Organophosphorus pesticides markedly inhibit the activities of natural killer, cytotoxic T lymphocyte and lymphokine-activated killer: a proposed inhibiting mechanism via granzyme inhibition

Abstract

We have previously found that diisopropyl methylphosphonate, an organophosphorus by-product generated during sarin synthesis in the Tokyo sarin disaster, significantly inhibited natural killer (NK) and cytotoxic T lymphocyte (CTL) activities. In the present study, to investigate whether organophosphorus pesticides (OPs) also affect NK and CTL activities, we firstly examined the effect of five OPs on human NK activity, and then the effect of Dimethyl 2,2-dichlorovinyl phosphate (DDVP), an OP on murine splenic NK, CTL and lymphokine-activated killer (LAK), and human LAK activities in vitro. To explore the underlying mechanism of decreased NK activity, we also investigated the effect of 4-(2-aminoethyl) benzenesulfonyl fluoride-HCl (p-ABSF), an inhibitor of serine proteases on NK, LAK and CTL activities, and the effect of DDVP on the activity of granzymes (serine proteases). We found that OPs significantly decreased human NK activity in a dose-dependent manner, but the degree of decrease in NK activity differed among the OPs investigated, and that DDVP significantly decreased NK, LAK and CTL activities in a dose-dependent manner, but the degree of decrease in these activities differed. p-ABSF showed a similar inhibitory pattern to DDVP, and had an additive inhibitory effect with DDVP on NK, LAK and CTL activities. We also found that DDVP significantly inhibited granzyme activity in a dose-dependent manner. These findings indicate that OPs significantly decrease NK, LAK and CTL activities in vitro via granzyme inhibition.

Introduction

Natural killer (NK), lymphokine-activated killer (LAK) and cytotoxic T lymphocyte (CTL) induce tumor or virus infected target cells death by two main mechanisms (Mori et al., 1997, Rodella et al., 1998, Sayers et al., 1998). The first mechanism is direct release of cytolytic granules that contain the pore-forming protein perforin, several serine proteases termed granzymes (Smyth and Trapani, 1995), and granulysin (Gamen et al., 1998) by exocytosis to kill target cells. The second mechanism is via the Fas ligand (Fas-L)/Fas pathway (Rodella et al., 1998, Sayers et al., 1998). Human NK, LAK and CTL have so far been demonstrated to express five granzymes. Granzyme A (GrA) and 3/K have a trypsin-like specificity, which cleave on the carboxyl side of basic residues such as arginine and lysine (Beresford et al., 1997, Sayers et al., 1996). GrB cleaves on the carboxyl side of aspartic acid residues (Harris et al., 1998), GrH prefers cleavage on the carboxyl side of hydrophobic residues such as phenylalanine (Edwards et al., 1999), and GrM cleaves on the carboxyl side of methionine, leucine or norleucine (Sayers et al., 2001).

During the course of immunotoxicologic investigations into several organophosphorus compounds, we found that diisopropyl methylphosphonate (DIMP) and diethyl methylphosphonate (DEMP), the by-products generated during sarin synthesis in the Tokyo sarin disaster (Minami et al., 1997a), significantly inhibited human and murine NK, and murine CTL activities in vitro (Li et al., 2000a). DIMP and DEMP have also been shown to be potent inhibitors of serine esterases, such as acetylcholinesterase and serum cholinesterase (Minami et al., 1997b). Thus, we speculate that the decrease in NK and CTL activities by DIMP and DEMP may be via the inhibition of serine proteases (granzymes), which are released from NK and CTL granules by exocytosis when target cells conjugate with the effector cells (Smyth and Trapani, 1995). To clarify whether organophosphorus pesticides (OPs) also affected NK and CTL activities like DIMP and DEMP, and the underlying mechanism, we examined the effect of several OPs on NK, LAK and CTL activities in vitro. We also investigated the effect of OPs on the enzyme activities of granzymes (A, B, 3, H and M) to clarify the inhibition mechanism of NK activity.