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Immunomodulatory effects of interferon-γ on autoreactive nephritogenic T-cell clones
Kidney International, Vol. 55 (1999), pp. 1395–1406
Immunomodulatory effects of interferon-g on autoreactive nephritogenic T-cell clones CATHERINE M. MEYERS and YOUKANG ZHANG Renal-Electrolyte and Hypertension Division, University of Pennsylvania School of Medicine, Department of Medicine, Philadelphia, Pennsylvania, USA, and the Institute of Nephrology, The First Teaching Hospital, Beijing Medical University, Beijing, China
Immunomodulatory effects of interferon-g on autoreactive nephritogenic T-cell clones. Background. We examined the immunomodulatory effects of interferon-g (IFN-g) on renal-derived CD41 a/b1 T cells, called mouse renal (MR) cells, isolated from animals with murine chronic graft-versus-host disease, a model of autoimmune glomerulonephritis. MR T cells express a Th2 cytokine profile, although IFN-g expression is also detected in a subset of clones that adoptively transfers renal disease to naive recipients. In view of disparate patterns of IFN-g expression, we evaluated the effects of exogenous IFN-g on nephritogenic (MR1.3) and nonnephritogenic (MR1.6) clonal activity. Methods. These studies examined IFN-g–mediated effects on clonal proliferation, cytokine production, nephritogenic potential, and IFN-g receptor expression. Results. IFN-g mediated dose-dependent inhibition of MR1.3 and MR1.6 proliferation. This cytostatic effect was not mediated by inhibiting cytokine genes, as expression of interleukin (IL)-4, IL-10, IL-13, and IFN-g after IFN-g treatment was not markedly altered in either clone, although baseline IL-13 expression was enhanced in MR1.6. IFN-g markedly altered the functional phenotype of MR1.6, as pretreated recipients developed severe mononuclear cell infiltrates and tubular damage following adoptive transfer of MR1.6. Neutralizing anti– IFN-g antibodies did not inhibit MR1.3 nephritogenicity, but did block MR1.6-induced disease in IFN-g–treated mice. Although both clones constitutively expressed the IFN-g receptor b chain, IFN-g exposure decreased its expression in MR1.3 cells, but did not markedly change its expression in MR1.6 cells. Conclusion. These studies describe an unusual permissive role for IFN-g in modulating nephritogenic Th2 activity in vivo, which facilitates the initiation of cell-mediated autoimmune renal injury. Apparent differential effects of IFN-g on distinct T-cell clones may be mediated in part by alterations in cytokine receptor expression.
Murine chronic graft-versus-host disease (cGVHD) is induced following the transfer of parental (DBA/2) T Key words: nephritis, T cell lymphocyte, graft vs. host disease, cytokines, autoimmunity, MHC. Received for publication February 6, 1998 and in revised form November 20, 1998 Accepted for publication November 23, 1998
1999 by the International Society of Nephrology
cells to (C57BL/6 3 DBA/2) F1 (B6D2 F1) hybrid recipients [1]. This multisystem autoimmune disorder comprises a spectrum of abnormalities such as vasculitis, polyarthritis, and mononuclear cell infiltration of multiple organs [2, 3]. A variety of circulating and deposited autoantibodies are also detected in affected animals, including anti-DNA, antihistone, and antilaminin antibodies, that are produced by autoreactive host B cells [4–7]. B6D2 F1 recipient animals with cGVHD develop an immune-complex glomerulonephritis within two to four weeks following the transfer of parental T cells, associated with progressive mononuclear (T cell) parenchymal infiltration, ultimately resulting in an end-stage renal lesion in all recipients [3, 6]. In this murine model of cGVHD, immunocompetent DBA/2 parental T cells are activated on transfer by B6D2 F1 host alloantigens, a process initiating a systemic autoimmune disease [8, 9]. Mechanisms by which host tissues are damaged, however, remain unclear. Studies from other investigators suggest that T-cell–derived proinflammatory cytokines, such as interferon-g (IFN-g), can mediate parenchymal damage and may play an important role in the development of renal disease in cGVHD [10]. Alterations in the cytokine network may provide a final common pathway of target organ damage in the setting of activation and expansion of autoreactive T-cell subsets [10]. Previous studies in our laboratory have characterized autoreactive B6D2 F1 CD41 a/b1 T cells derived from diseased kidneys of animals with cGVHD [11]. Two functional subsets of mouse renal (MR) T-cell clones were isolated. One subset is nephritogenic, restricted by Iad, and adoptively transfers tubulointerstitial inflammation and damage to syngeneic recipients [11]. The other subset does not transfer renal disease to syngeneic animals and is Iab reactive [11]. Both nephritogenic and nonnephritogenic MR T-cell clones express a Th2 cytokine pattern with marked interleukin (IL)-4, IL-6, and IL-10 expression on stimulation [11]. Further studies reveal,
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however, that MR T-cell clones that transfer renal disease also secrete low levels of IFN-g [11]. These T-cell clones are designated Th2 in view of their marked production of IL-4 and IL-10 on stimulation, with minimal IFN-g production [11]. We were particularly interested in the differential IFN-g expression in MR clones in view of the proscriptive influence of IL-10 on its synthesis in CD41 T-cell clones and its more characteristic expression in Th1 cells [12]. Interferon-g is a multifunctional lymphokine with inducible expression predominantly in T-cell lymphocytes as well as natural killer (NK) cells [10]. It is a proinflammatory cytokine that has a variety of stimulatory activities. IFN-g increases high-affinity Fcg receptor I expression on mononuclear phagocytes, augments expression of intercellular adhesion molecule-1 expression on various cell types, and increases B-7 costimulatory molecule expression on antigen-presenting cells (APCs) (monocytes) [13–15]. IFN-g is critical to differentiation and maturation of mononuclear phagocytes and activation of tissue macrophages [10]. Most notably, IFN-g increases class II major histocompatibility complex (MHC) gene transcription and cell-surface expression on APC and induces de novo class II MHC expression on various other cells, such as renal epithelial cells, perhaps converting them to “nonprofessional” APC [16–18]. Investigators also suggest that T-cell–derived IFN-g is an important mediator of inflammatory reactions mediated by CD41 T-cell clones [19]. Moreover, IFN-g–induced upregulation of class II MHC expression on renal epithelial cells in vivo may be a crucial event in local trafficking of autoimmune CD41 T cells in diseased kidneys [16–18]. In view of the disparate patterns of IFN-g expression in functional MR clonal subsets, we further evaluated the effects of exogenous IFN-g on nephritogenic (MR1.3) and non-nephritogenic (MR1.6) T-cell clones. In vitro studies indicate that IFN-g mediates dose-dependent inhibition of MR1.3 and MR1.6 proliferation, although cytokine gene expression of IL-4, IL-10, IL-13, and IFN-g after IFN-g exposure is not markedly altered in either T-cell clone. In vivo analyses, with adoptive transfers to IFNg–pretreated mice, demonstrate that the otherwise nonnephritogenic clone, MR1.6, induces cortical inflammation with focal mononuclear cell infiltrates and tubular dilatation and atrophy. Consistent with proinflammatory effects of IFN-g, renal class II MHC (I-Ab) expression was enhanced in kidneys of all IFN-g pretreated mice. Similar adoptive transfers with neutralizing anti–IFN-g antibodies, however, did not alter the nephritogenic potential of MR1.3, but did inhibit MR1.6 nephritogenicity in IFN-g–treated mice. Further analysis of IFN-g receptor b (IFN-gRb)-chain expression reveals constitutive expression in both MR1.3 and MR1.6, with distinct differences in expression levels on IFN-g exposure in these MR clones. These studies suggest an immunomodulatory
role for IFN-g in the cell-mediated renal lesion observed in this model of autoimmune glomerulonephritis, but also indicate that pathogenic T-cell lymphocytes can elicit parenchymal injury by other cytokine-mediated pathways. METHODS Mice B6D2 F1 (H-2b/d) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). T-cell clones Mouse renal T-cell clones, MR1.3 and MR1.6, were propagated by weekly passage with 10% MLA-144 supernatants as a source of IL-2 and other growth factors, renal antigen preparation derived from normal B6D2 F1 kidneys (10 mg/ml) and 5 3 106 irradiated (2500 rads) syngeneic B6D2 F1 spleen cells. T-cell culture medium consisted of RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with glutamine, antibiotics (penicillin, streptomycin, gentamicin), 10% decomplemented fetal calf serum (FCS), 5% NCTC (Whittaker Bioproducts, Inc., Walkersville, MD, USA), and 2 3 1025m 2-mercaptoethanol. Typically, wells became confluent at five to seven days and were carried every 10 to 14 days. All T cells were cultured at 378C in a 5% CO2 incubator. Proliferation assays Mouse renal T-cell clones (1 3 105) harvested from day 7 to 9 cultures were added to anti-CD3 antibodycoated plates (1 mg/ml) or were irradiated (2500 rads) B6D2 F1 splenocytes (5 3 104) with renal antigen preparation (10 mg/ml), with 200 ml T-cell medium with various doses of recombinant mouse IFN-g (PharMingen, Inc., San Diego, CA, USA) [11]. For all studies, cells were pulsed with 3HTdR at 48 hours and were then harvested at 72 hours for scintillation counting. Values represent the mean of triplicate samples 6 sd. Adoptive transfer of disease Renal subcapsular transfers were performed with naive 6- to 12-week-old B6D2 F1 mice. T cells were harvested at end passage (day 10 to 14) and were washed with phosphate-buffered saline (PBS), and aliquots of 10 3 106 in approximately 75 ml of PBS were injected under the kidney capsule with a 30-gauge needle. This volume uniformly lifted the capsule off from most of the parenchyma without bleeding. Some recipients were pretreated with i.p. IFN-g (1000 U; PharMingen) or PBS 24 hours prior to adoptive transfer. For some MR1.3 and MR1.6 adoptive transfers, anti-IFN-g (R4-6 A2; PharMingen) or rat IgG (Pierce, Rockford, IL, USA) antibody was coadministered to recipient mice. The
Meyers and Zhang: IFN-c and T-cell–mediated renal injury
doses of anti–IFN-g antibody selected, 2.0 mg and 6.0 mg, were sufficient to neutralize the antiviral activity of 670 U and 2000 U IFN-g activity, respectively (PharMingen). Three to seven days later, the kidneys were harvested and longitudinally sectioned with preservation of the subcapsular cell layer. Sections of kidney tissue for immunohistochemistry were embedded in OCTTM compound (Miles Inc., Elkhart, IN, USA), snap frozen in liquid nitrogen, and stored at 2708C. Other kidney sections were fixed in 10% buffered formalin, were paraffin embedded for staining (hematoxylin and eosin and periodic acid-Schiff), and examined for histological evidence of disease. Assessment of renal disease Semiquantitative methods assessed renal disease in affected kidneys. Sections of paraffin-embedded tissue (4 mm) were stained with hematoxylin and eosin and periodic acid-Schiff, and were examined for cellular infiltrates and tubular atrophy by an individual blinded to the experimental protocol (C.M.M.). Stained kidney sections were evaluated with two different grading scales. The severity of interstitial involvement was qualitatively assessed with a scale used in previous studies [20]: 0 5 no involvement from the subcapsular cell layer; 0.5 5 trace pathologic changes of cellular involvement in a focal pattern in the outermost cortical tubular area; 1 5 superficial, focal peritubular infiltration and tubular atrophy under the transferred cell layer; 2 5 focal, deeply extending, heavy cellular infiltrates with peritubular damage and tubular atrophy. The sections were also graded by approximating the tubular layer cell depth of the most advanced infiltrating front of mononuclear cells. Each layer equaled a tubular diameter and was given one point. The data from both methods were expressed as a mean for each group 6 sem. Immunohistochemistry Frozen kidney sections were stained for class II MHC (I-Ab; PharMingen, Inc.) using the avidin-biotin-peroxidase complex, ABC technique (Vector Laboratories, Burlingame, CA, USA). Frozen sections were fixed in chilled (2208C) acetone, air dried, rehydrated in PBS, and incubated with the avidin-biotin blocking reagent (Vector Laboratories) for 60 minutes. Appropriate dilutions of the primary antibody were applied, and the slides were incubated for 60 minutes. Slides were washed in PBS and then incubated with biotinylated antimouse IgG (Vector Laboratories) for 60 minutes. Following another PBS wash, slides were incubated with the Vectastain ABC reagent (Vector Laboratories) for an additional 60 minutes. Slides were then incubated with diaminobenzidine (Vector Laboratories) and counterstained with hematoxylin (Fisher Scientific, Medford, MA, USA). Control sections consisted of staining without primary
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antibody (PBS) and staining with an irrelevant primary antibody (normal mouse IgG). Northern blot hybridization Total RNA isolated from 10 3 106 T cells was fractionated on a 1.5% agarose-formaldehyde gel and then transferred to a Zetabind membrane (Cuno Laboratory Products, Meriden, CT, USA). Blots were hybridized to 32 P-labeled (Amersham Corp., Arlington Heights, IL, USA) murine probes (specific activity of 2.0 3 109 dpm/ mg) for 16 hours at 558C. Probes for IL-10 [21], IFN-g [22], IL-13 [23], IFN-gRb chain [24], and b-actin [25] were used in these studies. After hybridization, the blots were washed three times with 0.1 3 standard saline citrate/0.1% sodium dodecyl sulfate at 658C for 30 minutes. Autoradiography was performed with intensifying screens at 2708C. Some exposed films were scanned with a laser densitometer (Hoefer, San Francisco, CA, USA), and relevant mRNA levels were calculated relative to those of b-actin. Secreted cytokine measurements The production of IL-4, IL-10, IL-13, and IFN-g by T-cell clones was measured in cell-free culture supernatants by cytokine-specific enzyme-linked immunosorbent assay (ELISA). Supernatants were obtained from end-passage T-cell clones (1 to 2 3 106/ml) plated for 24 hours with fresh T-cell media in the presence or absence of immobilized anti-CD3 antibody (1 mg/ml) and various doses of IFN-g. To determine IL-4, IL-10, and IFN-g levels, purified anticytokine capture monoclonal antibody (PharMingen, Inc.) was diluted to 1.0 mg/ml in 0.1 m NaHCO3, pH 8.2, and 50 ml aliquots added to wells of an ELISA plate. Following overnight incubation at 48C and washing with PBS/0.05% Tween, wells were blocked with 200 ml PBS/10% FCS at room temperature for two hours, washed again with PBS/0.05% Tween, and then coated with cytokine standards and T-cell clone supernatants diluted in PBS/10% FCS in duplicate. Plates were incubated overnight at 48C, washed with PBS/0.05% Tween, and coated with 100 ml aliquots of biotinylated anticytokine-detecting antibody (PharMingen) diluted to 1 mg/ml in PBS/10% FCS. Following a 45-minute incubation at room temperature, the plates were washed with PBS/0.05% Tween, coated with 100 ml aliquots of avidin-peroxidase (Sigma, St. Louis, MO, USA) diluted in PBS/10% FCS, and incubated again at room temperature for 30 minutes. The plates were then washed with PBS/0.05% Tween, and 100 ml aliquots of peroxidase substrate reagent (Bio-Rad, Hercules, CA, USA) were added to each well, allowed to develop at room temperature for 5 to 10 minutes, and read on an ELISA plate reader at 405 nm. Sensitivity limits for the ELISA were IL-4, 30 pg/ml; IL-10, 30 pg/ml; and IFN-g, 0.3 U/ml. IL-13 levels were determined using the Quan-
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tikine M IL-13 assay kit (R&D Systems, Minneapolis, MN, USA) in accordance with manufacturer’s instructions. Sensitivity limits for the assay were 1.5 pg/ml. Statistical analysis Differences between experimental groups were determined, where appropriate, by Student’s t-test [26]. Statistical significance between groups of adoptively transferred renal lesions was determined by Kruskal–Wallis one-way analysis of variance by ranks and the Mann– Whitney U-test [26]. RESULTS Interferon-g–mediated effects on mouse renal T-cell clonal proliferation Recent reports indicate that IFN-g inhibits in vitro proliferation of CD41 Th2 clones, but not IFN-g– producing Th1 clones [27, 28]. To investigate whether IFN-g–producing nephritogenic Th2-like MR T-cell clones were also inhibited by this proinflammatory cytokine, we compared proliferative responses of MR1.3 with MR1.6, a nonnephritogenic Th2 clone, in the presence of exogenous IFN-g. For these studies, proliferation of MR1.3 and MR1.6 to immobilized anti-CD3 antibody was evaluated after incubation with varying doses of IFN-g. This route of T-cell stimulation was selected for these studies, as it eliminates potential non–T-cell receptor-mediated stimuli associated with spleen cells preparations [27]. In this setting, the response of distinct T-cell clones to triggering via the TCR complex can be uniformly examined. As anticipated, the proliferative response of nonnephritogenic Th2 clone MR1.6 was profoundly diminished by IFN-g (Fig. 1A). Studies presented in Figure 1A also reveal a similar dose-dependent inhibition of proliferation with the Th2-like nephritogenic clone MR1.3. Despite producing IFN-g, MR1.3 exhibits a typical cytostatic Th2 response to exogenously added IFN-g. Further proliferation studies conducted with MR1.3 and MR1.6 stimulated with irradiated B6D2 F1 splenocytes in the presence of various concentrations of IFN-g are shown in Figure 1B. These assays demonstrate that IFN-g inhibits both anti-CD3 antibody-mediated and APC-induced Th2 proliferation responses. Cytokine gene expression in interferon-g–treated mouse renal T-cell clones In view of the antiproliferative effects of IFN-g on both nephritogenic and non-nephritogenic clones, we also examined the impact of various levels of IFN-g exposure on MR T-cell clone cytokine gene expression. These studies addressed whether IFN-g blocked TCRstimulated proliferation by inhibiting lymphokine production. Immobilized anti-CD3 antibody-stimulated MR1.3 or MR1.6 T cells were incubated with various
antiproliferative doses of IFN-g (from 0.1 U/ml to 1000 U/ml). As shown in Figure 2, message expression of IL10, IL-13, and IFN-g was not markedly altered in either MR1.3 or MR1.6 after IFN-g treatment at doses that significantly inhibited proliferation. Of note, Northern hybridization studies in Figure 2 also revealed a significant difference in IL-13 expression between MR1.3 and MR1.6. Following stimulation, IL-13 was expressed at relatively low levels in MR1.3 cells, whereas the nonnephritogenic clone MR1.6 expressed much higher message levels. Additional Northern hybridization studies evaluating IL-4 expression in IFN-g–treated MR1.3 and MR1.6 did not reveal marked differences in IL-4 transcription between these T-cell clones (data not shown). Analysis of secreted cytokines in supernatants of IFNg–treated MR1.3 and MR1.6 T-cell clones corroborated initial Northern hybridization studies (Table 1). Quantitative assessment of secreted cytokines was performed, by ELISA, on a panel of anti-CD3 antibody-stimulated T-cell clones that were treated with various doses of IFN-g (0.1 to 1000 U/ml). As shown in Table 1, supernatants from nephritogenic clone MR1.3 contained significant amounts of IL-4, IL-10, and IL-13 and, consistent with our Northern hybridization data, also contained low levels of IFN-g. MR1.6, which does not induce renal inflammation, expressed IL-4, IL-10, and IL-13, but not IFN-g after stimulation. MR1.6 cells, however, consistently produced more IL-13 than MR1.3 cells with antiCD3 antibody stimulation. Also consistent with initial Northern analyses, no marked changes in IL-4, IL-10, IL-13, and IFN-g secretion were apparent after IFN-g treatment. These studies suggest that both MR1.3 and MR1.6 stimulated via the TCR complex, in the presence of IFN-g, are capable of producing relevant cytokines (particularly IL-4), but that they do not proliferate in response to autocrine or paracrine cytokine growth factors generated in this setting. Nephritogenic potential of MR1.3 and MR1.6 in interferon-g–treated recipients To assess IFN-g–mediated effects on the nephritogenic potential of renal-derived MR T-cell clones, we performed adoptive transfer studies with B6D2 F1 mice pretreated with IFN-g before renal subcapsular transfer. In previous studies, we have found that renal subcapsular transfer of immunocompetent T cells is a sensitive and reproducible means of determining the ability of discrete T-cell subsets to elicit inflammation in renal parenchyma [20, 29, 30]. We therefore used this technique to compare the nephritogenic potential of MR T cells under a variety of experimental conditions. As shown in Figure 3 A and B, IFN-g pretreatment did not alter the usual pattern of interstitial disease induced by nephritogenic clone MR1.3, which is characterized by focal cortical areas of mononuclear cell infiltration and areas of tubular dilata-
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Fig. 1. Proliferative responses of mouse renal (MR) clones. MR T-cell clones (1 3 105), either MR1.3 or MR1.6, were added to (A) anti-CD3 antibody-coated plates (1 mg/ml) or (B) irradiated B6D2F1 splenocytes with renal antigen (10 mg/ml), with various concentrations of interferon-g (IFN-g). Cells were pulsed with 3HTdR at 48 hours and were then harvested at 72 hours for scintillation counting. The values represent the mean of triplicate samples 6 sd.
tion and damage. In contrast, IFN-g pretreatment did alter the nephritogenicity of clone MR1.6. In this setting, MR1.6 induced cortical inflammation with focal mononuclear cell infiltrates and tubular dilation and atrophy (Fig. 3 C, D) in kidneys of syngeneic mice. The histologic features in all of these MR1.6-induced lesions are similar to those induced by the T-cell clone MR1.3. Formal histologic grading of these four groups, as well as IFN-g and PBS controls, is shown in Table 2. Analysis of adoptive transfers demonstrates that both the severity and depth of MR1.6-induced renal lesions in IFN-g–treated recipients are comparable to tubulointerstitial lesions elicited by MR1.3. Treatment with IFN-g or PBS alone did not induce renal inflammation or damage. As previously stated, proinflammatory effects of IFN-g on various subsets of immune cells may, in part, be mediated by its ability to up-regulate class II MHC
and costimulatory molecule expression on relevant APC [16]. In view of renal tubular epithelial cell responsiveness to these effects, we examined class II MHC expression shortly after adoptive transfer of MR1.3 and MR1.6 T cells to syngeneic mice. Kidney sections from animals that received MR1.3 cells exhibited an increase in renal tubular class II MHC (I-Ab) staining, whether animals were pretreated with PBS (Fig. 4A) or IFN-g (Fig. 4B). Adoptive transfer of MR1.6 with PBS did not increase I-Ab expression (Fig. 4C), but a marked increase in renal class II MHC expression was noted if animals were pretreated with IFN-g prior to MR1.6 transfer (Fig. 4D). These studies also indicate that pretreatment of animals with IFN-g alone (Fig. 4E), but not PBS (Fig. 4F), induced mild increases in renal class II MHC expression.
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Fig. 2. Cytokine expression in mouse renal (MR) clones following interferon-g (IFN-g) pretreatment. Nephritogenic (MR1.3) and nonnephritogenic (MR1.6) clones (3 3 106/ml) were plated on immobilized anti-CD3 antibody (1 mg/ ml) and various concentrations of IFN-g. After 24 hours, clonal RNA was harvested from 1 3 107 viable cells and evaluated for cytokine expression with cytokine-specific probes, interleukin (IL)-10, IL-13, and IFN-g.
Table 1. Secreted cytokines produced by MR1.3 and MR1.6 Cultured T cell clonea MR1.3 MR1.3 MR1.3 MR1.3 MR1.3 MR1.6 MR1.6 MR1.6 MR1.6 MR1.6
IFN-g dose U/ml 0 1 10 100 1000 0 1 10 100 1000
IL-4
IL-10
IL-13
ng/ml 8.0 7.0 10 9.0 14 10 14 12 12 11
1450 800 1700 1300 1400 600 800 250 750 350
21 22 20 19 20 110 115 115 110 110
IFN-g U/ml 2.5 2.5 4.5 14 2.5 ND ND ND ND ND
a MR T cell clones were harvested from day 10 cultures and plated in fresh medium at 1.5 3 106 cells/ml. Clones were then stimulated with anti-CD3 antibody-coated plates (1 mg/ml) and various doses of IFN-g for 24 hours. The levels of IL-4, IL-10, IL-13, and IFN-g in the resulting supernatants were determined by cytokine-specific ELISA. ND, not detected.
Effects of neutralizing anti–interferon-g antibody on adoptive transfer of renal disease As our nephritogenic clones secrete IFN-g following in vitro activation, we also evaluated the effects of neutralizing anti–IFN-g antibodies on adoptive transfer of renal disease. For these studies, MR1.3 cells were coadministered with anti–IFN-g antibody or with control rat IgG. As shown in the histology scores of Table 2, the severity and extent of tubulointerstitial lesions induced by MR1.3 were not markedly different in either of these treated groups. Transfer of MR1.3 to animals that were also treated with anti–IFN-g antibody or control rat IgG did not alter the nephritogenic potential of this T-cell clone. Moreover, renal class II MHC expression was still apparent in early lesions in all anti–IFN-g antibodytreated mice (data not shown).
In further studies, MR1.6 cells were administered to IFN-g–pretreated recipients with anti–IFN-g antibody or with control rat IgG. As delineated in Table 3, anti–IFN-g antibodies effectively inhibited the renal lesion typically induced by MR1.6 in IFN-g–pretreated mice. Following adoptive transfer of MR1.6, no evidence of tubulointerstitial damage was apparent in kidneys from animals that received the neutralizing anti–IFN-g antibody, whereas kidneys from the control IgG group of IFN-g–pretreated animals exhibited marked renal inflammation and injury (Table 3). Unlike the MR1.3 transfers, these studies suggest that IFN-g directly facilitates MR1.6 nephritogenicity. Interferon-gRb chain expression in mouse renal T-cell clones To investigate potential mechanisms of differential IFN-g–mediated effects in MR1.3 and MR1.6 cells, we examined the expression of the IFN-gRb chain in view of its critical signaling role in IFN-g–sensitive T cells [24, 31]. As shown in Figure 5, unstimulated MR1.3 and MR1.6 constitutively expressed IFN-gRb chain mRNA, but they responded differently to stimulation in the presence of IFN-g. Densitometric analysis of b-chain message levels revealed an approximate 50% decrease in MR1.3 cells, whereas levels in MR1.6 cells in this setting did not change markedly. Although both T-cell clones express the regulatory b chain following IFN-g exposure, differential regulation of the level of cytokine receptor expression may indeed effect distinct functional influences of IFN-g on these T-cell clones. DISCUSSION In the studies presented here, we evaluated the immunomodulatory effects of exogenous IFN-g on distinct
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Fig. 3. Effects of interferon-g (IFN-g) on adoptive transfers. For these studies, 5 3 106 mouse renal (MR) T-cell clones were injected beneath the renal capsule of B6D2 F1 mice 24 hours after pretreatment with either PBS or IFN-g (1000 U). Kidneys were harvested five to seven days after T-cell transfers and were evaluated by light microscopy. The interstitial lesion typically seen after transfer of MR1.3 cells is seen in both (A) (PBS pretreatment) and (B) (IFN-g pretreatment). Focal cortical areas of mononuclear cell infiltration are evident, as well as areas of tubular dilation. MR1.6 adoptive transfers are shown in (C) (PBS pretreatment) and (D) (IFN-g pretreatment). IFN-g or PBS pretreatment alone did not result in renal inflammation or damage (data not shown).
functional CD41 T cells derived from diseased kidneys of animals with cGVHD. Our studies indicate that IFN-g mediates dose-dependent inhibition of both nephritogenic MR1.3 and non-nephritogenic MR1.6 proliferation, although cytokine gene expression of IL-4, IL-10, and IFN-g after IFN-g treatment is not markedly altered in either T-cell clone following anti-CD3 Ab stimulation. We also noted a higher baseline level of IL-13 expression in MR1.6 compared with nephritogenic clone MR1.3. Of note, IFN-g markedly alters the nephritogenic potential of clone MR1.6, as pretreated naive recipients develop marked renal inflammation and damage after transfer
of MR1.6 T cells. The transfer of MR1.3 with neutralizing anti–IFN-g antibodies does not inhibit its ability to adoptively transfer renal inflammation and damage, whereas MR1.6 nephritogenicity in IFN-g–treated mice was blocked by these antibodies. In addition, both clones constitutively express the IFN-gRb chain, which is differentially expressed in MR1.3 and MR1.6 following IFN-g exposure. These studies describe a remarkably permissive role for IFN-g in modulating nephritogenic Th2 activity, perhaps by local up-regulation of renal class II MHC or costimulatory molecule expression, which facilitates the initiation of cell-mediated autoimmune renal injury.
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Table 2. Effects of IFN-g on adoptively transferred renal lesions Cells transferreda MR1.3 MR1.3 MR1.6 MR1.6 MR1.6 MR1.6 MR1.3 MR1.3
Recipient treatment
Severity of renal damage
Depth of lesion
PBS IFN-g PBS IFN-g PBS IFN-g Anti-IFN-g Ab Rat IgG
1.5 6 0.5b 1.4 6 0.3b 0 1.7 6 0.2b 0 0 1.5 6 0.1b 1.2 6 0.3b
22 6 7.5b 28 6 4.8b 0 25 6 3.0b 0 0 28 6 4.3b 20 6 2.9b
a Five days following transfer of 10 3 106 MR clones to normal B6D2 F1 mice, kidneys were harvested and evaluated by light microscopy, as detailed in the Methods section. Some recipients were injected with IFN-g (1000 U) or PBS IP 24 hours prior to subcapsular transfer. For some studies, MR cells were coadministered to recipient mice with anti-IFN-g antibody or an irrelevant antibody (Rat IgG) (N 5 4–6/group). b P , 0.001 vs. no cell/PBS control
The previous observation of differential IFN-g expression in the Th2-like MR clones was intriguing in view of the proscriptive influence of IL-10 on its synthesis in CD41 T-cell clones [12]. Although culture conditions may effect uncharacteristic changes in cytokine gene expression in cloned T-cell lines, similar Th2-like observations in other models of autoimmunity suggest that this particular CD41 T-cell phenotype may be relevant to disease expression [32–34]. Such observations include autoreactive T-cell clones isolated from MRL/lpr mouse kidneys and gold salt-injected Brown Norway rat lymph nodes, as well as allergen-specific human Th2 cells [32– 34]. These analyses indicate that IFN-g secretion by a Th2-like CD41 T cell may be of functional significance in vivo [32–34]. These MR clone studies indicate that IFN-g downregulates proliferation of IL-4–producing CD41 T cells by limiting their ability to respond to T-cell growthpromoting cytokines generated on activation via the TCR complex, rather than by inhibiting the production of relevant cytokines. It is intriguing, however, that despite the proscriptive influence on MR1.3 and MR1.6 proliferation in vitro, both T-cell clones affected renal inflammation and damage in IFN-g–treated recipients. IFN-g–induced up-regulation of class II MHC and relevant costimulatory molecule (that is, B7-1) expression, as well as cytokine production, in both professional and “nonprofessional” APCs may markedly alter patterns of T-cell activation in vivo. In addition to these proinflammatory effects, IFN-g also modulates T-cell biology through a number of antagonistic interactions with other T-cell–derived cytokines [10]. For instance, although IFN-g counteracts IL-4–induced proliferation, IL-4, inhibits transcriptional activation of IFN-g–inducible proteins, and blocks IFN-g–induced chemokine synthesis, it also inhibits Th1 IFN-g production [35–37]. Similarly, IFN-g suppresses Th2 cell IL-10 production, whereas IL10 inhibits Th1 cell IFN-g production [28, 38].
Our studies are noteworthy in that anti–IFN-g antibody does not block nephritogenicity of MR1.3, indicating that initiating renal disease by this T-cell clone is not critically dependent on IFN-g. Other inflammatory mediators produced by MR1.3, such as perforin (unpublished observation), may be more relevant to initiating this renal immune response. Anti–IFN-g antibody was effective, however, at blocking MR1.6 disease activity in IFN-g–pretreated mice, suggesting a primary initiating role for IFN-g in MR1.6 adoptively transferred lesions. Such observations further demonstrate functional differences between these clones, as they exhibit distinct responses to IFN-g exposure in the setting of renal injury. Recent studies on IFN-gR have shed light on IFNg–mediated effects on distinct T-cell subsets [24, 31]. T-cell responsiveness to IFN-g depends on IFN-gRb chain expression, the receptor subunit required for signaling, which is limited to Th2 cells and is virtually absent in Th1 cells [24, 31]. It is noteworthy that chronic Th2 cell exposure to high levels of IFN-g, greater than levels detected in our current studies, down-regulates IFN-gRb chain expression, and decreases Th2-responsiveness to exogenous IFN-g effects [24, 31]. Interestingly, ligandinduced receptor b-chain down-regulation does not occur in other cell preparations, such as fibroblast cell lines [31]. IFN-g appears to regulate expression of its own receptor b chain on certain cell types and thereby determines the responsiveness of these cells to subsequent IFN-g exposure [24, 39, 40]. Recent studies indicate that treatment of T cells with phorbol esters or with antiCD3 antibodies induces IFN-gRb chain mRNA [40]. These findings demonstrate that b-chain expression can be regulated either positively or negatively in a stimulusspecific manner [39]. Our observations of IFN-gRb chain expression also indicate distinct responses and regulation of this signaling chain in MR1.3 and MR1.6 cells, suggesting that different patterns of b-chain regulation in these T cells may indeed cause disparate functional responses in vivo. Different baseline IL-13 expression in distinct MR clones is worthy of further comment, in view of the distinct functions mediated by these T cells. MR1.3 is nephritogenic, adoptively transferring tubulointerstitial inflammation and damage to syngeneic recipients, and is restricted by Iad [11]. MR1.6, however, does not transfer renal disease to naive syngeneic animals and is restricted by Iab determinants [11]. Both MR1.3 and MR1.6 express a predominantly Th2 cytokine pattern with IL-4, IL-6, IL-10, and IL-13 expression, although our current studies suggest that MR1.6 expresses a higher level of IL-13 on stimulation. Sharing many properties with IL-4, IL-13 also has pleiotrophic effects on B cells, monocytes, macrophages, and neutrophils [41]. It largely suppresses T-cell–mediated immune responses [41]. The relatively low level of IL-13 expression in nephritogenic clone
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Fig. 4. Renal class II major histocompatibility complex (MHC) expression in adoptive transfers. Kidneys harvested from adoptive transfer experiments, three days after T cell transfers, were evaluated immunohistochemically for class II MHC (I-Ab) expression. Marked expression is seen in (A) (MR1.3 transfer after PBS pretreatment), (B) (MR1.3 transfer after IFN-g pretreatment), and (D) (MR1.6 transfer after IFN-g pretreatment), whereas class II MHC expression is not observed in (C) (MR1.6 transfer after PBS pretreatment) and (F) (PBS treatment control). A minimal increase in class II MHC staining is apparent in (E) (IFN-g treatment control).
Table 3. Effects of neutralizing anti-IFN-g antobodies on MR1.6-induced renal lesions Cells transferreda MR1.6 MR1.6 —
Recipient treatment
Severity of renal damage
Depth of lesion
IFN-g, Anti-IFN-g Ab IFN-g, Rat IgG PBS
0 1.3 6 0.2b 0
0 27 6 3.1b 0
a Five days following transfer of 10 3 106 MR clones to normal B6D2 F1 mice, kidneys were harvested and evaluated by light microscopy, as detailed in the Methods section. Some recipients were injected with IFN-g (1000 U) or PBS IP 24 hours prior to subcapsular transfer. For some studies, MR cells were coadministered to recipient mice with anti-IFN-g antibody or an irrelevant antibody (Rat IgG) (N 5 4–6/group). b P , 0.001 vs. no cell/PBS control
MR1.3 may facilitate the cell-mediated inflammatory renal lesion apparent on adoptive transfer, a response not significantly altered by IFN-g. Despite the apparent robust IL-13 expression in MR1.6, however, it is noteworthy that IFN-g pretreatment effectively alters its nephritogenic potential in vivo. This response suggests that
IFN-g–induced changes in host environment causes a dramatic change in the functional phenotype of MR1.6 on adoptive transfer. Although IL-13 has been detected in diseased kidneys in other models of immune-mediated renal disease, its immunomodulatory role in renal inflammation, however, has not been clearly defined [23]. A heterogeneity of IFN-g–mediated effects has been reported in a number of models of autoimmune disease. Previous studies conducted in this model of cGVHD examined the effects of exogenous IFN-g on induction of disease in B6D2 F1 mice [42]. Although IFN-g reduced serum IgE and IgG1 levels in affected mice, no difference in animal survival was noted [42]. The extent of renal involvement and degree of proteinuria, however, were not evaluated [42]. In murine models such as autoimmune diabetes, autoimmune thyroiditis, and lupus-prone (NZB/NZW) F1 mice, IFN-g facilitated disease induction, whereas administration of anti–IFN-g antibodies were disease protective [43–47]. Observations in experimental autoimmune encephalomyelitis (EAE) and adju-
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Fig. 5. Interferon (IFN)-gRb chain expression in mouse renal (MR) clones following IFN-g exposure. Clonal RNA was harvested from nonnephritogenic clone MR1.6 (A, B) and nephritogenic clone MR1.3 (C, D) and was evaluated for IFN-gRb chain expression by Northern hybridization. MR cells were either unstimulated (A, C) or plated on immobilized anti-CD3 antibody (1 mg/ml) in the presence of IFN-g (100 U/ml) for 24 hours (B, D). The findings are representative of three distinct experiments.
vant arthritis have been less straightforward [48, 49]. Earlier studies in EAE revealed that exogenous IFN-g reduced the incidence and severity of disease, and neutralizing antibodies augmented disease activity [48, 49]. Recent analysis of susceptible hosts suggests that IFN-g–deficient mice develop EAE as readily as wildtype controls following immunization with heterologous myelin basic protein, but have a much higher mortality rate [50]. IFN-g and its neutralizing antibody have disparate effects on disease activity in adjuvant arthritis, depending on the dose and timing of administration [51]. IFN-g administered prior to, or at least four days after, disease induction exacerbates disease activity. If given one to two days after disease induction, however, the disease is suppressed [51]. These disparate observations regarding activity of IFN-g in autoimmune tissue destruction further illustrate the complex, likely multistage nature of T-cell–mediated autoimmune responses. Bidirectional effects noted in various disease models may be due to the predominance of distinct T-cell subsets, with varying degrees of IFN-g responsiveness, at different stages of disease [51]. In addition to well-described effects on various relevant immune cells, recent investigations indicate that IFN-g induces changes in cells of nonimmune origin [16]. Most notably, IFN-g has a number of immunomodulatory effects on epithelial cells that may promote disease [16–18]. As observed in these MR clone studies, it induces de novo class II MHC expression on renal epithelial cells, perhaps converting them to “nonprofessional” APC in vivo [10, 16–18]. Studies in other models of systemic lupus erythematosus, syngeneic GVHD and cGVHD across minor histocompatibility differences
have also identified a correlation between class II MHC antigen induction and the pattern of lymphocytic parenchymal lesions observed in affected animals [52–54]. Increased renal class II MHC expression is likely not sufficient for inducing autoimmune injury, however, as class II MHC transgenic animals, with high levels of renal tubular epithelial cell class II MHC expression, do not develop spontaneous renal disease [55]. Recent in vitro observations also indicate that IFN-g alters epithelial cell permeability [56]. This cytokine-mediated change in epithelial permeability may be a major cause of altered epithelial barrier function in vivo. Such a change could be pathogenic, as it would facilitate access of relevant lymphocytic/inflammatory cells to epithelial surfaces, promoting inflammation [56]. The potential relevance of these IFN-g-tubular epithelial interactions is currently under investigation in our laboratory. ACKNOWLEDGMENTS This work was done during the tenure of a Grant-in-Aid Award from the American Heart Association (95012790). It was also supported in part by the Margaret Q. Landenberger Research Foundation, the Lupus Foundation of Philadelphia, Inc., National Institutes of Health DK-07006 and DK-49724, and the administrative/educational funds from the DCI RED Fund. The authors are grateful to Ms. Mary H. Foster for her comments and critical review of the manuscript and to Mr. Steven J. Birkos for his technical assistance. This work was presented in part at the annual meeting of the American Society of Nephrology, Orlando, Florida, November 1994. Reprint requests to Catherine M. Meyers, M.D., Renal-Electrolyte and Hypertension Division, University of Pennsylvania, 700 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: cGVHD, chronic graft vs. host disease; EAE, experimental autoimmune encephalomyelitis; FCS, fetal calf serum; IFN-g, interferon g; IFN-gRb, interferon g receptor b; IL, interleukin; MHC, major histocompatibility complex; MR, renal cells isolated from mice with chronic graft vs. host disease; MR1.3, nephritogenic T cell clone; MR1.6, non-nephritogenic T cell clone; PBS, phosphate buffered saline; Th2, T helper cell type 2.
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Immunomodulatory effects of interferon-g on autoreactive nephritogenic T-cell clones CATHERINE M. MEYERS and YOUKANG ZHANG Renal-Electrolyte and Hypertension Division, University of Pennsylvania School of Medicine, Department of Medicine, Philadelphia, Pennsylvania, USA, and the Institute of Nephrology, The First Teaching Hospital, Beijing Medical University, Beijing, China
Immunomodulatory effects of interferon-g on autoreactive nephritogenic T-cell clones. Background. We examined the immunomodulatory effects of interferon-g (IFN-g) on renal-derived CD41 a/b1 T cells, called mouse renal (MR) cells, isolated from animals with murine chronic graft-versus-host disease, a model of autoimmune glomerulonephritis. MR T cells express a Th2 cytokine profile, although IFN-g expression is also detected in a subset of clones that adoptively transfers renal disease to naive recipients. In view of disparate patterns of IFN-g expression, we evaluated the effects of exogenous IFN-g on nephritogenic (MR1.3) and nonnephritogenic (MR1.6) clonal activity. Methods. These studies examined IFN-g–mediated effects on clonal proliferation, cytokine production, nephritogenic potential, and IFN-g receptor expression. Results. IFN-g mediated dose-dependent inhibition of MR1.3 and MR1.6 proliferation. This cytostatic effect was not mediated by inhibiting cytokine genes, as expression of interleukin (IL)-4, IL-10, IL-13, and IFN-g after IFN-g treatment was not markedly altered in either clone, although baseline IL-13 expression was enhanced in MR1.6. IFN-g markedly altered the functional phenotype of MR1.6, as pretreated recipients developed severe mononuclear cell infiltrates and tubular damage following adoptive transfer of MR1.6. Neutralizing anti– IFN-g antibodies did not inhibit MR1.3 nephritogenicity, but did block MR1.6-induced disease in IFN-g–treated mice. Although both clones constitutively expressed the IFN-g receptor b chain, IFN-g exposure decreased its expression in MR1.3 cells, but did not markedly change its expression in MR1.6 cells. Conclusion. These studies describe an unusual permissive role for IFN-g in modulating nephritogenic Th2 activity in vivo, which facilitates the initiation of cell-mediated autoimmune renal injury. Apparent differential effects of IFN-g on distinct T-cell clones may be mediated in part by alterations in cytokine receptor expression.
Murine chronic graft-versus-host disease (cGVHD) is induced following the transfer of parental (DBA/2) T Key words: nephritis, T cell lymphocyte, graft vs. host disease, cytokines, autoimmunity, MHC. Received for publication February 6, 1998 and in revised form November 20, 1998 Accepted for publication November 23, 1998
1999 by the International Society of Nephrology
cells to (C57BL/6 3 DBA/2) F1 (B6D2 F1) hybrid recipients [1]. This multisystem autoimmune disorder comprises a spectrum of abnormalities such as vasculitis, polyarthritis, and mononuclear cell infiltration of multiple organs [2, 3]. A variety of circulating and deposited autoantibodies are also detected in affected animals, including anti-DNA, antihistone, and antilaminin antibodies, that are produced by autoreactive host B cells [4–7]. B6D2 F1 recipient animals with cGVHD develop an immune-complex glomerulonephritis within two to four weeks following the transfer of parental T cells, associated with progressive mononuclear (T cell) parenchymal infiltration, ultimately resulting in an end-stage renal lesion in all recipients [3, 6]. In this murine model of cGVHD, immunocompetent DBA/2 parental T cells are activated on transfer by B6D2 F1 host alloantigens, a process initiating a systemic autoimmune disease [8, 9]. Mechanisms by which host tissues are damaged, however, remain unclear. Studies from other investigators suggest that T-cell–derived proinflammatory cytokines, such as interferon-g (IFN-g), can mediate parenchymal damage and may play an important role in the development of renal disease in cGVHD [10]. Alterations in the cytokine network may provide a final common pathway of target organ damage in the setting of activation and expansion of autoreactive T-cell subsets [10]. Previous studies in our laboratory have characterized autoreactive B6D2 F1 CD41 a/b1 T cells derived from diseased kidneys of animals with cGVHD [11]. Two functional subsets of mouse renal (MR) T-cell clones were isolated. One subset is nephritogenic, restricted by Iad, and adoptively transfers tubulointerstitial inflammation and damage to syngeneic recipients [11]. The other subset does not transfer renal disease to syngeneic animals and is Iab reactive [11]. Both nephritogenic and nonnephritogenic MR T-cell clones express a Th2 cytokine pattern with marked interleukin (IL)-4, IL-6, and IL-10 expression on stimulation [11]. Further studies reveal,
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however, that MR T-cell clones that transfer renal disease also secrete low levels of IFN-g [11]. These T-cell clones are designated Th2 in view of their marked production of IL-4 and IL-10 on stimulation, with minimal IFN-g production [11]. We were particularly interested in the differential IFN-g expression in MR clones in view of the proscriptive influence of IL-10 on its synthesis in CD41 T-cell clones and its more characteristic expression in Th1 cells [12]. Interferon-g is a multifunctional lymphokine with inducible expression predominantly in T-cell lymphocytes as well as natural killer (NK) cells [10]. It is a proinflammatory cytokine that has a variety of stimulatory activities. IFN-g increases high-affinity Fcg receptor I expression on mononuclear phagocytes, augments expression of intercellular adhesion molecule-1 expression on various cell types, and increases B-7 costimulatory molecule expression on antigen-presenting cells (APCs) (monocytes) [13–15]. IFN-g is critical to differentiation and maturation of mononuclear phagocytes and activation of tissue macrophages [10]. Most notably, IFN-g increases class II major histocompatibility complex (MHC) gene transcription and cell-surface expression on APC and induces de novo class II MHC expression on various other cells, such as renal epithelial cells, perhaps converting them to “nonprofessional” APC [16–18]. Investigators also suggest that T-cell–derived IFN-g is an important mediator of inflammatory reactions mediated by CD41 T-cell clones [19]. Moreover, IFN-g–induced upregulation of class II MHC expression on renal epithelial cells in vivo may be a crucial event in local trafficking of autoimmune CD41 T cells in diseased kidneys [16–18]. In view of the disparate patterns of IFN-g expression in functional MR clonal subsets, we further evaluated the effects of exogenous IFN-g on nephritogenic (MR1.3) and non-nephritogenic (MR1.6) T-cell clones. In vitro studies indicate that IFN-g mediates dose-dependent inhibition of MR1.3 and MR1.6 proliferation, although cytokine gene expression of IL-4, IL-10, IL-13, and IFN-g after IFN-g exposure is not markedly altered in either T-cell clone. In vivo analyses, with adoptive transfers to IFNg–pretreated mice, demonstrate that the otherwise nonnephritogenic clone, MR1.6, induces cortical inflammation with focal mononuclear cell infiltrates and tubular dilatation and atrophy. Consistent with proinflammatory effects of IFN-g, renal class II MHC (I-Ab) expression was enhanced in kidneys of all IFN-g pretreated mice. Similar adoptive transfers with neutralizing anti–IFN-g antibodies, however, did not alter the nephritogenic potential of MR1.3, but did inhibit MR1.6 nephritogenicity in IFN-g–treated mice. Further analysis of IFN-g receptor b (IFN-gRb)-chain expression reveals constitutive expression in both MR1.3 and MR1.6, with distinct differences in expression levels on IFN-g exposure in these MR clones. These studies suggest an immunomodulatory
role for IFN-g in the cell-mediated renal lesion observed in this model of autoimmune glomerulonephritis, but also indicate that pathogenic T-cell lymphocytes can elicit parenchymal injury by other cytokine-mediated pathways. METHODS Mice B6D2 F1 (H-2b/d) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). T-cell clones Mouse renal T-cell clones, MR1.3 and MR1.6, were propagated by weekly passage with 10% MLA-144 supernatants as a source of IL-2 and other growth factors, renal antigen preparation derived from normal B6D2 F1 kidneys (10 mg/ml) and 5 3 106 irradiated (2500 rads) syngeneic B6D2 F1 spleen cells. T-cell culture medium consisted of RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with glutamine, antibiotics (penicillin, streptomycin, gentamicin), 10% decomplemented fetal calf serum (FCS), 5% NCTC (Whittaker Bioproducts, Inc., Walkersville, MD, USA), and 2 3 1025m 2-mercaptoethanol. Typically, wells became confluent at five to seven days and were carried every 10 to 14 days. All T cells were cultured at 378C in a 5% CO2 incubator. Proliferation assays Mouse renal T-cell clones (1 3 105) harvested from day 7 to 9 cultures were added to anti-CD3 antibodycoated plates (1 mg/ml) or were irradiated (2500 rads) B6D2 F1 splenocytes (5 3 104) with renal antigen preparation (10 mg/ml), with 200 ml T-cell medium with various doses of recombinant mouse IFN-g (PharMingen, Inc., San Diego, CA, USA) [11]. For all studies, cells were pulsed with 3HTdR at 48 hours and were then harvested at 72 hours for scintillation counting. Values represent the mean of triplicate samples 6 sd. Adoptive transfer of disease Renal subcapsular transfers were performed with naive 6- to 12-week-old B6D2 F1 mice. T cells were harvested at end passage (day 10 to 14) and were washed with phosphate-buffered saline (PBS), and aliquots of 10 3 106 in approximately 75 ml of PBS were injected under the kidney capsule with a 30-gauge needle. This volume uniformly lifted the capsule off from most of the parenchyma without bleeding. Some recipients were pretreated with i.p. IFN-g (1000 U; PharMingen) or PBS 24 hours prior to adoptive transfer. For some MR1.3 and MR1.6 adoptive transfers, anti-IFN-g (R4-6 A2; PharMingen) or rat IgG (Pierce, Rockford, IL, USA) antibody was coadministered to recipient mice. The
Meyers and Zhang: IFN-c and T-cell–mediated renal injury
doses of anti–IFN-g antibody selected, 2.0 mg and 6.0 mg, were sufficient to neutralize the antiviral activity of 670 U and 2000 U IFN-g activity, respectively (PharMingen). Three to seven days later, the kidneys were harvested and longitudinally sectioned with preservation of the subcapsular cell layer. Sections of kidney tissue for immunohistochemistry were embedded in OCTTM compound (Miles Inc., Elkhart, IN, USA), snap frozen in liquid nitrogen, and stored at 2708C. Other kidney sections were fixed in 10% buffered formalin, were paraffin embedded for staining (hematoxylin and eosin and periodic acid-Schiff), and examined for histological evidence of disease. Assessment of renal disease Semiquantitative methods assessed renal disease in affected kidneys. Sections of paraffin-embedded tissue (4 mm) were stained with hematoxylin and eosin and periodic acid-Schiff, and were examined for cellular infiltrates and tubular atrophy by an individual blinded to the experimental protocol (C.M.M.). Stained kidney sections were evaluated with two different grading scales. The severity of interstitial involvement was qualitatively assessed with a scale used in previous studies [20]: 0 5 no involvement from the subcapsular cell layer; 0.5 5 trace pathologic changes of cellular involvement in a focal pattern in the outermost cortical tubular area; 1 5 superficial, focal peritubular infiltration and tubular atrophy under the transferred cell layer; 2 5 focal, deeply extending, heavy cellular infiltrates with peritubular damage and tubular atrophy. The sections were also graded by approximating the tubular layer cell depth of the most advanced infiltrating front of mononuclear cells. Each layer equaled a tubular diameter and was given one point. The data from both methods were expressed as a mean for each group 6 sem. Immunohistochemistry Frozen kidney sections were stained for class II MHC (I-Ab; PharMingen, Inc.) using the avidin-biotin-peroxidase complex, ABC technique (Vector Laboratories, Burlingame, CA, USA). Frozen sections were fixed in chilled (2208C) acetone, air dried, rehydrated in PBS, and incubated with the avidin-biotin blocking reagent (Vector Laboratories) for 60 minutes. Appropriate dilutions of the primary antibody were applied, and the slides were incubated for 60 minutes. Slides were washed in PBS and then incubated with biotinylated antimouse IgG (Vector Laboratories) for 60 minutes. Following another PBS wash, slides were incubated with the Vectastain ABC reagent (Vector Laboratories) for an additional 60 minutes. Slides were then incubated with diaminobenzidine (Vector Laboratories) and counterstained with hematoxylin (Fisher Scientific, Medford, MA, USA). Control sections consisted of staining without primary
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antibody (PBS) and staining with an irrelevant primary antibody (normal mouse IgG). Northern blot hybridization Total RNA isolated from 10 3 106 T cells was fractionated on a 1.5% agarose-formaldehyde gel and then transferred to a Zetabind membrane (Cuno Laboratory Products, Meriden, CT, USA). Blots were hybridized to 32 P-labeled (Amersham Corp., Arlington Heights, IL, USA) murine probes (specific activity of 2.0 3 109 dpm/ mg) for 16 hours at 558C. Probes for IL-10 [21], IFN-g [22], IL-13 [23], IFN-gRb chain [24], and b-actin [25] were used in these studies. After hybridization, the blots were washed three times with 0.1 3 standard saline citrate/0.1% sodium dodecyl sulfate at 658C for 30 minutes. Autoradiography was performed with intensifying screens at 2708C. Some exposed films were scanned with a laser densitometer (Hoefer, San Francisco, CA, USA), and relevant mRNA levels were calculated relative to those of b-actin. Secreted cytokine measurements The production of IL-4, IL-10, IL-13, and IFN-g by T-cell clones was measured in cell-free culture supernatants by cytokine-specific enzyme-linked immunosorbent assay (ELISA). Supernatants were obtained from end-passage T-cell clones (1 to 2 3 106/ml) plated for 24 hours with fresh T-cell media in the presence or absence of immobilized anti-CD3 antibody (1 mg/ml) and various doses of IFN-g. To determine IL-4, IL-10, and IFN-g levels, purified anticytokine capture monoclonal antibody (PharMingen, Inc.) was diluted to 1.0 mg/ml in 0.1 m NaHCO3, pH 8.2, and 50 ml aliquots added to wells of an ELISA plate. Following overnight incubation at 48C and washing with PBS/0.05% Tween, wells were blocked with 200 ml PBS/10% FCS at room temperature for two hours, washed again with PBS/0.05% Tween, and then coated with cytokine standards and T-cell clone supernatants diluted in PBS/10% FCS in duplicate. Plates were incubated overnight at 48C, washed with PBS/0.05% Tween, and coated with 100 ml aliquots of biotinylated anticytokine-detecting antibody (PharMingen) diluted to 1 mg/ml in PBS/10% FCS. Following a 45-minute incubation at room temperature, the plates were washed with PBS/0.05% Tween, coated with 100 ml aliquots of avidin-peroxidase (Sigma, St. Louis, MO, USA) diluted in PBS/10% FCS, and incubated again at room temperature for 30 minutes. The plates were then washed with PBS/0.05% Tween, and 100 ml aliquots of peroxidase substrate reagent (Bio-Rad, Hercules, CA, USA) were added to each well, allowed to develop at room temperature for 5 to 10 minutes, and read on an ELISA plate reader at 405 nm. Sensitivity limits for the ELISA were IL-4, 30 pg/ml; IL-10, 30 pg/ml; and IFN-g, 0.3 U/ml. IL-13 levels were determined using the Quan-
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tikine M IL-13 assay kit (R&D Systems, Minneapolis, MN, USA) in accordance with manufacturer’s instructions. Sensitivity limits for the assay were 1.5 pg/ml. Statistical analysis Differences between experimental groups were determined, where appropriate, by Student’s t-test [26]. Statistical significance between groups of adoptively transferred renal lesions was determined by Kruskal–Wallis one-way analysis of variance by ranks and the Mann– Whitney U-test [26]. RESULTS Interferon-g–mediated effects on mouse renal T-cell clonal proliferation Recent reports indicate that IFN-g inhibits in vitro proliferation of CD41 Th2 clones, but not IFN-g– producing Th1 clones [27, 28]. To investigate whether IFN-g–producing nephritogenic Th2-like MR T-cell clones were also inhibited by this proinflammatory cytokine, we compared proliferative responses of MR1.3 with MR1.6, a nonnephritogenic Th2 clone, in the presence of exogenous IFN-g. For these studies, proliferation of MR1.3 and MR1.6 to immobilized anti-CD3 antibody was evaluated after incubation with varying doses of IFN-g. This route of T-cell stimulation was selected for these studies, as it eliminates potential non–T-cell receptor-mediated stimuli associated with spleen cells preparations [27]. In this setting, the response of distinct T-cell clones to triggering via the TCR complex can be uniformly examined. As anticipated, the proliferative response of nonnephritogenic Th2 clone MR1.6 was profoundly diminished by IFN-g (Fig. 1A). Studies presented in Figure 1A also reveal a similar dose-dependent inhibition of proliferation with the Th2-like nephritogenic clone MR1.3. Despite producing IFN-g, MR1.3 exhibits a typical cytostatic Th2 response to exogenously added IFN-g. Further proliferation studies conducted with MR1.3 and MR1.6 stimulated with irradiated B6D2 F1 splenocytes in the presence of various concentrations of IFN-g are shown in Figure 1B. These assays demonstrate that IFN-g inhibits both anti-CD3 antibody-mediated and APC-induced Th2 proliferation responses. Cytokine gene expression in interferon-g–treated mouse renal T-cell clones In view of the antiproliferative effects of IFN-g on both nephritogenic and non-nephritogenic clones, we also examined the impact of various levels of IFN-g exposure on MR T-cell clone cytokine gene expression. These studies addressed whether IFN-g blocked TCRstimulated proliferation by inhibiting lymphokine production. Immobilized anti-CD3 antibody-stimulated MR1.3 or MR1.6 T cells were incubated with various
antiproliferative doses of IFN-g (from 0.1 U/ml to 1000 U/ml). As shown in Figure 2, message expression of IL10, IL-13, and IFN-g was not markedly altered in either MR1.3 or MR1.6 after IFN-g treatment at doses that significantly inhibited proliferation. Of note, Northern hybridization studies in Figure 2 also revealed a significant difference in IL-13 expression between MR1.3 and MR1.6. Following stimulation, IL-13 was expressed at relatively low levels in MR1.3 cells, whereas the nonnephritogenic clone MR1.6 expressed much higher message levels. Additional Northern hybridization studies evaluating IL-4 expression in IFN-g–treated MR1.3 and MR1.6 did not reveal marked differences in IL-4 transcription between these T-cell clones (data not shown). Analysis of secreted cytokines in supernatants of IFNg–treated MR1.3 and MR1.6 T-cell clones corroborated initial Northern hybridization studies (Table 1). Quantitative assessment of secreted cytokines was performed, by ELISA, on a panel of anti-CD3 antibody-stimulated T-cell clones that were treated with various doses of IFN-g (0.1 to 1000 U/ml). As shown in Table 1, supernatants from nephritogenic clone MR1.3 contained significant amounts of IL-4, IL-10, and IL-13 and, consistent with our Northern hybridization data, also contained low levels of IFN-g. MR1.6, which does not induce renal inflammation, expressed IL-4, IL-10, and IL-13, but not IFN-g after stimulation. MR1.6 cells, however, consistently produced more IL-13 than MR1.3 cells with antiCD3 antibody stimulation. Also consistent with initial Northern analyses, no marked changes in IL-4, IL-10, IL-13, and IFN-g secretion were apparent after IFN-g treatment. These studies suggest that both MR1.3 and MR1.6 stimulated via the TCR complex, in the presence of IFN-g, are capable of producing relevant cytokines (particularly IL-4), but that they do not proliferate in response to autocrine or paracrine cytokine growth factors generated in this setting. Nephritogenic potential of MR1.3 and MR1.6 in interferon-g–treated recipients To assess IFN-g–mediated effects on the nephritogenic potential of renal-derived MR T-cell clones, we performed adoptive transfer studies with B6D2 F1 mice pretreated with IFN-g before renal subcapsular transfer. In previous studies, we have found that renal subcapsular transfer of immunocompetent T cells is a sensitive and reproducible means of determining the ability of discrete T-cell subsets to elicit inflammation in renal parenchyma [20, 29, 30]. We therefore used this technique to compare the nephritogenic potential of MR T cells under a variety of experimental conditions. As shown in Figure 3 A and B, IFN-g pretreatment did not alter the usual pattern of interstitial disease induced by nephritogenic clone MR1.3, which is characterized by focal cortical areas of mononuclear cell infiltration and areas of tubular dilata-
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Fig. 1. Proliferative responses of mouse renal (MR) clones. MR T-cell clones (1 3 105), either MR1.3 or MR1.6, were added to (A) anti-CD3 antibody-coated plates (1 mg/ml) or (B) irradiated B6D2F1 splenocytes with renal antigen (10 mg/ml), with various concentrations of interferon-g (IFN-g). Cells were pulsed with 3HTdR at 48 hours and were then harvested at 72 hours for scintillation counting. The values represent the mean of triplicate samples 6 sd.
tion and damage. In contrast, IFN-g pretreatment did alter the nephritogenicity of clone MR1.6. In this setting, MR1.6 induced cortical inflammation with focal mononuclear cell infiltrates and tubular dilation and atrophy (Fig. 3 C, D) in kidneys of syngeneic mice. The histologic features in all of these MR1.6-induced lesions are similar to those induced by the T-cell clone MR1.3. Formal histologic grading of these four groups, as well as IFN-g and PBS controls, is shown in Table 2. Analysis of adoptive transfers demonstrates that both the severity and depth of MR1.6-induced renal lesions in IFN-g–treated recipients are comparable to tubulointerstitial lesions elicited by MR1.3. Treatment with IFN-g or PBS alone did not induce renal inflammation or damage. As previously stated, proinflammatory effects of IFN-g on various subsets of immune cells may, in part, be mediated by its ability to up-regulate class II MHC
and costimulatory molecule expression on relevant APC [16]. In view of renal tubular epithelial cell responsiveness to these effects, we examined class II MHC expression shortly after adoptive transfer of MR1.3 and MR1.6 T cells to syngeneic mice. Kidney sections from animals that received MR1.3 cells exhibited an increase in renal tubular class II MHC (I-Ab) staining, whether animals were pretreated with PBS (Fig. 4A) or IFN-g (Fig. 4B). Adoptive transfer of MR1.6 with PBS did not increase I-Ab expression (Fig. 4C), but a marked increase in renal class II MHC expression was noted if animals were pretreated with IFN-g prior to MR1.6 transfer (Fig. 4D). These studies also indicate that pretreatment of animals with IFN-g alone (Fig. 4E), but not PBS (Fig. 4F), induced mild increases in renal class II MHC expression.
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Fig. 2. Cytokine expression in mouse renal (MR) clones following interferon-g (IFN-g) pretreatment. Nephritogenic (MR1.3) and nonnephritogenic (MR1.6) clones (3 3 106/ml) were plated on immobilized anti-CD3 antibody (1 mg/ ml) and various concentrations of IFN-g. After 24 hours, clonal RNA was harvested from 1 3 107 viable cells and evaluated for cytokine expression with cytokine-specific probes, interleukin (IL)-10, IL-13, and IFN-g.
Table 1. Secreted cytokines produced by MR1.3 and MR1.6 Cultured T cell clonea MR1.3 MR1.3 MR1.3 MR1.3 MR1.3 MR1.6 MR1.6 MR1.6 MR1.6 MR1.6
IFN-g dose U/ml 0 1 10 100 1000 0 1 10 100 1000
IL-4
IL-10
IL-13
ng/ml 8.0 7.0 10 9.0 14 10 14 12 12 11
1450 800 1700 1300 1400 600 800 250 750 350
21 22 20 19 20 110 115 115 110 110
IFN-g U/ml 2.5 2.5 4.5 14 2.5 ND ND ND ND ND
a MR T cell clones were harvested from day 10 cultures and plated in fresh medium at 1.5 3 106 cells/ml. Clones were then stimulated with anti-CD3 antibody-coated plates (1 mg/ml) and various doses of IFN-g for 24 hours. The levels of IL-4, IL-10, IL-13, and IFN-g in the resulting supernatants were determined by cytokine-specific ELISA. ND, not detected.
Effects of neutralizing anti–interferon-g antibody on adoptive transfer of renal disease As our nephritogenic clones secrete IFN-g following in vitro activation, we also evaluated the effects of neutralizing anti–IFN-g antibodies on adoptive transfer of renal disease. For these studies, MR1.3 cells were coadministered with anti–IFN-g antibody or with control rat IgG. As shown in the histology scores of Table 2, the severity and extent of tubulointerstitial lesions induced by MR1.3 were not markedly different in either of these treated groups. Transfer of MR1.3 to animals that were also treated with anti–IFN-g antibody or control rat IgG did not alter the nephritogenic potential of this T-cell clone. Moreover, renal class II MHC expression was still apparent in early lesions in all anti–IFN-g antibodytreated mice (data not shown).
In further studies, MR1.6 cells were administered to IFN-g–pretreated recipients with anti–IFN-g antibody or with control rat IgG. As delineated in Table 3, anti–IFN-g antibodies effectively inhibited the renal lesion typically induced by MR1.6 in IFN-g–pretreated mice. Following adoptive transfer of MR1.6, no evidence of tubulointerstitial damage was apparent in kidneys from animals that received the neutralizing anti–IFN-g antibody, whereas kidneys from the control IgG group of IFN-g–pretreated animals exhibited marked renal inflammation and injury (Table 3). Unlike the MR1.3 transfers, these studies suggest that IFN-g directly facilitates MR1.6 nephritogenicity. Interferon-gRb chain expression in mouse renal T-cell clones To investigate potential mechanisms of differential IFN-g–mediated effects in MR1.3 and MR1.6 cells, we examined the expression of the IFN-gRb chain in view of its critical signaling role in IFN-g–sensitive T cells [24, 31]. As shown in Figure 5, unstimulated MR1.3 and MR1.6 constitutively expressed IFN-gRb chain mRNA, but they responded differently to stimulation in the presence of IFN-g. Densitometric analysis of b-chain message levels revealed an approximate 50% decrease in MR1.3 cells, whereas levels in MR1.6 cells in this setting did not change markedly. Although both T-cell clones express the regulatory b chain following IFN-g exposure, differential regulation of the level of cytokine receptor expression may indeed effect distinct functional influences of IFN-g on these T-cell clones. DISCUSSION In the studies presented here, we evaluated the immunomodulatory effects of exogenous IFN-g on distinct
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Fig. 3. Effects of interferon-g (IFN-g) on adoptive transfers. For these studies, 5 3 106 mouse renal (MR) T-cell clones were injected beneath the renal capsule of B6D2 F1 mice 24 hours after pretreatment with either PBS or IFN-g (1000 U). Kidneys were harvested five to seven days after T-cell transfers and were evaluated by light microscopy. The interstitial lesion typically seen after transfer of MR1.3 cells is seen in both (A) (PBS pretreatment) and (B) (IFN-g pretreatment). Focal cortical areas of mononuclear cell infiltration are evident, as well as areas of tubular dilation. MR1.6 adoptive transfers are shown in (C) (PBS pretreatment) and (D) (IFN-g pretreatment). IFN-g or PBS pretreatment alone did not result in renal inflammation or damage (data not shown).
functional CD41 T cells derived from diseased kidneys of animals with cGVHD. Our studies indicate that IFN-g mediates dose-dependent inhibition of both nephritogenic MR1.3 and non-nephritogenic MR1.6 proliferation, although cytokine gene expression of IL-4, IL-10, and IFN-g after IFN-g treatment is not markedly altered in either T-cell clone following anti-CD3 Ab stimulation. We also noted a higher baseline level of IL-13 expression in MR1.6 compared with nephritogenic clone MR1.3. Of note, IFN-g markedly alters the nephritogenic potential of clone MR1.6, as pretreated naive recipients develop marked renal inflammation and damage after transfer
of MR1.6 T cells. The transfer of MR1.3 with neutralizing anti–IFN-g antibodies does not inhibit its ability to adoptively transfer renal inflammation and damage, whereas MR1.6 nephritogenicity in IFN-g–treated mice was blocked by these antibodies. In addition, both clones constitutively express the IFN-gRb chain, which is differentially expressed in MR1.3 and MR1.6 following IFN-g exposure. These studies describe a remarkably permissive role for IFN-g in modulating nephritogenic Th2 activity, perhaps by local up-regulation of renal class II MHC or costimulatory molecule expression, which facilitates the initiation of cell-mediated autoimmune renal injury.
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Table 2. Effects of IFN-g on adoptively transferred renal lesions Cells transferreda MR1.3 MR1.3 MR1.6 MR1.6 MR1.6 MR1.6 MR1.3 MR1.3
Recipient treatment
Severity of renal damage
Depth of lesion
PBS IFN-g PBS IFN-g PBS IFN-g Anti-IFN-g Ab Rat IgG
1.5 6 0.5b 1.4 6 0.3b 0 1.7 6 0.2b 0 0 1.5 6 0.1b 1.2 6 0.3b
22 6 7.5b 28 6 4.8b 0 25 6 3.0b 0 0 28 6 4.3b 20 6 2.9b
a Five days following transfer of 10 3 106 MR clones to normal B6D2 F1 mice, kidneys were harvested and evaluated by light microscopy, as detailed in the Methods section. Some recipients were injected with IFN-g (1000 U) or PBS IP 24 hours prior to subcapsular transfer. For some studies, MR cells were coadministered to recipient mice with anti-IFN-g antibody or an irrelevant antibody (Rat IgG) (N 5 4–6/group). b P , 0.001 vs. no cell/PBS control
The previous observation of differential IFN-g expression in the Th2-like MR clones was intriguing in view of the proscriptive influence of IL-10 on its synthesis in CD41 T-cell clones [12]. Although culture conditions may effect uncharacteristic changes in cytokine gene expression in cloned T-cell lines, similar Th2-like observations in other models of autoimmunity suggest that this particular CD41 T-cell phenotype may be relevant to disease expression [32–34]. Such observations include autoreactive T-cell clones isolated from MRL/lpr mouse kidneys and gold salt-injected Brown Norway rat lymph nodes, as well as allergen-specific human Th2 cells [32– 34]. These analyses indicate that IFN-g secretion by a Th2-like CD41 T cell may be of functional significance in vivo [32–34]. These MR clone studies indicate that IFN-g downregulates proliferation of IL-4–producing CD41 T cells by limiting their ability to respond to T-cell growthpromoting cytokines generated on activation via the TCR complex, rather than by inhibiting the production of relevant cytokines. It is intriguing, however, that despite the proscriptive influence on MR1.3 and MR1.6 proliferation in vitro, both T-cell clones affected renal inflammation and damage in IFN-g–treated recipients. IFN-g–induced up-regulation of class II MHC and relevant costimulatory molecule (that is, B7-1) expression, as well as cytokine production, in both professional and “nonprofessional” APCs may markedly alter patterns of T-cell activation in vivo. In addition to these proinflammatory effects, IFN-g also modulates T-cell biology through a number of antagonistic interactions with other T-cell–derived cytokines [10]. For instance, although IFN-g counteracts IL-4–induced proliferation, IL-4, inhibits transcriptional activation of IFN-g–inducible proteins, and blocks IFN-g–induced chemokine synthesis, it also inhibits Th1 IFN-g production [35–37]. Similarly, IFN-g suppresses Th2 cell IL-10 production, whereas IL10 inhibits Th1 cell IFN-g production [28, 38].
Our studies are noteworthy in that anti–IFN-g antibody does not block nephritogenicity of MR1.3, indicating that initiating renal disease by this T-cell clone is not critically dependent on IFN-g. Other inflammatory mediators produced by MR1.3, such as perforin (unpublished observation), may be more relevant to initiating this renal immune response. Anti–IFN-g antibody was effective, however, at blocking MR1.6 disease activity in IFN-g–pretreated mice, suggesting a primary initiating role for IFN-g in MR1.6 adoptively transferred lesions. Such observations further demonstrate functional differences between these clones, as they exhibit distinct responses to IFN-g exposure in the setting of renal injury. Recent studies on IFN-gR have shed light on IFNg–mediated effects on distinct T-cell subsets [24, 31]. T-cell responsiveness to IFN-g depends on IFN-gRb chain expression, the receptor subunit required for signaling, which is limited to Th2 cells and is virtually absent in Th1 cells [24, 31]. It is noteworthy that chronic Th2 cell exposure to high levels of IFN-g, greater than levels detected in our current studies, down-regulates IFN-gRb chain expression, and decreases Th2-responsiveness to exogenous IFN-g effects [24, 31]. Interestingly, ligandinduced receptor b-chain down-regulation does not occur in other cell preparations, such as fibroblast cell lines [31]. IFN-g appears to regulate expression of its own receptor b chain on certain cell types and thereby determines the responsiveness of these cells to subsequent IFN-g exposure [24, 39, 40]. Recent studies indicate that treatment of T cells with phorbol esters or with antiCD3 antibodies induces IFN-gRb chain mRNA [40]. These findings demonstrate that b-chain expression can be regulated either positively or negatively in a stimulusspecific manner [39]. Our observations of IFN-gRb chain expression also indicate distinct responses and regulation of this signaling chain in MR1.3 and MR1.6 cells, suggesting that different patterns of b-chain regulation in these T cells may indeed cause disparate functional responses in vivo. Different baseline IL-13 expression in distinct MR clones is worthy of further comment, in view of the distinct functions mediated by these T cells. MR1.3 is nephritogenic, adoptively transferring tubulointerstitial inflammation and damage to syngeneic recipients, and is restricted by Iad [11]. MR1.6, however, does not transfer renal disease to naive syngeneic animals and is restricted by Iab determinants [11]. Both MR1.3 and MR1.6 express a predominantly Th2 cytokine pattern with IL-4, IL-6, IL-10, and IL-13 expression, although our current studies suggest that MR1.6 expresses a higher level of IL-13 on stimulation. Sharing many properties with IL-4, IL-13 also has pleiotrophic effects on B cells, monocytes, macrophages, and neutrophils [41]. It largely suppresses T-cell–mediated immune responses [41]. The relatively low level of IL-13 expression in nephritogenic clone
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Fig. 4. Renal class II major histocompatibility complex (MHC) expression in adoptive transfers. Kidneys harvested from adoptive transfer experiments, three days after T cell transfers, were evaluated immunohistochemically for class II MHC (I-Ab) expression. Marked expression is seen in (A) (MR1.3 transfer after PBS pretreatment), (B) (MR1.3 transfer after IFN-g pretreatment), and (D) (MR1.6 transfer after IFN-g pretreatment), whereas class II MHC expression is not observed in (C) (MR1.6 transfer after PBS pretreatment) and (F) (PBS treatment control). A minimal increase in class II MHC staining is apparent in (E) (IFN-g treatment control).
Table 3. Effects of neutralizing anti-IFN-g antobodies on MR1.6-induced renal lesions Cells transferreda MR1.6 MR1.6 —
Recipient treatment
Severity of renal damage
Depth of lesion
IFN-g, Anti-IFN-g Ab IFN-g, Rat IgG PBS
0 1.3 6 0.2b 0
0 27 6 3.1b 0
a Five days following transfer of 10 3 106 MR clones to normal B6D2 F1 mice, kidneys were harvested and evaluated by light microscopy, as detailed in the Methods section. Some recipients were injected with IFN-g (1000 U) or PBS IP 24 hours prior to subcapsular transfer. For some studies, MR cells were coadministered to recipient mice with anti-IFN-g antibody or an irrelevant antibody (Rat IgG) (N 5 4–6/group). b P , 0.001 vs. no cell/PBS control
MR1.3 may facilitate the cell-mediated inflammatory renal lesion apparent on adoptive transfer, a response not significantly altered by IFN-g. Despite the apparent robust IL-13 expression in MR1.6, however, it is noteworthy that IFN-g pretreatment effectively alters its nephritogenic potential in vivo. This response suggests that
IFN-g–induced changes in host environment causes a dramatic change in the functional phenotype of MR1.6 on adoptive transfer. Although IL-13 has been detected in diseased kidneys in other models of immune-mediated renal disease, its immunomodulatory role in renal inflammation, however, has not been clearly defined [23]. A heterogeneity of IFN-g–mediated effects has been reported in a number of models of autoimmune disease. Previous studies conducted in this model of cGVHD examined the effects of exogenous IFN-g on induction of disease in B6D2 F1 mice [42]. Although IFN-g reduced serum IgE and IgG1 levels in affected mice, no difference in animal survival was noted [42]. The extent of renal involvement and degree of proteinuria, however, were not evaluated [42]. In murine models such as autoimmune diabetes, autoimmune thyroiditis, and lupus-prone (NZB/NZW) F1 mice, IFN-g facilitated disease induction, whereas administration of anti–IFN-g antibodies were disease protective [43–47]. Observations in experimental autoimmune encephalomyelitis (EAE) and adju-
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Fig. 5. Interferon (IFN)-gRb chain expression in mouse renal (MR) clones following IFN-g exposure. Clonal RNA was harvested from nonnephritogenic clone MR1.6 (A, B) and nephritogenic clone MR1.3 (C, D) and was evaluated for IFN-gRb chain expression by Northern hybridization. MR cells were either unstimulated (A, C) or plated on immobilized anti-CD3 antibody (1 mg/ml) in the presence of IFN-g (100 U/ml) for 24 hours (B, D). The findings are representative of three distinct experiments.
vant arthritis have been less straightforward [48, 49]. Earlier studies in EAE revealed that exogenous IFN-g reduced the incidence and severity of disease, and neutralizing antibodies augmented disease activity [48, 49]. Recent analysis of susceptible hosts suggests that IFN-g–deficient mice develop EAE as readily as wildtype controls following immunization with heterologous myelin basic protein, but have a much higher mortality rate [50]. IFN-g and its neutralizing antibody have disparate effects on disease activity in adjuvant arthritis, depending on the dose and timing of administration [51]. IFN-g administered prior to, or at least four days after, disease induction exacerbates disease activity. If given one to two days after disease induction, however, the disease is suppressed [51]. These disparate observations regarding activity of IFN-g in autoimmune tissue destruction further illustrate the complex, likely multistage nature of T-cell–mediated autoimmune responses. Bidirectional effects noted in various disease models may be due to the predominance of distinct T-cell subsets, with varying degrees of IFN-g responsiveness, at different stages of disease [51]. In addition to well-described effects on various relevant immune cells, recent investigations indicate that IFN-g induces changes in cells of nonimmune origin [16]. Most notably, IFN-g has a number of immunomodulatory effects on epithelial cells that may promote disease [16–18]. As observed in these MR clone studies, it induces de novo class II MHC expression on renal epithelial cells, perhaps converting them to “nonprofessional” APC in vivo [10, 16–18]. Studies in other models of systemic lupus erythematosus, syngeneic GVHD and cGVHD across minor histocompatibility differences
have also identified a correlation between class II MHC antigen induction and the pattern of lymphocytic parenchymal lesions observed in affected animals [52–54]. Increased renal class II MHC expression is likely not sufficient for inducing autoimmune injury, however, as class II MHC transgenic animals, with high levels of renal tubular epithelial cell class II MHC expression, do not develop spontaneous renal disease [55]. Recent in vitro observations also indicate that IFN-g alters epithelial cell permeability [56]. This cytokine-mediated change in epithelial permeability may be a major cause of altered epithelial barrier function in vivo. Such a change could be pathogenic, as it would facilitate access of relevant lymphocytic/inflammatory cells to epithelial surfaces, promoting inflammation [56]. The potential relevance of these IFN-g-tubular epithelial interactions is currently under investigation in our laboratory. ACKNOWLEDGMENTS This work was done during the tenure of a Grant-in-Aid Award from the American Heart Association (95012790). It was also supported in part by the Margaret Q. Landenberger Research Foundation, the Lupus Foundation of Philadelphia, Inc., National Institutes of Health DK-07006 and DK-49724, and the administrative/educational funds from the DCI RED Fund. The authors are grateful to Ms. Mary H. Foster for her comments and critical review of the manuscript and to Mr. Steven J. Birkos for his technical assistance. This work was presented in part at the annual meeting of the American Society of Nephrology, Orlando, Florida, November 1994. Reprint requests to Catherine M. Meyers, M.D., Renal-Electrolyte and Hypertension Division, University of Pennsylvania, 700 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: cGVHD, chronic graft vs. host disease; EAE, experimental autoimmune encephalomyelitis; FCS, fetal calf serum; IFN-g, interferon g; IFN-gRb, interferon g receptor b; IL, interleukin; MHC, major histocompatibility complex; MR, renal cells isolated from mice with chronic graft vs. host disease; MR1.3, nephritogenic T cell clone; MR1.6, non-nephritogenic T cell clone; PBS, phosphate buffered saline; Th2, T helper cell type 2.
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