bluuebunny/crossref_metadata_2025 huggingface dataset:
LIN-5 Is a Novel Component of the Spindle Apparatus Required for Chromosome Segregation and Cleavage Plane Specification in Caenorhabditis elegans
Authors: Lorson Monique A. , Horvitz H. Robert , van den Heuvel Sander
Published: July 2002

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.
CAM Kinase IV Regulates Lineage Commitment and Survival of Erythroid Progenitors in a Non-Cell–Autonomous Manner
Authors: Wayman Gary A. , Walters Melinda J. , Kolibaba Kathryn , Soderling Thomas R. , Christian Jan L.
Published: July 2002

Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.
Differential Roles for αMβ2 Integrin Clustering or Activation in the Control of Apoptosis via Regulation of Akt and ERK Survival Mechanisms
Authors: Whitlock Ben B. , Gardai Shyra , Fadok Valerie , Bratton Donna , Henson Peter M.
Published: July 2002

The role of integrins in leukocyte apoptosis is unclear, some studies suggest enhancement, others inhibition. We have found that β2-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of αMβ2 delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFα, or UV irradiation, ligation of active αMβ2 resulted in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins did not show this proapoptotic effect and continued to inhibit apoptosis. This discrepancy was attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive αMβ2 was capable of stimulating the kinases ERK and Akt. Activated αMβ2 stimulated Akt, but not ERK. When proapoptotic stimuli were combined with either αMβ2 clustering or activation, Akt activity was blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive αMβ2 continued to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, β2-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes.
The Sar1 Gtpase Coordinates Biosynthetic Cargo Selection with Endoplasmic Reticulum Export Site Assembly
Authors: Aridor Meir , Fish Kenneth N. , Bannykh Sergei , Weissman Jacques , Roberts Theresa H. , Lippincott-Schwartz Jennifer , Balch William E.
Published: July 2002

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.
Cvt9/Gsa9 Functions in Sequestering Selective Cytosolic Cargo Destined for the Vacuole
Authors: Kim John , Kamada Yoshiaki , Stromhaug Per E. , Guan Ju , Hefner-Gravink Ann , Baba Misuzu , Scott Sidney V. , Ohsumi Yoshinori , Dunn William A. , Klionsky Daniel J.
Published: July 2002

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.

We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.
A New Focal Adhesion Protein That Interacts with Integrin-Linked Kinase and Regulates Cell Adhesion and Spreading
Authors: Tu Yizeng , Huang Yao , Zhang Yongjun , Hua Yun , Wu Chuanyue
Published: July 2002

Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti–CH-ILKBP antibodies, and green fluorescent protein–CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP–ILK–PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.
Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans
Authors: Hannak Eva , Kirkham Matthew , Hyman Anthony A. , Oegema Karen
Published: July 2002

Centrosomes mature as cells enter mitosis, accumulating γ-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal α-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal γ-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1–dependent increase in centrosomal γ-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.
Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation
Authors: Zhao Ming , Harris Stephen E. , Horn Diane , Geng Zhaopo , Nishimura Riko , Mundy Gregory R. , Chen Di
Published: July 2002

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.
The aurora kinase AIR-2 functions in the release of chromosome cohesion in Caenorhabditis elegans meiosis
Authors: Rogers Eric , Bishop John D. , Waddle James A. , Schumacher Jill M. , Lin Rueyling
Published: July 2002

Accurate chromosome segregation during cell division requires not only the establishment, but also the precise, regulated release of chromosome cohesion. Chromosome dynamics during meiosis are more complicated, because homologues separate at anaphase I whereas sister chromatids remain attached until anaphase II. How the selective release of chromosome cohesion is regulated during meiosis remains unclear. We show that the aurora-B kinase AIR-2 regulates the selective release of chromosome cohesion during Caenorhabditis elegans meiosis. AIR-2 localizes to subchromosomal regions corresponding to last points of contact between homologues in metaphase I and between sister chromatids in metaphase II. Depletion of AIR-2 by RNA interference (RNAi) prevents chromosome separation at both anaphases, with concomitant prevention of meiotic cohesin REC-8 release from meiotic chromosomes. We show that AIR-2 phosphorylates REC-8 at a major amino acid in vitro. Interestingly, depletion of two PP1 phosphatases, CeGLC-7α and CeGLC-7β, abolishes the restricted localization pattern of AIR-2. In Ceglc-7α/β(RNAi) embryos, AIR-2 is detected on the entire bivalent. Concurrently, chromosomal REC-8 is dramatically reduced and sister chromatids are separated precociously at anaphase I in Ceglc-7α/β(RNAi) embryos. We propose that AIR-2 promotes the release of chromosome cohesion via phosphorylation of REC-8 at specific chromosomal locations and that CeGLC-7α/β, directly or indirectly, antagonize AIR-2 activity.
Negative Selection in the Thymus Includes Semimature T Cells
Authors: Kishimoto Hidehiro , Sprent Jonathan
Published: July 2002

The thymic medulla plays a key role in negative selection (self-tolerance induction) and contains differentiated T cells en route to the extrathymic environment. However, being relatively mature, medullary T cells are thought to be beyond the stage of tolerance induction. This paradox is resolved by the finding that medullary T cells (CD4+8− thymocytes) comprise two distinct subsets. Medullary thymocytes expressing a fully mature (HSAlo) phenotype are strongly resistant to tolerance induction, whereas cells with a semimature (HSAhi) phenotype are tolerance susceptible. These findings suggest that the differentiated T cells reaching the medulla from the cortex remain sensitive to tolerance induction for a brief period before acquiring a fully mature tolerance-resistant phenotype. The semimature subset of medullary T cells displays unique requirements for tolerance induction; depending upon the conditions used, tolerizing these cells can involve either a Fas (CD95)-dependent or a Fas-independent pathway.
CD40 Ligation on Human Cord Blood CD34+Hematopoietic Progenitors Induces Their Proliferation and Differentiation into Functional Dendritic Cells
Authors: Flores-Romo Leopoldo , Björck Pia , Duvert Valérie , van Kooten Cees , Saeland Sem , Banchereau Jacques
Published: July 2002

Human CD34+ multilineage progenitor cells (CD34HPC) from cord blood and bone marrow express CD40, a member of the tumor necrosis factor–receptor family present on various hematopoietic and nonhematopoietic cells. As hyper-IgM patients with mutated CD40 ligand (CD40L) exhibit neutropenia, no B cell memory, and altered T cell functions leading to severe infections, we investigated the potential role of CD40 on CD34HPC development. CD40activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells. DC generated via the CD40 pathway displayed strong major histocompatibility complex class II DR but lacked detectable CD1a and CD40 expression. These features were shared by a dendritic population identified in situ in tonsillar T cell areas. Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.
Inhibition of T Cell and Promotion of Natural Killer Cell Development by the Dominant Negative Helix Loop Helix Factor Id3
Authors: Heemskerk Mirjam H.M. , Blom Bianca , Nolan Garry , Stegmann Alexander P.A. , Bakker Arjen Q. , Weijer Kees , Res Pieter C.M. , Spits Hergen
Published: July 2002

Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.
Thalidomide Costimulates Primary Human T Lymphocytes, Preferentially Inducing Proliferation, Cytokine Production, and Cytotoxic Responses in the CD8+ Subset
Authors: Haslett Patrick A.J. , Corral Laura G. , Albert Matthew , Kaplan Gilla
Published: July 2002

The efficacy of thalidomide (α-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor α. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2–mediated T cell proliferation and interferon γ production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset.
Subsets of Transgenic T Cells That Recognize CD1 Induce or Prevent Murine Lupus: Role of Cytokines
Authors: Zeng Defu , Dick Michael , Cheng Lirong , Amano Masahiko , Dejbakhsh-Jones Sussan , Huie Philip , Sibley Richard , Strober Samuel
Published: July 2002

T cells with T cell receptor (TCR) transgenes that recognized CD1 on syngeneic B cells stimulated B cells to secrete immunoglobulins in vitro. The CD4+, CD8+, or CD4−CD8− T cells from the spleen of the TCR transgenic BALB/c donors induced lupus with anti–double stranded DNA antibodies, proteinuria, and immune complex glomerulonephritis in irradiated BALB/c nude mice reconstituted with nude bone marrow. Injection of purified CD4−CD8− T cells from the marrow of transgenic donors prevented the induction of lupus by the transgenic T cells. Transgenic T cells that induced lupus secreted large amounts of interferon (IFN)-γ and little interleukin (IL)-4, and those that prevented lupus secreted large amounts of IL-4 and little IFN-γ or IL-10.
Constant Mean Viral Copy Number per Infected Cell in Tissues Regardless of High, Low, or Undetectable Plasma HIV RNA
Authors: Hockett Richard D. , Michael Kilby J. , Derdeyn Cynthia A. , Saag Michael S. , Sillers Michael , Squires Kathleen , Chiz Scott , Nowak Martin A. , Shaw George M. , Bucy R. Pat
Published: July 2002

Quantitative analysis of the relationship between virus expression and disease outcome has been critical for understanding HIV-1 pathogenesis. Yet the amount of viral RNA contained within an HIV-expressing cell and the relationship between the number of virus-producing cells and plasma virus load has not been established or reflected in models of viral dynamics. We report here a novel strategy for the coordinated analysis of virus expression in lymph node specimens. The results obtained for patients with a broad range of plasma viral loads before and after antiretroviral therapy reveal a constant mean viral (v)RNA copy number (3.6 log10 copies) per infected cell, regardless of plasma virus load or treatment status. In addition, there was a significant but nonlinear direct correlation between the frequency of vRNA+ lymph node cells and plasma vRNA. As predicted from this relationship, residual cells expressing this same mean copy number are detectable (frequency <2/106 cells) in tissues of treated patients who have plasma vRNA levels below the current detectable threshold (<50 copies/ml). These data suggest that fully replication-active cells are responsible for sustaining viremia after initiation of potent antiretroviral therapy and that plasma virus titers correlate, albeit in a nonlinear fashion, with the number of virus-expressing cells in lymphoid tissue.
Cells Capable of Bone Production Engraft from Whole Bone Marrow Transplants in Nonablated Mice
Authors: Nilsson Susan K. , Dooner Mark S. , Weier Heinz-Ulrich , Frenkel Baruch , Lian Jane B. , Stein Gary S. , Quesenberry Peter J.
Published: July 2002

Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.
Presentation of Cytosolic Glycosylated Peptides by Human Class I Major Histocompatibility Complex Molecules in Vivo
Authors: Haurum John S. , Høier Ingelise Bjerring , Arsequell Gemma , Neisig Anne , Valencia Gregorio , Zeuthen Jesper , Neefjes Jacques , Elliott Tim
Published: July 2002

Antigens presented by class I major histocompatibility complex (MHC) molecules for recognition by cytotoxic T lymphocytes consist of 8–10-amino-acid-long cytosolic peptides. It is not known whether posttranslationally modified peptides are also presented by class I MHC molecules in vivo. Many different posttranslational modifications occur on cytoplasmic proteins, including a cytosolic O-β-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying the monosaccharide O-β-GlcNAc substitution on serine residues, we have shown that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide. Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-β-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-β-linked GlcNAc can be presented by class I MHC molecules to the immune system.
Antigen-Specific Signaling by a Soluble, Dimeric Peptide/Major Histocompatibility Complex Class II/Fc Chimera Leading to T Helper Cell Type 2 Differentiation
Authors: Casares Sofia , Zong Cong S. , Radu Dorel L. , Miller Alexander , Bona Constantin A. , Brumeanu Teodor-Doru
Published: July 2002

Interaction between a T cell receptor (TCR) and various ligands, i.e., anti-TCR antibodies, superantigens, peptides, or altered peptide ligands in the context of major histocompatibility complex (MHC) molecules can trigger different T helper cell (Th) effector functions. Herein, we studied the T cell response induced by a soluble, dimeric peptide/MHC class II chimera, namely hemagglutinin (HA)110-120/I-Edαβ/Fcγ2a (DEF). We have previously demonstrated that the soluble DEF molecule binds stably and specifically to HA110-120–specific TCRs expressed by a T cell hybridoma. Administration of DEF in vivo induced differentiation of resting and activated peptide-specific T cells toward a Th2 response, as indicated by the increase of interleukin (IL)-4, IL-10, and specific immunoglobulin (Ig)G1 antibodies and decrease of IL-2, specific IgG2a antibodies, and cytotoxic T lymphocyte activity. In contrast to HA110-120 peptide presented by the DEF molecule to T cells, the nominal synthetic peptide induced a predominant Th1 response, and the PR8 virus–derived HA110-120 peptides induced a mixed Th1/Th2 response. Independent of antigen processing, soluble DEF was almost 2 logs more potent in stimulating cognate T cells than the nominal peptide. Polarization of cognate T cells toward the Th2 response occurred upon interaction of soluble DEF with TCR and CD4 molecules followed by early activation of p56lck and ZAP-70 tyrosine kinases, and negative signaling of the signal transducer and activator of transcription (STAT)4 pathway of Th1 differentiation. DEF-like molecules may provide a new tool to study the mechanisms of signaling toward Th2 differentiation and may also provide a potential immunotherapeutic approach to modulate autoreactive T cells toward protective Th2 immune responses.
Epstein-Barr Virus–Infected Resting Memory B Cells, Not Proliferating Lymphoblasts, Accumulate in the Peripheral Blood of Immunosuppressed Patients
Authors: Babcock Gregory J. , Decker Lisa L. , Freeman Richard B. , Thorley-Lawson David A.
Published: July 2002

When Epstein-Barr virus (EBV) infects B cells in vitro, the result is a proliferating lymphoblast that expresses at least nine latent proteins. It is generally believed that these cells are rigorously controlled in vivo by cytotoxic T cells. Consistent with this, the latently infected cells in the peripheral blood of healthy carriers are not lymphoblasts. Rather, they are resting memory B cells that are probably not subject to direct immunosurveillance by cytotoxic T lymphocytes (CTLs). When patients become immunosuppressed, the viral load increases in the peripheral blood. The expansion of proliferating lymphoblasts due to the suppressed CTL response is believed to account for this increase and is considered to be a major risk factor for posttransplant lymphoproliferative disease (PTLD) and AIDS-associated B cell lymphoma. Here we show that there is an increase in the numbers of latently infected cells in the peripheral blood of immunosuppressed patients. However, the cells are not proliferating lymphoblasts. They are all latently infected, resting, memory B cells—the same population of infected cells found in the blood of healthy carriers. These results are discussed in the context of a model for EBV persistence that explains why PTLD is usually limited to the lymph nodes.
Human G Protein–Coupled Receptor Gpr-9-6/Cc Chemokine Receptor 9 Is Selectively Expressed on Intestinal Homing T Lymphocytes, Mucosal Lymphocytes, and Thymocytes and Is Required for Thymus-Expressed Chemokine–Mediated Chemotaxis
Authors: Zabel Brian A. , Agace William W. , Campbell James J. , Heath Heidi M. , Parent David , Roberts Arthur I. , Ebert Ellen C. , Kassam Nasim , Qin Shixin , Zovko Maria , LaRosa Gregory J. , Yang Li-Li , Soler Dulce , Butcher Eugene C. , Ponath Paul D. , Parker Christina M. , Andrew David P.
Published: July 2002

TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein–coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti–GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory α4β7high intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen–positive (CLA+) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic α4β7−CLA− memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti–GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.
T Helper 1 Cells and Interferon γ Regulate Allergic Airway Inflammation and Mucus Production
Authors: Cohn Lauren , Homer Robert J. , Niu Naiqian , Bottomly Kim
Published: July 2002

CD4 T helper (Th) type 1 and Th2 cells have been identified in the airways of asthmatic patients. Th2 cells are believed to contribute to pathogenesis of the disease, but the role of Th1 cells is not well defined. In a mouse model, we previously reported that transferred T cell receptor–transgenic Th2 cells activated in the respiratory tract led to airway inflammation with many of the pathologic features of asthma, including airway eosinophilia and mucus production. Th1 cells caused inflammation with none of the pathology associated with asthma. In this report, we investigate the role of Th1 cells in regulating airway inflammation. When Th1 and Th2 cells are transferred together into recipient mice, there is a marked reduction in airway eosinophilia and mucus staining. To address the precise role of Th1 cells, we asked (i), Are Th2-induced responses inhibited by interferon (IFN)-γ? and (ii) Can Th1 cells induce eosinophilia and mucus in the absence of IFN-γ? In IFN-γ receptor−/− recipient mice exposed to inhaled antigen, the inhibitory effects of Th1 cells on both airway eosinophilia and mucus production were abolished. In the absence of IFN-γ receptor signaling, Th1 cells induced mucus but not eosinophilia. Thus, we have identified new regulatory pathways for mucus production; mucus can be induced by Th2 and non-Th2 inflammatory responses in the lung, both of which are inhibited by IFN-γ. The blockade of eosinophilia and mucus production by IFN-γ likely occurs through different inhibitory pathways that are activated downstream of Th2 cytokine secretion and require IFN-γ signaling in tissue of recipient mice.
P-Selectin or Intercellular Adhesion Molecule (Icam)-1 Deficiency Substantially Protects against Atherosclerosis in Apolipoprotein E–Deficient Mice
Authors: Collins Robert G. , Velji Rizwan , Guevara Natalia V. , Hicks M. John , Chan Lawrence , Beaudet Arthur L.
Published: July 2002

The expression of leukocyte and endothelial cell adhesion molecules (CAMs) is essential for the emigration of leukocytes during an inflammatory response. The importance of the inflammatory response in the development of atherosclerosis is indicated by the increased expression of adhesion molecules, proinflammatory cytokines, and growth factors in lesions and lesion-prone areas and by protection in mice deficient in various aspects of the inflammatory response. We have quantitated the effect of deficiency for intercellular adhesion molecule (ICAM)-1, P-selectin, or E-selectin on atherosclerotic lesion formation at 20 wk of age in apolipoprotein (apo) E−/− (deficient) mice fed a normal chow diet. All mice were apo E−/− and CAM+/+ or CAM−/− littermates, and no differences were found in body weight or cholesterol levels among the various genotypes during the study. ICAM-1−/− mice had significantly less lesion area than their ICAM-1+/+ littermates: 4.08 ± 0.70 mm2 for −/− males vs. 5.87 ± 0.66 mm2 for +/+ males, and 3.95 ± 0.65 mm2 for −/− females vs. 5.59 ± 1.131 mm2 for +/+ females, combined P < 0.0001. An even greater reduction in lesion area was observed in P-selectin−/− mice: 3.06 ± 1.04 mm2 for −/− males vs. 5.09 ± 1.22 mm2 for +/+ males, and 2.85 ± 1.26 mm2 for −/− females compared with 5.60 ± 1.19 mm2 for +/+ females, combined P < 0.001. The reduction in lesion area for the E-selectin null mice, although less than that seen for ICAM-1 or P-selectin, was still significant (4.54 ± 2.14 mm2 for −/− males vs. 5.92 ± 0.63 mm2 for +/+ males, and 4.38 ± 0.85 mm2 for −/− females compared with 5.94 ± 1.44 mm2 for +/+ females, combined P < 0.01). These results, coupled with the closely controlled genetics of this study, indicate that reductions in the expression of P-selectin, ICAM-1, or E-selectin provide direct protection from atherosclerotic lesion formation in this model.
B-1 and B-2 Cell–Derived Immunoglobulin M Antibodies Are Nonredundant Components of the Protective Response to Influenza Virus Infection
Authors: Baumgarth Nicole , Herman Ometa C. , Jager Gina C. , Brown Lorena E. , Herzenberg Leonore A. , Chen Jianzhu
Published: July 2002

We have studied the role of secreted immunoglobulin (Ig)M in protection from infection with influenza virus and delineated the relative contributions of B-1 versus B-2 cell–derived IgM in this process. Mice deficient in secreted IgM but capable of expressing surface IgM and secreting other Ig classes show significantly reduced virus clearance and survival rates compared with wild-type controls. Irradiation chimeras in which only either B-1 or B-2 cells lack the ability to secrete IgM show mortality rates similar to those of mice in which neither B-1 nor B-2 cells secrete IgM. Dependence on both sources of IgM for survival is partially explained by findings in allotype chimeras that broadly cross-reactive B-1 cell–derived natural IgM is present before infection, whereas virus strain–specific, B-2 cell–derived IgM appears only after infection. Furthermore, lack of IgM secreted from one or both sources significantly impairs the antiviral IgG response. Reconstitution of chimeras lacking B-1 cell–derived IgM only with IgM-containing serum from noninfected mice improved both survival rates and serum levels of virus-specific IgG. Thus, virus-induced IgM must be secreted in the presence of natural IgM for efficient induction of specific IgG and for immune protection, identifying B-1 and B-2 cell–derived IgM antibodies as nonredundant components of the antiviral response.
Natural Killer T Cell Activation Inhibits Hepatitis B Virus Replication in Vivo
Authors: Kakimi Kazuhiro , Guidotti Luca G. , Koezuka Yasuhiko , Chisari Francis V.
Published: July 2002

We have previously reported that hepatitis B virus (HBV)–specific CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-γ when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of α-galactosylceramide (α-GalCer), a glycolipid antigen presented to Vα14+NK1.1+ T cells by the nonclassical major histocompatibility complex class I–like molecule CD1d. Within 24 h of α-GalCer injection, IFN-γ and IFN-α/β were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4+ and CD8+ T cells from the mice did not diminish the ability of α-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of α-GalCer was inhibited in mice that are genetically deficient for either IFN-γ or the IFN-α/β receptor, indicating that most of the antiviral activity of α-GalCer is mediated by these cytokines. Based on these results, we conclude that α-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.
Interleukin 12 P40 Production by Barrier Epithelial Cells during Airway Inflammation
Authors: Walter Michael J. , Kajiwara Naohiro , Karanja Peter , Castro Mario , Holtzman Michael J.
Published: July 2002

Human airway epithelial cells appear specially programmed for expression of immune response genes implicated in immunity and inflammation. To better determine how this epithelial system operates in vivo, we analyzed its behavior in mouse models that allow for in vitro versus in vivo comparison and genetic modification. Initial comparisons indicated that tumor necrosis factor α induction of epithelial intercellular adhesion molecule 1 required sequential induction of interleukin (IL)-12 (p70) and interferon γ, and unexpectedly localized IL-12 production to airway epithelial cells. Epithelial IL-12 was also inducible during paramyxoviral bronchitis, but in this case, initial IL-12 p70 expression was followed by 75-fold greater expression of IL-12 p40 (as monomer and homodimer). Induction of IL-12 p40 was even further increased in IL-12 p35-deficient mice, and in this case, was associated with increased mortality and epithelial macrophage accumulation. The results placed epithelial cell overgeneration of IL-12 p40 as a key intermediate for virus-inducible inflammation and a candidate for epithelial immune response genes that are abnormally programmed in inflammatory disease. This possibility was further supported when we observed IL-12 p40 overexpression selectively in airway epithelial cells in subjects with asthma and concomitant increases in airway levels of IL-12 p40 (as homodimer) and airway macrophages. Taken together, these results suggest a novel role for epithelial-derived IL-12 p40 in modifying the level of airway inflammation during mucosal defense and disease.
Epstein-Barr Virus Latent Membrane Protein 2a (Lmp2a) Employs the Slp-65 Signaling Module
Authors: Engels Niklas , Merchant Mark , Pappu Rajita , Chan Andrew C. , Longnecker Richard , Wienands Jürgen
Published: July 2002

In latently infected B lymphocytes, the Epstein-Barr virus (EBV) suppresses signal transduction from the antigen receptor through expression of the integral latent membrane protein 2A (LMP2A). At the same time, LMP2A triggers B cell survival by a yet uncharacterized maintenance signal that is normally provided by the antigen receptor. The molecular mechanisms are unknown as LMP2A-regulated signaling cascades have not been described so far. Using a novel mouse model we have identified the intracellular adaptor protein Src homology 2 (SH2) domain–containing leukocyte protein (SLP)-65 as a critical downstream effector of LMP2A in vivo. Biochemical analysis of the underlying signaling pathways revealed that EBV infection causes constitutive tyrosine phosphorylation of one of the two SLP-65 isoforms and complex formation between SLP-65 and the protooncoprotein CrkL (CT10 regulator of kinase like). This leads to antigen receptor-independent phosphorylation of Cbl (Casitas B lineage lymphoma) and C3G. In contrast, phospholipase C-γ2 (PLC-γ2) activation is completely blocked. Our data show that in order to establish a latent EBV infection, LMP2A selectively activates or represses SLP-65–regulated signaling pathways.
Notch 1–Deficient Common Lymphoid Precursors Adopt a B Cell Fate in the Thymus
Authors: Wilson Anne , MacDonald H. Robson , Radtke Freddy
Published: July 2002

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells.

In this report we show that Notch 1–deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1−/− BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1−/− precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1–deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.
Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum
Authors: Domínguez Mercedes , Moreno Inmaculada , López-Trascasa Margarita , Toraño Alfredo
Published: July 2002

In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement.
Targeted Disruption of LIGHT Causes Defects in Costimulatory T Cell Activation and Reveals Cooperation with Lymphotoxin β in Mesenteric Lymph Node Genesis
Authors: Scheu Stefanie , Alferink Judith , Pötzel Tobias , Barchet Winfried , Kalinke Ulrich , Pfeffer Klaus
Published: July 2002

The recently described tumor necrosis factor (TNF) family member LIGHT (herpes virus entry mediator [HVEM]-L/TNFSF14), a ligand for the lymphotoxin (LT)β receptor, HVEM, and DcR3, was inactivated in the mouse. In contrast to mice deficient in any other member of the LT core family, LIGHT−/− mice develop intact lymphoid organs. Interestingly, a lower percentage of LIGHT−/−LTβ−/− animals contain mesenteric lymph nodes as compared with LTβ−/− mice, whereas the splenic microarchitecture of LIGHT−/−LTβ−/− and LTβ−/− mice shows a comparable state of disruption. This suggests the existance of an additional undiscovered ligand for the LTβ receptor (LTβR) or a weak LTα3–LTβR interaction in vivo involved in the formation of secondary lymphoid organs. LIGHT acts synergistically with CD28 in skin allograft rejection in vivo. The underlying mechanism was identified in in vitro allogeneic MLR studies, showing a reduced cytotoxic T lymphocyte activity and cytokine production. Detailed analyses revealed that proliferative responses specifically of CD8+ T cells are impaired and interleukin 2 secretion of CD4+ T cells is defective in the absence of LIGHT. Furthermore, a reduced 3[H]-thymidine incorporation after T cell receptor stimulation was observed. This for the first time provides in vivo evidence for a cooperative role for LIGHT and LTβ in lymphoid organogenesis and indicates important costimulatory functions for LIGHT in T cell activation.