**4. Inflammation, COPD and heat shock proteins**

Chronic obstructive pulmonary disease is a slowly progressive condition, characterized by airflow inflammation, which is largely irreversible. It is suggested that the main etiological factor - cigarette smoking, produces inflammatory response in the lungs of all smokers and those who develop COPD have an abnormal or enhanced inflammation

### **4.1 Intracellular heat shock proteins – Antinflammatory molecules**

The heat shock response is one of the most evolutionary conserved protective mechanisms in cells. It involves a temporary modification of gene expression. Synthesis of different heat shock proteins helps the organism cope with environmental and physiological stresses. As anti-inflammatory effector the heat shock response modulates signal transduction and gene expression by inhibiting the translocation of transcriptional factor – nuclear factor kappa B to the nucleus and prevents the expression of inflammatory mediators (Wong et al, 1997; Sun et al, 2005; Malhotra and Wong, 2002). Initial observations in animals linked heat shock response to an altered inflammatory response and demonstrate that heat preconditioning confers survival in otherwise lethal endotoxin stress (Snyder et al, 1992; Ensor et al, 1994). Heat conditioned macrophages show decreased secretion of TNF-alpha induced by endotoxins. This decreased secretion was sustained as long as the cells had elevated HSP70 levels. Similar to these studies endotoxin induction of IL-6 was also unchanged in the heat-

Chronic Obstructive Pulmonary Disease - Chaperonopathology 79

by which HSP confer protection from cytokines are not clear but may involve the interplay of intracellular triggers related to cell survival, stress tolerance and inflammation. It is majorly accepted that this is cell type specific and is executed by the regulation of apoptosis.

**4.2 COPD and intracellular heat shock proteins – A mechanism of self-defence, a** 

There are a few studies that deal with the levels of expression of heat shock proteins in lung

Gal et al, 2011 showed for the first time that cigarette smoke extract stimulates the expression of HSP72 in alveolar epithelial cells. One possible mechanism for this could be the activation of cell preservation mechanisms, including decreased degradation of HSP72 in cells exposed to severely damaging substances. Heat shock protein induction is cytoprotective by preventing the onset of apoptosis. HSP72 has been shown to protect cells both from apoptosis and necrosis (Fekete et al. 2006; McConkey 1998). There are studies confirming other noxa - dimethylarsinic acid exposure-elevating intracellular HSP72 levels, changing the localization of the molecule and suppressing apoptosis of human alveolar cells

In addition HSP72 siRNA abolished the mRNA and protein increase in cells, in parallel apoptosis increased and less cells survived. These results confirm upregulation of HSP72 in the presence of CSE in order to ensure cell survival, and indicate key protecting role for HSP72 under this cellular stress condition. Moreover these authors show that the antiapoptotic effects of dexamethasone in alveolar epithelial cells are accomplished only after the upregulation of HSP72 that follows the cigarette smoke exposure. According to these experiments, increase in the inducible form of HSP70(HSP72) following CSE administration might enable proper action of administered dexamethasone, by increasing the assembly of GR and opening the steroid binding cleft. Glucocorticoid receptor (GR) function is dependent on the HSP90/HSP70 chaperone machinery. Initial GR interaction with HSP70 appears to be critical for the triage between HSP90 heterocomplex assembly and preservation of receptor function. HSP70 is required for the assembly of protein - HSP90 heterocomplexes, and the two chaperones interact directly with each other while opening

The results of Gal et al, are consistent to those reported by Chao-Jun Li et al, 2007. They found upregulation of HSP70 in human lung fibroblasts exposed to cigarette smoke. Similar is the data represented by Doz et al, 2008 who report that there is an increase in the levels of HSP70 in bronchoalveolar lavage of mice exposed to cigarette smoke. The inducible form of HSP70 is also upregulated in monocytes and endothelial cells of COPD patients. (Balsano, et

Ruicheng Hu et al, 2011 applied proteomics (MALDI-TOFF) to compare the expression profiles of proteins in cell lysates of lung tissue of 24 COPD smokers (6 in stage I and 18 in stage II, stable COPD), 24 non-COPD smokers, and 24 never-smokers. Age, gender distribution, body mass index did not differ significantly between the groups. Smoking index did not differ significantly between 11 COPD smokers and non-COPD smokers. Key spirometric parameters, including FEV1, FEV1%, FVC%, maximum predicted expiratory

**trigger for immune inflammation, or a chaperonopathology** 

the steroid binding cleft in the GR (Pratt and Toft 2003).

(Schett et al, 2003; Ran and Lu, 2004)

of COPD patients.

(Kato et al. 2000).

al, 1999; Ning et al, 2004)

conditioned cells. The decrease of cytokine production was associated with a decrease in cytokine mRNA, suggesting that cell stress response regulates cytokine transcription. (Snyder et al, 1992; Ensor et al, 1994)

The liver has been the most extensively studied tissue to delineate the heat stress response in animals, because of the massive accumulation of HSP70 following stress. There is compelling data to support a direct link between liver HSP70 accumulation and altered survival and inflammatory cytokine profile seen in heat-conditioned animals undergoing endotoxin stress. Human peripheral blood macrophages overexpressing HSP70 also inhibited lipopolysaccharide induced production of TNF-α, IL-1β, IL-10 and IL-12 (Ding et al, 2001; Dokladny et al, 2008). These data taken as a whole demonstrates, that HSP70 expression is sufficient to alter proinflammatory cytokine production and increase endotoxin tolerance. A number of potential explanations have been suggested for the HSP mediated inflammatory repression. Immune cells are stimulated by a number of incoming signals from cell surface, including ischaemia, oxidative stress, LPS etc and initiate an inflammatory response by the activation of signalling pathways and transcription factors. The NFκB transcription factors play a pivotal role, altering the expression of cytokines, chemokines, cell adhesion molecules, growth factors, anti-apoptotic proteins and immunoreceptors (Brasier et al, 2006). Inactive NFκB is normally found in the cytoplasm, bound to its inhibitory protein, IκB. NFκB is activated by a number of incoming signals. These activate IKK, which phosphorylates IκB and allows NFκB to translocate into the nucleus and bind its target genes – TNFα, IL-1β, IL-6, IL-12 (Zhang and Ghosh, 2000).

HSP70 interacts directly with the NFκB inhibitor protein – IκB-α, which appears to prevent NFκB dissociation. HSP70 blocks IκB-α degradation and up-regulates IκB-α mRNA. Another mechanism of inflammatory suppression is indirect mechanism - repression of MAPK activation that mediates the inhibition of NFκB cascade. In addition, HSP70 suppresses activation of Jun kinase (JNK) MAPK. This prevents the phosphorylation of c-JUN and the subsequent activation of transcription factor AP-1. (Wang et al, 2002)

High mobility group box 1 protein (HMGB1) can trigger MAPK pathway and subsequent NFκB mediated synthesis and release of inflammatory mediators. HMGB1 is another step in the inflammatory cascade that is regulated by heat shock response. (Tang et al, 2005). HSP 70 overexpression suppressed the release and translocation of HMGB1.

The anti-inflammatory function of the small heat shock proteins is also confirmed. They interfere with the signallingpathway through their ability to protect against oxidative stress (Mehlen et al, 1995) and through modulation of TAK-1 activity (Alford et al, 2007). Another mechanism is the ability of HSP27 to suppress NFκB activation by interaction with IKK-α and IKK-β (Kammanadiminti SJ and Chadee K, 2006). HSP27 is also needed for the activation of TAK1 and downstream signaling by p38 MAPK, JNK (Alford et al, 2007). Both HSP27 and αB-crystalline have crucial roles in the control of inflammatory processes.

Among the pathologies where anti-oxidative defence of HSP27 is important is the airway inflammation in asthma. It is observed that in the airway of asthmatic patients HSP27 has increased expression and generates protection against oxidative stress, accompanying the chronic inflammatory state of this tissue.

In addition to altering of cytokine production heat shock proteins are also capable to influence cell tolerance to cytokines (Kusher et al, 1990, Jattela et al, 1993). The mechanisms

conditioned cells. The decrease of cytokine production was associated with a decrease in cytokine mRNA, suggesting that cell stress response regulates cytokine transcription.

The liver has been the most extensively studied tissue to delineate the heat stress response in animals, because of the massive accumulation of HSP70 following stress. There is compelling data to support a direct link between liver HSP70 accumulation and altered survival and inflammatory cytokine profile seen in heat-conditioned animals undergoing endotoxin stress. Human peripheral blood macrophages overexpressing HSP70 also inhibited lipopolysaccharide induced production of TNF-α, IL-1β, IL-10 and IL-12 (Ding et al, 2001; Dokladny et al, 2008). These data taken as a whole demonstrates, that HSP70 expression is sufficient to alter proinflammatory cytokine production and increase endotoxin tolerance. A number of potential explanations have been suggested for the HSP mediated inflammatory repression. Immune cells are stimulated by a number of incoming signals from cell surface, including ischaemia, oxidative stress, LPS etc and initiate an inflammatory response by the activation of signalling pathways and transcription factors. The NFκB transcription factors play a pivotal role, altering the expression of cytokines, chemokines, cell adhesion molecules, growth factors, anti-apoptotic proteins and immunoreceptors (Brasier et al, 2006). Inactive NFκB is normally found in the cytoplasm, bound to its inhibitory protein, IκB. NFκB is activated by a number of incoming signals. These activate IKK, which phosphorylates IκB and allows NFκB to translocate into the nucleus and bind its target genes – TNFα, IL-1β, IL-6, IL-12 (Zhang and Ghosh, 2000).

HSP70 interacts directly with the NFκB inhibitor protein – IκB-α, which appears to prevent NFκB dissociation. HSP70 blocks IκB-α degradation and up-regulates IκB-α mRNA. Another mechanism of inflammatory suppression is indirect mechanism - repression of MAPK activation that mediates the inhibition of NFκB cascade. In addition, HSP70 suppresses activation of Jun kinase (JNK) MAPK. This prevents the phosphorylation of c-

High mobility group box 1 protein (HMGB1) can trigger MAPK pathway and subsequent NFκB mediated synthesis and release of inflammatory mediators. HMGB1 is another step in the inflammatory cascade that is regulated by heat shock response. (Tang et al, 2005). HSP

The anti-inflammatory function of the small heat shock proteins is also confirmed. They interfere with the signallingpathway through their ability to protect against oxidative stress (Mehlen et al, 1995) and through modulation of TAK-1 activity (Alford et al, 2007). Another mechanism is the ability of HSP27 to suppress NFκB activation by interaction with IKK-α and IKK-β (Kammanadiminti SJ and Chadee K, 2006). HSP27 is also needed for the activation of TAK1 and downstream signaling by p38 MAPK, JNK (Alford et al, 2007). Both

Among the pathologies where anti-oxidative defence of HSP27 is important is the airway inflammation in asthma. It is observed that in the airway of asthmatic patients HSP27 has increased expression and generates protection against oxidative stress, accompanying the

In addition to altering of cytokine production heat shock proteins are also capable to influence cell tolerance to cytokines (Kusher et al, 1990, Jattela et al, 1993). The mechanisms

HSP27 and αB-crystalline have crucial roles in the control of inflammatory processes.

JUN and the subsequent activation of transcription factor AP-1. (Wang et al, 2002)

70 overexpression suppressed the release and translocation of HMGB1.

chronic inflammatory state of this tissue.

(Snyder et al, 1992; Ensor et al, 1994)

by which HSP confer protection from cytokines are not clear but may involve the interplay of intracellular triggers related to cell survival, stress tolerance and inflammation. It is majorly accepted that this is cell type specific and is executed by the regulation of apoptosis. (Schett et al, 2003; Ran and Lu, 2004)

#### **4.2 COPD and intracellular heat shock proteins – A mechanism of self-defence, a trigger for immune inflammation, or a chaperonopathology**

There are a few studies that deal with the levels of expression of heat shock proteins in lung of COPD patients.

Gal et al, 2011 showed for the first time that cigarette smoke extract stimulates the expression of HSP72 in alveolar epithelial cells. One possible mechanism for this could be the activation of cell preservation mechanisms, including decreased degradation of HSP72 in cells exposed to severely damaging substances. Heat shock protein induction is cytoprotective by preventing the onset of apoptosis. HSP72 has been shown to protect cells both from apoptosis and necrosis (Fekete et al. 2006; McConkey 1998). There are studies confirming other noxa - dimethylarsinic acid exposure-elevating intracellular HSP72 levels, changing the localization of the molecule and suppressing apoptosis of human alveolar cells (Kato et al. 2000).

In addition HSP72 siRNA abolished the mRNA and protein increase in cells, in parallel apoptosis increased and less cells survived. These results confirm upregulation of HSP72 in the presence of CSE in order to ensure cell survival, and indicate key protecting role for HSP72 under this cellular stress condition. Moreover these authors show that the antiapoptotic effects of dexamethasone in alveolar epithelial cells are accomplished only after the upregulation of HSP72 that follows the cigarette smoke exposure. According to these experiments, increase in the inducible form of HSP70(HSP72) following CSE administration might enable proper action of administered dexamethasone, by increasing the assembly of GR and opening the steroid binding cleft. Glucocorticoid receptor (GR) function is dependent on the HSP90/HSP70 chaperone machinery. Initial GR interaction with HSP70 appears to be critical for the triage between HSP90 heterocomplex assembly and preservation of receptor function. HSP70 is required for the assembly of protein - HSP90 heterocomplexes, and the two chaperones interact directly with each other while opening the steroid binding cleft in the GR (Pratt and Toft 2003).

The results of Gal et al, are consistent to those reported by Chao-Jun Li et al, 2007. They found upregulation of HSP70 in human lung fibroblasts exposed to cigarette smoke. Similar is the data represented by Doz et al, 2008 who report that there is an increase in the levels of HSP70 in bronchoalveolar lavage of mice exposed to cigarette smoke. The inducible form of HSP70 is also upregulated in monocytes and endothelial cells of COPD patients. (Balsano, et al, 1999; Ning et al, 2004)

Ruicheng Hu et al, 2011 applied proteomics (MALDI-TOFF) to compare the expression profiles of proteins in cell lysates of lung tissue of 24 COPD smokers (6 in stage I and 18 in stage II, stable COPD), 24 non-COPD smokers, and 24 never-smokers. Age, gender distribution, body mass index did not differ significantly between the groups. Smoking index did not differ significantly between 11 COPD smokers and non-COPD smokers. Key spirometric parameters, including FEV1, FEV1%, FVC%, maximum predicted expiratory

Chronic Obstructive Pulmonary Disease - Chaperonopathology 81

Extracellular HSP, particularly HSP70, HSP90 and gp96, serve as antigen carriers and facilitate antigen uptake by dendritic or antigen presenting cells. Uptake is mediated by several mechanisms, including the α2-macroglobulin receptor (Binder et al, 2000). The HSPpeptide complex is more efficiently taken up by APCs than antigen alone. In addition HSP

HSP facilitate antigen processing and transfer to MHC – I complex for presentation to cytotoxic T-lymphocytes. They are also expressed on the surface of tumor cells in cell culture as well as in cells infected with viruses.(Multhoff et al,1997) The HSP expression on tumor cells correlates to direct natural killer cell induced cytotoxicity and can be blocked by incubating target cells with antibodies directed against HSP70 prior to NK cell exposure.

The molecular mechanisms by which cigarette smoke causes the inflammatory process and pathology of COPD remain poorly understood. Chronic bronchitis and lung emphysema are pathologic characteristics of COPD and both conditions result from progressive and amplified inflammation that destructs and remodels parenchyma. Immune activation is not restricted to the lungs and is systematic. It procedes even after smoking cessation. In contrast to other inflammatory conditions the inflammation in the lungs is severe even in the advanced stages. There is enough evidence that make it reasonable enough to hypothesize that COPD has some kind of autoimmiunity in its pathogenesis. Nowadays it is largely accepted that the oxidative stress, accompanying COPD leads to changes in cell structures that makes them antigenic, thus triggering an autoimmune inflammatory

It has been demonstrated that expression of HSPs is upregulated under stressful events in the lungs. Besides known intracellular chaperoning, it is possible that HSPs may also be released into the extracellular space following massive trauma or stress. This spillage of

The involvement of extracellular HSP in COPD inflammatory process was described by Chao-Jun Li, et al 2008. They show that lung fibroblast exposed to cigarette smoke extract (CSE) release IL-8. The secretion was HSP70 dependent. Marked induction of HSP70 was observed in fibroblast culture medium in response to CSE. Upon exogenous administration of recombinant HSP70 to CSE treated fibroblasts, IL-8 production also increased. These results suggest that HSP70 is secreted into the extracellular environment via an unidentified mechanism that stimulates the production of IL-8 in primary lung fibroblasts. To examine whether it is the extracellular HSP70 that leads to CSE-induced IL-8 production, they determined CSE-induced IL-8 production in the presence or absence of neutralizing antibodies against HSP70 in the medium. Fibroblasts subjected to neutralizing antibody to HSP70 in the medium, exhibited marked reduction of CSE induced IL-8 production but did not completely abrogate the response. These data suggest that the extracellular HSP70 plays a critical role in mediating CSE-induced IL-8 production but also point to an HSP70 independent pathway of IL-8 production by CSE stimulus. They identified a novel early molecular pathway that mediates chemokine IL-8 release by human primary fibroblasts after cigarette smoke exposure. Early growth factor -1 (EGR-1) can trigger the synthesis of

proteins serves as "danger signal" leading to cytokine transcription and release.

also stimulate APC maturation and activate NFκB signal pathways (Basu et al, 2000).

(Roigas et al, 1998)

**4.4 COPD and extracellular heat shock proteins** 

response in patients with a genetic susceptibility.

flow rate at 50% of vital capacity (MEF50%), and MEF25% were comparable between the non-COPD smokers and never-smokers.Twenty-four proteins were identified by MS as being differentially expressed among the three groups of subjects. The main functions of these proteins involve basic metabolism, oxidation/reduction, coagulation/fibrinolysis, protein degradation, signal transduction, inflammation, and cell growth/ differentiation/ apoptosis. Proteomic analysis revealed that the expression of HSP27 and CyPA was upregulated in smokers, and this upregulation was particularly marked in COPD smokers. The variation in expression of HSP27 and CyPA between the groups was confirmed by IHC and Western blotting. Based on the results from the present study and other studies that have shown a protective role for HSP27 against oxidative stress and apoptosis, it is suggested that induction of HSP27 protects the lung cells of smokers and COPD patients against oxidative stress and apoptosis.

In contrary to the other heat shock proteins Capello et al, 2006 found a decrease of tissue expression of HSP60 and HSP10 that were parallel to chronic obstructive pulmonary disease progression, but did not correlate to the severity of COPD in smoking patients with NSCLC. They detected a trend for a decrease of intracellular expression of this chaperone that correlated best to the degree of tissue dedifferentiation.

In conclusion most of the studies dedicated to the role of heat shock proteins in COPD pathology are concentrated on tissue cell lysates or epithelium. The up-regulation of chaperones in them seems to be a protective mechanism, providing survival through antiapoptotic and anti-oxidant role. Little is known about the expression of these cell proteins in neutrophils, lymphocytes and dendiritc cells and the way they influence the immune inflammation.
