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Background: Host-directed therapies are a comparatively new and promising method for the treatment of tuberculosis. A variety of host pathways, vaccines and drugs have the potential to provide novel adjunctive therapies for the treatment of tuberculosis. In this connection, we have earlier reported the immunotherapeutic potential of N-formylated N-terminal peptide of glutamine synthetase of Mycobacterim tuberculosis H37Rv (Mir SA and Sharma S, 2014). Now in the present study, we investigated the immunotherapeutic effect of N-terminally formylated internal-peptide 'f- MLLLPD' of mycobacterial glutamine synthetase (Rv2220) in mouse model of tuberculosis., Methods: The N-terminally formylated peptide, f-MLLLPD was tested for its potential to generate Reactive Oxygen Species (ROS) in murine neutrophils. Further, its therapeutic effect alone or in combination with anti-tubercular drugs was evaluated in mouse model of tuberculosis., Results: The f-MLLLPD peptide treatment alone and in combination with ATDs reduced the bacterial load (indicated as colony forming units) in lungs of infected mice by 0.58 (p<0.01) and 2.92 (p<0.001) log10 units respectively and in their spleens by 0.46 (p<0.05) and 2.46 (p<0.001) log10 units respectively. In addition, the observed histopathological results correlated well with the CFU data., Conclusion: The results of the current study show that f-MLLLPD peptide confers an additional therapeutic efficacy to the anti-tuberculosis drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Exposition of neutrophils (polymorphonuclear neutrophils, PMNs) to bacterial products triggers exacerbated activation of these cells, increasing their harmful effects on host tissues. We evaluated the possibility of interfering with the classic immune innate responses of human PMNs exposed to bacterial endotoxin (lipopolysaccharide, LPS), and further stimulated with bacterial formyl peptide (N-formyl-methionine-leucine-phenylalanine, fMLP). We showed that the low- molecular-weight fucoidan (LMW-Fuc), a polysaccharide extracted from brown algae, attenuated the exacerbated activation induced by fMLP on LPS-primed PMNs, in vitro, impairing chemotaxis, NET formation, and the pro-survival and pro-oxidative effects. LMW-Fuc also inhibited the activation of canonical signaling pathways, AKT, bad, p47phox and MLC, activated by the exposition of PMN to bacterial products. The activation of PMN by sequential exposure to LPS and fMLP induced the release of L-selectin+ microparticles, which were able to trigger extracellular reactive oxygen species production by fresh PMNs and macrophages. Furthermore, we observed that LMW-Fuc inhibited microparticle release from activated PMN. In vivo experiments showed that circulating PMN-derived microparticles could be detected in mice exposed to bacterial products (LPS/fMLP), being downregulated in animals treated with LMW-Fuc. The data highlight the autocrine and paracrine role of pro-inflammatory microparticles derived from activated PMN and demonstrate the anti-inflammatory effects of LMW-Fuc on these cells., (© 2018 The Author(s) Published by S. Karger AG, Basel.)

N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF's divergent effects on FPR1 phosphorylation as well as PMN function.

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47 phox , p67 phox , p40 phox , and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA 11,22,28 ]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47 phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the "MAPK-p47 phox phosphorylation-NOX2 activation" axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.

We have previously observed that neutrophils from neonates exhibit different migratory responses to intermediate and end-target chemoattractants compared to adults. The aim of this study was to investigate the effect of the chemoattractants IL-8 (intermediate) and formyl-methionyl-leucyl-phenylalanine (fMLP; end-target) on cell surface receptor expression involved in adhesion, migration and granule release of neutrophils from term newborn infants and adults. Heparinized cord blood from 16 healthy term newborn infants delivered by caesarean section and peripheral blood from 17 healthy adults were incubated with 1 μm IL-8 or 0.1 μm fMLP, previously defined as optimal inducers of neutrophil migration. The leukocytes were labelled with antibodies to cell surface receptors (CD11b, CD15S, CD18, CD35, CD44, CD64, CD65, CD88, CD162, CD181 and CD182). Receptor expression was quantified by flow cytometry analysis. Upregulation of CD11b and downregulation of CD88 and CD182 after stimulation with IL-8 were more pronounced in adults than in neonates (P < 0.05, P < 0.05 and P ≤ 0.001, respectively), whereas fMLP induced changes in receptor expression that were of the same magnitude in neutrophils from neonates as from adults. We observed similar expression of receptors that mediate adhesion, migration, granule activation and phagocytosis induced by fMLP in neutrophils from neonates and adults. In contrast, differences between neonates and adults, induced by IL-8, suggest that the neutrophil response to intermediate chemoattractants might lead to a compromised infectious response in newborn infants., (© 2016 The Foundation for the Scandinavian Journal of Immunology.)

Local infiltration of inflammatory cells is regulated by a number of biological steps during which the cells likely penetrate through subendothelial basement membranes that contain heparan sulfate proteoglycans. In the present study, we examined whether administration of heparastatin (SF4), an iminosugar-based inhibitor of heparanase, could suppress local inflammation and degradation of heparan sulfate proteoglycans in basement membranes. In a carrageenan- or formyl peptide-induced dorsal air pouch inflammation model, the number of infiltrated neutrophils and monocytes was significantly lower in mice after topical administration of heparastatin (SF4). The concentration of chemokines MIP-2 and KC in pouch exudates of drug-treated mice was similar to control. In a zymosan-induced peritonitis model, the number of infiltrated cells was not altered in drug-treated mice. To further test how heparastatin (SF4) influences transmigration of inflammatory neutrophils, its suppressive effect on migration and matrix degradation was examined in vitro. In the presence of heparastatin (SF4), the number of neutrophils that infiltrated across a Matrigel-coated polycarbonate membrane was significantly lower, while the number of neutrophils passing through an uncoated membrane was not altered. Lysate of bone marrow-derived neutrophils released sulfate-radiolabeled macromolecules from basement membrane-like extracellular matrix, which was suppressed by heparastatin (SF4). Heparan sulfate degradation activity was almost completely abolished after incubation of lysate with protein G-conjugated anti-heparanase monoclonal antibody, strongly suggesting that the activity was due to heparanase-mediated degradation. Taken together, in a dorsal air pouch inflammation model heparastatin (SF4) potentially suppresses extravasation of inflammatory cells by impairing the degradation of basement membrane heparan sulfate., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Peptide-based drugs are promising group of compounds which are characterized by specificity to their in vivo targets and high potency of action (antineoplastic, immunoregulatory, antibacterial). The peptides, however, involve a relatively high risk of allergic reactions that are not predictable on the basis of their sequence and chemical properties. In this study, peripheral blood was obtained from 53 patients including 38 hypersensitive patients and 15 control patients. Basophil activation stimulated by two antibacterial peptides (camel, citropin 1.1), and acetylsalicylic acid was assessed by means of BAT (basophil activation test). Basophil activation stimulated by camel occurred in 7 of 38 patients with hypersensitivity (18.42%) as well as in 2 of 15 control patients (13.33%). Basophils were activated by citropin 1.1 in 7 of 38 hypersensitive patients (18.42%) and in none of the control patients. Using the Structural Database of Allergenic Proteins, we confirmed that the examined peptides share some structural similarities with common environmental allergens. Therefore, the cross-reactivity between potentially present anti-allergen IgE with examined peptides cannot be excluded. Our study proved that BAT, together with other biological tests and specific databases of allergenic compounds, may serve as an initial selection of new active peptides and proteins., (© 2015 John Wiley & Sons A/S.)

Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton's tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Inflammation after trauma is thought to be aggravated by intramedullary nailing (IMN) and predisposes to acute respiratory distress syndrome. Polymorphonuclear granulocytes (PMNs) are the main effector cells in this process. However, in patients with a femur fracture, the injury severity was the decisive factor for the PMN phenotype. A tibia fracture is often caused by a more moderate injury and might allow for a window to assess the innate immune response caused by IMN., Methods: A consecutive series of patients with a tibia fracture were included. The innate immune response was measured before and after IMN by plasma interleukin 6, PMN Mac1, and active FcγRII (FcγRII*) expression both before and after fMLF (N-formylmethionyl-leucyl-phenylalanine) stimulation. Furthermore, HLA-DR on monocytes was analyzed., Results: Twenty-five consecutive patients were included. Polymorphonuclear granulocyte fMLF-induced Mac1 and FcγRII* were decreased. In concordance, HLA-DR expression on monocytes was decreased in patients compared with control subjects. Intramedullary nailing was associated with a further decrease of HLA-DR-positive monocytes, whereas no changes in PMN phenotype or plasma interleukin 6 levels were observed., Conclusion: Intramedullary nailing of a tibial fracture did not affect the PMN phenotype. The impact from injury determined the PMN phenotype. In contrast, the monocyte phenotype changed after the additional insult by IMN in patients with an isolated tibial fracture.

Background: Human neutrophil alloantigen-3a (HNA-3a) antibodies can induce transfusion-related acute lung injury (TRALI). The severity of TRALI varies largely among the affected patients. Severe comorbidity seems to increase the susceptibility for TRALI, potentially by priming of neutrophils. Thus, the impact of neutrophil priming on HNA-3a antibody-mediated neutrophil aggregation and CD11b surface expression was investigated., Study Design and Methods: Neutrophils were primed using formyl-methionyl-leucyl-phenylalanine (fMLP) or bacterial lipopolysaccharide (LPS). Granulocyte aggregation and CD11b surface expression were evaluated by the granulocyte agglutination test and by flow cytometry (FC), respectively. Priming-induced changes in the surface expression of choline transporter-like protein 2 (CTL2) and the CTL2 mRNA expression were assessed by FC and quantitative real-time polymerase chain reaction, respectively., Results: Priming of neutrophils lowered the amount of HNA-3a antibodies required for inducing granulocyte aggregation in a dose-dependent manner by 50% to 75%. The priming agent concentration necessary for this response differed between donors. Priming slightly enhanced binding of HNA-3a antibodies to neutrophils. However, CTL2 de novo synthesis was not induced after priming with LPS, indicating that increased HNA-3a antibody binding was likely caused by translocation of intracellular CTL2 to the surface or by increased affinity of HNA-3a antibodies to CTL2. HNA-3a antibodies influenced CD11b surface expression on neutrophils only marginally, which was also not potentiated by priming with fMLP or LPS., Conclusion: This study provides experimental evidence supporting the "threshold model" of TRALI. Priming of neutrophils with fMLP or LPS increases their aggregation response to HNA-3a antibodies by lowering the required antibody amount., (© 2014 AABB.)

It has been acknowledged that neutrophil granulocytes, the common mediators of immune responses against extracellular bacteria, can also intercede autoimmune reactions such as experimental autoimmune encephalomyelitis (EAE). Formyl-methionyl-leucyl-phenylalanine (fMLP) is a microbial peptide that can be well-tolerated when intravenously administered and can directly lead to activation and accumulation of neutrophils into the blood circulation. Here, this antigenic peptide was injected to the mice at the induction of EAE, and the immunological and pathological outcomes were assessed. As a peripheral immune organ, spleen of the animals that received fMLP contained considerably high percentages of Gr-1(hi)Ly7/4(hi) mature neutrophils. In the sera samples, only a slight difference was determined in Th1/Th2/Th17-related cytokine levels. Expression of CXCR1 or CXCR2 chemokine receptors was not significantly modulated in EAE with or without fMLP which might indicate a direct role for this antigenic peptide on neutrophil accumulation. Even though fMLP administration did not propone the clinical (symptomatic) onset of the disease, the animals showed severe body conditions with higher EAE scores. Accordingly, the expression of TNF-α and CXCL1 inflammatory mediators in the brain was increased in fMLP-EAE group. In conclusion, with its potent capacity to mobilize neutrophils and to stimulate innate immune cells, fMLP peptide can be used to aggravate immune reactions in EAE. This observation may indicate that the strength of innate immune responses particularly at the induction phase of EAE might influence the clinical course of the disease.

In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Post injury immune dysfunction can result in serious complications. Measurement of biomarkers may guide the optimal timing of surgery in clinically borderline patients and therefore prevent complications., Aim: peri-operative measurement of neutrophil oxidative burst capacity as an indicator of the immune response to major orthopaedic surgical procedures., Methods: Prospective cohort study of trauma patients aged ≥16 yrs with pelvic, acetabular, femoral shaft or tibial shaft fractures requiring surgical intervention. Blood samples were taken immediately pre-op and at 30 min, 7, 24 and 72-9 6 h post-operatively. Neutrophil oxidative burst capacity was measured both with and without stimulation by formyl-methionyl-leucyl-phenylalanine (fMLP, a chemotactic factor). Clinical outcomes measured were mortality, length of stay, MOF, pneumonia, acute respiratory distress syndrome (ARDS) and sepsis., Results: 100 consecutive orthopaedic trauma patients were enrolled over a 16 month period. 78% were male, with a mean age of 42 ± 18 years and an average ISS of 19 ± 13. Neutrophil oxidative burst capacity was significantly elevated at 7 h (p = 0.006) and 24 h (p = 0.022) post operatively. Patients who developed infective complications (pneumonia and sepsis) had higher levels of oxidative burst capacity pre-operatively (pneumonia: 1.52 ± 0.93 v 0.99 ± 0.66 p = 0.032, sepsis: 1.39 ± 0.86 v 0.97 ± 0.56 p = 0.024) and at 24 h post op (pneumonia: 2.72 ± 2.38 v 1.12 ± 0.63 p = < 0.001, sepsis: 2.16 ± 2.09 v 1.10 ± 0.54 p = < 0.001). When analysed by operation type, no statistical difference was seen between major and minor operations. No correlation was found between length of stay, length of ICU stay, ISS or age and neutrophil oxidative burst capacity at any time point., Conclusions: Neutrophil oxidative burst capacity response to orthopaedic trauma surgery is associated with the infective post injury complications. There was no correlation between magnitude of injury or operation and oxidative burst capacity. These results are promising for the development of tools for prediction of post-operative complications and guidance for optimal timing for surgical intervention., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A novel benzoylphloroglucinol derivative, garcimultiflorone G (1), was isolated from the fruits of Garcinia multiflora. The structure of 1 was determined through extensive 1D- and 2D-NMR, and MS analyses. Garcimultiflorone G (1) showed inhibitory effects against superoxide anion (O·2(-) generation and elastase release by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB), with IC50 values of 6.97 ± 1.56 and 11.70 ± 1.58 μM, respectively., (Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.)

The MHC class I family comprises both classical (class Ia) and non-classical (class Ib) members. While the prime function of classical MHC class I molecules (MHC class Ia) is to present peptide antigens to pathogen-specific cytotoxic T cells, non-classical MHC-I (MHC class Ib) antigens perform diverse array of functions in both innate and adaptive immunity. Vaccines against intracellular pathogens such as Mycobacterium tuberculosis need to induce strong cellular immune responses. Recent studies have shown that MHC class I molecules play an important role in the protective immune response to M. tuberculosis infection. Both MHC Ia-restricted and MHC class Ib-restricted M. tuberculosis -reactive CD8(+) T cells have been identified in humans and mice, but their relative contributions to immunity is still uncertain. Unlike MHC class Ia-restricted CD8(+) T cells, MHC class Ib-restricted CD8(+) T cells are constitutively activated in naive animals and respond rapidly to infection challenge, hence filling the temporal gap between innate and adaptive immunity. The present review article summarizes the general host immunity against M. tuberculosis infection highlighting the possible role of MHC class Ib molecule, H2-M3 and their ligands (N-formylated peptides) in protection against tuberculosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Staphylococcus aureus is the most common pathogen causing septic arthritis in humans. The affected joints are often rapidly and permanently damaged despite antibiotic treatment, indicating that the elicited host immune response contributes substantially to joint destruction. Bacterial formylated peptides are important chemotactic molecules mediating neutrophil recruitment into infected tissues as an important first step of host defense against invading bacteria. The role of formylated peptides in S. aureus infections has been unknown., Methods: Mice were intravenously inoculated with wild-type S. aureus strain RN4220 or its isogenic mutant strain (Δfmt) lacking the ability to produce formylated peptides. The development of arthritis was followed clinically and histopathologically., Results: Mice inoculated with the formyl peptide-producing wild-type strain showed a significantly increased frequency and severity of arthritis and subsequent joint destruction as compared with Δfmt mutant strain-inoculated mice. The wild-type S. aureus strain also induced significantly more weight loss than the Δfmt mutant strain. The recruitment of neutrophils into infected kidneys and synovial tissue was significantly higher in mice inoculated with the wild-type strain., Conclusions: Our data show that formylated peptides function as important virulence factors in S. aureus arthritis, partly by mediating neutrophil recruitment, which contributes substantially to the joint damage.

Objectives: Tigecycline is the prototype of the recently introduced, intravenously administered glycylcycline class of antibiotics, developed in response to the increasing problem of antibiotic resistance in Gram-positive bacteria, especially Staphylococcus aureus, as well as Gram-negative bacteria and anaerobes. However, relatively little is known about the immunomodulatory potential of tigecycline, specifically its interactions with human neutrophils. In the current study we investigated the effects of tigecycline at therapeutically relevant concentrations and greater (0.625-10 mg/L) on alterations in cytosolic Ca(2+) concentrations, generation of antimicrobial reactive oxygen species (ROS) and release of granule proteases [elastase, matrix metalloproteinase-8 (MMP-8) and matrix metalloproteinase-9 (MMP-9)] by human blood neutrophils activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 μM)., Methods: Cytosolic Ca(2+) concentrations were measured using fura-2/AM-based spectrofluorimetry and radiometric procedures, generation of ROS by oxygen consumption and myeloperoxidase-mediated auto-iodination, and protease release by ELISA procedures., Results: Exposure of the cells to fMLP resulted in activation of the generation of ROS, as well as release of the granule proteases, all of which were significantly increased by pre-incubation of the cells with tigecycline in a dose-dependent manner. Tigecycline-mediated enhancement of these neutrophil functions was associated with elevations in the concentrations of cytosolic Ca(2+), which appeared to result from the Ca(2+) ionophore activity of tigecycline., Conclusions: Tigecycline, by functioning as a Ca(2+) ionophore, and independent of antimicrobial activity, potentiates the pro-inflammatory functions of human neutrophils in vitro.

Polymorphonuclear neutrophils (PMN) play a key role in host defenses against invading microorganisms but also potentiate inflammatory reactions in case of excessive or misdirected responses. Release of the alarmin high-mobility group box 1 (HMGB1) by cells that die at an inflammatory site may act as an alert signal for the immune system. We studied the effect of HMGB1 on human PMN migration, using whole-blood samples to avoid cell activation associated with isolation procedures. HMGB1 50-100 ng/ml reduced baseline PMN migration as well as formyl-methionyl-leucyl-phenylalanine- and IL-8-induced PMN chemotaxis. This inhibitory effect was mediated by the RAGE receptor. In contrast, a higher HMGB1 concentration (5,000 ng/ml) had a chemoattractant effect on PMN through IL-8 production. This effect required the engagement of Toll-like receptors 2 and 4 in addition to the RAGE receptor. The A box component of HMGB1, which antagonizes the endogenous protein, reduced chemotaxis and also strongly inhibited the enhancement of PMN migration observed with the highest HMGB1 concentration. In contrast, the B box, reported to be the active form of HMGB1, exerted a chemoattractant effect. These results strongly point to a key regulatory role of HMGB1 in PMN recruitment to inflammatory tissues. The A box component could potentially serve to inhibit inappropriate PMN recruitment during chronic inflammatory disorders associated with excessive HMGB1 release., (Copyright © 2011 S. Karger AG, Basel.)

ATP and hydrolysis products of ATP like adenosine regulate the chemotaxis of neutrophils by activating purinoceptors and adenosine receptors. The present study was designed to examine exogenous ATP, activation of purinoceptors, and activation of A(3) adenosine receptor as key steps in the signal cascades that control cell orientation and migration of rat neutrophils. One or more of those steps might be potential therapeutic targets for treatment of inflammatory diseases. The chemotaxis of rat neutrophils was stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured with an EZ-TAXIScan apparatus. The effects of apyrase, exogenous ATP, suramin (P2X and P2Y blocker), PPADS (a P2X blocker), TNP-ATP (P2X(1) and P2X(3) antagonist), and Reactive Blue 2 (a P2Y blocker) on the chemotactic response were also investigated. Rat neutrophil chemotaxis was significantly suppressed by apyrase. fMLP induced rat neutrophil chemotaxis was potentiated by ATP, blocked by suramin, not affected by PPADS or TNP-ATP, and significantly inhibited by RB-2. Western blotting showed that A(3), P2Y(2), and P2Y(11) were expressed in rat neutrophils. The chemotactic response of rat neutrophils to fMLP stimulation is potentiated by ATP via P2Y(11) purinoceptors but not P2X purinoceptors or A(3) adenosine receptor, and that the response plays a critical role in host defense and pathogenicity.

Rationale: There are increased neutrophils in the lungs of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this is due to increased inflammatory signal or related to the inherent behavior of the neutrophils. This is critical, because inaccurate or excessive neutrophil chemotaxis could drive pathological accumulation and tissue damage., Objectives: To assess migratory dynamics of neutrophils isolated from patients with COPD compared with healthy smoking and nonsmoking control subjects and patients with α(1)-antitryspin deficiency., Methods: Migratory dynamics and structure were assessed in circulating neutrophils, using phase and differential interference contrast microscopy and time-lapse photography. The effect of COPD severity was studied. Surface expression of receptors was measured using flow cytometry. The in vitro effects of a phosphoinositide 3-kinase inhibitor (LY294002) were studied., Measurements and Main Results: COPD neutrophils moved with greater speed than cells from either control group but with reduced migratory accuracy, in the presence of IL-8, growth-related oncogene α, formyl-methionyl-leucyl-phenylalanine, and sputum. This was present across all stages of COPD. Structurally, COPD neutrophils formed fewer pseudopods during migration. There were no differences in surface expression of the receptors CXCR1, CXCR2, or FPR1. LY294002 reduced COPD neutrophil migratory speed while increasing chemotactic accuracy, returning values to normal. The inhibitor did not have these effects in healthy control subjects or patients with a similar degree of lung disease., Conclusions: COPD neutrophils are intrinsically different than cells from other studied populations in their chemotactic behavior and migratory structure. Differences are not due to surface expression of chemoattractant receptors but instead appear to be due to differences in cell signaling.

Recent in vitro data have suggested that the flavonoid quercetin (1) does not affect the functioning of neutrophils. Therefore, we evaluated in vivo and in vitro whether or not 1 affects neutrophil function, focusing on recruitment. The in vivo treatment with 1 inhibited in a dose-dependent manner the recruitment of neutrophils to the peritoneal cavity of mice induced by known chemotatic factors such as CXCL1, CXCL5, LTB(4), and fMLP. Furthermore, 1 also inhibited in a concentration-dependent manner the chemoattraction of human neutrophils induced by CXCL8, LTB(4), and fMLP in a Boyden chamber. In vitro treatment with 1 did not affect human neutrophil surface expression of CXCR1, CXCR2, BLT1, or FLPR1, but rather reduced actin polymerization. These results suggest that 1 inhibits actin polymerization, hence, explaining the inhibition of neutrophil recruitment in vivo and in vitro and highlighting its possible usefulness to diminish excessive neutrophil migration during inflammation.

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., (Copyright © 2010 S. Karger AG, Basel.)

In the host immune system, leukocytes are often exposed to multiple inflammation inducers. NF-κB is of considerable importance in leukocyte function because of its ability to activate the transcription of many proinflammatory immediate-early genes. Tremendous efforts have been made toward understanding how NF-κB is activated by various inducers. However, most research on NF-κB regulation has been focused on understanding how NF-κB is activated by a single inducer. This is unlike the situation in the human immune system where multiple inflammation inducers, including both exogenous and endogenous mediators, are present concurrently. We now present evidence that the formylated peptide f-Met-Leu-Phe (fMLP), a bacterial chemoattractant, synergizes with TNFα to induce NF-κB activation and the resultant inflammatory response in vitro and in vivo. The mechanism of synergistic activation of NF-κB by bacterial fMLP and TNFα may be involved in the induction of RelA acetylation, which is regulated by p38 MAPK. Thus, this study provides direct evidence for the synergistic induction of NF-κB-dependent inflammatory responses by both exogenous and endogenous inducers. The ability of fMLP to synergize with TNFα and activate NF-κB represents a novel and potentially important mechanism through which bacterial fMLP not only attracts leukocytes but also directly contributes to inflammation by synergizing with the endogenous mediator TNFα.

Local anesthetics possess a wide range of anti-inflammatory properties through their effects on neutrophils. However, limited information is available on the effects of ropivacaine (a new local anesthetic) on neutrophil function. The aim of this study was to investigate anti-inflammatory properties of ropivacaine with regard to its effects on the expression and function of CD11b in human neutrophils. CD11b expression was examined by flow cytometry and its function was determined by measuring adhesion of neutrophils to human umbilical vein endothelial cells (HUVECs). Ropivacaine inhibited CD11b expression in formyl-methionyl-leucyl-phenylalanine (fMLP)-activated neutrophils in a concentration-dependent manner, but not in a time-dependent manner. The inhibitory potency of ropivacaine was similar to that of bupivacaine and levobupivacaine, but was more potent than that of lidocaine. The up-regulation of CD11b induced by platelet-activating factor (PAF) priming was also inhibited by ropivacaine. fMLP increased adhesion of neutrophils to HUVECs, which was inhibited by ropivacaine. In addition, ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA), a chelator of extracellular Ca(2+). However, ropivacaine showed no effect on the fMLP-induced CD11b expression in the presence of butan-1-ol, a blocker of phospholipase D (PLD) pathway, which completely inhibited the fMLP-induced CD11b expression in neutrophils. Our results suggest that ropivacaine exerts anti-inflammatory activity, and this appears to be mediated through inhibiting the expression and function of adhesion molecule CD11b in neutrophils., (Copyright 2010 Elsevier B.V. All rights reserved.)

Injury causes a systemic inflammatory response syndrome (SIRS) that is clinically much like sepsis. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors. Similarly, cellular injury can release endogenous 'damage'-associated molecular patterns (DAMPs) that activate innate immunity. Mitochondria are evolutionary endosymbionts that were derived from bacteria and so might bear bacterial molecular motifs. Here we show that injury releases mitochondrial DAMPs (MTDs) into the circulation with functionally important immune consequences. MTDs include formyl peptides and mitochondrial DNA. These activate human polymorphonuclear neutrophils (PMNs) through formyl peptide receptor-1 and Toll-like receptor (TLR) 9, respectively. MTDs promote PMN Ca(2+) flux and phosphorylation of mitogen-activated protein (MAP) kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTDs can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These signal through innate immune pathways identical to those activated in sepsis to create a sepsis-like state. The release of such mitochondrial 'enemies within' by cellular injury is a key link between trauma, inflammation and SIRS.

Ca(2+) and Sr(2+) entry pathways activated by pro-inflammatory agonists FMLP, LTB(4) and PAF have been compared to thapsigargin in human neutrophils. 2-APB (10microM) increased Ca(2+) influx and to a greater extent in agonist than in thapsigargin stimulated neutrophils. This action of 2-APB was specific to Ca(2+) because 2-APB did not augment Sr(2+) entry in agonist and thapsigargin stimulated neutrophils. This suggests that Ca(2+) and Sr(2+) entry can be used to discriminate between receptor and non-receptor (store)-operated Ca(2+) influx. Our data show for the first time that Pyr3 whilst partially inhibiting agonist induced Ca(2+) influx almost completely abolished Ca(2+) influx after thapsigargin stimulation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Hepatic neutrophil adhesion during endotoxemia is an integrin-independent, CD44-dependent process. Because integrins function in other endotoxemic vasculatures, we used spinning disk confocal intravital microscopy to assess whether LPS down-modulated integrin functions in sinusoids. First, we applied fMLP onto the liver surface, and compared it with systemic LPS administration. Local fMLP caused neutrophil adhesion, crawling, and emigration for at least 2 h. Surprisingly, the number of adherent and crawling neutrophils was markedly reduced in Mac-1(-/-) and ICAM-1(-/-) mice, but not in mice treated with anti-CD44 mAb. By contrast, systemic LPS injection induced a robust accumulation of neutrophils in sinusoids, which was dependent on CD44, but not on integrins. Strikingly, local fMLP could not induce any integrin-dependent adhesion in endotoxemic mice treated with anti-CD44 mAb, indicating that Mac-1-dependent neutrophil adhesion was inhibited by LPS. This response was localized to the hepatic microvasculature because neutrophils still adhered via integrins in brain microvasculature. ICAM-1/ICAM-2 levels were not decreased, but following LPS treatment, Mac-1 was down-regulated in neutrophils localized to liver, but not in the circulation. Mac-1 down-regulation in neutrophils was not observed in IL-10(-/-) mice. In vitro neutrophil incubation with IL-10 induced direct decrease of Mac-1 expression and adhesivity in LPS-stimulated neutrophils. Therefore, our data suggest that Mac-1 is necessary for neutrophil adhesion and crawling during local inflammatory stimuli in sinusoids, but during systemic inflammation, neutrophils are exposed to high concentrations of IL-10, leading to a CD44-dependent, integrin-independent adhesion. This may be a mechanism to keep neutrophils in sinusoids for intravascular trapping.

In inflammatory diseases, circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not prevent neutrophil recruitment effectively. The Slit family of secreted proteins and their transmembrane receptor, Robo, repel axonal migration during CNS development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types toward a variety of chemotactic factors in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration but not random movement of neutrophils toward fMLP. Slit2 also inhibited neutrophil migration toward other chemoattractants, namely C5a and IL-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2 but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and MIP-2. In all instances, Slit2 reduced neutrophil recruitment effectively (P<0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis but not random motion of circulating neutrophils and point to Slit2 as a potential new therapeutic for preventing localized inflammation.

Background: Aggressive periodontitis (AgP) is associated with impaired polymorphonuclear leukocyte (PMN) chemotaxis toward bacterial N-formylpeptides. Formylpeptide receptors (FPRs) play a major role in guiding PMNs to infection sites. Previous work revealed a significant association between FPR1 single nucleotide polymorphism (SNP) 348T>C and AgP in African Americans. We tested the hypothesis that 348T impairs PMN chemotaxis by decreasing FPR mRNA expression, thereby increasing susceptibility to AgP., Methods: Blood samples were obtained from African American subjects (37 AgP cases and 38 controls). Chemotaxis to N-formyl-methionine-leucine-phenylalanine by freshly isolated PMNs was assayed in a modified Boyden chamber. RNA was isolated from PMNs, and FPR1 gene expression was quantified by real-time polymerase chain reaction (PCR). To detect FPR1 5' SNPs, genomic DNA was isolated, and four fragments spanning the FPR1 5' region were PCR-amplified and sequenced. Haplotype associations between SNP 348T>C and 5' SNPs were analyzed., Results: The homozygous 348T genotype was only found in AgP cases (P = 0.017; odds ratio, 18.9). Subjects with this genotype exhibited a significantly lower PMN chemotactic response relative to controls and to subjects with the 348C/C or 348T/C genotype (P <0.05). There were no significant differences in PMN FPR1 expression among subjects with the 348C/C, 348T/C, and 348T/T genotypes. Eleven FPR1 5' SNPs were detected, but none of the predicted haplotypes reflected associations with AgP or with 348T., Conclusions: Although the 348T/T genotype is relatively rare, it is associated with significantly impaired PMN chemotaxis and an increased risk for developing AgP in African Americans. These associations do not seem to be related to significant reductions in FPR1 transcripts in subjects expressing 348T.

Although green tea polyphenol catechin is considered as a potential anti-inflammatory agent, its effect on bacterial component-induced inflammation has been poorly investigated. We examined the capacity of epigallocatechin gallate (EGCG) to regulate leukocyte responses to bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), which is recognized by a human G protein-coupled receptor FPR on phagocytic leukocytes. Pretreatment of human monocytic cells or FPR-transfected rat basophilic leukemia cells (ETFR cells) with EGCG significantly inhibited fMLF-induced chemotaxis. Intraperitoneal administration of EGCG in mice suppressed fMLF-induced leukocyte infiltration into the air pouch created in the skin. Mechanistic studies revealed that EGCG dose-dependently suppressed fMLF-induced calcium flux in monocytic cells and ETFR cells. fMLF-induced ETFR cell migration was significantly inhibited by a specific MEK1/2 inhibitor, PD98059, which was associated with reduction in fMLF-induced ERK1/2 phosphorylation. These results suggest that EGCG inhibits FPR-mediated leukocyte activation thus is a promising anti-inflammatory compound.

The production of reactive oxygen species (ROS) in activated neutrophils is catalyzed by NADPH oxidase, a multiproteins complex. Various protein kinases including protein kinase C (PKC) and mitogen activated protein kinases (MAPKs) are involved in NADPH oxidase phosphorylation and activation. The main step in the NADPH oxidase activation is phosphorylation of its cytosolic protein p47(phox). We found previously that nitric oxide (NO) donors such as metabolite of molsidomine-SIN-1 and diethylamine/NO significantly impaired the ROS production in activated human neutrophils. In this study, we investigated the effects of both NO donors on NADPH oxidase-linked signaling proteins in activated neutrophils. We found that NO donors decreased the phosphorylation of p47(phox) on tyrosine and serine/threonine residues and PKC on serine residues in neutrophils. Both NO donors did not affect the phosphorylation of MAPKs. NO donors partially but significantly lost their ability to reduce ROS production in the presence of PKC but not MAPKs inhibitors. We show that whereas NO donors have no effect on MAPKs activity, they do decrease PMA- and/or fMLP-induced phosphorylation of p47(phox) and PKC as well as PMA- and fMLP-induced ROS production.

Neutrophils (polymorphonuclear leukocytes [PMNs]) are critical to the immune response, including clearance of infectious pathogens. Sepsis is associated with impaired PMN function, including chemotaxis. PMNs express peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a ligand-activated nuclear transcription factor involved in immune and inflammatory regulation. The role of PPAR-gamma in PMN responses, however, is not well characterized. We report that freshly isolated human PMNs constitutively express PPAR-gamma, which is up-regulated by the sepsis-induced cytokines TNF-alpha and IL-4. PMN chemotactic responses to formylmethionyl-leucyl-phenylalanine (fMLP) and IL-8 were dose-dependently inhibited by treatment with the PPAR-gamma ligands troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and by transfection of PMN-like HL-60 cells with a constitutively active PPAR-gamma construct. Inhibition of chemotaxis by PPAR-gamma ligands correlated with decreases in extracellular signal-regulated kinase-1 and -2 activation, actin polymerization, and adherence to a fibrinogen substrate. Furthermore, PMN expression of PPAR-gamma was increased in sepsis patients and mice with either of 2 models of sepsis. Finally, treatment with the PPAR-gamma antagonist GW9662 significantly reversed the inhibition of PMN chemotaxis and increased peritoneal PMN recruitment in murine sepsis. This study indicates that PPAR-gamma activation is involved in PMN chemotactic responses in vitro and may play a role in the migration of these cells in vivo.

Exposure of neutrophils to either lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) is associated with changes in the expression of cell adhesion molecules and elevation of intracellular calcium ions. Although dietary flavonoids are reported to possess anti-inflammatory properties, little is known regarding the effect of their metabolites. We have investigated the effects of quercetin and its major metabolites on LPS and fMLP-stimulated human neutrophils using concentrations comparable to those reported in feeding studies on human volunteers. The metabolite quercetin 3-glucuronide caused a significant reduction in fMLP-evoked calcium influx in human neutrophils (approximately 35%), while neither quercetin 3'-sulfate nor quercetin produced a similar change. Acute exposure of human neutrophils to LPS altered cell shape and surface expression of CD16, but neither of these events were significantly altered by quercetin, quercetin 3-glucuronide nor quercetin 3'-sulfate. In addition, LPS caused a fivefold up-regulation in the expression of beta(2)-integrin (CD11b/Mac 1) and a concomitant 70% down-regulation of L-selectin (CD62L) adhesion molecule expression in human neutrophils. Neither effect was altered by quercetin, quercetin 3-glucuronide or quercetin 3'-sulfate. In conclusion, we found that acute exposure to quercetin and quercetin 3'-sulfate does not affect either expression of cell adhesion molecules or the elevation of intracellular calcium ions in response to LPS and fMLP in human neutrophils. However, quercetin 3-glucuronide reduced fMLP-evoked calcium responses. While this study highlights that metabolites of quercetin may possess different biological properties, dietary ingestion of quercetin is unlikely to exert a major effect on neutrophil function in vivo.

The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1-10 nM and superoxide production at 10-100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are approximately 100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice.

Human neutrophil migratory responses to Toll-like receptor (TLR) agonists were studied using videomicroscopy. When challenged with lipopolysaccharide (LPS, TLR4 agonist) or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (P3CSK4, TLR2 agonist), neutrophils displayed enhanced motility, which was found to reflect increased random migration but not directed migration (chemotaxis). Enhanced neutrophil motility was detected within 10 min after stimulation with LPS or P3CSK4, and was sustained for more than 80 min. Stimulation of neutrophils with LPS or P3CSK4 resulted in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), which preceded neutrophil migration. TLR-mediated neutrophil migration was strongly suppressed by pretreatment of cells with U0126 (MAPK/ERK kinase inhibitor) but not with U0124 (an inactive analogue of U0126) or SB203580 (a p38 MAPK inhibitor), and was almost completely abolished by pretreatment of cells with U0126 and SB203580 in combination. Randomly migrating neutrophils in response to LPS or P3CSK4 displayed directed migration when further challenged with gradient concentrations of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF). These findings indicate that TLR agonists stimulate human neutrophil migration via the activation of ERK and p38 MAPK, and FMLP- or PAF-induced neutrophil chemotaxis is not affected by the pre-exposure of cells to TLR agonists.

Despite the development of new potent antibiotics, Streptococcus pneumoniae remains the leading cause of death from bacterial pneumonia. Polymorphonuclear neutrophil (PMN) recruitment into the lungs is a primordial step towards host survival. Bacterium-derived N-formyl peptides (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) and host-derived chemokines (KC and macrophage inflammatory protein 2 [MIP-2]) are likely candidates among chemoattractants to coordinate PMN infiltration into alveolar spaces. To investigate the contribution of each in the context of pneumococcal pneumonia, CD1, BALB/c, CBA/ca, C57BL/6, and formyl peptide receptor (FPR)-knockout C57BL/6 mice were infected with 10(6) or 10(7) CFU of penicillin/erythromycin-susceptible or -resistant serotype 3 or 14 S. pneumoniae strains. Antagonists to the FPR, such as cyclosporine H (CsH) and chenodeoxycholic acid, or neutralizing antibodies to KC and MIP-2 were injected either 1 h before or 30 min after infection, and then bronchoalveolar lavage fluids were obtained for quantification of bacteria, leukocytes, and chemokines. CsH was effective over a short period after infection with a high inoculum, while anti-CXC chemokine antibodies were effective after challenge with a low inoculum. CsH prevented PMN infiltration in CD1 mice infected with either serotype 3 or 14, whereas antichemokine antibodies showed better efficacy against the serotype 3 strain. When different mouse strains were challenged with serotype 3 bacteria, CsH prevented PMN migration in the CD1 mice only, whereas the antibodies were effective against CD1 and C57BL/6 mice. Our results suggest that fMLP and chemokines play important roles in pneumococcal pneumonia and that these roles vary according to bacterial and host genetic backgrounds, implying redundancy among chemoattractant molecules.

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni(2+)-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45-75 kDa species (peak M(r) approximately 60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of M(r) approximately 40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting approximately 60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337-346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Delta[Ca(2+)](i) and DeltapH(i)) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca(2+)](i), mostly from intracellular stores, simultaneously with a drop in pH(i); these are followed by a drop in [Ca(2+)](i) and a rise in pH(i), with the latter being due to a Na(+)/H(+) antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca(2+)](i) is restored, 10(-7) M fMLP, previously shown to elicit maximal Delta[Ca(2+)](i) but no bactericidal functions, did not prevent the cells' responses with Delta[Ca(2+)](i) to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 mug/ml IC, previously shown to elicit maximal Delta[Ca(2+)](i) and bactericidal functions, exhibited no subsequent Delta[Ca(2+)](i) or DeltapH(i) to either stimulus. While exposure to 10(-7) M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10(-5) M fMLP did, presumably via the low-affinity receptor, using a different Ca(2+) source.

The transmigration of peripheral human monocytes to the interstitium is a fundamental step in the host-defence mechanism against infections. Little is known about the state of function of in vivo transmigrated interstitial monocytes prior to differentiation into macrophages and dendritic cells. We hypothesized that newly recruited interstitial monocytes have a preserved responsiveness against bacterial-related peptides, giving them a specific role in the immediate defence against invading pathogens. In order to test this hypothesis, we explored the responsiveness of in vivo transmigrated as well as peripheral monocytes, in terms of CD11b expression and H(2)O(2) production towards the bacterial-related peptide formylmethionylleucylphenylalanine (fMLP) by the use of a skin chamber technique. In addition, we analysed the concentration of interleukin (IL)-8, monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor (TNF)-alpha in the skin blister exudates and in the circulation. We demonstrate that in vivo-transmigrated monocytes had a fivefold higher CD11b expression compared to monocytes obtained from the peripheral circulation. fMLP exposure induced a significantly higher CD11b expression on transmigrated cells compared to peripheral monocytes. In addition, newly recruited monocytes had a preserved H(2)O(2) production. The interstitial concentration of IL-8, MCP-1 and TNF-alpha was significantly higher in blister exudates compared to that in the peripheral circulation. Thus, in vivo transmigrated human monocytes preserve their capacity to respond towards bacterial peptides in terms of CD11b up-regulation and H(2)O(2) generation. These data strengthen a role for newly recruited interstitial human monocytes in the immediate defence against invading pathogens.

About 40 yr ago, two groups of investigators identified a new class of immunoglobulins, IgE. By exchanging their results and reagents, they proved that the immunoglobulin responsible for immediate hypersensitivity was IgE. From that day forward the science of allergy was greatly advanced. Within a few years of the IgE discovery, an assay for IgE was developed. This test was named the radio allergosorbent test. The specific IgE testing methodology has matured in the last four decades. Different means of detecting IgE bound to allergen is the subject of this review. We have included methods for measuring specific IgE using the ImmunoCAP 1000 instrument. The methodology for measuring basophile histamine release is also detailed in this chapter.

Neutrophil beta(2) integrins are activated by inside-out signaling regulating integrin affinity and valency; following ligand binding, beta(2) integrins trigger outside-in signals regulating cell functions. Addressing inside-out and outside-in signaling in hck(-/-)fgr(-/-) neutrophils, we found that Hck and Fgr do not regulate chemoattractant-induced activation of beta(2) integrin affinity. In fact, beta(2) integrin-mediated rapid adhesion, in static condition assays, and neutrophil adhesion to glass capillary tubes cocoated with ICAM-1, P-selectin, and a chemoattractant, under flow, were unaffected in hck(-/-)fgr(-/-) neutrophils. Additionally, examination of integrin affinity by soluble ICAM-1 binding assays and of beta(2) integrin clustering on the cell surface, showed that integrin activation did not require Hck and Fgr expression. However, after binding, hck(-/-)fgr(-/-) neutrophil spreading over beta(2) integrin ligands was reduced and they rapidly detached from the adhesive surface. Whether alterations in outside-in signaling affect sustained adhesion to the vascular endothelium in vivo was addressed by examining neutrophil adhesiveness to inflamed muscle venules. Intravital microscopy analysis allowed us to conclude that Hck and Fgr regulate neither the number of rolling cells nor rolling velocity in neutrophils. However, arrest of hck(-/-)fgr(-/-) neutrophils to >60 microm in diameter venules was reduced. Thus, Hck and Fgr play no role in chemoattractant-induced inside-out beta(2) integrin activation but regulate outside-in signaling-dependent sustained adhesion.

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease (LRTD) in infants. Eosinophils have been suggested to play a role in the disease pathogenesis of LRTD. Inflammation can induce functional and morphological alterations of peripheral blood granulocytes. In patients with RSV LRTD, we aimed to investigate the eosinophil activation status by analysing surface markers. In vitro stimulation of eosinophils with cytokines leads to up-regulation of CD11b and priming markers recognized by the recently developed priming markers A17 and A27, whereas interleukin (IL)-5Ralpha is being down-regulated. In 51 patients and 10 controls we examined the expression of these surface markers on eosinophils in moderate to severe RSV-induced LRTD patients at the time of admission and 6 weeks later during the convalescence phase. RSV-patients were characterized by a higher eosinophil CD11b expression compared to controls. Although basal A17 and A27 expression was not increased, we observed a significantly higher expression of these priming epitopes on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated cells of RSV patients compared with cells of controls, indicative of prior in vivo priming. Furthermore, IL-5Ralpha expression was down-regulated on peripheral blood eosinophils of these patients. Follow-up blood samples showed normalization of all markers but CD11b, which was persistently increased. Utilizing cellular markers, we observed that peripheral blood eosinophils from infants with RSV LRTD are in a more activated state compared to eosinophils of controls, which normalizes only partially during convalescence.

Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.

Objectives: The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca2+]i) during physiological pregnancy and labour., Methods: Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement., Results: fMLPRLs were detectable in amniotic fluid throughout pregnancy, and their levels did not vary during gestation. Labour significantly increased both the amniotic fMLPRL level and the expression of fMLP receptor in amnion tissue. The increased amniotic fMLPRL concentration noted during labour significantly increased neutrophil cAMP level and [Ca2+]i., Conclusions: These findings demonstrate for the first time the presence of fMLP receptor ligands in amniotic fluid, and indicate a modulation of the fMLP system by the events of physiological labour.

Association between staphylococcal infection and pathogenesis of upper airways disease has been reported. This study aimed to investigate the mechanisms underlying the rat pulmonary inflammation induced by airway exposure to staphylococcal enterotoxin A (SEA). SEA (0.3-10 ng trachea(-1)) caused dose-dependent neutrophil accumulation in BAL fluid, reaching maximal responses at 4 h (25-fold increase for 3 ng trachea(-1)). Significant accumulation of both lymphocytes and macrophages in BAL fluid was also observed at 4 h (2.1- and 1.9-fold increase, respectively, for 3 ng trachea(-1)). At later times (16 h), neutrophil counts in bone marrow (immature forms) and peripheral blood increased by 63 and 81%, respectively. SEA failed to directly induce chemotaxis and adhesion of isolated neutrophils. Analysis of mRNA expression for iNOS, COX-2 and CINC-2 in lung tissue showed an upregulation of these enzymes, which paralleled elevated levels of LTB4, PGE2, TNF-alpha, IL-6 and NO2- in BAL fluid. Expression of CINC-1 was unchanged, whereas CINC-3 was reduced in SEA-treated rats. Incubation of isolated alveolar macrophages with SEA (3 microg ml(-1)) resulted in significant elevations of TNF-alpha and NO2- levels in the cell supernatants. Dexamethasone (0.5 mg kg(-1)), celecoxib (3 mg kg(-1)) and compound 1400 W (5 mg kg(-1)) markedly reduced SEA-induced lung neutrophil influx and NO2- levels in BAL fluid. The lipoxygenase inhibitor AA-861 (100 microg kg(-1)) partly inhibited the neutrophil influx in SEA-treated rats without modifying the NO2- levels. None of these treatments reduced the number of mononuclear cells in BAL fluid (except of dexamethasone, which abolished the increased lymphocyte counts). Our study shows that airways exposure to SEA results in marked neutrophil influx through mechanisms involving increased expressions of CINC-2, iNOS and COX-2, as well as enhanced production of NO, PGE2, LTB4, TNF-alpha and IL-6.

The neutrophil is the first line of defense against infection. As a part of the innate immune response, neutrophils start to emigrate from blood to an affected site and their state is altered from passively circulating naïve to primed, and then to fully activated. The extent of neutrophil activation and their subsequent response varies depending on the stimuli and environment that neutrophils encounter. Because neutrophils can also induce deleterious effects on host tissues, tight regulation of recruitment and functions of neutrophils is required for efficient recovery. Galectin-3, a soluble beta-galactoside binding protein, of which expression is up-regulated during inflammation/infection, is suggested to be involved in various inflammatory responses. However, the precise roles of this lectin in innate immunity remain unknown, while it has been demonstrated that galectin-3 binds to naïve and primed neutrophils. Here we report that galectin-3 can induce L-selectin shedding and interleukin-8 production in naïve and primed neutrophils. These activities were shown to be dependent on the presence of the C-terminal lectin domain and the N-terminal nonlectin domain of galectin-3, which is involved in oligomerization of this lectin. We also found that, after galectin-3 binds to neutrophils, primed but not naïve neutrophils can cleave galectin-3, mainly through elastase, which results in the formation of truncated galectin-3 lacking the N-terminal domain. Together, these results suggest that galectin-3 activates naïve and primed neutrophils, and galectin-3-activated primed neutrophils have an ability to inactivate galectin-3.

G-protein-coupled receptors play a major role in the activation of the innate immune system, such as polymorphonuclear neutrophils. Members of the formyl peptide receptor family recognize chemotactic peptides as well the amyloïd-beta peptide and fragments of the human immunodeficiency virus envelope and may thus be implicated in major pathologies. The peptide WKYMVm-NH2 probably activates the receptor FPRL1 and its mouse orthologues Fpr-rs1 and Fpr-rs2. We examined the stimulation of C57BL6 mouse neutrophils by WKYMVm-NH2 and the effects of several inhibitors for intracellular signalling pathways (wortmannin, LY 294002, staurosporin, H-89, U 73122, thapsigargin and SKF 96365). We show here that WKYMVm-NH2 is a powerful stimulator of primary and secondary granule exocytosis as well as superoxide production. The signalling pathway involves phosphoinositide 3-kinase, protein kinase C, phospholipase C and store-operated calcium influx. Studies with peptide antagonists suggest that WKYMVm-NH2 preferentially activates exocytosis via FPRL1 and not FPR, the major receptor for N-formylated peptides such as fMLF. However, the signalling pathways activated by WKYMVm-NH2 in mouse neutrophils are similar to those activated by fMLF in human neutrophils. Thus, the effect and the signalling pathways of the two agonists and their receptors are at least partially overlapping.

During active intestinal inflammation granulocytes accumulate in the lumen of the gut where they damage the epithelium through the release of various products such as reactive oxygen species and proteolytic enzymes. Previously, using function blocking monoclonal antibodies, we showed that neutrophil migration across intestinal epithelial monolayers in response to various chemoattractants was partially beta(2) integrin Mac-1 (CD11b/CD18)-independent. Here, we show that treating neutrophils with intact monoclonal antibody (mAb) to CD18 activates the cells to express more CD11b. Thus our goal now was to determine whether neutrophil Mac-1-independent transepithelial migration proceeds independently of prior cell activation through Mac-1. We took two approaches, one using blocking Fab' fragments of mAb to CD18 and the second was to develop a neutrophil differentiated HL-60 cell line which is Mac-1 deficient to further study neutrophil/epithelial cell interaction. Anti-CD18 Fab' minimally activated neutrophils but inhibited approximately 75% of transepithelial migration to fMLP while having a minimal effect (