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The Journal of Immunology, 2003, 170: 5739-5747.
Copyright © 2003 by The American Association of Immunologists

Regulation of Lipopolysaccharide Sensitivity by IFN Regulatory Factor-21

Natalia Cuesta2, Cindy A. Salkowski2, Karen E. Thomas and Stefanie N. Vogel3

Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN regulatory factors (IRFs) are a family of transcription factors and include several members that regulate expression of pro- and anti-inflammatory genes. Mice with a targeted mutation in IRF-2 (IRF-2-/-) were studied after injection of LPS to evaluate the importance of IRF-2 in the regulation of endotoxicity. IRF-2-/- mice were highly refractory to LPS-induced lethality. Although hepatic TNF-{alpha} mRNA and circulating TNF-{alpha} were significantly elevated in LPS-challenged IRF-2-/- mice, levels of IL-1, IL-12, and IFN-{gamma} mRNA and protein, as well as IL-6 protein, were significantly lower than levels seen in LPS-challenged IRF-2+/+ mice. IRF-2-/- mice were also more refractory to TNF-{alpha} challenge than were control mice, which was consistent with their diminished sensitivity to LPS, yet no significant difference in the mRNA expression of TNFRs was observed. IL-12R{beta}2 mRNA levels from LPS-challenged IRF-2-/- mice were significantly different after 1, 6, and 8 h, suggesting that both diminished IL-12 and altered IL-12R expression contribute to the paucity of IFN-{gamma} produced. IRF-2 knockout mice also failed to sustain LPS-inducible levels of IRF-1 and IFN consensus sequence binding protein mRNA expression, two transacting factors required for IL-12 transcription, perhaps as a result of diminished IL-1{beta}, IL-6, and IFN-{gamma} levels. Liver sections from IRF-2+/+ and IRF-2-/- mice were analyzed 6 h after a typically lethal injection of LPS. IRF-2-/- mice exhibited greater numbers of apoptotic Kupffer cells than did wild-type mice, suggesting a novel anti-apoptotic role for IRF-2. Collectively, these findings reveal a critical role for IRF-2 in endotoxicity, and point to a previously unappreciated role for IRF-2 in the regulation of apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferons constitute a family of multifunctional cytokines that have been strongly implicated as primary mediators of the host defense against viral infection (1). The response to IFNs results in the transcriptional activation of target genes that are regulated by their interaction with DNA-binding proteins generated through the Janus kinase-STAT signaling pathway and/or the IFN regulatory factor (IRF) 4. The best characterized members of the IRF family of transcription factors, IRF-1 and IRF-2, were originally found to activate and repress transcription of the IFN-{beta} gene, respectively, by competing for the same DNA element, now termed the IRF-E (2, 3). IRFs may also interact to form protein complexes with other IRF family members, as well as other transacting factors. For example, IFN consensus sequence binding protein (ICSBP) interacts with both IRF-1 and IRF-2 (4, 5, 6). IRF-1 also interacts with other DNA-binding proteins, such as NF-{kappa}B and p53 (7, 8, 9, 10), and IRF-2 has been reported to bind to NF-{kappa}B in vitro (11). Thus, these transcription factors have the potential to mediate diverse functions in response to extracellular stimuli through their interactions with various promoter elements.

In addition to their opposing actions on IFN-{beta} gene expression, IRF-1 and IRF-2 have been shown to exert opposing effects on cyclooxygenase 2 gene expression (12). However, within the last few years, this relatively simplistic model of positive/negative regulation has been modified to account for the findings that IRF-2 has more recently been found to function as a transcriptional activator of the VCAM-1 (13) and histone H4 genes (14). Conversely, IRF-1 has been reported to act as a negative regulator of c-myb at the level of transcription (15).

Studies from mice with targeted mutations have revealed some other relevant IRF functions (16, 17). NK and Th1 cell development is impaired in IRF-1 knockout mice (18, 19, 20), and IL-12 is dysregulated in macrophages derived from both IRF-1 and IRF-2 knockout mice (21). Although induction of the inducible nitric oxide synthase gene is IRF-1-dependent (22), IRF-2 does not seem to have any effect in the transcriptional activation of this gene in macrophages, but it clearly affects release of NO (23, 24). IRF-1-/- mice are more susceptible to infection with Mycobacterium bovis (25), Brucella abortus (26), Leishmania major (18), and Toxoplasma gondii (27) than are wild-type mice, and IRF-2-/- mice are more susceptible to Listeria monocytogenes (24) and L. major (20). IRF-1 has also been implicated in the regulation of apoptosis (28, 29, 30); for example, macrophages from IRF-1 knockout mice are more resistant to LPS- and IFN-{gamma}-induced apoptosis (31, 32). In addition, IRF-1 regulates DNA damage-induced cell cycle arrest (8).

Some of these studies have focused on the cells in the liver, given the important immunological role that this organ plays in the first line of host defense. Many bacterial agents enter the liver via the portal vein (33), and most bacteria are entrapped by Kupffer cells and hepatocytes (34). In response to these stimuli, hepatocytes produce acute phase proteins and proteins of the complement cascade, whereas Kupffer cells produce many cytokines including IL-12, IL-10, and TNF-{alpha} (33, 35). IL-12 and other ILs, in turn, activate NK and Th1 cells to produce IFN-{gamma} (36). Both TNF-{alpha} and IFN-{gamma} are proinflammatory cytokines involved in LPS-induced toxicity (37, 38, 39, 40). Reporting that IRF-1 plays an important role in the pathogenesis of disease models mediated by TNF and IFN-{gamma} is evidenced by experiments in IRF-1-/- mice. IRF-1-/- mice are highly refractory to a dose of LPS that is lethal for wild-type mice (41), and the production of TNF-{alpha} and IFN-{gamma} is strikingly impaired, which is secondary to a down-regulation of gene expression in the liver and spleen or in the macrophages of IRF-1-/- mice (21, 41, 42).

In light of the close relationship that exists between IRF-1 and IRF-2 in many physiological responses, we sought to extend our original findings on the role of IRF-1 in LPS-induced toxicity to investigate the role of IRF-2 in a similar model of endotoxemia. Therefore, IRF-2-/- mice were studied after injection of LPS or TNF-{alpha} with the purpose of exploring the importance of IRF-2 in the pathogenesis of LPS-induced mortality. We have found that IRF-2-/- mice are more resistant to both LPS and recombinant TNF-{alpha} challenge than areIRF-2+/+ mice, and IRF-2-/- mice exhibit a significant inhibition of the expression of IL-12, IL-12R, and IFN-{gamma}, as well as IL-1{beta} and IL-6, while TNF-{alpha} levels are increased significantly. In contrast to IRF-1-/- macrophages that are resistant to LPS- and IFN-{gamma}-induced apoptosis, the number of apoptotic Kupffer cells is markedly increased in the livers of IRF-2-/- mice, both basally and in response to LPS or TNF-{alpha}. Thus, the loss of Kupffer cell function through apoptosis could result in decreased production of specific proinflammatory cytokines and attenuation of LPS-induced toxicity. Taken collectively, our data demonstrate the importance of IRF-2 as yet another critical transacting factor that regulates in vivo responses to LPS and have revealed an unexpected role for this DNA-binding protein in the regulation of apoptosis.


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

Protein-free (<0.008% protein) LPS was prepared from Escherichia coli K235 by phenol-water extraction (43). Recombinant human TNF-{alpha} was a kind gift of the Cetus Corporation (Emeryville, CA).

Mice

IRF-2-/- mice were generated by targeted disruption as previously described (44). Breeding pairs of IRF-2-/- and IRF-2+/- mice, backcrossed to C57BL/6 mice for three to five generations at the time we received them, were the kind gift of Dr. T. Mak (Amgen Institute, Toronto, Ontario, Canada). Colonies of background-matched IRF-2-/- and IRF-2+/+ mice used in this study have been since bred in our facility for over 5 years. Occasionally, when sufficient numbers of IRF-2+/+ mice were not available, C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were used as IRF-2+/+ controls. All IRF-2 mice bred in our colony were genotyped as previously described (23, 45). Mice were housed in cages with filter tops in a laminar flow hood and fed food and acid water ad libitum. For in vivo studies, mice were injected i.p. with a lethal dose of LPS (35 mg/kg) or i.v. with recombinant human TNF-{alpha} (2 mg/kg). All experiments were conducted with institutional (Institutional Animal Care and Use Committee) approval.

Serum assays

Mice were bled by cardiac puncture at the indicated times after injection, and serum samples were stored at -70°C. IL-12 p70 and IFN-{gamma} were detected using the Ab pairs and standards provided in the OPT-EIA ELISA kit (BD PharMingen, San Diego, CA) according to the manufacturer’s instructions. Total IL-12 (p40 monomer, p40 dimer, and p70) was detected by ELISA as previously described (42) using Ab pairs obtained from Genzyme (Cambridge, MA). To calculate IL-12 p40 protein levels, the values in the IL-12 p70 ELISA were subtracted from those obtained in the total IL-12 ELISA. TNF-{alpha}, IL-1{beta}, and IL-6 levels were measured using a commercial kit (R&D Systems, Minneapolis, MN), according to the directions provided by the manufacturer.

Quantification of mRNA by RT-PCR and Southern blot analysis

At the indicated time after LPS injection, liver samples from individual mice were isolated and frozen at -70°C. Total RNA was isolated and the relative quantities of mRNA for hypoxanthine-guanine phosphoribosyltransferase (HPRT), IL-12 p35, IL-12 p40, IL-12R{beta}1, IL-12R{beta}2, IFN-{gamma}, TNF-{alpha}, IRF-1, ICSBP, TNFR1, and TNFR2 were determined by RT-PCR as previously described (46). The primers and probe combinations for TNFR1 were 5'-CAGGGAGTGTGAAAAGGGCAC (sense), 5'-GTAGCGTTGGAACTGGTTCTC (antisense), 5'-ATCTCTCCTTGCCAAGCTGA (probe) and for TNFR2 were 5'-GCAAGCACAGATGCAGTCTG (sense), 5'-GGTCAGAGCTGCTACAGACG (antisense), 5'-GCCATCCCAAGGACACTCTA (probe). All other probe and primer combinations have been published (5, 42, 47, 48). Amplified products were electrophoresed and transferred to Hybond N+ membranes (Amersham, Arlington Heights, IL) in 10x SSC by standard Southern blotting techniques. DNA was cross-linked by exposure to UV light, baked onto the nylon membrane, and hybridized with an internal oligonucleotide probe. Labeling of the probe and subsequent detection of bound probe was conducted using an ECL system (Amersham).

Real-time PCR for mRNA quantification

For certain experiments, mRNA levels were measured using real-time PCR. The PCR was performed in a Sequence Detector System (ABI Prism 7900 Sequence Detection System and software; Applied Biosystems, Foster City, CA). Amplification was performed in a final volume of 25 µl, containing 30 ng cDNA from the reversed transcribed reaction, primer mixture (0.3 µM each of sense and antisense primers), and 12.5 µl 2x SYBR Green Master Mix (Applied Biosystems). The oligonucleotide primers were designed using Primer Express 1.5 software (Applied Biosystems). In addition, the sense and antisense sequences of each pair of primers were designed to overlap adjacent exon boundaries to exclude detection of genomic DNA.

The standard amplification program included 40 cycles of two steps each, comprised of heating to 95°C and heating to 60°C. Fluorescent product was detected at the last step of each cycle. The final mRNA levels of the genes studied were normalized according to the HPRT concentration of each sample. mRNA levels were reported as fold changes over background levels detected in control tissues.

The primers used were: IL-10 sense (5'-ATTTGAATTCCCTGGGTGAGAAG-3'); IL-10 antisense (5'-CACAGGGGAGAAATCGATGACA-3'); HPRT sense (5'-GCTGACCTGCTGGATTACATTAA-3'); HPRT antisense (5'-TGATCATTACAGTAGCTCTTCAGTCTGA-3'); IL-1{beta} sense (5'-ACAGAATATCAACCAACAAGTGATATTCTC-3'); IL-1{beta} antisense (5'-GATTCTTTCCTTTGAGGCCCA-3'); IL-6 sense (5'-TCAGGAAATTTGCCTATTGAAAATTT-3'); IL-6 antisense (5'-GCTTTGTCTTTCTTGTTATCTTTTAAGTTGT-3').

Immunohistochemistry

In vivo apoptosis was assessed using the TUNEL assay (49, 50). After the indicated treatments, mouse livers were resected and fixed overnight in 10% buffered formalin (Sigma-Aldrich, St. Louis, MO) and embedded in paraffin. Five micrometer thick tissue sections were adhered to Superfrost Plus slides (VWR, West Chester, PA). Sections were deparaffinized by heating at 60°C for 20 min and clearing in xylene for 30 min. The slides were then rehydrated through a graded series of ethanol concentrations (96, 70, and 50% ethanol), ending with a rinse in distilled water. To inactivate endogenous peroxidase, sections were treated with 3% H2O2 for 30 min at room temperature. After washing thoroughly in water, the slides were incubated in 10 mM Tris-HCl, pH 7.4 for 5 min and digested with 10 µg/ml proteinase K for 10 min at 37°C, washed in 10 mM Tris-HCl, then incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min at 4°C. After three washes in PBS, slides were incubated for 1 h at 37°C in reaction mixture (In Situ Cell Death Detection, POD kit; Roche Biomedical Laboratories, Mannheim, Germany) as instructed by the manufacturer. They were subsequently rinsed in PBS and incubated with converted POD (Roche Biomedical Laboratories) for 30 min at room temperature. The slides were washed in PBS and stained with the chromogen 3,3' diaminobenzidine tetrachloride (Vector Laboratories, Burlingame, CA).

The TUNEL assay was followed by immunostaining with a rat monoclonal anti-F4/80 Ab to identify Kupffer cells. For this purpose, TUNEL-stained sections were incubated with normal rabbit serum (5% in PBS; Vector Laboratories) for 30 min at room temperature and then with a primary rat anti-F4/80 Ab at 4°C overnight (1:200 dilution; Accurate Chemical and Scientific, Westbury, NY). Normal rabbit serum was used instead of the primary Ab as a negative control. After washing in PBS, biotinylated rabbit anti-rat Abs were applied for 30 min at room temperature (1:200 dilution; Vector Laboratories), followed by avidin-biotin alkaline phosphatase complexes for 30 min (1:1:100 dilution; Vector Laboratories). Red alkaline phosphatase substrate was used as the chromogen (Vector Laboratories). The slides were counterstained with Harris hematoxylin (VWR), dehydrated, cleared in xylene, and mounted in Permount (Fisher Scientific, Fair Lawn, NJ). Double staining was considered positive when the cells displayed a brown nuclear staining in the context of a surrounding red immunoreactive pattern.

Hepatic cells were counted under x1000 magnification. The areas scored were always selected randomly, and counts were conducted in a blinded fashion, revealing the code at the end of the study. The "apoptotic index" was determined by dividing the total number of Kupffer cells (F4/80-positive cells) that were exhibiting apoptosis (TUNEL-positive) by the total number of F4/80-positive cells in the fields examined (i.e., double-positive cells/total F4/80-positive cells) x 100.

Statistics

Results were analyzed using Student’s t test for comparisons between two groups or by one-way ANOVA, with Tukey’s post-hoc tests for comparisons between multiple treatment groups. Values for p <0.05 were accepted as the level of significance. All experiments were repeated at least twice with similar results. For immunohistochemical data, a minimum of 350 cells were enumerated per slide for analysis, with four slides per treatment derived from four separate mice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enhanced resistance of IRF-2-/- mice to LPS-induced lethality

IRF-2+/+ and IRF-2-/- mice were injected with 35 mg/kg LPS to assess whether loss of the transcription factor IRF-2 altered susceptibility to LPS. As shown in Fig. 1, IRF-2-/- mice were less susceptible to the lethal effects of LPS. By 48 h after LPS challenge, the mortality rate was 80% in IRF-2+/+ mice and only 10% in IRF-2-/- mice. Despite their enhanced resistance, IRF-2-/- mice developed clinical signs of endotoxemia, including lethargy, diarrhea, piloerection, cachexia, and conjunctivitis; these symptoms, however, were milder in IRF-2-/- than in IRF-2+/+ mice.



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  		FIGURE 1. IRF-2-/- mice exhibit enhanced resistance to LPS. IRF-2+/+ and IRF-2-/- mice were injected i.p. with 35 mg/kg LPS (10 mice/strain) and monitored for 72 h. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-.

 
IL-12 and IFN-{gamma} production are impaired in LPS-challenged IRF-2-/- mice

Numerous studies have demonstrated that lethality following LPS challenge is associated with overproduction of TNF-{alpha}, for which production is, in part, amplified by IL-12-induced IFN-{gamma}. In murine models of endotoxicity, loss of any of these principal mediators typically results in enhanced resistance to LPS-induced lethality (37, 38, 39, 51, 52). Because IRF-2-/- mice were more resistant to the lethal effects of LPS (Fig. 1), we next examined the in vivo regulation of IL-12 p35, IL-12 p40, IFN-{gamma}, and TNF-{alpha} mRNA expression in the livers of LPS-challenged IRF-2+/+ and IRF-2-/- mice (Fig. 2). Levels of circulating IL-12 p40, bioactive IL-12 p70, IFN-{gamma}, and TNF-{alpha} protein also were measured (Fig. 3). As shown in Fig. 2, IRF-2-/- mice had significantly heightened (~1.5- to 10-fold) levels of basal and LPS-induced IL-12 p40 mRNA expression in the liver than did IRF-2+/+ control mice. Whereas untreated IRF-2-/- mice had an ~2.5-fold higher level of circulating IL-12 p40, levels of circulating IL-12 p40 were not significantly different in the sera of IRF-2+/+ and IRF-2-/- mice after LPS challenge (Fig. 3). In contrast to IL-12 p40 mRNA expression, significantly lower levels of IL-12 p35 mRNA expression were observed in the livers of IRF-2-/- mice at 6 and 8 h (50% and 90% reduction, respectively) after LPS challenge (Fig. 2). These reductions in the level of IL-12 p35 mRNA were consistent with significantly lower levels of bioactive IL-12 p70 production in IRF-2-/- mice (Fig. 3). Specifically, IRF-2-/- mice produced 60–75% less circulating IL-12 p70 at 3, 6, and 8 h after LPS challenge than did IRF-2+/+ mice. Also consistent with the mitigated IL-12 p70 response was a profound reduction in hepatic IFN-{gamma} mRNA and serum protein levels observed in LPS-challenged IRF-2-/- mice (Figs. 2 and 3). IL-18 mRNA was not modulated by LPS, nor was it differentially expressed in IRF-2+/+ and IRF-2-/- mice (data not shown). Interestingly, IRF-2-/- mice not only had heightened levels of basal TNF-{alpha} mRNA expression (~2-fold increase), but also an ~4-fold increase in peak TNF-{alpha} mRNA expression 1 h after LPS challenge (Fig. 2). These increased levels of hepatic TNF-{alpha} mRNA expression were paralleled by significantly higher levels of circulating TNF-{alpha} protein in IRF-2-/- mice at 1 and 3 h after LPS challenge (Fig. 3).



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  		FIGURE 2. Regulation of IL-12 p40, IL-12 p35, IFN-{gamma}, and TNF-{alpha} mRNA expression in the livers of LPS-challenged IRF-2+/+ and IRF-2-/- mice. IRF-2+/+ and IRF-2-/- mice were injected i.p. with 35 mg/kg LPS. Data are expressed as the mean fold increase ± SEM from eight mice at each time point. Data were individually normalized to the housekeeping gene HPRT. Means are expressed relative to untreated IRF-2+/+ controls (time zero), which are arbitrarily assigned a value of 1. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-. Asterisk (*) indicates a significant difference (p < 0.05) between IRF-2+/+ and IRF-2-/- mice.

 


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  		FIGURE 3. Induction of IL-12 p40, IL-12 p35, IFN-{gamma}, and TNF-{alpha} protein levels in sera of LPS-challenged IRF-2+/+ and IRF-2-/- mice. Data are expressed as the mean ± SEM from four to eight mice per time point. Asterisk (*) indicates a significant difference (p < 0.05) between IRF-2+/+ and IRF-2-/- mice. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-.

 
Induction of IL-10 mRNA is not dysregulated in IRF-2-/- mice

Because IL-10 is a potent anti-inflammatory cytokine and an inhibitor of TNF-{alpha}, IL-12, and IFN-{gamma} production (53, 54, 55, 56), we also measured IL-10 gene expression in IRF-2-/- mice in response to LPS. Fig. 4 illustrates that there were no significant differences in IL-10 mRNA levels between IRF-2+/+ and IRF-2-/- mice at early time points. At 8 and 12 h after LPS i.p., IRF-2-/- mice produced slightly elevated levels of IL-10 mRNA, but this difference failed to achieve statistical significance (p = 0.059 and 0.054, respectively). Thus, IRF-2-dependent regulation of IL-10 gene expression cannot account for decreased early expression of IL-12 or IFN-{gamma} mRNA.



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  		FIGURE 4. Regulation of IL-10 mRNA expression in livers of LPS-challenged IRF-2+/+ vs IRF-2-/- mice. Data were obtained as described in Fig. 2. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-.

 
TNFR1 and TNFR2 hepatic mRNA expression is not dysregulated in IRF-2-/- mice

The heightened levels of both TNF-{alpha} mRNA and circulating TNF-{alpha} in IRF-2-/- mice were unexpected because IRF-2-/- mice were significantly more resistant to the lethal effects of LPS. This suggested the possibility that some component of the TNF signaling pathway might be disrupted in IRF-2-/- mice. Thus, hepatic TNFR1 and TNFR2 mRNA expression were examined to ascertain whether IRF-2-/- mice exhibited altered TNFR mRNA levels in vivo. By 1 h after LPS challenge, TNFR2 mRNA expression in the liver had increased ~4-fold in both IRF-2+/+ and IRF-2-/- mice and remained heightened (6- to 12-fold increase) for 12 h (Fig. 5). In contrast, no increase in TNFR1 mRNA expression in IRF-2+/+ and IRF-2-/- mice was observed until 12 h after LPS challenge (~2-fold increase). Importantly, no significant differences in either TNFR1 or TNFR2 mRNA levels were observed between IRF-2+/+ and IRF-2-/- mice.



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  		FIGURE 5. Regulation of TNFR1 and TNFR2 mRNA expression in livers of LPS-challenged IRF-2+/+ and IRF-2-/- mice. Data were obtained as described in Fig. 2. {square} and {blacksquare}, TNFR1 mRNA; {circ} and •, TNFR2 mRNA; open symbols, IRF-2+/+; closed symbols IRF-2-/-.

 
Enhanced resistance of IRF-2-/- mice to TNF-{alpha}-induced mortality

The refractory response of IRF-2-/- mice to LPS was surprising, given the elevated levels of TNF-{alpha} exhibited by IRF-2-/- mice, coupled with the apparent normality of their steady-state TNFR mRNA expression. Therefore, we next hypothesized that IRF-2-/- mice might also exhibit diminished sensitivity to TNF-{alpha}. To test this hypothesis, wild-type and IRF-2 knockout mice were challenged with recombinant TNF-{alpha}, and mortality was assessed over 72 h. Fig. 6 illustrates that IRF-2-/- mice were significantly more refractory to direct challenge with TNF-{alpha} than were the wild-type controls. Injection of TNF-{alpha} failed to elicit detectable IL-12 p70 or IFN-{gamma} in sera of IRF-2+/+ or IRF-2-/- mice in contrast to LPS-challenged mice (Table I).



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  		FIGURE 6. IRF-2-/- mice exhibit enhanced resistance to recombinant TNF-{alpha}. IRF-2+/+ and IRF-2-/- mice were injected i.v. with 2 mg/kg recombinant TNF-{alpha} (10 mice per strain) and were monitored for 72 h. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-.

 

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  		Table I. Recombinant TNF-{alpha} fails to induce IL-12 p70 and IFN-{gamma} in IRF-2+/+ and IRF-2-/- mice in vivoa

 
Regulation of IL-12R mRNA expression

Several observations indicated that the loss of LPS-induced IFN-{gamma} in IRF-2-/- mice may not be due entirely to reduced IL-12 p70 production. First, the initial induction of IL-12 p35 mRNA at 1 and 3 h following LPS challenge was similar in both IRF-2+/+ and IRF-2-/- mice, yet IFN-{gamma} mRNA levels in IRF-2-/-, but not IRF-2+/+ mice, remained uninduced at 3 h (Fig. 2). Moreover, while peak IL-12 p70 levels in the serum of IRF-2-/- mice were reduced by ~60%, peak IFN-{gamma} protein levels were reduced by >=96%. In a previous study we demonstrated that IL-12R mRNA expression was impaired in IRF-1-deficient mice (42). Because IRF-2 has been shown to interact with the same promoter element as IRF-1 (57), we postulated that IRF-2 may also regulate IL-12R expression. No significant differences in IL-12R{beta}1 subunit mRNA expression between IRF-2+/+ and IRF-2-/- mice were observed after LPS treatment; however, IL-12R{beta}2 subunit mRNA is down-regulated in IRF-2-/- mice 6 and 8 h after LPS challenge (Fig. 7). Of note, IL-12R{beta}2 mRNA levels are significantly higher in IRF-2-/- mice 1 h after LPS injection, when the levels of IL-12 p70 in serum are equivalent in both IRF-2+/+ and IRF-2-/- mice.



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  		FIGURE 7. Regulation of IL-12R{beta}1 and IL-12R{beta}2 mRNA expression in livers of LPS-challenged IRF-2+/+ vs IRF-2-/- mice. Data were obtained as described in Fig. 2. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-.

 
LPS-induced IRF-1 and ICSBP mRNA levels are not sustained in IRF-2-/- mice

We had previously demonstrated that IRF-1 is critical for regulation of both IL-12-dependent and -independent pathways of IFN-{gamma} production (42). Therefore, we decided to examine in vivo the regulation of IRF-1 mRNA following LPS challenge in IRF-2-/- mice. ICSBP mRNA expression was also analyzed, as it has been shown to interact with both IRF-1 and IRF-2 and to be necessary for IL-12 production (4, 5, 58). Both IRF-1 and ICSBP mRNA expression are down-regulated in IRF-2-/- mice 6 h after LPS injection, and these levels remain lowered 12 h after treatment (Fig. 8).



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  		FIGURE 8. Regulation of IRF-1 and ICSBP mRNA expression in livers of LPS-challenged IRF-2+/+ and IRF-2-/- mice. Data were obtained as described in Fig. 2. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-.

 
IL-6 and IL-1{beta} expression are dysregulated in IRF-2-/- mice after LPS challenge

Both IL-1 and IL-6 have long been implicated in endotoxicity. Moreover, both have been shown to induce IRF-1 gene expression (59, 60, 61). Therefore, we sought to investigate whether impaired production of these two cytokines could be responsible for the lowered levels of IRF-1 mRNA expression in LPS-challenged IRF-2-/- mice. In response to LPS, IRF-2-/- mice exhibited a significant decrease in both circulating IL-1{beta} and hepatic IL-1{beta} mRNA (Fig. 9, A and B).Although IL-6 mRNA gene expression was not dysregulated in IRF-2-/- mice, IL-6 protein levels were substantially reduced in IRF-2-/- mice after LPS treatment when compared with IRF-2+/+ mice (Fig. 9, C and D). The latter observation suggests a role for IRF-2 in the posttranscriptional regulation of IL-6.



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  		FIGURE 9. Regulation of IL-1{beta} and IL-6 hepatic mRNA and serum protein levels in LPS-challenged IRF-2+/+ and IRF-2-/- mice. IL-1{beta} mRNA (A), IL-1{beta} protein (B), IL-6 mRNA (C), and protein (D) were measured as described in Figs. 2 and 3. {square}, IRF-2+/+; {blacksquare}, IRF-2-/-.

 
IRF-2+/+ and IRF-2-/- mice exhibit differential sensitivity to LPS- and TNF-{alpha}-induced Kupffer cell apoptosis

Previous studies proposed that LPS-induced lethality is secondary to TNF-{alpha}-induced endothelial apoptosis that is detectable in various organs within 6 h of LPS administration (62). Macrophages derived from IRF-1-/- mice were found to resist LPS-induced apoptosis (45). Therefore, we next analyzed wild-type and IRF-2 knockout mice that had been injected with an LD100 (in wild-type mice) of LPS or TNF-{alpha} for hepatic sensitivity to apoptosis. A dual staining technique was used to identify cells that stained for the macrophage-specific marker, which used F4/80 to identify Kupffer cells and the TUNEL stain to identify cells undergoing apoptosis. Although total numbers of F4/80-positive cells (i.e., Kupffer cells) were not significantly different in wild-type vs IRF-2-/- liver sections (data not shown), IRF-2-/- mice exhibited significantly greater numbers of apoptotic Kupffer cells than did control mice, without or with injection of LPS (Fig. 10, A–F) or TNF-{alpha} (Fig. 10F). In contrast to the previous reports of increased endothelial apoptosis (62), this was not observed in livers of control or knockout mice.



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  		FIGURE 10. Increased numbers of apoptotic macrophages in IRF-2-/- mice challenged with LPS. Photomicrographs showing the double immunostaining TUNEL (brown)-F4/80 (red). A, Liver section of a C57BL/6J mouse 6 h after injection of saline, showing a paucity of apoptotic cells. B, Liver section of an IRF-2-/- mouse after saline injection. Note the presence of some positive cells for both TUNEL and F4/80 immunostaining. C, Section of a C57BL/6J mouse liver, 6 h after injection of LPS (35 mg/kg). The number of apoptotic cells is significantly higher than after saline treatment. D, Liver section of an IRF-2-/- mouse 6 h after LPS treatment. The number of apoptotic cells is considerably higher than in any other group. Note that some Kupffer cells have migrated to the hepatic vessels. E, Detail of a liver section showing the colocalization of TUNEL and F4/80 staining (arrows) in some cells. Arrowhead points to a TUNEL-positive, F4/80-negative cell in the hepatic sinusoid, and asterisks (*) mark the presence of a TUNEL-negative, F4/80-positive Kupffer cell. F, Graph showing the apoptotic index of livers from C57BL/6J and IRF-2-/- mice after the three different treatments. Values represent means ± SD, * = p < 0.05. Both LPS- and TNF-injected mice had significantly more apoptotic cells than saline-pretreated mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results from this study indicate that loss of the transcription factor IRF-2 renders mice more resistant to both LPS and TNF-{alpha} challenge (Figs. 1 and 6). Transfection experiments have shown that IRF-1 normally functions as an activator of transcription, whereas IRF-2 antagonizes the function of IRF-1 by working as a repressor (6). Any variation in the intracellular concentration of these transcription factors alters the ratio of IRF-1 to IRF-2 and may produce a dramatic change in the transcription pattern of cells (63). Our data show that LPS-induced IL-12, IL-12R, and IFN-{gamma} expression are all dysregulated in IRF-2-/- mice (Figs. 2, 3, and 6), indicating that IRF-2 plays a crucial role in endotoxicity by contributing to the regulation of both IL-12 and IL-12R expression, and subsequently, IFN-{gamma} production. Previous studies have shown that mice given Abs to IFN-{gamma} (39) are also refractory to LPS. Although levels of circulating IL-12 p40 were not significantly different in IRF-2+/+ and IRF-2-/- mice after LPS challenge, decreased IL-12 p70 levels in IRF-2-/- mice may be explained, in part, by significantly lower IL-12 p35 mRNA expression because IL-12 p35 is limiting in the formation of the bioactive IL-12 p40/p35 heterodimer (64). In addition, because IL-12 p40 homodimers have been previously shown to be competitive inhibitors of IL-12 p70 (65, 66), the relatively higher proportion of the inhibitory form to the bioactive form of IL-12 could also reasonably account for the severely curtailed IFN-{gamma} response observed in LPS-stimulated IRF-2 knockout mice.

One likely explanation for the observed paucity of IL-12 p35 mRNA expression is that IRF-2 knockout mice exhibit substantially reduced levels of IRF-1 and ICSBP mRNA in response to LPS. As IRF-1, IRF-2, and ICSBP form complexes to regulate transcription of IFN-inducible genes (4, 5, 6, 67), the loss of IRF-2 may not only affect the absolute levels of these transcription factors, but also their ability to interact with each other and other DNA-binding proteins. In this respect, Barber et al. (5) demonstrated that the relative expression of IRF-1 to IRF-2 mRNA is dramatically less in macrophages from the LPS-hyporesponsive (Toll-like receptor 4-defective) C3H/HeJ mouse strain and in macrophages rendered tolerant to endotoxin, when compared with the levels in normally LPS-responsive macrophages. Both IL-12 p40 and p35 promoters contain consensus sequences for a number of DNA-binding proteins, including NF-{kappa}B consensus sequences, NF-IL-6 binding sites, and others (68, 69, 70), and quite recently Maruyama et al. (71) demonstrated an IRF-1 binding site in the IL-12 p40 promoter. Cooperation and interaction between NF-{kappa}B and IRFs has been demonstrated for several genes (7, 11) and may also be involved in the regulation of murine IL-12.

We had previously shown in vitro that IL-12 p70 secretion is impaired in IRF-2-/- macrophages (21). In addition, we found that IRF-1, but not IRF-2, is involved in the induction of IL-12 p35 mRNA by LPS. The requirement for IRF-1 in IL-12 p35 gene expression has since been confirmed by promoter analysis (72, 73). We also found that ICSBP mRNA levels were lower in LPS-stimulated IRF-2-/- mice, but the deficit was apparently not sufficient to affect IL-12 p40 expression. This finding contrasts with previous studies showing that macrophage-like cells from ICSBP-/- mice could not induce IL-12 p40 transcripts (68), leading to the conclusion that ICSBP acts as a principal activator of this gene in macrophages. ICSBP binds to the IFN-stimulated regulatory element only weakly, but upon interactions with IRF-1 or IRF-2, its DNA-binding activity is dramatically increased (4). Other authors have also demonstrated that ICSBP may repress IRF-1-induced transcription (4, 74). Given the functional diversity of the IRFs, their capacity for interaction with other members of the family, and the changing levels of these proteins in stimulated cells, our data point to a more complex regulation of the production of IL-12 in vivo than in vitro in response to LPS.

A possible explanation for the down-regulation of IRF-1 mRNA expression after LPS challenge in IRF-2-/- mice, is the significantly lower levels of serum IFN-{gamma}, IL-1{beta}, and IL-6. The IRF-1 promoter contains a potential IFN-{gamma}-activated sequence (75). The profound reduction that is seen in IFN-{gamma} levels in IRF-2-/- mice when compared with IRF-2+/+ after LPS challenge would be predicted to result in an attenuation of IRF-1 transcription in the IRF-2-deficient mice. Both IL-1 and IL-6 have also been shown to induce IRF-1 (59, 60, 61), and our data show clearly that both are significantly reduced at the protein level in IRF-2-/- mice challenged with LPS. Therefore, diminished IRF-1 mRNA levels may well be secondary to decreases in these cytokines. It is interesting to note that work by Marecki et al. (76) showed that concurrent expression of IRF-1, IRF-2, and the Ets-like protein PU.1 with either IRF-4 or ICSBP synergize for maximal IL-1{beta} transcription. Thus, diminished levels of LPS-inducible IRF-1 mRNA are likely, in turn, to contribute to the diminished IL-1 expression. Taken collectively, our results reveal that IRF-2 is normally required for optimal expression of LPS-induced IL-1 and IL-6, and that there is cross-regulation between these cytokines and IRF-1.

In the same way, the down-regulation of ICSBP mRNA expression after LPS challenge in IRF-2-/- mice may be explained by the significant low levels of IFN-{gamma}, because it has been shown to induce ICSBP (74, 77). Our in vivo data indicate that the IL-12R{beta}2 mRNA expression is down-regulated between 6 to 8 h after LPS challenge in IRF-2-/- mice, whereas the IL-12R{beta}1 mRNA levels are not affected. The expression of these two genes is independently regulated (78, 79, 80), and although IRF-1 has been implicated in the regulation of both of them (42), our findings would suggest that IRF-2 normally contributes to the expression of the IL-12R{beta}2 subunit. IL-12R{beta}2 serves as the signal transduction component of this IL-12R heterodimeric receptor (79, 80). Disruption in the IL-12 pathway in IRF-2-/- mice was paralleled by a significant impairment in the ability of IRF-2 knockout mice to produce IFN-{gamma}. Although we have provided strong evidence for a disruption in this pathway at the levels of IL-12 p70 and IL-12R{beta}2 receptor mRNA expression, Lohoff et al. (20) also showed that NK and Th1 cell development is compromised in IRF-2-/- mice. As NK cells are the dominant IFN-{gamma}-secreting cell population in response to LPS (81), the combined paucity of IL-12 p70 and IL-12R expression in cells with mitigated NK function could readily account for the profound inhibition of IFN-{gamma} that we observed in the IRF-2-/- mice. Future studies will be required to demonstrate this directly.

The observed decrease in IFN-{gamma} expression might have been predicted to have resulted in lowered levels of TNF-{alpha} (82). Rather, circulating TNF-{alpha} was actually significantly higher in IRF-2-/- than IRF-2+/+ mice early after LPS injection, with no apparent TNFR mRNA dysfunction. In addition, heightened levels of TNF-{alpha} should have stimulated an increase in IRF-1 mRNA expression, as IRF-1 is also induced by many LPS-inducible cytokines (2, 83), as well as in LPS-challenged mice and macrophages (5, 42). Taken collectively, these data suggest that IRF-2 normally regulates expression of one or more components of the TNF-{alpha}-inducible signaling pathway.

Another factor that may contribute to the refractory response of IRF-2-/- mice to LPS is the observation that the number of apoptotic Kupffer cells is significantly higher than in control mice (both after saline injection and in response to LPS or TNF-{alpha}). Interestingly, it has been shown that Kupffer cell depletion results in an increase in circulating TNF-{alpha} (84). As TNF-{alpha} mediates apoptosis in Kupffer and endothelial cells (85, 86), increased levels of TNF-{alpha} in IRF-2-/- mice after LPS treatment may contribute to the significantly increased number of apoptotic Kupffer cells measured at 6 h after LPS challenge. This notion is supported by our observation that injection of recombinant TNF-{alpha} recapitulates this effect. However, IRF-2-/- mice also exhibited more apoptotic Kupffer cells after saline injection, suggesting that IRF-2 regulates basal levels of apoptosis as well. IRF-1 has been implicated in the regulation of cell cycle and apoptosis, and the target genes critical for apoptotic response may include caspases 1 and 7 (28, 87). These findings point to IRF-2 as a novel attenuator of apoptotic events in Kupffer cells. In addition, it has been reported that LPS-induced proinflammatory cytokine production by macrophages is significantly suppressed by coculture with apoptotic cells (88). Therefore, it is likely that the enhanced resistance of IRF-2-/- mice to LPS challenge may depend both on the removal of Kupffer cells and the active suppression of inflammatory mediators such as IL-12 and IFN-{gamma} by remaining cell types.


   Acknowledgments
 
We thank M. Joshua Cody for genotyping the mice used in this study and Dr. Ian Sabroe for his thoughtful comments on the revised version.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI-18797. Back

2 N.C. and C.A.S. contributed equally to the work presented in this paper. Back

3 Address correspondence to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, University of Maryland, Baltimore, 655 West Baltimore Street, 13-009, Baltimore, MD 21201. E-mail address: svogel{at}som.umaryland.edu Back

4 Abbreviations used in this paper: IRF, IFN regulatory factor; IRF-E, IRF element; ICSBP, IFN consensus sequence binding protein; HPRT, hypoxanthine-guanine phosphoribosyltransferase. Back

Received for publication October 9, 2002. Accepted for publication March 26, 2003.


   References
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 Materials and Methods
 Results
 Discussion
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