bluuebunny/crossref_metadata_2025 huggingface dataset:
The neuropeptide substance P activates transcription factor NF-kappa B and kappa B-dependent gene expression in human astrocytoma cells.
Authors: Lieb K , Fiebich B L , Berger M , Bauer J , Schulze-Osthoff K
Published: December 2022

The neuropeptide substance P is a major mediator of neurogenic inflammation and immunomodulatory activities within the central and peripheral nervous system. In several cell types, substance P induces the expression of proinflammatory cytokines that have been implicated in the pathogenesis of different neuropathologies. Substance P preferentially binds to NK-1, a receptor of the neurokinin family, but how the receptor-elicited signal is translated into inflammatory gene expression is not yet understood. In this work, we describe that in U373 MG astrocytoma cells, nanomolar concentrations of substance P potently triggered activation of NF-kappa B, a transcription factor involved in the control of cytokine expression and apoptosis. Substance P-induced NF-kappa B activation was associated with the increased mRNA expression and secretion of IL-8, an NF-kappa B-controlled target gene. The stimulatory effect of substance P was specific, since an NK-1-selective receptor antagonist completely prevented NF-kappa B activation in response to substance P, but not IL-1 beta. In addition, we show that the activity of substance P required mobilization of intracellular calcium and formation of reactive oxygen intermediates as second messengers. Our results suggest that NF-kappa B may be an important component controlling neurogenic inflammation within the peripheral and central nervous system.
Lipocortin 1 protects against splanchnic artery occlusion and reperfusion injury by affecting neutrophil migration.
Authors: Cuzzocrea S , Tailor A , Zingarelli B , Salzman A L , Flower R J , Szabó C , Perretti M
Published: December 2022

Splanchnic artery occlusion and reperfusion (SAO/R) shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (60-min reperfusion). Following this reperfusion period, rats developed a fall in mean arterial blood pressure, associated with a significant increase in tissue myeloperoxidase (MPO) activity in the intestine and a marked histologic injury to the distal ileum. Treatment of rats with a lipocortin-1 (LC1)-derived N-terminal peptide, peptide Ac(2-26), dose-dependently (0.125-0.5 mg/kg s.c.) reduced the progressive fall in blood pressure and prevented the infiltration of neutrophils into the reperfused intestine (reduced MPO activity). The LC1 peptide also reduced the degree of ischemia/reperfusion injury in the bowel as evaluated by histologic examination. The glucocorticoid dexamethasone (0.1 mg/kg s.c., -1 h) also produced a marked improvement in SAO/R shock (i.e., maintained mean arterial blood pressure and reduced tissue MPO activity), and this was reversed by pretreatment with two different antisera raised against the LC1 pharmacophore. Peptide Ac(2-26) (0.5 mg/kg s.c., -30 min) reduced (>60%) the extent of IL-1beta-induced cell emigration and significantly attenuated (approximately 45%) the number of adherent leukocytes in the rat mesenteric vascular bed, as assessed by video microscopy. These results suggest that LC1 inhibits neutrophil migration and accumulation into reperfused tissues, thereby ameliorating the outcome of SAO/R shock.
Immunosuppressive effects of human CTLA4Ig in a non-human primate model of allogeneic pancreatic islet transplantation.
Authors: Levisetti M G , Padrid P A , Szot G L , Mittal N , Meehan S M , Wardrip C L , Gray G S , Bruce D S , Thistlethwaite J R , Bluestone J A
Published: December 2022

Ag-specific T cell activation requires a CD28-mediated costimulatory interaction. This observation has suggested novel approaches to suppress donor-specific immunity, including the use of soluble CD28 antagonists, such as CTLA4Ig, which suppresses transplant rejection in small animal models. In this study, CTLA4Ig therapy was examined in a non-human primate model of allogeneic pancreatic islet transplantation. Two of five CTLA4Ig-treated monkeys showed prolonged graft survival, which correlated with donor-specific hyporesponsiveness in vitro. Humoral responses to the transplanted tissue were suppressed in all treated animals. These results suggest that CTLA4Ig is effective in suppressing both humoral and cellular immune responses in a non-human primate model of allogeneic transplantation.
Molecular cloning of leukotactin-1: a novel human beta-chemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3.
Authors: Youn B S , Zhang S M , Lee E K , Park D H , Broxmeyer H E , Murphy P M , Locati M , Pease J E , Kim K K , Antol K , Kwon B S
Published: December 2022

A new member of human beta-chemokine cDNA was isolated and named leukotactin-1 (Lkn-1). Lkn-1, along with murine macrophage inflammatory protein-related protein-1 and -2, defines a subgroup of beta-chemokines based on two conserved cysteines in addition to the four others conserved in all beta-chemokines. The putative mature Lkn-1 is composed of 92 amino acids with a calculated m.w. of 10,162. The Lkn-1 gene was mapped to human chromosome 17, region q12. Recombinant Lkn-1 was a potent chemoattractant for neutrophils, monocytes, and lymphocytes and induced calcium flux in these cells. Lkn-1 specifically induced calcium flux in CCR1- and CCR3-expressing HOS cell lines. Lkn-1 suppressed colony formation by human granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. Hence, we have isolated and characterized a human C6 beta-chemokine that is a potent agonist at CCR1 and CCR3 and shows broad biologic activities, including leukocyte chemoattraction.
Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase.
Authors: Yu C L , Jove R , Burakoff S J
Published: December 2022

The Lck protein, a Src family tyrosine kinase, plays a critical role in T cell maturation and activation. Dysregulation of Lck expression or Lck kinase activity has been implicated in T cell leukemias from mice to humans, although the mechanism underlying Lck-mediated oncogenesis is still largely unclear. We report here that both DNA binding activities and tyrosine phosphorylation of STAT3 and STAT5, but not STAT1, are constitutively enhanced in the mouse T cell lymphoma LSTRA, which is a well-characterized cell line that overexpresses Lck protein and exhibits high levels of Lck kinase activity. Furthermore, Janus kinase 1 (jak1) and Jak2 protein tyrosine kinases are constantly activated in these cells, as determined by their autophosphorylation in an in vitro kinase assay and increased levels of tyrosine phosphorylation on immunoblots. Therefore, like Src-transformed cells, Lck-overexpressing LSTRA cells also exhibit constitutive activation of distinct Jak and STAT proteins.
Insulin down-regulates the inducible nitric oxide synthase pathway: nitric oxide as cause and effect of diabetes?
Authors: Stevens R B , Sutherland D E , Ansite J D , Saxena M , Rossini T J , Levay-Young B K , Hering B J , Mills C D
Published: December 2022

Evidence in this paper indicates that insulin can down-regulate the inducible nitric oxide synthase (iNOS) pathway in vivo. The iNOS pathway is up-regulated in diabetes-prone rats and mice and is associated with an autoimmune process. However, the results presented here indicate that macrophage nitric oxide (NO) production and iNOS mRNA expression are also elevated in rats or mice made diabetic by streptozotocin injection in which there is no primary autoimmune component. Insulin administration reduces NO production in autoimmune-prone and streptozotocin-induced diabetic rodents. Finally, insulin decreases macrophage NO production in normal hosts. These results indicate that the autoimmune paradigm is inadequate to explain increased NO in diabetes. As a potential mechanism to explain insulin-mediated regulation of NO production, TGF-1 may be involved because 1) macrophages from diabetic mice produce less TGF-beta1 than macrophages from normal hosts; 2) the circulating TGF-beta1 level is lower in diabetic mice; and 3) insulin administration increases circulating TGF-beta1 in normal mice. Together, these results provide evidence that increased NO in diabetes is not only a cause but also an effect of beta-cell destruction and results in part from a heretofore unrecognized immunomodulatory activity of insulin.
Functional consequences of dysregulated B7-1 (CD80) and B7-2 (CD86) expression in B or T lymphocytes of transgenic mice.
Authors: Van Parijs L , Sethna M P , Schweitzer A N , Borriello F , Sharpe A H , Abbas A K
Published: December 2022

T cell activation and tolerance are regulated by interactions between CD28 or CTLA-4 on T cells and B7 costimulatory molecules on APCs. We have generated transgenic mouse strains that constitutively express B7-1 (CD80) at high levels on B cells or T cells or express B7-2 (CD86) on T lymphocytes to examine the consequences of dysregulated B7 expression on T cell responses. The transgene-derived B7 molecules are functional, because B7-1 transgenic B cells are more efficient APCs than are wild-type B cells, and the activation of B7 transgenic T cells is less dependent on exogenous costimulation than that of wild-type T cells. In vivo, constitutive expression of B7 molecules leads to the elimination of immature B cells. The expression of B7 molecules on thymocytes results in the down-regulation of CD28 expression. However, B7 transgenic mice have normal numbers of mature lymphocytes and mount normal T cell responses following immunization with protein Ag. Neither anergy induction nor superantigen-mediated deletion of T cells is altered by the dysregulated expression of B7-1 or B7-2 on B or T lymphocytes in these transgenic strains. Therefore, functionally significant levels of B7 expressed constitutively on mature lymphocytes are not, by themselves, sufficient to abrogate T cell tolerance or induce autoimmune disease.
TAP peptide transporter-independent presentation of heat-killed Sendai virus antigen on MHC class I molecules by splenic antigen-presenting cells.
Authors: Liu T , Chambers B , Diehl A D , Van Kaer L , Jondal M , Ljunggren H G
Published: December 2022

Immunization of C57BL/6 (B6) mice with heat-killed Sendai virus generates a Sendai virus-specific CD8+ T cell response. This suggests that APC have the capacity to take up and present exogenous (nonreplicative) Sendai virus Ag on MHC class I molecules. Little is known about the intracellular requirements for processing of this form of Ag and its presentation on MHC class I. Therefore, we have studied the processing and presentation of heat-killed Sendai virus Ag on MHC class I molecules in splenic APC. Heat-killed Sendai virus Ags were efficiently processed by normal B6 as well as by TAP-1(-/-) splenic APC. Presentation was MHC class I restricted, since no presentation was seen by APC from TAP-1/beta2m-/- mice that lack expression of MHC class I. Presentation occurred even in the presence of brefeldin A, but was blocked by cytochalasin D as well as chloroquine. Finally, B6 as well as TAP-1(-/-) splenic APC, loaded with heat-killed Sendai virus Ag in vitro, primed naive CD8+ T cells in vivo. These studies suggest the existence of a TAP-independent pathway for Ag presentation on MHC class I in normal splenic APC, bearing many similarities with the MHC class II pathway for Ag presentation. The present results are discussed in relation to the events underlying the processing and presentation of exogenous Ag on MHC class I, the molecular basis for CD8+ T cell priming during viral infections, and prospects for vaccine development.
Leech immunocytes contain proopiomelanocortin: nitric oxide mediates hemolymph proopiomelanocortin processing.
Authors: Salzet M , Salzet-Raveillon B , Cocquerelle C , Verger-Bocquet M , Pryor S C , Rialas C M , Laurent V , Stefano G B
Published: December 2022

This report establishes the presence of mammalian-like proopiomelanocotropic hormone (POMC), and six of its peptides, including adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH), in the immune tissues of the leech Theromyzon tessulatum. The 25.4-kDa protein was purified by high pressure gel permeation chromatography, anti-ACTH-affinity column, and reverse-phase HPLC. Its characterization was performed by Edman degradation, enzymatic treatments, and electrospray mass spectrometry. Leech POMC exhibits considerable amino acid sequence similarity to mammalian POMC. Of the six peptides, three showed high sequence similarity to their vertebrate counterparts met-enkephalin, alpha-MSH, and ACTH: 100, 84.6, and 70%, respectively; whereas gamma-MSH, beta-endorphin, and gamma-lipotropin hormone exhibited only 45, 20, and 10% sequence identity, respectively. No dibasic amino acid residues were found at the C terminus of the gamma- and beta-MSH peptides. In contrast, the leech alpha-MSH was flanked at its C-terminal by the Gly-Arg-Lys amidation signal. ACTH and corticotropin-like intermediary pituitary peptide were also C-terminally flanked by dibasic amino acid residues. The coding region of leech POMC was obtained by reverse transcription-PCR using degenerated oligonucleotide primers. Circulating levels of ACTH and MSH were 10 and 1 fmol/microl hemolymph, respectively. Morphine, in a dose-dependent manner, increased the levels of both peptides threefold; this effect was blocked by naloxone treatment. Similar results were found with the anandamide. Leech ACTH was processed to MSH by the enzymes neutral endopeptidase (24.11) and angiotensin-converting enzyme. Leech alpha-MSH had the same activity as authentic alpha-MSH in two bioasssay systems. Taken together, the study demonstrates that POMC is present in invertebrates and its immunoregulatory actions have been conserved during evolution.
Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1.
Authors: Castellanos M C , Muñoz C , Montoya M C , Lara-Pezzi E , López-Cabrera M , de Landázuri M O
Published: December 2022

The leukocyte Ag CD69, one of the earliest cell surface activation Ags, is up-regulated at the transcriptional level by proinflammatory stimuli involving the NF-kappaB/Rel family of transcription factors. However, promoter fragments lacking a critical kappaB motif respond to other stimuli such as phorbol esters and triggering Abs against TCR/CD3. Since the 5' promoter flanking region of the CD69 gene contains several putative binding sequences for transcription factor activating protein-1 (AP-1), we explored its role in the inducible expression of CD69. Stimuli that induce AP-1, but not NF-kappaB, such as pyrrolidine dithiocarbamate, augmented the cell surface expression of CD69 as well as its mRNA levels, and the promoter activity of the CD69 gene. This up-regulation is accompanied by an increased binding of jun and fos family members to a consensus AP-1 binding site of the proximal (-16) CD69 promoter region, which seems to be functionally responsive to different activation signals and is trans activated by c-jun expression vectors. Furthermore, cotransfection of a dominant negative version of c-jun, but not IkappaB, abolished the inducible transcriptional activity of the CD69 promoter. In conclusion, the inducible expression of the CD69 gene by mitogenic signals is regulated by the transcription factor AP-1.
A cryptic T cell epitope on the apical membrane antigen 1 of Plasmodium chabaudi adami can prime for an anamnestic antibody response: implications for malaria vaccine design.
Authors: Amante F H , Crewther P E , Anders R F , Good M F
Published: December 2022

We have investigated the proliferative and Th cell responses to the Plasmodium chabaudi adami DS homologue of the Plasmodium falciparum apical membrane Ag 1 (AMA-1), a leading malaria vaccine candidate. Immunodominant T cell epitopes were defined following immunization of BALB/c mice with Escherichia coli-expressed, refolded P. c. adami DS AMA-1 recombinant protein and testing cells from the draining lymph nodes for responses against a series of overlapping peptides spanning P. c. adami AMA-1. A limited number of major T cell sites were identified in both conserved and variable regions of the protein. Several cryptic epitopes that evoked T cell responses following immunization with peptides, but not after protein immunization, were also identified. Adoptive transfer of a T cell line specific for a conserved cryptic epitope (corresponding to residues 31-50) provided help for an anti-AMA-1 protein-specific Ab response following in vivo challenge with P. c. adami parasitized RBC, such that AMA-1-specific Abs appeared more rapidly in recipient mice than in controls. Furthermore, T cells specific for cryptic epitopes afforded partial protection against P. c. adami infection in nude mice. The identification of conserved cryptic Th cell epitopes has important implications for malaria vaccine design.
Aberrant cytokine expression and autocrine regulation characterize macrophages from young MRL+/+ and NZB/W F1 lupus-prone mice.
Authors: Alleva D G , Kaser S B , Beller D I
Published: December 2022

We investigated whether macrophages (Mphi) from young, lupus-prone MRL+/+ and NZB/W F1 mice expressed common defects in immunoregulatory cytokine production. Endotoxin-activated Mphi from both strains, obtained well before disease signs, had a markedly reduced capacity to maintain IL-1 production compared with Mphi from normal strains (BALB/c, A/J, and C57BL/6). Mphi from lupus-prone mice showed similar defects in IL-6 and TNF-alpha production, which preceded the IL-1 defect. In fact, defective TNF-alpha production appeared to be responsible for aberrant expression of the other cytokines because this defect was the first to be expressed, and treatment with exogenous TNF-alpha reduced the extent of defective IL-1 and IL-6. These "proinflammatory" cytokine defects appeared to be selective because the anti-inflammatory cytokine IL-10 was not expressed aberrantly in the lupus-prone strains. For this reason, and because anti-IL-10 mAb treatment did not correct defective proinflammatory cytokine production, IL-10 did not appear to be responsible for these defects. IFN-gamma was able to normalize TNF-alpha production in Mphi from lupus-prone mice, demonstrating a stimulus-specific induction of the proinflammatory defects. These studies also revealed that Mphi from the three normal strains studied here maintain a precise inverse relationship between levels of TNF-alpha and IL-10, a relationship not seen in Mphi from lupus-prone strains. These findings reveal shared elements of cytokine dysregulation in the two principal animal models of multigenic lupus, and suggest that the study of Mphi (and perhaps other cells of the innate immune system) may provide valuable insights into intrinsic functional defects associated with systemic autoimmunity.
CD94/NKG2 is the predominant inhibitory receptor involved in recognition of HLA-G by decidual and peripheral blood NK cells.
Authors: Söderström K , Corliss B , Lanier L L , Phillips J H
Published: December 2022

Prior studies demonstrated that NK cells isolated from adult peripheral blood kill the HLA-A-, HLA-B-, and HLA-C-deficient B lymphoblastoid cell line 721.221, but many are unable to kill 721.221 cells transfected with HLA-G, a molecule expressed preferentially on fetal cytotrophoblasts. To determine the biologic relevance of this recognition, we established NK cell clones from the placenta and demonstrate that these NK cells also were unable to kill 721.221 cells expressing HLA-G. Recognition of HLA-G by NK cells was prevented in the presence of anti-CD94 mAb, implicating CD94/NKG2 as the predominant inhibitory NK cell receptor for HLA-G used by decidual NK cells. In contrast, mAbs against the killer cell inhibitory receptors recognizing HLA-Cw3-related, HLA-Cw4-related, or HLA-Bw4 ligands did not affect NK cell killing of the HLA-G transfectants.
Differential association of phosphatidylinositol 3-kinase with insulin receptor substrate (IRS)-1 and IRS-2 in human thymocytes in response to IL-7.
Authors: Sharfe N , Roifman C M
Published: December 2022

IL-7 is central to T cell development, inducing proliferation and differentiation, but the signal transduction mechanisms by which it accomplishes these functions are poorly understood. We demonstrate that, in addition to activation of the Jak1 and Jak3 kinases, IL-7 stimulation of human thymocytes results in the rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-2 proteins. We show that the Jak1 and Jak3 kinases are associated with both IRS-1 and IRS-2 in thymocytes. However, the pool of Jak3 kinase associated with IRS-1 appears to be preferentially activated upon IL-7 stimulation. We further demonstrate that the 160- to 185-kDa IRS proteins associate with the p85 regulatory subunit of phosphatidylinositol 3 (PI3)-kinase in an IL-7-dependent manner, although significant differences were evident in the levels of PI3-kinase activity associating with each IRS protein. Although IRS-1 displays a higher degree of basal association, IL-7 induces a much greater increase in the activity associated with IRS-2. Lastly, we show that the PI3-kinase activity associated with the IRS proteins appears to be distinct from that bound to the IL-7R, suggesting the existence of separately regulated pools of PI3-kinase activity. This study suggests that IL-7R signaling diverges at the level of the IRS-1 and IRS-2 proteins, possibly with the two branches regulated by Jak3 and Jak1 activities, respectively.
Selective loss of the calcium ion signaling pathway in T cells maturing toward a T helper 2 phenotype.
Authors: Sloan-Lancaster J , Steinberg T H , Allen P M
Published: December 2022

In the CD4+ T cell lineage, two well-defined differentiated populations are the Th1 and Th2 cells, which stem from a common naive T helper precursor (Thp). In this study, we begin to dissect the signaling pathways selectively used by Th1 or Th2 cells as they mature from a common naive precursor in vitro. We show that the maturing Th1 cells mount a vigorous and specific Ca2+ transient upon contact with immunogenic ligand, which is enhanced over that of the naive progenitor cells. As the cells differentiate toward a Th2 phenotype, they quickly lose the ability to engage this pathway, indicating a developmental segregation of intracellular signaling utilization. Moreover, altered peptide ligand stimulation of the Th1 line stimulates a similar Ca2+ transient as native ligand stimulation of the naive precursors, consistent with a quantitative difference in intracellular signaling by these two peptides. These data provide a direct and sequential assessment of a signaling pathway utilization in peripheral T cells as they differentiate to their final functional states.
Involvement of protein tyrosine kinase p72syk and phosphatidylinositol 3-kinase in CD2-mediated granular exocytosis in the natural killer cell line, NK3.3.
Authors: Umehara H , Huang J Y , Kono T , Tabassam F H , Okazaki T , Bloom E T , Domae N
Published: December 2022

The granular exocytosis pathway is one mechanism by which NK cells and CTLs induce cytolysis of target cells. Triggering through adhesion molecules such as CD2 and LFA-1 as well as Fc gammaRIII (CD16) can invoke this pathway. CD2 is a cell surface glycoprotein present on CTLs and NK cells that plays an important role in both cellular adhesion and signal transduction. Here we report that cross-linking of CD2 as well as CD16 by immobilized Abs enhances granular exocytosis in an NK cell line, NK3.3. Herbimycin, a protein tyrosine kinase (PTK) inhibitor, or wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI 3-K), inhibited completely or almost completely CD2- or CD16-mediated granular exocytosis, suggesting the involvement of protein tyrosine kinases and PI 3-K in both CD2- and CD16-mediated granular exocytosis. We also observed that cross-linking of CD2 as well as CD16 enhances p72syk tyrosine kinase activity, and this enhancement correlated well with the increased tyrosine phosphorylation of several cellular proteins, including the adapter protein Shc. Furthermore, we have observed that cross-linking of CD2 as well as CD16 enhances the PI 3-K activity associated with the tyrosine-phosphorylated cellular proteins and Shc. These results provide insight into the signaling pathways by which triggering of CD2 and CD16 on NK cells leads to cytolysis of target cells.
Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells.
Authors: McGuire K L , Iacobelli M
Published: December 2022

CD28 is an important costimulatory molecule in the activation of human T cells. Costimulation of T cells through both the Ag receptor and CD28 leads to high level IL-2 production, which is vital to the development of an immune response in vivo. Previous reports have suggested the CD28 stimulation contributes to the activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including AP-1 and nuclear factor-kappaB (NF-kappaB)/Rel family members as well as an uncharacterized transcription factor called CD28 response complex. While several lines of investigation have suggested that NF-kappaB/Rel family members make up the CD28 response complex transcription factor, other work has not supported this conclusion. Recent studies suggest that the CD28 response element (CD28RE) does not function independently but works instead in conjunction with the adjacent promoter proximal AP-1-binding site and this hypothesis is confirmed here. Also in the current study, binding activity to the CD28RE/AP-1 sequence of the IL-2 promoter is evaluated. Although four specific complexes can be detected binding to this sequence, only one of these complexes is specific for both the CD28RE and the adjacent AP-1 site. Of the NF-kappaB/Rel family members tested, this CD28RE/AP-1-specific complex contains predominantly c-Rel, despite the fact that both p50 and RelA can efficiently bind to the CD28RE. c-Fos and c-Jun are also found in this CD28RE/AP-1-specific complex. These data indicate that functional complexes encompassing both the CD28RE and the AP-1-binding sites influence IL-2 promoter activity in CD28-costimulated T cells.
Differential effects of the T helper cell type 2-derived cytokines IL-4 and IL-5 on ligand binding to IgG and IgA receptors expressed by human eosinophils.
Authors: Bracke M , Dubois G R , Bolt K , Bruijnzeel P L , Vaerman J P , Lammers J W , Koenderman L
Published: December 2022

Increased numbers of eosinophilic granulocytes that exhibit an activated phenotype are found in bronchial tissue and bronchial alveolar lavage fluid of patients with allergic asthma. Little is known about the processes that lead to activation of eosinophils in vivo, but Igs might be important stimulants. In the present study we investigated the capacity of human eosinophils to interact with beads coated with human serum IgG or IgA. Binding of IgG/IgA-coated beads to eosinophils from normal donors appeared to be dependent on priming with Th2-derived cytokines. Priming with granulocyte-macrophage CSF, IL-4, or IL-5 is required for eosinophils to form rosettes with IgA-beads. IL-4 priming resulted in a fast and transient effect on binding of IgA-beads, whereas the effect of IL-5 priming was slower and longer lasting. The expression of Fc alphaR (CD89) was low compared with that on neutrophils, and experiments with the blocking mAb My43 (CD89) showed no inhibition of rosette formation between eosinophils and IgA-coated beads. However, polymeric myeloma IgA effectively inhibited the rosette formation of IgA-coated beads to eosinophils. Binding of IgG-beads could only be primed with granulocyte-macrophage CSF and IL-5, not with IL-4. These data are concurrent with the hypothesis that Th2-derived cytokines spatially produced at the side of an allergic inflammatory response can direct eosinophils to a rather restricted primed phenotype by IL-4 or to a more generalized primed phenotype by IL-5.
Soluble IL-6 receptor leads to a paracrine modulation of the IL-6-induced hepatic acute phase response in double transgenic mice.
Authors: Peters M , Odenthal M , Schirmacher P , Blessing M , Fattori E , Ciliberto G , Meyer zum Buschenfelde K H , Rose-John S
Published: December 2022

There is a growing number of soluble agonistic (IL-6, ciliary neurotropic factor, IL-11, and glia-derived neurotropic factor receptors) and antagonistic (IL-1 and TNF receptors) receptor proteins, modulating the biological functions of their cognate ligands. The physiologic role of these receptor molecules in vivo is unclear. In particular, it is not known how the specificity of function of soluble receptors after release into the blood stream is maintained. We addressed this question by studying the function of the soluble IL-6R (sIL-6R) at the cellular level in the liver. We have generated double transgenic mice coexpressing human sIL-6R and human IL-6 in the liver and have analyzed the expression patterns by in situ hybridization. The expression of the human sIL-6R, driven by the phosphoenolpyruvate carboxykinase promoter, is located mainly in periportal areas, whereas human IL-6 under the control of the metallothionein promoter is uniformly expressed throughout the liver. We show here by in situ hybridization that acute phase protein gene expression induced by human IL-6 and human sIL-6R correlated with the periportal expression of sIL-6R, indicating that sIL-6R acts mainly in an area where it is generated. We conclude that in a concentration-dependent manner, at low concentrations of sIL-6R, there is a predominantly paracrine action at the site of its generation, whereas at higher concentrations of the sIL-6R there are both local and systemic effects.
Expression of an anti-DNA-associated VH gene in immunized and autoimmune mice.
Authors: Ash-Lerner A , Ginsberg-Strauss M , Pewzner-Jung Y , Desai D D , Marion T N , Eilat D
Published: December 2022

The J558 family BW-16 VH gene is closely associated with the autoimmune response to DNA of lupus-prone mice. We have followed the expression of VHBW-16-encoded heavy chains in cDNA libraries prepared from unmanipulated normal (C3H, AKR) and autoimmune (New Zealand Black/New Zealand White F1) mice, and from mice immunized with a highly immunogenic peptide/DNA complex. The prevalence, clonal heterogeneity, and structural properties (somatic mutation, complementarity-determining region 3 composition) of these H chains were investigated, and the DNA affinity of VHBW-16-encoded hybridoma mAb was measured in solution. We find that H chains encoded by VHBW-16 are very rare in Igs of normal mice, but increase significantly in peptide/DNA-immunized mice, and dramatically in diseased mice. The experimentally induced VHBW-16-encoded H chains are clonally restricted, somatically mutated, partly switched from IgM to IgG, and give rise to anti-DNA Abs with low affinity. In contrast, the VHBW-16-encoded H chains from diseased New Zealand Black/New Zealand White mice are clonally heterogeneous, exclusively of the IgG isotype, and produce high affinity anti-DNA autoantibodies. We conclude that the experimentally induced and spontaneous immune responses to DNA are qualitatively similar, but quantitatively different, and may truly reflect the principles of self immunologic tolerance.
Bispecific humanized anti-IL-2 receptor alpha beta antibodies inhibitory for both IL-2- and IL-15-mediated proliferation.
Authors: Pilson R S , Levin W , Desai B , Reik L M , Lin P , Korkmaz-Duffy E , Campbell E , Tso J Y , Kerwin J A , Hakimi J
Published: December 2022

Humanized anti-Tac (HAT) and Mik beta1 (HuMik beta 1) Abs directed at IL-2R alpha and IL-2R beta, respectively, inhibit IL-2 binding and biological activity and together act synergistically in vitro. The Abs have been used successfully in primate models of allograft rejection, graft-vs-host disease, and autoimmunity. We produced bifunctional humanized anti-IL-2R alpha beta Abs (BF-IgG) to combine the specificity of the two Abs into one entity by fusing HAT-producing NSO cells and HuMik beta 1-producing Sp2/0 cells. BF-IgG was purified using protein G-Sepharose affinity chromatography, followed by IL-2R alpha and IL-2R beta affinity chromatography and hydrophobic interaction chromatography. BF-IgG exhibited both anti-IL-2R alpha and anti-IL-2R beta specificities in binding assays. While the Ab binds the IL-2R with intermediate affinity (Kd = 2.82 nM), it does not inhibit IL-15 binding to its high affinity IL-15R. In Kit225/K6 (IL-2R alpha beta gamma+) cells, BF-IgG was 10-fold more potent than a HAT/HuMik beta 1 equimolar mixture in blocking IL-2-induced proliferation and, unexpectedly, was at least 65-fold more active than the mixture in blocking IL-15-induced proliferation. This dual inhibitory activity may be due to cross-linking of the IL-2R alpha and IL-2R beta, thus blocking IL-2 binding and possibly impeding the association of IL-2R beta with IL-15R. BF-IgG has potent immunosuppressant activities against both IL-2- and IL-15-mediated responses, and this antagonist could be more efficacious than HAT and/or HuMik beta 1 for the treatment of autoimmunity and the prevention of allograft rejection.
Distinct mechanisms for the induction and maintenance of allograft tolerance with CTLA4-Fc treatment.
Authors: Tran H M , Nickerson P W , Restifo A C , Ivis-Woodward M A , Patel A , Allen R D , Strom T B , O'Connell P J
Published: December 2022

A murine CTLA4/Fc gamma2a heavy chain (mCTLA4-Fc) chimeric fusion molecule was used in B6AF1 recipients of BALB/c pancreatic islet allografts to study the induction and maintenance of tolerance following inhibition of the CD28-B7 pathway for T cell activation. Donor-specific tolerance was achieved by administering 100 microg of mCTLA4-Fc on alternate days for 14 days (8 total doses) or a single 500 microg dose of mCTLA4-Fc on day 2 after transplant. Tolerance was mediated by long-lived peripheral lymphocytes and showed features of organ and alloantigen specificity. Whereas tolerance could not be established in allograft recipients receiving simultaneous mCTLA4-Fc and rIL-2, previously tolerant animals did not reject their grafts when given IL-2, suggesting that the induction and maintenance phases of tolerance were distinct and separate. The maintenance of donor-specific tolerance was an active immunologic process that was CD4+ T cell dependent and could be adoptively transferred to naive lymphocytes, but could not be explained by apoptosis or deletion of alloreactive T cells. Although an IL-2-sensitive mechanism such as anergy may contribute toward the induction of tolerance, its maintenance involves active suppression.
Cultured NK1.1+ CD4+ T cells produce large amounts of IL-4 and IFN-gamma upon activation by anti-CD3 or CD1.
Authors: Chen H , Paul W E
Published: December 2022

NK1.1+ CD4+ T cells produce more IL-4 and IFN-gamma in response to stimulation with anti-CD3 than conventional CD4+ T cells that have been primed to be, respectively, Th2 or Th1 cells. Furthermore, NK1.1+ CD4+ T cells produce IL-4 even if cultured in the absence of IL-4, whereas conventional CD4+ T cells require IL-4 to develop into IL-4 producers. The IFN-gamma-producing capacity of NK1.1+ CD4+ T cells is enhanced by IL-12. In addition, NK1.1+ CD4+ T cells produce substantial amounts of IL-3, IL-5, and IL-10 upon anti-CD3 stimulation. NK1.1+ CD4+ T cells can be stimulated to produce IL-4 by culture with L cells expressing CD1 (L-CD1), but primary IL-4 production was rather slow and weak. Restimulation of L-CD1-activated NK1.1+ CD4+ T cells with L-CD1 gave rise to a much stronger and more rapid response, yielding IL-4 production comparable with anti-CD3 activation of cells initially primed with anti-CD3. L-CD1 stimulation of L-CD1-primed cells resulted in far less IFN-gamma than that elicited by anti-CD3 from cells that had been primed with anti-CD3, but such production is substantially increased by adding IL-12 to the culture. The differing patterns of cytokine production by NK1.1+ CD4+ T cells suggest that these cells may have complex effects on the priming of conventional T cells and may not simply drive such cells to the acquisition of a Th2 phenotype.
IL-7 activates alpha4beta1 integrin in murine thymocytes.
Authors: Kitazawa H , Muegge K , Badolato R , Wang J M , Fogler W E , Ferris D K , Lee C K , Candéias S , Smith M R , Oppenheim J J , Durum S K
Published: December 2022

IL-7, a cytokine produced by thymic epithelium, was shown to induce adhesion of murine thymocytes to gelatin-coated membranes. A major binding component of gelatin was identified as fibronectin. IL-7-induced adhesion was observed for all of the major thymocyte subsets, including double-negative, double-positive, and single-positive cells, and specific IL-7R were verified on each subset. Fibronectin binding was mediated via alpha4beta1 integrin (VLA-4), which is expressed at high levels on thymocytes. VLA-4 surface expression was not increased following IL-7 treatment, but was shown to undergo rapid tyrosine phosphorylation on the beta1 subunit. This tyrosine phosphorylation was blocked by genistein, which also blocked IL-7-induced adhesion. IL-7 was detected on the extracellular matrix of the thymus, suggesting that it could promote matrix association through an integrin pathway.
The proliferative in vivo activities of lpr double-negative T cells and the primary role of p59fyn in their activation and expansion.
Authors: Balomenos D , Rumold R , Theofilopoulos A N
Published: December 2022

To characterize the functional status of lpr T cells and determine whether activation is required for DN cell expansion, we performed in vivo labeling experiments in MRL-+/+, MRL-lpr, and p59fyn-/- MRL-lpr (Fyn-/-) mice. Multicolor FACS analysis of T cells from 8-wk-old mice receiving bromodeoxyuridine (BrdUrd) for 9 days showed that higher proportions of CD4+ and CD8+ lymph node cells were dividing (BrdUrd(high)) in lpr (15%) than in +/+ mice (3%), and the proportion of cycling cells was even higher in the DN (71%) and CD4+ B220+ (54%) lpr subsets. BrdUrd chase experiments documented that activation and division in most DN cells was initiated subsequent to their precursor CD8+ stage. Lymphadenopathy and other disease manifestations were greatly reduced in Fyn-/- lpr mice concomitant to decreased DN cells (from 77 to 20%). BrdUrd chase experiments showed that the division rate, signified by conversion of DN cells from BrdUrd(high) to BrdUrd(low), was severely reduced in Fyn-/- compared with conventional lpr mice, whereas division of all other T cell subsets (CD4+, CD8+, and CD4+ B220+) was equal in both types of mice. We conclude that 1) DN lpr T cells, rather than being end stage, retain the capacity to be activated and repeatedly divide before reaching an anergic or replicative senescence stage; 2) the CD3 zeta-chain-associated Fyn kinase is important to DN T cell signal transduction; 3) DN cells, as functional intermediates of the CD8+ subset, are highly dependent on Fas participation for apoptosis; and 4) DN cells contribute to the early development of the serologic and histologic features of the MRL-lpr lupus disease.
Convergence of Schiff base costimulatory signaling and TCR signaling at the level of mitogen-activated protein kinase ERK2.
Authors: Chen H , Hall S , Heffernan B , Thompson N T , Rogers M V , Rhodes J
Published: December 2022

Schiff base formation on specialized T cell surface amines provides a costimulatory signal to T cells through a mechanism that activates Na+ and K+ transport, substantially enhancing TCR-dependent IL-2 production. Schiff base-forming molecules that mimic the natural carbonyl donor potently enhance immune responses and provide the first mechanism-based, orally active immunopotentiatory agents. In the present study, costimulation by the Schiff base-forming molecule tucaresol was investigated at the level of mitogen-activated protein kinase (MAPK) in T cell lines. Both TCR-directed stimulation by anti-CD3 and Schiff base stimulation by tucaresol produced a distinct mobility shift in MAPK, characterized by direct immunoblotting of cell lysate proteins subjected to SDS-PAGE, that corresponded with increased phosphorylation. Combined TCR-CD3 and tucaresol stimulation substantially enhanced and prolonged the MAPK response, providing a biochemical basis for the costimulatory nature of the pathway utilized by Schiff base signaling. The MAPK affected was identified by immunoprecipitation as ERK2. Both the direct effects and the TCR signal-enhancing effects of tucaresol on MAPK activation were also demonstrated in a functional MAPK assay measuring substrate phosphorylation. Borohydride reduction of tucaresol's Schiff base-forming carbonyl group abolished both enhancement of MAPK phosphorylation and IL-2 production, as did a selective inhibitor of the MAPKK, MEK1. Tucaresol had no effect on TCR-mediated rises in intracellular free Ca2+ or inositol 1,4,5-triphosphate generation, while tucaresol signaling occurred normally in the lck-deficient J.CaM1.6 T cell line, consistent with convergence of tucaresol- and TCR-induced signals downstream of early TCR-mediated events.
Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo.
Authors: O'Reilly L A , Harris A W , Tarlinton D M , Corcoran L M , Strasser A
Published: December 2022

B lymphocyte differentiation proceeds through a series of alternating stages of proliferative expansion interspersed with noncycling stationary phases during which cells undergo either positive selection or apoptotic cell death. The molecular control of cell cycle progression and that of apoptosis appear to be interconnected. Overexpression of Bcl-2 in lymphocytes or fibroblasts antagonizes apoptosis and delays their transition from the quiescent state into the cell cycle. We have undertaken a systematic analysis of the impact of bcl-2 transgene expression on cell cycle distribution and turnover rate of developing B lymphocytes in normal mice and in mutant animals in which B cell differentiation is arrested at the pro-B/pre-BI or the pre-BII stage. These experiments revealed that overexpression of Bcl-2 reduces proliferation and slows turnover of B cells at all stages of development. This demonstrates that Bcl-2 can retard transition of B cells between the quiescent and the cycling state regardless of the mitogenic stimulus and the differentiation stage. The implications of these results for the normal control of B lymphopoiesis and for lymphomagenesis are discussed.
Chimeric peptides: a new approach to enhancing the immunogenicity of peptides with low MHC class I affinity: application in antiviral vaccination.
Authors: Tourdot S , Oukka M , Manuguerra J C , Magafa V , Vergnon I , Riché N , Bruley-Rosset M , Cordopatis P , Kosmatopoulos K
Published: December 2022

Recruitment of the CTL repertoire specific for subdominant epitopes that have a low MHC class I-binding affinity could be the way to achieve an efficient protective immunity against spontaneous tumors and viruses with high mutation rate. However, we have reported recently that subdominant peptides of influenza A Puerto Rico/8/34 (flu PR8) nucleoprotein (NP) with low Db affinity are only partially able to protect mice against lethal influenza infection. This seems to be due to their inability to recruit the specific CTL repertoire, and suggests that subdominant peptides could be used for vaccination only if they become highly immunogenic. In this work, we describe an approach that allows enhancement of the immunogenicity of every low affinity peptide presented by the Db molecule. It consists in producing chimeric peptides composed by amino acids from a high Db affinity peptide (NP366) in positions that interact with the MHC, and amino acids from low Db affinity nonimmunogenic influenza NP-derived peptides (NP17, NP97, NP330, and NP469) in positions that are exposed to the TCR. All chimeric peptides tested exhibited a high Db affinity and efficiently recruited the CTL repertoire specific for the corresponding low Db affinity peptide. Furthermore, vaccination with chimeric peptides that corresponded to subdominant NP17 and NP97 peptides induced a very potent anti-flu PR8 protective immunity.
Adsorption to aluminum hydroxide promotes the activity of IL-12 as an adjuvant for antibody as well as type 1 cytokine responses to HIV-1 gp120.
Authors: Jankovic D , Caspar P , Zweig M , Garcia-Moll M , Showalter S D , Vogel F R , Sher A
Published: December 2022

A series of protocols were tested to examine the adjuvant effects of IL-12 on humoral and type 1 cytokine responses elicited in mice by recombinant gp120 envelope protein from HIV-1. This Ag fails to induce detectable Ab responses when administered s.c. alone, but stimulates low Ab levels when combined with aluminum hydroxide (alum). Moreover, when i.p. injected rIL-12 was included in the immunization, no increase in Ab production was observed. Importantly, optimal gp120 Ab responses were achieved by immunizing mice s.c. with gp120 and rIL-12 simultaneously coadsorbed to alum. These animals displayed a highly polarized, type 1 cytokine profile, with the emergence of anti-gp120 Ig belonging to the IgG2 and IgG3 isotypes. In addition, a major increase occurred in Ab of the IgG1 subclass. The superior adjuvant activity of alum-adsorbed IL-12 compared with that of the free cytokine correlated with the prolonged detection of IFN-gamma in the sera of animals immunized using the former procedure. In related experiments, in vitro neutralization of IL-12 was shown to inhibit IFN-gamma production by spleen cells from mice immunized with gp120 plus alum, but not by splenocytes from mice primed in the presence of IL-12, suggesting that the latter protocol induces a stable type 1 phenotype. These studies demonstrate that presentation of IL-12 on alum enhances its immunomodulatory effects and establish a protocol for the use of the cytokine as an adjuvant for simultaneously promoting both humoral Ab and type 1 cytokine responses.
Induction of IL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages.
Authors: Hodge-Dufour J , Noble P W , Horton M R , Bao C , Wysoka M , Burdick M D , Strieter R M , Trinchieri G , Puré E
Published: December 2022

Components of the extracellular matrix (ECM) can regulate leukocyte activation and function at inflammatory sites. Low molecular weight fragments of the ECM glycosaminoglycan hyaluronan (LMW-HA) that accumulate in inflammation, but not the ubiquitous high molecular weight form of HA (HMW-HA), have been shown to induce cytokine and/or chemokine production by alveolar and bone-marrow derived macrophages. To determine the cellular requirements for responsiveness to HA, we compared the effects of HMW-HA and LMW-HA on resident and thioglycollate-elicited murine peritoneal macrophages. We demonstrate that treatment of elicited macrophages with LMW-HA, but not with HMW-HA, stimulated production of the chemokines RANTES and macrophage inflammatory protein-1alpha and -1beta. Further, we demonstrate that LMW-HA induced the production of biologically active IL-12, a proinflammatory cytokine not previously known to be regulated by cell-matrix interactions. The LMW-HA-induced production of IL-12 by elicited macrophages was inhibited by an anti-CD44 mAb that blocks HA binding. In contrast to elicited macrophages, freshly explanted resident peritoneal macrophages did not respond to LMW-HA. However, preculture in vitro before stimulation led to adhesion-dependent priming for LMW-HA-induced cytokine and chemokine production by resident macrophages. These results provide further evidence of the potential importance of CD44/LMW-HA interactions in regulating the immune response at sites of inflammation and demonstrate that the state of differentiation of macrophages may determine their sensitivities to matrix components.