©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Regulation of Integrin-dependent Binding to Domains 1 and 4 of Vascular Cell Adhesion Molecule-1 (*)

(Received for publication, November 14, 1994; and in revised form, January 13, 1995)

Gerda Kilger Lindsey A. Needham (1) Peter J. Nielsen (2) John Clements (1) Dietmar Vestweber (2) Bernhard Holzmann (§)

From the  (1)Institute for Medical Microbiology and Hygiene, Technical University, Trogerstrasse 4a, D-81675 Munich, Federal Republic of Germany, British Bio-technology Limited, Watlington Road, Cowley, Oxford, OX4 5LY, United Kingdom, and the (2)Max Planck Institute for Immunobiology, D-79108 Freiburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The vascular cell adhesion molecule-1 (VCAM-1) plays an important role in diverse physiological and pathological processes. The homologous first and fourth immunoglobulin-like domains of the seven domain form of VCAM-1 present binding motifs for alpha(4)beta(1) integrin. Using a panel of VCAM-1 domain deletion mutants we show that alpha(4)beta(7) integrin interacts with both domains 1 and 4. In contrast to their identical domain usage, alpha(4)beta(1) and alpha(4)beta(7) integrins differ in the activation states required for binding to domains 1 and 4 of VCAM-1. We show that integrin alpha(4)beta(1) required significantly higher concentrations of Mn than integrin alpha(4)beta(7) to support half-maximal adhesion to domain 4. Moreover, a clear difference in the capacity of integrins alpha(4)beta(1) and alpha(4)beta(7) to interact with domain 4 was detected in the presence of Ca and Mg cations. Adhesion to domain 1 of VCAM-1, however, was not affected by integrin heterodimer composition. Instead, the activity level of integrin alpha(4)beta(1) for domain 1 binding was regulated by CD24 expression. Binding to seven domain VCAM-1 was not altered significantly by beta(1) and beta(7) subunits or CD24. These data indicate that integrin heterodimer composition and CD24 expression differentially modulate integrin binding to domains 1 and 4 of VCAM-1. Mechanisms that alter integrin binding specificity or monovalent versus divalent interactions may affect the strength of adhesion as well as signal transmission in adherent cells and may therefore be critical to controlling the cellular response to integrin occupancy.


INTRODUCTION

The inducible vascular cell adhesion molecule-1 (VCAM-1) (^1)is a member of the immunoglobulin superfamily and may be involved in numerous physiological and pathological processes, including embryonic development of skeletal muscle, hematopoiesis, inflammatory reactions, and the development of autoimmune disorders(1, 2, 3) . In addition to the seven domain form of VCAM-1 (VCAM-7D), a six-domain form (VCAM-6D) that is generated by alternative splicing of domain 4, a glycolipid-anchored form containing the first three domains, and an eight-domain form of VCAM-1 have been described(1, 2) . The homologous first and fourth immunoglobulin-like domains of VCAM-1 (VCAM-7D) present binding motifs for alpha(4)beta(1) integrin(4, 5, 6, 7, 8, 9, 10) . Based on temperature sensitivity and phorbol 12-myristate 13-acetate activation, it has been suggested that alpha(4)beta(1) integrin requires different states of activation for binding to VCAM-1 domains 1 or 4 (11) . Recently, it has been demonstrated that integrin alpha(4)beta(7) functions as an alternative leukocyte receptor for VCAM-1(12, 13, 14, 15) . Integrins alpha(4)beta(1) and alpha(4)beta(7) also share binding specificity for the the CS-1 peptide derived from fibronectin(12, 13, 14, 15, 16) . Adhesion to the mucosal addressin MAdCAM-1, however, is predominantly mediated by integrin alpha(4)beta(7)(15, 17) .

The heat-stable antigen (CD24) has been described as a maturation marker of murine lymphocytes (18, 19, 20) and has co-stimulatory activity for the clonal expansion of CD4 T cells(21) . On B lymphocytes, cross-linking of CD24 results in a rapid rise in cytoplasmic calcium levels suggesting a role for CD24 in signal transduction(22) . Structurally, CD24 consists of a small glycolipid-anchored peptide core with a large number of potential N-linked and O-linked glycosylation sites(23, 24) . The carbohydrate moiety may be involved in homophilic CD24 interactions (25) and in presenting binding sites for P-selectin(26) . In addition, CD24 alters alpha(4)beta(1) integrin adhesion to fibronectin and TNF-stimulated endothelioma cells(27) .

In the present study we show that alpha(4)beta(7) integrin independently binds to domains 1 and 4 of VCAM-1. When compared with alpha(4)beta(1), adhesion of alpha(4)beta(7) integrin to domain 4 required significantly lower levels of activation, suggesting that domain 4 binding is regulated by the beta subunits of alpha(4) integrins. By contrast, efficient adhesion of alpha(4)beta(1) integrin to VCAM-1 domain 1 was dependent on CD24. Differential regulation of alpha(4) integrin binding to individual domains of VCAM-1 may affect the strength of adhesion as well as signal transmission in adherent leukocytes.


MATERIALS AND METHODS

Antibodies and Cell Lines

Antibodies used included rat-anti-murine integrin alpha(4), PS/2 (ATCC, Rockville, MD), rat-anti-murine LFA-1alpha, M17/4.2 (ATCC), mouse-anti-human VCAM-1 domain 1, 4B2(11) , mouse-anti-human VCAM-1 domain 6, 15D10(11) , rat-anti-murine CD24, J11d.2 (ATCC), and rat-anti-murine T cell receptor Vbeta14 (Dianova, Hamburg, Federal Republic of Germany). All antibodies were used at saturating concentrations as determined by flow cytometry analysis.

The C3H/He B cell lymphoma 38C13 was demonstrated to express membrane-bound alpha(4) subunits, but lacks detectable cell surface protein and RNA transcripts for integrin beta(1) and beta(7) subunits(28) . Integrin beta(1) or beta(7) chains were expressed de novo in 38C13 lymphoma cells by retrovirus-mediated gene transfer generating cell lines 38-beta(1) and 38-beta(7)(15) . TK1.cl14 is a spontaneous AKR/cum T cell lymphoma expressing integrin subunits alpha(4) and beta(7) but not beta(1), whereas RAW112 is an Abelson leukemia virus-induced BALB/c pre-B cell lymphoma that expresses alpha(4) and beta(1) but not beta(7) subunits(15) . CHO transfectants expressing the human VCAM-7D receptor and CHO/CDM8 control cells have been described(15) . The pre-B cell line N232.18 was generated by infection of bone marrow-derived cells from a CD24-deficient chimeric mouse with a replication defective A-MuLV(27) . 18H18 cells were obtained by transfection of N232.18 cells with the CD24 expression plasmid pHSEX62.8(27) .

Stable Expression of VCAM-1 Deletion Mutants

CHO cells were co-transfected with pCDM8 plasmids containing deletion mutants of the human VCAM-7D receptor lacking domains 1-3 (Delta1.2.3), domains 4-6 (Delta4.5.6), domains 1 and 4 (Delta1.4), domain 1 (Delta1), or domain 4 (Delta4) and with the plasmid pLXSN carrying the neomycin resistance gene. Clones expressing mutant VCAM-1 receptors were identified by flow cytometry analysis subsequent to selection in G418. Construction of VCAM-1 domain deletion mutants has been described(11) .

Flow Cytometry Analysis

Cells were incubated with saturating amounts of mAbs, washed, and stained with FITC-conjugated rabbit F(ab)(2) fragments reacting with mouse or rat Ig (Dianova, Hamburg, Federal Republic of Germany). Incubations were performed for 20 min at 4 °C in the presence of 0.05% NaN(3). Cells were fixed with 1% paraformaldehyde and analyzed on an EPICS Elite cytometer (Coulter Corp., Hialeah, FL).

Adhesion Assays

CHO transfectants (2-3 times 10^4/well) were added to 96-well plates and allowed to grow for 24 h prior to the assay. Confluent cell monolayers were washed with PBS and fixed for 10 min at room temperature with 0.05% glutaraldehyde. Recombinant soluble VCAM-1 was coated at 0.3 µg/well in PBS for 16 h at 4 °C. Plates were washed extensively with PBS containing 1 mM EDTA and subsequently with divalent cation-free cell adhesion buffer (24 mM Tris, pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 2 mM glucose, and 1% bovine serum albumin). 38-beta(1) and 38-beta(7) lymphoma cells were labeled for 30 min at 37 °C with 12 µg/ml H33342 dye (Calbiochem) in RPMI 1640 containing 1% bovine serum albumin and washed twice with PBS 1 mM EDTA. Cells (8 times 10^5/ml) were resuspended in cell adhesion buffer supplemented with various concentrations of Mn and saturating amounts of mAb PS/2 or M17/4.2. For some experiments, cells were resuspended in adhesion buffer containing 1.0 mM Ca and 1.0 mM Mg instead of Mn. After incubation for 10 min at room temperature cells (100 µl/well) were added to the plates and centrifuged for 10 min at 10 times g. Cells were allowed to adhere for 30 min at 37 °C, and nonadherent cells were removed by inverse centrifugation for 10 min at 50 times g. Adhesion assays were quantified by fluorimetry using a Cytofluor 2300 (Millipore, Bedford, MA). Specific adhesion of 38-beta(7) or 38-beta(1) cells was determined by subtracting the percent of cells adherent in the presence of mAb PS/2 from the percent of adherent cells treated with mAb M17/4.2. mAb PS/2 has been shown previously to completely inhibit ligand binding functions of murine alpha(4)beta(7) and alpha(4)beta(1) integrins(15) .


RESULTS

VCAM-1 Binding Sites for Integrin alpha(4)beta(7)

Both alpha(4)beta(1) and alpha(4)beta(7) integrins function as lymphocyte receptors for VCAM-1(12, 13, 14, 15, 16) . For integrin alpha(4)beta(1) to bind VCAM-1, epitopes present in domains 1 and 4 are required(4, 5, 6, 7, 8, 9, 10) . To identify VCAM-1 domains interacting with the alpha(4)beta(7) integrin, CHO cells were stably transfected with pCDM8 expression plasmids encoding VCAM-1 deletion mutants lacking domains 1-3 (Delta1.2.3), domains 4-6 (Delta4.5.6), domains 1 and 4 (Delta1.4), domain 1 (Delta1), or domain 4 (Delta4). Immunofluorescence staining and flow cytometry analysis using mAbs specific for domains 1 or 6 of VCAM-1 showed that the VCAM-7D receptor and deletion mutants were expressed at similar levels on CHO transfectants (data not shown).

38-beta(7) cells were allowed to adhere to CHO transfectants in the presence of 1.0 mM Mn to determine plateau levels of cell binding. To examine alpha(4)beta(7) integrin-dependent adhesion, the fraction of cells bound in the presence of mAb PS/2 (anti-alpha(4)) was subtracted from the percent of adherent cells treated with mAb M17/4.2 (anti-LFA-1). As is shown in Fig. 1A, plateau levels of 38-beta(7) cell binding to VCAM-1 were not altered significantly by Delta1.2.3 or Delta4.5.6 deletions. Plateau binding of 38-beta(7) cells was also not affected by deletion of either domain 1 or 4 (data not shown). By contrast, deletion of both domains 1 and 4 almost completely abrogated adhesion of 38-beta(7) cells (Fig. 1A), indicating that binding of integrin alpha(4)beta(7) to VCAM-1 requires the presence of either domain 1 or 4. Structural integrity of the Delta1.4 mutant was confirmed by reactivity with several mAbs specific for epitopes present in domains 2, 3, or 6 of VCAM-1 (data not shown). Consistent with previous reports on the domain requirements of alpha(4)beta(1) binding to VCAM-1 (11) , adhesion of Mn-treated 38-beta(1) cells was also abrogated by the Delta1.4 deletion, but was not affected by the Delta1.2.3 or Delta4.5.6 mutations (Fig. 1B). The results therefore indicate that domains 1 and 4 of VCAM-1 independently support binding of the alpha(4)beta(7) integrin.


Figure 1: Domain requirements for binding of integrin alpha(4)beta(7) to VCAM-1. Confluent monolayers of CHO cells transfected with VCAM-7D or Delta1.2.3, Delta4.5.6, and Delta1.4 domain deletion mutants were incubated with fluorescence-labeled 38-beta(7) (A) or 38-beta(1) (B) cells in the presence of 1 mM Mn and adherence was measured by fluorimetry. To determine alpha(4)beta(7) or alpha(4)beta(1) integrin-specific adhesion the percent of cells bound in the presence of mAb PS/2 was subtracted from the percent of adherent cells treated with mAb M17/4.2. The results are expressed as percent of specifically adherent cells and represent the mean ± S.D. from four to six independent experiments. Statistical analysis was performed by the Student's t test.



The Binding of alpha(4) Integrins to Domain 4 of VCAM-1 Is Modulated by beta(1) and beta(7) Subunits

To analyze alpha(4)beta(7) and alpha(4)beta(1) integrin activation states required for binding to individual domains of VCAM-1, adhesion of 38-beta(1) and 38-beta(7) cells was analyzed over a wide range of Mn concentrations, and the concentrations resulting in half-maximal adhesion (ED values) were determined. It has been shown previously that distinct activation states of alpha(4)beta(1) integrin can be distinguished by Mn titration experiments and that ED values are independent of integrin expression levels (29) . 38-beta(1) and 38-beta(7) cells differ exclusively in the expression of integrin beta(1) or beta(7) subunits(15) , thus excluding cell type-specific differences in the basal activation states of alpha(4)beta(1) and alpha(4)beta(7) integrins. The Mn titration experiments reveled that the average ED values for 38-beta(7) and 38-beta(1) adhesion to CHO/VCAM-7D cells did not differ significantly (19.2 ± 6.5 µMversus 22.8 ± 3.9 µM Mn, p > 0.05). In addition, similar ED values were obtained for binding of alpha(4)beta(7) (18.5 ± 7.7 µM Mn) and alpha(4)beta(1) integrins (14.4 ± 8.2 µM Mn) to recombinant-soluble VCAM-7D. These results therefore indicate that integrins alpha(4)beta(1) and alpha(4)beta(7) require the same level of activation for binding to the full-length VCAM-7D receptor.

The Mn titration profiles for adhesion of alpha(4)beta(7) and alpha(4)beta(1) integrins to distinct domains of VCAM-1 were further analyzed. As is depicted in Fig. 2A, the average ED values for binding of 38-beta(7) cells to full-length VCAM-7D or the Delta1.2.3 and Delta4.5.6 mutants did not differ significantly. Half-maximal adhesion was observed at 19-23 µM for each VCAM-1 construct (Table 1). By contrast, deletion of domains 1-3 or 4-6 differentially affected binding of 38-beta(1) cells. When compared with full-length VCAM-7D, the average ED values for 38-beta(1) binding to the Delta1.2.3 but not to the Delta4.5.6 mutant were significantly increased (Fig. 2B). Direct comparison of alpha(4)beta(7) and alpha(4)beta(1) integrins revealed a significant difference (p < 0.01) between the ED values for binding to domain 4 of VCAM-1 (Table 1). The average ED value for alpha(4)beta(7) integrin was 23.0 ± 2.2 µM, whereas the alpha(4)beta(1) receptor required 63.4 ± 27.9 µM Mn for half-maximal adhesion. Similarly, on 18H18 cells the ED values for adhesion of alpha(4)beta(1) integrin to domain 4 were 94.2 ± 10.0 µM Mn and 24.4 ± 5.7 µM Mn for domain 1 binding (Table 1). These results indicate that alpha(4)beta(1) requires a significantly higher level of activation than integrin alpha(4)beta(7) to bind domain 4 of VCAM-1.


Figure 2: Integrins alpha(4)beta(7) and alpha(4)beta(1) require distinct levels of activity for binding to domain 4 of VCAM-1. Adhesion of fluorescence-labeled 38-beta(7) (A) or 38-beta(1) (B) cells to CHO cells transfected with VCAM-7D or deletion mutants Delta1.2.3 and Delta4.5.6 was analyzed over a wide range of Mn concentrations. The percent of cells bound in the presence of mAb PS/2 was subtracted from the percent of adherent cells treated with mAb M17/4.2 for each Mn concentration and adherence was measured by fluorimetry. The ED values for alpha(4) integrin-dependent binding were calculated and average ED values were obtained from four to six independent experiments. The results are presented as mean ± S.D. Statistical analysis was performed by the Student's t test. NS = not significant.





We next asked whether different binding efficiencies of alpha(4)beta(1) and alpha(4)beta(7) integrins for VCAM-1 domain 4 are also detected under more physiological divalent cation conditions. When adhesion assays were performed in the presence of Ca and Mg at 1.0 mM each, deletion of VCAM-1 domains 1-3 strongly reduced binding of integrin alpha(4)beta(1) lymphoma lines 38-beta(1) and RAW112, but only minimally affected adhesion of alpha(4)beta(7) 38-beta(7) and TK1.cl14 cells (Fig. 3A). By contrast, both alpha(4)beta(1) and alpha(4)beta(7) lymphoma lines bound to the Delta4.5.6 mutant with the same efficiency as to the full-length VCAM-7D receptor (Fig. 3B). Thus, the Mn titration experiments revealed significant differences in the capacity of integrins alpha(4)beta(1) and alpha(4)beta(7) to interact with domain 4 of VCAM-1 that are also relevant for adhesion in the presence of Ca and Mg cations.


Figure 3: Binding of alpha(4)beta(1) and alpha(4)beta(7) integrins to VCAM-1 deletion mutants in the presence of Ca and Mg cations. Adhesion of fluorescence-labeled alpha(4)beta(1) lymphoma lines 38-beta(1) and RAW112 (solid bars) or alpha(4)beta(7) cell lines 38-beta(7) and TK1.cl14 (hatched bars) to CHO cells transfected with VCAM-1 deletion mutants Delta1.2.3 (A) or Delta4.5.6 (B) was analyzed in the presence of Ca and Mg at 1.0 mM each. The percent of cells bound to CHO/CDM8 control cells was subtracted from the percent of cells adherent to CHO/VCAM-7D, CHO/Delta1.2.3, or CHO/Delta4.5.6 transfectants. Adhesion to VCAM-1 deletion mutants is given as percent of VCAM-7D binding for each lymphoma line. The results were obtained from four to six independent experiments and are presented as mean ± S.D.



In control experiments, the Mn requirement for binding of alpha(4)beta(7) integrin to fibronectin was analyzed. When compared with recombinant soluble VCAM-1, plateau levels of fibronectin binding were similar (61.7 ± 5.8 versus 59.7 ± 5.5% specific adhesion; p > 0.05), whereas the average ED values were significantly increased (18.5 ± 7.7 versus 70.3 ± 27.2 µM Mn; p < 0.01). Therefore these data demonstrate that distinct activity levels of alpha(4)beta(7) integrin can be distinguished by Mn titration experiments.

The Binding of alpha(4)beta(1) Integrin to Domain 1 of VCAM-1 Is Regulated by CD24

Using N232.18 and 18H18 pre-B lymphoma cells that differ exclusively in the expression of CD24, it has been shown previously that CD24 alters binding of integrin alpha(4)beta(1) to endothelial cells and fibronectin (27) . Immunofluorescence staining and flow cytometry analysis confirmed the differential expression of CD24 by N232.18 and 18H18 cells (Fig. 4) and revealed that both cell lines do not express integrin beta(7) subunits (data not shown). Fig. 4also shows that lymphoma lines 38-beta(7), 38-beta(1), TK1.cl14, and RAW112 express high levels of CD24, with the expression on TK1.cl14 cells being more heterogeneous than on other lymphoma lines. To investigate the role of CD24 in regulating the VCAM-1 binding activity of alpha(4)beta(1) integrin, the Mn titration profiles were determined. Interestingly, the average ED values for adhesion of CD24 18H18 transfectants to recombinant soluble VCAM-7D or CHO/VCAM-7D cells (12.4 ± 5.9 and 26.5 ± 4.4 µM Mn; p < 0.01) were almost identical to those determined for 38-beta(1) cells. In addition, it was shown that the ED values for binding of CD24-deficient N232.18 cells to both purified and cellular VCAM-7D (31.3 ± 5.7 and 36.3 ± 6.0 µM Mn) were increased as compared with 18H18 cells. However, the average ED values for adhesion to CHO/VCAM-7D cells did not differ significantly and differences in binding purified VCAM-7D were of low statistical significance (p = 0.03). Thus, the expression of CD24 has only minor effects on the alpha(4)beta(1) integrin activation state required for binding to full length VCAM-7D.


Figure 4: Expression of CD24 on lymphoma lines and transfectants. Cell lines 38-beta(1), 38-beta(7), N232.18, 18H18, RAW112, and TK1.cl14 were incubated with mAb J11d.2 specific for CD24 (solid lines) or an isotype-matched control mAb directed against the T cell receptor Vbeta14 segment (dotted lines) followed by FITC-conjugated rabbit-anti-rat Ig serum.



The Mn titration profiles for adhesion of N232.18 and 18H18 cells to VCAM-1 domains 1 and 4 were further analyzed. Similar to the results obtained with 38-beta(1) cells, the average ED values for binding of 18H18 transfectants to full length VCAM-7D or the Delta4 mutant did not differ, whereas half-maximal adhesion to the Delta1 mutant required significantly higher Mn concentrations (Fig. 5A). By contrast, binding of CD24-deficient N232.18 cells to VCAM-1 was in addition sensitive to deletion of domain 4. As is shown in Fig. 5B, the average ED values for binding to the Delta1 as well as the Delta4 mutants were significantly increased. When N232.18 and 18H18 cells were compared directly, a clear difference between the ED values required for binding to the Delta4 (p < 0.0001) but not to the Delta1 mutant (p > 0.05) was observed. The average ED value for binding of 18H18 transfectants to CHO/Delta4 cells was 24.4 ± 5.7 µM, whereas N232.18 cells required 118.7 ± 20.1 µM Mn for half-maximal adhesion (Table 1). It therefore appears that the activation state of alpha(4)beta(1) for adhesion to domain 1 of VCAM-1 is controlled by CD24.


Figure 5: Integrin alpha(4)beta(1) binding to VCAM-1 domain 1 is regulated by CD24. Adhesion of fluorescence-labeled CD24 18H18 transfectants (A) or CD24-deficient N232.18 cells (B) cells to CHO cells transfected with VCAM-7D or deletion mutants Delta1 and Delta4 was analyzed over a wide range of Mn concentrations. The percent of cells bound in the presence of mAb PS/2 was subtracted from the percent of adherent cells treated with mAb M17/4.2 for each Mn concentration and adherence was measured by fluorimetry. The ED values for alpha(4)beta(1) integrin-dependent binding were calculated and average ED values were obtained from three to five independent experiments. The results are presented as mean ± S.D. Statistical analysis was performed by the Student's t test. NS = not significant.




DISCUSSION

Previous studies have shown that structurally related integrins may interact with non-homologous binding sites present in matrix proteins or cellular adhesion receptors. For example, LFA-1 and Mac-1 recognize distinct domains of ICAM-1(30) , and fibronectin contains separate binding sites for VLA-4 and VLA-5(31) . For binding of integrin alpha(4)beta(1) to its alternate counter-receptor VCAM-1, however, epitopes present in the homologous domains 1 and 4 are of critical importance(4, 5, 6, 7, 8, 9, 10) . In the present study, domain deletion mutants of VCAM-1 were used to localize the binding sites for integrin alpha(4)beta(7). The results indicate that integrins alpha(4)beta(7) and alpha(4)beta(1) share the domain requirements for adhesion to VCAM-1, as binding of both integrins was almost completely abolished by simultaneous deletion of domains 1 and 4. Structural integrity of the Delta1.4 mutant was confirmed by reactivity with several mAbs specific for VCAM-1 domains 2, 3, or 6. Amino acid residues in domains 1 and 4 have been identified that are critical to integrin alpha(4)beta(1) binding(6, 8, 9) . It will be interesting to determine whether the same residues are also involved in interactions of alpha(4)beta(7) with VCAM-1.

Incubation of cells with Mn induces the high avidity conformation of alpha(4) integrins even in the absence of other activating signals(29, 32) . Studies on mutant integrin alpha(4) subunits indicated that distinct levels of integrin binding activity can be distinguished by the concentrations of Mn required for half-maximal adhesion(29) . These studies also showed that ED values are independent of integrin expression levels and the overall adhesive state of the cell(29) . The results presented here extend these findings, as differences in the Mn titration profiles revealed a critical role of integrin heterodimer composition and CD24 expression for differentially regulating binding of alpha(4)beta(1) and alpha(4)beta(7) integrins to domains 1 and 4 of VCAM-1. Importantly, a clear difference in the capacity of alpha(4)beta(1) and alpha(4)beta(7) integrins to bind domain 4 of VCAM-1 was also detected in the presence of Ca and Mg cations. Taken together, these data suggest that Mn titration experiments may be applied to determine integrin activity states necessary for ligand binding.

The activity state of integrins can be influenced by numerous factors. Integrin phosphorylation (33, 34, 35) and glycosylation(36) , structural elements of alpha and beta subunit cytoplasmic tails(37, 38, 39) , divalent cation sites of alpha subunits(29) , expression of CD24(27) , composition of membrane phospholipids(40) , lipid factors(41) , putative signal transduction molecules(42) , and unknown cell type-specific factors (29) , all have been suggested to regulate the level of integrin activity. Whereas most of these mechanisms appear to alter ligand binding activity of integrins in general, the results reported here identify mechanisms that differentially modulate the binding activity of alpha(4) integrins for homologous binding sites in VCAM-1. It was demonstrated that integrin alpha(4)beta(7) binds much more efficiently to domain 4 of VCAM-1 than alpha(4)beta(1), whereas both integrins showed the same binding activity for domain 1. Previous results showing that phorbol 12-myristate 13-acetate induces binding of alpha(4)beta(1) integrin to domain 4, but not to domain 1, on human U937 cells support these findings(11) . In contrast to the effects of integrin heterodimer composition, the activation state of alpha(4)beta(1) integrin required for binding to domain 1 but not domain 4 was modulated by CD24 expression. Interestingly, flow cytometry analysis revealed that low levels of CD24 expressed by 18H18 transfectants are sufficient to enhance domain 1 adhesion of alpha(4)beta(1) integrin. Thus, the regulatory effects of CD24 appear to be largely independent of its cell surface expression levels. Since in these experiments defined sets of B lymphoma transfectants were used that differ exclusively in the expression of beta(1) and beta(7) subunits (38-beta(1) and 38-beta(7) cells) or CD24 (N232.18 and 18H18 cells), it seems highly unlikely that cell-specific differences can account for these results. In summary, we conclude from these data that beta(1) and beta(7) subunits are critical for modulating alpha(4) integrin binding to domain 4 of VCAM-1, whereas adhesion of alpha(4)beta(1) to domain 1 is regulated by CD24.

Triggering of lymphoid cells or monocytes through alpha(4)beta(1) may regulate gene expression and tyrosine phosphorylation of a 105-kDa protein, induce or inhibit cell death, deliver co-stimulatory signals for T cell proliferation, and promote cell migration(3) . As the full-length VCAM-1 receptor expresses two potential binding sites for both alpha(4)beta(1)(4, 5) and alpha(4)beta(7) integrin (Fig. 1), interaction of a single versus two integrin molecules with VCAM-1 may affect the strength of adhesion as well as signal transmission to adherent leukocytes. Therefore, mechanisms such as those described here that modulate binding to domains 1 and 4 differentially may be critical to controlling the intensity and/or chemical nature of signals and consequently the cellular response to alpha(4) integrin occupancy.

Previous studies have shown that the loss of CD24 expression on pre-B cells is associated with a strongly reduced binding activity of alpha(4)beta(1) integrin to fibronectin(27) . However, the same cells still bound to TNF-stimulated endothelioma cells via alpha(4)beta(1) integrin. Whereas adhesion of CD24 transfectants was blocked by anti-VCAM-1 mAb MK/2.7, binding of CD24-deficient cells was not sensitive to inhibition by mAb MK/2.7(27) . These data suggested that cells lacking CD24 may interact with a ligand distinct from VCAM-1 on TNF-stimulated endothelium. Alternatively, alpha(4)beta(1) on CD24-deficient cells may interact with alternate domains in VCAM-1 not inhibited by the domain 1 + 2-specific mAb MK/2.7. However, our results demonstrate that VCAM-1 binding of CD24-deficient N232.18 cells was completely dependent on the presence of domains 1 and 4 (not shown), and binding activity for domain 4 was decreased as compared with CD24 transfectants. Hence, it is highly unlikely that alpha(4)beta(1)-mediated binding of N232.18 cells to TNF-stimulated endothelium results from VCAM-1 interactions independent of domain 1. We therefore suggest that alternate endothelial ligands for alpha(4)beta(1) integrin may exist. Consistent with this interpretation, it has been reported that the adhesion of certain human B and T lymphoma cells to TNF-stimulated umbilical vein endothelial cells is completely inhibited by anti-alpha(4) integrin mAb but only partially by mAbs directed against VCAM-1(5) . The existence of alternate endothelial ligands may have important implications for current efforts to understand and manipulate alpha(4)beta(1) integrin functions in vivo.


FOOTNOTES

*
This work was supported by the Gerhard Hess Program of the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute for Medical Microbiology & Hygiene, Technical University, Trogerstr. 4a, D-81675 Munich, FRG. Tel.: 49-89-4140-4134; Fax: 49-89-4140-4868.

(^1)
The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; TNF, tumor necrosis factor; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Drs. C. Evans and K. Pfeffer for critically reading the manuscript and A. Lifka for expert technical assistance.


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