Role of chicken IL-2 on γδ T-cells and Eimeria acervulina-induced changes in intestinal IL-2 mRNA expression and γδ T-cells

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Abstract

Continuous culture of concanavalin A (Con A)-activated spleen cells in the presence of chicken recombinant IL-2 (rIL-2) promoted preferential growth of γδ T-cells. These cells displayed a high level of spontaneous cytotoxicity against LSCC-RP9 tumor cells, an avian NK cell target. Stimulation of IL-2-dependent γδ T-cells with Con A induced IFN-γ and IL-2 mRNA transcripts, whereas stimulation with rIL-2 induced only IFN-γ mRNA. Subcutaneous injection of 3-week-old chickens with IL-2 DNA increased splenic cells, expressing the CD8 and γδ TCR antigens. To investigate the role of IL-2 and γδ T-cells in parasitic infection, chickens were orally infected with Eimeria acervulina and the expression of IL-2 mRNA transcripts in the spleen and duodenum and the percentage of γδ T-cells in the duodenum were examined. Following both, the primary and secondary infections, a significant enhancement of IL-2 mRNA transcripts in the spleen and intestine and increased percentage of intraepithelial γδ T-cells in the duodenum were observed. These results indicate that host immune responses to E. acervulina involve an up-regulation of IL-2 secretion and an increased duodenum γδ T-cells.

Introduction

Interleukin-2 (IL-2) is a T-cell proliferation factor released from activated T lymphocytes and is an essential cytokine for expansion of activated T-cells (Grabstein et al., 1994). Recently, a chicken lymphokine, homologous to both mammalian IL-2 and IL-15, was described (Sundick and Gill-Dixon, 1997). This cytokine contained all four highly conserved cysteine residues which are highly characteristic of mammalian IL-15, and showed higher homology to mammalian IL-15 than to mammalian IL-2 based on the nucleic and amino acid sequences (Sundick and Gill-Dixon, 1997, Choi et al., 1999). Furthermore, in a manner analogous to mammalian IL-15, this cytokine stimulated the growth of γδ T-cells and had a wide tissue distribution as shown using RT-PCR (Choi et al., 1999). However, mRNA transcript of this cytokine was detected only in activated T lymphocytes using northern blot and possessed a short five region preceding the open reading frame which is characteristic of IL-2 (Sundick and Gill-Dixon, 1997).

IL-2 is an important cytokine which controls several immune mechanisms, including stimulation of lymphokine-activated killer (LAK) cells (Grimm et al., 1983) and natural killer (NK)-cells (Ortaldo et al., 1984, Trinchieri et al., 1984). IL-2 stimulates the proliferation and cytokine production by T-helper and NK-cells (Ortaldo et al., 1984, London et al., 1986, Talmadge et al., 1986). Our recent study showed that the chicken cytokine which is closely homologous to mammalian IL-15 and IL-2 promotes the growth of γδ T-cells in vitro (Choi et al., 1999). Although the role of γδ T-cells in chickens remains to be investigated, mammalian γδ T-cells play an important role in the defense against infectious agents (Rothenberg et al., 1996, Wallace et al., 1996, Kasper et al., 1996, Haas et al., 1993). In humans, γδ T-cells in the thymus and in extra-thymic tissues are CD4CD8 negative, although a small subpopulation in peripheral lymphoid organs may be CD8+ (Groh et al., 1989). Gamma-delta T-cells express the same granule mediators of cytotoxicity as NK-cells, including perforin and serine esterase 1 and 2 (Nakata et al., 1990, Guy-Grand et al., 1991). Furthermore, γδ T-cells can be stimulated to secrete a variety of lymphokines (Spits et al., 1990, Raziuddin et al., 1992). During Toxoplasma and Leishmania infections in mice, an expansion of γδ T-cells that has been linked to host immunity occurs (Kasper et al., 1996, Rosat et al., 1993). Following infection with Eimeria acervulina, an intestinal protozoan parasite which infects mainly the duodenum in chickens, significant increase in γδ T-cells occurs locally early after infection (Lillehoj, 1994). In view of our recent finding that chicken IL-2, which is homologous to mammalian IL-15, promotes the growth of γδ T-cells in vitro (Choi et al., 1999), we investigated the effect of Eimeria infection on local IL-2 production and intestinal γδ T-cell level following E. acervulina infection.

Section snippets

Infection of chickens with Eimeria

Embryonated eggs of SC chickens were purchased from the Hyline International Production Center (Dallas Center, IA), hatched at the Immunology and Disease Resistance Laboratory and kept in wire cages with water and food ad libitum. Special care was taken not to allow inadvertent exposure to pathogens such as Marek’s disease virus, mycoplasma or Eimeria. The wild-type strain of E. acervulina developed and maintained at the Livestock and Poultry Science Institute was used to infect chickens.

Spontaneous cytotoxicity mediated by γδ T lymphocytes

Continuous maintenance of Con A-stimulated splenic lymphoblasts in the presence of rIL-2 for 29 days generated a homogenous population (>99%) of lymphocytes expressing the CD3, CD8 and γδ TCR antigens (Fig. 1A). These CD8+ γδ T-cells were dependent on rIL-2 for continuous growth since they stop growing in the absence of rIL-2 in the growth medium (unpublished observation). Since mammalian γδ T-cells have been shown to mediate NK-cell activity (Haas et al., 1993), the ability of chicken γδ

Discussion

Three lines of evidence provided by this study establish that chicken IL-2 is a growth factor for γδ T-cells which are capable of mediating spontaneous cytotoxicity, and suggest an involvement of IL-2 and γδ T-cells in host immune response to E. acervulina infection. First, when cultured in the continual presence of rIL-2 for 29 days, splenic γδ T-cells demonstrated a high level of spontaneous cytotoxicity against an NK-cell target. Second, subcutaneous injection of the IL-2 DNA into 3-week-old

Acknowledgements

This work was supported by CSRS USDA NRI grant #98352046471. The authors also thank Drs. A. Guidry and E. Lillehoj for critical evaluation and editorial comments of the manuscript.

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