4 ± 0.6 (4 h)-, 3.7 ± 0.4 (6 h)-, 2.5 ± 0.4 (15 h)-, and 3.2 ± 0.3 (24 h)-fold (n = 4, P < 0.05 for the 2- and 4-h time points vs. 0 h). If ICAM-1 expression levels are not corrected for -actin expression levels in this experiment, actual mRNA values can be obtained. By 2 h, ICAM-1 mRNA had increased about eightfold from an unstimulated value of 3.9 ± 1.2 (10–7 pmol) to 31.8 ± 0.8 (10–7 pmol; n = 3, P < 0.001). By 4 h, ICAM-1 mRNA remained incremented by about fourfold above the control condition, i.e., 3.9 ± 1.2 (10–7 pmol) vs. 14.8 ± 1.2 (10–7 pmol; n = 3, P < 0.001). The 6-, 15-, and 24-h values were 6.3 ± 0.4, 6.0 ± 0.7, and 6.3 ± 0.2 (10–7 pmol), respectively, none of which were significantly different from the control value of 3.9 ± 1.2 (10–7 pmol).
In parallel experiments, CD154-induced ICAM-1 protein expression was explored by Western blot analysis (Fig. 3A). Compared with p38 MAPK (whose activity but not amount is stimulated by CD154 treatment), ICAM-1 expression increased by 1.2-fold at 24 h and 1.7-fold at 48 h (Fig. 3B). Later time points were not explored. Soluble ICAM-1 expression, as measured by quantitative sandwich-enzyme ELISA, was in keeping with these observations. As shown in Fig. 4A, soluble ICAM-1 expression peaked at 48 h of rhs CD154 (100 ng/ml + 1 μg/ml enhancer) exposure to a value of 49.4 ± 0.3 vs. 19.9 ± 0.3 ng/ml in the vehicle-treated group (n = 3, P < 0.001). Earlier and later time points showed lower levels of soluble ICAM-1 expression than at 48 h, but at each time point the CD154-stimulated value was significantly higher than the control value: 12 h, (ng/ml) 17.5 ± 0.3 (CD154) vs. 13.2 ± 0.3 (control); 24 h, 32.8 ± 0.4 (CD154) vs. 17.3 ± 0.4 (control); 72 h: 33.5 ± 0.7 (CD154) vs. 23.3 ± 0.4 (control).
A more physiologically relevant measurement of ICAM-1 expression might be the cell surface expression of the adhesion molecule, rather than a measurement of the soluble product outlined above. For this purpose, a cell-surface ELISA assay described in METHODS AND MATERIALS was used. As expected, early time points (up to 20 min) failed to detect any increase in cell-surface expression of ICAM-1 on PTCs vs. the control value (0 min; Fig. 4B). Similar to the soluble ICAM-1 data, cell surface expression of the adhesion molecule peaked at 48 h to about double the baseline value. Fluorescence-activated cell sorter (FACS) analysis further confirmed this observation (Fig. 5A). The number of cells labeled with ICAM-1-conjugated PE increased from 2,500 to 4,000 (Fig. 5B), and the expression of ICAM-1 on these cells, as measured by PE intensity, increased from 4,500 to 5,400 arbitrary units (Fig. 5C). In aggregate, these observations provide compelling evidence for CD154-mediated ICAM-1 upregulation in human PTCs.
CD154-mediated ICAM-1 expression promotes MC adhesion to PTCs. To establish the functional consequence of CD154-induced ICAM-1 expression by PTCs, we explored whether this maneuver might enhance MC adhesion to the monolayer. BCECF-AM-labeled human peripheral MCs were added to the monolayer under different conditions; after a 1-h coincubation period (and the appropriate washes), cells were visualized under phase and fluorescent microscopy, and MC cells were counted manually. As shown in Fig. 6, the untreated monolayer, visualized under phase microscopy (Fig. 6A and C, left), did not autofluoresce when examined under the fluorescent microscope (Fig. 6B), and only BCECF-labeled MCs were detected by fluorescent microscopy (Fig. 6C, right). After rhs CD154 (100 ng/ml + 1 μg/ml enhancer) treatment, the number of MCs that adhered to the PTC monolayer had increased at 24 h (Fig. 6E) and 48 h (Fig. 6F) compared with the untreated condition (Fig. 6D). MC adhesion to CD154-treated PTC monolayers was largely blunted in the presence of a neutralizing ICAM-1 antibody (10 μg/ml, 30 min) both at the 24-h (Fig. 6H) and 48-h (Fig. 6I) time points. The untreated condition with or without neutralizing antibody was comparable (Fig. 6, G and D). Finally, pretreatment with mouse IgG1 (isotype control, 10 μg/ml for 30 min) had no effect on MC adhesion to CD154-treated PTC monolayers at both the 24-h (Fig. 6K) and 48-h (Fig. 6L) time points compared with Fig. 6, E and F, respectively. The untreated condition with or without the isotype control was comparable (Fig. 6, J and D). The specificity of these observations suggests that CD154-mediated ICAM-1 expression by PTCs in monolayers enhances peripheral MC adhesion.
MC adhesion to PTCs is mediated through p38 MAPK. Because CD40 signals through MAPK pathways in PTCs (16), we explored whether CD154/CD40 activation of ICAM-1 proceeded in this manner. Confluent monolayers were challenged with vehicle or rhs CD154 (100 ng/ml + 1 μg/ml enhancer) in the absence and presence of the p38 inhibitor SB-203580 and the extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD-98059 and then subjected to Western blot analysis. The protein kinase C inhibitor calphostin C was included as a control. A representative experiment is depicted in Fig. 7 in the format of a Western blot (Fig. 7A) and the densitometric ratio of ICAM-1 to p38 MAPK (Fig. 7B). We have previously shown that the p38 inhibitor SB-203580 blunts p38 MAPK activity but not the level of protein expression (16). CD154 increased ICAM-1 expression at 24 h (lane 2) and 48 h (lane 6) vs. the control condition (lane 1). The ICAM-1-to-p38 ratio for these data was 0.88 at 24 h (bar 2) and 1.48 at 48 h (bar 6) vs. the control condition (0.66, bar 1). The ERK1/2 inhibitor PD-98059 somewhat decreased CD154-mediated ICAM-1 expression at 24 h (0.88 vs. 0.56; lane/bar 2 vs. 3) but was without effect at 48 h (1.48 vs. 1.33; lane/bar 6 vs. 7). In contrast, the p38 inhibitor SB-203580 markedly blunted CD154-evoked ICAM-1 expression at 24 h (0.88 vs. 0.20; lane/bar 2 vs. 4) and more impressively at 48 h (1.48 vs. 0.54; lane/bar 6 vs. 8). The protein kinase C inhibitor calphostin C was without effect at 24 h (0.88 vs. 0.78; lane/bar 2 vs. 5) but appeared to increase ICAM-1 expression at 48 h (1.48 vs. 2.03; lane/bar 6 vs. 9).
The functional relevance of this observation was explored using the cell attachment assay described earlier, using the 48-h data points. A representative experiment is shown in Fig. 8. Treatment of monolayers with rhs CD154 (100 ng/ml + 1 μg/ml enhancer) increased MC adhesion (Fig. 8D) compared with the vehicle-treated condition (Fig. 8A). The p38 inhibitor SB-203580 prevented MC attachment to CD154-treated monolayers (Fig. 8E). Indeed, MC attachment was no different from the control condition, where monolayers were similarly treated with SB-203580 (Fig. 8B). In contrast, the ERK1/2 inhibitor PD-98059 had no effect on MC adhesion to the monolayer (Fig. 8F) and was clearly different from the control experiment similarly treated with PD-98059 (Fig. 8C). In aggregate, these data are in keeping with the hypothesis that CD154-mediated ICAM-1 expression and the attendant adhesion of MC to the PTC are mediated through p38 MAPK.
DISCUSSION
The results presented demonstrate that CD154/CD40 ligation of human renal PTCs in primary culture upregulates 38 NF-B signal pathway genes by twofold at 1 h and that seven of these genes remain upregulated at 3 h (Tables 1 and 2). Of these upregulated genes, we further explored ICAM-1 (Fig. 1). Quantitative real-time PCR for ICAM-1 mRNA expression confirmed the gene array findings (Fig. 2). ICAM-1 protein expression was enhanced in parallel (Fig. 3), both in the format of soluble (Fig. 4A) and cell surface (Figs. 4B and 5) ICAM-1. CD154-mediated ICAM-1 expression by PTCs promoted MC adhesion to the monolayer (Fig. 6), which was mediated via activation of the p38 MAPK signal transduction pathway (Figs. 7 and 8).
CD40 was initially described as a B cell-specific receptor that, upon engagement by its cognate ligand CD154, resulted in B lymphocyte activation and proliferation and immunoglobulin production (6). It is now well recognized that CD40 is expressed on a wide variety of other cells, including dendritic cells, fibroblasts, and epithelial and endothelial cells, and plays a pivotal role not only in the regulation of immune responses but also in aspects of the inflammatory response in nonlymphoid cells (8, 14, 22). Because the transcription factor NF-B has been recognized as an early mediator of immune and inflammatory responses (25), it seemed logical to screen other genes of this signal transduction pathway for CD40/CD154 activation. We focused our efforts on ICAM-1 because we reasoned that adhesion of circulating monocytes to the PTC could potentially amplify the inflammatory response. Future studies may elaborate on the potential role of CD40-mediated upregulation of the other genes identified herein.
Precedent exists for CD40/CD154 stimulation of ICAM-1 production by nonlymphoid cells. For example, endothelial cells have been shown to acquire a proinflammatory and proatherogenic phenotype and express ICAM-1 and other leukocyte adhesion molecules in response to CD40/CD154 activation (13, 18). A similar type of scenario has been proposed in the setting of lupus nephritis where tubular lesions have been shown to be a good predictor of the renal outcome (9). In a retrospective study with immunohistochemistry performed on renal biopsy material obtained from 152 patients, the authors observed a high incidence of CD40 and ICAM-1 expression on damaged proximal (and distal) tubule cells. Interestingly, ICAM-1 immunostaining correlated with CD40 immunostaining. These observations suggest but do not prove that the epithelial cells of the proximal (and distal) tubule play an important role in recruiting circulating monocytes. In keeping with this concept is our demonstration that CD154-mediated ICAM-1 upregulation resulted in increased adhesion of MCs to the PTC in culture (Figs. 6 and 8). In preparatory studies, we have also demonstrated that prolonged exposure of PTCs to CD154 results in their transdifferentiation into mesenchymal cells (19). Future studies in an in vivo model may establish how these events might be interrelated.
The major intracellular signal transduction pathways involved in the regulation of ICAM-1 expression include the MAPKs, NF-B, protein kinase C, and the JAK/STAT pathway (23). In addition, a number of nuclear transcription factors have been shown to activate ICAM-1 expression (23), which enhances the complexity of the cell type-specific and stimulus-specific regulation of the ICAM-1 gene. For example, in cultured human umbilical vein endothelial cells, tumor necrosis factor (TNF)--induced ICAM-1 expression was mediated via p38 MAPK (11), whereas, in HTB-94 chondrosarcoma cells, TNF--increased ICAM-1 expression is independent on both p38 and ERK1/2 MAPK pathways (12). On the other hand, different agonists can stimulate ICAM-1 production via different MAPK activation in the same cell type. In A549 cells, both p38 and ERK1/2 MAPK pathways are involved in adenovirus-induced ICAM-1 expression (24), but neither of these pathways is involved in TNF-- and IL-1-increased ICAM-1 production (10). The current study shows that, in cultured human renal PTCs, p38 MAPK but not ERK1/2 MAPK is involved in CD40/CD154-induced ICAM-1 production (Fig. 7). The cell adhesion assay (Fig. 8) convincingly confirms this observation at the functional level.
Previous studies from this laboratory have demonstrated that CD40/CD154 ligation in human PTCs stimulates MCP-1 and IL-8 production via TRAF6 recruitment and MAPK activation (16). Using a similar approach, we now show that this proinflammatory agonist upregulates ICAM-1 expression. It is interesting to speculate whether CD40 activation might not directly enhance ICAM-1 expression and whether CD40-mediated MCP-1 and/or IL-8 production by the PTC may be the stimulus for ICAM-1 upregulation. Alternatively, CD40-mediated ICAM-1 expression may be amplified in an autocrine manner by MCP-1 and/or IL-8. These possibilities were not explored during the course of this project but form the basis for future studies.
In summary, we show that the human renal PTC, in response to CD154/CD40 ligation, upregulates a number of NF-B pathway genes and that enhanced expression of ICAM-1 correlates with increased adhesion of MCs to the epithelial cell. These findings are in keeping with the concept that the PTC, under defined conditions, produces proinflammatory chemokines and attracts leukocytes that may amplify the proinflammatory effect. Hence, the PTC appears to be a contributor to, rather than a victim of, interstitial inflammation.
GRANTS
This study was supported by a grant from the Paul Teschan Research Fund (Dialysis Clinics, Inc.).
ACKNOWLEDGMENTS
The expert secretarial assistance of Sherryl Krulik is gratefully acknowledged.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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