The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salkowski, C. A.
Right arrow Articles by Vogel, S. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salkowski, C. A.
Right arrow Articles by Vogel, S. N.
The Journal of Immunology, 1999, 163: 1529-1536.
Copyright © 1999 by The American Association of Immunologists

IL-12 Is Dysregulated in Macrophages from IRF-1 and IRF-2 Knockout Mice1

Cindy A. Salkowski, Karen Kopydlowski, Jorge Blanco, M. Joshua Cody, Ranney McNally and Stefanie N. Vogel2

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophages derived from IFN-regulatory factor-1 (IRF-1) and IRF-2 knockout (-/-) and wild-type (+/+) mice were utilized to examine the role of these transcription factors in the regulation of IL-12 mRNA and protein expression. Induction of IL-12 p40 mRNA by LPS was markedly diminished in both IRF-1-/- and IRF-2-/- macrophages. In contrast, IRF-1-/-, but not IRF-2-/-, macrophages exhibited impaired LPS-induced IL-12 p35 mRNA expression. The ability of IFN-{gamma} to augment LPS-induced IL-12 p40 mRNA further when both stimuli were present simultaneously was significantly diminished in both IRF-1-/- and IRF-2-/- macrophages, with the most profound impairment observed for IRF-1-/- macrophages. Reductions in IL-12 mRNA expression after stimulation with LPS or LPS plus IFN-{gamma} were accompanied by substantial reductions in IL-12 p40 and IL-12 p70 protein in both IRF-1-/- and IRF-2-/- macrophages. Priming IRF-1-/- and IRF-2-/- macrophages with IFN-{gamma} for 24 h before LPS treatment partially restored impaired IL-12 mRNA and protein production in both IRF-1-/- and IRF-2-/- macrophages. Depressed IL-12 levels were paralleled by significant reductions in IFN-{gamma} mRNA expression in IRF-1-/- and IRF-2-/- macrophages. These results indicate that both IRF-1 and IRF-2 are critical transcription factors in the regulation of macrophage IL-12 and consequently IFN-{gamma} production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a heterodimeric cytokine produced primarily by phagocytic cells and APC (1, 2, 3, 4, 5). IL-12 has been best characterized for its ability to induce IFN-{gamma} secretion from T cells and NK cells directly (6, 7), thereby facilitating Th1 responses (8, 9, 10) and regulating the balance of Th1 and Th2 cells. Studies using anti-IL-12 Ab or IL-12 knockout mice have demonstrated that increased resistance to a number of infectious agents requires production of endogenous IL-12 (reviewed in 11). In humans, severe mycobacterial and Salmonella infections have been observed in IL-12 receptor-deficient individuals (12). Thus, IL-12 plays a crucial role in linking innate and acquired immune responses, allowing phagocytic cells to facilitate the development of cell-mediated immunity to bacterial, viral, parasitic, and fungal pathogens (10, 13, 14, 15). However, for those diseases in which overproduction of IFN-{gamma} is deleterious, endogenous IL-12 can exacerbate disease processes. For example, endogenous IL-12 produced by LPS administration, by nematode infection, and during autoimmune disorders leads to enhanced mortality and/or accelerated disease (16, 17, 18).

Due to the critical role of IL-12 in the development of a protective immune response to pathogens, as well as its deleterious role in the response to LPS, it is important to understand how IL-12 biosynthesis is regulated. Bioactive IL-12 p70 is a heterodimer comprised of two disulfide-linked subunits, designated p35 and p40, which are regulated independently (19, 20). Whereas IL-12 p40 is secreted in monomeric and homodimeric forms (21), secretion of IL-12 p35 has not been reported to date (2). Typically, IL-12 p70 production in vitro and in vivo is accompanied by excess production of the p40 subunit (reviewed in 11), which functions as an antagonist of bioactive IL-12 p70 (21). A notable feature of IL-12 production by macrophages is the ability of a wide array of bacteria and microbial products like LPS, lipoteichoic acid, zymosan, exotoxins, and dsRNA to act as potent inducers of IL-12 secretion (5). Among the cytokines, IFN-{gamma} synergizes with bacteria and/or microbial products to augment IL-12 production (5, 22, 23), whereas IL-4, IL-10, IL-13, and TGF-{beta} exert negative regulatory effects (23, 24, 25, 26).

Induction of both IL-12 p40 and IL-12 p35 after stimulation with both LPS and IFN-{gamma} is controlled primarily at the transcriptional level (22). The promoter regions for the human and murine IL-12 p40 and IL-12 p35 genes have been cloned, and studies have begun to delineate the regulatory regions and transcription factors involved in IL-12 p40 mRNA induction (22, 27, 28, 29). Among the transcription factors up-regulated by LPS and IFN-{gamma} are several members of the IFN-regulatory factor (IRF)3 family (30, 31, 32), which were originally identified for their role in the regulation of IFN-{alpha}/{beta}-inducible genes. Although all IRF family members share the ability to bind a similar DNA motif known as an IFN-stimulated response element (ISRE), they differ remarkably in their abilities to regulate transcription. For example, IRF-1 is an activator of many IFN-{alpha}/{beta}-inducible genes (33), whereas IRF-2 and IFN consensus sequence-binding protein (ICSBP) function largely, but not exclusively, as negative regulators (34, 35, 36). Recently, ICSBP has been implicated in IL-12 regulation (28, 37, 38). Macrophages from ICSBP knockout (-/-) mice produced low levels of IL-12 p40 mRNA and 50–80% less IL-12 p40 protein in response to LPS and other microbial stimuli, a deficiency that was more pronounced in IFN-{gamma}-primed, LPS-activated macrophages (37). In response to a single high dose combination of LPS plus IFN-{gamma}, IRF-1-/- macrophages produced barely detectable levels of IL-12 p40 mRNA and undetectable IL-12 p70 protein (38). The role of other IRF family members in IL-12 regulation, as well as the ability of IRFs to regulate IL-12 p35 mRNA expression, has yet to be explored.

To elucidate further the role of IRF family members in the regulation of IL-12, we examined IL-12 mRNA and protein expression in LPS and/or IFN-{gamma}-activated macrophages from IRF-1-/- and IRF-2-/- mice. Results from this study indicate that loss of IRF-1 results in impaired production of not only IL-12 p40 but also IL-12 p35 mRNA after treatment with LPS or LPS plus IFN-{gamma}. These studies also demonstrate that IRF-2 plays an essential role in the up-regulation of IL-12 in activated macrophages. Specifically, IRF-2 was required for optimal expression of IL-12 p40 but not IL-12 p35 mRNA expression. Loss of IL-12 mRNA expression in activated macrophages from IRF-1-/- and IRF-2-/- mice was reflected at the level of protein synthesis as reductions in both IL-12 p40 and p70 secretion. Impaired IL-12 p70 protein secretion was associated with a loss in IFN-{gamma} mRNA expression in IRF-1-/- and IRF-2-/- macrophages, indicating the importance of IRF-1 and IRF-2 in regulating IFN-{gamma}-mediated host responses.


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

Murine rIFN-{gamma} was a kind gift from Genentech (South San Francisco, CA). LPS was prepared from Escherichia coli K235 by phenol-water extraction (39) and contained <0.008% protein. Zymosan was obtained from Sigma (St. Louis, MO). Staphylococcus aureus was grown to log phase, washed in water, and boiled. Heat-killed S. aureus (HKSA) was collected by centrifugation, dried by vacuum, and resuspended to 25 mg/ml in water. The HKSA preparation contained <0.3 endotoxin U/ml when tested by Limulus amebocyte lysate assay.

Mice

C57BL/6J mice were obtained from The Jackson Laboratory, Bar Harbor, ME. IRF-1-/- and IRF-2-/- mice were generated by targeted disruption in the embryonic stem cell using the neomycin resistance gene (40). Breeding pairs of IRF-1-/- and IRF-2-/- and IRF+/- mice, which were backcrossed to C57BL/6 mice for 3–5 generations, were a kind gift of Dr. Tak Mak (Amgen Institute, Toronto, Canada). Colonies of IRF-1 and IRF-2 mice used in this study were bred as described previously (41). IRF-1+/+ and IRF-2+/+ were also bred to provide background-matched controls; however, when unavailable, C57BL/6 mice were used. Additionally, all IRF-1 and IRF-2 mice were genotyped as described previously (41, 42). Mice were housed in cages with filter tops in a laminar flow hood and fed food and acid water ad libitum. IL-12 p40-/- and IL-12 p35-/- mice were a kind gift of Dr. Thomas Wynn and Dr. Alan Sher (National Institutes of Health, Bethesda, MD), respectively.

Macrophage cultures

Peritoneal exudate macrophages were obtained by lavage 4 days after injection of sterile 3% thioglycolate broth (3 ml i.p.). Cells were washed and resuspended in RPMI containing 2% FCS and standard supplements (43). Macrophages were plated in six-well tissue culture dishes (4 x 106 cells/well). After overnight incubation to allow for adherence of macrophages, monolayers were washed to remove nonadherent cells and incubated with the appropriate concentration of LPS and/or IFN-{gamma} in a final volume of 2 ml. Samples were harvested 6 h after treatment, the time that we had previously demonstrated as optimal for peak levels of both LPS-induced IL-12 p40 and IL-12 p35 mRNA expression (44).

Enzyme-linked immunosorbent assays

Supernatants from macrophage cultures were harvested at 6 h and stored at -70°C. IL-12 p70 was detected using the Ab pairs and standard provided in the OPT-EIA ELISA kit (PharMingen, San Diego, CA) according to the manufacturer’s instructions. For the total IL-12 (p40 monomer, p40 dimer, and p70) ELISA, Nunc-Immuno microtiter plates (Nunc, Roskilde, Denmark) were coated with 2 µg/ml monoclonal rat anti-mouse IL-12 Ab (clone C15.6, Genzyme, Cambridge, MA). The coating buffer was PBS, pH 7.5. After overnight incubation at 4°C, the plates were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and then blocked (37°C, 2 h) with PBS containing 1% BSA. Following four washes with PBS-T, 100 µl of either samples or rIL-12 standard (Genzyme) were added to the plates and incubated for 1 h at room temperature. The plates were washed again (four times) with PBS-T, followed by addition of 1 µg/ml biotinylated rat anti-mouse IL-12 Ab (clone C17.15, Genzyme). After four washes with PBS-T, the plates were incubated with HRP-streptavidin (1:50,000, Jackson ImmunoResearch Laboratories, West Grove, PA) for 15 min at room temperature. The plates were washed four times, followed by the addition of tetramethylbenzidine peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD), which was prepared according to the manufacturer’s instructions. After 20 min, color development was stopped by the addition of 1 M H3PO4 and the absorbance at 450 nm was read in an automated ELISA plate reader. Concentrations were calculated by regression analysis of a standard curve. Data are expressed as picograms per milliliter. 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 (p40/p70) ELISA. To verify the specificity of the IL-12 p70 ELISA used in this study, IL-12 p35-/- macrophages, which have been shown to produce no IL-12 p70 (45), were activated with LPS, IFN-{gamma}, or both, and supernatants were assayed for both IL-12 p40 and IL-12 p70. Although their IL-12 p40 protein production in response to LPS or LPS plus IFN-{gamma} was intact, IL-12 p35-/- macrophages produced no detectable IL-12 p70, thereby eliminating the possibility for IL-12 p40 to cross-react in the IL-12 p70 ELISA used in this study.

Analysis of mRNA

Total RNA was isolated from macrophage cultures, and the relative quantities of mRNA for hypoxanthine-guanine phosphoribosyltransferase (HPRT), IL-12 p35, IL-12 p40, IFN-{gamma}, IRF-1, IRF-2, and ICSBP were determined by RT-PCR, as described previously (46). The probes and primer combinations have been published (14, 30, 47). 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 enhanced chemiluminescence system (Amersham).

Statistics

Results were analyzed by Student’s t test for comparisons between two groups and analysis of variance. p values <0.05 were accepted as the level of significance. All experiments were repeated at least three times with similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dysregulation of IL-12 mRNA expression and protein secretion in IRF-1-/- macrophages

To examine the extent to which IRF-1 controls IL-12 mRNA expression and protein synthesis, peritoneal exudate macrophages from IRF-1+/+ and IRF-1-/- mice were treated with a broad range of concentrations of LPS, IFN-{gamma}, or both. Total RNA was isolated 6 h after treatment to assess levels of steady-state IL-12 p40 and IL-12 p35 mRNA expression by RT-PCR, whereas supernatants were analyzed for IL-12 p40 and IL-12 p70 protein by ELISA. Macrophages from IRF-1-/- mice produced detectable but substantially reduced levels of both IL-12 p40 and IL-12 p35 mRNA compared with IRF-1+/+ macrophages after activation with either LPS or LPS plus IFN-{gamma} (Fig. 1Go). In contrast to IRF-1+/+ macrophages, simultaneous treatment of IRF-1-/- macrophages with LPS plus IFN-{gamma} did not significantly augment IL-12 p40 mRNA expression above levels seen with LPS alone. Interestingly, increasing concentrations of exogenous IFN-{gamma} (5 and 25 U/ml) resulted in an inhibition of the level of IL-12 p35 mRNA expression induced by LPS in IRF-1-/- macrophages (Fig. 1Go and data not shown). IRF-1+/+ macrophages were less sensitive to inhibition of LPS-induced IL-12 p35 mRNA by IFN-{gamma} than IRF-1-/- macrophages, because >5 U/ml IFN-{gamma} was necessary to observe a significant inhibitory effect (data not shown).



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 1. Induction of IL-12 p40, IL-12 p35, and IFN-{gamma} mRNA in IRF-1+/+ and IFR-1-/- macrophages by LPS and IFN-{gamma}. Macrophage monolayers were treated with medium, LPS (0.1, 1, or 10 ng/ml), IFN-{gamma} (0.5 or 5 U/ml), or both LPS plus IFN-{gamma} for 6 h. A representative Southern blot of RT-PCR-amplified products is shown (n = 4). The cycle numbers were 22 for IL-12 p40 and IRF-1, 24 for HPRT, 28 for IL-12 p35, and 30 for IFN-{gamma}.

 
Defective IL-12 mRNA expression in IRF-1-/- macrophages was reflected in the significantly reduced levels of both IL-12 p40 and IL-12 p70 protein (Fig. 2Go). After treatment with LPS only, IRF-1-/- macrophages secreted 2- to 3-fold less IL-12 p40 and 10- to 20-fold less IL-12 p70 than IRF-1+/+ controls. Impaired IL-12 production was more evident in IRF-1-/- macrophages treated with LPS plus IFN-{gamma}, because treatment with LPS plus IFN-{gamma} failed to augment levels of IL-12 p40 or IL-12 p70 protein over that observed after LPS treatment alone. IRF-1-/- macrophages activated with LPS plus IFN-{gamma} secreted ~10-fold less IL-12 p40 and 30- to 100-fold less IL-12 p70 than IRF-1+/+ controls.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 2. IL-12 protein production by IRF-1-/- macrophages after treatment with LPS and/or IFN-{gamma}. Supernatants were harvested from the macrophage monolayers treated as described in Fig. 1Go. Data are expressed as the mean picograms per milliliter ± SEM from three independent experiments. All data from LPS- or LPS- plus IFN-{gamma}-treated IRF-1-/- () macrophages were significantly less (p < 0.05) than that from similarly treated IRF-1+/+ ({square}) macrophages.

 
LPS-induced IL-12 up-regulates IFN-{gamma} in peritoneal exudate macrophages

IL-12 has been shown to induce macrophage-derived IFN-{gamma} (48). Thus, we next used IL-12 p40 knockout mice, which produce no bioactive IL-12 (9), to assess the relative contribution of endogenous IL-12 to IFN-{gamma} mRNA production in our macrophage cultures. IL-12 p40-/- macrophages produced substantially less IFN-{gamma} mRNA after activation with either LPS or LPS plus IFN-{gamma} than C57BL/6 control macrophages (Fig. 3Go), indicating the importance of endogenous IL-12 in the autocrine production of IFN-{gamma} by activated macrophages. Impaired IL-12 mRNA and protein production in IRF-1-/- macrophages was accompanied by reduced levels of IFN-{gamma} mRNA (Fig. 1Go), suggesting that loss of endogenously produced, bioactive IL-12 (p70) by IRF-1-/- macrophages leads to impaired autocrine regulation of IFN-{gamma}. In a previous study, we reported that although LPS-stimulated macrophages produce a highly cell-associated IFN-{gamma}, secreted IFN-{gamma} could not be detected (47). Therefore, secretion of IFN-{gamma} into culture supernatants was not assessed in this study.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 3. Induction of IFN-{gamma} mRNA by LPS or both LPS and IFN-{gamma} is IL-12 dependent. Macrophage monolayers from IL-12 p40-/- and C57BL/6 (+/+) mice were treated with medium, LPS (10 ng/ml), IFN-{gamma} (5 U/ml), or both LPS and IFN-{gamma} for 6 h. A representative Southern blot of RT-PCR-amplified products is shown (n = 3).

 
IRF-2-/- macrophages also display impaired IL-12 expression

Because IRF-2 has been shown to interact with the same promoter element as IRF-1 (49), we postulated that IRF-2 may also regulate IL-12 production. Macrophages from IRF-2+/+ and IRF-2-/- mice were treated with increasing concentrations of LPS, IFN-{gamma}, or both. IRF-2-/- macrophages produced reduced levels of IL-12 p40 mRNA in response to activation with LPS or LPS plus IFN-{gamma}, and the ability of IFN-{gamma} to augment further LPS-induced IL-12 p40 mRNA remained partially intact in IRF-2-/- macrophages (Fig. 4Go). In contrast to IRF-1-/- macrophages, LPS-induced IL-12 p35 mRNA expression in IRF-2-/- macrophages was not dysregulated. The ability of increasing concentrations of exogenous IFN-{gamma} to reduce the level of LPS-induced IL-12 p35 mRNA expression, which was observed in IRF-2+/+ macrophages, was impaired in IRF-2-/- macrophages.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 4. Induction of IL-12 p40, IL-12 p35, and IFN-{gamma} mRNA in IRF-2+/+ and IRF-2-/- macrophages by LPS and IFN-{gamma}. Macrophage monolayers were treated with medium, LPS (0.1, 1 or 10 ng/ml), IFN-{gamma} (0.5 or 5 U/ml), or both LPS plus IFN-{gamma} for 6 h. A representative Southern blot of RT-PCR-amplified products is shown (n = 5).

 
We also assessed IL-12 p40 and p70 protein levels in culture supernatants to determine whether the lack of IRF-2 also dysregulated IL-12 protein production. As shown in Fig. 5Go, IRF-2-/- macrophages produced 2- to 5-fold less IL-12 p40 and IL-12 p70 protein in response to LPS than IRF-2+/+ macrophages. Although activation of IRF-2-/- macrophages with LPS plus IFN-{gamma} resulted in a synergistic increase in IL-12 p40 and IL-12 p70 protein, levels still remained 3- to 10-fold less than that observed for similarly treated IRF-2+/+ macrophages. Reductions in IL-12 p70 in supernatants from IRF-2-/- macrophages correlated with significant decreases in IFN-{gamma} mRNA expression (Fig. 4Go), once again demonstrating the importance of endogenous IL-12 in the autocrine/paracrine regulation of IFN-{gamma}.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 5. IL-12 protein production by IRF-2-/- macrophages following LPS and IFN-{gamma} treatment. Supernatants were harvested from the macrophage monolayers treated as described in Fig. 5Go. Data are expressed as the mean picograms per milliliter ± SEM from three experiments. {square}, IRF-2+/+ macrophages; , IRF-2-/- macrophages.

 
IFN-{gamma} priming partially restores defective IL-12 production in both IRF-1-/- and IRF-2-/- macrophages

IFN-{gamma} priming (i.e., pretreatment of macrophages before exposure to LPS) has been shown to increase IL-12 production by increasing transcription of both the IL-12 p40 and IL-12 p35 genes (22). Thus, we pretreated IRF-1-/-, IRF-2-/-, and control macrophages with medium or IFN-{gamma} (5 U/ml) for 24 h before the addition of LPS (1 ng/ml) to ascertain whether IFN-{gamma} priming could compensate for the defects in IL-12 mRNA and protein expression. IFN-{gamma}-primed, LPS-activated IRF-1-/- macrophages expressed increased levels of both IL-12 p40 and IL-12 p35 mRNA compared with IRF-1-/- macrophages simultaneously treated with LPS plus IFN-{gamma} (Fig. 6). Similarly, IFN-{gamma}-priming before LPS activation also resulted in increased levels of IL-12 p40 mRNA in IRF-2-/- macrophages (compared with IRF-2-/- macrophages treated simultaneously with LPS plus IFN-{gamma}). As shown in Tables I and II, IFN-{gamma} pretreatment resulted in an ~2- to 4-fold increase in IL-12 p40 and IL-12 p70 protein expression in LPS-stimulated IRF-1-/- and IRF-2-/- macrophages when compared with the same macrophages treated simultaneously with LPS plus IFN-{gamma}. Levels of IL-12 p40 and IL-12 p70 protein in supernatants from IRF-1-/- and IRF-2-/- macrophages, however, still remained well below those observed for similarly treated wild-type (+/+) controls.

Impaired IL-12 production in IRF-1-/- and IRF-2-/- macrophages after treatment with other microbial stimuli

IL-12 is also induced by a wide array of microbes and microbial products (5). We next assessed whether impaired IL-12 production by IRF-1-/- and IRF-2-/- macrophages was observed in response to other microbial stimuli. Macrophage monolayers were treated with HKSA or zymosan in the presence or absence of IFN-{gamma}. After treatment with HKSA or zymosan, IRF-1-/- macrophages had substantially reduced levels of both IL-12 p40 and IL-12 p70 protein (Table IIIGo). Moreover, these stimuli did not synergize with IFN-{gamma} to induce further increases in IL-12 protein in IRF-1-/- macrophages. The IL-12 response of IRF-2-/- macrophages to these alternate microbial stimuli was more complex. IL-12 p70 but not IL-12 p40 protein levels were significantly reduced in IRF-2-/- macrophages treated with HKSA or zymosan. Additionally, the ability of IFN-{gamma} to synergize with these stimuli to induce IL-12 p70 protein was partially reduced in IRF-2-/- macrophages, as was observed with LPS.


View this table:
[in this window]
[in a new window]
  		Table III. IL-12 production in response to various microbial products is defective in IRF-1-/- and IRF-2-/- macrophages

 
ICSBP is not dysregulated in IRF-1-/- and IRF-2-/- macrophages

A notable feature of IRF family members is their ability to cross-regulate expression of each other (50, 51). Because the promoter region of ICSBP contains an ISRE half-site (52) and ICSBP-/- macrophages have impaired IL-12 p40 mRNA expression (37), we next assessed whether loss of IRF-1 or IRF-2 resulted in altered ICSBP regulation in activated macrophages. After treatment with LPS or LPS plus IFN-{gamma}, IRF-1-/- and IRF-2-/- macrophages had levels of ICSBP mRNA comparable with that observed for +/+ controls (Fig. 7). Additionally, IRF-1-/- and IRF-1+/+ macrophages activated with LPS plus IFN-{gamma} had comparable levels of nuclear ICSBP protein (data not shown). These data suggest that the impaired IL-12 regulation observed in IRF-1-/- and IRF-2-/- macrophages is not due to an inability to produce ICSBP or its altered nuclear translocation. Finally, IRF-1 mRNA expression in LPS- or LPS plus IFN-{gamma}-treated IRF-2+/+ and IRF-2-/- macrophages was similar, as was IRF-2 mRNA expression in LPS- or LPS plus IFN-{gamma}-treated IRF-1+/+ and IRF-1-/- macrophages (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, IRF-1 and IRF-2 were found to be important transcription factors involved, directly and/or indirectly, in the up-regulation of IL-12 by LPS and other microbial Ags. Loss of either IRF-1 or IRF-2 was associated with profound reductions in IL-12 p40 mRNA inducibility by LPS, whereas loss of IRF-1, but not IRF-2, dysregulated IL-12 p35 mRNA inducibility by LPS. These defects became more pronounced on the addition of exogenous IFN-{gamma}. The capacity of IFN-{gamma} to augment LPS-induced increases in IL-12 p40 mRNA, as well as IL-12 protein, was also impaired in both IRF-1-/- and IRF-2-/- macrophages, suggesting that the IRFs also play an important role in regulating the synergy that is observed after macrophage activation with microbial Ags and IFN-{gamma}.

Our findings in IRF-1-/- macrophages confirm and extend the work of Taki et al. (38), who demonstrated that IRF-1-/- macrophages activated by 30 µg/ml LPS and 100 U/ml IFN-{gamma} produced barely detectable IL-12 p40 mRNA and no detectable IL-12 p70 protein. The inability of Taki et al. (38) to detect IL-12 p70, however, is in contrast to our findings in which IRF-1-/- macrophages consistently produced low, but reproducibly detectable levels of IL-12 p70 (Fig. 2Go and Table IGo). One likely explanation for the different observations is the extremely high concentration of IFN-{gamma} and LPS used in their study (38). IL-12 induction in +/+ macrophages was exquisitely sensitive to low concentrations of both stimuli. As little as 0.5 U/ml IFN-{gamma} and 1 ng/ml LPS were optimal for IL-12 p70 protein production, and further increases in the concentrations of IFN-{gamma} and LPS can result in reduced levels of IL-12 p70 protein. A second explanation is the relative sensitivity of the IL-12 p70 ELISAs used in each study. We routinely detected nanogram per milliliter levels of IL-12 p70 in +/+ macrophages optimally stimulated with LPS plus IFN-{gamma}, whereas only ~110 pg/ml IL-12 p70 were detected in supernatants from +/+ macrophages in the earlier study (38). The possibility that the heightened levels of IL-12 p70 we observed were the result of cross-reaction with IL-12 p40 protein was ruled out by the demonstration that IFN-{gamma}- plus LPS-stimulated IL-12 p35-/- macrophages produced no IL-12 p70 (data not shown).


View this table:
[in this window]
[in a new window]
  		Table I. LPS-induced IL-12 production by IRF-1-/- macrophages after IFN-{gamma} priming1

 
The murine IL-12 p40 promoter has been cloned (20) and contains consensus sequences for a number of cis-regulatory elements that are known to be involved in gene induction by LPS and IFNs. These potential regulatory sites include NF-IL-6, NF-{kappa}B, Ets-2, ISRE, and IFN-{gamma} activation sites (GAS) (20). The ISRE sites, the DNA binding domain of the IRF family, and NF-IL-6 sites are found in multiple copies. To date, the majority of murine IL-12 p40 promoter activity in response to LPS plus IFN-{gamma} has been localized to a region that contains a NF-{kappa}B site (-132 to -125 bp). This site bound both p50/p65 and p50/c-Rel complexes from IFN-{gamma}/LPS-activated J774 macrophages, and IFN-{gamma} augmented the ability of nuclear factors to bind this region (27). Our data support a role for both IRF-1 and IRF-2 in the regulation of IL-12 p40 gene expression. Although there is as yet no evidence for a functional ISRE in the murine IL-12 p40 promoter, no direct mutational analysis of these sites has been done. Cooperation between NF-{kappa}B and ISREs has been demonstrated for several genes including MHC class I, {beta}2-microglobulin, IL-6, IFN-{beta}, and VCAM-1 (36, 53, 54, 55), and this type of cooperative mechanism may be involved in murine IL-12 p40 regulation. Of interest, cotransfection of cells with constructs that encode NF-{kappa}B family members (p50, p65) and IRF-1 led to synergistic induction of MHC class I, VCAM-1, and IL-6 promoter activity (36, 53, 54), further indicating that members of the Rel and IRF families of transcription factors may act in a cooperative manner.

A second mechanism of IRF-1 and IRF-2 regulation of IL-12 p40 transcription may be through direct interaction with members of the NF-{kappa}B family. Drew et al. (36, 55) demonstrated that in vitro translated IRF-1 and IRF-2, as well as other IRFs, bound to both NF-{kappa}B p50 and p65. Only the amino-terminal 115 aa of IRF-1, which is involved in DNA binding and shares homology with other IRF family members, was necessary for this interaction. The ability of I{kappa}B{alpha} overexpression to ablate IRF-1-mediated IL-6 promoter activity provides further evidence for interaction between NF-{kappa}B and IRF-1 (54). Finally, two of the critical regions in the human IL-12 p40 promoter that are necessary for regulation by LPS plus IFN-{gamma} are an Ets and NF-{kappa}B site (28, 56). The Ets element bound a complex of nuclear proteins from activated macrophages that included Ets-2, IRF-1, and the NF-{kappa}B component, c-Rel (28). In contrast, this same Ets site incubated with nuclear extracts from EBV-transformed B cells bound Ets-2, c-Rel, and IRF-2, rather than IRF-1 (56).

It has been suggested that IL-12 p35 production is the rate-limiting factor in the synthesis of IL-12 p70 (23). Despite this, very little is known about IL-12 p35 regulation. This is likely due to early reports that IL-12 p35 was ubiquitously expressed and not highly inducible (2, 24). More recent studies have demonstrated that IL-12 p35 mRNA is strongly induced by LPS in macrophages and monocytes (23, 44). Like the murine IL-12 p40 promoter, the murine IL-12 p35 promoter contains consensus sequences for NF-IL6, as well as multiple NF-{kappa}B, GAS, and ISRE sites (20). This study indicates that IRF-1, but not IRF-2, is involved in the induction of IL-12 p35 mRNA by LPS and provides the first insights into which transcription factors may control murine IL-12 p35 promoter activity. Whether IRF-1 functions directly by binding to ISRE(s) in the murine IL-12 p35 promoter or by interaction with other transcription factors like NF-{kappa}B, or acts indirectly, is unknown. Finally, our studies indicate that IL-12 p35 availability alone may not limit IL-12 p70 production, as has been suggested (23), because impaired IL-12 p70 by IRF-2-/- macrophages was associated with diminished IL-12 p40 mRNA and protein, and not a reduction in IL-12 p35 mRNA expression.

Our data suggest that IRF-2 may act as a positive regulator in addition to its previously described activities as a negative regulator. The ability of the IRF family members, IRF-2 and ICSBP, to act as suppressors of gene regulation does not appear to be absolute. IRF-2 has been reported to up-regulate the histone H4 gene FO108 (57), whereas studies with ICSBP-/- macrophages have suggested that ICSBP is a positive regulator of IL-12 p40 (37).

Interestingly, as the concentration of IFN-{gamma} increased, LPS-induced IL-12 p35 mRNA expression in IRF-1+/+, IRF-1-/-, and IRF-2+/+ macrophages decreased (Figs. 1Go and 4Go, and data not shown). IRF-1+/+ macrophages were less sensitive to this inhibitory effect than IRF-1-/- macrophages, and the inhibition of LPS-induced IL-12 p35 mRNA by increasing concentrations of IFN-{gamma} was impaired in IRF-2-/- macrophages. We have observed similar findings for the regulation of the chemokine KC; specifically, the ability of IFN-{gamma} to suppress LPS-induced KC also is diminished in IRF-2-/- but not in IRF-1-/- macrophages (58). Whether the loss of this suppressive effect in our study is due to a direct action of the IRFs or is the result of the dysregulation of a negative regulator of IL-12 p35 is not known. To date, only IL-10 and TGF-{beta} have been shown to down-regulate IL-12 p35 mRNA expression (23, 59). IL-4, which suppressed LPS-induced IL-12 p40 mRNA expression, did not alter IL-12 p35 mRNA induction (23).

In addition to possible direct actions of IRF-1 and IRF-2 on IL-12 transcription, they may also act indirectly. For example, IRF-1 has been shown to up-regulate STAT1 expression (60). This suggested the possibility that the STAT1 pathway of IFN-{gamma} signaling might be impaired in IRF-1-/- macrophages, thereby accounting for the inability of IFN-{gamma} to synergize with LPS to induce either IL-12 (Fig. 2Go) or nitric oxide (42). Several observations suggest that STAT1 signaling, however, is not impaired in either IRF-1-/- or IRF-2-/- macrophages. The IRF-1 and ICSBP promoter regions contain GAS sequences that bind STAT1{alpha} (52, 61). Both IRF-1-/- and IRF-2-/- macrophages responded to IFN-{gamma} treatment with increased levels of ICSBP mRNA expression that was comparable to +/+ control levels (Fig. 7Go), and IFN-{gamma}-induced IRF-1 mRNA expression in IRF-2-/- macrophages also was intact (data not shown). Moreover, IP-10 and MCP-5, two other STAT1-dependent genes, are not dysregulated in either IRF-1-/- or IRF-2-/- macrophages after IFN-{gamma} treatment (58).



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 7. Loss of IRF-1 and IRF-2 does not alter ICSBP mRNA expression. Macrophage monolayers were treated with medium, LPS (1 ng/ml), IFN-g (5 U/ml), or both for 6 h. Data are from a representative Southern blot of RT-PCR-amplified products (n = 3).

 
An intriguing observation was the ability of IFN-{gamma} priming before LPS activation to partially restore impaired IL-12 mRNA and protein production in both IRF-1-/- and IRF-2-/- macrophages. IFN-{gamma} priming has been reported to enhance macrophage activation by three distinct mechanisms: 1) up-regulating and/or accelerating signal transduction pathways that utilize NF-{kappa}B and STAT1; 2) altering the production of autocrine/paracrine amplifiers and inhibitors of macrophage activation; and 3) enhancing mRNA stability (62, 63, 64, 65). The mechanism(s) whereby IFN-{gamma} priming enhances IL-12 production in IRF-1-/- and IRF-2-/- macrophages is unknown. One possibility is that other IRF family members, under the appropriate conditions, can partially compensate for a lack of IRF-1 or IRF-2 in knockout macrophages. This could occur either directly or indirectly, through the ability of "compensatory" IRFs to form protein-protein interactions with members of the Rel family or other transcription factors involved in IL-12 regulation. The ability of IRF-1 or IRF-2 to regulate other cytokine genes, which in turn, influence IFN-{gamma} priming of macrophages, is another likely possibility.

Although T cells and NK cells are considered the primary producers of IFN-{gamma}, macrophages have been shown to produce IFN-{gamma} in response to LPS, Mycobacterium tuberculosis, IL-12, and IL-18 (47, 48, 66, 67). Our data from IL-12 p40-/- macrophages suggests that the majority of LPS-induced IFN-{gamma} mRNA in peritoneal macrophage cultures is IL-12 mediated. Impaired IL-12 production in IRF-1-/- and IRF-2-/- macrophages was concomitant with reduced IFN-{gamma} mRNA expression. These data suggest that the IL-12 p70 measured by ELISA was bioactive and illustrate the importance of autocrine/paracrine production of IL-12 p70 in the up-regulation of macrophage IFN-{gamma}. Whether IRF-1 or IRF-2 is involved in IFN-{gamma} regulation directly is unknown.

Together, our findings indicate that IRF-1 and IRF-2 are important transcription factors in the regulation of IL-12, and as a consequence they have profound effects on IFN-{gamma} regulation. In vivo, IRF-1-/- mice, as do IL-12-/- mice, have impaired Th1 responses, have enhanced Th2 responses, and are more susceptible to intracellular pathogens like Leishmania major (45, 68). Understanding the molecular mechanism whereby IRF-1 and IRF-2 up-regulate IL-12 gene expression will provide new insights into approaches for enhancing host resistance as well as controlling autoimmune disorders and diseases like sepsis in which IL-12 promotes immunopathology.



View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 6. IFN-{gamma} pretreatment partially corrects impaired IL-12 mRNA expression in both IRF-1-/- and IRF-2-/- macrophages. To prime macrophages, monolayers were treated with medium or IFN-{gamma} (5 U/ml) for 24 h. Next, supernatants were removed and monolayers were treated with medium, IFN-{gamma} (5 U/ml), LPS (1 ng/ml), or both LPS plus IFN-{gamma} for 6 h as indicated in the figure. A representative Southern blot of RT-PCR-amplified products from IRF-1-/- and IRF-2-/- macrophages is shown in A and B, respectively (n = 3–5).

 

View this table:
[in this window]
[in a new window]
  		Table II. LPS-induced IL-12 production by IRF-2-/- macrophages after IFN-{gamma} priming1

 

   Acknowledgments
 
We thank Ms. Diana Miller for technical assistance.


   Footnotes
 
1 This work was supported by the Office of Naval Research and by National Institutes of Health Grant AI-18797. The opinions or assertions contained within are the private views of the authors and should not be construed as official or necessarily reflecting the views of Uniformed Services University of the Health Sciences or the Department of Defense. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council Department of Health and Human Services Publication (National Institutes of Health) 85-23. Back

2 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. Back

3 Abbreviations used in this paper: IRF, IFN-regulatory factor; ISRE, IFN-stimulated response element; ICSBP, IFN consensus sequence-binding protein; HKSA, heat-killed Staphylococcus aureus; HPRT, hypoxanthine-guanine phosphoribosyltransferase; GAS, IFN-{gamma} activation site. Back

Received for publication February 17, 1999. Accepted for publication May 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cassatella, M. A., L. Meda, S. Gasperini, A. D’Andrea, X. Ma, G. Trinchieri. 1995. Interleukin-12 production by human polymorphonuclear leukocytes. Eur. J. Immunol. 25:1.[Medline]
  2. D’Andrea, A., M. Rengaraju, N. M. Valiante, J. Chehimi, M. Kubin, M. Aste, S. H. Chan, M. Kobayashi, D. Young, E. Nickbarg, R. Chizzonite, S. F. Wolf, G. Trinchieri. 1992. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J. Exp. Med. 176:1387.[Abstract/Free Full Text]
  3. Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C. S. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, A. O’Garra. 1995. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J. Immunol. 154:5071.[Abstract]
  4. Kang, K., M. Kubin, K. D. Cooper, S. R. Lessin, G. Trinchieri, A. H. Rook. 1996. IL-12 synthesis by human Langerhans cells. J. Immunol. 156:1402.[Abstract]
  5. Skeen, M. J., M. A. Miller, T. M. Shinnick, H. K. Ziegler. 1996. Regulation of murine macrophage IL-12 production: activation of macrophages in vivo, restimulation in vitro, and modulation by other cytokines. J. Immunol. 156:1196.[Abstract]
  6. Murphy, E. E., G. Terres, S. E. Macatonia, C. S. Hsieh, J. Mattson, L. Lanier, M. Wysocka, G. Trinchieri, K. Murphy, A. O’Garra. 1994. B7 and interleukin 12 cooperate for proliferation and interferon {gamma} production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223.[Abstract/Free Full Text]
  7. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170:827.[Abstract/Free Full Text]
  8. Manetti, R., P. Parronchi, M. G. Giudizi, M. P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 (IL-12)) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177:1199.[Abstract/Free Full Text]
  9. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN-{gamma} production and type 1 cytokine responses. Immunity 4:471.[Medline]
  10. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, K. M. Murphy. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260:547.[Abstract/Free Full Text]
  11. Gately, M. K., L. M. Renzetti, J. Magram, A. S. Stern, L. Adorini, U. Gubler, D. H. Presky. 1998. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu. Rev. Immunol. 16:495.[Medline]
  12. de Jong, R., F. Altare, I. A. Haagen, D. G. Elferink, T. Boer, P. J. van Breda Vriesman, P. J. Kabel, J. M. Draaisma, J. T. van Dissel, F. P. Kroon, et al 1998. Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Science 280:1435.[Abstract/Free Full Text]
  13. Cooper, A. M., J. Magram, J. Ferrante, I. M. Orme. 1997. Interleukin 12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with Mycobacterium tuberculosis. J. Exp. Med. 186:39.[Abstract/Free Full Text]
  14. Wynn, T. A., I. Eltoum, I. P. Oswald, A. W. Cheever, A. Sher. 1994. Endogenous interleukin 12 (IL-12) regulates granuloma formation induced by eggs of Schistosoma mansoni and exogenous IL-12 both inhibits and prophylactically immunizes against egg pathology. J. Exp. Med. 179:1551.[Abstract/Free Full Text]
  15. Romani, L., A. Mencacci, L. Tonnetti, R. Spaccapelo, E. Cenci, P. Puccetti, S. F. Wolf, F. Bistoni. 1994. IL-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis. J. Immunol. 153:5167.[Abstract]
  16. Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, G. Trinchieri. 1995. Interleukin-12 is required for interferon-{gamma} production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
  17. Leonard, J. P., K. E. Waldburger, S. J. Goldman. 1995. Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J. Exp. Med. 181:381.[Abstract/Free Full Text]
  18. Finkelman, F. D., K. B. Madden, A. W. Cheever, I. M. Katona, S. C. Morris, M. K. Gately, B. R. Hubbard, W. C. Gause, Jr J. F. Urban. 1994. Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites. J. Exp. Med. 179:1563.[Abstract/Free Full Text]
  19. Schoenhaut, D. S., A. O. Chua, A. G. Wolitzky, P. M. Quinn, C. M. Dwyer, W. McComas, P. C. Familletti, M. K. Gately, U. Gubler. 1992. Cloning and expression of murine IL-12. J. Immunol. 148:3433.[Abstract]
  20. Yoshimoto, T., K. Kojima, T. Funakoshi, Y. Endo, T. Fujita, H. Nariuchi. 1996. Molecular cloning and characterization of murine IL-12 genes. J. Immunol. 156:1082.[Abstract]
  21. Ling, P., M. K. Gately, U. Gubler, A. S. Stern, P. Lin, K. Hollfelder, C. Su, Y. C. Pan, J. Hakimi. 1995. Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J. Immunol. 154:116.[Abstract]
  22. Ma, X., J. M. Chow, G. Gri, G. Carra, F. Gerosa, S. F. Wolf, R. Dzialo, G. Trinchieri. 1996. The interleukin 12 p40 gene promoter is primed by interferon {gamma} in monocytic cells. J. Exp. Med. 183:147.[Abstract/Free Full Text]
  23. Snijders, A., C. M. Hilkens, T. C. van der Pouw Kraan, M. Engel, L. A. Aarden, M. L. Kapsenberg. 1996. Regulation of bioactive IL-12 production in lipopolysaccharide-stimulated human monocytes is determined by the expression of the p35 subunit. J. Immunol. 156:1207.[Abstract]
  24. D’Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, G. Trinchieri. 1993. Interleukin 10 (IL-10) inhibits human lymphocyte interferon {gamma}-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041.[Abstract/Free Full Text]
  25. Muchamuel, T., S. Menon, P. Pisacane, M. C. Howard, D. A. Cockayne. 1997. IL-13 protects mice from lipopolysaccharide-induced lethal endotoxemia: correlation with down-modulation of TNF-{alpha}, IFN-{gamma}, and IL-12 production. J. Immunol. 158:2898.[Abstract]
  26. Du, C., S. Sriram. 1998. Mechanism of inhibition of LPS-induced IL-12 p40 production by IL-10 and TGF-{beta} in ANA-1 cells. J. Leukocyte Biol. 64:92.[Abstract]
  27. Murphy, T. L., M. G. Cleveland, P. Kulesza, J. Magram, K. M. Murphy. 1995. Regulation of interleukin 12 p40 expression through an NF-{kappa}B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
  28. Ma, X., M. Neurath, G. Gri, G. Trinchieri. 1997. Identification and characterization of a novel Ets-2-related nuclear complex implicated in the activation of the human interleukin-12 p40 gene promoter. J. Biol. Chem. 272:10389.[Abstract/Free Full Text]
  29. Hayes, M. P., F. J. Murphy, P. R. Burd. 1998. Interferon-{gamma}-dependent inducible expression of the human interleukin-12 p35 gene in monocytes initiates from a TATA-containing promoter distinct from the CpG-rich promoter active in Epstein-Barr virus-transformed lymphoblastoid cells. Blood 91:4645.[Abstract/Free Full Text]
  30. Barber, S. A., M. J. Fultz, C. A. Salkowski, S. N. Vogel. 1995. Differential expression of interferon regulatory factor 1 (IRF-1), IRF- 2, and interferon consensus sequence binding protein genes in lipopolysaccharide (LPS)-responsive and LPS-hyporesponsive macrophages. Infect. Immun. 63:601.[Abstract]
  31. Politis, A. D., J. Sivo, P. H. Driggers, K. Ozato, S. N. Vogel. 1992. Modulation of interferon consensus sequence binding protein mRNA in murine peritoneal macrophages: induction by IFN-{gamma} and down-regulation by IFN-{alpha}, dexamethasone, and protein kinase inhibitors. J. Immunol. 148:801.[Abstract]
  32. Kantakamalakul, W., A. D. Politis, S. Marecki, T. Sullivan, K. Ozato, M. J. Fenton, S. N. Vogel. 1999. Regulation of IFN consensus sequence binding protein expression in murine macrophages. J. Immunol. 162:7417.[Abstract/Free Full Text]
  33. Harada, H., K. Willison, J. Sakakibara, M. Miyamoto, T. Fujita, T. Taniguchi. 1990. Absence of the type I IFN system in EC cells: transcriptional activator (IRF-1) and repressor (IRF-2) genes are developmentally regulated. Cell 63:303.[Medline]
  34. Weisz, A., P. Marx, R. Sharf, E. Appella, P. H. Driggers, K. Ozato, B. Z. Levi. 1992. Human interferon consensus sequence binding protein is a negative regulator of enhancer elements common to interferon-inducible genes. J. Biol. Chem. 267:25589.[Abstract/Free Full Text]
  35. Nelson, N., M. S. Marks, P. H. Driggers, K. Ozato. 1993. Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferon-induced gene transcription. Mol. Cell. Biol. 13:588.[Abstract/Free Full Text]
  36. Drew, P. D., G. Franzoso, K. G. Becker, V. Bours, L. M. Carlson, U. Siebenlist, K. Ozato. 1995. NF{kappa}B and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class I gene expression. J. Interferon Cytokine Res. 15:1037.[Medline]
  37. Scharton-Kersten, T., C. Contursi, A. Masumi, A. Sher, K. Ozato. 1997. Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction. J. Exp. Med. 186:1523.[Abstract/Free Full Text]
  38. Taki, S., T. Sato, K. Ogasawara, T. Fukuda, M. Sato, S. Hida, G. Suzuki, M. Mitsuyama, E. H. Shin, S. Kojima, T. Taniguchi, Y. Asano. 1997. Multistage regulation of Th1-type immune responses by the transcription factor IRF-1. Immunity 6:673.[Medline]
  39. McIntire, F. C., H. W. Sievert, G. H. Barlow, R. A. Finley, A. Y. Lee. 1967. Chemical, physical, biological properties of a lipopolysaccharide from Escherichia coli K-235. Biochemistry 6:2363.[Medline]
  40. Matsuyama, T., T. Kimura, M. Kitagawa, K. Pfeffer, T. Kawakami, N. Watanabe, T. M. Kundig, R. Amakawa, K. Kishihara, A. Wakeham, et al 1993. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75:83.[Medline]
  41. Lakics, V., S. N. Vogel. 1998. Lipopolysaccharide and ceramide use divergent signaling pathways to induce cell death in murine macrophages. J. Immunol. 161:2490.[Abstract/Free Full Text]
  42. Salkowski, C. A., S. A. Barber, G. R. Detore, S. N. Vogel. 1996. Differential dysregulation of nitric oxide production in macrophages with targeted disruptions in IFN regulatory factor-1 and -2 genes. J. Immunol. 156:3107.[Abstract]
  43. Salkowski, C. A., S. N. Vogel. 1992. IFN-{gamma} mediates increased glucocorticoid receptor expression in murine macrophages. J. Immunol. 148:2770.[Abstract]
  44. Salkowski, C. A., G. R. Detore, S. N. Vogel. 1997. Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, {gamma}-interferon, and interleukin-10 mRNA production in murine macrophages. Infect. Immun. 65:3239.[Abstract]
  45. Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di Padova, R. Behin, M. K. Gately, J. A. Louis, G. Alber. 1996. Genetically resistant mice lacking interleukine-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26:1553.[Medline]
  46. Manthey, C. L., P. Y. Perera, C. A. Salkowski, S. N. Vogel. 1994. Taxol provides a second signal for murine macrophage tumoricidal activity. J. Immunol. 152:825.[Abstract]
  47. Fultz, M. J., S. A. Barber, C. W. Dieffenbach, S. N. Vogel. 1993. Induction of IFN-{gamma} in macrophages by lipopolysaccharide. Int. Immunol. 5:1383.[Abstract/Free Full Text]
  48. Puddu, P., L. Gantuzzi, P. Borghi, B. Varano, G. Rainaldi, E. Guillemard, W. Malorni, P. Nicaise, S. F. Wolf, F. Belardelli, S. Gessani. 1997. IL-12 induces IFN-{gamma} expression and secretion in mouse peritoneal macrophages. J. Immunol. 159:3490.[Abstract]
  49. Harada, H., T. Fujita, M. Miyamoto, Y. Kimura, M. Maruyama, A. Furia, T. Miyata, T. Taniguchi. 1989. Structurally similar but functionally distinct factors, IRF-1 and IRF- 2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell 58:729.[Medline]
  50. Harada, H., E. Takahashi, S. Itoh, K. Harada, T. A. Hori, T. Taniguchi. 1994. Structure and regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2 genes: implications for a gene network in the interferon system. Mol. Cell. Biol. 14:1500.[Abstract/Free Full Text]
  51. Cha, Y., A. B. Deisseroth. 1994. Human interferon regulatory factor 2 gene: intron-exon organization and functional analysis of 5'-flanking region. J. Biol. Chem. 269:5279.[Abstract/Free Full Text]
  52. Kanno, Y., C. A. Kozak, C. Schindler, P. H. Driggers, D. L. Ennist, S. L. Gleason, Jr J. E. Darnell, K. Ozato. 1993. The genomic structure of the murine ICSBP gene reveals the presence of the {gamma} interferon-responsive element, to which an ISGF3{alpha} subunit (or similar) molecule binds. Mol. Cell. Biol. 13:3951.[Abstract/Free Full Text]
  53. Neish, A. S., M. A. Read, D. Thanos, R. Pine, T. Maniatis, T. Collins. 1995. Endothelial interferon regulatory factor 1 cooperates with NF-{kappa}B as a transcriptional activator of vascular cell adhesion molecule 1. Mol. Cell. Biol. 15:2558.[Abstract]
  54. Faggioli, L., M. Merola, J. Hiscott, A. Furia, R. Monese, M. Tovey, M. Palmieri. 1997. Molecular mechanisms regulating induction of interleukin-6 gene transcription by interferon-{gamma}. Eur. J. Immunol. 27:3022.[Medline]
  55. Drew, P. D., M. Lonergan, M. E. Goldstein, L. A. Lampson, K. Ozato, D. E. McFarlin. 1993. Regulation of MHC class I and {beta}2-microglobulin gene expression in human neuronal cells: factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes. J. Immunol. 150:3300.[Abstract]
  56. Gri, G., D. Savio, G. Trinchieri, X. Ma. 1998. Synergistic regulation of the human interleukin-12 p40 promoter by NF{kappa}B and Ets transcription factors in Epstein-Barr virus-transformed B cells and macrophages. J. Biol. Chem. 273:6431.[Abstract/Free Full Text]
  57. Vaughan, P. S., F. Aziz, A. J. van Wijnen, S. Wu, H. Harada, T. Taniguchi, K. J. Soprano, J. L. Stein, G. S. Stein. 1995. Activation of a cell-cycle-regulated histone gene by the oncogenic transcription factor IRF-2. Nature 377:362.[Medline]
  58. Kopydlowski, K. M., C. A. Salkowski, M. J. Cody, J. Major, T. A. Hamilton, S. N. Vogel. 1999. Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J. Immunol. 163:0000.
  59. Toossi, Z., M. Mincek, E. Seeholtzer, S. A. Fulton, B. D. Hamilton, C. S. Hirsch. 1997. Modulation of IL-12 by transforming growth factor-{beta} (TGF-{beta}) in Mycobacterium tuberculosis-infected mononuclear phagocytes and in patients with active tuberculosis. J. Clin. Lab. Immunol. 49:59.[Medline]
  60. Nguyen, H., R. Lin, J. Hiscott. 1997. Activation of multiple growth regulatory genes following inducible expression of IRF-1 or IRF/RelA fusion proteins. Oncogene 15:1425.[Medline]
  61. Pine, R., A. Canova, C. Schindler. 1994. Tyrosine phosphorylated p91 binds to a single element in the ISGF2/IRF-1 promoter to mediate induction by IFN {alpha} and IFN {gamma}, and is likely to autoregulate the p91 gene. EMBO J. 13:158.[Medline]
  62. Donnelly, R. P., S. L. Freeman, M. P. Hayes. 1995. Inhibition of IL-10 expression by IFN-{gamma} up-regulates transcription of TNF-{alpha} in human monocytes. J. Immunol. 155:1420.[Abstract]
  63. Held, T. K., X. Weihua, L. Yuan, D. V. Kalvakolanu, A. S. Cross. 1999. {gamma} Interferon augments macrophage activation by lipopolysaccharide by two distinct mechanisms, at the signal transduction level and via an autocrine mechanism involving tumor necrosis factor {alpha} and interleukin-1. Infect. Immun. 67:206.[Abstract/Free Full Text]
  64. Brown, M. C., G. D. Tomaras, M. P. Vincenti, S. M. Taffet. 1997. Two forms of NF-{kappa} B1 (p105/p50) in murine macrophages: differential regulation by lipopolysaccharide, interleukin-2, and interferon-{gamma}. J. Interferon Cytokine Res. 17:295.[Medline]
  65. Shakhov, A. N., G. Woerly, B. D. Car, B. Ryffel. 1996. Interferon-{gamma} enhances tumor necrosis factor-{alpha} production by inhibiting early phase interleukin-10 transcription. Eur. Cytokine Network 7:741.[Medline]
  66. Fenton, M. J., M. W. Vermeulen, S. Kim, M. Burdick, R. M. Strieter, H. Kornfeld. 1997. Induction of {gamma}-interferon production in human alveolar macrophages by Mycobacterium tuberculosis. Infect. Immun. 65:5149.[Abstract]
  67. Munder, M., M. Mallo, K. Eichmann, M. Modolell. 1998. Murine macrophages secrete interferon {gamma} upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. 187:2103.[Abstract/Free Full Text]
  68. Lohoff, M., D. Ferrick, H. W. Mittrucker, G. S. Duncan, S. Bischof, M. Rollinghoff, T. W. Mak. 1997. Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 6:681.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
V. Vila-del Sol, C. Punzon, and M. Fresno
IFN-{gamma}-Induced TNF-{alpha} Expression Is Regulated by Interferon Regulatory Factors 1 and 8 in Mouse Macrophages
J. Immunol., October 1, 2008; 181(7): 4461 - 4470.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. A. Shirey, L. E. Cole, A. D. Keegan, and S. N. Vogel
Francisella tularensis Live Vaccine Strain Induces Macrophage Alternative Activation as a Survival Mechanism
J. Immunol., September 15, 2008; 181(6): 4159 - 4167.
[Abstract] [Full Text] [PDF]