Aspects Of Neutrophil Function Research Articles

The Sweet-Side of Leukocytes: Galectins as Master Regulators of Neutrophil Function.

Among responders to microbial invasion, neutrophils represent one of the earliest and perhaps most important factors that contribute to initial host defense. Effective neutrophil immunity requires their rapid mobilization to the site of infection, which requires efficient extravasation, activation, chemotaxis, phagocytosis, and eventual killing of potential microbial pathogens. Following pathogen elimination, neutrophils must be eliminated to prevent additional host injury and subsequent exacerbation of the inflammatory response. Galectins, expressed in nearly every tissue and regulated by unique sensitivity to oxidative and proteolytic inactivation, appear to influence nearly every aspect of neutrophil function. In this review, we will examine the impact of galectins on neutrophils, with a particular focus on the unique biochemical traits that allow galectin family members to spatially and temporally regulate neutrophil function.

Signalling in Neutrophils: A Retro Look

This review presents a summary of signalling events related to the activation of human polymorphonuclear neutrophils by a variety of soluble and particulate agonists. It is not intended as a comprehensive review of this vast field or as a presentation of the multiple new aspects of neutrophil functions that are being documented at an ever faster rate. Its aim is rather to focus on multiple aspects of major signalling pathways that, in the view of this reviewer, are currently shadowed by present trends and to provide the core evidence for their implication and the limitations of our present knowledge. More specifically, this review starts with cell surface receptors and some of their functional and biological properties and then moves on to downstream transducers (G proteins) and effectors (the phosphoinositide, tyrosine kinases, and cyclic nucleotide pathways). Classical second messengers (calcium, protein kinase C, polyphosphoinositides, and cyclic nucleotides) are emphasized. It is hoped that this presentation will not only remind present-day investigators of the central role these pathways play in the regulation of the functional responsiveness of neutrophils, but that it will also highlight some of the areas deserving additional investigation.

Francisella tularensis Inhibits the Intrinsic and Extrinsic Pathways To Delay Constitutive Apoptosis and Prolong Human Neutrophil Lifespan

Francisella tularensis is a facultative intracellular bacterium that infects many cell types, including neutrophils. We demonstrated previously that F. tularensis inhibits NADPH oxidase assembly and activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that F. tularensis significantly inhibited neutrophil apoptosis as indicated by morphologic analysis as well as annexin V and TUNEL staining. Thus, ∼80% of infected neutrophils remained viable at 48 h compared with ∼50% of control cells, and ∼40% of neutrophils that ingested opsonized zymosan. In keeping with this finding, processing and activation of procaspases-8, -9, and -3 were markedly diminished and delayed. F. tularensis also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of wbtA2 or FTT1236/FTL0708-genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate that F. tularensis profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation, and severe tissue damage that characterizes lethal pneumonic tularemia.

How has neutrophil research improved our understanding of periodontal pathogenesis?

Neutrophils are the predominant cells responsible for host defence against bacterial infection. Loss of neutrophil defence, due either to deficient number or function, strongly predisposes to bacterial infections such as periodontitis. Yet, the neutrophil oxidative and proteolytic arsenal has also been implicated in perpetrating periodontal tissue damage in periodontitis. In this review, we focus on recent developments that shed light on these two aspects of neutrophil function in periodontitis. Primary search: using PubMed search for "neutophil", "periodontal", and "periodontitis". Secondary search: using references from the articles found in the first stage. Early histological studies showed that infiltrating neutrophils form a wall of cells abutting the junctional epithelium in periodontal inflammatory lesions. The chronic standoff between these neutrophils and the bacterial community suggests that bacterial evasion of neutrophil clearance is a major characteristic of periodontitis. Indeed, not all functional neutrophil deficiencies increase the risk of periodontitis, an observation that points the way towards identification of particular anti-bacterial pathways essential for protection against periodontal pathogens. The net result in the majority of periodontitis patients who exhibit normal neutrophil number and function, is that neutrophils accumulate in the periodontal tissue where they are available to participate in tissue destruction. Diminished neutrophil clearance further contributes to the persistence of activated neutrophils in the periodontal tissue. Data on the role of neutrophils in the pathogenesis of periodontitis are mixed. Neutrophils are a critical arm of the defence against periodontitis, but bacterial evasion of the neutrophil microbicidal machinery coupled with delayed neutrophil apoptosis may transform the neutrophil from defender to perpetrator. At this stage of knowledge, attempts to induce host modulation through neutrophil suppression or activation are premature.

Neutrophil Immunosurveillance for Heart Transplant Rejection: A Prospective Study

Immunologic surveillance for rejection detection in human heart transplantation offers many potential advantages. To date, investigative efforts have focused primarily on the acquired immune system, particularly the lymphocyte. Little attention has been given to aspects of innate immune function. We have previously reported that perioperative neutrophil adhesion molecule expression is associated with early rejection episodes after human cardiac transplantation. Herein we have investigated the utility of neutrophil immunosurveillance in human heart transplant recipients at later time points. We recruited patients more than 3 months after transplantation. Neutrophil assessment was performed simultaneously with an endomyocardial biopsy that showed rejection. No significant relationship was seen between neutrophil maturity ( P = .622; n = 34), adhesion marker expression ( P = .567; n = 34), respiratory burst ( P = .604; n = 34), or apoptosis rates ( P = .662; n = 34) and contemporary rejection status at >3 months after transplantation. However, interesting relationships were noted between neutrophil adhesion markers at this late stage and historical rejection status. Higher levels of the adhesion protein CD11b observed at this late stage were significantly associated with a history of higher rejection grades in the first postoperative biopsy (Spearman rank coefficient 0.359; R = 0.304; P = .005; n = 62). Other aspects of neutrophil function and persistence were not significantly associated with rejection history. This finding, combined with the previously reported findings, supports a role for an individual phenotype in neutrophil function in early rejection episodes after transplantation.

Neutralization of monokine induced by interferon-γ during the early posttransplantation period prevents development of chronic allograft vasculopathy and graft fibrosis

Immunologic surveillance for rejection detection in human heart transplantation offers many potential advantages. To date, investigative efforts have focused primarily on the acquired immune system, particularly the lymphocyte. Little attention has been given to aspects of innate immune function. We have previously reported that perioperative neutrophil adhesion molecule expression is associated with early rejection episodes after human cardiac transplantation. Herein we have investigated the utility of neutrophil immunosurveillance in human heart transplant recipients at later time points. We recruited patients more than 3 months after transplantation. Neutrophil assessment was performed simultaneously with an endomyocardial biopsy that showed rejection. No significant relationship was seen between neutrophil maturity (P = .622; n = 34), adhesion marker expression (P = .567; n = 34), respiratory burst (P = .604; n = 34), or apoptosis rates (P = .662; n = 34) and contemporary rejection status at >3 months after transplantation. However, interesting relationships were noted between neutrophil adhesion markers at this late stage and historical rejection status. Higher levels of the adhesion protein CD11b observed at this late stage were significantly associated with a history of higher rejection grades in the first postoperative biopsy (Spearman rank coefficient 0.359; R = 0.304; P = .005; n = 62). Other aspects of neutrophil function and persistence were not significantly associated with rejection history. This finding, combined with the previously reported findings, supports a role for an individual phenotype in neutrophil function in early rejection episodes after transplantation.

Neutrophil defense in patients undergoing bone marrow transplantation: bactericidal/permeability-increasing protein (BPI) and defensins in graft-derived neutrophils.

Even after neutrophil counts return to near normal levels, patients undergoing myeloablative chemotherapy and bone marrow transplantation (BMT) are at risk for invasive bacterial infections, raising the possibility that their neutrophil function might be impaired. To assess potential qualitative defects in neutrophil function in patients undergoing BMT, we measured neutrophil content of the antimicrobial (poly)peptides BPI and defensins. Neutrophil extracts were analyzed for content of BPI by Western blotting and ELISA and for defensin peptides by acid-urea polyacrylamide gel electrophoresis (PAGE). Antibacterial activity of neutrophil extracts was measured against Escherichia coli K1/r, a clinical isolate sensitive to synergistic killing by BPI and defensins. Neutrophil extract BPI content on post-BMT days +20, +30, and +100 (169+/-35, 232+/-57, and 160+/-55 ng per 106 neutrophils, respectively) was similar to the neutrophil BPI content of normal controls (163+/-35 ng per 106 neutrophils). Neutrophil defensin content also did not vary during this time-span. Activity of neutrophil extracts against E. coli K1/r did not differ between BMT patients and controls. At post-BMT days +20 to +100, neutrophils derived from engrafted marrow contain normal quantities of BPI and defensins. Any deficiencies of neutrophil function during this phase are not due to inadequate expression of these antimicrobial (poly)peptides but could reflect abnormalities in other aspects of neutrophil function.

Coincidence detector for Rac activation

The monomeric guanosine triphosphatase Rac controls major aspects of neutrophil function including chemotaxis, phagocytosis, and production of reactive oxygen species. However the guanine-nucleotide exchange factors or GEFs that regulate Rac activity are not well described. Welch et al. purified a phosphatidylinositol(3,4,5) trisphopsphate [PtdIns(3,4,5)P 3 ]-activated Rac-GEF from pig neutrophils and then cloned the respective human gene encoding the protein they named P-Rex1. In vitro, P-Rex1 was not only directly activated by PtdIns(3,4,5)P 3 , but it was synergistically activated by direct interaction with G protein (heterotrimeric guanine nucleotide-binding protein) βγ subunits. Similar synergistic activation was observed in transfected insect SF9 cells. Such regulation of P-Rex fits nicely with its proposed role as a coincidence detector monitoring signals from G i -coupled receptors in neutrophils, which release G βγ subunits and activate G βγ -sensitive phophoinositide-3 kinase to generate PtdIns(3,4,5)P 3 . The authors anticipate the P-Rex1 may also function in other cells by stimuli that cause coinicident activation of type 1A phosphoinositide 3-kinase and G i or G o proteins. H. C. E. Welch, W. J. Coadwell, C. D. Ellson, G. J. Ferguson, S. R. Andrews, H. Erdjument-Bromage, P. Tempst, P. T. Hawkins, L. R. Stephens, P-Rex1, a PtdIns(3,4,5)P 3 - and G βγ -regulated guanine-nucleotide exchange factor for Rac. Cell 108 , 809-821 (2002). [Online Journal]

Peripheral and intrauterine neutrophil function in the cow: the influence of endogenous and exogenous sex steroid hormones

It has been accepted for many years that the susceptibility of the genital tract to infection is reduced during the follicular phase compared with the luteal phase of the estrous cycle. Since the role of intrauterine neutrophils is paramount in the elimination of bacteria, it can be hypothesized that these differences in resistance to infection could be mediated by differences in uterine-derived neutrophil function. In order to test this hypothesis two groups of cows were used in this study. Group 1 cows (n=5) were studied at estrus, diestrus, after ovariectomy, after exogenous estradiol and after progesterone treatment, at which time they underwent intrauterine infusion with 1% oyster glycogen (OG) and a bacterial-free filtrate (BFF) of Actinomyces pvogenes (BFF), the latter having been recovered from a clinical case of endometritis; neutrophils were harvested by flushing from the lumen 15 to 18 h later. A peripheral blood sample was collected at the time of flushing for the assay of estradiol and progesterone for a WBC and differential count and for the harvesting of neutrophils using a Percoll single-stage discontinuous gradient. After the recovery of the cells they were re-suspended in HBSS. Group 2 (n=4) were infused with BFF during during all reproductive states as Group 1, but with OG only after ovariectomy and after treatment with progesterone and estradiol. Neutrophil chemotaxis was assessed by measuring their migration using a modified Boyden chamber and Zymogen-activated serum as a chemoattractant. Phagocytic activity was measured by determining the number of Candida albicans ingested by each neutrophil after incubation. The percentage of kill was determined using a radiometric assay in which C. albicans was labeled with L-(5- 3H) Proline. Peripheral WBC concentration was not influenced by the reproductive state of the cow; however, the mean neutrophil concentration was significantly different between the reproductive states (P<0.001) and between individual cows (P<0.001). In Group 1, there was little difference in the function of the peripheral and uterine neutrophils, and while there were differences in all 3 aspects of neutrophil function from both sources between reproductive states and individual cows, of which some were statistically significant, there was no consistent pattern. In Group 2, neutrophils recovered after the infusion of BFF had poorer function compared with those recovered after the infusion of OG. There was no consistent influence of the reproductive state or individual animal. The hypothesis that the influence of the reproductive state of the cow on the resistance of the uterus to infection is mediated by the inherent differences in either peripheral or intrauterine neutrophil function was not supported by this study.

Neutrophils from term and preterm newborn infants express the high affinity Fcgamma-receptor I (CD64) during bacterial infections.

The high affinity Fcgamma-receptor I (FcgammaRI, CD64) is normally expressed only to a very low extent by neutrophils. During bacterial infections, however, neutrophils from adult patients significantly increase their expression of FcgammaRI. Stimulation through FcgammaRI is a highly effective way to improve various aspects of neutrophil function, including phagocytosis. In our study the expression of FcgammaRI on neutrophils from preterm (n = 9) and term (n = 3) newborn infants, children (n = 14), and adults (n = 6) during the early phase of an acute bacterial infection was investigated. Our results showed that neutrophils from newborn infants with bacterial infection expressed FcgammaRI to a significantly higher extent than both noninfected preterm (p < 0.001) and term (p < 0.001) newborn infants and that neutrophils from preterm neonates expressed FcgammaRI to the same extent as neutrophils from term neonates and older infants, children, and adults. No difference in the neutrophil cell surface expression of FcgammaRI during bacterial infections was found among newborn infants, children, and adults. Expression of FcgammaRI probably represents an important mechanism to improve neutrophil phagocytosis as well as other aspects of neutrophil function during bacterial infections, especially in preterm infants. Our study indicates that measurement of cell surface expression of FcgammaRI on neutrophils could be a useful indicator of severe bacterial infections in preterm and term neonates, as well as in older children and adults.

Paradoxic effect of ibuprofen on neutrophil accumulation in pulmonary and cutaneous inflammation.

Evidence exists to suggest that the local accumulation of neutrophils in pulmonary inflammation occurs by a different mechanism than in other microvascular beds. In the present study, this suggestion was investigated using the nonsteroidal antiinflammatory drug ibuprofen. This drug was used because in addition to inhibiting cyclooxygenase, it prevents several aspects of neutrophil function, including adhesion. Local inflammation in the lung and skin of the same rabbits was induced by the administration of C5a, and the effect of intravenous injection of ibuprofen on the accumulation of neutrophils at these two sites was examined. In the skin, neutrophil accumulation was inhibited by ibuprofen, and this appeared to be independent of cyclooxygenase blockade. A possible mechanism was prevention of neutrophil adherence to endothelium in postcapillary venules, and in vitro experiments showed that ibuprofen could entirely prevent neutrophil adherence, in addition to suppressing azurophil granule secretion and superoxide anion generation. However, the effect on adherence appeared to be independent of expression of the CD11/CD18 adhesion complex on the neutrophil. By contrast, in the pulmonary circulation of the same rabbit, C5a-induced neutrophil accumulation was enhanced by ibuprofen treatment. This was suggested to result from the prevention of thromboxane production, which normally serves to decrease local pulmonary blood flow to diminish delivery of neutrophils to the inflammatory site. Ibuprofen had no effect on retention of neutrophils in filters (models of pulmonary capillaries), suggesting that the drug was not enhancing the retention of neutrophils in capillaries on the first pass through the lung. Thus, it was possible that enhancement in the lung was caused by an increase in neutrophil supply. Because the accumulation of neutrophils in the skin is known to be dependent on the adherence phenomena, the paradoxic effects of ibuprofen on inflammation suggest that mechanisms responsible for the accumulation of neutrophils in cutaneous and pulmonary microcirculations are different.

Paradoxic effect of ibuprofen on neutrophil accumulation in pulmonary and cutaneous inflammation.

Evidence exists to suggest that the local accumulation of neutrophils in pulmonary inflammation occurs by a different mechanism than in other microvascular beds. In the present study, this suggestion was investigated using the nonsteroidal antiinflammatory drug ibuprofen. This drug was used because in addition to inhibiting cyclooxygenase, it prevents several aspects of neutrophil function, including adhesion. Local inflammation in the lung and skin of the same rabbits was induced by the administration of C5a, and the effect of intravenous injection of ibuprofen on the accumulation of neutrophils at these two sites was examined. In the skin, neutrophil accumulation was inhibited by ibuprofen, and this appeared to be independent of cyclooxygenase blockade. A possible mechanism was prevention of neutrophil adherence to endothelium in postcapillary venules, and in vitro experiments showed that ibuprofen could entirely prevent neutrophil adherence, in addition to suppressing azurophil granule secretion and superoxide anion generation. However, the effect on adherence appeared to be independent of expression of the CD11/CD18 adhesion complex on the neutrophil. By contrast, in the pulmonary circulation of the same rabbit, C5a-induced neutrophil accumulation was enhanced by ibuprofen treatment. This was suggested to result from the prevention of thromboxane production, which normally serves to decrease local pulmonary blood flow to diminish delivery of neutrophils to the inflammatory site. Ibuprofen had no effect on retention of neutrophils in filters (models of pulmonary capillaries), suggesting that the drug was not enhancing the retention of neutrophils in capillaries on the first pass through the lung. Thus, it was possible that enhancement in the lung was caused by an increase in neutrophil supply. Because the accumulation of neutrophils in the skin is known to be dependent on the adherence phenomena, the paradoxic effects of ibuprofen on inflammation suggest that mechanisms responsible for the accumulation of neutrophils in cutaneous and pulmonary microcirculations are different.

Effect of fluticasone propionate on neutrophil chemotaxis, superoxide generation, and extracellular proteolytic activity in vitro.

Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue.

Studies on Some Aspects of Neutrophil Functions and Uterine Defenses in Cows during the Estrous Cycle

Contents: Some aspects of the bovine uterine defense mechanisms were evaluated over two successive estrous cycles in five adult nonpregnant Friesian cows. Uterine flushings and blood samples were taken from each cow during the estrus and diestrus phases of each estrous cycle. A significant higher percentage of Candida albicans (C. albicans) were phagocytosed (P &lt; 0.01) and killed (P &lt; 0.05) by blood neutrophils during estrus than during diestrus. N‐acetyl‐β‐D‐glucosaminidase activity of uterine flushings, blood neutrophil alkaline phosphatase activity scores, and serum progesterone concentrations during diestrus increased significantly (P &lt; 0.01) compared to those at estrus. Mean total blood leucocyte and monocyte counts increased significantly (P &lt; 0.05) during estrus compared to diestrus. It is concluded, that the different hormonal status has at each stage of the estrous cycle a definite effect on uterine resistance against bacterial infection and a fall in serum progesterone concentrations is associated with an increased ability of blood neutrophils to phagocytose and kill C. albicans.

Activation of neutrophils by antigen-induced lymphokine, with emphasis on antibody-independent cytotoxicity.

Incubation of bovine neutrophils with antigen-stimulated mononuclear cell supernatant (lymphokine) caused an inhibition of neutrophil migration and an enhancement of the neutrophils' ability to adhere to plastic, reduce nitroblue tetrazolium, ingest Staphylococcus aureus, and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against chicken erythrocytes. Lymphokine-treated neutrophils also became cytotoxic for chicken, turkey and human erythrocytes in the absence of specific antibody but were not cytotoxic for bovine erythrocytes. The increase in antibody-independent neutrophil cytotoxicity (AINC) was not due to direct cytotoxic activity of the lymphokine or to a stable, soluble mediator released by the neutrophils. Enhancement of AINC, but not ADCC, required RNA and protein synthesis by the neutrophil. These results indicate that several aspects of neutrophil function can be altered by products secreted by antigen-stimulated mononuclear cells and that neutrophils can be induced to recognize and to have increased cytotoxic activity toward heterologous erythrocytes.

Flavonoid modulation of human neutrophil function

Flavonoids are naturally occurring plant compounds that have been demonstrated to possess a variety of anti-inflammatory effects. We studied the effects of flavonoids on three aspects of neutrophil function that are commonly considered to be associated with inflammation: the release of lysosomal enzymes, the chemiluminescence (CL) response, and the production of superoxide anion. Quercetin and eight other flavonoids at a 10 −5M concentration inhibited the neutrophil CL response to opsonized zymosan particles by approximately 60% or more. In contrast, the release of lysosomal β-glucuronidase from neutrophils stimulated with opsonized zymosan was only inhibited by two flavonoids, quercetin and chalcone, and only at concentrations of 1.5 × 10 −4M to 2 × 10 −4M. Quercetin also inhibited the generation of superoxide anion by neutrophils but to a lesser degree than its effect on CL. The present studies demonstrated that certain flavonoids are not uniformly active in inhibiting neutrophil CL, β-glucuronidase release, or superoxide generation. The effects of flavonoids on neutrophil functions probably depend on many variables including the response measured, the activating stimulus, and specific flavonoid structural features.

Human Milk-Reply

In Reply. —Dr Khan and his associates call attention to one aspect of neutrophil function, ie, chemotaxis, that we agree may be an important factor in host defense both for the mother in preventing breast abscesses and for the infant in providing neutrophils for antigen processing. Unfortunately, we have already imposed on theJournalby submitting an unusually lengthy article and so omitted details of this particular assay. We chose to focus on data more specific to Salmonella infection. Methods for chemotaxis under agarose were identical to those previously published 1 except that we quantitate the migrating cells with a micrometer disk (scale, 5 mm/100 units, type 9) eyepiece grid. After counting cells on the chemotactic side and control side of the neutrophil well, the chemotactic index is calculated using the following formula: chemotactic index (CI) = No. of cells on the chemotactic side/No. of cells on the control side. Our