bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation-Specific, Octamer-Dependent Costimulation of κ Transcription
Authors: Liberg David , Sigvardsson Mikael , Bemark Mats , Leanderson Tomas
Published: January 2023

By mutational analysis of the octamer-TATA box intervening region in the mouse SP6 κ promoter, we have mapped two octamer-dependent, costimulatory regions, A and B. The A region was active in late B cells only, while the B region was active throughout B cell differentiation. The B region was TATA proximal and contained a heptamer and an E box of the E2A type that is common in Vκ promoters. Mutation of the heptamer element did not decrease transcriptional stimulation from this region, but mutations in, or immediately 5′ of, the E box core sequence did. A protein binding to this region could be detected in nuclear extracts. The complex could only partially be competed with a μE5 binding site and could not be supershifted with Abs raised to E2A gene products, indicating that it may represent a novel E-box binding complex. The A region was located proximal to the octamer and contained a CCCT element that is conserved both with regard to position and sequence in human VκII promoters. By mutational analysis, the transcriptional stimulatory activity was mapped to the CCCT element that also is part of an early B cell factor (EBF) binding site. In late B cells, a novel protein (FA), which did not bind to the EBF binding site in the mb1 promoter, interacted with the A region. This protein was found to be expressed at lower levels in early B cells as well as in HeLa cells. Thus, the octamer-flanking sequence contains positive control elements that may act independently but that differ in the stage of B cell differentiation at which they are active. One of these factors is an example of an ubiquitously expressed transcription factor that participate in differentiation-specific transcriptional activation.
IFN-γ Induction of the Human Monocyte Chemoattractant Protein (hMCP)-1 Gene in Astrocytoma Cells: Functional Interaction Between an IFN-γ-Activated Site and a GC-Rich Element
Authors: Zhou Z-H Lucy , Chaturvedi Priya , Han Yu-long , Aras Sumer , Li Yi-shuan , Kolattukudy Pappachan E. , Ping Dongsheng , Boss Jeremy M. , Ransohoff Richard M.
Published: January 2023

We characterized regulation of the human monocyte chemoattractant protein-1 (hMCP-1) gene by IFN-γ in astrocytoma cells, because astroglial cells express chemokines in several central nervous system inflammatory states. It was found that IFN-γ-induced hMCP-1 transcription was rapid, transient, and mediated by a 213-bp promoter-proximal regulatory region of the gene. Our studies on both in vitro and in vivo states of the hMCP-1 regulatory region established requirement of an IFN-γ-activated site (GAS) and the presence of IFN-γ-inducible GAS-binding activity involving at least STAT-1α for IFN-γ-induced hMCP-1 expression. Unexpectedly, in vivo genomic footprinting of the proximal regulatory region of the IFN-γ-induced gene revealed protection of a GC-rich sequence (GC box) with the same temporal pattern as that seen at the GAS; in vitro, this GC-rich element is associated with nuclear factor Sp1. These observations suggested a cooperative interaction between the GAS and the GC box element. Interestingly, site-specific mutations that abolished GC-box or GAS-element function produced clearly disparate results. Disruption of the GC box did not affect fold induction by IFN-γ but reduced promoter-reporter expression by half. Conversely, GAS mutation abrogated induction but did not affect the magnitude of expression. These results establish the importance of the GAS element for induction of hMCP-1 and further our understanding of IFN-γ-mediated transcriptional induction by providing the first evidence in vivo for inducible signaling to the GC box by this cytokine.
Regulation of E-Cadherin-Mediated Adhesion in Langerhans Cell-Like Dendritic Cells by Inflammatory Mediators That Mobilize Langerhans Cells In Vivo
Authors: Jakob Thilo , Udey Mark C.
Published: January 2023

Adhesion of Langerhans cells (LC) to keratinocytes is mediated by E-cadherin. IL-1, TNF-α, and LPS mobilize LC from epidermis and presumably attenuate LC-keratinocyte adhesion. To determine whether these mediators modulated LC E-cadherin-dependent adhesion directly, we characterized their effects on LC-like dendritic cells expanded from murine fetal skin (FSDDC). FSDDC were propagated from day 16 C57BL/6 fetal skin and isolated as aggregates (FSDDC-A) in which homophilic adhesion was mediated by E-cadherin. IL-1, TNF-α, and LPS induced dissociation of FSDDC-A that began within 4 to 8 h and was complete within 20 h. Anti-IL-1RI mAb inhibited disaggregation caused by IL-1α and IL-1β, but not that induced by TNF-α or LPS. Anti-TNF-α mAb inhibited the effect of TNF-α and LPS, but not that caused by IL-1α or IL-1β. Flow cytometry of FSDDC-A revealed that IL-1, TNF-α, and LPS induced increased expression of MHC class II, CD40, and CD86 and decreased E-cadherin expression that was temporally related to dissociation of aggregates. IL-1 and TNF-α caused a rapid reduction in FSDDC E-cadherin mRNA levels that preceded the decrease in E-cadherin surface expression. These results demonstrate that cytokines that induce LC emigration in vivo act directly on LC-like cells in vitro, reduce E-cadherin mRNA levels, down-regulate E-cadherin surface expression, and induce a loss of E-cadherin-mediated adhesion.
Disparate Roles for TNF-α and Fas Ligand in Concanavalin A-Induced Hepatitis
Authors: Ksontini Riadh , Colagiovanni Dorothy B. , Josephs Michael D. , Edwards Carl K. , Tannahill Cynthia L. , Solorzano Carmen C. , Norman James , Denham Woody , Clare-Salzler Michael , MacKay Sally L. D. , Moldawer Lyle L.
Published: January 2023

Apoptosis is a physiologic process that serves to eliminate cells during development or in response to immunologic regulation. In acute inflammation, however, apoptosis triggered by the overproduction of “death factors” such as TNF-α or Fas ligand (FasL) may contribute to tissue injury. Both TNF-α and FasL are presumed to convey an apoptotic signal by activating a cascade of cysteine-aspartate proteases, which includes IL-1β-converting enzyme or caspase-1. In the present study, we evaluated the contribution of TNF-α and FasL, as well as the role of caspase-1, in Con A-induced hepatitis. We report here that TNF-α and FasL mRNA and protein levels are both increased in the livers of Con A-challenged mice. Using a novel inhibitor of TNF-α, we can confirm that Con A-induced hepatitis is primarily TNF-α dependent. Blockade of FasL with a soluble Fas immunoadhesin does not prevent liver injury in animals treated with Con A alone. However, administration of a matrix metalloproteinase inhibitor exacerbates liver injury, in part through a FasL-dependent process, since pretreatment with the soluble Fas immunoadhesin reduces liver injury in this model. In addition, mice lacking functional caspase-1 are resistant to Con A-induced hepatitis, even after pretreatment with a matrix metalloproteinase inhibitor. We conclude that TNF-α plays a predominant role in Con A-induced liver injury, although concomitant activation of FasL can also lead to apoptotic injury. Furthermore, Con A-induced hepatitis is caspase-1 dependent.
Dendritic Cells Are Required for the Development of Chronic Eosinophilic Airway Inflammation in Response to Inhaled Antigen in Sensitized Mice
Authors: Lambrecht Bart N. , Salomon Benoı̂t , Klatzmann David , Pauwels Romain A.
Published: January 2023

Asthma is characterized by chronic eosinophilic inflammation of the airways, and allergen-specific Th2 lymphocytes are thought to play a major role in the development and maintenance of this type of inflammation in allergic asthma. It is generally accepted that airway dendritic cells (DC) are essential for stimulating naive T cells in a primary immune response to inhaled Ag and for the development of allergic sensitization. We have examined the role of airway DC in stimulating memory T cells in a secondary response to inhaled Ag and the subsequent development of chronic airway inflammation. In our mouse model of asthma, OVA aerosol challenge in OVA-sensitized mice leads to CD4-dependent peribronchial and perivascular eosinophilic inflammation, lung Th2 cytokine production, and systemic IgE production. We have used conditional depletion of airway DC by treatment of thymidine kinase-transgenic mice with the antiviral drug ganciclovir to deplete DC during the secondary exposure to OVA. In sensitized thymidine kinase-transgenic mice, a significant decrease in the number of bronchoalveolar CD4 and CD8 T lymphocytes and B lymphocytes was seen after ganciclovir treatment. In addition, Th2 cytokine-associated eosinophilic airway inflammation was almost completely suppressed. These studies demonstrate for the first time that the DC is essential for presenting inhaled Ag to previously primed Th2 cells in the lung, leading to chronic eosinophilic airway inflammation. Altering the function of airway DC may therefore be an important target for new anti-asthma therapy.
Flt-3 Ligand Increases Microchimerism But Can Prevent the Therapeutic Effect of Donor Bone Marrow in Transiently Immunosuppressed Cardiac Allograft Recipients
Authors: Antonysamy Mary A. , Steptoe Raymond J. , Khanna Ajai , Rudert William A. , Subbotin Vladimir M. , Thomson Angus W.
Published: January 2023

C3H (H2k) mice received 50 × 106 B10 (H2b) bone marrow (BM) cells either alone or with flt-3 ligand (FL) (10 μg/day), tacrolimus (2 mg/kg/day), or both agents for 7 days. Donor MHC class II+ (IAb+) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR. Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group. Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group. This increase in donor cells was almost 500-fold compared with BM alone. C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0–13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days). Addition of FL (10 μg/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days). BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days). Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups. Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal. With FL, this may reflect striking augmentation of immunostimulatory dendritic cells.
Requirement for In Vivo Production of IL-4, But Not IL-10, in the Induction of Proliferative Suppression by Filarial Parasites
Authors: MacDonald Andrew S. , Maizels Rick M. , Lawrence Rachel A. , Dransfield Ian , Allen Judith E.
Published: January 2023

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-γ production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.
Cutting Edge: IL-4 Induces Functional Cell-Surface Expression of CXCR4 on Human T Cells
Authors: Jourdan Patrick , Abbal Claire , Nora Nelly , Hori Toshiyuki , Uchiyama Takashi , Vendrell Jean-Pierre , Bousquet Jean , Taylor Naomi , Pène Jérôme , Yssel Hans
Published: January 2023

Here we report that IL-4 specifically enhances cell surface expression of CXCR4 on resting peripheral and cord blood T cells. Whereas polarized Th2 clones express variable levels of CXCR4, expression of this receptor is undetectable on polarized Th1 clones but can be induced on the latter cells as well, following short-term culture in the presence of IL-4. The IL-4-induced CXCR4 is functional since interaction with its ligand, stromal-derived factor (SDF)-1, activates the p42 MAP-kinase ERK-2. In addition, although CXCR4 expression is down-regulated following stimulation of T cells and T cell clones via CD28 or CD3 and CD2 cell surface molecules, respectively, it is re-induced by IL-4. These data indicate an important role for IL-4 in rendering CD4+ T cells susceptible to infection with HIV via CXCR4, as well as in promoting SDF-1-induced migration of these cells.
Newborn Mice Develop Balanced Th1/Th2 Primary Effector Responses In Vivo But Are Biased to Th2 Secondary Responses
Authors: Adkins Becky , Du Rong-Qing
Published: January 2023

Newborn mice are impaired in their abilities to mount protective immune responses. For decades, it was generally held that the poor responses of newborns were largely due to the developmentally immature state of the T cells. In vitro studies showing that neonatal T cells were deficient in Th1 cytokine production, proliferation, and secondary responsiveness strongly supported this idea. Recently, several studies have challenged this view; animals exposed to Ag as neonates were shown to have mature Th1 responses in adulthood. However, it is not clear whether the mature immune responses were actually mounted by T cells generated after the neonatal stage. We have reexamined this issue by analyzing the capabilities of neonatal lymph node T cells to develop into Ag-specific effector cells during the actual neonatal period. Our results demonstrate that the capacity to develop a balanced Th1/Th2 primary effector response is fully mature within the first week of life. However, while neonatal and adult primary cytokine profiles were very similar, Th2 secondary responses predominated in animals first immunized as newborns. Moreover, we have observed other differences between adults and neonatal responses, including 1) the kinetics of cytokine production and responsiveness to adjuvant during the primary response, and 2) the contribution of spleen and lymph node to secondary responses. We propose that these differences reflect developmental regulation of effector cell function that has important consequences to neonatal immune function.
Matrix Metalloproteinases Produced by Rat IL-2-Activated NK Cells
Authors: Kitson Richard P. , Appasamy Pierette M. , Nannmark Ulf , Albertsson Per , Gabauer Megan K. , Goldfarb Ronald H.
Published: January 2023

We have previously documented that adoptively transferred IL-2-activated NK (A-NK) cells can accumulate within cancer metastases. Electron microscopic studies of pulmonary metastases have revealed that adoptively transferred A-NK cells that accumulate within metastases bind to endothelial cells and are able to traverse basement membranes. We have now extended these morphologic studies. We report that rat A-NK cells produce two matrix metalloproteinases: MMP-2 and MMP-9, as determined by SDS-PAGE gelatin zymography. These activities are inhibited following incubation with BB-94 (batimastat), a specific inhibitor of matrix metalloproteinases but not with 3,4-dichloroisocoumarin, an inhibitor of neutral serine proteases. The identity of MMP-2 was confirmed by Western blots using a polyclonal Ab against human MMP-2, whereas reverse transcriptase-PCR analysis of mRNA extracts of A-NK cells has confirmed the presence of MMP-9. In addition, we report for the first time that A-NK cells can migrate through a model basement membrane-like extracellular matrix. Moreover, the ability of A-NK cells to migrate through this model basement membrane was partially inhibited by BB-94; however, BB-94 has no effect on A-NK cell-mediated cytotoxicity, suggesting that matrix metalloproteinases do not contribute to cytolytic function of A-NK cells. In sum, our studies show that A-NK cells employ BB-94-inhibitable matrix metalloproteinases to degrade extracellular matrices. This suggests that matrix metalloproteinases may play a role in the accumulation of A-NK cells within cancer metastases.
Extensive Junctional Diversity of Ig Heavy Chain Rearrangements Generated in the Progeny of Single Fetal Multipotent Hematopoietic Cells in the Absence of Selection
Authors: Nourrit Françoise , Doyen Noëlle , Kourilsky Philippe , Rougeon François , Cumano Ana
Published: January 2023

We analyzed the progeny of individual multipotent hemopoietic cells, derived from the para-aortic splanchopleura, the earliest identified source of lymphocyte precursors in pre-liver mouse embryos. Single precursors were expanded in an in vitro culture system that permits both commitment and differentiation of B cell precursors. We show that from one single multipotent progenitor we could obtain large numbers of B cell precursors that rearrange the Ig heavy chain genes and generate a repertoire as diverse as that observed in adult populations. N region additions are present at V(D)J junctions, showing that terminal deoxynucleotidyl transferase expression has been switched on and is not, consequently, an intrinsic property of adult stem cells. Throughout the culture period, cells show a majority of DJ vs V(D)J rearrangements and a ratio of 2:1 of nonproductive to productive V(D)J rearrangements, which is close to the expected frequency in the absence of selection. In addition, counterselection for D-J rearrangements in reading frame 2 is observed in V(D)J joints, and allelic exclusion was consistently observed. We conclude that of the three events associated with heavy chain rearrangement, two of them, namely allelic exclusion and counterselection of cells in which the D segment is in reading frame 2, are intrinsic to the cell, while selection of productive heavy chain rearrangements is induced in the bone marrow environment.
Novel Glycosylation of HLA-DRα Disrupts Antigen Presentation Without Altering Endosomal Localization
Authors: Guerra Carolyn B. , Busch Robert , Doebele Robert C. , Liu Wendy , Sawada Tetsuji , Kwok William W. , Chang Ming-der Y. , Mellins Elizabeth D.
Published: January 2023

The HLA-DR hemizygous B lymphoblastoid cell line, 10.24.6, has a DRA mutation (Pro96→Ser) that creates a novel glycosylation site at Asn94. The mutant DR molecules are primarily associated with nested fragments of invariant chain (class II-associated invariant chain peptides), and their interaction with HLA-DM is impaired. Here we further analyzed the defect in 10.24.6 cells. Expressing Ser96 mutant DRA cDNA in DRA-null cells recapitulated the 10.24.6 phenotype, indicating that the mutation causes the Ag presentation defect. A mutation to Ala96α, which does not introduce an extra glycan, generated a normal phenotype; the critical role of the glycan was further supported by experiments in which N-glycosylation was blocked by tunicamycin. We also evaluated whether the 10.24.6 mutation affected DR3 maturation or trafficking. Metabolic labeling and subcellular fractionation showed that assembly, endosomal transport, and invariant chain proteolysis of mutant DR3 molecules were similar to wild-type. A slight delay in export from the endoplasmic reticulum to the Golgi apparatus in 10.24.6 cells probably did not contribute significantly to the Ag presentation defect, because the abundance of DM and mutant DR in peptide-loading compartments was normal at steady state. Our results indicate that proper localization of these molecules does not depend on their interaction.
IL-15 Enhances the Response of Human γδ T Cells to Nonpetide Microbial Antigens
Authors: García Verónica E. , Jullien Denis , Song Mark , Uyemura Koichi , Shuai Ke , Morita Craig T. , Modlin Robert L.
Published: January 2023

Human γδ T cells have the ability to rapidly expand and produce IFN-γ in response to nonpeptide Ags of microbial pathogens, in particular a class of compounds known as the prenyl phosphates. We investigated the ability of IL-15, a T cell growth factor, to modulate prenyl phosphate-induced γδ T cell proliferation and cytokine production. IL-15 significantly enhanced the expansion of γδ T cells in the peripheral blood after stimulation in vitro with isopentenyl pyrophosphate. Moreover, using γδ T cell clones, we determined that IL-15-induced T cell proliferation was dependent on the IL-2Rβ chain but not the IL-2Rα chain. We therefore studied the IL-15Rα chain expression in human γδ T cells in the presence or absence of nonpeptide Ags. We found IL-15Rα mRNA expression in IL-15-stimulated and Ag-stimulated human γδ T cells but not in resting γδ T cells. Although IL-15 itself had little effect on the production of IFN-γ, IL-15 plus IL-12 acted synergistically to augment IFN-γ production by γδ T cells. Moreover, we showed that this increase in IFN-γ could be explained by the dual activation of STAT1 and STAT4 by IL-15 and IL-12, respectively. Taken together, these results suggest that IL-15 may contribute to activation of human γδ T cells in the immune response to microbial pathogens.
Dominant Signals Leading to Inhibitor κB Protein Degradation Mediate CD40 Ligand Rescue of WEHI 231 Immature B Cells from Receptor-Mediated Apoptosis
Authors: Schauer Stephanie L. , Bellas Robert E. , Sonenshein Gail E.
Published: January 2023

Recently, we demonstrated maintenance of nuclear factor (NF)-κB/Rel factors plays a major role in B cell survival. Treatment of WEHI 231 immature B cells with an Ab against the surface IgM protein (anti-IgM) induces apoptosis that can be rescued by engagement of CD40 receptor. The dramatic decrease in high basal levels of NF-κB/Rel activity induced by anti-IgM treatment led to cell death. CD40 ligand (CD40L) treatment prevented the drop in NF-κB/Rel factor binding by inducing a sustained decrease in inhibitor (I) κB-α and transient decrease in IκB-β protein levels. In this study, we have investigated the regulation of these NF-κB/Rel-inhibitory proteins. In exponentially growing WEHI 231 cells, the IκB-α and IκB-β proteins decayed with an approximate t1/2 of 38 and 76 min, respectively, which was blocked effectively upon addition of the proteasome-specific inhibitor (benzylcarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO). Anti-IgM treatment stabilized IκB-α and IκB-β proteins. CD40L treatment resulted in a dramatic decrease in t1/2 (<5 min) for both IκB molecules, which was inhibited by addition of Z-LLF-CHO. CD40L treatment also caused a delayed increase in IκB-β mRNA levels, most likely contributing to the observed recovery of IκB-β levels. Microinjection of IκB-α-glutathione S-transferase fusion protein into nuclei of WEHI 231 cells ablated protection by CD40L from receptor-mediated killing. Furthermore, CD40L rescued apoptosis induced upon microinjection of a vector expressing wild-type IκB-α, but not a 32A/36A mutant form of IκB-α, unable to be phosphorylated and hence degraded. Thus, control of turnover of IκB proteins by CD40L plays a major role in maintenance of NF-κB/Rel and resultant rescue of WEHI 231 cells from apoptosis.
The 5′ Untranslated Region, Signal Peptide, and the Coding Sequence of the Carboxyl Terminus of IL-15 Participate in Its Multifaceted Translational Control
Authors: Bamford Richard N. , DeFilippis Andrew P. , Azimi Nazli , Kurys Gloria , Waldmann Thomas A.
Published: January 2023

We previously reported that the AUG-burdened 5′ untranslated region (UTR) of IL-15 mRNA impedes its translation. Here we demonstrate that the nucleotide or protein sequences of the IL-15 signal peptide and carboxyl terminus also contribute to the poor translation of IL-15 transcripts. In particular, the exchange of the IL-15 signal peptide coding sequence with that of IL-2 increased cellular and secreted levels of IL-15 protein 15- to 20-fold in COS cells, while IL-2 transcripts with the IL-15 signal peptide generated 30- to 50-fold less IL-2 protein than wild-type IL-2. Furthermore, the addition of an artificial epitope tag to the 3′ coding sequence of IL-15 increased its protein production 5- to 10-fold. Combining these two IL-15 message modifications, in addition to removing the 5′ UTR, increased IL-15 synthesis 250-fold compared with a wild-type construct with an intact 5′ UTR. These data suggest that IL-15 mRNA, unlike IL-2 mRNA, may exist in translationally inactive pools. By storing translationally quiescent IL-15 mRNA, cells might respond to intracellular infections or other stimuli by rapidly transforming IL-15 message into one that can be efficiently translated.
Thrombopoietin (TPO) Knockout Phenotype Induced by Cross-Reactive Antibodies Against TPO Following Injection of Mice with Recombinant Adenovirus Encoding Human TPO
Authors: Abina Mohammed-Amine , Tulliez Micheline , Duffour Marie-Thérèse , Debili Najet , Lacout Catherine , Villeval Jean-Luc , Wendling Françoise , Vainchenker William , Haddada Hedi
Published: January 2023

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.
Expression and Function of the Type 3 Complement Receptor in Tissues of the Developing Mouse
Authors: Hughes Derralynn A. , Gordon Siamon
Published: January 2023

Macrophage (Mφ) expression of the leukocyte integrins has been implicated in their adhesion and migration in the adult. Little is known, however, of the expression or function of these molecules during development. This study defines the spatial and temporal sequences of expression of the type 3 complement receptor (CR3) in the developing mouse; establishes the functional efficacy of this molecule in spreading, adhesion, and phagocytosis; and investigates its role in inflammatory and constitutive migration. Expression of CR3 on monocytes occurred early compared to Mφ-restricted glycoprotein F4/80, but expression on stellate tissue Mφ appeared later than F4/80 and was transient. Expression of CR3 on resident tissue Mφ is more widespread during development, being retained on only very specific Mφ populations in the adult. Neutrophil polymorphs expressed CR3 from day 17 of gestation onward. The anti-CR3 mAb 5C6 was used to investigate the role of CR3 in adhesion, spreading, and phagocytosis by neonatal Mφ. Neonatal macrophages were found to adhere, spread, and phagocytose by CR3-dependent mechanisms, and a CR3-independent system was implicated in the spreading of neonatal Mφ. The role of CR3 in migration during development was then investigated. 5C6 had potent effects on the early stages of the migration of myelomonocytic cells to an inflammatory stimulus in vivo. Despite efficient transplacental transfer of the Ab from pregnant mother to fetus, the process by which monocytes generate populations of resident tissue Mφ was undisrupted, indicating the existence of CR3-independent mechanisms of monocyte migration during development.
Cationic Lipids Enhance Cytokine and Cell Influx Levels in the Lung Following Administration of Plasmid: Cationic Lipid Complexes
Authors: Freimark Bruce D. , Blezinger H. Paul , Florack Valarie J. , Nordstrom Jeffrey L. , Long Scott D. , Deshpande Deepa S. , Nochumson Sam , Petrak Karel L.
Published: January 2023

Administration of plasmid/lipid complexes to the lung airways may be associated, in addition to expression of transgene, with a range of other responses. We report here the induction of cytokines and cellular influx in the lung airway following intratracheal administration of an N-[1-(2–3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol/plasmid positively charged complex in mice. We show that 1) the appearance of the Th1-associated cytokines IFN-γ and IL-12 in bronchoalveolar lavage fluid is caused by unmethylated CpG dinucleotide sequences present within the plasmid, and is enhanced by the lipid formulation; 2) cationic lipids by themselves do not induce IL-12 or IL-12p40; 3) TNF-α is rapidly induced by cationic lipids and plasmid/lipid complex, but not by plasmid alone; 4) an acute cellular influx is induced by cationic lipid alone and by a plasmid/lipid complex, but to a much lesser extent by plasmid alone; and 5) plasmid methylation does not influence the degree of inflammatory cell influx. The induction of the innate immune responses by plasmid/lipid complexes may be advantageous to gene therapy of lung diseases. In particular, induction of the Th1 cell-promoting cytokines by plasmid/lipid complexes could, in conjunction with an expressed transgene, be used to modulate immune responses in the lung airways in disease conditions that are deficient in Th1 cell responses or that have a dominant Th2 phenotype. Alternatively, the elimination of immunostimulatory sequences in plasmids may improve the tolerability and/or efficacy of nonviral gene therapy, especially for diseases requiring chronic administration.
Dendritic Cells as the Terminal Stage of Monocyte Differentiation
Authors: Palucka Karolina A. , Taquet Nicolas , Sanchez-Chapuis Francoise , Gluckman Jean Claude
Published: January 2023

Monocytes (MO) cultured for ≥5 days with either macrophage-CSF (M-CSF) or granulocyte macrophage (GM)-CSF and IL-4 differentiated without concomitant proliferation into CD14+ macrophages (Mφ) or CD1a+ dendritic cells (DC), respectively. When adherent and nonadherent CD14high Mφ from M-CSF cultures were separated and cultured further in cytokine-free medium or with GM-CSF/IL-4, most cells from both fractions that were exposed to GM-CSF/IL-4 acquired CD1a expression and DC morphology and function. Conversely, GM-CSF/IL-4 withdrawal at day 5 and additional culture of sorted CD1a+ DC for 2 to 7 days in cytokine-free medium led to cells rapidly becoming adherent CD1a−CD14+ Mφ. Replacing GM-CSF/IL-4 with M-CSF hastened the conversion of DC to Mφ without increasing cell numbers. CD1a+CD14−CD83+ mature DC were induced by a ≥2-day exposure to MO-conditioned medium, LPS, or TNF-α/IL-1β. Upon cytokine removal or culture with M-CSF, DC that had been pushed to maturation by conditioned medium or LPS remained stable or died in the new environment. TNF-α/IL-1β-driven DC displayed heterogeneous CD83 expression and could thus be sorted into CD83high and CD83low/− cells; in cytokine-free medium or in M-CSF, most CD83low/− cells converted to Mφ, whereas most CD83high cells remained nonadherent CD1a+CD14− or died and thus appeared truly terminally differentiated. Hence, MO are precursors of Mφ as well as of DC, with each cell type having the capability to convert into the other until late in the differentiation/maturation process. Accordingly, the cytokine environment and the presence of differentiation and/or other stimulatory signals may be the “final decision-making factors” determining whether these cells will acquire DC or Mφ characteristics and function.
Monocyte Chemoattractant Protein-1 Synthesis by Murine Lung Fibroblasts Modulates CD4+ T Cell Activation
Authors: Hogaboam Cory M. , Lukacs Nicholas W. , Chensue Stephen W. , Strieter Robert M. , Kunkel Steven L.
Published: January 2023

This study addressed the role of endogenous monocyte chemoattractant protein-1 (MCP-1) in Ag-stimulated lymphokine synthesis and proliferation by CD4+ T cells during their coculture with purified lung fibroblasts or splenic macrophages. Initial experiments showed that fibroblasts exposed to IL-4, TNF α, or IL-4 and TNF-α (all at 10 ng/ml) for 24 h released five- to eightfold more MCP-1 than similarly treated splenic macrophages. In 72-h coculture experiments, the synthesis of IL-4 by OVA-activated CD4+ T cells added to lung fibroblasts or splenic macrophages was significantly inhibited when endogenous MCP-1 was neutralized using polyclonal anti-MCP-1 antiserum. In these same cocultures, IFN-γ levels were significantly enhanced. Similarly, IFN-γ levels were significantly enhanced in 72-h cocultures of a purified peptide derivative-activated CD4+ Th1 clone and lung fibroblasts or splenic macrophages following immunoneutralization of MCP-1. In separate experiments, the selective inhibition of MCP-1 synthesis by lung fibroblasts and splenic macrophages using an MCP-1 antisense oligonucleotide significantly enhanced the proliferation of CD4+ T cells during a 96-h coculture. Taken together, these data suggest that MCP-1 exerts an immunomodulatory effect on CD4+ T cell-derived IL-4 and IFN-γ release and CD4+ T cell proliferation during cell-to-cell interactions.
In Vitro Stimulation of C1s Proteolytic Activities by C1s-Presenting Autoantibodies from Patients with Systemic Lupus Erythematosus
Authors: He Shiping , Lin Ya-Ling
Published: January 2023

Anti-C1s autoantibodies (IgG forms), which recognize the conjunction of C1s heavy chain and light chain (C1s-presenting autoantibodies) from patients with systemic lupus erythematosus (SLE), have been found to stimulate C1s enzymatic activities. This is due to acceleration of the proteolytic hydrolysis of the synthetic substrate C1-1 by C1s, enhancement of the complex formation of C1s with its natural pseudosubstrate, C1 inhibitor (C1 inh), and promotion of proteolytic activation of its natural substrate, C4. Seven of fifteen samples from patients with SLE were found to contain such autoantibodies. The hydrolysis of the synthetic substrate C1-1 catalyzed by C1s in 25 to 27 min in the presence of anti-C1s autoantibodies was equivalent to the hydrolysis of C1-1 catalyzed by C1s alone or C1s with control IgG from healthy sera in 110 min, approximately fourfold faster than the reaction in the absence of anti-C1s autoantibodies. Densitometry scanning data showed that the formation of the C1s-C1 inh complex in the presence of anti-C1s autoantibodies was three to four times greater than that with control IgG. It was also noticed that the autoantibodies convert almost all of the latent forms of C1s to an active form that binds to C1 inh. Another group of Western blots showed that C1s cleaved C4 α-chain three times faster in the presence of autoantibodies than of control IgG. It is likely that the overconsumption of complement components is common in the pathogenesis of tissue damage occurring in SLE.
Proteasomes Can Either Generate or Destroy MHC Class I Epitopes: Evidence for Nonproteasomal Epitope Generation in the Cytosol
Authors: Luckey Chance John , King Gina M. , Marto Jarrod A. , Venketeswaran Sunita , Maier Bernhard F. , Crotzer Victoria L. , Colella Teresa A. , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H.
Published: January 2023

Proteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results suggest that some epitopes are generated by nonproteasomal proteases in the cytosol.
A Partially Modified Retro-Inverso Pseudopeptide Modulates the Cytokine Profile of CTL Specific for an Influenza Virus Epitope
Authors: Ostankovitch Marina , Guichard Gilles , Connan Francine , Muller Sylviane , Chaboissier Aude , Hoebeke Johan , Choppin Jeannine , Briand Jean-Paul , Guillet Jean-Gérard
Published: January 2023

There is considerable evidence that peptides corresponding to MHC class I-restricted epitopes can be used as immunogens or immunomodulators. Pseudopeptides containing isosteric replacements of the amide bond provide more stable analogues, which may even have enhanced biologic activity. But there have been very few studies on the use of pseudopeptides to initiate or modulate the cellular immune response. This study describes the immunogenicity of a partially modified retro-inverso pseudopeptide of an influenza virus epitope and shows that this pseudopeptide modulates the cytokine profile expressed by CD8+CTL generated from primed precursors. Moreover, the pseudopeptide is much more efficient at low concentration than the wild-type epitope to stimulate IFN-γ secretion by CD8+ T effector cells. These results are analyzed with reference to changes in the conformation of the MHC molecule/peptide complex deduced from molecular modeling. The findings support the idea that partially modified retro-inverso analogues can be used as altered peptide ligands to enhance the stimulation of natural epitope-specific CTL and to modify their functional properties. Hence, pseudopeptide ligands might be promising tools for use in immunotherapy.
The N-Terminal Domains Target TNF Receptor-Associated Factor-2 to the Nucleus and Display Transcriptional Regulatory Activity
Authors: Min Wang , Bradley John R. , Galbraith Jennifer J. , Jones Sally J. , Ledgerwood Elizabeth C. , Pober Jordan S.
Published: January 2023

The subcellular localization of the TNF receptor-associated factor-2 (TRAF2) adaptor protein in human endothelial cells, which mediates proinflammatory responses of TNF, has been analyzed by confocal immunofluorescence microscopy and by Western blotting of fractionated cell extracts. Rabbit antisera reactive with either amino- or carboxyl-terminal TRAF2 peptides frequently but not uniformly stain nuclei of cultured HUVEC or the established human endothelial cell line, ECV304. However, Western blotting reveals significant heterogeneity in the reactivities of these polyclonal Abs. Transiently transfected HUVEC expressing FLAG epitope-tagged TRAF2 consistently show prominent nuclear localization, and deletion mutants of TRAF2 identify the portion of the molecule responsible for nuclear localization as the amino-terminal ring finger domain. TNF treatment does not appear to influence the localization of endogenous or transfected TRAF2 protein. Transfection of the amino-terminal half of the TRAF2 molecule, containing the ring and zinc finger domains, which localizes to the nucleus, results in activation of E-selectin but not of NF-κB promoter-reporter gene transcription or of c-Jun N-terminal kinase activation. These observations suggest that TRAF2 may reside in the nucleus and directly regulate transcription, independent of its role in cytoplasmic signal transduction.
Regulation of IL-6 Signaling by p53: STAT3- and STAT5-Masking in p53-Val135-Containing Human Hepatoma Hep3B Cell Lines
Authors: Rayanade Ravi J. , Ndubuisi MacKevin I. , Etlinger Joseph D. , Sehgal Pravin B.
Published: January 2023

The influence of p53 on cytokine-triggered Janus kinase-STAT signaling was investigated in human hepatoma Hep3B cell lines engineered to constitutively express the temperature-sensitive Val135 mutant of p53. In comparison to the parental p53-free Hep3B cells, these p53-Val135-containing Hep3B cell lines displayed a reduced response to IL-6 at the wild-type-like p53 temperature (32.5°C). In these cells, IL-6 induced a marked reduction in the immunologic accessibility of cytoplasmic and nuclear STAT3 and STAT5 within 20 to 30 min that lasted 2 to 4 h (STAT-masking) provided that the cells had been previously cultured at 32.5°C for at least 18 to 20 h. The onset of IL-6-induced STAT-masking required protein tyrosine kinase, protein tyrosine phosphatase, proteasomal, phospholipase C, and mitogen-activated protein kinase kinase 1 activities. The maintenance of IL-6-induced STAT-masking was dependent on continued signaling through the phosphatidylinositol-dependent phospholipase C pathway. Despite a reduction in IL-6-induced STAT3 DNA binding activity in the nuclear compartment during STAT-masking, there was increased and prolonged accumulation of tyrosine-phosphorylated STAT3 in both the cytoplasmic and nuclear compartments, indicating that the capacity of tyrosine-phosphorylated STAT3 to bind DNA was reduced during STAT-masking. Thus, IL-6-induced STAT-masking, as dramatically evident on immunomicroscopy, is a visible consequence of a novel cellular process by which a p53-Val135-induced gene product(s) regulates the association of masking protein(s) with and the DNA-binding capacity of STAT3.
Differences Between B Cell and Macrophage Transformation by the Bovine Parasite,Theileria annulata: A Clonal Approach
Authors: Sager Heinz , Bertoni Giuseppe , Jungi Thomas W.
Published: January 2023

Theileria annulata, a tick-transmitted protozoan parasite, infects and transforms cells of the hemopoietic system, particularly those of the B cell and monocyte/macrophage lineages. Here, the effect of infection/transformation on the resulting phenotype was studied using a clonal approach. Three phenotypes of transformed cell lines could be discerned. The first is characterized by surface expression of IgM, CD21, and the B cell epitopes, B-B2 and B-B8, Ig heavy chain gene rearrangement, and mRNA expression. Such lines were obtained from fresh and cultured PBMC and at increased frequency from purified B cells, but never from fetal bone marrow cells. The second phenotype can be distinguished from the first by the absence of Ig heavy chain expression and reduced surface expression of B cell markers (CD21, B-B2, B-B8). Clones with this phenotype were obtained from transformed fetal bone marrow cells only. The third phenotype showed an absence of all of the above B cell markers, including surface IgM, and a lack of Ig heavy chain gene rearrangement. The latter clones could be maintained for several weeks after elimination of T. annulata by BW720c treatment, and they reacquired a macrophage-like phenotype. This implies that parasite-induced dedifferentiation is restricted to monocyte/macrophage, and that B cell markers are indicative of cell lineage progeny. Demonstration of surface IgM on PBMC-derived B cell clones suggests that infection of B cells with T. annulata may be an epigenetic method to immortalize ruminant B cells of a defined Ag specificity.
Human Eosinophils Produce Biologically Active IL-12: Implications for Control of T Cell Responses
Authors: Grewe Markus , Czech Wolfgang , Morita Akimichi , Werfel Thomas , Klammer Michaela , Kapp Alexander , Ruzicka Thomas , Schöpf Erwin , Krutmann Jean
Published: January 2023

The present study assessed the capacity of eosinophils (EOS) to synthesize the cytokine IL-12. Blood-derived, highly purified human EOS from six atopic patients and two nonatopic individuals were treated in culture with IL-4, IL-5, granulocyte-macrophage CSF, IFN-γ, TNF-α, IL-1α, RANTES, and complement 5a, respectively. The expression of both IL-12 protein and mRNAs for the p35 and p40 IL-12 subunits was strongly induced in all donors by the Th2-like cytokines IL-4 and granulocyte-macrophage CSF and was also moderately induced by TNF-α and IL-1α. IL-5 treatment resulted in IL-12 synthesis in four atopic donors and one nonatopic donor, whereas IFN-γ induced IL-12 synthesis in only two atopic donors. In contrast, RANTES exclusively induced mRNA for the p40 subunit without detectable protein release, and complement 5a had no effect on IL-12 mRNA or protein expression. EOS-derived IL-12 was biologically active, because supernatants derived from IL-4-treated EOS superinduced the Con A-induced expression of IFN-γ by a human Th1-like T cell line. This activity was neutralized by anti-IL-12 Abs. In conclusion, EOS secrete biologically active IL-12 after treatment with selected cytokines, which mainly represent the Th2-like type. Consequently, EOS may promote a switch from Th2-like to Th1-like immune responses in atopic and parasitic diseases.