bluuebunny/crossref_metadata_2025 huggingface dataset:
Fc Receptor-Mediated Phagocytosis Makes a Significant Contribution to Clearance of Influenza Virus Infections
Authors: Huber Victor C. , Lynch Joyce M. , Bucher Doris J. , Le Jianhua , Metzger Dennis W.
Published: April 2014

Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent cell-mediated cytotoxicity. Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance in recovery from infection with intracellular pathogens is unclear. We have now investigated the role of opsonophagocytosis in protection against lethal influenza virus infection by using FcR γ−/− mice. Absence of the FcR γ-chain did not affect the expression of IFN-γ and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine. Titers of serum and respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR γ−/− mice were similar to levels seen in FcR γ+/+ mice. Nevertheless, FcR γ−/− mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from immune FcR γ+/+ mice. NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found to be capable of actively ingesting opsonized virus particles. We conclude that Fc receptor-mediated phagocytosis plays a pivotal role in clearance of respiratory virus infections.
γδ T Cell-Deficient Mice Have a Down-Regulated CD8+ T Cell Immune Response AgainstEncephalitozoon cuniculiInfection
Authors: Moretto Magali , Durell Brigit , Schwartzman Joseph D. , Khan Imtiaz A.
Published: April 2014

γδ T cells have been reported to play an essential effector role during the early immune response against a wide variety of infectious agents. Recent studies have suggested that the γδ T cell subtype may also be important for the induction of adaptive immune response against certain microbial pathogens. In the present study, an early increase of γδ T cells during murine infection with Encephalitozoon cuniculi, an intracellular parasite, was observed. The role of γδ T cells against E. cuniculi infection was further evaluated by using gene-knockout mice. Mice lacking γδ T cells were susceptible to E. cuniculi infection at high challenge doses. The reduced resistance of δ−/− mice was attributed to a down-regulated CD8+ immune response. Compared with parental wild-type animals, suboptimal Ag-specific CD8+ T cell immunity against E. cuniculi infection was noted in δ−/− mice. The splenocytes from infected knockout mice exhibited a lower frequency of Ag-specific CD8+ T cells. Moreover, adoptive transfer of immune TCRαβ+ CD8+ T cells from the δ−/− mice failed to protect naive CD8−/− mice against a lethal E. cuniculi challenge. Our studies suggest that γδ T cells, due to their ability to produce cytokines, are important for the optimal priming of CD8+ T cell immunity against E. cuniculi infection. This is the first evidence of a parasitic infection in which down-regulation of CD8+ T cell immune response in the absence of γδ T cells has been demonstrated.
Inhibition of Borrelia burgdorferi-Tick Interactions In Vivo by Outer Surface Protein A Antibody
Authors: Pal Utpal , Montgomery Ruth R. , Lusitani Denise , Voet Pierre , Weynants Vincent , Malawista Stephen E. , Lobet Yves , Fikrig Erol
Published: April 2014

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.
Gestational Attenuation of Lyme Arthritis Is Mediated by Progesterone and IL-4
Authors: Moro Manuel H. , Bjornsson Johannes , Marietta Eric V. , Hofmeister Erik K. , Germer Jeffrey J. , Bruinsma Elizabeth , David Chella S. , Persing David H.
Published: April 2014

Infection of different strains of laboratory mice with the agent of Lyme disease, Borrelia burgdorferi, results in arthritis, the severity of which has been correlated with the dominance of Th1 cytokines. In this study, we demonstrate that changes in B. burgdorferi-specific immunologic responses associated with pregnancy can alter the outcome of Lyme arthritis in mice. Whereas nonpregnant female C3H mice consistently developed severe Lyme arthritis, pregnant mice had a marked reduction in arthritis severity that was associated with a slight reduction in IFN-γ and markedly increased levels of IL-4 production by B. burgdorferi-specific T cells. Similar reductions in arthritis severity and patterns of cytokine production were observed in nonpregnant, progesterone-implanted mice. Ab neutralization of IL-4 in progesterone-implanted mice resulted in severe arthritis. Our results are consistent with the known shift toward Th2 cytokine expression at the maternal-fetal interface, and are the first to show a pregnancy-related therapeutic effect in an infectious model.
Transgenic Cystic Fibrosis Mice Exhibit Reduced Early Clearance of Pseudomonas aeruginosa from the Respiratory Tract
Authors: Schroeder Torsten H. , Reiniger Nina , Meluleni Gloria , Grout Martha , Coleman Fadie T. , Pier Gerald B.
Published: April 2014

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the ΔF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 ΔF508 Cftr mice, whereas ΔF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.
Suppression of Immune Response and Protective Immunity to a Japanese Encephalitis Virus DNA Vaccine by Coadministration of an IL-12-Expressing Plasmid
Authors: Chen Hsin-Wei , Pan Chien-Hsiung , Huan Hwei-Wen , Liau Ming-Yi , Chiang Jen-Ron , Tao Mi-Hua
Published: April 2014

IL-12 plays a central role in both innate and acquired immunity and has been demonstrated to potentiate the protective immunity in several experimental vaccines. However, in this study, we show that IL-12 can be detrimental to the immune responses elicited by a plasmid DNA vaccine. Coadministration of the IL-12-expressing plasmid (pIL-12) significantly suppressed the protective immunity elicited by a plasmid DNA vaccine (pE) encoding the envelope protein of Japanese encephalitis virus. This suppressive effect was associated with marked reduction of specific T cell proliferation and Ab responses. A single dose of pIL-12 treatment with plasmid pE in initial priming resulted in significant immune suppression to subsequent pE booster immunization. The pIL-12-mediated immune suppression was dose dependent and evident only when the IL-12 gene was injected either before or coincident with the pE DNA vaccine. Finally, using IFN-γ gene-disrupted mice, we showed that the suppressive activity of the IL-12 plasmid was dependent upon endogenous production of IFN-γ. These results demonstrate that coexpression of the IL-12 gene can sometimes produce untoward effects to immune responses, and thus its application as a vaccine adjuvant should be carefully evaluated.
Transmission Intensity Determines Lymphocyte Responsiveness and Cytokine Bias in Human Lymphatic Filariasis
Authors: King Christopher L. , Connelly Marc , Alpers Michael P. , Bockarie Moses , Kazura James W.
Published: April 2014

Humans living in areas where filariasis is endemic vary greatly in their exposure to mosquito-borne infective third-stage larvae (L3) of these parasitic helminths. Because the intensity of exposure to Ags affects T cell differentiation and susceptibility to parasitic infections in murine models, we compared T cell and cytokine responses in 97 residents of two villages in Papua New Guinea, where transmission intensity of Wuchereria bancrofti differed by 63-fold (37 vs 2355 L3 per person per year). Residents of the high transmission village had 4- to 11-fold lower proliferation and IFN-γ responses to filarial Ags, nonparasite Ag, and PHA by PBMC compared with the low transmission village (p < 0.01) even when subjects were matched for intensity of infection. In contrast, filarial Ag-driven IL-5 production was 5.5-fold greater (p < 0.001), and plasma IL-4 and TGF-β levels were 4-fold and 34% higher, respectively, in residents of the high transmission village. IL-4 and IL-10 responses by PBMC differed little according to village, and increased production of the counterregulatory cytokines IL-10 or TGF-β by PBMC did not correlate with weak proliferation and IFN-γ responses. Plasma IL-5, IFN-γ, and IL-10 levels were similar in the two villages. These data demonstrate that the intensity of exposure to L3 affects lymphocyte responsiveness and cytokine bias possibly by a mechanism that alters APC function.
Heterosubtypic Immunity to Influenza A Virus in Mice Lacking IgA, All Ig, NKT Cells, or γδ T Cells
Authors: Benton Kimberly A. , Misplon Julia A. , Lo Chia-Yun , Brutkiewicz Randy R. , Prasad Shiv A. , Epstein Suzanne L.
Published: April 2014

The mechanisms of broad cross-protection to influenza viruses of different subtypes, termed heterosubtypic immunity, remain incompletely understood. We used knockout mouse strains to examine the potential for heterosubtypic immunity in mice lacking IgA, all Ig and B cells, NKT cells (CD1 knockout mice), or γδ T cells. Mice were immunized with live influenza A virus and compared with controls immunized with unrelated influenza B virus. IgA−/− mice survived full respiratory tract challenge with heterosubtypic virus that was lethal to controls. IgA−/− mice also cleared virus from the nasopharynx and lungs following heterosubtypic challenge limited to the upper respiratory tract, where IgA has been shown to play an important role. Ig−/− mice controlled the replication of heterosubtypic challenge virus in the lungs. Acute depletion of CD4+ or CD8+ T cell subsets abrogated this clearance of virus, thus indicating that both CD4+ and CD8+ T cells are required for protection in the absence of Ig. These results in Ig−/− mice indicate that CD4+ T cells can function by mechanisms other than providing help to B cells for the generation of Abs. Like wild-type mice, CD1−/− mice and γδ−/− mice survived lethal heterosubtypic challenge. Acute depletion of CD4+ and CD8+ cells abrogated heterosubtypic protection in γδ−/− mice, but not B6 controls, suggesting a contribution of γδ T cells. Our results demonstrate that the Ab and cellular subsets deficient in these knockout mice are not required for heterosubtypic protection, but each may play a role in a multifaceted response that as a whole is more effective than any of its parts.
Immunogenicity of the Extracellular Domains of C-C Chemokine Receptor 5 and the In Vitro Effects on Simian Immunodeficiency Virus or HIV Infectivity
Authors: Lehner Thomas , Doyle Carl , Wang Yufei , Babaahmady Kaboutar , Whittall Trevor , Tao Louisa , Bergmeier Lesley , Kelly Charles
Published: April 2014

The C-C chemokine receptor CCR5 serves an important function in chemotaxis of lymphocytes, monocytes, and dendritic cells. CCR5 is also the major coreceptor in most macrophage-tropic HIV-1 infections. Immunization of rhesus macaques with a baculovirus-generated CCR5 construct or peptides derived from the sequences of the four extracellular domains of CCR5 elicited IgG and IgA Abs, inhibition of SIV replication, and CD4+ T cell proliferative responses to three of the extracellular domains of CCR5. The immune sera reacted with cell surface CCR5 expressed on HEK 293 cells. T and B cell epitope mapping revealed major and minor T and B cell epitopes in the N-terminal, first, and second loops of CCR5. The three C-C chemokines, RANTES, macrophage-inflammatory protein-1α, and macrophage-inflammatory protein-1β, were up-regulated by immunization with the CCR5-derived peptides, and the cell surface expression of CCR5 was decreased. The CCR5 Abs were complementary to the C-C chemokines in inhibiting HIV replication in vitro. Immunization with the four extracellular domains of CCR5 suggests that three of them are immunogenic, with maximal T cell responses being elicited by the second loop peptide. However, maximal Abs to the cell surface CCR5 or viral inhibitory Abs in vitro were induced by the N-terminal peptide. Up-regulation of the three C-C chemokines and down-modulation of cell surface CCR5 were elicited by the second loop, N-terminal, and first loop peptides. The data suggest that a dual mechanism of C-C chemokines and specific Abs may engage and down-modulate the CCR5 coreceptors and prevent in vitro HIV or SIV replication.
The Role of T Cell Subsets and Cytokines in the Pathogenesis ofHelicobacter pyloriGastritis in Mice
Authors: Eaton Kathryn A. , Mefford Megan , Thevenot Tracy
Published: April 2014

Gastritis due to Helicobacter pylori in mice and humans is considered a Th1-mediated disease, but the specific cell subsets and cytokines involved are still not well understood. The goal of this study was to investigate the immunopathogenesis of H. pylori-induced gastritis and delayed-type hypersensitivity (DTH) in mice. C57BL/6-Prkdcscid mice were infected with H. pylori and reconstituted with CD4+, CD4-depleted, CD4+CD45RBhigh, or CD4+CD45RBlow splenocytes from wild-type C57BL/6 mice or with splenocytes from C57BL/6IFN-γ−/− or C57BL/6IL-10−/− mice. Four or eight weeks after transfer, DTH to H. pylori Ags was determined by footpad injection; gastritis and bacterial colonization were quantified; and IFN-γ secretion by splenocytes in response to H. pylori Ag was determined. Gastritis and DTH were present in recipients of unfractionated splenocytes, CD4+ splenocytes, and CD4+CD45RBhigh splenocytes, but absent in the other groups. IFN-γ secretion in response to H. pylori Ags was correlated with gastritis, although splenocytes from all groups of mice secreted some IFN-γ. Gastritis was most severe in recipients of splenocytes from IL-10-deficient mice, and least severe in those given IFN-γ-deficient splenocytes. Bacterial colonization in all groups was inversely correlated with gastritis. These data indicate that 1) CD4+ T cells are both necessary and sufficient for gastritis and DTH due to H. pylori in mice; 2) high expression of CD45RB is a marker for gastritis-inducing CD4+ cells; and 3) IFN-γ contributes to gastritis and IL-10 suppresses it, but IFN-γ secretion alone is not sufficient to induce gastritis. The results support the assertion that H. pylori is mediated by a Th1-biased cellular immune response.
A Filarial Nematode-Secreted Phosphorylcholine-Containing Glycoprotein Uncouples the B Cell Antigen Receptor from Extracellular Signal-Regulated Kinase-Mitogen-Activated Protein Kinase by Promoting the Surface Ig-Mediated Recruitment of Src Homology 2 Domain-Containing Tyrosine Phosphatase-1 and Pac-1 Mitogen-Activated Kinase-Phosphatase
Authors: Deehan Maureen R. , Harnett William , Harnett Margaret M.
Published: April 2014

Unraveling the molecular mechanisms by which filarial nematodes, major human pathogens in the tropics, evade the host immune system remains an elusive goal. We have previously shown that excretory-secretory product-62 (ES-62), a homologue of phosphorylcholine-containing molecules that are secreted by human parasites and which is active in rodent models of filarial infection, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal transduction elements in B lymphocytes. Such activation mediates desensitization of subsequent B cell Ag receptor (BCR) ligation-induced activation of extracellular signal-regulated kinase-mitogen-activated protein (ErkMAP) kinase and ultimately B cell proliferation. We now show that the desensitization is due to ES-62 targeting two major regulatory sites of B cell activation. Firstly, pre-exposure to ES-62 primes subsequent BCR-mediated recruitment of SHP-1 tyrosine phosphatase to abolish recruitment of the RasErkMAP kinase cascade via the Igαβ-ShcGrb2Sos adaptor complex interactions. Secondly, any ongoing ErkMAP kinase signaling in ES-62-primed B cells is terminated by the MAP kinase phosphatase, Pac-1 that is activated consequently to challenge via the BCR.
Cytosolic Phospholipase A2 Participates with TNF-α in the Induction of Apoptosis of Human Macrophages Infected withMycobacterium tuberculosisH37Ra
Authors: Duan Lei , Gan Huixian , Arm Jonathan , Remold Heinz G.
Published: April 2014

Macrophage (MΦ) apoptosis, an important innate microbial defense mechanism induced by Mycobacterium tuberculosis (Mtb) H37Ra, depends on the induction of TNF-α synthesis. When protein synthesis is blocked, both infection with Mtb and addition of TNF-α are required to induce caspase 9 activation, caspase 3 activation and apoptosis. In this study, we show that the second protein synthesis-independent signal involves activation of group IV cytosolic phospholipase A2 (cPLA2). Apoptosis of Mtb-infected MΦ and concomitant arachidonic acid release are abrogated by group IV cPLA2 inhibitors (methyl arachidonyl fluorophosphate and methyl trifluoromethyl ketone), but not by inhibitors of group VI Ca2+-independent (iPLA2 ; bromoenol lactone) or of secretory low molecular mass PLA2. In MΦ homogenates, the predominant PLA2 activity showed the same inhibitor sensitivity pattern and preferred arachidonic acid over palmitic acid in substrates, also indicating the presence of one or more group IV cPLA2 enzymes. In concordance with these findings, MΦ lysates contained transcripts and protein for group IV cPLA2-α and cPLA2-γ. Importantly, group IV cPLA2 inhibitors significantly reduced MΦ antimycobacterial activity and addition of arachidonic acid, the major product of group IV cPLA2, to infected MΦ treated with cPLA2 inhibitors completely restored the antimycobacterial activity. Importantly, addition of arachidonic acid alone to infected MΦ significantly reduced the mycobacterial burden. These findings indicate that Mtb induces MΦ apoptosis by independent signaling through at least two pathways, TNF-α and cPLA2, which are both also critical for antimycobacterial defense of the MΦ .
Mannosylated Lipoarabinomannans Inhibit IL-12 Production by Human Dendritic Cells: Evidence for a Negative Signal Delivered Through the Mannose Receptor
Authors: Nigou Jérôme , Zelle-Rieser Claudia , Gilleron Martine , Thurnher Martin , Puzo Germain
Published: April 2014

IL-12 is a key cytokine in directing the development of type 1 Th cells, which are critical to eradicate intracellular pathogens such as Mycobacterium tuberculosis. Here, we report that mannose-capped lipoarabinomannans (ManLAMs) from Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis inhibited, in a dose-dependant manner, the LPS-induced IL-12 production by human dendritic cells. The inhibitory activity was abolished by the loss of the mannose caps or the GPI acyl residues. Mannan, which is a ligand for the mannose receptor (MR) as well as an mAb specific for the MR, also inhibited the LPS-induced IL-12 production by dendritic cells. Our results indicate that ManLAMs may act as virulence factors that contribute to the persistence of M. bovis bacillus Calmette-Guérin and M. tuberculosis within phagocytic cells by suppressing IL-12 responses. Our data also suggest that engagement of the MR by ManLAMs delivers a negative signal that interferes with the LPS-induced positive signals delivered by the Toll-like receptors.
Cooperative Regulation of Mcl-1 by Janus Kinase/STAT and Phosphatidylinositol 3-Kinase Contribute to Granulocyte-Macrophage Colony-Stimulating Factor-Delayed Apoptosis in Human Neutrophils
Authors: Epling-Burnette P. K. , Zhong Bin , Bai Fanqi , Jiang Kun , Bailey Ratna D. , Garcia Roy , Jove Richard , Djeu Julie Y. , Loughran Thomas P. , Wei Sheng
Published: April 2014

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.
Antibody-Mediated Phagocytosis of the Amyloid β-Peptide in Microglia Is Differentially Modulated by C1q
Authors: Webster Scott D. , Galvan Manuel D. , Ferran Erick , Garzon-Rodriguez William , Glabe Charles G. , Tenner Andrea J.
Published: April 2014

Microglial ingestion of the amyloid β-peptide (Aβ) has been viewed as a therapeutic target in Alzheimer’s disease, in that approaches that enhance clearance of Aβ relative to its production are predicted to result in decreased senile plaque formation, a proposed contributor to neuropathology. In vitro, scavenger receptors mediate ingestion of fibrillar Aβ (fAβ) by microglia. However, the finding that cerebral amyloid deposition in a transgenic mouse model of Alzheimer’s disease was diminished by inoculation with synthetic Aβ has suggested a possible therapeutic role for anti-Aβ Ab-mediated phagocytosis. Microglia also express C1qRP, a receptor for complement protein C1q, ligation of which in vitro enhances phagocytosis of immune complexes formed with IgG levels below that required for optimal FcR-mediated phagocytosis. The data presented here demonstrate FcR-dependent ingestion of Aβ-anti-Aβ complexes (IgG-fAβ) by microglia that is a function of the amount of Ab used to form immune complexes. In addition, C1q incorporated into IgG-fAβ enhanced microglial uptake of these complexes when they contained suboptimal levels of anti-Aβ Ab. Mannose binding lectin and lung surfactant protein A, other ligands of C1qRP, also enhanced ingestion of suboptimally opsonized IgG-fAβ, whereas control proteins did not. Our data suggest that C1qRP-mediated events may promote efficient ingestion of Aβ at low Ab titers, and this may be beneficial in paradigms that seek to clear amyloid via FcR-mediated mechanisms by minimizing the potential for destructive Ab-induced complement-mediated processes.
IL-12 Suppression During Experimental Endotoxin Tolerance: Dendritic Cell Loss and Macrophage Hyporesponsiveness
Authors: Wysocka Maria , Robertson Susan , Riemann Helge , Caamano Jorge , Hunter Christopher , Mackiewicz Agnieszka , Montaner Luis J. , Trinchieri Giorgio , Karp Christopher L.
Published: April 2014

Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experimental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phenomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of secondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11chigh DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-α, IFN-αβ, or nitric oxide, or the NF-κB family members p50, p52, or RelB.
Surfactant Protein D Regulates NF-κB and Matrix Metalloproteinase Production in Alveolar Macrophages via Oxidant-Sensitive Pathways
Authors: Yoshida Mitsuhiro , Korfhagen Thomas R. , Whitsett Jeffrey A.
Published: April 2014

Targeted ablation of the surfactant protein D (SP-D) gene caused progressive pulmonary emphysema associated with pulmonary infiltration by foamy alveolar macrophages (AMs), increased hydrogen peroxide production, and matrix metalloproteinase (MMP)-2, -9, and -12 expression. In the present study, the mechanisms by which SP-D influences macrophage MMP activity were assessed in AMs from SP-D−/− mice. Tissue lipid peroxides and reactive carbonyls were increased in lungs of SP-D−/− mice, indicating oxidative stress. Immunohistochemical staining of AMs from SP-D−/− mice demonstrated that NF-κB was highly expressed and translocated to the nucleus. Increased NF-κB binding was detected by EMSA in nuclear extracts of AMs isolated from SP-D−/− mice. Antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited MMP production by AMs from SP-D−/− mice. To assess whether increased oxidant production influenced NF-κB activation and production of MMP-2 and -9, AMs from SP-D−/− mice were treated with the NADPH oxidase inhibitors diphenylene iodonium chloride and apocynin. Inhibition of NADPH oxidase suppressed NF-κB binding by nuclear extracts and decreased production of MMP-2 and 9 in AMs from SP-D−/− mice. SN-50, a synthetic NF-κB-inhibitory peptide, decreased MMP production by AMs from SP-D−/− mice. Oxidant production and reactive oxygen species were increased in lungs of SP-D−/− mice, in turn activating NF-κB and MMP expression. SP-D plays an unexpected inhibitory role in the regulation of NF-κB in AMs.
A CD2-Green Fluorescence Protein-Transgenic Mouse Reveals Very Late Antigen-4-Dependent CD8+ Lymphocyte Rolling in Inflamed Venules
Authors: Singbartl Kai , Thatte Jayant , Smith Michael L. , Wethmar Klaus , Day Kathy , Ley Klaus
Published: April 2014

Intravital microscopy allows detailed analysis of leukocyte trafficking in vivo, but fails to identify the nature of leukocytes investigated. Here, we describe the development of a CD2-enhanced green fluorescence protein (EGFP)-transgenic mouse to characterize lymphocyte trafficking during inflammation in vivo. A CD2-EGFP plasmid construct including the CD2 promoter, the EGFP transgene, and the CD2 locus control region was injected into B6CBA/F1 pronuclei. EGFP+ offspring were backcrossed into C57BL/6 mice for six generations. Flow cytometry demonstrated that all peripheral blood EGFP+ cells were positive for CD2 and negative for the granulocyte Ag Ly 6-G (GR-1). EGFPhigh cells stained positive for CD2, CD3, CD8, TCR β-chain, and NK1.1 but did not express the B cell and monocyte markers CD45RA, CD19, and CD11b. In vitro stimulation assays revealed no difference in lymphocyte proliferation and IL-2 secretion between EGFP+ and EGFP− mice. Intravital microscopy of untreated or TNF-α-treated cremaster muscle venules showed EGFP+ cells in vivo, but these cells did not roll or adhere to the vessel wall. In cremaster muscle venules treated with both TNF-α and IFN-γ, EGFPhigh cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 μm/s) than that of neutrophils (10 μm/s). Blocking α4 integrin with a mAb increased rolling velocity to 24 μm/s. These findings show that CD8+ T cells roll in TNF-α/IFN-γ-pretreated vessels in vivo via an α4 integrin-dependent pathway.
Minocycline Provides Neuroprotection AgainstN-Methyl-d-aspartate Neurotoxicity by Inhibiting Microglia
Authors: Tikka Tiina M. , Koistinaho Jari E.
Published: April 2014

Glutamate excitotoxicity to a large extent is mediated through activation of the N-methyl-d-aspartate (NMDA)-gated ion channels in several neurodegenerative diseases and ischemic stroke. Minocycline, a tetracycline derivative with antiinflammatory effects, inhibits IL-1β-converting enzyme and inducible nitric oxide synthase up-regulation in animal models of ischemic stroke and Huntington’s disease and is therapeutic in these disease animal models. Here we report that nanomolar concentrations of minocycline protect neurons in mixed spinal cord cultures against NMDA excitotoxicity. NMDA treatment alone induced microglial proliferation, which preceded neuronal death, and administration of extra microglial cells on top of these cultures enhanced the NMDA neurotoxicity. Minocycline inhibited all these responses to NMDA. Minocycline also prevented the NMDA-induced proliferation of microglial cells and the increased release of IL-1β and nitric oxide in pure microglia cultures. Finally, minocycline inhibited the NMDA-induced activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells, and a specific p38 MAPK inhibitor, but not a p44/42 MAPK inhibitor, reduced the NMDA toxicity. Together, these results suggest that microglial activation contributes to NMDA excitotoxicity and that minocycline, a tetracycline derivative, represents a potential therapeutic agent for brain diseases.
Blockade of α5β1 Integrins Reverses the Inhibitory Effect of Tenascin on Chemotaxis of Human Monocytes and Polymorphonuclear Leukocytes Through Three-Dimensional Gels of Extracellular Matrix Proteins
Authors: Loike John D. , Cao Long , Budhu Sadna , Hoffman Stanley , Silverstein Samuel C.
Published: April 2014

Tenascin is an extracellular matrix protein found in adults in T cell-dependent areas of lymphoid tissues, sites of inflammation, and tumors. We report here that it inhibited chemotaxis of chemoattractant-stimulated human monocytes and chemoattractant-stimulated polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of collagen I or Matrigel, and chemotaxis of leukotriene B4-stimulated PMN through fibrin gels. The inhibitory effect of tenascin on monocyte or PMN chemotaxis through these matrices was reversed by Abs directed against α5β1 integrins or by a peptide (GRGDSP) that binds to β1 integrins. Tenascin did not affect leukotriene B4- or fMLP-stimulated expression of β1 or β2 integrins, but did exert a small inhibitory effect on PMN adhesion and closeness of apposition to fibrin(ogen)-containing surfaces. Thus, α5β1 integrins mediate the inhibitory effect of tenascin on monocyte and PMN chemotaxis, without promoting close apposition between these leukocytes and surfaces coated with tenascin alone or with tenascin bound to other matrix proteins. This contrasts with the role played by α5β1 integrins in promoting close apposition between fMLP-stimulated PMN and fibrin containing surfaces, thereby inhibiting chemotaxis of fMLP-stimulated PMN through fibrin gels. Thus, chemoattractants and matrix proteins regulate chemotaxis of phagocytic leukocytes by at least two different mechanisms: one in which specific chemoattractants promote very tight adhesion of leukocytes to specific matrix proteins and another in which specific matrix proteins signal cessation of migration without markedly affecting strength of leukocyte adhesion.
Hypoxia-Inducible Factor 1-Mediated Inhibition of Peroxisome Proliferator-Activated Receptor α Expression During Hypoxia
Authors: Narravula Sailaja , Colgan Sean P.
Published: April 2014

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone-binding proteins that regulate transcriptional responses to peroxisome proliferators and structurally diverse fatty acids. PPARs have been implicated in a wide variety of functions, including lipid homeostasis and inflammatory responses. In this study, we examined the expression of PPAR-α in response to ambient hypoxia. Initial studies using microarray analysis of intestinal epithelial mRNA revealed that hypoxia rapidly down-regulates PPAR-α mRNA and protein in epithelial cells in vitro and in vivo. Subsequent studies revealed that the PPAR-α gene bears a DNA consensus motif for the transcription factor hypoxia-inducible factor 1 (HIF-1). EMSA analysis revealed that ambient hypoxia induces HIF-1α binding to the HIF-1 consensus domain of PPAR-α in parallel to HIF-1 nuclear accumulation, and antisense depletion of HIF-1α resulted in a loss of PPAR-α down-regulation. The PPAR-α ligand pirinixic acid (WY14643) functionally promoted IFN-γ-induced ICAM-1 expression in normoxic epithelia, and this response was lost in cells pre-exposed to ambient hypoxia. Such results indicate that HIF-1-dependent down-regulation of PPAR-α may provide an adaptive response to proinflammatory stimuli during cellular hypoxia. These studies provide unique insight into the regulation of PPAR-α expression and, importantly, provide an example of a down-regulatory pathway mediated by HIF-1.
Non-Small Cell Lung Cancer Cells Induce Monocytes to Increase Expression of Angiogenic Activity
Authors: White Eric S. , Strom Scott R. B. , Wys Noel L. , Arenberg Douglas A.
Published: April 2014

Tumors are dependent on angiogenesis for survival and propagation. Accumulated evidence suggests that macrophages are a potentially important source of angiogenic factors in many disease states. However, the role(s) of macrophages in non-small cell lung cancer (NSCLC) have not been determined. We hypothesized that monocyte-derived macrophages are induced by NSCLC to increase expression of angiogenic factors. To define the role of macrophage-tumor cell interaction with respect to angiogenesis, human peripheral blood monocytes (PBM) were cocultured with A549 (human bronchoalveolar cell carcinoma) or Calu 6 (human anaplastic carcinoma) NSCLC cells. The resultant conditioned medium (CM) was evaluated for angiogenic potential and for expression of angiogenic factors. We found that endothelial cell chemotactic activity (as a measure of angiogenic potential) was significantly increased in response to CM from cocultures of PBM/NSCLC compared with PBM alone, NSCLC alone, or a combination of NSCLC and PBM CM generated separately. Subsequent analysis by ELISA reveals markedly increased CXC chemokine expression, with a lesser increase in vascular endothelial growth factor, in CM from PBM/NSCLC coculture. Neutralizing Ab to angiogenic CXC chemokines blocked the increase in endothelial cell chemotaxis. Furthermore, with separately generated CM as a stimulus, we found that macrophages are the predominant source of increased CXC chemokine expression. Finally, we found that NSCLC-derived macrophage migration-inhibitory factor is responsible for the increased expression of macrophage-derived angiogenic activity. These data suggest that the interaction between host macrophages and NSCLC cells synergistically increases angiogenic potential, and that this is due to an increased elaboration of angiogenic CXC chemokines.
Peripheral Blood Fibrocytes: Differentiation Pathway and Migration to Wound Sites
Authors: Abe Riichiro , Donnelly Seamas C. , Peng Tina , Bucala Richard , Metz Christine N.
Published: April 2014

Fibrocytes are a distinct population of blood-borne cells that display a unique cell surface phenotype (collagen I+/CD11b+/CD13+/CD34+/CD45RO+/MHC class II+/CD86+) and exhibit potent immunostimulatory activities. Circulating fibrocytes rapidly enter sites of tissue injury, suggesting an important role for these cells in wound repair. However, the regulatory processes that govern the differentiation of blood-borne fibrocytes and the mechanisms that underlie the migration of these cells to wound sites are currently not known. We report herein that ex vivo cultured fibrocytes can differentiate from a CD14+-enriched mononuclear cell population and that this process requires contact with T cells. Furthermore, we demonstrate that TGF-β1 (1–10 ng/ml), an important fibrogenic and growth-regulating cytokine involved in wound healing, increases the differentiation and functional activity of cultured fibrocytes. Because fibrocytes home to sites of tissue injury, we examined the role of chemokine/chemokine receptor interactions in fibrocyte trafficking. We show that secondary lymphoid chemokine, a ligand of the CCR7 chemokine receptor, acts as a potent stimulus for fibrocyte chemotaxis in vitro and for the homing of injected fibrocytes to sites of cutaneous tissue injury in vivo. Finally, we demonstrate that differentiated, cultured fibrocytes express α smooth muscle actin and contract collagen gels in vitro, two characteristic features of wound-healing myofibroblasts. These data provide important insight into the control of fibrocyte differentiation and trafficking during tissue repair and significantly expand their potential role during wound healing.
Ubiquitous Transgenic Expression of the IL-23 Subunit p19 Induces Multiorgan Inflammation, Runting, Infertility, and Premature Death
Authors: Wiekowski Maria T. , Leach Michael W. , Evans Ellen W. , Sullivan Lee , Chen Shu-Cheng , Vassileva Galya , Bazan J. Fernando , Gorman Daniel M. , Kastelein Robert A. , Narula Satwant , Lira Sergio A.
Published: April 2014

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-α and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.
Eotaxin (CCL11) Induces In Vivo Angiogenic Responses by Human CCR3+ Endothelial Cells
Authors: Salcedo Rosalba , Young Howard A. , Ponce M. Lourdes , Ward Jerrold M. , Kleinman Hynda K. , Murphy William J. , Oppenheim Joost J.
Published: April 2014

Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1α (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin’s lymphoma, nasal polyposis, endometriosis, and allergic diathesis.
Myelin Antigen-Specific CD8+ T Cells Are Encephalitogenic and Produce Severe Disease in C57BL/6 Mice
Authors: Sun Deming , Whitaker John N. , Huang Zhigang , Liu Di , Coleclough Christopher , Wekerle Hartmut , Raine Cedric S.
Published: April 2014

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35–55 from myelin oligodendrocyte glycoprotein (pMOG35–55) consistently activate a high proportion of CD8+ αβTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG35–55. CNS lesions in pMOG35–55 CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1−/− mice, but not in isogeneic β2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1−/− mice in which disease was induced adoptively with <1 × 106 T cells sensitized to pMOG35–55. It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.
MHC Class II-Regulated Central Nervous System Autoaggression and T Cell Responses in Peripheral Lymphoid Tissues Are Dissociated in Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis
Authors: Weissert Robert , de Graaf Katrien L. , Storch Maria K. , Barth Silvia , Linington Christopher , Lassmann Hans , Olsson Tomas
Published: April 2014

We dissected the requirements for disease induction of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis in MHC (RT1 in rat) congenic rats with overlapping MOG peptides. Immunodominance with regard to peptide-specific T cell responses was purely MHC class II dependent, varied between different MHC haplotypes, and was linked to encephalitogenicity only in RT1.Ba/Da rats. Peptides derived from the MOG sequence 91–114 were able to induce overt clinical signs of disease accompanied by demyelinated CNS lesions in the RT1.Ba/Da and RT1n haplotypes. Notably, there was no detectable T cell response against this encephalitogenic MOG sequence in the RT1n haplotype in peripheral lymphoid tissue. However, CNS-infiltrating lymphoid cells displayed high IFN-γ, TNF-α, and IL-4 mRNA expression suggesting a localization of peptide-specific reactivated T cells in this compartment. Despite the presence of MOG-specific T and B cell responses, no disease could be induced in resistant RT1l and RT1u haplotypes. Comparison of the number of different MOG peptides binding to MHC class II molecules from the different RT1 haplotypes suggested that susceptibility to MOG-experimental autoimmune encephalomyelitis correlated with promiscuous peptide binding to RT1.B and RT1.D molecules. This may suggest possibilities for a broader repertoire of peptide-specific T cells to participate in disease induction. We demonstrate a powerful MHC class II regulation of autoaggression in which MHC class II peptide binding and peripheral T cell immunodominance fail to predict autoantigenic peptides relevant for an autoaggressive response. Instead, target organ responses may be decisive and should be further explored.
Protein Kinase A RIβ Subunit Deficiency in Lupus T Lymphocytes: Bypassing a Block in RIβ Translation Reconstitutes Protein Kinase A Activity and Augments IL-2 Production
Authors: Khan Islam U. , Laxminarayana Dama , Kammer Gary M.
Published: April 2014

A profound deficiency of type I protein kinase A (PKA-I or RIα/β2C2) phosphotransferase activity occurs in the T lymphocytes of 80% of subjects with systemic lupus erythematosus (SLE), an autoimmune disorder of unknown etiology. This isozyme deficiency is predominantly the product of reduced or absent β isoform of the type I regulatory subunit (RIβ). Transient transfection of RIβ cDNAs from SLE subjects into autologous T cells that do not synthesize the RIβ subunit bypassed the block, resulting in RIβ subunit synthesis and restoration of the PKA-Iβ (RIβ2C2) holoenzyme. Transfected T cells activated via the T cell surface receptor complex revealed a significant increase of cAMP-activatable PKA activity that was associated with a significant increase in IL-2 production. These data demonstrate that a disorder of RIβ translation exists, and that correction of the PKA-I deficiency may enhance T lymphocyte effector functions in SLE.