IFN-␣-Dependent, IFN-␥ Secretion by Bone Marrow-Derived Macrophages Controls an Intracellular Bacterial Infection Antonio Gigliotti Rothfuchs,* Dulceaydee Gigliotti,*† Karin Palmblad,‡§ Ulf Andersson,‡§ Hans Wigzell,* and Martı´n E. Rottenberg1* Several reports have indicated that cell lineages apart from NK and T cells can also express IFN-␥. However, the biological relevance of this finding is uncertain. We show in this study that bone marrow-derived macrophages (BMMs) express IFN-␥ at the mRNA and protein level early after infection with Chlamydia pneumoniae. Increased IFN-␥ mRNA accumulation by infected BMMs is early, transient, and requires both bacterial and host protein synthesis. The induction of IFN-␥ mRNA levels is independent of IL-12 and was dramatically enhanced in IL-10ⴚ/ⴚ BMMs. Such IL-10ⴚ/ⴚ BMMs contained less bacteria than the wild-type controls, whereas IFN-␥Rⴚ/ⴚ BMMs showed increased C. pneumoniae load. Inducible NO synthase (iNOS) also participates in the control of bacterial load, as shown by the enhanced numbers of C. pneumoniae in iNOSⴚ/ⴚ BMMs. However, the increased accumulation of iNOS mRNA and NO in C. pneumoniae-infected BMMs depended on the presence of IFN-␣, but was independent of IFN-␥. Interestingly, IFN-␣ are also required for increased IFN-␥ mRNA accumulation in C. pneumoniaeinfected BMMs. Accordingly, IFN-␣Rⴚ/ⴚ BMMs showed higher levels of C. pneumoniae than wild-type BMMs. Our findings unravel an autocrine/paracrine macrophage activation pathway by showing an IFN-␣-dependent IFN-␥ and iNOS induction in response to infection, which protects macrophages against intracellular bacterial growth. The Journal of Immunology, 2001, 167: 6453– 6461. nterferon-␥ regulates a variety of immunological programs. It is the predominant cytokine during Th1-dominated immune reactions, and is the prototype macrophage-activating cytokine. Macrophages infected by different microorganisms secrete IL-12, which directs Th1 development and induces IFN-␥ production by NK and T cells (1). IFN-␥ does act on macrophages to augment IL-12 secretion (2) and to produce NO that eradicates intracellular microbes (3). NO release after IFN-␥ stimulation is the result of increased de novo synthesis of the inducible form of NO synthase (iNOS).2 Studies using neutralizing Abs against IFN-␥ and mice genomically deficient of IFN-␥, IFN-␥R, or IL-12 confirm the importance of these cytokines in innate immunity and Th1 development, and consequently, for controlling intracellular pathogens (4). A few studies show that IFN-␥ can be produced by macrophages in response to stimulation with live bacteria (5), bacterial components (5, 6), IL-12 (7, 8), a combination of IL-12 and IL-18 (9 –11), or IFN-␥ itself (12), suggesting presence of an autocrine loop. However, the biological relevance of these findings, at variance with the general assumption that NK and T cells are the sole IFN-␥ producers, is still unclear.
I
In the study presented in this work, we extend previous data by showing that bone marrow-derived macrophage (BMM) cultures produce IFN-␥ at the mRNA and protein level, after infection with Chlamydia pneumoniae, an obligate intracellular Gram-negative bacterium. IFN-␥ has been previously shown to play a protective role during the in vitro and in vivo infection with Chlamydia sp. (13–17). C. pneumoniae infects and grows in macrophages, but bacterial load is controlled after several days of infection (18, 19). We show in this study that IFN-␥ is transiently expressed early after in vitro infection of BMMs with live bacteria, and is involved in control of intracellular bacterial growth. Surprisingly, the expression of IFN-␥ did not depend on the presence of IL-12, but relied on IFN-␣R signaling. IFN-␣ have been shown to mediate defense mechanisms in an important number of viral infections and possess numerous immunoregulatory properties (reviewed in Ref. 20). In addition, IFN-␣ can in an IFN-␥-independent manner induce iNOS expression, adding to the control of intracellular bacterial growth. Thus, we suggest a novel autocrine/paracrine loop by which BMMs are induced by infection to produce IFN-␣, which in turn stimulates IFN-␥ secretion. In turn, this IFN-␥ mediates the control of the infecting bacteria.
Materials and Methods Mice *Microbiology and Tumorbiology Center, †Department of Microbiology, Pathology, and Immunology, Division of Biomedical Laboratory Technology, and Departments of ‡Rheumatology, Center of Molecular Medicine, and §Rheumatology, Astrid Lindgren’s Hospital, Karolinska Institutet, Stockholm, Sweden Received for publication June 1, 2001. Accepted for publication September 20, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Martin E. Rottenberg, Microbiology and Tumorbiology Center, Box 280, Karolinska Institutet 171 77 Stockholm, Sweden. E-mail address:
[email protected]
Mutant mouse strains without IFN-␥R (21), recombination-activating gene-1 (RAG-1), IL-10 (22), IL-12p40 (23), IFN-␣R (24), iNOS (25), and TNFR-I (26), generated by homologous recombination in embryonic stem cells, were bred and kept under specific pathogen-free conditions. RAG-1⫺/⫺, IL-10⫺/⫺, iNOS⫺/⫺, and IFN-␥R⫺/⫺ mice had been backcrossed with C57BL/6 mice, which were used as controls. The 129Sv/Ev mice were used as controls for experiments using IFN␣R- and TNFR-Ideficient mice. All animal experiments have been approved by the Stockholm’s Region Animal Research Ethical Board.
Cells
2
Abbreviations used in this paper: iNOS, inducible NO synthase; BMM, bone marrow-derived macrophage; IFU, inclusion-forming unit; RAG, recombination-activating gene; SPG, sucrose-phosphate-glutamate; WT, wild type. Copyright © 2001 by The American Association of Immunologists
BMMs were obtained as previously described (27). Briefly, mice were euthanized, and the femur and tibia of the hind legs were dissected. Bone 0022-1767/01/$02.00
IFN-␥ SECRETION BY BACTERIA-INFECTED MACROPHAGES
6454 marrow cavities were flushed with 5 ml cold, sterile PBS. The bone marrow cells were washed and resuspended in DMEM containing glucose and supplemented with 10% FCS, 20% L929 cell-conditioned medium (as a source of M-CSF), 100 g/ml streptomycin, and 100 U/ml penicillin. Bone marrow cells (1.2 ⫻ 107 cells; 2 ⫻ 106 cells/ml) were plated in six-well plates and incubated for 7 days at 37°C, 5% CO2. Before use, BMM cultures were washed vigorously to remove nonadherent cells. Cells from several wells were also harvested and counted by trypan blue exclusion. Typically, bone marrow cells from one mouse yield 2–3 ⫻ 106 BMMs after 7 days in culture.
Immunostainings BMMs were phenotypically characterized by FACS analysis. BMMs were detached from plates using a cell scraper, and incubated with PBS containing 10% normal goat serum (Sigma, St. Louis, MO) to block unspecific binding of the secondary Ab. Cells were then incubated with purified rat mAbs to F4/80, CD45, Mac-3, and CD80 for 30 min on ice. Cells were then washed and incubated with 1/200 biotinylated goat anti-rat serum for 30 min on ice. Anti-CD19, anti-CD11c, anti-CD8, and anti-CD40 Abs were directly labeled with FITC. Anti-CD3 and anti-CD4 Abs were directly labeled with PE. Anti-MHC class I, anti-MHC class II, and antiICAM-1 (MALA-2) Abs were biotinylated. Isotype-matched control rat Igs directly labeled with FITC, PE, or biotin were used as controls. When staining with biotinylated or directly labeled Abs, BMMs were preincubated with anti-CD16/CD32 to block Fc␥R. After incubation with biotinylated Abs, cells were washed and stained with 1/1500 Neutroavidin Alexa (Molecular Probes, Eugene, OR) before FACS analysis. All primary Abs were purchased from PharMingen (San Diego, CA), except for F4/80 (Serotec, Oxford, U.K.). When microscopic analysis was performed, BMMs grown on 13-mm2 coverslides were washed, fixed with 2% formaldehyde, and stained for the different cell surface markers, as indicated above. Thereafter, coverslides were washed, counterstained with 4⬘,6⬘-diamidino-2-phenylindole (5 g/ ml), mounted, and analyzed in a fluorescent microscope
Bacteria and infectivity assay Mycoplasma-free C. pneumoniae isolate Kajaani (28) was propagated in HL cells. Infected cells were sonicated, cell remnants were removed by centrifugation (6 min at 100 ⫻ g), and the bacteria were stored in small aliquots in sucrose-phosphate-glutamate (SPG) solution at ⫺70°C until used. The infectivity as measured by inclusion-forming units (IFU) of bacterial preparation was determined in HL cells. BMM cultures were inoculated with 106 C. pneumoniae (bacteria to cell ratio 1:3) and centrifuged for 1 h at 500 ⫻ g. At different time points after infection, cells were washed with PBS, and thereafter lysates using SPG solution were obtained. Lysates were then diluted 10- and 100-fold in DMEM containing 5% FCS and 100 g/ml streptomycin (DMEM 5% FCS). Infectious titers were assessed culturing 500 l duplicate dilutions of lysates on confluent HL cells grown on round 13-mm2 coverslides in a shell vial. Inoculated cells were centrifuged for 1 h at 500 ⫻ g. Thereafter, supernatants were removed, DMEM 5% FCS containing 0.5 g/ml cycloheximide was added, and cells were incubated at 35°C for 72 h, 5% CO2. Cells were then washed with PBS, fixed with methanol, and stained with a FITC-conjugated Chlamydia genus-specific mAb (Pathfinder Chlamydia Confirmation System; Kallestad Diagnostics, Chaska, MN). Inclusion bodies were counted by fluorescence microscopy. The existence of a linear regression between number of IFU recovered 3 days after infection and the number of infecting C. pneumoniae was proven in control experiments (data not shown).
Competitive RT-PCR assay The accumulation of IFN-␥, IFN-␣, IL-12p40, iNOS, the gp91 component of the phagocyte NADPH oxidase complex (gp91-phox), TNF-␣, IL-6, IL-1␣, and -actin mRNA in freshly extracted RNA from C. pneumoniaeinfected BMMs was visualized by RT-PCR and measured by competitive PCR assays, as previously described (29). Competitor fragments with a different length, but using the same primers as the target DNA were constructed using composite primers and an exogenous DNA fragment or by subcloning of cytokine cDNA sequences mutated by deletion or ligation (30). Competitors were purified (Qiagen, Studio City, CA) and quantified in a spectrophotometer. Three- or four-fold serial dilutions of the competitor were amplified in the presence of a constant amount of cDNA. Reactions were conducted for 28 – 45 cycles in a thermal cycler (PerkinElmer/Cetus, Norwalk, CT) using an annealing step at 60°C (except 65°C for IL-12, 56°C for gp91-phox, and 58°C for IL-10). The primer sequences for the amplification of the cDNA in competitive or noncompetitive RT-PCR are shown in Table I.
ELISA The amount of IFN-␥ in the culture supernatants of C. pneumoniae-infected BMMs was measured by ELISA, according to the instructions of the manufacturer (R&D Systems, Abingdon, U.K.).
C. pneumoniae DNA extraction and PCR amplification DNA was extracted from 150 l SPG BMM lysates, 5 days after infection with C. pneumoniae. Lysates were centrifuged for 10,000 ⫻ g, 35 min, at 4°C, and 0.5 ml lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, pH 7.6, 1% SDS) containing 200 g/ml proteinase K (Life Technologies, Paisley, U.K.) was added to the pellet. The samples were then incubated for 3 h at 50°C. Thereafter, DNA was extracted by use of Tris-saturated phenol, chloroform, and isoamyl alcohol (25:24:1; Sigma). DNA was then precipitated, washed, and quantified spectrophotometrically. The amplification of 16S RNA gene from C. pneumoniae was performed by PCR, as previously described (31). The primer sequences for the amplification of the C. pneumoniae DNA were: sense-16S RNA, 5⬘-TGA CAA CTG TAG AAA TAC AGC-3⬘; antisense-16S RNA, 5⬘-CGC CTC TCT CCT ATA AAT-3⬘. A PCR product of 465 bp was electrophoresed and visualized in a 1.5% ethidium bromide-stained agarose gel.
Detection of intracellular IFN-␥ protein by immunostaining BMMs were fixed at different time points after infection with C. pneumoniae with 2% (v/v) formaldehyde in PBS at pH 7.4 for 10 min and subsequently stored at ⫺70°C until required for staining. Detection of intracellular IFN-␥ production was made using indirect staining (32, 33). Briefly, permeabilization of cell membranes was performed using BSS (Earle’s BSS; Life Technologies) supplemented with 0.1% saponin in all subsequent washes and incubation steps. The slides were then blocked with BSS containing 2% FCS for 5 min at 37°C. Endogenous cellular biotin was blocked with avidin for 30 min and biotin for an additional 15 min (avidin/ biotin blocking kit; Vector, Burlingame, CA), both supplemented with 0.1% saponin. Subsequently, the cells were incubated overnight with 5 g/ml mouse IFN-␥-specific rat mAb (clone XMG1.2, kindly provided by J. Abrams, DNAX Research Institute, Palo Alto, CA). Slides were then washed and incubated for 30 min with a biotinylated second-step Ab (goat anti-rat; Vector) diluted 1/500. Finally, a developing step with 2 g/ml
Table I. PCR primers used in this studya Sequence
iNOS gp91-phox IFN-␣ IFN-␥ IL-10 IL-12p40 IL-1␣ IL-6 TNF-␣ -Actin a
Sense Primer (5⬘–3⬘)
CCC TTC CGA AGT TTC TGG CAG CAG CAG C CTT TGT CAT TCT GGT GTG GTT GG GAC TCA TCT GCT GCT TGG AAT GCA ACC CTC TGG ACC TGT GGG TTG TTG ACC TCA AAC TTG GC GAG AGC TCT GTC TAG GTC CGT GCT CAT GGC TGG TGC AAA G ATG GCC AAA GTT CCT GAC TTG TTT ATG AAG TTC CTC TCT GCA AGA GAC T ATG AGC ACA GAA AGC ATG ATC CGC GTG GGC CGC TCT AGG CAC CAA
Antisense Primer (5⬘–3⬘)
GGC TGT CAG AGC CTC GTG GCT TTG G CCC CAT TCT TCG ATT TTG TCT GC GAC TCA CTC CTT CTC CTC ACT CAG TCT TGC C TCG ATC TTG GCT TTG CAG CTC TTC CTC ATG GC CGG GAA GAC AAT AAC TG CTT CAT CTG CAA GTT CTT GGG C C CTT CAG CAA CAC GGG CTG GTC CA CTA GGT TTG CCG AGT AGA TCT C CC AAA GTA GAC CTG CCC GGA CTC CTC TTT GAT GTC ACG CAC GAT TTC
The IFN-␣ primer sequences recognize a consensus sequence present in IFN-␣1, IFN-␣2, and IFN-␣7 genes.
The Journal of Immunology Oregon Green coupled to avidin D (Molecular Probes) was applied for 30 min. The slides were air dried and mounted with PBS-buffered glycerol and examined in a Polyvar UV microscope (Reichert-Jung, Vienna, Austria). Specificity controls were based on parallel staining studies omitting the primary Ab or using primary isotype-matched Ig of irrelevant Ag specificity at the same concentration as the cytokine-detecting Abs.
Nitrite assay Nitrite concentrations were measured in BMM culture supernatants using the Greiss reagent in a previously described colorimetric assay (34). Fiftymicroliter aliquots of culture medium were mixed in 96-well plates with an equal volume of 0.5% sulfanilamide dihydrochloride and 0.05% naphthylethylenediamide dihydrochloride in 2.5% phosphoric acid. OD540 was determined using a Dynatech MR700 plate reader (Chantilly, VA). Sodium nitrite, dissolved in DMEM, was used to generate a standard concentration curve. The lower limit of detection of the assay was 1 M.
Results Infection of BMMs with C. pneumoniae induces the release of IFN-␥ protein and increases IFN-␥ mRNA levels We first phenotypically characterized uninfected BMMs by immunostaining, followed by FACS and/or microscopic analysis. BMMs were found to be homogeneously positive for macrophage markers as F4/80, Mac-3, and CD14. BMMs were also positive for CD45, and expressed low MHC class II, MHC class I, B7, and
6455 ICAM-1 levels. The T cell Ags CD3, CD4, and CD8, and the pan B cell Ag CD19 were uniformly negative (data not shown). BMMs were also negative for CD11c and CD40. In a second series of experiments, BMMs were infected with C. pneumoniae, and the presence of IFN-␥ mRNA was measured at several time points after infection. Infection of BMMs with C. pneumoniae resulted in increased IFN-␥ mRNA accumulation. Increased levels of IFN-␥ transcripts could be detected at 30 min and waned 24 h after infection (Fig. 1, A and B). IFN-␥ protein was also detected in supernatants from infected BMMs. IFN-␥ was detected at 6 h, peaked at 24 h, and was low or undetectable (⬍4 pg/ml) at later time points after infection (Fig. 1C). Moreover, BMMs after 18 h of infection with C. pneumoniae, but not BMMs 6 h and 26 h after infection or uninfected controls, were stained intracellularly with anti-IFN-␥ Abs (Fig. 1D). Infected RAG-1⫺/⫺ BMMs depleted or not of NK cells by anti-asialo-GM1 and complement treatment showed similar levels of IFN-␥ mRNA as wildtype (WT) controls, indicating that neither T, nor B, nor NK cells are necessary for chlamydial-induced IFN-␥ mRNA expression (Fig. 1F). Increased accumulation of IFN-␥ mRNA required infectious bacteria and host cell protein synthesis. UV or heat inactivation of C. pneumoniae dramatically diminished the steady state level of
FIGURE 1. Infection with C. pneumoniae induces the expression of IFN-␥ in BMMs. A, Total RNA from C. pneumoniae-infected BMMs was extracted at the indicated time points after infection. The presence of IFN-␥ and -actin mRNAs was evaluated by RT- PCR. A representative of at least six different experiments is shown. B, Total RNA was extracted from WT BMMs at the indicated time points after infection with C. pneumoniae. The accumulation of IFN-␥ and -actin mRNA in these samples was measured by competitive PCR. A representative from three independent experiments is shown. C, The content of IFN-␥ in supernatants from C. pneumoniae-infected BMMs obtained at the indicated time points after infection was evaluated by a specific ELISA. A representative from two independent experiments is shown. D, Videoprint photograph demonstrating an IFN-␥-producing cell with characteristic staining generated by accumulation of IFN-␥ in the Golgi organelle (⫻600). This staining pattern represents intracellular production and is never observed when exogenous IFN-␥ is added to the staining procedure (33). A specificity control using primary isotype-matched Ig of irrelevant Ag specificity is shown in E. F, BMMs were cultured from bone marrows of WT or RAG-1⫺/⫺ mice. In the latter case, cultures were treated with 1/100 anti-asialo-GM1 rabbit antiserum (Wako Pure Chemical, Osaka, Japan) and 1/10 complement (Low-Tox rabbit complement; Cedarlane, Ontario, Canada) or complement alone before infection with C. pneumoniae. Six hours after infection, total RNA from BMMs was extracted, and the amounts of IFN-␥ and -actin mRNA were quantified by competitive PCR. A representative from two independent experiments is shown.
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FIGURE 2. Host cell and bacterial protein synthesis are required for the increased IFN-␥ mRNA levels in BMMs. BMMs were coincubated for 1 h with untreated, UV irradiated (30 min, 15 cm), or heat-inactivated (75°C, 30 min) 106 IFU C. pneumoniae. In parallel experiments, BMMs were treated with 5 g/ml cycloheximide to inhibit eukaryotic protein synthesis or 10 g/ml chloramphenicol, an inhibitor of bacterial protein translation. Total RNA was extracted 6 h after coincubation with C. pneumoniae, and the presence of IFN-␥ and -actin mRNA was evaluated by RT-PCR (A). The accumulation of IFN-␥ and -actin mRNA in these samples was measured by competitive PCR (B). Comparable results were obtained in two separate experiments.
IFN-␥ mRNA in infected BMMs. The same was observed when coculture of C. pneumoniae and BMMs was done in presence of chloramphenicol, which inhibits bacterial protein synthesis (Fig. 2).
IFN-␥ SECRETION BY BACTERIA-INFECTED MACROPHAGES
FIGURE 3. IL-12 is not necessary for the enhanced IFN-␥ mRNA accumulation in C. pneumoniae-infected BMMs. Total RNA was extracted from WT and IL-12⫺/⫺ BMMs at the indicated time points after infection with C. pneumoniae. The accumulation of IFN-␥ and -actin mRNAs was visualized by RT-PCR (A). Comparable results were obtained in three separate experiments. The amount of IFN-␥ mRNA was quantitated in a competitive PCR using RNA samples from C57BL/6 and IL-12⫺/⫺ BMM at 0 and 6 h after infection with C. pneumoniae (B).
C. pneumoniae-induced IFN-␥ mRNA accumulation by BMMs is IL-12 independent and is controlled by IL-10 To determine whether host cell proteins are involved in the increased IFN-␥ mRNA in BMMs, cycloheximide, an eukaryotic protein synthesis inhibitor, was applied. Addition of cycloheximide during coculture of bacteria and BMMs reduced IFN-␥ mRNA levels (Fig. 2). IL-12 has been shown to increase IFN-␥ gene expression by NK and T cells (2). However, we found that C. pneumoniae-infected IL-12⫺/⫺ BMMs showed similar levels of IFN-␥ mRNA as WT controls (Fig. 3). Hence, IL-12 does not
FIGURE 4. Endogenous IL-10 hampers IFN-␥ and IL-12p40 mRNA accumulation in C. pneumoniae-infected BMMs. A, Total RNA was extracted from C57BL/6 BMMs at the indicated time points after infection with C. pneumoniae. The accumulation of IL-10 and -actin mRNA in these samples was measured by competitive PCR. B and C, Total RNA from C57BL/6 and IL-10⫺/⫺ BMMs was extracted at the indicated time points after infection with C. pneumoniae. The levels of IFN-␥ (B), IL-12p40 (C), and -actin (B, C) mRNA were measured by competitive PCR. Comparable results were obtained in two separate experiments.
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appear to mediate C. pneumoniae-induced IFN-␥ mRNA expression in BMMs. The temporary expression of IFN-␥ in infected BMMs was an unforeseen characteristic. IL-10 has been shown to inhibit IFN-␥ mRNA expression (35). IL-10 mRNA levels were increased in WT BMMs after infection with C. pneumoniae (Fig. 4A). Moreover, IFN-␥ mRNA levels were substantially increased in infected IL10⫺/⫺ BMMs as compared with WT BMMs (Fig. 4B). Presence of IL-10 also explains the low IL-12p40 mRNA levels measured after chlamydial infection, because IL-12p40 mRNA titers were significantly increased in C. pneumoniae-infected IL-10⫺/⫺ BMMs (Fig. 4C). IFN-␥ secreted by infected BMMs diminishes the intracellular bacterial growth We then investigated whether IFN-␥ secretion by infected BMMs could alter the course of the infection. For this purpose, we compared the susceptibility of IFN-␥R⫺/⫺, IL-10⫺/⫺, and WT BMMs to infection with C. pneumoniae. IFN-␥R⫺/⫺ BMMs showed higher numbers of bacteria (Fig. 5A) and bacterial DNA levels (Fig. 5B) than WT controls. On the contrary, IL-10⫺/⫺ BMMs showed diminished numbers of C. pneumoniae than WT BMMs (Fig. 5A). IFN-␥ and iNOS expression by C. pneumoniae-infected BMMs are dependent on IFN-␣ We next analyzed whether infection of BMMs with C. pneumoniae also affected the levels of IFN-␣, IL-1␣, IL-6, TNF-␣, iNOS, and IFN-␥-inducible gp91-phox transcripts. Increased levels of IFN-␣, IL-1␣, IL-6, TNF-␣, and iNOS, but not of gp91-phox mRNA, were observed in BMMs after infection with C. pneumoniae (Fig. 6). IFN-␣ have been shown to increase as well as decrease IFN-␥ gene expression in different viral infections. Because IFN-␣ mRNA levels were increased in infected BMMs, the expression of IFN-␥ mRNA and protein was studied in IFN-␣R⫺/⫺ BMMs. IFN-␥ mRNA and protein levels were not raised in IFN-␣R⫺/⫺ BMMs after infection with C. pneumoniae, indicating that IFN-␥ expression in infected BMMs is controlled by IFN-␣ (Fig. 7, A and B). TNF-␣ has been shown to act as a cofactor together with IL-12 in inducing IFN-␥ production (36, 37). However, signaling through the main receptor for TNF (TNFR-I) was not necessary for IFN-␥ expression, because infected TNFR-I⫺/⫺ BMMs showed unaltered IFN-␥ mRNA accumulation as compared with infected WT controls (Fig. 7A).
FIGURE 5. The expression of IFN-␥ by BMMs controls the intracellular growth of C. pneumoniae. A, IFN-␥R⫺/⫺, IL-10⫺/⫺, and C57BL/6 BMMs were infected with C. pneumoniae and lysed with SPG buffer at the indicated time points after infection. The number of C. pneumoniae IFU/well was quantified by infecting HL cells with 100 l BMM lysate. A representative from three independent experiments is shown. B, DNA was extracted from WT and IFN␥R⫺/⫺ BMM lysates 5 days after infection with C. pneumoniae from two separate experiments. The amplification of a chlamydial 16S RNA from individual DNA samples was performed, as described in Materials and Methods.
FIGURE 6. Enhanced accumulation of IL-1-␣, IL-6, iNOS, IFN-␥, IFN-␣ (1, 2, and 7 variants), and TNF-␣, but not gp91-phox mRNA during infection of BMMs with C. pneumoniae. Total RNA was extracted from WT BMMs at the indicated time points after infection with C. pneumoniae. The accumulation of the above mentioned transcripts and -actin mRNA in these samples was visualized by RT-PCR.
Taking into account that iNOS mRNA levels are enhanced during C. pneumoniae infection of BMMs, and that NO has been suggested as a potential chlamydiocidal molecule, we analyzed whether IFN-␥-mediated control of C. pneumoniae infection in BMMs was iNOS dependent. IFN-␥ was not necessary for increased iNOS mRNA and nitrite accumulation, because iNOS transcripts were not diminished in infected IFN-␥R⫺/⫺ BMMs (Fig. 8, A and B). Also, expression of IL-12 was not necessary for induction of iNOS mRNA and NO release (Fig. 8, A and B). On the contrary, iNOS mRNA and nitrite levels were dramatically diminished in infected IFN-␣R⫺/⫺ BMM as compared with infected controls (Fig. 8, C–E). IFN-␣ mediate control of infection with C. pneumoniae in BMMs Our results thus suggest an important role of IFN-␣-mediated mechanisms in the macrophage control of chlamydial infection. To test this hypothesis, IFN-␣R⫺/⫺ BMMs were infected and the bacterial load compared with that of WT controls. IFN-␣R⫺/⫺ BMMs showed higher numbers of intracellular C. pneumoniae (Fig. 9B). iNOS⫺/⫺ BMMs also showed diminished control of infection (Fig. 9A).
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IFN-␥ SECRETION BY BACTERIA-INFECTED MACROPHAGES
FIGURE 7. The increased IFN-␥ mRNA and protein levels in C. pneumoniae-infected BMMs depend on IFN-␣R, but not TNFR-I signaling. A, Total RNA was extracted from 129Sv/Ev, IFN-␣R⫺/⫺, and TNFR-I⫺/⫺ BMMs at the indicated time points after infection with C. pneumoniae. The levels of IFN-␥ and -actin mRNA were measured by competitive PCR. A representative from three independent experients is shown. B, The levels of IFN-␥ in supernatants from 129Sv/Ev or IFN-␣R⫺/⫺ BMMs at the indicated time points after infection with C. pneumoniae were quantified by a specific ELISA. Comparable results were obtained in two separate experiments.
Discussion
These experiments were stimulated by reports showing IFN-␥ secretion by cells other than T or NK cells (6, 38 – 40). We now report that in vitro infection of BMMs with C. pneumoniae induces both the
release of low levels of IFN-␥ protein and an early and transient increase in IFN-␥ mRNA levels. BMM-derived IFN-␥ probably functions in a paracrine/autocrine manner, because 1) IFN-␥ expression was not altered in BMM cultures lacking T or NK cells and 2) IFN-␥
FIGURE 8. The enhanced accumulation of iNOS in C. pneumoniae-infected BMMs is independent of IFN-␥ or IL-12 but dependent on IFN-␣R signaling. Total RNA was extracted from C57BL/6 (A), 129Sv/Ev (C), IFN-␥R⫺/⫺ (A), IFN-␣R⫺/⫺ (C), and IL-12⫺/⫺ (A) BMMs at the indicated time points after infection with C. pneumoniae. The accumulation of iNOS and -actin mRNA in these samples was visualized by RT-PCR. One of three independent experiments with every mouse strain is shown. The nitrite levels were measured in supernatants from C57BL/6 (B, E) 129Sv/Ev (D), IFN-␥R⫺/⫺ (B), IL-12⫺/⫺ (B), IFN-␣R⫺/⫺ (D), and iNOS⫺/⫺ (E) BMMs at different time points after infection were measured by the Griess assay. Mean ⫾ SEM from samples obtained from four independent experiments is shown.
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FIGURE 9. iNOS and IFN-␣ participate in the control of C. pneumoniae growth by BMMs. C57BL/6 (A), 129Sv/Ev (B), iNOS⫺/⫺ (A), and IFN-␣R⫺/⫺ (B) BMMs were infected with C. pneumoniae, and the number of IFU/well quantified by infection of HL cells at the indicated time points after the infection of BMMs. A representative from two independent experiments with each mouse strain is shown.
expression had a profound effect on chlamydial control by BMMs. Confirming our data, expression of IFN-␥ by macrophages from RAG-2⫺/⫺ mice has been recently observed (11). Acellular chlamydial components including whole chlamydial lysates, LPS, or heat shock proteins have all been shown to stimulate production of different cytokines and chemokines by mouse or human cells (41– 44). However, in our experimental model, infection with live bacteria and bacterial protein synthesis were required for the increased IFN-␥ mRNA accumulation in BMMs 6 h after infection. However, IFN-␥ could be induced after stimulation with different conentrations of inactivated bacteria or at later time points after coincubation. The observed secretion of BMM-derived IFN-␥ depended on host protein synthesis. IL-12, a cytokine produced by macrophages and dendritic cells in response to protozoan and bacterial infections, can induce IFN-␥ gene expression (45– 48). Exogenous IL-12 has been shown to induce expression of IFN-␥ in macrophages (7–11, 49). Surprisingly, IL-12 neither played a role in the chlamydial-induced enhancement of IFN-␥ mRNA levels in BMMs, nor in the control of bacterial load (data not shown). The low IL-12p40 mRNA and IL-12p70 protein levels (⬍40 pg/ml) secreted by C. pneumoniae-infected BMMs might not be adequate to induce the release of IFN-␥. A striking feature of chlamydial-induced IFN-␥ mRNA accumulation in BMMs was the early and transient expression. IL-10, a cytokine released by many cell types, including monocytes in response to activation with bacterial components, can inhibit IFN-␥ synthesis by T cells (50). Macrophages are also primary targets of IL-10 with prominent effects on morphology, phenotype, and cytokine secretion (35). Our data indicate that endogenous IL-10 diminishes IFN-␥ mRNA levels in Chlamydia-infected BMMs. IL-10 has been previously shown to reduce IFN-␥ expression in macrophages (8, 64). As IL-10 was also found to inhibit accumulation of IL-12p40 mRNA, IL-12 might account for the prolonged accumulation of IFN-␥ mRNA in infected IL-10⫺/⫺ BMMs. In contrast, infected IL-10⫺/⫺ BMMS showed no changes in IFN-␣ or iNDS mRNA and nitrite levels compared to WT controls (data not shown). IFN-␣ cytokines play essential roles in many antiviral immune responses. The role of IFN-␣ in the defense against nonviral agents is considerably less studied, but functional relationships have been issued (51). Produced by macrophages and other cells, IFN-␣ enhance NK cell cytotoxicity (52, 53), regulate lymphocyte proliferation (54), stimulate or inhibit macrophage activation (55), and regulate differentiation of Th cells into Th1 cells. In the latter case, IFN-␣ may inhibit or enhance IFN-␥ gene expression in murine and human T cells (56 – 60), a dichotomy probably reg-
ulated by STAT-1 activity (61). In accordance with previous studies on Chlamydia trachomatis-infected fibroblasts (62), we show that IFN-␣ mRNAs are induced after C. pneumoniae infection of BMMs. Our data also suggest that endogenous IFN-␣ participate in the control of intracellular chlamydial infection by allowing IFN-␥ release. NO produced after cell activation by IFN-␥ is important for killing or inhibiting growth of different microorganisms (3). iNOS mRNA and nitrite levels were increased in C. pneumoniae-infected BMMs, but BMM-derived IFN-␥ was not necessary for iNOS induction. Interestingly, iNOS mRNA induction and NO release depended on IFN-␣R signaling. We also showed that iNOS was necessary for effective chlamydial clearance by BMMs. This is in agreement with previous studies in which NO release was stimulated by coculture of IFN-␣ with Leishmania major-infected macrophages (55, 62). Infection of fibroblasts with C. trachomatis (57) or stimulation of macrophages with LPS (63) do also induce NO synthesis via an IFN-␣-mediated pathway. Thus, IFN-␣
FIGURE 10. Infection of BMMs with live C. pneumoniae enhances the steady state level of IFN-␣ mRNA. IFN-␣ in turn are necessary for increased IFN-␥ mRNA accumulation and protein secretion. IFN-␥ secreted by C. pneumoniae-infected BMMs participates in the control of the intracellular bacterial growth. IFN-␣ (but not IFN-␥) are also involved in the increased accumulation of iNOS mRNA. iNOS also contributes to reduce the intracellular proliferation of C. pneumoniae.
6460 signaling in infected BMMs appears to independently induce bothIFN-␥ and iNOS, also suggesting that a bidirectional IFN-␣ and IFN-␥ induction (65) does not occur in this experimental model. Both iNOS and IFN-␥ are necessary for the control of intracellular chlamydial growth. At this stage, it remains to unravel details of IFN-␥mediated mechanisms of chlamydial control in infected BMMs. In contrast to our results, endogenous IFN-␣ inhibited IFN-␥ release by macrophages when IL-12 was used as a stimulus (10). In summary, we propose that C. pneumoniae infection can stimulate an IFN-␥-mediated pathway of autocrine/paracrine macrophage activation. IFN-␥ expression is independent of IL-12 and TNFR-I, but depends on IFN-␣R signaling (Fig. 10). IFN-␣ are also involved in chlamydial control via inducing increased accumulation of iNOS transcripts. A biological role for macrophage release of IFN-␥ in the control of infection with Toxoplasma gondii has recently been shown (8). IFN-␥ release in such model was the result of exogenous addition of IL-12 and IL-18, and thereby strikingly different from the model proposed in this study. In a microbial infection, a pathway of macrophage activation via autocrine IFN-␥ release would be alternative or complementary to paracrine NK cell-secreted IFN-␥, and thus important for an early stage of innate immune response.
Acknowledgments We thank Berit Olsson for excellent technical assistance. IFN-␥R⫺/⫺ mice were provided by Dr. M. Aguet (Swiss Institute of Experimental Cancer Research, Lausanne, Switzerland).
References 1. Trinchieri, G. 1998. Immunobiology of interleukin-12. Immunol. Res. 17:269. 2. Ma, X., J. M. Chow, G. Gri, G. Carra, F. Gerosa, S. F. Wolf, R. Dzialo, and G. Trinchieri. 1996. The interleukin 12 p40 gene promoter is primed by interferon ␥ in monocytic cells. J. Exp. Med. 183:147. 3. MacMicking, J., Q. W. Xie, and C. Nathan. 1997. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323. 4. Boehm, U., T. Klamp, M. Groot, and J. Howard. 1997. Cellular responses to interferon-␥. Annu. Rev. Immunol. 15:749. 5. Fenton, M. J., M. W. Vermeulen, S. Kim, M. Burdick, R. M. Strieter, and H. Kornfeld. 1997. Induction of ␥ interferon production in human alveolar macrophages by Mycobacterium tuberculosis. Infect. Immun. 65:5149. 6. Fultz, M., S. Barber, C. Dieffenbach, and S. Vogel. 1993. Induction of IFN-␥ in macrophages by lipopolysaccharide. Int. Immunol. 5:1383. 7. Puddu, P., L. Fantuzzi, P. Borghi, B. Varano, G. Rainaldi, E. Guillemard, W. Malorni, P. Nicaise, S. F. Wolf, F. Belardelli, and S. Gessani. 1997. IL-12 induces IFN-␥ expression and secretion in mouse peritoneal macrophages. J. Immunol. 159:3490. 8. Fukao, T., D. M. Frucht, G. Yap, M. Gadina, J. J. O’Shea, and S. Koyasu. 2001. Inducible expression of Stat4 in dendritic cells and macrophages and its critical role in innate and adaptive immune responses. J. Immunol. 166:4446. 9. Munder, M., M. Mallo, K. Eichmann, and M. Modolell. 1998. Murine macrophages secrete interferon ␥ upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. 187:2103. 10. Fantuzzi, L., P. Puddu, B. Varano, M. Del Corno, F. Belardelli, and S. Gessani. 2000. IFN-␣ and IL-18 exert opposite regulatory effects on the IL-12 receptor expression and IL-12-induced IFN-␥ production in mouse macrophages: novel pathways in the regulation of the inflammatory response of macrophages. J. Leukocyte Biol. 68:707. 11. Schindler, H., M. B. Lutz, M. Rollinghoff, and C. Bogdan. 2001. The production of IFN-␥ by IL-12/IL-18-activated macrophages requires STAT4 signalling and is inhibited by IL-4. J. Immunol. 166:3075. 12. Di Marzio, P., P. Puddu, L. Conti, F. Belardelli, and S. Gessani. 1994. Interferon ␥ up-regulates its own gene expression in mouse peritoneal macrophages. J. Exp. Med. 179:1731. 13. Beatty, W. L., T. A. Belanger, A. A. Desai, R. P. Morrison, and G. I. Byrne. 1994. Tryptophan depletion as a mechanism of ␥ interferon-mediated chlamydial persistence. Infect. Immun. 62:3705. 14. Rottenberg, M., A. C. Gigliotti Rothfuchs, D. Gigliotti, C. Svanholm, L. Bandholtz, and H. Wigzell. 1999. Role of innate and adaptive immunity in the outcome of primary infection with Chlamydia pneumoniae, as analyzed in genetically modified mice. J. Immunol. 162:2829. 15. Perry, L. L., K. Feilzer, and H. D. Caldwell. 1997. Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-␥-dependent and -independent pathways. J. Immunol. 158:3344. 16. Mehta, S. J., R. D. Miller, J. A. Ramirez, and J. T. Summersgill. 1998. Inhibition of Chlamydia pneumoniae replication in Hep-2 cells by interferon-␥: role of tryptophan catabolism. J. Infect. Dis. 177:1326.
IFN-␥ SECRETION BY BACTERIA-INFECTED MACROPHAGES 17. Johansson, M., K. Schon, M. Ward, and N. Lycke. 1997. Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in ␥ interferon receptor-deficient mice despite a strong local immunoglobulin A response. Infect. Immun. 65:1032. 18. Pal, S., E. M. Peterson, and L. M. de La Maza. 2000. Role of Nramp1 deletion in Chlamydia infection in mice. Infect. Immun. 68:4831. 19. Gaydos, C. A., J. T. Summersgill, N. N. Sahney, J. A. Ramirez, and T. C. Quinn. 1996. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. Infect. Immun. 64:1614. 20. Biron, C. A. 1999. Initial and innate responses to viral infections: pattern setting in immunity or disease. Curr. Opin. Microbiol. 2:374. 21. Huang, S., W. Hendricks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, J. Vilcek, R. Zinkernagel, and M. Aguet. 1993. Immune response in mice that lack the interferon-␥ receptor. Science 259:1742. 22. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, and W. Muller. 1993. Interleukin10-deficient mice develop chronic enterocolitis. Cell 75:263. 23. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, and M. K. Gately. 1996. IL-12-deficient mice are defective in IFN ␥ production and type 1 cytokine responses. Immunity 4:471. 24. Muller, U., U. Steinhoff, L. F. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, and M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science 264:1918. 25. Laubach, V., E. Sheeley, O. Smithies, and P. Sherman. 1995. Mice lacking inducible nitric oxide synthase are not resistant to LPS-induced death. Proc. Natl. Acad. Sci. USA 92:10688. 26. Pfefffer, K., T. Matsuyama, T. Ku¨ ndig, A. Wakeham, K. Kishihara, A. Shahinian, K. Wiegmann, P. Ohashi, M. Kro¨ nke, and T. Mak. 1993. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxin shock, yet succumb to L. monocytogenes infection. Cell 73:457. 27. Racoosin, E. L., and J. A. Swanson. 1989. Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. J. Exp. Med. 170:1635. 28. Ekman, M., J. Grayston, R. Visakorpi, M. Kleemola, C. Kuo, and P. Saikku. 1993. An epidemic of infections due to Chlamydia pneumoniae in military conscripts. Clin. Infect. Dis. 17:420. 29. Rottenberg, M., E. Castan˜ os-Velez, R. Mesquita, O. Gon˜ i Laguardia, ¨ rn. 1996. Intracellular colocalization of inducible nitric P. Biberfeld, and A. O oxide synthase and Trypanosoma cruzi: evidence for a dual pathway of iNOS induction. Eur. J. Immunol. 26:3203. 30. Siebert, P. D., and J. W. Larrick. 1992. Competitive PCR. Nature 359:557. 31. Jantos, C. A., R. Roggendorf, F. N. Wuppermann, and J. H. Hegemann. 1998. Rapid detection of Chlamydia pneumoniae by PCR-enzyme immunoassay. J. Clin. Microbiol. 36:1890. 32. Sander, B., I. Hoiden, U. Andersson, E. Moller, and J. S. Abrams. 1993. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen: cytokine detection by immunoassay and intracellular immunostaining. J. Immunol. Methods 166:201. 33. Sander, B., J. Andersson, and U. Andersson. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol. Rev. 119:65. 34. Ding, A. H., C. F. Nathan, and D. J. Stuehr. 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J. Immunol. 141:2407. 35. Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, and T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11:165. 36. Tripp, C. S., S. F. Wolf, and E. R. Unanue. 1993. Interleukin 12 and tumor necrosis factor ␣ are costimulators of interferon ␥ production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725. 37. Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, and G. Trinchieri. 1995. Interleukin-12 is required for interferon-␥ production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672. 38. Ohteki, T., T. Fukao, K. Suzue, C. Maki, M. Ito, M. Nakamura, and S. Koyasu. 1999. Interleukin 12-dependent interferon ␥ production by CD8␣⫹ lymphoid dendritic cells. J. Exp. Med. 189:1981. 39. Yoshimoto, T., H. Okamura, Y. I. Tagawa, Y. Iwakura, and K. Nakanishi. 1997. Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon-␥ production from activated B cells. Proc. Natl. Acad. Sci. USA 94:3948. 40. Gupta, A. A., I. Leal-Berumen, K. Croitoru, and J. S. Marshall. 1996. Rat peritoneal mast cells produce IFN-␥ following IL-12 treatment but not in response to IgE-mediated activation. J. Immunol. 157:2123. 41. Kol, A., G. K. Sukhova, A. H. Lichtman, and P. Libby. 1998. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-␣ and matrix metalloproteinase expression. Circulation 98:300. 42. Netea, M. G., C. H. Selzman, B. J. Kullberg, J. M. Galama, A. Weinberg, A. F. Stalenhoef, J. W. Van der Meer, and C. A. Dinarello. 2000. Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells. Eur. J. Immunol. 30:541. 43. Rasmussen, S. J., L. Eckmann, A. J. Quayle, L. Shen, Y. X. Zhang, D. J. Anderson, J. Fierer, R. S. Stephens, and M. F. Kagnoff. 1997. Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis. J. Clin. Invest. 99:77.
The Journal of Immunology 44. Kalayoglu, M. V., and G. I. Byrne. 1998. A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide. Infect. Immun. 66:5067. 45. Trinchieri, G. 1998. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv. Immunol. 70:83. 46. Rottenberg, M. E., A. Gigliotti Rothfuchs, D. Gigliotti, M. Ceausu, C. Une, V. Levitsky, and H. Wigzell. 2000. Regulation and role of IFN-␥ in the innate resistance to infection with Chlamydia pneumoniae. J. Immunol. 164:4812. 47. Huang, J., M. D. Wang, S. Lenz, D. Gao, and B. Kaltenboeck. 1999. IL-12 administered during Chlamydia psittaci lung infection in mice confers immediate and long-term protection and reduces macrophage inflammatory protein-2 level and neutrophil infiltration in lung tissue. J. Immunol. 162:2217. 48. Geng, Y., K. Berencsi, Z. Gyulai, T. Valyi-Nagy, E. Gonczol, and G. Trinchieri. 2000. Roles of interleukin-12 and ␥ interferon in murine Chlamydia pneumoniae infection. Infect. Immun. 68:2245. 49. Wang, J., J. Wakeham, R. Harkness, and Z. Xing. 1999. Macrophages are a significant source of type 1 cytokines during mycobacterial infection. J. Clin. Invest. 103:1023. 50. Fiorentino, D. F., A. Zlotnik, P. Vieira, T. R. Mosmann, M. Howard, K. W. Moore, and A. O’Garra. 1991. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146:3444. 51. Bogdan, C. 2000. The function of type I interferons in antimicrobial immunity. Curr. Opin. Immunol. 12:419. 52. Senik, A., I. Gresser, C. Maury, M. Gidlund, A. Orn, and H. Wigzell. 1979. Enhancement of mouse NK cells by interferon. Transplant. Proc. 11:993. 53. Gidlund, M., A. Orn, H. Wigzell, A. Senik, and I. Gresser. 1978. Enhanced NK cell activity in mice injected with interferon and interferon inducers. Nature 273: 759. 54. Tough, D. F., P. Borrow, and J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272:1947. 55. Mattner, J., H. Schindler, A. Diefenbach, M. Rollinghoff, I. Gresser, and C. Bogdan. 2000. Regulation of type 2 nitric oxide synthase by type 1 interferons in macrophages infected with Leishmania major. Eur. J. Immunol. 30:2257.
6461 56. Sareneva, T., S. Matikainen, M. Kurimoto, and I. Julkunen. 1998. Influenza A virus-induced IFN-␣ and IL-18 synergistically enhance IFN-␥ gene expression in human T cells. J. Immunol. 160:6032. 57. Cousens, L. P., J. S. Orange, H. C. Su, and C. A. Biron. 1997. Interferon-␣ inhibition of interleukin 12 and interferon-␥ production in vitro and endogenously during viral infection. Proc. Natl. Acad. Sci. USA 94:634. 58. Orange, J. S., and C. A. Biron. 1996. Characterization of early IL-12, IFN-␣, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J. Immunol. 156:4746. 59. Hunter, C. A., K. E. Gabriel, T. Radzanowski, L. E. Neyer, and J. S. Remington. 1997. Type I interferons enhance production of IFN-␥ by NK cells. Immunol. Lett. 59:1. 60. Cousens, L. P., R. Peterson, S. Hsu, A. Dorner, J. D. Altman, R. Ahmed, and C. A. Biron. 1999. Two roads diverged: interferon ␣/- and interleukin 12-mediated pathways in promoting T cell interferon ␥ responses during viral infection. J. Exp. Med. 189:1315. 61. Nguyen, K., L. P. Cousens, L. A. Doughty, G. C. Pien, J. E. Durbin, and C. A. Biron. 2000. Interferon ␣/-mediated inhibition and promotion of interferon ␥: STAT1 resolves a paradox. Nat. Immun. 1:70. 62. Devitt, A., P. A. Lund, A. G. Morris, and J. H. Pearce. 1996. Induction of ␣/ interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine. Infect. Immun. 64:3951. 63. Gao, J. J., M. B. Filla, M. J. Fultz, S. N. Vogel, S. W. Russell, and W. J. Murphy. 1998. Autocrine/paracrine IFN-␣ mediates the lipopolysaccharide-induced activation of transcription factor Stat1␣ in mouse macrophages: pivotal role of Stat1␣ in induction of the inducible nitric oxide synthase gene. J. Immunol. 161:4803. 64. Salkowski, C. A., G. R. Detore, and S. N. Vogel. 1997. Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, ␥ interferon, and interleukin-10 mRNA production in murine macrophages. Infect. Immun. 65: 3239. 65. Fultz, M., and S. Vogel. 1995. Autoregulation by interferons provides an endogenous “priming” signal for LPS-responsive macrophages. J. Endotoxin Res. 2:77.
