Department of Medical Microbiology and Immunology, University of Aarhus, The Bartholin Building, DK-8000 Aarhus C, Denmark1
Author for correspondence: Svend Ellermann-Eriksen. Fax +45 8619 6128. e-mail ellermann{at}microbiology.au.dk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The importance of IL-12 production is well characterized for infections in mice with intracellular bacteria like Listeria monocytogenes (Hsieh et al., 1993 ; Tripp et al., 1994
) and intracellular parasites like Toxoplasma gondii (Gazzinelli et al., 1994
). These pathogens induce production of IL-12 which in turn is responsible for the early production of IFN-
by NK cells. Neutralization of IL-12 during such infections results in increased susceptibility and higher microbial burden, which shows that IL-12 is important for resistance against these intracellular bacteria and parasites. Much less is known about the production and function of IL-12 during virus infections, even though these pathogens also have an obligate intracellular habit. It has been best documented for infections with murine cytomegalovirus (MCMV) and influenza A virus that IL-12 is indeed produced during a virus infection and is responsible for the early production of IFN-
and primary control of the infection (Orange & Biron, 1996a
, b
; Carr et al., 1999
; Monteiro et al., 1998
). On the other hand, IL-12 is not produced during infection with lymphocytic choriomeningitis virus (LCMV) (Orange & Biron, 1996a
), and despite detection of IL-12 p40 mRNA during infection with murine hepatitis virus (MHV) (Schijns et al., 1996
, 1998
; Coutelier et al., 1995
), IL-12 knock-out mice exhibit an unaltered resistance against this infection (Schijns et al., 1998
). During an ocular herpes simplex virus (HSV)-1 infection of BALB/c mice, IL-12 p40 mRNA and protein was detected in lysates of cornea and local lymph nodes, and mRNA was detected in spleen and peritoneal cells infected in vitro (Kanangat et al., 1996
). Furthermore IL-12 p40 mRNA has also been detected in mice infected with lactate dehydrogenase elevating virus and adenovirus (Coutelier et al., 1995
).
IL-12 is a heterodimeric protein composed of the two subunits p35 and p40 (Kobayashi et al., 1989 ), encoded by separate genes. Expression of the biologically active heterodimer p70 is associated with a large excess of the monomer p40, whereas p35 only exists as part of the heterodimer (DAndrea et al., 1992
). Production of the p40 monomer is inducible by, e.g., lipopolysaccharide (LPS) via transcriptionally regulated mechanisms requiring de novo protein synthesis (Murphy et al., 1995
; Ma et al., 1996a
). Moreover, this induction is among other things dependent on interaction between transcription factors of the NF-
B family and an NF-
B halfsite element in the p40 promoter (Murphy et al., 1995
). Production of p35 is transcriptionally as well as translationally regulated (Ma et al., 1996a
; Babik et al., 1999
). Despite the fact that many cell types express p35 mRNA constitutively (DAndrea et al., 1992
), expression of this subunit might be limiting for the production of the heterodimer p70 (Snijders et al., 1996
; Babik et al., 1999
).
Here we show that infection with HSV-2 induces secretion of IL-12 p40 protein in murine macrophages and production of biologically active IL-12 p70, reflected by the inhibition of HSV-2-induced IFN- production by neutralization of IL-12. Furthermore, our data show that this induction requires de novo synthesis of viral and/or cellular intermediary factors, and that induction of IL-12 p40 expression by HSV-2 infection is dependent on NF-
B activation.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice.
Inbred, specific pathogen-free BALB/cABOM mice were obtained from Bomholtgaard Animal Breeding and Research Centre. Female mice were used at the age of 812 weeks, but for individual experiments only mice born within a single week were used.
Cell cultures.
The murine macrophage cell line J774A.1 (ATCC TIB 67) was grown in Dulbeccos modified Eagles medium with 1% Glutamax I (Life Technologies), supplemented with 5% LPS-free FCS, 200 IU/ml penicillin and 200 µg/ml streptomycin. Elicited peritoneal cells from BALB/c mice were obtained as previously described by injection of 2·0 ml 10% thioglycollate intraperitoneally 4 days before harvest of the cells by lavage of the peritoneum with cold PBS, pH 7·4, supplemented with 2% FCS and 200 IU/ml heparin (Baskin et al., 1997 ). After washing the cells were counted and grown in RPMI 1640 medium (BioWhittaker) supplemented with 10 mM glutamine, 2 mM HEPES and FCS and antibiotics as above.
For induction of cytokines cells were seeded in 96-well tissue culture plates to give a final concentration of 5x105 and 1·5x106 cells/ml in 200 µl RPMI medium and left to settle for 2 h and overnight for J774A.1 cells and peritoneal cells, respectively. After infection or treatment with cytokines for 24 h at 37 °C in a humidified atmosphere with 5% CO2 the supernatants were harvested for ELISA.
For isolation of total RNA J774A.1 cells were seeded in 10 cm2 tissue culture plates at a density of 2·5x106 cells per plate and allowed to settle for 2 h. The cultures were stimulated and infected 5 h before RNA was extracted.
Cytokines, antibodies and reagents.
Recombinant murine cytokines were used at the following concentrations: IL-12 p40 (PharMingen), 3·92000 pg/ml; IFN- for ELISA (R&D), 3·92000 pg/ml; IFN-
for stimulation of cells (PharMingen), 100 IU/ml (1·6 ng/ml); IFN-
/
(PBL Biomedical Laboratories, cat. no. 12100-1), 1000 IU/ml (232 ng/ml); TNF-
(Genzyme), 500 U/ml (12·4 ng/ml); IL-1
(R&D) 15 U/ml (75 ng/ml). The following neutralizing antibodies against murine cytokines were used: monoclonal rat anti IL-12 p40/p70 (clone C17.1, PharMingen), 100 NU/ml (10 µg/ml), and a corresponding purified rat IgG2a (PharMingen); polyclonal sheep anti-IFN-
/
(PBL Biomedical Laboratories), 1000 NU/ml; polyclonal goat anti-IL-1
/IL-1
(R&D) 100 NU/ml; polyclonal rabbit anti-TNF-
(Genzyme), 500 NU/ml. Antibodies against murine cytokines for ELISAs: monoclonal rat anti-IL-12 p40/p70 (clone C15.6, PharMingen); biotin-labelled monoclonal rat anti IL-12 p40/p70 (clone C17.1, PharMingen); rat anti-IFN-
(R&D), biotin-labelled goat anti-IFN-
(R&D).
N--tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-acetyl-L-cysteine (NAC), pyrrolidine dithiocarbamate (PDTC) and cycloheximide were purchased from Sigma. Trizol was from Gibco. Oligo(dT)15 primer, Expand reverse transcriptase, deoxynucleotide triphosphates and poly[d(I-C)] were from Boehringer Mannheim. Taq 2000 DNA polymerase and T4 polynucleotide kinase were purchased from Stratagene. The DNA oligonucleotides were provided by DNA Technology.
RNA extraction and RTPCR.
RNA was extracted with Trizol, following the recommendations of the manufacturer. Briefly, Trizol and chloroform were added and the phases were separated by centrifugation. RNA was pelleted by addition of 2-propanol and centrifugation. Finally, the RNA pellet was washed with ethanol and redissolved in RNase-free water. Using oligo(dT)15 as primer, the RNA (12 µg per reaction) was subjected to reverse transcription with Expand Reverse Transcriptase according to the manufacturers recommendations. To amplify specific cDNA the following primers were used for the PCR reactions: IL-12 p40, 5' CCA CTC ACA TCT GCT GCT CCA CAA 3' (sense), 5' CAG TTC AAT GGG CAG GGT CTC CTC 3' (antisense); -actin, 5' CCA ACC GTG AAA AGA TGA CC 3' (sense), 5' GCA GTA ATC TCC TTC TGC ATC C 3' (antisense). The products spanned 336 bp (IL-12 p40) and 616 bp (
-actin), respectively. For PCR amplification of cDNA 35 cycles and an annealing temperature of 55 °C were used for both p40 and
-actin.
Cytokine measurements.
Murine IL-12 p40 and IFN- were detected by ELISA. For detection of IL-12 p40 Maxisorp plates (Nunc) were coated overnight at 4 °C with anti-IL-12 p40/p70 (clone C15.6), 6 µg/ml in coating buffer [15 mM Na2CO3; 35 mM NaHCO3; 0·2% sodium azide, pH 9·6]. After blocking for 3 h at 20 °C with PBS pH 7·4 containing 1% (w/v) BSA samples and standard dilutions of IL-12 p40 (3·92000 pg/ml) were added to the wells and the plates were incubated at 4 °C overnight. Subsequently, the wells were incubated for 2 h at 20 °C with a biotin-labelled IL-12 p40 detection-antibody (clone C17.8) at a concentration of 2 µg/ml in blocking buffer. For development HRP-conjugated streptavidin, diluted 1:1000 in blocking buffer, was added and incubated for 1 h at 20 °C, after which 0·5 mg OPD/ml substrate buffer [7%, w/v, C6H8O7; 23·9%, w/v, Na2HPO4] supplemented with 0·03% (v/v) H2O2 was added to the wells. After 10 min the colour reaction was stopped with 5% (v/v) H2SO4. Between each step the plates were washed three times with PBS containing 0·05% (v/v) Tween 20. The detection limit of the ELISA assay was 3·9 pg/ml. ELISA for detection of IFN-
was performed with a monoclonal antibody Duoset (R&D Systems), and the protocol recommended by the manufacturer was followed. This assay also had a detection limit of 3·9 pg/ml.
Isolation of nuclear proteins.
To isolate nuclear proteins, cells were washed twice in ice-cold PBS, scraped into 5 ml PBS and centrifuged for 1 min at 2000 g. Thereafter, the cells were resuspended in hypotonic lysis buffer [20 mM HEPES, pH 7·9; 1·5 mM MgCl2; 10 mM KCl; 0·2 mM EDTA; 0·5 mM DTT; 0·2 mM PMSF; 0·2 mM leupeptin; 0·2 mM pepstatin A; 0·1 mM Na3VO4] and left for 15 min on ice, after which NP-40 was added to a final concentration of 0·6%, and the suspension was vortexed for 15 s. The nuclei were recovered by centrifugation (10000 g, 1 min) after which they were resuspended in 40 µl extraction buffer [20 mM HEPES, pH 7·9; 20% glycerol; 1·5 mM MgCl2; 420 mM NaCl; 0·2 mM EDTA; 0·5 mM DTT; 0·5 mM PMSF; 0·2 mM leupeptin; 0·2 mM pepstatin A; 0·1 mM Na3VO4; 0·2% NP-40] and left for 30 min. Supernatants containing nuclear proteins were clarified by centrifugation for 15 min at 10000 g.
Electrophoretic mobility shift assay (EMSA).
To assay for DNA-binding activity 3 µl nuclear extract was mixed with 3 µg poly[d(I-C)] and 20000 c.p.m. 32P-end-labelled probe in 25 µl. The final concentrations for the B assay were: 23·3 mM HEPES; 50 mM NaCl; 5 mM MgCl2; 0·8 mM EDTA; 4 mM Tris-HCl; 1·2 mM DTT; 14·2% glycerol. After incubation for 25 min at room temperature, the reaction mixture was subjected to electrophoresis in a non-denaturing 5% polyacrylamide gel in 0·5x TBE buffer [45 mM Tris base, 45 mM boric acid, 1 mM EDTA]. The gel was dried and analysed by autoradiography. Probes used were the
B halfsite of the murine IL-12 p40 promoter (5' CTT AAA ATT CCC CCA GAA TGT TTT G 3') and a mutant
B probe (5' CTT AAA CGG AAC CCA GAA TGT TTT G 3'; mutated sequence is underlined).
Statistical analysis.
Statistical analysis of numerical results was done with Studentss t-test for equal variances, and variance homogeneity was tested by the F-test. Synergism was tested as previously described (Ellermann-Eriksen, 1993 ) on values obtained from a culture minus the mean of controls (medium only). The distribution of values from double-treated cells was compared with the distribution derived from addition of the two distributions from single-treated cells.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Endogenous IFN-/
, TNF-
and IL-1
/
are not responsible for the induction of IL-12 p40 production
The intermediary proteins required for IL-12 p40 production could be host-derived factors such as transcription factors or cytokines, viral proteins or both. We have previously shown that virus-induced TNF- is responsible for expression of inducible nitric oxide synthase during an HSV-2 infection (Baskin et al., 1997
), and we therefore wanted to examine if macrophage-derived cytokines are also involved in induction of IL-12 p40. IFN-
/
, TNF-
and IL-1 are all produced by murine macrophages early during infection with HSV-2 (Ellermann-Eriksen et al., 1986
; Ellermann-Eriksen, 1993
; unpublished results). Their role in IL-12 p40 production was investigated by stimulation with recombinant cytokines and by neutralization of endogenously produced cytokines with specific antibodies. Of the three cytokines examined (Table 1
), only TNF-
had an IL-12 p40-inducing effect of its own (2P=0·01), which was not further enhanced by IFN-
treatment (2P=0·23). IFN-
did not significantly inhibit IL-12 p40 production (2P=0·35), as seen in previous studies (Cousens et al., 1997
, 1999
), and none of the three cytokines was able to synergize with HSV-2 infection or IFN-
treatment. The only synergism seen was that between HSV-2 and IFN-
(2P=0·03, 0·05 and 0·01 for the three experiments respectively). Addition of neutralizing antibodies against the three cytokines further substantiated that the ability of HSV-2 to induce IL-12 p40 production was not to any major extend dependent on intermediary production of these cytokines.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Macrophages represent one of the main sources of early cytokine production during virus infections. For instance, numerous reports have shown that IFN-/
, TNF-
and IL-1 are rapidly produced by macrophages in response to virus infections (Ellermann-Eriksen et al., 1986
; Macatonia et al., 1995
; Ellermann-Eriksen, 1993
; Sareneva et al., 1998
). By contrast, less is known about the production and function of another macrophage-produced cytokine, IL-12, during virus infections.
In this study we have shown that in vitro infection of murine peritoneal cells and the macrophage cell line J774A.1 with HSV-2 induces secretion of IL-12 p40, which is accompanied by secretion of biologically active IL-12 p70. Our experiments did not address the question of whether IL-12 is produced by infected macrophages or by uninfected bystanders. The secretion of IL-12 p40 protein in response to HSV-2 was regulated at the transcriptional level as seen from enhanced IL-12 p40 mRNA accumulation. Similar results have been obtained by Kanangat et al. (1996) during in vitro infection of spleen cells and peritoneal macrophages with HSV-1. In an in vivo infection model with MCMV Orange & Biron (1996a
, b
) were able to detect IL-12 both in serum of infected mice and in supernatants of spleen cell cultures established from infected mice. Essentially similar results have also been obtained in a murine model of influenza A virus infection (Monteiro et al., 1998
).
In addition we found that IFN- synergizes with HSV-2 in IL-12 p40 production. This is in agreement with studies using other IL-12-inducing stimuli such as LPS, poly(I-C), heat-killed L. monocytogenes and infection with live L. monocytogenes and Mycobacterium bovis BCG (Skeen et al., 1996
; Flesch et al., 1995
). The synergistic effect was maximal when IFN-
and HSV-2 were administered simultaneously and was not further enhanced by priming of the cells with IFN-
for up to 20 h before virus infection (data not shown). This is in contrast to the effect of IFN-
pretreatment on IL-12 production induced by other stimuli like LPS and BCG infection (Ma et al., 1996b
; Flesch et al., 1995
). Possibly this phenomenon might reflect a general difference in the mechanism of induction executed by bacteria or bacterial products versus a viral infection, requiring a lag time for expression of the inducing signal.
As to the functional implications of IL-12 expression during virus infection our data demonstrate that HSV-2-induced IL-12 is partly responsible for induction of IFN- production during infection of macrophages in vitro, since neutralizing antibodies against IL-12 p70 were able to reduce IFN-
secretion by at least 50%. Similar conclusions were reached by Orange & Biron (1996a
, b
) and Monteiro et al. (1998)
after in vivo infection of mice with MCMV and influenza A virus, respectively.
Infection of peritoneal and J774A.1 cells with UV-irradiated HSV-2 did not result in secretion of IL-12 p40. Similar findings with HSV-1 have been reported by Kanagat et al. (1996). This indicates that factors present in the virus particle, for example surface glycoproteins, are not sufficient to stimulate secretion of IL-12 p40, as has been reported for virus-induced secretion of IFN-/
(Ellermann-Eriksen, 1993
; Ankel et al., 1998
). Moreover, the ability of HSV-2 and IFN-
to activate IL-12 p40 transcription required de novo production of intermediary factors, as shown by sensitivity of the induction to cycloheximide. These factors could be either virus-induced cellular proteins and/or viral proteins. In either case they need to be produced early after infection, since IL-12 p40 mRNA can be detected after just 2 to 4 h (data not shown). In the light of this assumption early virus-induced cytokines represent good candidates for such intermediary factors. By stimulation with IFN-
, TNF-
and IL-1
and neutralization of endogenously produced cytokines by specific antibodies we concluded that none of the mentioned cytokines seem to play any major role as intermediary factors in virus-induced production of IL-12. Although it has been documented that IFN-
/
is able to inhibit IL-12 production both in vitro and in vivo (Cousens et al., 1997
, 1999
), we did not observe this effect either when stimulating with recombinant IFN-
or when neutralizing endogenously produced IFN-
/
during infection with HSV-2. The concentrations of IFN-
used in the studies of Cousens et al. (1997
, 1999
) and in our experiments were within the same range. Likewise, differences in the preparations of IFN-
used seems unlikely to be the explanation for the diverging results. The discrepancy could most likely reflect the different cell cultures in use, since we have used a macrophage cell line for our experiment whereas the other studies were done with mixed cell cultures like spleen leukocytes.
An alternative possibility would be that early produced viral factors, like products of the immediate early (IE) HSV-2 genes, are responsible for activation of IL-12 p40 expression. The HSV-1 IE proteins ICP4 and ICP27 have been reported to be required for sustained activation of NF-B following infection (Patel et al., 1998
). We are currently investigating this possibility. The requirement for de novo protein synthesis is also seen for activation of the IL-12 p40 promoter with other inducers like LPS or Staphylococcus aureus strain Cowan (SAC) (Aste-Amezaga et al., 1998
). This observation argues against the direct involvement of HSV-encoded factors and suggests a role for a common cellular signalling pathway in the IL-12 p40 induction by various stimuli.
The dissection of the molecular mechanisms in regulation of the IL-12 p40 promoter by pathogens and by cytokines has not been completed yet. Several promoter elements seem to be involved and activation is probably influenced by complex interactions between different transcription factors. Among these NF-B has been shown to play a central role. A number of independent studies have identified an NF-
B-responsive element in the murine and human IL-12 p40 promoter and have demonstrated protein-binding to this sequence during induction with bacteria or bacterial products like SAC, heat-killed L. monocytogenes and LPS (Murphy et al., 1995
; Plevy et al., 1997
; Gri et al., 1998
) as well as by a non-bacterial inducer, CD40 ligand (Yoshimoto et al., 1997
). By supershift assays some of these proteins were identified as members of the NF-
B/Rel family. Involvement of the NF-
B/Rel family of transcription factors has also been demonstrated functionally by use of inhibitors of NF-
B activation (Kang et al., 1999
; DAmbrosio et al., 1998
; Mazzeo et al., 1998
; Na et al., 1999
). In accordance with these studies we demonstrate virus-induced protein-binding to a sequence identical to the NF-
B element of the IL-12 p40 promoter. Specificity of this binding was confirmed by competition, where an identical cold
B-probe, but not a mutated cold
B-probe, could compete for binding. Furthermore, the functional relevance of the activation of NF-
B was tested by using different inhibitors of the activation. All three inhibitors (TPCK, NAC, PDTC), with different modes of action, effected a significant reduction in the virus-induced induction of IL-12 p40 production.
IFN- is an important component of the host defence against an invading virus, for instance by its ability to activate macrophages (Mogensen & Virelizier, 1987
). Specifically, IFN-
is known to play a pivotal role in host defence against HSV infections (Cantin et al., 1999
; Parr & Parr, 1999
; Yu et al., 1996
). Our data show that during in vitro infection HSV-2 is able to induce production of IL-12, which in turn is responsible for part of the IFN-
secretion. However, production of IL-12 during viral infection does not seem to be a general feature. Experimental murine infection with LCMV and infection of human blood cells with influenza A virus were not associated with secretion of IL-12 (Orange & Biron, 1996a
; Sareneva et al., 1998
). Furthermore, IL-12 produced endogenously during virus infections has been shown not to be essential for the late IFN-
production by T cells (Monteiro et al., 1998
; Orange & Biron, 1996a
). This indicates that other factors like IL-18 and IFN-
/
are involved in regulation of IFN-
production (Sareneva et al., 1998
; Cousens et al., 1997
, 1999
). The ability of IL-18 to synergize with both IL-12 and IFN-
/
for induction of IFN-
secretion (Sareneva et al., 1998
) supports the idea of redundancy between these cytokines in IFN-
induction.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aste-Amezaga, M., Ma, X., Sartori, A. & Trinchieri, G.(1998). Molecular mechanisms of the induction of IL-12 and its inhibition by IL-10.Journal of Immunology160, 5936-5944.
Babik, J. M., Adams, E., Tone, Y., Fairchild, P. J., Tone, M. & Waldmann, H.(1999). Expression of murine IL-12 is regulated by translational control of the p35 subunit.Journal of Immunology162, 4069-4078.
Baskin, H., Ellermann-Eriksen, S., Lovmand, J. & Mogensen, S. C.(1997). Herpes simplex virus type 2 synergizes with interferon- in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-
.Journal of General Virology78, 195-203.[Abstract]
Biron, C. A.(1998). Role of early cytokines, including and
interferons (IFN-
/
), in innate and adaptive immune responses to viral infections.Seminars in Immunology10, 383-390.[Medline]
Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. & Salazar-Mather, T. P.(1999). Natural killer cells in antiviral defense: function and regulation by innate cytokines.Annual Review of Immunology17, 189-220.[Medline]
Cantin, E., Tanamachi, B., Openshaw, H., Mann, J. & Clarke, K.(1999). Gamma interferon (IFN-) receptor null-mutant mice are more susceptible to herpes simplex virus type 1 infection than IFN-
ligand null-mutant mice. Journal of Virology73, 5196-5200.
Carr, J. A., Rogerson, J., Mulqueen, M. J., Roberts, N. A. & Booth, R. F.(1997). Interleukin-12 exhibits potent antiviral activity in experimental herpesvirus infections.Journal of Virology71, 7799-7803.[Abstract]
Carr, J. A., Rogerson, J., Mulqueen, M. J., Roberts, N. A. & Nash, A. A.(1999). The role of endogenous interleukin-12 in resistance to murine cytomegalovirus (MCMV) infection and a novel action for endogenous IL-12 p40.Journal of Interferon and Cytokine Research 19, 1145-1152.[Medline]
Cousens, L. P., Orange, J. S., Su, H. C. & Biron, C. A.(1997). Interferon-/
inhibition of interleukin 12 and interferon-
production in vitro and endogenously during viral infection. Proceedings of the National Academy of Sciences, USA94, 634-639.
Cousens, L. P., Peterson, R., Hsu, S., Dorner, A., Altman, J. D., Ahmed, R. & Biron, C. A.(1999). Two roads diverged: interferon /
- and interleukin-12-mediated pathways in promoting T cell interferon
responses during viral infection. Journal of Experimental Medicine189, 1315-1328.
Coutelier, J. P., Van Broeck, J. & Wolf, S. F.(1995). Interleukin-12 gene expression after viral infection in the mouse.Journal of Virology69, 1955-1958.[Abstract]
DAmbrosio, D., Cippitelli, M., Cocciolo, M. G., Mazzeo, D., Di Lucia, P., Lang, R., Sinigaglia, F. & Panina-Bordignon, P.(1998). Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-B downregulation in transcriptional repression of the p40 gene. Journal of Clinical Investigation101, 252-262.
DAndrea, A., Rengaraju, M., Valiante, N. M., Chehimi, J., Kubin, M., Aste, M., Chan, S. H., Kobayashi, M., Young, D. & Nickbarg, E.(1992). Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells.Journal of Experimental Medicine176, 1387-1398.[Abstract]
Ellermann-Eriksen, S.(1993). Autocrine secretion of interferon-/
and tumour necrosis factor-
synergistically activates mouse macrophages after infection with herpes simplex virus type 2.Journal of General Virology74, 2191-2199.[Abstract]
Ellermann-Eriksen, S., Liberto, M. C., Iannello, D. & Mogensen, S. C.(1986). X-linkage of the early in vitro /
interferon response of mouse peritoneal macrophages to herpes simplex virus type 2.Journal of General Virology67, 1025-1033.[Abstract]
Flesch, I. E., Hess, J. H., Huang, S., Aguet, M., Rothe, J., Bluethmann, H. & Kaufmann, S. H.(1995). Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon and tumor necrosis factor
.Journal of Experimental Medicine181, 1615-1621.[Abstract]
Gazzinelli, R. T., Wysocka, M., Hayashi, S., Denkers, E. Y., Hieny, S., Caspar, P., Trinchieri, G. & Sher, A.(1994). Parasite-induced IL-12 stimulates early IFN- synthesis and resistance during acute infection with Toxoplasma gondii.Journal of Immunology153, 2533-2543.
Gri, G., Savio, D., Trinchieri, G. & Ma, X.(1998). Synergistic regulation of the human interleukin-12 p40 promoter by NF-B and Ets transcription factors in Epstein-Barr virus-transformed B cells and macrophages.Journal of Biological Chemistry273, 6431-6438.
Hayes, M. P., Wang, J. & Norcross, M. A.(1995). Regulation of interleukin-12 expression in human monocytes: selective priming by interferon- of lipopolysaccharide-inducible p35 and p40 genes. Blood86, 646-650.
Heinzel, F. P., Schoenhaut, D. S., Rerko, R. M., Rosser, L. E. & Gately, M. K.(1993). Recombinant interleukin 12 cures mice infected with Leishmania major.Journal of Experimental Medicine177, 1505-1509.[Abstract]
Heise, M. T. & Virgin, H. W.(1995). The T-cell-independent role of interferon and tumor necrosis factor-
in macrophage activation during murine cytomegalovirus and herpes simplex virus infections.Journal of Virology69, 904-909.[Abstract]
Heufler, C., Koch, F., Stanzl, U., Topar, G., Wysocka, M., Trinchieri, G., Enk, A., Steinman, R. M., Romani, N. & Schuler, G.(1996). Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon- production by T helper 1 cells.European Journal of Immunology26, 659-668.[Medline]
Hsieh, C. S., Macatonia, S. E., Tripp, C. S., Wolf, S. F., OGarra, A. & Murphy, K. M.(1993). Development of TH1 CD4+T cells through IL-12 produced by Listeria-induced macrophages.Science260, 547-549.[Medline]
Kanangat, S., Thomas, J., Gangappa, S., Babu, J. S. & Rouse, B. T.(1996). Herpes simplex virus type 1-mediated up-regulation of IL-12 (p40) mRNA expression. Implications in immunopathogenesis and protection.Journal of Immunology156, 1110-1116.[Abstract]
Kang, B. Y., Chung, S. W., Im, S. Y., Hwang, S. Y. & Kim, T. S.(1999). Chloromethyl ketones inhibit interleukin-12 production in mouse macrophages stimulated with lipopolysaccharide.Immunology Letters70, 135-138.[Medline]
Kobayashi, M., Fitz, L., Ryan, M., Hewick, R. M., Clark, S. C., Chan, S., Loudon, R., Sherman, F., Perussia, B. & Trinchieri, G.(1989). Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes.Journal of Experimental Medicine170, 827-845.[Abstract]
Ma, X., Aste-Amezaga, M. & Trinchieri, G.(1996a). Regulation of interleukin-12 production.Annals of the New York Academy of Sciences 795, 13-25.[Medline]
Ma, X., Chow, J. M., Gri, G., Carra, G., Gerosa, F., Wolf, S. F., Dzialo, R. & Trinchieri, G.(1996b). The interleukin 12 p40 gene promoter is primed by interferon in monocytic cells. Journal of Experimental Medicine183, 147-157.[Abstract]
Macatonia, S. E., Hosken, N. A., Litton, M., Vieira, P., Hsieh, C. S., Culpepper, J. A., Wysocka, M., Trinchieri, G., Murphy, K. M. & OGarra, A.(1995). Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells.Journal of Immunology154, 5071-5079.
Mazzeo, D., Panina-Bordignon, P., Recalde, H., Sinigaglia, F. & DAmbrosio, D.(1998). Decreased IL-12 production and Th1 cell development by acetyl salicylic acid-mediated inhibition of NF-B.European Journal of Immunology28, 3205-3213.[Medline]
Mogensen, S. C.(1984). Host defences in mice against infections with herpes simplex virus.Microbiological Sciences1, 127-130.[Medline]
Mogensen, S. C. & Virelizier, J. L.(1987). The interferon-macrophage alliance.Interferon8, 55-84.[Medline]
Monteiro, J. M., Harvey, C. & Trinchieri, G.(1998). Role of interleukin-12 in primary influenza virus infection.Journal of Virology72, 4825-4831.
Murphy, T. L., Cleveland, M. G., Kulesza, P., Magram, J. & Murphy, K. M.(1995). Regulation of interleukin 12 p40 expression through an NF-B half-site.Molecular and Cellular Biology15, 5258-5267.[Abstract]
Na, S. Y., Kang, B. Y., Chung, S. W., Han, S. J., Ma, X., Trinchieri, G., Im, S. Y., Lee, J. W. & Kim, T. S.(1999). Retinoids inhibit interleukin-12 production in macrophages through physical associations of retinoid X receptor and NF-B.Journal of Biological Chemistry274, 7674-7680.
Orange, J. S. & Biron, C. A.(1996a). An absolute and restricted requirement for IL-12 in natural killer cell IFN- production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections.Journal of Immunology156, 1138-1142.[Abstract]
Orange, J. S. & Biron, C. A.(1996b). Characterization of early IL-12, IFN-/
, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. Journal of Immunology156, 4746-4756.
Parr, M. B. & Parr, E. L.(1999). The role of interferon in immune resistance to vaginal infection by herpes simplex virus type 2 in mice.Virology258, 282-294.[Medline]
Patel, A., Hanson, J., McLean, T. I., Olgiate, J., Hilton, M., Miller, W. E. & Bachenheimer, S. L.(1998). Herpes simplex type 1 induction of persistent NF-B nuclear translocation increases the efficiency of virus replication.Virology247, 212-222.[Medline]
Plevy, S. E., Gemberling, J. H., Hsu, S., Dorner, A. J. & Smale, S. T.(1997). Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins.Molecular and Cellular Biology17, 4572-4588.[Abstract]
Sareneva, T., Matikainen, S., Kurimoto, M. & Julkunen, I.(1998). Influenza A virus-induced IFN-/
and IL-18 synergistically enhance IFN-
gene expression in human T cells.Journal of Immunology160, 6032-6038.
Schijns, V. E., Wierda, C. M., van Hoeij, M. & Horzinek, M. C.(1996). Exacerbated viral hepatitis in IFN- receptor-deficient mice is not suppressed by IL-12.Journal of Immunology157, 815-821.[Abstract]
Schijns, V. E., Haagmans, B. L., Wierda, C. M., Kruithof, B., Heijnen, I. A., Alber, G. & Horzinek, M. C.(1998). Mice lacking IL-12 develop polarized Th1 cells during viral infection.Journal of Immunology160, 3958-3964.
Skeen, M. J., Miller, M. A., Shinnick, T. M. & Ziegler, H. K.(1996). Regulation of murine macrophage IL-12 production. Activation of macrophages in vivo, restimulation in vitro, and modulation by other cytokines. Journal of Immunology156, 1196-1206.[Abstract]
Snijders, A., Hilkens, C. M., van der Pouw Kraan, T. C., Engel, M., Aarden, L. A. & Kapsenberg, M. L.(1996). Regulation of bioactive IL-12 production in lipopolysaccharide-stimulated human monocytes is determined by the expression of the p35 subunit. Journal of Immunology156, 1207-1212.[Abstract]
Stern, A. S., Podlaski, F. J., Hulmes, J. D., Pan, Y. C., Quinn, P. M., Wolitzky, A. G., Familletti, P. C., Stremlo, D. L., Truitt, T. & Chizzonite, R.(1990). Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.Proceedings of the National Academy of Sciences, USA87, 6808-6812.[Abstract]
Trinchieri, G.(1995). Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity.Annual Review of Immunology13, 251-276.[Medline]
Tripp, C. S., Gately, M. K., Hakimi, J., Ling, P. & Unanue, E. R.(1994). Neutralization of IL-12 decreases resistance to Listeria in SCID and C.B-17 mice. Reversal by IFN-.Journal of Immunology152, 1883-1887.
Yoshimoto, T., Nagase, H., Ishida, T., Inoue, J. & Nariuchi, H.(1997). Induction of interleukin-12 p40 transcript by CD40 ligation via activation of nuclear factor-B. European Journal of Immunology27, 3461-3470.[Medline]
Yu, Z., Manickan, E. & Rouse, B. T.(1996). Role of interferon- in immunity to herpes simplex virus.Journal of Leukocyte Biology60, 528-532.[Abstract]
Received 15 June 2000;
accepted 31 August 2000.