Synergistic effects of IL-2, IL-12 and IL-18 on cytolytic activity, perforin expression and IFN-γ production of porcine natural killer cells

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Abstract

Natural killer (NK) cells are one of the main cellular components of the innate immune system. They play an important role in the immune response against infections as well as tumour cells and therefore have two major properties: production of immune regulatory cytokines and chemokines as well as cytolytic destruction of particular target cells.

The existence of NK cells in swine is well known as well as the phenotype of resting NK cells, but their response following activation by cytokines is still poorly understood. Therefore, we tested the influence of the immune regulatory cytokines IL-2, IL-12 and IL-18 on cytolytic activity, phenotype, IFN-γ production and the accumulation of perforin in cytoplasm of peripheral blood mononuclear cells (PBMC) as well as purified NK cells. NK cells were enriched from PBMC using a magnetic cell separation (MACS) strategy with monoclonal antibodies against CD3, CD21 and SWC3, thereby removing T-, B- and myeloid cells. Respective fractions were used in flow cytometry (FCM) based cytolytic assays with the human tumour cell line K562 as target. After stimulation with the cytokines described above, the NK cell enriched CD3CD21SWC3 fraction showed an evident increase in the cytolytic activity compared to PBMC. This enhanced cytolytic activity was accompanied by a strong enrichment of IFN-γ producing cells when a combination of all three cytokines (IL-2/IL-12/IL-18) was used; as determined in ELISPOT assays and intracellular staining of IFN-γ in FCM. Also, the combination of these three cytokines led to an accumulation of perforin in the cytoplasm and an up-regulation of CD25 compared to control cultures incubated in medium without cytokines.

The experiments performed clearly indicate a stimulatory role and strong synergistic effects of the investigated cytokines in the activation of porcine NK cells in vitro, inducing IFN-γ, perforin production and cytotoxicity against target 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+CD5CD8α+ (Pescovitz et al., 1988, Saalmüller et al., 1994) and extended by a recent study to perforin+CD2+CD3CD4CD5CD6CD8α+CD8βCD11b+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

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