Migration Of Neutrophil Granulocytes Research Articles

Expression and secretion of the pro‑inflammatory cytokine IL‑8 is increased in colorectal cancer cells following the knockdown of non‑erythroid spectrin αII.

Non‑erythroid spectrin αII (SPTAN1) expression is decreased in ~40% of cases of MLH1‑deficient colorectal cancer (CRC). SPTAN1 knockdown reduces cell viability, cellular mobility and cell‑cell contact formation, indicating that the SPTAN1 plays an important role in tumour growth, attachment and in regulating the tumour microenvironment. Changes in the tumour microenvironment can affect the immune response. Therefore, in the present study, proteome arrays were used to analyse the expression of 119 different chemokines and soluble receptors in CRC cell lines in which mutL homologue 1 (MLH1) or SPTAN1 were knocked down. The levels of interleukin (IL)‑8 were significantly increased in the cells in which SPTAN1 was knocked down, both at the mRNA and protein level. ELISA demonstrated that the cells in which SPTAN1 was knocked down secreted increased quantities of IL‑8, and chemotaxis assays revealed the enhanced trafficking of neutrophils, which was induced by media containing higher levels of IL‑8. The IL‑8 receptors, CRCX1 and CRCX2, were expressed in all the cell lines examined; however, their expression was not directly associated with IL‑8 expression. The results of the present study thus demonstrated that CRC cells in which SPTAN1 was knocked down secreted significantly higher levels of IL‑8, which in‑turn increased the migration of neutrophilic granulocytes. As MLH1‑deficient CRC exhibits an increased infiltration of cytotoxic T‑cells and is associated with a decreased SPTAN1 expression, it can thus be hypothesized that CRC with a low SPTAN1 expression may release increased quantities of IL‑8, resulting in increased immune cell infiltration.

Rol' arteriolo-venulyarnykh anastomozov v tsirkulyatsii neytrofil'nykh granulotsitov

Migration of neutrophilic granulocytes (NG), whose main function is phagocytosis, is primarily caused by the venule lumen. Nevertheless, NGs are often "blamed" for their ability to block microcirculation in the systemic circulation and thus to develop the no - reflow phenomenon in the cerebrum and myocardium. Apparently, NGs are a priori believed to enter the venous bed trans capillary, although their morphological features should prevent it. Are NGs able to circulate into venules via arterio-venous anastomoses (AVA)? The aim of the research is to elucidate the role of AVA in neutrophilic granulocyte circulation. The paper presents the study of the intestine, skeletal muscles, myocardium, cerebrum, peritoneum, and spleen in cats (n=7) and white rates (n=17). The research methods are intravascular impregnation by the Ranvier method, a universal method of impregnation with various silver salts, routine histological methods of staining paraffin sections. In intact animals, neutrophilic granulocytes enter the venules via AVAs only. In the myocardium and cerebrum, which have no AVAs, due to the blood separation phenomenon, NGs enter the epicardium exclusively in the former case and the facial soft tissues and pachymeninx in the latter.

Rol' arteriolo-venulyarnykh anastomozov v tsirkulyatsii neytrofil'nykh granulotsitov

Migration of neutrophilic granulocytes (NG), whose main function is phagocytosis, is primarily caused by the venule lumen. Nevertheless, NGs are often “blamed” for their ability to block microcirculation in the systemic circulation and thus to develop the no-reflow phenomenon in the cerebrum and myocardium. Apparently, NGs are a priori believed to enter the venous bed trans capillary, although their morphological features should prevent it. Are NGs able to circulate into venules via arterio-venous anastomoses (AVA)? The aim of the research is to elucidate the role of AVA in neutrophilic granulocyte circulation. The paper presents the study of the intestine, skeletal muscles, myocardium, cerebrum, peritoneum, and spleen in cats (n=7) and white rates (n=17). The research methods are intravascular impregnation by the Ranvier method, a universal method of impregnation with various silver salts, routine histological methods of staining paraffin sections. In intact animals, neutrophilic granulocytes enter the venules via AVAs only. In the myocardium and cerebrum, which have no AVAs, due to the blood separation phenomenon, NGs enter the epicardium exclusively in the former case and the facial soft tissues and pachymeninx in the latter.

Rac GTPase Activating Protein ARHGAP25 Regulates Leukocyte Transendothelial Migration in Mice.

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration.

Classically or alternatively activated bovine monocyte-derived macrophages in vitro do not resemble CD163/Calprotectin biased macrophage populations in the teat

The functional phenotype of resident macrophages significantly determines the character of an inflammatory response. In this study we identified two phenotypes of tissue macrophages in bovine teat tissue based on expression of Calprotectin and CD163. To investigate a possible link between the dichotomy in phenotype and functional properties of cells in association with different host mediators we set up an invitro model with bovine monocyte-derived macrophages (MdM). Invitro differentiated MdM invariably and uniformly expressed both antigens. Classically activated MdM (IFN-γ priming and LPS stimulation) showed a decreased CD163 expression while alternative activation (IL-4/IL-13 priming) did not change expression of CD163 and Calprotectin. Differently activated MdM showed a clearly distinct expression of genes related to classical (IL-12, inducible NO synthase) or alternative activation (IL-10, arginase I). The presence of the inflammatory host mediator prostaglandin E(2) (PGE(2)) neither influenced expression of Calprotectin and CD163 nor gene expression profiles in MdM generated in the presence of PGE(2) (PGE(2)-MdM). Supernatants of PGE(2-)MdM, however, significantly dampened the migration of neutrophilic granulocytes. The results of this study highlight the discrepancy between invivo and invitro obtained macrophages and point to the necessity to analyze the functional capacities of bovine tissue macrophages in situ.

Transcellular migration of neutrophil granulocytes through the blood-cerebrospinal fluid barrier after infection with Streptococcus suis

BackgroundA critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions.MethodsIn an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis) was used to stimulate porcine choroid plexus epithelial cells (PCPECs) specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1.ResultsHere, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome®-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process. Flow cytometric and transmigration inhibition studies with integrin-specific antibodies showed that PMN traversal is dependent on CD11b/CD18. Analysis of cell adhesion molecules in PCPECs revealed a significant increase of ICAM-1 and VCAM-1 expression after TNFα and S. suis stimulation.ConclusionOur data underline the relevance of the blood-CSF barrier as a gate for leukocyte entry into the CNS and suggest a novel transcellular migration step during the pathogenesis of bacterial meningitis.

Luminal and basal-like breast cancer cells show increased migration induced by hypoxia, mediated by an autocrine mechanism

BackgroundSome breast cancer patients receiving anti-angiogenic treatment show increased metastases, possibly as a result of induced hypoxia. The effect of hypoxia on tumor cell migration was assessed in selected luminal, post-EMT and basal-like breast carcinoma cell lines.MethodsMigration was assessed in luminal (MCF-7), post-EMT (MDA-MB-231, MDA-MB-435S), and basal-like (MDA-MB-468) human breast carcinoma cell lines under normal and oxygen-deprived conditions, using a collagen-based assay. Cell proliferation was determined, secreted cytokine and chemokine levels were measured using flow-cytometry and a bead-based immunoassay, and the hypoxic genes HIF-1α and CA IX were assessed using PCR. The functional effect of tumor-cell conditioned medium on the migration of neutrophil granulocytes (NG) was tested.ResultsHypoxia caused increased migratory activity but not proliferation in all tumor cell lines, involving the release and autocrine action of soluble mediators. Conditioned medium (CM) from hypoxic cells induced migration in normoxic cells. Hypoxia changed the profile of released inflammatory mediators according to cell type. Interleukin-8 was produced only by post-EMT and basal-like cell lines, regardless of hypoxia. MCP-1 was produced by MDA-MB-435 and -468 cells, whereas IL-6 was present only in MDA-MB-231. IL-2, TNF-α, and NGF production was stimulated by hypoxia in MCF-7 cells. CM from normoxic and hypoxic MDA-MB-231 and MDA-MB-435S cells and hypoxic MCF-7 cells, but not MDA-MB-468, induced NG migration.ConclusionsHypoxia increases migration by the autocrine action of released signal substances in selected luminal and basal-like breast carcinoma cell lines which might explain why anti-angiogenic treatment can worsen clinical outcome in some patients.

Effect of intraperitoneally administered plant lectins on leukocyte diapedesis and visceral organ weight in rats and mice

The effects of intraperitoneally administered plant lectins were examined in rats and mice. Intraperitoneally injected ConA transiently decreased the leukocyte count in the peritoneal cavity, due to the agglutination and attachment of cells to the peritoneal lining. Subsequently the total cell count was increased for hours, exceeding initial values. Peritoneal fluid aspartate transaminase (AST) concentration showed little change during the accumulation of ascitic fluid. The most marked histological alterations were found when wheat germ lectin was injected ip. (WGA, 10 mg/kg, 6 h). Neutrophil granulocytes migrated across the wall of both arterioles and venules, but the response was highly variable among adjacent vessels. The wall of the arterioles may have impeded the migration of neutrophil granulocytes, resulting in their accumulation in the muscular layer. Granulocyte accumulation was also observed in patches under the mesothelium and in other sites of the interstitium. Marked dilatation and thrombosis of a few venules were also observed. Kidney bean lectin (PHA) induced similar but less pronounced changes. The neutrophil diapedesis suggests the release of mediator(s) from mesothelial cells and/or peritoneal white cells. The cytokine-induced neutrophil chemoattractant CINC-1, injected as control, resulted in the diapedesis of predominantly mononuclear cells in the omentum within 40 minutes. In rats ip. injected ConA increased the wet weight of spleen and liver within 6 and 10 h, respectively, but kidney weight did not change. Intravascular clumping of red blood cells, thrombosis and organ weight changes also suggest the absorption of ConA into the circulation. The experiments show that plant lectins, used as models of bacterial lectins, can reproduce some aspects of peritonitis.

Expression of CXC Chemokine Receptors 1 and 2 in Human Bronchial Epithelial Cells

CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2) have been shown to play an important role in transepithelial migration of neutrophil granulocytes during inflammation in various tissues. This study investigated the regulation of gene expression and surface expression of CXCR1 and CXCR2 in a human bronchial epithelial cell line (BEAS-2B), as well as in primary bronchial epithelial cells (PBECs) from 10 COPD patients and 10 control subjects. The transcription expression of CXCR1 and CXCR2 was quantitatively assessed by means of real-time polymerase chain reaction (PCR) under various inflammatory conditions. Flow cytometry was used to measure CXCR1 and CXCR2 surface expression. There was a low baseline expression of CXCR1 and CXCR2 in real-time PCR in PBECs from COPD patients and control subjects as well as in BEAS-2B cells, and no significant regulation occurred under various inflammatory conditions in PBECs and BEAS-2B cells. Furthermore, unstimulated surface expression of CXCR1 and CXCR2 on BEAS-2B cells was very low, and no significant regulation was detectable under time-dependent inflammatory stimulation up to 24 h. Various inflammatory responses that are of potential relevance in COPD pathophysiology do not affect transcription regulation and surface expression of the interleukin-8 receptors CXCR1 and CXCR2 on human bronchial epithelial cells.

The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense.

The epidermal growth factor-seven transmembrane (EGF-TM7) family is a group of seven-span transmembrane receptors predominantly expressed by cells of the immune system. Family members CD97, EGF module-containing mucin-like receptor (EMR) 1, EMR2, EMR3, EMR4, and EGF-TM7-latrophilin-related protein are characterized by an extended extracellular region with a variable number of N-terminal EGF-like domains. EGF-TM7 receptors bind cellular ligands as demonstrated by the interaction of CD97 with decay accelerating factor (CD55) and dermatan sulfate. Investigating the effect of newly generated mAb on the migration of neutrophilic granulocytes, we here report for the first time in vivo data on the function of CD97. In dextran sulfate sodium-induced experimental colitis, we show that homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 mAb. The consequences of this defect in neutrophil migration for host defense are demonstrated in a murine model of Streptococcus pneumoniae-induced pneumonia. Mice treated with CD97 mAb to EGF domain 1 (1B2) and EGF domain 3 (1C5) displayed a reduced granulocytic inflammatory infiltrate at 20 h after inoculation. This was associated with a significantly enhanced outgrowth of bacteria in the lungs at 44 h and a strongly diminished survival. Together, these findings indicate an essential role for CD97 in the migration of neutrophils.

Locomotion of tumor cells: a molecular comparison to migrating pre- and postmitotic leukocytes

Increasing evidence has shown that the molecular regulation of active cell migration of slow-moving cells, e.g., tumor cells and fibroblasts, is different from fast-moving leukocytes, e.g., T lymphocytes and neutrophil granulocytes. Slow-moving cells develop focal adhesions as a crucial regulatory element during migration. These focal adhesions connect the extracellular matrix to the intracellular actin- and tubulin-cytoskeleton via integrins and enzymatically active proteins. Beside matrix-binding integrins, ligands of receptor tyrosine kinases and heterotrimeric G protein-coupled serpentine receptors initiate migration of slow-moving cells. Focal adhesions are not found in T lymphocytes and neutrophil granulocytes moving within three-dimensional matrices. In T lymphocytes, the T cell receptor is supposed to have a key regulatory function not only in antigen recognition, cell activation, and proliferation but also in cell migration. Regulatory molecules as well as the cytoskeleton are connected to the T cell receptor. The T cell receptor functionally combines elements of receptor tyrosine kinase signaling and of focal adhesions. In neutrophil granulocytes no multi-protein complexes regulating migration have been identified so far. Most potent activators of migration of neutrophil granulocytes, as those of T lymphocytes, are chemokines binding to heterotrimeric G protein-coupled serpentine receptors.

Chemotactic activity of LTB4 in man.

An improved skin window chamber technique has been developed and used for a quantitative study of the chemotactic effect of leukotriene B4 (LTB4). LTB4 (0.5 microM) was exposed to a skin window on the forearm of eight healthy volunteers, while phosphate buffered saline served as control in a skin window on the other forearm. Skin window exudates and samples of blood draining the skin window areas were collected after 1, 2, 4, 8, and 24 h. The samples were quantitated for the different types of leukocytes as well as the intra- and extracellular concentration of the eosinophilic cationic protein and lactoferrin as markers of eosinophil and neutrophil granulocytes. A significantly increased migration of neutrophil granulocytes into the skin window chamber containing LTB4 was found from the 2nd to the 8th hour after the initial LTB4 exposure. The eosinophils reached a significant peak at the 4th hour. The rise in the actual number of eosinophil cells did not reach significance, whereas measurements of the eosinophilic cationic protein in the cellular fraction of the exudate exhibited a significant increase as a reflection of the number of eosinophils. This highlights the potential clinical value of eosinophilic cationic protein measurements to reveal eosinophilia instead of the traditional eosinophil counts. Extracellular eosinophilic cationic protein and lactoferrin did not change significantly in the LTB4-exposed skin window, implying that LTB4 does not activate the eosinophils and neutrophils to exocytosis of their enzymes. The present in vivo results support the concept of LTB4 being a potent chemoattractant to neutrophil and less so to eosinophil granulocytes in humans, a chemoattractant that recruits the leukocytes but does not seem to activate them.

The chemotactic activity of normal and complement-activated serum as measured by the leading-front method using a Boyden chamber.

The chemotactic activity in serum, defined as the attractant effect of serum on the migration of neutrophil granulocytes (PMN) has been investigated for the purpose of characterizing the major chemotactic factors in serum as measured by the leading-front technique, using a modified Boyden chamber. The chemotactic activity was measured in fresh and heated normal and activated serum and in serum fractions thereof separated by gel filtration. By gel filtration on Sephacryl S-200 a partly heat-labile C3-C5-associated chemotactic factor with molecular weight between 70,000 and 150,000 was isolated from fresh normal serum. The heat-labile chemotactic activity was destroyed by pronounced complement activation. Gel filtration of complement-activated serum on a Sephacryl S-200 column showed the existence of one C5-associated chemotactic factor with approximately 70,000 molecular weight and one unidentified factor with approximately 150,000 molecular weight, whereas no low molecular weight chemotactic activity was demonstrated. On the other hand, gel filtration of activated serum on a Sephadex G-75 column demonstrated one C5-associated chemotactic factor of approximately 70,000 molecular weight and one 10,000-50,000 molecular weight factor active only in the presence of 2% normal serum. This investigation suggests that the chemotactic activity in fresh normal serum is mediated by a partly heat-labile C3-C5-associated complex. In activated serum three chemotactic factors were demonstrated, one unidentified factor with 150,000 mol wt and two C5-dependent factors with 70,000 and 10,000-50,000 mol wt, the latter probably corresponding to C5a desarg. Accordingly, this study also suggests that C5a is not the only chemotactic factor generated in serum.

Definition of a common leukocyte cell-surface antigen (Lp95-150) associated with diverse cell-mediated immune functions.

We have described a monoclonal antibody, designated 60.3, which reacts with a cell surface antigen expressed by most peripheral blood and bone marrow leukocytes. Immunoprecipitation showed at least three major components with relative m.w. of 95,000, 130,000, and 150,000 under reducing conditions. Antibody 60.3 inhibited several cell-mediated immune functions. The lytic activity of both cytotoxic T cells and natural killer cells was blocked in the presence of the antibody. The proliferative responses of T cells stimulated by soluble antigens, mitogens, or allogeneic cells were inhibited when antibody 60.3 was added at the initiation of culture, but not when added after 48 hr. Antibody 60.3 also blocked the migration of neutrophilic granulocytes. The antigen identified by antibody 60.3 thus appears to be involved in a membrane-dependent cell activation pathway that is common to diverse functional systems and is shared by both lymphoid and myeloid cells.