The avian lung-associated immune system

The lung is a major target organ for numerous viral and bacterial diseases of poultry. To control this constant threat birds have developed a highly organized lung-associated immune system. In this review the basic features of this system are described and their functional properties discussed. Most prominent in the avian lung is the bronchus-associated lymphoid tissue (BALT) which is located at the junctions between the primary bronchus and the caudal secondary bronchi. BALT nodules are absent in newly hatched birds, but gradually developed into the mature structures found from 6–8 weeks onwards. They are organized into distinct B and T cell areas, frequently comprise germinal centres and are covered by a characteristic follicle-associated epithelium. The interstitial tissue of the parabronchial walls harbours large numbers of tissue macrophages and lymphocytes which are scattered throughout tissue. A striking feature of the avian lung is the low number of macrophages on the respiratory surface under non-inflammatory conditions. Stimulation of the lung by live bacteria but not by a variety of bacterial products elicits a significant efflux of activated macrophages and, depending on the pathogen, of heterophils. In addition to the cellular components humoral defence mechanisms are found on the lung surface including secretory IgA. The compartmentalisation of the immune system in the avian lung into BALT and non BALTregions should be taken into account in studies on the host-pathogen interaction since these structures may have distinct functional properties during an immune response

NK and T cells constitute two major, functionally distinct intestinal epithelial lymphocyte subsets in the chicken

Non-mammalian NK cells have not been characterized in detail; however, their analysis is essential for the understanding of the NK cell receptor phylogeny. As a first step towards defining chicken NK cells, several tissues were screened for the presence of NK cells, phenotypically defined as CD8(+) cells lacking T- or B-lineage specific markers. By this criteria, approximately 30% of CD8(+) intestinal intraepithelial lymphocytes (IEL), but <1% of splenocytes or peripheral blood lymphocytes were defined as NK cells. These CD8(+)CD3(-) IEL were used for the generation of the 28-4 mAb, immunoprecipitating a 35-kDa glycoprotein with a 28-kDa protein core. The CD3 and 28-4 mAb were used to separate IEL into CD3(+) IEL T cells and 28-4(+) cells, both co-expressing the CD8 antigen. During ontogeny, 28-4(+) cells were abundant in the IEL and in the embryonic spleen, where two subsets could be distinguished according to their CD8 and c-kit expression. Most importantly, 28-4(+) IEL lysed NK-sensitive targets, whereas intestinal T cells did not have any spontaneous cytolytic activity. These results define two major, phenotypically and functionally distinct IEL subpopulations, and imply an important role of NK cells in the mucosal immune system

Synthesis of IFN-β by Virus-Infected Chicken Embryo Cells Demonstrated with Specific Antisera and a New Bioassay

Transcripts of interferon-α(IFN-α) and IFN-β genes are present in virus-infected chicken cells, but because of a lack of appropriate assays and reagents, it was unclear if biologically active IFN-β is secreted. We have established a nonviral bioassay for the sensitive detection of chicken IFN (ChIFN). This assay is based on a quail cell line that carries a luciferase gene that is controlled by the IFN-responsive chicken Mx promoter. Luciferase activity was strongly stimulated when the indicator cells were incubated with ChIFN-α, ChIFN-β, or ChIFN-γ but not with chicken interleukin-1β (ChIL-1β). Unlike the classic antiviral assay that preferentially detects ChIFN-α, the Mx-luciferase assay detected ChIFN-α and ChIFN-β with similar sensitivity. With the help of this novel assay and with rabbit antisera specific for either IFN-α or IFN-β, we analyzed the composition of IFN in supernatants of virus-infected chicken embryo cells. Virtually all IFN produced in response to Newcastle disease virus (NDV) was IFN-α. However, IFN produced in response to influenza A or vaccinia virus (VV) was a mixture of usually more than 80% IFN-α and up to 20% IFN-β. Thus, IFN-α and IFN-β both contribute to the cytokine activity in supernatants of virus-infected chicken cells. Furthermore, the infecting virus appears to determine the IFN subtype composition

Truncated Chicken Interleukin-1β with Increased Biologic Activity

Chicken interleukin-1β (ChIL-1β) is synthesized as a precursor molecule that unlike its mammalian counterpart, lacks a typical caspase-1 cleavage site. Therefore, it was unclear if proteolytic cleavage of ChIL-1β can occur and if cleavage might modulate the biologic activity of this cytokine. Using an avian indicator cell line that carries an NF-κB-regulated luciferase reporter gene, we established a sensitive and highly specific bioassay for ChIL-1β. Experiments with a rabbit antiserum indicated that the NF-κB-stimulating activity in supernatants of lipopolysaccharide (LPS)-treated chicken HD-11 macrophages is largely due to IL-1β and that proteolytic processing of natural and recombinant ChIL-1β is not very efficient. Functional analyses further revealed that cDNAs for either full-length or N-terminally truncated chicken ChIL-1β yielded active cytokine. A truncated molecule that closely resembled putative mature ChIL-1β exhibited more than 100-fold enhanced biologic activity after expression in mammalian cells, indicating that precursor cleavage is indeed of critical importance for maximal activity

Cytokines of Birds: Conserved Functions

Targeted disruptions of the mouse genes for cytokines, cytokine receptors, or components of cytokine signaling cascades convincingly revealed the important roles of these molecules in immunologic processes. Cytokines are used at present as drugs to fight chronic microbial infections and cancer in humans, and they are being evaluated as immune response modifiers to improve vaccines. Until recently, only a few avian cytokines have been characterized, and potential applications thus have remained limited to mammals. Classic approaches to identify cytokine genes in birds proved difficult because sequence conservation is generally low. As new technology and high throughput sequencing became available, this situation changed quickly. We review here recent work that led to the identification of genes for the avian homologs of interferon-a/b (IFNa/b) and IFN-g, various interleukins (IL), and several chemokines. From the initial data on the biochemical properties of these molecules, a picture is emerging that shows that avian and mammalian cytokines may perform similar tasks, although their primary structures in most cases are remarkably different

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease

Preliminary results on intersexual differences in gene expression of chemokine K203 in mononuclear cells of chicken

Sex steroid levels increase during sexual maturation and cause alterations in many physiological and morphological traits. Some of these changes may be connected with age-dependent and intersexual differences in the immune system. This topic is still insufficiently understood, especially in avian species, partially due to methodological limitations. In this study we measured the gene expression of proinflammatory cytokines (IL-1β, IL-6, IL-18) and chemokines [K60 (IL-8-like chicken chemokine — CXCLi1), CAF (IL-8-like chicken chemokine — CXCLi2), and K203] in mononuclear cells isolated from blood and spleen after in vitro stimulation with lipopolysaccharide (LPS). Samples were collected from chickens at two ages (from pullets before sexual maturity and from sexually mature egglaying hens). After LPS stimulation, a substantial increase was recorded in the gene expression of IL-6 and K203. All other measured genes were expressed at low levels in mononuclear cells irrespective of cell sources. We found a trend toward intersexual differences in K203 expression, but the expression of other cytokines and chemokines did not differ between the two sexes. The effect of stimulation was more pronounced in monocytes than in spleen macrophages, mainly in IL-6, IL-1β and K203 gene expression. Our findings represent a basis for further studies on the effects exerted by sexual hormones on the immune phenotype of birds

Chicken Toll-like Receptor 3 Recognizes Its Cognate Ligand When Ectopically Expressed in Human Cells

Recognition of pathogens by toll-like receptors (TLRs) causes activation of signaling cascades that trigger cytokine secretion and, ultimately, innate immunity. Genes encoding proteins with substantial homology to mammalian TLR1, TLR2, TLR3, TLR4, TLR5, and TLR7 are present in the chicken genome, whereas orthologs of TLR8, TLR9, and TLR10 seem to be defective or missing. Except for chicken TLR2 (ChTLR2), which was previously shown to recognize lipopeptides and lipopolysaccharides (LPS), the ligand specificity of ChTLRs had not been determined. We found that polyI:C, LPS, R848, S-28463, and ODN2006, which are specifically recognized by TLR3, TLR4, TLR7/8, and TLR9 in mammals, induced substantial amounts of type I interferon (IFN) and interleukin-6 (IL-6) in freshly prepared chicken splenocytes. To determine the ligand specificity of ChTLR3 and ChTLR7, we used a standard reporter assay frequently employed for analysis of mammalian TLRs. Neither S-28463 nor any other TLR ligand induced reporter activity in human 293 cells expressing ChTLR7. However, human 293 cells expressing ChTLR3 strongly and specifically responded to polyI:C, demonstrating that this chicken receptor represents a true ortholog of mammalian TLR3

IFNα and IFNγ Impede Marek’s Disease Progression

Marek’s disease virus (MDV) is an alphaherpesvirus that causes Marek’s disease, a malignant lymphoproliferative disease of domestic chickens. While MDV vaccines protect animals from clinical disease, they do not provide sterilizing immunity and allow field strains to circulate and evolve in vaccinated flocks. Therefore, there is a need for improved vaccines and for a better understanding of innate and adaptive immune responses against MDV infections. Interferons (IFNs) play important roles in the innate immune defenses against viruses and induce upregulation of a cellular antiviral state. In this report, we quantified the potent antiviral effect of IFNα and IFNγ against MDV infections in vitro. Moreover, we demonstrate that both cytokines can delay Marek’s disease onset and progression in vivo. Additionally, blocking of endogenous IFNα using a specific monoclonal antibody, in turn, accelerated disease. In summary, our data reveal the effects of IFNα and IFNγ on MDV infection and improve our understanding of innate immune responses against this oncogenic virus

Chicken BAFF

Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the chicken homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS). By searching a chicken EST database we identified two overlapping cDNA clones that code for the entire open reading frame of chicken BAFF (chBAFF), which contains a predicted transmembrane domain and a putative furin protease cleavage site like its mammalian counterparts. The amino acid identity between soluble chicken and human BAFF is 76%, considerably higher than for most other known cytokines. The chBAFF gene is most strongly expressed in the bursa of Fabricius. Soluble recombinant chBAFF produced by human 293T cells interacted with the mammalian cell-surface receptors TACI, BCMA and BAFF-R. It bound to chicken B cells, but not to other lymphocytes, and it promoted the survival of splenic chicken B cells in culture. Furthermore, bacterially expressed chBAFF induced the selective expansion of B cells in the spleen and cecal tonsils when administered to young chicks. Our results suggest that like its mammalian counterpart, chBAFF plays an important role in survival and/or proliferation of chicken B cells