bluuebunny/crossref_metadata_2025 huggingface dataset:
Staphylococcal Enterotoxin B Stimulates Expansion of Autoreactive T Cells That Induce Apoptosis in Intestinal Epithelial Cells: Regulation of Autoreactive Responses by IL-10
Authors: Ito Komei , Takaishi Hiromasa , Jin Yide , Song Fei , Denning Tim L. , Ernst Peter B.
Published: April 2014

T cell responses to self Ags and normal microbial flora are carefully regulated to prevent autoreactivity. Because IL-10-deficient mice develop colitis, and this response is triggered by luminal flora, we investigated whether IL-10 regulates the ability of microbial Ags to induce autoreactive T cells that could contribute to intestinal inflammation. T cells from wild-type mice were primed with staphylococcal enterotoxin B (SEB) in vitro, which induced an autoreactive proliferative response to syngeneic feeder cells. The cells were predominately CD3+ and CD4+. T cells from IL-10-deficient mice were constitutively autoreactive, and SEB priming enhanced this further. The autoreactive, proliferative response of T cells from wild-type mice was suppressed by IL-10 in the primary or secondary culture, and this effect was inhibited by neutralizing Abs to the IL-10R. To confirm that an autoreactive repertoire was expanded after SEB priming, we used CBA/J mice (Mls-1a) in which autoreactive T cells recognizing the endogenous viral superantigen are depleted (Vβ6, 7, 8.1 TCR-bearing cells). However, SEB rescued these autoreactive T cell repertoires. Adding anti-MHC class II Ab blocked the autoreactive response. SEB-primed splenic or colonic T cells also induced apoptosis in syngeneic intestinal epithelial cells that was blocked significantly by IL-10. Thus, microbial Ags have the potential to abrogate self tolerance by stimulating autoreactive T cells that become cytolytic to target cells. IL-10 plays a protective role in maintaining self tolerance after microbial stimulation by preventing the activation of T cells that contribute to epithelial cell damage.
In Vivo Survival of Autoreactive B Cells: Characterization of Long-Lived B Cells
Authors: Morris Suzanne C. , Moroldo Marta , Giannini Edward H. , Orekhova Tatyana , Finkelman Fred D.
Published: April 2014

To determine the effects of chronic Ag stimulation on B cell survival and phenotype, we compared survival and surface markers of hen egg lysozyme (HEL)-specific B cells in Ig transgenic (Tgn) mice, which lack HEL, and in HEL-Ig transgenic mice, which express soluble HEL. Serum HEL levels were maximized in HEL-Ig Tgn mice by feeding them zinc, which activates the metallothionein promoter that regulates HEL expression. B cell age was characterized by expression of heat-stable Ag, and B220 and B cell survival was studied by evaluating changes in B cell number when lymphopoiesis was suppressed with anti-IL-7 mAb and by identifying newly generated B cells through 5-bromo-2′-deoxyuridine incorporation. Our observations show that the mean B cell life span is considerably reduced in HEL-Ig Tgn compared with Ig Tgn mice, but also demonstrate that some HEL-Ig Tgn B cells survive to maturity. Some of these surviving B cells have undergone receptor editing (substitution of an endogenous Ig light chain for the transgenic Ig light chain), so that their ability to bind HEL is decreased or absent. Surviving HEL-Ig Tgn B cells that retain HEL specificity express decreased mIgD and little or no mIgM. mIgD expression progressively decreases with increasing HEL-Ig Tgn B cell age. These observations suggest that self Ag-specific B cells can survive in the presence of soluble self Ag by down-regulating mIg expression, which should limit B cell signaling by Ag that might otherwise cause deletion of these cells.
Mature CD4+ T Cells Perceive a Positively Selecting Class II MHC/Peptide Complex in the Periphery
Authors: Muranski Pawel , Chmielowski Bartosz , Ignatowicz Leszek
Published: April 2014

A repertoire of TCRs is selected in the thymus by interactions with MHC bound to self-derived peptides. Whether self peptides bound to MHC influence the survival of mature T cells in the periphery remains enigmatic. In this study, we show that the number of naive CD4+ T cells that developed in mice with class II MHC bound with endogenous peptides (Abwt) diminished when transferred into mice with Ab covalently bound with a single peptide (AbEp). Moreover, transfer of a mixture of naive CD4+ T cells derived from Abwt and from AbEp mice into AbEp mice resulted in the expansion of the latter and decline of the former. In contrast, when wild-type activated CD4+ T cells were transferred into AbEp or Abwt mice, these cells survived in both recipients for more than 4 wk, but further expanded in the Abwt host. We conclude that to survive, naive CD4+ T cells favor peripheral expression of the class II MHC/peptide complex(es) involved in their thymic selection, whereas some of activated CD4+ T cells may require them only for expansion.
A Putative 12 Transmembrane Domain Cotransporter Expressed in Thymic Cortical Epithelial Cells
Authors: Kim Moon Gyo , Flomerfelt Francis A. , Lee Kee-Nyung , Chen Chuan , Schwartz Ronald H.
Published: April 2014

We have isolated a full-length cDNA clone (thymic stromal origin (TSO)-1C12) from a SCID thymus library using a probe from a PCR-based subtractive library enriched for sequences from fetal thymic stromal cells. TSO-1C12 mRNA is expressed mainly in the thymic cortex and is highly enriched in SCID thymus. Expression per cell is highest during fetal thymus development and decreases after day 16. Antipeptide Abs immunoprecipitated a hydrophobic, plasma membrane glycoprotein (thymic stromal cotransporter, TSCOT) whose translated sequence has weak homology to bacterial antiporters and mammalian cation cotransporters with 12 transmembrane domains. TSCOT represents a new member of this superfamily that is highly expressed in thymic cortical epithelial cells.
TNF-α, IL-4, and IFN-γ Regulate Differential Expression of P- and E-Selectin Expression by Porcine Aortic Endothelial Cells
Authors: Stocker Claire J. , Sugars Katharine L. , Harari Olivier A. , Landis R. Clive , Morley Bernard J. , Haskard Dorian O.
Published: April 2014

P- and E-selectin are surface glycoproteins that mediate leukocyte rolling on the surface of endothelium in inflammation. We have cloned porcine P-selectin cDNA and generated a mAb, 12C5, with which to examine P-selectin expression by porcine aortic endothelial cells (PAEC) in comparison with that of E-selectin. Basal expression by PAEC of P-selectin was greater than that of E-selectin, whereas E-selectin expression was more prominently enhanced than that of P-selectin by stimulation with TNF-α or IL-1α. Both human or porcine IL-4 led to an increase in P-selectin expression, with kinetics that were delayed compared with those seen following stimulation with TNF-α or IL-1α, but IL-4 did not stimulate expression of E-selectin. When cells were stimulated with TNF-α in the presence of IL-4, we observed enhanced P-selectin expression with a parallel reduction in E-selectin expression. Finally, the increase in P-selectin expression due to human IL-4 was reduced in the presence of porcine but not human IFN-γ. These observations show that E-selectin and P-selectin expression are differentially regulated in PAEC, and that IL-4 leads to a shift in the relative surface density of the two molecules toward P-selectin. The ability of porcine IFN-γ to inhibit IL-4-induced P-selectin expression suggests that the balance between Th1 and Th2 cytokine production may determine the relative densities of the two selectins in chronic immune-mediated inflammation. Because the increased expression of P-selectin induced by human IL-4 was not inhibited by human IFN-γ, this balance may be shifted toward P-selectin expression in porcine xenografts infiltrated by human lymphocytes.
A Functional Role of IκB-ε in Endothelial Cell Activation
Authors: Spiecker Martin , Darius Harald , Liao James K.
Published: April 2014

The NF-κB inhibitor IκB-ε is a new member of the IκB protein family, but its functional role in regulating NF-κB-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, IκB-ε associates predominantly with the NF-κB subunit Rel A and to a lesser extent with c-Rel, whereas IκB-α and IκB-β associate with Rel A only. Following stimulation with TNF-α, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented IκB kinase-induced IκB-α, but not IκB-β or IκB-ε phosphorylation and degradation. Since the activation of NF-κB is required for the induction of adhesion molecule expression, we examined the role of IκB-ε in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of κB cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 κB promoter activity. By electrophoretic mobility shift assay, NF-κB heterodimers containing c-Rel specifically bind to the κB motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 κB promoter activity to a greater extent than that of E-selectin and overexpression of IκB-ε inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated IκB-ε is involved in the induction of ICAM-1 expression.
Fractalkine Is an Epithelial and Endothelial Cell-Derived Chemoattractant for Intraepithelial Lymphocytes in the Small Intestinal Mucosa
Authors: Muehlhoefer Andreas , Saubermann Lawrence J. , Gu Xuibin , Luedtke-Heckenkamp Kerstin , Xavier Ramnik , Blumberg Richard S. , Podolsky Daniel K. , MacDermott Richard P. , Reinecker Hans-Christian
Published: April 2014

Fractalkine is a unique chemokine that combines properties of both chemoattractants and adhesion molecules. Fractalkine mRNA expression has been observed in the intestine. However, the role of fractalkine in the healthy intestine and during inflammatory mucosal responses is not known. Studies were undertaken to determine the expression and function of fractalkine and the fractalkine receptor CX3CR1 in the human small intestinal mucosa. We identified intestinal epithelial cells as a novel source of fractalkine. The basal expression of fractalkine mRNA and protein in the intestinal epithelial cell line T-84 was under the control of the inflammatory mediator IL-1β. Fractalkine was shed from intestinal epithelial cell surface upon stimulation with IL-1β. Fractalkine localized with caveolin-1 in detergent-insoluble glycolipid-enriched membrane microdomains in T-84 cells. Cellular distribution of fractalkine was regulated during polarization of T-84 cells. A subpopulation of isolated human intestinal intraepithelial lymphocytes expressed the fractalkine receptor CX3CR1 and migrated specifically along fractalkine gradients after activation with IL-2. Immunohistochemistry demonstrated fractalkine expression in intestinal epithelial cells and endothelial cells in normal small intestine and in active Crohn’s disease mucosa. Furthermore, fractalkine mRNA expression was significantly up-regulated in the intestine during active Crohn’s disease. This study demonstrates that fractalkine-CX3CR1-mediated mechanism may direct lymphocyte chemoattraction and adhesion within the healthy and diseased human small intestinal mucosa.
Autoreactive B Cells Escape Clonal Deletion by Expressing Multiple Antigen Receptors
Authors: Kenny James J. , Rezanka Louis J. , Lustig Ana , Fischer Randy T. , Yoder Jeffrey , Marshall Shannon , Longo Dan L.
Published: April 2014

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2−/−) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2−/− knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.
A Human CTL Recognizes a Caspase-8-Derived Peptide on Autologous HLA-B*3503 Molecules and Two Unrelated Peptides on Allogeneic HLA-B*3501 Molecules
Authors: Mandruzzato Susanna , Stroobant Vincent , Demotte Nathalie , van der Bruggen Pierre
Published: April 2014

A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.
Calcitonin Gene-Related Peptide Induces IL-8 Synthesis in Human Corneal Epithelial Cells
Authors: Tran Mau T. , Ritchie Mary H. , Lausch Robert N. , Oakes John E.
Published: April 2014

Calcitonin gene-related peptide (CGRP), a neuropeptide with proinflammatory activities, is released from termini of corneal sensory neurons in response to pain stimuli. Because neutrophil infiltration of the clear corneal surface is a hallmark of corneal inflammation in the human eye, we determined whether CGRP can bind to human corneal epithelial cells (HCEC) and induce expression of the neutrophil chemotactic protein IL-8. It was found that HCEC specifically bound CGRP in a saturable manner with a Kd of 2.0 × 10−9 M. Exposure of HCEC to CGRP induced a significant increase in intracellular cAMP levels and enhanced IL-8 synthesis nearly 4-fold. The capacity of CGRP to stimulate cAMP and IL-8 synthesis was abrogated in the presence of the CGRP receptor antagonist CGRP8–37. CGRP stimulation had no effect on the half-life of IL-8 mRNA while increasing IL-8 pre-mRNA synthesis >2-fold. In contrast to IL-8, CGRP did not induce monocyte chemotactic protein-1 or RANTES synthesis, nor did the neuropeptide enhance detectable increases in steady state levels of mRNA specific for these two β-chemokines. The results suggest that HCEC possess CGRP receptors capable of initiating a signal transduction cascade that differentially activates expression of the IL-8 gene but not the genes for monocyte chemotactic protein-1 or RANTES. The capacity of CGRP to stimulate IL-8 synthesis in HCEC suggests that sensory neurons are involved in induction of acute inflammation at the eye surface.
Evidence for Human CD4+ T Cells in the CD1-Restricted Repertoire: Derivation of Mycobacteria-Reactive T Cells from Leprosy Lesions
Authors: Sieling Peter A. , Ochoa Maria-Teresa , Jullien Denis , Leslie David S. , Sabet Shereen , Rosat Jean-Pierre , Burdick Anne E. , Rea Thomas H. , Brenner Michael B. , Porcelli Steven A. , Modlin Robert L.
Published: April 2014

Both the CD4−CD8− (double negative) and CD4−CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-γ and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.
Phenotype and Functions of Brain Dendritic Cells Emerging During Chronic Infection of Mice withToxoplasma gondii
Authors: Fischer Hans-Georg , Bonifas Ursula , Reichmann Gaby
Published: April 2014

During chronic infection of mice with Toxoplasma gondii, gene message for IL-12p40, CD86, and the potassium channel Kv1.3 was detected in brain mononuclear cells, suggesting the presence of dendritic cells (DC) in the CNS. Consistently, cells bearing the DC markers CD11c and 33D1 were localized at inflammatory sites in the infected brain. The number of isolated CD11c+ brain cells increased until peak inflammation. The cells exhibited the surface phenotype of myeloid DC by coexpressing 33D1 and F4/80, little DEC-205, and no CD8α. These brain DC were mature, as indicated by high-level expression of MHC class II, CD40, CD54, CD80, and CD86. They triggered Ag-specific and primary allogeneic T cell responses at very low APC/T cell ratios. Among mononuclear cells from encephalitic brain, DC were the main producers of IL-12. Evidence for a parasite-dependent development of DC from CNS progenitors was obtained in vitro: after inoculation of primary brain cell culture with T. gondii, IL-12-secreting dendriform cells emerged, and DC marker genes were expressed. Different stimuli elicited the generation and maturation of brain DC: neutralization of parasite-induced GM-CSF prevented outgrowth of dendriform cells and concomitant release of IL-12. IL-12 production was up-regulated by external IFN-γ but was stopped by inhibiting parasite replication. Consistently, DC isolated from GM-CSF-treated brain cell culture were activated to secrete IL-12 by exposure to parasite lysate. In sum, these results demonstrate T. gondii-induced expansion and functional maturation of DC in the CNS and, thus, highlight a mechanism that may contribute to the chronicity of the host response.
Genetic Factors Determine the Contribution of Leukotrienes to Acute Inflammatory Responses
Authors: Goulet Jennifer L. , Byrum Robert S. , Key Mikelle L. , Nguyen MyTrang , Wagoner Victoria A. , Koller Beverly H.
Published: April 2014

Leukotrienes (LT) are potent lipid mediators synthesized by the 5-lipoxygenase pathway of arachidonic acid (AA) metabolism. LT have been implicated in a broad spectrum of inflammatory processes. To investigate the influence of genetic factors on the contribution of LT to acute inflammation, we generated congenic 5-lipoxygenase-deficient 129, C57BL/6 (B6), and DBA/1Lac (DBA) mouse lines. Topical application of AA evoked a vigorous inflammatory response in 129 and DBA mice, whereas only a modest response was seen in B6 animals. The response to AA in 129 and DBA strains is LT dependent. In contrast, LT make little contribution to this response in B6 mice. AA-induced inflammation in B6 mice is prostanoid dependent, since this response was substantially reduced by treating B6 mice with a cyclooxygenase inhibitor. These data suggest that prostanoids are essential for AA-induced cutaneous inflammation in B6 mice, whereas LT are the major mediators of this response in 129 and DBA strains. In contrast, the response to AA in the peritoneal cavity is robust in the 129 and B6 strains, but was significantly blunted in DBA mice, showing that strain differences in the response to AA are tissue specific. Variations in these and other experimental models of inflammation appear to correlate directly with the ability of a particular mouse strain and a specific tissue to respond to LT, specifically LTC4. Taken together, these findings indicate that the relative contribution of prostanoids and LT to inflammatory responses is variable not only between strains but also between different tissues within these inbred mouse lines.
Differential Role of Fas/Fas Ligand Interactions in Cytolysis of Primary and Metastatic Colon Carcinoma Cell Lines by Human Antigen-Specific CD8+ CTL
Authors: Bergmann-Leitner Elke S. , Abrams Scott I.
Published: April 2014

We have previously identified mutated ras peptides reflecting the glycine to valine substitution at position 12 as HLA-A2-restricted, CD8+ CTL neo-epitopes. CTL lines produced against these peptide epitopes lysed the HLA-A2+ Ag-bearing SW480 primary colon adenocarcinoma cell line, although IFN-γ treatment of the targets was necessary to achieve efficient cytotoxicity. Here, we compared the lytic phenotype of the SW480 cell line to its metastatic derivative, SW620, as an in vitro paradigm to further characterize the nature of a HLA class I-restricted, Ag-specific CTL response against neoplastic cell lines of primary and metastatic origin. Although both colon carcinoma cell lines were lysed by these Ag-specific CTL following IFN-γ pretreatment, the mechanisms of lysis were distinct, which reflected differential levels of sensitivity to the Fas pathway. Whereas IFN-γ pretreatment rendered SW480 cells sensitive to both Fas-dependent and -independent (perforin) pathways, SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. Moreover, pretreatment of SW480 cells with the anti-colon cancer agent, 5-fluorouracil (5-FU), led to enhanced Fas and ICAM-1 expression and triggered Ag-specific CTL-mediated lysis via Fas- and perforin-based pathways. In contrast, these phenotypic and functional responses were not observed with SW620 cells. Overall, these data suggested that 1) IFN-γ and 5-FU may enhance the lytic sensitivity of responsive colon carcinoma cells to immune effector mechanisms, including Fas-induced lysis; 2) the malignant phenotype may associate with resistance to Fas-mediated lysis in response to Ag-specific T cell attack; and 3) if Ag-specific CTL possess diverse lytic capabilities, this may overcome, to some extent, the potential “escape” of Fas-resistant carcinoma cells.
Inhibition of TNF-α-Induced Neutrophil Apoptosis by Crystals of Calcium Pyrophosphate Dihydrate Is Mediated by the Extracellular Signal-Regulated Kinase and Phosphatidylinositol 3-Kinase/Akt Pathways Up-Stream of Caspase 3
Authors: Tudan Christopher , Jackson John K. , Blanis Litsa , Pelech Steven L. , Burt Helen M.
Published: April 2014

The role of protein kinases in the inhibition of TNF-α associated apoptosis of human neutrophils by crystals of calcium pyrophosphate dihydrate (CPPD) (25 mg/ml) was investigated. We monitored the activities of the p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-α and CPPD crystals, separately and in combination, in parallel with the endogenous caspase 3 activity and DNA fragmentation. CPPD crystals were observed to induce a robust and transient activation of ERK1, ERK2, and Akt, whereas TNF-α produced only a modest and delayed activation of Akt. In the presence of TNF-α, Akt activity was enhanced, and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone, but TNF-α itself reduced the basal phosphotransferase activities of these MAP kinases. Preincubation with the MAP kinase kinase (MEK1) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM), or the PI3-K inhibitors wortmannin (100 nM) and LY294002 (50 μM) repressed the activation of ERK1, ERK2, and Akt in association with CPPD crystal incubation, in the absence or presence of TNF-α. Furthermore, the inhibition of the Mek1/Mek2→ERK1/ERK2 or PI3-K/Akt pathways reversed CPPD crystal-associated suppression of TNF-α-induced caspase 3 activation and neutrophil apoptosis. Together, these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/ERK2 and PI3-K/Akt-mediated stimulation of neutrophil activation and repression of apoptosis.
Mechanisms of Persistent NF-κB Activity in the Bronchi of an Animal Model of Asthma
Authors: Bureau Fabrice , Delhalle Sylvie , Bonizzi Giuseppina , Fiévez Laurence , Dogné Sophie , Kirschvink Nathalie , Vanderplasschen Alain , Merville Marie-Paule , Bours Vincent , Lekeux Pierre
Published: April 2014

In most cells trans-activating NF-κB induces many inflammatory proteins as well as its own inhibitor, IκB-α, thus assuring a transient response upon stimulation. However, NF-κB-dependent inflammatory gene expression is persistent in asthmatic bronchi, even after allergen eviction. In the present report we used bronchial brushing samples (BBSs) from heaves-affected horses (a spontaneous model of asthma) to elucidate the mechanisms by which NF-κB activity is maintained in asthmatic airways. NF-κB activity was high in granulocytic and nongranulocytic BBS cells. However, NF-κB activity highly correlated to granulocyte percentage and was only abrogated after granulocytic death in cultured BBSs. Before granulocytic death, NF-κB activity was suppressed by simultaneous addition of neutralizing anti-IL-1β and anti-TNF-α Abs to the medium of cultured BBSs. Surprisingly, IκB-β, whose expression is not regulated by NF-κB, unlike IκB-α, was the most prominent NF-κB inhibitor found in BBSs. The amounts of IκB-β were low in BBSs obtained from diseased horses, but drastically increased after addition of the neutralizing anti-IL-1β and anti-TNF-α Abs. These results indicate that sustained NF-κB activation in asthmatic bronchi is driven by granulocytes and is mediated by IL-1β and TNF-α. Moreover, an imbalance between high levels of IL-1β- and TNF-α-mediated IκB-β degradation and low levels of IκB-β synthesis is likely to be the mechanism preventing NF-κB deactivation in asthmatic airways before granulocytic death.
Attenuation of Experimental Autoimmune Demyelination in Complement-Deficient Mice
Authors: Nataf Serge , Carroll Steven L. , Wetsel Rick A. , Szalai Alexander J. , Barnum Scott R.
Published: April 2014

The exact mechanisms leading to CNS inflammation and myelin destruction in multiple sclerosis and in its animal model, experimental allergic encephalomyelitis (EAE) remain equivocal. In both multiple sclerosis and EAE, complement activation is thought to play a pivotal role by recruiting inflammatory cells, increasing myelin phagocytosis by macrophages, and exerting direct cytotoxic effects through the deposition of the membrane attack complex on oligodendrocytes. Despite this assumption, attempts to evaluate complement’s contribution to autoimmune demyelination in vivo have been limited by the lack of nontoxic and/or nonimmunogenic complement inhibitors. In this report, we used mice deficient in either C3 or factor B to clarify the role of the complement system in an Ab-independent model of EAE. Both types of complement-deficient mice presented with a markedly reduced disease severity. Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces of both wild-type and complement-deficient animals, in both C3−/− and factor B−/− mice there was little infiltration of the parenchyma by macrophages and T cells. In addition, compared with their wild-type littermates, the CNS of both C3−/− and factor B−/− mice induced for EAE are protected from demyelination. These results suggest that complement might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS.
CpG Oligodeoxynucleotides Can Reverse Th2-Associated Allergic Airway Responses and Alter the B7.1/B7.2 Expression in a Murine Model of Asthma
Authors: Serebrisky Denise , Teper Ariel A. , Huang Chih-Kang , Lee Soo-Young , Zhang Ten-Fei , Schofield Brian H. , Kattan Meyer , Sampson Hugh A. , Li Xiu-Min
Published: April 2014

CpG oligodeoxynucleotides (CpG-ODN) administered during Ag sensitization or before Ag challenge can inhibit allergic pulmonary inflammation and airway hyperreactivity in murine models of asthma. In this study, we investigated whether CpG-ODN can reverse an ongoing allergic pulmonary reaction in a mouse model of asthma. AKR mice were sensitized with conalbumin followed by two intratracheal challenges at weekly intervals. CpG-ODN was administered 24 h after the first Ag challenge. CpG-ODN administration reduced Ag-specific IgE levels, bronchoalveolar lavage fluid eosinophils, mucus production, and airway hyperreactivity. We found that postchallenge CpG-ODN treatment significantly increased IFN-γ concentrations and decreased IL-13, IL-4, and IL-5 concentrations in bronchoalveolar lavage fluids and spleen cell culture supernatants. Postchallenge CpG-ODN treatment also increased B7.1 mRNA expression and decreased B7.2 mRNA expression in lung tissues. These results suggest that CpG-ODN may have potential for treatment of allergic asthma by suppressing Th2 responses during IgE-dependent allergic airway reactions. The down-regulation of Th2 responses by CPG-ODN may be associated with regulation of the costimulatory factors B7.1 and B7.2.
Multiple Defects in FcεRI Signaling in Syk-Deficient Nonreleaser Basophils and IL-3-Induced Recovery of Syk Expression and Secretion
Authors: Kepley Christopher L. , Youssef Lama , Andrews Ronald P. , Wilson Bridget S. , Oliver Janet M.
Published: April 2014

Human basophils respond to Ag-induced cross-linking of their high affinity IgE receptor, FcεRI, by releasing histamine and other mediators from granules, producing IL-4 and other cytokines and, as shown in this study, by forming membrane ruffles and showing increased very late Ag-4 (VLA-4)-mediated adhesion to VCAM-1-expressing target cells. We have identified five blood donors whose basophils lack detectable levels of the FcεRI-associated protein tyrosine kinase, Syk. Despite showing no obvious ultrastructural differences from normal basophils, nonreleaser basophils fail to form membrane ruffles, to show increased VLA-4-mediated adhesive activity, or to produce IL-4 in response to FcεRI cross-linking. Although Syk protein levels are suppressed in basophils from all five donors, Syk mRNA is consistently present. Furthermore, culturing nonreleaser basophils for 4 days with IL-3 restores Syk protein expression and FcεRI-mediated histamine release. Understanding the reversible suppression of Syk protein expression in nonreleaser basophils, and learning to replicate this property in patients with allergic inflammation could be a powerful and specific way to limit symptomatic disease.
Triggering FCα-Receptor I (CD89) Recruits Neutrophils as Effector Cells for CD20-Directed Antibody Therapy
Authors: Stockmeyer Bernhard , Dechant Michael , van Egmond Marjolein , Tutt Alison L. , Sundarapandiyan Karuna , Graziano Robert F. , Repp Roland , Kalden Joachim R. , Gramatzki Martin , Glennie Martin J. , van de Winkel Jan G. J. , Valerius Thomas
Published: April 2014

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In 51Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcαRI) and CD20, a broad range of B cell lines were effectively killed. FcαRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcαRI × CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcγRI- or FcγRIII-directed BsAbs against CD20. Experiments with blood from human FcαRI/FcγRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcαRI × CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.
Leflunomide Suppresses TNF-Induced Cellular Responses: Effects on NF-κB, Activator Protein-1, c-Jun N-Terminal Protein Kinase, and Apoptosis
Authors: Manna Sunil K. , Mukhopadhyay Asok , Aggarwal Bharat B.
Published: April 2014

Leflunomide is a pyrimidine biosynthesis inhibitor that has recently been approved for treatment of rheumatoid arthritis. However, the mechanism of leflunomide’s antiarthritis activity and is not fully understood. The critical role that TNF plays in rheumatoid arthritis led us to postulate that leflunomide blocks TNF signaling. Previously, we have demonstrated that leflunomide inhibits TNF-induced NF-κB activation by suppressing I-κBα (inhibitory subunit of NF-κB) degradation. We in this study show that leflunomide also blocks NF-κB reporter gene expression induced by TNFR1, TNFR-associated factor 2, and NF-κB-inducing kinase (NIK), but not that activated by the p65 subunit of NF-κB, suggesting that leflunomide acts downstream of NIK. Leflunomide suppressed TNF-induced phosphorylation of I-κBα, as well as activation of I-κBα kinase-β located downstream to NIK. Leflunomide also inhibited TNF-induced activation of AP-1 and the c-Jun N-terminal protein kinase activation. TNF-mediated cytotoxicity and caspase-induced poly(ADP-ribose) polymerase cleavage were also completely abrogated by treatment of Jurkat T cells with leflunomide. Leflunomide suppressed TNF-induced reactive oxygen intermediate generation and lipid peroxidation, which may explain most of its effects on TNF signaling. The suppressive effects of leflunomide on TNF signaling were completely reversible by uridine, indicating a critical role for pyrimidine biosynthesis in TNF-mediated cellular responses. Overall, our results suggest that suppression of TNF signaling is one of the possible mechanisms for inhibitory activity of leflunomide against rheumatoid arthritis.
Disturbed Peripheral B Lymphocyte Homeostasis in Systemic Lupus Erythematosus
Authors: Odendahl Marcus , Jacobi Annett , Hansen Arne , Feist Eugen , Hiepe Falk , Burmester Gerd R. , Lipsky Peter E. , Radbruch Andreas , Dörner Thomas
Published: April 2014

In patients with active systemic lupus erythematosus (SLE), a marked B lymphocytopenia was identified that affected CD19+/CD27− naive B cells more than CD19+/CD27+ memory B cells, leading to a relative predominance of CD27-expressing peripheral B cells. CD27high/CD38+/CD19dim/surface Iglow/CD20−/CD138+ plasma cells were found at high frequencies in active but not inactive SLE patients. Upon immunosuppressive therapy, CD27high plasma cells and naive CD27− B cells were markedly decreased in the peripheral blood. Mutational analysis of V gene rearrangements of individual B cells confirmed that CD27+ B cells coexpressing IgD were memory B cells preferentially using VH3 family members with multiple somatic mutations. CD27high plasma cells showed a similar degree of somatic hypermutation, but preferentially employed VH4 family members. These results indicate that there are profound abnormalities in the various B cell compartments in SLE that respond differently to immunosuppressive therapy.
Delayed Onset of Inflammation in Protease-Activated Receptor-2-Deficient Mice
Authors: Lindner Jonathan R. , Kahn Mark L. , Coughlin Shaun R. , Sambrano Gilberto R. , Schauble Eric , Bernstein Daniel , Foy Dan , Hafezi-Moghadam Ali , Ley Klaus
Published: April 2014

Endothelial surface expression of P-selectin and subsequent leukocyte rolling in venules can be induced by mast cell-derived histamine and binding of thrombin to protease-activated receptor-1 (PAR1). We hypothesized that activation of endothelial PAR2 by mast cell tryptase or other proteases also contributes to inflammatory responses. Leukocyte rolling flux and rolling velocity were assessed by intravital microscopy of the cremaster muscles of wild-type mice following perivenular micropipette injections of a control (LSIGRL) or PAR2-activating (SLIGRL) oligopeptide. Injection of SLIGRL increased mean rolling leukocyte flux fraction from 34 ± 11 to 71 ± 24% (p < 0.05) and decreased mean rolling velocity from 63 ± 29 to 32 ± 2 μm/s (p < 0.05). No significant changes occurred with control peptide injection. To further evaluate the role of PAR2 in inflammatory responses, PAR2-deficient mice were generated by gene targeting and homologous recombination. Perivenular injections of SLIGRL resulted in only a small increase in rolling leukocyte flux fraction (from 21 ± 8 to 30 ± 2%) and no change in rolling velocity. Leukocyte rolling after surgical trauma was assessed in 9 PAR2-deficient and 12 wild-type mice. Early (0–15 min) after surgical trauma, the mean leukocyte rolling flux fraction was lower (10 ± 3 vs 30 ± 6%, p < 0.05) and mean rolling velocity was higher (67 ± 46 vs 52 ± 36 μm/s, p < 0.01) in PAR2-deficient compared with control mice. The defect in leukocyte rolling in PAR2-deficient mice did not persist past 30 min following surgical trauma. These results indicate that activation of PAR2 produces microvascular inflammation by rapid induction of P-selectin-mediated leukocyte rolling. In the absence of PAR2, the onset of inflammation is delayed.
Prostaglandin E2 Selectively Enhances the IgE-Mediated Production of IL-6 and Granulocyte-Macrophage Colony-Stimulating Factor by Mast Cells Through an EP1/EP3-Dependent Mechanism
Authors: Gomi Kaede , Zhu Fu-Gang , Marshall Jean S.
Published: April 2014

PGE2 is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE2 on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE2 alone or in the context of IgE-mediated activation. PGE2 treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-γ, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE2 stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE2, in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP1, EP3, and EP4 PGE receptor subtypes, including a novel splice variant of the EP1 receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE2 is mediated through EP1 and/or EP3 receptors. Our results suggest that PGE2 may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP1/EP3-dependent mechanism.
Hyporesponsiveness of Donor Cells to Lipopolysaccharide Stimulation Reduces the Severity of Experimental Idiopathic Pneumonia Syndrome: Potential Role for a Gut-Lung Axis of Inflammation
Authors: Cooke Kenneth R. , Hill Geoffrey R. , Gerbitz Armin , Kobzik Lester , Martin Thomas R. , Crawford James M. , Brewer Joanne P. , Ferrara James L. M.
Published: April 2014

Idiopathic pneumonia syndrome (IPS) is a major complication of allogeneic bone marrow transplantation (BMT). We have shown that experimental IPS is associated with increased levels of LPS and TNF-α in the bronchoalveolar lavage (BAL) fluid. We hypothesized that the deleterious effects of these inflammatory mediators in the lung may be linked to gut injury that develops after BMT. To test this hypothesis, we used mouse strains that differ in their sensitivity to LPS as donors in an experimental BMT model. Lethally irradiated C3FeB6F1 hosts received BMT from either LPS-sensitive or LPS-resistant donors. Five weeks after BMT, LPS-resistant BMT recipients had significantly less lung injury compared with recipients of LPS-sensitive BMT. This effect was associated with reductions in TNF-α secretion (both in vitro and in vivo), BAL fluid LPS levels, and intestinal injury. The relationship between TNF-α, gut toxicity, and lung injury was examined further by direct cytokine blockade in vivo; systemic neutralization of TNF-α resulted in a significant reduction in gut histopathology, BAL fluid LPS levels, and pulmonary dysfunction compared with control-treated animals. We conclude that donor resistance to endotoxin reduces IPS in this model by decreasing the translocation of LPS across the intestinal border and systemic and pulmonary TNF-α production. These data demonstrate a potential etiologic link between gut and lung damage after BMT and suggest that methods that reduce inflammatory responses to LPS, and specifically, those that protect the integrity of the gut mucosa, may be effective in reducing IPS after BMT.
Ad-Amino Acid Peptide Inhibitor of NF-κB Nuclear Localization Is Efficacious in Models of Inflammatory Disease
Authors: Fujihara Sheri M. , Cleaveland Jeffrey S. , Grosmaire Laura S. , Berry Karen K. , Kennedy Karen A. , Blake James J. , Loy James , Rankin Bruce M. , Ledbetter Jeffrey A. , Nadler Steven G.
Published: April 2014

The transcription factor NF-κB regulates many genes involved in proinflammatory and immune responses. The transport of NF-κB into the nucleus is essential for its biologic activity. We describe a novel, potent, and selective NF-κB inhibitor composed of a cell-permeable peptide carrying two nuclear localization sequences (NLS). This peptide blocks NF-κB nuclear localization, resulting in inhibition of cell surface protein expression, cytokine production, and T cell proliferation. The peptide is efficacious in vivo in a mouse septic shock model as well as a mouse model of inflammatory bowel disease, demonstrating that NF-κB nuclear import plays a role in these acute inflammatory disease models.
Isolation, Characterization, and Cloning of Porcine Complement Component C7
Authors: Agah Azin , Montalto Michael C. , Kiesecker Cheri L. , Morrissey Margaret , Grover Meera , Whoolery Kara L. , Rother Russell P. , Stahl Gregory L.
Published: April 2014

Activation of the complement system through the classical, alternative, or lectin pathway results in the formation of the terminal complement complex. C7 plays an integral role in the assembly of this complex with target cell membranes. To date, only human C7 has been cloned and characterized; thus, in this study, we characterized the porcine complement component C7. Porcine C7 was isolated by affinity chromatography as a single glycoprotein with an approximate molecular mass of 90 kDa and 100 kDa under reducing and nonreducing conditions, respectively. The full-length porcine C7 cDNA was isolated, and the predicted amino acid sequence exhibited 80% identity with human C7 with conservation of the cysteine backbone and two putative N-linked glycosylation sites. Porcine C7 mRNA expression was detected in all tissues investigated, except polymorphonuclear and mononuclear leukocytes. Addition of purified porcine C7 restored the hemolytic activity of C7-depleted human sera in a dose-dependent manner. A functionally inhibitory mAb against porcine C7 attenuated the hemolytic activity of human, rabbit, or rat sera, suggesting an important conserved C7 epitope among species. These data demonstrate that porcine and human C7 are highly conserved, sharing structural and functional characteristics.
TNF-α and IFN-γ Regulate Expression and Function of the Fas System in the Seminiferous Epithelium
Authors: Riccioli Anna , Starace Donatella , D’Alessio Alessio , Starace Giuseppe , Padula Fabrizio , De Cesaris Paola , Filippini Antonio , Ziparo Elio
Published: April 2014

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-α and IFN-γ markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-α-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-α and IFN-γ on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.
Engagement of CD153 (CD30 Ligand) by CD30+ T Cells Inhibits Class Switch DNA Recombination and Antibody Production in Human IgD+ IgM+ B Cells
Authors: Cerutti Andrea , Schaffer Andràs , Goodwin Raymond G. , Shah Shefali , Zan Hong , Ely Scott , Casali Paolo
Published: April 2014

CD153 (CD30 ligand) is a member of the TNF ligand/cytokine family expressed on the surface of human B cells. Upon exposure to IL-4, a critical Ig class switch-inducing cytokine, Ag-activated T cells express CD30, the CD153 receptor. The observation that dysregulated IgG, IgA, and/or IgE production is often associated with up-regulation of T cell CD30 prompted us to test the hypothesis that engagement of B cell CD153 by T cell CD30 modulates Ig class switching. In this study, we show that IgD+ IgM+ B cells up-regulate CD153 in the presence of CD154 (CD40 ligand), IL-4, and B cell Ag receptor engagement. In these cells, CD153 engagement by an agonistic anti-CD153 mAb or T cell CD30 inhibits Sμ→Sγ, Sμ→Sα, and Sμ→Sε class switch DNA recombination (CSR). This inhibition is associated with decreased TNFR-associated factor-2 binding to CD40, decreased NF-κB binding to the CD40-responsive element of the Cγ3 promoter, decreased Iγ3-Cγ3 germline gene transcription, and decreased expression of Ku70, Ku80, DNA protein kinase, switch-associated protein-70, and Msh2 CSR-associated transcripts. In addition, CD153 engagement inhibits IgG, IgA, and IgE production, and this effect is associated with reduced levels of B lymphocyte maturation protein-1 transcripts, and increased binding of B cell-specific activation protein to the Ig 3′ enhancer. These findings suggest that CD30+ T cells modulate CSR as well as IgG, IgA, and IgE production by inducing reverse signaling through B cell CD153.