The CD44-initiated pathway of T-cell extravasation uses VLA-4 but not LFA-1 for firm adhesion

Leukocytes extravasate from the blood in response to physiologic or pathologic demands by means of complementary ligand interactions between leukocytes and endothelial cells. The multistep model of leukocyte extravasation involves an initial transient interaction (“rolling” adhesion), followed by secondary (firm) adhesion. We recently showed that binding of CD44 on activated T lymphocytes to endothelial hyaluronan (HA) mediates a primary adhesive interaction under shear stress, permitting extravasation at sites of inflammation. The mechanism for subsequent firm adhesion has not been elucidated. Here we demonstrate that the integrin VLA-4 is used in secondary adhesion after CD44-mediated primary adhesion of human and mouse T cells in vitro, and by mouse T cells in an in vivo model. We show that clonal cell lines and polyclonally activated normal T cells roll under physiologic shear forces on hyaluronate and require VCAM-1, but not ICAM-1, as ligand for subsequent firm adhesion. This firm adhesion is also VLA-4 dependent, as shown by antibody inhibition. Moreover, in vivo short-term homing experiments in a model dependent on CD44 and HA demonstrate that superantigen-activated T cells require VLA-4, but not LFA-1, for entry into an inflamed peritoneal site. Thus, extravasation of activated T cells initiated by CD44 binding to HA depends upon VLA-4–mediated firm adhesion, which may explain the frequent association of these adhesion receptors with diverse chronic inflammatory processes.

Coordinated regulation and appropriate site-specific targeting of leukocyte extravasation results from various types of adhesion receptors acting in sequential fashion, allowing directed leukocyte exit from the blood into diverse tissues within the organism (1, 2). Thus, the elucidation of molecules mediating the extravasation of leukocytes into targeted sites is central to the understanding of the complex regulation of the host response to inflammation. Several families of adhesion receptors are thought to be essential in this multistep model of extravasation for both nonspecific effector cells and antigen-specific lymphocytes. The initial contact with and primary adhesion (“rolling”) of leukocytes on vascular endothelium has generally been attributed to the engagement of members of the selectin family by their carbohydrate ligands. Subsequent secondary (firm) adhesion is largely due to heterodimeric integrins interacting with their endothelial cell ligands, members of the immunoglobulin gene superfamily (3, 4). After arrest, leukocytes migrate through the endothelial and basement membrane barriers into underlying tissues.

Tissue-specific migration pathways of effector and memory T-lymphocyte subsets have been shown to be distinct, and in part attributable to adhesion receptors that they express (5–9). Our laboratory has been engaged in the description and characterization of a pathway of extravasation initiated by CD44 on lymphocytes activated to bind its major ligand, hyaluronan (HA). CD44 interactions with HA expressed on endothelial cells permit lymphocyte rolling under shear conditions simulating physiologic flow (10). Although the mechanism by which CD44 becomes activated to bind HA has not yet been completely elucidated, it has become clear that stimulation of normal T lymphocytes in vitro or in vivo through the T-cell receptor induces the activated form of CD44 and attendant primary adhesion on endothelium or HA substrate (11–13). These observations have established the HA-binding form of CD44 as an early activation marker on T cells after T-cell receptor stimulation, and support a role for this interaction during the course of an immune response. Moreover, CD44/HA interactions have been shown to be required for extravasation of superantigen-stimulated T cells into an inflamed peritoneal site in a mouse model (12). CD44 has been prominently associated with human arthritis and with a model of collagen-induced murine arthritis (14–17), and more recently with a model of multiple sclerosis, experimental allergic encephalomyelitis (18). CD44 interactions with HA are also thought to be important in allogeneic graft rejection (19, 20). We have proposed that this CD44-initiated pathway is used primarily by activated T cells for egress into a variety of inflamed tissues. Consistent with this suggestion, the presence of circulating T cells bearing the activated form of CD44 that enables HA-dependent rolling correlates highly with exacerbation of major autoimmune disease in humans (15). Evidence suggests that HA binding through CD44 can be regulated through mechanisms such as glycosylation and a variety of other posttranslational modifications (21–25), but the particular molecular basis for CD44 activation of this subset of T cells has not been clarified.

The predominant interactions observed in laminar flow assays between activated T cells bearing CD44 and an HA substrate have been those of primary adhesion. For physiologic relevance, these interactions would require subsequent firm adhesion and arrest to achieve extravasation, as predicted by our model and by in vivo evidence (12). Because secondary adhesion preceded by selectin-mediated primary adhesion is generally mediated by integrins, we chose to examine the 2 integrins that are most prominently expressed on most circulating T cells — VLA-4 (α4β1) and LFA-1 (αLβ2) — and their ligands as candidates for secondary adhesion subsequent to CD44-mediated rolling. We report that in these studies, VLA-4 is the major integrin used in this pathway both in vitro and in an in vivo model, whereas LFA-1 contributes little to this process.

The activated form of CD44 is associated with elevated VLA-4 expression on activated leukocytes. The 3 cell types used for these studies — the murine T cell line SAKRTLS12, the human lymphoblastoid line KCA, and normal HuPBT — were chosen based on their coexpression of the common leukocyte integrins α4β1 (VLA-4) and αLβ2 (LFA-1), as well as for their ability to convert CD44 to its activated (HA-binding) form after polyclonal stimulation (10, 32). Although CD44 is expressed at significant levels on resting HuPBT, it does not bind Fl-HA (Figure 1a). After overnight polyclonal stimulation, a significant percentage of the CD44⁺ cell population acquires the ability to bind Fl-HA (10, 32). As shown, the number of cells with the Fl-HA–binding phenotype was particularly increased among the VLA-4⁺ cells after stimulation, although overall VLA-4 levels diminished somewhat (Figure 1a). Thus, activation of CD44 to the HA-binding form is preferentially associated with cells expressing higher levels of VLA-4. This is further detailed in Figure 1b, where 3-color analysis demonstrates that CD44⁺ Fl-HA–binding cells express VLA-4 at higher levels than are seen in the total population (top). Likewise, it can be seen that CD44^hi/VLA-4^hi double-positive cells are predominantly positive for Fl-HA binding (bottom), a finding also seen in SEB-stimulated mouse lymphocytes (see Figure 4a).

Figure 1

Fl-HA binding by HuPBT. (a) Untreated cells (Unstim) or cells treated with PMA and ionomycin for 18 hours were stained with Fl-HA plus either CD44-PE or VLA-4/GaRIg-PE as indicated, and then analyzed by 2-color FACS^®. Few Fl-HA⁺ cells are seen among untreated HuPBT cells, whereas a distinct population of positive cells is seen after activation. Fl-HA–binding cells are found predominantly in both the CD44⁺ (top) and VLA-4⁺ (bottom) populations. Fl-HA binding by both CD44⁺ and VLA-4⁺ activated T cells is blocked by incubation with blocking anti-CD44 before staining. (b) Three-color immunofluorescence analysis of activated T cells demonstrates that the CD44^hi/Fl-HA⁺ population is also the VLA-4^hi population. Top: Histograms show the VLA-4–PE staining of cells stained with Fl-HA, VLA-4–PE, and CD44/SA-R670. The VLA-4–PE staining of gated CD44/Fl-HA double-positive cells (circled) is indicated by the solid histogram; the dashed histogram reflects the VLA-4–PE staining of the total population. Bottom: The CD44/VLA-4 double-positive population (red, circled) is contained predominantly in the CD44/Fl-HA double-positive population.

Figure 4

VLA-4 participates in the short-term homing of activated lymphocytes to an inflamed site. (a) Mesenteric lymph node cells from SEB-treated mice were stained with Fl-HA plus either CD44-PE, VLA-4/GaRIg-PE, or LFA-1/GaRIg-PE as indicated. Fl-HA–binding cells are present predominantly in both the CD44⁺ and VLA-4⁺ populations, but are distributed between both LFA-1⁺ and LFA-1^– populations. Fl-HA binding is blocked by incubation with blocking anti-CD44 before staining. (b) CFDA-labeled mesenteric lymph node cells from SEB- or PBS-treated mice were injected intravenously into recipient mice that had received an intraperitoneal injection of SEB 20 hours earlier. Cells from donor mice were injected without antibody or with HA-blocking anti-CD44 monoclonal antibody, blocking anti–VLA-4α, blocking anti–LFA-1, or isotype-matched control anti–H-2 monoclonal antibody. Two hours after donor cell injection, peritoneal cavities of recipient mice were lavaged, and recovered cells were analyzed by FACS^® for CFDA fluorescence. Data are shown as the number of fluorescent cells per 100,000 cells analyzed, and represent the mean ± SEM of 3 separate experiments (≥ 3 mice/group per experiment). No peritoneal accumulation of fluorescent cells was seen when either anti-CD44 or anti–VLA-4 antibody was added to the injected cell suspension. Anti–LFA-1 and control anti–H-2 had no effect on cell migration into peritoneal cavities.

CD44/HA–mediated primary adhesion is followed by VLA-4/VCAM-1–dependent secondary adhesion under shear forces. To examine firm adhesion directly under laminar flow after CD44-initiated rolling adhesion, plates were coated with HA simultaneously with the VLA-4 and LFA-1 integrin ligands sVCAM-1 and sICAM-1, respectively. Thus, activated murine and human lines and normal human T cells were analyzed for both primary and secondary adhesion under laminar flow on the substrates HA, sVCAM-1, HA plus sVCAM-1, and HA plus sICAM-1. Results of these analyses are shown in Figure 2. Activated SAKR cells exhibit primary adhesion on plates coated with HA alone, and this rolling can be completely inhibited with blocking anti-CD44 antibody, but not with isotype-matched control anti–LFA-1 (Figure 2a) (10). Both of the human cell populations (KCA and HuPBT) also rolled on HA after stimulation with PMA and ionomycin; this was also CD44 dependent (Figure 2, b and c). No substantial firm adhesion was noted with any of the cells tested on HA substrate alone, again confirming that the CD44/HA interaction is involved primarily in rolling adhesions.

Figure 2

Primary (rolling) and secondary (firm) adhesion of murine SAKR (a), human KCA (b), and HuPBT (c) cells on soluble substrates under physiologic shear stress. Cells were applied to feed solution already equilibrated under flow and then perfused over the indicated individual or combined substrates in the parallel plate flow assay. Rolling and sticking were analyzed as described in Methods, and the number of rolling cells/field per minute and the number of bound cells/field were determined for each substrate. Results are compared to control interactions (indicated with asterisks), and presented as a percentage of the total (100%) control. Mean absolute values (100%) for rolling (given in cells/field per minute) and sticking (given in cells/field), respectively, were: SAKR, 14.6 and 9.7; KCA, 40 and 35.5; HuPBT, 9.6 and 9.8. For blocking, anti-CD44, anti–VLA-4, anti–LFA-1 (Cont), or EDTA was added to the cell suspension before introduction into the flow system. With each cell type, firm adhesion occurred only on plates coated with HA plus sVCAM-1. All interactions were blocked with anti-CD44, whereas only anti–VLA-4 was able to block sticking without affecting rolling. EDTA also inhibited firm adhesion. (d) Expression of integrins VLA-4 and LFA-1 on SAKR, KCA, and HuPBT cells. Cells were stained with primary antibody plus GaRIg-PE or GaMIg-PE for FACS^® analysis. All 3 cell types were positive for both VLA-4 (dashed lines) and LFA-1 (solid lines). Stippled lines represent control staining. (e) Primary and secondary adhesion of anti-CD3–activated murine lymph node cells on soluble substrates under physiologic shear stress. Assays were performed and analyzed as above. Mean absolute values (100%) for rolling and sticking, respectively, were 15.1 cells/field per minute and 72.8 cells/field. Substr, substrate; Block, blocking antibody or EDTA.

On sVCAM-1 alone, detectable but minimal direct firm adhesion, and essentially no rolling, was observed (Figure 2). In contrast, when each of these cell populations was assayed on a substrate of HA plus sVCAM-1, significant numbers of firmly adherent cells were seen in addition to rolling cells (Figure 2, a–c), indicating that sVCAM-1 can function as a secondary adhesion ligand after primary adhesion mediated by CD44/HA. In addition, 22–46% of total adherent cells at 4 minutes were observed to convert from rolling to firmly adherent. To confirm that the lymphocyte receptor for this interaction was the integrin VLA-4, cells were pretreated with anti–VLA-4 before the shear stress assay. Anti–VLA-4 antibody treatment (M17/4 for mouse cells and L25 for human cells) completely inhibited firm adhesion in each case, but had little effect on CD44-dependent rolling. Control antibody also had no effect on either rolling or sticking. Also consistent with an integrin-mediated interaction (which requires the presence of divalent cations) is the fact that firm adhesion was preferentially inhibited by inclusion of 5 mM EDTA in the assay buffer (Figure 2, a and b). Moreover, the VLA-4 dependence of firm adhesion was not due to differences between expression levels of VLA-4 and LFA-1, because each cell type expressed comparable levels of both integrins, with SAKR cells expressing significantly more LFA-1 than VLA-4 (Figure 2d). In contrast, no firm adhesion was seen on plates coated with both HA and sICAM-1. Neither direct firm adhesion of cells to sICAM-1 alone nor firm adhesion after CD44-mediated primary adhesion on plates coated with both HA and sICAM-1 was observed, even at 10-fold higher sICAM-1 concentrations (data not shown). Thus, although expressed on all 3 cell types, LFA-1 does not appear to play a significant role in mediating firm adhesion under shear stress in the CD44-initiated pathway. However, it should be noted that LFA-1 on these populations was competent to bind ICAM-1 on coated plates in both static and nonstatic (rotational, low shear) adhesion assays (27, 33) (data not shown), consistent with prior reports (34–38). It is also of note that for these activated lymphocyte populations, rolling mediated by VLA-4/VCAM-1 was only minimally observed, although this capability has been demonstrated in previous studies (31, 39, 40). However, our observations are consistent with prior reports that rolling was not observed on isolated VCAM-1 substrate using activated lymphoid cells. Immediate firm adhesion was seen, but only at lower shear forces (0.3–0.7 dynes/cm²) (31). Direct firm adhesion was not observed on VCAM-1 under the conditions used in these studies. Therefore, using soluble substrates, primary adhesion mediated by activated CD44/HA appears to be followed by secondary adhesion mediated by VLA-4/VCAM-1 under physiologic shear stresses; this sequence is maintained in both mouse and human.

To determine whether a more physiologic stimulus resulted in a similar adhesion pathway, murine lymph node cells were stimulated directly through the T-cell receptor with anti-CD3. This alternative stimulation resulted in a pattern that was virtually identical to that seen with PMA and ionomycin. Stable arrest was overwhelmingly dependent on VLA-4 after CD44-initiated rolling adhesion (Figure 2e). Thus, a more physiologic protocol confirms the pattern found with the above populations.

VLA-4/VCAM-1–mediated firm adhesion follows CD44/HA primary adhesion on endothelial monolayers. We have previously demonstrated CD44-mediated rolling of T-cell populations on endothelial cell lines and primary endothelial cultures expressing HA. Moreover, endothelial HA levels are induced by proinflammatory stimuli; this results in increased CD44/HA–mediated adhesive interactions under conditions simulating physiologic shear stress (10, 27, 41). However, only primary adhesion was addressed in those experiments. Therefore, we sought to confirm the observations on isolated substrate, and to further examine the role of VLA-4–mediated secondary adhesion after CD44-mediated primary adhesion on endothelial monolayers expressing VCAM-1. To this end, we tested activated SAKR cells under laminar flow on monolayers of TNF-α–treated murine SVEC4-10 endothelial cells, which express both VCAM-1 and ICAM-1 (41). As shown in Figure 3, both primary and firm adhesion of SAKR cells occurred on this monolayer. Essentially all rolling interactions were blocked by anti-CD44, and very little firm adhesion was evident when CD44-mediated rolling was blocked, indicating a minimal role for VLA-4 in primary adhesion on these endothelial cells. Furthermore, very little firm adhesion was observed when CD44 interactions were blocked, indicating a minimal role of VLA-4/VCAM-1 in direct firm adhesion in this system. In contrast, firm adhesion was blocked more than 80% by anti–VLA-4, with compensatory increases in rolling interactions, whereas the control anti–LFA-1 (also expressed on SAKR cells, Figure 2d) had no significant effect on either sticking or rolling. A combination of anti-CD44 and anti–VLA-4 removed almost all interactions. Thus, the adhesion interactions observed using soluble substrates can be reproduced under more physiologic circumstances using endothelial cells expressing the appropriate ligands, HA and VCAM-1.

Figure 3

Primary and secondary adhesion of SAKR cells on SVEC4-10 endothelial cells under physiologic shear stress. SAKR cells were applied to feed solution already equilibrated under flow at a wall shear stress of 2.0 dynes/cm² and the solution was perfused over the monolayer in the parallel plate flow assay. Rolling and sticking were analyzed as described, and the number of cells/field per minute rolling and number of bound cells/field were determined. For blocking, anti-CD44, anti–VLA-4, or anti-CD44 together with anti–VLA-4 was added to the cell suspension before introduction into the flow system. Rolling and firm adhesion were both blocked by anti-CD44, whereas only anti–VLA-4 was able to block firm adhesion. Data shown are from a representative of 3 experiments.

VLA-4 participates in homing of superantigen-activated lymphocytes to an inflamed site in vivo. We showed previously that activated CD44 interactions with HA are critical to the traffic of superantigen-activated T cells from the bloodstream into inflamed peritoneal cavities in a short-term homing assay (13, 41). We conducted similar studies to determine if VLA-4 on lymphocytes also participates in this in vivo trafficking model. Draining mesenteric lymph node cells, from mice treated with SEB 20–24 hours before sacrifice, were used as donor cells. FACS^® analysis of these cells demonstrated a subpopulation bearing activated CD44, and a similar population expressing high levels of VLA-4 that preferentially bound Fl-HA (Figure 4a). Furthermore, as with activated normal human T cells (Figure 1), the HA-binding cells were concentrated in the population coexpressing high levels of both CD44 and VLA-4 (data not shown). In contrast, HA-binding cells were more evenly distributed across the expression levels of LFA-1, similar to results seen with activated human T cells (data not shown).

To assess homing capabilities, CFDA-labeled activated donor lymph node cells were injected intravenously into recipient mice treated intraperitoneally with SEB to create an inflamed peritoneal site. Migration of fluorescent donor cells from the blood into the inflamed site was assessed by FACS^® analysis of cells recovered by lavage of the peritoneal cavity 2 hours after intravenous injection. As shown in Figure 4b, significant numbers of fluorescent donor cells were found in recipient peritoneal cavities compared with either unactivated donor cells or activated cells injected into untreated recipients; these cells have previously been shown to be the activated Vβ8-bearing T-cell subset (12, 41). Fluorescent donor-cell migration into recipient peritoneal cavities is almost completely prevented by coadministration of either blocking anti-CD44 antibody or blocking anti–VLA-4 antibody (Figure 4b). However, neither anti–LFA-1 nor control anti–H-2 antibodies had any effect on cell migration. Thus, in an in vivo homing model that depends on CD44/HA interactions, VLA-4 also appears to be essential in the trafficking of activated lymphocytes into an inflamed peritoneal site, consistent with a model in which CD-44 and VLA-4 act sequentially.

VLA-4 is expressed on T cells, monocytes, and eosinophils (and according to some studies, on neutrophils), and has been shown to have a role in many inflammatory models involving all of these cell types (42–48). The VLA-4/VCAM-1 interaction has been shown to be important for lymphocyte/endothelial interactions at several inflammatory sites. Examples include entry of CD4⁺ T cells into the brain in experimental allergic encephalomyelitis (42, 49), in rheumatoid synovium (50, 51), and for the development of autoimmune diabetes (52). Our prior evidence in an antigen-specific contact hypersensitivity response suggested that this integrin operates at the level of effector T cells, which are in turn necessary for the recruitment of nonspecific effector cells (53). This is consistent with other contact hypersensitivity studies in mice and primates (54–56), while in a rat model of skin delayed-type hypersensitivity, both anti–VLA-4 and LFA-1 were required for maximal in vivo blocking (57). The studies presented here suggest that the lymphocyte-trafficking roles attributed to VLA-4 could in some situations follow CD44 tethering and rolling on endothelial HA.

Integrins containing the α4 chain have been demonstrated to be quite versatile with respect to the adhesion cascade. In addition to firm arrest (4), these integrins are able to mediate tethering and rolling. In contrast, we observed no measurable rolling, although, as noted, we did detect some direct attachment on VCAM-1 alone (Figure 2), consistent with earlier observations (31). In addition, α4β7 could mediate firm arrest but not rolling of preactivated lymph node cells in Peyer’s patch high endothelial venules in vivo (58). Analogous to the CD44-initiated pathway described here, VLA-4 has been reported to mediate firm adhesion of CD4⁺ T cells after P-selectin–driven primary adhesion (59). Moreover, in some in vivo models, rolling mediated by α4 and integrins/VCAM-1 has been shown to have a detectable but very small role compared with all selectin interactions (60). Thus, the CD44/HA primary adhesion interaction may be preferentially used in conjunction with VLA-4 by activated cell populations, whereas resting cells on which CD44 is not activated may use α4 integrins for both adhesion steps during extravasation, or may act in conjunction with selectins to effect firm adhesion.

The predilection for the use of VLA-4 but not LFA-1 in these experiments is striking. Very little contribution by LFA-1 under laminar flow (Figures 2 and 3) or for in vivo homing (Figure 4) could be detected, even though it was clear that LFA-1 was active after cell stimulation in static binding assays to purified mouse or human sICAM-1 (data not shown). This latter point is in agreement with previous reports showing active LFA-1 after cell stimulation (34–36, 61, 62). In addition, in laminar flow analysis applying half the shear used in other experiments, direct firm adhesion could be detected on ICAM-1 alone (data not shown). This further suggests that LFA-1 on recently activated cells could potentially participate in secondary adhesion after primary adhesion is initiated. LFA-1 is essential for the homing of naive lymphocytes into peripheral lymph nodes after L-selectin–mediated tethering and rolling (63). Thus, LFA-1 may generally be used for recruitment of naive rather than activated T cells. The mechanism for this selectivity requires further investigation; however, cross-talk between integrins has been reported. For example, interaction of LFA-1 selectively reduces adhesion mediated by α4β1, allowing coordination of integrin function on T cells (37), although our evidence suggests the reverse: that α4β1 may suppress LFA-1 in recently activated lymphoid cells.

In summary, these studies were undertaken to examine the mechanism by which CD44/HA–mediated tethering and rolling converts to firm adhesion. Both mouse and human activated T-cell populations demonstrate a dominant role for VLA-4 in this step under conditions of laminar flow, and superantigen-activated T cells show dependence on both VLA-4 and CD44 in an in vivo mouse model of peritoneal inflammation. LFA-1 does not appear to participate substantially in this pathway in these systems. These results help to clarify the molecular basis for extravasation of activated T cells, which are critical to the initiation and perpetuation of autoimmune and chronic inflammatory states. They may further explain the frequent association of both CD44 and VLA-4 interactions with chronic inflammatory states and suggest that therapeutic strategies targeting both of these receptors may be beneficial.

We thank Heather DeGrendele, and Todd Gonitzke for technical assistance. This work was supported by grants from the National Institutes of Health (R01 CA-57571 and HL-56746) and the Arthritis Foundation. M.H. Siegelman is an Established Investigator of the American Heart Association and a recipient of a Clinical Scientist Award from the Burroughs Wellcome Fund.

1. Butcher, EC, Picker, LJ. Lymphocyte homing and homeostasis. Science 1996. 272:60-66.
   View this article via: PubMed CrossRef Google Scholar
2. Springer, TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994. 76:301-314.
   View this article via: PubMed CrossRef Google Scholar
3. Kansas, GS. Selectins and their ligands: current concepts and controversies. Blood 1996. 88:3259-3287.
   View this article via: PubMed Google Scholar
4. Carlos, TM, Harlan, JM. Leukocyte-endothelial adhesion molecules. Blood 1994. 84:2068-2101.
   View this article via: PubMed Google Scholar
5. Mackay, CR. T-cell memory: the connection between function, phenotype and migration pathways. Immunol Today 1991. 12:189-192.
   View this article via: PubMed CrossRef Google Scholar
6. Koster, FT, McGregor, DD. The mediator of cellular immunity. III. Lymphocyte traffic from the blood into the inflamed peritoneal cavity. J Exp Med 1971. 133:864-876.
   View this article via: PubMed CrossRef Google Scholar
7. Guy, GD, Griscelli, C, Vassalli, P. The mouse gut T lymphocyte, a novel type of T cell. Nature, origin, and traffic in mice in normal and graft-vs.-host conditions. J Exp Med 1978. 148:1661-1677.
   View this article via: PubMed CrossRef Google Scholar
8. Issekutz, TB. Inhibition of in vivo lymphocyte migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody. A likely role for VLA-4 in vivo. J Immunol 1991. 147:4178-4184.
   View this article via: PubMed Google Scholar
9. Austrup, F, et al. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature 1997. 385:81-83.
   View this article via: PubMed CrossRef Google Scholar
10. DeGrendele, HC, Estess, P, Picker, LJ, Siegelman, MH. CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte/endothelial cell primary adhesion pathway. J Exp Med 1996. 183:1119-1130.
    View this article via: PubMed CrossRef Google Scholar
11. Lesley, J, Howes, N, Perschl, A, Hyman, R. Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response. J Exp Med 1994. 180:383-387.
    View this article via: PubMed CrossRef Google Scholar
12. DeGrendele, HD, Estess, P, Siegelman, MH. Requirement for CD44 in activated T cell extravasation into an inflamed site. Science 1997. 278:672-675.
    View this article via: PubMed CrossRef Google Scholar
13. DeGrendele, HC, Estess, P, Siegelman, MH. CD44 activation and associated primary adhesion is inducible via T cell receptor stimulation. J Immunol 1997. 159:2549-2553.
    View this article via: PubMed Google Scholar
14. Haynes, BF, Hale, LP, Patton, KL, Martin, ME, McCallum, RM. Measurement of an adhesion molecule as an indicator of inflammatory disease activity. Up-regulation of the receptor for hyaluronate (CD44) in rheumatoid arthritis. Arthritis Rheum 1991. 34:1434-1443.
    View this article via: PubMed Google Scholar
15. Estess, P, DeGrendele, HC, Pascual, V, Siegelman, MH. Functional activation of lymphocyte CD44 in peripheral blood is a marker of autoimmune disease activity. J Clin Invest 1998. 102:1173-1182.
    View this article via: JCI PubMed CrossRef Google Scholar
16. Mikecz, K, Brennan, FR, Kim, JH, Glant, TT. Anti-CD44 treatment abrogates tissue oedema and leukocyte infiltration in murine arthritis. Nat Med 1995. 1:558-563.
    View this article via: PubMed CrossRef Google Scholar
17. Verdrengh, M, Holmdahl, R, Tarkowski, A. Administration of antibodies to hyaluronanreceptor (CD44) delays the start and ameliorates the severity of collagen II arthritis. Scand J Immunol 1995. 42:353-358.
    View this article via: PubMed CrossRef Google Scholar
18. Brocke, S, Piercy, C, Steinman, L, Weissman, IL, Veromaa, T. Antibodies to CD44 and integrin alpha(4), but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment. Proc Natl Acad Sci USA 1999. 96:6896-6901.
    View this article via: PubMed CrossRef Google Scholar
19. Knoflach, A, et al. Immunomodulatory functions of low-molecular weight hyaluronate in an acute rat renal allograft rejection model. J Am Soc Nephrol 1999. 10:1059-1066.
    View this article via: PubMed Google Scholar
20. Knoflach, A, et al. Immunomodulatory functions of hyaluronate in the LEW-to-F344 model of chronic cardiac allograft rejection. Transplantation 1999. 67:909-914.
    View this article via: PubMed CrossRef Google Scholar
21. Hathcock, KS, Hirano, H, Murakami, S, Hodes, RJ. CD44 expression on activated B cells. Differential capacity for CD44-dependent binding to hyaluronic acid. J Immunol 1993. 151:6712-6722.
    View this article via: PubMed Google Scholar
22. Katoh, S, Zheng, Z, Oritani, K, Shimozato, T, Kincade, PW. Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J Exp Med 1995. 182:419-429.
    View this article via: PubMed CrossRef Google Scholar
23. Lesley, J, English, N, Perschl, A, Gregoroff, J, Hyman, R. Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation. J Exp Med 1995. 182:431-437.
    View this article via: PubMed CrossRef Google Scholar
24. Bennett, KL, et al. Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons. J Cell Biol 1995. 131:1623-1633.
    View this article via: PubMed CrossRef Google Scholar
25. Bartolazzi, A, Nocks, A, Aruffo, A, Spring, F, Stamenkovic, I. Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan. J Cell Biol 1996. 132:1199-1208.
    View this article via: PubMed CrossRef Google Scholar
26. Goldstein, LA, et al. A human lymphocyte homing receptor, the hermes antigen, is related to cartilage proteoglycan core and link proteins. Cell 1989. 56:1063-1072.
    View this article via: PubMed CrossRef Google Scholar
27. Mohamadzadeh, M, DeGrendele, HC, Estess, P, Siegelman, MH. Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA dependent primary adhesion. J Clin Invest 1998. 101:97-108.
    View this article via: JCI PubMed CrossRef Google Scholar
28. Cannistra, SA, et al. Binding of ovarian cancer cells to peritoneal mesothelium in vitro is partly mediated by CD44H. Cancer Res 1993. 53:3830-3838.
    View this article via: PubMed Google Scholar
29. Kansas, GS, Wood, GS, Dailey, MO. A family of cell-surface glycoproteins defined by a putative anti-endothelial cell receptor antibody in man. J Immunol 1989. 142:3050-3057.
    View this article via: PubMed Google Scholar
30. Clayberger, C, et al. Identification and characterization of two novel lymphocyte function-associated antigens, L24 and L25. J Immunol 1987. 138:1510-1514.
    View this article via: PubMed Google Scholar
31. Alon, R, et al. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J Cell Biol 1995. 128:1243-1253.
    View this article via: PubMed CrossRef Google Scholar
32. Lesley, J, Schulte, R, Hyman, R. Binding of hyaluronic acid to lymphoid cell lines is inhibited by monoclonal antibodies against Pgp-1. Exp Cell Res 1990. 187:224-233.
    View this article via: PubMed CrossRef Google Scholar
33. Alon, R, Rossiter, H, Wang, XH, Springer, TA, Kupper, TS. Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E selectin under physiological flow. J Cell Biol 1994. 127:1485-1495.
    View this article via: PubMed CrossRef Google Scholar
34. Schriever, F, et al. Human T lymphocytes bind to germinal centers of human tonsils via integrin alpha4/VCAM-1 and LFA-1/ICAM-1 and -2. Eur J Immunol 1997. 27:35-39.
    View this article via: PubMed CrossRef Google Scholar
35. Shimizu, Y, et al. Four molecular pathways of T cell adhesion to endothelial cells: roles of LFA-1, VCAM-1, and ELAM-1 and changes in pathway hierarchy under different activation conditions. J Cell Biol 1991. 113:1203-1212.
    View this article via: PubMed CrossRef Google Scholar
36. van Kooyk, Y, van de Wiel-van Kemenade, E, Weder, P, Huijbens, RJ, Figdor, CG. Lymphocyte function-associated antigen 1 dominates very late antigen 4 in binding of activated T cells to endothelium. J Exp Med 1993. 177:185-190.
    View this article via: PubMed CrossRef Google Scholar
37. Porter, JC, Hogg, N. Integrin cross talk: activation of lymphocyte function-associated antigen-1 on human T cells alters alpha4beta1- and alpha5beta1-mediated function. J Cell Biol 1997. 138:1437-1447.
    View this article via: PubMed CrossRef Google Scholar
38. Jones, DA, Mcintire, LV, Smith, CW, Picker, LJ. A two-step adhesion cascade for T cell endothelial cell interactions under flow conditions. J Clin Invest 1994. 94:2443-2450.
    View this article via: JCI PubMed CrossRef Google Scholar
39. Berlin, C, et al. Alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 1995. 80:413-422.
    View this article via: PubMed CrossRef Google Scholar
40. Johnston, B, Issekutz, TB, Kubes, P. The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J Exp Med 1996. 183:1995-2006.
    View this article via: PubMed CrossRef Google Scholar
41. Estess, PE, Nandi, A, Siegelman, MH. IL-15 induces endothelial hyaluronan expression in vitro and promotes activated T cell extravasation through a CD44 dependent pathway in vivo. J Exp Med 1999. 190:9-19.
    View this article via: PubMed CrossRef Google Scholar
42. Yednock, TA, et al. Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature 1992. 356:63-66.
    View this article via: PubMed CrossRef Google Scholar
43. Issekutz, AC, Issekutz, TB. Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms. J Exp Med 1995. 181:1197-1203.
    View this article via: PubMed CrossRef Google Scholar
44. Issekutz, TB, Issekutz, AC. T lymphocyte migration to arthritic joints and dermal inflammation in the rat: differing migration patterns and the involvement of VLA-4. Clin Immunol Immunopathol 1991. 61:436-447.
    View this article via: PubMed CrossRef Google Scholar
45. Yang, XD, Karin, N, Tisch, R, Steinman, L, McDevitt, HO. Inhibition of insulitis and prevention of diabetes in nonobese diabetic mice by blocking L-selectin and very late antigen 4 adhesion receptors. Proc Natl Acad Sci USA 1993. 90:10494-10498.
    View this article via: PubMed CrossRef Google Scholar
46. Issekutz, TB, Miyasaka, M, Issekutz, AC. Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation. J Exp Med 1996. 183:2175-2184.
    View this article via: PubMed CrossRef Google Scholar
47. Andersson, EC, Christensen, JP, Marker, O, Thomsen, AR. Changes in cell adhesion molecule expression on T cells associated with systemic virus infection. J Immunol 1994. 152:1237-1245.
    View this article via: PubMed Google Scholar
48. Reinhardt, PH, Elliott, JF, Kubes, P. Neutrophils can adhere via alpha(4)beta(1)-integrin under flow conditions. Blood 1997. 89:3837-3846.
    View this article via: PubMed Google Scholar
49. Baron, JL, Madri, JA, Ruddle, NH, Hashim, G, Janeway (Jr), CA. Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med 1993. 177:57-68.
    View this article via: PubMed CrossRef Google Scholar
50. van Dinther-Janssen, AC, et al. The VLA-4/VCAM-1 pathway is involved in lymphocyte adhesion to endothelium in rheumatoid synovium. J Immunol 1991. 147:4207-4210.
    View this article via: PubMed Google Scholar
51. Laffon, A, et al. Upregulated expression and function of VLA-4 fibronectin receptors on human activated T cells in rheumatoid arthritis. J Clin Invest 1991. 88:546-552.
    View this article via: JCI PubMed CrossRef Google Scholar
52. Baron, JL, Reich, EP, Visintin, I, Janeway (Jr), CA. The pathogenesis of adoptive murine autoimmune diabetes requires an interaction between alpha 4-integrins and vascular cell adhesion molecule-1. J Clin Invest 1994. 93:1700-1708.
    View this article via: JCI PubMed CrossRef Google Scholar
53. Catalina, MD, Estess, P, Siegelman, MH. Selective requirements for leukocyte adhesion molecules in models of acute and chronic cutaneous inflammation: participation of E- and P- but not L-selectin. Blood 1999. 93:580-589.
    View this article via: PubMed Google Scholar
54. Ferguson, TA, Kupper, TS. Antigen-independent processes in antigen-specific immunity. A role for alpha 4 integrin. J Immunol 1993. 150:1172-1182.
    View this article via: PubMed Google Scholar
55. Chisholm, PL, Williams, CA, Lobb, RR. Monoclonal antibodies to the integrin alpha-4 subunit inhibit the murine contact hypersensitivity response. Eur J Immunol 1993. 23:682-688.
    View this article via: PubMed CrossRef Google Scholar
56. Silber, A, et al. Recruitment of lymphocytes during cutaneous delayed hypersensitivity in nonhuman primates is dependent on E-selectin and vascular cell adhesion molecule 1. J Clin Invest 1994. 93:1554-1563.
    View this article via: JCI PubMed CrossRef Google Scholar
57. Issekutz, TB. Dual inhibition of VLA-4 and LFA-1 maximally inhibits cutaneous delayed-type hypersensitivity-induced inflammation. Am J Pathol 1993. 143:1286-1293.
    View this article via: PubMed Google Scholar
58. Bargatze, RF, Jutila, MA, Butcher, EC. Distinct roles of L-selectin and integrins a4b7 and LFA-1 in lymphocyte homing to Peyer’s Patch-HEV in situ: the multistep model confirmed and refined. Immunity 1995. 3:99-108.
    View this article via: PubMed CrossRef Google Scholar
59. Luscinskas, FW, Ding, H, Lichtman, AH. P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor alpha-activated vascular endothelium under flow. J Exp Med 1995. 181:1179-1186.
    View this article via: PubMed CrossRef Google Scholar
60. Jung, U, Ley, K. Mice lacking two or all three selectins demonstrate overlapping and distinct functions for each selectin. J Immunol 1999. 162:6755-6762.
    View this article via: PubMed Google Scholar
61. Oppenheimer-Marks, N, Davis, LS, Bogue, DT, Ramberg, J, Lipsky, PE. Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes. J Immunol 1991. 147:2913-2921.
    View this article via: PubMed Google Scholar
62. Hwang, ST, et al. GlyCAM-1, a physiologic ligand for L-selectin, activates beta 2 integrins on naive peripheral lymphocytes. J Exp Med 1996. 184:1343-1348.
    View this article via: PubMed CrossRef Google Scholar
63. Warnock, RA, Askari, S, Butcher, EC, von Andrian, UH. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J Exp Med 1998. 187:205-216.
    View this article via: PubMed CrossRef Google Scholar