bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes of liver-resident NK cells during liver regeneration in rats.
Authors: Vujanovic N L , Polimeno L , Azzarone A , Francavilla A , Chambers W H , Starzl T E , Herberman R B , Whiteside T L
Published: December 2022

To determine the role of NK cells in regulation of tissue growth, the phenotype and function of liver-resident NK cells were studied after 70% partial hepatectomy in rats. The process of liver regeneration was generally completed by day 14. In contrast, the number of liver-resident NK cells (NKR-P1bright) was restored as early as day 3 after partial hepatectomy. However, spontaneous functions of liver-resident NK cells, including killing of YAC-1 and P815 targets, Ab-dependent cellular cytotoxicity, and redirected killing via NKR-P1, were continuously suppressed throughout the entire period of liver regeneration (from 3 h to 14 days). Augmentation of NK cytotoxicity against P815 targets and induction of NK cell adherence to plastic following 24 h of IL-2 stimulation showed a similar pattern of suppression. However, IL-2-induced augmentation of YAC-1 killing, proliferation and generation of adherent NK cells, and LAK activity in 5- to 7-day cultures were found to be suppressed only during the first 24 h and increased between days 2 and 7 after hepatectomy. Sorted NK cells (> or = NKR-P1bright) from liver-resident mononuclear leukocytes 24 h after partial hepatectomy showed the same pattern of suppression as unsorted mononuclear leukocytes. In contrast to liver-resident NK cells, no significant changes were detected in peripheral blood or spleen NK cells of rats following partial hepatectomy. Of particular interest, in normal liver, hepatocytes were resistant to NK lysis, while resident NK cells were cytotoxic for various NK-sensitive targets. In contrast, during the early period of liver regeneration, when hepatocytes were sensitive to lysis by liver-resident NK cells of normal rats, NK cells obtained from regenerating liver tissues were unable to mediate cytotoxicity. At the final phase of liver regeneration (days 7-14 after hepatectomy), both resistance of hepatocytes to killing by NK cells and cytotoxicity of liver-resident lymphocytes against hepatocytes from regenerating liver were simultaneously restored. In vivo depletion of NK cells by injection of rats with anti-NKR-P1 mAb resulted in a significant augmentation of liver regeneration subsequent to partial hepatectomy. Our data suggest that liver-resident NK cells may be involved in regulation of the extent of liver regeneration.
Calcineurin activation protects T cells from glucocorticoid-induced apoptosis.
Authors: Zhao Y , Tozawa Y , Iseki R , Mukai M , Iwata M
Published: December 2022

In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin-independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca(2+)-dependent pathway.
Sequence analysis of anti-AChR antibodies in experimental autoimmune myasthenia gravis.
Authors: Graus Y , Meng F , Vincent A , van Breda Vriesman P , de Baets M
Published: December 2022

Autoantibodies directed against the acetylcholine receptor (AChR) lead to AChR loss and muscular weakness in myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis (EAMG). The role of different anti-AChR sequences and specificities in the pathogenesis of EAMG was investigated by sequencing a panel of 19 mouse mAbs, previously elicited against Torpedo and human AChR, that bound to at least four different epitope regions. The pathogenicity of eight mAbs that cross-reacted with mouse or rat AChR was tested. EAMG was induced by four mAbs against the main immunogenic region (MIR). Sequence analysis of different anti-AChR specificities showed a large diversity of H and L chain sequences. Highly homologous H chain sequences (> 90%) were found among some mAbs with similar specificities, whereas highly homologous L chain sequences were not restricted to Abs of a particular fine specificity. Sharing of a highly homologous VH gene or an identical DJH region was observed among three of four pathogenic anti-MIR mAbs, obtained by immunization with AChRs from different species. The VH genes of these three pathogenic mAbs were closely related to PC7183 germline genes indicating that some pathogenic Abs may already be present in the germline repertoire.
VH3 family antibodies bind domain D of staphylococcal protein A.
Authors: Roben P W , Salem A N , Silverman G J
Published: December 2022

Staphylococcal protein A (SpA) is a 45-kDa bacterial membrane protein that can interact with either Fc gamma, a constant region portion of IgG, or with the Fab portion that also mediates conventional Ag binding. In recent reports, SpA has been shown to specifically interact with Fab derived from the VH3 family and is little affected by VH CDR3, JH, or light chain usage. To identify a site on SpA responsible for VH3 Fab binding, we cloned and expressed in Escherichia coli the 61 amino acid sequence of SpA that represents domain D, and this small protein exhibited both the VH3 Fab and Fc gamma binding specificities. Surface plasmon resonance measurements demonstrated that domain D and native SpA had the strongest binding interactions with an IgM-kappa encoded by the germline configuration of the VH3 gene VH26c. In contrast, the apparent affinities for Fc gamma binding were at least fivefold weaker. A variant of domain D was also created that is devoid of the three-codon insertion that distinguishes domain D from all other domains in SpA. Although this deletion did not significantly affect the VH3 Fab-mediated SpA binding activity, it did improve the affinity of Fc gamma binding by an order of magnitude. These observations characterize a site on SpA responsible for binding interactions with B cell Ag receptors that are highly analogous to that of superantigens for T cell receptors.
Nitric oxide synthesis in rat peritoneal macrophages is induced by IgE/DNP complexes and cyclic AMP analogues. Evidence in favor of a common signaling mechanism.
Authors: Alonso A , Carvalho J , Alonso-Torre S R , Núñez L , Boscá L , Sánchez Crespo M
Published: December 2022

The production of nitric oxide was studied in rat adherent peritoneal cells stimulated with preformed IgE/DNP-BSA complexes made of IgE obtained from a secreting hybridoma. Stimulation with complexes at equivalence induced both the production of NO and an increased expression of the mRNA of the inducible isoform of NO synthase (iNOS). Production of NO was also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23. Because IgE/DNP-BSA complexes did not elicit Ca2+ mobilization and genistein did not influence the production of NO, cyclic AMP was considered as an alternative signaling molecule. Combination of a suboptimal concentration of dibutyryl cyclic AMP and IgE/DNP-BSA complexes showed an additive effect on NO production, whereas this was not observed when the agonists were used at supraoptimal doses. The inhibitor of cyclic AMP-specific phosphodiesterase IV, rolipram, which acts on the enzyme isoform predominantly expressed in inflammatory cells, also induced the production of NO. Furthermore, IgE/DNP-BSA complexes increased intracellular levels of cyclic AMP. Taken together, these data indicate that stimulation of mononuclear phagocytes via the low-affinity receptor Fc epsilon RII or rising intracellular concentrations of cyclic AMP leads to an enhanced expression of iNOS. Evidence in favor of the involvement of cyclic AMP in the signaling pathway linked to Fc epsilon RII is provided by the effect of IgE/DNP-BSA complexes on intracellular cyclic AMP levels and by the additive effect produced by dibutyryl cyclic AMP on NO production elicited by IgE/DNP-BSA complexes.
Matrilysin expression by human mononuclear phagocytes and its regulation by cytokines and hormones.
Authors: Busiek D F , Baragi V , Nehring L C , Parks W C , Welgus H G
Published: December 2022

Matrilysin is a recently described metalloproteinase with strong catalytic activity against a variety of extracellular matrix substrates including proteoglycans, elastin, laminin, fibronectin, gelatin, and entactin. Production of this metalloproteinase appears to be limited only to a few normal human cell types including glandular epithelium, mononuclear phagocytes, and renal mesangial cells. Furthermore, matrilysin expression in vivo has been demonstrated only in glandular epithelium, especially the endometrium. In the process of examining various cutaneous and lung inflammatory disorders for matrilysin expression by immunohistochemistry and in situ hybridization, we occasionally found monocytes within blood vessels and newly extravasated tissue-associated macrophages that exhibited matrilysin production. In specimens characterized by severe inflammation and, in particular, cystic fibrosis, this feature was commonly observed. We therefore studied the production of matrilysin by monocyte-derived macrophages in vitro in response to various physiologic signals such as endotoxin, phagocytosable material, cytokines, and hormones. We found that matrilysin expression was stimulated by LPS and opsonized zymosan. Up-regulation of matrilysin by LPS was PGE2-dependent, because indomethacin blocked production, an effect at least partially reversed by the addition of exogenous prostaglandin. LPS stimulated matrilysin production pretranslationally and, furthermore, when cultured cells were subjected to in situ hybridization after LPS exposure, considerable variability in matrilysin mRNA expression was observed on an individual cell basis, with some cells having strong signal and others being completely negative. We also found that matrilysin biosynthesis was inhibited by the lymphokines IL-4, IL-10, and IFN-gamma. Other cytokines such as IL-1, TNF-alpha, and IL-6 failed to modulate the production of matrilysin. Finally, matrilysin biosynthesis was suppressed by glucocorticoids and retinoids. Our studies indicate that matrilysin is produced in vivo by mononuclear phagocytes and is a highly regulated metalloproteinase whose production can be modified by a variety of physiologic and pharmacologic signals.
Involvement of protein kinase C during taxol-induced activation of murine peritoneal macrophages.
Authors: Jun C D , Choi B M , Kim H M , Chung H T
Published: December 2022

Taxol has been known to block cell division by stabilizing microtubules with promising anticancer activity. However, taxol has distinct cell cycle-independent effects. Recently, this novel drug has been shown to provide a second signal for murine macrophage activation to tumoricidal activity via L-arginine-dependent nitric oxide (NO) synthesis. To investigate the mechanism of taxol-induced NO synthesis, we evaluated the ability of protein kinase C (PKC) inhibitors such as staurosporine (STSN) or polymyxin B to block taxol-induced effects. Taxol alone had only a small effect, whereas taxol in combination with rIFN-gamma markedly increased NO synthesis in a dose-dependent manner. STSN and polymyxin B decreased NO synthesis, which had been induced by rIFN-gamma plus taxol. Furthermore, prolonged incubation of the cells with phorbol ester, which down-regulates PKC activity, abolished synergistic cooperative effect of taxol with rIFN-gamma on NO synthesis. Synergy between IFN-gamma and taxol was mainly dependent on taxol-induced TNF-alpha secretion because not only the increase of inducible NO synthase (iNOS) gene expression by rIFN-gamma plus taxol was associated with the increased expression of TNF-alpha gene but also taxol-induced NO production was decreased by the treatment of anti-murine TNF-alpha neutralizing Abs. STSN and polymyxin B potently inhibited taxol-induced TNF-alpha secretion and TNF-alpha gene expression as well as iNOS gene expression by rIFN-gamma plus taxol. However, rIFN-gamma plus TNF-alpha-induced NO synthesis was not blocked by STSN or polymyxin B. This result indicates that TNF-alpha-induced signaling for induction of NO synthesis is not dependent on PKC activation, and further suggests that the point at which TNF-alpha acts on the NO synthesis from rIFN-gamma-primed macrophages lies next to the point of PKC activation. In conclusion, the present results strongly suggest that the capacity of taxol to increase NO synthesis from rIFN-gamma-primed macrophages is the result of taxol-induced TNF-alpha secretion via the signal transduction pathway of PKC activation.
Increased cell-associated IL-8 in human exudative and A23187-treated peripheral blood neutrophils.
Authors: Kuhns D B , Gallin J I
Published: December 2022

IL-8 is a potent neutrophil chemoattractant that has been detected in high concentrations at acutely inflamed sites in vivo. Many cell types, including peripheral blood neutrophils, produce IL-8 that can be released by a variety of pro-inflammatory stimuli. However, the functional importance of neutrophil IL-8 during exudation is not yet known. We now report that neutrophils, harvested from skin lesions on the forearms of normal human volunteers (exudative neutrophils), expressed 100-fold higher levels of cell-associated IL-8 and spontaneously released up to 50-fold more IL-8 than freshly isolated peripheral blood neutrophils from the same donor. Furthermore, cell-associated IL-8 in peripheral blood neutrophils increased 20-fold during incubation at 37 degrees C in vitro and was increased over 200-fold after treatment with the Ca2+ ionophore A23187. More than 35% of the cell-associated IL-8 could be released by stimulation with either Ca2+ ionophore A23187 or phorbol myristate acetate. IL-8 was localized by sucrose gradient centrifugation to a subcellular fraction of heterogeneous, light membranous organelles. The accumulation of IL-8 within these organelles is inhibited by cycloheximide but not actinomycin D, suggesting that IL-8 accumulation is under translational, rather than transcriptional control. These studies indicate that peripheral blood neutrophils are capable of synthesis of large amounts of IL-8. Subsequent release of IL-8 during exudation may regulate neutrophil migration into sites of inflammation.
Identification and characterization of a novel protein associated with macrophage complement receptor 3.
Authors: Messika E J , Avni O , Gallily R , Yefenof E , Baniyash M
Published: December 2022

CR3 is a member of the beta 2 integrin family which functions as a bidirectional signaling receptor. The CR3 is composed of the alpha (CD11b) and beta (CD18) subunits, which contain a short intracytoplasmic domain devoid of catalytic activity. It was therefore postulated that CR3 is associated with intracellular molecules that link it to the cytoplasmic signal transduction apparatus. However, no direct association between such molecules and CR3 have been identified so far. We searched for CR3 co-associated molecules that might regulate the function of this receptor. For this purpose CR3 was immunoprecipitated from radiolabeled bone marrow macrophages using a combination of anti-CD11b and anti-CD18 mAbs. Two-dimensional isoelectric focusing analysis of the immunoprecipitates revealed the two CR3 subunits and an additional 16-kDa protein with an apparent isoelectric point of 5.1. This protein, designated p16/5.1, was intracellular, monomeric, nonglycosylated and noncovalently associated with CR3 but not with CR4. CR3-associated p16/5.1 was also detected in four of six macrophage lines as well as in thymic large macrophages, all of which express cell-surface CR3. We suggest that p16/5.1 may be involved in CR3-mediated function.
Ligand binding to Fc gamma R induces c-myc-dependent apoptosis in IL-2-stimulated NK cells.
Authors: Azzoni L , Anegon I , Calabretta B , Perussia B
Published: December 2022

The role of signals transduced via Fc gamma RIIIA in the modulation of the proliferative potential of NK cells has been investigated. Fc gamma R stimulation does not induce NK cell proliferation, and inhibits that induced by IL-2, but not by IL-12, as measured by [3H]TdR incorporation, without affecting entrance or progression through cell cycle. The inhibitory effect depends, at least in part, on induced apoptosis of the cells, detected by both light and electron microscopy examination. Fc gamma R stimulation induces apoptosis only in NK cells that have been previously activated by IL-2: this occurs within 3 h from receptor stimulation and is independent from de novo receptor-induced RNA or protein synthesis, but requires receptor-induced activation of protein tyrosine kinases and extracellular Ca2+ influx. IL-2 induces accumulation of c-myc mRNA in NK cells, and treatment of the cells with c-myc antisense oligodeoxyribonucleotides during the IL-2 stimulation phase inhibits the susceptibility to Fc gamma RIIIA-induced cell death, indicating that the induction of sustained levels of this proto-oncogene is necessary for the phenomenon. Thus, a two-step model is suggested for the Fc gamma R-induced apoptosis in IL-2 activated NK cells: the first step involves induced expression of c-myc, and possibly other permissive factors, upon IL-2 prestimulation; the second depends directly on the stimulation of the receptor, independently of additional gene induction. The evidence presented here suggests a mechanism of control of NK cell expansion at the latest stages of Ab-dependent immune responses.
Selective processing of exogenous antigens by antigen-presenting cells with deleted MHC genes.
Authors: Diment S , Shinde S
Published: December 2022

Recently, APCs expressing genes for the alpha- and beta-chains of MHC class II, but not other proteins encoded in the MHC class II locus, have been found to be defective in processing proteins by the MHC class II Ag presentation pathway. Ag presentation of hen egg OVA has been examined in one of these cell lines, T2.Ak. OVA was processed normally by T2.Ak when compared with OVA processed by T1.Ak, a cell line that expresses the MHC genes missing in T2.Ak. By contrast, native hen egg lysozyme (HEL) was not presented as Ag by T2.Ak, which is in agreement with earlier results. Digestion with thiol proteases is important for Ag processing of HEL. We therefore analyzed the expression of these enzymes in T1.Ak and T2.Ak by using a reagent that specifically radiolabels thiol proteases. In these experiments, the repertoire of proteases expressed in the microsomes of T1.Ak was found to be distinct from the repertoire expressed by T2.Ak. Finally, in in vitro digestion experiments, the group of thiol proteases active in T1.Ak microsomes digested HEL differently from the group identified in T2.Ak. These results provide evidence that the defect in Ag presentation that is encoded by MHC genes and manifests itself in defective processing of HEL is not absolute. Further, the mutation in T2.Ak coincides with altered activities of thiol proteases, a class of enzymes involved in processing exogenous Ags.
Fas antigen-mediated DNA fragmentation and apoptotic morphologic changes are regulated by elevated cytosolic Ca2+ level.
Authors: Oshimi Y , Miyazaki S
Published: December 2022

More than 80% of cells of the human B cell line FMO, which expresses the Fas Ag, underwent apoptosis within 2 h after addition of an anti-Fas mAb. Rises in cytosolic Ca2+ concentration ([Ca2+]i) and their effects were investigated by imaging individual cells continuously and introducing Ca2+ chelators to the outside and/or inside of the cell. The typical Ca2+ response consisted of 1) an early [Ca2+]i rise (basal [Ca2+]i, 85 to 110 nM; peak, 140 to 200 nM; duration, 15 to 20 min), 2) sustained elevation of [Ca2+]i at 140 to 150 nM, 3) a second Ca2+ rise (200 to 500 nM, 15 to 30 min) between 1.5 and 2 h, and 4) a large [Ca2+]i rise (1.2 to 2.0 microM) between 2 and 4 h after addition of mAb. Responses 1, 3, and 4 were mainly caused by Ca2+ entry, and response 2 involved intracellular Ca2+ release from stores. Apoptosis could be induced by mAb even in Ca(2+)-deprived external medium. Chelating cytosolic Ca2+ revealed that the [Ca2+]i rise is a prerequisite for fragmentation of DNA and chromatin, and is also necessary for fragmentation of cells. The critical [Ca2+]i was 140 to 150 nM and a sustained [Ca2+]i rise was more effective. A [Ca2+]i rise itself (without mAb) was ineffective. About 20% of mAb-treated cells showed chromatin condensation at the periphery of the nucleus (possibly an earlier stage of nuclear change) and a bubble-like cell shape even when [Ca2+]i was held below 100 nM. Thus, Ca2+ is mobilized immediately after Fas stimulation and functions as a key factor causing advanced apoptotic changes. Response 4 was related to secondary necrosis.
TGF-beta suppression of IFN-gamma-induced class II MHC gene expression does not involve inhibition of phosphorylation of JAK1, JAK2, or signal transducers and activators of transcription, or modification of IFN-gamma enhanced factor X expression.
Authors: Panek R B , Lee Y J , Benveniste E N
Published: December 2022

TGF-beta is a widely expressed immunoregulatory protein that exerts a diverse range of effects on many types of cells. One of the effects of TGF-beta is the inhibition of both constitutive and cytokine-inducible class II MHC gene expression. In this study, we demonstrate that TGF-beta inhibits expression of class II MHC surface protein, mRNA, and promoter activity in primary astrocytes, and that this inhibition is both dose and time dependent. TGF-beta does not act to inhibit IFN-gamma-induced gene expression in a global fashion, as induction of ICAM-1 and IRF-1 gene expression by IFN-gamma is unaffected by treatment with TGF-beta. Furthermore, TGF-beta does not affect events that are involved in IFN-gamma-induced intracellular signaling such as tyrosine phosphorylation of JAK1, JAK2, and STAT1 alpha, nor does it affect IFN-gamma induction of the class II X2 box binding protein IFN-gamma enhanced factor X. We speculate that TGF-beta may be exerting its effects by modulating the expression or function of constitutively expressed factors responsible for regulation of class II MHC gene expression in astrocytes.
Binding of a peptide antigen to multiple HLA alleles allows definition of an A2-like supertype.
Authors: del Guercio M F , Sidney J , Hermanson G , Perez C , Grey H M , Kubo R T , Sette A
Published: December 2022

Direct MHC binding assays with radiolabeled peptides and HLA class I-expressing mammalian cells such as EBV-transformed B cell lines and PHA-activated blasts have been developed. Significant binding of the radiolabeled probe could be obtained if the target cells were preincubated overnight at 26 degrees C in the presence of beta 2-microglobulin. Under these conditions, up to a few percent of the HLA molecules expressed by either cell type could be bound by the labeled peptides. With these assays, the degree of cross-reactivity of the A*0201-restricted hepatitis B virus core 18-27 peptide with other A2 subtypes was examined. It was determined that this peptide epitope also binds the A*0202, A*0205, and A*0206 but not A*0207 subtypes. Inhibition experiments with panels of synthetic peptide analogues underlined the similar ligand specificities of the HLA-A*0201, A*0202, and A*0205 alleles. Analysis of the polymorphic residues that help form the B and F pockets of various HLA alleles allowed prediction of binding of the hepatitis B virus core 18-27 epitope to two other HLA alleles (HLA-A*6802 and A*6901). Thus, it appears that a family of at least six different HLA-A molecules may share overlapping ligand specificities (aliphatic residues in position 2 and at the C termini). These results suggest that broadly cross-reactive peptide epitopes can be identified and greatly enhance the prospective feasibility of peptide-based vaccination approaches.
Fibrosarcoma cells expressing allogeneic MHC Class II antigens induce protective antitumor immunity.
Authors: Leach D R , Callahan G N
Published: December 2022

The initiation of effective immune responses usually requires presentation of Ags by MHC class I and class II molecules. Although most tumors express MHC class I molecules, MHC class II molecule expression is generally limited to specialized APCs. One reason spontaneous tumors may fail to elicit effective immune responses is that tumor Ags are inefficiently presented by APCs, and adequate T cell-mediated help is not generated. To test this hypothesis, we investigated the possibility of enhancing Th cell stimulation by inducing expression of MHC class II molecules on tumor cells. We transfected a murine fibrosarcoma, Sa1N, with the genes encoding allogeneic (I-Ad) or syngeneic (I-Ak) MHC class II molecules. We then compared the tumorigenic and immunogeneic potential of these transfectants to parental Sa1N tumor cells. Subcutaneous injection of allogeneic or syngeneic transfectants resulted in dramatically fewer tumors than injection of unmodified fibrosarcoma cells, and mice inoculated with MHC class II gene-transfected cells were resistant to subsequent challenge with parental Sa1N cells. Rejection of allogeneic MHC class II Ag+ tumor cells could be mediated by either CD4+ or CD8+ T cells, whereas rejection of secondary challenge with wild-type Sa1N tumor cells required both T cell subsets. These results demonstrate that allogeneic, as well as syngeneic, MHC class II Ag+ tumor cells can stimulate protective antitumor immunity.
The WI-1 antigen of Blastomyces dermatitidis yeasts mediates binding to human macrophage CD11b/CD18 (CR3) and CD14.
Authors: Newman S L , Chaturvedi S , Klein B S
Published: December 2022

Three genetically related strains of Blastomyces dermatitidis (Bd) yeasts that differ in their expression of WI-1, an immunodominant cell wall Ag, were tested for their capacity to bind to human macrophages (M phi) in the absence of serum. These strains included American Type Culture Collection (ATCC, Rockville, MD) ATCC 26199, which is virulent for mice; an attenuated mutant strain ATCC 60915, which expresses 2.5-fold more WI-1 than strain 26199; and an avirulent mutant strain, ATCC 60916, which has sixfold more WI-1 than strain 26199. Attachment of both mutant strains to M phi was rapid and was maximum after 10 min at 37 degrees C. Attachment of strain 26199 to M phi was approximately 40% of that obtained with the mutants. Binding of Bd to M phi was temperature- and Mg(2+)-dependent, and heat-killed yeasts bound to M phi as well as viable yeasts. Experiments with receptor-specific mAbs demonstrated that 26199 yeasts bound predominantly to the LPS binding site on CD11b/CD18 (CR3). However, the mutants bound to M phi CD14 as well as CR3. Fab anti-WI-1 inhibited the binding of all strains to M phi by 69 to 78%. Latex microspheres coated with purified WI-1 or a 25-amino acid tandem repeat located within WI-1 also bound to M phi CR3 and CD14. These data demonstrate that: 1) WI-1 is a major ligand on Bd that mediates attachment of yeasts to human M phi; 2) the binding activity of WI-1 is located within the 25-amino acid tandem repeat; and 3) binding of Bd yeasts to M phi is mediated through the LPS binding site on CR3 and CD14.
Synthetic hexa- and heptapeptides that inhibit IL-8 from binding to and activating human blood neutrophils.
Authors: Hayashi S , Kurdowska A , Miller E J , Albright M E , Girten B E , Cohen A B
Published: December 2022

IL-8 is a member of the chemokine alpha subfamily that activates and is chemotactic for neutrophils. In these studies, we have synthesized and characterized a hexapeptide inhibitor of IL-8. This peptide, with an acetylated amino terminus and an amidated carboxyl terminus (Ac-RRWWCR-NH2), inhibited the specific binding of 125I-IL-8 to neutrophils. The inhibition was biphasic and apparent Ki was estimated to be approximately 2.7 microM and 13 microM for two different IL-8 binding sites. The peptide inhibited neutrophil chemotaxis, beta-glucuronidase release from neutrophils, and rabbit skin edema induced by IL-8 with an EC50 of 90 microM, 0.8 microM, respectively. Ac-RRWWCR-NH2 also suppressed the binding of macrophage inflammatory protein (MIP) 2 beta to neutrophils. However, it did not inhibit the binding of MIP-1 alpha, C5a, or leukotriene B4 to neutrophils, chemotaxis induced by FMLP, or beta-glucuronidase release induced by FMLP, C5a, or leukotriene B4. Additional peptides were analyzed to identify a better inhibitor. Inhibition of binding by Ac-rrwwcrc-NH2 synthesized with all D-amino acids was almost four times more potent than Ac-RRWWCR-NH2. Small peptide homologues of the amino-terminal end of IL-8 failed to inhibit IL-8 binding to neutrophils. These studies have identified several peptides that significantly inhibit IL-8 function. Because IL-8 seems to be an important inflammatory mediator of several human illnesses, these peptides may have pharmacologic potential.
Chlorpromazine amplifies macrophage-dependent IL-10 production in vivo.
Authors: Tarazona R , González-García A , Zamzami N , Marchetti P , Frechin N , Gonzalo J A , Ruiz-Gayo M , van Rooijen N , Martínez C , Kroemer G
Published: December 2022

Chlorpromazine (CPZ) has potent immunomodulatory effects in vivo; it induces humoral autoimmunity in up to 50% of patients, inhibits delayed-type hypersensitivity reactions, and suppresses lethal immune hyperactivation in animal models of septic shock. Here, we show that in an in vivo model of acute superantigen-driven immune activation, CPZ independently down-regulates the production of various T cell-derived lymphokines (IL-2, IFN-gamma, IL-4, TNF, and GM-CSF) and up-regulates the secretion of IL-10. Whereas only low, if any, serum IL-10 levels are detectable by ELISA after injection of CPZ, bacterial LPS, or staphylococcal enterotoxin B (SEB) alone, simultaneous administration of CPZ + LPS or CPZ + SEB causes a significant increase in IL-10 production in vivo. CPZ-mediated amplification of the SEB-driven CPZ secretion is accompanied by an enhanced IL-10 mRNA accumulation, as shown by PCR analysis and in situ hybridization. Determination of IL-10 production in mice lacking T cells, B cells, or phagocytes revealed that SEB + CPZ-induced IL-10 was produced by phagocytic cells, but not by lymphocytes, a finding that is in accord with the distribution of splenic cells transcribing the IL-10 gene in response to SEB + CPZ. Moreover, these data indicate that bacterial superantigen can directly stimulate tissue phagocytes, even in the virtual absence of T lymphocytes. The blockade of dopamine type 1 (D1) but not type 2 (D2) receptors abolishes the CPZ effect on IL-10 production. Inhibition of Th1 and Th2 lymphokine production by CPZ is not mediated by dopamine receptors and is independent of IL-10 up-regulation. These findings may explain the mechanism by which CPZ and related drugs enhance humoral autoimmune reactions, block cellular immune responses, and prevent lethal septic shock in vivo.
Functional analysis of the TCR alpha- beta+ cells that accumulate in the pneumonic lung of influenza virus-infected TCR-alpha-/- mice.
Authors: Eichelberger M , McMickle A , Blackman M , Mombaerts P , Tonegawa S , Doherty P C
Published: December 2022

In mice homozygous (-/-) for a targeted TCR-alpha gene disruption, some thymocytes express a cell-surface TCR-beta chain on the cell surface in the absence of a TCR-alpha chain, and a few CD4+CD8- TCR-alpha-beta+ cells accumulate in the peripheral lymphoid organs. We have infected these mutant mice with an influenza A virus to show that large numbers of TCR-beta+ cells (most of which are CD4+) can be retrieved from the pneumonic lung. Both freshly isolated TCR-alpha-beta+ cells and TCR-alpha-beta+ hybridoma cell lines derived from influenza virus-infected mutant mice respond appropriately to stimulation with anti-CD3 epsilon or the Mls-1 superantigen. It thus seems that CD4+ TCR-alpha-beta+ cells in the peripheral lymphoid organs of TCR-alpha mutant mice can signal through their TCR surface complex. However, there are no indications that CD4+ TCR-alpha-beta+ lymphocytes can either recognize a complex between MHC and influenza virus peptide or act as effector or Th cells. The existence and function of such cells in wild-type mice remains to be established.
Dramatic hyperplasia of mtv-2+ lymph node grafts in mtv-2- recipients and selective stimulation of V beta 14+ T cells in recipients' lymph nodes in the DDD mouse.
Authors: Matsuzawa A , Nakano H , Sakamoto S , Yoshimoto T , Nariuchi H
Published: December 2022

DDD/1 (DDD) mice contrast strikingly with DDD-mtv-2/mtv-2 (DDD-mtv-2) congenics in their marked lymph node (LN) T cell paucity. To clarify the possible difference in LN function between them, reciprocal LN grafting experiments were conducted. DDD-mtv-2 LN grafts in DDD recipients underwent hyperplasia as dramatic as 10-to 20-fold increase in weight between 3 and 4 wk after implantation. Lymphoid cells in hyperplastic LN grafts were of recipient origin. Similar hyperplasia of mtv-2-heterozygous LN grafts also occurred on various hybrid backgrounds involving DDD mice. Moreover, LN grafts from BALB/c mice infected with mtv-2-derived exogenous mouse mammary tumor virus (MMTV) swelled in MMTV-free BALB/c recipients. Genetic analysis of DDD x (DDD x DDD-mtv-2)F1 backcross progeny demonstrated that LN hyperplasia was closely linked to mtv-2. The frequencies of V beta 5+ and V beta 8+ T cells unresponsive to mtv-2-encoded superantigen (SAg) changed with practically the same kinetics in both LN grafts and recipients' LN. Thus, the cells responsible for LN hyperplasia were polyclonal. V beta 14+ CD4+ cells responsive to mtv-2 SAg were specifically stimulated in recipients' LN but selectively deleted in hyperplastic LN grafts. DDD mice carrying hyperplastic mtv-2+ LN grafts or pretreated with mtv-2+ spleen cells developed an unresponsive state in terms of influx of mtv-2- lymphoid cells into mtv-2+ LN grafts. These results indicate that mtv-2 gene products including SAg may stimulate mtv-2- lymphoid cells of recipients and cause them to migrate into mtv-2+ LN grafts in a nonspecific manner with resulting LN hyperplasia.
Costimulatory signals can selectively modulate cytokine production by subsets of CD4+ T cells.
Authors: Shanafelt M C , Soderberg C , Allsup A , Adelman D , Peltz G , Lahesmaa R
Published: December 2022

Analysis of experimental animal models and human clinical samples has indicated that the selective activation of CD4+ T cell subsets with distinct profiles of cytokine production plays an important role in the pathogenesis of human inflammatory and allergic diseases. The possibility that differential activation of costimulatory pathways is a mechanism for selectively modulating cytokine production by CD4+ T cells was tested. The proliferative response and cytokines secreted by a panel of human CD4+ T cell clones, representative of Th1 or Th2/0 cells, in response to activation of different costimulatory pathways was measured. CD28-mediated costimulatory signals induced proliferation and IFN-gamma secretion by Th1 cells. Although CD28-ligation induced Th2/0 cells to proliferate, it did not trigger IL-4 production. Ligation of LFA-1 and CD45 isoforms also generated costimulatory signals activating cytokine secretion by the different types of T cell clones. Th1 cells secreted the same profile of cytokines, irrespective of which costimulatory pathway was engaged. However, the cytokine secreted by a subset of Th2/0 cells varied, depending upon which costimulatory pathways were activated. These results suggest that the costimulatory pathways activated by APCs can selectively influence cytokine production by CD4+ T cell subsets.
Stimulation of NK-like YT cells via leukocyte function-associated antigen (LFA)-1. Possible involvement of LFA-1-associated tyrosine kinase in signal transduction after recognition of NK target cells.
Authors: Sugie K , Minami Y , Kawakami T , Uchida A
Published: December 2022

The YTA-1 anti-LFA-1 alpha mAb activates protein tyrosine kinase (PTK), augments NK cytotoxicity, and induces proliferation of fresh CD3- large granular lymphocytes. We demonstrate here that LFA-1 is physically associated in the YT human NK-like cell line cells with a PTK(s) that is distinct from Src family PTKs such as Lck, Fyn, or Lyn. In vitro kinase assays revealed similar association of protein kinase activity with LFA-1 in normal CD3- large granular lymphocytes. Tyrosine phosphorylation of the proteins associated with LFA-1 drastically increased in YT cells after stimulation with NK-sensitive K562 cells but not with NK-resistant P815 cells. Furthermore, pretreatment of YT cells with TS1/22 anti-LFA-1 alpha and TS1/18 anti-LFA-1 beta mAbs abrogated not only the cytotoxicity against K562 cells but also an increase in tyrosine phosphorylation of LFA-1-associated molecules induced by K562 stimulation. These results provide biochemical evidence that the PTK(s) associated with LFA-1 is involved in the signal transduction that follows the recognition of NK target cells.
Lymphoid expression and regulation of A20, an inhibitor of programmed cell death.
Authors: Tewari M , Wolf F W , Seldin M F , O'Shea K S , Dixit V M , Turka L A
Published: December 2022

A20 is a cytokine-inducible primary response gene that encodes a protein that inhibits apoptosis. We cloned and characterized a murine A20 cDNA and mapped the gene to mouse chromosome 10, 3.5 centimorgans proximal to the c-myb locus. Using the murine cDNA, we conducted in situ hybridization studies to examine patterns of A20 expression in mouse embryos and postnatal tissues. Both temporally restricted and tissue-specific patterns of A20 expression were observed, with strikingly high levels in lymphoid organs, including the thymus, spleen, and gut-associated lymphoid tissue. Northern analysis using purified human lymphocyte populations demonstrated that A20 is constitutively expressed in both immature and mature thymocyte subpopulations, as well as in resting peripheral T cells. Activation leads to a down-regulation of A20 expression in both mature thymocytes and peripheral T cells. This pattern of expression and regulation differs markedly from that observed in prior studies with cell lines in which stimulation led to an induction of A20 expression. Additionally, this is the first demonstration of constitutive expression of A20 in primary cells. The data suggest a role for A20 in the function of the lymphoid system.
Molecular cloning of the mouse polymeric Ig receptor. Functional regions of the molecule are conserved among five mammalian species.
Authors: Piskurich J F , Blanchard M H , Youngman K R , France J A , Kaetzel C S
Published: December 2022

Transcytosis of polymeric Ig (pIg) by mucosal epithelial cells is mediated by the polymeric Ig receptor (pIgR). Here we describe the characterization of a 3095-bp mouse pIgR cDNA, which encodes a protein of 771 amino acids. Northern blot analysis detected a single mouse pIgR transcript of 3.9 kb, expressed at high levels in small intestine and liver, and at low levels in lung. Alignment of the amino acid sequences of mouse, rat, human, bovine, and rabbit pIgR revealed that functional regions of the molecule are conserved across species. In the extracellular region, conserved motifs include: a 23-amino acid pIg-binding site, 11 intradomain disulfide bonds, consensus sites for N-glycosylation, and a putative cleavage site at which the extracellular region of pIgR (secretory component) is released from the plasma membrane. A 10-amino acid sequence within the transmembrane region is highly conserved, possibly reflecting a mechanism for transmitting signals from the extracellular region to the cytoplasmic tail. Conservation within the cytoplasmic tail of pIgR is clustered in motifs that mediate polarized sorting, endocytosis, and transcytosis.
A mutated promoter of a human Ig V lambda gene segment is associated with reduced germ-line transcription and a low frequency of rearrangement.
Authors: Stiernholm N B , Berinstein N L
Published: December 2022

Several explanations for skewed variable (V) gene usage in the expressed Ig repertoire have been put forth, ranging from cellular selection to differences in the actual rearrangement frequencies of individual V gene segments. In this report we study V gene usage in human Ig lambda rearrangements in an in vitro system, thus largely avoiding selective forces. We find a significant bias in V lambda gene usage in these rearrangements. Through deletional mapping on Southern blots, the relative chromosomal organization of members of the V lambda I, V lambda V, V lambda VI, and V lambda X families was established, which revealed that the bias in V lambda gene usage cannot be readily explained by chromosomal location. We further found that the qualities of the recombination signal sequences, nucleotide sequence/regions of homology of the coding ends or the CpG methylation patterns, do not explain this bias either. We do, however, find a direct correlation between a low frequency of rearrangement and a reduced level of germ-line transcription of a particular V gene segment. We were able to identify three mutations in the octamer motif of the promoter region of this V gene segment. This is the first report showing that mutations in important transcriptional control motifs of individual V gene segments are associated with reduced levels of transcription in the germ line, which in turn may influence the frequency of rearrangement and the shaping of the expressed Ig repertoire.
CD4+ alpha beta T cell and gamma delta T cell responses to Mycobacterium tuberculosis. Similarities and differences in Ag recognition, cytotoxic effector function, and cytokine production.
Authors: Tsukaguchi K , Balaji K N , Boom W H
Published: December 2022

CD4+ and gamma delta T cells are activated readily by Mycobacterium tuberculosis. To examine their role in the human immune response to M. tuberculosis, CD4+ and gamma delta T cells from healthy tuberculin-positive donor were studied for patterns of Ag recognition, cytotoxicity, and cytokine production in response to M. tuberculosis-infected mononuclear phagocytes. Both T cell subsets responded to intact M. tuberculosis and its cytosolic Ags. However, CD4+ and gamma delta T cells differed in the range of cytosolic Ags recognized: reactivity to a wide m.w. range of Ags for CD4+ T cells, and a restricted pattern for gamma delta T cells, with dominance of Ags of 10 to 15 kDa. Both T cell subsets were equally cytotoxic for M. tuberculosis-infected monocytes. Furthermore, both CD4+ and gamma delta T cells produced large amounts of IFN-gamma: mean pg/ml of IFN-gamma in supernatants was 2458 +/- 213 for CD4+ and 2349 +/- 245 for gamma delta T cells. By filter-spot ELISA (ELISPOT), the frequency of IFN-gamma-secreting gamma delta T cells was one-half of that of CD4+ T cells in response to M. tuberculosis, suggesting that gamma delta T cells on a per cell basis were more efficient producers of IFN-gamma than CD4+ T cells. In contrast, CD4+ T cells produced more IL-2 than gamma delta T cells, which correlated with diminished T cell proliferation of gamma delta T cells compared with CD4+ T cells. These results indicate that CD4+ and gamma delta T cell subsets have similar effector functions (cytotoxicity, IFN-gamma production) in response to M. tuberculosis-infected macrophages, despite differences in the Ags recognized, IL-2 production, and efficiency of IFN-gamma production.
Fibrin enhances the expression of IL-1 beta by human peripheral blood mononuclear cells. Implications in pulmonary inflammation.
Authors: Perez R L , Roman J
Published: December 2022

Tissue injury is accompanied by increased vascular permeability and influx of plasma proteins including fibrinogen. Fibrinogen is converted into a fibrin matrix by procoagulants activated at the site of tissue injury and inflammation. This fibrin matrix is thought to participate early in the inflammatory response by providing a temporary protein "scaffold" for inflammatory cell adhesion and migration, and subsequent remodeling of the tissue with permanent extracellular matrix proteins such as collagen. Collagen and fibronectin have been shown to regulate the expression of inflammatory cytokines, but whether fibrin can regulate cytokine expression is not known. We hypothesized that fibrin induces the expression of the proinflammatory cytokine IL-1 beta and sought to explore mechanisms responsible for this induction. In this report, we demonstrate that fibrin stimulates human PBMCs to express IL-1 beta message and protein. We show that induction of IL-1 beta by fibrin is mediated partly by the integrin receptor CD11b/CD18 and modulated by cytoskeletal rearrangement. Fibrin also suppresses production of IL-1 receptor antagonist (IL-1ra) a non-bioactive competitor of IL-1 for IL-1Rs. We propose that injured tissues where the conditions favor coagulation and fibrin accumulation, the interaction between mononuclear cells and the newly formed fibrin matrices may elicit the production of the proinflammatory cytokine IL-1 beta. This proposal is supported by immunohistochemical studies which show the co-distribution of fibrin and IL-1 beta in granulomas in lung sections from patients with the systemic granulomatous disease, sarcoidosis.
Cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) by astrocytes and astrocytoma cell lines.
Authors: Rosenman S J , Shrikant P , Dubb L , Benveniste E N , Ransohoff R M
Published: December 2022

Astrocytes in the central nervous system (CNS) associate intimately with vascular endothelial cells and are integral to the blood-brain barrier (BBB). Leukocyte transmigration across the BBB is a cardinal feature of CNS inflammation, as observed in experimental autoimmune encephalomyelitis, and very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1) interactions have recently been proposed as essential for this process. VCAM-1 expression by astrocytes was recently reported. We addressed the regulation of VCAM-1 expression by inflammatory cytokines in primary human astrocytes and two human astrocytoma cell lines. Astrocytoma cells up-regulated surface VCAM-1 expression in response to cytokines in the following order: IFN-gamma plus TNF-alpha > IFN-gamma plus IL-1 beta > TNF-alpha > IFN-gamma. Cytokine-activated astrocytoma cells expressed 7-domain VCAM-1, as indicated by accumulation of 3.2-kb VCAM-1 mRNA and immunoprecipitation of a 100-kDa protein with anti-VCAM-1 mAb. Lymphoblast adhesion to cytokine-activated astrocytoma cell monolayers was significantly blocked by mAb specific for VCAM-1 and VLA-4, indicating that astrocytoma cell VCAM-1 was functional. Astrocytoma cell expression of VCAM-1 could be a constituent of the astrocyte phenotype. To support this possibility, we demonstrated that cytokine-treated adult human and rat primary astrocytes expressed VCAM-1, and the rank order of cytokine potency for VCAM-1 induction in primary and neoplastic astrocytes was strikingly similar. This is the first documentation of VCAM-1 expression by adult human astrocytes. Expression of VCAM-1 by astrocytes at the BBB could play a role in mononuclear leukocyte entry into the CNS.