bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum Amyloid P Component Binds to Fcγ Receptors and Opsonizes Particles for Phagocytosis
Authors: Bharadwaj Dwaipayan , Mold Carolyn , Markham Eric , Du Clos Terry W.
Published: April 2014

Serum amyloid P component (SAP) is a member of the pentraxin family of proteins. These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. We previously determined that the binding of CRP to leukocytes occurs through Fc receptors for IgG (FcγR). We now report that SAP also binds to FcγR and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to FcγRI, FcγRIIa, and FcγRIIIb expressed on transfected COS cells was detected using SAP-biotin and PE-streptavidin. Zymosan was used to test the functional consequences of SAP and CRP binding to FcγR. Both SAP and CRP bound to zymosan and enhanced its uptake by PMN. This enhanced phagocytosis was abrogated by treatment of PMN with wortmannin, a phosphatidylinositol-3 kinase inhibitor, or with piceatannol, a Syk inhibitor, consistent with uptake through FcγR. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove FcγRIIIb also decreased phagocytosis of SAP-opsonized zymosan, but not CRP-opsonized zymosan. These results suggest that SAP may function in host defense. In addition, as SAP binds to chromatin, a major immunogen in systemic lupus erythematosus, it may provide a clearance mechanism for this Ag through FcγR bearing cells.
Peroxisome Proliferator-Activated Receptor γ-Retinoid X Receptor Agonists Increase CD36-Dependent Phagocytosis ofPlasmodium falciparum-Parasitized Erythrocytes and Decrease Malaria-Induced TNF-α Secretion by Monocytes/Macrophages
Authors: Serghides Lena , Kain Kevin C.
Published: April 2014

Severe and fatal malaria is associated with the failure of host defenses to control parasite replication, excessive secretion of proinflammatory cytokines such as TNF-α, and sequestration of parasitized erythrocytes (PEs) in vital organs. The identification of CD36 as a major sequestration receptor has led to the assumption that it contributes to the pathophysiology of severe malaria and has prompted the development of antiadherence therapies to disrupt the CD36-PE interaction. This concept has been challenged by unexpected evidence that individuals deficient in CD36 are more susceptible to severe and cerebral malaria. In this study, we demonstrate that CD36 is the major receptor mediating nonopsonic phagocytosis of PEs by macrophages, a clearance mechanism of potential importance in nonimmune hosts at the greatest risk of severe malaria. CD36-mediated uptake of PEs occurs via a novel pathway that does not involve thrombospondin, the vitronectin receptor, or phosphatidylserine recognition. Furthermore, we show that proliferator-activated receptor γ-retinoid X receptor agonists induce an increase in CD36-mediated phagocytosis and a decrease in parasite-induced TNF-α secretion. Specific up-regulation of monocyte/macrophage CD36 may represent a novel therapeutic strategy to prevent or treat severe malaria.
A Retroviral-Derived Immunosuppressive Peptide Activates Mitogen-Activated Protein Kinases
Authors: Takahashi Akio , Day Noorbibi K. , Luangwedchakarn Voravich , Good Robert A. , Haraguchi Soichi
Published: April 2014

The highly conserved region within the retroviral transmembrane envelope proteins has been implicated in a number of retrovirus-associated mechanisms of immunosuppression. CKS-17, a synthetic peptide representing the prototypic sequence of the immunosuppressive domain, has been found to suppress numerous immune functions, disregulate cytokines, and elevate intracellular cAMP. In this report we show that using a human monocytic cell line THP-1, CKS-17 activates mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 and 2 (ERK1/2). Kinetic studies show that CKS-17 induces an acute increase of ERK1/2 activity followed by a rapid decrease and then a second sustained increase of ERK1/2. CKS-17 also activates MAP kinase/ERK kinase (MEK) with a similar induction pattern. Mutant THP-1 cells isolated in our laboratory, in which CKS-17 exclusively fails to activate cAMP, did not show the transient decrease of CKS-17-induced ERK1/2 phosphorylation. Pretreatment of THP-1 cells or mutant THP-1 cells with cAMP analog or forskolin followed by treatment with CKS-17 showed no activation of MEK or ERK1/2. These results indicate that CKS-17 activates the MEK/ERK cascade and that there is a cross-talk between CKS-17-mediated MEK/ERK cascade and cAMP in that the MEK/ERK cascade is negatively regulated by cAMP. These data present a novel molecular mechanism(s) by this highly conserved retroviral immunosuppressive component.
BALB/c Mice Bearing a Transgenic IL-12 Receptor β2 Gene Exhibit a Nonhealing Phenotype toLeishmania majorInfection Despite Intact IL-12 Signaling
Authors: Nishikomori Ryuta , Gurunathan Sanjay , Nishikomori Kanako , Strober Warren
Published: April 2014

In BALB/c mice infected with Leishmania major, early secretion of IL-4 leads to a Th2-type response and nonhealing. We explored the role of IL-4-induced down-regulation of the IL-12Rβ2 chain in the establishment of this Th2 response. First, we showed that the draining lymph nodes of resistant C57BL/6 mice infected with L. major were enriched in CD4+/IL-12Rβ2 chain+ cells producing IFN-γ. Next, we demonstrated that BALB/c background mice bearing an IL-12Rβ2-chain transgene manifested a nonhealing phenotype similar to wild-type littermates despite the persistence of their ability to undergo STAT4 activation. Finally, we found that such transgenic mice display more severe infection than wild-type littermates when treated with IL-12 7 days after infection, and under this condition, the mice display increased Leishmania Ag-induced IL-4 secretion. These studies indicate that although CD4+/IL-12Rβ2 chain+ T cells are important components of the Th1 response, maintenance of IL-12Rβ2 chain expression is not sufficient to change a Th2 response to a Th1 response in vivo and thus to allow BALB/c mice to heal L. major infection.
IκB Kinase Is Critical for TNF-α-Induced VCAM1 Gene Expression in Renal Tubular Epithelial Cells
Authors: Tu Zheng , Kelley Vicki Rubin , Collins Tucker , Lee Frank S.
Published: April 2014

The expression of VCAM1 is up-regulated in renal proximal tubular epithelial cells (TEC) in a variety of inflammatory renal diseases, a prominent example of which is acute renal allograft rejection. VCAM1 may play an important role in these diseases because it binds to the integrins very late Ag-4 and α4β7 on lymphocytes and monocytes, thereby providing a potential mechanism to recruit these leukocytes to sites of inflammation. The molecular mechanisms underlying VCAM1 regulation in renal TEC are essentially unknown. We now report that VCAM1 mRNA is dramatically up-regulated in C1, a cell line derived from renal TEC, on exposure to TNF-α. Two NF-κB binding sites in the VCAM1 promoter are critical for the TNF-α-induced VCAM1 transcriptional up-regulation, and both sites bind to p65-p50 NF-κB complexes. TNF-α induces activation of inhibitor of NF-κB (IκB) kinase-β (IKK-β), a protein kinase that phosphorylates the NF-κB inhibitor IκB, and thereby targets the latter for degradation via the ubiquitin-proteasome pathway. Moreover, dominant negative versions of IKK inhibit TNF-α activation of a VCAM1 promoter reporter. We conclude that the IKK/NF-κB pathway is critical in the TNF-α-induced up-regulation of VCAM1 mRNA in renal TEC.
Activation of Human Macrophages by Amyloid-β Is Attenuated by Astrocytes
Authors: Smits Hessel A. , van Beelen Astrid J. , de Vos N. Machiel , Rijsmus Annemarie , van der Bruggen Tjomme , Verhoef Jan , van Muiswinkel Freek L. , Nottet Hans S. L. M.
Published: April 2014

In Alzheimer’s disease, neuritic amyloid-β plaques along with surrounding activated microglia and astrocytes are thought to play an important role in the inflammatory events leading to neurodegeneration. Studies have indicated that amyloid-β can be directly neurotoxic by activating these glial cells to produce oxygen radicals and proinflammatory cytokines. This report shows that, using primary human monocyte-derived macrophages as model cells for microglia, amyloid-β1–42 stimulate these macrophages to the production of superoxide anions and TNF-α. In contrast, astrocytes do not produce both inflammatory mediators when stimulated with amyloid-β1–42. In cocultures with astrocytes and amyloid-β1–42-stimulated macrophages, decreased levels of both superoxide anion and TNF-α were detected. These decreased levels of potential neurotoxins were due to binding of amyloid-β1–42 to astrocytes since FACScan analysis demonstrated binding of FITC-labeled amyloid-β1–42 to astrocytoma cells and pretreatment of astrocytes with amyloid-β1–16 prevented the decrease of superoxide anion in cocultures of human astrocytes and amyloid-β1–42-stimulated macrophages. To elucidate an intracellular pathway involved in TNF-α secretion, the activation state of NF-κB was investigated in macrophages and astrocytoma cells after amyloid-β1–42 treatment. Interestingly, although activation of NF-κB could not be detected in amyloid-β-stimulated macrophages, it was readily detected in astrocytoma cells. These results not only demonstrate that amyloid-β stimulation of astrocytes and macrophages result in different intracellular pathway activation but also indicate that astrocytes attenuate the immune response of macrophages to amyloid-β1–42 by interfering with amyloid-β1–42 binding to macrophages.
The p38 Mitogen-Activated Protein Kinase Mediates Cytoskeletal Remodeling in Pulmonary Microvascular Endothelial Cells Upon Intracellular Adhesion Molecule-1 Ligation
Authors: Wang Qin , Doerschuk Claire M.
Published: April 2014

Changes in the cytoskeleton of endothelial cells (ECs) play important roles in mediating neutrophil migration during inflammation. Previous studies demonstrated that neutrophil adherence to TNF-α-treated pulmonary microvascular ECs induced cytoskeletal remodeling in ECs that required ICAM-1 ligation and oxidant production and was mimicked by cross-linking ICAM-1. In this study, we examined the role of ICAM-1-induced signaling pathways in mediating actin cytoskeletal remodeling. Cross-linking ICAM-1 induced alterations in ICAM-1 distribution, as well as the filamentous actin rearrangements and stiffening of ECs shown previously. ICAM-1 cross-linking induced phosphorylation of the p38 mitogen-activated protein kinase (MAPK) that was inhibited by allopurinol and also induced an increase in the activity of the p38 MAPK that was inhibited by SB203580. However, SB203580 had no effect on oxidant production in ECs or ICAM-1 clustering. ICAM-1 cross-linking also induced phosphorylation of heat shock protein 27, an actin-binding protein that may be involved in filamentous actin polymerization. The time course of heat shock protein 27 phosphorylation paralleled that of p38 MAPK phosphorylation and was completely inhibited by SB203580. In addition, SB203580 blocked the EC stiffening response induced by either neutrophil adherence or ICAM-1 cross-linking. Moreover, pretreatment of ECs with SB203580 reduced neutrophil migration toward EC junctions. Taken together, these data demonstrate that activation of p38 MAPK, mediated by xanthine oxidase-generated oxidant production, is required for cytoskeletal remodeling in ECs induced by ICAM-1 cross-linking or neutrophil adherence. These cytoskeletal changes in ECs may in turn modulate neutrophil migration toward EC junctions.
Gα16 Couples Chemoattractant Receptors to NF-κB Activation
Authors: Yang Ming , Sang Hairong , Rahman Arshad , Wu Dianqing , Malik Asrar B. , Ye Richard D.
Published: April 2014

The guanine nucleotide-binding regulatory protein α-subunit, Gα16, is primarily expressed in hemopoietic cells, and interacts with a large number of seven-membrane span receptors including chemoattractant receptors. We investigated the biological functions resulting from Gα16 coupling of chemoattractant receptors in a transfected cell model system. HeLa cells expressing a κB-driven luciferase reporter, Gα16, and the formyl peptide receptor responded to fMLP with a ∼7- to 10-fold increase in luciferase activity. This response was accompanied by phosphorylation of IκBα and elevation of nuclear κB-DNA binding activity, indicating activation of NF-κB. In contrast to Gα16, expression of Gαq, Gα13, and Gαi2 resulted in a marginal increase in κB luciferase activity. A GTPase-deficient, constitutively active Gα16 mutant (Q212L) could replace agonist stimulation for activation of NF-κB. Furthermore, expression of Gα16 (Q212L) markedly enhanced TNF-α-induced κB reporter activity. The Gα16-mediated NF-κB activation was paralleled by an increase in phospholipase C-β activity, and was blocked by pharmacological inhibitors of protein kinase C (PKC) and by buffering of intracellular Ca2+. The involvement of a conventional PKC isoform was confirmed by the finding that expression of PKCα enhanced the effect of Gα16, and a dominant negative PKCα partially blocked Gα16-mediated NF-κB activation. In addition to formyl peptide receptor, Gα16 also enhanced NF-κB activation by the C5a and C3a receptors, and by CXC chemokine receptor 2 and CCR8. These results suggest a potential role of Gα16 in transcriptional regulation downstream of chemoattractant receptors.
Chemokine Receptor CCR4 on CD4+ T Cells in Juvenile Rheumatoid Arthritis Synovial Fluid Defines a Subset of Cells with Increased IL-4:IFN-γ mRNA Ratios
Authors: Thompson Susan D. , Luyrink Lorie K. , Graham T. Brent , Tsoras Monica , Ryan Mary , Passo Murray H. , Glass David N.
Published: April 2014

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-γ mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4− cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-γ than CD4+/CCR4− cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.
Thiopalmitoylation of Myelin Proteolipid Protein Epitopes Enhances Immunogenicity and Encephalitogenicity
Authors: Greer Judith M. , Denis Bérangère , Sobel Raymond A. , Trifilieff Elisabeth
Published: April 2014

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP104–117 and PLP139–151) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.
IFN-γ-Inducible Protein-10 Is Essential for the Generation of a Protective Tumor-Specific CD8 T Cell Response Induced by Single-Chain IL-12 Gene Therapy
Authors: Pertl Ursula , Luster Andrew D. , Varki Nissi M. , Homann Dirk , Gaedicke Gerhard , Reisfeld Ralph A. , Lode Holger N.
Published: April 2014

The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murine neuroblastoma model depends entirely on the CXC chemokine IFN-γ-inducible protein 10 (IP-10). This was established by in vivo depletion of IP-10 with mAbs in mice vaccinated against NXS2 neuroblastoma by gene therapy with a linearized, single-chain (sc) version of the heterodimeric cytokine IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the effective abrogation of scIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. These findings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD8+ T cell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by two different anti-IP-10 mAbs revealed an abrogation of systemic-protective immunity against disseminated metastases. Second, CD8+ T cell-mediated MHC class I Ag-restricted tumor cell lysis was inhibited in such mice. Third, intracellular IFN-γ expressed by proliferating CD8+ T cells was substantially inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in mice depleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggest that IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12 gene therapy.
CD134L Expression on Dendritic Cells in the Mesenteric Lymph Nodes Drives Colitis in T Cell-Restored SCID Mice
Authors: Malmström Vivianne , Shipton Deborah , Singh Baljit , Al-Shamkhani Aymen , Puklavec Michael J. , Barclay A. Neil , Powrie Fiona
Published: April 2014

Transfer of CD45RBhigh CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin α4β7. Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.
Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit Expression of Fas Ligand in Activated T Lymphocytes by Regulating c-Myc, NF-κB, NF-AT, and Early Growth Factors 2/3
Authors: Delgado Mario , Ganea Doina
Published: April 2014

Activation-induced cell death in T cells, a major mechanism for limiting an ongoing immune response, is initiated by Ag reengagement and mediated through Fas/Fas ligand interactions. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two multifunctional neuropeptides, modulate innate and adaptive immunity. We reported previously that VIP/PACAP protect T cells from activation-induced cell death through down-regulation of Fas ligand (FasL). In this study, we investigate the molecular mechanisms involved in the protective effect of VIP and PACAP. VIP/PACAP reduce in a dose-dependent manner anti-CD3-induced apoptosis in 2B4.11 T cell hybridomas. The protective effect is mediated through the specific type 2 VIP receptor, and the cAMP/protein kinase A pathway. A functional study demonstrates that VIP/PACAP inhibit activation-induced FasL expression. VIP/PACAP inhibit the expression and/or DNA-binding activity of several transcriptional factors involved in FasL expression, i.e., c-myc, NF-κB, NF-ATp, and early growth factors (Egr) 2/3. The inhibition of NF-κB binding is due to the stabilization of I-κB (inhibitory protein that dissociates from NF-κB), through the inhibition of I-κB kinase α activity. Subsequently, p65 nuclear translocation is significantly reduced. The inhibition in NF-ATp binding results from a calcineurin-independent reduction in NF-ATp nuclear translocation. VIP/PACAP inhibit the expression of Egr2 and 3, but not of Egr1. The effects on the transcriptional factors are mediated through type 2 VIP receptor with cAMP as secondary messenger.
Histoplasma capsulatumYeasts Are Phagocytosed Via Very Late Antigen-5, Killed, and Processed for Antigen Presentation by Human Dendritic Cells
Authors: Gildea Lucy A. , Morris Randal E. , Newman Simon L.
Published: April 2014

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of world-wide importance. As the induction of cell-mediated immunity to Hc is of critical importance in host defense, we sought to determine whether dendritic cells (DC) could function as a primary APC for this pathogenic fungus. DC obtained by culture of human monocytes in the presence of GM-CSF and IL-4 phagocytosed Hc yeasts in a time-dependent manner. Upon ingestion, the intracellular growth of yeasts within DC was completely inhibited compared with rapid growth within human macrophages. Electron microscopy of DC with ingested Hc revealed that many of the yeasts were degraded as early as 2 h postingestion. In contrast to macrophages, human DC recognized Hc yeasts via the fibronectin receptor, very late Ag-5, and not via CD18 receptors. DC stimulated Hc-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of viable and heat-killed Hc yeasts, but greater proliferation was achieved after ingestion of viable yeasts. These data demonstrate that human DC can phagocytose and degrade a fungal pathogen and subsequently process the appropriate Ags for stimulation of lymphocyte proliferation. In vivo, such interactions between DC and Hc may facilitate the induction of cell-mediated immunity.
Bispecific Monoclonal Antibodies Mediate Binding of Dengue Virus to Erythrocytes in a Monkey Model of Passive Viremia
Authors: Hahn Chang S. , French Olivia G. , Foley Patricia , Martin Edward N. , Taylor Ronald P.
Published: April 2014

Dengue viruses (DEN), causative agents of dengue fever (DF) and more severe dengue hemorrhagic fever (DHF)/dengue shock syndrome, infect over 100 million people every year. Among those infected, up to one-half million people develop DHF, which requires an extensive hospital stay. Recent reports indicate that there is a significant correlation between virus titer in the bloodstream of infected individuals and the severity of the disease, especially the development of DHF. This suggests that if there is a procedure to reduce viremia in infected subjects, then the severity of the disease may be controlled during the critical early stages of the disease before it progresses to DHF. We have generated bispecific mAb complexes (heteropolymer(s), HP), which contain a mAb specific for the DEN envelope glycoprotein cross-linked with a second mAb specific for the primate E complement receptor 1. These HP facilitate rapid binding of DEN to human and monkey E in vitro, with ∼90% bound within 5 min. Furthermore, in a passive viremia monkey model established by continuous steady state infusion of DEN, injection of HP during the steady state promoted rapid binding of DEN to the E, followed by subsequent clearance from the vascular system. Moreover, HP previously infused into the circulation is capable of efficiently capturing a subsequent challenge dose of DEN and binding it to E. These data suggest that HP potentially can be useful for alleviating DEN infection-associated symptoms by reducing titers of free virus in the vascular system.
Mucosal Delivery of a Respiratory Syncytial Virus CTL Peptide with Enterotoxin-Based Adjuvants Elicits Protective, Immunopathogenic, and Immunoregulatory Antiviral CD8+ T Cell Responses
Authors: Simmons Cameron P. , Hussell Tracy , Sparer Tim , Walzl Gerhard , Openshaw Peter , Dougan Gordon
Published: April 2014

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8+ CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M282–90-specific CD8+ T cells were detected by IFN-γ enzyme-linked immunospot and 51Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-γ since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-γ did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8+ CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.
Highly Conserved Regions of the Immunodominant Major Surface Protein 2 of the Genogroup II Ehrlichial Pathogen Anaplasma marginale Are Rich in Naturally Derived CD4+ T Lymphocyte Epitopes that Elicit Strong Recall Responses
Authors: Brown Wendy C. , McGuire Travis C. , Zhu Daming , Lewin Harris A. , Sosnow Joshua , Palmer Guy H.
Published: April 2014

Genogroup II ehrlichia, including the agent of human granulocytic ehrlichiosis, Ehrlichia phagocytophila, and the bovine pathogen Anaplasma marginale, express a markedly immunodominant outer membrane protein designated major surface protein 2 (MSP2). MSP2 is encoded by a multigene family, resulting in the expression of variant B cell epitopes. MSP2 variants are sequentially expressed in the repeated cycles of rickettsemia that characterize persistent A. marginale infection and control of each rickettsemic cycle is associated with development of a variant-specific IgG response. Importantly, these persistent rickettsemic cycles are controlled at levels 100-1000 times lower than those responsible for clinical disease during acute infection. Control of rickettsemia during persistence could result from an anamnestic Th lymphocyte response to conserved regions of MSP2 that enhances the primary Ab response against newly emergent variants. Comparison of MSP2 variants reveals conserved N and C termini flanking the central, surface-exposed hypervariable region that represents the variant B lymphocyte epitopes. We demonstrate MSP2-specific CD4+ T lymphocyte recognition of epitopes common to several strains of A. marginale and the related pathogen A. ovis. Furthermore, T lymphocyte lines from three individuals identified six to nine overlapping peptides representing a minimum of four to seven dominant or subdominant epitopes in these conserved N and C termini. Immunodominant peptides induced high levels of IFN-γ, a cytokine associated with protection against ehrlichia and needed for rapid generation of variant-specific IgG2. The presented data support the potential importance of a strong Th lymphocyte response to invariant MSP2 epitopes in controlling rickettsemia during persistent infection to subclinical levels.
Nitric Oxide Synthase Inhibitors Have Opposite Effects on Acute Inflammation Depending on Their Route of Administration
Authors: Paul-Clark Mark J. , Gilroy Derek W. , Willis Dean , Willoughby Derek A. , Tomlinson Annette
Published: April 2014

The bulk of published data has shown that NO is proinflammatory. However, there also exists the conflicting notion that NO may be protective during an inflammatory insult. In an attempt to resolve this issue, we have compared the effects on inflammation of a range of NO synthase (NOS) inhibitors given either directly to the site of the inflammatory lesion or systemically. It was found that in the carrageenin-induced pleurisy, a single intrapleural injection of the selective inducible NO inhibitors S-(2-aminoethyl) isothiourea (AE-ITU; 3 and 10 mg/kg) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W; 10 mg/kg) or the selective endothelial cell NOS inhibitor l-N5(1-iminoethyl)-ornithine (10 mg/kg) not only exacerbated inflammation at the very early stages of the lesion (1–6 h), but also prevented inflammatory resolution. By contrast, administering NOS inhibitors systemically ameliorated the severity of inflammation throughout the reaction. To elucidate the mechanisms by which inhibition of NO synthesis locally worsened inflammation, we found an increase in histamine, cytokine-induced neutrophil chemoattractant, superoxide, and leukotriene B4 levels at the inflammatory site. In conclusion, this work shows that the local production of NO is protective by virtue of its ability to regulate the release of typical proinflammatory mediators and, importantly, that NOS inhibitors have differential anti-inflammatory effects depending on their route of administration.
Role of C5a in Multiorgan Failure During Sepsis
Authors: Huber-Lang Markus , Sarma Vidya J. , Lu Kristina T. , McGuire Stephanie R. , Padgaonkar Vaishalee A. , Guo Ren-Feng , Younkin Ellen M. , Kunkel Robin G. , Ding Jiabing , Erickson Richard , Curnutte John T. , Ward Peter A.
Published: April 2014

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O2, rising partial pressure of CO2). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.
Serum Amyloid P Component and C-Reactive Protein Mediate Phagocytosis Through Murine FcγRs
Authors: Mold Carolyn , Gresham Hattie D. , Du Clos Terry W.
Published: April 2014

The pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP) are acute-phase serum proteins in mice and humans, respectively. Although SAP binds to DNA and chromatin and affects clearance of these autoantigens, no specific receptor for SAP has been identified. CRP is an opsonin, and we have shown that it binds to FcγR. Mice deficient in FcγR were used to assess the role of these receptors in phagocytosis by pentraxins using zymosan as a ligand. Phagocytosis of zymosan by bone marrow macrophages (BMM) was enhanced by opsonization with SAP or CRP. BMM from mice deficient in all three FcγR or in γ-chain ingested unopsonized zymosan, but phagocytosis of SAP- or CRP-opsonized zymosan was not enhanced. SAP binding to BMM from γ-chain-deficient mice was also greatly reduced, indicating little or no binding of SAP to FcγRII. SAP and CRP opsonized zymosan for phagocytosis by BMM from mice deficient in FcγRII or FcγRIII. SAP, but not CRP, opsonized zymosan for uptake by neutrophils that express only low levels of FcγRI. Together these results indicate that FcγRI and FcγRIII are receptors for SAP in the mouse. Opsonization of zymosan by CRP is mediated through FcγRI. Pentraxins are major proteins of the innate immune system and arose earlier in evolution than Igs. The use of FcγR by the pentraxins links innate and adaptive immunity and may have important consequences for processing, presentation, and clearance of the self-Ags to which these proteins bind.
IL-4 Adenoviral Gene Therapy Reduces Inflammation, Proinflammatory Cytokines, Vascularization, and Bony Destruction in Rat Adjuvant-Induced Arthritis
Authors: Woods James M. , Katschke Kenneth J. , Volin Michael V. , Ruth Jeffrey H. , Woodruff Drew C. , Amin M. Asif , Connors Matthew A. , Kurata Hirokazu , Arai Ken-Ichi , Haines G. Kenneth , Kumar Pawan , Koch Alisa E.
Published: April 2014

IL-4 is a cytokine with anti-inflammatory properties on activated macrophages. Rheumatoid arthritis, an autoimmune inflammatory disease, is characterized by a paucity of IL-4 and an abundance of synovial macrophage-derived mediators. Herein, the effect of a single injection of adenovirus-producing rat IL-4 (AxCAIL-4) or a control virus with no inserted gene was compared with the effect of PBS injection into rat ankles. Ankles were injected before arthritis onset or at maximal inflammation. Preventatively, AxCAIL-4 reduced adjuvant-induced arthritis (AIA)- and/or AIA/adenoviral-induced ankle inflammation, decreasing articular index scores, ankle circumferences, paw volumes, radiographic scores, mean levels of monocyte chemoattractant protein-1, the number of inflammatory cells, and the number of synovial blood vessels. Therapeutically, AxCAIL-4 also decreased ankle circumferences and paw volumes in comparison with a control virus with no inserted gene and PBS groups. After arthritis onset, mean levels of TNF-α, IL-1β, macrophage inflammatory protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates compared with PBS-treated homogenates. Thus, increased expression of IL-4 via gene therapy administered in a preventative and/or therapeutic manner reduced joint inflammation, synovial cellularity, levels of proinflammatory cytokines, vascularization, and bony destruction in rat AIA, suggesting that a similar treatment in humans may be beneficial.
Rapid Up-Regulation of CXC Chemokines in the Airways after Ag-Specific CD4+ T Cell Activation
Authors: Knott Patrick G. , Gater Paul R. , Dunford Paul J. , Fuentes Maria Elena , Bertrand Claude P.
Published: April 2014

Ag-specific activation of CD4+ T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4+ T cell activation is important in lung chemokine production is currently not clear. Using αβ-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4+ T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11.10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4+ T cell activation was critical because CD4+ but not CD8+ T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4+ T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4+ T cells with APCs. A likely secondary mediator was TNF-α, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4+ T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.
Intratracheal Priming with Ovalbumin- and Ovalbumin 323–339 Peptide-Pulsed Dendritic Cells Induces Airway Hyperresponsiveness, Lung Eosinophilia, Goblet Cell Hyperplasia, and Inflammation
Authors: Sung Sun-sang J. , Rose C. Edward , Fu Shu Man
Published: April 2014

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA323–339 and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA323–339-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.
TNF-α-Induced Expression of Adhesion Molecules in the Liver Is Under the Control of TNFR1—Relevance for Concanavalin A-Induced Hepatitis
Authors: Wolf Dominik , Hallmann Rupert , Sass Gabriele , Sixt Michael , Küsters Sabine , Fregien Bastian , Trautwein Christian , Tiegs Gisa
Published: April 2014

TNF-α has been clearly identified as central mediator of T cell activation-induced acute hepatic injury in mice, e.g., Con A hepatitis. In this model, liver injury depends on both TNFRs, i.e., the 55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that the hepatic TNFRs are not transcriptionally regulated, but are regulated by receptor shedding. TNF directly mediates hepatocellular death by activation of TNFR1 but also induces the expression of inflammatory proteins, such as cytokines and adhesion molecules. Here we provide evidence that resistance of TNFR1−/− and TNFR2−/− mice against Con A hepatitis is not due to an impaired production of the central mediators TNF and IFN-γ. Con A injection results in a massive induction of ICAM-1, VCAM-1, and E-selectin in the liver. Lack of either one of both TNFRs did not change adhesion molecule expression in the livers of Con A-treated mice, presumably reflecting the fact that other endothelial cell-activating cytokines up-regulated adhesion molecule expression. However, treatment of TNFR1−/− and TNFR2−/− mice with murine rTNF revealed a predominant role for TNFR1 for the induction of hepatic adhesion molecule expression. Pretreatment with blocking Abs against E- and P-selectin or of ICAM−/− mice with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although accumulation of the critical cell population, i.e., CD4+ T cells was significantly inhibited. Hence, up-regulation of adhesion molecules during acute hepatitis unlikely contributes to organ injury but rather represents a defense mechanism.
A Deletion in the Gene Encoding the CD45 Antigen in a Patient with SCID
Authors: Tchilian Elma Z. , Wallace Diana L. , Wells R. Spencer , Flower Darren R. , Morgan Gareth , Beverley Peter C. L.
Published: April 2014

SCID is a heterogeneous group of hereditary diseases. Mutations in the common γ-chain (γc) of cytokine receptors, including those for IL-2, IL-4, IL-7, IL-9, and IL-15, are responsible for an X-linked form of the disease, while mutations of several other genes, including Janus-associated kinase-3, may cause autosomal recessive forms of SCID. We investigated the first SCID patient to be described with minimal cell surface expression of the leukocyte common (CD45) Ag. CD45 is an abundant transmembrane tyrosine phosphatase, expressed on all leukocytes, and is required for efficient lymphocyte signaling. CD45-deficient mice are severely immunodeficient and have very few peripheral T lymphocytes. We report here that a homozygous 6-bp deletion in the gene encoding CD45 (PTPRC, gene map locus 1q31–32), which results in a loss of glutamic acid 339 and tyrosine 340 in the first fibronectin type III module of the extracellular domain of CD45, is associated with failure of surface expression of CD45 and SCID. Molecular modeling suggests that tyrosine 340 is crucial for the structural integrity of CD45 protein. This is the second description of a clinically relevant CD45 mutation, provides direct evidence for the importance of CD45 in immune function in humans, and suggests that abnormalities in CD45 expression are a possible cause of SCID in humans.
Roles of TNF-Related Apoptosis-Inducing Ligand in Experimental Autoimmune Encephalomyelitis
Authors: Hilliard Brendan , Wilmen Andreas , Seidel Cheryl , Liu Tzu-Shang T. , Göke Rüdiger , Chen Youhai
Published: April 2014

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL receptor, we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were significantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells.
Thioether-Bonded Constructs of Fab′γ and Fcγ Modules Utilizing Differential Reduction of Interchain Disulfide Bonds
Authors: Kan Kwok S. , Anderson Valerie A. , Leong Weng S. , Smith Andrew M. , Worth Andrew T. , Stevenson George T.
Published: April 2014

We describe a two-stage preparation of chemically engineered Ab constructs, employing as modules Fab′γ from mAb or rAb, and Fc from human normal IgG1. A multivalent, optionally multispecific F(ab′)n core is formed in stage one, and one or more Fc modules added in stage two. Examples include bispecific Fab2Fc2 (for simplicity, primes and Greek letters are omitted from names of final constructs) and trivalent Fab3Fc2, which are designed to kill neoplastic cells. An essential element in the construction is the availability of the Fab′ in two reduced forms, Fab′(-sulfhydryl (SH))5 and Fab′-SH. The first is obtained by full reduction of the interchain disulfide bonds (SS) in the F(ab′)2 fragment of IgG. Fab′-SH is obtained by disulfide-interchange reactions on Fab′(-SH)5, whereby the γ-light SS is reconstituted, an unusual intrachain SS forms in the γ-chain hinge, and one hinge SH remains. F(ab′)2 and F(ab′)3 cores are built using partially reduced modules, being given intermodular thioether links that resist reduction. These cores are then fully reduced, making available SH groups for addition of the Fcγ modules. In the final constructs, all intermodular links embody tandem thioether bonds arising at hinge-region cysteines. Cytotoxic activities of representative constructs, and some enhancements deriving from multiple modules, are assessed. In guinea pigs, catabolism of Fab2Fc2 yielded a t1/2 similar to that of human IgG1, although the serum Fab2Fc2 revealed some proteolytic breakdown not shown by the IgG1. Immunotherapy of a guinea-pig leukemia confirmed the ability of these constructs to kill target cells in vivo.