bluuebunny/crossref_metadata_2025 huggingface dataset:
Potent Inhibition of Neutrophil Migration by Cryptococcal Mannoprotein-4-Induced Desensitization
Authors: Coenjaerts Frank E. J. , Walenkamp Annemiek M. E. , Mwinzi Pauline N. , Scharringa Jelle , Dekker Huberta A. T. , van Strijp Jos A. G. , Cherniak Robert , Hoepelman Andy I. M.
Published: April 2014

Cryptococcal capsular Ags induce the production of proinflammatory cytokines in patients with cryptococcal meningitis. Despite this, their cerebrospinal fluid typically contains few neutrophils. Capsular glucuronoxylomannan is generally considered to mediate the inhibition of neutrophil extravasation. In the current study, culture supernatant harvested from the nonglucuronoxylomannan-producing strain CAP67 was found to be as potent as supernatant from wild-type strains in preventing migration. We identified capsular mannoprotein (MP)-4 as the causative agent. Purified MP-4 inhibited migration of neutrophils toward platelet-activating factor, IL-8, and fMLP, probably via a mechanism involving chemoattractant receptor cross-desensitization, as suggested by its direct chemotactic activity. Supporting this hypothesis, MP-4 elicited Ca2+ transients that were inhibited by preincubation with either fMLP, IL-8, or C5a, but not platelet-activating factor, and vice versa. Moreover, MP-4 strongly decreased the neutrophil surface expression of L-selectin and induced shedding of TNF receptors p55/p75, whereas CD11b/18 increased. Finally, MP-4 was clearly detectable in both serum and cerebrospinal fluid of patients suffering from cryptococcal meningitis. These findings identify MP-4 as a novel capsular Ag prematurely activating neutrophils and desensitizing them toward a chemoattractant challenge.
Cross-Linking of Human FcγRIIIb Induces the Production of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor by Polymorphonuclear Neutrophils
Authors: Durand Véronique , Renaudineau Yves , Pers Jacques-Olivier , Youinou Pierre , Jamin Christophe
Published: April 2014

We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcγRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab′)2 to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab′)2 exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of β2 integrins, particularly CD11b, which is the α-chain of complement receptor type 3, but also CD18, which is its β-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcγRIIIb, which may occur in patients with PMN-binding anti-FcγRIIIb autoantibodies.
Human Mast Cells Release Metalloproteinase-9 on Contact with Activated T Cells: Juxtacrine Regulation by TNF-α
Authors: Baram Dana , Vaday Gayle G. , Salamon Pazit , Drucker Ilana , Hershkoviz Rami , Mekori Yoseph A.
Published: April 2014

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-α upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of β-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-α release peaked after only 6 h. Anti-TNF-α mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-α. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.
Immobilized IL-8 Triggers Progressive Activation of Neutrophils Rolling In Vitro on P-Selectin and Intercellular Adhesion Molecule-1
Authors: DiVietro Jeffrey A. , Smith McRae J. , Smith Bryan R. E. , Petruzzelli Lilli , Larson Richard S. , Lawrence Michael B.
Published: April 2014

The chemokine IL-8 is found on the luminal side of vascular endothelial cells, where it is postulated to be immobilized during inflammation. In this study, we observed that immobilized IL-8 can stimulate neutrophils to firmly adhere to a substrate containing ICAM-1 in a static adhesion assay. Soluble IL-8 was then perfused over neutrophils rolling on P-selectin (P-sel) and ICAM-1, confirming that IL-8 in solution can quickly cause rolling neutrophils to arrest. To mimic a blood vessel wall with IL-8 expressed on the luminal surface of endothelial cells, IL-8 was immobilized along with P-sel and ICAM-1 at defined site densities to a surface. Neutrophils rolled an average of 200 μm on surfaces of P-sel, ICAM-1, and IL-8 before firmly adhering through ICAM-1-β2 integrin interactions at 2 dynes/cm2 wall shear stress. Increasing the density of IL-8 from 60 to 350 sites/μm2 on the surface decreased by 50% the average distance and time the neutrophils rolled before becoming firmly adherent. Temporal dynamics of ICAM-1-β2 integrin interactions of rolling neutrophils following IL-8 exposure suggest the existence of two classes of β2 integrin-ICAM-1 interactions, a low avidity interaction with a 65% increase in pause times as compared with P-sel-P-sel glycoprotein ligand-1 interactions, and a high avidity interaction with pause times 400% greater than the selectin interactions. Based on the proportionality between IL-8 site density and time to arrest, it appears that neutrophils may need to sample a critical number of IL-8 molecules presented by the vessel wall before forming a sufficient number of high avidity β2 integrin bonds for firm adhesion.
Characterization of Mutations, Including a Novel Regulatory Defect in the First Intron, in Bruton’s Tyrosine Kinase Gene from Seven Korean X-Linked Agammaglobulinemia Families
Authors: Jo Eun-Kyeong , Kanegane Hirokazu , Nonoyama Shigeaki , Tsukada Satoshi , Lee Jae-Ho , Lim Kyu , Shong Minho , Song Chang-Hwa , Kim Hwa-Jung , Park Jeong-Kyu , Miyawaki Toshio
Published: April 2014

In this report, we describe seven mutations, including a novel single base pair substitution in intron 1, of the Bruton’s tyrosine kinase (Btk) gene found in 12 Korean patients with X-linked agammaglobulinemia. Various mutations, including three novel genetic alterations, were discovered using single-strand conformation polymorphism analysis and direct DNA sequencing. The effect of the intron 1 point mutation (intron 1 +5G→A) was further evaluated using reporter constructs. Using luciferase assay experiments, we showed that the transcriptional activity of the mutant was significantly lower than in normal counterparts, indicating that the intronic mutation was functional. In addition, DNase I footprinting analysis showed that a single protected region spanning the position +3 to +15 bp hybridized with a mutant-specific probe, but not with a wild-type probe. EMSA indicated that a distinct nuclear protein has the ability to bind the mutant oligonucleotides to produce a new DNA-protein complex. We also observed decreased expression of Btk proteins in monocytes of patients having the point mutation in intron 1. Taken together with the functional analysis, our results strongly suggest the existence of a novel cis-acting element, which might be involved in the down-regulation of Btk gene transcription. Precise definition of the regulatory defect in the Btk intron 1 may provide valuable clues toward elucidating the pathogenesis of X-linked agammaglobulinemia.
Soluble CD137 (4-1BB) Ligand Is Released Following Leukocyte Activation and Is Found in Sera of Patients with Hematological Malignancies
Authors: Salih Helmut R. , Schmetzer Helga M. , Burke Christine , Starling Gary C. , Dunn Robert , Pelka-Fleischer Renate , Nuessler Volkmar , Kiener Peter A.
Published: April 2014

Expression of CD137 ligand (4-1BBL), a member of the TNF family of proteins, has been reported on several types of APCs, various carcinoma cells, and can be induced on activated T cells. In this study, we report that the soluble ligand was released constitutively at low levels from leukocytes and at higher levels following cellular activation. Release from cells was blocked by addition of a metalloproteinase inhibitor which concomitantly caused the accumulation of 4-1BBL on the cell surface. In addition, we show that a soluble form of 4-1BBL was present at high levels in the sera of some patients with various hematological diseases, but only at low levels in healthy donors. Soluble 4-1BBL was active in that it competed with recombinant 4-1BBL for binding to the 4-1BB receptor and was able to costimulate IL-2 and IFN-γ release from peripheral T cells. These results indicate that the release of soluble 4-1BBL from the cell surface is mediated by one or more sheddases and likely regulates 4-1BB-4-1BBL interactions between cells in vivo. Cleavage of 4-1BBL to an active soluble form would alter both proximal and distal cellular responses, including cell survival and costimulatory or inflammatory responses, that are mediated through the 4-1BB pathway. This, in turn, would likely alter disease progression or outcome.
Role of Cyclin Kinase Inhibitor p21 in Systemic Autoimmunity
Authors: Santiago-Raber Marie-Laure , Lawson Brian R. , Dummer Wolfgang , Barnhouse Marlene , Koundouris Stefanos , Wilson Curtis B. , Kono Dwight H. , Theofilopoulos Argyrios N.
Published: April 2014

The cyclin kinase inhibitor protein p21 affects multiple processes relevant to the immune system, including cell cycle progression, replicative senescence, hemopoietic stem cell quiescence, and apoptosis. Therefore, malfunction of this protein may be a contributor to the pathogenesis of systemic autoimmunity. Here, we report that mixed background p21-deficient 129/Sv × C57BL/6 mice showed increased in vitro and in vivo T cell cycling and activation, moderate hypergammaglobulinemia and, at low penetrance, anti-chromatin autoantibodies. Homeostatic anti-self MHC/peptide ligand-induced proliferation of p21-deficient T cells was also enhanced. However, lymphoid organ enlargement was very mild, presumably due to increased apoptosis of the rapidly dividing cells. Moreover, the older p21-deficient mice had kidney pathology representing a similar, but slightly more advanced, state than that seen in the control mice. The timing and severity of the above serologic, cellular, and histologic manifestations in p21-deficient mice were unaffected by gender. Thus, p21 deficiency significantly enhances T cell activation and homeostatic proliferation, and can induce mild autoimmune manifestations at a low incidence without gender bias, but does not in itself generate the full spectrum of lupus-like disease.
HLA-DR Modulates Autoantibody Repertoire, But Not Mortality, in a Humanized Mouse Model of Systemic Lupus Erythematosus
Authors: Paisansinsup Tawatchai , Vallejo Abbe N. , Luthra Harvinder , David Chella S.
Published: April 2014

To evaluate the disease-modulating role of HLA-DR2 and DR3 molecules, which have been associated with systemic lupus erythematosus, a humanized mouse model was examined. HLA-DR2 (DRB1*1502)- and DR3 (DRB1*0301)-transgenic mice were backcrossed to the New Zealand Mixed 2410 (NZM 2410, H2z) strain. Seventh generation DR2 and DR3 transgene-positive animals along with their transgene-negative littermates and the parental strain NZM2410 were monitored for proteinuria, azotemia, autoantibody production, development of nephritis, and mortality. The results showed no significant differences in proteinuria, azotemia, or mortality between the backcrosses with and without HLA-DR2 or HLA-DR3. However, the genetic analysis of different backcrosses showed that heterozygosity at the endogenous H2-E locus (Ez/Eb) was strongly linked with acceleration of lupus nephritis in both HLA-DR2 and HLA-DR3 transgenics. More importantly, the presence of the HLA-DR2, but not the HLA-DR3, transgene significantly enhanced the production of anti-dsDNA, but not anti-ssDNA, anti-histone-dsDNA complex, or anti-histone, Abs. In contrast, neither HLA-DR2 nor HLA-DR3 influenced the development of glomerulonephritis or the degree of immune complex deposition. Moreover, nephritic kidneys from mice with and without HLA-DR2 or HLA-DR3 transgenes showed similar patterns of cytokine expression. Collectively, these findings provide molecular evidence that the association of HLA-DR2 or HLA-DR3 with lupus susceptibility is related to the type of autoantibody rather than to disease mortality. The use of a humanized mouse model provides a way of dissecting the roles of human MHC genes in systemic lupus erythematosus pathogenesis.
Neutralization of the Chemokine CXCL10 Reduces Inflammatory Cell Invasion and Demyelination and Improves Neurological Function in a Viral Model of Multiple Sclerosis
Authors: Liu Michael T. , Keirstead Hans S. , Lane Thomas E.
Published: April 2014

Intracerebral infection of mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a chronic demyelinating disease with clinical and histological similarities with the human demyelinating disease multiple sclerosis (MS). Following MHV infection, chemokines including CXC chemokine ligand (CXCL)10 (IFN inducible protein 10 kDa), CXCL9 (monokine induced by IFN-γ), and CC chemokine ligand 5 (RANTES) are expressed during both acute and chronic stages of disease suggesting a role for these molecules in disease exacerbation. Previous studies have shown that during the acute phase of infection, T lymphocytes are recruited into the CNS by the chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice with established demyelination were treated with antisera against these two chemokines, and disease severity was assessed. Treatment with anti-CXCL10 reduced CD4+ T lymphocyte and macrophage invasion, diminished expression of IFN-γ and CC chemokine ligand 5, inhibited progression of demyelination, and increased remyelination. Anti-CXCL10 treatment also resulted in an impediment of clinical disease progression that was characterized by a dramatic improvement in neurological function. Treatment with antisera against CXCL9 was without effect, demonstrating a critical role for CXCL10 in inflammatory demyelination in this model. These findings document a novel therapeutic strategy using Ab-mediated neutralization of a key chemokine as a possible treatment for chronic human inflammatory demyelinating diseases such as MS.
Up-Regulation of Fas Ligand Expression by Human Cytomegalovirus Immediate-Early Gene Product 2: A Novel Mechanism in Cytomegalovirus-Induced Apoptosis in Human Retina
Authors: Chiou Shih-Hwa , Liu Jorn-Hon , Hsu Wen-Ming , Chen Steve S.-L. , Chang Sui-Yuan , Juan Li-Jung , Lin Jung-Chun , Yang Yu-Ting , Wong Wing-Wai , Liu Cheng-Yi , Lin Young-Sun , Liu Wu-Tse , Wu Cheng-Wen
Published: April 2014

Human CMV (HCMV) is an important pathogen that causes widespread diseases in immunocompromised individuals. Among the opportunistic HCMV infections, HCMV retinitis is most common in transplant recipients and AIDS patients. It often leads to blindness if left untreated. The question as to how HCMV infection causes retinal pathogenesis remains unresolved. Here, we report that viral immediate-early gene product 2 (IE2), but not IE1, up-regulates the Fas ligand (FasL) expression in HCMV-infected human retinal pigment epithelium cells. Increased secretion of FasL from virally infected cells into cultured medium was observed upon HCMV infection. The capability of such cell-free medium to induce apoptosis of Fas (CD95)-expressing Jurkat cells further implies that Fas-FasL interaction might mediate cell death in the lesion of HCMV retinitis. To support this idea, we observed augmented soluble FasL levels in vitreous from AIDS patients with HCMV retinitis as compared with that from AIDS patients without HCMV infection. In addition, by in situ hybridization and immunohistochemistry, we detected enhanced signals of FasL, the existence of viral IE Ags and apoptotic cells at the same sites in the lesion of HCMV-infected retina. These results strongly suggest that IE2 induction of FasL expression in human retina might be an important event that takes place in the early stage of infection and finally leads to visual loss in individuals affiliated with HCMV retinitis.
IL-12 Administration Reveals Diabetogenic T Cells in Genetically Resistant I-Eα-Transgenic Nonobese Diabetic Mice: Resistance to Autoimmune Diabetes Is Associated with Binding of Eα-Derived Peptides to the I-Ag7 Molecule
Authors: Trembleau Sylvie , Gregori Silvia , Penna Giuseppe , Gorny Irmina , Adorini Luciano
Published: April 2014

Nonobese diabetic (NOD) and NOD-DRα transgenic (tg) mice, expressing Aαd:Aβg7 and Aαd:Aβg7 plus DRα:Eβg7 class II molecules, respectively, both develop insulin-dependent diabetes mellitus (IDDM), whereas NOD-Eα tg mice expressing Aαd:Aβg7 plus Eα:Eβg7 are protected. We show that IL-12 administration induces rapid IDDM onset in NOD-DRα but fails to provoke insulitis and diabetes in NOD-Eα tg mice. Nevertheless, T cells from IL-12-treated NOD-Eα tg mice secrete IFN-γ and transfer IDDM to NOD-SCID and NOD-Eα-SCID recipients, demonstrating the presence of peripheral diabetogenic Th1 cells in the protected mice. Surprisingly, regulatory cells were undetectable. Moreover, Eα:Eβg7 could substitute for DRα:Eβg7 in Ag presentation, arguing against mechanisms of protection involving capture of diabetogenic I-Ag7-restricted epitopes by Eα:Eβg7molecules. Interestingly, the expression of naturally processed epitopes derived from DRα- and Eα-chains bound to I-Ag7 is different in the two strains of tg mice, and the difference is enhanced by IL-12 administration. I-Ag7 molecules from both NOD-DRα and NOD-Eα tg mice present the conserved DRα/Eα 52-68 sequence, at high and low levels, respectively. In addition, only IDDM-resistant NOD-Eα tg mice possess APCs bearing Eα65-77/I-Ag7 complexes, which tolerize the specific T cells. This is associated with the selective inhibition of the response to insulinoma-associated protein 2 (IA-2), an autoantigen in IDDM. Our results support protective mechanisms based on I-Ag7 blockade by peptides unique to the Eα-chain, such as Eα65-77 and/or tolerance of diabetogenic T cells cross-reactive with Eα-peptide/I-Ag7 complexes.
Distinct Clonal Ig Diversification Patterns in Young Appendix Compared to Antigen-Specific Splenic Clones
Authors: Sehgal Devinder , Obiakor Harold , Mage Rose G.
Published: April 2014

The young rabbit appendix is a dynamic site for primary B cell repertoire development. To study diversification patterns during clonal expansion, we collected single appendix B cells from 3- to 9-wk-old rabbits and sequenced rearranged H and L chain genes. Single cells obtained by hydraulic micromanipulation or laser capture microdissection were lysed, PCR amplified, and products directly sequenced. Gene conversion-like changes occurred in rearranged H and L chain sequences by 3–4 wk of age. Somatic mutations were found in the D regions that lack known conversion donors and probably also occurred in the V genes. A few small sets of clonally related appendix B cells were found at 3–5 wk; by 5.5 wk, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found previously in splenic germinal centers where an immunizing Ag was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3, whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign Ags. This diversity of combining sites within B cell clones supports the proposed role of appendix in generating the preimmune repertoire.
CD40 Ligation Conditions Dendritic Cell Antigen-Presenting Function Through Sustained Activation of NF-κB
Authors: O’Sullivan Brendan John , Thomas Ranjeny
Published: April 2014

An understanding of the biochemical control of dendritic cell (DC) differentiation/activation is essential for improving T cell immunity by various immunotherapeutic approaches, including DC immunization. Ligation of CD40 enhances DC function, including conditioning for CTL priming. NF-κB, and particularly RelB, is an essential control pathway for myeloid DC differentiation. Furthermore, RelB regulates B cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L) and TNF-α, which differ in their capacity to condition DC, would also differ in their capacity to activate NF-κB. DC differentiated for 2 days from monocytes in the presence of GM-CSF and IL-4 were used as a model, as NF-κB activity was constitutively low. The capacity of DC to activate T cells following CD40L treatment was enhanced compared with TNF-α treatment, and this was NF-κB dependent. Whereas RelB/p50 translocation induced by TNF-α was attenuated after 6 h, RelB/p50 nuclear translocation induced by CD40L was sustained for at least 24 h. The mechanism of this difference related to enhanced degradation of IκBα following CD40L stimulation. However, NF-κB activation induced by TNF-α could be sustained by blocking autocrine IL-10. These data indicate that NF-κB activation is essential for T cell activation by DC, and that this function is enhanced if DC NF-κB activation is prolonged. Because IL-10 moderates DC NF-κB activation by TNF-α, sustained NF-κB activation can be achieved by blocking IL-10 in the presence of stimuli that induce TNF-α.
A Rational Strategy to Design Multiepitope Immunogens Based on Multiple Th Lymphocyte Epitopes
Authors: Livingston Brian , Crimi Claire , Newman Mark , Higashimoto Yuichiro , Appella Ettore , Sidney John , Sette Alessandro
Published: April 2014

Four HLA-DR-restricted HIV-derived Th lymphocyte (HTL) epitopes cross-reactive with the murine I-Ab class II molecule were used to evaluate different vaccine design strategies to simultaneously induce multiple HTL responses. All four epitopes were immunogenic in H-2b mice, demonstrating the feasibility of murine models to evaluate epitope-based vaccines destined for human use. Immunization with a pool of peptides induced responses against all four epitopes; illustrating immunodominance does not prevent the induction of balanced multispecific responses. When different delivery systems were evaluated, a multiple Ag peptide construct was found to be less efficient than a linear polypeptide encompassing all four epitopes. Further characterization of linear polypeptide revealed that the sequential arrangement of the epitopes created a junctional epitope with high affinity class II binding. Disruption of this junctional epitope through the introduction of a GPGPG spacer restored the immunogenicity against all four epitopes. Finally, we demonstrate that a GPGPG spacer construct can be used to induce HTL responses by either polypeptide or DNA immunization, highlighting the flexibility of the approach.
Id2 Negatively Regulates B Cell Differentiation in the Spleen
Authors: Becker-Herman Shirly , Lantner Frida , Shachar Idit
Published: April 2014

Early stages of B cell development occur in the bone marrow, resulting in formation of immature B cells. These immature cells migrate to the spleen where they differentiate into mature (B2 or marginal zone (MZ)) cells. This final maturation step is crucial for B cells to become responsive to Ags and to participate in the immune response. Id2 is a helix-loop-helix protein that lacks a DNA-binding region; and therefore, inhibits basic helix-loop-helix functions in a dominant negative manner. In this study, we show that Id2 expression is down-regulated during differentiation of immature B cells into mature B2 and MZ B cells. The high levels of Id2 expressed in the immature B cells result in inhibition of E2A binding activity to an E2 box site. Moreover, mice lacking Id2 show an elevation in the proportion of mature B2 cells in the spleen, while the MZ population in these mice is almost absent. Thus, Id2 acts as a regulator of the differentiation of immature B cells occurring in the spleen, it negatively controls differentiation into mature B2 cells while allowing the commitment to MZ B cells. In the absence of Id2 control, the unregulated differentiation is directed toward the mature B2 population.
The Mechanism Underlying T Cell Help for Induction of an Antigen-Specific In Vivo Humoral Immune Response to IntactStreptococcus pneumoniaeIs Dependent on the Type of Antigen
Authors: Wu Zheng-Qi , Shen Yi , Khan Abdul Q. , Chu Ching-Liang , Riese Richard , Chapman Harold A. , Kanagawa Osami , Snapper Clifford M.
Published: April 2014

Little is known concerning the role of T cells in regulating an anti-polysaccharide Ig response to an intact pathogen. We previously reported that the in vivo Ig responses to Streptococcus pneumoniae (strain R36A), specific for pneumococcal surface protein A (PspA) and for the phosphorylcholine (PC) determinant of C-polysaccharide, were both dependent on TCR-αβ+ T cells and B7-dependent costimulation, although only PspA-specific memory was generated. In this report, we show that the T cell help underlying these two Ag-specific Ig responses is distinct. Using H-Y-specific T cell transgenic mice made “nonleaky” by crossing with mice genetically deficient for TCR-α, we demonstrate that the T cell help for the anti-PC, in contrast to the anti-PspA, response is TCR-nonspecific and occurs normally in the absence of germinal center formation, although it is still dependent on B7-dependent costimulation. Consistent with these data, we demonstrate, using cathepsin S−/− mice, that although the anti-PC response is largely dependent on CD4+ T cells, there is a reduced (or lack of) dependence, relative to the anti-PspA response, on the generation of new peptide-MHC class II complexes. In this regard, the T cell help for an optimal anti-PC response is delivered more rapidly than that required for an optimal anti-PspA response. Collectively, these data demonstrate a novel accelerated TCR-nonspecific B7-dependent form of T cell help for augmenting a polysaccharide-specific Ig response to an intact bacterium without the generation of memory.
CD4 Cell Priming and Tolerization Are Differentially Programmed by APCs upon Initial Engagement
Authors: Higgins Amy D. , Mihalyo Marianne A. , McGary Patrick W. , Adler Adam J.
Published: April 2014

Bone marrow-derived APCs present both parenchymal-self and pathogen-derived Ags in a manner that elicits either T cell tolerization or immunity, respectively. To study the parameters that confer tolerogenic vs immunogenic APC function we used an adoptive transfer system in which naive TCR transgenic hemagglutinin (HA)-specific CD4+ T cells are either tolerized upon encountering HA expressed constitutively as a parenchymal self-Ag (self-HA) or primed to express effector function upon encountering transiently expressed vaccinia-derived HA (viral-HA). When the duration of viral-HA presentation was extended for the period required to elicit tolerization toward self-HA, CD4 cell tolerization to viral-HA did not occur. Furthermore, CD4 cells exhibited both phenotypic as well as functional differences during early stages of tolerization and priming, suggesting that these divergent differentiation processes are programmed soon after the initial APC-CD4 cell interaction. When mice expressing self-HA were infected with an irrelevant vaccinia, CD4 cell tolerization still occurred, indicating that priming vs tolerization cannot be explained by pathogen-induced third parties (i.e., non-APCs) that act directly on CD4 cells. Taken together, these results suggest that CD4 cell tolerization to parenchymal self-Ags and priming to pathogen-derived Ags are initiated by functionally distinct APCs.
Uptake of Apoptotic Antigen-Coupled Cells by Lymphoid Dendritic Cells and Cross-Priming of CD8+ T Cells Produce Active Immune Unresponsiveness
Authors: Ferguson Thomas A. , Herndon John , Elzey Bennett , Griffith Thomas S. , Schoenberger Steve , Green Douglas R.
Published: April 2014

The induction of immunologic unresponsiveness by i.v. administration of Ag-coupled lymphoid cells has been studied extensively, but the mechanisms remain unclear. We have further explored this model by examining the role of Fas/Fas ligand (FasL)-mediated apoptosis. Using i.v. injection of trinitrophenyl-coupled splenocytes (TNP-spl) as tolerogen, we found that Fas signaling for apoptosis in the spleen cells delivered by FasL in the recipient is the critical event. The requirement for Fas and FasL was overcome by prior induction of apoptosis in TNP-spl, making the tolerogen 100 times more potent. Prevention of apoptosis by a caspase inhibitor blocks tolerance. Interestingly, while blocking CD40/CD40 ligand interaction does not prevent tolerance induction, an agonist anti-CD40 Ab turns tolerogenic TNP-spl into an immunizing Ag. Studies further showed that tolerance is induced through cross-presentation of Ag in a class I MHC-dependent manner by CD8+CD11c+ lymphoid-derived dendritic cells to regulatory T cells. The results provide a mechanism for a well-established method of inducing immunologic unresponsiveness.
Impaired Light Chain Allelic Exclusion and Lack of Positive Selection in Immature B Cells Expressing Incompetent Receptor Deficient of CD19
Authors: Shivtiel Shoham , Leider Nira , Sadeh Orit , Kraiem Zaki , Melamed Doron
Published: April 2014

Positive signaling is now thought to be important for B cell maturation, although the nature of such signals has not yet been defined. We are studying the regulatory role of B cell Ag receptor (BCR) signaling in mediating positive selection of immature B cells. To do so, we use Ig transgenic mice (3-83Tg) that are deficient in CD19, thus generating a monoclonal immature B cell population expressing signaling-incompetent BCR. Immature 3-83Tg CD19−/− B cells undergo developmental arrest in the bone marrow, allowing maturation only to cells that effectively compensate for the compromised receptor by elevated levels of BCR. We find that developmentally arrested 3-83Tg CD19−/− B cells fail to impose L chain allelic exclusion and undergo intensive V(D)J recombination to edit their BCR. Furthermore, immature 3-83Tg CD19−/− B cells, which were grown in vitro, failed to undergo positive selection and to survive when adoptively transferred into normal recipients. However, elevation of BCR expression levels, obtained by transgene homozygosity, effectively compensated for the compromised BCR and completely restored BCR-mediated Ca2+ influx, allelic exclusion, and positive selection. Our results suggest that the BCR signaling threshold mediates positive selection of developing B cells, and that a receptor-editing mechanism has an important role in rescuing cells that fail positive selection because of incompetent receptors.
CD9 Is a Unique Marker for Marginal Zone B Cells, B1 Cells, and Plasma Cells in Mice
Authors: Won Woong-Jai , Kearney John F.
Published: April 2014

Marginal zone (MZ), follicular (FO), and B1 B cells form the long-lived naive B cell compartment. To identify surface markers that define MZ B cells in mice, we generated a panel of mAbs reactive with MZ but not FO B cells. One of these mAbs, MZ3, was found to recognize the tetraspanin CD9. CD9 expression not only distinguishes MZ B cells from FO B cells but also divided peritoneal cavity B1 cells into smaller subsets. After short-term in vitro stimulation with various mitogens, FO B cells failed to induce CD9 protein, while MZ B cells up-regulated the level of CD9 protein. However, after prolonged culture of FO B cells with LPS, surface CD9 was induced, together with syndecan 1, indicative of plasma cell differentiation. Following immunization with a T-independent-2 Ag, R36A, or a T-dependent Ag, SRBC, we found that CD9 is not expressed by germinal center B cells but is eventually expressed on plasma cells in response to both T-independent-2 and T-dependent Ags. Collectively, these results suggest that MZ B cells and B1 cell subsets are the immediate precursors of plasma cells in the primary response and that CD9 is acquired by T-dependent plasma cells.
AP2 Adaptor Complex-Dependent Internalization of CD5: Differential Regulation in T and B Cells
Authors: Lu Xianghuai , Axtell Robert C. , Collawn James F. , Gibson Andrew , Justement Louis B. , Raman Chander
Published: April 2014

CD5 is a key regulator of Ag receptor-mediated activation, selection, and differentiation in both T and B cells. Accumulating evidence indicates that lymphocyte activation and selection are sensitive to variations in levels of CD5 on the cell surface. We now show that CD5 expression on the surface of B and T cells is regulated posttranslationally by direct interaction with the μ2 subunit of the AP2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD5 is rapidly internalized from the cell surface in lymphoid cell lines, mature splenic T and B cells, and peritoneal CD5+ B cells following monovalent or bivalent ligation of the receptor. We mapped the μ2 subunit binding site on CD5 to Y429 and determined that the integrity of this site was necessary for CD5 internalization. Cross-linking of the Ag receptor with intact Abs inhibited CD5 internalization in B cells, but had the opposite effect in T cells. However, if F(ab′)2 Abs were used to stimulate the Ag receptor in B cells, the effect on CD5 internalization was now similar to that observed in T cells, indicating that signals through the Ag receptor and FcR regulate CD5 endocytosis in B cells. This was confirmed using an FcγRIIB1-deficient B cell line. The ability to differentially alter posttranslational CD5 expression in T and B cells is likely to be key in regulation of Ag receptor signaling and generation of tolerance in T and B lymphocytes.
Transcriptional Regulation of the HumanToll-Like Receptor 2Gene in Monocytes and Macrophages
Authors: Haehnel Viola , Schwarzfischer Lucia , Fenton Matthew J. , Rehli Michael
Published: April 2014

This report investigates the molecular basis for tissue-restricted and regulated expression of the pattern recognition receptor Toll-like receptor (TLR)2 in human monocytes and macrophages. To define the proximal promoter, the full 5′-sequence and transcriptional start sites of TLR2 mRNA were determined. The human TLR2 gene was found to consist of two 5′ noncoding exons followed by a third coding exon. Alternative splicing of exon II was detected primarily in human blood monocytes. The proximal promoter, exon I, and part of intron I were found to be located in a CpG island. Although CpG methylation of the proximal human TLR2 promoter in cell lines correlated with TLR2 repression, the promoter was unmethylated in primary cells, indicating that CpG methylation does not contribute to the cell-type specific expression of human TLR2 in normal tissues. The promoter sequence contains putative binding sites for several transcription factors, including Sp1 and Ets family members. Reporter gene analysis revealed a minimal promoter of 220 bp that was found to be regulated by Sp1, Sp3, and possibly PU.1. Interestingly, no sequence homology was detected between human and murine TLR2 promoter regions. In contrast to murine TLR2, expression of human TLR2 in monocytes/macrophages is not induced by the proinflammatory stimuli LPS or macrophage-activating lipopeptide-2, and reporter activity of the promoter was not enhanced by stimuli-induced NF-κB activation in THP-1 or MonoMac-6 cells. Our findings provide an initial definition of the human TLR2 promoter and reveal profound differences in the regulation of an important pattern recognition molecule in humans and mice.
Cloning and Characterization of the Homolog of Mammalian Lipopolysaccharide-Binding Protein and Bactericidal Permeability-Increasing Protein in Rainbow TroutOncorhynchus mykiss
Authors: Inagawa Hiroyuki , Honda Teruko , Kohchi Chie , Nishizawa Takashi , Yoshiura Yasutoshi , Nakanishi Teruyuki , Yokomizo Yuichi , Soma Gen-Ichiro
Published: April 2014

We cloned two cDNAs denoted as RT-LBP/BPI-1 and RT-LBP/BPI-2, respectively, which were derived from the mRNA of head kidney from rainbow trout. They showed structural homology with LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) in mammals. The full-length cDNA of RT-LBP/BPI-1 and RT-LBP/BPI-2 is 1666 and 1741 bp, respectively. Both cDNAs encoded 473 aa residues, including the amino acids conserved in mammalian LBP and BPI proteins that were assumed to be involved in LPS binding. The overall coding sequence of RT-LBP/BPI-1 has 33% amino acid homology to human LBP and 34% to human BPI, and RT-LBP/BPI-2 has 32% amino acid homology to human LBP and 33% to human BPI. Three-dimensional structure analysis by three-dimensional/one-dimensional (3D-1D) methods also demonstrated that RT-LBP/BPI-1 and RT-LBP/BPI-2 proteins showed significant similarity to human BPI, having a boomerang shape with N-terminal and C-terminal barrels. Phylogenetic analysis showed that the LBP and BPI genes seemed to be established after the divergence of mammals from teleosts. These results suggested that RT-LBP/BPI-1 and RT-LBP/BPI-2 may be a putative ortholog for mammalian LBP and/or BPI genes. This is the first study to identify the LBP family genes from nonmammalian vertebrates.
Characterization of a De Novo Conversion in Human Complement C4 Gene Producing a C4B5-Like Protein
Authors: Jaatinen Taina , Eholuoto Miia , Laitinen Tarja , Lokki Marja-Liisa
Published: April 2014

Complement C4 is a highly polymorphic protein essential for the activation of the classical complement pathway. Most of the allelic variation of C4 resides in the C4d region. Four polymorphic amino acid residues specify the isotype and an additional four specify the Rodgers and Chido determinants of the protein. Rare C4 allotypes have been postulated to originate from recombination between highly homologous C4 genes through gene conversions. Here we describe the development of a de novo C4 hybrid protein with allotypic and antigenic diversity resulting from nonhomologous intra or interchromosomal recombination of the maternal chromosomes. A conversion was observed between maternal C4A3a and C4B1b genes producing a functional hybrid gene in one of the children. The codons determining the isotype, Asp1054, Leu1101, Ser1102, Ile1105 and His1106, were characteristic of C4B gene, whereas the polymorphic sites in exon and intron 28 were indicative of C4A3a sequence. The protein produced by this hybrid gene was electrophoretically similar to C4B5 allotype. It also possesses reversed antigenicity being Rodgers 1, 2, 3 and Chido-1, -2, -3, 4, -5, and -6. Our case describes the development of a rare bimodular C4B-C4B haplotype containing a functional de novo C4 hybrid gene arisen through gene conversion from C4A to C4B. Overall the data supports the hypothesis of gene conversions as an ongoing process increasing allelic diversity in the C4 locus.
IFN Regulatory Factor 4 Participates in the Human T Cell Lymphotropic Virus Type I-Mediated Activation of the IL-15 Receptor α Promoter
Authors: Mariner Jennifer M. , Mamane Yael , Hiscott John , Waldmann Thomas A. , Azimi Nazli
Published: April 2014

IL-15Rα mRNA and protein levels are increased in human T cell lymphotropic virus type-I (HTLV-I)-associated adult T cell leukemia. Previously, we demonstrated that IL-15Rα expression was activated by HTLV-I Tax, in part, through the action of NF-κB. However, there appeared to be additional motifs within the IL-15Rα promoter that were responsive to HTLV-I Tax. In this study, we demonstrated that IL-15Rα mRNA expression was activated in human monocytes by IFN treatment, suggesting a role for IFN regulatory factors (IRFs) in IL-15Rα transcription. In addition, an IRF element within the Tax-responsive element of the IL-15Rα promoter was necessary for maximal Tax-induced activation of this promoter. Furthermore, we demonstrated that IRF-4, a transcription factor known to be elevated in HTLV-I-infected cells, activated the IL-15Rα promoter. Inhibition of IRF-4 action lead to reduced Tax-induced activation of the IL-15Rα promoter, while inhibition of both IRF-4 and NF-κB severely inhibited the Tax-induced activation of this promoter. These findings suggest a role for both NF-κB and IRF-4 in the transcriptional regulation of IL-15Rα by HTLV-I Tax. It is possible that the HTLV-I Tax-mediated induction of IL-15Rα and IL-15 may lead to an autocrine cytokine-mediated stimulatory loop leading to the proliferation of HTLV-I infected cells. This loop of proliferation may facilitate viral propagation and play a role in HTLV-I-mediated disease progression.
A Receptor for the Heterodimeric Cytokine IL-23 Is Composed of IL-12Rβ1 and a Novel Cytokine Receptor Subunit, IL-23R
Authors: Parham Christi , Chirica Madaline , Timans Jacqueline , Vaisberg Elena , Travis Marilyn , Cheung Jeanne , Pflanz Stefan , Zhang Rebecca , Singh Komal P. , Vega Felix , To Wayne , Wagner Janet , O’Farrell Anne-Marie , McClanahan Terrill , Zurawski Sandra , Hannum Charles , Gorman Daniel , Rennick Donna M. , Kastelein Robert A. , de Waal Malefyt Rene , Moore Kevin W.
Published: April 2014

IL-23 is a heterodimeric cytokine composed of the IL-12p40 “soluble receptor” subunit and a novel cytokine-like subunit related to IL-12p35, termed p19. Human and mouse IL-23 exhibit some activities similar to IL-12, but differ in their capacities to stimulate particular populations of memory T cells. Like IL-12, IL-23 binds to the IL-12R subunit IL-12Rβ1. However, it does not use IL-12Rβ2. In this study, we identify a novel member of the hemopoietin receptor family as a subunit of the receptor for IL-23, “IL-23R.” IL-23R pairs with IL-12Rβ1 to confer IL-23 responsiveness on cells expressing both subunits. Human IL-23, but not IL-12, exhibits detectable affinity for human IL-23R. Anti-IL-12Rβ1 and anti-IL-23R Abs block IL-23 responses of an NK cell line and Ba/F3 cells expressing the two receptor chains. IL-23 activates the same Jak-stat signaling molecules as IL-12: Jak2, Tyk2, and stat1, -3, -4, and -5, but stat4 activation is substantially weaker and different DNA-binding stat complexes form in response to IL-23 compared with IL-12. IL-23R associates constitutively with Jak2 and in a ligand-dependent manner with stat3. The ability of cells to respond to IL-23 or IL-12 correlates with expression of IL-23R or IL-12Rβ2, respectively. The human IL-23R gene is on human chromosome 1 within 150 kb of IL-12Rβ2.
BAD1, an Essential Virulence Factor ofBlastomyces dermatitidis, Suppresses Host TNF-α Production Through TGF-β-Dependent and -Independent Mechanisms
Authors: Finkel-Jimenez Beatriz , Wüthrich Marcel , Klein Bruce S.
Published: April 2014

We investigated how BAD1, an adhesin and virulence factor of Blastomyces dermatitidis, suppresses phagocyte proinflammatory responses. Wild-type yeast cocultured with murine neutrophils or macrophages prompted release of a soluble factor into conditioned supernatant that abolished TNF-α production in response to the fungus; isogenic, attenuated BAD1 knockout yeast did not have this effect. Phagocytes released 4- to 5-fold more TGF-β in vitro in response to wild-type yeast vs BAD1 knockout yeast. Treatment of inhibitory, conditioned supernatant with anti-TGF-β mAb neutralized detectable TGF-β and restored phagocyte TNF-α production. Similarly, addition of anti-TGF-β mAb into cultures of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-α production. Conversely, TGF-β treatment of phagocytes cultured with knockout yeast suppressed TNF-α production. Hence, TGF-β mediates BAD1 suppression of TNF-α by wild-type B. dermatitidis cultured in vitro with phagocytes. In contrast to these findings, neutralization of elevated TGF-β levels during experimental pulmonary blastomycosis did not restore BAD1-suppressed TNF-α levels in the lung or ameliorate disease. Soluble BAD1 was found to accumulate in the alveoli of infected mice at levels that suppressed TNF-α production by phagocytes. However, in contrast to yeast cell surface BAD1, which induced TGF-β, soluble BAD1 failed to do so and TNF-α suppression mediated by soluble BAD1 was unaffected by neutralization of TGF-β. Thus, BAD1 of B. dermatitidis induces suppression of TNF-α and progressive infection by both TGF-β-dependent and -independent mechanisms.
Sustained Impairment of IFN-γ Secretion in Suppressed HIV-Infected Patients Despite Mature NK Cell Recovery: Evidence for a Defective Reconstitution of Innate Immunity
Authors: Azzoni Livio , Papasavvas Emmanouil , Chehimi Jihed , Kostman Jay R. , Mounzer Karam , Ondercin Joe , Perussia Bice , Montaner Luis J.
Published: April 2014

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-γ and TNF-α-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV+ individuals had significantly lower proportions of mature CD3−/CD161+/CD56+ NK cells and of IFN-γ-producing NK cells compared with noninfected donors, independent of CD4+ T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3−/CD161+/CD56+ NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-γ. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-γ, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-γ secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.