Different signaling pathways stimulate a disintegrin and metalloprotease-17 (ADAM17) in neutrophils during apoptosis and activation

ADAM17 is a membrane-associated metalloprotease that cleaves proteins from the surface of neutrophils and modulates the density of various receptors and adhesion molecules. The protease activity of ADAM17 is highly inducible and occurs upon neutrophil activation as well as apoptosis. At this time, little is known about the signal transduction pathway that promotes ADAM17 activity in neutrophils upon the induction of apoptosis. We show that caspase-8 activation, Bid cleavage, and the release of mitochondrial reactive oxygen species are sequential transduction components of the Fas signaling cascade that induces ADAM17. This is different from ADAM17 stimulation upon overt neutrophil activation, which requires MAPK p38 or ERK, but not caspases and reactive oxygen species. ADAM17 activity in apoptotic neutrophils may serve to inactivate select effector molecules that promote the pro-inflammatory activity of recruited neutrophils. For instance, TNFα receptors TNF-RI and TNF-RII are substrates of ADAM17, and we show that they are shed during apoptosis, decreasing neutrophil sensitivity to TNFα. Altogether, our findings provide significant new insights into the signal transduction pathway that stimulates ADAM17 during induced neutrophil apoptosis. ADAM17 induction during apoptosis may rapidly diminish neutrophil sensitivity to the inflammatory environment, complementing other anti-inflammatory activities by these cells during inflammation resolution.     

Human Siglec-5 inhibitory receptor and immunoglobulin A (IgA) have separate binding sites in streptococcal beta protein

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors believed to be important for regulation of cellular activation and inflammation. Several pathogenic microbes bind specific Siglecs via sialic acid-containing structures at the microbial surface, interactions that may result in modulation of host responses. Recently, it was shown that the group B Streptococcus (GBS) binds to human Siglec-5 (hSiglec-5), an inhibitory receptor expressed on macrophages and neutrophils, via the IgA-binding surface β protein, providing the first example of a protein/protein interaction between a pathogenic microbe and a Siglec. Here we show that the hSiglec-5-binding part of β resides in the N-terminal half of the protein, which also harbors the previously determined IgA-binding region. We constructed bacterial mutants expressing variants of the β protein with non-overlapping deletions in the N-terminal half of the protein. Using these mutants and recombinant β fragments, we showed that the hSiglec-5-binding site is located in the most N-terminal part of β (B6N region; amino acids 1-152) and that the hSiglec-5- and IgA-binding domains in β are completely separate. We showed with BIAcore(TM) analysis that tandem variants of the hSiglec-5- and IgA-binding domains bind to their respective ligands with high affinity. Finally, we showed that the B6N region, but not the IgA-binding region of β, triggers recruitment of the tyrosine phosphatase SHP-2 to hSiglec-5 in U937 monocytes. Taken together, we have identified and isolated the first microbial non-sialic acid Siglec-binding region that can be used as a tool in studies of the β/hSiglec-5 interaction.     

Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2)

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of β-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.     

2-thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation

Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation.     

Control of secondary granule release in neutrophils by Ral GTPase

Neutrophil (polymorphonuclear leukocyte; PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocytosis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, we found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules but not primary or tertiary granules and caused dysregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, whereas chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Overexpression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, we found that Ral, which was associated with the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral translocation intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattractant stimulation.     

Osteopontin undergoes polymerization in vivo and gains chemotactic activity for neutrophils mediated by integrin alpha9beta1

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9β1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9β1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin α9 subunit in leukocytes, indicating that α9β1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.     

The importance of infiltrating neutrophils in SDF-1 production leading to regeneration of the thymus after whole-body X-irradiation

Our previous study demonstrated that neutrophils transiently infiltrated into a site where apoptosis had been induced. However, the role of infiltrating neutrophils has not been fully elucidated. In this study, we examined their role in regeneration of the thymus after whole-body X-irradiation by focusing on SDF-1 production. After X-irradiation, the thymus became severely atrophied presumably due to phagocytosis of apoptotic thymocytes. At that time, a significant number of neutrophils were detected in the thymus. The thymus was then partially regenerated on day 7, whereas the level of SDF-1 in it was significantly increased on days 3 and 5. Depletion of neutrophils greatly impaired SDF-1 production and the thymus regeneration. Moreover, administration of a CXCR4 antagonist also greatly suppressed the thymus regeneration. Furthermore, coculturing of a stromal cell line with infiltrating neutrophils increased SDF-1 production. These results suggest that infiltrating neutrophils play an auxiliary role in regeneration of the thymus after whole-body X-irradiation through augmentation of SDF-1 production.     

Amyloid Fibrils Trigger the Release of Neutrophil Extracellular Traps (NETs), Causing Fibril Fragmentation by NET-associated Elastase

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.     

The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

The specific detection of peroxidase activity in human granulocytes is essential to elucidate their role in innate immune responses, immune regulation, and inflammatory diseases. The halogenating activity of myeloperoxidase in neutrophils can be determined by the novel fluorescent probe aminophenyl fluorescein (APF). Thereby non-fluorescent APF is oxidized by HOCl to form fluorescein. We successfully verified that APF equally detects the hypobromous acid (HOBr)-producing activity of eosinophil granulocytes. This was revealed by three different approaches. First, we investigated the conversion of non-fluorescent APF into fluorescein by HOCl and HOBr by means of fluorescence and mass spectrometry approaches. Thereby comparable chemical mechanisms were observed for both acids. Furthermore in vitro kinetic studies were used to detect the halogenating activity of myeloperoxidase and eosinophil peroxidase by using APF. Here the dye well reflected the different substrate specificities of myeloperoxidase and eosinophil peroxidase regarding chloride and bromide. Finally, peroxidase activities were successfully detected in phorbol ester-stimulated neutrophils and eosinophils using flow cytometry. Thereby inhibitory studies confirmed the peroxidase-dependent oxidation of APF. To sum up, APF is a promising tool for further evaluation of the halogenating activity of peroxidases in both neutrophils and eosinophils.     

Clathrin Pit-mediated Endocytosis of Neutrophil Elastase and Cathepsin G by Cancer Cells

Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1⁺ endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized ¹²⁵I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (K_d) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.