Kidney International, Vol. 56 (1999), pp. 985–994

TGF-b1 down-regulates induced expression of both class IIMHC and B7-1 on primary murine renal tubular epithelial cells

NAZIFA BANU and CATHERINE M. MEYERS

University of Pennsylvania School of Medicine, Penn Center for the Molecular Studies of Kidney Diseases, Department ofMedicine, Renal-Electrolyte and Hypertension Division, Philadelphia, Pennsylvania, USA

TGF-b1 down-regulates induced expression of both class II The importance of T cells in initiating and amplifyingMHC and B7-1 on primary murine renal tubular epithelial cells. inflammatory renal lesions has been demonstrated in a

Background. We examined the immunomodulatory effects number of experimental models of autoimmune renalof transforming growth factor-b1 (TGF-b1) on the regulationdisease [1–4]. T cell-mediated events are initiated byof class II MHC and costimulatory molecule expression in arecognition of relevant antigen presented in the contextprimary renal tubular epithelial cell line, called F1K.

Methods. Class II major histocompatibility complex (MHC), of appropriate major histocompatibility complex (MHC)class II transactivator, B7-1, intercellular adhesion molecule-1 molecules [5]. Additional signals required for full devel-(ICAM-1) and interferon-g (IFN-g) receptor b chain were opment of T cell responses are provided by ligation ofevaluated in untreated and cytokine-treated F1K by Northern

cell-surface receptors by antigen presenting cell costimu-hybridization analysis and flow cytometry. T cell activationstudies were performed to assess TGF-b1-mediated effects on latory molecules [6, 7]. While expression of MHC mole-antigen presenting cell function of F1K. cules is critical for the recognition of antigen by T cells

Results. Pretreatment of F1K with TGF-b1 markedly inhib- studies indicate that B7 costimulatory molecules are alsoited IFN-g-induced class II MHC expression, by both FACS

requisite for activation of distinct CD41 T cells [5, 7].and Northern analysis. Total class II transactivator mRNARenal tubular epithelial cells upregulate class II MHClevels were also diminished by TGF-b1, indicating that class

II MHC modulation in F1K results from inhibition of this molecules in inflammatory renal disease and in culture,intermediate protein. As previous studies demonstrated that when stimulated by proinflammatory cytokines such ascotreatment of F1K cells with IFN-g 1 lipopolysaccharide (LPS) interferon-g (IFN-g) [8–10]. Moreover, recent in vitroinduces B7-1, we evaluated the potential regulatory effects of

and in vivo studies demonstrated that renal tubular epi-TGF-b1 exposure on B7-1 expression. Our studies revealedthelial cells, like professional antigen presenting cells,that B7-1 mRNA and cell-surface expression in IFN-g 1

LPS-treated F1K were decreased by TGF-b1 pretreatment. can be induced to express B7-1 costimulatory moleculesFunctional studies evaluating TGF-b1-mediated effects were under distinct experimental conditions [11]. Primary re-performed with IFN-g 1 LPS-treated F1K and MR1.3, a

nal tubular epithelial cells exposed to both IFN-g andnephritogenic CD41 Th2 clone derived from kidneys of animalslipopolysaccharide (LPS) express B7-1 and provide rele-with autoimmune glomerulonephritis. Interleukin (IL)-4 pro-

duction assays demonstrated activation of MR1.3 by IFN-g 1 vant costimulatory signals to nephritogenic CD41 T cellsLPS-treated cells, but not by IFN-g 1 LPS-treated cells pre- [11]. Renal expression of B7-1 is also apparent in kidneysviously exposed to TGF-b1, indicating that TGF-b1-mediated derived from similarly treated recipient mice [11]. Ininhibition of class II MHC and B7-1 expression alters the anti-

aggregate, these observations suggest that upregulatedgen presenting cell function of F1K.renal class II MHC and B7-1 expression may promoteConclusions. These studies describe the proscriptive influ-

ence of TGF-b1 on class II MHC and B7-1 expression in renal autoimmune injury by facilitating tubular epithelial celltubular epithelial cells. Such findings indicate that TGF-b1 presentation of self (renal-specific) antigens.alters the antigen presenting cell function of renal tubular epi-

Recent studies of cytokines, such as IFN-a/b, trans-thelial cells in vitro, and suggest a potential mechanism forforming growth factor-b (TGF-b), and interleukin (IL)-10,immunosuppression of T cell-mediated renal immune responses

in vivo. which exert suppressive activity on different aspects ofimmune responses, have explored the role of these en-dogenous peptides in immune regulation [12–15]. TGF-bKey words: T lymphocytes, transforming growth factor-b, lipopolysac-

charide, class II transactivator, renal immune response. has been shown to control growth, development, differ-entiation, apoptosis, and extracellular matrix remodelingReceived for publication December 22, 1998in a wide variety of cell types and tissues [16, 17]. Acti-and in revised form April 2, 1999

Accepted for publication April 12, 1999 vated TGF-b1 has multiple potent immunomodulatoryeffects on T cells in vitro, such as inhibiting growth, 1999 by the International Society of Nephrology

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Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression986

differentiation, and effector function, and also altering weeks following disease induction [23]. T cells weremaintained by weekly passage with 0.5 3 106 cells/mlT cell cytokine release [12, 18–20]. It has also been shown

that TGF-b1 promotes immune deviation by altering anti- irradiated (2500 rads) B6D2 F1 spleen cells, and 10 U/mlhuman recombinant IL-2 (Boehringer Mannheim Corp.,gen presenting cell accessory signals [13, 20]. Moreover,

recent studies of mice genetically deficient for TGF-b1, Indianapolis, IN, USA). T cell culture medium consistsof Dulbecco’s modified Eagle medium (DMEM)-highwhich die shortly after birth, have demonstrated aberrant

class II MHC expression in tissues prior to evidence of glucose supplemented with glutamine, antibiotics (peni-cillin, streptomycin, gentamicin), 10% decomplementeddiffuse inflammatory cell infiltration [21, 22].

In view of these immunosuppressive effects of TGF- FCS, 5% NCTC (Whittaker Bioproducts, Inc., Walk-ersville, MD, USA) and 2 3 10–5 m 2-ME. Cells wereb1, we investigated TGF-b1-mediated effects on antigen

presenting cell activity of renal tubular epithelial cells. cultured at 378C in a humidified 5% CO2 incubator.Our studies revealed TGF-b1 down-regulates induced

Additivesexpression of both class II MHC and B7-1 in a murinerenal tubular epithelial cell line, called F1K. Of note, Recombinant human TGF-b1 (R&D Systems, Minne-

apolis, MN, USA), recombinant murine IFN-g (Phar-induced B7-1 expression in F1K cells with IFN-g 1 LPScotreatment was significantly decreased by preincuba- Mingen, Inc., San Diego, CA, USA) and LPS from Esch-

erichia coli serotype 026:B6 (Sigma Chemical Co., St.tion with TGF-b1. Further analysis revealed that activa-tion of a nephritogenic CD41 Th2 clone, derived from Louis, MO, USA) were used for stimulation of F1K cells.kidneys of animals with chronic graft-vs-host disease by

RNA extraction and Northern hybridization analysisIFN-g 1 LPS-treated F1K cells was markedly inhibitedif the cells were previously exposed to TGF-b1 [23]. Total cellular RNA was isolated using Trizol reagent

(Gibco BRL) in accordance with the manufacturer’s in-These studies demonstrate that TGF-b1-mediated down-regulation of class II MHC and B7-1 alters the antigen structions. After assessing purity of final products by OD

ratios at 260/280 nm, which were typically 1.6 to 1.8,presenting cell function of proinflammatory cytokine-treated renal tubular epithelial cells in vitro, and suggest RNA samples were then used for Northern blot analysis.

Fifteen micrograms of total RNA per sample was elec-a potential mechanism for local down-regulation of cell-mediated renal immune responses in vivo. trophoresed through a 1.5% agarose-formaldehyde gel,

which was then transferred to a Zetabind membrane(Cuno Laboratory Products, Meriden, CT, USA). Blots

METHODSwere hybridized to 32P (NEN Life Science Products, Bos-

Mice ton, MA, USA) labeled murine probes (specific activityof 2.0 3 109 dpm/mg) for 16 hours at 658C. Murine probes,Female (C57BL/6 3 DBA/2) F1 (B6D2 F1) (H-2b/d)

were purchased from The Jackson Laboratory (Bar Har- B7-1 (kindly provided by Dr. Larry A. Turka, Univer-sity of Pennsylvania), I-Ad

a (kindly provided by Dr.bor, ME, USA).Ronald Germain, NIH/NIAI), class II transactivator

F1K epithelial cells [25], ICAM-1 (obtained from ATCC) [26], IFN-g recep-tor b chain [27] and b-actin [28] were used in thesePrimary renal tubular epithelial cells, called F1K cells,

were harvested from naive B6D2 F1 mouse kidneys fol- studies. After hybridization the blots were washed in0.1 3 standard sodium citrate (SSC)/0.1% sodium dode-lowing microdissection and brief collagenase digestion

[11, 24]. After Percoll-density sedimentation, F1K cells cyl sulfate (SDS) at 658C for one hour. Autoradiographywas performed with intensifying screens at 2708C. Ex-were grown on type I collagen-coated flasks in hormon-

ally defined K-1 serum-free medium [11, 24]. Expression posed films were scanned with a laser densitometer(Hoefer, San Francisco, CA, USA) and relevant mRNAof epithelial cell markers (cytokeratin and uvomorulin)

and a distal tubular marker (lotus lectin binding) were levels were calculated relative to those of b-actin.verified by immunofluorescence studies of subconfluent

Flow cytometrymonolayers (data not shown). F1K cells were maintainedin DMEM-high glucose medium (Gibco BRL, Gaithers- Single-cell suspensions were prepared by harvesting

F1K cells from T-75 flasks. Cells were then washed in ice-burg, MD, USA) with glutamine, antibiotics (penicillin,streptomycin), and 5% NuSerum (Beckton Dickinson cold phosphate buffered saline (PBS)/1% bovine serum

albumin (BSA) and 5 mm ethylenediaminetetraacetateLabware, Bedford, MA, USA). Cells were cultured at378C in a humidified 5% CO2 incubator. (EDTA), pH 8.0 (staining buffer). Aliquots of 1 3 106

cells were resuspended in 50 ml staining buffer. DirectT cells staining for B7-1 and ICAM-1 was performed with pri-

mary conjugated antibodies (16-10 A1 and 3E2; Phar-MR1.3 T cells were isolated from nephritic kidneys ofB6D2 F1 mice with chronic graft-vs-host disease 14 Mingen, Inc.) or similarly conjugated isotype-matched

Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression 987

control antibody (PharMingen, Inc.) for 20 minutes onice. Indirect staining for class II MHC was performedwith primary anti-I-Ab antibody (AF6–120.1, Phar-Mingen, Inc.), or isotype-matched control antibody(PharMingen, Inc.) for 20 minutes on ice. Washed cellswere incubated a second time on ice with appropriatesecondary antibody, goat antimouse IgG-FITC (Boeh-ringer Mannheim Corp.). Stained cells were then washedand resuspended in staining buffer. Propidium iodide(PI; Sigma Chemical Co.) 2 mg/ml was added to cellsuspensions, and fluorescence was recorded on a FACS-can cytofluorograph (Becton Dickinson). Data were ana-lyzed with CELL Quest software. In each run at least10,000 (PI-excluded) gated events were analyzed.

T cell activation assays

T cell stimulation was assessed by measuring IL-4 cyto-kine production in cell-free supernatants of CD41 Th2clone MR1.3 exposed to various antigen presenting cellpreparations. Day 10 MR1.3 T cells (1 to 1.5 3 106 cells/well) were cultured with renal antigen (10 mg/ml) in 24-well plates for 48 hours with various F1K cell preparations(0.5 3 106 cells/well), or T cell medium alone. Some F1Kpreparations were pretreated with IFN-g (100 U/ml) 1LPS (10 mg/ml) for 48 hours or TGF-b1 (5 ng/ml) for24 hours and IFN-g 1 LPS for an additional 48 hoursprior to exposure to T cells. IL-4 production by MR1.3T cells was measured in supernatants collected at 48hours by cytokine-specific ELISA [29]. Purified anti-IL-4capture monoclonal antibody (PharMingen, Inc.) wasdiluted to 1.0 mg/ml in 0.1 m NaHCO3, pH 8.2, and 50 mlaliquots were added to wells of an ELISA plate. Followingovernight incubation at 48C and washing with PBS/0.05%Tween, wells were blocked with 200 ml PBS/5% FCS at Fig. 1. Class II major histocompatibility complex (MHC) expression in

transforming growth factor-b1 (TGF-b1)-treated F1K cells. Confluentroom temperature for two hours. Plates were then washedF1K cells were incubated with no additives or with IFN-g (100 U/ml)with PBS/0.05% Tween and coated with 50 ml duplicatefor 48 hours. IFN-g-treated F1K cells were cultured with various concen-

aliquots of IL-4 standards diluted in PBS/5% FCS and trations of TGF-b1 for 24 hours prior to IFN-g incubation. (A) Har-vested RNA was evaluated for class II MHC mRNA expression withcell-free supernatants. Plates were incubated overnighta murine I-Ad

a cDNA probe. The b-actin signal reflects relative amountat 48C, washed with PBS/0.05% Tween, and coated withof RNA loaded in each lane. (B) Values for I-Ad

a mRNA were normal-100 ml aliquots of biotinylated anti-IL-4 detecting anti- ized to levels of b-actin mRNA expression, by densitometric analysis,

and plotted as a percentage of maximal response observed in IFN-g-body (1 mg/ml; PharMingen, Inc.). Following 45 minutestreated F1K cells at 48 hours. Presented findings are representative ofof incubation at room temperature, the plates werethree distinct experiments.

washed, coated with 100 ml aliquots of avidin-peroxidase(1:400; Sigma Chemical Co.), and incubated again atroom temperature for 30 minutes. After a final wash

RESULTSwith PBS/0.05% Tween, 100 ml aliquots of peroxidaseTGF-b1 inhibits IFN-g-induced class II MHC insubstrate reagent (Bio-Rad Laboratories, Hercules, CA)F1K cellswere added to each well, allowed to develop at room

temperature for 5 to 10 minutes, and read on an ELISA To evaluate the role of TGF-b1 in altering cell-surfaceplate reader at 415 nm. Sensitivity limit for the IL-4 determinants relevant to renal immune responses, weELISA was 30 pg/ml. examined the effect of TGF-b1 on class II MHC expres-

sion in the primary murine tubular epithelial cell lineStatistical analysis F1K. For these studies we exposed F1K cells to TGF-b1

Differences between experimental groups were deter- for 24 hours prior to a 48 hour incubation with IFN-g. Asshown in Figure 1A, F1K class II MHC (I-Ad

a) messagemined by Student’s t-test, where appropriate [30].

Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression988

not detected under basal conditions by Northern analysis(Fig. 3A). By contrast, Northern hybridization studies de-tected the class II transactivator mRNA transcript (5.2 kb)in IFN-g-stimulated cells (Fig. 3A). LPS stimulationalone did not induce class II transactivator mRNA ex-pression in F1K cells (data not shown). Figure 3(A)reveals that F1K class II transactivator message expres-sion was apparent 4 hours after stimulation, and mark-edly up-regulated 16 hours after incubation with IFN-g(100 U/ml). We then determined the effect of TGF-b1on F1K class II transactivator message expression. Asshown in Fig. 3(B), TGF-b1 pretreatment for 24 hoursat 0.1 ng/ml did not affect IFN-g-induced class II trans-activator mRNA expression, but pretreatment at levelsof 1, 5, and 10 ng/ml suppressed this induced expressionby 63%, 78% and 89%, respectively (densitometricFig. 2. Cell-surface expression of class II MHC in TGF-b1-treated F1Kquantitation of the blots shown in Fig. 3B). The degreecells. Confluent F1K cells were incubated with no additives or with

IFN-g (100 U/ml) for 48 hours. Some F1K cell preparations were pre- of TGF-b1 inhibition of IFN-g-induced I-Ada mRNA

treated with TGF-b1 (5 ng/ml) for 24 hours before exposure to IFN-g. and that of class II transactivator mRNA expression inHarvested F1K cells were then analyzed by flow cytometry using anF1K cells were comparable (Figs. 1A and 3B), suggestinganti-I-Ab antibody (AF6–120.1) or isotype control antibody. Presented

findings are representative of three separate experiments. Lines are: that class II MHC modulation in these cells results from(short dashed line) control Ig; (long dashed line) unstimulated F1K; inhibition of this intermediate protein.(thick solid line) IFN-g; (thin solid line) TGF-b1 1 IFN-g.

TGF-b1-mediated effects on costimulatory moleculeexpression in F1K cells

Induction of costimulatory molecule expression, suchexpression, which was not detected under basal condi-tions by Northern analysis, was markedly upregulated as B7-1 or B7-2, enhances antigen presenting cell func-

tion of nonprofessional antigen presenting cells in vitroby IFN-g (100 U/ml) at 48 hours. Pretreatment of F1Kcells with TGF-b1, however, inhibited IFN-g-induced [34, 35]. We have previously demonstrated that cotreat-

ment with IFN-g (100 U/ml) 1 LPS (10 mg/ml) inducesI-Ada message expression in a dose-dependent manner

(Fig. 1A). Densitometric analysis of I-Ada Northern blots B7-1 expression on F1K cells [11]. We therefore evalu-

ated the potential regulatory effects of TGF-b1 exposurerevealed that relative mRNA levels in IFN-g-treatedF1K cells, at 48 hours following exposure, were not al- on costimulatory molecule B7-1 expression. Our studies,

shown in Figure 4, reveal that TGF-b1 pretreatment fortered by TGF-b1 at a dose of 0.1 ng/ml, but decreased24%, 73%, and 83% at doses of 1, 5, and 10 ng/ml, 24 hours decreased B7-1 mRNA expression in IFN-g 1

LPS-treated F1K cells in a dose-dependent manner.respectively (Fig. 1B). This TGF-b1-mediated inhibitionof IFN-g-induced I-Ad

a mRNA accumulation was also Densitometric analysis of two independent experimentsdemonstrate that TGF-b1 inhibits B7-1 expression byevident if TGF-b1 (5 ng/ml) was added simultaneously

with IFN-g, suggesting that the 24 hour preincubation 70%, 85% and 90% at doses of 0.1, 1, and 5 ng/ml,respectively. Interestingly, the inhibitory effect of TGF-b1with TGF-b1 was not critical for blocking class II MHC

expression in F1K cells (data not shown). Further FACS on IFN-g 1 LPS-induced B7-1 expression, unlike IFN-g-induced class II MHC, was evident at lower doses, thatanalysis assessed the effect of TGF-b1 on F1K cell-sur-

face class II MHC expression. Consistent with our North- is, 0.1 ng/ml. FACS analysis further revealed a similarreduction in IFN-g 1 LPS-induced cell-surface B7-1 ex-ern hybridization data, IFN-g-induced class II MHC ex-

pression (I-Ab) was markedly inhibited at 48 hours by pression induced by 24 hour pretreatment with TGF-b1(5 ng/ml) (Fig. 5).pretreatment with TGF-b1 (5 ng/ml) for 24 hours (Fig. 2).

Previous studies of nonprofessional antigen presentingIFN-g-induced class II transactivator gene suppression cells indicate that TGF-b has stimulus-specific inhibitoryby TGF-b1 in F1K cells effects on ICAM-1 expression, another costimulatory

molecule relevant to cell-mediated interactions [36]. WeAs IFN-g-induced class II MHC expression requiresupregulation of class II transactivator, we investigated therefore examined the effect of TGF-b1 on IFN-g- or

IFN-g 1 LPS-induced ICAM-1 expression in F1K cells.TGF-b1-mediated effects on IFN-g-induced class IItransactivator mRNA expression in F1K cells [31–33]. Pretreatment of F1K with TGF-b1 (5 ng/ml) for 24 hours

did not markedly diminish IFN-g- or IFN-g 1 LPS-inducedSimilar to our observations of class II MHC expression,FIK cell class II transactivator mRNA expression was ICAM-1 message expression (Fig. 6). Further studies

Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression 989

Fig. 4. TGF-b1 down-regulates inducible B7–1 expression in F1K cells.F1K cells were cultured for 24 h with no additives, IFN-g (100 U/ml) 1LPS (10 mg/ml), or IFN-g 1 LPS after an initial 24 hour incubationwith various concentrations of TGF-b1. Total RNA harvested fromdiscrete F1K cell preparations was analyzed by Northern hybridizationwith a murine B7–1 cDNA probe. The b-actin signal reflects relativeamount of RNA loaded in each lane. Presented findings are representa-tive of three separate experiments.

Fig. 5. Cell-surface B7–1 expression in TGF-b1-treated F1K cells. Con-Fig. 3. Class II transactivator (CIITA) expression in cytokine-treatedfluent F1K cells were incubated in the presence or absence of IFN-gF1K cells. (A) Total RNA was harvested from untreated F1K cells or(100 U/ml) 1 LPS (10 mg/ml) for 48 hours. Some F1K cell preparationsF1K treated with IFN-g (100 U/ml) for 4, 8, 16, and 24 hours, andwere pretreated with TGF-b1 (5 ng/ml) for 24 hours before incubationanalyzed by Northern hybridization with a murine class II transactivatorwith IFN-g 1 LPS. Harvested F1K cells were then analyzed by flowcDNA probe. (B) Total RNA was harvested from F1K cells that werecytometry using an anti-B7–1 antibody (16-10A1) or isotype controltreated with various concentrations of TGF-b1 for 24 hours, before aantibody. Presented findings are representative of three separate experi-16 hour IFN-g (100 U/ml) exposure, and analyzed by Northern hybrid-ments. Lines are: (short dashed line) control Ig; (long dashed line)ization as in panel A. The b-actin signal reflects relative amount ofunstimulated F1K; (thick solid line) IFN-g 1 LPS; (thin solid line)RNA loaded in each lane. Presented findings are representative of threeTGF-b1 1 IFN-g 1 LPS.separate experiments.

Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression990

Fig. 6. Intercellular cell adhesion molecule-1 (ICAM-1) expression inTGF-b1-treated F1K cells. F1K cells were cultured for 24 hours withno additives, IFN-g (100 U/ml), and IFN-g 1 LPS (10 mg/ml). SomeF1K cell preparations were pretreated with TGF-b1 (5 ng/ml) for 24hours before incubation with IFN-g or IFN-g 1 LPS. Harvested F1K Fig. 7. Interferon-g (IFN-g) receptor b chain expression in cytokine-cell RNA was evaluated for ICAM-1 mRNA expression with a murine treated F1K cells. Total RNA was harvested from either untreated F1KICAM-1 cDNA probe. The b-actin signal reflects relative amount of cells or F1K cells 16 hours after exposure to IFN-g (100 U/ml), TGF-b1RNA loaded in each lane. Presented findings are representative of three (10 ng/ml), or both IFN-g 1 TGF-b1, and hybridized to a murinedistinct experiments. IFN-g receptor b chain cDNA probe. The b-actin signal reflects relative

amount of RNA loaded in each lane. Presented findings are representa-tive of three separate experiments.

conducted with higher doses of TGF-b1 (10 ng/ml), didnot markedly alter IFN-g-induced ICAM-1 mRNA ex-

the effect of TGF-b1 on IFN-g-induced gene expressionpression, although we observed a decrease, 40% by den-in F1K cells is not due to modulation of IFN-g receptorsitometry, in IFN-g 1 LPS-induced ICAM-1 messageb chain mRNA expression.expression at this dose of TGF-b1 (data not shown).

Further FACS analysis of F1K cells treated with IFN-gTGF-b1 inhibits the antigen presenting cell functionor IFN-g 1 LPS exposed to TGF-b1 (10 ng/ml), however,of IFN-g 1 LPS-stimulated F1K cellsdid not demonstrate a change in cell-surface ICAM-1

To examine the functional relevance of diminishedexpression (data not shown). Unlike the marked inhibi-B7-1 costimulatory molecule expression in the setting oftory effect on B7-1 expression observed in F1K cells,TGF-b1 pretreatment, we performed T cell activationTGF-b1 did not exert a similar influence on cytokine-assays with IFN-g 1 LPS-treated F1K cells and MR1.3,induced upregulation of ICAM-1 expression.a nephritogenic CD41 Th2 clone derived from kidneys

Effects of TGF-b1 on IFN-g receptor b chain of animals with chronic graft-vs-host disease, a model ofexpression in F1K cells autoimmune glomerulonephritis [23]. Previous studies

demonstrated that MR1.3 recognizes renal antigen inResponsiveness to IFN-g is conferred by expressionthe context of I-Ad, and adoptively transfers renal in-of the IFN-g receptor b chain, which is required forflammation and damage to naive syngeneic recipientsappropriate IFN-g-mediated signaling [37]. As TGF-b[23]. As shown in Figure 8, a fourfold increase in IL-4has previously been shown to decrease antigen present-production in assays with MR1.3 and IFN-g 1 LPS-ing cell expression of the IFN-g receptor, we examinedtreated F1K cells demonstrated activation of MR1.3the influence of TGF-b1 exposure on F1K cell IFN-gcells. By contrast, MR1.3 IL-4 production induced byreceptor b chain expression [38]. As shown in Fig. 7,IFN-g 1 LPS-treated F1K cells was markedly diminishedF1K cells constitutively express IFN-g receptor b chainwhen cells were previously exposed to TGF-b1. MR1.3mRNA, which is not upregulated by IFN-g (100 U/ml)IL-4 production was reduced to 38% of maximal levelsexposure. Exposure to TGF-b1 (10 ng/ml) in this setting,observed with IFN-g 1 LPS-treated F1K cells. Thesehowever, does not alter baseline F1K cell IFN-g receptor

b chain expression (Fig. 7). These results suggest that results indicate that TGF-b1-mediated downregulation

Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression 991

Fig. 8. TGF-b1 inhibits T cell activation byLPS 1 IFN-g-treated F1K cells. MR1.3 T cells(1 3 106 cells/well) and renal antigen (10mg/ml) were cultured for 48 hours with T cellmedium alone or with F1K cells (0.5 3 106

cells/well). Some F1K cells were pretreatedfor 48 hours with IFN-g (100 U/ml) 1 LPS(10 mg/ml), and others were pretreated withTGF-b1 (5 ng/ml) for 24 hours, followed bya 48 hour incubation with IFN-g 1 LPS.MR1.3 T cell activation was then evaluatedby measuring IL-4 production in cell-free su-pernatants. Results are presented as a percent-age of maximal IL-4 production observed inthe IFN-g 1 LPS-treated F1K cells (mean of3 experiments 6 sd).

of class II MHC and B7-1 expression alters the antigen T cell-mediated events [12]. These studies provide directevidence for an inhibitory effect of TGF-b1 on class IIpresenting cell function of F1K cells.MHC expression in renal tubular epithelial cells at boththe level of mRNA and cell-surface expression. This

DISCUSSION effect is evident whether or not TGF-b1 is added 24 hoursThe studies presented here describe TGF-b1-mediated prior to stimulation with IFN-g, or if TGF-b1 is coadminis-

downregulation of induced class II MHC and B7-1 ex- tered with IFN-g. Although not previously described inpression in murine renal tubular epithelial cells. TGF-b1, renal tubular epithelial cells, it is interesting that TGF-b1however, did not markedly alter induced ICAM-1 ex- has a similar regulatory influence on IFN-g-induced class IIpression in F1K cells under similar experimental condi- MHC expression in other nonprofessional antigen pre-tions. Further analysis revealed that inhibitory effects of senting cell lines [44, 45]. The impact of TGF-b exposureTGF-b1 on IFN-g-induced gene expression in F1K cells on class II MHC expression in professional antigen pre-was not due to downregulation of IFN-g receptor b chain senting cells, however, is less straightforward, with re-expression. These studies also demonstrated that TGF- ports of both inhibitory and stimulatory effects on mu-b1-mediated modulation of relevant cell-surface mole- rine B cell lymphocytes [46]. Such disparate effects oncules had a significant functional impact on F1K, as it class II MHC expression in response to TGF-b1 expo-decreased its ability to stimulate a nephritogenic CD41 sure may reflect inherent functional differences betweenTh2 clone. professional and nonprofessional antigen presenting cell

The antigen presenting cell capabilities of renal tubu- subsets.lar epithelial cells have been well-described [11, 39–41]. Recent investigations have delineated some of the rel-Multiple investigators have reported that renal tubular evant intracellular signaling events which mediate tran-epithelial cell preparations present both self and foreign scriptional responses to IFN-g, including that of class IIantigen to T cell hybridomas and CD41 T cell clones transactivator [31, 33]. Class II transactivator expression[11, 39–41]. Moreover, previous studies in our laboratory is induced by IFN-g, and its protein product is an obligatedemonstrated that proinflammatory cytokine-stimulated activator of class II MHC gene transcription in IFN-g-renal tubular epithelial cells upregulated class II MHC treated cells [31, 33]. In addition, both basal and inducedexpression, and expression of costimulatory molecules, class II transactivator expression correlates well withsuch as B7-1 and ICAM-1 [11]. The relevance of these the expression of class II MHC [32]. Our observationscell-surface markers to immune-mediated events in the demonstrate that TGF-b1 inhibits class II MHC expres-kidney has been further supported by their increased sion by limiting steady-state levels of class II transactivatorexpression on renal tubular epithelial cells in vivo, in mRNA, and suggest one mechanism of TGF-b1-mediatedsettings such as murine chronic-graft-vs-host disease, immune modulation of IFN-g-induced stimuli in renalIFN-g 1 LPS treatment, allograft rejection, and lupus tubular epithelial cells. Studies conducted by other inves-nephritis [11, 42, 43]. tigators are consistent with these findings, and indicate

Our current studies focused on the impact of TGF-b1 that TGF-b decreases class II transactivator activity inon antigen presenting cell function of renal tubular epi- distinct antigen presenting cell subsets [47, 48].

In addition to class II MHC expression, these studiesthelial cells, in view of its immunosuppressive effects on

Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression992

examined the role of TGF-b1 in modulating the expres- pression are not due to down-regulation of IFN-g recep-sion of costimulatory molecules on renal tubular epithe- tor b chain mRNA expression. Although down-regula-lial cells, as they provide critical second signals in cell- tion of IFN-g receptor expression in response to TGF-bmediated immune responses [7]. As previously stated, has been reported in studies of professional antigen pre-earlier studies from our laboratory demonstrated that senting cells, it has not previously been studied in non-proinflammatory mediators such as IFN-g and LPS not professional antigen presenting cell preparations [38].only induce class II MHC, but also B7-1 expression in In aggregate, our analyses further suggest the uniquerenal tubular epithelial cells [11]. Our current report antigen presenting cell characteristics of renal tubularextends these observations and is the first description epithelial cells, which may directly impact on the regula-of TGF-b1-mediated modulation of B7-1 expression in tion of renal immune responses in vivo.antigen presenting cells. These studies relate a significant It is noteworthy that TGF-b has been widely investi-immunomodulatory effect of TGF-b1 on renal tubular gated because of its pathogenic role in extracellularepithelial cells, in inhibiting induced B7-1 and abrogating matrix accumulation in both nonimmune and immune-stimulation of nephritogenic CD41 T cells in vitro. As mediated renal diseases [53, 54]. Indeed, studies of TGF-previous studies with LPS 1 IFN-g-stimulated renal tu- b1 transgenic mice suggest that increased circulatingbular epithelial cells demonstrated a similar decrease in levels of active TGF-b1 initiate a cascade of pathologicclass II MHC expression that, in the setting of upregu- events resulting in glomerular and interstitial fibrosislated B7-1, actually markedly enhanced their antigen [55]. Despite these observations, however, the potentialpresenting cell function, TGF-b1-mediated inhibition of immunoregulatory effects of TGF-b1 in vivo have alsoB7-1 may be a more critical event in blocking antigen been evaluated in different models of T cell-mediatedpresenting cell function than down-regulating class II

disease, such as experimental allergic encephalomyelitisMHC [11].

and rheumatoid arthritis [56, 57]. Of particular interest,Despite the significant inhibitory effects on class IIstudies in models of immune-mediated renal disease,MHC and B7-1 expression noted in our studies, we didsuch as chronic graft-vs-host disease and tubulointersti-not observe a similar influence of TGF-b1 on ICAM-1tial nephritis, have also revealed a disease-protective roleexpression in IFN-g or IFN-g 1 LPS-treated renal tubu-for TGF-b1 in susceptible animals (abstract; Matsuuralar epithelial cells, although some decrease in mRNA,et al, Lupus, 7:56, 1998) [12]. Exposure of nephritogenicbut not cell-surface expression was apparent in the high-T cells to TGF-b1 blocks their ability to transfer diseasedose (10 ng/ml) TGF-b1 studies. Reports of TGF-b-to naive animals (abstract; ibid) [12]. Our current experi-mediated effects on both professional and nonprofes-ments further extend these observations in the contextsional antigen presenting cell expression of costimulatoryof immune-mediated renal disease, and suggest thatmolecules, such as ICAM-1 and VCAM-1, have beenTGF-b1 exposure may alter the antigen presenting cellvariable [36, 49–52]. Despite reports of the inhibitoryfunction of resident renal parenchymal cells. TGF-b1-effects of TGF-b on proinflammatory cytokine-inducedmediated down-regulation of class II MHC and B7-1 onICAM-1 expression in antigen presenting cell prepara-renal tubular epithelial cells is functionally significant,tions, the refractoriness of induced ICAM-1 expressionas it alters the antigen presenting cell function of proin-to TGF-b has also been reported in recent publications

of both professional and nonprofessional antigen pre- flammatory cytokine-treated renal tubular epithelialsenting cells [36, 49, 51, 52]. Our current findings further cells in vitro. Such findings suggest a potential mechanismsupport the notion that TGF-b-mediated influences on for local down-regulation of cell-mediated renal immunecostimulatory molecule expression in antigen presenting responses that may underlie immunosuppressive effectscells may result from cell- or species-specific differences of TGF-b1 in vivo.in regulation.

Our observations regarding differential TGF-b1 mod- ACKNOWLEDGMENTSulation of class II MHC, B7-1, and ICAM-1 expression

This work was done during the tenure of a Grant-in-Aid Award fromindicate that TGF-b1 does not interfere with all IFN-g- the American Heart Association (95012790). It was also supported inmediated signaling pathways in renal tubular epithelial part by NIH DK-07006, DK-49724, the University of Pennsylvania

Research Foundation and by administrative/educational funds fromcells. Assessing IFN-g-induced stimuli following TGF-b1the DCI RED Fund. This work was presented in part at the annualexposure, we investigated IFN-g receptor b chain expres-meeting of the American Society of Nephrology, Philadelphia, PA,

sion in renal tubular epithelial cells, as this cell-surface October 1998.receptor is required for appropriate IFN-g-mediated sig-

Reprint requests to Catherine M. Meyers, M.D., University of Penn-naling [37]. We noted that renal tubular epithelial cellssylvania, Renal-Electrolyte and Hypertension Division, 700 Clinical Re-constitutively express the IFN-g receptor b chain, whichsearch Building, 415 Curie Boulevard, Philadelphia, Pennsylvania

is not altered by TGF-b1 treatment, suggesting that 19104.E-mail: meyersc@mail.med.upenn.eduTGF-b1-mediated effects on IFN-g-induced gene ex-

Banu and Meyers: TGF-b1 modulates class II MHC and B7-1 expression 993

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