In vivo analysis of uropod function during physiological T cell trafficking

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

Anti-HIV state but not apoptosis depends on IFN signature in CD4+ T cells

To gain insights into the molecular mechanisms underlying early host responses to HIV in the CD4(+) T cell target population, we examined gene expression in CD4(+) T cells isolated 24 h after ex vivo HIV infection of lymphocyte aggregate cultures derived from human tonsils. Gene profiling showed a distinct up-regulation of genes related to immune response and response to virus, notably of IFN-stimulated genes (ISGs), irrespective of the coreceptor tropism of the virus. This mostly IFN-alpha-dependent gene signature suggested the involvement of plasmacytoid dendritic cells, a principal component of the antiviral immune response. Indeed, depletion of plasmacytoid dendritic cells before HIV inoculation abrogated transcriptional up-regulation of several ISGs and resulted in increased levels of HIV replication. Treatment with a blocking anti-IFN-alphaR Ab yielded increased HIV replication; conversely, HIV replication was decreased in pDC-depleted cultures treated with IFN-alpha. Among up-regulated ISGs was also TRAIL, indicating a potential role of the IFN signature in apoptosis. However, a blocking anti-TRAIL Ab did not abrogate apoptosis of CD4(+) T cells in CXCR4-tropic HIV-infected cultures, suggesting the involvement of pathways other than TRAIL mediated. We conclude that acute HIV infection of lymphoid tissue results in up-regulation of ISGs in CD4(+) T cells, which induces an anti-HIV state but not apoptosis.

CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

CD4(+) T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)-transgenic (tg) CD4(+) T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3Kdelta(D910A/D910A) or PI3Kgamma-deficient TCR-tg CD4(+) T cells showed similar responsiveness to CCL21 costimulation as control CD4(+) T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4(+) T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca(2+) signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 degrees C, subsequently displayed a high thermotolerance, being able to grow at 42 degrees C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.

Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation.

The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)-restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC-lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer's patches to promote host-microbiota mutualism.

Microbial colonization of the gut induces the development of gut-associated lymphoid tissue (GALT). The molecular mechanisms that regulate GALT function and result in gut-commensal homeostasis are poorly defined. T follicular helper (Tfh) cells in Peyer's patches (PPs) promote high-affinity IgA responses. Here we found that the ATP-gated ionotropic P2X7 receptor controls Tfh cell numbers in PPs. Lack of P2X7 in Tfh cells enhanced germinal center reactions and high-affinity IgA secretion and binding to commensals. The ensuing depletion of mucosal bacteria resulted in reduced systemic translocation of microbial components, lowering B1 cell stimulation and serum IgM concentrations. Mice lacking P2X7 had increased susceptibility to polymicrobial sepsis, which was rescued by Tfh cell depletion or administration of purified IgM. Thus, regulation of Tfh cells by P2X7 activity is important for mucosal colonization, which in turn results in IgM serum concentrations necessary to protect the host from bacteremia.

T Cell Motility as Modulator of Interactions with Dendritic Cells

It is well established that the balance of costimulatory and inhibitory signals during interactions with dendritic cells (DCs) determines T cell transition from a naïve to an activated or tolerant/anergic status. Although many of these molecular interactions are well reproduced in reductionist in vitro assays, the highly dynamic motility of naïve T cells in lymphoid tissue acts as an additional lever to fine-tune their activation threshold. T cell detachment from DCs providing suboptimal stimulation allows them to search for DCs with higher levels of stimulatory signals, while storing a transient memory of short encounters. In turn, adhesion of weakly reactive T cells to DCs presenting peptides presented on major histocompatibility complex with low affinity is prevented by lipid mediators. Finally, controlled recruitment of CD8(+) T cells to cognate DC-CD4(+) T cell clusters shapes memory T cell formation and the quality of the immune response. Dynamic physiological lymphocyte motility therefore constitutes a mechanism to mitigate low avidity T cell activation and to improve the search for "optimal" DCs, while contributing to peripheral tolerance induction in the absence of inflammation.

In vivo TCR Signaling in CD4(+) T Cells Imprints a Cell-Intrinsic, Transient Low-Motility Pattern Independent of Chemokine Receptor Expression Levels, or Microtubular Network, Integrin, and Protein Kinase C Activity

Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4(+) T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48-72 h after activation of CD4(+) T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4(+) T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4(+) T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4(+) T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.

Sphingosine kinase 2 deficient mice exhibit reduced experimental autoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited.

Pulmonary hypoplasia and anasarca syndrome in Cika cattle

BACKGROUND: Hydrops foetalis is defined as excessive fluid accumulation within the foetal extravascular compartments and body cavities. It has been described in human and veterinary medicine, but despite several descriptive studies its aetiology is still not fully clarified. Pulmonary hypoplasia and anasarca (PHA) syndrome is a rare congenital abnormality in cattle that is characterised by hydrops foetalis including extreme subcutaneous oedema (anasarca) and undeveloped or poorly formed lungs (pulmonary hypoplasia). Until now, sporadic cases of PHA were reported in cattle breeds like Australian Dexter, Belted Galloway, Maine-Anjou, and Shorthorn. This report describes the first known cases of PHA syndrome in Slovenian Cika cattle. CASE PRESENTATION: A 13-year-old cow aborted a male calf in the seventh month of pregnancy, while a male calf was delivered by caesarean section on the due date from a 14-year-old cow. The pedigree analysis showed that the calves were sired by the same bull, the dams were paternal half-sisters and the second calf was the product of a dam-son mating. Gross lesions were similar in both cases and characterized by severe anasarca, hydrothorax, hydropericardium, ascites, hypoplastic lungs, absence of lymph nodes, and an enlarged heart. The first calf was also athymic. Histopathology of the second affected calf confirmed severe oedema of the subcutis and interstitium of the organs, and pulmonary hypoplasia. The lymph vessels in the subcutis and other organs were severely dilated. Histopathology of the second calf revealed also lack of bronchus associated lymphoid tissue and adrenal gland hypoplasia. CONCLUSIONS: The findings were consistent with known forms of the bovine PHA syndrome. This is the first report of the PHA syndrome occurring in the local endangered breed of Cika cattle. Observed inbreeding practice supports that this lethal defect most likely follows an autosomal recessive mode of inheritance. In the light of the disease phenotype it is assumed that a mutation causing an impaired development of lymph vessels is responsible for the hydrops foetalis associated malformations in bovine PHA.

Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows

Gene expression of adipose factors, which may be part of the mechanisms that underlie insulin sensitivity, were studied in dairy cows around parturition. Subcutaneous fat biopsies and blood samples were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. In the adipose tissue samples, mRNA was quantified by real-time reverse transcription polymerase chain reaction for tumour necrosis factor alpha (TNFalpha), insulin-independent glucose transporter (GLUT1), insulin-responsive glucose transporter (GLUT4), insulin receptor, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), regulatory subunit of phosphatidylinositol-3 kinase (p85) and catalytic subunit of phosphatidylinositol-3 kinase. Blood plasma was assayed for concentrations of glucose, beta-hydroxybutyric acid, non-esterified fatty acids (NEFA) and insulin. Plasma parameters followed a pattern typically observed in dairy cows. Gene expression changes were observed, but there were no changes in TNFalpha concentrations, which may indicate its local involvement in catabolic adaptation of adipose tissue. Changes in GLUT4 and GLUT1 mRNA abundance may reflect their involvement in reduced insulin sensitivity and in sparing glucose for milk synthesis in early lactation. Unchanged gene expression of IRS1, IRS2 and p85 over time may imply a lack of their involvement in terms of insulin sensitivity dynamics. Alternatively, it may indicate that post-transcriptional modifications of these factors came into play and may have concealed an involvement.

Inhibition of tissue transglutaminase sensitizes TRAIL-resistant lung cancer cells through upregulation of death receptor 5

Tissue transglutaminase (TG2) is implicated in cellular processes such as apoptosis and cell migration. Its acyl transferase activity cross-links certain proteins, among them transcription factors were described. We show here that the TG2 inhibitor KCC009 reversed resistance to tumor necrosis factor-related apoptosis-inducing factor (TRAIL) in lung cancer cells. Sensitization required upregulation of death receptor 5 (DR5) but not of death receptor 4. Upregulation of DR5 involved the first intron of the DR5 gene albeit it was independent from p53 and nuclear factor kappa B. In conclusion, inhibition of tissue transglutaminase provides an interesting strategy for sensitization to TRAIL-induced apoptosis in p53-deficient lung cancer cells.

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.