Role of lymph-borne cells in the early stages of scrapie agent dissemination from the skin

Scrapie is a natural transmissible spongiform encephalopathy (TSE) of sheep, infecting the animal via the gastrointestinal tract or the skin. This project tested the hypotheses that lymph-borne cells (especially dendritic cells) are crucial for the systemic dissemination of the infectious agent from the site of infection in the skin, that PrP genotype affects PrPSC association with dendritic cells and that PrPSC carriage by cells affects their expression of cytokines. Skin, of scrapie-susceptible VRQ/ARR and scrapie-resistant ARR/ARR PrP genotypes, was scarified with FITC-labelled PrPSC. Pseudoafferent lymphatic cannulation was then used to monitor the presence of FITC-PrPSC over time in different lymph cell populations and plasma in the draining afferent lymphatics. The major observation was that PrPSC did not associate significantly with any lymphocyte or dendritic cell population in the 5 days following PrPSC scarification. The only cells seen to associate with PrPSC were neutrophils. Furthermore, despite the quantity of PrPSC used for scarification being equivalent to a standard infectious dose (the VRQ/ARR sheep dying at approximately 260 days) the only PrP found in afferent lymph during the 0-5-day period was proteinase K sensitive (i.e. soluble PrPC). No differences were observed between the PrP genotypes. Analysis of the effects of PrPSC scarification of cellular cytokine mRNA expression (by a nuclease protection assay) showed raised levels of IL-1beta and IL-8 in the susceptible VRQ/ARR group and raised levels of IFNgamma in the resistant ARR/ARR animals.     

Steady state pig dendritic cells migrating in skin draining pseudo-afferent lymph are semi-mature

Dendritic cells (DC) in peripheral tissues are considered as immature cells that mature and migrate towards lymph nodes upon stimulation with pathogens. This commonly accepted paradigm is challenged by the fact that tolerance to peripheral self antigen is controlled by mature DC and that DC collected from afferent lymph draining different tissues from several species, in the absence of pathogen signaling, were inconsistently found to be either at a mature or semi-mature state. In order to better define the maturation state of DC that migrate in lymph in absence of pathogen stimulation, we compared skin lymph DC to resident and LPS (lipopolysaccharide)-activated skin DC thanks to the establishment of a mini-pig model of lymph duct cannulation. Based on their co-stimulatory molecules expression and endocytotic capacities, pig lymph skin DC were found at an intermediate state of maturation between resident and LPS-activated skin DC and were fully capable of allogeneic T cell stimulation. Furthermore, lymph skin DC could be further matured by LPS or influenza stimulation. Thus, using the pig skin model which is relevant to human, we show that skin-derived DC constantly migrate at an intermediate state of maturation that can be further enhanced upon appropriate stimulation.     

Antigen presenting cells in mucosal sites of veterinary species

The ability of antigen presenting cells, in particular dendritic cells, to integrate a variety of environmental signals, together with their ability to respond appropriately by initiating either tolerance or defensive immune responses make them cells of particular relevance and importance in the mucosal environment. They have been demonstrated in a variety of mucosal tissues in veterinary species and have been characterized to varying degrees, showing that fundamental immunological principles apply throughout all species, but also highlighting some species differences. A major advantage of carrying out immunological research in veterinary species is their size: it is possible to cannulate lymphatic ducts and obtain information about cell migration between different tissues. It is also possible to obtain pure populations of relatively rare cell types such as the plasmacytoid dendritic cells or mucosal dendritic cells ex vivo for the study of immune responses to diseases in their natural host and for other thorough functional studies. Two major myeloid antigen presenting cell (APC) (dendritic cells, DC) cell populations have been described in gut draining lymph and other mucosal sites in ruminants and pigs, characterised by the presence or absence of surface molecules, their enzyme profiles, their ability to phagocytose and their different potential as APC. There is evidence that one of these subsets has migrated from the diffuse mucosal tissue, where it is found as a phagocytic as well as stimulatory APC population, which in turn may be derived from blood macrophages. In addition, the presence and role in viral infection of the IFN-alpha producing plasmacytoid DC in mucosal tissue is discussed, based on studies in pigs.     

Dendritic cells in cattle: phenotype and function

Dendritic cells are professional antigen presenting cells derived from the bone marrow and distributed throughout body tissues where they are located in sites that are suitable for antigen uptake. They are central to the induction of immune responses in naive animals and thus have become targets in strategies that are aimed at modulating resistance to infection. Studies in cattle have shown that the dendritic cells are phenotypically heterogeneous and that the different phenotypes have different biological properties. The molecular basis for this variation has begun to be investigated and has led to the identification of a member of the SIRPalpha family of signal regulatory proteins (MyD1) on a subset of dendritic cells in afferent lymph. Uptake of antigen by cattle dendritic cells is by a number of mechanisms that can involve endocytosis via clathrin coated pits or via caveolae as well as macropinocytosis.     

ISVD‐1 Highlights of the skin immune system

Similar to intestine and pulmonary tissue, skin is in contact with the environment and hence, requires efficient immune surveillance. The concept of skin‐associated lymphoid tissue (SALT) was first introduced in 1978. The skin immune system (SIS) with its cellular and humoral components and the associated draining lymph nodes fulfills the three major functions of the innate and adaptive immune system: (1) induction of the primary immune response; (2) expression of immunity to previously encountered antigens (memory); and (3) avoidance of deleterious immune responses to nonthreatening antigens (tolerance). The cellular components of the SIS include dendritic antigen‐presenting cells (Langerhans cells and dermal dendritic cells), skin‐homing αβ‐T lymphocytes and γδ‐T lymphocytes, keratinocytes and their environment, the microvascular unit (endothelial cells of the dermal postcapillary venules), neural cells and dendritic neural processes, and the draining lymph node with its high endothelial venules. Humoral components of the SIS are defensins, complement components, immunoglobulins, cytokines, chemokines, fibrinolysin, eicosanoids and neuropeptides. Important characteristics and functions of these components and their interactions with each other will be discussed with regard to normal skin versus recruitment phase, retention phase and resolution phase associated with inflammatory conditions. The discussion will primarily focus on cutaneous dendritic antigen‐presenting cells, cutaneous lymphocytes and their homing into the skin, and keratinocytes. Moreover, the importance of the microvascular unit in relation to leukocyte recruitment and migration will be discussed.     

ISVD-2 Chytridiomycosis – an emerging and fatal infectious skin disease of amphibians

Similar to intestine and pulmonary tissue, skin is in contact with the environment and hence, requires efficient immune surveillance. The concept of skin-associated lymphoid tissue (SALT) was first introduced in 1978. The skin immune system (SIS) with its cellular and humoral components and the associated draining lymph nodes fulfills the three major functions of the innate and adaptive immune system: (1) induction of the primary immune response; (2) expression of immunity to previously encountered antigens (memory); and (3) avoidance of deleterious immune responses to nonthreatening antigens (tolerance). The cellular components of the SIS include dendritic antigen-presenting cells (Langerhans cells and dermal dendritic cells), skin-homing αβ-T lymphocytes and γδ-T lymphocytes, keratinocytes and their environment, the microvascular unit (endothelial cells of the dermal postcapillary venules), neural cells and dendritic neural processes, and the draining lymph node with its high endothelial venules. Humoral components of the SIS are defensins, complement components, immunoglobulins, cytokines, chemokines, fibrinolysin, eicosanoids and neuropeptides. Important characteristics and functions of these components and their interactions with each other will be discussed with regard to normal skin versus recruitment phase, retention phase and resolution phase associated with inflammatory conditions. The discussion will primarily focus on cutaneous dendritic antigen-presenting cells, cutaneous lymphocytes and their homing into the skin, and keratinocytes. Moreover, the importance of the microvascular unit in relation to leukocyte recruitment and migration will be discussed.     

Lymphocyte migration in the intestinal mucosa: entry, transit and emigration of lymphoid cells and the influence of antigen

Lymphocyte migration is important to transport immunological information between the different compartments of the intestinal immune system. Large numbers of lymphocytes emigrate from the Peyer's patches and reach the blood circulation after expansion and maturation within the mesenteric lymph nodes. So far the frequency of antigen specific lymphocytes emigrating from the Peyer's patches after oral stimulation is not known. After mesenteric lymph node resection those cells emigrating from the intestinal wall are accessible by calculating the major intestinal lymph duct. The first antigen specific cells draining from the intestines are obviously not lymphocytes but dendritic cells, thus the antigen is rapidly trapped in the parenchyma of the lymph nodes in vivo. When lymphocytes were taken from intestinal lymph, labeled in vitro and retransfused, marked numbers of B-cells were re-detected in intestinal lymph. Later preferentially T-cells recirculated through the gut wall. After immigration into the intestinal lamina propria the lymphocytes may enter the space between epithelial cells, where they are present as intraepithelial lymphocytes. Lymphoid cells in the S-phase of the cell cycle have been detected in all compartments of the intestinal wall. Apoptosis is probably a further important mechanism for the regulation of intestinal immunity in removing cells reacting against harmless dietary antigens to maintain oral tolerance.     

The perivascular spaces of ovine and canine brains

Abstract The specialised histology and anatomy of the central nervous system (CNS) is reflected in its pathology. This is particularly so for the perivenular/periarterial spaces in which inflammatory cells accumulate in inflammation. These spaces, although structurally not lymphatics, act as lymphatics draining fluid from brain to subarachnoid space and to lymphatics draining to deep cervical lymph nodes. Inflammatory cells may enter or leave the CNS by this route. In immune-based inflammation, they are colonised by subsets of immune cells allowing the processing of antigen, synthesis of antibody and development of cell mediated immune reactions.     

The perivascular spaces of ovine and canine brains

The specialised histology and anatomy of the central nervous system (CNS) is reflected in its pathology. This is particularly so for the perivenular/periarterial spaces in which inflammatory cells accumulate in inflammation. These spaces, although structurally not lymphatics, act as lymphatics draining fluid from brain to subarachnoid space and to lymphatics draining to deep cervical lymph nodes. Inflammatory cells may enter or leave the CNS by this route. In immune-based inflammation, they are colonised by subsets of immune cells allowing the processing of antigen, synthesis of antibody and development of cell mediated immune reactions.     

Patterns of cytokine gene expression of naïve and memory T lymphocytes in vivo

Large-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes via afferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferent lymphocytes have distinct phenotypes; afferent lymphocytes have a 'memory' phenotype, being CD62L(-)/CD45RA(-) and expressing high levels of CD2 and CD11a; efferent cells are largely 'na?ve', being CD62L(+)/CD45RA(+) with low levels of CD2 and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated in vitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can be stimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobe RNAase protection assay for 13 sheep cytokines (IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNgamma, TGFbeta and TNFalpha) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expression of cytokine mRNA in MACS-purified CD4+ and CD8+ T lymphocytes from both lymphoid compartments.     

The immune reactivity of the regional and distant lymph nodes to autochthonous ovine squamous cell carcinomata

Anti-tumor immune reactivity of lymphocytes derived from lymph nodes regional to and distant from tumor growth, as well as that of peripheral blood leucocytes, against autochthonous tumor cells, was investigated. Experiments were carried out in vitro using a 51Cr cytotoxic assay and in vivo by cannulating the afferent and efferent lymphatics of regional and distant lymph nodes and challenging via the afferent lymphatics with 10(7) cultivated autochthonous tumor cells. No anti-tumor cytotoxic reactivity was detected in vitro using lymphocytes derived from any of the sources studied. In vivo, while challenge with autochthonous tumor cells produced no response in the regional lymph node, significant blast cell response was obtained in the distant node. The response at the distant node was associated with the production of antibodies that could bind to tumor cells without causing their demise. The anergy observed at the regional lymph node, and the possibility of a relation between the events occurring at that node and those observed at the distant node, are discussed.     

Localized and disseminated histiocytic sarcoma of dendritic cell origin in dogs

Canine histiocytic proliferative disorders include a wide spectrum of diseases characterized by different biologic behaviors. The etiology and pathogenesis of these diseases are largely unknown. The clinicopathologic, morphologic and immunophenotypic characteristics of canine localized and disseminated histiocytic sarcoma were examined in 39 dogs. Rottweilers, Bernese Mountain Dogs, and retrievers were most commonly affected (79%). Localized histiocytic sarcomas (19 dogs) arose from a single site, and metastatic lesions were observed in draining lymph nodes. Predilection sites were subcutis and underlying tissues on extremities, but tumors occurred in other locations, including spleen, lung, brain, nasal cavity, and bone marrow. Disseminated histiocytic sarcomas (20 dogs), a multisystem disease previously described as malignant histiocytosis, primarily affected spleen, lungs, bone marrow, liver, and lymph nodes. Both localized and disseminated canine histiocytic sarcomas were composed of pleomorphic tumor cell populations. CD1+, CD4-, CD11c+, CD11d-, MHC II+, ICAM-1 +, Thy-1 +/- tumor cells were identified in all snap-frozen samples (31 dogs). This phenotype is characteristic for myeloid dendritic antigen-presenting cell lineage. Hence, canine localized and disseminated histiocytic sarcomas are likely myeloid dendritic cell sarcomas. Dendritic antigen-presenting cells are a heterogeneous cell population with regards to their ontogeny, phenotype, function, and localization. The exact sublineage of the proliferating dendritic antigen-presenting cells involved in canine histiocytic sarcomas remains to be determined. Phenotypic analysis of formalin-fixed tissues from eight dogs was limited by available markers. Morphologic features and the phenotype CD18+, CD3-, and CD79a- were the most useful criteria to indicate likely histiocytic origin.     

Total and Differential Leucocyte Counts and Lymphocyte Subpopulations in Lymph, Afferent and Efferent to the Supramammary Lymph Node, During Endotoxin-Induced Bovine Mastitis

Leucocyte trafficking in afferent and efferent mammary lymph and the supramammary lymph node in cows was examined during 4 h after intramammary infusion of endotoxin from Escherichia coli. Total and differential leucocyte counts were measured in milk, blood and lymph. The proportions of CD4(+), CD8(+), major histocompatibility complex (MHC) class II(+) and IgM(+) lymphocytes were examined in the lymph and lymph node. At post-infusion hour (PIH) 4, the flow rates of both lymph fluids had increased approximately eightfold. Total leucocyte concentration increased in afferent lymph, but decreased in efferent lymph. Neutrophils increased in afferent lymph at PIH 2 and in efferent lymph and milk at PIH 4. The predominant cell type in afferent lymph shifted from lymphocyte to neutrophil while lymphocyte was still at PIH 4 the predominant type in efferent lymph. Among the lymphocytes, B cells were predominant in afferent lymph and lymph node at PIH 4 while T cells, mainly CD4(+) cells, were predominant in efferent lymph both at PIH 0 and PIH 4. The CD4 : CD8 ratio was higher in efferent lymph and the challenged lymph node than in afferent lymph and the control node, respectively. There was a significant difference in proportions of each lymphocyte subpopulation except for IgM(+) cells, between afferent and efferent lymph after infusion. According to the results, there was already during the first hours of the immune response, a non-random trafficking of neutrophils and lymphocyte subpopulations resulting in a changed distribution of cells in afferent and efferent lymph and a difference in lymphocyte reactivity between the two lymph fluids.     

Inflammatory cytokines IL-6 and TNF-α regulate lymphocyte trafficking through the local lymph node

Lymphocyte trafficking from blood to lymph and back is a tightly regulated process. Given appropriate stimuli, trafficking of cells through the lymph node changes from a 'steady-state' to a bimodal flow. Initially, a 'shutdown' phase occurs, leading to a dramatic reduction in efferent cell output. This is followed by a 'recruitment' phase whereby the efferent cell output becomes greatly elevated before returning to baseline levels. The shutdown/recruitment process is hypothesised to promote encounters between Ag-specific lymphocytes and APCs in an environment conducive to immune response induction. Cytokines, such as TNF-α have been shown to play an important role in regulating lymphocyte trafficking. Here, we unravel the role of cytokines in the regulation of cell trafficking using an in vivo sheep lymphatic cannulation model whereby the prefemoral lymph nodes were cannulated and recombinant cytokines were injected subcutaneously into the draining area of the cannulated node. We demonstrate that local injection of purified IL-6 or TNF-α stimulates shutdown/recruitment in the draining lymph node. While the effect of IL-6 appears to be direct, TNF-α may mediate shutdown/recruitment through IL-6.     

Characterisation of immunosuppression in rabbits after infection with myxoma virus

Myxoma virus (MXV) causes the systemic disease myxomatosis in the European rabbit. Despite many in vitro studies on the function of MXV immunomodulatory proteins and detailed molecular knowledge of virus, little is known about the dynamics of interaction of the virus with the integrated host-immune system during infection. In this study changes in haematological profile, changes in lymphocyte subset distribution and non-specific proliferation activity of lymphocytes from different lymphoid compartments on the 2nd, 4th, 6th, 9th and 11th day after experimental infection of rabbits with MXV strain Lausanne was characterised. The relationship between alterations of immune parameters and dynamic of virus dissemination through the body was investigated. Haematological changes included moderate leucopenia with significant lymphopenia, neutrophilia, monocytosis and eosinopenia. A decrease of T cells including CD4+ and CD8+ and increase of CD79alpha+ were observed in draining popliteal lymph node 4 days after virus inoculation. From day 6, comparable changes were seen in collateral popliteal lymph node, spleen and peripheral blood. From day 9, the mentioned lymphocyte subsets tended to reach their original state in all of these lymphocyte compartments except draining popliteal lymph node. In thymus, MXV infection affected mainly CD4+CD8+ double positive thymocytes. On the other hand, proliferation activity of lymphocytes determined by the proliferation assay with plant-derived mitogens was significantly reduced from day 4 or 6 and remained reduced until the end of experiment in all observed lymphoid organs. Presence of MXV in respective lymphoid compartments preceded changes in lymphocyte subset distribution or lymphocyte activity.     

Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens.

The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the right udder with HKS at 1 month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral (draining) and contralateral (non-draining) suprammammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen-specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory cell migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, immunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals.     

Induction of T cell anergy by the treatment with IL-10-treated dendritic cells

In order to apply for reducing graft versus host disease in allogeneic stem cell transplantation, the study concerning the induction of specific T cell anergy was designed. Normal allogeneic lymphocytes, which were co-cultured with IL-10-treated immature dendritic cells in the first mixed leukocyte culture (MLC), were cultured with mature dendritic cells of the same origin as IL-10-treated immature dendritic cells in the second MLC. By co-culturing with IL-10-treated immature dendritic cells, the response of normal lymphocytes to mature dendritic cells cultured from the same individual as that of IL-10-treated dendritic cells was markedly reduced, compared with the lymphocytes cultured with non-treated dendritic cells or IL-10-treated dendritic cells from a third party individual. The present study demonstrated that antigen specific T cell anergy was generated by priming allogeneic lymphocytes with IL-10-treated immature dendritic cells. These data suggested the applicability of IL-10-treated recipient dendritic cells for the induction of recipient cell-specific donor T cell anergy in donor graft.     

鸡体内尿酸生物学功能的研究进展

尿酸是蛋白质以及核酸的代谢产物,在家禽体内含量较高。它是一种小分子抗氧化剂,能清除血浆和组织中 2/3 的自由基,尤其是活性较强的 OH-、ONOO-。家禽体内正常生理浓度的尿酸有效地防止了自由基对蛋白质、膜脂质以及 DNA的损伤,保护了动物细胞及组织,延长动物的寿命。同时,尿酸也是一种免疫增强物质,能促进树突状细胞成熟,增强其抗原呈递的作用。此外,在低盐营养水平下,高尿酸通过肾素依赖性机制维持动物的血压。因此,尿酸对鸡的健康具有重要意义。同时,了解尿酸的各种生物学功能,研究其作用机制,对于兽医工作者在临床、新药开发、疫苗研制过程中正确的利用尿酸也具有重要价值。     

Influence of betulinic acid on lymphocyte subsets and humoral immune response in mice

Betulinic acid is a pentacyclic triterpene found in many plant species, among others, in the bark of white birch Betula alba. Betulinic acid was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial, anticancer and anti-inflammatory activities. The effects of betulinic acid (50, 5, 0.5 mg/kg) administered orally five times at 24 hours intervals to non-immunized and red blood cells (SRBC)-immunized mice were determined. The present study examined the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes, and the percentage of subsets of T cells (CD4+CD8+, CD4CD8, CD4+, CD8+) in thymus,T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes, as well as white blood cell (WBC) and differential leukocyte counts in non-immunized mice, and humoral immune response in SRBC-immunized mice. SRBC was injected 24 hours after administration of the last dose of betulinic acid. It was found that betulinic acid administered orally five times at the dose of 0.5 mg/kg increased the total number of thymocytes, splenocytes, lymphocytes of mesenteric lymph node cells, and the weight ratio of the spleen and mesenteric lymph nodes in non-immunized mice. Betulinic acid also changed the percentage of T cell subsets in the thymus and T and B lymphocytes in peripheral lymphatic organs. The effects of betulinic acid on T and B cell subpopulations depended on the dose applied. The strongest stimulating effect of betulinic acid was observed when the drug was administered at the dose of 0.5 mg/kg. Five exposures to betulinic acid (0.5 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells with corresponding increases in the percentage and absolute count of mature, single-positive CD4+ thymocytes and decreased the percentage and total count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Multiple administration of betulinic acid at the investigated doses augmented the percentage and absolute count of CD19+ cells in the peripheral lymphatic organs. Moreover, betulinic acid at the dose of 5 mg/kg administered prior to SRBC immunization increased the number of plaque forming cells (PFC) but decreased the production of anti-SRBC antibodies on day 4 after priming. Thus, betulinic acid is a potential biological response modifier and may strengthen the immune response of its host.