key: cord-0007765-8tdiype6 authors: Charley, B.; Laverne, Sophie; Lavenant, Laurence title: Recombinant porcine interferon-gamma activates in vitro porcine adherent mononuclear cells to produce interleukin 1 date: 2002-11-12 journal: Vet Immunol Immunopathol DOI: 10.1016/0165-2427(90)90029-r sha: 408b334266cac4b8bdba738e806f4b49dc585a01 doc_id: 7765 cord_uid: 8tdiype6 The effect of recombinant porcine interferon-gamma (rPoIFNγ) on in vitro production of interleukin 1 (IL-1) by porcine blood monocytes was studied. Three-day-old cultures of porcine adherent blood mononuclear cells were treated by doses of rPoIFNγ for 3 or 6 days before lipopolysaccharide (LPS)-induction. While rPoIFNγ alone had no effect, a combined treatment by rPoIFNγ and LPS enhanced the IL-1 secretory potential of adherent mononuclear cells and, to a lesser extent, the level of cell-associated IL-1. The IL-1 activity was neutralized by anti porcine IL-1 α and β antisera. These results demonstrate that rPoIFNγ has immunomodulatory effects in vitro on porcine monokine production. Interferon-7 (IFNT) is a lymphokine produced by activated T lymphocytes. In addition to antiviral activity IFN), has various immunomodulatory effects on different cell types, including T-, B-and NK cells as well as neutrophils and macrophages (reviewed by Welsh, 1984; Trinchieri and Perussia, 1985; Virelizier and Arenzana-Seisdedos, 1985; Russell and Pace, 1987) . Expression of DNA-encoded IFN7 has recently enabled the evaluation of some of the biological activities of IFN~ in domestic animals. Thus, recombinant bovine IFNr (rBoIFN~) was shown to inhibit in vitro the replication of several viruses (Czarniecki et al., 1986) , and to modulate various neutrophil and lymphocyte functions, both in vitro and in vivo (Bielefeldt Ohmann and Babiuk, 1986). In the porcine species, the gene coding for IFN7 has been cloned by Genentech Inc. (U.S.A.) and E. coli-derived rPoIFN7 was produced. It was previously shown that this molecule exerted antiviral effects in vitro against vesicular 0165-2427/90/$03.50 stomatitis virus, the coronavirus transmissible gastroenteritis virus and African swine fever virus, and shared antigenic determinants with bovine but not human IFN)' (Charley et al., 1988; Esparza et al., 1988) . However, little is known about the immunomodulatory properties of rPoIFN),. In this report, we present results of a study on porcine monocyte activation in vitro by rPoIFN7. The functional criterion of monocyte activation analyzed was the production of interleukin 1 {IL-1 ) by aged monocyte cultures treated with IFNy and subsequently induced by endotoxin, as described for human IFN7 by Arenzana-Seisdedos et al. {1985) . They showed that aged monocyte cultures do not respond well to lipopolysaccharide stimulation and thus provide a sensitive model to use when evaluating monocyte activation by immunomodulating substances, such as IFNy. Additionally, the use of aged monocyte cultures provides assurance that endotoxin contamination does not yield confusing results in monocyte activation . We report that rPoIFN7 has enhancing effects on the IL-1 secretory potential of aged cultures of porcine blood adherent mononuclear cells. Porcine peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood by density gradient centrifugation {Lymphoprep, density 1.077; Nyegaard, Oslo, Norway). Blood samples were collected from 2-to 4month-old, conventionally reared animals. Mouse thymocytes were prepared from 3-week-old BALB/cJ mice maintained in our facilities. Cells were cultured in RPMI 1640 medium (Gibco, Paisley, Scotland) supplemented with 10% fetal bovine serum, antibiotics, 5 × 10-~ M 2-mercaptoethanol and 10 -:~ M pyruvate. Recombinant porcine IFN7 (lot no. 4648-58) was kindly provided by Ciba Geigy Ltd. (Basel, Switzerland); specific activity was 5 to 10× 106 antiviral units/mg. Porcine PBMC (10 v cells/ml) in RPMI 1640 complete medium ( 10% FBS ), supplemented with 10% autologous plasma, were plated in round-bottomed microtiter plates (Nunc, Roskild, Denmark) at 0.1 ml/well. Cultures were incubated for 3 days in a 95% air-5% CO2 humidified atmosphere at 37 ° C. After this initial incubation the cultures were washed three times with warm medium to remove non-adherent cells. The remaining cells consisted of 74 + 2.1% esterase-positive cells, therefore considered as being monocytes, while the other cells were non further characterized lymphocytes. Adherent PBMC cultures were incubated in a 95% air-5% CO~ humidified atmosphere at 37 °C for 3 days in control medium or in various concentrations of rPoIFN7 (0.1 ml/well). At the end of this incubation period, the medium was carefully removed, discarded and replaced by RPMI 1640 complete medium supplemented with 1 pg/ml of indomethacin (Sigma, St. Louis, U.S.A. ), to avoid possible interference with prostaglandin production, and 20 pg/ml of E. coli lipopolysaccharide (no. 0111 B4 from Sigma) (Cavaillon et al., 1989) . Following an overnight incubation at 37°C, supernatants were harvested and stored at -20 ° C. Cell lysates were obtained, after adding 50 pl of medium per well, by freezing and thawing three times. IL-1 activity was tested by the ability of supernatants and cell lysates to induce the proliferation of mouse thymocytes stimulated with submitogenic doses of phytohemagglutinin (PHA), as described (Charley et al., 1983; Arenzana-Seisdedos et al., 1985; Cavaillon et al., 1989) . Serial log._, dilutions of 0.1 ml samples were performed, in duplicate, in flat-bottom microtiter plates (Falcon, Oxnard, U.S.A.), in a final volume of 0.1 ml of RPMI 1640 complete medium. Mouse thymocytes (107 cells/ml) in RPMI 1640 complete medium, supplemented with 10 ttg/ml PHA-P (Difco, Detroit, U.S.A.) were added to each well. Cultures were incubated for 3 days at 37°C and pulsed overnight with 37 kBq/well of tritiated thymidine (CEA, Saclay, France). The incorporated radioactivity was collected on filter paper by an automated cell harvester. Interleukin 1 activity was expressed as cpm of tritiated thymidine incorporation. In order to assign the biological activity in the samples tested on mouse thymocytes to IL-1, seroneutralization assays were performed on positive supernatants with rabbit antisera directed against the two distinct forms of porcine IL-1. These antisera are able to specifically neutralize porcine IL-1 biological activities, such as cartilage resorption and thymocyte activation (Saklatvala et al., 1985 a,b) . Anti-IL-la and -fl, kindly provided by J. Saklatvala (Cambridge, Great Britain), were added at the non toxic dilution of l/ 640, to a 1.2 diluted test sample; normal rabbit serum was used as a control. These sera did not exert inhibitory effects on mouse thymocytes alone (Table 1 ) . IL-1 activity was assayed as previously described. When porcine adherent PBMC were cultured for 6 days in control medium before LPS induction, no IL-1 activity was detectable in supernatants (Fig. la) nor in cell lysates {Fig. lb). In contrast, 3-day-old cultures of adherent PBMC, when treated for a further 3 days with rPoIFN~ (doses ranging from 0.01 to 10 pg/ml ) before LPS, secreted high levels of IL-1 in their supernatants {Fig. la ). A similar dose-effect of rPolFN~ on the IL-1 activity associated with the cell lysates was observed but the IL-1 yield was lower than in supernatants (Fig. lb) . A seroneutralization test indicated that the mitogenic activity present in rPoIFN;~-treated PBMC supernatants was largely neutralized by antiporcine IL-la and, to a lesser extent, by anti-porcine IL-lfl specific antisera (Table 1) . Additionally, IL-2 titrations performed on IL-2-dependent mouse T-cell lines by C. Leclerc (Institut Pasteur, Paris ) indicated that the mitogenic activity was not related to IL-2 (data not shown). In the absence of LPS induction, no IL-1 activity was detected in the supernatants of rPoIFN),otreated adherent PBMC (Fig. 2) . A kinetic study indicated that a higher IL-1 yield was observed when adherent PBMC cultures were treated for 6 days, instead of 3, with IFN~ (Fig. 2) . Our data show that rPoIFN), is able to activate porcine adherent mononuclear cells to secrete IL-1 after stimulation with LPS. The experimental protocol used, which follows the description by Arenzana-Seisdedos et al. (1985) for human monocytes, allows the preparation of aged (3 days) cultures which poorly respond to LPS alone (Fig. 1 ) . Thus, 3-to 6-day-old porcine adherent PBMC cultures, containing a large majority (74%) of esterase-positive cells, provide a sensitive assay system to evaluate monocyte activation. A seroneutralization experiment conducted with anti-porcine IL-1 indicated that the mitogenic activity produced by rPoIFNT-treated cells, although lower than that shown in Figs. 1 and 2, could be assigned to IL-I~ and to a lesser extent to ILlfl (Table 1 ) . Since rPoIFNy alone is unable to induce IL-1 secretion (Fig. 2 ), it appears that rPoIFN;~ causes a modification to the cells that renders them responsive to LPS. IFNr has been shown, in other animal species, to exert numerous effects on several monocyte-macrophage functions, such as increased expression of membrane receptors and class II molecules, increased phagocytic and antimicrobial activities, or enhanced secretory potential, including cytokine production. In fact, IFN), clearly appears to be one of the macrophage-activating factors (MAF) (reviewed by Trinchieri and Perussia, 1985; Virelizier and Arenzana-Seisdedos, 1985; Russell and Pace, 1987) . Using IL-1 secretion as a functional parameter for monocyte activation, we provide experimental evidence that rPoIFN), has MAF properties in the porcine species. The complete state of activation requires a two step procedure, IFN~ providing, after a long incubation period (Fig. 2) , a first putatively differentiating signal whereas LPS provides the final triggering signal. The ability of rPoIFN7 to increase LPS-induced IL-1 secretion may have important potential implications. IL-1 is known to play a crucial role in the initiation of a specific immune response. It is likely, therefore, that rPoIFN7 could modulate in vivo the immune responsiveness of treated animals, leading to improved antibody production and/or T-cell functions following vaccination. In addition, since IFN~ has been shown to activate microbicidal and tumoricidal functions of rat and bovine alveolar macrophages in vivo (Badger et al., 1988; Bielefeldt Ohmann et al., 1987) , it will be very important to evaluate the ability of rPoIFN~ to activate porcine alveolar macrophages, in order to increase the lung defense mechanisms. Indeed, previous studies have indicated that porcine alveolar macrophages could respond to immunomodulatory compou,nds such as LPS or muramyldipeptide (Charley et al., 1983) and recently porcine somatotropin (Edwards et al., 1988) . The present report provides initial evidence for the potential imm~noenhancing effects of recently available recombinant PoIFNy. In vivo studies are, therefore, currently undertaken in our laboratory to evaluate the influence of rPoIFNy treatment in immunosuppressed pigs. Interferons as macrophage-activating factors. III. Preferential effects of interferon-7 on the interleukin 1 secretory potential of fresh or aged human monocytes Modulation of rat bronchoalveolar lavage cell function by the intratracheal delivery of interferon-7 Alteration of some leukocyte functions following in vivo and in vitro exposure to recombinant bovine alpha and gamma interferon Bovine interferon: its biology and application in veterinary medicine Dissociation of cellassociated interleukin-1 {IL-1 ) and IL-1 release induced by lipopolysaccharide and lipid A In vitro" effects of lipopolysaccharide and mycobacterial cell wall components on swine alveolar macrophages Antiviral and antigenic properties of recombinant porcine interferon gamma In vitro biological activities of Escherichia coli derived bovine interferon a, fl and A newly defined property of somatotropin: priming of macrophages for production of superoxyde anion Effect of interferon a, interferon ~, and tumor necrosis factor on African swine fever virus replication in porcine monocytes and macrophag~s The effects of interferons on macrophages and their precursors Pig interleukin 1. Purification of two immunologically different leukocyte proteins that cause cartilage resorption, lymphocyte activation, and fever An antiserum to pig IL-1 (catabolin) reacts with the acidic but not the neutral form of human IL-1 Immune interferon: a pleiotropic lymphokine with multiple effects Immunological functions of macrophages and their regulation by interferons Natural killer cells and interferon We gratefully acknowledge Dr. S. Martinod from Ciba Geigy for providing rPoIFNy, Dr. J. Saklatvala for providing anti-IL-1 and Dr. F. Blecha for revising the manuscript.