Porcine effector mechanisms: antibody-dependent cell-mediated cytotoxicity of pseudorabies-infected target cells

Antibody-dependent cell-mediated cytotoxicity (ADCC) against pseudorabies virus (PRV)-infected cells was determined in neonatal porcine pulmonary washout cells and in partially purified blood-derived leukocytes. The ADCC assays were performed at approximately 1, 2, 3 and 11 days after birth. It was found that pulmonary-derived cells had a low level of cytolytic activity against the PRV-infected target cells at about 1 day of age. This activity increased sharply during the next 2 days of age, but declined somewhat by 11 days of age. The increase in ADCC activity paralleled closely the increase in the relative and absolute numbers of macrophages in the alveolar washout fluids. ADCC activity was high at all times in blood-derived neutrophils, and present in blood-derived lymphocytes, but at a lower level of activity.     

Antibody-dependent cell-mediated cytotoxicity in sheep

The ability of sheep leukocytes to mediate antibody — dependent cell-mediated cytotoxicity (ADCC) and that of sheep serum IgG₁ and IgG₂ to induce ADCC were investigated. Partial characterization of effector cells was attempted. These investigations revealed that ADCC occurs in sneep. With chicken erythrocytes (CRBC) as the target cells, polymorphonu-cleated cells (PMN), and monocytes, were the most effective leukocytes. Ovine peripheral blood lymphocytes (PBL) also mediated ADCC, and within the PBL population, T-cells were capable of mediating ADCC. The T-cells were obtained by nylon wool fractionation and selective agglutination by peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA). Both nylon wool adherent and non-adherent fractions were active in ADCC, although the former were more active, implying heterogeneity in nylon wool adherence among ovine K-cells. Depletion of B (SIg⁺) cells did not affect ADCC activity of the remaining cells. Depletion of Fc⁺ cells markedly reduced cytotoxic activity of PBL. Both sheep IgG₁ and IgG₂ anti-CRBC immunoglobulins were able to induce ADCC.     

Antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against cells infected with porcine transmissible gastroenteritis virus.

The objective of this study was to determine whether porcine peripheral blood leukocytes and intestinal intraepithelial leukocytes can mediate antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against target cells infected with transmissible gastroenteritis virus. Peripheral blood leukocytes collected from six young adult pigs and intraepithelial leukocytes from a further five pigs were used as effector cells in chromium release assays against PK-15 cells persistently infected with transmissible gastroenteritis virus. Both peripheral blood leukocytes and intraepithelial leukocytes were capable of mediating antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against PK-15 transmissible gastroenteritis cells. While the peripheral blood leukocytes mediated lower levels of specific 51Cr release in spontaneous cell-mediated cytotoxicity than in antibody-dependent cell-mediated cytotoxicity, the intraepithelial leukocytes were more effective in spontaneous cell-mediated cytotoxicity than antibody-dependent cell-mediated cytotoxicity.     

Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.     

Cytotoxic capabilities of bovine lymphocytes after lectin stimulation

Bovine peripheral blood mononuclear leukocytes (PBML) were stimulated in vitro with the mitogenic lectins concanavalin A (Con A) and phytohemagglutinin. Their cytotoxic capabilities were evaluated in a 51Cr release assay. Lectin-activated bovine effector cells did not mediate antibody dependent cellular cytotoxicity (ADCC) nor direct killing against cultured tumor target cells. Nevertheless, activation of PBML with lectins consistently generated effector cells able to mediate lectin-dependent cellular cytotoxicity. Cultivation of Con A stimulated-PBML for 3 to 4 weeks in the presence of lymphokines-containing IL-2 generated cells with the ability to mediate lysis without using Con A-coated target cells. However, cytotoxic cultures capable of mediating direct lysis of target cells were not able to mediate ADCC.     

Effects of pseudorabies virus infection upon cytotoxicity and antiviral activities of porcine alveolar macrophages

Alveolar macrophages (AM) infected with Pseudorabies virus (PRV) were compared to noninfected AM for cytotoxicity against foreign or transformed cells and production of interferon (IFN). Five PRV strains were used to infect AM including strains that are known to be highly virulent for pigs, i.e. strain 4892 and strain S-62 as well as strains that are regarded as mild or nonvirulent, i.e. BUK and Bartha. The multiplicity of infection ranged from 0.005 to 0.05 TCID₅₀/cell. The target cells in the cytotoxicity assays were either chicken red blood cells, PRV-infected vero cells, or human myeloblastoma cells (K562 cell line). For the producton of IFN, AM cultures were treated with polyinosinic: polycytidylic acid (Poly I:C) diluted in tissue culture media at a concentration of 5 μg/10⁶ cells. Culture supernatants were collected at various times poststimulation and tested for antiviral activity using the Vesicular Stomatitis Virus replication inhibition test. Swine AM were able to lyse chicken red blood cells in an antibody-independent way but not in an antibody-dependent way, whereas lysis of PRV-infected vero cells was accomplished both ways. The cytotoxicity against chicken red blood cells was reduced in the PRV-infected AM as compared to noninfected cells, particularly in AM infected with virulent PRV strains. Specific ⁵¹Cr release values for AM infected with S-62 and 4892 strains were 14 and 19, while the noninfected AM had values of 36. Similarly, in the antibody-dependent cytotoxicity assay against PRV-infected vero cells there was no activity of AM against K562 cells. The production of IFN was readily stimulated with Poly I:C. The optimal time for supernatant collection was between 12 and 16h poststimulation. The antiviral activity was abrogated by treatment of the supernatant with antiserum against human leukocyte IFN; it was therefore considered to be due to interferon-alpha (IFN) released from the macrophages. The antiviral activity present in supernatants of PRV-infected AM was reduced compared to noninfected AM. The difference between AM cultures infected with virulent strains of PRV and noninfected AM cultures was statistically significant at P 0.025. The results provide support to the premise that the role of AM in lung defense can be compromised by PRV infection.     

Immunohistologic study of pulmonary and lymphatic tissues from gnotobiotic pigs inoculated with ara-T-resistant strain of pseudorabies virus

Ara-T-resistant strain of pseudorabies virus (PRV) was inoculated intranasally into six 2-week-old gnotobiotic pigs. Five inoculated pigs were sneezing and coughing. In pigs 1 to 4 killed on postinoculation days (PID) 3, 5, 7, and 9, respectively, PRV antigen was detected in respiratory epithelial cells, and pigs had severe pneumonitis. In pigs 5 and 6 killed on PID 11 and 13, respectively, PRV antigen was localized in macrophages in alveoli and necrotizing nodules. Immunoglobulin-containing cells (IgG, IgM, and IgA) were detected first in pneumonic lesions in pig 4 killed on PID 9. Detection of immunoglobulin-containing cells was coincident with pulmonary inflammation and regeneration of pneumonic lesions. The number of IgG-containing cells was greater than that of IgM- and IgA-containing cells. Corresponding to transient viral multiplication, IgG-, IgM-, and IgA-containing cells were demonstrated first in lymphatic tissues in pig 1 killed on PID 3 and their number was 5 to 10 times more than those in control pigs 7 and 8. Seemingly, PRV replication in lymphatic tissues stimulated the proliferative response of specific immunoglobulin-producing cells, and the appearance of immunoglobulin-containing cells in the lungs was associated with clearance of PRV and regeneration of pneumonic lesions.     

Cellular immunity shown in pseudorabies virus-infected pigs by leukocyte migration-inhibition procedure.

Cellular immunity in pigs inoculated with pseudorabies virus (PRV) was studied by the agarose plate technique of direct leukocyte migration-inhibition procedure. Migration of leukocytes from PRV-infected pigs was inhibited in the presence of PRV antigen, whereas migration of leukocytes from nonexposed pigs was not inhibited in the presence of the same antigen. The migration of leukocytes collected 4 days after intranasal exposure to PRV was inhibited; humoral antibodies could not be detected until 7 days after exposure. Cellular immunity was present in pigs 14 days after inoculation with inactivated PRV antigens; low concentrations of neutralizing and precipitating antibodies were present at this time. The leukocyte migration-inhibiton procedure was found to be a useful tool in studying the role of cellular immunity in PRV infections.     

Cellular immune responses in pigs fed a vitamin E- and selenium-deficient diet.

The effects of dietary restriction of vitamin E (Vit E) and selenium (Se) on lymphocyte proliferation, natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity (ADCC), and on burst respiratory response of stimulated granulocytes as measured by chemiluminescence (CL) were studied in pigs. Six male weanling pigs were maintained for 25 d on a torula yeast-based diet containing no measurable amount of alpha-tocopherol and less than .02 mg of Se per kilogram of feed. Six others received the same basal diet supplemented with 33 IU of DL-alpha-tocopheryl acetate and .2 mg of Se per kilogram of feed. All pigs were inoculated with Salmonella typhisuis on d 21 of the feeding period and killed on d 25. Tests to measure cellular immune functions were performed on cells isolated from blood samples taken on d 21 and 25. After 21 d of feeding, lymphocyte blastogenesis responses to phytohemagglutinin, concanavalin A, and pokeweed mitogen in pigs fed the Vit E- and Se-deficient diet were normal compared with the response in pigs fed the supplemented diet. Moreover, the cytotoxic activity of NK cells, the ADCC response, and the CL response of granulocytes were not affected. After 25 d, a marked suppression of lymphocyte response to mitogens occurred in pigs fed the Vit E- and Se-deficient diet when the cells were cultured in the presence of autologous serum. When fetal bovine serum replaced autologous serum in the cultures, no suppression was observed. No effect on NK activity and ADCC was observed, whereas the CL peak response of granulocytes tended to be higher in pigs fed the deficient diet.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Identification of feline monocytes and neutrophils as effector cells in antibody-dependent cellular cytotoxicity: sequential analysis, using light microscopy, histochemistry, and scanning electron microscopy

Feline monocytes and neutrophils functioned as effector cells in antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated chicken erythrocytes. Using light microscopy, effector cell populations were identified in effector-target cell interactions, with further characterization of these identical individual effector cells by histochemical evaluations and scanning electron microscopy. Monocytes and neutrophils, but not lymphocytes, were observed attacking target cells. Carbonyl iron depletion of monocytes and neutrophils from peripheral blood leukocytes caused a marked reduction from a mean of 62% to 3.6% lysis in ADCC as measured by a 4-hour 51Cr release assay. Effector cells functioning in the ADCC reaction were visualized, using sequential analysis and light microscopy, histochemistry, and scanning electron microscopy.     

Effects of canine distemper virus on natural killer cell activity in dogs

An in vitro 51Cr-release assay was developed to detect the cytotoxicity of natural killer cells (NK) of canine peripheral blood mononuclear leukocytes to canine distemper virus (CDV) target cell membrane-bound antigens. Leukocytes from 23 young (greater than or equal to 1 week of age), CDV-naive gnotobiotic dogs could discriminate between noninfected control and CDV-infected Vero target cells. However, the amount of preinfection NK activity did not positively correlate with the ultimate outcome of the disease process when these same dogs were given virulent R252-CDV. Evaluation of preinfection and postinfection CDV-specific NK activity indicated that infection-associated increases in cytolysis of CDV-infected or noninfected Vero targets did not occur. In vitro infection of peripheral blood leukocytes with CDV did not change the kinetics or magnitude of NK-mediated cytolysis of homologous virus-infected or other NK-susceptible target cells.     

Cytotoxic lymphocytes induced by African swine fever infection

The generation of lymphocytes cytotoxic to African swine fever virus infected testis cells during in vivo infection is described. Peripheral blood lymphocytes from 16 pigs developed cytotoxicity seven to eight days after infection but the lysis was not restricted to autologous cells.     

Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs

Replication of porcine circovirus type 2 (PCV2) in pigs, as measured by spliced capsid mRNA (Cap mRNA) and viral DNA, was investigated following experimental infection. Peripheral blood mononuclear cells (PBMCs), and tissue from bronchial lymph nodes (BLN), inguinal lymph nodes (ILN), tonsils, lungs, liver, kidneys, spleen and thymus from infected pigs on different days post-infection (DPI) were assessed. PCV2 replication differed dramatically between tissues from the same infected pig. The virus actively replicated in most tested tissues at 14DPI in association with increased PCV2 associated lesions and PCV2 antigen levels, although no clinical signs correlated with PCV2 associated disease were observed in infected pigs during the course of the study. The PCV2 Cap mRNA was detected only at 13DPI in PBMCs from infected pigs, suggesting replication of the virus in circulating blood is transient and not a major site for PCV2 replication in vivo. Evaluation of the Cap mRNA and viral DNA synthesis in T and B lymphocyte and monocyte populations from PBMCs and BLN at various intervals post-inoculation revealed replication of PCV2 in all cell subpopulations; however, viral replication in B lymphocytes was greater than observed in mononuclear cells isolated from BLN at 14DPI indicating that B lymphocytes may be an important cell population for PCV2 replication. These findings further our understanding of the cell types permissive for PCV2 replication and the pathogenesis of PCV2 infection in vivo.     

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.     

In vitro cytotoxicity of serum and peripheral blood leukocytes for equine herpesvirus type 1-infected target cells.

The immune response in horses following experimental infection with equine herpesvirus type 1 (EHV-1) was assessed by measuring cytotoxicity for EHV-1-infected target cells. A technique was developed, using [125I]5-iodo-2'-deoxyuridine ([125I]IUDR)-labeled equine fetal kidney cells infected with EHV-1 as the target cells. It was shown that peripheral blood leukocytes from a recovered horse were capable of lysing target cells, as measured by the loss of radio-active label. Following the experimental infection of specific-pathogen-free ponies with EHV-1, cytotoxicity was obtained with fresh autologous serum, peripheral blood leukocytes in autologous serum, and washed peripheral blood leukocytes. Cytotoxicity of the serum and peripheral blood leukocytes was detected as early as one day after infection. It is suggested that cytotoxic antibodies or cells could play an important part in restricting virus spread after infection of the horse with EHV-1.     

Effector mechanisms in the pig. Antibody-dependent cellular cytolysis of African swine fever virus infected cells

Antibody dependent cellular cytolysis (ADCC) against African swine fever virus infected nucleated cells was investigated in a porcine system. Of the peripheral blood components examined, only neutrophils acted as effectors. Lymph node derived cells displayed no ADCC activity. In vitro yield reduction assays suggested that neutrophil mediated ADCC may play a role in recovery from African swine fever virus infection.     

Immunomodulating effects of intestinal absorbed maternal colostral leukocytes by neonatal pigs.

Intestinal absorption of fluorescein isothiocyanate-labelled maternal colostral leukocytes (FITC-CL) was studied in 49 neonatal colostrum-deprived (CD) pigs from nine Minnesota miniature sows. Within 2 h postfeeding (pf), maternal FITC-CL were absorbed from the sibling's digestive tract and migrated into blood. The peak appearance of FITC-CL in blood occurred in samples at 5 and 7 h pf. By 24 h pf, cells were detected in liver, lung, lymph nodes, spleen and gastrointestinal tissues. To confirm intercellular migration of FITC-CL, gastrointestinal explant cultures from neonatal CD pigs were used. Maternal FITC-CL were observed to intercellularly migrate in 24 to 48 h pf between duodenal- and jejunal-epithelial cells to lamina propria cells and submucosal spaces. Fluorescein isothiocyanate-labelled maternal colostral leukocytes were not absorbed via ileal explant cultures. Unlike FITC-CL, maternal FITC-peripheral blood mononuclear leukocytes (FITC-PBL) were not absorbed either in vivo or in vitro by gastrointestinal tissues. When maternal FITC-PBL were intravenously administered to siblings they were distributed in blood and organs similar to FITC-CL. Following exposure to FITC-labelled cells, treated- and mock (untreated)-pigs were compared on the basis of PBL proliferative responses to phytomitogens. Sibling CD-pigs fed maternal FITC-CL showed higher PBL T-cell responses to phytohemagglutinin (PHA) and concanavalin A (ConA), and a significant stimulation (p < or = 0.01) of B-cell responses to pokeweed mitogen (PWM). Pigs fed FITC-PBL showed little PBL responses to PHA, ConA and PWM over PBL from mock pigs. Similarly, the influence of noncellular constituents of colostrum were also assessed by proliferative studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-mediated immunity in Marek's disease: cytotoxic responses in resistant and susceptible chickens and relation to disease

Two experiments were conducted to study the cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) from chickens inoculated with Marek's disease virus (MDV) against a Marek's disease-derived lymphoblastoid cell line (MSB-1) and to associate the cytotoxicity with incidence of disease. In experiment I, moderately susceptible random-bred, specific-pathogen-free chickens were inoculated with MDV (group 1), vaccinated with a herpesvirus of turkeys (HVT) and inoculated with MDV (group 2), vaccinated with HVT and inoculated with chicken kidney cells (CKC; group 3), and inoculated with CKC only (group 4). Cytotoxic activity in the PBL was detected initially during the first week after MDV inoculation and periodically throughout the observation period (groups 1, 2, and 3). Throughout the observation period, the magnitude of cytotoxic activity was similar in PBL from groups 1 and 2 chickens. The PBL from both surviving and fatally infected chickens (groups 1 and 2) were similarly cytotoxic when sampled during the first 16 days after MDV inoculation. In experiment II, inbred genetically susceptible (line 7) and resistant (line 6) chickens were used. Cytotoxic activity of PBL of significantly greater magnitude was associated with a lower mortality or incidence of gross lesions (or both) in MDV-inoculated line 6 (group B) and HVT-vaccinated and MDV-inoculated line 7 (group C) chickens compared with activity of PBL from MDV-inoculated line 7 (group A) chickens. The cytotoxic activity of PBL from individual inbred chickens did not correlate with the outcome of the infection.     

The role of specific immunoglobulins in antibody-dependent cell-mediated cytotoxicity assays during Babesia bovis infection

A stabilate prepared from Babesia bovis-infected Boophilus microplus ticks was used to infect intact adult cattle. Whole sera and immunoglobulin fractions from representative sera were tested by complement fixation (CF), indirect fluorescent antibody (IFA), and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The last test utilized 51Cr-labeled chicken erythrocytes coated with Babesia bovis antigen as targets. Mononuclear cell preparations, obtained from peripheral blood of normal donors and consisting of lymphocytes with 2--6% large monocytes, were used as the source of effector cells. Antibody activity was detected by all tests between 14 and 16 days following infection. Specific IgM and IgG1 were reactive in both CF and IFA tests, although the development of high titers was attributable to IgG, alone. The ADCC activity was restricted to IgG1 fractions and was greater in those sera or fractions with greater CF activity. No activity was demonstrated in IgG2 fractions by any test used.