**4.3 Extracellular heat shock proteins – Pro-inflammatory molecules**

Intracellular HSP confer anti-inflammatory state because they downregulate inflammatory cytokine production, increase cellular tolerance to cytokines mediated cytotoxicity and attenuate epithelial barrier permeability changes. Alongside heat shock proteins are involved in multifaceted inflammatory reactions when seen by immune effector cells in the extracellular environment. They serve as co-stimulator molecules for immune recognition and are among the major molecules, refered by Matzinger, as "danger" signals (Matzinger, 2002) The release of HSP in the extracellular environment is an area of current research. It is known however that HSP can be released either passively after cell necrosis, trauma of cells or viral infection, or actively – exercise, physiological stress, certain diseases (Basu et al, 2000, Caldwerwood et al, 2007; Hightower et al, 1989). In addition the immune effects of HSPs are different when released by necrotic cells or during physiological response. The immunogenic activity of HSPs is mediated by two main mechanisms: 1) cytokine reponse by the activation of Toll-like receptors 2) facilitation of antigen uptake and formation of HSPpeptide complexes by antigen presenting cells. (Asea, et al, 2007)

Extracellular HSP can bind to cell surface receptors like TLR2 and TLR4 and lead to signaling events and activation of antigen presenting cells (Srivastava et al,1994). This activation leads to a signalling cascade and includes the activation of IL-1 and NFκB signal transduction pathway. HSP70 for example signals through TLR2 and TLR4 with the involvement of CD14 of human monocytes. This is followed by an upregulation of inflammatory cytokine secretion (IL-1β, IL-6, TNF-α).

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

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

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

Intracellular HSP confer anti-inflammatory state because they downregulate inflammatory cytokine production, increase cellular tolerance to cytokines mediated cytotoxicity and attenuate epithelial barrier permeability changes. Alongside heat shock proteins are involved in multifaceted inflammatory reactions when seen by immune effector cells in the extracellular environment. They serve as co-stimulator molecules for immune recognition and are among the major molecules, refered by Matzinger, as "danger" signals (Matzinger, 2002) The release of HSP in the extracellular environment is an area of current research. It is known however that HSP can be released either passively after cell necrosis, trauma of cells or viral infection, or actively – exercise, physiological stress, certain diseases (Basu et al, 2000, Caldwerwood et al, 2007; Hightower et al, 1989). In addition the immune effects of HSPs are different when released by necrotic cells or during physiological response. The immunogenic activity of HSPs is mediated by two main mechanisms: 1) cytokine reponse by the activation of Toll-like receptors 2) facilitation of antigen uptake and formation of HSP-

Extracellular HSP can bind to cell surface receptors like TLR2 and TLR4 and lead to signaling events and activation of antigen presenting cells (Srivastava et al,1994). This activation leads to a signalling cascade and includes the activation of IL-1 and NFκB signal transduction pathway. HSP70 for example signals through TLR2 and TLR4 with the involvement of CD14 of human monocytes. This is followed by an upregulation of

against oxidative stress and apoptosis.

inflammation.

correlated best to the degree of tissue dedifferentiation.

**4.3 Extracellular heat shock proteins – Pro-inflammatory molecules** 

peptide complexes by antigen presenting cells. (Asea, et al, 2007)

inflammatory cytokine secretion (IL-1β, IL-6, TNF-α).

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

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. (Roigas et al, 1998)

### **4.4 COPD and extracellular heat shock proteins**

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 response in patients with a genetic susceptibility.

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

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

Chronic Obstructive Pulmonary Disease - Chaperonopathology 83

There was a significant increase of HSP27 in serum samples taken from the peripheral blood flow of patients suffering from COPD as compared to healthy smokers. Hacker et al, demonstrate a continuous increase of serum HSP27 concentrations with disease severity in their study. This effect may be due to increased tissue devastation especially in late stages of COPD and spreading of the inflammatory disease to other organs provoking a systemic spillage of HSP27 into the vascular bed. HSP27 generally acts as antiapoptotic mediator and can be seen as an endogenous immunosuppressive attempt to control excessive inflammation in COPD. Serum contents of HSP27 showed diagnostic potential to determine the occurrence of COPD in a logistic regression model and may serve as a marker for diagnosis and prediction of disease severity. Serum levels of HSP70 were elevated in patients at early and late stages of COPD. There was a four-fold increase in the GOLD I-II group compared to non-symptomatic smokers. Values were highest in the COPD I-II group, indicating a state of vast immune activation primarily at the early stages of the disease. Serum levels of HSP70 showed high sensitivity and specificity to define the occurrence of COPD in a logistic regression model and could serve as diagnostic marker. Because there was no significant difference between the COPD groups, HSP70 in comparison to HSP27 is unlikely to be a suitable marker for disease progression or

Rajagopal et al. characterized HSP90 as central factor in antigen presentation to T lymphocytes via major histocompatibility complex class II molecules (MHCII). Hacker et al, found soluble HSP90α was significantly upregulated in the peripheral blood of COPD groups as compared to healthy non-smokers. They hypothesize that elevated levels of extracellular HSP90α in COPD are essential in the adaptive immune system, triggering a possible autoreactive response to self-antigen. They suggest that HSP90α has immunomodulatory effects through cross-presentation of associated peptides in the context of major histocompatibility complex molecules. According to their results HSP60 is not a key element in the pathogenesis of COPD. Serum concentrations of HSP60 did not correlate with levels of other HSPs. Authors did not analyse whether there was a correlation between extracellular HSP and markers of inflammation – IL-6, CRP. They concluded that there were elevated serum concentrations of soluble heat shock proteins 27, 70 and 90α in patients with COPD. Their spillage into the vascular bed may be caused by continuous activation of the immune system in the deterioration of COPD through endogenous and exogenous trigger mechanisms. Furthermore, HSP27 and HSP70 showed statistical trends to serve as

Cherneva et al, compared the plasma levels of αB-crystalline in 63 COPD patients, 52 healthy-smokers and 48 smokers with inflammatory lung diseases. ELISA was applied as a method of detection. 43 of the COPD patients had severe disease (GOLD – III-IV) and 20 had mild disease (GOLD – I-II). The age distribution between the three groups was similar with a mean age of 67.24 (±8.06). All the patients had a comparable smoking exposure – 29.58 (±12.28) pack-years. In 26 of the COPD patients plasma levels of MMP-9 and CRP were also

The mean levels of αB-crystalline were respectively: COPD patients – 0.352 (±0.12); healthy smokers – 0.291 (±0.07); smokers with inflammatory lung diseases – 0.433 (±0.27). Statistically significant difference was established between the COPD patients and the healthy smoking volunteers - (p=0.010) and between smokers with inflammatory lung

response to therapy.

evaluated.

diagnostic markers or markers for disease progression.

HSP70. The HSP70 is then secreted into the extracellular environment and activates proinflammatory molecule (such as IL-8) production. They hypothesize that released HSP70 may activate cells through TLR-2, TLR-4, and CD14 and thereby mediate inflammation.

Similar are the results presented by Chase et al, 2007. Through their investigation, they have found evidence that supports the presence and biological activity of extracellular HSP72 in the lung. Chase et al, established that the airway epithelium itself is responsive to extracellular HSP72 and that this cytokine response is regulated through the TLR4 and NFκB pathways. Their data would suggest that extracellular HSP72 is responsible for inducing and propagating inflammation, a process that is central to the pathogenesis of lung injury. Ganter et al. found extracellular HSP72 to be a marker of improved alveolar fluid clearance and therefore recovery from lung injury. This and other clinical investigations presenting divergent effects of extracellular HSP72 would suggest that the mere presence of HSP72 in the extracellular milieu is not the only factor. Chase et al, hypothesize that there may be a threshold of extracellular HSP72 that is required to maintain adequate signaling, below which the cells are unprepared for the insult, and above which excessive inflammation and therefore increased injury occur.

Doz et al, 2008 show that cigarette smoke exposure of the airways induces acute inflammation in mice. They found that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signalling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4(-) and MyD88 (-), and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 (MMP-9) activity in the bronchoalveolar space. Cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro. The process was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activated the innate immune system via TLR4/MyD88 an resulted in airway inflammation.They concluded that acute cigarette exposure results in LPS-independent TLR4 activation. This led to the IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease.

Considering the presented data little is known about the clinical significance and mechanisms of secretion of extracellular heat shock proteins in COPD. There is scarce information about their relevance to the initiation and maintenance of the persistent inflammation.

#### **4.5 Extarcellular heat shock proteins – A diagnostic marker, a marker of inflammation or disease severity**

Hacker at al, 2009 investigated serum levels of heat shock proteins - HSP 27, 60, 70, 90alpha, 20S proteasomes, C-reactive protein (CRP), and interleukin-6 (IL-6). Serum levels were evaluated in healthy non-smoking volunteers (15), smokers without COPD (14), patients with mild to moderate COPD (19) and patients with severe or very severe COPD (16) were evaluated in four study groups. HSP27, HSP70 and HSP90α were significantly altered in patients suffering from COPD as compared to controls.

HSP70. The HSP70 is then secreted into the extracellular environment and activates proinflammatory molecule (such as IL-8) production. They hypothesize that released HSP70 may activate cells through TLR-2, TLR-4, and CD14 and thereby mediate

Similar are the results presented by Chase et al, 2007. Through their investigation, they have found evidence that supports the presence and biological activity of extracellular HSP72 in the lung. Chase et al, established that the airway epithelium itself is responsive to extracellular HSP72 and that this cytokine response is regulated through the TLR4 and NFκB pathways. Their data would suggest that extracellular HSP72 is responsible for inducing and propagating inflammation, a process that is central to the pathogenesis of lung injury. Ganter et al. found extracellular HSP72 to be a marker of improved alveolar fluid clearance and therefore recovery from lung injury. This and other clinical investigations presenting divergent effects of extracellular HSP72 would suggest that the mere presence of HSP72 in the extracellular milieu is not the only factor. Chase et al, hypothesize that there may be a threshold of extracellular HSP72 that is required to maintain adequate signaling, below which the cells are unprepared for the insult, and above which excessive inflammation and

Doz et al, 2008 show that cigarette smoke exposure of the airways induces acute inflammation in mice. They found that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signalling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4(-) and MyD88 (-), and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 (MMP-9) activity in the bronchoalveolar space. Cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro. The process was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activated the innate immune system via TLR4/MyD88 an resulted in airway inflammation.They concluded that acute cigarette exposure results in LPS-independent TLR4 activation. This led to the IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation

Considering the presented data little is known about the clinical significance and mechanisms of secretion of extracellular heat shock proteins in COPD. There is scarce information about their relevance to the initiation and maintenance of the persistent

**4.5 Extarcellular heat shock proteins – A diagnostic marker, a marker of inflammation** 

Hacker at al, 2009 investigated serum levels of heat shock proteins - HSP 27, 60, 70, 90alpha, 20S proteasomes, C-reactive protein (CRP), and interleukin-6 (IL-6). Serum levels were evaluated in healthy non-smoking volunteers (15), smokers without COPD (14), patients with mild to moderate COPD (19) and patients with severe or very severe COPD (16) were evaluated in four study groups. HSP27, HSP70 and HSP90α were significantly altered in

inflammation.

inflammation.

**or disease severity** 

therefore increased injury occur.

leading to chronic obstructive pulmonary disease.

patients suffering from COPD as compared to controls.

There was a significant increase of HSP27 in serum samples taken from the peripheral blood flow of patients suffering from COPD as compared to healthy smokers. Hacker et al, demonstrate a continuous increase of serum HSP27 concentrations with disease severity in their study. This effect may be due to increased tissue devastation especially in late stages of COPD and spreading of the inflammatory disease to other organs provoking a systemic spillage of HSP27 into the vascular bed. HSP27 generally acts as antiapoptotic mediator and can be seen as an endogenous immunosuppressive attempt to control excessive inflammation in COPD. Serum contents of HSP27 showed diagnostic potential to determine the occurrence of COPD in a logistic regression model and may serve as a marker for diagnosis and prediction of disease severity. Serum levels of HSP70 were elevated in patients at early and late stages of COPD. There was a four-fold increase in the GOLD I-II group compared to non-symptomatic smokers. Values were highest in the COPD I-II group, indicating a state of vast immune activation primarily at the early stages of the disease. Serum levels of HSP70 showed high sensitivity and specificity to define the occurrence of COPD in a logistic regression model and could serve as diagnostic marker. Because there was no significant difference between the COPD groups, HSP70 in comparison to HSP27 is unlikely to be a suitable marker for disease progression or response to therapy.

Rajagopal et al. characterized HSP90 as central factor in antigen presentation to T lymphocytes via major histocompatibility complex class II molecules (MHCII). Hacker et al, found soluble HSP90α was significantly upregulated in the peripheral blood of COPD groups as compared to healthy non-smokers. They hypothesize that elevated levels of extracellular HSP90α in COPD are essential in the adaptive immune system, triggering a possible autoreactive response to self-antigen. They suggest that HSP90α has immunomodulatory effects through cross-presentation of associated peptides in the context of major histocompatibility complex molecules. According to their results HSP60 is not a key element in the pathogenesis of COPD. Serum concentrations of HSP60 did not correlate with levels of other HSPs. Authors did not analyse whether there was a correlation between extracellular HSP and markers of inflammation – IL-6, CRP. They concluded that there were elevated serum concentrations of soluble heat shock proteins 27, 70 and 90α in patients with COPD. Their spillage into the vascular bed may be caused by continuous activation of the immune system in the deterioration of COPD through endogenous and exogenous trigger mechanisms. Furthermore, HSP27 and HSP70 showed statistical trends to serve as diagnostic markers or markers for disease progression.

Cherneva et al, compared the plasma levels of αB-crystalline in 63 COPD patients, 52 healthy-smokers and 48 smokers with inflammatory lung diseases. ELISA was applied as a method of detection. 43 of the COPD patients had severe disease (GOLD – III-IV) and 20 had mild disease (GOLD – I-II). The age distribution between the three groups was similar with a mean age of 67.24 (±8.06). All the patients had a comparable smoking exposure – 29.58 (±12.28) pack-years. In 26 of the COPD patients plasma levels of MMP-9 and CRP were also evaluated.

The mean levels of αB-crystalline were respectively: COPD patients – 0.352 (±0.12); healthy smokers – 0.291 (±0.07); smokers with inflammatory lung diseases – 0.433 (±0.27). Statistically significant difference was established between the COPD patients and the healthy smoking volunteers - (p=0.010) and between smokers with inflammatory lung

Chronic Obstructive Pulmonary Disease - Chaperonopathology 85

 A cell possessing a normal set of chaperones could potentially counteract the proteinopathies. However chaperones are themselves modified by the passage of time So it could be that both chaperonopathies and proteinopathies start in parallel and independently of one another. In that case chaperones would also play a major role, though not the primary one, demonstrating a failure of the cellular adaptation to maintain the protein homeostasis and functioning during senescence (age-related

 There could be an accumulation of damaged proteins due to high levels of stress, which would result in a widespread deficiency of otherwise normal chaperones and thus lead to an accelerated onset of degenerative or age-related diseases. In that case chaperonapathies seem to be a major trigger in the mechanisms of senescence (COPD); Another possibility – the presence of genetically defective chaperone system a chaperonopathy that can not counteract and keep the protein homeostasis even when there are no environmental challenges. Chaperone failure in that case would cause the initiation and progression of proteinopathies. Besides this the abnormal chaperones would not perform their physiological roles in cells, thus affecting essential cellular

 Chaperonopathies could be treated as a different characteristic of the process of senescence but not one of its mechanisms. However taking into account clinical,

Ignoring the dilemma, whether the chaperonapathies or the proteinopathies are first during the process of ageing, the immune system will inevitably response to what is going on in the organism. If this is the chaperonopathy 1) Chaperones could be spilled extracellularly as "danger" signals, activating the antigen-presenting cells; 2) chaperones could be associated with other peptides on the surface of cells announcing for a ''danger''cytotoxicity or; 3) chaperones can simply allow the accumulation of modified proteins that are accepted as

If this is simply a proteinpathy as Kirkow assumes the defects cause inflammatory reactions, which can themselves exacerbate existing damage, so that inflammatory and antiinflammatory factors can play a part in modulating the outcome of the ageing process. Thus, age-associated inflammation/structural change is a failure of elimination and/or

Ito and Barnes, 2009 define COPD as accelerated lung ageing disease. They find a lot of similarities between aged lung and COPD lung. These are not mere clinical characteristics –

1. Telomere length has been demonstrated to be significantly shorter in patients with emphysema than in asymptomatic nonsmokers. This is confirmed in alveolar type II cells and endothelial cells, (Tsuji et al, 2006) peripheral blood mononuclear cells,(Morla

2. There is ample evidence that oxidative stress plays a major role in COPD. Increase in nitrotyrosine deposition is also a feature seen in COPD lung as well as aged tissue. This is the evidence of an increase in nitrative/oxidative stress, and may contribute to the accumulation of nitrated and oxidized proteins. Superoxide dismutase enzyme activity is reported to be lower in long-term healthy smokers and in stable COPD patients than

pathological and experimental studies this is hardly reasonable.

accelerated decline of lung function but a number of molecular ones.

et al, 2006) and fibroblasts.(Muller et al, 2006)

emphysema);

self-antigenic.

failure of repair (DNA, protein).

processes (emphysema in COPD);

diseases and the healthy smoking volunteers (p=0.007). In comparison there was not a significant difference between the COPD patients and those with inflammatory pulmonary diseases - (p=0.158). No relation was established between αB-crystalline plasma levels and hsCRP and MMP-9 levels respectively (p=0.91 and p=0,76)

Authors concluded that αB-crystalline is not a specific diagnostic marker. It rather reflects oxidative stress, and inflammation that accompanies it.
