α4β1 integrin-dependent cell sorting dictates T-cell recruitment in oral submucous fibrosis

Abstract

Aim: The biological mechanism(s) that guide the immunological effectors of lymphocytes to sites of inflammatory response, a feature consistently seen in oral submucous fibrosis (OSF) was evaluated. It is envisaged that endothelial/lymphocyte adhesion cascades involving VCAM-1/α4β1 integrins control the migration of lymphocytes across the vascular endothelium resulting in their homing in these locales.

Materials and methods: The study group comprised 28 OSF cases (M:F = 12:16, age range 18-65 years; mean 55.4 ± 8.5 SD) divided into early (n=17) and advanced (n=11) disease groups. Biopsy specimens of normal buccal mucosa (site compatible) were obtained from 10 healthy volunteers (age and sex matched) who served as control. All the samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Immunolocalization of β1 subunit associated with α4 integrin was performed by a mouse heterodimer (clone 4B7R, Ig G, R & D Systems Inc., dilution 1:100) using a peroxidase labeled streptavidin-biotin technique. The immunocompetent cell density was expressed as the number of positive cells per mm(2). The Mann-Whitney U-test and Fischer exact test were used to evaluate differences. P<0.05 was considered to be significant.

Results: The median percentage of "T" lymphocytes with positive integrin α4β1 expression was 77.7 (an interquartile range of 73.3-83.4) for the test cases and for the controls, it was 28.2 (IQR 24.0-38.3). This difference was significant at 0.001 level. For the endothelial cells the positive expression was 82.8 (IQR 77-90.6) and 22.3 (IQR 18.3-29.2) respectively (P<0.001). When the intensity of integrin expression was considered 26/28 cases (96%) and 2/10 (20%) of controls showed intense expression of integrins α4β1 on T lymphocytes (P<0.001). Similarly, 27/28 cases (92.9%) and 2/10 (20%) of controls showed intense expression on endothelial cells (P<0.001). T lymphocyte-endothelial cell interactions were assessed by evaluating the overexpression of integrins on both the endothelial cells and lymphocytes together. The interaction was positive in 15/17 and 11/11 early and advanced OSF cases respectively (P=0.51).

Conclusion: Following leukocyte activation, the interaction between leukocyte integrin heterodimers and endothelial superfamily adhesion ligands results in a firm adherence of leukocytes to endothelium, leading to leukocyte migration and homing to sites of mucosal inflammation consistently seen in OSF.

Keywords: Cellular homing; T cells; endothelial cells; integrins; lymphocytes; oral submucous fibrosis; α4β1.

Figure 1

Integrin α4β1expression in normal mucosa, endothelial cells and occasional lymphocytes show immunoreactivity (×40)

Figure 2

Integrin α4β1expression in early OSF. The blood vessels are dilated and congested with endothelial staining uniformly seen. The stroma is intensely inflamed and more proportion of lymphocytes showed immunoreactivity for integrins. Stromal collagenisation is evident (×40)

Figure 3

Integrin α4β1expression in advanced OSF (×40)