Dysregulated expression of CD69 and IL-2 receptor alpha and beta chains on CD8+ T lymphocytes in flaky skin mice.

T-cell activation is considered to be an important element in the pathogenesis of psoriasis, a human skin disease characterized by keratinocyte hyperproliferation, altered keratinocyte differentiation and inflammation of the dermis and epidermis. Mice homozygous for the flaky skin (fsn) mutation develop a skin disorder that has histopathological and biochemical features resembling some forms of psoriasis. It has been reported recently that peripheral lymph nodes (PLN) in fsn/fsn mice exhibit various abnormalities in T-cell development suggestive of dysregulated T- and B-cell activation. In the present study, the expression of the inducible T-cell activation antigens CD69 and IL-2 receptor alpha chain (CD25) on PLN cells from fsn/fsn mice and their phenotypically normal littermates is examined. Expression of CD69 was significantly increased on PLN cells in fsn/fsn mice (mean +/- SD, 49.9 +/- 14.7% of cells) compared with control mice (14.6 +/- 4.2%). Analysis of CD4+ and CD8+ T cell subsets revealed that expression of CD69 in fsn/fsn PLN was significantly biased toward CD8+ cells. Although expression of CD25 was preferentially associated with CD4+ rather than CD8+ cells in both fsn/fsn and control PLN, with most CD4+ CD25+ cells being CD25hi, the proportion of CD4+ cells expressing CD25 was higher in fsn/fsn than control PLN. In contrast, CD25 was expressed by 2-3% of CD8+ PLN cells in both fsn/fsn and control mice and CD25hi cells accounted for < 1% of CD8+ cells in fsn/fsn PLN. The paucity of CD25 on CD8+ cells in fsn/fsn PLN did not appear to be due to a defect in the ability of these cells to upregulate CD25, because T cell receptor stimulation in vitro induced high expression of CD25 on both CD4+ and CD8+ cells. A striking and consistent finding was that most CD8+ cells in fsn/fsn PLN expressed high levels of IL-2R beta chain (CD122). In contrast, CD122 was expressed at low levels on CD8+ cells in control mice. Analysis of PLN cells from newborn fsn/fsn mice revealed that the high expression of CD122 on CD8+ cells was established by 2 weeks of age, prior to the appearance of clinical skin disease. These data indicate that large numbers of T cells in fsn/fsn mice are activated and reinforce the view that fsn is an important regulator of lymphocyte development and function. The relationship between T-cell activation and flaky skin disease in these mice remains to be established.

An evaluation of ontology exchange languages for bioinformatics.

Ontologies are specifications of the concepts in a given field, and of the relationships among those concepts. The development of ontologies for molecular-biology information and the sharing of those ontologies within the bioinformatics community are central problems in bioinformatics. If the bioinformatics community is to share ontologies effectively, ontologies must be exchanged in a form that uses standardized syntax and semantics. This paper reports on an effort among the authors to evaluate alternative ontology-exchange languages, and to recommend one or more languages for use within the larger bioinformatics community. The study selected a set of candidate languages, and defined a set of capabilities that the ideal ontology-exchange language should satisfy. The study scored the languages according to the degree to which they satisfied each capability. In addition, the authors performed several ontology-exchange experiments with the two languages that received the highest scores: OML and Ontolingua. The result of those experiments, and the main conclusion of this study, was that the frame-based semantic model of Ontolingua is preferable to the conceptual graph model of OML, but that the XML-based syntax of OML is preferable to the Lisp-based syntax of Ontolingua.

The peripheral lymphoid compartment is disrupted in flaky skin mice.

Flaky skin (fsn) is an autosomal recessive mutation on mouse chromosome 17 that causes severe anaemia, forestomach papillomatosis and a papulosquamous skin disease that resembles psoriasis in humans. In the present paper, it is reported that fsn causes peripheral lymphadenopathy, CD4/CD8 imbalance and hyperresponsiveness to T cell growth factors. Peripheral lymph nodes (PLN) of adult mutant (fsn/fsn) mice were found to contain almost 10-fold more leucocytes than PLN from phenotypically normal littermates (+/fsn or +/+, hereafter referred to as +/?). Analysis of PLN cells using mAbs and flow cytometry revealed that this predominantly lymphoid hyperplasia was characterized by approximately equivalent increases in the numbers of CD3+ T cells and CD19+ B cells. However, expansion within the T cell compartment was non-random, because fsn/fsn PLN had a considerably reduced ratio of CD4+ to CD8+ T cells (1.08 +/- 0.37) compared to +/? PLN (2.47 +/- 0.44, P < 0.0001). In vitro assays of cellular proliferation in response to T and B cell growth factors showed that fsn/fsn PLN cells were hyperresponsive to IL-2, IL-4 and IL-7 when compared with PLN cells from +/? mice. Studies using mesenteric lymph node and peripheral blood cells showed that hyperresponsive cells are widely distributed in fsn/fsn mice. Experiments in newborn mice showed that the lymphoid disturbances caused by fsn are established at least as early as 2 weeks of age, a time that precedes the onset of the earliest clinical skin lesions. These data implicate a role for the fsn gene product in regulating the size and content of the peripheral lymphoid compartment.

MHCWeb: converting a WWW database into a knowledge-based collaborative environment.

The World Wide Web (WWW) is useful for distributing scientific data. Most existing web data resources organize their information either in structured flat files or relational databases with basic retrieval capabilities. For databases with one or a few simple relations, these approaches are successful, but they can be cumbersome when there is a data model involving multiple relations between complex data. We believe that knowledge-based resources offer a solution in these cases. Knowledge bases have explicit declarations of the concepts in the domain, along with the relations between them. They are usually organized hierarchically, and provide a global data model with a controlled vocabulary. We have created the OWEB architecture for building online scientific data resources using knowledge bases. OWEB provides a shell for structuring data, providing secure and shared access, and creating computational modules for processing and displaying data. In this paper, we describe the translation of the online immunological database MHCPEP into an OWEB system called MHCWeb. This effort involved building a conceptual model for the data, creating a controlled terminology for the legal values for different types of data, and then translating the original data into the new structure. The OWEB environment allows for flexible access to the data by both users and computer programs.

Priming to mycobacterial antigen in vivo using antigen-pulsed antigen presenting cells generated in vitro is influenced by the dose and presence of IL-4 in APC cultures.

Antigen presenting cells (APC) similar to immature dendritic cells can be generated in vitro from bone marrow precursors. The authors have compared the yield, the phenotype and the function of murine bone marrow cells cultured for 7 or 11 days in either granulocyte macrophage colony stimulating factor alone (GM BMAPC) or in combination with interleukin-4 (GM/IL-4 BMAPC). The results showed that GM/IL-4 BMAPC expressed the highest levels of MHC Class 2 molecules, CD86/B7-2 and CD80/B7-1 co-stimulatory molecules and the lowest levels of F4/80 macrophage marker. However, when these APC were pulsed with BCG culture filtrate antigen or PPD they were not correspondingly more effective at stimulating activated T lymphocytes in vitro or priming naive T lymphocytes in vivo. Also, in contrast to GM BMAPC, high backgrounds recorded following injections of GM/IL-4 BMAPC without antigen were not consistently reduced by lowering the dose and irradiating the cells prior to administration. The authors conclude that the degree of maturity of BMAPC varies with culture conditions and that this may be an important consideration where BMAPC are to be used in vivo in immunotherapeutic regimens.

Standardized representations of the literature: combining diverse sources of ribosomal data.

We are building a knowledge base (KB) of published structural data on the 30s ribosomal subunit in prokaryotes. Our KB is distinguished by a standardized representation of biological experiments and their results, in a reusable format. It can be accessed by computer programs that exploit the rich interconnections within the data. The KB is designed to support the construction of 3D models of the 30S subunit, as well as the analysis and extension of relevant functional and phylogenetic information. Most published information about the structure of the ubiquitous ribosome focuses on E. coli as a model system. At the same time, thousands of RNA sequences for the ribosome have been gathered and cataloged. The volume and complexity of these data can complicate attempts to separate structural data peculiar to E. coli from data of universal relevance. We have written an application that dynamically queries the KB and the Ribosome Database Project, a repository of ribosomal RNA sequences from other organisms, in order to assess the relevance of structural data to particular organisms. The application uses the RDP alignment to determine whether a set of data refer primarily to conserved, mismatched, or gapped positions. For a set of 16 representative articles evaluated over 211 sequences, 73% of observations have unambiguous translations from E. coli to the other organisms, 21% have somewhat ambiguous translations, and 6% have no translations. There is a wide variation in these numbers over different articles and organisms, confirming that some articles report structural information specific to E. coli while others report information that is quite general.

Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors.

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II-restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T-cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM-derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.

Dendritic cell presentation of PPD and 19 kDa protein of Mycobacterium tuberculosis and emergent T helper cell phenotype.

Protection against infection with Mycobacterium tuberculosis is preferentially associated with the development of the T helper 1 subset, IFN-gamma production and a cell-mediated response, rather than with T helper 2 cells, 4 (IL-4) and antibody production. The type of APC interacting with T cells responsive to mycobacterial peptides may influence which of these responses predominates. This investigation focuses on the role of dendritic cells (DC) because they are the most potent APC in both primary and recall immune responses. Our results show that splenic DC-enriched suspensions prepared from C57BL/6 mice and pulsed with either purified protein derivative (PPD) or the immunodominant 19 kDa protein from M. tuberculosis, can activate antigen-primed T cells in vitro, whereas spleen cell suspensions depleted of DC cannot. DC pulsed with PPD or 19 kDa antigen are able to prime naive T cells in vivo. Supernatants collected from cultures containing T cells from mice injected with PPD-pulsed DC and then challenged in vitro with PPD-pulsed DC were found to contain more IL-2 and IFN-gamma than those from control mice which received either DC or PPD alone. No such antigen-specific IFN-gamma response occurred if DC pulsed with 19 kDa were used in place of PPD-pulsed DC. IL-4 was not detected in any of the culture supernatants. We conclude that DC can induce production of cytokines associated with a protective immune response when presenting peptides derived from heterogeneous mycobacterial antigens but not when exposed to the single 19 kDa immunodominant protein.

Antigen-independent maturation of CD2, CD11a/CD18, CD44, and CD58 expression on thymic emigrants in fetal and postnatal sheep.

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.

Changes in thymic export of gamma delta and alpha beta T cells during fetal and postnatal development.

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.

Changes in thymic export of L-selectin+ gamma delta and alpha beta T cells during fetal and postnatal development.

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.

CD45RA expression of gamma delta and alpha beta T cells emigrating from the fetal and postnatal thymus.

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.

Lymphatic drainage of hypertonic solution from peritoneal cavity of anesthetized and conscious sheep.

Lymphatic drainage of the peritoneal cavity may reduce ultrafiltration in continuous ambulatory peritoneal dialysis. We assessed lymphatic drainage of the peritoneal cavity in sheep under dialysis conditions by cannulation of the relevant lymphatic vessels and compared lymphatic drainage in anesthetized and conscious animals. Lymph was collected from the caudal mediastinal lymph node and the thoracic duct, both of which are involved in the lymphatic drainage of the ovine peritoneal cavity. Volumes of a hypertonic dialysis solution (50 ml/kg 4.25% Dianeal) containing 25 microCi 125I-human serum albumin were instilled into the peritoneal cavity, and lymph flows and the appearance of labeled protein in the lymphatic and vascular compartments were monitored for 6 h. Intraperitoneal pressures increased 4-5 cmH2O above resting levels after infusion of dialysate. On the basis of the appearance of tracer in the lymph, drainage of peritoneal fluid into the caudal lymphatic was calculated to be 3.09 +/- 0.69 and 14.14 +/- 2.86 ml/h in anesthetized and conscious sheep, respectively. Drainage of peritoneal fluid into the thoracic duct preparations was calculated to be 1.32 +/- 0.33 and 14.69 +/- 5.73 ml/h in anesthetized and conscious sheep, respectively. Significant radioactivity was found in the bloodstream, and at least a portion of this was likely contributed by the right lymph duct, which was not cannulated in our experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphatic removal of dialysate from the peritoneal cavity of anesthetized sheep.

Several investigators have suggested that the lymphatic circulation reduces ultrafiltration in continuous ambulatory peritoneal dialysis (CAPD). The purpose of this study was to assess lymphatic drainage of the peritoneal cavity directly in anesthetized sheep under dialysis conditions. Lymph was collected from the caudal mediastinal lymph node and the thoracic duct, both of which are involved in the lymphatic drainage of the ovine peritoneal cavity, and from the prescapular lymph node, which is not involved in peritoneal lymphatic drainage. Fifty ml/kg volumes of a mildly hypertonic dialysis solution (Dianeal 1.5%) containing 25 microCi 125I-human serum albumin were instilled into the peritoneal cavity, and lymph flows and the appearance of labeled protein in the lymphatic and vascular compartments were monitored for six hours. Following the instillation of dialysis fluid there was a tendency for lymph flow rates from the thoracic duct to increase but these changes were not significant. However, flow rates from the caudal lymphatic demonstrated significant increases, especially in the final three hours of the monitoring period. Only about 8% of the radiolabeled albumin was removed from the peritoneal cavity over six hours (that is, 92% was left in the peritoneal space). Of the albumin removed, approximately 17% of this was drained by abdominal visceral lymphatics into the thoracic duct. About 25% passed through the diaphragm into the caudal mediastinal lymph node and into efferent lymph. Since the efferent lymphatic duct of the caudal mediastinal node empties directly into the thoracic duct, about 42% of all protein removed from the peritoneal cavity of the sheep was ultimately transported to the thoracic duct.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphatic drainage of the peritoneal cavity in sheep.

Lymphatic drainage of the peritoneal cavity has been investigated in anesthetized sheep. Studies involving intraperitoneal administration of a complex of Evans blue dye and bovine serum albumin demonstrated the existence of three anatomically distinct pathways. In the first pathway, dye is removed from the peritoneal cavity by diaphragmatic lymphatics that pass into caudal sternal lymph nodes. Efferent lymphatics from these nodes transport the material to cranial sternal lymph nodes. Efferent cranial sternal lymphatics then convey the material either directly or indirectly, via tracheal lymphatic trunks, to the right lymph duct. In the second pathway, the complex is transported from the peritoneal cavity by diaphragmatic lymphatics that pass into the caudal mediastinal lymph node. Efferent lymphatic ducts from this node transport the material to the thoracic duct. The third pathway appears to involve transport of the dye across the mesothelial lining of the abdominal viscera and removal from the interstitium by afferent visceral lymphatics. Material taken up in this manner is ultimately transported to the thoracic duct by efferent visceral lymphatics. Experiments involving measurements of lymphatic absorption of 125I-labeled human serum albumin from the peritoneal cavity indicated that, over the 6-h period studied, 4.55 +/- 1.20 and 1.43 +/- 0.56% of the injected tracer could be recovered in thoracic duct lymph and caudal mediastinal efferent lymph, respectively, and the sum of these values represented 26% of the recovered radioactivity. On the other hand, 16.95 +/- 6.93% of the injected radioactivity could be found in the blood over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte subset-specific and tissue-specific lymphocyte-endothelial cell recognition mechanisms independently direct the recirculation of lymphocytes from blood to lymph in sheep.

Tissue-specific and lymphocyte subset-specific lymphocyte recirculation patterns have been analysed simultaneously. Lymphocytes obtained from one lymph compartment were directly labelled with fluorochrome in vitro and returned to the blood of the same animal. Over the next 48-72 h, the recirculation of these cells into both the same lymph compartment and at least one different lymph compartment was monitored. The cells in all of these lymph collections, as well as an aliquot of the cells used for direct fluorescent labelling, were then phenotyped with monoclonal antibodies (mAb) which define the mutually exclusive small CD4+ and CD8+ T-lymphocyte subsets in sheep. All cell samples were analysed by flow cytometry and CD4/CD8 ratios were determined for the recirculated, fluorochrome-labelled population in each lymph collection. The mean CD4/CD8 ratio calculated for each lymph compartment was then compared with the CD4/CD8 ratio calculated for each lymph compartment was then compared with the CD4/CD8 ratio of the transfused, starting population. In one experiment employing efferent prescapular lymph cells, three experiments employing efferent intestinal lymph cells, and two experiments employing afferent intestinal lymph cells, tissue-specific recirculation was observed. In all of these experiments, the pattern of recirculation of small CD4+ and CD8+ T lymphocytes was non-random. Moreover, in each experiment, this non-randomness was completely unrelated to tissue-specific phenomena, since the mean CD4/CD8 ratio of the recirculated population was higher than the CD4/CD8 ratio of the transfused, starting population regardless of the lymph compartment examined. These data are therefore consistent with the hypothesis that tissue-specific and lymphocyte subset-specific lymphocyte-endothelial cell recognition mechanisms independently direct the recirculation of small lymphocytes from blood to lymph.

The relevance of lymphoid cell migration to immunodeficiency syndromes.

It has been known for some time that antigen stimulation can alter lymphocyte traffic patterns and that viruses are particularly potent in this respect; such alterations may be a consequence of host-derived factors. The retention of lymphocytes in lymph nodes can be sustained for several hours with locally administered interferon (IFN)alpha. The extravasation of lymphocytes from blood into non-lymphoid tissues can be induced in the skin with IFN gamma and particularly tumor necrosis factor (TNF)alpha. Recent evidence supports the concept that the migratory capacity of CD4+ cells differs from the capacity of CD8+ cells. Agents (cytokines?) which differentially affect the traffic of these two sub-sets have not yet been described but such a possibility has not been adequately tested. Several new molecules have been defined which alter the interactions between lymphocytes and blood vascular endothelial cells, and these may be important in the critical adhesive event in lymphocyte traffic. In both rat and sheep, it has been possible to cultivate post-capillary endothelial cells from lymphoid tissue, and this may be a helpful approach to studying the mechanisms and molecules involved in adhesion. New cell tracking dyes recently available (Zynaxis Cell Science) permit more significant, long-term studies on the life span of lymphocyte sub-sets and their migratory status. In our experiments, labeled lymphocytes can be followed in vivo for over 30 days. Traffic alterations may explain some of the abnormalities in immunodeficiency states.

Non-random recirculation of small, CD4+ and CD8+ T lymphocytes in sheep: evidence for lymphocyte subset-specific lymphocyte- endothelial cell recognition.

The migratory properties of small, CD4+ and CD8+ T lymphocyte subsets have been examined in sheep under physiological conditions. Lymphocytes obtained free-floating in lymph were directly labeled with fluorochrome in vitro and returned to the blood of the same animal. Over the next 48 h the lymph collection bottles were replaced at various times. The cells in these collections, as well as an aliquot of the cells used for direct fluorescent labeling, were then phenotyped with monoclonal antibodies (mAbs) which define the mutually exclusive CD4+ and CD8+ T lymphocyte subsets in sheep. Binding of mAbs was detected by using secondary reagents labeled with a fluorochrome of a different colour. All cell samples were analyzed by flow cytometry and CD4/CD8 ratios were determined for the recirculated, fluorochrome-labeled population in each lymph collection. The mean CD4/CD8 ratio was then calculated and compared with the CD4/CD8 ratio of the intravenously infused starting population. In three experiments employing efferent prescapular lymph cells, three experiments employing efferent intestinal lymph cells and two experiments employing afferent intestinal lymph cells, the mean CD4/CD8 ratio of the recirculated, fluorochrome-labeled population was different from the CD4/CD8 ratio of the starting population, thereby indicating that non-random migration of these subsets had occurred. The finding that the CD4/CD8 ratio of the recirculated population was higher than the CD4/CD8 ratio of the transfused starting population in every case provides strong experimental support for the hypothesis that small, CD4+ T cells are extracted from the blood by specialized vascular endothelium with greater efficiency than small, CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, culture, and characterization of endothelial cells derived from the post-capillary venules of sheep mesenteric lymph nodes, Peyer's patches, and associated small bowel.

Lymphocyte recirculation is mediated principally by specialized endothelial cells which line the post-capillary venules of lymph nodes and other secondary lymphoid tissues. The ontogeny and physiology of this process have been characterized in sheep in considerable detail. To further enhance the analytical potential of this experimental system we have isolated endothelial cells from the post-capillary venules of ovine mesenteric lymph nodes by perfusion with small (37-74 microns diameter), sulfonated microcarrier beads. Cells isolated in this manner have been maintained in vitro for greater than 12 months through greater than 30 passages. The endothelial nature of these cells has been conclusively established on the basis of morphologic, metabolic, and immunologic criteria. Virtually all (greater than 99%) cells in primary and passaged cultures metabolized Dil-AC-LDL, a known marker for endothelial cells. Furthermore, nearly all (greater than 95%) cells expressed cell-surface von Willebrand factor and antithrombin III, which are known endothelial antigens. All cells expressed major histocompatibility class I antigens but no cells expressed class II antigens. In vitro lymphocyte-binding studies revealed that these cells bound lymphocytes in a dose-dependent fashion. The microcarrier perfusion technique was also used to isolate endothelial cells from the post-capillary venules of ileal Peyer's patches and associated small bowel in sheep. The majority (70%) of cells isolated in this manner resembled the cells isolated from mesenteric lymph nodes both morphologically and metabolically.