Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.
Integrins are functionally regulated by "inside-out" signaling, in that stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs, but the mechanisms that regulate inside out signaling are not completely understood. Previously, we identified gelsolin in a proteomics screen to identify proteins involved in inside-out control of integrins using the lymphocytic leukemia cell line L1210. Furthermore, we showed that gelsolin was involved in affinity regulation of ?1 -integrins in the leukemic cell line U937. Here, we examined how gelsolin regulates ?1 -integrin affinity in the leukemia cell line L1210. We show that gelsolin is mainly expressed at the cell membrane and is present near ?1 -integrins. The role for actin polymerization in integrin affinity regulation was examined using the actin modulating agent jasplakinolide, which decreased ?1 -integrin affinity. Similarly, knock-down of gelsolin in L1210 cells also decreased ?1 -integrin affinity and cell adhesion to collagen. These data suggest that increased actin polymerization through gelsolin regulates ?1 -integrin affinity and cell adhesion.
Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach was used as an unbiased approach to identify leptin-induced effects on neutrophils. Additionally chemotaxis and survival experiments were performed to reproduce results from literature showing putative effects of leptin on these neutrophil responses. Leptin did not induce any significant changes in the proteome provided leptin was added at physiologically relevant concentrations (250 ng). Our leptin batches were biologically active as they induced proliferation in LeptinR expressing Ba/F3 cells. At high concentrations (25000 ng) leptin induced a change in neutrophil proteome. Seventeen differently regulated spots were identified of which twelve could be characterized by mass spectrometry. Two of these identified proteins, SerpinB1 and p40 phox, were chosen for further analysis but leptin-induced expression analyzed by western blot were highly variable. Additionally leptin also induced neutrophil survival at these high concentrations. No leptin-induced chemotaxis of human neutrophils was detected at any concentration. In conclusion, physiological concentrations of leptin do not affect neutrophils. High leptin concentrations induced survival and changes in the neutrophils proteome, but this was most likely mediated by an indirect effect. However, it cannot be ruled out that the effects were mediated by a yet not-identified leptin receptor on human neutrophils.
Recent studies suggest that chemotherapy, in addition to its cytotoxic effects on tumor cells, can induce a cascade of host events to support tumor growth and spread. Here, we used an experimental pulmonary metastasis model to investigate the role of this host response in metastasis formation. Mice were pretreated with chemotherapy and after clearance of the drugs from circulation, tumor cells were administered intravenously to study potential "protumorigenic" host effects of chemotherapy. Pretreatment with the commonly used chemotherapeutic agents cisplatin and paclitaxel significantly enhanced lung metastasis in this model. This corresponded to enhanced adhesion of tumor cells to an endothelial cell monolayer that had been pretreated with chemotherapy in vitro. Interestingly, chemotherapy exposure enhanced the expression of VEGF receptor 1 (VEGFR-1) on endothelial cells both in vitro and in vivo. Administration of antibodies targeting VEGFR-1 reversed the early retention of tumor cells in the lungs, thereby preventing the formation of chemotherapy-induced pulmonary metastases. The data indicate that chemotherapy-induced expression of VEGFR-1 on endothelial cells can create an environment favorable to tumor cell homing. Inhibition of VEGFR-1 function may therefore be used to counteract chemotherapy-induced retention of tumor cells within the metastatic niche, providing a novel level of tumor control in chemotherapy.
Increased serum levels of TNF? and GM-CSF are found in various chronic inflammatory diseases and these cytokines affect the function of circulating and tissue neutrophils. TNF?- and GM-CSF-induced protein expression profiles could, therefore, serve as biomarker for the action of these cytokines in vivo. We stimulated human peripheral neutrophils with TNF? and GM-CSF in vitro and analyzed changes in their proteome by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). We report the differential expression of 3 and 18 protein spots following TNF? and GM-CSF stimulation, respectively. Differences in protein expression induced by TNF? were limited and did not show discriminatory power in a principal component analysis, whereas the profile induced by GM-CSF did. TNF?- and GM-CSF-induced both de novo IL-1? and sIL-1Ra protein expression as detected by Western blot analysis, which confirmed proper neutrophil activation by these cytokines in vitro. Mass spectrometry analysis of cytokine-regulated protein spots resulted in the identification of 8 proteins. Among the identified proteins, enolase 1 and annexin A1 might function as markers for peripheral neutrophil activation. In conclusion, a proteomic analysis of neutrophils by 2D-DIGE provides proof-of-principle that cytokine-induced protein profiles can serve as biomarkers for the action of individual cytokines in vivo.
Suppression of immune responses is necessary to limit damage to host tissue during inflammation, but it can be detrimental in specific immune responses, such as sepsis and antitumor immunity. Recently, immature myeloid cells have been implicated in the suppression of immune responses in mouse models of cancer, infectious disease, bone marrow transplantation, and autoimmune disease. Here, we report the identification of a subset of mature human neutrophils (CD11cbright/CD62Ldim/CD11bbright/CD16bright) as what we believe to be a unique circulating population of myeloid cells, capable of suppressing human T cell proliferation. These cells were observed in humans in vivo during acute systemic inflammation induced by endotoxin challenge or by severe injury. Local release of hydrogen peroxide from the neutrophils into the immunological synapse between the neutrophils and T cells mediated the suppression of T cell proliferation and required neutrophil expression of the integrin Mac-1 (?M?2). Our data demonstrate that suppression of T cell function can be accomplished by a subset of human neutrophils that can be systemically induced in response to acute inflammation. Identification of the pivotal role of neutrophil Mac-1 and ROS in this process provides a potential target for modulating immune responses in humans.
Inflammation, both local and systemic, is a hallmark of chronic obstructive pulmonary disease (COPD). Inflammatory mediators such as TNF? and GM-CSF are secreted by lung epithelium, alveolar macrophages and other inflammatory cells and are thought to be important contributors in the pathogenesis of COPD. Indeed, neutrophils are activated by these cytokines and these cells are one of the major inflammatory cell types recruited to the pulmonary compartment of COPD patients. Furthermore, these inflammatory mediators are found in the peripheral blood of COPD patients and, therefore, we hypothesized that TNF?/GM-CSF-induced protein profiles can be found in peripheral neutrophils of COPD patients.
NF-?B, an important transcription factor in the regulation of cellular inflammation, is one of the prime targets for novel anti-inflammatory therapeutics. Nowadays, anti-inflammatory therapies rely mostly on steroids, which among other effects, inhibit NF-?B activity. However, steroids have only limited efficacy in the treatment on neutrophil-driven diseases, such as COPD. Human neutrophils play an important role in the pathogenesis of COPD, and clearance of these cells by apoptosis is an effective pathway for resolution of inflammation. In this study, we tested the hypothesis that modulation of the NF-?B pathway in human neutrophils affects survival. Importantly, the pharmacological NF-?B inhibitor Bay 11-7082 inhibited NF-?B signaling in human neutrophils as expected. However, we found that complete inhibition of NF-?B activity with 10 ?M Bay 11-7082 prolonged neutrophil survival significantly, which was not observed with inhibitors for other signaling pathways. Bay 11-7082-induced neutrophil survival was dependent on p38-MAPK kinase activity, as the p38 kinase activity inhibitor SB203580 abrogated this response completely. Bay 11-7082 induced rapid and sustained p38 activation that correlated with inhibited NF-?B signaling and prolonged neutrophil survival. The precise role of NF-?B in regulation of p38-MAPK activation remains to be established. Under these conditions of survival, the stability of Bcl-xL but not Mcl-1 was enhanced. Although inhibition of NF-?B leads to down-regulation of inflammatory genes in many cell types, our results illustrate that interference with basal NF-?B signaling in neutrophils as a drug target should be used with caution.
Limited number of hematopoietic stem cells in umbilical cord blood (UCB) presents a problem when using UCB for stem cell transplantation. Improving their homing capacity could reduce the need for high initial cell numbers during transplantation procedures. Although it is evident that protein kinase B (PKB/c-Akt) plays an important role in regulation of migration of various cell types, a role for PKB in regulation of migration and homing of human hematopoietic stem and progenitor cells remains to be determined. PKB activity was found to be required for induction of adhesion to bone marrow-derived stromal cells and detrimental for migration of UCB-derived CD34(+) hematopoietic progenitors. In addition, PKB activity was found to positively regulate integrin expression. CD34(+) hematopoietic progenitors, and their capacity to form colonies in vitro, were not affected by transient inhibition of PKB. Finally, transplantation of ?2-microglobulin(-/-) nonobese diabetic/severe combined immunodeficient mice with CD34(+) cells ectopically expressing constitutively active PKB resulted in reduced migration to the bone marrow, whereas inhibition of PKB activity resulted in an induction in bone marrow homing and engraftment. These results indicate that transient inhibition of PKB activity may provide a means for ex vivo stem cell manipulation to improve bone marrow transplantation regimes.
The innate immune system and the blood haemostasis system function cooperatively in many pathological conditions such as acute respiratory distress syndrome, deep venous thrombosis, ischaemia/reperfusion injury and cardiovascular disease. Infiltration of neutrophils into thrombotic substrates such as fibrin clots supports fibrinolysis, tissue damage and inflammation. Despite the importance of integrins in neutrophil attachment to fibrin-coated surfaces under flow conditions, little is known about their role in migration processes in shear free two-dimensional (2D) and three-dimensional (3D) fibrin(ogen) environments. Therefore, the present study was designed to study the role of functional integrins in mediating neutrophil migration on and in fibrin matrices. Time lapse video sequences of neutrophil chemokinesis and chemotaxis were made under conditions of active- or non-active integrins. Interestingly, migration of neutrophils on 2D fibrinogen coated surfaces and 3D fibrin matrices is independent of integrins as the response is not sensitive to alphaM-(CD11b) and beta2-(CD18) blocking antibodies and/or chelation of Ca2+ and Mg2+ by EDTA in bivalent ion-free buffers. The blocking integrin antibodies were shown to be functionally active in regular adhesion assays. Our study shows that integrins are dispensable for migration on 2D and in 3D fibrin matrices, both when neutrophils enter into the fibrin matrix and when captured in the matrix.
Severe primary respiratory syncytial virus (RSV) infections are characterized by bronchiolitis accompanied by wheezing. Controversy exists as to whether infants suffer from virus-induced lung pathology or from excessive immune responses. Furthermore, detailed knowledge about the development of primary T-cell responses to viral infections in infants is lacking. We studied the dynamics of innate neutrophil and adaptive T-cell responses in peripheral blood in relation to the viral load and parameters of disease in infants admitted to the intensive care unit with severe RSV infection. Analysis of primary T-cell responses showed substantial CD8(+) T-cell activation, which peaked during convalescence. A strong neutrophil response, characterized by mobilization of bone marrow-derived neutrophil precursors, preceded the peak in T-cell activation. The kinetics of this neutrophil response followed the peak of clinical symptoms and the viral load with a 2- to 3-day delay. From the sequence of events, we conclude that CD8(+) T-cell responses, initiated during primary RSV infections, are unlikely to contribute to disease when it is most severe. The mobilization of precursor neutrophils might reflect the strong neutrophil influx into the airways, which is a characteristic feature during RSV infections and might be an integral pathogenic process in the disease.
Leukocyte integrins are functionally regulated by "inside-out" signaling, meaning that stimulus-induced signaling pathways act on the intracellular integrin tail and induce activation of the receptor at the outside. Both a change in conformation (affinity) and in clustering (avidity/valency) of the receptors has been described to occur. This inside-out signaling is essential for adequate migration of leukocytes to inflammatory sites; however, the exact underlying mechanism is not known. We used two variants of a mouse acute lymphocytic leukemia cell line (L1210), a suspension (L1210-S) and an adherent (L1210-A) variant that were characterized by nonactivated and activated integrins (beta(1), beta(2) and beta(3)), respectively. L1210-S and L1210-A cells were compared on protein expression profiles by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE). We found 86 protein spots that were more than 1.25-fold different between L1210-A and L1210-S. Only 4 protein spots were more than 2.5-fold different. We identified 29 proteins by mass spectrometry among which were gelsolin, L-plastin, and Rho GTPase dissociation inhibitor 2. These proteins were upregulated in the L1210-A cells versus L1210-S, which was verified by Western blot analysis. Overexpression of gelsolin in U937 resulted in increased high affinity integrin expression and cell adhesion. Comparison of functionally different cell lines from similar origin by 2D-DIGE might be a successful approach to identify regulatory proteins involved in integrin inside-out control.
Monocyte infiltration across the endothelium is part of the innate immune response, however it may contribute to severity of chronic conditions. We have investigated the effects of iron on the cytokine-mediated recruitment of monocytes to the endothelium, using a physiological flow model and a monocyte transendothelial migration model. Under flow, iron loading to endothelial cells promoted an increased number of tumor necrosis factor-alpha-mediated firm arrest of human monocytes. Similarly, an increased number of firmly adhered monocytes were observed in conditions in which monocytes were iron-loaded, compared to the non-iron-loaded conditions. In both iron-loaded and non-iron-loaded conditions, blockade of the alpha4 and beta2 integrins restored similar number and velocity of monocyte rolling, suggesting that iron did not modulate rolling interactions. However, with the integrin blockade, the number of firmly adhered cells remained higher in iron-loaded conditions than in control conditions, suggesting that iron could have modulated receptors other than the blocked integrins to promote firm arrest. Iron loading indeed upregulated expression of chemokine receptors, CC receptor-2 and CXC receptor-2, but not platelet endothelial cell adhesion molecule-1. This effect concomitantly promoted monocyte chemotactic protein-1-dependent transendothelial migration. In addition, iron-induced firm adhesion and transmigration were counteracted by iron chelation. These data reveal an immunomodulatory function of iron in the cascade of events of cytokine-mediated monocyte infiltration across endothelium, and therefore suggests the role for iron in inflammatory conditions underlying diseases like atherosclerosis and neurodegeneration.
Neutrophils are essential effector cells in host defense against invading pathogens. Regulation of adhesion, migration, and chemotactic processes is important in the homing and activation of these cells. We recently described three distinct subsets of circulating human neutrophils in peripheral blood during acute systemic inflammation. One subset, CD16(bright)/CD62L(dim), has immune suppressive characteristics because it can inhibit T-cell proliferation. The other two subsets consist of banded CD16(dim)/CD62L(bright) and phenotypically mature (normal) CD16(bright)/CD62L(bright) neutrophils. The current study was designed to determine the adhesion characteristics of these different neutrophil subsets. Analysis of adhesion to activated endothelium under flow conditions revealed that CD16(bright)/CD62L(dim) neutrophils adhered less compared with CD16(bright)/CD62L(bright) and CD16(dim)/CD62L(bright) neutrophils. This decrease in binding capacity could be mimicked in the other neutrophil subsets by blocking L-selectin. Chemotaxis of CD16(bright)/CD62L(dim) neutrophils to the end-target chemoattractant N-formylmethionine-leucine-phenylalanine was lower compared with that for the CD16(dim)/CD62L(bright) neutrophil subset, whereas chemotaxis to cell-derived chemoattractant CXCL8 was comparable. Our data indicate that capture on endothelium under flow conditions, a key mechanism necessary for extravasation, of CD16(bright)/CD62L(dim) neutrophils to inflammatory sites is attenuated, which may facilitate migration of these cells to other tissue localizations. Modulation of this process is a potential target to manipulate inflammation because potentiation of this immune suppression might aid in anti-inflammatory therapy.
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