Lymph Nodes Of Sheep Research Articles

Analysis of the CD1 cluster in sheep

The anti-CD1 monoclonal antibodies submitted to the 1st International Workshop on Leucocyte Differentiation Antigens of Cattle, Sheep and Goats were tested for their reactivity on sheep skin, thymus and lymph node and for their reactivity with sheep efferent and afferent lymph and peripheral blood. With the exception of 20–27 they all stained that same cell populations. The antibodies precipitated molecules with a heavy chain of 46 000 apparent molecular weight and a light chain of 14 000 apparent molecular weight. VPM5 and CC14 antigens were purified by affinity chromatography. All the antibodies cross-reacted with these molecules. The results show that 20–27 recognises the same molecules as the other antibodies and suggest that 20–27 is a pan CD1 monoclonal antibody and the other monoclonal antibodies are homologues of the human CD1b molecules.

Substance P increases and prolongs increased output of T4 (CD4) lymphocytes from lymph nodes of sheep in vivo: is it a mediator of immunological memory?

There are receptors on lymphocytes for substance P which are found both on small recirculating and on blast lymphocytes. The principal effect of substance P on lymphocytes appears to be a stimulating one, both in vitro and in vivo. The in vivo administration of substance P to sheep by acute infusion into cannulated afferent lymphatics of peripheral lymph nodes has been found to stimulate efferent lymph flow and the output into efferent lymph of both small recirculating and blast lymphocytes. We here report that substance P both enhances and prolongs the enhancement of the output of T4 (CD4) lymphocytes from lymph nodes of sheep in vivo. This output-stimulating effect appears to be specific to T4 (CD4) lymphocytes and is associated with a depressant effect on the output of T8 (CD8) and B lymphocytes. The output-stimulating effect on small T4 (CD4) lymphocytes is quite prolonged, lasting in excess of 96 h after a single 50 μg acute infusion. A brief post-infusion depression in T4 (CD4) lymphocyte output is associated with an equally brief, but marked, elevation in the output into efferent lymph of the arachidonic acid metabolite, thromboxane B2. The output-stimulating effect of substance P on blast T lymphocytes is confined to the T4 (CD4) blast lymphocytes. Substance P or a similar molecule may be of value when a specific T4 (CD4) lymphocyte output stimulant effect is desired. A single prior (6 days) acute infusion of substance P into a popliteal lymph node via its cannulated afferent lymphatic produced profound changes in the response to nodal drainage area immunization with killed S. muenchen bacteria. The latent period prior to increased antibody production was abolished, as was the standard post-immunization ‘shutdown’ period of decreased output of lymphocytes into efferent lymph. These changes were accompanied by a marked and progressive increase in antibody production. The findings reported here suggest substance P-induced long-term potentiation (LTP) of the immune response and raise the question of an involment of substance P as a major mediator of immunological memory.

Nerve‐mediated contractions of sheep mesenteric lymph node capsules.

1. Isometric tension was recorded in vitro from strips cut from the capsules of mesenteric lymph nodes of sheep. 2. One minute periods of field stimulation at frequencies of 1, 2, 4, 8 and 16 Hz (pulse duration, 0.3 ms) elicited tonic contractions of increasing force and duration. The stimulus frequency-response relationship began to flatten out at frequencies greater than 4 Hz, where the response was already 72% of that at 16 Hz. 3. The response to field stimulation was abolished by tetrodotoxin (1 microM). 4. Phentolamine, rauwolscine and prazosin (all 1 microM) reduced the response to field stimulation, while desipramine (1 microM), potentiated it. 5. Atropine (1 microM) was without effect on the response. 6. These results suggest that sheep mesenteric lymph node capsules have a noradrenergic innervation which modulates their tone via an action on alpha-adrenoceptors.

Extravasation of lymphocytes via paracortical venules in sheep lymph nodes: Visualization using an intracellular fluorescent label

In rodents and humans, lymphocytes extravasate into lymph nodes via specialized paracortical venules lined with high endothelium (HEV). Sheep and other ruminants do not have morphologically defined HEV in their lymph nodes. It has been assumed that lymphocyte extravasation in these species proceeds via analogous structures; i.e., paracortical venules lined with low to medium endothelium. In this study, lymphocyte suspensions were prepared from surgically excised lymph nodes of sheep and labeled with an intracellular fluorescent dye, H33342. Labeled cells were infused intravenously back into donors, and sheep were killed at various intervals after infusion. Frozen sections of lymph nodes were examined microscopically for the location of labeled cells. Ten minutes after infusion, labeled cells were seen in the lumen of venules located in the paracortical region of the nodes. At later time points, cells were seen apparently migrating through the venule walls and in the adjacent paracortical tissue. Similar experiments were performed in which H33342-labeled murine lymphocytes were infused into syngeneic mice. When equivalent cell numbers (based on animal size) were infused, no obvious differences were seen between location and kinetics of appearance of labeled cells in lymph nodes of sheep compared to those of mice. These results indicate that lymphocyte extravasation in sheep proceeds via paracortical venules in lymph nodes. The function of these venules appears to be analogous to HEV in nonruminant species.

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan

Afferent lymph vessels entering the popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan [HA; Mr of (0.85-1.2) x 10(5)] for up to 4 h at a rate of 17.4-23.1 micrograms/h. As much as 22.8 micrograms (99%) of infused [3H]HA was taken up by the node per h and degraded. During this interval it was observed that infused HA polymers of higher Mr were absorbed by the node to a greater degree than those of lower Mr. When proteoglycan monomer (PG; Mr 5 x 10(5); 400 micrograms of hexuronic acid/h) was infused concurrently with [3H]HA, the absolute amount of radioactivity appearing in efferent lymph (i.e. labelled material not absorbed by the node) increased, whereas the amount of labelled metabolites of low Mr was reduced considerably. During this period the Mr distribution of labelled HA in efferent outflow reverted to that of the infused material within 30-60 min. Our findings suggest that PG subunits and their chondroitin sulphate chains compete with HA for uptake into the peripheral lymph node of sheep. This indicates that PG, chondroitin sulphate and HA share the same pathway of elimination in this tissue, and is consistent with the view that the lymph node is involved in the metabolic turnover of normal intracellular matrix.

Characterization of sheep afferent lymph dendritic cells and their role in antigen carriage.

We have ablated peripheral lymph nodes in sheep and subsequently cannulated the pseudo-afferent lymphatic vessel that arises as a consequence of afferent lymphatic vessels reanastomosing with the former efferent duct. This technique allows the collection of lymph with a cellular composition that resembles true afferent fluid, and in particular, containing 1-10% dendritic cells. A 16-h collection of this lymph may contain between 10(6) and 10(7) dendritic cells. This dendritic cell population may be enriched to greater than 75% by a single-density gradient centrifugation step. We have generated a mAb that recognizes sheep CD1. This monoclonal not only reacts with afferent dendritic cells, but with dendritic cells in the skin and paracortical T cell areas of lymph nodes. The expression of CD1 suggests afferent dendritic cells are related to skin Langerhans' cells and other dendritic cells that act as accessory cells for T cell responses. Consistent with this is the high level of expression by dendritic cells of molecules involved in antigen recognition by T cells, including MHC class I and class II. Afferent dendritic cells express high levels of the cellular adhesion molecule LFA-3, and at the same time express a ligand for this molecule, namely CD2. The accessory functions of afferent dendritic cells resemble those displayed by mature Langerhans' cells and by lymph node interdigitating cells. These include clustering with resting T cells and stimulating their proliferation in a primary response to antigen. Afferent dendritic cells are capable of acquiring soluble protein antigen in vivo or in vitro and presenting the material directly to autologous T cells in an antigen-specific manner. We conclude that afferent dendritic cells represent a lymph-borne Langerhans' cell involved in antigen carriage to the lymph node.

Non‐random migration of cd4 and cd8 t cells: changes in the cd4: cd8 ratio and interleukin 2 responsiveness of efferent lymph cells following in vivo antigen challenge

In this investigation we have examined some of the cellular and molecular changes in efferent lymph that drains from an antigen-stimulated peripheral lymph node. Resting efferent lymph is characterized by a higher percentage of CD4+ cells and consequently, a higher CD4/CD8 ratio than peripheral blood. Following antigen stimulation of a cannulated peripheral lymph node in antigen-primed sheep, the percentage of CD4+ cells in efferent lymph increases above the resting level during days 1, 2 and 3 post antigen stimulation. This is followed on days, 3, 4, and 5 after antigen stimulation by an increase in the percentage of CD8+ cells above the resting level which occurs as the percentage of CD4+ cells returns to the resting level. These changes cause the CD4/CD8 ratio to first increase above the resting value during the CD4 phase and then decrease below the resting value during the CD8 phase. During the CD4 phase a lymphokine activity is present in cell-free lymph fluid. Lymph fluid collected at this time supports the proliferation of activated T cells. Supernatants generated from efferent cells collected at a similar time and cultured in vivo for 24 h without any further stimulation are capable of releasing this material. During the CD8 phase cells expressing functional interleukin(IL)2 receptors appear in lymph fluid. The data suggests a sequential exit of T cell subsets from an antigen-stimulated lymph node and that the appearance of IL2-like activity and IL2-responsive cells in efferent lymph fluid are temporally distinct events.

Blood vessels within lymph nodes: a comparison between pigs and sheep

In sheep, most blood vessels enter the lymph nodes at a discrete hilus, which is where the capsule overlies medullary tissue. In pigs, vessels within the node arise from an extensive network of arteries on the surface, and most vessels penetrate the capsule where it overlies diffuse tissue--the counterpart of the sheep medulla. In both species the blood vessels divide extensively as they approach and supply the dense lymphoid tissue which contains lymph nodules. The blood vessels within sheep lymph nodules are surrounded by a thick layer of amorphous material. The dense lymphoid tissue has an abundant network of venules which have smooth muscle cells in their walls. These venules are involved in lymphocyte migration and in pigs they are lined by high endothelium which is similar to that in most other species, while in sheep the endothelium is much lower. Fenestrated capillaries occur in both pig and sheep lymph nodes.

CD4+ lymphocytes are extracted from blood by peripheral lymph nodes at different rates from other T cell subsets and B cells.

The circulation of lymphocyte subsets through prescapular lymph nodes in sheep has been quantified using a panel of monoclonal antibodies against sheep lymphocyte surface antigens. Differences in the extraction of lymphocyte subsets from blood by the lymph node were found with CD4+ lymphocytes being extracted at a faster rate (1/2) than CD8+, SBU-T19+, major histocompatibility complex class II+ and B cells (1/4 to 1/5). In order to accommodate existing data on organ-specific adhesion molecules, one subset specific and one tissue specific, expressed on vascular endothelium could act jointly to regulate the migration of recirculating lymphocytes.

Uptake and degradation of hyaluronan in lymphatic tissue.

Afferent lymph vessels entering popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan of high Mr (4.3 x 10(6)-5.5 x 10(6)) and low Mr (1.5 x 10(5)). Analysis of efferent lymph and of residues in the nodes showed that hyaluronan presented by this route is taken up and degraded by lymphatic tissue. Labelled residues isolated in node extracts by gel chromatography and h.p.l.c. included N-acetylglucosamine, acetate, water and a fraction provisionally identified as N-acetylglucosamine 6-phosphate. Between 48 and 75% of the infused material was unrecovered, and had been presumably eliminated through the bloodstream as diffusible residues. Rates of degradation reached as high as 43 micrograms/h in a node of 2 g wt. infused with 56 micrograms/h. Some HA passed into efferent lymph and some was detected in the nodes, but fractions of Mr greater than 1 x 10(6) were not found in either. It is concluded that the amounts and Mr values of hyaluronan released from the tissues into peripheral lymph can be significantly underestimated by analysis of efferent lymph, i.e. lymph that has passed through lymph nodes. A substantial role in the normal metabolic turnover of at least one major constituent of intercellular matrix and connective tissue may now be added to the established functions of the lymphatic system.

Immune response of sheep to oral and subcutaneous administration of live aromatic-dependent mutant of Salmonella typhimurium (SL1479)

The major objective of the present study was to determine whether oral immunization with a live aromatic-dependent strain of Salmonella typhimurium (SL1479) was capable of stimulating an intestinal immune response in sheep similar to that induced by combined intraperitoneal injection followed by oral boosting. The results showed that repeated oral immunization was incapable of stimulating an anti-flagella antibody containing cell (ACC) response in the lamina propria of the intestine even though primary oral administration of 5×10 9 live SL1479 gave rise to an ACC response in intestinal lymph which was predominantly of the IgM isotype. ACC reached a peak 9–10 days after oral administration when ACC comprised 0.5–1% of total lymphocytes in lymph. An ACC response of similar isotope specificity also occurred in popliteal or prefemoral lymph of unprimed sheep following regional subcutaneous injection of SL1479. Oral administration of SL1479 to orally primed sheep did not reinvoke an ACC response in lymph although IgG 1-ACC were observed in medullary cords of mesenteric lymph nodes of sheep 6–8 days after the booster dose of SL1479. The results suggest that the protective immunity elicited by oral administration of SL1479 cannot be attributed to induction of a local intestinal antibody production.

Immunoglobulin isotype influences traffic of B lymphocytes through popliteal lymph nodes in sheep.

The antigen specificity of blast B lymphocytes was used to trace their passage from blood through antigenically naive popliteal lymph nodes in sheep. Ovine popliteal nodes selectively recruited lymphocytes containing IgG1 in preference to those containing IgG2. This selection appeared to operate at the level of the vascular endothelium. Stimulation of lymph nodes with killed S. aureus and dextran sulphate (DXS) (which promotes an antibody response in ovine lymph nodes biased in favour of IgG2) did not significantly alter this pattern.

Osseous choristoma of the ciliary body in guinea pigs.

ronts have many organisms. The multinucleate meronts of References the organisms in the calf are distinct from Toxoplasma and Besnoitia, because the latter divided by endodyogeny (division into two). No species of Isospora has been known to develop in tissues of ruminants (cattle, sheep, goat^).^ Thus, by exclusion, the organism may be Eimeria, although sexual stages were not seen. Lesions in the present study were considered to be due to the coccidium because the infection was localized in the small bile ducts and protozoa were seen in lesions. Moreover, the hepatobiliary lesions were similar to those caused by Eimeria hiepei in mink3 and Eimeria stiedae in rabbits.’ Intestinal coccidiosis due to Eimeria is among the most common of parasitic infections of cattle, sheep, and goats.5 Although species of Eimeria occasionally invade the gallbladder of goats’ and mesenteric lymph nodes of sheep and goats,6 none have been reported to do so in cattle. The parasite of cattle in the present case was similar to that in the gallbladder of a goat, but sexual stages were not observed.

Increased outputs of lymphocytes in lymph efferent from the lymph nodes of sheep during systemic arterial hypertension induced by phenylephrine or dopamine

Induced systemic arterial hypotension by intravenous nitroprusside administration and by acute arterial occlusion in sheep have been found to reduce lymphocyte traffic as mirrored in the output of lymphocytes into the efferent lymph of peripheral lymph nodes. In the present series of experiments in sheep with chronically cannulated efferent lymphatics of peripheral lymph nodes, induced and monitored systemic arterial hypertension with intravenous pump infusions of phenylephrine or dopamine both produced sharp increases in the output of lymphocytes into efferent lymph in all of 27 studies. The increases in lymphocyte output with dopamine were more sustained and less associated with evidence of lymphoid tissue damage than with phenylephrine. Phenylephrine infusions were attended by a high incidence of gross bleeding into the efferent lymph, of increased coagulability of efferent lymph in the absence of gross bleeding and of prolonged depression of lymphocyte outputs after the cessation of intravenous infusion.

Localized Deep Hyperthermia Increases the Traffic of Lymphocytes Through Peripheral Lymph Nodes of Sheep In Vivo

The production of localized deep hyperthermia by the radio frequency wave magnetic-loop induction method has been utilized to study the effects of deep hyperthermia on lymphocyte traffic in sheep in vivo. Deep hyperthermia has been applied both to primary peripheral lymph node drainage areas alone (popliteal and prefemoral) and directly over the study lymph node (popliteal). Deep tissue core temperatures were monitored in all studies and the findings were correlated with alterations in the levels of lymphocyte outputs into study node efferent lymph and the volume of efferent lymph flow. In all of 32 studies, there was a prompt and sharp increase in the output of lymphocytes into efferent lymph at 40-43 degrees C. Efferent lymph flow also was increased promptly but to a lesser degree than the output of lymphocytes. The two were not closely correlated. High deep temperature in the nodal area (45 degrees C) appeared to cause delayed nodal malfunction, which depressed lymphocyte output but did not affect lymph flow. Localized deep hyperthermia is an effective and noninvasive means for the local increase of lymphocyte traffic and lymph flow and may prove to be a useful means for the evaluation of the immunological consequences of altering both deep tissue temperature and lymphocyte traffic.

Effect of dextran sulphate on IgG subclass of antibody in efferent popliteal lymph of sheep

IgG1 and IgG2 participation in anti-hapten, anti-carrier and immunoglobulin-containing cell (Ig-cc) responses was studied in sheep immunised with killed Staphylococcus aureus-Dinitrophenyl (DNP) conjugates. The antigen was given with or without the polyanionic adjuvant dextran sulphate (DXS). Animals were immunised intracutaneously on the lateral hock of one hind leg with 1 X 10(9) S. aureus-DNP with or without 20 mg DXS. Six weeks later, primed sheep underwent surgery to cannulate an efferent popliteal lymphatic vessel in the immunised leg. Forty-eight hours after surgery, each sheep was given a further intracutaneous injection of 1 X 10(9) S. aureus-DNP with or without 20 mg DXS adjacent to the primary injection site. IgG1-cc and IgG2-cc in lymph were counted using indirect immunofluorescence. Total IgG1 and IgG2 anti-staphylococcal and anti-DNP antibody output in lymph was determined using enzyme-linked immunosorbent assays (ELISA's). DXS markedly increased antibody responses to S. aureus-DNP in the popliteal lymph node of sheep and was shown to strongly enhance immunological memory. The ratios of IgG2-cc:IgG1-cc and IgG2:IgG1 anti-staphylococcal antibody in lymph-draining popliteal lymph nodes of sheep stimulated with DXS and S. aureus-DNP was significantly greater than the same ratios in animals given antigen alone. The IgG subclass-specific immunomodulatory effects of DXS were exerted whether the adjuvant was given with primary and/or secondary inoculations. There was no difference in the ratio of IgG2:IgG1 anti-DNP antibody in lymph from animals given S. aureus-DNP with and without DXS.

Lymphocyte localization in lymph nodes of pubescent, prepartum, and postpartum sheep

It is generally accepted that lymphocytes associated with the mammary mucosal immune system of non-ruminants may be largely derived from gut-associated lymphoid tissue (GALT). The relationship between the mammary immune system and the GALT of ruminants has not been clearly defined. To address this question, we examined patterns of lymphocyte localization in sheep by 51Cr-labeled lymphocytes following infusion back into donor ewes. We found that lymphocytes taken from mammary lymph nodes of pubescent ewes returned preferentially to mammary nodes, while in prepartum and postpartum ewes, mammary node cells localized equally well in mammary and mesenteric lymph nodes. In contrast, ileal mesenteric lymph node cells from pubescent ewes localized equally well in mammary and mesenteric nodes, but in prepartum and postpartum ewes, localization in mammary nodes was markedly reduced. Comparison of the homing patterns of mammary, mesenteric, and peripheral lymph node cells indicated that mammary node cells behaved similarly to peripheral, rather than mesenteric node cells. This information may be relevant to the extent of communication between the gut and mammary gland in ruminants.

Localized deep hyperthermia increases the traffic of lymphocytes through peripheral lymph nodes of sheep in vivo.

The production of localized deep hyperthermia by the radio frequency wave magnetic-loop induction method has been utilized to study the effects of deep hyperthermia on lymphocyte traffic in sheep in vivo. Deep hyperthermia has been applied both to primary peripheral lymph node drainage areas alone (popliteal and prefemoral) and directly over the study lymph node (popliteal). Deep tissue core temperatures were monitored in all studies and the findings were correlated with alterations in the levels of lymphocyte outputs into study node efferent lymph and the volume of efferent lymph flow. In all of 32 studies, there was a prompt and sharp increase in the output of lymphocytes into efferent lymph at 40-43 degrees C. Efferent lymph flow also was increased promptly but to a lesser degree than the output of lymphocytes. The two were not closely correlated. High deep temperature in the nodal area (45 degrees C) appeared to cause delayed nodal malfunction, which depressed lymphocyte output but did not affect lymph flow. Localized deep hyperthermia is an effective and noninvasive means for the local increase of lymphocyte traffic and lymph flow and may prove to be a useful means for the evaluation of the immunological consequences of altering both deep tissue temperature and lymphocyte traffic.

Cellular composition of peripheral lymph and skin of sheep defined by monoclonal antibodies.

The surface phenotype of cells in peripheral lymph collected from afferent lymphatics leading to the popliteal lymph node of sheep was determined using a panel of monoclonal antibodies (mAbs). The majority of lymphocytes (83.5%) expressed the sheep pan-T cell antigen and only 13.3% bore surface immunoglobulin molecules. All peripheral T cell subsets occurring in sheep were detected; 50.2% of lymphocytes were positive for mAb SBU-T4 (T helper), 7.3% were positive for mAb ST-8 (T cytotoxic), and 8.4 and 43.0% expressed T subset markers recognized by mAbs 197 and T-80, respectively. Major histocompatibility complex (MHC) class I antigens were detected on 71.1% of lymphocytes and MHC class II antigens on 21.8%. The macrophage/veiled cells found in peripheral lymph did not express lymphocyte subset markers but were positive for MHC class I and II antigens, the sheep homologue of T6 antigen, leukocyte common antigen and mAb 175 (myeloid/erythroid). Macrophage-like cells occurring in the epidermis of skin taken from the lower hindleg gave positive staining reactions to the same mAbs which stained the macrophage/veiled cells in peripheral lymph. These results illustrate differences between the migration of lymphocyte subsets through nonlymphoid as compared to lymphoid tissues and point to a possible developmental or migratory relationship between the macrophage-like cells in skin and those in afferent popliteal lymph.

Lymphocyte traffic effects of systemic and local lidocaine in sheep

The traffic of lymphocytes through lymphoid tissues is of importance in both the initiation and development of the immune response. The effects of lidocaine on lymphocyte traffic through primary peripheral lymph nodes of sheep (popliteal and prefemoral) was investigated by three routes of lidocaine administration. Infiltration of lidocaine above the study node and as the sole anesthetic agent for study node efferent lymphatic cannulation produced mild depressions in lymphocyte outputs into study node efferent lymph. This depression was comparable to that encountered with regional epidural anesthesia for similar operations, but was significantly less than that associated with general anesthesia ( P < 0.000015). In animals with established chronic cannulation of study node efferent lymphatics, both study node drainage area injection of lidocaine and systemic administration of lidocaine (intravenously by bolus injection at a distant site) produced prompt and sharp depressions in lymphocyte traffic as mirrored in the output of lymphocytes into study node efferent lymph. The output of both small recirculating lymphocytes and of blast lymphocytes was affected.