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. 1998 Sep;57(9):550–558. doi: 10.1136/ard.57.9.550

Increased expression of human type IIa secretory phospholipase A2 antigen in arthritic synovium

O Jamal 1, P Conaghan 1, A Cunningham 1, P Brooks 1, V Munro 1, K Scott 1
PMCID: PMC1752736  PMID: 9849315

Abstract

OBJECTIVE—To determine the localisation and level of expression of human type IIa secretory phospholipase A2 (sPLA2) in the synovium of rheumatoid arthritis (RA), osteoarthritis (OA), and non-arthritic (NA) patients and to examine the relation between sPLA2 and histological features of inflammation.
METHODS—Immunoperoxidase staining using the anti-sPLA2 monoclonal antibody 9C1 was performed on frozen sections of knee synovium of 10 RA, 10 OA, and 10 NA patients. sPLA2 positive cells were scored on a scale of 0-3 in 10 fields of a representative tissue section from each case. Double labelling imunofluorescence confocal microscopy with antibodies to CD14 or CD45 and 9C1 was used to determine cell type specificity. Inflammation was assessed by semiquantitative scoring of lining layer thickness and mononuclear cell infiltrates (MC) and a cumulative inflammation score, generated by summing the two parameters. Scores in each group were compared using non-parametric statistical analysis.
RESULTS—sPLA2 was localised to endothelium (EC), vascular smooth muscle (VSM), and mast cells (M) in all tissue sections. In RA and OA sections, staining was seen in both macrophage-like and fibroblast-like cells in the synovial lining layer (LL) and subsynovial lining layer (SLL). Perineural cells stained positively. Subintimal lymphoid aggregates (LA) were negative in all sections. The RA group showed significantly greater staining in extravascular synovial tissue (median 3.6, range 1.5-6.0) than the OA (median 1.95, range 0-5.3) or NA (median 0, range 0-5.9) groups (p<0.05). LL staining was significantly higher in RA than both OA and NA sections (p<0.05). The OA group showed a trend to higher staining scores than the NA group that did not reach significance. There was a significant correlation between the sPLA2 staining score and inflammation score within the RA patient group (p<0.05).
CONCLUSIONS—The synovium is a site of increased expression of sPLA2 antigen in both RA and OA relative to NA. Its presence in both fibroblast and macrophage-like cells in the LL and SLL of synovial tissue in RA and OA, but not NA, indicates that the enzyme is specifically induced in these regions in both conditions with expression in the LL being particularly characteristic of RA. The widespread expression of sPLA2 in synovium suggests it is likely to play a significant part in synovial pathology

 Keywords: synovium; phospholipase A2; immunohistochemistry; confocal microscopy

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Figure 1  .

Figure 1  

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Immunolocalisation of sPLA2 in synovial tissue from RA (A-C), OA (D-E), and NA (F) patients. (A) Patient no 7 (table 1); synovial lining layer cells (LL) and subsynovial lining layer cells (SLL) stained strongly. No staining was observed on subintimal lymphoid aggregates (LA). (B) Control antibody 81193 demonstrates no significant staining in an adjacent section to that shown in A. (C) Patient no 8 (table 1) strong positive staining is seen in endothelial cells (EC) and mast cells (M). Vascular smooth muscle cells (VSM) were not positively stained in this field. (D) Patient no 15 (table 1): the majority of cells in SLL and one vessel stained positively for sPLA2 with no staining in the LL. (E) Patient no 15 (table 1) perineural cells (arrows) of a large nerve, and vascular smooth muscle cells (VSM) stained positively. (F) Patient no 24 (table 1) LL and SLL cells were not stained. Positive staining was observed in some endothelial cells (arrows). (Original magnification × 400 for A-C, × 200 for D-F). (Bar marker=25 µm for A-C; 50 µm for D-F).

Figure 2  .

Figure 2  

Figure 2  

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Double labelling immunofluorescence of rheumatoid synovial tissue for sPLA2 and cell type specific markers imaged by confocal microscopy. In each group of three images, A-C, D-F, G-I, and J-L, FITC (A,D,G,J) and TRITC (B,E,H,K) channels are each presented separately followed by the two channels viewed simultaneously (C,F,I,L). Immunofluorescence staining was performed on synovial tissue with CD14-FITC and sPLA2 detected with antimouse-rhodamine (A-F). CD14 and sPLA2 staining is seen in the lining layer (LL) and sublining layer (SLL) with sPLA2 also staining blood vessels(BV). D-F show a higher magnification (the region identified by the dotted lines in C). Both sPLA2 positive, CD14 negative cells (indicated by arrows in F) and sPLA2 positive, CD14 positive cells (indicated by arrowheads) were found in the LL and SLL. Rheumatoid synovium was also dual stained with CD45-FITC; and sPLA2-antimouse-rhodamine (G-I). Positive staining was seen in both the LL and SLL for both markers. sPLA2 positive, CD45 negative (thin arrow), sPLA2 positive, CD45 positive (arrowhead) and CD45 positive, sPLA2 negative cells (thick arrow) were identified. J-L shows a representative section from synovial tissue dual labelled with control antibodies IgG-FITC and murine monoclonal 81193 detected with antimouse-rhodamine. Space bars in F and L represent 40 µm in A-C and 20 µm in D-L.

Figure 3  .

Figure 3  

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Semiquantitative analysis of sPLA2 staining of synovial tissue from RA, OA, and NA patients. (A) Comparison of sPLA2 staining by site in synovial tissue of the three patient groups. EC, endothelium; VSM, vascular smooth muscle; SLL, subsynovial lining layer; LL, synovial lining layer. sPLA2 staining score is the median of the mean staining scores calculated from up to 10 fields per patient as described in Methods (table 1). RA group, (filled bars); OA group, (open bars); NA group (shaded bars). (B) Comparision of sPLA2 staining in extravascular tissue of the three patient groups. The extravascular sPLA2 score for each group is the median of the mean staining score for SLL and LL. Statistical analyses are as described in Methods. * Indicates significance of p<0.05.

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