Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.     

Mobilization and activation of nonspecific cytotoxic cells (NCC) in the channel catfish (Ictalurus punctatus) infected with Ichthyophthirius multifiliis

Channel catfish demonstrate a shift in the tissue distribution of nonspecific cytotoxic cells (NCC) when infected with the protozoan parasite, Ichthyophthirius multifiliis. NCC, isolated from head kidney (HK) tissues (hemopoietic organ) or peripheral blood leukocytes, were assessed for cytotoxic activity against NC-37 (a transformed mammalian cell line). NCC activity from HK tissue of moribund I. multifiliis-infected fish was depressed compared to HK-NCC activity in uninfected or I. multifillis-immune fish. The activity of NCC, isolated from the peripheral blood of moribund I. multifiliis-infected fish was significantly greater than the NCC activity in peripheral blood from either immune or uninfected fish. Chromium-51 release assays were combined with effector and target conjugate assays to determine killing capacity (Vmax) and affinity (Km) for target cells of peripheral blood NCC from moribund I. multifiliis-infected and uninfected fish. These experiments indicated that the peripheral blood from the moribund infected fish contained an increased percentage of active NCC with increased killing capacity and target cell affinity compared to peripheral blood NCC activity of uninfected fish.     

Vitamin A deficiency impairs cytotoxic T lymphocyte activity in Newcastle disease virus-infected chickens

The effect of vitamin A deficiency on cytotoxic T lymphocyte (CTL) activity was investigated during the acute phase of disease 7 days after primary inoculation and 1 day after secondary inoculation in chickens with or without Newcastle disease virus (NDV, La Sota strain) infection. Day-old chickens with limited vitamin A reserves were fed purified diets containing either marginal (ad libitum) or adequate (pair-fed) levels of vitamin A, and at 3 weeks of age half of the chickens in each group were infected with NDV. Cytotoxic activity was investigated during the acute phase of disease (7 days after primary inoculation) and 1 day after secondary inoculation, in an assay system with either peripheral blood lymphocytes (PBL) or nonadherent splenocytes as effector cells and adherent splenocytes from the same animal as target cells. After primary inoculation, cytotoxic activity could only be demonstrated in nonadherent splenocytes. Vitamin A deficiency resulted in significantly reduced CTL activity at all effector/target cell ratios tested. After reinfection CTL activity could also be demonstrated in PBL, but only from chickens fed the control diet, suggesting a diminished pool of CTL in vitamin A deficiency. The results of this study indicate that vitamin A deficiency impairs CTL activity - a part of the cell-mediated defense system - and this may have important implications for recovery from viral infection.     

Presence of adherent cytotoxic cells and non-adherent natural killer cells in progressive and regressive Marek's disease tumors

Marek's disease virus-induced progressive tumors and Marek's disease transplantable local regressive tumors were dispersed by treatment with collagenase and cells were examined in vitro for cytotoxic effector functions against target cells of tumor lines. Both types of tumors had adherent and non-adherent cytotoxic cells. The cytotoxicity of adherent cells was detected in an 18-hour but not in a 4-hour 51Cr-release assay. The adherent effector cells from progressive tumors were inactivated by pretreatment with carbonyl iron and carrageenan whereas the adherent effector cells from the regressive tumors were refractory to these treatments. In the progressive tumors, the 18-hour cytotoxic activity of cells of tumors and of spleens of tumor-bearing chickens was compared; the activity was higher in the tumor than in spleen. The nonadherent cell cytotoxicity detectable in a 4-hour 51Cr-release assay was associated with anti thymocyte serum-resistant natural killer cells. The incidence and levels of natural killer cell reactivity were greater in the regressive tumors than in the progressive tumors. In the regressive tumors, the natural cytotoxicity levels were higher in the tumor than in the spleen of tumor-bearing chickens. The differences in characteristics of adherent cytotoxic cells in virus-induced progressive tumors and transplantable regressive tumors and elevated levels of NK cells in regressive but not in progressive tumors may indicate a role for intratumoral immunity in tumor regression.     

A colorimetric assay to evaluate the cytotoxic activity of the intestinal intraepithelial lymphocytes of chickens

After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37±2.53 in a group of 5-week-old chickens and 80.2±3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethyl sulphoxide - E effector cells - GALT gut-associated lymphoid tissue - GM growth medium - iIELs intestinal intraepithelial lymphocytes - MTT 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide - NK cell natural killer cell - OD optical density - RPMI Rosewell Park Memorial Institute medium - T target cells     

Natural cell-mediated cytotoxicity to cells infected with infectious bovine rhinotracheitis virus

Cell-mediated cytotoxicity against viral-infected cells was demonstrated in a 6-hr 51Cr release assay. Peripheral blood mononuclear leukocytes from both infectious bovine rhinotracheitis virus (IBRV)-infected and noninfected cattle exhibited preferential lysis against IBRV-infected primary bovine embryonic kidney (BEK) cells compared to cells infected with pseudorabies virus and noninfected BEK cells. Addition of specific antibody to the assay did not enhance cytotoxicity. The effector cell was a nonadherent cell which was either spontaneously enriched or generated during in vitro cultivation. Maximal cytotoxic activity was detected in peripheral blood mononuclear cells cultured for 3 to 5 days. Several factors affected the magnitude of cytotoxicity during the assay: target cell type, concentration of viral inoculum, duration of effector and target cell contact. It is suggested that target cell lysis was a form of natural cell-mediated cytotoxicity mediated by a cell which has different characteristics from the typical human and murine NK cell.     

Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus.

Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.     

Characterization of porcine xenoreactive cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) against mouse P815 cells were detected after stimulation of porcine peripheral blood mononuclear cells (PBMC) with irradiated Balb/c splenocytes. In vivo priming prior to in vitro stimulation slightly enhanced CTL activity, but lysis of targets was undetectable from lymphocytes from non-immune or immune animals that were not cultured with mouse splenocytes. After primary culture with Balb/c (H-2d) splenocytes, specific killing of P815 (H-2d) targets and not L929 (H-2k) targets indicated that recognition was specific for the H-2 locus. Similarly, CTL primed by mouse cells from either of two congenic strains recognized targets with alleles homologous to the stimulating cells. The anti-murine CTL was confirmed to be a CD8+ T cell based on studies using specific monoclonal antibodies to the porcine CD4 or CD8 cells. The cells responsible for the cytotoxicity of P815 targets lacked the characteristics of non-specific NK cells because (1) naive PBMC were unable to lyse NK targets (K562 cells) during the 4 h cytotoxic assay and (2) CTL killing of P815 targets increased with time after primary stimulation, whereas killing of K562 cells remained low at all times. These results suggest that porcine CTL can be readily generated against the xenogeneic mouse major histocompatibility complex.     

不同感染量REV对鸡免疫反应和细胞毒性作用的影响

用不同剂量网状内皮增生症病毒（REV）感染肉鸡和SPF鸡后，检测血液中T淋巴细胞对ConA的反应和NK细胞、细胞毒T细胞（CTL）的细胞毒性作用以及NDV抗体生成变化等，观察REV感染对机体非特异性、特异性细胞免疫反应和体液免疫反应的影响。结果表明无论高剂量还是低剂量REV感染均造成体液免疫和非特异性细胞免疫抑制，而且高剂量比低剂量对免疫功能的抑制作用更强，但对NK细胞和细胞毒T细胞的细胞杀伤活性却有升高趋势，在抗肿瘤方面发挥一定的作用。这种结果说明REV感染对机体的免疫抑制是有选择性的，且抑制程度与感染病毒量有关。     

Interleukin-2 corrects defective NK activity of patients with leukemia

NK cells of patients with leukemia display low cytotoxic potential. Since the NK cells have been suggested to play a role in natural resistance to leukemia, we considered it of importance to investigate the approaches leading to the correction of NK defect of leukemic patients. Our studies demonstrate that culture of effector cells with interleukin-2 (IL-2) resulted in restoration of cytotoxic defect. This was indicated by normalization of tumor-binding as well as lytic NK activity, by normal frequency of cytotoxic cells and their ability to recycle. The NK cell nature of cytotoxic cells was shown by abrogation or depletion of cytotoxicity by antibody directed against NK cell-associated, but not T cell-associated antigen. The generation of NK cell activity against fresh leukemic cells suggests that adoptive transfer of IL-2 activated NK cells may be a new approach to leukemic treatment.     

重组鸡白细胞介素18(rChIL-18)对MDV感染SPF鸡细胞免疫的影响

用马立克氏病病毒（MDV）感染5日龄SPF鸡后,取21日龄和35日龄鸡的淋巴细胞,运用3H-TdR掺入法检测T淋巴细胞对ConA和重组鸡白细胞介素18（rChIL-18）的反应,并用MTT法检测NK细胞和CTL对MD肿瘤细胞系CU147的杀伤活性及rChIL-18和IFN-γ对它们的作用。结果显示,SPF鸡感染MDV后淋转水平显著下降,NK细胞、CTL杀伤活性在感染后21日龄时升高,而在35日龄时NK细胞杀伤活性显著下降。rChIL-18对对照组和感染组SPF鸡的淋转水平和杀伤活性均有提高作用,同样IFN-γ也具有提高NK细胞和CTL杀伤活性的作用。     

Natural immunity against ovarian tumors

We have analysed natural killer (NK) cytotoxic activity in peripheral blood and ascitic fluids of patients with advanced stage of ovarian epithelial carcinoma. All patients displayed low NK activity in peripheral blood and virtually no cytotoxicity in ascitic fluids. NK activity in ascitic fluids could be substantially augmented after regional administration of virus-modified tumor cell extracts (VMTE), and that in peripheral blood after culture of effector cells with interleukin-2 (IL-2) in vitro. Activated NK cells displayed cytotoxic activity against NK-sensitive and NK-resistant tumor cell lines as well as against fresh ovarian tumors. Parallelism was found between regional NK augmentation and regression of malignant ascites. The latter observation suggests possible NK cell role in defense against ovarian tumors.     

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.     

Morphological observation of canine natural killer cells mediated cytotoxicity.

The cytotoxic effects of canine NK cells on CL-1 target cells were examined by scanning electron microscopy (SEM). NK cell mediated cytotoxicity on CL-1 target cells was detected by 51Cr release assay. SEM showed that a canine NK cell extended projections to the CL-1 target cell. Furthermore, the surface of CL-1 target cells changed a mesh-like structure. Therefore, the cytotoxic effects of canine NK cells on CL-1 target cells were morphologically demonstrated.     

Flow cytometric analysis of chicken NK activity and its use on the effect of restraint stress

Immune system is organized by the influence of both neural and endocrine systems. NK activity plays an important role in the innate immunity. In this study, we observed the effects of restraint stress on chicken peripheral blood NK activity. Viability of FITC-labeled RP9 was measured with PI after treatment with the effector cells. Chicken peripheral blood CD8alpha+ cells expressed strong cytotoxic activity, in contrast to thrombocytes, while peripheral blood CD3+ CD8alpha+ cells and CD4+ cells had little cytotoxic activity. Con A supernatant enhanced the cytotoxic activity of CD8alpha+ cells. Therefore, it is considered that these cytotoxic activities measured by flow cytometry (FCM) analysis are NK activity. When chickens were exposed to restraint stress, the levels of serum corticosterone increased transiently over a short period of time while the NK activity decreased. The decreased NK activity, however, did not recover to the intact levels for a long time, even once the serum corticosterone levels had recovered. These data indicate that chicken NK activity is able to be measured by flow cytometric analysis and that restraint stress causes severe damage to the chicken NK activity.     

In vitro parameters for understanding the immunocompetence of chickens of different lineage. 1. The cytotoxicity of avian whole blood against Saccharomyces cells

A cytotoxicity test was performed as a parameter for the nonspecific defense capacity in chickens using blood and saccharomyces cells (SCT). The test has the special merit of frequent repetitions of the results by two parallel test arrangements. The results of individual animals were not altered during a period of 14 days, as well as relatively not being affected by actual day fluctuation as long as environmental changes or antigenic changes do not appear. The cytotoxic activity is due to cellular defense mechanisms, especially concerning the activity of granulocytes, thrombocytes and monocytes but not lymphocytes and erythrocytes. The lysozyme contents of plasma show a very space cytotoxic activity against saccharomyces cells.     

Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41

Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-β mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-β compared to uninfected birds.