The cellular response of lambs to Brucella ovis before and after birth.

The immunological response of lambs to Brucella ovis before and after birth was investigated. The establishment of indwelling cannulas in the efferent prescapular lymphatic ducts of foetal lambs allowed continual monitoring of the immune response of a single lymph node. Foetal lambs in the last trimester of pregnancy were shown to mount a strong cell-mediated immune response to B. ovis. Lymphocytes from the challenged lymph node stimulated with B. ovis in vitro usually first reacted significantly and had highest [3H]-thymidine incorporation between 4 and 6 days after primary and secondary challenge, whereas, lymphocytes from the unchallenged node did not exhibit significant [3H]-thymidine incorporation until some 24 h later. Lymphocytes from these lambs challenged as foetuses still exhibited significant [3H]-thymidine incorporation in response to B. ovis for 4 to 5 months after birth. The proportion of surface immunoglobulin-positive cells in efferent prescapular lymph of unchallenged lambs ranged from 0.5 to 2.0% but after B. ovis challenge this proportion ranged from 2.7 to 8.7% between 4 to 6 days after challenge. By 9 to 12 days after challenge, the proportion had declined to pre-challenge values.

The immune response of the sheep popliteal lymph node to a purified phenoloxidase from larval cuticle of the sheep ectoparasite, Lucilia cuprina.

The popliteal lymph node of the sheep can mount a strong immune response to a phenoloxidase, enzyme A, purified from larval cuticle of a major sheep ectoparasite, the sheep blowfly, Lucilia cuprina. Continuous sampling from a cannulated efferent lymphatic allowed monitoring of changes in both cellular output (total cells and large blast cells) and specific antibody production (by ELISA) following primary and secondary challenge with antigen. Anti-enzyme A antibodies in lymph selectively precipitated enzyme A but not a second cuticular phenoloxidase, enzyme B, a finding that will prove useful in immunolocalization of the enzymes and in elucidating their origins. The implications for immunization of sheep against L. cuprina are discussed.

Immunoelectron microscopy of cell populations in regional and central lymph of sheep.

The immunoreactivity and the ultrastructural localization of monoclonal anti-sheep lymphocyte antibodies conjugated with colloidal gold particles were examined in free-floating cells of sheep central lymph from the thoracic duct, postnodal lymph draining either the popliteal nodes or the mesenteric nodes, and prenodal lymph draining the pregnant uterus. The monoclonal antibodies used in this study were SBU-T1 (CD5), SBU-T4 (CD4), SBU-T8 (CD8), SBU-II (anti DR antibody), and E53 which are reported to be sheep homologues of human T1, T4, T8, HLA-DR, and pan B cell antibodies, respectively. Colloidal gold particles were evenly distributed or segmentally aggregated on the surfaces of lymphocytes and macrophages incubated with monoclonal antibodies and in vesicles in the cytoplasm of anti DR antibody labeled macrophages. Not only did CD5 labeled cells show a high percentage in each regional lymph examined, but the percentage of CD4 labeled cells was consistently higher than that of CD8 labeled cells. Moreover, the immunoreactivity of CD8 labeled cells was specific among lymph from the different regions. The sum of the percentages of CD4 and CD8 labeled cells was less than the percentage of CD5 labeled cells, indicating the presence of a minor T cell subpopulation which was CD5+, CD4-, and CD8-. A characteristic finding was a high percentage of CD8 labeled cells and many abnormal eosinophils in uterine prenodal lymph in pregnant sheep. Taken together the results showed that variously labeled immunoreactive cells are distributed somewhat differently in lymph derived from different organ sites.

Reassortment of cell populations within the lymphoid apparatus of the sheep.

As lymphocytes recirculate through the blood tissues and lymph they are sorted into populations which have varying morphological and functional characteristics. Lymphocytes are added, deleted and transformed within the lymphoid apparatus as a consequence of non-random migration and antigenic stimulation. There is evidence that the physiological characteristics of peripheral and central lymph nodes vary as a result of differences in the origins of the cells entering the nodes. Lymphocytes enter the lymph nodes from the blood and lymph in varying numbers; consequently the cell population in the efferent lymph of central and peripheral lymyph nodes contains different proportions of blood-borne and lymph-borne cells. Cells arriving in lymph nodes by way of the blood or the lymph migrate differently within the node. Those entering from the blood go principally to the paracortex and the follicular areas. Lymphocytes entering in the lymph are distributed through both the cortex and the medulla. In humoral antibody responses and in the response that occurs during the rejection of a renal allograft, lymph-borne cells populate the medullary cords, cortex and germinal centres of the nodes they enter. Within these nodes, new populations of cells are generated which have different functional attributes from the cells which provoked their formation.

Transfer of carbonic anhydrase-positive particles from the follicle-associated epithelium to lymphocytes of Peyer's patches in foetal sheep and lambs.

Carbonic anhydrase cytochemistry of the ileal Peyer's patch in foetal and neonatal lambs has indicated secretion from the follicle-associated epithelium to the follicles. Reaction for carbonic anhydrase in the follicle-associated epithelium was found in the luminal plasma membrane, in cytoplasmic vesicles, and in vacuoles containing 50-nm membrane-bounded particles that seemed to be shed to the intercellular space. The lateral plasma membrane was negative for carbonic anhydrase, indicating that formation of carbonic anhydrase-positive particles was restricted to vacuoles. Administration of ferritin to ileal loops of sheep foetuses showed ferritin localized in vesicles and vacuoles of the follicle-associated epithelium followed by exocytosis, together with carbonic anhydrase-positive particles, into the indentations of the lateral cell border. The carbonic anhydrase-positive particles seemed to be transported to the centres of lymphoid follicles where many were attached to the plasma membrane of lymphocytes. Carbonic anhydrase-positive particles were also seen in vesicles and sometimes free in the cytoplasm of the lymphocytes or attached to their nuclear envelope. Light microscopically, carbonic anhydrase reactivity of the follicle-associated epithelium was associated with the early formation of the ileal Peyer's patch at about 100 days gestation. At this time the follicle-associated epithelium showed a strong luminal but at most a weak lateral staining. With further foetal development there was a progressive increase in the amount of carbonic anhydrase-positive reaction product in extracellular particles, both along the lateral cell borders of the follicle-associated epithelium and among the lymphocytes of the follicle centres.

The role of gut-associated lymphoid tissues in the generation of immunoglobulin-bearing lymphocytes in sheep.

Experiments were designed to examine the relative contributions of Peyer's patches and mesenteric lymph nodes to the population of circulating immunoglobulin-bearing lymphocytes in sheep. The ileum, with more than 90% of the total Peyer's patches, the mesenteric lymph nodes, or both, were removed from lambs at different stages of development and the composition of the cell populations in lymph from different sources and in the blood was examined. Lambs which had had the ileum removed before or within a few days of birth were deficient in small lymphocytes bearing membrane immunoglobulin. This deficit remained for at least the first year of the animals' lives. Neither the removal of mesenteric lymph nodes nor removal of the ileum had any statistically significant effect on the total output of cells or on the population of IgA-producing cells in lymph draining from the gut.

Response of leucocyte populations in the illeal Peyer's patch of fetal lambs treated with ferritin per os.

A combination of immunohistochemical techniques, a panel of monoclonal antibodies, and computer-assisted morphometric analysis was used to examine the response of the ileal Peyer's patch of fetal lambs 7 days after treatment with ferritin per os. Consistent with previous studies in fetal lambs that have reported the ileal Peyer's patch to be indifferent to antigen, the present study did not find any significant changes in the size of the predominantly B-cell dome/follicle compartment or the predominantly T-cell interfollicular area, nor were differences identified in the distribution of IgM-positive (+), CD4+, and CD8+ cells in these two compartments. However, both compartments showed a significant increase (p < 0.05) in the percentage of area occupied by MHC II+ cells and a significant decrease (p < 0.05) in the percentage of area occupied by CD44+ and B5+ cells. These changes show that the ileal Peyer's patch of fetal lambs is not indifferent to antigen and may represent the transition of a purely primary lymphoid organ to an organ that has both primary and secondary lymphoid functions.

The physical and functional heterogeneity of circulating lymphocyte populations.

There is a considerable heterogeneity amongst populations of both fixed and circulating lymphocytes other than that determined by the organs from which these cells originate. The functional differences with which these cells are endowed during their primary differentiation are further modified by the changing environments they encounter during their life and by reassortitive processes occurring during their migration. The range of inductive and modulating stimuli applied to the free-floating and fixed lymphoid cell populations in different parts of the lymphatic apparatus is changing continually and this leads to patterns of cell differentiation which persist for variable periods of time. The experience of a variety of environmental interactions gives the lymphocyte a range of reactive options which are denied to those cells held in fixed relationships with other cells. As a consquence there is no predictable equivalence in the morphological or functional attributes of different lymphocyte populations present in different parts of the lymphatic system.

The lymph-borne response of foetal lamb lymph nodes to challenge with Brucella abortus in utero.

The cannulated prescapular lymph node of the foetal lamb was challenged with killed Brucella abortus. Usually, both efferent prescapular ducts were cannulated and one node left as a control. Nodes were given primary or secondary challenges with doses of 10(9) - 2 X 10(10) brucella organisms and the lymph-borne response of the nodes followed. The foetal lymph node produced a vigorous cellular response to the injected brucella, restricted to the challenged side. The total cell output and, more strikingly, large cell output increased to reach a peak value 4-5 days after challenge. This cellular response was seemingly dose-dependent. The output of antibody after a primary challenge was delayed considerably and could not be detected in the lymph until well after the cellular response had subsided. The concentrations of antibody produced were quite low and almost all mercaptoethanol-sensitive. With secondary challenges the output of antibody occurred much more quickly, in phase with the cellular response, and appreciable amounts of mercaptoethanol-resistant antibody were produced. Under exceptional circumstances, high titre, highly specific anti-brucella antibody has been produced in foetal lymph. The foetal lymph node provides a powerful tool for further studies of the ontogeny of the immune response, and might help elucidate the failure of newborn animals to respond to some important bacterial antigens.

The long-term collection of lymph from single lymph nodes of foetal lambs in utero.

Surgical techniques are described for the long-term collection of lymph from both prescapular efferent lymph ducts of foetal lambs in utero. Lymph ducts in foetal lambs 95 to 136 days post-conception were cannulated with a high rate of success. Lymph flow from the cannulas was not compromised by deliberate primary or secondary challenge of the lymph nodes with a wide variety of antigens, and often continued for long periods after the lambs were born naturally. The techniques allow cellular and humoral lymph-borne immune responses of foetal lambs to authentic primary antigenic challenge to be studied in utero, uncomplicated by any previous experience of antigens. Loss of lymphocytes from the cannulated ducts depleted the numbers of cells in the lymph, but lymph protein concentrations were unaffected by lymph drainage.