Antigen-specific IFN-gamma and IL-4 production in caprine herpesvirus infected goats

The analysis of cytokines secreted by antigen-specific lymphocytes is hampered in goats by the paucity of species-specific reagents yet it is crucial to study immune responses to infections. To overcome this limit, two commercial kits designed to measure soluble bovine IL-4 (by ELISA) and frequencies of bovine IFN-gamma secreting cells (by ELISPOT) were tested for cross-reactivity in goats. In addition, an ELISA specific to bovine/ovine IL-4 and employing two monoclonal antibodies, clones CC313 and CC314, was tested as well. Concanavalin A-stimulated caprine peripheral blood mononuclear cells (PBMCs) cultures were studied and they exhibited high levels of soluble IL-4 and high frequencies of IFN-gamma secreting cells. In addition, the two IL-4 ELISAs detected similar amounts of cytokine. To start defining the cytokine response triggered by caprine herpesvirus 1 (CpHV-1) infection, PBMC cultures were setup from goats naturally or experimentally infected with CpHV-1. High frequencies of IFN-gamma producing cells and low, when detectable, levels of soluble IL-4 were observed in CpHV-1-specific PBMC cultures from both groups of infected goats. Thus, the availability of cross-reactive research tools can expand cytokine studies in goats and can implement the research on immunity to other caprine infections.     

Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

To investigate cytokine alterations in pigs infected in-utero with porcine reproductive and respiratory syndrome virus (PRRSV), constitutive mRNA expression by peripheral blood mononuclear cells (PBMCs) was measured. PBMC from in-utero PRRSV-infected pigs displayed significantly increased IL-6, IL-10, and IFN-gamma mRNA expression at 0 and 14 days of age compared with age-matched control pigs. There were no significant differences in IL-2, IL-4, and IL-12 mRNA expression between in-utero PRRSV-infected and control pigs. However, the IL-10/IL-12 ratio was significantly increased in in-utero PRRSV-infected pigs at 0 and 14 days of age, suggesting the imbalance of IL-10 and IL-12 mRNA production. The abnormal mRNA expression of cytokines in in-utero PRRSV-infected pigs occurred concurrently with a significant decrease in the CD4(+)/CD8(+) T-cell ratio in peripheral blood. PRRSV was not isolated from the sera of pigs at 9 weeks of age that had been viremic at 0 and 14 days old. Delayed type hypersensitivity (DTH) responses to Tuberculin and analysis of cytokine mRNA expression by PBMC showed that cell-mediated immune response and cytokine message profiles in pigs infected in-utero with PRRSV had returned to levels similar to those of control pigs by 9 weeks of age. We conclude that in-utero infection with PRRSV results in significant alteration of cytokine mRNA expression that may cause transient immunomodulation. However, at 10 weeks of age the pigs' immune responses seemed to recover. This may help to understand the immunopathogenesis of in-utero PRRSV infection and the increased susceptibility to secondary bacterial pathogens in neonatal piglets.     

Characterization of specific antibodies and the establishment of sandwich ELISA and ELISPOT systems for swine IL-4

We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.     

Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses

Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in supernatants of mitogen stimulated equine peripheral blood mononuclear cells (PBMC). The sensitivity of the ELISA for detecting secreted IL-10 was found to be around 200pg/ml. The production of intracellular IL-10 was measured in equine PBMC by flow cytometry. PBMC were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of the secretion blocker Brefeldin A. All three anti-IL-10 mAbs detected a positive population in PMA stimulated lymphocytes which was absent in the medium controls. Around 80% of the IL-10(+) cells were CD4(+). Another 15% were CD8(+) cells. Double staining with IL-4 or interferon-gamma (IFN-gamma) indicated that PMA and ionomycin stimulation induced 80% IL-10(+)/IFN-gamma(+) lymphocytes, while only 5% IL-10(+)/IL-4(+) cells were observed. By calculation, at least 60% of the IL-10(+)/IFN-gamma(+) cells were CD4(+) lymphocytes. This expression profile corresponds to the recently described T regulatory 1 (T(R)1) cell phenotype. In summary, the new mAbs to equine IL-10 detected native equine IL-10 by ELISA and flow cytometry and can be used for further characterization of this important regulatory cytokine in horses.     

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV)

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.     

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.     

Immunostimulatory capacity of DNA vaccine vectors in porcine PBMC: a specific role for CpG-motifs?

With the development of DNA vaccines in pigs, the possibility was investigated that the nature and the amount of certain CpG-motifs present on plasmid DNA might have an effect on their immunostimulatory capacity. A panel of three CpG-oligodeoxynucleotides (ODN) and three eukaryotic expression vectors currently used in experimental DNA vaccines in pigs (pcDNA1, pcDNA3.1 and pCI) were screened for their immunostimulatory activity on porcine PBMC by evaluating in vitro the lymphocyte proliferative responses and cytokine profiles (IL-1alpha, IL-2, IL-4, IL-6, IL-10, IFN-gamma, TGF-beta, TNF-alpha). The vectors were chosen so that they differed in number and nature of certain CpG-motifs present on their backbone. CpG-ODN A (5'ATCGAT3') and to a lesser extend CpG-ODN C (5'AACGTT3') significantly enhanced the proliferation of porcine PBMC in contrast to CpG-ODN B (5'GACGTT3') where no effect was observed. Furthermore, CpG-ODN A significantly induced IL-6 and TNF-alpha together with elevated levels of IFN-gamma and IL-2 mRNA expression even though considerable heterogeneity was observed in the response of individual pigs. Comparison of the three vectors showed significantly increased proliferative responses for both pcDNA3.1 and pCI combined with a significant increase in IL-6 mRNA levels for pCI. For pcDNA1, proliferation was absent together with significantly decreased levels of IL-6 and IFN-gamma. CpG-ODN and plasmids both suppressed the TGF-beta and IL-1alpha mRNA expression. Taken together, these data confirm the identity of an optimal immunostimulating CpG-motif in pigs (5'-ggTGCATCGATGCAG-3') and demonstrates that the choice of the vector or the insertion of immunostimulatory motifs can be important in the future design of DNA vaccines in pigs, although further research is necessary to explore the possible link between certain CpG-motifs and the immunogenicity of DNA vaccines.     

Systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome (PMWS) as determined by semiquantitative RT-PCR and flow cytometric intracellular cytokine detection

Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease in pigs caused by porcine circovirus type 2 (PCV2). Development of this disease is presumably associated with an impairment of the immune system. We, therefore, investigated the systemic expression of relevant cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, TNF-alpha, IFN-gamma) and IL-2Ralpha at mRNA (semiquantitative RT-PCR) and at protein level (flow cytometric intracellular cytokine detection after short-time stimulation of peripheral blood mononuclear cells) in 10 feeder pigs aged 14 weeks suffering from natural PMWS and in 10 clinically healthy pen-mates. Hematological examination revealed a significant (p < 0.001) relative lymphopenia in the diseased animals when compared to reference pigs. IL-1alpha and IL-10 mRNA levels were notably increased in the affected pigs, whereas IL-2 and IL-2Ralpha (CD25) mRNA levels tended to be down-regulated. IL-8, TNF-alpha and IFN-gamma mRNA expressions appeared to be slightly increased. Intracellular cytokine levels as measured by flow cytometry revealed an increase of IL-1beta, IL-2, and IL-6, whereas IL-12 and TNF-alpha expressions were not affected. IFN-gamma was slightly decreased in the diseased animals. In conclusion, despite the assumption, that the cellular immune response to PMWS as a virus-induced disease should be characterized by either a Th1 driven cytokine profile or a cytokine profile indicative of T cell immunosuppression, our results did not support that hypothesis. Nevertheless, data from intracellular cytokine detection suggest an even increased percentage of the remaining lymphocytes capable to produce IL-2 upon in vitro stimulation, which is in contrast to the slightly diminished IL-2 mRNA levels reflecting the in vivo situation at least at the mRNA level.     

Butyrate differentially regulates cytokines and proliferation in porcine peripheral blood mononuclear cells

Although butyrate modulates proliferation and cytokine production by PBMC in some species, the role of butyrate as a regulator of immunocyte function in the pig has not been studied. Therefore, the primary objective of this study was to determine whether butyrate influences peripheral blood mononuclear cell (PBMC) proliferation, cytokine secretion and mRNA expression in the pig in vitro. We also sought to determine whether alterations in cytokine production attributable to butyrate were associated with changes in the expression of suppressor of cytokine signaling-3 (SOCS3). Porcine PBMC were isolated from venous blood and stimulated with concanavalin A (ConA) in the presence or absence of sodium butyrate at 0.2 or 2.0 mM. Butyrate at 2.0 mM suppressed (P<0.05) ConA-induced PBMC proliferation and led to a paradoxical increase (P<0.05) in IL-2 mRNA expression. The secretion and mRNA expression of interferon-gamma (IFN-gamma) by ConA-activated PBMC was increased (P<0.05) by butyrate at 2.0 mM. Exposing activated PBMC to butyrate at 2.0 mM decreased (P<0.05) the secretion of interleukin-10 (IL-10). In contrast, butyrate at 0.2 mM increased (P<0.05) both IL-10 secretion and mRNA expression. Activation of porcine PBMC with ConA increased (P<0.05) the expression of SOCS3 mRNA, and butyrate treatment further augmented (P<0.05) SOCS3 mRNA expression in a dose-dependent manner. Mechanistically, pretreatment with the adenyl cyclase inhibitor 2,5-dideoxyadenosine abolished (P<0.05) the inhibitory effect of 2.0 mM butyrate on IL-10 secretion, and partially reversed (P<0.05) the increase in IFN-gamma secretion induced by 2.0mM butyrate. These data indicate that the effect of butyrate on cytokine production by porcine PBMC is dose-dependent, and that butyrate increases the expression of SOCS3 in activated PBMC. In addition, we provide evidence that the effects of butyrate on IFN-gamma and IL-10 production are mediated in part via a cAMP-dependent mechanism.     

Characterization of Th1 activation by Bartonella henselae stimulation in BALB/c mice: Inhibitory activities of interleukin-10 for the production of interferon-gamma in spleen cells

This study was conducted to analyze cytokine production mechanisms in mice after Bartonella henselae stimulation. BALB/c mice were inoculated intraperitoneally with 3 x 10(6) colony forming units of B. henselae (Houston-1 strain) twice at 10-day interval. Spleen cells were harvested from the mice and stimulated with the organisms. Following the stimulation, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), IL-10, IL-12 and tumor necrosis factor-alpha (TNF-alpha) were measured in the culture supernatants of the spleen cells by ELISA. The spleen cells specifically secreted IFN-gamma, but not IL-4, indicating that T helper 1 (Th1) cells were activated following B. henselae stimulation. In addition, IL-10 and TNF-alpha productions were also detected in the culture supernatants of spleen cells. Neutralization of IL-10 in the culture supernatants significantly enhanced the production of IFN-gamma from the spleen cells stimulated with B. henselae. These results indicate that B. henselae predominantly stimulated Th1 cells and resulted in secreting IFN-gamma, however the production was partially inhibited by IL-10, which was produced simultaneously.     

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

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-gamma 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 CD3(-)CD21(-)SWC3(-) fraction showed an evident increase in the cytolytic activity compared to PBMC. This enhanced cytolytic activity was accompanied by a strong enrichment of IFN-gamma 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-gamma 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-gamma, perforin production and cytotoxicity against target cells.     

Interferon-gamma response of PBMC indicates productive pseudorabies virus (PRV) infection in swine

In Chinese Meishan/German Landrace cross-bred swine F2 generation interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) was determined directly ex vivo at different time points after survival of a virulent pseudorabies virus (PRV) infection. This reactivity was compared with the reactivity of na?ve PBMC. Significant IFN-gamma production was determined in ELISA and ELISPOT only after in vitro PBMC re-stimulation with PRV and not with the closely related bovine herpesvirus BHV-1. The PRV-specific IFN-gamma secretion from re-stimulated PBMC showed high levels 6 days after infection, before the presence of serum antibodies, and it persisted at a high level over a 3 months period. The response of a group of eight piglets infected intranasally with PRV varied. Only two animals showed the expected typical fever response. PRV specific IFN-gamma production by PBMC clearly indicated that infection had occurred. Early significant IFN-gamma production by primed PBMC turned out to be a reliable and specific ex vivo marker for cellular response against productive PRV infection in swine before antibody formation.     

Cell-mediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-gamma production

Two experimental approaches were used to investigate the immunological responses of chickens to a commercial killed Salmonella enteritidis (SE) vaccine. In the first, the effects of host age on antigen-specific proliferative responses and cytokine production were examined. Compared with non-vaccinated controls, 4-wk-old vaccinated chickens showed higher proliferation to SE LPS and flagella. The lymphoproliferation responses to these antigens of 8-mo-old vaccinated chickens were not different compared to the non-vaccinated controls. Increased production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by antigen-stimulated splenocytes following vaccination were, in general, more often observed in 4-wk-old compared with 8-mo-old chickens, whereas serum levels of these cytokines were consistently higher in the vaccinated birds compared with controls regardless of age. The second set of experiments were designed to determine the effects of SE vaccination on mitogen- or antigen-induced splenocyte proliferation and serum nitric oxide (NO) and cytokine levels. Splenocytes from vaccinated chickens stimulated with SE flagella showed significantly increased numbers of TCRgammadelta+ cells at 7 days post-vaccination compared with non-vaccinated birds. In contrast, no differences were noted with CD4+, CD8+, or TCRalphabeta+ cells at any time points examined. Higher levels of NO production were observed following stimulation with SE flagella at 4, 7, 11, and 14 days after SE vaccination while serum levels of IFN-gamma, IL-1, IL-6, and IL-8 were elevated only at day 7 post-vaccination. In conclusion, younger chickens mounted a more robust antigen-specific immune response to the SE vaccine compared with older birds and vaccination induced not only T-cell-mediated responses but also host innate and pro-inflammatory responses.