Myeloperoxidase Deficiency Alters the Process of the Regulated Cell Death of Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs) play a key role in host defense. However, their massive accumulation at the site of inflammation can delay regenerative healing processes and can initiate pathological inflammatory processes. Thus, the efficient clearance of PMNs mediated by the induction of regulated cell death is a key process preventing the development of these pathological conditions. Myeloperoxidase (MPO), a highly abundant enzyme in PMN granules, primarily connected with PMN defense machinery, is suggested to play a role in PMN-regulated cell death. However, the contribution of MPO to the mechanisms of PMN cell death remains incompletely characterized. Herein, the process of the cell death of mouse PMNs induced by three different stimuli - phorbol 12-myristate 13-acetate (PMA), opsonized streptococcus (OST), and N-formyl-met-leu-phe (fMLP) - was investigated. MPO-deficient PMNs revealed a significantly decreased rate of cell death characterized by phosphatidylserine surface exposure and cell membrane permeabilization. An inhibitor of MPO activity, 4-aminobenzoic acid hydrazide, did not exhibit a significant effect on PMA-induced cell death compared to MPO deficiency. Interestingly, only the limited activation of markers related to apoptotic cell death was observed (e.g. caspase 8 activation, Bax expression) and they mostly did not correspond to phosphatidylserine surface exposure. Furthermore, a marker characterizing autophagy, cleavage of LC3 protein, as well as histone H3 citrullination and its surface expression was observed. Collectively, the data show the ability of MPO to modulate the life span of PMNs primarily through the potentiation of cell membrane permeabilization and phosphatidylserine surface exposure.

Isolation of Human Neutrophils from Venous Blood.

Venous blood provides a ready source of large numbers of unstimulated granulocytes and mononuclear cells. Exploiting the differences in the relative densities of the leukocytes circulating in venous blood, one can separate leukocytes from erythrocytes as well as isolate the individual leukocyte populations in high purity for use in ex vivo studies. For selected functional studies, such as transcriptional analysis or cytokine quantitation, addition of an immunomagnetic negative selection step to the standard isolation protocol can yield highly purified human neutrophils.

Frontline Science: Staphylococcus aureus promotes receptor-interacting protein kinase 3- and protease-dependent production of IL-1ß in human neutrophils.

Microbial infection elicits robust immune responses that initially depend on polymorphonuclear neutrophils (PMN), which ingest and kill invading bacteria. However, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) remain viable within PMN and prompt their lysis with concomitant release of damage-associated molecular patterns and proinflammatory cytokines that promote additional inflammation. Here, we show that ultrapure human PMN (>99.8% pure) that have ingested CA-MRSA released interleukin (IL)-1ß but not IL-18. The ingested CA-MRSA needed to be viable, and phagocytosis alone was insufficient to stimulate IL-1ß secretion from PMN fed CA-MRSA. In contrast to PMN response to the canonical NLRP3 inflammasome agonist nigericin, IL-1ß secretion by PMN fed CA-MRSA occurred independently of NLRP3 inflammasome or caspase-1 activation and required instead active receptor-interacting protein kinase 3 (RIPK3) but not RIPK1. Furthermore, inhibition of neutrophil serine proteases blocked pro-IL-1ß cleavage in PMN fed CA-MRSA. Taken together, our data suggest that with respect to secretion of IL-1ß and IL-18, PMN differ from human macrophages and exhibit agonist-specific responses. After phagocytosis of CA-MRSA, human PMN secreted IL-1ß through a previously unrecognized mechanism dependent on RIPK3 and serine proteases but independent of canonical NLRP3 inflammasome and caspase-1 activation.

Lysis of human neutrophils by community-associated methicillin-resistant Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes infections associated with extensive tissue damage and necrosis. In vitro, human neutrophils fed CA-MRSA lyse by an unknown mechanism that is inhibited by necrostatin-1, an allosteric inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK-1). RIPK-1 figures prominently in necroptosis, a specific form of programmed cell death dependent on RIPK-1, RIPK-3, and the mixed-lineage kinase-like protein (MLKL). We previously reported that necrostatin-1 inhibits lysis of human neutrophils fed CA-MRSA and attributed the process to necroptosis. We now extend our studies to examine additional components in the programmed cell death pathway to test the hypothesis that neutrophils fed CA-MRSA undergo necroptosis. Lysis of neutrophils fed CA-MRSA was independent of tumor necrosis factor α, active RIPK-1, and MLKL, but dependent on active RIPK-3. Human neutrophils fed CA-MRSA lacked phosphorylated RIPK-1, as well as phosphorylated or oligomerized MLKL. Neutrophils fed CA-MRSA possessed cytoplasmic complexes that included inactive caspase 8, RIPK-1, and RIPK-3, and the composition of the complex remained stable over time. Together, these data suggest that neutrophils fed CA-MRSA underwent a novel form of lytic programmed cell death via a mechanism that required RIPK-3 activity, but not active RIPK-1 or MLKL, and therefore was distinct from necroptosis. Targeting the molecular pathways that culminate in lysis of neutrophils during CA-MRSA infection may serve as a novel therapeutic intervention to limit the associated tissue damage.

Lung Neutrophilia in Myeloperoxidase Deficient Mice during the Course of Acute Pulmonary Inflammation.

Systemic inflammation accompanying diseases such as sepsis affects primarily lungs and induces their failure. This remains the most common cause of sepsis induced mortality. While neutrophils play a key role in pulmonary failure, the mechanisms remain incompletely characterized. We report that myeloperoxidase (MPO), abundant enzyme in neutrophil granules, modulates the course of acute pulmonary inflammatory responses induced by intranasal application of lipopolysaccharide. MPO deficient mice had significantly increased numbers of airway infiltrated neutrophils compared to wild-type mice during the whole course of lung inflammation. This was accompanied by higher levels of RANTES in bronchoalveolar lavage fluid from the MPO deficient mice. Other markers of lung injury and inflammation, which contribute to recruitment of neutrophils into the inflamed lungs, including total protein and other selected proinflammatory cytokines did not significantly differ in bronchoalveolar lavage fluid from the wild-type and the MPO deficient mice. Interestingly, MPO deficient neutrophils revealed a decreased rate of cell death characterized by phosphatidylserine surface expression. Collectively, the importance of MPO in regulation of pulmonary inflammation, independent of its putative microbicidal functions, can be potentially linked to MPO ability to modulate the life span of neutrophils and to affect accumulation of chemotactic factors at the inflammatory site.

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix.

BACKGROUND: Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. METHODS: We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. RESULTS: MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. CONCLUSIONS: Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. GENERAL SIGNIFICANCE: Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.