It was recently reported that human neutrophils store abundant am

It was recently reported that human neutrophils store abundant amounts of resistin in granules, which is released extracellularly upon inflammatory stimulation by bacteria, such as Streptococcus pyogenes and Escherichia coli, or by selected bacterial components, such as streptococcal INK 128 cell line M protein and N-formyl-Met-Leu-Phe (Bostrom et al., 2009; Johansson et al., 2009; Kunnari et al., 2009). Aggregatibacter (Actinobacillus) actinomycetemcomitans, a Gram-negative facultative anaerobic coccobacillus, has been implicated

in periodontal diseases, especially aggressive periodontitis, and other infectious diseases, such as endocarditis (Zambon, 1985; Paturel et al., 2004; Haubek et al., 2008). It expresses several potential virulence factors thought to play roles in the modulation of inflammation, induction of tissue destruction, and inhibition of tissue repair (Wilson & Henderson, 1995). Leukotoxin, a virulence factor from A. actinomycetemcomitans, interacts with lymphocyte function-associated molecule 1 (LFA-1), which is a β2 integrin expressed on mammalian cells, and exhibits cytolytic activity towards polymorphonuclear leukocytes (PMNs) and macrophages of humans and primates (Taichman et al., 1980, 1987). Furthermore, leukotoxin has been reported to induce degranulation of PMNs independent

of LFA-1 (Johansson et al., 2000). In this study, we examined whether neutrophil-derived resistin was released extracellularly by stimulation with several A. actinomycetemcomitans strains that express differing levels of leukotoxin and whether it was released by cytolysis or degranulation. Aggregatibacter actinomycetemcomitans HK921 (strain JP2), HK912, and HK1604, which are minimally leukotoxic

strains, were grown in brain heart infusion broth (BHI; Difco Laboratories) at 37 °C in air plus 5% CO2. The three strains were a gift from Prof. Mogens Kilian, Department of Medical Microbiology and Immunology, Aarhus University, Aarhus, Denmark. Escherichia coli strains were grown in Luria–Bertani medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at 37 °C with aeration. When necessary for the selection of recombinant strains, the medium was supplemented with ampicillin (100 mg L−1) and/or kanamycin Idelalisib mouse (25 mg L−1). The ltxA gene was inactivated in the HK921 strain by insertional mutagenesis as described previously (Hayashida et al., 2002). Briefly, a fragment of the ltxA gene (positions 615–2978 in the ORF of ltxA from strain HK921) was amplified from 1 ng of whole-cell DNA by PCR using the following primers: 5′-ACAACTTAATAAGTTAGGTGAAGCAC-3′ (615–640) The amplicon was cloned into pGEM-T Easy Vector (Promega) using E. coli XL-1 Blue for propagation. The resulting plasmid was termed ‘pGEM-ltxA.’ The kanamycin resistance gene from the 1.7-kb transprimer transposon in pGPS1.1 was inserted into the ltxA gene fragment in pGEM-ltxA using TnsABC transposase. The purified plasmid was introduced into A.

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