Glycosylation might provide endothelial zip codes for organ-specific leukocyte traffic into inflammatory sites

Inflammatory diseases are characterized by the leukocyte infiltration into tissues. L-selectin on lymphocytes and its endothelial glycosylated ligands are instrumental in the initiation of lymphocyte extravasation. Immunohistochemical stainings with monoclonal antibodies against functionally active glycan-decorated L-selectin ligands, ie, sialyl-Lewis x (sLex, 2F3, and HECA-452) or sulfated extended core 1 lactosamine (MECA-79), were performed on more than 400 specimen representatives for thyroiditis, myocarditis, psoriasis, vasculitis, ulcerative colitis, and their corresponding noninflamed tissues. The endothelial expression of sLex or sulfo sLex glycans in postcapillary venules was either absent or low in control tissues. The de novo induction of endothelial expression of sLex or sulfo sLex glycans was detected in all inflamed tissues. Furthermore, each organ carried its own modification of sLex or sulfo sLex glycans, ie, zip code. Our results suggest that these zip code glycans may provide means for organ selective leukocyte traffic that could be used in selective leukocyte traffic inhibition.     

Selective expression of sialyl-Lewis x and Lewis a epitopes, putative ligands for L-selectin, on peripheral lymph-node high endothelial venules.

High endothelial venules (HEV) lined by the high endothelium are the sites where leukocytes enter into the lymph nodes from the blood. Lymphocyte homing into lymph nodes is organ-selective, i.e., different molecules are involved in the lymphocyte homing to peripheral nodes compared with mucosa associated lymphoid tissue. The traffic into peripheral nodes is regulated by the expression of L-selectin on leukocytes and its ligand on HEVs. The ligand for L-selectin is suggested to be a 50, 90, or 105 kd glycoprotein, which is sulfated, fucosylated, and sialylated. The two other members of the selectin family (E- and P-selectin) recognize sialyl-Lewis x and -Lewis a (sLex and sLea, respectively) carbohydrate motifs, and there is preliminary data suggesting that this would also be the case for L-selectin. We have initiated a study to identify the expression of these sialylated structures on endothelial surfaces. We present data that show that HEVs in peripheral nodes, but not in the mucosa-associated lymphoid tissue, express large quantities of sLex and sLea identified by MAbs in immunohistology. Endothelium in capillaries or larger vessels in non-lymphoid tissues do not react with anti-sLex or -Lea mAbs. Only 1-2% of the lymphocytes in the peripheral blood express sLex and so far only the skin-homing lymphocytes are known to be sLex positive in larger quantities. We show that in many occasions the B cells in the peripheral lymph-node germinal centers are also sLex-, but not sLea-positive, and provide evidence of the restricted pattern of sLex and sLea expression on peripheral lymph-node HEVs. We propose that they are at least parts of the ligand for L-selectin.     

Lymphocyte triggering via L-selectin leads to enhanced galectin-3-mediated binding to dendritic cells

For proper immune surveillance, naive lymphocytes are recruited from the blood into secondary lymphoid organs. L-selectin expressed on lymphocytes plays an important role in the initial attachment of these cells to high endothelial venules (HEV) in lymph nodes. Previously, we found that triggering via L-selectin resulted in activation of lymphocytes, followed by an alteration in their adhesion capacity. This suggested that L-selectin triggering might play a role in cell-cell interactions after lymph node entry. Here, we identify a novel adhesion mechanism involving L-selectin-triggered lymphocytes and dendritic cells, and we show that enhanced binding to dendritic cells is mediated by galectin-3 and not by integrins. Furthermore, it was shown that L-selectin-triggered T lymphocytes exhibited enhanced proliferation in an allogeneic mixed lymphocyte reaction. It is concluded that, in addition to a role for L-selectin in tethering and rolling on endothelium, triggering of the molecule on the lymphocyte surface leads to changes that are pertinent for the function of the cell after passing the HEV. We argue that the described adhesion mechanism plays a role in optimizing the initial interaction between dendritic cells and lymphocytes.     

The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.

The extravasation of blood-borne lymphocytes into organized lymphoid tissues and sites of chronic inflammation is directed in part by interactions of lymphocyte surface adhesion molecules, known as homing receptors, with tissue-selective endothelial ligands called vascular addressins. In mice and humans, lymphocyte L-selectin and the peripheral lymph node addressin (PNAd) form a homing receptor-endothelial ligand pair involved in lymphocyte traffic to peripheral lymph node (PLN). We have examined the tissue distribution and function of human PNAd, using monoclonal antibody MECA-79 and in vitro assays of L-selectin-dependent lymphocyte binding. We demonstrate that PNAd is expressed by human high endothelial venules (HEV) in lymphoid tissues which support lymphocyte adhesion via a PLN-associated recognition system. MECA-79 inhibits adhesion to these HEV of a cell line that binds predominantly via the PLN-homing receptor, L-selectin, but has no effect on adhesion by a mucosal HEV-binding cell line. Furthermore, MECA-79 blocks binding of human peripheral blood mononuclear cells to both PLN and tonsil HEV, but not significantly to HEV in the appendix. In addition, we demonstrate PNAd induction on venules at chronic inflammatory sites in humans, particularly sites with severe or long-standing chronic inflammatory involvement. These results confirm that PNAd functions as a PLN vascular addressin in humans, and that in addition to directing normal lymphocyte recirculation to lymph nodes and tonsils, this addressin likely participates in lymphocyte recruitment to sites of chronic inflammation.     

The cervical lymph node preparation: A novel approach to study lymphocyte homing by intravital microscopy

Objective Lymphocyte recirculation constitutes an integral part of the adaptive immune system. Blood-borne lymphocytes migrate into secondary lymphoid organs, crossing the vascular wall of site-specific high endothelial venules (HEVs). We created a preparation of the cervical lymph node in mice to study lymphocyte homing in vivo. Methods and Results Our novel approach allowed the detailed analysis of hemodynamics and lymphocyte-HEV endothelium interactions by means of intravital fluorescence microscopy. We confirm the key roles of L-selectin and LFA-1 for lymphocyte homing. Blockade of L-selectin function inhibited lymphocyte rolling and firm adhesion by 92% and 66%. In LFA-1-deficient mice, lymphocyte firm adhesion was reduced by 70%. In addition to the microcirculation studies, the cervical lymph node preparation allowed for visualization of afferent lymphatic transport, which is mainly derived from the oral mucosa. Conclusion This study reports a novel technical tool for the detailed in vivo analysis of adaptive immune responses. Received 28 September 2005; returned for revision 20 December 2005; accepted by M. Parnham 10 January 2006     

Endothelial L-Selectin Ligands Are Likely to Recruit Lymphocytes into Rejecting Human Heart Transplants

L-selectin-dependent lymphocyte extravasation is a hallmark of acute heart allograft rejection in rats. On screening over 600 endomyocardial biopsies (EMBs), taken at different time points after heart transplantation in man, we identified 91 samples with histological signs of acute rejection. Rejection and nonrejection EMBs were analyzed for the presence of properly glycosylated, ie, sulfated sialyl Lewis-x (sLex) decorated L-selectin ligands. Two anti-sLex (2F3 and HECA-452) and one anti-6- or 6'-sulfated and/or 6, 6'-bisulfation (MECA-79) monoclonal antibodies were used. Nonrejecting heart endothelium did not express, or expressed only weakly, sulfated and or sLex decorations of L-selectin ligands. On the contrary, these epitopes were readily detectable on endothelium of capillaries and venules at the onset and during acute rejection episodes. The more intense the sulfated sLex expression was, the more severe the rejection episode was in histological grading. The endothelial expression of L-selectin ligands decreased to background levels as the rejection resolved. Our data demonstrate a complete correlation between the level of expression of the sulfated sLex-decorated ligands on the one hand and the histological severity of acute heart allograft rejection on the other hand. These data suggest that functionally active endothelial L-selectin ligands are instrumental in lymphocyte extravasation into the human heart allografts at the onset and during acute rejection episodes.     

Regulation of L-selectin expression on cultured bone marrow leukocytes and their precursors.

L-selectin (LECAM-1, LAM-1, MEL-14 antigen, Dreg antigen) is one of the molecules controlling lymphocyte homing from the blood to peripheral lymph nodes and granulocyte adhesion to inflamed endothelium. In this work, regulation of L-selectin expression on mouse bone marrow cells was studied. L-selectin-negative cells were isolated by panning technique, cultured for 1-7 days with cytokines and mitogens, and L-selectin expression was analyzed by immunofluorescence staining. When cultured for 3 days with interleukin (IL) 1, IL 2, IL 5, IL 6, phytohemagglutinin, pokeweed mitogen or in the medium alone, 75%-85% of L-selectin-negative large cells (including granulocytes, macrophages/monocytes, blasts and their precursors) became L-selectin positive. In contrast, IL 3, IL 4, granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) prevented the induction of L-selectin in a time- and dose-dependent manner. GM-CSF was the most potent inhibitor and only 10%-15% of cells became L-selectin positive after 3 days of culture. Furthermore, L-selectin was down-regulated on cultured unselected bone marrow cells by IL 3, IL 4, GM-CSF and LPS stimulation. After culture, the relative molecular mass of L-selectin was 100 kDa, similar to the size of the granulocyte form of this antigen. Cultured cells adhered to high endothelial venules (HEV) only 10%-32% as effectively as freshly isolated bone marrow cells despite high levels of L-selectin expression. The phenotypic analysis and the HEV binding data indicate that after culturing L-selectin was almost exclusively expressed on bone marrow leukocytes of myeloid series, and on these cells it was not functional in mediating peripheral lymph node HEV binding. Overall, these results show that the expression of L-selectin can be modulated by regulating the maturation and differentiation of the cells in vitro. This supports the idea that different cytokines and mitogens may also be important in controlling migrational status of leukocytes in vivo.     

Endothelial sulfated sialyl Lewis x glycans, putative L-selectin ligands, are preferentially expressed in bronchial asthma but not in other chronic inflammatory lung diseases

Lymphocyte infiltrate is a hallmark of inflammatory responses. We have previously shown that de novo-induced endothelial sialyl Lewis x (sLex) expression guides lymphocytes in an L-selectin-dependent manner to sites of acute organ transplant rejections. In this research, we have analyzed five groups of chronic lung inflammations to determine the presence of properly glycosylated, i.e., sulfated, sLex-decorated, L-selectin ligands. Two anti-sLex (2F3 and HECA-452) and one anti-6- and/or 6'-sulfated and/or 6,6'-bisulfated (MECA-79) monoclonal antibodies (mAbs) were used. The control lung specimens did not express L-selectin ligands on endothelium. In contrast, the endothelial staining intensity and the number of positive peribronchial venules and capillaries with mAbs 2F3, HECA-452, and MECA-79 were significantly greater in bronchial biopsies from patients with asthma compared with normal specimens (P<0.003). However, no significant increase of peribronchial endothelial reactivity with these antibodies was observed in adult respiratory distress syndrome, chronic bronchitis, fibrosing alveolitis, and granulomatous inflammation compared with controls. These data suggest that sulfated sLex glycans, acting putatively as ligands for L-selectin, could be instrumental in lymphocyte extravasation into human peribronchial lung tissue during asthma, but not so important in several other inflammatory lung diseases.     

Altered patterns of T cell migration through lymph nodes and skin following antigen challenge.

Antigen challenge has profound effects on a regional lymph node (LN); it leads to an increase in blood flow to the node, and a marked increase in lymphocyte output through the efferent lymphatics. We used the isolated LN model developed in the sheep to see if antigen challenge in a LN resembled inflammation in peripheral tissues. Following stimulation with an antigen (purified protein derivative of tuberculin), lymphocyte output from the LN showed the typical periods of "lymphocyte shutdown" and "recruitment". The shutdown phase, when cell numbers in efferent lymph dropped by approximately 80%, affected almost exclusively the naive-type (adhesionlo, L-selectin+) T cell population. The large increase in T cell traffic through the node during the recruitment phase was mostly due to CD4+ memory-type T cells and, moreover, the majority of these T cells were L-selectin-, indicating that these cells were crossing from the blood by a molecular mechanism other than L-selectin interaction with its ligand, the "lymph node vascular addressin" (MECA-79). Examination of LN high endothelial venules revealed the presence of vascular cell adhesion molecule-1 (VCAM-1), an endothelial adhesion molecule which has been reported to bind preferentially memory-type T cells in inflammatory lesions. Within the skin, antigen challenge also induced the rapid expression of VCAM-1 on vascular endothelium. It was purely memory-type T cells (beta 1+, L-selectin+/-) that collected in lymph draining from this tissue. However within chronically inflamed skin, the MECA-79 determinant appeared on vascular endothelium, and a small proportion of T cells draining from chronically inflamed skin were of naive-type. The present results illustrate that there are similarities in the cellular and molecular events that characterize antigen stimulation of a LN and inflammation in a peripheral tissue.     

Characterization of a novel subset of T cells from human spleen that lacks L-selectin.

Human L-selection (LAM-1, Leu-8, TQ1, DREG 56) is a member of the 'selection' family of adhesion molecules. Antibodies to L-selectin have been shown to block the binding of T cells to peripheral lymph node high endothelial venules (HEV). Most unstimulated peripheral blood T cells express high levels of L-selectin whilst it is only weakly expressed on the majority of T cells from secondary lymphoid organs. We show here (a) that T cells from tonsil and lymph node up-regulate L-selectin when released from their microenvironment, (b) that in contrast, spleen contains a stable L-selectin negative subset, (c) that this subset remains surface L-selection negative after stimulation even though the T cells can respond by proliferation, (d) that this subset expresses minimal levels of LAM-1 mRNA and (e) that mucosal lymphocyte antigen (MLA) positive and T-cell receptor (TcR) gamma delta positive T cells found within the L-selectin negative population are similar to subsets of T cells found amongst lamina propria (LP) and intraepithelial lymphocytes (IEL) of the gut.     

Novel chondroitin sulfate-modified ligands for L-selectin on lymph node high endothelial venules

The migration of lymphocytes into lymph nodes via high endothelial venules (HEV) is dependent on the expression of L-selectin on the lymphocyte cell surface. HEV express several L-selectin ligands including CD34, GlyCAM-1, MAdCAM-1 and two sulfated glycoproteins (Sgp) of 200 kDa and 170 kDa which remain to be identified. In this investigation, labeling with sodium [35S]sulfate, which is incorporated into and forms part of the functional carbohydrate ligand, has been used to isolate and characterize macromolecular L-selectin ligands. High endothelial cells (HEC) cultured from rat lymph node HEV were shown to express ligands for L-selectin. HEC synthesized two groups of sulfated glycoproteins of 150 kDa and > 200 kDa, which were present in conditioned medium. These coeluted on anion exchange chromatography at 1.0-1.2 M NaCl and supported calcium-dependent L-selectin-mediated cell adhesion. In common with known L-selectin ligands, Sgp 150/> 200 were shown to be O-sialoglycoproteins; however, in contrast to other ligands, Sgp 150/> 200 contained chondroitin sulfate glycosaminoglycan modifications which were required for L-selectin recognition. Chondroitin sulfate-modified ligands for L-selectin were expressed at the HEC surface and by HEV in lymph nodes, suggesting that they may participate in lymphocyte interactions with HEV in vivo.     

Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein-1 (VAP-1)

The binding of leukocytes to vascular endothelium and their migration into tissues is mediated by adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein-1 (VAP-1) is a 170–180/90-kDa endothelial molecule expressed most prominently in high endothelial venules in peripheral lymph node (PLN) type lymphatic tissues. VAP-1 mediates lymphocyte binding to PLN, tonsil and synovium. The expression of VAP-1 is induced in inflammatory diseases such as arthritis and gut inflammation. We examined the expression, structure and function of VAP-1 in normal and inflamed skin and compared it to those of other adhesion molecules implicated in skin homing. In psoriasis, lichen ruber planus, pemphigoid and allergic lesions, VAP-1 was markedly up-regulated. The expression of VAP-1 was also increased in biopsies of healthy skin of the patients. The VAP-1 molecule induced in skin is decorated with abundant sialic acids. VAP-1 in inflamed skin is functional, since inhibition with anti-VAP-1 monoclonal antibodies caused a 60% reduction in lymphocyte adhesion to vascular endothelium. Antibodies against E-selectin, which has been regarded as the major vascular addressin directing cutaneous lymphocyte traffic, and, surprisingly, against peripheral lymph node addressin (PNAd), caused inhibitions of 30% and 60%, respectively, in the frozen section adhesion assay. These findings suggest important roles also for VAP-1 and PNAd in lymphocyte homing into inflamed skin.     

Rapid decrease in lymphocyte adherence to high endothelial venules in lymph nodes deprived of afferent lymphatic vessels

Occlusion of the afferent lymph flow to the lymph node (LN) results in both flattening of the endothelium of high endothelial venules (HEV) and a severe decrease in numbers of lymphocytes in transit across the walls of the flattened HEV. In the present study we have used the in vitro lymphocyte-binding assay to investigate the ability of HEV in rat LN to bind lymphocytes at various time points after occlusion of the afferent lymph flow. In addition the specificity of T and B lymphocyte adherence to HEV of such operated LN was studied. In normal LN, lymphocytes adhered to virtually all HEV using the in vitro binding assay. However, 1 and 2 weeks after operation lymphocytes bound to only 50-60% of the HEV and by 3-6 weeks 20-30%. The total numbers of lymphocytes bound to these HEV had also diminished to 10% of the control value 3-6 weeks after operation. Morphometric analysis showed that this was not only due to a reduction in the area of HEV endothelium available for lymphocyte adherence by flattening of the high endothelial cells, but also to a strong decrease in the numbers of bound lymphocytes per unit area high endothelium. In spite of the reduction in numbers of adhering lymphocytes the T/B cell ratio did not change. The results show that the reduction in lymphocyte binding of HEV in operated LN is a rapid event, probably due to loss of high endothelial cell determinants involved in binding of lymphocytes. The decrease in lymphocyte binding clearly precedes flattening of HEV endothelium suggesting that the height of high endothelial cells is of secondary importance to lymphocyte adherence.     

Prominent expression of sialyl Lewis X-capped core 2-branched O-glycans on high endothelial venule-like vessels in gastric MALT lymphoma

High endothelial venule (HEV)-like vessels have been observed in gastric B cell lymphoma of mucosa-associated lymphoid tissue type (MALT lymphoma), as well as in its preceding lesion, chronic Helicobacter pylori gastritis. Previously we reported that glycans on HEV-like vessels in the latter lesion served as L-selectin ligands, although their function is unclear. We have investigated sialyl Lewis X (sLeX)-related glycoepitopes and found that MECA-79(-) /HECA-452(+) /NCC-ST-439(+) HEV-like vessels preferentially mark gastric MALT lymphoma compared to chronic H. pylori gastritis. We then constructed CHO cell lines expressing potential MECA-79(-) /HECA-452(+) /NCC-ST-439(+) glycans, as well as other sLeX-type glycans, on CD34 and evaluated L-selectin binding to those cells, using L-selectin-IgM chimera binding and lymphocyte adhesion assays. L-selectin-IgM chimeras bound to CHO cells expressing 6-sulpho-sLeX attached to core 2-branched O-glycans with or without 6-sulpho-sLeX attached to extended core 1 O-glycans, but only marginally to other CHO cell lines. By contrast, CHO cells expressing 6-sulpho-sLeX attached to extended core 1 and/or core 2-branched O-glycans, as well as non-sulphated sLeX attached to core 2-branched O-glycans, showed substantial lymphocyte binding, while binding was negligible on lines expressing 6-sulpho- and non-sulphated sLeX attached to N-glycans and non-sulphated sLeX attached to extended core 1 O-glycans. These results indicate that MECA-79(-) /HECA-452(+) /NCC-ST-439(+) glycans, specifically, 6-sulpho- and non-sulphated sLeXs attached to core 2-branched O-glycans, expressed on HEV-like vessels in gastric MALT lymphoma function as L-selectin ligands and likely contribute to H. pylori-specific T cell recruitment in the progression of gastric MALT lymphoma.     

Structure and function of L-selectin.

The selectins are a newly described family of carbohydrate-binding adhesion molecules involved in the regulation of leukocyte traffic. Selectins are composed of an N-terminal C-type lectin domain, a single EGF domain, a variable number of short consensus repeat (SCR) domains, a transmembrane region and a cytoplasmic tail. L-selectin (LAM-1/LECAM-1/LECCAM-1) is the only selectin expressed on leukocytes, and mediates a number of leukocyte-endothelial interactions, including the binding of lymphocytes to HEV of peripheral lymph node high endothelial venules (HEV), neutrophil rolling, and leukocyte attachment to cytokine-treated endothelium in vitro. Stable transfectants expressing a series of chimeric selectins and deletion mutants were functionally analyzed in order to determine the molecular basis of adhesion mediated by L-selectin. The specificity of adhesion was found to reside entirely within the lectin domain, suggesting that this domain is the only domain of the protein to interact with the carbohydrate ligand. These results make previous observations that certain mAbs which block function map to each of the extracellular domains difficult to interpret. In addition, deletion of the cytoplasmic tail of L-selectin abolished adhesion, without affecting ligand recognition. Thus, each domain of the selectins has an important, but distinct, role in cell adhesion.     

Hepatic regulation of lymphocyte adhesion to, and activation on, syngeneic endothelial monolayers.

Endothelial monolayers were prepared from neonatal heart or liver tissue of Lewis (Le) rats. Cells in their first passage of culture were used to investigate the short-term (1 hr at 37 degrees) binding of 51Cr-labelled Le rat lymphocytes prepared from the mesenteric lymph node (MLN), peripheral lymph node (PLN) or Peyer's patches (PP) to those endothelia, or the activation by concanavalin A (Con A) or irradiated (Lewis x Brown Norway)F1 (LBNF1), of Le cells on the monolayers after 84 hr in culture. MLN and PP showed preferential binding to, and activation on, liver endothelium compared with heart endothelium (approximately twofold difference), while the converse was seen with PLN. No inhibition of binding was seen with antibodies to intracellular adhesion molecule-1 (ICAM-1) or lymphocyte function-associated antigen-1 (LFA-1). Preincubation of endothelial cells with plasma isolated from the portal or hepatic vein of normal adult mice (5% plasma, 37 degrees for 14 hr) caused a 1.5-2-fold stimulation of binding of MLN/PP to heart endothelium, which was inhibited (> or = 75%) by anti-ICAM-1 or anti-LFA-1, and a fourfold stimulation of binding to liver endothelium, which was not inhibited by these monoclonal antibodies (< or = 25% inhibition). In contrast, antibodies to tumour necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) caused inhibition of activation of liver endothelium (> or = 75%), while producing little affect on activation of heart endothelium. Similar results were seen when lymphocyte activation on endothelial cells rather than adhesion cells was investigated. Our data suggest a heterogeneity in lymphocyte-endothelial interactions, which is further regulated, under physiological conditions, by the liver.     

Blood Leucocyte Subsets of the Rat: Expression of Adhesion Molecules and Localization Within High Endothelial Venules

Although several distinct adhesion pathways are now well characterized, it is not clear whether analysis of adhesion molecule expression on leucocytes is sufficient to predict their interaction with endothelium in vivo. Therefore, in the present study this question was addressed by investigating the interaction between blood leucocyte subsets and high endothelial venules (HEV). The expression of different types of adhesion molecule (CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin) on lymphocytes, NK cells, monocytes and granulocytes of rat blood was determined by flow cytometry. In the same animals the numbers of blood leucocyte subsets present in the HEV of axillary lymph nodes and Peyer's patches were analysed using immunohistology. In the HEV of both axillary lymph nodes and of Peyer's patches lymphocytes (> 10.000 per mm²), as well as small numbers of NK cells and monocytes (< 500 per mm²), were found. In contrast, granulocytes were not detected here. Lymphocytes, NK cells, monocytes and granulocytes each expressed CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin in a pattern characteristic to cell type, but this did not correlate with the different ability of the leucocyte subsets to interact with the two types of HEV. In conclusion, determining the expression of CD44, α4-integrins, LFA-1, ICAM-1, CD2 and L-selectin on blood leucocytes alone is not sufficient to predict leucocyte/endothelium interaction in vivo     

Cloning and expression of rat fucosyltransferase VII at sites of inflammation

The sialyl Lewis x (NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium. The final step in the sialyl Lewis x synthesis is catalyzed by alpha1-3-fucosyltransferase, which transfers fucose to sialylated type 2 chain. We report the cloning of rat alpha1-3-fucosyltransferase gene (rFUT) isolated from rat lymph node and kidney allograft. The rFUT is expressed as two splice variants, but only the long one showed enzymatic activity towards sialylated lactosamine. Also flow cytometry analysis with the sLex mAbs indicated that the cloned rFuc-T was a functional enzyme and a member of the Fuc-TVII family. The rFuc-TVII mRNA expression level was strongly enhanced during acute inflammatory reaction induced by kidney allograft rejection, which could be detected by in situ hybridization and quantitative real-time PCR.     

Leukocyte migration is regulated by L-selectin endoproteolytic release

L-selectin mediates lymphocyte migration to peripheral lymph nodes and leukocyte rolling on vascular endothelium during inflammation. One unique feature that distinguishes L-selectin from other adhesion molecules is that it is rapidly cleaved from the cell surface after cellular activation. The biological significance of L-selectin endoproteolytic release was determined by generating gene-targeted mice expressing a modified receptor that was not cleaved from the cell surface. Blocking L-selectin cleavage on antigen-stimulated lymphocytes allowed their continued migration to peripheral lymph nodes and inhibited their short-term redirection to the spleen. Blocking homeostatic L-selectin cleavage also resulted in a constitutive 2-fold increase in overall L-selectin expression by leukocytes. As a result, neutrophils entered the inflamed peritoneum in greater numbers or for a longer duration. Thus, endoproteolytic cleavage regulates both homeostatic and activation-induced changes in cell surface L-selectin density, which directs the migration patterns of activated lymphocytes and neutrophils in vivo.     

Random entry of circulating lymphocyte subsets into peripheral lymph nodes and Peyer's patches: no evidence in vivo of a tissue-specific migration of B and T lymphocytes at the level of high endothelial venules.

Lymphocytes continuously migrate through the body and thus immune competent cells are constantly delivered to most tissues. They interact with high endothelial venules (HEV) via specific homing receptors and vascular addressins, and these molecules seem to be the reason for a preferential homing of B lymphocytes into Peyer's patches and of T lymphocytes into peripheral lymph nodes. When lymphocytes derived from lymph node cell suspensions were applied in the in vitro lymphocyte/endothelium binding assay, the well-known preference of mouse lymph node B lymphocytes for Peyer's patch HEV compared to peripheral lymph node HEV was confirmed in the rat (2.8 times). When in the same in vitro assay thoracic duct lymphocytes (TDL) were used this preference was far less obvious (1.4 times). However, by injecting rat TDL intravenously and by tracing them directly in HEV, B, T, CD4+ and CD8+ lymphocytes are seen to enter Peyer's patches and peripheral lymph nodes in vivo without preference. Thus, in contrast to lymphocytes from lymph node cell suspensions, no evidence was found of a tissue-specific migration of thoracic duct B, T, CD4+ and CD8+ lymphocytes at the HEV level. This finding demonstrates the importance of considering both experimental conditions and the cell source used when investigating lymphocyte traffic.