Clinical Science (1992) 83, 221-226 (Printed in Great Britain)

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Expression of adhesion molecules on circulating leucocytes in patients with inflammatory bowel disease S. M. GREENFIELD, A. HAMBLIN*, N. A. PUNCHARD and R. P. H. THOMPSON Gastrointestinal laboratory, The Rayne Institute, and *Department of Immunology, St Thomas’ Hospital, London, U.K. (Received 9 March 1992; accepted 2 April 1992)

1. The expression of leucocyte antigens CD11/CD18 and complement receptor 1 was studied on the circulating leucocytes of 13 patients with inflammatory bowel disease and 13 age- and sex-matched healthy control subjects. 2. Monoclonal antibodies against CDll/CDl$ and complement receptor 1 were added to leucocyte suspensions from patients and control subjects. Antibody binding was detected using a fluorescein-conjugated rabbit anti-mouse antibody and flow cytometry. The proportions of lymphocytes, monocytes and granulocytes expressing these molecules and the density of antigen expression, measured as mean fluorescence intensity, were determined. 3. There were no differences between patients and control subjects in the mean fluorescence intensity of antibody staining of surface molecules or in the proportion of cells expressing each molecule for any cell type. Analysis of subgroups of patients according to disease type, severity or treatment also showed no difference compared with control subjects. 4. We conclude that failure to identify a population of circulating leucocytes whose adhesion molecules or complement receptors are upregulated may arise because cells are only activated locally within the gut vasculature. Alternatively, structural changes in these molecules, rather than an increase in their number or the expression of other surface glycoproteins, may be more important in mediating adhesive interactions in inflammatory bowel disease. INTRODUCTION

The recruitment of leucocytes to inflammatory sites requires their prior adhesion to vascular endothelium, a process in part dependent on the expression of leucocyte adhesion molecules (Leu-CAMS). These glycoprotein heterodimers, present on leucocyte cell-surface membranes, consist of a common 8-chain (CD18) non-covalently associated with one of three distinct a-chains (CDlla, C D l l b or CDllc)

[l]. CDlla/CD18 is present on nearly all leucocytes and binds to at least two receptors on endothelium characterized as intercellular adhesion molecules 1 and 2 (ICAM1 and ICAM2) [2, 31. CDllb/CD18, expressed primarily on monocytes and granulocytes, binds to ICAM2, but the receptor for CDllc/CD18, found mainly on monocytes, is unknown. Leucocytes constitutively express the CD11/CD18 antigens, but cellular activation causes both quantitative and qualitative changes in their expression, enhancing cell adherence to complementary receptors [11. Quantitative increases in Leu-CAM expression arise either by mobilization of the intracellular storage pools of CDllb/CD18 and CDllc/CD18 to the plasma membrane [4, 51 or by increases in gene transcription and protein synthesis [6]. Such LeuCAM upregulation is associated with adhesiondependent phenomena, including leucocyte aggregation and adherence to endothelial monolayers ~571. Circulating leucocytes are activated in inflammatory bowel disease (IBD) with increased cytokine and free-radical production [S-111. The sera of these patients contain evidence of this activation in the form of increased levels of soluble interleukin-2 receptor and tumour necrosis factor-a (TNF-a), levels of which have been reported to correlate with disease activity [l2, 131. Since TNF-a is a powerful modulator of CD11/CD18 expression [14], it might be expected that this cytokine could cause LeuCAM upregulation on circulating leucocytes of patients with IBD. This could lead to increased cellular adhesion to the vascular endothelium with resultant emigration into the bowel wall and might account for the inflammatory infiltrate which characterizes this condition. Thus the principal aim of this study was to analyse CD11/CD18 expression on circulating leucocytes in patients with IBD in comparison with healthy control subjects. We also determined the expression of complement receptor 1 (CR1) on leucocytes, which, like CR3 (CDllb/ CDlS), is a marker of leucocyte activation.

Key words: adhesion molecule, flow cytometry, inflammatory bowel disease, leucocyte. Abbreviations: CDAI, Crohn’s disease activity index; CRI, complement receptor I; FCS, fetal calf serum; IBD, inflammatory bowel disease; ICAM, intercellular adhesion molecule; L e d a m , leucocyte adhesion molecule; MFI, mean fluorescence intensity; PBS, phosphate-buffered saline; TNF, tumour necrosis factor; T N F e , tumour necrosis factore; UC, ulcerative colitis. Correspondence: Dr S. M. Greenfield, Gastrointestinal Laboratory, The Rayne Institute, St Thomas’ Hospital, London SEI 7EH, U.K.

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Table 1. Demographic data of patients and control subjects. The two groups were well matched for age and sex.

Total Female Male Mean age (&EM) (years) UC (pancolitis, n =4; left-sided disease, n = 6 ; severe, n =5) Crohnf disease (terminal ileum, n=3; severe, n=2) Receiving steroids Receiving sulphasalazine/mesalazine N o t on treatment Severe UC/CDAl> 450

Patients with IBD

Control subjects

13 10

13

3

10 3

42.7 4.9

37.4 k4.2

10

-

3

-

3 5 8

7

-

MATERIALS A N D METHODS Patients and subjects

Thirteen patients with IBD [lo with ulcerative colitis (UC) and three with Crohn's disease] were recruited, their diagnoses being based on characteristic clinical, endoscopic, histological and radiological features. Thirteen age- and sex-matched healthy laboratory personnel, who had received no medication for the preceding 14 days, were also studied. The disease activity of patients was recorded according to the criteria of either Truelove & Witts [l5] for UC, or the Crohn's disease activity index (CDAI) [16]. The characteristics of patients and control subjects are shown in Table 1. Permission for venesection of patients was obtained from the Ethics Committee of St Thomas' Hospital. Antibodies

The mouse monoclonal antibodies UCHT1, UCHMl and 29 were used for leucocyte subpopulation identification, detecting CD3 on Tlymphocytes, CD14 on monocytes and CD15 on granulocytes, respectively. The antibodies MHM24, 44, 3.9, MHM23 and E l l , which recognize surface molecules CDlla, CDllb, CDllc, CD18 and CR1, respectively, were also used. Fluorescence labelling was performed using a fluorescein-conjugated F(ab)z rabbit anti-mouse antibody (Dakopatts, High Wycombe, Bucks, U.K.). The ascitic monoclonal antibodies MHM24 and MHM23 were titrated before use to find a satisfactory working dilution. All other antibodies were tissue culture supernatant and were used undiluted. Leucocyte preparation

Leucocytes were prepared for labelling using a procedure that we have described previously and which prevents artefactual increases in Leu-CAM expression during cell separation [171. Heparinized (10 units/ml) blood (0.5 ml) was fixed immediately in pre-warmed 0.13 mol/l formaldehyde/phosphate-

et al.

buffered saline (PBS-Oxoid), pH 7.4. at 37"C, before lysis of erythrocytes with 20 ml of 0.16 mol/l ammonium chloride/O.Ol mol/l Tris-methylamine buffer (4 min, 37°C). The cells were centrifuged, the supernatant discarded and leucocytes resuspended in PBS. After a second centrifugation the leucocyte suspensions were kept on ice until the antibody labelling procedure, performed within 30 min. Immunofluorescence labelling

The methods for antibody labelling of leucocytes and flow cytometry have been described previously [17]. Leucocyte suspension (25 pl) was added to the wells of a microtitre plate, containing 25pl of each monoclonal antibody in RPMI/5% (v/v) fetal calf serum (FCS). After 30 min, unbound antibody was removed by two washes with RPMI/FCS. Monoclonal antibody binding was detected using 25 pl of F(ab)z antibody (diluted 1:50), added to every well, and after a 30min incubation in the dark, this was washed out. The cells were then resuspended in 25 pl of RPMI/FCS and transferred to fluorescenceactivated cell sorting analysis tubes (Falcon 2054) containing 300 p1 of 1% (v/v) paraformaldehyde/ PBS. Leucocyte labelling was analysed by flow cytometry 24 h later using a FACScan analyser (BectonDickinson, Fort Collins, CO, U.S.A.) equipped with Consort 30 software (Hewlett-Packard, Mountain View, CA, U.S.A.). This time point was chosen to ensure that the cells were properly fixed and also to standardize the procedure. lmmunofluorescence flow cytometry

On each occasion, 10000 cells were analysed, and the lymphocytes, granulocytes and monocytes were separated into different populations according to their ability to scatter light in a forward and side direction. Each population was gated and the identity and purity of the cells were confirmed by staining with the cell-type-specific antibodies UCHT1, 29 and UCHM1. Cell purity was greater than 95% for lymphocytes and granulocytes, and greater than 90% for monocytes. Fluorescence data were collected on a four-decade logarithmic scale and markers were set using a control antibody which did not stain the cells (UCHM1 for lymphocytes and UCHTl for granulocytes and monocytes). All fluorescence to the right of this marker arose from the labelled cells and values of mean fluorescence intensity (MFI), a quantitative measurement of antigen density, were recorded by the FACScan analyser. The percentage of labelled or non-labelled cells was also recorded. Non-specific binding of the F(ab), fragment to leucocytes was negligible. Statistics

The mean MFI and mean percentage of stained cells for each cell type was compared between the

Leucocyte antigens in inflammatory bowel disease

A

223

I

A

'

4

.

I

50

8

0

IBD

-++ IBD

C

IBD

C

IBD

A

, C

IBD

C

IBD

-++

C

C

Lymphocytes Granulocytes Monocytes Monoclonal antibody t o CDI l a

Lymphocytes Granulocytes Monocytes Monoclonal antibody to CD18

9001 (4 0

I 500-

+++

Lymphocytes Granulocytes Monocytes Monoclonal antibody t o CDI I b

*

I

0

I

.

'+ + + IBD

C

IBD

C

IBD

C

Lymphocytes Granulocytes Monocyter Monoclonal antibody t o CDI Ic

Fig. 1. MFI of CD 18 (a), CDlla (b), CDllb (c) and CDllc (d)expression on leucocytes. Error bars represent mean and SEM. Abbreviation: C, control.

two groups for each antibody. Since the MFIs and the values for the percentages of stained cells were not normally distributed, the Wilcoxon rank sum (two-sample) test was used. Significance was taken to be at the 5% level. RESULTS Antibody stains

The MFIs of leucocytes of patients and control subjects stained with each antibody are depicted in Figs. l(a-d). CD18 and C D l l a were strongly

D

expressed on all cell types. Expression of C D l l b and C D l l c on all cells was generally weaker, except for granulocytes, which expressed CD1 l c most strongly. Granulocytes and monocytes expressed most CDllb. CR1 density was greatest on lymphocytes and granulocytes (Fig. 2). Individual donors varied in their expression of CD11/CD18 as reported previously by ourselves [17, 181 and others [19, 201 and accordingly represents variation within a normal population. The mean MFI and range of values for all molecules examined was similar in patients and control subjects and there was no significant difference between the mean MFI of both

S. M. Greenfield et al.

224 200

I

Most lymphocytes, monocytes and granulocytes expressed CDlla/CD18, a large proportion of granulocytes and monocytes expressed CD1 l b and just over 50% of monocytes expressed CDllc. CR1 was expressed on between 30 and 40% of granulocytes and monocytes and to a lesser extent on lymphocytes. There were no differences between patient and control data. There was also no significant difference in the MFIs of leucocytes from the 10 patients with UC compared with control subjects. In addition, the MFIs of leucocytes from those patients with either severe IBD or not receiving treatment were randomly scattered and were not significantly different from control values.

0 8 0

'

*+IBD

C

IBD

C

IBD

C

Lymphocytes Granulocytes Monocytes Monoclonal antibody to CRI

DISCUSSION

Fig.2. MFI of CRI expression on leucocytes. Error bars represent mean and SEM. Abbreviation: C. control.

Table 2. Percentage of leucocytes staining positive. Values are shown as mean (SEM).

Adhesion molecule

CDI la CDI Ib CDI Ic CD18 CR I

Lymphocytes (% staining positive) Patients with IBD

Control subjects

97.9 (0.4) 23.6 (2.2) l3.2( I .9) 88.2(4.0) 21.2(2.8)

98.4(0.5) 28.4(2.7) 13.1 (1.6) 89.8 (6.3) 20.5 (I .9)

Granulocytes (%staining positive)

CDlla CDllb CDI Ic CD18 CR I

Patients with IBD

Control subjects

95.0 (2.4) 63.4 (6.0) 30.9 (4.4) 90.7 (2.6) 40.0(5.0)

92.3 (2.3) 56.6(8.3) 25.4(6.1) 92.2 (9.6) 34.9 (7.7)

Monocytes (% staining positive)

CDI la CDI Ib CDI Ic CD18 CRI

Patients with IBD

Control subjects

99.5 (0.I) 89.0 (2.3) 54.6(4.1) 9442.2) 34.0 (2.7)

99.4 (0.2) 87.4 (4.3) 57.7 (4.2) 96.1 (7.5) 32.8(2.8)

groups for each molecule measured, including CR1 on granulocytes (P=0.051, Fig. 2). Table 2 depicts the mean (SEM) percentage of the leucocyte populations staining with each antibody.

The importance of Leu-CAM molecules in the initiation and maintenance of the inflammatory response is demonstrated in patients deficient in the expression of these molecules, who develop recurrent and often fatal bacterial infections [21]. Conversely, the expression of adhesion molecules is increased in conditions, in which circulating leucocyes are activated, such as systemic lupus erythematosus, sepsis, burns and during haemodialysis [5, 19, 221. Despite the previously documented cellular activation [8-111 and high levels of circulating TNF-a in IBD at concentrations shown to cause increased CD11/CD18 expression [23], we were unable to show upregulation of CD11/CDl8 or CR1 on leucocytes from patients with IBD. This might be because the patients reported here did not have TNF-a in their sera, despite the fact that seven patients, of whom five were not on treatment, had severe disease, and suggests that circulating TNF-a is not a consistent feature in IBD. Alternatively, TNF-a, if present, may not be biologically active; a tumour necrosis factor (TNF) inhibitor preventing cellular activation has been documented in the sera of patients with active Crohn's disease [24]. Such TNF inhibitors have been found in other studies and include the soluble form of the TNF receptor, the presence of which causes discrepancy between bioactivity and immunoassay [25]. Alternatively, our failure to demonstrate Leu-CAM upregulation may be due to drug treatment, although there was no difference in CD 11/CD 18 expression on the leucocytes of the eight patients not receiving either sulphasalazine or mesalazine when compared with healthy control subjects. Cellular activation may occur within the gut vasculature secondary to local cytokine production, resulting in adhesion molecule upregulation and leucocyte recruitment into the bowel. However, recent work has shown only a small increase in CD1 l a expression on colonic macrophages in

Leucocyte antigens in inflammatory bowel disease

patients with IBD [26]. This is in keeping with our results and suggests that it may not be the number of adhesion molecules on leucocytes that mediates binding to endothelial cell ligands, but an alteration in their conformation. Indeed, neutrophils pretreated with an anion-channel-blocking agent preventing an increase in CD11b/CD18 expression continue to exhibit increased adherence to endothelial cell monolayers after chemical stimulation ~271. Granulocytes from patients with IBD have increased adhesiveness to nylon fibre [ll], an assay that is comparable with adhesiveness to cultured endothelium [28]. Additionally, the adherence of mononuclear leucocytes to the vascular endothelium has been documented as an early lesion in Crohn’s disease [29]. Our findings imply that other leucocyte surface antigens are of prime importance in cellular emigration from the blood in IBD. For instance, the selectin MEL-14 and the integrin VLA4 are both involved in leucocyte adhesion to the endothelium [30, 311. Alternatively, an alteration in the state of the vascular endothelium, rather than Leu-CAM upregulation, could account for cellular recruitment in IBD; increased numbers of TNF-a-secreting cells have been described in the colonic mucosa of patients with IBD [32], as have raised concentrations of TNF-a in the stools of these patients [33], and it is possible that this cytokine could increase ICAM 1 expression on vessels, promoting leucocyte recruitment into the bowel. Cellular activation causes CR1 upregulation on cell surfaces [34], but was not demonstrated here on leucocytes from patients with IBD. In common with the CD11/CD18 molecules, it is likely that CR1 activation involves a structural change within the molecule rather than an increase in the number of molecules. Alternatively, previous findings of cellular activation may have arisen because cell-separation techniques, such as density gradients, were used in these studies [8-111 and these methods have been shown to cause adhesion molecule upregulation [14]. Such artefactual increases in cellular activation and adhesion molecule expression were prevented by the technique used in this study. Peripheral blood leucocytes in patients with IBD may be more readily activated by procedural manipulations than their normal counterparts, leading to differences between patients and control subjects. Accordingly, studies on peripheral cells should be performed with this in mind. Leucocyte+mdothelial cell interactions are central to the pathogenesis of IBD, where uncontrollable inflammation plays a significant role in causing severe tissue injury. From our findings it is likely that locally produced cytokines are responsible for leucocyte activation in IBD, resulting in a qualitative or quantitative change in adhesion molecules and hence cellular recruitment into the bowel wall.

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ACKNOWLEDGMENTS

We are grateful to the Special Trustees of St Thomas’ Hospital for their continuing supprt, to Ann Guyer who helped prepare the manuscript, and to Mr N. Taub for statistical advice. We thank Dr N. Hogg (Imperial Cancer Research Fund Laboratories) for the antibodies 29, 3.9, 44 and E l l , Professor A. McMichael (Institute of Molecular Medicine, John Radcliffe Hospital, Oxford) for the antibodies MHM24 and MHM23 and Professor P. Beverley (Ludwig Institute, University College and Middlesex Hospital Medical School) for the antibodies UCHTl and UCHM1. REFERENCES I. Arnaout, M.A. Structure and function of the leucocyte adhesion molecules CDII/CDI8. Blood 1990; 75, 1037-50. 2. Makgoba, M.W., Sanders, M.E., Ginther Luce, G.E. et al. ICAM-I: a ligand for LFA-ldependent adhesion of B, T and myeloid cells. Nature (London) 1988;

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