Inflammatory cell expression of Toll-like receptor-2 (TLR2) within refractory periapical granuloma

Abstract

Background: Toll-like receptor-2 (TLR2) is highly important within the immune system. Characterization of the expression of TLR2 within inflammatory cells in periapical lesions could help in diagnosis and management of refractory cases. The aim of the study is identification of Toll-like receptor (TLR2) through immunohistochemical and immunofluroscence expression in inflammatory cells within refractory periapical granuloma cases. Methods: Eight cases of refractory periapical granuloma were selected out of 772 cases. Histological examination and immunohistochemical staining with polyclonal rabbit antihuman TLR2, monoclonal mouse antihuman CD38, CD68 and CD83 primary antibodies, as well as immunofluorescence staining with goat anti-rabbit TLR2, donkey anti-mouse CD38, CD68 and CD83 primary antibodies was conducted. Positive controls, negative controls and experimental sections with no primary antibody were included in the study. Qualitative analysis and double immunofluorescence technique was used to characterize the TLR ⁺ cells. Results: In periapical granuloma, lymphocytes (CD38 cells) expressed the most amount of TLR reactivity followed by macrophages (CD68 cells), and odontogenic epithelial cells. Neutrophils, red blood cells (RBCs) and collagen ground substance were negative to TLR2. Conclusion: TLR2 was highly expressed by lymphocytes and plasma cells indicative of their major role in the inflammatory process and antigen recognition in refractory periapical granuloma. Dendritic cells expressing TLR2 were low in number suggesting a minor role in sustaining these lesions.

Keywords: Antigen presenting cells; chronic inflammatory cells; immune response; periapical granuloma; toll-like receptors..

Figure 1.

( a) A histopathology section of a selected refractory periapical granuloma showing areas of fibrous connective tissue (F), blood vessels, inflammatory cells (I) and interspersed odontogenic epithelium (Haematoxylin & Eosin staining x50), ( b) Proliferating epithelial cells (E) surrounded by chronic inflammatory cells (I) (Haematoxylin & Eosin staining x200), ( c) Fibrous connective tissue (F) with moderate chronic inflammatory cell infiltrate (I) (Haematoxylin & Eosin staining x200), ( d) Histopathology section of a periapical scar showing the un-inflamed, relatively acellular and avascular dense collagen tissue (Haematoxylin & Eosin staining x200).

Figure 2.

( a) Representative histopathology section of positive control (lingual tonsil) showing CD38+ cells (black arrows) in the germinal centres (CD38 Immunohistochemistry x400), ( b) Representative histopathology section from a periapical granuloma showing a cluster of CD38+ cells (red arrows) (CD38 Immunohistochemistry x400), ( c, d) Two images of CD38+ cells under high power magnification showing characteristic lymphocyte and plasma cell circular or oval shape with staining mainly on the cell membrane (CD38 Immunohistochemistry x1000).

Figure 3.

( a) Representative histopathology section of a mucocele showing positively stained macrophages (red arrows), cystic lining (CL) and cystic cavity (CC) (CD68 Immunohistochemistry x400), ( b) High power magnification of CD68+ cells in a mucocele histopathology section. Cytoplasmic vesicles (black arrows) and phagosomes (red arrows) are clearly demonstrated (CD68 Immunohistochemistry x1000), ( c) Representative histopathology section of a symptomatic periapical granuloma showing CD68+ cells which stained brown (black arrows) (CD68 Immunohistochemistry x400), ( d) High power magnification of the larger CD68+ cells showing spongy appearances and wavy cellular outlines which resembled macrophages. Positive immunostaining is located on the cell membrane as well as the cytoplasm. Unstained intracellular vesicles can be identified (black arrows) (CD68 Immunohistochemistry x1000), ( e) High power magnification of the smaller CD68+ cells showing a rounded cellular profile which resembled monocytes (black arrows) (CD68 Immunohistochemistry x1000).

Figure 4.

( a) Representative histopathology section of a germinal centre within lingual tonsil demonstrating CD83+ cells (black arrows) (CD83 Immunohistochemistry x400), ( b) High power magnification of a CD83+ cell showing a polygonal outline with little thorn-like or filamentous extensions (black arrows) characteristic of a dendritic cell (CD83 Immunohistochemistry x1000), ( c) Representative histopathology section from a periapical granuloma showing CD83+ cells (black arrows). Characteristically these cells were found in low numbers within a lesion (CD83 Immunohistochemistry x200), ( d, e) Two images of high power magnification of CD83+ cells in a periapical granuloma showing some examples of the variable cellular morphology they exhibited. Little thorn-like extensions can be seen on some cells (black arrows) indicative of dendritic cells (CD83 Immunohistochemistry x100).

Figure 5.

( a) Representative histopathology section of a traumatic oral ulcer showing an area of intact oral epithelium (OE) which was positively stained for TLR2. Mononuclear inflammatory cells (red arrows) in the connective tissue are also stained brown (TLR2 Immunohistochemistry x400), ( b): Representative histopathology section from a periapical granuloma showing numerous mononuclear inflammatory cells stained brown (red arrows), demonstrating TLR2 expression (TLR2 Immunohistochemistry x400), ( c) Representative histopathology section from a periapical granuloma showing TLR2+ immunostaining on numerous mononuclear inflammatory cells (red arrows) as well as interspersed odontogenic epithelium (E) (TLR2 Immunohistochemistry x400), ( d) Image of TLR2+ cells resembling immune cells of lymphoid lineage under high power magnification (red arrows). Note the cell membrane and cytoplasm are stained brown (TLR2 Immunohistochemistry x1000), ( e) High power magnification of a periapical granuloma histopathology section showing large foamy TLR2+ cells resembling macrophages (red arrows). Note that the neutrophils (black arrows), characterised by multi-lobed nuclei, were negative to TLR2 immunostaining (TLR2 Immunohistochemistry x1000).

Figure 6.

( a) Representative histopathology section of CD38 ⁺ cells in lingual tonsil tissue showing collections of red fluorescing cells (R) separated by areas of CD38 ^- cells that had no red fluorescence (N). RBCs had faint purple autoflorescence (white arrows) (CD38 Immunofluroscence x400). ( b) High power magnification of CD38 ⁺ cells in lingual tonsil tissue. These cells have a circular or oval profile with a narrow space of cytoplasm surrounding the nucleus, consistent with being lymphocytes. The red fluorescence is located on the cell membrane whereas the cytoplasm was negative. These cells are closely associated to one another. This image also shows a blood vessel containing RBCs which glowed in faint purple (white arrow) (CD38 Immunofluroscence x1000). ( c) Representative section of negative antibody control. ( d) Representative section of negative tissue control (periapical scar) (x400). Note the red fluorescing cells represent RBCs (white arrows) which exhibit autofluorescence and were scattered in the tissue or within the blood vessels.

Figure 7.

( a) Representative histopathology section showing CD68 ⁺ cells in a mucocele fluorescing in red. They are found in large number adjacent to the cystic lining (CC) and spread into the cystic cavity (CC). Characteristics of macrophage cells are evident e.g. irregular cell shape, intra-cellular vacuoles (x400). ( b) High power magnification of CD68 ⁺ cells in a mucocele showing their large spongy globular appearance. The red fluorescence is observed on the cell membrane and cytoplasm but not the intracellular vesicles or phagosomes (x1000). ( c) Representative section of negative antibody control. ( d) Representative section of negative tissue control (periapical scar) with autofluorescing RBCs (white arrows) (x400).

Figure 8.. High power magnification of CD83+ cells found in the lingual tonsil tissue.

The CD83 ⁺ cells exhibit several cellular shapes: circular, oval, teardrop and asymmetric polygons. Occasionally, little thorn-like projections could be detected on cell surfaces (white arrows). These characteristics are typical of dendritic cells (x1000).

Figure 9.. High power magnification images of a periapical granuloma captured under different emitting fluorescence.

Image A shows CD38+ cells with red fluorescing cell membrane and unstained cytoplasm. Image B shows TLR2+ cells displayed green fluorescence on the cell membrane as well as on the cytoplasm. The superimposed image C reveals CD38+/TLR2+ cells which have orange-red cell membrane and green cytoplasm (white arrows) (CD38/TLR2 Double Immunofluorescence x1000).

Figure 10.. Representative histopathology section of the same area in a periapical granuloma showing images captured under different emitting fluorescence’s.

Image A shows CD68+ cells fluorescing in red and B shows TLR2+ cells fluorescing in green. The superimposed image C reveals the CD68+/TLR2+ cells (white arrows) (CD68/TLR2 Double Immunofluorescence x400).

Figure 11.. High power magnification images of a periapical granuloma captured under individual emitting fluorescence.

Image A shows CD68+ cells fluorescing in red. Notice the cell membrane and cytoplasm are stained but not intracellular vesicles. Image B shows TLR2+ cells fluorescing in green. Notice the multi-lobular nuclear cells resembling neutrophils were negative to TLR2 staining (white arrows). The superimposed image C reveals CD68+/TLR2+ cells which have cell membrane and cytoplasm fluorescing in orange or a mixture of red and green speckles (white arrows) (CD68/TLR2 Double Immunofluorescence x1000).

Figure 12.. Representative histopathology section of the same area in a periapical granuloma showing images captured under individual fluorescence.

Image A shows CD83+ cells fluorescing in red. Notice the cells have variable outlines and cytoplasmic extension. They contributed as a minor cell population in the periapical granuloma. Image B shows TLR2+ cells fluorescing in green. The superimposed image C reveals the CD83+/TLR2+ cells in orange-red fluorescence (white arrows) (CD83/TLR2 Double Immunofluorescence x400).

Figure 13.. High power magnification images of an area in periapical granuloma captured under individual fluorescence.

Image A shows CD83+ cells fluorescing in red. Notice the cell membrane, cytoplasm and cytoplasmic extension (white arrows) are stained. Image B shows TLR2+ cells fluorescing in green. The superimposed image C reveals CD83+/TLR2+ cells (white arrows) which have cell membrane and cytoplasm fluorescing in orange or a mixture of red and green speckles (CD68/TLR2 Double Immunofluorescence x1000).