Syndrome and Rheumatoid Arthritis

806 Annals of the Rheumatic Diseases 1993; 52: 806-811

Vascular cell adhesion molecule 1 and a4 and a 1 Ann Rheum Dis: first published as 10.1136/ard.52.11.806 on 1 November 1993. Downloaded from integrins in lymphocyte aggregates in Sjogren's syndrome and rheumatoid arthritis

Jo C W Edwards, Linda S Wilkinson, Paul Speight, David A Isenberg

Abstract syndrome2 and in the synovium of those with Objectives-Interactions between vascu- rheumatoid arthritis,3 infiltrating lymphocytes lar cell adhesion molecule 1 (VCAM-1) form aggregates which take on a number of the and its ligand, the a4/01 integrin, have structural features ofthe follicles seen in lymph been shown to be important in a number nodes, tonsils, or the spleen. Small aggregates of cellular events in vitro. To assess the contain mostly T cells, but with increasing size importance of such interactions in the B cells appear at the aggregate centre, and in development of lymphocytic infiltration the largest aggregates in synovium true in diseased tissue the distribution germinal centre formation is seen. of the two ligands has been studied The subpopulation of lymphocytes infil- immunohistochemically. trating these tissues has been studied in detail. Methods-Cryostat sections of labial Most of the CD3+ (T cell) population in labial tissue from patients with Sjogren's syn- salivary gland tissue from patients with drome, normal labial tissues, rheumatoid Sjogren's syndrome is CD4+/CD8- and the synovia, and normal tonsils were stained ratio of 'primed' (CD45RA-/CD45RO+) to using antibodies to VCAM-1, o4 and 01 'naive' (CD45RA+/CD45RO-) cells is higher integrin chains, and markers for T cells, in focal than diffise areas of infiltrate.4 5 Most B cells, macrophages, and follicular den- ofthe T cells carry the ot3 T cell receptor.6 This dritic reticulum cells (FDRCs), visualised contrasts with findings in the peripheral blood using alkaline phosphatase and fast red. of patients with Sjogren's syndrome in which Results-Staining patterns for VCAM-1 the y6 receptor predominates.7 8 In rheuma- and integrin chains in lymphocyte aggre- toid synovium most of the T cells are also gates in synovial and labial tissues were CD4+/CD8- and primed cells predominate similar. VCAM-1 staining was found on over naive cells.9

both vascular and ramifying dendritic cells There is evidence of oligoclonality among http://ard.bmj.com/ at the centre of large T cell aggregates B cells in labial tissues in Sjogren's syndrome and in all aggregates where there was a on the basis of K and X chain expression.'0 central clustering of B cells. VCAM-1 The B cells in rheumatoid synovium appear to colocalised with, but also extended be polyclonal and have a wide range of anti- beyond, staining for the FDRC marker body affinities including rheumatoid factor R4/23. Staining for the a4 and 01 integrin activity."1 12 chains was more widespread than staining Despite this knowledge, the mechanisms on October 1, 2021 by guest. Protected copyright. for VCAM-1, with no significant increase responsible for the accumulation of lympho- in staining at sites of maximum VCAM-1 cytes in such lesions are still unclear. Recent staining. In tonsils VCAM-1 and R4/23 interest has focused on the possible role of the codistributed in germinal centres, but vascular cell adhesion molecule 1 (VCAM- 1) staining for the ct4 and ,B1 integrin chains and its ligand, the A4/13 1 integrin hetero- Department of was chiefly seen in T lymphocyte areas. dimer (VLA-4, CD29/49d),'3 in rheumatoid Medicine, Conclusions-VCAM-1 may be more arthritis.'4 15 VCAM-1 was first described as Faculty of Clinical It Sciences, important in determining the distribution being present on activated endothelium.'6 is University College of B than T lymphocytes in lymphocytic now known to be present on dendritic cells'7 London, infiltration ofnon-lymphoid tissue. Unlike and fibroblast-like synoviocytes."' It has London, follicles of been implicated in T cell emigration.'8 The United Kingdom the lymphoid tissue, ectopic J C W Edwards follicle-like structures in non-lymphoid adherence of lymphocytes to high endothelial L S Wilkinson tissues may form by immigration of venules in cryostat sections of rheumatoid D A Isenberg B cells via VCAM-1+ vessels at the centre synovia is dependent on an interaction between Department of Oral ofT cell aggregates. the 131 integrin chain CD49d and VCAM-1.'4 Pathology, VCAM-1 is also implicated in CD3 dependent Eastman Dental Hospital (Ann Rheum Dis 1993; 52: 806-81 1) T cell proliferation, in conjunction with the P Speight oA4/ 1 integrin,'9 and in B cell adherence to Correspondence to: dendritic cells (shown in cryostat sections of Dr J C W Edwards, In normal Arthur Stanley House, Lymphocytes pass in small numbers through lymphoid tissue).20 lymphoid tissue, Tottenham Street, normal non-lymphoid tissues and in chronic however, germinal centre B cells are negative London W1P 9PG, inflammation may accumulate in large or only weakly positive for both chains United Kingdom. the (ot and of the integrin Thus the Accepted for publication numbers.' In certain situations, including 1) ligand.2' 24 June 1993 salivary glands of patients with Sjogren's significance of VCAM-1 expression cannot VCAM-1 and integrins in Sjogren's syndrome and RA 807

entirely be explained on the basis of an inter- epitope on follicular dendritic reticulum cells action with ot4/01 integrin in vivo. (FDRCs),27 HP2/1 recognises CD49, the a4 Ann Rheum Dis: first published as 10.1136/ard.52.11.806 on 1 November 1993. Downloaded from This study aimed to investigate the integrin chain,28 and B-D 15 recognises CD29, distribution of VCAM-1 and the cx4 and 1 the , 1 integrin chain.29 integrin chains on cells within lymphocyte aggregates in non-lymphoid tissues to assess the likely significance of an interaction between IMMUNOHISTOCHEMISTRY these molecules in the development oflympho- Sections were removed from storage at -70°C cyte aggregates and to compare findings with and allowed to reach ambient temperature those in follicles in lymphoid tissue. before being unwrapped. Samples were fixed in cold acetone for ten minutes before incubation with primary monoclonal antibodies at opti- Materials and methods mum dilution for one hour at room tem- TISSUES perature, followed by washing in buffer. All Synovial tissue was obtained at arthroplasty dilutions and incubations were performed from the hip, knee, or wrist offive patients with in freshly prepared TRIS buffered saline, rheumatoid arthritis (definite or classical by the pH 7-6. For IgG monoclonal antibodies rabbit American Rheumatism Association criteria22). antimouse immunoglobulins (Dako, High Labial biopsy samples were obtained for Wycombe, Buckinghamshire, United Kingdom) diagnostic purposes. Of these, six were from in 20% normal human AB serum were then patients with Sjogren's syndrome and showed applied for 30 minutes, followed by alkaline characteristic histological features. Six were phosphatase-antialkaline phosphatase complexes from patients with unrelated symptoms and (Dako), also in 20% normal human serum, for were judged to be histologically normal. Of the 30 minutes. For R4/23, biotinylated goat anti- patients with Sjogren's syndrome two had no mouse IgM (Sera Lab) was applied, followed associated rheumatic disease (primary cases) by streptavidin alkaline phosphatase (Dako), and four had associated rheumatoid arthritis both in 20% normal human serum for 30 (secondary cases). Patients with rheumatoid minutes. Alkaline phosphatase conjugates were arthritis all had a disease duration of more than developed using naphthol-AS phosphate/fast five years. They had received various combi- red to yield a red product. Sections were lightly nations of non-steroidal anti-inflammatory counterstained in Harris's haematoxylin, drugs and disease modifying drugs, but not washed, and mounted in Apathy's medium. systemic corticosteroids. Two samples of tonsil The lymphocyte infiltration seen in labial were obtained from a routine tonsillectomy. tissue in Sjogren's syndrome occurs in the Where necessary, the tissues were trimmed of context of little or no other evidence of fat and fibrous tissue, snap frozen immediately inflammation. In rheumatoid synovium chronic after the operation, and stored at -70°C inflammatory changes such as macrophage until required for sectioning. Serial crysostat infiltration and fibrin deposition are promi-

sections (5 p,m thick) were cut on a Bright's nent. To gauge the importance of associated http://ard.bmj.com/ cryostat, taken up on to slides, air dried, and chronic inflammation to the pattern of stored individually wrapped in aluminium foil VCAM-1 and integrin chain expression the in a hermetically sealed box at -70°C until relative numbers of macrophages (CD68+) required for immunochemistry. and T cells (CD3+) were counted within 10 fields with a X20 objective for each tissue.

PRIMARY ANTIBODIES on October 1, 2021 by guest. Protected copyright. All monoclonal antibodies used were of Results the IgG class, with the exception of R4/23, The table gives an overview of the patterns of which is IgM. 1 4C3 recognises VCAM-1,23 staining. The normal labial tissues showed EBM1 1 recognises CD68 on macrophages,24 small numbers of T cells and macrophages. UCHT1 recognises CD3 on T lymphocytes,25 Staining for VCAM-1 and the integrin chains 4KB 128 recognises CD22 on B lymphocytes,26 was minimal. There was no staining with R4/23 recognises a non-CD designated R4/23.

Summary ofimmunohistochemicalfindings Tissue/area Stainingfor adhesion molecules VCAM-1 a4 integrin 31 integrin Normal labial tissue All sites Minimal Minimal Minimal Sj6gren's labial tissue Small T cell aggregates Central on small blood vessels and a few nearby cells Throughout Weakly throughout Larger aggregates Mesh in central B cell area extending beyond R4/23 Throughout Weakly throughout Tissue stroma A few scattered cells Most mononuclear cells Weakly throughout Rheumatoid synovium Small T cell aggregates Central on small blood vessels and a few nearby cells Weakly throughout Throughout Larger aggregates Mesh in central B cell area extending beyond R4/23 Weakly throughout Throughout Aggregates with germinal centres Mesh in central B cell area codistributing with R4/23 Weakly throughout Throughout Tissue stroma A few scattered cells Most mononuclear cells Throughout Synovial lining Prominent Minimal Prominent Tonsils T cell areas Scattered cells, venules Most cells Most cells Follicle centre Dense mesh throughout Minimal Minimal 808 Edwards, Wilkinson, Speight, Isenberg

Most of the infiltrating cells in rheumatoid VCAM-1 expressing cells. The 11 chain was,

synovium were macrophages, despite the on the other hand, expressed more strongly, Ann Rheum Dis: first published as 10.1136/ard.52.11.806 on 1 November 1993. Downloaded from presence of well developed lymphocytic particularly on endothelial cells, diffusely in aggregates. In the labial tissue from patients lymphoid aggregates, and within the synovial with Sjogren's syndrome T cells predominated intima. (figure A, B), though the aggregates tended to In tonsils oA and 13 integrin staining was be smaller than in the rheumatoid tissue. Mean absent from, or only weakly present in, the counts of CD68+ and CD3+ cells in 10 fields central VCAM- 1 rich areas of follicles. at X20 objective magnification were: normal labial tissue CD68+ 48, CD3+ 26-5; Sjogren's syndrome labial tissue CD68+ 108, CD3+ 153; Discussion and rheumatoid synovium CD68+ 558, The role of integrins and their ligands in CD3+ 156. B Lymphocytes made up less than human inflammatory and autoimmune pro- 10% of infiltrating mononuclear cells in all cesses remains uncertain. Initially, VCAM-1 instances, being present chiefly at the centres was chiefly implicated in the adherence of of larger lymphocyte aggregates (figure C). leucocytes to endothelium, but some studies The amount of staining for VCAM-1, the x4 suggest other possible roles, including the and 1P integrins, CD22, and R4/23 reflected promotion of the proliferation and maturation the numbers of infiltrating cells present. Thus of lymphocytes.'3"21 Direct evidence for the in lesional tissue from patients with either functional potential of adhesion molecules Sjogren's syndrome or rheumatoid arthritis, tends to come from in vitro studies, however. where there was a paucity of T cells and Immunohistochemical analysis plays an macrophages, little staining was seen. Where important complementary part in assessing the large lymphocyte aggregates were present in relevance of putative functional interactions to either type of tissue, however, VCAM-1 the whole tissue. appeared at the centre of the aggregate in an Dense lymphocyte aggregates can develop area occupied by B cells and R4/23 positive outside lymphoid tissues as part of a chronic FDRCs. In smaller aggregates VCAM-1 inflammatory response, which also includes staining was often limited to one or more events such as vascular proliferation, fibrin central blood vessels and a few nearby cells deposition, and macrophage and polymorph (figure D), but in larger aggregates VCAM-1 infiltration. This is perhaps best seen in formed a network throughout the whole central synovial tissue in rheumatoid arthritis. Similar B cell containing area (figure E). R4/23 staining lymphocyte aggregates also occur in the for FDRCs was sparse in the Sjogren's syn- salivary glands of patients with Sjogren's syndrome labial tissues, with the exception of the drome in the absence of significant inflam- tissue with the largest T cell infiltrate. In mation. In this situation there may be a specific Sjogren's syndrome labial tissues and rheu- disorder of lymphocyte traffic, possibly related matoid synovial tissues VCAM- 1 staining to a low grade lymphoproliferative process.'0 colocalised with, but extended beyond, R4/23 The findings of this study indicate that large http://ard.bmj.com/ staining in many smaller aggregates. Where lymphocyte aggregates developing in non- fully formed germinal centres were present in lymphoid tissues, in either of the above rheumatoid arthritic tissue, however, the two contexts, mimic the development of lymphoid markers showed a complete and precise follicles in terms of the central accumulation codistribution. of B cells and the presence of associated VCAM- 1 expression in tonsils was seen VCAM-1 and R4/23 positive cells. These latter most strongly within follicle centres and co- cells are probably FDRCs, which, unlike other on October 1, 2021 by guest. Protected copyright. distributed with R4/23, forming a continuous 'dendritic cells', are macrophage marker and reticulum in the B cell rich area. Individual class II negative.30 The implication is that VCAM-1 positive cells were also present in the FDRCs can be differentiated from local peri- outer T cell zone of follicles and VCAM-1 was vascular fibroblastic cells in non-lymphoid present on small venules. tissues subject to chronic T cell infiltration. Although the distribution of VCAM- 1 VCAM- 1 expression within central areas of closely paralleled that of CD22 and R4/23, lymphocyte aggregates often extended beyond there was no relation between the distributions that of R4/23 and was often present of VCAM-1 and either a4 or 13 integrin where R4/23 was absent. This suggests that chains. In the Sjogren's syndrome labial tissue VCAM- 1 expression appears on FRDCs or there was expression of cx4 within the central other supporting cells at an earlier stage of areas of lymphocyte aggregates, but extending differentiation than R4/23, which is in keeping beyond the VCAM-1 staining and at a similar with ontological studies3' which suggest level to that on macrophages (figure F) and T R4/23 is a 'late' FDRC marker. (Other markers cells scattered within the tissue. Expression of of FDRCs which might clarify this are, the 131 integrin was ubiquitous on infiltrating unfortunately, unhelpful in a histological study cells but weak, being barely detectable in four such as this because they are also present on of the tissues. the adjacent B cell.) Although the factors In rheumatoid synovium little ac4 integrin responsible for T cell infiltration in chronic was present diffusely in the lymphocyte synovitis and in the labial tissue of patients aggregates, but the intensity of staining was with Sjogren's syndrome are thought to be weaker than in Sjogren's syndrome labial different, it appears that the cellular patterns tissue, though the pattern was similar, with no within lymphocyte aggregates follow the same preferential expression in areas containing rules. VCAM-1 and integrins in Sjdgren's syndrome and RA 809

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a.&. sa. lw§ -., -se. ... ,. -.9 tk Sections oflabial glandfrom patients with Sjdgren 's syndrome stained using an indirect alkaline phosphatase-antialkaline phosphatase technique, developed with fast red, and counterstained with haematoxylin. (A) Stainingfor CD3 showing that most lymphocytes in aggregates found between salivary acina are T cells. Bar = 20 ,um. (B) Stainingfor CD68 showing macrophages scattered within the tissue stroma. Bar = 20 ,um. (C) Staining for CD22 showing an area ofpositive cells in the centre of a lymphocyte aggregate. Bar = 40 ,um. (D) A small T cell cluster showing stainingfor VCAM-1 on afew central cells, probably including a small vessel. Bar = 20 ,um. (E) Extensive VCAM-l staining at the centre of a large lymphocyte aggregate. Bar = 40 jim. (F) Stainingfor a4 integrin chain showing positive cells diffusely within a lymphocyte aggregate, and also scattered among the tissue stroma. Bar = 20 ,um. 810 Edwards, Wilkinson, Speight, Isenberg

Despite the demonstration that VCAM- 1 lymphocytes use the a4/1 1 heterodimer as an

binding to the T cell surface of oA4/1 1 integrin assistance to cell migration. The c4/1 1 com- Ann Rheum Dis: first published as 10.1136/ard.52.11.806 on 1 November 1993. Downloaded from can increase the adhesion of T cells to bination also binds fibronectin and it may be endothelial cells in vitro, it seems unlikely that that the T cell responds to the binding ofoA4/ 1 VCAM-1 expression is important in T cell integrin to a ligand by increased movement infiltration in the tissues studied. In samples rather than by continued attachment. In where lymphocyte infiltrates were composed contrast, when oA4/ 1 integrin on B cells comes almost exclusively of T rather than B cells, into contact with VCAM-1, the B cell may VCAM- 1 expression on the vessels was respond by a reduction or end of movement minimal. and, if concomitant antigen specific signals are In contrast, B cells were not seen at the appropriate, by proliferation. There is also an centre of aggregates unless VCAM-1 positive outside possibility that an alternative ligand cells were also present. There is evidence that for VCAM- 1, not involving either c4 or 131 in lymphoid tissues B cells migrate to the integrin chains, could be preferentially centre of follicles from the surrounding tissue expressed on B lymphocytes. Either way it through the outer T cell rich zone.32 In would appear that B cells allow VCAM- 1 inflamed tissues, however, it seems possible positive cells to envelope them, giving close that B cells appearing at the centre of correspondence between the extent of staining aggregates have entered the tissue through for VCAM- 1 and the B cell marker. VCAM-1 expressing venules at the centre of The absence of prominent a-4 and 1 the aggregate. This difference from lymphoid integrin chain staining from the B cell rich tissue would not, perhaps, be surprising. In areas in the tissues studied may not mean that lymph nodes lymphocyte entry is thought to the B cells'did not carry larger amounts of the occur chiefly through the specialised high integrin on arrival at the site. a4 and 1 endothelial venules of the paracortical T cell integrin chains have been found to be absent areas. High endothelial venules are not present or only weakly expressed on the B cells in constitutively in synovium, but appear in lymphoid tissue of normal human tonsils, rheumatoid arthritis vessels with similar Peyer's patches, and peripheral lymph nodes,2' properties, though only in areas rich in T but circulating B cells and B cells activated in cells.33 It seems reasonable to suggest that once vitro express more of o4 and 13. The fact that T cells have accumulated in sufficient numbers staining for the integrin chains was present in to form substantial aggregates they induce any amount in the B cell rich areas of diseased VCAM-1 expression on cells in and around the tissue (in contrast with a virtual absence in B walls of venules lying within the aggregates, cell rich areas of tonsils) suggests that in and that the influx of B cells is then facilitated diseased tissue a higher proportion of B cells at this site. may have recently immigrated from the The most unexpected finding of the study circulation or be undergoing activation. was the lack of correlation between the VCAM- 1 is expressed strongly on fibro-

distribution ofVCAM- 1 expression and that of blastic synoviocytes, where it may interact with http://ard.bmj.com/ the integrin chains. This could suggest that the integrin on the surface of the closely associated degree of expression of oA and 13 integrin macrophages. The function of such an inter- chains on lymphocytes does not determine action is unknown. Thus it appears that a whether or not these cells associate with molecule initially implicated specifically in VCAM-1 positive cells, raising the question of leucocyte/endothelial interactions has a num- the function ofVCAM- 1 expression at this site. ber of roles in diseased non-lymphoid tissues

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