Celiac disease (CD) is an immunologically mediated enteropathy of the small intestine, characterized by lifelong intolerance to gliadin and related prolamines (1). The essential function of the intestinal epithelium is the maintenance of a selective barrier through which nutrients and electrolytes can permeate, whereas potentially harmful agents are excluded (2). In CD the structure of this barrier is damaged, which leads to the leakage of cereal proteins across the intestinal epithelium. Wheat gliadin then activates both the innate and the adaptive immune system (3–5) and increases the apoptosis (6) of enterocytes.
Heat shock protein (HSP) 72 (7–8) was first described as a molecular chaperone induced by increased temperature. Later it was demonstrated that several other physiological or pathological stress factors can induce the synthesis of HSP72. HSP72 may alter diverse biological functions such as the inflammatory and the apoptotic processes.
Increased intracellular HSP72 is expressed on the cell surface and is subsequently released into the circulation (9). Circulating HSP72 binds to antigen-presenting cells (APCs) and stimulates proinflammatory cytokine synthesis (10), chemokine (11), and reactive oxygen species release (12). Recent findings demonstrate that HSP72s are ligands for Toll-like receptors (TLRs), which play a crucial role in the defense mechanism of the innate immune system (13). HSP72 may exert immunoregulatory effects by binding especially to TLR2 and TLR4 on APCs (14).
Moreover, using human colonic Caco-2/bbe (C2) cells the activation of caspase-9, a key early step leading to apoptosis, occurs earlier when cells express low levels of HSP72 (15). It suggests that HSP72 may also have a protective function through its antiapoptotic effect (16).
On the basis of the interaction of increased mucosal expression of TLR2 and TLR4 in CD (17) and potential immunostimulatory and antiapoptotic features of HSP72 we investigated the modulatory role of this protein in CD.
PATIENTS AND METHODS
Duodenal biopsy samples from 16 children with untreated (boys 6, girls 10; median age 6.7 years [3.7–13.9]) and 9 with treated (boys 4, girls 5; median age 6.7 years [4.9–12.7]) CD were collected. Biopsy samples of 7 children with untreated CD were taken at the time of diagnosis, before the introduction of a gluten-free diet. From the other 9 children duodenal biopsies were obtained before (untreated CD) and 1.5 years (range 1.1–2.5) after exclusion of gluten from the diet (treated CD) (Table 1). The diagnosis of CD was based on the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition criteria (18). All of the untreated patients with CD had anti-endomysium IgA positivity and subtotal villous atrophy of the intestinal mucosa. In cases of treated patients with CD full clinical remission was observed on the diet and no serum anti-endomysium antibodies were detected. The control group consisted of 10 children (boys 4, girls 6; age 8 years [1.7–13]) who were investigated for either growth retardation or chronic diarrhea, and an upper gastrointestinal endoscopy was part of their diagnostic procedure. The intestinal mucosa was normal in all of them and no significant age- or sex-related differences were observed among children with untreated CD, treated CD, and controls (P = NS). Biopsy samples were immediately frozen and stored at −80°C until further analysis. Written informed consent was obtained from parents of each participant before the procedure, and the study was approved by the Semmelweis University Regional and Institutional Committee of Science and Research Ethics (TUKEB: 73/2003).
RNA Isolation and Real-time RT-PCR
Total RNA was isolated from the samples by RNeasy RNA isolation kit (Qiagen GmbH, Hilden, Germany). One microgram of RNA was reverse transcribed using SuperScript II RNase H- (Gibco/BRL, Eggenstein, Germany) to generate first-stranded cDNA. HSP72 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression were determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) using SYBR Green (for HSP72) or fluorescence resonance energy transfer hybridization probes (for GAPDH) on a Light Cycler system (Roche Diagnostics, Mannheim, Germany). The reaction mix for HSP72 contained 4 mmol/L MgCl2, 500 nmol/L of each PCR primers (TibMolBiol, Berlin, Germany), 2 μL of FastStart DNA Master SYBR Green enzyme mix (Roche Diagnostics), and 1 μL of cDNA sample. The conditions were 1 cycle at 95°C for 8 minutes, 50 cycles at 95°C for 5 seconds, 61°C for 5 seconds, and 72°C for 20 seconds with a single fluorescence detection point at the end of the extension. The GAPDH-PCR contained 2 mmol/L MgCl2, 0.17 nmol/L of each hybridization probe, 0.5 nmol/L of each primer, 2 μL of the Light-Cycler FastStart DNA Master HybProbe (Roche Diagnostics), and 1 μL of cDNA. The conditions were 1 cycle at 95°C for 8 minutes, 50 cycles at 95°C for 4 seconds, 55°C for 8 seconds, and 72°C for 22 seconds with a single fluorescence detection point at the end of the annealing segment. Results were analyzed by Light-Cycler software version 3.5.3 (Roche Diagnostics). The mRNA expression of HSP72 was determined by comparison with GAPDH as an internal control from the same sample (Table 2).
Protein Isolation and Western Blotting
Biopsy specimens were lysed in buffer containing leupeptin, aprotinin, Triton X-100, Tris-HCl, ethylene glycol-bis (2-aminoethylether),N,N,N′,N′-tetraacetic-acid, sodium fluoride, phenylmethylsulfonylfluoride, and Na-orthovanadate (each substance from Sigma-Aldrich Co, St Louis, MO) and centrifuged (10,000g, 10 minutes, 4°C) to pellet nuclei and large cellular fragments. Protein concentration of the supernatants was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Twenty micrograms was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis at 120 V (∼40 mA, 90 minutes) (Penguin Dual-Gel Water Cooled Systems, Owl, NH). Prestained protein mixture (BenchMark, Gibco/BRL, Eggenstein, Germany) was used as a marker of molecular mass. The separated proteins were transferred to nitrocellulose membrane (Hybond ECL, AP Biotech, Buckinghamshire, UK) at 70 V (∼220 mA, 90 minutes) (MiniTank electroblotter, Owl, NH). Nonspecific binding sites were blocked (2 hours, room temperature [RT]) in 5% nonfat dry milk containing blot solution. Membranes were incubated with rabbit monoclonal antibodies to HSP72 (donated by Dr L. Laszlo, Eötvös University, Budapest, Hungary) (19) diluted to 1:10,000. Blots were washed and incubated (30 minutes, RT) with peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich Co) diluted to 1:5000. Immunoreactive bands were visualized using enhanced chemiluminescence Western blotting detection protocol (AP Biotech, Buckinghamshire, UK). Negatives were analyzed by computerized densitometry (Gel-Pro Analyzer 3.1 software, Media Cybernetics, Bethesda, MD).
Duodenal biopsy samples were immediately snap frozen for immunofluorescent analysis and embedded in Shandon cryomatrix (ThermoElectron Co, Waltham) and then cut to 5-μm slides. Slides were incubated (1 hour, RT) with anti-HSP72 rabbit polyclonal IgG antibody (same antibody as in Western blot) diluted to 1:1000. After washing, slides were incubated (30 minutes, RT) with Alexa Fluor 488 F (ab′)2 fragment of goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) diluted to 1:100. DNA was stained (10 minutes, RT) with Hoechst 33342 (Sigma-Aldrich Co) diluted to 1:1000. Finally slides were coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA). Appropriate controls were performed omitting the primary antibodies to ensure their specificity and avoid autofluorescence. To visualize the stained tissues on the sections, Zeiss LSM 510 Meta confocal laser scanning microscope (Carl Zeiss, Jena, Germany) was used equipped with an inverted Axiovert 200 M microscope, 20× Plan Apochromat (NA = 0.80), and 63× Plan Apochromat oil immersion DIC objectives (NA = 1.4).
Data were analyzed using Statistica 7.0 software (StatSoft Inc, Tulsa, OK). After testing the normality with the Shapiro-Wilk test, the Mann-Whitney U test was used to determine the differences among all groups. Data are shown in a form of scatterplots. P values ≤0.05 were considered statistically significant.
HSP72 mRNA Expression in the Duodenal Mucosa
Figure 1 shows the mRNA expression of HSP72 using real-time PCR as detected in the duodenal mucosa of samples from children with untreated CD, children with treated CD, and controls. HSP72 mRNA expression was significantly increased in the duodenal mucosa of children with untreated CD as well as children with treated CD compared with that in controls (P = 0.0002 and P = 0.023, respectively). In the duodenal mucosa of children with treated CD, the HSP72 mRNA level was decreased in comparison to children with untreated CD (P = 0.003).
HSP72 Protein Levels in the Duodenal Mucosa
Western blot analysis of duodenal biopsy specimens from children with untreated CD, children with treated CD, and controls using anti-HSP72 rabbit polyclonal antibody revealed distinct bands at 72 kDa (Fig. 2A). Significantly elevated HSP72 protein levels were detected in the duodenal mucosa of children with untreated and also with treated CD compared with that in controls (P = 0.0001 and P = 0.003). In the duodenal mucosa of children with treated CD, HSP72 protein levels were markedly higher than in untreated CD (P = 0.002) (Fig. 2B).
Localization of HSP72 in the Duodenal Mucosa
The cellular distribution of HSP72 proteins was determined by immunofluorescent staining. In the duodenal villi of children with untreated CD, strong HSP72 staining intensity was found in the villous enterocytes and immune cells of the lamina propria compared with controls. In the duodenal villi of children with treated CD, weaker HSP72 signal was found in villous enterocytes and immune cells of the lamina propria compared with children with untreated CD. In the normal duodenum of controls only weak HSP72 immunoreactivity was observed. HSP72 was not present at detectable levels in the nuclear fractions (Fig. 3).
In the present study we first demonstrated the increased expression of HSP72 in the duodenal mucosa of children with CD. CD is a complex small intestinal disorder affecting 1% of the society and caused by intolerance against wheat gliadin and related proteins (20). Deregulated immune response and disturbance in the epithelial barrier function leads to the inflammation of the lamina propria (21) and increased enterocyte apoptosis (6).
In the intestinal mucosa the expression of HSP72 has been sparsely examined. There are only a few data about the importance of HSP72 in inflammatory bowel diseases. Cuzzocrea et al (22) have revealed that the intraperitoneal administration of cyclopentenone prostaglandin (15-deoxy-Δ [12,14]-PGJ2)] stimulates the activation of HSP72 in the inflamed colon of dinitrobenzene sulfonic acid–induced experimental rat colitis and causes substantial reduction of the degree of colonic injury (22).
In the present study we demonstrated significant upregulation of HSP72 in the villous enterocytes and immune cells of the lamina propria of children with untreated CD. In the duodenal mucosa of children maintained on gluten-free diet, the expression of HSP72 was markedly decreased compared with that of untreated children; however; its level remained higher than that in controls.
The main and most well-known function ascribed to HSP72 is that they can be intracellular molecular chaperones of naïve, aberrantly folded, or mutated proteins (23). It has been shown that, besides its intracellular chaperone functions, HSP72 has extracellular, cytokine-like effects as well (24).
Our present data may indicate the mucosal stress induced in the mucosa of CD; however; on the basis of the previous studies we suggest that HSP72 may also participate in the protection of the duodenal mucosa integrity by different mechanisms (15).
Tao et al (25) have found that the probiotic Lactobacillus GG (LGG-CM) treatment of intestinal epithelial cells induces HSP72 expression, which contributes to the beneficial clinical effects attributed to this probiotic through preservation of cytoskeletal integrity (25). HSP72 can bind and stabilize key cytoskeleton-associated proteins, such as α-actinin, or tight junction-associated proteins, such as the zonula occludens (ZO) (26). Other probiotics have similar effects. Bifidobacterium lactis treatment of C2 cells protected the tight junctions against the toxic effects of gliadin, as evinced by the pattern of ZO-1 expression (27).
Another site of action is the protection of mitochondrial function; HSP72 may stabilize mitochondrial membranes and proteins required for structural integrity and function of the cell. Moreover, using human colonic C2 cells it has been found that activation of caspase-9 occurs earlier in cells that express low levels of HSP72 (15), suggesting that HSP72 may also have a protective function through its antiapoptotic effect (16).
Certain gliadin peptides are able to induce innate immune response through TLRs and other pattern-recognition receptors (4,28). We have found elevated TLR2 and TLR4 expression in the duodenal mucosa of children with CD (17). It has been shown that HSP70s are ligands for TLRs (13). HSP72 can specifically activate both the TLR2 and TLR4 molecules on APCs and exert immunoregulatory effects, including upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release (13,14). It has been demonstrated that TLR2 and TLR4 are localized on the villous enterocytes and on the immune cells of the lamina propria (17,29). Now we have demonstrated similar tissue localization of the HSP72 in CD. Taken together, these data suggest that the HSP72-TLR2/TLR4 signaling may alter the integrity of the enterocytes and the immune reaction against gluten toxicity.
Cario and Podolsky (29) and Cario et al (30) have shown that stimulation of TLR2 and TLR4 by their ligands can preserve ZO-1–associated intestinal epithelial barrier integrity. Moreover, Galloway et al have found that in cells of the innate immune system (eg, in monocytes), HSP72 uses TLR2 and TLR4 to induce proinflammatory cytokine production (24,31,32), supporting its potential role as an endogenous signal (33,34), which promotes immune responses and improves host defense.
In summary, the elevated level of HSP72 in the duodenal mucosa of children with CD found in the present study indicates that this molecule may have a role in defense against the gliadin-mediated cytotoxicity. Because of its antiapoptotic effects it may foster surveillance of the epithelial cells and helps to retain their integrity, potentially diminishing villous atrophy, which is a major symptom of the disease. HSP72 as a cellular chaperone activated due to stressors may serve as a “danger signal” for the cells of the innate immune system to promote their protection against injury. Additional studies are needed to clarify the precise role of HSP72 in CD. Furthermore, due to the protective effects of HSP72 it could be regarded as a potential therapeutic target to treat this gastrointestinal disease.
We are grateful to Mária Bernáth for excellent technical assistance.
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