LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the β2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized β2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated β2 variants, we showed that the subregion Q23-D300 of the β2 subunit is sufficient to combine with the αL and αM subunits intracellularly. However, only the β2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the β2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0.5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the β2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.

The integrins are a family of cell adhesion molecules that mediate a wide repertoire of biological functions (1). The β2 integrin subfamily is comprised of four members sharing a common β subunit associated noncovalently with the following four distinct but structurally homologous α subunits: LFA-1 (αLβ2, CD11a/CD18), Mac-1 (αMβ2, CD11b/CD18), p150,95 (αXβ2, CD11c/CD18), and αDβ2 (2, 3). In the present study, we focus on LFA-1 and Mac-1. LFA-1 is expressed on all leukocytes and is involved in a broad range of immunological processes including leukocyte extravasation, Ag presentation, and T-lymphocyte alloantigen-induced proliferation (4). The three well-characterized LFA-1 counterreceptors are ICAM-1 (CD54), -2 (CD102), and -3 (CD50), all of which are members of the Ig superfamily (5, 6, 7). Mac-1 is expressed mainly on cells of myeloid lineage and plays an important role in the phagocytosis of infectious agents (8), the transendothelial migration of phagocytes (9, 10), and the activation of neutrophils and monocytes (11). Mac-1 recognizes a wide spectrum of ligands, which include ICAM-1 (12), fibrinogen (13), the blood-clotting factor X (14), the complement fragment iC3b (8), the hookworm neutrophil inhibitory factor (15), and denatured proteins (16).

The extracellular domain of the common β2 subunit of LFA-1 and Mac-1 is linearly organized into a short N-terminal cysteine-rich region (CRR),5 a highly conserved region (HCR) predicted to have an Inserted (I)-domain-like fold, a mid-region, and a major CRR followed by a transmembrane segment and a cytoplasmic tail (Fig. 1). The N-terminal CRR is predicted to form a plexin-semaphorin-integrin (PSI) domain (17). Functional roles have been assigned to the HCR and the major CRR. Mutations of selected residues in the HCR abolished the ligand-binding capacity of LFA-1 and Mac-1 (18). In addition, of the 10 missense mutations reported to date for patients with leukocyte adhesion deficiency type-1, nine reside in the HCR, the exception being the R593C mutation, which is located in the CRR. These mutations either abrogate the expression of the CD11/CD18 integrins or lead to the expression of dysfunctional integrins (19, 20). These observations underscore the importance of the HCR in the expression and ligand-binding capacity of the β2 integrins. The major role of the CRR appears to be regulation of adhesion functions. Replacing the major CRR of the β2 subunit with that of β1, which does not form heterodimers with either the αL or αM subunits, results in a chimeric β21(CRR) subunit that forms a constitutively active LFA-1 with respect to ICAM-1-binding (21). Furthermore, the epitopes of β2 integrin-activating mAbs KIM185, KIM127, MEM48, and CBR LFA-1/2 reside in the major CRR of the β2 subunit (22, 23, 24, 25). Thus, perturbation of the major CRR affects the activity of the β2 integrins. The β2 cytoplasmic tail interacts with a number of actin-binding proteins through which the adhesion properties of LFA-1 and Mac-1 are regulated (26, 27, 28, 29, 30, 31, 32).

FIGURE 1.

Schematic map of the integrin β2 subunit (CD18)-truncated variants. The integrin β2 subunit is linearly organized into a N-terminal CRR, a HCR with two suggested boundaries as indicated (Ref), a mid-region, a major CRR, a transmembrane segment, and a cytoplasmic tail. The 56 conserved cysteine residues are indicated by vertical lines under the main diagram. The two predicted long-range disulfide bridges (C25-C447) and (C420-C662) are indicated. The β2 variants are named according to the amino acid at which the stop codon is introduced. The initiation methionine is assigned number “1” in the protein sequence. Illustrated below the main diagram is the region A430-C459, encompassing the cysteine residues C445, C447, and C449, and the predicted disulfide bond arrangements.

FIGURE 1.

Schematic map of the integrin β2 subunit (CD18)-truncated variants. The integrin β2 subunit is linearly organized into a N-terminal CRR, a HCR with two suggested boundaries as indicated (Ref), a mid-region, a major CRR, a transmembrane segment, and a cytoplasmic tail. The 56 conserved cysteine residues are indicated by vertical lines under the main diagram. The two predicted long-range disulfide bridges (C25-C447) and (C420-C662) are indicated. The β2 variants are named according to the amino acid at which the stop codon is introduced. The initiation methionine is assigned number “1” in the protein sequence. Illustrated below the main diagram is the region A430-C459, encompassing the cysteine residues C445, C447, and C449, and the predicted disulfide bond arrangements.

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It was previously reported that an integrin β3 variant lacking most of the major CRR, transmembrane segment, and cytoplasmic tail combined with a truncated version of the integrin αIIb subunit and formed a functional soluble receptor (33). The extracellular domains of LFA-1, Mac-1, α1β1 and α3β1 have also been expressed as soluble molecules and shown to be able to bind ligands (34, 35, 36, 37). In this article, we report the determination of a minimal fragment of the integrin β2 subunit required for the surface expression of LFA-1 and Mac-1, and the effects of β2 subunit truncations on the functions of the resultant variants.

The following mAbs were generous gifts from various sources: MHM23 (dimer specific) and MHM24 (anti-αL; Refs. 38 and 39) were obtained from A. J. McMichael (John Radcliffe Hospital, Oxford, U.K.); LPM19c (anti-αM; Ref. 40) was obtained from the Leukaemia Research Fund Diagnostic Unit (John Radcliffe Hospital); KIM185 (anti-β2, activating mAb; Ref. 41) was obtained from Dr. M. K. Robinson (Celltech, Slough, U.K.); mAb 24 (directed against an activation reporter epitope on β2 integrins; Ref. 42) was obtained from Dr. N. Hogg (Imperial Cancer Research Fund, London, U.K.); MEM148 (anti-β2; Ref. 43) was obtained from Dr. V. Horejsí (Institute of Molecular Genetics, Prague, Czech Republic); and MRC OX33 and MRC OX34 were obtained from M. J. Puklavec (Medical Research Council Cellular Immunology Unit, Oxford, U.K.). The mAb H52 was previously described (44). Purified IgGs of MHM23, MHM24, LPM19c, and H52 were prepared from hybridoma supernatants using HiTrap protein-G columns (Pharmacia Biotech, Uppsala, Sweden). ICAM-1/Fc was prepared as described previously (45).

The αL cDNA in expression vector pcDNA3 (Invitrogen, Groningen, The Netherlands) was described previously (46). Full-length αM cDNA was obtained by tailoring partial cDNA clones screened from a HPB-ALL library (45) using probes constructed by PCR based on a published sequence of αM (47). The β2 cDNA J8.1E (21) in pBluescript KS− (Stratagene, La Jolla, CA was used as a template for the construction of the truncated β2 variants. They were named according to the amino acids at which stop codons (*) were introduced through site-directed mutagenesis using PCR or the QuikChange Site-Directed Mutagenesis kit (Stratagene). In all cases, the modified PCR fragments were re-introduced into pBS-J8.1E. The authenticity of the truncated β2 variants was confirmed by DNA sequencing before subsequent subcloning into expression vector pcDNA3. The initiation methionine is assigned number “1” in the protein sequence.

COS-7 cells were cultured in complete media containing the following: RPMI 1640 with l-glutamine (Life Technologies, Paisley, U.K.), 10% (v/v) heat-inactivated FCS (Sigma, St. Louis, MO), and 100 μg/ml kanamycin. Transfections were performed using the DEAE-dextran method (45). COS-7 cells grown to ∼90% confluency in 80 cm2 tissue culture flasks were washed twice with RPMI 1640 followed by incubation in 10 ml of RPMI 1640 containing 0.4 mg/ml DEAE-dextran, 0.1 mM chloroquine, and 5 μg each of the respective expression vectors for the αL, αM, and β subunits for 4 h at 37°C in a 5% CO2 incubator. Thereafter, cells were washed once in PBS, shocked in 5 ml of 10% (v/v) DMSO in PBS for 3 min, and washed twice in PBS. The cells were then returned to fresh complete media. The following day, cells were detached with trypsin and transferred to new tissue culture flasks for another 24 h. Cells were harvested by detachment with 0.5 mM EDTA in PBS for subsequent analyses.

Twenty-four hours after transfection, COS-7 transfectants were detached with trypsin and ∼1 × 106 cells were transferred into wells of six-well tissue culture plates, and cultured for 24 h at 37°C in a 5% CO2 incubator. Cell surface labeling was conducted by washing adherent cells twice in PBS followed by incubation in sulfo-NHS-biotin (Pierce, Rockford, IL) at 0.5 mg/ml in PBS for 20 min on ice. The reaction was quenched by washing the surface-labeled cells once in PBS containing 10 mM Tris-HCl (pH 8.0) and 0.1% (w/v) BSA, and cell lysates were prepared.

For metabolic labeling, adherent cells were washed twice with PBS and incubated for 30 min in Met/Cys-free RPMI 1640 (ICN Biomedicals, Irvine, CA) at 37°C. Thereafter, cells were incubated in 1.5 ml of Met/Cys-free RPMI 1640 containing 300 mg/L l-glutamine, 5% (v/v) dialyzed heat-inactivated FCS, and 120 μCi/ml of Translabel 35S-labeled Met/Cys (ICN Biomedicals). After 2 h, 1.5 ml of complete media was added to each well. After 4 h at 37°C, the cells were washed twice in PBS and cell lysates were prepared.

For detecting soluble β2 variants, COS-7 transfectants were incubated for 30 min in Met/Cys-free RPMI 1640 (ICN Biomedicals) at 37°C, washed with PBS, and incubated for 6 h in 3 ml of Met/Cys-free RPMI 1640 containing 300 mg/L l-glutamine, 5% (v/v) dialyzed heat-inactivated FCS, and 120 μCi/ml of Translabel 35S-labeled Met/Cys (ICN Biomedicals) at 37°C in a 5% CO2 incubator. The spent medium was collected and centrifuged at 12,000 × g for 5 min to remove cell debris. A total of 1 ml spent medium was used for immunoprecipitation.

Labeled cells were lysed by incubating on ice for 30 min in lysis buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5 mM MgCl2, 0.15 mM CaCl2, 1 mg/ml BSA, 0.5 mM PMSF, 75 μg/ml aprotinin, 2.5 mM iodoacetamide, and 3 mM NaN3). Cell nuclei were removed by centrifugation for 15 min, 12,000 × g at 4°C. Cell lysate (200 μl) was precleared with an irrelevant Ab, either MRC OX33 (IgG1) or OX34 (IgG2a; 50 μl of hybridoma culture supernatant), and rabbit anti-mouse IgG (Sigma) was coupled to protein A-Sepharose beads (Sigma) before immunoprecipitation with the desired mAb (3 μg) for 1 h at 4°C. Beads were washed once with lysis buffer containing 500 mM NaCl, followed by another wash with lysis buffer, and then were loaded onto a 30% (w/v) sucrose cushion. After centrifugation for 3 min at 12,000 × g at 4°C, beads were washed once with 10 mM Tris-HCl (pH 8.0) containing 0.05% (w/v) SDS. Bound proteins were eluted with SDS-sample buffer containing 30 mM DTT at 85°C for 5 min. Protein samples were subjected to SDS-PAGE. For surface labeling studies, proteins were transferred to immobilon P membranes (Millipore, Bedford, MA) by electrophoresis and biotinylated protein bands were detected with streptavidin-HRP followed by ECL-plus (Amersham Life Sciences, Little Chalfont, U.K.). For metabolic labeling analysis, the protein gel was fixed in 10% (v/v) acetic acid and 20% (v/v) isopropanol for 20 min, incubated in Amplify (Amersham Life Sciences) for 15 min, vacuum-dried at 80°C onto a 3 MM Whatman chromatography paper, and exposed to Fuji (London, U.K.) x-ray film for 48 h at −70°C.

For analysis of LFA-1-mediated adhesion to ICAM-1, the wells of Polysorb microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μl/well of goat anti-human IgG (Fc specific) at 5 μg/ml in 50 mM sodium bicarbonate buffer (pH 9.2) for 16–20 h at 4°C. Nonspecific binding sites were blocked with 0.5% (w/v) BSA (Sigma) in PBS for 30 min at 37°C. Thereafter, 50 μl of 1 μg/ml ICAM-1/Fc in PBS containing 0.1% (w/v) BSA was added to each well of the coated plates and incubated for 2 h at room temperature. This coating was shown to be saturating for detection of LFA-1-mediated adhesion (21). For analysis of Mac-1-mediated adhesion to denatured proteins, plates were coated with 100 μl/well of 50 μg/ml BSA in sodium bicarbonate buffer for 24 h at 4°C. Nonspecific binding sites were blocked with 150 μl/well of 0.2% (w/v) polyvinyl-pyrrolidone (m.w. 10,000; Sigma) in PBS for 30 min at 37°C. In all cases, coated plates were washed twice with wash buffer (RPMI 1640 containing 5% (v/v) heat-inactivated FCS, 10 mM HEPES (pH 7.4)) before being used. Transfected cells were harvested and labeled with 3.0 mM 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester fluorescent dye (Molecular Probes, Eugene, OR) by incubation for 20 min at 37°C. Labeled cells were washed once in wash buffer, resuspended, and dispensed into each well of the ligand-coated plates at 1–3 × 104 cells/well followed by incubation for 30 min at 37°C in a 5% CO2 incubator. For different adhesion assays, cells were incubated with various combinations of activating and blocking reagents. The mAbs MHM24, LPM19c, and/or KIM185 were used at 10 μg/ml. Mg2+/EGTA (5 mM MgCl2 and 1 mM EGTA) and 0.5 mM MnCl2 were used to activate LFA-1- and Mac-1-mediated adhesion, respectively. Total cell fluorescence was determined using a fluorescence plate reader (CytoFluor 4000; PerSeptive Diagnostics, Framingham, MA). Plates were washed three times with wash buffer and the fluorescence of bound cells was determined.

Cells were incubated with 20 μg/ml primary mAb in wash buffer for 1 h at 4°C unless otherwise stated. Thereafter, cells were washed twice and incubated with FITC-conjugated sheep anti-mouse F(ab′)2 secondary Ab (1:400 dilution; Sigma) for 45 min at 4°C. Stained cells were washed once and fixed in 1% (v/v) formaldehyde in PBS. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Data were analyzed using CellQuest Software (Becton Dickinson). Expression index was calculated by (% cells gated positive × geo-mean fluorescence intensity).

The initial set of truncated β2 variants are shown in Fig. 1 (upper part of the diagram). β2(I701*) contains the entire extracellular domain of the β2 subunit with both the transmembrane and cytoplasmic domains removed. β2(C481*) corresponds to a truncated β3 subunit which contained the first 469 expressed residues and was reported to be capable of forming a functionally active platelet integrin αIIbβ3 heterodimer complex (33). β2(C459*) was designed to terminate at the putative N-terminal boundary of the CRR. However, as will be shown later, this boundary assignment may have to be revised. β2(A430*): During the characterization of a β integrin subunit from sea sponge (phylum Porifera), a splice variant was found to code for a truncated β subunit (48). β2(A430*) was constructed accordingly. β2(K414*) and β2(Y301*): The HCR of the β2 subunit was postulated to have an I-domain-like fold. However, two different boundaries of the HCR were predicted to have the same N terminus but different C termini (49, 50). β2(Y301*) was constructed such that it contains the shorter version of the HCR, and β2(K414*) was generated encompassing the longer version. β2(K414*) also included a portion of the mid-region to which the epitope of mAb H52 was mapped (44). Construction of the β2(R450*) and β2(C445*) variants will be described in later sections.

COS-7 cells transfected with αL or αM in combination with the β2 variants were analyzed by flow cytometry using the dimer-specific mAb MHM23 (Fig. 2). Single αL or αM transfectants showed minimal staining with MHM23. Similarly, the MHM23 epitope is not expressed on single β2 transfectants (data not shown), although the β2 subunit is expressed at high levels in COS-7 transfectants (21). Positive staining was detected for transfectants expressing αL or αM in combination with wild-type β2 or the variants β2(I701*), β2(C481*), and β2(C459*), although the expression levels of the integrins with the truncated β2 variants were lower than that with wild-type β2. Minimal MHM23 staining was detected on cells transfected with αL or αM, or with β2(A430*), β2(K414*), and β2(Y301*), suggesting that these variants fail to support heterodimer expression on the cell surface.

FIGURE 2.

Flow cytometric analyses of COS-7 cells transfected with LFA-1 and Mac-1 variants. COS-7 transfectants were analyzed by flow cytometry using the dimer-specific mAb MHM23. The unshaded histogram represents MHM23 staining of COS-7 cells transfected with single αL or αM. %GP, Percentage of cells gated positive; MFI, geo-mean fluorescence intensity; and EI, expression index.

FIGURE 2.

Flow cytometric analyses of COS-7 cells transfected with LFA-1 and Mac-1 variants. COS-7 transfectants were analyzed by flow cytometry using the dimer-specific mAb MHM23. The unshaded histogram represents MHM23 staining of COS-7 cells transfected with single αL or αM. %GP, Percentage of cells gated positive; MFI, geo-mean fluorescence intensity; and EI, expression index.

Close modal

This conclusion was confirmed by surface-labeling experiments. Transfectants were surface biotinylated and cell lysates were immunoprecipitated with mAb MHM24 (αL specific) or mAb LPM19c (αM specific). Both αL and β2 wild-type or variant subunits were precipitated by MHM24 (Fig. 3,A, left panel) from the cell lysates of αLβ2wt, αLβ2(I701*), αLβ2(C481*), and αLβ2(C459*) transfectants. In contrast, only low levels of αL were precipitated by MHM24 from lysates of αL, αLβ2(A430*), αLβ2(K414*), and αLβ2(Y301*) transfectants. An identical profile was obtained using the heterodimer-specific mAb 6.5e (not shown), whose epitope on the β2 subunit has been mapped to a region within the shortest of the β2-truncated subunits (i.e., β2(Y301*); Ref. 24). A similar profile was observed for Mac-1 (Fig. 3 A, right panel) using LPM19c with the exception that the αM band was prominent in all lanes. This is concordant with the expression properties of the αL and αM subunits as monomers on COS-7 cell transfectants; whereas the αL subunit is expressed at a suboptimal level (21), the expression level of the αM subunit is high (51) as compared with αLβ2 and αMβ2 transfectants, respectively.

FIGURE 3.

Immunoprecipitation of LFA-1- and Mac-1-truncated variants. COS-7 cells were transfected with the indicated cDNA expression constructs followed by cell surface (A) or metabolic labeling (B). Immunoprecipitations were conducted with mAbs MHM24 (anti-αL) and LPM19c (anti-αM). Immunoprecipitated complexes were resolved on 7.5–10% acrylamide gradient gels under reducing conditions.

FIGURE 3.

Immunoprecipitation of LFA-1- and Mac-1-truncated variants. COS-7 cells were transfected with the indicated cDNA expression constructs followed by cell surface (A) or metabolic labeling (B). Immunoprecipitations were conducted with mAbs MHM24 (anti-αL) and LPM19c (anti-αM). Immunoprecipitated complexes were resolved on 7.5–10% acrylamide gradient gels under reducing conditions.

Close modal

The lack of cell surface expression of β2(A430*), β2(K414*), and β2(Y301*) variants in association with αL and αM might be due to defects in protein biosynthesis or heterodimer assembly. To examine these possibilities, the same set of transfectants was metabolically labeled with [35S]-labeled Met/Cys and cell lysates were immunoprecipitated with MHM24 or LPM19c. In all cases, the β2 variants coprecipitated with the αL or αM subunits (Fig. 3 B). Hence, β2(A430*), β2(K414*), and β2(Y301*) are expressed and can combine with the αL or αM subunit, but they do not promote cell surface expression of the resultant heterodimers.

The size of each of the following β2 variants precipitated was in agreement with their predicted molecular mass: β2wt (∼95 kDa), β2(I701*) (∼83 kDa), β2(C481*) (∼50 kDa), β2(C459*) (∼48 kDa), β2(A430*) (∼46 kDa), β2(K414*) (∼43 kDa), and β2(Y301*) (∼38 kDa).

LFA-1-mediated adhesion was assayed on immobilized ligand ICAM-1 (Fig. 4 A). The wild-type αLβ2 transfectants showed minimal adhesion to ICAM-1 but adhesion was augmented in the presence of Mg2+/EGTA (42) or the activating mAb KIM185 (41). In contrast, αLβ2(I701*), αLβ2(C481*), and αLβ2(C459*) transfectants adhered strongly to ICAM-1 irrespective of the presence or absence of Mg2+/EGTA. KIM185 was only included for the analysis of αLβ2wt and αLβ2(I701*) because its epitope was mapped to the β2 major CRR and, therefore, was absent from the remaining β2 variants (41). In all cases, adhesion was specific because it was abrogated in the presence of the function-blocking mAb MHM24. Consistent with the lack of cell surface expression, αLβ2(A430*), αLβ2(K414*), and αLβ2(Y301*) transfectants showed minimal adhesion to ICAM-1 under all conditions (data not shown).

FIGURE 4.

Ligand-binding properties and mAb 24 staining of LFA-1 variants. Cell adhesion assays were performed as described in Materials and Methods. A, Adhesion of transfectants expressing LFA-1 variants to ICAM-1. The forward bars represent adhesion in the presence of function-blocking mAb MHM24 (anti-αL). B, COS-7 transfectants expressing the indicated constructs were stained with mAb 24 under different conditions and analyzed by flow cytometry. Taking into account the difference in expression levels between wild-type LFA-1 and the variants, the expression level of mAb 24 was calculated by the following equation: (expression index of mAb 24/expression index of mAb MHM23) × 100.

FIGURE 4.

Ligand-binding properties and mAb 24 staining of LFA-1 variants. Cell adhesion assays were performed as described in Materials and Methods. A, Adhesion of transfectants expressing LFA-1 variants to ICAM-1. The forward bars represent adhesion in the presence of function-blocking mAb MHM24 (anti-αL). B, COS-7 transfectants expressing the indicated constructs were stained with mAb 24 under different conditions and analyzed by flow cytometry. Taking into account the difference in expression levels between wild-type LFA-1 and the variants, the expression level of mAb 24 was calculated by the following equation: (expression index of mAb 24/expression index of mAb MHM23) × 100.

Close modal

The mAb 24 is an activation reporter for LFA-1 (42). Thus we performed flow cytometric analyses on the expression of the mAb 24 epitope on the αLβ2(I701*), αLβ2(C481*), and αLβ2(C459*) transfectants. αLβ2wt transfectant only showed positive staining with mAb 24 in the presence of Mg2+/EGTA at 37°C (Fig. 4 B). In contrast, αLβ2(I701*), αLβ2(C481*), and αLβ2(C459*) transfectants expressed the mAb 24 epitope in the absence of Mg2+/EGTA at 4°C. Taken together, these observations indicate that αLβ2(I701*), αLβ2(C481*), and αLβ2(C459*) are expressed in an active conformation and, therefore, display constitutive ICAM-1 adhesion.

We next examined the adhesion properties of Mac-1 variants on denatured BSA (Fig. 5). αMβ2wt transfectant showed a low level of adhesion, which was augmented in the presence of 0.5 mM Mn2+. Adhesion was specific because it was abolished by the function-blocking mAb LPM19c. A similar adhesion profile was observed for the αMβ2(I701*), αMβ2(C481*), and αMβ2(C459*) transfectants. In addition, αMβ2wt can also be activated to adhere to denatured BSA with the mAb KIM185. It should be noted that αMβ2(I701*) cannot be activated with the mAb. Thus, unlike LFA-1 variants, the Mac-1 variants did not show any constitutive ligand-binding properties. Similar results were obtained for adhesion of the Mac-1 variants on iC3b (data not shown).

FIGURE 5.

Ligand-binding properties of Mac-1 variants. Adhesion of transfectants expressing Mac-1 variants to denatured BSA. The forward bar represents adhesion in the presence of function-blocking mAb LPM19c (anti-αM). Binding is activated either by 0.5 mM MnCl2 (upper panel) or KIM185 (lower panel).

FIGURE 5.

Ligand-binding properties of Mac-1 variants. Adhesion of transfectants expressing Mac-1 variants to denatured BSA. The forward bar represents adhesion in the presence of function-blocking mAb LPM19c (anti-αM). Binding is activated either by 0.5 mM MnCl2 (upper panel) or KIM185 (lower panel).

Close modal

The above results showed that there is a clear distinction between the two sets of β2 truncation variants; the variants longer than β2(C459*) support LFA-1 and Mac-1 surface expression and those shorter than β2(A430*) do not. Therefore, there may be critical residues within the A430-L458 region of β2 that are important for LFA-1 and Mac-1 cell surface expression. The prime candidates are the three highly conserved cysteine residues found in all integrin β subunits sequentially arranged in the pattern of CXCXC. In the case of β2 subunit, they are C445, C447, and C449 (Fig. 1, lower part of the diagram). This led us to construct the β2(C445*) and β2(R450*) variants which flank the boundaries of the CECRC sequence.

Both β2(C445*) and β2(R450*) support LFA-1 and Mac-1 cell surface expression (Fig. 6,A). In addition, the LFA-1 variants are constitutively active with respect to ICAM-1 adhesion, and the Mac-1 variants require Mn2+ for adhesion to denatured BSA (Fig. 6 B). Thus, the conserved cysteines C445, C447, and C449 of the β2 subunit are not required for LFA-1 and Mac-1 cell surface expression. Furthermore, deletion of these cysteines did not alter the functional properties of the resultant variant β2(C445*) as compared with β2(R450*) or to longer versions of the β2 variants.

FIGURE 6.

Properties of the β2(R450*) and β2(C445*) variants. COS-7 cells were transfected with β2(R450*) and β2(C445*) in combination with αL and αM. A, Transfectants were analyzed by flow cytometry using the dimer-specific mAb MHM23. B, Adhesion of transfectants expressing the resultant LFA-1 variants to ICAM-1 (upper panel) and Mac-1 variants to denatured BSA (lower panel). %GP, Percentage of cells gated positive; MFI, geo-mean fluorescence intensity; EI, expression index.

FIGURE 6.

Properties of the β2(R450*) and β2(C445*) variants. COS-7 cells were transfected with β2(R450*) and β2(C445*) in combination with αL and αM. A, Transfectants were analyzed by flow cytometry using the dimer-specific mAb MHM23. B, Adhesion of transfectants expressing the resultant LFA-1 variants to ICAM-1 (upper panel) and Mac-1 variants to denatured BSA (lower panel). %GP, Percentage of cells gated positive; MFI, geo-mean fluorescence intensity; EI, expression index.

Close modal

We next examined whether the β2 variants that support LFA-1 and Mac-1 cell surface expression could be secreted into the tissue culture media (Fig. 7). Single β2 variant transfectants were metabolically labeled with [35S]-labeled Met/Cys and the spent media collected. β2(I701*), β2(C481*), β2(C459*), β2(R450*), and β2(C445*) were immunoprecipitated from the spent media of the respective transfectants with the mAbs H52 and MEM148. However, only β2(I701*) was precipitated with the mAb KIM185, which was consistent with the KIM185 epitope mapping to the major CRR of the β2 subunit (41).

FIGURE 7.

Expression of soluble β2 variants. COS-7 cells were transfected with the indicated β2 variants in the absence of any α subunit. An αL transfectant was included as control. Transfectants were metabolically labeled and spent media was immunoprecipitated with the β2-specific mAbs H52, MEM148, and KIM185. Immunoprecipitated complexes were resolved on 7.5–10% polyacrylamide gradient gels under reducing conditions.

FIGURE 7.

Expression of soluble β2 variants. COS-7 cells were transfected with the indicated β2 variants in the absence of any α subunit. An αL transfectant was included as control. Transfectants were metabolically labeled and spent media was immunoprecipitated with the β2-specific mAbs H52, MEM148, and KIM185. Immunoprecipitated complexes were resolved on 7.5–10% polyacrylamide gradient gels under reducing conditions.

Close modal

We have constructed a series of truncated β2 variants and analyzed for their capacity to support expression of functional LFA-1 and Mac-1. A summary of the results is shown in Table I.

Table I.

Properties of LFA-1 and Mac-1 variants with truncated β2 integrin subunit

LFA-1 ICAM-1 BindingMac-1 BSA Binding
αβaExpbRPMI 1640cMg/EdαβaExpbRPMI 1640cMne
β2 wt − +/− 
β2 1701* +/− 
β2 C481* +/− 
β2 C459* +/− 
β2 R450* +/− 
β2 C445* +/− 
β2 A430* − − − − − − 
β2 K414* − − − − − − 
β2 Y301* − − − − − − 
LFA-1 ICAM-1 BindingMac-1 BSA Binding
αβaExpbRPMI 1640cMg/EdαβaExpbRPMI 1640cMne
β2 wt − +/− 
β2 1701* +/− 
β2 C481* +/− 
β2 C459* +/− 
β2 R450* +/− 
β2 C445* +/− 
β2 A430* − − − − − − 
β2 K414* − − − − − − 
β2 Y301* − − − − − − 
a

Intracellular αβ complexes detected by metabolic labeling followed by immunoprecipitation.

b

Surface expression of αβ complexes detected by flow cytometry using heterodimer-specific mAb, and by surface labeling followed by immunoprecipitation.

c

LFA-1 adhesion to ICAM-1 and Mac-1 adhesion to BSA performed in RPMI 1640 wash buffer.

d

LFA-1 adhesion to ICAM-1 performed in RPMI 1640 wash buffer supplemented with 5 mM MgCl2 and 1 mM EGTA.

e

Mac-1 adhesion performed in RPMI 1640 wash buffer supplemented with 0.5 mM MnCl2.

The current model of an integrin heterodimer is based on the electron microscopic studies of two integrins, α5β1 and αIIbβ3 (52, 53). The N-terminal regions of both subunits interact with each other to form an ovoid head from which their respective C-terminal regions extend. The biochemical data presented in this paper suggest that the structures of LFA-1 and Mac-1 are in general agreement with this model. The truncated constructs can be segregated into two groups. The shorter constructs, β2(Y301*), β2(K414*), and β2(A430*) can only interact with the αL and αM subunit intracellularly but the longer constructs (i.e., β2(I701*), β2(C481*), β2(C459*), β2(R450*), and β2(C445*)) also support cell surface expression of LFA-1 and Mac-1. The expression indices of the LFA-1 and Mac-1 variants, as determined by the expression of the epitope of the dimer-specific mAb MHM23 (Figs. 2 and 6), are generally lower than that of their wild-type counterparts. Exactly how different truncations affect the level of LFA-1 and Mac-1 expression remains to be investigated. As a general trend, the truncated β2 variants support LFA-1 expression better than Mac-1. In both cases, the most ineffective variant is β2(I701*), which is composed of the entire extracellular domain of the β2 subunit. There is no gross structural change in the β2(I701*) variant, because the conformational epitopes of the four mAbs, KIM185 (22), KIM127 (22), MEM48 (23), and MEM148 (43) are expressed (data not shown).

It is intriguing that the shorter β2 subunits can interact with the αL and αM subunits intracellularly but cannot support cell surface expression of LFA-1 and Mac-1. The evidence for intracellular interaction came from the immunoprecipitation of the biosynthetically labeled cell lysates using mAbs specific for the αL and αM subunits. The shorter β2 subunits were found in the immunoprecipitate in addition to the α subunits (Fig. 3,B). The evidence that the shorter subunits cannot support surface expression of LFA-1 and Mac-1 came from two sets of experiments. First, the epitope of heterodimer-specific mAb MHM23 was not detected by flow cytometry (Fig. 2). Second, the shorter β2 subunits were not coprecipitated with the α subunits from surface-labeled cell lysates using the α subunit-specific mAbs (Fig. 3,A). Similar flow cytometry and immunoprecipitation profiles were obtained using the heterodimer-specific mAbs IB4 (54) and 6.5e (Ref. 24 ; data not shown). It is unlikely that the shorter β2 subunits are not detected on the cell surface because they are not biotinylated. There is no difference in the number of lysine residues of the shortest β2 subunit detected by surface labeling (i.e., β2(C459*)) and the longest β2 subunit not detected (i.e., β2(A430*)). Furthermore, when we permeabilized the αLβ2(A430*) transfectant before labeling, we were able to detect the intracellular biotinylated β2(A430*) subunit by coprecipitation with the αL subunit using the αL-specific mAb MHM24 (data not shown). It is interesting to note that most of the mAbs mapped to the N-terminal region of the human β2 subunit only recognize the αβ heterodimer but not the free β2 subunit (24). For this reason we are at present unable to determine the fate of the shorter truncated β2 subunits in particular, whether they are secreted as monomers into the tissue culture medium. In contrast, the longer variants can be found in the tissue culture medium of their respective transfectants by immunoprecipitation with the mAbs H52 and MEM148 (Fig. 7). These soluble β2 variants will be most useful for future studies on the structure and function of the β2 integrin subunit.

A characteristic feature of the integrin β subunits is the very conserved pattern of cysteine residues in their extracellular domains. A disulfide-bond model has been proposed for the integrin β3 subunit (55). Of relevance to this investigation are the three cysteines in positions 445, 447, and 449 in the β2 subunit. On the assumption that the disulfide bonding patterns are identical in the β2 and β3 integrin subunits, C445 and C449 engage in a disulfide bond, and C447 bonds to C25 near the N terminus of the subunit. This arrangement suggests that these cysteines are likely to have an important role in the overall structure and function of the integrin heterodimers. Intriguingly, the β2(C445*) variant in which the three cysteine residues were deleted retains the ability to assemble with αL and αM. Furthermore, the resultant LFA-1 and Mac-1 molecules did not exhibit any significant alteration in cell surface expression and ligand-binding properties as compared with other longer β2 variants (R450*, C459*, C481*, and I701*). Therefore, the importance of these three cysteine residues and their possible disulfide bond engagements needs to be re-evaluated.

We have stated that the extracellular domain of the integrin β subunit is composed of four regions. However, the boundaries between these regions are not well defined. Based on secondary structure analyses, it was suggested that the HCR has an I-domain-like fold. Two C-terminal boundaries have been proposed (49, 50). The experiments presented in this paper did not provide any distinction between the two possibilities because both β2(Y301*) (containing the N-terminal PSI domain and the short version of the HCR) and β2(K414*) (extending beyond the long version of the HCR) behaved identically in all experiments, and in particular, both failed to support LFA-1 or Mac-1 cell surface expression. Correspondingly the mid-region has two alternative N termini. The CRR is generally described as having four repeating elements of about 45 residues, each containing 8 cysteines (56, 57). Again, the boundaries of these repeating elements are uncertain. According to the disulfide bonding pattern proposed for the β3 integrin (54), the repeating element of the CRR starts at C459 (β2 sequence numbering), thus we constructed the variant β2(C459*) which contains the N-terminal PSI, the HCR (either version), and the mid-region. The β2(C459*) variant supports LFA-1 and Mac-1 surface expression, but so do the shorter constructs of β2(C445*) and β2(R450*). If we argue that the breakpoint, the point at which the constructs differ in their ability to support integrin heterodimer expression, must lie between discrete subdomains of the β subunit, then the boundary between the mid-region and the CRR must lie between the residues A430 and Q444. Secondary structure prediction indicated that there is one β strand within this region (50). Experiments are under way to determine whether there is a single residue break in this region to pinpoint the boundary between the mid-region and the CRR.

The cell surface-expressed LFA-1 variants (i.e., LFA-1 with the β2(C445*), β2(R450*), β2(C459*), β2(C481*), and β2(I701*) subunits) bind ICAM-1 more effectively than the wild type. In particular, whereas the transfectants expressing wild-type LFA-1 require either the activating mAb KIM185 or Mg2+/EGTA for adhesion to immobilized ICAM-1, the transfectants expressing the LFA-1 variants are constitutively active with respect to ICAM-1 adhesion, which cannot be promoted with either KIM185 or Mg2+/EGTA. This cannot be due to the difference in the level of LFA-1 expression on the cell surface because all variants are expressed at lower levels as compared with the wild type. These variants also expressed the active-LFA-1 reporter epitope of mAb 24 in the absence of Mg2+/EGTA and elevated temperature, suggesting that they are expressed in a conformational active state. It has been reported from different laboratories that removal of the cytoplasmic tail of the β2 subunit led to the expression of constitutively active LFA-1 (27, 28). This is possibly due to a constraint imposed on the wild-type LFA-1 by interactions between the α and β subunits. In the platelet integrin αIIbβ3, the opposing charges of an arginine residue (R995) in the αIIb subunit and an aspartic residue (D723) in the β3 subunit are important in maintaining the integrin in a resting state (58). It was proposed that disruption of this salt bridge may be a critical feature of integrin activation via inside-out signaling. There are no transmembrane and cytoplasmic domains in the truncated β2 subunits described in this paper, thus the absence of a restraining salt bridge may have been the cause of the constitutive active properties of the LFA-1 variants. However, Mac-1 with the same set of β2 variants is not constitutively active with respect to adhesion to denatured BSA but, like the wild-type Mac-1, adhesion could be promoted with 0.5 mM Mn2+. Because αL and αM only share ∼35% sequence homology, it is reasonable to postulate that the β2 subunit interacts differently with the αL and αM subunits. Recently, we have found that two mutations, A341P in the HCR and R593C in the major CRR, of the integrin β2 subunit of patients with leukocyte adhesion deficiency type-1, which can support LFA-1 but not Mac-1 expression (S. K. A. Law, manuscript in preparation). Together with the results presented in this article, these observations suggest that the β2 subunit may have different roles in the function of LFA-1 and Mac-1.

We thank Dr. E. C. Mathew for careful review of the manuscript and B. R. Micklem for constructing the β2(C445*) and β2(R450*) variants.

1

This work was supported in part by Grants L0509 and L0524 from the Arthritis Research Campaign, U.K.

5

Abbreviations used in this paper: CRR, cysteine-rich region; HCR, highly conserved region; I, inserted; PSI, plexin-semaphorin-integrin.

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