Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5838-5842, June 1993 Immunology Ligand intercellular adhesion molecule 1 has a necessary role in activation of integrin lymphocyte function-associated molecule 1 (adhesion/activation epitope/induced fit) CARLOS CABANAS AND NANCY HOGG* Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom Communicated by Walter F. Bodmer, March 16, 1993 (receivedfor review December 10, 1992) ABSTRACT The signaling that causes the leukocyte inte- MATERIALS AND METHODS grin lymphocyte function-associated molecule (LFA-1) to bind firmly to its ligand intercellular adhesion molecule 1 (ICAM-1) mAbs and ICAM-1. Production, isolation, and character- is transduced indirectly through other T-cell receptors and is ization of mAb 24 has been described (6). Other mAbs used termed inside-out signaling. We show here that the high- were mAb 38 (6) and MHM24 (9), both recognizing the LFA-1 affinity state of LFA-1 is characterized by expression of the a subunit (CDiia); RRi/1, recognizing ICAM-1 (CD54) (10); LFA-1 epitope detected by monoclonal antibody 24. This CD3 mAb UCHT1 (11); and control mAbs 52U (IgGl) and 4U epitope is expressed not in response to the initial agonist- (IgG2a) (unpublished data). mAbs 24, 38, RRi/1, 52U, and mediated signal but when LFA-1 binds to ICAM-1, indicating 4U were used as purified immunoglobulin and the other that ligand binding induces an alteration in LFA-1. As would mAbs were used as tissue culture supernatant. Fab fragments be predicted, the monoclonal antibody 24 epitope is confined to of mAb 24 were prepared by pepsin digestion (1%; wt/wt) of the LFA-1, which is located at the site ofcontact between T cells intact IgGl in order to generate F(ab')2 fragments, which and ICAM-1-expressing transfectants. When a fixation proto- were subsequently reduced with 0.01 M cysteine for 1 h at col for "freezing" receptors is used, only T cells that are fixed room temperature and blocked for a further hour with 0.03 M after prior exposure to ICAM-1 bind frmly to ICAM-1 a iodoacetamide (8). Any intact IgG and F(ab')2 fragments second time. This suggests that, in addition to the inside-out were separated from Fab 24 by extensive adsorption with signaling, a previously unrecognized requirement for full ac- protein A-coupled Sepharose followed by fractionation using tivation ofthe leukocyte integrin LFA-1 is the initial interaction gel-filtration chromatography. Analysis of 10 ,ug ofFab 24 by with its ligand ICAM-1. Thus, activation of LFA-1 is in part SDS/PAGE showed it to be free of any such contaminants achieved by an induced fit imposed from without by interaction (results not shown). Direct fluorescein isothiocyanate (FITC) with ligand. conjugates of mAb 24 were prepared as described and were used at 10 p,g/ml (8, 12). Recombinant ICAM-1-Fc protein consisted of the first The integrin lymphocyte function-associated molecule 1 three domains of ICAM-1 and the hinge, CH2, and CH3 (LFA-1) and immunoglobulin-like intercellular adhesion mol- domains ofIgGl (Fc) (13). The expression plasmid containing ecule 1 (ICAM-1) form a receptor/ligand pair of adhesion this construct was a gift from A. Craig and A. Berendt and molecules required for many leukocyte functions (1, 2). was used to produce ICAM-1-Fc protein by transient expres- Regulation of LFA-1/ICAM-1 adhesion resides with LFA-1, sion in COS-1 cells and protein A chromatography. The which, under normal circumstances, binds only weakly to ICAM-1-CHO transfectant was a gift from J. Trowsdale and ICAM-1 (3). On T cells, the affinity ofLFA-1 for ICAM-1 can D. Sansom and was maintained in selection medium contain- be increased transiently by cross-linking other membrane ing geneticin (0.5 mg/ml). receptors such as CD3 (XL-CD3) associated with the T-cell T Cells. Peripheral blood mononuclear cells (PBMCs) were receptor for longer periods oftime by treatment with phorbol prepared from freshly drawn heparinized blood by centrifu- esters such as phorbol dibutyrate (PdBu) (3, 4) or by incu- gation over Ficoll/Hypaque (Pharmacia). As previously re- bating T cells with the divalent cations required by LFA-1 for ported, activated T cells were function (5). We have described an epitope recognized by expanded from unstimulated monoclonal antibody (mAb) 24 that is present on the CD11 a PBMCs by culture in RPMI 1640 medium containing 10% subunit and is expressed in a manner that correlates with fetal calf serum (FCS) plus treatment with phytohemagglu- Mg2+ (and Mn2+) binding (5-7). This epitope has been tinin (10 pg/ml) for 48 h (5, 8). Cells were then washed and thought to be a "reporter" of the activated state of LFA-1 in cultured in 10% FCS plus recombinant interleukin 2 (20 that its expression parallels receptor activity (5-8). A role for ng/ml) (Cetus). the mAb 24 epitope in LFA-1 activation was further sug- T-Cell Binding Assay. Standard T-cell assay. The LFA-1- gested by the ability of mAb 24 to inhibit some LFA-1 dependent adhesion of cultured T cells to immobilized mediated functions, possibly by locking the integrin in acti- ICAM-1-Fc has been described (5, 8). Briefly, 40 ul of vated form and preventing a return to the low binding state ICAM-1-Fc per well in phosphate-buffered saline (PBS) (5 (8). In the present study, we show that in addition to signals ,g/ml) was incubated overnight at 4°C, followed by satura- such as XL-CD3, another necessary step in the induction of tion of any remaining plastic sites with 2% bovine serum fully active LFA-1 is fulfilled by the initial binding of ligand albumin in PBS. T cells (2-5 x 107 cells per ml) were labeled ICAM-1 to LFA-1. This causes exposure of the activation with 200 ,uCi (1 Ci = 37 GBq) of 51Cr for 1 h at 37°C. marker or epitope detected by mAb 24, which is character- Cross-linking of CD3 was accomplished by incubating an istic of LFA-1 when it is able to complex with ICAM-1 in a aliquot of the T cells on ice for 20 min with UCHT1 mAb (1:5 stable manner. Abbreviations: FITC, fluorescein isothiocyanate; ICAM-1, intercel- lular adhesion molecule 1; LIBS, ligand-induced binding site(s); The publication costs ofthis article were defrayed in part by page charge LFA-1, lymphocyte function-associated molecule-i; mAb, mono- payment. This article must therefore be hereby marked "advertisement" clonal antibody; PdBu, phorbol dibutyrate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5838 Downloaded by guest on October 6, 2021 Immunology: Cabaiias and Hogg Proc. Natl. Acad. Sci. USA 90 (1993) 5839 dilution of culture supernatant; IgG2a isotype), followed by the latter samples undergoing an additional incubation at 0°C a second incubation period of 20 min on ice with goat with tetramethylrhodamine B isothiocyanate (TRITC)- anti-mouse IgG2a in RPMI 1640 medium (1:100 dilution; conjugated goat anti-mouse IgGl antiserum (1:50 dilution). Nordic, Maidenhead, U.K.). Either the XL-CD3- or PdBu The cells were then fixed with 5% formaldehyde in PBS for (50 nM)-treated or control T cells (2 x 105 cells per well) were 20 min at room temperature. For double-antibody staining, added to the ICAM-1-Fc-precoated wells, centrifuged at 75 x PdBu-treated T cells were incubated with 25 /4 of directly g for 2 min, and incubated at 37°C for the periods of time conjugated FITC-mAb 24 (IgGl) at 10 ug/ml in RPMI 1640 indicated. After washing four times with warm RPMI 1640 medium followed by a second incubation with the anti-CD11a medium to remove unbound T cells, the radioactivity of the mAb 38 (IgG2a) at 10 ,ug/ml and a final incubation with bound T cells was assayed with a Betaplate counter (LKB). TRITC-conjugated goat anti-mouse IgG2a antiserum (1:50 Fixed T-cell assay. Briefly, [3H]thymidine-labeled T cells dilution in RPMI 1640 medium). Incubations with various were incubated at 2.5 x 105 cells per well in flexiwell plates control mAbs showed the observed staining patterns to be for 20 min at 37°C in 40 nM PdBu/RPMI 1640 medium in specific. either the presence or absence of anti-ICAM-1 mAb RR1/1 at 40 pg/ml. After two washes, cells were fixed with the indicated percentages of freshly prepared paraformaldehyde RESULTS in Hepes buffer for 10 min at 37°C and the fixation reaction Expression of mAb 24 Epitope Correlates with Activation of was terminated with 0.1% glycine for 10 min at room tem- LFA-1. Although previous experiments suggested that the perature. Because the T cells were now fixed, Mg2+/EGTA presence of the mAb 24 epitope paralleled LFA-1 activity, its was used as a means of activating receptors from outside the expression had never been assessed in a system that mim- cell. After three washes, the fixed T cells were transferred icked physiological T-cell activation. Therefore, we triggered into ICAM-1-Fc-containing wells precoated at 60 pg/ml and cultured T cells in several ways and tested the activation incubated a second time in Hepes buffer containing 1 mM MgCl2 and 1 mM EGTA for 30 min at 37°C. Finally, the T cells status of LFA-1 by its ability to bind to ICAM-1 using the were washed three times with warm Hepes buffer and the following two assays.