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The Journal of Immunology, 2002, 169: 5941-5947.
Copyright © 2002 by The American Association of Immunologists

Dysregulation of IFN-{gamma} Signaling Pathways in the Absence of TGF-{beta}1

Nancy L. McCartney-Francis1 and Sharon M. Wahl

Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Deficiency of TGF-{beta}1 is associated with immune dysregulation and autoimmunity as exemplified by the multifocal inflammatory lesions and early demise of the TGF-{beta}1 null mice. Elevated NO metabolites (nitrite and nitrate) in the plasma of these mice suggest a participatory role of NO in the pathogenic inflammatory response. To determine the mechanism for this dysregulation, we examined upstream elements that could contribute to the overexpression of NO, including inducible NO synthase (iNOS) and transcription factors Stat1{alpha} and IFN-regulatory factor-1 (IRF-1). The coincident up-regulation of IFN-{gamma}, an iNOS inducer, and iNOS, before the appearance of inflammatory lesions, suggests that failed regulation of the IFN-{gamma} signaling pathway may underlie the immunological disorder in TGF-{beta}1 null mice. In fact, IFN-{gamma}-driven transcription factors IRF-1 and Stat1{alpha}, both of which act as transcriptional activators of iNOS, were elevated in the null mice. Treatment of mice with a polyclonal anti-IFN-{gamma} Ab reduced expression and activity not only of transcription factors Stat1{alpha} and IRF-1 but also of iNOS. Furthermore, anti-IFN-{gamma} treatment delayed the cachexia normally seen in TGF-{beta}1 null mice and increased their longevity. The global nature of immune dysregulation in TGF-{beta}1 null mice documents TGF-{beta}1 as an essential immunoregulatory molecule.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide, a highly reactive diatomic free radical, functions as a first line of defense against invading pathogens and tumor cells (1, 2). High levels of NO are generated from L-arginine through the actions of inducible NO synthase (iNOS).2 Although vital to effective host defense, overproduction of NO can be cytotoxic. Indeed, the association of iNOS expression and elevated NO with certain autoimmune and inflammatory diseases implicates a pathogenic role (1, 2, 3). Furthermore, inhibition of NO production in animal models of inflammatory pathology attenuates the response (3, 4, 5, 6). Balance between these toxic and protective properties is required to maintain homeostasis (7). One molecule that plays an important role in the regulation of NO is TGF-{beta}, a potent suppressor of iNOS (8).

Consistent with the role of TGF-{beta} in regulating NO, TGF-{beta}1 null mice express elevated serum NO and iNOS protein in tissues (9). Distinguishing features of the TGF-{beta}1 null mice include multifocal inflammation, wasting, and death by 3–4 wk of age (10, 11). Infiltration of lymphocytes and macrophages, particularly in heart and lungs, is associated with cardiopulmonary failure. Increased MHC Ag, cytokines, adhesion molecules, and autoantibody expression suggest an autoimmune origin of the pathological lesions (12, 13, 14, 15, 16, 17). Recent studies have shown enhanced expression of IFN-{gamma} RNA before the appearance of inflammatory lesions in TGF-{beta}1 null mice (13), consistent with disruption of immunoregulatory circuits in the absence of TGF-{beta}1. Because IFN-{gamma} acts singularly as well as synergistically with inflammatory stimuli to induce NO production (reviewed in Ref. 18), the coincidence of enhanced NO and IFN-{gamma} expression in the TGF-{beta}1 null mice implicates an in vivo role for IFN-{gamma} in the induction of iNOS. The promoter region of iNOS contains enhancer elements known to bind NF-{kappa}B and IFN-{gamma}-responsive transcription factors, Stat1{alpha} and IFN-regulatory factors (IRF) IRF-1 and IRF-2 (19, 20). The transcription factor IRF-1 mediates the effects of IFN-{gamma} by binding to response elements (IFN-stimulated response element (ISRE)) within the promoters of IFN-{gamma}-inducible genes, such as iNOS, and activating transcription, whereas IRF-2 inhibits transcription (reviewed in Ref. 21). Promoter deletion and mutation studies have demonstrated that IRF-1 as well as Stat1{alpha} and NF-{kappa}B play critical roles in the transcriptional regulation of iNOS (19, 20, 22). Furthermore, IRF-1, Stat1{alpha}, and NF-{kappa}B (c-rel) knockout mice display defective NO production, supporting an in vivo role for these transcription factors in the regulation of iNOS (23, 24, 25, 26). Because TGF-{beta} also regulates NO by an ill-defined pathway, we used the TGF-{beta}1 knockout mouse as an in vivo model to study its regulation of iNOS expression and the transcription factors involved in this pathway. In the absence of TGF-{beta}1, the augmented expression of iNOS occurs via increased expression of Stat1{alpha} and also IRF-1, which may be secondary to unrestricted IFN-{gamma} production. The resulting toxic levels of NO contribute to the lethal phenotype of the TGF-{beta}1 null mice, and targeting of upstream factors such as IFN-{gamma} interrupts this autotoxic pathway to delay the lethal phenotype of the TGF-{beta}1 null mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

TGF-{beta}1-deficient mice have been described previously (10). Heterozygous mice were interbred to generate TGF-{beta}1 null offspring. Genotyping was performed by PCR of tail biopsies (15). Mice were maintained on standard mouse chow supplemented with a liquid diet (Bioserv, Frenchtown, NJ). All animal experiments were performed in accordance with institutional guidelines and with approval from the Institutional Animal Care and Use Committee.

Histopathology and immunohistochemistry

Tissues were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. Sections (6 µm) were stained with H&E for histopathology or processed for immunohistochemistry. Macrophages were detected by avidin-biotin-peroxidase complex immunohistochemistry, using anti-Mac2 Ab (generous gift of Dr. S. Vogel, University of Maryland School of Medicine, Baltimore, MD) or anti-iNOS Ab (Upstate Biotechnology, Lake Placid, NY) and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). For electron microscopy, tissues were fixed in 2% paraformaldehyde, 2% glutaraldehyde and embedded in plastic.

Nitrite plus nitrate determination in plasma

Plasma was filtered using an Ultrafree-MC microcentrifuge filter unit (14,000 rpm for 15 min; Millipore, Bedford, MA) and treated with nitrate reductase (Sigma-Aldrich, St. Louis, MO) in the presence of NADPH (Sigma-Aldrich) to convert nitrate to nitrite (27, 28). On the addition of 2,3-diaminonaphthalene (Sigma-Aldrich), which reacts with nitrite under acidic conditions to form a fluorescent product (1(H)-naphthotriazole), fluorescence intensity was measured with a fluorescence microplate reader with excitation at 365 nm and emission at 450 nm and nitrite quantitated by comparison with a standard curve of NaNO2.

IFN assay

Plasma levels of IFN-{gamma} were measured using a commercial ELISA kit (Genzyme, Boston, MA).

Semiquantitative RT-PCR

Total RNA was isolated from tissues with Trizol reagent (Invitrogen, Gaithersburg, MD) and 2 µg were reverse transcribed. The cDNA was amplified by PCR using appropriate oligonucleotide primers and predetermined conditions. The primers (5' to 3') included the following: hypoxanthine phosphoribosyltransferase (HPRT) (162 bp), 5'-GTTGGATACAGGCCAGACTTTGTTG, 3'-GATTCAACTTGCGCTCATCTTAGGC; iNOS (270 bp), 5'-TTGGGTCTTGTTCACTCCACGGAG, 3'-ATTCTGTGCTGTCCCAGTGAGGAG; IRF-1 (478 bp), 5'-TTCCAGATTCCATGGAAGCACGC, 3'-AGACTGCTGCTGACGACACACG; IRF-2 (322 bp), 5'-AACAACGCCTTCAGAGTCTACCG, 3'-CACTCTCAGTGGTCACTTCTAC; IFN-{gamma} (460 bp), 5'-TGAACGCTACACACTGCATCTTGG, 3'-CGACTCCTTTTCCGCTTCCTGAG; Stat1{alpha} (645 bp), 5'-GCCCGACCCTATTACAAAAA, 3'-CTGCCAACTCAACACCTCTG.

Samples were heated at 94°C for 10 min and amplified using cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1–2 min (HPRT, IRF-1, 28 cycles; Stat 1{alpha}, IRF-2, 31 cycles; IFN-{gamma}, 42 cycles; iNOS, 35 cycles). The amplified products were analyzed by ethidium bromide staining after agarose (1.5% in 0.5x Tris borate EDTA buffer) gel electrophoresis. Bands were scanned and quantitated by a fluoroimager using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Western blot analyses

Tissues were homogenized in lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 2 mM EDTA) containing protease and phosphatase inhibitors (100 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/ml soybean trypsin inhibitor, 20 µM sodium vanadate). The lysate was clarified by centrifugation (10,000 rpm, 20 min at 4°C), and protein content was determined (DC Protein Assay; Bio-Rad, Hercules, CA). Proteins were separated by electrophoresis on a 7.5% or 10% SDS-polyacrylamide gel and then electrophoretically transferred to a polyvinylidene fluoride Immobilon-P membrane (Millipore). After blocking in 1% chicken albumin (Sigma-Aldrich) at room temperature for 1 h, the membrane was washed in TBS-T buffer (100 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C with anti-Stat1{alpha} (generous gift of Dr. G. Feldman, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD) or biotinylated anti-phosphotyrosine Abs (Leinco, St. Louis, MO). After washing, the blots were incubated with protein A HRP (Amersham Pharmacia Biotech, Piscataway, NJ) or avidin-HRP (Neutralite; Southern Biotechnology Association, Birmingham, AL) for 1 h at room temperature and developed with enhanced chemiluminescence detection system (Renaissance ECL; New England Nuclear, Boston, MA).

Immunoprecipitation

Tissue lysates (250–350 µg in 500 µl) were precleared by incubation with 20 µl of protein G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C. The lysates were then incubated overnight at 4°C with anti-Stat1{alpha} Ab and protein G beads. The beads were washed four times in lysis buffer, and the immunoprecipitates were resuspended in SDS-Laemmli loading buffer, boiled for 5 min, and used for Western blot analysis as described above.

EMSA

Tissues were homogenized in lysis buffer (20 mM Tris (pH 7.6), 120 mM NaCl, 1% Nonidet P-40, 10% glycerol, 10 mM sodium pyrophosphate, 100 mM NaF, 2 mM sodium orthovanadate, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 5 µg/ml leupeptin) and total protein determined. Protein extracts (5 µg) were incubated with polynucleotide kinase-radiolabeled DNA probes (0.05 pmol) in reaction buffer (10 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, 4% glycerol, 80 µg/ml salmon sperm DNA, with a final adjusted concentration of 0.08 M NaCl) for 30 min at room temperature. The reaction mixture was electrophoresed in a 6% nondenaturing acrylamide gel containing 0.25 M Tris borate EDTA buffer which was then vacuum dried and analyzed with a PhosphorImager (Molecular Dynamics). To assess specificity, binding of protein to the DNA probe was prevented by a 30-min incubation at 4°C with 50-fold excess of unlabeled oligonucleotide. IRFs were detected using the ISRE probe (5'-CTGTCAATATTTCACTTTCATAAT-3') and Stat 1{alpha} using the IFN-{gamma} activated site (GAS) probe (5'-TGTTTGTTCCTTTTCCCCTAACA-3') (20).

Ab treatment

For longevity studies, mice received i.p. injections of polyclonal anti-IFN-{gamma} (1 mg, gift of Dr. R. Seder, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD), normal rat IgG, or PBS every 2–5 days beginning on postnatal days 12–14. Body weights were recorded daily, and time of death was noted. For analytical studies, mice received a single i.p. injection on day 8, and tissues were analyzed on day 13.

Statistical analysis

Data are presented as mean ± SEM and analyzed for significance by the one-tailed unpaired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presence of inflammatory cells expressing iNOS and producing NO in heart tissue of TGF-{beta}1 null mice

A distinguishing histological feature of the TGF-{beta}1 null mouse is the overwhelming influx of inflammatory cells into nonlymphoid organs including the heart, lung, and salivary glands. The degree of cellular infiltration into these organs severely affects their structure, contributing to the faulty function and early demise of the null mice. For example, immunohistochemical staining of heart tissue from TGF-{beta}1 null mice reveals a large number of mononuclear cells that stain positive for the macrophage-specific marker Mac2 and exhibit morphological evidence of activation (Fig. 1Go). By electron microscopy, large, vacuolated macrophages are located in contact with or in close proximity to myocytes (Fig. 1GoC). Also present are lymphocytes and large granulated lymphocytes (29). Cellular damage, apoptotic cells, and active phagocytosis are evident in the heart tissue of the TGF-{beta}1 null mice, thus contributing to the structural dissolution of the heart muscle and eventual heart failure.



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  		FIGURE 1. Mononuclear cell infiltration in heart tissue of TGF-{beta}1 null mice. Tissue sections of heart tissue from TGF-{beta}1 null were stained with H&E (A) or anti-Mac2 Ab (B) (magnification, x200). Pronounced mononuclear cell infiltration and tissue disruption was evident in heart tissue of TGF-{beta}1 null mice. A substantial number of the infiltrating cells were macrophages (Mac2+). Macrophage (M) and lymphocyte (L) infiltration was confirmed by electron microscopy (C) (magnification, x9000).

 
One macrophage product regulated by TGF-{beta} and autotoxic is NO. Immunohistochemical staining of heart tissue from TGF-{beta}1 null mice for iNOS protein revealed increased expression of the enzyme in areas associated with cellular infiltration and tissue destruction (Fig. 2GoA). Constitutive low level staining with the iNOS Ab was also evident in noninflamed areas of heart tissue from null mice as well as from wild-type littermates (Fig. 2Go, A and C). In the absence of the primary Ab, no staining was observed (Fig. 2Go, B and D), suggesting that the basal expression of iNOS in myocytes of both wild-type and null hearts may be physiologically relevant (30, 31). To confirm the overexpression of iNOS, total RNA from heart tissue of TGF-{beta}1 null and wild-type littermates was analyzed by semiquantitative RT-PCR for iNOS mRNA expression. Consistent with the in situ expression of iNOS protein, abundant levels of iNOS mRNA were detected in heart tissue of TGF-{beta}1 null mice as compared with wild-type littermates (Fig. 3GoA). iNOS mRNA was elevated not only in symptomatic mice, (i.e., >=12 days of age and displaying visible signs of wasting), but also in asymptomatic null mice (<12 days of age) and message levels were consistently elevated throughout the short life span of the mice. Thus, expression of iNOS enzyme is augmented in TGF-{beta}1 null mice and, in the absence of the suppressive background of TGF-{beta}1, induced levels of iNOS protein and mRNA are sustained.



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  		FIGURE 2. Inducible NOS is expressed in heart tissue of TGF-{beta}1 null mice. Tissue sections from TGF-{beta}1 null (A and B) and wild-type (C and D) mice were stained with rabbit anti-mouse iNOS Ab (A and C) or normal serum (B and D). Hearts from TGF-{beta}1 null mice contained many iNOS-positive cells within the inflammatory infiltrates (A). Low level iNOS staining was evident in myocardial tissue in both null and wild-type mice (A and C). In the absence of iNOS Ab, no staining was observed (B and D) (magnification, x400).

 


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  		FIGURE 3. iNOS and IFN-{gamma} RNA levels are elevated in TGF-{beta}1 null mice. A, Total RNA from heart tissue of TGF-{beta}1 null (KO) and wild-type (WT) littermates was analyzed by semiquantitative RT-PCR for iNOS, IFN-{gamma}, and the housekeeping gene HPRT mRNA expression. PCR products were electrophoresed and visualized by ethidium bromide staining. iNOS and IFN-{gamma} mRNA levels were elevated in heart samples from both asymptomatic (<12 days) and symptomatic (>=12 days) TGF-{beta}1 null mice. B, NO production was assessed by measuring total nitrite and nitrate in plasma using the fluorescent microplate assay. Data are expressed as mean ± SEM of TGF-{beta}1 null (n = 27) and wild-type mice (n = 25), age 12–33 days. Plasma samples of TGF-{beta}1 null mice (n = 17, age >=12 days) and wild-type littermates (n = 16) were assayed for IFN-{gamma} content by ELISA, and data were expressed as mean ± SEM. TGF-{beta}1 null mice expressed significantly higher levels of NO (p = 0.0001) and IFN-{gamma} (<=0.02) as compared with wild-type mice (one-tailed unpaired t test).

 
In addition to increased iNOS expression in heart tissue as well as lung and salivary gland (not shown), circulating levels of NO, as measured by nitrite and nitrate content in the plasma, were significantly higher in TGF-{beta}1 null mice than in wild-type littermates, suggesting spillover of NO from the affected organs into the circulation (Fig. 3GoB) (9). Whereas asymptomatic null mice did not express increased levels of circulating NO, an average of 4-fold more NO was detected in the plasma of symptomatic null mice (>=12 days of age) than in plasma of wild-type littermates (157.5 ± 27.9 µM vs 41.7 ± 4.3, p = 0.0001).

Up-regulation of IFN-{gamma} expression in heart tissue and plasma of TGF-{beta}1 null mice

Because transcriptional induction of iNOS occurred early in the life of the TGF-{beta}1 null mouse, we sought to identify stimulatory molecules that could contribute to the signaling pathways leading to the induction of iNOS transcription and NO production. One potential inducer of iNOS in mouse macrophages is IFN-{gamma}. Local IFN-{gamma} mRNA levels were increased between 4- and 9-fold in the heart tissue of symptomatic and asymptomatic TGF-{beta}1-deficient mice as compared with wild-type littermates (Fig. 3GoA) and, as was the case for iNOS expression, IFN-{gamma} mRNA levels remained elevated throughout the life span of the mice. In addition, circulating levels of IFN-{gamma} were significantly elevated in TGF-{beta}1 null mice as compared with wild-type littermates (32.9 ± 11.2 pg/ml vs 4.4 ± 0.9, p < 0.02) (Fig. 3GoB), consistent with the elevation of IFN-{gamma} mRNA in the tissues (Fig. 3AGo and Refs. 13 and 32). The coordinated expression of iNOS and IFN-{gamma} provide evidence for activation of signaling pathways leading to the transcriptional activation of iNOS.

Up-regulation of IRF-1 RNA and protein in heart tissue of TGF-{beta}1 null mice

Induction of iNOS by IFN-{gamma} occurs through the activation of transcription factors that bind to response elements in the iNOS promoter. In particular, two juxtaposed ISREs are present in the iNOS promoter which bind the transcription factor IRF-1 and regulate iNOS gene expression (20, 23). To determine whether IRF-1 expression was altered in the TGF-{beta}1 null mice, we first examined total mRNA from heart tissue by semiquantitative RT-PCR for IRF-1 gene expression. IRF-1 mRNA was significantly elevated in TGF-{beta}1 null mice as compared with wild-type littermates (2- to 4-fold increase; Fig. 4GoA). As was observed for both IFN-{gamma} and iNOS, IRF-1 gene expression was elevated not only in older, symptomatic mice but also strikingly in asymptomatic mice, before histological evidence of inflammatory pathology. In contrast, constitutive expression of IRF-2 mRNA, a transcription factor with inhibitory activity that also binds to the ISRE, was similar between null and wild-type littermates (Fig. 4GoB). To confirm the expression of IRF in null mice, electrophoretic mobility shift assays were performed using a probe containing the 24-base ISRE. Increased amounts of DNA-protein complexes were observed with heart tissue homogenates of TGF-{beta}1 null mice (Fig. 4GoC). This binding could be competed with 50-fold molar excess of cold oligonucleotide, confirming the specificity for the ISRE site (not shown).



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  		FIGURE 4. IRF-1 mRNA and protein are elevated in TGF-{beta}1 null mice. A, Total RNA from heart tissue of TGF-{beta}1 null (KO) mice and wild-type (WT) littermates was subjected to semiquantitative RT-PCR using oligonucleotides specific for IRF-1, IRF-2, and HPRT. After electrophoresis and ethidium bromide staining, PCR products were quantitated by a fluoroimager (B). B, Densitometric units of IRF-1 and -2 PCR products were normalized to HPRT, and values were reported as a ratio of null to wild-type (x100%). C, Proteins from heart tissue of TGF-{beta}1 null (n = 16) and wild-type (n = 14) mice were analyzed by EMSA using a radiolabeled oligonucleotide probe containing the ISRE-binding site.

 
Increased expression of Stat1{alpha} protein in TGF-{beta}1 null mice

The IRF-1 and iNOS promoters both contain GAS elements that bind homodimers of phosphorylated Stat1{alpha}. Because IFN-{gamma} activates Stat1{alpha} through tyrosine phosphorylation, we looked for evidence of Stat1{alpha} expression and activation in TGF-{beta}1 null mice which overexpress IFN-{gamma}. Western blot analysis of proteins isolated from heart tissue of null mice between the ages of 7 and 28 days revealed striking elevations in expression of Stat1{alpha} protein within the first week after birth as compared with wild-type littermates (Fig. 5GoA). To determine the phosphorylation state of the Stat1{alpha}, the protein was immunoprecipitated with anti-Stat1{alpha} and then analyzed by Western blot using anti-phosphotyrosine Ab (Fig. 5GoB). In contrast to the low level expression of Stat1{alpha} and pStat1{alpha} in wild-type littermates, a notable proportion of the immunoprecipitated Stat1{alpha} in heart tissue of null mice was phosphorylated, indicating that a portion of the Stat1{alpha} was already activated. To confirm the activation state of the Stat1{alpha} protein, we next performed EMSAs. Increased binding of protein to a radiolabeled oligonucleotide probe containing the GAS-binding site was observed in heart tissue from the TGF-{beta}1 null mice as compared with the wild-type littermates (Fig. 5GoC), thus confirming the phosphorylation of the Stat1{alpha} protein.



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  		FIGURE 5. Up-regulation and activation of Stat1{alpha} protein in TGF-{beta}1 null mice. A, Proteins (5 µg) extracted from heart tissue of null and wild-type mice were electrophoresed in 5% acrylamide gels containing SDS and transferred to nitrocellulose, and Western blot analysis was performed using anti-Stat1{alpha} Ab. B, Stat1{alpha} proteins were immunoprecipitated and analyzed by Western blot using anti-phosphotyrosine Ab to detect phosphorylated Stat1{alpha} (pStat1{alpha}). C, Proteins isolated from hearts of TGF-{beta}1 null and wild-type mice were subjected to gel shift analysis using a radiolabeled oligonucleotide containing the GAS-binding site. Binding could be competed with excess cold oligonucleotide, confirming the presence of specific GAS-binding proteins (data not shown).

 
Temporal expression of IFN-{gamma} signaling elements

To determine the temporal relationship between the IFN-{gamma} signaling elements expressed in the TGF-{beta}1 null mice, heart tissue from null mice and wild-type littermates, age 3–7 days, was examined by RT-PCR for iNOS, IFN-{gamma}, Stat1{alpha}, and IRF-1 (Fig. 6Go). IFN-{gamma} mRNA expression was evident in the null mice by day 5 and, as expected, preceded that of iNOS mRNA which became detectable by day 7. Both IRF-1 and Stat1{alpha} were highly expressed by days 5 and 7, coincident with IFN-{gamma} and iNOS expression. Of surprise was the detection of IRF-1 and Stat1{alpha} transcripts, albeit low levels, in heart tissue of 3-day-old mice, suggesting the possibility that IFN-{gamma}-independent as well as IFN-{gamma}-dependent signaling pathways may contribute to the overexpression of iNOS in the TGF-{beta}1 null mice.



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  		FIGURE 6. Sequential dysregulation of gene expression in TGF-{beta}1 null mice. Total RNA from heart tissue of TGF-{beta}1 null (KO, n = 9) and wild-type (WT) mice (n = 6) (age 3–7 days) was analyzed by RT-PCR for IRF-1, Stat1{alpha}, IFN-{gamma}, iNOS, and HPRT gene expression.

 
Modulation of transcription factor and iNOS mRNA expression and NO production by anti-IFN-{gamma} treatment

The up-regulation of elements of the IFN-{gamma} signaling pathway leading to iNOS transcription suggested that IFN-{gamma} is a potential target for interrupting the signaling cascade culminating in toxic NO production in the TGF-{beta}1 null mice. To confirm the IFN-{gamma} dependency of this signaling pathway, TGF-{beta}1 null mice were treated with 1 mg polyclonal anti-IFN-{gamma}, normal rat IgG, or PBS, and tissues and plasma were examined 5 days later (Fig. 7Go, A and B). By RT-PCR analysis of total heart RNA, elevations in transcription factors IRF-1 and Stat1{alpha} RNA observed in untreated or normal IgG treated null mice were reversed by treatment with anti-IFN-{gamma} (Fig. 7GoA). Both IRF-1 and Stat1{alpha} RNAs were reduced by 53–86%, and in most cases expression levels were comparable with that of untreated or IgG-treated wild-type littermates. This reduction in RNA levels was confirmed by gel shift analysis using the ISRE and GAS oligonucleotide probes. Binding of proteins from heart tissue of anti-IFN-{gamma}-treated null mice to the ISRE oligonucleotide probe was reduced compared with binding from untreated or IgG-treated null mice (data not shown). Concomitant with the reductions in transcription factor expression, reduced NO activity was reflected in the heart tissue by decreased expression of iNOS mRNA (67–73% reduction) in anti-IFN-{gamma}-treated null mice as compared with IgG-treated null mice (Fig. 7GoA). Furthermore, plasma levels of NO metabolites nitrite and nitrate were significantly reduced in anti-IFN-{gamma}-treated null mice as compared with IgG-treated null mice (p < 0.05) (Fig. 7GoB).



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  		FIGURE 7. Anti-IFN-{gamma} treatment reduces transcription factor expression and NO production and delays death in TGF-{beta}1 null mice (KO). A and B, TGF-{beta}1 null mice received one i.p. injection of 1 mg anti-IFN-{gamma} (n = 5) or normal rat IgG (n = 4) on day 8, and plasma and heart tissues were collected on day 13. IRF-1, Stat1{alpha}, iNOS, and HPRT mRNA expression in heart tissue from null and wild-type (WT) mice were measured by semiquantitative RT-PCR (A). Plasma nitrite plus nitrate levels were determined using the fluorescent microplate assay (B). C, TGF-{beta}1 null mice were treated with polyclonal anti ({alpha})-IFN-{gamma} (1 mg) or normal rat IgG (1 mg) on day 12 and every 2–5 days thereafter, and the time of death was noted. Data represent a summary of life span data of null mice treated with anti-IFN-{gamma} (n = 3) or normal IgG (n = 2).

 
Treatment with anti-IFN-{gamma} increases the life span of the TGF-{beta}1 null mice

To determine whether the anti-IFN-{gamma} inhibition of NO influenced the health and well-being of the null mice, TGF-{beta}1 null mice were treated with polyclonal anti-IFN-{gamma} or normal rat IgG (1 mg) beginning on day 12 when inflammatory symptoms begin to manifest and every 2–5 days thereafter. As a measure of the wasting syndrome that predicts lethality, body weight was recorded daily. Null mice treated with normal rat IgG experienced the typical wasting syndrome which ensued at ~12–14 days of age (data not shown and Ref. 15) and died by 15–19 days. In comparison, mice treated with anti-IFN-{gamma} maintained a constant weight or increased in weight, and life span was extended by nearly 2-fold to days 29–32 (p = 0.004 as compared with IgG-treated null mice) (Fig. 7GoC). However, the anti-IFN-{gamma} could not sustain the complete reduction in IFN-{gamma} and NO, and eventually the null mice succumbed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The overexpression of iNOS in TGF-{beta}1-deficient mice implicates TGF-{beta}1 as a pivotal link in the regulation of this signaling pathway. Induction of iNOS expression results from a signal transduction cascade that may involve several transcription factors including Stat1{alpha} and IRF-1, and this study assessed the in vivo role of TGF-{beta}1 in the signaling cascade leading to NO toxicity.

The suppressive activity of TGF-{beta} toward iNOS reportedly involves decreased stability and translation of iNOS mRNA and increased degradation of iNOS protein (8). In the absence of TGF-{beta}1, as in the TGF-{beta}1 null mouse, unrestrained expression of iNOS mRNA in several tissues including heart and lung is associated with tissue pathology and compromised function. Moreover, iNOS enzymatic activity is evident by elevation of NO metabolites nitrite and nitrate in the circulation. Whereas NO is classically defined as a protective agent for the host against bacterial or tumor invasion, overproduction of NO and other reactive nitrogen species including peroxynitrite can have autotoxic effects (3, 4, 5, 33, 34). These toxic effects can be manifested in the immune system as promotion of DNA damage; induction of cell injury and apoptosis; and enhancement of leukocyte adhesion, recruitment, and inflammatory mediator synthesis (35, 36).

Although the precipitating events that activate the signaling pathway leading to expression of iNOS have yet to be determined, trans-activating factors Stat1{alpha} and IRF-1 provide a link to overproduction of NO in target organs. Binding of IFN-{gamma} to the cell receptors initiates a Jak-Stat signaling pathway involving the tyrosine phosphorylation of Stat1{alpha} and its subsequent dimerization and mobilization into the nucleus, where it binds to the GAS site in the iNOS promoter, as well as promoters of other IFN-{gamma}-regulated genes, to activate transcription (22). Furthermore, Stat1{alpha} also trans-activates the IRF-1 gene (37) and the IRF-1 protein then binds to the ISRE site in the iNOS promoter to induce transcription (20). Macrophages isolated from mice deficient in Stat1{alpha} or IRF-1 make little or no NO or iNOS mRNA in response to LPS and IFN-{gamma} (23, 25, 26) and, importantly, susceptibility to autoimmune diseases is reduced in these mice (38, 39). Such data are in accord with our findings, demonstrating a linkage among overabundant Stat1{alpha} and IRF-1 activation, excess NO, and autoimmune lesions in the absence of regulatory control by TGF-{beta}1.

Beyond their roles as IFN-{gamma}-responsive proteins, Stat1{alpha} and IRF-1 can also be induced/activated by type I IFNs (IFN-{alpha}{beta}) and by agents that activate NF-{kappa}B (40, 41, 42). The appearance of low levels of Stat1{alpha} and IRF-1 mRNA in the TGF-{beta}1 null mice before detection of IFN-{gamma} mRNA may reflect the sensitivity of the assay and/or suggest the involvement of additional IFN-{gamma}-independent pathways. Whether the induction of this alternative pathway(s) occurs via environmental or inflammatory stimuli is unknown but may involve IFN-{alpha}{beta} in the regulation of iNOS via induction of transcription factors Stat1{alpha} as well as IRF-1 (40, 42). In this regard, increased levels of IFN-{beta} transcripts are evident by day 5 in TGF-{beta}1 null mice but absent in wild-type littermates and in vivo treatment with Ab directed against IFN-{alpha} and IFN-{beta} resulted in reduction in circulating levels of NO (N. McCartney-Francis, unpublished observations), suggesting a participatory role for type I IFNs in the overexpression of NO in these mice.

Based on the anti-IFN-{gamma} treatment studies, it is clear that IFN-{gamma} plays a significant role in the activation of the signaling pathway leading to iNOS expression. Not only did the anti-IFN-{gamma} alter Stat1{alpha} and IRF-1 transcription factor expression, but also reduced iNOS expression in the heart and returned circulating NO to baseline levels. Importantly, by reducing toxic levels of NO, the anti-IFN-{gamma} treatment extended the life span of the TGF-{beta}1 null mice. Although we had anticipated a more prolonged protective effect of the Ab based on the significant annulment of NO production, treatment with the NO inhibitor NG-monomethyl-L-arginine (9) or N-imimoethyl-L-lysine (N. McCartney-Francis, unpublished observations) which also reduced NO levels did not further prolong survival. The eventual death of these mice may be a reflection of the limitations of the i.p. administration of the Ab, contribution of alternative signaling pathways, or multiple contributing lethal factors in the null mice. Even attempts to rescue the mice with exogenous TGF-{beta}1, whether administered orally, i.p., i.v., or by gene transfer, have failed, suggesting that there is an inherent immune defect in the TGF-{beta}1 null mouse (43).

The increased iNOS expression detected within the heart tissue of TGF-{beta}1 null mice correlated with the density of infiltrated Mac-2+ cells, similar to that seen in experimental models of autoimmune myocarditis (44). In addition to inflammatory cells in the heart, cardiomyocytes have been shown to express iNOS (30, 31), consistent with the basal expression we observed in both TGF-{beta}1 null and wild-type myocytes. Although endothelial NO synthase is the predominantly expressed isoform in the heart, basal expression of iNOS by myocytes may provide for endogenous production of NO important for maintaining myocardial function such as regulation of vascular tone and immune defense (45). Whereas submicromolar NO concentrations improve myocardial contraction, excess intracellular expression of iNOS and NO inhibits myocardial contraction (46), and this suppressive effect of NO can be antagonized by TGF-{beta} (30). Thus, in the absence of TGF-{beta}1, deleterious production of NO by both myocytes and recruited inflammatory cells may contribute to cardiovascular failure. Prolonged activation of transcription factors Stat1{alpha} and IRF-1 coincident with overexpression of IFN-{gamma} may lead to iNOS-mediated heart failure and tissue damage. These studies confirm the role of TGF-{beta}1 in the regulation of iNOS gene expression and provide an in vivo model with which to study the dysregulation of signaling pathways and novel strategies for therapeutic intervention.


   Acknowledgments
 
We thank Dr. Robert Seder, Dr. Gerald Feldman, and Dr. Stefanie Vogel for their generous gifts of Abs; Dr. Jan Orenstein for the electron micrographs; and Diane Mizel and George McGrady for excellent technical assistance. We also thank Dr. Larry Wahl and Dr. Nancy Vázquez-Maldonado for reviewing the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Nancy L. McCartney-Francis, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 30, Room 326, 30 Convent Drive, Bethesda, MD; 20892-4352. E-mail address: nfrancis{at}dir.nidcr.nih.gov Back

2 Abbreviations used in this paper: iNOS, inducible NO synthase; IRF, IFN-regulatory factor; ISRE, IFN-stimulated response element; HPRT, hypoxanthine phosphoribosyltransferase; Gas, IFN-{gamma} activated site. Back

Received for publication July 18, 2002. Accepted for publication September 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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