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Originally published online as doi:10.1189/jlb.0105035 on June 10, 2005

Published online before print June 10, 2005
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(Journal of Leukocyte Biology. 2005;78:686-695.)
© 2005 by Society for Leukocyte Biology

IL-2 induces expression and secretion of IFN-{gamma} in murine peritoneal macrophages

Patrizia Puddu, Maria Carollo, Immacolata Pietraforte, Francesca Spadaro, Marina Tombesi, Carlo Ramoni, Filippo Belardelli and Sandra Gessani1

Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy

1Correspondence: Istituto Superiore di Sanità, Department of Cell Biology and Neurosciences, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: gessani{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the effect of interleukin (IL)-2, a T cell growth factor capable of activating certain macrophage functions, on interferon (IFN)-{gamma} expression in resting mouse peritoneal macrophages (PM). IL-2 addition to PM from different mouse strains up-modulated IFN-{gamma} mRNA and protein secretion. It is notable that endogenous type I and II IFNs did not play any role in the IL-2-mediated effect, as comparable levels of secreted IFN-{gamma} were observed upon IL-2 stimulation of PM from deficient mice. In contrast, endogenous IFN-{gamma} was requested for the IL-12-induced IFN-{gamma} production. It is interesting that blocking of each component of the IL-2 receptor (IL-2R) by neutralizing antibodies almost completely abolished IL-2-induced IFN-{gamma} production, suggesting that all IL-2R chains contribute to the PM biological response to IL-2. The simultaneous treatment of PM with IL-2 and IL-12 resulted in a higher IFN-{gamma} secretion with respect to that obtained upon treatment with IL-2 or IL-12 alone. It is notable that IFN-{gamma} protein was expressed intracellularly in the majority of cells exhibiting a macrophage phenotype (i.e., F4/80+) and was secreted upon IL-2 stimulation. Overall, these findings demonstrate that IL-2 regulates at different levels IFN-{gamma} expression in macrophages, highlighting the crucial role of these cells and their regulated responsiveness to key cytokines in the cross-talk between innate and adaptive immunity.

Key Words: cytokine secretion • mouse • gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interferon (IFN)-{gamma} is a pleiotropic cytokine endowed with potent immunomodulatory effects on a variety of immune cells in vitro and in vivo [1 , 2 ]. Several studies carried out in mice lacking the expression of IFN-{gamma} or its receptor have pointed out that IFN-{gamma} is one of the most important factors in the host defense against infectious agents [3 ]. In particular, IFN-{gamma} exerts important activities on monocytes/macrophages and lymphocytes, which generally result in macrophage activation and T cell differentiation toward a T helper cell type 1 (Th1) of immune response [4 , 5 ]. Although this cytokine does not act as a Th differentiation-like factor, it serves as a cofactor in Th differentiation by up-regulating interleukin-12 receptor (IL-12R) expression and IL-12 production by antigen-presenting cells (APCs). The exploitation of such a variety of effects by a single cytokine is achieved by complex patterns of cell-specific gene regulation [6 ]. IFN-{gamma} expression was long considered to be restricted to activated natural killer (NK) cells and T lymphocytes [3 ]. It is now well established, however, that IFN-{gamma} expression can also occur in other human and murine cell types, including macrophages and dendritic cells (DCs) under physiological and pathological conditions [1 , 7 , 8 ]. In this regard, it has been previously reported that the IFN-{gamma} gene is constitutively expressed in unstimulated, resting peritoneal macrophages (PM) explanted from normal mice [9 ]. Macrophages can be further stimulated to produce IFN-{gamma} by lipopolysaccharide (LPS) treatment, which results in increased steady-state levels of IFN-{gamma} mRNA as well as cell-associated IFN-{gamma} protein [10 ]. In addition, the expression of IFN-{gamma} in resting PM can be up-regulated by IFN-{gamma} itself as well as by IL-12 stimulation [9 , 11 ]. It is interesting that the IL-12-induced expression of IFN-{gamma} in PM can be further stimulated or inhibited by IL-18 and IFN-{alpha}, respectively [12 ].

IL-2, a cytokine mainly produced by activated T lymphocytes, plays a fundamental role in sustaining the growth of T, B, and NK cells [13 ]. During the last phases of antigen-specific T cell response, this cytokine contributes to the maintenance of T cell homeostasis by promoting activation-induced cell death of effector T lymphocytes [14 ]. In keeping with the pivotal regulatory role exerted by IL-2 in the immune system, IL-2-deficient mice show a generalized immune system deregulation [15 ]. The subsequent recognition of the monocyte/macrophage-activating properties of IL-2 has broadened our knowledge about the biological effects of this lymphokine from a T cell growth factor to a molecule with pleiotropic effects [16 ]. The detailed analysis of the mechanisms of action of IL-2, including its biological effects on different cell types and the regulation of its receptors, has therefore increased the spectrum of the biological responses induced by this cytokine. Among APCs, murine macrophages express functional IL-2Rs and biologically respond to IL-2 by activating specific functions or up-modulating some cytokine genes [16 ]. Particularly, IL-2, alone or in combination with tumor necrosis factor (TNF)-{alpha}, has been reported to enhance macrophage tumoricidal activity [17 ]. Moreover, the combination of IFN-{gamma} and IL-2 cooperatively activates nuclear factor-{kappa}B in murine PM, leading to TNF-{alpha} and IFN-inducible protein 10 gene expression [18 ]. Likewise, IL-2 can also act as a cofactor, together with IFN-{gamma}, in inducing macrophage resistance to intracellular parasite infection [19 ]. It is notable that it has also been reported that the simultaneous treatment of mature, splenic DCs with IL-2 and IL-12 enhances the IL-12-induced IFN-{gamma} production [20 ]. Thus, the recognized macrophage and DC responsiveness to IL-2 along with the recently suggested role of this cytokine in bridging innate and acquired immunity [21 ] make this cytokine an attractive candidate for the regulation of IFN-{gamma} expression in macrophages.

In the attempt to identify new immune mediators involved in the control of IFN-{gamma} expression in macrophages, we therefore focused our attention on IL-2, showing that this cytokine is capable of stimulating mRNA accumulation and secretion of IFN-{gamma} in resting PM harvested from different mouse strains. Blockade of IL-2R chains by specific neutralizing antibodies almost completely abrogates IFN-{gamma} production, thus indicating that each component of IL-2R is indispensable to the biological response of PM to IL-2. The IL-2 response of PM was not mediated by the endogenous type I and type II IFNs, as PM harvested from IFN receptor (IFNR) knockout (KO) mice fully responded to IL-2 by producing IFN-{gamma}. In contrast, the effect of IL-12 on IFN-{gamma} expression was dependent on IFN-{gamma} endogenously expressed in PM. Furthermore, IL-2 synergized with IL-12 in the production of IFN-{gamma}. Finally, we report that the majority of F4/80+ cells intracellularly express IFN-{gamma}, which is secreted upon IL-2 treatment.

On the whole, these findings demonstrate that IL-2 regulates IFN-{gamma} expression in murine macrophages by acting at different levels, thus widening the spectrum of biological activities of this T cell cytokine to novel effects on macrophages, which suggests the existence of an active cross-talk between cells of the innate immunity and proliferating Th1 lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C3H/HeN and 129sv were purchased from Harlan (Horst, The Netherlands), whereas C3H/HeJ, Balb/c, and Balb/c-background nu/nu mice were obtained from Charles River, Italia S.p.A. (Milan, Italy). Breeding pairs of IFN-{gamma} R–/– and IFN-{alpha}/ß R–/– KO mice (strain 129/Sv-Ifngrtml) were obtained through the courtesy of Dr. Ion Gresser (Institute de Reherche Scientifiques sur le Cancer, Villejuif, France). A colony was then bred and maintained at the Istituto Superiore di Sanità (Rome, Italy). Mice were kept under pathogen-free (C3H/HeN, C3H/HeJ, 129Sv, Balb/c) or germ-free conditions (IFN-{alpha}/ß R–/– and IFN-{gamma} R–/– KO mice, Balb/c-background athymic nu/nu mice). All mice were 5–6 weeks old and were used within 2 weeks from their arrival.

PM cultures
Total peritoneal cells were harvested by washing the peritoneal cavity of mice with cold saline (5 ml per mouse). Total peritoneal cells were centrifuged and suspended in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (complete medium). Cells were cultured in 24 (1x106 cells/well, 500 µl)- or 48-well tissue-culture plates (5x105 cells/well, 250 µl; Costar, Cambridge, MA) at 37°C in 5% CO2 humidified air. After 1 h, nonadherent cells were removed by three washes with medium without FBS, and then complete medium was added. Experiments were undertaken when the cells were firmly adherent to the culture wells after vigorous washing. Over 95% of the cells stained for nonspecific esterase [22 ] and were positively stained with phycoerythrin-conjugated antibody against F4/80, as described previously [11 ].

Reagents and antibodies
RPMI-1640 medium was supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), and 10% FBS. All tissue reagents were purchased from Hyclone Laboratories (Logan, UT) as endotoxin-free lots and further assessed by the Limulus amebocyte assay.

Murine recombinant (mr)IL-2 (5x106 U/mg) was obtained from Peprotech (London, UK). mrIL-12 (1.2x106 U/mg) was kindly provided by the Genetics Institute, Inc. (Cambridge, MA). The following purified rat anti-mouse, preservative-free antibodies to IL-2R components were used: CD25 (clone PC61), CD122 (clone TM-ß1), and CD132 (clone TUGm2) against the {alpha}, ß, and {gamma} chains, respectively. The matched isotype antibody was used as a negative control. For confocal microscopy experiments, macrophages, DCs, T lymphocytes, and NK cells were stained with rat anti-mouse F4/80, hamster anti-mouse CD11c (HL3 clone), hamster anti-mouse CD3{varepsilon} chain (clone 145-2C11), and mouse anti-mouse Ly-49C/I (clone 5E6) biotin-conjugated monoclonal antibodies (mAb), respectively. Cells stained with biotin-conjugated antibodies were then incubated with Alexa Fluor 594 streptavidin-conjugated antibody (Molecular Probes Europe BV, The Netherlands). The cell-associated IFN-{gamma} protein was detected by fluorescein isothiocyanate (FITC)-conjugated rat immunoglobulin G 1 (IgG1; clone XMG1.2) and purified rat IgG1 (clone R4-6A2) antibodies specific for mouse IFN-{gamma}. The matched isotype antibody was used as a negative control. All antibodies, except F4/80 and its matched control isotype (Serotec, Oxford, UK), were purchased from BD Biosciences (San Jose, CA).

RNA-polymerase chain reaction (PCR)
Total peritoneal cells (3x106) were seeded in six-well cluster plates, and total RNA was extracted from firmly adherent macrophages by using RNeasy mini kit (Qiagen S.p.A., Italy). RNA was reverse-transcribed in a 20-µl reaction containing 0.5 µg total RNA, 2.5 µM oligo-dt (15 mer), 2.5 U/µl Moloney murine leukemia virus reverse transcriptase (RT), 1 mM each deoxy-adenosine 5'-triphosphate, -cytidine 5'-triphosphate, -guanosine 5'-triphosphate, and [thymidine 5'-triphosphate, 5 mM MgCl2, and 1x PCR buffer II and incubated for 15 min at 42°C, 5 min at 99°C, and 5 min at 5°C. The cDNA product was amplified in a 100-µl reaction containing 2 mM MgCl2, 1x PCR buffer II, 5 ng/µl each of the gene-specific upstream and downstream primers [11 ], and 2.5 U AmpliTaq DNA polymerase (Perkin Elmer Cetus Corp., Norwalk, CT), using a PTC-100 DNA thermal cycler (M. J. Research, Watertower, MA). The amplification program comprised an initial enzyme activation step consisting of 105 s at 95°C, followed by 35 cycles of denaturation for 15 s at 95°C and a primer annealing-extension passage for 30 s at 60°C. At the end of the amplification program, samples were incubated at 72°C for 7 min. A negative control, lacking template RNA or RT, was included in each experiment. The PCR products were run on 2.5% agarose gel and visualized by ethidium bromide staining.

Assay for induction of antiviral state in macrophages: vesicular stomatitis virus (VSV) yield
Adherent macrophages were cultured with IL-2 (100 U/ml) in the presence or in the absence of neutralizing antibodies to IFN-{gamma} (clone R4-6A2) for 3 days or left untreated. At day 3, supernatants were collected for IFN-{gamma} titration, and 0.2 ml of a viral dilution (multiplicity of infection of ~2) was added to each well. After 1 h of incubation at 37°C, the cell monolayer was washed thoroughly, and 1 ml complete medium was added. After 18 h incubation at 37°C in a 5% CO2 air incubator, the cell-free supernatant was assayed for virus titer in mouse L929 cells.

Detection of secreted and intracellular IFN-{gamma}
Firmly adherent macrophages were stimulated with cytokines soon after cell seeding, and supernatants were collected 18–24 h post-treatment. In some experiments, cells were cultured for 3 days in the presence of cytokines, and at day 3, culture supernatants were replaced with fresh complete medium; then cells were further stimulated with cytokines. After 24 h, culture supernatants were collected for IFN-{gamma} titration, and titration of intracellular IFN-{gamma} was performed in samples obtained as follows: Adherent macrophages were stimulated with IL-2 for 3 h in 24-well tissue-culture plates, and then monensin (GolgistopTM, BD Biosciences) was added following manufacturing instructions. After overnight incubation, supernatants were collected for IFN-{gamma} titration, and the cell monolayer was washed twice with cold phosphate-buffered saline (PBS) and incubated for 30 min on ice in 100 ml of 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol, and 1% Nonidet P-40 buffer, containing protease inhibitors. Samples were centrifuged for 20 min at 14,000 rpm, and the supernatant was stored at –20°C.

Cell-associated and secreted IFN-{gamma} was titrated by enzyme-linked immunosorbent assay (ELISA) using the Mouse IFN-{gamma} MiniKitTM containing mouse rIFN-{gamma}, coating and detecting antibodies. Mouse IFN-{gamma} MiniKitTM, poly-horseradish peroxidase streptavidin, and 3,3',5,5'-tetramethylbenzidine substrate solution were all purchased from Endogen (Woburn, MA). Ninety-six-well plastic plates (maxi-sorb F8) were obtained from Nunc (Roskilde, Denmark). The ELISA kit described above exhibits a high sensitivity of IFN-{gamma} detection (15–30 pg/ml). As a control, some supernatants were also titrated by using the R4-6A2 antibody as a coating antibody.

Confocal laser-scanning microscopy (CLSM) analysis
For CLSM analysis, total peritoneal cells were seeded in 24-well cluster plates on cover glasses (12 mm diameter; 5x105 cell/well, 500 µl), and after 1 h, nonadherent cells were removed by washing, and only firmly adherent cells were treated with IL-2 (100 U/ml) or left untreated. To block intracellular transport processes and favor accumulation of IFN-{gamma} into the Golgi complex, some CLSM experiments were carried out in cells treated with GolgistopTM containing monensin (BD Biosciences), which was added 3 h after IL-2 stimulation and maintained for the entire length of the experiment (15 h). At the indicated time-points, cells were washed with PBS, stained with lineage-specific surface marker, biotin-conjugated antibodies for 30 min at 4°C, washed twice with PBS, and subsequently stained with Alexa Fluor 594 streptavidin-conjugated antibodies for 30 min at 4°C. Cells were then fixed with methanol for 10 min at –20°C, stained with FITC-conjugated anti-IFN-{gamma} antibody for 30 min at 37°C, and finally incubated with Hoechst-33258 for 10 min at room temperature. The extensively rinsed cover glasses were then mounted on the microscope slide with the antifade Vectashield reagent (Vector Laboratories, Burlingame, CA). CLSM observations were performed by using a Leica TSC SP2 AOBS apparatus, equipped with a diode laser 405, an argon laser 488, and a He/Ne laser 543/594. Image acquisition and processing were conducted by using Leica confocal software multicolor analysis (Leica Lasertechnik Ambh, Heidelberg, Germany) and the Adobe Photoshop software program. Several fields were analyzed for each labeling condition and a 200- to 300-nm central optical section of cells is presented.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-2 induces IFN-{gamma} mRNA expression and protein secretion in PM from different mouse strains
We performed initial experiments aimed at verifying whether IL-2 per se was capable of stimulating IFN-{gamma} production in PM harvested from normal mice and at identifying the optimal experimental conditions for such induction. As shown in Figure 1A , treatment of freshly isolated PM with 100 units/ml IL-2 induced a time-dependent secretion of IFN-{gamma}. The minimal time requested for achieving a detectable secretion of IFN-{gamma} was 12 h; then, the level of secreted protein increased with time and reached the plateau level at 48 h. No further increase, but rather a slight decrease, was observed at later time-points (72 h, data not shown). We previously demonstrated that PM cultured in vitro for 3 days produce IFN-{gamma} in response to exogenous IL-12 [11 ]. However, this response occurred, to a greater extent, when PM, cultured for 3 days in the presence of IL-12, were restimulated with IL-12 for an additional 24-h period. On the basis of these results and of the observation that freshly isolated PM express all IL-2R components [22 ], freshly isolated PM were treated with different IL-2 concentrations soon after cell seeding and restimulated at day 3 with the same IL-2 concentrations. As shown in Figure 1B , a single IL-2 treatment of freshly isolated PM resulted in a significant production of IFN-{gamma}, only when 100 units of this cytokine were used. However, when PM were cultured for 3 days in the continuous presence of IL-2 and then restimulated with IL-2 for a further 24-h period, a markedly higher secretion of IFN-{gamma} was observed with respect to the single treatments, which were already significant with 10 units of IL-2, and further increased at the concentration of 100 units. To evaluate whether the IFN-{gamma} secreted upon IL-2 stimulation of PM was biologically active, we assessed the capacity of the secreted protein to protect cells from infection with the VSV. As shown in Figure 1C , control PM replicated the virus at high levels. However, a marked reduction in viral replication was observed in IL-2-treated PM with respect to control cultures. This protective effect was almost completely abolished when neutralizing antibodies to IFN-{gamma} were added to IL-2-stimulated cultures.



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  		Figure 1. Concentration- and treatment-dependent IFN-{gamma} production in IL-2-stimulated C3H/HeN PM. Peritoneal cells harvested from C3H/HeN mice were seeded at the concentration of 1 x 106/well in 24-well cluster plates as described in Materials and Methods. (A) Adherent PM were treated with IL-2 (100 U/ml) soon after cell seeding. At different time-points, cell supernatants were collected and IFN-{gamma} measured by ELISA. Values represent the mean of three independent experiments in duplicate ± SD. (B) Adherent PM were treated with different concentrations of IL-2 soon after cell seeding or were left untreated. Supernatants were collected for IFN-{gamma} titration 24 h later (day 0). Some cultures were treated with different concentrations of IL-2 at day 0 and left in the presence of IL-2 for 3 days. On day 3, medium was replaced, and cultures were further stimulated with IL-2 (day 0+day 3). On day 4, aliquots of the culture medium were collected and tested by ELISA for the presence of IFN-{gamma}. Values represent the mean of three independent experiments in duplicate ± SD. (C) Adherent PM were treated with IL-2 (100 U/ml) in the presence or in the absence of neutralizing antibodies to IFN-{gamma} (1 µg/ml) at day 0 and further stimulated in the same manner at day 3. Twenty-four hours later, cells were infected with VSV. Supernatants were collected 18 h post-infection, and the virus yields titrated on L929 cells as described in Materials and Methods. Values represent the mean of three independent experiments in duplicate ± SD.

 
Experiments were then carried out to evaluate whether the IL-2-stimulated IFN-{gamma} production was also observed in PM harvested from other mouse strains. The addition of IL-2 to PM harvested from three different mouse strains resulted in a clear-cut accumulation of IFN-{gamma} mRNA, although to a different extent, depending on the mouse strain used (Fig. 2A ). Consistently with this result, remarkable levels of IFN-{gamma} protein were found in the culture medium of IL-2-treated PM, and no detectable cytokine production was observed in control cultures (Fig. 2B) . The different levels of IFN-{gamma} secreted apparently reflected differences in the amount of mRNA accumulated in PM upon IL-2 stimulation. Notably, IFN-{gamma} secretion was also observed after IL-2 stimulation of PM harvested from Balb/c T cell-deficient nu/nu mice (Fig. 2B) .



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  		Figure 2. Induction of IFN-{gamma} mRNA and protein secretion in IL-2-treated PM from different mouse strains. Induction of IFN-{gamma} mRNA and protein secretion in IL-2-treated PM. (A) Peritoneal cells freshly explanted from C3H/HeN, C3H/HeJ, and Balb/c mice were seeded in six-well cluster plates (3x106/well, three wells for experimental condition) and cultured as described in Materials and Methods. Adherent PM were then treated with IL-2 (100 U/ml) or left untreated, and total RNA was extracted 3 h later and processed for RT-PCR analysis of IFN-{gamma} mRNA accumulation, as described in Materials and Methods. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase. (B) Peritoneal cells harvested from C3H/HeN, C3H/HeJ, Balb/c, and Balb/c-background athymic nu/nu mice were seeded at the concentration of 1 x 106/well in 24-well cluster plates as described in Materials and Methods. Adherent PM were treated with IL-2 (100 U/ml) or left untreated. On day 3, medium was replaced, and IL-2 was further added to cultures. On day 4, aliquots of the culture medium were collected and tested by ELISA for the presence of IFN-{gamma}. Values represent the mean of three independent experiments in duplicate ± SD.

 
Endogenous type I and II IFNs do not affect the IL-2-mediated IFN-{gamma} production in PM
We previously demonstrated that IFN-{gamma} is capable of inducing its own synthesis in an autocrine manner in PM [9 ]. We also showed that type I IFN negatively regulates the expression of IFN-{gamma} induced in PM by IL-12 treatment [12 ]. As freshly harvested PM express low levels of endogenous type I and type II IFNs [9 , 23 ], experiments were carried out to assess their possible role on the IL-2-mediated IFN-{gamma} production by measuring the cytokine levels in the supernatant of PM obtained from IFN-{gamma} R–/– and IFN-{alpha}/ß R–/– KO mice. As shown in Figure 3 , IL-2 treatment of PM from IFN-{gamma} R–/– and IFN-{alpha}/ß R–/– KO mice resulted in a remarkable production of IFN-{gamma}, similar to that obtained in PM from control 129Sv mice, indicating that type I and type II endogenous IFNs do not play any role in the regulation of IL-2-induced IFN-{gamma} production in PM.



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  		Figure 3. Effect of endogenous type I or II IFN on IL-2-induced IFN-{gamma} expression in PM. Peritoneal cells (1x106), harvested from control mice (129Sv) or mice genetically deficient for the expression of type I (IFN-{alpha}/ß R–/–) or type II (IFN-{gamma} R–/–) IFNRs, were seeded as described in the legend to Figure 1B , treated with IL-2 (100 U/ml), or left untreated. At day 3, supernatants were replaced with fresh medium, and cells were restimulated with the cytokine. Aliquots of the supernatants were collected for ELISA determination of IFN-{gamma} after 24 h. Values represent the mean of three independent experiments in duplicate ± SD.

 
The IL-2-mediated IFN-{gamma} production is dramatically reduced upon blocking of IL-2R components
IL-2R components display variable affinity for its own ligand in human and mouse cell models [24 ]. To determine the contribution of single components of the IL-2R complex to the biological response of PM to this cytokine, we measured the IFN-{gamma} secretion in PM stimulated for 24 h with IL-2 in the absence or in the presence of neutralizing antibodies against the {alpha}, ß, or {gamma} receptor chains. As shown in Figure 4 , blocking of single receptor components resulted in an almost complete abrogation of IL-2-induced IFN-{gamma} production with respect to IL-2-stimulated control culture. In contrast, no differences in the levels of IFN-{gamma} production were observed in the presence of a matched control antibody (Fig. 4) .



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  		Figure 4. Effect of IL-2R components blocking on the IL-2-induced IFN-{gamma} production. Peritoneal cells (1x106) harvested from C3H/HeN mice were seeded as described in the legend to Figure 1 and treated with IL-2 soon after cell seeding in the presence or in the absence of specific blocking antibodies (Ab) directed to the {alpha}, ß, or {gamma} chains of the IL-2R (10 µg/ml). Some cultures were treated with a matched control isotype. Aliquots of the supernatants were collected for ELISA determination of IFN-{gamma} after 24 h. Values represent the mean of three independent experiments in duplicate ± SD.

 
Synergistic effect of a combined treatment with IL-2 and IL-12 on IFN-{gamma} production in PM: role of endogenous IFN-{gamma} on the IL-12-induced IFN-{gamma} production
It has been demonstrated previously that the simultaneous treatment of mature splenic DCs with IL-2 and IL-12 enhanced the IL-12-induced IFN-{gamma} production in these cells [20 ]. To investigate whether IL-2 could exert a similar enhancing effect in IL-12-stimulated PM, the extent of IFN-{gamma} production induced by IL-2 and IL-12 alone was compared with that induced upon a combined treatment. As shown in Figure 5 , a strong enhancement in the secretion of IFN-{gamma} was observed in PM cultures simultaneously treated with IL-2 and IL-12 with respect to the single treatments. It is interesting that although endogenous IFN-{gamma} expression was not needed for the IL-2-induced IFN-{gamma} production (Fig. 3) , we observed that it played an important role in the secretion of IFN-{gamma} induced by IL-12. In fact, as shown in Table 1 , no IFN-{gamma} production was observed when PM from IFN-{gamma} R –/– mice were stimulated with IL-12, and PM from the same animals secreted normal levels of IFN-{gamma} in response to IL-2. In contrast, only a modest reduction in the production of IFN-{gamma} was detected when defective mice were simultaneously stimulated with IL-12 and IL-2 with respect to control PM, suggesting that endogenous IFN-{gamma} was dispensable for the synergistic effect induced by the combined treatment.



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  		Figure 5. Synergystic effect of a combined treatment with IL-2 and IL-12 on the production of IFN-{gamma} in PM. Peritoneal cells (1x106) harvested from C3H/HeN mice were seeded as described in the legend to Figure 1 and treated with IL-2 (100 U/ml) and IL-12 (1 ng/ml), alone or with a combination of the two cytokines, or left untreated. On day 3, PM cultures were restimulated as described. Aliquots of the supernatants were collected for ELISA determination of IFN-{gamma} after 24 h. Values represent the mean of three independent experiments in duplicate ± SD.

 

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  		Table 1. Endogenous IFN-{gamma} Is Involved in the IL-12 but Not in the IL-2-Induced IFN-{gamma} Production

 
Immunoreactive IFN-{gamma} is intracellularly expressed in F4/80+ PM and is extracellularly released upon IL-2 treatment
We have reported previously that IFN-{gamma} protein can be detected in peritoneal cells, clearly exhibiting a macrophage phenotype (i.e., F4/80+), by using a sensitive fluorescent-staining protocol [11 ]. To further characterize the macrophage as the IL-2-responsive cell type in the peritoneum, CLSM analysis was carried out to directly analyze the intracellular distribution of IFN-{gamma} protein in individual cells, exhibiting different phenotypes, as assessed by the concomitant surface expression of lineage-specific phenotypic markers, i.e., F4/80, and intracellular staining of IFN-{gamma}. Initial experiments were carried out in PM, in which intracellular trafficking processes have been blocked to allow intracellular accumulation of proteins in the Golgi complex. As shown in Figure 6A , IFN-{gamma} protein was constitutively expressed in PM cultures maintained in the presence of monensin. It is notable that the majority of cells expressing IFN-{gamma} exhibited a macrophage phenotype, as they were positively stained with anti-F4/80 antibody. However, when PM were stimulated to produce IFN-{gamma} by IL-2 treatment, no increased intracellular accumulation of this cytokine was observed, despite the blocking of intracellular trafficking. In parallel, no staining was obtained with the matched control isotypes for IFN-{gamma} and F4/80, in control PM or in IL-2-treated PM (data not shown). To explore the possibility that the lack of intracellular accumulation of IFN-{gamma} upon IL-2 treatment could be a result of the fact that IL-2 was somehow favoring IFN-{gamma} release, CLSM experiments were therefore carried out under experimental conditions, fully allowing intracellular transport processes. As shown in Figure 6B , PM cultured under normal conditions exhibited a slightly different morphology with respect to cells cultured in the presence of monensin. However, a clear-cut expression of IFN-{gamma} protein was detected in unstimulated PM at comparable levels with respect to PM treated with monensin. Likewise, the majority of cells expressing IFN-{gamma} exhibited a macrophage phenotype, as they were positively stained with anti-F4/80 antibody. It is interesting that the intracellular content of IFN-{gamma} appeared to be slightly decreased in IL-2-treated cultures upon 15 h of stimulation, with respect to control-untreated cultures, and a significant secretion of IFN-{gamma} was detected in the supernatants collected from the same IL-2-treated cultures 15 h post-treatment, but not in control-unstimulated cultures (Fig. 6C) . At earlier time-points (6 h), the intracellular content of IFN-{gamma} protein was comparable with that observed after 15 h of culture and did not change upon IL-2 treatment (data not shown). In contrast, no IFN-{gamma} secretion was observed in PM cultures stimulated with IL-2 in the presence of monensin (Fig. 6C) . Parallel experiments were carried out to precisely quantify the intracellular content of IFN-{gamma}. As shown in Figure 6D , comparable levels of IFN-{gamma} protein were detected in the cell pellet of PM, untreated or IL-2-treated in the absence of monensin. In contrast, a remarkable secretion of IFN-{gamma} was detected in the supernatant of the same cultures stimulated with IL-2. In keeping with the results shown in Figure 6B , the IL-2-induced secretion of IFN-{gamma} was blocked completely when monensin was added to the culture, whereas no significant changes in the intracellular IFN-{gamma} content were observed under these experimental conditions.



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  		Figure 6. Detection by immunostaining of intracellular IFN-{gamma} protein in F4/80+ PM. (A) PM from C3H/HeN mice were treated with IL-2 (100 U/ml) for 3 h, and then, Golgistop-containing monensin was added. After 15 h of culture, cells were double-stained with antibodies against F4/80 (red) and IFN-{gamma} (green) . Each panel shows a 200- to 300-nm central section of analyzed cells. Original scale bar: 40 µM. (B) PM were double-stained with antibodies against F4/80 (red) and IFN-{gamma} (green) after 15 h of culture, in the presence or in the absence of IL-2. (C) Supernatants collected from the cultures, shown in A and B at 15 h, were analyzed for the presence of IFN-{gamma} by ELISA. Values represent the mean of duplicate samples. Sample variability did not exceed 10%. (D) PM were treated with IL-2 (100 U/ml) for 3 h, and then, Golgistop-containing monensin was added. After 15 h of culture, supernatants and pellets were collected for IFN-{gamma} titration (as described in Materials and Methods). ELISA was carried out by using rat-purified R4-6A2 as coating antibody. Similar results have been obtained by using antibody pairs of Mouse IFN-{gamma} MiniKitTM (Endogen). Values represent mean of duplicates. The results are representative of two independent experiments. Ctr, Control.

 
Parallel experiments were then carried out to evaluate whether contaminant cells, possibly present in the macrophage cultures, could contribute to IFN-{gamma} production induced by IL-2. As shown in Figure 7 , CD3+ T cells were not found in the macrophage cultures. Few cells stained with CD11c or Ly-49C/I antibodies were occasionally found after observation of an elevated number of fields, thus confirming the high level of purity of our PM cultures. The presence of cells in each field was confirmed by Hoechst-33258 staining of the nuclei. Although some intracellular accumulation of IFN-{gamma} was observed in the rare, unstimulated CD11c+ cells found in the macrophage cultures, no intracellular accumulation of this cytokine was detected in the occasionally found NK cells under basal conditions or upon IL-2 stimulation (Fig. 7) . Consistently with the results already shown in Figure 6 , the large majority of IFN-{gamma}-expressing cells was positively stained for the macrophage-specific marker F4/80.



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  		Figure 7. Detection by immunostaining of intracellular IFN-{gamma} protein in individual peritoneal cells. Adherent peritoneal cells from C3H/HeN mice were superficially stained with anti F4/80, anti-CD3, anti-CD11c, and anti-Ly-49C/I antibodies (red) or intracellular IFN-{gamma} (green) after 15 h of culture, in the presence or in the absence of IL-2 (100 U/ml). Each panel shows a three-dimensional reconstitution of analyzed cells. Staining of nuclei by Hoechst-33258 is shown in blue. Original scale bar: 40 µM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IFN-{gamma} is a pleiotropic cytokine endowed with potent immunomodulatory effects, whose expression was long-considered to be restricted to T and NK cells. It has now become evident that IFN-{gamma} production can also occur in other cell types, including monocytes/macrophages [1 , 7 , 8 ]. Macrophage-derived IFN-{gamma} has been described to serve as an autocrine stimulator of macrophages, which induces effector mechanisms such as enhancement of antiviral and microbicidal activity and induction of nitric oxide [11 , 25 26 27 ]. Although the in vivo role of the IFN-{gamma} produced by myeloid cells is still poorly characterized, it has been reported recently that APC-derived IFN-{gamma} contributes to the early host defense against intracellular pathogens [28 ]. However, a chronic overproduction of IFN-{gamma}, in particular, physiological conditions, has been reported to be detrimental for the host’s health and involved in the establishment of chronic inflammatory diseases, such as murine lupus [29 ]. Of interest, Carvalho-Pinto and colleagues [30 ] reported that autocrine production of IFN-{gamma} by macrophages controls their recruitment to kidney and the development of glomerulonephritis in mixed leukocyte reaction/lpr mice.

The production of IFN-{gamma} by macrophages is modulated by a number of cytokines. In this regard, we previously demonstrated that resting PM constitutively express the IFN-{gamma} gene, but its mRNA undergoes a rapid turnover [9 ]. Of note, PM treatment with IFN-{gamma} itself or IL-12 results in a clear-cut accumulation of IFN-{gamma} transcripts and protein secretion [9 , 11 ]. Furthermore, the IL-12-induced IFN-{gamma} production is positively or negatively regulated upon treatment of PM with IL-18 and type I IFN, respectively [12 ].

In this study, we report that IL-2 stimulates in a dose-dependent manner IFN-{gamma} protein secretion in resting PM harvested from different mouse strains. It is interesting that IFN-{gamma} production was also detected in PM from Balb/c mice, wild-type or athymic nu/nu, although to a much lesser extent than in PM from other mouse strains, including C3H/HeN and C3H/HeJ mice. Of note, Balb/c mice have been shown to easily induce Th2 responses in several infection models. Although the mechanisms involved in the induction of this type of response in Balb/c mice remain to be clarified, a recent study by Kuroda and co-workers [31 ] has correlated the scarce production of IFN-{gamma} in PM from Balb/c, in response to the simultaneous treatment with IL-12 and IL-18, with reduced levels of signal transducer and activator of transcription (STAT)4 expression in PM. Notably, STAT4 and IFN-{gamma} production by other cell types, such as B and T lymphocytes, was absolutely normal [31 ]. In keeping with these findings, we observed that PM harvested from Balb/c mice exhibit a markedly reduced response to IL-2 with respect to PM from other mouse strains, as assessed by a lower IFN-{gamma} mRNA accumulation and protein secretion. Our results completely exclude a role for LPS in the IL-2-mediated effect, as IFN-{gamma} secretion was detected at comparable levels in the culture supernatants of cytokine-stimulated PM from LPS-hyporesponsive (C3H/HeJ) and LPS-responsive (C3H/HeN) mice.

In this study, we also report that endogenous expression of type I and type II IFNs in PM does not influence the capacity of these cells to respond to IL-2, as PM harvested from mice genetically deficient for IFNR expression are fully responsive to the IL-2-induced IFN-{gamma} production. Moreover, in keeping with the effect observed in mature splenic DCs [20 ], we report that IL-2 enhances the IL-12-induced IFN-{gamma} production in PM. It is interesting that whereas endogenous IFN-{gamma} is not required for the IL-2-stimulated IFN-{gamma} secretion, it turned out to be essential for the IL-12-mediated effect. However, the enhanced production of IFN-{gamma} observed upon a combined treatment with IL-2 and IL-12 was only modestly reduced in PM from IFN-{gamma} R–/– mice, indicating that endogenous IFN-{gamma} is no longer needed for the IL-12-mediated effect when IL-2 is present in the culture medium. These results suggest that the IL-2 and IL-12 might enhance IFN-{gamma} expression in PM through different mechanisms. Furthermore, IFN-{gamma} and IL-2 might favor the expression/activation of factor(s) essential for the stimulating effect of IL-12 on IFN-{gamma} production.

The expression of functional IL-2Rs was originally thought to be restricted to T lymphocytes. However, other lymphoid cell types, including activated B and NK cells, were subsequently shown to express functional IL-2R [13 ]. It is now well established that human and mouse cells, belonging to the myeloid lineage, including blood monocytes, alveolar macrophages, PM, DCs, and a variety of monocytic cell lines, express functional IL-2Rs and biologically respond to IL-2 [16 17 18 19 20 ]. However, some differences have been found between the mouse and human IL-2R complex. In fact, it has been demonstrated that the mouse IL-2R complex differs from the human complex in the {alpha} chain requirement for the functional mouse receptor [24 ]. The expression of IL-2R chains has been previously analyzed in PM. Although, it was reported that all receptor chains are expressed in these cells, {alpha} and ß chains are expressed at markedly lower levels with respect to the {gamma} chain [22 ]. Of note, we have found that blocking of single IL-2R chains by specific mAb completely abrogates the effect of IL-2 on the IFN-{gamma} production, thus suggesting that all receptor chains, despite the different level of expression, are involved in the IL-2 biological response of PM. Moreover, IL-2 itself did not induce any significant change in the surface expression of the IL-2R components in PM.

Our data provide convincing evidence that macrophages are indeed the cell type responsible for IFN-{gamma} production triggered by IL-2. In fact, by using the CLSM technique, we have been able to show that the majority of PM, positively stained by the F4/80 antibody, intracellularly express considerable levels of IFN-{gamma} protein. It has been reported recently that minute numbers of contaminating CD8+ T cells or NK cells are the source of IFN-{gamma} in IL-12/IL-18-stimulated peritoneal exudate cells harvested from C57BL/6 or Balb/c mice [32 ]. In this regard, it is worth mentioning that in our study, we have been unable to detect the presence of contaminating cells exhibiting the phenotype described by Schleicher and colleagues [32 ]. This is likely a result of the fact that the composition of the peritoneal cells, recruited by thioglycolate-induced sterile inflammation, differs from that present in the peritoneum under resting conditions and may vary among mouse strains. Likewise, the more activated phenotype exhibited by PM recruited in the course of inflammation may at least in part account for a lower capacity to produce IFN-{gamma}. In fact, it is reasonable to speculate that the low levels of macrophage-derived IFN-{gamma} might play a role in the macrophage biology in physiological conditions, whereas under inflammatory conditions, the massive production of this cytokine could be a distinct feature of the cell of the lymphoid compartment.

The results of our study indicate that multiple mechanisms are likely underlying the capacity of IL-2 to modulate the production of IFN-{gamma}. In fact, we report that IL-2 stimulation of PM from different mouse strains resulted in the accumulation of IFN-{gamma} transcripts. The extent of this effect was dependent on the mouse strain and correlated with the level of IFN-{gamma} secreted. Conversely, we also report that when PM cultures previously stimulated with IL-2 were treated with monensin, the intracellular accumulation of IFN-{gamma} was not enhanced. Moreover, the intracellular levels of IFN-{gamma} did not significantly change, and the cytokine was detected extracellularly by ELISA after 15 h when PM were stimulated with IL-2 in the absence of monensin. These results were confirmed by quantifying the intracellular content of IFN-{gamma} by ELISA. Although additional studies are needed to define the precise mechanisms involved in the IL-2-enhancing effect on the production of IFN-{gamma}, the ensemble of our results suggests that IL-2 regulates IFN-{gamma} expression by acting at different levels. In fact, IL-2 treatment of PM resulted in an increased accumulation of IFN-{gamma} mRNA. However, this effect did not parallel a concomitant accumulation of the corresponding protein, suggesting that IL-2 could contribute to the replenishment of a pool of IFN-{gamma} transcripts, highly unstable [9 , 11 ], needed for the maintenance of the basal level of IFN-{gamma} protein expression. Conversely, the observation that IL-2 does not increase IFN-{gamma} protein accumulation under conditions in which its release is blocked by monensin suggests that IL-2 could also act at a post-transcriptional level, somehow favoring IFN-{gamma} release. Furthermore, although the early accumulation of IFN-{gamma} mRNA (3 h) and the relative short time requested for initial IFN-{gamma} secretion (12 h) observed upon IL-2 stimulation argue for a direct effect of this cytokine, it cannot be ruled out that some factor(s), whose expression/secretion might be blocked by monensin, may contribute to the IL-2-mediated IFN-{gamma} production.

Overall, our results suggest that F4/80+ PM homogenously express intracellular IFN-{gamma} and are the main IFN-{gamma}-producing cell in response to IL-2. The finding that PM can respond to IL-2 by producing IFN-{gamma} adds a new component to the puzzle of the complex role of macrophages in the interactions between natural antimicrobial defense and generation of T cell immunity. Moreover, these results unravel a novel role for IL-2 at the early phases of the innate immune response. The constitutive accumulation of IFN-{gamma} in PM and the direct amplification of the IFN-{gamma} production in response to a variety of Th1 cytokines reflect the biological function of macrophages as early players in the host innate response to pathogens. We suggest that in the presence of microbial products or cytokines released early upon pathogen encountering, IFN-{gamma} "prearmed" macrophages can be stimulated to secrete remarkable amounts of IFN-{gamma}, which can affect macrophage function in a paracrine or autocrine manner, thus contributing to activate T cells toward a Th1 type of immune response.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the European Commission, Contract No. QLK2-CT-2001-02103. We thank Anna Maria Fattapposta and Cinzia Gasparrini for excellent editorial assistance, Massimo Venditti for his technical assistance, and Alessandro Spurio for preparing drawings. We thank Giovanna Schiavoni and all participants of the INVADERS project for helpful discussion.

Received January 21, 2005; revised April 29, 2005; accepted May 12, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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