COMPARATIVE ESTIMATION OF HEAT SHOCK PROTEIN 70 ( HSP 70 ) EXPRESSION IN HEALTH , GINGIVITIS AND PERIODONTITIS

Background: Although periodontal bacteria are the causative agents of periodontitis, subsequent progression and disease severity are thought to be determined by a host's immune responses. HSPs may be expressed during periodontal diseases. Objective: To compare the HSP 70 expression within layers of epithelium and connective tissue and correlate the expression of HSP 70 with the severity of periodontal disease. Materials & Methods: The study population comprised of 25 patients with periodontitis, and 23 patients with gingivitis and 21 controls with healthy gingival; tissue samples collected from the Department of Periodontology /Oral and Maxillofacial Surgery respectively. An immunohistochemical analysis was done for HSP 70 expression. Results: One way ANOVA results indicate a statistically significant expression in basal layer for mild inflammation (mean=2.36) and intensity in stratum spinosum for mild (mean=2.27) and minimum (mean=2.39) inflammation. An overall greater HSP 70 expression is noticed in periodontitis group (total IRIDI = 25.41) however not statistically significant Conclusion: We elucidated that there was significant HSP 70 expression in stratum basale and spinosum for mild inflammation that did not increase with severity of inflammation. The overall expression of HSP70 is insignificantly elevated in periodontitis compared to health & gingivitis. Statistical analysis used: One way Analysis of Variance (ANOVA), Mann Whitney U test and Kruskal Wallis Test.


Introduction
Periodontitis is defined as "an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms or groups of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with pocket formation, recession, or both 1 . When exposed to a wide range of environmental stresses (temperature, pH, redox potential, etc.), prokaryotic and eukaryotic cells respond by inducing or accelerating the synthesis of specific proteins known as stress proteins, including heat-shock proteins (HSPs). HSPs are expressed constitutively under normal conditions but are quickly up-regulated under stressful conditions. HSPs act as molecular chaperones in the assembly and folding of proteins, and as proteases when damaged or toxic proteins have to be degraded 2,3 . Heat shock proteins (Hsp) are highly conserved proteins found in all prokaryotes and eukaryotes. In response to a wide variety of stressful stimuli, there is a marked increase in total Hsp synthesis 3 , known as the cellular stress response. The stress response is designed to enhance the ability of the cell to cope with increasing concentrations of unfolded or denatured proteins. Of all heat shock proteins, the Hsp70 family constitutes the most conserved and best-studied class.
Heat shock proteins play an essential role in the etiology of numerous diseases, with a rapidly increasing role in clinical practice 4,5,6 . HSPs may be expressed during periodontal diseases 7 . Although periodontal bacteria like Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans are the causative agents in Periodontitis, the progression of the disease and the amount of severity is known to be controlled by host immune responses 8 . HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes 9 .Therefore, the present study aimed to determine the expression of HSP70 molecule and their possible role in the etiopathogenesis of periodontal disease.
In periodontal disease as per the current evidence the host immuno inflammatory response to pathogenic bacteria play a key role in disease progression. Salivary HSP 70 induces such autoimmune mechanisms that contribute to the initiation of gingivitis 10 and also since mainly secreted in a lipid bound form and so protein bound lipid molecules are known to form nucleus of calcium-phosphate deposition leading to calculus formation so that takes part in the acquired pellicle formation on tooth surfaces and gingival inflammation under certain conditions 11,12 . Molecular mimicry between GroEL of the periodontopathic Porphyromonas gingivalis and autologous human HSP 60 may play a role in immune mechanisms of periodontal disease 13 .

Inclusion criteria
Sixty nine individuals aged 20-55 years were included in the study with a minimum of ≥ 20 teeth in the dentition and categorized into three groups.  The clinical parameters recorded are gingival index, pocket probing depth & clinical attachment level.

Exclusion criteria
Individuals excluded include who had undergone periodontal /antibiotic therapy within past six months, previous history of cardiovascular disease, inflammatory conditions, hepatic or thyroid disease, use of drugs, pregnant and lactating mothers.

Tissue collection
Biopsies were obtained (one biopsy per patient) by making two parallel vertical incisions (approximately 4 mm apart) connected by a horizontal incision made at approximately 1 mm below the base of the gingival sulcus with no-15 blade and BP handle. For the healthy and gingivitis group, gingival biopsies were obtained at the time of extraction.
For the periodontitis category, biopsies were obtained at the time of flap surgery, done to eliminate periodontal pockets. All the tissue specimens were fixed in 10% neutral buffered formalin. The tissues were embedded in Paraffin wax for the histopathological examination and IHC.

Antibodies
The primary antibody is Anti-Heat Shock Protein (HSP-70), BioGenex Laboratories, San Ramon, CA, (AM289-5M). The secondary antibody is Mouse/Rabbit polydetector HRP/DAB detection kit BioSB (BSB-0201). For all the tissues H and E stained sections were obtained. The H and E stained sections were reviewed for the adequacy of the epithelium and CT.

Histopathological grading of IHC slides
The IHC slides were viewed under 10X and 20X magnification objective by a senior oral pathologist and PI. All the clinical findings were blinded. The grading was done separately for the basal layer, parabasal layer & stratum spinosum. The grading was done using ProgResCapture Pro 2.8.8 JENOPTIK OPTICAL SYSTEMS software. The slides were focused on 20x and live image was obtained in the computer screen. 100 cells were marked on the computer for each layer for each field. Out of 100 cells marked, positive cells were counted to obtain the percentage positivity. This procedure was repeated for 10 (20x) fields.
The percentage positivity was graded as Grade 0 -≤10% positive or immunoreactive cells, Grade 1 -11% to 25% positive cells, Grade 2 -26% to 50% positive cells, Grade 3 -51% to 75% positive cells; Grade 4 -≥76% positive cells14. The intensity of staining was recorded as score 1 for light yellow or mild staining, score 2 for yellow or moderate staining, and score 3 for brown or intense staining 15 ( figure 1). In the connective tissue, the percentage positivity was counted in 10 (40x) fields and mean value was obtained. The cells counted were the inflammatory cells and fibroblasts. Immunohistochemical evaluation was performed according to Chaiyarit et al 1999 14 . The kappa statistic valve for interobserver variability was almost a perfect agreement (0.85).

IRI D I c al cu lat io n
Immunoreactivity intensity distribution index (IRIDI) scores were obtained by multiplying the grade of positive cells and intensity of staining. The final scores ranged from 0 to 12. A median score was then calculated. The tissue with scores equal to or more than median were defined as the high expression group and those with scores less than median were defined as the low expression group 16 . IRIDI scores were calculated for the basal, parabasal layer, stratum spinosum and for the connective tissue of all the patient samples as well as control samples.

Resu lt s
A statistical evaluation was performed. Comparison of mean values of two groups is done using independent samples t-tests. Comparison of mean values of more than two groups is done using oneway Analysis of Variance (ANOVA). To compare the equality of proportions among the two groups, Mann Whitney U test has been applied and to compare the equality of proportions among more than two groups, Kruskal Wallis Test has been applied.
There is a greater intensity and staining in all the epithelium layers compared to connective tissue (figure 1a,1b,1c) although not statistically significant this may be because of smaller sample size. More hyperplastic epithelium is seen with age which is significant (.024) ( Table 1).  Oneway ANOVA results indicate a statistically significant expression in basal layer for mild inflammation (mean=2.36) and intensity in stratum spinosum for mild (mean=2.27) and minimum (mean=2.39) inflammation (Table 2). The overall HSP 70 expression is more in periodontitis group (mean= 25.41) compared to gingivitis (mean= 22.96) or health (mean = 21.64) but not statistically significant (p>0.01) ( Table 3).

D isc u s si on
Periodontitis is a chronic inflammatory disease that affects the tooth supporting tissue-the periodontium. It is the most frequent cause of tooth loss in the adult population. The prevalence of the disease is high, with the moderate form affecting 50% and the progressive form 5-15% of the adult population 17 . The prevalence of cardiovascular diseases in patients with periodontitis is 25-50% higher than in healthy individuals.
Heat shock proteins have important functions in the cell such as folding, assembly, and translocation of polypeptides across membranes and play a main role in protein repair after cell damage. The human and bacterial cognates of heat shock proteins are similar, sharing more than 50% sequence homology at the amino acid level 3 . Heat shock protein production by several periodontopathic micro-organisms has been extensively documented. The presence of these stress proteins has been demonstrated in tissue samples from periodontitis lesions 18,7 . 19 examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. They demonstrated that Hsp60-specific T cells accumulated in the gingival lesions of periodontitis patients but not in gingivitis patients and that the T cell clones with an identical specificity to those in peripheral blood existed in periodontitis lesions. 20 showed that Porphyromonas gingivalis Hsp reactive T cell immune response might be involved in immunopathogenesis of periodontal disease. They suggested that T cells in the circulating peripheral blood may home to periodontal lesions where Porphyromonas gingivalis have infiltrated potentially leading to T cell response cross reactive to mammalian HSP of gingival fibroblasts. Of all heat shock proteins, the Hsp70 family constitutes the most conserved and best studied class. Earlier studies by Amina Gamal el Din et al (2010) 16 evaluated the differential immunehistochemical expression of HSP 70 in psoriatic skin. Antibodies to HSP70 were analyzed immunehistochemically. Immunoreactivity intensity distribution index (IRIDI) scores including the proportion of immunoreactive cells and their staining intensity were calculated in the basal, suprabasal, superficial as well as the whole epidermal layers of patients and controls. HSP70 was not immunohistochemically detected in the dermis of normal skin. Intense HSP70 immunostaining was found in psoriatic lesions, predominantly in basal and suprabasal epidermal cell layers. 9 have hypothesized that HSP 70 upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Due to their high conservation among various microbial pathogens and their ability to induce very strong immune responses, HSP 70s are thought to play a role as candidate antigens in periodontal disease.

Asea et al (2000)
In a study by Sergio Sanchez-Rodriguez et al (2011) 21 looked for the expression of Hsp70, Hsp90 and P53 in the tissue of patients with chronic periodontal disease and gingivitis. There was an increase of Hsp70 and 90, and a slight increase in p53 levels. By PCR Hsp70 expression was increased in initial and progressive periodontitis and in a low level in advanced.
Our study aimed at comparing the HSP 70 expression within layers of epithelium and connective tissue and to correlate intensity of HSP 70 expression with severity of periodontal disease.
This study was of its first kind where detailed estimation of HSP 70 expression was seen in layers of epithelium and connective tissue.
Our results suggest that there is a greater intensity and staining in all the epithelium layers compared to connective tissue although not statistically significant. A statistically significant HSP70 expression in basal layer for mild inflammation IRIDI (µ= 8.95) and intensity in stratum spinosum for mild (µ = 8.73) and minimum inflammation (µ = 9.29). The stratum spinosum has a significant HSP70 intensity and IRIDI score for mild and minimum inflammation group. The HSP70 expression in stratum spinosum is higher in normal epithelium compared to hyperplastic epithelium.
A statistically insignificant expression and intensity in different groups of periodontal disease. The total IRIDI score of HSP 70 expression in gingivitis group (µ = 22.96) was greater than health group (µ = 21.64) respectively and also comparatively greater than both health and gingivitis group in periodontitis (µ = 25.41) even though it is not statistically significant due to smaller sample size.
This denotes the possible correlation exists in disease progression in periodontal disease. Even though intervention of immune response during the molecular mimicry between the bacterial and human HSP 70 is not feasible at this stage. A hand of heat shock proteins in the pathophysiology of periodontal disease helps us to intervene in the immune responses, by future studies to search for immunotherapeutic approach at suppressing the inflammatory response which is useful for therapeutic approaches of periodontal disease.

Con c lu s ion
We elucidated that there was significant HSP expression in stratum basale and spinosum for mild inflammation that did not increase with severity of inflammation. The overall expression of HSP is insignificantly elevated in periodontitis compared to health & gingivitis which shows the role of it in disease initiation and progression.