Neutrophil Chemotactic Response Research Articles

Immune phenotype is differentially affected by changing the type of bovine respiratory disease vaccine administered at revaccination in beef heifers.

During preconditioning, modified-live vaccines are frequently administered to beef calves before weaning. In this study, we began to characterize the immune phenotype of calves that received a modified-live vaccination at 3-4 months of age and then either received the same modified-live or an inactivated vaccine upon arrival at the feedlot (weaning) and 28 days post-arrival (booster). Innate and adaptive immune measures were assessed before revaccination and 14 and 28 days post. Heifers that received three doses of the modified-live vaccine exhibited a relatively balanced immune response based on increases in mean cytokine concentrations (IL-17, IL-21) and total immunoglobulin-G (IgG) and subsets IgG1 and IgG2, which are related to both arms of the adaptive immune system. Conversely, heifers that received one dose of modified live and two doses of the inactivated vaccine had a more robust neutrophil chemotactic response and greater serum-neutralizing antibody titers, resulting in an enhanced innate immune and a skewed proinflammatory response. These results indicate that the revaccination protocol used after initial vaccination with a modified-live vaccine differentially influences the immune phenotype of beef calves, with three doses of modified live inducing potentially immune homeostasis and a combination of modified live and inactivated vaccines inducing a skewed immune phenotype. However, more research is needed to determine the protective efficacy of these vaccination protocols against disease.

A novel CXCL8-IP10 hybrid protein is effective in blocking pulmonary pathology in a mouse model of Klebsiella pneumoniae infection

Klebsiella pneumoniae (K. pneumoniae) is a hospital-acquired infectious agent that causes a range of diseases. Herein we have developed a novel CXCL8-IP10 hybrid protein and evaluated its efficacy in an animal model of K. pneumoniae infection. Neutrophil chemotaxis data revealed that CXCL8-IP10 could inhibit human neutrophil chemotactic responses induced by the ELR-CXC chemokine CXCL8. To evaluate the effect of CXCL8-IP10 on K. pneumoniae infection, C57BL/6 mice were challenged with K. pneumoniae followed by treatment with CXCL8-IP10 (500 μg/kg, i.p.), or dexamethasone (0.8 mg/kg, s.c.), or ceftazidime (200 mg/kg, s.c.) individually. CXCL8-IP10, dexamethasone or ceftazidime markedly inhibit Klebsiella-induced pulmonary inflammation as assessed by gross examination and histopathology. Moreover, the chemotactic responses of neutrophils was blocked by CXCL8-IP10 rather than dexamethasone or ceftazidime. Furthermore, the levels of inflammatory factors IL-1β, IFN-γ and TNF-α were decreased after CXCL8-IP10, dexamethasone or ceftazidime treatment. Together, these results suggest that CXCL8-IP10 may provide a novel strategy for treating K. pneumoniae infection.

Chronic Low-Dose Cadmium Exposure Impairs Cutaneous Wound Healing With Defective Early Inflammatory Responses After Skin Injury.

Impairment of the immune system is a developing concern in evaluating the toxicity of cadmium (Cd). In the present study, we investigated if Cd could impair cutaneous wound healing through interfering with inflammation after injury. We found that exposure of mice to CdCl2 through drinking water at doses of 10, 30, and 50 mg/l for 8 weeks significantly impaired cutaneous wound healing. Chronic 30 mg/l CdCl2 treatment elevated murine blood Cd level comparable to that of low dose Cd-exposed humans, had no effect on blood total and differential leukocyte counts, but reduced neutrophil infiltration, chemokines (CXCL1 and CXCL2), and proinflammatory cytokines (TNFα, IL-1β, and IL-6) expression in wounded tissue at early stage after injury. Wounded tissue homogenates from CdCl2-treated mice had lower chemotactic activity for neutrophils than those from untreated mice. Mechanistic studies showed that chronic Cd treatment suppressed ERK1/2 and NF-κB p65 phosphorylation in wounded tissue at early stage after injury. Compared with neutrophils isolated from untreated mice, neutrophils from CdCl2 treated mice and normal neutrophils treated with CdCl2 invitro both had lower chemotactic response, calcium mobilization and ERK1/2 phosphorylation upon chemoattractant stimulation. Collectively, our study indicate that chronic low-dose Cd exposure impaired cutaneous wound healing by reducing neutrophil infiltration through inhibiting chemokine expression and neutrophil chemotactic response, and suppressing proinflammatory cytokine expression. Cd may suppress chemokine and proinflammatory expression through inactivating ERK1/2 and NF-κB, and inhibit neutrophil chemotaxis by attenuating calcium mobilization and ERK1/2 phosphorylation in response to chemoattractants.

Effects of K11R and G31P Mutations on the Structure and Biological Activities of CXCL8: Solution Structure of Human CXCL8(3-72)K11R/G31P.

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), the expression levels of which are elevated in many inflammatory diseases, binds to both the CXCR1 and CXCR2 receptors with high affinity. Recently, an analogue of human CXCL8, CXCL8(3–72)K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both the CXCR1 and CXCR2. Herein, we have determined the solution structure and the CXCR1 N-terminal peptide binding sites of hG31P by NMR spectroscopy. We have found that the displacement within the tertiary structure of the 30 s loop and the N-terminal region and more specifically change of the loop conformation (especially H33), of hG31P may affect its binding to the CXCR1 receptor and thereby inhibit human neutrophil chemotactic responses induced by ELR-CXC chemokines. Our results provide a structural basis for future clinical investigations of this CXCR1/CXCR2 receptor antagonist and for the further development of CXCL8 based antagonists.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

Differential neutrophil chemotactic response towards IL-8 and bacterial N-formyl peptides in term newborn infants

Background: A prerequisite for an effective innate immunity is the migrative ability of neutrophils to respond to inflammatory and infectious agents such as the intermediate interleukin (IL)-8 and the end-target formyl-methionyl-leucyl-phenylalanine (fMLP) chemoattractants. The aim was to study the chemotactic capacity of neutrophils from newborn infants and adults in response to IL-8 and the bacterial peptide fMLP.Methods: In the under-agarose cell migration assay, isolated leukocytes from healthy adults and from cord blood of healthy term newborn infants were studied with dose responses towards IL-8 and fMLP. The same number of leukocytes (1 × 105 cells), with the same distribution of neutrophils and monocytes, were analyzed in neonates and adults. Chemotaxis was distinguished from randomly migrating neutrophils, and the neutrophil pattern of migration, i.e. the migration distance and the number of migrating neutrophils per distance, was evaluated.Results: In comparison to adults, fewer neutrophils from newborn infants migrated towards IL-8 and for a shorter distance (P < .01, respectively). The number of neutrophils migrating to different gradients of fMLP, the distance they migrated, and the correlation between the number and the distance were the same for neonates and adults. Random migration did not differ in any instance.Conclusion: Chemotaxis of neutrophils from newborn infants was as co-ordinated as neutrophils from adults in response to fMLP, whereas the response to IL-8 was reduced. The differential response of neutrophils from neonates to intermediate and end-target chemoattractants could indicate a reduced infectious response.

Evidence for chemokine synergy during neutrophil migration in ARDS

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening condition characterised by pulmonary oedema, respiratory failure and severe inflammation. ARDS is further characterised by the recruitment of neutrophils into the lung...

Melatonin in sperm biology: breaking paradigms.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

Technical Advance: Introducing a novel metric, directionality time, to quantify human neutrophil chemotaxis as a function of matrix composition and stiffness

A direct consequence of cellular movement and navigation, migration incorporates elements of speed, direction, and persistence of motion. Current techniques to parameterize the trajectory of a chemotaxing cell most commonly pair migration speed with some measure of persistence by calculating MSD, RMS speed, TAD, and/or CI. We address inherent limitations in TAD and CI for comparative analysis by introducing two new analytical tools to quantify persistence: directionality index and directionality time. With the use of these tools, we show that the mechanical properties of the underlying substrate contribute significantly to the regulation of human neutrophil chemotaxis toward fMLP on Fgn-, Col-, and Fn-coated gels of varying elasticity. The β₁-integrin ligand Col demonstrated mechanosensitive speed. In contrast, β₂-integrin ligand Fgn supported mechanosensitive persistence. Fn, recognized by β₁ and β₂ integrins, mechanoregulated speed and persistence. Blocking β₂ integrins of cells migrating on Fn identified an underlying β₂-integrin-directed modulation of persistence. These data demonstrate that individual components of the neutrophil chemotactic response show integrin dependence and are finely tunable with different ligand, mechanotactic, and chemotactic cues, underscoring the need for sensitive analytical methods.

Neutrophil chemotaxis in granulomatosis with polyangiitis (Wegener's) and idiopathic pulmonary fibrosis

The presence of antineutrophil cytoplasmic antibodies in granulomatosis with polyangiitis (Wegener's) (GPA) implicates the neutrophil as a key effector cell. Previous studies have reported elevated neutrophil counts in the lung, although the determinants of neutrophil chemotaxis in the GPA lung are unknown. Bronchoalveolar lavage fluid (BALF) cell counts, myeloperoxidase (MPO) and chemokines were measured in 27 patients with GPA, 20 disease controls with idiopathic pulmonary fibrosis (IPF) and six healthy controls. CXC chemokine ligand (CXCL)8, interleukin (IL)-1β, epithelial neutrophil-activating protein 78, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor were measured by ELISA. The neutrophil chemotactic potential of BALF was investigated using the under-agarose method, and specific antibodies were used to examine the role of CXCL8 and IL-1β. GPA BALF had an increased neutrophil percentage, and elevated MPO, CXCL8 and G-CSF concentrations compared with healthy controls. Chemotaxis of control neutrophils towards BALF from patients with active (p=0.006) and remission (p=0.077) GPA, and IPF (p=0.001) patients was increased compared with normal controls. BALF-induced chemotaxis correlated with BALF IL-1β (r=0.761, p=0.001) and CXCL8 (r=0.640, p=0.012) in GPA, and was inhibited by anti-CXCL8 (85%; p<0.001) and anti-IL-1β (69%; p<0.001). Our study confirms a neutrophilia and pro-inflammatory alveolar milieu that persists in clinical remission. CXCL8 and IL-1β appear to play important roles in the neutrophil chemotactic response to BALF.

The role of the ribosomal protein S19 C-terminus in altering the chemotaxis of leucocytes by causing functional differences in the C5a receptor response

Ribosomal protein S19 (RP S19) oligomers have been discovered as the first chemoattractant of migrating monocytes/macrophages to apoptotic cells via the C5a receptor (C5aR). In contrast to C5a, a fusion of the C-terminus (I(134)-H(145)) of RP S19 to C5a, the C5a/RP S19 chimera, substitutes for the RP S19 oligomers and is able to replicate C5aR antagonist-induced and agonist-induced dual effects on neutrophil and monocyte chemotactic responses, respectively. We recently discovered a gain of binding affinity when the I(134)-H(145) inhibited the activation of neutrophil C5aR-mediated chemotactic pathways. However, the opposing ligand-dependent chemotactic mechanisms are not fully understood. In this study, a loss of this additional binding affinity appeared to cause the monocyte C5aR to activate an alternative signalling pathway. The p38 mitogen activated-protein kinase (MAPK) pathway was linked to cell migration rather than a classical extracellular-regulated kinase 1/2 pathway commonly used by C5a. C5aR internalization was not involved in the alternative chemotactic pathway. We propose a model of activation involving a C5aR co-molecule that interferes with the C5aR-Gi protein interaction upon binding to the I(134)-H(145) in neutrophils; however, a free I(134)-H(145) from the C5aR co-molecule can guide the alternative activation of the chemotactic p38MAPK pathway in monocytes/macrophages.

Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice

Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses. K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF(-/-) , and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation. K/BxN serum-transfer arthritis was markedly attenuated in MIF(-/-) mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF(-/-) mice. Similarly, MIF(-/-) mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF(-/-) mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases. These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment.

The effect of essential oil extracted from Nigella sativa (L.) seeds on human neutrophil functions

This study was aimed to investigate the effect of essential oil on human neutrophil (HN) functions. The neutrophils were isolated on percoll gradients, counted, and tested for viability using the trypan blue exclusion method. The chemotactic response was based on a multiple blind well assay system. The control movement and chemotactic response of neutrophils to 0.1 µM fMLP were reduced at a dose-dependent manner. The essential oil significantly inhibited neutrophil chemotaxis from 0.05 to 0.5 mg mL−1. The inhibitory concentrations (IC50) showing 50% inhibition to induced neutrophil chemotaxis, and control movement were 0.08 and 0.07 mg mL−1, respectively. The human neutrophil elastase secretion was inhibited by essential oil at a concentration dependent manner from 0.5 to 2.5 mg mL−1. The components of essential oil are potent inhibitors for polymorpho nuclear leukocytes functions. The observed inhibition of neutrophil functions occurred via intracellular pathway. Active serine protease could be essential for neutrophil responding process and/or signal transduction pathways.

Neutrophil hyperchemotaxis in Behçet’s disease: a possible role for monocytes orchestrating bacterial-induced innate immune responses

Increased neutrophil chemotaxis and hyperresponsiveness to streptococci have been considered to play a role in Behçet's disease (BD) pathogenesis. Our aim was to correlate TLR2 expression and chemotactic responses stimulated by bacterial lipoteichoic acid (LTA) in BD neutrophils. Thus, we assessed expressions of TLR2 and the correlate receptors CD14, CD114 (G-CSF receptor), CD116 (GM-CSF receptor) and also TLR4 on circulating neutrophils and monocytes of patients with active BD. Serum concentration of soluble CD14 (sCD14) was also measured. Neutrophil chemotactic responses from BD patients and healthy controls under LTA stimulation were assessed. Disease activity was evaluated by Behçet's Disease Current Activity Form (BDCAF). Receptor expressions were measured by flow cytometry, neutrophil chemotaxis was assessed in a Boyden chamber and sCD14 was measured by enzyme-linked immunosorbent assay. TLR2 expression was higher only on BD monocytes compared with healthy controls (39.9 +/- 13.1 vs. 33.6 +/- 5.3, p = 0.019). Expressions of all other receptors were similar in BD and control group. Of particular interest, TLR2 expression on neutrophils was similar in both groups and, when stimulated with LTA, BD neutrophils showed chemotactic responses similar to the controls. Neutrophils exhibited increased chemotaxis only when incubated with BD plasma. Serum sCD14 concentration was higher in BD patients than in controls (1,920.8 +/- 563.6 vs. 1,623.2 +/- 391.3 ng/ml, p = 0.008), and it was positively correlated with both CD14 expression on circulating monocytes membrane (p = 0.035) and BDCAF scores (p = 0.025). In conclusion, isolated BD neutrophils do not overexpress TLR2 neither overreact to LTA. However, because TLR2 expression was higher on BD monocytes, sCD14 from monocyte origin correlated with disease activity and neutrophil hyperchemotaxis occurred on strict dependence of plasmatic soluble factors, we suggest a possible role for bacterial stimulation of monocytes via TLR2 producing neutrophil-stimulating pro-inflammatory factors in BD.

ELR-CXC Chemokine Receptor Antagonism Targets Inflammatory Responses at Multiple Levels

The ELR-CXC chemokines play important roles in neutrophilic inflammation. We report in this study that a fully human ELR-CXC chemokine antagonist that we have generated, CXCL8((3-72))K11R/G31P (G31P), has potent anti-inflammatory effects that arise through its actions at multiple levels. G31P inhibited CXCL8-induced chemotactic responses and intracellular Ca(2+) flux in CXCR1-transfected HEK cells and neutrophils, and responses of neutrophils to CXCR2-exclusive ligands. G31P desensitized heterologous G protein-coupled receptors on neutrophils, 52-86% reducing their Ca(2+) flux and chemotactic responses to leukotriene B(4), C5a, and the bacterial tripeptide fMLP. G31P also 60-90% blocked neutrophil chemotactic responses to mediators present in 10 of 12 sputum samples from cystic fibrosis or bronchiectasis subjects with bacterial pneumonia. Moreover, whereas A549 bronchial epithelial cells (which expressed CXCR1) secreted approximately 29,000 pg/ml CXCL8 in response to in vitro endotoxin challenge, G31P reduced this response by up to 98%, presumably by interrupting an autocrine inflammatory loop. The anti-inflammatory effects of G31P extended also to reversing the antiapoptotic influence of ELR-CXC chemokines on neutrophils. That these effects were relevant in vivo was confirmed in a guinea pig model of airway endotoxemia, wherein the human form of G31P >95% blocked neutrophil infiltration into and activation within the airways, as determined by airway levels of the neutrophil primary, secondary, and tertiary granule markers myeloperoxidase, lactoferrin, and matrix metalloproteinase-9, respectively, and the epithelial cell marker matrix metalloproteinase-2. These data suggest that the beneficial effects of ELR-CXC chemokine antagonism arise through effects that occur at multiple levels, including epithelial cells, neutrophils, and alternate G protein-coupled receptors.

THE ROLE OF NEUTROPHILS IN SEVERE SEPSIS

Neutrophils are key effectors of the innate immune response. Reduction of neutrophil migration to infection sites is associated with a poor outcome in sepsis. We have demonstrated a failure of neutrophil migration in lethal sepsis. Together with this failure, we observed more bacteria in both peritoneal exudates and blood, followed by a reduction in survival rate. Furthermore, neutrophils obtained from severe septic patients displayed a marked reduction in chemotactic response compared with neutrophils from healthy subjects. The mechanisms of neutrophil migration failure are not completely understood. However, it is known that they involve systemic Toll-like receptor activation by bacteria and/or their products and result in excessive levels of circulating cytokines/chemokines. These mediators acting together with LPS stimulate expression of iNOS that produces high amounts of NO, which in turn mediates the failure of neutrophil migration. NO reduced expression of CXCR2 on neutrophils and the levels of adhesion molecules on both endothelial cells and neutrophils. These events culminate in decreased endothelium-leukocyte interactions, diminished neutrophil chemotactic response, and neutrophil migration failure. Additionally, the NO effect, at least in part, is mediated by peroxynitrite. In this review, we summarize what is known regarding the mechanisms of neutrophil migration impairment in severe sepsis.

Chemotactic effect of melatonin on leukocytes

Melatonin seems to be an important stimulatory factor of the immune system. This indolamine is capable of inducing activation of leukocytes. Tissue leukocyte infiltration is a key feature of inflammatory and immune responses; however, there is no information about the effect of melatonin on leukocyte chemotaxis. Therefore, the aim of this study was to examine the in vitro and in vivo effects of melatonin on leukocyte chemotaxis, on modulation of leukocyte chemotaxis to other chemoattractants and on the in vivo induction of leukocyte chemokines. Neutrophils and mononuclear leukocytes (PBMC) were isolated by a discontinuous gradient on Hystopaque. Chemotaxis was performed in blind well Boyden's chambers. In vivo chemotaxis was determined after intraperitoneal injection of melatonin into rats. Leukocyte chemotactic response and leukocyte chemokine expression were determined in human volunteers treated with 20 mg daily of melatonin. Increased neutrophils and PBMC chemotaxis in response to 1.2 nm melatonin was observed in vitro. Peritoneal leukocytes were found increased after melatonin injection. Humans treated with melatonin showed an increased neutrophil chemotactic response to a physiological chemoattractant and increased expression of intracellular chemokines; however, decreased chemotactic response and no chemokine expression were observed in PBMC. These data suggest that melatonin could have a relevant role during the tissue leukocyte infiltration in inflammatory and immune responses.

STAT3 governs distinct pathways in emergency granulopoiesis and mature neutrophils

AbstractGranulocyte colony-stimulating factor (G-CSF) is essential for the host response to bacterial infection by controlling neutrophil production in the bone marrow. The G-CSF receptor (G-CSFR) activates the Jak/STAT pathway, although little is understood about how these signals regulate basal and stress-induced granulopoiesis. We examined STAT3 function in granulocytes using a bone marrow conditional knockout mouse model. Our results show that STAT3 has a crucial role in emergency granulopoiesis and mature neutrophil function. STAT3-deficient mice have an aberrant response to G-CSF in vivo, characterized by failure to accumulate immature granulocytes and an increased ratio of mature to immature neutrophils in the bone marrow, peripheral blood, and spleen. Acute neutrophil mobilization is impaired in STAT3-deficient mice as judged by their failure to up-regulate circulating neutrophils following short-term G-CSF exposure. STAT3 also controls neutrophil chemotactic responses to natural ligands for CXCR2 and regulates the magnitude of chemoattractant-induced actin polymerization. These functions of STAT3 are independent of its principal target gene Socs3, which encodes a crucial feedback inhibitor of cytokine signaling. Our results demonstrate the existence of distinct STAT3 target pathways in neutrophils required for granulopoiesis and innate immunity.

Impaired Neutrophil Chemotaxis in Chronic Obstructive Pulmonary Disease

Neutrophilic airway inflammation is considered to be a major factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), with sputum and bronchoalveolar lavage neutrophil counts broadly correlating with disease severity. The mechanisms responsible for neutrophil accumulation are poorly understood, but they could involve increased influx and/or survival of these cells. To investigate whether neutrophil chemotactic responsiveness and/or chemotactic activity in airway secretions are increased in subjects with COPD. Chemotaxis experiments were performed using induced sputum supernatants from subjects with and without COPD as a source of chemotactic activity, and neutrophils from healthy donors as responder cells. In addition, chemotactic responses to N-formyl-Met-Leu-Phe (fMLP) and interleukin-8 (IL-8/CXCL8) were studied using neutrophils from healthy subjects and subjects with COPD. As reported in the literature, sputum neutrophil counts were significantly increased in subjects with COPD compared with healthy subjects. However, this was associated with reduced chemotactic activity in sputum in COPD, as judged by reduced chemotaxis to the fluid phase of sputum from subjects with COPD compared with healthy subjects. Furthermore, whereas neutrophils from subjects with stage I COPD had normal responses to fMLP and IL-8, subjects with more severe stage II-IV COPD showed reduced levels of spontaneous migration and chemotaxis to fMLP and IL-8. Neither increased chemotactic activity in the airways nor increased chemotactic responsiveness of neutrophils explains the increased number of these cells in subjects with stable COPD. The implications of the observed reduction in neutrophil chemotactic activity remain to be established.

Effect of docosahexaenoic acid-rich fish oil supplementation on human leukocyte function

The effect of a docosahexaenoic acid (DHA)-rich fish oil (FO) supplementation on human leukocyte function was investigated. Ten male volunteers were supplemented with 3g/day FO containing 26% eicosapentaenoic acid (EPA, 20:5, n-3) and 54% DHA (22:6, n-3) for 2 months. FO supplementation changed the fatty acid (FA) composition of leukocytes resulting in an increase of n-3/n-6 ratio from 0.18 to 0.62 in lymphocytes and from 0.15 to 0.70 in neutrophils. DHA-rich FO stimulated an increase in phagocytic activity by 62% and 145% in neutrophils and monocytes, respectively. Neutrophil chemotactic response was increased by 128%. The rate of production of reactive oxygen species by neutrophils was also increased, as it was with lymphocyte proliferation. These changes were partially reversed after a 2-month wash out period. With respect to cytokine production by lymphocytes, interleukin (IL)-4 release was not altered, whereas secretions of IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha were raised. These results are in contrast to those described by others using EPA-rich FO supplementation. Lymphocyte pleiotropic gene expression was analyzed by a macroarray technique. Of the analyzed genes (588 in total), 77 were modified by the supplementation. FO supplementation resulted in up-regulation of 6 genes (GATA binding protein 2, IL-6 signal transducer, transforming growth factor alpha, TNF, heat shock 90kDa protein 1-alpha and heat shock protein 70kDa 1A) and a down regulation of 71 genes (92.2% of total genes changed). The largest functional group of altered genes was that related to signaling pathways (22% of the total modified genes). Therefore, although EPA and DHA are members of n-3 FA family, changes in the proportion of DHA and EPA exert different effects on neutrophil, monocyte and lymphocyte function, which may be a result of specific changes in gene expression.