bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of major heat shock protein hsp70 inhibits tumor necrosis factor-induced activation of phospholipase A2.
Authors: Jäättelä M
Published: December 2022

We have recently shown that major heat shock protein (hsp70) protects WEHI-S tumor cells from the cytotoxicity mediated by TNF. In the present study, the mechanism of hsp70-associated TNF resistance was investigated. Overexpression of human hsp70 or inhibition of endogenous hsp70 synthesis by expression of antisense hsp70 RNA did not change the ability of WEHI-S tumor cells to bind TNF or internalize and degrade the receptor-bound TNF. Moreover, TNF-induced activation of NF-kappa B-like transcription factors was unaffected by altered levels of hsp70 as tested by electrophoretic mobility shift assay. Thus, it is unlikely that the resistance is due to changes in TNF receptors or in their ability to transduce signals leading to the regulation of genes, whose expression is regulated by NF-kappa B-like transcription factors. The idea that hsp70-associated TNF resistance is independent of regulation of TNF-induced gene expression was further supported by the results showing that hsp70 protected WEHI-S cells from TNF-mediated killing also in the presence of inhibitors of either translation or transcription. Interestingly, TNF-induced activation of arachidonic acid metabolism correlated directly with their sensitivity to TNF and inversely with the amount of hsp70 in the cells. Furthermore, TNF-induced activation of arachidonic acid metabolism was inhibited in WEHI-S cells and two TNF-sensitive human cell lines by induction of the synthesis of endogenous heat shock proteins by heat shock. Even stronger inhibition of arachidonic acid metabolism was seen in WEHI cells rendered TNF-resistant by culturing them in the presence of increasing concentrations of TNF. These cells also had reduced numbers of type 1 TNF receptors. Overexpression of a low molecular weight heat shock protein hsp27 in WEHI-S cells had no effect on any of the parameters studied. These results show that both hsp70-mediated and TNF-induced TNF resistance are associated with a reduced activation of phospholipase A2 suggesting that phospholipase A2 plays an essential role in TNF-mediated cytotoxicity and that hsp70 interferes with the signal transduction pathway leading to its activation.
Mouse Crry/p65. Characterization of monoclonal antibodies and the tissue distribution of a functional homologue of human MCP and DAF.
Authors: Li B , Sallee C , Dehoff M , Foley S , Molina H , Holers V M
Published: December 2022

In previous studies we have demonstrated that mouse Crry/p65 regulates complement component C3 deposition on self membranes, a functional property that both human decay-accelerating factor (DAF) and membrane cofactor protein (MCP) exhibit. We have proposed that Crry/p65 has a similar biologic role in mouse as MCP and perhaps DAF and is the mouse analogue of one or both of these proteins. In order to address this hypothesis and further study Crry/p65, we have prepared rat mAb and a rabbit polyclonal Ab to this protein. Using these reagents we demonstrate that, like human MCP and DAF, the tissue distribution of Crry/p65 is very broad. Most if not all cells of nonneuronal origin express this protein. In addition, by immunohistochemical analysis, Crry/p65 is shown to be more highly expressed in some tissues at potential sites of immune complex deposition and damage, such as the mesangium of the renal glomerulus and the arterial vessel endothelium. By Western blot analysis, protein isoforms can be demonstrated. Unlike human DAF, however, no phosphatidylinositol-specific phospholipase C-sensitive Crry/p65 protein form can be demonstrated on lymphocytes or erythrocytes. Five of six anti-Crry/p65 mAb can partially or completely reverse the capacity of Crry/p65 to block C3 deposition on cell membranes. Analysis of four IgG rat anti-Crry/p65 mAb demonstrates that two major independent epitopes can be detected. Overall, Crry/p65 retains many of the major features of human MCP and DAF, and the use of these reagents should further the understanding of the biologic roles of this class of complement regulatory proteins.
Cytokine expression after the topical administration of substance P to human nasal mucosa. The role of substance P in nasal allergy.
Authors: Okamoto Y , Shirotori K , Kudo K , Ishikawa K , Ito E , Togawa K , Saito I
Published: December 2022

Human nasal mucosal samples exposed in vitro to substance P or allergenic Ag were tested for the mRNA of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, and IFN-gamma using specific reverse transcriptase-polymerase chain reaction assays. After the administration of substance P, at dosages ranging from 10(-6) to 10(-9) M, an enhanced expression of the mRNA for IL-1 beta, -3, -5, -6, TNF-alpha, and IFN-gamma was observed in all mucosal samples of allergic subjects and in half of the nonallergic subjects. The expression of IL-2 and IL-4 was low. Mucosal samples of allergic subjects showed an increased expression of mRNA for cytokines after administration of specific Ag, whereas no enhancement was observed in samples from nonallergic subjects. Our data suggest that substance P may regulate allergic reactions via enhanced production of certain regulatory cytokines.
Superantigenic properties of a novel mitogenic substance produced by Yersinia pseudotuberculosis isolated from patients manifesting acute and systemic symptoms.
Authors: Uchiyama T , Miyoshi-Akiyama T , Kato H , Fujimaki W , Imanishi K , Yan X J
Published: December 2022

A novel product from Yersinia pseudotuberculosis exhibiting mitogenic activity for human PBMC (designated Y. pseudotuberculosis-derived mitogen, YPM) was examined for its biologic activities for human lymphocytes. YPM induced a substantial proliferative response and IL-2 production at 0.1 ng/ml or more in whole PBMC but not in T-depleted PBMC. IL-2 production occurred within 12 h after YPM stimulation. T cells from PBMC produced IL-2 in the presence of L cells transfected with HLA DR genes or DP genes but not in the presence of control L cells used as accessory cells. Paraformaldehyde fixation did not abolish the AC activity of the DR+ L cells. The results suggest that YPM bind directly to HLA class II molecules. Analysis of the TCR V beta element using the polymerase chain reaction revealed that YPM selectively activated human T cells bearing V beta 3, V beta 9, V beta 13.1, and V beta 13.2 in TCR. These results indicate that YPM is a potent T cell activator and has superantigenic properties (abilities to bind directly to MHC class II molecules and selectively stimulate T cell populations bearing particular V beta elements in TCR). The role of YPM in the mechanism of pathogenesis of Y. pseudotuberculosis infections manifesting acute and systemic clinical symptoms is discussed.
Stimulation of mitogen-activated protein kinase activity by different secretory stimuli in rat basophilic leukemia cells.
Authors: Offermanns S , Jones S V , Bombien E , Schultz G
Published: December 2022

Ag-induced cross-linking of IgE bound to its high affinity receptor (Fc epsilon RI) at the surface of basophils or mast cells triggers a number of biochemical events culminating in the release of several inflammatory mediators. In rat basophilic leukemia (RBL-2H3) cells expressing the G protein-coupled m1 muscarinic receptor, Ag/IgE-induced cross-linking of Fc epsilon RI, calcium ionophore A23187, and carbachol through M1 receptors stimulated tyrosine phosphorylation of several proteins, including two of 42 and 44 kDa. Proteins of identical molecular masses were recognized by anti-MAP-kinase antibodies, and these immunoreactive proteins exhibited in part a slightly increased molecular mass on SDS polyacrylamide gels after incubation of cells with secretory stimuli. All stimuli led to the activation of MAP kinase, which co-purified on Mono Q chromatography with 42- and 44-kDa proteins, which were tyrosine phosphorylated in response to secretory stimuli and reacted with anti-(MAP kinase) antibodies. Finally, 42- and 44-kDa proteins immunoprecipitated by anti-MAP-kinase antibodies and anti-phosphotyrosine antibodies were recognized by anti-phosphotyrosine and anti-MAP-kinase antibodies, respectively. Primarily threonine and tyrosine residues were found to be phosphorylated in 42- and 44-kDa proteins immunoprecipitated from [32P]phosphate-labeled cells that had been treated with secretory stimuli. The dose dependence of secretagogue-induced MAP kinase activation correlated with that of increases in serotonin release from activated cells, and the maximum of MAP kinase activation coincided with the maximum rate of secretion. Down-regulation or inhibition of protein kinase C as well as incubation of cells with the tyrosine kinase inhibitor genistein markedly inhibited MAP kinase activation in parallel with serotonin release. Taken together, these findings demonstrate that 42- and 44-kDa MAP kinases are activated in response to secretory stimuli and provide some evidence for a functional link between MAP kinase activation and signaling events leading to mediator release in RBL cells.
Signal transduction mechanisms for leukotriene B4 induced hyperadhesiveness of endothelial cells for neutrophils.
Authors: Palmblad J , Lerner R , Larsson S H
Published: December 2022

We have previously demonstrated that leukotriene B4 (LTB4) induces in vitro a transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC) for neutrophils (PMN). The magnitude of this response is intermediate of that conferred by thrombin and by platelet-activating factor (PAF). This report shows that the LTB4 response was neither related to HUVEC expression of PAF (because it could not be blocked by the PAF receptor antagonist WEB-2086), nor to access to LTB4 receptors on neutrophils (as shown by LTB4 receptor desensitization experiments). However, it could be partly blocked by treating HUVEC with an LTB4 receptor antagonist (SC-41930). LTB4 evoked a rise of intracellular calcium concentrations, [Ca2+]i, in the HUVEC, and the hyperadhesive HUVEC response to LTB4 was abrogated by buffering of [Ca2+]i by Quin-2. The response was not inhibited by treating HUVEC with pertussis toxin before LTB4. Neutrophils showed no signs of activation when adhering to LTB4-treated HUVEC because they did not i) release lactoferrin, or ii) react with an increase of [Ca2+]i, and iii) they bound equally well to the stimulated endothelial cells after having been treated with pertussis toxin so that up-regulation of PMN adhesion to LTB4 was abolished. LTB4-treated HUVEC did not shed factors that modulated neutrophil adherence or chemotaxis. Thus, LTB4 promotes HUVEC hyperadhesiveness for PMN, and the transduction mechanism involves calcium ions, may depend on a surface receptor for LTB4, but does not involve pertussis toxin-sensitive G proteins or PMN activation.
HIV-1 infection of human CD4+ T cells in vitro. Differential induction of apoptosis in these cells.
Authors: Martin S J , Matear P M , Vyakarnam A
Published: December 2022

The H9 and CEM CD4+ T cell lines were infected with HIV-1 (NY5/LAV-1 isolate) and monitored for losses in cell viability, syncytium formation, and internucleosomal DNA cleavage (a marker for apoptosis). H9 cells were found to undergo cell death via apoptosis as a result of HIVNY5 infection, but this effect was not apparent in CEM cell cultures. The differential effects of HIV-1NY5 in terms of its apoptosis-inducing properties correlated with the relative abilities of H9 and CEM cells in supporting replication of this HIV-1 isolate, since infected CEM cell cultures produced 10-fold lower levels of HIV-1 p24 protein, and very few of these cells stained positive for cell-associated p24 by comparison with H9 cell cultures infected at the same multiplicity of infection. Furthermore, a different HIV-1 isolate (RF), which replicated equally efficiently in both H9 and CEM cells, produced similar levels of apoptosis in these cultures. HIV-1NY5 was also found to be capable of inducing apoptosis in purified peripheral blood CD4+ T cells as well as inhibiting anti-CD3-driven proliferation of these cells. In contrast, incubation of purified CD8+ T cells with HIV-1NY5 under similar conditions produced no cytopathic effects. Substantial levels of apoptosis were also recorded in HIV-1NY5-infected PHA blasts cell cultures. Soluble rHIV-1IIIB type CHO-derived gp120 was found to mimic the effects of HIV in terms of inhibition of anti-CD3/TCR mAb-induced proliferation of T cells, but apoptosis was not detected in gp120-treated T cell cultures whether cross-linked or used in conjunction with anti-CD3 mAb or not. We conclude therefore that both HIV-1NY5 and HIV-1RF isolates have the capacity to directly trigger apoptotic cell death in CD4+ T cells and that this appears to be at least partly associated with the efficiency of virus replication in these cells.
Inhibition of IgE production and normalization of airways responsiveness by sensitized CD8 T cells in a mouse model of allergen-induced sensitization.
Authors: Renz H , Lack G , Saloga J , Schwinzer R , Bradley K , Loader J , Kupfer A , Larsen G L , Gelfand E W
Published: December 2022

The functional role of CD8 T cells in in vivo IgE production, immediate cutaneous reactivity, and altered airways responsiveness (AR) was examined in a murine model of allergen-induced sensitization. Exposure of BALB/c mice to nebulized OVA triggered an IgE anti-OVA response in the serum, immediate-type skin test responses to OVA, and the development of increased AR (as measured by nonspecific reactivity to electrical field stimulation). In spleens of sensitized mice, analysis of the distribution of CD4/CD8 T cell subpopulations revealed an increase in total numbers of CD8 T cells. Transfer of purified spleen CD8 T cells from OVA-sensitized mice (CD8OVA) to sensitized recipients reduced serum IgE anti-OVA production by roughly 50%. Furthermore, studies of in vitro Ig production indicated that mononuclear cells from recipients of CD8 cells (CD8OVA > CD8PBS) produced less IgE and IgG1 antibodies, whereas in vitro IgG2a production was enhanced. The suppression of IgE production in recipients of CD8OVA T cells was associated with the failure to respond to intradermal challenge with OVA. The increase in AR found in sensitized mice was prevented after transfer of CD8OVA cells. When CD8 T cells from nonimmunized animals (CD8PBS) were used for the transfer into sensitized recipients, serum anti-OVA IgE was decreased by only 20%, whereas skin test reactivity and AR were not significantly affected. The ex vivo analysis of the pattern of cytokine-producing lymphocytes by immunofluorescence microscopy indicated that the sensitization procedure increased the fraction of IFN-gamma- and IL-4-positive cells in the spleen. Further in vitro analysis demonstrated that a high percentage of CD8 T cells were positive for IFN-gamma, whereas IL-4 was produced mainly by CD4 T cells. These data suggest that CD8 T cells may play an important role in the negative regulation of IgE production and AR and that IFN-gamma may be a relevant mediator of the functions of CD8 T cells in this model.
Regulation of cytokine gene expression in T helper cell subsets.
Authors: Lederer J A , Liou J S , Todd M D , Glimcher L H , Lichtman A H
Published: December 2022

We have examined the transcriptional regulation of the Th1-specific IL-2 gene and the Th2-specific IL-4 gene by transient transfection of promoter-chloramphenicol acetyl transferase (CAT) constructs into T cell clones and by electrophoretic mobility shift assays. Transfection of the Th2 clone D10.G4 with IL-4 promoter-CAT constructs demonstrated anti-CD3 inducible IL-4 promoter activity. In contrast, CAT constructs containing murine IL-2 promoter sequences were not inducible in D10.G4 cells. Transfection analyses in the Th1 clone D1.1 demonstrated inducible IL-2 promoter activity but no IL-4 promoter activity. Electrophoretic mobility shift assays using well-defined regulatory elements in the murine IL-2 gene promoter showed that anti-CD3 stimulation of Th2 clones failed to induce a characteristic increase in the ratio of p65-p50:p50-p50 NF-kappa B binding complexes in the nucleus that did occur in IL-2-producing clones. In addition, other protein complexes with NF-kappa B binding sequences were seen using lysates from Th1 and Th0 cells but not Th2 cells. Thus, expression of the promoter-CAT constructs directly correlates with endogenous IL-2 and IL-4 gene expression in Th1 and Th2 clones, confirming that the differential expression of IL-2 and IL-4 genes in these T cells is transcriptionally controlled. Furthermore, the lack of IL-2 transcription in activated Th2 cells is associated with the failure to generate required IL-2 gene promoter binding proteins, particularly NF-kappa B in the nucleus.
CD23/IgE-mediated regulation of the specific antibody response in vivo.
Authors: Gustavsson S , Hjulström S , Liu T , Heyman B
Published: December 2022

We have recently reported that IgE Abs specific for TNP are able to enhance the specific IgG response in mice via the low affinity receptor for IgE, Fc epsilon RII, or CD23. In this study we show that IgE can up-regulate IgM, IgG1, IgG2a, and the IgE response, thereby indicating the possibility of a viscious circle in the maintenance of an allergic response. One of the suggested modes of action of IgE/CD23 is to increase the ability of B cells to present Ag to T cells. The involvement of T cells in IgE-mediated enhancement of the Ab response was studied in several ways: nude mice were resistant to the effect of IgE and a dramatic effect on the induction of immunologic memory was seen, both by in situ secondary immunizations and in adoptive transfer systems. Basic conditions for the ability of IgE to induce enhancement were established, demonstrating critical importance of factors such as type of Ag and temporal relationship between administration of IgE and Ag. Finally, no evidence for the requirement for CD23 for a normal (non-IgE induced) Ab response was found, although modulation of the receptor completely abrogated the IgE-induced Ab response.
Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis.
Authors: Mower D A , Peckham D W , Illera V A , Fishbaugh J K , Stunz L L , Ashman R F
Published: December 2022

Mature resting mouse spleen B cells progress stochastically into apoptosis at a uniform rate over the first 16 h in vitro in 3 stages. In stage 1, early apoptotic B cells decreased the normal phospholipid packing of their plasma membranes, detected as increased binding of the lipophilic dye merocyanine 540, and also decreased in volume, detected as decreased forward scatter. In stage 2 there was abrupt internucleosomal cleavage of DNA, quantitated as hypodiploid nuclei by flow cytometry. Some stage 2 cells entered stage 3, where the plasma membrane became permeable to propidium iodide. B cells in later stages of this sequence retained the characteristics of earlier stages, whereas nonapoptotic B cells remained in their original state. Cycloheximide increased the progression of B cells through these three stages, whereas dextran sulfate inhibited stage 1 more effectively than stages 2 or 3. Increased orthogonal scatter also occurred late in some of the cells that had passed through stage 1, but did not correlate well with propidium iodide permeability. Fresh small dense spleen B cells contained 5% to 7% stage 1 cells but only about 1% stage 2 cells. Macrophages have been reported to destroy preferentially apoptotic thymocytes by recognizing plasma membrane alterations deriving from loose packing of phospholipid head groups. The recognition of stage 1 rather than stage 2 B cells by macrophages may help to keep the proportion of apoptotic cells low in vivo.
A nonimmunodominant nucleoprotein-derived peptide is presented by influenza A virus-infected H-2b cells.
Authors: Oukka M , Riché N , Kosmatopoulos K
Published: December 2022

Influenza A virus-infected H-2b mice mount a CTL response directed against the nucleoprotein (NP) 366-374 but not against the NP 55-63 peptide, although both peptides fulfill the prerequisites for having high binding affinity toward the Db molecule. Two hypotheses have been proposed to explain the inability of B6 mice to respond to the NP 55-63 peptide: 1) B6 mice are tolerant to the NP 55-63 peptide and 2) NP 55-63 peptide is not naturally processed by H-2b cells. Our results show that 1) B6 mice possess a NP 55-63-specific CTL repertoire because their immunization with the NP 55-63 peptide itself recruits specific CTL; 2) NP 55-63 peptide is naturally processed by the virus-infected H-2b cells but its efficient presentation by the Db molecule requires higher amounts of NP than presentation of the NP 366-374 peptide; 3) NP 55-63 peptide is naturally presented in virus infected B6 mice, however, the quantity of Db/NP 55-63 complexes at the cell surface is sufficient to tolerize but not to recruit and stimulate specific CTL; and 4) NP 55-63 peptide binds to the Db molecule with a lower affinity than NP 366-374 does and this difference could explain the inefficient presentation of the NP 55-63 peptide by B6 cells. The involvement of the self-protein-derived nonimmunodominant peptides in self-tolerance and the possibility of using nonimmunodominant peptides of viral proteins for peptide vaccination are discussed.
B cells from M167 mu kappa transgenic mice fail to proliferate after stimulation with soluble anti-Ig antibodies. A model for antigen-induced B cell anergy.
Authors: Sieckmann D G , Holmes K , Hornbeck P , Martin E , Guelde G , Bondada S , Longo D L , Kenny J J
Published: December 2022

The transgenic (TG) mouse strain 207-4, carries mu a + kappa transgenes ligated to the anti-phosphocholine (PC) VH1 and V kappa 24 V region genes from the MOPC-167 myeloma. Although B cells from mice carrying these transgenes respond both in vivo and in vitro to thymus-dependent Ags, they failed to proliferate in response to soluble goat anti-mu Ab or other soluble anti-Ig reagents. On the other hand, B cells from the Sp6 mu kappa anti-trinitrophenyl TG mouse line proliferated normally after stimulation with soluble anti-mu. However, the 207-4 anti-PC transgene positive (TG+) splenic B cells proliferated when stimulated with anti-mu, anti-idiotype, anti-allotype, or PC-conjugated to Sepharose beads. TG+ B cells were also induced to proliferate when stimulated with anti-Lyb-2; thus, their defect may be restricted to signaling through sigM. The lack of response to soluble anti-mu could not be reversed by addition of IL-4, by removal of T cells, by addition of anti-FcR Ab, or by stimulation with F(ab')2 anti-mu. Thus, the failure to proliferate was not caused by active T cell suppression or FcR-mediated inhibition. In mixed cultures of TG+ and transgene negative (TG-) spleen cells, the TG- cells were able to proliferate normally to soluble anti-mu, indicating that suppressive factors were not involved in the unresponsiveness of the TG+ anti-PC-specific B cells. These studies suggest that B cells in the 207-4 anti-PC TG mice exhibit a defect in activation through their sIgM receptors, and this unresponsiveness may reflect a form of Ag-induced tolerance.
Maternal anti-placental reactivity in natural, immunologically-mediated fetal resorptions.
Authors: Tangri S , Wegmann T G , Lin H , Raghupathy R
Published: December 2022

Observations on maternal recognition of the fetus and the demonstration of the effects of cytokines on reproductive events led to the "immunotrophism" model, which suggests that maternal immune recognition of fetally-derived Ags results in the release of cytokines that promote the growth of the placenta; any disturbance in this balance of cytokines could result in deleterious consequences for the placenta and, in turn, the fetus. We have focused our attention on the murine CBA/J x DBA/2 model of spontaneous abortions and compared them with normal CBA x BALB/c pregnancies. Our results indicate that the extent of stimulation of maternal strain lymphocytes in response to stimulator placental cells in mixed lymphocyte-placenta reactions (MLPR) was much higher in the normal mating combination compared with the abortion-prone mating combination. Cytokine analysis of the supernatants from MLPR indicates that there is significantly higher production of TNF-alpha, IFN-gamma, and IL-2 in supernatants from the abortion-prone combination than in supernatants from the normal combination. Furthermore, MLPR-stimulated cells induce resorptions in normal pregnant mice; maternal strain lymphocytes stimulated by placentas from the abortion-prone combination induce high rates of fetal resorptions, but lymphocytes stimulated with placentas from the normal combination do not. Together, these results suggest that immunologically mediated fetal resorptions probably result from improper or inappropriate maternal responses to placental Ags. Our observations also suggest that such effects are probably mediated by cytokines.
Lipopolysaccharide LPS-mediated soluble TNF receptor release and TNF receptor expression by monocytes. Role of CD14, LPS binding protein, and bactericidal/permeability-increasing protein.
Authors: Leeuwenberg J F , Dentener M A , Buurman W A
Published: December 2022

Previously we demonstrated that two soluble(s) tumor necrosis factor receptors, TNF-R55 as well as sTNF-R75, are constitutively released in vitro by monocytes, and that this release was markedly enhanced after activation. Because LPS is an important activator of monocytes, we investigated the effect of LPS on sTNF-R release by monocytes. It was found that release of sTNF-R75, but not (or minimally) release of sTNF-R55, was enhanced after activation with LPS, reaching plateau levels after approximately 2 days. CD14, one of the membrane receptors for LPS, is an intermediate in this process, as shown in experiments using mAb directed against CD14. Under serum-free conditions, LPS-induced sTNF-R75 release was less as compared with release in the presence of serum, suggesting involvement of serum proteins. Addition of LPS binding protein (LBP) enhanced the LPS-induced sTNF-R75 release under serum-free conditions, but had no effect in the presence of serum. On the other hand, bactericidal/permeability-increasing protein (BPI), known to possess LPS neutralizing activity, inhibited LPS-induced sTNF-R75 release. Furthermore, cell surface expression of both types of TNF-R was shown to be controlled by LPS, LBP, and BPI. LPS caused, within 1 h, a complete reduction of TNF-R55 as well as TNF-R75 expression, followed by enhanced re-expression of both receptors after 24 h. The down-modulation of expression was increased by LBP, whereas BPI counteracted the LPS-induced down-regulation. The LPS-enhanced release of sTNF-R75, capable of inactivation of TNF, as well as LPS-induced initial down-modulation of TNF-R expression leading to postulated temporary unresponsiveness to TNF may share in a physiological mechanism to carefully control the effects of TNF.
IFN-gamma up-regulates platelet-activating factor receptor gene expression in human monocytes.
Authors: Ouellet S , Müller E , Rola-Pleszczynski M
Published: December 2022

Human monocytes constitutively express the platelet-activating factor receptor (hPAF-R). In this report, we have investigated the modulation of hPAF-R by IFN-gamma. Treatment of monocytes with IFN-gamma caused a time- and concentration-dependent accumulation of hPAF-R mRNA. In contrast, IFN-alpha was inactive. The effect of IFN-gamma was rapid, evident by 1 h of stimulation and reaching a maximum after 2 h. The high level of hPAF-R mRNA was maintained for at least 24 h. Flow cytometry analysis revealed that monocytes treated with IFN-gamma had a two- to sixfold increase in PAF receptor expression at the cell surface, when compared with untreated cells. The increase in hPAF-R expression was associated with an augmented response of IFN-gamma-treated cells to PAF in terms of cytosolic calcium ([Ca2+]i) variations. The IFN-gamma-dependent accumulation of hPAF-R mRNA was not due to the stabilization of hPAF-R mRNA, as shown by unchanged hPAF-R mRNA t1/2. Pretreatment of monocytes with actinomycin D, however, completely abrogated the effect of IFN-gamma, suggesting a transcriptional regulation. Moreover, the up-regulation of hPAF-R mRNA by IFN-gamma was independent of de novo protein synthesis since cycloheximide, an inhibitor of protein synthesis, did not affect this up-regulation. These studies are the first report showing that IFN-gamma regulates hPAF-R gene expression in human monocytes, by a mechanism suggesting transcriptional regulation. This may represent a prototypic example of regulation by lymphocyte-derived cytokines of lipid mediator receptors in myeloid cells, thus adding a novel element in the interrelationship between immune and inflammatory responses.
A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.
Authors: Yamamoto N , Lindsay D D , Naraparaju V R , Ireland R A , Popoff S N
Published: December 2022

Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages.
Inhibition of T cell activation by the extracellular matrix protein tenascin.
Authors: Hemesath T J , Marton L S , Stefansson K
Published: December 2022

Tenascin (TN) is an extracellular matrix protein that is expressed widely in the fetus and sparingly in the adult, but reappears at high levels in certain areas of tissue insult such as tumor matrices and sites of wound healing. We show here that soluble TN inhibits proliferation of human T cells in response to alpha CD3 Ab co-immobilized with the extracellular matrix protein fibronectin (FN). TN also inhibits proliferation driven by alpha CD3/IL-2 or by phorbol ester/IL-2, and it prevents high level induction of IL-2R. The presence of TN in culture medium does not detectably alter the pattern of tyrosine phosphorylation resulting from T cell triggering with alpha CD3, but at later time points prevents the appearance of functional NF-AT1 transcription factor complexes in T cell nuclear extracts. These findings are consistent with the postulated role for TN as a natural antagonist to FN action, and suggest that T cell responses occurring at tissue sites in which TN is expressed could be influenced by its presence.
Cell surface distribution of high-avidity LFA-1 detected by soluble ICAM-1-coated microspheres.
Authors: Pyszniak A M , Welder C A , Takei F
Published: December 2022

Murine recombinant soluble ICAM-1 (sICAM-1) was immobilized on polystyrene microspheres. The binding of sICAM-1-coated microspheres to splenic T cells required previous activation of the cells and was inhibited by antibody to LFA-1 (CD11a/CD18), indicating that sICAM-1-coated microspheres bind exclusively to high-avidity LFA-1. The cytoskeleton inhibitor cytochalasin B did not inhibit the binding of sICAM-1-coated microspheres to PMA-activated splenic T cells, whereas their adhesion to sICAM-1 immobilized on microtiter wells was almost completely inhibited. The murine T hybridoma T28 cells on activation with PMA also bound sICAM-1-coated microspheres, and the binding sites on the cell surface seemed localized on some of the cells, whereas fluorescence staining showed an even distribution of LFA-1 on the cell surface. In contrast, the murine B cell line A20A8 and monocytic line P388 showed a more even distribution of sICAM-1 binding sites. To further investigate the distribution of high-avidity LFA-1, murine fibroblast L cells expressing LFA-1 were generated by gene transfer. The transfected L cells constitutively expressed high-avidity LFA-1 and bound sICAM-1-coated microspheres without previous activation. Interestingly, the binding sites seemed highly localized on most cells. In contrast, the binding sites for anti-LFA-1 Ab-coated microspheres were randomly distributed on the transfected L cells. Furthermore, fluorescence staining also revealed a uniform punctate distribution of LFA-1 on the surfaces of these cells. These results show that 1) sICAM-1-coated microspheres represent a useful tool in identifying high-avidity LFA-1, 2) the binding of sICAM-1-coated microspheres to high-avidity LFA-1 does not require an intact cytoskeleton, and 3) the cell surface distribution of high-avidity LFA-1 can be different from that of LFA-1 in general, and the former seems highly localized on some cells.
Cytokine regulation of early human lymphopoiesis.
Authors: Ryan D H , Nuccie B L , Ritterman I , Liesveld J L , Abboud C N
Published: December 2022

An in vitro culture system in which bone marrow-derived fibroblast-like cells support the growth of TdT+ colonies derived from CD34+/CD10- human bone marrow progenitor cells has recently been described. The regulatory role of cytokines during early B lineage commitment was investigated using this culture system. Expression of IL-7, a cytokine that induces proliferation of B cell precursors, was detectable in the adherent layer by PCR and bioassay. Lymphoid progenitor colonies were inhibited by neutralizing anti-IL-7 Ab, suggesting that IL-7 produced by the adherent layer was required even in the earliest recognizable stages of human B cell lymphopoiesis. IL-1 alpha, IL-4, and TNF-alpha inhibited lymphoid progenitor colonies in a dose-dependent fashion. Neutralizing Ab to IL-1 alpha, IL-4, or TNF-alpha did not increase lymphoid progenitor colonies, suggesting that inhibitory concentrations of these cytokines are not constitutively elaborated in the adherent layer. Recombinant Steel factor and IL-6 as well as neutralizing Abs to these cytokines did not significantly affect lymphoid progenitor colonies, arguing against an important role for these cytokines in early human B lymphopoiesis. These results indicate that IL-7 provided by the bone marrow microenvironment is a critical growth factor at the earliest recognizable stages of human lymphopoiesis. IL-1 alpha, IL-4, and TNF-alpha have been shown to indirectly stimulate release of myeloid growth factors. The inhibition of lymphopoiesis by these cytokines suggests a possible mechanism for the observed reciprocal relationship between lymphoid and myeloid supportive bone marrow microenvironments.
Prostaglandin E2 inhibits T cell-dependent Ig secretion by neonatal but not adult lymphocytes.
Authors: Splawski J B , Lipsky P E
Published: December 2022

Prostaglandin E2 (PGE2) inhibits Ig production by adult B cells stimulated with Staphylococcus aureus and IL-2, and inhibits the production of IL-2 by anti-CD3 stimulated T cells. By contrast, PGE2 did not inhibit T cell-dependent Ig production by adult B cells. However, T cell-dependent Ig secretion by neonatal B cells was markedly inhibited by PGE2 even in the presence of supplemental IL-2. Forskolin, a direct activator of adenylate cyclase, and dibutyryl cAMP caused similar effects. PGE2 inhibited T cell-dependent Ig secretion by both neonatal CD5+ and CD5- B cells but did not inhibit Ig secretion by either CD5+ or CD5- adult peripheral blood B cells. PGE2 did not inhibit IL-2-dependent anti-CD3 stimulated neonatal T cell proliferation or T cell-dependent neonatal B cell proliferation. PGE2 inhibited neonatal B cell Ig production when addition was delayed, suggesting that initial B cell activation and proliferation were not blocked, but that PGE2 prevented subsequent differentiation. In addition, PGE2 inhibited neonatal T cell help independent of cytokine production. PGE2-induced cAMP levels in neonatal B and T cells were not different from adult levels. These results indicate that production of Ig by neonatal lymphocytes is uniquely sensitive to the inhibitory effects of agents such as PGE2 that elevate cAMP levels. This sensitivity may contribute to the suppressed in vivo responses of human neonatal B cells.
Functionally important amino acids in the TCR revealed by immunoselection of membrane TCR-negative T cells.
Authors: Caspar-Bauguil S , Arnaud J , Gouaillard C , Hou X , Geisler C , Rubin B
Published: December 2022

A spontaneous TCR cell surface variant (3P11) of the Jurkat T cell line is described and characterized. 3P11 was selected by incubation of Jurkat cells with anti-TCR mAb followed by passage through Ig anti-Ig columns and cloning. 3P11 contained mRNA for both Ti alpha and Ti beta and CD3 gamma, delta, epsilon and zeta. Biochemical analyses demonstrated that all of the TCR components were produced in 3P11 cells. The Ti alpha beta/CD3 gamma delta epsilon zeta complex was assembled in the endoplasmic reticulum but the zeta did not associate with this complex. Epitopes recognized by the Ti beta chain specific mAb beta F1 and JOVI as well as anti-V beta 8 were affected in the 3P11 Ti beta chain indicating that the 3P11 Ti beta chain was mutated. Transfection of a wild-type Ti beta cDNA into 3P11 cells reconstituted TCR expression. Sequence analyses of the 3P11 Ti beta chain demonstrated a guanine to adenine change in the second nucleotide of the triplet coding for cysteine191 resulting in a cysteine to tyrosine exchange. Cysteine191 is the C-terminal cysteine involved in the intrachain disulfide bond in the C domain of the Ti beta chain; thus, the 3P11 Ti beta chain did not contain this disulfide bond. Transfection of a site-directed Ti beta chain containing the 3P11 mutation into a Ti beta negative variant of the Jurkat cell line resulted in a TCR phenotype identical with 3P11 demonstrating that the mutation identified in the 3P11 Ti beta chain was the sole cause for the 3P11 defect.
Role of differential mRNA stability in the regulated expression of IgM and IgD.
Authors: Reed D J , Hawley J , Dang T , Yuan D
Published: December 2022

The expression of IgM and IgD heavy chain mRNA in resting vs activated B cells offers a unique tool for the assessment of the effect of translation on mRNA stability because mu and delta mRNA have identical VDJ sequences but differ substantially in the rest of the molecule. We have shown that despite the 5' UTR identity that allows equal access to the translation machinery, mu mRNA has a significantly higher turnover rate than delta mRNA. However, the short t1/2 of mu mRNA increases significantly after B cell activation. Furthermore, the induction of microS mRNA after B cell activation provides yet another related molecule for comparison. Thus, despite the fact that microS and microM mRNA differ at their 3' ends, they have identical turnover rates in activated B cells. In addition, because the turnover rates of delta mRNA and beta 2 and GAPDH mRNA remain unchanged, these experiments suggest that B cell activation results in the induction of regulatory factor(s) that target specific sequences within mRNA-mu to confer greater stability. They also argue against a more passive regulation of mRNA stability that is a consequence of alterations in the secretory machinery.
Stimulation of macrophage Fc gamma RIIIA activates the receptor-associated protein tyrosine kinase Syk and induces phosphorylation of multiple proteins including p95Vav and p62/GAP-associated protein.
Authors: Darby C , Geahlen R L , Schreiber A D
Published: December 2022

The role of tyrosine phosphorylation during signal transduction by the phagocytic macrophage (Mphi) FcR Fc gamma RIIIA was investigated. Cross-linking Fc gamma RIIIA on pulmonary Mphi or cultured monocytes used as in vitro model for differentiated Mphi induced rapid and transient phosphorylation of multiple protein substrates. The lymphocyte/mast cell 72-kDa protein tyrosine kinase (PTK) designated PTK72 or Syk, whose expression in Mphi was previously unknown, was identified as a major substrate by immunoprecipitation and phosphopeptide mapping. Activation of Fc gamma RIIIA stimulated a fourfold increase in Syk kinase activity assessed by autophosphorylation. Under mild lysis conditions several specific proteins were co-precipitated with the gamma subunit of Fc gamma RIIIA. Tyrosine kinase activity was also co-precipitated with gamma, as shown by in vitro phosphorylation of gamma. One of these kinases was identified as Syk by phosphopeptide mapping, suggesting a physical association between this PTK and the receptor. Two additional phosphoproteins induced by Fc gamma RIIIA cross-linking were identified: the hematopoietic proto-oncogene product p95Vav, previously implicated in B lymphocyte and mast cell signaling; and p62, a protein associated with p21Ras-GAP. Our results also establish that there are important functional similarities in signal transduction between a phagocytic Mphi FcR and other multi-subunit Ig gene family receptors in diverse cell lineages.
CD11b/CD18 (Mac-1) is required for degranulation of human eosinophils induced by human recombinant granulocyte-macrophage colony-stimulating factor and platelet-activating factor.
Authors: Horie S , Kita H
Published: December 2022

Recent evidence suggests adhesion molecules play an important role in the function of leukocytes. Because human eosinophils are known to express beta 2 integrins, we hypothesized that these adhesion molecules mediate the effector function of eosinophils. Normal human eosinophils incubated in albumin-coated polystyrene plates released granule protein and produced superoxide anion when stimulated with human recombinant granulocyte-macrophage CSF (rGM-CSF), platelet-activating factor (PAF), or PMA. Simultaneous monitoring of eosinophil adhesion and degranulation showed that degranulation was always preceded by cellular adhesion regardless of stimuli. Furthermore, eosinophil degranulation induced by human rGM-CSF and PAF was abolished in suspension culture of the cells when the cell suspensions were gently stirred. To identify the molecules involved in this adhesion-dependent degranulation, we have investigated the effects of mAbs (mAb) against beta 2 integrins. mAb reactive with CD18 markedly inhibited the eosinophil adhesion and degranulation induced by PAF and human rGM-CSF. mAb reactive with CD11b also moderately inhibited the adhesion and degranulation. In contrast, mAb reactive with CD11a slightly enhanced or showed no effect on the adhesion and degranulation by human rGM-CSF or PAF. Superoxide production induced by human rGM-CSF and PAF was also abolished by the treatment of cells with anti-CD18 mAb. mAb against CD11b and CD18 had little effect on degranulation and superoxide production induced by PMA. These results indicate that CD11b/CD18 (Mac-1)-dependent cellular adhesion plays an important role in the degranulation and superoxide production of eosinophils induced by human rGM-CSF and PAF, and that these mechanisms may be employed in vivo where eosinophils contact with stromal cells and/or proteins.
Antigenic surface of the bee venom allergen phospholipase A2. Structural functional analysis of human IgG4 antibodies reveals potential role in protection.
Authors: Dudler T , Schneider T , Annand R R , Gelb M H , Suter M
Published: December 2022

In the human immune response to the main bee venom allergen phospholipase A2 (PLA2), Abs of the IgG4 isotype are associated with protection. The antigenic sites of PLA2 that are recognized by two mAb of this isotype have been analyzed at the molecular level. The interaction of PLA2 with these two Abs has previously been shown to depend on the conformation of the Ag and to be sensitive to lysine modifications. Therefore, genetically engineered Ag displaying single point mutations of lysine residues were generated. Nine out of twelve surface-exposed lysine residues were substituted by glutamate or glutamine, and the effect of individual mutations on mAb binding was analyzed in the context of the folded Ag. Substitution of lysine at position 25 with either glutamate or glutamine completely abrogates binding to both mAb. All other mutants do not show a difference in binding when compared with wild-type Ag. Probing of sera from bee keepers with lysine 25 mutants reveals that a significant proportion of serum Abs of the IgG4 isotype display specificity for this epitope, suggesting that this represents a major B cell-antigenic site in hyperimmune individuals. We further found that both mAbs inhibit the catalytic activity of PLA2, a property that may account for the protective role of IgG4 Abs in hyperimmune individuals.
LFA-1-mediated antigen-independent T cell adhesion is regulated by CD4 and p56lck tyrosine kinase.
Authors: Mazerolles F , Barbat C , Meloche S , Gratton S , Soula M , Fagard R , Fischer S , Hivroz C , Bernier J , Sekaly R P
Published: December 2022

We examined the role of CD4 and p56lck in the regulation of LFA-1-dependent T cell adhesion to B cells and to fibroblasts expressing ICAM-1 and HLA-DR by using various transfectant constructions. Although CD4 transfection in CD4low HUT78 T cell lines did not significantly modify their maximal binding to B cells and fibroblasts, it made the LFA-1-dependent adhesion sensitive to inhibition by anti-CD4 Ab, HIV-1 (env) gp 160, and a 12-mer peptide encompassing the 35-46 sequence of the beta 1 domain of the MHC class II molecule. CD4low HUT78 T cell adhesion to B cells was stable over 60 min, whereas expression of CD4 led to a transient adhesion. In addition, adhesion of CD4+ T cells to MHC class II- B cells was also stable. The CD4-dependent alteration of adhesion required the association of CD4 with p56lck because expression of mutant forms of CD4 unable to bind p56lck resulted in a lack of CD4-dependent regulation of adhesion. Herbimycin A, an inhibitor of tyrosine kinase activity, reversed the effect of CD4 transfection on adhesion. These results indicate that ligand binding to CD4 delivers a signal-inducing cell dissociation by activating p56lck tyrosine kinase. This regulatory pathway may provide a quick and reliable way for multiple and subsequent Ag-independent adhesion events of CD4+ T cells.