Limited effect of recombinant porcine interleukin-12 on porcine lymphocytes due to a low level of IL-12 beta2 receptor.

The cytokine interleukin-12 (IL-12) is a key molecule in the regulation of CD4 + T cell development and specifically potentiates T helper 1 responses in mouse and man. However, biological effects mediated by IL-12 have not been well defined in pigs. Herein, recombinant porcine IL-12 (rPoIL-12) was expressed in a swine poxvirus system as a biologically active heterodimer and used to stimulate bovine or swine lymphoblast cells. After 3 days of incubation, only bovine blasts were responsive to the rPoIL-12 treatment as monitored by cell proliferation in several independent trials. Similarly, i.m. administration of rPoIL-12 in the hind leg of 3-week-old pigs indicated a reduction in the number of interferon-gamma (IFN-gamma) producing lymphocytes isolated from inguinal lymph nodes. The porcine IL-12R beta2 (IL-12Rbeta2) sequence was cloned and results generated by reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that the expression of IL-12R on porcine blasts as measured by the relative levels of IL-12Rbeta2 mRNA was less than that in bovine blasts and are in agreement with the reduced proliferation response of swine blast cells to rPoIL-12 treatment. Real time PCR analysis demonstrated that after PBMC stimulation, bovine blasts had an 11-fold increase in IL-12Rbeta2 mRNA levels while porcine blasts had almost no change. These data support a mechanism for IL-12 stimulation in swine inconsistent with that observed in conventional models.

Cytokine and lymphocyte profiles in miniature swine after oral infection with Toxoplasma gondii oocysts.

Pigs are considered an important source of Toxoplasma gondii infection for humans. A major strategy for immune prophylaxis of toxoplasmosis in swine is the understanding of the immune response against T. gondii infection. The phenotype of peripheral blood mononuclear cells (PBMC) and the kinetics of interferon-gamma (IFN-gamma), interleukin-12 (IL-12) and interleukin-10 (IL-10) transcriptional changes were characterised in miniature swine following infection. A total of 66, 4-9-month-old miniature swine were used for three experiments performed over a period of 2 years. All pigs were fed iota1000 oocysts of the VEG strain of T. gondii and blood samples were obtained on the day of inoculation and at days 3, 6, 10, 17, 25, 32 and 40 after infection. An increase in expression of activation markers CD25 and SLA-DQ was detected in the first week of infection. A significant increase in the percentage of CD8+cells was observed in the second week of infection. Relative competitive RT-PCR analysis indicated an increase in IFN-gamma mRNA as well as a reduction in IL-10 mRNA during the second week post infection. Increase in IL-12 transcription was not observed until the fourth week of infection. The ability of the pigs to respond to T. gondii infection by simultaneously inducing pro-inflammatory cytokines early and anti-inflammatory cytokines later is a likely indication of the requirement to strike a balance between controlling parasite growth and avoiding cytokine toxicity.

Characterization of lymphocyte subsets from mucosal tissues in neonatal swine.

Monitoring differences in lymphocytes during neonatal development constitutes a key to understanding the developing piglet's natural and pathological immune responses. A survey was conducted to accumulate information on the phenotype of lymphocytes isolated from blood, lymph nodes, and lymphoid associated structures of the pig small intestine of conventional pigs from day 1 to 47 of age and inbred miniature pigs between 12 and 82days. The effect of weaning, and age before and after weaning, were also evaluated. Weaning had a significant effect on the number of CD4(+), CD8(+), double positive CD4(+)/CD8(+), CD21(+), deltagammaTCR(+), SWC3(+) and SLA-DQ(+) cells. Aging of the pig before and after weaning resulted in significant changes in lymphocytes isolated from mesenteric lymph nodes and ileal sites. These results constitute an important baseline for studying mucosal immune response of neonatal pigs and identifying factors that influence the ability of the neonate to respond to the stresses and antigenic exposure associated with weaning.

Isolation and purification of lymphocyte subsets from gut-associated lymphoid tissue in neonatal swine.

The characterization of lymphoid subsets isolated from different anatomical sites is of great importance for understanding the mechanisms and interactions of normal and pathological immune reactions in the pig. The objective of this study was to standardize a protocol for the isolation of lymphocytes from mucosal tissues of neonatal pigs. Specific protocols for the isolation of lymphocytes from Peyer's patches of jejunum (jejPP) and ileum (ilPP), the Intraepithelial (IE) and lamina propria (LP) compartments of the jejunum and ileum, the mesenteric lymph nodes (MLN), and the peripheral blood (PBMC) are described in detail. The analysis of the cells isolated indicated a high viability (>90%). The histological sections from fragments collected from the intestine demonstrated that in nursing young pigs, the recovery of IE and LP lymphocytes may be limited because of the low numbers of lymphocytes present in early age. In addition, the presence of large intracytoplasmic vacuoles and hyaline droplets between the columnar epithelial cells during the first week of age interferes with the isolation of pure lymphocytes from the IE and LP compartments. Optimal lymphocyte yields for all the samples analyzed was confirmed by immunostaining with the pan-lymphocyte marker, CD45. The successful isolation and comparison of large numbers of pure populations from compartmentalized areas of the intestine and associated lymphoid tissues opens up a broad area for the investigation of mucosal immune responses of pigs.