A New Method for Isolation of Salivary Neutrophils and Determination of Their Functional Activity

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Abstract
The purposes of this study were to develop a new method for isolating salivary neutrophils (SPMNs), and to determine their functional activity. Studies of neutrophils in the oral cavity have been largely limited to crevicular PMNs, because of difficulties in obtaining viable SPMNs free of epithelial cells. A method to obtain SPMNs is presented. Donors performed rapid sequential rinsings by placing in their mouths 15 mL of Hanks' balanced salt solution [free of calcium or magnesium ions (HBSS-CMF)], which contained 0.1% gelatin, then swishing the solution for 30 s, and expectorating into a polypropylene receptacle containing 400 mL 4°C HBSS-CMF. This sequence was repeated for 20 min. The collected solution was stirred for 10 min, the cells were washed, and the re-suspended pellet was passed sequentially through a 20-μm and a 10-μm nylon mesh. The cells consisted of 97.7 ± 1.7% SPMNs, only 2.3% epithelial cells, and were almost free of oral debris. The SPMNs were studied for CDllb expression, H2O2 production, and F-actin polymerization. SPMNs had significantly higher resting values for CDIIb, H2O 2 production, and F-actin polymerization compared with blood neutrophils (BPMNs). SPMNs responded to stimulation by chemotactic peptide or phorbol ester in a dose-dependent fashion, with levels of CDllb, H2O 2, and F-actin comparable with BPMNs at optimal stimulant concentrations. The elevated resting levels of CD11b, H2O2, and F-actin for SPMNs were probably caused by exposure to gingival and oral bacteria. The ability of in vitro-stimulated SPMNs to increase their levels of CDllb, H2O2, and F-actin to levels comparable with BPMNs indicates that these cells can function after leaving the gingival crevices. The method described is relatively simple and provides a high number (4-30 × 10 6) of purified and functional SPMNs.