CD4 cytotoxic and dendritic cells in the immunopathologic lesion of Sjögren's syndrome

Abstract

The existence of CD4+ T lymphocytes with cytotoxic activity in minor salivary gland (MSG) biopsies from Sjögren's syndrome (SS) patients was investigated using in situ double immunohistochemistry technique. The presence of dendritic cells (DC) in SS lesions was examined by using single and double immunohistochemistry methods and a panel of different MoAbs to specific cell surface markers (i.e. CD3, CD11c, DRC). Furthermore, the ultrastructural morphology of DC was characterized by electron microscopy (EM). Immunogold labelling technique using the DRC surface marker was also applied. Finally, we investigated the existence of germinal centres (GC) in the salivary gland lesions of SS patients. Seven patients with primary SS and five patients with non-specific sialadenitis were the subjects of this study. Our results indicate the existence of a CD4+ cytotoxic cell population that utilizes perforin-mediated cell destructions as they expressed perforin mRNA. Quantitative analysis of these cells revealed that they comprised approximately 20% of the existing T lymphocytes. We also identified a population of CD4+ T cells that expressed the CD11c activation marker. Furthermore, we observed a distinct cell subtype which expressed the DRC cell surface marker. These cells had the characteristic ultrastructural morphology of DC and were DRC+ when examined by immunoelectron microscopy. Finally, the formation of GC structures in the histopathologic lesions of the salivary glands was observed. The above findings indicate that both CD4+ cytotoxic T lymphocytes (CTL) and DC may be involved in the initiation and perpetuation of SS pathogenesis. Moreover, the formation of GC in the lesions reveals a possible mechanism for in situ differentiation and proliferation of activated B lymphocytes.

Fig. 1

(a) In situ double immunohistochemistry technique on a cryostat section from a minor salivary gland biopsy of a Sjögren's syndrome (SS) patient using the anti-sense probe of perforin mRNA. CD4⁺ T lymphocytes (red colour) which express perforin mRNA (purple colour) were observed (arrows) (× 400). No counterstain was applied on sections. (b) In situ double immunohistochemistry technique using the sense probe of perforin mRNA. No background staining was detected and only CD4⁺ T lymphocytes (red colour) were stained (× 400).

Fig. 2

Serial cryostat section from a minor salivary gland biopsy (MSG) of a Sjögren's syndrome (SS) patient using the anti-CD11c (a) and anti-CD4 MoAbs (b). A large population of CD4⁺ T lymphocytes expressed the activation marker CD11c (× 200).

Fig. 3

(a) Double immunohistochemical staining showing CD4⁺ T lymphocytes (brown colour) and DRC⁺ cells (arrows) (red colour). DRC⁺ cells were located in periacinar and periductal areas near a CD4⁺ large lymphocytic infiltration (× 200). (b) Minor salivary gland biopsy from a patient with non-specific sialadenitis stained with anti-DRC MoAb. No staining was identified on the existing mononuclear cells (× 200).

Fig. 4

(a) Transmission electron micrograph of a dendritic cell (DC) with characteristic cytoplasmic processes (arrow), lobulated nucleus with perinuclear condensed heterochromatin and multivesicular bodies (arrowheads) in the cytoplasm. Few cytoplasmic organelles were observed inside the DC (× 35 000). (b) Transmission electron micrograph showing a DC in close contact with a lymphocyte (LC). Desmosomes (arrow) were identified between interacting cells (× 46 000). (c) Immunogold labelling of a DC with the anti-DRC MoAb with no counterstain. Gold particles (silver enhanced) can be observed on the cell membrane (arrows) (× 18 200).

Fig. 5

(a) The classical dark (arrow) and light zones of a mature germinal centre (GC) in a minor salivary gland (MSG) biopsy from a Sjögren's syndrome (SS) patient identified with the anti-Ki-67 MoAb (× 100). (b) Serial section stained with the anti-CD3 MoAb showing T cells located inside the light zone and in the outer zone of a GC. The observed T lymphocytes were Ki-67⁻ and represent non-dividing cells (× 100).