Histopathologic and histomorphometric studies and determination of IL-8 in patients with periodontal disease.

BACKGROUND: Periodontitis is an inflammatory disease characterized by connective tissue breakdown and alveolar bone resorption.
OBJECTIVE: The aim of this study was to make a quantitative evaluation of the gingival tissue components in biopsies from patients with different clinical states of periodontal disease and to determine the relationship between the presence of interleukin-8 and the tissue destruction.
MATERIALS AND METHODS: The study group consisted of 33 biopsies from adult subjects. A total of 25 periodontal patients were sub-classified on the basis of the clinical and radiographic criteria in mild, moderate and advanced periodontitis. Gingival samples were obtained from patients in the course of basic periodontal surgeries as a muco-periostic collate. Data were analyzed by the SPSS system.
RESULTS: Collagen fibres decreased as non-specific chronic states increased. A lymphocyte inflammatory infiltrate changed to a lymphoplasmocytary form. Reactivity to interleukin-8 was detected with the severity of the histopathologic diagnoses; however there was no association with the clinical diagnoses.
CONCLUSIONS: Histopathology and histomorphometry of gingival tissue changed with the degree of inflammation. No evidence of interleukin-8 as a biomarker for clinical diagnoses of periodontitis was obtained.

INTRODUCTION

Periodontitis is one of the most common oral diseases and is characterized by gingival inflammation and alveolar bone resorption.[1] Inflammatory periodontal diseases are initiated and maintained by plaque bacteria and their metabolic products, which trigger the local infiltration of inflammatory cells associated with the degradation of extracellular matrix macromolecules.[23] Among these macromolecules, the major component is collagen, which has an important role in the structure of the gingival connective tissue. Changes in collagen may thus reflect the severity of the periodontal disease, which can be clinically classified as mild (LP), moderate (MP), and advanced periodontitis (AP).[45]

The immune and inflammatory responses are critical to understanding the pathogenesis of periodontal disease and they are orchestrated by a number of host-related factors, either intrinsic or induced.[6] Major tissue destruction in established periodontal lesions results from the recruitment of host cells through the activation of mononuclear phagocytes, including monocytes and macrophages.[7] Selective migration and accumulation of leukocytes is determined by chemokines, a family of low-molecular-weight cytokines with cell-type-specific chemo-attractant properties.[8] In recent years, considerable interest has been focused on the critical role of cytokines in the pathogenesis of periodontal destruction, not only as important mediators of host defence against the microbial challenge, but also as mediators of periodontal tissue destruction.[9] Interleukin-8 (IL-8) a member of the neutrophil-specific CXC subfamily of chemokines is a potent neutrophil chemotactic and activating factor that has a crucial role in the selective recruitment and activation of neutrophils and in routing them to the gingival sulcus.[10] It can be secreted from many different host cells, including monocytes/macrophages, lymphocytes, fibroblasts, endothelial cells, and epithelial cells.[11] IL-8 has been detected in the gingival crevicular fluid and gingival tissue sections of patients with periodontitis.[10] To date, the information regarding the role of IL-8 in the initiation and progression of chronic periodontal disease is not clear, and sometimes contradictory.

The aim of our study was to conduct a quantitative evaluation of the gingival tissue components in biopsies from patients with different clinical states of periodontal disease.

MATERIALS AND METHODS

Clinical parameters

Periodontal diagnoses were performed on all teeth by one calibrated examiner, who recorded the following clinical parameters at the sample sites: Gingival index (GI),[12] Plaque index (PI),[13] bleeding on probing (BOP), probing depth (PD) and clinical attachment loss (CAL), and radiographic criteria. Periapical radiographs were taken using a standardized long-cone paralleling technique, and bone resorption in chronic adult periodontitis was determined. The monitored sites in periodontitis had PD ≥ 4 mm as determined manually, and CAL evidenced by a distance ≥3 mm measured from the cement-enamel junction to the most apical penetration by manual probing. Clinical considerations for patients with periodontitis and controls (C) were made based on an adaptation by our working group[5] from Lindhe[4] and the clinical classification from the American Dental Association (ADA). Periodontal sub-classification included: Mild periodontitis (LP): GI > 1, PI > 20%, BOP, and PD and CAL between 3 and 5 mm; Moderate periodontitis (MP): GI > 1, PI > 20%, BOP, and PD and CAL between 5 and 6 mm; Advanced periodontitis (AP): GI > 1, PI > 20%, BOP, and PD and CAL ≥ 7mm; C subjects: GI < 1, PI < 20%, no BOP, PD ≤ 3 mm and no CAL.

Study population

The study group consisted of 33 adult subjects. Patients were classified according to the clinical diagnosis as LP (n = 8), aged 36.8 ± 3.8 years, MP (n = 8) aged 41.7 ± 12.8 years, and AP (n = 9), aged 49.2 ± 6.9 years. The C group consisted of eight periodontally clinically healthy patients aged 38.5 ± 2.3 years. All subjects were selected from patients visiting the School of Dentistry at the National University of Tucumán for periodontal consultation. Written informed consent was obtained from all patients prior to participation.

Exclusion criteria for all subjects were: Smoking, systemic diseases, surgical and not surgical periodontal therapy, use of antibiotics, steroidal or non-steroidal anti-inflammatory agents, all in the 6 months prior to the study. Inclusion criteria were: A minimum of 20 natural teeth other than third molars.

Tissue preparation

The gingival tissue samples were obtained from patients a week later than the basic periodontal therapy. Biopsies were obtained as a mucoperiosteal flap; incisions were made 1 to 2 mm into the subgingival. Biopsies included external gingiva and internal epithelium of the periodontal pocket. Control biopsies were obtained from periodontally clinically healthy patients during the surgical removal of third molars.

Biopsies were fixed in 4% phosphate buffered formalin, pH 7.0, and processed by routine laboratory techniques for paraffin embedding. Histological analyses were performed using a light microscope (Axiostar plus, Carl Zeiss, Göttingen, Germany).

Histological and histomorphometric evaluation

In order to evaluate changes in the gingival connective tissue in controls and periodontally diseased patients, the surface area with collagen fibres, inflammatory cells and blood vessels was determined by an oral specialized calibrated pathology researcher. Collagen fibres were identified with hematoxylin and eosin stain on 6-8 μm gingival tissue sections obtained from each specimen. The degree of histological inflammation in the connective tissue was quantified according to the density and distribution of the inflammatory cells.

Histomorphometric analyses were carried out using a 111-point grid that divided the field into 100 equal parts following the technique of Weibel et al.[14] Fibers, inflammatory cells, and blood vessels were counted in the total number of intersections of the total area, with ×400 magnification. The number of units was divided by the total number of intersections and expressed as a percentage of fibers, inflammatory cells and blood vessels and the mean ± standard deviation (X ± SD) was determined for each group of patients. Clinically and radiographically classified periodontal patients were histologically considered based on the percentage of the whole area occupied by inflammatory infiltrate and then classified as mild, moderate, or severe non-specific chronic gingivitis groups.

Immunohistochemistry

Immunostaining for IL-8 was performed on deparaffinized tissue sections 4-6 μm thick. Non-specific tissue binding was blocked by incubation with 1.5% normal blocking serum in PBS for 30 min. To unmask antigens, tissue sections were placed in a microwave oven in 100 mM citrate buffer, pH 5-7, for 5 × 2 min at the highest potency. Specimen were incubated overnight at 4°C with the primary antibody, goat polyclonal anti-human antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA) (1:20 dilution) followed by the biotinylated anti-goat immunoglobulin G (1:200 dilution) during 30 min. Endogenous peroxidase activity was quenched by exposures for 10 min with hydrogen peroxide 0.3% v/v in methanol. Sections were finally incubated for 45 min at room temperature with avidin-biotin horseradish peroxidase conjugate, (ABS Staining System Santa Cruz Biotechnology, Santa Cruz, California, USA), followed by three baths in PBS, pH 7.2, for 10 min each. The peroxidase activity was revealed using 3’, 3’ diaminobenzidine tetrahydrochloride with hydrogen peroxide for 30s to 10 min. The sections were counterstained with hematoxylin for 5-10 s. Positive and negative tissue section controls were included using biopsies belonging to the archives of Department of Pathology School of Dentistry, National University of Tucumán.

The distribution of the positively stained cells in the infiltrating connective and epithelial tissues was considered with a scale of 0 to 4, as previously described by Gamonal et al.[8] 0 = not detectable, 1+ = less than 10%; 2+ =10%-25%; 3+ =25%−50% and 4+ = more than 50%.

Data analysis

The data were analyzed using Statistical Package for Social Science (SPSS) version 11.5. Tissue response differences among mild, moderate and severe non-specific chronic gingivitis groups were assessed using the analysis of variance (ANOVA) test. The P–value was taken as significant when less than 0.05 (confidence interval of 95% was taken).

RESULTS

Table 1 shows the mean values for the characteristics and clinical parameters of patients with periodontitis and periodontally healthy subjects. Subjects were classified on the basis of their clinical and radiographic diagnoses and sub-classified as described above.[5] Groups did not differ in age or sex. Their clinical parameters gradually rose from LP to AP; PI, GI, BOP, PD, and CAL were higher in LP, MP and AP than in the C group.

Table 1

Mean±standard deviation (X±SD) of characteristics and clinical parameters of periodontal patients and clinically healthy subjects

Table 2 shows the histopathologic findings from all biopsies. Collagen fibres decreased significantly from the mild, non-specific chronic gingivitis group to the severe form. Conversely, the lymphocyte-predominant inflammatory infiltrate in the mild group increased and changed to a lymphoplasmocyte type of cell infiltrate in severe non-specific chronic gingivitis blood vessels showed no statistically significant difference.

Table 2

Mean±standard deviation (X±SD) values of the inflammatory infiltrate in the non-specific chronic gingivitis groups

Table 3 shows the relationship between the histopathologic and the clinical radiographic diagnoses. It can be seen that the most frequent histopathologic diagnosis corresponds to the mild non-specific chronic gingivitis in the C, LP, and MP groups. Epithelial hyperplasia and collagen fiber conservation with a mild inflammatory infiltrate of lymphocytic cells can be observed [Figure 1a]. Only patients with clinical diagnosis of AP mostly showed a moderate non-specific chronic gingivitis with hyperplasia, spongiosis and epithelial atrophy; the collagen fibers of the connective tissue were replaced by an inflammatory infiltrate and a proliferation of vascular structures [Figure 1b]. In severe non-specific chronic gingivitis, there was epithelial atrophy, hyperplasia, and spongiosis. Major lymphoplasmocytic infiltration was observed [Figure 1c].

Table 3

Relationship between the histopathologic and the clinical and radiographic diagnoses

Figure 1

(a) Histopathologic characteristics in a biopsy section of the epithelial and connective tissues of mild non-specific chronic gingivitis: Epithelial hyperplasia, mild inflammatory lymphocyte infiltrate. HE. Original magnification ×100. (b) Histopathologic characteristics in a biopsy section of the epithelial and connective tissues of moderate non-specific chronic gingivitis: Proliferation of epithelial crests, moderate inflammatory lymphocyte infiltrate, congestion. HE. Original magnification ×100. (c) Histopathologic characteristics in a biopsy section of the epithelial and connective tissues of severe non-specific chronic gingivitis: Epithelial atrophy, spongiosis, severe lymphoplasmocytic infiltration. HE. Original magnification ×200

Table 4 shows the IL-8 (+) immunoreactivity in relation to the histopathologic diagnoses and the topographic localization of stained cells. 77.5% of the biopsy sections with mild non-specific chronic gingivitis showed IL-8 (+); less than 10% was distributed in the basal and spinous layers of the epithelium [Figure 2a]. 64.5% of the biopsy sections with moderate non-specific chronic gingivitis showed IL-8 (+); 10-25% included epithelial cells [Figure 2b] and about 10% corresponded to connective tissue cells [Figure 2c]. 66.4% of the biopsies with severe non-specific chronic gingivitis showed positive reactivity to IL-8; the stained epithelial cells were between 10 to 25% [Figure 2d], and about 10% corresponded to connective tissue cells [Figure 2e]. [Figure 3a and b] correspond to positive controls of pyogenic granuloma and maxillary epithelial inflammatory cyst, respectively.

Table 4

Relationship between the histopathologic diagnoses, the IL.8 (+) percentage and the tissue location in the biopsy sections

Figure 2

(a) Immunohistochemical staining of IL-8 (+) cells in the squamous epithelial tissue of mild non-specific chronic gingivitis. Original magnification ×200. (b) Immunohistochemical staining of IL-8 (+) cells in the basal layer of the epithelial tissue of moderate non-specific chronic gingivitis. Original magnification ×200. (c) Immunohistochemical staining of IL-8 (+) cells in the connective tissue of moderate non-specific chronic gingivitis. Original magnification ×400. (d) Immunohistochemical staining of IL-8 (+) cells in the epithelial tissues of severe non-specific chronic gingivitis. Original magnification ×400. (e) Immunohistochemical staining of IL-8 (+) cells in the connective tissues of severe non-specific chronic gingivitis. Original magnification ×400

Figure 3

(a) Immunohistochemical staining of IL-8 (+) cells in pyogenic granuloma surface area. Original magnification ×200. (b) Immunohistochemical staining of IL-8 (+) cells in maxillary epithelial inflammatory cyst. Original magnification ×400

DISCUSSION

The histopathologic components of gingival tissue samples from patients at different stages of periodontal disease were quantified and analyzed and the immunoreactivity for IL-8 was evaluated. Samples were obtained during basic periodontal therapy, which consisted of eliminating the pocket, scaling and root planning, with the aim of markedly reducing the degree of inflammation.

Biopsies from periodontally healthy subjects were used as controls; however, microscopic studies revealed they were not pristine and they belonged to the gingivitis histopathologic diagnosis.

Results showed a marked decrease in collagen fibers as the degree of inflammation increased. Thus, in cases of mild non-specific chronic gingivitis, the percentage area occupied by collagen fibers was 66% in the mild, 37.5% in the moderate and only 3.4% in the severe form, which was inversely proportional to the inflammatory infiltrate.

There was no association between the clinical and the histopathologic and histomorphometric diagnoses. Our results for moderate non-specific chronic gingivitis were similar to those of Seguier et al.; the area of collagen fibers was about 27% and the collagen fibers of the upper connective tissue were highly disorganized.

Gamonal et al.[8] found that biopsies of tissues from healthy patients showed low inflammation and poorly defined infiltrate. A significantly higher degree of inflammation was observed in the gingiva of patients with periodontal disease with well-organized, dense cell infiltration in the connective tissue. In our study, biopsies from the control group showed mild to severe non-specific chronic gingivitis.

Fitzgerald et al.[15] Mathur et al.[16] and Tsai et al.[17] reported that the concentration of IL-8 in gingiva and gingival crevicular fluid of patients with active periodontal disease is greater than in healthy subjects, Gemmell et al.[18] reported that the local increase in IL-8 may indicate the initial stage of peri-implantitis. However, other authors[1920] showed increased IL-8 in gingival crevicular fluid in control patients. In our study we found that the expression of IL-8, in epithelial tissue increased from 10% in the mild non-specific chronic gingivitis group to 10-25% in the moderate and severe non-specific chronic gingivitis. In connective tissue IL-8 increased with the degree of inflammation. The immunoreactivity in the control group may be associated with the natural inflammation stage and was comparable to that of mild non-specific chronic gingivitis as observed in our histopathologic results. In all groups, the most common topographic distribution of IL-8 was the epithelial location. As well as our results, Kebschull et al.[21] has shown that IL-8 is expressed in the periodontal pocket epithelium in both periodontal health and disease in order to maintain a continuous neutrophil migration into the sulcus/pocket. Data in the literature show that the maximal expression for IL-8 was found in the epithelial regions with massive polymorphonuclear infiltration.[10]

In moderate and severe non-specific chronic gingivitis, the presence of reactivity in the stromal inflammatory cells was also observed. The level of this cytokine did not increase with the severity of clinical diagnoses; this may be explained by the fact that these classifications do not reflect active periodontal disease.

The histopathologic, histomorphometric, and immun-ohistochemical studies of gingival tissue biopsies from patients with periodontal disease showed changes that have been related to the degree of gingival inflammation. There is a no clear relationship between the clinical diagnoses and the degree of gingival inflammation. These results may be linked to the fact that sampling was conducted during basic therapy and not in the pre-treatment, in addition to the fact that the clinical parameters used for classifying patients do not reflect an active disease.