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The effect of aging and inflammation on heat shock protein 27 in human monocytes and lymphocytes
Experimental Gerontology 41 (2006) 312–319 www.elsevier.com/locate/expgero
The effect of aging and inflammation on heat shock protein 27 in human monocytes and lymphocytes R. Njemini a,b, M. Lambert a, C. Demanet c, T. Mets a,b,* a Geriatric Unit, Academic Hospital, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium Gerontology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium c HLA and Immunology laboratory, Academic Hospital, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium b
Received 5 August 2005; received in revised form 19 January 2006; accepted 20 January 2006
Abstract Heat shock proteins (Hsp) are highly conserved proteins and their synthesis is ubiquitous in virtually every species in which they have been sought. In the present study we have investigated the effect of age and inflammation on the induction of Hsp27 in human peripheral blood mononuclear cells, using flow cytometry. Sixty-six healthy control subjects or patients suffering from inflammation participated in the study. In both heat shocked (HS) and non-HS conditions, the percentage of Hsp27 producing lymphocytes as well as the intensity of Hsp27 in lymphocytes and monocytes were negatively influenced by age. The basal levels and also the levels of Hsp27 production after HS were higher for monocytes compared to lymphocytes. In addition, we found that HS resulted in a small but significant increase in the levels of Hsp27 in lymphocytes whereas a significant decrease in Hsp27 was noticed for monocytes. In conclusion, results presented herein provide evidence in support of an age-related decrease in the level of Hsp27, which disappeared in the presence of inflammation. Several relationships between the circulating levels of CRP, IL-6 and TNF-a with the various Hsp27 determinations were observed, indicating that cytokines are able to influence the production of Hsp27. q 2006 Elsevier Inc. All rights reserved. Keywords: Elderly; Cytokines; Mononuclear blood cells; Heat shock; Heat shock protein 27
1. Introduction Heat shock proteins (Hsp) are present in cells of all organisms and the genes encoding them are highly conserved (Rattan et al., 2004). Distinction has been made between Hsp that are almost absent under non-stressed conditions but synthesised after cellular stress, and Hsp that are constitutively synthesised in cells. The former are called inducible proteins, while the latter are known as cognate proteins. Constitutive and stress-inducible Hsp play diverse roles in cellular function. Under normal physiological conditions constitutively synthesised Hsp act as molecular chaperones modulating protein folding, assembly, intracellular localisation, secretion, and
Abbreviations: HS, Heat shock(ed); Hsp, Heat shock protein(s); MFI, Mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species. * Corresponding author. Address: Geriatric Unit, Academic Hospital, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium. Tel.: C32 2 477 63 66; fax: C32 2 477 63 64. E-mail address: [email protected] (T. Mets). 0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.01.006
degradation (Ellis and van der Vies, 1991; Hartl, 1996). When cells endure stress such as high temperature, exercise, oxidative stress, osmotic stress, and inflammation, the expression of inducible Hsp is increased (Cotto and Morimoto, 1999; Fehrenbach and Niess, 1999; Jolly and Morimoto, 2000; Njemini et al., 2002, 2003b, 2005), and these proteins participate in protein refolding and protection, in dissolving aggregated proteins, and in targeting them for degradation (Mathew and Morimoto, 1998; Jolly and Morimoto, 2000). Hsp are classified into families based on their molecular weights. The human Hsp27 belongs to the family of small Hsp (Ciocca et al., 1993). Hsp27, like other Hsp, has been implicated as a molecular chaperone; it is also a regulator of the cytoskeleton by binding to the actin filament (Jakob et al., 1993; Wieske et al., 2001) and it is able to induce an increase in cellular glutathione levels, which works together with ascorbic acid and co-enzyme Q as a redox buffer for cellular protection (Preville et al., 1999). Hsp27 is composed of four distinct isoforms (A, B, C, and D), which have been reported to represent post-translational modifications of the same protein (Kim et al., 1984; Welch, 1985). Cumulative findings support a role for Hsp27 in inflammation (Hastie et al., 1997). This protein has been reported to
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protect cells from noxious stimuli such as cytokines and reactive oxygen species (ROS) (Mehlen et al., 1995a,b) and to prolong cellular survival in murine models of ischemiareperfusion injury (Yokoyama et al., 2001). It has been demonstrated that Hsp27 controls inflammation by regulating the expression of the pro- and anti-inflammatory genes (De AK et al., 2000; Park et al., 2003). Moreover, some authors have shown that the expression and phosphorylation of Hsp27 are directed by cytokines (Hatakeyama et al., 2002). Oxidative damage to cellular macromolecules, particularly to DNA and proteins, induced by ROS, progressively accumulates with aging and age-related diseases. Several reports have linked oxidative damage to the decline in tissue function, and there is strong evidence suggesting that oxidative stress and damage may be one of the causal factors in the aging process. It has been shown that cells isolated from aged tissues and cells undergoing replicative senescence in vitro, have a reduced HS response and a higher incidence of death when subjected to severe stress (Lee et al., 1996; Liu et al., 1996). Because Hsp27 is thought to protect cells from oxidative stress, alterations in the production of Hsp27 can further promote aging by enhancing the accumulation of damaged proteins. The aim of our study was to assess the effect of age on Hsp27 production in vivo in human peripheral blood mononuclear cells (PBMC) and to determine the influence of HS and inflammation, two different types of stressors. Results presented in this report provide evidence in support of an agerelated decrease in the level of Hsp27, which disappeared in the presence of inflammation. We also found that monocytes showed an important constitutive production of Hsp27 while lymphocytes produced much less Hsp27. 2. Participants, materials and methods 2.1. Participants Fifteen elderly (11 women and 4 men, aged between 73 and 95 years, mean age 82.7 years (SD 7.2)) and 16 young (10 women and 6 men, aged between 25 and 47 years, mean age 37.9 years (SD 6.7)) patients with inflammation due to acute infectious diseases, hospitalized at the Academic Hospital, Vrije Universiteit Brussel (VUB), were included in the study. Patients were hospitalized for acute pyelonephritis (2), angina (1), appendicitis (1), bronchopneumonia (11), cholangitis (1), cutaneous infection (3), diverticulitis of the colon (4), influenza (1), osteomylitis (1), sinusitis (1), or urinary tract infection (6); and were selected during the first three days of their admission. Patients were excluded if other causes of inflammation were present, if they were treated with glucocorticosteroids (other than inhalation steroids) or non-steroidal anti-inflammatory drugs (other than low dose acetylsalicylic acid, given for antiaggregating purposes) in the week before admission, or had severe hepatic (serum bilirubin O1.3 mg/dL) or renal insufficiency (serum creatinine O1.5 mg/dL). A control group consisted of 17 apparently healthy young subjects (12 women and 5 men, aged between 21 and 48 years, mean age 36.4 years (SD 8.1)) who were recruited from the blood
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transfusion centre of the Academic Hospital, VUB, and eighteen apparently healthy elderly subjects (6 women and 12 men, aged between 67 and 88 years, mean age 75.0 years (SD 6,3)) recruited on a voluntary basis from different senior organizations following informative lectures about the study. In agreement with recent guidelines for studies on elderly patients, the presence of other co-morbidity was not an exclusion criterion, thus resulting in a commonly seen casemix for hospitalized elderly patients (Ferrucci et al., 2004). On average, the number of relevant co-morbidity conditions was 1.1 (SD 1.6) for young patients, 3.9 (SD 1.4) for elderly patients and 1.1 (SD 1.3) for elderly controls; there was no pathology in the case of young controls. All participants gave an informed consent. 3. Methods 3.1. Blood sampling Venous blood was sampled in the morning after overnight fasting. After separation from blood cells, serum was aliquoted and stored at K70 8C. EDTA-anticoagulated blood was used for flow cytometric studies. (Beckman-Coulter Epics XL, USA). 3.1.1. Reagents and antibodies Lymphoprep was from Nycomed (Oslo, Norway). Bovine serum albumin (BSA) was from SIGMA (Bornem, Belgium). Fetal calf serum (FCS) was from Biochrom (International Medical, Belgium). Culture medium (RPMI 1640), phosphate buffered saline (PBS), trypsin-EDTA (0.05% trypsin, 0.02% EDTA), L-glutamine, N-2-hydroxyethylpiperazine -N’-2-ethanesulphonic acid (HEPES) buffer, penicillin and streptomycin were purchased from Life Technologies (Gibco, Paisley, Scotland). Anti-CD14 phycoerythrin (PE) conjugated was from Becton Dickinson (Erembodegen, Belgium). The monoclonal antibodies directed against Hsp27 and Hsp70 (clone G3.1 isotype IgG1, SPA-800F and clone C92F3A-5 isotype IgG1, SPA-810, respectively) were from StressGen (Vic., Canada); according to the manufacturer, these antibodies are specific for their respective proteins and do not exhibit reactivity with other proteins. Pure mouse IgG (Isotype matched control) was from Becton Dickinson. Antibodies for cytokine determination were obtained from Biosource International (Nijvel, Belgium). Reagents for the determination of CRP were from DADE BEHRING (Marburg, Germany). The products were applied according to the manufacturers’ guidelines. 3.1.2. Cell preparation PBMC were recovered as described previously (Njemini et al., 2002). Briefly, EDTA blood was diluted 2 times with PBS and carefully layered over lymphoprep in a 10 ml test tube. After centrifugation the cells were washed twice in PBS containing 1% BSA (PBS/BSA) at 900g for 3 min, and resuspended in RPMI 1640 supplemented with 10% FCS, 2 mM HEPES buffer, 2 mM L- glutamine, penicillin and
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streptomycin. To evaluate the Hsp27 concentration as it is present in cells in vivo, we determined in a rapid procedure the presence of Hsp27, without extended incubation. On the other hand, to investigate the effect of HS, cells were plated in 35 mm diameter petri dishes (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) and incubated in a 5% CO2 incubator at 37 8C. After 4 h the supernatants in the petri dishes were collected, centrifuged and the pellets re-suspended in supplemented RPMI. These suspensions were re-introduced in their respective petri dishes containing adherent cells. The petri dishes containing the cells were then surrounded with a parafilm band and placed in a thermostatically regulated water bath (VEL, Leuven, Belgium) at 42 8C for 1 h. Thereafter, cells were allowed to recover overnight in a 5% CO2 incubator at 37 8C. The following morning, the cells were detached from the petri dishes with 0.5 ml trypsin-EDTA and stained for their intracellular levels of Hsp27. It is known that cell preparation and culture conditions can influence the expression of certain proteins. However, in previous work done on Hsp70, another family of Hsp (Njemini et al., 2002), we found only small differences when the MFI of Hsp70 for monocytes as well as lymphocytes was determined immediately or after recovery during prolonged incubation at 37 8C. 3.1.3. Intracellular staining Hundred microlitres of the cell suspension was incubated with 10 ml of anti-CD14 PE-conjugated antibody for 15 min at 4 8C. Cells were washed at 900g for 3 min in 1 ml PBS/BSA and fixed at room temperature with 100 ml of PBS containing 4% paraformaldehyde for 15 min. After washing in PBS/BSA, the cells were permeabilized with 100 ml of PBS containing 0.1% saponin and 1% BSA (saponin/BSA) and at the same time incubated with 10 ml of the Hsp-specific antibody diluted (1:5 for Hsp27, and 1:20 for Hsp70) in PBS/BSA. In negative controls, an isotype matched mouse IgG was used as the primary antibody. Incubation was carried out for 15 min at room temperature and cells were washed in PBS/BSA. In the case of Hsp70 and the negative controls, labeled cells were resuspended again in 100 ml of saponin/BSA and 10 ml of the secondary antibody (FITC-conjugated F(ab’)2 fragment of goat anti-mouse IgG) diluted 1:10 in PBS/BSA, and further incubated for 15 min in the dark at room temperature. After washing, 500 ml of facsflow solution were added. The samples were analyzed by flow cytometry immediately or within a few hours (stored at 4 8C). 3.1.4. Flow cytometry analysis The labelled samples were analysed with a Coulter Epics XL-MCL flow cytometer (Coulter, Miami, FL, USA). Data acquisition was performed using the Coulter System II 3.0 software (Epics). The FITC and PE dyes were excited with a 15 MW argon laser of wavelength 488 nm. Analysis was done in listmode, for the green fluorescence through a 525 nm filter and for the red fluorescence through a 575 nm filter. Fig. 1 portrays two typical Facs-analyses as histograms. The monocyte and lymphocyte subpopulations were differentiated
Fig. 1. Detection of Hsp27 in monocytes and lymphocytes. Cells were incubated with Hsp 27 antibody conjugated with FITC. Background staining was determined by substituting the Hsp27 antibody with an isotype matched mouse IgG. Displayed are the corresponding histograms of the monocytic and lymphocytic cell populations of a sample. HS, heat shock; C, negative control.
according to granularity and size in the forward versus side scattergram, and were gated. For both gates, data were represented as mean fluorescence intensity (MFI, measured in log scale; Figs. 1 and 2), and percentage of positive cells (Fig. 3), which was corrected for background fluorescence with the negative controls. Dead cells were excluded by electronic gating. A total of 3000 cells were collected in the monocyte gate. A pure monocyte cluster was obtained from the monocyte gate by selecting only cells reacting with the monoclonal antibody CD14. 3.1.5. Cytokine assay Sera were assayed for interleukin (IL)-6 using a commercially available ELISA kit (Biosource international, Nijvel, Belgium), according to the instructions provided by the manufacturer. IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha were determined by an optimized ELISA method using Human Cytokine Cytosetse 10-plate format (Biosource international, Nijvel, Belgium). Briefly, plates were coated with 100 ml of coating antibody (1 mg/ml of clone 945A 5D11 for IL-10, 1.6 mg/ml of clone 350B 10G6 for IFN-gamma and 8 mg/ml of clones-mix 68B 6A3 and 68B 2B3 for TNF-alpha) diluted in PBS (pH 7.4). After overnight incubation at 4 8C, the coated plates were washed six times with a sodium chloride solution containing 0.1% tween 20 (wash buffer) and non-specific binding sites blocked by incubation with 300 ml of PBS containing 0.5% BSA (PBS/BSA) for 2 h on a shaker. After washing, 100 ml of the
R. Njemini et al. / Experimental Gerontology 41 (2006) 312–319
100
A (Lymphocytes)
90 %of positive lymphocytes
15.0 12.5 MFI of Hsp27
315
10.0 7.5 5.0
80 70 60 50 40 30 20 10
2.5 0.0
0
YC EC YP EP Without HS
YC EC YP EP After HS
B (Monocytes) 200 175
YC EC YP EP Without HS
YC EC YP EP After HS
Fig. 3. The effect of age, inflammation and temperature on the percentage of Hsp27 producing lymphocytes. YC, young control; EC, elderly control; YP, young patient; EP, elderly patient. Peripheral blood mononuclear cells were isolated and lymphocytes were identified as mentioned in the methods section. The percentage of Hsp27 producing cells was obtained by flow cytometry. Horizontal bars indicate mean values. The statistical analysis of the data in this figure is presented in Table 2.
MFI of Hsp27
150
nephelometry using commercial kits from DADE BEHRING (Marburg, Germany). CRP concentrations below 3.11 mg/L were undetectable.
125 100 75 50 25 0 YC EC YP EP Without HS
YC EC YP EP After HS
Fig. 2. The effect of age, inflammation and temperature on Hsp27 production by lymphocytes (A) and monocytes (B). YC, young control; EC, elderly control; YP, young patient; EP, elderly patient. Peripheral blood mononuclear cells were isolated and monocytes and lymphocytes were identified as mentioned in the methods section. The mean fluorescence intensity (MFI) of Hsp27 was obtained by flow cytometry. Horizontal bars indicate mean values. The statistical analysis of the data in this figure is presented in Table 2.
reference preparation or samples were added and the plate incubated for 2 h on a shaker. Plates were washed six times and 100 ml of the biotinylated detection antibody (clone 945A 5A10, 1/1000 for IL-10; clone 67F 12A8, 1/1000 for IFNgamma; and clone 68B 3C5, 1/300 for TNF-alpha) diluted in PBS/BSA containing 0.1% tween 20 (PBS/BSA/T) was added. After 2 h on shaker, plates were washed and incubated with 100 ml of streptavidin horse raddish peroxidase (1/2500 for IL10, and IFN-gamma; and 1/625 for TNF-alpha) in PBS/BSA/T for 2 h on shaker. Plates were then washed and 200 ml of o-phenylenediamine dihydrochloride substrate was added. After 30 min on shaker the absorbance was determined at 490 nm with background subtraction at 620 nm using a microplate reader (CERES 900C, BIO-TEC instruments, Inc, Belgium). Cytokine concentrations were detected by comparing sample absorbance with the absorbance of a reference purified recombinant cytokine. CRP was determined by
3.1.6. Statistical analysis Prism 3.0 statistical softwareR and Analyse-itR were used to analyze the results. Parameters were tested for distribution to normality. Averages were compared using Student’ t or ANOVA test. For data that were not normally distributed, the nonparametric Wilcoxon signed rank test and the Mann– Whitney-test were applied. Continuous variables were compared by Pearson or Spearman correlation. A p-value (2 sided) !0.05, was considered as statistically significant. 4. Results Analysis of freshly isolated human PBMC by flow cytometer revealed small but detectable Hsp27 above background level in lymphocytes and abundant Hsp27 in monocytes (Fig. 2, Table 1). A correlation was found between the concentrations of Hsp27, without any HS, in lymphocytes and monocytes (rZ0.49; p!0.001) when the entire population of patients and controls was considered. This relationship remained when the participants were divided into patients (rZ0.533; pZ0.002) and controls (rZ0.467; pZ0.005) as well as into young (rZ0.551; p!0.001) and elderly (rZ0.478; pZ0.005). The levels of Hsp27 in PBMC were independent of the gender of the participants. We found a differential HS response of Hsp27 in lymphocytes compared to monocytes in both controls and patients (Fig. 2). Whereas HS resulted in a small but significant increase in the levels of Hsp27 in lymphocytes in both controls (pZ0.001 and p!0.001 for young and elderly controls respectively) and patients (pZ0.001 and pZ0.003 for young and elderly patients, respectively), a significant decrease in
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Table 1 Heat shock protein 27 levels in monocytes and lymphocytes for young (YC) and elderly controls (EC), and for young (YP) and elderly patients (EP) No heat shock MFI (SD) Monocytes YC EC YP EP Lymphocytes YC EC YP EP
91.5 (34.6) 72.2 (31.2) 78.0 (29.9) 94.1 (21.9) 3.3 (1.1) 2.4 (0.6) 2.5 (0.7) 3.1 (1.9)
Y/E
HS/nHS
Heat shock at 42 8C MFI (SD) Y/E
ns
ns ns p!0.001 p!0.001
61.3 (18.8) 45.7 (14.2) 47.3 (21.7) 57.7 (22.2)
pZ0.003 p!0.001 p!0.001 p!0.001
5.1 3.8 5.2 6.1
%PC (SD) Y/E
HS/nHS
pZ0.017
p!0.001 p!0.001 pZ0.001 p!0.001
99.5 (0.5) 99.3 (0.7) 97.9 (2.9) 98.0 (2.1)
pZ0.001 p!0.001 pZ0.001 pZ0.003
78.4 (19.3) 55.9 (17.0) 33.6 (16.7) 40.8 (22.8)
ns
pZ0.008 ns
ns
p!0.001 ns
(1.1) (0.8) (2.6) (3.2)
%PC (SD)
99.7 (0.3) 99.6 (0.4) 99.7 (0.1) 99.7 (0.2)
Y/E
pZ0.006 ns
pZ0.001
91.6 (7.7) 81.0 (8.1) 84.0 (10.2) 84.2 (10.1)
ns
ns ns
p!0.001 ns
Data represent mean (SD) of the mean fluorescence intensity (MFI) or percentage of positive cells (%PC). Y/E: difference between YC and EC or between YP and EP; HS/nHS, difference between heat shocked (HS) and non-HS conditions. ns, not significant.
Hsp27 was noticed for monocytes in both controls (p!0.001 for both young and elderly controls) and patients (pZ0.001 and p! 0.001 for young and elderly patients, respectively). For the percentage of positive cells a significant increase was noticed for lymphocytes in both controls (pZ0.003 and p!0.001, for young and elderly controls respectively) and patients (p!0.001 for both young and elderly patients) (Fig. 3). The percentage of positive monocytes was very high for young and old participants, as well as for those without and with inflammation (all values O90%; data not shown). There was a positive correlation between the levels of Hsp27 in non-HS monocytes compared to the levels attained when these cells were exposed to a HS (rZ0.57; p!0.001). As a control we determined the MFI of Hsp70 and found that on average the values increased in monocytes from 15.3 without HS to 124.8 with HS; for lymphocytes these values were, respectively 2.6 and 6.8. An inverse relationship with the age of the control subjects was found, both for the percentage of Hsp27 producing lymphocytes, as well without as with HS (p!0.001 for both) and for the intensity of their production (pZ0.008 without HS and pZ0.001 after HS). An age-related decline was also noticed in the MFI of Hsp27 producing monocytes (pZ0.017 without HS and pZ0.006 after a HS) (Figs. 2 and 3). On the other hand, there was no significant difference in the HSinduced changes in the MFI of Hsp27 (values obtained after subtracting results without HS from those with HS) between young and elderly control subjects (data not shown). These differences were not found in the patients. To clarify the relationship between Hsp27 production and inflammation, patients presenting inflammation of acute infectious origin were included. The levels of Hsp27 in HSlymphocytes were higher in patients compared to control subjects (pZ0.022, Fig. 1). On the other hand, the percentages of Hsp27 producing monocytes (pZ0.001) and lymphocytes (p!0.001) were higher in controls compared to patients (Fig. 3) in the absence of HS. There were no other significant differences between patients and controls with respect to Hsp27 production. The relationship of inflammatory parameters with the production of Hsp27 was investigated, considering the entire
population of patients and controls (see Table 2). Negative correlations were found between the circulating levels of both CRP and IL-6 with the percentage of Hsp27 producing non-HS lymphocytes and monocytes. On the other hand, positive correlations were found between TNF-a and the percentage of Hsp27 producing lymphocytes as well as the intensity of their production after HS. A similar relationship was noticed for TNF-a and the MFI of non-HS monocytes. There were no relationships between the percentage of positive cells or the intensities of Hsp27 and the serum concentrations of IL-10 and IFN-g. Noteworthy, there was no significant difference in the serum levels of cytokines between the elderly and young control subjects. 5. Discussion The cellular response to stress ensures survival of the cell in the presence of otherwise lethal forms of stress such as HS and Table 2 Relationship between the inflammatory parameters and the various determinations of Hsp27 Inflammatory parameter Percentage of positive cells Non-HS lymphocytes HS lymphocytes Non-HS monocytes HS monocytes MFI Non-HS lymphocytes HS lymphocytes Non-HS monocytes HS monocytes
TNF-a
CRP
IL-6
ns
rZ-0.575 p!0.001 ns
rZ-0.496 p!0.001 ns
rZ-0.515 p!0.001 ns
rZ-0.359 pZ0.003 ns
ns ns
ns ns
ns
ns
ns
ns
rZ0.261 pZ0.034 ns ns ns rZ0.268 pZ0.029 rZ0.252 pZ0.042 ns
ns, not significant; HS, heat shocked.
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inflammation (Cotto and Morimoto, 1999). It has been demonstrated that with aging, there is a general decline in the capacity of cells to respond to stressors including HS (Njemini et al., 2002), hypoxia (Kim et al., 2003) and oxidative insult (van der Vlies et al., 2003). While some studies refer to the reduced activity and binding of HS transcription factor (HSF) to the heat shock element (HSE) as being the cause of the age-related decrease in Hsp production (Heydari et al., 1994; Locke and Tanguay, 1996), other studies point to the role of cytokines (Stephanou et al., 1997; Njemini et al., 2002, 2003a,b, 2004) which can influence the HSE through transcription factors (Stephanou et al., 1998, 1999a,b). Thus far, the overwhelming majority of studies have been carried out on Hsp70. Studies on Hsp27, which is another potential downregulator of ROS, have been largely limited. In the current study, we have examined the production of Hsp27 in circulating human PBMC. We have found that the basal level of Hsp27, measured as MFI or as percentage of Hsp27 producing cells, was inversely related to the age of the control subjects, both for lymphocytes and for monocytes. The effect of age in the production of Hsp is very controversial. Some investigators have reported an increase in the basal levels of Hsp with age (Maiello et al., 1998), which is indicative of an adaptive response to cumulative intracellular stress during aging and may be associated with increased oxidative stress (Fonager et al., 2002; McArdle et al., 2002). On the other hand, a decrease (Bonelli et al., 1999; Verbeke et al., 2001), or no effect (Wu et al., 1993) of age on Hsp basal levels have also been reported. Differences in experimental conditions and the health status of the subjects as well as the type of sample employed may explain these divergent results. A decrease in Hsp27 levels with aging after HS was found in this study, possibly reflecting the lower levels that already existed in the elderly subjects. It has been reported that different basal levels of expression contribute to a high individual variability of Hsp inducibility in leukocytes (Boshoff et al., 2000). In addition, there was no significant difference in the HS-induced changes in Hsp27 levels with respect to the age of the control subjects. As we described previously for Hsp70 (Njemini et al., 2002, 2003b) and Hsp32 (Njemini et al., 2005), markedly higher concentrations of Hsp27 were observed in monocytes than in lymphocytes. Others (Beresford et al., 1998; Fehrenbach et al., 2000) also have demonstrated that the expression of Hsp27 as well as Hsp70 is high in monocytes compared to other leukocyte subsets. Because Hsp27 is up-regulated following oxidative stress (Arata et al., 1995), a likely explanation for this phenomenon is the higher capacity of monocytes to induce ROS (Orie et al., 1999) and thus to promote oxidative stress (Devadas et al., 2002). Studies by Beresford et al. (1998) as well as our present communication support the view that HS up-regulates the production of Hsp27 in lymphocytes. We found a roughly twofold increase, 16 h after a HS of 42 8C during 1 h; Beresford et al. (1998) reported a higher induction (13-fold). In the present study, HS did not induce Hsp27 production in monocytes as it has been shown to do by these authors, but,
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on the contrary, resulted in a significant decrease. Beresford et al. (1998), however, did not provide details of their cell culture conditions. We co-cultured all PBMC, allowing the much more abundant lymphocytes to influence monocytes, most likely through secretion of inflammatory cytokines (Stephanou et al., 1998). In separate experiments (unpublished data) we have, indeed, found that without lymphocyte coculture, an increase of Hsp27 production after HS is seen in monocytes. As a control for our HS technique, we evaluated simultaneously the Hsp70 production after HS; as in previous experiments (Njemini et al., 2003b), we found a clear increase after HS in monocytes (8.1-fold), and in lymphocytes (2.6fold). Some investigators have reported an up-regulation of Hsp27 production with inflammation (Hastie et al., 1997), leading to the suggestion that Hsp27 may have some anti-inflammatory or immune modulatory capacities on leukocytes (Hashiguchi et al., 2001). To investigate the effect of inflammation on Hsp27 production, we included patients suffering from acute infection. Contrary to the controls, in this patient population no age related decline of the Hsp27 levels was found, neither for lymphocytes or monocytes nor with or without HS. The tendency of an increase, which could be noticed, appeared not to be significant. These findings implicate that inflammation results in the neutralisation of the age induced Hsp27 repression. Acute phase factors, which mediate the regulation of Hsp genes by interacting with several signalling pathways (Stephanou et al., 1998), are most likely involved in this process. TNF-a might be one such factor, since we found a positive, albeit weak (r2Z0.06–0.07) correlation with the Hsp27 production in monocytes without HS and in lymphocytes after HS. Both CRP and IL-6 showed a larger (r2Z0.13– 0.33), negative correlation with the percentages of non-HS lymphocytes and monocytes producing Hsp27; no such relationship was found with the levels of Hsp27. This observation is compatible with the known pro-inflammatory tendency that is observed during aging, and might explain the lower values for Hsp27 we found in the elderly compared to the younger control subjects (Ferrucci et al., 1999; Kuller, 1999). Both increased circulating levels of CRP and IL-6 have been described in elderly normal subjects. In our study, however, we could not demonstrate such differences between the young and elderly control participants. Possibly, these might have shown up when using high sensitive determinations for these parameters. No relationships between IL-10 or IFN-g and the levels of Hsp27 were found. Although from our results it appears likely that cytokines can influence the production of Hsp27, no clear pattern could be identified, entailing that other inflammatory parameters than those tested might be implicated. In conclusion, we provide evidence in support of an agerelated decrease in the basal levels of Hsp27. This difference disappeared when inflammation was present. The level of Hsp27 was higher in monocytes compared to lymphocytes in both HS and non-HS cells. In addition, we found that HS resulted in a small but significant increase in the levels of Hsp27 in lymphocytes whereas a significant decrease in
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The effect of aging and inflammation on heat shock protein 27 in human monocytes and lymphocytes R. Njemini a,b, M. Lambert a, C. Demanet c, T. Mets a,b,* a Geriatric Unit, Academic Hospital, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium Gerontology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium c HLA and Immunology laboratory, Academic Hospital, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium b
Received 5 August 2005; received in revised form 19 January 2006; accepted 20 January 2006
Abstract Heat shock proteins (Hsp) are highly conserved proteins and their synthesis is ubiquitous in virtually every species in which they have been sought. In the present study we have investigated the effect of age and inflammation on the induction of Hsp27 in human peripheral blood mononuclear cells, using flow cytometry. Sixty-six healthy control subjects or patients suffering from inflammation participated in the study. In both heat shocked (HS) and non-HS conditions, the percentage of Hsp27 producing lymphocytes as well as the intensity of Hsp27 in lymphocytes and monocytes were negatively influenced by age. The basal levels and also the levels of Hsp27 production after HS were higher for monocytes compared to lymphocytes. In addition, we found that HS resulted in a small but significant increase in the levels of Hsp27 in lymphocytes whereas a significant decrease in Hsp27 was noticed for monocytes. In conclusion, results presented herein provide evidence in support of an age-related decrease in the level of Hsp27, which disappeared in the presence of inflammation. Several relationships between the circulating levels of CRP, IL-6 and TNF-a with the various Hsp27 determinations were observed, indicating that cytokines are able to influence the production of Hsp27. q 2006 Elsevier Inc. All rights reserved. Keywords: Elderly; Cytokines; Mononuclear blood cells; Heat shock; Heat shock protein 27
1. Introduction Heat shock proteins (Hsp) are present in cells of all organisms and the genes encoding them are highly conserved (Rattan et al., 2004). Distinction has been made between Hsp that are almost absent under non-stressed conditions but synthesised after cellular stress, and Hsp that are constitutively synthesised in cells. The former are called inducible proteins, while the latter are known as cognate proteins. Constitutive and stress-inducible Hsp play diverse roles in cellular function. Under normal physiological conditions constitutively synthesised Hsp act as molecular chaperones modulating protein folding, assembly, intracellular localisation, secretion, and
Abbreviations: HS, Heat shock(ed); Hsp, Heat shock protein(s); MFI, Mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species. * Corresponding author. Address: Geriatric Unit, Academic Hospital, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium. Tel.: C32 2 477 63 66; fax: C32 2 477 63 64. E-mail address: [email protected] (T. Mets). 0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.01.006
degradation (Ellis and van der Vies, 1991; Hartl, 1996). When cells endure stress such as high temperature, exercise, oxidative stress, osmotic stress, and inflammation, the expression of inducible Hsp is increased (Cotto and Morimoto, 1999; Fehrenbach and Niess, 1999; Jolly and Morimoto, 2000; Njemini et al., 2002, 2003b, 2005), and these proteins participate in protein refolding and protection, in dissolving aggregated proteins, and in targeting them for degradation (Mathew and Morimoto, 1998; Jolly and Morimoto, 2000). Hsp are classified into families based on their molecular weights. The human Hsp27 belongs to the family of small Hsp (Ciocca et al., 1993). Hsp27, like other Hsp, has been implicated as a molecular chaperone; it is also a regulator of the cytoskeleton by binding to the actin filament (Jakob et al., 1993; Wieske et al., 2001) and it is able to induce an increase in cellular glutathione levels, which works together with ascorbic acid and co-enzyme Q as a redox buffer for cellular protection (Preville et al., 1999). Hsp27 is composed of four distinct isoforms (A, B, C, and D), which have been reported to represent post-translational modifications of the same protein (Kim et al., 1984; Welch, 1985). Cumulative findings support a role for Hsp27 in inflammation (Hastie et al., 1997). This protein has been reported to
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protect cells from noxious stimuli such as cytokines and reactive oxygen species (ROS) (Mehlen et al., 1995a,b) and to prolong cellular survival in murine models of ischemiareperfusion injury (Yokoyama et al., 2001). It has been demonstrated that Hsp27 controls inflammation by regulating the expression of the pro- and anti-inflammatory genes (De AK et al., 2000; Park et al., 2003). Moreover, some authors have shown that the expression and phosphorylation of Hsp27 are directed by cytokines (Hatakeyama et al., 2002). Oxidative damage to cellular macromolecules, particularly to DNA and proteins, induced by ROS, progressively accumulates with aging and age-related diseases. Several reports have linked oxidative damage to the decline in tissue function, and there is strong evidence suggesting that oxidative stress and damage may be one of the causal factors in the aging process. It has been shown that cells isolated from aged tissues and cells undergoing replicative senescence in vitro, have a reduced HS response and a higher incidence of death when subjected to severe stress (Lee et al., 1996; Liu et al., 1996). Because Hsp27 is thought to protect cells from oxidative stress, alterations in the production of Hsp27 can further promote aging by enhancing the accumulation of damaged proteins. The aim of our study was to assess the effect of age on Hsp27 production in vivo in human peripheral blood mononuclear cells (PBMC) and to determine the influence of HS and inflammation, two different types of stressors. Results presented in this report provide evidence in support of an agerelated decrease in the level of Hsp27, which disappeared in the presence of inflammation. We also found that monocytes showed an important constitutive production of Hsp27 while lymphocytes produced much less Hsp27. 2. Participants, materials and methods 2.1. Participants Fifteen elderly (11 women and 4 men, aged between 73 and 95 years, mean age 82.7 years (SD 7.2)) and 16 young (10 women and 6 men, aged between 25 and 47 years, mean age 37.9 years (SD 6.7)) patients with inflammation due to acute infectious diseases, hospitalized at the Academic Hospital, Vrije Universiteit Brussel (VUB), were included in the study. Patients were hospitalized for acute pyelonephritis (2), angina (1), appendicitis (1), bronchopneumonia (11), cholangitis (1), cutaneous infection (3), diverticulitis of the colon (4), influenza (1), osteomylitis (1), sinusitis (1), or urinary tract infection (6); and were selected during the first three days of their admission. Patients were excluded if other causes of inflammation were present, if they were treated with glucocorticosteroids (other than inhalation steroids) or non-steroidal anti-inflammatory drugs (other than low dose acetylsalicylic acid, given for antiaggregating purposes) in the week before admission, or had severe hepatic (serum bilirubin O1.3 mg/dL) or renal insufficiency (serum creatinine O1.5 mg/dL). A control group consisted of 17 apparently healthy young subjects (12 women and 5 men, aged between 21 and 48 years, mean age 36.4 years (SD 8.1)) who were recruited from the blood
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transfusion centre of the Academic Hospital, VUB, and eighteen apparently healthy elderly subjects (6 women and 12 men, aged between 67 and 88 years, mean age 75.0 years (SD 6,3)) recruited on a voluntary basis from different senior organizations following informative lectures about the study. In agreement with recent guidelines for studies on elderly patients, the presence of other co-morbidity was not an exclusion criterion, thus resulting in a commonly seen casemix for hospitalized elderly patients (Ferrucci et al., 2004). On average, the number of relevant co-morbidity conditions was 1.1 (SD 1.6) for young patients, 3.9 (SD 1.4) for elderly patients and 1.1 (SD 1.3) for elderly controls; there was no pathology in the case of young controls. All participants gave an informed consent. 3. Methods 3.1. Blood sampling Venous blood was sampled in the morning after overnight fasting. After separation from blood cells, serum was aliquoted and stored at K70 8C. EDTA-anticoagulated blood was used for flow cytometric studies. (Beckman-Coulter Epics XL, USA). 3.1.1. Reagents and antibodies Lymphoprep was from Nycomed (Oslo, Norway). Bovine serum albumin (BSA) was from SIGMA (Bornem, Belgium). Fetal calf serum (FCS) was from Biochrom (International Medical, Belgium). Culture medium (RPMI 1640), phosphate buffered saline (PBS), trypsin-EDTA (0.05% trypsin, 0.02% EDTA), L-glutamine, N-2-hydroxyethylpiperazine -N’-2-ethanesulphonic acid (HEPES) buffer, penicillin and streptomycin were purchased from Life Technologies (Gibco, Paisley, Scotland). Anti-CD14 phycoerythrin (PE) conjugated was from Becton Dickinson (Erembodegen, Belgium). The monoclonal antibodies directed against Hsp27 and Hsp70 (clone G3.1 isotype IgG1, SPA-800F and clone C92F3A-5 isotype IgG1, SPA-810, respectively) were from StressGen (Vic., Canada); according to the manufacturer, these antibodies are specific for their respective proteins and do not exhibit reactivity with other proteins. Pure mouse IgG (Isotype matched control) was from Becton Dickinson. Antibodies for cytokine determination were obtained from Biosource International (Nijvel, Belgium). Reagents for the determination of CRP were from DADE BEHRING (Marburg, Germany). The products were applied according to the manufacturers’ guidelines. 3.1.2. Cell preparation PBMC were recovered as described previously (Njemini et al., 2002). Briefly, EDTA blood was diluted 2 times with PBS and carefully layered over lymphoprep in a 10 ml test tube. After centrifugation the cells were washed twice in PBS containing 1% BSA (PBS/BSA) at 900g for 3 min, and resuspended in RPMI 1640 supplemented with 10% FCS, 2 mM HEPES buffer, 2 mM L- glutamine, penicillin and
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streptomycin. To evaluate the Hsp27 concentration as it is present in cells in vivo, we determined in a rapid procedure the presence of Hsp27, without extended incubation. On the other hand, to investigate the effect of HS, cells were plated in 35 mm diameter petri dishes (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) and incubated in a 5% CO2 incubator at 37 8C. After 4 h the supernatants in the petri dishes were collected, centrifuged and the pellets re-suspended in supplemented RPMI. These suspensions were re-introduced in their respective petri dishes containing adherent cells. The petri dishes containing the cells were then surrounded with a parafilm band and placed in a thermostatically regulated water bath (VEL, Leuven, Belgium) at 42 8C for 1 h. Thereafter, cells were allowed to recover overnight in a 5% CO2 incubator at 37 8C. The following morning, the cells were detached from the petri dishes with 0.5 ml trypsin-EDTA and stained for their intracellular levels of Hsp27. It is known that cell preparation and culture conditions can influence the expression of certain proteins. However, in previous work done on Hsp70, another family of Hsp (Njemini et al., 2002), we found only small differences when the MFI of Hsp70 for monocytes as well as lymphocytes was determined immediately or after recovery during prolonged incubation at 37 8C. 3.1.3. Intracellular staining Hundred microlitres of the cell suspension was incubated with 10 ml of anti-CD14 PE-conjugated antibody for 15 min at 4 8C. Cells were washed at 900g for 3 min in 1 ml PBS/BSA and fixed at room temperature with 100 ml of PBS containing 4% paraformaldehyde for 15 min. After washing in PBS/BSA, the cells were permeabilized with 100 ml of PBS containing 0.1% saponin and 1% BSA (saponin/BSA) and at the same time incubated with 10 ml of the Hsp-specific antibody diluted (1:5 for Hsp27, and 1:20 for Hsp70) in PBS/BSA. In negative controls, an isotype matched mouse IgG was used as the primary antibody. Incubation was carried out for 15 min at room temperature and cells were washed in PBS/BSA. In the case of Hsp70 and the negative controls, labeled cells were resuspended again in 100 ml of saponin/BSA and 10 ml of the secondary antibody (FITC-conjugated F(ab’)2 fragment of goat anti-mouse IgG) diluted 1:10 in PBS/BSA, and further incubated for 15 min in the dark at room temperature. After washing, 500 ml of facsflow solution were added. The samples were analyzed by flow cytometry immediately or within a few hours (stored at 4 8C). 3.1.4. Flow cytometry analysis The labelled samples were analysed with a Coulter Epics XL-MCL flow cytometer (Coulter, Miami, FL, USA). Data acquisition was performed using the Coulter System II 3.0 software (Epics). The FITC and PE dyes were excited with a 15 MW argon laser of wavelength 488 nm. Analysis was done in listmode, for the green fluorescence through a 525 nm filter and for the red fluorescence through a 575 nm filter. Fig. 1 portrays two typical Facs-analyses as histograms. The monocyte and lymphocyte subpopulations were differentiated
Fig. 1. Detection of Hsp27 in monocytes and lymphocytes. Cells were incubated with Hsp 27 antibody conjugated with FITC. Background staining was determined by substituting the Hsp27 antibody with an isotype matched mouse IgG. Displayed are the corresponding histograms of the monocytic and lymphocytic cell populations of a sample. HS, heat shock; C, negative control.
according to granularity and size in the forward versus side scattergram, and were gated. For both gates, data were represented as mean fluorescence intensity (MFI, measured in log scale; Figs. 1 and 2), and percentage of positive cells (Fig. 3), which was corrected for background fluorescence with the negative controls. Dead cells were excluded by electronic gating. A total of 3000 cells were collected in the monocyte gate. A pure monocyte cluster was obtained from the monocyte gate by selecting only cells reacting with the monoclonal antibody CD14. 3.1.5. Cytokine assay Sera were assayed for interleukin (IL)-6 using a commercially available ELISA kit (Biosource international, Nijvel, Belgium), according to the instructions provided by the manufacturer. IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha were determined by an optimized ELISA method using Human Cytokine Cytosetse 10-plate format (Biosource international, Nijvel, Belgium). Briefly, plates were coated with 100 ml of coating antibody (1 mg/ml of clone 945A 5D11 for IL-10, 1.6 mg/ml of clone 350B 10G6 for IFN-gamma and 8 mg/ml of clones-mix 68B 6A3 and 68B 2B3 for TNF-alpha) diluted in PBS (pH 7.4). After overnight incubation at 4 8C, the coated plates were washed six times with a sodium chloride solution containing 0.1% tween 20 (wash buffer) and non-specific binding sites blocked by incubation with 300 ml of PBS containing 0.5% BSA (PBS/BSA) for 2 h on a shaker. After washing, 100 ml of the
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100
A (Lymphocytes)
90 %of positive lymphocytes
15.0 12.5 MFI of Hsp27
315
10.0 7.5 5.0
80 70 60 50 40 30 20 10
2.5 0.0
0
YC EC YP EP Without HS
YC EC YP EP After HS
B (Monocytes) 200 175
YC EC YP EP Without HS
YC EC YP EP After HS
Fig. 3. The effect of age, inflammation and temperature on the percentage of Hsp27 producing lymphocytes. YC, young control; EC, elderly control; YP, young patient; EP, elderly patient. Peripheral blood mononuclear cells were isolated and lymphocytes were identified as mentioned in the methods section. The percentage of Hsp27 producing cells was obtained by flow cytometry. Horizontal bars indicate mean values. The statistical analysis of the data in this figure is presented in Table 2.
MFI of Hsp27
150
nephelometry using commercial kits from DADE BEHRING (Marburg, Germany). CRP concentrations below 3.11 mg/L were undetectable.
125 100 75 50 25 0 YC EC YP EP Without HS
YC EC YP EP After HS
Fig. 2. The effect of age, inflammation and temperature on Hsp27 production by lymphocytes (A) and monocytes (B). YC, young control; EC, elderly control; YP, young patient; EP, elderly patient. Peripheral blood mononuclear cells were isolated and monocytes and lymphocytes were identified as mentioned in the methods section. The mean fluorescence intensity (MFI) of Hsp27 was obtained by flow cytometry. Horizontal bars indicate mean values. The statistical analysis of the data in this figure is presented in Table 2.
reference preparation or samples were added and the plate incubated for 2 h on a shaker. Plates were washed six times and 100 ml of the biotinylated detection antibody (clone 945A 5A10, 1/1000 for IL-10; clone 67F 12A8, 1/1000 for IFNgamma; and clone 68B 3C5, 1/300 for TNF-alpha) diluted in PBS/BSA containing 0.1% tween 20 (PBS/BSA/T) was added. After 2 h on shaker, plates were washed and incubated with 100 ml of streptavidin horse raddish peroxidase (1/2500 for IL10, and IFN-gamma; and 1/625 for TNF-alpha) in PBS/BSA/T for 2 h on shaker. Plates were then washed and 200 ml of o-phenylenediamine dihydrochloride substrate was added. After 30 min on shaker the absorbance was determined at 490 nm with background subtraction at 620 nm using a microplate reader (CERES 900C, BIO-TEC instruments, Inc, Belgium). Cytokine concentrations were detected by comparing sample absorbance with the absorbance of a reference purified recombinant cytokine. CRP was determined by
3.1.6. Statistical analysis Prism 3.0 statistical softwareR and Analyse-itR were used to analyze the results. Parameters were tested for distribution to normality. Averages were compared using Student’ t or ANOVA test. For data that were not normally distributed, the nonparametric Wilcoxon signed rank test and the Mann– Whitney-test were applied. Continuous variables were compared by Pearson or Spearman correlation. A p-value (2 sided) !0.05, was considered as statistically significant. 4. Results Analysis of freshly isolated human PBMC by flow cytometer revealed small but detectable Hsp27 above background level in lymphocytes and abundant Hsp27 in monocytes (Fig. 2, Table 1). A correlation was found between the concentrations of Hsp27, without any HS, in lymphocytes and monocytes (rZ0.49; p!0.001) when the entire population of patients and controls was considered. This relationship remained when the participants were divided into patients (rZ0.533; pZ0.002) and controls (rZ0.467; pZ0.005) as well as into young (rZ0.551; p!0.001) and elderly (rZ0.478; pZ0.005). The levels of Hsp27 in PBMC were independent of the gender of the participants. We found a differential HS response of Hsp27 in lymphocytes compared to monocytes in both controls and patients (Fig. 2). Whereas HS resulted in a small but significant increase in the levels of Hsp27 in lymphocytes in both controls (pZ0.001 and p!0.001 for young and elderly controls respectively) and patients (pZ0.001 and pZ0.003 for young and elderly patients, respectively), a significant decrease in
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Table 1 Heat shock protein 27 levels in monocytes and lymphocytes for young (YC) and elderly controls (EC), and for young (YP) and elderly patients (EP) No heat shock MFI (SD) Monocytes YC EC YP EP Lymphocytes YC EC YP EP
91.5 (34.6) 72.2 (31.2) 78.0 (29.9) 94.1 (21.9) 3.3 (1.1) 2.4 (0.6) 2.5 (0.7) 3.1 (1.9)
Y/E
HS/nHS
Heat shock at 42 8C MFI (SD) Y/E
ns
ns ns p!0.001 p!0.001
61.3 (18.8) 45.7 (14.2) 47.3 (21.7) 57.7 (22.2)
pZ0.003 p!0.001 p!0.001 p!0.001
5.1 3.8 5.2 6.1
%PC (SD) Y/E
HS/nHS
pZ0.017
p!0.001 p!0.001 pZ0.001 p!0.001
99.5 (0.5) 99.3 (0.7) 97.9 (2.9) 98.0 (2.1)
pZ0.001 p!0.001 pZ0.001 pZ0.003
78.4 (19.3) 55.9 (17.0) 33.6 (16.7) 40.8 (22.8)
ns
pZ0.008 ns
ns
p!0.001 ns
(1.1) (0.8) (2.6) (3.2)
%PC (SD)
99.7 (0.3) 99.6 (0.4) 99.7 (0.1) 99.7 (0.2)
Y/E
pZ0.006 ns
pZ0.001
91.6 (7.7) 81.0 (8.1) 84.0 (10.2) 84.2 (10.1)
ns
ns ns
p!0.001 ns
Data represent mean (SD) of the mean fluorescence intensity (MFI) or percentage of positive cells (%PC). Y/E: difference between YC and EC or between YP and EP; HS/nHS, difference between heat shocked (HS) and non-HS conditions. ns, not significant.
Hsp27 was noticed for monocytes in both controls (p!0.001 for both young and elderly controls) and patients (pZ0.001 and p! 0.001 for young and elderly patients, respectively). For the percentage of positive cells a significant increase was noticed for lymphocytes in both controls (pZ0.003 and p!0.001, for young and elderly controls respectively) and patients (p!0.001 for both young and elderly patients) (Fig. 3). The percentage of positive monocytes was very high for young and old participants, as well as for those without and with inflammation (all values O90%; data not shown). There was a positive correlation between the levels of Hsp27 in non-HS monocytes compared to the levels attained when these cells were exposed to a HS (rZ0.57; p!0.001). As a control we determined the MFI of Hsp70 and found that on average the values increased in monocytes from 15.3 without HS to 124.8 with HS; for lymphocytes these values were, respectively 2.6 and 6.8. An inverse relationship with the age of the control subjects was found, both for the percentage of Hsp27 producing lymphocytes, as well without as with HS (p!0.001 for both) and for the intensity of their production (pZ0.008 without HS and pZ0.001 after HS). An age-related decline was also noticed in the MFI of Hsp27 producing monocytes (pZ0.017 without HS and pZ0.006 after a HS) (Figs. 2 and 3). On the other hand, there was no significant difference in the HSinduced changes in the MFI of Hsp27 (values obtained after subtracting results without HS from those with HS) between young and elderly control subjects (data not shown). These differences were not found in the patients. To clarify the relationship between Hsp27 production and inflammation, patients presenting inflammation of acute infectious origin were included. The levels of Hsp27 in HSlymphocytes were higher in patients compared to control subjects (pZ0.022, Fig. 1). On the other hand, the percentages of Hsp27 producing monocytes (pZ0.001) and lymphocytes (p!0.001) were higher in controls compared to patients (Fig. 3) in the absence of HS. There were no other significant differences between patients and controls with respect to Hsp27 production. The relationship of inflammatory parameters with the production of Hsp27 was investigated, considering the entire
population of patients and controls (see Table 2). Negative correlations were found between the circulating levels of both CRP and IL-6 with the percentage of Hsp27 producing non-HS lymphocytes and monocytes. On the other hand, positive correlations were found between TNF-a and the percentage of Hsp27 producing lymphocytes as well as the intensity of their production after HS. A similar relationship was noticed for TNF-a and the MFI of non-HS monocytes. There were no relationships between the percentage of positive cells or the intensities of Hsp27 and the serum concentrations of IL-10 and IFN-g. Noteworthy, there was no significant difference in the serum levels of cytokines between the elderly and young control subjects. 5. Discussion The cellular response to stress ensures survival of the cell in the presence of otherwise lethal forms of stress such as HS and Table 2 Relationship between the inflammatory parameters and the various determinations of Hsp27 Inflammatory parameter Percentage of positive cells Non-HS lymphocytes HS lymphocytes Non-HS monocytes HS monocytes MFI Non-HS lymphocytes HS lymphocytes Non-HS monocytes HS monocytes
TNF-a
CRP
IL-6
ns
rZ-0.575 p!0.001 ns
rZ-0.496 p!0.001 ns
rZ-0.515 p!0.001 ns
rZ-0.359 pZ0.003 ns
ns ns
ns ns
ns
ns
ns
ns
rZ0.261 pZ0.034 ns ns ns rZ0.268 pZ0.029 rZ0.252 pZ0.042 ns
ns, not significant; HS, heat shocked.
R. Njemini et al. / Experimental Gerontology 41 (2006) 312–319
inflammation (Cotto and Morimoto, 1999). It has been demonstrated that with aging, there is a general decline in the capacity of cells to respond to stressors including HS (Njemini et al., 2002), hypoxia (Kim et al., 2003) and oxidative insult (van der Vlies et al., 2003). While some studies refer to the reduced activity and binding of HS transcription factor (HSF) to the heat shock element (HSE) as being the cause of the age-related decrease in Hsp production (Heydari et al., 1994; Locke and Tanguay, 1996), other studies point to the role of cytokines (Stephanou et al., 1997; Njemini et al., 2002, 2003a,b, 2004) which can influence the HSE through transcription factors (Stephanou et al., 1998, 1999a,b). Thus far, the overwhelming majority of studies have been carried out on Hsp70. Studies on Hsp27, which is another potential downregulator of ROS, have been largely limited. In the current study, we have examined the production of Hsp27 in circulating human PBMC. We have found that the basal level of Hsp27, measured as MFI or as percentage of Hsp27 producing cells, was inversely related to the age of the control subjects, both for lymphocytes and for monocytes. The effect of age in the production of Hsp is very controversial. Some investigators have reported an increase in the basal levels of Hsp with age (Maiello et al., 1998), which is indicative of an adaptive response to cumulative intracellular stress during aging and may be associated with increased oxidative stress (Fonager et al., 2002; McArdle et al., 2002). On the other hand, a decrease (Bonelli et al., 1999; Verbeke et al., 2001), or no effect (Wu et al., 1993) of age on Hsp basal levels have also been reported. Differences in experimental conditions and the health status of the subjects as well as the type of sample employed may explain these divergent results. A decrease in Hsp27 levels with aging after HS was found in this study, possibly reflecting the lower levels that already existed in the elderly subjects. It has been reported that different basal levels of expression contribute to a high individual variability of Hsp inducibility in leukocytes (Boshoff et al., 2000). In addition, there was no significant difference in the HS-induced changes in Hsp27 levels with respect to the age of the control subjects. As we described previously for Hsp70 (Njemini et al., 2002, 2003b) and Hsp32 (Njemini et al., 2005), markedly higher concentrations of Hsp27 were observed in monocytes than in lymphocytes. Others (Beresford et al., 1998; Fehrenbach et al., 2000) also have demonstrated that the expression of Hsp27 as well as Hsp70 is high in monocytes compared to other leukocyte subsets. Because Hsp27 is up-regulated following oxidative stress (Arata et al., 1995), a likely explanation for this phenomenon is the higher capacity of monocytes to induce ROS (Orie et al., 1999) and thus to promote oxidative stress (Devadas et al., 2002). Studies by Beresford et al. (1998) as well as our present communication support the view that HS up-regulates the production of Hsp27 in lymphocytes. We found a roughly twofold increase, 16 h after a HS of 42 8C during 1 h; Beresford et al. (1998) reported a higher induction (13-fold). In the present study, HS did not induce Hsp27 production in monocytes as it has been shown to do by these authors, but,
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on the contrary, resulted in a significant decrease. Beresford et al. (1998), however, did not provide details of their cell culture conditions. We co-cultured all PBMC, allowing the much more abundant lymphocytes to influence monocytes, most likely through secretion of inflammatory cytokines (Stephanou et al., 1998). In separate experiments (unpublished data) we have, indeed, found that without lymphocyte coculture, an increase of Hsp27 production after HS is seen in monocytes. As a control for our HS technique, we evaluated simultaneously the Hsp70 production after HS; as in previous experiments (Njemini et al., 2003b), we found a clear increase after HS in monocytes (8.1-fold), and in lymphocytes (2.6fold). Some investigators have reported an up-regulation of Hsp27 production with inflammation (Hastie et al., 1997), leading to the suggestion that Hsp27 may have some anti-inflammatory or immune modulatory capacities on leukocytes (Hashiguchi et al., 2001). To investigate the effect of inflammation on Hsp27 production, we included patients suffering from acute infection. Contrary to the controls, in this patient population no age related decline of the Hsp27 levels was found, neither for lymphocytes or monocytes nor with or without HS. The tendency of an increase, which could be noticed, appeared not to be significant. These findings implicate that inflammation results in the neutralisation of the age induced Hsp27 repression. Acute phase factors, which mediate the regulation of Hsp genes by interacting with several signalling pathways (Stephanou et al., 1998), are most likely involved in this process. TNF-a might be one such factor, since we found a positive, albeit weak (r2Z0.06–0.07) correlation with the Hsp27 production in monocytes without HS and in lymphocytes after HS. Both CRP and IL-6 showed a larger (r2Z0.13– 0.33), negative correlation with the percentages of non-HS lymphocytes and monocytes producing Hsp27; no such relationship was found with the levels of Hsp27. This observation is compatible with the known pro-inflammatory tendency that is observed during aging, and might explain the lower values for Hsp27 we found in the elderly compared to the younger control subjects (Ferrucci et al., 1999; Kuller, 1999). Both increased circulating levels of CRP and IL-6 have been described in elderly normal subjects. In our study, however, we could not demonstrate such differences between the young and elderly control participants. Possibly, these might have shown up when using high sensitive determinations for these parameters. No relationships between IL-10 or IFN-g and the levels of Hsp27 were found. Although from our results it appears likely that cytokines can influence the production of Hsp27, no clear pattern could be identified, entailing that other inflammatory parameters than those tested might be implicated. In conclusion, we provide evidence in support of an agerelated decrease in the basal levels of Hsp27. This difference disappeared when inflammation was present. The level of Hsp27 was higher in monocytes compared to lymphocytes in both HS and non-HS cells. In addition, we found that HS resulted in a small but significant increase in the levels of Hsp27 in lymphocytes whereas a significant decrease in
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