bluuebunny/crossref_metadata_2025 huggingface dataset:
The state of maturation of monocytes into macrophages determines the effects of IL-4 and IL-13 on HIV replication.
Authors: Naif H M , Li S , Ho-Shon M , Mathijs J M , Williamson P , Cunningham A L
Published: December 2022

The molecular mechanisms of the effects of IL-4 and IL-13 on HIV infection in human monocytes as they matured into monocyte-derived macrophages over 7 days were investigated using HIV-1(BaL), and low passage clinical strains. IL-4 and IL-13 up-regulated the expression of both genomic and spliced HIV mRNA in monocytes cultured on Teflon, as determined by Northern analysis and p24 Ag assay. Using a nuclear run-on assay, IL-4 stimulation was shown to enhance transcription by two- to threefold. IL-4 stimulated nuclear factor-kappaB nuclear translocation and binding before enhancement of HIV RNA expression. Conversely, IL-4 and IL-13 markedly and significantly inhibited HIV replication at the transcriptional level in monocyte-derived macrophages, and this occurred whether these cytokines were added before or after HIV infection. The reversal from stimulation to inhibition occurred after 3 to 5 days of adherence to plastic. IL-4 had no significant effect on HIV reverse transcription. The effect of both cytokines on the monocyte maturation/differentiation (CD11b, CD13, and CD26) and other macrophage markers (CD14 and CD68) was examined. IL-4 enhanced CD11b, but inhibited CD26 expression and delayed CD13 loss. IL-13 had similar effects on CD11b and CD13, but no effect on CD26. Hence, these cytokines do not simply enhance monocyte differentiation, but have complex and slightly divergent effects that impact on HIV replication probably through cell signaling pathways and nuclear factor-kappaB translocation.
CD28-costimulation activates cyclic AMP-responsive element-binding protein in T lymphocytes.
Authors: Hsueh Y P , Liang H E , Ng S Y , Lai M Z
Published: December 2022

Cyclic AMP-responsive element binding protein (CREB) mediates gene expression in response to cAMP stimulation. The transcriptional activity of CREB depends on both the phosphorylation of Ser133 and the recruitment of cofactor for assembly of transcriptional complex. Extensive Ser133 phosphorylation of CREB was induced during T cell activation. This phosphorylation event is essential for IL-2 gene expression. However, phosphorylation of CREB at Ser133 was not sufficient for transcriptional activity by CREB. The presence of a second signal from CD28, a potent costimulatory molecule on T cells, stimulated CREB-mediated gene expression. CD28, an effective costimulator of T cell activation and IL-2 gene expression, is shown to induce CREB activation in the presence of anti-CD3 or O-tetradecanoylphorbol 13-acetate. These two signals together stimulated a CRE-dependent reporter gene, the proliferating cell nuclear Ag promoter, and transactivation by the GAL4-CREB fusion protein. Thus optimal induction of CREB, similar to the full activation of T lymphocytes, may be mediated by two distinct signal transductions. Using the specific kinase inhibitor, one of the two pathways appeared to involve mitogen-activated protein kinase kinase but not protein kinase C, protein kinase A, or p70 S6 kinase.
Neutrophils as a source of putative restriction proteases: degradation of mammalian and yeast proteins monitored by two-dimensional gel electrophoresis.
Authors: Lefkovits I , Frey J R , Kuhn L , Sadovnikov V B , Pleshakova O V , Sukharev S A
Published: December 2022

We have compared the ability of intact neutrophils to degrade a complex substrate of proteins from mammalian and yeast origin. The substrate was obtained by biosynthetic labeling, and subsequent lysis of K562 cells (leukemic cell line) and of yeast culture. The mammalian substrate consisted of 619 and the yeast substrate of 185 different polypeptides, as visualized and represented on two-dimensional gel patterns. Upon incubation of the mammalian substrate with neutrophils, the bulk of spots disappeared so rapidly that after 240 min of incubation only 21 spots were detectable. Just one spot remained unaltered in its intensity throughout the whole period of incubation. About 440 spots reveal a t1/2 shorter than 8 min. Yeast substrate is represented by a smaller number of the starting polypeptides (185) from which 55 spots "survive" the neutrophil treatment. About 30 spots have a t1/2 shorter than 8 min. We conclude that neutrophils are equipped with a potent proteolytic apparatus, and this is capable of eliminating various proteins in a highly efficient manner. The system is much less effective in eliminating proteins from distant species, like yeast. Although the cells governing and regulating the immune system are clearly of lymphoid origin, it might well be that the preimmune task of eliminating self antigens in a manner as predicted in the restriction protease hypothesis is performed by neutrophils.
Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor.
Authors: Apasov S G , Koshiba M , Chused T M , Sitkovsky M V
Published: December 2022

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.
N-acetylcysteine and alpha-tocopherol reverse the inflammatory response in activated rat Kupffer cells.
Authors: Fox E S , Brower J S , Bellezzo J M , Leingang K A
Published: December 2022

Activation of the resident macrophage populations of the reticuloendothelial system is a key component of the complex pathophysiology of sepsis. Macrophage activation leads to production and secretion of inflammatory mediators such as cytokines, vasoactive substances, free radicals, and chemokines, which have been associated with high morbidity and mortality in the septic patient. The goal of the present study was to determine whether antioxidants could suppress Kupffer cell activation at points beyond the initiation of activation. Kupffer cells were studied since they are central to the clearance of bacteria and endotoxins, and have been associated with hepatocellular dysfunction in sepsis. Cells were activated with 10 ng/ml LPS for various times whereupon N-acetylcysteine (30 mM) and alpha-tocopherol (50 microM) were added. Steady state levels of cytokine mRNA, activation of nuclear factor-kappaB, and TNF-alpha secretion were determined when expression was maximal in control cells. The results of this study show that antioxidants can be used to suppress Kupffer cell activation at points beyond the initiation of activation. Furthermore, we show that N-acetylcysteine-mediated inhibition of activation requires secondary protein synthesis, but does not modulate IkappaB-alpha mRNA expression. The inhibitory effect of these drugs occurs at the very earliest steps of the LPS signal transduction cascade as it is currently understood. The results of the present study suggest that the inflammatory response to sepsis may be controlled through appropriate antioxidant therapy.
Up-regulation of cytokine mRNA, adhesion molecule proteins, and MHC class II proteins in salivary glands of TGF-beta1 knockout mice: MHC class II is a factor in the pathogenesis of TGF-beta1 knockout mice.
Authors: Nakabayashi T , Letterio J J , Geiser A G , Kong L , Ogawa N , Zhao W , Koike T , Fernandes G , Dang H , Talal N
Published: December 2022

Mice homozygous for a disrupted TGF-beta1 allele develop multiple lymphoproliferative disorders similar to those seen in the pseudolymphoma of Sjögren's syndrome. At 2 wk of age, these TGF-beta1 mutant mice begin to develop wasting syndrome and die at around 4 to 5 wk of age. We studied salivary glands from symptomatic mutant mice >14 days of age. Reverse transcriptase-PCR analysis showed up-regulation of proinflammatory cytokine genes such as IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, and IFN-gamma in these mutant mice. Enhanced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and MHC class II as well as CD4-positive T lymphocyte infiltration was detected by immunostaining. To elucidate the role of MHC class II, salivary glands from TGF-beta1/MHC class II double knockout mice were used to investigate the expression of adhesion molecules and MHC class II. In spite of the existence of basal intercellular adhesion molecule-1 expression on vessels, there was neither MHC class II expression, enhanced vascular cell adhesion molecule-1 expression, nor lymphocytic infiltration in the salivary glands. These results suggest that MHC class II plays a significant role in the pathogenesis of TGF-beta1 mutant mice. Although the mechanism that initiates multiple inflammatory diseases in these mice remains unclear, the context reported here would provide insight into the immunopathology of Sjögren's syndrome.
Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59.
Authors: Powell M B , Marchbank K J , Rushmere N K , van den Berg C W , Morgan B P
Published: December 2022

We have previously described the isolation and cloning of the rat analogue of the human complement inhibitor CD59 (hCD59). Using the rat CD59 (rCD59) coding region as probe, we have isolated positive cDNAs from a mouse kidney cDNA library. Sequence analysis of these clones indicated that they contained an open reading frame encoding a 124 amino acid protein. Comparisons with the known sequences of hCD59 and rCD59 suggested that the clones contained a full-length cDNA encoding the mouse analogue of CD59 (mCD59). The cDNA encoded a 81-bp 5'-flanking region, a 23 amino acid NH2-signal peptide, a 101 amino acid coding region including putative N-glycosylation sites and a glycosyl phosphatidylinositol (GPI) anchoring signal, and approximately 0.8 kb 3'-untranslated flanking region. Reverse transcriptase PCR revealed the presence of mCD59 mRNA in all mouse tissues examined. The gene for mCD59 was mapped by fluorescence in situ hybridization to the E2-E4 region of mouse chromosome 2, a region that includes areas syntenous with the location of the human CD59 gene on chromosome 11p13. Expression of mCD59 in a CD59-negative human cell line conferred protection against lysis by complement from rodent, human, and several other species, confirming that mCD59 was the functional analogue of hCD59 and that function was not species restricted. The expressed protein was glycosyl phosphatidylinositol anchored as demonstrated by its partial removal from U937 cells on treatment with phosphatidylinositol-specific phospholipase C. Abs raised against the expressed protein demonstrated the presence of mCD59 on all mouse blood cell types and on several mouse cell lines and neutralized function of mCD59 on mouse E and expressed on U937. Western blot analysis revealed that both expressed and endogenous mCD59 had a molecular mass of 22 to 24 kDa.
Response to local inflammation of IL-1 beta-converting enzyme- deficient mice.
Authors: Fantuzzi G , Ku G , Harding M W , Livingston D J , Sipe J D , Kuida K , Flavell R A , Dinarello C A
Published: December 2022

IL-1 beta-converting enzyme (ICE) cleaves pro-IL-1 beta to the mature, released form. Although other proteases can process pro-IL-1 beta, ICE-deficient (ICE -/-) mice do not release mature IL-1 beta in response to endotoxin. The purpose of our study was to investigate the response of ICE -/- mice in two models of local inflammation, turpentine-induced tissue damage and zymosan-induced peritonitis. No differences were observed in the development of the systemic acute phase response after turpentine administration between wild-type and ICE -/- mice, but this response was completely impaired in IL-1 beta -/- mice. Accordingly, the levels of mature IL-1 beta produced in response to turpentine did not differ between wild-type and ICE -/- mice. In contrast, following zymosan-induced peritonitis, the levels of mature IL-1 beta were significantly lower in ICE -/- mice. This was associated with a 50% decrease in cellular infiltrate in ICE -/- mice compared with that in wild-type controls. The reduced production of zymosan-induced mature IL-1 beta in ICE -/- mice was also observed from cultured peritoneal or spleen cells. Our results demonstrate that in turpentine-induced tissue necrosis, precursor IL-1 beta is processed by non-ICE proteases, but in complement-mediated inflammation, ICE participates in the processing of the IL-1 beta precursor.
Regulation of IFN-alpha/beta, MxA, 2',5'-oligoadenylate synthetase, and HLA gene expression in influenza A-infected human lung epithelial cells.
Authors: Ronni T , Matikainen S , Sareneva T , Melén K , Pirhonen J , Keskinen P , Julkunen I
Published: December 2022

The epithelial cells of the respiratory tract are the primary sites of virus replication in influenza A virus infections. We infected human alveolar epithelium-like A549 cells and fibroblast-like human fetal lung (HFL1) cells with a pathogenic influenza A virus (A/Beijing/353/89), and studied the kinetics of infection and the expression of host IFN-alpha/beta, MxA, OAS (2',5'-oligoadenylate synthetase), and HLA class I and II genes. Viral mRNA and protein synthesis was clearly seen in virus-infected lung cells. A549 and HFL1 cells produced only small amounts of IFN-alpha/beta, whereas virus-infected macrophages produced type I IFN very efficiently. The kinetics of IFN-beta gene expression in A549 cells was rapid, as shown by reverse-transcriptase PCR, and IFN-beta mRNA expression levels correlated well to the kinetics of nuclear factor-kappa B transcription factor activation. In influenza A virus-infected A549 and HFL1 cells, MxA gene induction was mediated by IFN-alpha/beta released into the cell culture supernatant, and was prevented by anti-type I IFN Abs. HLA class I Ag expression, which could be activated by IFN in noninfected A549 and HFL1 cells, was not induced in these cells by virus infection. The results suggest that type I IFN are essential for the activation of the antiviral response in lung epithelial cells.
A novel role for B cells in early protective immunity to an intracellular pathogen, Francisella tularensis strain LVS.
Authors: Culkin S J , Rhinehart-Jones T , Elkins K L
Published: December 2022

Normal BALB/cByJ mice given a sublethal infection of Francisella tularensis strain LVS survived 10(6) LD50s of lethal challenge given only 3 days later. Here, we determine the cell types responsible for this very strong early protective immunity. Early protection is observed in athymic nu/nu mice but not fully immunodeficient scid mice, implicating a lymphocyte in this response. Using scid mice that are reconstituted with various purified cell subpopulations, as well as mice with genetically targeted disruptions in lymphocyte subpopulations (knockout mice), we demonstrate that strong early protection is highly dependent on B cells. This protective mechanism, which limits bacterial growth in the organs of the reticuloendothelial system very quickly after infection, requires IFN-gamma but is unlikely to involve specific Ab.
Calpain, an upstream regulator of thymocyte apoptosis.
Authors: Squier M K , Cohen J J
Published: December 2022

Apoptosis is the common phenotype of programmed or physiologic cell death, the process used to remove excess or defunct cells during normal tissue maintenance. One of the most studied cell types with respect to apoptosis is the immature T cell from the thymus, which activates its death program in response to an enormous variety of agents. Previously, our group implicated the calcium-dependent cytosolic protease calpain as a participant in thymocyte apoptosis initiated by glucocorticoids or irradiation. We found that the calpain inhibitors N-acetyl-leu-leu-norleucinal (calpain inhibitor I) and carbenzoxy-val-phe-H (MDL 28,170) prevented dexamethasone-induced apoptosis of thymocytes; in this study, we show that two additional calpain active site inhibitors, L-3-carboxy-trans-2,3-epoxypropionyl-leu-amido-(4-guanidinio )butane ethyl ester (E64d) and carbenzoxy-leu-leu-tyr-CHN2 (ZLLY-CHN2), also prevent apoptosis in this model. Three compounds that inhibit lysosomal cysteine proteases, carbenzoxy-tyr-ala-CHN2 (ZYA-CHN2), ammonium chloride, and chloroquine, do not block apoptosis, indicating that the effect of the calpain inhibitors is not due to cross-inhibition of lysosomal proteases. In addition, I-benzyl-CH=C(SH)COOH (PD150606), a calpain inhibitor directed toward the calcium binding sites of calpain, also prevents apoptosis. Calpain is necessary for other models of programmed cell death that require new gene expression (induction models), those following treatment of thymocytes with the calcium ionophore A23187, ionomycin, or forskolin. However, two models of thymocyte apoptosis that do not require new gene expression (transduction models), those triggered by heat shock and by valinomycin, are calpain independent, as is calcium-triggered DNA fragmentation in isolated thymocyte nuclei. These experiments suggest an upstream regulatory role for calpain in a pathway to thymocyte apoptosis common to several inducers.
Mast cell Fc epsilonRI expression in the rat intestinal mucosa and tongue is enhanced during Nippostrongylus brasiliensis infection and can be up-regulated by in vivo administration of IgE.
Authors: Shaikh N , Rivera J , Hewlett B R , Stead R H , Zhu F G , Marshall J S
Published: December 2022

The activation of rodent and human mast cells can occur through the cross-linking of tetrameric IgE receptors each containing single alpha- and beta- and two gamma-subunits. However, the factors that regulate the in vivo expression of Fc epsilonRI are poorly understood. We have examined the expression of the Fc epsilonRI beta-subunit in the Nippostrongylus brasiliensis (Nb)-induced mode l of rat intestinal inflammation. We developed a double-staining technique for mast cell granules (Alcian blue) and the beta-subunit of Fc epsilonRI. The intensity of immunohistochemical staining per mast cell was quantified using an image analysis system. Jejunal and tongue mast cells of Lewis rats were visible by Alcian blue staining before Nb infection, but they expressed very low levels of beta-subunit as assessed by immunohistochemical staining. These levels were increased by day 11 postinfection and reached a maximum at day 14. Since serum IgE levels correlated well with the degree of beta-subunit expression, we investigated whether the observed enhancement of receptor expression might occur through the stabilization of receptor complexes by IgE. Therefore, Lewis rats were treated with myeloma IgE, and beta-subunit expression was examined. In both tongue and jejunal tissue, a significant rise in beta-subunit expression was observed in response to IgE injection, although levels of beta-subunit expression were not as high as those observed in Nb-infected animals. The increase in beta-subunit expression was accompanied by an increase in the amount of mast cell-associated IgE. These observations may have important implications for the regulation of IgE receptor expression during disease.
Cytokine-in-adjuvant steering of the immune response phenotype to HIV-1 vaccine constructs: granulocyte-macrophage colony-stimulating factor and TNF-alpha synergize with IL-12 to enhance induction of cytotoxic T lymphocytes.
Authors: Ahlers J D , Dunlop N , Alling D W , Nara P L , Berzofsky J A
Published: December 2022

To enhance and steer immune responses to synthetic peptide vaccines toward selected functional types and to understand the cooperative action of cytokines in fine-tuning the immune response, we attempted to influence the in vivo cytokine environment by delivering cytokines directly to the microenvironment in which the immune response is initiated. Here we study the effects of IL-2, IL-4, IL-7, IL-1beta, IL-12, IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) incorporated with peptide in adjuvant on a variety of responses elicited: CTL, T cell proliferation, cytokine production and message, and Ab isotype. We show GM-CSF to be the single most effective cytokine for enhancing both cellular and humoral immunity to two previously characterized HIV-1 MN vaccine constructs. Novel synergies were also detected. GM-CSF synergized with IL-12 for CTL induction in BALB/c mice concomitant with suppression of Th2 cytokines IL-4 and IL-10. TNF-alpha also synergized with IL-12, but by a different mechanism, inducing IFN-gamma production in BALB/c mice and thus shifting the response to a Th1 phenotype. The results presented here suggest that in addition to IL-2, optimum induction of CD8+ CTL in vivo requires a combination of cytokines, including GM-CSF (probably acting to enhance Ag presentation and CD4+ cell help) and IL-12 (steering the Th response toward Th1 cytokines).
Presence of B cell progenitors in the thymus.
Authors: Mori S , Inaba M , Sugihara A , Taketani S , Doi H , Fukuba Y , Yamamoto Y , Adachi Y , Inaba K , Fukuhara S , Ikehara S
Published: December 2022

We characterize B cell progenitors in the thymus. Although mature thymic B cells are surface Ig+, B220(high), but CD43-, B cell progenitors in the thymus were found to be slg-, B220(med), and CD43+. Most progenitors showed rearranged Ig D-J and V-D-J patterns when DNA from sorted B cell progenitors was amplified by PCR, blotted, and probed. When B cell progenitors were purified from the thymus and injected intrathymically into Ig heavy chain (Igh) allotype-disparate recipients, B cells bearing donor-type Igh6 were detected in the thymus but not in the periphery. Thymic B cells generated from these progenitors were CD5+ cells, although there was a broad range of expression of CD5 molecules. Furthermore, B cells purified from thymus that had been inoculated with B cell progenitors were able to differentiate into Ab-forming cells under the influence of CD40 ligand plus IL-10, and secreted IgM with donor Igh6 allotype. We thus clarified the existence of B cell progenitors in the thymus and also characterized the surface markers and their developmental functions.
Kinase-independent potentiation of B cell antigen receptor-mediated signal transduction by the protein tyrosine kinase Src.
Authors: Lin J , Tao J , Dyer R B , Herzog N K , Justement L B
Published: December 2022

Signal transduction mediated by the B cell Ag receptor involves the activation of multiple protein tyrosine kinases that are members of the Src family (i.e., Fyn, Lyn, Blk, Lck). To determine whether members of the Src family possess common physical and/or enzymatic properties that enable them to potentiate signal transduction via the B cell Ag receptor, we expressed the protein tyrosine kinase Src in the B lymphoma cell line K46-17 mu m lambda. Based on coprecipitation analysis and two-color immunofluorescence, this heterologous Src family kinase was observed to physically associate with the B cell Ag receptor. Additional experiments demonstrated that B cell Ag receptor cross-linking results in increased tyrosine phosphorylation and activation of Src. Several parameters of B cell activation, including tyrosine phosphorylation of intracellular substrates, calcium mobilization, and transcription factor activation, were potentiated in cells that expressed Src when compared with control cells. To determine whether potentiation of Ag receptor-mediated signaling by Src was dependent on its catalytic activity, a kinase-deficient form of Src was expressed in K46-17 mu m lambda cells. Transfectants expressing kinase-deficient Src exhibited an enhanced responsiveness to stimulation through the B cell Ag receptor that was comparable with transfectants expressing wild-type Src. Additionally, kinase-deficient Src was observed to associate with the endogenous kinase Lyn in an activation-dependent manner. These findings indicate that members of the Src family may potentiate Ag receptor-mediated signaling via a kinase-independent mechanism(s) that involves amplification of kinase recruitment to the Ag receptor activation complex.
A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules.
Authors: Borges L , Hsu M L , Fanger N , Kubin M , Cosman D
Published: December 2022

Leukocyte Ig-like receptors (LIRs) are a newly discovered family of immunoreceptors expressed on monocytes and B cells and at lower levels on dendritic cells and NK cells. The amino acid sequences in the extracellular regions of eight of these receptors show between 63 and 84% identity to the prototypic LIR-1 sequence. LIRs contain either two or four Ig domains and fall into three classes: those with cytoplasmic domains containing two, three, or four immunoreceptor tyrosine-based inhibitory motif-like motifs; those with a short cytoplasmic domain and no immunoreceptor tyrosine-based inhibitory motif-like motifs; and those with no transmembrane domain represented by a single LIR molecule that is presumably secreted. The LIRs are structurally related to the human Fc(alpha)R and the killer inhibitory receptors and map to the same region of chromosome 19 as these genes. Like killer inhibitory receptors, at least two LIRs bind to MHC class I Ags, but their different cellular distribution suggests a distinct role in immune system modulation.
A functional B cell receptor transgene allows efficient IL-7-independent maturation of B cell precursors.
Authors: Melamed D , Kench J A , Grabstein K , Rolink A , Nemazee D
Published: December 2022

IL-7 supports the proliferation of B cell precursors, but inhibits their maturation to mature surface IgM+ (sIgM+) B cells. This inhibition is thought to occur by direct or indirect down-regulation of recombinase genes, preventing the B cells from undergoing Ig light chain rearrangements. To directly analyze the IL-7 inhibitory effects, we studied B cell development and maturation in B cells bearing a transgenic (Tg) B cell receptor (BCR). We show here that proliferation of Tg B cell precursors is IL-7 dependent both in vivo and in vitro and is comparable to that of non-Tg B cell precursors. Tg B cell precursors grown on stroma and IL-7 expressed sIgM on >90% of the cells, and a large proportion of these cells coexpressed additional maturation markers such as IgD, CD23, CD21, and L-selectin, indicating that IL-7 does not inhibit maturation of Tg B cell precursors. The presence of the Tg inhibited V(D)J recombination in the cultured cells, as very low levels of recombination activating genes 2 (RAG-2) expression and endogenous V-Jkappa DNA rearrangements were found. Expression levels of RAG mRNAs were not significantly changed after removal of IL-7 from the in vitro Tg B cell cultures. In contrast, we found that IL-7 inhibited maturation of non-Tg B cell precursors and that removal of IL-7 resulted in a significant increase in RAG-2 expression and kappa rearrangements, thus allowing the B cells to express sIgM and to mature. These results suggest that IL-7-mediated inhibition of Ig gene rearrangement blocks maturation of B cell precursors and that the presence of Tg BCR efficiently circumvents this inhibition.
The effect of the proteasome inhibitor lactacystin on the presentation of transporter associated with antigen processing (TAP)-dependent and TAP-independent peptide epitopes by class I molecules.
Authors: Bai A , Forman J
Published: December 2022

Cells were treated with two proteolytic inhibitors, N-acetyl-leucyl-leucyl-norleucinal and lactacystin, the latter reported to be a specific inhibitor for the proteasome. Both inhibitors retarded the maturation of endo-H-resistant forms of murine and human class I molecules from their endo-H-sensitive precursors in cell lines with functional TAP proteins. HLA-A2 maturation readily occurs in TAP-deficient T2 cells, and it has been shown that the peptides associated with A2 are derived from the leader segment of proteins in the secretory pathway. This maturation is inhibited by N-acetyl-leucyl-leucyl-norleucinal but not lactacystin, indicating that the proteasome is not required for the generation of HLA-A2 binding peptides in these cells. The murine class Ib molecule Qa-1b presents a leader peptide derived from D-end class I molecules to alloreactive CTL. Since this presentation is dependent on the expression of TAP proteins, we determined if this requirement reflects a need for the proteasome to process this peptide. We found that lactacystin did not inhibit the maturation of endo-H-resistant forms of Qa-1b that are dependent on this leader peptide for its maturation, nor did it inhibit the expression of this peptide-Qa-1b complex in a functional assay. Thus, unlike conventional cytosolic peptides, leader peptides (regardless of whether they are dependent on TAP for their presentation) do not require the proteasome for processing.
IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade.
Authors: Ogata A , Chauhan D , Teoh G , Treon S P , Urashima M , Schlossman R L , Anderson K C
Published: December 2022

IL-6 mediates growth of some human multiple myeloma (MM) cells and IL-6-dependent cell lines. Although three IL-6 signaling pathways (STAT1, STAT3, and Ras-dependent MAPK cascade) have been reported, cascades mediating IL-6-triggered growth of MM cells and cell lines are not defined. In this study, we therefore characterized IL-6 signaling cascades in MM cell lines, MM patient cells, and IL-6-dependent B9 cells to determine which pathway mediates IL-6-dependent growth. IL-6 induced phosphorylation of JAK kinases and gp130, regardless of the proliferative response of MM cells to this growth factor. Accordingly, we next examined downstream IL-6 signaling via the STAT3, STAT1, and Ras-dependent mitogen-activated protein kinase (MAPK) cascades. IL-6 triggered phosphorylation of STAT1 and/or STAT3 in MM cells independent of their proliferative response to IL-6. In contrast, IL-6 induced phosphorylation of Shc and its association with Sos1, as well as phosphorylation of MAPK, only in MM cells and B9 cells that proliferated in response to IL-6. Moreover, MAPK antisense, but not sense, oligonucleotide inhibited IL-6-induced proliferation of these cells. These data suggest that STAT1 and/or STAT3 activation may occur independently of the proliferative response to IL-6, and that activation of the MAPK cascade is an important distal pathway for IL-6-mediated growth.
Bridging of neutrophils to target cells by opsonized zymosan enhances the cytotoxicity of neutrophil-produced H2O2.
Authors: Jiang X , Wu T H , Rubin R L
Published: December 2022

Hydrogen peroxide (H2O2) is a well-established cytotoxic agent released by activated neutrophils into the extracellular environment. However, a maximum of only 5 microM H2O2 was detected in the medium when 10(6) neutrophils/ml were activated with opsonized zymosan (OZ), more than 50-fold lower than the concentration of exogenous H2O2 required to produce equivalent killing of a cell line. In addition PMA-activated neutrophils were noncytotoxic, despite the capacity of PMA to generate two- to fourfold as much H2O2 for five times longer. The basis for this discrepancy was explored. NaN3 increased cytotoxicity to >90% only when neutrophils were activated with OZ due in part to inhibition of myeloperoxidase-mediated hydrolysis of H2O2, while catalase completely prevented cytotoxicity of OZ-activated neutrophils. These results indicate that H2O2 was solely responsible for the observed cytotoxicity. OZ-mediated cytotoxicity was prevented by intermittent agitation of the cultures or by the addition of soluble complement receptor type 1, suggesting that a physical association between neutrophils and target cells mediated by OZ was required to generate a cytotoxic environment. Significant numbers of neutrophil-target cell aggregates were observed by microscopic examination only under low hydrodynamic shear conditions. We conclude that the cytotoxic potency of H2O2 produced by neutrophils activated with OZ was due to a localized high concentration of H2O2 to which the target cells were exposed as a result of their labile adherence to OZ. This phenomenon may reflect a mechanism that neutrophils have acquired for maximizing the antimicrobial power of extracellular oxidants toward microbes that escape phagocytotosis.
Redundant expression but selective utilization of nuclear factor of activated T cells family members.
Authors: Timmerman L A , Healy J I , Ho S N , Chen L , Goodnow C C , Crabtree G R
Published: December 2022

Nuclear factor of activated T cells (NF-AT) complexes regulate the induction of many early T cell activation molecules. Four related proteins can function as the cytoplasmic subunit of NF-AT, and their overlapping expression patterns and the mild phenotype of the NF-ATp null mice suggest that they may be functionally redundant. We characterized the distribution and activation of cytoplasmic NF-AT proteins in mature lymphocytes and found that NF-ATc, NF-ATp, and NF-AT4/x/c3 are co-expressed and co-regulated in mature T and B cells. Each protein forms independent DNA binding complexes, and at physiologic concentrations, NF-ATc and NF-ATp complexes out-compete NF-AT4/x/c3 for occupancy of NF-AT sites from the IL-2, IL-3/granulocyte-macrophage CSF, IL-4, and CD40 ligand genes. This predicts heavily redundant immune regulatory functions of NF-ATp and NF-ATc, but distinct activities for NF-AT4/x/c3. Additionally, Ab interaction with NF-ATp induces high affinity NF-kappaB site interaction, suggesting that nuclear partners may dramatically vary the specificity of the NF-AT family.
DBA/1 mice expressing the human TNF-alpha transgene develop a severe, erosive arthritis: characterization of the cytokine cascade and cellular composition.
Authors: Butler D M , Malfait A M , Mason L J , Warden P J , Kollias G , Maini R N , Feldmann M , Brennan F M
Published: December 2022

Arthritis spontaneously develops in mice expressing a human TNF-alpha transgene modified with the 3' untranslated region of beta-globin. We have backcrossed these mice onto the arthritis-susceptible DBA/1 background and found an acceleration of the onset of arthritis with successive generations of interbreeding. Bioactive TNF-alpha in primary synovial membrane cell cultures was significantly higher in the DBA/1 transgenic mice than in transgenic mice on the original background. Elevated levels of human TNF-alpha were accompanied by increases in synovial cell expression of murine IL-1beta and IL-6, but murine granulocyte-macrophage CSF, IFN-gamma, and IL-4 could not be detected. Interestingly, the anti-inflammatory cytokine IL-10 could be detected, but levels were not modulated by expression of the transgene. Analysis of the synovial membrane cell composition revealed that >50% of synovial cells were CD45-negative cells, presumably fibroblasts and endothelial cells, and the majority of CD45-expressing cells were neutrophils. Peritoneal macrophages and lymphocytes from the spleen, bone marrow, and lymph nodes required LPS stimulation to produce human TNF-alpha, indicating that, when activated, cells of these lineages were capable of expressing the transgene; however, few were found in synovial tissues. In contrast, fibroblasts derived from synovial tissue spontaneously released human TNF-alpha, and using immunohistochemical techniques, this cytokine was localized to fibroblast-like cells and chondrocytes. We propose that arthritis in DBA/1 human TNF-alpha transgenic mice is driven in part through the spontaneous expression of transgene by connective tissue cells, and there is little evidence of the participation of lymphocytes in this model.
Protective effects of IL-4, IL-10, IL-12, and IL-13 in IgG immune complex-induced lung injury: role of endogenous IL-12.
Authors: Mulligan M S , Warner R L , Foreback J L , Shanley T P , Ward P A
Published: December 2022

Using the IgG immune complex (BSA-anti-BSA) model of acute lung injury in rats, we have compared four intratracheally administered cytokines for their protective effects on parameters of injury (albumin leak and hemorrhage) and on neutrophil accumulation (lung content of myeloperoxidase). The descending rank order of protective effects was: IL-10 > or = IL-13 > IL-4 > > IL-12. In animals receiving an intratracheal instillation of 1.0 microg murine rIL-4, IL-10, IL-12 or IL-13, the levels of TNF-alpha in bronchoalveolar (BAL) fluids after intrapulmonary deposition of IgG immune complexes were reduced by 98, 98, 34, and 97%, respectively, implying a corresponding reduction in up-regulation of lung vascular intercellular adhesion molecule-1. The unexpected findings with IL-12 were further evaluated. In spite of reduced BAL levels of TNF-alpha in IL-12-treated animals, BAL levels of IFN-gamma were elevated sixfold, indicative of the expected biologic response to IL-12. Alveolar macrophages obtained from the same animals showed a 68% reduction in formation in vitro of NO2-/NO3-. When rats undergoing intrapulmonary deposition of IgG immune complexes were treated either i.v. or intratracheally with blocking Ab to murine IL-12, there were significant increases in lung permeability and myeloperoxidase values, suggesting that in this model intrinsic IL-12 functions in a regulatory manner. In homogenates of injured lungs, this Ab detected heterodimeric complex, consistent with rat IL-12. These data confirm the ability of certain cytokines to suppress in vivo lung inflammatory responses and underscore the unexpected anti-inflammatory activities of IL-12.
Surface Tension of Blood Serum in Syphilis
Authors: Yagle Elizabeth M.
Published: December 2022

A short review is given of some of the recent work on the surface tension of blood serum. The present study was undertaken to find if differentiation by means of surface tension could be made between sera giving positive and negative complement-fixation and precipitation reactions for syphilis. The technic of measuring the surface tension of serum is described. Results of surface tension estimations of serum in different dilutions show 1:5000 to be the optimum dilution for the maximum time drop. The difference between initial and static surface tension is shown. Average time drops of 25 negative and 25 positive sera, heated and unheated show the time drop to be slightly greater in positive than in negative sera. Likewise the time drop is slightly greater in heated than in unheated sera. Study of individual sera shows that there may be a greater difference in time drop between individual sera of the positive or of the negative group than between positive and negative sera. The drop of surface tension is not correlated with positive or negative complement-fixation and precipitation reactions. The effect of heating at 55°C. for thirty minutes is doubtful.
Natural Killer Cell Depletion Fails to Influence Initial CD4 T Cell Commitment In Vivo in Exogenous Antigen-Stimulated Cytokine and Antibody Responses
Authors: Wang MingDong , Ellison Cynthia A. , Gartner John G. , HayGlass Kent T.
Published: December 2022

The role played by NK- and NK1.1-expressing T cells in CD4 T cell activation and induction of immune responses in vivo is controversial. These effector cells of the innate immune response are hypothesized to play a pivotal role in shaping initial T cell activation, with some groups reporting that classical NK cells are required for optimal Th1-like T cell activation, and others supporting a role for NK1.1+ αβ T cells in Th2 generation. Here, we examine the impact of in vivo NK cell depletion on the development of exogenous Ag-specific cytokine and Ab responses using a murine model of human immediate hypersensitivity. OVA-specific immune responses were induced in 1) C57Bl/6 bg/bg and bg/+ mice, 2) BALB/c mice pretreated with anti-asialoGM1 or control Ab, and 3) C57Bl/6 mice depleted of NK1.1-expressing cells by in vivo administration of anti-NK1.1 mAb PK136. Depletion efficacy was assessed by functional assays and flow cytometric analysis. Each of these approaches indicated that depletion of NK cells and NK1.1+CD4+ T cells fails to alter the Th1:Th2 balance of Ag-driven cytokine synthesis, as indicated by OVA-stimulated cytokine synthesis in primary bulk culture. Similarly, the kinetics and intensity of effector responses such as OVA-specific IgG2a and IgE synthesis were neither increased nor decreased in any of the three models examined. The results argue that NK cells and peripheral NK1.1+ T cells do not play an essential role in shaping the induction of Ag-specific immune responses to soluble exogenous Ags, the most common class of inhalant allergen.
Polymorphism Within a TCRAV Family Influences the Repertoire Through Class I/II Restriction
Authors: Sim Bee-Cheng , Wung Jay L. , Gascoigne Nicholas R. J.
Published: December 2022

Antibody-staining experiments have shown that closely related members of the TCRAV3 family are reciprocally selected into the CD4 or CD8 peripheral T cell subsets. This has been attributed to the individual AV3 members interacting preferentially with either MHC class I or MHC class II molecules. Single amino acid residues present in the complementarity-determining regions (CDR) CDR1α and CDR2α are important in determining MHC class specificity. We have now extended these observations to survey the expressed repertoire of the AV3 family in C57BL/6 mice. Three of the four expressed AV3 members are preferentially selected into the CD4+ subset of T cells. These share the same amino acid residue in both CDR1α and CDR2α that differ from the only CD8-skewed member. Preferential expression of an individual AV3 is not caused by other endogenous α- or β-chains, by any conserved CDR3 sequence, or by the usage of TCRAJ regions. This study shows that residues in the CDR1 and CDR2 regions are primary determinants for MHC class discrimination and suggests that polymorphism found within a TCRAV family has an important effect on the overall shaping of the T cell repertoire.
Cytokine Production and Surface Marker Expression in Acute and Stable Multiple Sclerosis: Altered IL-12 Production and Augmented Signaling Lymphocytic Activation Molecule (SLAM)-Expressing Lymphocytes in Acute Multiple Sclerosis
Authors: Ferrante Pasquale , Fusi Maria Luisa , Saresella Marina , Caputo Domenico , Biasin Mara , Trabattoni Daria , Salvaggio Antonino , Clerici Enrico , de Vries Jan E. , Aversa Gregorio , Cazzullo Carlo L. , Clerici Mario
Published: December 2022

Ag-stimulated IL-2 production and mitogen-stimulated type 1 and type 2 cytokine production by PBMC, as well as expression of Th1- and Th2-associated phenotypical markers, of B7-1, B7-2, and CD95 (Fas) on the surface of immune cells, and the serum concentration of soluble Apo-1/Fas were evaluated in multiple sclerosis (MS) patients with either acute (AMS) or stable (SMS) disease and in healthy controls (HC). Results showed that 1) Ag-stimulated IL-2 production is reduced in MS patients compared with that in HC; 2) mitogen-stimulated type 1 cytokine production is increased, and IL-10 production is reduced in MS patients compared with those in HC, and in AMS patients compared with those in SMS; 3) whereas production of the metabolically active p70 heterodimers is comparable in SMS, AMS, and HC, production of the p70 heterodimer and the p40 chains (total IL-12) is increased in SMS compared with that in AMS and HC; 4) CD4+, CD4+SLAM+, and CD4+CD7+ lymphocytes (preferentially type 1 cytokine-producing lymphocytes) are increased in MS compared with levels in HC; 5) B7-2- as well as Fas+-expressing monocytes are augmented in MS compared with those in HC, and serum soluble Apo-1/Fas is augmented in AMS compared with SMS and HC. These results confirm that a complex imbalance in both cytokine production and the Fas system is present in MS and indicate that different cytokine profiles may be observed in patients with acute or stable disease. The data also suggest that peculiar phenotypic populations are over-represented in MS patients, and for the first time show that SLAM expression is correlated with dysregulation of type 1 and type 2 cytokine production in human pathology.
Elf-1 Regulates Basal Expression from the T Cell Antigen Receptor ζ-Chain Gene Promoter
Authors: Rellahan Barbara L. , Jensen Jane P. , Howcroft Thomas K. , Singer Dinah S. , Bonvini Ezio , Weissman Allan M.
Published: December 2022

In mature T cells, limited synthesis of the TCR-ζ subunit is primarily responsible for regulating surface expression of TCRs. Transcription of ζ is directed by a complex promoter that includes two potential binding sites for the Ets family of transcription factors at −52 (zEBS1) and −135 (zEBS2). Mutation of these two sites results in a marked reduction of transcription from this promoter. Using electrophoretic mobility shift analysis, Elf-1 was demonstrated to be the Ets family member that binds to these sites. One site, zEBS1, matches the optimal Elf-1 consensus sequence in eight of nine bases, making it the best match of any known mammalian Elf-1 binding site. A role for Elf-1 in TCR-ζ trans-activation was confirmed by ectopic expression of Elf-1 in COS-7 cells. This resulted in an increase in TCR-ζ promoter activity that mapped to zEBS1 and zEBS2. Additional support for the involvement of Elf-1 in TCR-ζ trans-activation derives from the finding that a GAL4-Elf-1 fusion protein trans-activated TCR-ζ promoter constructs that had been modified to contain GAL4 DNA binding sites. These results demonstrate that Elf-1 plays an essential role in the trans-activation of a constitutively expressed T cell-specific gene, and that trans-activation occurs in the context of the native promoter in both lymphoid and nonlymphoid cells. Taken together with the existing literature, these data also suggest that the requirement for inducible factors in Elf-1-mediated trans-activation may decrease as the affinity and number of Elf-1 sites increase.
Involvement of C-Abl Tyrosine Kinase in Lipopolysaccharide- Induced Macrophage Activation
Authors: Le Quan , Daniel Rene , Chung Siu-Wah , Kang Anthony D. , Eisenstein Toby K. , Sultzer Barnet M. , Simpkins Henry , Wong Peter M. C.
Published: December 2022

LPS endotoxin-induced macrophage activation is recognized to be important in both nonspecific immunity and endotoxin-induced sepsis when excessive macrophage stimulation occurs. In this study, we showed that reduction of c-Abl in macrophages prevented LPS-induced growth arrest, nitric oxide production and TNF-α secretion by ANA-1 macrophages. These cells continued to grow but later underwent apoptosis. Reduction of c-Abl in these cells led to reduced c-Abl kinase activity associated with Ran, which recently has been shown to be an LPS-responsive gene product. Our data suggest that c-Abl tyrosine kinase is one of the intermediates downstream of the initial signal transduction event related to activation of macrophages by LPS.