Soluble ICAM-1 by Divalent Metal Cations Leukocyte Function ...

Molecular Regulation of the Interaction Between Leukocyte Function-Associated Antigen-1 and Soluble ICAM-1 by Divalent Metal Cations Mark E. Labadia, Deborah Durham Jeanfavre, Gary O. Caviness, and Maurice M. Morelock1 Surface plasmon resonance (SPR) was used to investigate and characterize the interaction between LFA-1 and sICAM-1 (a soluble form of ICAM-1). Full-length LFA-1 was immobilized on a hydrophobic surface, and sICAM-1 binding was monitored in a flow cell format. The binding of sICAM-1 to LFA-1 was specific and dependent upon Mg21; Abs to both sICAM-1 and LFA-1 blocked the interaction, and EDTA abolished all binding. Association and dissociation rate constants (ka and kd, respectively) for sICAM-1 were 2.24 3 105 M21 s21 and 2.98 3 1022 s21, respectively, giving a calculated KICAM of 133 nM. Since the LFA-1/ICAM-1 interaction is highly sensitive to the presence of metal cations, SPR was also used to probe the affinity of the metal binding sites. The KMg values were 160 and 12 mM in the absence (EGTA) and the presence of Ca21 (100 mM), respectively; in addition, KMn was 2 mM in the presence of Ca21 (100 mM). Increasing Ca21 into the millimolar concentration range, however, resulted in a competitive displacement of Mg21/Mn21 and decreased sICAM-1 binding. Based on these data, a synergistic model for the molecular regulation of LFA-1 by divalent metal cations is proposed, and implications to cellular adhesion are discussed. The Journal of Immunology, 1998, 161: 836 – 842.

C

ell adhesion plays a fundamental role in many cellular processes, including embryonic development, malignant transformation, and regulating the immune response (1). Adhesion events are regulated through families of transmembrane receptors, one of which is the integrin supergene family (2, 3). The integrin receptors have evolved to recognize a variety of substrates, including extracellular matrix proteins and cell surface receptors (4, 5). The b2 integrin LFA-1 and its cognate ligand ICAM-1 are instrumental in T cell processes such as natural killing, B cell responses, Ag presentation, and lymphocyte extravasation (6). The b2 integrins are metallo-proteins composed of an a-chain (CD11, at least four known) noncovalently associated with a common b2-chain (CD18) (7). The a-chains of the b2 integrins contain three metal-binding sequences that resemble the Ca21-binding EFhand motif. Recently, an additional metal binding site was identified on some a-chains in an inserted region, referred to as the I domain (8). The I domain appears to play a significant role in ligand binding. Abs that block as well as activate LFA-1 function have been mapped to the I domain (9, 10). Mutations within this domain significantly affect the affinity of ICAM-1 for LFA-1 (11, 12). The crystal structure of the I domain revealed a Mg21 (Mn21) coordination site and was termed the metal ion-dependent adhesion site (13, 14). This site is defined by a DXSXS binding motif along with two noncontiguous T and D residues. Interestingly, this motif is also found in the b-chain (14). Additional studies have shown that mutations within the EF-hand (15) or metal ion-dependent adhesion site binding motifs within the a-chain (11, 12) sigDepartment of Biology, Boehringer Ingelheim Pharmaceuticals, Research and Development Center, Ridgefield, CT 06877 Received for publication January 29, 1998. Accepted for publication March 23, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Maurice M. Morelock, Department of Biology, Boehringer Ingelheim Pharmaceuticals, Research and Development Center, 900 Ridgebury Rd., P.O. Box 368, Ridgefield, CT 06877-0368.

Copyright © 1998 by The American Association of Immunologists

nificantly affect ligand binding, confirming the critical role for divalent cations observed at the cellular level. The proposed mechanism(s) by which LFA-1 is activated for cellular adhesion is dependent upon at least two events, multimerization and conformational change. Clustering of LFA-1, which appears to be mediated exclusively by calcium, has been implicated in intracellular signal transduction and results in increased cellular adhesion (presumably due to avidity). In addition to clustering, the conformation of LFA-1 has been shown to play an important role in ICAM-1 binding. Interestingly, certain Abs that recognize LFA-1 have been shown to promote ICAM-1 binding, rather than inhibit it. Although magnesium and manganese do not promote LFA-1 clustering, they do support cellular adhesion (16). Although the divalent cation requirements for the ICAM-1/ LFA-1 interaction have been extensively studied (5, 16 –23), the specific role(s) of divalent cations remains unclear. Recent studies suggest that the binding of magnesium and manganese to LFA-1 induces a high affinity conformation of the receptor (19, 21). This metal binding event causes a conformational change in LFA-1 such that an epitope (a signature of the high affinity LFA-1 state) is exposed, being recognized by mAb 24 (19). Calcium, on the other hand, does not induce the presentation of the mAb 24 epitope. It does, however, enhance magnesium-mediated cellular adhesion at submillimolar concentrations (5, 17), but attenuates this same phenomena in the millimolar (physiologic) concentration range (23). These observations suggest that the affinity of LFA-1 for ICAM-1 is finely tuned through a subtle interplay between magnesium/(manganese) and calcium. In this study, surface plasmon resonance (SPR)2 (see Ref. 24 for a review) was used to investigate and characterize the interaction between LFA-1 and sICAM-1 (a soluble form of ICAM-1). LFA-1 was immobilized on a hydrophobic surface, and sICAM-1 binding was monitored in a flow cell format. On and off rate constants (kon 2 Abbreviations used in this paper: SPR, surface plasmon resonance; sICAM-1, soluble ICAM-1; kon and koff, on and off rate constants; RU, resonance units; Rmax, maximum response.

0022-1767/98/$02.00

The Journal of Immunology

FIGURE 1. Immobilization of LFA-1 onto a hydrophobic surface (BIAcore HPA chip). Arrows indicate injection points of specific reagents along the sensorgram: OG, n-octly-b-D-glucopyranoside.

and koff, respectively) for sICAM-1 were determined. Since the LFA-1/ICAM-1 interaction is highly sensitive to the presence of metal cations, SPR was also used to probe the affinity of the metal binding sites. Based on these data, a synergistic model for the molecular regulation of LFA-1 by Mg21 and Ca21 is proposed, and implications to cellular adhesion are discussed.

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FIGURE 2. Specificity of the LFA-1/sICAM-1 interaction. Top curve, sICAM-1 was injected with no Ab; middle curve, sICAM-1 was coinjected with R6.5 (10 mM); bottom curve, the LFA-1-immobilized surface was pretreated with R3.1 (67 nM) before sICAM-1 was injected. The concentration of sICAM-1 for all sensorgrams was 1 mM; baselines were normalized.

(BIAcore). Derivative plots of the sensorgrams were used to distinguish regions of mass transport and changes in bulk refractive index due to sample/running buffer differences. The edited data were then analyzed by a global analysis method (see Appendix).

Materials and Methods

Determination of binding constants for Mg21, Mn21, and Ca21

Proteins

To determine the binding constants of the divalent metal cations to sites on LFA-1, the binding of sICAM-1 to LFA-1 was used as the reporter. To minimize effects due to contaminating metals, the introduced metals and buffer salts used in these experiments were of the highest quality (Alfa AESAR, Ward Hill, MA). EGTA (20 mM) was included in the sample and running buffer to measure the Kd for Mg21 in the absence of Ca21 ions. A typical Mg21 titration experiment consisted of a coinject protocol: the LFA-1 surface was equilibrated (4 –5 min) with the Mg21 concentration of interest, immediately followed by an injection of 1 mM sICAM-1 in the same buffer. The data were analyzed using one- and two-site metal binding models (see Appendix). Experiments involving the effects of Ca21 and Mn21 were conducted in a similar manner.

LFA-1 was prepared as previously described (25), and sICAM-1 was provided by the biotechnology group at Boehringer Ingelheim Pharmaceuticals (Indianapolis, IN). The construct for a soluble form of ICAM-1 containing domains 1 and 2 expressed in baculovirus (26) was the gift of Dr. Timothy Springer. The mAb CA7 (directed against domain 5 of ICAM-1) was provided by Dr. Robert Rothlein.

LFA-1 immobilization All SPR experiments were conducted on a BIAcore-1000 instrument (BIAcore, Piscataway, NJ). HPA (hydrophobic surface) chips were purchased from BIAcore and stored at 4°C until use. Briefly, the surface was pretreated with 40 mM n-octly-b-D-glucopyranoside (50 ml in H2O) at a flow rate of 10 ml/min (see Fig. 1). The flow rate was then decreased to 2 ml/min, and LFA-1 at 100 mg/ml in 10 mM Tris and 1 to 2 mM MgCl2, pH 7.5, was adsorbed onto the surface until saturation (;3000 resonance units (RU)). The surface was then blocked with 0.1 mg/ml BSA at a flow rate of 2 ml/min for 25 min. Several cycles of sICAM-1 at 1 mM were then injected at a flow rate of 30 ml/min over the LFA-1 surface to assess the binding capacity of the surface. The activity of the surface (as determined by stochiometry of mass binding) ranged from 40 to 60% (presumably due to improperly orientated and/or denatured LFA-1). The maximum response (Rmax) for all the LFA-1-immobilized surfaces ranged from 30 to 180 RU. In an alternative protocol, the LFA-1/sICAM-1 interaction was evaluated by immobilizing LFA-1 with an Ab. The TS2/4 mAb (27) was immobilized by a standard amine-coupling protocol. Briefly, a carboxymethyl-cellulose surface (CM5 chip purchased from BIAcore) was activated with EDC/NHS for 7 min according to the manufacturer’s protocol. Ab at 50 mg/ml in 10 mM sodium acetate, pH 4.5, was then injected over the activated surface for 8 min at a rate of 5 ml/min. The surface was blocked with 1 M ethanolamine. This protocol resulted in approximately 3000 to 4000 RU of immobilized Ab. LFA-1 at 50 mg/ml in 10 mM Tris and 1 to 2 mM MgCl2, pH 7.5, was adsorbed onto the TS2/4 surface at a flow rate of 2 ml/min until saturation was achieved (;1000 RU). The binding capacity (Rmax) of the surface ranged between 100 and 200 RUs.

Determination of binding constants for sICAM-1 Binding data for the sICAM-1/LFA-1 interaction were obtained by injecting various concentrations of sICAM-1 over an LFA-1 surface (see above) at a flow rate of 30 ml/min. Experiments were conducted in buffer consisting of the following reagents: 10 mM Tris, 150 mM NaCl, and 1 to 2 mM MgCl2 at pH 7.5. The running buffer always contained 1 to 2 mM Mg21 to prolong the functional capacity of the immobilized LFA-1. Surface densities (Rmax) of LFA-1 ranged from 30 to 180 RU. Association, dissociation, and equilibrium data were collected using the BIAevaluation software

Results Several control experiments were conducted to evaluate the specificity of sICAM-1 binding to LFA-1 immobilized on a hydrophobic surface (see Fig. 2). Injection of sICAM-1 over a surface loaded with a nonrelevant protein such as BSA results in negligible binding. Injection over a surface loaded with LFA-1 and then blocked with BSA, gives the typical SPR sensorgram (see Fig. 2); injection in 10 mM EDTA, however, results in no binding, in accordance with the requirement of metals for binding. Abs against either sICAM-1 or LFA-1 can be used to delineate the specificity of this interaction. For example, R3.1 binds LFA-1 and has previously been shown to block the sICAM-1/LFA-1 interaction (28). Consequently, injection of sICAM-1 (see Fig. 2) across the R3.1bound LFA-1 surface gives a sensorgram exhibiting minimal binding. In other experiments, Abs against sICAM-1 can be used to either block or enhance the sICAM-1/LFA-1 interaction. For example, R6.5 recognizes domains 1 and 2 of sICAM-1, which have been shown to block ICAM-1 binding to LFA-1 (29). Coinjection of R6.5 and sICAM-1 results in ablation of sICAM-1/LFA-1 binding (see Fig. 2). In contrast, when nonblocking CA7 (directed against domain 5 of ICAM) is used, a dimeric sICAM-1 species, i.e., CA7-(sICAM-1)2, is formed. Injection of this complex results in a high affinity interaction with LFA-1 (due to an avidity effect), defined by an extremely slow off rate (data not shown) (30). Thus, sICAM-1 binds in a specific manner to LFA-1 immobilized on the hydrophobic surface, in agreement with previous assays and observations.

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REGULATION OF LFA-1 BINDING BY DIVALENT METAL CATIONS

FIGURE 4. Mg21 binding to immobilized LFA-1. The KMg values in the absence of (100 mM EGTA) and the presence of Ca21 (100 mM) were 2.78 (60.31) 3 1025 and 2.41 (60.45) 3 1024 M, respectively, using Equation 4 with sICAM-1 (1 mM) as the reporter (the values in parentheses are the SEs). These data have been normalized to each other by converting RU data to the fraction u of LFA-1 bound with Mg21, i.e., uMg (see Appendix).

FIGURE 3. Determination of kinetic rate constants for the interaction between sICAM-1 and immobilized LFA-1. Nonlinear regression global analysis (applying Equation 2, see Appendix) converged to the following solution: ka 5 2.17 (60.02) 3 105 M21 s21, kd 5 4.70 (60.07) 3 1022 s21, Rmax 5 1.59 (0.01) 3 102 RU, and Rmin 5 7.37 (62.33) 3 1021 RU, where the values in parentheses are the SEs.

In an alternative protocol, the LFA-1/sICAM-1 interaction was evaluated by immobilizing LFA-1 with an Ab. Since the mAb TS2/4 (31) was used to affinity-purify LFA-1, this Ab was selected to immobilize LFA-1. Although the kinetic rate constants obtained were similar to those determined on the hydrophobic surface, the off-rate of sICAM-1 was significantly faster for this surface. The accelerated off-rate may be a common problem encountered with immobilization techniques using Abs that are partially blocking and/or possess only moderate affinities. Since the activity and the integrity of the LFA-1 surface must be maintained throughout the SPR experiment, the hydrophobic surface was chosen for all subsequent studies. Figure 3 shows families of curves for the association (top panel) and dissociation (bottom panel) reactions involving the sICAM1/LFA-1 interaction. Since cellular adhesion has been reported to occur in the presence of physiologic concentrations of magnesium, the kinetic experiment was conducted with 1 to 2 mM Mg21. The parameters ka and kd were determined to be 2.24 (60.69) 3 105 M21 s21 and 2.98 (60.69) 3 1022 s21, respectively (where the number in the parentheses is the SD for n 5 6). The calculated Mg KICAM (5kd/ka) of 1.33 3 1027 M is in excellent agreement with the value obtained previously using ELISA methodology (1.30 3 1027 M) (30); experiments conducted at higher concentrations of Mg21 (10 mM) gave similar results. The amplitude of the reaction (Rmax 2 Rmin) ranged from approximately 35 to 190 RU. Since the ka and kd values varied little over this range, it can be concluded that these data are not affected by artifacts associated with high density surfaces (typically .200 RU), such as mass transport and rebinding phenomena. Truncated sICAM-1 containing only domains 1 and 2 was found to bind LFA-1 with slightly less affinity

(ka 5 1.56 (60.23) 3 105 M21 s21 and kd 5 6.28 (60.32) 3 1022 s21 (n 5 3); calculated Kd 5 4.03 3 1027 M), primarily the result of a faster off rate. Since Mn21 has also been implicated in cellular adhesion, the sICAM-1/LFA-1 interaction was studied in the presence of 1 mM Mn21. The results are similar with ka 5 1.69 (60.42) 3 105 M21 s21 and kd 5 4.59 (60.86) 3 1022 s21 (n 5 Mn 4), giving a calculated KICAM of 2.72 3 1027 M. Thus, the sICAM-1/LFA-1 binding (defined by the presence of Mg21 or Mn21) is characterized by an on rate of approximately 2 3 105 Mg,Mn M21 s21 and an off-rate of 3 to 5 3 1022 s21 (KICAM 5 ;2 3 1027 M). A much lower affinity was observed in the presence of saturating Ca21 (30 mM). Since SPR bulk effects are very large for these experimental conditions, equilibrium (instead of kinetic) data were acquired for the determination of binding affinity (see ApCa pendix), giving a KICAM 5 1.14 (60.29) 3 1025 M (n 5 3). This affinity is nearly 2 orders of magnitude weaker than that characterized in the presence of saturating Mg21 or Mn21. Figure 4 shows the determination of the equilibrium constant for Mg21 using a one-site model (see Appendix). In the absence of Ca21, KMg is 1.60 (61.07) 3 1024 M (n 5 4). In the presence of Ca21, KMg drops an order of magnitude to 1.24 (60.33) 3 1025 M (n 5 2); since EGTA preferentially binds Mn21, KMn was only determined in the presence of Ca21 and was 2.46 (61.39) 3 1026 M (n 5 2). Obviously, Ca21 binds at a separate site, enhancing the affinity of LFA-1 for Mg21 at its preferred site. Increasing concentrations of Ca21 into the millimolar concentration range, however, results in inhibition of sICAM-1/LFA-1 binding. This inhibition was competitive, since increasing concentrations of Mg21/ Mn21 shifted the midpoint of a Ca21 titration curve to higher concentrations (data not shown). The working model for the regulation of sICAM-1/LFA-1 binding by Ca21 and Mg21/Mn21 is shown in Figure 5. This is a two-site metal binding model for LFA-1 in which site 1 (S1) preferentially binds Ca21, and site 2 (S2) preferentially binds Mg21 (or Mn21). Ca21 binding at site 1 induces a conformational change, resulting in a structure that has an increased affinity for Mg21 at site 2. Mg21 binding at site 2 also induces a conformational change in LFA-1, but results in a structure with a high affinity for sICAM-1. Although Ca21 binds preferentially to site 1, titration into the millimolar concentration range (in the presence of Mg21) leads to competitive displacement of Mg21 at site 2 and thereby a


839

Discussion

FIGURE 5. Proposed synergistic model for the molecular regulation of LFA-1 by Ca21 at Site-1 and Mg21 (Mn21) at Site-2.

Mg decrease in sICAM-1 binding, the result of KICAM (133 nM) 3 Ca KICAM (11 mM). The KMetal values (i.e., K1–K5, see Fig. 5) can be approximated by selecting reactant concentrations that isolate each equilibrium. First, K1 and K3 can be determined by setting [Mg21]T and [Ca21]T 5 0, respectively, and titrating the other metal. K2 can then be approximated by holding [Ca21]T at 10 3 K1 and titrating Mg21, and K5 can be approximated by holding [Mg21]T at 10 3 K2 and titrating Ca21. Since the KMetal values depend upon one another (i.e., K1zK2 5 K3zK4), this procedure must be performed in an iterative manner. Since preliminary experiments showed K1 (low micromolar concentration) ,, K5 (low millimolar concentration), the concentration of Ca21 was adjusted to saturate site 1 (100 mM), while minimizing the population of site 2. This approach allowed the equilibrium involving K2 to be isolated and evaluated (see Fig. 4). The constant determined with the most certainty is K3, since Ca21 can be removed from the system using EGTA (see Fig. 4). Knowing, then, K2 and K3 with a high degree of certainty, the equilibria involving K4 and K5 could be studied setting [Mg21] . K2 and titrating Ca21 (see right panel of Fig. 6). All data in Figure 6 were fitted simultaneously (global analysis), holding K2 (1.24 3 1025 M) and K3 (1.60 3 1024 M) constant and fitting for K4 5 1.13 (60.07) 3 1026 M and K5 5 1.54 (60.73) 3 1023 M, where the values in parentheses are the SDs for n 5 2. See Figure 5 for a summary of the KMetal affinities using the twosite metal binding model.

FIGURE 6. Mg21 and Ca21 binding to immobilized LFA-1. Left panel, Titration of Mg21 in the absence of Ca21 (20 mM EGTA); center panel, titration of Mg21 in the presence of Ca21; right panel, titration of Ca21 in the presence of Mg21. sICAM-1 (1 mM) was used as the reporter in each experiment. Nonlinear regression global analysis (applying Equation 6, given in Appendix, with K2 and K3 held constant at 1.24 3 1025 and 1.60 3 1024 M, respectively) converged to the following solution: K4 5 1.18 (60.23) 3 1026 M and K5 5 1.02 (60.15) 3 1023 M, where the values in parentheses are the SEs.

A hydrophobic surface was used to immobilize the membrane protein LFA-1 for SPR analysis (see Fig. 1). The binding of sICAM-1 to LFA-1 was specific (see Fig. 2). Abs that bind to sICAM-1 (R6.5) or LFA-1 (R3.1) blocked the interaction. In addition, EDTA inhibited sICAM-1 binding, demonstrating the dependence upon divalent cations. The observed rate constants (kon and koff) were 2.24 3 105 M21 s21 and 2.98 3 1022 s21, respectively (see Fig. 3). The rate constants were independent of surface LFA-1 surface densities, indicating that the data are free of experimental artifacts. The calculated affinity constant (KICAM) for the LFA-1/ICAM-1 interaction is 133 nM, in agreement with previously published data (30). Although ICAM-1 consists of five domains, recent studies have reported that only domains 1 and 2 are required for binding to LFA-1. A soluble construct consisting of domains 1 and 2 bound to immobilized LFA-1 with an observed kon 5 1.56 3 105 M21 s21 and a koff 5 6.28 3 1022 s21, giving a calculated KD1D2 of 300 nM. This observation corroborates the findings of previous studies (30). The divalent cation dependence of the ICAM-1/LFA-1 interaction has been extensively documented (5, 16 –23). Until now, however, the proposed models of regulation have been based on cellular phenomena. In this study, SPR was used to evaluate purified LFA-1 isolated from the complications of cellular events such as signal transduction and clustering. In the metal binding experiments reported herein, sICAM-1 was held constant, while metal ions were titrated. Since Mg21 (or Mn21) is required for LFA-1/ sICAM-1 interaction, the observed sICAM-1 binding is directly proportional to the fraction of LFA-1 bound with Mg21. In the absence of Ca21, the measured KMg using a one-site model (see Appendix) was 240 mM; in the presence of Ca21 (100 mM), the KMg was shifted to 28 mM (see Fig. 4). It should be pointed out that the equilibration of Mg21 and Ca21 with LFA-1 was nearly instantaneous, indicating that the on and off rates of these metals are very fast. Thus, sICAM-1 binding to the surface-immobilized LFA-1 could not only be switched on and off in a reversible manner, it could also be attenuated by adjusting the concentrations of Mg21 and Ca21. Although micromolar concentrations of Ca21 were found to enhance sICAM-1 binding, increasing Ca21 into the millimolar concentration range resulted in decreased sICAM-1 binding. This inhibition was competitive, i.e., higher concentrations of Mg21 (or Mn21) required higher concentrations of Ca21 to effect the same response. Since the enhancement of Mg21-mediated sICAM-1

840


binding was observed at low Ca21 concentrations and attenuated at higher concentrations, a one-site metal-binding model is not applicable. The role of Ca21 in the regulation of the ICAM-1/LFA-1 interaction has been controversial. Depending upon cell type, activation status, and culture conditions, Ca21 has been shown to be synergistic with Mg21 and enhance binding (5, 17), inhibit binding (21, 23), or have no affect on binding (16). Recent reports have demonstrated that both Mg21 and Ca21 binding to LFA-1 induce distinct epitopes recognized by specific Abs (19, 20). This suggests that metal binding can regulate LFA-1 affinity through conformational changes in the protein structure. Based on this premise and the data presented herein, a two-site model for metal ion regulation of LFA-1 is proposed (see Fig. 5 and Appendix). Site 1 is a Ca21 binding site, while site 2 is a Mg21 (Mn21) binding site required for competent sICAM-1 binding. This model allows for either metal to bind first, with subsequent binding of the other metal to constitute the native LFA-1 receptor. The binding is synergistic, in that binding of Ca21 at site 1 increases the affinity for Mg21 at site 2 and vice versa (hence, the observed log difference of KMg affinities in the absence and the presence of Ca21; see above). At higher concentrations, however, Ca21 competitively displaces Mg21 from site 2 and leads to a form of LFA-1 with lowered Mg Ca affinity for sICAM-1, the result of KICAM (133 nM) 3 KICAM (11 mM). All data in Figure 6 were fitted simultaneously to this twosite model (see Fig. 6). See Figure 5 for a summary of the KMetal affinities. In addition to the model presented herein for LFA-1 (ab2), two models have been proposed for the regulation of other members of the integrin family (a5b1 (32) and aVb3 (33)) by divalent metal ions. All three models include effector (Ca21) and ligand-competent (Mg21, Mn21) metal binding sites. Binding of Ca21 at the effector site increases the affinity of Mg21 at the ligand-competent site, thereby increasing the binding of the complementary ligand. Increasing the Ca21 concentration, however, results in decreased ligand binding, presumably due to displacement of the ligand-competent metal. Ca21 was competitive with Mg21 for the b1 and b2 integrins, but not for the b3 integrin (suggesting that Ca21 acts as an allosteric inhibitor in this system). An additional metal binding site (Mn21) was proposed for the b1 system, since Ca21 was competitive with Mg21 but not with Mn21. In contrast, Ca21 was shown in this study to be competitive with both Mg21 and Mn21, being adequately defined by a two-site model. Thus, regulation of the integrin family by divalent metal cations is characterized by a effector binding site (Ca21) accompanied by a ligand-competent binding site(s) (Mg21/Mn21), with subtle differences defining the specific receptor-ligand interaction. The Kmetal values determined herein suggest that purified LFA-1 is always in a high affinity (Mg21-mediated ligand-competent) state under physiologic conditions (1 mM Mg21 and 1 mM Ca21; i.e., [Mg21]/K2 ; 100 and [Ca21]/K5 ; 1). The question then arises as to how the binding properties determined for LFA-1 in an isolated state correlate with the activity of LFA-1 in a cell membrane. Resting or nonactivated leukocytes exist primarily in a nonadhesive state (18, 20). However, once the leukocyte is activated (either through cell surface receptors or soluble stimuli such as phorbol esters), the leukocyte is transformed into a highly adhesive cell (18, 20). While this activation promotes LFA-1 clustering, it does not enhance the affinity of the LFA-1 for ICAM-1 (23). Current paradigms suggest that LFA-1 present on resting T cells exist in a low affinity/low avidity state, whereas LFA-1 present on activated T cells exist in a high affinity/high avidity state (23, 34). Recent studies (35, 36) have shown that the LFA-1 deletion mutant a1090* (truncated just before the conserved intracellular GFFKR

motif) constitutively binds ICAM-1 with high affinity, but without the ability to mediate cellular adhesion; conversely, a mutant with truncation immediately after the motif (a1095*) neither binds ICAM-1 constitutively nor has the ability to effect cellular adhesion upon stimulation. In addition, mAb 24 (which recognizes an extracellular epitope on activated LFA-1 (19, 21)) binds mutants lacking the binding motif (a1090* and aDGFFKR), but not wildtype a subunit transfectants. It is clear from these data that if cellular interactions with the a subunit tail are eliminated, then LFA-1 will constitutively exist in a high affinity state. The results reported herein are consistent with these data. LFA-1 was characterized in its purified state, unrestrained by intracellular interactions with the a and b subunit tails. Consequently, purified LFA-1 was always observed to be in a high affinity state. What is not clear, however, is whether constitutive LFA-1 exists in a low affinity/low avidity state or whether it exists in a high affinity/low avidity state due to improper presentation to ICAM-1. Interestingly, studies involving MAC-1 (CD11b/ CD18) have shown that this receptor displays high affinity, very low affinity, or no affinity; in addition, no energetic difference between these two states was observed, indicating that activation was not dependent upon a conformational change in the protein (37). Consistent with these observations is the fact that the mAb 24 recognizes a pre-existing epitope on the a subunit (38). Based on these findings and our own, it is not unreasonable to suspect that cell surface LFA-1 always exists in a high affinity state of readiness but constitutively restrained from binding ICAM-1 by intracellular interactions.

Appendix Mathematical models have previously been reported for the analysis of SPR kinetic and equilibrium data (39, 40). These models have been modified and extended to analyze the data presented herein.

Determination of rate constants for the sICAM-1/LFA-1 interaction Equation 1 is a real-time expression for the amount of sICAM-1 bound to the surface-immobilized LFA-1 in association and dissociation reactions.

R5

H

J

ka z @ICAM#T z ~Rmax 2 Rmin! a

H

1 R0 2

J

ka z @ICAM#T z ~Rmax 2 Rmin! a

z e2~ka z @ICAM#T 1 kd! z t 1 Rmin .

(1)

R is the relative response, i.e., corrected for baseline and bulk (refractive index due to sample buffer and protein) in resonance units at time t. R0 refers to the relative response at the point on the respective reaction curve where t is defined to be zero; Rmax is the maximum value of R observed when all surface-immobilized LFA-1 is saturated with sICAM-1; and Rmin is the minimum value associated with no sICAM-1 bound. Equation 1 is globally applied to all kinetic data simultaneously (global analysis), estimating the parameters ka, kd, Rmax, and Rmin, with [sICAM-1]T and t as the independent variables and R as the dependent variable.


841

Determination of equilibrium constants for the sICAM-1/LFA-1 interaction The plateau values (R[ICAM]T) reached in association reactions can be used in the determination of equilibrium constants. The response amplitude (R[ICAM]T 2 Rmin) is always some fractional value (ubound) of the quantity (Rmax 2 Rmin), i.e., uICAM 5 (R[ICAM]T 2 Rmin)/(Rmax 2 Rmin) 5 [ICAM*LFA]/[LFA]T. Rearranging

R@ICAM#T 5 uICAM z ~Rmax 2 Rmin! 1 Rmin

(2)

and substituting for ubound (derived from equilibrium expressions) gives

R@ICAM#T 5

@ICAM#T z ~Rmax 2 Rmin! 1 Rmin , KICAM 1 @ICAM#T

(3)

where KICAM is the dissociation constant for the LFA-1/sICAM-1 interaction. The parameters KICAM, Rmax, and Rmin are estimated by nonlinear regression using Equation 3 with [ICAM]T as the independent variable and R[ICAM]T as the dependent variable. Determination of equilibrium constants for Mg21 and Ca21 Equation 3 is also applicable to equilibrium data involving effector elements, such as metal ions. If the concentration of sICAM-1 is held constant and the concentration of metal ion (now the response-dependent reactant) varied, the signal (due to sICAM-1 binding) will increase in a sigmoid manner with increasing metal concentration. Since [sICAM-1] is being held constant, the Rmax observed is really an apparent maximum response at that particular Mg sICAM-1 concentration (a function of KICAM ) under metal satuMetal rating conditions, i.e., RApparent 5 u z max ICAM Rmax. Equation 3 is then rewritten as

R@Metal#T 5

@Metal#T Metal z @~uICAM z Rmax! 2 Rmin# 1 Rmin . KMetal 1 @Metal#T

(4)

The parameters KMetal, Rmax, and Rmin are estimated by nonlinear regression using Equation 4 with [Metal]T as the independent variable and R[Metal]T as the dependent variable. The working model for the regulation of sICAM-1/LFA-1 binding by Ca21 and Mg21 is shown in Figure 5. This is a two-site model for LFA-1 in which site 1 (S1) preferentially binds Ca21, and site 2 (S2) preferentially binds Mg21. When there are multiple metals, sites, and affinities, the fraction of each species (us1s2) must be determined. By definition, K1 z K2 5 K3 z K4; thus, a knowledge of three constants defines the fourth. Substitutions of equilibrium expressions into mass balance expressions and rearrangement gives the following expression for the fraction of metal-free LFA-1:

uS1~2!S2~2! 5

1 . @Ca#2T @Ca#T @Ca#T z @Mg#T @Mg#T 11 1 1 1 K1 K1 z K2 K3 K1 z K 5

(5)

Equation 5 can then be used to determine us1s2 for each of the metal-bound species. The metal-dependent response for sICAM1/LFA-1 binding can now be written in a manner analogous to Equations 2 and 4 as follows: Ca R@Ca,Mg#T 5 ~uS1~Ca!S2~2! 1 uS1~Ca!S2~Ca!! z @~uICAM z Rmax! 2 Rmin# Mg 1 ~uS1~Ca!S2~Mg! 1 uS1~2!S2~Mg!! z @~uICAM z Rmax! 2 Rmin# 1 Rmin .

(6)

Substituting the appropriate equilibrium expressions for the metalbound us1s2 values, the parameters KMetal (i.e., K1 2 K5), Rmax, and Rmin are estimated by nonlinear regression using Equation 6 with [Ca21]T and [Mg21]T as the independent variables and R[Ca,Mg]T as the dependent variable.

Acknowledgments We thank Joseph R. Woska, Jr. and Drs. Robert Rothlein, T. Kei Kishimoto, and James Stevenson for many helpful discussions and critical reading of the manuscript.

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