Immunology and Pathology

Central Role for Interferon-γ Receptor in the Regulation of Renal MHC Expression

YUTAKA TAKEI, TASHA N. SIMS, JOAN URMSON and PHILIP F. HALLORAN
JASN February 2000, 11 (2) 250-261;

Abstract

The role of the interferon-γ (IFN-γ) receptor 1 (IFN-γR1) was investigated in the regulation of MHC expression in kidney in the basal state, in response to potent inflammatory stimuli, and after renal injury. In this study, MHC regulation in mice lacking IFN-γR due to targeted disruption of the IFN-γR1 gene (GRKO mice) was compared with regulation in 129Sv/J mice with wild-type IFN-γR1 genes. Basal class I expression was reduced by approximately 45% in kidneys of GRKO mice, while basal class II expression was confined to interstitial cells and was not reduced in GRKO kidneys. Recombinant IFN-γ administration induced widespread expression of class I and II in renal tubules, arterial endothelium, and glomeruli of 129Sv/J mice, but produced no change in kidneys of GRKO mice. Potent systemic inflammatory stimuli (injections of allogeneic cells, skin sensitization with oxazolone, and injection of bacterial lipopolysaccharide) significantly induced both class I and class II expression in 129Sv/J mice, but not in GRKO mice. Acute renal injury increased local expression of class I and II in both 129Sv/J and GRKO mice, but the induction in GRKO mice was reduced compared with 129Sv/J mice. Thus, the IFN-γ receptor plays a unique and nonredundant role in the regulation of renal MHC in the response to inflammation, in the response to renal injury, and in the basal state.

The MHC products are key components in antigen presentation and are thus relevant to graft rejection, autoimmunity, and host defense. MHC molecules play essential roles in T cell development and T cell survival in the periphery (1), in presenting antigen during immune responses, and as a target for immune effector mechanisms. The expression of MHC genes is regulated transcriptionally (2) and is massively increased by inflammatory stimuli and moderately increased by local injury (3). Interferon-γ (IFN-γ) potently induces the expression of MHC genes and thus affects antigen presentation and potential immune targets in disease states. MHC induction by inflammatory stimuli is reduced or absent in mice lacking IFN-γ (3). IFN-γ or IFN-γR has been shown to regulate a variety of renal diseases including autoimmune systemic lupus erythematosus in mice (4,5), but not anti-glomerular basement membrane crescentic nephritis (6). IFN-γ also affects both early and late events in organ transplants (7, 8, 9).

IFN-γ signals via the IFN-γ receptor (IFN-γR) complex composed of a ligand-binding chain R1 and an accessory chain R2, which are members of the type II cytokine receptor family that includes IFN-α/β R and IL-10R (10, 11, 12, 13). In the IFN-γ/IFN-γR structure, there is possible contact between the IFN-γR2 accessory chain and the IFN-γ homodimer (14). To date, in vitro studies have not shown persistent IFN-γ-dependent effects through R2 when R1 is disrupted. However, the issue has not been addressed in vivo. Humans and mice with disrupted genes for IFN-γ, IFN-γR1, or IFN-γR2 display severe defects in IFN-γ-mediated responses and some defects in host defense (15, 16, 17). However, there has been no systematic comparison of the phenotypes produced by disruption of the genes for IFN-γ, IFN-γR1, and IFN-γR2 in vivo. Each of these phenotypes should be established independently in vivo, particularly because the IFN-γR is widely distributed in most resting cells and has potential for multiple and massive effects on a variety of cell types. One reported discrepancy between IFN-γR1 deficiency versus IFN-γ deficiency is that mice with disrupted IFN-γR1 genes (GRKO mice) express MHC class II on their renal tubules in vivo in response to bacterial lipopolysaccharide (LPS) (18), whereas mice with disrupted IFN-γ gene (GKO mice) show little class II induction after LPS (3). This apparent discrepancy could reflect either phenotypic differences between the GRKO and GKO mice due to previously unsuspected redundancies between IFN-γ and its R1 and R2 mutations induced by the gene disruption protocol, and/or background genetic differences or potential variance in experimental design.

Since GRKO mice are becoming important models for immunologic renal disease and transplantation (4, 5, 6), it is important to establish their renal phenotypes and to explore potential differences between GRKO and GKO mice. In the present study, we examined the MHC phenotype of kidneys of GRKO mice compared with wild-type mice and the previously described phenotype of GKO mice (3,19). We studied three states of MHC expression: the basal state without stimulation, after potent inflammatory stimulation or recombinant IFN-γ (rIFN-γ), and after local renal injury. Basal class I but not class II expression was reduced in kidneys from GRKO mice. Systemic inflammatory stimuli induced little or no MHC mRNA or products, and GRKO mice showed no response to injections of rIFN-γ. Renal injury induced MHC expression in GRKO mice, but the level of induction was reduced compared with control 129Sv/J mice. Our results also suggest that class II induction is greatly reduced in GRKO mice given bacterial LPS, consistent with our previous studies of GKO mice. Thus, the IFN-γ/IFN-γR pathway plays an essential role in basal class I expression, in the intense systemic MHC expression that accompanies inflammation, and in the local MHC induction in injured tissue. The results are consistent with the exclusive and essential roles of IFN-γ and IFN-γR1 in basal and induced renal MHC expression in vivo, but also confirm that acute renal injury can induce MHC expression by a pathway independent of IFN-γ or IFN-γR1.

Materials and Methods

GRKO Mice

The original GRKO (129/Sv/Ev) mouse strain with disrupted IFN-γR1 genes was generated by gene targeting in murine embryonic stem cells (17). The gene was disrupted by inserting the neomycin-resistance gene (neor) into exon V, which encodes an extracellular domain. Homozygous 129Sv/Ev mice were provided by Dr. Michel Aguet (University of Zurich, Zurich, Switzerland).

Control Mice with Wild-Type IFN-γR1 Genes

129Sv/J mice with wild-type IFN-γR1 genes were obtained from Jackson Laboratories (Bar Harbor, ME) and used as controls. The mice were maintained in the Health Sciences Laboratory Animal Services at the University of Alberta and were kept on acidified water. All experiments conformed to approved animal care protocols.

rIFN-γ Treatment

The rIFN-γ was generously provided by Genentech, Inc. (South San Francisco, CA). A dose of 50,000 IU in saline, or saline alone as a control, was intraperitoneally injected in each animal on day 0 and day 1, and mice were sacrificed on day 3.

P815 Tumor Allografts

Murine mastocytoma tumor cell line P815 (American Type Culture Collection, Rockville, MD) was passaged in DBA/2(H-2d) mice from Jackson Laboratories (Bar Harbor, ME). After P815 cells were collected from the ascites fluid, approximately 20 × 106 cells, or an equivalent volume of saline as a control, were injected intraperitoneally into mice. The tissues of experimental mice were examined 7 d later.

Oxazolone Skin Painting

A total of 100 μl of 25% oxazolone (4-ethoxymethylene-2 phenol-2 oxazolin-5-one) in acetone was painted onto the shaved back of mice on day 0. On day 4, 25 μl of 5% oxazolone in acetone was again applied to the same area. Sham mice were treated with 100 μl of acetone onto the shaved back on day 0 and then repeated with 25 μl of acetone on day 4. Mouse tissues were harvested on day 6.

LPS Injections

LPS of Salmonella minnesota dissolved in sterile saline at 250 μg/ml and heated at 56°C for 5 h, or a saline control, was injected intraperitoneally on day 0, and the mice were sacrificed on day 3.

Acute Toxic Injury

Mice were subcutaneously injected with gentamicin (250 mg/kg three times a day) or with a sham saline injection on two consecutive days. Mice were killed and their tissues were examined on day 7.

Acute Tubular Necrosis

Unilateral ischemic injury has been widely reported (3,20,21). Briefly, mice 8 to 12 wk of age were anesthetized with 2,2,2-tribromoethanol in tert-butyl alcohol (Avertin, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada) by intraperitoneal injection. The left renal pedicle was identified through a midline incision and occluded with a micro-bulldog clamp for 60 min. Before closure, the kidney was inspected to ensure reperfusion, and the abdominal cavity was filled with warm saline. Sham-operated control mice underwent a simple laparotomy under identical conditions. Each group had between 5 and 10 mice, and the tissues were harvested at day 7.

Antibodies

Monoclonal antibodies (mAb) were purified in our laboratory from supernatants of hybridoma cell lines. The lines were AF 6-120.1.2 (mouse IgG against mouse I-Ab), 20-8-4S (mouse IgG against mouse H-2KbDb), M1/42.3.9.8 (rat IgG2a against all mouse H-2 haplotypes), and M5/114.15.20 (rat IgG2b against mouse I-Ab,d,q and I-Ed,k), obtained from American Type Culture Collection (Rockville, MD). Briefly, the hybridoma cell lines were maintained in tissue culture, and the supernatants containing AF 6-120.1.2 (anti-I-Ab) and 20-8-4S (anti-H-2KbDb) were purified by protein A chromatography. The supernatants containing M1/42.3.9.8 (anti-H-2 antigens, all haplo-types) and M5/114.15.20 (anti-I-Ab,d,q and I-Ed,k) were ammonium sulfate-precipitated, and then the antibodies were purified with a DE52 anion exchanger column (Whatman, Hillsboro, OR) and by concentration with Amicon ultrafiltration. The protein concentration was adjusted to 1 mg/ml and maintained at -70°C. Peroxidase-conjugated goat IgG against rat IgG and peroxidase-conjugated goat IgG against mouse IgG were supplied by Organon Teknika (Scarborough, Ontario, Canada).

Radiolabeled Antibody Binding Assay

Anti-H-2KbDb mAb and anti-I-Ab mAb were radiolabeled with [125I]-iodide using the Iodogen method (Pierce Chemical Co., Rockford, IL) (22). Tissues of individual mice were prepared as described previously (22,23). The tissue concentration was adjusted to 20 mg/ml. A total of 5 mg of kidney tissue was aliquoted in triplicate and spun. The pellets were incubated on ice with 125I-labeled mAb in 10% normal mouse serum (100,000 cpm per 100 μl) with agitation for 1 h. After washing, the pellets were counted in a gamma counter and the nonspecific binding of a negative tissue was subtracted.

Staining of Tissue Sections

Flash-frozen cryostat sections were fixed in acetone, then incubated with normal goat serum. The slides were incubated with rat mAb against class I (M1) and class II (M5) or controls. The slides were then incubated with affinity-purified peroxidase-conjugated goat anti-rat IgG F(ab′)2 fragment. Antibody binding was visualized by the use of 3′3-diaminobenzidine tetrahydrochloride and hydrogen peroxide for the color reaction and then counterstained with hematoxylin.

Northern Blot Analysis

Total RNA was extracted from pooled samples of 4 M guanidinium isothiocyanate followed by centrifugation in 5.7 M cesium chloride as described previously (3). Total RNA (10 μg) was electrophoresed in a 1.5% agarose gel in the presence of 2.2 M formaldehyde, transferred to a nitrocellulose filter, and hybridized with 32P-labeled cDNA probes for H-2K (class I) or α chain of I-A (class II) and with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin for loading control. After hybridization, the blots were exposed to Kodak X-Omat AR film at -70°C with an intensifying screen.

Statistical Analyses

Statistical significance between experimental groups was performed using ANOVA, and MHC fold increases were determined from cpm counts using a standard curve.

Results

Basal MHC Expression in Kidney from 129Sv/J and GRKO Mice

We examined basal expression of MHC class I and class II products in kidney homogenates of 47 age- and gendermatched GRKO and 47 129Sv/J (WT) mice by radiolabeled antibody binding assay (RABA) (Figure 1). The binding of the class I mAb (as assessed by cpm) was significantly lower in GRKO mice than in 129Sv/J mice (P < 0.05). On a standard curve, this corresponded to a 45% decrease in class I expression in GRKO mice. Basal expression of MHC class II in GRKO mice was not significantly reduced.

Figure 1.
Figure 1.

Basal MHC expression in kidney of 129Sv/J (WT) and GRKO mice. MHC class I (H-2KbDb) and II (I-Ab) detection in normal kidney homogenates using radiolabeled monoclonal antibody (mAb). One kidney of each mouse was tested individually, and all of the individual results were used to calculate a single mean ± SEM. The results are representative of eight experiments with five to 10 mice per experiment (total, n = 47). *, significant differences between 129Sv/J and GRKO. Comparisons between the means of the pooled experiments were performed using two-factor ANOVA.

Response to rIFN-γ

We assessed the renal response to IFN-γ in GRKO and 129Sv/J mice by measuring MHC mRNA and protein levels after injecting 100,000 U of rIFN-γ or saline (control) intraperitoneally. The rIFN-γ treatment increased the steady-state levels of MHC class I and II mRNA significantly in 129Sv/J, but not in GRKO kidneys (Figure 2A). IFN-γ also increased expression of MHC products in tissue homogenates in 129Sv/J but not GRKO kidneys (Figure 2B). When plotted on a standard curve, the changes in 129Sv/J kidneys corresponded to an approximate 14-fold increase in class I expression and a two-fold increase in class II expression after IFN-γ treatment. Thus, kidneys of GRKO mice have no MHC changes in response to rIFN-γ.

Figure 2.
Figure 2.

The effect of recombinant IFN-γ (rIFN-γ) administration on expression of MHC expression in kidneys from 129Sv/J (control) and GRKO mice. The rIFN-γ was administered on days 0 and 1, and mice were sacrificed on day 3. (A) Pooled mRNA from five kidneys were subjected to Northern blot analysis for MHC class I, class II, and GAPDH (loading control). (B) MHC class I and II expression in kidney homogenates of 129Sv/J and GRKO mice was assessed by radiolabeled antibody binding assay (RABA). *P < 0.05, sham versus the rIFN-γ-injected groups. Comparisons were performed using ANOVA.

Effect of Remote Proinflammatory Stimuli on Renal MHC Expression in GRKO Mice

MHC expression is induced in kidney during inflammation at remote sites, due to systemic circulation of cytokines. We compared these MHC responses in GRKO and 129Sv/J mice.

Allogeneic Stimulation. Mice inoculated with allogeneic P815 ascites tumor cells develop intense IFN-γ-dependent MHC expression in kidney (3). GRKO and control mice were challenged with an intraperitoneal injection of allogeneic P815 cells. The steady-state MHC mRNA (Figure 3A) increased in 129Sv/J mice but not in GRKO mice. As assessed by RABA (Figure 3B), MHC product expression was increased 28-fold (class I) and 12-fold (class II) in kidneys of 129Sv/J mice. There was no increase in MHC expression in GRKO mice.

Figure 3.
Figure 3.

Expression of steady-state mRNA and molecules for MHC class I and II in kidney from of 129Sv/J (control) and GRKO mice in response to allogeneic cells. P815 was injected on day 0, and animals were harvested on day 7. (A) Pooled mRNA of five kidneys was subjected to Northern blot analysis for MHC class I, class II, and β-actin (loading control). (B) MHC class I and II expression in kidney homogenates of 129Sv/J and GRKO mice was assessed by RABA. *P < 0.05, sham versus the gentamicin-injected groups. Comparisons were performed using ANOVA.

Skin Sensitization with Oxazolone. We induced a local skin hypersensitivity response to oxazolone and assessed the induction of MHC class I and class II mRNA expression and products by Northern blot analysis and RABA, respectively. Oxazolone induced less skin inflammation in GRKO mice, as reported previously (24) (data not shown). Kidneys of 129Sv/J mice with oxazolone skin reactions exhibited increased steady-state mRNA levels for class I and class II. GRKO kidneys showed essentially no increase of MHC mRNA expression after oxazolone treatment (Figure 4A). Similarly, the RABA showed a striking increase in MHC products in 129Sv/J but not GRKO kidney homogenates (Figure 4B). This corresponded with approximate six- and threefold increases in class I and II expression, respectively.

Figure 4.
Figure 4.

Expression of steady-state mRNA and molecules for MHC class I and II in kidney from of 129Sv/J (129) and GRKO mice after skin sensitization by oxazolone or in response to lipopolysaccharide (LPS). Skin sensitization: The shaved back of mice was painted with oxazolone (100 μl of 25% oxazolone) on day 0 and received 25 μl of 5% oxazolone applied to the same area on day 4. Sham mice were treated with acetone on the same schedule. Experimental animals were harvested at day 7. LPS treatment: Mice were injected intraperitoneally with LPS (25 μg) or saline on day 0 and then sacrificed on day 3. (A) Pooled mRNA of six kidneys was subjected to Northern blot analysis for MHC class I, class II, and GAPDH (loading control). (B) MHC class I and II expression in kidney homogenates of 129Sv/J and GRKO mice was assessed by RABA. *P < 0.05, sham versus oxazolone-treated groups. Comparisons were performed using ANOVA. (C) MHC class I and II expression in kidney homogenate of 129Sv/J and GRKO mice was assessed by RABA. *P < 0.05, sham versus LPS-treated groups. The comparisons were performed using ANOVA, P < 0.05.

Lipopolysaccaride. LPS induces MHC products in kidney by an IFN-γ-dependent pathway (25). Haas et al. (18) reported that LPS induced MHC class II in renal tubular epithelium of GRKO mice. In the present experiments, LPS induced expression of class I and class II mRNA in kidney of 129Sv/J mice but not GRKO mice (Figure 4A). We also examined the expression of class I and class II molecules in kidney of 129Sv/J and GRKO mice in response to LPS treatment (Figure 4C). LPS induced class I and class II products in kidney of 129Sv/J—a fivefold increase in class I expression and a two-fold increase in class II expression in 129Sv/J mice on a standard curve—but no increase in GRKO mice.

Induction of Renal MHC Expression in GRKO Mice after Renal Injury

Renal injury induces local MHC expression (26), which is only partially dependent on IFN-γ (3,21). We induced renal injury with high toxic doses of gentamicin and assessed MHC expression. Class I mRNA levels in GRKO mice were less than in 129Sv/J mice (Figure 5A), and class I product was increased in kidney of 129Sv/J and GRKO mice after toxic renal injury (Figure 5B). The induction of class II in GRKO and 129Sv/J mice was not significantly different (Figure 5, A and B). Both the class II mRNA and the RABA were increased after toxic renal injury. The changes in 129Sv/J mice tended to be greater than in the GRKO mice. Acute injury by ischemia also induced class I expression by RABA but had less effect in GRKO mice (Figure 5C). Thus, GRKO mice displayed reduced MHC induction in response to injury, particularly ischemic injury.

Figure 5.
Figure 5.

Expression of steady-state mRNA and molecules for MHC class I and II in kidney from 129Sv/J (Wild-type) and GRKO mice after acute renal injury. Injury was induced by gentamicin (subcutaneous injection on day 0 and day 1) or unilateral ischemia (the left kidney pedicle was clamped for 60 min and released), both harvested on day 7. (A) Effect of gentamicin (Gent) injury on MHC mRNA levels. Pooled mRNA of five kidneys was subjected to Northern blot analysis for MHC class I, class II, and GAPDH. (B) Effect of gentamicin injury on class I and II expression by RABA. MHC class I and II expression was measured in kidney homogenates of 129Sv/J and GRKO mice. *P < 0.05, saline versus the gentamicin-injected groups. Comparisons were performed using ANOVA. (C) Effect of renal ischemic injury on the expression of class I and II products in 129Sv/J and GRKO kidneys by RABA. Acute ischemic injury was performed on the left renal pedicle in 129Sv/J or GRKO mice. Right kidneys serve as contralateral controls. n = 5 or 6 mice per group.

Tissue Staining for MHC Class I and II in Kidney

We assessed patterns of MHC expression in kidney sections from 129Sv/J and GRKO mice by indirect immunoperoxidase staining in four states: the basal state, after rIFN-γ administration, after systemic inflammatory stimuli, and after toxic injury. The staining for basal class I and II in kidney sections was not significantly different between 129Sv/J and GRKO mice except that class I staining of the arterial endothelium was stronger in 129Sv/J than in GRKO mice (reported previously in reference (27) (data not shown). Basal class II staining was confined to interstitial cells in 129Sv/J and GRKO mice (Table 1, Figure 6, A and B). The rIFN-γ strongly induced class I staining of tubules, arterial endothelium, and glomeruli in 129Sv/J mice but not in GRKO mice (Table 1, Figure 7, C and D). The rIFN-γ induced class II expression in tubules of 129Sv/J but not GRKO mice (Table 1, Figure 6, C and D).

View this table:
Table 1.

Immunoperoxidase staining for MHC class I and II molecules in kidney from 129Sv/J and GRKO mice in the basal and induced statesa

Figure 6.
Figure 6.

Photomicrographs showing indirect immunoperoxidase staining for MHC class II in kidneys from wild-type 129Sv/J (A: sham-treated; C: rIFN-γ treatment; E: P815 stimulation; G: oxazolone skin painted; I: LPS-treated, K: gentamicin treatment) and GRKO mice (B: sham; D: rIFN-γ; F: P815; H: oxazolone; J: LPS; L: gentamicin treatment) in the basal (sham) and induced states. Arrows show staining of tubular epithelium. The photomicrographs were taken at the same magnification (×160).

Figure 7.
Figure 7.

Photomicrographs showing indirect immunoperoxidase staining for MHC class I in kidneys from wild-type 129Sv/J (A: sham-treated; C: rIFN-γ treatment; E: P815 stimulation; G: oxazolone skin painted; I: LPS-treated, K: gentamicin treatment) and GRKO mice (B: sham; D: rIFN-γ; F: P815; H: oxazolone; J: LPS; L: gentamicin treatment) in the basal (sham) and induced states. Arrows show staining of tubular epithelium. The photomicrographs were taken at the same magnification (×160).

Remote and systemic inflammatory stimuli induced MHC in a pattern identical to that of rIFN-γ in kidneys of 129Sv/J mice (i.e., class I in tubules, arterial endothelium, and glomeruli and class II in kidney tubules) (Table 1, Figure 7, Figure 7 [class I], and Figure 6 [class II], E, G and I). These stimuli had little effect in GRKO kidney (Table 1, Figure 7, Figure 7 [class I], and Figure 6 [class II], F, H and J). In particular, heat-inactivated LPS of S. minnesota did not induce class II staining in the tubules of GRKO mice, in contrast to a previous study (18). Acute injury by gentamicin induced more staining for class I in kidney tubules of 129Sv/J than in GRKO mice (Table 1, Figure 7, K and L). In gentamicin-treated 129Sv/J mice, MHC class II antigens were induced in kidney tubules (Table 1, Figure 6K). Gentamicin treatment also induced MHC class II expression in GRKO mice. Class II-positive interstitial cell staining was also increased in both GRKO and 129Sv/J mice (Table 1, Figure 6L) after gentamicin injection.

Discussion

The present study demonstrates the essential role of IFN-γR1 in the regulation of renal MHC expression. GRKO mice had reduced MHC class I expression in the basal state, whereas basal class II expression was independent of IFN-γR. MHC expression in GRKO mice was unchanged in response to rIFN-γ. However, there remained the potential for other mechanisms that could bypass the lack of IFN-γR, particularly during complex inflammatory states in which numerous cytokines with potential for MHC regulation (i.e., IFN-α/β or tumor necrosis factor-α) are produced. The induction of renal MHC class I and II expression by systemic inflammatory stimuli, which are potent inducers of many cytokines, was essentially lost in GRKO mice. In contrast, acute renal injury by gentamicin induced MHC expression in GRKO mice, albeit at reduced levels. Thus, IFN-γR1 in kidney is essential for normal class I expression in the basal state, for class I and II expression after rIFN-γ treatment, and after systemic stimuli that induce IFN-γ in vivo. Kidney injury induces at least some MHC expression by an IFN-γR1-independent mechanism (3). The regulation of MHC gene expression in kidney is becoming a major example of the complex yet precise mechanisms that regulate gene expression in response to local and remote influences.

The close agreement of the present studies with earlier results in GKO mice indicate that both IFN-γ and IFN-γR1 have essential roles in regulating the expression of MHC genes in the kidney (3,28). The principal difference was that GKO responded to rIFN-γ and GRKO did not, as expected. These studies confirm that some renal MHC expression is independent of IFN-γ and IFN-γR1: basal class II, a component of basal class I expression, and some of the class I and II changes during acute renal injury. However, the high MHC expression that occurs in response to systemic inflammation is dependent on IFN-γ. This does not suggest that IFN-γ is the only cytokine operating, because synergy with other cytokines increases the effect of IFN-γ on MHC expression (29,30). Nevertheless, the high cytokine levels in these inflammatory states apparently cannot bypass the absence of IFN-γ or IFN-γR1. Because gene-disrupted mice sometimes reveal unsuspected redundancies, it was conceivable that in vivo other cytokines might stimulate the IFN-γR in GKO mice or IFN-γ might stimulate noncognate receptors in GRKO mice or have weak interactions with IFN-γR2 chains. IFN-γR2 does not bind ligand but is required for signal transduction in vitro (31). GRKO mice presumably have R2, but on the basis of these results R2 cannot compensate for the loss of R1 and makes no contribution to MHC regulation in the absence of R1 in vivo. Thus, IFN-γ and its receptor play nonredundant roles in their MHC regulating functions, unlike many other cytokines.

The surprising role of IFN-γ in renal MHC expression in the basal state has several implications. First, IFN-γ must be constantly present in sufficient quantities in unstimulated mice to induce genes in the arterial endothelium, raising the question of whether this is also a feature of human endothelium. However, some class I is expressed in the absence of IFN-γ or its receptor, either due to constitutive expression or to induction by non-IFN-γ stimuli. Mouse class II is more difficult to induce by IFN-γ than class I, and this may explain (as illustrated here in the response to rIFN-γ) why class I is induced but class II is not. This may be relevant to the human kidney, in which class II is present on many endothelial cells in vivo. We suggest that class II expression in human endothelium, but not mouse endothelium, reflects either higher IFN-γ production in the basal state in humans or greater sensitivity of human class II genes to basal IFN-γ production. Because IFN-γ potentiates atherosclerosis in the apolipoprotein E knockout mouse (32), the fact that basal IFN-γ production can affect gene expression in renal endothelium is potentially relevant to renal atherosclerosis.

Renal injury induces increased expression of MHC genes by mechanisms both dependent on and independent of IFN-γ/IFN-γR1, confirming our previous conclusions (3,32) and raising the question of the mechanisms. We presume that the IFN-γ/IFN-γR1-dependent component is mediated by natural killer cells, which are major sources of IFN-γ production and will be recruited to injured areas by the chemokine response. Antigen nonspecific recruitment and activation of T cells may also play a role. In contrast, we suspect that the IFN-γ/IFN-γR1-independent component of this response seen in our gentamicin toxicity experiments may represent an intrinsic response of the stressed and regenerating epithelium (i.e., to changes in integrin signals from extracellular matrix), but may also reflect non-IFN-γ cytokines from inflammatory cells. In short, the mechanisms of injury-mediated inflammation and the immune response have yet to be well defined (33). The ability of injury to induce renal MHC expression bears no obvious relationship to the nature of the inducing stimulus. For example, it is not dependent on reperfusion injury as we first proposed when we and others observed this effect after renal ischemia (20,21,34). The injury-induced increases in antigen presentation could be a link between renal injury and both autoimmunity and transplant rejection (33,35).

The differences between our results with LPS (class II induction was IFN-γ-dependent) and those of Haas et al. (18) (class II induction was independent of IFN-γ) probably reflect variations in experimental protocol. Haas et al. studied MHC induction by staining tissues 4 d after injection of 50 μg of LPS from Escherichia coli, noting that LPS induced MHC class II expression in renal tubules of GRKO mice. They concluded that IFN-γ is not absolutely required for induction of class II after LPS treatment. We found little or no class II induction by S. minnesota LPS in GRKO tubules, compared with 129Sv/J controls. One possible mechanism for the induction of class II by LPS in GRKO mice in the studies of Haas et al. is that they used an LPS protocol that induced some renal injury and thus led to the type of class II expression that follows acute tubular injury. We selected heat-inactivated LPS from S. minnesota to avoid the stress response and injury, and thus the only effects we elicited were those related to IFN-γ production.

The congruence between the phenotypes of GKO and GRKO mice confirms the unique dependency of IFN-γ on IFN-γR1 and vice versa, and the unique role of the IFN-γ/IFN-γR1 unit in renal MHC regulation in vivo. Certain effects of IFN-γ on immune regulation as well as the host defense problems encountered by humans and mice with IFN-γ and IFN-γR deficiencies may reflect the defects in antigen presentation and MHC regulation, rather than direct effects on lymphocytes. The role of the tubular epithelium in antigen presentation has been explored elsewhere (36, 37, 38, 39) but remains unresolved. Regulation is not limited to class II, since class I and many other components of the antigen presenting system are also induced in kidney (40). It will thus be instructive to explore how the high responsiveness of the kidney to stimuli, both through the IFN-γ/IFN-γR system and independent of it, impacts the propensity toward autoimmune diseases (41).

Acknowledgments

We are grateful to Dr. Michel Aguet (University of Zurich, Zurich, Switzerland) for arranging for us to receive the GRKO mice and to Genentech for providing us with rIFN-γ. We thank Dr. Marjan Afrouzian for reviewing the pathology and Ms. Lina Kung for reviewing the manuscript. We also wish to thank Ms. Pam Publicover for her skilful secretarial assistance.

This work is supported by operating grants from The Medical Research Council of Canada, The Kidney Foundation of Canada, The Muttart Chair in Clinical Immunology, the Royal Canadian Legion, Fujisawa Canada, Novartis Pharmaceuticals Canada, and Hoffmann-La Roche Canada; Dr. Yutaka Takei was supported by a grant from the International Council for Canadian Studies; Tasha N. Sims is supported by a Studentship from the Alberta Heritage Foundation for Medical Research.

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Central Role for Interferon-γ Receptor in the Regulation of Renal MHC Expression
YUTAKA TAKEI, TASHA N. SIMS, JOAN URMSON, PHILIP F. HALLORAN
JASN Feb 2000, 11 (2) 250-261;
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