Summary
Granules are essential for the ability of neutrophils to fulfill their role in innate immunity. Granule membranes contain proteins that react to environmental cues directing neutrophils to ...sites of infection and initiate generation of bactericidal oxygen species. Granules are densely packed with proteins that contribute to microbial killing when liberated to the phagosome or extracellularly. Granules are, however, highly heterogeneous and are traditionally subdivided into azurophil granules, specific granules, and gelatinase granules in addition to secretory vesicles. This review will address issues pertinent to formation of granules, which is a process intimately connected to maturation of neutrophils from their precursors in the bone marrow. We further discuss possible mechanisms by which decisions are made regarding sorting of proteins to constitutive secretion or storage in granules and how degranulation of granule subsets is regulated.
Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these ...subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents a common mechanism for modulating innate or adaptive immunity.
Determination of the distribution of proteins in isolated granule subsets, secretory vesicles, and plasma membrane of human neutrophils.
Neutrophils are indispensable in the innate immune defense ...against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four‐layer Percoll gradient. Granule subsets were pooled and subjected to SDS‐PAGE, and gel pieces were in‐gel‐digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes.
ABSTRACTThe innate immune system of mammals provides a rapid response to repel assaults from numerous infectious agents including bacteria, viruses, fungi, and parasites. A major component of this ...system is a diverse combination of cationic antimicrobial peptides that include the α‐ and β‐defensins and cathelicidins. In this study, we show that 1,25‐dihydroxyvitamin D3 and three of its analogs induced expression of the human cathelicidin antimicrobial peptide (CAMP) gene. This induction was observed in acute myeloid leukemia (AML), immortalized keratinocyte, and colon cancer cell lines, as well as normal human bone marrow (BM) ‐derived macrophages and fresh BM cells from two normal individuals and one AML patient. The induction occurred via a consensus vitamin D response element (VDRE) in the CAMP promoter that was bound by the vitamin D receptor (VDR). Induction of CAMP in murine cells was not observed and expression of CAMP mRNA in murine VDR‐deficient bone marrow was similar to wild‐type levels. Comparison of mammalian genomes revealed evolutionary conservation of the VDRE in a short interspersed nuclear element or SINE in the CAMP promoter of primates that was absent in the mouse, rat, and canine genomes. Our findings reveal a novel activity of 1,25‐dihydroxyvitamin D3 and the VDR in regulation of primate innate immunity. Gombart, A. F., Borregaard, N., Koeffler, H. P. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up‐regulated in myeloid cells by 1,25‐dihydroxyvitamin D3. FASEB J. 19, 1067–1077 (2005)
Neutrophils are produced in the bone marrow from stem cells that proliferate and differentiate to mature neutrophils fully equipped with an armory of granules. These contain proteins that enable the ...neutrophil to deliver lethal hits against microorganisms, but also to cause great tissue damage. Neutrophils circulate in the blood as dormant cells. At sites of infection, endothelial cells capture bypassing neutrophils and guide them through the endothelial cell lining whereby the neutrophils are activated and tuned for the subsequent interaction with microbes. Once in tissues, neutrophils kill microorganisms by microbicidal agents liberated from granules or generated by metabolic activation. As a final act, neutrophils can extrude stands of DNA with bactericidal proteins attached that act as extracellular traps for microorganisms.
Background Neutrophil gelatinase-associated lipocalin (NGAL) is proposed as an early marker of kidney injury. We report the association of urinary NGAL with indexes of intraoperative renal ...hypoperfusion (cardiopulmonary bypass time and aortic cross-clamp time) and acute kidney injury (AKI) after adult cardiac surgery. Study Design Diagnostic test accuracy. Setting & Participants Adult cardiac surgical patients (n = 426) in a single center from 2004 to 2006. Index Test Urinary NGAL immediately and 3, 18, and 24 hours after cardiac surgery, using an enzyme-linked immunosorbent assay. Reference Test or Outcome Serum creatinine–based definition for AKI (increase in serum creatinine from preoperative values by >50% or >0.3 mg/dL within 48 hours). Results Mean urinary NGAL level was 165 ± 663 (SD) ng/mL preoperatively, peaked immediately after cardiac surgery at 1,490 ± 102 ng/mL, and remained significantly higher 3, 18, and 24 hours after surgery. 85 patients (20%) developed AKI. Areas under the receiver operating characteristic curve for urinary NGAL immediately after and 3, 18, and 24 hours later as a predictor for AKI were 0.573 (95% confidence interval CI, 0.506 to 0.640), 0.603 (95% CI, 0.533 to 0.674), 0.611 (95% CI, 0.544 to 0.679), and 0.584 (95% CI, 0.510 to 0.657), respectively. Urinary NGAL, but not serum creatinine, level correlated significantly with cardiopulmonary bypass and aortic cross-clamp times. Areas under receiver operating characteristic curves for cardiopulmonary bypass time and aortic cross-clamp time to predict AKI were 0.592 (95% CI, 0.518 to 0.666) and 0.593 (95% CI, 0.523 to 0.665), respectively. Limitations Limited sensitivity of changes in serum creatinine levels for kidney injury. Conclusions Urinary NGAL has limited diagnostic accuracy to predict AKI defined by change in serum creatinine after cardiac surgery.
Lipocalin‐2 (LCN2) was originally isolated from human neutrophils and termed neutrophil gelatinase‐associated lipocalin (NGAL). However, the functions of LCN2 and the cell types that are primarily ...responsible for LCN2 production remain unclear. To address these issues, hepatocyte‐specific Lcn2 knockout (Lcn2Hep–/–) mice were generated and subjected to bacterial infection (with Klesbsiella pneumoniae or Escherichia coli) or partial hepatectomy (PHx). Studies of Lcn2Hep–/– mice revealed that hepatocytes contributed to 25% of the low basal serum level of LCN2 protein (∼62 ng/mL) but were responsible for more than 90% of the highly elevated serum LCN2 protein level (∼6,000 ng/mL) postinfection and more than 60% post‐PHx (∼700 ng/mL). Interestingly, both Lcn2Hep–/– and global Lcn2 knockout (Lcn2–/–) mice demonstrated comparable increases in susceptibility to infection with K. pneumoniae or E. coli. These mice also had increased enteric bacterial translocation from the gut to the mesenteric lymph nodes and exhibited reduced liver regeneration after PHx. Treatment with interleukin (IL)‐6 stimulated hepatocytes to produce LCN2 in vitro and in vivo. Hepatocyte‐specific ablation of the IL‐6 receptor or Stat3, a major downstream effector of IL‐6, markedly abrogated LCN2 elevation in vivo. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that STAT3 was recruited to the promoter region of the Lcn2 gene upon STAT3 activation by IL‐6. Conclusion: Hepatocytes are the major cell type responsible for LCN2 production after bacterial infection or PHx, and this response is dependent on IL‐6 activation of the STAT3 signaling pathway. Thus, hepatocyte‐derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration. (Hepatology 2015;61:692‐702)
Neutrophils are essential for host defense at the oral mucosa and neutropenia or functional neutrophil defects lead to disordered oral homeostasis. We found that neutrophils from the oral mucosa ...harvested from morning saliva had released neutrophil extracellular traps (undergone NETosis) in vivo. The NETosis was mediated through intracellular signals elicited by binding of sialyl LewisX present on salival mucins to l-selectin on neutrophils. This led to rapid loss of nuclear membrane and intracellular release of granule proteins with subsequent neutrophil extracellular trap (NET) release independent of elastase and reduced NAD phosphate-oxidase activation. The saliva-induced NETs were more DNase-resistant and had higher capacity to bind and kill bacteria than NETs induced by bacteria or by phorbol-myristate acetate. Furthermore, saliva/sialyl LewisX mediated signaling enhanced intracellular killing of bacteria by neutrophils. Saliva from patients with aphthous ulcers and Behçet disease prone to oral ulcers failed to induce NETosis, but for different reasons it demonstrated that disordered homeostasis in the oral cavity may result in deficient saliva-mediated NETosis.
•Saliva induces bactericidal and DNase resistant NETs in the oral cavity via sialyl LewisX- L-selectin signaling.•Disordered homeostasis in the oral cavity may lead to deficient saliva-mediated NETosis.
Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the ...generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.
The inflammasome generates IL-1 family proteins, but its role in neutrophils is poorly understood.
Neutrophils store key inflammasome components in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33.
Neutrophils store inflammasome components in intracellular compartments.
Targeting the inflammasome in neutrophils represents a future anti-inflammatory strategy.
The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs ...primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D(3) (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D(2) twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D "insufficient" conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.
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