Ionita Ghiran

Human Bone Marrow Stromal Cells Express a Distinct Set of Biologically Functional Chemokine Receptors

Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum‐free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface‐expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen‐activated protein kinase (e.g., extracellular signal–related kinase [ERK]‐1 and ERK‐2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)‐5 whereas CCL5 activates STAT‐1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F‐actin polymerization. Studies of culture‐expanded BMSCs, for example, 12–16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM‐1, ICAM‐2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity

The ulcer-causing gastric pathogen Helicobacter pylori is the only bacterium known to colonize the harsh acidic environment of the human stomach. H. pylori survives in acidic conditions by producing urease, which catalyzes hydrolysis of urea to yield ammonia thus elevating the pH of its environment. However, the manner in which H. pylori is able to swim through the viscoelastic mucus gel that coats the stomach wall remains poorly understood. Previous rheology studies on gastric mucin, the key viscoelastic component of gastric mucus, indicate that the rheology of this material is pH dependent, transitioning from a viscous solution at neutral pH to a gel in acidic conditions. Bulk rheology measurements on porcine gastric mucin (PGM) show that pH elevation by H. pylori induces a dramatic decrease in viscoelastic moduli. Microscopy studies of the motility of H. pylori in gastric mucin at acidic and neutral pH in the absence of urea show that the bacteria swim freely at high pH, and are strongly constrained at low pH. By using two-photon fluorescence microscopy to image the bacterial motility in an initially low pH mucin gel with urea present we show that the gain of translational motility by bacteria is directly correlated with a rise in pH indicated by 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH sensitive fluorescent dye. This study indicates that the helicoidal-shaped H. pylori does not bore its way through the mucus gel like a screw through a cork as has previously been suggested, but instead achieves motility by altering the rheological properties of its environment.

Malaria-Infected Erythrocyte-Derived Microvesicles Mediate Cellular Communication within the Parasite Population and with the Host Immune System

Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system.

Airway Eosinophils: Allergic Inflammation Recruited Professional Antigen-Presenting Cells

The capacity of airway eosinophils, potentially pertinent to allergic diseases of the upper and lower airways, to function as professional APCs, those specifically able to elicit responses from unprimed, Ag-naive CD4+ T cells has been uncertain. We investigated whether airway eosinophils are capable of initiating naive T cell responses in vivo. Eosinophils, isolated free of other APCs from the spleens of IL-5 transgenic mice, following culture with GM-CSF expressed MHC class II and the costimulatory proteins, CD40, CD80, and CD86. Eosinophils, incubated with OVA Ag in vitro, were instilled intratracheally into wild-type recipient mice that adoptively received i.v. infusions of OVA Ag-specific CD4+ T cells from OVA TCR transgenic mice. OVA-exposed eosinophils elicited activation (CD69 expression), proliferation (BrdU incorporation), and IL-4, but not IFN-γ, cytokine production by OVA-specific CD4+ T cells in paratracheal lymph nodes (LN). Exposure of eosinophils to lysosomotropic NH4Cl, which inhibits Ag processing, blocked each of these eosinophil-mediated activation responses of CD4+ T cells. By three-color fluorescence microscopy, OVA Ag-loaded eosinophil APCs were physically interacting with naive OVA-specific CD4+ T cells in paratracheal LN after eosinophil airway instillation. Thus, recruited luminal airway eosinophils are distinct allergic “inflammatory” professional APCs able to activate primary CD4+ T cell responses in regional LNs.

Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels

This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.

Eosinophil granules function extracellularly as receptor-mediated secretory organelles

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-γ, and G protein–coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Human Eosinophils Secrete Preformed, Granule-Stored Interleukin-4 Through Distinct Vesicular Compartments

Secretion of interleukin‐4 (IL‐4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL‐4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)‐inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin‐elicited IL‐4 secretion, we identified at the ultrastructural level distinct vesicular IL‐4 transport mechanisms. Interleukin‐4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL‐4 and IL‐4‐loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin‐induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin‐elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL‐4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.

Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Human Complement Receptor Type 1/CD35 Is an Epstein-Barr Virus Receptor

Epstein-Barr virus (EBV) attachment to primary B cells initiates virus entry. Although CD21 is the only known receptor for EBVgp350/220, a recent report documents EBV-infected B cells from a patient genetically deficient in CD21. On normal resting B cells, CD21 forms two membrane complexes: one with CD19 and another with CD35. Whereas the CD21/CD19 complex is widely retained on immortalized and B cell tumor lines, the related complement-regulatory protein CD35 is lost. To determine the role(s) of CD35 in initial infection, we transduced a CD21-negative pre-B cell and myeloid leukemia line with CD35, CD21, or both. Cells expressing CD35 alone bound gp350/220 and became latently infected when the fusion receptor HLA II was coexpressed. Temporal, biophysical, and structural characteristics of CD35-mediated infection were distinct from CD21. Identification of CD35 as an EBV receptor uncovers a salient role in primary infection, addresses unsettled questions of virus tropism, and underscores the importance of EBVgp350/220 for vaccine development.

EliCell: a gel-phase dual antibody capture and detection assay to measure cytokine release from eosinophils

Eosinophils contain many preformed cytokines and chemokines, which are stored in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesicular transport. We have developed a sensitive method to detect and quantitate eosinophil vesicular transport-mediated release of specific eosinophil proteins. Our EliCell assay is based on microscopic observations of individual viable eosinophils embedded in an agarose matrix that contains immobilized antibody to the protein of interest. Following stimulation of eosinophils, released protein is bound by the capture antibody at its site of release and is detected by a fluorochrome-conjugated detection antibody. We have validated this assay by evaluating interferon-gamma-induced release of RANTES from eosinophils. Extracellularly released RANTES was visualized as focal immunoflourescent staining and was quantitated by scoring the numbers of eosinophils releasing RANTES and by measuring the fluorescent intensity over individual eosinophils. In comparison with ELISA assays of RANTES released into supernatant fluids by interferon-gamma-stimulated eosinophils, EliCell assays were more sensitive enabling detection of RANTES release at earlier times and at lower levels of interferon-gamma stimulation. The EliCell assay provides a sensitive method to study the regulated release of eosinophil-derived cytokines, chemokines and other granule proteins.