The leukotriene B4 receptor (BLT1) is required for effector CD8+ T cell-mediated, mast cell-dependent airway hyperresponsiveness

Studies in both humans and rodents have suggested that CD8+ T cells contribute to the development of airway hyperresponsiveness (AHR) and that leukotriene B4 (LTB4) is involved in the chemotaxis of effector CD8+ T cells (T(EFF)) to the lung by virtue of their expression of BLT1, the receptor for LTB4. In the present study, we used a mast cell-CD8-dependent model of AHR to further define the role of BLT1 in CD8+ T cell-mediated AHR. C57BL/6+/+ and CD8-deficient (CD8-/-) mice were passively sensitized with anti-OVA IgE and exposed to OVA via the airways. Following passive sensitization and allergen exposure, C57BL/6+/+ mice developed altered airway function, whereas passively sensitized and allergen-exposed CD8-/- mice failed to do so. CD8-/- mice reconstituted with CD8+ T(EFF) developed AHR in response to challenge. In contrast, CD8-/- mice reconstituted with BLT1-deficient effector CD8+ T cells did not develop AHR. The induction of increased airway responsiveness following transfer of CD8+ T(EFF) or in wild-type mice could be blocked by administration of an LTB4 receptor antagonist confirming the role of BLT1 in CD8+ T cell-mediated AHR. Together, these data define the important role for mast cells and the LTB4-BLT1 pathway in the development of CD8+ T cell-mediated allergic responses in the lung.     

CC chemokine receptor-2 is not essential for the development of antigen-induced pulmonary eosinophilia and airway hyperresponsiveness

Monocyte chemoattractant proteins-1 and -5 have been implicated as important mediators of allergic pulmonary inflammation in murine models of asthma. The only identified receptor for these two chemokines to date is the CCR2. To study the role of CCR2 in a murine model of Ag-induced asthma, we compared the pathologic and physiological responses of CCR2(-/-) mice with those of wild-type (WT) littermates following immunization and challenge with OVA. OVA-immunized/OVA-challenged (OVA/OVA) WT and CCR2(-/-) mice developed significant increases in total cells recovered by bronchoalveolar lavage (BAL) compared with their respective OVA-immunized/PBS-challenged (OVA/PBS) control groups. There were no significant differences in BAL cell counts and differentials (i.e., macrophages, PMNs, lymphocytes, and eosinophils) between OVA/OVA WT and CCR2(-/-) mice. Serologic evaluation revealed no significant difference in total IgE and OVA-specific IgE between OVA/OVA WT mice and CCR2(-/-) mice. Lung mRNA expression and BAL cytokine protein levels of IL-4, IL-5, and IFN-gamma were also similar in WT and CCR2(-/-) mice. Finally, OVA/OVA CCR2(-/-) mice developed increased airway hyper-responsiveness to a degree similar to that in WT mice. We conclude that following repeated airway challenges with Ag in sensitized mice, the development of Th2 responses (elevated IgE, pulmonary eosinophilia, and lung cytokine levels of IL-4 and IL5) and the development of airway hyper-responsiveness are not diminished by a deficiency in CCR2.     

CXCL10 Can Inhibit Endothelial Cell Proliferation Independently of CXCR3

CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10) is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10''s angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10''s glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.     

Enhanced inhibition of human immunodeficiency virus type 1 by Met-stromal-derived factor 1beta correlates with down-modulation of CXCR4

CXCR4 is a chemokine receptor used by some strains of HIV-1 as an entry coreceptor in association with cell surface CD4 on human cells. In human immunodeficiency virus type 1 (HIV-1)-infected individuals, the appearance of viral isolates with a tropism for CXCR4 (T tropic) has been correlated with late disease progression. The presumed natural ligands for CXCR4 are SDF-1alpha and SDF-1beta, which are proposed to play a role in blocking T-tropic HIV-1 cell entry. Here, we demonstrate that addition of an N-terminal methionine residue to SDF-1beta (Met-SDF-1beta) results in a dramatically enhanced functional activity compared to that of native SDF-1beta. Equivalent concentrations of Met-SDF-1beta are markedly more inhibitory for T-tropic HIV-1 replication than SDF-1beta. A comparison of the biological activities of these two forms of SDF-1beta reveals that Met-SDF-1beta induces a more pronounced intracellular calcium flux yet binds with slightly lower affinity to CXCR4 than SDF-1beta. Down-modulation of CXCR4 is similar after exposure of cells to either chemokine form for 2 h. However, after a 48-h incubation, the surface expression of CXCR4 is much lower for cells treated with Met-SDF-1beta. The enhanced blocking of T-tropic HIV-1 by Met-SDF-1beta appears to be related to prolonged CXCR4 down-modulation.     

Distal enhancer of the mouse FGF-4 gene and its human counterpart exhibit differential activity: critical role of a GT box

Previous studies have shown that there is a strict requirement for fibroblast growth factor-4 (FGF-4) during mammalian embryogenesis, and that FGF-4 expression in embryonic stem (ES) cells and embryonal carcinoma (EC) cells are controlled by a powerful downstream distal enhancer. More recently, mouse ES cells were shown to express significantly more FGF-4 mRNA than human ES cells. In the work reported here, we demonstrate that mouse EC cells also express far more FGF-4 mRNA than human EC cells. Using a panel of FGF-4 promoter/reporter gene constructs, we demonstrate that the enhancer of the mouse FGF-4 gene is approximately tenfold more active than its human counterpart. Moreover, we demonstrate that the critical difference between the mouse and the human FGF-4 enhancer is a 4 bp difference in the sequence of an essential GT box. Importantly, we demonstrate that changing 4 bp in the human enhancer to match the sequence of the mouse GT box elevates the activity of the human FGF-4 enhancer to the same level as that of the mouse enhancer. We extended these studies by examining the roles of Sp1 and Sp3 in FGF-4 expression. Although we demonstrate that Sp3, but not Sp1, can activate the FGF-4 promoter when artificially tethered to the FGF-4 enhancer, we show that Sp3 is not essential for expression of FGF-4 mRNA in mouse ES cells. Finally, our studies with human EC cells suggest that the factor responsible for mediating the effect of the mouse GT box is unlikely to be Sp1 or Sp3, and this factor is either not expressed in human EC cells or it is not sufficiently active in these cells.     

Multiple chemokine receptors, including CCR6 and CXCR3, regulate antigen-induced T cell homing to the human asthmatic airway

Human allergic asthma is a chronic inflammatory disease of the airways thought to be driven by allergen-specific Th2 cells, which are recruited into the lung in response to inhaled allergen. To identify chemoattractant receptors that control this homing pattern, we used endobronchial segmental allergen challenge in human atopic asthmatics to define the pattern of chemoattractant receptor expression on recruited T cells as well as the numbers of recruited CD1d-restricted NKT cells and levels of chemokines in the bronchoalveolar (BAL) fluid. CD1d-restricted NKT cells comprised only a small minority of BAL T cells before or after Ag challenge. BAL T cells were enriched in their expression of specific chemoattractant receptors compared with peripheral blood T cells prechallenge, including CCR5, CCR6, CXCR3, CXCR4, and BLT1. Surprisingly, following segmental allergen challenge, no chemoattractant receptor was specifically increased. However, CCR6 and CXCR3, which were expressed on virtually all CD4(+) BAL T cells prechallenge, were markedly decreased on all recruited BAL T cells following Ag challenge, suggesting that these receptors were internalized following encounter with ligand in the airway. Our data therefore suggests a role for CCR6 and CXCR3, in conjunction with other chemoattractant receptors, in the recruitment of inflammatory T cells into the BAL during the allergic asthmatic response.     

Movement within and movement beyond: Synaptotagmin-mediated vesicle fusion during chemotaxis

Leukocyte chemotaxis plays an essential role in generating and delivering immune responses and is a critical component of inflammation. In order to identify novel genes and pathways important for regulating chemotaxis, we performed an RNAi-based screen and identified several genes involved with vesicle movement and fusion as mediators of chemotaxis. Our recently published data¹ show that during chemotaxis vesicle trafficking proteins are required for lysosome fusion, uropod release and efficient directed cell migration.Key words: chemotaxis, synaptotagmin, vesicle trafficking, lysosome, uropod, rabLeukocyte chemotaxis is an essential process that directs the immune system and drives inflammatory responses.² The processes whereby an extracellular chemical signal induces a program that results in morphological changes to the cell and induces chemotaxis remain incompletely understood. Since the earliest days of the study of chemotaxis, a number of investigators have found clues suggesting that membrane flow and vesicle trafficking play roles in the movement of cells toward a chemotactic stimulus. As early as 1962 it was suggested that cell migration involves a membrane cycle.³ Data emerged suggesting that the plasma membrane flowed toward the rear of the migrating cell.^4–6 More recently, it was suggested the membrane actually flowed toward the front of the migrating cell.⁷ This later hypothesis was supported by the finding that recycling transferrin receptors were routed to the plasma membrane of the leading edge lammellopodia.⁸ Finally, a potential role for lysosome fusion with the uropod has been suggested.^9–12 Despite these observations, a functional correlation of vesicle trafficking in chemotaxis had not been established.We performed an RNAi-based genetic screen to identify novel regulators of CXCR4-mediated lymphocyte chemotaxis. In the screen, we found two genes in the synaptotagmin family of calcium sensing vesicle fusion proteins. Synaptotagmin-2 (Syt2) was identified as a negative regulator of lymphocyte chemotaxis and Synaptotagmin-like protein-5 (Sytl5) was identified as a positive regulator of chemotaxis. Lymphocytes in which SYT2 expression was reduced using RNAi demonstrated enhanced migration in a CXCL12 gradient. In contrast, cells in which SYTL5 mRNA expression was reduced demonstrated decreased migration in a CXCL12 gradient. The knock-down of these genes had similar effects in the migration of a monocyte cell line to a different chemokine, CCL2.In order to determine the extent of the role of synaptotagmin family member proteins in chemotaxis, we extended the screen to the remaining synaptotagmin and synaptotagmin-like family member genes and identified Synaptotagmin-7 (Syt7) as encoding a positive regulator of chemotaxis. The identification of SYT7 provided potential insight into the mechanistic role of these proteins in chemotaxis as it is expressed on the lysosomal membrane of many different cell types and has been shown to be important in lysosome-cell membrane fusion as well as in membrane repair. Additionally, an SYT7-deficient mouse line was available to facilitate further experiments to analyze the function of SYT7 in chemotaxis. Lymphocytes and neutrophils obtained from SYT7-deficient mice demonstrated decreased chemotaxis in response to chemokine gradients. By analyzing cell migration tracks of cells migrating in a Zigmond chemotaxis chamber, we found that the cells lacking SYT7 had both a defect in migration velocity, as well as in migration direction. In an in vivo model of monosodium urate (MSU) crystal induced gout-like inflammation, we found that SYT7-deficient female mice demonstrated decreased neutrophil recruitment into an experimentally produced airpouch containing MSU crystals. The decreased neutrophil recruitment was not due to decreased IL-1 or neutrophil-active chemokine release as the airpouches contained similar amounts of these cytokines. These results confirmed and extended the hypothesis that synaptotagmin family member proteins have a role in chemotaxis.In addition to synaptotagmin family members, the process of vesicle trafficking within the cell also requires SNARE proteins. Indeed, a role for the SYT7 interacting SNARE protein, VAMP7, has been shown in epithelial cell migration.¹³ As the interaction of SYT7 and VAMP7 has also been shown to be important for lysosome fusion with the cell membrane and SYT2 has been shown to be a negative regulator of lysosomal exocytosis,^14,15 we investigated whether lysosome fusion was important for chemotaxis. Using FACS analysis, we found that chemoattractant stimulation of lymphocytes and neutrophils resulted in the expression of the lysosomal membrane protein LAMP-1 (CD107a) on the cell surface. Expression of LAMP-1 was dependent on the expression of SYT7 as well as the release of intracellular calcium. Further, confocal micrographs of podocytes, large cells of renal origin that express CXCR3, show that podocytes express the lysosomal marker LAMP-2 on their surface following stimulation with the CXCR3 ligand CXCL11. These data support the hypothesis that chemoattractant-mediated calcium influx induces the fusion of lysosomes with the cell membrane in an SYT7-dependent manner. To further investigate the role of lysosomes in chemotaxis, we analyzed the migration of lysotracker-stained wild-type and Syt7^-/- lympohocytes in a CXCL10 gradient in a Dunn chamber. We found that lysotracker accumulated in the uropods of Syt7^-/- cells. Additionally, these cells were adherent to the fibronectin-coated substrate at their uropods. Taken together, these data suggested that Syt7-mediated fusion of lysosomes is required for efficient uropod release during chemotaxis.During the 1970s, chemoattractant stimulation of neutrophils was shown to induce lysosomal enzyme release^10,11 and a role for lysosomal fusion was speculated to play a role in neutrophil adhesion.⁹ However, it was difficult to identify a direct role of lysosomal enzyme release or to separate lysosome membrane fusion with that of enzyme release in chemotaxis.We speculate that the migration and fusion of lysosomes is required to deliver cargo during chemotaxis that allows uropod release. It is possible that the fusion of lysosomes is required to deliver a specific protein required for uropod release. Alternatively, the cargo may be lipids required for membrane cycling during chemotaxis or for the delivery of a specific lipid or protein needed in the chemotaxis signaling pathway. Finally, it is possible that the contents of the lysosome, perhaps enzymes, are required on the outside of the cell to facilitate the release of uropods from the extracellular matrix. The identification of these factors is of great interest.While our data specifically implicate a role for lysosomal trafficking and fusion in chemotaxis, it is likely that the flow of other vesicles and membrane is important in directed cell migration. Sytl5 was originally identified in a yeast two-hybrid screen as a binding partner for Rab27a.¹⁶ Using this information, we used RNAi-mediated knockdowns of Rab27a and Rab3a mRNA and found similar to the synaptotagmins, these proteins had a role in lymphocyte chemotaxis. We have recently extended this genetic screen and have identified additional Rab family members that regulate chemotaxis (Fig. 1). The identified Rabs have broad functions with regard to vesicle trafficking, suggesting a more general requirement for vesicle trafficking during chemotaxis. These data support a role for the trafficking of other types of vesicles in addition to lysosomes during chemotaxis.Open in a separate windowFigure 1Transwell chemotaxis of SupT1 cells infected with viruses containing shRNA targeting genes encoding the indicated members of the Rab family or EGFP as a control. The number of cells migrating to the lower chamber was determined using CyQUANT as previously described.¹ Reductions in the chemokine dose response were significant using ANOVA (p < 0.01 for each of the knock-down cell lines).Future studies are needed to determine the roles of vesicle trafficking in regulating chemotaxis. We have presented experiments specifically demonstrating a role for lysosome or lysosome-like vesicle fusion in uropod release during chemotaxis (Fig. 2). However, it is likely that there will be many more roles for vesicle trafficking in chemotaxis. For example, we have found that the knock-down of additional Rab family member proteins not specifically associated with lysosomal movement also reduced chemotaxis in lymphocytes. It is likely that vesicle trafficking is essential for delivering lipid membrane to the cell surface as the cell shape changes.¹⁷ Additionally, chemokine receptor recycling during chemotaxis depends on vesicle trafficking and SYT3.¹⁸ Specific cargo, including integrins and other receptors, are likely to be delivered to the leading and trailing edges by vesicles.¹⁹ It is likely that that the movement of vesicles within the cell is essential for many aspects of cell movement beyond the cell.Open in a separate windowFigure 2Model of SYT7 activity in chemotaxis. Following the binding of a chemoattractant (eg. chemokine) to its G protein-coupled seven transmembrane spanning receptor, calcium is released. Binding of calcium to Syt7 on the lysosome surface induces Vamp7 on the lysosome surface to interact with Syn-4 and SNAP-23 at the cell membrane thereby facilitating the fusion of the lysosome with the cell membrane.     

Population genomic analyses reveal a history of range expansion and trait evolution across the native and invaded range of yellow starthistle (Centaurea solstitialis)

Identifying sources of genetic variation and reconstructing invasion routes for non‐native introduced species is central to understanding the circumstances under which they may evolve increased invasiveness. In this study, we used genome‐wide single nucleotide polymorphisms to study the colonization history of Centaurea solstitialis in its native range in Eurasia and invasions into the Americas. We leveraged this information to pinpoint key evolutionary shifts in plant size, a focal trait associated with invasiveness in this species. Our analyses revealed clear population genomic structure of potential source populations in Eurasia, including deep differentiation of a lineage found in the southern Apennine and Balkan Peninsulas and divergence among populations in Asia, eastern Europe and western Europe. We found strongest support for an evolutionary scenario in which western European populations were derived from an ancient admixture event between populations from eastern Europe and Asia, and subsequently served as the main genetic ‘bridgehead’ for introductions to the Americas. Introductions to California appear to be from a single source region, and multiple, independent introductions of divergent genotypes likely occurred into the Pacific Northwest. Plant size has evolved significantly at three points during range expansion, including a large size increase in the lineage responsible for the aggressive invasion of the California interior. These results reveal a long history of colonization, admixture and trait evolution in C. solstitialis, and suggest routes for improving evidence‐based management decisions for one of the most ecologically and economically damaging invasive species in the western United States.     

Coinfection modulates inflammatory responses and clinical outcome of Helicobacter felis and Toxoplasma gondii infections

The host immune response plays a critical role in determining disease manifestations of chronic infections. Inadequate immune response may fail to control infection, although in other cases the specific immune response may be the cause of tissue damage and disease. The majority of patients with chronic infections are infected by more than one organism yet the interaction between multiple active infections is not known, nor is the impact on disease outcome clear. Using the BALB/c strain of mice, we show that Toxoplasma gondii infection in a host infected with Helicobacter felis alters the natural outcome of T. gondii infection, allowing uncontrolled tachyzoite replication and severe organ damage. Survival rates decrease from 95% in T. gondii infection alone to 50% in dual-infected mice. In addition, infection with T. gondii alters the specific H. felis immune response, converting a previously resistant host to a susceptible phenotype. Gastric mucosal IFN-gamma and IL-12 were significantly elevated and IL-10 substantially reduced in dual-infected mice. These changes were associated with severe gastric mucosal inflammation, parietal cell loss, atrophy, and metaplastic cell changes. These data demonstrate the profound interactions between the immune response to unrelated organisms, and suggest these types of interactions my impact clinical disease.