Species specialization in cytokine biology: Is interleukin-4 central to the TH1–TH2 paradigm in swine?

Developmental and Comparative Immunology 33 (2009) 344–352

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Species specialization in cytokine biology: Is interleukin-4 central to the TH1–TH2 paradigm in swine? Michael P. Murtaugh *, Craig R. Johnson, Zhengguo Xiao 1, Ronald W. Scamurra 2, Yaling Zhou 3 Department of Veterinary & Biomedical Sciences, University of Minnesota, St. Paul, MN 55108, USA

A R T I C L E I N F O

A B S T R A C T

Article history:

The TH1–TH2 paradigm provides an elegant model of directed response to infectious pathogens. Developed in the mouse, the model has provided a framework for systematic and mechanistic studies of immune regulation, protective immunity, and vaccine development in swine. Interleukin-4 (IL-4) plays a central role in the paradigm as a regulatory molecule directing development of the TH2 phenotype, as a developmental cytokine essential for antibody production, and as a soluble diagnostic marker of the TH2 cell type. In contrast, while characterizing the biological properties of porcine IL-4, we discovered that it was not a stimulatory factor for porcine B cells. Rather, it blocked antibody and IL-6 secretion and suppressed antigen-stimulated proliferation of B cells. Inhibition was not reversed by treatment with IL-2 and IL-6 treatment. IL-4 did not stimulate T lymphocyte proliferation, but induced cell growth in lymphoblasts in a dose-dependent fashion. These results suggest that IL-4 plays a different role in pigs than in mice and humans, in which it stimulates B cells and is essential for antibody production. Furthermore, the functions of IL-4 in swine cannot be inferred from results in model systems such as the mouse. General models of disease resistance show substantial variation between pigs and mice at the cellular and molecular level. Advances in somatic cell technologies and animal engineering to enable gene knockouts in pigs, in combination with a continuously expanding immunological toolkit, promise an exciting future for pig immunology, detailed mechanistic elucidation of the TH1–TH2 paradigm, and an improved understanding of the role of IL-4 in porcine immunity to infectious disease. ß 2008 Elsevier Ltd. All rights reserved.

Available online 28 August 2008 Keywords: Swine Interleukin Cytokine Immunology T-cell B-cell

1. Introduction In our view, the immune system evolved to resist rapidly proliferating pathogens that, in the absence of an immediate response, had the potential to overwhelm and kill a host. Slowly replicating pathogens, by comparison, posed a less immediate threat and so required less of an immediate response. The cells and molecules responsible for immunological function are most easily explained as arising at single points in evolutionary history. The sensing receptors of innate immunity are ancient, and lymphocytes first appeared in the cartilaginous fishes. Because similar cells and

* Corresponding author. Tel.: +1 612 625 6735; fax: +1 612 625 5203. E-mail addresses: [email protected] (M.P. Murtaugh), [email protected] (Z. Xiao), [email protected] (R.W. Scamurra), [email protected] (Y. Zhou). 1 Current address: Center for Immunology, Department of Laboratory Medicine and Pathology, MMC 334, University of Minnesota, Minneapolis, MN 55455, USA. 2 Current address: Boehringer Ingelheim Vetmedica Inc., 2621 North Belt Highway, St. Joseph, MO 64506, USA. 3 Current address: Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, DC 20307, USA. 0145-305X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2008.06.014

molecules of immunity can be recognized through evolutionary time and across vertebrate species, knowledge obtained in one species is widely disseminated and assumed to be relevant to other species. In this way, comparative immunology has been employed widely to guide vaccine development in food and companion animals since the tools and resources for direct interrogation of basic immunology in these species often is limited or lacking. Pioneering studies at DNAX by the Mosmann and Coffman laboratories laid the foundation of the TH1–TH2 paradigm, a simple concept summarized in Fig. 1. It is based on patterns of cytokine secretion in activated helper T cells that provided a conceptual and mechanistic basis for development of immune responses against intracellular and extracellular pathogens [1,2]. TH1 cells secreted IL-2 and IFNg that activated cytotoxic T cells and macrophages, cell types actively involved in the control of infection by intracellular pathogens. TH2 T cells secreted IL-4, which stimulated antibody production, and controlled infections by extracellular pathogens. IFNg and IL-4 were not only activating molecules of cellular and humoral immunity, they also were key negative regulatory molecules suppressing TH2 and TH1 cells, respectively. The relevance of a murine immunoregulatory paradigm in pigs is related to the degree that the underlying assumptions are valid.

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Fig. 1. General characteristics of TH1 and TH2 immunity. Experimental models and reagents are needed that will shed light on each of these characteristics to facilitate a better understanding of the regulation of immune responses to infectious disease in swine.

Little is known about IL-4 in swine. Here, we introduce the basic function of IL-4 in the TH1–TH2 paradigm and provide new data on its genomic organization, regulation of expression and its biological properties. The purpose is to address what, to us, is a fundamental contradiction in IL-4 function that we observed in the pig when trying to replicate the original observation that IL-4 stimulated Bcell growth. IL-4 can easily be a central character in reviews of antiparasite immunity, asthma, allergy, T-cell differentiation, and cytokine signaling. Here, we focused on its regulatory role in Bcell differentiation and function. The findings indicate that the gene and protein are conserved through mammalian evolution, but that its role in regulation of adaptive immune responses in the pig is different than in mouse or human. One consequence of these differences is that the key TH2-type immunoregulatory molecule may be different in pigs or the pathways of signal transduction may be different from those in mouse and human. 2. Porcine interleukin-4 Interleukin-4 (IL-4) was originally recognized as a co-stimulatory factor for the anti-IgM antibody-induced proliferation of resting B cells [3]. It plays an important role in promoting humoral immune responses against extracellular pathogens, and its expression is characteristic of TH2 cells, where it is primarily produced [2,4]. Gene knockout experiments demonstrated in mice that IL-4 is essential for TH2 cytokine responses [5] and antibody production [6]. In addition to its B-cell stimulating properties, IL-4 possesses anti-inflammatory effects on monocytes and macrophages [7–12]. Expression of IL-4 is relatively low compared to other cytokines, at both protein and mRNA levels [13]. The IL-4 gene is present in pigs on chromosome 2 in a locus also containing IL-13 and, in other species, a large family of TH2 cytokines that appear to have arisen by gene duplications (Supplementary Fig. 1 and [14,61–63]). Porcine IL-4 expression can be induced by IL-4, TNF, lipopolysaccharide (LPS) and T cell mitogenic lectins in mononuclear cell cultures (Supplementary Fig. 2 and [15,16]). The expression of IL-4 mRNA is low or undetectable in vivo [17,18]. In vitro, IL-4 mRNA in PBMC treated with IL-4, TNF, and LPS was detected only after Southern blot analysis of PCR amplicons

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(Supplementary Fig. 2C–E). In PBMC stimulated with Con A, the most potent activator, the level was calculated at less than one molecule of IL-4 transcript per cell after stimulation using a competitive RT-PCR analysis (Zhou and Murtaugh, data not shown). Levels of IL-4 protein also are low in serum, and show little correlation with mRNA levels [19,20]. Discordant results also were observed in cells cultured from various lymphoid tissues, in which protein was undetectable in bone marrow cells by ELISA yet IL-4 secreting cells were most abundant by ELISPOT (Supplementary Fig. 3). Similarly, IL-4 protein was detected at relatively high levels in mesenteric lymph node cultures, but the number of IL-4 secreting cells in mesenteric lymph node was no different than in spleen or inguinal lymph node cells (Supplementary Fig. 3). The findings show that data obtained by one method may not provide a full picture of IL-4 production. Because of the general inability to detect circulating IL-4 in pigs and the extensive overlap in their functions and properties in other species [14,21], IL-13 was proposed to have the functions of IL-4 in swine [22]. The chromosomal organization of the locus is similar in pigs and mice, as shown in Supplementary Fig. 1, and both promoter regions contain multiple GATA-3 transcription factor binding sites. Interestingly, the predicted transcription factor binding sites in murine IL-4 appear more similar to porcine IL-13 and murine IL-13 appear more similar to porcine IL-4 (Supplementary Fig. 1). The promoter elements vary between pigs and cattle, suggesting the possibility of differential regulation in these two artiodactyl species [23]. Mitogenic stimulation had little effect on IL-13 mRNA levels in PBMC. As shown in Supplementary Fig. 4A and B, IL-13 mRNA levels were low, at the same level as HPRT and substantially less than TGFb1, in the presence or absence of PHA, and similar to the levels observed in human PBMC cultured under the same conditions (Supplementary Fig. 4D). Culture for 5 days with or without stimulation also resulted in very low levels of mRNA, requiring 40 cycles of amplification for detection (Supplementary Fig. 4C). The findings indicate that, at this level of analysis, IL-13 is expressed at very low levels similar to the level of IL-4 expression. IL-4 appears able to control macrophage inflammatory activities in the pig. As shown in Supplementary Fig. 2, it is induced by inflammatory mediators. It directly suppresses induction of inflammatory cytokine and NADPH oxidase gene expression, and abolished LPS-induced TNF production [24,25]. With GM-CSF, it mediates differentiation of monocytes and macrophages into dendritic cells [26,27]. The findings are consistent with a role for IL-4 in control of inflammatory responses and macrophage differentiation. Evidence pointing to a role for IL-4 in modulating or directing immune response phenotypes in a TH1–TH2 model is lacking, but models of parasitic infection may provide an opportunity to address this issue [28]. Below, we present findings which address the putative function of IL-4 as a B-cell stimulatory factor by specifically testing if it promotes antibody production and IL-6 production in porcine B-cells and macrophages. These activities are central to the role of IL-4 in regulation of immune responses in mice. 3. Effects of IL-4 on B-cell proliferation in the pig IL-4 was discovered by its potentiation of anti-IgM induced Bcell proliferation [3]. Thus, we examined this effect of IL-4 in swine using the same approach. In contrast to the marked stimulation of B-cell proliferation observed in mice, neither porcine nor human IL-4 stimulated B-cell proliferation in the pig (Fig. 2). Instead, there appeared to be a reduced response to antigen receptor engagement by anti-porcine IgM antibodies, and there was no proliferation in response to IL-4 alone. The IL-4 effect was observed over a wide

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Fig. 2. Species-specific effect of IL-4 on B-cell proliferation. (A) Stimulation of anti-IgM-dependent B-cell proliferation of murine B-cells grown three days in media with (*) or without (*) IL-4 (Fig. 1 of [3]). (B) Pig PBMC, labeled with CFSE, were stimulated to proliferate with 10 mg/ml goat anti-swine IgM alone or with 10 ng/ml human IL-4 or with 10 ng/ml porcine IL-4. After 5 days culture, cells were analyzed by FACS for B-cell proliferation using goat anti-swine IgM and phycoerythrin-conjugated rabbit anti-goat IgG to gate on B-cells and CFSE to determine proliferation events. No proliferation was observed in cells cultured with IL-4 alone or without anti-IgM. (data not shown).

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stimulation. In the absence of IL-4 there were few cells, whereas in the presence of IL-4, B-cells survived and had undergone, in most cases, one or two cell divisions (Fig. 5). These results indicated that in the pig IL-4 does not provide a proliferative signal to B-cells, but gives a survival signal in cells that are transiently stimulated to divide. In addition, the evidence for an anti-proliferative effect in sentinel antigen-specific B-cells is novel. 4. Effects of IL-4 on B-cell antibody and cytokine secretion in the pig

Fig. 3. IL-4 inhibition of B-cell proliferation is dose-dependent. CFSE-treated porcine peripheral blood mononuclear cells were cultured for 5 days in the presence of antiswine IgM and varying concentrations of IL-4. Cells were washed and stained with goat anti-swine IgM and phycoerythrin-conjugated anti-goat IgG. No proliferation was observed in culture media or media containing IL-4 alone. Number of divided cells was calculated as the number of CFSE-positive cells in the culture well that divided one time or more.

range of concentrations, with maximum suppression observed at 5 ng/ml (Fig. 3). The suppressive effect also was apparent if cells were pretreated for 24 h with IL-4, washed and then stimulated with anti-IgM (Fig. 4). In all of these experiments carried out with PBMC, IL-4 by itself stimulated no proliferation. The only positive effect that was observed occurred when PBMC were stimulated with anti-IgM for 24 h, washed, then incubated for 5 days without further antigenic

It was possible that IL-4 was acting in a stimulatory manner on B-cells in other stages of differentiation that we could not detect by staining for the B-cell antigen receptor, or that responding cells were resident in lymphoid tissue rather than in the circulation. To address these possibilities, we determined its effects on immunoglobulin and IL-6 secretion, which are commensurate with a TH2-type immune response, in B cells isolated from the spleen in a model of antigen-specific recall. Pigs were immunized with keyhole limpet hemocyanin (KLH) and splenocytes were harvested two weeks later. Splenic B-cells at this time were not actively secreting antibody, but were differentiated to express the surface anti-KLH receptor, as demonstrated by the secretory response to KLH antigen (Table 1). Presence of antigen in cultures containing lymphocytes and macrophages provided signals for robust antibody secretion. However, removal of CD4+ T cells, but not CD8+ T cells or macrophages and dendritic cells, abrogated the response (Table 1). To determine if lympho-proliferative cytokines could replace CD4+ T cell help, splenocyte cultures depleted as described above were stimulated with antigen with various combinations of IL-2, IL-6 and IL-4. IL-2 and IL-6 alone and in combination fully restored the antigen-dependent stimulation of anti-KLH antibody production. But surprisingly, IL-4 completely blocked antibody production by itself and profoundly suppressed the effects of IL-2 and IL-6 (Table 1).

Fig. 4. IL-4 pretreatment inhibits anti-IgM-dependent B-cell proliferation. PBMC were cultured for one day in the absence or presence of porcine IL4, washed, treated with CFSE, and cultured in goat anti-porcine IgM (10 mg/ml). After 5 days, cells were washed and stained with anti-porcine IgM (A). (B) FACS analysis of cells without exposure to IL-4. (C) FACS analysis of cells exposed to IL-4 for 24 h, then stimulated with anti-IgM. No proliferation was observed when IL-4 was present throughout the incubation period. FACS analysis was performed as described in Fig. 2 legend.

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Table 1 IL-4 inhibits antigen-specific antibody secretiona Treatment

Splenocyte population Total

–CD4

–CD8

–Mø/DC

Control KLH KLH + IL-6 KLH + IL-2 KLH + IL-6 + IL-2 KLH + IL-4 KLH + IL-6 + IL-4 KLH + IL-6 + IL-2 + IL-4

0 28.7 nd nd nd nd nd nd

0.5 9.4 17.0 29.4 23.4 0.8 1.6 2.1

0.3 28.3 35.3 30.6 48.3 2.1 6.9 2.5

0 27.6 23.8 30.2 30.2 5.0 5.8 5.0

a Pigs were immunized intramuscularly with KLH in incomplete Freund’s adjuvant. After 2 weeks splenocytes were isolated and cultured in triplicate directly or after magnetic selection to remove CD4+ T cells, CD8+ T cells, or macrophages and dendritic cells. Antigen-specific B cells were restimulated in the presence of the indicated cytokines. After 96-h incubation, anti-KLH antibody secreting cells were quantified by ELISPOT. Data shown are mean number of antibody forming cells/105 cells. Antibodies for negative selection were used as ascited from hybridomas HB147 (anti-porcine CD4), HB143 (anti-porcine CD8), obtained from the American Type Culture Collection (Rockville, MD), and 74-22-15, specific for macrophages, monocytes dendritic cell subset, and neutrophils (Mø/DC), kindly provided by Joan Lunney, USDA (Beltsville, MD). Antibodies were mixed with splenocytes on ice for 1 h, washed three times, and incubated with Biomag goat anti-mouse IgG magnetic particles (PerSeptive Diagnostics, Cambridge, MA) at a particle to target ratio of 20:1. Particles were magnetically separated for 5 min two times. Non-selected cells were recovered, plated, and analyzed by FACS. Negative selection removed >97% of target cell populations.

Similarly, induction of a primary antibody response to KLH in vitro was dependent on the presence of T cells and macrophages (adherent splenocytes) (Fig. 6). IL-6 and IL-2 replaced the requirement for T cells and macrophages; IL-6 by itself partially restored B-cell differentiation and antibody secretion, and IL-2 alone or in combination with IL-6 was as good or better than mixed leukocyte culture. However, cultures that contained IL-4 under any of a variety of conditions showed almost no induction of specific antibody production. IL-6 has a major role promoting B-cell proliferation and differentiation [29] and is actively secreted by porcine lymphocytes [30]. Splenocyte cultures spontaneously secreted IL-6, an effect that disappeared if CD4+ or CD8+ T cells or macrophages were removed and was increased if specific KLH antigen was added (Table 2). IL-6 secretion was completely restored in T cell-depleted populations by the addition of IL-2, suggesting that cytokine stimulation of B cells, or of the remaining non-depleted T cells, was sufficient to restore IL-6 secretion. However, addition of IL-4, which was originally discovered as the second B-cell stimulatory factor after IL-6 [3], not only did not restore IL-6 secretion, it completely suppressed it below the detection limit of the assay (Table 2). 5. Effect of IL-4 on T cell proliferation The negative effects of IL-4 on B-cell function did not appear to be due to non-specific toxicity insofar as human recombinant IL-4

Fig. 5. IL-4 promotes B-cell survival when the growth signal is removed. Porcine peripheral blood mononuclear cells were cultured for 1 day in the presence of 10 mg/ml antiswine IgM, washed, treated with CFSE, and cultured 5 more days in media alone (A, top panels) or in porcine IL-4 at 10 ng/ml (B, bottom panels). Cells were washed and stained with anti-porcine IgM. No proliferation was observed when anti-IgM was omitted in the first 5-day culture period, or if IL-4 was present throughout. FACS analysis was performed as described in Fig. 2 legend.

M.P. Murtaugh et al. / Developmental and Comparative Immunology 33 (2009) 344–352 Table 2 IL-4 inhibits IL-6 secretiona Splenocyte population Treatment

Total

–CD4

–CD8

–Mø/DC

Control KLH KLH + IL-2 KLH + IL-4

13  11 22  15 nd nd

52 83 26  16 3

3 14  6 36  22 3

3 3 41 3

a Experimental design was the same as in Table 1. Antigen-specific B cells were restimulated in the presence of 5 mg/ml KLH alone, or with 10 ng/ml rhIL-2 or rhIL4. After 96-h incubation, supernatants were collected, and IL-6 bioactivity was assayed by the B9 proliferation assay [30]. Data shown are IL-6 U/105 cells.

also suppressed B-cell proliferation, as shown in Fig. 2. To further demonstrate that the findings were biologically relevant and not due to a nonspecific toxicity, we examined the effect of IL-4 on T lymphocyte proliferation. Alone, it had no effect on cell proliferation in PBMC. However, mitogenic stimulation of PBMC for 5 day to produce lymphoblasts made both CD4+ and CD8+ T cells proliferate in a dose-dependent fashion in the presence of IL-4 (Fig. 7). 6. Porcine IL-4 and the TH1–TH2 paradigm Our findings and published data show that in swine, IL-4 suppresses macrophage inflammatory responses [24,31], suppresses natural killer cell activity [32], is involved in anti-parasite responses [33], helps to differentiate macrophages and monocytes into dendritic cells [26,27], and stimulates T lymphoblast proliferation (Fig. 7). In dendritic cell differentiation, IL-4 appears to facilitate cell survival but not differentiation [26], and in B cells it promotes survival of activated B cells (Fig. 5). It also protects endothelial cells against injury by human complement in xenotransplantation [34,35]. However, except for a possible role in class switching and IgM expression in the gut, there is little evidence that IL-4 is necessary for B-cell function in the pig [36]. The elegance of the TH1–TH2 model is based on the convergence of multiple lines of data and logic that together account for a large

Fig. 6. IL-6 and IL-2 fail to reverse IL-4 suppression of primary B-cell antibody production. Splenocytes were isolated from non-immune pigs and cultured in 24well plates directly or after negative magnetic selection to remove CD4+ T cells, CD8+ T cells, or macrophages and dendritic cell subsets as described in Table 1. Cells were stimulated with or without KLH antigen and IL-6, IL-2, and IL-4 for 4 days. Specific antibody-secreting cells were determined in plates coated with monoclonal anti-KLH antibody. After 16–20 h, plates were processed for ELISPOT. Antigenspecific antibody secreting cells were detected using anti-swine IgG+M heavy chains, and positive spots were counted.

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body of observed immunological phenomena. IFNg or IL-4, secreted by antigen-specific helper T cells, stimulate effector responses, primarily cytotoxic T cells and macrophages or neutralizing antibody, appropriate for the pathogen, either intracellular or extracellular, respectively. Fig. 1 illustrates the key characteristics of the TH1–TH2 paradigm of immune response regulation, while recent reviews provide deeper levels of complexity with regard to cytokines, cell types, and effector molecules and more recent immunoregulatory pathways involving TH17 and regulatory T cells, that contribute to a full immunological response [37–40]. As summarized in Fig. 1, each cytokine, either IFNg or IL-4, inhibits development of the opposing helper T cell. At the molecular level, transcription factors tie together IFNg or IL-4 secretion with effector and regulatory gene expression profiles that are reflective of TH1 and TH2-type immune responses. At the genetic level, susceptibility or resistance of various mouse strains is associated with the predicted TH1 or TH2 bias. Knockout experiments further validate the model. In particular, a functional IL-4 response is essential for a TH2-type response and antibody production [5,6]. At about the same time, immunologists became aware of surveillance molecules sensitive to various classes of pathogens that further solidified a rational basis for the biased evolution of immune responses depending on the nature of the infecting pathogen [41,42]. With links now evident between innate recognition of invading pathogens that could skew adaptive immune responses toward a favorable and successful response, it became possible to understand the mechanisms of protective immunity in a directed and intelligent fashion. The application and analysis of cytokine expression patterns in response to antigens and pathogens proliferated rapidly. It became apparent that the fine details of the Mosmann and Coffman model were more complex [43], and differed between mice and humans. Nevertheless, the central elements of the paradigm held firm over time and have been widely applied to vaccine development in the expectation that more effective vaccines would be readily produced. Even though our knowledge of immunity and disease resistance has increased greatly in the last two decades, the pace and methods for developing swine vaccines has not changed markedly. Novel adjuvants based on Toll-like receptor ligands and cytokines have had little impact, antigen discovery for subunit vaccines has languished, and delivery systems still are in a stage of experimental development. We assume that the TH1–TH2 concept is relevant to swine since it explains so well the overall mechanisms of disease resistance. If so, it will be necessary to identify the key molecules or their surrogates that direct the development of appropriate immune responses. We expect that the key molecule should have the central functional properties of B-cell stimulation and differentiation as does IL-4 in the mouse. IL-10 is an antiinflammatory cytokine but does not provide the signals necessary for specific T cell development. Perhaps another molecule in the IL4 locus of pigs might be the central regulator. Notably in other species, a large group of cytokines including IL-4, IL-5, IL-9, and IL13 are located in this region and, at least in the mouse, are coordinately regulated [43–45]. PBMC are the most common window to swine immunity since blood is readily accessible and facilitates longitudinal studies in live animals. However, the key events in immune induction and development occur in lymph nodes, which can give a very different picture of B-cell responses compared to PBMC [46]. Little is known about the cellular and molecular events surrounding the generation of an adaptive immune response in porcine lymph nodes. There can be considerable regional variation [46] and immune responses to the same antigen are different in mucosal and systemic immune tissues [47].

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Fig. 7. IL-4 induction of T-blast proliferation is dose-dependent and equal for helper and cytotoxic T-cells. No proliferation was observed when mitogen was omitted from the first 5-day culture period, or if IL-4 was present in the first 5 days (data not shown). Single cell suspensions (5  105 cells in 100 ml PBS) were incubated with specific antibodies for 30 min at 4 8C, washed twice with PBS, and examined in a FACSCalibur flow cytometer (BD Biosciences, San Jose CA). Antibodies were anti-pig CD4 (74-12-4) and anti-pig CD8 (76-2-11) conjugated to FITC or PE (BD Pharmingen).

Reliance on cytokine expression in PBMC or cytokine detection in serum may not be a reliable indicator of an immune response bias. As cited previously, detection of an IL-4 response by any method is difficult in PBMC [19,20], which has led some investigators to infer a type 1 or type 2-like immune response depending on the presence or absence of IFNg. IL-10 levels also have been used, though a rational basis for this practice has not been established. IL-13 has been proposed as an alterative to IL-4, but at present there is no evidence one way or the other that its expression is necessary for B-cell development or can bias an immune response [22]. A key tenet of the TH1–TH2 model is that the cytokines are secreted by helper T cells in response to specific antigen stimulation. In pigs, establishment of antigen-specific CD4+ T cell lines by isolation and cloning of lymphocytes has been difficult [48,49]. Alternative approaches using fluorescent labeling of cell surface antigens and intracellular cytokine staining to identify IL-4 secreting cells in mixed populations is like looking for the proverbial needle in a haystack since the frequency of responding cells may be less than the background in FACS analysis. The issue of cell source is particularly significant since IFNg, the hallmark cytokine of a TH1 response, is produced by a wide variety of lymphocytes including, natural killer T cells [50], activated CD8+ T cell [51], and gd T cells [52], in addition to CD4+ helper T cells. Regulatory T cells have now been described in swine; their cytokine expression potential has not been determined [53]. Pigs have a substantial population of mature CD4+ CD8+ doublepositive T cells whose cytokine expression profiles are not known [54,55]. Without a more acute knowledge of the cell type and antigen-specificity of T cells secreting IFNg, it will be difficult to describe even a TH1 response. 7. Re-evaluation of the TH1–TH2 paradigm in porcine immunoregulation Pigs have a number of immunological characteristics that are different from mice in addition to T lymphocyte subpopulations. For example, lymph node architecture is inverted relative to most

other species [56]; development of antibody diversity involves gene conversion [57]; and gd receptor features are unusual [58]. Pigs are comprised of outbred populations with diverse MHC class I and class II compositions, in contrast to inbred mouse strains widely used in immunological research. Due to these and other differences, we expect that pigs might have evolved other molecules to execute immune responses within the TH1–TH2 paradigm. The greatest need in resolving these outstanding questions is methods for isolation and cloning of antigen-specific helper, cytotoxic and regulatory T cells in the pig. With antigenspecific T cells in hand, it will be possible to characterize peptide antigenicity for T-cells in pigs and their stimulatory or inhibitory consequences, patterns of cytokine expression, the nature of responding cell populations, and effects of the cytokine environment. Porcine pathogens, like those of other species, express multiple proteins, all of which have numerous peptides that can be expressed in patterns that are influenced by the MHC milieu of host presenting cells. It remains possible that IL-4 or IL-13 is the key cytokine in TH2like immune responses. Inhibition of proliferation and antibody secretion might be secondary to induction of a class switch [36]. We feel this is unlikely since the suppressive effect of IL-4 was profound, even in the presence of IL-2 and IL-6, and was observed in assays of proliferation and antibody secretion in both primary and recall response models. Alternatively, there may be a genetic difference similar to the variation previously observed in IL-6 secretion by macrophages [30] or concentration-dependent effects that are not yet characterized. These issues need to be clarified. IL-4 might play a critical role in immunity to parasites, whose study was a primary motivation in the discovery of the paradigm [59]. Gene knockouts by homologous recombination, which have helped clarify mechanisms of immunoregulation in the mouse, are now feasible in pigs through nuclear transfer [60]. Advances in somatic cell technologies, animal engineering, RNA interference, and other in vivo and in vitro techniques, in combination with a continuously expanding immunological toolkit, promise an exciting future for pig immunology and detailed elucidation of the TH1– TH2 paradigm.

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