Defective Expression of Neutrophil Serine Proteases in Myeloid Progenitors of Congenital Neutropenia Patients Carrying Either ELA2 or HAX1 Mutations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3299-3299 ◽  
Author(s):  
Julia Skokowa ◽  
John-Paul Fobiwe ◽  
Dan Lan ◽  
Manuela Germeshausen ◽  
Karl Welte
Keyword(s):  
Mrna Expression ◽  
Neutrophil Elastase ◽  
Cathepsin D ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Proteinase 3 ◽  
Congenital Neutropenia ◽  
Expression Levels ◽  
Patient Groups ◽  
Mrna Expression Levels

Abstract Severe congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis with two major types of inheritance: autosomal dominant CN defined by mutations in ELA2 gene encoding neutrophil elastase (NE) (Horwitz M., et al., Nat Genet.1999;23:433) and autosomal recessive CN (including Kostmann syndrome) carrying HAX-1 mutations (Klein C., et al., Nat Genet.2007;39:86), both characterized by a maturation arrest of granulopoiesis at the level of promyelocytes. In the present study we aimed to evaluate the expression profile of genes specifically expressed in the CD33+ bone marrow promyelocytes of both patient groups harbouring ELA2 or HAX1 mutations. In healthy individuals mRNA expression levels of neutrophil serine proteases (neutrophil elastase (ELA2), cathepsin G, cathepsin D, proteinase 3 and azurocidin) as well as of myeloperoxidase (MPO) and defensins reached highest levels in the azurophil granules at the promyelocytic stage of neutrophil differentiation (Borregaard N., et al., Curr Opin Hematol.2001;8:23). We found downregulation of mRNA expression levels of ELA2 (8.9 fold), cathepsin G (7.6 fold), cathepsin D (11.2 fold), proteinase 3 (9.2 fold) and defensin B1 (6.5 fold) in both groups of CN patients (with ELA2 or HAX1 mutations), in comparison to G-CSF-treated patients with idiopathic neutropenia (IN) and G-CSF-treated healthy controls. In contrast, there were no difference in mRNA expression levels of azurocidin and only slight decrease in the expression of MPO mRNA in CN patients. Additionally, we found significantly reduced protein levels of neutrophil elastase (NE) in plasma of CN patients irrespective of “ELA2 or HAX1” inheritance, in comparison to cyclic neutropenia (CyN) patients, IN patients and G-CSF-treated healthy controls. Taken together, both ELA2 and HAX1 mutations are associated with defective expression of neutrophil serine proteases such as NE, cathepsin G, cathepsin D, proteinase 3 as well as of defensin B1 in CD33+ myeloid progenitor cells of CN patients, suggesting a common pathway for both patient groups. Intriguingly, ELA2 expression is directly regulated by LEF-1, suggesting that abrogated LEF-1 expression in CN promyelocytes (Skokowa J., et al., Nat. Med.2006;12:1191) may be responsible for defective serine proteases expression in both groups, since all are regulated by a similar mechanism.

Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1752-1758 ◽  
Author(s):  
Frank El Ouriaghli ◽  
Hiroshi Fujiwara ◽  
J. Joseph Melenhorst ◽  
Giuseppe Sconocchia ◽  
Nancy Hensel ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Neutrophil Elastase ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Proteinase 3 ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Macrophage Colony ◽  
Serum Free Suspension

There is evidence that neutrophil production is a balance between the proliferative action of granulocyte–colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro–proteinase 3 inhibits granulocyte macrophage–colony-forming unit (CFU-GM) growth, and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3, elastase, azurocidin, and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells, elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase, G-CSF, but not GM-CSF or SCF, was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF.

scholarly journals Local structural plasticity of the Staphylococcus aureus evasion protein EapH1 enables engagement with multiple neutrophil serine proteases

2020 ◽  
Vol 295 (22) ◽  
pp. 7753-7762 ◽  
Author(s):  
Timothy J. Herdendorf ◽  
Daphne A. C. Stapels ◽  
Suzan H. M. Rooijakkers ◽  
Brian V. Geisbrecht
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Neutrophil Elastase ◽  
Serine Proteases ◽  
Noncovalent Complex ◽  
Structural Plasticity ◽  
Binding Mode ◽  
Cathepsin G ◽  
Proteinase 3 ◽  
High Affinity

Members of the EAP family of Staphylococcus aureus immune evasion proteins potently inhibit the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin-G, and proteinase-3. Previously, we determined a 1.8 Å resolution crystal structure of the EAP family member EapH1 bound to neutrophil elastase. This structure revealed that EapH1 blocks access to the enzyme's active site by forming a noncovalent complex with this host protease. To determine how EapH1 inhibits other NSPs, we studied here the effects of EapH1 on cathepsin-G. We found that EapH1 inhibits cathepsin-G with a Ki of 9.8 ± 4.7 nm. Although this Ki value is ∼466-fold weaker than the Ki for EapH1 inhibition of neutrophil elastase, the time dependence of inhibition was maintained. To define the physical basis for EapH1's inhibition of cathepsin-G, we crystallized EapH1 bound to this protease, solved the structure at 1.6 Å resolution, and refined the model to Rwork and Rfree values of 17.4% and 20.9%, respectively. This structure revealed a protease-binding mode for EapH1 with cathepsin-G that was globally similar to that seen in the previously determined EapH1–neutrophil elastase structure. The nature of the intermolecular interactions formed by EapH1 with cathepsin-G differed considerably from that with neutrophil elastase, however, with far greater contributions from the inhibitor backbone in the cathepsin-G–bound form. Together, these results reveal that EapH1's ability to form high-affinity interactions with multiple NSP targets is due to its remarkable level of local structural plasticity.

Olfactomedin 4 Down-Regulates Neutrophil Killing of Gram-Positive and Gram-Negative Bacteria.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3777-3777
Author(s):  
Wenli Liu ◽  
Ming Yan ◽  
Yueqin Liu ◽  
William G Coleman ◽  
Griffin P. Rodgers
Keyword(s):  
Neutrophil Elastase ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Proteinase 3 ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Cathepsin C ◽  
E Coli ◽  
Deficient Mice

Abstract Abstract 3777 Introduction: Olfactomedin 4 (OLFM4) is a member of olfactomedin-related glycoprotein family, which is specifically expressed in neutrophils and gastrointestinal tract. OLFM4 expression is upregulated in gastrointestinal cancer and inflammatory diseases such as chronic inflammatory bowel disease and Helicobacter pylori infection. It has been shown that OLFM4 is a target gene of NF-kB and Notch pathways and that OLFM4 down-regulates innate immunity to H. pylori infection. However, its potential biological functions in neutrophils still remain to be defined. The goal of this study is to determine whether OLFM4 is involved in the bactericidal activity of neutrophils using an OLFM4 deficient mouse model. Results: 1. OLFM4 expression in neutrophils is upregulated in response to Staphylocococus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. 2. We have shown that neutrophils from OLFM4 deficient mice have increased intracellular killing of S. aureus and E. coli bacteria in vitro. 3. The OLFM4 deficient mice displayed enhanced bacterial clearance in vivo when the mice were challenged with intra-peritoneal injection of S. aureus and E. coli. 4. To elucidate the molecular mechanisms that mediate these effects, we performed a yeast 2-hybrid screen and found that OLFM4 interacts with cathepsin C (dipeptidyl peptidase I or DPPI), a lysosomal cysteine protease that has a degraditive function as an exopeptidase and is essential for activation of neutrophil granule-associated serine proteases including neutrophil elastase, cathepsin G and proteinase 3. The direct association of OLFM4 with cathepsin C was confirmed in human primary neutrophils. 4. We have demonstrated that OLFM4 is a direct substrate of DPPI and inhibits DPPI activity in transfected 293T cells. 5. The cathepsin C activity in neutrophils from OLFM4 deficient mice was significantly higher than that in neutrophils from wild-type littermate mice in the absence or presence of bacterial infection, suggesting that OLFM4 is an endogenous inhibitor of cathepsin C in neutrophils. 6. We have also demonstrated increased activities of neutrophil elastase, cathepsin G and proteinase 3 (whose processing and maturity require cathepsin C activity) in OLFM4 deficient neutrophils compared with wild-type neutrophils. 7. Activation of NADPH oxidase, myeloperoxidase (MPO) activity, and neutrophil phagocytosis were not altered in OLFM4 deficient neutrophils compared with wild-type neutrophils. Conclusion: These results suggest that OLFM4 is an important regulator of neutrophil bacterial killing activity via negative regulation of cathepsin C activity and its down stream granule-associated serine proteases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Potent and Broad but not Unselective Cleavage of Cytokines and Chemokines by Human Neutrophil Elastase and Proteinase 3

2020 ◽  
Vol 21 (2) ◽  
pp. 651 ◽  
Author(s):  
Zhirong Fu ◽  
Srinivas Akula ◽  
Michael Thorpe ◽  
Lars Hellman
Keyword(s):  
Mast Cell ◽  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Serine Proteases ◽  
General Pattern ◽  
Neutrophil Activation ◽  
Cathepsin G ◽  
Human Neutrophil Elastase ◽  
Proteinase 3 ◽  
Cytokines And Chemokines

In two recent studies we have shown that three of the most abundant human hematopoietic serine proteases—mast cell chymase, mast cell tryptase and neutrophil cathepsin G—show a highly selective cleavage of cytokines and chemokines with a strong preference for a few alarmins, including IL-18, TSLP and IL-33. To determine if this is a general pattern for many of the hematopoietic serine proteases we have analyzed the human neutrophil elastase (hNE) and human proteinase 3 (hPR-3) for their cleavage of a panel of 69 different human cytokines and chemokines. Our results showed that these two latter enzymes, in sharp contrast to the two previous, had a very potent and relatively unrestrictive cleavage on this panel of targets. Almost all of these proteins were cleaved and many of them were fully degraded. In light of the proteases abundance and their colocalization, it is likely that together they have a very potent degrading activity on almost any protein in the area of neutrophil activation and granule release, including both foreign bacterial or viral proteins as well as various self-proteins in the area of inflammation/infection. However, a few very interesting exceptions to this pattern were found indicating a high resistance to degradation of some cytokines and chemokines, including TNF-α, IL-5, M-CSF, Rantes, IL-8 and MCP-1. All of these are either important for monocyte-macrophage, neutrophil or eosinophil proliferation, recruitment and activation, suggesting that cytokines/chemokines and proteases may have coevolved to not block the recruitment of monocytes–macrophages, neutrophils and possibly eosinophils during an inflammatory response involving neutrophil activation.

scholarly journals The role of CXCR3 and its ligands expression in Brucellar spondylitis

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Xin Hu ◽  
Xiaoqian Shang ◽  
Liang Wang ◽  
Jiahui Fan ◽  
Yue Wang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Inflammatory Infiltration ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Inflammatory Damage ◽  
Ifn Γ ◽  
Mrna Expression Levels

Abstract Aim Brucellar spondylitis (BS) is one of the most serious complications of brucellosis. CXCR3 is closely related to the severity of disease infection. This research aimed to study the degree of BS inflammatory damage through analyzing the expression levels of CXCR3 and its ligands (CXCL9 and CXCL10) in patients with BS. Methods A total of 29 BS patients and 15 healthy controls were enrolled. Real-Time PCR was used to detect the mRNA expression levels of IFN-γ, CXCR3, CXCL9 and CXCL10 in peripheral blood mononuclear cells (PBMCs) of BS patients and healthy controls. Hematoxylin-Eosin staining was used to show the pathological changes in BS lesion tissues. Immunohistochemistry staining was used to show the protein expression levels of Brucella-Ab, IFN-γ, CXCR3, CXCL9 and CXCL10 in BS lesion tissues. At the same time, ELISA was used to detect the serum levels of IFN-γ, CXCL9 CXCL10 and autoantibodies against CXCR3 in patients with BS. Results In lesion tissue of BS patients, it showed necrosis of cartilage, acute or chronic inflammatory infiltration. Brucella-Ab protein was abundantly expressed in close lesion tissue. And the protein expression levels of IFN-γ, CXCR3 and CXCL10 were highly expressed in close lesion tissue and serum of BS patients. At the same time, the mRNA expression levels of IFN-γ, CXCR3 and CXCL10 in PBMCs of BS patients were significantly higher than those in controls. Conclusion In our research, the expression levels of IFN-γ, CXCR3 and its ligands were significantly higher than those in controls. It suggested that high expression levels of IFN-γ, CXCR3 and its ligands indicated a serious inflammatory damage in patients with BS.

Unilateral labyrinthectomy induced changes on GDNF receptor complex proteins in the rat medial vestibular nuclei

2007 ◽  
Vol 16 (4-5) ◽  
pp. 171-177
Author(s):  
Adrian Lozada ◽  
Kaj Karlstedt ◽  
Pertti Panula ◽  
Antti A. Aarnisalo
Keyword(s):  
Mrna Expression ◽  
Vestibular Nucleus ◽  
Medial Vestibular Nucleus ◽  
Receptor Complex ◽  
Trophic Effect ◽  
Expression Levels ◽  
Protein Levels ◽  
Unilateral Labyrinthectomy ◽  
Ganglion Neurons ◽  
Mrna Expression Levels

In the auditory periphery, GDNF has been shown to have a trophic effect to spiral ganglion neurons, both during development and in adult animals. We have studied the effect of unilateral labyrinthectomy (UL) on protein levels and expression of GDNF multicomponent receptor complex: the ret tyrosine kinase and coreceptor GFRα-1 in the medial vestibular nucleus of the adult rat. GFRα-1 protein levels display an increasing trend in ipsilateral medial vestibular nucleus culminating at 48 h post UL. On the other hand, GFRα-1 mRNA expression levels in ipsi- and contralateral medial vestibular nucleus show a steadily decreasing trend that is significant at 1 week post-lesion. Protein levels for c-Ret isoforms also show an initial bilateral decreasing trend that ceases at 48 h in ipsilateral medial vestibular nucleus but persists on the contralateral side. c-Ret mRNA expression levels show a significant decrease at 4 h post UL followed by another significant decrease 1 week post UL. Our data would suggest that neurotrophins belonging to the GDNF family are involved in this model of post-lesional CNS plasticity.

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