bluuebunny/crossref_metadata_2025 huggingface dataset:
C1qRP Is a Heavily O-Glycosylated Cell Surface Protein Involved in the Regulation of Phagocytic Activity
Authors: Nepomuceno R. R. , Ruiz S. , Park M. , Tenner A. J.
Published: January 2023

C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) interact with human monocytes and macrophages, resulting in the enhancement of phagocytosis of suboptimally opsonized targets. mAbs that recognize a cell surface molecule of 126,000 Mr, designated C1qRP, have been shown to inhibit C1q- and MBL-mediated enhancement of phagocytosis. Similar inhibition of the SPA-mediated enhancement of phagocytosis by these mAbs now suggests that C1qRP is a common component of a receptor for these macromolecules. Ligation of human monocytes with immobilized R3, a IgM mAb recognizing C1qRP, also triggers enhanced phagocytic capacity of these cells in the absence of ligand, verifying the direct involvement of this polypeptide in the regulation of phagocytosis. While the cDNA for C1qRP encodes a 631 amino acid membrane protein, Chinese hamster ovary cells transfected with the cDNA of the C1qRP coding region express a surface glycoprotein with the identical 126,000 Mr in SDS-PAGE as the native C1qRP. Use of glycosylation inhibitors, cleavage of the mature C1qRP with specific glycosidases, and in vitro translation of C1qRP cDNA demonstrated that both posttranslational glycosylation and the nature of the amino acid sequence of the protein contribute to the difference between its predicted m.w. and its migration on SDS-PAGE. These results verify that the cDNA cloned codes for the mature C1qRP, that C1qRP contains a relatively high degree of O-linked glycoslyation, and that C1qRP cross-linked directly by monoclonal anti-C1qRP or engaged as a result of cell surface ligation of SPA, as well as C1q and MBL, enhances phagocytosis.
Modulation of Formyl Peptide Receptor Expression by IL-10 in Human Monocytes and Neutrophils
Authors: Thivierge Maryse , Parent Jean-Luc , Stankova Jana , Rola-Pleszczynski Marek
Published: January 2023

IL-10, originally described as a cytokine synthesis inhibitory factor, is secreted by a number of cells of the immune system, including monocytes and T cells. Although IL-10 is being assigned as an immunosuppressive cytokine, our study showed that FMLP-R mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of this cytokine, with maximal (three- to fourfold) stimulation with 10 ng/ml. The effect was rapid, being observable as early as 1 h of treatment with IL-10, maximal between 2 and 4 h, and still evident after 24 h and was associated with an increase of receptor expression on the cell surface as assessed by flow cytometry analysis. Pretreatment of monocytes with actinomycin D completely abrogated the effect of IL-10, suggesting a transcriptional regulation. Moreover, IL-10-treated monocytes showed a significantly enhanced functional responsiveness to FMLP with enhanced (three- to fourfold) chemotaxis and augmented (twofold) intracellular calcium mobilization. In polymorphonuclear neutrophils (PMN), IL-10 also mediated a twofold augmentation of FMLP-R expression. In parallel experiments, we observed that IL-10 could differentially modulate other chemotactic receptors. Hence, we observed that IL-10 augmented two- to threefold platelet-activating factor receptor (PAF-R) expression, whereas it had no significant effect on the fifth component of complement (C5a) receptor (C5a-R) expression. Collectively, our results demonstrate that IL-10 may play an important role in inflammatory processes through modulation of chemotactic receptor expression.
EBV Gene Expression Not Altered in Rheumatoid Synovia Despite the Presence of EBV Antigen-Specific T Cell Clones
Authors: Edinger James W. , Bonneville Marc , Scotet Emmanuel , Houssaint Elisabeth , Schumacher H. Ralph , Posnett David N.
Published: January 2023

T cells infiltrating the rheumatoid arthritis (RA) joint are oligoclonal, implicating an Ag-driven process, but the putative joint-specific Ags remain elusive. Here we examine expression of selected EBV genes in RA synovia and find no abnormal expression in RA. DNA of CMV and EBV was detectable by PCR in the synovial tissue of RA. RNA of several latent and lytic EBV genes was also detectable. However, there were no differences in EBV gene expression in synovial tissues or peripheral blood when comparing RA with osteoarthritis, Gulf War syndrome, and other disease controls. RA synovia with highly expanded CD8 T cell clones reactive with defined EBV peptide Ags presented by HLA class I alleles lacked evidence of abnormal mRNA expression for the relevant EBV Ag (BZLF1) or lacked amplifiable mRNA (BMLF1). Thus, local production of EBV Ags in synovial tissues may not be the cause of the accumulation of T cell clones specific for these Ags. Instead, APCs loaded with processed EBV peptides may migrate to the synovium. Alternatively, EBV-specific T cell clones may be generated in other tissues and then migrate to synovia, perhaps due to cross-reactive joint-specific Ags or because of expression of homing receptors.
Differential Effects of CD40 Ligand/Trimer Stimulation on the Ability of Dendritic Cells to Replicate and Transmit HIV Infection: Evidence for CC-Chemokine-Dependent and -Independent Mechanisms
Authors: McDyer John F. , Dybul Mark , Goletz Theresa J. , Kinter Audrey L. , Thomas Elaine K. , Berzofsky Jay A. , Fauci Anthony S. , Seder Robert A.
Published: January 2023

The role of exogenous stimulation of CD40 by CD40 ligand (CD40L) in dendritic cell (DC) maturation, CC-chemokine production, and CCR5 receptor expression was examined using a soluble trimeric CD40L agonist protein (CD40LT). Stimulation of monocyte-derived DCs with CD40LT enhanced the production of the CC-chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES and diminished surface expression of CCR5. Based on these findings, the functional role of CD40LT stimulation on the ability of DCs to replicate and transmit HIV viral infection was studied. The addition of CD40LT to cocultures of naive CD4+ T cells and autologous DCs (T/DC) infected with the macrophage-tropic isolate, HIVBaL, caused a striking reduction in reverse transcriptase (RT) activity after 10 and 14 days of culture. The addition of a mixture of Abs against CC-chemokines abrogated the decrease in RT activity, demonstrating that the inhibitory effect mediated by CD40LT was CC-chemokine-dependent. In contrast, the presence of CD40LT in T/DC cocultures infected with the T cell-tropic isolate, HIVIIIB, caused an increase in RT activity that was CC-chemokine-independent. Of note, CD40LT stimulation also inhibited RT activity in cultures containing macrophage-tropic virus (HIVBaL)-infected DC only. However, in contrast to the results seen in the T/DC cocultures, CD40LT stimulation inhibited RT activity in cultures of DCs alone in a CC-chemokine-independent manner. Together, these results show that CD40LT stimulation of DCs suppresses HIV replication and transmission to CD4+ T cells by two potentially different mechanisms.
Molecular and Functional Analysis of a Conserved CTL Epitope in HIV-1 p24 Recognized from a Long-Term Nonprogressor: Constraints on Immune Escape Associated with Targeting a Sequence Essential for Viral Replication
Authors: Wagner Ralf , Leschonsky Bernd , Harrer Ellen , Paulus Christina , Weber Christine , Walker Bruce D. , Buchbinder Susan , Wolf Hans , Kalden Joachim R. , Harrer Thomas
Published: January 2023

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression of HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.
Cutting Edge: Toll-Like Receptor 4 (TLR4)-Deficient Mice Are Hyporesponsive to Lipopolysaccharide: Evidence for TLR4 as the Lps Gene Product
Authors: Hoshino Katsuaki , Takeuchi Osamu , Kawai Taro , Sanjo Hideki , Ogawa Tomohiko , Takeda Yoshifumi , Takeda Kiyoshi , Akira Shizuo
Published: January 2023

The human homologue of Drosophila Toll (hToll), also called Toll-like receptor 4 (TLR4), is a recently cloned receptor of the IL-1/Toll receptor family. Interestingly, the TLR4 gene has been localized to the same region to which the Lps locus (endotoxin unresponsive gene locus) is mapped. To examine the role of TLR4 in LPS responsiveness, we have generated mice lacking TLR4. Macrophages and B cells from TLR4-deficient mice did not respond to LPS. All these manifestations were quite similar to those of LPS-hyporesponsive C3H/HeJ mice. Furthermore, C3H/HeJ mice have, in the cytoplasmic portion of TLR4, a single point mutation of the amino acid that is highly conserved among the IL-1/Toll receptor family. Overexpression of wild-type TLR4 but not the mutant TLR4 from C3H/HeJ mice activated NF-κB. Taken together, the present study demonstrates that TLR4 is the gene product that regulates LPS response.
Cutting Edge: Species Specificity of the CC Chemokine 6Ckine Signaling Through the CXC Chemokine Receptor CXCR3: Human 6Ckine Is Not A Ligand for the Human or Mouse CXCR3 Receptors
Authors: Jenh Chung-Her , Cox Mary Ann , Kaminski Heather , Zhang Meng , Byrnes Heather , Fine Jay , Lundell Daniel , Chou Chuan-Chu , Narula Satwant K. , Zavodny Paul J.
Published: January 2023

The CC chemokine known as 6Ckine (SLC, Exodus-2, or TCA4) has been identified as a ligand for CCR7. Mouse 6Ckine has also been shown to signal through mouse CXCR3 and share some of the activities of IFN-γ inducible protein 10 and monokine induced by IFN-γ. Nonetheless, human 6Ckine has not been shown to bind CXCR3 receptor or have angiostatic activity. In this study, we report that human 6Ckine does not induce a calcium flux in either human CXCR3 or mouse CXCR3 transfected cells, although it is an equally potent agonist as mouse 6Ckine and human macrophage inflammatory protein-3β in human CCR7 transfected cells. Mouse 6Ckine (but not human 6Ckine) is capable of competing with radiolabeled IFN-γ inducible protein 10 for human CXCR3. In addition, radiolabeled human 6Ckine does not bind to either human CXCR3 or mouse CXCR3. Together these data suggest that human CC chemokine 6Ckine is not a ligand for the human or mouse CXC chemokine receptor CXCR3.
Mice with IFN-γ Receptor Deficiency Are Less Susceptible to Experimental Autoimmune Myasthenia Gravis
Authors: Zhang Guang-Xian , Xiao Bao-Guo , Bai Xue-Feng , van der Meide Peter H. , Örn Anders , Link Hans
Published: January 2023

IFN-γ can either adversely or beneficially affect certain experimental autoimmune diseases. To study the role of IFN-γ in the autoantibody-mediated experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis in humans, IFN-γR-deficient (IFN-γR−/−) mutant C57BL/6 mice and congenic wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus CFA. IFN-γR−/− mice exhibited significantly lower incidence and severity of muscle weakness, lower anti-AChR IgG Ab levels, and lower Ab affinity to AChR compared with wild-type mice. Passive transfer of serum from IFN-γR−/− mice induced less muscular weakness compared with serum from wild-type mice. In contrast, numbers of lymph node cells secreting IFN-γ and of those expressing IFN-γ mRNA were strongly augmented in the IFN-γR−/− mice, reflecting a failure of negative feedback circuits. Cytokine studies by in situ hybridization revealed lower levels of lymphoid cells expressing AChR-reactive IL-1β and TNF-α mRNA in AChR + CFA-immunized IFN-γR−/− mice compared with wild-type mice. No differences were found for AChR-reactive cells expressing IL-4, IL-10, or TGF-β mRNA. These results indicate that IFN-γ promotes systemic humoral responses in EAMG by up-regulating the production and the affinity of anti-AChR autoantibodies, thereby contributing to susceptibility to EAMG in C57BL/6-type mice.
Chemokine Receptor Responses on T Cells Are Achieved Through Regulation of Both Receptor Expression and Signaling
Authors: Rabin Ronald L. , Park Matthew K. , Liao Fang , Swofford Ruth , Stephany David , Farber Joshua M.
Published: January 2023

To address the issues of redundancy and specificity of chemokines and their receptors in lymphocyte biology, we investigated the expression of CC chemokine receptors CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4 and responses to their ligands on memory and naive, CD4 and CD8 human T cells, both freshly isolated and after short term activation in vitro. Activation through CD3 for 3 days had the most dramatic effects on the expression of CXCR3, which was up-regulated and functional on all T cell populations including naive CD4 cells. In contrast, the effects of short term activation on expression of other chemokine receptors was modest, and expression of CCR2, CCR3, and CCR5 on CD4 cells was restricted to memory subsets. In general, patterns of chemotaxis in the resting cells and calcium responses in the activated cells corresponded to the patterns of receptor expression among T cell subsets. In contrast, the pattern of calcium signaling among subsets of freshly isolated cells did not show a simple correlation with receptor expression, so the propensity to produce a global rise in the intracellular calcium concentration differed among the various receptors within a given T cell subset and for an individual receptor depending on the cell where it was expressed. Our data suggest that individual chemokine receptors and their ligands function on T cells at different stages of T cell activation/differentiation, with CXCR3 of particular importance on newly activated cells, and demonstrate T cell subset-specific and activation state-specific responses to chemokines that are achieved by regulating receptor signaling as well as receptor expression.
Reconciling Repertoire Shift with Affinity Maturation: The Role of Deleterious Mutations
Authors: Shannon Michele , Mehr Ramit
Published: January 2023

The shift in Ab repertoire, from Abs dominating certain primary B cell responses to genetically unrelated Abs dominating subsequent “memory” responses, challenges the accepted paradigm of affinity maturation. We used mathematical modeling and computer simulations of the dynamics of B cell responses, hypermutation, selection, and memory cell formation to test hypotheses attempting to explain repertoire shift. We show that repertoire shift can be explained within the framework of the affinity maturation paradigm, only when we recognize the destructive nature of hypermutation: B cells with a high initial affinity for the Ag are less likely to improve through random mutations.
Stable Peptide Binding to MHC Class II Molecule Is Rapid and Is Determined by a Receptive Conformation Shaped by Prior Association with Low Affinity Peptides
Authors: Natarajan Sateesh K. , Assadi Masoumeh , Sadegh-Nasseri Scheherazade
Published: January 2023

Formation of stable class II MHC/peptide complex involves conformational changes and proceeds via an intermediate. Although this intermediate complex forms and dissociates in minutes, its conversion to a stable complex is a very slow process, taking up to a few days to reach completion. Here, we investigate the different steps of this binding and demonstrate that the conformational changes necessary to generate a receptive molecule is the rate-determining slow step in the process, while formation of the stable MHC/peptide complex is very rapid. With HLA-DR1 as our model class II molecule, we first used low affinity variants of hemagglutinin peptide (HA306–318), which lack the principal anchor, to shape the conformation of the MHC and then studied the kinetics of stable binding of HA306–318 to such an induced conformation. We found that the apparent association rate of HA306–318 is equivalent to the dissociation rate of the low affinity peptide. A 4- to 18-fold enhancement in the binding rates of HA306–318 was observed depending on the dissociation rates of the low affinity peptides. These results establish that 1) formation of stable MHC/peptide complexes is very rapid and 2) prior binding of low affinity peptide induces a receptive conformation in MHC for efficient stable peptide binding. Furthermore, in the absence of any free peptide, this receptive molecule rapidly reverts to slow binding behavior toward the subsequently offered peptide. These results have important implications for the roles of low affinity MHC/peptide complexes in Ag presentation.
Inhibition of Matrix Metalloproteinases Prevents Allergen-Induced Airway Inflammation in a Murine Model of Asthma
Authors: Kumagai Katsunori , Ohno Isao , Okada Shinji , Ohkawara Yuichi , Suzuki Ko , Shinya Takashi , Nagase Hideaki , Iwata Kazushi , Shirato Kunio
Published: January 2023

Although matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components in vitro, the role of MMPs in the in vivo accumulation of the cells to the site of inflammation in bronchial asthma is still obscure. In this study, we investigated the role of MMPs in the pathogenesis of bronchial asthma, using a murine model of allergic asthma. In this model, we observed the increase of the release of MMP-2 and MMP-9 in bronchoalveolar lavage fluids after Ag inhalation in the mice sensitized with OVA, which was accompanied by the infiltration of lymphocytes and eosinophils. Administration of tissue inhibitor of metalloproteinase-2 to airways inhibited the Ag-induced infiltration of lymphocytes and eosinophils to airway wall and lumen, reduced Ag-induced airway hyperresponsiveness, and increased the numbers of eosinophils and lymphocytes in peripheral blood. The inhibition of cellular infiltration to airway lumen was observed also with tissue inhibitor of metalloproteinase-1 and a synthetic matrix metalloproteinase inhibitor. These data suggest that MMPs, especially MMP-2 and MMP-9, are crucial for the infiltration of inflammatory cells and the induction of airway hyperresponsiveness, which are pathophysiologic features of bronchial asthma, and further raise the possibility of the inhibition of MMPs as a therapeutic strategy of bronchial asthma.
Cutting Edge: Phorbol Ester Induction of IFN-γ Receptors Leads to Enhanced DRα Gene Expression
Authors: Sakamoto Shuji , Nie Jing , Taniguchi Taketoshi
Published: January 2023

We observed that IFN-γ-inducible expression of the DRα gene was enhanced when THP-1 cells are differentiated into macrophage-like cells by phorbol ester treatment. Here, we observed that class II MHC trans-activator and STAT1α mRNA, mediators of the signaling cascade from the IFN-γ receptor to the DRα induction, were markedly increased by IFN-γ stimulation in phorbol ester-activated THP-1 cells; however, both mRNAs were not increased by phorbol ester treatment alone. Then, we demonstrated that the mRNA and proteins of the IFN-γ receptor α- and β-chains were amplified by phorbol ester treatment in THP-1 cells. Consequently, these results indicate that the enhancement of DRα gene expression by IFN-γ treatment in phorbol ester-activated THP-1 cells is due to the phorbol ester-induced up-regulation of IFN-γ receptor α- and β-chains. As a result, the amplification of STAT1α and the increment of class II MHC trans-activator results in enhancement of DRα expression.
The Functional Synergy Between IL-12 and IL-2 Involves p38 Mitogen-Activated Protein Kinase and Is Associated with the Augmentation of STAT Serine Phosphorylation
Authors: Gollob Jared A. , Schnipper Claudia P. , Murphy Erin A. , Ritz Jerome , Frank David A.
Published: January 2023

IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-γ, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways.
CD28 Costimulation Is Crucial for the Development of Spontaneous Autoimmune Encephalomyelitis
Authors: Oliveira-dos-Santos Antonio J. , Ho Alexandra , Tada Yoshifumi , Lafaille Juan J. , Tonegawa Susumu , Mak Tak W. , Penninger Josef M.
Published: January 2023

Multiple sclerosis (MS) is a severe central nervous system disease. Experimental autoimmune encephalomyelitis (EAE) mimics MS in mice. We report that spontaneous development of EAE in RAG-1-deficient mice transgenic for a myelin basic protein (MBP)-specific TCR (TgMBP+/RAG-1−/−) requires expression of the T cell costimulatory molecule CD28. Surprisingly, T cells from CD28−/−TgMBP+/RAG-1−/− mice proliferate and produce IL-2 in response to MBP1–17 peptide in vitro, excluding clonal anergy as the mechanism of CD28-regulated pathogenesis. Proliferation of autoaggressive T cells was dependent on the concentration of the MBP peptide, as was the development of MBP-induced EAE in CD28-deficient PL/J mice. These results provide the first genetic evidence that CD28 costimulation is crucial for MBP-specific T cell activation in vivo and the initiation of spontaneous EAE.
Modulation of Allospecific CTL Responses During Pregnancy in Equids: An Immunological Barrier to Interspecies Matings?
Authors: Baker Jessica M. , Bamford Anona I. , Antczak D. F.
Published: January 2023

Maternal immune recognition of the developing conceptus in equine pregnancy is characterized by the strongest and most consistent alloantibody response described in any species, a response directed almost exclusively against paternal MHC class I Ags. This work investigated the cellular immune response to paternal MHC Ags in pregnant and nonpregnant horses and donkeys, and in horses carrying interspecies hybrid mule conceptuses. We observed profound decreases in classical, MHC-restricted, CTL activity to allogeneic paternal cells in peripheral blood lymphocytes from both horse mares and donkey jennets carrying intraspecies pregnancies, compared with cells from nonpregnant controls. This is the first evidence in a randomly bred species for a generalized systemic shift of immune reactivity away from cellular and toward humoral immunity during pregnancy. Surprisingly, mares carrying interspecies hybrid mule conceptuses did not exhibit this transient, pregnancy-associated decrease in CTL activity. The failure of interspecies pregnancy to down-regulate cellular immune responses may be a heretofore-unrecognized, subtle barrier to reproductive success between species.
Expression of CD1d2 on Thymocytes Is Not Sufficient for the Development of NK T Cells in CD1d1-Deficient Mice
Authors: Chen Yi-Hua , Wang Bin , Chun Taehoon , Zhao Lillian , Cardell Susanna , Behar Samuel M. , Brenner Michael B. , Wang Chyung-Ru
Published: January 2023

CD1 is an MHC class I-like molecule that has been conserved throughout mammalian evolution. Unlike MHC class I molecules, CD1 can present unique nonprotein antigens to T cells. The murine CD1 locus contains two highly homologous genes, CD1d1 and CD1d2. CD1d1 is essential for the development of a major subset of NK T cells that promptly secrete IL-4 following activation. However, the function of CD1d2 has not yet been demonstrated. In the present study, we examined the expression of CD1d2 in CD1d1-deficient (CD1d1°) mice with the anti-CD1 Ab 3H3. Unlike CD1d1, which is expressed by all lymphocytes, CD1d2 can be detected only on the surface of thymocytes. To determine whether CD1d2 can select a unique subset of NK T cells, we compared the remnant population of NK T cells in CD1d1° and CD1d1, CD1d2-double deficient (CD1d1°CD1d2°) mice. No significant difference in the number of NK T cells and cytokine secretion capacity can be detected between CD1d1° and CD1d1°CD1d2° mice, indicating that CD1d2 cannot substitute for CD1d1 in NK T cell development. The inability of CD1d2 to select NK T cells is not due to the structural constraints of CD1d2 since CD1d2-transfected cells can be recognized by both NK T cell hybridomas and freshly isolated NK T cells. Given the structural similarities, it is possible that the low levels of surface expression and limited tissue distribution of CD1d2 may prevent it from functioning in the selection and expansion of NK T cells.
Complex Conserved Organization of the Mammalian Leukemia Inhibitory Factor Gene: Regulated Expression of Intracellular and Extracellular Cytokines
Authors: Haines Bryan P. , Voyle Roger B. , Pelton Tricia A. , Forrest Regan , Rathjen Peter D.
Published: January 2023

Leukemia inhibitory factor (LIF) is a member of the IL-6 family of pleiotropic cytokines, which are extensively involved in modulating hematopoiesis and immunity. We have undertaken a detailed analysis of LIF genomic organization and gene transcription and investigated the proteins expressed from alternate transcripts. Previously unidentified LIF transcripts, containing alternate first exons spliced onto common second and third exons, were cloned from murine embryonic stem cells, human embryonal carcinoma cells, and primary porcine fibroblasts. Based on sequence homology and position within the genomic sequence, this confirmed the existence of the LIF-M transcript in species other than the mouse and identified a new class of transcript, designated LIF-T. Thus, a complex genomic organization of the LIF gene, conserved among eutherian mammals, results in the expression of three LIF transcripts (LIF-D, LIF-M, and LIF-T) differentially expressed from alternate promoters. The first exon of the LIF-T transcript contained no in-frame AUG, causing translation to initiate downstream of the secretory signal sequence at the first AUG in exon two, producing a truncated LIF protein that was localized within the cell. Enforced secretion of this protein demonstrated that it could act as a LIF receptor agonist. Regulated expression of biologically active intracellular and extracellular LIF cytokine could thus provide alternate mechanisms for the modulation of hematopoiesis and immune system function.
Dramatic Influence of Vβ Gene Polymorphism on an Antigen-Specific CD8+ T Cell Response In Vivo
Authors: Bour Hélène , Michielin Olivier , Bousso Philippe , Cerottini Jean-Charles , MacDonald H. Robson
Published: January 2023

According to recent crystallographic studies, the TCR-αβ contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1β (CDR1β) and the hypervariable (D)J-encoded CDR3β and CDR3α domains. To evaluate directly the relative importance of CDR1β polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic Vβa and Vβb mouse strains that differ by a CDR1 polymorphism in the Vβ10 gene segment. The Vβ10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in Vβa compared with Vβb mice, as measured either by the expansion of Vβ10+ cells or by the binding of MHC-peptide tetramers. These data indicate that Vβ polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1β-peptide interactions.
Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Prevent Inducible Nitric Oxide Synthase Transcription in Macrophages by Inhibiting NF-κB and IFN Regulatory Factor 1 Activation
Authors: Delgado Mario , Munoz-Elias Ernesto J. , Gomariz Rosa P. , Ganea Doina
Published: January 2023

High-output nitric oxide (NO) production from activated macrophages, resulting from the induction of inducible NO synthase (iNOS) expression, represents a major mechanism for macrophage cytotoxicity against pathogens. However, despite its beneficial role in host defense, sustained high-output NO production was also implicated in a variety of acute inflammatory diseases and autoimmune diseases. Therefore, the down-regulation of iNOS expression during an inflammatory process plays a significant physiological role. This study examines the role of two immunomodulatory neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), on NO production by LPS-, IFN-γ-, and LPS/IFN-γ-stimulated peritoneal macrophages and the Raw 264.7 cell line. Both VIP and PACAP inhibit NO production in a dose- and time-dependent manner by reducing iNOS expression at protein and mRNA level. VPAC1, the type 1 VIP receptor, which is constitutively expressed in macrophages, and to a lesser degree VPAC2, the type 2 VIP receptor, which is induced upon macrophage activation, mediate the effect of VIP/PACAP. VIP/PACAP inhibit iNOS expression and activity both in vivo and in vitro. Two transduction pathways appear to be involved, a cAMP-dependent pathway that preferentially inhibits IFN regulatory factor-1 transactivation and a cAMP-independent pathway that blocks NF-κB binding to the iNOS promoter. The down-regulation of iNOS expression, together with previously reported inhibitory effects on the production of the proinflammatory cytokines IL-6, TNF-α, and IL-12, and the stimulation of the anti-inflammatory IL-10, define VIP and PACAP as “macrophage deactivating factors” with significant physiological relevance.
A New Member of the Ig Superfamily and a V-ATPase G Subunit Are Among the Predicted Products of Novel Genes Close to the TNF Locus in the Human MHC
Authors: Neville Matt J. , Campbell R. Duncan
Published: January 2023

It is becoming increasingly apparent that many of the genes in the class III region of the human MHC encode proteins involved in the immune and inflammatory responses. Furthermore, genetic studies have indicated that genes within the class III region, particularly the telomeric segment containing the TNF gene, could contribute to susceptibility to diseases of immune-related etiology. We have sequenced an 82-kb segment of DNA around the TNF gene to identify candidate disease susceptibility genes in this region. The 10 known genes in this region have been precisely positioned with the order allograft inflammatory factor 1, G1, 1C7, leukocyte-specific transcript 1 (B144), lymphotoxin B, TNF, lymphotoxin A, NB6, IKBL, BAT1 (centromere to telomere), and their genomic structures have been defined. Comparison of the G1 genomic region with previously described cDNA and genomic sequences, together with the results of reverse transcriptase-PCR, indicates that three alternative transcripts, G1, allograft inflammatory factor 1, and IFN-γ-responsive transcript, are all derived from this gene. The completion of the sequence of 1C7 (D6S2570) has revealed that this gene encodes a putative novel member of the Ig superfamily. A number of alternatively spliced transcripts of 1C7 were identified by reverse transcriptase-PCR, all of which are expressed in immune-related cell lines. Alternative splicing within the Ig domain-encoding region was seen to result in possible set switching between an IgV domain and an IgC2 domain. Lastly, a previously unidentified gene, homologous to a number of V-ATPase G subunits, has been located 1 kb telomeric of IKBL.
The Origin and Function of Soluble CD14 in Experimental Bacterial Meningitis
Authors: Cauwels Anje , Frei Karl , Sansano Sebastiano , Fearns Colleen , Ulevitch Richard , Zimmerli Werner , Landmann Regine
Published: January 2023

Murine experimental meningitis models induced by either Escherichia coli LPS, live Streptococcus pneumoniae, or Listeria monocytogenes were used to study the origin and potential function of soluble CD14 (sCD14) in the brain during bacterial meningitis. Whereas intracerebral infection caused only a minor and/or transient increase of sCD14 levels in the serum, dramatically elevated concentrations of sCD14 were detected in the cerebrospinal fluid. Reverse-transcriptase PCR and FACS analysis of the leukocytes invading the subarachnoid compartment revealed an active amplification of CD14 transcription and concomitant surface expression. These findings were confirmed by in situ hybridization and immunohistochemical analysis. In contrast, parenchymal astrocytes and microglial cells were shown not to significantly contribute to the elevated levels of sCD14. Simultaneous intracerebral inoculation of rsCD14 and S. pneumoniae resulted in a markedly increased local cytokine response. Taken together, these data provide the first evidence that sCD14 can act as an inflammatory co-ligand in vivo. Thus, during bacterial meningitis, sCD14 is massively released by intrathecal leukocytes, and the sCD14 found in the cerebrospinal fluid can play an important role in the pathogenesis of this disease.
CD14 Plays No Major Role in Shock Induced by Staphylococcus aureus but Down-Regulates TNF-α Production
Authors: Haziot Alain , Hijiya Naoki , Schultz Karine , Zhang Fan , Gangloff Sophie C. , Goyert Sanna M.
Published: January 2023

Recent in vitro studies have suggested that CD14, a major receptor for LPS, may also be a receptor for cell wall components of Gram-positive bacteria and thus play a role in Gram-positive shock. To analyze the in vivo role of CD14 in responses to Gram-positive bacteria, CD14-deficient and control mice were injected with Staphylococcus aureus, and the effects on lethality, bacterial clearance, and production of cytokines were analyzed. Survival of CD14-deficient and control mice did not differ significantly after administration of various doses of either unencapsulated or encapsulated S. aureus; furthermore, mice in both groups displayed similar symptoms of shock. In addition, inflammatory cytokines such as TNF-α and IL-6 were readily detectable in the serum of CD14-deficient mice injected with live or antibiotic-killed S. aureus. Surprisingly, the serum concentration of TNF-α in CD14-deficient mice was at least threefold higher than in control mice after injection of either unencapsulated or encapsulated S. aureus, suggesting that CD14 down-regulates TNF-α. A similar increase in serum TNF-α occurred when CD14-deficient animals were injected with gentamicin-killed bacteria even though no symptoms of shock were observed. These studies indicate that CD14, in contrast to its key function in responses to the Gram-negative bacterium, Escherichia coli 0111, does not play a prominent role in septic shock induced by S. aureus, and that the symptoms of S. aureus shock are not due solely to TNF-α.
Inflammation Alone Evokes the Response of a TCR-Invariant Mouse γδ T Cell Subset
Authors: Mukasa Akiko , Born Willi K. , O’Brien Rebecca L.
Published: January 2023

Whether γδ T lymphocytes respond to microbial Ags or to inducible host Ags remains a matter of controversy. Using several different disease models and mouse strains, we and others have seen that Vγ6/Vδ1 γδ T cells preferentially increase among the γδ T cells infiltrating inflamed tissues. However, it was not clear whether bacteria are necessary to bring about this response. Therefore, we have reexamined this question using a disease model in which inflammation is induced by a purely autoimmune process involving no bacteria, bacterial products, or other foreign material: testicular cell-induced autoimmune orchitis. Using this model we found that γδ T cells were still plentiful among the infiltrating T lymphocytes, being 9- to 10-fold more prevalent than in spleen, and that Vγ6/Vδ1+ cells again represented the predominant γδ T cell type. This finding shows that the response of the Vγ6/Vδ1+ subset does not, in fact, depend upon the presence of bacteria or bacterial products. The stimulus triggering the response of the Vγ6/Vδ1 γδ T cells appears to be neither foreign nor organ-specific in origin, but instead consists of a self-derived host Ag or signal induced during the inflammatory process.
Administration of mAb Against αEβ7 Prevents and Ameliorates Immunization-Induced Colitis in IL-2−/− Mice
Authors: Lúdvíksson Björn R. , Strober Warren , Nishikomori Ryuta , Hasan Syed K. , Ehrhardt Rolf O.
Published: January 2023

We previously demonstrated that 2,4,6-trinitrophenol (TNP)-OVA immunization leads to a transmural colitis in the IL-2−/− mouse that is caused by IL-12-driven CD4+ Th1 T cells and resembles human Crohn’s disease. The integrin αEβ7 is highly expressed on colonic intraepithelial lymphocytes and has been suggested to function as a homing or retention molecule for intraepithelial lymphocytes. To evaluate the role of αEβ7 in colitis, we administered a mAb against αEβ7 to IL-2−/− mice that were immunized at the same time with TNP-OVA in CFA. To our surprise, this treatment resulted in a significantly reduced colitis severity score, 0–2 vs 3–4, that was associated with a significant reduction in CD4+ lamina propria lymphocyte subpopulation (p < 0.01). In contrast, the total number of splenic CD4+ T cells of treated animals was significantly elevated compared with that of untreated animals (3.2 ± 0.6 × 107 vs 1.2 ± 0.2 × 107; p < 0.05). Similarly, functional studies revealed that IFN-γ production by lamina propria lymphocytes isolated from IL-2−/− TNP-OVA-immunized mice treated with anti-αEβ7 was significantly lower than in untreated IL-2−/− TNP-OVA-immunized mice. In contrast, IFN-γ production by splenic cells isolated from treated IL-2−/− TNP-OVA-immunized mice was significantly higher than in untreated mice. Finally, TNP-OVA-immunized IL-2−/− mice that were treated after the colitis had been established also showed a significant decrease in mucosal inflammation after αEβ7 mAb administration. Thus, the above findings demonstrate that the onset and maintenance of inflammatory bowel disease depends on the colonic localization of lamina propria CD4+ lymphocytes expressing αEβ7.
Induction of Mucosal Immunity by Inactivated Poliovirus Vaccine Is Dependent on Previous Mucosal Contact with Live Virus
Authors: Herremans Tineke M. P. T. , Reimerink Johan H. J. , Buisman Annemarie M. , Kimman Tjeerd G. , Koopmans Marion P. G.
Published: January 2023

The inactivated poliovirus vaccine (IPV) is used for protection against poliomyelitis in The Netherlands. It is not clear, however, whether IPV vaccination can lead to priming of the mucosal immune system and the induction of IgA. It has been demonstrated that IPV vaccination is able to induce strong memory IgA responses in the serum of persons who have been naturally exposed to wild-type poliovirus. This has led to the hypothesis that IPV vaccination is able to induce poliovirus-specific IgA at mucosal sites in persons who have been previously primed with live poliovirus at mucosal sites. To test this hypothesis, the kinetics of the IgA response in serum and saliva after IPV vaccination were examined in persons previously vaccinated with oral poliovirus vaccine (OPV) or IPV. ELISA and enzyme-linked immunospot assays were used for the detection of poliovirus-specific IgA responses. In addition, B cell populations were separated on the basis of the expression of mucosal (α4β7 integrin) and peripheral homing receptors (L-selectin). Parenteral IPV vaccination was able to boost systemic and mucosal IgA responses in previously OPV-vaccinated persons only. None of the previously vaccinated IPV recipients responded with the production of IgA in saliva. In agreement with this finding, a large percentage of the poliovirus-specific IgA-producing lymphocytes detected in previous OPV recipients expressed the α4β7 integrin. It is concluded that IPV vaccination alone is insufficient to induce a mucosal IgA response against poliovirus. In mucosally (OPV-) primed individuals, however, booster vaccination with IPV leads to a strong mucosal IgA response.
Cutting Edge: Recognition of Gram-Positive Bacterial Cell Wall Components by the Innate Immune System Occurs Via Toll-Like Receptor 2
Authors: Yoshimura Atsutoshi , Lien Egil , Ingalls Robin R. , Tuomanen Elaine , Dziarski Roman , Golenbock Douglas
Published: January 2023

Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-κB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.
Invariant or Highly Conserved TCR α Are Expressed on Double-Negative (CD3+CD4−CD8−) and CD8+ T Cells
Authors: Han Mei , Harrison Lisa , Kehn Patricia , Stevenson Kimberly , Currier Jeffrey , Robinson Mary Ann
Published: January 2023

TCR α (TCRA) expression was examined in RNA samples from PBMC and isolated populations of CD4+, CD8+, and DN T cells from 15 healthy individuals. The expressed TCR repertoire was surveyed using spectratype analysis, a technique that displays the distribution of complementarity determining region 3 (CDR3) lengths for each TCRAV gene family. The results revealed the presence of unusual populations of double-negative (DN; CD4−CD8−CD3+) T cells that express invariant or conserved TCRAV4A, AV7, AV19, and AV24 chains. Each of the conserved TCRA families was over-represented in >70% of the individuals studied, and all individuals expressed at least one of the over-represented TCRAV families. Over-represented conserved AV4A or AV7 sequences were also present in CD8+ T cells from most donors. The extent of TCRA sequence conservation is unparalleled. TCRAV4A, AV19, and AV24 sequences were invariant, although AV4A and AV19 transcripts contained N region additions. TCRAV24 transcripts derived from the direct juxtaposition of V and J gene segments. TCRAV7 sequences showed some diversity in two amino acids encoded at junctions of V and J gene segments. Although derivation of DN T cells with conserved TCRA chains is puzzling, the wide-spread expression of these unusual cells suggests an important function.
Characterization of Eosinophil Adhesion to TNF-α-Activated Endothelium Under Flow Conditions: α4 Integrins Mediate Initial Attachment, and E-Selectin Mediates Rolling
Authors: Ulfman Laurien H. , Kuijper Philip H. M. , van der Linden Jan A. M. , Lammers Jan-Willem J. , Zwaginga Jaap Jan , Koenderman Leo
Published: January 2023

The multistep model of leukocyte adhesion reveals that selectins mediate rolling interactions and that integrins mediate firm adhesion processes. In this study, the interaction between eosinophils and TNF-α-activated HUVEC (second or third passage) was studied under flow conditions (0.8 and 3.2 dynes/cm2). Especially the role of α4 integrins on eosinophils and E-selectin on HUVEC was studied. Inhibition of the integrin α4 chain on eosinophils reduced the number of firmly adhered resting eosinophils to TNF-α-stimulated endothelium by 43% whereas the percentage rolling cells increased 2.2-fold compared with untreated control eosinophils. Blocking of E-selectin on the endothelium reduced the number of adherent eosinophils by only 23% and 16%. In this situation, however, hardly any rolling adhesion was observed, and the few rolling cells showed a low rolling velocity. Blocking both α4 integrin on eosinophils and E-selectin on HUVEC reduced the number of adhered eosinophils by 95%. P-selectin did not significantly participate in eosinophil adhesion to TNF-α-activated HUVEC. Inhibition of both α4 integrins and β2 integrins on eosinophils resulted in a reduction of adhered cells by 65% and a 3-fold increase in percentage rolling cells. Taken together, these results clearly show that resting eosinophils preferentially use constitutively active α4 integrins (α4β1, α4β7) for the first attachment to TNF-α-activated HUVEC. In addition, α4 integrins and E-selectin work synergistically in eosinophil adherence to TNF-α-activated HUVEC. Although E-selectin is important for eosinophil rolling under these conditions, P-selectin plays only a minor role.