bluuebunny/crossref_metadata_2025 huggingface dataset:
Human CD38 (ADP-Ribosyl Cyclase) Is a Counter-Receptor of CD31, an Ig Superfamily Member
Authors: Deaglio Silvia , Morra Massimo , Mallone Roberto , Ausiello Clara M. , Prager Elisabeth , Garbarino Giovanni , Dianzani Umberto , Stockinger Hannes , Malavasi Fabio
Published: January 2023

Human CD38 is a cell surface molecule involved in the regulation of lymphocyte adhesion to endothelial cells. This suggests that HUVEC bear a ligand(s) for CD38 on the cell surface. By means of the mAb Moon-1, which specifically inhibits CD38-mediated cell adhesion, we have identified a trans-membrane 130-kDa molecule acting as a ligand for CD38. Here, we report that the molecule recognized by the Moon-1 mAb is CD31, a member of the Ig superfamily. This conclusion is based on 1) cross-inhibition assays between Moon-1 and reference anti-CD31 mAbs; 2) sequential immunoprecipitation experiments using Moon-1 and known anti-CD31 mAbs, and 3) reactivity of the Moon-1 mAb with CD31 transfectants. Further, CD31 and CD38 cognate interactions were found to modulate heterotypic adhesion as well as to implement cytoplasmic calcium fluxes identical to those obtained by means of agonistic anti-CD38 mAbs. Other effects tested included the synthesis of messages for a panel of cytokines, markedly increased upon receptor-ligand interactions. These results suggest that the interplay between CD38 and its ligand CD31 is an important step in the regulation of cell life and of the migration of leukocytes (and CD38+ cancer cells) through the endothelial cell wall.
Regulated Production of Type I Collagen and Inflammatory Cytokines by Peripheral Blood Fibrocytes
Authors: Chesney Jason , Metz Christine , Stavitsky Abram B. , Bacher Michael , Bucala Richard
Published: January 2023

We recently described a novel population of blood-borne cells, termed fibrocytes, that display a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and contribute to scar formation. To further characterize the role of these cells in vivo, we examined the expression of type I collagen and cytokine mRNAs by cells isolated from wound chambers implanted into mice. Five days after chamber implantation, CD34+ fibrocytes but not CD14+ monocytes or CD90+ T cells expressed mRNA for type I collagen. Fibrocytes purified from wound chambers also were found to express mRNA for IL-1β, IL-10, TNF-α, JE/MCP, MIP-1α, MIP-1β, MIP-2, PDGF-A, TGF-β1, and M-CSF. The addition of IL-1β (1–100 ng/ml), a critical mediator in wound healing, to fibrocytes isolated from human peripheral blood induced the secretion of chemokines (MIP-1α, MIP-1β, MCP-1, IL-8, and GROα), hemopoietic growth factors (IL-6, IL-10, and macrophage-CSF), and the fibrogenic cytokine TNF-α. By contrast, IL-1β decreased the constitutive secretion of type I collagen as measured by ELISA. Additional evidence for a role for fibrocytes in collagen production in vivo was obtained in studies of livers obtained from Schistosoma japonicum-infected mice. Mouse fibrocytes localized to areas of granuloma formation and connective matrix deposition. We conclude that fibrocytes are an important source of cytokines and type I collagen during both the inflammatory and the repair phase of the wound healing response. Furthermore, IL-1β may act on fibrocytes to effect a phenotypic transition between a repair/remodeling and a proinflammatory mode.
A Model of Peptide-Induced Lupus Autoimmune B Cell Epitope Spreading Is Strain Specific and Is Not H-2 Restricted in Mice
Authors: James Judith A. , Harley John B.
Published: January 2023

Anti-Sm is a common and specific autoantibody found in systemic lupus erythematosus. The peptide PPPGMRPP from Sm B/B′ is an early target of the autoimmune response in some anti-Sm-positive human patients. After immunization with this peptide on a MAP backbone, rabbits develop anti-Sm autoantibodies with B cell epitope spreading of the autoimmune response as well as other features of lupus autoimmunity. Various strains of inbred mice have been immunized with peptide PPPGMRPP or PSQQVMTP (nonantigenic region of Sm B/B′) in Freund’s adjuvant or with no peptide. All peptide-immunized mouse strains eventually develop high titers of specific anti-peptide of immunization Abs. Mice immunized with Freund’s adjuvant alone have no measurable Ab binding to the PPPGMRPP peptide. With time, nearly half the mouse strains tested develop Abs that react with additional regions of Sm B/B′ and Sm D. All the regions bound by mouse serum are major epitopes of the human systemic lupus erythematosus anti-Sm response. These same strains also develop significant anti-Sm and anti-nuclear ribonucleoprotein titers. In addition, some of these strains demonstrate positive anti-nuclear Abs and anti-dsDNA Abs. Experiments with congenic H-2 mice demonstrate that the H-2 region does not play a role in spreading the immune response from the peptide of immunization to other epitopes of the spliceosome. These results present a new murine model of B cell epitope spreading and lupus autoimmunity induced by peptide immunization that is strain specific and not apparently dependent upon the loci at H-2.
Cutting Edge: Immature Dendritic Cells Generated from Monocytes in the Presence of TGF-β1 Express Functional C-C Chemokine Receptor 6
Authors: Yang De , Howard O. M. Zack , Chen Qian , Oppenheim Joost J.
Published: January 2023

Although CD34+ progenitor-derived immature dendritic cells (DCs) express CCR6, several recent studies reported that monocyte-derived immature DCs do not do so. We observed that DCs generated from monocytes in the presence of GM-CSF, IL-4, and TGF-β1 consistently responded to liver and activation-regulated chemokine (LARC, also known as macrophage inflammatory protein-3α). These immature DCs expressed one class of high-affinity binding sites for LARC, and expressed both CCR6 mRNA and protein. Therefore, LARC-CCR6 interaction presumably also contributes to the regulation of trafficking of monocyte-derived DCs, and utilization of TGF-β can potentially provide a ready source of CCR6+ monocyte-derived DCs for therapeutic purposes.
Cutting Edge: Association of Phospholipase C-γ2 Src Homology 2 Domains with BLNK Is Critical for B Cell Antigen Receptor Signaling
Authors: Ishiai Masamichi , Sugawara Hitoshi , Kurosaki Mari , Kurosaki Tomohiro
Published: January 2023

To explore the mechanism(s) by which phospholipase C (PLC)-γ2 participates in B cell Ag receptor (BCR) signaling, we have studied the function of PLC-γ2 mutants in B cells deficient in PLC-γ2. Mutation of the N-terminal Src homology 2 domain [SH2(N)] resulted in the complete loss of inositol 1,4,5-trisphosphate generation upon BCR engagement. A possible explanation for the SH2(N) requirement was provided by findings that this mutation abrogates the association of PLC-γ2 with an adaptor protein BLNK. Moreover, expression of a membrane-associated form (CD16/PLC-γ2) with SH2(N) mutation required coligation of BCR and CD16 for inositol 1,4,5-trisphosphate generation. Together, our results suggest a central role for the SH2(N) domain in directing PLC-γ2 into the close proximity of BCR signaling complex by its association with BLNK, whereby PLC-γ2 becomes tyrosine phosphorylated and thereby activated.
Cutting Edge: Dueling TCRs: Peptide Antagonism of CD4+ T Cells with Dual Antigen Specificities
Authors: Robertson Jennifer M. , Evavold Brian D.
Published: January 2023

T cells expressing two different TCRs were generated by interbreeding 3A9 and AND CD4+ TCR transgenic mice specific for the hen egg lysozyme (HEL) peptide 48–62:I-Ak and moth cytochrome c (MCC) peptide 88–103:I-Ek peptide:MHC ligands, respectively. Peripheral T cells in the offspring express two TCR Vβ-chains and respond to HEL and MCC. We observed minimal or no additive effects upon simultaneous suboptimal stimulation with both agonist peptides; however, an antagonist peptide for the 3A9 TCR was able to inhibit the response of the dual receptor T cells to MCC, the AND TCR agonist. This HEL antagonist peptide did not affect AND single transgenic T cells, indicating that the antagonism observed in the dual TCR cells is dependent on the presence of the HEL-specific 3A9 TCR. In contrast, anti-TCR Abs mediate receptor-specific antagonism. These results demonstrate that peptide antagonism exerts a dominant effect.
Strain Variation in Autoimmunity: Attempted Tolerization of DA Rats Results in the Induction of Experimental Autoimmune Encephalomyelitis
Authors: Lenz Derek C. , Wolf Norbert A. , Swanborg Robert H.
Published: January 2023

This paper reports that DA rats develop experimental autoimmune encephalomyelitis (EAE) when immunized with encephalitogenic myelin basic protein (MBP) peptide (MBP63–81) in IFA. In contrast, most rodent strains are tolerized by this procedure. Doses as low as 5 μg peptide + IFA induced EAE in DA rats. Lewis (LEW) rats did not develop EAE, even after immunization with 100 μg encephalitogenic peptide (MBP68–86) + IFA, but were rendered tolerant to EAE. DA rat T cells proliferated to peptide, and proliferation was inhibited by CTLA4Ig, and by anti-B7.1 and anti-B7.2 mAbs. This indicates that the ease of induction of EAE in this strain does not reflect a decreased requirement for T cell costimulation through the B7/CD28 costimulatory pathway. The inhibitory effect of CTLA4Ig was abrogated in the presence of anti-TGF-β-neutralizing Ab. An encephalitogenic DA T cell line expressed mRNA for the Th1 cytokines IFN-γ and TNF-α, as well as IL-10, and secreted these cytokines. In contrast, a T cell line from peptide + IFA-immunized LEW rats (which did not develop EAE) failed to secrete these cytokines. Although this line did not express TNF-α or IL-10 mRNA, IFN-γ mRNA was detected, suggesting posttranscriptional regulation of IFN-γ expression. Attempts to induce unresponsiveness in DA rats with encephalitogenic peptide-coupled splenocytes were also unsuccessful.
NK Lytic-Associated Molecule: A Novel Gene Selectively Expressed in Cells with Cytolytic Function
Authors: Kozlowski Miroslaw , Schorey Jeff , Portis Toni , Grigoriev Vitalii , Kornbluth Jacki
Published: January 2023

NK cells are most effective in killing a broad spectrum of primary tumor cells after stimulation with cytokines. We have cloned a novel gene, designated NKLAM (for NK lytic-associated molecule), whose expression is associated with this cytokine-enhanced process. NKLAM expression is up-regulated in NK cells by IL-2 and IFN-β. NKLAM is also selectively expressed by activated macrophages and CTL. Treatment of NK cells and CTL with NKLAM antisense oligonucleotides specifically decreases their cytolytic activity, while having no effect on cell growth. The NKLAM gene encodes a 62-kDa ring finger-containing protein that localizes to the cytoplasmic granules in NK cells. Further study of this gene may add to our understanding of cytotoxic processes common to NK cells, CTL, and activated macrophages.
Insulin in Oral Immune “Tolerance”: A One-Amino Acid Change in the B Chain Makes the Difference
Authors: Homann Dirk , Dyrberg Thomas , Petersen Jacob , Oldstone Michael B. A. , von Herrath Matthias G.
Published: January 2023

Oral administration of self-Ags can dampen or prevent autoimmune processes by induction of bystander suppression. Based on encouraging results from experiments in nonobese diabetic (NOD) mice, clinical trials have been initiated in type 1 diabetes using human insulin as an oral Ag. However, neither the precise antigenic requirements nor the mechanism of bystander suppression are currently understood in detail. Here we report that 1) a 1-aa difference in position 30 of the insulin B chain abrogated the ability of insulin to confer protection in both NOD as well as a virus-induced transgenic mouse model for type 1 diabetes. In the latter model transgenic mice express the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under the control of the rat insulin promotor (RIP) in the pancreatic β cells and develop diabetes only following LCMV infection; and 2) protection could be transferred with insulin B chain-restimulated but not LCMV-restimulated splenocytes from RIP-NP transgenic mice, demonstrating that the mechanism of diabetes prevention in the RIP-NP model is mediated by insulin B chain-specific, IL-4-producing regulatory cells acting as bystander suppressors.
Protective Antiviral Cytotoxic T Cell Memory Is Most Efficiently Maintained by Restimulation Via Dendritic Cells
Authors: Ludewig Burkhard , Oehen Stephan , Barchiesi Federica , Schwendener Reto A. , Hengartner Hans , Zinkernagel Rolf M.
Published: January 2023

Dendritic cells (DC) play a key role in the initiation of T cell-mediated immune responses and may therefore be successfully used in antiviral and antitumor vaccination strategies. Because both strength and duration of an immune response determines the outcome of a vaccination protocol, we evaluated the life span of DC-induced antiviral CTL memory against systemic and peripheral challenge infections with lymphocytic choriomeningitis virus (LCMV). We found that expansion and activation of CTL by DC was transient. Protection against systemic LCMV infection after DC immunization was relatively long-lived (>60 days), whereas complete protection against peripheral infection via intracerebral infection or infection into the footpad with LCMV, where rapid recruitment of effector T cells to the site of infection and elimination of viral pathogen plays a major role, was short-lived (<30 days). Protective immunity was most efficiently restored by administration of antigenic peptides via DC, rather than in combination with IFA or in liposomes. These results suggest that Ag presentation by DC may be crucial for both initiation and maintenance of protective CTL-mediated immunity against viruses infecting solid organs or against peripheral mesenchymal or epithelial tumors.
The Ectoenzyme γ-Glutamyl Transpeptidase Regulates Antiproliferative Effects of S-Nitrosoglutathione on Human T and B Lymphocytes
Authors: Henson Sarah E. , Nichols Timothy C. , Holers V. Michael , Karp David R.
Published: January 2023

Expression of the ectoenzyme γ-glutamyl transpeptidase (GGT) is regulated on T lymphocytes. It is present at a low level on naive T cells, at a high level on activated T cells, and at an intermediate level on resting memory T cells. GGT cleaves the glutamyl group from glutathione, which is the first step in the uptake of extracellular glutathione. In vitro, purified GGT also metabolizes the naturally occurring nitrosothiol, S-nitrosoglutathione (GSNO). Because of this relationship, the effects of cellular GGT on the metabolism of and cellular response to GSNO were tested. The GGT-negative lymphoblasts Ramos and SupT1 were transfected with cDNA for human GGT. In the presence of cells lacking GGT, GSNO is extremely stable. In contrast, GGT-expressing cells rapidly metabolize GSNO leading to nitric oxide release. The nitric oxide causes a rapid (<2-h) inhibition of DNA synthesis. There is a concomitant decrease in the concentration of intracellular deoxyribonucleotides, suggesting that one effect of the nitric oxide generated from GSNO is the previously described inactivation of the enzyme ribonucleotide reductase. GSNO also caused a rapid, GGT-dependent cytostatic effect in Hut-78, a human T cell lymphoma, as well as in activated peripheral blood T cells. Although DNA synthesis was decreased to 16% of control values in anti-CD3-stimulated Hut-78, the production of IL-2 was unchanged by GSNO. These data show that GGT, a regulated ectoenzyme on T cells, controls the rate of nitric oxide production from GSNO and thus markedly affects the physiological response to this biologically active nitrosothiol.
Inflammatory Cytokines Synergize with the HIV-1 Tat Protein to Promote Angiogenesis and Kaposi’s Sarcoma Via Induction of Basic Fibroblast Growth Factor and the αvβ3 Integrin
Authors: Barillari Giovanni , Sgadari Cecilia , Palladino Clelia , Gendelman Rita , Caputo Antonella , Morris Cindy Bohan , Nair Bala C. , Markham Philip , Nel Andrè , Stürzl Michael , Ensoli Barbara
Published: January 2023

The Tat protein of HIV-1, a transactivator of viral gene expression, is released by acutely infected T cells and, in this form, exerts angiogenic activities. These have linked the protein to the pathogenesis of Kaposi’s sarcoma (KS), a vascular tumor frequent and aggressive in HIV-1-infected individuals (AIDS-KS). In this study, we show that a combination of the same inflammatory cytokines increased in KS lesions, namely IL-1β, TNF-α, and IFN-γ, synergizes with Tat to promote in nude mice the development of angioproliferative KS-like lesions that are not observed with each factor alone. Inflammatory cytokines induce the tissue expression of both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), two angiogenic molecules highly produced in primary KS lesions. However, bFGF, but not VEGF, synergizes with Tat in vivo and induces endothelial cells to migrate, to adhere, and to grow in response to Tat in vitro. Tat angiogenic effects correlate with the expression of the αvβ3 integrin that is induced by bFGF and binds the arginine-glycine-aspartic acid (RGD) region of Tat. In contrast, no correlation is observed with the expression of αvβ5, which is promoted by VEGF and binds Tat basic region. Finally, KS lesion formation induced by bFGF and Tat in nude mice is blocked by antagonists of RGD-binding integrins. Because αvβ3 is an RGD-binding integrin that is highly expressed in primary KS lesions, where it colocalizes with extracellular Tat on vessels and spindle cells, these results suggest that αvβ3 competitors may represent a new strategy for the treatment of AIDS-KS.
Intradermal Delivery of IL-12 Naked DNA Induces Systemic NK Cell Activation and Th1 Response In Vivo That Is Independent of Endogenous IL-12 Production
Authors: Watanabe Morihiro , Fenton Robert G. , Wigginton Jon M. , McCormick Kathryn L. , Volker Kirk M. , Fogler William E. , Roessler Philip G. , Wiltrout Robert H.
Published: January 2023

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-γ and IFN-γ-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-γ proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o− plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o− to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o− into BALB/c IL-12 p40−/− mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-γ protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.
Partial IgA-Deficiency with Increased Th2-Type Cytokines in TGF-β1 Knockout Mice
Authors: Ginkel Frederik W. van , Wahl Sharon M. , Kearney John F. , Kweon Mi-Na , Fujihashi Kohtaro , Burrows Peter D. , Kiyono Hiroshi , McGhee Jerry R.
Published: January 2023

Though it has been shown that TGF-β1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-β1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-β1 gene-disrupted (TGF-β1−/−) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-β1−/− mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-β1−/− mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-β1−/− mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.
APCs Express DCIR, a Novel C-Type Lectin Surface Receptor Containing an Immunoreceptor Tyrosine-Based Inhibitory Motif
Authors: Bates Elizabeth E. M. , Fournier Nathalie , Garcia Eric , Valladeau Jenny , Durand Isabelle , Pin Jean-Jacques , Zurawski Sandra M. , Patel Sejal , Abrams John S. , Lebecque Serge , Garrone Pierre , Saeland Sem
Published: January 2023

We have identified a novel member of the calcium-dependent (C-type) lectin family. This molecule, designated DCIR (for dendritic cell (DC) immunoreceptor), is a type II membrane glycoprotein of 237 aa with a single carbohydrate recognition domain (CRD), closest in homology to those of the macrophage lectin and hepatic asialoglycoprotein receptors. The intracellular domain of DCIR contains a consensus immunoreceptor tyrosine-based inhibitory motif. A mouse cDNA, encoding a homologous protein has been identified. Northern blot analysis showed DCIR mRNA to be predominantly transcribed in hematopoietic tissues. The gene encoding human DCIR was localized to chromosome 12p13, in a region close to the NK gene complex. Unlike members of this complex, DCIR displays a typical lectin CRD rather than an NK cell type extracellular domain, and was expressed on DC, monocytes, macrophages, B lymphocytes, and granulocytes, but not detected on NK and T cells. DCIR was strongly expressed by DC derived from blood monocytes cultured with GM-CSF and IL-4. DCIR was mostly expressed by monocyte-related rather than Langerhans cell related DC obtained from CD34+ progenitor cells. Finally, DCIR expression was down-regulated by signals inducing DC maturation such as CD40 ligand, LPS, or TNF-α. Thus, DCIR is differentially expressed on DC depending on their origin and stage of maturation/activation. DCIR represents a novel surface molecule expressed by Ag presenting cells, and of potential importance in regulation of DC function.
The Leukocyte Integrin αDβ2 Binds VCAM-1: Evidence for a Binding Interface Between I Domain and VCAM-1
Authors: Van der Vieren Monica , Crowe David T. , Hoekstra Denise , Vazeux Rosemay , Hoffman Patricia A. , Grayson Mitchell H. , Bochner Bruce S. , Gallatin W. Michael , Staunton Donald E.
Published: January 2023

The trafficking of leukocytes through tissues is supported by an interaction between the β2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the β2 integrins, αDβ2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that αDβ2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of αDβ2 in a lymphoid cell that does not express α4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of αDβ2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of αD contains a binding site for VCAM-1 since recombinant αD I domain binds specifically to VCAM-1. In addition, αD mAb that block cellular binding to VCAM-1 bind the αD I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for αDβ2 overlaps with that of α4 integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to αDβ2 and abrogates binding to the αD I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to αDβ2 binding. Finally, we demonstrate that αDβ2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by α4β1. These results indicate that VCAM-1 can bind to an I domain and that the binding of αDβ2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express αDβ2.
Localization on a Physical Map of the NKC-Linked Cmv1 Locus Between Ly49b and the Prp Gene Cluster on Mouse Chromosome 6
Authors: Brown Michael G. , Zhang Jun , Du Ying , Stoll Janis , Yokoyama Wayne M. , Scalzo Anthony A.
Published: January 2023

The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of ∼390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.
Specific Inhibition of Macrophage TNF-α Expression by In Vivo Ribozyme Treatment
Authors: Kisich Kevin O. , Malone Robert W. , Feldstein Paul A. , Erickson Kent L.
Published: January 2023

The overproduction of the cytokine TNF-α is associated with inflammatory and autoimmune diseases. We have developed a means to block TNF-α production with ribozymes directed against TNF-α mRNA to selectively inhibit its production in vitro and in vivo. Following cationic lipid-mediated delivery to peritoneal murine macrophages in culture, anti-TNF-α ribozymes were more effective inhibitors of TNF-α secretion than catalytically inactive ribozyme controls. Inhibition of TNF-α secretion was proportional to the concentration of ribozyme administered, with an IC50 of ∼10 nM. After i.p. injection of cationic lipid/ribozyme complexes, elicited macrophages accumulated ∼6% of the administered ribozyme. The catalytically active ribozyme suppressed LPS-stimulated TNF-α secretion by ∼50% relative to an inactive ribozyme control without inhibiting secretion of another proinflammatory cytokine produced by macrophages, IL-1α. Ribozyme-specific TNF-α mRNA degradation products were found among the mRNA extracted from macrophages following in vivo ribozyme treatment and ex vivo stimulation. Thus, catalytic ribozymes can accumulate in appropriate target cells in vivo; once in the target cell, ribozymes can be potent inhibitors of specific gene expression.
Antiinflammatory Effects of Glucocorticoids in Brain Cells, Independent of NF-κB
Authors: Bourke Emer , Moynagh Paul N.
Published: January 2023

Glucocorticoids are potent antiinflammatory drugs. They inhibit the expression of proinflammatory cytokines and adhesion molecules. It has recently been proposed that the underlying basis to such inhibition is the induction of the protein IκB, which inhibits the transcription factor NF-κB. The latter is a key activator of the genes encoding cytokines and adhesion molecules. The present study shows that the synthetic glucocorticoid, dexamethasone, inhibits the induction of the proinflammatory cytokine IL-8 and the adhesion molecules VCAM-1 and ICAM-1 in human 1321N1 astrocytoma and SK.N.SH neuroblastoma cells. However, dexamethasone failed to induce IκB or inhibit activation of NF-κB by IL-1 in the two cell types. EMSA confirmed the identity of the activated NF-κB by demonstrating that an oligonucleotide, containing the wild-type NF-κB-binding motif, inhibited formation of the NF-κB-DNA complexes whereas a mutated form of the NF-κB-binding motif was ineffective. In addition, supershift analysis showed that the protein subunits p50 and p65 were prevalent components in the activated NF-κB complexes. The lack of effect of dexamethasone on the capacity of IL-1 to activate NF-κB correlated with its inability to induce IκB and the ability of IL-1 to cause degradation of IκB, even in the presence of dexamethasone. The results presented in this paper strongly suggest that glucocorticoids may exert antiinflammatory effects in cells of neural origin by a mechanism(s) independent of NF-κB.
Initial Characterization of the Vitamin D Binding Protein (Gc-Globulin) Binding Site on the Neutrophil Plasma Membrane: Evidence for a Chondroitin Sulfate Proteoglycan
Authors: DiMartino Stephen J. , Kew Richard R.
Published: January 2023

The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.
Monocyte Chemoattractant Protein-1 Mediates Cockroach Allergen-Induced Bronchial Hyperreactivity in Normal But Not CCR2−/− Mice: The Role of Mast Cells
Authors: Campbell Emma M. , Charo Israel F. , Kunkel Steven L. , Strieter Robert M. , Boring Landin , Gosling Jennifa , Lukacs Nicholas W.
Published: January 2023

Bronchial eosinophil and mononuclear cell infiltrates are a hallmark of the asthmatic lung and are associated with the induction of reversible airway hyperreactivity. In these studies, we have found that monocyte chemotactic protein-1 (MCP-1), a CC (β) chemokine, mediates airway hyperreactivity in normal and allergic mice. Using a murine model of cockroach Ag-induced allergic airway inflammation, we have demonstrated that anti-MCP-1 Abs inhibit changes in airway resistance and attenuate histamine release into the bronchoalveolar lavage, suggesting a role for MCP-1 in mast cell degranulation. In normal mice, instillation of MCP-1 induced prolonged airway hyperreactivity and histamine release. In addition, MCP-1 directly induced pulmonary mast cell degranulation in vitro. These latter effects would appear to be selective because no changes were observed when macrophage-inflammatory protein-1α, eotaxin, or MCP-3 were instilled into the airways of normal mice or when mast cells were treated in vitro. Airway hyperreactivity was mediated by MCP-1 through CCR2 because allergen-induced as well as direct MCP-1 instilled-induced changes in airway hyperreactivity were significantly attenuated in CCR2 −/− mice. The neutralization of MCP-1 in allergic animals and instillation of MCP-1 in normal animals was related to leukotriene C4 levels in the bronchoalveolar lavage and was directly induced in pulmonary mast cells by MCP-1. Thus, these data identify MCP-1 and CCR2 as potentially important therapeutic targets for the treatment of hyperreactive airway disease.
A Putative Susceptibility Locus on Chromosome 18 Is Not a Major Contributor to Human Selective IgA Deficiency: Evidence from Meiotic Mapping of 83 Multiple-Case Families
Authors: Vořechovský Igor , Blennow Elisabeth , Nordenskjöld Magnus , Webster A. David B. , Hammarström Lennart
Published: January 2023

Previous reports of an association between constitutional chromosome 18 abnormalities and low levels of IgA suggested that this chromosome contains a susceptibility locus for selective IgA deficiency (IgAD), the most frequent Ig deficiency in humans. IgAD is genetically related to common variable immunodeficiency (CVID), characterized by a lack of additional isotypes. Our previous linkage analysis of 83 multiple-case IgAD/CVID families containing 449 informative pedigree members showed a significantly increased allele sharing in the chromosome region 6p21 consistent with allelic associations in family-based and case-control studies and provided the evidence for a predisposing locus, termed IGAD1, in the proximal part of the MHC. We have typed the same family material at 17 chromosome 18 marker loci with the average intermarker distance of 7 cM. A total of 7633 genotypes were analyzed in a nonparametric linkage analysis, but none of the marker loci exhibited a significantly increased allele sharing in affected family members. In addition, reverse painting and deletion mapping of a panel of constitutional chromosome 18 deletions/translocations showed the presence of IgA-deficient and IgA-proficient patients with the same abnormality and did not reveal a region commonly deleted. The linkage analysis of chromosome 8 and 21 regions involved in reciprocal translocations t(8;18) and t(18;21), which were identified in two patients lacking IgA, did not disclose a significant allele sharing. Although these results do not exclude the presence of a minor predisposing locus on this chromosome, such a putative locus would confer a population risk of developing IgAD/CVID much lower than IGAD1.
Sequence Polymorphisms in the Chemokines Scya1 (TCA-3), Scya2 (Monocyte Chemoattractant Protein (MCP)-1), and Scya12 (MCP-5) Are Candidates for eae7, a Locus Controlling Susceptibility to Monophasic Remitting/Nonrelapsing Experimental Allergic Encephalomyelitis
Authors: Teuscher Cory , Butterfield Russell J. , Ma Runlin Z. , Zachary James F. , Doerge R. W. , Blankenhorn Elizabeth P.
Published: January 2023

Experimental allergic encephalomyelitis (EAE), the principal animal model of multiple sclerosis, is genetically controlled. To date, 13 disease-modifying loci have been identified in the mouse by whole genome scanning using an F2 intercross between EAE-susceptible SJL/J and EAE-resistant B10.S/DvTe mice. Two quantitative trait loci (QTL), eae6 and eae7, on chromosome 11 were identified by classical marker-specific linkage analysis and interval mapping. Both QTL were reported to be associated with severity and duration of clinical signs. eae7 was subsequently shown to be a unique locus controlling the development of monophasic remitting/nonrelapsing EAE. In this study, composite interval mapping resolved eae6 into two linked QTL: eae6a at 0–13 cM is associated with disease severity, and eae6b at 19–28 cM associated with the duration of clinical signs. Additionally, composite interval mapping significantly refined the locations of eae6a, eae6b, and eae7, thereby facilitating systematic candidate gene screening by cDNA sequencing of SJL/J and B10.S/DvTe alleles. Sequence polymorphisms were not seen in Lif and IL12β, candidate genes for eae6a and eae6b, respectively. Similarly, cDNA sequence polymorphisms in Nos2, Scya3, Scya4, Scya5, Scya6, Scya7, Scya9, Scya10, and Scya11 were excluded as candidates for eae7. However, multiple sequence polymorphisms resulting in significant amino acid substitutions were identified in Scya1 (TCA-3), Scya2 (monocyte chemoattractant protein (MCP)-1), and Scya12 (MCP-5). Given the role of chemokines in EAE, these sequence polymorphisms are promising candidates for eae7, a locus associated with severity of clinical signs and susceptibility to the shorter, less severe monophasic remitting/nonrelapsing form of disease.
Generation of Anti-p53 Fab Fragments from Individuals with Colorectal Cancer Using Phage Display
Authors: Coomber David W. J. , Hawkins Nicholas J. , Clark Michelle A. , Ward Robyn L.
Published: January 2023

Although many individuals with malignancy develop Abs against p53, little is currently known of the structural features, V gene usage, and degree of somatic mutation of these Abs. Such information is critical to any meaningful understanding of the nature and significance of this humoral immune response to p53. We have constructed phage display libraries from six individuals with colorectal cancer and a demonstrable serum immune response against p53. Following panning with recombinant p53, a total of 43 binding Fab were identified. Four of these Abs bound with high affinity to wild-type denatured p53 (1.19 × 10−8 − 1.57 × 10−8), as determined by BIAcore analysis, and were highly specific for both recombinant and cell line-derived p53, as determined by ELISA and immunoprecipitation. Epitope mapping showed they were reactive with the N terminus of human p53 between residues 27 and 44. Sequence analysis showed that the heavy chains were derived from the VH1 gene family, and the light chains from VL4. The pattern of replacement and silent mutations in the Fab sequence indicated that negative selection had occurred in the framework regions of all the VH genes. We show that lymphocytes from individuals with cancer represent a valuable source of high affinity human Abs against p53. This approach provides an opportunity to examine the genetic structure of these naturally occurring Abs, and to draw inferences regarding the nature of the immune response that produced them. Abs identified in this way have a number of potential therapeutic applications.
Isolation and Characterization of Human Monoclonal Antibodies to Digoxin
Authors: Ball William J. , Kasturi Rama , Dey Purabi , Tabet Michael , O’Donnell Susan , Hudson Debra , Fishwild Dianne
Published: January 2023

Fab preparations of sheep polyclonal anti-digoxin Abs have proven useful for reversal of the toxic effects of digoxin overdoses in patients. Unfortunately, the use of foreign species proteins in humans is limited because of the potential for immunological responses that include hypersensitivity reactions and acute anaphylaxis. Immunization of recently developed transgenic mice, whose endogenous μ heavy and κ light chain Ig genes are inactivated and which carry human Ig gene segments, with a digoxin-protein conjugate has enabled us to generate and isolate eight hybridoma cell lines secreting human sequence anti-digoxin mAbs. Six of the mAbs have been partially characterized and shown to have high specificity and low nanomolar affinities for digoxin. In addition, detailed competition binding studies performed with three of these mAbs have shown them to have distinct differences in their digoxin binding, and that all three structural moieties of the drug, the primary digitoxose sugar, steroid, and five-member unsaturated lactone ring, contribute to Ab recognition.
High T Cell Epitope Sharing Between Two HLA-B27 Subtypes (B*2705 and B*2709) Differentially Associated to Ankylosing Spondylitis
Authors: García-Peydró Marina , Martí Mercè , López de Castro José A.
Published: January 2023

HLA-B*2705 is strongly associated with ankylosing spondylitis (AS) and reactive arthritis. In contrast, B*2709 has been reported to be more weakly or not associated to AS. These two molecules differ by a single amino acid change: aspartic acid in B*2705 or histidine in B*2709 at position 116. In this study, we analyzed the degree of T cell epitope sharing between the two subtypes. Ten allospecific T cell clones raised against B*2705, 10 clones raised against B*2703 but cross-reactive with B*2705, and 10 clones raised against B*2709 were examined for their capacity to lyse B*2705 and B*2709 target cells. The anti-B*2705 and anti-B*2703 CTL were peptide dependent as demonstrated by their failure to lyse TAP-deficient B*2705-T2 transfectant cells. Eight of the anti-B*2705 and five of the anti-B*2703 CTL clones lysed B*2709 targets. The degree of cross-reaction between B*2705 and B*2709 was donor dependent. In addition, the effect of the B*2709 mutation (D116H) on allorecognition was smaller than the effect of the other naturally occurring subtype change at this position, D116Y. These results demonstrate that B*2705 and B*2709 are the antigenically closest HLA-B27 subtypes. Because allospecific T cell recognition is peptide dependent, our results imply that the B*2705- and B*2709-bound peptide repertoires are largely overlapping. Thus, to the extent to which linkage of HLA-B27 with AS is related to the peptide-presenting properties of this molecule, our results would imply that peptides within a relatively small fraction of the HLA-B27-bound peptide repertoire influence susceptibility to this disease.
Immune Responses in Asymptomatic HIV-1-Infected Patients After HIV-DNA Immunization Followed by Highly Active Antiretroviral Treatment
Authors: Calarota Sandra A. , Leandersson Ann-Charlotte , Bratt Göran , Hinkula Jorma , Klinman Dennis M. , Weinhold Kent J. , Sandström Eric , Wahren Britta
Published: January 2023

Intensive chemotherapy is capable of reducing the viral load in HIV-1-infected individuals while infected cells are still present. A special property of DNA immunization is to induce both new CTL and Ab responses. We evaluated the possibility of inducing new immune responses in already infected individuals by means of DNA constructs encoding the nef, rev, or tat regulatory HIV-1 genes. Significant changes in viral loads and CD4+ counts were observed in four patients who started highly active antiretroviral treatment (HAART) during the immunization study. The DNA immunization induced Ag-specific T cell proliferation, which persisted up to 9 mo after the last DNA injection, and cytolytic activities but did not, by itself, reduce viral load. Increased levels of CTL precursor cells were induced in all nine DNA-immunized patients. The profile of IFN-γ secretion observed when human PBMC were transfected with the nef, rev, and tat DNA resembled that found in the CTL activity (nef > tat > rev). Ab responses that occurred after immunizations were of a low magnitude. In accordance with the high IL-6 production induced by the nef DNA plasmid, IgG titers were highest in patients immunized with nef DNA. The initiation of HAART appears to contribute to the induction of new HIV-specific CTL responses, but by itself did not cause obvious re-induction of these activities.
Cutting Edge: Mouse IgG1 Antibodies Comprise Two Functionally Distinct Types That Are Differentially Regulated by IL-4 and IL-12
Authors: Faquim-Mauro Eliana L. , Coffman Robert L. , Abrahamsohn Ises A. , Macedo Mahasti S.
Published: January 2023

IL-4-dependent and -independent IgG1 Abs differ in their ability to induce mast cell degranulation as measured by passive cutaneous anaphylaxis (PCA). Mice immunized with OVA or PIII (fraction of Ascaris suum) produced high titers of IgG1 as shown by ELISA and PCA. In contrast, another A. suum fraction, PI, elicited IgG1 Abs with no PCA activity. IgG1 with anaphylactic activity required IL-4, as IgG1 responses to OVA and PIII in IL-4−/− mice gave no PCA. PI-specific IgG1 was IL-4-independent, because no difference was found between the responses of IL-4−/− and IL-4+/+ mice. Significant PCA reactions were elicited, however, with PI-specific IgG1 from IL-12−/− or anti-IFN-γ Ab-treated mice, although less Ab was measured by ELISA. These results indicate that one type of IgG1 has anaphylactic activity and its synthesis is IL-4-dependent, being inhibited by IL-12 or IFN-γ; the other lacks this activity and its synthesis is stimulated by IL-12 or IFN-γ.
Cutting Edge: Thymic Selection and Autoreactivity Are Regulated by Multiple Coreceptors Involved in T Cell Activation
Authors: Page Dawne M.
Published: January 2023

Immune responses are shaped by several processes that promote responses to pathogens and hinder responses to self. One mechanism that contributes to this polarization in response is negative selection, in which thymocytes that can respond to self-peptide/MHC complexes are deleted from the T cell repertoire. I found here that several coreceptors known to contribute to mature T cell activation also participate in negative selection. Interestingly, these molecules appeared to act in a cooperative fashion. Blocking the contribution of these molecules in fetal thymus organ culture not only prevented negative selection in the CD4+ lineage, but also induced the appearance of autoreactive thymocytes. This is the first demonstration that blocking coreceptor interactions during thymic development can produce autoreactive T cells. The contribution of negative selection to the mature T cell repertoire and to autoimmunity is discussed in light of these results.
Unexpected Reactivities of T Cells Selected by a Single MHC-Peptide Ligand
Authors: Singh Nagendra , Van Kaer Luc
Published: January 2023

In H2-DM mutant mice, most MHC class II molecules are bound by a single peptide, CLIP, derived from the class II-associated invariant chain. Previous studies showed that H2-DM− cells are defective in presenting synthetic peptides to class II-restricted T cells. In sharp contrast, however, the same peptides elicited strong CD4+ T cell responses in H2-DM− animals. We now provide an explanation for this apparent discrepancy. Peptide-specific CD4+ T cells from wild-type mice were efficiently stimulated by H2-DM+, but not by H2-DM− cells pulsed with the cognate peptide. In sharp contrast, CD4+ T cells from mutant animals specific for the same MHC-peptide combination recognized peptide-pulsed H2-DM+ and H2-DM− cells equally well. In addition, unlike Ag-specific T cells from wild-type animals, the reactivities of peptide-specific T cells from mutant animals could not be efficiently blocked by Abs specific for the cognate MHC class II-peptide combination. We further demonstrated that the distinct reactivities of CD4+ T cells from H2-DM+ and H2-DM− mice are due to differences in thymic selection. Collectively, these findings indicate that the CD4+ T cell repertoires of H2-DM+ and H2-DM− mice are remarkably different.