bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha 4 beta 1 (CD49d/CD29) integrin costimulation of human T cells enhances transcription factor and cytokine induction in the absence of altered sensitivity to anti-CD3 stimulation.
Authors: Udagawa T , Woodside D G , McIntyre B W
Published: December 2022

The integrin alpha 4 beta 1 can provide a costimulus to induce IL-2 secretion and IL-2R expression leading to enhanced proliferation of purified, peripheral blood T cells. Similar to expression of IL-2, we demonstrated that recombinant vascular-cell adhesion molecule-1, when co-immobilized with anti-CD3 mAb, significantly enhanced the induction of transcription factors NF-AT, AP-1, and NF-kappa B as determined by electromobility shift assays. alpha 4 beta 1 ligation alone had no effect on transcription factor binding. The requirements for induction of transcription factors reflected the requirements for the secretion of multiple cytokines, including IL-2, TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF. In contrast to freshly isolated T cells, in vitro-cultured T cells did not require costimulation for cytokine secretion in response to anti-CD3 alone. Comparison of the dose response to anti-CD3 stimulation demonstrated that half-maximal induction of IL-2 was achieved using the same dose of anti-CD3 for both freshly isolated and cultured T cells. Furthermore, the dose of OKT3 required to achieve half-maximal activation was the same using PMA or different concentrations of alpha 4 beta 1 ligands. Therefore, costimulation by alpha 4 beta 1 ligands was not due to stabilization of the interaction of the cells with its substrate. We conclude, rather, that alpha 4 beta 1 in freshly isolated T cells delivers a distinct signal that synergizes early with signals initiated by TCR/CD3 ligation to induce DNA binding of multiple transcription factors required for cytokine gene induction.
TCR-mediated adhesion of T cell hybridomas to planar bilayers containing purified MHC class II/peptide complexes and receptor shedding during detachment.
Authors: Dustin M L , Miller J M , Ranganath S , Vignali D A , Viner N J , Nelson C A , Unanue E R
Published: December 2022

T cell recognition of foreign Ag/MHC class II complexes is sensitive down to approximately 100 complexes per cell or approximately 0.2 complexes/micron2. To better understand the physical basis of the recognition stage of Ag presentation, we examined adhesion of the lysozyme- specific T cell hybridoma, 3A9, to artificial bilayers containing covalent MHC class II/peptide complexes or adhesion molecules. Adhesion of 3A9 cells required a superphysiologic density of the MHC class II/peptide complex and was partly dependent on CD4; cells adhered but did not crawl. No adhesion was observed to bilayers containing MHC class II molecules without the lysozyme peptide. Activated 3A9 cells adhered and crawled on bilayers containing ICAM-1. The physical strength of contacts was tested with fluid shear. 3A9 cells adherent to bilayers containing MHC class II/peptide complexes shed their contact, which remained on the substrate and contained TCR. In contrast, 3A9 cells peeled from the ICAM-1 bilayer, and held firmly on LFA-1 bilayers; in a manner dependent on filamentous actin. When ICAM-1 and the MHC/peptide complexes were combined, the 3A9 cells adhered tightly and spread, but did not crawl, on the bilayers and TCR clustered at the center of the contact area. Physiologically, the TCR is unlikely to directly initiate adhesion. TCR clusters formed with the assistance of adhesion mechanisms may have to be shed to allow de-adhesion, and this may contribute to TCR down-regulation.
In vitro induction of MUC-1 peptide-specific type 1 T lymphocyte and cytotoxic T lymphocyte responses from healthy multiparous donors.
Authors: Agrawal B , Reddish M A , Longenecker B M
Published: December 2022

We have recently provided evidence that there is a natural immunization against human cancer-associated MUC-1 mucin epitopes during pregnancy by studying MUC-1 Ag-specific T cell lines established from multiparous women. Using this experimental model system, we now report that MUC-1 peptide-specific MHC class I-restricted CTLs can be generated in vitro using T cells from multiparous women stimulated with synthetic MUC-1 peptide-loaded, autologous APCs. The complexity of cytokines produced in response to the MUC-1 peptide by anti-MUC-1 T-cells was examined. IFN-gamma was generated by MUC-1-specific T cell lines in long term cultures, whereas in short term cultures, both IFN-gamma and IL-4 were produced. The presence of MUC-1-reactive T cells in multiparous women is consistent with their potential role in immune surveillance and provides a rationale for the use of certain synthetic MUC-1 peptides for active specific immunotherapy of human carcinomas.
Receptors for immune complexes activate gene expression and synthesis of matrix proteins in cultured rat and human mesangial cells: role of TGF-beta.
Authors: López-Armada M J , Gómez-Guerrero C , Egido J
Published: December 2022

Most human glomerulonephritis are induced by the deposition and/or formation of immune complexes in the glomerular region. Recently, it has been demonstrated that cultured glomerular mesangial cells (MC) express Fc receptors for IgA and IgG (Fc-alpha and Fc-gamma receptors). In this work, we studied whether the interaction of IgA and IgG complexes with MC induces accumulation of mesangial matrix, the histologic hallmark of progressive glomerular diseases. The exposure of MC to IgA and IgG complexes increased extracellular matrix components, such as fibronectin (FN) and collagens, at both the mRNA and protein levels in a time- and dose-dependent manner. Monomeric IgA or F(ab')2 fragments did not increase FN production, indicating that a constant region of IgA and cross-linking of Fc-alpha receptors are required. We also explored the role of TGF-beta, a profibrogenic cytokine, in the regulation of matrix synthesis. Both IgA and IgG complexes caused in MC an augmentation in TGF-beta1 mRNA and TGF-beta activity and the conversion of latent TGF-beta to the biologically active form. The coincubation of cells with complexes and a neutralizing Ab to TGF-beta significantly reduced the FN synthesis. These results indicate that the Fc receptor occupancy of MC increases the production of extracellular matrix proteins. The autocrine TGF-beta synthesis appears to be largely responsible for this effect. These findings could have implications for a better understanding of the glomerulosclerosis process in immune complex nephritis.
Pleiotropic effects of immobilized versus soluble recombinant HIV-1 Tat protein on CD3-mediated activation, induction of apoptosis, and HIV-1 long terminal repeat transactivation in purified CD4+ T lymphocytes.
Authors: Zauli G , Gibellini D , Celeghini C , Mischiati C , Bassini A , La Placa M , Capitani S
Published: December 2022

CD3 mAb and HIV-1 Tat protein co-immobilized on plastic were able to induce a strong proliferation of resting human CD4 T cells, cultured in a serum-free chemically defined medium. Blocking studies performed with heparin or peptides containing the RGD sequence demonstrated that the heparin-binding basic domain of Tat plays a predominant role in CD4+ T cell activation. Moreover, the enhanced proliferative response of CD4+ T cells to immobilized Tat appeared to be mediated by alpha 5, beta 1, and alpha v subunits of surface integrin receptors. In contrast, soluble Tat showed a dose-dependent inhibitory activity on the proliferative response of resting CD4+ T cells stimulated by CD3 mAb co-immobilized with Tat or fibronectin, but not with CD28 mAb. In transient transfection assays performed with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) plasmid CD3 mAb co-immobilized with Tat or fibronectin or CD28 mAb significantly stimulated CAT activity over the background. On the other hand, while immobilized Tat alone had no effects on LTR transactivation, soluble Tat was able to transactivate LTR-CAT in a dose-dependent manner. When CD4+ T cells activated by CD3 mAb co-immobilized with Tat were recovered, cultured for 7 days with 25 U/ml recombinant IL-2, and given an additional activation signal by recross-linking CD3 mAb, a marked increase of apoptosis was observed with respect to cells not subjected to CD3 mAb recross-linking. While co-immobilized Tat plus CD3 mAb did not show any significant effect on activation-induced cell death, high concentrations of soluble Tat synergized with immobilized CD3 mAb in the induction of apoptosis.
Characterization of the human C6 promoter: requirement of the CCAAT enhancer binding protein binding site for C6 gene promoter activity.
Authors: González S , López-Larrea C
Published: December 2022

The sixth complement component (C6) is a late-acting complement protein that participates in the assembly of the membrane attack complex. C6 and most of the complement proteins are mainly synthesized in the liver. However, the human hepatoma-derived cell line Hep-G2, which produces the majority of complement proteins, synthesizes traces of C6. Here, we have isolated and characterized the human C6 promoter. Approximately 1 kb of C6 upstream sequence is shown to be sufficient to achieve tissue-specific expression of a luciferase reporter gene in two hepatic (Hep-G2 and Hep-3B) and two extrahepatic cell lines (fibroblast M1 and HeLa) in a manner similar to endogenous C6. There are wide differences in C6 mRNA expression among the four cell lines, whereas Hep-3B expresses high levels of C6, Hep-G2 and M1 poorly synthesize C6, and HeLa completely lacks C6 expression. Deletional and mutational analysis demonstrates that a C/EBP (CCAAT/enhancer binding protein) site located at -67 is required for C6 expression in Hep-3B cells, but it has little effect in M1 and Hep-G2 cells. Electrophoretic mobility shift assays show that this sequence binds to a C/EBP alpha using Hep-3B nuclear extract, but a negligible activity is detected using a Hep-G2 extract. To further investigate whether C/EBP alpha is the limiting factor for C6 expression, we have transfected a C/EBP alpha expression vector into Hep-G2 and Ml cells. C/EBP alpha expression vector dramatically trans-activates the luciferase reporter gene controlled by the C6 promoter, and it partially restores C6 mRNA expression in Hep-G2 cells.
T cell-mediated terminal maturation of dendritic cells: loss of adhesive and phagocytotic capacities.
Authors: Kitajima T , Caceres-Dittmar G , Tapia F J , Jester J , Bergstresser P R , Takashima A
Published: December 2022

Dendritic cells (DC) are a specific subset of APC characterized by the potent ability to activate immunologically naive T cells. We have observed previously that the murine epidermis-derived DC line XS52 undergoes a set of profound changes upon Ag-specific interaction with T cells, including IL-1 beta secretion acquired expression of CD86, and lost expression of CD115 (CSF-1 receptor) and proliferative responsiveness to CSF-1. These changes, which appear to reflect a critical transition during Ag presentation, have been termed T cell-mediated "terminal maturation" of DC, Here we report that XS52 cells also lose their adhesive and phagocytotic capacities during this event. XS52 cells, ordinarily adhere to petri dishes and phagocytose latex heads, as has been reported for DC freshly procured from spleen and skin. Importantly, XS52 cells lose both capacities after 3 to 24 h of incubation with HDK-1 T cells (keyhole limpet hemocyanin-specific TH1 clone) or with 5S8 T cells (dinitrobenzene sulfonate specific Th0 clone) in the presence of Ag. By contrast, incubation with T cells alone or with Ag alone has minimal effects, indicating that this regulation required both T cells and Ag. With respect to mechanisms, several lines of evidence suggest this IFN-gamma, which is secreted by T cells, serves as the primary mediator in down-regulating both capacities. Our observations illustrate a unique mechanism by which responding T cells upon Ag-specific activation by DC, suppress the machinery of Ag uptake through the elaboration of IFN-gamma.
Role of reactive oxygen intermediates in TCR-induced death of T cell blasts and hybridomas.
Authors: Williams M S , Henkart P A
Published: December 2022

The functional role of reactive oxygen intermediates (ROI) in activation-induced death of mature T lymphocytes and hybridomas was tested using antioxidants and inhibitors of enzymes that generate oxidants. These agents were shown to inhibit TCR-triggered death in a concentration-dependent manner, suggesting a possible role of ROI in this death. Since the TCR-induced death of both human T blasts and the murine T cell hybridoma 2B4 involve an initial step of TCR-induced Fas ligand (FasL) up-regulation followed by a lethal step induced by Fas cross-linking, both steps were examined separately for ROI dependence. The thiol antioxidants N-acetyl cysteine and glutathione blocked Fas-induced death triggered via cross-linking either by IgM anti-Fas or cell-bound FasL, while the other inhibitors of activation-induced death did not block this late lethal step. None of the agents used blocked early events after TCR ligation, as seen by the lack of inhibition of IL-2 secretion or CD69 up-regulation. However, the nonthiol agents that blocked activation-induced death all blocked FasL up-regulation induced by TCR signals in the hybridoma, as measured by a functional assay. Agents inhibiting FasL up-regulation also inhibited activation-induced ROI generation in the hybridoma, as detected by flow cytometry using dihydrorhodamine oxidation. Furthermore, a good correlation was found between the extent of ROI generation and functional FasL expression in 2B4 cells. Thus, while ROI do not appear to act as downstream mediators of apoptotic death induced by steroid or Fas cross-linking in T cells, they are generated by TCR signaling and appear to participate in FasL up-regulation.
Differential activation of the nuclear factor-kappa B by TNF muteins specific for the p60 and p80 TNF receptors.
Authors: Chainy G B , Singh S , Raju U , Aggarwal B B
Published: December 2022

Human TNF is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors, viz p60 and p80, which transmit their signals independently of each other. Activation of the transcription factor NF-kappa B is one of the earliest events induced by TNF, but whether it is mediated through one or both forms of the TNF receptor is controversial. in the present studies, we examined the role of each receptor in the activation of NF-kappa B by using TNFs that has been designed by site-specific mutagenesis to bind either the p60 (R32W; S86T) or the p80 (D143N; A145R) form of the receptor. Human myelogenous leukemic ML-1 a cells known to express almost equal amounts of the two receptors were used. The binding of TNF to these cell could be inhibited equally by either TNF(p60) or TNF(p80) mutein. Treatment of these cells with TNF(p60) mutein activate NF-kappa B within 30 min, whereas TNF(p80) mutein, even at a 1000-fold excess, had no effect, suggesting that the activation of NF-kappa B is differentially regulated through the p60 receptor. Consistent with these results, treatment with either anti-p80 monoclonal or polygonal Abs blocked the binding of TNF to the p80 receptor without affecting TNF-mediated activation of NF-kappa B TNF(p60) mutein was also effective in cell killing, but the TNF(p80) mutein was totally ineffective. The effect was not cell type-specific, since other p80-expressing cell lines were also unresponsive. Overall, our results clearly demonstrate that the activation of NF-kappa B and cytotoxicity by TNF is differentially regulated through the p60 receptor.
Characterization of three promoter elements and cognate DNA binding protein(s) necessary for IFN-gamma induction of gp91-phox transcription.
Authors: Eklund E A , Luo W , Skalnik D G
Published: December 2022

The cytochrome b558 heavy chain (gp9l-phox) is expressed in terminally differentiated myelomonocytic cells. Three cis-elements located between -450 and -100 bp of the gp91-phox promoter are required for IFN-gamma induced transcription. Mutations that disrupt individual cis-elements incrementally decrease gp9l-phox promoter activity, and one of the two proximal elements must be present for an IFN-gamma response. The DNA-binding activities that interact with each of the cis-elements exhibit similar gel mobility and binding site specificity, although a consensus binding site common to the three elements is not apparent. An increased level of each DNA/protein complex is observed in myeloid cells following treatment with PMA, retinoic acid/dimethylformamide, or IFN-gamma, but not in similarly treated HeLa cells. The myeloid-specific increase in the intensity of each complex is delayed 12 to 24 h following IFN-gamma treatment, and the complexes are not immunoreactive with antisera directed against IFN-responsive factors such as IRF-1, IRF-2, IFN consensus sequence binding protein, Stat1, and IFN-stimulated gene factor-3 gamma, although IRF-2 is additionally detected as binding to the middle cis-element. These results reveal cis-elements and a DNA-binding factor(s) that participate in a common pathway in response to various stimuli that induce gp9l-phox transcription.
Use of phage surface expression to analyze regions of human V4-34(VH4-21)-encoded IgG autoantibody required for recognition of DNA: no involvement of the 9G4 idiotope.
Authors: Mockridge C I , Chapman C J , Spellerberg M B , Isenberg D A , Stevenson F K
Published: December 2022

The V4-34 gene encodes the majority of autoanti-red cell Abs of I/i specificity. It also encodes a proportion of autoanti-DNA Abs found in patients with systemic lupus erythematosus. Nucleotide sequence analysis of mAbs that use this gene has indicated a role for CDR3 in discrimination between these autoantigens. Specifically, anti-DNA activity may require basic amino acids, such as arginine, found in this region. To investigate this requirement, we have expressed VH and VL sequences from a patient's IgG anti-DNA mAb, as Fab molecules at the surface of phage. Expressed Fab bound strongly to DNA, whereas control VH and VL pairs from an anti-red cell mAb did not. Replacement of the homologous mutated V4-34 sequence by germ-line sequence did not affect binding, indicating that somatic mutations in VH did not contribute significantly. In contrast, replacement of the basic CDR3 by an anti-red cell CDR3 abrogated anti-DNA activity, confirming its major role. However, an influence of VL was revealed by replacing homologous mutated V kappa IIIb by an unmutated V kappa IIIb sequence, reducing binding by approximately 50%. This influence was apparent only with homologous VH since the mutated V kappa was unable to generate anti-DNA activity when combined with anti-red cell VH. The 9G4 idiotope, which arises from FWR1, was expressed by all constructs. Substitution of Trp by Ser at position 7 in FWR11 caused complete loss of idiotope expression, with no effect on recognition of DNA, indicating no influence of idiotope expression on anti-DNA activity. Phage surface expression provides a powerful and rapid technique for assessing sequences relevant for Ab specificity or idiotope expression.
Interval mapping of quantitative trait loci controlling humoral immunity to exogenous antigens: evidence that non-MHC immune response genes may also influence susceptibility to autoimmunity.
Authors: Wu J , Longmate J A , Adamus G , Hargrave P A , Wakeland E K
Published: December 2022

IgG Ab titers elicited to bovine rhodopsin in CFA differ 8- to 10-fold between H2s identical inbred strains A.SW/snJ (high responder) and SJL/snJ (low responder). This variation in IgG Ab titer resulted from a dramatic difference in the rise in Ab titer occurring during the maturation of the T-dependent humoral immune response. To determine the positions of non-MHC genes controlling this quantitative variation in T-dependent humoral immune responsiveness, 206 reciprocal (A.SW/snJ x SJL/snJ)F2 female progeny were immunized and assayed for anti-rhodopsin responsiveness. The genomes of these progeny were screened with 115 polymorphic simple sequence repeat markers covering >90% of the mouse genome. interval mapping analysis localized the positions of these non-MHC immune response genes to genomic intervals on chromosomes 1, 5, and 13. Interestingly, these three intervals coincide exactly with three intervals recently shown to contain genes contributing to susceptibility to systemic lupus erythematosus and/or the production of autoimmune anti-dsDNA Abs. These results suggest that some genes affecting levels of humoral immune responsiveness to exogenous Ag may also play a role in genetic susceptibility to humoral autoimmune diseases. Analyses of the modes of inheritance demonstrated that high responder alleles were inherited from both parental genomes, indicative of epistatic interactions among genes influencing humoral immune responsiveness.
Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha.
Authors: Garofalo R , Mei F , Espejo R , Ye G , Haeberle H , Baron S , Ogra P L , Reyes V E
Published: December 2022

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.
In vivo fate of the inflammatory macrophage during the resolution of inflammation: inflammatory macrophages do not die locally, but emigrate to the draining lymph nodes.
Authors: Bellingan G J , Caldwell H , Howie S E , Dransfield I , Haslett C
Published: December 2022

The resolution of acute inflammation requires bulk clearance of extravasated inflammatory cells in an ordered manner. Neutrophils undergo apoptosis and are ingested by macrophages (M psi) via a novel recognition mechanism that fails to provoke proinflammatory responses. Thereafter, the fate of inflammatory M psi themselves remains unclear. We investigated this in vivo, developing a semiallogeneic adoptive transfer system to track the fate of inflammatory M psi in a murine model of resolving peritonitis. Fluorescently labeled M psi from H-2k/d mice were transferred into the peritoneal cavity of H-2k mice at the same stage of resolving inflammation as the donor mice. Dual color flow cytometry permitted discrimination among donor cells, recipient cells, and donor cells that had been phagocytosed by recipient M psi. Despite the absence of significant local phagocytosis, the number of transferred M psi free in the peritoneum of recipient mice declined rapidly, being undetectable by 96 h. These data suggest that inflammatory M psi normally emigrate rapidly from the peritoneal cavity during the resolution of inflammation, contrasting with resident M psi, which persist in the noninflamed peritoneum for weeks. Accordingly, labeled nonphagocytosed cells were detected in the draining lymph nodes, but not in a variety of other tissues. Thus, unlike the polymorphonuclear leukocyte, which dies by apoptosis and is ingested by M psi, the inflammatory M psi itself does not die locally. Having performed its acute inflammatory and scavenging roles, it emigrates in a nonrandom fashion to the draining lymph node, where it may play an important part in the presentation of Ags from the inflamed site.
p161, a murine membrane protein expressed on mast cells and some macrophages, is mouse CD13/aminopeptidase N.
Authors: Chen H , Kinzer C A , Paul W E
Published: December 2022

pl6l is a membrane glycoprotein expressed on mast cells and on activated macrophages but on few if any other cells of hematopoietic lineages. Its lack of expression on basophils makes it useful to distinguish mast cells from basophils and aids in the analysis of mast cells and their precursors. p161 was purified from the mast cell line CFTL-12 by affinity chromatography and subjected to limited proteolysis. The sequences of the resultant peptides indicated that p161 is homologous with rat and human CD13/aminopeptidase N. Using oligonucleotide primers derived from rat CD13 cDNA, a mouse cDNA was obtained. Its deduced amino acid sequence displays 87% identity with rat CD13 and 76 % identity with human CD13. Expression of the mouse cDNA in M12 cells, which are p161 negative, renders these cells positive for staining with the monoclonal anti-p161 Ab, K-1. Furthermore, a mAb raised against partially purified mouse intestinal aminopeptidase N specifically blocked the binding of K-1 to both CFTL-12 cells and the transfected M12 cells. These results strongly indicate that mouse p161 is CD13/aminopeptidase N. Northern blot analysis shows that p161 mRNA is most abundantly expressed in the intestinal tract and kidney and is present in liver, lymph node, spleen, and brain.
IL-6 is an in vitro and in vivo autocrine growth factor for middle T antigen-transformed endothelial cells.
Authors: Giraudo E , Arese M , Toniatti C , Strasly M , Primo L , Mantovani A , Ciliberto G , Bussolini F
Published: December 2022

Murine endothelial cells immortalized with the middle-size Ag of polyomavirus (PmT) cause vascular tumors in syngenic mice by recruitment of host normal endothelial cells. This pathogenic process is similar to that occurring in Kaposi's sarcoma, in which the core of the lesion is constituted by "spindle cells," which recruit normal vascular mesenchymal cells. In murine endothelial cells, PmT induces modification of the expression of genes, including that of IL-6. Since IL-6 is a pleiotrophic cytokine that also regulates endothelial cell functions related to angiogenesis, we studied the relevance of IL-6 in the tumorigenicity of PmT-endothelial cells. In vitro studies demonstrated that the spontaneous PmT-endothelial cell proliferation rate was slow during the first 6 days of culture and then increased rapidly and paralleled the IL-6 release. The addition of recombinant IL-6 during the first days of culture induced a marked proliferation in a dose-dependent manner. PmT-endothelial cells expressed on their surface a high-affinity binding site for IL-6 constituted by both IL-6Ralpha and gp130 transmembrane receptors. The growth-promoting effect of exogenous IL-6 or that released by PmT-endothelial cells was abrogated by mAbs anti-IL-6Ralpha, whereas a mAb recognizing the endothelial cell CD31 molecule was inactive. 15A7 mAb anti-murine IL-6Ralpha was also active in vivo, reducing the number of metastases forming after transplantation of PmT-endothelial cells in DBA/2 mice. 15A7 mAb also increased the survival of mice bearing vascular tumors. We conclude that IL-6 is involved in the progression of vascular tumors induced by PmT, and that the blockage of IL-6-mediated intercellular circuits could be useful in the management of human vascular tumors, including Kaposi's sarcoma.
Induction of the TNF-alpha promoter in the murine dendritic cell line 18 and the murine mast cell line CPII is differently regulated.
Authors: Preischl E E , Pendl G G , Elbe A , Serfling E , Harrer N E , Stingl G , Baumruker T
Published: December 2022

While it was recently shown that activation of dendritic cells (DC) results in the production of a number of cytokines, the signal pathways and transcription factors involved in this process have not been described. To address this issue we compared the events resulting in the activation of the human TNF-alpha promoter occurring in the fetal dendritic cell line 18 (DC18) with those in the well-characterized murine mast cell line CPII. As stimuli we employed the protein kinase C inducer, PMA, and the Ca2+ ionophore, ionomycin, both of which are known to activate a large variety of intracellular signaling pathways. In the DC18 cells, PMA alone induces the TNF-alpha promoter in a macrolide-insensitive manner. In contrast, in the mast cell line CPII, both stimuli (PMA plus ionomycin) are necessary for promoter activation which, in addition, is sensitive to immunosuppressive drugs. Mapping of the TNF-alpha promoter showed that in both cell types the so-called kappa factor binding site is the crucial promoter element for the induction. We show that in DC18 cells, this sequence is bound to and controlled by NF-kappaB proteins p50 (NF-kappaB1) and p65 (ReIA), whereas in CPII mast cells, NF-AT and AN factors are the predominant proteins that bind to and control the kappaB element of the TNF-alpha promoter. These and further experimental data indicate that in DC, NF-kappaB factors play a predominant role in the activation of the TNF-alpha promoter and, possibly, of other cytokine promoters.
T helper 2 cytokines differently regulate monocyte chemoattractant protein-1 production by human peripheral blood monocytes and alveolar macrophages.
Authors: Yano S , Yanagawa H , Nishioka Y , Mukaida N , Matsushima K , Sone S
Published: December 2022

Th2 cytokines, such as IL-4, IL-10, and IL-13, suppress proinflammatory cytokine production by monocytes/macrophages. Since monocyte chemoattractant protein-1 (MCP-1) is presumed to play an important role in monocyte recruitment and activation during inflammatory and immune responses, we examined here the effects of these Th2 cytokines on MCP-1 production by human blood monocytes and alveolar macrophages. Unstimulated, highly purified blood monocytes did not produce MCP-1 spontaneously, while LPS treatment induced the production of MCP-1 and its mRNA expression. All Th2 cytokines tested suppressed LPS-induced MCP-1 production and its mRNA expression, although the suppressive effect of IL-13 was weaker than that of IL-4 or IL-10. In contrast, IL-10, but neither IL-4 nor IL-13, induced unstimulated peripheral blood monocytes to produce biologically active MCP-1 protein within 4 h, reaching a maximal level at 12 h. IL-10-induced MCP-1 production was reduced by pretreatment of IL-10 with anti-IL-10 Ab, negating the involvement of contaminated endotoxin. Moreover, IL-10 induced MCP-1 mRNA expression in unstimulated monocytes, independent of de novo protein synthesis. Furthermore, human alveolar macrophages produced MCP-1 spontaneously, and the production was inhibited by IL-4 or IL-13, but was augmented by IL-10. These findings suggest that IL-10 regulates MCP-1 production by monocytes/macrophages in a different way from other Th2 cytokines, such as IL-4 and IL-13, and contributes to host defense responses.
Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 response.
Authors: Caspi R R , Silver P B , Chan C C , Sun B , Agarwal R K , Wells J , Oddo S , Fujino Y , Najafian F , Wilder R L
Published: December 2022

This study examines whether genetic susceptibility vs genetic resistance to experimental autoimmune uveoretinitis (EAU) are connected to a predisposition to mount a Th1-dominated (IFN-gamma high, IL-4 low) vs a Th2-dominated (IL-4 high, lFN-gamma low) response. Lewis rats developed disease with high incidence after immunization with the uveitogenic peptide R16, whereas F344 rats were resistant. Primed lymph node cells from both strains proliferated in culture in response to R16. However, while the Lewis cultures transferred EAU to syngeneic recipients, those of F344 did not. The Lewis cultures produced substantially more IFN-gamma mRNA and protein in response to R16, than did those of F344. Both strains made low levels of IL-10 mRNA and IL-4 mRNA. Unlike the primary cultures, long-term (R16-specific) T cell lines derived from each of the strains transferred EAU equally well to their respective recipients, and produced similar, high levels of IFN-gamma mRNA and protein. Treatment of F344 with Bordetella pertussis toxin concurrently with immunization abrogated its resistance, enhanced Ag-specific IFN-gamma production in culture, and yielded a primed cell population capable of transferring EAU. Conversely, immunization of Lewis rats with R16 in IFA induced little or no disease; the primed cells produced minimal amounts of IFN-gamma and did not transfer EAU. However, addition of IL-12 into the culture resulted in a highly pathogenic, IFN-gamma-producing cell population. We conclude that genetic susceptibility to ocular autoimmunity in this model is connected to an elevated Th1 response. Genetic resistance, however, does not seem to involve an elevated Th2 response, but rather an inhibited development of Th1-like effector cells.
Disturbed myeloperoxidase-dependent activity of neutrophils in cystic fibrosis homozygotes and heterozygotes, and its correction by amiloride.
Authors: Witko-Sarsat V , Allen R C , Paulais M , Nguyen A T , Bessou G , Lenoir G , Descamps-Latscha B
Published: December 2022

The present study addresses the question of a possible linkage between the cystic fibrosis (CF) genetic autosomal recessive disorder and disturbance in neutrophil function. Neutrophil-dominated chronic airway inflammation is present at an early age in children with CF, even in the absence of detectable infection. As evidenced by extracellular superoxide anion release (measured by lucigenin luminescence) or intracellular hydrogen peroxide production (measured by 2',7'-dichlorofluorescein (DCF) fluorescence), no significant difference in the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity of isolated neutrophils was observed in noninfected CF children (homozygotes), their mothers or fathers (CF heterozygotes), and controls. In contrast, both myeloperoxidase (MPO)-dependent oxygenation activity (measured by luminol luminescence) and chloramine release were increased significantly in both CF homozygotes and heterozygotes as compared with controls. In the presence of either amiloride (a sodium channel inhibitor and sodium/proton antiport blocker) or EIPA (5-ethyl-N-isopropyl-amiloride, a specific inhibitor of the antiport), or choline buffer, intracellular MPO activity was decreased significantly in controls and in the CF homozygotes and heterozygotes, thus bringing intracellular MPO-dependent activity in CF subjects back to the level of controls. Extracellular release of MPO, measured by an ELISA to provide an activity-independent assessment of the enzyme, was increased only in CF homozygotes, and was decreased by amiloride and choline buffer, but not by EIPA. We conclude that a modification of intracellular pH and/or ionic concentrations may be related to the altered MPO enzymatic activity observed in CF neutrophils.
Clonotypic differences in signaling from CD94 (kp43) on NK cells lead to divergent cellular responses.
Authors: Brumbaugh K M , Pérez-Villar J J , Dick C J , Schoon R A , López-Botet M , Leibson P J
Published: December 2022

Ligation of MHC class I-recognizing receptors on NK cells dramatically modulates their secretory and cytotoxic function. This study focuses on characterizing key signaling events regulating these activities after ligation of the C-type lectin superfamily member, CD94. We isolated separate clonal populations of human NK cells in which ligation of CD94 (kp43) either triggered cell-mediated cytotoxicity (group A clones) or potently inhibited NK cell activation (group B clones). We then evaluated the proximal signaling events that regulate these alternative responses. CD94 stimulation of group A clones induced the rapid activation of intracellular protein tyrosine kinases (i.e., lck and ZAP-70), phospholipase C, and phosphatidylinositol 3-kinase. In contrast, CD94 ligation on group B clones had none of the above noted effects and instead inhibited the FcR-induced tyrosine phosphorylations of ZAP-70 and phospholipase C-gamma 2, the formation of phospho-zeta/ZAP-70 complexes, and the release of inositol phosphates. These results define distinct proximal signaling events initiated after CD94 ligation and suggest that clonotypic differences in signaling generate fundamentally different NK cell-mediated responses.
IL-12 p40 messenger RNA expression in target organs during acute graft-versus-host disease. Possible involvement of IFN-gamma.
Authors: Kichian K , Nestel F P , Kim D , Ponka P , Lapp W S
Published: December 2022

The onset of acute graft-vs-host disease (aGVHD) is accompanied by macrophage (M phi) priming and the presence of bacteria-derived LPS in the sera of transplanted animals. Priming of M phi occurs during aGVHD despite the suppression of T cell function. We have investigated whether IL-12 mediates the continued production of IFN-gamma during the state of T cell immunosuppression that accompanies aGVHD. Acute GVHD was induced in nonirradiated AxC57BL/6F1 mice by the injection of C57BL/6 lymphoid cells. Despite T cell immunosuppression, M phi became primed, as shown by their expression of inducible nitric oxide synthase mRNA and their production of nitric oxide in response to LPS. Continual exposure to IFN-gamma was required to maintain a primed state in M phi during aGVHD. IL-12 p40 peptide mRNA was increased in M phi purified from animals undergoing aGVHD 14 days after transplantation. Target organs of aGVHD, including thymus, salivary gland, and lung, showed increased IFN-gamma mRNA between days 7 and 14 after transplantation. The increase was accompanied by an induction of mRNA for the p40 peptide of IL-12 and inducible nitric oxide synthase within the target organs. These results provide evidence for localized production of IFN-gamma within aGVHD target organs and suggest that it is mediated by LPS-induced production of IL-12 by M phi. Our data elucidate the mechanism of activation of M phi during aGVHD that results in TNF-alpha and nitric oxide production and delineates the effector role of M phi in the pathology of aGVHD.
Differential effects of a monoclonal antibody to cis-urocanic acid on the suppression of delayed and contact hypersensitivity following ultraviolet irradiation.
Authors: Moodycliffe A M , Bucana C D , Kripke M L , Norval M , Ullrich S E
Published: December 2022

Urocanic acid (UCA) occurs naturally in the stratum corneum of the skin as the trans-isomer and, upon exposure to UVB radiation, converts to cis-UCA. It has been proposed that trans-UCA is the photoreceptor for and, following its isomerization to cis-UCA, a mediator of the suppressive effects of UVB irradiation on systemic T cell-mediated immune responses, such as contact hypersensitivity (CH) and delayed-type hypersensitivity (DTH). To address this question directly, we studied the consequence of deleting the in vivo function of cis-UCA on systemic suppression of CH and DTH, by injecting mice with a anti-cis-UCA mAb several hours before exposure to UVB radiation. We found that while DTH responses were completely restored, the anti-cis-UCA Ab had no effect on UV-induced immunosuppression of the CH response, even though suppressor cell formation was inhibited in both cases. Further, the kinetics of IL-10 expression in the skin of irradiated mice injected with the anti-cis-UCA mAb was altered and the diminished APC function of spleen-adherent cells from UVB-irradiated mice was totally reversed by the Ab. These findings suggest that cis-UCA acts as a mediator for some but not all of the systemic suppressive effects of UVB irradiation. They also suggest that cis-UCA may act indirectly via IL-10 to modulate immune function.
Type 1 CD8+ T cell responses during infection with the helminth Schistosoma mansoni.
Authors: Pedras-Vasconcelos J A , Pearce E J
Published: December 2022

IL-4 promotes the development of type 2 CD8+ T cells. In mice infected with the helminth Schistosoma mansoni, the Th response is overtly Th2-like, creating an environment rich in IL-4. Consequently, we examined whether CD8+ subset development in schistosome-infected mice is biased in a type 2 direction; this is of interest because CD8+ cells have been proposed to play an immunoregulatory role during schistosomiasis. Contrary to expectation, our data indicate that the CD8+ cell response in infected mice is strongly type 1-like. Thus, infection with S. mansoni leads to the development of concurrent Th2 and type 1 CD8+ cell responses. Cytokine production by type 1 CD8+ cells is dependent upon help from CD4+ cells; this helper activity can be substituted by exogenous IL-2 or IL-4. Since Th cells from infected mice make little IL-2 but large amounts of IL-4, we propose that IL-4 is likely to be the physiologic mediator of help in infected animals, a view supported by the ability of mAbs against IL-4R and IL-4 to reduce IFN-gamma production by splenocytes in vitro. CD8+ cells from infected mice are able to produce IFN-gamma in response to schistosome Ag presented by bone marrow-derived APC. A regulatory role for the CD8+ cells is implied by the observation that CD8+ cell-depleted splenocytes from infected mice exhibit increased proliferative responses and IL-4 production in response to mAb anti-CD3. These findings suggest that in mice infected with schistosomes there exists a regulatory pathway in which type 1 CD8+ cells, under the control of IL-4, dampen immunopathologic type 2 responses.
NK cell trafficking and cytokine expression in splenic compartments after IFN induction and viral infection.
Authors: Salazar-Mather T P , Ishikawa R , Biron C A
Published: December 2022

Studies were undertaken to characterize mechanisms for NK cell cytokine delivery in vivo. Conditions of systemic IFN-beta expression elicited by polyinosinic-polycytidylic acid (poly(I:C)) treatment or IFN-alpha beta production during lymphocytic choriomeningitis virus or murine cytomegalovirus infections resulted in profound splenic histologic changes, with relocalization of nucleated cells from red to white pulp regions. Cell-trafficking experiments, with fluorescently labeled populations, showed that poly(I:C) induced T/B cell-dependent leukocyte migration into white pulp regions. Splenic leukocytes prepared from severe combined immunodeficient (SCID) and bone marrow cells prepared from SCID or normal C57BL/6 mice revealed a unique poly(I:C)-induced accumulation of non-T/non-B cells along splenic red and white pulp region borders characteristic of marginal zones. Lymphocytic choriomeningitis virus and murine cytomegalovirus infections also induced this trafficking pattern. Ab treatments of normal and SCID cell donor mice to eliminate specific cell subsets demonstrated that the unique migration of non-T/non-B cells was dependent upon cells with the NK phenotype, NK1.1+ AGM1+F4/80-. In situ hybridization showed that, at early times after infections, small proportions of cells along red and white pulp divisions expressed high levels of IFN-gamma mRNA, and that these cells were depleted by protocols eliminating NK cells. The results suggest an exciting model for induction of NK cell trafficking from bone marrow to secondary compartments. This migratory pattern may localize NK cells to receive additional activation signals and/or to precisely deliver NK cell-produced cytokines.
Reduced susceptibility to collagen-induced arthritis in mice deficient in intercellular adhesion molecule-1.
Authors: Bullard D C , Hurley L A , Lorenzo I , Sly L M , Beaudet A L , Staite N D
Published: December 2022

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the firm adhesion of leukocytes to venular endothelium and facilitates leukocyte extravasation from the vasculature into inflamed tissue. In addition, ICAM-1 is an important costimulatory molecule during Ag presentation to lymphocytes. Using mice deficient in ICAM-1, we have investigated the role of this molecule in the development of collagen-induced arthritis. After immunization with type II collagen, 71% of wild-type mice developed arthritis compared with 50% of ICAM-1 heterozygote mutants and 18% of ICAM-1 homozygous mutants. In those ICAM-1 mutants that developed arthritis, the mean day of onset, the mean number of involved paws, and the severity of paw inflammation were not significantly different from those in wild-type mice. The reduced incidence of arthritis in the ICAM-1 homozygous mutant mice was not due to lack of immunity to type II collagen, since these mice developed similar levels of anti-type II collagen IgG compared with wild-type mice and had a positive delayed-type hypersensitivity reaction to type II collagen. The reduction of arthritis in heterozygous as well as homozygous deficient mice indicates that expression of ICAM-1 can be a pivotal variable in the pathogenesis of collagen-induced arthritis in mice. The results suggest that naturally occurring genetic variation in the expression of ICAM-1 or related inflammatory cell adhesion molecules might influence susceptibility to the complex disease of rheumatoid arthritis in humans and support the concept that pharmacologic approaches to chronic reduction in the expression or the function of ICAM-1 may be of therapeutic value.
Isolation and characterization of murine fra-1: induction mediated by CD40 and surface Ig is protein kinase C dependent.
Authors: Huo L , Rothstein T L
Published: December 2022

The murine fra-1 gene, encoding Fos-related Ag 1, was isolated from a splenic cDNA library and sequenced. Murine fra-1 was highly homologous to rat and human fra-1. Oligonucleotide primers based on the murine sequence were used to construct a quantitative reverse transcription-PCR assay for gene expression. B lymphocyte stimulation via both CD40 and surface Ig (sIg) receptors substantially induced fra-1 expression, and for both receptors, induction was protein kinase C (PKC) dependent. This contrasts with induction of c-fos by both CD40 and sIg, which is PKC independent and indicates that CD40 is capable of signaling through PKC or a closely related kinase. Induction of fra-1 following engagement of CD40 did not require protein synthesis, suggesting that the PKC-dependent linkage between CD40 and fra-1 is direct. CD40-mediated fra-1 induction did require tyrosine kinase activity. These results demonstrate that CD40, like sIg, may employ PKC in producing select outcomes, that individual B cell receptors may signal downstream events via both PKC-dependent and PKC-independent pathways, and that multiple signal transduction pathways may be used to activate the expression of closely related genes.