bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: The Induction of Acute Phase Proteins by Lipopolysaccharide Uses a Novel Pathway That Is CD14-Independent
Authors: Haziot Alain , Lin Xing Y. , Zhang Fan , Goyert Sanna M.
Published: December 2022

LPS (endotoxin) and proinflammatory cytokines (IL-6, IL-1, and TNF-α) are potent inducers of acute phase proteins (APP). Since LPS induces high levels of these cytokines after its interaction with CD14, a protein expressed on the surface of monocytes and neutrophils, it has been assumed that CD14 mediates the LPS induction of APP expression. To test this hypothesis, CD14-deficient and control mice were injected with low doses of LPS, and the expression of several APP that are normally up-regulated by LPS was measured. CD14-deficient mice showed no alteration in the induction of APP, including serum amyloid A, LPS-binding protein, fibrinogen, or ceruloplasmin; in contrast, C3H/HeJ mice, which carry a mutation in the Lps gene, do not up-regulate the expression of these proteins. These studies show that the up-regulation of APP by LPS utilizes a non-CD14 receptor and requires a functional Lps gene.
Induction of Autoimmune Arthritis in HLA-DR4 (DRB1*0401) Transgenic Mice by Immunization with Human and Bovine Type II Collagen
Authors: Rosloniec1 Edward F. , Brand David D. , Myers Linda K. , Esaki Yukio , Whittington Karen B. , Zaller Dennis M. , Woods Andrea , Stuart John M. , Kang Andrew H.
Published: December 2022

Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263–270 and a minor determinant within CII286–300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.
Intracellular Pathway for the Generation of Functional MHC Class II Peptide Complexes in Immature Human Dendritic Cells
Authors: Saudrais Cédric , Spehner Danièle , de la Salle Henri , Bohbot Alain , Cazenave Jean-Pierre , Goud Bruno , Hanau Daniel , Salamero Jean
Published: December 2022

Binding of antigenic peptides to MHC class II (MHC-II) molecules occurs in the endocytic pathway. From previous studies in B lymphocytes, it is believed that most but not all of the newly synthesized MHC-II molecules are directly targeted from the trans-Golgi network to endosomal compartments. By using pulse-chase metabolic labeling followed by cell surface biotinylation, we show here that in contrast to an EBV-transformed B cell line and human monocytes, the majority of newly synthesized MHC-II molecules (at least 55 ± 13%) are first routed to the plasma membrane of dendritic cells derived from human monocytes. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC-II to the endosomal/lysosomal system. Following rapid internalization and degradation of Ii, these αβIi complexes are converted into αβ-peptide complexes as shown by their SDS stability. These SDS-stable dimers appear as soon as 15 to 30 min after internalization of the αβIi complexes. More than 80% of αβ dimers originating from internalized αβIi complexes are progressively delivered to the cell surface within the next 2 h. Depolymerization of microtubules, which delays the transport to late endosomal compartments, did not affect the kinetics of conversion of surface αβIi into SDS-stable and -unstable αβ dimers. Altogether, these data suggest that newly liberated class II αβ heterodimers may bind peptides in different compartments along the endocytic pathway in dendritic cells derived from human monocytes.
Production of IL-10 by Human Natural Killer Cells Stimulated with IL-2 and/or IL-12
Authors: Mehrotra Priti T. , Donnelly Raymond P. , Wong Susan , Kanegane Hirokazu , Geremew Amare , Mostowski Howard S. , Furuke Keizo , Siegel Jay P. , Bloom Eda T.
Published: December 2022

Human NK cell activity can be augmented in vitro by stimulation with IL-2 or IL-12, both of which also induce the production of IFN-γ, TNF-α, and granulocyte-macrophage CSF by NK cells. For the first time, we demonstrate that freshly purified NK cells stimulated with IL-2 proliferated and produced IL-10 in a dose-dependent manner. IL-10 mRNA expression, as detected by semiquantitative reverse transcription-PCR, reached peak levels at 24 h. IL-10 protein was detectable on day 2 and further increased on days 3 and 6 as measured by ELISA. However, IL-12 alone induced neither substantial proliferation nor detectable IL-10 production by fresh NK cells, but it synergized with IL-2 in inducing IL-10 mRNA expression and protein synthesis. IL-10 production by activated NK cells was confirmed by intracytoplasmic cytokine staining by three-color immunofluorescence of CD16+ and/or CD56+ NK cells with anti-IL-10 antibody. IL-10 production by NK cells was further confirmed in the NK-like cell line, YT, which constitutively expressed IL-10 mRNA and protein. IL-12 alone did not induce NK proliferation, but it inhibited IL-2-induced proliferation. Neutralization of endogenously produced IL-10 with anti-IL-10 antibodies did not overcome the inhibition of IL-2-induced proliferation by IL-12. Together, these results demonstrate that IL-2 and IL-12 synergize to induce IL-10 production by human NK cells and that IL-12 inhibits IL-2 induced NK cell proliferation by an IL-10-independent mechanism.
A Role for Perforin in Activation-Induced Cell Death
Authors: Spaner David , Raju Kaliannan , Radvanyi Laszlo , Lin Yunping , Miller Richard G.
Published: December 2022

The granule exocytosis pathway of T cell cytotoxicity is absent in mice whose perforin gene has been ablated by targeted mutagenesis. The ability of activated naive T cells to undergo apoptosis in vitro following reaggregation of the TCR complex with anti-TCR mAbs via a Fas-independent pathway was found to be defective in the absence of perforin. Protection from death was most marked in CD8+ T cells. In wild-type cells, perforin was expressed at the same time that apoptosis occurred, and blockade of perforin expression by either incubation with perforin antisense oligonucleotides or with anti-IL-2 Abs resulted in increased viability of activated T cells. The role of perforin was not via perforin-dependent fratricidal killing. The results suggest a model in which perforin acts internally to cause a form of activation-induced T cell death distinct from that caused by members of the TNFR superfamily.
A Phosphorylcholine-Containing Filarial Nematode-Secreted Product Disrupts B Lymphocyte Activation by Targeting Key Proliferative Signaling Pathways
Authors: Deehan Maureen R. , Frame Mhairi J. , Parkhouse R. Michael E. , Seatter Sandra D. , Reid Steven D. , Harnett Margaret M. , Harnett William
Published: December 2022

Filarial nematodes infect more than 100 million people in the tropics, causing elephantiasis, chronic skin lesions, and blindness. The parasites are long-lived as a consequence of being able to evade the host immune system, but an understanding of the molecular mechanisms underlying this evasion remains elusive. In this study, we demonstrate that ES-62 (2 μg/ml), a phosphorylcholine (PC)-containing glycoprotein released by the rodent filarial parasite Acanthocheilonema viteae, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal-transduction elements in B lymphocytes. Although this interaction is insufficient to cause B lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide-3-kinase and Ras mitogen-activating protein kinase pathways, and hence also to proliferation, via the Ag receptor. The active component of ES-62 appears to be PC, a molecule recently shown to act as an intracellular signal transducer, as the results obtained with ES-62 are broadly mimicked by PC alone. As PC-containing secreted products (PC-ES) are also released by human filarial parasites, our data suggest that PC-ES, by interfering with B cell function, could play a role in prolonging filarial infection in parasitized individuals.
Generation of Antigen-Specific Th2 Cells from Unprimed Mice In Vitro: Effects of Dexamethasone and Anti-IL-10 Antibody
Authors: Dozmorov Igor M. , Miller Richard A.
Published: December 2022

We describe a system for the in vitro production of Ag-specific mouse CD4 cell lines from unprimed mice. Purified CD4+CD45RBhigh T cells were exposed to Ag-pulsed accessory cells in serum-free medium for 24 h; cultured in the absence of Ag and in the presence of serum, IL-2, dexamethasone, and Abs to IL-10 for an additional 4 days; and then re-exposed to the original sensitizing Ag. The presence of dexamethasone and Abs to IL-10 during the initial expansion stage appeared to be critical for the ability of the stimulated and expanded T cells to respond to restimulation with the same Ag. Repeated cycles of in vitro stimulation led to increased specificity for the sensitizing Ag (in the current case, pigeon cytochrome c), a decline in production of IL-2 and IFN-γ, and increased production of IL-4, IL-5, and IL-10. This culture protocol provides a test system for exploration of factors that regulate the conversion of naïve cells to memory cells and the development of specific immune responses to protein Ags. The data are consistent with models that implicate glucocorticoids as regulators of immune response specificity.
Nucleic Acid Vaccine-Induced Immune Responses Require CD28 Costimulation and Are Regulated by CTLA4
Authors: Horspool James H. , Perrin Peter J. , Woodcock Juliana B. , Cox Josephine H. , King Christopher L. , June Carl H. , Harlan David M. , St. Louis Daniel C. , Lee Kelvin P.
Published: December 2022

Immunization with plasmids expressing specific genes (DNA or nucleic acid vaccination (NAV)) elicits robust humoral and cell-mediated immune responses. The mechanisms involved in T cell activation by NAV are incompletely characterized. We have examined the costimulatory requirements of NAV. CD28-deficient mice did not mount Ab or CTL responses following i.m. immunization with eukaryotic expression plasmids encoding the bacterial gene β-galactosidase (βgal). Because these mice retained their ability to up-regulate the CTLA4 receptor (a negative regulator of T cell activation), we examined CTLA4’s role in the response of wild-type BALB/c mice to NAV. Intact anti-CTLA4 mAb but not Fab fragments suppressed the primary humoral response to pCIA/βgal without affecting recall responses, indicating CTLA4 activation inhibited Ab production but not T cell priming. Blockade of the ligands for CD28 and CTLA4, CD80 (B7-1) and CD86 (B7-2), revealed distinct and nonoverlapping function. Blockade of CD80 at initial immunization completely abrogated primary and secondary Ab responses, whereas blockade of CD86 suppressed primary but not secondary responses. Simultaneous blockade of CD80 + CD86 was less effective at suppressing Ab responses than either alone. Enhancement of costimulation via coinjection of B7-expressing plasmids augmented CTL responses but not Ab responses, and without evidence of Th1 to Th2 skewing. These findings suggest complex and distinct roles for CD28, CTLA4, CD80, and CD86 in T cell costimulation following nucleic acid vaccination.
Homeostasis Limits the Development of Mature CD8+ But Not CD4+ Thymocytes
Authors: van Meerwijk Joost P. M. , Marguerat Samuel , MacDonald H. Robson
Published: December 2022

The involvement of a variety of clonal selection processes during the development of T lymphocytes in the thymus has been well established. Less information, however, is available on how homeostatic mechanisms may regulate the generation and maturation of thymocytes. To investigate this question, mixed radiation bone marrow chimeras were established in which wild-type T cell precursors capable of full maturation were diluted with precursors deficient in maturation potential because of targeted mutations of the RAG1 or TCR-α genes. In chimeras in which the majority of thymocytes are blocked at the CD4−CD8−CD25+ stage (RAG1 deficient), and only a small proportion of T cell precursors are of wild-type origin, we observed no difference in the maturation of wild-type CD4−CD8−CD25+ cells to the CD4+CD8+ stage as compared with control chimeras. Therefore, the number of cell divisions occurring during this transition is fixed and not subject to homeostatic regulation. In contrast, in mixed chimeras in which the majority of thymocytes are blocked at the CD4+CD8+ stage (TCR-α deficient), an increased efficiency of development of wild-type mature CD8+ cells was observed. Surprisingly, the rate of generation of mature CD4+ thymocytes was not affected in these chimeras. Thus, the number of selectable CD8 lineage thymocytes apparently saturates the selection mechanism in normal mice while the development of CD4 lineage cells seems to be limited only by the expression of a suitable TCR. These data may open the way to the identification of homeostatic mechanisms regulating thymic output and CD4/CD8 lineage commitment, and the development of means to modulate it.
Induction of Jak/STAT Signaling by Activation of the Type 1 TNF Receptor
Authors: Guo DanQun , Dunbar James D. , Yang Chuan He , Pfeffer Lawrence M. , Donner David B.
Published: December 2022

Cellular responses to TNF are initiated by either of two cell surface receptors, the type 1 TNF receptor (TNFR1) and the type 2 TNF receptor (TNFR2). Although neither receptor contains an intrinsic protein tyrosine kinase, such activity has been implicated in TNF action. In this study, we show that murine TNF induces the tyrosine phosphorylation and activation of the intracellular Janus tyrosine kinases Jak1, Jak2, and Tyk2 in murine 3T3-L1 adipocytes. Activation of Jak kinases by TNF was associated with tyrosine phosphorylation of STAT1, STAT3, STAT5, and STAT6, but not STAT2 or STAT4, showing that TNF acts on a specific subset of these latent cytoplasmic transcription factors in 3T3-L1 adipocytes. Agonist antiserum to TNFR1 induced Jak kinase and STAT protein phosphorylation. Phosphorylation of Jak proteins was also induced by human TNF, which selectively binds to TNFR1 on murine cells. 35S-labeled Jak kinases were precipitated from a cell-free system and from lysates of 3T3-L1 adipocytes by a glutathione S-transferase fusion protein containing the cytoplasmic domain of TNFR1. These results suggest that the cytoplasmic domain of TNFR1 can directly interact with and form signaling complexes with Jak kinases. Jak2 was precipitated from HeLa cells by antiserum to TNFR1, directly demonstrating their association in vivo. Thus, TNF activates a Jak/STAT signal-transduction cascade by acting through TNFR1.
The A-Myb Transcription Factor Is a Marker of Centroblasts In Vivo
Authors: Golay Josée , Broccoli Vania , Lamorte Giuseppe , Bifulco Carlo , Parravicini Carlo , Pizzey Arnold , Thomas N. Shaun B. , Delia Domenico , Ferrauti Paola , Vitolo Domenico , Introna Martino
Published: December 2022

The A-Myb transcription factor is structurally related to the c-myb proto-oncogene and is involved in the control of proliferation and/or differentiation of mature B lymphocytes. We have shown previously by PCR analysis that A-myb is preferentially expressed in CD38+CD39−sIgM− mature B cells. We demonstrate here, using in situ hybridization, that A-mybexpression is restricted to the dark zone of human tonsils and lymph nodes. Furthermore, we show that A-Myb expression is cell cycle regulated both in tonsillar B cells and in Burkitt’s lymphoma cell lines, being detectable only in the S and G2/M phases of the cell cycle and not in G0/G1 phase. Strong proliferation of resting human B cells induced in vitro by a variety of physiologic signals, including anti-μ, CD40 ligand, IL-2, IL-4, IL-6, IL-13, IFN-γ, TNF-α, anti-CD19, and anti-CD20, failed to induce A-myb expression, suggesting that proliferation alone is not sufficient for A-myb expression in the absence of induction of a true centroblast phenotype. Finally, we show that differentiation of germinal center B cells in vitro toward either memory or plasma cells is accompanied by rapid down-regulation of A-myb expression. We conclude that A-myb is a marker of centroblasts generated in vivo.
The Lipophosphoglycan ofLeishmania donovaniUp-Regulates HIV-1 Transcription in T Cells Through the Nuclear Factor-κB Elements
Authors: Bernier Richard , Barbeau Benoı̂t , J. Tremblay Michel , Olivier Martin
Published: December 2022

We have recently demonstrated that the parasite Leishmania donovani and its surface molecule, lipophosphoglycan (LPG), can activate HIV-1 replication in monocytoid cells. Our present interest was to determine whether LPG could also up-regulate HIV-1 transcription in T cells. Using a CD4-positive human lymphoid T cell line (1G5) containing a stably integrated HIV-1 long terminal repeat (LTR)-luciferase construct, we found that LPG is a potent inducer of HIV-1 LTR activity. Treatment of 1G5 cells with signaling antagonists revealed that protein tyrosine kinase- and protein kinase A-dependent pathways were actively participating in the LPG-induced enhancement of HIV-1 LTR-driven activity. Transfection of Jurkat E6.1 cells with plasmids containing wild-type and nuclear factor-κB (NF-κB)-mutated HIV-1 LTR-luciferase constructs has suggested a role for NF-κB binding sites in the LPG-mediated induction of HIV-1 LTR activity. An LPG-induced binding factor specific to the NF-κB consensus sequences could be observed using electrophoretic mobility shift assay. Finally, transfection experiments performed with a vector containing HIV-1 κB binding sites only showed similar LPG-mediated induction, which was abrogated by sodium salicylate, a known NF-κB inhibitor. We thus demonstrate that the LPG-mediated induction of HIV-1 LTR activity in T cells involves several second messengers culminating in activation of HIV-1 LTR-driven transcription via NF-κB-binding consensus sequences. In conclusion, these results reinforce the idea that L. donovani is a putative cofactor in HIV-1 pathogenesis.
CTL Effector Function Within the Central Nervous System Requires CD4+ T Cells
Authors: Stohlman Stephen A. , Bergmann Cornelia C. , Lin Mark T. , Cua Daniel J. , Hinton David R.
Published: December 2022

CTL responses induced during most viral infections are independent of help derived from the CD4+ T cell population. However, clearance of virus from the central nervous system (CNS) during infection with the neurotropic JHM strain of mouse hepatitis virus is inhibited in the absence of CD4+ T cells. Adoptive transfer of activated CD8+ T cells with virus-specific cytolytic activity into CD4+ T cell-depleted hosts demonstrated that CD4+ T cells were one component of the host response required for expression of CTL effector function(s) within the CNS. Analysis of mice infected with the JHM strain of mouse hepatitis virus demonstrated that, in contrast to CD8+ T cells, few CD4+ T cells entered the brain parenchyma. Although fewer CD8+ T cells entered the brain parenchyma in mice depleted of CD4+ T cells, access of CTL was not inhibited in the absence of CD4+ T cells. The number of apoptotic lymphocytes in the CNS increased in the absence of CD4+ T cells, suggesting that CTL enter the CNS during viral infection in a CD4-independent manner. However, these cells rapidly undergo apoptosis, indicating that expression of CTL effector function with the parenchyma of the CNS is CD4 dependent. These data raise the possibility that programmed cell death of CD8+ T cells within the CNS is due to the increased Ag present in the CNS of infected CD4 depleted mice or that autocrine cytokines, which maintain CTL activity within peripheral tissues, are inhibited in the microenvironment of the CNS.
Resistance to Melanoma in Mice Immunized with Semiallogeneic Fibroblasts Transfected with DNA from Mouse Melanoma Cells
Authors: de Zoeten Edwin F. , Carr-Brendel Victoria , Cohen Edward P.
Published: December 2022

Tumor-associated Ags (TAA) that characterize a population of malignant cells are recognized by CTLs in the context of determinants specified by the MHC class I locus. Nevertheless, most progressively growing neoplasms do not induce antitumor immune responses that can control tumor cell growth. The TAA may be insufficiently antigenic. We found previously that immunization of mice with a cellular immunogen prepared by transfecting tumor DNA into allogeneic mouse fibroblasts resulted in strong antitumor immune responses that were specific for the type of tumor from which the DNA was obtained. Since the fibroblasts differed at the MHC from the immunized mice, we postulated that the immunogenic properties of the allogeneic transfected cells might be enhanced if the cells were modified to express syngeneic class I determinants. In a mouse melanoma model system, the H-2Kb gene was introduced into LM mouse fibroblasts (H-2k). Afterward, the cells were transfected with DNA from B16 melanoma cells (H-2b). The transfected cells were tested for their immunotherapeutic properties in C57BL/6J mice (H-2b) with melanoma. Mice with melanoma treated solely by immunization with the semiallogeneic transfected cells developed strong, long-term resistance to the growth of the tumor. In some instances, the mice survived indefinitely. Intact rather than disrupted transfected cells were required to induce the antimelanoma response, consistent with direct presentation of TAA by the transfected cells. The augmented resistance to melanoma in mice treated with the semiallogeneic transfected cells points toward an analogous form of therapy for cancer patients.
Sea Urchin Coelomocytes Specifically Express a Homologue of the Complement Component C3
Authors: Al-Sharif Walid Z. , Sunyer J. Oriol , Lambris John D. , Smith L. Courtney
Published: December 2022

A homologue of complement component C3 (SpC3) has been cloned and sequenced from the purple sea urchin, Strongylocentrotus purpuratus. The preprocessed, deduced protein size is estimated to be 186 kDa with a short leader and two chains, α and β. There are cysteines in conserved positions for interchain disulfide bonding, and there is a conserved thioester site in the α-chain with an associated histidine. There are five consensus N-linked glycosylation sites, and putative cleavage sites for factor I and C3 convertase. Partially purified SpC3 on protein gels shows a nonreduced size of 210 kDa and, under reducing conditions, reveals an α-chain of 130 kDa and a β-chain of 80 kDa. These sizes are larger than the deduced sizes, suggesting that the protein has carbohydrates added to most of the consensus N-linked glycosylation sites. Phylogenetic analysis of SpC3 compared with other members of the thioester protein family, which includes C3, C4, C5, and α2-macroglobulin, shows that SpC3 is the first divergent complement protein, falling at the base of the complement protein clade. Transcripts from the SpC3 gene (Sp064) are 9 kb, and the gene is expressed specifically in coelomocytes, which are the immunocytes in the sea urchin. Genome blots suggest that SpC3 is encoded by a single copy gene per haploid genome. This is the first identification of a complement component in an invertebrate, and suggests homology of the innate immune system within the deuterostome lineage of animals.
Regulation of Hyaluronan-Induced Chemokine Gene Expression by IL-10 and IFN-γ in Mouse Macrophages
Authors: Horton Maureen R. , Burdick Marie D. , Strieter Robert M. , Bao Clare , Noble Paul W.
Published: December 2022

Turnover of the extracellular matrix (ECM), activation of macrophages, and accumulation of chemokines/cytokines are all hallmarks of chronic inflammation. Extracellular matrix components, such as hyaluronan (HA), have recently been shown to influence macrophage effector functions, such as the release of inflammatory chemokines and cytokines. Although low m.w. fragments of the glycosaminoglycan HA induce macrophages to secrete numerous inflammatory mediators, the mechanisms regulating ECM-induced macrophage activation are poorly understood. We have examined the effects of IL-10 and IFN-γ on HA-induced chemokine gene expression in primary mouse macrophages. We found that IL-10 and IFN-γ independently inhibit HA-induced expression of macrophage inflammatory protein-1α (MIP-1α), MIP-1β, and KC at both the mRNA and protein levels. Whereas IL-10 inhibited most of the HA-induced chemokines tested, IFN-γ selectively inhibited only MIP-1α, MIP-1β, and KC. This inhibition did not require prestimulation and occurred even when the cytokines were added up to 3 h after stimulation with HA. For MIP-1α, the inhibition by IFN-γ occurred at the level of transcription, whereas IL-10 predominantly decreased the stability of MIP-1α mRNA. IFN-γ and IL-10 equally inhibited macrophage expression of MIP-1β mRNA at the level of transcription, but MIP-1β mRNA stability was decreased to a greater extent by IL-10. These data identify a previously unrecognized role for IL-10 and IFN-γ as regulators of ECM-induced macrophage expression of inflammatory chemokines.
Treatment with an Anti-IL-4 Monoclonal Antibody Blocks Suppression of Collagen-Induced Arthritis in Mice by Oral Administration of Type II Collagen
Authors: Yoshino Shin
Published: December 2022

Oral administration of type II collagen (CII) has been shown to suppress collagen-induced arthritis (CIA) in experimental animals. However, the exact mechanism by which CIA is suppressed following administration of CII remains to be investigated, although it was demonstrated that active suppression by regulatory T cells might be involved in the suppression. Therefore, we have examined whether the inhibitory cytokine IL-4 plays a role in the suppression of CIA, by using an anti-IL-4 mAb (11B11 mAb). Mice were fed daily with CII over a period of 10 days before immunization with CII. 11B11 mAb was i.p. injected 30 min before each oral administration of CII. The results showed that treatment with 11B11 mAb markedly blocked suppression of CIA by the oral Ag. The blockade of suppression of CIA by the anti-IL-4 mAb was associated with the blockade of augmentation of IL-4 secretion in CII-fed mice. The treatment with 11B11 mAb also resulted in the prevention of decreases in anti-CII IgG2a Ab production, DTH responses to CII, proliferation of lymphoid cells to CII, and IFN-γ secretion in mice given CII orally. Thus, the neutralization of IL-4 by an anti-IL-4 Ab appears to be effective in blocking suppression of CIA by oral administration of CII, suggesting that IL-4 may be critically involved in its suppression.
Distinct Peptide Loading Pathways for MHC Class II Molecules Associated with Alternative Ii Chain Isoforms
Authors: Bikoff Elizabeth K. , Kenty George , Van Kaer Luc
Published: December 2022

Mutant mouse strains expressing either p31 or p41 Ii chain appear equally competent with respect to their class II functional activities including Ag presentation and CD4+ T cell development. To further explore possibly divergent roles provided by alternative Ii chain isoforms, we compare class II structure and function in double mutants also carrying a null allele at the H2-DM locus. As for DM mutants expressing wild-type Ii chain, AαbAβb dimers present in DM-deficient mice expressing either Ii chain isoform appear equally occupied by class II-associated Ii chain-derived peptides (CLIP). Surprisingly, in functional assays, these novel mouse strains exhibit strikingly different phenotypes. Thus, DM-deficient mice expressing wild-type Ii chain or p31 alone are both severely compromised in their abilities to present peptides. In contrast, double mutants expressing the p41 isoform display markedly enhanced peptide-loading capabilities, approaching those observed for wild-type mice. The present data strengthen evidence for divergent class II presentation pathways and demonstrate for the first time that functionally distinct roles are mediated by alternatively spliced forms of the MHC class II-associated Ii chain in a physiologic setting.
CD8+ Memory T Cells (CD44high, Ly-6C+) Are More Sensitive than Naive Cells (CD44low, Ly-6C−) to TCR/CD8 Signaling in Response to Antigen
Authors: Curtsinger Julie M. , Lins Debra C. , Mescher Matthew F.
Published: December 2022

Memory CD8+ T cells from mice previously primed with alloantigen (alloAg) can respond in vitro to IL-2 and purified class I alloAg presented on microspheres, while no response can be detected using cells from naive mice. Similar results have been obtained using cells from OT-1 mice expressing a transgenic TCR that is specific for OVA257–264 (SIINFEKL) peptide bound to H-2Kb. A population of resting memory cells (defined on the basis of low forward scatter and CD44high, Ly-6C+, CD25−, CD69− surface phenotype) that is present in the OT-1 mice exhibits a substantially higher sensitivity to Ag-stimulation than do naive cells (CD44low, Ly-6C−) expressing the same TCR. CD44high cells respond vigorously to H-2Kb immobilized on microspheres and pulsed with peptide, while CD44low cells respond weakly and only at high class I density and peptide concentration. The Ag-presenting surface only has ligands for TCR and CD8 (class I and peptide), thus ruling out the possibility that differences are due to ligand binding by other adhesion or costimulatory receptors that are expressed at high levels on the memory cells. Experiments using anti-TCR mAb as the stimulus and coimmobilized non-Ag class I as a ligand for CD8 suggest that the difference between naive and memory cells may be at the level of stimulation through the TCR. Thus, in addition to expressing increased levels of adhesion receptors that may enhance responses to Ag on APCs, memory CD8+ T cells appear to be intrinsically more sensitive than naive cells to stimulation through the TCR/CD8 complex.
Ontogeny of the Heavy Chain Immunoglobulin Repertoire in Fetal Liver and Bone Marrow
Authors: Delassus Sylvie , Darche Sylvie , Kourilsky Philippe , Cumano Ana
Published: December 2022

We studied the kinetics of maturation of B cell progenitors in the mouse embryo, from day 15 of development to birth, both in liver and bone marrow. The analysis of Ig heavy chain rearrangements at different time points of late fetal development shows that oligoclonal patterns of VH-D-JH rearrangements are detected by day 15 in fetal liver. The pattern is polyclonal and diverse by day 17; however, 80% of the rearrangements are nonproductive. In bone marrow, the pattern of rearrangements is less diverse at birth, although the percentage of nonproductive rearrangements approaches adult bone marrow levels (35–40%). After day 17 in fetal liver, there is a sudden reversal in the percentage of nonproductive rearrangements that reaches 33% at day 19 (birth). Maturation of B cells, as measured by the fraction of surface Ig+ in total B220+ cells and the presence of N sequence additions in VH-D-JH joints, occurs in the marrow before fetal liver. These results demonstrate that the lymphopoietic environment in fetal liver and bone marrow of animals at the same stage of development is functionally distinct.
Stimulation of Human T Lymphocytes by LPS Is MHC Unrestricted, But Strongly Dependent on B7 Interactions
Authors: Mattern Taila , Flad Hans-Dieter , Brade Lore , Rietschel Ernst T. , Ulmer Artur J.
Published: December 2022

Recently, we have shown that LPS is a potent inducer of human T cell proliferation and lymphokine production. However, the activation of T cells by LPS has been demonstrated to be monocyte dependent and to require direct cell-to-cell contact. Here, we investigated the role of monocytes as accessory cells and the requirement for costimulatory signals in more detail. We found that the accessory cell activity of monocytes during LPS-induced T cell proliferation is characterized by the following features: LPS-primed monocytes are competent stimulators of T cell proliferation; interaction of LPS with monocytes during the priming step is dependent on CD14 and is sensitive to ammonia; monocyte/T cell interactions are not MHC restricted but are strongly dependent on interactions of CD28 and/or CTLA-4 on T cells and their ligands CD80 and/or CD86 on monocytes. CD80 seems to be crucial for the activation of T cells by monocytes, since monocytes expressing CD86 but not CD80 after LPS stimulation were unable to stimulate T cells; IL-12, at least as a costimulatory factor, but not IL-15, is important in LPS-induced T cell proliferation. Taken together, our results indicate that LPS acts neither as a mitogen, nor as a superantigen, nor as an Ag. The activation of human T cells by LPS requires the help of accessory functions by primed monocytes and is MHC unrestricted but needs costimulatory signals via CD28 and/or CTLA-4.
Crucial Role of TNF Receptor Type 1 (p55), But Not of TNF Receptor Type 2 (p75), in Murine Toxoplasmosis
Authors: Deckert-Schlüter Martina , Bluethmann Horst , Rang Andrea , Hof Herbert , Schlüter Dirk
Published: December 2022

TNF-α exerts its biologic activity through two distinct receptors, TNF receptor type 1 (TNFR1, p55) and TNF receptor type 2 (TNFR2, p75). To analyze their function in toxoplasmosis, we orally infected mice genetically deficient for TNFR1 (TNFR10/0), TNFR2 (TNFR20/0), or both TNF receptors (TNFR1/20/0), as well as wild-type (wt) mice with a low-virulent strain of Toxoplasma gondii. TNFR1/20/0 and TNFR10/0 mice succumbed to toxoplasmosis within 17 and 27 days, respectively, whereas TNFR20/0 and wt mice were equally resistant to acute toxoplasmosis. Histopathology attributed death of TNFR1/20/0 and TNFR10/0 mice to a fulminant necrotizing encephalitis. In addition, pneumonia contributed to the fatal outcome. The poor prognosis of TNFR1/20/0 and TNFR10/0 mice was reflected by a significantly increased parasitic load in the brain and lung as compared with TNFR20/0 and wt mice. Immunohistochemistry demonstrated a remarkable reduction of inducible nitric oxide synthase protein in brain and lung of TNFR1/20/0 and TNFR10/0 as compared with TNFR20/0 and wt mice. Reverse-transcribed PCR showed that in contrast to TNFR20/0 and wt mice, TNFR10/0 mice were unable to up-regulate inducible nitric oxide synthase mRNA transcripts in the course of infection, whereas intracerebral levels of IFN-γ, TNF-α, and IL-1β mRNA transcripts, recruitment of immune cells to the brain, and the amount of apoptotic cells in inflammatory foci did not differ significantly among the various experimental groups. These results illustrate that in Toxoplasma encephalitis, TNF-α-mediated immune responses are of crucial importance and that signaling through TNFR1, but not TNFR2, provides the stimulus required for the induction of protective nitric oxide.
A Critical Role for IL-13 in Resistance to Intestinal Nematode Infection
Authors: Bancroft Allison J. , McKenzie Andrew N. J. , Grencis Richard K.
Published: December 2022

Mice in which either the IL-4 or the IL-13 gene has been disrupted (IL-4 KO and IL-13 KO) were susceptible to infection with the intestinal nematode Trichuris muris, whereas their wild-type littermates were highly resistant and expelled the parasite. IL-4 KO mice showed diminished Th2-type responses with T. muris infection and also failed to produce parasite-specific IgG1 Abs. Although IL-13 KO mice made reduced Th2-type responses early in infection, they were capable of generating strong Th2-type responses at later time points and were unable to regulate the magnitude of their Ab isotype response. These results confirm the importance of IL-4 in resistance to T. muris and provide the first demonstration of an important role for IL-13 in resistance to helminth infection. The IL-13 KO mouse had a separate phenotype to that of the IL-4 KO mouse, suggesting that both IL-4 and IL-13 play important yet different roles in mediating immunity to intestinal helminths.
Activation of Caspase 3 (CPP32)-Like Proteases Is Essential for TNF-α-Induced Hepatic Parenchymal Cell Apoptosis and Neutrophil-Mediated Necrosis in a Murine Endotoxin Shock Model
Authors: Jaeschke Hartmut , Fisher Michael A. , Lawson Judy A. , Simmons Carol A. , Farhood Anwar , Jones David A.
Published: December 2022

Endotoxin (ET)-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. Members of the caspase family have been implicated in the signal transduction pathway of apoptosis. The aim of this study was to characterize ET-induced hepatic caspase activation and apoptosis and to investigate their effect on neutrophil-mediated liver injury. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine (Gal) and 100 μg/kg Salmonella abortus equi ET increased caspase 3-like protease activity (Asp-Val-Glu-Asp-substrate) by 1730 ± 140% at 6 h. There was a parallel enhancement of apoptosis (assessed by DNA fragmentation ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). In contrast, activity of caspase 1 (IL-1β-converting enzyme)-like proteases (Tyr-Val-Ala-Asp-substrate) did not change throughout the experiment. Caspase 3-like protease activity and apoptosis was not induced by Gal/ET in ET-resistant mice (C3H/HeJ). Furthermore, only murine TNF-α but not IL-1αβ increased caspase activity and apoptosis. Gal/ET caused neutrophil-dependent hepatocellular necrosis at 7 h (area of necrosis, 45 ± 3%). Delayed treatment with the caspase 3-like protease inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD) (10 mg/kg at 3 h) attenuated apoptosis by 81 to 88% and prevented liver cell necrosis (≤5%). Z-VAD had no effect on the initial inflammatory response, including the sequestration of neutrophils in sinusoids. However, Z-VAD prevented neutrophil transmigration and necrosis. Our data indicate that activation of the caspase 3 subfamily of cysteine proteases is critical for the development of parenchymal cell apoptosis. In addition, excessive hepatocellular apoptosis can be an important signal for transmigration of primed neutrophils sequestered in sinusoids.
Regulation of Macrophage Phagocytosis of Apoptotic Cells by cAMP
Authors: Rossi Adriano G. , McCutcheon Judith C. , Roy Noémi , Chilvers Edwin R. , Haslett Christopher , Dransfield Ian
Published: December 2022

Regulation of macrophage capacity to remove apoptotic cells may control the balance of apoptotic and necrotic leukocytes at inflamed foci and the extent of leukocyte-mediated tissue damage. Although the molecules involved in the phagocytic process are beginning to be defined, little is known about the underlying regulatory and signaling mechanisms controlling this process. In this paper, we have investigated the effects of treatment of human monocyte-derived macrophages with PGs and other agents that elevate intracellular cAMP on phagocytosis. PGE2 and PGD2 specifically reduced the proportion of macrophages that phagocytosed apoptotic cells. Similar results were obtained with the membrane-permeable cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP but not with the cGMP analogue dibutyryl-GMP. Consistent with the observation that phagocytosis was inhibited by cAMP elevation, treatment of monocyte-derived macrophages with PGE2 resulted in rapid, transient increase in levels of intracellular cAMP. These effects were not due to nonspecific inhibition of monocyte-derived macrophage phagocytosis given that ingestion of Ig-opsonized erythrocytes was unaffected. Elevation of cAMP induced morphologic alterations indicative of changes in the adhesive status of the macrophage, including cell rounding and disassembly of structures that represent points of contact with substrate containing actin and talin. These results strongly suggest that rapid activation of cAMP signaling pathways by inflammatory mediators regulates processes that limit tissue injury and that modulation of cAMP levels represents an additional therapeutic target in the control of resolution of inflammation.
Cutting Edge: Role of the Inositol Phosphatase SHIP in B Cell Receptor-Induced Ca2+ Oscillatory Response
Authors: Okada Hidetaka , Bolland Silvia , Hashimoto Akiko , Kurosaki Mari , Kabuyama Yukihito , Iino Masamitsu , Ravetch Jeffrey V. , Kurosaki Tomohiro
Published: December 2022

Src homology-2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP) is a recently identified protein that has been implicated as an important signaling molecule. Although SHIP has been shown to participate in the FcγRIIB-mediated inhibitory signal, the functional role of SHIP in activation responses by immunoreceptor tyrosine-based activation motif-bearing receptors such as B cell receptor (BCR) remains unclear. Indeed, it has been proposed that SHIP serves as a linking molecule for the regulation of the extracellular signal-regulated kinase pathway in BCR signaling, because SHIP associates with Shc. We now report that SHIP-deficient DT40 B cells display enhanced Ca2+ mobilization in response to BCR ligation, whereas extracellular signal-regulated kinase activation is unaffected. This Ca2+ enhancement is due to a sustained intracellular Ca2+ increase or to long-lasting Ca2+ oscillations by loss of SHIP, as revealed by single-cell Ca2+ imaging analysis. These results demonstrate the importance of SHIP in B cell activation by the modulation of Ca2+ mobilization.
Cutting Edge: LFA-1 Interaction with ICAM-1 and ICAM-2 Regulates Th2 Cytokine Production
Authors: Salomon Benoı̂t , Bluestone Jeffrey A.
Published: December 2022

The role of CD28/B7 and LFA-1/ICAM costimulation in proliferation and Th1/Th2 differentiation of naive CD4+ T cells was addressed using T cells from DO11.10 TCR transgenic mice stimulated by dendritic cells. The blockade of either CD28/B7 or LFA-1/ICAM interactions partially inhibited T cell proliferation. By comparison, blocking CD28/B7 costimulation inhibited IL-4 and IL-5 (Th2 cytokine) production, whereas blocking LFA-1/ICAM-1 or LFA-1/ICAM-2 led to a significant increase (15- to 40-fold) of Th2 cytokines. The combination of anti-ICAM-1 and anti-ICAM-2 mAbs had a synergistic effect with a 100- to 1000-fold increase of Th2 cytokine production. Thus, these two costimulatory pathways have opposing roles in the regulation of Th2 development.
Anti-CD4 Monoclonal Antibody-Induced Tolerance to MHC-Incompatible Cardiac Allografts Maintained by CD4+ Suppressor T Cells That Are Not Dependent upon IL-4
Authors: Hall Bruce M. , Fava Lisa , Chen Juchuan , Plain Karren M. , Boyd Rochelle A. , Spicer S. Timothy , Berger Manuela F.
Published: December 2022

Anti-CD4 mAb-induced tolerance to transplanted tissues has been proposed as due to down-regulation of Th1 cells by preferential induction of Th2 cytokines, especially IL-4. This study examined the role of CD4+ cells and cytokines in tolerance to fully allogeneic PVG strain heterotopic cardiac allografts induced in naive DA rats by treatment with MRC Ox38, a nondepleting anti-CD4 mAb. All grafts survived >100 days but had a minor mononuclear cell infiltrate that increased mRNA for the Th1 cytokines IL-2, IFN-γ, and TNF-β, but not for Th2 cytokines IL-4 and IL-6 or the cytolytic molecules perforin and granzyme A. These hosts accepted PVG skin grafts but rejected third-party grafts, which were not blocked by anti-IL-4 mAb. Cells from these tolerant hosts proliferated in MLC and produced IL-2, IFN-γ, and IL-4 at levels equivalent to naive cells. Unfractionated and CD4+ T cells, but not CD8+ T cells, transferred specific tolerance to irradiated heart grafted hosts and inhibited reconstitution of rejection by cotransferred naive cells. This transfer of tolerance was associated with normal induction of IL-2 and delayed induction of IFN-γ, but not with increased IL-4 or IL-10 mRNA. Transfer of tolerance was also not inhibited by anti-IL-4 mAb. This study demonstrated that tolerance induced by a nondepleting anti-CD4 mAb is maintained by a CD4+ suppressor T cell that is not associated with preferential induction of Th2 cytokines or the need for IL-4; nor is it associated with an inability to induce Th1 cytokines or anergy.
HSV-1 Glycoprotein I-Reactive TCRγδ Cells Directly Recognize the Peptide Backbone in a Conformationally Dependent Manner
Authors: Sciammas Roger , Bluestone Jeffrey A.
Published: December 2022

Despite the description of numerous antigenic ligands recognized by TCRγδ cells, detailed information concerning the structural nature of these antigenic epitopes is lacking. In addition, the recent descriptions of human TCRγδ cells recognizing mycobacterium-derived low m.w. lipid molecules confirms that the spectrum and nature of biologic structures that are capable of being recognized by TCRγδ cells are unclear. We have previously described a murine TCRγδ cell clone, TgI4.4, that is reactive to herpes simplex virus (HSV)-1 glycoprotein I (gI). Unlike TCRαβ-mediated, MHC-restricted Ag recognition but similar to Ig Ag recognition, TgI4.4 recognizes purified gI directly, in the absence of Ag processing or presentation. Since gI is a complex glycoprotein, the nature of the antigenic epitope was investigated. First, gI recognition by TgI4.4 is conformationally dependent, as revealed by denaturation and proteolytic experiments. Secondly, the epitope recognized by TgI4.4 was mapped to the amino terminus by using insertion mutants of gI. Lastly, TgI4.4 recognizes the gI protein directly since completely deglycosylated forms of gI are efficiently recognized. Therefore, TCRγδ cells are capable of recognizing a variety of molecular structures, including proteins. The ability of TgI4.4 to recognize a nonglycosylated form of gI suggests that HSV-1 recognition by TCRγδ cells in vivo is not limited by cell-specific glycosylation patterns or glycosylation-dependent conformational influences.
The Nephritogenic T Cell Response in Murine Chronic Graft-Versus-Host Disease
Authors: Meyers Catherine M. , Tomaszewski John E. , Glass Joan D. , Chen Clarice W.
Published: December 2022

To investigate mechanisms of cell-mediated events in chronic glomerulonephritis, T cell clones were isolated from kidneys of animals with murine chronic graft-vs-host disease. This systemic disorder is induced in normal (C57BL/6 × DBA/2)F1 recipients (H-2b/d) following transfer of parental (DBA/2) T cells (H-2d). These studies demonstrate that mouse renal (MR) T cells isolated from nephritic kidneys of diseased recipients are host-derived CD4+ α/β+ T cells. Adoptive transfer of a panel of MR clones to naive (C57BL/6 × DBA/2)F1 recipients reveals distinct functional subsets. One subset does not transfer renal disease, and one induces severe renal inflammation and damage. In vitro proliferative responses of nephritogenic MR clones reveal predominant reactivity toward autologous class II MHC (I-Ed/I-Ad) determinants, and selected nephritogenic MR clones preferentially recognize renal Ag preparations derived from normal (C57BL/6 × DBA/2)F1 kidneys. In addition, cytokine profile analysis of MR clones indicates a Th2 pattern with IL-4 and IL-10 expression, although nephritogenic T cell clones also express IFN-γ. These data suggest that the nephritogenic T cell response in chronic graft-vs-host disease is autoreactive in nature and may be restricted by determinants shared by both graft and host (Iad).