In vivo two-photon laser-scanning microscopy of Ca2+ dynamics in visual motion-sensitive neurons

We applied two-photon laser-scanning microscopy (TPLSM) to motion-sensitive visual interneurons of the fly to study Ca(2+) dynamics in vivo at a higher spatial and temporal resolution than possible with conventional fluorescence microscopy. Based on a custom-built two-photon microscope, we performed line scans to measure changes in presynaptic Ca(2+) concentrations elicited by visual stimulation. We used a fast avalanche photodiode (APD) with a high quantum efficiency to detect even low levels of emitted fluorescence. Our experiments show that our in vivo preparation is amenable to TPLSM: with excitation intensities low enough not to cause photodamage, activity-dependent fluorescence changes of Ca(2+)-sensitive dyes can be detected in small neuronal branches. The performance of two-photon and conventional Ca(2+) imaging carried out consecutively at the same neuron is compared and it is demonstrated that two-photon imaging allows us to detect differences in Ca(2+) dynamics between individual neurites.     

Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

The angiotensin converting enzyme 2 (ACE2) has been identified as a receptor for the severe acute respiratory syndrome associated coronavirus (SARS-CoV). Here we show that ACE2 expression on cell lines correlates with susceptibility to SARS-CoV S-driven infection, suggesting that ACE2 is a major receptor for SARS-CoV. The soluble ectodomain of ACE2 specifically abrogated S-mediated infection and might therefore be exploited for the generation of inhibitors. Deletion of a major portion of the cytoplasmic domain of ACE2 had no effect on S-driven infection, indicating that this domain is not important for receptor function. Our results point to a central role of ACE2 in SARS-CoV infection and suggest a minor contribution of the cytoplasmic domain to receptor function.     

Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor.     

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

Dual function of interleukin-1beta for the regulation of interleukin-6-induced suppressor of cytokine signaling 3 expression

Interleukin-6 (IL-6) exerts pro- as well as anti-inflammatory activities in response to infection, injury, or other stimuli that affect the homeostasis of the organism. IL-6-induced expression of acute-phase protein genes in the liver is tightly regulated through both IL-6-induced feedback inhibitors and the activity of pro-inflammatory cytokines such as tumor necrosis factor alpha and interleukin-1beta. In previous studies mechanisms for how IL-1beta counteracts IL-6-dependent acute-phase protein gene induction have been proposed. Herein we analyzed IL-1beta-mediated regulation of IL-6-induced expression of the feedback inhibitor SOCS3. In hepatocytes IL-1beta alone does not induce SOCS3 expression, but it counteracts SOCS3-promoter activation in long term studies. Surprisingly, short term stimulation revealed IL-1beta to be a potent enhancer of SOCS3 expression in concert with IL-6. This activity of IL-1beta does not depend on IL-1beta-dependent STAT1-serine phosphorylation but on NF-kappaB-dependent gene induction. Such a regulatory network allows IL-1beta to counteract IL-6-dependent expression of acute-phase protein genes without inhibiting IL-6-induced SOCS3 expression and provides a reasonable mechanism for the IL-1beta-dependent inhibition of acute-phase gene induction, because reduced SOCS3 expression would lead to enhanced IL-6 activity.     

Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.     

Pneumolysin-induced lung injury is independent of leukocyte trafficking into the alveolar space

Pneumolysin (PLY) is a major virulence factor released by Streptococcus pneumoniae and has been implicated in the pathogenesis of pneumococcal pneumonia. In this study, we evaluated the contribution of newly recruited neutrophils and monocytes and resident alveolar macrophages to the pathogenesis of PLY-induced lung injury. Mice received either adhesion-blocking Abs to inhibit alveolar leukocyte trafficking or liposomal clodronate to deplete alveolar macrophages before intratracheal application of native PLY or its noncytotoxic derivative PdB. We found that treatment with PLY but not PdB resulted in increased lung vascular permeability. In addition, PLY also induced a decrease in the resident alveolar macrophage population, and the recruitment of peripheral blood neutrophils and monocytes into the alveolar space. Blockade of PLY-induced alveolar leukocyte trafficking by pretreatment of mice with anti-CD18 plus anti-CD49d Abs or depletion of circulating neutrophils did not attenuate the increase in lung permeability observed in response to intratracheal PLY. In addition, depletion of resident alveolar macrophages with clodronated liposomes did not reduce alveolar injury developing in response to PLY. PLY-induced lung injury was associated with only a small increase in bronchoalveolar lavage concentrations of cytokines. These data indicate that PLY-induced lung injury results from direct pneumotoxic effects on the alveolar-capillary barrier and is independent of both resident and recruited phagocytic cells.     

Partial activation of neonatal CD11c+ dendritic cells and induction of adult-like CD8+ cytotoxic T cell responses by synthetic microspheres

Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8(+) cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8(+) T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-gamma-secreting CD8(+) splenocytes. Neonatal CD8(+) T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c(+) dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c(+) DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4(+) T cell help. The effective induction of neonatal CD8(+) T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c(+) DC may mature sufficiently to stimulate naive CD8(+) neonatal T cells, even in the absence of strong maturation signals.     

Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages

Macrophages (MF) are the final host cells for multiplication of the intracellular parasite Leishmania major (L. major). However, polymorphonuclear neutrophil granulocytes (PMN), not MF, are the first leukocytes that migrate to the site of infection and encounter the parasites. Our previous studies indicated that PMN phagocytose but do not kill L. major. Upon infection with Leishmania, apoptosis of human PMN is delayed and takes 2 days to occur. Infected PMN were found to secrete high levels of the chemokine MIP-1beta, which attracts MF. In this study, we investigated whether MF can ingest parasite-infected PMN. We observed that MF readily phagocytosed infected apoptotic PMN. Leishmania internalized by this indirect way survived and multiplied in MF. Moreover, ingestion of apoptotic infected PMN resulted in release of the anti-inflammatory cytokine TGF-beta by MF. These data indicate that Leishmania can misuse granulocytes as a "Trojan horse" to enter their final host cells "silently" and unrecognized.