Proc. Nati. Acad. Sci. USAVol. 88, pp. 3106-3110, April 1991Immunology

Expression of a soluble and functional form of the human f82integrin CD11b/CD18

(recombinant ,32 integrins/complement receptors/leukocyte-endothelial adhesion/inflammatory reactions/reperfuison injury)

NAVA DANA*, DEHMANI M. FATHALLAH, AND M. AMIN ARNAOUTRenal Unit and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston MA 02114

Communicated by Kurt J. Isselbacher, December 28, 1990

ABSTRACT Polymorphonuclear cells and monocytes(phagocytes) are a critical component of host defense againstinfections. However, these cells also play a sigifiant role inhost tissue damage in many noninfectious diseases, such asischemia-reperfusion n'jury syndromes and rejection of trans-planted organs. The leukocyte adhesion molecule family CD11/CD18 (P2 integrins) is critical to the function of polymorpho-nuclear cells and monocytes in inflammation and Injury.Inherited deficiency of CD11/CD18 impairs phagocyte chemo-taxis, adhesion and transmigration across endothelium, andclearance of invading microorganisms through phagocytosisand cell-mediated killing. Furthermore, murine monoclonalantibodies directed against the CD11b/CD18 (CR3) het-erodimer have been shown to reduce, by 50%-80%, phago-cyte-mediated ischemia-reperfusion injury in several organsystems, such as the myocardium, liver, and gastrointestinaltract and to Inhibit development of insulin-dependent diabetesmellitus in nonobese diabetic (NOD) mice. Expression ofCD11b/CD18 in a soluble and functional form might thereforebe potentially useful as an anti-inflammatory agent. We havenow expressed a recombinant soluble heterodimeric form ofthis human 132 integrin, normally expressed as two nonco-valently associated membrane-bound subunits. The secretedreceptor exhibited direct and specific binding to its ligand,iC3b, the major complement C3 opsonin, and inhibited bindingof polymorphonuclear cells to recombinant interleukin 1-acti-vated endothelium.

There is increasing awareness of the critical role of phago-cytic cells, polymorphonuclear cells (PMN), and monocytesin tissue destruction in many clinical disorders includingmyocardial infarction, certain immune-complex glomerulo-nephritides, blistering skin diseases, allograft rejection, andulcerative colitis (1). Interventions aimed at attenuating thisharmful role may have great therapeutic benefit. However,given the myriad of proteinases, oxidants, and cationicproteins produced by phagocytes after activation withchemoattractants and cytokines and during phagocytosis (2),this goal has proven elusive.

Recently, another approach has been suggested by unrav-eling the molecular basis of an inherited disease (leukocyteadhesion molecule deficiency) manifested clinically by life-threatening recurrent bacterial infections in which affectedindividuals have a global deficiency in phagocyte-mediatedacute inflammatory responses due to lack of expression ofCD11/CD18 (J32 integrins) (3, 4). CD11/CD18 are adhesionreceptors that promote interaction of leukocytes with eachother (5, 6), with endothelial cells (during transmigration)(7-9), and with specific opsonins deposited on invadingbacteria and rejected or hypoxic tissues (10, 11). The CD11/CD18 family consists of three heterodimeric surface-

membrane glycoproteins, each with a distinct a subunit(CD11a, b, or c) noncovalently associated with an identical /3subunit (CD18) (12, 13). As in other integrins, association ofthe CD11 and CD18 subunits is required for normal surface-membrane expression and function of these receptors (14,15). The CD11b/CD18 (CR3) heterodimer is a major (2integrin on PMN and monocytes and mediates many of theproinflammatory functions in these cells (3, 16-18). Murinemonoclonal antibodies (mAbs) directed against CD11b/CD18 reduce the degree of ischemia-reperfusion injury by50-80% in several animal models of phagocyte-dependentacute tissue injury (19-21) and prevent development ofinsulin-dependent diabetes mellitus in susceptible mousestrains (22) through prevention of phagocyte accumulation indamaged tissues and their interaction with complement iC3b.These antibodies, however, usually elicit an antiglobulinresponse in humans and are thus of limited therapeuticusefulness. We therefore considered that antagonism ofphagocyte interactions with inflamed endothelium and withiC3b might also be achieved by using a soluble and functionalform of human CD11b/CD18.

In this paper we report the expression of a recombinantsoluble form of CD11b/CD18 in mammalian cells. The sol-uble receptor remains complexed as a heterodimer as re-vealed by immunoprecipitation using subunit-specific mAbs.Furthermore the soluble heterodimer binds specifically toiC3b in a divalent cation-dependent manner similar to that ofthe wild-type receptor and inhibits binding of PMN to re-combinant interleukin 1 (rIL-1)-activated endothelium.

MATERIALS AND METHODSReagents. Activated thiol-Sepharose (ATS) was bought

from Sigma. Human complement C3 and factors I and H werepurified as described (23, 24). Purified human C3 was ex-posed to ATS in the presence of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Worthing-ton) to convert it to C3b covalently linked to ATS (ATS-C3b).The concentration of C3b was 4.5 mg/ml of settled Sepha-rose. ATS-iC3b was generated by proteolytic cleavage ofATS-C3b with purified human complement factors H and I(24, 25). Conversion of C3b to iC3b was confirmed bySDS/PAGE analysis under reducing conditions. HumanPMN were isolated from EDTA-anticoagulated blood bycentrifugation over Ficoll/Hypaque as described (26). Puri-fied cells were surface labeled with 125I (New EnglandNuclear) using Iodo-Gen (Pierce) as described (27). rIL-1 wasprovided by Biogen.

Abbreviations: PMN, polymorphonuclear cells; rIL-1, recombinantinterleukin 1; CR3, complement receptor type 3; ATS, activatedthiol-Sepharose; mAb, monoclonal antibody; HUVE, human umbil-ical vein endothelial cells; mCDllb/CD18, membrane-bound recep-tor CD11b/CD18; sCDllb/CD18, sCDllb, and sCD18, secretedCD11b/CD18, CD11b, and CD18, respectively.*To whom reprint requests should be addressed.

3106

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 88 (1991) 3107

Generation of In-Frame Stop Codons in CD11b and CD18cDNAs. We used site-directed mutagenesis to introduce in-frame translational stop codons at the predicted extracellularboundaries of the corresponding transmembrane domains ofCDllb and CD18 (Fig. 1). Asn-700 in CD18 and Leu-1090 inCD11b were replaced by translational stop codons in thewild-type cDNAs. In-frame stop codons in full-length CDlbcDNA and CD18 cDNA (29-31) were generated by using thegapped-duplex mutagenesis technique (32), modified as de-scribed (29). Briefly, the PAT-X plasmid containing thepartial CD18 cDNA clones J19 [provided by Alex Law,Oxford University (31)] was linearized with HindIII or di-gested with Nco I [to generate a 1331-base-pair (bp) gap].These two plasmids were mixed with an excess of thesynthetic and 5'-end-phosphorylated 18-mer (0-18 N/st;5'-aggccccTaGatcgccgc) containing desired nucleotide sub-stitutions (uppercase letters). The mixture was denatured byboiling and renatured by stepwise cooling. RehybridizedDNA (containing the single-stranded region to which themutant 18-mer is hybridized) was primer-extended to fill thegap and used to transform Escherichia coli strain BMH 71-18mut L (29). Plasmids containing the mutation were identifiedby differential hybridization with 32P-labeled wild-type ormutant 18-mers, and the DNA was used to retransform E. coliJM109. Positive colonies were identified after rehybridiza-tion, sequenced to verify the mutation, and then used toreplace the corresponding fragment in wild-type full-lengthCD18 cDNA cloned in the 1TH3M expression vector (29). Astop codon was similarly introduced in CD11b cDNA byusing a Bluescript plasmid vector containing the full codingregion of membrane-bound CD11b (30). A mixture of KpnI-linearized and gapped (by removing a Sma I fragment,1048-bp long) CD11b cDNAs were mixed with an excess ofthe synthetic mutant 18-mer (0-18 L/st; 5'-caaccccTAgc-cgctcat), processed as detailed above, and the desired cDNA(confirmed by sequencing) was subcloned into the ffH3Mexpression vector.

Production of Mutant CDllb and CD18 in COS Cells. Eachplasmid was transfected into COS cells using DEAE-dextranas described (29, 33). Forty to 50o confluent COS M6

Wild TvpeCD] | ~Ala Glv Pro Asn Ile AlaGCA GCC CCC AAC AlT CCC

Ala Glv Pro *Secreted CDI8:

GCA GGC CCC TAG A_TCC

CD18 COI

C011b la COOH

2 A-DOMAIN 1 4 5 6

Asn Pro Leu Proreu IleWild Type CDlIb: AAC ccc cCrG CCG CTC ATC

Aso Pro SSecreted CD]lb: AAC CCC TAG CCG CTC ATC

FIG. 1. Schematic diagram showing the structure of normalCDllb/CD18 heterodimer and positions in the immediate extracel-lular region where stop codons were introduced. The extracellularregion ofCD1lb contains seven short repeats, three of which containputative metal-binding regions (A), and a large domain (A-domain)containing several epitopes for functionally active mAbs (28). Thetransmembrane (TM) and cytoplasmic tail (C) are shown. Theextracellular region of CD18 contains four cysteine-rich repeats anda more N-terminal region (outlined) involved in heterodimer forma-tion (29).

monkey kidney cells in 100-mm tissue-culture dishes werewashed in serum-free medium and transfected using DEAE-dextran/chloroquine and supercoiled mutated CD11b cDNAand mutated CD18 cDNA constructs, each at 2 pug/ml. Insome transfections, COS cells were transfected with eithermutated CD1lb or CD18 cDNA. Sham-transfected cells wereused as controls. To detect secreted cell products, cells fromeach 100-mm dish, representing -3 x 106 cells, were pulsedwith 0.1 mCi (1 Ci = 37 GBq) of [35S]methionine for 16 hr at370C in a humidified 5% Co2 incubator. Supernatants werecentrifuged to remove nonadherent cells and debris and thenstored at 40C or used for immunoprecipitations, ligand bind-ing, or inhibition studies.Binding of Radiolabeled Membrane-Bound and Truncated

Receptors to ATS-iC3b. This binding was done as described(25) with minor modifications. Briefly, 1 x 108 1251-surface-labeled PMN (a source of radiolabeled-membrane-boundreceptor, mCDllb/CD18) were solubilized in 1 ml of phos-phate-buffered saline/0.5% Nonidet P-40/2 mM phenyl-methylsulfonyl fluoride. The detergent-soluble fraction wasthen diluted 3-fold in distilled water/0.5% Nonidet P.40/2mM MgCl2/0.33 mM CaCl2. One milliliter of the dilutedlysate was then mixed with either 200 1,u of settled ATS-C3bor ATS-iC3b. After 90 min at room temperature, each samplewas transferred to a 10-ml Econo column (Bio-Rad) andwashed with 10 ml of 5 mM Tris/24mM NaCl/1 mM KCI, pH7.4/Ca2+-Mg2+ (washing buffer) containing 0.5% NonidetP-40. The column was then eluted in 1-ml fractions with 0.4M NaCI in the same buffer. Fractions containing peakradioactivity were pooled, dialyzed extensively against wa-ter, lyophilized, and then analyzed on SDS/PAGE underreducing conditions (34). Transfected cell supernatants wereprocessed similarly. The recombinant CDllb/CD18 wasradiolabeled by metabolically labeling CDllb/CD18 cDNA-transfected COS cells (and COS cells transfected with mu-tated CD11b, or mutated CD18 cDNA or sham-transfectedcells) with [35S]methionine. Supernatants from labeled trans-fected COS cells were collected (each pooled from twosemi-confluent 100-mm petri dishes each containing -3 x 106cells) and used as sham control, a source of secreted CDllb/CD18 (sCDllb/CD18) secreted CD11b (sCDllb), or se-creted CD18 (sCD18). Each pool (15 ml) was concentrated to1 ml using collodion bags (10,000 Mr cut-off; Schleicher &Schuell). One-hundred microliters was used for immunopre-cipitations to detect the radiolabeled sCDllb/CD18 het-erodimer, sCDllb, or sCD18 monomers. The rest was diluted3-fold in distilled water/2 mM MgCl2/0.33 mM CaCl2 or 10mM EDTA, and 1 ml was incubated with ATS-C3b orATS-iC3b. Samples were then processed as describedabove. ATS elution was accomplished by using washingbuffer containing 0.1% Nonidet P40 and 0.4 M NaCl.

Immunoprecipitations and Quantitation of RecombinantSecreted Receptor. Subunit-specific mAbs 44a (anti-CD11b)(10, 35) or TS18 (anti-CD18) (36) were used to immunopre-cipitate CDllb/CD18 from PMN or COS cell supernatant.Typically, 100 ,ul of 251I-labeled PMN detergent lysate or 100jl of metabolically labeled COS cell supernatant (following a10-fold concentration) were used in each case. The 44a andTS18 mAbs were provided by R. F. Todd III (University ofMichigan, Ann Arbor) and S. Burakoff(Dana-Farber CancerInstitute, Boston), respectively. Immunoprecipitations weredone as described (37). To determine the fraction of thesecreted receptor expressed as a heterodimer, intensity ofCD11b and CD18 bands immunoprecipitated from equalvolumes of supernatants by the anti-CD11b and anti-CD18mAbs was compared by densitometry with an LKB Ultro-scan XL densitometer (Pharmacia LKB).The concentration of secreted receptor in supernatants from

two separate transfections was quantified by using an RIA.Five-tenths milliliter ofCDllb/CD18 receptor-containing super-

Immunology: Dana et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

natant, a sham control, and dilutions of the detergent-solublePMN lysate from an individual with a known number ofCD11b/CD18 receptors [6 x 106 receptors/calcium ionophore-activatedPMN (38)] were slot-blotted onto nitrocellulose filters by using aMinifold II slot-blot system (Schleicher& Schuell). The filterwasprobed with a monospecific rabbit anti-CD11b antibody (1:400dilution) and developed with an 15I-labeled goat anti-rabbitimmunoglobulin (New England Nuclear). The autoradiographwas then scanned with an LKB Ultroscan, and the amount ofsoluble CD11b was quantified from the PMN-lysate-derivedstandard curve.PMN-Adhesion to rIL-1-Treated Endothelium. Adherence

of purified human PMN to confluent monolayers of humanumbilical vein endothelial cells (HUVE) pretreated withrIL-1 (10 units/ml for 4 hr at 370C) was done as described (8)with the following modifications: PMN were labeled withcarboxyfluorescein (Molecular Probes) by incubating 4 x 106cells with 30 jig of carboxyfluorescein in 1 ml of Tris-bufferedsaline for 10 min on ice, followed by three washes. HUVEwere preincubated with undiluted supernatants from trans-fected COS cells, in triplicate wells for 10 min at 370C, beforelabeled PMN (5 x 105 cells per well) in an equal volume wereadded, and incubation was continued for an additional 10min. Some human PMN were preincubated with NS1 (anegative control ascites), 44a mAb, or TS18 mAb ascites (at1:100 dilution) for 5 min at room temperature before use. Atthe end of the adhesion assay, HUVE were washed twice,and HUVE-associated fluorescein was harvested in distilledwater/0.1% SDS/0.1 M NaOH and measured (at excitatorywavelength, 490 nm; emitted wavelength, 300 nm) using afluorometer (SLM 8000; SLM Aminco, Urbana, IL). Typi-cally, 25-32% of added PMN adhered to endothelium inbuffer alone or the NS1 control mAb under these conditions(ref. 8 and data not shown). PMN binding with NS1 orsupernatant from sham-transfected COS cells was normal-ized to 100 arbitrary units, and relative binding in the pres-ence of mAb 44a, mAb TS18, or sCDllb/CD18 was thencalculated. The data are expressed as the mean of fourseparate experiments representing three separate transfec-tions and the variance as the SEM. Significance was deter-mined by the Student's t test.

RESULTSExpression of a Soluble CD11b/CD18 Heterodimer in COS

Cells. To determine whether COS cells can express a secretedprotein heterodimer, we cotransfected the mutated humanCD11b and CD18 cDNA constructs into COS cells. Aftermetabolic labeling, harvested supernatants were used forimmunoprecipitation with anti-CD11b or anti-CD18 mAbs.Fig. 2 shows that both subunits of the receptor were immu-noprecipitated from mutated CDllb/CD18-transfected butnot from sham-transfected COS cell supernatants. The de-tergent-soluble fraction of the pulsed COS cells containedlittle or no radiolabeled recombinant CD11b/CD18 (data notshown), indicating that the bulk of the synthesized receptorwas secreted. The sCDllb/CD18 heterodimer had an appar-ent molecular mass of 149 kDa and 84 kDa for its CD11b andCD18 subunits, respectively (Fig. 2), compared with 155 kDaand 94 kDa for the membrane-associated forms (10, 35), inagreement with an expected loss of 45 and 69 amino acidsfrom CD11b and CD18, respectively. mAbs directed againsteither the sCDllb or sCD18 subunits immunoprecipitatedboth subunits (Fig. 2). Quantitative analysis of the immuno-precipitated bands by using densitometry indicated that-69% of the secreted material is present in an af complex.In two separate transfections, the total amount of sCDllbdetected in 1 ml of culture supernatant was 20 ng + 1.sCDllb/CD18 Binds to iC3b. Both 44a and TS18 mAbs,

which reacted with sCDllb/CD18, are known to be directed

El l)

kDa

200 ]> at.

%I _

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69 A

L' d

At's kDa

¶6 *O 0

-4 92.5

.9-q-I-

-S(-.vaS ----^.r.ZZ.i.

--a 69

1-4Q 4(b

FIG. 2. Autoradiograph of a SDS/7.0%o PAGE showing immuno-precipitates from [35S]methionine-labeled COS cell supernatantsafter cotransfection of COS cells with the mutated CD11b and CD18cDNA constructs. A negative control ascites (NS1) (lanes a and d),an anti-CD18 mAb (TS18) (lane b), and an anti-CD11b mAb (44a)(lane c) were used. Arrowheads represent migration of molecularmass standards myosin, phosphorylase b, bovine serum albumin,and ovalbumin.

to functional epitopes in membrane-bound receptor(mCDllb/CD18) (13, 16, 36), suggesting that the recombi-nant secreted heterodimer may be functional. We thereforemeasured the ability of sCDllb/CD18 to bind to its well-characterized ligand iC3b, the major opsonic fragment ofcomplement C3, using mCDllb/CD18 as a positive control.The 1251-labeled mCDllb/CD18 bound specifically to iC3b(Fig. 3A, lanes b and d) but not to C3b (Fig. 3A, lanes a andc), confirming reports (39, 40). Under the same conditions,metabolically labeled supernatants containing the sCDllb/CD18 heterodimer bound to ATS-iC3b (Fig. 3B, lane b).Binding was specific in that no binding was observed toATS-C3b (Fig. 3B, lane c). No comparable binding activity toiC3b was detected in supernatants from the sham-transfectedCOS cells (Fig. 3B, lane a). Binding of mCDllb/CD18 andsCDllb/CD18 to iC3b was inhibited in the presence ofEDTA (data not shown). Binding of sCDllb/CD18 to iC3bwas also critically dependent on the presence of the recom-binant receptor as a heterodimer because neither sCDllb orsCD18 subunits, expressed separately, bound to iC3b-Sepharose (Fig. 4).sCDllb/CD18 Inhibits PMN Binding to Inflamed Endothe-

lium. The ability of sCDllb/CD18 to inhibit binding ofhuman PMN to inflamed endothelium was also examined andcompared with levels of inhibition found using anti-CD1lb oranti-CD18 mAbs (8, 9). 44a and TS18 mAbs inhibited adhe-sion of PMN to rIL-1-treated endothelium by 32 ± 9.3% and72 ± 6.4 % (mean + SEM, n = 4), respectively (Fig. 5), inagreement with our previous data (8, 9). Supernatants con-taining sCDllb/CD18 (-7 ng of sCDllb added to each wellcontaining 2.7 ng of surface-expressed receptor, or 3-foldmolar excess of secreted to membrane-bound receptor) werealso effective in blocking PMN adhesion to rIL-1-inducedendothelium (58 + 7.2% inhibition, P < 0.001 compared tosupernatants from sham-transfected cells) (Fig. 5).

3108 Immunology: Dana et al.

-.* s C., DI1. 1

.-. 406.

'T

46 'x-

Proc. Natl. Acad. Sci. USA 88 (1991) 3109

B

A a b c (l

C ee

CR1---

a b c- .* 4T.kn

* * 4. -4200

kDa sCDlb--->- fl

22100 , 92.5

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FiG. 3. Binding of membrane (A) and secreted (B) CD11b/CD18to iC3b. (A) Autoradiograph of a SDS/6% PAGE of PMN-derived125I-surface-labeled glycoproteins eluted from ATS-C3b (lane a) andATS-iC3b (lane b). Eiuants from ATS-C3b (lane a) contained com-

plement receptor type 1 (CRI) (250 kDa) and the C3-binding regu-

latory protein gp45/70 (45-70 kDa) (39. 40). Eluants from ATS-iC3b(lane b) contained two additional proteins at 155 kDa and 94 kDa.representing mCD11b/CD18 heterodimer. CD11b/CD18 was immu-noprecipitated with 44a mAb from material eluted from ATS-iC3b(lane d) but not from ATS-C3b (lane c). Molecular mass standards(arrowheads) were myosin. phosphorylase b, and bovine serum

albumin. (B) Autoradiograph of an SDS/8% PAGE showing specificbinding of metabolically labeled sCD11b/CD18 heterodimer to iC3b.sCD11b/CD18 was eluted from ATS-iC3b (lane b) but not fromATS-C3b (ane c). No specific radiolabeled material was present in

eluant of ATS-iC3b exposed to sham-transfected COS cells (lane a).Molecular mass standards (arrowheads) are as in Fig. 2. Gel expo-sure to x-ray film (with an intensifying screen) was at -80'C for 5days. Similar results were seen with supernatants from two othertransfections.

DISCUSSIONThis report demonstrates the synthesis and expression of a

functional and soluble form of a recombinant integrin het-erodimer. The evidence that the recombinant 82 integrinCD11b/CD18 can be expressed in a water-soluble form issupported by the observations that a truncated form of thereceptor of the expected molecular size is present in super-natants from the transfected cells. That the bulk of theexpressed receptor is in a heterodimeric form is shown by theability of mAbs directed against either the CD11b or CD18subunits to immunoprecipitate both subunits (Fig. 2). Thesefindings also suggest that neither the cytoplasmic end nor thetransmembrane regions of the CD11b or CD18 subunits are

required for heterodimer formation. We cannot exclude thepossibility, however, that these regions may improve thestability of this complex.Our ability to express a recombinant sCDllb/CD18 het-

erodimer contrasts with the significant difficulties beingencountered in expressing heterodimeric forms of otherreceptors (41) and, perhaps, reflects special features in therequirements for formation of an a/3 complex in integrins.The present findings agree with recent molecular cloningstudies (29, 42, 43) and EM studies of integrins (44) thatsuggest that a/3 association requires direct binding of theN-terminal halves of the two subunits, forming a mushroom-like head with the remainder of each subunit forming a freetail embedded in the plasma membrane.The secreted receptor reacted with mAbs directed against

functional epitopes or domains (13, 16, 36) suggesting that theoverall conformation of the native form of the receptor ismaintained. This suggestion was directly confirmed by ex-

43 >

FIG. 4. Autoradiograph of a SDS/10%o PAGE evaluating bindingof COS cell supernatants containing metabolically labeled sCDllb,sCD18, or sCDllb/CD18 heterodimer to iC3b. Binding activity wasobserved only in supernatants from COS cells expressing sCDllb/CD18 but not in those expressing either sCDllb or sCD18 alone.Culture supernatants contained equivalent amounts of sCDllb,sCD18, or sCDllb/CD18, as determined by immunoprecipitationstudies (data not shown). Molecular mass standards (arrows) are thesame as for Fig. 2.

amination of the ligand-binding properties of the recombinantreceptor. sCDllb/CD18 bound to its ligand, iC3b, undersimilar conditions to those previously used to show direct andspecific binding of mCD1lb/CD18 (Fig. 3). Binding ofsCDllb/CD18 to iC3b was divalent cation-dependent andspecific in that no binding was detected to C3b. Binding alsorequired expression of the secreted receptor in its het-erodimeric form because no binding activity was displayed byits secreted subunits when expressed separately (Fig. 4). The

150

50 _

To

t csCs H b\

FIG. 5. Histogram showing relative binding of PMN to rIL1-treated HUVE in the presence of sCDllb/CD18 and mAbs 44a andTS18. Data points are mean ± SEM of triplicate determinations fromfour independent experiments. * and **, P <0.05 and P <0.001,respectively (when compared with adhesion in the presence of NS1or sham-transfected cell supernatants).

- 92.5CD18

-' 0(94 kE~a)

gp45/70 -a-

a

92.5-b

Immunology: Dana et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

secreted heterodimer also significantly inhibited binding ofPMN to rIL-1-activated endothelium.

,82 integrins have been suggested to require the cytoplas-mic portion for enhanced adhesion to various ligands. Stim-ulus-induced phosphorylation of CD18 has been directlydemonstrated (45) and occurs with kinetics similar to those ofstimulus-induced adhesive interactions (46). The ability ofsCDllb/CD18 to block f32-integrin-mediated cell adhesionindicates that the ligand-binding properties themselves areintact at least with regard to interaction with iC3b andrIL-1-activated endothelium. Similar findings have also beenobserved in other adhesion receptor families such as thecadherins (47). The observed posttranslational modificationsin mCD11/CD18 receptors may enhance their affinity toligands through interactions with cytoskeleton and/orexpression of conformational epitopes or binding sites thatmay also become expressed in the recombinant receptor.Comparison of the binding affinities of secreted vs. mem-brane-bound integrins can now directly address this question.The purified sCDllb/CD18 described in this report should

be useful in identifying other putative ligands for this receptorthat appear to mediate binding of phagocyte to endothelium(8, 9) and to a variety of microorganisms (48). It should alsohelp in better delineation ofthe areas involved in heterodimerformation. Generation of larger quantities of this receptorfrom stably transfected cell lines should also permit a detailedevaluation of its functional activity in a large number ofadhesive interactions mediated by CD11b/CDI8 (e.g.,phagocytosis, chemotaxis, binding to clotting factors, op-sonized-zymosan-induced oxygen free radicals and prote-olytic-enzyme release) (3). The anti-adhesive and comple-ment iC3b-binding activities of sCDllb/CD18 demonstratedin this report make it (or a derivative) an attractive anti-inflammatory candidate, which could effectively block phag-ocyte emigration into inflamed organs as well as complement-dependent tissue injury. These effects may be additive whenused in combination with the secreted form of the monomericreceptor CR1 (49). This approach should also apply to otherintegrins important in inflammatory and thrombo-occlusivedisorders (50-52).

We thank Dr. Vibeke Videm, Ms. Maria Giovino, and Mr. CraigNelson for expert technical assistance and Ms. Lisa Firicano forsecretarial help. This work was supported by the National Institutesof Health, the Arthritis Foundation, and the March of Dimes BirthDefects Foundation.

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