bluuebunny/crossref_metadata_2025 huggingface dataset:
EBI1/CCR7 Is a New Member of Dendritic Cell Chemokine Receptor That Is Up-Regulated upon Maturation
Authors: Yanagihara Shigehiro , Komura Emiko , Nagafune Jun , Watarai Hiroshi , Yamaguchi Yasunori
Published: December 2022

Dendritic cells (DC) that are stimulated with inflammatory mediators can maturate and migrate from nonlymphoid tissues to lymphoid organs to initiate T cell-mediated immune responses. This migratory step is closely related to the maturation of the DC. In an attempt to identify chemokine receptors that might influence migration and are selectively expressed in mature DC, we have discovered that the chemokine receptor, EBI1/CCR7, is strikingly up-regulated upon maturation in three distinct culture systems: 1) mouse bone marrow-derived DC, 2) mouse epidermal Langerhans cells, and 3) human monocyte-derived DC. The EBI1/CCR7 expressed in mature DC is functional because ELC/MIP-3β, recently identified as a ligand of EBI1/CCR7, induces a rise in intracellular free calcium concentrations and directional migration of human monocyte-derived mature DC (HLA-DRhigh, CD1alow, CD14−, CD25+, CD83+, and CD86high) in a dose-dependent manner, but not of immature DC (HLA-DRlow, CD1ahigh, CD14−, CD25−, CD83−, and CD86−). In contrast, macrophage inflammatory protein-1α (MIP-1α), monocyte chemotactic protein-3 (MCP-3), and RANTES are active on immature DC but not on mature DC. Thus, it seems likely that MIP-1α, MCP-3, and RANTES can mediate the migration of immature DC located in peripheral sites, whereas ELC/MIP-3β can direct the migration of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of EBI1/CCR7, to lymphoid organs. It is postulated that different chemokines and chemokine receptors are involved in DC migration in vivo, depending on the maturation state of DC.
c-Fos Induces Apoptosis in Germinal Center B Cells
Authors: Inada Kunimasa , Okada Seiji , Phuchareon Janyaporn , Hatano Masahiko , Sugimoto Toyohiko , Moriya Hideshige , Tokuhisa Takeshi
Published: December 2022

We examined the role of c-Fos in the differentiation of mature B cells into IgG-producing cells using transgenic mice carrying the c-fos gene under the control of the IFN-α/β-inducible Mx promoter (Mx-c-fos) or the constitutive H-2Kb promoter (H2-c-fos). Splenic B cells from Mx-c-fos mice were cultured with LPS and rIL-4, and IgG1+ B cells were developed in the culture after day 3. When IFN-α/β was added to the culture from day 2, development of IgG1+ B cells was perturbed, and the number of apoptotic cells increased within 24 h, suggesting that c-Fos induces apoptosis in Ig class-switching B cells. To confirm the effect of c-Fos on B cell differentiation in vivo, H2-c-fos mice were immunized with DNP-OVA. The mice produced primary IgM, but not IgG, anti-DNP Ab in serum and failed to generate germinal centers in spleen. The perturbation of germinal center formation in H2-c-fos mice was rescued by mating them with transgenic mice carrying the bcl-2 gene with the Ig promoter. However, primary IgG1 anti-DNP Ab production was still suppressed in doubly transgenic mice, suggesting that Bcl-2 can delay the time of c-Fos-induced apoptosis in Ig class-switching B cells but cannot rescue the death. Since c-Fos is induced in mature B cells reacted with Ags, and clonal deletion of self-reactive B cells in germinal centers is insensitive to Bcl-2, these results suggest that c-Fos plays a causal role in clonal deletion of germinal center B cells.
Therapy with Monoclonal Antibodies. II. The Contribution of Fcγ Receptor Binding and the Influence of CH1 and CH3 Domains on In Vivo Effector Function
Authors: Isaacs John D. , Greenwood Judith , Waldmann Herman
Published: December 2022

An in vivo model is used to define Fc motifs engaged by mAbs to deplete target cells. Human IgG1 and human IgG4 were very potent, and mutations within a motif critical for FcγR binding (glutamate 233 to proline, leucine/phenylalanine 234 to valine, and leucine 235 to alanine) completely prevented depletion. Mouse IgG2b was also potent, and mutations to prevent complement activation did not impair depletion with this isotype, as previously shown for human IgG1. In contrast, a mutation that impaired binding to mouse FcγRII (glutamate 318 to alanine) eliminated effector function of mouse IgG2b and also reduced the potency of human IgG4. To reveal potential contributions of domains other than CH2, domain switch mutants were created between human IgG1 and rat IgG2a. Two hybrid mAbs were generated with potencies exceeding anything previously seen in this model. While their mechanism of depletion was not defined, their activity appeared dependent upon interdomain interactions in the Fc region.
Comparisons of the Ability of Human IgG3 Hinge Mutants, IgM, IgE, and IgA2, to Form Small Immune Complexes: A Role for Flexibility and Geometry
Authors: Roux Kenneth H. , Strelets Lioudmila , Brekke Ole Henrik , Sandlie Inger , Michaelsen Terje E.
Published: December 2022

Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb, and the resultant immune complexes were analyzed by negative stain immunoelectron microscopy. Complexes were scored for their geometry (linear versus ring complexes) and size (dimer, trimer, etc.). Ring dimers are the thermodynamically most favorable configuration, unless inhibited by steric and/or flexibility constraints. We found ring dimerization to correlate with the length of the upper, but not middle or lower, hinge. In contrast, the geometry and size of complexes of those molecules lacking formal hinges were unpredictable. A hingeless IgG mutant and native IgE readily formed ring dimers. Remarkably, monomeric IgM formed more ring dimers than any of the other Igs tested, including IgG3. We also tagged the Fab arms and measured the mean Fab-Fab angles and the degree of angular variation for each type of Ig. Surprisingly, IgM proved the most flexible by this assay. In hinged Igs, there was a correlation between length of the upper hinge and Fab-Fab flexibility. In contrast, we found no correlation between the mean Fab-Fab angle in uncomplexed Igs and their ability to dimerize with anti-Id mAb. These data suggest that the physicochemical methods typically used to evaluate molecular flexibility are often of low predictive value when tested in a functional assay.
Heat-KilledListeria monocytogenesas an Adjuvant Converts Established Murine Th2-Dominated Immune Responses into Th1-Dominated Responses
Authors: Yeung V. Peter , Gieni Randall S. , Umetsu Dale T. , DeKruyff Rosemarie H.
Published: December 2022

We investigated the capacity of heat-killed Listeria monocytogenes (HKL), a potent stimulator of the innate immune system, as a vaccine adjuvant to modify both primary and secondary Ag-specific immune responses. Mice immunized with the Ag keyhole limpet hemocyanin (KLH) mixed with HKL generated a KLH-specific primary response characterized by production of Th1 cytokines and large quantities of KLH-specific IgG2a Ab. Moreover, administration of KLH with HKL as an adjuvant reversed established immune responses dominated by the production of Th2 cytokines and high levels of KLH-specific IgE and induced a Th1-type response with high levels of IFN-γ and IgG2a and low levels of IgE and IL-4. Neutralization of IL-12 activity at the time of HKL administration blocked the enhancement of IFN-γ and reduction of IL-4 production, indicating that IL-12, induced by HKL, was responsible for the adjuvant effects on cytokine production. These results suggest that HKL as an adjuvant during immunization can successfully bias the development of Ag-specific cytokine synthesis toward Th1 cytokine production even in the setting of an ongoing Th2-dominated response. Thus, HKL may be clinically effective in vaccine therapies for diseases such as allergy and asthma, which require the conversion of Th2-dominated immune responses into Th1-dominated responses.
Oleic Acid Increases Cell Surface Expression and Activity of CD11b on Human Neutrophils
Authors: Mastrangelo Anthony M. , Jeitner Thomas M. , Eaton John W.
Published: December 2022

Traumatic bone injury frequently results in the release of marrow-derived fatty material into the circulation. This may lead to the syndrome of fat embolism, associated with the generation of free fatty acids, the sequestration of neutrophils in the lungs, and the subsequent development of acute respiratory distress. Neutrophil accumulation in tissues requires their adherence to vascular endothelial cells and involves the β2 integrin, CD11b/CD18 (Mac-1). We now report that the exposure of isolated human neutrophils to oleic acid causes a rapid increase in the cell surface expression and affinity state of CD11b, particularly under acidic conditions that are typical of inflammatory sites. Oleic acid also triggers neutrophil aggregation and neutrophil adherence to both fibrinogen-coated surfaces and confluent cultures of HUVEC. These processes are blocked by CD11b-specific inhibitors, including neutrophil-inhibitory factor and mAbs to CD11b. These observations may help explain the etiology of so-called fat embolism wherein trauma-induced release of fatty material causes pulmonary neutrophil accumulation and the development of acute respiratory distress.
Evidence for a Critical Role for IL-2 in CD40-Mediated Activation of Naive B Cells by Primary CD4 T Cells
Authors: Johnson-Léger Caroline , Christenson Jillian R. , Holman Mary , Klaus Gerry G. B.
Published: December 2022

The interaction of CD40 on B cells with the CD40 ligand (CD40L) on preactivated CD4 T cells is critical for the initiation of T-dependent Ab responses. It is believed that signals via CD40 synergize with cytokines (e.g., IL-4 and IL-5) to drive B cell activation. However, primary T cells preactivated via CD3 alone cannot induce B cell proliferation; we have shown previously that costimulation of T cells via CD3 and CD28 stabilizes the expression of the CD40L, which we propose contributes to their capacity to act as competent helper-effector cells. Here we show that an additional, critical reason why CD3-stimulated CD40L-bearing T cells are incompetent helper cells is because they secrete insufficient IL-2. In contrast, CD28/CD3-activated T cells induce B cells to become IL-2 responsive via a combination of CD40L and IL-2-mediated signals, and these two stimuli subsequently drive B cell proliferation and IgM secretion. We therefore propose that T cells must first encounter Ag in conjunction with CD80/86 on APCs. This leads to the stable expression of CD40L and maximal secretion of IL-2, which together render primary T cells competent to activate B cells in an IL-2-dependent fashion.
Role of T Cells and Germinal Center Formation in the Generation of Immune Responses to the Thymus-Independent Carbohydrate Dextran B512
Authors: Sverremark Eva , Fernandez Carmen
Published: December 2022

Immunization with the thymus-independent (TI) Ag native dextran (DX) B512 induces germinal center (GC) formation in the spleen. However, despite this GC formation, the anti-DX response is poor, and no affinity maturation can be observed. Using cholera toxin (CT) as an adjuvant, splenic as well as humoral responses to DX are improved. In this study, we investigated immune responses against DX in mice lacking TNF receptor I and in athymic mice. The adjuvant effect of CT on these responses was also evaluated. Mice lacking the TNF receptor I allowed us to investigate the role of follicular dendritic cell networks and GC formation in the spleen for the generation of Ab responses to DX, whereas we could investigate the role of T cells in GC development to TI Ags using athymic mice. We found that the humoral immune response to TI DX B512 was not dependent upon T cells or the presence of GCs, although GC development occurred after DX immunization. However, T cells were required for this GC formation, since athymic mice could not develop GCs after immunization with DX. We also show that even if CT is able to directly activate B cells when administered as an adjuvant, the major effect may require T cell participation; this is also the case for TI Ags. In contrast, CT adjuvancy is independent of GC formation.
Human Endothelial Cells Induce and Regulate Cytolytic T Cell Differentiation
Authors: Biedermann Barbara C. , Pober Jordan S.
Published: December 2022

We compared the capacity of cultured human endothelial cells (EC) vs B lymphoblastoid cells (BLC) from the same donor to stimulate allogeneic CD8+ T cells to differentiate into CTL, assaying for allorestricted cytotoxicity, T cell growth, IFN-γ secretion, and perforin expression. The input cell number affected specificity and potency of the resulting CTL. At low input (<105 cells/well), anti-EC CTL were rarely detected. At high input (>106 cells/well), anti-EC CTL developed that displayed unrestricted, low-titer killing and an unstable phenotype. At intermediate input (1.0–2.5 × 105 cells/well), classical class I MHC-restricted, CD8+, and perforin-positive anti-EC CTL developed with reproducible frequencies. However, under all conditions EC were less efficient stimulators than BLC from the same donor. Anti-EC CTL did not kill BLC, whereas anti-BLC CTL killed BLC and EC from the same donor with comparable efficiency. When CD8+ T lymphocytes were grown in the presence of EC and BLC together, the differentiation of anti-BLC CTL was completely suppressed, while the anti-EC response was intact. The inhibition of the allogeneic anti-BLC CTL response was independent of T cell-EC contact, and proliferation of CD8+ T cells was inhibited by EC-conditioned medium. We conclude that EC are competent but less efficient activators of CTL differentiation than are BLC and that EC actively regulate differentiation and/or expansion of allospecific CTL.
Ligation of Cell Surface CD38 Protein with Agonistic Monoclonal Antibody Induces a Cell Growth Signal in Myeloid Leukemia Cells
Authors: Konopleva Marina , Estrov Zeev , Zhao Shourong , Andreeff Michael , Mehta Kapil
Published: December 2022

CD38 is expressed during early stages of differentiation in normal and leukemic myeloid cells. Recently, CD38 has been shown to participate in intracellular signal transduction pathways following its ligation with CD38-specific mAbs. In this study we report that ligation of CD38 by one such agonistic mAb (IB4) induced proliferation of cultured leukemic cells in vitro. In HL-60, KG-1A, NB4, and OCI-AML-3 myeloid leukemia cell lines, IB4 mAb induced an increase in the proliferating cell fraction as determined by cell number, clonogenic assay, and flow cytometric analysis. The presence of Ab caused a dose-dependent increase in the number of CFU and an increase in cell divisions. HL-60-Dox cells (a HL-60-doxorubicin-resistant cell line), which have no detectable CD38 expression, failed to respond to IB4 mAb. The effect of CD38 ligation on cell growth was also evaluated in freshly isolated leukemic cells from patients with acute myelogenous leukemia (AML). A significant increase in the proliferating cell fraction (S+G2M) was observed in 50% of the patients incubated with IB4 mAb. In five of the six AML patients, anti-CD38 mAb stimulated the proliferation of AML colony-forming cells. These results suggest that ligation of CD38 can induce the proliferation of leukemic cells and may play a role in the propagation of leukemic cell clones in certain cohorts of AML patients.
A Molecular Basis for How a Single TCR Interfaces Multiple Ligands
Authors: Boesteanu Alina , Brehm Michael , Mylin Lawrence M. , Christianson Gregory J. , Tevethia Satvir S. , Roopenian Derry C. , Joyce Sebastian
Published: December 2022

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.
Two Diverged Complement Factor B/C2-Like cDNA Sequences from a Teleost, the Common Carp (Cyprinus carpio)1, 2
Authors: Nakao Miki , Fushitani Yuko , Fujiki Kazuhiro , Nonaka Masaru , Yano Tomoki
Published: December 2022

Mammalian complement components factor B and C2 act as proteolytic subunits of the C3 convertases in the alternative and the classical activation pathways, respectively, and are believed to have diverged from a common ancestor by gene duplication. However, it is unclear when the B/C2 duplication occurred. Here, we describe two diverged B/C2-like cDNA clones (B/C2-A and B/C2-B) isolated from a bony fish, the common carp (Cyprinus carpio). B/C2-A shares the same domain structure as the factor B and C2 complement components of vertebrates reported so far and shows a close similarity to zebrafish B and medaka fish B/C2. These teleost sequences show almost the same degree of similarity to C2 and B of higher vertebrates. In contrast, B/C2-B has a novel structural feature in that it contains four short consensus repeat modules and does not have a close relative upon phylogenetic analysis. Northern blotting revealed the presence of two transcripts with different sizes for both the B/C2-A and B/C2-B in the hepatopancreas of the carp. Southern blotting suggested the presence of multiple genes for B/C2-A and a single gene for B/C2-B. Although structural features of B/C2-B are slightly more C2-like than B-like, B/C2-B has a crucial amino acid substitution in the serine protease domain, which makes it unlikely that B/C2-B functions as a C3 convertase. A possible phylogenetic relationship between the two carp sequences and mammalian C2 and B is discussed.
Mechanism of Catecholamine-Mediated Destabilization of Messenger RNA Encoding Thy-1 Protein in T-Lineage Cells
Authors: Wajeman-Chao Sophie A. , Lancaster Susan A. , Graf Jr. Lloyd H. , Chambers Donald A.
Published: December 2022

The Ig superfamily cell surface glycoprotein Thy-1 expressed on immune cells and neurons of rodents and humans is hypothesized to function in cell adhesion and signal transduction in T cell differentiation, proliferation, and apoptosis. This study analyzes effects of cAMP and catecholamines on transcriptional Thy-1 gene expression. Incubation of murine thymocytes or S49 mouse thymoma cells with dibutyryl-cAMP, 8-bromo-cAMP, cholera toxin, norepinephrine, or isoproterenol caused time- and concentration-dependent decreases in levels of Thy-1 mRNA assayed by Northern hybridization or T2 nuclease protection. After 4 h of treatment with 500 μM dibutyryl-cAMP or 8-bromo-cAMP, 1 nM cholera toxin, 100 μM norepinephrine, or 100 μM isoproterenol, Thy-1 mRNA levels were 60 to 96% lower than those of controls. Norepinephrine-mediated decreases in Thy-1 mRNA levels were prevented by the β-adrenergic receptor antagonist propranolol (10 μM). Dibutyryl-cAMP and norepinephrine decreased the apparent half-life of S49 cell Thy-1 mRNA from ≫6 h to 2 to 3 h, whereas nuclear run-on assays showed no cAMP or norepinephrine effect on de novo transcription of the Thy-1 gene. In mutant S49 cells lacking cAMP-dependent protein kinase A, neither dibutyryl cAMP nor norepinephrine affected Thy-1 mRNA levels. These observations show that exogenous cAMP and norepinephrine can induce decreases in steady state Thy-1 mRNA levels in T-lineage cells through posttranscriptional destabilization of Thy-1 mRNA, associated with protein kinase A-mediated protein phosphorylation. Catecholamine-mediated β-adrenergic protein kinase A-dependent Thy-1 mRNA destabilization may be an example of a more general mRNA decay system regulating cellular responses to stress.
The IL-1 Receptor-Related T1 Antigen Is Expressed on Immature and Mature Mast Cells and on Fetal Blood Mast Cell Progenitors
Authors: Moritz Dirk R. , Rodewald Hans-Reimer , Gheyselinck Jacqueline , Klemenz Roman
Published: December 2022

Expression of the T1 gene, also known as ST2, DER4, and Fit-1, has been shown to be associated with cell proliferation. It gives rise to two different mRNAs that encode a receptor-like protein and a soluble molecule representing the ectodomain of the receptor form. Although T1 is a member of the IL-1R family, its biologic function is currently unknown. In this study, we have analyzed the expression of the T1 surface Ag in murine hemopoietic organs. Mast cells (MCs) were shown to be the only identifiable cell lineage that expressed T1 at high levels. T1 expression was found on cultured bone marrow-derived immature MCs. Similarly, freshly isolated connective tissue-type MCs from the i.p. cavity were also shown to express high levels of T1. Interestingly, the earliest detectable committed MC precursor isolated from fetal blood (FB) at day 15.5 of gestation, but not circulating hemopoietic stem cells in FB, also expresses high level of T1. Since FB promastocytes lack expression of the high affinity IgE receptor (FcεRI), T1 expression precedes expression of FcεRI in MC ontogeny. The finding that the T1 Ag is selectively expressed at several stages during development of the MC lineage suggests that this cell surface molecule, in combination with the well-established markers c-Kit and FcεRI, should be valuable for studying the MC lineage.
A His19 to Ala Mutant of Melanoma Growth-Stimulating Activity Is a Partial Antagonist of the CXCR2 Receptor
Authors: Baly Deborah L. , Horuk Richard , Yansura Daniel G. , Simmons Laura C. , Fairbrother Wayne J. , Kotts Claire , Wirth Cindy M. , Gillece-Castro Beth L. , Toy Karen , Hesselgesser Joseph , Allison David E.
Published: December 2022

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.
Activation of the RON Receptor Tyrosine Kinase Inhibits Inducible Nitric Oxide Synthase (iNOS) Expression by Murine Peritoneal Exudate Macrophages: Phosphatidylinositol-3 Kinase Is Required for RON-Mediated Inhibition of iNOS Expression
Authors: Chen Yi-Qing , Fisher James H. , Wang Ming-Hai
Published: December 2022

RON (recepteur d’origine nantais) is a receptor tyrosine kinase expressed in murine peritoneal resident macrophages and activated by macrophage-stimulating protein (MSP). The objectives of this investigation were to study the RON expression in exudate macrophages and the mechanisms by which RON inhibits inducible nitric oxide synthase (iNOS) expression induced by LPS and IFN-γ. We found that mouse peritoneal resident and Con A-elicited macrophages collected on day 3 or day 5 express RON. Acute exudate macrophages collected on day 1 did not express RON. Activation of RON inhibited LPS- and IFN-γ-induced macrophage nitric oxide production and iNOS mRNA accumulation. Similar inhibition was observed also in Raw264.7 macrophage cell lines transfected with human RON cDNA. In these cells, MSP induced RON phosphorylation concomitant with reduced iNOS mRNA expression and protein synthesis. Further, we show that activated RON inhibited the iNOS gene transcription activity as assessed by chloramphenicol acetyltransferase activity in Raw264.7 cells expressing RON. Wortmannin, a specific inhibitor of phosphatidylinositol-3 (PI-3) kinase, prevented the inhibitory effect of RON on the iNOS gene promoter activity and on the nitric oxide production induced by LPS and IFN-γ. These effects were confirmed further by introducing a dominant-inhibitory PI-3 kinase p85 subunit in RON-expressing Rwa264.7 cells. Taken together, our results suggest that RON is expressed in peritoneal macrophages at later stages of inflammation. Activation of RON by MSP in mature exudate macrophages inhibits LPS- and IFN-γ-induced iNOS synthesis. PI-3 kinase is an important effector molecule required for RON-mediated inhibition of iNOS expression in macrophages.
HLA-DQ6/8 Double Transgenic Mice Develop Auricular Chondritis Following Type II Collagen Immunization: A Model for Human Relapsing Polychondritis
Authors: Bradley David S. , Das Pritam , Griffiths Marie M. , Luthra Harvinder S. , David Chella S.
Published: December 2022

We have generated transgenic (tg) mice expressing HLA-DQ8αβ (DQA1*0301/DQB*0302) or HLA-DQ6αβ (DQA1*0103/DQB1*0601) molecules lacking endogenous murine class II expression (Aβ0) to investigate the ability of these HLA class II to present type II collagen (CII) and induce collagen-induced arthritis. The DQ8αβ tg mice responded strongly to CII, developing severe arthritis, while DQ6αβ tg mice were nonresponsive to CII. The addition of the mixed haplotype DQ8α6β molecule did not significantly influence CII reactivity. To examine the interaction of DQ6αβ and DQ8αβ molecules in vivo, we generated double tg DQ6αβ/8αβ (Aβ0) mice expressing both the α- and β-chains of DQ6 and DQ8 molecules by mating DQ6αβ (Aβ0) and DQ8αβ (Aβ0) tg mice. CII-immunized DQ6αβ/8αβ tg mice developed severe experimental polychondritis, exhibiting both polyarthritis and auricular chondritis. The clinical, serologic, and histologic manifestations of experimental polychondritis are similar to those symptoms in human relapsing polychondritis. The susceptibility of DQ6αβ/8αβ tg mice compared with resistance in the parental strains suggests that expression of both the DQ6αβ and DQ8αβ tgs, unique to the DQ6αβ8αβ tg strain, is important in susceptibility to experimental polychondritis. The DQ6αβ/8αβ tg mice provide a model to investigate putative autoantigens and the mechanisms of pathogenesis involved in relapsing polychondritis as well as the influence of the expression of multiple HLA class II molecules on the disease process.
Cutting Edge: Immunoregulation of Th Cells by Naturally Processed Peptide Antagonists
Authors: Carson Richard T. , Desai Dharmesh D. , Vignali Kate M. , Vignali2 Dario A. A.
Published: December 2022

Th cells recognize protein Ags as short peptides bound to MHC class II molecules. Altered peptide ligands can antagonize (inhibit) T cell responses to stimulatory peptides. Peptides generated by APC may contain peptide flanking residues (PFR), which lie outside the minimal binding epitope and can be recognized by the TCR. Our data show that PFR-dependent T cells were found to be potently antagonized by peptides that lack PFR and responded poorly to native protein or the immunogenic epitope delivered by a recombinant influenza virus. These data provide the first evidence that Ag processing generates both stimulatory and antagonist peptides from a single immunogenic epitope, an observation that may have important implications for T cell immunoregulation and autoimmunity.
Use of Intranasal IL-12 to Target Predominantly Th1 Responses to Nasal and Th2 Responses to Oral Vaccines Given with Cholera Toxin
Authors: Marinaro Mariarosaria , Boyaka Prosper N. , Jackson Raymond J. , Finkelman Fred D. , Kiyono Hiroshi , Jirillo Emilio , McGhee Jerry R.
Published: December 2022

We have investigated the effects of IL-12 and cholera toxin (CT) on the immune response to tetanus toxoid (TT) given by intranasal or oral routes. CT inhibited IL-12-induced IFN-γ secretion both in vivo and in vitro. Intranasal administration of IL-12 to mice nasally immunized with the combined vaccine of TT and CT resulted in increased TT-specific IgG2a and IgG3 Abs, while IgG1 and IgE Ab responses were markedly reduced. This shift of the CT-induced immune response toward Th1 type was associated with TT-specific CD4+ T cells secreting IFN-γ and reduced levels of Th2-type cytokines (i.e., IL-4, IL-5, IL-6, and IL-10). In contrast, intranasal IL-12 enhanced the CT-induced serum IgG1 and IgE Ab responses in mice given the combined vaccine orally. IFN-γ secretion by TT-specific CD4+ T cells was also enhanced; however, Th2-type cytokine responses were predominant. Mucosal secretory IgA responses to oral or nasal vaccines were not affected by intranasal IL-12. Thus, intranasal IL-12 delivery influences Th cell subset development in mucosal inductive sites that are dependent on the route of vaccine delivery.
Structural Features of Autoreactive TCR That Determine the Degree of Degeneracy in Peptide Recognition
Authors: Hausmann Stefan , Martin Margarita , Gauthier Laurent , Wucherpfennig Kai W.
Published: December 2022

Structural aspects of human TCRs that allow the activation of autoreactive T cells by diverse microbial peptides were examined using two human myelin basic protein (MBP)-specific T cell clones. The TCR sequences of these clones differed only in the N region of TCR-α and -β since the clones had the same Vα-Jα and Vβ-Jβ rearrangements. The two clones had a similar fine specificity for the MBP peptide, except for the P5 position of the peptide (lysine). In the crystal structure of the HLA-DR2/MBP peptide complex, P5 lysine is a prominent, solvent-exposed residue in the center of the DR2/MBP peptide surface. Five microbial peptides with conservative or nonconservative changes at the P5 position (lysine to arginine, serine, or proline) activated one of these clones. In contrast, the other clone was activated only by three of these peptides which had a conservative lysine to arginine change at P5. The degree of specificity/degeneracy in recognition of the P5 side chain was the key difference between these TCRs since the Escherichia coli/Haemophilus influenzae peptide stimulated both clones when the P5 position was substituted from serine to arginine. These results demonstrate that the complementarity-determining region 3 loops contribute to the degree of degeneracy in peptide recognition by human MBP-specific TCRs.
Characterization of the Signal Transduction Pathway Activated in Human Monocytes and Dendritic Cells by MPIF-1, a Specific Ligand for CC Chemokine Receptor 1
Authors: Nardelli Bernardetta , Tiffany H. Lee , Bong Gary W. , Yourey Pamela A. , Morahan Diana K. , Li Yuling , Murphy Philip M. , Alderson Ralph F.
Published: December 2022

The receptor specificity and signal transduction pathway has been identified and characterized for a truncated form of myeloid progenitor inhibitory factor-1 (MPIF-124–99). MPIF-1 binds specifically to sites, in particular CCR1, shared with macrophage inflammatory protein-1α (MIP-1α) on the surface of human monocytes and dendritic cells, as inferred by its ability to compete for [125I]MIP-1α, but not for [125I]MIP-1β or [125I]monocyte chemotactic protein-1(MCP-1) binding to intact cells. Based on calcium flux, MPIF-1 is an agonist on CCR1-transfected HEK-293 cells, monocytes, and dendritic cells, but not on CCR5-, CCR8-, or CX3CR1-transfected cells. The inhibitory effect of guanosine 5′-O-(3-thio-triphosphate) (GTP-γS) or pertussis toxin pretreatment on MPIF-1 binding and calcium mobilization, respectively, indicates the involvement of G proteins in the interaction of MPIF-1 and its receptor(s). The increase in intracellular free calcium concentration following MPIF-1 treatment is mainly due to the influx of calcium from an extracellular pool. However, a portion of the intracellular free calcium concentration is derived from a phospholipase C inhibitor-sensitive intracellular pool. MPIF-1 induces a rapid dose-dependent release of [3H]arachidonic acid from monocytes that is dependent on extracellular calcium and is blocked by phospholipase A2 (PLA2) inhibitors. Furthermore, PLA2 activation is shown to be necessary for filamentous actin formation in monocytes. Thus, the MPIF-1 signal transduction pathway appears to include binding to CCR1; transduction by G proteins; effector function by phospholipase C, protein kinase C, calcium flux, and PLA2; and cytoskeletal remodeling.
Inhibition of Allergic Airway Inflammation in Mice Lacking Nitric Oxide Synthase 2
Authors: Xiong Yelin , Karupiah Gunasegaran , Hogan Simon P. , Foster Paul S. , Ramsay Alistair J.
Published: December 2022

We have used mice rendered deficient for nitric oxide synthase 2 (NOS2) production to study the role of inducible nitric oxide (NO) in the pathogenesis of allergic airways disease. Using a model with OVA as aeroallergen, we show that the manifestations of disease, including infiltration of inflammatory cells, particularly eosinophils, loss of structural integrity of the airway walls, microvascular leakage, pulmonary edema, and airway occlusion are markedly less severe in the NOS2 mutants than in wild-type animals. Indeed, NOS2-deficiency resulted in a 55–60% reduction in both circulatory and pulmonary eosinophil numbers following aeroallergen treatment, although eosinophil maturation or efflux from the bone marrow was not suppressed. There were no obvious differences in levels of airway hyperreactivity recorded in OVA-treated wild-type and NOS2-deficient mice. Interestingly, the suppression of allergic inflammation was accompanied by marked increases in T cell production of IFN-γ but not by any obvious reduction in the secretion of either IL-4 or IL-5, nor by major changes in the IgG1 and IgE OVA-specific serum Ig profiles in the mutants. The markedly enhanced production of IFN-γ in NOS2−/− mice was apparently responsible for the suppression of both eosinophilia and disease, as in vivo depletion of this factor restored allergic pathology in these animals. Our data indicate that NOS2 promotes allergic inflammation in airways via down-regulation of IFN-γ activity and suggest that inhibitors of this molecule may represent a worthwhile therapeutic strategy for allergic diseases including asthma.
Regulation of IL-6 Synthesis in Human Peripheral Blood Mononuclear Cells by C3a and C3adesArg
Authors: Fischer Wolfgang H. , Jagels Mark A. , Hugli Tony E.
Published: December 2022

The anaphylatoxin C3a has been reported to have immunomodulatory effects on a number of different cell types. In this study we investigated the effects of C3a and C3adesArg on gene expression and protein secretion of IL-6 in human PBMCs, either alone or in combination with LPS or IL-1β. C3a or C3adesArg alone exhibited no effect on the expression or secretion of IL-6. However, when PBMC were stimulated with LPS or IL-1β, both C3a and C3adesArg were found to enhance IL-6 release by PBMC in a dose-dependent manner. Since C3a has been shown to induce PGE2 production by monocytes, and PGE2 has been shown to influence cytokine production, we investigated the potential role of PGE2 in C3a-mediated enhancement of LPS- and IL-1β-induced IL-6 production. Indomethacin blocked PGE2 release, but had no influence on the observed effects of C3a, suggesting that the effects of C3a on IL-6 production are independent of PGE2 formation by monocytes. Northern blot analysis showed that C3a as well as C3adesArg enhanced LPS-induced mRNA levels for IL-6. Pretreatment of PBMCs with pertussis toxin blocked the functions of C3a and C3adesArg, indicating that the actions of these two molecules are mediated by a G protein-coupled pathway. Furthermore, we investigated the effects of C3a and C3adesArg on induction of NF-κB and activating protein-1 binding. Both molecules enhanced LPS-induced NF-κB and activating protein-1 binding activity. These results demonstrate the capacity of intact C3a and its circulating des-Arg form to exert immunmodulatory effects in vitro.
Effect of Taxol and Taxotere on Gene Expression in Macrophages: Induction of the Prostaglandin H Synthase-2 Isoenzyme
Authors: Moos Philip J. , Muskardin D. T. , Fitzpatrick F. A.
Published: December 2022

Induction of genes encoding cytokines or other, unidentified proteins may contribute to the pharmacological effects of taxol. We hypothesized that prostaglandin H synthase-2 (PGHS-2) was one of the unidentified genes induced by taxol. Taxol alone or taxol plus IFN-γ increased PGE2 formation, PGHS-2 protein expression, and PGHS-2 mRNA expression in RAW 264.7 murine macrophages. The kinetics for mRNA induction, protein expression, and catalysis were self-consistent. A selective inhibitor of PGHS-2 blocked PGE2 formation by cells incubated with taxol; a selective inhibitor of PGHS-1 had no effect. A glucocorticoid blocked the induction of mRNA, the expression of PGHS-2 protein, and the formation of PGE2. Neither taxol alone nor taxol plus IFN-γ altered the expression of the PGHS-1 isoenzyme in RAW 264.7 cells. Taxotere, an analogue that stabilizes microtubules as potently as taxol, did not alter the expression of PGHS-2, implying that its induction in RAW 264.7 murine macrophages did not originate from microtubule stabilization. Taxol and taxotere each induced PGHS-2 expression in human monocytes suspended in 10% human serum. However, human monocytes suspended in 10% bovine serum responded only to LPS, not to taxol or taxotere, implying that they act independently of the LPS-mimetic process that is prominent in mice. Taxol induced PGHS-2 in human and murine monocytes via a p38 mitogen-associated protein kinase pathway. The inclusion of PGHS-2 among the early response genes induced in leukocytes may be relevant to the beneficial and adverse effects encountered during taxol administration.
IL-13 Production by NK Cells: IL-13-Producing NK and T Cells Are Present In Vivo in the Absence of IFN-γ
Authors: Hoshino Tomoaki , Winkler-Pickett Robin T. , Mason Anna T. , Ortaldo John R. , Young Howard A.
Published: December 2022

In this study, we demonstrate that human NK cells, human NK clones, the human NK cell line (NK3.3), and a population of murine NK cells can produce the type 2 cytokine IL-13 in response to IL-2 or phorbol myristate acetate plus ionomycin. IL-2 rapidly induced new IL-13 mRNA and protein synthesis in the NK3.3 cell line. Six of 12 human NK clones tested produced IL-13 protein in response to IL-2 or phorbol myristate acetate and ionomycin. Intracellular analysis revealed that ∼2% of human peripheral NK cells produced IL-13 protein in response to IL-2. Isolated NK cells from SCID and RAG-2 knockout (−/−) mice that lack T and B cells as well as normal mice also can produce IL-13 mRNA and protein in response to IL-2. We hypothesized that in the absence of IFN-γ, IL-13-producing NK cells may predominate in vivo. Utilizing IFN-γ knockout (−/−) mice as a model system, IL-2-activated liver NK and T cells expressed 10-fold more IL-13 and IL-5 mRNA and protein than normal controls following IL-2 treatment in vitro. These results suggest that in the absence of IFN-γ, an IL-13- and IL-5-producing NK and T cells predominate in vivo. The existence of this cell type has important implications in innate immunity given that the balance between IFN-γ and IL-13/IL-5-producing NK cells may influence the early development of a cell-mediated or humoral immune response.
Interaction of Sulfonamide Derivatives with the TCR of Sulfamethoxazole-Specific Human αβ+ T Cell Clones
Authors: von Greyerz Salome , Zanni Martin P. , Frutig Karin , Schnyder Benno , Burkhart Christoph , Pichler Werner J.
Published: December 2022

Drugs like sulfamethoxazole (SMX) or lidocaine can be presented to specific human αβ+ T cell clones (TCC) by undergoing a noncovalent association with MHC-peptide complexes on HLA-matched APCs. For a better understanding of the molecular basis of the recognition of such drugs by specific TCC, we investigated 1) the fine specificity of the recognizing TCR, 2) the dose-response relationship for the induction of proliferation or cytokine production, and 3) the mechanism of TCR triggering. For that purpose, we tested the reactivity of 11 SMX-specific CD4+ TCC and 2 SMX-specific CD8+ TCC to a panel of 13 different sulfonamide derivatives bearing the same core structure. Five of 13 clones recognized only SMX, while all other clones were responding to as many as 6 different compounds. Some of the compounds needed up to two orders of magnitude higher concentrations than SMX to stimulate TCC, thereby displaying features of weak agonists. Different clones showed clear differences in the minimal drug concentration required for the induction of a proliferative response. Therefore, weaker or stronger agonistic properties were not a characteristic of a given sulfonamide derivative but rather an intrinsic property of the reacting TCR. Finally, the number of down-regulated TCRs was a logarithmic function of the ligand concentration, implicating that specific T cells were activated by serial TCR engagement. Our data demonstrate that, despite the special way of presentation, nonpeptide Ag like drugs appear to interact with the TCR of specific T cells in a similar way as peptide Ags.
Mice Lacking Mature T and B Lymphocytes Develop Arthritic Lesions after Immunization with Type II Collagen
Authors: Plows David , Kontogeorgos George , Kollias George
Published: December 2022

Collagen-induced arthritis in DBA/1 mice is a widely used experimental model of rheumatoid arthritis. The induction phase of the disease is thought to be dependent upon MHC-restricted T and B cell-mediated immune responses to type II collagen, but an influence of additional non-MHC-restricted mechanisms has also been proposed. In this study, we report that type II collagen immunization of DBA/1 mice lacking mature T and B lymphocytes resulted in the development of arthritic lesions, which were characterized by synovial hyperplasia with occasional inflammation as well as cartilage and bone destruction. The specificity of disease induction to type II collagen was confirmed, because arthritis could not be induced when control preparations of OVA or adjuvant alone were administered. A delay in clinical disease onset and a reduction in severity between lymphocyte-positive and -negative DBA/1 mice confirmed that lymphocytes play an important role in disease; however, similar pathologic features and normal incidence suggest that lymphocyte-independent mechanisms of disease induction also operate in the standard collagen-induced arthritis model. We conclude that adaptive immune responses are not the only arthritogenic mechanism and hypothesize that the nonantigenic properties of type II collagen can also lead to arthritis.
Inhibition of Experimental Autoimmune Encephalomyelitis by a Tyrosine Kinase Inhibitor
Authors: Constantin Gabriela , Laudanna Carlo , Brocke Stefan , Butcher Eugene C.
Published: December 2022

Migration of lymphocytes from the blood into the brain is a critical event in the pathogenesis of experimental autoimmune encephalomyelitis. Lymphocyte adhesion to brain endothelium is the first step in lymphocyte entry into the central nervous system, leading subsequently to myelin damage and paralysis. In this paper we show that the tyrosine kinase inhibitor, tyrphostin AG490, prevents binding of freshly isolated mouse lymph node cells and of in vivo activated lymphocytes to endothelium of inflamed brain in Stamper-Woodruff adhesion assays. Moreover, AG490 inhibits adhesion of encephalitogenic T cell lines to purified ICAM-1 and VCAM-1, molecules implicated in T cell recruitment into the central nervous system. In contrast, 2-h treatment of T cell lines with high doses of tyrphostin AG490 have no effect on the viability, intracellular calcium elevation induced by Con A or TCR cross-linking, proliferation, or TNF production by Ag-stimulated T cell lines. Systemic administration of AG490 prevents the accumulation of leukocytes in the brain and the development of experimental autoimmune encephalomyelitis induced by proteolipid protein, peptide 139–151-specific T cell lines in SJL/J mice. Blood leukocytes isolated from mice treated with tyrphostin AG490 are less adhesive on purified very late Ag-4 ligands compared with adhesion of leukocytes from control animals. Our results suggest that inhibition of signaling pathways involved in lymphocyte adhesion may represent a novel therapeutic approach for demyelinating diseases.
Identification of HLA-A3 and -B7-Restricted CTL Response to Hepatitis C Virus in Patients with Acute and Chronic Hepatitis C
Authors: Chang Kyong-Mi , Gruener Norbert H. , Southwood Scott , Sidney John , Pape Gerd R. , Chisari Francis V. , Sette Alessandro
Published: December 2022

The inverse relationship between peripheral blood CTL responsiveness to multiple hepatitis C virus (HCV) epitopes and viral titer in patients with persistent HCV infection suggests that enhancement of the CTL response might result in viral clearance. Since several HLA-A2-restricted HCV CTL epitopes are already known, we aimed to identify CTL epitopes restricted by other HLA types in an effort to expand the epitope repertoire available for T cell-mediated therapeutic vaccine development. Scanning of 14 different HCV genome sequences for the presence of conserved peptides containing the HLA-A3 and -B7 motifs revealed 9- to 10-mer peptides that were synthesized and assayed for binding to HLA-A3, -B7 supertype molecules. Peptides with good HLA-binding affinities and cross-reactivities with at least three of five most common molecules of each supertype were tested for the ability to stimulate a memory CTL response in the peripheral blood from selected HCV-infected patients and normal seronegative donors in vitro. We identified eight HLA-A3 supertype-restricted CTL epitopes and one HLA-B7 supertype-restricted CTL epitope that were recognized by infected patients but not by healthy seronegative donors. HLA class I serotyping of 158 chronically infected patients revealed that 80% expressed one or more of HLA molecules belong to either the A2, A3, or B7 supertypes. In conclusion, the epitopes, herein identified combined with previously defined HLA-A2-restricted CTL epitopes, should be useful for the design of an ethnically unbiased, therapeutic CTL vaccine for the treatment of patients with chronic HCV infection.