Ross N. P. Cahill

The kinetics of hyaluronan in normal and acutely inflamed synovial joints: Observations with experimental arthritis in sheep

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)

Efficacy of DNA vaccination by different routes of immunisation in sheep.

DNA vaccination, delivered through various routes, has been used extensively in laboratory animals. Few studies have focused on veterinary species and while results obtained in laboratory animals can often be extrapolated to veterinary species this is not always the case. In this study we have compared the effect of the route of immunisation with DNA on the induction of immune responses and protection of sheep to challenge with live Corynebacterium pseudotuberculosis. Intramuscular injection of plasmid DNA encoding an inactivated form of the phospholipase D (PLD) antigen linked to CTLA4-Ig resulted in the induction of a strong memory response and sterile immunity following challenge in 45% of the animals. In contrast, gene gun delivery or subcutaneous (SC) injection of the DNA vaccine induced comparatively poor responses and insignificant levels of protection. Thus, DNA vaccine efficacy in sheep is strongly influenced by the route of vaccination. Amongst intramuscular vaccinates, protected sheep had significantly elevated IgG2 responses compared to unprotected animals, while both subgroups had equivalent IgG1 levels. This suggests that the presence of IgG2 antibodies and hence a Th1-like response, induced by the DNA vaccine gave rise to protective immunity against C. pseudotuberculosis.

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX

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.

The kinetics of antigen-reactive cells during lymphocyte recruitment

Abstract Lymphocyte recruitment, the increased traffic of lymphocytes from blood to lymph which occurs within antigenically stimulated lymph nodes, was monitored in the efferent lymph of single lymph nodes in sheep after immunization with allogeneic lymphocytes or purified protein derivative. Specific antigen-reactive cells were assayed by their ability to proliferate in vitro in the presence of the priming antigen. During lymphocyte recruitment such cells were no longer detected in the efferent lymph draining either the immunized node or a nonstimulated node remote from the region of antigen administration. These results probably reflect the selective removal of specific lymphocytes from the recirculating pool. Alternatively, the findings could involve a state of specific unresponsiveness of the cells.

Induction of lymphocyte recruitment in the absence of a detectable immune response.

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.

Behaviour of Sheep-Immunoglobulin-Bearing and Non-Immunoglobulin-Bearing Lymphocytes Isolated by Nylon Wool Columns

The technique of separating lymphocytes on nylon wool columns has been applied to sheep lymphocytes obtained from efferent lymph. The non-immunoglobulin-bearing (sIg-) lymphocytes which pass through the column were MLR-reactive, responsive only to T cell mitogens, and migrated in vivo like T lymphocytes described in rodents. Immunoglobulin-bearing (sIg+) lymphocytes were MLR-non-reactive, responsive to lipopolysaccharide and migrated in vivo like B lymphocytes in rodents. It is considered that the majority of sIg- lymphocytes obtained in this way are T lymphocytes and the majority of sIg+ lymphocytes are B lymphocytes.

Thymic export in aged sheep: a continuous role for the thymus throughout pre- and postnatal life.

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.

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.

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.