Effect of leukotoxin of Mannheimia haemolytica and LPS of E. coli on secretory response of bovine neutrophils in vitro

To evaluate the role of leukotoxin (LKT) of Mannheimia haemolytica and lipopolysaccharide (LPS) of E. coli 055:B5 in pathogenesis of bovine respiratory disease (BRD) we investigated their in vitro effects on cultured bovine neutrophils. Functional parameters of neutrophils including degranulation, generation of superoxide, and nitric oxide were distorted in response to both toxins. The most essential reaction of neutrophils was found in respect to release of elastase after addition of LKT as well as LPS at concentration of 300 microg/ml. Moreover, we observed an increased release of myeloperoxidase (MPO) and alkaline phosphatase (ALK-P) from polymorphonuclear cells (PMN) after addition of LKT and LPS. We also found enhanced superoxide generation by bovine neutrophils after exposure to different concentrations of LKT and LPS. In cultures of PMN treated with LKT, concentration of nitrite increased with growing concentrations of LKT. Lower values of nitrite were obtained in cultures exposed to LPS. Partial lysis of PMN, determined by LDH (lactate dehydrogenase) leakage, started at concentration of 300 microg/ml for both toxins, meanwhile LKT concentration above 300 microg/ml was lethal. Our study has revealed that neutrophils in response to both toxins exaggerate release of analysed substances, which participate in worsening the course of the disease and play a role in lung injury during BRD. Toxins introduced to the cultural medium stimulate release of studied constituents from neutrophils by combined activation and lysis of neutrophils.     

Proliferative response of mammary gland mononuclear cells to recombinant bovine interleukin-2.

Methods of augmenting bovine mononuclear cell responsiveness during physiological transitions of the udder may enhance resistance of the mammary gland to intramammary infections. Interleukin-2 is required for proliferation of T-lymphocytes and may contribute to B-lymphocyte proliferation. Recombinant bovine interleukin-2 (rBoIL-2) was evaluated as a potential immunoenhancer of bovine mammary gland mononuclear cells. Bovine mononuclear cells were isolated from five primiparous Holstein cows at 14-18 and 28-32 days of involution and at 7-13 days prior to parturition. Bovine blood and mammary gland mononuclear cells were highly responsive to rBoIL-2. Response of mammary gland mononuclear cells to rBoIL-2 was comparable with response of blood mononuclear cells. These data suggest that rBoIL-2 may be an effective immunoenhancer of bovine mononuclear cells during the non-lactating and prepartum periods.     

Detection of bovine herpesvirus-1 in peripheral blood mononuclear cells eight months postinfection.

Peripheral blood mononuclear cells (PBMCs) from 5 calves (3 controls and 2 vaccinates) used in a bovine herpesvirus 1 (BHV-1) vaccine study with a BHV-1 Cooper strain challenge were collected 6 months after challenge. The PBMCs from the control animals were positive by immunofluorescence for the BHV-1 glycoprotein D (gD) while the vaccinates were negative. The PBMC samples from 4 of the 5 animals were examined for BHV-1 DNA by polymerase chain reaction (PCR) and for gD immunofluorescence at 8 months after challenge. The BHV-1 DNA and viral antigen were detected in PBMC samples at 8 months postinfection, but no virus was isolated.     

Response of bovine and porcine peripheral blood mononuclear cells to human recombinant interleukin 2(125)

Bovine and porcine peripheral blood mononuclear cells (PBMC) were tested for their response to human recombinant interleukin 2(125) (rIL 2(125)). The rIL 2(125) used in these experiments was purified to homogeneity from Escherichia coli, contained a site-specific modification at amino acid #125 replacing a cysteine with a serine residue and had a specific activity of 4 X 10(6) units/mg. Human rIL 2(125) was shown to be directly mitogenic for bovine and porcine PBMC and was able to maintain the long-term growth of mitogen-activated PBMC of both species. Long-term cultures were highly sensitive to low levels of rIL 2(125) and showed dose-dependent responses when used in short-term IL 2 assays. Bovine and porcine PBMC preincubated with human rIL 2(125) for 1 and 5 days demonstrated enhanced levels of cell-mediated cytotoxicity against both allogeneic and xenogeneic cell lines.     

Expression dynamics of Toll-like receptors mRNA and cytokines in porcine peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide

The Toll-like receptor (TLR)4 is critical for the recognition of Gram-negative bacterial lipopolysaccharide (LPS) but in porcine peripheral blood mononuclear cells (PBMCs) it may cooperate with other TLRs and lead to the production of inflammatory cytokines. Therefore, we analyzed TLR1-10 mRNA expression in porcine PBMCs stimulated with LPS over time (1-48 h) by using quantitative real-time PCR and cytokine proteins level by ELISA in culture supernatant. TLR1-10 mRNA was detectable in porcine PBMCs. When compared with the control (non-stimulated), TLR1 mRNA were increased (p<0.05) at 3 h after challenge with 1 μg/ml LPS, whereas TLR1 and TLR2 mRNA were increased (p<0.01) at 6 h after challenge with 10 μg/ml LPS. TLR4 increased (p<0.001) at 3h after challenge with LPS and remained constant. TLR5 and TLR6 mRNA increased (p<0.05) at 9 h and 1 h after of LPS stimulation, respectively. The mRNA of CD14 and MD2 were increased (p<0.001) at 1h after LPS stimulation. Additionally, at most of the time analyzed, the mRNA expression increased with the dose of LPS. The LPS concentration had influence (p<0.05) on all the TLRs expression except TLR10; whereas time had effect (p<0.05) on all TLRs expression except TLR2, 3, 6 and 10. When compared to the control, the cytokines IL1b, IL8 and TNFα proteins were increased (p<0.001) immediately at 1 h after LPS stimulation and remained constant till 48 h. IL12b was increased (p<0.001) 12 h after challenge with 10 μg/ml of LPS. Although IL8 level was the highest, the higher (p<0.05) expression of all these inflammatory cytokines indicate that upon interacting with TLRs, LPS exerted inflammatory response in PBMCs through the production of Th1 type cytokines. The production of cytokines was influenced (p<0.001) by both the dose of LPS and the stimulation time. Hence, the porcine PBMCs are likely able to express all members of TLRs.     

Effect of interleukin-1 receptor antagonist on concanavalin A response of peripheral blood mononuclear cells from newborn calves

The coexistence of interleukin (IL)-1beta with IL-1 receptor antagonist (ra) in bovine colostrum and the possibility of simultaneous transfer of these cytokines to neonates via colostrum have been demonstrated. In the present study, we investigated the effect of IL-1ra on the mitogenic response of calf peripheral blood mononuclear cells (PBMC) stimulated by concanavalin A (ConA), which was mediated by IL-1. Pretreatment of PBMC with recombinant bovine (rb) IL-1ra alone significantly suppressed the proliferation of ConA-stimulated cells. However, in the presence of rbIL-1beta, the suppressive activity of rbIL-1ra was counteracted. These results suggest that coexistence of IL-1ra with IL-1 in colostrum may have no effect on the activation of the neonatal immune system by IL-1beta.     

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.     

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.     

The cytokine response of circulating peripheral blood mononuclear cells is changed after intravenous injection of lipopolysaccharide in cattle

The aim of the study was to investigate lipopolysaccharide (LPS)-induced short and long term changes in capacity for intracellular cytokine-production of bovine circulating peripheral blood mononuclear cells (PBMCs). Eight dairy cows each received three intravenous injections of Escherichia coli LPS (10, 100 and 1000ng/kg, consecutively) at 3week intervals. Intracellular cytokine production was determined by flow cytometry in PBMCs obtained 0, 2, 6 and 24h after each LPS challenge. After LPS administration, proportions of monocytes producing tumour necrosis factor (TNF) alpha, interleukin (IL)-1beta and IL-8, as well as proportions of circulating lymphocytes producing interferon (IFN) gamma, decreased significantly. Within 24h, proportions had returned to or increased above pre-injection levels. Proportions of lymphocytes producing IL-4 and IL-10 increased significantly after injection of 1000ng LPS/kg. This study demonstrated that cytokine profiles shift quickly, but temporarily, to favour the anti-inflammatory response immediately after LPS exposure. The long term response to LPS was opposite to the immediate response, as cytokine profiles shifted in the 3weeks between challenges towards a pro-inflammatory response. Proportions of monocytes producing IL-1beta and TNFalpha determined immediately before the second and/or third LPS injection were higher than proportions determined before the first injection, whereas pre-injection proportions of lymphocytes producing IL-4 decreased with each challenge. These changes may result in a quicker host response to invading pathogens.     

2-Mercaptoethanol influences the in vitro function of bovine peripheral blood mononuclear cells.

The effects of 2-mercaptoethanol (2-ME) on some in vitro functions of bovine peripheral blood mononuclear cells (PBMC) were examined. It was shown that 2-ME enhanced, in a dose-dependent manner, the production of antibodies to bovine coronavirus. In this test the optimal concentration of 2-ME was 50 microM. This molarity of 2-ME was also optimal for the pokeweed mitogen (PWM)-induced proliferation of PBMC obtained from the 7 cattle tested. Similarly, the spontaneous proliferation of PBMC from 4 out of these cattle was enhanced. Thus, 2-ME evoked an increase (up to 2.5 times) or a decrease (at most 10 times) of the quota between the PWM-induced and the spontaneous proliferation (stimulation index). In general, the presence of 50 microM 2-ME enhanced the in vitro production of interferon by bovine PBMC. On the contrary, the highest proliferative response of PBMC to stimulation with bovine virus diarrhoea virus was achieved in cultures without 2-ME or in cultures with 0.5 or 5 microM 2-ME. Since the effects of 2-ME varied, for different tests as well as for cattle tested, attention should be paid to the use of 2-ME in cultures of bovine PBMC.     

CD14+ cells are required for IL-12 response in bovine blood mononuclear cells activated with Toll-like receptor (TLR) 7 and TLR8 ligands

Single-stranded viral RNA (ssRNA) was recently identified as the natural ligand for TLR7 and TLR8. ssRNA sequences from viruses, as well as their synthetic analogues stimulate innate immune responses in immune cells from humans and mice, but their immunostimulatory activity has not been investigated in ruminants. In the present investigations, we tested whether synthetic RNA oligoribonucleotides (ORN) can activate immune cells from cattle. In vitro incubation of bovine peripheral blood mononuclear cells (PBMCs) with ORN-induced production of IL-12, IFN-gamma and TNF-alpha. No significant induction of IFN-alpha was observed. Depletion of CD14+ cells from PBMC abrogated the IL-12 response and consequently the IFN-gamma response, suggesting that CD14+ cells are required for PBMC immune activation with ORN. Consistent with these findings, the putative receptors for ORN (TLR7 and TLR8) were expressed at higher levels in the CD14+ fraction than the CD14- PBMC fraction. Pre-treatment of PBMC with bafilomycin (an inhibitor of phagosomal acidification) prior to stimulation with ORN abolished the cytokine responses, confirming that the receptor(s) which mediate the ORN-induced responses are intracellular. These results demonstrate for the first time that the TLR7/8 agonist ORN's have strong immune stimulatory effects in cattle, and suggest that further investigation on the potential of TLR7/8 ligands to activate innate and adaptive immune responses in domestic animals are warranted.     

Nonspecific immune response of peripheral blood neutrophils in two horse breeds (Anglo-Arabian and Spanish-Arabian): response to exercise

The aim of the present paper was: (1) to find out if there were any differences in the nonspecific immunological pattern of peripheral blood neutrophil between two breeds of horses (AA and SA); (2) to evaluate the effects of an exercise in the aerobic-anaerobic threshold. This has been observed in a group of 11 untrained horses (6 SA and 5 AA) of 2.5 years old. No statistically significant differences were found in the different stages of immune response between the rest and immediately after physical exercise to two breeds. However, the chemotaxis was significant higher at rest in the AA than SA breed. A positive correlation was found at rest between the phagocytic and oxidative metabolism activity for AA breed and a negative correlation too between the adherence and chemotaxis with phagocytic capacity, immediately after exercise test, for the same breed.     

Effect of progesterone on the in vitro response of peripheral blood mononuclear cells stimulated by Escherichia coli in mares

Escherichia coli(E. coli) isolated from the uterus of a Thoroughbred mare with bacterial endometritis was used to evaluate the effect of progesterone (P(4)) on the immune response of mares. Peripheral blood mononuclear cells (PBMCs) were collected from 10 nonpregnant clinically healthy adult mares (range, 4-12 years) during diestrus, four Thoroughbreds and six Hokkaido native horses. Cell proliferation and expression of cytokine mRNA, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-10, of PBMCs stimulated with E. coli and P(4) were examined in vitro. P(4) was shown to have significantly inhibited E. coli induced proliferation and expression of IFN-γ in PBMCs. These results indicate that P(4) inhibits the immune response to E. coli in mares.     

In vitro activation of bovine macrophage and peripheral blood mononuclear cells by Nocardia rubra cell wall skeleton (N-CWS)

Nocardia rubra cell wall skeleton (N-CWS) was used for immunotherapy to bovine leukemia virus (BLV)-positive cattle with enlarged subcutaneous lymphatic nodules. Electron microscopical observations showed the infiltration of macrophage and T cells around N-CWS treated lesions. Antitumor effect induced by N-CWS was examined in vitro. The maximum cytolytic activity of macrophage was observed, when the cells were incubated with 10 micrograms/ml of N-CWS. Chemiluminescence response of peripheral blood mononuclear cells (PBMC) using N-CWS as stimulant was observed with a high level of activity for a long period, 5 hr. Mitogenic effect of N-CWS to PBMC was observed in the presence of macrophages but not without macrophages. Furthermore, interleukin 2 activity was recognized in supernatant of PBMC cultured with N-CWS. Maximum cytotoxic T lymphocyte response was induced when PBMC were cultured with 0.1 micrograms/ml of N-CWS.     

Cell-mediated cytotoxicity of peripheral blood mononuclear cells stimulated in vitro for infectious bovine rhinotracheitis virus-infected cells

Peripheral blood mononuclear cells (PBMC) from calves infected with and hyperimmunized to infectious bovine rhinotracheitis virus (IBRV) were stimulated in vitro with viral antigens to evaluate their cytotoxicity for a variety of cells. The 51-Cr release assay was used to measure cytotoxicity. Cytotoxicity was not present in fresh nonstimulated cells, but was detected in cultured, IBRV-stimulated cells at day 3, was maximal at day 7, and declined thereafter. PBMC stimulated in vitro with IBRV expressed a preference for killing IBRV-infected cells compared to pseudorabies virus (PRV)-infected cells. IBRV-infected, but not PRV-infected, cold target cells inhibited lysis of IBRV-labeled target cells. High concentrations of IBRV hyperimmune serum partially blocked cytotoxicity. Cells expressing a viral preference for cytotoxicity showed no preference for lysis of autologous compared to heterologous bovine cells. PBMC from calves that were either IBRV-immune or not immune were cultured without IBRV stimulation and had similar levels of cytotoxicity for IBRV-infected cells as cells from IBRV-infected cattle.     

Stimulation and killing of bovine mononuclear leukocytes by bacterial lipopolysaccharide (endotoxin)

Bovine adherent mononuclear leukocytes were incubated with bacterial lipopolysaccharides (LPS) in vitro, and these cells produced a factor that increased the blastogenic reaction of mouse thymocytes to concanavalin A. This factor most resembles interleukin 1. The LPS were also cytotoxic for bovine adherent mononuclear leukocytes in a dose-and time-dependent manner. Cytotoxicosis was determined by the release of cytoplasmic lactic dehydrogenase. This cytotoxicosis was blocked by treating the cells with corticosteroids. Variation in the reaction to LPS occurred in cells collected from the same cow on different days and from cells collected from different cows.     

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.     

麻鸭白细胞介素17的原核表达及鉴定

为表达麻鸭白介素17(duIL-17),本研究提取ConA刺激的麻鸭脾脏淋巴细胞总RNA,RT-PCR扩增duIL-17基因片段,将该片段插入克隆载体pCR2.1中构建重组质粒pCR2.1-duIL17.将duIL-17基因片段亚克隆到原核表达载体pGEX-6P-1中,构建重组质粒pGEX-duIL17,将其转化到大肠杆菌BL21中,IPTG诱导表达.测序结果显示,duIL-17与参考序列(EU366165.1)相比较第174位碱基由C突变为T,并且为沉默突变.SDS-PAGE和western blot分析显示,诱导表达的重组蛋白GST-duIL17主要以包涵体形式存在,分子量大小约为45 ku,duIL-17与抗鸡IL-17抗体具有良好反应性.本研究为制备duIL-17单克隆抗体提供了实验依据.     

Equine herpesvirus-1-specific interferon gamma (IFNgamma) synthesis by peripheral blood mononuclear cells in thoroughbred horses

REASONS FOR PERFORMING STUDY: An assay has been developed that measures EHV-1 specific interferon gamma synthesis (IFNgamma), a cytokine produced following the activation of memory T lymphocytes and therefore a measure of cell mediated immunity. The method requires validation in the field. OBJECTIVES: To measure the frequency of EHV-1 specific, IFNgamma synthesising peripheral blood mononuclear cells (PBMC) in a population of Thoroughbred horses, and examine its relationship with age, gender, premises and history of vaccination or field infection with EHV-1. METHODS: Lymphocytes from 200 Thoroughbred horses were stimulated with EHV-1 in vitro, and IFNgamma detected using a monoclonal antibody and indirect immunofluorescence. Percent positive cells were enumerated by flow cytometric analysis and the results described and compared statistically between groups. RESULTS: The frequency of IFNgamma+ PBMC was significantly higher in animals age >5 years compared with 2-4 years, in females vs. males, on stud farms vs. training yards and following vaccination of 2-year-olds with inactivated virus compared with nonvaccinates. Age strongly confounded all these associations and care must therefore be taken interpreting these results. Mares exposed to a field infection with EHV-1 also had higher frequencies of IFNgamma+ PBMC than other vaccinated horses. CONCLUSIONS: The frequency of EHV-1 specific, IFNgama+ PBMC among the sample Thoroughbred population was diverse but lowest in young, unvaccinated horses-in-training. POTENTIAL RELEVANCE: The frequency of EHV-1 specific lymphocytes synthesising IFNgamma in this population may be associated with its susceptibility to infection with this virus. This easy technique may be applied to monitor the antigenicity of vaccines and their effectiveness at stimulating cellular immunity.