bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-2
Authors: Caughey Gillian E. , Cleland Leslie G. , Penglis Peter S. , Gamble Jennifer R. , James Michael J.
Published: April 2014

The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH2, the common substrate for thromboxane A2 (TXA2), prostacyclin (PGI2), and PGE2 synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1β caused induction of COX-2. TXA2 was the predominant COX-1-derived product, and TXA2 synthesis changed little with up-regulation of COX-2 by IL-1β (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI2 and PGE2 (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished PGI2 and PGE2 synthesis, but had little effect on TXA2 synthesis. The up-regulation of COX-2 by IL-1β was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA2 synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI2 and PGE2 synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI2 and PGE2 linkage with COX-2 activity may be explained by a temporal increase in total COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition.
CD18 Dependency of Transendothelial Neutrophil Migration Differs During Acute Pulmonary Inflammation
Authors: Mackarel A. Jill , Russell Kenneth J. , Ryan Clodagh M. , Hislip Shirley J. , Rendall Jacqueline C. , FitzGerald Muiris X. , O’Connor Clare M.
Published: April 2014

Neutrophil extravasation during inflammation can occur either by a mechanism that requires the neutrophil integrin complex, CD18, or by an alternative CD18-independent route. Which of the two pathways is used has been shown to depend on the site and nature of the inflammatory insult. More recent evidence suggests that selection may also depend on whether inflammation is chronic or acute, but why this is the case remains unknown. Using an in vitro model that supports both migratory mechanisms, we examined the CD18 dependency of migration of neutrophils isolated from patients with either chronic or acute pulmonary infection. Chronic neutrophils were found to behave like normal neutrophils by migrating to IL-8 and leukotriene B4 using the CD18-independent pathway, but to the bacterial product, FMLP, using the CD18-dependent route. In contrast, migration of acute neutrophils to all of these stimuli was CD18 dependent. Normal neutrophils could be manipulated to resemble acute neutrophils by exposing them to FMLP before migration, which resulted in a “switch” from the CD18-independent to -dependent mechanism during migration to IL-8 or leukotriene B4. Although treatment of normal neutrophils with FMLP caused selective down-regulation of the IL-8 receptor, CXCR2, and acute neutrophils were found to have less CXCR2 than normal, a functional relationship between decreased CXCR2 and selection of CD18-dependent migration was not demonstrated. Results indicate that selection of the CD18-dependent or -independent migration mechanism can be controlled by the neutrophil and suggest that the altered CD18 requirements of acute neutrophils may be due to priming in the circulation during acute infection.
A Novel Function of Phosphorothioate Oligodeoxynucleotides as Chemoattractants for Primary Macrophages
Authors: Baek Kwan Hyuck , Ha Sang Jun , Sung Young Chul
Published: April 2014

Phosphorothioate cytosine-guanine oligodeoxynucleotides (CpG PS-ODNs) has been reported to induce Th1 immune responses against coadministered Ags more efficiently than phosphodiester CpG ODNs (CpG PO-ODNs). Here, we demonstrated that PS-ODNs, but not PO-ODNs, have a chemotactic effect on primary macrophages, which is independent of the CpG motif. In addition, the conjugation of a hexameric dG run (dG6 run) at the 3′ terminus reduced the concentration required for the optimal chemotactic activity of PS-ODNs by ∼10-fold. Endosomal maturation blockers, such as monensin and chloroquine, inhibited the chemotactic effect of PS-ODNs. The inhibition of the activities of p38 mitogen-activated protein (MAP) kinase, and extracellular signal-related kinases (ERKs) as well as phosphoinositide 3-kinase with their specific inhibitors also resulted in suppressing the chemotaxis of primary macrophages induced by PS-ODNs. These results indicate that the PS-ODN-mediated chemotaxis requires the activation of ERKs, p38 MAP kinase, and phosphoinositide 3-kinase as well as endosomal maturation. In addition, the phosphorylations of the p38 MAP kinase, ERKs, and protein kinase B, Akt, were induced by PS-ODN, which were further enhanced by the presence of both a dG6 run and CpG motifs. Our findings suggest that the chemotactic activity of PS-ODNs may be one of the mechanisms by which PS-ODNs exhibit stronger immunomodulatory activities than PO-ODNs in vivo.
IL-13 and IL-4 Up-Regulate Cysteinyl Leukotriene 1 Receptor Expression in Human Monocytes and Macrophages
Authors: Thivierge Maryse , Staňková Jana , Rola-Pleszczynski Marek
Published: April 2014

The cysteinyl (Cys) leukotrienes (LT)C4, LTD4, and LTE4, are lipid mediators that have been implicated in the pathogenesis of asthma. The human LTD4 receptor (CysLT1R) was recently cloned and characterized. The present work was undertaken to study the potential modulation of CysLT1R expression by the Th2 cytokines IL-13 and IL-4. In this study, we report that IL-13 up-regulates CysLT1R mRNA levels, with consequently enhanced CysLT1R protein expression and function in human monocytes and monocyte-derived macrophages. CysLT1R mRNA expression was augmented 2- to 5-fold following treatment with IL-13 and was due to enhanced transcriptional activity. The effect was observed after 4 h, was maximal by 8 h, and maintained at 24 h. IL-4, but not IFN-γ, induced a similar pattern of CysLT1R up-regulation. Monocytes pretreated with IL-13 or IL-4 for 24 h showed enhanced CysLT1R protein expression, as assessed by flow cytometry using a polyclonal anti-CysLT1R Ab. They also showed enhanced responsiveness to LTD4, but not to LTB4, in terms of Ca2+ mobilization, as well as augmented chemotactic activity. Our findings suggest a possible mechanism by which IL-13 and IL-4 can modulate CysLT1R expression on monocytes and macrophages, and consequently their responsiveness to LTD4, and thus contribute to the pathogenesis of asthma and allergic diseases.
Human IgA Activates the Complement System Via the Mannan-Binding Lectin Pathway
Authors: Roos Anja , Bouwman Lee H. , van Gijlswijk-Janssen Daniëlle J. , Faber-Krol Maria C. , Stahl Gregory L. , Daha Mohamed R.
Published: April 2014

The recently identified lectin pathway of the complement system, initiated by binding of mannan-binding lectin (MBL) to its ligands, is a key component of innate immunity. MBL-deficient individuals show an increased susceptibility for infections, especially of the mucosal system. We examined whether IgA, an important mediator of mucosal immunity, activates the complement system via the lectin pathway. Our results indicate a dose-dependent binding of MBL to polymeric, but not monomeric IgA coated in microtiter plates. This interaction involves the carbohydrate recognition domain of MBL, because it was calcium dependent and inhibited by mannose and by mAb against this domain of MBL. Binding of MBL to IgA induces complement activation, as demonstrated by a dose-dependent deposition of C4 and C3 upon addition of a complement source. The MBL concentrations required for IgA-induced C4 and C3 activation are well below the normal MBL plasma concentrations. In line with these experiments, serum from individuals having mutations in the MBL gene showed significantly less activation of C4 by IgA and mannan than serum from wild-type individuals. We conclude that MBL binding to IgA results in complement activation, which is proposed to lead to a synergistic action of MBL and IgA in antimicrobial defense. Furthermore, our results may explain glomerular complement deposition in IgA nephropathy.
Fibrinogen Induces IL-8 Synthesis in Human Neutrophils Stimulated with Formyl-Methionyl-Leucyl-Phenylalanine or Leukotriene B4
Authors: Kuhns Douglas B. , Nelson Edward L. , Alvord W. Gregory , Gallin John I.
Published: April 2014

Human exudative neutrophils have greatly increased stores of the neutrophil chemoattractant IL-8 compared with peripheral blood cells, but the mechanism for the increase is not defined. In this report, we show that treatment of peripheral blood neutrophils with the chemotactic peptide fMLP or with leukotriene B4 or fibrinogen results in little increase in the production of IL-8 by peripheral blood neutrophils. However, a chemotactically active dose of fMLP (5 × 10−9 M) or leukotriene B4 (1 × 10−7 M) in the presence of a physiological concentration (2 mg/ml) of fibrinogen results in a receptor-mediated, pertussis toxin-sensitive, synergistic 30-fold increase in IL-8 synthesis. The levels of IL-8 attained are comparable to those observed in exudative cells. Higher concentrations of fMLP (1 × 10−7 M) are associated with reduced IL-8 protein synthesis without IL-8 degradation, indicating a sensitive regulatory mechanism for IL-8 production. Treatment of neutrophils with fibrinogen and fMLP resulted in minimal changes in the steady state levels of mRNA for macrophage inflammatory protein-1α and -1β and monocyte chemoattractant protein-1. In contrast, in the presence of fibrinogen, the steady-state level of neutrophil IL-8 mRNA increased 8-fold with 5 × 10−9 M fMLP but was not decreased with 1 × 10−7 M fMLP, suggesting that neutrophils are specifically adapted to modulate neutrophil IL-8 synthesis through transcriptional and posttranscriptional mechanisms. The data indicate that fibrinogen can function not only as a substrate in the clotting cascade, but also as an important effector during the evolution of the innate immune response.
A Role for IL-18 in Neutrophil Activation
Authors: Leung Bernard P. , Culshaw Shauna , Gracie J. Alastair , Hunter David , Canetti Claudio A. , Campbell Carol , Cunha Fernando , Liew Foo Y. , McInnes Iain B.
Published: April 2014

IL-18 expression and functional activity has been identified in several autoimmune and infectious diseases. To clarify the potential role of IL-18 during early innate immune responses, we have explored the capacity of IL-18 to activate neutrophils. Human peripheral blood-derived neutrophils constitutively expressed IL-18R (α and β) commensurate with the capacity to rapidly respond to IL-18. IL-18 induced cytokine and chemokine release from neutrophils that was protein synthesis dependent, up-regulated CD11b expression, induced granule release, and enhanced the respiratory burst following exposure to fMLP, but had no effect upon the rate of neutrophil apoptosis. The capacity to release cytokine and chemokine was significantly enhanced in neutrophils derived from rheumatoid arthritis synovial fluid, indicating differential responsiveness to IL-18 dependent upon prior neutrophil activation in vivo. Finally, IL-18 administration promoted neutrophil accumulation in vivo, whereas IL-18 neutralization suppressed the severity of footpad inflammation following carrageenan injection. The latter was accompanied by reduction in tissue myeloperoxidase expression and suppressed local TNF-α production. Together, these data define a novel role for IL-18 in activating neutrophils and thereby promoting early innate immune responses.
Fibrinogen Stimulates Macrophage Chemokine Secretion Through Toll-Like Receptor 4
Authors: Smiley Stephen T. , King Jennifer A. , Hancock Wayne W.
Published: April 2014

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1α (MIP-1α), MIP-1β, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-γ and differentiation with vitamin D3, respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.
Differential Role of CD18 Integrins in Mediating Lung Neutrophil Sequestration and Increased Microvascular Permeability Induced byEscherichia coliin Mice
Authors: Gao Xiao-pei , Xu Ning , Sekosan Marin , Mehta Dolly , Ma Shuang Y. , Rahman Arshad , Malik Asrar B.
Published: April 2014

The in vivo contributions of CD18 integrin-dependent and -independent mechanisms in mediating the increases in lung neutrophil (polymorphonuclear leukocyte; PMN) sequestration and microvascular permeability are not well understood. We determined the time course of these responses to Gram-negative sepsis in the mouse lung and addressed the specific contributions of CD18 integrins and ICAM-1. PMN sequestration in the lung was assessed by morphometric analysis, and transalveolar PMN migration was assessed by bronchoalveolar lavage. Lung tissue PMN number increased by 6-fold within 1 h after i.p. Escherichia coli challenge; this value peaked at 3 h (7-fold above control) and decreased at 12 h (3.5-fold above control). PMN migration into the airspace was delayed; the value peaked at 6 h and remained elevated up to 12 h. Saturating concentrations of anti-CD18 and anti-ICAM-1 mAbs reduced lung tissue PMN sequestration and migration; however, peak responses at 3 and 6 h were inhibited by 40%, indicating that only a small component of PMN sequestration and migration was CD18 dependent at these times. In contrast to the time-dependent decreased role of CD18 integrins in mediating PMN sequestration and migration, CD18 and ICAM-1 blockade prevented the increase in lung microvascular permeability and edema formation at all times after E. coli challenge. Thus, Gram-negative sepsis engages CD18/ICAM-1-independent mechanisms capable of the time-dependent amplification of lung PMN sequestration and migration. The increased pulmonary microvascular permeability induced by E. coli is solely the result of engagement of CD18 integrins even when PMN accumulation and migration responses are significantly CD18 independent.
Bystander Activation Involving T Lymphocytes in Herpetic Stromal Keratitis
Authors: Deshpande Shilpa , Zheng Mei , Lee Sujin , Banerjee Kaustuv , Gangappa Shivaprakash , Kumaraguru Udayasankar , Rouse Barry T.
Published: April 2014

Herpes simplex virus infection of mouse corneas can lead to the development of an immunopathological lesion, termed herpetic stromal keratitis (HSK). Such lesions also occur in TCR-transgenic mice backcrossed to SCID (TgSCID) that are unable to mount detectable HSV-specific immune responses. The present study demonstrates that lesion expression in such mice depends on continuous viral replication, whereas in immunocompetent mice, lesions occurred even if virus replication was terminated at 4 days after infection. The continuous replication in TgSCID mice was considered necessary to produce an activating stimulus to CD4+ T cells that invade the cornea. Lesions in TgSCID were resistant to control by cyclosporin A, but were inhibited by treatment with rapamycin. This result was interpreted to indicate that T cell activation involved a non-TCR-mediated cytokine-driven bystander mechanism. Bystander activation was also shown to play a role in HSK lesions in immunocompetent mice. Accordingly, in immunocompetent DO11.10 mice, lesions were dominated by KJ1.26+ OVA-specific CD4+ T cells that were unreactive with HSV. In addition, KJ1.26+ HSV nonimmune cells parked in ocularly infected BALB/c mice were demonstrable in HSK lesions. These results provide insight for the choice of new strategies to manage HSK, an important cause of human blindness.
Methotrexate Suppresses NF-κB Activation Through Inhibition of IκBα Phosphorylation and Degradation
Authors: Majumdar Sekhar , Aggarwal Bharat B.
Published: April 2014

Methotrexate (MTX), a folate antagonist, is a commonly used anti-inflammatory, antiproliferative, and immunosuppressive drug whose mode of action is not fully established. Due to the central role of NF-κB in these responses, we postulated that MTX must mediate its effects through suppression of NF-κB activation. We investigated the effects of MTX on NF-κB activation induced by TNF in Jurkat cells. The treatment of these cells with MTX suppressed TNF-induced NF-κB activation with optimum effects occurring at 10 μM MTX for 60 min. These effects were not restricted to Jurkat cells because other cell types were also inhibited. Besides TNF, MTX also suppressed the NF-κB activation induced by various other inflammatory stimuli. The suppression of TNF-induced NF-κB activation by MTX correlated with inhibition of IκBα degradation, suppression of IκBα phosphorylation, abrogation of IκBα kinase activation, and inhibition of NF-κB-dependent reporter gene expression. Because ecto 5′ nucleotidase inhibitor (α,β-methylene adenosine-5′-diphosphate) blocked the effect of MTX, adenosine mimicked the effect of MTX, and adenosine A2b receptor antagonist (3,7-dimethyl-1-propargylxanthine) reversed the inhibitory effect of MTX, we suggest that MTX suppresses NF-κB activation by releasing adenosine. A partial reversal of MTX-induced NF-κB suppression by thymidine and folinic acid indicates the role of the thymidylate synthase pathway also. Overall, our results clearly demonstrate that MTX suppresses NF-κB activation through the release of adenosine, which may contribute to the role of MTX in anti-inflammatory, immunomodulatory, and antiproliferative effects.
Lactoferrin Binds CpG-Containing Oligonucleotides and Inhibits Their Immunostimulatory Effects on Human B Cells
Authors: Britigan Bradley E. , Lewis Troy S. , Waldschmidt Mari , McCormick Michael L. , Krieg Arthur M.
Published: April 2014

Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge-charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection.
Enhanced Dendritic Cell-Driven Proliferation and Anti-HIV Activity of CD8+ T Cells by a New Phenothiazine Derivative, Aminoperazine
Authors: Lu Wei , Achour Amar , Arlie Marine , Cao Li , Andrieu Jean-Marie
Published: April 2014

T cell anergy, apoptosis, and chronic activation of T lymphocytes are prevailing features of HIV infection. The inability to develop an efficient natural antiviral activity in infected patients might be the consequence of a failure of the Ag presentation by dendritic cells (DCs) in chronically activated lymphoid tissues. We have identified a new phenothiazine derivative aminoperazine (APR; 2-amino-10-[3′-(1-methyl-4-piperazinyl)propyl]phenothiazine, C20H26N4S; m.w. 354.51) able to increase (effective dose from 0.1 to 100 nM) the Ag-specific DC-driven proliferation and differentiation of in vitro HIV-infected and uninfected normal donor T cells and of T cells from HIV-1-infected patients. The immunomodulatory effect of APR-sensitized DCs were ascribed to soluble factors derived from DCs. APR was also capable of increasing HIV gag-p24-specific proliferation and anti-HIV cytotoxic activity of patients’ CD8+ T cells against autologous B-lymphoblastoid cell lines expressing a HIV gag gene, resulting in the suppression of both proviral DNA and supernatant viral RNA in the HIV-1-infected patients’ T cell culture. This new phenothiazine derivative (APR) might be used for boosting the immune response of vaccinated individuals and for restoring the immunity of immunocompromised patients.
Accumulation and Activation-Induced Release of Preformed Fas (CD95) Ligand During the Pathogenesis of Experimental Graft-Versus-Host Disease
Authors: Wasem Christoph , Frutschi Corina , Arnold Diana , Vallan Claudio , Lin Tesu , Green Douglas R. , Mueller Christoph , Brunner Thomas
Published: April 2014

Fas (CD95/APO-1) ligand (FasL)-mediated cytotoxicity has been implicated in tissue destruction in a variety of diseases, including acute graft-vs-host disease (GVHD). In this study, we have analyzed FasL expression and regulation during the course of experimental murine acute GVHD. Although activation-induced FasL-mediated cytotoxicity in control T cells was sensitive to the immunosuppressant cyclosporin A, we observed that functional FasL expression of GVHD T cells became increasingly cyclosporin A unresponsive. This was found to be the result of a massive in vivo accumulation and intracellular storage of FasL protein and its release in a transcription- and protein synthesis-independent manner. Immunohistochemistry analysis of FasL expression in situ revealed accumulation of FasL-expressing cells in the spleen, the liver, and small intestine, with a typical cytoplasmic and granular expression pattern. Thus, we conclude that the release of preformed FasL by infiltrating donor T cells may contribute to recipient tissue damage during the pathogenesis of acute GVHD.
Prevention of Experimental Autoimmune Encephalomyelitis in the Common Marmoset (Callithrix jacchus) Using a Chimeric Antagonist Monoclonal Antibody Against Human CD40 Is Associated with Altered B Cell Responses
Authors: Boon Louis , Brok Herbert P. M. , Bauer Jan , Ortiz-Buijsse Antonio , Schellekens Marc M. , Ramdien-Murli Seema , Blezer Erwin , van Meurs Marjan , Ceuppens Jan , de Boer Mark , ’t Hart Bert A. , Laman Jon D.
Published: April 2014

Inhibition of CD40-CD40 ligand interaction is a potentially effective approach for treatment of autoimmune diseases, such as multiple sclerosis. We have investigated this concept with a chimeric antagonist anti-human CD40 mAb (ch5D12) in the marmoset monkey experimental autoimmune encephalomyelitis (EAE) model. Marmosets were immunized with recombinant human myelin oligodendrocyte glycoprotein (rMOG) and treated from the day before immunization (day −1) until day 50 with either ch5D12 (5 mg/kg every 2–4 days) or placebo. On day 41 after the induction of EAE, four of four placebo-treated monkeys had developed severe clinical EAE, whereas all animals from the ch5D12-treated group were completely free of disease symptoms. High serum levels of ch5D12 associated with complete coating of CD40 on circulating B cells were found. At necropsy placebo- and ch5D12-treated animals showed similar MOG-specific lymphoproliferative responses in vitro, but ch5D12 treatment resulted in strongly reduced anti-MOG IgM Ab responses and delayed anti-MOG IgG responses. Most importantly, treatment with ch5D12 prevented intramolecular spreading of epitope recognition. Postmortem magnetic resonance imaging and immunohistologic analysis of the CNS showed a markedly reduced lesion load after ch5D12 treatment. In conclusion, the strong reduction of clinical, pathological, and radiological aspects of EAE by ch5D12 treatment in this preclinical model points to a therapeutic potential of this engineered antagonist anti-CD40 mAb for multiple sclerosis.
Plasmid Vaccination with Insulin B Chain Prevents Autoimmune Diabetes in Nonobese Diabetic Mice
Authors: Bot Adrian , Smith Dan , Bot Simona , Hughes Anna , Wolfe Tom , Wang Lilin , Woods Catherine , von Herrath Matthias
Published: April 2014

The insulin B (InsB) chain bears major type 1 diabetes-associated epitopes of significance for disease in humans and nonobese diabetic (NOD) mice. Somatic expression of InsB chain initiated early in life by plasmid inoculation resulted in substantial protection of female NOD mice against disease. This was associated with a T2 shift in spleen, expansion of IL-4-producing and, to a lesser extent, of IFN-γ-secreting T cells in pancreatic lymph nodes, as well as intermolecular Th2 epitope spreading to glutamic acid decarboxylase determinants. A critical role of IL-4 for the Ag-specific protective effect triggered by plasmid administration was revealed in female IL-4−/− NOD mice that developed diabetes and higher Th1 responses. Coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the unfavorable response of male NOD mice to DNA vaccination with InsB chain. Thus, plasmid-mediated expression of the InsB chain early in diabetes-prone mice has the potential to prevent transition to full-blown disease depending on the presence of IL-4.
Immunoreactivity of Organic Mimeotopes of the E2 Component of Pyruvate Dehydrogenase: Connecting Xenobiotics with Primary Biliary Cirrhosis
Authors: Long S. Alice , Quan Chao , Van de Water Judy , Nantz Michael H. , Kurth Mark J. , Barsky Daniel , Colvin Michael E. , Lam Kit S. , Coppel Ross L. , Ansari Aftab , Gershwin M. Eric
Published: April 2014

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.
Absence of Macrophage-Inflammatory Protein-1α Delays Central Nervous System Demyelination in the Presence of an Intact Blood-Brain Barrier
Authors: McMahon Eileen J. , Cook Don N. , Suzuki Kinuko , Matsushima Glenn K.
Published: April 2014

Chemokines are small chemotactic cytokines that modulate leukocyte recruitment and activation during inflammation. Here, we describe the role of macrophage inflammatory protein-1α (MIP-1α) during cuprizone intoxication, a model where demyelination of the CNS features a large accumulation of microglia/macrophage without T cell involvement or blood-brain barrier disruption. RNase protection assays showed that mRNA for numerous chemokines were up-regulated during cuprizone treatment in wild-type, C57BL/6 mice. RANTES, inflammatory protein-10, and monocyte chemoattractant protein-1 showed greatest expression with initiation of insult at 1–2 wk of treatment, whereas MIP-1α and β increased later at 4–5 wk, coincident with peak demyelination and cellular accumulation. The function of MIP-1α during demyelination was tested in vivo by exposing MIP-1α knockout mice (MIP-1α−/−) to cuprizone and comparing pathology to wild-type mice. Demyelination at 3.5 wk of treatment was significantly decreased in MIP-1α−/− mice (∼36% reduction), a result confirmed by morphology at the electron microscopic level. The delay in demyelination was correlated to apparent decreases in microglia/macrophage and astrocyte accumulation and in TNF-α protein levels. It was possible that larger effects of the MIP-1α deficiency were being masked by other redundant chemokines. Indeed, RNase protection assays revealed increased expression of several chemokine transcripts in both untreated and cuprizone-treated MIP-1α−/− mice. Nonetheless, despite this possible compensation, our studies show the importance of MIP-1α in demyelination in the CNS and highlight its effect, particularly on cellular recruitment and cytokine regulation.
Predominant Th2/Tc2 Polarity of Tumor-Infiltrating Lymphocytes in Human Cervical Cancer
Authors: Sheu Bor-Ching , Lin Rong-Hwa , Lien Huang-Chun , Ho Hong-Nerng , Hsu Su-Ming , Huang Su-Cheng
Published: April 2014

Cytotoxic T lymphocytes (Tc) play a central role in cellular immunity against cancers. The cytotoxic potential of freshly isolated tumor-infiltrating lymphocytes (TILs) is usually not expressed. This suggests the possible existence of as yet unspecified and perhaps complex immunosuppressive factors or cytokines that affect the anti-tumor capacity of these TILs in the tumor milieu. In the present study, we demonstrated for the first time that TILs derived from human cervical cancer tissue consist mainly of Th2/Tc2 phenotypes. In vitro kinetic assays further revealed that cancer cells could direct the tumor-encountered T cells toward the Th2/Tc2 polarity. Cancer cells promote the production of IL-4 and down-regulate the production of IFN-γ in cancer-encountered T cells. The regulatory effects of cervical cancer cells are mediated mainly by IL-10, and TGF-β plays only a synergistic role. The cancer-derived effects can be reversed by neutralizing anti-IL-10 and anti-TGF-β Abs. IL-10 and TGF-β are present in cancer tissue and weakly expressed in precancerous tissue, but not in normal cervical epithelial cells. Our study strongly suggests important regulatory roles of IL-10 and TGF-β in cancer-mediated immunosuppression.
Early Chemokine Cascades in Murine Cardiac Grafts Regulate T Cell Recruitment and Progression of Acute Allograft Rejection
Authors: Morita Ken , Miura Masayoshi , Paolone David R. , Engeman Tara M. , Kapoor Anil , Remick Daniel G. , Fairchild Robert L.
Published: April 2014

The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2a) and syngeneic heart grafts in C57BL/6 (H-2b) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene α (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1β and 1α. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN-γ-inducible protein-10 and monokine induced by IFN-γ, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts.
Deficiency in β2-Microglobulin, But Not CD1, Accelerates Spontaneous Lupus Skin Disease While Inhibiting Nephritis in MRL-FaslprMice: An Example of Disease Regulation at the Organ Level
Authors: Chan Owen T. M. , Paliwal Vipin , McNiff Jennifer M. , Park Se-Ho , Bendelac Albert , Shlomchik Mark J.
Published: April 2014

When mutations that inactivate molecules that function in the immune system have been crossed to murine lupus strains, the result has generally been a uniform up-regulation or down-regulation of autoimmune disease in the end organs. In the current work we report an interesting dissociation of target organ disease in β2-microglobulin (β2m)-deficient MRL-Faslpr (MRL/lpr) mice: lupus skin lesions are accelerated, whereas nephritis is ameliorated. β2m deficiency affects the expression of classical and nonclassical MHC molecules and thus prevents the normal development of CD8- as well as CD1-dependent NK1+ T cells. To further define the mechanism by which β2m deficiency accelerates skin disease, we studied CD1-deficient MRL/lpr mice. These mice do not have accelerated skin disease, excluding a CD1 or NK1+ T cell-dependent mechanism of β2m deficiency. The data indicate that the regulation of systemic disease is not solely governed by regulation of initial activation of autoreactive lymphocytes in secondary lymphoid tissue, as this is equally relevant to renal and skin diseases. Rather, regulation of autoimmunity can also occur at the target organ level, explaining the divergence of disease in skin and kidney in β2m-deficient mice.
Critical Involvement of OX40 Ligand Signals in the T Cell Priming Events During Experimental Autoimmune Encephalomyelitis
Authors: Ndhlovu Lishomwa C. , Ishii Naoto , Murata Kazuko , Sato Takayuki , Sugamura Kazuo
Published: April 2014

OX40 ligand (OX40L) expressed on APCs, and its receptor, OX40 present on activated T cells, are members of the TNF/TNFR family, respectively, and have been located at the sites of inflammatory conditions. We have observed in OX40L-deficient mice (OX40L−/−) an impaired APC capacity and in our recently constructed transgenic mice expressing OX40L (OX40L-Tg), a markedly enhanced T cell response to protein Ags. Using these mice, we demonstrate here the critical involvement of the OX40L-OX40 interaction during the T cell priming events in the occurrence of experimental autoimmune encephalomyelitis (EAE). In OX40L−/− mice, abortive T cell priming greatly reduced the clinical manifestations of actively induced EAE, coupled with a reduction in IFN-γ, IL-2, and IL-6 production in vitro. Adoptive transfer experiments however revealed an efficient transfer of disease to OX40L−/− mice using wild-type donor T cells, indicating an intact capacity of OX40L−/− mice to initiate effector responses. On the other hand, OX40L−/− donor T cells failed to transfer disease to wild-type recipient mice. Furthermore, OX40L-Tg mice developed a greater severity of EAE despite a delayed onset, while both OX40L-Tg/CD28−/− and OX40L-Tg/CD40−/− mice failed to develop EAE demonstrating a requisite for these molecules. These findings indicate a pivotal role played by OX40L in the pathogenesis of EAE.
Control of the Autoimmune Response by Type 2 Nitric Oxide Synthase
Authors: Shi Fu-Dong , Flodström Malin , Kim Soon Ha , Pakala Shyam , Cleary Mary , Ljunggren Hans-Gustaf , Sarvetnick Nora
Published: April 2014

Immune defense against pathogens often requires NO, synthesized by type 2 NO synthase (NOS2). To discern whether this axis could participate in an autoimmune response, we immunized NOS2-deficient mice with the autoantigen acetylcholine receptor, inducing muscle weakness characteristic of myasthenia gravis, a T cell-dependent Ab-mediated autoimmune disease. We found that the acetylcholine receptor-immunized NOS2-deficient mice developed an exacerbated form of myasthenia gravis, and demonstrated that NOS2 expression limits autoreactive T cell determinant spreading and diversification of the autoantibody repertoire, a process driven by macrophages. Thus, NOS2/NO is important for silencing autoreactive T cells and may restrict bystander autoimmune reactions following the innate immune response.
Cutting Edge: Altered Pulmonary Eosinophilic Inflammation in Mice Deficient for Clara Cell Secretory 10-kDa Protein
Authors: Chen Li-Chen , Zhang Zhongjian , Myers Allen C. , Huang Shau-Ku
Published: April 2014

Clara cell secretory protein (CC10) is a steroid-inducible protein, and its in vivo function is currently unclear. The role of CC10 in modulation of pulmonary allergic inflammation was examined in mice deficient for the CC10 gene. Wild-type and homozygous CC10-deficient mice were sensitized with an Ag, OVA, and challenged with either OVA or saline. When compared with that seen in wild-type mice, a significantly higher level of pulmonary eosinophilia was found in Ag-sensitized and challenged CC10-deficient mice. Significantly increased levels of Th2 cytokines IL-4, IL-5, IL-9, and IL-13 were also found in CC10-deficient mice. In addition, an increased level of eotaxin, but not RANTES, was also seen in CC10-deficient mice. No significant difference was observed in the level of a Th1 cytokine, IFN-γ, between different groups of mice. These results provided the first in vivo evidence that CC10 plays a role in the modulation of pulmonary allergic inflammation.
Cutting Edge: The Spirochetemia of Murine Relapsing Fever Is Cleared by Complement-Independent Bactericidal Antibodies
Authors: Connolly Sean E. , Benach Jorge L.
Published: April 2014

Abs are the major effectors of host defense against infections with Borrelia. Bactericidal murine mAbs and their Fabs destroy B. burgdorferi, the agent of Lyme disease, and relapsing fever Borrelia in the absence of complement. These in vitro observations led to the expansion of a search for functionally similar Abs in vivo. In this study, we demonstrate that functionally unique IgM Abs develop in vivo and are responsible for the elimination of spirochetemia in murine models of relapsing fever, without the assistance of complement. Mice deficient in the fifth or third component of complement can clear the spirochetemia, whereas B cell-deficient mice cannot. The B cell-deficient mice developed spirochetemia that was an order of magnitude higher and persisted for a longer period of time in comparison to the wild-type mice. Additionally, B cell-deficient mice passively immunized with immune IgM and with immune serum were protected from challenge.
Relatively Normal Human Lymphopoiesis but Rapid Turnover of Newly Formed B Cells in Transplanted Nonobese Diabetic/SCID Mice
Authors: Rossi Maria Isabel D. , Medina Kay L. , Garrett Karla , Kolar Grant , Comp Phillip C. , Shultz Leonard D. , Capra J. Donald , Wilson Patrick , Schipul Arthur , Kincade Paul W.
Published: April 2014

Human B lineage lymphocyte precursors in chimeric nonobese diabetic/SCID mice transplanted with umbilical cord blood cells were directly compared with those present in normal bone marrow. All precursor subsets were represented and in nearly normal proportions. Cell cycle activity and population dynamics were investigated by staining for the Ki-67 nuclear Ag as well as by incorporation experiments using 5-bromo-2′-deoxyuridine. Again, this revealed that human B lymphopoiesis in chimeras parallels that in normal marrow with respect to replication and progression through the lineage. Moreover, sequencing of Ig gene rearrangement products showed that a diverse repertoire of VH genes was utilized by the newly formed lymphocytes but there was no evidence for somatic hypermutation. The newly formed B cells frequently acquired the CD5 Ag and had a short life span in the periphery. Thus, all molecular requirements for normal B lymphocyte formation are present in nonobese diabetic/SCID mice, but additional factors are needed for recruitment of B cells into a fully mature, long-lived pool. The model can now be exploited to learn about species restricted and conserved environmental cues for human B lymphocyte production.
Dissociation of Hemopoietic Chimerism and Allograft Tolerance After Allogeneic Bone Marrow Transplantation
Authors: Umemura Akihisa , Morita Hirofumi , Chang Li Xian , Tahan Steven , Monaco Anthony P. , Maki Takashi
Published: April 2014

Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.
Regulation of Fas Ligand-Induced Apoptosis by TNF
Authors: Elzey Bennett D. , Griffith Thomas S. , Herndon John M. , Barreiro Ramon , Tschopp Jurg , Ferguson Thomas A.
Published: April 2014

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas+ lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2−/− T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1−/− T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1−/− 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2−/− 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.
Gene Microarrays Reveal Extensive Differential Gene Expression in Both CD4+ and CD8+ Type 1 and Type 2 T Cells
Authors: Chtanova Tatyana , Kemp Roslyn A. , Sutherland Andrew P. R. , Ronchese Franca , Mackay Charles R.
Published: April 2014

An important subdivision of effector T cells can be made based on patterns of cytokine production and functional programs. Type 1 T cells produce IFN-γ and protect against viral pathogens, whereas type 2 cells produce cytokines such as IL-4 and IL-5 and protect against large extracellular parasites. Both CD4+ and CD8+ T cells can be polarized into type 1 or type 2 cytokine-secreting cells, suggesting that both populations play a regulatory role in immune responses. In this study, we used high-density oligonucleotide arrays to produce a comprehensive picture of gene expression in murine CD4+ Th1 and Th2 cells, as well as CD8+ type 1 and type 2 T cells. Polarized type 1 and 2 cells transcribed mRNA for an unexpectedly large number of genes, most of which were expressed in a similar fashion between type 1 and type 2 cells. However, >100 differentially expressed genes were identified for both the CD4+ and CD8+ type 1 and 2 subsets, many of which have not been associated with T cell polarization. These genes included cytokines, transcription factors, molecules involved in cell migration, as well as genes with unknown function. The program for type 1 or type 2 polarization was similar for CD4+ and CD8+ cells, since gene expression patterns were roughly the same. The expression of select genes was confirmed using real-time PCR. The identification of genes associated with T cell polarization may give important insights into functional and phenotypic differences between effector T cell subsets and their role in normal responses and inflammatory disease.