Porcine Blood Mononuclear Cells Research Articles

Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production

Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated the induction of IFN-α, IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam 3Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24 h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN-α and IL-12 p40, and PGN, LPS and Pam 3Cys inducing varying amounts of IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10. Surprisingly, the ssRNA-mimic poly-U induced IL-6 and IL-1β only. Using CpG, PGN and LPS, the kinetics of cytokine production measured as mRNA (reverse transcription (RT)-qPCR) and protein (ELISA), respectively, correlated well, mRNA responses preceding protein responses. With the exception of IL-1β and IL-6, mRNA-responses were transient, whereas protein responses, except for TNF-α, followed saturation kinetics. Remarkably, LPS-induced TNF-α mRNA was not followed by a protein response. These results provide guidelines concerning the timing and use of protein and mRNA determinations for the characterization of porcine cytokine responses to PAMPs, although given the low number of animals used here results are preliminary and need confirmation in a larger study.

Steering orally induced deviation of xenogeneic immunity with exogenous cytokines in rats fed porcine proteins

Immune response to xenoantigens is a barrier to xenotransplantation. The objective of this study was to determine if rat cell-mediated immunity (CMI) and antibody (Ab) to discordant porcine xenoantigens could be suppressed by oral administration of porcine protein with adjunct systemic cytokine therapy. Based on principles of oral tolerance, it was hypothesized that: a. Feeding proteins from porcine blood mononuclear cells (PBMC) would induce a type 2 response, inhibiting CMI and type 1 Ab (associated with xenograft rejection) but increasing the amount of type 2 Ab. b. IL-4, a type 2 cytokine would exaggerate type 2 bias, enhancing immune deviation. c. IFN-γ, a type 1 cytokine was expected to down-regulate overall Ab production, but increase CMI and type 1 Ab. DA rats fed porcine proteins with or without subcutaneous and intraperitoneal injections of IFN-γ or IL-4 received PBMC by subcutaneous injection. Dermal delayed-type hypersensitivity to PBMC was the indicator of CMI. The IgG Ab to porcine proteins was quantified and type 2 (IgG 1 + IgG 2a) to type 1 (IgG 2b) Ab ratios indicated IR bias. Unfed, PBMC-challenged controls had a type 1 response bias but feeding PBMC induced a type 2 bias that suppressed CMI and type 1 Ab and increased type 2 Ab but not total IgG Ab. Contrary to the T H1/T H2 paradigm, IL-4 decreased oral-induced type 2 IR deviation and did not provide significant benefits relative to feeding alone. Treatment with IFN-γ prevented a switch to type 2 IR but feeding-induced suppression of CMI was not significantly compromised. The IFN-γ potently suppressed Ab, including, surprisingly, type 1 IgG isotypes. Contrary to the hypothesis tested, feeding Ag in combination with systemic delivery of recombinant IFN-γ apparently created an immunoregulatory environment most favorable to xenograft survival.

Products of Fourth-Stage Larvae of Oesophagostomum dentatum Induce Proliferation in Naïve Porcine Mononuclear Cells

Infection of pigs with Oesophagostomum dentatum is a major cause of economic losses in pig productions. Whether infection with this nematode results in a protective immunity is still in debate and information about immune-modulating properties of O. dentatum are lacking. The present study investigated the question whether products of O. dentatum larvae modulate the proliferative response of porcine blood mononuclear cells (poMNC) in vitro. The poMNC of naïve and O. dentatum-infected pigs were cultured for 72 h in the presence of products (total homogenates and culture supernates) derived from third- (L3) and fourth-stage larvae (L4) of O. dentatum. Numbers of vital cells and blast-transformed cells were determined flow cytometrically. No larvae product induced an accelerated death of poMNC in vitro. In contrast, products of L4 (but not L3) significantly increased the numbers of vital poMNC in vitro (up to 187 %). In addition, L4 products (homogenates and supernates, 0.1–10 μg/ml) but not those of L3 induced significant blastogenesis of poMNC. This was seen with poMNC from naïve and from O. dentatum-infected animals. In spite of these effects, the larvae products were not able to modulate the mitogen-induced (Concanavalin A) poMNC proliferation of naïve and infected animals. In summary, larvae of O. dentatum contain and secrete products with potential immunomodulatory capacity for porcine peripheral blood mononuclear cells. The differential effects of L3 and L4 indicate that the parasite alters its set of immunomodulatory substances during its development. This has to be considered in further studies and may help to identify the mediators involved.

Deviation of xenogeneic immune response and bystander suppression in rats fed porcine blood mononuclear cells

Reducing or deviating xenogeneic immune response prior to xenotransplantation may enhance the efficacy of conventional immunosuppressive therapies in prolonging xenograft survival. The potential to suppress or steer immune responses by oral administration of xenoantigens was evaluated. Based on knowledge of oral tolerance, hypotheses tested were that feeding xenoantigens would inhibit cell-mediated immune response (CMIR) and production of antibodies associated with graft rejection and induce bystander suppression. DA and LEW rats, high and low responders to xenoantigens, respectively, were fed dead porcine blood mononuclear cells (PBMC) and subsequently received live PBMC and hen egg-white lysozyme (HEWL, a third-party antigen) by subcutaneous injection. Delayed-type hypersensitivity (DTH) to PBMC was an indicator of CMIR. Quantification of T H1 (IgG 2b) and T H2 (IgG 1)-associated antibodies and their ratio measured magnitude and bias of the antibody-mediated response to PBMC and HEWL. Feeding PBMC reduced IgG 2b antibody production by 90% (DA) and 71% (LEW) and increased IgG 1 antibodies by 116% in DA but not LEW rats ( p ≤ 0.05). DTH was unaffected (DA) or increased (LEW) in antigen-fed rats. Bystander suppression was demonstrated by inhibition of antibodies to HEWL in DA rats. LEW rats did not respond to HEWL. Evaluation of tertiary immune response to PBMC revealed DTH and antibodies to xenoantigens was inhibited. These data demonstrated orally-induced immune deviation and bystander suppression in rats given PBMC that may favor discordant xenograft survival and suggests a method to suppress xenogeneic immunity in high responders by repeated immunization with xenoantigens.

Rat genotype influences quantitative and qualitative aspects of xenogeneic immune response to pig blood mononuclear cells

Xenotransplantation seeks to have cells with discordant genotypes co-exist. The hypothesis that host genotype modulates xenogeneic immune response (IR) was tested. Two inbred rat strains [Dark Agouti (DA) and Lewis (LEW)] representing diverse IR phenotypes were immunized with porcine blood mononuclear cells (BMC). Delayed-type hypersensitivity (DTH), immunoglobulin (Ig), antibody (Ab) and isotype bias of Ab response were evaluated. DTH to pig BMC was greater in DA than in LEW rats. Natural Ab was qualitatively different between strains (IgM and IgA predominated in DA, IgM and IgG(2a) predominated in LEW). Twice as much IgG was elicited from DA than LEW rats and DA utilized all isotypes whereas LEW did not use IgG(2a) or IgG(2c). IR bias was diametrically opposed; type 1 in DA but type 2 in LEW. Strains even differed in Ig profiles and dermal responses to saline injections and mitogen. The DA rats were the higher responders to pig BMC. Recipient genotype had significant and broad effects on IR to porcine BMC and may influence xenograft rejection and xenotolerance induction. Moreover, this study suggests that caution should prevail when interpreting data derived from a single inbred strain, particularly given that humans, the target species, are genetically diverse.

Porcine peripheral blood dendritic cells and natural interferon-producing cells.

Peripheral blood contains two major particular infrequent dendritic cells (DC) subsets linking the innate and specific immune system, the myeloid DC and plasmacytoid DC equivalent to the natural interferon-producing cells (NIPC). The functional characterization of these cells demands large volumes of blood, making a large animal model more appropriate and beneficial for certain studies. Here, two subsets of porcine blood mononuclear cells expressing swine workshop cluster 3 (SWC3, a SIRP family member), are described and compared to monocytes. The blood DC specialized in T-cell stimulation were major histocompatibility complex (MHC) class II+, CD80/86+, CD1+/-, CD4-, and in contrast to monocytes CD14-. A CD16- and a CD16+ subset could be discriminated. Granulocyte-macrophage colony-stimulating factor and interleukin-3 were survival factors for this DC subset, and culture induced an up-regulation of MHC class II and CD80/86. The second subset described, are porcine NIPC, typically CD4++, MHC class IIlow, CD80/86low, CD1-, CD8-/low, CD16-/low and CD45RA-/low. Porcine NIPC had high interleukin-3 binding capacity, and survived in response to this cytokine. Their unique function was strong interferon type I secretion after virus stimulation. Both subsets were endocytically active when freshly isolated, and down-regulated this activity after in vitro maturation. Taken together, the present report has delineated porcine blood DC and NIPC, permitting a more detailed understanding of innate immune defences, particularly in response to infections.

Products of fourth-stage larvae of Oesophagostomum dentatum induce proliferation in naïve porcine mononuclear cells.

Infection of pigs with Oesophagostomum dentatum is a major cause of economic losses in pig productions. Whether infection with this nematode results in a protective immunity is still in debate and information about immune-modulating properties of O. dentatum are lacking. The present study investigated the question whether products of O. dentatum larvae modulate the proliferative response of porcine blood mononuclear cells (poMNC) in vitro. The poMNC of naïve and O. dentatum-infected pigs were cultured for 72 h in the presence of products (total homogenates and culture supernates) derived from third- (L3) and fourth-stage larvae (L4) of O. dentatum. Numbers of vital cells and blast-transformed cells were determined flow cytometrically. No larvae product induced an accelerated death of poMNC in vitro. In contrast, products of L4 (but not L3) significantly increased the numbers of vital poMNC in vitro (up to 187%). In addition, L4 products (homogenates and supernates, 0.1-10 microg/ml) but not those of L3 induced significant blastogenesis of poMNC. This was seen with poMNC from naïve and from O. dentatum-infected animals. In spite of these effects, the larvae products were not able to modulate the mitogen-induced (Concanavalin A) poMNC proliferation of naïve and infected animals. In summary, larvae of O. dentatum contain and secrete products with potential immunomodulatory capacity for porcine peripheral blood mononuclear cells. The differential effects of L3 and indicate that the parasite alters its set immunomodulatory substances during its development. This has to be considered in further studies and may help to identify the mediators involved.

FACTORS DETERMINATING THE ONSET OF HYPERACUTE REJECTION FOLLOWING DISCORDANT PORCINE TO CYNOMOLGUS MONKEY KIDNEY XENOTRANSPLANTATION

521 In the discordant xenotransplantation setting especially in the transplantation of gal alpha 1-3 gal positive organs into gal alpha 1-3 gal negative recipients hyperacute graft rejection (HAR) mediated by preformed natural antibodies is believed to occur invariably in xenografts thus leading to rapid destruction and complete thrombosis of the graft. Recent data however have shown, that in the porcine to cynomolgus setting, HAR is not inevitably seen after porcine kidney or cardiac transplantation. In this study, by using nontransgenic large white porcine donor-kidneys, we have analyzed the influence of preoperative antiporcine antibody levels in the recipient, cold ischemia time, and donor organ weight on the onset of HAR. Material and methods: Porcine kidney xenotransplantation was performed in either a non life supporting model (n=7) or in a life supporting model (n=5) using cynomolgus monkeys of 3400 g to 8000 g body weight as recipients. The donor body weights varied between 4200 g and 25000 g, the corresponding donor kidney weights varied between 20 and 90 g. Cold ischemia times were between 75 minutes and 310 minutes. Preoperative antiporcine antibody levels were quantified using flow cytometry on aliquotated cryopreserved porcine blood mononuclear cells. In the explanted graft or biopsies appropriate immunohistochemistry (complement deposition, deposition of IgM and IgG) were performed. Results: HAR was defined as complete thrombosis of the graft after 2 hours with large vessels open and technical failure excluded. No correlation was found between cold ischemia time or preoperative antibody levels on the onset of HAR. In contrast, while 3 out of 9 grafts with an organ weight between 20 and 33 g and donor/recipient weight ratio below 2.0 experienced HAR, none out of three grafts with a donor organ weight > 70g and a donor to recipient weight ratio > 2.0 showed signs of HAR. In addition, all three larger grafts showed intraoperative and postoperative urine production, while in none of the 9 grafts weighting between 20 and 33 g primary graft function was observed. In one animal, a second kidney (23 g) was successfully (without HAR) transplanted immediately after HAR and subsequent removal of his first porcine kidney (20 g). Conclusion: By increasing the donor/recipient weight ratio and thus the organ weight, in the porcine to cynomolgus kidney transplantation model, the frequency of the onset of HAR can be at least reduced. This is most likely due to immunoabsorption of the recipients preformed antibodies in the porcine kidney without lethal damage for the graft. Studies determining the extent of antibody absorption or complement deposition in the grafts as well as antibody depletion from the recipients circulation are currently underway. This study was funded by Imutran/Novartis

Reactivity of workshop monoclonal antibodies on paraformaldehyde-fixed porcine blood mononuclear cells

One hundred sixty-four monoclonal antibodies (mAbs) of the second international swine CD workshop were tested for their reactivity with porcine blood mononuclear cells before and after fixing the cells with varying concentrations of paraformaldehyde (PFA) (1, 5 and 10 g l −1). A total of 38 (out of 134) positive reacting mAbs were significantly affected in their binding behavior on fixed cells. Modulation was seen as reduction in binding (staining intensity and/or % positive cells, n=18) or in elevated values ( n=20). Modified mAb binding occurred after fixing cells with 5 to 10 g l −1 PFA.

Human CD8 responder cells can be directly activated to proliferate and to produce IL‐2 following stimulation by allogeneic, but not by xenogeneic, porcine blood mononuclear cells

Abstract: In order to determine the precise nature of human T lymphocytes reactivity against porcine stimulator cells, purified CD4+ and CD8+ human peripheral T lymphocytes have been tested for their responsiveness against porcine stimulator cells. In a xenogeneic mixed lymphocyte reaction (MLR), CD4+ T cells were capable of proliferating as a result of the recognition of porcine peripheral blood lymphocytes (PBL), whereas CD8+ T cells were unresponsive. A proliferative response of CD8+ T cells could be restored by treatment with human IL‐2, but not by IL‐lα, IL‐lβ, or IL‐6. Production of IL‐2 was not detected in the xenostimulated CD8+ responder cells, nor could IL‐2 production be restored by the addition of IL‐lα, IL‐1β, or IL‐6. The presence of human CD4+ responder cells was crucial both for a xenoproliferative response and for IL‐2 synthesis. However, when the expression of the IL‐2 receptor (CD25) on the CD8+ T cells was analyzed, no difference was detected between xenostimulated and allostimulated CD8+ T cells. When the development of cytotoxic T cells in xenogeneic and allogeneic MLRs was compared, the cytotoxic activity exhibited by purified CD8+ T cells in xenogeneic MLR was significantly lower than that in the allogeneic combination. In the xenogeneic combination, exogenous IL‐2 reconstituted the cytotoxicity by purified CD8+ T cells; however, IL‐lα, IL‐lβ, or IL‐6 did not.Our results show that purified human CD4+ T cells respond directly against pig PBMCs, whereas purified CD8+ T cells do not. Furthermore, responsiveness in CD8+ T cells is completely restored by the addition of human IL‐2.

Age-related increase of porcine natural interferon α producing cell frequency and of interferon yield per cell

Porcine blood mononuclear cells (PBMC) were shown to secrete interferon α (IFN-α) after induction by a coronavirus, the transmissible gastroenteritis virus (TGEV). IFN-α producing cells, referred to as natural interferon α producing (NIP) cells, were detected by an ELISPOT assay using anti-porcine IFN-α monoclonal antibodies. The frequency of NIP cells among blood cells is low, at most 40–110 per 10 5 PBMC and each NIP cell was found to produce several units of IFN. We have shown that NIP cell frequency and IFN yield per cell gradually increased with the age of the donor animals, from the neonatal period to the adult age, with a significant increase around puberty. Our present results also indicate that NIP cells may be influenced by physiological and genetic factors; thus (1) NIP cell frequency and IFN yield per cell were decreased during lactation; (2) Chinese (Meishan) pigs were found to have higher NIP cell frequency and IFN yield per cell than European (Large White) animals.

Characterization of blood mononuclear cells producing IFNα following induction by coronavirus-infected cells (porcine transmissible gastroenteritis virus)

Porcine blood mononuclear cells (PBMC) were shown to produce interferon-α (IFNα) following incubation with cells infected by a coronavirus, transmissible gastroenteritis virus. Monoclonal antibodies (mAb) with specificities for leukocyte subsets and major histocompatibility complex (MHC) antigens were used to characterize IFNα producer cells. The production of IFNα was found to be a function of non-phagocytic, non-adherent, non-T, non-B, CD4 + (and to a lesser extent CD8 +) MHC-class-II-positive cells. Furthermore, addition of anti-MHC (class II) mAb during PBMC incubation with virus-infected cells reduced IFN yields, suggesting that masking of these surface antigens alters PBMC responsiveness to IFN induction.