Flow cytometry analysis of the expression of neutrophil FMLP receptors

Journal o]'hnmunoh~gica/l~h'thod~. 14~)( 1~9~921159-It'~4

15,~

~i'~1992 ElsevierSciencePublishers13.V.All rights reservedI1tl22-175~/~2/$(15.1111

JIM(16261

Flow cytometry analysis of the expression of neutrophil FMLP receptors C.A. Allen, M.F. B r o o m and V.S. Chadwick Ih'partment of Experimenttd Methcme. Umt'er~it~,,of Otugo ~,h'dical School I)mtethn. Nclv Zeahmd

(Received 24 June It;It/I,revisedreceived311()ct()bcr Iqgl. ;iccepletl Ih I)ccembcr 1901)

The number of cell surfilcc FMLP receptors cxprcsscd by peripheral blot~d ncutrophils taken from healthy individuals was quantitated using a direct fluorescence label analysed on the FACScan, Receptor numbers, assayed on cells from hepariniscd blood preparcd at 4°C or 37°C or in the presence of LPS (311 n g / m l ) were 12,5411 + SD 2413, 18,588 + 4938 and 45,041 + 11L744 respectively. Anticoagulation with EGTA resulted in 25-30% greater basal receptor numbers but had no effect on receptor level after maximal stimulation with LPS. Double labelling with a ncutrophil-spccific mAb showed that the entire neutrophil population expressed FMLP receptors. Kt'y =wJrds: Flowcytometry: Ncutrophil; lq)rmyl-mcthionlnc-lcucinc-phcnyhda;.increceptor

Introduction The pro-inflammatory bactcrial peptidc formyl-methioninc-lcucinc-phcnylalaninc (FMLP) is a potent chemotactic factor for human ncutrophils. The binding of FMLP to its specific cell surface receplor induces a series of coordinated biochemical and cellular rcsponscs, i.c., altcration of ion fluxes, morphological polarization,

(,'orre~pemdence to: V.S. Chadwick, l)epartment of Experimental Medicine. Universityof Otago Medical Schot~l. P.O. Box 913, Dunedin, New Zealand. Ahhretiations: FMLP, formyl-methioninc-leucinc-phcnylalanine; FACScan,fluorescenceactivatedcell seamier:EGTA. ethylene glycol-bis(lJ-aminocthylcthcr)-N.N,N ',N '-tetraacetic acid; LPS, lipopolysaccharidc:FIT(', fluorcsceinisothiocyanate: fNLPNTL-FITC, fluoresccin conjugated formyl Nlcu-Leu-Phe-Nlcu-Tyr-Lys; DMSO, dimuthyl sulphoxidc: MFCN, mean fluorescence channel number; mAb. monoclonal antibody: PEgaMIg. phycocrythrin-labcllcdgc~at antimouse immun~globulin.

locomotion, secretion of [ysosomal contents, superoxide anion production - resulting in neutrophil activation (Gallin an0 =qeligmann, 1984; Painter et al., 1984; Foy and Simchowitz, 19891. This complex process cause'; the generation of agents that will destroy micro-organisms and, if triggcrcd inappropriately, can cause tissue injury (Wciss, 1989). This type of interaction between bacterial products and host cells has been implicated in the pathogenesis of di:,orders such as inflammatory bowel disease (An'3n et al., 19891 and scptic shock (Tennenbcrg and Solomkin, 1988) whcrc ncutrophils are known to play a prominent part in the inflammatory response. The reported values for FMLP receptor numbers pcr neutrophil in healthy individuals varies widely 1581111-24,11001 (Tcnncnberg and Solomkin, 1988; Tenncnbcrg et al., 1988; Anton ctal., 1989; Colts ct al., 19891. This variability may be duc to inadvertcnt ccll activation during cell preparation. The enumeration of cell surface neutrophil

I:MI,P receptors has been accompli,,.hcd by the use of tritiatcd FMLP lal~cls (Tcnnenberg and Solomkin, 1988: Tenncnbcrg ct al., 1988: Anton ctal., 19,',;9: ('olcs ct ill., 1989), although qualitative studies with fluorcsccinatcd ligands have bccn described (Van Epps ctal.. 19901. This papc, describes a quantitative method for the estimation of ncutrophil FMLP receptor numbers by FACScan analysis using a fluorcsccin-labcllcd (FITC) peptidc analoguc fNleuLcu-I:'hc-Nlcu-Tyr-Lys (fNLPNTL-FITC). The effects of different anticoagulants and thc effects of pre-incuhation with cndotoxin (lipopolysaccharidc, LPS). a possiblc contaminant in vitro (Haslctt ctal.. 19851 and an important inflammatory mediator in vivo (Bayston and Cohcn, 1990), wcrc cvaluatcd in order to study the dynamics of receptor uprcgulation.

Materials and methods

Cell preparation Hcparinizcd venous blood was drawn from normal volunteers. For comparative purposes, some blood samples were also anti-coagulated with EGTA. A ncutrophil-enrichcd fraction was dcrivcd from sedimentation of whole blood (one part) with RPMi 1640 mcdium (Gibco, onc part, to which was addcd I% foetal calf scrum, 1.25 mM EGTA and 1).115e/c sodium azide) and 6% dcxtran (Hcspan, Dupont onc part). Sedimentation was performed for 311 rain at 4°C. Alternativcly, for thc purposes of double labelling cxpcrimcnts neutrophils wcre prepared by ccntrifugation of whole blood for 30 rain at 400 x g at room temperature on a Ficoll d.'nsity gradient (MonoPoly Resolving Medium, Flow).

Endotoxm treatment of neutrophils Frcshly drawn anti-coagulated whole blood was incubated at 4°C or 37°C with or without 311 n g / m l lipopolysaccharidc (E. coil 055:B5, LPS, Sigma) for 1 h prior to cell separation.

Preparation of Jbnnyl Nle-Leu-Phe-Nle-Tyr-LysFITC conjugate Fluoroisothiocyanate (0.5 rag) and fNle-LeuPhc-NIc-Tyr-Lys 1211{) ~g) were dissolved in 1).5

ml dimcthyl sulphoxidc (DMSO) and adjustcd to pH 9.5 by addition of l.l} ml of Na2CO 3 0.2 M, pH 9.5. After overnight incubation at 37°C, 2110 ~1 aliquots of the reaction mixture were adjusted to pH 7.6 with 0.1 N HCI and fractionated by reverse phase chromatography using a Waters tt BONDAPAK CN column (buffer A milli-Q water, buffer B 100% acctonitrile, flow rate 11.5 m l / m i n , linear gradient over 30 mink Free FITC eluted in the void whcrcas the more hydrophobic coujugatc cluted mid-gradient. After multiple runs. the pooled conjugate peak was lyophilised and rcsuspended in phosphatc-buffercd saline pH 7.2. The concentration of conjugate was calculated assuming that the optical density at 495 nm for 1 m g / m l FITC={}.I75 U (from the molar cxtinction coefficicnt), and that FITC binds to fNLPNTL in a 1 : I ratio and adjusting for fluoresccnce quenching duc to coupling (sec below). Equimolar concentrations of fNLPNTL-FITC (from amino acid analysis) and frcc FITC were cxcitcd at 492 nm in a spcctrofluorimctcr (Pcrkin Elmer LS5I}, UK). Thc cmission spcctra (518 nm) indicated that the quenching of the fluorescent signal due to fNLPNTL-FITC coupling was 19.9%.

Re('eptor assay Following separation (see above), cells were washed in mcdium and kept at 4°C. Cells were finally rcsuspended at a concentration of 5 x 10"/ml in the above medium with the addition of 11)7~ autologous plasma. Differing amounts of FITC-labelled fNlcu-Leu-Phe-Nleu-Tyr-Lys (final concentration 0-18.7 x 10- '~ M) were added to aliquots containing 2.5 x 11)s granulocytcs. Nonspecific binding was dctermincd by the addition of excess unlabellcd FMLP (10 ~ M) in a parallel set of tubes. In all cases the total assay volume was 0.25 ml, i.e., a final cell concentration of 1 × 10~'/ml. After 30 rain incubation at 4°C thc cells were washed, rcsuspendcd in the same volumc and analysed immediately using the FACScan flow cytometer (Becton Dickinson). Calibration beads with 7.2 x 103-1.8 x 11~' FITC mclecules/particlc (Flow Cytometry Standards, USA) were run under the same conditions as the test samples. Live gatcs were set on the forward a n g l c / 9 0 ° scatter distribution to selectively anal-

161 yse n e u t r o p h i l s in the c c l l p r c p a r a t i o n and 2(1(1t) events were acquired. From a calibration graph of mean fluorescence channel number (MFCN)

7o. 6o

I

v e r s u s log. F I T C m o l e c u l e s / p a r t i c l e , the test M F C N could be related to receptor n u m b e r / c e l l . A f t e r a p p r o p r i a t e a d j u s t m e n t for non-specific binding, a final receptor n u m b e r / c e l l was obtained. T h i s figure was a d j u s t e d to allow for the q u e n c h i n g of the fluorescent signal d u e to the f N L P N T L - F I T C coupling.

Double labelling o f neutrophils with anti-neutrophil mAb29 and FNLPNTL-FITC Single cell s u s p e n s i o n s (50 ~1) c o n t a i n i n g 5 × 105 n e u t r o p h i l s p r e p a r e d over a Fieoll g r a d i e n t a n d > 95% pure, were i n c u b a t e d at 4°C for 31) m i n with 50 #1 m A b 29 ( a n t i - n e u t r o p h i l ( B e r n s t e i n a n d Self, 1986), C D 15 kindly provided by Dr. N. Hogg). A f t e r w a s h i n g t h e cells twice with m e d i u m , 5 p,I p h y c o e r y t h r i n - c o n j u g a t e d goat a n t i - m o u s e K light c h a i n s a n d s a t u r a t i n g a m o u n t s o f F N L P N T L - F I T C were a d d e d for a f u r t h e r 30 rain at 4°C. Finally the cells were w a s h e d a n d analysed with t h e F A C S c a n . T h e relevant controls, excess F M L P ; no m A b 29; no P E c o n j u g a t e or F N L P N T L - F I T C were also analysc:! :~, check t h e specificity o f binding.

INLPNTL.FITC(nM) Fig. 1. Saluration curvet; ()f the FMLP receptor,i on neutrophils derived from healthy individuals (n = 8, mean +SD)

.,,howing number of FMLP receptors per cell at various conccntrations of fNLPNTL-FITC (0-18.7 nM). Whole bitted was preincubated (at ~nt40(` • • , (b) at 370C • •. (c) at 37~(" with 3(I ng/ml LPS o - - - - o followed by cell preparation and assay at 4°C.

without LPS ( p = 0.(111115, up to a four-fold incrcase). T h i s uprcgulation can bc cxpresscd as a stimulation inde~ (Table I). T h e r e was a consistent iitcrcase in cell receptor n u m b e r on the surface of n e u t r o p h i l s p r e i n c u b a t e d at 37°C withTABLE I

Results

T h e cell surface F M L P r e c e p t o r s were satur a t e d by t h e fluorescent ligand at a c o n c e n t r a t i o n o f 8.3 n M (or 10 n g / m l ) in all instances (Fig. 1). T h e m e a n cell r e c e p t o r n u m b e r was 12,540 _+ SD 2413 ( n = 8) for cells p r e p a r e d at 4°C, 18,588 + S D 4938 for blood p r e i n c u b a t c d for I h at 37°C before cell s e p a r a t i o n at 4°C a n d 45,041 + SD 10,744 for blood p r e i n c u b a t e d for I h with 30 n g / m l LPS at 37°C prior to cell s e p a r a t i o n at 4°C (Table 11. T h e s t i m u l a t o r y in vitro effect of 30 n g / m t LPS on whole blood t a k e n from n o r m a l volunt e e , s was dramatic. T h e r e was no c h a n g e in the receptor n u m b e r o f n e u t r o p h i l s i n c u b a t e d with 311 n g / m l LPS at 4°C (data not shown). T h e r e was a m a r k e d increase in the n u m b e r of receptors exp r e s s e d by n e u t r o p h i l s e x p o s e d to LPS at 37°C as c o m p a r e d to those p r e p a r e d either at 4°C or 37°C

EFF[-(.'I" ()N FMI.P RECEPTC;R FXPRESSION OF I21FFERENT PREIN('UBATION CONDITIONS OF WIIOLE BLOOD PRIOR TO ('ELL PREPARATION AND ASSAY

AT ,INC. Preincubation conditions (I h) (at 40( . (b) 37°C (c) 37°C + LPS

Stimulat(on index (c)/(u)

Receptor number/ cell (n = S)

i r~.217

26,055

63.889

3.9

8.639

12.147 14.782 22.453 17,135 14,557 17.959 23,614 18.588 " 4,938 1,745

311268 44,757 53.758 46,336 34.207 39.376 47.7411 45.(141h 10.7,14 3,7q9

3.5 3.3 3.t~ 4.2 3.1 3.0 4.1

13.664 14.875 11.1142 11,11811 13.1t71 11.735

Mean SD SE

12.5411 2.413 853

" Sludcnt's "1' le:,( ,'?.~ 0.(1'02 versus 4°C. t~p = 0.1}01)5 ver~;ur, -1'(" and 37~C.

out LPS its c o m p a r e d to those p r c i n e u b a t e d at 40(. ` ( p = 0.(1()2). Neutrophils

stimuhttcd lron~

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hit numbers

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drawn showed marked differences in FMLP receptor number. The receptor number for cells anticoagulated with EGTA was consistently 253(1% higher than that obtained with heparin. Double labelling of ncutrophils prepared on a Ficoll density gradient (> 95% pure) with neutrophil-specific mAb 29 and FITC labelled FNLPNTL, illustrated that all antibody-labelled cells expressed receptors for the FMLP analogue

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FiI~. 2. FAl'Scan dot plot~, of neutrophih, live gated on fl+r~ard angle/L)() ° scatter after ( a ) no hdlel. ( b ) labelling with m A b 29 and ph],met~thrin conjugated goat anti-mouse lgG (PEgaMlg). to) double labelling v,'ith m A b 2tJ. P E g a M I g and f N L P N T L - F I T C , ( d ) double lahclhng "~ith m A b 29+ PEgaMlg, F M L P and f N L P N T L - F I T C . Fluorescence 1 = fluoreseein. Fluorescence 2 = ph~ cocr'~thrin.

TABLE It NUMBER OF FMLP RECEPTORS EXPRESSED P['R CELL AFTER ANTICOAGULATION WITII EITIIER HEPARIN OR EGTA FOLLOWED BY PREINCUBATION OF WIIOLE BLOOD (u) AT 4°C, (b) AT 37°(". (c) a,T 37°C WITH 30 ng/ml LPS. Preincubation wa~; followed by cell freparalion and a~,say at 4~C. no.

Anti-coagulation Preincubation conditions ( l h) conditions (a)4°C (b137~C (c) 370(_"with 3(1 ng/ml LPS

I

Heparin EGTA

13,(171 17,946 211.437 21.3115

39,376 37,64(1

2

tleparin EGTA

111,175 17,559 15,805 19.494

36,392 411,674

3

Heparin EGTA

11,742 15,705

37,828 33,327

16,848 18,708

and expressed these receptors to a similar extent, i,e., the population was h o m o g e n e o u s (Fig. 2).

prevented temperature dependent neutrophil activation (Fearon and Collins, 19831. Furthermore, preparcd cells must be maintained at 4°C, since it is known that the preparation of neutrophils at 4°C followed by a warming of cells to 37°C, inducts a profound increase in the surface expression of integrin molecules (Forsyth and Levinsky, 199(I) and F M L P receptors (Tcnncnbcrg and Solomkin, 1988}. The effects of different anticoagt;!ants (hcparin vcrsus E G T A ) were apparent only at 4°C with increased values in E G T A anticoagulatcd blood. The nature of the anticoagulant must therefore be specified when reporting values for basal F M L P receptor numbers. Neutrophils stimulated by LPS demonstrated a 3-5 times increase in receptor number over the 4°C baseline level. The stimulation indices wcrc calculated to give an idca of the range of responses to stimulation in normal subjects. A comparison between 'basal' and LPS stimulated reccptor numbers might be an appropriate way of asscssing this aspect of neutrophil function in disease states.

Discussion References We have described in this p a p e r a quick and simple m e t h o d for the estimation of cell surface F M L P receptors. Double-labelling experiments with pure cell preparations showed that ncutrophils obtained from a single d o n o r all expressed similar levels of F M L P receptors. Thus, a single p r e p a r a t o r y step involving dcxtran s(dimentation of the bulk of the erythrocytes is adequate for flow cytometry analysis since the FACScan will gatc out irrclcvant cells. An additional advantage is that the cell surface expression of F M L P receptors on monocytes can be assayed on the same mixed cell preparation. T e m p e r a t u r e can have marked and variablc effects on F M L P receptor expression ( I . l - 2 . 0 times upregulation when whole blood is kept at 37°C for l h before cell separation at 4°C). For this reason, immediate plunging of whole blood into ice to rapidly halt cell metabolism before any in vitro procedure, followed by cell p r c p a r a t i o n also at 4°C, was d e e m e d a more reliable method of assaying basal FMLP receptor number. This

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Gallin. J.I. and ,~cligmann. B.E. (1984) MohilizaHm~ ar, d adaptati=.~n of human neutrophil chemoattractant .~vletLeu-Phe receplors. Fed. Pro¢. 43, 2732. Ila~,lctl. C.. Gulhrie, A.. Kopaniak. M.M.. Johnston, R.B. and tlenson. P.M. (19,%~ Modulation of multiple ncutrophil funelions hy preparalivc meth.~ds or trace eonecntrati~)n'~ of haclerial lipapolysaccharidc. Am. J. Pathol. 119. 101. Painter. R.G.. Sklar. L.A.. Jesaitis. AJ.. Schmitt, M. and ('ochrane. ('.G. (19X4) Activation of neutrophils by N-form~.'l ehcmotactlc pcptides. Fed. Proc. 43, 2737. Tenncnherg. 5.1). and Solnmkin. J.S. (198H) Ncuirophil activalion in sepsis. Arch. ,~urg. 123. 1"71.

Tennenherg. S.D., Zemlan, F.P. and Solomkin. J.S. (1988) Characterisation of N-formyl-methionyl-lcucyl-phcnylalanine receptors on human neu!rophils. J. Immunol. 141, 3937. Van Epps, D.E., Simpson, S., Bender, J.G. and Chenoweth, D.E. (199[)) Regulation of C5a and formyl peptide receptor expression on human polymorphonuelear leukocytes. J. lmmunol. 144, 1062. Weiss, S.J. (1989) Tissue destruction by neutrophils. New EngL J. Med. 320, 365.