bluuebunny/crossref_metadata_2025 huggingface dataset:
Transfer of Central Nervous System Autoantigens and Presentation in Secondary Lymphoid Organs
Authors: de Vos Alex F. , van Meurs Marjan , Brok Herbert P. , Boven Leonie A. , Hintzen Rogier Q. , van der Valk Paul , Ravid Rivka , Rensing Susanne , Boon Louis , Hart Bert A. ’t , Laman Jon D.
Published: April 2014

Dendritic cells are thought to regulate tolerance induction vs immunization by transferring Ags and peripheral signals to draining lymph nodes (LN). However, whether myelin Ag transfer and presentation in LN occurs during demyelinating brain disease is unknown. In this study, we demonstrate redistribution of autoantigens from brain lesions to cervical LN in monkey experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Immunohistochemical analysis revealed significantly more cells containing myelin Ags in cervical LN of monkeys with EAE compared with those of healthy control monkeys. Myelin Ags were observed in cells expressing dendritic cell/macrophage-specific markers, MHC class II, and costimulatory molecules. Moreover, these cells were directly juxtaposed to T cells, suggesting that cognate interactions between myelin-containing APC and T cells are taking place in brain-draining LN. Indeed, myelin Ag-reactive T cells were observed in cervical LN from marmosets and rhesus monkeys. Importantly, these findings were paralleled by our findings in human tissue. We observed significantly more myelin Ag-containing cells in LN of individuals with MS compared with those of control individuals. These cells expressed APC markers, as observed in marmosets and rhesus monkeys. These findings suggest that during MS and EAE, modulation of T cell reactivity against brain-derived Ags also takes place in cervical LN and not necessarily inside the brain. A major implication is that novel therapeutic strategies may be targeted to peripheral events, thereby circumventing the blood-brain barrier.
Functional Activity of Natural Antibody is Altered in Cr2-Deficient Mice
Authors: Reid Russell R. , Woodcock Sean , Shimabukuro-Vornhagen Alexander , Austen William G. , Kobzik Lester , Zhang Ming , Hechtman Herbert B. , Moore Francis D. , Carroll Michael C.
Published: April 2014

The major source of natural IgM Abs are B-1 cells, which differ from conventional B cells in their anatomic location, cell surface phenotype, restricted usage of particular VH genes and limited use of N-region addition during V-D-J rearrangement. The origin of B-1 cells is unclear. However, they are capable of self-renewal and their development is sensitive to signaling via the B cell receptor, as genetic defects that impair the strength of the signal often result in limited development. These findings suggest that B-1 cells require either an intrinsic signal, or contact with Ag, for positive selection and expansion and/or maintenance in the periphery. In support of interaction with cognate Ag, deficiency in the complement receptors CD21/CD35 results in a 30–40% decrease in the CD5+ B-1 population. To determine whether this reduction reflects a loss of certain specificities or simply a proportional decline in the repertoire, we examined peritoneal B cells isolated from Cr2+ and Cr2def mice for recognition of a B-1 cell Ag, i.e., phosphatidylcholine, and assayed for injury in an IgM natural Ab-dependent model of reperfusion injury. We found a similar frequency of phosphatidylcholine-specific CD5+ B-1 cells in the two strains of mice. By contrast, the Cr2def mice have reduced injury in the IgM-dependent model of reperfusion injury. Reconstitution of the deficient mice with pooled IgM or adoptive transfer of Cr2+ peritoneal B cells restored injury. These results suggest that complement receptors CD21/CD35 are important in maintenance of the B-1 cell repertoire to some, but not all, specificities.
Evidence That SHIP-1 Contributes to Phosphatidylinositol 3,4,5-Trisphosphate Metabolism in T Lymphocytes and Can Regulate Novel Phosphoinositide 3-Kinase Effectors
Authors: Freeburn Robin W. , Wright Karen L. , Burgess Steven J. , Astoul Emmanuelle , Cantrell Doreen A. , Ward Stephen G.
Published: April 2014

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5′-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3; the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P3 and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P3 or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P3 and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.
IgE Cross-Linking or Lipopolysaccharide Treatment Induces Recruitment of Th2 Cells to the Lung in the Absence of Specific Antigen
Authors: Stephens Robin , Chaplin David D.
Published: April 2014

We previously showed that Th1 cells can increase recruitment of Th2 cells to the lungs even in the absence of the Th2-specific Ag. The fact that Th2 recruitment is independent from the Th2 cell Ag suggested that Th1 cells may support Th2 cell recruitment using their Ag-nonspecific proinflammatory functions. To investigate the potential for inflammatory stimuli that are distinct from Ag-specific signals to affect the recruitment of T cells, we tested whether cross-linking of IgE or treatment with LPS modulated influx of Th2 cells into the airways in the presence or absence of inhaled Ag. When naive mice that had been treated with OVA-specific Th2 cells and passively sensitized with anti-DNP IgE were challenged by intranasal administration of either DNP-haptenated OVA or DNP-BSA, increased numbers of Th2 cells were recruited to the lung compared with mice challenged intranasally with OVA alone. Intranasal administration of LPS also increased recruitment of Th2 cells to the airways. These two distinct inflammatory stimuli increased the numbers of recruited Th2 cells equally with or without concurrent challenge using the cognate Th2 Ag. This Ag-independent recruitment of Th2 cells to the lung was not associated with localization of these cells to the regional lymph nodes and was independent of Th2 cell activation. Interestingly, P- or E-selectin contributed to Th2 cell recruitment to the lung. These data suggest that Th2 cells of the adaptive immune response are similar to cells of the innate immune response in their lack of requirement for protein Ag to initiate cell recruitment. They demonstrate further that recruitment can occur independently of Ag-dependent activation.
Macrophage-Derived Complement Component C4 Can Restore Humoral Immunity in C4-Deficient Mice
Authors: Gadjeva Mihaela , Verschoor Admar , Brockman Mark A. , Jezak Heather , Shen Li Ming , Knipe David M. , Carroll Michael C.
Published: April 2014

Mice with a disrupted C4 locus (C4−/−) have an impaired immune response to thymus-dependent Ags. To test the role of bone marrow-derived C4 in humoral immunity, we reconstituted deficient animals with wild-type bone marrow or an enriched fraction of bone marrow-derived macrophages. C4 chimeras were immunized with 4-hydroxy-3-nitrophenyl5 conjugated to keyhole limpet hemocyanin (NP5- KLH) or infected with HSV-1, and the Ab response was evaluated. Wild-type bone marrow rescued the humoral immune response to both Ags, i.e., the soluble Ag and HSV-1, demonstrating that local C4 production is sufficient for humoral responses. Although the C4 chimeric animals lacked detectable C4 in their sera, C4 mRNA was identified in splenic sections by in situ hybridization, and C4 protein deposits were identified in the germinal center areas of splenic follicles by immunofluorescence staining. Macrophages derived from bone marrow produced sufficient C4 protein to restore the humoral response to NP5-KLH in C4-deficient animals when administered along with Ag. Cell-sorting experiments, followed by C4-specific RT-PCR, identified splenic macrophages (CD11b+, CD11c−) as a cellular source for C4 synthesis within the spleen.
Organ-Specific Cytokine Polarization Induced by Adoptive Transfer of Transgenic T Cells
Authors: Zhang Lei , Lizzio Elaine F. , Gubina Elena , Chen Trina , Mostowski Howard , Kozlowski Steven
Published: April 2014

There are two distinct phenotypes of T cell cytokine responses that lead to different effector functions and different outcomes in disease processes. Although evidence suggests a possible role of the local microenvironment in the differentiation or localization of T cells with these phenotypes, there are no examples of divergent T cell cytokine phenotypes with the same Ag specificity concurrently existing in different tissue compartments. Using a CD8+ T cell adoptive transfer model for graft-vs-host disease, we demonstrate that a potent type 2 cytokine response develops in the spleen while a potent type 1 cytokine response simultaneously develops in the testis. These experiments demonstrate for the first time that cytokine production can be oppositely polarized in different organs of the same individual. This may have important implications for organ-specific pathology in infection or autoimmunity: infections or autoimmune diseases that affect multiple organs may have heterogeneity in tissue cytokine responses that is not revealed in systemic lymphocyte cytokine responses. Therefore, attempts to modulate the immune response phenotype may ameliorate pathology in one organ while exacerbating pathology in another.
HLA-DQ Determines the Response to Exogenous Wheat Proteins: A Model of Gluten Sensitivity in Transgenic Knockout Mice
Authors: Black Kay E. , Murray Joseph A. , David Chella S.
Published: April 2014

We have investigated the genetic basis of the immune response to dietary gluten in HCD4/DQ8 and HCD4/DQ6 double transgenic mice. Mice were immunized with gluten i.p. or individual peptides s.c. and spleen or draining lymph node T cells were challenged in vitro. Strong proliferative responses to gluten were seen in the HCD4/DQ8 mice, whereas the HCD4/DQ6 mice responded to gluten poorly. A series of overlapping peptides spanning gliadin were synthesized. The HCD4/DQ8 mice reacted to many of the individual peptides of gliadin, while the HCD4/DQ6 mice were relatively unresponsive. T cells isolated from HCD4/DQ8 mice also responded well to modified (deamidated) versions of the gliadin peptides, whereas HCD4DQ6 mice did not. The T cell response to gluten was CD4 dependent and DQ restricted and led to the production of cytokines IL-6, TGF-β, and IL-10. Finally, intestinal lymphocytes isolated from gluten-fed HCD4/DQ8 mice displayed an activated phenotype. These data suggest that this HLA class II transgenic murine model of gluten sensitivity may provide insight into the initiation of the MHC class II-restricted gluten sensitivity in celiac disease.
The Adjuvant Effects ofMycobacterium tuberculosisHeat Shock Protein 70 Result from the Rapid and Prolonged Activation of Antigen-Specific CD8+ T Cells In Vivo
Authors: Harmala Lisa A. E. , Ingulli Elizabeth G. , Curtsinger Julie M. , Lucido Michelle M. , Schmidt Clint S. , Weigel Brenda J. , Blazar Bruce R. , Mescher Matthew F. , Pennell Christopher A.
Published: April 2014

Heat shock protein 70 (hsp70) is a potent adjuvant that links innate and adaptive immune responses. To study how hsp70 activates naive CD8+ T cells in vivo, we tracked Ag-specific CD8+ T cells in mice immunized with a fusion protein containing chicken OVA linked to hsp70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). On a molar basis, OVA.TBhsp70 was several hundred times more effective than OVA peptide plus CFA in eliciting specific CD8+ T cell responses. Immunization with OVA.TBhsp70 activated >90% of detectable OVA-specific CD8+ T cells within 3 days and led to the persistence of cytotoxic effectors for at least 17 days. These studies demonstrate that the potent adjuvant effect of M. tuberculosis hsp70 results from the relatively complete, rapid, and durable activation of Ag-specific CD8+ T cells.
Dendritic Cell-Associated Lectin-1: A Novel Dendritic Cell-Associated, C-Type Lectin-Like Molecule Enhances T Cell Secretion of IL-4
Authors: Ryan Elizabeth J. , Marshall Aaron J. , Magaletti Dario , Floyd Helen , Draves Kevin E. , Olson N. Eric , Clark Edward A.
Published: April 2014

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12–13 just 3′ to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a+ DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4+ CD45RA+ T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4+ T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4+ T cell proliferation or IL-4 and IFN-γ secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4+ T cells toward a Th2 response by enhancing their secretion of IL-4.
Absence of L-Selectin Delays Mucosal B Cell Responses in Nonintestinal Effector Tissues
Authors: Csencsits Keri L. , Pascual David W.
Published: April 2014

Previous studies suggest that lymphocyte trafficking to head and neck lymph nodes, also referred to as cranial-, oral-, nasal-associated lymphoid tissue (CONALT), is L-selectin (L-Sel) dependent, despite coexpression of α4β7, resulting in their marked reduction in L-Sel-deficient (L-Sel−/−) mice. Consequently, early phase (16 days) Ab responses to cholera toxin (CT) are diminished. The following studies reveal that lack of mucosal effector responses is not caused by loss of inductive immune responses in the L-Sel−/− CONALT. Indeed, there was an increased accumulation of total IgA, but not Ag-specific IgA Ab-forming cells (AFC) in L-Sel−/− CONALT. This increased accumulation was not evident in L-Sel+/+ CONALT. Identification of lymphocyte-homing receptors on L-Sel−/− and L-Sel+/+ CONALT lymphocytes revealed no significant differences in expression of α4β7, which might contribute to lymphocyte homing in the absence of L-Sel. Studies of CONALT responses during the late phase (6 wk post-intranasal immunization) revealed the number of lymphocytes recovered from L-Sel−/− CONALT was less than L-Sel+/+ CONALT; however, L-Sel−/− CT-specific and total AFC did not vary from 16-day responses, suggesting a defect in CT-specific B cell export. No significant differences in α4β7 expression between L-Sel−/− and L-Sel+/+ CONALT were noted. Yet, these increases in CONALT AFC correlated with restoration of immunity in L-Sel−/− nasal passages and reproductive tracts.
Peptide-Independent Folding and CD8αα Binding by the Nonclassical Class I Molecule, Thymic Leukemia Antigen
Authors: Weber Dominique A. , Attinger Antoine , Kemball Christopher C. , Wigal Jerrod L. , Pohl Jan , Xiong Yi , Reinherz Ellis L. , Cheroutre Hilde , Kronenberg Mitchell , Jensen Peter E.
Published: April 2014

The nonclassical class I molecule, thymic leukemia (TL), has been shown to be expressed on intestinal epithelial cells and to interact with CD8+ intraepithelial T lymphocytes. We generated recombinant soluble TL (T18d) H chains in bacteria as inclusion bodies and refolded them with β2-microglobulin in the presence or absence of a random peptide library. Using a mAb, HD168, that recognizes a conformational epitope on native TL molecules, we observed that protein folds efficiently in the absence of peptide. Circular dichroism analysis demonstrated that TL molecules have structural features similar to classical class I molecules. Moreover, thermal denaturation experiments indicated that the melting temperature for peptide-free TL is similar to values reported previously for conventional class I-peptide complexes. Our results also show that CD8αα binding is not dependent on either TL-associated peptide or TL glycosylation.
Differential Regulation of IL-12 and IL-10 Gene Expression in Macrophages by the Basic Leucine Zipper Transcription Factor c-Maf Fibrosarcoma
Authors: Cao Shanjin , Liu Jianguo , Chesi Marta , Bergsagel Peter Leif , Ho I-Cheng , Donnelly Raymond P. , Ma Xiaojing
Published: April 2014

IL-12 is a principal activator of both innate and adaptive immunity against infectious agents and malignancies. Regulation of proinflammatory IL-12 gene expression in phagocytes by the anti-inflammatory cytokine IL-10 represents a major homeostatic process underlying host-pathogen and host-self interactions. Delineation of the signaling pathway of IL-10 is crucial to the understanding of immunological regulatory networks. In this study, we report that IL-10 and c-musculoaponeurotic fibrosarcoma (Maf) induce their mutual expression in inflammatory macrophages. We demonstrate that c-Maf is one of the physiological mediators of IL-10’s immunosuppressive activities. When overexpressed, c-Maf selectively inhibits transcriptional activation of IL-12 p40 and p35 genes while potently activating IL-10 and IL-4 expression, potentially contributing to the development of a state of anti-inflammation and dichotomy of immunologic polarization. c-Maf induces changes in nuclear DNA-binding activities at multiple sites including the ets, GA-12, NF-κB, C/EBP, and AP-1 elements. Nonetheless, the essential c-Maf-responsive element appears to be located elsewhere. Inhibition of IL-12 p40 gene expression by c-Maf requires the N-terminal transactivation domain, suggesting an indirect mechanism of transcriptional inhibition involving the induction of an unidentified repressor. In c-Maf-deficient murine macrophages, IL-10 production is impaired. However, IL-10-mediated inhibition of IL-12 production remains intact, indicating the existence of alternative mediators in the absence of c-Maf, consistent with the observation that a functional AP-1 is required for this pathway.
Small Interfering RNA-Mediated Gene Silencing in T Lymphocytes
Authors: McManus Michael T. , Haines Brian B. , Dillon Christopher P. , Whitehurst Charles E. , van Parijs Luk , Chen Jianzhu , Sharp Phillip A.
Published: April 2014

Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8α specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.
Involvement of Classical and Novel Protein Kinase C Isoforms in the Response of Human Vγ9Vδ2 T Cells to Phosphate Antigens
Authors: Cipriani Barbara , Knowles Heather , Chen Lanfen , Battistini Luca , Brosnan Celia F.
Published: April 2014

Human γδ T cells expressing the Vγ9Vδ2 gene segments are activated polyclonally by phosphoantigens found on a wide variety of pathogenic organisms. After ligand exposure, Vγ9Vδ2 T cells proliferate and rapidly secrete large amounts of cytokines and chemokines that contribute to the innate immune response to these pathogens. Neither APCs nor costimulatory molecules are required. In this study we examined whether these phosphoantigens activate protein kinase Cθ (PKCθ). This novel PKC isoform is essential for Ag signaling through the αβ TCR in a costimulation-dependent fashion. The results showed that isopentenyl pyrophosphate (IPP), a soluble phospholigand released by mycobacteria, led to the rapid and persistent activation of PKCθ in γδ T cells, as determined by evidence of translocation and phosphorylation. In contrast, no ligand-dependent response was detected for PKCα/β or PKCδ. Using the inhibitors Gö6976 and rottlerin, a role for both conventional and novel PKC isoforms in IPP-induced proliferation, CD25 expression, and cytokine and chemokine production was demonstrated. Gel-shift assays indicated that the transcription factors NF-κB and AP-1 were downstream targets of PKC activation. IPP also induced the rapid and persistent phosphorylation of extracellular signal-regulated kinases 1 and 2, p38 mitogen-activated kinase, and stress-activated kinase/c-Jun N-terminal kinase, but only an inhibitor of conventional PKCs blocked these responses. We conclude that the γδ T cell response to phosphoantigens is regulated by both novel and conventional PKC isoforms, with PKCθ being more responsive to ligand stimulation and PKCα/β to growth-factor availability.
The Protective Immune Response to Heat Shock Protein 60 ofHistoplasma capsulatumIs Mediated by a Subset of Vβ8.1/8.2+ T Cells
Authors: Scheckelhoff Mark , Deepe George S.
Published: April 2014

Immunization with recombinant heat shock protein 60 (rHsp60) from Histoplasma capsulatum or a region of the protein designated fragment 3 (F3) confers protection from a subsequent challenge in mice. To determine the T cell repertoire involved in the response to Hsp60, T cell clones from C57BL/6 mice immunized with rHsp60 were generated and examined for Vβ usage by flow cytometry and RT-PCR. Vβ8.1/8.2+ T cells were preferentially expanded; other clones bore Vβ4, -6, or -11. When Vβ8.1/8.2+ cells were depleted in mice, Vβ4+ T cell clones were almost exclusively isolated. Measurement of cytokine production demonstrated that nine of 16 Vβ8.1/8.2+ clones were Th1, while only three of 13 non-Vβ8.1/8.2+ clones were Th1. In mice immunized with rHsp60, depletion of Vβ8.1/8.2+, but not Vβ6+ plus Vβ7+, T cells completely abolished the protective efficacy of Hsp60 to lethal and sublethal challenges. Examination of the TCR revealed that a subset of Vβ8.1/2+ clones that produced IFN-γ and were reactive to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell clones into TCR α/β−/− or IFN-γ−/− mice significantly improved survival, while transfer of other Vβ8.1/8.2+ clones that were F3 reactive but were Th2 or clones that were not reactive to F3 but were Th1 did not confer protection. These data indicate that a distinct subset of Vβ8.1/8.2+ T cells is crucial for the generation of a protective response to rHsp60.
A Unique Mechanism for Innate Cytokine Promotion of T Cell Responses to Viral Infections
Authors: Pien Gary C. , Nguyen Khuong B. , Malmgaard Lene , Satoskar Abhay R. , Biron Christine A.
Published: April 2014

The kinetics of CD8 T cell IFN-γ responses as they occur in situ are defined here during lymphocytic choriomeningitis virus (LCMV) infections, and a unique mechanism for the innate cytokines IFN-αβ and IL-18 in promoting these responses is defined. Infections of mice with Armstrong or WE strains of LCMV induced an unexpectedly early day 4 IFN-γ response detectable in serum samples and spleen and liver homogenates. Production of IFN-γ was MHC class I/CD8 dependent, but did not require IL-12, NK cells, TCR-γδ T cells, MHC class II, or CD4 T cells. Peak response required specific Ag recognition, as administration of antagonist peptide partially impaired day 4 IFN-γ induction, and viral peptide stimulation enhanced CD8 T cell IFN-γ expression in culture. The IFN-γ response was associated with IL-18 and IFN-αβ expression. Furthermore, both factors augmented peptide-driven IFN-γ production in culture, and mice lacking IL-18 or IFN-αβ functions had reduced day 4 IFN-γ. Collectively, these results demonstrate that during viral infections, there is a dramatic in vivo CD8 T cell response preceding maximal expansion of these cells, and that the mechanism supporting this response is dependent on endogenous innate cytokines. Because stimulation by microbial products is linked to innate cytokine expression, the studies also suggest a pathway for precisely limiting T cell functions to times of need.
Development of Inflammation in Proteoglycan-Induced Arthritis Is Dependent on FcγR Regulation of the Cytokine/Chemokine Environment
Authors: Kaplan Charles D. , O’Neill Shannon K. , Koreny Tamas , Czipri Matyas , Finnegan Alison
Published: April 2014

FcγRs are specialized cell surface receptors that coordinately regulate immune responses. Although FcγR expression is a prerequisite for the development of several immune complex-mediated diseases, the mechanism responsible for FcγR-dependent regulation in autoimmunity remains unclear. Therefore, we assessed FcγR-dependent regulation of inflammation in proteoglycan-induced arthritis (PGIA) using FcγR−/− mice. FcγRIIb−/− mice developed arthritis at an earlier time point and with a greater severity than wild-type (WT) mice. In γ-chain−/− (FcγRI−/− and FcγRIII−/−) mice, no clinical or histological evidence of inflammation was observed. Exacerbation of arthritis in FcγRIIb−/− mice correlated with enhanced PG-specific Ab production, but did not significantly affect PG-specific T cell priming. In γ-chain−/− mice, the absence of arthritis did not correlate with serum Ab responses, as PG-specific Ab production was normal. Although PG-specific T cell proliferation was diminished, spleen cells from γ-chain−/− mice successfully adoptively transferred arthritis into SCID mice. Our studies indicated that the mechanism responsible for FcγR regulation of PGIA development was at the level of inflammatory cytokine and β-chemokine expression within the joint. FcγRIIb regulated the development of PGIA by controlling the initiation of cytokine and chemokine expression within the joint before the onset of arthritis, whereas the expression of FcγRI and or FcγRIII controlled cytokine and chemokine expression late in the development of PGIA during the onset of disease. These results suggest that FcγRs are critical for the development of inflammation during PGIA, possibly by maintaining or enhancing inflammatory cytokine and β-chemokine production.
Enhanced Recruitment of Th2 and CLA-Negative Lymphocytes by the S128R Polymorphism of E-Selectin
Authors: Rao Ravi M. , Haskard Dorian O. , Landis R. Clive
Published: April 2014

E-selectin is a cytokine-inducible endothelial cell adhesion molecule that binds a restricted population of T lymphocytes consisting of Th1 memory cells bearing the cutaneous lymphocyte Ag (CLA). A serine to arginine (S128R) polymorphism in E-selectin has been reported at increased frequency in patients with systemic lupus erythematosus and atherosclerosis. Here we tested the hypothesis that the S128R substitution may contribute to increased vascular disease by altering the number and/or phenotype of lymphocytes interacting with E-selectin under shear flow. We observed that CHO cell monolayers transfected with S128R recruited significantly greater numbers of unfractionated lymphocytes than monolayers expressing an equivalent density of wild-type (WT) E-selectin. Depletion of the CLA+ subpopulation or generation of CLA− lymphoblasts abolished rolling and arrest on WT E-selectin, but left a residual population that interacted with S128R. Generation of Th subsets revealed preferential interaction of Th0 and Th2, but not Th1, cells with S128R compared with WT. However, only T cells of a memory phenotype interacted with S128R, since neither monolayer supported rolling of CD45RA+ cells. Our results demonstrate that the S128R polymorphism extends the range of lymphocytes recruited by E-selectin, which may provide a mechanistic link between this polymorphism and vascular inflammatory disease.
A Comparison of Reactive Oxygen Species Generation by Rat Peritoneal Macrophages and Mast Cells Using the Highly Sensitive Real-Time Chemiluminescent Probe Pholasin: Inhibition of Antigen-Induced Mast Cell Degranulation by Macrophage-Derived Hydrogen Peroxide
Authors: Swindle Emily J. , Hunt John A. , Coleman John W.
Published: April 2014

Mast cells and macrophages live in close proximity in vivo and reciprocally regulate one another’s function in various ways. Although activated macrophages possess a powerful reactive oxygen species (ROS) generating system, there is conflicting evidence regarding whether mast cells can produce ROS. We used the highly sensitive real-time chemiluminescent probe Pholasin to examine ROS release by peritoneal macrophages and mast cells isolated from OVA-sensitized rats. Macrophages stimulated with PMA (0.8 μM) or ionomycin (1 μM), but not OVA (1 μg/ml), released high-level ROS, levels of which peaked after 3–7 min and declined to baseline levels within 1 h. Superoxide was identified as the major ROS species induced by PMA but not by ionomycin. In contrast, purified mast cells stimulated with PMA released low-level ROS, which was entirely due to the contaminating (2%) macrophages, and did not release any detectable ROS in response to ionomycin or OVA at concentrations that induced degranulation. Stimulation of mixed cell populations with PMA to induce macrophage ROS release led to 50% inhibition of serotonin release from mast cells stimulated 5 min later with OVA. The PMA-induced inhibitory factor was identified as hydrogen peroxide. In conclusion, activated rat peritoneal macrophages but not mast cells produce ROS, and macrophage-derived hydrogen peroxide inhibits mast cell degranulation. The latter could be an important mechanism whereby phagocytic cells regulate mast cell activation and promote resolution of IgE-mediated inflammation.
Toll-Like Receptor 4 and Toll-IL-1 Receptor Domain-Containing Adapter Protein (TIRAP)/Myeloid Differentiation Protein 88 Adapter-Like (Mal) Contribute to Maximal IL-6 Expression in Macrophages
Authors: Schilling Dagmar , Thomas Karen , Nixdorff Kathryn , Vogel Stefanie N. , Fenton Matthew J.
Published: April 2014

Previous studies have shown that engagement of Toll-like receptors (TLR) 2 and 4 can induce macrophages to express a variety of proinflammatory cytokines. We have recently demonstrated that TLR2 agonists poorly induce a subset of TLR4-inducible proinflammatory genes (e.g., inducible protein (IP)-10, inducible NO synthase (iNOS), monocyte chemoattractant protein-5, IL-12p40), due in part to differential activation of IFN-β production and phosphorylation of the transcription factor STAT1. TLR4, but not TLR2, agonists can induce IFN-β expression via a mechanism that requires the adapter protein Toll-IL-1R domain-containing adapter protein (TIRAP)/myeloid differentiation protein 88 (MyD88) adapter-like (Mal), but not the adapter protein MyD88. Thus, the failure of TLR2 agonists to induce STAT1-dependent genes results, in part, from their failure to induce the expression of IFN-β. In this study, we show that IL-6 expression is also preferentially induced by activation of TLR4. TLR4-dependent induction of IL-6 expression did require Toll-IL-1R domain-containing adapter protein (TIRAP)/MyD88 adapter-like (Mal), but unlike iNOS and IP-10, it did not require the expression of IFN-β. Although exogenous IFN-β and IFN-γ could synergize with TLR2 agonists to restore high levels of iNOS expression and NO production, these IFNs could not synergize with TLR2 agonists to induce high levels of IL-6. Similarly, neutralizing anti-IFN Abs could block iNOS gene expression in LPS-stimulated murine macrophages, whereas these Abs had little effect on IL-6 gene expression in these cells. Together, these studies demonstrate that IL-6, like iNOS and IP-10, is differentially expressed in macrophages stimulated via TLR2 vs TLR4, although these differences appear to arise from distinct signaling mechanisms.
Adhesion of B Cell Lines to Endothelial Cells from Human Lymphoid Tissue Modulates Tyrosine Phosphorylation and Endothelial Cell Activation
Authors: Reyes Lilian I. , Escobar Paula , Bono María R. , Rosemblatt Mario
Published: April 2014

Through the production of cytokines and growth factors the endothelium of secondary lymphoid organs plays a crucial role in controlling lymphocyte migration to the lymphoid microenvironment, an essential step in the initiation of the immune response. Here we demonstrate that direct contact of B cell lines with tonsil-derived human endothelial cells resulted in changes in the phosphorylation state of endothelial cells, causing their functional activation. We found a rapid (<15-s) and transient dephosphorylation, followed by a rapid rephosphorylation of tyrosine residues of the focal adhesion kinase, paxillin, and ERK2. Maximal rephosphorylation occurred after 15–30 min of B cell contact. Preincubation of lymphoid B cells with an adhesion-blocking Ab directed against α4β1 integrin abrogated adhesion-mediated changes of endothelial cell tyrosine phosphorylation, suggesting that cell contact was essential. Similar patterns of tyrosine phosphorylation, but with slightly different kinetics were induced after cross-linking of β1 integrin or CD40 on endothelial cells. Functional activation of endothelial cells by B cell adhesion was confirmed by the production of IL-6, IL-8, monocyte chemoattractant protein-1, M-CSF, and macrophage inflammatory protein-1β mRNA. However, direct cross-linking of β1 integrin and CD40 failed to accomplish the same functional activation. These data indicate that direct contact of lymphoid B cells with the endothelium from lymphoid tissue induce endothelial cell signaling, resulting in chemokine and cytokine production. This phenomenon may provide a mechanism for the remodeling of the endothelium from lymphoid tissues, thus contributing to the free migration of lymphocytes and other cells into the lymphoid organs.
Selective Eosinophil Adhesion to Fibroblast Via IFN-γ-Induced Galectin-9
Authors: Asakura Hirofumi , Kashio Yumiko , Nakamura Kazuhiro , Seki Masako , Dai Shuyan , Shirato Yukako , Abedin Mohammad J. , Yoshida Naoko , Nishi Nozomu , Imaizumi Tadaatsu , Saita Naoki , Toyama Yoshihiro , Takashima Hitoshi , Nakamura Takanori , Ohkawa Motoomi , Hirashima Mitsuomi
Published: April 2014

Among galectin family members, galectin-9 was first described as a potent eosinophil chemoattractant derived from Ag-stimulated T cells. In the present study a role of galectin-9 in the interaction between eosinophils and fibroblasts was investigated using a human lung fibroblast cell line, HFL-1. RT-PCR, real-time PCR, and Western blot analyses revealed that both galectin-9 mRNA and protein in HFL-1 cells were up-regulated by IFN-γ stimulation. On the one hand, IL-4, known as a Th2 cytokine, did not affect the galectin-9 expression in HFL-1 cells. We further confirmed that IFN-γ up-regulated the expression of galectin-9 in primary human dermal fibroblasts. Flow cytometric analysis revealed that IFN-γ up-regulated surface galectin-9 expression on HFL-1 cells. Stimulation of HFL-1 cells with IFN-γ up-regulated adhesion of eosinophils, but not neutrophils, to HFL-1 cells. This adherence of eosinophils to HFL-1 cells was inhibited by both lactose and anti-galectin-9 Ab. These findings demonstrate that IFN-γ-induced galectin-9 expression in fibroblasts mediates eosinophil adhesion to the cells, suggesting a crucial role of galectin-9 in IFN-γ-stimulated fibroblasts as a physiological modulator at the inflammatory sites.
Bax is Crucial for IFN-γ-Induced Resolution of Allergen- Induced Mucus Cell Metaplasia
Authors: Tesfaigzi Yohannes , Fischer Mark J. , Daheshia Massoud , Green Francis H. Y. , De Sanctis George T. , Wilder Julie A.
Published: April 2014

Allergic airway responses cause proliferation of epithelial cells and mucus cell metaplasia (MCM), and the resolution of MCM involves reduction of cell numbers. The role of inflammation and apoptosis on this process was investigated in P-selectin +/+ and −/− mice sensitized and challenged with OVA by analyzing the expression and the role of regulators of apoptosis in metaplastic mucus cells. No differences were observed in MCM at 5 days of allergen exposure between +/+ and −/− mice, despite reduced IL-13 levels in −/− mice. Although IL-4 levels were similar in both −/− and +/+ mice, IL-13 and IL-5 levels had decreased and IFN-γ levels were increased earlier in −/− compared with +/+ mice. MCM levels were decreased 4-fold at 7 days of allergen exposure in −/− mice and at 15 days in +/+ mice. The percentage of Bax-expressing mucus cells increased significantly at 7 days in −/− mice and at 10 days in +/+ mice. The Bax-positive mucus cells exhibited caspase-specific cleavage of cytokeratin 18. IFN-γ caused Bax expression in IL-13-induced MCM in microdissected airway cultures. MCM remained significantly elevated in Bax −/− mice following 15 days of allergen exposure compared with +/+ mice, while the number of eosinophils was reduced in both Bax +/+ and −/− mice at 15 days. Together, these data demonstrate that reduced IL-13 levels were sufficient to elicit maximum MCM, that IFN-γ induces Bax in metaplastic mucus cells, and that Bax plays a critical role in the resolution of MCM, but not in the resolution of eosinophils.
Anticancer Activity of Sodium Stibogluconate in Synergy with IFNs
Authors: Yi Taolin , Pathak Manas K. , Lindner Daniel J. , Ketterer Michael E. , Farver Carol , Borden Ernest C.
Published: April 2014

Cancer cell resistance limits the efficacy of IFNs. In this study, we show that sodium stibogluconate (SSG) and IFN-α synergized to overcome IFN-α resistance in various human cancer cell lines in culture and eradicated IFN-α-refractory WM9 human melanoma tumors in nude mice with no obvious toxicity. SSG enhanced IFN-α-induced Stat1 tyrosine phosphorylation, inactivated intracellular SHP-1 and SHP-2 that negatively regulate IFN signaling, and induced cellular protein tyrosine phosphorylation in cancer cell lines. These effects are consistent with inactivation of phosphatases as the basis of SSG anticancer activity. Characterization of SSG by chromatography revealed that only selective compounds in SSG were effective protein tyrosine phosphatase inhibitors. These observations suggest the potential of SSG as a clinically usable protein tyrosine phosphatase inhibitor in cancer treatment and provide insights for developing phosphatase-targeted therapeutics.
Differential Expression of TRAIL and TRAIL Receptors in Allergic Asthmatics Following Segmental Antigen Challenge: Evidence for a Role of TRAIL in Eosinophil Survival
Authors: Robertson Noreen M. , Zangrilli James G. , Steplewski Andrzej , Hastie Annette , Lindemeyer Rochelle G. , Planeta Maria A. , Smith Mary K. , Innocent Nathalie , Musani Ali , Pascual Rodolfo , Peters Stephen , Litwack Gerald
Published: April 2014

Asthma is a chronic lung disease exhibiting airway obstruction, hyperresponsiveness, and inflammation, characterized by the infiltration of eosinophils into the airways and the underlying tissue. Prolonged eosinophilic inflammation depends on the balance between the cell’s inherent tendency to undergo apoptosis and the local eosinophil-viability enhancing activity. TRAIL, a member of the TNF family, induces apoptosis in most transformed cells; however, its role in health and disease remains unknown. To test the hypothesis that Ag-induced inflammation is associated with TRAIL/TRAIL-R interactions, we used a segmental Ag challenge (SAC) model in ragweed-allergic asthmatics and nonasthmatic patients and analyzed bronchoalveolar lavage (BAL) material for 2 wk. In asthmatic patients, the level of TRAIL in BAL fluid dramatically increased 24 h after SAC, which significantly correlated with BAL eosinophil counts. Immunohistochemical analysis of bronchial biopsies from asthmatic patients demonstrated that TRAIL staining was increased in epithelial, airway smooth muscle, and vascular smooth muscle cells and throughout the interstitial tissue after SAC. This was confirmed by quantitative immunocytochemical image analysis of BAL eosinophils and alveolar macrophages, which demonstrated that expression levels of TRAIL and DcR2 increased, whereas expression levels of the TRAIL-Rs DR4 and DR5 decreased in asthmatic subjects after SAC. We also determined that TRAIL prolongs eosinophil survival ex vivo. These data provide the first in vivo evidence that TRAIL expression is increased in asthmatics following Ag provocation and suggest that modulation of TRAIL and TRAIL-R interactions may play a crucial role in promoting eosinophil survival in asthma.
A Key Role for Prostaglandin I2 in Limiting Lung Mucosal Th2, But Not Th1, Responses to Inhaled Allergen
Authors: Jaffar Zeina , Wan Kong-Sang , Roberts Kevan
Published: April 2014

The cellular events that serve to regulate lung mucosal Th2 responses and limit allergic inflammatory reactions are unclear. Using the DO11.10 TCR transgenic mouse, we developed a model of T cell-mediated pulmonary inflammation and demonstrated that high levels of PGI2 are produced in the airways following OVA inhalation. Selective inhibition of cyclooxygenase-2 in vivo specifically reduced PGI2 synthesis and resulted in a marked increase in Th2-mediated, but not Th1-mediated, lung inflammation. The elevated Th2-mediated inflammatory response elicited by the cyclooxygenase-2 inhibitor was associated with enhanced airway hyperreactivity and was coincident with a marked increase in the levels of IL-4, IL-5, and IL-13 in the airways, but a reduction in IL-10 production. In keeping with these observations, we found that the mRNA for the PGI2 receptor was expressed by Th2, but not Th1, cells, and transcripts for the PGI2 receptor were induced by IL-4 and OVA peptide stimulation. Interestingly, treatment with PGI2 or its stable analog, carbaprostacyclin, augmented IL-10 production by Th2 cells. Collectively, our findings reveal a key role for PGI2 in differentially limiting Th2 responses, possibly by promoting production of the immunosuppressive cytokine IL-10 at the site of allergic lung inflammation. These results indicate an important role for prostanoids generated during inflammation in regulating mucosal T cell responses and highlight a potential risk in the use of cyclooxygenase-2-specific inhibitors by allergic asthmatics.
The Apoptotic Ligands TRAIL, TWEAK, and Fas Ligand Mediate Monocyte Death Induced by Autologous Lupus T Cells
Authors: Kaplan Mariana J. , Lewis Emily E. , Shelden Eric A. , Somers Emily , Pavlic Robert , McCune William J. , Richardson Bruce C.
Published: April 2014

Individuals with systemic lupus erythematosus show evidence of a significant increase in monocyte apoptosis. This process is mediated, at least in part, by an autoreactive T cell subset that kills autologous monocytes in the absence of nominal Ag. We have investigated the apoptotic pathways involved in this T cell-mediated process. Expression of the apoptotic ligands TRAIL, TNF-like weak inducer of apoptosis (TWEAK), and Fas ligand on lupus T cells was determined, and the role of these molecules in the monocyte apoptotic response was examined. We report that these apoptotic ligands mediate the autologous monocyte death induced by lupus T cells and that this cytotoxicity is associated with increased expression of these molecules on activated T cells, rather than with an increased susceptibility of lupus monocytes to apoptosis induced by these ligands. These results define novel mechanisms that contribute to increased monocyte apoptosis characterizing patients with lupus. We propose that this mechanism could provide a source of potentially antigenic material for the autoimmune response and interfere with normal clearing mechanisms.
DNA Vaccination with Heat Shock Protein 60 Inhibits Cyclophosphamide-Accelerated Diabetes
Authors: Quintana Francisco J. , Carmi Pnina , Cohen Irun R.
Published: April 2014

Nonobese diabetic (NOD) mice spontaneously develop diabetes as a consequence of an autoimmune process that can be inhibited by immunotherapy with the 60-kDa heat shock protein (hsp60), with its mycobacterial counterpart 65-kDa (hsp65), or with other Ags such as insulin and glutamic acid decarboxylase (GAD). Microbial infection and innate signaling via LPS or CpG motifs can also inhibit the spontaneous diabetogenic process. In addition to the spontaneous disease, however, NOD mice can develop a more robust cyclophosphamide-accelerated diabetes (CAD). In this work, we studied the effect on CAD of DNA vaccination with constructs encoding the Ags human hsp60 (phsp60) or mycobacterial hsp65 (phsp65). Vaccination with phsp60 protected NOD mice from CAD. In contrast, vaccination with phsp65, with an empty vector, or with a CpG-positive oligonucleotide was not effective, suggesting that the efficacy of the phsp60 construct might be based on regulatory hsp60 epitopes not shared with its mycobacterial counterpart, hsp65. Vaccination with phsp60 modulated the T cell responses to hsp60 and also to the GAD and insulin autoantigens; T cell proliferative responses were significantly reduced, and the pattern of cytokine secretion to hsp60, GAD, and insulin showed an increase in IL-10 and IL-5 secretion and a decrease in IFN-γ secretion, compatible with a shift from a Th1-like toward a Th2-like autoimmune response. Our results extend the role of specific hsp60 immunomodulation in the control of β cell autoimmunity and demonstrate that immunoregulatory networks activated by specific phsp60 vaccination can spread to other Ags targeted during the progression of diabetes, like insulin and GAD.