Effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils

JOURNAL OF

MWOGGICAL

ELSEVIER

Journal of Immunological Methods 186(1995) 217-224

Effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils Peter P. Youssef apb,d~+, Basil X. Mantzioris b, Peter J. Roberts-Thomson Michael J. Ahern ‘TV,Malcolm D. Smith c,d

a,b,

a Rheumatology Unit, Flinders Medical Centre, Flinders Drive, Bedford Park, Adelaide, South Australia 5042, Australia h Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, Adelaide, South Australia 5042, Australia ’ Department of Medicine, Flinders University, Bedford Park, Adelaide, South Australia 5042, Ausiralia ’ Rheumatology Unit, Repatriation General Hospital, Daws Road, Daw Park, SOL& Australia 5042, Australia Received 9 February 1995; revised 27 April

1995; accepted 22 May 1995

Abstract In vitro studies of ncutrophil adhesion generally utilise purified populations and are often pcrformcd at 37°C. This study determines the effects of temperature changes and neutrophil separation procedures on the expression of ccl1 adhesion molecules on neutrophils. We found that neutrophil separation procedures involving erythrocyte sedimentation and hypotonic lysis are associated with a significant increase in the expression of both a structural and functional epitope of the & integrin CDllb, an increase in the expression of sialyl Lewi? (CDlSs) and the hyaluronate receptor (CD44) as well as a significant decrease in L-selectin (CD62L) expression. Separated neutrophils are also more resistant than unseparated neutrophils to PMA induced upregulation of a functional epitope of CDllb. Incubating neutrophils at 37°C is associated with increases in the expression of structural and functional epitopes of CDllb. Neutrophil separation is also associated with increases in the expression of both structural and functional epitopes of CDllb which is greater when neutrophil separation is performed at room temperature compared with neutrophil separation at 0-4°C. However, this difference is lost when the latter are incubated at 37°C. Furthermore, neutrophil separation at both 0-4°C and room temperature is associated with a significant increase in CD& expression. This increase is less when separation is performed at room temperature. These findings suggest that neutrophil separation should be performed at room temperature unless the cells are going to be used at 0-4°C. Researchers using purified neutrophil populations need to be aware of these significant structural and functional changes when extrapolating in vitro results to in vivo situations.

Keywords:

Neutrophil;

* Corresponding Adelaide,

author.

South Australia

0022-1759/95/$09.50

SSDI 0022-

Leukocyte integrin; Selectin; Sialyl Lewi?;

I759(95)00

0

At:

Department

5042, Australia.

1995 Elsevier 146-8

of Clinical

Immunology

Tel.: (081-204-4722;

,

CD44

Flinders

Fax: (08)-204-5450.

Science B.V. All rights reserved

Medical

Centre,

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P.P. Youssef et nl./Jowm~l

218

of Immunologicnl Methods 186 (1995) 217-223

neutrophils (Fearon and Collins, 1983; Berger et al., 1984; Hed et al., 1988; Forsyth and Levinsky, 1990, Macey et al., 1992). As a result of this, a number of workers have recommended that neutrophil separation be performed at 0-4°C (Fearon and Collins, 1983; Hed et al., 19881. However, others have recommended that neutrophil preparation be performed at room temperature because cooling and then rewarming increases neutrophil adherence to human umbilical vein endothelial cells and makes them more resistant to FMLP induced CDllb upregulation suggesting that these cells are both activated and ‘exhausted’ (Forsyth and Levinsky, 1990). The effects of such procedures on other cell adhesion molecules have been less well studied. L-selectin is expressed at high levels on circulating blood neutrophils and is involved in neutrophil ‘rolling’, the initial step in neutrophil-endothelial interaction, during which the neutrophil samples the vascular endothelium (Kishimoto et al., 1989, Tedder et al., 1989; Von Andrian et al., 1991). It is rapidly shed in response to a number of neutrophil activating factors (Kishimoto et al., 19891, a process which needs to occur prior to neutrophil transmigration. Although the major ligands for the selectins

1. Introduction

Neutrophils are important effector cells in many inflammatory states. Their migration from the vasculature across the vascular endothelium and into tissues is essential for the normal host response to infections (Weiss, 1989). Molecules on the neutrophil surface which may be important for adhesion to the vascular endothelium and subsequent trafficking into inflamed tissues Linclude the p2 integrins CDlla-c/CD18, selectin (CD62L), sialyl LewisX (CD%) and the hyaluronate receptor (CD44) (Springer, 1990; Butcher, 1991; Haynes et al., 1991; Springer and La&y, 1991). Many in vitro studies of neutrophil adhesion use purified neutrophil populations and are often performed at 37°C especially if & integrin mediated adhesion is being studied (Lawrence and Springer, 1991). Extrapolating the results of these assays to the in vivo situation is complicated by changes in neutrophil surface phenotype and function associated with the manipulations required for neutrophil separation. Erythrocyte sedimentation, hypotonic lysis, centrifugation and temperature increases have all been reported to quantitatively upregulate CDllb expression on Table 1 Monoclonal

antibodies

used in this study

-

Antibody

Isotype

Ligand

Comments

Source

Ref

WM20

IgG2

CDLlb

Dr. K. Bradstock

Favaloro et al., 1988 Diamond and Springer, 1993 Pilarski et al., 1991 Fukushima et al., 1984 Shimizu et al., 1989 Zola et al., 1981 Kohler and Milstein, 1975 O’Connor and Ashman, I982

CBRM1/5

IgG2

CDllb

FMC46

IgGl

CD62L

Recognises structural epitope on o(, chain of p2 integrins Recognises functional epitope on (Y, chain of pa integrins Recognises L-selectin

CSLex

IgM

CDISs

Recognises

NIH44

IgGl

CD44

FMCIO

IgM

CD15

Recognises the hyaluronate receptor Positive control recognises

X63

IgGl

Undetermined

Sal4

IgG2

FMC41

IgM

Salmonella antigen Blood group A associated antigen

” ATCC:

American

Type Culture

Collection,

sialyl Lewis”

Mouse myeloma negative control Negative control Negative

Lewis”

protein

Flinders Centre ATCC

Medical

Dr. L. Ashman (Adelaide) Flinders Medical Ccntrc

control

12301 Parklnwn

(Sydney) Dr. T. Springer (Boston, MA) Flinders Medical Centre Kamiya Biomed (Thousand Oaks, CA) ATCC

Drive.

Rockville,

MD 2OS52, USA

P.P. Yortssefet al. / Jowrral of Imtmnologkal M&oh

are yet to be determined, all selectins bind CD& or structures bearing this oligosaccharide (Springer and Lasky, 1991). CDlSs is found at high levels on circulating neutrophils and its expression may be decreased on neutrophils that have migrated into inflamed tissues (Munro et al., 1992). It is also shed by activated neutrophils in vitro and can be measured in the surrounding medium (Walz et al., 1990). CD44 is broadly expressed on leukocytes and may have a role in lymphocyte binding to HEV (Jalkanen et al., 1987). However, its role in neutrophil adhesion remains to be elucidated. It appears to be shed during the migration of neutrophils through activated vascular endothelium (Haynes et al., 1991) and upon treatment of neutrophils with PMA and ionomycin (Bazil and Horejsi, 1992). This study determines the effects of temperature and neutrophil separation procedures on the expression of CDllb, L-selectin, CDlSs and CD44 on neutrophils by comparing the results of fluorescent flow cytometry of whole blood neutrophils with that of purified neutrophil populations.

2. Materials

and methods

2. I. Monoclonal antibodies

The monoclonal antibodies (mAb) used in this study are detailed in Table 1. We used two antibodies for CDllb: WM20 which recognises a structural epitope (Favaloro et al., 1988) and CBRMl/S which is not detected on resting neutrophils but becomes apparent on neutrophil activation (Diamond and Springer, 1993). 2.2. Whole blood (WB) preparation and activation Peripheral blood was anticoagulated with acid citrate dextrose (vol:vol 1:lO) and either immediately: 1. placed on ice (WB cold) or 2. incubated at 37°C for 15 min (WB 370 or 3. incubated with 100 mM phorbol myristate (PMA; Sigma, St. Louis, MO) at 37°C for 15 min (WB PMA).

156 (1995) 217-224

219

Aliquots of cell suspensions (100 ~1 of lo7 cells/ml) were then incubated with previously titrated saturating concentrations of primary antibody at 4°C for 30 min, washed with PBS plus 0.01% sodium azide (PBS azide) and then incubated with saturating concentrations of F(ab’), fragments of fluorescein isothiocyanate-conjugated sheep anti-mouse Ig (DDAF, Silenus, Melbourne, Australia; dilution l/50 of stock with PBS azide) for 30 min at 4°C. Red cells were then lysed with FACS Lysing Solution (Becton Dickinson, l/10 dilution of stock with distilled water), washed with PBS azide and resuspended in FACS fixative buffer (PBS containing 2% D-glucosan and 2.6% formaldehyde) and kept at 0-4°C until analysed. 2.3. Neutrophil preparation and nctivation Whole blood was anticoagulated with acid citrate dextrose and either placed immediately on ice or kept at room temperature. All separation procedures were performed under sterile conditions either at 0-4°C or room temperature. Neutrophil separation was performed as described by Boyurn (1968) with minor modifications. Briefly, 3 ml of sterile 1% (w/v> methylcellulose (Sigma, St. Louis, MO) was added to anticoagulated whole blood and the red cells allowed to sediment over 30 min. The supernatant was then collected and underlaid with Lymphroprep (Nycomed) and centrifuged at 150 X g for 20 min. Red cell lysis was then performed using 0.2% NaCl and stopped by adding an equal volume of 1.6% NaCl. Cells were washed twice with PBS and resuspended in PBS supplemented with 1 mM calcium and magnesium. Separated (SEP) neutrophils were either 1. kept at 4°C (SEP cold) or 2. incubated at 37°C for 15 min (SEP 37C) or 3. incubated with 100 mM PMA at 37°C for 15 min (SEP PMA). Preparation for flow cytometry was as for whole blood except that the first wash was with PBS azide containing 5% heat inactivated human AB serum and the second wash with PBS azide containing 1% heat inactivated Serum was used for blocking to Fc receptors.

foetal calf serum. non-specific binding

P.P. Youssef et 01. /Jourr~nl of Immwzologicnl Methods 186 (1995) 217-224

220

Extreme care was taken to limit endotoxin contamination of fluids during neutrophil separation. Disposable plasticware was used wherever possible and glassware soaked with E-Toxaclean (Sigma, St. Louis, MO). All solutions were prepared using endotoxin free water and filtered through Zetapore filters (Cuno, Meriden, CT). Levels of LPS less than 50 pg/ml in all reagents were confirmed by Limulus lysate assay (Whittaker Bioproducts, Walkersville, MA). 2.4. Flow cytometric

EFFECTS OF TEMPERATURE, SEPARATION AND PMA ON WHOLE BLOOD NEUTROPHILS

analysis

Flow cytometric analysis was performed using the Lysis II programme on a FACScan cytofluorometer (Becton Dickinson). Neutrophils were identified from forward scatter and side scatter characteristics on dot plot profiles and were analysed for fluorescence intensity. Mean fluorescence intensity was measured in arbitrary units and for analysis was transformed to a linear scale from the log,,, channel number of mean fluorescence. Isotype-specific mAb of irrelevant specificity were used as the negative controls. Specific linear immunofluorescence was obtained by subtracting the background linear fluorescence of the isotype controls. All tubes were read within 48 h. 2.5. Statistical analysis Paired t tests were used to compare the data at various time points. Results are given as mean (standard error of the mean @EM)) and the associated significance levels of the mean differences (p values).

3. Results Incubating whole blood at 37°C for 15 min results in an increase in the expression of WM20 by 148% and CBRMl/S by 98% (Fig. 11 when compared with whole blood kept at 0-4°C without any significant effect on L-selectin, CD15s or CD44 (Fig. 2). The results for CBRMl/5 are given as the ratio of the negative isotype control

WMZO

CBRUl/S ANTIBODY

Fig. 1. Effects of temperature, separation and PMA on whole blood neutrophil expression of CDllb. WM2O recognises a structural epitope whereas CBRMl/S recognises a functional epitope. Results for WM20 are given as a ratio of mean fluorescence of whole blood kept at O-4°C (WB cold). Results for CBRMI/S are given as a ratio of mean fluorescence of the negative control as their is no expression of the epitope recognised by CBRMl/S on WB cold. Results given as means and standard error of the mean of seven experiments. Paired t test used to compare differences with WB COLD. * corresponds to p < 0.05.

as there was no expression of the epitope recognised by this mAb on whole blood kept at 0-4°C. Neutrophil separation was associated with a greater than 400% increase in both WM20 and CBRMl/S which was similar in magnitude to the effect of PMA on whole blood neutrophils (Fig. 1). There were also significant increases in the expression of CD15s (increased by 91%) and CD44 (increased by 130%) in contrast to a 30% decrease in L-selectin expression (Fig. 2). Separated neutrophils were more resistant to PMA induced CDllb upregulation than whole blood neutrophils (Fig. 3). Whole blood neutrophils increased their expression of the epitope recognised by WM20 (structural epitope) by a mean of 122% compared with only 2% on separated cells (p < 0.05). In addition, whole blood neutrophils increased their expression of the epi-

P. P. Youssef et nl. /Journal EFFECTS OF TEMPERATURE AND SEPARATION ON NEUTROPHIL SURFACE PHENOTYPE

L-selectin

CD1 5s

CD44 ANTIBODY

Fig. 2. Effects of temperature and separation on whole blood neutrophil expression of L-selectin, CD44 and CDlSs. All results are given as a ratio of mean fluorescence of whole blood kept at 64°C (WB cold). Results given as means and standard error of the mean of 4-7 experiments. WB 37C is whole blood incubated at 37°C. SEP cold are neutrophils separated at II-4°C. Paired 1 test used to compare differences with WB cold.

EFFECTS OF PMA ON CD 11 b EXPRESSION NEUTROPHILS

q

4

q

I

I

711

of Immunological Methods 186 (1995) 217-224

tope recognised by CBRMl/S (functional epitope) by 230% as compared with only 12% on separated cells. Neutrophil separation at room temperature was associated with a 30% increase in both WM20 and CBRMl/S when compared with neutrophils separated at 0-4°C (Fig. 4). This difference was lost when the neutrophils separated at 0-4°C were incubated at 37°C for 15 min. Neutrophil separation at both room temperature and 0-4°C significantly increased the expression of CDlSs. This increase was less when neutrophil separation was performed at room temperature ( Figs. 2 and 4). There were no differences in L-selectin and CD44 expression between neutrophils separated 0-4°C or at room temperature. EFFECTS OF TEMPERATURE ON SURFACE PHENOTYPE OF SEPARATED NEUTROPHILS

ON

WE 37C OR SEP 37C WEI PMA SEP PMA

WM20

CBRMl/5

L-selectin

CD44

CD1 5s

ANTIBODY

WMZD

CBRMl/5 ANTIBODY

Fig. 3. Comparison of the effects of PMA on whole blood neutrophils kept at 37°C (WB PMA) with the effects of PMA on separated neutrophils kept at 37°C (SEP PMA). Results of WB PMA given as a ratio of whole blood kept at 37°C (WB 37C). Results of SEP PMA given as a ratio of separated neutrophils kept at 37°C (SEP 370 Note that baseline expression of CDllb is higher on separated neutrophils than whole blood neutrophils. Results given as means and standard error of the mean of 3 or 4 experiments.

Fig. 4. Effects of temperature on separated neutrophil expression of CDllb (WM20 recognises a structural epitope; CBRMl/S recognises a functional epitope), I.-selectin, CD44 and CD&. All results are given as a ratio of the mean fluorescence of neutrophils separated at 0-4°C (SEP cold). Results given as means and standard error of the mean of 4-7 experiments. SEP RT are neutrophils separated at room temperature. SEP cold to 37C are neutrophils separated at 0-4°C incubated at 37°C. Paired ! test used to compare differences with SEP cold. ’ corresponds to significantly different from SEP COLD an<1 SEP.RT al ~1< 0.05.

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of Imm~mological Methods 156 (1995) 217-224

In preliminary studies, we found the reproducibility of flow cytometry using the whole blood technique to be within 5% for WM20, 3% for CBRMl/S, 9% for FMC46, 9% for NIH44 and 6% for CSLex (data not shown).

4. Discussion The major findings of this study are that neutrophil separation and warming are associated with significant changes in structural and functional epitopes of multiple neutrophil cell adhesion molecules. As these changes are likely to reflect changes in neutrophil function, they need to be considered when working with purified neutrophil populations. The results of this study confirm those of other workers that CDllb is quantitatively upregulated during neutrophil preparation (Fearon and Collins, 1983; Berger et al., 1984; Hed et al., 1988; Macey et al., 1992). We also report an associated upregulation of a functional epitope of CDllb as demonstrated by the increased expression of the epitope recognised by CBRMl/S. This epitope is not expressed on whole blood neutrophils kept at 0-4°C but becomes apparent on purified neutrophils. Antibodies to this epitope have been reported to inhibit neutrophil adherence to vascular endothelium (Diamond and Springer, 1993). Neutrophil separation is also associated with a significant decrease in r_-selectin expression. The combination of these phenotypic changes are seen when neutrophils are incubated with activating factors (Kishimoto et al., 1989) thereby suggesting that neutrophils may be activated during separation procedures. The finding of increased expression of CD44 and CDlSs on separated neutrophils is unexpected as there is evidence that neutrophils actually shed these receptors upon activation (Walz et al., 1990; Haynes et al., 1991; Munro et al., 1992). It may be that there is increased production or decreased shedding of these molecules by purified neutrophils when compared with whole blood neutrophils. The mechanism of shedding is unknown although it may be due to a plasma factor from which the cells are isolated during prepara-

tion. Another possibility is that neutrophil preparative procedures uncover the epitopes recognised by NIH44 and CSLex. The combination of these multiple effects need to be considered when extrapolating results from in vitro experiments using purified neutrophil populations to the in vivo situation. This study confirms the findings of other studies that neutrophil preparation at room temperature is associated with a quantitative increase in CDllb expression when compared with separation at 0-4°C (Fearon and Collins, 1983; Hed et al., 1988). In addition, this study demonstrates an associated increase in the expression of a functional epitope of CDllb on neutrophils prepared at room temperature. However, these differences are lost when neutrophils prepared at 0-4°C are incubated at 37°C suggesting that there is no advantage in preparing neutrophils at 0-4°C if they are going to be used at 37°C. Furthermore, there is a significant increase in CD15s expression on neutrophils prepared at either 0-4°C or room temperature when compared with whole blood neutrophils maintained at 0-4°C. This increase was minimised when neutrophils were separated at room temperature. We therefore recommend that neutrophil preparation be performed at room temperature unless the cells are going to be maintained and used at 0-4°C. There were no differences in CD44 and r_-selectin expression between neutrophils prepared at 0-4°C or at room temperature. This study confirms previous reports that neutrophils incubated at 37°C quantitatively increase their expression of CDllb (Fearon and Collins, 1983; Hed et al., 1988). However, we also found an associated increase in the expression of the functional epitope of CDllb recognized by CBRMl/S suggesting that there may be associated functional upregulation. This contrasts with the findings of Philips et al. (1988) who found no increase in neutrophil aggregation on warming neutrophils despite an increase in CDllb expression, and those of Diamond and Springer (1993) who found that warming cells to 37°C did not increase the expression of the epitope recognised by CBRMl/S despite increasing the expression of a structural epitope on CDllb. These studies,

P.P. Youssef et 01. /.Jourtd

of lmmunologicol Metllotls 156 (1995) 217-224

however, assessed the effects of temperature on purified neutrophil populations which are already functionally activated when compared with their in vivo state and which may be resistant to further functional upregulation. This is supported by our findings that purified neutrophil populations are more resistant to PMA induced CDllb functional upregulation (as measured by the expression of the epitope for CBRM1/5) than whole blood neutrophils. In summary, neutrophil separation procedures are associated with significant increases in structural and functional epitopes on CDllb, increases in CDlSs and CD44 expression as well as decreases in L-selectin expression. Separated neutrophils are also more resistant to PMA induced CDllb functional upregulation than unseparated neutrophils. Neutrophil separation at room temperature is associated with a greater increase in structural and functional epitopes of CDllb than separation at 0-4°C although this difference is lost when the latter are incubated at 37°C. Neutrophil separation at both 0-4°C and room temperature is associated with a significant increase in CD15s expression which is minimised when separation is performed at room temperature. This suggests that neutrophil separation should be performed at room temperature unless the cells are going to be used at 0-4°C. Finally, incubating neutrophils at 37°C is also associated with increased expression of both structural and functional epitopes of CDllb. As these changes are likely to reflect changes in neutrophil function, researchers using purified neutrophil populations need to be aware of these significant changes induced by neutrophil separation when extrapolating in vitro results to the in vivo situation.

Acknowledgements

We thank Professors Tim Springer and Heddy Zola for kindiy providing monoclonal antibodies. Dr. Peter Youssef is supported by National Health and Medical Research Council Postgraduate Research Scholarship no. 933222.

223

References Bazil, V. and Horejsi, V. (7992) Shedding of the CD44 adhesion molecules from leukocytes induced by anti-CD44 monoclonal antibody simulating the effect of a natural receptor ligand. J. Immunol. 149, 747. Berger, M., O’Shea, J., Cross, A.S., Folka, T.M., Chused, T.M., Brown, E.J. and Frank, MM. (1984) Human neutrophils increase expression of C3bi as well as C3b receptors upon activation. J. Clin. Invest. 74, 1566. Boyurn, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at lg. Stand. J. Clin. Lab. Invest. 21 (suppl.97), 77. Butcher, E.C. (1991) Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67. 1033. Diamond, MS. and Springer, T.A. (1993) A subpopulation of Mac-l (CDllb/CDlB) molecules mediates neutrophil adhesion to ICAMand fibrinogen. J Cell Biol. 120, 545. Favaloro, E.J., Bradstock, K.F., Kabral, A., Grimsley, P.. Zowtyj. H. and Zola, H. (19881 Further characterization of human myeloid antigens (gp 160,95; gp 150; gp671, investigation of epitopic heterogeneity and non-haemopoietic disribution using panels of monoclonal antibodies belonging to CD-llb. CD-13 and CD33. Br. J. Haematol. 69, 163. Fearon, D.T. and Collins, L.A. (1983) Increased expression ot C3b receptors on polymorphonuclear leukocytes induced by chcmotactic factors and by purification procedures J. Immunol. 130, 370. Forsyth, K. and Levinsky, R. (1990) Preparative procedures ot cooling and re-warming increase integrin expression and function on neutrophils. J. Immunol. Methods 125. 159. Fukushima, K., Hirota, M., Terasaki, PI., Wakisaka, A., Togashi, H., Chia, D., Suyama, N.. Fukushi, Y., Nudelman. E. and Hakomori, S-i. (1984): Characterization of sialosylated Lewis” as a new tumor-associated antigen. Cancer Res. 44, 5279. Haynes, B.F., Hale, L.P., Patton, K.L., Martin, M.E. and McCallum, R.M. (1991) Measurement of an adhesive molecule as an indicator of inflammatory disease activity. Upregulation of the receptor for hyaluronidate (CD44) in rheumatoid arthritis. Arthritis Rheum. 34. 1434. Hed, J., Berg, O., Forslid. J., Hallden, G. and Larks-Rafner. G. (1988) The expression of CR1 and CR3 on non-modulated granulocytes of healthy blood donors as measured by flow cytofluorometry. Stand. J. Immunol. 28, 339. Jalkanen, S.T., Bargatze, R.F.. De 10s Toyos, J. and Butcher, E.C. (1987) Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an S5-0.5 kD glycoprotein antigen differentially inhibit lymphocyte hinding to lymph node, mucosal or synovial endothelial cells. J. Cell Biol. 105, 983. Kishimoto, T.K., Jutila, M.A., Berg, EL. and Butcher, EC. (1989) Neutrophil Mac-l and MEL-14 adhesion protcin\

224

P. P. Youssef et al. /Journal

of Immunological Methods 186 (I 995) 217-224

inversely regulated by chemotactic factors. Science 245, 1238. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256,495. Lawrence, M.B. and Springer, T.A. (1991) Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65, 859. Macey, M.G., Jiang, X.P., Veys, P., McCarthy, D. and Newland, A.C. (1992) Expression of functional antigens on neutrophils. Effects of preparation. J. Immunol. Methods 149, 31. Munro, J.M., Lo, S.K., Corless, C.,.Robertson, M.J., Lee, NC., Barnhill, R.L., Weinberg, D.S. and Bevilacqua, M.P. (1992) Expression of sialyl-Lewis x, an E-selectin ligand, in inflammation, immune processes, and lymphoid tissues. Am. J. Pathol. 141, 1397. O’Connor, C.G. and Ashman, L.K. (1982) Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated anti-immunoglobulin for determination of the specificity of monoclonal antibodies for protein mixtures. J. Immunol. Methods 54, 267. Philips, M.R., Buyon, J.P., Winchester, R., Weissmann, G. and Abramson, S.B. (1988) Up-regulation of the iC3b receptor (CR3) is neither necessary nor sufficient to promote neutrophil aggregation. J. Clin. Invest. 82, 495. Pilarski, L.M., Turley, E.A., Shaw, A.R.E., Gallatin W.M., Laderoute, M.P., Gillitzer, R., Beckman, I.G.R. and Zola,

H. (1991) FMC46, a cell protrusion-associated leukocyte adhesion molecule-l epitope on human lymphocytes and thymocytes. J. Immunol. 147, 136. Shimizu, Y., van Seventer, G-A., Siraganian, R., Wahl, L. and Stephen, S. (1989) Dual role of the CD44 molecule in T cell adhesion and activation. J. Immunol. 143, 2457. Springer, T.A. (1990) Adhesion receptors of the immune system. Nature 346, 425. Springer, T.A. and Lasky, L.A. (1991) Cell adhesion. Sticky sugars for selectins. Nature 349, 196. Tedder, T.F., Isaacs, C.M., Ernst, T.J., Demetri, G.D., Adler, D.A., Disteche, C.M. (1989) Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule LAM-l. J. Exp. Med. 170, 123. Von Andrian, U.H,. Chambers, J.D., McEvoy, L.M., Bargatze, R.F., Arfors, K.E. and Buthcher, E.C. (1991) Two-step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and the leukocyte p2 integrins in vivo. Proc. Natl. Acad. Sci. USA 88, 7538. Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M. and Seed, B. (1990) Recognition by ELAM-1 of the sialyl-Le” determinant on myeloid and tumor cells. Science 250, 1132. Weiss, S.J. (1989) Tissue destruction by neutrophils. New Engl. J. Med. 320, 365. Zola, H., McNamara, P., Thomas, M., Smart, I.J. and Bradley, J. (1981) The preparation and properties of monoclonal antibodies against human granulocyte membrane antigens. Br. J. Haematol. 48, 481.