bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Regulation of the Metalloproteinase ADAM-8 during Human Neutrophil Pathophysiological Activation and Its Catalytic Activity on L-Selectin Shedding
Authors: Gómez-Gaviro Maria , Domínguez-Luis Maria , Canchado Javier , Calafat Jero , Janssen Hans , Lara-Pezzi Enrique , Fourie Anne , Tugores Antonio , Valenzuela-Fernández Agustín , Mollinedo Faustino , Sánchez-Madrid Francisco , Díaz-González Federico
Published: April 2014

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.
Regulated Shedding of Transmembrane Chemokines by the Disintegrin and Metalloproteinase 10 Facilitates Detachment of Adherent Leukocytes
Authors: Hundhausen Christian , Schulte Alexander , Schulz Beate , Andrzejewski Michael G. , Schwarz Nicole , von Hundelshausen Philipp , Winter Ulrike , Paliga Krzysztof , Reiss Karina , Saftig Paul , Weber Christian , Ludwig Andreas
Published: April 2014

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.
Histamine 4 Receptor Activation Induces Recruitment of FoxP3+ T Cells and Inhibits Allergic Asthma in a Murine Model
Authors: Morgan Ross K. , McAllister Brian , Cross Lillian , Green Daniel S. , Kornfeld Hardy , Center David M. , Cruikshank William W.
Published: April 2014

Histamine has an important role in regulation of immune response which is mediated by differential expression of four distinct receptors, H1R–H4R. H1R and HR2 have previously been shown to be involved with modulation of lung inflammation. H4R is also expressed on inflammatory cells; therefore, we investigated the potential role of H4R in development of allergic asthma in a murine model. We determined that the H4R agonist 4-methylhistamine when delivered intratracheally before Ag challenge mitigated airway hyperreactivity and inflammation. This was associated with an increase in IL-10 and IFN-γ, but not TGF-β or IL-16, as well as a decrease in IL-13 in the bronchoalveolar lavage fluid. We also observed that H4R agonist instillation resulted in accumulation of FoxP3+ T cells suggesting a direct effect on T regulatory cell recruitment. To investigate this further, we determined the in vitro effect of H4R stimulation on human T cell migration. The H4R agonist induced a 2- to 3-fold increase in T cell migration, similar to that seen for H1R agonists. Cells transmigrating to the H4R agonist, but not H1R, were skewed toward a CD4 cell expressing CD25 and intracellular FoxP3. H4R-responsive cells suppressed proliferation of autologous T cells, an effect that was dependent on IL-10 production. We conclude that H4R stimulation enriches for a regulatory T cell with potent suppressive activity for proliferation. These findings identify a novel function for H4R and suggest a potential therapeutic approach to attenuation of asthmatic inflammation.
TNF-α Inhibits Macrophage Clearance of Apoptotic Cells via Cytosolic Phospholipase A2 and Oxidant-Dependent Mechanisms
Authors: McPhillips Kathleen , Janssen William J. , Ghosh Moumita , Byrne Aideen , Gardai Shyra , Remigio Linda , Bratton Donna L. , Kang Jihee L. , Henson Peter
Published: April 2014

Removal of apoptotic cells from inflammatory sites is an important step in the resolution of inflammation. Both murine and human macrophages stimulated with TNF-α or directly administered arachidonic acid showed an impaired ability to ingest apoptotic cells (efferocytosis). The inhibition was shown to be due to generation of reactive oxygen species, was blocked with a superoxide dismutase mimetic, MnTBAP, and was mimicked by direct addition of H2O2. To determine the mechanism of TNF-α-stimulated oxidant production, bone marrow-derived macrophages from gp91phox-deficient mice were examined but shown to still produce oxidants and exhibit defective apoptotic cell uptake. In contrast, a specific cytosolic phospholipase A2 inhibitor blocked the oxidant production and reversed the inhibited uptake. The suppressive effect of endogenous or exogenous oxidants on efferocytosis was mediated through activation of the GTPase, Rho. It was reversed in macrophages pretreated with C3 transferase to inactivate Rho or with an inhibitor of Rho kinase. During maturation of human monocyte-derived macrophages, only mature cells exhibited TNF-α-induced suppression of apoptotic cell clearance. The resistance of immature macrophages to such inhibition was shown to result not from defective generation of oxidants, but rather, from lack of response of these cells to the oxidants. Overall, the data suggest that macrophages in a TNF-α- and oxidant-rich inflammatory environment are less able to remove apoptotic cells and, thereby, may contribute to the local intensity of the inflammatory response.
Identification of Ectonucleotidases CD39 and CD73 in Innate Protection during Acute Lung Injury
Authors: Eckle Tobias , Füllbier Lars , Wehrmann Manfred , Khoury Joseph , Mittelbronn Michel , Ibla Juan , Rosenberger Peter , Eltzschig Holger K.
Published: April 2014

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5′-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39−/− mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39−/− or cd73−/− mice with soluble apyrase or 5′-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5′-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.
Shiga Toxin Produced by Enterohemorrhagic Escherichia coli Inhibits PI3K/NF-κB Signaling Pathway in Globotriaosylceramide-3-Negative Human Intestinal Epithelial Cells
Authors: Gobert Alain P. , Vareille Marjolaine , Glasser Anne-Lise , Hindré Thomas , de Sablet Thibaut , Martin Christine
Published: April 2014

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli (EHEC) binds to endothelial cells expressing globotriaosylceramide-3 (Gb-3) and induces cell death by inhibiting translation. Nonetheless, the effects of Stx on human enterocytes, which lacks receptor Gb-3, remain less known. In this study, we questioned whether EHEC-derived Stx may modulate cellular signalization in the Gb-3-negative human epithelial cell line T84. Stx produced by EHEC was fixed and internalized by the cells. A weak activation of NF-κB was observed in T84 cells after EHEC infection. Cells infected with an isogenic mutant lacking stx1 and stx2, the genes encoding Stx, displayed an increased NF-κB DNA-binding activity. Consequently, the NF-κB-dependent CCL20 and IL-8 gene transcription and chemokine production were enhanced in T84 cells infected with the Stx mutant in comparison to the wild-type strain. Investigating the mechanism by which Stx modulates NF-κB activation, we showed that the PI3K/Akt signaling pathway was not induced by EHEC but was enhanced by the strain lacking Stx. Pharmacological inhibition of the PI3K/Akt signalization in EHEC ΔStx-infected T84 cells yielded to a complete decrease of NF-κB activation and CCL20 and IL-8 mRNA expression. This demonstrates that the induction of the PI3K/Akt/NF-κB pathway is potentially induced by EHEC, but is inhibited by Stx in Gb-3-negative epithelial cells. Thus, Stx is an unrecognized modulator of the innate immune response of human enterocytes.
IFN-γ-Induced Chemokines Synergize with Pertussis Toxin to Promote T Cell Entry to the Central Nervous System
Authors: Millward Jason M. , Caruso Maria , Campbell Iain L. , Gauldie Jack , Owens Trevor
Published: April 2014

Inflammation of the CNS, which occurs during multiple sclerosis and experimental autoimmune encephalomyelitis, is characterized by increased levels of IFN-γ, a cytokine not normally expressed in the CNS. To investigate the role of IFN-γ in CNS, we used intrathecal injection of a replication-defective adenovirus encoding murine IFN-γ (AdIFNγ) to IFN-γ-deficient (GKO) mice. This method resulted in stable, long-lived expression of IFN-γ that could be detected in cerebrospinal fluid using ELISA and Luminex bead immunoassay. IFN-γ induced expression in the CNS of message and protein for the chemokines CXCL10 and CCL5, to levels comparable to those seen during experimental autoimmune encephalomyelitis. Other chemokines (CXCL2, CCL2, CCL3) were not induced. Mice lacking the IFN-γR showed no response, and a control viral vector did not induce chemokine expression. Chemokine expression was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. IFN-γ-induced chemokine expression did not lead to inflammation. However, when pertussis toxin was given i.p. to mice infected with the IFN-γ vector, there was a dramatic increase in the number of T lymphocytes detected in the CNS by flow cytometry. This increase in blood-derived immune cells in the CNS did not occur with pertussis toxin alone, and did not manifest as histologically detectable inflammatory pathology. These results show that IFN-γ induces a characteristic glial chemokine response that by itself is insufficient to promote inflammation, and that IFN-γ-induced CNS chemoattractant signals can synergize with a peripheral infectious stimulus to drive T cell entry into the CNS.
lgtC Expression Modulates Resistance to C4b Deposition on an Invasive Nontypeable Haemophilus influenzae
Authors: Ho Derek K. , Ram Sanjay , Nelson Kevin L. , Bonthuis Paul J. , Smith Arnold L.
Published: April 2014

We have previously shown that C3 binding to serum-resistant nontypeable Haemophilus influenzae (NTHi) strain R2866 is slower than C3 binding to a serum-sensitive strain. Ab-dependent classical pathway activation is required for complement-dependent killing of NTHi. To further characterize the mechanism(s) of serum resistance of R2866, we compared binding of complement component C4b to R2866 with a serum-sensitive variant, R3392. We show that C4b binding to R2866 relative to R3392 was delayed, suggesting regulation of the classical pathway of complement. Increased C4b deposition on R3392 was independent of the amount and subclass of Ab binding, suggesting that an impediment to C4b binding existed on R2866. Immunoblotting and mass spectrometry indicated that lipooligosaccharide and outer membrane proteins P2 and P5 were targets for C4b. P2 and P5 sequences and expression levels were similar in both strains. Insertional inactivation of the phase-variable lipooligosaccharide biosynthesis gene lgtC in R2866 augmented C4b deposition to levels seen with R3392 and rendered the bacteria sensitive to serum and whole blood. These results suggest a direct role of lgtC expression in the inhibition of C4b deposition and consequent serum resistance of R2866. Alteration of surface glycans of NTHi may be a critical event in determining the ability of a strain to evade host defenses and cause disseminated infection.
Targeted Antireceptor Therapy with Monoclonal Antibodies Leads to the Formation of Inactivated Tetrameric Forms of ErbB Receptors
Authors: Furuuchi Keiji , Berezov Alan , Kumagai Toru , Greene Mark I.
Published: April 2014

mAbs capable of disabling heterodimeric kinase complexes of the epidermal growth factor receptor (EGFR) and human EGFR type 2/neu have therapeutic relevance to various human cancers. In this study, we demonstrate that in addition to the dimer, EGFR and human EGFR type 2 can associate as homo- and heterotetramers. EGF-induced phosphorylation of the tetramers was significantly lower than that of the dimers, indicating that the tetrameric receptor complexes have impaired signaling activity. Targeting v-erb-b2 erythroblastic leukemia viral oncogene homolog (erbB) receptors with mAbs promoted erbB tetrameric assembly, suggesting that a component of the antitumor activity may be mediated by the ability of Abs to shift the equilibrium from active dimeric to impaired tetrameric receptor complex states. This study suggests a novel therapeutic approach to disable signaling of erbB and potentially other receptors in tumors by biologic agents capable of inducing receptor tetramerization.
Coordinated Control of Immunity to Muscle StageTrichinella spiralisby IL-10, Regulatory T Cells, and TGF-β
Authors: Beiting Daniel P. , Gagliardo Lucille F. , Hesse Matthias , Bliss Susan K. , Meskill Diana , Appleton Judith A.
Published: April 2014

We previously demonstrated that IL-10 is critical in the control of acute inflammation during development of Trichinella spiralis in the muscle. In this study, we use gene-targeted knockout mice, adoptive transfer of specific T cell populations, and in vivo Ab treatments to determine the mechanisms by which inflammation is controlled and effector T cell responses are moderated during muscle infection. We report that CD4+CD25− effector T cells, rather than CD4+CD25+ regulatory T cells, suppress inflammation by an IL-10-dependent mechanism that limits IFN-γ production and local inducible NO synthase induction. Conversely, we show that depletion of regulatory T cells during infection results in exaggerated Th2 responses. Finally, we provide evidence that, in the absence of IL-10, TGF-β participates in control of local inflammation in infected muscle and promotes parasite survival.
Distinct NKT Cell Subsets Are Induced by Different Chlamydia Species Leading to Differential Adaptive Immunity and Host Resistance to the Infections
Authors: Joyee Antony George , Qiu Hongyu , Wang Shuhe , Fan Yijun , Bilenki Laura , Yang Xi
Published: April 2014

We investigated the role of NKT cells in immunity to Chlamydia pneumoniae and Chlamydia muridarum infections using a combination of knockout mice and specific cellular activation approaches. The NKT-deficient mice showed exacerbated susceptibility to C. pneumoniae infection, but more resistance to C. muridarum infection. Activation of NKT reduced C. pneumoniae in vivo growth, but enhanced C. muridarum infection. Cellular analysis of invariant NKT cells revealed distinct cytokine patterns following C. pneumoniae and C. muridarum infections, i.e., predominant IFN-γ in the former, while predominant IL-4 in the latter. The cytokine patterns of CD4+ and CD8+ T cells matched those of NKT cells. Our data provide in vivo evidence for a functionally diverse role of NKT cells in immune response to two intracellular bacterial pathogens. These results suggest that distinct NKT subsets are induced by even biologically closely related pathogens, thus leading to differential adaptive immune response and infection outcomes.
Innate Immune Responses to Endosymbiotic Wolbachia Bacteria in Brugia malayi and Onchocerca volvulus Are Dependent on TLR2, TLR6, MyD88, and Mal, but Not TLR4, TRIF, or TRAM
Authors: Hise Amy G. , Daehnel Katrin , Gillette-Ferguson Illona , Cho Eun , McGarry Helen F. , Taylor Mark J. , Golenbock Douglas T. , Fitzgerald Katherine A. , Kazura James W. , Pearlman Eric
Published: April 2014

The discovery that endosymbiotic Wolbachia bacteria play an important role in the pathophysiology of diseases caused by filarial nematodes, including lymphatic filariasis and onchocerciasis (river blindness) has transformed our approach to these disabling diseases. Because these parasites infect hundreds of millions of individuals worldwide, understanding host factors involved in the pathogenesis of filarial-induced diseases is paramount. However, the role of early innate responses to filarial and Wolbachia ligands in the development of filarial diseases has not been fully elucidated. To determine the role of TLRs, we used cell lines transfected with human TLRs and macrophages from TLR and adaptor molecule-deficient mice and evaluated macrophage recruitment in vivo. Extracts of Brugia malayi and Onchocerca volvulus, which contain Wolbachia, directly stimulated human embryonic kidney cells expressing TLR2, but not TLR3 or TLR4. Wolbachia containing filarial extracts stimulated cytokine production in macrophages from C57BL/6 and TLR4−/− mice, but not from TLR2−/− or TLR6−/− mice. Similarly, macrophages from mice deficient in adaptor molecules Toll/IL-1R domain-containing adaptor-inducing IFN-β and Toll/IL-1R domain-containing adaptor-inducing IFN-β-related adaptor molecule produced equivalent cytokines as wild-type cells, whereas responses were absent in macrophages from MyD88−/− and Toll/IL-1R domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) deficient mice. Isolated Wolbachia bacteria demonstrated similar TLR and adaptor molecule requirements. In vivo, macrophage migration to the cornea in response to filarial extracts containing Wolbachia was dependent on TLR2 but not TLR4. These results establish that the innate inflammatory pathways activated by endosymbiotic Wolbachia in B. malayi and O. volvulus filaria are dependent on TLR2-TLR6 interactions and are mediated by adaptor molecules MyD88 and TIRAP/Mal.
Opsonization of HIV with Complement Enhances Infection of Dendritic Cells and Viral Transfer to CD4 T Cells in a CR3 and DC-SIGN-Dependent Manner
Authors: Bouhlal Hicham , Chomont Nicolas , Réquena Mary , Nasreddine Nadine , Saidi Héla , Legoff Jérôme , Kazatchkine Michel D. , Bélec Laurent , Hocini Hakim
Published: April 2014

In the present study, we demonstrated that opsonization of primary HIV-1 with human complement enhances infection of immature monocyte-derived dendritic cells (iDC) and transmission in trans of HIV to autologous CD4+ T lymphocytes. Infection of iDC by opsonized primary R5- and X4-tropic HIV was increased 3- to 5-fold as compared with infection by the corresponding unopsonized HIV. Enhancement of infection was dependent on CR3 as demonstrated by inhibition induced by blocking Abs. The interaction of HIV with CCR5 and CXCR4 on iDC was affected by opsonization. Indeed, stromal-derived factor-1 was more efficient in inhibiting infection of iDC with opsonized R5-tropic HIV-1BaL (45%) than with heat-inactivated complement opsonized virus and similarly RANTES inhibited more efficiently infection of iDC with opsonized X4-tropic HIV-1NDK (42%) than with heat-inactivated complement opsonized virus. We also showed that attachment of complement-opsonized virus to DC-specific ICAM-grabbing nonintegrin (DC-SIGN) molecule on iDC and HeLa DC-SIGN+ CR3− cells was 46% and 50% higher compared with heat-inactivated complement opsonized virus, respectively. Hence, Abs to DC-SIGN suppressed up to 80% and 60% the binding of opsonized virus to HeLa cells and iDC, respectively. Furthermore, Abs to DC-SIGN inhibited up to 70% of the infection of iDC and up to 65% of infection in trans of autologous lymphocytes with opsonized virus. These results further demonstrated the role of DC-SIGN in complement opsonized virus uptake and infection. Thus, the virus uses complement to its advantage to facilitate early steps leading to infection following mucosal transmission of HIV.
Vasoactive Intestinal Peptide Balances Pro- and Anti-Inflammatory Cytokines in the Pseudomonas aeruginosa-Infected Cornea and Protects against Corneal Perforation
Authors: Szliter Elizabeth A. , Lighvani Shahrzad , Barrett Ronald P. , Hazlett Linda D.
Published: April 2014

Corneal infection with Pseudomonas aeruginosa perforates the cornea in susceptible C57BL/6 (B6), but not resistant BALB/c, mice. To determine whether vasoactive intestinal peptide (VIP) played a role in development of the resistant response, protein expression levels were tested by immunocytochemistry and enzyme immunoassay in BALB/c and B6 corneas. Both mouse strains showed constitutive expression of corneal VIP protein and nerve fiber distribution. However, disparate expression patterns were detected in the cornea after infection. VIP protein was elevated significantly in BALB/c over B6 mice at 5 and 7 days postinfection. Therefore, B6 mice were injected with rVIP and subsequently demonstrated decreased corneal opacity and resistance to corneal perforation compared with PBS controls. rVIP- vs PBS-treated B6 mice also demonstrated down-regulation of corneal mRNA and/or protein levels for proinflammatory cytokines/chemokines: IFN-γ, IL-1β, MIP-2, and TNF-α, whereas anti-inflammatory mediators, IL-10 and TGF-β1, were up-regulated. Treatment with rVIP decreased NO levels and polymorphonuclear neutrophil (PMN) number. To further define the role of VIP, peritoneal macrophages (Mφ) and PMN from BALB/c and B6 mice were stimulated with LPS and treated with rVIP. Treatment of LPS-stimulated Mφ from both mouse strains resulted in decreased IL-1β and MIP-2 protein levels; PMN responded similarly. Both cell types also displayed a strain-dependent differential response to rVIP, whereby B6 Mφ/PMN responded only to a higher concentration of VIP compared with cells from BALB/c mice. These data provide evidence that neuroimmune regulation of the cytokine network and host inflammatory cells functions to promote resistance against P. aeruginosa corneal infection.
PDE4 Inhibition Prevents Preterm Delivery Induced by an Intrauterine Inflammation
Authors: Schmitz Thomas , Souil Evelyne , Hervé Roxane , Nicco Carole , Batteux Frédéric , Germain Guy , Cabrol Dominique , Evain-Brion Danièle , Leroy Marie-Josèphe , Méhats Céline
Published: April 2014

The aim of this study was to explore the anti-inflammatory properties of phosphodiesterase-4 (PDE4) inhibitors in vivo and their potential ability to prevent inflammation-induced preterm delivery. Indeed, intrauterine inflammation is the major etiology of very preterm delivery, the leading cause of neonatal mortality and morbidity. Intrauterine injection of Escherichia coli LPS in 15-day-pregnant mice induced an increase of PDE4 activity and PDE4B expression at the maternofetal interface, a rise of amniotic fluid levels of TNF-α, IL-1β, IL-6, and IL-10 and provoked massive preterm delivery and fetal demise. Selective PDE4 inhibition by rolipram prevented the rise in the proinflammatory cytokines. Following the nuclear translocation of the transcription factor NFκB, as a marker of cellular activation after the inflammatory challenge, showed a time-dependent sequential activation of the gestational tissues, from the uterine mesometrial to the fetal compartment, particularly in the glycogen-trophoblastic cells of the placenta. This activation was disrupted by PDE4 inhibition, and inflammation-induced preterm delivery and fetal demise were prevented. PDE4 selective inhibitors may thus represent a novel effective treatment to delay inflammation-induced preterm delivery and to prevent adverse outcomes in infants.
Lipopolysaccharide-Induced Up-Regulation of Triggering Receptor Expressed on Myeloid Cells-1 Expression on Macrophages Is Regulated by Endogenous Prostaglandin E2
Authors: Murakami Yousuke , Kohsaka Hitoshi , Kitasato Hidero , Akahoshi Tohru
Published: April 2014

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified cell surface molecule that is expressed by neutrophils and monocytes. TREM-1 expression is modulated by various ligands for TLRs in vitro and in vivo. However, the influence of PGE2, a potential mediator of inflammation, on TREM-1 expression has not been elucidated. In this study, we examined the effects of PGE2 on LPS-induced TREM-1 expression by resident murine peritoneal macrophages (RPM) and human PBMC. PGE2 significantly induced murine TREM-1 (mTREM-1) expression by RPM. Up-regulation of TREM-1 expression was specific to PGE2 among arachidonic acid metabolites, while ligands for chemoattractant receptor-homologous molecule expressed on Th2 cells and the thomboxane-like prostanoid receptor failed to induce mTREM-1 expression. PGE2 also increased expression of the soluble form of TREM-1 by PBMC. LPS-induced TREM-1 expression was regulated by endogenous PGE2 especially in late phase (>2 h after stimulation), because cyclooxygenase-1 and -2 inhibitors abolished this effect at that points. A synthetic EP4 agonist and 8-Br-cAMP also enhanced mTREM-1 expression by RPM. Furthermore, protein kinase A, PI3K, and p38 MAPK inhibitors prevented PGE2-induced mTREM-1 expression by RPM. Activation of TREM-1 expressed on PGE2-pretreated PBMC by an agonistic TREM-1 mAb significantly enhanced the production of IL-8 and TNF-α. These findings indicate that LPS-induced TREM-1 expression on macrophages is mediated, at least partly, by endogenous PGE2 followed by EP4 and cAMP, protein kinase A, p38 MAPK, and PI3K-mediated signaling. Regulation of TREM-1 and the soluble form of TREM-1 expression by PGE2 may modulate the inflammatory response to microbial pathogens.
IL-8 and IDO Expression by Human Gingival Fibroblasts via TLRs
Authors: Mahanonda Rangsini , Sa-Ard-Iam Noppadol , Montreekachon Pattanin , Pimkhaokham Atiphan , Yongvanichit Kosol , Fukuda Mark M. , Pichyangkul Sathit
Published: April 2014

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-α enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-γ enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-γ, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.
IFN-γ Alters the Response ofBorrelia burgdorferi-Activated Endothelium to Favor Chronic Inflammation
Authors: Dame Tarah M. , Orenzoff Barbara L. , Palmer Lance E. , Furie Martha B.
Published: April 2014

Borrelia burgdorferi, the agent of Lyme disease, promotes proinflammatory changes in the endothelium that lead to the recruitment of leukocytes. The host immune response to infection results in increased levels of IFN-γ in the serum and lesions of Lyme disease patients that correlate with greater severity of disease. Therefore, the effect of IFN-γ on the gene expression profile of primary human endothelial cells exposed to B. burgdorferi was determined. B. burgdorferi and IFN-γ synergistically augmented the expression of 34 genes, 7 of which encode chemokines. Six of these (CCL7, CCL8, CX3CL1, CXCL9, CXCL10, and CXCL11) attract T lymphocytes, and one (CXCL2) is specific for neutrophils. Synergistic production of the attractants for T cells was confirmed at the protein level. IL-1β, TNF-α, and LPS also cooperated with IFN-γ to induce synergistic production of CXCL10 by the endothelium, indicating that IFN-γ potentiates inflammation in concert with a variety of mediators. An in vitro model of the blood vessel wall revealed that an increased number of human T lymphocytes traversed the endothelium exposed to B. burgdorferi and IFN-γ, as compared with unstimulated endothelial monolayers. In contrast, addition of IFN-γ diminished the migration of neutrophils across the B. burgdorferi-activated endothelium. IFN-γ thus alters gene expression by endothelia exposed to B. burgdorferi in a manner that promotes recruitment of T cells and suppresses that of neutrophils. This modulation may facilitate the development of chronic inflammatory lesions in Lyme disease.
The Postnatal Maturation of the Immunoglobulin Heavy Chain IgG Repertoire in Human Preterm Neonates Is Slower than in Term Neonates
Authors: Zemlin Michael , Hoersch Gabriele , Zemlin Cosima , Pohl-Schickinger Anja , Hummel Michael , Berek Claudia , Maier Rolf F. , Bauer Karl
Published: April 2014

During the perinatal period the development of the IgH chain CDR3 (CDR-H3) repertoire of IgM transcripts is maturity-dependent and not influenced by premature exposure to Ag. To study whether maturity-dependent restrictions also predominate in the perinatal IgG repertoire we compared 1000 IgG transcripts from cord blood and venous blood of extremely preterm neonates (24–28 wk of gestation) and of term neonates from birth until early infancy with those of adults. We found the following. First, premature contact with the extrauterine environment induced the premature development of an IgG repertoire. However after preterm birth the diversification of the IgG repertoire was slower than that after term birth. Second, the IgG repertoire of preterm neonates retained immature characteristics such as short CDR-H3 regions and overrepresentation of DH7–27. Third, despite premature exposure to the extrauterine environment, somatic mutation frequency in IgG transcripts of preterm infants remained low until they reached a postconceptional age corresponding to the end of term gestation. Thereafter, somatic mutations accumulated with age at similar rates in preterm and term neonates and reached 30% of the adult level after 6 mo. In conclusion, class switch was inducible already at the beginning of the third trimester of gestation, but the developing IgG repertoire was characterized by similar restrictions as those of the developing IgM repertoire. Those B cells expressing more “mature” H chain sequences were not preferentially selected into the IgG repertoire. Therefore, the postnatal IgG repertoire of preterm infants until the expected date of delivery differs from the postnatal repertoire of term neonates.
Monocyte-Astrocyte Networks Regulate Matrix Metalloproteinase Gene Expression and Secretion in Central Nervous System Tuberculosis In Vitro and In Vivo
Authors: Harris James E. , Nuttall Robert K. , Elkington Paul T. , Green Justin A. , Horncastle Donna E. , Graeber Manuel B. , Edwards Dylan R. , Friedland Jon S.
Published: April 2014

CNS tuberculosis (CNS-TB) is the most deadly form of tuberculous disease accounting for 10% of clinical cases. CNS-TB is characterized by extensive tissue destruction, in which matrix metalloproteinases (MMPs) may play a critical role. We investigated the hypothesis that Mycobacterium tuberculosis activates monocyte-astrocyte networks increasing the activity of key MMPs. We examined the expression of all human MMPs and the tissue inhibitors of metalloproteinases (TIMPs) in human astrocytes stimulated by conditioned medium from M. tuberculosis-infected monocytes (CoMTB). Real-time RT-PCR showed that gene expression of MMP-1, -2, -3, -7, and -9 was increased (p < 0.05). MMP-9 secretion was significantly up-regulated at 24 h and increased over 120 h (p < 0.01). MMP-1, -3, and -7 secretion was not detected. Secretion of MMP-2 was constitutive and unaffected by CoMTB. Astrocyte gene expression and secretion of TIMP-1 was not affected by CoMTB although TIMP-2 secretion increased 3-fold at 120 h. Immunohistochemical analysis of human brain biopsies confirmed that astrocyte MMP-9 secretion is a predominant feature in CNS-TB in vivo. Dexamethasone inhibited astrocyte MMP-9, but not TIMP-1/2 secretion in response to CoMTB. CoMTB stimulated the nuclear translocation of NF-κB, inducing a 6-fold increase in nuclear p65 and a 2-fold increase in nuclear p50. This was associated with degradation of IκBα and β within 30 min, persisting for 24 h. In summary, networks active between monocytes and astrocytes regulate MMP-9 activity in tuberculosis and astrocytes are a major source of MMP-9 in CNS-TB. Astrocytes may contribute to a matrix degrading environment within the CNS and subsequent morbidity and mortality.
Cutting Edge: KIR3DS1, a Gene Implicated in Resistance to Progression to AIDS, Encodes a DAP12-Associated Receptor Expressed on NK Cells That Triggers NK Cell Activation
Authors: Carr William H. , Rosen David B. , Arase Hisashi , Nixon Douglas F. , Michaelsson Jakob , Lanier Lewis L.
Published: April 2014

The killer cell Ig-like receptor (KIR) gene, KIR3DS1, has been implicated in slowing disease progression in HIV infection; however, little is known about its expression, function, or ligand specificity. Using retrovirally transduced NKL cells and peripheral blood NK cells from KIR3DS1-positive donors we assessed expression of this gene by flow cytometry and its function by in vitro assays measuring KIR3DS1-induced cell-mediated cytotoxicity and cytokine production. In the present study, we demonstrate that KIR3DS1 is expressed on peripheral blood NK cells and triggers both cytotoxicity and IFN-γ production. Using cotransfection and coimmunoprecipitation, we found that KIR3DS1 associates with the ITAM-bearing adaptor, DAP12. Soluble KIR3DS1-Ig fusion proteins did not bind to EBV-transformed B lymphoid cell lines transfected with HLA-Bw4 80I or 80T allotypes, suggesting that if KIR3DS1 does recognize HLA-Bw4 ligands, this may be peptide dependent.
Cutting Edge: Distinct NK Receptor Profiles Are Imprinted on CD8 T Cells in the Mucosa and Periphery during the Same Antigen Challenge: Role of Tissue-Specific Factors
Authors: Laouar Amale , Manocha Monika , Wan Meimei , Yagita Hideo , van Lier Rene A. W. , Manjunath N.
Published: April 2014

NK cell receptors (NKRs) modulate T lymphocyte responses by modifying the Ag activation threshold. However, what governs their expression on T cells remains unclear. In this study we show that different NKRs are imprinted on CD8 T cells in the gut mucosa and periphery during the same Ag challenge. After a viral, bacterial, and tumor challenge, most CD8 peritoneal exudate lymphocytes expressed NKG2A but not 2B4. In contrast, most CD8 intraepithelial lymphocytes exhibited 2B4 but not NKG2A. Our data suggest that tissue-specific factors may determine the pattern of NKR expression. In the gut, CD70 licensing appears to promote 2B4 induction on mucosal CD8 T cells. Conversely, retinoic acid produced by the intestinal dendritic cells may suppress NKG2A expression. Thus, tissue-specific factors regulate NKR expression and may confer T cells with differing effector functions in a tissue and site-specific manner.
Augmented Lipopolysaccharide-Induced TNF-α Production by Peritoneal Macrophages in Type 2 Diabetic Mice Is Dependent on Elevated Glucose and Requires p38 MAPK
Authors: Sherry Christina L. , O’Connor Jason C. , Kramer Jason M. , Freund Gregory G.
Published: April 2014

Dysregulated inflammation is a complication of type 2 diabetes (T2D). In this study, we show that augmented LPS-induced TNF-α production by resident peritoneal macrophages (PerMφ) in type 2 diabetic (db/db) mice is dependent on elevated glucose and requires p38 MAPK. Intraperitoneal LPS administered to db/db and nondiabetic (db/+) mice induced 3- and 4-fold more TNF-α in the peritoneum and serum, respectively, of db/db mice as compared with db/+ mice. Examination of the TLR-4/MD2 complex and CD14 expression showed no difference between db/db and db/+ PerMφ. Ex vivo stimulation of PerMφ with LPS produced a similar 3-fold increase in TNF-α production in db/db PerMφ when compared with db/+ PerMφ. PerMφ isolated from db/+ mice incubated in high glucose (4 g/L) medium for 12 h produced nearly 2-fold more TNF-α in response to LPS than PerMφ incubated in normal glucose medium (1 g/L). LPS-dependent stimulation of PI3K activity, ERK1/2 activation, and p38 kinase activity was greater in PerMφ from db/db mice as compared with db/+ mice. Only inhibition of p38 kinase blocked LPS-induced TNF-α production in PerMφ from db/db mice. Taken together, these data indicate that augmented TNF-α production induced by LPS in macrophages during diabetes is due to hyperglycemia and increased LPS-dependent activation of p38 kinase.
Prostaglandin I2 Analogs Inhibit Proinflammatory Cytokine Production and T Cell Stimulatory Function of Dendritic Cells
Authors: Zhou Weisong , Hashimoto Koichi , Goleniewska Kasia , O’Neal Jamye F. , Ji Shaoquan , Blackwell Timothy S. , FitzGerald Garret A. , Egan Karine M. , Geraci Mark W. , Peebles R. Stokes
Published: April 2014

Signaling through the PGI2 receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI2. In this study, we determined that PGI2 analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI2 analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI2 analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-α, IL-1α, IL-6) and chemokines (MIP-1α, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-κB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI2 signaling through the IP may exert anti-inflammatory effects by acting on DC.
Guanosine Inhibits CD40 Receptor Expression and Function Induced by Cytokines and β Amyloid in Mouse Microglia Cells
Authors: D’Alimonte Iolanda , Flati Vincenzo , D’Auro Mariagrazia , Toniato Elena , Martinotti Stefano , Rathbone Michel P. , Jiang Shucui , Ballerini Patrizia , Di Iorio Patrizia , Caciagli Francesco , Ciccarelli Renata
Published: April 2014

Growing evidence implicates CD40, a member of the TNFR superfamily, as contributing to the pathogenesis of many neurodegenerative diseases. Thus, strategies to suppress its expression may be of benefit in those disorders. To this aim, we investigated the effect of guanosine, a purine nucleoside that exerts neurotrophic and neuroprotective effects. CD40 expression and function are increased by exposure of mouse microglia cultures or the N9 microglia cell line to IFN-γ (10 ng/ml) plus TNF-α (50 ng/ml) or β amyloid (Aβ) peptide (Aβ1–42; 500 nM). Culture pretreatment with guanosine (10–300 μM), starting 1 h before cytokine or Aβ addition, dose-dependently inhibited the CD40-induced expression as well as functional CD40 signaling by suppressing IL-6 production promoted by IFN-γ/TNF-α challenge in the presence of CD40 cross-linking. Moreover, guanosine abrogated IFN-γ-induced phosphorylation on Ser727 and translocation of STAT-1α to the nucleus as well as TNF-α-/Aβ-induced IκBα and NF-κB p65/RelA subunit phosphorylation, thus inhibiting NF-κB-induced nuclear translocation. Guanosine effects were mediated by an increased phosphorylation of Akt, a PI3K downstream effector, as well as of ERK1/2 and p38 in the MAPK system, because culture pretreatment with selective ERK1/2, p38 MAPK, and PI3K antagonists (U0126, SB203580, or LY294002, respectively) counteracted guanosine inhibition on IFN-γ/TNF-α-induced CD40 expression and function as well as on STAT-1α or NF-κB nuclear translocation. These findings suggest a role for guanosine as a potential drug in the experimental therapy of neuroinflammatory/neurodegenerative diseases, particularly Alzheimer’s disease.
IL-21 Counteracts the Regulatory T Cell-Mediated Suppression of Human CD4+ T Lymphocytes
Authors: Peluso Ilaria , Fantini Massimo Claudio , Fina Daniele , Caruso Roberta , Boirivant Monica , MacDonald Thomas T. , Pallone Francesco , Monteleone Giovanni
Published: April 2014

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4+CD25+ regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4+CD25+ T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4+CD25− T cells and counteracts the suppressive activities of CD4+CD25+ T cells on CD4+CD25− T cells without affecting the percentage of Foxp3+ cells or survival of Treg. Additionally, CD4+CD25+ T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8+CD25− T cells but does not revert the CD4+CD25+ T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4+ T cells resistant to suppression rather than inhibiting CD4+CD25+ T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4+CD25+ T cells. Data indicate that IL-21 renders human CD4+CD25− T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.
Klotho—a Common Link in Physiological and Rheumatoid Arthritis-Related Aging of Human CD4+ Lymphocytes
Authors: Witkowski Jacek M. , Soroczyńska-Cybula Monika , Bryl Ewa , Smoleńska Żaneta , Jóźwik Agnieszka
Published: April 2014

Human CD4+ T lymphocytes undergo aging-related changes leading to decreased immunity to infections and neoplasms, and to increased frequency of autoimmune diseases including rheumatoid arthritis (RA). Certain changes, observed in the CD4+ cells of RA patients, resemble those observed during physiological aging, but occur at earlier age. Underlying cellular mechanism(s) of these similarities are so far largely unknown. Here we show that KLOTHO, a β-glucuronidase gene whose activity changes are associated with aging phenotype, is down-regulated at the mRNA, protein, and enzymatic (β-glucuronidase) activity levels both in the healthy elderly and especially in RA CD4+ lymphocytes. Although the exact role of Klotho activity for CD4+ cell function is unknown, we propose here that it might be involved in anti-inflammatory processes occurring in the young and healthy individuals, but reduced in both healthy elderly and RA patients. To support this hypothesis, we show here that the reduction of Klotho expression and activity in both elderly and patients’ lymphocytes occurs in concert with the down-regulation of T cell costimulatory molecule CD28, the latter known to be dependent on increased levels of TNF-α. Thus, a common mechanism of KLOTHO down-regulation, but executed at various times in life, may underlie both physiological and disease-related T cell aging. Klotho activity might become a target of anti-RA drug development as well as a tool to help increase the immune system efficiency in the elderly.