bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-17 Promotes Bone Erosion in Murine Collagen-Induced Arthritis Through Loss of the Receptor Activator of NF-κB Ligand/Osteoprotegerin Balance
Authors: Lubberts Erik , van den Bersselaar Liduine , Oppers-Walgreen Birgitte , Schwarzenberger Paul , Coenen-de Roo Christina J. J. , Kolls Jay K. , Joosten Leo A. B. , van den Berg Wim B.
Published: April 2014

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-κB, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.
Anti-idiotype × Anti-LFA-1 Bispecific Antibodies Inhibit Metastasis of B Cell Lymphoma
Authors: Cohen Sharon , Haimovich Joseph , Hollander Nurit
Published: April 2014

Abs to adhesion molecules can block tumor metastasis. However, they may also block the function of normal cells. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. We therefore produced a bispecific Ab with specificity to the adhesion molecule LFA-1 and to the Id of the murine B cell lymphoma 38C-13. Here we demonstrate that this Ab blocked liver metastasis in mice carrying primary s.c. tumors and partially inhibited lymph node metastasis. Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis resulted at least in part from reduced homing to these organs. In contrast to anti-LFA-1 monospecific Abs, the anti-Id × anti-LFA-1 bispecific Ab did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave much of the immune system intact.
High Expression of Fas Ligand by Synovial Fluid-Derived γδ T Cells in Lyme Arthritis
Authors: Roessner Karen , Wolfe Julie , Shi Cuixia , Sigal Leonard H. , Huber Sally , Budd Ralph C.
Published: April 2014

γδ T cells accumulate at epithelial barriers and at sites of inflammation in various infectious and autoimmune diseases, yet little is understood about the function of tissue-infiltrating γδ T cells. We observe that γδ T cells of the Vδ1 subset accumulate in synovial fluid of human Lyme arthritis and are intensely cytolytic toward a wide array of target cells. Particularly striking is that the cytolytic activity is highly prolonged, lasting for at least 3 wk after stimulation of the γδ T cells with Borrelia burgdorferi. Cytolysis is largely Fas dependent and results from very high and prolonged expression of surface Fas ligand, which is transcriptionally regulated. This also manifests in a substantial level of self-induced apoptosis of the γδ T cells. In this capacity, certain γδ T cell subsets may serve as cytolytic sentinels at sites of inflammation, and perhaps at epithelial barriers.
Concomitant Induction of CD4+ and CD8+ T Cell Responses in Volunteers Immunized with Salmonella enterica Serovar Typhi Strain CVD 908-htrA
Authors: Salerno-Gonçalves Rosângela , Wyant Timothy L. , Pasetti Marcela F. , Fernandez-Viña Marcelo , Tacket Carol O. , Levine Myron M. , Sztein Marcelo B.
Published: April 2014

Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a ΔaroC ΔaroD ΔhtrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4+ and CD8+ S. Typhi immune responses. Potent CTL responses and IFN-γ secretion by CD8+ T cells were detected following immunization with CVD 908-htrA in high (4.5 × 108 CFU) and low (5 × 107 CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN-γ spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 ± 41 SFC/106 PBMC and 233 ± 87 SFC/106 PBMC, in the high and low dose groups, respectively. Strong CD4+ T cell responses were also observed. Increases in the IFN-γ production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN-γ SFC to stimulation with S. Typhi-infected cells and IFN-γ released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN-γ production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.
Fine-Scale Mapping at IGAD1 and Genome-Wide Genetic Linkage Analysis Implicate HLA-DQ/DR as a Major Susceptibility Locus in Selective IgA Deficiency and Common Variable Immunodeficiency
Authors: Kralovicova Jana , Hammarström Lennart , Plebani Alessandro , Webster A. David B. , Vorechovsky Igor
Published: April 2014

Selective IgA deficiency (IgAD) and common variable immunodeficiency (CVID) are the most common primary immunodeficiencies in humans. A high degree of familial clustering, marked differences in the population prevalence among ethnic groups, association of IgAD and CVID in families, and a predominant inheritance pattern in multiple-case pedigrees have suggested a strong, shared genetic predisposition. Previous genetic linkage, case-control, and family-based association studies mapped an IgAD/CVID susceptibility locus, designated IGAD1, to the MHC, but its precise location within the MHC has been controversial. We have analyzed a sample of 101 multiple- and 110 single-case families using 36 markers at the IGAD1 candidate region and mapped homozygous stretches across the MHC shared by affected family members. Haplotype analysis, linkage disequilibrium, and homozygosity mapping indicated that HLA-DQ/DR is the major IGAD1 locus, strongly suggesting the autoimmune pathogenesis of IgAD/CVID. This is supported by the highest excess of allelic sharing at 6p in the genome-wide linkage analysis of 101 IgAD/CVID families using 383 marker loci, by previously reported restrictions of the T cell repertoires in CVID, the presence of autoantibodies, impaired T cell activation, and a dysregulation of a number of genes in the targeted immune system. IgAD/CVID may thus provide a useful model for the study of pathogenesis and novel therapeutic strategies in autoimmune diseases.
Cutting Edge: Toll-Like Receptor 9 Expression Is Not Required for CpG DNA-Aided Cross-Presentation of DNA-Conjugated Antigens but Essential for Cross-Priming of CD8 T Cells
Authors: Heit Antje , Maurer Tobias , Hochrein Hubertus , Bauer Stefan , Huster Katharina M. , Busch Dirk H. , Wagner Hermann
Published: April 2014

Covalent linkage of immunostimulatory CpG DNA to OVA results in CpG DNA-aided cross-presentation of OVA by dendritic cells (DCs). In vivo, cross-presentation is conditional for cross-priming of OVA-specific CD8 T cells. In this study, we investigated the involvement of the CpG DNA receptor Toll-like receptor (TLR)9 in CpG DNA-aided cross-presentation and cross-priming. Although CpG DNA-aided cross-presentation is not altered in TLR9-deficient cells, TLR9 is required for maturation of APC allowing cross-priming, as resulting in CTL function. These findings imply that TLR9 does not trigger endocytosis of CpG-OVA conjugates, but activates DCs downstream of endocytosis.
Cutting Edge: Mycobacterium tuberculosis Blocks Ca2+ Signaling and Phagosome Maturation in Human Macrophages Via Specific Inhibition of Sphingosine Kinase
Authors: Malik Zulfiqar A. , Thompson Christopher R. , Hashimi Samad , Porter Brandon , Iyer Shankar S. , Kusner David J.
Published: April 2014

One-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca2+ that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca2+-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.
IL-1 Receptor-Associated Kinase 1 Regulates Susceptibility to Organ-Specific Autoimmunity
Authors: Deng Caishu , Radu Caius , Diab Asim , Tsen May F. , Hussain Rehana , Cowdery John S. , Racke Michael K. , Thomas James A.
Published: April 2014

Infections often precede the development of autoimmunity. Correlation between infection with a specific pathogen and a particular autoimmune disease ranges from moderately strong to quite weak. This lack of correspondence suggests that autoimmunity may result from microbial activation of a generic, as opposed to pathogen-specific host-defense response. The Toll-like receptors, essential to host recognition of microbial invasion, signal through a common, highly conserved pathway, activate innate immunity, and control adaptive immune responses. To determine the influence of Toll/IL-1 signaling on the development of autoimmunity, the responses of wild-type (WT) mice and IL-1R-associated kinase 1 (IRAK1)-deficient mice to induction of experimental autoimmune encephalomyelitis were compared. C57BL/6 and B6.IRAK1-deficient mice were immunized with MOG 35–55/CFA or MOG 35–55/CpG DNA/IFA. WT animals developed severe disease, whereas IRAK1-deficient mice were resistant to experimental autoimmune encephalomyelitis, exhibiting little or no CNS inflammation. IRAK1-deficient T cells also displayed impaired Th1 development, particularly during disease induction, despite normal TCR signaling. These results suggest that IRAK1 and the Toll/IL-1 pathway play an essential role in T cell priming, and demonstrate one means through which innate immunity can control subsequent development of autoimmunity. These findings may also help explain the association between antecedent infection and the development or exacerbations of some autoimmune diseases.
Low Activation Threshold As a Mechanism for Ligand-Independent Signaling in Pre-T Cells
Authors: Haks Mariëlle C. , Belkowski Stanley M. , Ciofani Maria , Rhodes Michele , Lefebvre Juliette M. , Trop Sebastién , Hugo Patrice , Zúñiga-Pflücker Juan Carlos , Wiest David L.
Published: April 2014

Pre-TCR complexes are thought to signal in a ligand-independent manner because they are constitutively targeted to lipid rafts. We report that ligand-independent signaling is not a unique capability of the pre-TCR complex. Indeed, the TCRα subunit restores development of pTα-deficient thymocytes to the CD4+CD8+ stage even in the absence of conventional MHC class I and class II ligands. Moreover, we found that pre-TCR and αβTCR complexes exhibit no appreciable difference in their association with lipid rafts, suggesting that ligand-independence is a function of the CD4−CD8− (DN) thymocytes in which pre-TCR signaling occurs. In agreement, we found that only CD44−CD25+ DN thymocytes (DN3) enabled activation of extracellular signal-regulated kinases by the pre-TCR complex. DN thymocytes also exhibited a lower signaling threshold relative to CD4+CD8+ thymocytes, which was associated with both the markedly elevated lipid raft content of their plasma membranes and more robust capacitative Ca2+ entry. Taken together these data suggest that cell-autonomous, ligand-independent signaling is primarily a property of the thymocytes in which pre-TCR signaling occurs.
T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation
Authors: Sanjuán Miguel A. , Pradet-Balade Bérengère , Jones David R. , Martínez-A Carlos , Stone James C. , Garcia-Sanz Jose A. , Mérida Isabel
Published: April 2014

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGKα, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGKα, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGKα to the membrane fraction. At early time points, DGKα translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGKα activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGKα mutants. We show that membrane localization of DGKα is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGKα, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.
A Pool of Central Memory-Like CD4 T Cells Contains Effector Memory Precursors
Authors: Blander J. Magarian , Sant’Angelo Derek B. , Metz Daniela , Kim Sang-Won , Flavell Richard A. , Bottomly Kim , Janeway Charles A.
Published: April 2014

The L51S mutation in the D10.G4.1 TCR α-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the β-chain of I-Ak, and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44highCD62LnegCD45RBneg CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2Rα only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.
The Role of the CD134-CD134 Ligand Costimulatory Pathway in Alloimmune Responses In Vivo
Authors: Yuan Xueli , Salama Alan D. , Dong Victor , Schmitt Isabela , Najafian Nader , Chandraker Anil , Akiba Hisaya , Yagita Hideo , Sayegh Mohamed H.
Published: April 2014

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-γ-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 ± 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 ± 2.8 days) as did CTLA4Ig (MST: 21.5 ± 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 ± 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.
Secretion of IL-2 and IFN-γ, But Not IL-4, by Antigen-Specific T Cells Requires Extracellular ATP
Authors: Langston Heather P. , Ke Yong , Gewirtz Andrew T. , Dombrowski Kenneth E. , Kapp Judith A.
Published: April 2014

Extracellular ATP and other nucleotides transmit signals to cells via surface-associated molecules whose binding sites face the extracellular milieu. Ecto-nucleoside triphosphate diphosphohydrolase is such an ATP-binding enzyme that is expressed by activated lymphocytes. We have previously shown that nonhydrolyzable ATP analogs block the lytic activity of NK cells and CD8+ T cells as well as their E-NTPDase activity. These results suggest that the hydrolysis of ATP may play a role in lymphocyte function. Here we report that E-NTPDase activity is up-regulated within 15 min of T cell stimulation and that reversible and irreversible enzyme inhibitors profoundly reduce secretion of IL-2 and IFN-γ, but not IL-4. TNF-α, IL-10, and IL-5 production showed intermediate sensitivity to these ATP analogs. Depletion of extracellular ATP also inhibited secretion of IFN-γ, but not IL-4, supporting the interpretation that extracellular ATP is required for secretion of some, but not all, cytokines. E-NTPDase antagonists reduced transcription of IL-2 mRNA and inhibited TCR-mediated intracellular calcium flux. These results suggest that extracellular ATP plays an essential role in the TCR-mediated signal transduction cascade for expression of certain cytokine genes.
Blockade of CD40-Mediated Signaling Is Sufficient for Inducing Islet But Not Skin Transplantation Tolerance
Authors: Phillips Nancy E. , Markees Thomas G. , Mordes John P. , Greiner Dale L. , Rossini Aldo A.
Published: April 2014

Treatment of mice with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb to block CD40-mediated signaling uniformly induces donor-specific transplantation tolerance. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. The nature of the cellular mechanisms involved and the basis for the difference in survival of islet vs skin allografts are not known. In this study, we used CD40 knockout mice to investigate the role of CD40-mediated signaling in each component of the tolerance induction protocol: the DST, the graft, and the host. When CD40-mediated signaling was eliminated in only the DST or the graft, islet allografts were rapidly rejected. However, when CD40 signaling was eliminated in the host, ∼40% of the islet allografts survived. When CD40 signaling was eliminated in the DST, the graft, and the host, islet grafts survived long term (>84 days), whereas skin allografts were rapidly rejected (∼13 days). We conclude that transplantation tolerance induction in mice treated with DST and anti-CD154 mAb requires blockade of CD40-mediated signaling in the DST, the graft, and the host. Blockade of CD40-mediated signaling is necessary and sufficient for inducing islet allograft tolerance and is necessary but not sufficient for long-term skin allograft survival. We speculate that a requirement for regulatory CD4+ T cells in skin allograft recipients could account for this differential response to tolerance induction.
The Roles of Toll-Like Receptor 9, MyD88, and DNA-Dependent Protein Kinase Catalytic Subunit in the Effects of Two Distinct CpG DNAs on Dendritic Cell Subsets
Authors: Hemmi Hiroaki , Kaisho Tsuneyasu , Takeda Kiyoshi , Akira Shizuo
Published: April 2014

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNAs) can function as powerful immune adjuvants by activating APC. Compared with conventional phosphorothioate-backbone CpG DNAs, another type of CpG DNAs, called an A or D type (A/D-type), possesses higher ability to induce IFN-α production. Conventional CpG DNAs can exert their activity through Toll-like receptor 9 (TLR9) signaling, which depends on a cytoplasmic adapter, MyD88. However, it remains unknown how A/D-type CpG DNAs exhibit their immunostimulatory function. In this study we have investigated murine dendritic cell (DC) responses to these two distinct CpG DNAs. Not only splenic, but also in vitro bone marrow-derived, DCs could produce larger amounts of IFN-α in response to A/D-type CpG DNAs compared with conventional CpG DNAs. This IFN-α production was mainly due to the B220+ DC subset. On the other hand, the B220− DC subset responded similarly to both CpG DNAs in terms of costimulatory molecule up-regulation and IL-12 induction. IFN-α, but not IL-12, induction was dependent on type I IFN. However, all activities of both CpG DNAs were abolished in TLR9- and MyD88-, but were retained in DNA-PKcs-deficient DCs. This study demonstrates that the TLR9-MyD88 signaling pathway is essential for all DC responses to both types of CpG DNAs.
CD40-Mediated Transcriptional Regulation of the IL-6 Gene in B Lymphocytes: Involvement of NF-κB, AP-1, and C/EBP
Authors: Baccam Mekhine , Woo So-Youn , Vinson Charles , Bishop Gail A.
Published: April 2014

Engagement of CD40 by its ligand CD154 induces IL-6 production by B lymphocytes. We previously reported that this IL-6 production is dependent upon binding of the adapter protein TNF receptor-associated factor 6 to the cytoplasmic domain of CD40, while binding of TNF receptor-associated factors 2 and 3 is dispensable, as is the activation-induced nuclear translocation of NF-κB. The present study was designed to characterize CD40-mediated transcriptional control of the IL-6 gene in B cells. CD40 engagement on B lymphocytes activated the IL-6 promoter, and mutations in the putative binding sites for AP-1 and C/EBP transcription factors reduced this activation. Interestingly, a mutation in the putative NF-κB binding site completely abrogated the basal promoter activity, thus also rendering the promoter unresponsive to CD40 stimulation, suggesting that this site is required for binding of NF-κB constitutively present in the nucleus of mature B cells. The expression of dominant negative Fos or C/EBPα proteins, which prevent binding of AP-1 or C/EBP complexes to DNA, also reduced CD40-mediated IL-6 gene expression. Furthermore, CD40 stimulation led to phosphorylation of c-Jun on its activation domain, implicating CD40-mediated Jun kinase activation in the transcriptional regulation of IL-6 production.
Human IgG2 Can Form Covalent Dimers
Authors: Yoo Esther M. , Wims Letitia A. , Chan Lisa A. , Morrison Sherie L.
Published: April 2014

Unlike IgA and IgM, IgG has not yet been shown to form covalent polymers. However in the presence of specific Ag, murine IgG3 has been shown to polymerize through noncovalent interactions. In contrast to the noncovalent oligomers found with murine IgG3, we have detected covalent dimers in three different recombinant human IgG2 Abs produced in myeloma cells. Both IgG2,κ and IgG2,λ can form dimers. In addition, analysis of pooled human γ globulin and several normal sera revealed the presence of IgG2 dimers. The IgG2 dimers are in contrast to the noncovalent IgG dimers found in pooled sera of multiple donors resulting from idiotype/anti-idiotype (Id/anti-Id) interactions. Cyanogen bromide cleavage analysis suggests that one or more Cys residues in the γ2 hinge are involved in dimer assembly. The potential role of IgG2 dimers in immunity against carbohydrate Ags is discussed.
Perforin-Mediated Effector Function Within the Central Nervous System Requires IFN-γ-Mediated MHC Up-Regulation
Authors: Bergmann Cornelia C. , Parra Beatriz , Hinton David R. , Chandran Ramakrishna , Morrison Maureen , Stohlman Stephen A.
Published: April 2014

CD8+ T cells infiltrating the CNS control infection by the neurotropic JHM strain of mouse hepatitis virus. Differential susceptibility of infected cell types to clearance by perforin or IFN-γ uncovered distinct, nonredundant roles for these antiviral mechanisms. To separately evaluate each effector function specifically in the context of CD8+ T cells, pathogenesis was analyzed in mice deficient in both perforin and IFN-γ (PKO/GKO) or selectively reconstituted for each function by transfer of CD8+ T cells. Untreated PKO/GKO mice were unable to control the infection and died of lethal encephalomyelitis within 16 days, despite substantially higher CD8+ T cell accumulation in the CNS compared with controls. Uncontrolled infection was associated with limited MHC class I up-regulation and an absence of class II expression on microglia, coinciding with decreased CD4+ T cells in CNS infiltrates. CD8+ T cells from perforin-deficient and wild-type donors reduced virus replication in PKO/GKO recipients. By contrast, IFN-γ-deficient donor CD8+ T cells did not affect virus replication. The inability of perforin-mediated mechanisms to control virus in the absence of IFN-γ coincided with reduced class I expression. These data not only confirm direct antiviral activity of IFN-γ within the CNS but also demonstrate IFN-γ-dependent MHC surface expression to guarantee local T cell effector function in tissues inherently low in MHC expression. The data further imply that IFN-γ plays a crucial role in pathogenesis by regulating the balance between virus replication in oligodendrocytes, CD8+ T cell effector function, and demyelination.
Activation of STAT3 by IL-6 and IL-10 in Primary Human Macrophages Is Differentially Modulated by Suppressor of Cytokine Signaling 3
Authors: Niemand Claudia , Nimmesgern Ariane , Haan Serge , Fischer Patrick , Schaper Fred , Rossaint Rolf , Heinrich Peter C. , Müller-Newen Gerhard
Published: April 2014

On human macrophages IL-10 acts as a more potent anti-inflammatory cytokine than IL-6, although both cytokines signal mainly via activation of the transcription factor STAT3. In this study we compare IL-10 and IL-6 signaling in primary human macrophages derived from blood monocytes. Pretreatment of macrophages with PMA or the proinflammatory mediators LPS and TNF-α blocks IL-6-induced STAT3 activation, whereas IL-10-induced activation of STAT3 remains largely unaffected. Although LPS induces the feedback inhibitor suppressor of cytokine signaling 3 (SOCS3) in macrophages, inhibition of IL-6 signal transduction by LPS occurs rapidly and does not depend on gene transcription. We also found that pretreatment of macrophages with IL-10 inhibits subsequent STAT3 activation by IL-6, whereas IL-10-induced STAT3 activation is not affected by preincubation with IL-6. This cross-inhibition is dependent on active transcription and might therefore be explained by different sensitivities of IL-10 and IL-6 signaling toward the feedback inhibitor SOCS3, which is induced by both cytokines. In contrast to the IL-6 signal transducer gp130, which has been previously shown to recruit SOCS3 to one of its phosphotyrosine residues (Y759), peptide precipitation experiments suggest that SOCS3 does not interact with phosphorylated tyrosine motifs of the IL-10R. Taken together, different sensitivities of IL-10 and IL-6 signaling toward mechanisms that inhibit the Janus kinase/STAT pathway define an important mechanism that contributes to the different anti-inflammatory potencies of these two cytokines.
Monocytes Are Potent Facilitators of Alveolar Neutrophil Emigration During Lung Inflammation: Role of the CCL2-CCR2 Axis
Authors: Maus Ulrich A. , Waelsch Katharina , Kuziel William A. , Delbeck Tim , Mack Matthias , Blackwell Timothy S. , Christman John W. , Schlöndorff Detlef , Seeger Werner , Lohmeyer Jürgen
Published: April 2014

Coordinated neutrophil and monocyte recruitment is a characteristic feature of acute lung inflammatory responses. We investigated the role of monocyte chemotactic protein-1 (CCL2, JE) and the chemokine receptor CCR2 in regulating alveolar leukocyte traffic. Groups of wild-type (WT) mice, CCR2-deficient mice, lethally irradiated CCR2-deficient and WT mice that were reciprocally bone marrow transplanted (chimeric CCR2 deficient and WT, respectively), chimeric CCR2-deficient mice with an enriched CCR2+ alveolar macrophage population, and CCR2-deficient mice transfused with CCR2+ mononuclear cells were treated with intratracheal CCL2 and/or Escherichia coli endotoxin. Our data show that alveolar monocyte recruitment is strictly dependent on CCR2. LPS-induced neutrophil migration to the lungs is CCR2 independent. However, when CCR2-bearing blood monocytes are present, alveolar neutrophil accumulation is accelerated and drastically amplified. We suggest that this hitherto unrecognized cooperativity between monocytes and neutrophils contributes to the strong, coordinated leukocyte efflux in lung inflammation.
Secretory Phospholipases A2 Activate Selective Functions in Human Eosinophils
Authors: Triggiani Massimo , Granata Francescopaolo , Balestrieri Barbara , Petraroli Angelica , Scalia Giulia , Del Vecchio Luigi , Marone Gianni
Published: April 2014

Secretory phospholipases A2 (sPLA2s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA2. We have examined the effects of two structurally different sPLA2s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA2 induced a concentration-dependent release of β-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA2s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA2s examined induced the production of IL-5, the de novo synthesis of leukotriene C4 and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA2s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes’ effects. In addition, inactivation of sPLA2 enzymatic activity by bromophenacyl bromide did not influence the release of β-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA2s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA2s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.
Engineered Recombinant Peanut Protein and Heat-Killed Listeria monocytogenes Coadministration Protects Against Peanut-Induced Anaphylaxis in a Murine Model
Authors: Li Xiu-Min , Srivastava Kamal , Huleatt James W. , Bottomly Kim , Burks A. Wesley , Sampson Hugh A.
Published: April 2014

Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods. Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions. We investigated the effects of s.c. injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy. PN-allergic C3H/HeJ mice were treated s.c. with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1–3 plus HKLM). The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined. Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1–3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5. Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced. This protective effect was markedly more potent than in the mAra h 1–3 protein alone-treated group. HKLM alone did not have any protective effect. Reduced IL-5 and IL-13, and increased IFN-γ levels were observed only in splenocytes cultures from mAra h 1–3 plus HKLM-treated mice. These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA.
Neutrophils Promote Mononuclear Cell Infiltration During Viral-Induced Encephalitis
Authors: Zhou Jiehao , Stohlman Stephen A. , Hinton David R. , Marten Norman W.
Published: April 2014

Neutrophils are the first infiltrating cell population to appear within the CNS during infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). To determine whether neutrophils play a role in limiting acute JHMV infection, mice were depleted of neutrophils. Infection of neutropenic animals resulted in increased levels of virus replication and mortality compared with control mice. Furthermore, neutropenia resulted in significantly reduced mononuclear leukocyte infiltration possibly due to reduced loss of blood brain barrier integrity during acute JHMV infection. These data suggest that infiltrating neutrophils are crucial for limiting virus replication during acute JHMV infection, contribute to the loss of blood brain barrier integrity and play a role in shaping adaptive immunity within the CNS.
The Cytokine Macrophage Migration Inhibitory Factor Reduces Pro-Oxidative Stress-Induced Apoptosis
Authors: Nguyen Mai Tuyet , Lue Hongqi , Kleemann Robert , Thiele Michael , Tolle Gabriele , Finkelmeier Doris , Wagner Eva , Braun Andrea , Bernhagen Jürgen
Published: April 2014

The cytokine macrophage migration inhibitory factor (MIF) exhibits pro- and anti-inflammatory activities and regulates cell proliferation and survival. We investigated the effects of MIF on apoptosis. As MIF exhibits oxidoreductase activity and participates in regulating oxidative cell stress, we studied whether MIF could affect oxidative stress-induced apoptosis. We demonstrated that MIF exhibits antiapoptotic activity in various settings. MIF suppressed camptothecin-induced apoptosis in HeLa and Kym cells and HL-60 promyeloblasts. Both exogenous MIF and endogenous MIF, induced following overexpression through tetracycline (tet) gene induction, led to significant suppression of apoptosis. Apoptosis reduction by MIF was also observed in T cells. A role for MIF in redox stress-induced apoptosis was addressed by comparing the effects of rMIF with those of the oxidoreductase mutant C60SMIF. Endogenous overexpression of C60SMIF was similar to that of MIF, but C60SMIF did not suppress apoptosis. Exogenous rC60SMIF inhibited apoptosis. A role for MIF in oxidative stress-induced apoptosis was directly studied in HL-60 leukocytes and tet-regulated HeLa cells following thiol starvation or diamide treatment. MIF protected these cells from redox stress-induced apoptosis and enhanced cellular glutathione levels. As overexpressed C60SMIF did not protect tet-regulated HeLa cells from thiol starvation-induced apoptosis, it seems that the redox motif of MIF is important for this function. Finally, overexpression of MIF inhibited phosphorylation of endogenous c-Jun induced by thiol starvation, indicating that MIF-based suppression of apoptosis is mediated through modulation of c-Jun N-terminal kinase activity. Our findings show that MIF has potent antiapoptotic activities and suggest that MIF is a modulator of pro-oxidative stress-induced apoptosis.
Role of the Chemokine Stromal Cell-Derived Factor 1 in Autoantibody Production and Nephritis in Murine Lupus
Authors: Balabanian Karl , Couderc Jacques , Bouchet-Delbos Laurence , Amara Ali , Berrebi Dominique , Foussat Arnaud , Baleux Françoise , Portier Alain , Durand-Gasselin Ingrid , Coffman Robert L. , Galanaud Pierre , Peuchmaur Michel , Emilie Dominique
Published: April 2014

In normal mice, stromal cell-derived factor 1 (SDF-1/CXCL12) promotes the migration, proliferation, and survival of peritoneal B1a (PerB1a) lymphocytes. Because these cells express a self-reactive repertoire and are expanded in New Zealand Black/New Zealand White (NZB/W) mice, we tested their response to SDF-1 in such mice. PerB1a lymphocytes from NZB/W mice were exceedingly sensitive to SDF-1. This greater sensitivity was due to the NZB genetic background, it was not observed for other B lymphocyte subpopulations, and it was modulated by IL-10. SDF-1 was produced constitutively in the peritoneal cavity and in the spleen. It was also produced by podocytes in the glomeruli of NZB/W mice with nephritis. The administration of antagonists of either SDF-1 or IL-10 early in life prevented the development of autoantibodies, nephritis, and death in NZB/W mice. Initiation of anti-SDF-1 mAb treatment later in life, in mice with established nephritis, inhibited autoantibody production, abolished proteinuria and Ig deposition, and reversed morphological changes in the kidneys. This treatment also counteracted B1a lymphocyte expansion and T lymphocyte activation. Therefore, PerB1a lymphocytes are abnormally sensitive to the combined action of SDF-1 and IL-10 in NZB/W mice, and SDF-1 is key in the development of autoimmunity in this murine model of lupus.
Prothrombin Binds to the Surface of Apoptotic, But Not Viable, Cells and Serves as a Target of Lupus Anticoagulant Autoantibodies
Authors: D’Agnillo Paolo , Levine Jerrold S. , Subang Rebecca , Rauch Joyce
Published: April 2014

Anti-phospholipid Ab (aPL) are a heterogeneous group of autoantibodies directed against various combinations of phospholipids (PL) and PL-binding proteins. Lupus anticoagulant (LA) Ab, a subset of aPL, exhibit anticoagulant properties in vitro, but are procoagulant in vivo. Most LA Ab are specific for either β2-glycoprotein I (β2GPI) or prothrombin (PT), two PL-binding proteins. We have previously shown that β2GPI and β2GPI-dependent aPL bind specifically to apoptotic, but not viable, thymocytes. In this study, we demonstrate that PT, like β2GPI, binds selectively to the surface of apoptotic, but not viable, Jurkat cells. Furthermore, PT supports the binding of systemic lupus erythematosus-derived polyclonal and murine monoclonal LA Ab to apoptotic cells. Two LA mAb, which differed dramatically in their relative affinities for PT, were studied. Although one mAb (29J3-62) had a high affinity for PT alone, the other (29I4-24) showed minimal reactivity with PT alone and required PL for elevated binding. Monovalent fragments of 29I4-24 reacted with PL-bound PT with high affinity, suggesting that this mAb recognizes a PL-dependent epitope. Despite these differences, PT-dependent binding of both mAb to apoptotic cells was 30-fold greater than that to viable cells. Moreover, binding of PT to apoptotic cells was, itself, increased in the presence of bivalent, but not monovalent, forms of either mAb. In summary, our data demonstrate the following: 1) specific binding of PT to apoptotic cells, an effect enhanced by PT-dependent LA Ab; 2) heterogeneity of PT-dependent LA Ab; and 3) potential pathogenicity of Ab of either low or high affinity for PT.
Cytokine Polymorphisms Play a Role in Susceptibility to Ultraviolet B-Induced Modulation of Immune Responses after Hepatitis B Vaccination
Authors: Sleijffers Annemarie , Yucesoy Berran , Kashon Michael , Garssen Johan , De Gruijl Frank R. , Boland Greet J. , Van Hattum Jan , Luster Michael I. , Van Loveren Henk
Published: April 2014

UVB exposure can alter immune responses in experimental animals and humans. In an earlier human volunteer study, we demonstrated that hepatitis B-specific humoral and cellular immunity after vaccination on average were not significantly affected by UVB exposure. However, it is known that individuals differ in their susceptibility to UVB-induced immunomodulation, and it was hypothesized that polymorphisms in specific cytokines may play a role in this susceptibility. In this respect, we previously demonstrated that immune responses after hepatitis B vaccination are influenced by the minor allelic variant of IL-1β in the general population. For all volunteers, single nucleotide polymorphisms were determined for the following UV response-related cytokines: IL-1 receptor antagonist (+2018), IL-1α (+4845), IL-1β (+3953), TNF-α (−308), and TNF-α (−238). Exposure to UVB significantly suppressed Ab responses to hepatitis B in individuals with the minor variant for the IL-1β polymorphism. Increased minimal erythema dose values (just perceptible), which resulted in higher absolute UVB exposures, were observed in the same individuals. There were no associations observed between UVB-induced immunomodulation and the other cytokine polymorphisms examined. This study indicates that individual susceptibility to UVB radiation needs to be considered when studying the effects of UVB in humans.
CD4+ T Cells Pass Through an Effector Phase During the Process of In Vivo Tolerance Induction
Authors: Huang Ching-Tai , Huso David L. , Lu Zhenbing , Wang Tianhong , Zhou Gang , Kennedy Eugene P. , Drake Charles G. , Morgan David J. , Sherman Linda A. , Higgins Amy D. , Pardoll Drew M. , Adler Adam J.
Published: April 2014

An important process in the generation of tolerance to peripheral self-Ags is the induction of unresponsiveness in mature specific T cells. Although the end stage of this process, termed anergy, is well defined, the pathway by which naive T cells become anergic remains to be elucidated. Using an in vivo self-tolerance model, we demonstrate that CD4+ T cells pass through a significant effector stage on their way to an anergic state. This stage is characterized by production of effector cytokines, provision of help for CD8+ T cells, and induction of in vivo pathology within organs that express cognate Ag. These results suggest that the initial activation stage in T cell tolerance is similar to that seen in memory induction. They also suggest that autoimmune pathology can result during the natural process of tolerance induction rather than requiring that tolerance be broken.
T Cell Tolerance to a Neo-Self Antigen Expressed by Thymic Epithelial Cells: The Soluble Form Is More Effective Than the Membrane-Bound Form
Authors: Zhang Meifen , Vacchio Melanie S. , Vistica Barbara P. , Lesage Sylvie , Egwuagu Charles E. , Yu Cheng-Rong , Gelderman Monique P. , Kennedy Michael C. , Wawrousek Eric F. , Gery Igal
Published: April 2014

We have previously shown that transgenic (Tg) mice expressing either soluble or membrane-bound hen egg lysozyme (sHEL or mHEL, respectively) under control of the αA-crystallin promoter develop tolerance due to thymic expression of minuscule amounts of HEL. To further address the mechanisms by which this tolerance develops, we mated these two lines of Tg mice with the 3A9 line of HEL-specific TCR Tg mice, to produce double-Tg mice. Both lines of double-Tg mice showed deletion of HEL-specific T cells, demonstrated by reduction in numbers of these cells in the thymus and periphery, as well as by reduced proliferative response to HEL in vitro. In addition, the actual deletional process in thymi of the double-Tg mice was visualized in situ by the TUNEL assay and measured by binding of Annexin V. Notably, the apoptosis localized mainly in the thymic medulla, in line with the finding that the populations showing deletion and increased Annexin V binding consisted mainly of single- and double-positive thymocytes. Interestingly, the thymic deletional effect of sHEL was superior to that of mHEL in contrast to the opposite differential tolerogenic effects of these HEL forms on B cells specific to this Ag. Analysis of bone marrow chimeras indicates that both forms of HEL are produced by irradiation-resistant thymic stromal cells and the data suggest that sHEL is more effective in deleting 3A9 T cells due mainly to its higher accessibility to cross-presentation by dendritic APC.