IL-18 Stimulates the Proliferation and IFN-γ Release of CD4+ T Cells in the Chicken: Conservation of a Th1-Like System in a Nonmammalian Species

Thomas W. Göbel, Kirsten Schneider, Beatrice Schaerer, Iris Mejri, Florian Puehler, Steffen Weigend, Peter Staeheli and Bernd Kaspers
J Immunol August 15, 2003, 171 (4) 1809-1815; DOI: https://doi.org/10.4049/jimmunol.171.4.1809

Abstract

The phylogeny of Th1 and Th2 subsets has not been characterized mainly due to the limited information regarding cytokines in nonmammalian vertebrates. In this study, we characterize a Th1-like regulatory system focusing on the IL-18-regulated IFN-γ secretion. Stimulation of splenocytes with chicken IL-18 induced high levels of IFN-γ secretion. Depletion of either macrophages or CD4+ T cells from the splenocyte cultures caused unresponsiveness to IL-18. In contrast, PBL were unresponsive to IL-18 in the presence or absence of macrophages, but IFN-γ secretion was stimulated by suboptimal anti-TCR cross-linking combined with IL-18. Splenocytes from five different chicken lines responded equally well to the IL-18 treatment. LSL chicken splenocytes, however, responded only to IL-18 when stimulated either with optimal TCR cross-linking alone or suboptimal TCR cross-linking combined with IL-18. IL-18 not only induced IFN-γ secretion, but also stimulated splenocyte proliferation. This IL-18-induced proliferation was compared with the effects observed with IL-2. Both cytokines activated the splenocytes as demonstrated by increased size and MHC class II Ag up-regulation in the case of IL-18. Phenotypic analyses following 6 days of culture revealed that IL-2 mainly affected the proliferation of CD8+ cells, whereas IL-18 had an opposite effect and stimulated the proliferation of CD4+ cells. Taken together, these results demonstrate the conservation of Th1-like proinflammatory responses in the chicken; they characterize IL-18 as a major growth factor of CD4+ T cells and identify two distinct mechanisms of IL-18-induced IFN-γ secretion.

Immune responses against various pathogens are modulated by at least two distinct Th cell subsets. According to the Th1-Th2 paradigm, strong cellular immune responses against intracellular pathogens are mediated by IFN-γ-secreting Th1 cells; whereas Th2 cells produce IL-4, IL-5, and IL-13 and are critically involved in responses to parasitic pathogens and in the development of allergies (1, 2). Although this concept has been confirmed by numerous studies in mice and humans, there is only limited information about nonmammalian cytokines in general, and as a consequence the Th1-Th2 dichotomy has not been established (3). In particular, the cytokines involved in Th1 and Th2 differentiation, IL-12 and IL-4, respectively, have not been identified in nonmammalian vertebrates. The lack of appropriate methods for long-term T cell cultures and T cell cloning in nonmammalian vertebrates has been an additional problem in the analyses of T cell-derived cytokines. In contrast, acquired immune responses have been intensively studied in the chicken and have provided important information regarding the phylogeny of immune responses. These studies have been instrumental for investigating host pathogen interactions in a number of economically important poultry diseases (4).

Progress in avian cytokine research has only been made recently due to improved molecular methods to identify and characterize chicken cytokine homologues (3). In particular, the increasing numbers of chicken-expressed sequence tag database entries have been a rich source of nonidentified cytokine homologues (5, 6). Identified chicken cytokines include the IFNs, the proinflammatory cytokines IL-1, IL-6, and IL-18 as well as IL-2 and IL-15.

Mammalian IL-18 represents an additional member of the IL-1 family (7, 8). IL-18 is produced by a variety of nonimmune and immune cells, including Kupffer cells, macrophages, dendritic cells, lymphocytes, astrocytes, and microglia (8). It is synthesized as an inactive precursor and requires processing by the intracellular cysteine protease caspase-1 to mature to the biologically active, secreted form (9). IL-18 was initially characterized as a factor inducing IFN-γ secretion by Th1 cells and NK cells in the presence of IL-12 (10). Although IL-18 does not by itself induce Th1 cell development, it up-regulates the IL-12Rβ expression on Th1 cells and thereby promotes the IL-12-mediated Th1 cell development (11). IL-18 alone, however, can also induce the production of IL-4 and IL-13 by T and NK cells (12, 13). IL-18 thus represents a potent cytokine that enhances the IL-12-driven Th1 development, but can also stimulate Th2 responses.

It is now well established, that a fully functional IFN system including type I and type II IFN is present in the chicken (14, 15). As in mammals, chicken IFN-γ is an essential cytokine in the host defense against a variety of pathogens as demonstrated in a number of infection models. IFN-γ was shown to inhibit the Marek’s disease virus replication by its ability to induce NO synthesis (16), to reduce the replication of coccidian parasites (17), and to inhibit src oncogene-induced tumor growth (18). The regulation of IFN-γ secretion has not been clarified in the chicken. The two major pathways in mammals include the so-called acquired T cell activation pathway mediated by TCR engagement and IL-12 and the innate pathway requiring the combination of IL-12 and IL-18 (19).

Chicken IL-18 has recently been cloned and expressed following its identification in a chicken-expressed sequence tag database (20). It shares ∼30% amino acid identity to human IL-18, including a conserved caspase-1 cleavage site. Initial experiments have further suggested a role of chicken IL-18 in the IFN-γ release of splenocytes (20).

In this report, we identify two pathways of IL-18-mediated IFN-γ induction, either by IL-18 and TCR cross-linking or by IL-18 treatment in the presence of macrophages. Remarkably, IL-18 also stimulated T cell proliferation, but in contrast to IL-2 which caused an increased frequency of CD8+ cells, IL-18 increased the frequency of CD4+ cells and caused the up-regulation of MHC class II Ags. These data identify IL-18 as a major regulator of IFN-γ secretion in chickens and characterize IL-18 as an important growth factor of CD4+ T cells.

Materials and Methods

Animals

H.B19 (B19/B19 haplotype) chickens were raised at the Institute for Animal Physiology, University of Munich (Munich, Germany). Fertilized eggs of commercial LSL birds were obtained from Lohmann, Cuxhaven; and eggs of CC (B4/B4) and CB (B12/B12) congenic chicken lines were kindly provided by J. Plachy (Institute for Molecular Genetics, Czechoslovakia Academy of Sciences, Prague, Czech Republic). Lines R11 (B15/B15) and M11 ((B2/B2) have been maintained at the Institute for Animal Science, Mariensee and are described elsewhere (21). All birds were hatched at the Institute and the animals were used for experiments at the age of 3–10 wk.

Cytokines

Histidine-tagged recombinant chicken IL-18 was expressed in a prokaryotic expression system and purified using nickel-chelate affinity chromatography as described previously (20). Chicken IL-2 was cloned by PCR according to the published sequence (22) and expressed in a myeloma-based system (23). Recombinant chicken IFN-γ used as a control in all bioassays was obtained from transiently transfected COS-7 cells and supernatants harvested 48 h after transfection were used at a 1/500 dilution (15).

Cell isolation and culture

Chicken splenocytes were separated by density centrifugation on Ficoll-Paque (Amersham Pharmacia Biotech, Freiburg, Germany) and PBL by slow-speed centrifugation as previously described (24) or when indicated by density centrifugation. All primary cells were cultured in RPMI 1640 supplemented with 10% FCS, 60 μg/ml penicillin, and 50 μg/ml streptomycin (standard medium; Life Technologies, Grand Island, NY) at 40°C and 5% CO2.

HD11 cells (25) were cultured in RPMI 1640 supplemented with 8% FCS, 2% chicken serum (Sigma-Aldrich, Deisenhofen, Germany), 60 μg/ml penicillin, and 50 μg/ml streptomycin at 40°C and 5% CO2 in 75-cm2 tissue culture flasks (Nunc, Wiesbaden, Germany). Confluent cell layers were harvested by treatment with PBS-EDTA for 5 min at 40°C, washed twice, and resuspended in the same medium.

Induction of IFN-γ synthesis and quantification of IFN-γ

Chicken splenocytes were seeded at a density of 2.5 × 105 cells/well of 96-well flat-bottom microtiter plates and incubated with the indicated amounts of IL-18. For TCR stimulation, the TCR1 mAb (26) or TCR2 mAb (27) recognizing γδ and αVβ1 T cells, respectively, were coated on the flat-bottom microtiter plates at the concentrations indicated. Supernatants were harvested 48 h after incubation and assayed for the presence of IFN-γ using the HD11 cell bioassay described previously (28). Briefly, HD11 cells were adjusted to 1 × 105 cells/well of 96-well microtiter plates and supernatants of IL-18-stimulated cells were added at different concentrations. A rabbit anti-IFN-γ polyclonal antiserum was used for neutralization. After 24 h, the supernatants were harvested and IFN-γ-induced nitrite accumulation was measured by the Griess reaction (29). Standard curves were included in all assays using recombinant chicken IFN-γ.

Lymphocyte proliferation assay

Lymphocyte proliferation was measured by a standard [3H]TdR incorporation assay. Splenocytes were cultured in standard medium at 2.5 × 105 cells/well in 96-well flat-bottom plates at 40°C and 5% CO2. IL-18 or IL-2 was added in triplicates at the concentrations indicated and cultures were maintained for 5 days. [3H]TdR (1 μCi) was added for the final 16 h of culture, cells were harvested on filters using a cell harvester, and incorporation of radioactivity was determined on a Microplate Scintillation Counter (Canberra; Packard, Dreieich, Germany). Data are presented as stimulation index defined as cpm of stimulated sample/cpm of unstimulated control.

Negative selection of lymphocyte populations

For the depletion experiments, the anti-CD4 mAb 2-4 (30), anti-CD8 mAb 3-298 (31), or the macrophage- and thrombocyte-specific K1 mAb (32) were used to stain splenocytes, followed by a goat anti-mouse IgG-FITC conjugate (Sigma-Aldrich). Labeled cells were further incubated with paramagnetic anti-FITC-microbeads and depletion of CD4+, CD8+, or K1+ cells was performed on an LD separation column according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of sorted cell preparations was analyzed by flow cytometry.

RT-PCR analysis

The depleted cells were treated with 250 ng/ml IL-18 or with a control protein that was expressed and purified identically to IL-18 (MxA). Following 72 h of stimulation, total RNA of the cells was prepared using TRIzol reagent (Invitrogen, Karlsruhe, Germany). cDNA was prepared using the RevertAid H Minus first-strand cDNA syntheses kit (MBI Fermentas, St. Leon-Rot, Germany). The PCR primers used for actin were 5′-TACCACAATGTACCCTGGC-3′ and 5′-CTCGTCTTGTTTTATGCGC-3′ and for IFN-γ 5′-GATCAAGAGTCGACGCATACTGCAAGTAGTCTA-3′ (nucleotide for cloning and restriction site underlined) and 5′-GAGAAGATCTGGTCATAAGATGCCATTA-3′ that amplify a 454-bp IFN-γ fragment. The PCR was performed for 35 rounds at 65°C annealing temperature and the products were analyzed on an ethidium bromide-stained 1% agarose gel.

Cell staining and analysis

Staining of cells for flow cytometric analysis was performed according to standard procedures. Cells were stained with either unlabeled or biotinylated mAb against CD4 (2-4) (30), CD8 (3-298) (31), and MHC class II (2G11) (33), followed by an anti-mouse Ig-FITC conjugate, and in the case of double staining streptavidin-PE (Sigma-Aldrich), and analyzed with a FACScan (BD Biosciences, Heidelberg, Germany) using CellQuest software.

Results

IL-18 induces IFN-γ secretion in splenocytes, but not in PBL

The induction of IFN-γ synthesis by NK cells and T cells is a major biological activity of IL-18 in mammals. We therefore examined the IFN-γ-inducing activity of recombinant chicken IL-18 on PBL and splenocytes. To quantify the IL-18-induced IFN-γ activity in cell culture supernatants, we made use of a well-established IFN-γ assay (28). The supernatants of IL-18-stimulated cells were added to cultures of the chicken macrophage cell line HD11 which responds to IFN-γ with the synthesis of NO, but not to IL-18 (data not shown).

Splenocytes from 8-wk-old H.B19 birds strongly responded to IL-18 with a dose-dependent secretion of IFN-γ (Fig. 1⇓A). In contrast, IL-18 did not induce any detectable IFN-γ secretion in PBL under the same culture conditions (Fig. 1⇓A). The NO production was induced by IFN-γ, since preincubation of the supernatants with a neutralizing antiserum completely abrogated the response of HD11 cells (Fig. 1⇓B). This indicated a major difference in the IL-18 reactivity between lymphocytes derived from PBL and spleen.

FIGURE 1.
FIGURE 1.

Different pathways of IL-18-mediated IFN-γ release in splenocytes and PBL. H.B19-derived splenocytes or PBL (2.5 × 105 cells/well) were cultured in the presence of the indicated concentrations of IL-18. A, Supernatants were harvested after 72 h and assayed for IFN-γ release. B, Supernatants from wells stimulated with 250 ng/ml IL-18 or recombinant chicken IFN-γ (rChIFN-γ) were pretreated with an anti-chicken IFN-γ antiserum before addition to the HD11 macrophage cell line. Data represent mean ± SD of triplicate cultures.

IL-18-induced IFN-γ secretion is dependent on CD4+splenocytes and macrophages

To further characterize the major target cells for IL-18 in the spleen, cells were separated according to their CD4 and CD8 expression (Fig. 2⇓A). Negative magnetic sorting was chosen, mainly to prevent any stimulatory effect of the mAb used for staining. The efficiency of the depletion was consistently >95% (data not shown). Splenocytes depleted of CD8 cells, secreted IFN-γ upon IL-18 treatment in amounts comparable to unseparated cells. In contrast, CD4 depletion caused a complete loss in IL-18-mediated IFN-γ secretion (Fig. 2⇓A). These results were confirmed by RT-PCR analyses with IFN-γ-specific primers using either cells treated with an irrelevant control protein or IL-18 following CD4 or CD8 depletion. Whereas the IFN-γ message could be detected in CD8-depleted cells, no IFN-γ signal was obtained in CD4-depleted cells (Fig. 2⇓B).

FIGURE 2.
FIGURE 2.

The IL-18-mediated IFN-γ release in splenocytes is dependent on CD4+ T cells and macrophages. Cell depletions by MACS were used to remove >95% of respective cell types. Sorted and unsorted cell preparations were adjusted to 5 × 105 cells/well of 96-well microtiter plates, cultured for 72 h, and analyzed for IFN-γ secretion. A, CD4-depleted (○) and CD8-depleted (▾) cell preparations were compared for their IL-18 responsiveness. One representative experiment of five is shown. B, The stimulated cells were harvested and analyzed for IFN-γ transcripts using RT-PCR. Controls (Ctr.) were stimulated with an irrelevant protein expressed and purified identically to IL-18. Actin transcripts were used as control of cDNA integrity. C, Macrophages were depleted from splenocytes and stimulated with IL-18. The mean ± SD of three individual experiments is depicted.

Interestingly, when positive sorting was used instead of depletion, purified CD4+ splenocytes were unreactive to IL-18 (data not shown). These experiments indicated that other cells like B cells or macrophages that were not depleted had some effect on the IL-18-induced IFN-γ secretion by CD4+ T cells. To test this hypothesis, splenocytes were depleted from myeloid cells by MACS. Depletion of myeloid cells resulted in drastically diminished IFN-γ secretion following IL-18 treatment (Fig. 2⇑C). Taken together, the depletion experiments reveal that the IL-18-induced IFN-γ secretion by CD4+ splenocytes is dependent on macrophages.

TCR cross-linking induces the IL-18-mediated IFN-γ release in PBL

The insensitivity of PBL to IL-18 (Fig. 1⇑) could be due to several reasons, including the activation status of CD4+ T cells or differences between the PBL and splenocyte preparations. To characterize the mechanisms controlling the IFN-γ production in PBL, lymphocytes were isolated by two different protocols either exactly like splenocytes with density centrifugation or slow-speed centrifugation which removes most of the thrombocytes and part of the macrophages. Both PBL preparations, however, did not respond to IL-18 in contrast to total splenoytes (Fig. 3⇓A).

FIGURE 3.
FIGURE 3.

TCR cross-linking restores IL-18 reactivity of PBL. A, Two distinct PBL preparations by slow-speed centrifugation density centrifugation were treated with IL-18 and their IFN-γ secretion was compared with IL-18-treated splenocytes from the same chicken. Data represent mean ± SD of three individual experiments. B, PBL were treated with the IL-18 amounts indicated in the presence of plate-bound TCR or control mAb (0.5 μg/ml).

In mammals, effective IFN-γ secretion is dependent on costimulation with either TCR cross-linking or IL-12. Since IL-12 has not been cloned in the chicken, TCR cross-linking was used as costimulatory signal. When PBL were incubated with plate-bound TCR2 mAb (anti-αVβ1) in combination with various amounts of IL-18, PBL responded with dose-dependent secretion of IFN-γ (Fig. 3⇑B) that was comparable to the amounts released by IL-18-treated splenocytes (Fig. 1⇑). In conclusion, in contrast to splenocytes, the IFN-γ secretion in PBL is dependent on combined signals by IL-18 and TCR cross-linking.

LSL chicken-derived splenocytes are nonresponsive to IL-18 treatment unless activated by TCR cross-linking

The initial experiments were performed with splenocytes obtained from the inbred chicken line H.B19. To test whether splenocytes from other chicken lines also produce IFN-γ upon IL-18 treatment, six different chicken lines, R11, M11, CB, CC, H.B19, and the commercial LSL birds, were screened. With the exception of LSL-derived splenocytes, which did not secrete IFN-γ, the splenocytes from the other five chicken lines responded equally well to IL-18 treatment with the secretion of significant amounts of IFN-γ (Fig. 4⇓A and data not shown for CB and CC lines).

FIGURE 4.
FIGURE 4.

LSL-derived chicken splenocytes release IFN-γ by TCR cross-linking combined with IL-18. A, Splenocytes of different chicken lines were stimulated with the indicated amounts of IL-18 for 72 h. IFN-γ concentrations were subsequently analyzed in the HD11 cell bioassay. Data represent mean values from five birds per chicken line. B, LSL splenocytes were stimulated with immobilized TCR1 mAb (anti-γδ) or TCR2 mAb (anti-αVβ1) at the indicated concentrations for 72 h with mAb alone or with TCR2 mAb in combination with 250 ng/ml IL-18 (C).

This unresponsiveness toward IL-18 treatment observed in LSL chickens could neither be overcome by stimulation with larger amounts of IL-18 nor by prolonged activation. Two alternative explanations for this lack of IL-18 response would include a general block in IFN-γ gene induction or an insensitivity of LSL splenocytes in response to the IL-18 treatment. Splenocytes of LSL origin were therefore stimulated with high-dose TCR cross-linking. Whereas the TCR1 mAb directed against γδ T cells had no effect on the secretion of IFN-γ, the αVβ1-specific TCR2 mAb induced secretion of IFN-γ by LSL splenocytes in amounts comparable to IL-18 stimulation (Fig. 4⇑B).

Since PBL of all chicken lines are dependent on costimulation for the IL-18-mediated IFN-γ release, we tested whether the IFN-γ secretion by LSL splenocytes could also be induced with suboptimal TCR stimulation in combination with IL-18. In this assay, TCR cross-linking in combination with 250 ng of IL-18/ml potently induced IFN-γ secretion (Fig. 4⇑C). These results indicate that the LSL-derived splenocytes can be stimulated to secrete IFN-γ and identify optimal TCR-mediated signaling as an additional pathway to induce IFN-γ secretion.

IL-18 induced the proliferation and activation of splenocytes

In initial experiments, where different incubation times for the maximum IL-18-induced IFN-γ production were tested, an IL-18 effect on the proliferation of cells was observed. This IL-18-induced splenocyte proliferation was further analyzed and compared with the effect caused by IL-2, which has been the only characterized chicken cytokine causing T cell proliferation.

IL-18 induced a dose-dependent splenocyte proliferation after a 5-day stimulation period (Fig. 5⇓). Heat inactivation of IL-18 totally abrogated the effect on splenocytes, thus excluding the possibility of a potential LPS-mediated effect. A control protein expressed and purified identically to IL-18 also had no effect on the proliferation (data not shown). The direct comparison of IL-2 and IL-18 on the proliferation of splenocytes revealed, that the maximum proliferation induced by IL-2 (stimulation index of 14) was about twice as high as the IL-18-mediated effect (stimulation index of 7; Fig. 5⇓). Thus, both IL-2 and IL-18 caused, without additional costimulation, vigorous splenocyte proliferation; however, IL-2 was more potent than IL-18.

FIGURE 5.
FIGURE 5.

IL-18 is a potent inducer of lymphocyte proliferation. Triplicates of freshly isolated splenocytes were cultivated 5 days in the presence of the indicated amounts of IL-18 or IL-2 (final dilution of eukaryotic expressed IL-2). As a control for potential LPS effects, heat-inactivated IL-18 (100°C for 5 min) was included. One representative experiment of five is shown.

Phenotypic changes induced by IL-2 and IL-18 treatment

IL-2 and IL-18 caused different levels of splenocyte proliferation. This effect may be explained by the preferential stimulation of different splenocyte subsets by the two cytokines. Therefore, splenocytes treated for 6 days with either IL-2 or IL-18 were phenotypically analyzed using a panel of different mAb and compared with the cells before culture.

The analysis of the forward scatter profile as indicator of the cell growth clearly revealed that both cytokines caused increased cell size as compared with freshly isolated cells (Fig. 6⇓A). Further detailed phenotypic analyses revealed that both cytokines promoted the growth of CD3+ T lymphocytes, whereas B cells were not detectable after 6 days of culture (data not shown). IL-2 caused an increase in the frequency of CD8+ splenocytes, whereas the frequency of CD4+ cells decreased. IL-18 had the opposite effect on the splenocytes, resulting in a high frequency of CD4+ cells following 6 days of culture and a marked decrease of CD8+ cells (Fig. 6⇓B).

FIGURE 6.
FIGURE 6.

Phenotypic analysis of IL-2- or IL-18-stimulated splenocytes. Splenocytes were cultured for 6 days in either IL-2 or IL-18 and analyzed by flow cytometry in comparison to freshly isolated cells for forward scatter as indication of cell size (A), with the mAb indicated (B), or MHC class II and CD4 double staining (C). Numbers indicate the percentage of positive cells as compared with control staining. One representative experiment of six is shown.

In the chicken, MHC class II Ags are constitutively expressed on B cells and monocytes, but they are up-regulated as a consequence of T cell activation (34). Therefore, the MHC class II Ag levels were analyzed on the IL-18-stimulated splenocytes. Double immunofluorescence staining revealed that in all experiments (n = 6) >95% of the CD4+ cells in the IL-18-treated cultures coexpressed MHC class II Ags (Fig. 6⇑C). Taken together, IL-2 and IL-18 both function as potent T cell growth factors; however, IL-2 mainly induced CD8+ T cell proliferation, whereas IL-18 stimulated the CD4+ T lymphocyte subset.

Discussion

The recent progress in chicken cytokine research (3) has finally enabled studies on immunoregulatory mechanisms and thus allows, for the first time, the ability to address the phylogeny of the Th1-Th2 dichotomy. In this study, we report that a fully functional IL-18-IFN-γ system already developed before the divergence of birds and mammals from a common ancestor ∼300–350 million years ago (35). This conclusion is based on our findings that an IL-18 homologue is present in birds (20) and that chicken IL-18 is a potent activator of chicken Th cells through the induction of 1) cytokine secretion, 2) T cell proliferation; and 3) MHC class II Ag up-regulation.

IL-18 was initially identified as a factor promoting IFN-γ production by T and NK cells when costimulated by IL-12 (10). Successive experiments have further supported the finding that the IFN-γ secretion by IL-18 is dependent on additional costimulators like IL-12, immobilized anti-CD3, or Ag (36). In this study, we reveal two distinct IL-18-mediated pathways that induce IFN-γ secretion in lymphocytes. In the presence of macrophages, IL-18 by itself stimulated chicken splenocytes to secrete large amounts of IFN-γ, whereas TCR costimulation combined with IL-18 was essential to induce similar amounts of IFN-γ in PBL. The role of macrophages in the splenocyte cultures is currently not known. They could either stimulate the T cells by direct cognate interaction or more likely by secretion of soluble factors, such as IL-12. Since chicken IL-12 has not been identified so far, its costimulatory properties in these assays could not be analyzed in more detail.

Contrary to splenocytes, IFN-γ production by PBL was dependent on IL-18 and TCR stimulation, even when PBL were prepared identically to splenocytes. This closely resembles the mammalian situation (8) and indicates that naive cells need costimulatory signals to become IL-18 sensitive. Regulation of the IL-18R is known to play a critical role in the IL-18 response of mammalian lymphocytes (37). It was shown that IL-12 up-regulates the IL-18R and thus renders uncommitted Th cells responsive to IL-18 (11, 37). In the chicken that lacks lymph nodes, the spleen is the major secondary lymph organ and is thus expected to contain a mixture of naive and previously activated cells. These cells could express the IL-18R due to recent activation and therefore react to IL-18. In contrast, PBL that do not respond to IL-18 may not have received appropriate signals for IL-18R expression. The TCR stimulation of PBL most likely induced the IL-18R up-regulation, rendering these PBL IL-18 sensitive (Fig. 3⇑B). Although the experiments were performed with the TCR2 mAb, both αVβ1 and αVβ2 cells were stimulated due to the known cross-reactivity of the TCR2 mAb with αVβ2 cells when using higher amounts of the TCR2 mAb. γδ T cells secreted small amounts of IFN-γ when stimulated with IL-18; however, there were variations of yet unknown reasons between individual chickens and therefore the effect of IL-18 on γδ T cells has to be analyzed in more detail in future experiments.

Interestingly, splenocytes of one of six different chicken lines were not responsive to the IL-18 treatment alone (Fig. 4⇑A). However, TCR cross-linking by itself or in combination with IL-18 treatment induced IFN-γ secretion (Fig. 4⇑). A direct comparison of the IL-18R expression between splenocytes of different chicken lines is not possible due to the lack of appropriate reagents; however, defects of constitutive IL-18R expression on a fraction of splenocytes that is overcome by TCR signaling is a potential explanation for the IL-18 insensitivity. Alternatively, unknown costimulatory factors like IL-12 derived from macrophages in the cultures could be nonfunctional or absent in the LSL-derived chickens. Future binding assays with epitope-tagged IL-18 may further clarify this issue.

The IL-18-mediated transcription and translation of IFN-γ genes was restricted to the CD4+ T cell population, while no response was observed by CD8+ cells (Fig. 2⇑, A and B). This is in contrast to the situation in mammals where CD4+ Th cells and CD8+ NK cells are both reactive to IL-18 (8). The chicken NK cells are included in the CD8+ cell population; however, they are present in a frequency of <1% (38), which may not be sufficient for the secretion of detectable amounts of IFN-γ. In addition, the possibility remains that the CD8+ cells require the help of CD4+ cells to respond to IL-18. B cells that were still present after the CD4 cell depletion did not secrete detectable amounts upon IL-18 treatment, either because of species-specific differences or, more likely, due to the lack of costimulation. As in mammals (39), chicken B cells may require anti-CD40 stimulation to become sensitive to IL-18.

Most strikingly, IL-18 also stimulates the proliferation and activation of splenic T cells (Figs. 5⇑ and 6⇑). As observed for the IFN-γ induction, this IL-18 activity was independent of further costimulatory signal, unless already present in the cultures. The analyses of the IL-18-treated splenocytes revealed that this IL-18 effect was mainly restricted to the CD4+ subpopulation (Fig. 6⇑B). The comparison of the IL-2 with the IL-18-induced proliferation revealed a more pronounced effect of IL-2 and in contrast to IL-18, IL-2 mainly stimulated the CD8+ population. Mammalian IL-18 induces the proliferation of T cells only in the presence of costimulatory signals similar to the IFN-γ induction (36). In addition, IL-18 is also involved in the expansion of distinct NK cell subsets (40). IL-12 and IL-18 stimulate NK1.1−CD3− cells, whereas the IL-2- and IL-18-reactive cells were characterized as NK1.1+CD3− cells (40). In contrast, the splenocytes proliferating in response to chicken IL-18 were identified as CD4+ T lymphocytes, but not NK cells. These IL-18-stimulated cells displayed an increased size and high levels of MHC class II surface Ag, both typical signs of T cell activation (Fig. 6⇑). The up-regulation of MHC class II Ag could either be caused directly by IL-18 or indirectly by the secretion of IFN-γ, which is known to increase MHC class II levels (15). It seems likely that the combined effects of IL-18 and the secreted IFN-γ potently stimulate the CD4+ T lymphocyte subset. In future experiments, the possible mechanisms for the differences observed between mammals and birds will be addressed; however, this will only be possible after the generation of additional tools including identification, cloning and expression of chicken IL-12, and components of the IL-18 receptors.

The phylogenetic origin of Th1 and Th2 responses is currently unresolved. The IL-18-induced IFN-γ system described here is the first indication of a Th1-like regulatory network in nonmammalian species. Interestingly, cytokines indicative of Th1 and proinflammatory responses such as IL-1, IL-6, and IL-18 have been well conserved in lower vertebrates (3). The recent cloning of cartilaginous fish IL-1β further illustrates the conservation of proinflammatory cytokine responses (41). Proinflammatory cytokines are released upon activation through the highly conserved pattern recognition receptors exemplified by the Toll-like receptor family (42). Toll-like receptors have been conserved from arthropods to mammals. Moreover, the Toll-like receptors utilize a signaling pathway shared with the IL-1R and IL-18R. Therefore, the IL-18/IL-18R system may represent a phylogenetically old part of immune responses that later developed in a potent Th1 or Th2 promoting system. Taken together, these data suggest that a cytokine system including the IL-18-regulated IFN-γ response has been present for at least 230 million years, the time span between the divergence of birds and mammals.

Acknowledgments

We thank Drs. O. Vainio, C. Chen, K. Hala, and J. Salomonsen for providing mAb and J. Plachy for providing fertilized eggs of the CC and CB chicken lines and B. Amann for expert technical assistance.

Footnotes

  • ↵1 This work was supported by grants from the European Union (to B.K.) and Deutsche Forschungsgemeinschaft (to T.G. and P.S.).

  • ↵2 Address correspondence and reprint requests to Dr. Bernd Kaspers, Institute for Animal Physiology, Veterinàrstrasse 13, 80539 Munich, Germany. E-mail address: kaspers{at}tiph.vetmed.uni-muenchen.de

  • Received December 11, 2002.
  • Accepted June 10, 2003.

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