Modulation of macrophages by infectious bursal disease virus.

Infectious bursal disease is one of the most important naturally occurring viral diseases of chickens worldwide. The causative agent, infectious bursal disease virus (IBDV), belongs to the family Birnaviridae. This virus causes an acute, highly contagious and immunosuppressive disease in chickens. The virus infects and destroys actively dividing IgM-bearing B cells. Although B cells are the principal targets for IBDV, recent data show that the virus also infects macrophages. IBDV-infected macrophages produce various cytokines and chemokines which may play an important role in the protection and/or pathogenesis of IBDV. In this review, the modulatory effects of IBDV on macrophages will be discussed.

In vivo activation of chicken macrophages by infectious bursal disease virus.

Infectious bursal disease virus (IBDV) infects and replicates in the dividing B lymphocytes of chickens. In the present study, the in vivo effect of IBDV infection on chicken macrophage populations and macrophage activation were examined. Specific-pathogen-free chickens were exposed to virulent IBDV and splenic macrophages were recovered during the acute phase of the disease. At 3 and 5 days post-infection (dpi), spleens of virus-exposed chickens had fewer macrophages than those of virus-free controls (p < 0.05). Confocal microscopic examination revealed cells that were positive for both KUL01 (macrophage surface marker) and R63 (IBDVVP2), indicating presence of the virus in macrophages. MQ-NCSU cells, an avian macrophage cell line, were susceptible to replication of IBDV. In addition, splenic macrophages were activated and had temporarily increased levels of mRNA transcripts of pro-inflammatory mediators, including IL-1beta, IL-6, IL-18, and iNOS. The robust expression of proinflammatory cytokine transcripts, along with a decrease in macrophage numbers, suggest that IBDV activates and may lead to a reduction of resident macrophages in vivo.

Presence of natural killer cells in specific-pathogen-free chickens.

Spleen cells of normal specific-pathogen-free N, P, and 15 X 7 chickens were cytotoxic in vitro for target cells of Marek's disease lymphoma line MSB-1. The natural killer (NK) cell activity, best expressed in chickens over 7 weeks old, varied among several genetic lines of chickens tested. The NK cells were thermolabile, and incubation of the cells at 37 degrees C for 30--60 minutes resulted in substantial loss of cytotoxicity. The specificity of NK effector cells was directed against common antigen(s) on tumor cell lines of diverse origin but not on normal adult or embryonic cells.

Detection of T-cell surface antigens in a Marek's disease lymphoblastoid cell line.

Indirect membrane immunofluorescence was applied in the study of a lymphoblastoid cell line derived from Marek's disease lymphoma. The cell line tacked surface immunoglobulins and reacted specifically with antisera prepared against chicken thymus cells. Cells transformed by Marek's disease virus were of thymus origin.

Cell-mediated cytotoxic response to cells bearing Marek's disease tumor-associated surface antigen in chickens infected with Marek's disease virus.

In a microcytotoxicity test in which 51Cr-labeled cells of a Marek's disease (MD) lymphoblastoid line (MSB-1 line) were used, cell-mediated cytotoxicity of spleen cell suspensions prepared from chickens inoculated with MD virus was demonstrated. This cytotoxic response, presumably directed against MD tumor-associated surface antigen, was detectable briefly after virus infection and paralleled the appearance of early lymphoproliferative lesions characteristic of MD.

Development of cell-mediated immunity to Marek's disease tumor cells in chickens inoculated with Marek's disease vaccines.

Chickens inoculated with herpesvirus of turkeys or with apathogenic or attenuated vaccine strains of Marek's disease virus (MDV) developed a T-cell-mediated immune response to Marek's disease (MD) tumor cells. This immune response was detected in a 4-hour 51Cr-release assay in which effector cells obtained from spleens of vaccinated chickens were reacted with 51Cr-labeled target cells of an MD lymphoblastoid cell line (MSB-1). The cytotoxic effector cells generated by the vaccine viruses had characteristics similar to those noted previously for anti-MSB-1 effector cells generated by MDV. The immune response was specific to MSB-1 cells, because another target cell line (TLT) antigenically unrelated to MSB-1 cells was not lysed by the effector cells nor did the unrelated target cells inhibit the cytotoxicity of effector cells against MSB-1 target in a cold-target inhibition assay. Because MSB-1 cells contain MD tumor-associated surface antigen, we postulated that the immune response detected in the vaccinated chickens may be directed against this antigen and that the antitumor antigen immunity may play a role in the mechanism of vaccine protection against lymphoma development by pathogenic MDV.

Reduced incidence of Marek's disease gross lymphomas in T-cell-depleted chickens.

Chickens of line 7, highly susceptible to Marek's disease (MD), were depleted of T-cells by neonatal thymectomy, total-body gamma-irradiation, and multiple injections with antithymocyte serum. In two replicate experiments, significantly fewer gross lymphomas were present in T-cell-depleted chickens than in intact or in T-cell-depleted, reconstituted hatchmates; these findings provided evidence that T-cells may be the principal target for MD virus (MDV) transformation, T-cell depletion was not complete, and the presence of microscopic lesions in T-cell-depleted chickens was attributed to residual T-cells. Ten lymphomas from intact chickens and 2 lymphomas from a T-cell-depleted chicken were examined for cellular composition. All lymphomas consisted predominantly of T-cells. The results of this and other published studies indicated that T-cells may have a dual role in MD; They may serve as a target for lymphoma formation by MDV and also may participate in immune surveillance against the disease in resistant chickens.

Characteristics of JMV Marek's disease tumor: a nonproductively infected transplantable cell lacking in rescuable Virus.

Cells of the JMV Marek's disease (MD) tumor, originally produced by rapid serial passage of MD lymphoma cells in chickens, were characterized to determine whether they were of host or donor origin and to ascertain certain virus-host cell interrelationships. Differences noted in blood group B surface alloantigens between tumor cells and host lymphocytes indicated a probable nonhost origin (i.e., transplantability) of the tumor. JMV spleen tumors contained predominantly large lymphoblasts bearing MD tumor-associated surface antigen. DNA from JMV tumor cell suspensions hybridized significantly with MD virus cRNA, which indicated that JMV cells contained at least a portion of the MD virus genome. No MD virus was rescued from JMV tumors by techniques suitable for rescue of virus from MD lymphomas. The JMV tumor cells were also devoid of MD virus-specific antigens. These properties differed markedly from those of MD lymphoma cells and make the JMV tumor cell a unique, potentially valuable, tool for further study of oncogenic herpesvirus infection and tumor immunity in the chicken.

The effect of B-cell immunosuppression on age-related resistance of chickens to Marek's disease.

Chickens were bursectomized by cyclophosphamide treatment at hatching. At 8 or 9 weeks of age, bursectomized and unbursectomized hatchmates, free from prior infection, were challenged with pathogenic Marek's disease virus. Oncogenicity of the virus inoculum was confirmed by inoculating 1-day-old susceptible chickens. At the time of virus challenge, blood cells from the cyclophosphamide-treated chickens were able to mount a vigorous graft-versus-host reaction in allogeneic embryos. This ability indicated that the thymus function was intact. There were no significant differences in Marek's disease response of bursectomized and unbursectomized chickens, in spite of a severe defect in the bursa-dependent functions in the bursectomized chickens. Some bursa-deficient chickens had non-proliferating, presumably regressing lesions in peripheral nerves. Because these lesions lacked plasma cells, it was concluded that the plasma cell may not play a functional role in recovery from Marek's disease.

In vivo and In vitro interferon induction in chickens by S -28828, an imidazoquinolinamine immunoenhancer.

Imiquimod and its analogs belonging to a class of imidazoquinolinamines, activate immune system via cytokine induction, and have antitumor and antiviral effects in mammals. In this study, we showed that a related analog, designated S-28828, induced interferon (IFN) and macrophage activating cytokine(s) (macrophage activating factor, MAF) in chickens in vivo, ex vivo, and in vitro. IFN and MAF were detectable in the serum of chickens following oral administration. Serum IFN levels were the highest at 2 h after treatment. Although there was no detectable IFN in sera of chickens at 8, 24, and 48 h after treatment, high levels of interferon inducible enzyme, 2'-5' oligoadenylate synthase (2'5'OAS) were present at these time points. In vitro and ex vivo studies showed that spleen cells, bone marrow (BM) cells, and peripheral blood leukocytes (PBL) were capable of producing IFN and MAF, although spleen cells produced the highest levels. Our results suggest that S-28828 administered orally may be a useful immunoenhancing and antiviral agent for chickens.

In vitro effects of recombinant chicken interferon-gamma on immune cells.

We used the recombinant chicken interferon-gamma (ChIFN-gamma) to determine its in vitro effects on chicken immune cells. We found that ChIFN-gamma induced nitric oxide (NO) production, upregulated Ia expression on the cell surface, and inhibited the replication of Newcastle disease virus in NCSU and HD11 cells (chicken macrophage cell lines). In addition, ChIFN-gamma had an antiproliferative effect on RP9 cells, a chicken B cell line. Finally, ChIFN-gamma inhibited mitogenic proliferation of normal chicken spleen cells and induced the cells to generate NO. Inhibition of viral replication and mitogenic proliferation of normal cells were correlated with NO production. We conclude that recombinant chicken ChIFN-gamma modulates chicken immune cells.

Nitric oxide inducing factor as a measure of antigen and mitogen-specific T cell responses in chickens.

We describe here an assay to measure responses of T cells to in vitro stimulation with antigens and a T cell mitogen (ConA). Spleen cells from chickens immunized with live viruses and an inactivated antigen produced macrophage activating factors (MAF) in response to in vitro stimulation with homologous antigens. The production of MAF, quantitated by the induction of NO in a retrovirus transformed macrophage cell line, HD11 (Beug et al., 1979, Cell 18, 375) was antigen-specific and correlated well with T cell proliferation. Further studies showed that production of MAF was abrogated by cyclosporin A, anti-CD4 and anti-CD8 monoclonal antibodies. These data suggested that production of MAF required T cell activation and can be used as measure of antigen and mitogen-specific T cell responses in chickens.

Molecular cloning and sequence analysis of the penton base genes of type II avian adenoviruses.

We describe here the identification of the penton base gene of hemorrhagic enteritis virus (HEV), a type II avian adenovirus, in a 2477-base pair (bp)-EcoRI fragment of the viral DNA by sequence analysis. Identification is based on an extensive amino acid homology between the HEV-open reading frame and the penton base of a fowl adenovirus (FAV-10) and various human adenoviruses. The 1344 bp-penton base gene of HEV encodes a 448-amino acid polypeptide of molecular weight of 50,843 Da. The nucleotide sequences of penton base genes of HEV and marble spleen disease virus (MSDV) are identical. The HEV penton base lacks the RGD motif, present in most human adenoviruses (Ad2, Ad3, Ad4, and Ad 12) suggesting that HEV may not use alpha v integrins to gain entry into host cells. Further sequence analysis revealed the presence of a Leu-Asp-Val (LDV) motif in the HEV penton base amino acid sequence similar to most of the human adenoviruses. LDV motif on the fibronectin has been shown to interact with the alpha 4 beta 1 integrins on cells, which includes lymphocytes and monocytes. The presence of LDV motif in the penton base of HEV implicates the involvement of alpha 4 beta 1 integrins in the viral internalization into host cells.

Immunopathogenesis of haemorrhagic enteritis virus (HEV) in turkeys.

Infection of turkeys with the haemorrhagic enteritis virus (HEV), a type II avian adenovirus, results in varying rates of morbidity and mortality. The disease is characterised by splenomegaly, intestinal haemorrhage, sudden death and immunosuppression. The mechanisms of HEV immunopathogenesis and immunosuppression are not fully understood. Recent studies indicate that immune responses play a central role in disease pathogenesis. HEV infects B cells and macrophages and induces necrosis as well as apoptosis in infected and possibly in by-stander cells. The ability of the infected birds to mount an optimum humoral immune response as well as normal macrophage functions such as phagocytosis may be impaired. Elevated numbers of splenic CD4(+) cells during the acute phase of infection may be associated with viral clearance. Types I and II interferons (IFN) and pro-inflammatory cytokines such as interleukin-6 and tumour necrosis-like factors (TNF) are released at the peak of the infection. Cytokines may play a protective as well as a destructive role. While a massive release of proinflammatory cytokines may lead to systemic shock associated with haemorrhagic enteritis and death, release of IFNs may protect turkeys from the disease. Treatment with thalidomide, which is a potent TNF down-regulatory drug, prevented HEV-induced intestinal haemorrhage and treatment with an IFN-inducing chemical prevented HEV-replication and inhibited HEV-induced pathological and histopathological lesions.

Immunological tolerance in chickens hatching from eggs injected with cell-associated herpesvirus of Turkey (HVT).

Cell-associated herpesvirus of turkeys (HVT) was inoculated in ovo at various stages of incubation. Chickens hatching from these eggs were tested for anti-HVT antibodies by several serologic procedures including enzyme-linked immunosorbent assay, indirect immunofluorescence assays, and western blot. Viremic chickens that remained free of detectable antibodies were considered tolerant to HVT. Chickens exposed to HVT at embryonation day 14 or earlier had 6-33% incidence of tolerance. Tolerant chickens developed persistent HVT viremia. A preliminary challenge experiment provided circumstantial evidence that tolerant to HVT may be associated with reduced resistance to virulent Marek's disease virus. Tolerance to HVT did not influence the ability of the chickens to produce antibodies against an extraneous antigen or respond to a T cell mitogen.

Blastogenic response of whole blood cells of turkeys to a T-cell mitogen.

Cellular immune mechanisms in the turkey are not well understood because adequately standardized, reproducible in vitro assays to measure cellular immunity are not available. Our purpose was to optimize conditions for a whole blood mitogenic assay that would facilitate quantitative assessment of the ability of circulating T cells of turkeys to respond to Con A. Heparinized peripheral blood from normal turkeys was examined. Data indicated that diluting the blood 1:20 or 1:40 and incubating the test cultures at 39 degrees C or 41 degrees C gave the best mitogenic stimulation. Presence of 7.5% turkey serum but not chicken or fetal bovine serum in the culture medium substantially enhanced the blastogenic response. Ontogeny of the whole blood mitogenic response was examined by repeat observations on a group of turkeys at various age levels starting at 1 week of age. Whole blood cells from 1-week-old turkeys responded poorly to Con A, although by 2 weeks of age, the response was well developed. Tests at subsequent ages revealed variable levels of activity. There was a considerable individual variation in the level of blastogenic response of turkeys within the same age group.

Studies on lymphocyte subpopulations and the effect of age on immune competence in turkeys.

We characterized the lymphocyte subpopulations and investigated the effect of age on cellular and humoral immunity, development of lymphoid organs, and the relative proportions of CD4+ and CD8+ cells in turkeys. The mitogenic responses of peripheral T cells were poorly developed at hatch but developed rapidly after hatch and reached adult levels by 2 weeks-of-age. The average percentage of CD4+ cells was 45, 29.8, and 26.3 in the thymi, peripheral blood, and spleens, respectively, in turkeys. The mean percentage of CD8+ cells in the thymi, peripheral blood, and spleens of turkeys was 53.8, 13.6, and 15.5, respectively. Age did not influence the relative proportions of CD4+ and CD8+ T cells in the spleens and peripheral blood of turkeys. The mean percentages of IgM+ cells in the bursae and spleens were 78.5 and 26.8, respectively. Day-old turkeys did not develop detectable antibodies to either thymus dependent or independent antigens. However, 2 week or older turkeys showed good humoral responses. Inoculation of BSA at hatch induced tolerance, whereas injection of SRBC did not. Analysis of relative organ weights of turkey lymphoid organs showed that spleens and thymi developed rapidly during the first week-of-age.

Bioactivities of a tumour necrosis-like factor released by chicken macrophages.

To test for tumour necrosis-like factor (TNF) of chickens, supernatants of a lipopolysaccharide (LPS)-stimulated chicken macrophage cell line MQ-NCSU were analysed. A sequence of ion-exchange and gel-permeation chromatography was utilised to isolate TNF-like activity from the culture supernatant. The peak of TNF-like cytotoxic activity corresponded to the fractions with a molecular weight of 81 kDa or higher. Polyclonal anti-human TNF-alpha antiserum cross-reacted by Western blotting with a 17 kDa protein in the TNF-containing fraction under denaturing conditions. This result indicated that chicken TNF-like factor in the biologically active form may be a protein multimer of monomers of about 17 kDa. The molecular weight of these monomers is similar to the molecular weight of mammalian TNF-alpha. Chicken TNF-like factor stimulated macrophages by inducing morphological changes, enhancing Ia-expression, nitric oxide (NO) production and by synergising with interferon (IFN)-gamma in the induction of NO release from macrophages. The biological activities were not neutralised by anti-human TNF antiserum. These data suggest that LPS-stimulated chicken macrophages produced a functional homologue to mammalian TNF-alpha. This may be structurally quite different from the mammalian TNF molecule. Other factors may have been co-purified with the chicken TNF-like factor having overlapping functions and molecular weight. However, co-purification of chemokines and interleukin-1, major macrophage derived factors, with the chicken TNF-like factor can be excluded based on the purification strategies.

Infectious bursal disease virus of chickens: pathogenesis and immunosuppression.

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus of chickens. The virus is ubiquitous and, under natural conditions, chickens acquire infection by the oral route. IgM+ cells serve as targets for the virus. The most extensive virus replication takes place in the bursa of Fabricius. The acute phase of the disease lasts for about 7-10 days. Within this phase, bursal follicles are depleted of B cells and the bursa becomes atrophic. Abundant viral antigen can be detected in the bursal follicles and other peripheral lymphoid organs such as the cecal tonsils and spleen. CD4(+) and CD8(+) T cells accumulate at and near the site of virus replication. The virus-induced bursal T cells are activated, exhibit upregulation of cytokine genes, proliferate in response to in vitro stimulation with IBDV and have suppressive properties. Chickens may die during the acute phase of the disease although IBDV induced mortality is highly variable and depends, among other factors, upon the virulence of the virus strain. Chickens that survive the acute disease clear the virus and recover from its pathologic effects. Bursal follicles are repopulated with IgM(+) B cells. Clinical and subclinical infection with IBDV may cause immunosuppression. Both humoral and cellular immune responses are compromised. Inhibition of the humoral immunity is attributed to the destruction of immunoglobulin-producing cells by the virus. Other mechanisms such as altered antigen-presenting and helper T cell functions may also be involved. Infection with IBDV causes a transient inhibition of the in vitro proliferative response of T cells to mitogens. This inhibition is mediated by macrophages which are activated in virus-exposed chickens and exhibit a marked enhancement of expression of a number of cytokine genes. We speculate that T cell cytokines such as interferon (IFN)-gamma may stimulate macrophages to produce nitric oxide (NO) and other cytokines with anti-proliferative activity. Additional studies are needed to identify the possible direct immunosuppressive effect of IBDV on T cells and their functions. Studies are also needed to examine effects of the virus on innate immunity. Earlier data indicate that the virus did not affect normal natural killer (NK) cell levels in chickens.

The inhibitory effect of the imidazoquinolinamine S-28828 on the pathogenesis of a type II adenovirus in turkeys.

In this study we show that a type I-IFN inducing compound, S-28828, modulated the pathogenesis of an avian type II adenovirus in turkeys. By itself, S-28828 induced a strong reaction in the spleen characterized by hyperplasia of the red and white pulps as well as an increase in lymphoid cell aggregations. Oral administration of S-28828 before the time of virus inoculation suppressed significantly (P<0.05) the replication of hemorrhagic enteritis virus (HEV) in turkeys. Two doses of 5 or 50 mg of S-28828 administered at 2 days before and at the day of virus inoculation inhibited HEV-induced pathological and histopathological lesions. Virus-induced apoptosis and reduced IgM-surface expression of B cells were suppressed by low dose S-28828 treatment. These results are of interest because mammalian adenoviruses were shown to be resistant to antiviral effects of type I IFN, the major effector cytokine induced by S-28828.