Behavior Of Neutrophils Research Articles

Fever-Like Temperatures Affect Neutrophil NF-κB Signaling, Apoptosis, and ANCA-Antigen Expression

The neutrophil is pivotal to ANCA vasculitis pathogenesis. Fever frequently complicates ANCA diseases. This study investigated the effects of short-term heat exposure on apoptosis in neutrophils that were treated with LPS, GM-CSF, IL-8, and dexamethasone. All compounds delayed apoptosis. Heat abrogated the apoptosis-delaying effect of LPS without affecting constitutive apoptosis or delayed apoptosis by GM-CSF, IL-8, or dexamethasone. The heat effect was dose dependent over the 39 to 42 degrees C range. NF-kappaB but not extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or phosphatidylinositol 3-kinase/Akt controlled LPS-delayed apoptosis. Furthermore, LPS-induced IkappaBalpha degradation, DNA binding, and NF-kappaB-dependent gene transcription activation were abrogated by short-term heat. When core temperatures were raised to 40.5 degrees C for 30 min in mice, LPS-induced neutrophil NF-kappaB activation also was prevented. Short-term heat removed heat-shock protein 90 from the IkappaB kinase complex, resulting in failure of LPS-induced IkappaB kinase activation. Despite delayed apoptosis, ANCA antigen expression was increased in LPS-treated neutrophils. ANCA antigen increase was prevented by p38 MAPK inhibition and by heat exposure. Heat exposure did not inhibit LPS-induced p38 MAPK phosphorylation. Instead, apoptosis-mediated p38 MAPK degradation was accelerated, thereby decreasing the p38 MAPK that was available for LPS-mediated ANCA antigen upregulation. These data suggest that fever-like temperatures modulate neutrophil behavior in this disease.

Sequence Variability of Human Cytomegalovirus UL146 and UL147 Genes in Low-Passage Clinical Isolates

Objectives: Human cytomegalovirus (HCMV) infects a number of organs and cell types in vivo. The different symptoms and tissue tropisms of HCMV infection perhaps result from the genetic polymorphism. A new region of DNA containing at least 19 open reading frames (ORFs – denoted UL133–151) was found in the low-passage HCMV clinical strain Toledo and several other low-passage clinical isolates, but not present in the HCMV laboratory strain AD169. Two of these genes, UL146 and UL147, encode proteins with sequence characteristics of CXC (α) chemokines, suggesting that they might influence the behavior of neutrophils during infection. This research was to study the sequence variability of UL146 and UL147 ORFs in HCMV clinical isolates and examine the possible associations between gene variability and the outcome of HCMV infection. Methods: UL146 and UL147 genes from strains obtained from suspected congenitally HCMV-infected infants were PCR amplified and sequenced. Results: High variability was found in UL146 and UL147 gene among HCMV clinical strains. However, the α chemokine motif in UL146 and UL147 genes was conserved in almost all sequences. According to the phylogenetic analysis, all sequences of UL146 in clinical isolates could be divided into three groups. All strains from congenital megacolon infants existed in G2A only, and all from asymptomatic infants existed in G2B peculiarly. Conclusions: Sequence variability among HCMV clinical strains may affect the ability of UL146 and UL147 to attract human neutrophils and influence viral dissemination. No obvious linkage was observed between UL146 polymorphisms and outcome of suspected congenital HCMV infection.

Neutrophils may contribute to the morbidity and mortality of claudicants.

Peripheral arterial occlusive disease is a common cause of morbidity in middle-aged men; 5 per cent of those aged over 50 years suffer from intermittent claudication. While claudication itself is not fatal, claudicants have a mortality rate approximately three times that of non-claudicating men of the same age, mainly from cardiovascular disease. This review examines the evidence for involvement of the neutrophil in this increased mortality and describes the possible pathogenesis. It also discusses how treatment of claudication may modify neutrophil behaviour, reducing subsequent mortality and morbidity rates.

LEGO® bricks used as chemotactic chambers: evaluation by a computer‐assisted image analysis technique

One of the main techniques used to explore neutrophil motility, employs micropore filters in chemotactic chambers. Many new models have been proposed, in order to perform multiple microassays in a rapid, inexpensive and reproducible way. In this work, LEGO® bricks have been used as chemotactic chambers in the evaluation of neutrophil random motility and chemotaxis and compared with conventional Boyden chambers in a “time‐response” experiment. Neutrophil motility throughout the filters was evaluated by means of an image‐processing workstation, in which a dedicated algorithm recognizes and counts the cells in several fields and focal planes throughout the whole filter; correlates counts and depth values; performs a statistical analysis of data; calculates the true value of neutrophil migration; determines the distribution of cells; and displays the migration pattern. By this method, we found that the distances travelled by the cells in conventional chambers and in LEGO® bricks were perfectly identical, both in random migration and under chemotactic conditions. Moreover, no interference with the physiological behaviour of neutrophils was detectable. In fact, the kinetics of migration was identical both in random migration (characterized by a gaussian pattern) and in chemotaxis (characterized by a typical stimulation peak, previously identified by our workstation). In conclusion, LEGO® bricks are extremely precise devices. They are simple to use and allow the use of small amounts of chemoattractant solution and cell suspension, supplying by itself a triplicate test. LEGO® bricks are inexpensive, fast and suitable for current diagnostic activity or for research investigations in every laboratory.

The chemotaxis defect of Shwachman-Diamond Syndrome leukocytes.

Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive, multisystem disorder presenting in childhood with intermittent neutropenia and pancreatic insufficiency. It is characterized by recurrent infections independent of neutropenia, suggesting a functional neutrophil defect. While mutations at a single gene locus (SBDS) appear to be responsible for SDS in a majority of patients, the function of that gene and a specific defect in SDS neutrophil behavior have not been elucidated. Therefore, employing 2D and 3D computer-assisted motion analysis systems, we have analyzed the basic motile behavior and chemotactic responsiveness of individual polymorphonuclear leukocytes (PMNs) of 14 clinically diagnosed SDS patients. It is demonstrated that the basic motile behavior of SDS PMNs is normal in the absence of chemoattractant, that SDS PMNs respond normally to increasing and decreasing temporal gradients of the chemoattractant fMLP, and that SDS PMNs exhibit a normal chemokinetic response to a spatial gradient of fMLP. fMLP receptors were also distributed uniformly through the plasma membrane of SDS PMNs as in control PMNs. SDS PMNs, however, were incapable of orienting in and chemotaxing up a spatial gradient of fMLP. This unique defect in orientation was manifested by the PMNs of every SDS patient tested. The PMNs of an SDS patient who had received an allogenic hematopoietic stem cell transplant, as well as PMNs from a cystic fibrosis patient, oriented normally. These results suggest that the defect in SDS PMNs is in a specific pathway emanating from the fMLP receptor that is involved exclusively in regulating orientation in response to a spatial gradient of fMLP. This pathway must function in parallel with additional pathways, intact in SDS patients, that emanate from the fMLP receptor and regulate responses to temporal rather than spatial changes in receptor occupancy.

Granulocyte colony-stimulating factor receptor signals for β 1-integrin expression and adhesion in bladder cancer

Objectives To examine the association of granulocyte colony-stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) expression with increased β 1-integrin expression and determine the ability of autocrine G-CSFR signaling to promote bladder cancer cell adhesion by way of β 1-integrin. β 1-integrin is expressed at higher levels in more invasive bladder carcinoma cells and participates in the process of tissue invasion. Cancer cell invasion and metastasis in some ways mimic normal neutrophil behavior. In neutrophils, G-CSF acts through its receptor to enhance adhesion and migration. A significant fraction of bladder carcinoma has been reported to express both G-CSF and G-CSFR. Methods We examined bladder carcinoma tissue samples obtained from segmental or radical cystectomy specimens for expression of G-CSF, G-CSFR, and β 1-integrin using reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical methods. We determined the G-CSFR–mediated β 1-integrin adhesion using a static adhesion assay and the bladder cancer cell line 5637. Results Eleven of 14 bladder cancer samples expressed G-CSFR. All 11 G-CSFR positive tumors also expressed G-CSF, and the G-CSF/G-CSFR positive tumors had elevated β 1-integrin protein levels. All but one G-CSFR negative tumor demonstrated low β 1-integrin protein levels. In four G-CSF/G-CSFR positive tumors for which distant urothelium was available for examination, G-CSF, G-CSFR, and β 1-integrin expression was also increased. In the 5637 cell line, we demonstrated G-CSFR–mediated upregulation of β 1-integrin–dependent adhesion to fibronectin and laminin. Conclusions G-CSF/G-CSFR expression in some bladder cancers appears to be an early event during malignant transformation that increases β 1-integrin expression and adhesion and thereby may promote tissue invasion.

Letters to the Editor

The contribution of ICAM-1 to neutrophil-SEC interaction is under controversy. In a recent issue, we reported that ICAM-1 plays a crucial role in neutrophil adhesion to and migration through the SEC monolayer in vitro (1). Jaeschke et al. (2,3) previously reported that anti-ICAM-1 had no significant effect on hepatic accumulation of neutrophils in endotoxic liver injury, and speculated that neutrophil accumulation is caused by SEC and Kupffer cell swelling and the stiffness of neutrophil cell membrane when exposed to endotoxin. In this letter, they stated that the inhibition of ICAM-1 had only a limited effect in our in vitro study, and that the data obtained under our experimental conditions could not be applied to the behavior of neutrophils in hepatic sinusoid in vivo. However, in contrast to their observation, a few experiments in vivo showed that ICAM-1 was involved in liver injury during endotoxemia (4,5), which is consistent with our findings. We can propose two reasons why their data in vivo differs from ours in vitro. First, they investigated the role of ICAM-1 in hepatic infiltration of neutrophils only 90 min after LPS exposure in mice. As we stated in the issue, this time frame would be too early for them to estimate the effect of anti-ICAM-1 antibody on neutrophil-SEC interaction because overexpression of ICAM-1 on SEC was not observed until 6 to 8 h after LPS exposure in vivo (6,7). In fact, Xu et al. (4) reported that hepatic infiltration of neutrophils was significantly inhibited 24 h after LPS injection in ICAM-1-deficient mice, but was not inhibited 2 h afterward. This in vivo data clearly showed that ICAM-1 was involved in neutrophil accumulation in ICAM-1-overexressing liver. We can agree with the data of Jaeschke that hepatic neutrophil accumulation was not mediated by ICAM-1 90 min after LPS exposure when ICAM-1 was not enough upregulated on SEC. However, our experiments suggest that ICAM-1 plays an important role in hepatic infiltration in a later stage, when the expression of ICAM-1 was enhanced on SEC. The next reason is that they estimated neutrophil accumulation by histochemical analysis. To investigate neutrophil-SEC interaction, we used an in vitro flow system (IVFS) (1,8), which allows of the dynamic analysis of cell-to-cell interaction (9). IVFS enables us to investigate the each step of infiltration and to distinguish adhered cells from floating or rolling cells on SEC monolayers. In other words, we can count the actually adhered or migrated cells by using our device, but by means of their methods, it is impossible to distinguish strictly neutrophil adhesion from leukostasis, which was secondarily induced by circulatory disturbance. Vollmar et al. (10) reported that the number of nonperfused sinusoids correlated with the number of neutrophils in sinusoids during endotoxemia. Indeed, our in vitro data showed that once a neutrophil adhered to SEC, the next adhesive reaction occurred around the adhered neutrophil at first. This finding may reveal that the adhered cells change the flow rate and make turbulent flow, which enhances additional adhesive reaction (preliminary data). Besides, neutrophil-SEC interaction mediated by ICAM-1 induces SEC injury by activated neutrophils (8). We do not deny the possibility that neutrophil accumulation is caused by passive trapping due to SEC swelling or active vasoconstriction; however, our findings revealed that anti-ICAM-1, in part but at least significantly, inhibited an initial adhesive reaction that induces the secondary leukostasis and SEC injury in hepatic sinusoids in vivo. Considering these points, even if anti-ICAM-1 inhibits the adhesion of only 50%–60% of neutrophils, we can conclude that ICAM-1 plays a major role in the hepatic infiltration of neutrophils during endotoxic liver injury. Dr. Takeshi Okanoue Dr. Shinichi Sakamoto

Selectin-mediated rolling of neutrophils is essential for their activation and retention in the reperfused coronary system.

Neutrophil adhesion to coronary endothelium is a key event for cardiac reperfusion injury. Adhesion is proposed to be a multi-step event, consisting of selectin-mediated rolling, chemotactic activation, and subsequent integrin-mediated firm attachment. However, it is not clear whether this sequence also occurs in the coronary circulation with its unique hemodynamic properties (turbulent flow, flow reversal). We have studied neutrophil adhesion in the coronary system of isolated perfused guinea pig hearts under basal and reperfusion conditions (15 min global ischemia). Adhesion was manipulated by an anti-CD18 antibody (blocking firm adhesion) and fucoidin (reducing rolling). Neutrophil behavior during coronary passage was assessed by measurement of CD11b expression, forward scatter (FSC, indicating polarization), and sideward scatter (SSC, measure for granularity) via flow cytometry. Adhesion rose from 21 % (basal) to 35 % after ischemia. Anti-CD18 decreased adhesion to 11 % and 14 %, respectively; fucoidin altered only the postischemic increase (23 %). CD11b was unchanged by passage through the non-ischemic coronaries, but rose postischemically (139 % increase). CD18 blockade did not reduce the postischemic rise of CD11b, while fucoidin was inhibitory (24 % increase). FSC did not differ between controls and ischemic hearts in any group, while SSC decreased most in postischemic hearts after CD18 blockade. Blockade of rolling and of firm attachment both reduce neutrophil retention, while only inhibition of rolling reduces intracoronary activation. Thus, rolling seems to be mandatory for endothelial-leukocyte communication in the coronary system.

Topographical control of human neutrophil motility on micropatterned materials with various surface chemistry

Controlling cell responses to an implantable material is essential to tissue engineering. Because the surface is in direct contact with cells, both chemical and topographical properties of a material surface can play a crucial role. In this study, parallel ridges/grooves were micropatterned on glass surfaces using photosensitive polyimide to create transparent substrates. The migratory behavior of live human neutrophils on the patterned surfaces was observed using a light microscope with transmitted light source. The width (2 μm) and length (400 μm) of the ridges were kept constant. The height (5 or 3 μm) and the repeat spacing (6–14 μm) of the ridges were systematically changed to investigate the effect of microgeometry on neutrophil migration. In addition, the effect of surface chemistry on neutrophil migration was studied by deposition of a thin layer of “inert”, biocompatible metal such as Au–Pd alloy and titanium on patterned substrates. More than 95% of neutrophils moved in the direction of the long axis of ridges/grooves regardless of the topographical geometry and chemistry, consistent with a phenomenon termed “contact guidance”. Therefore, cell migration was characterized using a one-dimensional persistent random walk. The rate of cell movement was strongly dependent on the topographical microgeometry of the ridges. The random motility coefficient μ, 9.8×10 −9 cm 2/s, was the greatest at a ridge height of 5 μm and spacing of 10 μm, about 10 times faster than on smooth glass surface. The Au–Pd coating did not change neutrophil migratory behavior on patterned surfaces, whereas titanium decreased cell motility substantially. The results of this study suggest that optimization of both surface chemistry and topography may be important when designing biomaterials for tissue engineering. In addition, parallel ridges/grooves can be used to control the direction and rate of cell migration on the surface.

Role of the neutrophil in the development of systemic inflammatory response syndrome and sepsis following abdominal aortic surgery.

There is evidence to suggest that the polymorphonuclear neutrophil (PMN) plays a critical early step in the development of the ischaemia-reperfusion syndrome, the systemic inflammatory response syndrome (SIRS) and sepsis. The PMN receptor CD16 plays an important role in phagocytosis, cell-mediated cytotoxicity and the release of free radicals and proteolytic enzymes. The aim of this study was to determine whether there is any relationship between PMN CD16 expression, phagocytosis and the development of sepsis. Fifty patients who underwent elective infrarenal abdominal aortic aneurysm repair were studied. Venous blood was taken before operation, throughout surgery and for 7 days after operation. CD16 expression was measured, unstimulated and following further stimulation, by means of flow cytometry. Phagocytosis was determined using flow cytometry. Some 36 patients had an uncomplicated recovery; 14 developed SIRS or sepsis. There was no difference between the two groups with respect to nutritional, co-morbid or technical factors. In the group that developed septic complications after operation, the level of PMN CD16 expression was significantly higher before surgery (mean channel fluorescence (MCF) 30.2 versus 10.4; P < 0.05, Mann-Whitney U test) and throughout the postoperative period. Surgery produced no change in CD16 expression. After operation, stimulation of PMNs in the septic group resulted in a fall in CD16 expression (40.8 versus 20.4 MCF; P < 0.05, Mann-Whitney U test); surgery produced no change in the level of expression in the uncomplicated group. This study provides evidence of phenotypic and functional differences in neutrophil behaviour in patients who develop sepsis following aneurysm surgery.

Changes in the mechanical and adhesive behaviour of human neutrophils on cooling in vitro.

Changes in the rheological properties of neutrophils may influence flow in microvessels that are cooled below normal body temperature. We investigated the effects of temperature on the mechanical and adhesive properties of human neutrophils by measuring transit times for individual cells flowing through 8-microm-pores in filters, and adhesion to P-selectin for cells perfused over a monolayer of activated platelets. Pore transit time increased as temperature was decreased from 37 degrees C to 0 degrees C. Upon rapid cooling, there was an instantaneous increase attributable to changes in aqueous viscosity. Interestingly, at 10 degrees C specifically, there was an additional increase in transit time, which was abolished by the inhibitor of actin polymerization, cytochalasin B. This meant that by 15 min, transit time at 10 degrees C was greater than at 0 degrees C. Most adherent cells on P-selectin were rolling, rather than stationary, at 10, 26 or 37 degrees C. The velocity of rolling slowed with decreasing temperature. The total number of adherent cells decreased with increasing wall shear rate, but for a given shear rate there was relatively little effect of temperature on attachment. However, when adhesion at 10, 26 or 37 degrees C was compared at equal shear stress (taking into account fluid viscosity), adhesion was greatest at 10 degrees C. Measurements of immunofluorescence showed that exposure to 10 degrees C gradually increased expression of beta2-integrin CD11b/CD18, but this did not cause transformation to stationary adhesion with time in the flow assay. Thus, neutrophils show an anomalous rheological response around 10 degrees C, which may impair local microcirculation in the cold. On rewarming, "activated" cells might inhibit recovery or become released into the systemic circulation.

Neutrophil recruitment, chemokine receptors, and resistance to mucosal infection

Neutrophil migration to infected mucosal sites involves a series of complex interactions with molecules in the lamina propria and at the epithelial barrier. Much attention has focussed on the vascular compartment and endothelial cells, but less is known about the molecular determinants of neutrophil behavior in the periphery. We have studied urinary tract infections (UTIs) to determine the events that initiate neutrophil recruitment and interactions of the recruited neutrophils with the mucosal barrier. Bacteria activate a chemokine response in uroepithelial cells, and the chemokine repertoire depends on the bacterial virulence factors and on the specific signaling pathways that they activate. In addition, epithelial chemokine receptor expression is enhanced. Interleukin (IL)-8 and CXCR1 direct neutrophil migration across the epithelial barrier into the lumen. Indeed, mIL-8Rh knockout mice showed impaired transepithelial neutrophil migration, with tissue accumulation of neutrophils, and these mice developed renal scarring. They had a defective antibacterial defense and developed acute pyelonephritis with bacteremia. Low CXCR1 expression was also detected in children with acute pyelonephritis. These results demonstrate that chemokines and chemokine receptors are essential to orchestrate a functional antimicrobial defense of the urinary tract mucosa. Mutational inactivation of the IL-8R caused both acute disease and chronic tissue damage.

Altered migratory capacity of polymorphonuclear leucocytes as an effect of TNF-alpha blockade in patients with rheumatoid arthritis

Rheumatoid Arthritis (RA) is associated with progressive joint destruction, functional disability and decreased life expectancy. Althought the underlying cause of RA is unknown, TNF-alpha, a proinflammatory cytokine, contributes to the pathogenesis of synovitis and joint destruction. Anti-TNF-biological response modifiers, such as Etanercept a recombinant human TNF receptor fusion protein, suggest that TNF-alpha-inhibition is a viable approach to control disease activity in RA. Considering the pivotal role TNF alpha plays in the first line defense against bacterial and fungal infections by polymorphonuclear leucocytes (PMN) it seems important to focus on the PMN function in RA patients, treated with Etanercept. Since a 29% increase in infections of the upper respiratory tract under TNF-alpha blockade has been reported, we investigated inflammatory parameters related to PMN-activity in 6 RA-patients, before and after 3-months of Etanercept treatment without altering their methotrexate and/or glucocorti-coid-medication. The migration of the neutrophils was measured with a standardized whole blood membrane filter assay with and without stimulation by the chemoattractant fMLP. The percentage of migrating PMN (total migration index,TMI) and the relative penetration depth into the filters (distribution characteristics, DC) served to characterize the migratory behaviour of neutrophils. To estimate in vivo granulocyte activation, neutrophil elastase was measured in plasma from RA patients. In addition, PMN-blood count and C-reactive protein as a marker of disease activity, were analysed. TNF-alpha blockade significantly (p < 0,03) reduced the spontaneous and fMLP stimulated median TMI (15.2 vs. 10.9 and 16.5 vs. 11,4, respectively). Moreover the spontaneous and the fMLP-stimulated median DC was reduced under Etanercept-treatment (21.1 vs. 5.9, P < 0.03 and 20.5 vs. 6.6, P < 0.06 respectively). Interestingly, these values were still within the normal range of migratory activity. Furthermore, TNF blockade significantly reduced the median plasma elastase levels (252 vs. 108, p <0,04), as well as the median C reactive protein levels (21 vs. 7 P < 0,035) and the median number of polymorphonuclear leucocytes (7.73 vs. 4.58, P < 0.0001). Of note, plama elastase levels as a sign of systemic PMN-activation were still above the normal range. During the observed treatment period all patients showed an improvement in the inflammatory symptoms of RA (2 had a 70% ACR-response, 3 a 50% and 1 a 20% ACR response). No patients had signs of bacterial or fungal infections. In conclusions, TNF alpha blockade did not suppress PMN migratory activities to levels that are assosciated with higher incidences of infections.

Deficiency of Src homology 2-containing phosphatase 1 results in abnormalities in murine neutrophil function: studies in motheaten mice.

Neutrophils, an essential component of the innate immune system, are regulated in part by signaling pathways involving protein tyrosine phosphorylation. While protein tyrosine kinase functions in regulating neutrophil behavior have been extensively investigated, little is known about the role for specific protein tyrosine phosphatases (PTP) in modulating neutrophil signaling cascades. A key role for Src homology 2 domain-containing phosphatase 1 (SHP-1), a PTP, in neutrophil physiology is, however, implied by the overexpansion and inappropriate activation of granulocyte populations in SHP-1-deficient motheaten (me/me) and motheaten viable (me(v)/me(v)) mice. To directly investigate the importance of SHP-1 to phagocytic cell function, bone marrow neutrophils were isolated from both me/me and me(v)/me(v) mice and examined with respect to their responses to various stimuli. The results of these studies revealed that both quiescent and activated neutrophils from motheaten mice manifested enhanced tyrosine phosphorylation of cellular proteins in the 60- to 80-kDa range relative to that detected in wild-type congenic control neutrophils. MOTHEATEN: neutrophils also demonstrated increased oxidant production, surface expression of CD18, and adhesion to protein-coated plastic. Chemotaxis, however, was severely diminished in the SHP-deficient neutrophils relative to control neutrophils, which was possibly attributable to a combination of defective deadhesion and altered actin assembly. Taken together, these results indicate a significant role for SHP-1 in modulating the tyrosine phosphorylation-dependent signaling pathways that regulate neutrophil microbicidal functions.

Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes

Fluorescent dyes assist visualisation of leukocytes for intravital studies of adhesion or for in vitro studies utilising whole blood. We have used in vitro flow-based assays to investigate the effects of three fluorescent dyes (acridine orange, AO, 5–100 μg/ml; calcein-AM, C-AM, 5–20 μg/ml; rhodamine 6G, R6G, 10–100 μg/ml) on adhesion and migration of isolated neutrophils and mononuclear cells. AO had little effect on the number or velocity of neutrophils rolling on P-selectin presented by a surface coated with platelets. However, AO did cause a dose- and time-dependent conversion of rolling to immobilisation. Pretreatment of neutrophils with an antibody against CD18 prevented this conversion to stationary adhesion, indicating that β 2 integrins were activated by AO. C-AM had little effect on neutrophil behaviour, but tended to cause some immobilisation at the highest concentration. R6G did not affect the number of neutrophils that bound to the platelet monolayer or the percentage rolling, but the rolling velocity of the neutrophils was increased in a dose-dependent manner. None of the dyes impaired the ability of neutrophils to respond to formyl peptide by converting from rolling to stationary adhesion. Neither C-AM nor R-6G reduced the number of flowing neutrophils or mononuclear cells binding to endothelial cells stimulated with tumour necrosis factor. Interestingly, R-6G inhibited transendothelial migration of mononuclear cells but not neutrophils, while C-AM did not affect transmigration of either cell type. The dose-dependent effects of dyes should be taken into consideration when designing any experimental protocol. AO does not appear to be a suitable dye for adhesion studies. R6G and C-AM can be used at ∼10 μg/ml (a concentration at which cells can be clearly visualised) although R-6G specifically inhibits the migratory response of mononuclear cells.

Techniques for measuring and manipulating free Ca 2+ in the cytosol and organelles of neutrophils

Ca 2+ signalling in neutrophils is important for triggering and coordinating the behaviour of neutrophils. Fluorescent probes for cytosolic free Ca 2+ concentration, e.g., fura2 and fluo3, have been widely used in neutrophils. These probes can be used to monitor Ca 2+ in the cytosol, the nucleus, near the plasma membrane and theoretically within Ca 2+ storage organelles. The longer wavelength indicators, e.g., fluo3 and calcium green, can be used confocally to monitor subcellular Ca 2+ changes in the cytosol of neutrophils and in the nucleus. Confocal techniques also permit “impossible views” imaging of Ca 2+ and newer scanning techniques promise very fast temporal resolution. Techniques using chlortetracycline (CTC) and DiOC 6(3) are also described for monitoring the position of Ca 2+ storage sites in neutrophils and for manipulating their activity. Thus, in this review, a spectrum of new (and older) optical techniques are presented which are useful for measuring, monitoring and manipulating cytosolic free Ca 2+ concentration and Ca 2+ storage in neutrophils. With these techniques, it is hoped that more insight will be gained into both the mechanism of and the consequences of Ca 2+ signalling in neutrophils.

Leukocyte L-selectin is up-regulated after mechanical trauma in adults.

Infection and multiple organ failure remain the principal causes of late mortality after trauma despite advances in the resuscitation of injured patients. Because a better understanding of postinjury leukocyte trafficking is essential to the development of possible therapeutic measures aimed at preventing these complications, we have performed a study of one factor in the early posttrauma endothelial adhesion behavior of monocytes, lymphocytes, and neutrophils: their cell surface expression of L-selectin (CD62L). We have also studied the plasma levels of soluble L-selectin in these patients. Two venous blood samples were taken from each of 41 trauma patients at median times of 1 and 20 hours after injury. The study group included 16 patients with major (Injury Severity Score (ISS) > or = 16), 17 with moderate (ISS = 9-15), and 8 with minor (ISS < 9) trauma. Cell surface L-selectin was measured on leukocyte subsets by staining with specific fluorescent-labeled monoclonal antibodies to CD62L and using flow cytometry. Both the percentage of cells expressing the molecule and the mean channel fluorescence were measured. Levels of soluble L-selectin were measured in the plasma, sampled concurrently, by enzyme-linked immunosorbent assay. Monocytes, lymphocytes, and neutrophils all showed an early increase in cell surface L-selectin expression as measured by mean channel fluorescence (p < 0.0001, p < 0.001, and p < 0.0001, respectively), and this persisted in later samples taken at a median 20 hours after injury (p < 0.0001,p < 0.0001, and p < 0.01). Only monocytes showed an increased percentage of cells expressing the molecule in the early phase (p < 0.02), and this remained in the later phase (p < 0.001). Monocytes also showed a further significant increase in mean channel fluorescence (p < 0.02) between the two periods. No significant changes in levels of plasma soluble L-selectin were found at either stage. An increase in the expression of L-selectin on each of three leukocyte populations has been demonstrated in the early phase after trauma. This would tend to promote rolling behavior of leukocytes and increase their contact with the vascular endothelium. There were marked differences in the later responses of the three populations, which may represent differential control of their behavior.

Passive Mechanical Behavior of Human Neutrophils: Effects of Colchicine and Paclitaxel

The role of microtubules in determining the mechanical rigidity of neutrophils was assessed. Neutrophils were treated with colchicine to disrupt microtubules, or with paclitaxel to promote formation of microtubules. Paclitaxel caused an increase in the number of microtubules in the cells as assessed by immunofluorescence, but it had no effect on the presence or organization of actin filaments or on cellular mechanical properties. Colchicine at concentrations <1.0 μM caused disruption of microtubular structures, but had little effect on either F-actin or on cellular mechanical properties. Higher concentrations of colchicine disrupted microtubular structure, but also caused increased actin polymerization and increases in cell rigidity. Treatment with 10 μM colchicine increased F-actin content by 17%, the characteristic cellular viscosity by 30%, the dependence of viscosity on shear rate by 10%, and the cortical tension by 18%. At 100 μM colchicine the corresponding increases were F-actin, 25%; characteristic viscosity, 50%; dependence of viscosity on shear rate, 20%; and cortical tension, 21%. These results indicate that microtubules have little influence on the mechanical properties of neutrophils, and that increases in cellular rigidity caused by high concentrations of colchicine are due to a secondary effect that triggers actin polymerization. This study supports the conclusion that actin filaments are the primary structural determinants of neutrophil mechanical properties.

Cytosolic Ca2+ signalling in inflammatory neutrophils: implications for rheumatoid arthritis (Review).

Recognition of the ways in which neutrophil behaviour is regulated may be crucial for a full understanding of their role in inflammation and in rheumatoid arthritis. Although it is well established that changes in cytosolic free Ca2+ play a central role in triggering neutrophil responses, only recently has evidence accumulated which points strongly to the existence of two distinct Ca2+ pathways in neutrophils. One pathway is mediated by conventional agonists, such as formylated peptides, IL-8, C5a and PAF, and the other by cross-linking and immobilisation of surface receptors, such as integrins, and the Fc receptors, CD32 and CD16. In this review, we give evidence for these two signalling pathways in neutrophils, highlighting the roles of two Ca2+ storage and release organelles, one centrally located and stationary, and the other peripheral and mobile. We point out the significance of these two routes of Ca2+ signalling for the correct sequence of neutrophil responses, and suggest that aberration of this sequence could result in pathogenic neutrophil activation.

Neutrophil behavior following exposure to in vivo or in vitro zinc in normal and acutely-inflamed rats: studies on lysozyme secretion, superoxide anion release and platelet adhesion.

The mechanism was studied of the anti-inflammatory effect of oral zinc (114 mg/kg/day of elemental metal, given for 14 days) on the development of the carrageenan-induced paw oedema of the rat, and the impact of in vivo treatment on the activity of neutrophils isolated from the blood of inflamed and non-inflamed animals. The effects of the in vitro incubation with the metal on either non-inflamed or inflamed neutrophils coming from zinc-untreated rats were also examined. It was found that the administration of oral zinc inhibited markedly the process of ex vivo adhesion of the cells obtained from the inflamed rats (an observation confirmed by the in vitro experiments). In vitro release of lysozyme and superoxide anion productions were measured: in the absence of zinc, the 30' of pre-incubation carried out before stimulating with PMA did not influence the cell's reactivity of the non-inflamed neutrophils. It was, on the contrary, capable of significantly reducing that of the inflamed ones. As a consequence, it is quite difficult to properly interpret the data obtained studying the activity of the cells exposed to the metal in vitro.