The cytoplasmic domains of LFA-1 (CD11a/CD18) are thought to play an important role in the regulation of LFA-1 function. To further elucidate the role of the LFA-1 cytoplasmic domains, we transfected chimeric proteins consisting of the extracellular domain of CD4 fused with the transmembrane and cytoplasmic domains of LFA-1 into T and B cell lines, EL-4 and A20, respectively, and examined their effects on LFA-1-mediated cell adhesion. The CD4/18, but not CD4/11a, chimera profoundly inhibited LFA-1-mediated cell adhesion to ICAM-1, as well as cell spreading following cell adhesion. Unexpectedly, cell adhesion to fibronectin was also inhibited by the CD4/18 chimera. The CD4/18 chimera did not affect the expression of endogenous LFA-1 or the association of CD11a and CD18. Truncation of the carboxyl-terminal 13 amino acid residues of the CD18 cytoplasmic domain of the chimera completely abrogated the inhibitory effect on LFA-1. Among these amino acid residues, the carboxyl-terminal six residues were dispensable for the inhibitory effect in EL-4 cells, whereas it significantly reduced the inhibitory activity of CD4/18 in A20 cells. A larger truncation of the CD18 cytoplasmic domain was needed to fully abrogate the inhibitory effects of CD4/18 on the adhesion to fibronectin. These results show that 1) the CD4/18 chimera has dominant-negative effects on cell adhesion mediated by LFA-1 as well as fibronectin receptors, and 2) amino acid residues of the CD18 cytoplasmic domain involved in the inhibition of LFA-1 seem to be different from those for fibronectin receptors.

Lymphocyte function-associated Ag-1 (LFA-1)4 (CD11a/CD18; αLβ2) is a member of the leukocyte integrin subfamily of adhesion receptors (1, 2). By interacting with its ligands, the intercellular adhesion molecules (ICAM-1 (CD54) (3), ICAM-2 (CD102) (4, 5, 6), and ICAM-3 (CD50)) (7, 8), LFA-1 participates in several important functions in the immune system (9). LFA-1-mediated cell adhesion is regulated by cell activation. Through the process of “inside-out” signaling, low avidity LFA-1 is converted into an active form capable of mediating adhesion. This reversible conversion correlates with cell activation and does not involve an increase in the cell surface expression of LFA-1 (10, 11). Thus, a qualitative rather than quantitative change in LFA-1 is responsible for up-regulating adhesion to ICAM-1. The prevailing theories to explain this phenomenon are that LFA-1 undergoes a conformational change upon cell activation, that LFA-1 is redistributed at the cell surface, or both (12, 13). Evidence for the former comes from the existence of Abs that recognize epitopes strictly on activated integrins (11, 14, 15, 16). Other studies have suggested that a change in the cell surface distribution influencing the clustering and subsequent adhesion of LFA-1 may be important (17, 18, 19, 20).

The intracellular events leading to increased adhesion are currently poorly understood. Recent studies have suggested that the integrin cytoplasmic domains play crucial roles in the regulation of integrin function. Studies with integrins containing truncated cytoplasmic domains or heterologous cytoplasmic domains from other integrins support such a model (12). For LFA-1, the CD18 subunit has been shown to be particularly important in regulating adhesiveness (20, 21, 22). For example, truncation of the β2, but not that of the αL subunit, significantly reduces binding of LFA-1 to ICAM-1 (20). A cluster of three threonine residues (positions 758–760) and a phenylalanine (position 766) located 10 and 4 amino acids, respectively, from the carboxyl terminus of human CD18 are necessary for ligand binding (22) as well as for transmission of signals leading to postreceptor events such as formation of focal adhesions, reorganization of the cytoskeleton, and cell spreading (20). Thus, interaction of integrin cytoplasmic domains with the cytoskeleton may be important for regulation of LFA-1 binding. Modulation of adhesion through cytoplasmic domains may also occur upon interaction of these domains with other cytoplasmic factors (23, 24, 25, 26).

In this study, the possibility that overexpression of LFA-1 cytoplasmic domains may disrupt the regulation of LFA-1 on lymphocytes was examined. Chimeric transmembrane proteins consisting of the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of LFA-1 were constructed and used to examine their effects on leukocyte adhesion to ICAM-1, as well as on cell spreading following adhesion. Expression of the exogenous CD18, but not the CD11a, cytoplasmic domain abrogated both adhesion to ICAM-1 and cell spreading following adhesion. Furthermore, the CD18 cytoplasmic domain inhibited the adhesion of leukocytes to fibronectin, suggesting a common regulatory pathway for fibronectin receptors and β2 integrins. To identify amino acid residues responsible for the dominant-negative effect of the CD18 cytoplasmic domain, various truncation mutants of CD18 were expressed in both T and B cells. In vitro binding assays identified amino acid residues at the carboxyl terminus of CD18 that are likely involved in inhibiting leukocyte binding to ICAM-1 and to fibronectin.

The murine B cell line A20A8 (27) and the T cell line EL-4 (28) have been described. All cell lines were cultured in DMEM supplemented with 5% FCS. Rat hybridomas producing anti-LFA-1α (TIB 213:FD441.8), anti-CD4 (TIB 207:GK1.5), and the mouse hybridoma producing anti-rat Ig 54 (TIB 169:RG11/39.4) were obtained from the American Type Culture Collection (Rockville, MD). Purification of mAbs as well as FITC conjugation of RG11/39.4 mAb have been described (27). FITC-conjugated anti-murine CD4 was purchased from Boehringer Mannheim (Indianapolis, IN).

The cDNA fragment encoding the transmembrane and cytoplasmic domains of murine CD11a and CD18 were amplified by PCR. The cDNA fragment encoding the extracellular domain of murine CD4 was also amplified by PCR, and both PCR products were ligated to generate the chimeric constructs (Fig. 1,A). Truncated CD4/18 chimeras (C1, C2, and C3) consisting of the extracellular domain of CD4 and transmembrane and truncated cytoplasmic domains of CD18 were generated by PCR using the CD4/18 construct as template (see Fig. 5 A). All PCR products were subcloned into pBluescript and were verified by nucleotide sequencing. The resulting chimeras were subcloned into the mammalian expression vector pBCMGSneo (29).

FIGURE 1.

Design and expression of LFA-1 cytoplasmic domain chimeras. A, Schematic illustrations of constructs containing the extracellular domain of CD4 fused to the transmembrane and cytoplasmic domains of LFA-1 are shown. Amino acid sequences of CD4 present at the junction with LFA-1 chains are also shown. Constructs were generated by PCR and electroporated into EL-4 and A20A8 cell lines. B, Flow cytometric analysis of transfected A20A8 and EL-4 cells. The expression levels of the CD4/LFA-1 chimeras, as well as of endogenous LFA-1, were determined by direct immunofluorescence using, respectively, anti-CD4 and anti-β2 Abs conjugated to FITC. C, Immunoprecipitation of LFA-1 and of CD4/LFA-1 chimeras. Proteins were immunoprecipitated from surface-biotinylated A20A8 transfectants using anti-αL (L) or anti-CD4 (4) primary Abs, followed by rat anti-mouse Ig secondary Abs conjugated to Affigel 10 beads. After separation by SDS-PAGE and electroblotting, proteins were detected by enzyme chemiluminescence using peroxidase-conjugated streptavidin. Molecular weight markers are shown on the left. EX, extracellular domain; TM, transmembrane domain; CY, cytoplasmic domain; VEC, vector alone; CD4ΔCY and 4ΔC, CD4 extracellular domain; 4/11, CD4/CD11a; 4/18, CD4/18; L, α- and β-chains of LFA-1. 4, CD4/LFA-1 chimeras.

FIGURE 1.

Design and expression of LFA-1 cytoplasmic domain chimeras. A, Schematic illustrations of constructs containing the extracellular domain of CD4 fused to the transmembrane and cytoplasmic domains of LFA-1 are shown. Amino acid sequences of CD4 present at the junction with LFA-1 chains are also shown. Constructs were generated by PCR and electroporated into EL-4 and A20A8 cell lines. B, Flow cytometric analysis of transfected A20A8 and EL-4 cells. The expression levels of the CD4/LFA-1 chimeras, as well as of endogenous LFA-1, were determined by direct immunofluorescence using, respectively, anti-CD4 and anti-β2 Abs conjugated to FITC. C, Immunoprecipitation of LFA-1 and of CD4/LFA-1 chimeras. Proteins were immunoprecipitated from surface-biotinylated A20A8 transfectants using anti-αL (L) or anti-CD4 (4) primary Abs, followed by rat anti-mouse Ig secondary Abs conjugated to Affigel 10 beads. After separation by SDS-PAGE and electroblotting, proteins were detected by enzyme chemiluminescence using peroxidase-conjugated streptavidin. Molecular weight markers are shown on the left. EX, extracellular domain; TM, transmembrane domain; CY, cytoplasmic domain; VEC, vector alone; CD4ΔCY and 4ΔC, CD4 extracellular domain; 4/11, CD4/CD11a; 4/18, CD4/18; L, α- and β-chains of LFA-1. 4, CD4/LFA-1 chimeras.

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FIGURE 5.

Design and expression of truncated CD4/18 chimeras. A, Amino acid sequences of the cytoplasmic domains of the truncated CD4/18 chimeras, C1, C2, and C3. B, Flow cytometry of transfected A20A8 and EL-4 cells. Expression of endogenous LFA-1, as well as truncated CD4/18 chimeras, is shown. C, Immunoprecipitation of endogenous LFA-1 and of truncated CD4/18 chimeras was conducted as in Figure 1 C. Molecular weight markers are shown on the left. VEC, vector alone. CD4ΔCY, extracellular CD4 cytoplasmic domain.

FIGURE 5.

Design and expression of truncated CD4/18 chimeras. A, Amino acid sequences of the cytoplasmic domains of the truncated CD4/18 chimeras, C1, C2, and C3. B, Flow cytometry of transfected A20A8 and EL-4 cells. Expression of endogenous LFA-1, as well as truncated CD4/18 chimeras, is shown. C, Immunoprecipitation of endogenous LFA-1 and of truncated CD4/18 chimeras was conducted as in Figure 1 C. Molecular weight markers are shown on the left. VEC, vector alone. CD4ΔCY, extracellular CD4 cytoplasmic domain.

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A20A8 and EL-4 cells were transfected with 10 μg of either CD4/18, CD4/CD11a, C1, C2, or C3 chimeric cDNA in pBCMGSneo by electroporation. Cells were also transfected with vector alone or with the truncated CD4 cDNA encoding the extracellular and transmembrane domains of CD4 as controls (Fig. 1 A). The transfectants were selected and subsequently maintained in DMEM + 5% FCS containing 0.3 mg/ml (EL-4) or 0.5 mg/ml (A20A8) G418 (Life Technologies, Grand Island, NY). In cases of low expression of chimeras, cells were sorted by FACStar (Becton Dickinson, Mountain View, CA) using anti-CD4 mAb.

Expression of LFA-1 and chimeric CD4 on transfected cells was determined by flow cytometric analysis. Cells (0.5 × 106) were incubated in HBSS containing 2% FCS and 30 μg/ml anti-LFA-1 (TIB 213) Ab for 30 min on ice. After washing with HBSS containing 2% FCS, cells were incubated for 30 min on ice with 5 μg/ml anti-rat Igκ (TIB 169)-FITC-conjugated secondary Ab. Finally, cells were washed in HBSS containing 2% FCS and 0.1% sodium azide. CD4 expression was determined by direct staining of cells with 5 μg/ml anti-CD4 FITC (Boehringer Mannheim), for 30 min at 4°C. Analysis of stained cells was conducted on a FACScan Flow Cytometer (Becton Dickinson).

EL-4 cells (4 × 106) were surface biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce, Rockford, IL) as described (30). Cells were lysed for 10 min at 4°C in HBSS containing 1% Triton X-100, 2% BSA, 5 μg/ml PMSF, and 5 μg/ml leupeptin. After microcentrifugation, supernatants were precleared with 30 μl TIB 169-coupled Affigel-10 beads (Bio-Rad, Richmond, CA) for 90 min at 4°C. Cleared lysates were then incubated with 20 μl of GK1.5 or TIB 213 culture supernatant for 60 min at 4°C, followed by TIB 169-Affigel-10 for a further 60 min with continuous mixing. Beads were washed extensively with 1% Triton X-100/HBSS; bound proteins were eluted with SDS-PAGE sample buffer containing 4% SDS, and then separated on a 10% SDS-PAGE gel. Proteins were then electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) and detected by enzyme chemiluminescence (Pierce, Rockford, IL).

A20A8 or EL-4 cells were stimulated with 50 ng/ml PMA (Sigma, St. Louis, MO) for 25 min at 37°C. Stimulated and unstimulated cells were then labeled with 1 μg/ml 2′, 7′-bis-(2-carboxyethyl)-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, OR) in HBSS for 10 min at 37°C. After washing, cells were resuspended in HBSS containing 2% FCS, and adhesion to immobilized ICAM-1 or fibronectin (Sigma, St. Louis, MO) was assayed essentially as described (31). To test for specificity of integrin binding, A20A8 cells were incubated with peptide Gly-Arg-Gly-Asp-Ser-Pro (RGD) or control peptide Gly-Arg-Gly-Glu-Ser-Pro (RGE) (Life Technologies). The degree of cell adhesion was quantitated using a Cytofluor 2300 microplate reader (Millipore), and was expressed as the percentage of the fluorescence remaining in the wells after washing away unbound cells. To assay cell spreading, unlabeled A20A8 cells were taken through adhesion assays as described above, and then incubated at 37°C for a further 20 min. The bound cells were fixed with 0.5% glutaraldehyde in HBSS and photographed on an inverted microscope using a ×20 objective.

To test whether isolated LFA-1 cytoplasmic domains disrupt the regulation of endogenous LFA-1 function, we generated chimeras consisting of the transmembrane and cytoplasmic domains of CD11a and CD18 joined to the extracellular domain of CD4 (Fig. 1,A). As a control, a truncated form of CD4 lacking all but the first four amino acids of the cytoplasmic domain was also generated. The constructs were transfected into murine B (A20A8) and T (EL-4) cell lines, and the transfected cell lines expressing the chimeric molecules were established by cell sorting using anti-CD4 Abs. Flow cytometric analysis of the resulting lines indicated that all the lines expressed equivalent levels of control or chimeric molecules. The expression of endogenous LFA-1 was unaffected by the presence of the chimeras, and was similar among all the lines (Fig. 1,B). Based on the amino acid sequence of the chimeric molecules, the expected molecular masses were calculated to be approximately 51, 55, and 46 kDa for CD4/18, CD4/CD11a and CD4ΔC chimeras, respectively. Immunoprecipitation of the chimeras from the various lines demonstrated that the sizes of the proteins were higher than the expected sizes, consistent with post-translational modifications (e.g., glycosylation) of the chimeras (Fig. 1 C). The apparent size of the CD4/CD11a chimera is considerably larger than expected, probably due to the constitutive phosphorylation of the CD11a cytoplasmic domain (32) in addition to glycosylation. Importantly, no association of the chimeras with endogenous LFA-1 subunits was observed by immunoprecipitation with anti-CD4 or anti-LFA-1 mAbs.

The effects of CD4/LFA-1 chimeras on LFA-1-mediated cell adhesion to immobilized ICAM-1 were examined. Both EL-4 and A20A8 cells transfected with vector alone bound to ICAM-1, and PMA up-regulated this adhesion significantly (Fig. 2, A and B). Cells transfected with the CD4/11a chimera, as well as those transfected with the truncated CD4 construct, bound to a similar extent. In contrast, cells expressing the CD4/18 chimera bound to ICAM-1 at significantly reduced levels regardless of PMA stimulation. Thus the CD4/18 chimera exhibited a dominant-negative effect on LFA-1-mediated adhesion to ICAM-1 in both B cells and T cells. The lack of binding to ICAM-1 of cells expressing CD4/18 chimera was not due to a defect of LFA-1 molecule. Treatment of those cells with Mn2+ before the binding assay resulted in complete reversal of the dominant-negative effect, suggesting that LFA-1 on CD4/18 transfected cells was functionally intact (Fig. 2 C).

FIGURE 2.

Adhesion of transfected cells to ICAM-1. EL-4 (A) or A20A8 (B) cells were unstimulated (open bars), treated with PMA (hatched bars), or treated with PMA plus anti-LFA-1 (black bars). C, Cells were incubated with medium alone (open bars), with PMA alone (black bars), with PMA plus either anti LFA-1 (gray bars), or with 10 mM MnCl2 (bars with diagonal lines). Cells were labeled with BCECF-AM and added to microwells coated with soluble ICAM-1. After removing unbound cells, the fluorescence emission of bound cells was determined. Results are expressed as the mean of triplicate measurements in each condition ± SEM, and are representative of three independent experiments with similar results. BCMGS, vector alone.

FIGURE 2.

Adhesion of transfected cells to ICAM-1. EL-4 (A) or A20A8 (B) cells were unstimulated (open bars), treated with PMA (hatched bars), or treated with PMA plus anti-LFA-1 (black bars). C, Cells were incubated with medium alone (open bars), with PMA alone (black bars), with PMA plus either anti LFA-1 (gray bars), or with 10 mM MnCl2 (bars with diagonal lines). Cells were labeled with BCECF-AM and added to microwells coated with soluble ICAM-1. After removing unbound cells, the fluorescence emission of bound cells was determined. Results are expressed as the mean of triplicate measurements in each condition ± SEM, and are representative of three independent experiments with similar results. BCMGS, vector alone.

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To examine whether the dominant-negative effect of the CD18 cytoplasmic domain was specific for adhesion to ICAM-1, the binding of A20A8 control and transfected cells to fibronectin in microwells was assayed. Adhesion of A20A8 cells to fibronectin was generally of lower degree than their binding to ICAM-1, but interestingly, the CD4/18 chimera significantly reduced this adhesion whereas CD4/11a had minimal effect (Fig. 3). Binding of A20A8 cells to fibronectin was not inhibited by anti-LFA-1 mAb TIB 213 (data not shown). Thus, this cell adhesion is not mediated by LFA-1 and most likely involves other integrins on the surface of A20A8 cells. Adhesion to fibronectin was completely inhibited by RGD peptide, but not by control RGE peptide, suggesting that this adhesion is mediated by integrins. These results suggest that common mechanisms regulate the function of β2 and fibronectin receptors. Whether overexpression of either LFA-1 cytoplasmic domain affects postbinding events such as cell spreading following adhesion to ICAM-1 was also examined.

FIGURE 3.

Adhesion of transfected A20A8 cells to fibronectin. Cells were unstimulated (open bars), PMA-activated (vertically hatched bars), and PMA activated and treated with either RGD (black bars) or RGE peptide (horizontally hatched bars). Assays were performed as described in Figure 2. Results are the mean of triplicate determinations for each condition ± SEM and are representative of three independent experiments with similar results. BCMGS, vector alone.

FIGURE 3.

Adhesion of transfected A20A8 cells to fibronectin. Cells were unstimulated (open bars), PMA-activated (vertically hatched bars), and PMA activated and treated with either RGD (black bars) or RGE peptide (horizontally hatched bars). Assays were performed as described in Figure 2. Results are the mean of triplicate determinations for each condition ± SEM and are representative of three independent experiments with similar results. BCMGS, vector alone.

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Adhesion assays using A20A8 cells were done, and after washing away unbound cells, the remaining cells were incubated for an additional 20 min at 37°C. Cells transfected with either vector alone, CD4/11a, or CD4ΔCY readily spread on ICAM-1. In contrast, cells expressing CD4/18 did not spread (Fig. 4).

FIGURE 4.

Spreading of A20A8 transfectants on ICAM-1. Cells transfected with vector alone (top left), CD4/18 chimera (top right), CD4/11a chimera (bottom left), or CD4 extracellular domain (bottom right) were taken through conventional plate adhesion assays as described in Materials and Methods except that cells were not labeled with BCECF-AM. After washing away unbound cells, the remaining cells were incubated for a further 20 min. Cells were photographed on an inverted microscope using a ×20 objective.

FIGURE 4.

Spreading of A20A8 transfectants on ICAM-1. Cells transfected with vector alone (top left), CD4/18 chimera (top right), CD4/11a chimera (bottom left), or CD4 extracellular domain (bottom right) were taken through conventional plate adhesion assays as described in Materials and Methods except that cells were not labeled with BCECF-AM. After washing away unbound cells, the remaining cells were incubated for a further 20 min. Cells were photographed on an inverted microscope using a ×20 objective.

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To investigate further the mechanisms by which expression of CD4/18 induces a dominant negative effect on LFA-1-mediated binding to ICAM-1, a group of chimeras termed C1, C2, and C3 were generated (Fig. 5,A). In C1, amino acids PKFAES from the C terminus of the CD18 cytoplasmic domain were deleted. This truncation targeted a phenylalanine at position 764 previously shown to be important for LFA-1 function (22). In mutant C2, amino acids PKFAES and the adjacent ATTTVMN motif, which includes three threonines at positions 758–760, were deleted. These threonines were also previously shown to be required for normal LFA-1 function (20, 22). Lastly, mutant C3 lacked the amino acids deleted in C1 and C2 plus 14 amino acids immediately amino terminal of C2, including a serine at position 756 found previously to be highly phosphorylated in activated LFA-1 (22). Mutants were electroporated into A20A8 and EL-4 cells and those expressing chimeric molecules were selected by panning and FACS sorting using anti-CD4 Abs. Flow cytometric analysis indicated that all cell lines expressed similar levels of endogenous LFA-1 (Fig. 5,B). Similarly, the transfected cell lines expressed the chimeric CD4/18 to the same degree (Fig. 5,B). Expression of CD4/18 chimeras was further analyzed by immunoprecipitation with anti-LFA-1 (FD441.8) or anti-CD4 (GK 1.5) Abs. Bands with the expected molecular masses of 50 kDa for the chimeras and 180 and 90 kDa for the αL- and β2-chains, respectively, of endogenous LFA-1 were identified in all transfectants as determined by SDS-PAGE (Fig. 5 C).

Resting and PMA-stimulated A20A8 and EL-4 cells expressing the C1, C2, or C3 chimeras were tested for binding to immobilized ICAM-1. As shown in Figure 6,A, EL-4 cells expressing C1 lacking the PKFAES sequence, did not bind to immobilized ICAM-1 regardless of PMA stimulation. These results were similar to those for the chimeric CD4/18 containing the intact CD18 cytoplasmic domain (Fig. 6,A). These findings suggest that the PKFAES sequence is not required for the dominant-negative effect of CD4/18 chimera in EL-4 cells. In contrast, EL-4 cells expressing C2 lacking both PKFAES and ATTTVMN bound normally to ICAM-1 and this was significantly enhanced by PMA stimulation (Fig. 6,A). Similarly, cells expressing C3 bound to ICAM-1 to a degree similar to that observed for control cells expressing the extracellular domain of CD4 (Fig. 6 A). In all cases, anti-LFA-1 Ab (FD441.8) abrogated binding to ICAM-1, indicating that binding was LFA-1 specific. These data suggest that the ATTTVMN sequence is critical for the negative regulation of LFA-1 binding to ICAM-1 in EL-4 cells overexpressing the CD18 cytoplasmic domain.

FIGURE 6.

Adhesion to ICAM-1 of EL-4 (A) and A20A8 (B) cells transfected with truncated forms of CD4/18. Cells were unstimulated (open bars), PMA activated (black bars), or PMA activated and treated with anti-LFA-1 mAb (gray bars). Cell adhesion assays were performed as in Figure 2. The results are the mean ± SEM of triplicate determinations for each condition and are representative of three independent experiments with similar results. C, Adhesion to fibronectin of A20A8 cells transfected with truncated CD4/18 chimeras. Cells were unstimulated (open bars), PMA-activated (black bars), and PMA-activated and treated either with RGD (bars with horizontal lines) or RGE peptide (stippled bars). Binding of PMA-activated cells to BSA-coated microtiter wells is also shown (bars with diagonal lines). Results are the mean ± SEM of triplicate determinations for each condition and are representative of three independent experiments with similar results.

FIGURE 6.

Adhesion to ICAM-1 of EL-4 (A) and A20A8 (B) cells transfected with truncated forms of CD4/18. Cells were unstimulated (open bars), PMA activated (black bars), or PMA activated and treated with anti-LFA-1 mAb (gray bars). Cell adhesion assays were performed as in Figure 2. The results are the mean ± SEM of triplicate determinations for each condition and are representative of three independent experiments with similar results. C, Adhesion to fibronectin of A20A8 cells transfected with truncated CD4/18 chimeras. Cells were unstimulated (open bars), PMA-activated (black bars), and PMA-activated and treated either with RGD (bars with horizontal lines) or RGE peptide (stippled bars). Binding of PMA-activated cells to BSA-coated microtiter wells is also shown (bars with diagonal lines). Results are the mean ± SEM of triplicate determinations for each condition and are representative of three independent experiments with similar results.

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Similar results were obtained with A20A8 cells expressing C1, C2, or C3 chimeras (Fig. 6,B). However, unlike EL-4 cells, the C1 chimera only partially inhibited the adhesion of A20A8 cells to ICAM-1, whereas C2 had no inhibitory effect. Therefore, not only the carboxyl-terminal six amino acids (PKFAES), but also the adjacent seven residues (ATTTVMN) seem to be involved in the inhibition of adhesion of A20A8 to ICAM-1. As expected, cells expressing the intact CD18 cytoplasmic domain (CD4/18) did not bind to ICAM-1 (Fig. 6 B).

The effects of C1, C2, or C3 on the adhesion of A20A8 cells to fibronectin were also examined. Binding of A20A8 cells to fibronectin was significantly inhibited upon expression of CD4/18, C1, and C2, but not C3 (Fig. 6,C). The inhibition by C1 was comparable with that observed with CD4/18 whereas C2 was a slightly less effective. Binding to fibronectin was inhibited by RGD, but not by RGE (Fig. 6 C).

Previous studies demonstrated that truncation of either of the cytoplasmic domains of LFA-1 significantly alters LFA-1-mediated cell adhesion to ICAM-1 (21, 33, 34). These findings suggested that LFA-1 function is regulated by interactions between cytosolic regulatory molecules with the cytoplasmic domains of CD11a, CD18, or both. Based on these observations, we hypothesized that expression of exogenous LFA-1 cytoplasmic domains may competitively inhibit interactions between putative regulatory molecules with endogenous LFA-1 cytoplasmic domains and disrupt the regulation of LFA-1-mediated cell adhesion. To test this hypothesis, chimeric transmembrane proteins consisting of the cytoplasmic and transmembrane domains of LFA-1 and the extracellular domain of CD4 were generated and transfected into the murine T lymphoma line EL-4 and the B lymphoma line A20A8. The CD4 portion of these chimeras was used to select transfectants expressing the chimeric protein. Results from adhesion assays showed that the chimera containing the cytoplasmic domain of CD18, but not CD11a, profoundly inhibited LFA-1-mediated cell adhesion to ICAM-1, as well as cell adhesion to fibronectin. Treatment of the transfectants with phorbol ester, which normally up-regulates integrin function, failed to overcome this inhibitory effect of the CD4/18 chimera. This dominant-negative effect of the CD4/18 chimera was diminished by truncation of carboxyl-terminal residues of the CD18 cytoplasmic domain. Truncated CD4 (CD4ΔCY) lacking the most of its cytoplasmic domain also had no effect on LFA-1. Therefore, the dominant-negative effect appears to be mediated by the cytoplasmic domain of CD18. This effect does not seem to be due to competition between endogenous CD18 and the CD4/18 chimera for association with the CD11a subunit since immunoprecipitation of CD4/18 showed no association of CD11a with the chimera. Similarly, immunoprecipitation with anti-CD11a mAbs detected coprecipitated CD18, but not the CD4/18 chimera. Therefore, it is more likely that the exogenous CD18 cytoplasmic domain disrupts the mechanisms that regulate endogenous LFA-1.

Our results are consistent with a previous report demonstrating that expression of integrin β1 or β3 cytoplasmic domains reduced the binding affinity of platelet glycoprotein αIIbβ3 in transfected Chinese hamster ovary cells (23). However, it was unexpected that the CD4/18 chimera in our study inhibited adhesion mediated not only by LFA-1, but also by fibronectin receptors. Adhesion of A20A8 cells to fibronectin was effectively inhibited by RGD, but not by RGE, tripeptide, suggesting that the receptor is likely an integrin molecule. A blocking anti-murine β1 mAb (35) did not inhibit the adhesion of A20A8 to fibronectin (data not shown), and the identity of the fibronectin receptors on A20A8 is currently unknown. Nevertheless, the dominant-negative effect of CD4/18 on cell adhesion to fibronectin suggests that a common pathway regulates different integrins.

Results with the deletion mutants of the CD4/18 chimera showed that the carboxyl-terminal 13 amino acid residues of the CD18 cytoplasmic domain are critical for the dominant-negative effect on adhesion of LFA-1 to ICAM-1. Truncated CD4/18 lacking these 13 amino acid residues failed to inhibit adhesion of both EL-4 and A20A8 to ICAM-1. Among these amino acid residues, the role of the carboxyl-terminal hexapeptide (PKFAES) varied between the two cell lines tested. In EL-4 cells, the hexapeptide is dispensable for the dominant-negative effect, whereas truncation of the hexapeptide resulted in a significant reduction of the dominant-negative effect of CD4/18 on A20A8 cell adhesion. Interestingly, the hexapeptide was dispensable for the inhibition of adhesion of A20A8 to fibronectin. Even truncation of the carboxyl-terminal 13 amino acid residues of the CD18 cytoplasmic domain did not completely reverse the inhibitory effect of CD4/18 on the adhesion to fibronectin. Therefore, the dominant-negative effect of the CD4/18 chimera on LFA-1 and on fibronectin receptors appears to require different regions of the CD18 cytoplasmic domain. The results further suggest that multiple regulatory molecules may interact with the CD18 cytoplasmic domain.

Hibbs et al. reported that human LFA-1 lacking the carboxyl-terminal five amino acids of the CD18 cytoplasmic domain failed to bind to ICAM-1 (22). Mutational analysis suggested that phenylalanine at position 766 and three threonines at positions 758–760 are critical for the binding of LFA-1 to ICAM-1. Mutations of the threonine residues also impaired postreceptor functions, including alteration of cell spreading, disruption of cytoskeletal stress fiber formation, and localization of receptors to focal contacts (20). Our results are consistent with these observations and confirm the importance of the carboxyl-terminal 13 amino acid residues, including the three threonines and phenylalanine, in the regulation of LFA-1 binding to ICAM-1. The cytoplasmic domains of some integrin β-chains are conserved at the carboxyl termini. In particular, threonine residues at positions 758–760 in CD18 are highly conserved among β1, β2, and β3 subunits (22). How this region of the CD18 cytoplasmic domain is involved in the regulation of LFA-1 and other integrins is currently unknown.

The CD4/18 chimera likely disrupts the interaction of cytosolic regulatory molecules with the CD18 cytoplasmic domain. To date, multiple proteins have been reported to interact with the CD18 cytoplasmic domain, including the cytoskeletal proteins α-actinin (36), talin (17), and filamin (37). However, α-actinin and filamin bind to the CD18 cytoplasmic domain at positions 724–747 and 728–745, respectively, which is 10 amino acid residues upstream of the carboxyl-terminal 13 amino acids implicated in our studies. It is unlikely that interaction with these cytoskeletal proteins is responsible for the dominant-negative effect of CD4/18, although an involvement of other unidentified cytoskeletal proteins cannot be ruled out. Another cytosolic protein possibly interacting with the CD4/18 chimera is cytohesin-1, which has been reported to bind to the CD18 cytoplasmic domain through its SEC7 domain and to induce activation of LFA-1 (25). CD4/18 may compete with endogenous CD18 for the binding of cytohesin-1 and inhibit activation of LFA-1. The cytohesin-1-binding site in the CD18 cytoplasmic domain is currently unknown, and further studies are needed to determine whether cytohesin-1 is involved in the dominant-negative effect of the CD4/18 chimera.

It has become apparent in recent years that the regulation of integrin function is a complex process that is dependent on the individual integrins and cell types studied. However, similarities may exist in the regulation of different integrins, because most of them can be activated in a similar fashion in what is known as “integrin cross-talk.” In some cases, the ligand binding of one integrin can suppress the function of other integrins (trans-dominant inhibition). This is thought to be caused by a blockade of the target integrin signaling processes that control integrin affinity (inside out signaling) (38), suggesting again that integrins may share common regulatory mechanisms. Our results not only support this notion, but also may have important practical implications. Adhesion of leukocytes to endothelial cells seems to involve multiple integrins, including LFA-1, very late antigen-4, and α4β7 (39, 40, 41, 42). It may be possible to inhibit functions of these integrins by introducing CD18 cytoplasmic domain or synthetic homologues, which may provide an effective therapy in modulating this component of the inflammatory response.

We thank Dr. Neil E. Reiner for critically reading the manuscript.

1

This work was supported by the Medical Research Council of Canada and the Arthritis Society of Canada with core support from the BC Cancer Agency.

4

Abbreviations used in this paper: LFA-1, lymphocyte function-associated antigen-1; CD4/18, chimera consisting of the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of CD18; CD4/CD11a, chimera consisting of the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of CD11a; CD4ΔCY, construct containing the extracellular and transmembrane domains of CD4; CD4/LFA-1, chimeras containing the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of either CD18 or CD11a; RGD, Gly-Arg-Gly-Asp-Ser-Pro; RGE, Gly-Arg-Gly-Glu-Ser-Pro.

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