bluuebunny/crossref_metadata_2025 huggingface dataset:
Positive Regulation of c-Jun N-Terminal Kinase and TNF-α Production But Not Histamine Release by SHP-1 in RBL-2H3 Mast Cells
Authors: Xie Zhi-Hui , Zhang Juan , Siraganian Reuben P.
Published: April 2014

The SH2-containing protein tyrosine phosphatase1 (SHP-1) is important for signaling from immune receptors. To investigate the role of SHP-1 in mast cells we overexpressed the wild-type and the phosphatase-inactive forms of SHP-1 in rat basophilic leukemia 2H3 (RBL-2H3) mast cell line. The phosphatase-inactive SHP-1 (C453S or D419A) retains its ability to bind tyrosine phosphorylated substrates and thereby competes with the endogenous wild-type enzyme. Overexpression of wild-type SHP-1 decreased the FcεRI aggregation-induced tyrosine phosphorylation of the β and γ subunits of the receptor whereas the dominant negative SHP-1 enhanced phosphorylation. There were also similar changes in the tyrosine phosphorylation of Syk. However, receptor-induced histamine release in the cells expressing either wild-type or dominant negative SHP-1 was similar to that in the parental control cells. In contrast, compared with the parental RBL-2H3 cells, FcεRI-induced c-Jun N-terminal kinase phosphorylation and the level of TNF-α mRNA was increased in the cells overexpressing wild-type SHP-1 whereas the dominant negative SHP-1 had the opposite effect. The substrate-trapping mutant SHP1/D419A identified pp25 and pp30 as two major potential substrates of SHP-1 in RBL-2H3 cells. Therefore, SHP-1 may play a role in allergy and inflammation by regulating mast cell cytokine production.
NF-κB Modulates TNF-α Production by Alveolar Macrophages in Asymptomatic HIV-Seropositive Individuals
Authors: Mathys Jean-Marie , Melanson Suzanne M. , Schiffer-Alberts Deborah J. , Ioannidis John P. A. , Koziel Henry , Skolnik Paul R.
Published: April 2014

Local TNF-α production in different organs may affect HIV replication and pathogenesis. Alveolar macrophages (AMs) obtained by bronchoalveolar lavage from asymptomatic HIV-seropositive and HIV-seronegative individuals did not spontaneously release TNF-α, but LPS stimulation of these cells significantly increased TNF-α production. We tested whether NF-κB affects TNF-α production by AMs using N-tosyl-l-phenylalanine chloromethylketone (TPCK) or N-benzoyl-l-tyrosine ethyl ester (BTEE), which inhibit the degradation of IκB, or tricyclodecan-9-yl-xanthogenate-potassium (D609), which inhibits phospholipase C. Alveolar macrophages were exposed to LPS alone and with the chemical protease inhibitors TPCK, BTEE, and D609. NF-κB DNA binding induced by LPS treatment of AMs was inhibited by TPCK, BTEE, and D609. These agents also inhibited TNF-α mRNA and TNF-α protein production. After 24 h, the levels of TNF-α mRNA reached equilibrium, as assessed by RT-PCR. The levels of NF-κB mRNA remained constant under all conditions. The levels of IκB-α mRNA were similar after 30, 60, and 180 min, but the IκB-β mRNA concentration was initially low and increased over time under all conditions. IκB-α and IκB-β protein production was not affected by the chemical protease inhibitors. Our data show that TNF-α production by LPS-stimulated AMs from asymptomatic HIV-seropositive and -seronegative individuals is regulated via the phospholipase C pathway and by NF-κB DNA binding activity without obvious changes in IκB-α or IκB-β protein concentrations.
Delineation of a CpG Phosphorothioate Oligodeoxynucleotide for Activating Primate Immune Responses In Vitro and In Vivo
Authors: Hartmann Gunther , Weeratna Risini D. , Ballas Zuhair K. , Payette Paul , Blackwell Sue , Suparto Irma , Rasmussen Wendy L. , Waldschmidt Marianella , Sajuthi Dondin , Purcell Robert H. , Davis Heather L. , Krieg Arthur M.
Published: April 2014

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5′ end followed by three 6 mer CpG motifs (5′-GTCGTT-3′) separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.
MEK Activity Regulates Negative Selection of Immature CD4+CD8+ Thymocytes
Authors: Bommhardt Ursula , Scheuring Yvonne , Bickel Chrisitan , Zamoyska Rose , Hünig Thomas
Published: April 2014

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3ε-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.
The Density of Peptides Displayed by Dendritic Cells Affects Immune Responses to Human Tyrosinase and gp100 in HLA-A2 Transgenic Mice
Authors: Bullock Timothy N. J. , Colella Teresa A. , Engelhard Victor H.
Published: April 2014

Several HLA-A*0201-restricted peptide epitopes that can be used as targets for active immunotherapy have been identified within melanocyte differentiation proteins. However, uncertainty exists as to the most effective way to elicit CD8+ T cells with these epitopes in vivo. We report the use of transgenic mice expressing a derivative of HLA-A*0201, and dendritic cells, to enhance the activation of CD8+ T cells that recognize peptide epitopes derived from human tyrosinase and glycoprotein 100. We find that by altering the cell surface density of the immunizing peptide on the dendritic cells, either by pulsing with higher concentrations of peptide, or by changing the MHC-peptide-binding affinity by generating variants of the parent peptides, the size of the activated CD8+ T cell populations can be modulated in vivo. Significantly, the density of peptide that produced the largest response was less than the maximum density achievable through short-term peptide pulsing. We have also found, however, that while some variant peptides are effective at eliciting both primary and recall CD8+ T cell responses that can recognize the parental epitope, other variant epitopes lead to the outgrowth of CD8+ T cells that only recognize the variant. HLA-A*0201 transgenic mice provide an important model to define which peptide variants are most likely to stimulate CD8+ T cell populations that recognize the parental, melanoma-specific peptide.
Sensitivity Difference to the Suppressive Effect of Prostaglandin E2 Among Mouse Strains: A Possible Mechanism to Polarize Th2 Type Response in BALB/c Mice
Authors: Kuroda Etsushi , Sugiura Tsutomu , Zeki Kazuya , Yoshida Yasuhiro , Yamashita Uki
Published: April 2014

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-γ production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-γ production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-γ production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-γ production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-γ and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.
Role of IL-6 in Directing the Initial Immune Response to Schistosome Eggs
Authors: La Flamme Anne Camille , MacDonald Andrew S. , Pearce Edward J.
Published: April 2014

The eggs of Schistosoma mansoni are strong inducers of a Th2 response, and previous work has shown that Ag-specific IL-6 is produced within 24 h after the injection of eggs into mice. Investigations to determine the role of IL-6 in orchestrating the early response to schistosome eggs have revealed that IL-12 is rapidly produced in lymph node cell cultures from egg-injected mice. This “early” IL-12 primes for the production of IL-6 and IFN-γ, for in IL-12−/− mice egg injection fails to stimulate increased production of either of these cytokines. Furthermore, IL-6 also up-regulates IL-10 production which, together with IL-6, negatively regulates IL-12 and IFN-γ production. Finally, IL-10 down-regulates the production of its inducer, IL-6. These data indicate that the anti-inflammatory role of IL-6 may be effected through negative regulation of type 1 (IFN-γ) and type 1-associated (IL-12) cytokines either directly (by IL-6) or indirectly (through the induction of IL-10) and suggest that one mechanism by which eggs may support the development of Th2 responses is through the negative regulation of the type 1 response.
Low CD86 Expression in the Nonobese Diabetic Mouse Results in the Impairment of Both T Cell Activation and CTLA-4 Up-Regulation
Authors: Dahlén Eva , Hedlund Gunnar , Dawe Kim
Published: April 2014

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune insulin-dependent diabetes mellitus and serves as a model for human type I diabetes. NOD spleen cells proliferate to a lesser extent than those from C57BL/6 and BALB/c mice in response to anti-CD3. To investigate the cause of this reduced T cell proliferation, costimulatory molecule expression was investigated. It was found that NOD macrophages, dendritic cells, and T cells, but not B cells, expressed lower basal levels of CD86, but not CD80, CD28, or CD40, compared with C57BL/6 and BALB/c. This low CD86 expression was not dependent on the MHC haplotype or on diabetes development since the NOD-related, diabetes-free mouse strains NON (H-2nb1) and NOR (H-2g7) exhibited similar low levels of CD86 expression and proliferation. Furthermore, following activation, the relative up-regulation of CTLA-4, as compared with CD28, was more pronounced on C57BL/6 and BALB/c T cells as shown by an increased CTLA-4/CD28 ratio. This activation-induced increase in the CTLA-4/CD28 ratio was markedly reduced on NOD T cells compared with the other two strains. The low CD86 expression in NOD mice may account for the reduced increase in both proliferation and the CTLA-4/CD28 ratio, since reducing CD86 expression in C57BL/6 and BALB/c cultures to NOD levels significantly reduces the proliferation and the CTLA-4/CD28 ratio. Therefore, we propose that a low level of CD86 expression in the NOD mouse contributes to a defective regulation of autoreactive T cells by preventing the full activation of T cells and therefore the up-regulation of CTLA-4.
Maturation and Trafficking of Monocyte-Derived Dendritic Cells in Monkeys: Implications for Dendritic Cell-Based Vaccines
Authors: Barratt-Boyes Simon M. , Zimmer Michael I. , Harshyne Larry A. , Meyer E. Michael , Watkins Simon C. , Capuano Saverio , Murphey-Corb Michael , Falo Louis D. , Donnenberg Albert D.
Published: April 2014

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1α (MIP-1α), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3β and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.
The IL-2 Receptor Promotes Lymphocyte Proliferation and Induction of the c-myc, bcl-2, and bcl-x Genes Through the trans-Activation Domain of Stat5
Authors: Lord James D. , McIntosh Bryan C. , Greenberg Philip D. , Nelson Brad H.
Published: April 2014

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R β-chain (IL-2Rβ). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rβ except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.
Molecular Analysis of the Autoantibody Response in Peptide-Induced Autoimmunity
Authors: Putterman Chaim , Deocharan Bisram , Diamond Betty
Published: April 2014

Immunization of nonautoimmune BALB/c mice with multimeric DWEYSVWLSN, a peptide mimotope of DNA, induces anti-DNA and other lupus-associated Abs. To further investigate the pathogenesis of the autoantibody response induced by peptide immunization, we generated hybridomas from peptide-immunized mice that bound peptide, dsDNA, cardiolipin, Sm/ribonucleoprotein (RNP), or some combination of these Ags. Analysis of 24 IgM Abs led to the identification of three groups of Abs: 1) Abs reactive with peptide alone, 2) anti-peptide Abs cross-reactive with one or more autoantigens, and 3) autoantibodies that do not bind to peptide. The gene families and particular VH-VL combinations used in those hybridomas binding DNA were similar to those used in the anti-DNA response in spontaneous murine lupus. Another similarity to the spontaneous anti-DNA response was the generation of arginines in the complementarity-determining region-3 of DNA-binding hybridomas. Interestingly, one Ab had the VH-VL combination present in the original R4A anti-DNA Ab used to select the DWEYSVWLSN peptide from a phage display library. Many of the heavy and light chains displayed evidence of somatic mutation, suggesting that they were made by Ag-activated B cells. Analysis of the Ab repertoire in peptide-induced autoimmunity may provide insights into the generation of anti-DNA Abs following exposure to foreign Ag. Furthermore, the recovery of an Ab with the heavy and light chain combination of the Ab originally used to isolate the immunizing peptide confirms the utility of phage display peptide libraries in generating true molecular mimics.
Regulation of the Calcium/NF-AT T Cell Activation Pathway by the D2 Domain of CD45
Authors: Wang Ying , Liang Lianzhu , Esselman Walter J.
Published: April 2014

CD45 contains tandem repeated protein tyrosine phosphatase (PTP) domains and is essential for the initiation of the earliest activation events resulting from Ag ligation of the TCR. The second PTP domain (D2) contains four CK2 phosphorylation sites in a unique 19-aa insert, which are targets of CK2 phosphorylation. This study was designed to evaluate the roles of these Ser residues in T cell activation. Transient transfection of the CD45− T cell line, J45.01, with CD45 cDNA incorporating four Ser to Ala (S/A) mutations in the 19-aa insert did not affect the magnitude of NF-AT activation resulting from TCR ligation. However, the basal level of NF-AT activity in unstimulated cells expressing the CD45 S/A mutation was elevated 9- to 10-fold. Increased basal NF-AT was dependent on extracellular Ca2+ stores as judged by EGTA treatment. In additional experiments, isolation of stable clones derived from transfection of the CD45 S/A mutant into CD45− H45.01 cells showed sustained calcium flux after TCR engagement. The sustained calcium flux returned to baseline levels after addition of EGTA, suggesting that the expression of the CD45 S/A mutant may have prevented deactivation of plasma membrane calcium channels. Consideration of both transient and stable transfection systems suggests that in addition to being essential for initial events in T cell triggering, the intact CD45 D2, 19-aa insert is necessary for regulation of TCR-mediated calcium signaling pathways.
Lipopolysaccharide Induces Scavenger Receptor A Expression in Mouse Macrophages: A Divergent Response Relative to Human THP-1 Monocyte/Macrophages
Authors: Fitzgerald Michael L. , Moore Kathryn J. , Freeman Mason W. , Reed Guy L.
Published: April 2014

Gene deletion studies indicate that the macrophage scavenger receptor A (SR-A) protects mice from LPS-induced endotoxemia. Paradoxically, cultured human monocyte-derived macrophages down-regulate SR-A expression when exposed to LPS. We found that human THP-1 monocyte/macrophages decrease SR-A expression in response to LPS independent of their differentiation status. In contrast, primary and elicited mouse peritoneal macrophages as well as the J774A.1 and RAW264.7 mouse macrophage lines increase SR-A expression in response to LPS. Exposure to LPS caused J774A.1 and RAW264.7 cells to increase SR-A transcripts by 3- and 5-fold, respectively. LPS caused a concomitant 3-fold increase in SR-A protein levels and increased cell membrane expression of the receptor. RAW264.7 cells increased SR-A transcript levels in response to LPS at concentrations as low as 1 ng/ml, and the response was saturated at 10 ng/ml. The LPS induction of SR-A transcripts required continual protein synthesis and began at 8 h, peaked by 16 h, and persisted for at least 48 h. LPS induction did not increase SR-A gene transcription or affect alternative transcript splicing, but mildly increased mature transcript stability and proceeded in the presence of actinomycin D. Finally, treatment of RAW264.7 cells with TNF-α did not induce SR-A transcript levels, indicating that a TNF-α autocrine/paracrine signaling mechanism alone is not sufficient to recapitulate the LPS induction of SR-A transcripts. The induction of SR-A expression by LPS-stimulated mouse macrophages is the opposite of the down-regulation of SR-A reported in human monocyte-derived macrophages and may have implications for the observed resistance mice show toward endotoxemia.
IFN-γ-Inducing Factor (IL-18) Increases Allergic Sensitization, Serum IgE, Th2 Cytokines, and Airway Eosinophilia in a Mouse Model of Allergic Asthma
Authors: Wild James S. , Sigounas Anastasia , Sur Nilanjana , Siddiqui Mohammed S. , Alam Rafeul , Kurimoto Masashi , Sur Sanjiv
Published: April 2014

We investigated the effects of IFN-γ-inducing factor (IL-18) in a ragweed (RW) mouse model of allergic asthma. Administration of IL-18 in conjunction with allergic sensitization and challenge in wild-type, but not IFN-γ −/− mice, inhibited the bronchoalveolar lavage (BAL) eosinophilia induced by RW challenge, and increased serum levels of RW-specific IgG2a and production of IFN-γ from splenocytes cultured with RW, indicating a critical role for IFN-γ in mediating these effects. Paradoxically, the same treatment schedule in WT mice increased serum levels of RW-specific IgE and IgG1, and production of IL-4 and IL-5 from splenocytes cultured with RW. When the effects of the same IL-18 treatment schedule were allowed to mature for 3 wk, the inhibition of lung eosinophil recruitment was replaced by augmentation of lung eosinophil recruitment. In another experiment, IL-18 administered only with allergic sensitization increased BAL eosinophilia and lung expression of IL-5 and IFN-γ, while IL-18 administered only with RW challenge decreased BAL eosinophilia and increased lung IFN-γ expression, while lung expression of IL-5 remained unchanged. IL-18 administered without RW or adjuvant to naive mice increased total serum IgE levels. Finally, intrapulmonary administrations of IL-18 plus RW in naive mice dramatically increased Th2 cytokine production, IgE levels, eosinophil recruitment, and airway mucus, demonstrating induction of allergic sensitization. This is the first report demonstrating that IL-18 promotes a Th2 phenotype in vivo, and potently induces allergic sensitization. These results suggest that IL-18 may contribute to the pathogenesis of allergic asthma.
A Conserved Mycobacterial Heat Shock Protein (hsp) 70 Sequence Prevents Adjuvant Arthritis upon Nasal Administration and Induces IL-10-Producing T Cells That Cross-React with the Mammalian Self-hsp70 Homologue
Authors: Wendling Uwe , Paul Liesbeth , van der Zee Ruurd , Prakken Berent , Singh Mahavir , van Eden Willem
Published: April 2014

Immunization with Mycobacterium tuberculosis heat shock protein (hsp) 60 has been shown to protect rats from experimental arthritis. Previously, the protection-inducing capacity was shown to reside in the evolutionary conserved parts of the molecule. Now we have studied the nature of the arthritis suppressive capacity of a distinct, antigenically unrelated protein, M. tuberculosis hsp70. Again, a conserved mycobacterial hsp70 sequence was found to be immunogenic and to induce T cells that cross-reacted with the rat homologue sequence. However, in this case parenteral immunization with the peptide containing the critical cross-reactive T cell epitope did not suppress disease. Upon analysis of cytokines produced by these peptide-specific T cells, high IL-10 production was found, as was the case with T cells responsive to whole hsp70 protein. Nasal administration of this peptide was found to lead to inhibition of subsequent adjuvant arthritis induction. The data presented here shows the intrinsic capacity of conserved bacterial hsp to trigger self-hsp cross-reactive T cells with the potential to down-regulate arthritis via IL-10.
Systemic Activation and Antigen-Driven Oligoclonal Expansion of T Cells in a Mouse Model of Colitis
Authors: Matsuda Jennifer L. , Gapin Laurent , Sydora Beate C. , Byrne Fergus , Binder Scott , Kronenberg Mitchell , Aranda Richard
Published: April 2014

Transfer of CD4+CD45RBhigh T cells into immunodeficient mice results in both the expansion of the transferred T cells and colitis. Here we show that colitis pathogenesis requires expression of MHC class II molecules by the immune-deficient host. Analysis of the TCRβ repertoire of the cells found in the large intestine of diseased mice revealed a population with restricted TCR diversity. Furthermore, nucleotide sequence analysis demonstrated the selection for particular CDR3β amino acid sequence motifs. Collectively, these data indicate that the expansion of T cells in the intestine and colitis pathogenesis are likely to require the activation of Ag-specific T cells, as opposed to nonspecific or superantigen-mediated events. There is relatively little overlap, however, when the TCR repertoires of different individuals are compared, suggesting that a number of Ags can contribute to T cell expansion and the generation of a T cell population in the intestine. Surprisingly, many of the expanded clones found in the large intestine also were found in the spleen and elsewhere, although inflammation is localized to the colon. Additionally, donor-derived T cells appear to be activated in both the intestine and the spleen at early time points after cell transfer. Together, these results strongly suggest that disease induction in this model involves either the early and systemic activation of antigen-specific T cells or the rapid dispersal of T cells activated at a particular site.
Dendritic Cells in the Induction of Protective and Nonprotective Anticryptococcal Cell-Mediated Immune Responses
Authors: Bauman Sean K. , Nichols Kasie L. , Murphy Juneann W.
Published: April 2014

Dendritic cells (DC) can be divided into three subsets, Langerhans cells, myeloid DC (MDC), and lymphoid DC (LDC), based upon phenotypic and functional differences. We hypothesized that different DC subsets are associated with the development of protective vs nonprotective cell-mediated immune (CMI) responses against the fungal pathogen, Cryptococcus neoformans. To test this, mice were immunized with protective and/or nonprotective immunogens, and DC subsets in draining lymph nodes were assessed by flow cytometry. The protective immunogen (cryptococcal culture filtrate Ag-CFA), in contrast to the nonprotective immunogen (heat-killed cryptococci-CFA), the nonprotective immunogen mixed with the protective immunogen (cryptococcal culture filtrate Ag + heat-killed cryptococci-CFA), or controls, stimulated significant increases in total leukocytes, Langerhans cells, MDC, LDC, and activated CD4+ T cells in draining lymph nodes. The protective immune response resulted in significantly increased levels of anticryptococcal delayed-type hypersensitivity reactivity and activated CD4+ T cells at the delayed-type hypersensitivity reaction site. Draining lymph node LDC:MDC ratios induced by the protective immunogen were significantly lower than the ratios induced by either immunization in which the nonprotective immunogen was present. In contrast, mice given the nonprotective immunogen had LDC:MDC ratios similar to those of naive mice. Our data indicate that lymph node Langerhans cells and MDC are APC needed for induction of the protective response. The predominance of LDC in mice undergoing nonprotective responses suggests that lymph node LDC, like splenic LDC, are negative regulators of CMI responses. In addition to showing DC subsets associated with functional differences, our data suggest that the LDC:MDC balance, which can be modulated by the Ag, determines whether protective or nonprotective anticryptococcal CMI responses develop.
TCR/CD3-Induced Activation and Binding of Emt/Itk to Linker of Activated T Cell Complexes: Requirement for the Src Homology 2 Domain
Authors: Ching Keith A. , Grasis Juris A. , Tailor Pankaj , Kawakami Yuko , Kawakami Toshiaki , Tsoukas Constantine D.
Published: April 2014

Expressed in mast and T cells/inducible T cell tyrosine kinase (Emt/Itk), a Tec family protein tyrosine kinase, is critical for the development and activation of T lymphocytes. The mechanism through which Emt/Itk mediates its effector functions is poorly understood. In this study, we show that the Emt/Itk Src homology 2 (SH2) domain is critical for the transphosphorylation and activation of Emt/Itk catalytic activity that is mediated by TCR/CD3 engagement. Furthermore, we find that the Emt/Itk SH2 domain is essential for the formation of TCR/CD3-inducible Emt/Itk-LAT complexes, whereas the SH3 domain and catalytic activity are not required. The Emt/Itk-linker of activated T cells (LAT) complexes are biologically important because Jurkat T cells with deficient LAT expression (JCaM2) fail to increase Emt/Itk tyrosine phosphorylation upon TCR/CD3 stimulation. Confocal microscopy reveals that in activated cells, LAT complexes colocalize with TCR/CD3. The present data suggest that upon TCR/CD3 engagement, the Emt/Itk SH2 domain mediates the formation of a molecular complex containing Emt/Itk, LAT, and TCR/CD3; this complex is essential for Emt/Itk activation and function.
Members of the Ikaros Gene Family Are Present in Early Representative Vertebrates
Authors: Haire Robert N. , Miracle Ann L. , Rast Jonathan P. , Litman Gary W.
Published: April 2014

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologues of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates.
Induction of Permanent Mixed Chimerism and Skin Allograft Tolerance Across Fully MHC-Mismatched Barriers by the Additional Myelosuppressive Treatments in Mice Primed with Allogeneic Spleen Cells Followed by Cyclophosphamide
Authors: Tomita Yukihiro , Yoshikawa Masahiro , Zhang Qi-Wei , Shimizu Ichiro , Okano Shinji , Iwai Toshiro , Yasui Hisataka , Nomoto Kikuo
Published: April 2014

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 × 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 × 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE−) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vβ11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 × 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).
Δ-9-Tetrahydrocannabinol Inhibits Antitumor Immunity by a CB2 Receptor-Mediated, Cytokine-Dependent Pathway
Authors: Zhu Li X. , Sharma Sherven , Stolina Marina , Gardner Brian , Roth Michael D. , Tashkin Donald P. , Dubinett Steven M.
Published: April 2014

In this study, we show that Δ-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, suppresses host immune reactivity against lung cancer. In two different weakly immunogenic murine lung cancer models, intermittent administration of THC (5 mg/kg, four times/wk i.p. for 4 wk) led to accelerated growth of tumor implants compared with treatment with diluent alone. In contrast to our findings in immunocompetent mice, THC did not affect tumor growth in tumor-bearing SCID mice. The immune inhibitory cytokines, IL-10 and TGF-β, were augmented, while IFN-γ was down-regulated at both the tumor site and in the spleens of THC-treated mice. Administration of either anti-IL-10- or anti-TGF-β-neutralizing Abs prevented the THC-induced enhancement in tumor growth. Both APC and T cells from THC-treated mice showed limited capacities to generate alloreactivity. Furthermore, lymphocytes from THC-treated mice transferred the effect to normal mice, resulting in accelerated tumor growth similar to that seen in the THC-treated mice. THC decreased tumor immunogenicity, as indicated by the limited capacity for tumor-immunized, THC-treated mice to withstand tumor rechallenge. In vivo administration of a specific antagonist of the CB2 cannabinoid receptor also blocked the effects of THC. Our findings suggest the THC promotes tumor growth by inhibiting antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway.
T Cell Vaccination in Mice with Invasive Pulmonary Aspergillosis
Authors: Cenci Elio , Mencacci Antonella , Bacci Angela , Bistoni Francesco , Kurup Viswanath P. , Romani Luigina
Published: April 2014

Aspergillus fumigatus, an opportunistic fungal pathogen, is responsible for multiple airway diseases of an allergic and a nonallergic nature. In a murine model of invasive pulmonary aspergillosis, resistance is associated with a decreased lung inflammatory pathology and the occurrence of an IL-12-dependent Th1-type reactivity that are both impaired by IL-4. In the present study we assess the ability of Aspergillus crude culture filtrate Ags and the recombinant allergen Asp f 2 to induce protective antifungal responses in mice with invasive pulmonary aspergillosis. Similar to what occurred upon nasal exposure to viable A. fumigatus conidia, treatment of immunocompetent mice with Aspergillus crude culture filtrate Ags resulted in the development of local and peripheral protective Th1 memory responses, mediated by Ag-specific CD4+ T cells producing IFN-γ and IL-2 capable of conferring protection upon adoptive transfer to naive recipients. Protective Th1 responses could not be observed in mice deficient of IFN-γ or IL-12 and did not occur in response to Asp f 2, which, on the contrary, elicited high level production of inhibitory IL-4. The results show that Ags of Aspergillus exist with the ability to induce both Th1- and Th2-type reactivity during infection, a finding that suggests a possible mechanism through which potentially protective immune responses are inhibited in mice with the infection. However, the occurrence of Th1-mediated resistance upon vaccination with Aspergillus crude culture filtrate Ags, suggests the existence of fungal Ags useful as a candidate vaccine against invasive pulmonary aspergillosis.
The Chemokine Fractalkine Inhibits Fas-Mediated Cell Death of Brain Microglia
Authors: Boehme Stefen A. , Lio Francisco M. , Maciejewski-Lenoir Dominique , Bacon Kevin B. , Conlon Paul J.
Published: April 2014

Fractalkine is a CX3C-family chemokine, highly and constitutively expressed on the neuronal cell surface, for which a clear CNS physiological function has yet to be determined. Its cognate receptor, CX3CR-1, is constitutively expressed on microglia, the brain-resident macrophages; however, these cells do not express fractalkine. We now show that treatment of microglia with fractalkine maintains cell survival and inhibits Fas ligand-induced cell death in vitro. Biochemical characterization indicates that this occurs via mechanisms that may include 1) activation of the phosphatidylinositol-3 kinase/protein kinase B pathway, resulting in phosphorylation and blockade of the proapoptotic functions of BAD; 2) up-regulation of the antiapoptotic protein Bcl-xL; and 3) inhibition of the cleavage of BH3-interacting domain death agonist (BID). The observation that fractalkine serves as a survival factor for primary microglia in part by modulating the protein levels and the phosphorylation status of Bcl-2 family proteins reveals a novel physiological role for chemokines. These results, therefore, suggest that the interaction between fractalkine and CX3CR-1 may play an important role in promoting and preserving microglial cell survival in the CNS.
Macrophage-Induced Neutrophil Apoptosis
Authors: Meszaros Adriana J. , Reichner Jonathan S. , Albina Jorge E.
Published: April 2014

Macrophages (Mφ) contribute to the resolution of early inflammation by recognizing and ingesting apoptotic polymorphonuclear neutrophils (PMN). In addition, experiments reported here demonstrated that Mφ can actively induce PMN apoptosis. Coculture of cells from 2- or 5-day-old wounds in rats, or of Mφ purified from such preparations, with PMN-rich wound cell populations obtained 1 day after wounding increased PMN apoptosis by >3-fold. Neither resident- nor Proprionibacterium acnes-elicited peritoneal Mφ-induced PMN apoptosis. Apoptosis was not mediated by a soluble factor and required E:T contact. Fixed wound-Mφ and membrane isolates from viable Mφ were as effective as intact cells in inducing PMN apoptosis. Mφ-induced apoptosis was inhibited by peptide Arg-Gly-Asp-Ser, anti-β3 (CD61) Ab, CD36 peptide, or anti-TNF-α Ab. Soluble TNF-α did not induce PMN apoptosis. In additional studies, K562 cells (negative for β3, TNF-α, and Fas ligand) transfected to express either αvβ3 integrin, an uncleavable membrane form of TNF-α, or both were used in cocultures with wound PMN. Only the double transfectants were able to induce PMN apoptosis, an effect inhibited by anti-β3 (CD61) or anti-TNF-α Abs. These results demonstrate that wound Mφ induce PMN apoptosis through a constitutive effector mechanism requiring both intercellular binding through integrin-ligand interactions and membrane-bound TNF-α.
IL-12 Responsiveness and Expression of IL-12 Receptor in Human Peripheral Blood Monocyte-Derived Dendritic Cells
Authors: Nagayama Hitomi , Sato Katsuaki , Kawasaki Hiroshi , Enomoto Makoto , Morimoto Chikao , Tadokoro Kenji , Juji Takeo , Asano Shigetaka , Takahashi Tsuneo A.
Published: April 2014

We analyzed the expression of IL-12Rβ1 and IL-12Rβ2 and the role of IL-12 in the activation of monocyte-derived dendritic cells (DCs) via IL-12Rβ1-mediated signaling events. Flow cytometric analysis revealed that IL-12Rβ1 was expressed in T cells, Con A blasts, and monocyte-derived DCs, but not in monocytes, while its transcript was detected in all of these cell types. Transcriptional expression of IL-12Rβ2 was observed in T cells, Con A blasts, and monocyte-derived DCs, but not monocytes. The ligation of DCs as well as Con A blasts by IL-12 induced the production of GM-CSF, IL-1β, IL-6, TNF-α, and IFN-γ at the transcription levels. Furthermore, stimulation of DCs with IL-12 induced IL-12p40 transcript, but not IL-12p35 transcript, whereas this stimulation caused the expressions of both transcripts in Con A blasts. Stimulation of DCs with IL-12 caused a tyrosine phosphorylation of several intracellular proteins, and the pattern of these events were distinct from those of IL-12-stimulated Con A blasts. IL-12 also induced tyrosine phosphorylation of IL-12Rβ1 as well as recruitment of several tyrosine-phosphorylated proteins to IL-12Rβ1 in DCs and Con A blasts. Receptor engagement of DCs as well as Con A blasts by IL-12 resulted in activation of Janus kinase 2 and Tyk2 kinases and Stat3 and Stat4 transcription factors and the association of these proteins to IL-12Rβ1. Stimulation with IL-12 caused a tyrosine phosphorylation and enzymatic activity of a family of mitogen-activated protein kinases, p38mapk. These results suggest that IL-12 acts directly on DCs to induce their functional activation via IL-12Rβ1-mediated signaling events.
An Amiloride-Sensitive and Voltage-Dependent Na+ Channel in an HLA-DR-Restricted Human T Cell Clone
Authors: Lai Zhong-Fang , Chen Yu-Zhen , Nishimura Yasuharu , Nishi Katsuhide
Published: April 2014

We investigated changes in voltage-gated Na+ currents and effects of extracellular Na+ on proliferation in HLA-DR-restricted human CD4+ αβ T cells after stimulation with a non-self antigenic peptide, M12p54–68. In the absence of antigenic peptide, neither single (n = 80) nor APC-contacted (n = 71) T cells showed voltage-gated inward currents recording with whole-cell patch-clamp techniques, even with Ca2+ and Na+ ions present in the perfusion solution. However, with the same recording conditions, 31% (26 of 84) of APC-contacted T cells stimulated with the antigenic peptide showed voltage-dependent inward currents that were elicited from −60 mV. The inward currents were not inhibited in extracellular Ca2+-free conditions or in the presence of 1 mM NiCl2. However, they were completely inhibited in extracellular Na+-free conditions, which were made by replacing Na+ with iso-osmotic N-methyl-d-glucamine or choline. The Na+ currents were insensitive to tetrodotoxin, a classical blocker of Na+ channels, but were dose-dependently inhibited by amiloride, a potassium-sparing pyrazine diuretic. Furthermore, the Ag-specific proliferative response of T cells was completely inhibited in Na+-free Tyrode’s solution and was suppressed by amiloride in a dose-dependent manner. Our findings suggest that activation of amiloride-sensitive and voltage-gated Na+ channels would be an important step to allow an adequate influx of Na+ and maintain a sustained high Ca2+ level during T cell activation.
Cutting Edge Commentary: A Copernican Revolution? Doubts About the Danger Theory
Authors: Vance Russell E.
Published: April 2014

The immune system is often said to function by “self-nonself” discrimination. Recently, some have argued that it actually detects “danger” or “strangers.” There are problems with all of these points of view. Given that the immune system has been cobbled together throughout evolution and uses a diverse array of innate and adaptive defense mechanisms, it may not be possible to account for immunity within one “paradigm” or another.
Cutting Edge: Contribution of NK Cells to the Homing of Thymic CD4+NKT Cells to the Liver
Authors: Miyamoto Mamiko , Emoto Masashi , Brinkmann Volker , van Rooijen Nico , Schmits Rudolf , Kita Eiji , Kaufmann Stefan H. E.
Published: April 2014

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.
Cutting Edge: Essential Role of Phospholipase C-γ2 in B Cell Development and Function
Authors: Hashimoto Ari , Takeda Kiyoshi , Inaba Muneo , Sekimata Masayuki , Kaisho Tsuneyasu , Ikehara Susumu , Homma Yoshimi , Akira Shizuo , Kurosaki Tomohiro
Published: April 2014

Cross-linking of the B cell Ag receptor (BCR) induces the tyrosine phosphorylation of multiple cellular substrates, including phospholipase C (PLC)-γ2, which is involved in the activation of the phosphatidylinositol pathway. To assess the importance of PLC-γ2 in murine lymphopoiesis, the PLC-γ2 gene was inducibly ablated by using IFN-regulated Cre recombinase. Mice with a neonatally induced loss of PLC-γ2 function displayed reduced numbers of mature conventional B cells and peritoneal B1 cells and defective responses in vitro to BCR stimulation and in vivo to immunization with thymus-independent type II Ags. In contrast, T cell development and TCR-mediated proliferation were normal. Taken together, PLC-γ2 is a critical component of BCR signaling pathways and is required to promote B cell development.