Isolation of human salivary polymorphonuclear leukocytes and their stimulation-coupled responses

Abstract

A simple method was developed to isolate viable human salivary polymorphonuclear leukocytes (SPMN) from the oral cavity, and stimulation-coupled responses of these cells were examined. From morphological characteristics and the presence of neutrophil-specific annexin protein (39-kDa protein), we found that these cells seemed to be very similar to human peripheral polymorphonuclear leukocytes (PPMN), although they were in rather young stages. Stimulation-coupled responses of these cells were observed in terms of superoxide (O 2 ⨪) generation, luminol chemiluminescence response (LCL), membrane depolarization, and changes in intracellular calcium ion concentration ([Ca 2+] i). The rates of superoxide generation by various stimuli, such as formylmethionylleucylphenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) were different. Superoxide generation and strong chemiluminescence response were observed without addition of any stimuli. This endogenous LCL was inhibited by azide and superoxide dismutase (SOD), but not by uric acid (UA). The intensity of the endogenous LCL decreased with time after isolation from the oral cavity. This decrease was accompanied by the appearance of a FMLP-coupled response. Furthermore, the endogenous activity which produced active oxygen species was maintained in the medium at 4 °C for a long period after isolation. From these results, it is suggested that SPMN have the ability to show characteristic responses to various stimuli, and that SPMN play important roles in the defense mechanisms in the oral cavity.