Formyl-methionyl-leucyl-phenylalanine Research Articles

Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis

Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The present study aimed to determine the extracellular ROS production in both sperm and PMN. With this purpose, (1) donkey sperm were exposed to reductive and oxidative stresses, through adding different concentrations of reduced glutathione (GSH) and hydrogen peroxide (H2O2), respectively; and (2) PMN were subjected to NETosis in the presence of the whole semen, sperm, seminal plasma (SP) or other activators such as formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular ROS production (measured as H2O2 levels) was determined with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. Donkey sperm showed more resilience to oxidative stress than to the reductive one, and GSH treatments led to greater H2O2 extracellular production. Moreover, not only did SP appear to be the main inducer of NETosis in PMN, but it was also able to maintain the extracellular H2O2 levels produced by sperm and NETosis.

Seminal Plasma, Sperm Concentration, and Sperm-PMN Interaction in the Donkey: An In Vitro Model to Study Endometrial Inflammation at Post-Insemination.

In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work intended to set an in vitro model through harvesting PMN from the peripheral blood of jennies. Thereafter, how PMN, which require a triggering agent like formyl-methionyl-leucyl-phenylalanine (FMLP) to be activated, are affected by donkey semen was interrogated. Finally, we tested how four concentrations of spermatozoa (100 × 106, 200 × 106, 500 × 106 and 1000 × 106 spermatozoa/mL) affected their interaction with PMN. We observed that semen, which consists of sperm and SP, is able to activate PMN. Whereas there was a reduced percentage of spermatozoa phagocytosed by PMN, most remained attached on the PMN surface or into a surrounding halo. Spermatozoa not attached to PMN were viable, and most of those bound to PMN were also viable and showed high tail beating. Finally, only sperm concentrations higher than 500 × 106 spermatozoa/mL showed free sperm cells after 3 h of incubation, and percentages of spermatozoa not attached to PMN were higher at 3 h than at 1 h, exhibiting high motility. We can thus conclude that semen activates PMN in the donkey, and that the percentage of spermatozoa phagocytosed by PMN is low. Furthermore, because percentages of spermatozoa not attached to PMN were higher after 3 h than after 1 h of incubation, we suggest that PMN-sperm interaction plays an instrumental role in the reproductive strategy of the donkey.

Increased oxygen consumption observed in phorbol 12-myristate 13-acetate stimulated human cultured promonocytic U937 cell lines treated with calcitriol and retinoic acid

ObjectiveTo investigate the effect of phorbol 12-myristate 13-acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (FMLP) on oxygen consumption of differentiated and non-differentiated immune cell lines by retinoic acid and calcitriol treatment which might be useful in subsequent elicitation of immunological action during immunosuppressive states. MethodsPMA and FMLP were used to artificially stimulate reactive oxygen production in cultured promonocytic U937 cell line. Paralleled samples of the cultured cells were separately prepared with calcitriol (1, 25- dihydroxyvitamin D3) and retinoic acid followed by a 72-hour incubation period. The rate of respiratory burst was measured using the Clark oxygen electrode. ResultsThe average increase in cell concentrations per mL observed was significantly higher in retinoic acid-treated cells (9×106 cells/mL) when compared with calcitriol-treated samples (4×106 cells/mL). There was a marked increase in oxygen consumption of the calcitriol-treated cell lines against the retinoic acid-treated ones. Exposure of differentiated U937 cells to PMA and FMLP increased significantly (P<0.05) in their oxygen consumption when compared with the control. PMA calcitriol-treated cells resulted in 55% oxygen consumption more than the control while FMLP oxygen consumption increased 78% by comparison with the control. ConclusionsThe result demonstrated that calcitriol may serve as a physiological promoter of normal differentiation of precursor cells which may exert an immunological action. This effect could elicit a marker potential and increase immune cell activity of the host especially in immunosuppressed diseased states.

Immunopharmacological Activity of Calcitriol Pre-Treated RAW264.7 Cell Line Stimulated by Phorbol 12-Myristate13-Acetate (PMA) and Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) Agonist

The study investigates the effect of calcitriol treatment on oxygen consumption rate as generated by agonist stimulation of RAW 264.7 cell lines and its usefulness in eliciting reduced oxygen consumption in high respiratory burst-dependent disease state. Phorbol 12-myristate 13-acetate (PMA) and Formyl-methionyl-leucyl-phenylalanine (FMLP) were used to artificially stimulate oxygen consumption in cultured pro-monocytic RAW264.7 cell line. Samples of the cultured cells were previously prepared with calcitriol (1, 25- Dihydroxyvitamin D3) followed by a 72-hour incubation period. The percentage oxygen consumption was measured using the Clark oxygen electrode. There was a significant increase in oxygen consumption in FMLP treated cells (P<0.05) when compared with the PMA and the control groups. The PMA calcitriol-treated cells showed 24% oxygen consumption rate more than the control while FMLP treated cells was 57% higher. The result demonstrated that calcitriol, a known stimulant used to prep most cells for agonists stimulation of oxygen consumption may serve as a physiological moderator of oxygen consumption in immune cells when co-administered with agonist (PMA and/or FMLP). This may result to increased pathogen attack in a diseased state.

Evaluation of the impact of leucocytospermia on semen oxidative status by chemiluminescence technique in infertile men

The presence of high reactive oxygen species (ROS) levels in semen is a major factor involved in the decline of male fertility. In seminal plasma, ROS are mainly produced by activated leucocytes. Spermatozoa were the first cell type reported to show a potential susceptibility to oxidative damage. The aim of our study was to evaluate the impact of leucocytospermia on basal and FMLP (Formyl-Methionyl-Leucyl-Phenylalanine) induced oxidative status in semen of infertile men. We also analyzed the correlations of the spermatic parameters with amounts of ROS in semen. Our study included 50 semen samples of infertile men. Sperm analysis was performed using WHO standardized method. Seminal leucocytes were quantified using peroxidase technique. The measurement of ROS levels in semen was made by chemiluminescence assay. We measure respectively ROS amounts in neat semen and in washed sperm cells suspension from the same ejaculate. We also applied the test of provocation of leucocytes by FMLP on neat and washed samples to assess the spermatic oxidative status after leucocyte stimulation. Our results showed significant correlations between ROS levels in neat semen and many sperm parameters: motility, sperm concentration, leucocytes concentration and the rate of sperm cytoplasmic droplets. The studied samples were divided into 2 groups: (G1) composed of 36 samples without leucocytospermia and (G2) composed of 14 leucospermic samples. ROS levels were significantly lower in G1 than in G2 (p = 0.002). ROS production was significantly increased after application of FMLP in washed leucospermic samples (p = 0.001). The measurement of ROS in neat semen is a considerable contribution to explore the impairment of semen quality in infertile men. ROS levels in washed semen reflect the oxidative status generated by sperm preparation techniques used in assisted reproductive procedures. Levels of ROS are highly influenced by the presence of leucocytes and associated with decreased seminal parameters.

Equine platelets inhibit E. coli growth and can be activated by bacterial lipopolysaccharide and lipoteichoic acid although superoxide anion production does not occur and platelet activation is not associated with enhanced production by neutrophils

Activated platelets can contribute to host defense through release of products with bactericidal actions such as antimicrobial peptides and reactive oxygen species (ROS), as well as by forming heterotypic aggregates with neutrophils and enhancing their antimicrobial properties. Whilst release of vasoactive mediators from equine platelets in response to stimuli including bacterial lipopolysaccharide (LPS) has been documented, neither ROS production, nor the effects of activated platelets on equine neutrophil ROS production, have been reported. This study first sought evidence that activated equine platelets inhibit bacterial growth. Platelet superoxide production in response to stimuli including Escherichia coli-derived LPS and lipoteichoic acid (LTA) from Staphylococcus aureus was then determined. The ability of LPS and LTA to up-regulate platelet P-selectin expression and induce platelet–neutrophil aggregate formation was investigated and the effect of co-incubating activated platelets with neutrophils on superoxide production measured.Growth of E. coli was inhibited in a time-dependent manner, and to a similar extent, by addition of platelet rich plasma (PRP) or platelet poor plasma (PPP) obtained by centrifugation of PRP. Activation of platelets in PRP by addition of thrombin led to a significant increase in the inhibitory action between 0.5 and 2h.Although phorbol myristate acetate (PMA) caused superoxide production by equine platelets in a protein kinase C-dependent manner, thrombin, platelet activating factor (PAF), LPS, LTA and formyl-methionyl-leucyl phenylalanine (FMLP) were without effect. LPS and LTA did induce platelet activation, measured as an increase in P-selectin expression (% positive cells: 17±3 (un-stimulated); 63±6 (1μg/ml LPS); 64±6 (1μg/ml LTA); n=5) but not platelet superoxide production or heterotypic aggregate formation. Co-incubation of activated platelets with neutrophils did not increase neutrophil superoxide production.This study has demonstrated for the first time that when activated, equine platelets, like those of other species, are capable of releasing ROS that could assist in bacterial killing. However, the findings suggest that neither superoxide production by platelets nor enhancement of production by neutrophils is likely to play a significant role. Nevertheless, as has been reported in man, equine PPP and PRP did inhibit E. coli growth in vitro, and addition of thrombin significantly increased the inhibitory effect of PRP. This suggests that products released from activated platelets could contribute to antimicrobial activity in the horse. The factors in equine plasma and released by activated platelets that are responsible for inhibiting bacterial growth have yet to be determined.

Epinephrine, Cortisol, Endotoxin, Nutrition, and the Neutrophil

Neutrophil dysfunction has been documented after injury in animals and human beings. This review evaluates the relative effects of the hormonal and endotoxin response to injury on immune resistance. Review of the pertinent English-language literature. In volunteers given total parenteral nutrition, neutrophils demonstrate a robust response to leukotriene B4 but none to zymosan/activated serum or the bacterial metabolite formyl-methionyl-leucyl-phenylalanine (FMLP). This finding suggests subclinical exposure to activated complement and FMLP that does not occur during enteral feeding. Additional evidence of neutrophil activation is the release of lactoferrin to the same degree with the two routes of feeding. When normal volunteers are challenged with endotoxin, uniform impairment of the neutrophil response to chemotactic stimuli except LTB4 is demonstrated. Epinephrine increases the total circulating neutrophil pool for a few hours, whereas when cortisol is administered, the neutrophil counts continue to increase through 6 h. A combined epinephrine and cortisol infusion extends the half-life of neutrophils. The role of genomic and central nervous system control through the vagus nerve also is reviewed. Normal volunteers have provided insight into the stress response to infection that is understood only partially.

IgE-Dependent and IgE-Independent Stimulation of Human Basophils Increases the Presence of Immature FcεRIα by Reversing Degradative Pathways

Background: Expression of the high-affinity IgE receptor, FcεRI, on mast cells and basophils has previously been shown to be sensitive to the presence of IgE or cytokines. The current study examined whether stimulation of human basophils resulted in a change in the expression of FcεRI. Methods: Changes in the well-expressed immature intracellular form of the receptor, FcεRIα (p46), were examined by quantitative PCR, Western blot and the pulse-chase method. Results: Both IgE-dependent (anti-IgE antibody) and IgE-independent stimulation [formyl-methionyl-leucyl-phenylalanine (FMLP) and C5a] led to increased accumulation of p46. The p46 form of FcεRIα increased 1.52 ± 0.09-, 2.58 ± 0.09- and 1.47 ± 0.07-fold following stimulation with anti-IgE, FMLP and C5a, respectively. There were no changes in the steady-state levels of mRNA for FcεRIα. The kinetics of the increase in p46 was slow following stimulation with anti-IgE antibody, with the earliest increases observed after 8 h. The p46 form was degraded in a bafilomycin A (lysosomal inhibitor)-sensitive process. There was no synergy between treatment with bafilomycin A and anti-IgE or FMLP stimulation, suggesting that the 2 methods of enhancement operate on the same pathway. Pulse-chase studies corroborated this conclusion. In contrast, IL-3 and bafilomycin A synergistically increased p46, suggesting that IL-3 increased synthesis of FcεRIα. Conclusions: Taken together, these results suggest that secretagogue stimulation results in an increase in p46 due to reversal of degradative pathways rather than increased synthesis of FcεRIα. Nevertheless, a decrease in the degradation of FcεRIα at an intermediate step in its processing by non-FcεRI-dependent stimulation may still influence expression of this important receptor.

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren's syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.

Ca2+ Signaling Occurs via Second Messenger Release from Intraorganelle Synthesis Sites

SummaryCyclic ADP-ribose is an important Ca2+-mobilizing cytosolic messenger synthesized from β-NAD+ by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCβ and ARCγ are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a β-NAD+ transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca2+ wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.

Differential involvement of cyclooxygenase isoforms in neutrophil migration in vivo and in vitro

Pretreatment using celecoxib, a cyclooxygenase (COX) 2 inhibitor, or indomethacin, a nonselective COX inhibitor, reduced lypopolyssaccharide (LPS)-induced leukocyte migration to the rat peritoneal cavity. The effect of celecoxib (12 mg/kg) or indomethacin (2 mg/kg) on neutrophil chemotaxis induced by formyl-methionyl-leucyl-phenylalanine (FMLP) in an in vitro chemotactic assay (Boyden chamber) was investigated. Celecoxib and indomethacin inhibited chemotaxis induced by FMLP (Control = 26.6 ± 1.45, Celecoxib = 12.8 ± 3.04, Indomethacin = 6.26 ± 2.19 cells/field). When observed under intravital microscopy, a mouse cremaster preparation was used to assess the microvasculature to further investigate which step of cell recruitment was affected by these drugs. Celecoxib and indomethacin inhibited leukocyte migration induced by 0.05 μg/kg LPS injected into the cremaster muscle. However, the effect of celecoxib was associated with reduced cell rolling and adhesion, whereas indomethacin was only effective at inhibiting cell adhesion. Furthermore, SC560 pretreatment (a COX-1 selective inhibitor) of normal or LPS-challenged tissues did not alter leukocyte migration or cell adhesion, but it did enhance leukocyte rolling activity in both cases. Taken together, these results indicate that: 1) COX-1 activity is mainly related to leukocyte traffic under physiological conditions, and 2) COX-2 activity is mainly related to cell traffic under inflammatory conditions in vascular beds, suggesting a possible effect of selective COX-2 inhibitors on the expression of adhesion molecules.

PLUNC is a secreted product of neutrophil granules

Airway epithelia and neutrophils are frequently recruited to release host defense factors in response to a variety of pulmonary pathogens. One abundant product of airway epithelia is palate, lung, nasal epithelium clone (PLUNC), a proposed innate immune protein expressed in submucosal glands and surface airway epithelia. In this study, we report the expression of PLUNC in human neutrophils, a previously unrecognized source of this protein. Immunoblots performed on polymorphonuclear cell (PMN) lysates and PMN subcellular fractions indicated that PLUNC was present in the specific granules of the neutrophil. Furthermore, secretion assays demonstrated that PLUNC protein was released by neutrophils upon stimulation with secretogogues, including formyl methionyl leucyl phenylalanine and the calcium ionophore A23187. Although recombinant PLUNC protein failed to exhibit antibacterial activity in our studies, its storage and secretion by a professional phagocytic cell support the hypothesis that PLUNC participates in an aspect of the inflammatory response that contributes to host defense. These studies suggest that PLUNC expression is less restricted than previously believed, and highlight new avenues of research for the study of PLUNC function.

Cyclic ADP-Ribose Mediates Formyl Methionyl Leucyl Phenylalanine (fMLP)-Induced Intracellular Ca2+Rise and Migration of Human Neutrophils

Although cyclic ADP-ribose (cADPR), a novel Ca2+-mobilizing mediator, is suggested to be involved in the functions of neutrophils in rodents, its role in human neutrophils remains unclear. The present study examined the ability of cADPR to mobilize Ca2+and mediate formyl methionyl leucyl phenylalanine (fMLP)-stimulated increase in cytosolic free Ca2+concentration ([Ca2+]i) and migration in human neutrophils. cADPR induced Ca2+release from digitonin-permeabilized neutrophils, and the release was blocked by 8Br-cADPR, an antagonist of cADPR. Immunophilin ligands, FK506 and rapamycin, but not cyclosporine A, inhibited cADPR-induced Ca2+release. 8Br-cADPR partially reduced fMLP-induced [Ca2+]irise and abolished the rise in combination with 2APB, an IP3-receptor antagonist. Anti-CD38Ab and NADase that interfere with cADPR formation, reduced the fMLP-induced [Ca2+]irise. When β-NAD+, a substrate of ADP-ribosyl cyclase, and cADPR were added to the medium, the former gradually increased [Ca2+]iand the latter potentiated the fMLP-induced [Ca2+]irise. The β-NAD+–induced [Ca2+]irise in Ca2+-free medium was inhibited by anti-CD38Ab, 8Br-cADPR, FK506, ruthenium red, and thapsigargin. mRNAs of nucleoside transporter (NT), ENT1, ENT2, CNT, and CNT3 were expressed in neutrophils; and their inhibitors, inosine, uridine, and s-(4-nitrobenzyl)-6-thioinosine, reduced the [Ca2+]irise induced by β-NAD+and fMLP. fMLP-stimulated migration was inhibited by the removal of Ca2+from the medium or by the addition of 8Br-cADPR, anti-CD38Ab, NADase, and NT inhibitors. These results suggest that cADPR was synthesized extracellularly by CD38, transported into the cells through NTs, and then Ca2+was mobilized by FK506-binding protein–dependent process. This process may be involved in fMLP-induced intracellular Ca2+signaling and migration in human neutrophils.

Spreading of Neutrophils: From Activation to Migration

Neutrophils rely on rapid changes in morphology to ward off invaders. Time-resolved dynamics of spreading human neutrophils after activation by the chemoattractant fMLF (formyl methionyl leucyl phenylalanine) was observed by RICM (reflection interference contrast microscopy). An image-processing algorithm was developed to identify the changes in the overall cell shape and the zones of close contact with the substrate. We show that in the case of neutrophils, cell spreading immediately after exposure of fMLF is anisotropic and directional. The dependence of spreading area, A, of the cell as a function of time, t, shows several distinct regimes, each of which can be fitted as power laws ( A ∼ t b). The different spreading regimes correspond to distinct values of the exponent b and are related to the adhesion state of the cell. Treatment with cytochalasin-B eliminated the anisotropy in the spreading.

Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation

AbstractThe deregulation of inflammatory response during sepsis seems to reflect the overproduction of mediators, which suppress leukocyte functions. We investigated the intracellular mechanisms underlying the inability of neutrophils from severe septic patients to migrate toward chemoattractants. Patients with sepsis (52) and 15 volunteers were prospectively enrolled. Patients presented increased circulating levels of tumor necrosis factor-α, interferon-γ, interleukin (IL)–8, and IL-10. Patients showed reduced neutrophil chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or IL-8. No difference in the transcription or expression of the IL-8 receptor, CXCR1, was detected in neutrophils from controls and patients. However, septic neutrophils failed to increase tyrosine phosphorylation and actin polymerization in response to IL-8 or LTB4. In contrast, septic neutrophils, similar to controls, showed phagocytic activity that induced actin polymerization and augmented phosphotyrosine content. Treatment of control neutrophils with cytokines and lipopolysaccharide (LPS) to mimic endogenous septic environment inhibited actin polymerization and tyrosine phosphorylation in response to IL-8 or LTB4. High expression of G protein–coupled receptor kinase 2 (GRK2) and GRK5 was detected in septic neutrophils and control cells treated with cytokines plus LPS. Data suggest that endogenous mediators produced during sepsis might continually activate circulating neutrophils, leading to GRK activation, which may induce neutrophil desensitization to chemoattractants.

Impaired neutrophil functions in the pathogenesis of an outbreak of recurrent furunculosis caused by methicillin-resistant Staphylococcus aureus among mentally retarded adults

Staphylococcus aureus is an important cause of skin infections. We recently described an outbreak of recurrent furunculosis involving methicillin-resistant S. aureus among mentally retarded adults. We sought to determine the role of impaired neutrophil functions in its pathogenesis. Blood neutrophil functions were determined during both the outbreak (1997) and a disease-free period (2000). Chemotaxis was measured by migration toward formyl-methionyl-leucyl-phenylalanine (FMLP), specifically and randomly; phagocytosis of opsonized zymosan (OZ) was assessed by microscopy; superoxide production was determined by cytochrome c reduction in unstimulated neutrophils and after stimulation with 50 ng/ml phorbol myristate acetate, 1 mg/ml OZ or 5 × 10 −7 M FMLP. Functions were compared between recurrent furunculosis ( n = 10) and non-recurrent furunculosis patients ( n = 13). During 2000, functions were normal among the 23 subjects, except for specific/nonspecific chemotaxis (mean 68% ± 26 and 69% ± 28). During infection, recurrent furunculosis patients had a significantly increased basal superoxide production as compared to disease-free period (10.5 ± 4.7 vs. 4.9 ± 1.9 nmol O 2 −/10 6 cells/min, p = 0.003). During the disease-free period, recurrent furunculosis patients had lower basal superoxide production (4.9 ± 1.9 vs. 7.7 ± 3.5, p = 0.067) and impaired specific chemotaxis (57% ± 28 vs. 76% ± 21, p < 0.05) as compared to non-recurrent furunculosis patients. Only specific chemotaxis was an independent risk factor for recurrent furunculosis. Mentally retarded adults have impaired chemotaxis, with recurrent furunculosis cases having an even greater impairment. Abnormal specific chemotaxis is an independent risk factor for recurrent furunculosis. Impaired neutrophil functions thus have a role in the pathogenesis of outbreaks of recurrent furunculosis.

Effects of CNI-1493 on human granulocyte functions

During acute bacterial infections such as sepsis and meningitis, activation of inflammatory mediators such as nitric oxide (NO) plays a crucial role in both pathogenesis and host defense. We have previously reported that CNI-1493, a macrophage deactivator, reduced mortality in infant rats infected with Haemophilus influenzae type b (Hib) with associated decrease in the number of granulocytes in the infected tissue. The aim of the present study was to investigate how CNI-1493 affects granulocytes and macrophages in vitro. Murine macrophages (RAW 264.7) pre-incubated with CNI-1493 prior to activation with lipopolysaccharide (LPS)/interferon gamma (IFN γ) had decreased NO production measured as NO 2 − /NO 3 − levels and reduction in inducible NO-synthase (iNOS) expression. Reactive oxygen species (ROS) production was increased in formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated granulocytes following CNI-1493 treatment, whereas F-actin content, motility and chemotaxis were decreased under the same conditions. The effects of CNI-1493 on both NO production in LPS/IFN γ-activated macrophages and ROS production, F-actin content, motility and chemotaxis in granulocytes, may contribute to the reduced inflammatory response and increased survival in Hib-infected animals treated with CNI-1493.

Inappropriate neutrophil activation in venous disease.

Neutrophil oxygen radical production was studied in 18 limbs with class 2 or 3 venous disease and compared with that of nine normal limbs. Neutrophils were isolated from arm and leg venous samples. Free radical production was determined using chemiluminescence after stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) or the ester phorbol myristate acetate (PMA). The ratio of leg to arm luminescence was greater after FMLP stimulation in patients with venous disease (median 1.52 (95 per cent confidence interval (c.i.) 1.27-2.60)) than in controls (median 0.97 (95 per cent c.i. 0.70-1.12); P < 0.01). These changes were not observed with PMA (venous disease 1.16 (95 per cent c.i. 1.05-1.40); controls 0.95 (95 per cent c.i. 0.78-1.24)). There were fewer FMLP receptors on activated leg neutrophils (median 20.19 (95 per cent c.i. 3.58-51.42) fluorescence units) than arm neutrophils (median 36.03 (95 per cent c.i. 13.00-65.28) fluorescence units; P < 0.05), indicating an amplification of signal transduction. Intracellular calcium imaging demonstrated a larger release of calcium after stimulation of leg neutrophils (median 25.0 per cent (95 per cent c.i. 15.7-43.9 per cent)) compared with neutrophils from the arm (median 8.0 per cent (95 per cent c.i. 5.6-16.1 per cent); P = 0.04), demonstrating calcium-dependent activation. Neutrophils in patients with chronic venous disease inappropriately produce more oxygen free radical as a result of amplification of a calcium-dependent signal pathway.

Anthrax Lethal Toxin Paralyzes Neutrophil Actin‐Based Motility

Bacillus anthracis causes high-level bacteremia, strongly suggesting paralysis of the innate immune system. We have examined the effects of anthrax lethal toxin (LT) on human neutrophil chemotaxis, a process that requires actin filament assembly. Polymorphonuclear neutrophils (PMNs) treated with a sublethal concentration of LT (50 ng/mL) for 2 h demonstrated insignificant apoptosis or necrosis. However, this same concentration slowed human PMN formylmethionylleucylphenylalanine (FMLP)-stimulated chemokinesis by >60%, markedly reduced polar morphology, and rendered PMNs incapable of responding to a chemotactic gradient. These changes were accompanied by a >50% reduction in FMLP-induced actin filament assembly. One hour of exposure to LT failed to impair polarity or actin assembly, and the effects of LT were independent of mitogen-activated protein kinase kinase 1 inhibition. We conclude that 2 h of exposure to LT markedly impairs PMN actin assembly, and reductions in actin filament content are accompanied by a profound paralysis of PMN chemotaxis.

Characterization of Two Classes of Pancreatic Shock Factors: Functional Differences Exhibited by Hydrophilic and Hydrophobic Shock Factors

Pancreatic tissue homogenate induces a powerful pathophysiologic response sufficient to produce lethal shock in a rat. However, limited progress has been made in the biochemical characterization of these pancreas-derived active factors or their mechanisms of action. It has been shown that the pancreas is a major source of these shock-inducing factors and that they are generated by pancreatic proteinases. Porcine pancreas was homogenized and the filtered homogenate was subjected to organic extraction both before and after incubation for 2.5 h at 37 degrees C. The aqueous and lipid extracts of pancreatic homogenates were collected and analyzed for their ability to activate human neutrophils and to induce lethal shock in the rat. Neutrophil activation, a presumed hallmark of shock, was determined by chemiluminescence and myeloperoxidase (MPO) release. Only the intact homogenate and lipid extracts stimulated the neutrophils, and the aqueous extracts proved to be inactive. Neutrophils exhibited enhanced cellular activation when exposed to substimulatory levels of either formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF) followed by substimulatory levels of the lipid extracts, but not by the aqueous extracts. Both the lipid and aqueous extracts induced dramatic decreases in heart rate and blood pressure when injected in the rat, often resulting in lethal shock. In all cases, incubation of the homogenates at 37 degrees C enhanced the potency of the extracts. Our results demonstrated that the pancreas-derived homogenate and lipid factors were capable of inducing both neutrophil activation and shock. These results support the hypothesis that shock is produced via neutrophils that have been activated by inflammatory components. However, the shock-inducing factors in the aqueous extracts (i.e., hydrophilic fraction of the homogenate) apparently function via a pathway independent of neutrophil activation. This is the first evidence that there are both hydrophobic and hydrophilic factors generated in tissue homogenates capable of inducing shock, and that these different chemical classes of factors appear to function via separate mechanisms.