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Rheumatology 2001; 40: 1033-1037
© 2001 British Society for Rheumatology

Original Papers

Increased expression of the blood group-related Lewis Y antigen on synovial fluid granulocytes of patients with arthritic joint diseases

M. Dettke, G. Pálfi1, E. Pursch1, M. B. Fischer and H. Loibner1

Department of Blood Group Serology and Transfusion Medicine, AKH Vienna, University Hospital of Vienna and
1 NOVARTIS Forschungsinstitut, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To evaluate the expression of the carbohydrate structures Lewis Y (LeY), sialyl-LeX (sLeX) and Lewis X (LeX) on paired peripheral blood (PB) and synovial fluid (SF) granulocytes in patients with arthritic diseases.

Methods. Ten patients with rheumatoid arthritis (RA), seven patients with spondyloarthritis (SA) and eight patients with osteoarthritis (OA) were studied. Granulocyte expression of the Le oligosaccharides was analysed by fluorescence-activated cell sorting.

Results. SF granulocytes of patients with RA, SA and OA expressed higher levels of the LeY oligosaccharide than PB granulocytes. Increases in LeY on SF granulocytes were similar in all three underlying diseases. No differences in the expression of the Le antigens were detected between PB granulocytes of patients and healthy individuals. Expression of sLeX and LeX showed no variation between SF and PB neutrophils.

Conclusion. The selective increase in LeY antigen on SF granulocytes in RA, SA and OA suggests a role of the LeY oligosaccharide in granulocyte traffic and inflammatory responses.

KEY WORDS: Lewis antigens, Granulocytes, Joint disease.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The migration of granulocytes to sites of inflammation is a multistep process that involves the reciprocal interaction of neutrophils with various adhesion molecules expressed on the activated endothelium [1]. The initial contact with the vessel wall occurs via the transient adhesion of neutrophils to the selectin family of adhesion molecules, including L-selectin and vascular E- and P-selectins [2]. The cellular ligands for E- and P-selectins have both been identified as blood group-related carbohydrate structures of the Lewis (Le) family, in particular the lacto series type 2-based tetrasaccharide sialyl-LeX (sLeX) [3, 4]. As an integral part of the inflammatory response, binding of sLeX and related oligosaccharide structures to selectin receptors precedes the activation of the integrin-mediated attachment and, subsequently, the extravasation of peripheral blood cells to the inflammatory tissue [5, 6].

The expression of the difucosylated LeY antigen on circulating blood cells was described recently [7–9]. The LeY antigen is expressed at a low density on circulating granulocytes of healthy individuals, as determined by the LeY-specific monoclonal antibody (mAb) ABL 364 [7]. LeY expression is up-regulated rapidly on peripheral granulocytes upon activation with the chemotactic peptide fMLP in vitro [7]. Although the function of LeY expression on granulocytes is not known, the close structural relationship between LeY and the proadhesive carbohydrate structures LeX and sLeX implies the involvement of the LeY determinant in the process of granulocyte adhesion and mobilization.

As modulation of granulocyte Le antigens may be an important factor in the initial phase of granulocyte recruitment into the affected joint, we analysed changes in membrane densities of the carbohydrate antigens LeY, sLeX and LeX on the surface of synovial fluid (SF) and peripheral blood (PB) granulocytes obtained from patients with distinct rheumatoid diseases.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients and healthy volunteers
Ten patients with rheumatoid arthritis (RA; four females, six males; median age 49 yr, range 33–75 yr), seven patients with spondyloarthritis (SA; two females, five males; median age 45 yr, range 32–62 yr) and eight patients with osteoarthritis (OA; five females, three males; median age 63 yr, range 56–78 yr) were studied. Patients with RA fulfilled the criteria of the American College of Rheumatology for the diagnosis of RA [10], presented with active disease (erythrocyte sedimentation rate >40 mm/h; more than three joint regions swollen; morning stiffness with tender joints for >1 h), and nine of the 10 patients were positive for rheumatoid factor (latex test). The group of patients with SA met the criteria of the European Spondyloarthropathy Study Group [11] and consisted of three patients with psoriatic arthritis, three patients with ankylosing spondylitis and one patient with Reiter's disease. All patients received non-steroidal anti-inflammatory drugs, two patients with RA and one patient with SA additionally received low-dose corticosteroid therapy (<10 mg prednisolone/day). Paired samples of 10 ml heparinized peripheral blood and 10 ml of knee joint effusions were collected at the times of arthrocentesis for therapeutic or diagnostic purposes after informed consent had been obtained. As a control, the expression pattern of Le antigens on peripheral blood granulocytes obtained from 15 age- and sex-matched healthy volunteers with no known history of inflammatory joint disease was analysed. Specimens were cooled immediately to 4°C and analysed within 4 h after collection.

Monoclonal anti Le antibodies
The characterization and purification of the mIgG3 anti-LeY mAb ABL 364 and its respective F(ab)'2 fragment have been described [12, 13]. Anti-sialyl-LeX mAb CESLEX-1 was purchased from Becton Dickinson (Mountain View, CA, USA) and anti-LeX mAb PM-81 was obtained from Medarex, West Lebanon, NH, USA [14, 15].

Immunofluorescence staining
Immunofluorescence staining was performed according to a method described previously [7]. In brief, after lysis of contaminating erythrocytes with ammonium chloride, 1x106 whole-blood leucocytes or SF leucocytes were labelled with three concentrations (0.5, 1 and 2 µg/sample) of the respective anti-Le mAbs. Isotype-specific antibodies (Becton Dickinson) served as the negative control. Bound mAbs were visualized by counterstaining with fluorescein isothiocyanate (FITC)-conjugated sheep F(ab)'2 anti-mouse mAb (An der Grub, Austria). The mean fluorescence intensity (MFI) of stained cells was recorded on a FACScan cytofluorimeter (Becton Dickinson). Forty thousand cells were counted in each sample. Between assays, the sensitivity of fluorescence detection was maintained by using FITC-labelled fluorospheres as a control (Becton Dickinson).

Statistical analysis
Data are expressed as median MFI and the 95% confidential interval (CI). Data were subjected to analysis of variance (ANOVA). A P value of <0.05 was considered to indicate a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Expression of LeY, sialyl-LeX and LeX on paired PB and SF granulocytes
The expression pattern of the lactosamine structures LeY, sLeX and LeX on the surface of paired PB and SF granulocytes of 10 patients with RA, seven patients with SA and eight patients with OA was analysed by flow cytometry. Circulating blood granulocytes of both patients and healthy individuals showed strong expression of LeX and sLeX but only modest expression of the LeY antigen, confirming previous results [7]. Expression of the LeY hapten was restricted to granulocytes, and there were no differences in the membrane densities of LeY, sLeX and LeX expressed on PB granulocytes from patients with RA, SA or OA and from controls (data not shown).

The analysis of individual SF samples of patients with RA, SA and OA revealed increased expression of the LeY antigen on SF neutrophils (Fig. 1Go). SF granulocytes expressed the LeY antigen at an MFI of 128.6 (CI 104.2–176.2) compared with 44.8 (CI 27.04–54.9) for PB neutrophils, corresponding to an approximately threefold increase in LeY expression on SF granulocytes (P=0.002). In contrast, the membrane density of sLeX was modestly decreased on SF granulocytes compared with circulating neutrophils [SF granulocytes, MFI 157.6 (CI 134.5–178.4); PB granulocytes, MFI 104.1 (CI 89.5–127.2); P=0.055]. No change in the expression pattern of the monofucosylated LeX structure was observed between SF and PB granulocytes [SF granulocytes, MFI 296 (CI 269.8–316.3); PB granulocytes, 301.0 (271.8–326.1); P=0.652] (Fig. 1Go).



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  		FIG. 1. Neutrophil expression of LeY, sLeX and LeX in individual paired samples of whole-blood granulocytes and SF granulocytes from a total of 25 patients with distinct arthropathies (10 patients with RA, seven with SA and eight with OA). Data are expressed as MFI.

 
Increased staining of anti-LeY mAb ABL 364 was specific for increased expression of LeY on SF granulocytes, as staining with the F(ab)'2 fragment of anti-LeY mAb ABL 364 instead of the complete mAb molecule revealed similar increases in LeY on SF granulocytes and PB granulocytes (data not shown). Thus, the increased reactivity of mAb ABL 364 towards SF granulocytes was not related to increased non-specific binding to granulocyte Fc receptors known to be up-regulated on SF leucocytes [16, 17].

Relative increases in LeY on SF granulocytes in different disease groups
To assess disease-specific differences in the up-regulated LeY density between RA, SA and OA, the relative increases in LeY on SF granulocytes were compared in the different disease groups. With increases in the median value of 3.1 (CI 2.1–5.0) for RA, 3.0 (CI 2.4–3.5) for SA and 2.7 (CI 2.1–3.5) for patients with OA, no significant differences in the relative increase in LeY on SF granulocytes were detected between the disease groups (Fig. 2Go).



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  		FIG. 2. Relative increase in granulocyte LeY expression on SF neutrophils obtained from patients with RA, SA and OA. The relative increase was calculated from individual paired samples by dividing the MFI of LeY expression on SF granulocytes by the MFI of LeY expression on PB granulocytes. There were no significant differences between the three disease groups.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Oligosaccharides of the Le family play a crucial role in the adhesion and migration of leucocytes to the inflammatory joint [1]. Here, we demonstrate that granulocytes within the synovial compartment of patients with RA, SA and OA express a higher membrane density of the LeY carbohydrate antigen compared with PB neutrophils. Accumulation of neutrophils expressing a high level of LeY (LeYhigh granulocytes) was specific for granulocytes within the synovial compartment, as no difference in LeY expression was detected between PB granulocytes of patients with RA, SA or OA and healthy controls.

The functional relevance of an increased level of LeY expression on SF granulocytes remains to be determined. On the basis of recent findings demonstrating a role for LeY in adhesion and cellular contact mechanisms [18–20], one explanation includes the participation of LeY in granulocyte traffic into the affected joint. This view is supported by our previous results demonstrating that the LeY hapten is attached to granulocyte CD66 antigens [7]. As a group of activation-associated adhesion molecules described as being up-regulated on SF granulocytes [21–23], the CD66 antigens contribute to the activation of L-selectin-mediated adhesion [24]. One role for LeY can therefore be related to the adhesion-promoting activity of the CD66 antigens, in which LeY may support adhesion by binding to other carbohydrate structures, i.e. the proposed interaction between LeY and the H antigen [25]. Alternatively, LeY may contribute to neovascularization within the inflamed joint. Interactions between leucocytes and synovial tissue can promote angiogenesis [26, 27], and recent findings have identified the LeY/H glycoconjugate as an angiogenic mediator in the SF and synovial tissue of patients with RA [28]. Hypothetically, the specific compartmentalization of LeYhigh granulocytes within the SF may therefore support inflammatory angiogenesis through the described chemotactic activity of LeY/H on endothelial cells [28].

The pronounced expression of LeY on granulocytes within the synovial compartment separates the LeY oligosaccharide from the closely related carbohydrate structures LeX and sLeX. Membrane expression of sLeX and LeX was unchanged on SF granulocytes compared with PB granulocytes, confirming previous findings [29]. However, the fact that we did not detect any significant variation in granulocyte sLeX expression does not accord with the up-regulation of sLeX on SF monocytes of patients with RA [30]. Whether this discrepancy reflects cell-type-specific differences in the carbohydrate pattern involved in cell–cell or cell–matrix interactions remains to determined [31, 32]. Alternatively, cell-specific variations in the glycosylation pattern of membrane proteins may account for the discrepancy in sLeX expression between SF granulocytes and SF monocytes [33, 34].

The results of the present study provide the first in vivo evidence for up-regulation of the LeY antigen on accumulated granulocytes within the affected joints of patients with RA, SA and OA. The finding that increased LeY expression was observed to a similar extent on SF granulocytes of patients with RA, SA and OA supports the idea of a common role for increased LeY expression on activated granulocytes, independently of the underlying joint disease. The physiological function and possible relevance of increased granulocyte LeY expression, however, remain to be defined. This information may lead to the identification of the LeY oligosaccharide as a possible target structure for therapeutic intervention.


   Acknowledgments
 
This work was supported by grant L 0033/MED (to M.D.) from the Österreichischen Fond zur Förderung der Wissenschaftlichen Forschung, Vienna, Austria.


   Notes
 
Correspondence to: M. Dettke, AKH Vienna, Department of Blood Group Serology and Transfusion Medicine, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria Back


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 20 July 2000; Accepted 27 March 2001


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