|Year : 2014 | Volume
| Issue : 5 | Page : 567-571
|Immunolocalization of heme oxygenase-1 in periodontal diseases
G Gayathri1, S Muthukumar2, Leena Dennis Joseph3, R Suresh2
1 Department of Periodontics, Sri Venkateswara Dental College, Chennai, Tamil Nadu, India
2 Department of Periodontics, Sri Ramachandra Medical College, Chennai, Tamil Nadu, India
3 Department of Pathology, Sri Ramachandra Medical College, Chennai, Tamil Nadu, India
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|Date of Submission||22-Oct-2013|
|Date of Decision||01-Mar-2014|
|Date of Acceptance||22-Mar-2014|
|Date of Web Publication||16-Dec-2014|
|Date of Print Publicaton||16-Dec-2014|
| Abstract|| |
Context: Chronic periodontitis is an inflammatory condition of supporting tissues initiated by organisms in dental plaque. The reactive oxygen species and free radicals mediate connective tissue destruction in periodontitis. In order to counteract the free radical mediated tissue damage, numerous antioxidant mechanisms exist within the host. One such system is heme oxygenase enzymes. Heme oxygenase is the key enzyme involved in catabolism of heme. It cleaves the heme molecule to yield equimolar amounts of biliverdin, carbon monoxide, and iron. These end products act as important scavengers of reactive oxygen metabolites. Increased heme oxygenase expression has been identified in inflammatory condition, such as pancreatitis, diabetes, nephritis, and atherosclerosis. Since chronic periodontitis is one such inflammatory condition, we assessed the expression of heme oxygenase-1, in smokers and periodontitis group using immunohistochemistry technique.
Aims: The aim of this study is to compare the expression of heme oxygenase-1 in patients with healthy periodontium, periodontitis and smokers.
Materials and Methods: Gingival tissue samples were taken from 30 patients, who were divided into three groups healthy controls (n = 10), chronic periodontitis (n = 10), and smokers with chronic periodontitis (n = 10). All the samples were subjected to immunohistochemical staining using the antiheme oxygenase-1 antibody and were tested for efficiency by staining a positive control (prostate cancer tissue sections) and a negative control. The results were tabulated and analyzed.
Results: Our results showed increased expression of heme oxygenase-1 in the gingival tissue samples taken from smokers compared with periodontitis and healthy tissue.
Conclusion: The results of our study is an increasing evidence of involvement of antioxidant enzymes like heme oxygenase-1 in periodontal inflammation and their implication for treatment of chronic periodontitis.
Keywords: Antioxidants, chronic periodontitis, heme oxygenase, reactive oxygen species
|How to cite this article:|
Gayathri G, Muthukumar S, Joseph LD, Suresh R. Immunolocalization of heme oxygenase-1 in periodontal diseases
. Indian J Dent Res 2014;25:567-71
Mouth acts as a window to lot of systemic diseases and serves as a port of entry for various infections that can alter and affect the immune status of the person. 
|How to cite this URL:|
Gayathri G, Muthukumar S, Joseph LD, Suresh R. Immunolocalization of heme oxygenase-1 in periodontal diseases
. Indian J Dent Res [serial online] 2014 [cited 2019 May 22];25:567-71. Available from: http://www.ijdr.in/text.asp?2014/25/5/567/147080
Chronic periodontitis denotes an inflammatory disease that affects the tooth supporting structures namely, the gingiva, periodontal ligament, cementum, and alveolar bone. The disease occurs as a consequence of improper oral hygiene measures that lead to excessive accumulation of dental plaque around the gingival margins. Virulent micro-organisms that colonize the plaque biofilm produce toxins that kindle the host immune response, which is characterized by influx of immune and inflammatory cells into the periodontal tissues. These cells produce cytokines, inflammatory mediators, free radicals, and reactive oxygen species that mediate tissue destruction characterized clinically as pocket formation, pus discharge, tooth mobility, and eventually tooth loss. 
Periodontal disease is multifactorial, that is, not one, but several risk factors are implicated. Among these, tobacco smoking has been found to be associated with severe periodontal destruction. Deleterious components such as nicotine from tobacco smoke, affect critical pathways in the immune response, and deregulates oxidant-antioxidant balance thereby causing tissue destruction. 
To mitigate the effect of oxidative stress the body has evolved potent antioxidant mechanisms. In this regard, the role of heme oxygenase-1 is important to be understood. This enzyme is involved in the catabolism of heme into bilirubin, carbon monoxide, and iron. Bilirubin and carbon monoxide are efficient free radical scavengers and thereby exert antioxidant functions. Elevated heme oxygenase levels are a feature of inflammatory conditions such as acute pancreatitis, diabetes mellitus type 2, glomerulonephritis, and atherosclerosis. Since, periodontitis is also an inflammatory disease; we sought to assess the immunohistochemical expression of heme oxygenase-1 in gingival tissue samples of healthy subjects and chronic periodontitis patients (smokers and nonsmokers).
| Materials and methods|| |
A total of 30 patients attending the outpatient clinic, of the Department of Periodontology, of the faculty of dental sciences, in a tertiary care hospital were recruited for this study, after obtaining their informed consent. The study was approved by the Institutional Ethics Committee of Sri Ramachandra University. The study comprised of three groups:
- Healthy controls subjects (n = 10)
- Chronic periodontitis patients (n = 10)
- Smokers with chronic periodontitis (n = 10).
Gingival tissue samples were obtained from healthy control subjects and chronic periodontitis patients, according to the method used by Tamilselvan et al.;  healthy control gingival tissue were obtained from subjects undergoing orthodontic treatment, while chronic periodontitis samples were obtained from patients undergoing flap surgery in the Department of Periodontics, faculty of dental sciences in a tertiary care hospital.
The gingival tissue samples (healthy) were obtained from the control subjects from sites without clinical evidence of gingival inflammation or bleeding on probing, and with a probing depth of < 4 mm. All Chronic periodontitis samples expressed clinical signs of inflammation and were taken from sites with probing depth more than 6 mm. In the smoker group alone current smokers with chronic periodontitis, who have smoked for a minimum period of 2 years were chosen for tissue sampling.
Pregnant and lactating women and patients on anti-inflammatory, antioxidants and antibiotics were excluded from the study. Patients with any existing systemic diseases were also excluded from the study.
All the tissue samples were obtained by a single trained operator. The protocol of tissue sample collection was carried out under local anesthesia (xylocaine 2% with 1 in 1 lakh units adrenaline). A portion of the interdental gum tissue was excised using a surgical blade during periodontal treatment. The tissue samples were immediately washed in sterile saline to remove blood and were fixed in 10% buffered formalin solution for 24 h. Following fixation, the samples were dehydrated in alcohol (100%) and embedded in paraffin and the blocks were prepared. All the samples were subjected to immunohistochemical staining using the antiheme oxygenase-1 antibody. The antibody was tested for efficiency by staining a positive control (prostate cancer tissue sections) [Figure 1] and a negative control.
After immunohistochemical staining, all the slides were interpreted by a single trained pathologist, who graded the staining intensity and performed cell counting. The results were tabulated [Table 1] and statistically analyzed [Figure 2].
| Results|| |
Immunohistochemistry, revealed positivity for heme oxygenase-1 in all the 30 samples analyzed. In the healthy control group, 8 out of 10 (80%) samples showed intense membrane positivity in the basal layer of the epithelium; occasional endothelial cells and inflammatory cells also showed positive staining [Figure 3].
In chronic periodontitis samples, the staining showed full thickness cytoplasmic positivity of epithelium in 9 out of 10 subjects (90%) [Figure 4]. The connective tissue showed staining for few endothelial cells (40%) [Figure 5] and inflammatory cells (80%) [Figure 6].
In smokers with chronic periodontitis, immunoreactivity to heme oxygenase-1 was detected in the epithelium [Figure 7] and connective tissue. Nine out of 10 subjects showed positivity in the epithelium (90%). Marked staining of fibroblasts [Figure 8] was noted in seven subjects (70%). The fibroblast staining was more significant in smokers with periodontitis compared with the other two groups.
| Discussion|| |
Chronic periodontitis is a well-recognized chronic inflammatory condition that affects the tooth supporting apparatus. The involvement of free radicals and reactive oxygen species in mediating tissue damage in chronic periodontitis is an important mechanism in the pathogenesis of this condition. Equally important is the counter effect of the host's antioxidant mechanism that mitigates and nullifies the deleterious effect of reactive oxygen species. In this regard, the role of heme oxygenase-1 in controlling cellular and tissue damage during oxidative stress has been understood.
In this regard, we are justified in assessing the expression of heme oxygenase-1 in the gingival tissue of healthy subjects and chronic periodontitis patients. Since smoking is dual risk factor for chronic periodontitis and oxidative injury, we also included smokers with chronic periodontitis as group in our study.
The results of our study showed that heme oxygenase-1 was expressed in all the samples tested with varying intensities. In healthy control subjects, we found heme oxygenase-1 staining in the basal layer of the epithelium in 8 out of 10 samples (80%). This can be explained based on the fact that heme oxygenase-1 is constitutively expressed in many tissues of the body like the spleen and liver.  The renal and pulmonary epithelium also expresses heme oxygenase-1 in small amounts. , In a similar manner, the gingival epithelial cells could also express heme oxygenase-1.
Clinically, healthy gingiva of healthy subjects cannot be regarded as "pristine". The word "pristine gingiva" refers to gingival tissues, histologically free of inflammatory cell infiltrate. This is never encountered in a normal clinical scenario. All our healthy samples hence, had a low grade inflammatory cell infiltrate. This cellular infiltrate is a source of proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). Studies by Cantoni et al.,  has shown that IL-1β and TNF-α are inducers of heme oxygenase-1 expression in hepatic cell lines. In our healthy samples also, low levels of IL-1β and TNF-α could have induced heme oxygenase-1 expression in the epithelium.
Our study results on chronic periodontitis tissue samples, showed enhanced heme oxygenase-1 expression in the gingival tissues, which included epithelial cells (90%) and inflammatory cells in the connective tissue (90%). The staining intensity in these was greater than control. Chronic periodontitis is an inflammatory condition in which microbes, cytokines, free radicals, and reactive oxygen species mediate tissue destruction. With regard to the microbial etiology of chronic periodontitis Porphyromonas gingivalis, Bacteriodes forsythus, Treponema denticola hich are Gram-negative anaerobic bacteria, are implicated. The lipopolysaccharide of Gram-negative anaerobic organisms have been found to be prototypic activators of heme oxygenase-1.  This could be a solid reason for elevated heme oxygenase-1 expression in chronic periodontitis.
With regard to the cytokine profile in chronic periodontitis IL-1β and TNF-α have been found to be elevated in gingival crevicular fluid (GCF) samples, serum and tissue homogenates. These proinflammatory cytokines are also switch factors for elevating heme oxygenase-1 expression in periodontitis samples. Another feature of the immune response in chronic periodontitis is the role of toll like receptors (TLR). Our lab has previously demonstrated elevated TLR-2 and TLR-4 expression in chronic periodontitis.  Study by Mohri et al. 2006,  has shown that TLR bound lipopolysaccharide is an enhancer of heme oxygenase-1 expression. In our samples too, this phenomenon could have upregulated heme oxygenase-1. However, oxidative stress is an important mechanism in chronic periodontal disease. Elevated levels of markers of oxidative injury have been seen in saliva, serum and GCF in chronic periodontitis patients; oxidative injury is the most potent enhancer of heme oxygenase-1. In our chronic periodontitis samples also, elevated oxidative stress could have upregulated heme oxygenase-1 expression.
With regard to the smokers with chronic periodontitis, there was marked heme oxygenase-1 expression in gingival epithelium and connective tissue. In addition, the gingival fibroblasts were also found to express the protein in 70% of the samples. These results can be justified based on the fact that oxidative injury is higher in smokers compared to nonsmokers.  Enhanced production of reactive oxygen species metabolites like peroxynitrite  and superoxide  have been shown in smokers. These oxidative species could serve as enhancers of heme oxygenase-1 expression in the smoker tissue samples. Smoking alters critical features of the immune response like neutrophil chemotaxis and phagocytosis. Increased production of IL-1β and TNF-α has been shown in GCF samples from smokers.  Since these cytokines can upregulate heme oxygenase-1 expression, we could find more intense staining in these samples compared to the other two groups.
We could find fibroblastic staining only in the smokers with chronic periodontitis group. This can also be explained based on the fact that nicotine exposure upregulates heme oxygenase-1 expression in fibroblast cell lines. ,
The other unique feature of periodontal disease pathogenesis in smokers, is lowered oxygen tension  and lowered subgingival temperature da Silva et al.  Both hypoxia and hypothermia are switch-on factors for heme oxygenase-1 ex pression as documented by the studies of Jordi-Racine et al. 1998  and Akhalaya et al. 2006.  These factors could also explain elevated heme oxygenase-1 expression in smokers with chronic periodontitis.
From all the above accumulated evidence, we have shown elevated expression of heme oxygenase-1 in chronic periodontitis (both in smokers and nonsmokers) compared to healthy individuals. This finding throws light on the role of the host antioxidant mechanism in countering the effect of oxidative stress.
Based on our study findings, we can understand the pathogenesis of periodontal disease, which would help evolve better therapeutic strategies, to treat the disease.
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Department of Periodontics, Sri Ramachandra Medical College, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
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