bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the IL-12 Receptor β1 and β2 Subunits in Human Tuberculosis
Authors: Zhang Ming , Gong Jianhua , Presky David H. , Xue Wanfen , Barnes Peter F.
Published: January 2023

To determine whether the Th1 response in tuberculosis correlated with IL-12R expression, we measured expression of the IL-12Rβ1 and IL-12Rβ2 subunits, as well as IL-12Rβ2 mRNA expression in tuberculosis patients and healthy tuberculin reactors. In tuberculosis patients, IFN-γ production by Mycobacterium tuberculosis-stimulated PBMC was reduced, the percentages of T cells expressing IL-12Rβ1 and IL-12Rβ2 were significantly decreased, and IL-12Rβ2 mRNA expression was also markedly reduced. In contrast, in pleural fluid and lymph nodes at the site of disease in tuberculosis patients, in which IFN-γ production is enhanced, IL-12Rβ2 mRNA expression was also increased. In M. tuberculosis-stimulated peripheral blood T cells from tuberculosis patients, anti-IL-10 and anti-TGF-β enhanced IL-12Rβ1 and IL-12Rβ2 expression, and IFN-γ production. In M. tuberculosis-stimulated peripheral blood T cells from healthy tuberculin reactors, recombinant IL-10 and TGF-β reduced IL-12Rβ1 and IL-12Rβ2 expression, as well as IFN-γ production. In combination with prior studies showing increased production of TGF-β by blood monocytes from tuberculosis patients, this suggests that increased TGF-β production is the underlying abnormality that reduces IL-12Rβ1 and IL-12Rβ2 expression in tuberculosis. Our findings provide evidence that IL-12R expression correlates well with IFN-γ production in human tuberculosis, and that expression of IL-12Rβ1 and IL-12Rβ2 may play a central role in mediating a protective Th1 response.
Expression of Functional Selectin Ligands on Th Cells Is Differentially Regulated by IL-12 and IL-4
Authors: Lim Yaw-Chyn , Henault Lori , Wagers Amy J. , Kansas Geoffrey S. , Luscinskas Francis W. , Lichtman Andrew H.
Published: January 2023

Immune responses may be qualitatively distinct depending on whether Th1 or Th2 cells predominate at the site of Ag exposure. T cell subset-specific expression of ligands for vascular selectins may underlie the distinct patterns of recruitment of Th1 or Th2 cells to peripheral inflammatory sites. Here we examine the regulation of selectin ligand expression during murine T helper cell differentiation. Large numbers of Th1 cells interacted with E- and P-selectin under defined flow conditions, while few Th2 and no naive T cells interacted. Th1 cells also expressed more fucosyltransferase VII mRNA than naive or Th2 cells. IL-12 induced expression of P-selectin ligands on Ag-activated naive T cells, even in the presence of IL-4, and on established Th2 cells restimulated in the presence of IL-12 and IFN-γ. In contrast, Ag stimulation alone induced only E-selectin ligand. Interestingly, restimulation of established Th2 cells in the presence of IL-12 and IFN-γ induced expression of P-selectin ligands but not E-selectin ligands; IFN-γ alone did not enhance expression of either selectin ligand. In summary, functional P- and E-selectin ligands are expressed on most Th1 cells, few Th2 cells, but not naive T cells. Furthermore, selectin ligand expression is regulated by the cytokine milieu during T cell differentiation. IL-12 induces P-selectin ligand, while IL-4 plays a dominant role in down-regulating E-selectin ligand.
Negative Regulation of Myeloid Cell Proliferation and Function by the SH2 Domain-Containing Tyrosine Phosphatase-1
Authors: Dong Qin , Siminovitch Katherine A. , Fialkow Lea , Fukushima Takeyasu , Downey Gregory P.
Published: January 2023

The SH2 domain containing tyrosine phosphatase SHP-1 has been implicated in the regulation of a multiplicity of signaling pathways involved in hemopoietic cell growth, differentiation, and activation. A pivotal contribution of SHP-1 in the modulation of myeloid cell signaling cascades has been revealed by the demonstration that SHP-1 gene mutation is responsible for the overexpansion and inappropriate activation of myelomonocytic populations in motheaten mice. To investigate the role of SHP-1 in regulation of myeloid leukocytes, an HA epitope-tagged dominant negative (interfering) SHP-1 (SHP-1C453S) was expressed in the myelo-monocytic cell line U937 using the pcDNA3 vector. Overexpression of this protein in SHP-1C453S transfectants was demonstrated by Western blot analysis and by detection of decreased specific activity. Growth, proliferation, and IL-3-induced proliferative responses were substantially increased in the SHP-1C453S-overexpressing cells relative to those in control cells. The results of cell cycle analysis also revealed that the proportion of cells overexpressing SHP-1C453S in S phase was greater than that of control cells. The SHP-1C453S-expressing cells also displayed diminished rates of apoptosis as detected by flow cytometric analysis of propidium iodide-stained cells and terminal deoxynucleotidyltransferase-mediated fluorescein-dUTP nick end-labeling assay. While motility and phagocytosis were not affected by SHP-1C453S overexpression, adhesion and the oxidative burst in response to PMA were enhanced in the SHP-1C453S compared with those in the vector alone transfectants. Taken together, these results suggest that SHP-1 exerts an important negative regulatory influence on cell proliferation and activation while promoting spontaneous cell death in myeloid cells.
Overexpression of Protein Kinase C Isoforms Protects RAW 264.7 Macrophages from Nitric Oxide-Induced Apoptosis: Involvement of c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase, p38 Kinase, and CPP-32 Protease Pathways
Authors: Jun Chang-Duk , Oh Chun-Do , Kwak Hyun-Jeong , Pae Hyun-Ock , Yoo Ji-Chang , Choi Byung-Min , Chun Jang-Soo , Park Rae-Kil , Chung Hun-Taeg
Published: January 2023

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-βII, -δ, and -η) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.
Induction of Ig Somatic Hypermutation and Class Switching in a Human Monoclonal IgM+ IgD+ B Cell Line In Vitro: Definition of the Requirements and Modalities of Hypermutation
Authors: Zan Hong , Cerutti Andrea , Dramitinos Patricia , Schaffer András , Li Zongdong , Casali Paolo
Published: January 2023

Partly because of the lack of a suitable in vitro model, the trigger(s) and the mechanism(s) of somatic hypermutation in Ig genes are largely unknown. We have analyzed the hypermutation potential of human CL-01 lymphocytes, our monoclonal model of germinal center B cell differentiation. These cells are surface IgM+ IgD+ and, in the absence of T cells, switch to IgG, IgA, and IgE in response to CD40:CD40 ligand engagement and exposure to appropriate cytokines. We show here that CL-01 cells can be induced to effectively mutate the expressed VHDJH-Cμ, VHDJH-Cδ, VHDJH-Cγ, VHDJH-Cα, VHDJH-Cε, and VλJλ-Cλ transcripts before and after Ig class switching in a stepwise fashion. In these cells, induction of somatic mutations required cross-linking of the surface receptor for Ag and T cell contact through CD40:CD40 ligand and CD80:CD28 coengagement. The induced mutations showed intrinsic features of Ig V(D)J hypermutation in that they comprised 110 base substitutions (97 in the heavy chain and 13 in the λ-chain) and only 2 deletions and targeted V(D)J, virtually sparing CH and Cλ. These mutations were more abundant in secondary VHDJH-Cγ than primary VHDJH-Cμ transcripts and in V(D)J-C than VλJλ-Cλ transcripts. These mutations were also associated with coding DNA strand polarity and showed an overall rate of 2.42 × 10−4 base changes/cell division in VHDJH-CH transcripts. Transitions were favored over transversions, and G nucleotides were preferentially targeted, mainly in the context of AG dinucleotides. Thus, in CL-01 cells, Ig somatic hypermutation is readily inducible by stimuli different from those required for class switching and displays discrete base substitution modalities.
Two Constituents of the Initiation Complex of the Mannan-Binding Lectin Activation Pathway of Complement Are Encoded by a Single Structural Gene
Authors: Stover Cordula M. , Thiel Steffen , Thelen Marcus , Lynch Nicholas J. , Vorup-Jensen Thomas , Jensenius Jens C. , Schwaeble Wilhelm J.
Published: January 2023

Mannan-binding lectin (MBL) forms a multimolecular complex with at least two MBL-associated serine proteases, MASP-1 and MASP-2. This complex initiates the MBL pathway of complement activation by binding to carbohydrate structures present on bacteria, yeast, and viruses. MASP-1 and MASP-2 are composed of modular structural motifs similar to those of the C1q-associated serine proteases C1r and C1s. Another protein of 19 kDa with the same N-terminal sequence as the 76-kDa MASP-2 protein is consistently detected as part of the MBL/MASP complex. In this study, we present the primary structure of this novel MBL-associated plasma protein of 19 kDa, MAp19, and demonstrate that MAp19 and MASP-2 are encoded by two different mRNA species generated by alternative splicing/polyadenylation from one structural gene.
Differential Regulation of 4E-BP1 and 4E-BP2, Two Repressors of Translation Initiation, During Human Myeloid Cell Differentiation
Authors: Grolleau Annabelle , Sonenberg Nahum , Wietzerbin Juana , Beretta Laura
Published: January 2023

Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-γ or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway.
A Novel Receptor Tyrosine Kinase, Mer, Inhibits TNF-α Production and Lipopolysaccharide-Induced Endotoxic Shock
Authors: Camenisch Todd D. , Koller Beverly H. , Earp2 H. Shelton , Matsushima Glenn K.
Published: January 2023

The regulation of monocyte function and the inhibition of TNF-α production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia. To study the function of a novel receptor tyrosine kinase, mer, that is expressed in monocytes, we generated mice (merkd) that lack the signaling tyrosine kinase domain. Upon LPS challenge, merkd animals died of endotoxic shock (15/17, 88.2%), whereas control wild-type mice survived (1/15, 6.7% died). Susceptible merkd mice exhibited edema, leukocyte infiltration, and signs of endotoxic shock that correlated with higher levels of TNF-α found in the serum of merkd mice as compared with wild-type control animals. Death due to LPS-induced endotoxic shock in merkd mice was blocked by administration of anti-TNF-α Ab, suggesting that overproduction of this cytokine was principally responsible for the heightened suseptibility. The increase in TNF-α production appeared to be the result of a substantial increase in the LPS-dependent activation of NF-κB nuclear translocation resulting in greater TNF-α production by macrophages from merkd mice. Thus, Mer receptor tyrosine kinase signaling participates in a novel inhibitory pathway in macrophages important for regulating TNF-α secretion and attenuating endotoxic shock.
Nasopharyngeal-Associated Lymphoreticular Tissue (NALT) Immunity: Fimbriae-Specific Th1 and Th2 Cell-Regulated IgA Responses for the Inhibition of Bacterial Attachment to Epithelial Cells and Subsequent Inflammatory Cytokine Production
Authors: Yanagita Manabu , Hiroi Takachika , Kitagaki Noriko , Hamada Shigeyuki , Ito Hiro-o , Shimauchi Hidetoshi , Murakami Shinya , Okada Hiroshi , Kiyono Hiroshi
Published: January 2023

To investigate the antibacterial activity of mucosal Th1 and Th2 immune responses induced nasally and orally, mice were immunized with mucosal vaccine containing fimbrial protein of Porphyromonas gingivalis, a causative agent for a destructive chronic inflammation in the periodontium, and cholera toxin (CT) as mucosal adjuvant. Nasal vaccine containing low doses of fimbriae (10 μg) and CT (1 μg) induced Ag-specific Th1/Th2-type response in CD4+ T cells in mucosal effector tissues, including nasal passage and submandibular glands, which accounted for the generation of Ag-specific IgA-producing cells. In contrast, oral immunization required higher amounts of fimbriae and CT for the induction of Ag-specific IgA responses. Fimbriae-specific IgA mAbs generated from submandibular glands of nasally immunized mice inhibited P. gingivalis attachment to and reduced subsequent inflammatory cytokine production from epithelial cells. These findings suggest that nasal vaccination is an effective immunization regimen for the induction of Ag-specific Th1 and Th2 cell-driven IgA immune responses that possess the ability to inhibit bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.
Amplification of the Antibody Response by C3b Complexed to Antigen Through an Ester Link
Authors: Villiers Marie-Bernadette , Villiers Christian L. , Laharie Anne-Marie , Marche Patrice N.
Published: January 2023

Complement C3 has been described as playing an important role in the cell-mediated immune response. C3b has the capacity to covalently bind Ag and then to stimulate in vitro Ag presentation to T lymphocytes. To verify this observation in vivo, we prepared and purified covalent human C3b-Ag complexes using lysozyme (HEL) as Ag. The characterization of these HEL-C3b complexes indicates that they are representative of those susceptible to be generated in physiological conditions. Mice were immunized with 0.1 to 0.6 μg of either free HEL, HEL + C3b, HEL-C3b, or HEL + CFA. Response was assessed after two i.p. injections by quantification of specific Ab production. Immunization with either HEL-C3b complexes or HEL + CFA leads to anti-HEL IgG production whereas free HEL or HEL + C3b was ineffective. Either HEL-C3b or HEL + CFA immunizations led to a similar Ig subclasse patterns, including IgG1, IgG2a, IgA, and IgM.

Our experiments provide the first evidence for modulation of specific Ab response by C3b when it is bound to Ag through a physiological-like link. Taken together with previous data concerning Ab response following recombinant HEL-C3d immunization, cellular events such as processing of C3b-Ag by APC and recognition by T lymphocytes, this present result underlines the importance of C3b and its fragments in stimulation of the immune system, through the multiplicity and complementarity of its interactions.
Colitis-Inducing Potency of CD4+ T Cells in Immunodeficient, Adoptive Hosts Depends on Their State of Activation, IL-12 Responsiveness, and CD45RB Surface Phenotype
Authors: Claesson Mogens H. , Bregenholt Søren , Bonhagen Kerstin , Thoma Stefan , Möller Peter , Grusby Michael J. , Leithäuser Frank , Nissen Mogens H. , Reimann Jörg
Published: January 2023

We studied the induction, severity, and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6–12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12–16 wk posttransfer. SCID mice transplanted with STAT-4−/− CD4+ T cells showed a late-onset IBD manifest >20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-γ and TNF-α. In SCID mice transplanted with IL-12-unresponsive STAT-4−/− CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-γ but abundant levels of TNF-α. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency.
TCR Signaling Thresholds Regulating T Cell Development and Activation Are Dependent upon SHP-1
Authors: Johnson Kenneth G. , LeRoy Frances G. , Borysiewicz Leszek K. , Matthews R. James
Published: January 2023

An examination of thymocytes and peripheral T cells from SHP-1-deficient motheaten mice possessing a transgenic MHC class I-restricted TCR has implicated SHP-1 in regulating TCR signaling thresholds at three checkpoints in T cell development and activation. First, in the population of CD4−CD8− double negative thymocytes, SHP-1 appears capable of regulating signals from TCR complexes that control the maturation and proliferation of double negative thymocytes. Second, the loss of SHP-1 increased the number of CD4+CD8+ double positive thymocytes capable of maturing as TCRhigh single positive thymocytes. Third, the loss of SHP-1 altered the basal level of activation of naive lymph node T cells. Accordingly, SHP-1-deficient lymph node T cells bearing the transgenic TCR demonstrated a hyperresponsiveness to stimulation with cognate peptide. However, the loss of SHP-1 did not alter the cytolytic ability of mature effector cytotoxic T lymphocytes. Together these results suggest that SHP-1 contributes to establishing thresholds for TCR signaling in thymocytes and naive peripheral T cells.
Extracellular Signal-Regulated Kinase, Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase, and p38mapkAre Involved in IL-10-Mediated Selective Repression of TNF-α-Induced Activation and Maturation of Human Peripheral Blood Monocyte-Derived Dendritic Cells
Authors: Sato Katsuaki , Nagayama Hitomi , Tadokoro Kenji , Juji Takeo , Takahashi Tsuneo A.
Published: January 2023

TNF-α or IL-10 has been implicated to reversibly regulate physiological states of dendritic cells (DCs). However, little is known about dual stimulations of these cytokines on DC properties and the intracellular signaling events that are responsible for the regulation of these states. Here, we show that a family of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 2 (ERK2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38mapk, are potentially involved in IL-10-mediated selective suppression of TNF-α-induced changes of the monocyte-derived DC properties. TNF-α induced the cluster formation of the cells and the enhancement of cell surface expression levels of CD83, CD86, and HLA-DR, and T cell stimulatory capacity, whereas the capacities for the endocytosis and the chemotactic migration were suppressed in these cells. Treatment of monocyte-derived DCs with IL-10 resulted in the reduction of the cell surface expression levels of CD86, HLA-DR, and T cell stimulatory capacity, whereas both endocytic and chemotactic migratory capacities were increased by IL-10. Dual stimulations of monocyte-derived DCs with TNF-α and IL-10 selectively antagonized their respective effects on these DC properties. TNF-α induced tyrosine phosphorylation and enzymatic activation of ERK2, SAPK/JNK, and p38mapk, whereas IL-10 did not induce these events. Dual stimulations of TNF-α plus IL-10 abolished TNF-α-induced changes of these MAPKs in DCs. These results suggest that the blockage in the MAPKs cascades contributes to IL-10-mediated repression of TNF-α-induced changes of DC properties.
Endotoxin-Mediated S-Nitrosylation of p50 Alters NF-κB-Dependent Gene Transcription in ANA-1 Murine Macrophages
Authors: delaTorre Andrew , Schroeder Rebecca A. , Punzalan Cecile , Kuo Paul C.
Published: January 2023

Nitric oxide (NO) regulates cellular function, in part, by S-nitrosylating active site thiol groups of proteins. Ex vivo S-nitrosylation of NF-κB p50 significantly decreases its capacity for DNA binding. To determine the cellular relevance of this observation, we utilized the ANA-l murine macrophage model of endotoxin (LPS)-mediated NO synthesis. In selected instances, the NO synthase inhibitor, l-arginine methyl ester (L-NAME; 100 μM), or the NO donor, S-nitroso-N-acetylcysteine (SNAC; 100 μM), was added. In contrast to that of LPS cells, nuclear extracts from LPS + L-NAME cells demonstrated increased NF-κB DNA binding on gel shift analysis. Addition of SNAC to LPS + L-NAME cells restored binding to a level equivalent to that of LPS cells. Spectrophotometric analysis of NF-κB p50 immunoprecipitates demonstrated S-NO bonds exclusively in LPS cells; these p50 protein isolates retained the same DNA binding characteristics as that of the nuclear extracts. Transfection assays utilizing NF-κB-dependent promoter-reporter constructs demonstrated increased activity in LPS + L-NAME cells compared with LPS cells; nuclear run-on assays confirmed increased transcription of the corresponding genes. These results suggest that LPS-mediated NO synthesis is associated with S-nitrosylation of NF-κB p50 and inhibition of NF-κB-dependent DNA binding, promoter activity, and gene transcription. We conclude that NO can regulate gene transcription by S-nitrosylation of NF-κB.
IL-16 as an Anti-Inflammatory Cytokine in Rheumatoid Synovitis
Authors: Klimiuk Piotr A. , Goronzy Jörg J. , Weyand Cornelia M.
Published: January 2023

T lymphocytes are a major component of the inflammatory infiltrate in rheumatoid synovitis, but their exact role in the disease process is not understood. Functional activities of synovial T cells were examined by adoptive transfer experiments in human synovium-SCID mouse chimeras. Adoptive transfer of tissue-derived autologous CD8+ T cells induced a marked reduction in the activity of lesional T cells and macrophages. Injection of CD8+, but not CD4+, T cells decreased the production of tissue IFN-γ, IL-1β, and TNF-α by >90%. The down-regulatory effect of adoptively transferred CD8+ T cells was not associated with depletion of synovial CD3+ T cells or synovial CD68+ macrophages, and it could be blocked by Abs against IL-16, a CD8+ T cell-derived cytokine. In the synovial tissue, CD8+ T cells were the major source of IL-16, a natural ligand of the CD4 molecule that can anergize CD4-expressing cells. The anti-inflammatory activity of IL-16 in rheumatoid synovitis was confirmed by treating synovium-SCID mouse chimeras with IL-16. Therapy for 14 days with recombinant human IL-16 significantly inhibited the production of IFN-γ, IL-1β, and TNF-α in the synovium. We propose that tissue-infiltrating CD8+ T cells in rheumatoid synovitis have anti-inflammatory activity that is at least partially mediated by the release of IL-16. Spontaneous production of IL-16 in synovial lesions impairs the functional activity of CD4+ T cells but is insufficient to completely abrogate their stimulation. Supplemental therapy with IL-16 may be a novel and effective treatment for rheumatoid arthritis.
Increased Peroxynitrite Activity in AIDS Dementia Complex: Implications for the Neuropathogenesis of HIV-1 Infection
Authors: Boven Leonie A. , Gomes Lucio , Hery Christiane , Gray Françoise , Verhoef Jan , Portegies Peter , Tardieu Marc , Nottet Hans S. L. M.
Published: January 2023

Oxidative stress is suggested to be involved in several neurodegenerative diseases. One mechanism of oxidative damage is mediated by peroxynitrite, a neurotoxic reaction product of superoxide anion and nitric oxide. Expression of two cytokines and two key enzymes that are indicative of the presence of reactive oxygen intermediates and peroxynitrite was investigated in brain tissue of AIDS patients with and without AIDS dementia complex and HIV-seronegative controls. RNA expression of IL-1β, IL-10, inducible nitric oxide synthase, and superoxide dismutase (SOD) was found to be significantly higher in demented compared with nondemented patients. Immunohistochemical analysis showed that SOD was expressed in CD68-positive microglial cells while inducible nitric oxide synthase was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes and in equal amounts in microglial cells. Approximately 70% of the HIV p24-Ag-positive macrophages did express SOD, suggesting a direct HIV-induced intracellular event. HIV-1 infection of macrophages resulted in both increased superoxide anion production and elevated SOD mRNA levels, compared with uninfected macrophages. Finally, we show that nitrotyrosine, the footprint of peroxynitrite, was found more intense and frequent in brain sections of demented patients compared with nondemented patients. These results indicate that, as a result of simultaneous production of superoxide anion and nitric oxide, peroxynitrite may contribute to the neuropathogenesis of HIV-1 infection.
Autoantibodies to T Cell Costimulatory Molecules in Systemic Autoimmune Diseases
Authors: Matsui Toshihiro , Kurokawa Manae , Kobata Tetsuji , Oki Shinji , Azuma Miyuki , Tohma Shigeto , Inoue Tetsufumi , Yamamoto Kazuhiko , Nishioka Kusuki , Kato Tomohiro
Published: January 2023

To determine whether antilymphocyte Abs to T cell costimulatory molecules are generated in patients with autoimmune diseases and, if they exist, to clarify the mechanism of their production and pathological roles, we investigated the presence of autoantibodies to CTLA-4 (CD152), CD28, B7-1 (CD80), and B7-2 (CD86) in serum samples obtained from patients with various autoimmune diseases and from normal subjects using recombinant fusion proteins. In ELISAs, anti-CD28, anti-B7-1, and anti-B7-2 Abs were rarely seen, whereas anti-CTLA-4 Abs were detected in 8.2% of the patients with systemic lupus erythematosus, 18.8% of those with rheumatoid arthritis, 3.1% of those with systemic sclerosis, 31.8% of those with Behçet’s disease, 13.3% of those with Sjögren’s syndrome, and 0% of healthy donors. This reactivity was confirmed by immunoblotting. More importantly, the purified anti-CTLA-4 Abs reacted with CTLA-4 expressed on P815 cells by flow cytometry. In addition, we found at least three epitopes on the CTLA-4 molecule. Furthermore, among the patients with Behçet’s disease, uveitis was seen significantly less frequently in the anti-CTLA-4 Ab-positive patients. Taken collectively, these data indicate that anti-CTLA-4 autoantibodies are generated in systemic autoimmune diseases by an Ag-driven mechanism and may modulate the immune response in vivo by binding to CTLA-4 on T cells.
Accelerated Emigration of B Lymphocytes in the Xid Mouse
Authors: Cariappa Annaiah , Kim Tae Jin , Pillai Shiv
Published: January 2023

The B cell receptor is required for the emigration of newly generated B lymphocytes and for their maintenance in the periphery. A specific maintenance defect was noted in fraction I (IgDhighIgMlow) B cells in Xid mice (which harbor a mutation in Btk). Although Bcl-2 levels in fractions I and II (IgDhighIgMhigh) are equivalent in normal and Xid B cells, a novel peak of Bcl-2low fraction III (IgDlowIgMhigh) B cells was noted in the Xid mouse. Since this B cell population resembled bone marrow immature B cells, we examined the emigration of newly formed B cells in normal and Xid mice. These studies revealed the accelerated emigration of newly formed Xid B cells. We conclude that distinct Btk-independent and Btk-dependent signals mediate emigration and maintenance events during peripheral B cell maturation.
Negative Regulation of c-kit-Mediated Cell Proliferation by FcγRIIB
Authors: Malbec Odile , Fridman Wolf H. , Daëron Marc
Published: January 2023

FcγRIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They are widely expressed by cells of hematopoietic origin. We investigated here whether FcγRIIB could also negatively regulate protein tyrosine kinase receptor (RTK)-dependent cell proliferation. As an experimental model, we used growth factor-dependent mast cells that constitutively express FcγRIIB and c-kit, an RTK prototype. We found that anti-c-kit Abs mimicked the effect of stem cell factor and induced thymidine incorporation in FcγRIIB−/−, but not in wild-type (wt) mast cells unless FcγRIIB were blocked or anti-c-kit F(ab′)2 were used. When coaggregated with c-kit by intact Abs in wt mast cells, FcγRIIB inhibited thymidine incorporation, as well as cell proliferation, and inhibition was correlated with an arrest of cells in G1 during the cell cycle. The coaggregation of c-kit with FcγRIIB did not affect ligand-induced c-kit phosphorylation and induced the tyrosyl-phosphorylation of FcγRIIB, which selectively recruited the Src homology 2 domain-bearing inositol 5-phosphatase SHIP. Our results indicate that IgG Abs to growth factors or growth factor receptors may control RTK-dependent proliferation of a variety of cells that express FcγRIIB.
Differential Cytokine and Chemokine Gene Expression by Human NK Cells Following Activation with IL-18 or IL-15 in Combination with IL-12: Implications for the Innate Immune Response
Authors: Fehniger Todd A. , Shah Manisha H. , Turner Matthew J. , VanDeusen Jeffrey B. , Whitman Susan P. , Cooper Megan A. , Suzuki Kazuhiro , Wechser Mark , Goodsaid Frederico , Caligiuri Michael A.
Published: January 2023

NK cells constitutively express monocyte-derived cytokine (monokine) receptors and secrete cytokines and chemokines following monokine stimulation, and are therefore a critical component of the innate immune response to infection. Here we compared the effects of three monokines (IL-18, IL-15, and IL-12) on human NK cell cytokine and chemokine production. IL-18, IL-15, or IL-12 alone did not stimulate significant cytokine or chemokine production in resting NK cells. The combination of IL-18 and IL-12 induced extremely high amounts of IFN-γ protein (225 ± 52 ng/ml) and a 1393 ± 643-fold increase in IFN-γ gene expression over those in resting NK cells. IL-15 and IL-12 induced less IFN-γ protein (24 ± 10 ng/ml; p < 0.007) and only a 45 ± 19-fold increase in IFN-γ gene expression over those in resting NK cells. The CD56bright NK cell subset produced significantly more IFN-γ following IL-18 and IL-12 compared with CD56dim NK cells (p < 0.008). However, the combination of IL-15 and IL-12 was significantly more potent than that of IL-18 and IL-12 for NK cell production of IL-10, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, and TNF-α at the protein and transcript levels. Granulocyte-macrophage CSF was optimally induced by IL-15 and IL-18. Resting CD56+ NK cells expressed IL-18R transcript that was up-regulated by IL-12 or IL-15. Our results show that distinct cytokine and chemokine patterns are induced in NK cells in response to different costimulatory signals from these three monokines. This suggests that NK cell cytokine production may be governed in part by the monokine milieu induced during the early proinflammatory response to infection and by the subset of NK cells present at the site of inflammation.
Differential Expression and Regulation of Cyclooxygenase Isozymes in Thymic Stromal Cells
Authors: Rocca Bianca , Spain Lisa M. , Ciabattoni Giovanni , Patrono Carlo , FitzGerald Garret A.
Published: January 2023

Prostaglandins (PGs) are lipid-derived mediators of rapid and localized cellular responses. Given the role of PG in supporting thymic T cell development, we investigated the expression of the PG synthases, also known as cyclooxygenases (COX)-1 and -2, in the biosynthesis of PGs in thymic stromal cell lines. The predominant isozyme expressed in cortical thymic epithelial cells was COX-1, while COX-2 predominated in the medulla. IFN-γ up-regulated expression and activity of COX-2 in medullary cells, in which COX-2 was expressed constitutively. In contrast, IFN-γ down-regulated COX-1 activity, but not expression, in cortical cells. Stromal cells support T cell development in the thymus, although the mediators of this effect are unknown. Selective inhibition of COX-2, but not COX-1, blocked the adhesion of CD4+CD8+ and CD4+CD8− thymocytes to medullary cell lines. No effect of the inhibitors was observed on the interactions of thymocytes with cortical epithelial lines. These data further support the differential regulation of COX-1 and COX-2 expression and function in thymic stromal cells. PGs produced by COX-2 in the medullary thymic stroma may regulate the development of thymocytes by modulating their interaction with stromal cells.
Two Mechanisms for the Non-MHC-Linked Resistance to Spontaneous Autoimmunity
Authors: Verdaguer2 Joan , Amrani2 Abdelaziz , Anderson Brad , Schmidt Dennis , Santamaria Pere
Published: January 2023

Genetic susceptibility and resistance to most autoimmune disorders are associated with highly polymorphic genes of the MHC and with non-MHC-linked polygenic modifiers. It is known that non-MHC-linked polymorphisms can override or enhance the susceptibility to an autoimmune disease provided by pathogenic MHC genes, but the mechanisms remain elusive. In this study, we have followed the fate of two highly diabetogenic β cell-specific T cell receptors (Kd and I-Ag7 restricted, respectively) in NOR/Lt mice, which are resistant to autoimmune diabetes despite expressing two copies of the diabetogenic MHC haplotype H-2g7. We show that at least two mechanisms of non-MHC-linked control of pathogenic T cells operate in these mice. One segregates as a recessive trait and is associated with a reduction in the peripheral frequency of diabetogenic CD8+ (but not CD4+) T cells. The other segregates as a dominant trait and is mediated by IL-4- and TGF-β1-independent immune suppressive functions provided by lymphocytes that target diabetogenic CD4+ and CD8+ T cells, without causing their deletion, anergy, immune deviation, or ignorance. These results provide explanations as to how non-MHC-linked polymorphisms can override the susceptibility to an autoimmune disease provided by pathogenic MHC haplotypes, and demonstrate that protective non-MHC-linked genes may selectively target specific lymphoid cell types in cellularly complex autoimmune responses.
DNA Binding by the VH Domain of Anti-Z-DNA Antibody and Its Modulation by Association of the VL Domain
Authors: Chen Yan , Stollar B. David
Published: January 2023

mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.
Specific Proteolytic Cleavages Limit the Diversity of the Pool of Peptides Available to MHC Class I Molecules in Living Cells
Authors: Serwold Thomas , Shastri Nilabh
Published: January 2023

MHC class I molecules display peptides selected from a poorly characterized pool of peptides available in the endoplasmic reticulum. We analyzed the diversity of peptides available to MHC class I molecules by monitoring the generation of an OVA-derived octapeptide, OVA257–264 (SL8), and its C-terminally extended analog, SL8-I. The poorly antigenic SL8-I could be detected in cell extracts only after its conversion to the readily detectable SL8 with carboxypeptidase Y. Analysis of extracts from cells expressing the minimal precursor Met-SL8-I by this method revealed the presence of SL8/Kb and the extended SL8-I/Kb complexes, indicating that the peptide pool contained both peptides. In contrast, cells expressing full length OVA generated only the SL8/Kb complex, demonstrating that the peptide pool generated from the full length precursor contained only a subset of potential MHC-binding peptides. Deletion analysis revealed that SL8-I was generated only from precursors lacking additional C-terminal flanking residues, suggesting that the generation of the C terminus of the SL8 peptide involves a specific endopeptidase cleavage. To investigate the protease responsible for this cleavage, we tested the effect of different protease inhibitors on the generation of the SL8 and SL8-I peptides. Only the proteasome inhibitors blocked generation of SL8, but not SL8-I. These findings demonstrate that the specificities of the proteases in the Ag-processing pathway, which include but are not limited to the proteasome, limit the diversity of peptides available for binding by MHC class I molecules in the endoplasmic reticulum.
Enhancement of the Listeria monocytogenes p60-Specific CD4 and CD8 T Cell Memory by Nonpathogenic Listeria innocua
Authors: Geginat Gernot , Nichterlein Thomas , Kretschmar Marianne , Schenk Simone , Hof Herbert , Lalic-Mülthaler Mio , Goebel Werner , Bubert Andreas
Published: January 2023

The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.
DNA Vaccination Against the Idiotype of a Murine B Cell Lymphoma: Mechanism of Tumor Protection
Authors: Syrengelas Athanasia D. , Levy Ronald
Published: January 2023

Several studies have shown that immunization with DNA, which encodes the idiotypic determinants of a B cell lymphoma, generates tumor-specific immunity. Although induction of antiidiotypic Abs has correlated with tumor protection, the effector mechanisms that contribute to tumor protection have not been clearly identified. This study evaluated the tumor protective effects of humoral and cellular immune mechanisms recruited by idiotype-directed DNA vaccines in the 38C13 murine B cell lymphoma model. Antiidiotypic Abs induced by DNA vaccination supported in vitro complement-mediated cytotoxicity of tumor cells, and simultaneous transfer of tumor cells and hyperimmune sera protected naive animals against tumor growth. However, in vitro stimulation of immune splenocytes with tumor cells failed to induce idiotype-specific cytotoxicity, and following vaccination, depletion of CD4 or CD8 T cell subsets did not compromise protection. Furthermore, protection of naive recipients against tumor challenge could not be demonstrated either by a Winn assay approach or by adoptive transfer of spleen and lymph node cells. Thus, in this experimental model, current evidence suggests that the tumor-protective effects of DNA vaccination can be largely attributed to idiotype-specific humoral immunity.
Endotoxin-Stimulated Monocytes Release Multiple Forms of IL-1β, Including a ProIL-1β Form Whose Detection Is Affected by Export
Authors: Wewers2 Mark D. , Winnard Alissa V. , Dare Heidi A.
Published: January 2023

The processing and release of 31-kDa proIL-1β to the mature 17-kDa form of IL-1β are still poorly understood. To help elucidate the mechanisms involved in IL-1β processing and release, we measured IL-1β forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1β. Our studies demonstrate that in addition to the 17-kDa mature IL-1β, IL-1β is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1β represented 20–40% of the total released IL-1β, as measured by SDS-PAGE with densitometry. This released proIL-1β was susceptible to ICE processing; however, this proIL-1β was not detectable by either a mature or proIL-1β-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1β was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1β could complex the type II IL-1R, released proIL-1β did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1β binding to another protein. These findings imply that IL-1β is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1β.
IL-3 and IL-4 Activate Cyclic Nucleotide Phosphodiesterases 3 (PDE3) and 4 (PDE4) by Different Mechanisms in FDCP2 Myeloid Cells
Authors: Ahmad Faiyaz , Gao Guang , Wang Ling Mei , Landstrom Tova Rahn , Degerman Eva , Pierce Jacalyn H. , Manganiello Vincent C.
Published: January 2023

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-α, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-α also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-α, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-α could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).