**3. Protein chip analysis**

#### **3.1 ECM degradation**

A RayBio biotin label-based human antibody array I (RayBiotech, Inc.) was used to measure the expression levels of 507 proteins in culture medium conditioned by MH7A cells. Table 1 presents the results of the biotin label-based antibody array analysis of secreted proteins that categorized interleukins, and molecules involved in ECM metabolism. IL-12 p70 and IL-8 were

Profiling Inflammatory Genes

**4. Medical treatments for RA** 

quality of life of RA patients.

as possible.

cardiovascular system (Suissa et al., 2006).

**4.2 Proposal of RA therapy using linear polarized infrared light** 

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 161

Several disease-modifying anti-rheumatic drugs (DMARDs) have been used to control the progression of RA. While the majority of these drugs act as immunomodulatory agents, some also act by inhibiting cytokines and endothelial cell proliferation (Cozzolino et al., 1993, Volin et al., 1999). Newly developed biological response modifiers (biologics) offer even more hope, having a greater potential to suppress disease activity, and improve the

**4.1 Disease-modifying anti-rheumatic drugs, TNF- blockers, and other agents** 

Guidelines advocate treatment with DMARDs as soon as RA is diagnosed to control symptoms and delay disease progression (Smolen & Steiner, 2003, O'Dell, 2004, Saag et al., 2008). DMARDs slow the progression of the joint damage that leads to loss of function, whereas drugs such as nonsteroidal antiinflammatory drugs and corticosteroids only control the symptoms (Kirwan & Power, 2007). On the other hand, targeted inhibition of TNF- is an effective therapy widely used against RA and other rheumatic diseases. The advent of TNF blockers has led to substantial improvement in the management of active RA refractory to conventional DMARDs. While on a patient-population level, the efficacy of the TNF blockers infliximab, etanercept, and adalimumab seems comparable, on an individual level, there are no clear-cut methods for assessing whether there will be a response to anti-TNF therapy, let alone which agent would yield the best outcome. The updated consensus statement on biological agents for the treatment of rheumatic diseases states that the optimal treatment for anti-TNF refractory RA remains to be determined. However, most importantly, it is not yet possible to identify prior to therapy which patients will fail to respond or who are at increased risk for adverse drug reactions (Mendonça et al., 2011). Given the important role that TNF-α antagonists play in managing rheumatoid arthritis and the concern for safety during long-term therapy, such factors need to be determined as soon

Glucocorticoids have potent immunosuppressive effects and have been widely used in the management of chronic inflammatory diseases such as severe RA. A number of studies have focused on the cellular and molecular mechanisms underlying the anti-inflammatory effects of glucocorticoids such as dexamethasone (Dex). Glucocorticoids exert their effects via intracellular receptors that act as potent transcriptional activators of genes that possess glucocorticoid responsive elements. By regulating gene expression, glucocorticoids suppress the production of pro-inflammatory proteins, such as cytokines, chemokines and some enzymatic mediators. However, the therapeutic management of long-term pathological conditions with steroids is often linked to unwanted side effects involving the

It is unclear whether early treatment with biologics should continue to be recommended, given their potential to slow disease progression and extend productivity on the one hand, while causing unwanted side effects and risks due to drug-associated health care utilization on the other. Therefore, we examined the potential of photodynamic therapy using a linear polarized infrared light (LPIL). Laser therapy is a new arthroscopic technique to treat inflammation of the synovial membrane. Following gallium-aluminum-arsenide (Ga-Al-As)

both abundantly expressed in the IL-1-induced MH7A cells (57.8- and 12.6-fold increases, respectively). IL-12 is a heterodimer expressed in RA synovial tissue (Morita et al., 1998); dendritic cells and macrophages produce IL-12 within minutes of activation, and it plays a central role in the development of Th1 responses (Hunter, 2005). The biological functions of these cells are not yet fully understood, but they are known to play a role in the differentiation and proliferation of T cells of the Th1 phenotype. Th1 responses in turn are deemed to be of particular importance in inflammatory arthritis, including RA. This induction of IL-12p70 by IL-1 in MH7A cells may be the first *in vitro* evidence of IL-12 expression in RA. This is noteworthy, because Connell & McInnes (Connell & McInnes, 2006) suggested in their review that IL-12 likely contributes to the pathology of inflammatory joint disease. If this is the case, it may represent a potentially useful therapeutic target.


RayBio biotin label-based human antibody array I (RayBiotech, Inc.) was used to measure the expression levels of 507 proteins in culture medium conditioned by MH7A cells. The cell were cultured for 16 h and then treated with IL-1 (0.1 U/ml) for 3 h. The cultured medium from five wells was then combined and dialyzed against PBS (pH 8.0), and an internal control protein was added to the 400 µl of dialyzed sample medium. The procedure used for biotin labeling, blocking and incubation of the antibody array, as well as fluorescence detection were carried out according to the operating manual from RayBiotech. The images were captured using an Axon GenePix laser scanner. Biotinylation of the internal control protein yielded a positive control signals that was used to identify the orientation of the results and help normalize them for comparison of those from the untreated and IL-1-treated MH7A cell arrays. Values below 0.01 were set to 0.001.

Table 1. IL-1-induced protein.

On the other hand, the levels of two MMP family enzymes, MMP-10, and MMP-11 were upregulated by IL-1, while the levels of TIMP-1 and -3 were downregulated. As the overall MMP activity reflects a balance between the amount of MMPs and TIMPs present, the reduction in TIMP-1 and TIMP-3 levels seen in MH7A cells would be expected to shift the balance in favor of increased MMP activity. Taken together, these data suggest that IL-1 elicits a series of responses in the synovial fluid, leading to the pathology seen in RA, including enhanced expression of inflammatory cytokines and enzymes, and a reduction in the ability to protect against the destruction of the tissue ECM.
