Synergistic effects of IL-2, IL-12 and IL-18 on cytolytic activity, perforin expression and IFN-γ production of porcine natural killer cells
Introduction
NK cells form an important cellular component of the innate immune system. They are specialized lymphocytes, which in humans and mice comprise approximately 5–20% of total lymphocytes in spleen, liver and peripheral blood and are present at lower frequencies in the bone marrow, thymus and lymph nodes (Lian and Kumar, 2002). Research on NK cells over the last decades has shown that they possess two major properties: production of immunostimulatory cytokines and cytolytic activity against particular target cells.
NK cells are triggered to kill or to ignore target cells depending on a delicate balance of inhibitory and activating signals received through ligands on potential targets (Ghiringhelli et al., 2006). For example, the missing of self-proteins that are expressed by normal cells, but down regulated on infected or transformed cells, can elicit cytolytic activity (Vivier and Biron, 2002, Moretta and Moretta, 2004). Indeed, this mode of action is supposed to take place in the lysis of MHC class I deficient tumour cell lines, often used as target cells in the assessment of cytolytic activity of NK cells in in vitro experiments. Two main pathways of NK cells are known to induce apoptosis of target cells: death-receptor engagement (Takeda et al., 2001, Smyth et al., 2001) and granule exocytosis (Trapani and Smyth, 2002). The involved cytolytic granules contain serine proteases like granzymes and perforin, the latter one being well described for its importance in inducing cell death of virally infected target cells (Tay and Welsh, 1997, Loh et al., 2005).
Cytokine production is another key effector function of NK cells, which is controlled by distinct biochemical signalling pathways. NK-cell subsets can be grouped according to the cytokines they produce. Similar to the Th1/Th2 paradigm, research in mice has shown that NK-cell precursors can differentiate into NK cells that produce either type 1 (NK1) or type 2 (NK2) cytokines (Peritt et al., 1998, Loza and Perussia, 2001). For example, NK1 cells produce IFN-γ and are generated when IL-12 dominates in the local cytokine milieu (Shi and Van Kaer, 2006). In fact, in both viral and bacterial models of infection, IFN-γ production by NK cells has been shown to be a key event in successful resolution of infection (Orange et al., 1995, Byrne et al., 2004). Besides IL-12, NK cells respond to a number of further cytokines such as IL-2, IL-15, IL-18, IL-21 and type I interferons. The latter ones can induce NK cell survival and proliferation, and all the mentioned cytokines promote cytotoxic function and/or production of IFN-γ in vitro (Biron et al., 1999). It is also assumed that these cytokines participate in the activation of NK cell responses in vivo (Andoniou et al., 2005).
Swine possess only a small number of classical cytotoxic T-cells but large numbers of lymphocytes with the potential for innate cytotoxic activity like NK and TCR-γδ T-cells (Denyer et al., 2006). The phenotype of porcine NK cells was previously described as CD2+CD5−CD8α+ (Pescovitz et al., 1988, Saalmüller et al., 1994) and extended by a recent study to perforin+CD2+CD3−CD4−CD5−CD6−CD8α+CD8β−CD11b+CD16+ (Denyer et al., 2006). Cytolytic activity of porcine NK cells has also been reported. Studies of in vitro NK activity against targets infected with pseudorabies virus (Martin and Wardley, 1984) and transmissible gastroenteritis virus (Cepica and Derbyshire, 1986) were accomplished. Increased NK activity in swine chronically exposed to infectious and parasitic diseases was also observed (Richerson and Misfeldt, 1989), whereas lethal infection with classical swine fever virus led to a decrease of the NK cell activity during course of disease (Pauly et al., 1998). Despite these data obtained in disease models, little is known about the fundamental mechanisms involved in activation of porcine NK cells. Therefore, in this study we tested the influence of recombinant cytokines IL-2, IL-12 and IL-18, which all play an important role in NK cell function, on cytolytic activity, expression of perforin, phenotypic changes and IFN-γ production of porcine NK cells.
Section snippets
Cells
Heparinized blood was collected from 6-month-old pigs from an abattoir. PBMC were isolated by gradient centrifugation and resuspended in cell culture medium consisting of RPMI 1640 (Invitrogen, Austria) supplemented with 10% heat-inactivated FCS, 100 IU/ml penicillin and 0.1 mg/ml streptomycin (PAA Laboratories, Austria).
K562 cells, a human erythroleukemic cell line (Lozzio and Lozzio, 1975), were used as target cells in cytolytic assays and propagated in the same medium as PBMC.
Antibodies
Table 1
Analysis of cytolytic activity of MACS separated porcine NK cells
To study various in vitro functions of porcine NK cells, a MACS depletion strategy with mAb against CD21 (B cells), CD3 (T cells) and SWC3 (myeloid cells) was used to enrich NK cells from PBMC. In a first set of experiments, the cytolytic activity of PBMC and MACS enriched NK cells against K562 target cells was tested, including control groups of antibody-labelled PBMC and positive selected CD3+CD21+SWC3+ cells. After MACS-separation, cell fractions were either used directly in cytolytic assays
Discussion
In this study we investigated the influence of the cytokines IL-2, IL-12 and IL-18 on phenotype, cytolytic activity, IFN-γ production and perforin accumulation of porcine NK cells in vitro. In the past, a large number of studies showed that dendritic cells (DC) cells produce IL-12 and IL-18 which enhance NK cell cytokine production (IFN-γ). In addition, although T cells are well known to be major producers of IL-2, it has recently been shown that DCs also secrete small amounts of IL-2 following
References (60)
- et al.
A monoclonal antibody recognising an epitope associated with pig interleukin-2 receptors
J. Immunol. Meth.
(1992) - et al.
Summary of workshop findings for porcine B-cell markers
Vet. Immunol. Immunopathol.
(2001) - et al.
Characterization of the effector cells in antibody-dependent and spontaneous cell-mediated cytotoxicity in swine against target cells infected with transmissible gastroenteritis virus
Res. Vet. Sci.
(1986) - et al.
Enhancement of porcine natural killer cell activity by recombinant human and murine IL-12
Cell. Immunol.
(1996) - et al.
A triggering structure recognized by G7 monoclonal antibody on porcine lymphocytes and granulocytes
Cell. Immunol.
(1992) - et al.
Establishment and characterization of porcine cytolytic cell lines and clones
Vet. Immunol. Immunopathol.
(1997) - et al.
Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells
Vet. Immunol. Immunopathol.
(2006) - et al.
Porcine interleukin-12 fusion protein and interleukin-18 in combination induce interferon-γ production in porcine natural killer and T cells
Vet. Immunol. Immunopathol.
(2002) - et al.
Response of bovine and porcine peripheral blood mononuclear cells to human recombinant interleukin 2
Vet. Immunol. Immunopathol.
(1986) - et al.
Characterization of monoclonal antibodies directed against swine leukocytes
Vet. Immunol. Immunopathol.
(1986)
Murine natural killer cell progenitors and their requirements for development
Semin. Immunol.
Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome
Blood
Natural cytotoxicity detected in swine using Aujeszky's disease virus infected targets
Res. Vet. Sci.
Regulation of interferon-γ production by IL-12 and IL-18
Curr. Opin. Immunol.
Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop
Vet. Immunol. Immunopathol.
Host environment as a modulating factor of swine natural killer cell activity
Vet. Immunol. Immunopathol.
Resting porcine T lymphocytes expressing class II major histocompatibility antigen
Immunobiology
T-helper cells from naive to committed
Vet. Immunol. Immunopathol.
Perforin and interferon-γ activities independently control tumor initiation, growth, and metastasis
Blood
Porcine γδ T cells: possible roles on the innate and adaptive immune response following virus infection
Vet. Immunol. Immunopathol.
Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop
Vet. Immunol. Immunopathol.
Analyses of monoclonal antibodies reactive with porcine CD44 and CD45
Vet. Immunol. Immunopathol.
Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity
Nat. Immunol.
Natural killer cells in antiviral defense: function and regulation by innate cytokines
Annu. Rev. Immunol.
Depletion of NK cells results in disseminating lethal infection with Bordetella pertussis associated with a reduction of antigen-specific Th1 and enhancement of Th2, but not Tr1 cells
Eur. J. Immunol.
IL-10 enhances NK cell proliferation, cytotoxicity and production of IFN-γ when combined with IL-18
Eur. J. Immunol.
Induction of interferon γ production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers
J. Exp. Med.
The costimulatory effect of IL-18 on the induction of antigen-specific IFN-γ production by resting T cells is IL-12 dependent and is mediated by up-regulation of the IL-12 receptor β2 subunit
Eur. J. Immunol.
Differential effects of human and porcine interleukin-2 on natural killing (NK) activity of newborn piglets and adult pigs lymphocytes
Ann. Rech. Vet.
The role of regulatory T cells in the control of natural killer cells: relevance during tumor progression
Immunol. Rev.
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