bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency of IL-5 Receptor α-Chain Selectively Influences the Development of the Common Mucosal Immune System Independent IgA-Producing B-1 Cell in Mucosa-Associated Tissues
Authors: Hiroi Takachika , Yanagita Manabu , Iijima Hideki , Iwatani Kouichi , Yoshida Toshimi , Takatsu Kiyoshi , Kiyono Hiroshi
Published: December 2022

Deletion of IL-5Rα-chain (IL-5Rα−/−) selectively influenced the mucosal IgA responses in vivo. While levels of IgA in mucosal secretions were more reduced in IL-5Rα−/− mice than in wild-type mice, the levels of IgA in serum were not changed. The frequency of IgA-producing cells was reduced in mucosal effector sites (e.g., intestinal lamina propria and nasal passage), but not in inductive sites such as Payer’s patches and nasal-associated lymphoreticular tissues in IL-5Rα−/− mice. IgA-committed (surface IgA+; sIgA+) B-1 cells mainly resided in mucosal effector tissues, while conventional sIgA+ B (B-2) cells formed in mucosal inductive sites of wild-type mice. In contrast, in the effector tissue of IL-5Rα−/− mice, sIgA+ B-1 cells, but not sIgA+ B-2 cells in the inductive site, were significantly reduced. IL-5Rα was more expressed on sIgA+ B-1 cells than was IL-6R, while both IL-5Rα and IL-6R were expressed on sIgA+ B-2 cells in wild-type mice. sIgA+ B-1 cells produced high levels of IgA with rIL-5 rather than of rIL-6 in vitro. Taken together, the findings suggest that the IL-5/IL-5R signaling pathway is critically important for the development of common mucosal immune system independent sIgA+ B-1 cell in mucosal effector tissues in vivo.
T Cell Priming Enhances IL-4 Gene Expression by Increasing Nuclear Factor of Activated T Cells
Authors: Cron Randy Q. , Bort Susan J. , Wang Yunxia , Brunvand Mark W. , Lewis David B.
Published: December 2022

The repetitive activation of T cells (priming) enhances the expression of many cytokines, such as IL-4, but not others, such as IL-2. Molecular mechanisms underlying selective expression of cytokines by T cells remain poorly understood. Here we show that priming of CD4 T cells selectively enhances IL-4 expression relative to IL-2 expression by a transcriptional mechanism involving nuclear factor of activated T cells (NFAT) proteins. As detected by in vivo footprinting, priming markedly increases the activation-dependent engagement of the P0 and P1 NFAT-binding elements of the IL-4 promoter. Moreover, each proximal P element is essential for optimal IL-4 promoter activity. Activated primed CD4 T cells contain more NFAT1 and support greater NFAT-directed transcription than unprimed CD4 T cells, while activator protein 1 binding and activator protein 1-mediated transcription by both cell types is similar. Increased expression of wild-type NFAT1 substantially increases IL-4 promoter activity in unprimed CD4 T cells, suggesting NFAT1 may be limiting for IL-4 gene expression in this cell type. Furthermore, a truncated form of NFAT1 acts as a dominant-negative, reducing IL-4 promoter activity in primed CD4 T cells and confirming the importance of endogenous NFAT to increased IL-4 gene expression by effector T cells. NFAT1 appears to be the major NFAT family member responsible for the initial increased expression of IL-4 by primed CD4 T cells.
Alternative Splicing and Hypermutation of a Nonproductively Rearranged TCR α-Chain in a T Cell Hybridoma
Authors: Marshall Brendan , Schulz Ruth , Zhou Min , Mellor Andrew
Published: December 2022

Like Ig genes, TCR genes are formed by somatic rearrangements of noncontiguous genomic V, J, and C regions. Unlike Ig genes, somatic hypermutation of TCR V regions is an infrequent event. We describe the occurrence of spontaneous hypermutation in a nonproductively rearranged TCR α-chain gene in a clonal T cell hybridoma that had lost its productively rearranged α-chain. The mutating hybridoma was eventually supplanted in culture by a nonmutating variant that had restored an open reading frame in the nonproductively rearranged TCR α-chain through the use of cryptic splice sites in the Vα region. Evidence is presented for the presence of cDNA reverse transcripts of the TCR α-chain within the hybridoma, suggesting a role for reverse transcriptase in the generation of mutations.
The Extracellular Domain of the ζ-Chain Is Essential for TCR Function
Authors: Johansson Britt , Palmer Ed , Bolliger Luca
Published: December 2022

The ζ-chain homodimer is a key component in the TCR complex and exerts its function through its cytoplasmic immunoreceptor-tyrosine activation motif (1). The ζ-chain extracellular (EC) domain is highly conserved; however, its functional and structural contributions to the TCR signaling have not been elucidated. We show that the EC domain of the ζ homodimer is essential for TCR surface expression. To gain a more detailed structural and functional information about the ζ-chain EC domain, we applied a cysteine scanning mutagenesis to conserved amino acids of the short domain. The results showed that the interchain disulfide bridge can be displaced by seven or eight amino acids along the EC domain. The TCR signaling efficacy was dramatically reduced during peptide/MHC engagement in the ζ mutants containing the displaced disulfide bond. These signaling defective ζ mutants produced an unconventional early tyrosine phosphorylation pattern. While the tyrosine phosphorylated forms of ζ (p21 and p23) could be observed during Ag stimulation, downstream signaling events such as the generation of phospho-p36, higher m.w. forms of phospho-ζ, and phospho-ζ/ZAP-70 complexes were impaired. Together these results suggest an important function of the phylogenetically conserved ζ-EC domain.
IL-1β Inhibits IFN-γ-Induced Class II MHC Expression by Suppressing Transcription of the Class II Transactivator Gene
Authors: Rohn Wolfgang , Tang Li Ping , Dong Yuanshu , Benveniste Etty N.
Published: December 2022

Class II MHC Ags are critical for the initiation of immune responses by presenting Ag to T lymphocytes, leading to their activation and differentiation. The transcriptional activation of class II MHC genes requires the induction of the class II transactivator (CIITA) protein, a master regulator that is essential for both constitutive and IFN-γ-inducible class II MHC expression. The cytokine IL-1β has been shown to inhibit IFN-γ-induced class II MHC expression in various cell types. We investigated the underlying mechanism of this inhibitory effect of IL-1β using human astroglioma cell lines. Our findings demonstrate that IL-1β prevents the expression of class II MHC mRNA and protein upon treatment with IFN-γ. Furthermore, we demonstrate that IFN-γ induction of CIITA mRNA expression is inhibited by treatment of cells with IL-1β. IL-1β suppressed IFN-γ activation of the type IV CIITA promoter in astroglioma cells, indicating that the inhibitory influence of IL-1β is mediated by inhibition of CIITA transcription. IL-1β did not interfere with IFN-γ receptor signal transduction, since tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT-1α to an IFN-γ activation sequence of the type IV CIITA promoter were not affected by IL-1β. As well, IL-1β treatment did not affect the ability of IFN-γ-induced interferon-regulatory factor-1 (IRF-1) to bind the IRF-1 element within the type IV CIITA promoter. This study suggests that IL-1β may play a role in regulating immunoreactivity by inhibiting transcription of the CIITA gene, thereby reducing subsequent class II MHC expression.
IL-13 Is a Key Regulatory Cytokine for Th2 Cell-Mediated Pulmonary Granuloma Formation and IgE Responses Induced bySchistosoma mansoniEggs
Authors: Chiaramonte Mónica G. , Schopf Lisa R. , Neben Tamlyn Y. , Cheever Allen W. , Donaldson Debra D. , Wynn Thomas A.
Published: December 2022

Schistosoma mansoni egg-induced pulmonary granuloma formation is a cell-mediated inflammatory response associated with dominant Th2-type cytokine expression, tissue eosinophilia, and high levels of serum IgE. In the present study, we show that in vivo blockade of the Th2 cytokine IL-13, using soluble IL-13R α2-Fc fusion protein, significantly reduced the size of pulmonary granulomas in unsensitized as well as egg-sensitized mice. Blocking IL-13 also significantly reduced total serum IgE levels. Interestingly, however, IL-13 blockade did not affect the evolving egg-induced Th2-type cytokine response. IL-4, IL-5, as well as IL-13 responses were indistinguishable in control-Fc- and soluble IL-13R α2-Fc fusion protein-treated animals. The smaller granulomas were also phenotypically like the control Fc-treated mice, displaying a similar eosinophil content. Additional studies in IL-4-deficient mice demonstrated that IL-13 was produced, but at much lower levels than in wild-type mice, while IL-4 expression was completely independent of IL-13. Moreover, while granuloma formation was partially reduced in IL-4-deficient mice, blocking IL-13 in these animals almost completely abrogated granuloma development and the pulmonary eosinophilia, while it simultaneously increased IFN-γ production. Together, these data demonstrate that IL-13 serves as an important mediator of Th2-mediated inflammation and plays a role in eliciting IgE responses triggered by schistosome eggs.
Development of an Animal Model for Neurocysticercosis: Immune Response in the Central Nervous System Is Characterized by a Predominance of γδ T Cells
Authors: Cardona Astrid E. , Restrepo Blanca I. , Jaramillo Juan M. , Teale Judy M.
Published: December 2022

Neurocysticercosis is the most common parasitic disease of the central nervous system worldwide. It is caused by the metacestode form of the helminth Taenia solium. Study of the immune response in the human brain has been limited by the chronic progression of the disease, the influence of corticosteroid treatment, and the scarcity of patients who undergo surgical intervention. To better understand the immune response and associated pathology in neurocysticercosis, a mouse model was developed by intracranial infection of BALB/c mice with Mesocestoides corti, a cestode organism related to T. solium. The immune response reveals the presence of abundant inflammatory infiltrates appearing as early as 2 days postinfection in extraparenchymal regions. In contrast, infiltration of immune cells into parenchymal tissue is significantly delayed. There is a natural progression of innate (neutrophils and macrophages), early induced (NK cells and γδ T cells), and adaptive immune responses (αβ T cells and B cells) in infected mice. γδ T cells are the predominant T cell population. A cell-mediated Th1 pathway of cytokine expression is evident in contrast to the previously described Th2 phenotype induced in the periphery.
Processing and Release of IL-16 from CD4+ But Not CD8+ T Cells Is Activation Dependent
Authors: Wu David M. H. , Zhang Yujun , Parada Nereida A. , Kornfeld Hardy , Nicoll John , Center David M. , Cruikshank William W.
Published: December 2022

IL-16 is synthesized as a precursor molecule of 68 kDa (pro-IL-16) that is processed by caspase-3, a member of the IL-1 converting enzyme (ICE) family. This cleavage results in a 13-kDa carboxy terminal peptide, which constitutes the bioactive secreted form of IL-16. We have previously reported constitutive IL-16 mRNA expression and pro-IL-16 protein in CD4+ and CD8+ T cells. Although bioactive IL-16 protein is present in unstimulated CD8+ T cells, there is no bioactive IL-16 present in CD4+ T cells. Along these lines, unstimulated CD8+ T cells contain active caspase-3. In the current studies we investigated the regulation of IL-16 protein and mRNA expression in CD4+ T cells and determined the kinetics of secretion following stimulation of the TCR. CD4+ T cells release IL-16 protein following antigenic stimulation, and this release is accelerated in time by costimulation via CD28. However, CD3/CD28 costimulation did not alter IL-16 mRNA appearance or stability in either CD4+ or CD8+ T cells. The secretion of bioactive IL-16 from CD4+ T cells correlated with the appearance of cleavage of pro-caspase-3 into its 20-kDa active form. Thus, resting CD8+ T cells contain active caspase-3 that is capable of cleaving pro-IL-16, whereas CD4+ T cells require activation for the appearance of active caspase-3. The mechanism of release or secretion of bioactive IL-16 is currently unknown, but does not correlate with cellular apoptosis.
Processing of HIV-1 Envelope Glycoprotein for Class I-Restricted Recognition: Dependence on TAP1/2 and Mechanisms for Cytosolic Localization
Authors: Ferris Robert L. , Hall Christopher , Sipsas Nikolaos V. , Safrit Jeffrey T. , Trocha Alicja , Koup Richard A. , Johnson R. Paul , Siliciano Robert F.
Published: December 2022

Processing of viral proteins for recognition by CTL involves degradation of the proteins in the cytosol of an infected cell followed by transport of the resulting peptides into the endoplasmic reticulum (ER) by the TAP1/2 complex. Uncertainty exists over the site of processing of viral envelope (env) proteins since the extracellular domains of env proteins are not present in the cytosol where the class I Ag-processing pathway begins. Rather, the ectodomains of env proteins are cotranslationally translocated into the ER during biosynthesis. To analyze env protein processing, we used the herpes simplex virus protein ICP47 to block peptide transport by TAP1/2 and examined the effects of TAP blockade on the processing of the HIV-1 env protein. For the majority of env-specific CD8+ CTL, the processing pathway required TAP1/2-mediated transport of cytosolic peptides into the ER. To determine how env peptides are generated in the cytosol, we analyzed the processing of two TAP1/2-dependent epitopes containing N-linked glycosylation sites. In each case, processing involved glycosylation-dependent posttranslational modification of asparagine residues to aspartic acid. These results are consistent with cotranslational translocation of env into the ER, where glycosylation occurs. This is followed by export of a fraction of the newly synthesized protein into the cytosol, where it is deglycosylated, with conversion of the asparagines to aspartic acid residues. Following cytoplasmic proteolysis, env peptides are retransported by TAP1/2 into the ER, where association with class I occurs. Thus, the env protein can enter the class I pathway through multiple distinct processing mechanisms.
Lymphotoxin αβ Is Expressed on Recently Activated Naive and Th1-Like CD4 Cells but Is Down-Regulated by IL-4 During Th2 Differentiation
Authors: Gramaglia Irene , Mauri Davide N. , Miner Kent T. , Ware Carl F. , Croft Michael
Published: December 2022

Lymphotoxin (LT) is a cytokine that orchestrates lymphoid neogenesis and formation of germinal center reactions. LT exists as a membrane heterotrimer of α and β subunits and is secreted as a homotrimer, LTα3. Using LTβR.Fc, expression of LTαβ on CD4 T cell subsets was investigated in a TCR transgenic model. LTαβ was evident 24–72 h after activation of naive T cells with specific Ag, and declined thereafter. Early expression was independent of IFN-γ and IL-12, however, IL-12 prolonged expression. LTαβ was reinduced within 2–4 h after Ag restimulation, but declined by 24 h regardless of IL-12 or IFN-γ priming. Exposure of naive T cells to IL-4 did not affect early LTαβ expression at 24 h, but resulted in subsequent down-regulation. IL-4-differentiated Th2 effectors did not re-express LTαβ, and LTαβ was transiently found on Th1 clones but not Th2 clones. LTα3 and TNF were immunoprecipitated from supernatants and lysates of IL-12 primed cells but not IL-4 primed cells. These studies demonstrate that LTαβ is expressed by activated naive CD4 cells, unpolarized IL-2-secreting effectors, and Th1 effectors. In contrast, loss of surface LTαβ and a lack of LTα3 and TNF secretion is associated with prior exposure to IL-4 and a Th2 phenotype.
TCR, LFA-1, and CD28 Play Unique and Complementary Roles in Signaling T Cell Cytoskeletal Reorganization
Authors: Sedwick Caitlin E. , Morgan Margaret M. , Jusino Lismaida , Cannon Judy L. , Miller Jim , Burkhardt Janis K.
Published: December 2022

T cells interacting with APCs undergo rearrangement of surface receptors and cytoskeletal elements to face the zone of contact with the APC. This polarization process is thought to affect T cell signaling by organizing a specialized domain on the T cell surface and to direct T cell effector function toward the appropriate APC. We have investigated the contribution of TCR, CD28, and LFA-1 signaling to T cell cytoskeletal polarization by assaying the response of an Ag-specific Th1 clone toward a panel of transfected APCs expressing MHC class II alone or in combination with ICAM-1 or B7-1. We show that polarization of talin, an actin-binding protein, occurs in response to integrin engagement. In contrast, reorientation of the T cell microtubule-organizing center (MTOC) is dependent on and directed toward the site of TCR signaling, regardless of whether integrins or costimulatory molecules are engaged. MTOC reorientation in response to peptide-MHC complexes is sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. CD28 coengagement overcomes this sensitivity, as does activation via Ab cross-linking of the TCR or via covalent peptide-MHC complexes, suggesting that phosphatidylinositol 3-kinase is not required per se but rather plays a role in signal amplification. Engagement of TCR in trans with LFA-1 results in separation of MTOC reorientation and cortical cytoskeletal polarization events, indicating that the two processes are not directly mechanistically linked. These studies show that T cells mobilize individual cytoskeletal components in response to distinct and specific cell surface interactions.
Monocytes Stimulate Expression of the Bcl-2 Family Member, A1, in Endothelial Cells and Confer Protection Against Apoptosis
Authors: Noble Karen E. , Wickremasinghe R. Gitendra , DeCornet Chris , Panayiotidis Panayiotis , Yong Kwee L.
Published: December 2022

We have investigated the molecular mechanisms underlying the ability of peripheral blood monocytes to block apoptosis induction in endothelial cells. Monocytes stimulated the expression of the bcl-2 homologue A1 in serum-starved endothelial cells after 6 h of coincubation, with elevated A1 levels persisting for up to 21 h. IL-1 and TNF also stimulated A1 expression at 6 h, but A1 transcript levels fell by 21 h. Direct cellular contact with monocytes was required for stimulation of A1 mRNA in endothelial cells. Stimulation of endothelial cell A1 mRNA by monocytes was not inhibited by anti-β2 integrin Abs, but anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) mAb reduced A1 transcript levels at 21 h. Studies employing either TNF on its own, or anti-TNF in endothelium/monocyte cocultures showed that TNF plays a role in the early (6-h) stimulation of A1, but is less important for the sustained elevation of A1 levels at 21 h. Serum-starved endothelial cells demonstrated increased survival and decreased apoptosis after coculture with monocytes. IL-10 reduced A1 mRNA expression in, as well as survival of, endothelial cells that were cocultured with monocytes. In comparison with A1, Bcl-2 was expressed at low levels and was up-regulated by monocytes only at 21 h, while neither Bax nor Bcl-xL levels were altered by monocytes. The interaction of monocytes with endothelium during the course of an inflammatory reaction may provide survival signals to endothelial cells.
Distinct Effects of Jak3 Signaling on αβ and γδ Thymocyte Development
Authors: Eynon Elizabeth E. , Livák Ferenc , Kuida Keisuke , Schatz David G. , Flavell Richard A.
Published: December 2022

Janus kinase 3 (Jak3) plays a central role in the transduction of signals mediated by the IL-2 family of cytokine receptors. Targeted deletion of the murine Jak3 gene results in severe reduction of αβ and complete elimination of γδ lineage thymocytes and NK cells. The developmental blockade appears to be imposed on early thymocyte differentiation and/or expansion. In this study, we show that bcl-2 expression and in vivo survival of immature thymocytes are greatly compromised in Jak3−/− mice. There is no gross deficiency in rearrangements of the TCRδ and certain γ loci in pre-T cells, and a functional γδ TCR transgene cannot rescue γδ lineage differentiation in Jak3−/− mice. In contrast, a TCRβ transgene is partially able to restore αβ thymocyte development. These data suggest that the signals mediated by Jak3 are critical for survival of all thymocyte precursors particularly during TCRβ-chain gene rearrangement, and are continuously required in the γδ lineage. The results also emphasize the fundamentally different requirements for differentiation of the αβ and γδ T cell lineages.
Interaction of Murine MHC Class I Molecules with Tapasin and TAP Enhances Peptide Loading and Involves the Heavy Chain α3 Domain
Authors: Suh Woong-Kyung , Derby Michael A. , Cohen-Doyle Myrna F. , Schoenhals Gary J. , Früh Klaus , Berzofsky Jay A. , Williams David B.
Published: December 2022

In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin. We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222 as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.
In Vivo IL-12 Production and IL-12 Receptors β1 and β2 mRNA Expression in the Spleen Are Differentially Up-Regulated in Resistant B6 and Susceptible A/J Mice During Early Blood-StagePlasmodium chabaudiAS Malaria
Authors: Sam Hakeem , Stevenson Mary M.
Published: December 2022

As previously reported, blood-stage Plasmodium chabaudi AS malaria is lethal by days 10–12 postinfection in susceptible A/J mice that mount an early, predominantly Th2 response. In contrast, resistant C57BL/6 (B6) mice clear the infection by 4 wk with an early Th1 response. In this study, we analyzed in vivo production of IL-12, a potent Th1-inducing cytokine, during the first 5 days after P. chabaudi AS infection in these mice. By day 2, serum IL-12 p70 levels were significantly increased in B6 mice over basal levels and were also significantly higher compared with A/J mice that showed no significant changes in serum p70 levels after infection. Splenectomy of resistant B6 mice before infection demonstrated that the spleen is the major source of systemic IL-12 in these hosts. Splenic mRNA levels of both p40 and p35 were significantly higher in A/J mice; however, the ratios of p40/p35 mRNA levels were similarly up-regulated in both strains. Furthermore, B6 but not A/J mice showed significant up-regulation of splenic IL-12R β2 mRNA over basal levels by days 3 and 4, coincident with sustained up-regulation of splenic IFN-γ mRNA levels on days 3–5. However, IL-12R β1 mRNA levels in the spleen were similarly up-regulated in both mouse strains by day 3. Taken together, these data suggest that high systemic IL-12 production, accompanied by an early and sustained up-regulation of both IL-12R β1 and β2 mRNA levels in the spleen, as occurs in resistant B6 mice, appears to preferentially induce protective Th1 responses against blood-stage malaria.
Biased TCR Repertoire in Infiltrating Lesional T Cells in Human Bancroftian Filariasis
Authors: Freedman David O. , Plier David Adam , Almeida Adriana de , Miranda Janaina , Braga Cynthia , Maia e Silva Maria C. , Tang Jianming , Furtado Andre
Published: December 2022

To investigate the hypothesis that T cells recognizing specific Ags localize to the site of disease activity in human bancroftian filariasis, we have compared the repertoire of TCR Vβ gene segments in lesions vs blood in individual patients by RT-PCR ELISA. Vβ14 and Vβ24 were overrepresented (5% greater in tissue compared with PBMCs and/or tissue/PBMC ratios in the highest 5% of all tissue/PBMC ratios for all Vβs for all subjects) in 50% and 40% of study subjects, respectively. Overrepresentation of these two Vβs did not occur in any control subject. In comparing three patient groups, the proportion of individuals meeting at least one criterion for Vβ14 overrepresentation was shown to increase in tandem with our current concepts of disease progression (asymptomatic filariasis = 25%; clinical filariasis with active infection = 60%; clinical filariasis without active infection = 71%). In 6 of the 10 individuals with Vβ14 overrepresentation, Vβ14 represented >20% of the entire lesional Vβ repertoire. All but one of the 20 study subjects had at least one Vβ gene segment that was overrepresented in tissue compared with PBMCs. Only a small number of Vβs, usually three or less, were overrepresented in any single filariasis patient. However, in the same tissue, no differences between patient groups were found when IFN-γ, TNF-α, IL-4, IL-5, and IL-12 mRNA expression were examined. Taken together, these findings suggest that, in principle, in essentially all patients, whether with subclinical or with clinical filariasis, distinct and limited T cell populations are concentrated in affected tissue.
Cutting Edge: Clustered AU-Rich Elements Are the Target of IL-10-Mediated mRNA Destabilization in Mouse Macrophages
Authors: Kishore Raj , Tebo Julie M. , Kolosov Mikhail , Hamilton Thomas A.
Published: December 2022

In the present study we show that IL-10-mediated inhibition of inflammatory gene expression can be mediated by an AU-rich element (ARE) cluster present in the 3′ untranslated region (3′UTR) of sensitive genes. A series of chloramphenicol acetyl transferase (CAT) reporter gene constructs were prepared in which different fragments from the IL-10-sensitive KC mRNA 3′UTR were placed downstream of the coding region of the reporter gene CAT. CAT mRNA containing the KC 3′UTR was markedly destabilized as compared with the control CAT mRNA, and the decay rate was further increased in cells stimulated with IL-10. The KC 3′UTR contains an ARE cluster and three isolated ARE motifs. The ARE cluster spanning nucleotides 378–399 appeared to be both necessary and sufficient to mediate sensitivity to IL-10 because a 116-nucleotide fragment that contains the cluster conferred sensitivity, while mutation of the sequence between positions 378 and 399 eliminated sensitivity. The destabilizing effect of IL-10 was relatively selective, as the stability of chimeric CAT mRNAs was not modulated in cells treated with IFN-γ or IL-4.
N-Acetyl-l-Cysteine Inhibits Primary Human T Cell Responses at the Dendritic Cell Level: Association with NF-κB Inhibition
Authors: Verhasselt Valérie , Vanden Berghe Wim , Vanderheyde Nathalie , Willems Fabienne , Haegeman Guy , Goldman Michel
Published: December 2022

N-acetyl-l-cysteine (NAC) is an antioxidant molecule endowed with immunomodulatory properties. To investigate the effect of NAC on the induction phase of T cell responses, we analyzed its action on human dendritic cells (DC) derived from adherent PBMC cultured with IL-4 and granulocyte-macrophage CSF. We first found that NAC inhibited the constitutive as well as the LPS-induced activity of the transcription factor NF-κB. In parallel, NAC was shown to down-regulate the production of cytokines by DC as well as their surface expression of HLA-DR, CD86 (B7-2), and CD40 molecules both at the basal state and upon LPS activation. NAC also inhibited DC responses induced by CD40 engagement. The inhibitory effects of NAC were not due to nonspecific toxicity as neither the viability of DC nor their mannose receptor-mediated endocytosis were modified by NAC. Finally, we found that the addition of NAC to MLR between naive T cells and allogeneic DC resulted in a profound inhibition of alloreactive responses, which could be attributed to a defect of DC as APC-independent T cell responses were not inhibited by NAC. Altogether, our results suggest that NAC might impair the generation of primary immune responses in humans through its inhibitory action on DC.
IL-4 and IL-10 Are Both Required for the Induction of Oral Tolerance
Authors: Rizzo Luiz Vicente , Morawetz Renate A. , Miller-Rivero Nancy E. , Choi Rosan , Wiggert Barbara , Chan Chi-Chao , Morse Herbert C. , Nussenblatt Robert B. , Caspi Rachel R.
Published: December 2022

Protection from the development of experimental autoimmune uveitis (EAU) can be induced by feeding mice interphotoreceptor retinoid binding protein before uveitogenic challenge with the same protein. Two different regimens are equally effective in inducing protective tolerance, although they seem to do so through different mechanisms: one involving regulatory cytokines (IL-4, IL-10, and TGF-β), and the other with minimal involvement of cytokines. Here we studied the importance of IL-4 and IL-10 for the development of oral tolerance using mice genetically engineered to lack either one or both of these cytokines. In these animals we were able to protect against EAU only through the regimen inducing cytokine-independent tolerance. When these animals were fed a regimen that in the wild-type animal is thought to predominantly induce regulatory cells and is associated with cytokine secretion, they were not protected from EAU. Interestingly, both regimens were associated with reduced IL-2 production and proliferation in response to interphotoreceptor retinoid binding protein. These findings indicate that both IL-4 and IL-10 are required for induction of protective oral tolerance dependent on regulatory cytokines, and that one cytokine cannot substitute for the other in this process. These data also underscore the fact that oral tolerance, manifested as suppression of proliferation and IL-2 production, is not synonymous with protection from disease.
CD38 Signaling in B Lymphocytes Is Controlled by Its Ectodomain but Occurs Independently of Enzymatically Generated ADP-Ribose or Cyclic ADP-Ribose
Authors: Lund Frances E. , Muller-Steffner Hélène M. , Yu Naixuan , Stout C. David , Schuber Francis , Howard Maureen C.
Published: December 2022

CD38 is a type II transmembrane glycoprotein that is expressed by many cell types including lymphocytes. Signaling through CD38 on B lymphocytes can mediate B cell activation, proliferation, and cytokine secretion. Additionally, coligation of CD38 and the B cell Ag receptor can greatly augment B cell Ag receptor responses. Interestingly, the extracellular domain of CD38 catalyzes the conversion of NAD+ into nicotinamide, ADP-ribose (ADPR), and cyclic ADPR (cADPR). cADPR can induce intracellular calcium release in an inositol trisphosphate-independent manner and has been hypothesized to regulate CD38-mediated signaling. We demonstrate that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme activity. In contrast, independent point mutations in the extracellular domain of CD38 dramatically impaired signal transduction. However, no correlation could be found between CD38-mediated signaling and the capacity of CD38 to catalyze an enzyme reaction and produce cADPR, ADPR, and/or nicotinamide. Instead, we propose that CD38 signaling and coreceptor activity in vitro are regulated by conformational changes induced in the extracellular domain upon ligand/substrate binding, rather than on actual turnover or generation of products.
Coordinate Involvement of Invasin and Yop Proteins in aYersinia pseudotuberculosis-Specific Class I-Restricted Cytotoxic T Cell-Mediated Response
Authors: Falgarone Géraldine , Blanchard Hervé S. , Virecoulon François , Simonet Michel , Breban Maxime
Published: December 2022

Yersinia pseudotuberculosis is a pathogenic enteric bacteria that evades host cellular immune response and resides extracellularly in vivo. Nevertheless, an important contribution of T cells to defense against Yersinia has been previously established. In this study we demonstrate that Lewis rats infected with virulent strains of Y. pseudotuberculosis, mount a Yersinia-specific, RT1-A-restricted, CD8+ T cell-mediated, cytotoxic response. Sensitization of lymphoblast target cells for cytolysis by Yersinia-specific CTLs required their incubation with live Yersinia and was independent of endocytosis. Although fully virulent Yersinia did not invade those cells, they attached to their surface. In contrast, invasin-deficient strain failed to bind to blast targets or to sensitize them for cytolysis. Furthermore, an intact virulence plasmid was an absolute requirement for Yersinia to sensitize blast targets for cytolysis. Using a series of Y. pseudotuberculosis mutants selectively deficient in virulence plasmid-encoded proteins, we found no evidence for a specific role played by YadA, YopH, YpkA, or YopJ in the sensitization process of blast targets. In contrast, mutations suppressing YopB, YopD, or YopE expression abolished the capacity of Yersinia to sensitize blast targets. These results are consistent with a model in which extracellular Yersinia bound to lymphoblast targets via invasin translocate inside eukaryotic cytosol YopE, which is presented in a class I-restricted fashion to CD8+ cytotoxic T cells. This system could represent a more general mechanism by which bacteria harboring a host cell contact-dependent or type III secretion apparatus trigger a class I-restricted CD8+ T cell response.
Mutations of the Type A Domain of Complement Factor B That Promote High-Affinity C3b-Binding
Authors: Hourcade Dennis E. , Mitchell Lynne M. , Oglesby Teresa J.
Published: December 2022

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes “gain of function” mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the βA-α1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.
IL-12 Gene as a DNA Vaccine Adjuvant in a Herpes Mouse Model: IL-12 Enhances Th1-Type CD4+ T Cell-Mediated Protective Immunity Against Herpes Simplex Virus-2 Challenge
Authors: Sin Jeong-Im , Kim Jong J. , Arnold Renee L. , Shroff Khushroo E. , McCallus Don , Pachuk Cathy , McElhiney Sue P. , Wolf Mary W. , Pompa-de Bruin Sylvia J. , Higgins Terry J. , Ciccarelli Richard B. , Weiner David B.
Published: December 2022

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-γ) and chemokines (RANTES and macrophage inflammatory protein-1α) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.
TGF-β Does Not Inhibit IL-12- and IL-2-Induced Activation of Janus Kinases and STATs
Authors: Sudarshan Chitra , Galon Jérôme , Zhou Yong-jie , O’Shea John J.
Published: December 2022

The immune system is an important target for the cytokine TGF-β1, whose actions on lymphocytes are largely inhibitory. TGF-β has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-γ production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-β blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-γ have not found such an inhibition. The effect of TGF-β on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-β1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-β1 inhibited IL-2- and IL-12-induced IFN-γ production. Similarly, but in contrast to previous reports, we found that TGF-β1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-β1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-β on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-β.
Ligation of ICAM-1 on Endothelial Cells Leads to Expression of VCAM-1 Via a Nuclear Factor-κB-Independent Mechanism
Authors: Lawson Charlotte , Ainsworth Mark , Yacoub Magdi , Rose Marlene
Published: December 2022

ICAM-1 is an Ig-like cell adhesion molecule expressed by several cell types, including the endothelium. Cross-linking of ICAM-1 on the surface of different cell types has previously been shown to cause an increase in cellular activation within the cytoplasm. In this study, we have compared signaling events following ligation of ICAM-1 by cross-linking with mAbs with events after activation of HUVEC by TNF. ICAM-1 cross-linking caused activation of Erk-1 and the AP-1 transcription factor complex, without any increase in NF-κB activity, in contrast to TNF stimulation. Transcription of VCAM-1 mRNA was observed by reverse-transcriptase PCR after ICAM-1 cross-linking, with no associated transcription of E-selectin. This was reflected by the presence of VCAM-1 protein after immunoprecipitation, without E-selectin expression, in ICAM-1 cross-linked cells. In contrast, mRNA and protein for both VCAM-1 and E-selectin were observed in TNF-treated HUVEC, as expected. Addition of the MEK (MAP/Erk kinase) inhibitor PD98059 reduced expression of VCAM-1 after ICAM-1 cross-linking, suggesting that the Erk pathway is involved in ICAM-1-mediated VCAM-1 expression. In conclusion, ICAM-1-induced expression of VCAM-1 represents a pathway for adhesion molecule up-regulation that is distinct from the TNF-induced pathway. It may be similar to the IL-4 pathway or it may represent a novel pathway.
A Phosphatidylcholine-Specific Phospholipase C Regulates Activation of p42/44 Mitogen-Activated Protein Kinases in Lipopolysaccharide-Stimulated Human Alveolar Macrophages
Authors: Monick Martha M. , Carter Aaron Brent , Gudmundsson Gunnar , Mallampalli Rama , Powers Linda S. , Hunninghake Gary W.
Published: December 2022

This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC ζ, and this activation was inhibited by D609. LPS-activated PKC ζ phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS.
Strain-Dependent Induction of Allergic Sensitization Caused by Peanut Allergen DNA Immunization in Mice
Authors: Li Xiu-min , Huang Chih-Kang , Schofield Brian H. , Burks A. Wesley , Bannon Gary A. , Kim Kawn-Hyoung , Huang Shau-Ku , Sampson Hugh A.
Published: December 2022

To investigate the potential application of allergen gene immunization in the modulation of food allergy, C3H/HeSn (C3H) mice received i.m. injections of pAra h2 plasmid DNA encoding one of the major peanut allergens, Ara h2. Three weeks following pDNA immunization, serum Ara h2-specific IgG2a, IgG1, but not IgE, were increased significantly in a dose-dependent manner. IgG1 was 30-fold higher in multiply compared with singly immunized mice. Ara h2 or peanut protein injection of immunized mice induced anaphylactic reactions, which were more severe in multiply immunized mice. Heat-inactivated immune serum induced passive cutaneous anaphylaxis, suggesting that anaphylaxis in C3H mice was mediated by IgG1. IgG1 responses were also induced by intradermal injection of pAra h2, and by i.m. injection of pOMC, the plasmid DNA encoding the major egg allergen protein, ovomucoid. To elucidate whether the pDNA immunization-induced anaphylaxis was a strain-dependent phenomenon, AKR/J and BALB/c mice also received multiple i.m. pAra h2 immunizations. Injection of peanut protein into these strains at weeks 3 or 5 following immunization did not induce reactions. Although IgG2a was increased significantly from week 2 in AKR/J mice and from week 4 in BALB/c mice and remained elevated for at least 6 wk, no IgG1 or IgE was detected. These results indicate that the type of immune responses to pDNA immunization in mice is strain dependent. Consequently, models for studying human allergen gene immunization require careful selection of suitable strains. In addition, this suggests that similar interindividual variation is likely in humans.
Genetic Analysis of Disease Subtypes and Sexual Dimorphisms in Mouse Experimental Allergic Encephalomyelitis (EAE): Relapsing/Remitting and Monophasic Remitting/Nonrelapsing EAE Are Immunogenetically Distinct
Authors: Butterfield Russell J. , Blankenhorn Elizabeth P. , Roper Randall J. , Zachary James F. , Doerge R. W. , Sudweeks Jayce , Rose John , Teuscher Cory
Published: December 2022

Experimental allergic encephalomyelitis (EAE) is the principal animal model of multiple sclerosis (MS), the major inflammatory disease of the central nervous system. Murine EAE is generally either an acute monophasic or relapsing disease. Because the clinical spectrum of MS is more diverse, the limited range of disease subtypes observed in EAE has raised concern regarding its relevance as a model for MS. During the generation of a large F2 mapping population between the EAE-susceptible SJL/J and EAE-resistant B10.S/DvTe inbred lines, we identified four distinct subtypes of murine EAE resembling clinical subtypes seen in MS. We observed acute progressive, chronic/nonremitting, remitting/relapsing, and monophasic remitting/nonrelapsing EAE. An additional subtype, benign EAE, was identified after histologic examination revealed that some mice had inflammatory infiltrates of the central nervous system, but did not show clinical signs of EAE. Genome exclusion mapping was performed to identify the loci controlling susceptibility to each disease subtype. We report three novel EAE-modifying loci on chromosomes 16, 7, and 13 (eae11–13, respectively). Additionally, unique loci with gender-specific effects govern susceptibility to remitting/relapsing (eae12) and monophasic remitting/nonrelapsing (eae7 and 13) EAE.
Cutting Edge: Generation of IL-18 Receptor-Deficient Mice: Evidence for IL-1 Receptor-Related Protein as an Essential IL-18 Binding Receptor
Authors: Hoshino Katsuaki , Tsutsui Hiroko , Kawai Taro , Takeda Kiyoshi , Nakanishi Kenji , Takeda Yoshifumi , Akira Shizuo
Published: December 2022

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and Th1 cell response. Recently IL-1R-related protein (IL-1Rrp) has been cloned as the receptor for IL-18. However, the functional role of IL-1Rrp is still controversial due to its low affinity to IL-18 as well as the possibility of the presence of another high-affinity binding receptor. In the present study, we have generated and characterized IL-1Rrp-deficient mice. The binding of murine rIL-18 was not detected in Th1-developing splenic CD4+ T cells isolated from IL-1Rrp-deficient mice. The activation of NF-κB or c-Jun N-terminal kinase were also not observed in the Th1 cells. NK cells from IL-1Rrp-deficient mice had defects in cytolytic activity and IFN-γ production in response to IL-18. Th1 cell development was also impaired in IL-1Rrp-deficient mice. These data demonstrate that IL-1Rrp is a ligand-binding receptor that is essential for IL-18-mediated signaling events.