Chemokinetic Response Research Articles

DISSECTING THE ROLE OF ODORANT RECEPTORS IN NAVIGATING HUMAN SPERM

In addition to odorant receptor (OR) expression in sensory neurons of the olfactory epithelium, some OR proteins are also found in ectopic tissues, e.g. in testis. In mammals, such testicular ORs have been attributed a potential function as molecular switches of unique sperm swimming behaviors. Using a combination of recombinant receptor expression in HEK293 cells, single cell calcium imaging, sperm accumulation assays, flagellar beating and video motion analysis, we recently identified and functionally characterized a human testicular OR, OR1D2 (a.k.a. hOR17-4), that specifically responds to a subset of structurally similar floral odors (e.g. bourgeonal) and triggers chemotactic and chemokinetic sperm responses in vitro. In addition, we here report functional description of two novel putative sperm ORs. With respect to their individual physiological roles in vivo, however, several crucial questions remain. First, are these ORs actually functional in human sperm? Second, do they trigger common or distinct signaling pathways, evoking similar or OR-specific behavioral responses? Third, is the popular “one cell - one receptor” rule that applies to olfactory neurons also applicable to sperm? Comparative analysis of receptor-specific activation profiles reveals non-overlapping molecular receptive fields as well as distinct susceptibility to antagonistic odors. Activation of individual ORs in human sperm mediates distinct behavioral response patterns. Our findings thus support the notion that OR-dependent signaling pathways could play a substantial role in various pre-fusion aspects of sperm physiology.

Movement of the parasitic nematode Phasmarhabditis hermaphrodita in the presence of mucus from the host slug Deroceras reticulatum

Abstract Phasmarhabditis hermaphrodita is a parasitic nematode capable of killing several species of slugs including Deroceras reticulatum, the most widespread slug pest in the world. This nematode can control slug infestations in a wide range of crops such as wheat, lettuce and strawberries. Optimization of this biocontrol agent depends on a proper understanding of the interaction between the host and parasite. In this paper, we investigate the response of P. hermaphrodita to the presence of slug mucus on plates of agar. We define an attraction index and find that the nematodes are significantly attracted by filter paper impregnated with slug mucus compared to paper impregnated with water. Second, nematode trails were recorded on a homogeneous layer of technical agar, with or without the presence of the slug mucus. Mucus was applied in two treatments comprising localization on a piece of filter paper and a uniform distribution across the plate. The different mucus treatments induced significantly different effects on the speed of nematode movement and the distribution of the turning angles, as well as the fractal dimension of nematode foraging trail. These results are consistent with the hypothesis that the nematodes exhibit both a chemotactic and chemokinetic response to a signal emanating from slug mucus.

Modeling of Granulocyte Cytoskeletal Responses Following fMLP Challenging

Formyl peptides released from Gram-negative bacteria ligate a group of specific mammalian receptors, expressed mainly on granulocytes, monocytes, and macrophages. Receptor ligation activates different transduction cascades, eventually leading to the release of reactive oxygen species and other bactericidal chemical species, and the activation of the actin cytoskeleton with extension of lamellipodia and migration toward the sites of maximal formyl peptide concentration. In vitro, under conditions of nongradient formyl peptide concentrations, lamellipodia form all around the cell contour (chemokinesis). In granulocytes challenged under these conditions with N-formyl-methionyl-leucyl-phenylalanine, (i) the power spectrum of the contour of activated cells shows a peak at a specific periodicity, indicating that the lamellipodial extension is not completely random but stochastically conforms to a deterministic scheme, and (ii) the morphological response (percent of cells exhibiting chemokinesis) tends to reach a maximum at certain drug concentrations, then declining at higher concentrations. Accordingly, the logarithm of the drug concentration-polarizing effect curve is bell-shaped. Herein we illustrate theoretical models for the simulation of these two components of the chemokinetic responses. We show that the main traits of the general morphology and arrangement of lamellipodia may be simulated by an algorithm that starting from a situation of random distribution of active receptors on the cell membrane, encompasses in the successive calculation cycles both a local autocatalytic enhancement of the actin polymerization and a relative inhibition of the actin polymerization at some distance from the more active polymerization foci. In addition, a drug log concentration-polarizing effect bell-shaped curve may be simulated by assuming that the N-formyl-methionyl-leucyl-phenylalanine, while binding with high affinity to the specific receptor, is also able to bind to another lower affinity receptor that may effect depolarizing actions or, more generally, metabolic blocking effects. Under these conditions, at low drug concentrations the polarizing effect brought about by the ligation of the specific receptor is largely predominant. However, as the drug concentration increases and the specific receptors approach saturation, the inhibitory effects become more and more powerful and the net polarizing effect is reduced.

Entamoeba histolyticacell movement: A central role for self-generated chemokines and chemorepellents

Entamoeba histolytica cells, the cause of amoebic dysentery, are highly motile, and this motility is an essential feature of the pathogenesis and morbidity of amoebiasis. However, the control of E. histolytica motility within the gut and during invasion is poorly understood. We have used an improved chemotaxis assay to identify the key extracellular signals mediating Entamoeba chemotaxis. The dominant responses we observe are caused by factors generated by E. histolytica cells themselves. Medium that has been conditioned by E. histolytica growth causes both chemokinesis and negative chemotaxis. The speed of random movement is more than doubled in conditioned compared with fresh medium, and cells move efficiently away from conditioned medium by negative chemotaxis. Ethanol, the product of Entamoeba glucose metabolism, is the principal component of the chemokinetic response. The closely related but nonpathogenic Entamoeba dispar shows no change in motility in response to conditioned medium implying that these responses are central to E. histolytica pathogenesis.

Selective priming of peripheral blood eosinophils in patients with idiopathic hypereosinophilic syndrome

The idiopathic hypereosinophilic syndrome (HES) is characterised by blood eosinophilia associated with organ involvement. Elevated numbers of blood neutrophils have been observed during episodes of active HES. However, an increased responsiveness of eosinophils to chemotactic and chemokinetic stimuli may explain the selective eosinophil infiltration of the tissue. We have studied the migratory responses of blood eosinophils and neutrophils from 9 patients with HES and from 13 healthy control subjects. Chemokinetic and chemotactic responses to factors acting on both cell types were analysed by means of a modification of the Boyden chamber technique. We found increased migratory responses of the eosinophils, but not of the neutrophils, from the patients with HES. Increased blood neutrophil counts in three of the patients did not coincide with alterations of the neutrophil migratory responses. Our finding of increased migratory responses of eosinophils from patients with HES towards non-specific chemoattractants suggests selective priming of eosinophils in this disease. Interleukin (IL)-5 has previously been shown to prime eosinophils for migratory responses, and successful anti-IL-5 therapy of patients with HES indicates an important role for this cytokine in the development of hypereosinophilia.

Fluid Shear Stress Modulates Cell Migration Induced by Sphingosine 1-Phosphate and Vascular Endothelial Growth Factor

The rational design of drug delivery systems requires the ability to predict the environment-specific responses of target cells to the delivered drug. Here we describe the in vitro effects of fluid shear stress, vascular endothelial growth factor (VEGF), and sphingosine 1-phosphate (S1P) on the migration of human umbilical vein endothelial cells (HUVEC). Endothelial cell migration into a scrape wound was enhanced in S1P- or VEGF-stimulated HUVEC by the addition of fluid shear stress. In both cases, scrape wound closure rates were near a maximal value that was not exceeded when cells were exposed to all three factors. We also found that cell migration into a scrape wound due to S1P stimulation was correlated with the S1P1 mRNA concentration, in systems where cell migration was not already near maximal. The present work represents our initial steps toward predicting cell migration based upon the activation state of the receptors and enzymes involved in the chemokinetic response. These results also illustrate the importance of context-dependent analysis of cell signaling cascades.

Impaired dendritic-cell homing in vivo in the absence of Wiskott-Aldrich syndrome protein

AbstractRegulated migration and spatial localization of dendritic cells (DCs) are critical events during the initiation of physiologic immune responses and maintenance of tolerance. Here we have used cells deficient in the Wiskott-Aldrich syndrome protein (WASp) to demonstrate the importance of dynamic remodeling of the actin cytoskeleton for these trafficking processes to occur in vitro and in vivo. On fibronectin-coated surfaces, WASp-null immature murine DCs exhibited defects both of attachment and detachment, resulting in impaired net translocation compared with normal cells. The chemokinetic response to CCL21, which is critical for normal lymphatic trafficking, was also abrogated in the absence of WASp. In vivo in both fluorescein isothiocyanate (FITC) and oxazolone contact hypersensitivity models, WASp-null Langerhans cell (LC) migration was compromised, as judged by exit from the skin as well as by homing to the draining lymph node (LN). Furthermore, following systemic challenge with lipopolysaccharide (LPS) or toxoplasma-derived antigen, WASp-null DCs showed incomplete redistribution to T-cell areas in the spleen. Instead, they were retained ectopically in the marginal zone. DC trafficking in vivo is therefore dependent on a normally regulated actin cytoskeleton, which performs an essential function during maintenance of physiologic immunity and when disturbed may contribute significantly to the immunopathology of Wiskott-Aldrich Syndrome.

Transendothelial migration of human basophils.

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1beta significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that beta2 integrins play the primary role in basophil TEM, but beta1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.

A new interpretation of the Keller-Segel model based on multiphase modelling

In this paper an alternative derivation and interpretation are presented of the classical Keller-Segel model of cell migration due to random motion and chemotaxis. A multiphase modelling approach is used to describe how a population of cells moves through a fluid containing a diffusible chemical to which the cells are attracted. The cells and fluid are viewed as distinct components of a two-phase mixture. The principles of mass and momentum balance are applied to each phase, and appropriate constitutive laws imposed to close the resulting equations. A key assumption here is that the stress in the cell phase is influenced by the concentration of the diffusible chemical. By restricting attention to one-dimensional cartesian geometry we show how the model reduces to a pair of nonlinear coupled partial differential equations for the cell density and the chemical concentration. These equations may be written in the form of the Patlak-Keller-Segel model, naturally including density-dependent nonlinearities in the cell motility coefficients. There is a direct relationship between the random motility and chemotaxis coefficients, both depending in an inter-related manner on the chemical concentration. We suggest that this may explain why many chemicals appear to stimulate both chemotactic and chemokinetic responses in cell populations. After specialising our model to describe slime mold we then show how the functional form of the chemical potential that drives cell locomotion influences the ability of the system to generate spatial patterns. The paper concludes with a summary of the key results and a discussion of avenues for future research.

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.

Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B(4) (LTB(4)) is one of the most potent PMN chemoattractants. LTB(4) exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB(4) signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6(-/-) mice. In vitro, GRK6(-/-) PMN showed increased chemokinetic and chemotactic responses to LTB(4). GRK6(-/-) PMN respond to LTB(4) with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB(4) receptor desensitization in the absence of GRK6. However, pre-exposure to LTB(4) renders both GRK6(-/-) as well as wild-type PMN refractory to restimulation with LTB(4), indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.

Chemotactic and chemokinetic activities of Saprolegnia parasitica toward different metabolites and fish tissue extracts

The chemotactic and chemokinetic activities in zoospores of Saprolegnia parasitica NJM 8604 (= H2) were examined using various amino acids, carbohydrates, fatty acids, and fish tissue extracts to estimate one of the important factors for attachment of zoospores to their host. All the tested six amino acids showed strong chemotactic reactions whereas carbohydrates and fatty acids caused moderate or strong chemotactic reactions. The chemokinetic activities against amino acids and carbohydrates were moderate or weak, whereas almost all fatty acids showed negative chemokinetic responses. Almost all tested fish tissue showed moderate chemotactic response and weak or moderate chemokinetic responses. Generally, chemotactic activity was strong in the amino acids, and the strongest activity was observed in alanine. Based on these facts, we considered that zoospores may react against amino acids of the fish body to attach and establish their colonization.

Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting. Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils. An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor. In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.

Mobilization of endothelial and hematopoietic stem and progenitor cells by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and angiopoietin-1.

The chemokine stroma-derived factor-1 (SDF-1) is produced within the bone marrow and mediates chemokinesis and chemotaxis on a variety of cell types that express the CXCR4 receptor. SDF-1-responsive cell types include monocytes and macrophages, B and T lymphocytes, platelets and megakaryocytes, and CD34+ cells, including both hematopoietic progenitors and stem cells. We have used intravenous injection of a replication-incompetent adenovector expressing the SDF-1 gene to elevate serum levels of SDF-1 in Balb/c and SCID mice. Within 3 to 5 days there was a marked leukocytosis, predominantly involving monocytes, and a three-fold increase in platelets. In addition, AdSDF-1 mobilized CFU-GM, CFU-s, and cells with long-term repopulating potential. We have identified a bone marrow-derived, circulating endothelial stem cell characterized by expression of the VEGFR2 (Flk-1/KDR). This cell exhibits a chemotactic and chemokinetic response to SDF-1 and VEGF. We have elevated serum levels of VEGF165 using intravenous adenovector gene delivery and compared this to an adenovector expressing angiopoietin-1 alone or in combination with VEGF. VEGF elevation was associated with rapid mobilization of hematopoietic stem and progenitor cells and a population of Flk-1-positive endothelial progenitors. In contrast angiopoietin induced a delayed mobilization of endothelial and hematopoietic progenitors. The combination of VEGF and angiopoietin produced a more prolonged elevation of these progenitors in the circulation with increased proliferation of capillaries and expansion of sinusoidal spaces in the marrow.

Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo.

Recruitment of macrophages to sites of cell death is critical for induction of an immunologic response. Calcium concentrations in extracellular fluids vary markedly, and are particularly high at sites of injury or infection. We hypothesized that extracellular calcium participates in modulating the immune response, perhaps acting via the seven-transmembrane calcium-sensing receptor (CaR) on mature monocytes/macrophages. We observed a dose-dependent increase in monocyte chemotaxis in response to extracellular calcium or the selective allosteric CaR activator NPS R-467. In contrast, monocytes derived from mice deficient in CaR lacked the normal chemotactic response to a calcium gradient. Notably, CaR activation of monocytes bearing the receptor synergistically augmented the transmigration response of monocytes to the chemokine MCP-1 in association with increased cell-surface expression of its cognate receptor, CCR2. Conversely, stimulation of monocytes with MCP-1 or SDF-1alpha reciprocally increased CaR expression, suggesting a dual-enhancing interaction of Ca(2+) with chemokines in recruiting inflammatory cells. Subcutaneous administration in mice of Ca(2+), MCP-1, or (more potently) the combination of Ca(2+) and MCP-1, elicited an inflammatory infiltrate consisting of monocytes/macrophages. Thus extracellular calcium functions as an ionic chemokinetic agent capable of modulating the innate immune response in vivo and in vitro by direct and indirect actions on monocytic cells. Calcium deposition may be both consequence and cause of chronic inflammatory changes at sites of injury, infection, and atherosclerosis.

Modulation of eosinophil chemotactic activities to leukotriene B 4 by n-3 polyunsaturated fatty acids

Eosinophil accumulation induced by leukotriene B 4 appears to be involved in the pathogenesis of allergic diseases. We evaluated the effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on chemotaxis to leukotriene B 4 in guinea pig peritoneal eosinophils. Guinea pigs that were sensitized to polymyxin B were administered an intraperitoneal injection of polymyxin B (1 mg/animal) alone or combined with DHA (15 or 50 mg/kg, i.p.), EPA (50 or 100 mg/kg, i.p.), or with linoleic acid (LA) (100 mg/kg, i.p.). Forty hours later, eosinophils were obtained from the intraperitoneal lavage fluid and purified. The chemotactic and chemokinetic responses of eosinophils to leukotriene B 4 were measured using a 96-well microchemotaxis chamber. DHA significantly decreased the chemotactic and chemokinetic responses of eosinophils in a dose-dependent fashion. A higher dose of EPA also significantly inhibited both of those responses, whereas LA had no effect. Our results suggested a possible mechanism for the improvement of allergic diseases by dietary supplementation with n-3 PUFA.

CD19-SELECTED B LYMPHOCYTES SYNTHESIZE, SECRETE AND MIGRATE IN THE PRESENCE OF IL-8. TNF-α AND γIP-10 ARE ALSO B LYMPHOCYTE MIGRATORY FACTORS

B lymphocytes are responsible for antigen uptake and presentation, as well as antibody production. These reactions require close cell-to-cell contact between B lymphocytes and monocytes. In this study we demonstrate that interleukin 8 (IL-8), γ-immune protein 10 (γIP-10) and tumour necrosis factor α (TNF-α) all induce a significant chemokinetic response of human B lymphocytes. Among the cytokines tested, rIL-8 was the strongest B lymphocyte migratory factor with a migratory index (MI) of 2.03±0.32, (P<0.002), followed by rTNF-α (MI=1.89±0.17,P<0.001) and rγIP-10 (MI=1.63±0.17,P<0.001). We did not observe B lymphocyte migration towards rIL-1α, rIL-2, rIL-4, rIL-10, interferon γ (rINF-γ) or transforming growth factor β (rTGF-β). Furthermore, we report that human B lymphocytes have a constitutive IL-8 mRNA expression and protein secretion in vitro. Resting as well as stimulated B lymphocytes secrete on average 1.5 ng IL-8/ml medium/24 h (2×106B lymphocytes). Our data indicate a possible mechanism by which B lymphocytes make contact with other cells, during immuno-inflammatory processes.

MIGRATION RESPONSES OF HUMAN MONOCYTIC CELL LINES TO α- AND β-CHEMOKINES

The β-chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), MIP-1β and regulated on activation, normal T cells, expressed and secreted (RANTES) induced the in vitro migration of the monocytic cell line MonoMac-6. MCP-1 exhibits the most potent chemotactic effect on this cell line while MIP-1α, RANTES and to a lesser extent MIP-1β were more moderate inducers of cell migration. MonoMac-6 migration in response to chemokines was shown to be a chemotactic and not a chemokinetic response, which was inhibited by pertussis and cholera toxins suggesting a role for G proteins in chemokine receptor-mediated signalling in these cells; chemotaxis of MonoMac-6 cells in response to MCP-1 was abrogated by the addition of anti-MCP-1 antibody. The response of MonoMac-6 cells to the α-chemokines IL-8, IP-10, growth-related peptide (Gro) α and MIP-2β was substantially weaker than to the β-chemokines. MCP-1 caused an alteration in cellular morphology by increasing ruffling at the cell membrane and the number of cells exhibiting extended pseudopodia. The chemotactic response of MonoMac-6 cells to β-chemokines was compared with less well-differentiated myelomonocytic cell lines. THP-1 showed a similar, but weaker response to the β-chemokines while both HL60 and U937 failed to respond to any member of this subfamily when tested under the same conditions. These results suggest that the differentiation status of cells of monocytic lineage may affect their response to β-chemokines.

Effects of in vivo administration of G-CSF on neutrophil functions in healthy volunteers.

The aim of the present study was to investigate the in vivo effects of granulocyte colony-stimulating factor (G-CSF) on neutrophil (PMN) function. G-CSF was administered once daily as s.c. injection for 6 d (d1-6) to healthy male volunteers. PMN migration (modified Boyden chamber), chemiluminescence (CL), adherence to nylon fibers and phagocytosis of IgG- and IgG-C3-coated particles were investigated before (d1), during (d2, d5) and 3 wk after G-CSF 7.5-10 micrograms/kg/d (n = 12). PMN surface expression of adhesion- and Fc gamma-receptors was measured on d1, d5, d8 and 3 wk after G-CSF 3-5 micrograms/kg (n = 12). Results obtained after G-CSF were compared to baseline using Wilcoxon's signed rank test. G-CSF induced PMNs showed a significantly (p < 0.05) decreased chemokinetic response (d5) as well as a reduced chemotaxis towards zymosan activated serum, FMLP and IL-8, respectively. Chemotaxis was reduced both at d2 and d5. Neutrophil adherence, phagocytosis and luminol-enhanced CL increased, whereas G-CSF had no effect on lucigenin-enhanced CL. G-CSF (3-5 micrograms/kg) caused an enhanced expression of CD11b, CD18, CD35, CD64 (Fc gamma RI) and CD32 (Fc gamma RII), respectively. We conclude that neutrophils produced in response to G-CSF have a reduced chemotaxis but an enhanced adherence and phagocytic capacity. G-CSF in vivo does not stimulate the respiratory burst.

Glucocorticoid-mediated inhibition of neutrophil emigration in an endotoxin-induced rat pulmonary inflammation model occurs without an effect on airways MIP-2 levels.

In an intratracheal endotoxin lipopolysaccharide (LPS) challenge model of acute lung injury, we recently reported that while dexamethasone (DEX) at 2 to 4 mg/kg reduced neutrophil (PMN) emigration into the airways, bronchoalveolar lavage (BAL) fluid contained substantial amounts of immunoreactive macrophage inflammatory protein-2 (MIP-2). In the present study, DEX in quantities up to 30 mg/kg further reduced PMN influx, but MIP-2 levels were unaffected 3 h following LPS challenge. MIP-2 in 3-h BAL samples from DEX- or vehicle-pretreated animals was bioactive and approximately 75% of the ex vivo chemotactic activity was neutralized by polyclonal antirat MIP-2. In contrast to the in vivo studies, DEX significantly suppressed MIP-2 synthesis from LPS-stimulated rat alveolar macrophages in vitro. Ex vivo chemotactic activity was comparable between BAL samples from DEX- and vehicle-pretreated rats. Chemotaxis of rat PMN to recombinant MIP-2 exhibited a bell-shaped concentration-response profile in vitro with optimal activity at 17.6 ng/ml and this was shifted 16-fold to the right by antirat MIP-2. Three-hour BAL MIP-2 (10.73 +/- 0.45 ng/ml) correlated with the ascending limb of the recombinant rat MIP-2 concentration-response profile. In addition to inhibiting chemotaxis, antirat MIP-2 also reduced the chemokinetic response to 3-h BAL fluid by 84%. The present study demonstrates that in vivo MIP-2 is bioactive, and because its synthesis within the rat lung is resistant to DEX it is likely to play a significant role in glucocorticoid refractory PMN influx within the airways during acute lung injury.