Zerumbone`s Effect on Major Histocompatibility Complex Type II Cells in Synovial Membrane of OsteoArthritic Joint

MATERIALS AND METHODS

Animals and Treatment
Thirty eight adult Sprague Dawley rats were used in this study. All animals will have free access to water and pellet food. The study was conducted in April 2007 at the Faculty of Veterinary Medicine, Universiti Putra Malaysia. Osteoarthritis was induced by injecting collagenase into the right stifle joint at D1 and at D4 (approved by animal ethic committee animal utilization protocol No. 07R7). The rats were supplemented orally with either 0.5 mL corn oil (control group; n = 19) or 0.5 mL of 0.02% w/v zerumbone in corn oil (experimental group; n = 19) from D7-D28. At D29 the rats were sacrificed and synovial membrane samples from the right stifle joint and contralateral joint (left stifle joint) were processed for immunohistochemistry work.

Light Microcopy Immunohistochemistry
The synovial membrane samples were fixed in 4% paraformaldehyde overnight and then processed following a standard immunohistochemistry method. Briefly the samples were frozen in liquid nitrogen cooled in isopenthane and sectioned at 6 μm. The sections were dehydrated in ethanol, rinsed in 0.1 M Phosphate Buffered Saline (PBS) and then incubated in monoclonal antisera against MHC II (a kind gift from Dr. C. Vaillant, University of Liverpool), to mark the antigen presenting cells. Sections were then incubated in secondary antiserum and subsequently with avidin- biotinylated HRP complex. Samples were rinsed in PBS after each step. Finally, sections were immersed in glucose diaminobenzidine nickel substrate, washed in distilled water and stained with hematoxyline.

Preembedding Electron Microscopy Immunohistochemistry
Electron microscopic technique was used to study the type of synovial cells that express MHC II. A pre-embedding technique was used. Briefly samples were dehydrated in 50% ethanol for 30 min, then in PBS before immersing in 1% sodium borohydride (NaBH₄) in PBS for 40 min. Incubation in primary, secondary and tertiary were the same as the light microscopy staining technique but for a longer period of time. Samples were post-fixed in 1% osmium tetraoxide (OsO₄) in 0.1 M sodium cacodylate [(CH₃)₂As(O)O.Na.xH₂O)] buffer for 1 h, then dehydrated in a varying concentration of ethanol and then followed by acetone. The samples were then incubated in a mixture of 50% TAAB resin in acetone at room temperature, then in 100% TAAB resin for 5 h at room temperature for better penetration of the resin before polymerizing at 60°C overnight. Semithin sections were stained with toluidine blue and examined under the light microscope. Areas containing labelled cells were selected for ultrathin sectioning. These sections were examined under transmission electron microscope, stained with lead citrate (C₁₂H₁₀O₁₄Pb₃) for 5 min, rinsed in distilled water and then stained with uranyl acetate [UO₂(CH₃COO)₂.2H₂O)] for 5 min before rinsing again in distilled water. Finally sections were examined under transmission electron microscope.

Statistical Analysis
ANOVA was used to analyse the data.

RESULTS

The synovial membrane of control group (corn oil) joints exhibited varying degrees of mononuclear cell infiltration (Fig. 1-4). The synovial cell hyperplasia with 3-4 lining cells was observed. However, inflammatory changes in zerumbone treated group were less prominent than corn oil group.

Staining for MHC class II antigens, revealed that most of the synovial lining cells in the corn oil treated were positive for MHC II. In the zerumbone treated group, the numbers of MHC II + cells were significantly lower compared to the corn oil treated group (Table 1). When compared to the synovial membrane samples from the contralateral (left stifle) joint, the MHC II + cells were significantly lower for both corn oil and zerumbone treated groups (Table 1).

In the synovial membrane from zerumbone treated group, there was a marked decrease in the number of lining cells but in the corn oil treated joints, there was a marked increase in the number of synovial lining cells and virtually all the cells stained positive for MHC II (Fig. 1). In the zrumbone treated synovial membrane, the proportion of lining cells stained varied between different regions of the sections. In some regions the majority of lining cells showed positive labeling for MHC II, whereas in other regions only the outer layer of cells was positive. Furthermore in the subintimal layer of zerumbone and corn oil treated group, a small number of scattered cells were also positively stained (Fig. 2). The MHC II + cells decreased in number compared to the corn oil group. Most of the MHC II + scattered cells in the subintimal layers of both corn oil and zerumbone samples had dendritic cell morphology with spine-like projections.

Fig. 1:	Immunostaining for MHC class II in synovial membrane obtained from zerumbone (A) treated group. There were very few number MHC II + cells. The MHC II+ cells in the subintimal layer had dendritic cell morphology (→) (x670)

Fig. 2:	Immunostaining of corn oil treated group. Hyperplasia of the lining cells is very clear and almost all of these cells are positive for MHC II (x670)

Fig. 3:	Electron micrograph of rat type B cells with prominent rough endoplasmic reticulum (x25000)

Fig. 4:	MHC II labeling of rat synovial membrane from corn oil treated group. More type A cells are present. Only type A cells (A) are labelled for MHC II. Notice also that some of the type A cells are not labelled (x10000)

Immunoperoxidase staining at the electron microscopy level, revealed two types of synovial cells: the type A cell which is phagocytic cell and type B cell which is a fibroblast-like cells (Fig. 3, 4). The type A cell contained a prominent Golgi apparatus and some lysosomes but little rough endoplasmic reticulum, whereas the type B cells contained abundant rough endoplasmic reticulum (Fig. 3, 4).

Table 1:	The number of MHC II+ cells in the synovial membrane from the corn oil (control) and zerumbone treated group
abMean with different superscript within the row are significantly different (p<0.01); xyMean with different superscript within the column are significantly different (p<0.01)

In zerumbone treated samples, there were more type B than type A cells, whereas in the corn oil samples type A cells predominated. In this type A cells, there is an increase in the number of lysosomes compared to type A cells in zerumbone treated synovial membrane. More type A cells were observed in corn oil treated group compared to the zerumbone treated group. In both corn oil and zerumbone treated synovial membrane, only type A cells were MHC II positive. Type B cells did not show any positive labeling for MHC II (Fig. 3, 4).

DISCUSSION

The results of the current study showed that supplementation of zerumbone reduce the percentage of inflammatory cells in the synovial membrane of OA. This was clearly demonstrated by the reduction of MHC class II expressing cells in the synovial membrane of zerumbone treated group. The labelled cells in this study had the morphology of type A cells. Thus, type B cells were not induced to express MHC II. In man, type A cells are known to be antigen presenting cells (Burmester et al., 1983). The present study reports that rat type A cells express MHC II and are, therefore antigen presenting cells in this species. The number of MHC II expressing cells increased in synovial membrane from corn oil treated samples. In humans, an increase in MHC II (HLA-DR) expressing cells has been reported in synovial membrane from cases of osteoarthritis and rheumatoid arthritis (Thomas et al., 1994; Athanasou et al., 1988). A number of studies have shown that MHC II + cells increase in the synovial membrane in human rheumatoid arthritis where they play a major role in arthritic changes by activating T- lymphocytes in the synovial membrane (Trautwein et al., 1999; Kumar Bhardwaj, 1995; Klareskog, 1991; Athanasou et al., 1988). The factors stimulating this increase are not known but the sensory neuropeptides, substance P is known to modulate synoviocyte function and exert chemotactic effects on antigen presenting cells (Shanthi et al., 2004; Matucci Cerinic et al., 1995).

The current study also shows that zerumbone treatment reduces the number of MHC II expressing cells in the synovial membrane and thus, reduces the number of type A cells which are present during arthritis. The reduction of these cells clearly demonstrates that zerumbone does have some anti-inflammatory effect that acts on these cells that are present during inflammation.

The anti-inflammatory action of zerumbone has been claimed to be the basis of a positive effect on symptoms in rheumatic disorders (Srivasta and Mustafa, 1992). However, the anti-inflammatory mechanism of action of zerumbone on the OA joint is beyond the scope of the current study but it may be due to high content of HMP which is a dual inhibitor of cyclooxygenase on 5-lipoxygenase which makes zerumbone even more interesting in the field of rheumatology (Murakami et al., 2002, 2005; Sharma et al., 1994).

The current research also showed a positive correlation between MHC class II cells and arthritis. This finding can be used to indicate inflammation in osteoarthritis. However, the 28 days periods therapy in this study might not have been sufficient for all effects of zerumbone to be discovered. Future studies might look into dose-response and duration of therapy.

ACKNOWLEDGMENT

The author wish to thank Universiti Putra Malaysia, as this study was supported by E Science grant (Project number: 02-01-04-SF0882) awarded by Ministry of Science, Technology and Innovation.