A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been hindered by the lack of distinctive methods to specifically quantify NET release in time. Here, we validate and refine a semi-automatic live imaging approach for quantification of NET release. Importantly, our approach is able to correct for neutrophil input and distinguishes NET release from neutrophil death by other means, aspects that are lacking in many NET quantification methods. Real time visualization shows that opsonized S. aureus rapidly induces cell death by toxins, while actual NET formation occurs after 90 minutes, similar to the kinetics of NET release by immune complexes and PMA. Inhibition of SYK, PI3K and mTORC2 attenuates NET release upon challenge with physiological stimuli but not with PMA. In contrast, neutrophils from chronic granulomatous disease patients show decreased NET release only in response to PMA. With this refined method, we conclude that NET release in primary human neutrophils is dependent on the SYK-PI3K-mTORC2 pathway and that PMA stimulation should be regarded as mechanistically distinct from NET formation induced by natural triggers.

Myeloid cells play a crucial role in controlling infection. Activation of these cells needs to be tightly regulated, because their potent effector functions can damage host tissue. Inhibitory receptors expressed by immune cells play an important role in restricting immune cell activation. In this study, we have characterized a hitherto unidentified ITIM-bearing receptor that is highly expressed on human neutrophils and monocytes: signal inhibitory receptor on leukocytes-1 (SIRL-1). The chromosomal location of SIRL-1 is adjacent to the human leukocyte receptor complex on chromosome 19q13.4 and contains two ITIMs in its cytoplasmic tail. As a classical ITIM-bearing receptor, SIRL-1 is capable of inhibiting FcεRI-mediated signaling and can recruit the Src homology 2 domain-containing phosphatases Src homology region 2 domain-containing phosphatases 1 and 2. To investigate the specific involvement of the individual ITIMs in this study, mutational analysis was performed, which revealed that both ITIMs are crucial for SIRL-1 inhibitory function and phosphatase recruitment. When primary cells were stimulated in vitro, SIRL-1high monocytes produce less TNF-α than SIRL-1low monocytes. Thus, SIRL-1 is a novel inhibitory immune receptor belonging to the growing family of ITIM-bearing receptors that is implied in the regulation of phagocytes.

Our data indicate that insufficient help of CD4(+) T cells may cause loss of IFN- gamma -producing CD8(+) T cells and loss of control of CMV dissemination. Increasing CMV-infected cell counts in the face of high CMV-specific perforin- and granzyme B-expressing CD8(+) T cell counts may explain the immune pathological characteristics of CMV disease.

Increased NET release is associated with autoimmunity and inflammation in SLE and APS. Inhibition of NET release thus could be of potential benefit in a subset of patients with SLE and APS.

ROS production is an important effector mechanism mediating intracellular killing of microbes by phagocytes. Inappropriate or untimely ROS production can lead to tissue damage, thus tight regulation is essential. We recently characterized signal inhibitory receptor on leukocytes-1 (SIRL-1) as an inhibitory receptor expressed by human phagocytes. Here, we demonstrate that ligation of SIRL-1 dampens Fc receptor-induced ROS production in primary human phagocytes. In accordance, SIRL-1 engagement on these cells impairs the microbicidal activity of neutrophils, without affecting phagocytosis. The inhibition of ROS production may result from reduced ERK activation, since co-ligation of Fc receptors and SIRL-1 on phagocytes inhibited phosphorylation of ERK. Importantly, we demonstrate that microbial and inflammatory stimuli cause rapid downregulation of SIRL-1 expression on the surface of primary neutrophils and monocytes. In accordance, SIRL-1 expression levels on neutrophils in bronchoalveolar lavage fluid from patients with neutrophilic airway inflammation are greatly reduced. We propose that SIRL-1 on phagocytes sets an activation threshold to prevent inappropriate production of oxygen radicals. Upon infection, SIRL-1 expression is downregulated, allowing microbial killing and clearance of the pathogen.Keywords: Inhibitory receptor r Phagocytes r ROS production r SIRL-1 Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionNeutrophils, monocytes, and macrophages are professional phagocytes and have a crucial role in host defense through Correspondence: Dr. Linde Meyaard e-mail: l.meyaard@umcutrecht.nl recognition, phagocytosis, and elimination of invading pathogens [1]. Phagocytes are equipped with a range of receptors that recognize microbes and facilitate their uptake. These PRR include TLR and C-type lectins, which are critical in detecting invading microorganisms, resulting in activation of phagocytes and leading to production of inflammatory cytokines and chemokines to recruit and activate additional effector cells.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 1298 Tessa A. M. Steevels et al. Eur. J. Immunol. 2013. 43: 1297-1308 Besides pathogen detection, microbicidal activity is a key function of phagocytes and is achieved through phagocytosis of the infectious agent, followed by fusion of the intracellular phagosome with lysosomal granules and ROS production [2]. The NADPH oxidase complex is responsible for ROS production in phagocytes, generating superoxide anion radicals (O 2 − •), which are dismutated to form H 2 O 2 . The lowered pH leads to enzyme activation in the phagosome [3]. Hypochlorous acids are formed by peroxidase enzymes that catalyze H 2 O 2 -dependent oxidation of chloride and bromide [4,5]. This phagocytic response is often initiated through activation of Fc receptors (FcRs) by Ig-opsonized bacteria [6]. The key role of ROS in microbial killing is most apparent from the recurrent bacterial infec...