High synovial expression of the inhibitory FcgammaRIIb in rheumatoid arthritis

Abstract

Activating Fc gamma receptors (FcgammaRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcgammaRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcgammaRIIb isoform. FcgammaRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcgammaRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcgammaRI, FcgammaRII and FcgammaRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcgammaRII, FcgammaRIII but not FcgammaRI, all activating FcgammaRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcgammaRIIb and the activating FcgammaRs. Anti-inflammatory treatment with glucocorticoids reduced FcgammaR expression in arthritic joints, particularly that of FcgammaRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcgammaRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcgammaRIIb and activating FcgammaRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcgammaRs may represent valuable therapeutics in this disease.

Figure 1

The monoclonal antibody (mAb) GB3 is specific for human Fc gamma receptor (FcγR)IIb. (a-c) Human B cell (Raji) or monocyte (U937) cell lines were stained with either the anti-FcγRIIb mAb GB3, the anti-FcγRIIa mAb IV.3 or the pan anti-FcγRII mAb KB61. Filled graphs represent the specific antibody and dotted graphs the isotype control antibody. Raji cells were clearly stained positive for FcγRIIb by the GB3 mAb, while U937 cells were negatively stained, thus demonstrating the FcγRIIb specificity of the GB3 mAb (a). Raji cells were, somewhat surprisingly, stained positive by IV.3 mAb and, as expected, so were the U937 cells (b). This indicates that the IV.3 mAb detects FcγRIIa but is also cross-reactive to FcγRIIb. Both cell lines were positively stained by the pan anti-FcγRII mAb, detecting both the a and b isoforms (c). (d) The epitope specificity of the GB3 mAb as demonstrated by ELISA. Serially diluted GB3 mAb show binding activity to recombinant sFcγRIIb whereas no reactivity to recombinant sFcγRIIa.

Figure 2

Expression of Fc gamma receptor (FcγR)IIb in healthy and rheumatoid arthritis (RA) synovia. (a) FcγRIIb was sparsely expressed in the few healthy synovial biopsies that were positively stained by GB3. The arrows indicate FcγRIIb positive cells in the sub-lining synovial layer and tissue. (b) The IgG1 isotype control antibody did not stain the healthy synovium. (c,e) Positive FcγRIIb expression was found in all the stained RA synovial biopsies. FcγRIIb staining was observed in the synovial lining and sub-lining layers, perivasculary and inside lymphocyte infiltrates (e). (d) No staining of the RA synovium was observed when the anti-FcγRIIb monoclonal antibody was bound by recombinant human soluble FcγRIIb prior to staining. (f) Synovial FcγRIIb expression was significantly up-regulated in RA patients (n = 10) compared to healthy individuals (n = 5) (mean values of two independent observers, SEM and SK with standard error of the mean, *p = 0.0104). DAB, diaminobenzidine. (Original magnification ×125.)

Figure 3

Increased Fc gamma receptor (FcγR)I, II and III expression in rheumatoid arthritis (RA) synovia. (a) Sequentially sectioned synovial biopsy from a healthy individual demonstrating FcγRII and III, but not FcγRI, positive cells. The same expression pattern was seen in all healthy volunteers studied (n = 10). (Original magnification ×250.) (b) Sequentially sectioned synovial tissue from a RA patient demonstrating FcγRI, II and III positive cells in the synovial lining and sub-lining layers and perivasculary. Note the dense staining of FcγRIII in the synovial lining layer. The FcγR stainings were representative of all other RA tissues stained (n = 26). IgG1 isotype control stainings were included for all the patient material used. (Original magnification ×125.) (c) The expression of FcγRs in healthy (n = 10), early (n = 11) and late RA (n = 15) synovial tissue was evaluated and compared. Note that FcγRI was not detected in healthy synovial tissue and that FcγRI, FcγRII and III were significantly more expressed in both early and late RA synovial tissue compared to healthy synovia (mean values of two independent observers, SEM and SK with standard error of the mean; **p < 0.01; ***p < 0.001). DAB, diaminobenzidine.

Figure 4

Co-localisation of CD163 with Fc gamma receptor (FcγR)I, II, IIb and III. (a-d) Rheumatoid arthritis synovial tissue showed overlapping expression of the macrophage marker CD163 with the expression of FcγRI (a), FcγRII (b), FcγRIIb (c) and FcγRIII (d). The majority of the CD163 positive macrophages expressed FcγRIIb. However, note that a CD163 positive cell lacked FcγRIIb expression. (Original magnification ×400.)

Figure 5

Fc gamma receptor (FcγR) expression correlates with the presence of T cells in rheumatoid arthritis (RA) synovium. (a) Sequentially sectioned synovial tissue from a late stage RA patient demonstrating FcγRI, II and III positive cells perivasculary. The FcγR positive cells are located in the same area as CD3 positive T cells. Note the FcγRIII positive cell inside the vessel. This staining was representative of all other late RA synovial tissues stained in the same way (n = 26). The IgG1 isotype control did not show any background (data not shown). (Original magnification ×250.) (b) The expression of FcγRI, II and III was significantly correlated with the expression of CD3 in RA synovium (each symbol represents one patient, n = 26).

Figure 6

Decreased Fc gamma receptor (FcγR) expression after local treatment with glucocorticoids. (a) Immunohistochemical staining of FcγRs and macrophages (CD163) in arthritic synovial tissue (n = 9) before and after local glucocorticoid treatment of the joint was analysed. The FcγRI expression was significantly decreased after treatment (*p < 0.05) and there was a trend towards decreased expression of FcγRII (p = 0.074). (b,c) Staining of FcγRI on arthritic synovial biopsy, taken before and after one intraarticular injection of glucocorticoids, from the same biopsy area and patient. Note the reduced FcγRI staining after treatment (c). DAB, diaminobenzidine.