bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the B7-1a molecule, an alternatively spliced form of murine B7-1 (CD80), on T cell activation.
Authors: Inobe M , Aoki N , Linsley P S , Ledbetter J A , Abe R , Murakami M , Uede T
Published: December 2022

B7 molecules (CD80 (B7-1) and CD86 (B7-2/B70)) on APCs provide costimulatory signals for T cell proliferation. We previously described the presence of an alternatively spliced form of murine CD80 (previously termed MB7-2 and renamed as B7-1a) that completely lacks the second Ig-like domain coded by exon 3 in activated murine B cells. in this study, we first examined whether B7-1a mRNA can be detected in vivo by RNase protection assay. The expression of B7-1a mRNA was only detected in lymphoid organs although the level of expression was lesser than that of CD80 mRNA. However, we demonstrated that the expression of B7-1a mRNA like CD80 mRNA was considerably augmented in spleen cells treated with either LPS in vitro or OVA/CFA conjugate in vivo. We next determined the functional activity of B7-1a using Chinese hamster ovary (CHO) cells transfected by B7 genes. When resting T cells were cocultured with CHO cells expressing B7-1a molecules in the presence of PMA/ionomycin, T cell proliferation was not detected, while CHO cells either expressing CD80 or CD86 could promote the proliferation of resting T cells. in contrast to resting T cells, CHO cells expressing B7-1a could support the proliferation of activated T cells. Thus, costimulatory activity of B7-1a molecules was dependent upon the activation stage of T cells. Therefore the IgV-like region of CD80 contains a critical region for functional interaction with its ligands and can transduce a costimulatory signal for T cell proliferation.
Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo.
Authors: Schutze-Redelmeier M P , Gournier H , Garcia-Pons F , Moussa M , Joliot A H , Volovitch M , Prochiantz A , Lemonnier F A
Published: December 2022

The homeodomain of the Antennapedia molecule (AntpHD) spontaneously crosses cellular membranes and can be used to deliver up to 50 additional amino acids to the cytoplasm. We exploited this approach to deliver antigenic peptides to the MHC class I processing and presentation pathway. AntpHD-based fusion peptides expressing the 170-179 HLA-Cw3 CTL epitope (pCw3) were produced in bacteria. Incubation of these fusion peptides with H-2d target cells resulted in efficient delivery to the cytosol as indicated by protease resistance and confocal microscopy. Moreover, this introduction of an exogenous Ag resulted in sensitization of the cell to lysis by a CTL clone specific for the 170-179 HLA-Cw3-derived peptide. Sensitivity of the Ag processing to brefeldin A but not to chloroquine is consistent with the delivery of AntpHD fusion peptides to the conventional class I-associated processing pathway. Immunization of DBA/2 (H-2d) mice with AntpHD pCw3 fusion peptide in the presence of SDS primed H-2Kd-restricted HLA-Cw3-specific CTL. Similar results were obtained with AntpHD fusion peptides expressing the 147-156 influenza nucleoprotein peptide. The strategy outlined in this paper provides a new approach for introducing molecules into the MHC class I Ag-presenting pathway. This approach has clear relevance to the design of synthetic peptide-based vaccines.
Self-MHC-restricted peptides recognized by an alloreactive T lymphocyte clone.
Authors: Udaka K , Wiesmüller K H , Kienle S , Jung G , Walden P
Published: December 2022

Alloreactive T lymphocytes are readily detected in unprimed animals although they have never encountered the alloantigen before. This well-established phenomenon is usually explained with the assumption that a self-MHC molecule complexed with a defined peptide resembles the allo-MHC molecule with another peptide and induces the corresponding T cell specificities. Here, for the first time and in support of this hypothesis, self-MHC-restricted peptides are described for a T cell clone that was induced with allo-MHC. The allo-MHC-specific CTL clone 2C was derived from a H-2b mouse and recognizes H-2Ld complexed with the naturally occurring endogenous peptide LSPFPFDL. H-2Kb was shown to be involved in positive selection of its TCR, and peptides associated with this MHC molecule are implicated in the process. To identify such peptides, positional scanning with random peptide libraries combined with an iterative approach was employed. Several active peptides were found and the most efficient, SIYRYYGL, was chosen for further studies. Recognition by 2C of the two MHC-peptide adducts H-2Ld + LSPFPFDL and H-2Kb + SIYRYYGL is mediated by the same TCR and appears to be similarly efficient as concluded from inhibition experiments with an Id-specific Ab. CTLs from SIYRYYGL-primed H-2b mice respond to H-2Ld + LSPFPFDL. This reciprocal cross-reactivity suggests that structural features are shared by the two MHC-peptide complexes.
IL-12 is a central mediator of acute graft-versus-host disease in mice.
Authors: Williamson E , Garside P , Bradley J A , Mowat A M
Published: December 2022

Distinct forms of graft-vs-host disease (GVHD) occur in (C57B1/6 x DBA/2)F1 (BDF1) mice inoculated with either C57B1/6 or DBA/2 parental spleen cells, and it has been suggested that this reflects differential activation of CD4+ Th cell subsets. Transfer of B6 cells produces an acute GVHD, during which an early period of lymphoid hyperplasia precedes immunosuppression, weight loss, and mortality, and a Th1 pattern of cytokines is produced. Conversely, transfer of DBA/2 cells induces a chronic GVHD, in which no weight loss or mortality is observed, but an autoimmune, SLE-like GVHD develops in association with a Th2 pattern of cytokines. Recent work indicates that IL-12 plays a central role in the polarization of Th cell-dependent responses, and here we have examined its role in polarizing GVHD, by administering or depleting IL-12 during the afferent phase of both the acute and chronic forms of GVHD in BDF1 mice. In vivo neutralization of endogenous IL-12 ameliorated acute GVHD, in association with reduced splenic NK cell activity, IFN-gamma production, immunosuppression, weight loss, and mortality. Conversely, administration of exogenous murine rIL-12 exacerbates this disease and converts the chronic GVHD into a lethal acute GVHD-like syndrome. These results indicate that IL-12 plays an important role in the development of acute, but not chronic, GVHD and suggest that differential production of IL-12 early in the disease may underlie these distinct outcomes of the GVHD in BDF1 mice injected with different parental cells.
Requirement of Fas(CD95), CD45, and CD11a/CD18 in monocyte-dependent apoptosis of human T cells.
Authors: Wu M X , Ao Z , Hegen M , Morimoto C , Schlossman S F
Published: December 2022

Our previous studies demonstrated that upon activation, monocytes (Mo) were able to sensitize peripheral blood T (PBT) cells to apoptosis induced by treatment with PMA. However, it is unknown what gene products provide the death signal to the sensitized PBT cells and how activated Mo enable PBT cells to become susceptible to apoptosis. Here, we show that PBT cells, but not Mo, express functional Fas ligand upon treatment with PMA. Moreover, this Mo-dependent T cell apoptosis could be blocked by a Fas-Ig fusion protein, as well as by a nonlytic mAb against Fas molecule. These results strongly suggest involvement of Fas-Fas ligand interaction in the death of PBT cells. Unlike Fas-induced apoptosis, however, Mo-dependent T cell death was completely inhibited by overexpression of the Bcl-2 protein, and PMA alone was sufficient to trigger apoptosis in T cells when Mo were included in culture. Furthermore, anti-CD11a, anti-CD18, or anti-CD45/CD45RA mAbs; could prevent PBT cells from death triggered by PMA plus Mo, suggesting that these Ags participate in the apoptotic process. The participation of CD45RA in the death of PBT cells was further demonstrated by the observation that the J45.01 cell line, a CD45-deficient variant of Jurkat cells, did not undergo apoptosis by this Mo-dependent mechanism. When transfected with cDNA encoding CD45RA, J45.01 cells acquired apoptotic response to PMA stimulation in the presence of Mo to a similar, but lesser, degree than normal Jurkat cells.
Non-T cell-derived IL-4 plays an important role in IgE production induced by antigen resensitization and is resistant to FK506.
Authors: Yamashita M , Yamaoka M , Seki N , Fujii T
Published: December 2022

The present study investigates the role of non-T cell-derived IL-4 in IgE production. It is well known that IL-4 is generally produced by T cells and induces IgE production. We have demonstrated that non-T cells also produce IL-4 in vivo by injection with Ag following passive sensitization of mice with IgE. In contrast to its known potent immunosuppressive effects on T cells, FK506 only partially inhibited IL-4 produced by non-T cells; therefore, FK506 was found to be a useful tool for identifying a cell source of IL-4 production. We used this observation to examine the role of non-T cell-derived IL-4 in IgE production. The production of a high titer of IgE was induced by priming BALB/c mice with Ascaris suum extract and further enhanced by secondary immunization. The IgE production in this model was dependent on IL-4, since both primary and secondary IgE production were completely suppressed by injection with anti-IL-4 Ab. Although primary IgE production was completely inhibited by FK506, secondary IgE production was not inhibited. Furthermore, IL-4 induction in plasma was only partially inhibited by FK506 when the A. suum extract-primed mice were resensitized with Ag. These results indicate that IL-4 produced by non-T cells plays a crucial role in secondary IgE production.
High affinity antibodies against Lex and sialyl Lex from a phage display library.
Authors: Dinh Q , Weng N P , Kiso M , Ishida H , Hasegawa A , Marcus D M
Published: December 2022

Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.
Single chain Ig/gamma gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle lytic capacity.
Authors: Weijtens M E , Willemsen R A , Valerio D , Stam K , Bolhuis R L
Published: December 2022

To enable construction of CTL with known predefined Ab specificity for adoptive immunotherapy, we constructed a chimeric scFv/gamma gene composed of the variable regions of a mAb joined to the Fc(epsilon)RI signaling receptor gamma-chain of mast cells. Introduction of this chimeric receptor into CTL rendered these lymphocytes specific for renal cell carcinoma. This approach combines the specificity of tumor-selective Abs with the efficacy of CTL to destroy tumor cells. We not only demonstrated that the transduced CTL functionally express the scFv/gamma receptor for a prolonged period of time (4.5 mo of in vitro culture), but also showed high levels of Ab-dictated lysis of renal cell carcinoma similar to that of normal CTL, and importantly, we demonstrated that these CTL can recycle their lytic activity. Moreover, these scFv/gamma-expressing T lymphocytes produce cytokines upon stimulation with the relevant target cell. These results together with the donor independence of our gene transduction protocol demonstrate the feasibility of redirecting T lymphocytes for cancer treatment.
Human IgG production in vivo: determination of synthetic rate by nonradioactive tracer incorporation.
Authors: Thornton C A , Welle S , Griggs R C , Abraham G N
Published: December 2022

Exposure of Ab-secreting cells to selected hormones, cytokines, and drugs alters the rate of Ig production in vitro. Whether these effects are important clinically is unknown because there are no safe, reproducible, and appropriate techniques for measuring Ig synthesis in vivo. We developed a stable isotope tracer method to measure IgG secretion into plasma. L-[1-13C]Leucine was given as a priming dose followed by a constant i.v. infusion over 8 h. Tracer accrual in IgG, determined by mass spectrometry, was linear from 2 to 8 h of the infusion. The normal rate of IgG synthesis into plasma assessed in 21 healthy subjects was 860 +/- 310 mg/day (mean +/- SD). The synthetic rate measurements were remarkably reproducible (coefficient of variation = 10.5 %). Simultaneous analysis of leucine kinetics allows, for the first time, Ig secretion to be studied in the context of whole body protein economy. IgG secretion into plasma accounts for 0.3% of whole body protein synthesis. Experimental support for a key metabolic assumption, that tracer enrichment in plasma and that at the site of IgG synthesis were similar, came from a comparison of synthetic rates derived from low dose and high dose tracer infusions. Measurement of Ig secretion by tracer incorporation is rapid and reproducible. In contrast to older methods that rely on radioisotope disappearance, the tracer incorporation method is safe for serial measurements in an individual and will be useful for quantitative studies of treatment effects and immune regulation in vivo.
Eosinophil granule proteins activate human heart mast cells.
Authors: Patella V , de Crescenzo G , Marinò I , Genovese A , Adt M , Gleich G J , Marone G
Published: December 2022

Eosinophilia in humans is often associated with heart disease and cardiac localization of eosinophil granule proteins, and several results suggest that granule proteins mediate endomyocardial damage. Here we investigated the in vitro effects of the four principal eosinophil granule proteins (eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin, and eosinophil peroxidase (EPO)) on the activation of effector cells of inflammation (mast cells) isolated from human heart tissue (HHMC). ECP and, to a lesser extent, MBP (0.3-3 microM), but not eosinophil-derived neurotoxin and eosinophil peroxidase stimulated the release of preformed (histamine and tryptase) and the de novo synthesis of vasoactive and proinflammatory mediators (PGD2) from HHMC. Activation of HHMC by ECP and MBP was Ca2+- and temperature-dependent and was abolished by preincubation (15 min, 37 degrees C) with 2-deoxy-D-glucose (10 mM) and antimycin A (1 microM). There was a significant correlation between the maximal percentage of histamine release induced by ECP and anti-IgE from HHMC (rs = 0.73; p < 0.005), by MBP and anti-IgE (rs = 0.79; p < 0.001), and by ECP and MBP (rs = 0.65; p < 0.005). A positive correlation was also found between histamine and tryptase secretion (rs = 0.71; p < 0.001) and between histamine and PGD2 release induced by ECP from HHMC (rs = 0.85; p < 0.001). This is the first demonstration that some eosinophil cationic proteins, namely ECP and MBP, found at the site of heart damage in patients with eosinophilia, act as complete secretagogues on HHMC. This observation indicates another mechanism by which infiltrating eosinophils and their metabolic products cause inflammatory reactions and thus endomyocardial lesions in patients with eosinophilia.
Quantitative thresholds of MHC class II I-E expressed on hemopoietically derived antigen-presenting cells in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection.
Authors: Hanson M S , Cetkovic-Cvrlje M , Ramiya V K , Atkinson M A , Maclaren N K , Singh B , Elliott J F , Serreze D V , Leiter E H
Published: December 2022

Two homozygous lines of transgenic NOD/Lt mice expressing MHC class II I-E molecules at quantitatively different levels were utilized to study mechanisms of I-E-mediated diabetes prevention. In line 12, I-E expression on APC at levels comparable with that in BALB/cByJ controls conferred only partial diabetes resistance. In line 5, greater than normal I-E levels on APC correlated with nearly complete resistance. Levels of endogenously encoded I-Ag7 correlated inversely with transgene-induced I-E expression. T cell transfer experiments into NOD/severe combined immunodeficient mice demonstrated the presence of pathogenic T cells in I-E+ donors, and that continuous expression of I-E on hemopoietically derived APC was required to block their pathogenic function. T cells from transgenic and nontransgenic NOD/Lt mice primed in vivo against the beta cell autoantigen 65-kDa isoform of glutamic acid decarboxylase (GAD65) and two peptides derived from this protein proliferated when restimulated in vitro. However, reverse-transcription PCR and ELISA measurements of cytokine mRNA and protein levels showed that the GAD65-reactive T cells from both line 5 and line 12 mice produced higher levels of IL-4 and lower levels of IFN-gamma than similar T cells from standard NOD/Lt mice. Thus, the inverse relationship between I-E and I-Ag7 expression was associated with qualitative differences in T cell responses to putative beta cell autoantigens. Collectively, these data indicate quantitative increases in I-E expression on APC may block insulin-dependent diabetes mellitus by altering the balance of cytokines produced by beta cell autoreactive T cells.
Altered cytokine production and accessory cell function after HIV-1 infection.
Authors: Yoo J , Chen H , Kraus T , Hirsch D , Polyak S , George I , Sperber K
Published: December 2022

We investigated cytokine production and accessory cell function in human macrophage hybridoma cell lines and primary monocytes after infection with HIV-1. HIV-1 infection induced IL-10 production in the macrophage hybridoma cell line with loss of IL-12 1 wk after infection. There were also significant increases in production of IL-10 (537 +/- 521 vs 687 +/- 625 pg/ml) while there was a reduction in IL-12 (6.3 +/- 3.1 vs 1.2 +/- 1.0 pg/ml, p = 0.021) in the primary monocytes 5 days after HIV-1 infection. In addition, the hybridoma cell lines and primary monocytes failed to support PHA, Con A, PWM, or anti-CD3- induced T cell proliferation 1 wk after infection. The viability of the T cells cocultured with the HIV-1-infected macrophage cell lines or the primary monocytes as determined by propidium iodide staining was unaltered and there was no increase in apoptosis-specific DNA strand breaks or increased expression of Bcl-2 in the T cells. No soluble suppressor factor was present, since UV-inactivated supernatants from the hybridoma cell line and primary monocytes failed to inhibit mitogen- and anti-CD3-induced T cell proliferation. Early events in T cell activation, including calcium flux and phosphotyrosine kinase activity, were intact in the T cells cocultured with the HIV-1- infected hybridomas and monocytes but there was reduced IL-2 production. Addition of exogenous IL-2 restored the proliferative responses. Taken together, these data suggest that alteration of cytokine production and accessory cell function for mitogens and anti-CD3-induced T cell proliferation independent of induction of apoptosis, suppressor factor production, or inhibition of T cell signaling occurs very early after HIV-1 infection and may contribute to the global immunosuppression observed in AIDS.
Naive murine neonatal T cells undergo apoptosis in response to primary stimulation.
Authors: Adkins B , Chun K , Hamilton K , Nassiri M
Published: December 2022

The cellular mechanisms controlling deficient immune responses in newborn animals are not well understood. Our earlier studies showed that developmental regulation of Th cell activity may be one of the major causes of immunodeficiency in neonatal animals. Naive murine neonatal T cells showed poor Th1 activity but robust Th2 activity in response to primary stimulation in vitro. Although they produced high levels of IL-4, neonatal T cells proliferated poorly, suggesting that neonatal T cell survival in primary cultures may be limited. We show here that, unlike adult T cells, naive neonatal T cells undergo apoptosis in response to primary TCR-mediated stimulation. Ligation of the TCR alone, in the absence of accessory cell costimulation, is sufficient to induce apoptosis. Moreover, CD4+ and CD8+ T cells show equivalent levels of apoptosis. Lastly, this apoptosis can be prevented by the addition of excess IL-2 or by conditions promoting a high level of IL-2 production (TCR-independent stimulation, anti-CD28 mAb, or exogenous IL-6) by neonatal T cells. However, IL-2 alone is not sufficient to support functional rescue from apoptosis; only IL-6 supports the ability of these cells both to survive and to mount vigorous secondary responses. The identification of conditions allowing functional rescue from apoptosis in vitro has important implications for enhancing vaccine responsiveness in vivo.
A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes.
Authors: Haire R N , Ota T , Rast J P , Litman R T , Chan F Y , Zon L I , Litman G W
Published: December 2022

Xenopus laevis is a unique model for studying the ontogenetic development of immune functions. A short primer PCR amplification method was employed to amplify fragments from Xenopus genomic DNA that are related to Ig light chains and TCR. One fragment was identified that appeared to represent a novel type of light chain and was used as a probe to recover the corresponding cDNA from a spleen cDNA library. We designate these light chains type III. Using an iterative screening procedure, six families of VL genes, two distinct JL and two distinct CL sequences, were identified. In a comparison of phylogenetically diverse light chains, the type III genes align with higher vertebrate lambda genes. Southern blot hybridization analyses with genomic DNA from different animals showed the VL and CL sequences to be both diverse and polymorphic. Intrafamily sequence comparisons of VL genes revealed additional diversity. Collectively, these studies confirm the existence of a third type of light chain gene in Xenopus, establish a high degree of genetic variation in the sequences encoding the light chain V regions, and provide the most significant evidence to date for the presence of a lambda-like light chain gene at the phylogenetic level of the amphibians.
Rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism.
Authors: Gern J E , Vrtis R , Kelly E A , Dick E C , Busse W W
Published: December 2022

There is evidence that rhinovirus (RV) infections are frequent causes of increased asthmatic symptoms and can specifically enhance allergic inflammation in the airway. To further define effects of RV infection on cellular immunity, we have begun to develop in vitro models for study. When human PBMC were incubated with 35S-labeled RV16, specific binding via ICAM-1 on monocytes was observed. Incubation of PBMC with RV also led to a dose-related increase in the expression of the early activation marker CD69 on 30 to 70% of T cells. The RV16-induced increases in CD69 were blocked by anti-ICAM-1 mAb, and were not elicited by UV-inactivated (noninfectious) virus. The degree of CD69 enhancement correlated with the number of monocytes in mixtures of PBMC, did not occur in monocyte-depleted cultures, and was mediated by one or more soluble factor(s). RV also induced secretion of IFN-gamma from both peripheral blood T cells and NK cells, and IFN-gamma mRNA was greatest in T cells that were CD69+. Finally, supernatant from RV-activated CD3+CD69+ cells had biologic activity that promoted eosinophil survival in vitro; this RV16-associated activity was blocked when co-incubations were performed with IFN-gamma mAbs. These observations suggest that RV nonspecifically activates a large proportion of T cells through a monocyte-dependent mechanism. Such changes in vivo could enhance airway inflammation, and this may include effects on inflammatory cells in the airways of allergic individuals.
In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation.
Authors: Blackwell T S , Blackwell T R , Holden E P , Christman B W , Christman J W
Published: December 2022

We hypothesized that endotoxin injection in rats would stimulate in vivo nuclear factor-kappa B (NF-kappa B) activation in lung tissue and that antioxidant treatment before endotoxin injection would attenuate endotoxin-induced NF-kappa B activation, chemokine gene expression, and neutrophilic lung inflammation. We studied NF-kappa B activation in rat lung tissue following a single i.p. injection of endotoxin (6 mg/kg). After in vivo endotoxin treatment, lung NF-kappa B activation peaked at 2 h and temporally correlated with the expression of cytokine-induced neutrophil chemoattractant mRNA in lung tissue. Treatment with the antioxidant N-acetylcysteine (NAC) 1 h before endotoxin resulted in decreased lung NF-kappa B activation in a dose-dependent manner (from 200-1000 mg/kg) and diminished cytokine-induced neutrophil chemoattractant mRNA expression in lung tissue. Treatment with NAC significantly suppressed endotoxin-induced neutrophilic alveolitis. The average total lung lavage neutrophil count was 5.5 x 10(6) with endotoxin treatment vs 0.9 x 10(6) with NAC treatment before endotoxin. The NF-kappa B pathway represents an attractive therapeutic target for strategies to control neutrophilic inflammation and lung injury.
Phospholipase A2 triggers the first phase of the thermal stress response and exhibits cell-type specificity.
Authors: Jurivich D A , Pangas S , Qiu L , Welk J F
Published: December 2022

To understand the relationship of inflammatory and cellular stress responses, phospholipase A2 (PLA2) was examined for its role in the first phase of the transcriptional response to cellular stress. Electromobility shift analysis revealed heat shock transcription factor (HSF1)-DNA binding when HeLa S3 and Jurkat cells were exposed to exogenous PLA2. Although PLA2-inducible HSF1-DNA binding was comparable to thermal stress, it did not induce maximal heat shock gene expression. PLA2-induced HSF1 was not hyperphosphorylated relative to the heat-inducible form, thus suggesting that exogenous PLA2 affects the signal for HSF1 multimerization but not its phosphorylation. Because inflammation often involves elevated temperatures, the effect of PLA2 on thermal regulation of HSF1-DNA binding activity was examined. PLA2 exposure altered the thermal threshold for HSF1 activation, and pore-gradient gel analysis indicated that either conformational changes or other modifications of HSF1 are being induced when cells are treated by PLA2, thus creating a synergistic environment for HSF1 activation into its DNA-bound state. Surprisingly, the monocyte-like cell line, U-937, was insensitive to the action of exogenous PLA2. Neither HSF1-DNA binding or lowering of the temperature threshold for HSF1 activation was observed in PLA2-treated U-937 cells. These data suggest that inflammatory mediators such as PLA2 partially affect transcriptional switches mediating thermal stress in some cell types but not others. The purpose of HSF1 activation during inflammation and its differential induction are discussed relative to these observations.
C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway.
Authors: Nilsson G , Johnell M , Hammer C H , Tiffany H L , Nilsson K , Metcalfe D D , Siegbahn A , Murphy P M
Published: December 2022

Mast cells are known to accumulate at sites of inflammation, however, the chemotaxins involved are undefined. Since most natural leukocyte secretagogues also induce cell migration, and since the anaphylatoxins C3a and C5a are mast cell secretagogues, we hypothesized that both C3a and C5a are also mast cell chemotaxins. Here we report that C3a and C5a are, in fact, potent chemotaxins for the human mast cell line HMC-1. The optimal concentrations, half-maximal effective concentrations (a measure of agonist potency) and the efficacy (response at the optimal concentration) compared with medium control were, for C3a: 10 nM, 0.5 nM, and 256%, respectively; for C5a: 1 nM, 10 pM and 145%. Chemotaxis of HMC-1 cells to both C3a and C5a was blocked by pertussis toxin, suggesting that Gi-coupled receptors are involved in signal transduction. C3a and C5a also induced transient pertussis toxin-inhibitable increases in [Ca2+]i (ED50 = 1 nM for both) that could be homologously but not heterologously desensitized, suggesting that the receptors for C3a and C5a are distinct. These results make C3a the most effective mast cell chemotaxin identified to date. The chemotactic potency described here for C3a is also 100- to 1000-fold greater than for all of its previously described cellular actions. Direct chemoattraction of mast cells by C3a and C5a may help explain the rapid accumulation of mast cells at sites of inflammation.
Mouse bone marrow-derived mast cells and mast cell lines constitutively produce B cell growth and differentiation activities.
Authors: Tkaczyk C , Frandji P , Botros H G , Poncet P , Lapeyre J , Peronet R , David B , Mécheri S
Published: December 2022

The present report describes a novel function of mast cells that consists of a B cell growth activity. The B cell response occurred without any stimulation or preactivation of mast cells. A small number of mast cells was required, since mast cell/B cell ratios as low as 1/100 to 1/10,000 lead to effective B cell activation. Mast cell-dependent B cell activation resulted, within 48 h of incubation, in blast formation, proliferation, and IgM production. Both low and high density B cells were responsive to mast cells. Supernatants from unstimulated mast cells could also activate B cells, suggesting that a B cell-stimulating activity (MC-BSA) is mediated by a soluble factor(s). The addition of anti-IL-4 or anti-IL-6 mAbs or even proteases to the mast cell-derived supernatants did not alter B cell activation. However, treatment of mast cells with mitomycin C or actinomycin D, or paraformaldehyde fixation totally abrogated MC-BSA. Fractionation of mast cell supernatant by gel filtration chromatography resulted in four peaks, ranging from > 200 to 15 kDa, all of which were biologically active on B cells. Because mast cells are known to continuously release proteoglycans, MC-BSA was subjected to chondroitinase and heparinase treatment, but no significant inhibition of B cell activation was obtained. This direct T cell-independent stimulatory effect of mast cells on B cells could account for a mechanism by which plasma cells are continuously produced in lymphoid organs and particularly in bone marrow.
Role of vitamin D3-binding protein in activation of mouse macrophages.
Authors: Yamamoto N , Naraparaju V R
Published: December 2022

When mouse peritoneal nonadherent (lymphocytes) cells were treated with lysophosphatidylcholine (lyso-Pc) and cultured with adherent cells (macrophages) in 1% fetal calf serum (FCS)- or adult mouse serum (AMS)-supplemented medium for 3 h, markedly enhanced phagocytic and superoxide-generating capacities of macrophages were observed. Stepwise cultivation of lyso-Pc-treated B cells and untreated T cells with an FCS-supplemented medium generated a macrophage-activating factor (MAF), whereas cultivation of lyso-Pc-treated B cells alone in AMS-supplemented medium was sufficient to generate the MAF. The accumulated evidence suggests that lyso-Pc-inducible beta-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes modified the bovine serum vitamin D3-binding protein (DBP) to yield the MAF, a protein with N-acetylgalactosamine as the remaining sugar. In contrast, the lyso-Pc-inducible beta-galactosidase of B cells alone modified mouse DBP to yield the MAF. These observations led us to conclude that bovine DBP carries a trisaccharide composed of N-acetylgalactosamine, galactose, and sialic acid, whereas mouse DBP carries a disaccharide composed of N-acetylgalactosamine and galactose. Thus, macrophages of a T-cell-deficient nude (BALB/c nu/nu) mouse and a T-cell Neu-1 sialidase-deficient SM/J mouse were efficiently activated by administration of lyso-Pc.
Biochemical characterization and microsequencing of a 205-kDa synovial protein stimulatory for T cells and reactive with rheumatoid factor containing sera.
Authors: Hain N A , Stuhlmüller B , Hahn G R , Kalden J R , Deutzmann R , Burmester G R
Published: December 2022

Synovial fluid (SF) was found to possess stimulatory capacity for the proliferation of T cell clones derived from patients with rheumatoid arthritis (RA) when cultured together with IL-2. Using chromatography technique and gel electrophoresis, a synovial fluid protein with an apparent m.w. of 205 kDa (p205) was isolated that demonstrated a bioactivity analogous to that obtained with native synovial fluid. After electroelution, p205 dissociated into 70-kDa fragment(s). Upon IEF, it appeared as a single band with an isoelectric point of 6.5, suggesting a noncovalently bound trimer complex. Amino acid sequences of the whole protein and of tryptic peptides were determined by N terminal sequencing. The N terminal amino acid sequence of the 70-kDa fragment and of the tryptic peptides showed no identity to recently described protein sequences. One peptide matched, in 11 amino acid residues, with the human IgG1-4 constant heavy chain and rheumatoid factor (RF) binding region. The p205 induced the proliferation of peripheral blood T cells and long term T cell cultures that had been raised by alternate stimulation with IL-2 and p205. In a similar approach, synovial lining cells were shown to release a protein with biochemical characteristics similar to the synovial fluid-derived p205. Western blot analysis revealed the binding of RF-containing sera to p205, which was diminished by absorption with an RF reagent. These observations suggest that p205 is expressed by synovial cells and may be a target for T and B cells in RA.
T-T cellular interaction between CD4-CD8- regulatory T cells and T cell clones presenting TCR peptide. Its implication for TCR vaccination against experimental autoimmune encephalomyelitis.
Authors: Kozovska M F , Yamamura T , Tabira T
Published: December 2022

Regulatory T cells recognizing TCR determinants presumably play a critical role in the control of experimental autoimmune encephalomyelitis, a prototype tissue-specific autoimmune disease. This study was initiated to determine whether regulatory T cells can be induced against a V beta 17a CDR2 peptide (residues 50-68) in SJL/J mice. Although the TCR peptide showed regulatory effects in vivo, the presence of T cells specific for the peptide could not be proven with conventional proliferation assays. Unexpectedly, in the presence of myelin basic protein-specific T clone cells (Tcc), the sensitized spleen cells vigorously proliferated in response to the TCR peptide. The subsequent experiment showed that this was due to the outstanding capability of the Tcc as APC for the exogenous TCR peptide. Using the Tcc as APC, we were able to establish V beta 17a50-68-specific T cell lines from in vivo primed spleen cells. The line cells were MHC class I restricted and dominated by T cells with a distinct surface phenotype (CD4-CD8-V beta 17a+). Presentation of the peptide by the Tcc was inhibited by treatment with gelonin that could block a MHC class I presentation pathway. The ability of T cells to present the TCR peptide was not related to their Ag specificity, but correlated with the expression levels of MHC class I molecules and adhesion molecules such as intercellular adhesion molecule-1 and B7-1 on their surface. The TCR peptide-specific T cells produced a soluble mediator(s) that is inhibitory for T cell activation and were protective against actively induced experimental autoimmune encephalomyelitis. These results show that V beta 17a50-68 vaccination induces regulatory CD4-CD8- T cells that could interact with T cells presenting relevant TCR fragments.
Reduced IL-12 production by monocytes upon ultraviolet-B irradiation selectively limits activation of T helper-1 cells.
Authors: Kremer I B , Hilkens C M , Sylva-Steenland R M , Koomen C W , Kapsenberg M L , Bos J D , Teunissen M B
Published: December 2022

The capacity of APC to stimulate the proliferation of human peripheral blood T cells decreases upon ultraviolet-B (UVB) irradiation. The aim of this study was to investigate whether all T cell subsets are equally sensitive to this reduced APC function. Established human Th1, Th2, and Th0 clones were stimulated with monocytes in a soluble CD3 mAb-mediated assay that is dependent on the presence of APC. Monocytes were exposed to low nonlethal doses of UVB radiation before coculture with T cells. UVB irradiation inhibited the capacity of monocytes to stimulate the proliferation and IFN-gamma production of Th1 cells in a dose-related fashion. In contrast, UVB-treated monocytes induced normal proliferation and IL-4 production in Th2 cells. Stimulation of Th0 cell proliferation by UVB-irradiated monocytes was normal, but a preferential suppression of IFN-gamma production was observed, thus leading to a more Th2-like cytokine response. The loss of Th1 proliferation upon stimulation with UVB-irradiated monocytes could be overcome by rIL-2; however, IFN-gamma production remained suppressed. IFN-gamma production could be completely restored by rIL-12, whereas the addition of IL-1 beta, TNF-alpha, or indomethacin had no such effect, nor did the addition of mAb to CD28, added to compensate for the reduced B7 expression of UVB-irradiated monocytes. Monocytes exposed to UVB radiation exhibited reduced expression of mRNA for the IL-1 2 subunits p35 and p40 and suppressed production of the IL-12 p70 protein. Our results thus indicate that UVB irradiation of APC selectively impairs Th1-like responses, a phenomenon caused by the UVB-induced suppression of monocyte IL-12 production.
Defective transcription of the IL-2 gene is associated with impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells.
Authors: Mondino A , Whaley C D , DeSilva D R , Li W , Jenkins M K , Mueller D L
Published: December 2022

Anergic CD4+ Th cells do not produce IL-2 when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical IL-2 DNA enhancer elements: NF-AT (nuclear factor of activated T cells; a sequence that binds a heterotrimeric NFATp, Fos, and Jun protein complex) and Activator Protein-1 (AP-1) (that binds Fos and Jun heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible AP-1 family members c-Fos, FosB, and JunB. However, the reduced expression of these proteins was not the result of a global TCR/CD3-signaling defect because CD3 cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as NFATp dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells. Thus, defective IL-2 gene transcription appears to be due, at least in part, to a selective block in the expression of the AP-1 Fos and Jun family members in anergic T cells.
Beta-chemokine TCA3 binds to and activates rat vascular smooth muscle cells.
Authors: Luo Y , D'Amore P A , Dorf M E
Published: December 2022

The present study compares the activity of TCA3 with other beta-chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and monocyte chemoattractant protein (MCP)-1) on rat vascular smooth muscle cells. TCA3, MIP-1 alpha, and MCP-1 (but not MIP-1 beta) treatment stimulates chemotaxis of vascular smooth muscle cells. TCA3-mediated chemotactic responses are sensitive to treatment with pertussis toxin, suggesting that G alpha-i proteins are involved in TCA3 signaling of smooth muscle. In addition, TCA3, MIP-1 alpha, and MCP-1 increase vascular smooth muscle cell adhesiveness to type III collagen. In contrast, stimulation with TCA3, but not other beta-chemokines, induces proliferation of vascular smooth muscle cells. TCA3 receptors were identified on rat vascular smooth muscle cells by direct binding of radiolabeled ligand. TCA3 binds to this receptor with high affinity (3 nM). Rat vascular smooth muscle cells display approximately 75,000 binding sites/cell. Competitive inhibition studies indicated that murine MIP-1 alpha, murine MCP-1, and human RANTES are weak partial competitors of TCA3 binding, demonstrating the existence of a unique receptor for TCA3. Murine MIP-1 beta, which fails to stimulate any biologic functions in vascular smooth muscle cells, also does not inhibit TCA3 binding. The combined data demonstrate that TCA3 and other beta-chemokines can modulate vascular smooth muscle cell function.
Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145-kDa inositol polyphosphate 5-phosphatase, SHIP.
Authors: Chacko G W , Tridandapani S , Damen J E , Liu L , Krystal G , Coggeshall K M
Published: December 2022

Stimulation of the B cell Ag receptor (BCR) has been reported to induce tyrosine phosphorylation of a 145-kDa protein and its association with the adapter protein Shc. We have identified this protein as the novel inositol polyphosphate 5-phosphatase (SHIP). Further analysis revealed that both maximal phosphorylation of SHIP and its association with Shc require co-clustering with the Fc receptor for IgG (Fc gamma RII) rather than stimulation of the BCR alone. Since co-clustering of the BCR and Fc gamma RII also down-regulates proliferation induced by Ag receptor stimulation, we hypothesize that tyrosine phosphorylation of SHIP and its association with Shc contribute to negative signaling through effects on inositol and phosphatidylinositol metabolism.
Phosphatidylinositol 3 kinase activity is not essential for B7-1-mediated costimulation of proliferation or development of cytotoxicity in murine T cells.
Authors: Ni H T , Deeths M J , Mescher M F
Published: December 2022

It has been suggested that induction of phosphatidylinositol (PI) 3 kinase activity upon CD28 costimulation may contribute to CD28-mediated signaling. In this report, T cell stimulation by microspheres bearing coimmobilized anti-TCR mAb and purified B7-1 ligand was examined. This approach allows study of cosignaling mediated by CD28 interaction with its native ligand in the absence of potentially confounding contributions from other receptor-ligand interactions. For murine CD4+ and CD8+ T cells, costimulation with B7-1 up-regulated PI3 kinase activity assayed in vitro, and this was blocked by treatment of the cells with wortmannin, a specific inhibitor of PI3 kinase, before stimulation. However, wortmannin failed to inhibit B7-1-dependent T cell proliferation or development of cytotoxicity in CD8+ cells. These results indicate that the enzymatic activity of PI3 kinase is not essential in the CD28-mediated signaling involved in the costimulation of proliferation or induction of CTL activity in precursor CTL.
Critical role of Ras in the proliferation and prevention of apoptosis mediated by IL-2.
Authors: Gómez J , Martínez-A C , Fernández B , García A , Rebollo A
Published: December 2022

Activation of the Ras small guanine nucleotide-binding proteins upon IL-2 stimulation of T lymphocytes has been described previously. However, the relevance of the Ras molecules in the cellular events triggered by IL-2 still remained to be clarified. To analyze the effects of Ras inhibition in early proliferative responses to IL-2, we have transiently expressed a Ras dominant negative mutant carrying a point mutation in codon 17 (Ser17Asn or N17) under a tetracycline-controlled expression system. We have found that Ras has a crucial role in both proliferation and prevention of apoptosis through IL-2R, while IL-4 promotes proliferation and inhibits apoptosis by Ras-independent signals. The use of flow cytometry has allowed us to correlate the effects in proliferation and apoptosis with the expression level of the Ras mutant among the transfected population. Furthermore, the availability of a murine T cell line that can be maintained independently in the presence of IL-2 or IL-4 has provided specificity controls, since IL-4 does not trigger Ras activation.
Activation of the mitogen-activated protein kinase cascade is suppressed by low concentrations of dexamethasone in mast cells.
Authors: Rider L G , Hirasawa N , Santini F , Beaven M A
Published: December 2022

Antigen stimulation of mast cells via the IgE receptor, Fc epsilon RI, results in recruitment of the cytosolic tyrosine kinases, Lyn and Syk, and the phosphorylation of proteins. We examined the effects of the glucocorticoid dexamethasone on these events in a cultured (RBL-2H3) mast cell line. Nanomolar concentrations of dexamethasone suppressed phosphorylation of proteins that were associated with the activation of the mitogen-activated protein (MAP) kinase/phospholipase A2 pathway without inhibiting initial events. For example, tyrosine phosphorylation of the subunits of Fc epsilon RI, Lyn, or Syk or of the Ras-guanine nucleotide exchange factor, Vav, was not suppressed in cells treated with up to 1 microM dexamethasone. In contrast, phosphorylation of Raf1, MEK1, p42mapk, and cytosolic phospholipase A2, as well as the associated increase in MAP kinase activity and release of arachidonic acid, were markedly inhibited in cells treated with as little as 10 nM dexamethasone--a concentration that only partially inhibited hydrolysis of inositol phospholipids or release of secretory granules. Prolonged exposure to dexamethasone also resulted in a partial decrease in expression of MEK1, p42mapk, and cytosolic phospholipase A2, which may contribute further to the effects of dexamethasone on this pathway. Activation of the MAP kinase/phospholipase A2 pathway by the calcium-mobilizing agent thapsigargin was similarly suppressed in dexamethasone-treated cells. These findings suggested that an early step in the pathway, possibly a step immediately before the activation of Raf1, was suppressed by low concentrations of dexamethasone.
MHC class I-processed pseudogenes in New World primates provide evidence for rapid turnover of MHC class I genes.
Authors: Cadavid L F , Hughes A L , Watkins D I
Published: December 2022

The MHC class I genes of the New World primate, the cotton-top tamarin (Saguinus oedipus), are an exception to the high polymorphism and variability displayed by this multigene family. We report the isolation of the first two processed pseudogenes from the MHC region in primates. These two MHC class I-processed pseudogenes (MHC-PS1 and -PS2) were found in several species of New World primates, suggesting a possible explanation for the cotton-top tamarin's limited MHC class I diversity. The pattern of synonymous and nonsynonymous substitutions in PS1 suggests that the gene that gave rise to this processed pseudogene was once subject to selection for variability in the peptide binding region and might, therefore, have been functional. Additionally, PSI is not closely related to the expressed cotton-top tamarin's MHC class I genes, but does show some similarity to So-N1, a tamarin pseudogene from which no transcript has been found. Thus, PS1 may represent a remnant of a once active MHC class I gene that is no longer functional in the cotton-top tamarin. The MHC class I loci in primates, therefore, appear to be evolving by a continual process of duplication and inactivation. This process seems to be exaggerated in New World primates and may in part be responsible for the cotton-top tamarin's limited MHC class I diversity.