©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Energetics of Leukocyte Integrin Activation (*)

Tian-Quan Cai , Samuel D. Wright (§)

From the (1)Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cell adhesion mediated by leukocyte integrin CR3 (CD11b/CD18, ) may be rapidly modulated without changes in receptor number, and transient changes in adhesivity are thought to be driven by reversible alteration of the affinity of CR3 for ligand. Here we measure the binding affinity of CR3 using purified active and inactive receptor and the ligand, C3bi, coupled to alkaline phosphatase. Immobilized, active CR3 bound saturably and with high affinity (12.5 ± 4.7 nM). In contrast, inactive CR3 exhibited no measurable binding. High affinity binding could be restored by the addition of the activating anti-CR3 monoclonal antibody KIM-127 to inactive CR3. Since the affinity of KIM-127 for active and inactive receptor was identical, it cannot contribute the energy to convert a low affinity receptor into a high affinity receptor. Rather, KIM-127 appears to facilitate binding of C3bi by lowering the activation energy for the shift from an inactive to an active state. These results suggest that CR3-mediated binding and detachment of cells is not driven by a reversible change in affinity but by two mechanistically distinct processes, an energetically neutral activation step for binding and an energy-dependent step that reverses binding of ligand.


INTRODUCTION

The leukocyte integrin, complement receptor type 3 (CR3,()Mac-1, CD11b/CD18, ) is expressed by polymorphonuclear leukocytes (PMN), monocytes, and natural killer cells and mediates the binding of cells to surfaces coated with a wide variety of ligands, including C3bi (Wright et al., 1983), fibrinogen (Wright et al., 1988), intercellular adhesion molecule-1 (Diamond et al., 1990), and unknown ligands on endothelial cells (Lo et al., 1989a; Diamond et al., 1990). CR3 thereby plays a critical role in the adhesion of PMN to the vasculature, to opsonized particles, and to extracellular matrix.

The ability of CR3 to bind its ligands is low in resting cells, but it is transiently enabled by stimulation (Wright and Meyer, 1986; Lo et al., 1989a, 1989b). For example, stimulation of PMN with phorbol ester causes a dramatic rise in adhesivity after 20 min and a return to base line by 40 min (Wright and Meyer, 1986). Similar activation-dependent binding has also been found in several other integrins (Dustin and Springer, 1989; O'Toole et al., 1990; Shaw et al., 1990), and various models have been proposed to explain this phenomenon (Hynes, 1992; Smyth et al., 1993; Diamond and Springer, 1994). Since changes in adhesivity may occur without a change in receptor number, the ``affinity modulation model'' has received a significant amount of study. In this model, the avidity of individual receptors is thought to shift reversibly from low to high. Shifting of the affinity to the high level allows adhesion, and shifting back to low affinity allows detachment. Reliable estimates of CR3 affinity, however, have not been made, and studies with other integrins have suggested that the affinity may be too low to measure readily (Horwitz et al., 1985; Lollo et al., 1993). In the present study, we describe a novel, soluble, monomeric ligand for CR3 and an enzymatic microassay to study the affinity of individual receptors. Our data make the surprising finding that the affinity of CR3 for C3bi is very high. Moreover, inactive receptor exhibited a kinetic block in binding, not a change in binding energetics. The implications of these findings for the affinity modulation model are discussed.


MATERIALS AND METHODS

Reagents and Monoclonal Antibodies

Calf intestinal alkaline phosphatase (AP), n-succinimidyl 3-(2-pyridithio)propionate (SPDP), and bovine serum albumin were purchased from Pierce. Iodoacetamide, n-octyl -D-glucopyranoside, Sepharose CL-4B-200, activated thiol-Sepharose, and cyanogen bromide-activated Sepharose were from Sigma. Dulbecco's phosphate-buffered saline (PBS) was from Biowhittaker (Walkersville, MD). Attophos was from JBL Scientific, Inc. (San Luis Obispo, CA). All protease inhibitors were from Calbiochem-Novabiochem Co. (La Jolla, CA).

IB4 (anti-CD18, Wright et al.(1983)), OKM-1 (anti-CD11b, Wright et al.(1983)), 44a (anti-CD11b, Dana et al.(1986)), TS1/22 (anti-CD11a, Davignon et al. (1981)), W6/32 (anti-class 1 histocompatability Ag, Barnstable et al.(1978)), and 3C10 (anti-CD14, Van Voorhis et al. (1983)) were as described previously, and are all available from American Type Culture Collection (Rockville, MD). KIM-127 (anti-CD18, Robinson et al.(1992)) was a gift from Dr. M. Robinson (Celltech Ltd., United Kingdom). Anti-C3bi was from Quidel (San Diego, CA). Alkaline phosphatase-conjugated goat anti-mouse IgG was from Bio-Rad.

Constructing a Soluble, Monomeric Probe: C3bi-Alkaline Phosphatase

C3bi was conjugated to calf intestinal AP through a disulfide bridge. A sulfhydryl was first introduced into AP with the bifunctional cross-linker, SPDP. 2-Pyridyldisulfide-AP was prepared by incubating 5 mg of AP in 1 ml of PBS with 25 µl of 20 mM SPDP diluted in MeSO. After a 30-min incubation at 37 °C, excess cross-linker was removed with a NAP-25 (Pharmacia LKB Biotechnology, Uppsala, Sweden) desalting column equilibrated with PBS, 1 mM EDTA. The extent of derivitization was measured using an molar extinction coefficient of 8.08 10M cm at 343 nm for released 2-pyridyldisulfide (Carlsson et al., 1978) and was found to be 1.24 mol of 2-pyridyldisulfide/mol of AP.

C3bi with a single free sulfhydryl was purified from normal human plasma by modifying the method reported by Ross et al. (1987). Briefly, 50 ml of normal human plasma was treated with a final concentration of 20 mM iodoacetamide for 1 h at 37 °C to block all free sulfhydryl groups. After extensive dialysis, the plasma was incubated with 2 g of activated thiol-Sepharose for 2 h at 37 °C. The Sepharose activates the complement cascade, and because proteolytic activation of C3 leads to the liberation of a free sulfhydryl (Pangurn, 1986), the C3 is captured in disulfide linkage with the Sepharose. After thorough washing, C3bi was eluted with 10 mML-cysteine in 20 mM Tris, pH 7.4. C3bi was further purified with a Mono Q column eluted with a gradient (0-1 M) of NaCl in 20 mM Tris, 5 mM EDTA, 0.02% NaN, pH 7.4. Purified C3bi was then mixed with equimolar 2-pyridyldisulfide-AP and rotated at room temperature for 24 h. Approximately 80% of C3bi became bound to AP. Conjugated and free proteins were separated with a Mono Q column eluted with a gradient (0-1 M) of NaCl in 20 mM Tris, 0.02% NaN, pH 7.4, and results of conjugation were analyzed by electrophoresis on a 8-25% native gels (see Fig. 1). The presence of a single band of conjugate suggests stoichiometric coupling of C3bi and AP, and this inferrence is confirmed by the observation that the mobility of the conjugate is slower than either of the two reactants, and its Ris consistent with a 1:1 complex. The purity of the conjugate was also verified by immunoprecipitation. Incubation of conjugate with immobilized mAb against C3bi removed over 95% of AP activity, while with irrelevant mAb, AP in the supernatant remained unchanged (data not shown).


Figure 1: Conjugation of C3bi to AP. C3bi purified from normal human plasma was conjugated to calf intestinal alkaline phosphatase as described under ``Materials and Methods.'' Proteins were analyzed on native gels (8-25%) stained with Coomassie Brilliant Blue. LaneA, molecular mass markers (Thy, thyroglobulin, 669 kDa; Fer, ferritin, 440 kDa; Cat, catalase, 232 kDa; LD, lactase dehydrogenase, 140 kDa; BSA, bovine serum albumin, 67 kDa); laneB, purified AP; laneC, purified C3bi; laneD, purified C3bi-AP conjugate.



Purification of CR3

CR3 was purified from PMN lysates by one of the following two methods.

Antibody Affinity Chromatography

Pellets of PMN, prepared as described previously (Van Strijp et al., 1993), were lysed at 2 10 cells/ml with lysis buffer (100 mM Tris, pH 8.0, 150 mM NaCl, 2 mM MgCl, 1% Triton X-100, 0.02% NaN, protease inhibitors (1 µg/ml each of antipain, benzamidine, chymostatin, leupeptin, and pepstatin, 0.24 units/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 5 mM pefablock)) for 1 h at 4 °C with stirring. The lysate was then centrifuged at 35,000 g for 30 min. The supernatant was passed over an IB4 (anti-CD18) immunoaffinity column that was prepared by coupling IB4 to cyanogen bromide-activated Sepharose. The column was prewashed with equilibration buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM MgCl, 0.1% Triton X-100). The column was loaded at 1 ml/min and sequentially washed with equilibration buffer and buffer composed of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM MgCl, 1% n-octyl -D-glucopyranoside. CR3 was eluted with buffer containing 50 mM triethylamine, pH 11.0, 150 mM NaCl, 2 mM MgCl, 1% n-octyl -D-glucopyranoside into tubes with neutralizing buffer (15% by volume 1 M Tris-HCl, pH 7.4). Aliquots were made and stored at -80 °C. The purity of CR3 was analyzed on SDS-polyacrylamide gels (4-15%) stained with silver. Strong bands corresponding to the and chain of CR3 were observed at 165 and 95 kDa, and the identity of chain (165 kDa band) was confirmed with Western blot using polyclonal anti-CR3 (data not shown). Although PMN contain low amounts of CD11a/CD18 and CD11c/CD18, our preparation showed no CD11c and only a weak CD11a band (<5% of the density of the CD11b band). This preparation will bind C3bi and is referred to as ``active'' CR3.

Ligand Affinity Chromatography

CR3 was purified on immobilized C3bi as described previously (Van Strijp et al., 1993). Briefly, C3bi-coated Sepharose beads were prepared by incubating normal human plasma with Sepharose CL-4B-200 for 1 h at 37 °C. The beads were incubated with PMN lysate for 20 min, and, after thorough washing, CR3 bound to C3bi-coated beads was eluted using 10 mM EDTA. Receptor isolated in this way fails to bind C3bi and is referred to as ``inactive'' CR3.

Binding of C3bi-AP to Immobilized CR3

To measure the binding of C3bi-AP to purified CR3, 72-well terasaki plates (Robbins Scientific, Mountain View, CA) were coated with 10 µl/well (10 µg/ml) of purified CR3 diluted in PBS, 0.02% NaN for 2 h at 4 °C. Wells were aspirated and then blocked with 10% dry milk diluted in PBS for 1 h at 4 °C. After four washes with PBS, a total of 10 µl of C3bi-AP diluted in PBS with 0.1% of milk was added to each well. Plates were incubated at 37 °C for 20 min, unless indicated otherwise. Following four washes with saline, 5 µl/well of a fluorogenic substrate for AP, Attophos, was added. The fluorescence signal generated at 21 °C was detected at time intervals with a Cytofluor 2300 fluorescence plate reader (Millipore Corp., Bedford, MA). The amount of C3bi-AP bound to CR3-coated plates was expressed as the change in arbitrary fluorescence units/time interval. The actual value of fmol of C3bi-AP bound per well was calculated from a standard curve relating enzyme activity to µg of conjugate. Each sample was assayed in triplicate.

Quantitation of CR3 on Plates

The method for quantitating the amount of CR3 bound to coated terasaki plates was as described previously (Van Strijp et al., 1993). Briefly, CR3-coated plates were incubated with I-labeled mAb anti-CR3 (I-44a, iodinated with NaI by the Iodogen method (Fraker and Speck, 1978)) at 37 °C for 60 min. After thorough washing, plates were dried. The amount of I-44a bound was determined by counting individual wells in a -counter. Results were expressed in cpm, and the actual value of fmol of I-44a bound per well was calculated from the specific activity of the I-44a.

Protein Concentration Assay and Data Analysis

Protein concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA). Bovine serum albumin was used as a standard. Data are presented as the mean of three wells ± S.D. of a representative experiment (n = 4).


RESULTS

C3bi-AP Binds CR3 Specifically

C3 binds covalently to surfaces through an ester link (Law and Levine, 1977; Law et al., 1979). In this process, an intrachain thiol ester in C3 undergoes a transesterification reaction yielding an ester link to a hydroxyl (or to water) and a free thiol (Abbas et al., 1991). We have used this free thiol to conjugate alkaline phosphatase to C3bi. Since the free thiol residue of C3bi is only four amino acids away from the normal site of attachment to surfaces (Khan and Erickson, 1982), it represents a sterically favorable site to attach a bulky group. Analysis of the conjugate indicates that it is a soluble monomeric molecule with a 1:1 ratio between C3bi and AP (Fig. 1). We measured binding of C3bi-AP to purified CR3 by incubating C3bi-AP in plates coated with purified CR3. The CR3 used for this study was purified using mAb affinity chromatography, and this purification produces a population of receptors that avidly bind C3bi (see below).

C3bi-AP bound to surfaces coated with CR3 but not to uncoated surfaces (Fig. 2A, first and lastcolumns). The CR3-dependent binding of C3bi-AP was completely inhibited by mAb IB4 (anti-CD18) and 44a or OKM1 (both anti-CD11b), while mAb W6/32 (anti-HLA class-1 antigen) and TS1/22 (anti-CD11a) were ineffective. The binding of C3bi-AP conjugates to CR3 was also dose-dependently blocked by unlabeled C3bi (Fig. 2B). C3bi-AP binds at least as avidly as unlabeled C3bi, and attachment of AP thus appears not to decrease the affinity of C3bi for CR3. Additional studies showed that unconjugated AP failed to bind to CR3, and the amount of C3bi-AP binding varied with the dose of CR3 used to coat the surfaces (data not shown). These studies established that C3bi-AP bound specifically to CR3.


Figure 2: C3bi-AP specifically binds CR3. CR3-coated plates were incubated with C3bi-AP (15 nM) for 20 min at 37 °C in the presence of the indicated mAbs (10 µg/ml) (A) or in the presence of increasing concentrations of unconjugated C3bi (B, plates coated with () or without () CR3). These incubations were done with buffer containing 1 mM Ca and 1 mM MgCl. The requirement for divalent cations was measured in parallel incubation with buffer containing 1 mM each of the indicated divalent cations (C). After thorough washing, the binding of C3bi-AP to plates was quantitated by enzyme assay as described under ``Materials and Methods.'' Parallel experiment showed the binding rate to be linear at this time point.



Binding of C3bi-AP to CR3 Is Divalent Cation-dependent

To determine the cation dependence of binding of C3bi-AP to CR3, binding studies were conducted in the presence of 1 mM each of Ca, Mg, and Mn at 37 °C for 20 min (Fig. 2C). Omission of divalent cations completely inhibited the binding of C3bi-AP to purified CR3. In the presence of 1 mM Ca or Mg, binding was partially restored, but the most efficient binding was observed in the presence of Mn or the combination of all three divalents. These results are consistent with our previous studies showing that binding of C3bi-coated particles to CR3 on cells (Wright and Silverstein, 1982) and plastic (Van Strijp et al., 1993) is divalent cation-dependent and are also consistant with the known divalent cation binding properties of CR3 (Michishita et al., 1993).

Binding of C3bi-AP to CR3 Is Temperature-dependent and Requires a High Activation Energy

We have previously shown that binding of C3bi-coated erythrocytes to CR3-bearing cells (Wright and Jong, 1986) or CR3-bearing plastic surfaces (Van Strijp et al., 1993) could not be observed at low temperature. To determine if this temperature dependence was a property of CR3 or the C3bi-coated erythrocytes, binding of C3bi-AP to CR3 coated plates was studied at various temperatures with 1 mM each of Ca and Mg. The rate of binding of C3bi-AP to CR3 was greatest at 37 °C, but was nearly absent when the temperature was lowered to 17 °C (Fig. 3A) and could not be observed at 0 °C (data not shown). The strong temperature dependence thus suggests a high activation energy for the reaction between C3bi and CR3. Arrhenius analysis of the data in Fig. 3A yielded an activation energy (E) of 15 ± 2 kcal/mol (Fig. 3B).


Figure 3: Effects of incubation temperature on the binding of C3bi-AP to CR3. A, plates coated with () or without () CR3 were incubated with C3bi-AP (15 nM) for 20 min at the indicated temperatures. Parallel experiments showed that the binding rate was linear at this time point. After thorough washing, the binding of C3bi-AP to plates was quantitated by enzyme assay at 21 °C as described under ``Materials and Methods.'' B, data in A were plotted by the method of Arrhenius.



Binding of C3bi-AP to CR3 Is Time-dependent and High Affinity

The specificity, divalent cation, and temperature dependence for C3bi-AP binding to CR3-coated plates indicated that the soluble monomeric probe, C3bi-AP, had binding properties identical to those of immobilized C3bi (Wright and Silverstein, 1982; Van Strijp etal., 1993). We may thus use soluble C3bi-AP to estimate the binding constant of CR3 for C3bi. To ensure the accuracy of our estimation, the kinetics of C3bi binding to CR3 were first studied by incubating 12 nM C3bi-AP with CR3-coated plates for increasing times. Binding was time-dependent with an association rate (k) of 6 10Mmin (Fig. 4). Binding reached steady state at 60 min.


Figure 4: Kinetics of C3bi-AP binding to CR3. Plates coated with () or without () CR3 were incubated with C3bi-AP (12 nM) at 37 °C for increasing times. After thorough washing, the binding of C3bi-AP to plates was quantitated by enzyme assay. k was calculated using this kinetic data as described by Lauffenburger and Linderman (1993).



To measure the affinity of CR3 for C3bi, increasing concentrations of C3bi-AP were incubated with plates coated with or without CR3 for 60 min at 37 °C. Binding of C3bi-AP to CR3 was dose-dependent and saturable (Fig. 5A). For affinity estimation, values from plates without CR3 coating were treated as nonspecific binding and were subtracted to yield specific binding. Nearly identical nonspecific values were obtained when excess unlabeled C3bi or mAb anti-CR3 was used for competing the specific binding of C3bi-AP to CR3-coated plates (data not shown). Scatchard analysis (Fig. 5B) revealed a K of 12.5 ± 4.7 nM.


Figure 5: Dose-dependent binding of C3bi-AP to CR3. A, plates coated with () or without () CR3 were incubated with increasing concentrations of C3bi-AP for 60 min at 37 °C. After thorough washing, the binding of C3bi-AP to plates was quantitated by enzyme assay. B, the data in A were plotted by the method of Scatchard after subtraction of background.



The binding of C3bi-AP to CR3 appears to occur in a 1:1 ratio. When 50 nM C3bi-AP (a saturating concentration) was incubated with CR3-coated plates for 60 min, the amount of C3bi-AP bound to CR3 was estimated to be 7 fmol/well, while the number of CR3 molecules coated on the plate was 10 fmol/well as determined by I-labeled anti-CR3 (I-44a) (data not shown).

C3bi-AP Does Not Bind Inactive CR3

Cells may express CR3 that is active or inactive in mediating cell adhesion (Wright and Meyer, 1986; Diamond and Springer, 1993; Elemer and Edgington, 1994). The CR3 used in the above studies was isolated by method using mAb affinity chromatography that yielded constitutively active receptors, and this activity was demonstrated here by the avid binding of C3bi-AP (Fig. 5A). We have previously described a method for purification of inactive CR3 using affinity chromatography on C3bi-Sepharose (Van Strijp et al., 1993). This procedure yields receptor that neither binds C3bi-coated particles (Hermanowski-Vosatka et al., 1992; Van Strijp et al., 1993) nor CBRM1/5,()a monoclonal antibody that detects an activation epitope of CR3 (Diamond and Springer, 1993). Studies using this inactive receptor preparation showed that 12 nM C3bi-AP failed to bind above background (Fig. 6, firstcolumn). Similar results were obtained in experiments using higher concentrations of C3bi-AP (100 nM) or incubations as long as 18 h (data not shown). These findings indicate that inactive CR3 either fails to bind C3bi or binds with a very low affinity.


Figure 6: Binding of C3bi-AP to inactive CR3. Plates coated with inactive CR3 (purified by ligand affinity chromatography) were incubated with C3bi-AP (15 nM) at 37 °C for 30 min in the presence of the indicated mAbs (10 µg/ml except IB4 that was 50 µg/ml). After thorough washing, the binding of C3bi-AP to plates was quantitated by enzyme assay.



To verify that our inactive CR3 is functional and capable of being activated, we employed the monoclonal antibody KIM-127. This antibody against CD18 strongly increases CR3-mediated PMN adhesion to protein-coated surfaces without influencing the expression of integrin (Robinson et al., 1992), a result we have confirmed (data not shown). To determine if the effects of KIM-127 are caused by direct action of the antibody on the receptor, plates coated with inactive CR3 were incubated with C3bi-AP in the presence or absence of KIM-127. The addition of KIM-127 led to strong binding of C3bi-AP. The binding enabled by KIM-127 was totally blocked by mAb IB4 (anti-CD18) (Fig. 6). This finding indicates that our inactive CR3 retained the capacity to bind C3bi, and that KIM-127 acted directly on CR3 to change its binding properties. The ability of KIM-127 to enhance binding of C3bi-AP to inactive CR3 was dose-dependent, with a maximal effect at 1 nM (data not shown). The curve for enhanced binding of C3bi-AP to CR3 closely matched the curve for binding of KIM-127 to CR3 (see Fig. 7below).


Figure 7: Effect of mAb KIM-127 on binding of C3bi-AP to CR3. A, binding of C3bi-AP to CR3. Plates coated with (, ) or without () CR3 were incubated with increasing concentrations of C3bi-AP for 20 min at 37 °C in the presence (, ) or absence () of KIM-127 (10 µg/ml). After a thorough washing, the binding of C3bi-AP to plates was quantitated by enzyme assay. B, the data in A were plotted by the method of Scatchard after subtraction of background.



KIM-127 Increases Binding of C3bi to CR3 without Changing Affinity

Results from the above experiments indicate that the capacity of inactive CR3 to bind monomeric ligand may be dramatically altered by changes, presumably conformational in nature, induced by binding of KIM-127. Equilibrium measurement of the binding in the presence of 10 µg/ml of KIM-127 yielded K = 15.9 ± 3.4 nM. This value is not significantly different from the K of active CR3 in the absence of KIM-127 (12.5 ± 4.7 nM). This result suggests that KIM-127 does not influence the energetics of the interaction of CR3 and C3bi. To further ask if KIM-127 alters the affinity of active receptors, increasing concentrations of C3bi-AP were incubated with plates coated with an active CR3 preparation in the presence or absence of KIM-127, and binding was measured (Fig. 7). The addition of KIM-127 to CR3-coated plates increased the number of binding sites for C3bi-AP to CR3, but the shape of the binding curve indicated a single population of CR3 with similar affinity in all cases. Further studies showed identical temperature dependence of binding in the presence and absence of KIM-127 (data not shown). These observations suggest first that the active CR3 preparation contains two populations, a population of active receptors that bind C3bi, and an inactive population that only binds after treatment with KIM-127. In four experiments, the increase in C3bi binding sites caused by KIM-127 varied from 30 to 100%, suggesting that 15-50% of the receptors in this preparation were inactive but susceptible to activation by KIM-127. More importantly, the results suggest that KIM-127 does not modulate the binding affinity of CR3 for C3bi. It is either high (12 16 nM) or unmeasurable, with no middle value.

KIM-127 Does Not Contribute to the Energetics of Binding of C3bi-AP to CR3

Inactive CR3 has unmeasurable affinity for C3bi, while the active receptor has an affinity of 12 nM, a change consonant with a G of approximately -11 kcal/mol. To test the hypothesis that KIM-127 contributes this energy, we measured the binding affinity of KIM-127 for active and inactive CR3. The affinities, roughly estimated from concentrations for half-maximal binding, were practically identical (1 nM) for active and inactive CR3 (Fig. 8). If KIM-127 contributed the 11 kcal/mol needed for binding, its affinity would change from 1 nM for the active receptor to 80 mM for the inactive receptor. This large change would have been easily observed in our studies. Additional studies showed that ligation of CR3 with C3bi also had no effect on the binding of KIM-127 (not shown). These results suggests that KIM-127 affects binding of C3bi to CR3 without contributing to the energetics of binding.


Figure 8: Binding of mAb KIM-127 to active and inactive CR3. Plates coated with active (, purified by mAb affinity chromatography), inactive CR3 (, purified by ligand affinity chromatography), or without CR3 () were incubated with increasing concentrations of mAb KIM-127 for 30 min at 37 °C. After four washes with PBS, alkaline phosphatase-conjugated goat anti-mouse IgG (1:2000 dilutions, Bio-Rad) was added, and plates were incubated for another 30 min at 37 °C. After a thorough washing, the binding of mAb KIM-127 to plates was quantitated by enzyme assay.




DISCUSSION

To determine the binding properties of leukocyte integrin CR3, we developed a novel, soluble, monomeric probe, C3bi-AP, and a binding assay using purified immobilized CR3. Our procedures obviate problems such as pinocytic uptake and degradation of ligand that are inherent in cell-based assays, especially with interactions that require warm temperatures. They further obviate problems inherent in measurements of multimeric ligands and yield the first equilibrium measurements on CR3 suitable for rigorous quantitative analysis. Using these reagents, we determined that the stoichiometry of the binding of C3bi to CR3 is 1:1, that binding shows an absolute requirement for divalent cations, and that binding is of high affinity.

Previous work has shown that CR3 can be isolated in a form that is capable (Diamond etal., 1990) or incapable (Van Strijp et al., 1993) of binding multimeric ligand, and we confirmed these findings here using our monomeric probe. We further showed that addition of mAb KIM-127 to purified CR3 caused a shift from an inactive to an active state. This result indicates that KIM-127 acts directly on CR3 to enable its ligand binding properties. Importantly, KIM-127-induced binding exhibited an affinity for C3bi that is identical with the affinity for active receptor preparation, suggesting that the site exposed by this mAb is similar to that exposed during activation of receptors by physiological stimuli.

KIM-127 could enable binding either by contributing sufficient energy to change receptor affinity or by lowering the activation energy required for binding. Studies reported here suggest that KIM-127 does not modulate affinity. If KIM-127 drove a conformational change that raised the affinity of the C3bi binding site, then the binding energy for C3bi conferred by binding of mAb would be balanced by a decrease in the binding energy for KIM-127. Such reciprocal changes in binding energy are observed in allosteric proteins (e.g. hemoglobin) for ligands (e.g. 2,3-diphosphoglycerate and O (Kilmartin, 1976)). To determine if KIM-127 makes such an energetic contribution, we measured its affinity for both active and inactive receptors. No difference in affinity was observed (Fig. 8). It is thus clear that KIM-127 mediates alterations in binding activity without significant energy input, and we may draw active CR3 and inactive CR3 at equivalent positions on an energy diagram (Fig. 9).


Figure 9: Energetics of leukocyte integrin CR3. Inactive CR3 and active CR3 are isoenergetic, but equilibrium is not established because of a high activation energy for conversion of these two species. Inactive CR3, once formed, may thus persist for long periods, even in the presence of C3bi. KIM-127 has the same binding affinity for both active and inactive CR3, but it facilitates the interconversion of these two species. Thus the equilibrium distribution of inactive and active CR3 is not changed by KIM-127, but the nonequilibrium distribution can be changed by KIM-127 acting to catalyze conversion of inactive CR3 to active CR3. Binding of C3bi to active CR3 is energetically very favorable and will draw the reaction to the right. We speculate that inactive and active CR3 at the cell surface are also isoenergetic, that they are also separated by a high activation energy barrier, and that receptor activation is occasioned by facilitation of the interconversion of these forms.



An important consequence of the above finding is that cells may alter the binding properties of CR3 without a need for transmitting a large amount of energy from the cytoplasmic side of the membrane to the exoplasmic surface. Indeed, it is difficult to imagine a propagated conformational change carried by the two membrane-spanning peptides of CR3 delivering an energetically large signal to the exoplasmic domain. While the shift from inactive to active receptor is energetically neutral, the transition appears to have a very high activation energy since it does not occur to a significant degree during a prolonged incubation (18 h, 37 °C) in the presence of ligand. Under these conditions, binding of ligand would be expected to ``trap'' any receptor that may become active during the incubation. We have thus drawn a high activation energy profile for the transition from inactive to active CR3 and have depicted KIM127 as lowering this barrier (Fig. 9). The action of KIM-127 thus resembles that of a catalyst and could be mediated by exposure of a physically hidden binding site for C3bi.

The most surprising finding of the study is that the binding affinity of CR3 for C3bi is very high (K 10M). We further showed that the activation energy for binding is also high (E 15 kcal/mol), and we may thus draw the energy diagram in Fig. 9. Several conclusions derive from this rendering of binding energetics. The first is that, while switching from inactive to active receptors is energetically neutral, removal of ligand bound to receptor is energetically expensive. A consequence of this is that spontaneous disassociation of ligand from receptor will be slow. From the on rate (Fig. 4) and the affinity (Fig. 5), we calculate that more than 20 min is required for spontaneous disassociation of one-half of the ligand, and preliminary studies of the disassociation of C3bi-AP in the presence of excess unlabeled C3bi are consistent with this estimate (not shown). The slow spontaneous disassociation of ligand from CR3 stands in contrast with the observation that during chemotaxis of PMN, CR3 may bind substrate-bound ligand and release the ligand in less than 1 min (Nahas et al., 1971). We have also observed that C5a-stimulated PMN may bind to endothelial cells via CR3 and may fully detach within 7 min (Lo et al., 1989b), that C5a-stimulated PMN may bind C3bi-coated erythrocytes and release the erythrocytes within 5 min,()and that PMN bind C3bi-coated epithelial monolayers via CR3 but promptly detach upon stimulation (Cramer et al., 1986). These observations indicate that detachment of PMN from a ligand-coated surface is not likely to be caused by spontaneous release of ligand from CR3 and is likely to require an energy-dependent step.

The above studies necessitate a revision of current thinking regarding control of integrin-mediated cell adhesion. The widely held affinity modulation model calls for increases in integrin affinity to enable cell adhesion and for a reversal of this process to cause detachment. Data in this paper suggest that neither of the two propositions of this model are tenable. Studies with our model system show that the activation of receptors does not result from changes in affinity of CR3 but from a change in the activation energy for shifting receptor from inactive to active state. A second revision concerns the mechanism by which adhesion is reversed. Since our data indicate that release of bound ligand requires significant energy input, cell detachment must involve a process mechanistically distinct from receptor activation and cannot result from a reversal of receptor activation. One potential mechanism for reversing adhesion may involve removal of divalents, since divalent cations are required for binding of C3bi-AP to CR3 (Fig. 2C) and removal of divalent cations reverses binding.()An alternative mechanism to reverse binding could involve proteolytic degradation of the receptor or ligand. The nature of the process by which ligand is removed from receptor is currently under study.

The final observation derived from Fig. 9is that receptors might be incapable of binding ligand and mediating adhesion for two distinct reasons. They may be in the inactive state or in a ligand-occupied state. Experiments are currently ongoing to determine the state of receptors on living PMN.


FOOTNOTES

*
This work was supported by American Cancer Society Grant CB102. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Cellular Physiology and Immunology, Box 303, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8110; Fax: 212-327-7901.

The abbreviations used are: CR3, complement receptor type 3; PMN, polymorphonuclear leukocytes; AP, alkaline phosphatase; SPDP, n-succinimidyl 3-(2-pyridithio)propionate; PBS, phosphate-buffered saline; mAb, monoclonal antibody.

K. P. M. van Kessel, M. Diamond, T. A. Springer, and S. D. Wright, unpublished observations.

S. D. Wright, unpublished observations.

T. Q. Cai and S. D. Wright, unpublished observations.


ACKNOWLEDGEMENTS

We thank D. S. Miller for assistance in preparation of inactive CR3. We also thank Dr. M. K. Robinson (Celltech Ltd., UK) for the generous gift of mAb KIM-127, Dr. P. A. Detmers for critical reading of the manuscript, and our colleagues for valuable discussions.


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