4b and c). These results suggest that A. actinomycetemcomitans-expressed leukotoxin induced the release Volasertib price of resistin by degranulation of the neutrophils before cytolysis. To examine the possible involvement of LFA-1, which is the receptor for leukotoxin, and an Src family

tyrosine kinase in the release of resistin and elastase, we pretreated neutrophils with monoclonal TS1/22 (CD11a) or TS1/18 (CD18) antibody against LFA-1 subunits at a final concentration of 5 μg mL−1 and with 10 μM PP1 for 15 min, followed by incubation with wild-type or mutant HK921 for 2 h (Fig. 5a and b). In contrast to pretreatment with TS1/22, the level of resistin released from neutrophils pretreated with TS1/18 or PP1 was significantly lower than that from untreated neutrophils after incubation with wild-type HK921,

as was the release of elastase. Additionally, the inhibitory effect of pretreatment with TS1/18 or PP1 on the levels of resistin and elastase released from neutrophils after incubation with mutant HK921 for 2 h was lower than those with wild-type HK921, but significant (P<0.05) (Fig. 5a and b). Among the several virulence factors expressed by A. actinomycetemcomitans, leukotoxin is thought to play a major role in disease progression (Henderson et al., 2003). Leukotoxin has been reported to activate cytolysis of human leukocytes, including neutrophils and monocytes CX-5461 manufacturer (Taichman et al., 1980), and to induce caspase-1 activation and bioactive IL-1β secretion in human macrophages (Kelk et al., 2003). In the present study,

significantly more resistin was released from neutrophils incubated with wild-type HK921, which is characterized by a 530-bp deletion of the promoter region of the leukotoxin gene, than from neutrophils incubated with minimally leukotoxic strains. The ability of the wild-type strain to produce leukotoxin was confirmed by Western blot analysis using a leukotoxin-specific antiserum, and its cytotoxic activity against neutrophils was demonstrated by LDH release. Furthermore, the mutant strain, medroxyprogesterone which is incapable of producing leukotoxin, released a significantly lower level of resistin (P<0.05). Whereas resistin is derived exclusively from adipose tissue in mice, leukocytes are the major source of resistin in humans. Neutrophils store abundant amounts of resistin in their granules, and the extracellular release of resistin via degranulation may be stimulated by bacterial or inflammatory stimuli (Bostrom et al., 2009; Johansson et al., 2009; Kunnari et al., 2009). This study demonstrated that the release of resistin from neutrophils in the presence of a highly leukotoxic strain, which is strongly associated with aggressive periodontitis in certain susceptible human populations (Haubek et al., 2008), was significantly higher than in the presence of a leukotoxin-deficient isogenic mutant (P<0.05).