bluuebunny/crossref_metadata_2025 huggingface dataset:
Negative Regulation by Phosphatidylinositol 3-Kinase of Inducible Nitric Oxide Synthase Expression in Macrophages
Authors: Díaz-Guerra María J. M. , Castrillo Antonio , Martín-Sanz Paloma , Boscá Lisardo
Published: January 2023

Triggering of the macrophage cell line RAW 264.7 with LPS promotes a transient activation of phosphatidylinositol 3-kinase (PI3-kinase). Incubation of activated macrophages with wortmannin and LY294002, two inhibitors of PI3-kinase, increased the amount of inducible nitric oxide synthase (iNOS) and the synthesis of nitric oxide. Treatment with wortmannin promoted a prolonged activation of NF-κB in LPS-treated cells as well as an increase in the promoter activity of the iNOS gene as deduced from transfection experiments using a 1.7-kb fragment of the 5′ flanking region of the iNOS gene. Cotransfection of cells with a catalytically active p110 subunit of PI3-kinase impaired the responsiveness of the iNOS promoter to LPS stimulation, whereas transfection with a kinase-deficient mutant of p110 maintained the up-regulation in response to wortmannin. These results indicate that PI3-kinase plays a negative role in the process of macrophage activation and suggest that this enzyme might participate in the mechanism of action of antiinflammatory cytokines.
Tyrphostin B42 Inhibits IL-12-Induced Tyrosine Phosphorylation and Activation of Janus Kinase-2 and Prevents Experimental Allergic Encephalomyelitis
Authors: Bright John J. , Du Caigan , Sriram Subramaniam
Published: January 2023

IL-12 is a macrophage-derived cytokine that induces proliferation, cytokine production, and cytotoxic activity of T and NK cells. Signaling through its receptor, IL-12 induces these cellular responses by tyrosine phosphorylation and activation of Janus kinase-2 (Jak-2), Tyk-2, Stat3, and Stat4. We have used tyrphostin B42 (AG490), a Jak-2 inhibitor, to determine the role of Jak-2 kinase in IL-12 signaling and IL-12-induced T cell functions. Treatment of activated T cells with tyrphostin B42 inhibited the IL-12-induced tyrosine phosphorylation and activation of Jak-2 without affecting Tyk-2 kinase. In contrast, treatment with tyrphostin A1 inhibited the tyrosine phosphorylation of Tyk-2 but not that of Jak-2 kinase. Inhibition of either Jak-2 or Tyk-2 leads to a decrease in the IL-12-induced tyrosine phosphorylation of Stat3, but not of Stat4, protein. While inhibition of Jak-2 lead to programmed cell death, the inhibition of Jak-2 or Tyk-2 resulted a decrease in IFN-γ production. We have further tested the in vivo effects of tyrphostin B42 in experimental allergic encephalomyelitis, a Th1 cell-mediated autoimmune disease. In vivo treatment with tyrphostin B42 decreased the proliferation and IFN-γ production of neural Ag-specific T cells. Treatment of mice with tyrphostin B42 also reduced the incidence and severity of active and passive EAE. These results suggest that tyrphostin B42 prevents EAE by inhibiting IL-12 signaling and IL-12-mediated Th1 differentiation in vivo.
Inflammatory Cytokines and HIV-1-Associated Neurodegeneration: Oncostatin-M Produced by Mononuclear Cells from HIV-1-Infected Individuals Induces Apoptosis of Primary Neurons
Authors: Ensoli Fabrizio , Fiorelli Valeria , DeCristofaro Maria , Santini Muratori Donatella , Novi Arianna , Vannelli Barbara , Thiele Carol J. , Luzi Giuseppe , Aiuti Fernando
Published: January 2023

Neurologic abnormalities are common in HIV-1-infected patients and often represent the dominant clinical manifestation of pediatric AIDS. The neurological dysfunction has been directly related to CNS invasion by HIV-1 that is principally, if not exclusively, supported by blood-derived monocytes/macrophages and lymphocytes. By using primary long term cultures of human fetal sensory neurons as well as sympathetic precursors-like neuronal cells, we determined that blood-derived mononuclear cells from HIV-1-infected individuals spontaneously release soluble mediators that can potently inhibit the growth and survival of developing neurons as well as the viability of postmitotic neuronal cells by inducing apoptotic cell death. Analysis of the cytokines produced by lymphomonocytic cells, HIV-1 infected or activated, indicated that oncostatin M (oncM) is a major mediator of these effects. Since low TGF-β1 concentrations were capable of enhancing oncM-mediated neuronal alterations, our data indicate that by acting in concert with other cytokines, oncM may induce neuronal demise in both the developing and the mature brain. Thus, this cytokine may contribute to the setting of the neuronal cell damage observed in HIV-1-infected individuals.
Highly Cross-Reactive T Cell Responses to Myelin Basic Protein Epitopes Reveal a Nonpredictable Form of TCR Degeneracy
Authors: Loftus Christine , Huseby Eric , Gopaul Priya , Beeson Craig , Goverman Joan
Published: January 2023

We identified two nonoverlapping epitopes in myelin basic protein presented by I-Au that are responsible for mediating tolerance induction to this self-Ag. A large number of T cells expressing diverse TCRs are strongly cross-reactive to both epitopes. Surprisingly, the TCR contact residues in each peptide are highly dissimilar. Furthermore, functional TCR contacts cannot be interchanged between the two epitopes, indicating that the TCR contacts in each peptide can only be recognized within the context of the other amino acids present in that peptide’s sequence. This observation indicates that both buried and exposed residues of each peptide contribute to the sculpting of completely distinct antigenic surfaces. We propose that the cross-reactive TCRs adopt mutually exclusive conformations to recognize these dissimilar epitopes, adding a new dimension to TCR degeneracy. This unpredictable TCR plasticity indicates that using just the TCR contacts on a single epitope to define other cross-reactive peptides will identify only a subset of the complete repertoire of cross-reactive epitopes.
Murine Cytomegalovirus Infection Down-Regulates MHC Class II Expression on Macrophages by Induction of IL-10
Authors: Redpath Stella , Angulo Ana , Gascoigne Nicholas R. J. , Ghazal Peter
Published: January 2023

Herpesviruses utilize many strategies for weakening the host immune response. For CMV, this includes avoidance of NK clearance and inhibition of MHC class I and class II presentation pathways. In this study, we report that mouse CMV (MCMV) specifically causes a premature and transient activation of host IL-10 very early in the course of infection, resulting in a dramatic and selective reduction in MHC class II surface expression. The expression of IL-10 is normally late in the immune response to a pathogen, serving to dampen the response by suppression of the production of inflammatory cytokines. In infection of macrophages, we show that MCMV induces the production of IL-10, leading to an early and selective reduction in the expression of MHC class II on the surface of the cells. Inhibition of MHC class II expression was not observed in the presence of neutralizing Abs to IL-10 or in macrophages from IL-10-deficient mice. Moreover, MCMV-infected IL-10-deficient mice developed an early and significantly more robust macrophage MHC class II induction than normal mice. Altogether, our results demonstrate that viral induction of an IL-10 autocrine pathway plays an essential early role in selectively reducing MHC class II expression on the surface of APC prior to stimulation by IFN-γ.
CD38-Mediated Signaling Events in Murine Pro-B Cells Expressing Human CD38 With or Without Its Cytoplasmic Domain
Authors: Kitanaka Akira , Suzuki Toshio , Ito Chikako , Nishigaki Hikari , Coustan-Smith Elaine , Tanaka Terukazu , Kubota Yoshitsugu , Campana Dario
Published: January 2023

To elucidate the signaling mechanism of CD38 (a transmembrane molecule highly expressed in immature hemopoietic cells), we transfected Ba/F3 murine pro-B cells with a cDNA encoding human CD38. CD38 ligation with anti-CD38 Abs caused rapid, transient, dose-dependent tyrosine phosphorylation of several proteins, including the tyrosine kinase TEC and the adaptor molecule CBL, and association of tyrosine-phosphorylated proteins with phosphatidylinositol 3-kinase p85. Exposure to anti-CD38 Abs or their F(ab′)2 and Fab also induced tight aggregation of CD38-transfected Ba/F3 cells, which appeared to be Ca2+ and Mg2+ independent and did not involved LFA-1. Aggregation was abrogated by addition of the tyrosine kinase inhibitor herbimycin A and was delayed by the phosphatidylinositol 3-kinase inhibitor wortmannin, suggesting a link between biochemical events and cellular effects induced by CD38. Cell aggregation was accompanied by a decrease in cell recovery. After 3 days of culture on bone marrow-derived stroma, the mean (±SD) cell recovery in the presence of anti-CD38 (T16) was 10.5 ± 9.2% (n = 7) of that in parallel cultures with an isotype-matched nonreactive Ab. Finally, CD38 ligation in Ba/F3 cells expressing a mutant human CD38 lacking the cytoplasmic domain induced tyrosine phosphorylation with intensity and kinetics similar to those seen with the entire protein. It also induced cell aggregation and decreased cell recovery. We conclude that CD38 triggers remarkably similar signaling pathways in human and murine immature B cells. This signaling is independent of the CD38 cytoplasmic domain, suggesting the existence of accessory transmembrane molecules associated with CD38.
Molecular Basis of Human Complement C1s Deficiency
Authors: Endo Yuichi , Kanno Kazuko , Takahashi Minoru , Yamaguchi Ken-ichi , Kohno Yoichi , Fujita Teizo
Published: January 2023

This is the first report on the molecular basis of human complement C1s deficiency. Two abnormalities in the C1s gene were identified in a Japanese family, including one patient, by using exon-specific PCR, single-strand conformation polymorphism analysis, and nucleotide sequencing. A deletion of 4 bp, TTTG, was identified in exon X when using genomic DNA from the patient, his father, and his paternal grandmother. They were all heterozygous for the mutation. The mutant gene encodes a truncated C1s from the N terminus to the short consensus repeat domain. By further sequencing the PCR products, a nonsense mutation from G to T was identified at codon 608 in exon XII in the patient, his mother, and his sister. They were all heterozygous for the nonsense mutation. The mutant gene encodes a truncated form of C1s that lacks the C-terminal 80 amino acids. These results indicate that the patient was a compound heterozygote with the 4-bp deletion on the paternal allele and the nonsense mutation on the maternal allele. The levels of serum C1s seem to be correlated to the genotypes of the C1s gene in which no C1s was detected in the patient, and one-half of the normal level in the family members who are heterozygous for either mutation. The present study demonstrates that the disease is inherited in an autosomal recessive mode.
Anti-KS: Identification of Autoantibodies to Asparaginyl-Transfer RNA Synthetase Associated with Interstitial Lung Disease
Authors: Hirakata Michito , Suwa Akira , Nagai Sonoko , Kron Michael A. , Trieu Edward P. , Mimori Tsuneyo , Akizuki Masashi , Targoff Ira N.
Published: January 2023

Autoantibodies to five of the aminoacyl-transfer RNA (tRNA) synthetases have been described, and each is associated with a syndrome of inflammatory myopathy with interstitial lung disease (ILD) and arthritis. Serum KS, from a patient with ILD and inflammatory arthritis without evidence of myositis, immunoprecipitated a tRNA that was distinct from that precipitated by any described anti-synthetase or other reported tRNA-related Abs, along with a protein of 65 kDa. KS serum and IgG fraction each showed significant (88%) inhibition of asparaginyl-tRNA synthetase (AsnRS) activity, but not of any of the other 19 aminoacyl-tRNA synthetase activities. Among 884 patients with connective tissue diseases tested, only two other sera were found to immunoprecipitate tRNAs and proteins of identical gel mobility. These two and KS showed identical immunodiffusion lines using HeLa cell extract. The new sera significantly inhibited AsnRS without significant effects on other synthetases tested. Both patients had ILD but neither had evidence of myositis. These data strongly suggest that these three sera have autoantibodies to AsnRS, representing a sixth anti-synthetase. Anti-KS was more closely associated with ILD than with myositis. Further study of this Abs might prove useful in dissecting the stimuli responsible for the genesis of anti-synthetase autoantibodies.
Determinant Spreading Associated with Demyelination in a Nonhuman Primate Model of Multiple Sclerosis
Authors: McFarland Hugh I. , Lobito Adrian A. , Johnson Michele M. , Nyswaner Jeffrey T. , Frank Joseph A. , Palardy Gregory R. , Tresser Nancy , Genain Claude P. , Mueller John P. , Matis Louis A. , Lenardo Michael J.
Published: January 2023

Definition of the immune process that causes demyelination in multiple sclerosis is essential to determine the feasibility of Ag-directed immunotherapy. Using the nonhuman primate, Callithrix jacchus jacchus (common marmoset), we show that immunization with myelin basic protein and proteolipid protein determinants results in clinical disease with significant demyelination. Demyelination was associated with spreading to myelin oligodendrocyte glycoprotein (MOG) determinants that generated anti-MOG serum Abs and Ig deposition in central nervous system white matter lesions. These data associate intermolecular “determinant spreading” with clinical autoimmune disease in primates and raise important issues for the pathogenesis and treatment of multiple sclerosis.
Recruitment of Pleckstrin and Phosphoinositide 3-Kinase γ into the Cell Membranes, and Their Association with Gβγ After Activation of NK Cells with Chemokines
Authors: Al-Aoukaty Ala , Rolstad Bent , Maghazachi Azzam A.
Published: January 2023

The role of phosphoinositide 3 kinases (PI 3-K) in chemokine-induced NK cell chemotaxis was investigated. Pretreatment of NK cells with wortmannin inhibits the in vitro chemotaxis of NK cells induced by lymphotactin, monocyte-chemoattractant protein-1, RANTES, IFN-inducible protein-10, or stromal-derived factor-1α. Introduction of inhibitory Abs to PI 3-Kγ but not to PI 3-Kα into streptolysin O-permeabilized NK cells also inhibits chemokine-induced NK cell chemotaxis. Biochemical analysis showed that within 2–3 min of activating NK cells, pleckstrin is recruited into NK cell membranes, whereas PI 3-Kγ associates with these membranes 5 min after stimulation with RANTES. Recruited PI 3-Kγ generates phosphatidylinositol 3,4,5 trisphosphate, an activity that is inhibited upon pretreatment of NK cells with wortmannin. Further analysis showed that a ternary complex containing the βγ dimer of G protein, pleckstrin, and PI 3-Kγ is formed in NK cell membranes after activation with RANTES. The recruitment of pleckstrin and PI 3-Kγ into NK cell membranes is only partially inhibited by pertussis toxin, suggesting that the majority of these molecules form a complex with pertussis toxin-insensitive G proteins. Our results may have application for the migration of NK cells toward the sites of inflammation.
Inhibition of Inducible Nitric Oxide Synthase Exacerbates Chronic Cerebral Toxoplasmosis inToxoplasma gondii-Susceptible C57BL/6 Mice But Does Not Reactivate the Latent Disease inT. gondii-Resistant BALB/c Mice
Authors: Schlüter Dirk , Deckert-Schlüter Martina , Lorenz Elke , Meyer Timothy , Röllinghoff Martin , Bogdan Christian
Published: January 2023

Infection of C57BL/6 mice with Toxoplasma gondii leads to progressive and ultimately fatal chronic Toxoplasma encephalitis (TE). Genetic deletion or inhibition of inducible nitric oxide synthase (iNOS) from the beginning of infection increased the number of T. gondii cysts in the brain and markedly reduced the time-to-death in this mouse strain. In the present study, we addressed whether iNOS also contributes to the control of intracerebral parasites in a clinically stable latent infection that develops in T. gondii-resistant BALB/c mice after resolution of the acute phase of TE. iNOS was expressed in the inflammatory cerebral infiltrates of latently infected BALB/c mice, but the number of iNOS+ cells was significantly lower than in the brains of chronically infected T. gondii-susceptible C57BL/6 mice. In BALB/c mice with latent TE (>30 days of infection), treatment with the iNOS inhibitors l-N6-iminoethyl-lysine or l-nitroarginine-methylester for ≤40 days did not result in an increase of the intracerebral parasitic load and a reactivation of the disease, despite the presence of iNOS-suppressive inhibitor levels in the brain. However, l-nitroarginine-methylester treatment had remarkably toxic effects and induced a severe wasting syndrome with high mortality. In contrast to BALB/c mice, l-N6-iminoethyl-lysine treatment rapidly exacerbated the already established chronic TE of C57BL/6 mice. Thus, the containment of latent toxoplasms in T. gondii-resistant BALB/c mice is independent of iNOS, whereas the temporary control of intracerebral parasites in T. gondii-susceptible C57BL/6 mice with chronic TE requires iNOS activity.
Glucocorticoids Promote Nonphlogistic Phagocytosis of Apoptotic Leukocytes
Authors: Liu Yuqing , Cousin Joanne M. , Hughes Jeremy , Van Damme Jo , Seckl Jonathan R. , Haslett Christopher , Dransfield Ian , Savill John , Rossi Adriano G.
Published: January 2023

Phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis promote the resolution of inflammation. This study assessed the effects of anti-inflammatory glucocorticoids on this leukocyte clearance mechanism. Pretreatment of “semimature” 5-day human monocyte-derived macrophages (Mφ) for 24 h with methylprednisolone, dexamethasone, and hydrocortisone, but not the nonglucocorticoid steroids aldosterone, estradiol, and progesterone, potentiated phagocytosis of apoptotic neutrophils. These effects were specific in that the potentiated phagocytosis of apoptotic neutrophils was completely blocked by the glucocorticoid receptor antagonist RU38486, and glucocorticoids did not promote 5-day Mφ ingestion of opsonized erythrocytes. Similar glucocorticoid-mediated potentiation was observed with 5-day Mφ uptake of alternative apoptotic “targets” (eosinophils and Jurkat T cells) and in uptake of apoptotic neutrophils by alternative phagocytes (human glomerular mesangial cells and murine Mφ elicited into the peritoneum or derived from bone marrow). Importantly, methylprednisolone-mediated enhancement of the uptake of apoptotic neutrophils did not trigger the release of the chemokines IL-8 and monocyte chemoattractant protein-1. Furthermore, longer-term potentiation by methylprednisolone was observed in maturing human monocyte-derived Mφ, with greater increases in 5-day Mφ uptake of apoptotic cells being observed the earlier glucocorticoids were added during monocyte maturation into Mφ. We conclude that potentiation of nonphlogistic clearance of apoptotic leukocytes by phagocytes is a hitherto unrecognized property of glucocorticoids that has potential implications for therapies aimed at promoting the resolution of inflammatory diseases.
Increased Generation of CD8+ T Cell Clones in p53 Mutant Mice
Authors: Zhou Xianzheng , Wong Stacey , Walter Jurgen , Jacks Tyler , Eisen Herman N.
Published: January 2023

Very few cultured CD8+ T cell clones can normally be obtained from a single mouse and maintained in long-term culture. To improve the yield, we immunized p53 mutant mice with peptides of Sendai virus (FAPGNYPAL) and influenza virus (ASNENMETM) origin. Substantially more clones could be derived from p53−/− mice than from similarly treated wild-type mice (p53+/+); an intermediate yield was obtained from heterozygous mice (p53+/−). CTL lines or clones from p53−/− mice exhibited greater proliferative activity and resistance to γ-irradiation than those from p53+/+ mice, and were cytolytically potent.
Gene Conversion and Hypermutation During Diversification of VH Sequences in Developing Splenic Germinal Centers of Immunized Rabbits
Authors: Schiaffella Enrico , Sehgal Devinder , Anderson Arthur O. , Mage Rose G.
Published: January 2023

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine γ-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.
Cloning and Modeling of the First Nonmammalian CD4
Authors: Koskinen Riitta , Lamminmäki Urpo , Tregaskes Clive A. , Salomonsen Jan , Young John R. , Vainio Olli
Published: January 2023

We have cloned and sequenced the first nonmammalian CD4 cDNA from the chicken using the COS cell expression method. Chicken CD4 contains four extracellular Ig domains that, in analogy to mammalian CD4, are in the order V, C2, V, and C2. The molecule is 24% identical with both human and mouse sequences. The extracellular domains were modeled using human and rat CD4 crystal structures as templates. In the first domain there are two extra Cys residues that are at suitable distance to form an intra-β-sheet disulfide bridge in addition to the canonical one in the V domain. The region responsible for the interaction with MHC class II is relatively nonconserved in chicken. However, there are positively charged amino acids in the C″ region of the N-terminal domain that may mediate the association to the negatively charged residues of the MHC class II β-chain. Molecular modeling also implies that the membrane-proximal domain mediates dimerization of chicken CD4 in a similar way as it does for human CD4. Furthermore, the cytoplasmic tail is highly conserved, containing the protein tyrosine kinase p56lck recognition site that is preceded by an adjacent di-leucine motif for the internalization of the molecule. Interestingly, there are no Ser residues in the cytoplasmic part, which may explain the slow down-regulation of chicken CD4 after phorbol ester stimulation.
Regulation of Cytosolic COX-2 and Prostaglandin E2 Production by Nitric Oxide in Activated Murine Macrophages
Authors: Patel Rajesh , Attur Mukundan G. , Dave Mandar , Abramson Steven B. , Amin Ashok R.
Published: January 2023

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and ∼10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 × g indicates that the COX-2 is distributed both in the 100,000 × g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor l-NMMA at concentrations that inhibit nitrite accumulation by ≤80% is inadequate to augment PGE2 production. However, inhibition of nitrite accumulation by ≥85% with higher concentrations of l-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 × g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10–15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.
Ligation of FcγRII (CD32) Pivotally Regulates Survival of Human Eosinophils
Authors: Kim Jin-Tack , Schimming Andrew W. , Kita Hirohito
Published: January 2023

The low-affinity IgG Fc receptor, FcγRII (CD32), mediates various effector functions of lymphoid and myeloid cells and is the major IgG Fc receptor expressed by human eosinophils. We investigated whether FcγRII regulates both cell survival and death of human eosinophils. When cultured in vitro without growth factors, most eosinophils undergo apoptosis within 96 h. Ligation of FcγRII by anti-CD32 mAb in solution inhibited eosinophil apoptosis and prolonged survival in the absence of growth factors. Cross-linking of human IgG bound to FcγRII by anti-human IgG Ab or of unoccupied FcγRII by aggregated human IgG also prolonged eosinophil survival. The enhanced survival with anti-CD32 mAb was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated survival. In fact, mRNA for GM-CSF was detected in eosinophils cultured with anti-CD32 mAb. In contrast to mAb or ligands in solution, anti-CD32 mAb or human IgG, when immobilized onto tissue culture plates, facilitated eosinophil cell death even in the presence of IL-5. Cell death induced by these immobilized ligands was accompanied by DNA fragmentation and was inhibited when eosinophil β2 integrin was blocked by anti-CD18 mAb, suggesting that β2 integrins play a key role in initiating eosinophil apoptosis. Thus, FcγRII may pivotally regulate both survival and death of eosinophils, depending on the manner of receptor ligation and β2 integrin involvement. Moreover, the FcγRII could provide a novel mechanism to control the number of eosinophils at inflammation sites in human diseases.
Augmentation of Type I IL-1 Receptor Expression and IL-1 Signaling by IL-6 and Glucocorticoid in Murine Hepatocytes
Authors: Ito Atsushi , Takii Takemasa , Matsumura Takayuki , Onozaki Kikuo
Published: January 2023

IL-1 signal is transduced through type I receptor (IL-1RI). We have recently reported that LPS augments IL-1RI mRNA expression in the hepatocytes of mice in vivo, and the augmentation is mediated by the interaction of IL-1, IL-6, and glucocorticoid (GC). In this study, we examined whether IL-1RI mRNA expression level in the hepatocytes reflects those of cell surface molecule and IL-1 signaling. When primary cultured murine hepatocytes were treated with dexamethasone (Dex) or IL-6, these two reagents synergistically up-regulated IL-1RI mRNA expression in the cells. 125I-labeled IL-1 binding experiment showed that the level of binding was also up-regulated by the treatment with Dex and IL-6. Scatchard analysis revealed that the number of IL-1R increased. The increased binding of IL-1 was completely inhibited by an Ab against murine IL-1RI, indicating that Dex and IL-6 augmented the expression of cell surface IL-1RI molecule. When hepatocytes were pretreated with Dex and IL-6, the activation of IL-1R-associated kinase was augmented in response to IL-1, indicating that IL-1 signaling was also augmented. In addition, IL-1 treatment following administration of the combination of Dex and IL-6 into mice markedly increased the serum level of serum amyloid A. These results indicate that GC and IL-6 augment the expression of cell surface IL-1RI in hepatocytes, as well as IL-1 signaling and IL-1R-associated kinase activation, through up-regulation of IL-1RI mRNA level, which represents a novel regulatory network between IL-1, GC, and IL-6.
Evidence of Alternative or Concomitant Use of Perforin- and Fas-Dependent Pathways in a T Cell-Mediated Negative Regulation of Ig Production
Authors: Majlessi Laleh , Bordenave Guy
Published: January 2023

To study the possible involvement of perforin (Pfp)- and/or Fas-dependent cytotoxicity pathways in a T cell-mediated negative regulation of Ig production, we used the T cell-induced Ig-allotype suppression model. T splenocytes from Igha/a mice, when neonatally transferred into histocompatible Igha/b F1 or Ighb/b congenic hosts, are intrinsically able to totally, specifically, and chronically suppress the production of IgG2a of the Ighb haplotype (IgG2ab). It has not been established whether the suppression effectors, which are anti-IgG2ab MHC class I-restricted CD8+ T cells, cytolyse IgG2ab+ B targets or whether they only silence Ig production. In this study, using T cells from Igha/a Pfp+/+ or Pfpo/o mice, the latter obtained by crossbreeding, and B cells from Ighb/b Fas+/+ or Faslpr/lpr (lymphoproliferation) mice in appropriate adoptive transfer models, we demonstrated that: 1) under blockage of the Pfp-mediated pathway, Igha/a T cells were still able to induce suppression against wild-type IgG2ab+ B cells, 2) IgG2ab+ B cells with impaired Fas expression were also subjected to suppression by WT Igha/a T splenocytes, and 3) the suppression establishment was totally inhibited when both Pfp- and Fas-dependent mechanisms were simultaneously blocked, i.e., when Igha/a Pfpo/o T cells were used to induce suppression against Ighb/b Faslpr/lpr B cells. These results provide the first demonstration of the existence of alternative or simultaneous use of the major cytotoxic mechanisms in a T cell-mediated down-regulation of an Ig production.
Phosphorylation of CD19 Y484 and Y515, and Linked Activation of Phosphatidylinositol 3-Kinase, Are Required for B Cell Antigen Receptor-Mediated Activation of Bruton’s Tyrosine Kinase
Authors: Buhl Anne Mette , Cambier John C.
Published: January 2023

Bruton’s tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD19 mutant in which tyrosine residues previously shown to mediate CD19 interaction with PI3-K, Y484 and Y515, were changed to phenylalanine. Taken together, the data presented indicate that BCR aggregation-driven CD19 phosphorylation functions to promote Btk activation via recruitment and activation of PI3-K. Resultant phosphatidylinositol 3,4,5-trisphosphate probably functions to localize Btk for subsequent phosphorylation and activation by Src and Syk family kinases.
TGF-β1 Prevents the Noncognate Maturation of Human Dendritic Langerhans Cells
Authors: Geissmann Frederic , Revy Patrick , Regnault Armelle , Lepelletier Yves , Dy Michel , Brousse Nicole , Amigorena Sebastian , Hermine Olivier , Durandy Anne
Published: January 2023

TGF-β1 is critical for differentiation of epithelial-associated dendritic Langerhans cells (LC). In accordance with the characteristics of in vivo LC, we show that LC obtained from human monocytes in vitro in the presence of TGF-β1 1) express almost exclusively intracellular class II Ags, low CD80, and no CD83 and CD86 Ags and 2) down-regulate TNF-RI (p55) and do not produce IL-10 after stimulation, in contrast to dermal dendritic cells and monocyte-derived dendritic cells. Surprisingly, while LC exhibit E-cadherin down-regulation upon exposure to TNF-α and IL-1, TGF-β1 prevents the final LC maturation in response to TNF-α, IL-1, and LPS with respect to Class II CD80, CD86, and CD83 Ag expression, loss of FITC-dextran uptake, production of IL-12, and Ag presentation. In sharp contrast, CD40 ligand cognate signal induces full maturation of LC and is not inhibited by TGF-β1. The presence of emigrated immature LCs in human reactive skin-draining lymph nodes provides in vivo evidence that LC migration and final maturation may be differentially regulated.

Therefore, due to the effects of TGF-β1, inflammatory stimuli may not be sufficient to induce full maturation of LC, thus avoiding potentially harmful immune responses. We conclude that TGF-β1 appears to be responsible for both the acquisition of LC phenotype, cytokine production pattern, and prevention of noncognate maturation.
Two Independent Calcineurin-Binding Regions in the N-Terminal Domain of Murine NF-ATx1 Recruit Calcineurin to Murine NF-ATx1
Authors: Liu Jie , Masuda Esteban S. , Tsuruta Lisako , Arai Naoko , Arai Ken-ichi
Published: January 2023

Intracellular calcium regulates events controlling nuclear translocation of nuclear factor of activated T cells (NF-AT). Calcium-dependent phosphatase calcineurin (CN) plays a central role in this process. Structural and functional analyses of the N-terminal domain of murine NF-ATx1, a member of the NF-AT family, have defined two distinct CN binding regions (CNBRs), CNBR1 and CNBR2, which are located in the region preceding the SP boxes of serine/proline-rich sequences and the region between the SP boxes and Rel similarity domain, respectively. The binding of murine NF-ATx1 (mNF-ATx1) to CN was abolished by deletion of these two regions, yet was unaffected by the individual deletion. In contrast, the nuclear translocation of mNF-ATx1 was much reduced when only CNBR2 was removed. Luciferase assay revealed that both regions are required for mNF-ATx1-dependent activation of the murine IL-2 promoter. Most importantly, recombinant CNBR2 bound CN with a higher affinity, and when expressed in Jurkat cells, it functioned as a dominant negative mutant that prevented the transcription driven by exogenous mNF-ATx1, probably by interfering with the function of CN. We propose that activation of mNF-ATx1 can be modulated through two distinct CN target regions. Our findings provide a new opportunity for pharmacological intervention with Ca2+-dependent signaling events.
Actinobacillus actinomycetemcomitans Immunosuppressive Protein Is a Member of the Family of Cytolethal Distending Toxins Capable of Causing a G2 Arrest in Human T Cells
Authors: Shenker Bruce J. , McKay Terry , Datar Sugandha , Miller Mark , Chowhan Rohini , Demuth Donald
Published: January 2023

We have previously shown that Actinobacillus actinomycetecomitans produces an immunosuppressive factor (ISF) capable of impairing human lymphocyte function by perturbing cell cycle progression. We now report that ISF is the product of the cdtB gene, one of three genes encoding the family of cytolethal distending toxins (Cdt). The ISF polypeptide exhibits ≥95% identity with Hemophilus ducreyi CdtB protein and ≤60% homology with Escherichia coli or Campylobacter jejuni CdtB. Pretreatment of PHA-activated lymphocytes with 5–25 ng ISF results in G2 arrest of CD4+ and CD8+ T cells. Similarly, treatment of HeLa cells results in G2 arrest and cell elongation and distension. However, lymphocytes are at least 5 times more sensitive to ISF than HeLa cells and do not undergo the elongation and distension that characterizes interactions of Cdts with cell lines. ISF-treated lymphocytes express normal cyclin A and B1 levels, but contain reduced levels of cell cycle-dependent kinase-1 (Cdk1). Additionally, the majority of Cdk1 is in the hyperphosphorylated, inactive, form. In contrast, PHA-induced G2 cells contain elevated levels of the hypophosphorylated, active Cdk1. Failure of ISF-treated cells to dephosphorylate Cdk1 is not associated with decreased availability of Cdc25. These studies suggest that the CdtB protein alone is capable of inducing G2 arrest in lymphocytes and cell cycle arrest, elongation, and distension of HeLa cells. Our studies also suggest that lymphocytes may be primary targets for A. actinomycetemcomitans CdtB (ISF) and possibly for other Cdt family members as well. Thus, Cdts may function to impair host immunity and contribute to the pathogenesis of disease associated with Cdt-producing organisms.
CD8+ T Cells Become Nonresponsive (Anergic) Following Activation in the Presence of Costimulation
Authors: Deeths Matthew J. , Kedl Ross M. , Mescher Matthew F.
Published: January 2023

CD8+ T cells stimulated in vitro with anti-TCR mAb and B7-1 or ICAM-1 produce IL-2 and clonally expand. Effector function is acquired within 3 days, but proliferation ceases and the cells begin to die by apoptosis. Stimulation in vivo with B7-1-expressing allogeneic tumor results in the same sequence of events with a comparable time course. In both cases, the cells become anergic within 3 or 4 days of responding; they can no longer respond by producing IL-2 and proliferating, but can still be stimulated to proliferate in response to exogenous IL-2. This activation-induced nonresponsiveness (AINR) is not simply a consequence of ongoing cell death; cytokines that promote survival (IL-7 or IFN-α) or proliferation (human IL-2) do not restore the ability to produce IL-2 in response to costimulation. Although similar to the anergy described for CD4+ T cell clones, AINR differs in that it results from an initial stimulation with both signal 1 and signal 2. AINR appears to be an aspect of the normal differentiation of fully stimulated CD8+ T cells. It is probably important in regulating CTL responses; it limits the initial T helper-independent response and converts it to a response that requires T cell help to be sustained and further expanded. When the initial helper-independent response is not sufficient to clear Ag, and if help is not available, AINR likely results in tolerance to the Ag.
Bacterial DNA or Oligonucleotides Containing Unmethylated CpG Motifs Can Minimize Lipopolysaccharide-Induced Inflammation in the Lower Respiratory Tract Through an IL-12-Dependent Pathway
Authors: Schwartz David A. , Wohlford-Lenane Christine L. , Quinn Timothy J. , Krieg Arthur M.
Published: January 2023

To determine whether the systemic immune activation by CpG DNA could alter airway inflammation, we pretreated mice with either i.v. bacterial DNA (bDNA) or oligonucleotides with or without CpG motifs, exposed these mice to LPS by inhalation, and measured the inflammatory response systemically and in the lung immediately following LPS inhalation. Compared with non-CpG oligonucleotides, i.v. treatment with CpG oligonucleotides resulted in higher systemic concentrations of polymorphonuclear leukocytes, IL-10, and IL-12, but significantly reduced the concentration of total cells, polymorphonuclear leukocytes, TNF-α, and macrophage inflammatory protein-2 in the lavage fluid following LPS inhalation. The immunoprotective effect of CpG-containing oligonucleotides was dose-dependent and was most pronounced in mice pretreated between 2 and 4 h before the inhalation challenge, corresponding to the peak levels of serum cytokines. bDNA resulted in a similar immunoprotective effect, and methylation of the CpG motifs abolished the protective effect of CpG oligonucleotides. The protective effect of CpG oligonucleotides was observed in mice with either a disrupted IL-10 or IFN-γ gene, but release of cytokines in the lung was increased, especially in the mice lacking IFN-γ. In contrast, CpG DNA did not protect mice with a disrupted IL-12 gene against the LPS-induced cellular influx, even though CpG DNA reduced the release of TNF-α and macrophage inflammatory protein-2 in the lung. These findings indicate that CpG-containing oligonucleotides or bDNA are protected against LPS-induced cellular airway inflammation through an IL-12-dependent pathway, and that the pulmonary cytokine and cellular changes appear to be regulated independently.
The Specificity of a Weak γδ TCR Interaction Can Be Modulated by the Glycosylation of the Ligand
Authors: Hampl Johannes , Schild Hansjörg , Litzenberger Christa , Baron Miriam , Crowley Michael P. , Chien Yueh-hsiu
Published: January 2023

The γδ T cell clone LBK5 recognizes the MHC molecule IEk. Here, we demonstrate that the affinity of this interaction is weaker than those typically reported for αβ TCRs that recognize peptide/MHC complexes. Consistent with our previous finding that peptide bound to the IE molecule does not confer specificity, we show that the entire epitope for LBK5 is contained within the polypeptide chains of the molecule, centered around the polymorphic residues 67 and 70 of the IE β-chain. However, LBK5 recognition is profoundly influenced by the N-linked glycosylation at residue 82 of the IE α-chain. Since infected, stressed, or transformed cells often change the posttranslational modifications of their surface glycoproteins, this finding suggests a new way in which γδ T cell Ag recognition can be regulated.
Protective Immunity from Naive CD8+ T Cells Activated In Vitro with MHC Class I Binding Immunogenic Peptides and IL-2 in the Absence of Specialized APCs
Authors: Hauser Conrad , Zipprich Frank , Leblond Isabelle , Wirth Susanne , Hügin Ambros W.
Published: January 2023

Ag-specific CTL can protect against tumors and some viral infections and may be useful for adoptive immunotherapy. Here, we show that purified CD8+ T cells from naive C57BL/6 mice can be primed in vitro with different immunogenic peptides, which bind to MHC class I gene products, and IL-2 to exhibit specific and MHC-restricted effector function in vitro and in vivo protection against lymphocytic choriomeningitis virus infection and B16.F10 melanoma lung metastases. Limiting dilution assays in the absence of feeder cells with highly purified CD8+ T cells from two transgenic mice strains, each expressing a different MHC class I-restricted TCR, indicated that only peptide and IL-2, but not TCR− cells, were required for the growth of naive CD8+ T cells. These alternative minimal requirements for the activation and expansion of specific CD8+ T lymphocytes, without the need for professional APC, may be exploited for adoptive immunotherapy.
Involvement of Thioredoxin in Rheumatoid Arthritis: Its Costimulatory Roles in the TNF-α-Induced Production of IL-6 and IL-8 from Cultured Synovial Fibroblasts
Authors: Yoshida Shinichi , Katoh Tetsuji , Tetsuka Toshifumi , Uno Kazuko , Matsui Nobuo , Okamoto Takashi
Published: January 2023

Thioredoxin (TRX) is a cellular reducing catalyst induced by oxidative stress and is involved in the redox regulation of transcription factors such as NF-κB. We found that the serum TRX concentration was elevated in patients with rheumatoid arthritis (RA) as compared with values from healthy individuals and patients with osteoarthritis (33.6 ± 35.1 vs 11.8 ± 6.6 ng/ml, p < 0.01). Moreover, the TRX concentration in the synovial fluid (SF) was much more elevated in RA patients than in osteoarthritis patients (103.4 ± 53.3 vs 24.6 ± 17.4 ng/ml, p < 0.001). Multiple regression analysis revealed that the serum C-reactive protein value was better correlated with the linear combination of SF TNF-α and SF TRX values than with SF TNF-α alone, suggesting that TRX might play a subsidiary role in the rheumatoid inflammation. We thus examined the effect of TRX on the TNF-α-induced IL-6 and IL-8 production using rheumatoid synovial fibroblast cultures. The extents of IL-6 and IL-8 production in response to TNF-α were greatly augmented by TRX as compared with TNF-α alone. TRX alone did not have such effects. We also found that TRX appeared to accelerate the nuclear translocation of NF-κB, a major transcriptional regulator for production of IL-6 and IL-8 on stimulation with TNF-α. Consistent with these findings, the IκBα phosphorylation at Ser32 and its subsequent degradation in response to TNF-α was facilitated by TRX. These findings indicate that the elevated TRX concentration in SF of RA patients might be involved in the aggravation of rheumatoid inflammation by augmenting the NF-κB activation pathway.
Osteoclast Differentiation Factor Acts as a Multifunctional Regulator in Murine Osteoclast Differentiation and Function
Authors: Jimi Eijiro , Akiyama Shuichi , Tsurukai Taro , Okahashi Nobuo , Kobayashi Kanichiro , Udagawa Nobuyuki , Nishihara Tatsuji , Takahashi Naoyuki , Suda Tatsuo
Published: January 2023

Osteoclast differentiation factor (ODF), a novel member of the TNF ligand family, is expressed as a membrane-associated protein by osteoblasts/stromal cells. The soluble form of ODF (sODF) induces the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here, the effects of sODF on the survival, multinucleation, and pit-forming activity of murine osteoclasts were examined in comparison with those of M-CSF and IL-1. Osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts and bone marrow cells expressed mRNA of RANK (receptor activator of NF-κB), a receptor of ODF. The survival of OCLs was enhanced by the addition of each of sODF, M-CSF, and IL-1. sODF, as well as IL-1, activated NF-κB and c-Jun N-terminal protein kinase (JNK) in OCLs. Like M-CSF and IL-1, sODF stimulated the survival and multinucleation of prefusion osteoclasts (pOCs) isolated from the coculture. When pOCs were cultured on dentine slices, resorption pits were formed on the slices in the presence of either sODF or IL-1 but not in that of M-CSF. A soluble form of RANK as well as osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF, blocked OCL formation and prevented the survival, multinucleation, and pit-forming activity of pOCs induced by sODF. These results suggest that ODF regulates not only osteoclast differentiation but also osteoclast function in mice through the receptor RANK.
In Situ T Cell Responses Against Melanoma Comprise High Numbers of Locally Expanded T Cell Clonotypes
Authors: thor Straten Per , Guldberg Per , Grønbæk Kirsten , Hansen Mia Riise , Kirkin Alexei F. , Seremet Tina , Zeuthen Jesper , Becker Jürgen C.
Published: January 2023

It is well established that melanoma cells express Ags that are recognized by autologous T cells in vitro. Tumor-infiltrating lymphocytes in situ comprise clonotypic T cells, suggesting that their expansion is driven by Ag stimulation. Still, little is known about the detailed characteristics of the in situ T cell response. In the present study, we scrutinized this response by analyzing multiple metastatic lesions for the presence of clonotypic T cells. This approach was chosen to distinguish whether the clonal T cell expansion occurs as a systemic or localized phenomenon. TCR clonotype mapping of six s.c. metastases from two patients revealed the presence of multiple (from 40 to >60) clonotypic T cells in all lesions. Clonotypic T cells were present in TCR β-variable regions expressed both at high and low levels. Comparison of the T cell clonotypes present in different lesions from individual patients demonstrated that, in general, clonotypes were exclusively detected in a single lesion. Hence, anti-melanoma T cell responses are much more heterogeneous than previously anticipated and accommodate a predominance of strictly localized T cell clonotypes.
Increased Apoptosis in Patients with Major Depression: A Preliminary Study
Authors: Eilat Eran , Mendlovic Shlomo , Doron Adiel , Zakuth Vera , Spirer Zvi
Published: January 2023

Apoptosis is a programmed cell death that can be observed in normal cells. Major depression poses a combination of a depressed and destructive autoimmune reaction. We measured apoptosis in the PBLs of seven patients with major depression and in age- and sex-matched controls. We observed significantly increased apoptosis in the PBLs of depressive patients (p < 0.05). These preliminary results could contribute to an understanding of the interactions of the CNS with the immune system, which could lead to the increased vulnerability of the CNS in depressive disorders. Further studies are needed to establish these results.