462 Journal of Leukocyte Biology Volume 53, April 1993

Endocytosis of � integrins by stimulated human neutrophilsanalyzed by flow cytometry

J. David Chambers� Scott I. Simon,’ Elaine M. Berger� Larry A. Sklar,t and KarI-E. Arfors**La Jolla Institute for Experimental Medicine, La Jolla, California, and TCytometry, University of New Mexico School of

Medicine, Albuquerque

Abstract: Flow cytometry and fluorescently labeled mono-

clonal antibodies were used to investigate endocytosis ofhuman neutrophil f32 integrins following cellular activa-

tion. CD18 initially present on the cell surface cycled in

two phases after exposure to formyl peptide or platelet-

activating factor. The first phase lasted 3 mm at 37#{176}C;af-ter a lag, CD18 was specifically internalized at approxi-

mately 20%/mm. Subsequently a second phase was detec-table consisting of exponential reduction of internal

fluorescence with a half-time of approximately 2 mm,representing probe reexpression. At peak endocytosis ap-

proximately 40% of CD18 was internalized. All of the in-

ternalized CD18 was associated with aM (CR3); no en-

docytosis of aL (LFA-1) was observed. When neutrophilswere stimulated with phorbol esters or calcium iono-

phore, CD18 was internalized much more slowly (ti/2 =

5 mm) and probe was not reexpressed. Endocytosis of

CD18 may participate in regulating neutrophil adhesive-

ness, removing activated receptors, or permitting recep-

tor recycling. J. Leukoc. Rio!. 53: 462-469; 1993.

Key Words: CDJ8 . internalization . monoclonal antibody. fluorescent probe . receptor cycling

INTRODUCTION

The f32 integrins comprise a family ofheterodimeric molecules

found on the surface of leukocytes. These molecules share a

common 13 chain (f32, CD18) but have distinct a chains.

There are three known members in this family: a�J32

(CD11a/CD18, LFA-1), aM/32 (CD11b/CD18, CR3, Mae-i,

Mol), and aXI32 (CD11e/CD18, p150,95). /-�2 integnins func-

tion as cell adhesion molecules, playing a central role in leu-

kocyte function, particularly of neutrophils. The importance

off32 integrin molecules on the cell surface is dramatically il-

lustrated by the consequences of their absence, which occurs

in the disease leukocyte adhesion deficiency (LAD). Leuko-

cytes of LAD patients express very low quantities of all mem-

bers of the /32 family because of genetic errors in 132 chain

synthesis on export. Such patients have incompetent

neutrophil-mediated immunity and are characterized by

nonpurulent abscesses with no neutrophil infiltration despite

pronounced neutrophilia. Their neutrophils are unable to

undergo diapedesis and in vitro manifest defective locomo-

tion and aggregatory responses [1-3]. Studies of LAD and

use of anti-CD18 monoclonal antibodies (mAbs) have rev-

ealed that /32 integnins are predominantly responsible for

firm cellular adhesion during processes such as diapedesis

and extravasation, phagocytosis, and locomotion (reviewed

in ref. 4).

Unstimulated neutrophils appear to express on their sun-face low levels (tens of thousands) of /32 integnin molecules,

mainly of type aM/32 (CR3, CDilb/CD18) [4]. Among the

sequelae of polymorphonuclear neutrophil (PMN) stimula-

tion is transloeation of large quantities of /32 integnins from

preformed cellular stores to the cell surface [5]. This up-

regulation is a rapid event and results in at least a 10-fold in-

crease in I�2 integrin expression. Large numbers of the up-regulated molecules remain on the PMN surface for cx-

tended periods of time after stimulation [3, 5]. Another con-

sequence of PMN activation is that the cells become sticky

and adhere to one another (homotypie aggregation) and to

other cells (heterotypic aggregation) and surfaces. These

adhesive responses are /32 integrin dependent because theycan be blocked by anti-CD18 mAbs and are not manifested

by LAD PMNs. Early neutrophil adhesive responses do not

require up-regulation [6-8], and it is thought that basally cx-

pressed 132 integrins are converted to an actively adhesive

form upon cell stimulation [9]. The precise role of up-

regulated molecules in the early stages of adhesion is still un-

certain; however, it is reasonable to suspect that they might

play some part in the adhesive process at some time.

The ability of neutnophils to undergo processes such as di-

apedesis, which must require well-orchestrated variations in

adhesiveness, and to disaggregate in a homotypic aggrega-

tion model despite exaggerated surface expression of /32 inte-grins [10, 11] raises the question ofhow adhesion is regulated.

Regulation of adhesion might be accomplished by changes in

molecular conformation [9, 12], receptor clustering, and

deelustering resulting in changes in avidity [13] or by en-

docytosis of some of the molecules, thereby physically

preventing them from interacting with counter ligands.Phagocytosis is one of the primary neutrophil functions and

leads to endocytosis, and although CR3 is implicated [14] in

the process, it appears to be driven primarily by complement

receptors type 1 (CR1), Fe receptors, and the presence of

ligands on particles or surfaces. In addition, phagocytosis in-

volves invagination of relatively large areas of membrane andis unlikely to contribute any specific regulation of adhesiveness.

This study was designed to investigate the possibility of

specific 132 integnin endocytosis. Flow eytometry allows

Abbreviations: CR1, complement receptor type 1; DMSO, dimethyl sul-

foxide; DPBS, Dulbecco-Vogt’s PBS; Flit, fluorescein isothiocyanate;

IMLP, formylmethionyl-leucyl-phenylalanine; LAD, leukocyte adhesion

deficiency; mAb, monoclonal antibody; MHC, major histocompatibility

complex; PAF, platelet-activating factor; PBS, phosphate-buffered saline;

PE, phycoerythrin; PMA, phorbol myristate acetate; PMN, polymorpho-

nuclear neutrophil; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide

gel electrophoresis; SMCC, succinimidyl tmns-4-(N-maleimidylmethyl)cyclo-

hexane-l-carboxylate; SPDP, 3-(2-pyridyldithio)propionic acid N-hydroxy-

succinimide ester.

Reprint requests: J.D. Chambers, La Jolla Institute for Experimental

Medicine, 11077 North Torrey Pines Road, La Jolla, CA 92037.Received August 31, 1992; accepted December 11, 1992.

Chambers et al. Endocytosis of neutrophil 132 integrins 463

simultaneous automated analysis of many cellular

parameters on an individual cell basis. Rapid kinetics can be

studied in real time because measurements can be madewithout perturbing the system under investigation [15]. Us-

ing a flow cytometric assay for endocytosis, we studied inter-

nalization of basally expressed surface f32 integnins after cx-

posure of human neutrophils to several stimuli, finding

evidence for rapid specific endocytosis of CR3 and for reex-

terionization of a certain amount of the endocytosed fluores-

cent probe.

MATERIALS AND METHODS

Phosphate-buffered saline (PBS), pH 7.4, was made from

solid ingredients and low-endotoxin deionized water accord-

ing to the standard formulation, then autoelaved. Dulbeeco-

Vogt’s PBS (DPBS) was PBS supplemented with 0.9 mM

Ca2� and 0.5 mM Mg2�. Acid buffer was PBS adjusted to pH

2.5 with HC1. Formylmethionyl-leucyl-phenylalanine

(fMLP), platelet-activating factor (PAF, i-O-alkyl-2-aeetyl-sn-glyeeryl-3-phosphorylehohine), and phorbol mynistate ace-

tate (PMA) (Sigma) were dissolved in anhydrous dimethyl

sulfoxide (DMSO) at 102, 10�, and 102 M, respectively,

and stored in aliquots at - 70#{176}C until use. Individual ali-

quots were used for 1 day’s experiment only, then discarded.

Isolation of neutrophils

Heparinized human venous blood was collected from appar-

ently healthy volunteers. Neutrophils were isolated by a

modification ofthe method ofHjorth et al. [16]. Whole blood

was sedimented for 40 mm at room temperature with 2%

dextran 70 (Macrodex, Pharmacia, Piseataway, NJ). Isotonic

Pereoll (Pharmacia) was diluted with pynogen-free sterile iso-

tonic saline. Discontinuous Pereoll gradients were prepared

in 16 x 100 mm polystyrene tubes by layering 2.5 ml of 55%

Peneoll onto 2.5 ml of 74% Pereoll. Leukocyte-nich superna-

tant was layered on top of the gradients, which were cen-trifuged for 20 mm at 400g at 20#{176}C.Neutrophils aecumu-

lated at the 55%/74% Pereoll interface and were removed

with a plastic transfer pipette. Cells were washed with PBS

to remove residual Pereoll and were stored at room tempera-

ture. Preparations contained >90% neutrophils with

>95% viability, assessed by exclusion of propidium iodide.

Monoclonal antibodies

1B4 mAb (IgG2a), directed against human CD18 [17] was

extracted from hybnidoma tissue culture supennatant using

protein A and protein G columns (Pharmacia). LM2/i

(IgG2a), TS1/22 (IgG1), N418 (IgG1), and W6/32 (IgG2a) were

purified from tissue culture supernatants using protein G.

Fluorescence labeling

Fluorescein isothiocyanate (FI1�C) labeling of mAbs was per-

formed by a modification of the technique of Forni and DcPetnis [17a]. The mAb buffer was exchanged for 0.05 M ear-

bonate buffer, pH 9.6, on a Sephadex G25 column. FITCisomer I (Molecular Probes, Eugene, OR) was dissolved at

a concentration of 1.5 mg/ml in anhydrous DMSO (silyla-

tion grade; Pierce, Roekford, IL) and was added to the mAb

in the proportions of 0.1 ml FITh-DMSO per 1 ml mAb at3 mg/ml in carbonate buffer. The mixture was incubated in

the dark at room temperature for 1.5 h with periodic agita-

tion. Free FIIC was removed and the pH corrected by cx-

tensive dialysis against PBS at 4#{176}Cin the dark.

R-Phyeoerythnin (PE) labeling of 1B4 was performed as

follows (modified from refs. 18 and 19). One milligram of R-

PE, 4 mg/ml in 60% ammonium sulfate (Molecular Probes),

was centrifuged at 8500g for 10 mm. The supernatant wasdiscarded, and the pellet was reconstituted with 0.25 ml of

PBS and then dialyzed twice against PBS to remove residual

ammonium sulfate. R-PE was converted to a pynidylsulfide

derivative: 16 �Ll of 3-(2-pyridyldithio)propionie acid N-hydroxy-

suceinimide ester (SPDP, Sigma), 1.3 mg/ml in methanol,

was added to 0.7 ml of R-PE, 1.4 mg/ml in PBS, and allowedto react at room temperature for 2.5 h; then 30 �tl of dithiothreitol

(77 mg/ml in PBS) was added and the reaction was allowed

to proceed for a further 30 mm. MAb IB4 was activated with

suecinimidyl trans-4-(N-maleimidylmethyl)cyclohexane-1-

earboxylate (SMCC, Molecular Probes): 20 jzl ofSMCC, 1.7

mg/ml in DMSO, was added to 0.5 ml of 1B4 (3.0 mg/ml inPBS) and allowed to react at room temperature for 1 h. This

reaction was timed to finish coincident with the end of the

PE modification. Modified PE and activated mAb were

separated from the reaction mixtures on Sephadex GlO

columns (volume increased to 2 ml each), admixed, and in-

cubated for 18 h at 4#{176}Cwith constant end-over-end mixing.Further reaction was then stopped by adding 80 �il ofO.i mM

N-ethylmaleimide. After concentration to 0.5 ml on Centri-

eon mieroconeentrators (Amicon), conjugated antibody was

separated from free PE and uneonjugated antibody by gel

filtration on a 117 x 1 cm Sephacryl 5-300 column. Con-

jugated mAb eluted first, followed by free PE and then un-conjugated antibody. Aliquots of 1 ml were collected and the

process was monitored by measuring optical density (OD) at

280 and 565 nm. Fractions were tested by flow cytometry for

binding to PMNs and the fractions showing the most ac-

tivity, corresponding to the first 565-nm peak, were pooled

and concentrated using Centriprep and Centricon coneen-

trators (Amicon).

Preparation of Fab fragments

Fab fragments were prepared from FIIC-labeled IB4 mAb

using a commercial kit (Pierce Immunopure): antibody was

digested with immobilized papain in a cysteine-containing

buffer for 5 h at 37#{176}Cwith shaking. Fab fragments were

separated from Fe fragments and undigested IgG on a pro-

tein A column. Aggregates and particles were removed by

centrifuging at i0,000g followed by filtration through a

0.22-jim filter. Purity ofthe Fab fragments was verified usingSDS-PAGE; Fe or IgG contamination was not detected by

this method.

Flow cytometry

Flow cytometnie measurements were made using a Becton-

Dickinson FAC Scan cytometer (Becton-Dickinson, Moun-

tam View, CA) equipped with an argon laser emitting 15 mWlight at 488 nm. Data were collected and stored in list mode

pending off-line analysis. The linear fluorescence amplifier

gain at constant photomultiplier voltage was adjusted as

necessary for each sample to position the signal on scale.

Mean fluorescence channel numbers were calculated off line

and normalized to a gain of 1.0; the corrected mean channel

number (CMCN) was computed from the formula

CMCN =

observed mean channel number

amplifier gain

0 300 600 900

464 Journal of Leukocyte Biology Volume 53, April 1993

TABLE I . Intracellular Compartments Are Protected for an ExtenPeriod from Quenching by Extracellular Acid’

ded

Time (mm)

Fluorescence remaining (%)

FITC PE

0

I

10

20

100

99

86

72

100

100

96

92

‘PMNs were labeled with saturating concentrations (2.5 �g/ml) of fluores-

cein (FITC)- or phycoerythrin (PE)-labeled intact mAb 1B4 or its Fab frag-

ments (5 �g/ml) for 30 mm at 0#{176}C.Cells were washed, equilibrated to 37#{176}C

for 5 mm, and then stimulated with l06 M formyl peptide, fMLP. After

3 mm an aliquot was added to buffer at pH 2.5 to quench and remove cx-

tracellular fluorescence. Tubes were held on ice and samples were removed

immediately and at 1 , 10, and 20 mm thereafter and read on the flow cytometer

to investigate quenching of internal FITC probe by the extracellular acid.

Internalized fluorescent antibody could be measured as cell-associated fluores-cence. Figures are expressed as percentage fluorescence remaining, relative

to 100% at time 0.

Endocytosis of CD18

Endocytosis of surface-bound IB4 was measured using an

acid stripping technique. Before flow cytometnie analysis, la-

beled PMNs were subjected to a low-pH environment that

removed antibody bound to the cell surface. PMNs were la-

beled at 0#{176}Cwith a saturating concentration of mAb (2.5

�g/ml for IB4, 5 �g/ml for its Fab fragments), washed,

resuspended at 3 x 106/ml in DPBS, and equilibrated to

37#{176}C for 5 mm. Two aliquots of0.05 ml were removed to es-

tablish baseline values; one (“unquenched”) was added to

0.25 ml of ice-cold PBS in order to assess total cell-bound

fluorescence, and the other (“quenched”) was mixed with

0.25 ml of ice-cold acid buffer. The acidic environment

rapidly removed and/or quenched all extracellular fluores-

cenee, but intracellular compartments were protected for an

extended time when the samples were kept on ice (Table 1).

Residual cell-associated fluorescence could therefore be at-tnibuted to internalized antibody. Tubes were immediatelyread on the flow cytometer as the experiment progressed.

Stimulus was added at time 0 and the tube was rapidly

mixed. Internalization kinetics were investigated by remov-

ing 0.05-ml samples into 0.25-mi aliquots of ice-cold acid

buffer at 30, 60, 90, 120, 180, 240, 300, 360, 420, 600, and

900 s. Unless otherwise noted, stimulus was 5 x 10� M

fMLP. Internalized fluorescence was expressed as a peneen-

tage of unquenched cellular fluorescence at time 0.

Up-regulation of CD18

Up-regulation of CD18 following PMN stimulation was

measured using 1B4 as a probe. FITC-1B4-labeled washedPMNs at 3 x 106/ml in DPBS were equilibrated to 37#{176}Cfor

5 mm and then stimulated with 5 x 10� M fMLP. Samples

of 0.05 ml were removed at various times and added to

0.45-mi aliquots of ice-cold DPBS containing a saturating

concentration of FI’TC-1B4 (2.5 �g/m1). The rapid reduction

in temperature to 0#{176}Cprevented further cellular response.Samples were analyzed by flow cytometry after 30 mm of in-

cubation on ice to ensure consistent antibody binding. Inves-

tigation of the binding kinetics of IB4 at this concentration

showed that binding was 99% complete within 5 mm (data

not shown).

Neutrophil aggregation

Homotypic neutrophil aggregation was measured as previ-

ously reported [ii] using a neal-time flow cytometnic assay.

RESULTS

Endocytosis of CD18 after stimulation

In order to investigate the fate of surface CD18 following

PMN activation, we studied internalization of FITC-IB4

bound to CD18. PMNs were labeled with a saturating eon-

centration of FITC-IB4 or its Fab fragments, equilibrated to

37#{176}C,and stimulated with 5 x 10� M fMLP. Endocytosis of

CD18 was quantitated on the flow cytometer as nonqueneha-

ble cellular fluorescence. Internal fluorescence was expressed

as a percentage of total cell surface fluorescence measuredwithout quenching at time 0. In this way it was possible to

estimate the proportion internalized of the CD18 initially

present on the cell surface.

Endocytosis of surface-bound fluoresceinated intact anti-

body or Fab fragments by IMLP-stimulated PMNs was ob-

served. The fact that Fab fragments were endocytosed cx-

eludes the possibility that the phenomenon was due to

receptor cross-linking or interactions with Fe receptors. Ki-

neties ofthe phenomenon revealed two phases (Fig. 1). After

a lag, cells rapidly acquired nonquenehable (internal)

�0a)N

a)

C

a)C

a)

CD

C

Q�1o

Time after stimulation (sec)Fig. 1. Internalization ofCDl8 basally expressed before stimulation. PMNs

were labeled with saturating concentrations of fluorescein-labeled intact

mAb 1B4 (2.5 �tg/ml) or its Fab fragments (5 �g/ml) for 30 mm at 0#{176}C.Cellswere washed, equilibrated to 37#{176}Cfor 5 mm, and then stimulated with 106

M formyl peptide, fMLP. Samples were removed at intervals, added to

buffer at pH 2.5 tO quench and remove extracellular fluorescence, and thenimmediately read on the flow cytometer. Internalized fluorescent antibody

could be measured as cell-associated fluorescence. Results are expressed as

percentage internalized relative to unquenched antibody binding at time 0.

Filled symbols, cells stimulated with fMLP. (#{149})Intact 1B4; (#{149})Fab frag-ments. Open symbols, results obtained when the cells were preblocked with

excess unlabeled antibody. (0) Intact 1B4; (0) Fab fragments. The lack of

cell-associated fluorescence in the latter samples excludes pinocytosis as a

mechanism for internalization. Data represent the mean of two experi-

ments, repeated eight times with similar results. Unstimulated cells

produced results similar to those for preblocked stimulated cells (not shown).

Inset: FITC- and PE-labeled probes return similar results. PMNs were la-beled with saturating concentrations of fluorescein- or phycoerythrin-

labeled intact 1B4 mAb and then washed. Cells were equilibrated to 37#{176}C

for 5 mm and then stimulated with 106 M formyl peptide, fMLP. Internal-

ized antibody was measured by quenching/stripping extracellular fluores-

cence at low pH. (0) Cells labeled with FITh-IB4; (A) cells labeled with PE-

1B4. Data are means of two separate experiments, each run in triplicate.

60

�0a)N

CD

C1.�

a)4�JC

a)0)CD

Ca)C-)

a)0�

�0a)

250�

200a)0)CD

Ca)

I 3U C-)5.-

a)

0

30

0 300 600 900

Time after stimulation (sec)

Specificity of response

To exclude the possibility that the observed endocytosis was

300

Chambers et aL Endocytosis of neutrophil /32 integrins 465

fluorescence at 21%/mm for intact antibody probe or

14%/mm for Fab probe. Internalization peaked at around 3mm (36% for intact antibody or 26% for Fab); thereafter,

nonquenchable fluorescence was lost with half-times of 2 and

2.5 mm for intact IgG and Fab probe, respectively. At 15 mm

internal fluorescence remained constant, representing en-

docytosis of 20% of the initial surface CD18. The Fab frag-

ments were subjected to SDS-PAGE analysis, which did not

reveal any heavy chain contamination. In addition, flow

cytometrie analysis of binding returned a kd of approxi-

mately 0.5 nM for intact antibody and 5.7 nM for Fab frag-

ments. Studies of dissociation of labeled antibody from

PMNs were also conducted. Antibody rebinding was

prevented by 10-fold dilution of the sample with 100-fold cx-

cess of unlabeled antibody; these studies indicated that half-

times for dissociation were 40 mm for Fab fragments and 76

mm for intact IB4 (k0ff = 2.9 x 10� and 1.5 x i0� s1,

respectively). Taken together, these data strongly support the

monovalence of the Fab preparation.

In order to exclude the possibility that the observed inter-

nalization was due to pinocytosis, the experiment was

repeated with blocked cells in the presence of labeled anti-

body. If pinoeytosis of the fluorescent extracellular medium

took place, the cells would be expected to acquire internal

fluorescence. PMNs were incubated for 30 mm with 100-fold

excess unlabeled IB4; then FITC-IB4 (2.5 �tg/ml) or its Fab

fragments (5 �tg/ml) was added and incubation proceeded for

a further 30 mm. Wash steps were omitted, ensuring that theassay was performed in the presence of excess labeled and

unlabeled antibody. The observation that the cells did not ac-

#{149}0a)N

CDCL..

C

0)0)CD

4-I

C0)05.-

0)

Fig. 2. Preferential internalization of CDI8 over nonintegrin surface anti-

gens. PMNs were labeled with saturating concentrations of fluorescein-

labeled intact antibodies IB4 (anti-fl2), LM2/l (anti-aM), TS1/22 (anti-aL),

W6/32 (anti-HLA), or N4l8 (nonbinding) for 30 mm at 0#{176}Cand then

washed. Cells were equilibrated to 37#{176}Cfor 5 mm and then stimulated with

lO6 M formyl peptide, fMLP. Internalized antibody was measured by

quenching/stripping extracellular fluorescence at low pH. Symbols

represent cells labeled with (#{149})1B4, (0) LM2/l, (�) TS1/22, (A) W6/32,

or (0) N418. It is apparent that there is preferential internalization of IB4over W6/32 and N4l8, arguing against 1B4 endocytosis being due to simple

membrane turnover. In addition, it is apparent that all of the

�2 integrin endocytosed is associated with aM (IB4 and LM2/l lines) be-cause there is little or no endocytosis of a1 (TS1/22 line). Data shown from

one representative experiment, repeated three times with similar results.

�100300 600 900

Time after stimulation (sec)Fig. 3. Comparison of kinetics of CD18 internalization and up-regulation.

PMNs were labeled with a saturating concentration of fluorescein-labeledmAb 1B4, washed and equilibrated to 37#{176}Cfor 5 mm, and then stimulated

with 5 x l0� M formyl peptide, IMLP. Internalized antibody was measured

by quenching/stripping extracellular fluorescence at low pH. CDI8 up-

regulation was measured by removing aliquots of the reaction mixture and

mixing with ice-cold buffer containing FI1t-IB4 in order to produce a

saturating concentration (2.5 �sg/ml). These samples were analyzed on the

flow cytometer after 30 minutes to ensure antibody binding. (#{149}):Internali-zation. (0): upregulation. Data from one representative experiment,

repeated twice with similar results.

quire internal fluorescence (Fig. 1) argues against pinocytosis

as a mechanism for the internalization of surface-bound an-

tibody.

Validation of the use of fluoresceinated probe

The endocytosis assay used here functions by acid stripping

of extracellular fluorescent probe. Fluoresecin-labeled probes

are, however, very sensitive to pH, and we were concerned

about the possible effects of intracellular pH changes result-ing from cellular metabolism or leakage into the cell of the

extracellular acid used in the final step of the assay. We there-

fore performed internalization experiments using both

FITC- and PE-labeled 1B4. PE was used as a control because

it does not exhibit variations in fluorescence emission as the

pH of the environment changes. Cells at peak endocytosis (3

mm after stimulation) were added to ice-cold acid buffer and

aliquots were analyzed at intervals in order to study possible

acid leakage into the cell. As time progressed, a small

amount of fluorescence was lost from FITC- and PE-probed

cells (Table 1) but the rate ofloss was very low (1.4%/mm for

FITC probe, 0.4%/mm for PE probe), indicating that the in-tracellular environment was well protected from extracellu-

lar acid for a prolonged period. In all other experimentssamples were always read within 1 mm of acidification;

therefore acid leakage was not a serious problem.

Simultaneous kinetic experiments were performed using

FITC- and PE-labeled probe. The inset in Figure 1 showsthat there was little difference between the two probes,

confirming that under these conditions intracellular pH

changes resulting from cellular activation did not adversely

affect the fluonescein probe’s performance.

50

a)N

a)

C

a)C

a)

a)4-.Ca)0

a)0�

Cl)

a)

0.4 �Co

0

x0.3 �

a)aCl)a)

4-.

a)C)a)

C)C)

0.2

0.1

0 60 120 180 240 300 360

Time after stimulation (sec) The kinetics of CD18 endocytosis were found to be dramati-

eally influenced by variations in stimulus concentration.

Figure 5 shows curves obtained when the stimulating fMLP

concentration was varied between 10� and 106 M. At the

lowest concentration tested (109 M), internalization of CD18

was virtually indistinguishable from that of unstimulated

cells. With 106 and i0� M fMLP, a biphasic curve resulted,

as described above. With the intermediate concentration of108 M, however, the process was much slower and the loss

of internal fluorescence did not occur. The higher coneentra-

tions of stimulus produced by 15 mm an apparently stable

internalized CD18 level of about 15%.

PMN response to different stimuli

PMNs were stimulated with fMLP, PAF, or PMA (Figure

6). PAF- and fMLP-dniven kinetics were similar, but PMA

resulted in a slower, more prolonged response with a

pronounced lag phase. Phorbol dibutyrate and the calcium

40

35

30

25

�0a)N

CDC5.-

a)4-IC

20

0)CD

Ca)05.-a)

10

Comparison of endocytosis and up-regulation kinetics

The kinetics of endocytosis and up-regulation of CD18 werecompared (Fig. 3). Up-regulation after stimulation with

5 x 10�� M fMLP followed a simple exponential curve and

the cells were observed to increase surface expression of

CD18 almost threefold. Half-maximal response was 152 s

0 300 600 900

Time after stimulation (sec)Fig. 5. Internalization of CDI8 by human neutrophils after stimulation by

different concentrations of fMLP. Washed PMNs labeled with a saturating

concentration of fluorescein-labeled mAb IB4 were equilibrated to 37#{176}Cfor

5 mm and then stimulated with � l0�, 107, or l06 M formyl peptide,

fMLP, or PBS as control. Internalized antibody was measured by quenching

extracellular fluorescence at low pH. (0) PBS control; (0) l0� M fMLP;(i�5) 108 M fMLP; (#{149})10� M fMLP; (U) 106 M fMLP. Data are means

of five separate experiments induding all concentrations, each run in

triplicate.

466 Journal of Leukocyte Biology Volume 53, April 1993

Fig. 4. Comparison of kinetics of CD18 internalization and homotypic

PMN aggregation. PMNs were labeled with a saturating concentration of

fluorescein-labeled mAb 1B4, washed and equilibrated to 37#{176}Cfor 5 mm,

and then stimulated with 5 x 10� M formyl peptide, fMLP. Internalized

antibody was measured by quenching/stripping extracellular fluorescence at

low pH. Homotypic PMN aggregation was measured after fMLP stimula-

tion by a real-time flow cytometric assay. (#{149})Internalization; (0) aggrega-

tion. Aggregation is expressed as total aggregates (doublets + triplets +

quartets and higher) per 5 x 106 cells.

due simply to gross nonspecific membrane turnover after

stimulation, a control monoclonal antibody was used. W6/32

I20] recognizes monomorphic determinants in major

histocompatibihity complex (MHC) class I molecules. The

FITC-iabcied antibody bound to PMNs in a specific and

saturable manner (not shown). When the internalization cx-

pcniment was repeated with this antibody, much smallerproportions became internalized (Fig. 2), suggesting

preferential and specific internalization of CD18. If the ob-

served endocytosis had been due to nonspecific membrane

turnover, for example, invagination of large areas of mem-

branc, similar proportions of both antibodies should havebeen internalized, assuming a reasonably homogeneous dis-

tnibution of receptors in the unstimulated cell.

In order to investigate the relative contributions of the in-

dividual a subunits associated with CD18, we used three ad-

ditional mAbs. LM2/1 [21] is directed against aM (CR3)and TS1/22 [22] against aL (LFA-1); N418 (hamster an-

timouse ax [23]) did not show any specific binding to hu-

man PMNs and was included as a nonbinding control. Data

shown in Figure 2 indicate that essentially all ofthe internali-

zation of CD18 (probed with IB4) was attributable to inter-

nalization of CR3 (probed with LM2/1). Neither the anti-

aL probe nor the nonbinding probe showed any significantendocytosis.

Using these fluorescent mAbs, we estimated that the un-

stimulated preparation of PMNs shown in Figure 2 cx-

pressed about 115,000 CD18 per cell, 90,000 CR3, 36,000

LFA-1, and 105,000 MHC class I. Upon stimulation, thecells up-regulated CD18 and CR3 about fourfold, but there

was no change in the expression of aL or MHC class I.

0.5 and coincided with peak internalization. The initial burst of

endocytosis proceeded more rapidly and reached half-

maximum by 73 5.

Comparison of endocytosis and aggregation kinetics

The kinetics of fMLP-stimulatcd endocytosis and homotypic

aggregation are compared in Figure 4. It is apparent thatmost of the internalization takes place during the plateau

phase of the aggregatory response, between 50 and 150 s af-ter stimulation. This is consistent with the possibility that

endocytic removal of activated adhesive receptors contrib-

utes to progressive destabilization of aggregates until, after

150 s, insufficient bonds remain between the cells and theyrapidly disaggregate.

0.0 Effect of different stimulus concentrations

I I #{149}FITC-lB4

80 � U PE-IB4

60�\

40 -

20 -

60

50

40

30

20

#{149}0a)N

CDC5.-

a)4-IC

a)a)CD

4-’

Ca)05-.

a)3-

0 300 600 900

Time after stimulation (sec)

1 00�

Chambers et aL Endocytosis of neutrophil �2 integrins 467

ionophore A23187 produced kinetics similar to those ob-

tamed with PMA (not shown).

Receptor recycling

The known ability of PMN to up-regulate surface /32 inte-

grins leads to the possibility that some of the endoeytosed

receptors might be recycled to the surface. The shape of the

observed endocytosis curve (Fig. 1) describes a rapid rise to

a peak followed by an exponential reduction in internal

fluorescence. In order to further investigate the second

phase, PMNs were labeled with FITh-IB4 or PE-IB4 and

stimulated with 10� M fMLP at 37#{176}C.After 3 mm, external

surface fluorescence was removed by adding the cell suspen-

sion to icc-cold acid buffer, incubating for 15 s, and thenwashing twice with a large excess of ice-cold DPBS. If the

cells were subsequently held on ice, their fluorescence re-

mained nonqucnehable, indicating only internal probe. The

cells were warmed to 37#{176}Cwithout further stimulation and

aliquots were removed at intervals and analyzed with or

without acid quenching. Figure 7 shows that fluorescence

became progressively more susceptible to quenching, in-

dicating that internal probe was transported to the cell surface.

DISCUSSION

Flow cytometnie analysis has been used to investigate the

characteristics of endocytosis of f32 integrins (CDI8 adhesiveglycoprotcins) in activated human neutrophils. There is one

report to date of endocytosis of f32 intcgrins unrelated to

phagocytosis [24]; however, this describes a slow process of

small magnitude in cultured cell lines rather than in human

blood leukocytes. There are reports of endocytosis of inte-

grins ofelass I3� [24-26] but these also were not in leukoeytcs.

The 132 integrin family (LFA-1: a�j32; Mae-1/Mol/CR3:

aM$2; and p150,95: crx/32) is of paramount importance for

Fig. 6. Internalization of CD18 after stimulation by a variety of stimuli.

Washed PMNs labeled with a saturating concentration of fluorescein labeled

mAb IB4 were equilibrated to 37#{176}Cfor 5 mm and then stimulated with

fMLP, PAF (l0� M), or PMA (l0� M). Internalized antibody was meas-

ured by quenching extracellular fluorescence at low pH. (#{149})fMLP; (0)

PAF; (0) PMA. Data are means of three separate experiments including all

stimuli, each run in triplicate.

CDC5.-

a)4-IC

0)

C

C

CD

Ea)5.-

4�I

Ca)05.-

a)3-

00 300 600 900

Time after stimulation (sec)Fig. 7. Recyding of internalized fluorescent probe. Washed PMNs labeled

with a saturating concentration of fluorescein or phycoerythrin labeled mAb

1B4 were equilibrated to 37#{176}Cfor 5 mm and then stimulated with 5 x 10�

M fMLP. At 3 mm the cells were added to ice-cold acid buffer such that the

final pH was 2.5 and were incubated for 15 s. A large excess of ice-cold PBS

was added and the cells were washed twice. Cells were rewarmed to 37#{176}C

and internal fluorescence was measured at intervals by subjecting aliquotsto low pH and immediately analyzing on the flow cytometer. Results showthat some of the fluorescent probe endocytosed after stimulation is reex-

pressed on the cell surface. (#{149})FITC-IB4; (U) PE-IB4. Data are means of

two separate experiments, each run in duplicate.

adhesive responses in all lcukocytes (reviewed in ref. 27).

Neutrophils exhibit rapid responses oflarge magnitude when

stimulated, including homotypie aggregation and transloca-

tion to the surface of preformed receptors (“up-regulation”),

particularly aMfl2 (CD11b/CD18). It was initially thoughtthat up-regulation was the primary mechanism for increased

neutrophil adhesiveness. This notion has been challenged

[6-8] and it now seems that CD18-mediated neutrophil

adhesiveness results from activation of molecules already

present on the surface. The fate of PMN surface CD18 after

cellular stimulation is not well understood. Data presented in

this paper show that in addition to being translocated to the

PMN surface after stimulation, CD18 expressed on the sun-

face before stimulation can be rapidly endocytosed. Specific

endocytosis of CD18 after formyl peptide stimulation was

sustained at a relatively high rate for almost 3 mm followed

by recycling to the surface of 50% of fluorescent probe mi-tially endocytosed.

The endocytosis assay used here is similar to that

described by Finney and Sklar [28] because it exploits in part

the pH sensitivity of fluorescein emission. Acid quenches the

fluorescence of probe on the surface of the cell; consequently,

this is no longer detected. Probe residing inside the cell is

protected for an extended time from extracellular acid

quenching and can therefore be measured by flow cytometry.

Phycocrythnin, unlike fluorescein, does not exhibit variations

in fluorescence efficiency as pH is changed. Results obtained

with PE-labeled probe indicate that the assay used in this

study functions primarily by acid stripping: antibody bound

to the cell surface dissociates rapidly upon exposure to low

pH; therefore cell-associated fluorescence detected by the

flow cytometer is due only to internalized antibody, pro-

teeted from the acid.

468 Journal of Leukocyte Biology Volume 53, April 1993

Use of fluorescein-labeled probe in this assay is convenientand appropriate; FI1C conjugation is technically simple,

conjugated antibody is easy to prepare, and this method of

labeling does not result in a significant increase in probe

molecular mass. The assay works well both in theory and in

practice because residual surface antibody not immediately

stripped off by exposure to acid will certainly be quenched;therefore only internal probe can generate a fluorescence sig-

nal. Although pH sensitivity of probe fluorescence on the

one hand reinforces the assay, it might also represent a

significant weakness if the probe becomes associated with

one of the neutrophil compartments known to change pH af-

ten stimulation [29]. Under these circumstances an errone-

ous signal could be generated. To investigate this possibility,

we performed simultaneous internalization experiments us-

ing FITC- and PE-labeled 1B4. Little difference was found

between the two fluorescent probes (Fig. 1, inset), validating

use of a fluorescein-labeled probe and suggesting that either

the probe does not become associated with a rapidly acidifiedcompartment or the compartment undergoes only very small

on slow pH changes.

Endocytosis of Fab fragments bound to CD18 on the PMN

surface proceeded at an initial rate of 14%/mm. Intact IgG

probe was initially internalized at 21%/mm. The slightly

higher rate observed for whole antibody may indicatepreferential internalization of cross-linked receptors or

receptors cross-linked to Fe receptors by the intact Ig

molecule. Linkage of receptors to Fe receptors resulting in

cellular activation or modulation of function has been

described [30, 31] and this phenomenon could explain the

results reported here. Reexpression of fluorescent probe onthe cell surface took place with a half-time of approximately

2 mm and was similar whether the probe was intact IgG or

Fab fragments.

When compared with endocytosis of formyl peptide recep-

ton, endocytosis of CD18 has considerably different eharac-

tenistics. At 37#{176}C,internalization of formyl peptide receptor

proceeds rapidly, with a half-time of approximately 3 mm,

and continues until most of the surface receptor has been in-

ternalized [32]. Most PMN batches studied here internal-

ized a maximum of 40% of CD18 initially present on the

PMN surface under similar conditions. Furthermore, ap-

proximately half of the internalized fluorescent probe was

subsequently reexpressed on the cell surface. These observa-

tions further support the idea that PMN CD18 exists in a

heterogeneous state. It has been noted that CD18-mediated

adhesion does not necessarily correlate with quantitative cell

surface expression [6-8J and aggregated PMNs are able to

disaggregate despite significantly up-regulated CD18 [11],

suggesting that CD18 exists in an actively adhesive and anonadhesive state. Other workers have studied phosphoryla-

tion of the $2 chain and have found both constitutive and

activation-dependent phosphorylation states [33, 34].

Although PMA-dniven phosphorylation correlated with

aggregation, LMLP-dniven phosphorylation was either un-

detectable [33] on correlated with aggregation but was anorder of magnitude weaker [34, 35], leading Merrill et al. to

suggest two independent phosphorylation pathways. CD18

has also been reported to be present in the membrane in a

mobile and an immobile form [36]. The current study adds

to the growing list of CD18 heterogeneity; we observe a mini-

mum of two populations of receptor: (1) basally expressed

and endocytosed after stimulation and (2) basally expressed

but not endocytosed. Exactly what happens to the endocy-

tosed receptors remains to be determined. The observation

that about half of the internalized fluorescent probe reap-

pears on the cell surface suggests that some receptors may

recycle to the surface (in which ease these represent a third

population); however, it is not possible to ascertain from the

current data whether the probe remains bound to receptor

after endocytosis.The functional significance of adhesive receptor cycling

may be understood in the light of the kinetics of homotypie

neutrophil aggregation. We estimated an average lifetime of

approximately 60 s for CD18 adhesive bonds [11], which sug-gests a turnover of adhesive sites within the cell-cell contact

region during the plateau phase of aggregation. When en-

doeytosis and aggregation kinetics are compared (Fig. 4) it

appears that most of the endocytosis takes place during this

plateau phase; therefore it is possible that endoeytosis of ae-

lively adhesive CD18 may contribute to disaggregation ofPMN aggregates. It is also possible that this endocytosis

represents functional inactivation of unused activated adhe-

sive molecules.

Neutrophils express primarily CR3 (aM/32) and LFA-1

(a�J32) [4], and it is pertinent to consider what relative eon-

tnibutions these molecules make to the observed CD18 inter-

nalization. When we conducted experiments using fluores-

cently labeled anti-aM and anti-aL mAbs (LM2/1 andTS1/22 respectively, Fig. 2), we observed that all of the CD18

endocytosed was associated with CR3. Neutrophil LFA-1

was neither up-regulated (data not shown) nor endocytosed

(Fig. 2) in response to fMLP stimulation.

This study demonstrates that human neutrophils upon

stimulation rapidly internalize a large proportion of the

CD18 molecules initially present on their surface and defines

the kinetics of the phenomenon using flow cytometry. This

endocytosis may be important in the control of cellular adhe-

sive activity. Whether active or inactivated receptors become

internalized, by what mechanisms, and whether these recep-tors are recycled are important questions that remain to beanswered.

ACKNOWLEDGMENTS

J.D.C. holds a Research Fellowship from the American Heart

Association, California Affiliate, with funds contributed by

the Orange County Chapter. This work was supported in

part by NIH grants HL43026 and RR01315 and by the La

Jolla Institute for Experimental Medicine. The authors wish

to thank Drs. Uli von Andrian and Darey Wilson for their

helpful critique.

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