ICAM-1-expressing pocket epithelium, LFA-1-expressing T cells in gingival tissue and gingival crevicular fluid as features characterizing inflammatory cell invasion and exudation in adult periodontitis

Activated T lymphocytes constitute a major component of inflammatory cells in the early periodontal lesion, and also appear in the gingival crevicular fluid. In an attempt to clarify the relationship between the ICAM-1 (CD54) expression of pocket epithelium in gingiva and the infiltrating lymphocyte population, we carried out an analysis of CD11a⁺ (LFA-lα), CD25⁺ (IL-2Rα) and CD4⁺ (Th) cells subjacent to ICAM-1-expressing pocket epithelia and CD11a⁺ CD25⁺CD4⁺ cells in gingival crevicular fluid (GCF). GCF was collected by crevicular washing from 16 patients with periodontitis (P group) and 3 subjects with healthy gingiva (H group). Peripheral blood (PB) was collected at the same time. Mononuclear cells were isolated by Ficoll-paque gradient centrifugation from GCF and PB. Monoclonal antibodies (mAb) to CD11a, CD25, and CD4 were used for three-color flow cytometry. Gingival biopsies were obtained from 7 patients in P group and 3 subjects in H group. Serial cryostat sections (6 μm in thickness) were prepared from each biopsy, on which a double staining was performed. The number of CD11a⁺ CD25⁺ CD4⁺ cells and the fluorescence intensity of FITC conjugated anti-CD 11a were significantly higher in GCF than in PB (p≤0.001 to p≤0.01). CD11a⁺ CD25⁺CD4⁺ cells were not detected in GCF in H group. The pocket epithelia expressed CD54 in P group, but not in H group. The number of CD11a⁺, CD25⁺ and CD4⁺cells infiltrating the connective tissue subjacent to the upper, middle and lower parts of the CD54 positive pocket epithelium (n=16) was 141±26, 38±13, 144±29 (cells/0.04 mm²), respectively, whereas in the CD54 negative pocket epithelium, it was (n=5) 9±2, 3±1, 8±3. In P group, the CD11a⁺CD25⁺CD4⁺cell number in GCF correlated with CD25⁺, CD11a⁺cells in the connective tissue subjacent to the CD54⁺pocket epithelium. These results indicate that expression of ICAM-1 in pocket epithelium is relevant to the migration of CD11a, CD25, CD4 positive cells in connective tissue subjacent to the pocket epithelium into the periodontal pocket. Assessing the relationship of our findings and other adhesion molecules would offer important clues to the understanding of T cell migration in affected gingiva.     

Polymorphonuclear neutrophils and their mediators in gingival tissues from generalized aggressive periodontitis.

BACKGROUND: Impaired polymorphonuclear neutrophil (PMN) functions were generally considered to be related to the onset of generalized aggressive periodontitis (GAgP). However, some research has indicated that the hyperreactivity of PMN seems to be involved in the inflammatory response of GAgP. The present study's main purpose was to provide more evidence about the role of PMN in the pathogenesis of GAgP by surveying PMN infiltration in gingiva and its relationship with the expression of their mediators including intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). The inflammatory response in GAgP was also compared with that in adult periodontitis (AP) and periodontally healthy subjects. Since these PMN mediators were reported to be produced mainly by macrophages, the association between the expression of these PMN mediators and the distribution of macrophages was also investigated. METHODS: A total of 25 gingival specimens were obtained from 10 GAgP patients, 10 AP patients, and 5 periodontally healthy subjects. Serial sections were obtained from each specimen, and the following techniques were adopted to investigate the distribution and interrelation of different cells and cytokines. Infiltration of PMN was observed by using hematoxylin and eosin staining. Distribution of the macrophages, identified as CD68+, was shown by using immunohistochemistry. Immunohistochemistry and in situ hybridization were used to detect the expression of ICAM-1, IL-8, IL-1beta, and TNF-alpha in gingival tissues. These techniques were performed in serial sections from each individual specimen. RESULTS: Large numbers of infiltrating PMNs were observed in gingiva from GAgP. In gingiva from both GAgP and AP, the strongest protein and mRNA expression of IL-8, ICAM-1, IL-1beta, and TNF-alpha were located in pocket epithelium and adjacent connective tissue with large numbers of infiltrating PMNs. In tissues without abundant PMN infiltration, the appearance of positive cells expressing IL-8, ICAM-1, IL-1beta, and TNF-alpha was scattered. CD68+ was distributed sparsely in connective tissue and was hardly seen in pocket epithelium with large numbers of PMN infiltration. The degree of leukocyte infiltration and connective tissue destruction in gingiva from GAgP patients was not distinctly different from that in gingiva from AP. The gingival specimens with heavy PMN infiltration from both GAgP and AP patients presented strong expressions of IL-1beta and TNF-alpha; showed more extensive inflammatory cell infiltration; had severe connective tissue destruction; and presented severe elongation and ulceration of pocket epithelium. In gingiva from healthy subjects, inflammation was minor with visually no PMN, CD68+, or the positive cells of IL-8, ICAM-1, IL-1beta and TNF-alpha expression. CONCLUSIONS: Enhanced accumulation of PMN, which is associated with the upregulation of IL-8, ICAM-1, IL-1beta, and TNF-alpha expression, relates to the severity and activity of GAgP. In addition to macrophages, PMN and/or epithelial cells might also be important sources of IL-8, IL-1beta, and TNF-alpha production in gingiva.     

Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites

Interleukin-l (IL-1) molecules, IL-lα and IL-lβ are cytokines involved in the acute-phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL-1 receptor antagonist (IL-1ra) is effective in reducing the inflammatory reactions mediated by IL-1. However, the relationship between these three naturally occurring IL-1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL-1 molecules and the clinical status of patients with different severities of periodontitis. IL-lα, IL-1β, IL-1ra and the total IL-1/IL-1ra ratio (IL-1 activity index; IL-1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non-inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL-lα, IL-1bT and IL-1ra were measured by specific non-cross-reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL-lα, IL-1β and the IL-1AI, but not total IL-1ra, were found to be correlated with alveolar bone loss score. Three IL-1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL-1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL-1 and IL-1AI are closely associated with periodontal disease severity.     

Extensive expression of TGF-b1 in chronically-inflamed periodontal tissue

The host immune response in chronic marginal periodontitis (CMP) raised against bacteria colonizing the dentogingival area is modulated by cytokines. This study examines the distribution of the transforming growth factor-beta1 containing (TGF-beta1+) cells in formalin-fixed and paraffin-embedded gingival specimens from 11 patients with chronic marginal periodontitis and 7 persons with healthy gingiva. Inflamed periodontal tissue contained a 100-fold more TGF-beta1+ cells than healthy gingiva. Diverse morphological TGF-beta1+ cell types were discerned. Double immuno-enzymatic and -fluorescence staining revealed that TGF-beta1+ cells comprised 21-29% macrophages 2-3% T-cells, 3-9% B-cells, 34-35% neutrophilic granulocytes and 7-10% mast cells. The densities of all TGF-beta1+ cell types in CMP were strongly increased in the connective tissue adjacent to the pocket epithelium, in the lamina propria and adjacent to the oral epithelium. In lesions with extensive inflammation, expression was also marked in pocket epithelium. TGF-beta1 is an immunosuppressive cytokine that stimulates wound healing. Upregulation of the cytokine in inflamed gingiva may counterbalance for destructive gingival inflammatory responses that are simultaneously taking place in patients with CMP.     

A histochemical study of glycosidases in hydantoin induced hyperplastic, healthy and inflamed human gingiva

Fresh samples of hydantoin induced hyperplastic, healthy and inflamed human gingiva were studied histochemically using various azo dyes for β-glucuronidase (EC 3.2.1.31), for β- and β-glucosidase (EC 3.2.1.20 and 3.2.1.21) as well as β-galactosidase (EC 3.2.1.23) and for N-acetylglucosaminidase (EC 3.2.1.30), in order to add support to the hypothesis that hydantoin induced hyperplasia is always connected with inflammation.
Moderate β-glucuronidase activity was observed in healthy, inflamed and hydantoinhyperplastic gingiva. The distribution of the enzyme activity was similar in all types of the tissue except the stratum corneum. The healthy gingiva did not reveal this activity whereas the inflamed and hydantoinhyperplastic gingiva did. The stratum basale and spinosum of the epithelium, the fibroblasts and the inflammatory cells, especially the macrophages, revealed enzyme activity in all types of tissues. In the healthy tissue only a few inflammatory cells were seen and thus the β-glucuronidase activity was low when compared to inflamed or hyperplastic gingiva.
Weak β-galactosidase, N-asetylglucosaminidase and β-glucosidase activity was seen in all types of gingival samples. Enzyme activity was observed in the same structures as β-glucuronidase with the exception of the stratum corneum, which revealed no activity.
The relatively strong β-glucuronidase activity in the keratinized cell layer of the epithelium of inflamed and hydantoinhyperplastic tissue may be due to the microbial enzyme diffusion into the keratinized cell layer of the injured tissue.     

Localization of mRNA for inflammatory cytokines in radicular cyst tissue by in situ hybridization, and induction of inflammatory cytokines by human gingival fibroblasts in response to radicular cyst contents

The expression of mRNA encoding the inflammatory cytokines interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) have been examined in radicular cysts by in situ hybridization. Furthermore, the biological activity of the contents of radicular cysts (RCC) has been assayed by adding extracts of RCC to cultured human gingival fibroblasts (HGFs) and analyzing the culture medium for the release of inflammatory cytokines. In the epithelial layer, keratinocytes expressed all cytokine mRNAs examined at various levels. Basal layer cells expressed mRNA for each cytokine. In the subepithelial granulation tissue of the cysts, fibroblasts and macrophages expressed mRNA for IL-6, IL-8, IL-1β and TNF-α mRNA at varying levels; especially clear expression of TNF-α and IL-1β mRNA was detected on macrophages. The infiltrating lymphoid cells, largely composed of T cells and plasma cells, expressed these cytokine mRNAs, especially those encoding IL-6 and IL-8, at various levels. In vitro analysis indicated dose-dependent release of both IL-6 and IL-8 by HGFs in response to RCC. After heating to 100°C for 10 min, RCC almost completely failed to stimulate IL-6 release from HGFs. Furthermore, anti-IL-1β antibody (neutralization test) did not prevent the stimulation of IL-6 release by RCC. Significant amounts of IL-6 and IL-8 were detected in RCC in two cases, and a trace amount of IL-1β was detected in one case. This study demonstrated the wide expression of mRNA encoding inflammatory cytokines in radicular cyst tissues, and RCC itself was capable of stimulating 1L-6 and 1L-8 production from HGFs.     

The gingival crevicular fluid Interleukin-1β and tumour necrosis factor-α levels in patients with rapidly progressive periodontitis

Cytokines are believed to play an important role in the pathogenesis of periodontal diseases. In the present study, gingival crevicular fluid (GCF) levels of two important cytokines, interleukin 1-/3 (IL-1β) and tumour necrosis factor-α (TNF-α) and, in addition, serum IL-1β levels, were determined in patients with severe and rapid periodontal breakdown by use of ELISA. While IL-1β was detected in all of the GCF samples studied, TNF-α could only be detected in about half the samples. The mean GCF IL-1β level was 38.45 ± 13.99 pg/mL, and the mean TNF-α level was 3.20 ± 1.39 pg/mL, respectively. The GCF IL-1β levels also presented a strong positive correlation with the mean pocket depths. Although weak, both of the cytokines also presented correlations with the presence of bleeding on probing. Additionally GCF samples contained increased IL-1β levels when compared with the serum samples suggesting local production mechanisms. The findings of the present study suggest that these cytokines may be involved in the pathogenesis of periodontal diseases (IL-1β being more significant), and also may help in defining the active phase of periodontal breakdown.     

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin-labeled cRNA probes (386 and 496 bp). TIMP-1 and -2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP-1 and -2 mRNA-expressing cells showed significantly different localization. TIMP-1 mRNA was broadly observed in the gingival connective tissue while TIMP-2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP-1 and TIMP-2 mRNA was detected differentially and site-specifically in periodontal diseased gingival tissue.     

纤维调节素在大鼠和人类牙龈组织中的差异性表达

目的:研究纤维调节素在大鼠和人类健康和牙龈组织中的分布与表达。方法:用免疫组织化学的方法研究纤维调节素在大鼠牙龈及人类健康和炎性牙龈组织中的分布。与之比较,I型胶原纤维在鼠磨牙牙龈组织中的分布也做了研究。结果:在大鼠牙龈中,纤维调节素分布在牙龈上皮基底上层,在腭侧牙龈结缔组织有强表达。I型胶原纤维也有类似的分布。比较而言,纤维调节素在颊侧牙龈结缔组织表达较弱。对于人类,纤维调节素在炎性牙龈结缔组织中表达明显,然而在健康牙龈结缔组织中表达较弱。结论:与Ⅰ型胶原纤维关系密切的纤维调节素在颊侧和腭侧牙龈中有差异性表达,并且在人类炎性牙龈组织中表达增强。     

Immunohistochemical demonstration of the plasminogen activator system in human gingival tissues and gingival fibroblasts

The relative distribution of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) was studied in cultured human gingival fibroblasts, healthy gingival tissues and inflamed gingival tissues by immunohistochemistry. In cultured gingival fibroblasts t-PA, u-PA and PAI-1 were expressed in cytoplasm; u-PA and PAI-1 were more intensely stained than t-PA; PAI-2 was not detectable in gingival fibroblasts. Following interleukin 1β (IL-1β) stimulation, the intensity of intracellular staining for t-PA was increased and a number of cells staining strongly for PAI-2 were seen; no difference in the intensity of immunostaining level was noted for the expression of u-PA and PAI-1 between IL-1β stimulated cells and unstimulated cells. In healthy gingival tissues, u-PA and PAI-1 displayed a wide distribution throughout all the connective tissue and epithelium; t-PA localized mainly in the connective tissue while PAI-2 showed little association with the connective tissue but did faintly stain in the epithelial layer. In inflamed gingival tissues, staining for t-PA was significantly increased in the extracellular matrix of the connective tissue, whereas staining for u-PA, PAT-I and PAI-2 was found to be slightly increased, but no significant difference was noted for staining when compared with the healthy gingival tissues. A granular distribution of t-PA, u-PA, PAI-1 and PAI-2 was noted around areas of inflammatory cell infiltration. These immunohistochemical findings indicate that the plasminogen activator system produced by fibroblasts may be influenced by the presence of the inflammatory mediator IL-1β. In addition, the significant increase of t-PA in inflamed connective tissue and the wide expression of these components around inflamed cells may contribute to connective tissue degradation and may relate to the migration and localization of monocytes/macrophages in inflamed tissue.     

Disturbances of gingival fibroblast population homeostasis due to experimentally induced inflammation in the Cynomolgus monkey (Macaca fascicularis): potential mechanism of disease progression

We have studied the relationship between perturbations of fibroblast turnover in inflamed gingiva of different severities. To perform detailed spatial analyses of gingival fibroblast progenitor cells, inflammatory cell infiltrates and blood vessels, 3 Cynomolgus monkeys with healthy periodontium and 2 with naturally occurring gingivitis and ligature-induced periodontitis were pulse-labeled with ³H-thymidine. Morphometric analyses of radioautographs from mid-sagittal supra-alveolar gingival connective tissues of incisors were performed in sites subjacent to junctional, sulcular and oral epithelium, in the body of the lamina propria and just superior to the alveolar crest. The percentage of fibroblasts incorporating ³H-thymidine label, expressed as the labeling index (LI), was higher subjacent to the sulcular epithelium in periodontitis (1.73±0.37) than in healthy sites (1.06±0.22). This was not statistically significant (0.05 < p < 0.1) due to the small number of animals used. The sites subjacent to the sulcular epithelium also exhibited the largest increase in lymphocyte density from health to gingivitis (p < 0.01). In contrast, the LI of fibroblasts subjacent to the oral epithelium was 5-fold higher in healthy (0.82±0.17) compared to periodontitis sites (0.13 ± 0.09; p < 0.05). Labeled fibroblasts were found close to blood vessels in all compartments and in all disease states; distance to blood vessels was reduced in inflamed sites (p < 0.10). There were increased numbers of blood vessels per unit area in the lamina propria of gingivitis compared to healthy sites. However, there were no regional differences with respect to blood vessel numbers or area in sites subjacent to junctional epithelium with different disease states. The results indicate that: 1) experimentally-induced inflammation in the gingiva of Cynomolgus monkeys is associated with site-specific perturbations of cell turnover; 2) fibroblast progenitors are preferentially situated adjacent to blood vessels as in the periodontal ligament; 3) the vascular response to inflammation is a generalized increase in blood vessel numbers, but not their size; 4) reactive proliferation of fibroblasts may compensate for cell death in the lamina propria but is not detectable at the site of connective tissue attachment loss subjacent to the junctional epithelium. Failure to maintain the fibroblast progenitor population may be an important component of attachment loss in progressive periodontitis lesions.     

Immunohistochemical localization of very late activation integrins in healthy and diseased human gingiva

The β1-integrins (VLA family) are cellular adhesion molecules (CAM) that play a major role in cell-cell and cell-matrix interactions. The expression pattern of CAM was studied in 5 clinically normal volunteers with healthy gingiva and in 18 patients with clinically different stages of periodontitis. In healthy human gingiva α2. α3 and α6 integrin chains were found in a characteristic distribution, showing a broad continuous expression on the junctional and sulcular epithelium sites. The expression of these integrins was demonstrated primarily on the basal cell layers and in some cells of the stratum spinosum. Inflammatory stages of periodontitis revealed further upregulation of α2, α3 and α6 integrins into the junctional and sulcular epithelial cells, which correlated with the stage of the periodontitis and the extent of the cellular infiltration. α4 and α6 were found to be the predominant β1 integrin chains on inflammatory cells. The amount of δ4 and ş6 positive infiltrative cells increased with the number of inflammatory cells. VCAM-1. the corresponding cell-cell ligand of VLA-4 (α4) was present on the majority of subepithelial vessels in all stages of gingivitis and periodontitis. The α5 subunit was expressed on both endothelium and gingival connective tissue cells. Samples from advanced periodontitis cases showed a higher number of a5 positive mononuclear cells. In comparison to normal epidermis, a human gingival epithelial cells express higher levels of integrins. This expression is further upregulated in advanced stages of periodontitis, indicating changes of the β1 integrin organization.     

Endothelial cell leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression in gingival tissue during health and experimentally-induced gingivitis

The changes in vascular adhesion molecule expression and numbers of infiltrating leukocytes during a 21-day experimental gingivitis episode were investigated immunohistochemically. Monoclonal antibodies to ELAM-1 (1.2B6), ICAM-1 (6.5B5), CD3 (OKT3-pan-T cell) and neutrophils (PMN-elastase) were used to identify positive vessels and leukocytes within gingival biopsies taken on d 0, 7, 14 and 21. Vascular endothelium expressed ELAM-1 and ICAM-1 both in clinically 'healthy' tissue (d 0) and in experimentally inflamed tissue (d 7 to 21). Positive vessels were found mainly in the connective tissue subjacent to the junctional epithelium where the highest numbers of T cells and neutrophils were also seen. Although T cells were found in all tissue areas studied, neutrophils were largely concentrated in the junctional epithelium and the subjacent connective tissue but were absent from the oral epithelial region. As the experimental gingivitis developed, the number of T cells or neutrophils in the different tissue regions did not change significantly although the most intense vascular ICAM-1 and ELAM-1 staining redistributed to the CT adjacent to the junctional epithelium. A prominent feature was the intense ICAM-1 positive staining of the junctional epithelium and its absence in the closely adjacent oral epithelium, in both clinically 'healthy' and inflamed tissue. The gradient of ICAM-1 in junctional epithelium, with the strongest staining on the crevicular aspect plus the vascular expression of ELAM-1 and ICAM-1 in both clinically 'healthy' and inflamed tissue may be crucial processes which direct leukocyte migration towards the gingival crevice.     

The use of extracted teeth to evaluate clinical measurements of periodontal disease.

Clinical indicators of periodontal disease, Gingivitis Index, Gingival crevicular fluid and pocket depth measurements were obtained from the gingiva surfaces of 30 teeth. The gingival margins were marked on the surfaces of the teeth prior to extraction. The extracted teeth were stained with hematoxylin and air dried, and the distances from the groove to the base of the calculus, plaque, and connective tissue attachment were obtained. The plaque-free zone was also measured. Comparisons were made between clinical and tooth surface measurements. A high correlation was found between clinical pocket depth measurements and tooth surface parameters. The correlations between all tooth surface parameters and GCF were statistically significant. The G.I. was significantly correlated only with the penetration of calculus into the pocket. The clinical pocket depth was statistically the same as the distance from the gingival groove to the coronal connective tissue attachment. The plaque-free zone appeared to represent the junctional epithelium.     

lnterleukin-1β and phenytoin reduce α1 (1) procollagen mRNA expression in human gingival fibroblasts

Effects of and interactions between interleukin-1β (IL-1 β) and phenytoin (PHT) on α1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E₂ (PGE₂) formation were studied. IL-1β (300 pg/ ml) reduced the steady-state level of αl(I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 μg/ml) reduced the level of α1(I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1β, PHT potentiated the inhibitory effect of IL-1β on αl(I) procollagen mRNA level that was accompanied by an increased PGE₂ formation. Preincubation with indomethacin (10^-6m) partially reduced the inhibitory effect of IL-1β as well as of IL-1β in combination with PHT on the mRNA level of αl(I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE₂ (≥10 nm) dose-dependently reduced steady-state level of α1(I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of αl(I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces α1(I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.     

The gingival plasma cell infiltrate in HIV-positive patients with periodontitis is disorganized

Patients infected with the human immunodeficiency virus (HIV) are highly susceptible to chronic marginal periodontitis (CMP) and the lesion is generally characterized by abundant plasma cell infiltration. HIV-induced reduction of CD4+ T cells may indirectly affect local production of immunoglobulins (Ig). Gingival biopsies taken from 10 HIV+ and 12 HIV- control patients with CMP were washed, fixed in ethanol and embedded in paraffin. Sections were examined after immunohistochemical staining with monoclonal antibodies against IgA, IgA1-2, IgG, IgG1-4, IgM and IgE. Ig-containing cells were counted in 3 separate connective tissue zones (subjacent to pocket epithelium, central zone and subjacent to oral epithelium). HIV+ patients showed a remarkably increased density of all Ig-containing cells in the connective tissue zone subjacent to the oral epithelium (p<0.05) and a lower % of IgG2+ cells in the entire gingival section (p<0.05). In HIV+ patients, the density of IgG-containing cells in the gingiva was strongly correlated with the serum IgG concentration. The altered topical distribution might imply impaired restriction of the inflammatory lesion, additional antigenic challenges by unusual microorganisms in the oral cavity, or be secondary to HIV-induced dysregulation of the B-cell system.     

Immunohistochemical localization of Toll-like receptors 2 and 4 in gingival tissue from patients with periodontitis

The present study investigated the expression of Toll-like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2-, TLR4-, CD14- and CD1a-positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2-positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4-positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14-positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down-regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.