bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulatory Effects of B7-Related Protein-1 on Cellular and Humoral Immune Responses in Mice
Authors: Guo Jane , Stolina Marina , Bready James V. , Yin Songmei , Horan Tom , Yoshinaga Steven K. , Senaldi Giorgio
Published: April 2014

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-γ, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.
Fcα Receptor Cross-Linking Causes Translocation of Phosphatidylinositol-Dependent Protein Kinase 1 and Protein Kinase Bα to MHC Class II Peptide-Loading-Like Compartments
Authors: Lang Mark L. , Shen Li , Gao Hong , Cusack William F. , Lang Gillian A. , Wade William F.
Published: April 2014

A20 IIA1.6 B cells cotransfected with FcαR and wild-type γ-chain (wt-ITAM (immunoreceptor tyrosine-based activation motif)) or FcαR and γ-chain, in which the wt-ITAM was substituted with the FcγRIIA ITAM (IIA-ITAM), were used to investigate cell signaling events influencing presentation of FcαR-targeted exogenous Ag in the context of MHC class II. wt-ITAM cells presented FcαR-targeted OVA more efficiently than IIA-ITAM transfectants to OVA-specific T cell hybridomas. Phosphatidylinositol 3-kinase (PI 3-kinase) inhibition abrogated Ag presentation, suggesting that FcαR may trigger a PI 3-kinase-dependent signal transduction pathway, and thus phosphatidylinositol-dependent protein kinase (PDK1) and protein kinase B α (PKBα) activation. Cross-linking FcαR on wt-ITAM or IIA-ITAM cells triggered equivalent PI 3-kinase-dependent activation of PKBα. Furthermore, FcαR cross-linking triggered recruitment of PDK1 and serine-phosphorylated PKBα to capped cell surface FcαR irrespective of the γ-chain ITAM. Although FcαR endocytosis was accompanied by translocation of PDK1 and phospho-PKBα to FcαR-containing vesicles in both transfectants, this was decreased in IIA-ITAM cells, and a significant proportion of PDK1 and PKBα remained at the plasma membrane. In wt-ITAM cells, PDK1 and serine-phosphorylated PKBα translocated to lysosomal-associated membrane glycoprotein 1- and cathepsin B-containing vesicles, consistent with MHC class II peptide-loading compartments (MIIC) described by other groups. Our data indicate that translocation of signal transduction mediators to MIIC-like compartments accompanies efficient presentation of receptor-targeted Ag, and suggest a mechanism connecting signaling to the Ag-processing pathway.
Neonatal Exposure to Antigen Induces a Defective CD40 Ligand Expression that Undermines Both IL-12 Production by APC and IL-2 Receptor Up-Regulation on Splenic T Cells and Perpetuates IFN-γ-Dependent T Cell Anergy
Authors: Min Booki , Legge Kevin L. , Bell J. Jeremiah , Gregg Randal K. , Li Lequn , Caprio Jacque C. , Zaghouani Habib
Published: April 2014

T cell deletion and/or inactivation were considered the leading mechanisms for neonatal tolerance. However, recent investigations have indicated that immunity develops at the neonatal stage but evolves to guide later T cell responses to display defective and/or biased effector functions. Although neonatal-induced T cell modulation provides a useful approach to suppress autoimmunity, the mechanism underlying the biased function of the T cells remains unclear. In prior studies, we found that exposure of newborn mice to Ig-PLP1, a chimera expressing the encephalitogenic proteolipid protein (PLP) sequence 139–151, induced deviated Th2 lymph node cells producing IL-4 instead of IL-2 and anergic splenic T cells that failed to proliferate or produce IFN-γ yet secreted significant amounts of IL-2. However, if assisted with IFN-γ or IL-12, these anergic splenic T cells regained full responsiveness. The consequence of such biased/defective T cells responses was protection of the mice against experimental allergic encephalomyelitis. In this study, investigations were performed to delineate the mechanism underlying the novel form of IFN-γ-dependent splenic anergy. Our findings indicate that CD40 ligand expression on these splenic T cells is defective, leading to noneffective cooperation between T lymphocytes and APCs and a lack of IL-12 production. More striking, this cellular system revealed a requirement for IL-2R expression for CD40 ligand-initiated, IL-12-driven progression of T cells into IFN-γ production.
Antigen Recognition and Immunomodulation by γδ T Cells in Bovine Tuberculosis
Authors: Rhodes Shelley G. , Hewinson R. Glyn , Vordermeier H. Martin
Published: April 2014

This report describes the in vitro proliferative responses of peripheral blood γδ T cells to defined mycobacterial protein Ags and the immunomodulatory effect of γδ T cells in cattle infected with Mycobacterium bovis. γδ T cell responses were specific to M. bovis infection because they were detected in cattle either experimentally or naturally infected with M. bovis, but were not present in uninfected controls. Proliferating γδ T cell cultures produced enhanced levels of IFN-γ and TGF-β, but not IL-2 in response to the more immunodominant mycobacterial Ags. Depletion of γδ T cells from PBMC resulted in an increased Ag-specific proliferation in half the animals tested, indicating a suppressive effect of γδ T cells upon other (αβ) T cell responses. Because γδ T cells constitute a major T cell population in the peripheral blood of cattle, the activities of γδ T cells described in this report could make a significant contribution to the immune response in bovine tuberculosis.
Sanglifehrin A, a Novel Cyclophilin-Binding Immunosuppressant, Inhibits IL-2-Dependent T Cell Proliferation at the G1 Phase of the Cell Cycle
Authors: Zhang Ling-Hua , Liu Jun O.
Published: April 2014

Sanglifehrin A (SFA) is a novel immunosuppressive natural product that binds to cyclophilin but is structurally distinct from cyclosporin A (CsA). We have investigated the cellular and molecular mechanisms of the action of SFA in T lymphocytes. We show that SFA inhibits T cell proliferation induced by IL-2 with an IC50 of 200 nM. Distinct from CsA, which also binds to cyclophilin, SFA does not affect calcium-dependent IL-2 production, although SFA enhanced IL-2 gene transcription in the same cells. SFA blocks T cell proliferation induced by IL-2 in G1 with no appreciable effect on IL-2 receptor expression in a manner similar to that of the immunosuppressant rapamycin. Unlike rapamycin, however, SFA has no effect on the phosphorylation or enzymatic activity of p70s6k kinase, distinguishing SFA from rapamycin in their mode of action. SFA inhibits hyperphosphorylation of Rb and the activity of cyclin E-cyclin-dependent kinase 2 on IL-2 signaling. These results suggest that SFA has a novel mode of action in comparison with CsA, FK506, and rapamycin, and that its use as a molecular probe may lead to the discovery of a novel target involved in T cell activation.
IL-12 Antagonism Enhances Apoptotic Death of T Cells Within Hepatic Allografts from Flt3 Ligand-Treated Donors and Promotes Graft Acceptance
Authors: Li Wei , Lu Lina , Wang Zhiliang , Wang Lianfu , Fung John J. , Thomson Angus W. , Qian Shiguang
Published: April 2014

Mouse livers are accepted across MHC barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, livers from donors treated with Flt3 ligand, which dramatically increases hepatic interstitial dendritic cells, are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating cells. We hypothesized that IL-12 production by enhanced numbers of donor APC might inhibit apoptosis, promote expansion of Th1 cells, and play a key role in liver rejection. Therefore, C3H (H2k) recipients of liver grafts from Flt3 ligand-treated B10 donors were given neutralizing anti-IL-12 mAb (200 or 500 μg) on days 0 and 2 after transplant. Graft survival was markedly prolonged at the higher mAb dose, with 50% of grafts surviving >100 days. This effect was associated with reductions in IFN-γ gene transcripts within the graft-infiltrating cell population and with reductions in circulating IFN-γ and IL-10 levels, donor-specific CTL and NK cell activities, and circulating alloantibody levels. At the same time, there were marked increases in apoptotic (TUNEL+) CD4+ and especially CD8+ cells, both within the grafts and in spleens of anti-IL-12 mAb-treated mice. In vitro, exogenous IL-12 inhibited apoptotic death induced in naive allogeneic T cells by liver nonparenchymal cells. These findings suggest that suppression of rejection by IL-12 antagonism, linked to restoration of apoptotic activity within the peripheral alloreactive T cell population, is important for liver allograft survival and tolerance induction.
Epitope Mapping of Antibodies to the C-Terminal Region of the Integrin β2 Subunit Reveals Regions that Become Exposed Upon Receptor Activation
Authors: Lu Chafen , Ferzly Mazen , Takagi Junichi , Springer Timothy A.
Published: April 2014

The cysteine-rich repeats in the stalk region of integrin β subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the β2 subunit, one, KIM127, preferentially bound to αLβ2 that was activated by mutations in the cytoplasmic domains, and by Mn2+. KIM127 also bound preferentially to the free β2 subunit compared with resting αLβ2. Activating β2 mutations also greatly enhanced binding of KIM127 to integrins αMβ2 and αXβ2. Thus, the KIM127 epitope is shielded by the α subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated αLβ2 and bound equally well to resting and activated αLβ2, differentially recognized resting αMβ2 and αXβ2, and bound fully to activated αMβ2 and αXβ2. The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344–432 and 432–487, respectively. We thus define five different β2 stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.
Mice Triallelic for the Ig Heavy Chain Locus: Implications for VHDJH Recombination
Authors: Barreto Vasco , Meo Tommaso , Cumano Ana
Published: April 2014

VHDJH recombination has been extensively studied in mice carrying an Ig heavy chain rearranged transgene. In most models, inhibition of endogenous Ig rearrangement occurs, consistently with the feedback model of IgH recombination. Nonetheless, an incomplete IgH allelic exclusion is a recurrent observation in these animals. Furthermore, transgene expression in ontogeny is likely to start before somatic recombination, thus limiting the use of Ig-transgenic mice to access the dynamics of VHDJH recombination. As an alternative approach, we challenged the regulation of somatic recombination with the introduction of an extra IgH locus in germline configuration. This was achieved by reconstitution of RAG2−/− mice with fetal liver cells trisomic for chromosome 12 (Ts12). We found that all three alleles can recombine and that the ratio of Ig allotype-expressing B cells follows the allotypic ratio in trisomic cells. Although these cells are able to rearrange the three alleles, the levels of Ig phenotypic allelic exclusion are not altered when compared with euploid cells. Likewise, we find that most VDJ rearrangements of the silenced allele are unable to encode a functional μ-chain, indicating that the majority of these cells are also genetically excluded. These results provide additional support for the feedback model of allelic exclusion.
Isoforms of Jun Kinase Are Differentially Expressed and Activated in Human Monocyte/Macrophage (THP-1) Cells
Authors: Dreskin Stephen C. , Thomas Gregory W. , Dale Sara N. , Heasley Lynn E.
Published: April 2014

Ten isoforms of c-jun N-terminal kinase (JNK) have been described that arise by differential mRNA splicing of three genes. In that the relative expression and function of these different JNK proteins in human monocytic cells is not known, we have examined the JNK isoforms in THP-1 monocyte/macrophage cells. Differentiation of THP-1 cells by exposure to 10−8 M PMA for 42–48 h enhances cellular responses to LPS, including enhanced activation of total JNK activity and increased phosphorylation of p54 JNK as well as p46 JNK. Examination of JNK proteins on Western blots reveals a predominance of p46 JNK1 and p54 JNK2 proteins. Clearing of lysates by immunoprecipitation of JNK1(99% effective) removes 46% of the JNK enzymatic activity (p < 0.01), whereas clearing of JNK1 plus JNK2 (70% effective) depletes the sample of 72% of the JNK activity (p < 0.01). Further analysis, undertaken with real-time RT-PCR, revealed that 98% of the JNK messages code for three isoforms: JNK1β1, JNK2α1, and JNK2α2. The p54 JNK that is phosphorylated in LPS-stimulated, PMA-differentiated THP-1 cells is most likely JNK2α2 because 97% of the p54 JNK-encoding messages code for JNK2α2. By analogous reasoning, the p46 JNKs that are not heavily phosphorylated, but account for approximately half of the N-terminal c-jun kinase enzymatic activity, are most likely either JNK1β1 or JNK2α1 because they account for 98% of the messages that can code for 46kDa JNKs.
ZAP-70 and SLP-76 Regulate Protein Kinase C-θ and NF-κB Activation in Response to Engagement of CD3 and CD28
Authors: Herndon Thomas M. , Shan Xiaochuan C. , Tsokos George C. , Wange Ronald L.
Published: April 2014

The transcription factor NF-κB is a critical regulator of T cell function that becomes strongly activated in response to coengagement of TCR and CD28. Although events immediately proximal to NF-κB activation are well understood, uncertainty remains over which upstream signaling pathways engaged by TCR and CD28 lead to NF-κB activation. By using Jurkat T cell lines that are deficient or replete for either the protein tyrosine kinase ZAP-70 or the cytosolic adapter molecule SLP-76, the role of these proteins in modulating NF-κB activation was examined. NF-κB was not activated in response to coengagement of TCR and CD28 in either the ZAP-70- or SLP-76-negative cells, whereas stimuli that bypass these receptors (PMA plus A23187, or TNF-α) activated NF-κB normally. Protein kinase C (PKC) θ activation, which is required for NF-κB activation, also was defective in these cells. Reexpression of ZAP-70 restored PKCθ and NF-κB activation in response to TCR and CD28 coengagement. p95vav (Vav)-1 tyrosine phosphorylation was largely unperturbed in the ZAP-70-negative cells; however, receptor-stimulated SLP-76/Vav-1 coassociation was greatly reduced. Wild-type SLP-76 fully restored PKCθ and NF-κB activation in the SLP-76-negative cells, whereas 3YF-SLP-76, which lacks the sites of tyrosine phosphorylation required for Vav-1 binding, only partially rescued signaling. These data illustrate the importance of the ZAP-70/SLP-76 signaling pathway in CD3/CD28-stimulated activation of PKC θ and NF-κB, and suggest that Vav-1 association with SLP-76 may be important in this pathway.
Protein Kinase C-θ Participates in the Activation of Cyclic AMP-Responsive Element-Binding Protein and Its Subsequent Binding to the −180 Site of the IL-2 Promoter in Normal Human T Lymphocytes
Authors: Solomou Elena E. , Juang Yuang-Taung , Tsokos George C.
Published: April 2014

IL-2 gene expression is regulated by the cooperative binding of discrete transcription factors to the IL-2 promoter/enhancer and is predominantly controlled at the transcriptional level. In this study, we show that in normal T cells, the −180 site (−164/−189) of the IL-2 promoter/enhancer is a p-cAMP-responsive element-binding protein (p-CREB) binding site. Following activation of the T cells through various membrane-initiated and membrane-independent pathways, protein kinase C (PKC)-θ phosphorylates CREB, which subsequently binds to the −180 site and associates with the transcriptional coactivator p300. Rottlerin, a specific PKC-θ inhibitor, diminished p-CREB protein levels when normal T cells were treated with it. Rottlerin also prevented the formation of p-CREB/p300 complexes and the DNA-CREB protein binding. Cotransfection of fresh normal T cells with luciferase reporter construct driven by two tandem −180 sites and a PKC-θ construct caused a significant increase in the transcription of the reporter gene, indicating that this site is functional and regulated by PKC-θ. Cotransfection of T cells with a luciferase construct driven by the −575/+57 region of the IL-2 promoter/enhancer and a PKC-θ construct caused a similar increase in the reporter gene transcription, which was significantly limited when two bases within the −180 site were mutated. These findings show that CREB plays a major role in the transcriptional regulation of IL-2 and that a major pathway for the activation of CREB and its subsequent binding to the IL-2 promoter/enhancer in normal T cells is mediated by PKC-θ.
Cloning, Expression, and Function of BLAME, a Novel Member of the CD2 Family
Authors: Kingsbury Gillian A. , Feeney Lee Ann , Nong Yuhua , Calandra Susan  A , Murphy Curran J. , Corcoran Justin M. , Wang Yanjun , Prabhu Das Mercy R. , Busfield Samantha J. , Fraser Christopher C. , Villeval Jean Luc
Published: April 2014

The CD2 family is a growing family of Ig domain-containing cell surface proteins involved in lymphocyte activation. Here we describe the cloning and expression analysis of a novel member of this family, B lymphocyte activator macrophage expressed (BLAME). BLAME shares the structural features of the CD2 family containing an IgV and IgC2 domain and clusters with the other family members on chromosome 1q21. Quantitative PCR and Northern blot analysis show BLAME to be expressed in lymphoid tissue and, more specifically, in some populations of professional APCs, activated monocytes, and DCs. Retroviral forced expression of BLAME in hematopoietic cells of transplanted mice showed an increase in B1 cells in the peripheral blood, spleen, lymph nodes, and, most strikingly, in the peritoneal cavity. These cells do not express CD5 and are CD23lowMac1low, characteristics of the B1b subset. BLAME may therefore play a role in B lineage commitment and/or modulation of signal through the B cell receptor.
Uncoordinated HLA-D Gene Expression in a RFXANK-Defective Patient with MHC Class II Deficiency
Authors: Lennon-Duménil Ana-Maria , Barbouche Mohamed-Ridha , Vedrenne Jocelyn , Prod’Homme Thomas , Béjaoui Mohamed , Ghariani Salma , Charron Dominique , Fellous Marc , Dellagi Koussay , Alcaïde-Loridan Catherine
Published: April 2014

We describe the analysis of a patient, JER, presenting classical immunological features of MHC class II deficiency. Unexpectedly, some HLA transcripts (HLA-DRA, HLA-DQA, and HLA-DMA) were found to be expressed in the JER cell line at nearly wild-type levels, while HLA-DPA and the HLA-D β-chain transcripts were not detected. Gene reporter experiments confirmed the differential transcriptional activities driven by the HLA-D promoters in the JER cells. A defect in RFXANK was first suggested by genetic complementation analyses, then assessed with the demonstration of a homozygous mutation affecting a splice donor site downstream exon 4 of RFXANK. Because the severe deletion of the resulting protein cannot account for the expression of certain HLA-D genes, minor alternative transcripts of the RFXANK gene were analyzed. We thereby showed the existence of a transcript lacking exon 4, encoding a 28-aa-deleted protein that retains a transcriptional activity. Altogether, we characterize a new type of mutation in the RFXANK gene in a MHC class II-defective patient leading to an uncoordinated expression of the HLA-D genes, and propose that this phenotype is ensured by severely limited amounts of an active, although truncated RFXANK protein.
Endotoxin-Induced Maturation of MyD88-Deficient Dendritic Cells
Authors: Kaisho Tsuneyasu , Takeuchi Osamu , Kawai Taro , Hoshino Katsuaki , Akira Shizuo
Published: April 2014

LPS, a major component of the cell wall of Gram-negative bacteria, can induce a variety of biological responses including cytokine production from macrophages, B cell proliferation, and endotoxin shock. All of them were completely abolished in MyD88-deficient mice, indicating the essential role of MyD88 in LPS signaling. However, MyD88-deficient cells still show activation of NF-κB and mitogen-activated protein kinase cascades, although the biological significance of this activation is not clear. In this study, we have examined the effects of LPS on dendritic cells (DCs) from wild-type and several mutant mice. LPS-induced cytokine production from DCs was dependent on MyD88. However, LPS could induce functional maturation of MyD88-deficient DCs, including up-regulation of costimulatory molecules and enhancement of APC activity. MyD88-deficient DCs could not maturate in response to bacterial DNA, the ligand for Toll-like receptor (TLR)9, indicating that MyD88 is differentially required for TLR family signaling. MyD88-dependent and -independent pathways originate at the intracytoplasmic region of TLR4, because both cytokine induction and functional maturation were abolished in DCs from C3H/HeJ mice carrying the point mutation in the region. Finally, in vivo analysis revealed that MyD88-, but not TLR4-, deficient splenic CD11c+ DCs could up-regulate their costimulatory molecule expression in response to LPS. Collectively, the present study provides the first evidence that the MyD88-independent pathway downstrem of TLR4 can lead to functional DC maturation, which is critical for a link between innate and adaptive immunity.
Human Cytomegalovirus pp65- and Immediate Early 1 Antigen-Specific HLA Class I-Restricted Cytotoxic T Cell Responses Induced by Cross-Presentation of Viral Antigens
Authors: Tabi Zsuzsanna , Moutaftsi Magdalena , Borysiewicz Leszek K.
Published: April 2014

Dendritic cells (DCs) play a pivotal role in the development of anti-viral CD8+ CTL responses. This is straightforward if they are directly infected with virus, but is less clear in response to viruses that cannot productively infect DCs. Human CMV (HCMV) shows strain-specific cell tropism: fibroblast (Fb)-adapted laboratory strains (AD169) and recent clinical isolates do not infect DCs, whereas endothelial cell-adapted strains (TB40/E) result in productive lytic DC infection. However, we show here that uninfected DCs induce CD8+ T cell cytotoxicity and IFN-γ production against HCMV pp65 and immediate early 1 Ags following in vitro coculture with HCMV-AD169-infected Fbs, regardless of the HLA type of these Fbs. CD8+ T cell stimulation was inhibited by pretreatment of DCs with cytochalasin B or brefeldin A, indicating a phagosome/endosome to cytosol pathway. HCMV-infected Fbs were not apoptotic as measured by annexin V binding, and induction of apoptosis of infected Fbs in vitro did not augment CTL induction by DCs, suggesting a mechanism other than apoptosis in the initiation of cross-presentation. Furthermore, HCMV-infected Fbs provided a maturation signal for immature DCs during coculture, as evidenced by increased CD83 and HLA class II expression. Cross-presentation of HCMV Ags by host DCs enables these professional APCs to bypass some of the evasion mechanisms HCMV has developed to avoid T cell recognition. It may also serve to explain the presence of immediate early 1 Ag-specific CTLs in the face of pp65-induced inhibition of Ag presentation at the level of the infected cell.
Neonatal T Cells in an Adult Lung Environment Are Competent to Resolve Pneumocystis carinii Pneumonia
Authors: Qureshi Mahboob H. , Garvy Beth A.
Published: April 2014

Initiation of the pulmonary inflammatory response to Pneumocystis carinii is delayed by 3 wk in mice infected as neonates compared with adults. There was no difference in the proliferative response of draining lymph node T cells from mice infected as neonates compared with adults when stimulated in vitro with either Con A or anti-CD3 mAb. However, TNF-α and IFN-γ mRNA expression in the lungs of P. carinii-infected neonates was significantly lower than in adults indicating a lack of appropriate activation signaling in the local environment. This may have been due to active suppression because TGF-β mRNA expression was significantly elevated in neonatal lungs compared with adults. To determine whether T cells from 10-day-old mice would effect resolution of P. carinii if harbored in an adult lung environment, cells were adoptively transferred to SCID mice with established P. carinii infections. There was no difference in the kinetics of T cell migration into the lungs or of clearance of P. carinii organisms when SCID mice were reconstituted with splenocytes from young mice as compared with adult mice. Furthermore, splenocytes from young mice stimulated both TNF-α and IFN-γ mRNA expression to levels that were similar to that in the lungs of SCID mice reconstituted with adult cells. These data indicate that neonatal lymphocytes are competent to resolve P. carinii infection when harbored in an adult lung environment, suggesting that the neonatal lung environment, and not the T cells, is ineffective at responding to P. carinii infection.
A Heritable Defect in IL-12 Signaling in B10.Q/J Mice. I. In Vitro Analysis
Authors: Ortmann Robert , Smeltz Ronald , Yap George , Sher Alan , Shevach Ethan M.
Published: April 2014

B10.Q mice are normally susceptible to the induction of collagen-induced arthritis. We noted that one subline of B10.Q mice, B10.Q/J, was completely resistant to disease induction when immunized with collagen in CFA. B10.Q/J mice have a global defect in the generation of Th1 responses, and Ag-specific T cells derived from this strain failed to produce IFN-γ. Because T cells from these mice could produce normal amounts of IFN-γ when activated by IL-12/IL-18-independent stimuli, the defect appeared to be a failure to respond to IL-12. This defect extended to NK cells, which also failed to produce IFN-γ when stimulated by IL-12. The capacity of NK cells, but not activated T cells, to produce IFN-γ in response to IL-12 could be partially restored by IL-18. The expression of the IL-12R β1- and β2-chains on T cells and NK cells from B10.Q/J mice was normal. However, activated T cells from B10.Q/J mice did not signal normally through the IL-12R and manifested a defect in their capacity to phosphorylate Stat4. This defect was partial in that it could be overcome by increasing both the concentration of IL-12 and the incubation times in the Stat4 phosphorylation assays. Because Stat4 function is apparently intact in B10.Q/J mice, the defect in IL-12 signaling can be localized between the IL-12R complex and Stat4. This subtle abnormality in IL-12 responsiveness results in a profound defect in the generation of Th1 cells and the development of autoimmune disease.
A Heritable Defect in IL-12 Signaling in B10.Q/J Mice. II. Effect on Acute Resistance to Toxoplasma gondii and Rescue by IL-18 Treatment
Authors: Yap George S. , Ortmann Robert , Shevach Ethan , Sher Alan
Published: April 2014

This study documents a defect in IL-12-dependent IFN-γ responses in a substrain (B10.Q-H2-q/SgJ) of B10.Q mice that manifests as an acute susceptibility to infection by the intracellular protozoan pathogen, Toxoplasma gondii. Despite robust systemic production of IL-12, infected B10.Q/J animals fail to mount an early IFN-γ response after parasite inoculation. Genetic experiments revealed that the host resistance and IFN-γ production defects are determined by a single autosomal recessive locus distinct from the Stat4 gene. Nonetheless, a delayed IL-12-mediated IFN-γ response emerges in later stages of acute infection but is unable to prevent host mortality. IL-18 administration restores, in an IL-12-dependent manner, the early IFN-γ response and host resistance of B10.Q/J animals. These in vivo studies indicate that the partially impaired IL-12 responsiveness in B10.Q/J mice can result in defective host resistance and demonstrate a therapeutic function for IL-18 in reversing a genetically based immunodeficiency in IL-12-dependent IFN-γ production.
A Role for CD44 in the Production of IFN-γ and Immunopathology During Infection with Toxoplasma gondii
Authors: Blass Sarah L. , Puré Ellen , Hunter Christopher A.
Published: April 2014

The interaction of activated CD44 with its ligand, low m.w. hyaluronan, is involved in inflammation, but no role has been identified for this interaction in the regulation of an immune response to infection. In these studies, infection of C57BL/6 mice with Toxoplasma gondii resulted in increased expression of CD44 on T cells, B cells, NK cells, and macrophages, and a small percentage of CD4+ T cells express an activated form of CD44. Administration of anti-CD44 to infected mice prevented the development of a CD4+ T cell-dependent, infection-induced inflammatory response in the small intestine characterized by the overproduction of IFN-γ. The protective effect of anti-CD44 treatment was associated with reduced production of IFN-γ, but not IL-12, in vivo and in vitro. Furthermore, the addition of low m.w. hyaluronan to cultures of splenocytes or purified CD4+ T cells from infected mice resulted in the production of high levels of IFN-γ, which was dependent on IL-12 and TCR stimulation. Together, these results identify a novel role for CD44 in the regulation of IFN-γ production by CD4+ T cells during infection and demonstrate a role for CD44 in the regulation of infection-induced immune pathology.
Improving Vaccine Potency Through Intercellular Spreading and Enhanced MHC Class I Presentation of Antigen
Authors: Hung Chien-Fu , Cheng Wen-Fang , Chai Chee-Yin , Hsu Keng-Fu , He Liangmei , Ling Morris , Wu T.-C.
Published: April 2014

The potency of naked DNA vaccines is limited by their inability to amplify and spread in vivo. VP22, a HSV-1 protein, has demonstrated the remarkable property of intercellular transport and may thus provide a unique approach for enhancing vaccine potency. Therefore, we created a novel fusion of VP22 with a model Ag, human papillomavirus type 16 E7, in a DNA vaccine that generated enhanced spreading and MHC class I presentation of Ag. These properties led to a dramatic increase in the number of E7-specific CD8+ T cell precursors in vaccinated mice (around 50-fold) and converted a less effective DNA vaccine into one with significant potency against E7-expressing tumors. In comparison, nonspreading VP221–267 mutants failed to enhance vaccine potency. Our data indicated that the potency of DNA vaccines may be dramatically improved through intercellular spreading and enhanced MHC class I presentation of Ag.
Salmonella Pathogenicity Island 2-Encoded Type III Secretion System Mediates Exclusion of NADPH Oxidase Assembly from the Phagosomal Membrane
Authors: Gallois Annabelle , Klein Joanna R. , Allen Lee-Ann H. , Jones Bradley D. , Nauseef William M.
Published: April 2014

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b558, was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.
Expression of Stromal-Derived Factor-1 Is Decreased by IL-1 and TNF and in Dermal Wound Healing
Authors: Fedyk Eric R. , Jones Dan , Critchley Hilary O. D. , Phipps Richard P. , Blieden Timothy M. , Springer Timothy A.
Published: April 2014

Stromal-derived factor-1 (SDF-1) is a CXC chemokine that is believed to be constitutively expressed by stromal cells of numerous tissues. In this report, we demonstrate that dermal fibroblasts and vessels of noninflamed tissues express SDF-1. Unexpectedly, we found that expression of SDF-1 is regulated by inflammation. Expression of SDF-1 by primary cultures of human gingival fibroblasts is potently inhibited by activated macrophages via secretion of IL-1α and TNF-α. Levels of SDF-1 mRNA also decrease in acutely inflamed mouse dermal wounds. We propose that SDF-1 functions as a homeostatic regulator of tissue remodeling, whose expression stabilizes existing dermal architecture.
Combinatorial Model of Chemokine Involvement in Glomerular Monocyte Recruitment: Role of CXC Chemokine Receptor 2 in Infiltration During Nephrotoxic Nephritis
Authors: Zernecke Alma , Weber Kim S. C. , Erwig Lars P. , Kluth David C. , Schröppel Bernd , Rees Andrew J. , Weber Christian
Published: April 2014

A sequential model involving chemokines has been proposed for leukocyte extravasation into areas of inflammation; however, site-specific aspects remain to be elucidated. Hence, we studied the role of chemokines produced by mesangial (MC) or glomerular endothelial cells (GEC) and their receptors in glomerular recruitment of monocytes. Stimulation of MC with TNF-α up-regulated mRNA and protein of CC and CXC chemokines but not constitutive expression of the CX3C chemokine fractalkine. While growth-related activity (GRO)-α was immobilized to MC proteoglycans, monocyte chemotactic protein (MCP)-1 was secreted into the soluble phase. Firm adhesion and sequestration of monocytes on activated MC was supported by the GRO-α receptor CXCR2 and to a lesser extent by CX3CR, whereas the MCP-1 receptor CCR2 contributed to their transendothelial chemotaxis toward activated MC. In contrast, fractalkine mRNA and protein was induced by TNF-α in transformed rat GEC, and both CXCR2 and CX3CR mediated monocyte arrest on GEC in shear flow. The relevance of these mechanisms was confirmed in a rat nephrotoxic nephritis model where acute glomerular macrophage recruitment was profoundly inhibited by blocking CXCR2 or CCR2. In conclusion, our results epitomize a combinatorial model in which chemokines play specialized roles in driving glomerular monocyte recruitment and emphasize an important role for CXCR2 in macrophage infiltration during early phases of nephrotoxic nephritis.
Eosinophils Are a Major Source of Nitric Oxide-Derived Oxidants in Severe Asthma: Characterization of Pathways Available to Eosinophils for Generating Reactive Nitrogen Species
Authors: MacPherson Jennifer C. , Comhair Suzy A. A. , Erzurum Serpil C. , Klein Dennis F. , Lipscomb Mary F. , Kavuru Mani S. , Samoszuk Michael K. , Hazen Stanley L.
Published: April 2014

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO2Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 ± 198 μmol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 ± 40.7 μmol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 ± 457 μmol BrY/mol tyrosine; 161 ± 88 μmol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 ± 14.5 μmol BrY/mol tyrosine; 65 ± 69 μmol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO2Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO2−, NO3−, or NO2Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO2− or an exogenous NO source. At low, but not high (e.g., >2 μM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.
Rapid Depletion of Peripheral Antigen-Specific T Cells in TCR-Transgenic Mice After Oral Administration of Myelin Basic Protein
Authors: Meyer Abbie L. , Benson Jacqueline , Song Fei , Javed Najma , Gienapp Ingrid E. , Goverman Joan , Brabb Thea A. , Hood Leroy , Whitacre Caroline C.
Published: April 2014

In myelin basic protein (MBP)-specific TCR-transgenic (Tg) mice, peripheral T cells express the Vα2.3/Vβ8.2-Tg TCR, demonstrate vigorous proliferative responses to MBP in vitro, and can exhibit experimental autoimmune encephalomyelitis (EAE) within 5 days of pertussis toxin injection. We explored the effects of oral administration of MBP on the cellular trafficking of the MBP-specific TCR-Tg cells and the ability of oral MBP to protect Tg mice from EAE. Tg mice were fed MBP, OVA or vehicle and sacrificed at various times after feeding. An immediate and dramatic decrease in Vα2.3/Vβ8.2+-Tg cells was observed in the periphery within 1 h after feeding. By 3 days after feeding, the percentage of Tg cells increased to near control levels, but decreased again by 10 days. When MBP or vehicle-fed Tg mice were challenged for EAE at this point, disease was severe in the vehicle-fed mice and reduced in the MBP-fed mice over the 40-day observation period. In vitro studies revealed a biphasic pattern of MBP proliferative unresponsiveness and an induction of Th1 cytokines. Immunohistochemical staining showed that the number of Tg cells found in the intestinal lamina propria increased dramatically as the number of Tg cells in the periphery decreased. There was no apparent proliferation of Tg cells in the lamina propria, indicating that Tg cells trafficked there from the periphery. Taken together, these results suggest that T cell trafficking into the site of Ag deposition acts to protect the TCR-Tg mouse from EAE.
Carcinogen-Induced Inflammation and Immunosuppression Are Enhanced in Xeroderma Pigmentosum Group A Model Mice Associated with Hyperproduction of Prostaglandin E2
Authors: Miyauchi-Hashimoto Hiroko , Kuwamoto Kazue , Urade Yoshihiro , Tanaka Kiyoji , Horio Takeshi
Published: April 2014

Xeroderma pigmentosum group A (XPA) gene-deficient mice easily developed skin cancers by the application of topical chemical carcinogens as well as by UV irradiation. As certain chemical carcinogens have been shown to be immunosuppressive, we examined the inflammatory and immunosuppressive effects of dimethylbenz(a)anthracene (DMBA) on XPA mice. Compared with wild-type mice, XPA mice showed greater ear swelling and reduction of epidermal Langerhans cells after DMBA application. Topical application of DMBA impaired the induction of contact hypersensitivity, initiated either locally or at distant sites. These DMBA-induced local and systemic immunosuppressions were more greatly enhanced in XPA mice than in wild-type mice. DMBA application induced pronounced production of PGE2, IL-10, and TNF-α in the skin of XPA mice. Treatment with indomethacin, a potent inhibitor of PG biosynthesis, inhibited DMBA-induced inflammation and local immunosuppression. In XPA mice, increased serum IL-10 was detected after DMBA treatment. Excess production of PGE2, TNF-α, and IL-10 after DMBA application may be involved in the enhanced local and systemic immunosuppression in DMBA-treated XPA mice. Susceptibility to DMBA-induced skin tumors in XPA mice may be due to easy impairment of the immune system by DMBA in addition to a defect in the repair of DMBA-DNA adduct. Enhanced immunosuppression by chemical carcinogens as well as the mutagenicity of these mutagens might be associated with the high incidence of internal malignancies seen in XP patients. Moreover, these results supported the hypothesis that persistent DNA damage is a trigger for the production of immunoregulatory cytokines.
A Murine IL-4 Receptor Antagonist That Inhibits IL-4- and IL-13-Induced Responses Prevents Antigen-Induced Airway Eosinophilia and Airway Hyperresponsiveness
Authors: Tomkinson A. , Duez C. , Cieslewicz G. , Pratt J. C. , Joetham A. , Shanafelt M.-C. , Gundel R. , Gelfand E. W.
Published: April 2014

The closely related Th2 cytokines, IL-4 and IL-13, share many biological functions that are considered important in the development of allergic airway inflammation and airway hyperresponsiveness (AHR). The overlap of their functions results from the IL-4R α-chain forming an important functional signaling component of both the IL-4 and IL-13 receptors. Mutations in the C terminus region of the IL-4 protein produce IL-4 mutants that bind to the IL-4R α-chain with high affinity, but do not induce cellular responses. A murine IL-4 mutant (C118 deletion) protein (IL-4R antagonist) inhibited IL-4- and IL-13-induced STAT6 phosphorylation as well as IL-4- and IL-13-induced IgE production in vitro. Administration of murine IL-4R antagonist during allergen (OVA) challenge inhibited the development of allergic airway eosinophilia and AHR in mice previously sensitized with OVA. The inhibitory effect on airway eosinophilia and AHR was associated with reduced levels of IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid as well as reduced serum levels of OVA-IgE. These observations demonstrate the therapeutic potential of IL-4 mutant protein receptor antagonists that inhibit both IL-4 and IL-13 in the treatment of allergic asthma.
Possible Involvement of EBV-Mediated α-Fodrin Cleavage for Organ-Specific Autoantigen in Sjogren’s Syndrome
Authors: Inoue Hiroko , Tsubota Kazuo , Ono Masafumi , Kizu Yasuhiro , Mizuno Fumio , Takada Kenzo , Yamada Koichi , Yanagi Kumiko , Hayashi Yoshio , Saito Ichiro
Published: April 2014

A cleavage product of α-fodrin may be an important organ-specific autoantigen in the pathogenesis of Sjogren’s syndrome (SS), but the mechanisms of α-fodrin cleavage remain unclear. Since EBV has been implicated in the pathogenesis of SS, we determined whether EBV activation could induce the SS-specific 120-kDa autoantigen α-fodrin. ZEBRA mRNA expression, a marker for activation of the lytic cycle of EBV, was found in the salivary gland tissues from SS patients, but not in those from control individuals. ZEBRA-expressing lymphoid cells were also found in the SS glands in double-stained immunohistochemistry. Furthermore, a significant link between production of Abs against 120-kDa α-fodrin and reactivated EBV Ag was found in sera from patients with SS, but not in those from control individuals. EBV-activated lymphoid cells showed specific α-fodrin cleavage to the expected 120-kDa fragments in vitro. Pretreatment with caspase inhibitors inhibited cleavage of α-fodrin. Thus, an increase in apoptotic protease activities induced by EBV reactivation may be involved in the progression of α-fodrin proteolysis in the development of SS.
SIN-1, a Nitric Oxide Donor, Ameliorates Experimental Allergic Encephalomyelitis in Lewis Rats in the Incipient Phase: The Importance of the Time Window
Authors: Xu Ling-Yun , Yang Jian-She , Link Hans , Xiao Bao-Guo
Published: April 2014

NO is involved in the regulation of immune responses. The role of NO in the pathogenesis of experimental allergic encephalomyelitis (EAE) is controversial. In this study, 3-morpholinosydnonimine (SIN-1), an NO donor, was administered to Lewis rats on days 5–7 postimmunization, i.e., during the incipient phase of EAE. SIN-1 reduced clinical signs of EAE compared with those in PBS-treated control rats and was accompanied by reduced ED1+ macrophages and CD4+ T cell infiltration within the CNS. Blood mononuclear cells (MNC) obtained on day 14 postimmunization revealed that SIN-1 administration enhanced NO and IFN-γ production by blood MNC and suppressed Ag- and mitogen-induced proliferative responses. MHC class II, B7-1 and B7-2 were down-regulated in SIN-1-treated EAE rats. Simultaneously, frequencies of apoptotic cells among blood MNC were increased. In vivo, SIN-1 is likely to behave as an NO donor. Administration of SIN-1 induced NO production, but did not affect superoxide and peroxynitrite formation. Enhanced NO production during the priming phase of EAE thus promotes apoptosis, down-regulates disease-promoting immune reactivities, and ameliorates clinical EAE, mainly through SIN-1-derived NO, without depending on NO synthase.
Kinetics of TCR Use in Response to Repeated Epitope-Specific Immunization
Authors: Monsurrò Vladia , Nielsen Mai-Britt , Perez-Diez Ainhoa , Dudley Mark E. , Wang Ena , Rosenberg Steven A. , Marincola Francesco M.
Published: April 2014

Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209–217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient’s kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN-γ ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies.