bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential modulation of astrocyte cytokine gene expression by TGF-beta.
Authors: Benveniste E N , Kwon J , Chung W J , Sampson J , Pandya K , Tang L P
Published: January 2023

In this study, we demonstrate that TGF-beta inhibits TNF-alpha expression, and induces/enhances IL-6 expression by primary rat astrocytes. Treatment of astrocytes with TGF-beta alone had no effect on TNF-alpha mRNA or protein expression; however, TGF-beta suppressed induction of TNF-alpha expression by three different stimuli (IFN-gamma/LPS, IFN-gamma/IL-1 beta, TNF-alpha) at both the protein and mRNA level. The extent of TGF-beta-mediated inhibition was greatest when astrocytes were pretreated with TGF-beta for 6 to 24 h, then exposed to the inducing stimuli. Inhibition of TNF-alpha mRNA steady-state levels by TGF-beta was a result of inhibition of TNF-alpha gene transcription, rather than degradation of the TNF-alpha message. In contrast, TGF-beta alone induced expression of IL-6 by astrocytes and synergized with two other cytokines, IL-1 beta and TNF-alpha, for enhanced IL-6 expression. TGF-beta-induced/enhanced IL-6 expression was mediated by transcriptional activation of the IL-6 gene. These results indicate that TGF-beta is an important regulator of cytokine production by astrocytes under inflammatory conditions in the brain.
Novel cell-surface Ag expressed on rat osteoclasts regulating the function of the calcitonin receptor.
Authors: Kukita T , Kukita A , Nagata K , Maeda H , Kurisu K , Watanabe T , Iijima T
Published: January 2023

Osteoclasts are known to be hematopoietic in origin. However, the detailed mechanisms of their differentiation and activation are not known. Cell-surface molecules preferentially expressed on cells of the osteoclast lineage may play some important roles in these processes. We prepared a mAb that recognizes a unique cell-surface membrane protein specifically expressed on rat osteoclasts. Expression of this Ag, designated as Kat1 Ag, was markedly stimulated by a factor secreted by the osteoblastic cell line ROS 17/2.8. Binding studies of 125I-labeled calcitonin (CT) showed that the Ag was not the CT receptor (CTR). However, interestingly, studies of the biologic activity of this mAb that recognizes Kat1-antigen (mAb Kat1) revealed possible regulatory functions of this Ag in osteoclasts. Firstly, mAb Kat1 significantly elevated the binding affinity of the CTR expressed on osteoclast-like cells without altering the number of receptors. Secondly, CT-sensitivity of the osteoclast progenitor cells in the system of osteoclast differentiation showed marked augmentation on treatment of these cells with this mAb. Even a very low concentration of CT (0.1 ng/ml) significantly inhibited osteoclast differentiation in the presence of mAb Kat1, whereas a higher concentration of CT (10 ng/ml) was required to inhibit their differentiation in the absence of this mAb. Thirdly, mAb Kat1 inhibited dentin-resorbing activity of osteoclast-like cells. Furthermore, the inhibitory effects of CT on osteoclast-mediated dentin resorption was augmented by the presence of this mAb. These observations strongly suggest that Kat1-antigen is a unique cell-surface protein regulating the affinity of the CTR expressed on osteoclasts and also the bone-resorbing function of these cells.
Inhibitors of serotonin synthesis and antagonists of serotonin 1A receptors inhibit T lymphocyte function in vitro and cell-mediated immunity in vivo.
Authors: Aune T M , Golden H W , McGrath K M
Published: January 2023

The serotonin (5HT1A) receptor subtype is one member of the 5HT1 receptor family and is constitutively expressed on Jurkat cells and is elevated on human T lymphocytes after mitogenic activation. Published reports show that human T lymphocytes and monocytes also release 5HT after stimulation with PHA or IFN-gamma. In lymphocytes and the central nervous system, the 5HT1A receptor is coupled to regulation of adenylate cyclase. The 5HT1A receptor agonists inhibit activation of adenylate cyclase. The purpose of the experiments reported here was to investigate further the role 5HT and the 5HT1A receptor may play in the regulation of human and murine T cell activity. For this purpose, human PBMC or murine spleen cells were used for experimental purposes rather than Jurkat cells. The results show that inhibition of 5HT synthesis inhibits IL-2-stimulated human T cell proliferation and that addition of 5-hydroxytryptophan, a precursor of 5HT, reverses inhibition of T cell proliferation. The 5HT1 receptor antagonist, metitepine, and the 5HT1A selective antagonist, pindobind-5HT1A, also block T cell proliferation. Inhibition by metitepine is reversed by 5HT and by the selective 5HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) (8-OH-DPAT). Selective 5HT1A receptor antagonists cause elevation of cAMP in human T cells. In a murine model, selective 5HT1A receptor antagonists inhibit contact sensitivity responses but not Ab responses to oxazalone in vivo. Inhibition is reversed by 8-OH-DPAT. In addition, production of Th1 cytokines, such as IL-2 and IFN-gamma, by Ag-stimulated, immune murine spleen cells is inhibited by 5HT1A receptor antagonists in vitro but not by 5HT1C/2 receptor antagonists.
Human T cell lymphotropic virus type I-induced T cell activation. Resistance to TGF-beta 1-induced suppression.
Authors: Höllsberg P , Ausubel L J , Hafler D A
Published: January 2023

T cell proliferation is potently suppressed by TGF-beta during the G1 phase of the cell cycle. The mechanism appears to involve inhibition of cell cycle kinases that phosphorylate the retinoblastoma protein (pRb), a key regulator of cell cycle progression from G1 to S phase. Although productive infection with the human T cell lymphotropic virus type I (HTLV-I) induces T cell activation, it also, paradoxically, leads to increased production of TGF-beta. To investigate whether infection by HTLV-I conferred resistance to TGF-beta in non-immortalized T cells, we generated T cell clones from patients with HTLV-I myelopathy by direct single cell cloning. Here we report that HTLV-I-infected but not uninfected T cell clones have hyperphosphorylated pRb consistent with viral-induced T cell activation. Furthermore, the HTLV-I-infected T cells were resistant to growth suppression by rTGF-beta 1 and this correlated with the inability of TGF-beta 1 to prevent hyperphosphorylation of pRb. However, when spontaneously proliferating HTLV-I-infected T cell clones were further stimulated by cross-linking of the CD3/TCR complex, the superimposed proliferation was significantly inhibited by TGF-beta 1, suggesting that the TGF-beta 1 signaling pathway was intact. Together these findings suggest that HTLV-I induces T cell activation through a pathway that is insensitive to TGF-beta 1. This may have implications for the altered immune regulation in patients with HTLV-I myelopathy.
NF-kappa B regulates IL-1 beta transcription through a consensus NF-kappa B binding site and a nonconsensus CRE-like site.
Authors: Cogswell J P , Godlevski M M , Wisely G B , Clay W C , Leesnitzer L M , Ways J P , Gray J G
Published: January 2023

In these studies, we show that NF-kappa B induces transcription from the human pro-IL-1 beta (IL-1 beta) gene. A recombinant plasmid pIL-1(-4000)-CAT, containing 4 kb of the IL-1 beta gene upstream regulatory sequence was transactivated by the p65 subunit of NF-kappa B or by treatment of the cells with a combination of NF-kappa B inducers including LPS, PMA, and dibutyryl cyclic AMP (L+P+C) in U937 cells. Coexpression of p65 with L+P+C treatment led to a synergistic response, whereas coexpression of the I kappa B alpha/MAD-3 protein, in place of p65, blocked L+P+C induction. A series of 5' deletion mutants of the IL-1 beta promoter were used to define two p65 response regions: region I located between -2800 to -2720 bp and region II located between -512 and -133 bp. Electrophoretic mobility shift assays confirmed that NF-kappa B-like proteins could bind to two consensus binding sites in region II. A site-specific mutation in only one of these NF-kappa B sites (-296/-286 bp) caused a specific loss of induction by p65 or L+P+C. A cyclic AMP response element (CRE) site (-2761/-2753 bp) in region I has been shown previously to be critical for L+P+C induction. Mutation of the CRE in an enhancerless test plasmid containing two copies of region I blocked transactivation by p65. Likewise, coexpression of I kappa B alpha inhibited CRE-dependent L+P+C induction of the wild-type counterpart. These data show that NF-kappa B regulates a nonconsensus CRE site in addition to the consensus binding site at -296/-286 bp and suggest that NF-kappa B may play multiple roles in the induction of IL-1 beta transcription.
Identification of a structural epitope by using a peptide library displayed on filamentous bacteriophage.
Authors: Hoess R H , Mack A J , Walton H , Reilly T M
Published: January 2023

The screening of phage-displayed random peptide libraries has recently emerged as a powerful technique for probing Ab-Ag interactions. We have used this method to identify the epitope recognized by a mAb, CB5B10, raised against plasminogen activator inhibitor type-1 (PAI-1). Two phage libraries, displaying random hexapeptides with or without flanking cysteine residues, were screened for binding to mAb CB5B10. The selected phages were shown to contain similar peptide sequences, all of which were flanked by cysteines. When compared with the crystal structure of PAI-1, the selected peptides closely resemble the sequence of a solvent-exposed loop connecting the COOH-terminal of an alpha-helix at Phe114 to a beta-sheet at Ser119. Because of the constraints imposed by the flanking cysteine residues, the selected peptides appear to mimic the structure and the sequence of the PAI-1 epitope. Specific contacts between the amino acids displayed by the phage and the mAb were explored using site-directed mutants of the phage peptide. The effects of these substitutions on binding to the mAb correlated well with the accessibility of the corresponding residues in the PAI-1 epitope. This is the first example of the use of phage-displayed peptide libraries to identify a structural epitope.
The murine Ig 3' alpha enhancer is a target site with repressor function for the B cell lineage-specific transcription factor BSAP (NF-HB, S alpha-BP).
Authors: Neurath M F , Strober W , Wakatsuki Y
Published: January 2023

Using electrophoretic mobility shift assays (EMSAs) we have identified several target sites for nuclear proteins in the murine heavy chain Ig 3' alpha enhancer. Two of these sites, denoted oligo-H and oligo-K, were shown by several criteria, including cell distribution and stimulation experiments, EMSA cross-competition studies, and proteolytic clipping bandshift assays, to bind to the same protein identical to the transcription factor B cell lineage-specific activator protein (BSAP) (NF-HB, S alpha-BP). To assess the possible functional role of these BSAP binding sites in the 3' alpha enhancer, we transiently transfected a construct containing a 314-bp 3' alpha enhancer fragment upstream of a luciferase reporter gene in MOPC-315 cells, a plasmacytoma line lacking BSAP. In these cells, co-transfection with a vector expressing recombinant BSAP led to significant reduction in the activity of the 3' alpha enhancer fragment. Conversely, in the mature B lymphoma cell line CH12.LX, a cell line that expresses BSAP and has a less active 3' alpha enhancer, selective BSAP down-regulation by an antisense phosphorothioate oligonucleotide was sufficient to considerably up-regulate 3' alpha enhancer activity, as were mutations of both binding sites that prevented binding of BSAP to the 3' alpha enhancer. Our findings thus suggest that the natural loss of BSAP expression in terminally differentiated plasma cells contributes to the activation of the murine Ig 3' alpha enhancer.
Molecular cloning, characterization, and tissue distribution of rat lipopolysaccharide binding protein. Evidence for extrahepatic expression.
Authors: Su G L , Freeswick P D , Geller D A , Wang Q , Shapiro R A , Wan Y H , Billiar T R , Tweardy D J , Simmons R L , Wang S C
Published: January 2023

LPS binding protein (LBP) is a glycoprotein present in normal serum that becomes markedly elevated during acute phase responses. LBP has been reported to greatly potentiate host responses to endotoxin or LPS. Therefore, LBP may play a critical role in the body's response to injury and infection. Little is known about the factors regulating production of LBP. To investigate the regulation of LBP expression, we have cloned the full-length cDNA for rat LBP. The deduced amino acid sequence of rat LBP was highly homologous with that reported for rabbit and human LBP. The sequence of rat LBP further refines the conserved regions found within the family of proteins that bind LPS; this family is comprised of bactericidal permeability-increasing protein and LBP from multiple species. Use of the rat LBP cDNA clone for Northern blot analysis reveals that LBP mRNA levels are markedly up-regulated in liver during acute phase responses. However, in contrast to previous reports, we also find evidence of extrahepatic expression of LBP under these induced conditions. The presence of LBP mRNA in activated tissues other than liver suggests that LBP may play a larger role in local tissue responses to LPS than previously appreciated.
Identification of an N-terminally acetylated encephalitogenic epitope in myelin proteolipid apoprotein for the Lewis rat.
Authors: Zhao W , Wegmann K W , Trotter J L , Ueno K , Hickey W F
Published: January 2023

Proteolipid apoprotein (PLP) is a major component of the central nervous system myelin. As such, it is capable of inducing experimental allergic encephalomyelitis (EAE) in many subhuman species. On the basis of a putative MHC class II binding motif in Lewis rats (RT-1B1) recently identified in our laboratory, the present study identifies one pathogenic T cell epitope of PLP for the Lewis rat, located in the area between amino acid residues 217 and 240. Four overlapping synthetic peptides derived from this region were tested for their antigenicity and encephalitogenicity. Although the longer peptides could not induce EAE in the Lewis rats in their "theoretically" native form after immunization, they were endowed with encephalitogenic ability when modified by N-terminal acetylation. All animals immunized with N-acetylated peptides PLP 217-233 and PLP 224-240 developed inflammation in the lower spinal cord, but with very low incidence of clinical EAE (1 of 12). In contrast, none of the animals immunized with nonacetylated peptides developed either clinical or histologic EAE. Mild inflammation of the spinal cord was also found in two of four rats immunized with N-acetylated peptide PLP 220-234. The animals immunized with the decapeptide, N-acetylated PLP 224-233, did not develop inflammation of the spinal cord. Despite the low incidence of clinical disease, it was possible to generate vigorous T cell lines against all the peptides synthesized from this region of PLP.(ABSTRACT TRUNCATED AT 250 WORDS)
Dendritic epidermal T cells inhibit T cell proliferation and may induce tolerance by cytotoxicity.
Authors: Love-Schimenti C D , Kripke M L
Published: January 2023

Hapten-conjugated cells of the dendritic epidermal T cell (DETC) line AU16 induce specific immunologic tolerance in vivo and inhibit the proliferation of native T cells in response to hapten-bearing dendritic cells in vitro. The purpose of this study was to test the hypothesis that these activities of DETC are mediated by a cytotoxic mechanism. In addition, because AU16 cells, unlike most DETC, express a TCR-alpha beta, we compared their activity in the in vivo and in vitro assays with a TCR(+)-gamma delta DETC line (T245) and an TCR(+)-alpha beta, FITC-specific T cell line (T4/28). Hapten-conjugated AU16 and T245 cells, but not T4/28 cells, induced tolerance to FITC in vivo. In addition, the proliferative response of naive T lymphocytes to gamma-irradiated, hapten-bearing APCs was inhibited by the addition of gamma-irradiated, FITC-conjugated DETC to the cultures on day 0. To determine whether the T cell or the APC was the target, AU16 cells were added to the cultures on day 4, when few APCs remained. FITC-conjugated AU16 and T245 cells profoundly inhibited [3H]thymidine incorporation by the T cells, indicating that the T cell was the target of the DETC. Furthermore, AU16 cells reduced the number of T cells in the in vitro proliferation assay, suggesting that inhibition of [3H]thymidine incorporation was a result of the cytotoxic activity of DETC. We speculate that cytotoxicity may also account for the ability of DETC to induce tolerance in vivo.
Functional role and signal-induced modulation of proteins recognizing the conserved TCATTT-containing promoter elements in the murine IL-5 and GM-CSF genes in T lymphocytes.
Authors: Naora H , van Leeuwen B H , Bourke P F , Young I G
Published: January 2023

The TCATTT-containing element extending from -61 to -41 of the mouse IL-5 gene is highly conserved in the corresponding region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and has been previously shown to be involved in regulating inducible GM-CSF gene expression. By using stable transfection assays in the mouse Th2 clone D10.G4.1, we show that the TCATTT-containing element is also involved in the regulation of inducible IL-5 gene expression. The mouse IL-5 and GM-CSF homologues of this element were found by gel shift analysis to form DNA-nuclear protein complexes of similar electrophoretic mobility under conditions in which expression of these genes is induced. However, comparative studies using extracts of D10.G4.1 cells treated with the cellular activators Con A and PMA and the inhibitors cycloheximide and cyclosporin A indicated that the binding activities to the conserved elements in the IL-5 and GM-CSF genes (designated NF-IL-5A and NF-GM-CSFA, respectively) are regulated by different signaling pathways. In addition, NF-IL-5A is not induced in the Th1 clone HDK-1 which does not express the IL-5 gene. The strong correlation between the signal-dependent and cell-specific modulation of IL-5 and GM-CSF gene expression patterns and the binding activities of NF-IL-5A and NF-GM-CSFA suggests that these nuclear proteins are involved in the transduction of T cell activation signals to the transcriptional machinery of these genes through their interactions with their respective TCATTT-containing elements.
Beta 2-microglobulin induces a conformational change in an MHC class I H chain that occurs intracellularly and is maintained at the cell surface.
Authors: Danliczyk U G , Delovitch T L
Published: January 2023

beta 2-microglobulin (beta 2-m) can regulate the cell surface conformation and expression of a class I MHC H chain. To further determine how the intracellular association of beta 2-m with a class I H chain influences the subsequent cell surface conformation and expression of the H chain, we examined the expression of HLA-A3 H chains in transgenic mice. We found that the overall conformation and level of expression of a transgenic HLA-A3 H chain is determined by its previous intracellular association with beta 2-m during its maturation in the endoplasmic reticulum, and that this conformation is not altered appreciably after the H chain reaches the cell surface. This conclusion is based on two observations. First, the conformation-dependent W6/32 mAb reacts with an HLA-A3 H chain bound to human beta 2-m (h beta 2-m) on the surface of lymphocytes from (A3 x h beta 2-m)F1 double transgenic mice in which the two chains associate intracellularly after synthesis and are then co-transported to the surface membrane. Second, this W6/32 mAb does not react with an HLA-A3 H chain/h beta 2-m surface complex formed by the exchange of H chain bound mouse beta 2-m for exogenously added h beta 2-m on lymphocytes from A3 single transgenic mice in which the HLA-A3 H chain is synthesized and transported to the cell surface in association with mouse beta 2-m.
IL-12 regulates T helper type 1 cytokine responses in human infectious disease.
Authors: Sieling P A , Wang X H , Gately M K , Oliveros J L , McHugh T , Barnes P F , Wolf S F , Golkar L , Yamamura M , Yogi Y
Published: January 2023

We investigated the role of IL-12 in regulating T cell and cytokine responses in human infectious disease by using the spectrum of leprosy as a model. Tuberculoid patients mount strong T cell responses to Mycobacterium leprae, with production of the type 1 cytokines IL-2 and IFN-gamma in lesions; whereas lepromatous patients manifest weak T cell responses to M. leprae, with production of the type 2 cytokines IL-4 and IL-10 in lesions. We found expression of IL-12 p40 mRNA, as measured by PCR amplification, and IL-12 p70, as measured by immunohistochemistry, to be 10-fold greater in tuberculoid lesions than in lepromatous lesions. The ability of M. leprae to stimulate release of IL-12 from monocytes was inhibited by rIL-4 and rIL-10. M. leprae-induced T cell proliferation in tuberculoid patients was blocked by the addition of neutralizing Abs to IL-12. Furthermore, rIL-12 stimulated proliferation of CD4+ type 1 T cell clones from tuberculoid lesions, but not CD8+ type 2 T cell clones from lepromatous lesions; however, both responded to rIL-2, rIL-12 augmented M. leprae-specific T cell proliferation in lepromatous patients, thereby causing the selective expansion of CD4+ T cells and increasing T cell IFN-gamma production. These data indicate that IL-12 is an important mediator in the generation of the type 1 cytokine response in human infectious disease.
Chimeric zeta-receptors direct human natural killer (NK) effector function to permit killing of NK-resistant tumor cells and HIV-infected T lymphocytes.
Authors: Tran A C , Zhang D , Byrn R , Roberts M R
Published: January 2023

Chimeric receptors in which a signaling component of the TCR complex such as zeta is fused directly to the ligand binding domain of a heterologous receptor or Ab have been shown to redirect the specific effector activity of T lymphocytes. We previously described the ability of two classes of such chimeric zeta-receptors bearing extracellular domains derived from either the HIV receptor CD4 (CD4 zeta) or an HIV-specific single chain Ab to redirect primary human CD8+ T cells to kill HIV-infected T cells. In this report we demonstrate that human NK cells can be genetically modified to express high levels of CD4 zeta using retroviral transduction. The CD4 zeta chimeric receptor is biochemically active, as cross-linking of CD4 zeta on NK cells results in tyrosine phosphorylation of CD4 zeta and multiple cellular proteins. More importantly, the CD4 zeta chimeric receptor is functionally active and can direct NK cells to specifically and efficiently lyse either NK-resistant tumor cells expressing the relevant ligand, gp120, or CD4+ T cells infected with HIV. These results show that human NK cells can be readily activated via zeta-based chimeric receptors to target both tumor and virally infected cells, and suggest a novel approach to the treatment of disease.
Requirement for natural killer cells in the induction of cytotoxic T cells.
Authors: Kos F J , Engleman E G
Published: January 2023

Cell-mediated immunity involves the participation of both regulatory and cytotoxic cells. The conversion of precursors to effector CD8+ cytotoxic T (Tc) cells requires cell-cell collaboration in which CD4+ T cells are traditionally viewed as helper cells. An in vitro system was used here to demonstrate that the generation of human alloantigen-specific CD8+ Tc cells requires the participation of CD3-CD16+CD56+ NK cells but not CD4+ T helper cells. Depletion of NK cells from responders abolished the induction of alloantigen-specific Tc cells in mixed lymphocyte cultures (MLC). Purified CD5+CD8+ T cells stimulated with alloantigen proliferated but did not differentiate into fully functional effector Tc cells. Coculture of responder CD5+CD8+ T cells with NK cells promoted the conversion of CD8+ Tc cell precursors (pTc) into effector Tc cells. Anti-CD56 mAbs blocked Tc cell induction in MLC, suggesting a role for CD56 molecules expressed on NK cells in either alloantigen recognition or delivery of accessory signals to pTc cells. These findings suggest a novel critical link between the natural and specific immune responses.
Temporal and spatial expression of HLA-G messenger RNA in extraembryonic tissues of transgenic mice.
Authors: Schmidt C M , Chen H L , Chiu I , Ehlenfeldt R G , Hunt J S , Orr H T
Published: January 2023

HLA-G, a nonclassical class I molecule, is expressed by trophoblasts, the only fetal cells in direct contact with maternal tissue. Results of previous experiments suggested that a 244-bp region located over 1 kb 5' from exon 1 is critical for extraembryonic expression of HLA-G in transgenic mice. We report here the production of HLA-G transgenic lines with a 6.0-kb HLA-G transgene that includes the 244-bp region. These lines exhibit copy number-dependent and developmentally appropriate transgene expression. One HLA-G transgenic line, G.3.2, exhibits extraembryonic HLA-G mRNA expression levels similar to those seen in human extraembryonic tissues. Studies of the cell-type-specific localization of HLA-G mRNA in placentas from the G.3.2 HLA-G transgenic mouse line show predominant localization of the HLA-G message in the spongiotrophoblast layer. This layer is in a similar anatomic location to the HLA-G-expressing human cytotrophoblast shell. The G.3.2 HLA-G transgenic mouse line should serve as an appropriate model for the study of HLA-G function at the maternal-fetal interface.
Immunoreceptor tyrosine-based activation motif is required to signal pathways of receptor-mediated growth arrest and apoptosis in murine B lymphoma cells.
Authors: Yao X R , Flaswinkel H , Reth M , Scott D W
Published: January 2023

The B cell Ag receptor is a multimeric protein complex consisting of the ligand binding mlg and the Ig alpha/lg beta heterodimer. The cytoplasmic tails of Ig alpha and Ig beta both contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif is believed to play a critical role in the receptor-mediated signal transduction. To explore the role of ITAM in signaling for B cell death (apoptosis), we transfected CH31 cells, an immature B lymphoma cell line, with expression vectors encoding for the CD8 extracellular/transmembrane domains and the cytoplasmic signal-transducing domain (ITAM) of Ig alpha or Ig beta, respectively. Here, we demonstrate that cross-linking of CD8:Ig alpha or CD:Ig beta with anti-CD8 mAb effectively induced cell growth arrest and apoptosis characterized by [3H]thymidine release and DNA fragmentation; in contrast, CD8:gamma 2a or truncated CD8:Ig alpha lacking the ITAM could not do so. Moreover, selective point mutation of either of the two conserved tyrosine residues within the ITAM, but not the nonconserved tyrosine, completely abrogated the ability of this motif to mediate cell death signals. These findings clearly indicate that ITAM is a critical component required for transmitting growth arrest and apoptotic signals, and that these functions of ITAM are positively regulated by tyrosine phosphorylation.
Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumonia.
Authors: Greenberger M J , Strieter R M , Kunkel S L , Danforth J M , Goodman R E , Standiford T J
Published: January 2023

Effective host defense against bacterial infection is dependent upon the vigorous recruitment and activation of neutrophils and macrophages. We hypothesized that IL-10 is produced in the setting of bacterial pneumonia, and this cytokine may attenuate host defense by inhibiting the expression of important activating and chemotactic cytokines. CD-1 mice were challenged with either 30 microliters of saline or saline containing 10(3) CFUs of Klebsiella pneumoniae intratracheally (i.t.) and lungs were harvested at 8, 24, and 48 h. The i.t. inoculation with K. pneumoniae resulted in a 13-, 14-, and 8-fold increase in lung homogenate TNF, macrophage inflammatory protein-2 (MIP-2), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) levels, respectively, as compared with control animals. In addition, we observed an increase in IL-10 mRNA and protein levels in lung homogenates, maximal at 48 h postinoculation. To establish the biologic relevance of IL-10 in Klebsiella pneumonia, we passively immunized CD-1 mice with 0.5 ml of rabbit anti-murine IL-10 serum or preimmune serum i.p. 2 h before i.t. administration of K. pneumoniae. Treatment of animals with anti-IL-10 serum resulted in increased levels of TNF, MIP-2, and MIP-1 alpha, respectively, within lung homogenates at 24 and 48 h, as compared with preimmune-treated animals. Furthermore, neutralization of IL-10 resulted in a significant decrease in K. pneumoniae CFU in both lung homogenates and plasma harvested at 48 h, as well as a significant increase in survival in these animals. Our studies indicate that 1) IL-10 is produced during Klebsiella pneumonia; and 2) inhibition of IL-10 bioactivity in vivo results in enhanced bacterial clearance, increased expression of proinflammatory cytokines, and prolonged survival.
Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice.
Authors: Heinzel F P , Rerko R M , Ahmed F , Pearlman E
Published: January 2023

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.
Staphylococcal enterotoxin B-activated T cells can be redirected to inhibit multicycle virus replication.
Authors: Moran T M , Toy E , Kuzu Y , Kuzu H , Isobe H , Schulman J L
Published: January 2023

Cell-mediated immunity is a crucial part of recovery from virus infections. Adoptive transfer of T cells into infected animals is restricted by the need for Ag-specific and MHC-restricted T cells. One way to overcome these limitations is to use bifunctional Abs to redirect the T cells against virus-infected cells. We have demonstrated that bifunctional Abs can inhibit virus replication in the presence of activated T cells. To generate a large number of activated T cells in a short time, we tested the ability of the superantigen, staphylococcal enterotoxin B (SEB), to activate T cells. We demonstrate that SEB-activated T cells are effective killers when bridged to Fc receptor-bearing target cells using anti-CD3 Abs. SEB T cells can lyse virus-infected target cells in the presence of HHA6, a bifunctional Ab specific for the V beta 8 TCR product and the H1 hemagglutinin of influenza A/PR/8/34 virus. In addition, bifunctional Ab and SEB T cells can inhibit multicycle virus replication in vitro. In a conventional 4-h chromium release assay, SEB-activated CD8 T cells are efficient killers, whereas CD4 T cells are not. Yet both subpopulations have the ability to inhibit multicycle replication in vitro. Superantigens may represent a potent method for generation of effector cells for use in redirected immunotherapy protocols.
HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers.
Authors: Wizel B , Houghten R , Church P , Tine J A , Lanar D E , Gordon D M , Ballou W R , Sette A , Hoffman S L
Published: January 2023

CD8+ CTL specific for the Plasmodium yoelii sporozoite surface protein 2 (PySSP2) protect mice against malaria. For this reason, vaccines designed to induce CTL against P. falciparum SSP2 (PfSSP2) are under development. Optimal development of PfSSP2 as a component of human malaria vaccines requires characterization of HLA class I-restricted CTL against this Ag. For this purpose, PBMC from four HLA-A2+ human volunteers immunized with P. falciparum irradiated sporozoites were stimulated with a recombinant vaccinia virus expressing PfSSP2 and with 35 PfSSP2-derived 15-amino acid peptides containing sequences conforming to HLA-A2 binding motifs. Ag-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity against autologous target cells transfected with the PfSSP2 gene was demonstrated in the four volunteers. Twelve of the 35 peptides sensitized HLA-A2-matched target cells for lysis by peptide-stimulated effectors. Three volunteers had CTL against 9 of the 12 peptides, and one had no peptide-specific CTL. HLA-A*0201 restriction was confirmed by demonstrating that effectors from the three responders could be stimulated with six different peptides to lyse HLA-A*0201+ T2 cells incubated with the homologous peptides. Peptide-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity was also demonstrated against two peptides using unstimulated PBMC as effectors. Available data indicate that the motif-bearing sequences in 6 of the 12 positive peptides are conserved among P. falciparum isolates and clones. Demonstration of HLA-A2-restricted CTL responses to multiple PfSSP2-derived peptides, and of circulating activated CTL against PfSSP2 in immune volunteers provide important information for optimal design and evaluation of vaccines containing this pre-erythrocytic stage Ag.
Characterization of a hypoxia-responsive adhesion molecule for leukocytes on human endothelial cells.
Authors: Ginis I , Mentzer S J , Li X , Faller D V
Published: January 2023

Decreased oxygen tension is one important component of ischemic episodes, which are characterized by leukocyte accumulation at the site of infarction. We have reported recently that cultured endothelial and muscle RD cells transferred from pO2 of 120 torr to pO2 of 50 torr for 3 h dramatically increased their adhesiveness for lymphocytes and neutrophils. In the present study, we identify and characterize a new endothelial cell adhesion molecule that mediates hypoxia-induced adhesion to leukocytes. We have generated a mAb, HAL 1/13, that recognizes a cell surface epitope common to both RD and endothelial cells and inhibits lymphocyte and neutrophil adhesion to muscle and endothelial cell monolayers under normoxic conditions in a dose-dependent manner. The contribution of the HAL 1/13 ligand to leukocyte adhesion significantly increased (more than twofold) when the muscle or endothelial cells had been exposed previously to a hypoxic environment. HAL 1/13 mAb completely abolished the enhancing effect of hypoxia on RD and endothelial cell adhesiveness for leukocytes. HAL 1/13 mAb immunoprecipitated a protein with Mr approximately 85,000 Da from cell surface-iodinated muscle and endothelial cells. The Mr, CD cluster dendrogram analysis, and lack of induction by IL-1 of HAL 1/13 distinguish it from most other known endothelial cell adhesion molecules. We suggest that this hypoxia-sensitive adhesion receptor on mesenchymal cells participates in neutrophil adhesion and consequent diapedesis during hypoxia-associated pathologic conditions.
Inhibition of TNF-triggering activity of lipopolysaccharide by a proteinaceous factor from normal mouse liver extract.
Authors: Satoh M , Matsumura T , Nakamoto M , Yamazaki M
Published: January 2023

Recent studies have revealed that animals have evolved a wide range of protective systems against the deleterious effects of LPS, but the molecular mechanisms in the barrier functions of liver against enterobacterial LPS, particularly in mammals, are poorly understood. In this study, we extracted a soluble fraction from the liver of normal mice (normal liver extract, NLE) and examined its effect on biologic activities of LPS. Preincubation of NLE and LPS suppressed serum-dependent TNF induction (TNF-triggering activity) of LPS; the effect was dose-dependent and overcome by increasing LPS concentration, but treatment of macrophages with NLE showed no effect. Separation by ultrafiltration and protease sensitivity demonstrated that the factor(s) in NLE was a protein(s). We tentatively called this liver LPS-inactivating factor (LLIF). LPS inactivation by LLIF was temperature-dependent and required the coexistence of divalent cations. LLIF also suppressed a synthetic lipid A analogue, ONO-4007. Pretreatment of LPS with serum rendered LPS refractory to the action of LLIF. However, LLIF was unable to inhibit limulus amebocyte lysate activation by LPS. Direct interaction of LLIF and lipid A was evident by the method of [1-14C]ONO-4007 binding to solid-phase LLIF, but treatment of [1-14C]ONO-4007 with LLIF generated no degradative products. These results suggest that LLIF probably interacts with the lipid A portion of LPS and interferes with the association of LPS and LBP (LPS-binding protein) in serum, and that LLIF may be one of the protective molecules in liver against the gastrointestine-derived LPS.
Exogenous nitric oxide regulates IFN-gamma plus lipopolysaccharide-induced nitric oxide synthase expression in mouse macrophages.
Authors: Sheffler L A , Wink D A , Melillo G , Cox G W
Published: January 2023

This study was performed to determine the effects of nitric oxide (NO) on the expression of inducible NO synthase (iNOS) in mouse macrophages. We used the NO donor diethylamine dinitric oxide (DEA/NO) and the mouse macrophage cell line ANA-1 in these experiments. ANA-1 macrophages did not express iNOS mRNA either constitutively or following exposure to 100 U/ml IFN-gamma alone, to 10 ng/ml LPS alone, or to 200 microM DEA/NO alone. Similarly, ANA-1 macrophages did not express detectable levels of iNOS mRNA following treatment with 100 U/ml IFN-gamma plus 200 microM DEA/NO. However, IFN-gamma (100 U/ml) plus LPS (10 ng/ml) induced high levels of iNOS mRNA in ANA-1 macrophages after 6 h. Low concentrations of DEA/NO (approximately 1 to 12 microM) caused up to a 2.5-fold augmentation of IFN-gamma plus LPS-induced iNOS mRNA expression. In contrast, 200 microM DEA/NO suppressed IFN-gamma plus LPS-induced iNOS mRNA expression (60% decrease). The effects of DEA/NO were gene-specific because DEA/NO did not affect the IFN-gamma plus LPS-induced expression of TNF-alpha mRNA. Moreover, the biphasic effects of DEA/NO were specifically due to released NO. Diethylamine and nitrite were unable to regulate IFN-gamma plus LPS-induced gene expression in ANA-1 macrophages. Time-response experiments suggested that the effects of NO were short-lived and occurred early during the induction of iNOS gene expression. The effects of NO were not limited to iNOS mRNA expression but were apparent at the level of iNOS protein expression and enzymatic activity. Overall, these results suggest that NO has immunoregulatory effects and may control the extent and duration of cytokine- and/or endotoxin-induced iNOS expression in macrophages.
Transcriptional activation of the human TNF-alpha promoter by superantigen in human monocytic cells: role of NF-kappa B.
Authors: Trede N S , Tsytsykova A V , Chatila T , Goldfeld A E , Geha R S
Published: January 2023

We have studied the transcriptional activation of the human TNF-alpha gene by the superantigen staphylococcal enterotoxin A (SEA) in the human premonocytic cell line THP-1. Nuclear proteins from SEA-stimulated THP-1 cells bound strongly to kappa 3, the most proximal of three putative NF-kappa B binding sites (kappa 1-kappa 3) found in the 5' regulatory region of the TNF-alpha gene, but only weakly to kappa 1, the most distal of the NF-kappa B binding sites, and showed no binding to kappa 2. The mobility of the kappa 3-nucleoprotein complex was identical to that of complexes formed between nuclear proteins and the consensus NF-kappa B seuqence. Moreover, both 5' and 3' mutants of kappa 3 were unable to displace kappa 3 binding, suggesting that the kappa 3 binding complex induced by SEA has the characteristics of NF-kappa B. Studies using Abs directed against the NF-kappa B subunits p50 and p65 suggested that both p50 and p65 bind to the kappa 3 sequence. Reporter gene assays showed that deletion of kappa 3 (-99 to -89 bp) and point mutation of the three 5' guanine bases in the kappa 3 sequence reduced the inducibility of the TNF-alpha promoter by SEA and LPS. These results indicate that superantigen induces NF-kappa B in human monocytic cells and suggest that binding of NF-kappa B to the kappa 3 site of the TNF-alpha promoter plays an important role in the transcriptional activation of the TNF-alpha gene by superantigen.
Engineered anti-CD38 monoclonal antibodies for immunotherapy of multiple myeloma.
Authors: Ellis J H , Barber K A , Tutt A , Hale C , Lewis A P , Glennie M J , Stevenson G T , Crowe J S
Published: January 2023

Multiple myeloma is a malignancy of plasma cells for which there is no effective treatment. To develop an immunotherapeutic agent, we have raised a high affinity mAb (AT13/5) against CD38, one of the few well-characterized surface Ags present on myeloma cells. Since murine monoclonals have many disadvantages as human therapeutics, we prepared two engineered forms of the Ab: a CDR-grafted humanized IgG1 and a chimeric FabFc2 (mouse Fab cross-linked to two human gamma 1 Fc). To retain affinity in the humanized Ab, a number of changes were required to the human framework regions of the heavy chain. In particular, through systematic mutagenesis and computer modeling, we identified a critical interaction between the side chains of residues 29 and 78, which may be important for the humanization of other Abs. The properties of the humanized IgG1 and FabFc2 constructs were compared in a series of in vitro tests. Both constructs efficiently directed Ab-dependent cellular cytotoxicity against CD38-positive cell lines, but C was activated only poorly. Neither construct caused down-modulation of CD38, nor did they affect the NADase activity of CD38. Despite their differing structures, both Abs showed similar activity in most assays, although the humanized IgG1 was more potent at inducing monocyte cytotoxicity. These data represent the first direct comparison of CDR-grafted and chimeric FabFc2 forms of the same Ab, and offer no support for the perceived advantages of the FabFc2. These Abs show promise for therapy of multiple myeloma and other diseases involving CD38-positive cells.
Immunization of a rabbit with beta 2-glycoprotein I induces charge-dependent crossreactive antibodies that bind anionic phospholipids and have similar reactivity as autoimmune anti-phospholipid antibodies.
Authors: Kouts S , Wang M X , Adelstein S , Krilis S A
Published: January 2023

A rabbit immunized with beta 2-glycoprotein I (beta 2-GPI) produced Abs that bind to negatively charged phospholipids and to beta 2-GPI. After affinity purification of the Abs to beta 2-GPI, the dual reactivity could still be detected. Adsorption studies with a phosphatidylserine affinity column depleted phospholipid-reactive Abs, but beta 2-GPI reactivity was retained. The same pattern of reactivity was found with culture supernatants from rabbit anti-beta 2-GPI splenocytes fused with an immortalized rabbit cell line. The reactivity to negatively charged phospholipids is likely to involve ionic interactions, as high ionic strength buffers eliminated binding to anionic phospholipids, but not to beta 2-GPI. Affinity-purified anti-phospholipid (aPL) Abs from four of seven autoimmune patients bound anionic phospholipids in the absence of beta 2-GPI. However, in high ionic strength buffer, this binding was abolished in three patients and significantly reduced in the fourth. In contrast, affinity-purified aPL Abs from seven autoimmune patients bound to beta 2-GPI-coated plates, and binding in high ionic strength buffer was reduced only moderately in three patients. Therefore, autoimmune-type aPL Abs display anti-beta 2-GPI reactivity and charge-dependent binding to anionic phospholipids similar to affinity-purified rabbit anti-beta 2-GPI Abs.
Selective involvement of proteasomes and cysteine proteases in MHC class I antigen presentation.
Authors: López D , Del Val M
Published: January 2023

CTL recognize peptides derived from protein Ags bound to MHC-class I molecules. Proteasomes probably participate in the generation of these peptide epitopes. We investigated the role of proteasomes in the presentation of endogenously synthesized short viral proteins. To this end, we employed proteasome and cysteine protease inhibitors and two closely related recombinant vaccinia viruses that code for 17- and 19-amino acid-long products encompassing murine CMV 9pp89 epitope. Presentation of both minigene products required processing to shorter peptides and was independent of ubiquitination. Proteasomes were necessary for processing the 17-mer product, and cysteine proteases were not required. In contrast, the 19-mer product could be processed in parallel either by proteasomes or by cysteine proteases independently. These results highlight the diversity of alternative processing pathways even for short peptidic Ags, provide evidence for the involvement of cysteine proteases in MHC class I presentation, and show that cleavage by cysteine proteases is governed by sequences flanking the epitope.
Immunohistochemical analysis of in vivo patterns of TRAF-3 expression, a member of the TNF receptor-associated factor family.
Authors: Krajewski S , Zapata J M , Krajewska M , VanArsdale T , Shabaik A , Gascoyne R D , Reed J C
Published: January 2023

An immunohistochemical approach was used to explore the in vivo expression of TNF receptor-associated factor 3 (TRAF-3), a putative signaling protein that binds to the cytosolic domains of CD30, CD40, and lymphotoxin-beta receptors. TRAF-3 immunostaining was detected in many types of cells throughout the human body. TRAF-3 immunostaining was only rarely present in thymocytes but was found in the thymic epithelioreticular cells. Lymphocytes in the bone marrow were also typically TRAF-3 immunonegative, whereas myeloid progenitor cells and megakaryocytes were often TRAF-3 positive. Peripheral blood lymphocytes were mostly TRAF-3 immunonegative, while granulocytes were TRAF-3 immunopositive. Monocytes were strongly immunostained for TRAF-3, but macrophages in nodes typically contained little or no TRAF-3 immunoreactivity. Some lymphocytes within the germinal centers of secondary lymphoid follicles in normal and reactive nodes were TRAF-3 immunopositive, as were occasional interfollicular lymphocytes in the T cell regions of these organs, but most lymphocytes appeared to be TRAF-3 immunonegative or stained only weakly. Plasma cells, however, were strongly TRAF-3 positive. Stimulation of PBLs with anti-CD3 Ab induced marked increases in the steady state levels of TRAF-3 protein in vitro as determined by immunoblotting, while levels of TRAF-2 were unchanged, implying a dynamic regulation of TRAF-3 expression. The findings establish for the first time the cell type- and differentiation-specific patterns of expression of a member of the TRAF family of proteins.