Roles of {alpha}4 integrins/VCAM-1 and LFA-1/ICAM-1 in the binding and transendothelial migration of T lymphocytes and T lymphoblasts across high endothelial venules

Christelle Faveeuw1,3, Mary E. Di Mauro2, Abigail A. Price1 and Ann Ager1

1 Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Immunology Research Group, Biological Sciences, University of Manchester, Manchester M13 9PT, UK

Correspondence to: A. Ager


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Several cell adhesion molecules that mediate the binding of lymphocytes to high endothelial venules (HEV) from flowing blood have been identified but the regulation of lymphocyte migration across the HEV wall into the lymph node (LN) is far from understood. In this study we have used an in vitro model of lymphocyte migration across HEV, and analysed the roles of two integrins in the binding and transendothelial migration of T lymphocytes and T lymphoblasts. The adhesion of T lymphocytes to high endothelial cells (HEC) cultured from rat LN HEV differed from that of T lymphoblasts since the percentage of T lymphoblasts that adhered and transmigrated was higher and was not increased by IFN-{gamma} pretreatment of HEC. Antibodies to {alpha}4 integrins, VCAM-1 or LFA-1 maximally inhibited T lymphocyte adhesion by 40–50%, whereas antibodies to ICAM-1 were less effective (<20% inhibition). The effects of {alpha}4 integrin and LFA-1 antibodies were additive, giving >90% inhibition. T lymphocytes which adhered in the presence of LFA-1 antibody showed reduced levels of transmigration and, in the presence of {alpha}4 integrin antibody, slightly increased transmigration. Antibodies to {alpha}4 integrins, VCAM-1, LFA-1 or ICAM-1 had little effect on T lymphoblast adhesion (maxima of 10–30% inhibition) and T lymphoblasts transmigrated normally in the presence of either {alpha}4 integrin or LFA-1 antibodies. However, the effects of {alpha}4 integrin and LFA-1 antibodies on T lymphoblast adhesion were synergistic, giving >90% inhibition of adhesion. These results suggest that the majority of T lymphoblasts use either {alpha}4 integrins or LFA-1 to bind and transmigrate HEV, and the roles of these integrins on activated T cells are overlapping and redundant. In contrast, either integrin supports half-maximal binding of unactivated T lymphocytes to the surface of HEV and LFA-1 makes a larger contribution than {alpha}4 integrins to transendothelial migration.

Keywords: {alpha}4 integrins, high endothelial venules, ICAM-1, LFA-1, T lymphocytes, transendothelial migration, VCAM-1


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The initiation of immune responses occurs in secondary lymphoid organs, such as lymph nodes (LN), where rare, antigen-specific naive T cells migrating in from the blood encounter antigen on specialized presenting cells migrating in from the surrounding tissues (1). Following engagement of the antigen receptor lymphocytes divide and differentiate, and the progeny, either as lymphoblasts or memory/effector cells, exit the node, re-enter the bloodstream and are distributed around the body. Early studies in sheep suggested that memory/effector T lymphocytes which are CD45Rlo do not migrate into LN directly from the bloodstream (2); however, recent studies in rodents have demonstrated clearly that CD4+ T cells expressing low mol. wt isoforms of CD45 in rats (CD45RClo) (3) and mice (CD45RBlo) (4) do migrate from the bloodstream into LN. The migration of antigen-activated T lymphoblasts directly from the bloodstream into LN has also been shown clearly in rodents (5). The migration of T lymphoblasts and memory/effector T cells to lymphoid organs will contribute to maintenance of an immune repertoire, peripheral tolerance and the dissemination of immunological memory (6,7).

The migration of thoracic duct lymphocytes from the bloodstream into peripheral LN was originally demonstrated to be via specialized high endothelial venules (HEV) (8). Subsequent studies have demonstrated clearly that this is the point of entry for T and B lymphocytes (9) as well as T lymphoblasts (5) and naive and memory/effector CD4+ T lymphocytes (3). The first stage of lymphocyte migration, that of binding to the surface of HEV from flowing blood, requires a coordinated sequence of adhesive interactions in which lymphocytes first tether and roll on the luminal surface of HEV, and are then arrested from blood flow (10,11). The subsequent directed migration of lymphocytes across the HEV wall and into the LN (diapedesis) is a complex, poorly understood process (12). The vessel wall comprises two cell types: high endothelial cells (HEC), which form a continuous layer lining the vessel, and pericytes or reticular cells, which together with the basal lamina form a sheath surrounding the endothelial lining. Migration across the HEV wall can therefore be divided into two stages: transendothelial migration and migration across the pericytic sheath. The adhesion molecules, chemoattractants and activation events that regulate these stages remain to be identified.

Transendothelial migration of lymphocytes across HEV has been modelled in vitro using HEC cultured from peripheral LN of the rat (13,14). A detailed kinetic analysis of lymphocytes interacting with cultured HEC (15) and the effects of cytokine pretreatment of cultured HEC (16) have shown that binding to the surface of HEC and transendothelial migration are separately regulated. {alpha}4 integrins are activated on lymphocytes following transendothelial migration and we have proposed that {alpha}4 integrin activation may be required for transmigration (15). Until recently (17), studies in mice had failed to implicate {alpha}4 integrins or VCAM-1 in lymphocyte migration to peripheral LN; however, we have shown that antibodies to {alpha}4 integrins and VCAM-1 partially inhibit the overall adhesion of lymphocytes to rat peripheral LN HEC (18), but effects on binding and transendothelial migration were not distinguished. In this study the precise role of {alpha}4 integrins in the separate events of binding and transendothelial migration of T lymphocytes have been determined. LFA-1/ICAM interactions, which play a major role in lymphocyte migration to peripheral LN of mice (19) and have been implicated in the transendothelial migration of lymphocytes across other types of EC (2022), have also been studied. Activated T cells utilize different integrins than unactivated lymphocytes to bind to EC (23,24). The roles of {alpha}4 integrins and LFA-1 in the binding and transendothelial migration of antigen-activated T lymphoblasts across HEC have therefore been determined.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Inbred LOU rats (RT1u), 8–12 weeks old, bred and maintained under specific pathogen-free conditions at the NIMR, London, were used in this study.

HEC culture
Individual strains of HEC were isolated from cervical LN of single AO (RT1u) rats and cultured as previously described (25). Confluent primary cultures of HEC were serially subcultured and plated at 50% of confluent density. Two different strains of HEC were used between the 11th and 24th passage in this study. Previous studies have shown that the interactions between lymphocytes and cultured HEC are independent of passage number (13).

Antibodies and reagents
The mAb used in this study were: HP2/1 (mouse anti-human {alpha}4 integrin subunit which cross-reacts on rat {alpha}4; IgG1), generously provided by F. Sanchez-Madrid (Madrid), and 5F10 (mouse anti-rat VCAM-1; IgG2a), generously provided by R. Lobb (Biogen, Cambridge, MA). MRC-OX1 (mouse anti-rat CD45; IgG1), MRC-OX6 (mouse anti-rat MHC class II; IgG1), MRC-OX12 (mouse anti-rat Ig{kappa}; IgG2a), MRC-OX49 (mouse anti-rat CD44; IgG2a), W6/32 (mouse anti-human HLA-ABC; IgG2a) and R73 (mouse anti-rat {alpha}ßT cell receptor; IgG1) hybridomas were obtained from ECACC (Porton Down, UK), and WT1 (mouse anti-rat CD11a; IgG2a) and 1A29 (mouse anti-rat ICAM-1; IgG1) hybridomas were generously provided by M. Miyasaka (Osaka). mAb were purified from hybridoma culture supernatants or ascites fluid by affinity to Protein G and used either unconjugated or conjugated to biotin or FITC. FITC-conjugated anti-rat CD11a (WT1 clone) was purchased from PharMingen Ltd (Cowley, UK). FITC-conjugated anti-BrdU was purchased from Becton Dickinson Ltd (Cowley, UK). Streptavidin–FITC (Southern Biotechnology Associates, Birmingham, AL) and FITC-conjugated goat anti-mouse Ig (PharMingen) were used as second-stage reagents. Fusion proteins were generously provided by Peter Lane (University of Birmingham, UK) (CTLA-4–Ig) and Gerry Klaus (National Institute for Medical Research, London) (CD40–Ig).

Purification of T lymphocytes
LN cells (LNC) were pooled from cervical, brachial, axillary and mesenteric LN of LOU (RT1u) rats, and T lymphocytes prepared by depletion of B cells and macrophages. LNC were incubated (30 min, 4°C) with 100 µg each of MRC-OX12 and MRC-OX6 to stain B cells and macrophages. After washing in PBS plus 1% FCS, lymphocytes were incubated with sheep anti-mouse IgG coupled Dynabeads M-450 according to manufacturer's instructions (Dynal A.S., Oslo, Norway) on a roller-rocker for 30 min at 4°C and beads with attached cells removed using a magnet. The purity of T lymphocytes as assessed using FITC-conjugated R73 ({alpha}ß TCR) was >97%.

Generation of T lymphoblasts using a bispecific antibody (BsAb)
T lymphoblasts were generated using a BsAb (26) comprising chemically cross-linked Fab' fragments of mAb to rat {alpha}ß TCR (R73) and rat CD2 (MRC-OX54), generously provided by Dr M. Glennie (Tenovus, Southampton, UK). LNC or purified T lymphocytes were cultured in RPMI 1640 supplemented with 5% FCS at 37°C either in 96-well plates (5x105 cells; 0.2 ml/well) or in bulk culture (5x107 cells; 5 ml/25 cm2 flask) in the presence of BsAb (0–1000 ng/ml) for up to 4 days. Proliferation was assessed in 96-well trays by a final 24 h pulse with 1 µCi/well [methyl-3H]thymidine (185 GBq/mmol; Amersham International, Little Chalfont, UK). Incorporated radioactivity was harvested using a Skatron Micro 96 Harvester (Skatron Instruments A.S., Lier, Norway) and counted on a LKB Wallac ß-counter. The effect of antibodies and fusion proteins on T cell proliferation was determined by pre-incubating lymphocytes for 30 min prior to addition of BsAb and incubation for 3 days. Results are expressed as mean ± SD of replicate wells (n = 3–4). For adhesion assays, blasts in 3 day 300 ng/ml BsAb-stimulated bulk cultures were either labelled with [3H]thymidine (see below) or were purified prior to assay. Blasts were first enriched by centrifugation on a discontinuous 50:70:85% Percoll gradient at 800 g for 20 min at 4°C. Blasts harvested from the interface between 50 and 70% Percoll were then sorted by size using a FACStar (Becton Dickinson). Enrichment of T lymphoblasts was assessed by anti-BrdU staining (see below). Lymphoblasts were enriched from 20 to 50% after the Percoll gradient and to >70% after sorting.

Anti-BrdU staining of T lymphoblasts
LNC were incubated for 3 days with 300 ng/ml of BsAb in the presence of 40 µM BrdU and stained as previously described (27). Briefly, after two washes cells were resuspended in 25 µl of ice-cold 0.15 M NaCl, 70 µl ice-cold 95% ethanol was added dropwise while mixing and cells incubated on ice for 30 min. Cells were washed with 100 µl of PBS and incubated at room temperature with PBS/1% paraformaldehyde/0.01% Tween 20 for 30 min. After washing, cells were resuspended in 200 µl of 0.15 M NaCl, 4.2 mM MgCl2, pH 5.0 containing 50 Konitz units/ml DNase I (Sigma, St Louis, MO) and incubated for 45 min at 37°C. Cells were washed, incubated with FITC–anti-BrdU in PBS/5% FCS/0.5% Tween 20 for 30 min on ice and analysed by flow cytometry.

Lymphocyte adhesion to HEC
HEC were plated at 5x103/well of 96-well trays (Nunc, Life Technologies, Paisley, UK) and pretreated in triplicate with IFN-{gamma} (200 units/ml; 72h). Adhesive interactions with lymphocytes were optimized by calculating the number of T cells required to give a monolayer covering HEC (13); for T lymphoblasts 10-fold fewer cells were required. T lymphocyte and T lymphoblast adhesion was compared by expressing the number of adherent T cells as a percentage of the number plated. For the assay, T lymphocytes were labelled with [3H]leucine as described previously (18) and lymphoblasts were labelled at 25x106 BsAb stimulated cells/ml for 60 min at 37°C with 10 µCi/ml [methyl-3H]thymidine (185 GBq/mmol; Amersham International). Lymphoblasts in BsA6-stimulated cultures were distinguished by size and counted in a haemocytometer. Cells were resuspended at 107 T lymphocytes/ml or 106 T lymphoblasts/ml in assay medium (HEPES-buffered RPMI 1640 plus 1% FCS), and incubated in triplicate on HEC for 60 min at 37°C (0.1 ml/well). Non-adherent lymphocytes were removed by washing 5 times using Dulbecco's/PBS plus 1% FCS. HEC and adherent lymphocytes were solubilized in 25 µl 95% formic acid, the samples transferred to filters and counted on a LKB Wallac ß-counter. HEC-associated radioactivity was expressed as a percentage of the total plated to give percent lymphocyte adhesion; results are means ± SD (n = 4–6). The effects of mAb to adhesion molecules on HEC were determined by pre-incubating HEC with 50 µl mAb at twice the final concentration prior to addition of an equal volume of lymphocytes (2x107 T cells/ml or 2x106 T lymphoblasts/ml) and the assay continued as described above. The effects of mAb to adhesion molecules on lymphocytes were determined by pre-incubating lymphocytes with mAb for 30 min at 4°C and warming to 37°C for 15 min, prior to the assay in the continued presence of mAb. Results are expressed as percent inhibition of adhesion with the specific antibody relative to that obtained with an isotype-matched control antibody and are means ± SD (n = 4–6).

Microscope assay to distinguish between surface-bound and migrated lymphocytes
Light microscope analysis of HEC-adherent lymphocytes can distinguish between lymphocytes bound to the surface of HEC and lymphocytes which have migrated below the HEC layer (15). HEC were plated at 5x103/well in 96-well trays and treated with IFN-{gamma} (200 units/ml; 72 h). T lymphocytes were resuspended at 107/ml and T lymphoblasts at 106/ml in assay medium, and 0.1 ml of the suspension was incubated in triplicate on HEC for 1 h at 37°C. The wells were washed 5 times using Dulbecco's/PBS plus 1% FCS, HEC layers fixed for 30 min with 1% glutaraldehyde, washed and stored in PBS/azide. Cells were viewed on a phase-contrast microscope (CK-2 Olympus) using x20 objective and surface-bound (type I) lymphocytes which were small, round and phase light were distinguished from migrated (type II) cells which were larger, flattened and phase dark. Lymphocytes which were seen in transition from surface bound to migrated were recorded as migrated. A graticule of 0.137 mm2 was used to count numbers of type I and type II lymphocytes in replicate wells. Results are expressed as mean number of lymphocytes/mm2 ± SEM (n = 4–12). The migration index was calculated as follows: no. type II lymphocytes/no. type I + type II lymphocytes.

Lymphocyte adhesion to recombinant soluble human VCAM-1
Triplicate wells of 96-well trays were coated overnight at 4°C with 10 µg/ml VCAM-1 (generously provided by Dr R. Lobb, Biogen) in PBS. Non-specific binding sites were blocked for a minimum of 1 h with 10 mg/ml heat-inactivated BSA (85°C; 10 min) in PBS at room temperature and the wells washed twice with assay medium. [3H]leucine-labelled lymphocytes resuspended at 107/ml or [3H]thymidine-labelled T lymphoblasts at 106/ml were incubated in triplicate (100 µl/well), in the presence or absence of mAb, for 1 h at 37°C. Non-adherent cells were removed and the wells washed 3 times with PBS plus 1% FCS. Adherent cells were solubilized in 25 µl 95% formic acid, processed for ß-scintillation counting and the percent lymphocyte adhesion calculated as described using HEC. Adhesion to BSA-coated wells was typically <1% and was subtracted from adhesion to VCAM-1-coated wells to give percent specific adhesion. The effects of antibodies were expressed as percent inhibition of adhesion, which was calculated as described using HEC.

Staining of HEC and lymphocytes for flow cytometric analysis
HEC plated at 105/well in 35 mm diameter six-well cluster trays (Nunc) were pretreated with IFN-{gamma} (200 units/ml; 72 h) and incubated with or without lymphocytes (5x106 T lymphocytes/ml or 5x105 T lymphoblasts/ml; 2 ml/well) for 60 min at 37°C. The wells were washed 5 times using Dulbecco's/PBS plus 1% FCS and HEC or HEC plus adherent lymphocytes were detached using 0.1% EDTA in PBS and stained as described previously (18). Briefly, cells were washed twice with PBS plus 1% FCS, resuspended in 50 µl of primary antibody diluted in PBS/0.1% BSA/0.1% azide and incubated on ice for 30 min. After washing, cells were stained with 50 µl of FITC-conjugated goat anti-mouse Ig plus 10% normal rat serum for 30 min on ice. After three washes, cells were fixed with 1% formaldhedyde in PBS and stained cells analyzed on a Becton Dickinson FACStar Plus or FACS Vantage. Data were analysed using FACSplot software developed by John Green (Computing Laboratory, NIMR) or using WinMDI (Joseph Trotter, Scripps Institute, CA).

Statistical analysis
Student's t-test was used to compare groups of data.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Characterization of BsAb generated T lymphoblasts
BsAb stimulated proliferation of LN lymphocytes in a dose-dependent manner with maximal proliferation at >100 ng/ml after 3 days (Fig. 1Go). The lymphoblast population generated was identified by increased forward and side scatter profiles, CD25 expression, and by BrdU incorporation (Fig. 2Go). Lymphoblasts were enriched in CD8+ over CD4+ cells, and the majority expressed {alpha}ß TCR, CD5 and CD25. BsAb-stimulated [3H]thymidine uptake was partially inhibited by anti-LFA-1 (mAb WT1) or CTL4-A–Ig fusion protein and completely inhibited by the combination (Fig. 1Go). Antibodies to ICAM-1 (mAb 1A29) maximally inhibited [3H]thymidine uptake by 51%, which was similar to the level of inhibition by anti-LFA-1, whereas antibodies to {alpha}4 integrins (mAb HP2/1) had no effect (data not shown). Purified T lymphocytes were not stimulated to proliferate by BsAb; however, PdBu/ionomycin stimulated similar levels of proliferation in T cells and LNC (Fig. 1Go). Together these results suggest that BsAb-stimulated T cell proliferation is dependent on accessory cells, analogous to antigen receptor-driven proliferation. We have therefore used BsAb generated T lymphoblasts as a source of antigen-activated T cells for studies of interactions with cultured HEC.



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Fig. 1. The proliferation of lymphocytes stimulated by {alpha}ß TCRxCD2 BsAb. LNC were incubated with (A) a range of concentrations of BsAb for 72 h or (B) 100 ng/ml BsAb (closed symbols) or no BsAb (open symbols) for up to 120 h. The proliferation of LNC and purified T lymphocytes to 100 ng/ml BsAb or 100 nM PdBu + 500 ng/ml ionomycin after 72 h is shown in (C). The effects of 10 µg/ml anti-LFA-1 mAb and CTL4-A–Ig on BsAb-induced proliferation of LNC are shown in (D). Isotype-matched mAb MRC-OX12 and CD40–Ig were used as controls. Results are mean c.p.m. ± SD (n = 3–6). *P < 0.001 compared to proliferation in the absence of BsAb.

 


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Fig. 2. Analysis of BsAb generated lymphoblasts. Lymphoblasts were generated using 300 ng/ml BsAb for 72 h, and enriched by density gradient centrifugation and FACS sorting. LN cells (A) and lymphoblasts (B) were analysed for size, subset composition and integrin expression by flow cytometry. Forward and side scatter profiles are presented as dot-plots; the composition and integrin expression of each population is given below. For subset analysis, results are mean percent positive cells ± SEM (n = 3–10). For integrin expression results are percent positive cells with mean fluorescence intensity in parentheses from one representative experiment. n.d., not determined. (C) Expression of CD25 by lymphoblasts (open histogram) and LNC (solid histogram), and (D) staining of lymphoblasts for incorporated BrdU (open histograms) in comparison with controls (solid histogram) are also shown for a single representative experiment.

 
T lymphocyte and T lymphoblast adhesion to HEC
Previous studies have shown that IFN-{gamma} pretreatment of cultured HEC increases the overall adhesion of T and B lymphocytes, and that the numbers of surface-bound and transmigrated lymphocytes are both increased (16). Although the role of cytokines in regulating HEC activity in vivo is not well understood (28), systemic administration of IFN-{gamma} in rats increases the migration of T and B lymphocytes from the blood into LN via HEV (29). We have therefore used IFN-{gamma} to increase T cell–HEC interactions to facilitate analysis of the separate events of lymphocyte binding and transendothelial migration (Table 1Go). VCAM-1 and ICAM-1 expression by HEC were both up-regulated by IFN-{gamma} pretreatment. T lymphocyte adhesion was increased 2.5-fold by IFN-{gamma} pretreatment of HEC and the percentage of adherent T cells that transmigrated was increased from 28 to 44%. The adhesion and transmigration of T lymphoblasts on unactivated HEC were higher than for T lymphocytes, and were not increased further by IFN-{gamma} pretreatment of HEC. The increased binding and transmigration of T lymphoblasts was not simply dependent on carry-over of BsAb since addition of up to 1 µg/ml BsAb had no effect on interactions of either T lymphoblasts or T lymphocytes with HEC (data not shown). The lack of effect of IFN-{gamma} pretreatment of HEC was not due to saturation of binding sites since the percentage of T lymphoblasts adhering to HEC was similar if the number of cells plated was reduced by 75% (data not shown).


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Table 1. Effect of IFN-{gamma} on lymphocyte interactions and adhesion molecule expression by HEC
 
Antibodies to {alpha}4 integrins and VCAM-1 partially inhibit adhesion of T lymphocytes and T lymphoblasts to HEC
mAb against several different cell surface molecules expressed by lymphocytes (CD45), by HEC (MHC class II), by both cell types (CD44, MHC class I) or non-cross-reacting antibodies (anti-human HLA) either alone or in combination had no effect on T lymphocyte or T lymphoblast adhesion to HEC (data not shown) and were therefore used as control reagents. Antibodies to either {alpha}4 integrins or VCAM-1 showed dose-dependent inhibition of adhesion of T lymphocytes and T lymphoblasts; however, the levels of inhibition were greater for T lymphocytes than T lymphoblasts (Fig. 3Go). For example, inhibition of T lymphoblast adhesion by anti-{alpha}4 integrin saturated using 30 µg/ml at 23 ± 8%, whereas inhibition of T lymphocytes was higher at 40 ± 2% (P < 0.001). Similar effects were found using anti-VCAM-1. A combination of anti-{alpha}4 integrin and anti-VCAM-1 antibodies did not inhibit adhesion further. Although the adhesion of T lymphocytes to unactivated HEC was lower, antibodies to {alpha}4 integrins and anti-VCAM-1 gave similar levels of inhibition to those measured using cytokine activated HEC (data not shown).



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Fig. 3. The effect of antibodies to {alpha}4 integrins and VCAM-1 on adhesion of T lymphocytes and T lymphoblasts to HEC and VCAM-1. The adhesion of T lymphocytes (open symbols) and T lymphoblasts (closed symbols) to cultured HEC (A and B) or immobilized VCAM-1 (C and D) was measured in the presence of antibodies to {alpha}4 integrin (A and D), VCAM-1 (B) or in the absence of antibody (C). Effects of antibodies were calculated as percent inhibition of adhesion relative to isotype-matched control antibodies. Results are mean percent inhibition of adhesion (A, B and D) or mean percent adhesion (C) ± SD (n = 4–6). *P < 0.05 between T lymphoblasts and T lymphocytes.

 
The low levels of inhibition by antibodies to {alpha}4 integrins and VCAM-1, particularly of T lymphoblast adhesion, raised the possibility that rat T cells may not utilize this adhesion pathway. However, as shown in Fig. 3Go, T lymphoblasts bound to VCAM-1 and the level of binding was 3-fold higher than that of unactivated T lymphocytes. T lymphocyte and T lymphoblast adhesion to VCAM-1 was inhibited by antibodies to {alpha}4 integrins, and maximal inhibition occurred at 1–3 µg/ml, which was 10-fold lower than the dose required to block adhesion to HEC.

Antibodies to LFA-1 partially inhibit the adhesion of T lymphocytes and T lymphoblasts; antibodies to ICAM-1 have little effect
Antibodies to LFA-1 inhibited the adhesion of T lymphocytes and T lymphoblasts to HEC in a dose-dependent manner (Fig. 4Go). The effect was greater on T lymphocytes than T lymphoblasts and maximal inhibition was similar to that found using anti-{alpha}4 integrin antibody (43% for T lymphocytes and 23% for T lymphoblasts). Antibodies to ICAM-1 had little effect on the adhesion of T lymphocytes or T lymphoblasts (Fig. 4Go). At 60 µg/ml, the highest dose tested, T lymphocyte adhesion was maximally inhibited by 18 ± 8% and T lymphoblast adhesion by 12 ± 6%. A combination of anti-LFA-1 and anti-ICAM-1 antibodies did not inhibit adhesion further. Antibodies to LFA-1 and ICAM-1 gave similar levels of inhibition using unactivated HEC (data not shown).



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Fig. 4. The effect of antibodies to LFA-1/ICAM-1 and {alpha}4 integrins/VCAM-1 on T lymphocyte and T lymphoblast adhesion to HEC. The adhesion of T lymphocytes (open symbols) and T lymphoblasts (closed symbols) to cultured HEC was determined in the presence of antibodies to LFA-1 (A), ICAM-1 (B), LFA-1 plus {alpha}4 integrins (C) or ICAM-1 plus VCAM-1 (D). Effects of antibodies were calculated as percent inhibition of adhesion relative to isotype-matched control antibodies and results are mean percent inhibition ± SD (n = 4–6). *P < 0.05 between T lymphoblasts and T lymphocytes.

 
Since antibodies to {alpha}4 integrins, VCAM-1, LFA-1 or ICAM-1 had partial effects on T lymphocyte and T lymphoblast adhesion to HEC, the expression of these adhesion molecules before and after the adhesion assay was determined. The majority of T lymphocytes and T lymphoblasts expressed {alpha}4 integrins and LFA-1; however, the level of expression was higher on T lymphoblasts (Fig. 2Go). In comparison with the starting population (Fig. 2Go), the number of cells expressing {alpha}4 integrins and the level of expression was not altered on T lymphocytes (98% positive; MFI 452) or T lymphoblasts (99%; MFI 645) that adhered to HEC. Similarly, the number of cells expressing LFA-1 and the level of expression was not altered on adherent T lymphocytes (96%; MFI 485) or T blasts (99%; MFI 692). The number of HEC expressing VCAM-1 or ICAM-1 and the levels of expression were not affected by T lymphocyte or T lymphoblast adhesion.

A combination of antibodies to {alpha}4 integrins and LFA-1 completely inhibits adhesion of T lymphocytes and T lymphoblasts to HEC
To determine whether the roles of {alpha}4 integrins and LFA-1 in T lymphocyte interactions with HEC are additive or synergistic, a combination of antibodies specific for both integrins was used (Fig. 4Go). The combination of HP2/1 and WT1 showed dose-dependent inhibition of binding of T lymphocytes and T lymphoblasts to HEC, and adhesion of both cell populations was inhibited by >90% at 30 µg/ml antibody mixture; however, T lymphocyte adhesion was more sensitive to inhibition by the combination of antibodies. The combined effect of antibodies to {alpha}4 integrins and LFA-1 on T lymphocyte adhesion equalled the sum of their individual effects, whereas the combined effect of antibodies to {alpha}4 integrins and LFA-1 on T lymphoblast adhesion was greater than the sum of their individual effects (Table 2Go). A combination of antibodies to VCAM-1 and ICAM-1 showed dose-dependent inhibition of T lymphocyte and T lymphoblast adhesion which was greater than that with either antibody alone (Fig. 4Go); however, at saturating doses of antibodies, inhibition was not complete and the effect was greater on T lymphocytes (79%) than T lymphoblasts (63%).


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Table 2. Inhibition of T cell adhesion to HEC by antibodies to {alpha}4 integrins and LFA-1
 
Effects of antibodies to {alpha}4 integrins and LFA-1 on transmigration of T lymphocytes and T lymphoblasts across HEC
The transendothelial migration assay is based on microscopic analysis of surface-bound and transmigrated lymphocytes which differ according to phase contrast appearance and size. BsAb-stimulated cultures contained a mixture of large and small lymphocytes which would be difficult to distinguish after transmigration. Highly enriched populations of lymphoblasts were therefore used for the migration assay. The percentage of T lymphoblasts plated that bound to HEC was higher than T lymphocytes, as found using radiolabelled lymphocytes; however, the number of adherent T lymphoblasts was lower (Fig. 5Go) since 10-fold fewer cells were incubated with HEC (see Methods). As shown in Fig. 5Go, the effects of antibodies to {alpha}4 integrins and LFA-1 on the total number of adherent cells (surface bound and transmigrated) were similar to data obtained using radiolabelled lymphocytes. T lymphocytes transmigrated HEC in the presence of antibodies to {alpha}4 integrins or LFA-1 (Fig. 5Go); however, the migration index was slightly increased in the presence of anti-{alpha}4 integrin antibody and significantly reduced in the presence of anti-LFA-1. Antibodies to {alpha}4 integrins or LFA-1 had no effect on T lymphoblast transmigration.



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Fig. 5. The effect of antibodies to {alpha}4 integrins and LFA-1 on transendothelial migration of T lymphocytes and T lymphoblasts. T lymphocytes (A) and T lymphoblasts (B) were incubated with cultured HEC in the presence of antibodies to {alpha}4 integrins and LFA-1, and the numbers of surface-bound and migrated cells determined by microscopy. Results presented are mean numbers of T cells/mm2 ± SEM (n = 4–12). Bars give total number of adherent cells and within each bar surface-bound (hatched area) and migrated (filled area) cells are presented. Percent inhibition and migration index for each antibody treatment is given below. Results are means ± SEM (n = 5). *P < 0.05 compared to control antibodies.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report we have used primary cultures of HEC derived from rat peripheral LN HEV to start to identify the cell adhesion molecules that regulate transendothelial migration of lymphocytes across HEV. Since transendothelial migration is preceded by binding to the surface of HEC (15), the numbers of lymphocytes that bind (type I cells) and the numbers that transmigrate (type II cells) are measured to determine if effects on transendothelial migration are specific or simply reflect altered binding to the surface of HEC. If binding and transendothelial migration are regulated by the same cell adhesion molecules they will not be distinguishable in this assay.

The overall adhesion (i.e. binding and transmigration) of T lymphocytes or T lymphoblasts was inhibited by >90% using a combination of antibodies to {alpha}4 integrins and LFA-1. However, there were clear differences in the use of {alpha}4 integrins and LFA-1 by T lymphocytes and T lymphoblasts in binding and transendothelial migration. The majority of T lymphoblasts used either {alpha}4 integrins or LFA-1 to bind to HEC, and T lymphoblasts transmigrated normally when either integrin was blocked, suggesting overlapping and redundant roles for {alpha}4 integrins and LFA-1 in binding and transendothelial migration. In contrast, the roles of {alpha}4 integrins and LFA-1 on unactivated cells were distinguishable since either integrin supported half-maximal binding of T lymphocytes to HEC and LFA-1 made a larger contribution to transendothelial migration of T lymphocytes than {alpha}4 integrins. Since both integrins mediated, at least partial, binding of unactivated or activated T cells to the surface of HEC we cannot formally exclude the possibility that transendothelial migration is regulated by additional adhesion molecules. In addition, T lymphocytes using LFA-1 to bind to HEC may represent a distinct, highly migratory subpopulation and those using {alpha}4 integrins a less migratory population. Further experiments will be required to resolve these possibilities.

Antibodies to VCAM-1 gave similar levels of inhibition as antibodies to {alpha}4 integrins, suggesting that VCAM-1 is the major ligand on HEC for {alpha}4 integrins, as reported previously (18). Identification of the {alpha}4 integrin awaits generation of suitable reagents to distinguish between {alpha}4ß1 and {alpha}4ß7 in the rat. Further studies will be required to identify the ligands for LFA-1 on peripheral LN HEV since antibodies to ICAM-1 had little effect on T lymphocyte or T lymphoblast adhesion to cytokine-activated HEC, even though ICAM-1 expression was substantially up-regulated by IFN-{gamma}. Antibody blockade of ICAM-1 in mice has little effect on lymphocyte migration to peripheral LN (A. Hamann, pers. commun.) indicating the use of alternative ligands for LFA-1, such as ICAM-2. Recent studies in ICAM-2 knockout mice have shown that lymphocyte migration into peripheral LN is normal (30), raising the possibility that ICAM-1 and ICAM-2 play redundant roles in lymphocyte recruitment by HEV.

The assay of T cell–HEC interactions used in this study does not take into account shear stress at the HEV–blood interface and therefore it is difficult to extrapolate these results to the in vivo situation. However, recent elegant studies of lymphocytes interacting with HEV in mice using intravital microscopy have confirmed that LFA-1 mediates the pertussis toxin-sensitive activation-dependent arrest of rolling lymphocytes on the inner surface of the vessel wall (11). Antibody blockade of LFA-1 in rats (31) and mice (19) maximally inhibited lymphocyte migration to peripheral LN by 60%, suggesting a role for other adhesion molecules. Early reports failed to detect VCAM-1 expression by HEV in mice (32), and antibodies to {alpha}4 integrins and VCAM-1 had no effect on migration to peripheral LN (33). In contrast, in the rat VCAM-1 is expressed by peripheral LN HEV (18), the migration of CD4+CD45RClo T cells to peripheral LN is partially blocked by antibodies to VCAM-1 (34), and the migration of T blasts (35), small peritoneal exudate lymphocytes (35) and CD4+CD45RClo T cells (34) to peripheral LN are partially blocked by antibodies to {alpha}4 integrins. Recent studies in mice using LFA-1–/– lymphocytes have demonstrated a clear role for {alpha}4 integrin/VCAM-1 interactions in lymphocyte migration to peripheral LN and confirmed the expression of VCAM-1 by HEV in these organs (17). Together these results suggest that {alpha}4 integrin/VCAM-1 interactions regulate the migration of unactivated and activated T lymphocytes to peripheral LN.

The integrins used by activated T cells and their ability to transmigrate EC vary significantly according to the activating stimulus used (24,36,37 and this report). We chose to use BsAb-induced T lymphoblasts since the mechanism of their generation was similar to that following antigen engagement of the TCR. The higher proportion of T lymphoblasts adhering to HEC may be simply due to the higher level of expression of {alpha}4 integrin and LFA-1 by T lymphoblasts and increased affinity of these integrins following TCR engagement. The 2- to 3-fold increase in binding of T lymphoblasts to immobilized VCAM-1 supports this hypothesis. However, the adhesion of T lymphoblasts to HEC was not simply dependent on VCAM-1/ICAM-1 interactions since the levels of adhesion and transmigration were not affected by IFN-{gamma} treatment of HEC, which increases both VCAM-1 and ICAM-1 expression. Trafficking experiments in mice have shown that activated T lymphocytes localize less efficiently than unactivated lymphocytes in LN, suggesting that they have lost the ability to bind and/or transmigrate HEV. Studies using isolated, perfused rat LN have shown clearly that activated cells migrate more efficiently than unactivated cells (38), in agreement with the results reported here using cultured HEC. Activated lymphocytes enter non-lymphoid organs such as the lungs and liver; it is therefore possible that reduced migration to LN in intact animals reflects preferential migration to other organs.

The expression and functions of {alpha}4 integrins and LFA-1 are down-regulated on T cells that have transmigrated other types of EC (39); however, we did not find any alterations in expression of {alpha}4 integrins or LFA-1 by T lymphocytes and T lymphoblasts following binding and transmigration across HEC. We have previously suggested that {alpha}4 integrin activation may be required for lymphocyte transmigration across HEC (15); however, the antibody used in this study inhibits {alpha}4 integrins irrespective of activation state; reagents that selectively inhibit activated {alpha}4 integrins are required to test this hypothesis. Thus far, studies using several different sources of EC have not implicated {alpha}4 integrins or VCAM-1 in lymphocyte transendothelial migration (22,37,40); however, a single report using rat epididymal fat pad EC has shown {alpha}4 integrins to be required for the transmigration, but not the binding, of T cell clones (41).

In contrast, LFA-1 and/or ICAM-1 have been implicated in the transendothelial migration of unactivated and activated T lymphocytes across all types of EC studied including human umbilical vein EC (21,22), rat retinal capillary EC (37) and mouse brain capillary EC (40). The transendothelial migration of activated T lymphocytes across human EC promoted by activating antibodies to ICAM-3 or by chemokines is also LFA-1 dependent (42). Therefore, HEC are distinguishable from other types of EC in that the transendothelial migration of T cells was not exclusively dependent on LFA-1 and/or ICAM-1. The molecular mechanisms utilized by lymphocytes to transmigrate HEC may differ from those used to transmigrate other types of EC, particularly with respect to the use of chemokine receptors and the route of transendothelial migration (i.e. intercellular versus transcellular) (43). It is also possible that distinct subpopulations of T lymphocytes interact with HEC and with other types of EC using different adhesion (44) and signalling molecules (45) to bind and transmigrate. In conclusion, these results show that T cell transmigration across HEV into LN is not exclusively dependent on LFA-1/ICAM-1 and that {alpha}4 integrin/VCAM-1 interactions and possibly other adhesion molecules regulate this stage of lymphocyte diapedesis.


    Acknowledgments
 
We thank Graham Preece for help with adhesion assays, Chris Atkins for assistance with flow cytometry and the Photographics Department, NIMR for the figures. We would like to thank Martin Glennie, Masa Miyasaka, Paco Sanchez-Madrid and Roy Lobb for their generous gifts of antibodies. This work was funded by the Medical Research Council (UK). M. Di M. was the recipient of a PhD studentship from Zeneca.


    Abbreviations
 
BsAb bispecific antibody
EC endothelial cell
HEC high endothelial cell
HEV high endothelial venule
LN lymph node
LNC lymph node cells
MFI mean fluorescence intensity

    Notes
 
3 Present address: Centre d'Immunologie et de Biologie Parasitaire, INSERM U167, Institut Pasteur de Lille, 59019 Lille Cedex, France. Back

Transmitting editor: T. Hünig

Received 14 June 1999, accepted 1 November 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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