Flow cytometric detection of salivary mononuclear phagocytes: a new window on leucocyte extravasation

S.Z. Rajbally, P.J. Bergin and N.J. Goulding. William Harvey Research Institute, Queen Mary’s School of Medicine & Dentistry, Charterhouse Square, London, UK

Polymorphonuclear leucocytes (PMN) in saliva are a major contributor to innate defence against microbial infection. Emanating from the gingival crevice, significant numbers of PMN are found in the saliva of healthy individuals. Thus far there has been no definitive study of the existence of salivary mononuclear phagocytes (MNP). We therefore investigated the prevalence of MNP, lymphocytes and PMN in saliva as a possible tool for the non-invasive study of leucocyte extravasation in humans.
Twelve healthy volunteers (6 male, 6 female; mean age 35, range 24-58) were recruited to this study with informed consent. Individuals had no evidence of either overt periodontal disease or infection. Subjects provided 6 samples of saliva by rinsing with 20ml buffered saline over 10 min. Venous blood was also collected and mononuclear leucocytes and PMN isolated by density-gradient centrifugation over ficoll-hypaque. Saliva samples were pooled and leucocytes separated by centrifugation with 2 washing steps in RPMI1640 culture medium. Following cell counting, blood or salivary leucocytes were resuspended to 5x107 / ml in RPMI1640 medium and 1 x 106 cells plated into microtitre plates. Cells from blood or saliva were reacted on ice with monoclonal antibodies to the pan-phagocyte marker CD11b and to markers present on T lymphocytes (CD3), monocytes (CD14), macrophages (CD163) and PMN (CD16). Following wash steps, cells were reacted on ice with FITC-conjugated Fab’2 anti-mouse IgG. Stained cells were then analysed by flow cytometry with lymphocytes, monocytes, macrophages and PMN discriminated by forward / side scatter. Fluorescence was reported in mean units of fluorescence intensity (MFI).

Whilst the greatest proportion of salivary leucocytes were PMN (81 +3%), a significant percentage of MNP (12 +2%) and lymphocytes (7 +3%) were detectable based on scatter characteristics. This profile was different to that found in peripheral blood (PMN 39 +4%; MNP 10 +2%; lymphocytes 51 +5%). Surface CD11b was unchanged between blood and saliva on both MNP and PMN (Table 1). Expression of CD16, a marker of macrophage differentiation, was higher in salivary MNP compared to blood (*p<0.01, Wilcoxon paired test) but otherwise surface marker expression of salivary leucocytes was strikingly lower than blood counterparts (**p<0.01), possibly due to proteolytic cleavage during extravasation or within salivary fluid.

ND = not detectable

Salivary lymphocytes were only detectable in 4/12 individuals and meaningful comparison of CD3 expression between blood and saliva was not possible.
In conclusion, we believe this is the first flow cytometric demonstration of MNP in the saliva of healthy individuals. These cells show a surface marker profile characteristic of mature, infiltrating phagocytes, indicating that the oral cavity may provide a novel, non-invasive site for the study of extravasation of both mononuclear and polymorphonuclear.