Mouse Polymorphonuclear Neutrophils Research Articles

Recombinant GM-CSF enhances the bactericidal ability of PMNs by increasing intracellular IL-1β and improves the prognosis of secondary Pseudomonas aeruginosa pneumonia in sepsis.

This study tested the hypothesis that recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances polymorphonuclear neutrophils (PMNs) via interleukin (IL)-1β to improve the prognosis of secondary infection in sepsis. The latter stage of sepsis is prone to induce immunosuppression, resulting in secondary fatal infections. Recombinant GM-CSF has become a way for sepsis-induced immunosuppression due to its immunomodulatory effect. However, the functional impact of GM-CSF on PMNs in sepsis remains obscure. This study aimed to study the role of recombinant GM-CSF on the bactericidal ability of PMNs in septic mice, assessing its effect on the prognosis of secondary pneumonia, and explore the mechanism of recombinant GM-CSF by intervening PMNs in patients with sepsis. The C57BL/6J sepsis mouse model was induced by cecal ligation and puncture. Recombinant murine GM-CSF (rmGM-CSF) was used in vivo when mice developed immunosuppression, which was characterized by abnormal bactericidal function of PMNs in peripheral blood. rmGM-CSF improved the prognosis of secondary pneumonia and reversed the function of PMNs. PMNs isolated by Percoll from septic patients were treated by recombinant human GM-CSF (rhGM-CSF) in vitro. The expression of CD11b, reactive oxygen species, phagocytosis, and neutrophil extracellular trap release in PMNs were enhanced by rhGM-CSF treatments. Whole-transcriptomic sequencing of mouse PMNs indicated that recombinant GM-CSF increased the expression of Il1b gene in PMNs. Blocking and inhibiting IL-1β release effectively counteracted the enhancing effect of GM-CSF on the bactericidal function of PMNs. rmGM-CSF enhances the bactericidal function of PMNs in vivo and improves the prognosis of secondary pneumonia in septic mice, and recombinant GM-CSF increases IL-1β precursor reserves, which, if stimulated, can rapidly enhance the bactericidal capacity of PMNs.

Optimized flow cytometry assays to monitor neutrophil activation in human and mouse whole blood samples

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples.Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions.Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively.Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.

Recombinant Mouse GM-CSF Enhances the Bactericidal Ability of Pmns and Improves the Prognosis of Secondary Pseudomonas Aeruginosa Pneumonia By Intracellular IL-1β

Objective: Sepsis is one of the most common clinical emergency and critical illness. The later stage of sepsis is prone to induce immunosuppression and results in secondary fatal infections. Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes the proliferation, differentiation and functional maturation of bone marrow hematopoietic stem cells, and has the function of enhancing immunity, so recombinant GM-CSF becomes an important direction for the treatment of sepsis-induced immunosuppression. However, the effect of recombinant GM-CSF on abnormal polymorphonuclear neutrophils (PMNs) in sepsis remains elusive. This study aimed to study the role of recombinant GM-CSF on the bactericidal ability of PMNs in septic mice, assessing its effect on the prognosis of secondary pneumonia. Methods: The C57BL/6J septic mouse model was induced by cecal ligation and puncture (CLP). Recombinant mouse GM-CSF (rmGM-CSF) was used in vivo when mice developed immunosuppression, then PMNs CD11b expression, reactive oxygen species (ROS), phagocytosis and neutrophil extracellular trap release (NETs) were detected by flow cytometry and fluoresce microplate reader. Pseudomonas aeruginosa pneumonia was induced by intratracheal injection after rmGM-CSF intervention in CLP mice, and the survival of mice were monitored. Meanwhile, PMNs from mouse peripheral blood were isolated for whole-transcriptomic sequencing. Combining with the results of bioinformatics analysis, anti-GM-CSF antibody and Caspase-1 inhibitor (Ac-YVAD-cmk) were treated into peripheral blood PMNs from septic patients in vitro, and then bactericidal ability of PMNs and intracellular IL-1β concentration were evaluated by flow cytometry and ELISA. Results: 1. The septic mice were developed 5 days after CLP, which manifested as decreased lymphocytes in bone marrow, spleen and peripheral blood. The septic mice showed declined expression of MHC-II molecules and abnormal bactericidal function of PMNs in peripheral blood. 2. The expression of MHC-II in monocytes and the bactericidal function of PMNs were improved when septic mice were treated with rmGM-CSF in vivo. 3. RmGM-CSF improved the prognosis of secondary pneumonia in septic mice. 4. Percoll-isolated PMNs from septic patients were treated with rhGM-CSF in vitro. The expression of CD11b, ROS, phagocytosis and NETs release in PMNs were enhanced compared with those without rhGM-CSF treatments. 5. The whole-transcriptomic sequencing of mouse PMNs indicated that recombinant GM-CSF increased the expression of IL-1β in PMNs. 6. Caspase-1 is an important enzyme for the cleavage of pro form IL-1β into mature form. Ac-YVAD-cmk effectively inhibited the activation of IL-1β. treatments reduced the ROS, phagocytosis and release of NETs of PMNs, suggesting that rmGM-CSF enhanced the bactericidal ability of PMNs by IL-1β. Conclusion: RmGM-CSF enhances the bactericidal function of PMNs in vivo, and improves the prognosis of secondary pneumonia in septic mice. In vitro experimental data showed rhGM-CSF strengthen PMNs bactericidal ability by stimulating active IL-1β expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

The Enzymatic and Non-Enzymatic Function of Myeloperoxidase (MPO) in Inflammatory Communication.

Myeloperoxidase is a signature enzyme of polymorphonuclear neutrophils in mice and humans. Being a component of circulating white blood cells, myeloperoxidase plays multiple roles in various organs and tissues and facilitates their crosstalk. Here, we describe the current knowledge on the tissue- and lineage-specific expression of myeloperoxidase, its well-studied enzymatic activity and incoherently understood non-enzymatic role in various cell types and tissues. Further, we elaborate on Myeloperoxidase (MPO) in the complex context of cardiovascular disease, innate and autoimmune response, development and progression of cancer and neurodegenerative diseases.

Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice.

In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities.

TBK1/IKKε Negatively Regulate LPS-Induced Neutrophil Necroptosis and Lung Inflammation.

Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that controls cell release of inflammatory mediators from innate immune cells, such as polymorphonuclear neutrophils (PMNs), and critically regulates the progress of inflammation. Cell necroptosis features receptor-interacting protein (RIPK) 1 activation and necroptosome formation. This leads to loss of plasma membrane integrity, the release of cell contents into the extracellular space, and subsequent increased inflammation. Here, we report an intra-PMN mechanism of negative regulation of necroptosis mediated through TBK1/IKKε. Using an in vivo mouse model of intratracheal injection (i.t.) of LPS and in vitro LPS stimulation of mouse PMN, we found that LPS-TLR4 signaling in PMNs activates and phosphorylates TBK1 and IKKε, which in turn suppress LPS-induced formation of the RIPK1-RIPK3-MLKL (necrosome) complex. TBK1 dysfunction by knockdown or inhibitor significantly increases the phosphorylation of RIPK1 (∼67%), RIPK3 (∼68%), and MLKL (∼50%) and promotes RIPK1-RIPK3 and RIPK3-MLKL interactions and increases PMN necroptosis (∼83%) in response to LPS, with subsequent augmented lung inflammation. These findings suggest that the LPS-TLR4-TBK1 axis serves as a negative regulator for PMN necroptosis and might be a therapeutic target for modulating PMN death and inflammation.

Analysis of leukocyte transepithelial migration using an in vivo murine colonic loop model.

Molecular mechanisms that control leukocyte migration across the vascular endothelium (transendothelial migration; TEndoM) have been extensively characterized in vivo, but details of leukocyte transepithelial migration (TEpM) and its dysregulation (a pathologic feature of many mucosal diseases) are missing due to the lack of suitable animal models. Here, we describe a murine model that utilizes a vascularized proximal colonic segment (pcLoop) and enables quantitative studies of leukocyte trafficking across colonic epithelium. Consistent with previous in vitro studies, intraluminal injection of antibodies against integrin CD11b/CD18 reduced recruitment of polymorphonuclear neutrophils (PMN) into the lumen of pcLoops, and it increased subepithelial accumulation of PMN. We extended studies using the pcLoop to determine contributions of Junctional Adhesion Molecule-A (JAM-A, or F11R) in PMN TEpM and confirmed that mice with total loss of JAM-A or mice with intestinal epithelial selective loss of JAM-A had increased colonic permeability. Furthermore, there was reduced PMN migration into the colonic lumen that paralleled subepithelial accumulation of PMN in global-KO mice, as well as in intestinal epithelial-targeted JAM-A-deficient mice. These findings highlight a potentially novel role for JAM-A in regulating PMN TEpM in vivo and demonstrate utility of this model for identifying receptors that may be targeted in vivo to reduce pathologic intestinal inflammation.

Effect of multiple sclerosis materials on polymorphonuclear neutrophils of mice.

An assessment was made of a bioassay for a multiple sclerosis (MS)-associated agent claimed to cause a depression in the proportion of circulating polymorphonuclear neutrophils (PMN) in mice. Serum (1 sample), supernate from brain homogenates (four samples) and cerebrospinal fluid (four samples), and corresponding control materials, were tested under code by intraperitoneal injection into BALB/c mice. Results were plotted of proportions and absolute numbers of PMN over observation periods up to 2 weeks. Handling, injection and bleeding of mice did not significantly influence "baseline" counts of PMN. However, all samples from MS patients and controls caused an initial increase in PMN over 1--4 days, and then a later fall towards baseline. The claimed effect of MS materials in depressing counts of PMN in mice was not observed in these experiments, and variations in counts of PMN did not discriminate between samples from MS patients and controls.

Effects of rhubarb polysacchrides on apoptosis of colonic epithelial cells and peripheral blood polymorphonuclear neutrophils in mice with ulcerative colitis

Effects of rhubarb polysacchrides on apoptosis of colonic epithelial cells and peripheral blood polymorphonuclear neutrophils in mice with ulcerative colitis

Anti-LcrV antibody inhibits delivery of Yops by Yersinia pestis KIM5 by directly promoting phagocytosis.

LcrV of Yersinia pestis is a major protective antigen proposed for inclusion in subunit plague vaccines. One way that anti-LcrV antibody is thought to protect is by inhibiting the delivery of toxins called Yops to host cells. The present study characterizes the relation between this inhibition and the phagocytosis of the bacteria. J774A.1 cells were infected with Y. pestis KIM5 in the presence of a protective polyclonal anti-LcrV antibody or a nonprotective polyclonal anti-YopM antibody, and delivery of YopH and YopE into the cytoplasm was assayed by immunoblotting. The ability to inhibit the delivery of these Yops depended upon having antibody bound to the cell surface; blocking conditions that prevented the binding of antibody to Fc receptors prevented the inhibition of Yop delivery. Anti-LcrV antibody also promoted phagocytosis of the yersiniae, whereas F(ab')(2) fragments did not. Further, anti-LcrV antibody could not inhibit the delivery of Yops into cells that were unable to phagocytose due to the presence of cytochalasin D. However, Yops were produced only by extracellular yersiniae. We hypothesize that anti-LcrV antibody does not directly inhibit Yop delivery but instead causes phagocytosis, with consequent inhibition of Yop protein production in the intracellular yersiniae. The prophagocytic effect of anti-LcrV antibody extended to mouse polymorphonuclear neutrophils (PMNs) in vitro, and PMNs were shown to be critical for protection: when PMNs in mice were ablated, the mice lost all ability to be protected by anti-LcrV antibody.

Histamine synthesis in mouse polymorphonuclear neutrophils.

Histamine synthesis in mouse polymorphonuclear neutrophils.

Cutting Edge: Chemotactic Activity of Soluble Fas Ligand Against Phagocytes

AbstractA recombinant soluble form of human Fas ligand (sFasL) was tested for its chemotactic activity against human and mouse polymorphonuclear neutrophils (PMN) by the Boyden chamber method. sFasL exhibited a potent chemotactic activity against both human and mouse PMN and HL-60 cells when differentiated into neutrophils or monocytes. A neutralizing anti-FasL mAb abolished the chemotactic activity, while control mAb did not. Ligation of Fas by either IgM- or IgG-type anti-Fas mAb also induced PMN migration. PMN derived from lpr mice that express few Fas molecules did not respond to sFasL. In contrast, those derived from lprcg mice that express Fas molecules with a mutated death domain normally responded to sFasL chemotaxis. These results directly indicated a chemotactic activity of sFasL against PMN and suggest a novel signaling function of Fas, which appears to be independent of the death domain-mediated apoptosis.

Expression and biologic characterization of the murine chemokine KC.

KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was expressed in Escherichia coli by using a maltose binding protein vector and biochemically characterized as a ligand for both murine and human polymorphonuclear neutrophils (PMN). On murine PMN, KC is both a potent chemoattractant and up-regulator of Mac-1 cell surface expression. On human PMN, in contrast, KC exhibits dissociation of its chemoattractant and Mac-1 up-regulatory activities. Although KC strongly increases Mac-1 expression on human PMN, it does not induce chemotaxis in vitro. 125I-KC-Tyr binds to both mouse and human PMN with two classes of binding sites, including high affinity sites of 0.8 and 2 nM, with approximately 9,000 and 10,000 sites per cell, respectively. On mouse PMN, human macrophage inflammatory protein (MIP)-2 alpha and MIP-2 beta compete for 125I-KC-Tyr binding with high affinity, whereas the murine beta-chemokine TCA-3 does not compete. KC binds to human PMN by the IL-8 type B receptor and to murine PMN by a murine IL-8 type B receptor homologue. 125I-KC-Tyr also binds to human RBC with a single class of high affinity sites. KC mRNA is constitutively expressed in multiple murine tissues. With human IL-8 and KC cDNA as probes, a mouse neutrophil exudate library was screened: KC and MIP-2 were the dominant chemokine species found. Thus, KC appears to be intimately involved in murine inflammation and its constitutive expression may have a role in the basal trafficking of neutrophils.

The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization.

KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was synthesized as a recombinant protein in Escherichia coli and binds with 0.8 nM affinity to mouse neutrophils. Human neutrophils also bind recombinant KC at a site competitive with human interleukin (IL8) and Gro-alpha/MGSA, consistent with binding at the IL8 type B receptor (IL8RB). The cDNA corresponding to human IL8RB hybridizes strongly with two restriction fragments in murine genomic DNA, representing candidate receptor genes for KC. Molecular cloning of both mouse genomic DNA and neutrophil exudate cell cDNA libraries yielded a receptor with approximately 68% sequence identity to both the human IL8 type A and B receptors. Transient expression of the murine receptor cDNA in COS cells conferred binding ability to KC and a related gene product, macrophage inflammatory protein-2 (MIP-2) with high affinity (approximately 5 nM). Human IL8 was a poor agonist for this expressed receptor (Kd = approximately 400 nM). The potent activity of human IL8 on mouse polymorphonuclear neutrophils is not consistent with binding on the cloned receptor and suggests that murine homologues of IL8 and an IL8 type A receptor remain to be identified. Our data indicate that KC is the murine homologue of human Gro-alpha, and the KC receptor is an IL8 type B receptor homologue capable of binding both KC and macrophage inflammatory protein-2 with high affinity.

Lipopolysaccharide restores anti-Candida albicans growth inhibition activity of polymorphonuclear neutrophils from retrovirus-immunosuppressed mice

It has been documented that the immune function of leukocytes may be markedly suppressed after infection of mice with the murine retrovirus Friend leukemia virus (FLV). Antimicrobial activity of polymorphonuclear neutrophils (PMNs) against Candida albicans is impaired after retrovirus infection of mice, and this occurs as early as 3 days after infection of genetically susceptible BALB/c mice. By 2 weeks after infection, there was essentially very little growth inhibition of C. albicans by PMNs from the FLV-infected mice. However, when bacterial lipopolysaccharide (LPS), a known activator of macrophages and PMNs, was added to PMNs from the FLV-infected mice, anti-C. albicans activity was restored to normal levels. This restoration of anti-C. albicans activity of FLV-infected mouse PMNs was observed after stimulation with as little as 0.01 micrograms of LPS per ml. The data obtained show that the impaired antimicrobial function of PMNs from retrovirus-infected mice can be readily restored by a biological response modifier such as bacterial LPS.

Presence of IL-1 receptors on human and murine neutrophils. Relevance to IL-1-mediated effects in inflammation.

Inflammatory responses are characterized by the infiltration of polymorphonuclear neutrophils (PMN) at the involved site. IL-1 may have an important role in mediating this response, but whether IL-1 acts directly on PMN is controversial. In this study, we examined PMN for the presence of IL-1R and determined the effect of IL-1 on PMN migration in vivo. Thioglycollate, proteose-peptone, or IL-1 elicited peritoneal exudate cells were found to bind 125I-IL-1 alpha in a specific and saturable manner. This binding was localized to the PMN in the exudate. Scatchard plot analysis indicates the presence of approximately 1700 receptors per PMN and an apparent dissociation constant of 3.0 x 10(-10) M. Binding sites for 125I-IL-1 alpha were also found on human PMN prepared from peripheral blood. There are approximately 900 receptors per cell on human PMN with a dissociation constant similar to that observed for elicited murine PMN. Binding of 125I-IL-1 alpha to the mouse and human PMN is inhibited by both recombinant human IL-1 alpha and IL-1 beta, indicating that both IL-1 proteins bind to the same receptor on these cells. Human PMN were able to internalize radioiodinated IL-1. We conclude that PMN possess receptors for IL-1 and that these binding sites may be important in mediating IL-1 effects on granulocytes that are involved in the inflammatory response.

Multiple sclerosis associated agent (MSAA): failure to confirm an association with multiple sclerosis.

Recent reports indicate that a multiple sclerosis agent (MSAA) has been isolated. This agent was detected in mice by a depression in the percentage of mouse polymorphonuclear neutrophils (PMNs). We attempted to repeat these studies using coded specimens. The test, itself, was extremely variable and difficult to reproduce. In the first study, three of three multiple sclerosis (MS) patients and one of two control patients' specimens showed some PMN depression. In a repeat study of these same samples, one of three MS patients and one of two controls were associated with PMN depression. With a second set of coded specimens, one of four MS patients, one of three patients with neurologic diseases, and none of three controls showed PMN depression. We were unable to confirm the presence of MSAA and conclude that the results were random.