Peluso and colleagues (Peluso et al., 2007) analysed the differences in the salivary protein profiles of primary SS and secondary SS patients, and of control subjects using HPLC-ESI-MS. The authors collected whole saliva specimens from 9 primary SS patients, 9 secondary SS patients (3 Rheumatoid Arthritis-2° SS; 3 systemic sclerosis-2° SS; 3 systemic lupus erythematosus-2° SS), and 10 healthy controls, and they analysed the levels and frequencies of 62 proteins. The analysis focused mainly on low molecular weight proteins represented by acid and basic proline-rich proteins, statherins, histatins, cystatins, lysozyme, and defensins. In the second part of the study, the authors examined the effect of pilocarpine on the salivary peptide and protein profiles in a subgroup of 6 primary SS patients. They found that the basic and acid proline-rich proteins and the statherins had the best response to the pilocarpine treatment, while the salivary cystatin and histatin protein classes were modified less. In the comparison between primary and secondary SS salivary profiles, the researchers outlined that patients with secondary SS showed a protein profile that was intermediate between that of the primary SS patients and the healthy subjects. In particular, salivary cystatins (C, S, S2, SA, and SN) and histatins (2, 3, 4, 7, 9, 11 and 12) were less frequently identifiable in primary and secondary SS patients versus controls. On the other hand, 3 proteins (IB-6, P-B Des1-4, and -defensin 2) were identifiable in a significantly higher percentage of secondary SS patients than in the controls. In particular, -defensin 2 was found in 6 of the primary SS patients, in 3 of the 9 secondary SS patients, and in none of the controls. Finally, IB-1 and statherin showed significantly lower levels in secondary SS than in the controls.

Additional information was reported by Flesseig and colleagues (Flesseig et al., 2009), who in a preliminary individual saliva sample analysis showed that SS patients (six SS patients as well as one symptomatic subject not fulfilling the criteria completely, and one who had developed follicular lymphoma) exhibited two patterns of protein expression with an indirect relation to the clinical serological or histological severity of disease.

Recently, Hu and co-workers (Hu et al., 2011) have demonstrated the potential of the highthroughput protein microarray approach in the discovery of autoantibody biomarkers for the non-invasive diagnosis of SS. Saliva autoantibodies present in patients with SS or systemic lupus erythematosus and healthy control subjects were profiled with protein microarrays. After comparison with controls (systemic lupus erythematosus and healthy subjects), statistical analysis of the microarray data revealed 24 autoantibody biomarkers that could differentiate SS from both control groups. A validation of four of these autoantibodies (anti-SSA, anti-SSB, anti-transglutamine, anti-histone) was performed using commercial ELISA kits. Although these are known autoantibodies in SS, they were usually tested in serum samples. The authors suggest that testing these autoantibodies in saliva may be valuable for the diagnosis of SS. Therefore, up to now a wide spectrum of proteins has been identified that might include both "true" disease biomarkers, as well as specific markers of tissue damage (i.e., actin) or inflammation (calgranulins). Therefore, we can hypothesise that further studies might shed some light on this aspect.

Sjögren's Syndrome: The Proteomic Approaches 205

lymphomas. Therefore, we speculate that during the follow-up of patients with lymphomas, proteomic analysis might be able to use the salivary biomarkers as early predictors of treatment response. From the perspective of the research, the analysis of biomarker signatures in saliva could also help to clarify the pathogenetic pathways underlying lymphoproliferation in SS, leading to the development of new methods in early diagnosis

Fig. 1. Protein-protein interaction network of regulated pSS-associated proteins identified in at least two proteomic studies found differentially expressed in saliva. The STRING tool (http://string-db.org/) was used making the network with the following proteins: -2 microglobulin, polymeric Ig receptor (PIGR), salivary acidic proline-rich phosphoprotein 1 (PRH1) and 2 (PRH2), -amylase, carbonic anhydrase VI, Cystatin SA (CST2), Cystatin SN (CST1), Cystatin C (CST3), Cystatin S (CST4), Cystatin D (CST5), LCN1, Calgranulin A (S100A8), Calgranulin B (S100A9), Psoriasin (S100A7), Fatty acid-binding protein, epidermal (FABP5), IGKC (LOC652493), Ig -1 chain C region (IGHG1), -Actin (ACTB), Fc fragment of IgG, receptor, transporter (FCGRT), leukocyte immunoglobulin-like receptor (LILRB1), HLA

class I histocompatibility antigen, alpha chain G (ENSG00000237216) , major

indicates the level of confidence.

histocompatibility complex class IA (HLA-A) major histocompatibility complex class IB (HLA-B) and glycoprotein hormones, alpha polypeptide (CGA). In the figure is shown the potential interaction with additional proteins with score values ranging from 0.993 to 0.999. The different clusters are indicated by the same colour. The thickness of the connecting lines

and curative therapies.

#### **4.3.1 Our results**

In 2005, we began to study rheumatic diseases using a proteomic approach. In our studies, whole saliva was chosen as the biological fluid to discover specific disease biomarkers for primary and secondary manifestations of SS and also of other correlated rheumatic diseases such as systemic sclerosis (Giusti et al., 2007; Baldini et al., 2008), fybromialgia (Bazzichi et al., 2009) and rheumatoid arthritis (Giusti et al., 2010). In our work on SS, we analysed the whole saliva of 12 primary SS patients in comparison with 12 healthy controls by using quantitative 2DE experiments combined with MALDI-TOF-MS for protein identification (Giusti et al., 2007). In particular, in this study, by comparing the SS with control classes, we found that 4 proteins were unique to the control samples (carbonic anhydrase VI, cystatins S, C precursor and cystatin D), and 6 proteins were unique to the SS samples (calgranulin B, cyclophilin A, lipocalin-1 precursor, phosphatidyl ethanolammine binding protein, Ig kappa chain C region (IGKC), protein and Zinc--2 glycoprotein precursor). Moreover, in evaluating the mean ± SD of the percentage of the volume of each single protein of the analytical (not synthetic) gels, the authors also discovered that 10 protein spots were upregulated with > 2-fold changes (fatty acid binding protein (E-FABP), ACTB protein, -actin fragment, leukocyte elastase inhibitor, glutathione-S-transferase (GST), and 5 unidentified proteins). On the other hand, 4 were down regulated (-amylase precursor, cystatin SN precursor, keratin 6L, prolactin-inducible protein precursor) in SS patients compared with controls. These results confirmed the decrease of some of the typical acinar proteins and the increase of many inflammatory proteins. Moreover, they outlined the relevance of proteins not previously described i.e., PIP, keratin 6L, and lipocalin, as markers of acinar damage and oral environment alteration. This study was the basis for further investigations aimed at characterising possible differences in salivary protein profiles in patients who have connective tissue diseases associated with secondary SS. Therefore, we extended the study to refine the diagnostic power of a panel of candidate salivary biomarkers described in SS with respect to both healthy volunteers and pathological controls (sicca syndrome). Moreover, the aim of the study was also to explore the biological and pathogenetic functions of the putative salivary proteomic biomarkers, both in the local exocrinopathy and in the systemic inflammatory autoimmune systemic processes of SS. Our preliminary results, to be published, suggest that novel, non-invasively-collected salivary proteomic biomarkers might be helpful in an early and accurate characterisation of primary and secondary SS. In addition, some of the secondary SS identified biomarkers apparently reflected not only the SS component, but also the concomitant systemic autoimmune disorders, shedding new light on the potential diagnostic role of saliva in autoimmune diseases irrespectively of salivary gland involvement.

The capacity of whole saliva to reflect systemic conditions was also suggested from the preliminary unpublished results of a case study on the salivary proteome of non-Hodgkin' lymphomas. We observed that clinical and functional changes of the salivary glands driven by autoimmune and lymphoproliferative processes might be reflected in patients' whole saliva proteins, and that there was a specific correspondence between clinical improvement and proteomic changes of the salivary peptide complex. These observations indicate the potential usefulness of proteomic analysis in discovering not only diagnostic but also prognostic and therapeutic biomarkers for patients with primary SS and non-Hodgkin's

In 2005, we began to study rheumatic diseases using a proteomic approach. In our studies, whole saliva was chosen as the biological fluid to discover specific disease biomarkers for primary and secondary manifestations of SS and also of other correlated rheumatic diseases such as systemic sclerosis (Giusti et al., 2007; Baldini et al., 2008), fybromialgia (Bazzichi et al., 2009) and rheumatoid arthritis (Giusti et al., 2010). In our work on SS, we analysed the whole saliva of 12 primary SS patients in comparison with 12 healthy controls by using quantitative 2DE experiments combined with MALDI-TOF-MS for protein identification (Giusti et al., 2007). In particular, in this study, by comparing the SS with control classes, we found that 4 proteins were unique to the control samples (carbonic anhydrase VI, cystatins S, C precursor and cystatin D), and 6 proteins were unique to the SS samples (calgranulin B, cyclophilin A, lipocalin-1 precursor, phosphatidyl ethanolammine binding protein, Ig kappa chain C region (IGKC), protein and Zinc--2 glycoprotein precursor). Moreover, in evaluating the mean ± SD of the percentage of the volume of each single protein of the analytical (not synthetic) gels, the authors also discovered that 10 protein spots were upregulated with > 2-fold changes (fatty acid binding protein (E-FABP), ACTB protein, -actin fragment, leukocyte elastase inhibitor, glutathione-S-transferase (GST), and 5 unidentified proteins). On the other hand, 4 were down regulated (-amylase precursor, cystatin SN precursor, keratin 6L, prolactin-inducible protein precursor) in SS patients compared with controls. These results confirmed the decrease of some of the typical acinar proteins and the increase of many inflammatory proteins. Moreover, they outlined the relevance of proteins not previously described i.e., PIP, keratin 6L, and lipocalin, as markers of acinar damage and oral environment alteration. This study was the basis for further investigations aimed at characterising possible differences in salivary protein profiles in patients who have connective tissue diseases associated with secondary SS. Therefore, we extended the study to refine the diagnostic power of a panel of candidate salivary biomarkers described in SS with respect to both healthy volunteers and pathological controls (sicca syndrome). Moreover, the aim of the study was also to explore the biological and pathogenetic functions of the putative salivary proteomic biomarkers, both in the local exocrinopathy and in the systemic inflammatory autoimmune systemic processes of SS. Our preliminary results, to be published, suggest that novel, non-invasively-collected salivary proteomic biomarkers might be helpful in an early and accurate characterisation of primary and secondary SS. In addition, some of the secondary SS identified biomarkers apparently reflected not only the SS component, but also the concomitant systemic autoimmune disorders, shedding new light on the potential diagnostic role of saliva in autoimmune diseases irrespectively of

The capacity of whole saliva to reflect systemic conditions was also suggested from the preliminary unpublished results of a case study on the salivary proteome of non-Hodgkin' lymphomas. We observed that clinical and functional changes of the salivary glands driven by autoimmune and lymphoproliferative processes might be reflected in patients' whole saliva proteins, and that there was a specific correspondence between clinical improvement and proteomic changes of the salivary peptide complex. These observations indicate the potential usefulness of proteomic analysis in discovering not only diagnostic but also prognostic and therapeutic biomarkers for patients with primary SS and non-Hodgkin's

**4.3.1 Our results**

salivary gland involvement.

lymphomas. Therefore, we speculate that during the follow-up of patients with lymphomas, proteomic analysis might be able to use the salivary biomarkers as early predictors of treatment response. From the perspective of the research, the analysis of biomarker signatures in saliva could also help to clarify the pathogenetic pathways underlying lymphoproliferation in SS, leading to the development of new methods in early diagnosis and curative therapies.

Fig. 1. Protein-protein interaction network of regulated pSS-associated proteins identified in at least two proteomic studies found differentially expressed in saliva. The STRING tool (http://string-db.org/) was used making the network with the following proteins: -2 microglobulin, polymeric Ig receptor (PIGR), salivary acidic proline-rich phosphoprotein 1 (PRH1) and 2 (PRH2), -amylase, carbonic anhydrase VI, Cystatin SA (CST2), Cystatin SN (CST1), Cystatin C (CST3), Cystatin S (CST4), Cystatin D (CST5), LCN1, Calgranulin A (S100A8), Calgranulin B (S100A9), Psoriasin (S100A7), Fatty acid-binding protein, epidermal (FABP5), IGKC (LOC652493), Ig -1 chain C region (IGHG1), -Actin (ACTB), Fc fragment of IgG, receptor, transporter (FCGRT), leukocyte immunoglobulin-like receptor (LILRB1), HLA class I histocompatibility antigen, alpha chain G (ENSG00000237216) , major histocompatibility complex class IA (HLA-A) major histocompatibility complex class IB (HLA-B) and glycoprotein hormones, alpha polypeptide (CGA). In the figure is shown the potential interaction with additional proteins with score values ranging from 0.993 to 0.999. The different clusters are indicated by the same colour. The thickness of the connecting lines indicates the level of confidence.

Sjögren's Syndrome: The Proteomic Approaches 207

Bayetto, K. & Logan, R.M. Sjögren's syndrome: a review of aetiology, pathogenesis,

Bazzichi, L., Ciregia, F., Giusti, L., Baldini, C., Giannaccini, G., Giacomelli, C., Servissi, F.,

Binard, A., Devauchelle-Pensec, V., Fautrel, B., Jousse, S., Youinou, P. & Saraux, A.

Bolstad, A.I., Eiken, H.G., Rosenlund, B., Alarcón-Riquelme, M.E. & Jonsson, R. Increased

Carlsson, A., Wuttge, D.M., Ingvarsson, J., Bengtsson, A.A., Sturfelt, G., Borrebaeck, C.A. &

Chang, X., Cui, Y., Zong, M., Zhao, Y., Yan, X., Chen, Y. & Han, J. Identification of proteins

Dai, Y., Hu, C., Wang, L., Huang, Y., Zhang, L, Xiao, X. & Tan, Y. Serum peptidome patterns

*Rheumatology,* Vol. 36, No. 5, (May 2009), pp. 872-880, ISSN 0315-162X. Dai, Y., Hu, C., Huang, Y., Huang, H., Liu, J. & Lv, T. A proteomic study of peripheral blood

*Rheumatology*, Vol. 39, No. 3, (May 2010), pp. 240-246, ISSN 0300-9742. De Ceuninck, F., Marcheteau, E., Berger, S., Caliez, A., Dumont, V., Raes, M., Anract, P.,

*Techniques,* Vol. 16, No. 3, (September 2005), pp. 256-265, ISSN: 1524-0215. de Seny, D., Fillet, M., Meuwis, M.A., Geurts, P., Lutteri, L., Ribbens, C., Bours, V.,

de Seny, D., Fillet, M., Ribbens, C., Marée, R., Meuwis, M.A., Lutteri, L., Chapelle, J.P.,

(September 2008), pp. 799-804, ISSN 0961-2033.

2010), pp. 39-47, ISSN 0045-0421

2009), pp. 191-199, ISSN 0770-3198.

0004-3591.

9476.

3591.

pp.1066-1075, ISSN 0009-9147.

11, (November 2009), pp. 1296-1304, ISSN 1862-8346.

diagnosis and management. (2010). *Australian Dental Journal*, Vol. 55, No 1, (June

Bombardieri, S. & Lucacchini, A. (2009). Detection of potential markers of primary fibromyalgia syndrome in human saliva. *Proteomics Clinical Applications*, Vol. 3, No.

Epidemiology of Sjögren's syndrome: where are we now? (2007). *Clinical and Experimental Rheumatology*, Vol. 25, No. 1, (January 2007), pp. 1-4, ISSN 0392-856X. Bo, G.P., Zhou, L.N., He, W.F., Luo, G.X., Jia, X.F., Gan, C.J., Chen, G.X., Fang, Y.F., Larsen,

P.M. & Wu, J. (2009). Analyses of differential proteome of human synovial fibroblasts obtained from arthritis. *Clinical Rheumatology*, Vol. 28, No. 2, (February

salivary gland tissue expression of Fas, Fas ligand, cytotoxic T lymphocyteassociated antigen 4, and programmed cell death 1 in primary Sjögren's syndrome. (2003). *Arthritis and Rheumatism*, Vol. 48, No.1, (January 2003), pp. 174-185, ISSN

Wingren, C. Serum protein profiling of systemic lupus erythematosus and systemic sclerosis using recombinant antibody microarrays. (2011). *Molecular Cellular Proteomics*, Vol. 10, No. 5, (May 2011), doi: 10.1074/mcp.M110.005033, ISSN 1535-

with increased expression in rheumatoid arthritis synovial tissues. (2009). *Journal of*

mononuclear cells in systemic lupus erythematosus. (2008). *Lupus,* , Vol. 17, No. 9,

of human systemic lupus erythematosus based on magnetic bead separation and MALDI-TOF mass spectrometry analysis. (2010). *Scandinavian Journal of*

Leclerc, G., Boutin, J.A. & Ferry, G. (2005). Assessment of some tools for the characterization of the human osteoarthritic cartilage proteome. *Journal Biomolecular*

Wehenkel, L., Piette, J., Malaise, M. & Merville, M.P. (2005). Discovery of new rheumatoid arthritis biomarkers using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry ProteinChip approach. *Arthritis and Rheumatism,* Vol.52, No.12, (December 2005), pp. 3801-3812, ISSN 0004-

Wehenkel, L., Louis, E., Merville, M.P. & Malaise, M. Monomeric calgranulins measured by SELDI-TOF mass spectrometry and calprotectin measured by ELISA as biomarkers in arthritis. (2008). *Clinical Chemistry*, Vol. 54, No. 6, (June 2008),

#### **5. Conclusions**

SS lacks any true diagnostic criteria for primary and secondary manifestations as well as a set of activity criteria. The risk of misdiagnosis is still quite high, and it highlights the need for a more definitive set of tests and criteria to classify these patients. One possible solution is the proteomic approach, which might represent a promising tool to explore biomarkers for diagnostic aims. Until now, the studies performed have been carried out on parotid biopsies, tears, and saliva. Saliva represents an attractive medium for proteomic analysis because its composition is not complex, and it reflects more accurately the current state of the organism at any moment. Moreover, it presents many logistical advantages because the collection is not invasive, and may be repeated for monitoring over time. However, the identification of true biomarkers of primary and secondary SS is still in its infancy. The results obtained from different studies have not yet defined a conclusive panel of biomarkers useful in diagnostic purposes. Nonetheless, some conclusions can be drawn: SS patients showed a decrease of proteins of glandular origins and an increase of inflammatory proteins, while the salivary profile of secondary SS is intermediate between that of primary SS patients and healthy subjects. Figure 1 shows a representative interactive network obtained by STRING analysis among the proteins found differentially expressed in the proteomic studies performed in saliva. In addition to identifying proteins, we enlarged the network to obtain a large interactive network with more nodes. Interestingly, the figure shows that -2 microglobulin, which is the invariant chain of the Major Histocompatibility Complex class I molecules, considered as a marker of B cell activation, is the key node of the main cluster.

#### **6. Acknowledgement**

The authors wish to thanks Dr Laura Fatuzzo for her valuable contribution in reviewing the text.

#### **7. References**

SS lacks any true diagnostic criteria for primary and secondary manifestations as well as a set of activity criteria. The risk of misdiagnosis is still quite high, and it highlights the need for a more definitive set of tests and criteria to classify these patients. One possible solution is the proteomic approach, which might represent a promising tool to explore biomarkers for diagnostic aims. Until now, the studies performed have been carried out on parotid biopsies, tears, and saliva. Saliva represents an attractive medium for proteomic analysis because its composition is not complex, and it reflects more accurately the current state of the organism at any moment. Moreover, it presents many logistical advantages because the collection is not invasive, and may be repeated for monitoring over time. However, the identification of true biomarkers of primary and secondary SS is still in its infancy. The results obtained from different studies have not yet defined a conclusive panel of biomarkers useful in diagnostic purposes. Nonetheless, some conclusions can be drawn: SS patients showed a decrease of proteins of glandular origins and an increase of inflammatory proteins, while the salivary profile of secondary SS is intermediate between that of primary SS patients and healthy subjects. Figure 1 shows a representative interactive network obtained by STRING analysis among the proteins found differentially expressed in the proteomic studies performed in saliva. In addition to identifying proteins, we enlarged the network to obtain a large interactive network with more nodes. Interestingly, the figure shows that -2 microglobulin, which is the invariant chain of the Major Histocompatibility Complex class I molecules, considered as a marker of B cell activation, is the key node of the

The authors wish to thanks Dr Laura Fatuzzo for her valuable contribution in reviewing the

Aden, N., Shiwen, X., Aden, D., Black, C., Nuttall, A., Denton, C.P., Leask ,A., Abraham, D.

Baillet, A., Trocmé, C., Berthier, S., Arlotto, M., Grange, L., Chenau, J., Quétant, S.,Sève, M.,

Baldini, C., Giusti, L., Bazzichi, L., Ciregia, F., Giannaccini, G., Giacomelli, C., Doveri, M.,

Baraniuk, J.N., Casado, B., Maibach, H., Clauw, D.J., Pannell, L.K. & Hess, S.S. (2005). A

Vol. 47, No. 12, (December 2008), pp. 1754-1760, ISSN 1462-0324.

Vol. 35, No. 9, (September 2009), pp.1820-1824, ISSN 0315-162X.

*Neurology*, Vol. 5, No. 22, (December 2005), ISSN 1471-2377.

4 (April 2010), pp. 671-682, ISSN 1462-0324.

& Stratton, R. (2008). Proteomic analysis of scleroderma lesional skin reveals activated wound healing phenotype of epidermal cell layer. *Rheumatology (Oxford),*

Berger, F., Juvin, R., Morel, F. & Gaudin, P. (2010). Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases. *Rheumatology (Oxford),* Vol. 49, No.

Del Rosso, M., Bombardieri, S. & Lucacchini, A. (2008). Association of psoriasin (S100A7) with clinical manifestations of systemic sclerosis: is its presence in whole saliva a potential predictor of pulmonary involvement? *Journal of Rheumatology*,

Chronic Fatigue Syndrome-related proteome in human cerebrospinal fluid. *BMC*

**5. Conclusions**

main cluster.

**7. References**

text.

**6. Acknowledgement**

Sjögren's Syndrome: The Proteomic Approaches 209

Hjelmervik, T.O., Jonsson, R.& Bolstad, A.I. The minor salivary gland proteome in Sjögren's

Hu, S., Loo, J.A. & Wong, D.T. Human saliva proteome analysis. (2007a), *Annals of the New York Academy of Sciences,* Vol. 1098, (March 2007), pp. 323-329, ISSN 0077-8923. Hu, S., Wang, J., Meijer, J., Ieong, S., Xie, Y., Yu, T., Zhou, H., Henry, S., Vissink, A., Pijpe, J.,

Hu, S., Zhou, M., Jiang, J., Wang, J., Elashoff, D., Gorr, S., Michie, S.A., Spijkervet, F.K.,

Hueber, W., Kidd, B.A., Tomooka, B.H., Lee, B.J., Bruce, B., Fries, J.F., Sønderstrup, G.,

Hueber, W., Tomooka, B.H., Zhao, X., Kidd, B.A., Drijfhout, J.W., Fries, J.F., van Venrooij,

*Rheumatic Diseases*, Vol. 66, No. 6, (June 2007), pp. 712-719, ISSN 0003-4967. Kassan, S.S. & Moutsopoulos, H.M. Clinical manifestations and early diagnosis of Sjögren's

Kim, C.W., Cho, E.H., Lee, Y.J., Kim, Y.H., Hah, Y.S. & Kim, D.R. Disease-specific proteins

Lambrecht, S., Verbruggen, G., Verdonk, P.C., Elewaut, D. & Deforce, D. Differential

Li, T., Huang, Z., Zheng, B., Liao, Z., Zhao, L.& Gu, J. Serum disease-associated proteins of

Li, T., Zheng, B., Huang, Z., Lu, H., Lin, Q., Liao, Z., Lin, Z., Zhao, L., Wang, X. & Gu, J.

(January 1998), pp. 20-24, ISSN 0003-4967.

(January 2009), pp. 81-92, ISSN 0004-3591.

No. 3 (June 2006), pp. 478-484, ISSN 1011-8934.

(February 2008), pp. 163-173, ISSN 1063-4584.

523X.

ISSN 1615-9853.

2655, ISSN 0004-3591.

1275-1284, ISSN 0003-9926.

207. ISSN 0392-856X.

from a population based study. (1998). *Annals of Rheumatic Diseases.* Vol. 57, No.1,

syndrome. (2009). *Oral Diseases*, Vol. 15, No. 5, (July 2009), pp. 342-353, ISSN 1354-

Kallenberg, C., Elashoff, D., Loo, J.A. & Wong, D.T. Salivary proteomic and genomic biomarkers for primary Sjögren's syndrome. (2007b). *Arthritis and Rheumatism,* Vol. 56, No.11, (November 2007), pp. 3588-3600, ISSN 0004-3591. Hu, S., Vissink, A., Arellano, M., Roozendaal, C., Zhou, H., Kallenberg, C.G. & Wong, D.T.

Identification of autoantibody biomarkers for primary Sjögren's syndrome using protein microarrays. (2011). *Proteomics,* Vol. 11, No. 8, (April 2011), pp. 1499-1507,

Bootsma, H., Kallenberg, C.G., Vissink, A., Horvath, S. & Wong, D.T. Systems biology analysis of Sjögren's syndrome and mucosa-associated lymphoid tissue lymphoma in parotid glands. (2009). *Arthritis and Rheumatism,* Vol. 60, No. 1,

Monach, P., Drijfhout, J.W., van Venrooij, W.J., Utz, P.J., Genovese, M.C. & Robinson, W.H. Antigen microarray profiling of autoantibodies in rheumatoid arthritis. (2005). *Arthritis and Rheumatism,* Vol. 52, No. 9 (September 2009), pp. 2645-

W.J., Metzger, A.L., Genovese, M.C.& Robinson, W.H. Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is associated with up regulation of proinflammatory cytokines. (2007). *Annals of*

syndrome. (2004). *Archives of Internal Medicine,* Vol. 164, No. 12, (June 2004), pp.

from rheumatoid arthritis patients. (2006). *Journal of Korean Medical Science,* Vol. 21,

proteome analysis of normal and osteoarthritic chondrocytes reveals distortion of vimentin network in osteoarthritis. (2008). *Osteoarthritis and Cartilage,* Vol. 16, No. 2,

ankylosing spondylitis: results of a preliminary study by comparative proteomics. (2010). *Clinical and Experimental Rheumatology*, Vol. 28, No. 2, (March 2010), pp. 201-

Over-expression of talin 1 and integrin-linked kinase in PBMCs of patients with ankylosing spondylitis: a proteomic study. (2010). *Clinical and Experimental Rheumatology*, Vol. 28, No. 6, (November 2010), pp. 828-835, ISSN 0392-856X.

de Seny, D., Sharif, M., Fillet, M., Cobraiville, G., Meuwis, M.A., Marée, R., Hauzeur, J.P.,

Drynda, S., Ringel, B., Kekow, M., Kühne, C., Drynda, A., Glocker, M.O., Thiesen, H.J. &

Fietta, A., Bardoni, A., Salvini, R., Passadore, I., Morosini, M., Cavagna, L., Codullo, V.,

Fleissig, Y, Deutsch, O., Reichenberg, E., Redlich, M., Zaks, B., Palmon, A. & Aframian, D.J.

Giusti, L., Baldini, C., Bazzichi, L., Ciregia, F., Tonazzini, I., Mascia, G., Giannaccini, G.,

Giusti, L., Baldini, C., Ciregia, F., Giannaccini, G., Giacomelli, C., De Feo, F., Delle Sedie, A.,

Godezie, R., Kho, A., Krastins, B., Saracino, D.A., Thornhill, T.S., Chase, M., Millett, P.J. &

Gottenberg, J.E., Busson, M., Loiseau, P., Cohen-Solal, J., Lepage, V., Charron, D., Sibilia, J. &

Gran, J.T. Diagnosis and definition of primary Sjögren's syndrome. *Scandinavian Journal of Rheumatology*, Vol. 31, No. 2, (February 2002), pp. 57-59 (2002), ISSN 0300-9742. Guo, D., Tan, W., Wang, F., Lv, Z., Hu, J., Lv, T., Chen, Q., Gu ,X., Wan, B. & Zhang, Z.

Hay, E.M., Thomas, E., Pal, B., Hajer, A., Chambers, H. & Silman, A.J. Weak association

*Application,* Vol. 4, No. 3, (March 2010), pp. 315-324, ISSN 1862-8346. Giusti, L., Bazzichi, L., Baldini, C., Ciregia, F., Mascia, G., Giannaccini, G., Del Rosso, M.,

pp.1144-1152, ISSN 0003-4967.

No. 2, pp. 165-171, ISSN 0344-0338.

(June 2006), pp. R160, ISSN 1478-6354.

No. 10, (May 2007), pp. 1634-1643, ISSN 1615-9853.

10, (October 2007), pp. 2063-2069, ISSN 0315-162X.

(July 2008), pp. 439-444, ISSN 1297-319X.

ISSN 0140-6736.

R36, ISSN 1478-6354.

3591.

Wehenkel, L., Louis, E., Merville, M.P., Kirwan, J., Ribbens, C. & Malaise, M. Discovery and biochemical characterisation of four novel biomarkers for osteoarthritis. (2011). *Annals of Rheumatic Diseases.* Vol. 70, No. 6 (June 2011),

Kekow, J. Proteome analysis reveals disease-associated marker proteins to differentiate RA patients from other inflammatory joint diseases with the potential to monitor anti-TNF-alpha therapy. (2004). *Pathology Research and Practice,* Vol. 200,

Pozzi, E., Meloni, F. & Montecucco, C. (2006). Analysis of bronchoalveolar lavage fluid proteome from systemic sclerosis patients with or without functional, clinical and radiological signs of lung fibrosis. *Arthritis Research & Therapy,* Vol. 8, No 6,

Different proteomic protein patterns in saliva of Sjögren's syndrome patients. (2009). *Oral Diseases*, Vol. 15, No. 1, (January 2009), pp. 61-68, ISSN 1354-523X. Fox, R.I. Sjogren's Syndrome. (2005). *Lancet,* Vol. 366, No. 9482, (July 2005), pp. 321-331,

Bombardieri, S.& Lucacchini, A. Proteome analysis of whole saliva: a new tool for rheumatic diseases--the example of Sjögren's syndrome. (2007) *Proteomics,* Vol. 7,

Riente, L., Lucacchini, A., Bazzichi, L, & Bombardieri, S. Is GRP78/BiP a potential salivary biomarker in patients with rheumatoid arthritis? (2010). *Proteomics Clinical*

Bombardieri, S. & Lucacchini, A. Specific proteins identified in whole saliva from patients with diffuse systemic sclerosis. (2007). *Journal of Rheumatology,* Vol. 34, No.

Lee, D.M. High abundance synovial fluid proteome: distinct profiles in health and osteoarthritis. (2007). *Arthritis Research & Therapy,* Vol. 9, No. 2, (February 2007), pp.

Mariette, X. In primary Sjögren's syndrome, HLA class II is associated exclusively with autoantibody production and spreading of the autoimmune response. (2003). *Arthritis and Rheumatism,* Vol. 48, No. 8, (August 2003), pp. 2240-2245, ISSN 0004-

Proteomic analysis of human articular cartilage: identification of differentially expressed proteins in knee osteoarthritis. (2008). *Joint Bone Spine*, Vol. 75, No. 4,

between subjective symptoms and objective testing for dry eyes and mouth: results

from a population based study. (1998). *Annals of Rheumatic Diseases.* Vol. 57, No.1, (January 1998), pp. 20-24, ISSN 0003-4967.

Sjögren's Syndrome: The Proteomic Approaches 211

Ramos-Casals, M., Tzioufas, A.G.& Font, J. Primary Sjögren's syndrome: new clinical and

Ramos-Casals, M., Brito-Zerón, P., Yagüe, J., Akasbi, M., Bautista, R., Ruano, M., Claver, G.,

Rosenthal, A.K., Gohr, C.M., Ninomiya, J. & Wakim, B.T. Proteomic analysis of articular

Ryu, O.H,, Atkinson, J.C., Hoehn, G.T., Illei, G.G. & Hart, T.C. Identification of parotid

Scambi, C., La Verde, V., De Franceschi, L., Barausse, G., Poli, F., Benedetti, F., Sorio, M.,

Schulz, M., Dotzlaw, H., Mikkat, S., Eggert, M. & Neeck, G. Proteomic analysis of peripheral

Seror, R., Ravaud, P., Bowman, S.J., Baron, G., Tzioufas, A., Theander, E., Gottenberg, J.E.,

Sinz, A., Bantscheff, M., Mikkat, S., Ringel, B., Drynda, S., Kekow, J., Thiesen, H.J. & Glocker,

Stea, E.A., Routsias, J.G., Samiotaki, M., Panayotou, G., Papalambros, E., Moutsopoulos,

Stone, J.H., Rajapakse, V.N., Hoffman, G.S., Specks, U., Merkel, P.A., Spiera, R.F., Davis, J.C.,

*Diseases*, Vol. 69, No. 6, (June 2010), pp. 1103-1109, ISSN 0003-4967.

Vol. 147, No. 1, (January 2007), pp. 81-89, ISSN 0009-9104.

*Rheumatism,* Vol. 63, No. 2, (February 2011), pp. 401-411, ISSN 0004-3591. Ruiz-Romero, C., López-Armada, M.J. & Blanco, F.J. Proteomic characterization of human

*(Oxford),* Vol. 44, No. 1, (January 2005), pp. 89-94, ISSN 1462-0324.

(September 2006), pp. 1077-1086, ISSN 1462-0324.

2007), pp. 3752-3759, ISSN 1535-3893.

2005), pp. 347-354, ISSN 0003-4967.

ISSN 1615-9853.

ISSN 1932-6203.

3456, ISSN 0173-0835.

therapeutic concepts. (2005a). *Annals of Rheumatic Diseases*, Vol. 64, No. 3, (March

Gil, V. & Font, J. Hypocomplementaemia as an immunological marker of morbidity and mortality in patients with primary Sjogren's syndrome. (2005b). *Rheumatology*

cartilage vesicles from normal and osteoarthritic cartilage. (2011). *Arthritis and*

normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases. (2005). *Proteomics,* Vol. 5, No. 12, (August 2005), pp. 3048 -3059,

salivary biomarkers in Sjögren'ssyndrome by surface-enhanced laser desorption/ionization time-of flight mass spectrometry and two-dimensional difference gel electrophoresis. (2006). *Rheumatology (Oxford),* Vol. 45, No. 9,

Deriu, F., Roncada, P., Bortolami, O., Turrini, F., Caramaschi, P., Stranieri, C., Bambara, L.M. & Biasi, D. Comparative proteomic analysis of serum from patients with systemic sclerosis and sclerodermatous GVHD. Evidence of defective function of factor H. (2010). *PLoS One,* Vol. 13, No. 5, suppl. 8(August 2010), pp. e12162,

blood mononuclear cells: selective protein processing observed in patients with rheumatoid arthritis. (2007). *Journal of Proteome Research,* Vol. 6, No. 9, (September

Bootsma, H., Mariette, X., Vitali, C. & EULAR Sjögren's Task Force. EULAR Sjogren's syndrome disease activity index: development of a consensus systemic disease activity index for primary Sjogren's syndrome. (2010). *Annals of Rheumatic*

M.O. Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. (2002). *Electrophoresis,* Vol. 23, No. 19, (September 2002), pp. 3445-

H.M. & Tzioufas, A.G. (2007). Analysis of parotid glands of primary Sjögren's syndrome patients using proteomic technology reveals altered autoantigen composition and novel antigenic targets. (2007). *Clinical Experimental Immunology,*

St Clair, E.W., McCune, J., Ross, S., Hitt, B.A., Veenstra, T.D., Conrads, T.P., Liotta, L.A. & Petricoin, E.F. 3rd. Wegener's Granulomatosis Etanercept Trial Research Group. A serum proteomic approach to gauging the state of remission in

Li, T.W., Zheng, B.R., Huang, Z.X., Lin, Q., Zhao, L.K., Liao, Z.T., Zhao, J.J., Lin, Z.M. & Gu,

Liao, H., Wu, J., Kuhn, E., Chin, W., Chang, B., Jones, M.D., O'Neil, S., Clauser, K.R., Karl, J.,

Liu, J., Zhu, P., Peng, J., Li, K., Du, J., Gu, J.& Ou, Y. Identification of disease-associated

Lood, C., Amisten, S., Gullstrand, B., Jönsen, A., Allhorn, M., Truedsson, L., Sturfelt, G.,

No. 116, suppl. 11, (September 2010), pp. 1951-1957, ISSN 0006-4971. Ma, W.J., Guo, X., Liu, J.T., Liu, R.Y., Hu, J.W., Sun, A.G., Yu, Y.X. & Lammi, M.J. Proteomic

*Proteomics,* Vol*.*11, No. 14, (July 2011), pp. 2881-2890, ISSN 1615-9853. Manganelli, P., Fietta, P. & Quaini, F. Hematologic manifestations of primary Sjögren's

Matsuo, K., Arito, M., Noyori, K., Nakamura, H., Kurokawa, M.S., Masuko, K., Okamoto, K.,

Matsuo, K., Xiang, Y., Nakamura, H., Masuko, K., Yudoh, K., Noyori, K., Nishioka, K., Saito, T. & Kato, T. (2006). Identification of novel citrullinated autoantigens of synovium in

Mosley, K., Tam, F.W., Edwards, R.J., Crozier, J., Pusey, C.D. & Lightstone, L. Urinary

Pavón, E.J,, Muñoz, P., Lario, A., Longobardo, V., Carrascal, M., Abián, J., Martin, A.B.,

Peluso, G., De Santis, M., Inzitari, R., Fanali, C., Cabras, T., Messana, I., Castagnola & M.,

*Rheumatism,* Vol. 56, No. 7, (July 2007), pp. 2216-2222, ISSN 0004-3591.

5, No. 123, Suppl. 5, (March 2010), pp. 537-43. ISSN 0366-6999.

(December 2004), pp. 3792-3803, ISSN 0004-3591.

536, ISSN 0006-291X.

pp. 438-448, ISSN 0392-856X.

8, No. 6, (June 2006), pp. R175, ISSN 1478-6354.

suppl. 1, (April 2006), pp. S282-92, ISSN 1615-9853.

0003-4967.

0324.

J.R. Screening disease-associated proteins from sera of patients with rheumatoid arthritis: a comparative proteomic study. (2010). *Chinese Medical Journal (Engl).* Vol.

Hasler, F., Roubenoff, R., Zolg, W.& Guild, B.C. Use of mass spectrometry to identify protein biomarkers of disease severity in the synovial fluid and serum of patients with rheumatoid arthritis. (2004). *Arthritis and Rheumatism,* Vol. 50, No. 12

proteins by proteomic approach in ankylosing spondylitis. (2007). *Biochemical and Biophysical Research Communications,* Vol. 1, No. 357, suppl. 2, (June 2007), pp. 531-

Erlinge, D.& Bengtsson, A.A. Platelet transcriptional profile and protein expression in patients with systemic lupus erythematosus: up-regulation of the type I interferon system is strongly associated with vascular disease. (2010). *Blood.* Vol. 16,

changes in articular cartilage of human endemic osteoarthritis in China. (2011).

syndrome. (2006). *Clinical and Experimental Rheumatology*, Vol 24, N0. 4, (July 2006),

Nagai, K., Suematsu, N., Yudoh, K., Beppu, M., Saito, T. & Kato, T. Arthritogenicity of annexin VII revealed by phosphoproteomics of rheumatoid synoviocytes. (2011). *Annals of Rheumatic Diseases*, Vol. 70, No. 8, (August 2011), pp. 1489-1495, ISSN

rheumatoid arthritis using a proteomic approach. *Arthritis Research & Therapy,* Vol.

proteomic profiles distinguish between active and inactive lupus nephritis. (2006). *Rheumatology (Oxford),* Vol. 45, No. 12, (December 2006), pp. 1497-1504, ISSN 1462-

Arias, S.A., Callejas-Rubio, J.L., Sola, R., Navarro-Pelayo, F., Raya-Alvarez, E., Ortego-Centeno, N., Zubiaur, M. & Sancho, J. Proteomic analysis of plasma from patients with systemic lupus erythematosus: increased presence of haptoglobin alpha2 polypeptide chains over the alpha1 isoforms. (2006). *Proteomics,* Vol. 6,

Ferraccioli, G.F. Proteomic study of salivary peptides and proteins in patients with Sjögren's syndrome before and after pilocarpine treatment. (2007). *Arthritis and*

**Part 3**

**Psychosocial Considerations**

**in Rheumatic Disease**

Wegener's granulomatosis. (2005). *Arthritis and Rheumatism,* Vol. 52, No. 3, (March 2005), pp. 902-910, ISSN 0004-3591.

## **Part 3**

**Psychosocial Considerations in Rheumatic Disease**

212 Insights and Perspectives in Rheumatology

Streckfus, C.F. & Dubinsky, W.P. Proteomic analysis of saliva for cancer diagnosis. (2007), *Expert Review of Proteomics,* Vol. 4, No. 3, (June 2007), 329-332, ISSN 1478-9450. Tilleman, K., Van Beneden, K., Dhondt, A., Hoffman, I., De Keyser, F., Veys, E., Elewaut, D.

Tomosugi, N., Kitagawa, K., Takahashi, N., Sugai, S. & Ishikawa, I. Diagnostic potential of

Tzioufas, A.G. & Voulgarelis, M. Update on Sjögren's syndrome autoimmune epithelitis:

*Rheumatic Diseases*, Vol. 61, No. 6, (June 2002), pp. 554-558, ISSN 0003-4967. Voulgarelis, M., Dafni, U.G., Isenberg, D.A.& Moutsopoulos, H.M. Malignant lymphoma in

*Rheumatism,* Vol. 42, No. 8, (August 1999), pp. 1765-1772, ISSN 0004-3591. Wang, L., Dai, Y., Qi, S., Sun, B., Wen, J., Zhang, L. & Tu, Z. Comparative proteome analysis

Wright, C., Edelmann, M., diGleria, K., Kollnberger, S., Kramer, H., McGowan, S., McHugh,

Wu, J., Liu, W., Bemis, A., Wang, E., Qiu, Y., Morris, E.A., Flannery, C.R. & Yang, Z.

Zhang, X., Jin, M., Wu, H., Nadasdy, T., Nadasdy, G., Harris, N., Green-Church, K.,

Zheng, X., Wu, S.L., Hincapie, M. & Hancock, W.S. Study of the human plasma proteome of

No. 11, (November 2007), pp. 3675-3684, ISSN 0004-3591.

Vol. 74, No. 6, (September 2008), pp. 799-807, ISSN 0085-2538.

Vol. 4, No. 3, (May 2005), pp. 820-825, ISSN 1535-3893.

10.1007/s00296-010-1625-9, ISSN 0172-8172.

16, (April 2009), 3538-3545, ISSN 0021-9673.

1626-1632, ISSN 0003-4967.

2005), pp. 902-910, ISSN 0004-3591.

2257, ISSN 1615-9853.

Wegener's granulomatosis. (2005). *Arthritis and Rheumatism,* Vol. 52, No. 3, (March

& Deforce, D. Chronically inflamed synovium from spondyloarthropathy and rheumatoid arthritis investigated by protein expression profiling followed by tandem mass spectrometry. (2005). *Proteomics,* Vol. 5, No. 8, (May 2005), pp. 2247-

tear proteomic patterns in Sjögren's syndrome. (2005). *Journal of Proteome Research,*

from classification to increased neoplasias. (2007). *Best Practice & Research in Clinical Rheumatology.* Vol. 21, No. 6, (December 2007), pp. 989-1010, ISSN 1521-6942. Vanarsa, K. & Mohan, C. Proteomics in rheumatology: the dawn of a new era. (2010). *F1000 Medicine Report,* Vol. 8, No. 2, (December 2010), pp. 87, ISSN 1757-5931. Vitali, C., Bombardieri, S., Jonsson, R., Moutsopoulos, H.M., Alexander, E.L., Carsons, S.E.,

Daniels, T.E., Fox, P.C., Fox, R.I., Kassan, S.S., Pillemer, S.R., Talal, N., Weisman, M.H.& European Study Group on Classification Criteria for Sjögren's Syndrome. Classification criteria for Sjögren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group.(2002). *Annals of*

primary Sjögren's Syndrome: a multicenter, retrospective, clinical study by the European Concerted Action on Sjögren's Syndrome. (1999). *Arthritis and*

of peripheral blood mononuclear cells in systemic lupus erythematosus with iTRAQ quantitative proteomics. (2010). *Rheumatology International*, doi

K., Taylor, S., Kessler, B.& Bowness, P. Ankylosing spondylitis monocytes show upregulation of proteins involved in inflammation and the ubiquitin proteasome pathway. (2009). *Annals of Rheumatic Diseases*, Vol. 68, No. 10, (October 2009), pp.

Comparative proteomic characterization of articular cartilage tissue from normal donors and patients with osteoarthritis. (2007). *Arthritis and Rheumatism,* Vol. 56,

Nagaraja, H., Birmingham, D.J., Yu, C.Y., Hebert, L.A. & Rovin, B.H. Biomarkers of lupus nephritis determined by serial urine proteomics. (2008) *Kidney International,*

rheumatoid arthritis. (2009). *Journal of Chromatography A,* Vol. 17, No. 1216, suppl.

**12**

*Germany*

**The Pathogenetic Link Between Stress and Rheumatic Diseases**

*Rheumatology Unit, Medical Clinic II, University Hospital Leipzig*

Hans Selye was the first to define stress as a non-specific response of the body to any demand made upon it (1936)*.* It has been demonstrated that Selye's theory about the effects of stress is applicable to any type of stress (Stojanovich & Marisavljevich, 2008). Key organ of stress processes is the brain. It determines what individuals will experience as stressful, it orchestrates how individuals will cope with stressful experiences, and it changes both functionally and structurally as a result of stressful experiences (McEwen & Gianaros, 2011). Stress begins with a stimulus (stressor) that causes a reaction in the brain (stress perception), which subsequently activates physiologic systems in the body (stress response). The stress response system reacts with the release of neurotransmitters (norepinephrine etc.) and hormones (cortisol etc.) which serve to send an efferent message from the brain to the

It is clear that stress, originating in the cortex of the brain, must be converted into somatic signals, which can modulate the immune system or pain perception. These somatic signals typically start off in the highest centres of the autonomic nervous system (ANS) and the endocrine system within the hypothalamus (Cutolo & Straub, 2006). Selye was the first to describe the system by which the body copes with stress: the hypothalamus–pituitary– adrenal system (HPA) (Stojanovich & Marisavljevich, 2008). The hypothalamus has close connections to the limbic system, which is responsible for the emotionalisation of psychological stress (whether it is perceived as positive eustress or negative distress). The major neuroendocrine response mediating stress adaptation relies on the activation of the HPA axis by stimulating corticotropin releasing hormone (CRH) and vasopressin (VP) from parvocellular neurons of the hypothalamic paraventricular nucleus. This leads in turn to stimulation of pituitary aderocorticotropic hormone (ACTH) secretion and increases in glucocorticoid secretion from the adrenal cortex. Basal production of glucocorticoids and its hypothalamic regulators together with transient increases during stress are essential for neuronal plasticity and normal brain function. While activation of the HPA axis is essential for survival during stress, chronic exposure to stress hormones can predispose to

periphery in order to modulate the stressful event (Cutolo & Straub, 2006).

psychological, metabolic and immune alterations (Aguilera et al., 2011).

**1. Introduction**

**1.1 Definition of stress**

O. Malysheva and C.G.O. Baerwald

### **The Pathogenetic Link Between Stress and Rheumatic Diseases**

O. Malysheva and C.G.O. Baerwald *Rheumatology Unit, Medical Clinic II, University Hospital Leipzig Germany*

#### **1. Introduction**

#### **1.1 Definition of stress**

Hans Selye was the first to define stress as a non-specific response of the body to any demand made upon it (1936)*.* It has been demonstrated that Selye's theory about the effects of stress is applicable to any type of stress (Stojanovich & Marisavljevich, 2008). Key organ of stress processes is the brain. It determines what individuals will experience as stressful, it orchestrates how individuals will cope with stressful experiences, and it changes both functionally and structurally as a result of stressful experiences (McEwen & Gianaros, 2011).

Stress begins with a stimulus (stressor) that causes a reaction in the brain (stress perception), which subsequently activates physiologic systems in the body (stress response). The stress response system reacts with the release of neurotransmitters (norepinephrine etc.) and hormones (cortisol etc.) which serve to send an efferent message from the brain to the periphery in order to modulate the stressful event (Cutolo & Straub, 2006).

It is clear that stress, originating in the cortex of the brain, must be converted into somatic signals, which can modulate the immune system or pain perception. These somatic signals typically start off in the highest centres of the autonomic nervous system (ANS) and the endocrine system within the hypothalamus (Cutolo & Straub, 2006). Selye was the first to describe the system by which the body copes with stress: the hypothalamus–pituitary– adrenal system (HPA) (Stojanovich & Marisavljevich, 2008). The hypothalamus has close connections to the limbic system, which is responsible for the emotionalisation of psychological stress (whether it is perceived as positive eustress or negative distress). The major neuroendocrine response mediating stress adaptation relies on the activation of the HPA axis by stimulating corticotropin releasing hormone (CRH) and vasopressin (VP) from parvocellular neurons of the hypothalamic paraventricular nucleus. This leads in turn to stimulation of pituitary aderocorticotropic hormone (ACTH) secretion and increases in glucocorticoid secretion from the adrenal cortex. Basal production of glucocorticoids and its hypothalamic regulators together with transient increases during stress are essential for neuronal plasticity and normal brain function. While activation of the HPA axis is essential for survival during stress, chronic exposure to stress hormones can predispose to psychological, metabolic and immune alterations (Aguilera et al., 2011).

The Pathogenetic Link Between Stress and Rheumatic Diseases 217

Cytokines are important factors connecting and modulating the immune and neuroendrocrine systems (Elenkov, 2005). Furthermore, cytokines and their receptors are expressed in the neuroendocrine system and exert their effects both centrally and peripherally (Benveniste et al., 1998). Systemic cytokines can affect the brain through several mechanisms, including active transport across the blood–brain barrier, through leaky areas in the blood–brain barrier in the circumventricular organs or through the activation of neural pathways such as the vagal nerve (Eskandari, 2005). For example, IL-6 and tumour necrosis factor (TNF)-a, released during an immune response and inflammation activate the central components of the stress system, alter neurotransmitter networks activity and induce fever, sleepiness, fatigue, loss of appetite and decreased libido. In addition, they activate the hepatic synthesis of acute phase proteins—changes referred to as 'sickness behavior' and

Otherwise, administration of interleukin (IL)-1 in the periphery increases the turnover of norepinephrine in the hypothalamus; intracerebroventricular and peripheral injection of interferon (IFN)-α or IL-1β produces a long-lasting increase of the sympathetic activity of the splenic nerve and an increased turnover of norepinephrine in the spleen (Elenkov, 2000). Furthermore, it was shown that monoamine neurotransmitters, released in stress situations, represent a tonic sympathetic control on cytokine production and on the balance of pro-

Autoimmune diseases are characterized by common alterations of the T-helper (Th)1 versus Th2 cells and a shift towards pro-inflammatory cytokines. In RA the balance is skewed towards Th1 and an excess of IL-12 and TNF-α production, whereas Th2 activity and the

Neurotransmitters, through stimulation of the beta 2-adrenoreceptors (b2AR)-cAMP-protein kinase A pathway, inhibit the production of type 1/proinflammatory cytokines, such as IL-12, TNF-alpha, and IFN-gamma by antigen-presenting cells and Th1 cells, whereas they stimulate the production of type 2/anti-inflammatory cytokines such as IL-10 and transforming growth factor-beta. Through these mechanisms endogenous catecholamines may cause a selective suppression of Th1 responses and cellular immunity and a Th2 shift towards dominance of humoral immunity (Madden et al., 1995, Elenkov et al., 2000). On the other hand, in certain local responses and under certain conditions, catecholamines may actually boost regional immune responses through induction of IL-1, TNF-α, and IL-8 production. Thus, activation of the sympathetic nervous system during an immune response might be aimed to confine the inflammatory response on the local level through induction of neutrophil accumulation and stimulation of more specific humoral immune responses. However, systemically the SNS may suppress Th1 responses and thus protect the organism from the detrimental effects of pro-inflammatory cytokines and other products of activated macrophages (Elenkov et al., 2000). Otherwise, norepinephrine and epinephrine, through stimulation of classic cytoplasmic/nuclear glucocorticoid receptors and β2-AR, respectively, suppress the production of IL-12 by antigen presenting cells (APCs) being the main inducer of Th1 responses (Elenkov et al., 2005). It was shown that an increase in disease activity among RA patients was associated with an increase in the level of stress experienced by these patients, suggesting that a stress induced increase in norepinephrine release might

'acute-phase response', respectively (Elenkov, 2005).

inflammatory/anti-inflammatory cytokines (Szelényi & Vizi, 2007).

activate naive Th1 cells and/or increase their activity (Kin & Sanders, 2007).

production of IL-10 appear to be deficient (Segal et al., 1998).

Selye is considered to be the first to demonstrate the existence of a particular stress disease, the stress syndrome, or the general adaptation syndrome (Stojanovich & Marisavljevich, 2008). These processes can be adaptive in the short term and may contribute to the dysregulation of the hormone status if persisting for a longer time. Moreover, they are involved in bidirectional signalling between the brain and the body as well as in stress-related mental and physical health conditions (McEwen & Gianaros, 2011). The chronic stress-induced dysregulation appears to be of sufficient magnitude to impact health (Gouin et al., 2008).

Recent advances in psychoneuroimmunology have provided insight into the complex mechanisms by which stressors might affect the homeostasis and the body's immune system (de Brouwer et al., 2010). Stress challenges the organism's homeostatic mechanisms, triggering a cascade of events that should, normally, maintain or allow a return to equilibrium (Pêgo et al., 2010). Psychological stress can influence immunoregulatory circuits and the course of an inflammatory disease (Marshall GD Jr, 1997). Furthermore, cytokines generated by the immune system influence hormonal secretion and the central nervous system, producing specific behavioural changes accompanying infectious and inflammatory diseases (Straub et al., 2008). Psychological distress and immune dysregulation have been linked to each other in chronic stress, in disease and other major life events.

It was shown that psychological stress is also thought to contribute to the aetiology, maintenance and exacerbation of rheumatic diseases (de Brouwer et al., 2010). Stress has been studied in several autoimmune rheumatic diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) (Cutolo & Straub, 2006). Growing evidence supports the hypothesis that alterations of the stress response and interactions between the neuroendocrine and immune systems contribute to the pathogenesis of rheumatic diseases (Chikanza et al., 1992).

#### **2. Neurotransmitter-cytokine relationship**

The stress response is mediated by the sympathetic nervous system and it results in the release of some neurotransmitters (epinephrine, norepinephrine) that regulates the immune system at regional, local, and systemic levels (Felten et al., 1988).

Immune organs including thymus, spleen, and lymph nodes are innervated by sympathetic nerves (Felten et al., 1988). Interruption of sympathetic innervation of immune organs has been shown to modulate the outcome of, and susceptibility to, inflammatory and infectious disease. Denervation of noradrenergic fibres of lymph nodes is associated with exacerbation of inflammation (Lorton et al., 1996), whereas systemic sympathectomy or denervation of joints is associated with decreased severity of inflammation (Eskandari et al., 2005).

Immune cells also express neurotransmitter receptors, such as adrenergic receptors (alphaor beta- subtype) on lymphocytes that allow them to respond to sympathetic neurotransmitters (Elenkov & Chrousos, 1999). Neural influences on the immune response may be due to direct synapse-like nerve-lymphocyte connections (Mix et al., 2007). Through stimulation of adrenergic receptors locally released norepinephrine or circulating catecholamines affect lymphocyte traffic, circulation, and proliferation, and, in turn, modulate cytokine production as well as functional activity of various lymphoid cells (Elenkov et al., 2000; Straub et al., 2000; Madden et al., 2001).

Selye is considered to be the first to demonstrate the existence of a particular stress disease, the stress syndrome, or the general adaptation syndrome (Stojanovich & Marisavljevich, 2008). These processes can be adaptive in the short term and may contribute to the dysregulation of the hormone status if persisting for a longer time. Moreover, they are involved in bidirectional signalling between the brain and the body as well as in stress-related mental and physical health conditions (McEwen & Gianaros, 2011). The chronic stress-induced dysregulation

Recent advances in psychoneuroimmunology have provided insight into the complex mechanisms by which stressors might affect the homeostasis and the body's immune system (de Brouwer et al., 2010). Stress challenges the organism's homeostatic mechanisms, triggering a cascade of events that should, normally, maintain or allow a return to equilibrium (Pêgo et al., 2010). Psychological stress can influence immunoregulatory circuits and the course of an inflammatory disease (Marshall GD Jr, 1997). Furthermore, cytokines generated by the immune system influence hormonal secretion and the central nervous system, producing specific behavioural changes accompanying infectious and inflammatory diseases (Straub et al., 2008). Psychological distress and immune dysregulation have been

It was shown that psychological stress is also thought to contribute to the aetiology, maintenance and exacerbation of rheumatic diseases (de Brouwer et al., 2010). Stress has been studied in several autoimmune rheumatic diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) (Cutolo & Straub, 2006). Growing evidence supports the hypothesis that alterations of the stress response and interactions between the neuroendocrine and immune systems

The stress response is mediated by the sympathetic nervous system and it results in the release of some neurotransmitters (epinephrine, norepinephrine) that regulates the immune

Immune organs including thymus, spleen, and lymph nodes are innervated by sympathetic nerves (Felten et al., 1988). Interruption of sympathetic innervation of immune organs has been shown to modulate the outcome of, and susceptibility to, inflammatory and infectious disease. Denervation of noradrenergic fibres of lymph nodes is associated with exacerbation of inflammation (Lorton et al., 1996), whereas systemic sympathectomy or denervation of

Immune cells also express neurotransmitter receptors, such as adrenergic receptors (alphaor beta- subtype) on lymphocytes that allow them to respond to sympathetic neurotransmitters (Elenkov & Chrousos, 1999). Neural influences on the immune response may be due to direct synapse-like nerve-lymphocyte connections (Mix et al., 2007). Through stimulation of adrenergic receptors locally released norepinephrine or circulating catecholamines affect lymphocyte traffic, circulation, and proliferation, and, in turn, modulate cytokine production as well as functional activity of various lymphoid cells

joints is associated with decreased severity of inflammation (Eskandari et al., 2005).

appears to be of sufficient magnitude to impact health (Gouin et al., 2008).

linked to each other in chronic stress, in disease and other major life events.

contribute to the pathogenesis of rheumatic diseases (Chikanza et al., 1992).

system at regional, local, and systemic levels (Felten et al., 1988).

**2. Neurotransmitter-cytokine relationship**

Cytokines are important factors connecting and modulating the immune and neuroendrocrine systems (Elenkov, 2005). Furthermore, cytokines and their receptors are expressed in the neuroendocrine system and exert their effects both centrally and peripherally (Benveniste et al., 1998). Systemic cytokines can affect the brain through several mechanisms, including active transport across the blood–brain barrier, through leaky areas in the blood–brain barrier in the circumventricular organs or through the activation of neural pathways such as the vagal nerve (Eskandari, 2005). For example, IL-6 and tumour necrosis factor (TNF)-a, released during an immune response and inflammation activate the central components of the stress system, alter neurotransmitter networks activity and induce fever, sleepiness, fatigue, loss of appetite and decreased libido. In addition, they activate the hepatic synthesis of acute phase proteins—changes referred to as 'sickness behavior' and 'acute-phase response', respectively (Elenkov, 2005).

Otherwise, administration of interleukin (IL)-1 in the periphery increases the turnover of norepinephrine in the hypothalamus; intracerebroventricular and peripheral injection of interferon (IFN)-α or IL-1β produces a long-lasting increase of the sympathetic activity of the splenic nerve and an increased turnover of norepinephrine in the spleen (Elenkov, 2000). Furthermore, it was shown that monoamine neurotransmitters, released in stress situations, represent a tonic sympathetic control on cytokine production and on the balance of proinflammatory/anti-inflammatory cytokines (Szelényi & Vizi, 2007).

Autoimmune diseases are characterized by common alterations of the T-helper (Th)1 versus Th2 cells and a shift towards pro-inflammatory cytokines. In RA the balance is skewed towards Th1 and an excess of IL-12 and TNF-α production, whereas Th2 activity and the production of IL-10 appear to be deficient (Segal et al., 1998).

Neurotransmitters, through stimulation of the beta 2-adrenoreceptors (b2AR)-cAMP-protein kinase A pathway, inhibit the production of type 1/proinflammatory cytokines, such as IL-12, TNF-alpha, and IFN-gamma by antigen-presenting cells and Th1 cells, whereas they stimulate the production of type 2/anti-inflammatory cytokines such as IL-10 and transforming growth factor-beta. Through these mechanisms endogenous catecholamines may cause a selective suppression of Th1 responses and cellular immunity and a Th2 shift towards dominance of humoral immunity (Madden et al., 1995, Elenkov et al., 2000). On the other hand, in certain local responses and under certain conditions, catecholamines may actually boost regional immune responses through induction of IL-1, TNF-α, and IL-8 production. Thus, activation of the sympathetic nervous system during an immune response might be aimed to confine the inflammatory response on the local level through induction of neutrophil accumulation and stimulation of more specific humoral immune responses. However, systemically the SNS may suppress Th1 responses and thus protect the organism from the detrimental effects of pro-inflammatory cytokines and other products of activated macrophages (Elenkov et al., 2000). Otherwise, norepinephrine and epinephrine, through stimulation of classic cytoplasmic/nuclear glucocorticoid receptors and β2-AR, respectively, suppress the production of IL-12 by antigen presenting cells (APCs) being the main inducer of Th1 responses (Elenkov et al., 2005). It was shown that an increase in disease activity among RA patients was associated with an increase in the level of stress experienced by these patients, suggesting that a stress induced increase in norepinephrine release might activate naive Th1 cells and/or increase their activity (Kin & Sanders, 2007).

The Pathogenetic Link Between Stress and Rheumatic Diseases 219

Perception of an external stressful stimulus prompts the activation of various physiological systems that together define the body's stress response, which is aimed at re-establishing homeostasis (S. de Brouwer, 2010). Central to the maintenance of homeostasis is an appropriate response of the HPA axis as well as the sympathetic nervous system (Dunn & Swiergiel, 2008; Eskandari et al., 2003,). From animal models of arthritis as well as from clinical studies in RA patients a central role of the HPA axis in onset and severity of RA was suggested (Chikanza, 1992). In these patients an inadequate secretion of cortisol in the setting of a sustained inflammatory disease has been observed (Vassilopoulos & Mantzoukis, 2006). A hypoactive stress system may facilitate or sustain the Th1 shift in Th1 mediated RA. A suboptimal production of cortisol is involved in the onset and/or progression of RA. Patients with RA have ''inappropriately normal'' or low plasma cortisol levels in the setting of severe, chronic inflammation, characterized by increased production of TNF-a, IL-1 and IL-6. This may actually facilitate or sustain the pro-inflammatory shift in this disease (Elenkov, 2005). The HPA axis has been evaluated in multiple forms in former studies of RA patients. Assuming that an inflammatory process up-regulates HPA axis function, cortisol levels are inappropriately normal in RA patients when cytokine levels are increased (Straub et al., 2008). The proposed defect could reside at various levels of the HPA axis, i.e. the hypothalamic level, the pituitary, and the adrenal gland. However, results of HPA axis tests in RA patients were inconclusive since steroid treatment, drugs including disease modifying antirheumatic drugs (DMARD) and disease duration might interfere with the results leading to conflicting results for ACTH levels and cortisol response in

Experimental psychosocial stress consistently increased autonomic activity, blood pressure and catecholamine levels in patients with RA and SLE, and increased neuroendocrine variables (cortisol and ACTH]) in patients with RA. Furthermore, after such stress exposure an increase in IL-4 level in patients with SLE, an increase in TNF-α in RA patients, and inconsistent findings for IL-6 were observed (S. de Brouwer, 2010). Exercise stress was characterised by decreased levels of cortisol whereas ACTH levels and growth hormone levels increased. Moreover, this stress elicited a different cytotoxic T cell response in patients

The stress hormones, supposedly acting via β- and α-adrenergic as well as glucocorticoid receptors, down-regulate immune and inflammatory processes; however, these processes also influence the central nervous system (Ader et al., 1995; MacEwen, 1998). Circulating cytokines and activated immune cells, markers of inflammation, activate both (intermediates of) the HPA axis and the autonomic nervous system. Chronically elevated levels of cytokines, as occur during long-term inflammation, might lead to changes in HPA axis and ANS activity (Chrousos, 1995). Otherwise, circulating hormones or locally released neurotransmitters regulate major immune functions, such as antigen presentation, antibody production, lymphocyte activity, proliferation and traffic, and the secretion of cytokines including the modulation of Th1 or Th2 cytokine responses. During inflammation, activation of the stress system, through induction of a Th2 shift protects the organism from systemic "overshooting" with Th1/pro-inflammatory cytokines. Under certain conditions, however, stress hormones, substance P, adenosine triphosphate (ATP) and the activation of CRH/substance P-histamine axis may actually facilitate inflammation, through induction of

**3. Integration of stress hormones, cytokines and neurotransmitters**

RA patients (Eijsbouts, 2005).

with RA and SLE (Pool et al., 2004; Kurtais et al., 2006).

Since β2-ARs are expressed on Th1 cells, but not on Th2 cells (Sanders et al., 1997), catecholamines do not affect directly the cytokine production by Th2 cells. In murine and human systems β2-AR agonists inhibit IFN-gamma production by Th1 cells, but do not affect IL-4 production by Th2 cells (Borger et al., 1998; Wahle et al., 2006). However, neurotransmitters through stimulation of β2-AR up-regulate the production of the antiinflammatory cytokine IL-6 and IL-10 by APCs (Elenkov, 2005). Administration of a β2AR antagonist or a β2AR agonist prior to or during the development of arthritis inhibits or exacerbates disease pathology, suggesting that NE stimulating β2AR on naive T and/or Th1 cells modulate IFN-gamma production. As with the in vitro findings, the in vivo effects were found to depend on the time point when norepinephrine was released during the course of a disease (Kin & Sanders, 2007). The increased responsiveness of T cells from RA patients to β2AR stimulation following NE exposure might exacerbate IFN-gamma production and promote conditions for RA development and progression. Therefore, NE, which normally participates in a CD4- T cell response to maintain homeostasis by enhancing the baseline immune cell-regulated IFN-gamma response, may cause disease progression when the regulatory conditions controlling sympathetic nervous system/ β2AR activity have changed (Kin & Sanders, 2007).

Otherwise, between RA patients and controls there was a highly significant distortion in the distribution of the beta2AR polymorphisms at codon 16 and to a lower extent at codon 27 contributing together with the human leuokocyte antigen (HLA)-DR alleles to the genetic background of RA. Furthermore, polymorphisms of the beta2R gene may modulate the expression of anti citrullinated peptide (anti-CCP) antibodies further supporting the close interplay between the autonomic nervous system and the immune system (Malysheva et al., 2008). Single nucleotide polymorphisms have been found to be associated with the β2AR expressed by cells of RA patients, suggesting that a change in the β2AR structure on a naive T and/or Th1 cell may heighten the level of cell responsiveness to NE (Kin & Sanders, 2007).

In patients with RA, experimental stress did not change levels of inflammatory markers (IL-2, soluble IL-2 receptor, IL-4, IL-10 and IFN-γ) but TNF-α. Results for IL-6 were inconsistent (S.de Brouwer, 2010). In patients with SLE, experimental stress did not change levels of cytokines and other inflammatory markers (IL-2, IL-6, IL-10, IFN-γ, βadrenoceptors), although levels of IL-4 increased after stress (Jocobs et al., 2001). Patients with SLE differed from controls in their response to stress, having a larger increase in IL-4, smaller IL-10 and IFN-γ responses, and fewer β-adrenoceptors on monocytes (S. de Brouwer, 2010). In patients with JIA, stress did not affect IL-8 production, but increased IL-6 production (Roupe et al., 2000). However, in healthy subjects stress increased IFN gamma and IL-10 levels (Jacobs et al., 2001; Motivala et al., 2008). Thus, although experimental stress does not seem to influence levels of certain cytokines and inflammatory markers in patients with rheumatic diseases (for example, sIL-2, IL-8, IL-10, IFN-γ, β-adrenoceptors), it does cause specific changes in c-reactive protein (CRP) and TNF-α in patients with RA, changes in IL-4 in patients with SLE, and changes in IL-6 in patients with JIA. These changes are not observed in controls (S. de Brouwer, 2010). Finally, the pharmacological manipulation of the sympathetic-immune interface is reviewed with focus on new therapeutic strategies using selective alpha(2)- and beta(2)-AR agonists and antagonists and inhibitors of phosphodiesterase type IV in the treatment of experimental models of autoimmune diseases (Elenkov et al., 2000).

Since β2-ARs are expressed on Th1 cells, but not on Th2 cells (Sanders et al., 1997), catecholamines do not affect directly the cytokine production by Th2 cells. In murine and human systems β2-AR agonists inhibit IFN-gamma production by Th1 cells, but do not affect IL-4 production by Th2 cells (Borger et al., 1998; Wahle et al., 2006). However, neurotransmitters through stimulation of β2-AR up-regulate the production of the antiinflammatory cytokine IL-6 and IL-10 by APCs (Elenkov, 2005). Administration of a β2AR antagonist or a β2AR agonist prior to or during the development of arthritis inhibits or exacerbates disease pathology, suggesting that NE stimulating β2AR on naive T and/or Th1 cells modulate IFN-gamma production. As with the in vitro findings, the in vivo effects were found to depend on the time point when norepinephrine was released during the course of a disease (Kin & Sanders, 2007). The increased responsiveness of T cells from RA patients to β2AR stimulation following NE exposure might exacerbate IFN-gamma production and promote conditions for RA development and progression. Therefore, NE, which normally participates in a CD4- T cell response to maintain homeostasis by enhancing the baseline immune cell-regulated IFN-gamma response, may cause disease progression when the regulatory conditions controlling sympathetic nervous system/ β2AR activity

Otherwise, between RA patients and controls there was a highly significant distortion in the distribution of the beta2AR polymorphisms at codon 16 and to a lower extent at codon 27 contributing together with the human leuokocyte antigen (HLA)-DR alleles to the genetic background of RA. Furthermore, polymorphisms of the beta2R gene may modulate the expression of anti citrullinated peptide (anti-CCP) antibodies further supporting the close interplay between the autonomic nervous system and the immune system (Malysheva et al., 2008). Single nucleotide polymorphisms have been found to be associated with the β2AR expressed by cells of RA patients, suggesting that a change in the β2AR structure on a naive T and/or Th1 cell may heighten the level of cell responsiveness to NE (Kin & Sanders, 2007). In patients with RA, experimental stress did not change levels of inflammatory markers (IL-2, soluble IL-2 receptor, IL-4, IL-10 and IFN-γ) but TNF-α. Results for IL-6 were inconsistent (S.de Brouwer, 2010). In patients with SLE, experimental stress did not change levels of cytokines and other inflammatory markers (IL-2, IL-6, IL-10, IFN-γ, βadrenoceptors), although levels of IL-4 increased after stress (Jocobs et al., 2001). Patients with SLE differed from controls in their response to stress, having a larger increase in IL-4, smaller IL-10 and IFN-γ responses, and fewer β-adrenoceptors on monocytes (S. de Brouwer, 2010). In patients with JIA, stress did not affect IL-8 production, but increased IL-6 production (Roupe et al., 2000). However, in healthy subjects stress increased IFN gamma and IL-10 levels (Jacobs et al., 2001; Motivala et al., 2008). Thus, although experimental stress does not seem to influence levels of certain cytokines and inflammatory markers in patients with rheumatic diseases (for example, sIL-2, IL-8, IL-10, IFN-γ, β-adrenoceptors), it does cause specific changes in c-reactive protein (CRP) and TNF-α in patients with RA, changes in IL-4 in patients with SLE, and changes in IL-6 in patients with JIA. These changes are not observed in controls (S. de Brouwer, 2010). Finally, the pharmacological manipulation of the sympathetic-immune interface is reviewed with focus on new therapeutic strategies using selective alpha(2)- and beta(2)-AR agonists and antagonists and inhibitors of phosphodiesterase type IV in the treatment of experimental models of autoimmune diseases

have changed (Kin & Sanders, 2007).

(Elenkov et al., 2000).

#### **3. Integration of stress hormones, cytokines and neurotransmitters**

Perception of an external stressful stimulus prompts the activation of various physiological systems that together define the body's stress response, which is aimed at re-establishing homeostasis (S. de Brouwer, 2010). Central to the maintenance of homeostasis is an appropriate response of the HPA axis as well as the sympathetic nervous system (Dunn & Swiergiel, 2008; Eskandari et al., 2003,). From animal models of arthritis as well as from clinical studies in RA patients a central role of the HPA axis in onset and severity of RA was suggested (Chikanza, 1992). In these patients an inadequate secretion of cortisol in the setting of a sustained inflammatory disease has been observed (Vassilopoulos & Mantzoukis, 2006). A hypoactive stress system may facilitate or sustain the Th1 shift in Th1 mediated RA. A suboptimal production of cortisol is involved in the onset and/or progression of RA. Patients with RA have ''inappropriately normal'' or low plasma cortisol levels in the setting of severe, chronic inflammation, characterized by increased production of TNF-a, IL-1 and IL-6. This may actually facilitate or sustain the pro-inflammatory shift in this disease (Elenkov, 2005). The HPA axis has been evaluated in multiple forms in former studies of RA patients. Assuming that an inflammatory process up-regulates HPA axis function, cortisol levels are inappropriately normal in RA patients when cytokine levels are increased (Straub et al., 2008). The proposed defect could reside at various levels of the HPA axis, i.e. the hypothalamic level, the pituitary, and the adrenal gland. However, results of HPA axis tests in RA patients were inconclusive since steroid treatment, drugs including disease modifying antirheumatic drugs (DMARD) and disease duration might interfere with the results leading to conflicting results for ACTH levels and cortisol response in RA patients (Eijsbouts, 2005).

Experimental psychosocial stress consistently increased autonomic activity, blood pressure and catecholamine levels in patients with RA and SLE, and increased neuroendocrine variables (cortisol and ACTH]) in patients with RA. Furthermore, after such stress exposure an increase in IL-4 level in patients with SLE, an increase in TNF-α in RA patients, and inconsistent findings for IL-6 were observed (S. de Brouwer, 2010). Exercise stress was characterised by decreased levels of cortisol whereas ACTH levels and growth hormone levels increased. Moreover, this stress elicited a different cytotoxic T cell response in patients with RA and SLE (Pool et al., 2004; Kurtais et al., 2006).

The stress hormones, supposedly acting via β- and α-adrenergic as well as glucocorticoid receptors, down-regulate immune and inflammatory processes; however, these processes also influence the central nervous system (Ader et al., 1995; MacEwen, 1998). Circulating cytokines and activated immune cells, markers of inflammation, activate both (intermediates of) the HPA axis and the autonomic nervous system. Chronically elevated levels of cytokines, as occur during long-term inflammation, might lead to changes in HPA axis and ANS activity (Chrousos, 1995). Otherwise, circulating hormones or locally released neurotransmitters regulate major immune functions, such as antigen presentation, antibody production, lymphocyte activity, proliferation and traffic, and the secretion of cytokines including the modulation of Th1 or Th2 cytokine responses. During inflammation, activation of the stress system, through induction of a Th2 shift protects the organism from systemic "overshooting" with Th1/pro-inflammatory cytokines. Under certain conditions, however, stress hormones, substance P, adenosine triphosphate (ATP) and the activation of CRH/substance P-histamine axis may actually facilitate inflammation, through induction of

The Pathogenetic Link Between Stress and Rheumatic Diseases 221

disease activity of rheumatic diseases. These influences include factors such as emotional distress, coping, and familial factors (Anthony & Schanberg, 2007), diet, and some socioeconomic factors (such as level of education, area of residence and income) may affect levels of pain and physical disability experienced by rheumatic patients (Symmons, 2003;

It was shown that the family environment is important in juvenile rheumatic diseases. Thus, diagnosis of a rheumatic disease in children was associated with depression in the mother and a composite measure of parental (mother and father) distress and passive coping. Children's emotional and behavioural functioning was not related to medical diagnosis, however, a mother's depression and parental distress were associated with a child's behavioural problems. This kind of distress was associated with child functioning and interventions to ameliorate parental distress may have beneficial effects on the children's behaviour and on parent's reactions to their children (Frank et al, 1998). Furthermore, the social environment was found to operate on the core health outcome, i.e. pain severity in rheumatic diseases. Not only the status of being married but also the quality of the relationship in terms of long-term stress and emotional support may be useful prognostic factors in rheumatic diseases. Patient reports of negative spouse behaviour (such as avoidance and critical remarks) and baseline depression predicted worse pain outcome, and this association remained significant in analyses controlling for baseline pain. The level of formal education showed a weak correlation to disability, emotional support, and pain. Daily emotional support and social life domains associated with positive affect had an indirect influence on outcome. The absence of strong rather than weak social ties was the component of the loneliness construct linked to pain. These associations between social prognostic factors and pain severity, however, were mediated by psychological functioning

One more important factor of environment is socioeconomic status. Although RA occurrence is more frequent in lower socioeconomic parts of the population, it was not identified as a significant risk factor in RA (Damjanovic et al., 2009). Otherwise, there is a lack of good quality studies of the epidemiology of rheumatic diseases in developing countries. It appears that a threshold level where higher socio-economic status is associated with reduced prevalence of rheumatic disease is not reached in developing countries. Therefore, differences in the prevalence between socio-economic groups can be undetected. A similar case can be made for overcrowding (Steer et al., 2002). However, the education level as risk factor is significantly related to RA occurrence (Damjanovic et al., 2009). Furthermore, socioeconomic disparities in patients with chronic musculoskeletal complains leading to increased pain may be attributable to both greater frequency of stressful financial events as well as greater vulnerability to economic hardship for those at the lower end of the socioeconomic spectrum. Conditions of economic hardship and daily ratings of financial worry both had significant detrimental effects on daily pain. Participants with greater levels of economic hardship reported greater pain severity in response to daily financial worries than their counterparts with little or no economic hardship. Further, participants in the

Kobayshi et al., 2008).

**4.1 Family factors**

at baseline (Waltz et al., 1999).

**4.2 Socioeconomic status**

IL-1, IL-6, IL-8, IL-18, TNF-alpha and CRP production. Thus, a dysfunctional neuroendocrine-immune interface associated with abnormalities of the 'systemic antiinflammatory feedback' and/or 'hyperactivity' of the local pro-inflammatory factors may play a role in the pathogenesis of autoimmune diseases (Elenkov, 2008).

Cytokines signalling to the brain can not only activate the HPA axis but also facilitate pain and induce a series of mood and behavioural responses generally termed sickness behaviour (Watkins & Maier, 2000). Cytokines, such as IL-1, IL-6, and TNF-alpha, are also produced in the brain (Sebire, 1993). These brain-derived cytokines can stimulate the HPA axis. For example, IL-1 stimulates the expression of the gene encoding CRH and thereby the release of the hormone from the hypothalamus, the release of AVP from the hypothalamus, and the release of ACTH from the anterior pituitary, IL-2 stimulates AVP secretion from the hypothalamus, IL-6 and TNF-alpha also stimulate ACTH secretion. Otherwise, in chronic inflammation there seems to be a shift from CRH-driven to arginine vasopressin (AVP) driven HPA axis response (Eskandari, 2005).

CRH is the strongest known activator of the HPA axis which has been functionally implicated in the regulation of many endocrine functions, such as glucocorticoid release from the adrenal gland and associated endocrine-immune responses (Elenkov, 2008). CRH not only activates the pituitary-adrenal axis but also sets in motion a coordinated series of behavioural and physiologic responses, suggesting that the central nervous system may coordinate both behavioural and immunologic adaptation during stressful situations (Stermberg, 1992; O'Connor, 2000). CRH is positively regulated by catecholaminergic system (Eskandari, 2005). During an immune response certain cytokines, such as IL-1, IL-6, and TNF-a can signal the brain, which via a complex CRH-dependent pathway, triggers activation of both the SNS and the HPA axis (Elenkov, 2000).

 Recently several polymorphisms in the highly conserved regulatory region of the CRH gene were described with the gene being located on chromosome 8q13. Three polymorphisms cosegregated absolutely resulting in two alleles *A1* and *A2* with a further polymorphism being assigned as alleles *B1* and *B2*. Population investigations demonstrated a distortion in the distribution of the resulting compound alleles A1B1, A2B1 and A2B2 between RA patients and healthy controls (Baerwald et al., 1997). In vitro studies revealed a different regulation of the CRH gene by these polymorphisms (Wagner et al., 2006). Furthermore, it was shown that the stress induced response of cortisol is differentially modulated by CRH promoter polymorphisms in RA patients. Thus, during insulin hypoglycemia test RA patients bearing the A2B2 allele exhibited an earlier CRH as well as ACTH peak compared to A1B1 positive patients. The integrated cortisol response to hypoglycemia expressed as area under the curve was significantly lower in RA patients with the A1B1 allele than in RA patients with the A2B2 allele. Moreover, there was a significant difference in the molar ratio cortisol to ACTH between controls and RA patients underlining the dissociation of the pituitary and the adrenal gland in patients with RA (Malysheva et al., 2011).

#### **4. Environmental factors in stress – disease interaction**

The concept of environment included different factors that could disturb the homeostatic state of health (Parniapour, 1990). A growing body of research in rheumatic diseases highlights the importance of environmental influences to the contribution of stress on disease activity of rheumatic diseases. These influences include factors such as emotional distress, coping, and familial factors (Anthony & Schanberg, 2007), diet, and some socioeconomic factors (such as level of education, area of residence and income) may affect levels of pain and physical disability experienced by rheumatic patients (Symmons, 2003; Kobayshi et al., 2008).

#### **4.1 Family factors**

220 Insights and Perspectives in Rheumatology

IL-1, IL-6, IL-8, IL-18, TNF-alpha and CRP production. Thus, a dysfunctional neuroendocrine-immune interface associated with abnormalities of the 'systemic antiinflammatory feedback' and/or 'hyperactivity' of the local pro-inflammatory factors may

Cytokines signalling to the brain can not only activate the HPA axis but also facilitate pain and induce a series of mood and behavioural responses generally termed sickness behaviour (Watkins & Maier, 2000). Cytokines, such as IL-1, IL-6, and TNF-alpha, are also produced in the brain (Sebire, 1993). These brain-derived cytokines can stimulate the HPA axis. For example, IL-1 stimulates the expression of the gene encoding CRH and thereby the release of the hormone from the hypothalamus, the release of AVP from the hypothalamus, and the release of ACTH from the anterior pituitary, IL-2 stimulates AVP secretion from the hypothalamus, IL-6 and TNF-alpha also stimulate ACTH secretion. Otherwise, in chronic inflammation there seems to be a shift from CRH-driven to arginine vasopressin (AVP)-

CRH is the strongest known activator of the HPA axis which has been functionally implicated in the regulation of many endocrine functions, such as glucocorticoid release from the adrenal gland and associated endocrine-immune responses (Elenkov, 2008). CRH not only activates the pituitary-adrenal axis but also sets in motion a coordinated series of behavioural and physiologic responses, suggesting that the central nervous system may coordinate both behavioural and immunologic adaptation during stressful situations (Stermberg, 1992; O'Connor, 2000). CRH is positively regulated by catecholaminergic system (Eskandari, 2005). During an immune response certain cytokines, such as IL-1, IL-6, and TNF-a can signal the brain, which via a complex CRH-dependent pathway, triggers

 Recently several polymorphisms in the highly conserved regulatory region of the CRH gene were described with the gene being located on chromosome 8q13. Three polymorphisms cosegregated absolutely resulting in two alleles *A1* and *A2* with a further polymorphism being assigned as alleles *B1* and *B2*. Population investigations demonstrated a distortion in the distribution of the resulting compound alleles A1B1, A2B1 and A2B2 between RA patients and healthy controls (Baerwald et al., 1997). In vitro studies revealed a different regulation of the CRH gene by these polymorphisms (Wagner et al., 2006). Furthermore, it was shown that the stress induced response of cortisol is differentially modulated by CRH promoter polymorphisms in RA patients. Thus, during insulin hypoglycemia test RA patients bearing the A2B2 allele exhibited an earlier CRH as well as ACTH peak compared to A1B1 positive patients. The integrated cortisol response to hypoglycemia expressed as area under the curve was significantly lower in RA patients with the A1B1 allele than in RA patients with the A2B2 allele. Moreover, there was a significant difference in the molar ratio cortisol to ACTH between controls and RA patients underlining the dissociation of the pituitary and

The concept of environment included different factors that could disturb the homeostatic state of health (Parniapour, 1990). A growing body of research in rheumatic diseases highlights the importance of environmental influences to the contribution of stress on

play a role in the pathogenesis of autoimmune diseases (Elenkov, 2008).

driven HPA axis response (Eskandari, 2005).

activation of both the SNS and the HPA axis (Elenkov, 2000).

the adrenal gland in patients with RA (Malysheva et al., 2011).

**4. Environmental factors in stress – disease interaction**

It was shown that the family environment is important in juvenile rheumatic diseases. Thus, diagnosis of a rheumatic disease in children was associated with depression in the mother and a composite measure of parental (mother and father) distress and passive coping. Children's emotional and behavioural functioning was not related to medical diagnosis, however, a mother's depression and parental distress were associated with a child's behavioural problems. This kind of distress was associated with child functioning and interventions to ameliorate parental distress may have beneficial effects on the children's behaviour and on parent's reactions to their children (Frank et al, 1998). Furthermore, the social environment was found to operate on the core health outcome, i.e. pain severity in rheumatic diseases. Not only the status of being married but also the quality of the relationship in terms of long-term stress and emotional support may be useful prognostic factors in rheumatic diseases. Patient reports of negative spouse behaviour (such as avoidance and critical remarks) and baseline depression predicted worse pain outcome, and this association remained significant in analyses controlling for baseline pain. The level of formal education showed a weak correlation to disability, emotional support, and pain. Daily emotional support and social life domains associated with positive affect had an indirect influence on outcome. The absence of strong rather than weak social ties was the component of the loneliness construct linked to pain. These associations between social prognostic factors and pain severity, however, were mediated by psychological functioning at baseline (Waltz et al., 1999).

#### **4.2 Socioeconomic status**

One more important factor of environment is socioeconomic status. Although RA occurrence is more frequent in lower socioeconomic parts of the population, it was not identified as a significant risk factor in RA (Damjanovic et al., 2009). Otherwise, there is a lack of good quality studies of the epidemiology of rheumatic diseases in developing countries. It appears that a threshold level where higher socio-economic status is associated with reduced prevalence of rheumatic disease is not reached in developing countries. Therefore, differences in the prevalence between socio-economic groups can be undetected. A similar case can be made for overcrowding (Steer et al., 2002). However, the education level as risk factor is significantly related to RA occurrence (Damjanovic et al., 2009). Furthermore, socioeconomic disparities in patients with chronic musculoskeletal complains leading to increased pain may be attributable to both greater frequency of stressful financial events as well as greater vulnerability to economic hardship for those at the lower end of the socioeconomic spectrum. Conditions of economic hardship and daily ratings of financial worry both had significant detrimental effects on daily pain. Participants with greater levels of economic hardship reported greater pain severity in response to daily financial worries than their counterparts with little or no economic hardship. Further, participants in the

The Pathogenetic Link Between Stress and Rheumatic Diseases 223

The role of other types of environmental stress was studied in patients with different autoimmune diseases as well. For example, cognitive stressors elicited changes in leukocyte/lymphocyte counts, subsets of lymphocytes, and CRP levels in patients with RA (Geenen, 1998; Veldhuijzen van Zanten, 2005), and changes in leucocyte counts and cytoxic T cell numbers in patients with SLE (Hinrichsen, 1992). It has recently been demonstrated that thermal stress (Spa therapy) induces a reduction in the circulating levels of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), IL-1β and TNF-α, important mediators of inflammation and pain in patients with rheumatic diseases (Fioravanti, 2011). It was shown that environmental desiccating stress can be involved in the pathogenesis of chronic dry eye syndrome by exposing shared epitopes in the cornea, conjunctiva, and the lacrimal gland that induce pathogenic CD4+T cells leading to lacrimal keratoconjunctivitis, which under normal circumstances is restrained by CD4(+)CD25(+) forkhead/winged helix transcription

Some other environmental factors such as weather conditions and climate could be also important in rheumatic pain. For example, persons with osteoarthritis in urban Chicago exhibited more weather sensitivity than their rural counterparts in Grand Forks, North Dakota. Multiple regression analysis revealed that precipitation affected pain for urban subjects who identified weather as a pain-generating factor; barometric pressure, relative humidity and sunshine were significant factors influencing pain-related stress. Wind speed correlated with pain and pain-related stress; relative humidity and precipitation correlated with pain-related stress for urban subjects who did not perceive weather as a problem. Specific weather variables were not identified as affecting rural subjects' pain. However, temperature and barometric pressure affected pain-related stress in rural subjects who perceived weather as a problem (Laborde et al., 1986). However, one systematic review of nine longitudinal observational studies (up to September 2009) dealing with the association of weather variables (temperature, relative humidity and atmospheric pressure) and severity of pain in RA could not demonstrate any significant effects. Individual analyses from two studies indicate that pain reporting in a minority (< 25%) of RA patients is influenced by temperature, relative humidity or atmospheric pressure. The studies to date do not show any consistent group effect of weather conditions on pain in RA. There is, however, evidence suggesting that pain in some individuals is more affected by the weather than in others, and that patients react in different ways to various weather conditions. Thus, the hypothesis that weather changes might significantly influence pain reporting in clinical care and research in some patients with RA cannot be rejected (Smedslund & Hagen, 2011). Otherwise, one population-based study demonstrated that pain is not an inevitable consequence of climatic conditions. Persons were less likely to report pain on days with > 5.8 h of sunshine and with average temperature of > 17.5°C underlining a strong relationship between lack of sunshine, lower temperatures and pain reporting (Macfarlane et al, 2010). Interestingly, 84% of RA patients believe in an association between weather and rheumatism, while 57% claimed to have ability to forecast weather. The maximum contribution of weather on symptoms was 17.1 %. Of interest, the belief about a presence of an association between weather and arthritis was found to be stronger than its statistical

factor(+) regulatory T cells (Niederkorn et al., 2006).

**4.4 Climate conditions**

power (Cay et al., 2011).

sample who were not employed and who reported higher levels of economic hardship exhibited the most pain reactivity in response to daily financial worries. Economic hardship was associated not only with greater exposure to daily financial worries but also with greater vulnerability to pain on days when daily financial worries were experienced (Rios & Zautra, 2011). A prospective cohort study (1958 British Birth Cohort Study) demonstrated that the prevalence of shoulder, forearm, low back, knee and chronic widespread pain at 45 years generally increased with lower social class. Persons in the lowest social class (compared to the highest) experienced nearly a threefold increase in the risk of chronic widespread pain (relative risk: 2.9, 95% CI 1.8 to 4.6). Social class during childhood also demonstrated a relationship with most regional pain and chronic widespread pain. With the exception of forearm pain, the magnitude of effect of childhood social status on reporting of pain in adulthood was less than that of adult social status. On multivariable analysis these relationships were partly explained by poor adult mental health, psychological distress, adverse life events and lifestyle factors (Macfarlane, 2009). Furthermore, there was an association between socioeconomic status, occupation, and hospitalization for RA. A total of 13,820 male and 14,509 female hospitalizations for RA were analysed during the study. Men and women with an education level > 12 years had a significantly decreased incidence ratio. Occupation also played a role in these items studied with an increased incidence ratio among farmers, miners and quarry workers, electrical workers, other construction workers, and engine and motor operators for men. Among women, assistant nurses and religious, juridical, and other social-science-related workers had significantly increased incidence ratio in all 3 cohorts (Li et al, 2008).

#### **4.3 Education and life style**

It was proposed that such environmental factor as level of education was significantly inversely associated with the risk of RA, with a 2-fold lower risk of RA among those with the highest education compared with those having the lowest level of education. On the other hand, it is hypothesised that RF-positive and RF-negative RA have different "aetiologies" with factors related to educational level predominantly associated with the risk of RF-positive RA (Pedersem et al., 2006).

Diet and food life style could also be an important environmental factor contributing to rheumatic diseases. For example, diet, nutrition, and weight loss have shown promise in alleviating some of rheumatic burden. These lifestyle changes may give patients a feeling of control over their disease as well as a non-pharmacologic means of treatment. In particular the role of the Mediterranean diet has to be recognised as a protective factor against RA (Li & Micheletti, 2011). The goals of dietary therapy in rheumatic diseases are alleviation of under- and malnutrition, inhibition of inflammation, prophylaxis of osteoporosis, as well as recognition and treatment of nutrient sensitivities or intolerances. Inhibition of inflammation in these patients is improved by modulating the omega-3/omega-6 fatty acids ratio in the diet. Reduction of dietary arachidonic acid is recommended. This polyunsaturated fatty acid is the main precursor of pro-inflammatory mediators which interact with chemokines und cytokines. Simultaneously, intake of anti-inflammatory omega-3 fatty acids is increased. Studies have shown that this dietary regimen results in an amelioration of symptoms in patients with inflammatory rheumatic diseases (Adam et al., 2009).

The role of other types of environmental stress was studied in patients with different autoimmune diseases as well. For example, cognitive stressors elicited changes in leukocyte/lymphocyte counts, subsets of lymphocytes, and CRP levels in patients with RA (Geenen, 1998; Veldhuijzen van Zanten, 2005), and changes in leucocyte counts and cytoxic T cell numbers in patients with SLE (Hinrichsen, 1992). It has recently been demonstrated that thermal stress (Spa therapy) induces a reduction in the circulating levels of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), IL-1β and TNF-α, important mediators of inflammation and pain in patients with rheumatic diseases (Fioravanti, 2011). It was shown that environmental desiccating stress can be involved in the pathogenesis of chronic dry eye syndrome by exposing shared epitopes in the cornea, conjunctiva, and the lacrimal gland that induce pathogenic CD4+T cells leading to lacrimal keratoconjunctivitis, which under normal circumstances is restrained by CD4(+)CD25(+) forkhead/winged helix transcription factor(+) regulatory T cells (Niederkorn et al., 2006).

#### **4.4 Climate conditions**

222 Insights and Perspectives in Rheumatology

sample who were not employed and who reported higher levels of economic hardship exhibited the most pain reactivity in response to daily financial worries. Economic hardship was associated not only with greater exposure to daily financial worries but also with greater vulnerability to pain on days when daily financial worries were experienced (Rios & Zautra, 2011). A prospective cohort study (1958 British Birth Cohort Study) demonstrated that the prevalence of shoulder, forearm, low back, knee and chronic widespread pain at 45 years generally increased with lower social class. Persons in the lowest social class (compared to the highest) experienced nearly a threefold increase in the risk of chronic widespread pain (relative risk: 2.9, 95% CI 1.8 to 4.6). Social class during childhood also demonstrated a relationship with most regional pain and chronic widespread pain. With the exception of forearm pain, the magnitude of effect of childhood social status on reporting of pain in adulthood was less than that of adult social status. On multivariable analysis these relationships were partly explained by poor adult mental health, psychological distress, adverse life events and lifestyle factors (Macfarlane, 2009). Furthermore, there was an association between socioeconomic status, occupation, and hospitalization for RA. A total of 13,820 male and 14,509 female hospitalizations for RA were analysed during the study. Men and women with an education level > 12 years had a significantly decreased incidence ratio. Occupation also played a role in these items studied with an increased incidence ratio among farmers, miners and quarry workers, electrical workers, other construction workers, and engine and motor operators for men. Among women, assistant nurses and religious, juridical, and other social-science-related workers had significantly increased incidence ratio

It was proposed that such environmental factor as level of education was significantly inversely associated with the risk of RA, with a 2-fold lower risk of RA among those with the highest education compared with those having the lowest level of education. On the other hand, it is hypothesised that RF-positive and RF-negative RA have different "aetiologies" with factors related to educational level predominantly associated with the

Diet and food life style could also be an important environmental factor contributing to rheumatic diseases. For example, diet, nutrition, and weight loss have shown promise in alleviating some of rheumatic burden. These lifestyle changes may give patients a feeling of control over their disease as well as a non-pharmacologic means of treatment. In particular the role of the Mediterranean diet has to be recognised as a protective factor against RA (Li & Micheletti, 2011). The goals of dietary therapy in rheumatic diseases are alleviation of under- and malnutrition, inhibition of inflammation, prophylaxis of osteoporosis, as well as recognition and treatment of nutrient sensitivities or intolerances. Inhibition of inflammation in these patients is improved by modulating the omega-3/omega-6 fatty acids ratio in the diet. Reduction of dietary arachidonic acid is recommended. This polyunsaturated fatty acid is the main precursor of pro-inflammatory mediators which interact with chemokines und cytokines. Simultaneously, intake of anti-inflammatory omega-3 fatty acids is increased. Studies have shown that this dietary regimen results in an amelioration of symptoms in patients with inflammatory rheumatic diseases (Adam et al.,

in all 3 cohorts (Li et al, 2008).

**4.3 Education and life style**

2009).

risk of RF-positive RA (Pedersem et al., 2006).

Some other environmental factors such as weather conditions and climate could be also important in rheumatic pain. For example, persons with osteoarthritis in urban Chicago exhibited more weather sensitivity than their rural counterparts in Grand Forks, North Dakota. Multiple regression analysis revealed that precipitation affected pain for urban subjects who identified weather as a pain-generating factor; barometric pressure, relative humidity and sunshine were significant factors influencing pain-related stress. Wind speed correlated with pain and pain-related stress; relative humidity and precipitation correlated with pain-related stress for urban subjects who did not perceive weather as a problem. Specific weather variables were not identified as affecting rural subjects' pain. However, temperature and barometric pressure affected pain-related stress in rural subjects who perceived weather as a problem (Laborde et al., 1986). However, one systematic review of nine longitudinal observational studies (up to September 2009) dealing with the association of weather variables (temperature, relative humidity and atmospheric pressure) and severity of pain in RA could not demonstrate any significant effects. Individual analyses from two studies indicate that pain reporting in a minority (< 25%) of RA patients is influenced by temperature, relative humidity or atmospheric pressure. The studies to date do not show any consistent group effect of weather conditions on pain in RA. There is, however, evidence suggesting that pain in some individuals is more affected by the weather than in others, and that patients react in different ways to various weather conditions. Thus, the hypothesis that weather changes might significantly influence pain reporting in clinical care and research in some patients with RA cannot be rejected (Smedslund & Hagen, 2011). Otherwise, one population-based study demonstrated that pain is not an inevitable consequence of climatic conditions. Persons were less likely to report pain on days with > 5.8 h of sunshine and with average temperature of > 17.5°C underlining a strong relationship between lack of sunshine, lower temperatures and pain reporting (Macfarlane et al, 2010). Interestingly, 84% of RA patients believe in an association between weather and rheumatism, while 57% claimed to have ability to forecast weather. The maximum contribution of weather on symptoms was 17.1 %. Of interest, the belief about a presence of an association between weather and arthritis was found to be stronger than its statistical power (Cay et al., 2011).

The Pathogenetic Link Between Stress and Rheumatic Diseases 225

the onset of specific diseases including rheumatic diseases, and premature death (Boscarino

From results of 27 studies evaluating more than 3,000 patients with RA it was recognised that stress is an important risk factor in the pathogenesis of autoimmune diseases. Major life events (e.g. death of a spouse, severe long-term illness of a spouse, loss of a parent, divorce of parents, death of a parent or severe disease of a parent) lead to an intense release of stress mediators (large time integral of released neurotransmitters and hormones), whereas in minor life events (daily hassles with small intensity) only short-lived surges of neurotransmitters and hormones are expected. Therefore, it is suggested that neurotransmitters such as norepinephrine or stress hormones such as cortisol might have different effects on immune/inflammatory responses at high and low concentrations present during short or extended periods of time, respectively. Long-lasting (chronic) stress may lead to pro-inflammatory effects since no adequate long-term responses of stress axes (anti-inflammatory) are to be expected (Cutolo & Straub, 2006). Furthermore, major life events and chronic minor stress seem to be important factors in juvenile chronic arthritis and are significantly associated with the onset of the rheumatic disease. With respect to RA, stress may be a provoking factor. However, during the course of the disease, minor stress could aggravate SLE and RA (Herrmann et al., 2000). In contrast, strong major stress, which is likely accompanied by a large and long-lived release of stress axes mediators, was

One study (follow up 1980 till 1996) examining the risk of RA in parents after the death of a child does not support an association between severe psychological stress and RA. The relative risk of first hospitalisation for RA was 0.88 [95% CI 0.63-1.24]. The risk was close to 1 throughout the 18 years of follow-up (Li et al., 2005). Recent findings are consistent on the impact of early life stress on subsequent inflammatory responses. A predominant role for parent distress in children's adjustment to juvenile rheumatic disease was shown (Ryan et al., 2010). In one retrospective cohort study (follow–up 1995/1997 through 2005) of 15,357 adults it could be demonstrated that 64% adults reported early life stress with at least one adverse childhood experience (ACE), including childhood physical, emotional, or sexual abuse; witnessing domestic violence; growing up with household substance abuse, mental illness, parental divorce, and/or an incarcerated household member. The event rate (per 10,000 person-years) for a first hospitalization with any autoimmune disease was 31.4 in women and 34.4 in men. First hospitalizations for any autoimmune disease increased with increasing number of ACEs (p < 0.05). Persons with equal or more than 2 ACEs were at a 100 % increased risk for rheumatic diseases compared to persons without any ACE (p < 0.05) (Dube et al., 2009). Patients with juvenile idiopathic arthritis reported having pain, stiffness, and fatigue on > 70% of days, with significant variability in symptom levels. Furthermore, significant same-day relationships between stress, mood, and disease symptoms were revealed. Specifically, daily fluctuations in both stress and mood were predictive of increased pain, stiffness, and fatigue. Increases in daily stress, mood, and disease symptoms were also significantly related to decreased participation in social

et al., 2004; Sommershof et al., 2009).

**5.1.1 Early life stress**

**5.1 Stress as pathogenetic factor of autoimmune diseases**

associated with a decrease in disease activity (Cutolo & Straub, 2006).

#### **4.5 Cultural difference**

In addition, cultural differences in experiencing individual stress in RA patients were observed. In one study covering Polish and German RA patients it was demonstrated that in both countries, mental as well as physical health deteriorated resulting in about 50% of patients requiring support in everyday activities. 95% of Polish and 62% of German patients felt rejected from social activities. For the psychological stress perceived, functional capacity class 3 and male gender were shown to be predictive in Polish patients and living in a small town in German patients. In the Polish group, the tertiary/bachelor level of education was linked with lower distress level. RA has a serious impact on the mental health owing to a great disease burden (Bugajska et al., 2010).

#### **4.6 Other factors**

Interestingly, during a 15-year follow-up study of 74 female patients with RA two categories of RA were identified: a disease form less connected with genetic factors and more influenced by major psychodynamic conflict situations ('major conflict group') and a second form more associated with hereditary predisposition and less influenced by environmental psychosocial changes ('non-conflict group') (Rimón & Laakso,1985). In addition, a patient's perspective of the causes and consequences of RA could underline the role of environmental factors in disease. Two descriptive categories of patient's understanding of disease could be identified: the category 'consequences beyond personal control' comprised not having a clue, being exposed to climatic change, being genetically exposed and unexpected effects of events; the category 'overloaded circumstances' involved work and family-related strain. Consequences beyond personal control implied that the patients could not prevent the disease and expressed their lack of understanding as to why they contracted it. Overloaded circumstances were described as strained situations that were both work and family factors related and could be influenced by the patient. Reasoning along these lines it is concluded that understanding the patients' own view of disease is needed in order to achieve a more successful medical care model (Bergsten et al., 2009). Identifying of how these factors contribute to the development of autoimmune diseases may further lead to better explaining the pathogenesis of rheumatic diseases (Symmons, 2003; Kobayshi et al., 2008).

Finally, in one recent study of patients with psoriatic arthritis environmental factors such as lifting heavy loads of at least 100 pounds/hr (OR 2.8, 95% CI 1.51-5.05), infections that required antibiotics (OR 1.7, 95% CI 1.00-2.77), and injuries (OR 2.1, 95% CI 1.11-4.01) were associated with the occurrence of arthritis. No association was found between psoriasis arthritis and alcohol consumption, vaccination, and female hormonal exposures (Eder et al., 2011).

#### **5. Rheumatic diseases as a model of stress induced disease**

Despite more than twenty years of stress research in rheumatology, it is not completely understood whether stress induced neuroimmune dysfunctions play a causal role in the aetiology of rheumatic diseases or, in turn, rheumatic disease could change the stress reaction and thereby changing disease activity and worsening the outcome. However, epidemiological research increasingly suggests that exposure to traumatic stressors and psychological trauma is related to increased health care utilisation, adverse health outcomes, the onset of specific diseases including rheumatic diseases, and premature death (Boscarino et al., 2004; Sommershof et al., 2009).

#### **5.1 Stress as pathogenetic factor of autoimmune diseases**

From results of 27 studies evaluating more than 3,000 patients with RA it was recognised that stress is an important risk factor in the pathogenesis of autoimmune diseases. Major life events (e.g. death of a spouse, severe long-term illness of a spouse, loss of a parent, divorce of parents, death of a parent or severe disease of a parent) lead to an intense release of stress mediators (large time integral of released neurotransmitters and hormones), whereas in minor life events (daily hassles with small intensity) only short-lived surges of neurotransmitters and hormones are expected. Therefore, it is suggested that neurotransmitters such as norepinephrine or stress hormones such as cortisol might have different effects on immune/inflammatory responses at high and low concentrations present during short or extended periods of time, respectively. Long-lasting (chronic) stress may lead to pro-inflammatory effects since no adequate long-term responses of stress axes (anti-inflammatory) are to be expected (Cutolo & Straub, 2006). Furthermore, major life events and chronic minor stress seem to be important factors in juvenile chronic arthritis and are significantly associated with the onset of the rheumatic disease. With respect to RA, stress may be a provoking factor. However, during the course of the disease, minor stress could aggravate SLE and RA (Herrmann et al., 2000). In contrast, strong major stress, which is likely accompanied by a large and long-lived release of stress axes mediators, was associated with a decrease in disease activity (Cutolo & Straub, 2006).

#### **5.1.1 Early life stress**

224 Insights and Perspectives in Rheumatology

In addition, cultural differences in experiencing individual stress in RA patients were observed. In one study covering Polish and German RA patients it was demonstrated that in both countries, mental as well as physical health deteriorated resulting in about 50% of patients requiring support in everyday activities. 95% of Polish and 62% of German patients felt rejected from social activities. For the psychological stress perceived, functional capacity class 3 and male gender were shown to be predictive in Polish patients and living in a small town in German patients. In the Polish group, the tertiary/bachelor level of education was linked with lower distress level. RA has a serious impact on the mental health owing to a

Interestingly, during a 15-year follow-up study of 74 female patients with RA two categories of RA were identified: a disease form less connected with genetic factors and more influenced by major psychodynamic conflict situations ('major conflict group') and a second form more associated with hereditary predisposition and less influenced by environmental psychosocial changes ('non-conflict group') (Rimón & Laakso,1985). In addition, a patient's perspective of the causes and consequences of RA could underline the role of environmental factors in disease. Two descriptive categories of patient's understanding of disease could be identified: the category 'consequences beyond personal control' comprised not having a clue, being exposed to climatic change, being genetically exposed and unexpected effects of events; the category 'overloaded circumstances' involved work and family-related strain. Consequences beyond personal control implied that the patients could not prevent the disease and expressed their lack of understanding as to why they contracted it. Overloaded circumstances were described as strained situations that were both work and family factors related and could be influenced by the patient. Reasoning along these lines it is concluded that understanding the patients' own view of disease is needed in order to achieve a more successful medical care model (Bergsten et al., 2009). Identifying of how these factors contribute to the development of autoimmune diseases may further lead to better explaining

the pathogenesis of rheumatic diseases (Symmons, 2003; Kobayshi et al., 2008).

**5. Rheumatic diseases as a model of stress induced disease**

Finally, in one recent study of patients with psoriatic arthritis environmental factors such as lifting heavy loads of at least 100 pounds/hr (OR 2.8, 95% CI 1.51-5.05), infections that required antibiotics (OR 1.7, 95% CI 1.00-2.77), and injuries (OR 2.1, 95% CI 1.11-4.01) were associated with the occurrence of arthritis. No association was found between psoriasis arthritis and alcohol consumption, vaccination, and female hormonal exposures (Eder et al.,

Despite more than twenty years of stress research in rheumatology, it is not completely understood whether stress induced neuroimmune dysfunctions play a causal role in the aetiology of rheumatic diseases or, in turn, rheumatic disease could change the stress reaction and thereby changing disease activity and worsening the outcome. However, epidemiological research increasingly suggests that exposure to traumatic stressors and psychological trauma is related to increased health care utilisation, adverse health outcomes,

**4.5 Cultural difference**

**4.6 Other factors**

2011).

great disease burden (Bugajska et al., 2010).

One study (follow up 1980 till 1996) examining the risk of RA in parents after the death of a child does not support an association between severe psychological stress and RA. The relative risk of first hospitalisation for RA was 0.88 [95% CI 0.63-1.24]. The risk was close to 1 throughout the 18 years of follow-up (Li et al., 2005). Recent findings are consistent on the impact of early life stress on subsequent inflammatory responses. A predominant role for parent distress in children's adjustment to juvenile rheumatic disease was shown (Ryan et al., 2010). In one retrospective cohort study (follow–up 1995/1997 through 2005) of 15,357 adults it could be demonstrated that 64% adults reported early life stress with at least one adverse childhood experience (ACE), including childhood physical, emotional, or sexual abuse; witnessing domestic violence; growing up with household substance abuse, mental illness, parental divorce, and/or an incarcerated household member. The event rate (per 10,000 person-years) for a first hospitalization with any autoimmune disease was 31.4 in women and 34.4 in men. First hospitalizations for any autoimmune disease increased with increasing number of ACEs (p < 0.05). Persons with equal or more than 2 ACEs were at a 100 % increased risk for rheumatic diseases compared to persons without any ACE (p < 0.05) (Dube et al., 2009). Patients with juvenile idiopathic arthritis reported having pain, stiffness, and fatigue on > 70% of days, with significant variability in symptom levels. Furthermore, significant same-day relationships between stress, mood, and disease symptoms were revealed. Specifically, daily fluctuations in both stress and mood were predictive of increased pain, stiffness, and fatigue. Increases in daily stress, mood, and disease symptoms were also significantly related to decreased participation in social

The Pathogenetic Link Between Stress and Rheumatic Diseases 227

stressors few days prior to the visit (Cutolo & Straub, 2006). In addition, it was shown that stress is a potentially important risk factor for the onset of adult Still's disease. Stressful life events (OR 2.56; 95% CI 1.18 to 5.52) in the year preceding the onset was significantly associated with increased risk for adult Still's disease (Sampalis et al., 1996). Likewise, a longitudinal study over a period of 5 years showed that RA patients with a higher daily stress level at baseline had a poorer outcome and significantly more bony erosions after 5 years. Therefore, long-lasting (chronic) stress may lead to proinflammatory effects because no adequate long-term responses of stress axes (HPA – anti-inflammatory) are to be expected. Only 5 studies on about 150 RA patients did not support the link between minor stress and disease flares (Cutolo & Straub, 2006). However, it was shown that psychological distress and social support are more important than objectively assessed disease status in determining marital and sexual satisfaction in patients with RA (vam Lankfeld et al., 2004). In RA patients pain was the predominantly perceived stressor followed by limitation in mobility, difficulties in carrying out activities of daily living, helplessness, dependency on others, threat to self-esteem, interference in social activity, interference in family relationships, difficulties performing at work, and discomfort of the treatment (Mahat, 1997). Interestingly, perceived stress had the strongest relationship with psychological well-

In a large population-based case-control study (1996–2003) with incident cases of RA (1,221 cases and 1,454 controls) it could be shown that high psychological stress of job demands tended to be associated with a decreased risk of RA (OR = 0.8; 95 % CI = 0.6 – 1.0). Interestingly, low decision latitude was associated with an increased risk of RA (selfreported data: OR = 1.6; 95 % CI = 1.2 – 2.2). Self-reported job strain was associated with a 30% higher risk of RA, compared with relaxed working conditions (Benggsson et al., 2009). Similarly, in patients with osteoarthritis the relationship between social support, stress and functional status was of major importance. Thus, physical disability was associated with being older and having less tangible support; psychological disability was correlated with being younger, caucasian, and having less support; and pain was associated with being younger, caucasian and having less education. Self-esteem appeared to be the most, and appraisal the least, consistent social support dimension when predicting functional status. While exposure to stressors negatively affected functional status, its impact was greatest

Rheumatic diseases represent an important public health burden. To reduce the social and economic impact of these pathologies, an appropriate management of these conditions should be encouraged based on the use of established intervention strategies, including

In a recent metaanalysis of efficacy of psychological interventions in the treatment of RA patients 31 studies with 2021 patients could be included. There is consistent supportive evidence for the efficacy of disclosure therapy and cognitive behavioural therapy followed

being of rheumatic patients (Treharne et al., 2007).

with respect to psychological disability (Weinberger et al., 1990).

**6. Therapeutic interventions**

**6.1 Psychological approach**

stress reduction approaches (Ottonello, 2007).

activities on a day-to-day basis (Schanberg et al., 2005). However, one study does not support the hypothesis that stressful life events and adverse childhood experiences play an aetiologic role in the development of rheumatic disease. The number and timing of occurrence of stressful life events, as well as their subjective immediate impact, did not differ between participants who developed RA and their matched controls. Termination of pregnancy was the only specific event individually associated with a higher risk of developing RA (OR 3.74; 95% CI 1.4-9.9). Negative childhood experiences were not associated with the risk of RA. However, RA cases reported significantly slower adaptation to the effects of adverse events than controls (Carette et al., 2000).

#### **5.1.2 Posttraumatic stress disorder**