Search

Background and Aim: The development and use of experimental models using lymphatic cannulation techniques have been hampered by the lack of high-quality colour imaging of lymphatic vessels in situ. Most descriptions of lymphatic anatomy in sheep have historically depended on schematic diagrams due to limitations in the ability to publish colour images of the lymphatic vessels with decent resolution. The aim of this work was to encourage more widespread use of the ovine cannulation model by providing clear photographic images identifying the location and anatomical layout of some major lymphatic ducts and their in situ relationship to surrounding tissues., Methods: The cadavers of the sheep were collected after they had been euthanized at the end of animal trials not associated with this study. The lymphatics were dissected and exposed to show their appearance in the surrounding tissues and their relationship to other organs. Patent Blue was used to locate lymphatic vessels in exploratory preparations. However, in order to present the natural appearance of the vessels, we used minimal dissection and dye was not used for the photographed examples. Instead, we have indicated the course of the vessels with lines where their position is less clear., Results and Conclusion: In this paper, we have used sheep specimens as examples to show characteristic images of lymphatic vessels. The images of in situ lymphatics and lymph nodes combined with schematic summaries provide a concise illustration of the lymphatic drainage scheme in sheep., Competing Interests: The authors have declared that no competing interests exist.

Lymphatic cannulation models are useful tools for studying the immunobiology of the lymphatic system and the immunopathology of specific tissues in diseases. Sheep cannulations have been used extensively, as models for human physiology, fetal and neonatal development, human diseases, and for studies of ruminant pathobiology. The development of new and improved cannulation techniques in recent years has meant that difficult to access sites, such as mucosal associated tissues, are now more readily available to researchers. This review highlights the new approaches to cannulation and how these, in combination with advanced omics technologies, will direct future research using the sheep model., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of increasing prevalence marked by poor prognosis and limited treatment options. Ca 2+ -activated K Ca 3.1 potassium channels have been shown to play a key role in the aberrant activation and responses to injury in both epithelial cells and fibroblasts, both considered key drivers in the fibrotic process of IPF. Pharmacological inhibition of IPF-derived fibroblasts is able to somewhat prevent TGF-β- and basic fibroblast growth factor-dependent profibrotic responses. In the current study, we investigated whether blockade of the K Ca 3.1 ion channel in vivo with a selective inhibitor, Senicapoc, was able to attenuate both histological and physiological outcomes of early fibrosis in our large animal (sheep) model for pulmonary fibrosis. We also determined whether treatment was targeting the profibrotic activity of sheep lung fibroblasts. Senicapoc was administered in established fibrosis, at 2 weeks after bleomycin instillation, and drug efficacy was assessed 4 weeks after treatment. Treatment with Senicapoc improved pre-established bleomycin-induced changes compared with vehicle control, leading to improved lung compliance, reduced extracellular matrix and collagen deposition, and a reduction in both α-smooth muscle actin expression and proliferating cells, both in vivo and in vitro. These studies show that inhibiting the K Ca 3.1 ion channel is able to attenuate the early fibrogenic phase of bleomycin-dependent fibrosis and inhibits profibrotic behavior of primary sheep lung fibroblasts. This supports the previous research conducted in human IPF-derived fibroblasts and suggests that inhibiting K Ca 3.1 signaling may provide a novel therapeutic approach for IPF.

Background: The purpose of this study was to investigate the therapeutic efficacy of intravenously administered immunoselected STRO-3 + mesenchymal precursor cells (MPCs) on clinical scores, joint pathology and cytokine production in an ovine model of monoarthritis., Methods: Monoarthritis was established in 16 adult merino sheep by administration of bovine type II collagen into the left hock joint following initial sensitization to this antigen. After 24 h, sheep were administered either 150 million allogeneic ovine MPCs (n = 8) or saline (n = 8) intravenously (IV). Lameness, joint swelling and pain were monitored and blood samples for leukocytes and cytokine levels were collected at intervals following arthritis induction. Animals were necropsied 14 days after arthritis induction and gross and histopathological evaluations were undertaken on tissues from the arthritic (left) and contralateral (right) joints., Results: MPC-treated sheep demonstrated significantly reduced clinical signs of lameness, joint pain and swelling compared with saline controls. They also showed decreased cartilage erosions, synovial stromal cell activation and angiogenesis. This was accompanied by decreased infiltration of the synovial tissues by CD4 + lymphocytes and CD14 + monocytes/macrophages. Over the 3 days following joint arthropathy induction, the numbers of neutrophils circulating in the blood and plasma concentrations of activin A were significantly reduced in animals administered MPCs., Conclusions: The results of this study have demonstrated the capacity of IV-administered MPCs to mitigate the clinical signs and some of the inflammatory mediators responsible for joint tissue destruction in a large animal model of monoarthritis.

Background and Aim: Mesenchymal precursor cells (MPC) are reported to possess immunomodulatory properties that may prove beneficial in autoimmune and other inflammatory conditions. However, their mechanism of action is poorly understood. A collagen-induced arthritis model has been previously developed which demonstrates local joint inflammation and systemic inflammatory changes. These include not only increased levels of inflammatory markers, but also vascular endothelial cell dysfunction, characterised by reduced endothelium-dependent vasodilation. This study aimed to characterise the changes in systemic inflammatory markers and endothelial function following the intravenous administration of MPC, in the ovine model., Methods: Arthritis was induced in sixteen adult sheep by administration of bovine type II collagen into the hock joint following initial sensitisation. After 24h, sheep were administered either 150 million allogeneic ovine MPCs intravenously, or saline only. Fibrinogen and serum amyloid-A were measured in plasma to assess systemic inflammation, along with pro-inflammatory and anti-inflammatory cytokines. Animals were necropsied two weeks following arthritis induction. Coronary and digital arterial segments were mounted in a Mulvaney-Halpern wire myograph. The relaxant response to endothelium-dependent and endothelium-independent vasodilators was used to assess endothelial dysfunction., Results and Conclusion: Arthritic sheep treated with MPC demonstrated a marked spike in plasma IL-10, 24h following MPC administration. They also showed significantly reduced plasma levels of the inflammatory markers, fibrinogen and serum amyloid A, and increased HDL. Coronary arteries from RA sheep treated with MPCs demonstrated a significantly greater maximal relaxation to bradykinin when compared to untreated RA sheep (253.6 ± 17.1% of pre-contracted tone vs. 182.3 ± 27.3% in controls), and digital arteries also demonstrated greater endothelium-dependent vasodilation. This study demonstrated that MPCs given intravenously are able to attenuate systemic inflammatory changes associated with a monoarthritis, including the development of endothelial dysfunction.

Collagen induced arthritis (CIA) is the most studied and used rheumatoid arthritis (RA) model in animals, as it shares many pathological and immunological features of the human disease. The aim of this study was to characterize clinical and immunological aspects of the ovine CIA model, and develop lameness and histopathological scoring systems, in order to validate this model for use in therapeutic trials. Sheep were sensitized to bovine type II collagen (BCII), arthritis was induced by injection of bovine collagen type II into the hock joint and the response was followed for two weeks. Clinical signs of lameness and swelling were evident in all sheep and gross thickening of the synovium surrounding the tibiotarsal joint and erosion on the cartilage surface in the arthritic joints. Leucocyte cell counts were increased in synovial fluid and there was synovial hyperplasia, thickening of the intimal layer, inflammation and marked angiogenesis in the synovial tissue. There was a large influx of monocytes and lymphocytes into the synovial tissue, and increased expression of TNF-α and IL-1β in arthritic intima, angiogenesis and upregulation of VCAM-1. CIA in sheep appears to be an excellent large animal model of RA and has the potential for testing biological therapeutics for the treatment of rheumatoid arthritis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Rheumatoid arthritis (RA) induces systemic inflammation, producing a range of co-morbidities including cardiovascular disease. An early vascular change is endothelial dysfunction, characterized by reduced endothelium-dependent vasodilation. The aim of this study was to assess endothelial function in isolated coronary and digital arteries using an ovine model of collagen-induced RA., Methods: Sheep were culled following induction of arthritis, and their endothelial function was compared to that of normal sheep. Paired arterial segments were mounted in a wire myograph and dilated with endothelium-dependent vasodilators [bradykinin, serotonin, carbachol and adenosine diphosphate (ADP); linked to either Gi or Gq signalling pathways] and endothelium-independent dilators (adenosine and sodium nitroprusside) to construct cumulative concentration-response curves., Results: Coronary arteries from arthritic sheep exhibited a significantly greater EC50 value for bradykinin-induced relaxation compared to non-arthritic controls (2.9 × 10(-8) M for arthritic sheep vs. 8.6 × 10(-9) M for controls). Digital arteries from arthritic sheep also exhibited a significantly greater EC50 for relaxation to ADP and a significant decrease in the carbachol maximal response. Responses to sodium nitroprusside were unchanged in both coronary and digital arteries., Conclusion: Sheep with RA demonstrated attenuated arterial relaxation to endothelium-dependent vasodilators. This may provide a useful model of endothelial dysfunction in chronic inflammatory conditions. The dysfunction did not appear to be associated with one specific G-protein signalling pathway.

Background: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis., Methodology/principal Findings: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression., Conclusions/significance: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection.

Viral infections in chickens pose a major health threat to the poultry industry. Infectious bronchitis virus (IBV) usually causes respiratory disease; however, the disease severity is influenced by the genotype of the chicken and the IBV strain involved. Nephropathogenic strains of IBV, such as the Australian T strain, can cause high mortalities due to kidney failure characterized by mononuclear cell infiltration and inflammation. In a previous study, a line of specific pathogen-free chickens, the S-line, was shown to be susceptible to high mortalities from IBV infection. The cause of these high mortalities is unknown but it is suspected that differential cytokine expression may play a role. With this in mind, we decided to study the role of the proinflammatory cytokine interleukin (IL)-6 during infection to determine its contribution to nephritis and influence on disease susceptibility. To investigate this, we infected the susceptible S-line and the more disease-resilient HWL line with the T strain of IBV and measured their cytokine response levels. In both lines of birds, IL-6 mRNA levels were elevated in the kidneys at 4 d postinfection. However, in S-line chickens, these levels were 20 times higher than those in the HWL chickens. In addition, S-line birds also showed three times higher serum IL-6 levels than HWL birds after IBV infection. These findings suggest that IL-6 may play a role in IBV-induced nephritis and may open an avenue to develop alternative strategies, such as the use of antiinflammatory cytokines, to overcome the nephropathogenic effects of IBV.

In the fetus the peripheral T cell pool expands as the fetus grows, but the mechanisms that regulate T cell homeostasis during fetal life are unknown. Here, we show that the peripheral T cell pool in the sheep fetus is established by the export from the fetal thymus of twice as many CD8+ as CD4+ thymic emigrants every day. Clonal deletion of CD4+ thymocytes in the fetal thymus appeared to be more stringent than was the case for CD8+ thymocytes because only 1 in 35 single-positive CD4 (SPCD4) thymocytes was exported from the thymus whereas the majority (2/3) of the single-positive CD8 (SPCD8) thymocytes were exported from the fetal thymus each day. Furthermore, within the thymus, the number of apoptotic SPCD4 thymocytes was 40 times greater than the number of apoptotic SPCD8 thymocytes. A tissue-specific migration of CD8+ emigrants localizing in the spleen was also established in the fetus in contrast to CD4+ emigrants, which migrated randomly to spleen and LN.

The worldwide trend towards a reduced reliance on in-feed antibiotics has increased the pressure to develop alternative strategies to manage infectious diseases in poultry. With this in mind, there is a great emphasis on vaccine use and the enhancement of existing vaccines to provide long-term protection. Currently existing adjuvants for poultry can have deleterious side-effects, such as inflammation, resulting in the down-grading of meat quality and a subsequent reduction in profits. Therefore, to enhance the use of vaccination, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants in poultry is attracting considerable attention, and their potential role as such has been addressed by several studies. The recent identification of a number of chicken cytokine genes has provided the possibility to study their effectiveness in enhancing the immune response during infection and vaccination. This review focuses on the recent studies involving the assessment of cytokines as vaccine adjuvants.

Cytokines, as immune activators, have been investigated in mammalian systems as natural adjuvants and therapeutics. In particular, interleukin-2 (IL-2) has been studied widely as a vaccine adjuvant and immuno-enhancer because of its role in activating T cell proliferation. We show here that the first nonmammalian IL-2 gene cloned, chicken IL-2 (ChIL-2), exhibits similar biologic activities to those of mammalian IL-2. To assess the activities of ChIL-2 in vivo, we injected birds with recombinant ChIL-2 (rChIL-2) protein. rChIL-2 treatment induced peripheral blood lymphocytes to express cell surface IL-2 receptors (IL-2R) within 48 h and resulted in an increase in the proportion of peripheral blood CD4+ and CD8+ T cells. Using bromodeoxyuridine (BrdU) incorporation as a measurement of cell proliferation, we showed the increase in T cell populations to be due to cell proliferation. The ability of ChIL-2 to cause both activation and proliferation of T cells in vivo indicates that it has the potential to be used as an immune activator.

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

In this study the role of the thymus in the development of sessile T cell populations resident in spleen and lymph nodes (LN) was contrasted with the development of recirculating T cell populations trafficking between blood and lymph. Extensive analysis of the composition and the rate of growth of the secondary lymphoid tissues and recirculating lymphocyte pool coupled with neonatal thymectomy revealed that the sessile and recirculating T cell populations showed different degrees of thymic dependency and increased in size at different rates, suggesting these two populations might be under separate homeostatic control. Neonatal thymectomy also resulted in a much greater depletion of CD8+ and gammadelta TCR+ T cell subsets compared with CD4+ T cells in the sessile and recirculating T cell pools, and greatly reduced the number of T cells homing to peripheral lymph nodes compared with those homing to the gut.

A diverse repertoire among peripheral T cells is established in early life by thymic export when the naive T cell pool is first formed. In contrast, during adult life the thymus has been thought to play only a minor role in T cell homeostasis. As individuals age there is an increasing proportion of peripheral T cells bearing a memory phenotype, as well as a corresponding decrease in the number of naive T cells. The change in the composition of the peripheral T cell pool with age is thought to occur as a result of reduced or completely curtailed thymic export following thymic involution at puberty together with the antigen-driven expansion of naive T cells in the periphery. We examined thymic export throughout life in fetal, neonatal and aged sheep. We found that the thymus in adult animals showed an efficiency of production and export on a per gram basis equivalent to that observed for much younger animals, and continued to export substantial numbers of T cells long after puberty. The data demonstrate that naive T cells constantly enter the peripheral T cell pool at the same rate throughout fetal, neonatal and adult life, and that one in every 50 T cells in the peripheral lymphoid tissues of aged sheep had emigrated from the thymus in the previous 24 h. The data suggest that restoration by the thymus of a normal peripheral T cell repertoire in chronic T cell-depleting conditions should be possible in adult patients, provided the thymus is not damaged by disease or therapy.

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels. The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [(3)H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [(3)H] acetate. Perfusion of lymph nodes with [(3)H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24 h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19 h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4-5x10(5)D; heparan sulfate proteoglycan was 2.8x10(4) to 9.4x10(5)D; heparin 7.9x10(4)D and chondroitin sulfate 1.3x10(4)D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation. It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.

Before parturition the fetal lamb develops a large pool of long-lived recirculating T cells which provides a large population of naive T cells with a diverse TcR repertoire. After birth and concomitant with exposure to environment antigens, fetal T cells are rapidly replaced by short-lived cells formed postnatally. The majority of thymic emigrants homing to spleen in postnatal lambs are short-lived, in contrast to emigrants targeting lymph nodes where a population appears to be long-lived. The lifespan of thymic emigrants in the fetus is unknown as in the relative importance of antigen-driven processes versus developmental programming in regulating T cell homeostasis in early postnatal life.

We have studied the appearance and phenotype of recent thymic emigrants in blood, spleen and lymph nodes (LN) of neonatal lambs. Using in situ labelling of thymocytes with fluoroscein isothiocyanate (FITC), we examined the expression of the LN homing receptor L-selectin on alphabeta and gammadelta subsets of recent thymic emigrants 24 hr after labelling. There were marked differences in the proportions of CD4+, CD8+ and gammadelta T-cell receptor (TCR+) cells exported from the thymus to spleen compared to lymph nodes. Spleen was enriched in CD8+ and gammadelta TCR+ emigrants while LN were enriched in CD4+ emigrants. There were also marked differences in the expression of L-selectin by emigrants homing to spleen compared with those homing to lymph nodes. While the majority of thymic emigrants in LN expressed L-selectin, considerably fewer emigrants in spleen were L-selectin+. The presence of large numbers of CD8+ L-selectin- and gammadelta TCR+ L-selectin- thymic emigrants homing to spleen raises the possibility that unique homing receptor specificities underpin the migration of T cells to spleen as distinct from lymph nodes.

Studies on the ontogeny of the immune system in the sheep foetus, which remains immunologically naive until after birth, indicate that a large scale recirculation of T cells is just as much a feature of the foetal immune system as it is in animals after birth. An extensive recirculation of T cells and dendritic cells through peripheral tissues-including the gastrointestinal tract and skin-develops early in foetal life, although a population of gut-homing memory T cells does not develop until postnatal life. Current evidence suggests that two populations of thymic emigrants with distinct tissue-homing specificities to spleen and lymph node contribute to the development of the foetal peripheral T cell pool. CD8(+) thymic emigrants mostly target spleen while CD4(+) thymic emigrants home predominantly to lymph nodes. The lifespan of thymic emigrants is uncertain, although cells entering lymph are long-lived and form the basis of a diverse pre-immune repertoire of recirculating T cells. The life history and growth rates of non-recirculating T cells in spleen and lymph nodes during foetal life are at present unknown., (Copyright 1999 Academic Press.)

Lymphocyte recirculation is an essential element in the integration of immune responses and is an absolute requirement for the development of systemic memory in postnatal animals. During foetal life a large pool of recirculating T cells develops and migration pathways of naive T cells to skin and peripheral tissues as well as LN are established. At birth a process is triggered whereby naive fetal T cells are rapidly lost from the circulating pool and are replaced by newly arriving T cells which have been formed since birth. At present our data suggest that the thymic export in the fetus creates a pool of long-lived naive T cells of wide diversity. The situation in neonatal lambs is more complex since the thymus is exporting large numbers of short-lived thymic emigrants which enter a peripheral T-cell population where many T cells are dividing., (Copyright 1997 Academic Press Limited.)

Requirements for the activation and proliferation of gammadelta T cells were investigated. Maximum numbers of gammadelta T cells expressed the IL-2R alpha-chain after 6-h Con A stimulation in peripheral blood, efferent lymph, and afferent lymph. In comparison, IL-2R alpha-chain expression on CD4 T cells only reached maximum levels in response to Con A stimulation in peripheral blood and afferent lymph populations. Analysis of enriched gammadelta T cells demonstrated that Con A-induced expression of the IL-2R alpha-chain was independent of APC. Together, these data suggest that the requirements for gammadelta T cell activation are less stringent than those for alphabeta T cell activation. Unfractionated peripheral blood, efferent lymph, and afferent lymph cell populations proliferated in response to Con A alone. In contrast, enriched gammadelta T cells (CD4/CD8 depleted) from efferent lymph did not proliferate in response to Con A alone, but required the addition of IL-2. This requirement for exogenous IL-2 could be overcome by the addition of dendritic cells purified from afferent lymph. These results suggested that gammadelta T cells required costimulatory signals provided by APC to ensure the production of sufficient IL-2 to drive proliferation. CD28 and CTLA-4 mRNA were detected in efferent lymph and afferent lymph populations containing CD4 and CD8 T cells stimulated with Con A and IL-2 or with Con A alone, respectively. In contrast, negligible levels of these mRNA species were detected in efferent and afferent lymph populations devoid of CD4 and CD8 T cells. These results suggest that ovine gammadelta T cells may use alternative costimulatory pathways.

Lymphocyte recirculation is critical to maximize the efficiency of immunological surveillance and is an absolute requirement for the development of systemic memory. The consensus view of the lifespan of peripheral T cells holds that naive T cells are long-lived cells and most memory T cells are short-lived cells, although the question of the lifespan of peripheral T cells is not yet fully resolved. We have studied the lifespan of T cells circulating in efferent lymph draining lymph nodes (LN) in the immunologically naive sheep fetus and in postnatal lambs immediately following birth by examining the in vivo incorporation of [3H]thymidine by newly formed T cells during continuous administration of [3H]thymidine. We report that authentically naive fetal T cells are long-lived cells which continue to recirculate between blood and lymph during fetal life. At birth, however, a process is triggered whereby fetal T cells circulating through LN are rapidly lost from the peripheral T cell pool and are replaced by freshly arriving T cells which have been formed since birth. Our results indicate that by the end of the first week of postnatal life, around three-quarters of the T cells circulating through peripheral LN have been formed since birth.

Ruminant gamma delta T cells are concentrated at epithelial surfaces and share many features in common with species such as mice and humans which contain relatively fewer gamma delta T cells than ruminants. To date no gamma delta T cells with invariant TcR have been found in sheep and the generation of gamma delta TcR diversity which is thymic dependent follows a developmentally regulated sequence. Analysis of thymic export of gamma delta T cells shows that emigration of gamma delta T-cell subsets increases markedly during fetal life and after birth suggesting intrathymic processes leading to mature gamma delta T cells may change during development. Skin homing gamma delta T cells acquire their tissue tropism inside the thymus and pathways of recirculation of gamma delta T cells to skin are laid down during fetal development independent of antigen and remain stable through into adult life.

Apoptosis plays a crucial role in both the development and the control of the immune system. During T lymphocyte development, thymocytes undergo apoptosis as part of the process of elimination of self-reactive clones. Mature T cells also undergo apoptosis following antigen-stimulated proliferation as part of a mechanism that controls the immune response. Apoptosis also provides a defense mechanism against viruses whereby the rapid death of virus-infected cells reduces virus spread. Viruses, on the other hand, often express proteins that inhibit apoptosis of their host cells, thereby enhancing their infectivity. We have isolated a novel gene, ita (inhibitor of T cell apoptosis), which is a vertebrate homologue of the viral apoptosis inhibitor IAP. Expression of ita appears to be restricted to cells of the T lymphocyte lineage, and high levels of ita mRNA are induced within 4-8 hr of T cell activation. Immunohistologic studies show that medullary and cortical thymocytes express detectable levels of ITA. ITA is a 69 kDa protein that contains a C-terminal ring-finger motif that is found in several oncogenic proteins and N-terminal repeat elements that have only been reported in other apoptosis inhibitors. These findings suggest that ITA may play a role in controlling apoptosis in T cells.

Tissue-specific circulation of T cells is a critical element in the integration of systemic immune responses. Current models of T-cell migration suggest that homing specificities of T cells for tissues such as gut and skin are generated outside the thymus as a result of activation of virgin T cells by antigen in lymph nodes. We have used the sheep fetus (which is immunologically virgin and contains no memory or effector T-cell subsets) to examine the migration of 51Cr-labelled T cells in vivo. We report that gut-homing T cells are not present in the fetus and that gut-homing T cells from postnatal lambs home normally to fetal gut. Fetal thymectomy performed immediately prior to birth failed to prevent the development of gut-homing T cells in postnatal life. Gut-homing specificities on T cells are thus acquired extrathymically.

Current models of T cell migration place severe restrictions on the recirculation of virgin T cells, condemning them to migrate exclusively via high endothelial venules in lymph nodes until they either die or acquire the capacity to migrate to skin and peripheral tissues as memory cells following stimulation with antigen. We have demonstrated in the sheep fetus (which is immunologically virgin until after birth) that virgin T cells and dendritic cells circulate through skin and peripheral tissues during fetal life in the same non-random manner as adult T cells but in much larger numbers than they do in adult animals. Our data also showed that T cells do not discriminate between peripheral tissues and skin or lymph nodes on the basis of virgin or memory CD45R phenotype, or CD2, CD58 or CD44 phenotype, and with the possible exception of CD11a/CD18, that it is not mandatory for lymphocytes to be activated to adhesion moleculehi status in order to home to fetal skin. Our results indicate that unique tissue-homing specificities for extra-lymphoid tissues can be imprinted on virgin T cells independent of foreign antigen. Virgin T cells have previously been thought to be denied access to peripheral tissues; however, the large-scale traffic of virgin T cells through extra-lymphoid tissues in the fetus reported here provides a mechanism whereby direct virgin T cell interactions with self-antigens expressed only on tissues outside the thymus can occur repeatedly during development of the fetal immune system.

Current models of lymphocyte traffic suggest that homing specificities of T cells to tissues such as skin are generated outside the thymus as a result of activation of naive T cells by antigen in lymph nodes. Virgin T cells are thought to home to high endothelial venules in lymph nodes, but are thought to be unable to home to extra-lymphoid tissues such as skin. We used the technique of in situ labeling of the thymus with fluorescein isothiocyanate to examine the homing specificities of authentically naive T cells in vivo, immediately after their export from the thymus. We report that homing specificities for skin as well as lymph node are imprinted on T cells inside the thymus, independent of antigen. We also show that both alpha beta and gamma delta emigrant T cells exhibit homing patterns to skin and lymph nodes which are identical to those of mature T cells. Our findings demonstrate a key role for the thymus in the induction of skin-homing specificities on T cells indicating that skin-homing specificities of T cells are not generated solely outside the thymus as a result of the activation of virgin T cells by antigen. The migration of thymic emigrants to extra-lymphoid tissues within a few hours of leaving the thymus may have implications for mechanisms of peripheral self-tolerance. This pathway provides an opportunity for direct virgin T cell interactions with self components only expressed in the periphery at a time when emigrants may be more susceptible to tolerance induction than mature circulating T cells.

We have compared the expression of CD2, CD11a/CD18, CD44, and CD58 and alpha beta and gamma delta T cells emigrating from the fetal and postnatal thymus. We report that both gamma delta and the CD4+CD8- and CD4-CD8+ subsets of alpha beta T cells express mature levels of the adhesion molecules CD11a/CD18, CD44, and CD58 upon emigration from the thymus. Whereas CD44 is up-regulated on gamma delta + thymocytes prior to export, down-regulation of both CD11a/CD18 and CD58 occurs prior to emigration from the thymus, suggesting that down-regulation of these molecules may be a final maturational step taken by developing gamma delta T cells before their export from the thymus. In contrast, there is continued up-regulation of CD2 on gamma delta and alpha beta T cells upon emigration from the thymus and as they move into the mature peripheral T-cell pool. There was also a marked reduction in the number of CD2+ gamma delta T cells exported during fetal development that was associated with a marked reduction in the percentage of CD2+ gamma delta thymocytes exported. The postthymic maturation of CD2 and the other changes in adhesion-molecule expression appear to be independent of extrinsic antigen, as the same changes were observed in the antigen-free environment of the fetus as in the postnatal lamb, which has been exposed to extrinsic antigen. It thus appears that these changes in adhesion-molecule expression are as a result of the normal maturation pathway from a developing thymocyte to a mature peripheral T cell.

Tissue-selective streaming of T cells is considered to be a critical element in the integration of normal immune responses in intact animals. The results presented in this paper show that while there were major subsets of gut-homing T cells present in intestinal lymph, there were considerable differences in the tissue tropism of T cell populations circulating in lymph draining gut and peripheral lymph nodes. Thus, while CD4+ cells returned preferentially to their tissue of origin, gamma delta T cells showed a strong migratory preference for peripheral lymph nodes regardless of their tissue of origin. In contrast, although a population of gut-homing CD8+ cells was present in ileal lymph, CD8+ T cells from peripheral lymph nodes homed equally well to gut and lymph nodes. There were also considerable differences in the expression of L-selectin on T cells circulating in the two compartments. L-selectin was down-regulated on alpha beta T cells present in ileal lymph but not on gamma delta T cells which expressed the highest levels of L-selectin of all T cell subsets. It is suggested that gut-homing alpha beta T cells which have down-regulated L-selectin are formed in the gut-associated lymphoid tissues in response to gut antigens while the migratory properties of gamma delta T cells are ontogenetically determined, independent of antigen.

Sheep gamma delta T cells have been shown serologically to express T19, a membrane protein of 180-200 kDa which is a member of the scavenger receptor superfamily. Previous work from this laboratory resulted in the detection of a multigene family of T19-like genes in the sheep genome. In this study nucleotide sequences from several T19 genes were determined and are reported along with the corresponding segments of a number of expressed mRNA molecules. A segment of a single sheep T19-like gene was sequenced and these data, along with the corresponding sequences from cloned T19-like cDNA molecules from sheep and cow, were used to design an oligonucleotide primer system suitable for amplification of corresponding segments of many T19 genes and their cDNAs. Between 30 and 40% of cloned T19 genes were amenable to amplification using the selected primers, and sequence analysis of cloned PCR products confirmed that different T19 genes encode unique amino acid sequences. The expression of multiple T19 genes was established using cDNA molecules obtained from a single sample of sheep lymphocyte mRNA. The possible role of the T19 family of genes is discussed.

The thymus plays an essential role in the generation and selection of T cells and exports approximately 0.5-1% of thymocytes per day in young animals and considerably fewer in older animals. To date there have been no studies directly examining fetal thymic export in any species. Using the technique of intrathymic injection of fluorescein isothiocyanate, followed by an assay for green fluorescent cells in the periphery and for the expression of cell surface antigens on these cells, we have compared directly the export of T cells from the fetal and postnatal ovine thymus. While the thymus exports both alpha beta and gamma delta T cells, our results demonstrate that the proportion of thymic gamma delta T cells that are exported per day is much higher than that of thymic alpha beta T cells. Moreover, the export rate of gamma delta T cells increased from approximately 1 in every 60 gamma delta thymocytes per day emigrating from the fetal thymus to 1 in every 20 from the postnatal thymus. In addition, we identify a population of CD5+CD4-CD8-gamma delta-. T cells emigrating from the fetal thymus but greatly reduced among thymic emigrants after birth. These findings have several implications regarding the mechanisms and control of selection of both gamma delta and alpha beta T cells.

Following T cell activation with antigen or mitogens, there is an up-regulation of interleukin-2 receptor alpha (IL-2R alpha) chain expression. A high proportion of the IL-2R alpha chain is shed from the surface of the T cell in a soluble form following proteolytic cleavage, and thus determination of soluble IL-2R alpha (sIL-2R alpha) chain is an excellent measure of lymphocyte activation. A sandwich immunoassay for the detection of ovine sIL-2R alpha chain has been developed. Three monoclonal antibodies (mAbs) with specificity for the IL-2R alpha chain, demonstrated by immunoprecipitation of a 50 kDa protein from an ovine IL-2R alpha chain cDNA transfected Chinese hamster ovarian (CHO IL-2R) cell line, were analysed for additive and competitive binding to CHO IL-2R cells and Concanavalin A (Con A) activated ovine lymphocytes, respectively. Two non-competitive ovine IL-2R alpha chain specific mAbs were then used in a sandwich immunoassay to detect native sIL-2R alpha chain in the supernatant (SN) of Con A activated ovine lymphocytes and recombinant sIL-2R alpha chain in the SN of CHO IL-2R cells. Soluble IL-2R alpha chain could also be detected in complex biological fluid. In the efferent lymph of a cannulated ovine popliteal lymph node (LN), an increase in the level of sIL-2R alpha chain following local alloantigen LN activation was observed. This increase correlated with an increase in the output of activated T cell blasts.

We have used the technique of in situ intrathymic injection of fluorescein isothiocyanate to examine L-selectin expression on gamma delta and alpha beta T cells immediately after emigrating from the thymus of fetal and postnatal animals. We found that the percentage of L-selectin+ thymocytes exported per day decreased by half after birth and that the export of T cells from the thymus does not rely on expression of the peripheral lymph node homing receptor, L-selectin. Analysis of L-selectin on emigrant and mature T cell subsets revealed a remarkable heterogeneity of expression, both in terms of the numbers of cells expressing this molecule as well as the level of expression. gamma delta T cells, reportedly not having a propensity for homing to lymph nodes, not only contained the highest proportion of L-selectin+ cells, but also expressed far more of this molecule than either CD4+CD8- or CD4-CD8+ alpha beta T cells. Furthermore, those emigrant T cells expressing L-selectin are somewhat immature in their expression of this molecule. Subsequent maturation resulted in up-regulation of L-selectin on mature peripheral blood T cells, maturation that was clearly independent of extrinsic antigen. This antigen-independent post-thymic maturation appeared to occur as part of the normal progression from immature thymocyte to mature peripheral T cell in both fetal and postnatal animals.

We have compared the expression of CD45RA on alpha beta and gamma delta T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA+ and CD45RA- T cells. The number of gamma delta +CD45RA+ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5-8% of gamma delta thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to gamma delta T cells, up to one quarter of both fetal and postnatal alpha beta emigrants expressed CD45RA. Post-thymic maturation of CD45RA expression of alpha beta emigrants, which occurred both before and after birth, appeared to be antigen independent.

Levels of the N-terminal propeptide of type III collagen (PIIINP) in bronchoalveolar lavage fluid (BALF) are thought to reflect type III collagen production by the lungs, and increased levels have been reported in patients with pulmonary fibrosis. We wanted to know more about the relative proportions of these peptides in normal BALF, whether they altered in pulmonary fibrosis, and whether lymphoid tissue is capable of clearing PIIINPs. In this study, we used a radioimmunoassay which detects the different forms of PIIINP-related antigens with equal specificity, to measure PIIINPs in serum and BALF of patients with cryptogenic fibrosing alveolitis (CFA). To investigate why PIIINP profiles in BALF differed from serum, the absolute concentration and relative proportion of PIIINPs in lymph afferent and efferent to the popliteal lymph node of a sheep were also determined. PIIINP concentrations were significantly greater in serum and BALF of patients with CFA, compared with controls. Gel chromatography indicated that serum antigen distribution, both of patients and controls, contained approximately 20% Col 1-3; the remainder being Col 1. In contrast, BALF contained Col 1-3 and Col 1, together with an antigen of high molecular weight (> 150 kD). The relative proportion of each antigen varied quite widely, but there were no apparent differences between patients and controls. The concentration of PIIINPs in afferent lymph was 295 ng.ml-1 and in efferent lymph was 104 ng.ml-1. Gel chromatography demonstrated that a significant amount of Col 1-3, together with a high molecular weight peptide, had been cleared during passage through the node.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic half-life of hyaluronan (HA) in synovial fluid was estimated in sheep from the rate of appearance of 3H2O in plasma after injection of highly polymerized labeled HA. This material is substituted with 3H in its acetyl group and is rapidly and almost completely degraded in sheep and other species to yield 3H2O. Previously sensitized sheep were studied before and after induction of acute monoarticular arthritis by intraarticular challenge with type II collagen. In both circumstances 3H was released from the joint in a monophasic exponential pattern and appeared in plasma only as 3H2O. Before challenge, the mean metabolic half-life of [3H]HA was 20.8 hours (range, 15.8 to 27.9 hours, n = 5); an estimate in a single unsensitized sheep (27.0 hours) fell within this range. After challenge, swelling occurred around the joint without frankly increased synovial fluid. The mean half-life fell to 11.5 hours (range, 9.0 to 16.8 hours), with a corresponding increase in mean fractional turnover from 3.5%/h to 6.3%/h; an increased amount of the label was also retained within the peripheral tissues. It is concluded that a relatively mild acute inflammation can induce major changes in the metabolic turnover of synovial HA without the development of gross effusions. In the course of this study, mean synovial fluid volume in the normal sheep hock joint was estimated to be 1.54 mL; the concentration and content of HA were 0.54 mg/mL and 0.84 mg, respectively. These data add to other evidence that the volume and HA content of normal synovial fluid vary widely in different joints and species.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the distribution of alpha beta and gamma delta T cells, together with B cells, in a range of lymph nodes and blood before and after birth. The proportions of blood-borne alpha beta and gamma delta T cells changed markedly during development with a wave of increasing numbers of blood-borne gamma delta T cells occurring in the fetus during the last month of gestation and early postnatal life. gamma delta T cells constituted 18% of T cells in the blood of fetal lambs one month before birth and 60% of T cells in the blood of lambs one month after birth. There were also changes in the numbers of alpha beta T cells circulating through lymph nodes after birth. The proportion of CD4+ and CD8+ cells in mesenteric nodes increased substantially after birth, whereas that of gamma delta T cells decreased. Although B cells were present in much larger numbers in ileal lymph nodes in both the fetus and lamb, there was a large increase in the concentration of B cells in all lymph nodes in lambs after birth. In addition to the differences in the distribution of lymphocyte subsets circulating in fetal and postnatal lambs, markedly different growth patterns were also observed between lymph nodes before and after birth.

Type II native bovine collagen was used to induce arthritis in sheep. The sheep received a subcutaneous injection of collagen in Freund's adjuvant, and some sheep also received an intra-articular injection of collagen. The response to the collagen injections was monitored by measurement of antibody levels to bovine and ovine collagen in serum, synovial fluid and afferent lymph samples. There were antibodies in the normal sheep serum which reacted with native and denatured type II collagen, but there was a marked rise in serum and synovial fluid reactivity after subcutaneous injection of native collagen. In these sensitised sheep there was a further rise in antibody levels in the synovial fluid and afferent lymph samples after an intra-articular injection of collagen. There was little effect on antibody levels in the non-injected contralateral limb. Histological and immunohistochemical studies showed that the rise in antibody levels was associated with pathological changes to the synovium which were consistent with the development of arthritis. These changes were most severe in those joints of sensitized sheep which had received an intra-articular injection of collagen and were characterized by increased numbers of T cells in the synovial tissue, mainly of the CD4 phenotype. There was a marked increase in B cell expression by cells on the surface of the synovial intima, and accumulations of B and T cells were seen in the subsynovial tissues. Collagen-immune complex arthritis in sheep appears to be a useful large animal model of arthritis, with significant similarities to the arthropathies of other species, including man.(ABSTRACT TRUNCATED AT 250 WORDS)

Marked differences were found in the percentage and distribution of lymphocyte subsets (CD5, CD4, CD8, T19, CD45, and CD45R cells) in hemal nodes compared to those in mesenteric lymph nodes and blood. There were a greater number of T19 (gamma/delta)+ lymphocytes and fewer CD8+ lymphocytes in hemal nodes than in mesenteric nodes and a higher percentage of CD4+ in hemal and mesenteric lymph nodes than in blood. T19 (gamma/delta)+ lymphocytes were found throughout hemal nodes but were absent from the follicles. Of special interest was the finding that 20% of hemal node lymphocytes appeared not to express CD45 (leucocyte common antigen). According to these results, because of the unique distribution of lymphocyte subsets and the presence of germinal centers, hemal nodes may have a specialized role in the immune system.