bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired Antibody Responses in H-2Ab Mice
Authors: Gustavsson Susanne , Hjulström-Chomez Susanna , Lidström Bo-Marcus , Ahlborg Niklas , Andersson Roland , Heyman Birgitta
Published: December 2022

In murine in vivo systems, Ags administered in physiologic solutions together with specific IgE induce a significantly higher Ab response than Ags administered alone. In vitro, IgE in complex with Ag enhances B cell-mediated presentation of the Ag to T cells. Both phenomena require an intact low affinity receptor for IgE (FcεRII/CD23), suggesting that the effect on in vivo Ab responses is caused by increased Ag presentation. We here show that mice carrying the MHC class II Ab molecule (e.g., C57BL/6 and 129/Sv) do not produce Abs to BSA when immunized with BSA-2,4,6-trinitrophenyl (TNP) in complex with monoclonal IgE anti-TNP. In contrast, strains of all other MHC haplotypes tested (H-2d, H-2k, H-2p, H-2q, and H-2s) respond vigorously to IgE/BSA-TNP complexes, with Ab responses several hundred-fold higher than the responses in H-2b mice. C57BL/6 mice were unable to produce a carrier-specific response also after immunization with IgE/OVA-TNP, IgE/diphtheria toxoid-TNP, or IgE/tetanus toxoid-TNP. Although the low responsiveness mapped to the Ab region, responsiveness was not restored in C57BL/6 mice carrying transgenic Ak, suggesting that a nonclassical A-region-encoded gene product is involved. Most importantly, our data call attention to the fact that the C57BL/6 and 129 mouse strains, which are widely used for producing transgenic animals, have defective immune responses.
TGF-β Inhibits IL-12-Induced Activation of Jak-STAT Pathway in T Lymphocytes
Authors: Bright John J. , Sriram Subramaniam
Published: December 2022

IL-12 is a macrophage-derived heterodimeric cytokine, capable of inducing proliferation, cytokine production, and cytotoxic activity of NK cells and T cells, and is critical for the development of Th1 responses. TGF-β is an immunosuppressive cytokine that inhibits IL-12-mediated responses in NK and T cells. To determine the mechanism of action of TGF-β, we examined its inhibitory effect on IL-12 signal-transduction pathway in T cells. Stimulation of activated T cells with IL-12 leads to tyrosine phosphorylation and activation of Jak-2 and Tyk-2 kinases and STAT3 and STAT4 transcription factors. Treatment of activated T cells with TGF-β blocked IL-12-induced tyrosine phosphorylation and activation of both Jak-2 and Tyk-2 kinases. Furthermore, inhibition of Jak kinases by TGF-β was associated with a decrease in tyrosine phosphorylation of STAT3 and STAT4 proteins. Abrogation of IL-12-induced Jak-Stat pathway by TGF-β resulted in decreased T cell proliferation and IFN-γ production, and increased apoptotic cell death. These findings highlight that TGF-β inhibits IL-12-mediated responses by blocking IL-12 signal transduction in T cells.
Characterization of a Mobile Stat6 Activation Motif in the Human IL-4 Receptor
Authors: Ryan John J. , McReynolds Lisa J. , Huang Hua , Nelms Keats , Paul William E.
Published: December 2022

The IL-4R induces proliferation and gene expression through the use of conserved tyrosine residues located in growth and gene regulation domains, respectively. We demonstrate that residues surrounding these conserved tyrosines (juxtatyrosine residues) are essential for the proper activation of the signaling molecules IRS-2 and Stat6, as well as for IL-4-induced gene expression. Further, we found that the IL-4R gene regulation domain (amino acids 557–657) contains a tyrosine-based sequence (EAGYKAF) that can convey Stat6 DNA binding and gene expression activities to a minimally active IL-4R mutant, Δ557. Thus, this tyrosine-based sequence can function as a mobile Stat6 activation cassette. However, mutants bearing this sequence induced CD23 expression much less efficiently than did wild-type IL-4R, requiring 150-fold more IL-4 to reach maximal CD23 expression. Our results indicate the importance of juxtatyrosine residues in IL-4R signaling and argue for an essential role of extended domain structure in the recognition and function of juxtatyrosine sequences.
Translational Control of MHC Class II I-A Molecules by IFN-γ
Authors: Goñalons Eduard , Barrachina Marta , García-Sanz José A. , Celada Antonio
Published: December 2022

MHC class II molecules are expressed in a limited number of cell types, including B lymphocytes and macrophages (Mφ). IFN-γ increases the surface expression of class II molecules in a murine B cell line without inducing detectable changes in either I-A or I-A mRNA levels. In bone marrow-derived Mφ, IFN-γ causes an increase in class II expression at both the mRNA and surface levels. In addition to the increase in transcription rates described for Mφ, IFN-γ increases the rate of synthesis of IAα and IAβ proteins and the ribosome loading for both mRNA molecules in both cell types. Interestingly, there is a significant peak of free I-A mRNA in noninduced cells. Therefore, IFN-γ regulates the expression of MHC class II molecules at the translational level in both B cells and Mφ and, as already reported, at the transcriptional level only in Mφ. The actual mechanism of regulation causes changes in the translation initiation rates in both cell types, as demonstrated by an increase in ribosome loading in polysome gradients.
Signal-Regulatory Protein Is Selectively Expressed by Myeloid and Neuronal Cells
Authors: Adams Susan , van der Laan Luc J. W. , Vernon-Wilson Elizabeth , Renardel de Lavalette Chantal , Döpp Ed A. , Dijkstra Christine D. , Simmons David L. , van den Berg Timo K.
Published: December 2022

Signal-regulatory proteins (SIRP) are transmembrane glycoproteins with three extracellular Ig-like domains, closely related to Ag receptors Ig, TCR, and MHC, and a cytoplasmic domain with two immunoreceptor with tyrosine-based inhibition motifs that can interact with src homology 2 domain-containing phosphatases. SIRP have previously been shown to inhibit signaling through receptor tyrosine kinases, but their physiologic function is unknown. Here we demonstrate by expression cloning that the mAbs ED9, ED17, and MRC-OX41 recognize rat SIRP. In addition, we show for the first time that rat SIRP is selectively expressed by myeloid cells (macrophages, monocytes, granulocytes, dendritic cells) and neurons. Moreover, SIRP ligation induces nitric oxide production by macrophages. This implicates SIRP as a putative recognition/signaling receptor in both immune and nervous systems.
Lung Injury Induced by Alloreactive Th1 Cells Is Characterized by Host-Derived Mononuclear Cell Inflammation and Activation of Alveolar Macrophages
Authors: Clark Joan G. , Madtes David K. , Hackman Robert C. , Chen Wei , Cheever Martin A. , Martin Paul J.
Published: December 2022

We have investigated a murine model of acute lung injury caused by i.v. administration of a T cell clone (CD4+, Th1 phenotype) that recognizes Ly5, a polymorphic cell surface glycoprotein expressed on hemopoietic cells. Alloreactive cloned T cells, specific for host Ly5 Ag, cause a mononuclear cell pulmonary vasculitis and interstitial pneumonitis. In further studies of the cellular mechanisms involved in this model, we found that mature host T cells or B cells are not required, since lung injury was comparable in transgenic host mice that lack these cells (RAG-1 knockout). Cloned T cells labeled in vitro with bromodeoxyuridine were localized in inflammation foci in lung, but the majority of cells in the foci were not labeled. Using transgenic mice that constitutively express lacZ, we determined that the mononuclear cell vasculitis is of host cell origin. Alveolar macrophages (AM) from T cell-treated mice spontaneously secreted TNF-α in culture, whereas TNF-α was not detected in AM cultures from control mice. TNF-α production in response to LPS stimulation was significantly higher in AM cultures derived from T cell-treated mice than in those from control mice. Challenge with sublethal doses of LPS resulted in 50% mortality in T cell-treated mice and was associated with augmented AM TNF-α production and protein in bronchoalveolar lavage fluid. We conclude that immune activation of T cells of the Th1 phenotype can initiate lung injury characterized by a host-derived mononuclear cell inflammation and activation of AM.
The Differential Production of Three Forms of IL-1 Receptor Antagonist by Human Neutrophils and Monocytes
Authors: Malyak Mark , Smith Michael F. , Abel Ashley A. , Hance Kenneth R. , Arend William P.
Published: December 2022

IL-1R antagonist (IL-1Ra) exists as three well-characterized isoforms. The 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1RaI) arise by alternative transcription of the same IL-1Ra gene. The recently described 16-kDa intracellular IL-1Ra (icIL-1RaII) is formed by alternative translation initiation of sIL-1Ra mRNA. Transcription and translation of IL-1Ra isoforms were examined in LPS-stimulated human neutrophils and PBMC using RT-PCR, ELISA, and Western blot analysis. LPS stimulation of neutrophils resulted in elevated sIL-1Ra mRNA levels by 1 h, whereas icIL-1RaI mRNA remained undetectable through 22 h of culture. Extracellular glycosylated sIL-1Ra protein and intracellular icIL-1RaII were observed in LPS-stimulated neutrophils by 3 h of culture; no icIL-1RaI protein was detected by immunoblot. LPS stimulation of PBMC resulted in elevated sIL-1Ra mRNA levels by 1 h and detectable icIL-1RaI mRNA at 8 h of culture. LPS-stimulated PBMC demonstrated extracellular glycosylated sIL-1Ra protein and intracellular icIL-1RaII within 3 h of stimulation, whereas detection of icIL-1RaI protein was delayed until 15 h of culture. Subcellular localization experiments established that both icIL-1RaI and icIL-1RaII were present primarily within the cytoplasmic compartment, as expected by their lack of a signal peptide. These results demonstrate that although both LPS-stimulated neutrophils and PBMC synthesize sIL-1Ra and icIL-1RaII, only PBMC transcribe and translate icIL-1RaI. Furthermore, sIL-Ra transcription and translation (and translation of icIL-1RaII) are early events, whereas icIL-1RaI transcription in PBMC is delayed.
IFN-α Priming of Human Monocytes Differentially Regulates Gram-Positive and Gram-Negative Bacteria-Induced IL-10 Release and Selectively Enhances IL-12p70, CD80, and MHC Class I Expression
Authors: Hermann Patrice , Rubio Manuel , Nakajima Toshi , Delespesse Guy , Sarfati Marika
Published: December 2022

Administration of IFN-γ and IFN-α may protect or induce autoimmune diseases. Although the in vitro regulation of monokine secretion by IFN-γ have been extensively studied, the regulatory function of IFN-α has not yet been elucidated. We compared IFN-α and IFN-γ, added alone or simultaneously before bacterial stimulation, for the control of monokine release and the expression of costimulatory molecules by human monocytes. Our data show that: 1) IFN-α primes monocytes for increased production of IL-10 in response to Staphylococcus aureus Cowan I strain (SAC) but not to LPS, leading to a lack of IFN-α priming for TNF-α secretion; 2) pretreatment of monocytes with IFN-α inhibits LPS- or SAC-induced IL-12p40 production but unexpectedly enhances the release of the biologically active form of IL-12 (IL-12p70); 3) IFN-α and IFN-γ exert an antagonistic effect on LPS- and SAC-induced IL-10 as well as IL-12p40 release, whereas they further enhance IL-12p70 production when added simultaneously; 4) in contrast to IFN-α, IFN-γ primes monocytes to enhance LPS- or SAC-induced TNF-α and IL-12 production, but surprisingly, it increases IL-10 production by monocytes following LPS but not SAC stimulation; and finally, 5) IFN-α pretreatment selectively up-regulates CD80 and MHC class I expression on monocytes. It is proposed that the outcome of the immune response at the site of inflammation may depend on both the type of bacterial injury (Gram-positive or -negative) and of locally produced IFNs, and that the differential and opposite effects of type I and type II IFNs on monocytes may account for the beneficial or detrimental effects of IFN-α therapy.
Anti-β2-Glycoprotein I Autoantibodies from Patients with the “Antiphospholipid” Syndrome Bind to β2-Glycoprotein I with Low Affinity: Dimerization of β2-Glycoprotein I Induces a Significant Increase in Anti-β2-Glycoprotein I Antibody Affinity
Authors: Sheng Yonghua , Kandiah David A. , Krilis Steven A.
Published: December 2022

“Antiphospholipid” autoantibodies are associated with arterial and venous thrombosis, recurrent fetal loss, and thrombocytopenia. At present, the best-characterized antigenic target for these autoantibodies (or Abs) is the phospholipid-binding protein β2-glycoprotein I (β2GPI). These Abs bind β2GPI only in the presence of negatively charged phospholipids or microtiter polystyrene plates that have been specially treated to give the surface a negative charge. To determine whether the binding of these Abs to β2GPI on negatively charged surfaces is dependent on increased density or neo-epitopes formed as a consequence of a conformational change on β2GPI, we generated mutants of β2GPI by site-directed mutagenesis and assessed the binding characteristics of anti-β2GPI Abs to these mutants. Our results demonstrate that mutant F307*, which spontaneously forms significant dimerization, is bound best by all the anti-β2GPI Abs in an anti-β2GPI ELISA using irradiated polystyrene microtiter plates. In addition, these Abs bound mutant F307* coated onto standard polystyrene microtiter wells in the absence of phospholipid, whereas there was minimal binding with wild-type and mutant F307*/C288A, which formed minimal dimerization. Affinity-purified anti-β2GPI Abs from patients with the antiphospholipid syndrome demonstrated significantly higher binding affinity for mutant F307* in fluid phase than for wild-type or mutant F307*/C288A of β2GPI. These results demonstrate that autoantibody binding to β2GPI is intrinsically of low affinity and that the binding is dependent on the density of the Ag and not on neo-epitope formation.
Primary Th1 Cell Immunization Against HIVgp160 in SCID-hu Mice Coengrafted with Peripheral Blood Lymphocytes and Skin
Authors: Delhem Nadirah , Hadida Fabienne , Gorochov Guy , Carpentier Françoise , de Cavel Jean-Pierre , Andréani Jean-François , Autran Brigitte , Cesbron Jean-Yves
Published: December 2022

SCID-hu mouse models are of interest in the pathologic investigation of HIV infection, but obtaining a T cell response in SCID-hu-PBL mice is still controversial. We have developed a SCID model by engrafting human skin and autologous PBLs from HIV-seronegative individuals. The study describes the ability of this human-mouse chimera to generate in vivo a primary T lymphocyte response against HIV Ag. The injection of human autologous PBLs was performed 4 to 5 wk after the skin engraftment. Two weeks after injection of PBLs, chimeric mice were immunized with recombinant canary pox virus expressing HIV-1 LAIgp160 (vCP-LAIgp160) and supplemented or not with rIL-2. Intradermal vCP-LAIgp160 injection induced an intradermal perivascular human lymphocytic infiltrate and an epidermic network of CD1a+, CD80+, and CD86+ cells. We derived CD4+ T cell lines (STLs) from the human skin graft of immunized mice, showing that STLs mediated an MHC class II-restricted cytolytic activity directed against HIV-LAIgp160 Ags. Cytokine gene expression in both human skin cells and in STLs showed a predominance of IL-2, IFN-γ, and IL-12 transcripts. Finally, the T cell repertoire analysis using the immunoscope technique showed a very limited CDR3 length polymorphism in the skin infiltrating lymphocytes suggesting an Ag-specific repertoire. The ability to induce a primary Th1 cell response in vivo affords a useful preclinical model for testing vaccine strategies.
MHC Class II Transport from Lysosomal Compartments to the Cell Surface Is Determined by Stable Peptide Binding, But Not by the Cytosolic Domains of the α- and β-Chains
Authors: Théry Clotilde , Brachet Valérie , Regnault Armelle , Rescigno Maria , Ricciardi-Castagnoli Paola , Bonnerot Christian , Amigorena Sebastian
Published: December 2022

Inside APCs, MHC class II molecules associate with antigenic peptides before reaching the cell surface. This association takes place in compartments of the endocytic pathway, more related to endosomes or lysosomes depending on the cell type. Here, we compared MHC class II transport from endosomal vs lysosomal compartments to the plasma membrane. We show that transport of MHC class II molecules to the cell surface does not depend on the cytosolic domains of the α- and β-chains. In contrast, the stability of the αβ-peptide complexes determined the efficiency of transport to the cell surface from lysosomal, but not from endosomal, compartments. In murine B lymphoma cells, SDS-unstable and -stable complexes were transported to the cell surface at almost similar rates, whereas after lysosomal relocalization or in a cell line in which MHC class II molecules normally accumulate in lysosomal compartments, stable complexes were preferentially addressed to the cell surface. Our results suggest that when peptide loading occurs in lysosomal compartments, selective retention and lysosomal degradation of unstable dimers result in the expression of highly stable MHC class II-peptide complexes at the APC surface.
Critical Role of IL-12 in Dendritic Cell-Induced Differentiation of Naive B Lymphocytes
Authors: Dubois Bertrand , Massacrier Catherine , Vanbervliet Béatrice , Fayette Jérome , Brière Francine , Banchereau Jacques , Caux Christophe
Published: December 2022

Dendritic cells (DC) are potent APCs initiating immune responses. In a previous report, we demonstrated that DC directly enhance both proliferation and differentiation of CD40-activated naive and memory B cells. The present study deciphers the molecular mechanisms involved in DC-dependent regulation of B cell responses. Herein, we have identified IL-12 as the mandatory molecule secreted by CD40-activated DC that promote the differentiation of naive B cells into plasma cells secreting high levels of IgM. In fact, IL-12 synergizes with soluble IL-6R α-chain (sgp80), produced by DC, to drive naive B cell differentiation. IL-12 is critical for the differentiation of naive B cells into IgM plasma cells, whereas IL-6R signaling mainly promotes Ig secretion by already differentiated B cells. The differentiation of naive B cells in cocultures of B cells, T cells, and DC is IL-12 dependent, definitely demonstrating that the role of DC in humoral responses is not confined to the activation of T cells and further extending the physiologic relevance of DC/B cell interaction. Finally, this study also identifies differential requirements for DC-dependent naive and memory B cell differentiation, the latter being IL-12 independent. Altogether these results indicate that, in addition to prime T cells toward Th1 development, DC, through the production of IL-12, may also directly signal naive B cell during the initiation of the immune response.
Substance P (Neurokinin-1) Receptor Is a Marker of Human Mucosal But Not Peripheral Mononulear Cells: Molecular Quantitation and Localization
Authors: Goode Triona , O’Connell Joe , Sternini Catia , Anton Peter , Wong Helen , O’Sullivan Gerald C. , Collins J. Kevin , Shanahan Fergus
Published: December 2022

Reciprocal communication between the immune sytem and the neuroendocrine system is mediated via a common chemical language of shared ligands and receptors. The neuropeptide substance P (SP) has been implicated as a mediator of immunomodulation. The evidence for substance P receptors on human lymphocytes is, however, controversial. The aims of the present study are to investigate substance P receptor (SPR) expression in human peripheral and mucosal mononuclear cells and to identify cellular sites of expression in human colonic mucosa. Using reverse-transcriptase PCR, we demonstrate that PBMC isolations are negative for SPR mRNA expression, whereas lamina propria mononuclear cell (LPMC) isolations express on average eight SPR mRNA transcripts per cell. In situ hybridization performed on surgically resected colonic tissue confirms the expression of SPR mRNA in LPMC in vivo. SPR mRNA signal was detected in LPMC, lymphoid follicles, and epithelium. The complementary technique of immunohistochemistry gave a similar distribution of SPR expression that colocalized with CD45 immunoreactivity. Dual-fluorochrome flow cytometry revealed SPR expression by CD4, CD45RO, CD45RA, CD8, CD19, and CD14 LPMC subsets, but not PBMC. Our findings suggest that SPR expression is distinctive of human colonic mucosal mononuclear cells and support a direct role for SP in mucosal immunomodulation.
A Plasmid Encoding Murine Granulocyte-Macrophage Colony-Stimulating Factor Increases Protection Conferred by a Malaria DNA Vaccine
Authors: Weiss Walter R. , Ishii Ken J. , Hedstrom Richard C. , Sedegah Martha , Ichino Motohide , Barnhart Kerry , Klinman Dennis M. , Hoffman Stephen L.
Published: December 2022

Using the murine parasite Plasmodium yoelii (Py) as a model for malaria vaccine development, we have previously shown that a DNA plasmid encoding the Py circumsporozoite protein (PyCSP) can protect mice against sporozoite infection. We now report that mixing a new plasmid PyCSP1012 with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) increases protection against malaria, and we have characterized in detail the increased immune responses due to GM-CSF. PyCSP1012 plasmid alone protected 28% of mice, and protection increased to 58% when GM-CSF was added (p < 0.0001). GM-CSF plasmid alone did not protect, and control plasmid expressing inactive GM-CSF did not enhance protection. GM-CSF plasmid increased Abs to PyCSP of IgG1, IgG2a, and IgG2b isotypes, but not IgG3 or IgM. IFN-γ responses of CD8+ T cells to the PyCSP 280–288 amino acid epitope increased but CTL activity did not change. The most dramatic changes after adding GM-CSF plasmid were increases in Ag-specific IL-2 production and CD4+ T cell proliferation. We hypothesize that GM-CSF may act on dendritic cells to enhance presentation of the PyCSP Ag, with enhanced IL-2 production and CD4+ T cell activation driving the increases in Abs and CD8+ T cell function. Recombinant GM-CSF is already used in humans for medical purposes, and GM-CSF protein or plasmids may be useful as enhancers of DNA vaccines.
Differential Regulation of Lipopolysaccharide (LPS) Activation Pathways in Mouse Macrophages by LPS-Binding Proteins
Authors: Amura Claudia R. , Kamei Takayuki , Ito Noriko , Soares Michael J. , Morrison David C.
Published: December 2022

LPS binding to its receptor(s) on macrophages induces the synthesis of inflammatory mediators involved in septic shock. While the signaling mechanism(s) remains to be fully defined, the human LPS-binding protein (LBP) is known to regulate responses to LPS by facilitating its binding to CD14 on human monocytes. The structurally related bactericidal permeability increasing protein (BPI) differs from LBP by inhibiting LPS-induced human monocyte activation. We have demonstrated that, unlike the human monocyte response to LPS, both LBP and BPI inhibited LPS-stimulated TNF-α production in mouse peritoneal macrophages. In contrast, LPS-dependent nitric oxide release was not affected by LBP. LPS induces the phosphorylation of a number of proteins in a dose and time-dependent manner, however, the pattern of LPS-induced phosporylation was not reduced by either LBP or BPI under conditions that result in selective TNF-α inhibition. Further, activation of the transcription factor NF-κB in response to LPS was also not modified by either LBP or BPI. Finally, no differences were detected in TNF-α or inducible nitric oxide synthase mRNA accumulations induced by LPS in the presence or absence of either protein, whereas a slight decreased mRNA stability was observed in the group with LPS treatment. These results would suggest that many of the early signaling events contribute to LPS-induced macrophage signaling at a point preceding the divergence of pathways that differentially regulate TNF-α and NO production.
The Up-Regulation of IL-6 and IL-8 in Human Endothelial Cells by Activated Protein C
Authors: Hooper W. Craig , Phillips Donald J. , Renshaw Mary A. , Evatt Bruce L. , Benson Jane M.
Published: December 2022

The protein C/protein S anticoagulant pathway has been proposed to be a common link between coagulation and inflammation. Studies have suggested that a component of the anticoagulant pathway, activated protein C (APC), may play a role in the inflammatory response by modulating the effects of cytokines such as TNF and by blocking neutrophil activation. Cytokines are known to be intimately involved in the inflammatory response and to function in part to restore hemostatic balance. To begin to delineate what role APC may have in the inflammatory response, we have investigated the effect of APC on the production of the proinflammatory cytokines IL-6 and IL-8 in primary HUVEC, human microvascular endothelial cells, and human coronary artery endothelial cells. Our results have demonstrated that physiologic concentrations of APC significantly up-regulated the production of both IL-6 and IL-8. This increase, which was seen at both the RNA and protein level, was not due to either thrombin or LPS contamination of the APC preparation. Additional studies also showed that the APC-mediated up-regulation of IL-6 and IL-8 was IL-1 independent. Although neither purified protein C nor protein S alone had an effect on cytokine production, protein S, the cofactor for APC, significantly enhanced the ability of APC to up-regulate IL-6/IL-8 production. These results provide further evidence for a role for APC in the inflammatory response.
The Heat Shock Response Inhibits RANTES Gene Expression in Cultured Human Lung Epithelium
Authors: Ayad Onsy , Stark James M. , Fiedler Michael M. , Menendez Ingrid Y. , Ryan Marnie A. , Wong Hector R.
Published: December 2022

The chemokine RANTES is thought to be involved in the pathophysiology of inflammation-associated acute lung injury. Although much is known regarding signals that induce RANTES gene expression, relatively few data exist regarding signals that inhibit RANTES gene expression. The heat shock response, a highly conserved cellular defense mechanism, has been demonstrated to inhibit a variety of lung proinflammatory responses. We tested the hypothesis that induction of the heat shock response inhibits RANTES gene expression. Treatment of A549 cells with TNF-α induced RANTES gene expression in a concentration-dependent manner. Induction of the heat shock response inhibited subsequent TNF-α-mediated RANTES mRNA expression and secretion of immunoreactive RANTES. Transient transfection assays involving a RANTES promoter-luciferase reporter plasmid demonstrated that the heat shock response inhibited TNF-α-mediated activation of the RANTES promoter. Inhibition of NF-κB nuclear translocation with isohelenin inhibited TNF-α-mediated RANTES mRNA expression, indicating that RANTES gene expression is NF-κB dependent in A549 cells. Induction of the heat shock response inhibited degradation of the NF-κB inhibitory protein, I-κBα but did not significantly inhibit phosphorylation of I-κBα. We conclude that the heat shock response inhibits RANTES gene expression by a mechanism involving inhibition of NF-κB nuclear translocation and subsequent inhibition of RANTES promoter activation. The mechanism by which the heat shock response inhibits NF-κB nuclear translocation involves stabilization of I-κBα, without significantly affecting phosphorylation of I-κBα.
Increased Susceptibility to Postoperative Sepsis in Patients with Impaired Monocyte IL-12 Production
Authors: Hensler Thorsten , Heidecke Claus-Dieter , Hecker Heike , Heeg Klaus , Bartels Holger , Zantl Niko , Wagner Hermann , Siewert Jörg-Rüdiger , Holzmann Bernhard
Published: December 2022

IL-12 is a potent immunoregulatory cytokine that is essential for the development of protective immunity, as demonstrated by numerous animal models of infection. Here, we provide evidence for a critical role of IL-12 in human sepsis. The results of a prospective study of 184 patients undergoing major elective surgery of the upper and lower gastrointestinal tract revealed that, in contrast to patients showing uneventful recovery, monocyte IL-12 production was severely and selectively impaired in patients developing postoperative sepsis. Moreover, the extent of monocyte IL-12 suppression correlated with the severity of postoperative sepsis. Monocyte IL-12 secretion was suppressed before surgery and remained low until the onset of sepsis. Therefore, the suppression of IL-12 secretion preceded the onset of postoperative sepsis but did not occur as a consequence of major surgery. In contrast, IL-1β production was only reduced during the late postoperative course in patients developing postoperative sepsis, and TNF-α release was even increased at different time intervals before the onset of sepsis. Thus, reduced IL-12 release does not reflect a general defect in monocyte cytokine production. Consequently, these results establish a critical role for IL-12 in early resistance to postoperative infection and may allow for the development of novel therapeutic strategies designed to stimulate host defense mechanisms and to reduce the incidence and severity of septic complications.
Prostaglandin E2 and Dexamethasone Inhibit IL-12 Receptor Expression and IL-12 Responsiveness
Authors: Wu Chang-You , Wang Kening , McDyer John F. , Seder Robert A.
Published: December 2022

Regulation of the factors governing IL-12R expression and IL-12 responsiveness has been shown to be important in the generation and stability of Th1- and Th2-type responses. In this regard, cytokines have been shown to have a prominent role in regulating IL-12R expression. In this study, the role that PGE2 and dexamethasone (DXM) have in regulating IL-12R expression was evaluated. Addition of PGE2 or DXM to human PBMCs stimulated with immobilized anti-CD3 plus IL-12 inhibited the production of IFN-γ in a dose-responsive manner. Moreover, PBMCs stimulated with immobilized anti-CD3 in the presence of PGE2 or DXM for 3 days, washed extensively, and restimulated in the presence of IL-12 still did not produce IFN-γ. This lack of IL-12 responsiveness from cells cultured in either PGE2 or DXM was correlated with diminished surface expression of IL-12Rβ1, IL-12Rβ2 mRNA expression, and IL-12 binding. Finally, the PGE2- and DXM-mediated inhibition of IL-12R expression was not affected significantly by addition of neutralizing Abs against either IL-4, IL-10, or TGF-β. By contrast, addition of dibutyryl cAMP, 8-bromoadenosine 3:5 cAMP (8-Br-cAMP), or cholera toxin substantially reduced IL-12R expression, suggesting that PGE2 may be mediating its effects through enhancement of cAMP.
The Susceptibility of Mice to Immune-Mediated Neurologic Disease Correlates with the Degree to Which Their Lymphocytes Resist the Effects of Brain-Derived Gangliosides
Authors: Irani David N.
Published: December 2022

SJL mice develop immune-mediated disorders of the central nervous system (CNS) when infected with certain neurotropic viruses or when immunized with myelin Ags. Other strains including BALB/c are more resistant to these diseases. During Sindbis virus-induced encephalitis, both mice are easily infected and elicit rapid mononuclear cell inflammation in the brain. However, only SJL mice develop immune-mediated paralysis; BALB/c mice remain asymptomatic. To understand how the same stimulus produces such divergent immunologic effects on the host, the present study investigated lymphocytes that were isolated from the brains of Sindbis virus-infected animals. Cells from the brains of SJL mice exhibited more proliferation, produced more IL-2, maintained a higher viability, and expressed less bax mRNA (a proapoptotic mediator) than did lymphocytes from the brains of BALB/c mice. Since the central nervous system is enriched in gangliosides that regulate T cell proliferation and IL-2 production in vitro, purified brain-derived gangliosides were tested on peripheral lymphocytes from both strains. These lipids had less of an effect on the mitogen-induced proliferation, IL-2 production, activation-induced cell death, and up-regulation of bax mRNA in lymphocytes from SJL mice compared with those from BALB/c mice. Thus, gangliosides may inhibit various T cell effector functions and induce T cell apoptosis to a greater degree in the brains of BALB/c mice compared with the brains of SJL mice. This relative deficiency in local lymphocyte regulation may enhance the susceptibility of SJL mice to immune-mediated neurologic disease.
Granzymes D, E, F, and G Are Regulated Through Pregnancy and by IL-2 and IL-15 in Granulated Metrial Gland Cells
Authors: Allen Melissa P. , Nilsen-Hamilton Marit
Published: December 2022

Granulated metrial gland (GMG) cells are NK cells that proliferate and differentiate within the murine uterus during pregnancy. They have been predicted to play important roles in nurturing the embryo, normal placentation, and uterine tissue remodeling. GMG cell differentiation is manifested by the accumulation of the cytolytic mediators, perforin, granzyme A, and granzyme B, within cytoplasmic granules. The signaling mechanisms required for GMG cell differentiation are largely unknown, although recent in vitro assays have implicated IL-15 in these events. In this report, we demonstrate that granzymes D, E, F, and G (granzymes D–G) are also expressed in GMG cells but at a later stage in pregnancy when compared with granzyme A expression. Whereas granzyme A is expressed in early to mid-gestation, the expression of granzymes D–G peak in mid- to late gestation. In addition, we show that the expression patterns of IL-2Rβ and the IL-2Rγ mRNAs overlap with that of granzyme D–G mRNAs in the pregnant uterus. Finally, we demonstrate that granzymes D–G are up-regulated by IL-2 and IL-15 in primary cultures containing GMG cells. Taken together, these results suggest that IL-2 and/or IL-15 may regulate GMG cell differentiation in vivo, and that granzymes D–G may have different functions than granzyme A during pregnancy.
Prostaglandin E2 Induces the Final Maturation of IL-12-Deficient CD1a+CD83+ Dendritic Cells: The Levels of IL-12 Are Determined During the Final Dendritic Cell Maturation and Are Resistant to Further Modulation
Authors: Kaliński Paweł , Schuitemaker Joost H. N. , Hilkens Catharien M. U. , Kapsenberg Martien L.
Published: December 2022

Activation of immature dendritic cells (DC) in peripheral tissues induces their migration to lymph nodes and their maturation into CD83+ DC, which are able to prime naive T cells. The inflammatory cytokines IL-1β and TNF-α induce mature DC, which can secrete IL-12 and promote the development of Th0/Th1-biased cells. DC maturation factors with a Th2-promoting function have not been described. Here we show that PGE2, although it does not induce final DC maturation by itself, synergizes with IL-1β and TNF-α, and allows their effectiveness at 100-fold lower concentrations. While being phenotypically identical with the DC matured in the presence of high concentrations of IL-1β and TNF-α alone, DC matured in the additional presence of PGE2 show impaired IL-12 production and bias naive Th cell development toward the Th2. The ability of DC to produce IL-12 is also suppressed by IL-10, which in contrast to PGE2, inhibits their maturation. The differences in the ability to produce IL-12, established during the final DC maturation, are stable after the removal of modulatory factors. Importantly, fully mature DC become unsusceptible to PGE2 and IL-10. This indicates that the levels of IL-12 production in vivo, in mature DC interacting with Th cells within the lymph nodes, are mainly predetermined at the stage of immature DC in peripheral tissues. These data imply that the character of pathogen-induced local inflammatory reaction can “instruct” local DC to initiate Th1 or Th2-biased responses.
Molecular Modeling of an Anti-DNA Autoantibody (V-88) and Mapping of Its V Region Epitopes Recognized by Heterologous and Autoimmune Antibodies
Authors: Hobby Paul , Ward Francis J. , Denbury Andrew N. , Williams D. Gwyn , Staines Norman A. , Sutton Brian J.
Published: December 2022

Anti-DNA autoantibodies are a characteristic feature of human systemic lupus erythematosus (SLE) and lupus diseases in the mouse. V-88 is an IgG1/κ ssDNA-binding Ab, derived from a lupus mouse, that bears a cross-species, cross-reactive Id (CRI) that has been implicated in the pathogenesis of both human and murine disease. A linear epitope map of V-88 has been determined with anti-idiotypic antisera obtained from rabbits, and candidate sequences for the idiotopes of the CRI have been proposed. We now report the modeling of the three-dimensional structure of the V regions of Ab V-88, to map the location of these idiotopes. The V region framework structure was derived from those of crystallographically determined Ab structures, and the complementarity determining region (CDR) structures were based upon the set of canonical structures adopted by these loop regions in Abs of known structure. One of the idiotopes is an extensive, highly accessible epitope consisting of framework regions spatially adjacent to CDR2 in the heavy chain. Epitopes recognized by an anti-idiotypic rabbit antiserum were compared with those recognized by autoimmune sera from SLE-prone mice, and common features were identified. By analogy with the crystal structure of an anti-DNA Ab BV04-01 complexed with a trinucleotide, the modeled structure also suggests a mode of binding of ssDNA to V-88. The location of the candidate CRI, although within the framework region of VH, is such that it could influence Ag specificity.
Human T Cell Leukemia Virus-I (HTLV-I) Tax-Mediated Apoptosis in Activated T Cells Requires an Enhanced Intracellular Prooxidant State
Authors: Los Marek , Khazaie Khashayarsha , Schulze-Osthoff Klaus , Baeuerle Patrick A. , Schirrmacher Volker , Chlichlia Katerina
Published: December 2022

We have shown that an estradiol-dependent activation of human T cell leukemia virus-I Tax leads to the inhibition of cell proliferation and to the induction of apoptosis. The present study demonstrates that a hormone-dependent activation of Tax promotes an enhanced prooxidant state in stably transfected Jurkat cells as measured by changes in the intracellular levels of glutathione and H2O2; these changes are followed by apoptotic cell death. Additional stimulation of the CD3/TCR pathway enhances the oxidative and apoptotic effects. Both Tax-mediated apoptosis and oxidative stress can be potently suppressed by antioxidants, as is seen with the administration of recombinant thioredoxin (adult T cell leukemia-derived factor) or pyrrolidine dithiocarbamate. Hormone-induced Tax activation induces a long-lasting activation of NF-κB, which is a major target of reactive oxygen intermediates. The long-term exposure of Jurkat cells to hormone eventually results in a selection of cell clones that have lost Tax activity. A subsequent transfection of these apparently “nonresponsive” clones allows the recovery of Tax responses in these cells. Our observations indicate that changes in the intracellular redox status may be a determining factor in Tax-mediated DNA damage, apoptosis, and selection against the long-term expression of Tax function.
Treatment ofParacoccidioides brasiliensis-Infected Mice with a Nitric Oxide Inhibitor Prevents the Failure of Cell-Mediated Immune Response
Authors: Bocca Anamélia L. , Hayashi Emília E. , Pinheiro Artur G. , Furlanetto Alessandra B. , Campanelli Ana P. , Cunha Fernando Q. , Figueiredo Florêncio
Published: December 2022

The activation of the nitric oxide (NO) production system and its involvement in the control of the lung fungal burden and in immunosuppression mechanisms were studied during the course of Paracoccidioides brasiliensis-infected mice. Mice that had been infected with the fungus were treated daily with a specific inhibitor of NO synthesis, Nω-nitro-l-arginine, or with buffered saline (control); NO production was assessed on the basis of spontaneous NO2− production by bronchoalveolar and peritoneal macrophages (Mφ) and of serum NO3− levels. The infection coursed with an elevation of NO3− levels. The Mφ produced NO2− and released TNF-α only after stimulation with LPS. In addition, the immunoproliferative responses of spleen cells that had been stimulated with the fungus Ag or with Con A were depressed. An examination of the lungs of infected animals showed a progressive increase in the size of the lesions. Treatment of the animals, which resulted in an inhibition of NO2− production by Mφ and a reduction of serum NO3− levels, caused the spontaneous release of TNF-α from infected animals and prevented the failure of the lymphoproliferative capacity of spleen cells. Furthermore, the treatment resulted in less pulmonary damage despite the fact that the lung fungal burden increased. It was also demonstrated that the NO donors S-nitroso-acetyl penicillamine and 3-morpholino-sydnonimine-hydrochloride were able to inhibit the growth of P. brasiliensis in vitro. These results suggest that although NO is important for the killing of the fungi, the activation of NO production in P. brasiliensis infection contributes to the occurrence of the immunosuppression observed during the course of the infection.
Tissue and T Cell Distribution of Precursor and Mature IL-16
Authors: Chupp Geoffrey L. , Wright Eric A. , Wu David , Vallen-Mashikian Margaret , Cruikshank William W. , Center David M. , Kornfeld Hardy , Berman Jeffrey S.
Published: December 2022

IL-16 is a novel cytokine, which is chemoattractant for CD4+ T cells, macrophages, and eosinophils. Recently, it was reported that IL-16 is synthesized as an approximately 80-kDa precursor molecule, pro-IL-16. Since little is known about the processing and tissue distribution of IL-16 and pro-IL-16, we investigated the distribution of IL-16 mRNA and protein in human lymphoid tissue. Northern blotting identified IL-16 mRNA predominantly in normal lymphoid organs, including PBMC, spleen, and thymus. Immunohistochemistry of human lymph node localized IL-16 protein to lymphocyte cytoplasm within T cell zones and occasionally in lymphocytes in B cell zones. Flow cytometric detection of intracellular IL-16 showed that >70% of CD4+ and CD8+ T cells constitutively expressed IL-16 protein. Western blot analysis of PBMC revealed nearly all of this protein to be approximately 80-kDa pro-IL-16 in unstimulated PBMC, and upon cell activation, the amino terminus of pro-IL-16 is processed into multiple fragments. These results show that pro-IL-16 is widely and constitutively expressed and suggest that the amino terminus of the protein can be processed upon cell activation.
TNFR80-Dependent Enhancement of TNFR60-Induced Cell Death Is Mediated by TNFR-Associated Factor 2 and Is Specific for TNFR60
Authors: Weiss Tilo , Grell Matthias , Siemienski Katrin , Mühlenbeck Frank , Dürkop Horst , Pfizenmaier Klaus , Scheurich Peter , Wajant Harald
Published: December 2022

Costimulation of TNFR80 can strongly enhance TNFR60-induced cell death. In this study, we show that this enhancement is TNFR60 selective, as neither TNF-related apoptosis-inducing ligand/Apo2 ligand-, Apo1/Fas-, ceramide-, nor daunorubicin-mediated cell death was affected by costimulation of TNFR80. We further demonstrate that TNFR-associated factor 2 (TRAF2) is critically involved in both negative and positive regulation of TNF-induced cell death. Overexpression of TRAF2 and of a TRAF2 mutant, deficient in nuclear factor-κB activation, selectively desensitized and enhanced, respectively, TNFR60-induced cell death in HeLa cells. However, upon costimulation of TNFR80, which mediates activation of nuclear factor-κB and the c-Jun amino-terminal kinase via TRAF2, TNF-induced cell death is drastically enhanced in parental and TRAF2-transfected, but not in TRAF2 (87–501)-transfected cells. These data point to a critical role of TRAF2 in the apoptotic TNFR cross talk, whereby the TNFR80-dependent enhancement of TNFR60-induced cell death is due to TNFR80-mediated negative regulation of TRAF2 function(s). An interference with TRAF2 function was confirmed independently by analysis of c-Jun amino-terminal kinase activation via TNFR60 upon prestimulation of TNFR80. We propose that the apoptotic TNFR cross talk is based on TNFR80-mediated abrogation of antiapoptotic TRAF2-dependent signaling pathways initiated by TNFR60, but not Apo1/Fas or the apoptotic TNF-related apoptosis-inducing ligand receptors.
Functional Analysis of Peripheral Blood B Cells in Patients with X-Linked Agammaglobulinemia
Authors: Nonoyama Shigeaki , Tsukada Satoshi , Yamadori Tomoki , Miyawaki Toshio , Jin Yin Zhu , Watanabe Chiaki , Morio Tomohiro , Yata Jun-Ichi , Ochs Hans D.
Published: December 2022

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations of Bruton tyrosine kinase (Btk); Btk plays an essential role in the development of mature B cells. However, small numbers of B cells (“leaky B cells”) are present in the peripheral blood of most XLA patients. In this study, we analyzed the function of these leaky B cells obtained from XLA patients. Enough numbers of B cells were available for analysis from five of nine XLA patients originally screened. Sequence analysis revealed missense mutations of Btk in four of the five XLA patients. No mutation was found in the coding region of Btk in one patient. Western blotting and/or flow cytometric analysis failed to detect Btk protein in all five patients. B cells isolated from peripheral blood of these XLA patients were CD5−, CD20+, CD19+, and CD21−. If stimulated with anti-CD40 and IL-4, XLA B cells proliferated normally and produced significant amounts of IgE. Anti-CD40 stimulation of XLA B cells resulted in normal expression of CD23. In addition, three of the five XLA patients studied were immunized with bacteriophage φX174 and produced low but detectable levels of antiphage-specific Ab. Similarly, X-linked immunodeficiency mice, which carry a missense mutation in Btk, produced substantial amounts of antiphage Ab. These results indicate that CD40 signaling is intact in B cells lacking demonstrable Btk, and that leaky B cells in XLA patients can proliferate, undergo isotype switching, and differentiate into specific Ab-producing cells.
FLIP Prevents Apoptosis Induced by Death Receptors But Not by Perforin/Granzyme B, Chemotherapeutic Drugs, and Gamma Irradiation
Authors: Kataoka Takao , Schröter Michael , Hahne Michael , Schneider Pascal , Irmler Martin , Thome Margot , Froelich Cristopher J. , Tschopp Jürg
Published: December 2022

FLICE-inhibitory protein, FLIP (Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT), which contains two death effector domains and an inactive caspase domain, binds to FADD and caspase-8, and thereby inhibits death receptor-mediated apoptosis. Here, we characterize the inhibitory effect of FLIP on a variety of apoptotic pathways. Human Jurkat T cells undergoing Fas ligand-mediated apoptosis in response to CD3 activation were completely resistant when transfected with FLIP. In contrast, the presence of FLIP did not affect apoptosis induced by granzyme B in combination with adenovirus or perforin. Moreover, the Fas ligand, but not the perforin/granzyme B-dependent lytic pathway of CTL, was inhibited by FLIP. Apoptosis mediated by chemotherapeutic drugs (i.e., doxorubicin, etoposide, and vincristine) and gamma irradiation was not affected by FLIP or the absence of Fas, indicating that these treatments can induce cell death in a Fas-independent and FLIP-insensitive manner.