Immunology 1989 67 502-508

CD11/CD18-independent neutrophil adherence to inducibleendothelial-leucocyte adhesion molecules (E-LAM) in vitro

A. DOBRINA,* B. R. SCHWARTZ, T. M. CARLOS, H. D. OCHS,t P. G. BEATTY & J. M. HARLAN*Institute of General Pathology, University of Trieste, Trieste, Italy and Departments of Medicine and

tPediatrics, University of Washington, Seattle, Washington, U.S.A.

Acceptedfor publication 5 April 1989

SUMMARY

We examined the mechanisms involved in neutrophil adherence to cultured human umbilical veinendothelial cells (HEC) induced by direct stimulation of the neutrophils by phorbol myristate acetate(PMA), formylmethionyl-leucyl-phenylalanine (FMLP), or the calcium ionophore A23187 (neutro-phil-dependent adherence), or by pretreatment of HEC with interleukin-1 (IL-1), tumour necrosisfactor (TNF) or lipopolysaccharide (LPS) (endothelial-dependent adherence). Two distinct mecha-nisms for neutrophil adherence to HEC were demonstrated by performing adherence assays: (i) at 370versus 40; (ii) in the presence of Ca2+ only versus Mg2+ only; and (iii) in the presence or absence ofmonoclonal antibodies (mAb) to the CD1I/CD 18 adhesion complex of neutrophils. A CD1I/CD 18-dependent mechanism (i.e. inhibited by anti-CD18 mAb) was identified that was active in thepresence of Mg2+ only but not of Ca2+ only, and at 370 but not at 4°. A CD1 1/CD18-independentmechanism (i.e. not inhibited by anti-CD 18 mAb) was active at 40 and at 370, and in the presence ofCa2+ only and of Mg2+ only. Neutrophil-dependent adherence induced by FMLP or PMA occurredsolely via the CD I l /CD 18-dependent mechanism, whereas endothelial-dependent adherenceinduced by a 4-hr pretreatment with IL- 1, TNF, or LPS involved both CD I/CD 18-dependentand/independent mechanisms. CD1i/CD18-deficient neutrophils isolated from a patient withleucocyte adherence deficiency (LAD) maintained the ability to adhere to LPS-pretreated HEC in thepresence of Ca2+ only, indicating that this mechanism of adherence involves a receptor on theneutrophil distinct from CD1 i/CD 18. Furthermore, the disappearance of the CDI 1/CD 18-independent, but not of the CD II/CD 1 8-dependent mechanism of adherence, in HEC treated withTNF for 24 hr suggests that the two mechanisms of neutrophil adherence also involve distinctinducible endothelial-leucocyte adhesion molecules (E-LAM).

INTRODUCTION

Neutrophil adherence to endothelium can occur by neutrophil-dependent or endothelial-dependent mechanisms. Neutrophil-dependent adherence involves direct stimulation of the neutro-phil by activating agents such as chemotactic peptides, tumournecrosis factors, or platelet-activating factor. Neutrophil depen-dent adherence is mediated by the interaction of the CD II/CD18 neutrophil membrane adhesion complex (Cobbold, Hale& Waldmann, 1987), specifically the CDlib/CDi8 (Mac-1)subunit (Wallis et al., 1986), with its ligand on the endothelial

Abbreviations: A23187, calcium ionophore; E-LAM, endothelial-leucocyte adhesion molecule; FMLP, N-formylmethionyl-leucyl-phenylalanine; HEC, human umbilical vein endothelial cells; IL-1,recombinant human interleukin- 1; LAD, leucocyte adherence deficien-cy; LPS, lipopolysaccharide; PMA, phorbol-1 2-myristate-1 3-acetate;TNFx, recombinant human tumour necrosis factor-alpha.

Correspondence: Dr A. Dobrina, Institute of General Pathology,University of Trieste, Via A. Fleming, 22 34127 Trieste, Italy.

cell surface. The ligand for CD lIb/CD 18 has recently beenreported to be ICAM-1 (intercellular adherence molecule-i)(Smith et al., 1988), originally identified as the ligand forCD IIa/CD 18 (LFA- 1) (Rothlein et al., 1986).

Endothelial-dependent adherence involves expression ofendothelial cell surface molecules that promote neutrophiladherence. Endothelial-dependent mechanisms include therapid, protein synthesis-independent alteration in endothelialcell membrane adhesiveness induced by thrombin (Zimmerman,McIntyre & Prescott, 1985), leukotriene C4, and leukotriene D4(McIntyre, Zimmerman & Prescott, 1986), and the newlysynthesized surface proteins induced by interleukin-1 (IL-1)(Bevilacqua et al., 1985), tumour necrosis factor-alpha (TNFoc)(Gamble et al., 1985) and lipopolysaccharide (LPS) (Schleimer& Rutledge, 1986). The latter have been designated endothelial-leucocyte adhesion molecules (E-LAM) (Bevilacqua et al.,1987). Neutrophil adherence to E-LAM is in part dependentupon neutrophil CDI I /CD 18 (Pohlman et al., 1986; Rampart &Williams, 1988), whereas neutrophil adherence induced by

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thrombin treatment of endothelium is CDI 1/CD18-indepen-dent (Zimmerman & McIntyre, 1988).

In this study we examine further the contribution ofneutrophil and endothelial cell adhesion molecules to theprocess of neutrophil adherence to cultured human endothe-lium. We demonstrate that neutrophil binding to E-LAMinvolves two distinct receptors on neutrophils which appear tointeract with different E-LAM.

MATERIALS AND METHODS

Cell cultureHuman umbilical vein endothelial cells (HEC) were prepared bycollagenase treatment ofthe vessels as described elsewhere (Jaffeet al., 1973) and maintained in endotoxin-free RPMI-1640medium (M.A. Bioproducts, Walkersville, MD) supplementedwith 10% adult bovine serum (Hyclone Sterile Systems, Logan,UT) and 10% normal calf serum (Armour Pharmaceutical Co.,Kankakee, IL) (ABS-NCS). Passaged HEC were maintained inRPMI-1640 supplemented with 20% ABS-NCS containingheparin (90 pg/ml; Sigma Chemical Company, St Louis, MO)and endothelial cell growth factor (50 pg/ml) as described byThornton, Mueller & Levine (1983). Endothelial cell growthfactor was prepared from bovine hypothalamus according tothe method of Maciag et al. (1979).

Neutrophil isolation and labellingPeripheral blood was obtained by venepuncture from healthydonors and a patient with leucocyte adhesion deficiency afterinformed consent according to protocols approved by theHuman Subjects Review Board of the University of Washing-ton. The blood was collected in syringes containing heparin(10 U/ml), and the neutrophils were isolated by Ficoll-Hypaque(Pharmacia Fine Chemicals, Picataway, NJ) gradient centri-fugation, 3% dextran sedimentation, and hypotonic saline lysis(Boyum, 1968). This procedure resulted in a preparation greaterthan 95% neutrophils, which exceeded 95% viability by trypanblue dye exclusion. Isolated peripheral blood neutrophils weresuspended in phosphate-buffered saline (PBS) (Gibco Labora-tories, Grand Island, NY) without Ca2+ or Mg2+ at 50 x 106cells/ml and were labelled for 60 min at 370 with 50 pCi/ml 5'Cras sodium chromate 1 yCi/ml, 200-500 Ci/g (New EnglandNuclear, Boston, MA) (Gallin, Clark & Kimball, 1973). Afterlabelling, the neutrophils were washed three times with PBS andsuspended at a final concentration of 2 x 106 cells/ml in PBScontaining 5 mM glucose and containing 0 7 mm CaC12 and0-7 mm MgCI2 unless otherwise stated.

Adherence assayFirst to fourth passage HEC were harvested with 0-05% trypsinand 0-02% EDTA in balanced salt solution (Gibco Laborator-ies). The cells were then plated in 11 mm diameter wells (CostarCluster, Cambridge, MA) at 5 x 104 cells/well in RPMI-1640supplemented with 20% ABS-NCS. Visually confluent mono-layers were formed after overnight incubation. Cells werepretreated with reagents for 4 hr unless otherwise stated, and themonolayers were then washed with two-well volumes of PBS.For the adherence assays at 40, after the incubation at 370 HECwere incubated for 30 min in a cold room (2-4°) and thenwashed with a two-well volume of ice-cold PBS. 5'Cr-labelledneutrophils were then added (250 p1/well). Neutrophils for the

40 adherence assay were incubated for 30 min on ice prior toaddition to the wells. Neutrophils and HEC were then incubatedfor 30 min at 370 or in the cold room. After incubation, themonolayers were washed with a single-well volume of PBS orice-cold PBS, to remove non-adherent neutrophils. The celllayers with remaining adherent 5'Cr-labelled neutrophils weresolubilized by addition of IN NH40H, and the lysates werecounted in a gamma counter (Micromedic Systems, Inc.,Horsham, PA). Neutrophil adherence was expressed as:

% adherence=c.p.m. lysate x 100/c.p.m. added.

Monoclonal antibodiesMAb 60 3 (Beatty et al., 1983) and MAb 1B4 (Wright et al.,1983) are of IgG2a subclass and recognize the CD18 subunit(common beta-chain) of the CD 1 /CD1 8 antigen complex. Theantigen recognized by mAb 60-3 and 1B4 is present on allperipheral blood leucocytes. MAb 1B4 was kindly provided byKarl-E. Arfors, Pharmacia Experimental Medicine, La Jolla,CA.

Immunofluorescenceflow cytometryImmunofluorescence flow cytometry was performed as pre-viously described (Hickstein et al., 1987). Neutrophils (5 x 105)suspended in 100 pI PBS were incubated in polypropylene,round-bottomed tubes (Becton Dickinson and Co., LincolnPark, NJ) with 50 p1 of heat-inactivated, dialysed human ABserum at 40 for 20 min. A saturating concentration ofmAb 60 3was then added to the cells, followed by the addition of CaCI2(0 7 mM) and/or MgCI2 (0-7 mM) or PBS, to a final volume of200 p1. The cells were incubated for 30 min at 370 or 40, washedfree of unbound antibody and incubated again at 40 for 30 minwith 50 ul of a 1:50 dilution of FITC-goat anti-mouse IgG(Tago, Burlingame, CA). After washing, the cells were sus-pended in 500 p1 of 1% paraformaldehyde in PBS and stored at40. The mean fluorescence ofeach cell population was quantifiedby a fluorescence-activated cell sorter (FACS) 440 (BectonDickinson and Co., Sunnyvale, CA) with quantitative deter-mination ofpeak fluorescence intensity. Data on 5000 cells werecollected from each sample. By using this system, immunofluor-escence intensity served as quantitative measure of antigenexpression.

ReagentsEscherichia coli 055: B5 lipopolysaccharide (LPS), extracted byphenol/water, was obtained from Sigma Chemical Company.The LPS preparation was suspended in PBS at concentration of1-5 mg/ml, dispersed by sonication for 5 min at 40 and stored inaliquots at -35° until used. Phorbol-12-myristate-13-acetate(PMA), and N-formylmethionyl-leucyl-phenylalanine (FMLP)were purchased from Sigma Chemical Company. Calciumionophore A23187 was purchased from Calbiochem, La Jolla,CA. PMA (1 mg/ml), FMLP (100pM) and A23 187 (100 gM stocksolutions were stored in dimethylsulphoxide (Eastman KodakCo., Rochester, NY) at -20°. Purified recombinant humaninterleukin-l-alpha (1000 U/ml) (IL-1) was purchased fromGenzyme, Boston, MA. Human recombinant TNF-alpha(TNFax) was kindly provided by Genentech, Inc., San Francisco,

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36-8% + 10 2 and 54 9% + 7 2, respectively (mean + SE). Whenthe adherence assay was performed at 40, however, mAb 60 3was without significant effect. It is noteworthy that in the sameexperiments mAb 60-3 inhibited FMLP-stimulated neutrophiladherence at 370 by 89% + 4 3 (mean + SE), and that FMLP didnot increase neutrophil adherence at 4° in the presence or in theabsence of mAb 60-3.

Similar results were obtained with mAb 1B4, which is alsodirected to an epitope on CD 18. Mab 1B4 inhibited neutrophiladherence to LPS-, TNF-, and IL-l-pretreated HEC by 43-8%,66 5% and 44-6%, respectively, when the assay was conductedat 370, but inhibited only by 3 0%, 1[7% and 0%, respectively,when the assay was performed at 4° (not shown).

Figure 1. Effect of temperature on (a) neutrophil-dependent and (b)endothelial-dependent adherence. (a) 51Cr-labelled neutrophils sus-pended in PBS containing 5 mm glucose, 0 7 mm CaCl2 and 0 7 mMMgCl2 were added to HEC monolayers with medium (control) or withPMA (100 yg/ml), FMLP (10-5 mM), or A23187 (5 x 10-7 M). For theadherence assay at 40, neutrophils were incubated on ice and HEC wereincubated in a cold room (2-40) for 30 min prior to the assay. (b). HECmonolayers were pretreated at 37° for 4 hr with medium alone (control)or with medium containing E. Coli LPS (100 ,g/ml), TNF (500 U/ml) orIL-l (10 U/ml) and with or without a final 30 min incubation at 2-4°.Percentage neutrophil adherence was determined after a 30 minincubation at 370 or at 4°. Values are means + SE of five experiments,with three to four replicate wells in each experiment. Statisticalsignificance of differences from control values (paired t-test): *P < 0 05.**P<0-01. Other results were not significantly different from controlvalues.

CA. The preparation assayed (by the supplier) for its cytolyticactivity on actinomycin D-treated L929 mouse fibroblast cells,contained 2 x 107 U/ml.

StatisticsSignificance was determined by paired, two-tailed t-test.

RESULTS

Influence of temperature on neutrophil adherence to HEC

Adherence assays were performed at 370 or 40. Neutrophiladherence to HEC was increased either by stimulating theneutrophil directly with PMA, FMLP or A23 187 (Harlan et al.,1985) or, alternatively, by preincubating HEC monolayers withLPS, TNFax or IL-la, agents that induce synthesis of E-LAM(Bevilacqua et al., 1987).

Stimulation with PMA, A23 187 or FMLP markedlyincreased neutrophil adherence to HEC when the assay wasperformed at 370, but not at 40 (Fig. 1 a). In contrast, neutrophiladherence to LPS-, TNF-, or IL-1-pretreated HEC increasedsignificantly at 40 as well as at 370 (Fig. lb).

Role of neutrophil CD11/CD18 complex in endothelial-dependentadherence

To define the role of neutrophil CD11/CD 18 complex inendothelial-dependent adherence, adherence assays were per-formed at 370 and at 40 in the presence or absence of the anti-CD1 8 mAb 60 3. As shown in Table 1, when the adherence assaywas performed at 37°, mAb 60 3 inhibited neutrophil adherenceto LPS-, TNF- and IL-1-pretreated HEC by 50-1% +77,

Divalent cation requirements for neutrophil-dependent andendothelial-dependent mechanisms of adherence

In the preceding experiments neutrophils were suspended in amedium containing both Ca2+ and Mg2+. Additional experi-ments were performed to determine the divalent cation require-ments for the neutrophil-dependent and the endothelial-depen-dent mechanisms ofadherence. In the absence ofboth Mg2 + andCa2+, adherence was less than 5% (data not shown). As shownin Table 2a, when the assay was performed at 370 and in thepresence of Ca2+ only, neutrophils adhered to LPS-pretreatedHEC, but the adherence of PMA-stimulated neutrophils tountreated HEC was abolished. Moreover, mAb 60 3 did notinhibit the adherence of neutrophils to LPS-pretreated HEC inmedium containing only Ca2+. In the presence of Mg2+ only,neutrophils adhered to LPS-pretreated HEC, but in thiscondition mAb 603 significantly inhibited neutrophil adher-ence. Finally, in the presence of Mg2+, neutrophil adherence toHEC was stimulated by PMA, and PMA-induced adherencewas completely inhibited by mAb 60 3.

At 40 neutrophils adhered to LPS-pretreated HEC in thepresence of either Ca2+ only or Mg2+ only (Table 2b). Adher-ence to LPS-pretreated HEC in the presence of Ca2+ only at 40was comparable to that observed at 37°. Adherence to LPS-pretreated HEC at 40 in the presence of Mg2+ only wassomewhat lower than adherence at 37°. No inhibition by mAb60 3 was observed at 40 with either Ca2+ only or Mg2+ only, thusconfirming the results obtained at 40 with both Ca2+ and Mg2+present (Table 1).

Immunofluorescence flow cytometry assays were performedto evaluate the binding ofmAb 60-3 to neutrophils at 370 and at40 and in the presence or absence of Ca2+ and/or Mg2+. Theresults (not shown) indicated identical binding in all conditions,thereby excluding the possibility that the failure of the anti-CD1 8 mAb to inhibit neutrophil adherence to HEC at 40 or inthe absence of Mg2+ was due to changes in the affinity of themAb for neutrophils.

Mg2+-independent adherence involves a neutrophil receptordistinct from CD11/CD18The observation that neutrophil adherence to LPS-, TNF- orIL-1-pretreated HEC was inhibited by mAb 60 3 or Mab 1 B4 byonly 50% suggested that there was a second, CDll/CD18-independent mechanism. An alternative explanation was thatneutrophil binding to HEC under these conditions involved anepitope(s) on CD18 that was not recognized by mAb 60-3 or

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Table 1. Effect of mAb 60-3 on adherence at 370 and at 40

% adherence

Pretreatment Addition to % inhibition % inhibitionof HEC neutrophils 370 by mAb 60 3 40 by mAb 60 3

Medium Medium (5) 6 1+11 3 3+11

LPS Medium (4) 27 8+2-1 19-9+4-9LPS mAb 60-3 (4) 14-0+2-7 501 +7-7* 18 2+5-0 8 5+9 8 NS

TNF Medium (5) 32-3+3 5 18-3+3 1TNF mAb 60-3 (5) 201+38 368± 10-2t 174+30 37+94NS

IL-I Medium (4) 29-8+3-2 22-6+8-6IL-I mAb 60 3 (4) 13 4+2-5 54.9+7 2* 23-6+9 3 2-3+33 NS

Medium FMLP (4) 21 0+1*4 5 3+1 6Medium FMLP+mAb 60 3 (4) 2 4+1.1 89 1 +4-3t 4-4+1 6 18 0+13 8 NS

HEC monolayers were pretreated for 4 hr with medium alone or medium containing E. coli LPS(100 ng/ml), TNFa (500 U/ml), or IL-i (10 U/ml). 51Cr-labelled neutrophils suspended in PBScontaining 5 mM glucose, 0 7 mM CaCI2 and 0 7 mM MgCl2 were incubated for 15 min at roomtemperature and in the presence or absence ofmAb 60 3 (40 pg/ml). For the adherence assays at 40,neutrophils were further incubated on ice and HEC were incubated in a cold room for 30 min beforethe assay. Neutrophils were then added to HEC monolayers and medium or FMLP (10-5M) wasadded to the neutrophils. Percentage neutrophil adherence was determined after a 30 min incubationat 370 or in the cold room (2-4°). Values are means + SE of (n) experiments with three to four replicatewells in each experiment. Statistical significance of difference of adherence in the absence or presenceof mAb 60 3 (paired t-test): *P< 0 01; tP< 0-05; NS, not significant.

Table 2. Effect of Ca2+ and Mg2+ on neutrophil-dependent and endothelial-dependentadherence at 370 and at 40

% adherence

Pretreatment Addition to % inhibition % inhibitionof HEC neutrophils Ca2+ by mAb 60-3 Mg2+ by mAb 60-3

(a) 370Medium Medium 2-0+0-6 1-7+0 3

LPS Medium 15-0+1-4 16-4+38LPS mAb60-3 154±1-7 Noinhibition 8-2+2-9 53+11-1*

Medium PMA 0-8+0-1 53-8+12-1Medium PMA-mAb 60-3 0 4+001 1-3+0 4 97+ 0.9*

(b) 40Medium Medium 1-2+0 1 1-2+0-2

LPS Medium 17-5+2-2 12-5+1-8LPS mAb 60-3 21-5 + 1-4 No inhibition 16-0 + 2-9 No inhibition

Medium PMA 1[0+0-2 1-4+0 3

HEC monolayers were pretreated for 4 hr with medium alone or medium containing E. coliLPS (100 ng/ml). 51Cr-labelled neutrophils suspended in PBS containing 5mM glucose and either0-7 mm CaC12 or 0 7mM MgCl2 were incubated for 15 min at room temperature, in the presence orabsence ofmAb 60-3 (40 pg/ml). For adherence assays at 4°, neutrophils were further incubatedon ice and HEC were incubated in a cold room for 30 min before the assay. Neutrophils were thenadded to HEC monolayers and PBS (medium) or PMA (100 ng/ml) was added to the neutrophils.Percentage neutrophil adherence was determined after a 30 min incubation at 370 (a) or in thecold room (2-4°) (b). Values are means + SE of three experiments with three to four replicatewells in each experiment. Statistical significance of difference of adherence in the absence orpresence ofmAb 60 3 (paired t-test): *P<0.05.

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Table 3. Adherence of LAD neutrophils to LPS-pretreated HEC

% adherence

Normal donor LADPretreatment Addition toof HEC neutrophils Ca2+ Mg2+ Ca2+/Mg2+ Ca2+ Mg2+ Ca2+/Mg2+

Medium Medium ND ND 11-2+1-4 ND ND 3-1+0-6LPS Medium 22-1+69 20-3+1-9 26-1+1 5 29-3+6-2 93+0-8 21-5+5-4Medium PMA ND ND 33-4+2-6 ND ND 6-2+0-6Medium PMA+mAb 60 3 ND ND 8-5+ 12 ND ND 7 0+224

HEC monolayers were pretreated for 4 hr with medium alone or medium containing E. coli LPS (100 ng/ml).51Cr-labelled neutrophils from a normal donor or from a LAD patient deficient in CDlI/CD18 were suspended inPBS containing either 0 7 mM CaC12 or 0 7 mm MgCl2 or both cations and were incubated for 15 min at roomtemperature in the presence or absence ofmAb 60-3 (40 pg/ml). Neutrophils were then added to HEC monolayersand PBS (medium) or PMA (100 ng/ml) was added to the neutrophils. Percentage neutrophil adherence wasdetermined after a 30 min incubation at 37°. Values are means + SD ofthree replicate wells in one experiment. ND,not done.

mAb 1 B4. This latter possibility was excluded by use ofneutrophils isolated from a patient with LAD (Bowen et al.,1982), the syndrome resulting from congenital deficiency ofCDl 1/CD18 (Anderson & Springer, 1987; Todd & Freyer1988). As shown in Table 3, the adherence ofLAD neutrophilsto LPS-pretreated HEC was significantly greater than adher-ence to untreated HEC.

Furthermore, LAD neutrophils adhered to LPS-pretreatedHEC in the presence of Ca2+ only, demonstrating that someother structure besides CDl 1/CD 1 8 must function as thereceptor for Mg2+-independent binding.

As expected, direct stimulation ofLAD neutrophils byPMAfailed to increase adherence to untreated HEC. PMA caused

Table 4. Effect of Ca2+ and Mg2+ on the adherence of neutrophils toendothelial cells pretreated with TNF for 4 or 21 hr

0/ adherencePretreatment Addition toof HEC neutrophils Ca2+ Mg2+

TNF (21 hr) Medium 45+09 16.9+2.3*TNF (21 hr) mAb 60 3 ND 1-8 +0 2TNF (4 hr) Medium 17-7+3.1* 20.1+3 2*TNF (4 hr) mAb 603 ND 81 +2lItMedium Medium 3 9+009 3 9 +0 7Medium mAb 60 3 ND 2 0+0 4

HEC monolayers were pretreated for 4 hr or for 20-22 hr withmedium alone (control) or medium containing TNF (500 U/ml). 5"Cr-labelled neutrophils suspended in PBS containing 5 mm glucose andeither 0 7mm CaCl2 or 0-7 mM MgCl2 were incubated for 15 min at roomtemperature, in the presence or absence of mAb 60 3 (40 pg/ml).Neutrophils were then added to HEC monolayers and percentageneutrophil adherence was determined after a 30 min incubation at 37°.Values are means + SE of four experiments with three to four replicatewells in each experiment. Statistical significance of differences fromcontrol values of medium only by paired t-test: tP <0-05; *P< 002.Other results were not significantly different from control values. hrhours of treatment. ND not done.

normal neutrophils to adhere to HEC when Mg2+ was presentin the incubation medium. This adherence was blocked bymAb 60 3.

Mg2+-dependent and -independent neutrophil adherence involvesdistinct receptors on endothelial cellsHaving established that the two mechanisms of neutrophiladherence to LPS-pretreated HEC involved distinct receptorson neutrophils, we then investigated whether the two neutrophilreceptors recognized different ligands on HEC. In the experi-ments reported above, pretreatment of HEC for 4 hr with LPS,TNF or IL-l always induced both mechanisms of neutrophiladherence. This suggested that the same E-LAM might beshared by both mechanisms. This possibility, however, wasexcluded by experiments in which HEC were treated with TNFfor either 4 hr or 20-22 hr. As shown in Table 4, when HEC weretreated with TNF for 4 hr, a significant increase in PMNadherence above control values (untreated HEC) was obtainedboth in the presence of Ca2+ only and of Mg2+ only. MAb 60 3inhibited neutrophil adherence by 50% in the presence of Mg2+only. In contrast, when HEC were treated with TNF for 20-22hr, neutrophil adherence in the presence of Mg2+ only remainedsignificantly increased above control values, whereas neutrophiladherence in the presence of Ca2+ only had returned to controlvalues. Moreover, mAb 60 3 completely inhibited neutrophiladherence at 20-22 hr in the presence of Mg2+ only, indicatingthat only the CD I 1/CD 18-dependent mechanism that requiresMg2+ was involved in neutrophil binding to HEC.

Similar results were obtained when HEC were pretreatedwith IL-l (10 U/ml) for 3 hr or 21 hr. With Ca2+ only neutrophiladherence was 11-2%+21 at 3 hr versus 16%+0-2 at 21 hr.With Mg2+ only neutrophil adherence was 19 9% + 1 1 at 3 hrversus 17-1 +20 at 21 hr. MAb 60 3 inhibited adherence inMg2+ only at 3 hr by 65% and completely abolished adherenceat 21 hr (means + SE of four replicate wells in one experiment).

DISCUSSION

These results demonstrate that neutrophil adherence stimulatedby neutrophil-activating agents such as PMA, FMLP, or

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A23187 (neutrophil-dependent adherence) is mediated solely byCD1I/CD18, requires Mg2+ (Ca2+ alone is not sufficient), anddoes not occur at 4°. The adherence of neutrophils to LPS-.TNF-, or IL-1-pretreated HEC (endothelial-dependent adher-ence), however, involves two distinct mechanisms. One endo-thelial-dependent mechanism requires Mg2+ (Ca2+ alone is notsufficent) and is temperature-dependent (it is abolished at 4°).This endothelial-dependent mechanism of adherence involvesthe CD 1 /CD18 complex of neutrophils, since it is inhibited byanti-CD18 mAb, and accounts for approximately 50% ofneutrophil binding to E-LAM at 4 hr. The second endothelial-dependent mechanism operates equally well in the presence ofCa2+ or Mg2+ and is temperature-independent (it is notinhibited at 4°). The failure of anti-CD18 mAb to inhibitbinding to LPS-, IL-I, or TNF-treated HEC at 40 and thebinding of LAD cells to LPS-treated HEC in the presence ofCa2+ only confirm that this second endothelial-dependentmechanism is not dependent on the CDII/CD18 adhesioncomplex of neutrophils, but involves a second, as yet unidenti-fied neutrophil receptor. The variable inhibition of neutrophilbinding to LPS-, TNF-, or IL-l-treated HEC by anti-CD18mAb (50-70%) and the variable binding of LAD cells (5% inPohlman et al., 1986 vs. 20-30% in this study) can be explainedby our observation that the neutrophil receptor for CDI I/CD18-independent adherence to E-LAM is down-regulatedfollowing neutrophil activation (A. Dobrina, T. Carlos, B.Schwartz and J. Harlan, manuscript in preparation). The twomechanisms of neutrophil adherence to LPS-, IL-I-, or TNF-pretreated HEC involve two distinct E-LAM. This conclusion isbased on the observation that the CD1 1/CD18-dependent andCD1 1/CD1 8-independent mechanisms of binding to E-LAMexhibited different time-course. Neutrophil adherence in thepresence of Ca2+ only was greatly reduced after 20-22 hrcompared to 3-4 hr pretreatment with TNF. In contrast,neutrophil adherence in the presence of Mg2+ only wasminimally reduced after 20-22 hr compared to 3-4 hr pretreat-ment with TNF. Furthermore, the increased binding of neutro-phils in the presence of Mg2+ only at 24 hr was completelyabolished by mAb 60-3, indicating that the CD1 1/CD18-dependent mechanism was exclusively responsible for theneutrophil binding at that time.

From these results it seems likely that E-LAM-1 describedby Bevilacqua et al. (1987) corresponds to the CDll/CD18-independent mechanism that does not require Mg2+, since thekinetics of neutrophil binding in the presence of Ca2+ onlyparallels that reported for E-LAM-1 antigen expression by IL- I -treated HEC (Bevilacqua et al., 1987). This conclusion issupported by the recent abstract by Luscinskas et al. (1988),reporting that neutrophil adherence to IL-i-treated endothelialcells was only partially inhibited either by anti-E-LAM-1 mAbalone or by anti-CD 1 1/CD 1 8 mAb alone, but was completelyinhibited in the presence of both mAb. Their results and ourssuggest that E-LAM-I and CDl 1/CD 18 are not acting as anobligate receptor-ligand pair.

The identity of the endothelial surface molecule induced byIL-I, TNF or LPS that promotes CDII/CD18-dependentbinding is unknown. Recently, Smith et al. (1988) reported thatmAb to endothelial ICAM-I abolished neutrophil adherenceinduced by FMLP and inhibited neutrophil adherence to IL-1-treated HEC by 50%, with no additive effect observed with co-incubation with an anti-CD 18 mAb. These studies indicate that

ICAM-l is the endothelial ligand for neutrophil CDl l/CD18.In contrast to E-LAM-1, which is expressed by HEC only uponstimulation (Bevilacqua et al., 1987), ICAM-1 is constitutivelyexpressed on the HEC surface, although its expression isincreased by IL-I or TNF (Dustin et al., 1986; Pober et al.,1986). Since neutrophils do not adhere to ICAM-l of untreatedHEC unless they are directly stimulated (neutrophil-dependentadherence), it seems unlikely that increased expression ofICAM-alone is responsible for CD 1 /CD 18-dependent neutro-phil adherence to IL-i- or TNF-treated HEC. The existence ofan induced surface molecule on TNF-, IL-1- or LPS-treatedHEC that stimulates neutrophils to adhere via CDIl/CD18-ICAM-1 must be considered. This molecule could be newlysynthetized ICAM-1, perhaps processeed or glycosylated differ-ently, or a distinct molecule (i.e. a second E-LAM) yet to beidentified.

Finally, having established that two different mechanismsunderlie neutrophil adherence to TNF-, LPS- or IL-i-stimu-lated endothelial cells in vitro, we then question whether thesetwo mechanisms are equally relevant in the adherence ofneutrophils to the vessel wall at inflammatory sites in vivo.Observations in LAD patients argue against any major physio-logical relevance of the CD1 1/CD18-independent mechanism,since LAD neutrophils are apparently incapable of emigratingfrom the bloodstream to inflamed tissues. However, our in vitrostudies indicate that LAD neutrophils maintain the ability tobind to LPS-pretreated HEC by the CD 1I/CD1 8-independentmechanism. One possible explanation for the discrepancybetween our in vitro results and the findings in LAD is that theCDI I/CDl 8-independent mechanism is functionally inactive ordown-regulated at sites of inflammation in vivo. However, in arecent study we found that pretreatment of rabbits with anti-CD18 mAb failed to inhibit neutrophil emigration into lungalveoli, although in the same animal the anti-CD 18 mAbabolished neutrophil emigration into a subcutaneous site ofinflammation induced by the same inflammatory agents (Doers-chuk et al., 1988). Therefore, under some circumstances theCD18-independent mechanism of adherence alone may besufficient for neutrophil emigration.

ACKNOWLEDGMENTSThis work was supported in part by grants from the USPHS (HL 18645,Al 07073, CA 18029), the March of Dimes, Birth Defects Foundation(6-273) and the CNR of Italy and the Italian Association for CancerResearch (AIRC). Dr Dobrina is a recipient of a Fulbright grant. DrHarlan is a recipient ofan Established Investigatorship Award from theAmerican Heart Association. We thank Kathe E. Stannes and PennyThompson for technical assistance and Lou Limtiaco for her successfulword processing.

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