bluuebunny/crossref_metadata_2025 huggingface dataset:
cDNA cloning of a NGFI-B/nur77-related transcription factor from an apoptotic human T cell line.
Authors: Okabe T , Takayanagi R , Imasaki K , Haji M , Nawata H , Watanabe T
Published: December 2022

A human T lymphoid cell line, PEER, dies by apoptosis in the presence of PMA and calcium ionophore. A new gene, TINUR, was cloned from apoptotic PEER cells. The expression of the TINUR gene is induced within 1 h after the cross-linking of the T cell Ag receptor complex. TINUR belongs to the NGFI-B/nur77 family of the steroid receptor superfamily and is an orphan receptor. TINUR binds to the same DNA sequence as NGFI-B/nur77. We also propose that the NGFI-B/nur77 family can be classified into two subtypes.
Transcription of the TCR-beta locus initiates in adult murine bone marrow.
Authors: Soloff R S , Wang T G , Lybarger L , Dempsey D , Chervenak R
Published: December 2022

Successful expression of the TCR beta-chain gene is a multistep process that involves: 1) initial transcription of multiple, unrearranged gene segments, 2) rearrangement of V, D, and J gene segments to form a complete beta-chain gene, and 3) transcription of the fully rearranged beta gene. All of these events have been shown to occur in the thymus, where the majority of T cell development takes place; however, the extent to which any of these events may occur prethymically has not been established. To examine prethymic TCR-beta gene expression, RNA was isolated from a precursor T cell-enriched population (Thy 1low CD3-) of C58/J mouse bone marrow, and analyzed by reverse transcriptase-PCR. A transcript containing TCR-beta constant (C) region sequences but not variable (V) region sequences was amplified, suggesting that an unrearranged TCR-beta gene locus is transcriptionally active in this bone marrow population. The same product was detected in Thy 1+ CD3- bone marrow cells from nude mice, indicating that the thymic microenvironment is not necessary for initiation of TCR-beta gene transcription. This C beta transcript is not confined to pre-B cells, as it was identified in RNA isolated from Thy 1low CD3- B220- bone marrow cells. Germline V beta transcripts were also detected in RNA from this bone marrow population. Furthermore, Sca-1+ Lin- and Sca-1+ Lin+ bone marrow populations from both C58/J mice and nude mice also expressed the C beta transcript. DNA-PCR analyses with D beta-J beta primer sets revealed that partial rearrangement of the beta locus had occurred in all bone marrow populations analyzed. These data suggest that both transcription and partial rearrangement of the TCR-beta locus can initiate in bone marrow cells of adult mice, before exposure of these cells to the thymus.
T lymphocytes infiltrating sites of tumor rejection and progression display identical V beta usage but different cytotoxic activities.
Authors: Kurt R A , Park J A , Panelli M C , Schluter S F , Marchalonis J J , Carolus B , Akporiaye E T
Published: December 2022

Most tumors grow progressively and overwhelm the host. The rare but documented cases of spontaneous regression of primary tumors are indicative of the potential of tumor-bearing hosts to develop a significant antitumor response. Because most tumors grow progressively in the host, it is not surprising that the majority of studies have focused on T lymphocytes that infiltrate these tumors. Although these studies have generated significant and useful information during the period of tumor growth, they can only speculate on the mechanisms that are involved in tumor rejection. We have used a well developed sponge model of concomitant tumor immunity that allows us to compare the immunologic events that occur during tumor progression vs rejection. In this model, an animal harboring a primary EMT6 mammary tumor is challenged with a secondary tumor implant through a pre-implanted gelatin sponge. During the manifestation of concomitant tumor immunity, the secondary tumor is rejected and the effector cells mediating the response are retained within the sponge matrix. Using this model we analyzed the TCR usage, cytotoxic activity of lymphocytes, and cytokine production at both tumor sites. The data revealed that tumor-rejecting lymphocytes isolated from the site of secondary tumor implant were cytotoxic toward EMT6 cells, whereas tumor-infiltrating lymphocytes isolated from the progressing primary tumor were not. Interestingly, the TCR-V beta repertoire of the tumor-infiltrating lymphocytes and tumor-rejecting lymphocytes were identical with V beta 1 and V beta 8 being predominant at both sites. Furthermore, the rejection site showed higher gene expression of IFN-gamma, TNF-alpha, and IL-10 whereas TGF-beta expression was slightly higher in the progressing tumors. These findings suggest that the disparate effector functions observed during tumor progression vs rejection are not caused by different T cell phenotypes but may be due instead to influences exerted by cytokines produced at the tumor sites.
Differentiation-dependent effects of IL-1 and TGF-beta on human articular chondrocyte proliferation are related to inducible nitric oxide synthase expression.
Authors: Blanco F J , Geng Y , Lotz M
Published: December 2022

This study analyzed the effect of chondrocyte differentiation on iNOS expression and responses to IL-1 and TGF-beta. During subculturing of chondrocytes, the growth-stimulatory effects of TGF-beta decreased, and cells in later passages even were growth inhibited by TGF-beta. IL-1 beta responses showed an inverse pattern. The antiproliferative effects of IL-1 beta decreased, and, after passage 6, IL-1 beta became a growth stimulator for chondrocytes. This change in growth factor response pattern was associated with a decrease in type II collagen expression. To determine whether these changes in the growth regulatory effects of IL-1 beta and TGF-beta were related to nitric oxide (NO), inducible nitric oxide synthase (iNOS) expression and NO release were analyzed. In primary chondrocytes, TGF-beta did not stimulate iNOS mRNA expression or NO release, and, during co-incubation, it did not detectably alter the IL-1 beta effect. Preincubation with TGF-beta resulted in a time-dependent increase in IL-1-induced NO. With increasing passage number, the IL-1 beta effects decreased, and, after passage 6, IL-1 beta no longer detectably stimulated iNOS expression or NO release. However, TGF-beta increased NO production synergistically with IL-1 beta during the same culture period when it lost its growth-stimulatory effects. The antiproliferative effects of TGF-beta in late passage chondrocytes were reversed by the NO synthase inhibitor NG-monomethylarginine. These results suggest a novel pattern of iNOS regulation by IL-1 and TGF-beta and show that the factors that modulate iNOS expression and proliferation are dependent on the differentiation status of the cells.
Exogenous and endogenous nitric oxide attenuates tumor necrosis factor synthesis in the murine macrophage cell line RAW 264.7.
Authors: Eigler A , Moeller J , Endres S
Published: December 2022

Effects of TNF on nitric oxide (NO) production have been well documented in a variety of experimental and clinical settings, as for example, in septic shock. Investigations focusing on an inverse relation of NO on TNF synthesis are rare. Previously we could demonstrate that exogenous NO-releasing agents suppress LPS-induced TNF production in human PBMC. We now investigate whether a regulatory role on TNF synthesis could also be ascribed to endogenous NO. This was studied in the murine macrophage cell line RAW 264.7, which is able to express both TNF and the inducible NO synthase. No production was determined by measuring nitrite with Griess reagents. TNF formation was quantified by L929 cytotoxicity. We found a suppression of LPS-induced TNF synthesis by the exogenous addition of NO-releasing agents in the murine cell line, as previously observed in human cells. The application of NO synthase inhibitors led to a decrease in NO production, associated with an increase in TNF synthesis. TNF production increased from a base line (stimulation with 1 microgram/ml LPS alone) of 20.8 ng/ml to 36.3 ng/ml (means of six experiments) in the presence of the NO synthase inhibitor NG-monomethyl L-arginine (100 microM). Similar results were obtained with another NO synthase inhibitor, NG-nitro L-arginine-methylester. Lack of L-arginine in the medium resulted in a threefold increase in LPS-stimulated TNF synthesis compared with medium containing the usual concentration of 1 mM L-arginine. Restitution of L-arginine but not of D-arginine reversed this increase in TNF synthesis in a dose-dependent manner. To our knowledge these results indicate for the first time a negative feedback by endogenous NO on TNF synthesis in vitro. This finding may be relevant in pathophysiologic processes in which both TNF and NO are formed and in experimental therapies aiming at changes of NO concentrations.
Inducible expression of type 2 IL-1 receptors by cultured human keratinocytes. Implications for IL-1-mediated processes in epidermis.
Authors: Groves R W , Giri J , Sims J , Dower S K , Kupper T S
Published: December 2022

Two species of cell surface receptor for IL-1 (IL-1R) have been characterized. Only one of these, the type 1 IL-1R, transduces a signal after ligand binding. Whereas mRNA for the nonsignal transducing type 2 IL-1R seems to have a broad tissue distribution, functional type 2 IL-1R has been carefully studied only in leukocytes and related cell lines. Because normal human keratinocytes, which are IL-1 alpha-producing epithelial cells, inducibly express large numbers of IL-1R, we have studied their putative type 1 and type 2 IL-1R at the level of RNA, protein, and biologic function. At the level of function, gene expression of the IL-1-inducible cytokine granulocyte-macrophage-CSF by keratinocytes was mediated entirely by low numbers of type 1 IL-1R, although type 2 IL-1R were more numerous on both resting and activated keratinocytes. Type I IL-1R mRNA was detected only at very low levels, whereas a marked induction of type 2 IL-1R mRNA was readily observed in activated keratinocytes. A sensitive and specific ELISA demonstrated shed type 2 IL-1R in the conditioned medium of IFN-gamma or PMA-activated keratinocytes. Keratinocyte type 2 IL-1R bound IL-1 alpha with higher affinity (Kd approximately 3 x 10(-9) M) than type 2 IL-1R on leukocytes; however, the intracellular epithelial form of the IL-1R antagonist was bound 100-fold less avidly. These findings demonstrate that a normal nontransformed epithelial cell may express large numbers of the nonsignal transducing type 2 IL-1R that binds IL-1 with high affinity and can be shed into the pericellular environment. This receptor may function as an IL-1 antagonist in autocrine, juxtacrine, and paracrine cutaneous pathways.
Hyaluronic acid enhances cell proliferation during eosinopoiesis through the CD44 surface antigen.
Authors: Hamann K J , Dowling T L , Neeley S P , Grant J A , Leff A R
Published: December 2022

We examined the effect of hyaluronic acid in promoting proliferation of undifferentiated progenitor cells through the CD44 receptor during eosinopoiesis in vitro. Undifferentiated umbilical cord blood cells were purified on the first day to isolate primitive progenitor cells expressing the CD34 hemopoietic surface marker. Culture in wells coated with 100 micrograms/ml hyaluronic acid caused a 198 +/- 28.7% augmentation of proliferation of CD34+ progenitor cells at 3 wk (p < 0.01). By contrast, concentrations of hyaluronic acid > 10 micrograms/ml inhibited proliferation of unfractionated cord blood mononuclear cells. The augmented proliferation of precursor cells caused by hyaluronic acid was associated with complete (93.0 +/- 5.12%) differentiation to eosinophil morphology. By contrast, concentrations of hyaluronic acid > or = 10 micrograms/ml inhibited eosinophilic differentiation of unfractionated mononuclear cells. Wright-Giemsa staining demonstrated 95.4 +/- 2.92% eosinophils for CD34+ cells cultured for 3 wk without hyaluronic acid (control) and 93.8 +/- 5.11% for CD34+ cells cultured in hyaluronic acid-coated wells (100 micrograms/ml); for unfractionated cells, 94.0 +/- 3.02% demonstrated eosinophilic morphology in control wells at 3 wk vs 55.4 +/- 8.34% in hyaluronic acid-coated (100 micrograms/ml) wells (p < 0.05). Augmented proliferation caused by hyaluronic acid was attenuated completely by the anti-CD44 mAbs, 212.3 and IM7.8.1. Pretreatment of CD34+ cells with 5 micrograms/ml 212.3 inhibited the augmented proliferation caused by the optimal concentration of hyaluronic acid (100 micrograms/ml) from 260 +/- 39.2% of control growth to 114 +/- 16.4% of control growth (p = 0.02). Inhibition was comparable for IM7.8.1. Control mAb (LM2) to the beta 2 integrin subunit CD11b had no effect on proliferation induced by hyaluronic acid. We demonstrate that hyaluronic acid stimulates the growth of CD34+ selected umbilical cord blood cells into specifically differentiated mature eosinophils. This process is modulated by the CD44 receptor on the progenitor cell population.
Effect of IL-6 receptor antisense oligodeoxynucleotide on in vitro proliferation of myeloma cells.
Authors: Keller E T , Ershler W B
Published: December 2022

IL-6 stimulates proliferation of various tumors, including lymphoma and myeloma; thus, inhibiting IL-6 may decrease the growth of these tumors. Accordingly, we examined the effect of IL-6 and IL-6R antisense phosphorothioated oligodeoxynucleotides (ODNs) on proliferation of IL-6 responsive (U266) and nonresponsive (RPMI 8226) myeloma cell lines. Cells were grown in the presence or absence of IL-6, with added antisense to either IL-6 or IL-6R. Cells were evaluated for proliferation ([3H]thymidine uptake) and steady state levels of both IL-6 and IL-6R mRNA by competitive PCR (C-PCR). Proliferation of U266 cells was decreased markedly by IL-6 antisense ODN in the absence of IL-6, but not in its presence. In contrast, IL-6R antisense ODN inhibited proliferation of U266 cells in both the presence and absence of IL-6. As anticipated, neither IL-6 nor IL-6R antisense ODN had an effect on RPMI 8226 proliferation. C-PCR demonstrated a marked and specific decrease of IL-6 and IL-6R mRNA in cells exposed to IL-6 ODN and IL-6R ODN, respectively. These results suggest that IL-6R antisense ODN may be a more effective inhibitor of IL-6-stimulated cells than IL-6 antisense in a therapeutic setting.
5-Oxo-6,8,11,14-eicosatetraenoic acid is a potent stimulator of human eosinophil migration.
Authors: Powell W S , Chung D , Gravel S
Published: December 2022

Human neutrophils and monocytes contain a highly specific dehydrogenase which converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). We have previously shown that 5-oxo-ETE is a potent stimulus of neutrophil calcium levels and migration and have now investigated its effects on human eosinophils. 5-Oxo-ETE is a potent stimulus of eosinophil migration, with significant effects being detected at concentrations as low as 1 nM and a maximal response at 1 microM. The responses elicited by 5-oxo-ETE were about two to three times greater than those to platelet-activating factor (PAF) and 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE) at all concentrations tested between 10 nM and 1 microM. Leukotrienes B4 and D4 also significantly stimulated eosinophil migration, but the maximal responses to these agonists were only about 4% of the maximal response to 5-oxo-ETE. A low concentration of 5-oxo-ETE (1 nM) potentiated eosinophil migration in response to PAF. Eosinophils were capable of converting 5-HETE to 5-oxo-ETE, and this reaction was enhanced by phorbol myristate acetate. Stimulation of eosinophils with A23187 in the presence of low concentrations of arachidonic acid and phorbol 12-myristate 13-acetate led to the formation of 5-oxo-ETE and 5-oxo-15-hydroxy-ETE, but the amounts were considerably less than those of other eicosanoids such as leukotriene C4, cysteine-containing lipoxins, and 5,15-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid. In summary, of all the lipid mediators tested, 5-oxo-ETE was the most effective in stimulating migration of human eosinophils. Although eosinophils are capable of synthesizing 5-oxo-eicosanoids, the amounts detected were relatively small, and other leukocytes such as neutrophils, monocytes, or macrophages may be more important sites for the synthesis of this compound.
Monocyte chemotactic protein MCP-2 activates human basophil and eosinophil leukocytes similar to MCP-3.
Authors: Weber M , Uguccioni M , Ochensberger B , Baggiolini M , Clark-Lewis I , Dahinden C A
Published: December 2022

It has been shown that CC chemokines activate basophil and eosinophil leukocytes with different selectivities and patterns of activity. The most effective are monocyte chemotactic protein-1 (MCP-1), a potent stimulus of mediator release in basophils without effects on eosinophils, RANTES, a weak stimulus of release and strong chemoattractant for basophils and eosinophils, and MCP-3, which combines the activities of MCP-1 and RANTES. We have now compared MCP-2, which has 62 and 60% of sequence identity with MCP-1 and MCP-3, respectively, with the other CC chemokines. MCP-2 induced mediator release by human basophils with lower efficacy and potency than MCP-1 and MCP-3. It promoted transient changes of cytosolic-free calcium concentration ([Ca2+]i) and chemotactic responses in both basophils and eosinophils, however somewhat less efficiently than MCP-3 and RANTES. Desensitization studies indicate that MCP-2 interacts with receptors recognizing MCP-1 as well as RANTES. These results demonstrate that MCP-2 and MCP-3 exert qualitatively similar biologic activities on basophils and eosinophils. In basophils that had not been treated with IL-3, MCP-2 induced minimal exocytosis only, but desensitized the cells toward MCP-1 and MCP-3, suggesting that MCP-2 may act as a functional inhibitor of CC chemokine actions. The results of this study further indicate that MCP analogues display partially distinct, partially overlapping bioactivities toward eosinophils and basophils, and may thus regulate inflammatory processes involving these effector cell types.
CTLs from lymphoid organs recognize an optimal HLA-A2-restricted and HLA-B52-restricted nonapeptide and several epitopes in the C-terminal region of HIV-1 Nef.
Authors: Hadida F , Haas G , Zimmermann N , Hosmalin A , Spohn R , Samri A , Jung G , Debre P , Autran B
Published: December 2022

In a previous analysis of HIV-1-specific CTLs in lymphoid organs from HIV-seropositive patients, we reported high frequencies of in vivo differentiated CTLs directed against two immunodominant regions in the central and in the C-terminal part of the HIV-1 Nef protein. The present study analyzes the epitopes recognized by CTLs in the carboxyl terminus of Nef (amino acids 182-205). In addition to several epitopes that are recognized in association with different HLA molecules (A1, A2, A25(10), B35, B52), we defined an optimal nonapeptide (190-198). This nonapeptide was recognized by CTLs down to nanomolar concentrations in the context of at least two HLA molecules, HLA-B52 and HLA-A2, including three HLA-A2 subtypes: HLA-A2.1, -A2.2, and -A2.4. We also determined the relative frequencies of effector CTLs directed against peptide 190-198 to be as high as 10(-4), as opposed to lower frequencies ranging between 5 x 10(-5) and 5 x 10(-6) observed for the other peptides recognized in the same region, thus confirming the optimal presentation of this nonapeptide in vivo. Molecular modeling of the interactions between HLA-A2.1 and Nef peptide 190-198 suggests the formation of a stable complex and allowed us to study sequence motifs that are important for the binding of the HIV-1 peptide in the pockets of the HLA-A2.1 molecule.
Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in specific allergen-stimulated T cell cultures.
Authors: Jutel M , Pichler W J , Skrbic D , Urwyler A , Dahinden C , Müller U R
Published: December 2022

The mechanisms of bee venom immunotherapy (VIT) are largely unknown. The aim of this study was to follow the changes of T cell cytokine secretion during the course of VIT. Ten bee venom-allergic patients with a history of severe systemic reactions, positive skin tests, and bee venom (BV)-specific serum IgE Abs were treated as follows: on the first day, a cumulative dose of 111 micrograms, starting with 0.1 microgram, was administered s.c. under intensive care conditions. Further injections of 100 micrograms BV were given on day 7, day 21, and thereafter at intervals of 4 wk. Blood samples were obtained just before the initiation of VIT, after the last injection on the same day, and before the subsequent BV injections on days 7, 21, and 50 of VIT. Peripheral blood mononuclear cells (PBMC) were stimulated with phospholipase A (PLA), the major BV allergen, or with a control Ag tetanus toxoid (TT). Cytokine secretion was measured 24 h after restimulation of the cultures with solid-phase bound OKT3 F(ab')2 mAbs after 7 days of culture. In PLA-stimulated cultures, VIT resulted in decreased IL-4 and IL-5 and increased IFN-gamma secretion. In TT-stimulated cultures, we observed similar levels of cytokines before and during VIT. We conclude that ultra-rush VIT changes allergen-specific T cell reactivity.
Functional differences in idiotypically defined IgG1 anti-polysaccharide antibodies elicited by vaccination with Haemophilus influenzae type B polysaccharide-protein conjugates.
Authors: Lucas A H , Granoff D M
Published: December 2022

We investigated the relationship between the form of the Haemophilus influenzae type B (Hib) polysaccharide (PS)-protein conjugate vaccine, Id expression, and Ab quality. Two post-vaccination pools were prepared from sera of infants vaccinated with either Hib PS oligomers coupled to CRM197, a mutant diphtheria toxin (HbOC), or with higher m.w. Hib PS coupled to an outer membrane protein complex of Neisseria meningitidis group B (Hib-OMP). The mean anti-Hib PS Ab avidity of the serum pool from the infants vaccinated with HbOC was threefold higher than that of the pool from infants vaccinated with Hib-OMP. Using sequential immunoabsorption, three IgG1 idiotypically-defined anti-Hib PS fractions were isolated from each of the serum pools: Hibld-1, Hibld-2, and a Hibld-1/-2-depleted population, designated Hibld-0. Hibld-1 and Hibld-2 are idiotypic markers for anti-Hib PS Abs expressing kappa II-A2 and lambda VII V regions, respectively. Hibld-1 anti-Hib PS Abs had significantly higher avidity, 2- to 19-fold higher in vitro bactericidal activity, and were more protective against Hib bacteremia in infant rats, than the respective Hibld-2 Abs isolated from each of the pools. Comparing the two vaccines, Hibld-1 anti-Hib PS Abs elicited by HbOC had significantly higher avidity and 10-fold higher bactericidal and rat protective activity than the Hibld-1 Abs elicited by Hib-OMP. These findings demonstrate that the molecular form of the Hib PS immunogen dictates both V region usage and quality of Ab function.
Differential expression of alternate mB7-2 transcripts.
Authors: Borriello F , Oliveros J , Freeman G J , Nadler L M , Sharpe A H
Published: December 2022

The murine B7-2 (mB7-2) costimulatory molecule is expressed on APCs early during the course of an immune response, suggesting that it may play a pivotal role in the decision between T cell activation and anergy. Murine B7-2 mRNA displays a restricted pattern of expression; it is inducible in B cells, T cells, NK cells, and dendritic cells, but constitutively expressed in unstimulated monocytes. The constitutive and inducible expression of mB7-2 on distinct cell types indicates that mB7-2 is regulated differentially. To further characterize mB7-2 transcripts, we employed 5' rapid amplification of cDNA ends and reverse transcriptase-PCR to examine transcripts expressed in a variety of types of APCs and analyzed the genomic organization of the mB7-2 gene. We report here that the mB7-2 locus consists of 12 exons and demonstrate that exons 1 through 5 can be used in alternative fashions to produce five distinct transcripts, differing in their 5' untranslated and signal regions. The expression of these transcripts differs in distinct types of APCs and is modulated by stimuli that activate B cells. These results demonstrate that mB7-2 transcripts are differentially regulated in a tissue-specific fashion and in response to activation stimuli.
Alloantigen-induced human lymphocytes rendered nonresponsive by a combination of anti-CD80 monoclonal antibodies and cyclosporin-A suppress mixed lymphocyte reaction in vitro.
Authors: Comoli P , Montagna D , Moretta A , Zecca M , Locatelli F , Maccario R
Published: December 2022

Induction of a state of long-term, alloantigen-specific T cell nonresponsiveness has significant implications for human transplantation. It has been previously described that alloantigen-specific anergy may be induced by addition of cyclosporin-A together with anti-CD80(B7-1) mAb to a MLR. In this study we endeavored to verify whether alloantigen-induced PBL rendered anergic by the addition of a combination of anti-B7 mAb and cyclosporin-A during a MLR had a suppressive effect when added to autologous lymphocytes activated in MLR. We found that: 1) the addition of cells rendered anergic by this procedure to a MLR suppress both proliferative and cytotoxic response of autologous responsive PBL to either the same or third-party stimulator cells; 2) the suppressive effect is limited to alloantigen-induced T cell activation, as addition of anergic cells does not influence mitogen- or antigen-induced proliferation of autologous responsive T cells; 3) nonresponsiveness of suppressed cells cannot be reversed by either subsequent restimulation with allogeneic cells or addition of exogenous IL-2 to the cultures; 4) the suppressive effect is apparently not due to secretion of anergic cell-derived soluble factors, but it seems to be dependent on cell-cell contact between anergic, responsive, and stimulator cells. These data suggest that: 1) the delivery of a direct signal mediated by anergic lymphocytes through a cell-cell contact is likely to be the mechanism responsible for the suppressive effect here described; 2) anergic cells may propagate alloantigen-specific tolerance to potentially responsive autologous lymphocytes. Preliminary experiments indicate that anti-CD86(B7-2) mAb may play a similar role in the generation of alloantigen-induced nonresponsiveness.
Cross-linking of the Fas/APO-1 antigen suppresses the CD3-mediated signal transduction events in human T lymphocytes.
Authors: Kovacs B , Tsokos G C
Published: December 2022

The Fas/APO-1 Ag, a member of the TNF-R family, mediates apoptosis (programmed cell death, PCD). PCD has been recognized to be important in the regulation of normal and autoimmune responses. However, the underlying molecular mechanisms are not fully known. To elucidate the role of the Fas Ag in lymphocyte activation, we investigated the influence of Fas Ag cross-linking on CD3/TCR-mediated signal transduction in IL-2-dependent human T cell lines. Early signal transduction events were evaluated in 10-day-old T cell lines using anti-Fas and anti-CD3 mAbs. Incubation of Indo-1-loaded cells with anti-Fas mAb (10 micrograms/ml), but not with an isotypic control mAb almost completely inhibited the anti-CD3-mediated Ca2+ influx. This inhibition was mAb concentration dependent and was detected after incubation for at least 15 min. Cross-linking of the Fas Ag did not affect the anti-CD3-mediated release of Ca2+ from the internal stores or the thapsigargin-mediated Ca2+ influx. Anti-Fas mAb-incubated cells also showed decreased CD3-mediated inositol 1,4,5-trisphosphate production. Preincubation of cells with anti-Fas mAb inhibited the anti-CD3-mediated tyrosine phosphorylation of multiple cellular proteins. Cross-linking of the Fas Ag for up to 6 h did not alter the expression of CD3 on the surface of the T cells. Preincubation of Indo-1-loaded EBV-transformed B cells with anti-Fas mAb did not affect the anti-mu Ab-induced increase in intracellular calcium ([Ca2+]i). Our results show that cross-linking of the Fas Ag can suppress the receptor-mediated signaling process in human T but not B cells.
Modulation of TCR-mediated signaling pathway by thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2).
Authors: Saitoh S , Kosugi A , Noda S , Yamamoto N , Ogata M , Minami Y , Miyake K , Hamaoka T
Published: December 2022

Thymic shared antigen-1 (TSA-1) is a glycosyl-phosphatidylinositol (GPI)-anchored differentiation Ag expressed on murine lymphocytes, and is identical to stem cell Ag-2 (Sca-2). Using newly established mAb against TSA-1/Sca-2, we have previously shown that surface TSA-1 expression is induced upon activation in T cells, and that anti-TSA-1 inhibits IL-2 production induced by anti-CD3 stimulation in T cell hybridomas. In the present study, we have analyzed the functional role of TSA-1 during T cell activation using normal T cells, T cell hybridomas, and transfected Jurkat cell lines that expressed either GPI-anchored or transmembrane form of TSA-1. Anti-TSA-1 inhibited IL-2 production from normal T cells stimulated with soluble anti-CD3 plus accessory cells. Anti-TSA-1 exhibited the inhibitory effect on T cells, but not on accessory cells, because anti-TSA-1 inhibited IL-2 production in Jurkat cells transfected with TSA-1 cDNA, but not in control transfectant. A transmembrane form of TSA-1 was expressed in Jurkat cells by fusing the extracellular portion of TSA-1 to the transmembrane and cytoplasmic regions of the class 1 Db. The analysis using this transfectant revealed that anti-TSA-1-mediated inhibition of IL-2 production did not require the GPI anchor of TSA-1. Finally, in addition to the inhibition of IL-2 production, tyrosine phosphorylation of CD3 zeta-chains observed following TCR stimulation, one of the important early activation events, was markedly reduced by anti-TSA-1. These results imply that TSA-1/Sca-2 plays an important regulatory role in the TCR signaling pathway of activated T cells in addition to its role in T cell differentiation.
Bacterial lipoproteins may substitute for cytokines in the humoral immune response to T cell-independent type II antigens.
Authors: Snapper C M , Rosas F R , Jin L , Wortham C , Kehry M R , Mond J J
Published: December 2022

Bacterial lipoproteins share a common structural motif that has been shown to stimulate proliferation and Ig secretion of murine B cells, in a manner distinct from that mediated by LPSs. Studies of lipoprotein-mediated B cell activation utilized heterogeneous populations of lymphoid cells, leaving unresolved their ability to directly activate resting B cells, as well as their ability to interact with other B cell stimuli. Using highly enriched and/or sort-purified resting murine B cells, we demonstrate that, in contrast to previous reports, lipoproteins (lipoprotein-D, lipoprotein-OspA, and/or the synthetic analogue Pam3Cys) stimulate little, if any, proliferation or Ig secretion in resting B cells. However, when combined with a multivalent membrane (m)Ig-mediated cross-linking signal, dextran-conjugated anti-IgD Abs (alpha delta-dex), lipoproteins mediate up to 10,000-fold inductions in IgM secretion and up to 25-fold enhancements in cellular proliferation relative to that observed with alpha delta-dex alone, in the absence of added cytokines. This mIg-mediated enhancement of Ig secretion was not observed when B cells were stimulated with bivalent, unconjugated anti-Ig. CD40 ligand (CD40L), shows a similar, although somewhat more moderate, synergy with lipoproteins for induction of proliferation and IgM secretion. By contrast, lipoproteins by themselves are relatively ineffective at costimulating Ig secretion in the presence of various combinations of cytokines. These data suggest that bacteria may induce Ag-specific humoral immunity through the action of bacterial polysaccharides that mediate an Ag-specific multivalent mIg signal, in concert with bacterial lipoproteins that deliver ancillary signals, without a requirement for recruitment of non-B cell types.
Identification of IFN-gamma receptor binding sites for JAK2 and enhancement of binding by IFN-gamma and its C-terminal peptide IFN-gamma(95-133).
Authors: Szente B E , Subramaniam P S , Johnson H M
Published: December 2022

The tyrosine kinase JAK2 is an integral part of the signal transduction pathways of a number of cytokines and growth factors, including IFN-gamma. Previously, we identified a species-nonspecific binding site for the C terminus of IFN-gamma, encompassed by IFN-gamma peptide IFN-gamma(95-133), on the membrane proximal region of the cytoplasmic domain of the IFN-gamma R alpha-chain. Using both a radioligand binding assay and coimmunoprecipitation with antireceptor antiserum, we were able to demonstrate specific interaction of JAK2 with the murine IFN-gamma R(MIR) alpha-chain. Furthermore, this interaction is increased by the addition of murine IFN-gamma or its C-terminal peptide, muIFN-gamma(95-133). We also identified two regions of the cytoplasmic domain of the receptor that interact with JAK2 using synthetic peptides of the MIR alpha-chain in receptor competition studies. These regions are encompassed by receptor peptide MIR(283-309), which is adjacent to the membrane proximal region at which the C terminus of IFN-gamma binds, and receptor peptide MIR(404-432), which lies near the C terminus of the receptor, encompassing a potentially important phosphorylation site. These data show site-specific interaction between JAK2 and IFN-gamma with the IFN-gamma R and have broader implications for the role of the IFN-gamma ligand in the IFN-gamma signal transduction pathway. Furthermore, the data support previous studies that demonstrated that intracellular IFN-gamma plays a role in cell activation.
Reconstitution ratio is critical for alloreactive T cell deletion and skin graft survival in mixed bone marrow chimeras.
Authors: Taniguchi H , Abe M , Shirai T , Fukao K , Nakauchi H
Published: December 2022

Although allogeneic bone marrow (BM) transplantation is an effective way to induce donor-specific tolerance, its clinical application is hampered because of the risk associated with vigorous myeloablative pretransplant conditioning. One approach to overcome this problem is to establish a lower chimeric state by mild myeloablation. It is not clear, however, whether there is a threshold in the extent of chimerism that is required for tolerance induction. In this study, we establish a mixed BM chimera system to examine the correlation between the reconstitution ratio of BM chimerism, donor-reactive T cell deletion, and skin graft acceptance using I-E alpha transgenic C57BL/6 mice as the BM and skin graft donors and Ly5 congenic C57BL/6 mice as the recipients. In this system, the class II MHC molecule I-E was the transplantation Ag, and the extent of I-E-reactive T cell deletion was determined by flow cytometry using a mAb specific for the V beta 11 TCR. The degree of BM chimerism was measured by examining the expression of donor-derived Ly5.2 and host-derived Ly5.1 on peripheral blood cells. Transplantation of I-E+ transgenic donor BM cells resulted in deletion of V beta 11+CD4+ T cells in recipient's PBL, and the extent of deletion was proportional to the degree of chimerism. When mice of different degrees of chimerism were tested for skin graft survival, we found that recipient mice with > 30% chimerism could accept skin grafts from I-E+ donor mice, whereas those with < 10% chimerism showed prolonged but not permanent graft survival. These findings revealed the sequence of events for induction of tolerance. First, the degree of BM chimerism determines the number of I-E+ cells in the thymus, which then elicits negative selection of I-E-reactive T cells in a form of clonal deletion. The extent of T cell deletion ultimately determines the mode of tolerance. These data provide experimental evidence for the potential use of partial chimerism by bone marrow transplantation for the induction of donor-specific tolerance in clinical settings.
Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1.
Authors: Kühn S , Skerka C , Zipfel P F
Published: December 2022

The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.
Involvement of natural killer cells in nitric oxide production by spleen cells after stimulation with Mycobacterium bovis BCG. Study of the mechanism of the different abilities of viable and killed BCG.
Authors: Yang J , Kawamura I , Zhu H , Mitsuyama M
Published: December 2022

Viable and killed BCG were compared for the ability to induce nitric oxide (NO) production in normal spleen cells and peritoneal exudate macrophages. Stimulation of spleen cells with viable BCG resulted in a strong expression of inducible NO synthase followed by an enhanced production of nitrite. However, those responses were not induced after stimulation with killed BCG or when macrophages were stimulated with killed and viable BCG. The same level of TNF-alpha mRNA expression was observed in reverse transcription-PCR after stimulation of spleen cells with viable and killed BCG. However, IFN-gamma production was induced only when spleen cells were stimulated with viable BCG. Concurrent stimulation of rIFN-gamma with either viable or killed BCG resulted in a strong nitrite production by macrophages. Neutralization of IFN-gamma and TNF-alpha caused a complete inhibition of nitrite production. Furthermore, anti-asialo GM1 Ab plus complement treatment abolished IFN-gamma production after stimulation with viable BCG, indicating that the NK cell was the major source of IFN-gamma, and its production was triggered only by stimulation with viable BCG. The present study showed that the ability of viable BCG to induce NO production is superior to that of killed BCG, and both IFN-gamma and TNF-alpha are essential for BCG-induced NO production by spleen cells. NK cells appeared to be important as a source of IFN-gamma, and the insufficient NO induction by killed BCG was due to the inability to induce IFN-gamma from NK cells.
Inducible binding of bioactive cathepsin G to the cell surface of neutrophils. A novel mechanism for mediating extracellular catalytic activity of cathepsin G.
Authors: Owen C A , Campbell M A , Boukedes S S , Campbell E J
Published: December 2022

Catalytically active cathepsin G that is bound to the cell surface of human neutrophils may play a variety of roles in normal neutrophil biology and in pathobiology associated with inflammation. In this study, we describe expression of neutrophil cell surface-bound cathepsin G in response to TNF-alpha and platelet-activating factor (PAF) under conditions in which minimal free release of cathepsin G is detected. TNF-alpha and PAF alone induced modest (two- to threefold) increases in cell surface-bound cathepsin G, but exhibited a marked dose- and time-dependent priming effect for subsequent chemoattractant-induced responses (up to 15- to 25-fold increases in cell surface expression). When optimally primed (TNF-alpha, 100 U/ml, or PAF, 10(-9) M), neutrophils expressed five- to sixfold more cell surface-bound cathepsin G, in comparison with cells exposed to FMLP alone. Priming responses were more rapid with PAF (15 s to 5 min) than with TNF-alpha (1 to 60 min). Optimally primed and FMLP-stimulated neutrophils express approximately 160 ng of catalytically active cathepsin G per 10(6) cells, which represents approximately 11% of the cellular content of unstimulated cells. Cathepsin G binds to the cell surface by a charge-dependent mechanism since: 1) incubation of cells with highly positively charged molecules abrogated agonist-induced up-regulation of the cell surface expression of cathepsin G and 2) cathepsin G was eluted from the cell surface by high concentrations of NaCl. These data indicate that interactions between biologically relevant pro-inflammatory cytokines and chemoattractants serve to markedly up-regulate cell surface-bound cathepsin G. The focused catalytic activity of cell surface-bound cathepsin G may alter endothelial and epithelial barriers, promote thrombogenesis, injure extracellular matrix, and/or facilitate directed migration of neutrophils during inflammation.
Fas/Apo-1 (CD95) receptor lacking the intracytoplasmic signaling domain protects tumor cells from Fas-mediated apoptosis.
Authors: Cascino I , Papoff G , De Maria R , Testi R , Ruberti G
Published: December 2022

FAS/Apo-1 (CD95) is an apoptosis-signaling cell surface receptor belonging to the TNF receptor family. Tumor cells resistant to Fas-mediated apoptosis have been described, but to date, the mechanisms responsible for this resistance are not well understood. We found that a series of apoptosis-resistant clones from human HUT78 lymphoma cells express a splicing variant coding for a truncated Fas molecule that lacks the intracellular death-signaling domain. The mutation responsible for the FasExo8Del expression was identified as a deletion-insertion in the intron 7/exon 8 region of the Fas gene. Moreover this mutation affects the phenotype in a dominant negative fashion, i.e., even in the presence of the normal receptor.
Promoter region of the human gene coding for beta-chain of C4b binding protein. Hepatocyte nuclear factor-3 and nuclear factor-I/CTF transcription factors are required for efficient expression of C4BPB in HepG2 cells.
Authors: Arenzana N , Rodríguez de Córdoba S
Published: December 2022

Differential expression of the human genes coding for the alpha and beta polypeptides of the human C component C4b binding protein (C4BP) modulates the levels of C4BP molecules containing C4BP beta polypeptides, providing a mechanism to avoid the potential harmful effects of elevated concentrations of C4BP beta in plasma. To understand how the expression of the C4BPB gene is controlled, we have examined, in the major promoter of the human C4BP B gene, potential regulatory elements. A region from nucleotide -126 to +25 was able to drive high expression of a reporter gene in the human hepatoma cell line HepG2. A small subfragment of this region (from -126 to -90) is responsible for more than 90% of the promoter activity. Electrophoretic mobility shift assays revealed that transcription factors of the hepatocyte nuclear factor-3 (HNF-3) and nuclear factor-I (NFI/CTF) families were able to bind to this region in a sequence-specific manner. We have characterized binding sites for these transcription factors and determined their relative contribution to the activity of the C4BPB promoter. The results suggest that cooperative interaction between HNF-3 and NF-I/CTF is required to obtain a full C4BPB promoter activity. Comparison of the structures of the C4BPA and C4BPB promoters reveals significant differences that could explain the differential transcription of the C4BP alpha and C4BP beta polypeptides during the acute phase response.
Surface T cell Fas receptor/CD95 regulation, in vivo activation, and apoptosis. Activation-induced death can occur without Fas receptor.
Authors: Tucek-Szabo C L , Andjelić S , Lacy E , Elkon K B , Nikolić-Zugić J
Published: December 2022

Fas-mediated apoptosis is a form of cell death that operates through a receptor-ligand interaction. The FasR has been implicated directly in peripheral T cell tolerance and activation-induced apoptosis of T cells in vitro, although to date its expression on murine peripheral T cells has been characterized incompletely. In this study, we document substantial expression of FasR on the vast majority of recent thymic emigrants and resting peripheral T lymphocytes. FasR ligation can induce death in a minor (approximately 5%) subset of these cells. By contrast to rather slow activation-mediated FasR up-regulation in vitro, we demonstrate that in vivo T cell activation by alpha CD3 mAb or superantigen results in rapid up-regulation of the FasR. This up-regulation is paralleled by the kinetics of activation-induced apoptosis in lymph node T cells. However, we demonstrate that the FasR is not necessary for activation-induced cell death. Lymph node T cells from young, healthy, FasR expression-deficient MRL-Ipr/Ipr and animals could be activated in vivo through the TCR-CD3 complex. Most importantly, MRL-Ipr/Ipr T cells underwent massive activation-induced apoptosis in response to high and intermediate doses of alpha CD3. At a low alpha CD3 dose, however, both MRL-Ipr/Ipr and MRL +/+ T cells were activated similarly, but only the latter underwent adequate apoptosis. Taken together, these findings suggest that in vivo, the Fas pathway may not be the only regulator of activation-induced T cell death, but that this pathway may be critical in regulating responses to weak stimuli.
Ceramide inhibits IgE-mediated activation of phospholipase D, but not of phospholipase C, in rat basophilic leukemia (RBL-2H3) cells.
Authors: Nakamura Y , Nakashima S , Ojio K , Banno Y , Miyata H , Nozawa Y
Published: December 2022

Ceramide, a product of sphingomyelin hydrolysis by sphingomyelinase, elicits various cellular functions and has recently been regarded as a second messenger. To investigate the role of ceramide in rat basophilic leukemia (RBL-2H3) cells, the effects of a cell-permeable analogue, N-acetylsphingosine (C2-ceramide), on Ag-mediated cellular responses were examined. C2-Ceramide inhibited Ag- or PMA-induced activation of phospholipase D (PLD), whereas Ca2+ ionophore A23187-induced PLD activation was not affected. C2-Ceramide failed to inhibit PLD activity in two different in vitro assay systems. Since PLD activity is known to be regulated by several factors, the effects of C2-ceramide on these factors were examined. We have previously reported the possible involvement of protein tyrosine kinase in Ag-mediated PLD activation. However, C2-ceramide had no effect on Ag-induced protein tyrosine phosphorylation, including mitogen-activated protein (MAP) kinases. In fura-2-loaded RBL-2H3 cells, C2-ceramide suppressed Ag-induced Ca2+ influx, leaving initial Ca2+ increase and inositol phosphate production unaffected. Western blot analysis revealed that Ag caused translocation of protein kinase C (PKC) alpha, beta 1, beta 2, delta and epsilon isozymes from cytosol to membrane fraction. Translocation of alpha, beta 1, and beta 2 isozymes was specifically prevented by C2-ceramide. Moreover, C2-ceramide suppressed Ag-induced serotonin release. In the absence of extracellular Ca2+, Ag-induced PLD activation and release reaction were greatly reduced. The inhibitory profile was nearly the same as that obtained in C2-ceramide-treated cells. These results suggest that C2-ceramide inhibits Ag-induced PLD activation and serotonin release, probably through the blockage of Ca2+ influx and translocation of Ca(2+)-dependent PKC isozymes in RBL-2H3 cells.