Elizabeth A. Washington

Embryonic age influences the capacity for cytokine induction in chicken thymocytes

Thymocyte responses to functional activation are of relevance to the evaluation of the efficacy of in ovo immunotherapies and vaccines in chickens. In this study we have demonstrated differences in chicken thymocyte responses according to developmental age. RNA samples from stimulated and unstimulated chicken thymocytes were assayed for messenger RNA encoding the cytokines interleukin‐1β (IL‐1β), IL‐2, interferon‐α (IFN‐α), IFN‐β, IFN‐γ and transforming growth factor‐β4 (TGF‐β4), and also components of the major histocompatibility complex (MHC), β2‐microglobulin (β2M) and the MHC class I α‐chain (MHC IA). At embryonic day 14 thymocytes were least responsive to functional activation and differences existed even between thymocyte populations at embryonic day 18 and day 1 post‐hatch. The duration of proliferation in response to stimulation was found to increase with increasing embryonic age. Mitogen stimulation of embryonic day 18 and day 1 post‐hatch thymocytes induced up‐regulation of IFN‐γ, IL‐1β and TGF‐β transcripts, and down‐regulation of IFN‐α, IFN‐β and IL‐2 transcripts, with a higher induction of IFN‐γ, IL‐1β and TGF‐β transcripts in more immature T‐cell‐receptor‐negative (TCR−) than TCR+ (TCR1+, TCR2+, or TCR3+) subsets. In contrast, in the mouse and human, both mature and immature thymocytes respond to mitogen stimulation with up‐regulation of IL‐2. Thymocytes from embryonic day 14 chicks responded to mitogen with a short burst of unsustained proliferation, and transcriptional down‐regulation of the cytokines IL‐2, IL‐1β, IFN‐α, IFN‐β and IFN‐γ. These results suggest that embryonic day 14 thymocytes are largely unresponsive to mitogen. Transcripts encoding TGF‐β and type I interferons (IFN‐α and IFN‐β) were constitutively expressed at high levels in very early thymocytes at embryonic day 14. Thymocytes at embryonic days 14 and 18 and day 1 post‐hatch responded to mitogen stimulation with up‐regulation of MHC IA transcript. The pattern of β2M transcription following mitogen stimulation was distinct from that of the globally up‐regulated MHC IA transcript, with up‐regulation of β2M transcription observed at embryonic day 18 and day 1 post‐hatch but not at embryonic day 14. In thymocyte subsets, up‐regulation of β2M transcription was found to be specific to the CD8+ TCR+ population. The balance of responses in the embryonic thymus suggests that at all stages thymocytes have a reduced capacity for activation in comparison to mature thymocyte populations.

L-selectin expression on thymic emigrants defines two distinct tissue-migration pathways.

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 αβ and γδ subsets of recent thymic emigrants 24 hr after labelling. There were marked differences in the proportions of CD4+, CD8+ and γδ T‐cell receptor (TCR+) cells exported from the thymus to spleen compared to lymph nodes. Spleen was enriched in CD8+ and γδ 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 γδ 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.

Nonrandom migration of CD4+, CD8+, and γδ+T19+ lymphocyte subsets following in vivo stimulation with antigen

Abstract We report here the results of experiments in which the migration of three T cell subsets (CD4+, CD8+, and γδ+ T19+ cells) through antigen-stimulated lymph nodes and subcutaneous granulomas has been compared with that through normal skin and resting lymph nodes. The percentage of γδ+T19+ lymphocytes was halved and the percentage of CD8+ lymphocytes was doubled in lymph draining stimulated compared with control tissues, and all lymphocyte subsets except γδ+T19+ lymphocytes had higher hourly outputs in lymph draining antigen-stimulated compared with control tissues. Antigen also resulted in a higher percentage of CD8+ lymphoblasts and a lower percentage of γδ+T19+ lymphoblasts in efferent lymph draining antigen-stimulated lymph nodes. The data indicate that lymphocyte subsets leave the blood with differing efficiencies in different vascular beds and raise the possibility that antigen can influence the rate at which tissues extract individual T cell subsets from the blood.

Clinical and histopathological characterization of a large animal (ovine) model of collagen-induced arthritis.

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.

Effect of Mesenchymal Precursor Cells on the Systemic Inflammatory Response and Endothelial Dysfunction in an Ovine Model of Collagen-Induced Arthritis

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.

Endothelial Dysfunction in an Ovine Model of Collagen-Induced Arthritis

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 × 10-8M for arthritic sheep vs. 8.6 × 10-9M 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.

Regulation of T cell homeostasis during fetal and early postnatal life

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.

Changes in the distribution of αβ and γδ T cells in blood and in lymph nodes from fetal and postnatal lambs

We have examined the distribution of αβ and γδ T cells, together with B cells, in a range of lymph nodes and blood before and after birth. The proportions of blood-borne αβ and γδ T cells changed markedly during development with a wave of increasing numbers of blood-borne γδ T cells occurring in the fetus during the last month of gestation and early postnatal life. γδ 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 αβ 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 γδ 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.

STAGE-SPECIFIC ANTIGENS OF NEMATOSPIROIDES DUBIUS BAYLIS, 1926 (NEMATODA: HELIGMOSOMIDES)

Antigens were identified from Nematospiroides dubius recovered from outbred Quackenbush mice between 4 and 10 days postinoculation (PI). Parasite surface proteins were radioiodinated and extracts of the whole worms were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and reacted with normal and immune mouse sera followed by an avidin-biotin-peroxidase assay. Antigens ranged between 250,000 and 20,000 molecular weight (MW). A major surface antigen, 60,000 MW, which appeared to be a complex of different antigens, and a 250,000-MW internal antigen were found on fourth-stage (L4) and fifth-stage (L5) larvae 5-10 days PI but not earlier. A group of minor surface antigens (24,000-30,000 MW) were also expressed as larvae molted from L4 to L5, 6 and 7 days PI, but they differed from antigens of similar MW expressed by adult worms. An antigen, 45,000 MW, was detected in worms 5-10 days PI, but it was only expressed on the surface of L5 worms 9 and 10 days PI. We suggest that the antigen(s) common to adults and larvae may account for protective immunity.

LATE-TERM FETAL THYMECTOMY DOES NOT PREVENT THE DEVELOPMENT OF GUT-HOMING T CELLS AFTER BIRTH

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.