Regulation and function of fibronectin receptors expressed by natural killer cells.
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Animals↗
Biomedical subjects
L Frati
Publications and source records attributed to L Frati.
Evidence for three binding sites for C3 (hemolytically inactive), C3b and C3d on a CR2-positive Burkitt lymphoma-derived cell line (Raji).
The present investigation shows that C3 (hemolytic inactive) as well as C3b and C3d bind Raji, a CR2-positive Burkitt lymphoma-derived cell line. Pretreatment of the cells with OKB-7 inhibited the binding of C3, whereas pretreatment with HB-5 inhibited the binding of C3b. Furthermore, the cells coated either with OKB-7 or HB-5 bound high amounts of C3d. TPA-treated cells showed binding for C3b and weak binding for C3 and C3d. Taken together, the data suggest that Raji cells may express three binding sites for C3, C3b and C3d which can be differently modulated by anti-CR2 MoAbs and TPA.
Antibodies, Monoclonal↗
Human B-cell immune response to the polymorphic epithelial mucin.
Human antibodies generated by Epstein-Barr virus immortalized B-cells from tumor-draining lymph nodes of an ovarian cancer patient were screened for reactivity in enzyme-linked immunosorbent assay with a synthetic peptide corresponding to the protein core of the polymorphic epithelial mucin. Epitopes within this region are in fact considered tumor specific since they are selectively exposed in tumor cells due to aberrant glycosylation. Human antibody BB5, thus selected, reacts in enzyme-linked immunosorbent assay and immunohistochemistry with polymorphic epithelial mucin-expressing tumor cells. This is the first demonstration of the existence of a B-cell immune response to selected epitopes of polymorphic epithelial mucin and, together with the cytotoxic T-cell response already demonstrated, constitutes the basis for the use of synthetic peptides as a vaccine in cancer patients.
Adult↗
Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: relationship to diacylglycerol and phosphatidic acid accumulation.
Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [3H]choline administration, resulted in an enhanced release of [3H]phosphocholine (PCho) into the external medium; enhanced [3H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [3H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [3H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [3H]phosphatidylethanol in cells prelabelled with [3H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [3H]myristic acid and [14C]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2, whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.
Carbachol↗
Transcriptional regulation of interleukin-2 gene expression by CD69-generated signals.
The 5' flanking region of the human interleukin (IL)-2 gene was investigated for enhancer activity in response to CD69-generated signals, using a chloramphenicol acetyltransferase (CAT)-driven transient expression system in Jurkat cells. The region extending from -317 to +47 relative to the initiation site of IL-2 gene transcription was shown to contain sequences able to respond to CD69 cross-linking, by enhancing by about 100% a phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin stimulation of CAT activity. A similar increase in CAT activity produced by PMA-plus-anti-CD3 mAb was induced by CD69 cross-linking, while a 200% increase over that obtained by PMA-plus-anti-CD28 mAb stimulation was seen. Analysis of enhancer deletion mutants revealed that proximal AP-1, OCT-1/octamer-associated protein and nuclear factor of activated T cells (NFAT) binding regions were all necessary to allow CD69-mediated enhancement of CAT activity. By gel mobility shift analysis, cyclosporin A-sensitive NFAT-binding induction and enhancement of AP-1 binding activity could be detected in nuclear extracts of both Jurkat and peripheral blood T cells after simultaneous CD69 and protein kinase C stimulation. Finally, CD69-mediated signals could increase NFAT and AP-1 binding activity following PMA and ionomycin stimulation in peripheral blood T cells. Collectively, these data suggest that CD69-generated signals participate in the control of the IL-2 gene expression at the transcriptional level, likely acting through NFAT and AP-1 transcription factor complexes.
Antigens, CD↗
Continuous in vivo activation and transient hyporesponsiveness to TcR/CD3 triggering of human gut lamina propria lymphocytes.
Three-color immunofluorescence and flow cytometric analysis showed that the vast majority of normal human T-lamina propria lymphocytes (LPL) expressed high levels of the early activation antigen CD69, together with CD45R0, irrespective of their CD4, CD8 or gamma/delta-TcR phenotype, indicating that they are continuously stimulated in vivo. Importantly, measurement of cytoplasmic [Ca2+]i showed that T-LPL had significantly higher basal [Ca2+]i levels, compared to autologous peripheral blood lymphocytes (PBL). Both cytoplasmic [Ca2+]i elevation and inositol (1,4,5) trisphosphate generation following CD3 cross-linking by monoclonal antibodies in vitro were essentially abolished in T-LPL, as compared to autologous T-PBL. Moreover, freshly isolated LPL could be induced to proliferate by CD2- or CD28-mediated signals, but not by CD3-mediated signals. Surprisingly however, impairment in TcR/CD3-mediated early signaling and proliferation in T-LPL could be completely reversed by 24 h incubation of the cells at 37 degrees C in culture medium, a condition which allowed basal intracellular [Ca2+]i to return to levels comparable to peripheral T cells. Our data suggest that selective hyporesponsiveness to TcR/CD3-mediated signaling may represent a transient event during continuous in vivo activation of mucosal lymphocytes.
Calcium↗
Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells.
Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15-22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA.
Animals↗
Relationship between epidermal growth factor and its receptor in human benign prostatic hyperplasia.
Human benign prostatic hyperplasia (BPH) samples were analyzed to evaluate the presence of immunoreactive epidermal growth factor (irEGF) and EGF receptor (EGFR). In all BPH samples examined both peptide and its receptor were present. Scatchard analysis of binding data of [125I]EGF showed two classes of binding sites with high and low affinity. Intratissular irEGF concentrations showed a significant inverse linear correlation with EGFR levels. Two groups of samples can be identified: the first showing high irEGF concentrations and low levels of EGF binding sites; the second low irEGF and high concentrations of EGFR. The simultaneous presence of EGF and its receptor in BPH samples indicates that this growth factor may act in an autocrine/paracrine manner in human prostatic tissue. The inverse relationship between EGF and the two sites of EGFR lead one to hypothesize that EGF itself could play a central role in determining receptor cell surface availability.
Aged↗
Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription.
Interaction between protein kinase C (PKC)- and glucocorticoid receptor (GR)-mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to inhibit GR-dependent transcription of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Here we report that this interference is cell specific, as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts. TPA-GR synergism was determined to have occurred at the GR-responsive element (GRE) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression. Synergism required an intact GR DNA-binding domain, whereas amino- or carboxyl-terminal domains were dispensable. The effect was abrogated by the PKC inhibitor staurosporine, suggesting a role for PKC. Increased c-jun, jun-B, and jun-D expression above basal levels and increased transcriptional activity of AP-1/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone. The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun, jun-B, or jun-D expression vectors, which augmented GR-dependent transcription from either MMTV LTR or GRE. Conversely, c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells. The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells, but not in T cells. Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells. We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE, selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment. In this regard, intracellular levels of c-fos appear to be influential.
3T3 Cells↗
[5-fluorouracil, cisplatin and retinol palmitate in the management of advanced cancer of the oral cavity. Phase II study].
23 patients, with advanced oral cavity cancer, radically unresectable or relapsing after surgery and/or radiotherapy, were treated with a chemotherapeutic regimen, including 5-Fluorouracil 1000 mg/m2 and cis-platinum 20 mg/m2 for 5 consecutive days. Retinol palmitate was administered at the dose of 15.000 IU b.i.d. in chemotherapy intervals. In complete or partial responders, radiotherapy was planned on primary tumor. Chemotherapy treatment was well tolerated with G3 haematological toxicity in 30% of patients and G2 gastrointestinal toxicity in 20% of patients. Seven patients (31.8%) had a complete response (CR), 7 patients had a partial response (PR) (31.8%), stable disease was observed in 3 patients (SD) (13.7), while 5 patients progressed (PD) (22.7%). Median time to treatment failure was 5.6 months. Median survival, from start of chemotherapy, was 8 months 237 days (range 8 days-4 years). 12 patients received consolidation chemotherapy on the primary disease site. No patient developed a second tumor. A combined approach, in the treatment of oral cavity cancer, may give prolonged responses, with acceptable toxicity, without interfering on the quality of life.
Adult↗
[The why of a reform].
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Education, Medical↗
Triggering through CD16 or phorbol esters enhances adhesion of NK cells to laminin via very late antigen 6.
Very late antigens VLA-1, VLA-2, VLA-3, and VLA-6, belonging to the beta 1 subfamily of integrins, have been identified as receptors for different binding domains of laminin (LM). We have detected VLA-6, but not VLA-1 and VLA-2 on a subset (50-70%) of fresh peripheral blood CD3-, CD16+, CD56+ human natural killer (NK) cells by immunofluorimetric and biochemical analysis. Binding assays performed on LM-coated plates showed that 10-15% of NK cells spontaneously adhere to LM, and this adhesion is mediated by VLA-6. Activation of NK cells through CD16 triggering or by phorbol ester results in a rapid increase of adhesion to LM, which is still mediated by VLA-6. The enhanced adhesiveness is not associated with changes in beta 1 LM receptor expression, while it correlates with changes in the phosphorylation status of alpha 6 subunit. The expression of VLA-6 on NK cells and the modulation of its avidity by activating stimuli may be relevant for NK cell migration and tissue location during inflammation or immune response.
Animals↗
4-Hydroxynonenal, a product of cellular lipid peroxidation, which modulates c-myc and globin gene expression in K562 erythroleukemic cells.
Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Here, we show that 4-hydroxynonenal (HNE) concentrations close to the level found in normal cells (in the range of 1 and 3 microM) can specifically induce changes in the expression of c-myc and gamma-globin mRNA in K562 cells, without inducing any toxic effects or affecting cell viability. Since we have determined that K562 cells have undetectable levels of endogenous lipid peroxidation, all these effects can be assigned to the exogenous HNE treatment. After a 1-h treatment with 1 microM HNE, c-myc mRNA levels decrease transiently during the first 4 h, rebounding later to higher levels, and normalizing to basal expression after 4 days. Run-on experiments show a transient transcriptional block 20 min after HNE treatment and subsequent posttranscriptional regulation. According to S1 mapping, mRNA changes are exerted on c-myc transcripts initiated from both the principal constitutive start sites (P1 and P2). gamma-Globin mRNA levels concomitantly increase 3- to 4-fold, but no significant changes of housekeeping gene expression are observed. On the basis of these results it appears that the restoration in human erythroleukemic K562 cells of HNE concentrations closer to the level in normal cells can modulate the expression of specific genes.
Aldehydes↗
Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation.
CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.
Antigens, CD↗
Neuromodulatory loop mediated by nerve growth factor and interleukin 6 in thymic stromal cell cultures.
Neural crest cell derivatives have been suggested to be involved in thymus development. We established nonlymphoid thymic stromal cell cultures capable of supporting T-cell differentiation. In these nonlymphoid cell cultures, we identified cells with phenotypic and biochemical markers specific for neuronal cells. Neurofilament mRNA and 68- and 160-kDa neurofilament proteins, as well as 74-kDa synapsin I isoform, were expressed in many of the cultured cells. For example, neurofilament immunoreactivity was detected in 20-30% of the cells. To see whether thymic neuronal-like cells were involved in a neural differentiation pathway, we investigated the effect of nerve growth factor (NGF) and interleukin 6 (IL-6), two known neurotrophic factors. The expression of the above-described neural markers was enhanced by NGF and IL-6, which we report to be produced in an autocrine way by thymic stromal cell cultures. Finally, we found that IL-6 gene expression in these cell cultures was enhanced by NGF. Evidence is thus offered of a neuromodulatory loop within the thymic stromal cell population supported by local production of NGF and IL-6 and involving neural cell elements. Interestingly, IL-6, which is known to be implicated in thymocyte differentiation, also displays a neuromodulatory activity on thymic stromal cells, suggesting a multivalent role for this cytokine within the thymus.
Animals↗
Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression through impairment of the cooperativity between nuclear factor of activated T cells and AP-1 enhancer elements.
The immunosuppressant hormone dexamethasone (Dex) interferes with T cell-specific signals activating the enhancer sequences directing interleukin 2 (IL-2) transcription. We report that the Dex-dependent downregulation of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and calcium ionophore-induced activity of the IL-2 enhancer are mediated by glucocorticoid receptor (GR) via a process that requires intact NH2- and COOH-terminal and DNA-binding domains. Functional analysis of chloramphenicol acetyltransferase (CAT) vectors containing internal deletions of the -317 to +47 bp IL-2 enhancer showed that the GR-responsive elements mapped to regions containing nuclear factor of activated T cells protein (NFAT) (-279 to -263 bp) and AP-1 (-160 to -150 bp) motifs. The AP-1 motif binds TPA and calcium ionophore-induced nuclear factor(s) containing fos protein. TPA and calcium ionophore-induced transcriptional activation of homo-oligomers of the NFAT element were not inhibited by Dex, while AP-1 motif concatemers were not stimulated by TPA and calcium ionophore. When combined, NFAT and AP-1 motifs significantly synergized in directing CAT transcription. Such a synergism was impaired by specific mutations affecting the trans-acting factor binding to either NFAT or AP-1 motifs. In spite of the lack of hormone regulation of isolated cis elements, TPA/calcium ionophore-mediated activation of CAT vectors containing a combination of the NFAT and the AP-1 motifs became suppressible by Dex. Our results show that the IL-2-AP-1 motif confers GR sensitivity to a flanking region containing a NFAT element and suggest that synergistic cooperativity between the NFAT and AP-1 sites allows GR to mediate the Dex inhibition of IL-2 gene transcription. Therefore, a Dex-modulated second level of IL-2 enhancer regulation, based on a combinatorial modular interplay, appears to be present.
Base Sequence↗
Differential expression of granzyme A and granzyme B proteases and their secretion by fresh rat natural killer cells (NK) and lymphokine-activated killer cells with NK phenotype (LAK-NK).
Granzymes A-G are a family of serine proteases localized in the cytoplasmic granules of cytotoxic T lymphocytes (Masson, D. et al. Cell 1987. 49: 679) and granzyme A is secreted by T lymphocytes in response to antigenic stimulation. Granzyme A is also expressed by natural killer (NK) and lymphokine-activated killer (LAK) cells. Here we show that fresh rat NK cells constitutively express granzyme B and that granzyme A and granzyme B are differentially regulated in unstimulated NK cells vs. LAK cells with NK phenotype (LAK-NK cells). We also show that both granzymes A and B are secreted in a calcium-dependent manner, by NK and LAK-NK cells in response to stimuli which trigger NK cell functions.
Animals↗
The interaction between exogenous DNA and sperm cells.
Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.
Animals↗