bluuebunny/crossref_metadata_2025 huggingface dataset:
Primed T Cells Are More Resistant to Fas-Mediated Activation-Induced Cell Death than Naive T Cells
Authors: Inaba Motoko , Kurasawa Kazuhiro , Mamura Mizuko , Kumano Kotaro , Saito Yasushi , Iwamoto Itsuo
Published: December 2022

Memory T cells respond in several functionally different ways from naive T cells and thus function as efficient effector cells. In this study we showed that primed T cells were more resistant to Fas-mediated activation-induced cell death (AICD) than naive T cells using OVA-specific TCR transgenic DO10 mice and Fas-deficient DO10 lpr/lpr mice. We found that apoptosis was efficiently induced in activated naive T cells at 48 and 72 h after Ag restimulation (OVA peptide; 0.3 and 3 μM), whereas apoptosis was not significantly increased in activated primed T cells at 24–72 h after Ag restimulation. We further showed that the resistance to AICD in primed T cells was due to the decreased sensitivity to apoptosis induced by Fas-mediated signals, but TCR-mediated signaling equally activated both naive and primed T cells to induce Fas and Fas ligand expressions. Furthermore, we demonstrated that primed T cells expressed higher levels of Fas-associated death domain-like IL-1β-converting enzyme inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, at 24–48 h after Ag restimulation than naive T cells. In addition, Bcl-2 expression was equally observed between activated naive and primed T cells after Ag restimulation. Thus, these results indicate that naive T cells are sensitive to Fas-mediated AICD and are easily deleted by Ag restimulation, while primed/memory T cells express higher levels of FLIP after Ag restimulation, are resistant to Fas-mediated AICD, and thus function as efficient effector cells for a longer period.
Thymus-Derived Glucocorticoids Set the Thresholds for Thymocyte Selection by Inhibiting TCR-Mediated Thymocyte Activation
Authors: Vacchio Melanie S. , Lee Jan Y. M. , Ashwell Jonathan D.
Published: December 2022

Selection processes in the thymus eliminate nonfunctional or harmful T cells and allow the survival of those cells with the potential to recognize Ag in association with self-MHC-encoded molecules (Ag/MHC). We have previously demonstrated that thymus-derived glucocorticoids antagonize TCR-mediated deletion, suggesting a role for endogenous thymic glucocorticoids in promoting survival of thymocytes following TCR engagement. Consistent with this hypothesis, we now show that inhibition of thymus glucocorticoid biosynthesis causes an increase in thymocyte apoptosis and a decrease in recovery that are directly proportional to the number of MHC-encoded molecules present and, therefore, the number of ligands available for TCR recognition. Expression of CD5 on CD4+CD8+ thymocytes, an indicator of TCR-mediated activation, increased in a TCR- and MHC-dependent manner when corticosteroid production or responsiveness was decreased. These results indicate that thymus-derived glucocorticoids determine where the window of thymocyte selection occurs in the TCR avidity spectrum by dampening the biological consequences of TCR occupancy and reveal that glucocorticoids mask the high percentage of self-Ag/MHC-reactive thymocytes that exist in the preselection repertoire.
On the Role of Feedback in Promoting Conflicting Goals of the Adaptive Immune System
Authors: Segel Lee A. , Bar-Or Ruth Lev
Published: December 2022

We explored here the implications of two premises. 1) In their response over days or weeks to pathogen invasion, cells of the immune system combine several overlapping and perhaps contradictory goals. 2) The immune system has ways to monitor progress toward these goals via receptors that bind chemicals whose concentrations are related to such progress. We illustrate with simple mathematical models how such monitoring can lead to feedbacks that improve the efficiency of a given effector type in accomplishing its specialized task, and also how feedbacks can shift the balance among a variety of effectors toward a preponderance of the more effective. Specific suggestions are given for feedback molecules.
Human Blood Dendritic Cell-Like B Cells Isolated by the 5G9 Monoclonal Antibody Reactive with a Novel 220-kDa Antigen
Authors: Zhong Rui-Kun , Donnenberg Albert D. , Zhang Hai-Fan , Watkins Simon , Zhou Jie-Hua , Ball Edward D.
Published: December 2022

We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40–60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either κ- or λ-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9− PBMC, 5G9− B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.
A CD1a+/CD11c+ Subset of Human Blood Dendritic Cells Is a Direct Precursor of Langerhans Cells
Authors: Ito Tomoki , Inaba Muneo , Inaba Kayo , Toki Junko , Sogo Shinji , Iguchi Tomoko , Adachi Yasushi , Yamaguchi Kazuyuki , Amakawa Ryuichi , Valladeau Jenny , Saeland Sem , Fukuhara Shirou , Ikehara Susumu
Published: December 2022

Based on the relative expression of CD11c and CD1a, we have identified three fractions of dendritic cells (DCs) in human peripheral blood, including a direct precursor of Langerhans cells (LCs). The first two fractions were CD11c+ DCs, comprised of a major CD1a+/CD11c+ population (fraction 1), and a minor CD1a−/CD11c+ component (fraction 2). Both CD11c+ fractions displayed a monocyte-like morphology, endocytosed FITC-dextran, expressed CD45RO and myeloid markers such as CD13 and CD33, and possessed the receptor for GM-CSF. The third fraction was comprised of CD1a−/CD11c− DCs (fraction 3) and resembled plasmacytoid T cells. These did not uptake FITC-dextran, were negative for myeloid markers (CD13/CD33), and expressed CD45RA and a high level of IL-3Rα, but not GM-CSF receptors. After culture with IL-3, fraction 3 acquired the characteristics of mature DCs; however, the expression of CD62L (lymph node-homing molecules) remained unchanged, indicating that fraction 3 can be a precursor pool for previously described plasmacytoid T cells in lymphoid organs. Strikingly, the CD1a+/CD11c+ DCs (fraction 1) quickly acquired LC characteristics when cultured in the presence of GM-CSF + IL-4 + TGF-β1. Thus, E-cadherin, Langerin, and Lag Ag were expressed within 1 day of culture, and typical Birbeck granules were observed. In contrast, neither CD1a−/CD11c+ (fraction 2) nor CD1a−/CD11c− (fraction 3) cells had the capacity to differentiate into LCs. Furthermore, CD14+ monocytes only expressed E-cadherin, but lacked the other LC markers after culture in these cytokines. Therefore, CD1a+/CD11c+ DCs are the direct precursors of LCs in peripheral blood.
IL-10 Transgenic Mice Present a Defect in T Cell Development Reminiscent to SCID Patients
Authors: Rouleau Matthieu , Cottrez Françoise , Bigler Mike , Antonenko Sevtlana , Carballido José M. , Zlotnik Albert , Roncarolo Maria-Grazia , Groux Hervé
Published: December 2022

To analyze the effect of IL-10 overexpressed by APCs as observed in some SCID patients, we have expressed the human IL-10 cDNA under the control of the murine MHC class II promoter in transgenic mice. Similar to SCID patients, these mice presented a defect in T cell maturation characterized by a rapid thymic aplasia that started after birth. The blockage in T cell maturation was strictly restricted to TCR-αβ T cells as the absolute number of thymic dendritic, TCR-γδ and NK1.1 T cells were equivalent to control littermates. Crossing IL-10 transgenic mice with TCR transgenic mice or treatment with staphylococcal enterotoxin B showed that the defect was not related to the impairment of positive or negative selection. However, repopulating of IL-10 transgenic mouse-fetal thymic organ culture with different stages of triple negative T cells isolated from control mice showed that the blockage occurred specifically at the pre-T cell stage and was reverted by treatment with blocking anti-IL-10 mAbs. These results demonstrate that IL-10 regulates T cell maturation and that dysregulation of IL-10 expression can lead to severe T cell immunodeficiency.
A Novel CD8 T Cell-Restricted CD45RB Epitope Shared by CD43 Is Differentially Affected by Glycosylation
Authors: Carlow Douglas A. , Ardman Blair , Ziltener Hermann J.
Published: December 2022

The mAb 1B11 has been characterized as recognizing the activation-associated glycoform of murine CD43, a heavily O-glycosylated protein implicated in leukocyte homing. When hemopoietic cells from CD43−/− mice were stained with 1B11, CD43-independent binding of 1B11 was observed on peripheral CD8 T cells and at low levels on thymocytes, while no binding was detected on CD4 T cells, B cells, or bone marrow cells. Levels of 1B11 staining were comparable in lymph node CD8+ T cells from both CD43−/− mice and CD43+/+ mice. We sought to identify the CD43-independent target of 1B11 expressed on CD8 T cells. Previous work had demonstrated that neuraminidase treatment of lymph node cells (LNC) enhanced 1B11 binding on CD43+/+ LNC; this enhancement was also observed in CD43−/− LNC. We show that neuraminidase-enhanced 1B11 binding in CD43−/− LNC and EL4 thymoma cells is CD43 independent and that 1B11 detects a novel target of apparent mass of ∼200 kDa identified as a hyposialylated form of CD45RB preferentially expressed on peripheral CD8, but not CD4, T cells. Our data also show that the recognition of CD43 and CD45RB by 1B11 is differentially affected by O-linked glycosylation and sialic acid. Whereas 1B11 recognition of CD43 on activated T cells required both core 2 O-glycan branching and sialic acid, 1B11 recognition of CD45 only occurred in the absence of both core 2 glycosylation and sialic acid.
Protective Immune Responses Induced by Vaccination with an Expression Genomic Library ofLeishmania major
Authors: Piedrafita David , Xu Damo , Hunter David , Harrison Robert A. , Liew Foo Y.
Published: December 2022

To develop an effective vaccine against the intracellular protozoan parasite Leishmania spp., we investigated the feasibility of expression library immunization (ELI) in the mouse. Genomic expression libraries of L. major were constructed and used to immunize mice. One of the three libraries (L1, with 105 clones) induced a significant protective immune response and delayed the onset of lesion development in highly susceptible BALB/c mice after i.m. immunization, compared with control mice immunized with the empty vector (EV). L1 was then divided into five sublibraries of ∼2 × 104 clones each. Mice immunized with one of the sublibraries (SL1A) developed an even stronger protective effect than that induced by L1. SL1A was further divided into 20 sublibraries (SL2) of ∼103 clones each. One of the SL2 libraries (SL2G) induced a strong protective effect against L. major infection. In direct comparative studies, the protective effect of the sublibraries was in the order of SL2G > SL1A > L1. Lymphoid cells from mice vaccinated with SL2G produced more IFN-γ and NO, compared with cells from control mice injected with EV. Serum from the vaccinated mice also contained more parasite-specific IgG2a Ab, compared with controls. Therefore, these data demonstrate that ELI is feasible against this complex intracellular parasitic infection, by preferentially inducing the development of Th1 responses. Furthermore, by sequential division of the libraries, this approach may be used to enrich and identify protective genes for effective gene vaccination against other parasitic infections.
Cellular Immune Responses Are Essential for the Development of Helicobacter felis-Associated Gastric Pathology
Authors: Roth Kevin A. , Kapadia Sharookh B. , Martin Steven M. , Lorenz Robin G.
Published: December 2022

The bacteria Helicobacter pylori is a major human pathogen that infects over half of the world’s population. Infection initiates a series of changes in the gastric mucosa, beginning with atrophic gastritis and leading in some patients to peptic ulcer disease, mucosa-associated lymphomas, and gastric adenocarcinoma. Although this cascade of events clearly occurs, little is known about the role of the host immune response in disease progression. We have utilized the C57BL/6 Helicobacter felis mouse model to critically analyze the role of the adaptive immune response in the development of Helicobacter-associated gastric pathology. Infection of B and T cell-deficient RAG-1−/− mice or T cell-deficient TCRβδ−/− mice with H. felis resulted in high levels of colonization, but no detectable gastric pathology. Conversely, infection of B cell-deficient μMT mice resulted in severe gastric alterations identical with those seen in immunocompetent C57BL/6-infected mice, including gastric mucosal hyperplasia and intestinal metaplasia. These results demonstrate that the host T cell response is a critical mediator of Helicobacter-associated gastric pathology, and that B cells and their secreted Abs are not the effectors of the immune-mediated gastric pathology seen after H. felis infection. These results indicate that in addition to specific Helicobacter virulence factors, the host immune response is an important determinant of Helicobacter-associated disease.
Regulation of Macrophage Chemokine Expression by Lipopolysaccharide In Vitro and In Vivo
Authors: Kopydlowski Karen M. , Salkowski Cindy A. , Cody M. Joshua , van Rooijen Nico , Major Jennifer , Hamilton Thomas A. , Vogel Stefanie N.
Published: December 2022

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-γ-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1α, MIP-1β, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-γ selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1α, and MIP-1β mRNA and/or protein. Like the response to IFN-γ, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-γ-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1α, MIP-1β, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.
Normal Immune Function of Monocyte-Derived Dendritic Cells from HIV-Infected Individuals: Implications for Immunotherapy
Authors: Chougnet Claire , Cohen Sandra S. , Kawamura Tatsuyoshi , Landay Alan L. , Kessler Harold A. , Thomas Elaine , Blauvelt Andrew , Shearer Gene M.
Published: December 2022

Dendritic cells (DC) are the most potent cells involved in the generation of primary and secondary immune responses. To assess the feasibility of using autologous DC as immunotherapy for HIV disease, we analyzed a variety of immune parameters using DC isolated from HIV-infected (HIV+) individuals, as well as DC obtained from HIV-uninfected (HIV−) individuals infected in vitro with HIV. After stimulation with recombinant CD40 ligand (CD40LT), cytokine and β-chemokine production were similar by DC from HIV− donors infected in vitro with the CCR5-using HIV Ba-L strain (n = 8) compared with uninfected DC from the same donors. Production of β-chemokines, but not of cytokines, was increased by a CXCR4-using IIIB strain-infected DC (n = 7). Stimulation of HIV-infected DC with CD40LT decreased infection in Ba-L-infected DC, but had no effect on IIIB-infected DC. Consistent with this finding, CD40LT down-regulated CCR5 and up-regulated CXCR4 expression on DC. Monocyte-derived DC were also propagated from 15 HIV+ and 13 HIV− donors. They exhibited similar expression of costimulatory molecules and produced similar amounts of IL-12, IL-10, and β-chemokines, following stimulation. By contrast, stimulated PBMC from HIV+ patients exhibited decreased IL-12 and increased IL-10 production. In summary, phenotype, cytokine secretion, and β-chemokine production by DC from HIV+ individuals were normal. These cells may prove useful in boosting cellular immune responses in HIV+ individuals.
Abnormal NF-κB Activity in T Lymphocytes from Patients with Systemic Lupus Erythematosus Is Associated with Decreased p65-RelA Protein Expression
Authors: Wong Henry K. , Kammer Gary M. , Dennis Greg , Tsokos George C.
Published: December 2022

Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-κB contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-κB protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-κB binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-κB activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-κB binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-κB activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-κB activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.
Cutting Edge: Developmental Switches in Chemokine Responses During T Cell Maturation
Authors: Campbell James J. , Pan Junliang , Butcher Eugene C.
Published: December 2022

We show that developmental transitions during thymocyte maturation are associated with dramatic changes in chemotactic responses to chemokines. Macrophage-derived chemokine, a chemokine expressed in the thymic medulla, attracts thymocytes only during a brief window of development, between the late cortical and early medullary stages. All medullary phenotypes (CD4 or CD8 single positive) but not immature thymocytes respond to the medullary stroma-expressed (and secondary lymphoid tissue-associated) chemokines secondary lymphoid-tissue chemokine and macrophage inflammatory protein-3β. The appearance of these responses is associated with the phenotypic stage of cortex to medulla migration and with up-regulation of mRNA for the receptors CCR4 (for macrophage-derived chemokine and thymus and activation-regulated chemokine) and CCR7 (for secondary lymphoid-tissue chemokine and macrophage inflammatory protein-3β). In contrast, most immature and medullary thymocytes migrate to thymus-expressed chemokine, an ability that is lost only with up-regulation of the peripheral homing receptor L-selectin during the latest stages of thymocyte maturation associated with export to the periphery. Developmental switches in chemokine responses may help regulate critical migratory events during T cell development.
Cutting Edge: Activation of NK T Cells by CD1d and α-Galactosylceramide Directs Conventional T Cells to the Acquisition of a Th2 Phenotype
Authors: Singh Nagendra , Hong Seokmann , Scherer David C. , Serizawa Isao , Burdin Nicolas , Kronenberg Mitchell , Koezuka Yasuhiko , Van Kaer Luc
Published: December 2022

NK T cells recognize glycolipid Ags such as α-galactosylceramide (α-GalCer) presented by the MHC class I-like molecule CD1d. In this paper we have studied the in vivo effects of α-GalCer on the generation of adaptive immune responses. Treatment of mice with α-GalCer resulted in rapid activation of NK T cells and production of the cytokines IL-4 and IFN-γ. However, after this initial stimulation, NK T cells became polarized for the production of IL-4. Further, as soon as 6 days after α-GalCer injection, a marked increase in serum IgE levels was observed. Administration of α-GalCer at the time of priming of mice with protein Ag resulted in the generation of Ag-specific Th2 cells and a profound increase in the production of IgE. Collectively, these findings indicate that α-GalCer may be useful for modulating immune responses toward a Th2 phenotype during prophylaxis and therapy.
A Mathematical Model on Germinal Center Kinetics and Termination
Authors: Keşmir Can , De Boer Rob J.
Published: December 2022

We devise a mathematical model to study germinal center (GC) kinetics. Earlier models for GC kinetics are extended by explicitly modeling 1) the cell division history of centroblasts, 2) the Ag uptake by centrocytes, and 3) T cell dynamics. Allowing for T cell kinetics and T-B cell interactions, we study the role of GC T cells in GC kinetics, GC termination, and B cell selection. We find that GC T cells play a major role in GC formation, but that the maintenance of established GC reactions requires very few T cells only. The results therefore suggest that the termination of a GC reaction is largely caused by lack of Ag on the follicular dendritic cells and is hardly influenced by Th cells. Ag consumption by centrocytes is the major factor determining the decay rate of the antigenic stimulus during a GC reaction. Investigating the effect of the Ag dose on GC kinetics, we find that both the total size of the GC and its duration are hardly influenced by the initial amount of Ag. In the model this is due to a buffering effect by competition for limited T cell help and/or competition between proliferating centroblasts.
Assessment of the Role of Determinant Selection in Genetic Control of the Immune Response to Insulin in H-2b Mice
Authors: Thayer Wesley P. , Kraft Jennifer R. , Tompkins S. Mark , Moore Joseph C. , Jensen Peter E.
Published: December 2022

The immune response to insulin is regulated by MHC class II genes. Immune response (Ir) gene-linked low responsiveness to protein Ags can be mediated by the low affinity of potential antigenic determinants for MHC molecules (determinant selection) or by the influence of MHC on the functional T cell repertoire. Strong evidence exists that determinant selection plays a key role in epitope immunodominance and Ir gene-linked unresponsiveness. However, the actual measurement of relative MHC-binding affinities of all potential peptides derived from well-characterized model Ags under Ir gene regulation has been very limited. We chose to take advantage of the simplicity of the structure of insulin to study the mechanism of Ir gene control in H-2b mice, which respond to beef insulin (BINS) but not pork insulin (PINS). Peptides from these proteins, including the immunodominant A(1–14) determinant, were observed to have similar affinities for purified IAb in binding experiments. Functional and biochemical experiments suggested that PINS and BINS are processed with similar efficiency. The T cell response to synthetic pork A(1–14) was considerably weaker than the response to the BINS peptide. We conclude that the poor immunogenicity of PINS in H-2b mice is a consequence of the T cell repertoire rather than differences in processing and presentation.
mdr1a-Encoded P-Glycoprotein Is Not Required for Peripheral T Cell Proliferation, Cytokine Release, or Cytotoxic Effector Function in Mice
Authors: Eisenbraun Michael D. , Miller Richard A.
Published: December 2022

The plasma membrane transport protein P-glycoprotein (P-gp) is expressed by subsets of both CD4+ and CD8+ T cells in mice. The proportion of T cells that express P-gp goes up with age, and the P-gp-expressing subset of the CD4 memory population is hyporesponsive in many in vitro assays. The significance of P-gp expression for T cell function has not been well established, although several reports have suggested that it may promote cytokine export and/or cytotoxic T cell function. To elucidate which T cell functions may require P-gp, we have compared a variety of responses using T cells from wt and P-gp knockout mice. Protein expression and rhodamine-123 efflux studies revealed that peripheral T cells exclusively utilize the mdr1a-encoded isoform rather than the homologous mdr1b or mdr2 isoforms. Comparisons of T cells from mdr1a+/+ and mdr1a−/− mice showed no differences in proliferation or in secretion of IL-2, IL-4, IL-5, IL-10, or IFN-γ in response to polyclonal stimulation. Moreover, mdr1a−/− T cells produced strong allospecific cytotoxic responses comparable to those of wt T cells. Our results show that P-gp is not a necessary component of peripheral T cell functional responses. Further investigation will be needed to determine the significance of P-gp expression in T lymphocytes.
The Role of CTLA-4 in Regulating Th2 Differentiation
Authors: Oosterwegel Mariette A. , Mandelbrot Didier A. , Boyd Scott D. , Lorsbach Robert B. , Jarrett Delma Y. , Abbas Abul K. , Sharpe Arlene H.
Published: December 2022

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4−/−) mouse strains: DO11.10 CTLA-4−/− mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4−/− B7.1/B7.2−/− ). When purified naive CD4+ DO11.10 T cells from CTLA-4−/− and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4−/− DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4−/− CD4+ T cells from mice lacking CTLA-4, B7.1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4−/− CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2−/− mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4−/− B7.1/B7.2−/− mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2−/− mice produced IFN-γ. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4−/− CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4−/− B7.1/B7.2−/− but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.
Analysis of a 26-kb Region Linked to theMhcin Zebrafish: Genomic Organization of the Proteasome Component β/Transporter Associated with Antigen Processing-2 Gene Cluster and Identification of Five New Proteasome β Subunit Genes
Authors: Murray Brent W. , Sültmann Holger , Klein Jan
Published: December 2022

Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and of cDNA clones has led to the identification of five new loci coding for β subunits of proteasomes (PSMB). Together with the four genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment, and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be inducible by IFN-γ. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became associated with it.
Immunization with Treponema pallidum Outer Membrane Vesicles Induces High-Titer Complement-Dependent Treponemicidal Activity and Aggregation of T. pallidum Rare Outer Membrane Proteins (TROMPs)
Authors: Blanco David R. , Champion Cheryl I. , Lewinski Michael A. , Shang Ellen S. , Simkins Stephen G. , Miller James N. , Lovett Michael A.
Published: December 2022

The purpose of this study was to determine whether immunization with purified outer membrane vesicles (OMV) from Treponema pallidum (T.p.) could elicit Abs capable of killing this organism. It is well established that the immunization of rabbits or mice with killed T.p. or with recombinant T.p. Ags has failed to generate serum killing activity comparable with that of infection-derived immunity. Because of the small amount of T.p. OMV obtainable, a single mouse was immunized with purified OMV. The mouse anti-OMV serum and infection-derived immune rabbit serum (IRS) were compared by reactivities on two-dimensional T.p. immunoblots and by the T.p. immobilization test, a complement-dependent killing assay. Whereas IRS detected >40 Ags, the anti-OMV serum identified only 6 Ags corresponding to proteins identified previously in the outer membrane. T.p. immobilization testing showed that IRS had a 100% killing titer of 1:44 and a 50% killing titer of 1:662. By comparison, the mouse anti-OMV serum had a significantly greater 100% killing titer of 1:1,408 and a 50% killing titer of 1:16,896. Absorption of the anti-OMV serum to remove Ab against outer membrane-associated lipoproteins did not change the 100% killing titer. Freeze-fracture analysis of T.p. incubated in IRS or anti-OMV serum showed that T.p. rare membrane-spanning outer membrane proteins were aggregated. This is the first demonstration of high-titer killing Abs resulting from immunization with defined T.p. molecules; our study indicates that the targets for these Abs are T.p. rare outer membrane proteins.
Enhanced Antigen Transport Across Rat Tracheal Epithelium Induced by Sensitization and Mast Cell Activation
Authors: Yang Ping-Chang , Berin M. Cecilia , Perdue Mary H.
Published: December 2022

Ag challenge to the apical surface of tracheal epithelium results in a rapid ion secretory response due to the activation of mast cells. The aim of this study was to examine the impact of sensitization and specific Ag challenge on the timing, route, and quantity of Ag transported across tracheal epithelium. After sensitization of rats to a model protein, HRP, tracheal tissues were excised and mounted in Ussing chambers. Tracheas from HRP-sensitized rats, but not naive or OVA-sensitized rats, responded to apical HRP challenge with a rise in short-circuit current (beginning at ∼2 min). Photomicrographs of tissues fixed at 2 min showed that initial transepithelial HRP transport occurred via endosomes and was significantly enhanced in HRP-sensitized rats compared with both control groups. In addition, nonciliated cells, the proportion of which increased after sensitization, contained significantly more HRP than ciliated cells. The hypersensitivity response occurred only in HRP-sensitized and challenged rats and was associated with increased conductance of tracheal epithelium and overall flux of HRP across the tissue. This increased flux of Ag and elevated conductance was not observed in mast cell-deficient Ws/Ws rats. Photomicrographs of tissues fixed 90 min after challenge also showed HRP in the paracellular spaces between adjacent epithelial cells. We conclude that sensitization increases uptake of specific Ag initially via an endosomal transcellular pathway across tracheal epithelium and that, after the hypersensitivity reaction, mast cell-dependent recruitment of the paracellular pathway further augments Ag influx into airway tissue.
Lipopolysaccharide Binding Protein and Serum Amyloid A Secretion by Human Intestinal Epithelial Cells During the Acute Phase Response
Authors: Vreugdenhil Anita C. E. , Dentener Mieke A. , Snoek A. M. Patricia , Greve Jan-Willem M. , Buurman Wim A.
Published: December 2022

The acute phase proteins LPS binding protein (LBP) and serum amyloid A (SAA) are produced by the liver and are present in the circulation. Both proteins have been shown to participate in the immune response to endotoxins. The intestinal mucosa forms a large surface that is continuously exposed to these microbial products. By secretion of antimicrobial and immunomodulating agents, the intestinal epithelium contributes to the defense against bacteria and their products. The aim of this study was to explore the influence of the inflammatory mediators TNF-α, IL-6, and IL-1β on the release of LBP and SAA by intestinal epithelial cells (IEC). In addition, the induction of LBP and SAA release by cell lines of intestinal epithelial cells and hepatic cells was compared. The data obtained show that in addition to liver cells, IEC also expressed LBP mRNA and released bioactive LBP and SAA upon stimulation. Regulation of LBP and SAA release by IEC and hepatocytes was typical for class 1 acute phase proteins, although differences in regulation between the cell types were observed. Endotoxin did not induce LBP and SAA release. Glucocorticoids were demonstrated to strongly enhance the cytokine-induced release of LBP and SAA by IEC, corresponding to hepatocytes. The data from this study, which imply that human IEC can produce LBP and SAA, suggest a role for these proteins in the local defense mechanism of the gut to endotoxin. Furthermore, the results demonstrate that tissues other than the liver are involved in the acute phase response.
Stem Cell Factor Is Localized in, Released from, and Cleaved by Human Mast Cells
Authors: de Paulis Amato , Minopoli Giuseppina , Arbustini Eloisa , de Crescenzo Gennaro , Dal Piaz Fabrizio , Pucci Piero , Russo Tommaso , Marone Gianni
Published: December 2022

Stem cell factor (SCF) is the most important cytokine regulating human mast cell growth and functions. The immunogold technique showed SCF in the secretory granules of skin mast cells and in lung parenchymal mast cells (HLMC). Immunoreactive SCF (iSCF) was detected in cell lysates of HLMC, but not in basophils; iSCF and histamine were detected in supernatants of HLMC 3 min after challenge with anti-FcεRI or anti-IgE, and iSCF in supernatants rapidly declined after 30 min, whereas histamine remained unchanged for 120 min. HPLC and electrospray mass spectrometry (ES/MS) analysis of recombinant human SCF1–166 (18,656.9 ± 0.9 Da) treated with chymase showed a polypeptide of 17,977.1 ± 0.6 Da and a minor component of 697.4 ± 0.1 Da generated by specific cleavage at Phe159. SCF1–166 and SCF1–159 similarly activated HLMC, potentiated anti-IgE-induced activation of these cells, and stimulated HLMC chemotaxis. SCF159–166 had no effect on mast cells. Western blot analysis of supernatants of anti-IgE-activated HLMC incubated with recombinant human SCF1–166 showed that SCF1–166 was rapidly cleaved to SCF1–159 and SCF1–144. Experiments with supernatants of anti-IgE-activated HLMC incubated with SCF1–166 yielded similar results. In conclusion, SCF is stored in mast cell secretory granules and is immunologically released by human mast cells. SCF1–166 is rapidly and specifically cleaved to SCF1–159 by chymase, which retains its biological effect on mast cells. SCF is also cleaved by other proteases to several SCF species whose possible biological activities remain to be established.
Inhibition of Murine Neutrophil Recruitment In Vivo by CXC Chemokine Receptor Antagonists
Authors: McColl Shaun R. , Clark-Lewis Ian
Published: December 2022

In this study, we have examined the ability of chemokine receptor antagonists to prevent neutrophil extravasation in the mouse. Two murine CXC chemokines, macrophage-inflammatory protein (MIP)-2 and KC, stimulated the accumulation of leukocytes into s.c. air pouches, although MIP-2 was considerably more potent. The leukocyte infiltrate was almost exclusively neutrophilic in nature. A human CXC chemokine antagonist, growth-related oncogene (GRO)-α(8–73), inhibited calcium mobilization induced by MIP-2, but not by platelet-activating factor in leukocytes isolated from the bone marrow, indicating that this antagonist inhibits MIP-2 activity toward murine leukocytes. Pretreatment of mice with GROα(8–73) inhibited, in a dose-dependent manner, the MIP-2-induced influx of neutrophils to levels that were not significantly different from control values. Moreover, this antagonist was also effective in inhibiting the leukocyte recruitment induced by TNF-α, LPS, and IL-1β. Leukocyte infiltration into the peritoneal cavity in response to MIP-2 was also inhibited by prior treatment of mice with GROα(8–73) or the analogue of platelet factor 4, PF4(9–70). The results of this study indicate 1) that the murine receptor for MIP-2 and KC, muCXCR2, plays a major role in neutrophil recruitment to s.c. tissue and the peritoneal cavity in response to proinflammatory agents and 2) that CXCR2 receptor antagonists prevent acute inflammation in vivo.
Acceleration and Increased Severity of Collagen-Induced Arthritis in P-Selectin Mutant Mice
Authors: Bullard Daniel C. , Mobley James M. , Justen James M. , Sly Laurel M. , Chosay John G. , Dunn Colin J. , Lindsey J. Russell , Beaudet Arthur L. , Staite Nigel D.
Published: December 2022

P-selectin plays an important role in leukocyte adherence to microvascular endothelium and is expressed in synovial tissue from patients with rheumatoid arthritis (RA). However, the contribution of P-selectin to the initiation and chronicity of joint inflammation is not well understood. In these studies, collagen-induced arthritis (CIA) was induced in P-selectin mutant (−/−) mice to explore the role of P-selectin in the development of joint inflammation. Surprisingly, CIA onset was accelerated and severity was increased in P-selectin mutant mice, compared with wild-type mice (+/+). Increased levels of anti-type II collagen IgG were detected in both nonarthritic and arthritic P-selectin mutant mice from days 14–91. In addition, splenocytes isolated from immunized and nonimmunized P-selectin mutant mice produced significantly less IL-2 and IL-4, but significantly higher levels of IL-10 and IL-5 than splenocytes from wild-type mice. These observations show that P-selectin-mediated leukocyte rolling is not required for the development of murine CIA and that P-selectin expression exerts a controlling effect on the development of Ag-driven inflammatory joint disease, possibly by mediating the recruitment and/or trafficking of specific leukocyte subtypes into lymphoid tissue or inflammatory foci.
Endothelial α2,6-Linked Sialic Acid Inhibits VCAM-1- Dependent Adhesion Under Flow Conditions
Authors: Abe Yasunori , Smith C. Wayne , Katkin Julie P. , Thurmon Lisa M. , Xu Xudong , Mendoza Leonardo H. , Ballantyne Christie M.
Published: December 2022

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Galβ1-4GlcNAc α2,6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Galβ1-4GlcNAc/Galβ1-3GalNAc α2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both α2,6- and α2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing α2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with α2,6- but not α2,3-linked sialic acids. However, overexpression of α2,6-sialyltransferase did not increase α2,6-linked sialic acid on VCAM-1 but did increase α2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that α2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.
Eotaxin Potentiates Antigen-Dependent Basophil IL-4 Production
Authors: Devouassoux Gilles , Metcalfe Dean D. , Prussin Calman
Published: December 2022

Basophils are a major source of IL-4, which is a critical factor in the generation of allergic inflammation. Eotaxin induces chemotaxis mediated through the CC chemokine receptor 3 (CCR3) present on basophils as well as eosinophils and Th2 cells, thereby promoting cell recruitment. To determine whether eotaxin has other proinflammatory activity, we examined the effect of eotaxin on basophil IL-4 expression by flow cytometry. Eotaxin alone had no effect on basophil IL-4 production, but further increased allergen-stimulated IL-4 expression. Eotaxin also enhanced IL-4 release from purified basophils 2- to 4-fold, as determined by ELISA (p < 0.01). Addition of eotaxin to cultures resulted in a 40-fold left shift in the dose response to Ag. This effect was obtained with physiologic concentrations of eotaxin (10 ng/ml), was abrogated by an Ab to the CCR3 receptor, and was noted with other chemokine ligands of CCR3. Additionally, eotaxin augmented IL-3 priming of basophil IL-4 production in a synergistic manner (p < 0.01). In contrast, no priming was observed with either IL-5 or GM-CSF. These results establish a novel function for eotaxin and other chemokine ligands of CCR3: the potentiation of Ag-mediated IL-4 production in basophils, and suggest a potential nonchemotactic role for CC chemokines in the pathogenesis and amplification of inflammation.
Inducible Expression and Regulation of the α1-Acid Glycoprotein Gene by Alveolar Macrophages: Prostaglandin E2 and Cyclic AMP Act as New Positive Stimuli
Authors: Fournier Thierry , Bouach Najet , Delafosse Christian , Crestani Bruno , Aubier Michel
Published: December 2022

We have reported that α1-acid glycoprotein (AGP) gene expression was induced in lung tissue and in alveolar type II cells during pulmonary inflammatory processes, suggesting that local production of this immunomodulatory protein might contribute to the modulation of inflammation within the alveolar space. Because AGP may also be secreted by other cell types in the alveolus, we have investigated the expression and the regulation of the AGP gene in human and rat alveolar macrophages. Spontaneous AGP secretion by alveolar macrophages was increased 4-fold in patients with interstitial lung involvement compared with that in controls. In the rat, immunoprecipitation of [35S]methionine-labeled cell lysates showed that alveolar macrophages synthesize and secrete AGP. IL-1β had no effect by itself, but potentiated the dexamethasone-induced increase in AGP production. RNase protection assay demonstrated that AGP mRNA, undetectable in unstimulated cells, was induced by dexamethasone. Conditioned medium from LPS-stimulated macrophages as well as IL-1β had no effect by themselves, but potentiated the dexamethasone-induced increase in AGP mRNA levels. In addition to cytokines, PGE2 as well as dibutyryl cAMP increased AGP mRNA levels in the presence of dexamethasone. When AGP expression in other cells of the monocyte/macrophage lineage was examined, weak and no AGP production by human blood monocytes and by rat peritoneal macrophages, respectively, were observed. Our data showed that 1) AGP expression is inducible specifically in alveolar macrophages in vivo and in vitro; and 2) PGE2 and cAMP act as new positive stimuli for AGP gene expression.
Signaling Functions of L-Selectin in Neutrophils: Alterations in the Cytoskeleton and Colocalization with CD18
Authors: Simon Scott I. , Cherapanov Vera , Nadra Imad , Waddell Tom K. , Seo Scott M. , Wang Qin , Doerschuk Claire M. , Downey Gregory P.
Published: December 2022

Ligation and clustering of L-selectin by Ab (“cross-linking”) or physiologic ligands results in activation of diverse responses that favor enhanced microvascular sequestration and emigration of neutrophils. The earliest responses include a rise in intracellular calcium, enhanced tyrosine phosphorylation, and activation of extracellular signal-regulated kinases. Additionally, cross-linking of L-selectin induces sustained shape change and activation of β2 integrins, leading to neutrophil arrest under conditions of shear flow. In this report, we examined several possible mechanisms whereby transmembrane signals from L-selectin might contribute to an increase in the microvascular retention of neutrophils and enhanced efficiency of emigration. In human peripheral blood neutrophils, cross-linking of L-selectin induced alterations in cellular biophysical properties, including a decrease in cell deformability associated with F-actin assembly and redistribution, as well as enhanced adhesion of microspheres bound to β2 integrins. L-selectin and the β2 integrin became spatially colocalized as determined by confocal immunofluorescence microscopy and fluorescence resonance energy transfer. We conclude that intracellular signals from L-selectin may enhance the microvascular sequestration of neutrophils at sites of inflammation through a combination of cytoskeletal alterations leading to cell stiffening and an increase in adhesiveness mediated through alterations in β2 integrins.