Flotillins are involved in the polarization of primitive and mature hematopoietic cells.

BACKGROUND: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present evidence that raft-associated endocytic proteins (flotillins) are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions. CONCLUSIONS: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.

Soluble CD146 is generated by ectodomain shedding of membrane CD146 in a calcium-induced, matrix metalloprotease-dependent process.

CD146 is a cell adhesion molecule localized at the endothelial junction and is involved in the control of cell-cell cohesion. In this study, we showed that calcium influx in human microvascular lung endothelial cells results in the loss of surface CD146 and the release of soluble CD146. This calcium-induced CD146 shedding could be prevented with inhibitors of matrix metalloproteases indicating a central role of matrix metalloproteases in this process. We also investigated if CD146 shedding influences vascular permeability. Endothelial cell monolayers cultured on filter membranes showed an increased permeability for albumin when stimulated with ionomycin. This calcium-induced increase in permeability was inhibited when CD146 shedding was prevented by a matrix metalloprotease inhibitor. Our data indicate that surface CD146 plays a central role in the regulation of vascular permeability and demonstrate that CD146 and matrix metalloproteases are potential targets to modify endothelial barrier function.

Substantial reduction of naïve and regulatory T cells following traumatic stress.

Posttraumatic stress disorder (PTSD) is associated with an enhanced susceptibility to various somatic diseases. However, the exact mechanisms linking traumatic stress to subsequent physical health problems have remained unclear. This study investigated peripheral T lymphocyte differentiation subsets in 19 individuals with war and torture related PTSD compared to 27 non-PTSD controls (n=14 trauma-exposed controls; n=13 non-exposed controls). Peripheral T cell subpopulations were classified by their characteristic expression of the lineage markers CD45RA and CCR7 into: naïve (CD45RA(+) CCR7(+)), central memory (T(CM): CD45RA(-) CCR7(+)) and effector memory (T(EM): CD45RA(-) CCR7(-) and T(EMRA): CD45RA(-) CCR7(-)) cells. Furthermore, we analyzed regulatory T cells (CD4(+)CD25(+)FoxP3(+)) and ex vivo proliferation responses of peripheral blood mononuclear cells after stimulation with anti-CD3 monoclonal antibody. Results show that the proportion of naïve CD8(+) T lymphocytes was reduced by 32% (p=0.01), whereas the proportions of CD3(+) central (p=0.02) and effector (p=0.01) memory T lymphocytes were significantly enhanced (+22% and +34%, respectively) in PTSD patients compared to non-PTSD individuals. To a smaller extent, this effect was also observed in trauma-exposed non-PTSD individuals, indicating a cumulative effect of traumatic stress on T cell distribution. Moreover, PTSD patients displayed a 48% reduction in the proportion of regulatory T cells (p<0.001). Functionally, these alterations were accompanied by a significantly enhanced (+34%) ex vivo proliferation of anti-CD3 stimulated T cells (p=0.05). The profoundly altered composition of the peripheral T cell compartment might cause a state of compromised immune responsiveness, which may explain why PTSD patients show an increased susceptibility to infections, and inflammatory and autoimmune diseases.

Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry.

Brown algae of the Laminariales (kelps) are the strongest accumulators of iodine among living organisms. They represent a major pump in the global biogeochemical cycle of iodine and, in particular, the major source of iodocarbons in the coastal atmosphere. Nevertheless, the chemical state and biological significance of accumulated iodine have remained unknown to this date. Using x-ray absorption spectroscopy, we show that the accumulated form is iodide, which readily scavenges a variety of reactive oxygen species (ROS). We propose here that its biological role is that of an inorganic antioxidant, the first to be described in a living system. Upon oxidative stress, iodide is effluxed. On the thallus surface and in the apoplast, iodide detoxifies both aqueous oxidants and ozone, the latter resulting in the release of high levels of molecular iodine and the consequent formation of hygroscopic iodine oxides leading to particles, which are precursors to cloud condensation nuclei. In a complementary set of experiments using a heterologous system, iodide was found to effectively scavenge ROS in human blood cells.

Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury.

BACKGROUND: Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration. METHODS: Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10(6) cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals. RESULTS: We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats. CONCLUSION: Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflammatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.

CD21 and CD62L shedding are both inducible via P2X7Rs.

Neutrophils and lymphocytes are recruited to sites of inflammation and require the adhesion molecule L-selectin (CD62L) for adherence to endothelial cells. Nucleotides released from activated or dying cells at sites of inflammation can mediate signaling through purinergic receptor family II, resulting in CD62L shedding. Activation of B lymphocytes requires the complement receptor type II (CD21) and at the same time leads to shedding of CD21. Both CD62L and CD21 shedding possibly depends on the same families of proteases. In the present study, we characterized peripheral blood naive and memory cells and neutrophils for CD62L surface expression and analyzed benzoyl-benzoyl triphosphate (BzATP)-induced shedding. BzATP is able to induce CD62L shedding in naive and memory lymphocytes, but not in neutrophils. CD21 shedding can be induced through activation of the B cell receptor (BCR) or with mitogens. Here we show that CD21 is also susceptible to BzATP-induced shedding on peripheral B cells. In addition, using receptor inhibitors, we show that shedding of CD21 and CD62L is mediated via the P2X7R. P2X7R-mediated CD62L and CD21 shedding could occur as a result of extracellular accumulated ATP and may have an influence on leukocyte migrational behavior and BCR-mediated signaling.

Early events in the perception of lipopolysaccharides in the brown alga Laminaria digitata include an oxidative burst and activation of fatty acid oxidation cascades.

This study provides evidence that bacterial lipopolysaccharides can be strong triggers of early events of defence reactions in the brown algal kelp Laminaria digitata, constituting the first report of a biological activity of this class of macromolecules in a marine alga. The early events include an oxidative burst, release of free saturated and unsaturated fatty acids (FFAs) and accumulation of oxylipins such as 13-hydroxyoctadecatrienoic acid and 15-hydroxyeicosapentaenoic acid. The formation of reactive oxygen species can be inhibited by diphenylene iodonium, suggesting that the source is an NAD(P)H oxidase and is similar to the oxidative burst in neutrophils and terrestrial plants. In addition and besides triggering an oxidative burst, the hypolipidemic drug clofibrate also induces the release of FFAs, to a lesser extent than lipopolysaccharides, but it does not induce oxylipin production. Other strong inducers of the oxidative burst in Laminaria such as oligoguluronates could not induce the release of FFAs nor oxylipin production. These results suggest that different signalling pathways are involved in the induction of the oxidative burst and oxylipin production.

Possible tetramerisation of the proteasome maturation factor POMP/proteassemblin/hUmp1 and its subcellular localisation.

The proteasome is a multisubunit complex with a central role in non-lysosomal proteolysis and the processing of proteins for presentation by the MHC class I pathway. The 16kDa proteasome maturation protein POMP (also named proteassemblin or hUmp1) acts as a chaperone and is essential for the maturation of the 20S proteasome proteolytic core complex. However, the exact mechanism, timing and localisation of mammalian proteasome assembly remains elusive. We sought to investigate the localisation of POMP within the cell and therefore purified the protein and produced a polyclonal antibody. For immunisation, POMP was overexpressed and purified from a bacterial GST-system. Interestingly, after removal of the GST-tag, POMP was hardly detectable by Coomassie blue- and Ponceau red-staining. However, with a reverse zinc-staining, the protein could easily be visualised. POMP was gel-filtrated and eluted from a calibrated chromatography column with an apparent molecular weight of approximately 64kDa, suggesting that it forms tetramers. Moreover, localisation studies by immunofluorescence stainings and confocal microscopy revealed that POMP is present in the cytoplasm as well as in the nucleus.

Preformed reggie/flotillin caps: stable priming platforms for macrodomain assembly in T cells.

T cell activation after contact with an antigen-presenting cell depends on the regulated assembly of the T cell receptor signaling complex, which involves the polarized assembly of a stable, raft-like macrodomain surrounding engaged T cell receptors. Here we show that the preformed reggie/flotillin caps present in resting T cells act as priming platforms for macrodomain assembly. Preformed reggie-1/flotillin-2 caps are exceptionally stable, as shown by fluorescence recovery after photobleaching (FRAP). Upon T cell stimulation, signaling molecules are recruited to the stable reggie/flotillin caps. Importantly, a trans-negative reggie-1/flotillin-2 deletion mutant, which interferes with assembly of the preformed reggie/flotillin cap, impairs raft polarization and macrodomain formation after T cell activation. Accordingly, expression of the trans-negative reggie-1 mutant leads to the incorrect positioning of the guanine nucleotide exchange factor Vav, resulting in defects in cytoskeletal reorganization. Thus, the preformed reggie/flotillin caps are stable priming platforms for the assembly of multiprotein complexes controlling actin reorganization during T cell activation.

D3 dopamine receptor mRNA is elevated in T cells of schizophrenic patients whereas D4 dopamine receptor mRNA is reduced in CD4+ -T cells.

The expression of dopamine receptors was examined in purified human neutrophils, monocytes, B cells, natural killer cells and CD4+ - and CD8+ -T lymphocytes by RT-PCR. In healthy subjects, D1 and D2 receptors were not expressed in leukocytes. Real Time PCR for dopamine receptors D3 and D4 disclosed that D3 receptors are expressed in T cells and natural killer cells and D4 receptors in CD4+ -T cells. The comparison of schizophrenic patients with sex- and age-matched controls revealed a significantly higher expression of D3 receptor mRNA in T cells of schizophrenic patients, whereas D4 receptor mRNA in CD4+ -T cells was downregulated.

Granulocyte colony-stimulating factor (filgrastim) treatment primes for increased ex vivo inducible prostanoid release.

We investigated whether anti-inflammatory effects of treatment with granulocyte colony-stimulating factor (G-CSF, filgrastim) are mediated via prostaglandin E(2) (PGE(2)) induction. In a double-blind crossover study, 10 healthy volunteers received 300 microg of filgrastim or saline 1 week apart. This was repeated after oral administration of 50 mg of flurbiprofen 1 h before injection. The increase in neutrophilic granulocytes initiated by G-CSF was augmented significantly by flurbiprofen. Lipopolysaccharide-induced PGE(2) and thromboxane (TxB(2)) release were increased 8 h after G-CSF treatment. This increase was abrogated by flurbiprofen. However, flurbiprofen did not affect G-CSF-mediated decrease in tumor necrosis factor-alpha or interferon-gamma release. Of the volunteers treated with G-CSF, eight reported side effects (headache and bone pain) against none in the saline group. When flurbiprofen was given before injection, one volunteer each reported side effects in the G-CSF and in the saline group. These data show that G-CSF primes for increased PGE(2) and TxB(2) release. Cyclooxygenase inhibition counteracts neither the hematopoietic nor the anti-inflammatory activity of G-CSF but reduces side effects.

Generation of three-dimensional pannus-like tissues in vitro from single cell suspensions of synovial fluid cells from arthritis patients.

Using single cell suspensions from synovial fluid cells of arthritis patients, we observed differentiation of three-dimensional tissues in vitro. This new model of pannus-like tissue (PLT) might be useful to study pannus tissue formation and differentiation. In the PLT cultures, we observed two cell types, fibroblast-like and macrophage-like cells, defined by their distinct morphology and major histocompatibility complex (MHC) by human leukocyte antigen (HLA) class II expression. We could discriminate several intermediate steps of differentiation which finally led to 3D villi-like structures. Secretion of interferon gamma, interleukin-10, and tumor necrosis factor alpha was measured in the culture supernatants. Using methotrexate at various concentrations, the growth of PLT could be inhibited. We describe definite intermediate steps of differentiation. The present approach could be a suitable model for the in vitro study of pannus tissue formation.

Febrile temperatures attenuate IL-1 beta release by inhibiting proteolytic processing of the proform and influence Th1/Th2 balance by favoring Th2 cytokines.

We investigated possible feedback mechanisms of febrile temperatures on LPS- and staphylococcal enterotoxin B (SEB)-induced cytokine release in human whole blood. LPS-induced IL-1beta release was inhibited at temperatures >38 degrees C, whereas intracellular proIL-1beta formation as well as the release of other cytokines except IL-18 were only attenuated above 42 degrees C, indicating that febrile temperatures impair the proteolytic processing of proIL-1beta. This attenuated processing is not due to either heat inactivation of caspase-1 or structural changes in proIL-1beta produced at higher temperatures. Instead, we propose that febrile conditions change cytosolic compartmentation or trafficking, so that synthesized proIL-1beta cannot encounter caspase-1. Febrile temperatures also influenced Th1/Th2 cytokine balance. We observed a 3-fold increase in the Th2-cytokines IL-5 and IL-13 and a reduction to 15% of the Th1-cytokine IL-2 when SEB-stimulated whole blood was incubated at 40 degrees C compared with 37 degrees C. These results indicate that fever limits the production of the fever-inducing IL-1beta and also influences the adaptive immune response, favoring Th2 cytokine production.

Granulocyte colony-stimulating factor attenuates LPS-stimulated IL-1beta release via suppressed processing of proIL-1beta, whereas TNF-alpha release is inhibited on the level of proTNF-alpha formation.

In the presence of granulocyte colony-stimulating factor (G-CSF), the release of IL-1beta and TNF-alpha by LPS-stimulated human whole blood was suppressed. Via measurement of cytokine mRNA, inactive precursor and mature protein, we investigated whether this inhibition occurs at the transcriptional, translational or post-translational level of cytokine production. G-CSF inhibited IL-1beta release, but the formation of proIL-1beta was not attenuated, indicating that G-CSF interferes with the proteolytic processing of proIL-1beta. Since the release of IL-1beta in LPS-stimulated whole blood was blocked by the caspase-1 inhibitor YVAD-cmk, processing of proIL-1beta appears to depend on caspase-1 activity. The conclusion that G-CSF inhibits caspase-1 activity was supported bythe finding that the release of IL-18 was also inhibited by G-CSF, similar to IL-1beta release. Intracellular caspase-1 activity in monocytes was measured by flow cytometry with the cell-permeablecaspase substrate Asp(2)-rhodamine. In the presence of G-CSF the cleavage of this substrate was inhibited by more than 50%. G-CSF had no effect on LPS-induced doubling of caspase-1 mRNA, indicating that G-CSF affects caspase-1 activation and not its formation. For TNF-alpha another mechanism of G-CSF action was identified: TNF-alpha as well as proTNF-alpha formation were inhibited by G-CSF, butG-CSF had no influence on LPS-induced TNF-alpha mRNA level. We therefore suggest that G-CSF causes translational silencing of LPS-induced TNF-alpha mRNA.