**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 as possible.

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 cardiovascular system (Suissa et al., 2006).

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

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)

Profiling Inflammatory Genes

CXCL1 chemokine (C-X-C motif) ligand 1,

CXCL2 chemokine (C-X-C motif) ligand 2,

CXCL3 chemokine (C-X-C motif) ligand 3,

**Gene Title Affy** 

pulse energy.

al., 2003, Mattioli et al., 2004).

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 163

IL8 interleukin 8, CXCL8 20.2 0.86/0.04 (P/A) 5.6 7.6

IL6 Interleukin 6 2.7 5.51/2.07 (P/P) 1.0 2.2

IL1B Interleukin 1, beta 2.5 2.33/0.92 (P/M) 1.4 1.5

Table 2. Inhibitory effects of anti-inflammatory treatments on IL-1-induced gene expression inhibitory effects of LPIL irradiation on IL-1 inducible inflammation, we next used an ELISA to monitor IL-1 induced IL-8 synthesis and secretion from MH7A cells. We found that IL-1 strongly induced IL-8 production, and that the effect was similarly suppressed by Dex or LPIL (Fig. 6). We compared the effects of one-time LPIL irradiation and Dex administration on the production of IL-8. Even one period of exposure to LPIL irradiation diminished the IL-1-induced production of IL-8 to the same extent as administration of 1 µM Dex to the cells. Furthermore, using end-point PCR and real-time PCR, we determined that there were corresponding decreases in the expression of IL-8 mRNA (Fig. 6). It is also noteworthy that the inhibitory effects on IL-8 transcription increased with increasing optical

The genes whose products are involved in NF-B signaling (e.g., NFKB1, RelA, and C/EBP-) were not induced by IL-1 stimulation as determined by the microarray analysis. On the other hand, increasing evidence has highlighted the importance of NF-B modification in the regulation of its transcriptional activity. For instance, it appears that NF-B activity can be modulated by phosphorylation of the RelA subunit. Recent evidence indicates that the phosphorylation of the RelA subunit on S276 and S536 is physiologically induced by various proinflammatory stimuli. Zhong *et al*. (2002) found that LPS stimulates protein kinase A-dependent phosphorylation of RelA on S276 and subsequently recruits the transcription coactivator CREB binding protein (CBP)/p300 to stimulate NF-B-mediated transcription. Furthermore, it appears that S536 of endogenous cytoplasmic RelA is phosphorylated in response to a wide variety of NF-B stimuli and is then rapidly dephosphorylated in the nucleus (Sakurai et al., 1999, Sakurai, 2003, Yang et

Because transduction of the IL-1 signal is tightly regulated in MH7A cells via a network involving NF-B, we tried to measure the levels of NF-B phosphorylation. As shown in Fig. 7, the total levels of NF-B-bound RelA were nearly the same in all of the treated MH7A cells (right panel). Phosphorylation of the RelA subunit on both S276 and S536 was significantly increased by IL-1 stimulation, and was decreased in the presence of Bay11-

**Fold Normalized intensity** 

GRO1 4.1 3.36/0.82 (P/P) 3.2 1.8

GRO2 4.0 3.78/0.94 (P/P) 2.4 3.1

GRO3 2.7 0.85/0.32 (M/A) 3.2 2.1

**IL-1/None IL-1+Dex** 

**Raw** 

**/None** 

**Data Fold Fold** 

**IL-1+LPIL /None** 

laser treatment, histological examination of the irradiated synovial membranes showed flattening of the epithelial cells, decreased villous proliferation, narrower vascular lumens and less infiltration by inflammatory cells than was seen in non-irradiated synovia. An alternative approach makes use of a linear polarized infrared light (LPIL) instrument (Super Lizer™, Tokyo Iken Co., Ltd), which has been used clinically with good effects in several patients with inflammatory disease. LPIL can be applied during physical therapy to relieve pain, and several studies have shown that such irradiation also relieves temporomandibular joint pain (Yokoyama & Oku, 1999), improves the structure of stored human erythrocytes (Yokoyama & Sugiyama, 2003), and improves the flexibility of shoulder and ankle joints (Demura et al., 2002). A fuller understanding of the anti-inflammatory mechanism of LPIL in rheumatoid synoviocytes could serve as the basis for improved treatment of RA patients in the future.

We treated MH7A cells IL-1 (0.1 U/ml) and/or Dex (1 µM) or LPIL (3.8 J/cm2). Microarray experiments were then carried out using the Affymetrix Focus Array HG-8500 GeneChip. As shown in Fig. 5A, the transcription of numerous genes was up- or down-regulated to a greater degree in cells treated with Dex than in those treated with LPIL. Likewise, the effects of LPIL irradiation on the transcription levels of many genes were smaller than those of IL-1. In total, 120 (IL-1), 561 (Dex), 877 (Dex/IL-1), 88 (LPIL) and 90 (LPIL/IL-1) genes were up- or downregulated by the different treatments (Fig. 5B).

The global gene expression in MH7A cells. The normalized gene expression is shown. MH7A cells were treated with Dex or LPIL in the presence or absence of IL-1 before the extraction of their RNA. N, untreated cells; IL, IL-1-treated cells; D, Dex-treated cells. **B.** The numbers of up- and down-regulated genes. Genes whose expression was increased or decreased by over two-fold are indicated. [This figure is modified from (Shibata et al., 2009b)]

Fig. 5. Microarray data analysis.

We focused on the genes that were up-regulated by at least 2.0-fold in cells treated with IL-1 and then suppressed by the anti-inflammatory treatments. IL-8 transcripts showed the largest increase (20.8 fold), and LPIL and Dex each suppressed this gene expression. Others genes, including CXCL1, 2, 3, IL6, and IL1B, which respectively encode Gro-, Gro-, Gro-, IL-6 and IL-1, were also suppressed by LPIL to the same extent as by Dex (Table 2). Thus, although for the most part LPIL irradiation did not alter the transcription levels to the same degree as Dex did, IL-1 inducible genes, such as the CXCL type chemokines, IL6 and IL1B, were clearly suppressed by LPIL irradiation. To evaluate the

laser treatment, histological examination of the irradiated synovial membranes showed flattening of the epithelial cells, decreased villous proliferation, narrower vascular lumens and less infiltration by inflammatory cells than was seen in non-irradiated synovia. An alternative approach makes use of a linear polarized infrared light (LPIL) instrument (Super Lizer™, Tokyo Iken Co., Ltd), which has been used clinically with good effects in several patients with inflammatory disease. LPIL can be applied during physical therapy to relieve pain, and several studies have shown that such irradiation also relieves temporomandibular joint pain (Yokoyama & Oku, 1999), improves the structure of stored human erythrocytes (Yokoyama & Sugiyama, 2003), and improves the flexibility of shoulder and ankle joints (Demura et al., 2002). A fuller understanding of the anti-inflammatory mechanism of LPIL in rheumatoid synoviocytes could serve as the basis for improved treatment of RA patients

We treated MH7A cells IL-1 (0.1 U/ml) and/or Dex (1 µM) or LPIL (3.8 J/cm2). Microarray experiments were then carried out using the Affymetrix Focus Array HG-8500 GeneChip. As shown in Fig. 5A, the transcription of numerous genes was up- or down-regulated to a greater degree in cells treated with Dex than in those treated with LPIL. Likewise, the effects of LPIL irradiation on the transcription levels of many genes were smaller than those of IL-1. In total, 120 (IL-1), 561 (Dex), 877 (Dex/IL-1), 88 (LPIL) and 90 (LPIL/IL-1) genes

The global gene expression in MH7A cells. The normalized gene expression is shown. MH7A cells were treated with Dex or LPIL in the presence or absence of IL-1 before the extraction of their RNA. N, untreated cells; IL, IL-1-treated cells; D, Dex-treated cells. **B.** The numbers of up- and down-regulated genes. Genes whose expression was increased or decreased by over two-fold are indicated. [This figure

We focused on the genes that were up-regulated by at least 2.0-fold in cells treated with IL-1 and then suppressed by the anti-inflammatory treatments. IL-8 transcripts showed the largest increase (20.8 fold), and LPIL and Dex each suppressed this gene expression. Others genes, including CXCL1, 2, 3, IL6, and IL1B, which respectively encode Gro-, Gro-, Gro-, IL-6 and IL-1, were also suppressed by LPIL to the same extent as by Dex (Table 2). Thus, although for the most part LPIL irradiation did not alter the transcription levels to the same degree as Dex did, IL-1 inducible genes, such as the CXCL type chemokines, IL6 and IL1B, were clearly suppressed by LPIL irradiation. To evaluate the

were up- or downregulated by the different treatments (Fig. 5B).

is modified from (Shibata et al., 2009b)] Fig. 5. Microarray data analysis.

in the future.


Table 2. Inhibitory effects of anti-inflammatory treatments on IL-1-induced gene expression

inhibitory effects of LPIL irradiation on IL-1 inducible inflammation, we next used an ELISA to monitor IL-1 induced IL-8 synthesis and secretion from MH7A cells. We found that IL-1 strongly induced IL-8 production, and that the effect was similarly suppressed by Dex or LPIL (Fig. 6). We compared the effects of one-time LPIL irradiation and Dex administration on the production of IL-8. Even one period of exposure to LPIL irradiation diminished the IL-1-induced production of IL-8 to the same extent as administration of 1 µM Dex to the cells. Furthermore, using end-point PCR and real-time PCR, we determined that there were corresponding decreases in the expression of IL-8 mRNA (Fig. 6). It is also noteworthy that the inhibitory effects on IL-8 transcription increased with increasing optical pulse energy.

The genes whose products are involved in NF-B signaling (e.g., NFKB1, RelA, and C/EBP-) were not induced by IL-1 stimulation as determined by the microarray analysis. On the other hand, increasing evidence has highlighted the importance of NF-B modification in the regulation of its transcriptional activity. For instance, it appears that NF-B activity can be modulated by phosphorylation of the RelA subunit. Recent evidence indicates that the phosphorylation of the RelA subunit on S276 and S536 is physiologically induced by various proinflammatory stimuli. Zhong *et al*. (2002) found that LPS stimulates protein kinase A-dependent phosphorylation of RelA on S276 and subsequently recruits the transcription coactivator CREB binding protein (CBP)/p300 to stimulate NF-B-mediated transcription. Furthermore, it appears that S536 of endogenous cytoplasmic RelA is phosphorylated in response to a wide variety of NF-B stimuli and is then rapidly dephosphorylated in the nucleus (Sakurai et al., 1999, Sakurai, 2003, Yang et al., 2003, Mattioli et al., 2004).

Because transduction of the IL-1 signal is tightly regulated in MH7A cells via a network involving NF-B, we tried to measure the levels of NF-B phosphorylation. As shown in Fig. 7, the total levels of NF-B-bound RelA were nearly the same in all of the treated MH7A cells (right panel). Phosphorylation of the RelA subunit on both S276 and S536 was significantly increased by IL-1 stimulation, and was decreased in the presence of Bay11-

Profiling Inflammatory Genes

the NF-kB RelA subunit

**6. Acknowledgements** 

22592078).

**7. References** 

interact with the promoter region of the IL8 gene.

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 165

MH7A cells were cultured for 16 h in 96 well plates and then used to assess the phosphorylation of the NFkB RelA subunit. Cells were irradiated with LPIL for 680 s immediately after stimulation with IL-1b for 2 h, or Dex was added to cells at the same time as IL-1b. Some cells were pretreated with Bay11-7085 for 1 h before addition of the IL-1b. After stimulation with IL-1b, the culture medium was removed, the cells were fixed by microwaving, and the levels of phosphorylation of the NF-kB RelA subunit on S276 and S536 were measured using a Cellular Activating of Signaling ELISA CASE™ kit. The results are expressed as the means ± SD (n=6); \*P < 0.005 vs. untreated. [This figure is modified from (Shibata et al., 2009a)] Fig. 7. The effects of Bay11-7085, LPIL or Dex on the phosphorylation of S536 and S276 of

IL-8, originally called monocyte-derived neutrophil chemotactic factor, is a potent chemokine, causing recruitment and infiltration of neutrophils and T cells into local inflammatory sites. The infiltration of neutrophils contributes to inflammation and has been implicated in various diseases. The transcription factors NF-B, AP-1, and NF-IL-6 are all involved in the stimulus-induced expression of IL-8. Subsequent IPA revealed that NF-B signaling was likely important for the synthesis and release of the IL-8 protein. We then found that IL-1-induced IL-8 release from MH7A cells requires the activation of the canonical NF-B pathway and kinases catalyzing its phosphorylation. Furthermore, treatments with Dex or LPIL suppressed the phosphorylation of S276 of the RelA subunit, which would in turn inhibit the translocation of NF-B into the nucleus, where it could

We gratefully acknowledge the assistance of Mrs Asayo Imaoka with the microarray technology. This research was supported in part by "Academic Frontier" Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology in 2007-2011, the Grants-in-Aid for Scientific Research (Kakenhi: 21390497,

Agro, A., Langdon, C., Smith, F. & Richards, C. D. (1996). Prostaglandin E2 enhances

interleukin 8 (IL-8) and IL-6 but inhibits GMCSF production by IL-1 stimulated

7085 (left and center panels). Interestingly, LPIL irradiation suppressed the phosphorylation of both S276 to the same degree as Dex in IL-1-stimulated MH7A cells.

The extent to which the risk of side-effects may be driven by drugs that decrease immune surveillance (notably, DMARDs including TNF- blockers, and glucocorticoid agents), is not completely clear. This issue has gained interest in light of reports of serious infections in patients receiving newer RA therapies.

The inhibitory effects of LPIL or Dex on the production of IL-8 in MH7A cells. An end-point PCR analysis shows that LPIL irradiation inhibits IL8, but not GAPDH, gene expression. The power level of the irradiation was changed by adjusting the power dial on the Super Lizer™ to 30%, 50%, or 80%. The duration of the irradiation was the same for all wells. A real-time PCR analysis shows the dose-dependent inhibitory effects of LPIL on IL-8 gene expression. [This figure is modified from (Shibata et al., 2009a)]

Fig. 6. The transcriptional regulation of IL-8 by LPIL and Dex in IL-1-stimulated MH7A cells.

#### **5. Conclusion**

To study the pathophysiological mechanisms involved in chronic inflammatory disorders such as RA, *in vitro* culture systems using IL-1-activated FLSs from RA patients have become useful models of the first step in RA-induced joint deterioration. Our findings demonstrate the utility of profiling the gene expression in IL-1 stimulated-MH7A cells, with the supposition that it is representative of the early disease stage of RA. By employing GeneChips and IPA, we were able to show that autocrine induction of IL-1 gene expression may underlie the strong expression of several inflammatory cytokines, including IL-8, IL-1 and IL-6, and suggest that continuous IL-1 production has an exacerbating effect on RA. In addition, we have identified upregulation of the BIRC3 gene as a candidate mediator of the FLS overproliferation in RA. BIRC3 encodes cIAP, which is a key negative regulator of apoptotic signaling in FLSs. The resulting inhibition of FLS apoptosis in the inflamed area would be expected to contribute to the formation of the pannus in the RA joint. It is our expectation that further studies of the comprehensive information provided by the GeneChip/IPA analysis and antibody array systems will provide a means to ease the suffering caused by RA.

7085 (left and center panels). Interestingly, LPIL irradiation suppressed the phosphorylation

The extent to which the risk of side-effects may be driven by drugs that decrease immune surveillance (notably, DMARDs including TNF- blockers, and glucocorticoid agents), is not completely clear. This issue has gained interest in light of reports of serious infections in

The inhibitory effects of LPIL or Dex on the production of IL-8 in MH7A cells. An end-point PCR analysis shows that LPIL irradiation inhibits IL8, but not GAPDH, gene expression. The power level of the irradiation was changed by adjusting the power dial on the Super Lizer™ to 30%, 50%, or 80%. The duration of the irradiation was the same for all wells. A real-time PCR analysis shows the dose-dependent inhibitory effects of LPIL on IL-8 gene expression. [This figure is modified from (Shibata et al., 2009a)] Fig. 6. The transcriptional regulation of IL-8 by LPIL and Dex in IL-1-stimulated MH7A

To study the pathophysiological mechanisms involved in chronic inflammatory disorders such as RA, *in vitro* culture systems using IL-1-activated FLSs from RA patients have become useful models of the first step in RA-induced joint deterioration. Our findings demonstrate the utility of profiling the gene expression in IL-1 stimulated-MH7A cells, with the supposition that it is representative of the early disease stage of RA. By employing GeneChips and IPA, we were able to show that autocrine induction of IL-1 gene expression may underlie the strong expression of several inflammatory cytokines, including IL-8, IL-1 and IL-6, and suggest that continuous IL-1 production has an exacerbating effect on RA. In addition, we have identified upregulation of the BIRC3 gene as a candidate mediator of the FLS overproliferation in RA. BIRC3 encodes cIAP, which is a key negative regulator of apoptotic signaling in FLSs. The resulting inhibition of FLS apoptosis in the inflamed area would be expected to contribute to the formation of the pannus in the RA joint. It is our expectation that further studies of the comprehensive information provided by the GeneChip/IPA analysis and antibody array systems will provide a means to ease the

of both S276 to the same degree as Dex in IL-1-stimulated MH7A cells.

patients receiving newer RA therapies.

cells.

**5. Conclusion** 

suffering caused by RA.

MH7A cells were cultured for 16 h in 96 well plates and then used to assess the phosphorylation of the NFkB RelA subunit. Cells were irradiated with LPIL for 680 s immediately after stimulation with IL-1b for 2 h, or Dex was added to cells at the same time as IL-1b. Some cells were pretreated with Bay11-7085 for 1 h before addition of the IL-1b. After stimulation with IL-1b, the culture medium was removed, the cells were fixed by microwaving, and the levels of phosphorylation of the NF-kB RelA subunit on S276 and S536 were measured using a Cellular Activating of Signaling ELISA CASE™ kit. The results are expressed as the means ± SD (n=6); \*P < 0.005 vs. untreated. [This figure is modified from (Shibata et al., 2009a)]

Fig. 7. The effects of Bay11-7085, LPIL or Dex on the phosphorylation of S536 and S276 of the NF-kB RelA subunit

IL-8, originally called monocyte-derived neutrophil chemotactic factor, is a potent chemokine, causing recruitment and infiltration of neutrophils and T cells into local inflammatory sites. The infiltration of neutrophils contributes to inflammation and has been implicated in various diseases. The transcription factors NF-B, AP-1, and NF-IL-6 are all involved in the stimulus-induced expression of IL-8. Subsequent IPA revealed that NF-B signaling was likely important for the synthesis and release of the IL-8 protein. We then found that IL-1-induced IL-8 release from MH7A cells requires the activation of the canonical NF-B pathway and kinases catalyzing its phosphorylation. Furthermore, treatments with Dex or LPIL suppressed the phosphorylation of S276 of the RelA subunit, which would in turn inhibit the translocation of NF-B into the nucleus, where it could interact with the promoter region of the IL8 gene.

## **6. Acknowledgements**

We gratefully acknowledge the assistance of Mrs Asayo Imaoka with the microarray technology. This research was supported in part by "Academic Frontier" Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology in 2007-2011, the Grants-in-Aid for Scientific Research (Kakenhi: 21390497, 22592078).
