bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of isoproterenol and phosphodiesterase inhibitors on platelet-activating factor biosynthesis in the human neutrophil.
Authors: Fonteh A N , Winkler J D , Torphy T J , Heravi J , Undem B J , Chilton F H
Published: December 2022

Increasing cellular levels of cAMP inhibit eicosanoid production in the human neutrophil; however, little is known about the effects of cAMP on platelet-activating factor (PAF) biosynthesis in this cell. In the current study, the beta-adrenergic receptor agonist isoproterenol, alone or in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), was used to increase cAMP in neutrophils. The incorporation of [3H]acetate into PAF and the synthesis of leukotrienes in response to ionophore A23187 were significantly inhibited by 10 microM isoproterenol. The inhibitory effect on PAF was potentiated by the addition of 10 microM IBMX. The effects of IBMX were mimicked by rolipram, an inhibitor of the cAMP-specific phosphodiesterase IV. Mass spectrometric analysis of the PAF molecular species in stimulated neutrophils indicated that the combination of isoproterenol and IBMX inhibited (> 50%) ionophore- and fMLP-induced production of PAF. To better understand the mechanism involved in the inhibition of PAF formation, the major biosynthetic steps were examined in the presence and absence of a maximally effective concentration of isoproterenol and IBMX. Isoproterenol alone or in the presence of IBMX had no measurable effect on the ionomycin-induced increase in cytosolic calcium concentration, as assessed by fura-2 fluorescence. Treating intact neutrophils with a combination of isoproterenol and IBMX did not inhibit acetyltransferase activity when assayed in a subsequent broken cell preparation. Finally, increasing cellular cAMP with these drugs did not influence the ability of the neutrophil to catabolize PAF. Phospholipase A2-like activity was assayed in the whole cell by measuring the mobilization of phospholipase A2 products, PAF, lyso PAF, and arachidonic acid, from cellular phosphoglycerides. Treatment of neutrophils with isoproterenol and IBMX significantly reduced the production of lyso PAF and PAF from 1-alkyl-2-arachidonoyl sn-glycero-3-phosphocholine. Similarly, increasing cellular levels of cAMP inhibited the cell's ability to mobilize arachidonic acid upon cell activation. These data suggest that increasing cellular levels of cAMP leads to the inhibition of PAF and leukotriene biosynthesis, at least in part, by regulation of phospholipase A2 activity.
Differential activation of Th1 and Th2 CD4+ cells by murine brain microvessel endothelial cells and smooth muscle/pericytes.
Authors: Fabry Z , Sandor M , Gajewski T F , Herlein J A , Waldschmidt M M , Lynch R G , Hart M N
Published: December 2022

CD4+ Th cell infiltration into the brain and the activation by cellular elements of the central nervous system (CNS) are thought to be important steps in the initiation of CNS autoimmune diseases. T cell activation requires Ag-specific stimulation and additional costimulatory signals provided by the APC. Here we describe how murine brain microvessel endothelial (En) cells and smooth muscle/pericytes (SM/P) selectively induce the Ag-specific activation of different Th1 and Th2 CD4+ T cell clones. Th1 and Th2 cell clones were used that were specific for the same peptide Ag in the context of the same class II allotype. SM/P preferentially activated Th1 cell clones, whereas En cells activated Th2 cell clones better, as reflected by cell proliferation and production of IL-2 by SM/P-activated Th1 clones and IL-4 by Th2 clones. There was no difference in the level of expression of CD4, CD2, or LFA-1 molecules between these Th cell clones, and anti-CD4, CD2, LFA-1 or ICAM-1 mAb did not differentially affect Ag-induced proliferation among the clones. Moreover, antibody to CD28 did not influence Ag presentation by brain microvessel En or SM/P cells to Ag-specific Th1 and Th2 clones. These results suggest that: 1) different The subsets might require different signals for their activation; 2) different APC might provide different costimulatory signals for Th cell subsets; and 3) brain microvessel En and SM/P might play a differential role in induction of autoreactive T cell responses in the CNS.
Release of vesicles enriched in complement receptor 1 from human erythrocytes.
Authors: Pascual M , Lutz H U , Steiger G , Stammler P , Schifferli J A
Published: December 2022

Vesicles released from human E by Ca(2+)-loading, ATP-depletion, or storage are enriched in several glycosylphosphatidylinositol-anchored proteins such as acetylcholinesterase (AchE) and decay-accelerating factor (DAF). As a result of this, the remaining E are depleted of these proteins. We analyzed whether vesiculation induced by ATP-depletion in vitro was also responsible for a loss of C receptor 1 (CR1), which is a transmembrane protein arranged predominantly in small clusters. ATP-depleted E had lost 15.4% to 33.9% of their CR1. This loss was similar to that of AchE and DAF. The released vesicles contained CR1. The number of CR1 per band 3 protein was 1.7 to 2.7 that in the original E, indicating an enrichment of CR1 in vesicles. This enrichment was similar to that observed for AchE and DAF (1.83- and 2.6-fold, respectively). The capacity of the vesicles and the ATP-depleted E to bind C3b-coated immune complexes correlated with the CR1 number, suggesting that there was no preferential loss of CR1 clusters. Vesicles released from human E during C attack also contained CR1. In conclusion, in vitro aging induced by ATP-depletion is responsible not only for a loss of glycosylphosphatidylinositol-anchored proteins, but also of CR1. Whether vesiculation explains the loss of CR1 from aging E in vivo and from E of patients with SLE or AIDS remains to be studied.
Differential modulation of the effects of lipopolysaccharide on macrophages by a major outer membrane protein of Proteus mirabilis.
Authors: Weber G , Link F , Ferber E , Munder P G , Zeitter D , Bartlett R R , Nixdorff K
Published: December 2022

We previously showed that a major protein isolated from purified cell walls of Proteus mirabilis (39-kDa protein) is a strong modulator of the specific immune responses to LPS from this bacterium in mice. When mixed with LPS before immunization, this protein enhances T cell-dependent, IgG antibody-producing cell responses specific for LPS. Furthermore, complexes of the 39-kDa protein with LPS drastically inhibit the production of oxygen radicals by murine macrophages activated with LPS, as measured in a chemiluminescence assay. In the present report, we have further investigated possible modulating effects of the protein at the level of LPS-macrophage interaction. When mixed with LPS, the 39-kDa protein inhibited IL-1 production by murine macrophages derived from bone marrow in a dose-dependent manner, as determined in an IL-2-dependent IL-1 assay. On the other hand, the protein had little effect on LPS-mediated suppression of MHC class II expression on the surface of macrophages induced with IFN-gamma. Some abrogation of suppression was observed, but the amounts of protein needed for this effect were quite large, in comparison with the amounts rendering inhibition of IL-1 production. In contrast, the 39-kDa protein enhanced the LPS-induced cytotoxicity of macrophages against L929 target cells, primarily as the result of production of TNF. These results show that the 39-kDa protein is a potent modulator of the interaction of LPS with macrophages, exerting its effects in a differential manner with respect to various parameters of LPS-induced activation of macrophages.
Identification of a new neutralizing epitope conformationally affected by the attachment of CD4 to gp120.
Authors: Kang C Y , Hariharan K , Posner M R , Nara P
Published: December 2022

We have developed a strategy to purify and characterize various anti-gp120 antibody populations in HIV+ sera by using anti-Id mAb. One preparation of human anti-gp120 antibody (ES+ Ab) isolated on an anti-Id mAb (ES)-conjugated immunoabsorbent exhibited a novel neutralizing epitope specificity. The ES+ Ab bound only to the native form of recombinant gp120SF2 and gp120IIIB and not to the third hypervariable region (V3) loop peptide. In contradistinction to other CD4-gp120-inhibiting and V3-specific neutralizing antibodies, ES+ Ab exhibited a dose-dependent enhancement of binding to recombinant gp120 in the presence of recombinant soluble CD4. In addition, flow cytometric analysis revealed a similar increase in the binding of ES+ Ab to the native form of gp120 expressed on the HIV-infected cells. The ES+ Ab competed with CD4 binding site- and V3-specific antibodies in binding to gp120, suggesting that the ES+ Ab epitope is located near the CD4 binding site epitope and the V3 region. The ES+ Ab neutralized six genetically distinct HIV-1 strains. The neutralizing activity of ES+ Ab on HIVIIIB was significantly increased in the presence of human anti-CD4 binding site mAb. These data suggest that the ES+ Ab epitope represents a conserved, conformational, neutralization target on gp120 that may be involved in viral infection in an event after the CD4-gp120 interaction and that is distinct from previously defined neutralizing epitopes of gp120. This finding may be important for the development of an AIDS vaccine and immunotherapy.
T cell epitopes of type II collagen that regulate murine collagen-induced arthritis.
Authors: Myers L K , Seyer J M , Stuart J M , Terato K , David C S , Kang A H
Published: December 2022

Chick type II collagen (CII), a protein commonly found in joint cartilage, induces an autoimmune arthritis when administered to susceptible strains of mice. A cyanogen bromide fragment of CII, CB11, contains the requisite epitopes critical for inducing collagen-induced arthritis. If administered as a tolerogen, however, before immunization, CB11 prevents the onset of disease. Therefore, delineation of structural elements of CB11 that can regulate autoreactive T cells became the goal of this study. To delineate the structural elements of CB11 antigenic to T cells, 14 peptides containing overlapping sequences of CB11 were generated. Mononuclear cells from CII-immunized DBA/1 mice were cultured with these peptides and the resulting supernatants examined for the production of IFN-gamma. Two peptides, CII 181-209 and CII 245-270, generated the greatest responses. The ability of these two peptides to regulate arthritis was tested by administering them to neonatal DBA/1 mice as tolerogens before immunization with CII. Both peptides suppressed the incidence of arthritis whereas no other peptide used as a tolerogen significantly altered the course of the disease. T cells from four arthritis-resistant murine strains did not recognize either peptide when immunized with CII, whereas cells from the disease-susceptible B10.Q mice responded well to both. Thus, the coincidence of T cell responses to CII 181-209 and CII 245-270 in CIA-susceptible mice and the lack of response in disease-resistant strains or CII-tolerized mice identify these two peptides as containing important T cell epitopes that regulate CIA.
bcl-2 proto-oncogene expression during human T cell development. Evidence for biphasic regulation.
Authors: Gratiot-Deans J , Ding L , Turka L A , Nuñez G
Published: December 2022

To ensure that the mature T cell repertoire is MHC-restricted yet not autoreactive, cortical thymocytes that express low levels of the TCR/CD3 complex along with CD4 and CD8 (double positive cells) are subjected to positive and negative selection. Surviving cells lose either CD4 or CD8 (single positive cells) and are located primarily in the thymic medulla. bcl-2, a novel proto-oncogene that promotes cell survival by inhibiting programmed cell death (apoptosis), may be an important protein in regulating cell survival during thymocyte development. We have examined the expression of bcl-2 during T cell development by using human thymocytes. Consistent with previous studies, human thymic tissue sections stained for bcl-2 revealed occasional bcl-2+ cells within the thymic cortex and intense staining of virtually all medullary thymocytes. More quantitative western blot analysis and S1 nuclease protection assay revealed that single positive thymocytes contained approximately 2 to 3 times the level of bcl-2 protein and 3 to 4 times the level of bcl-2 mRNA as double positive thymocytes. Flow cytometric analysis of purified double positive thymocytes revealed that minimal amounts of bcl-2 protein was in fact detectable in most cells, although a small subpopulation (10-20%) contained higher levels. In contrast, brighter staining for bcl-2 was observed in virtually all single positive thymocytes. Surprisingly, CD4-CD8- thymocytes (both CD3- and CD3+) expressed the same amount of bcl-2 as did the single positive thymocytes. Because a large percentage of CD3-CD4-CD8- cells are cycling, we examined the effect of mitogenic stimulation on bcl-2 expression by double positive thymocytes by using western blot analysis. bcl-2 expression in double positive thymocytes could not be induced by cell cycle entry following stimulation with PMA and ionomycin. Our data demonstrate that bcl-2 expression is biphasic during T cell development. Both CD3-CD4-CD8- and CD3+CD4+ and CD3+CD8+ thymocytes express high levels of bcl-2. Therefore, diminished bcl-2 expression in double positive thymocytes seems to be the result of specific down-regulation in order to facilitate the selection CD4+CD8+ thymocytes.
Cross-linking of HLA class II antigens modulates the release of tumor necrosis factor-alpha by the EBV-B lymphoblastoid cell line JY.
Authors: Altomonte M , Pucillo C , Damante G , Maio M
Published: December 2022

In addition to their functional role as peptide-binding proteins HLA class II Ag can also act as signal-transducing molecules. The present study showed that cross-linking of HLA class II Ag by the anti-HLA-DR mAb L243 or by the anti-HLA-DR,-DP mAb IVA12 significantly (p < 0.05) increased the release of TNF-alpha by the EBV-B lymphoblastoid cell line JY. In contrast, the anti-HLA-DR mAb 2.06 or the superantigens staphylococcal exotoxin toxic shock syndrome toxin-1 and staphylococcal enterotoxin B that bind to HLA-DR,-DQ Ag did not affect the release of TNF-alpha by JY cells. The accumulation of TNF-alpha in the culture medium of JY cells peaked at 24 h, decreased thereafter, and was found to be dependent on the dose of mAb L243 or mAb IVA12 used to cross-link HLA class II Ag. mAb L243 or staphylococcal exotoxin toxic shock syndrome toxin-1 enhanced the spontaneous homotypic aggregation of JY cells and mediated a dose-dependent inhibition of JY cell proliferation. These phenomena were not mediated by TNF-alpha released in response to cross-linking of HLA class II Ag; polyclonal anti-TNF-alpha neutralizing antibody did not affect JY cell aggregation and the inhibition of JY cell proliferation mediated by mAb L243. In contrast, TNF-alpha secreted by JY cells enhanced a nuclear factor-kB-like activity through the binding to the 75-kDa TNF-alpha receptor. These results demonstrate an additional role of HLA class II Ag as signal-transducing molecules regulating the production of bioactive TNF-alpha by EBV-B cells. The release of TNF-alpha after the triggering of HLA class II molecules could be relevant to different aspects of B cell biology and might play a role in the pathogenesis of human diseases in which antibodies cross-reactive to HLA class II Ag have been identified.
Cloning of mouse Ox40: a T cell activation marker that may mediate T-B cell interactions.
Authors: Calderhead D M , Buhlmann J E , van den Eertwegh A J , Claassen E , Noelle R J , Fell H P
Published: December 2022

A cDNA library was prepared from the murine Th cell line Th2 D.10 and used to clone the murine homologue of Ox40 by polymerase chain reaction. Comparison of the mouse sequence with the rat revealed greater than 90% homology between the two sequences at both the DNA and protein level. Northern blot analysis found that, as in the rat, Ox40 expression appears to be restricted to activated T cells. A chimeric receptor globulin was prepared to include the mouse Ox40 extracellular domain coupled to the hinge-CH2-CH3 domains of human IgG1 (Ox40-Ig). This soluble form of the molecular was then used to identify cells bearing a ligand for Ox40. FACS analysis revealed that Ox40-Ig bound to a subset of peritoneal B cells as well as to a fraction of LPS-activated splenic B cells. Immunostaining of spleen sections using an Ag-specific conjugate and Ox40-Ig found a significant proportion of antibody-forming cells co-stained with Ox40-Ig. Immunoprecipitation of cell-surface radiolabeled peritoneal B cells suggests a specific interaction with a protein of 70 kDa.
T cell receptor V alpha gene segment with alternate splicing in the junctional region.
Authors: Marche P N , Six A , Gahéry H , Gris-Liebe C , Cazenave P A , Jouvin-Marche E
Published: December 2022

The locus encoding mouse TCR-alpha chain includes approximately 100 V alpha gene segments that can be organized in about 20 structural subfamilies. Southern blot analysis of a T cell line derived from the BALB/c strain, M5T, has indicated that both alpha loci were rearranged, as assessed by the deletion of the delta locus, and that the V alpha gene segment involved in one of the rearrangements did not belong to any of the V alpha subfamilies already described. Transcripts of TCR-alpha chains from the M5T line were cloned after cDNA synthesis and anchored-polymerase chain reaction, revealing a V alpha gene segment of an as yet unidentified subfamily, V alpha 5T. Molecular cloning of germ-line V alpha 5T gene segments has shown that this subfamily contained two members, one of them being a pseudogene. The two members were located to each extremity of the alpha locus associated with a member of the V alpha 13 and V alpha BWB subfamilies. Analysis of transcripts bearing the V alpha 5T gene segment in the M5T line as well as in thymocytes has revealed that J alpha are frequently absent. This is due to an alternate donor splice site generated at the V alpha 5T-J alpha junction that leads to a splicing from the end of V alpha 5T to C alpha instead of the J alpha to C alpha conventional splicing. The impact of J alpha spliced-out transcripts on the allelic exclusion process is discussed.
Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells.
Authors: Mathew P A , Garni-Wagner B A , Land K , Takashima A , Stoneman E , Bennett M , Kumar V
Published: December 2022

We have recently described a signal transducing molecule, 2B4, expressed on all NK and T cells that mediate non-MHC-restricted killing. The gene encoding this molecule was cloned and its nucleotide sequence determined. The encoded protein of 398 amino acids has a leader peptide of 18 amino acids and a transmembrane region of 24 amino acids. The predicted protein has eight N-linked glycosylation sites, suggesting that it is highly glycosylated. Comparison of 2B4 with sequences in the databanks indicates that 2B4 is a member of Ig supergene family, and it shows homology to murine and rat CD48 and human LFA-3. Northern blot analysis has shown at least three transcripts for 2B4 in adherent lymphokine-activated killer cells of several mouse strains and TCR-gamma/delta dendritic epidermal T cell lines but not in allospecific T cell clones. These three mRNA are the products of differential splicing of heterogeneous nuclear RNA. Southern blot analysis of genomic DNA from several mouse strains revealed that 2B4 belongs to a family of closely related genes. The 2B4 gene has been mapped to mouse chromosome 1 by analysis of 2B4 expression in recombinant inbred mouse strains.
Homogeneous antigen receptor beta-chain genes in cloned CD4- CD8- alpha beta T suppressor cells.
Authors: Schmidt-Wolf I G , Liang O , Dejbakhsh-Jones S , Wang H , Cheng L , Holm B , Bell R , Strober S
Published: December 2022

The rearrangements of beta-chain genes of the T cell Ag receptor were examined in 12 CD4- CD8- alpha beta + T cell lines derived from the spleen or thymus of neonatal or adult BALB/c mice. Eleven of the lines were cloned and established from six independent cloning procedures from different mice. Five cloned lines used V beta 9, four cloned lines used V beta 15, and two cloned lines used V beta 7. Nucleotide sequencing of the beta-chain genes showed that clones that used a given V beta were identically rearranged even when they were derived from independent cloning procedures. In the case of V beta 7 and V beta 15 all nucleotides in the V-D-J joining region were in the germ line configuration without N region additions. Rearrangements of the V beta 7, V beta 9, and V beta 15 genes were functional. Each V beta 15 clone also had a homogeneous rearrangement of the V beta 13 gene, which was nonfunctional. The predicted amino acid sequence of the joining regions of the V beta 7, V beta 9, and V beta 15 rearrangements showed homology in four of seven amino acids in the peptide contact region.
Binding of peptides lacking consensus anchor residue alters H-2Ld serologic recognition.
Authors: Solheim J C , Carreno B M , Smith J D , Gorka J , Myers N B , Wen Z , Martinko J M , Lee D R , Hansen T H
Published: December 2022

CTL recognize class I MHC/peptide complexes on the surface of target cells. Crystallographic and serologic data have indicated that peptide ligands can influence the conformation of class I molecules and hence T cell recognition. How the binding of peptides with disparate sequence motifs affects the conformation of distinct regions within a class I molecule remains unknown. A series of site-directed mutants of the murine class I molecule H-2Ld was studied to address this question. These mutants were generated by in vitro mutagenesis and used to map the serologic epitopes recognized by a panel of Ld-reactive mAb. The influence of six different ligands on serologic recognition by these mAb was then examined. Of 12 mAb tested, only one, B22/249, was found to be significantly influenced by the bound peptide. Peptide discrimination by B22/249 was observed at the cell surface and in immunoprecipitates of Ld after incubation with two of the six ligands. The two peptides that caused suboptimal B22/249 recognition of Ld/peptide lack a proline at position 2, which is present in the other four peptides and has previously been defined as an anchor residue for Ld ligands. The epitope on Ld detected by mAb B22/249 includes residues 63 to 70 on the alpha 1 domain helix. Two of these residues are in pocket B, which computer modeling predicts to be in contact with the second residue of Ld-binding peptides. Therefore, these data imply that a mAb to a class I molecule can distinguish peptides with different motifs, possibly reflecting peptide-dependent conformational changes in the class I molecule.
Effect of granulocyte/macrophage colony-stimulating factor on human monocytes infected with influenza A virus. Enhancement of virus replication, cytokine release, and cytotoxicity.
Authors: Bender A , Amann U , Jäger R , Nain M , Gemsa D
Published: December 2022

The activating properties of granulocyte/macrophage (GM)-CSF were studied in vitro with human monocytes infected by influenza A virus. When monocytes were pretreated for 8 h with GM-CSF (100 U/ml) and then exposed to influenza A virus, de novo virus protein synthesis was enhanced, more virus particles were released, and cells were killed at a higher rate. In virus-infected monocytes, GM-CSF induced a more rapid IFN-alpha release and potentiated production of TNF-alpha, IL-1 beta, and IL-6. Although GM-CSF or influenza A virus were each capable of independently activating TNF-alpha, IL-1 beta, and IL-6 gene transcription, a combination of both induced a massive cytokine mRNA accumulation which was readily translated into bioactive protein. Thus, GM-CSF may display a Janus-like action by accelerating virus infection but also by priming monocytes for elevated cytokine production. Whether the facilitated influenza A virus replication caused by GM-CSF may be counterbalanced by an improved cytokine response remains to be studied under more complex in vivo conditions.
70 kDa heat shock cognate protein is a transformation-associated antigen and a possible target for the host's anti-tumor immunity.
Authors: Tamura Y , Tsuboi N , Sato N , Kikuchi K
Published: December 2022

We previously investigated a novel heat-inducible transformation-associated cell surface Ag that is expressed on the activated H-ras oncogene-transformed rat fibrosarcoma W31, but not its parental nontransformed fibroblast WFB. This Ag was detected by mAb 067. Herein, we characterized the molecular nature of the Ag by using anti-heat shock protein (HSP) mAb. The accumulated data indicated that the cell surface expression of Ag was clearly enhanced by several stressors, such as TNF, L-azetidine-2-carboxylic acid, and sodium arsenite. The immunoprecipitate made with mAb 067 and W31 cell lysates reacted with anti-rat 70 kDa heat shock cognate (HSC) mAb, TG5E, indicating that 067-defined Ag may be a rat 70 kDa HSC. Because this Ag seemed to be one of the transformation-associated Ag of WFB, we further studied whether it could play an important role in the host's anti-tumor immunity. Peripheral T cells of rats primed with live BCG showed cytotoxicity to W31 but not to WFB. Because the possibility existed that HSP may interact with certain populations of T cells, we focused on the reactivity of CD4-CD8- double negative T (DNT) cells against 067-defined molecule. DNT cells from spleen and PBL of live BCG-primed rats showed the cytotoxicity against W31 cells. This cytotoxicity was completely blocked by mAb 067 and anti-CD3 mAb. However, it was not blocked by mAb R48B1 and 109, which detect the MHC class I nonpolymorphic determinant and a target molecule of the cytolysis by poly I:C-induced NK cells, respectively. Furthermore, brefeldin A was able to block the cytotoxicity against W31 targets by DNT cells, but not by NK cells. These data suggest that 70 kDa HSC may be a tumor Ag and may act as a presenting molecule perhaps complexed with cellular peptides to certain DNT cells.
Protein Fv produced during vital hepatitis is a novel activator of human basophils and mast cells.
Authors: Patella V , Bouvet J P , Marone G
Published: December 2022

Protein Fv is found in the normal liver and is released in the stools of patients suffering from viral hepatitis. Protein Fv isolated from five patients stimulated the release of histamine and sulfidopeptide leukotriene C4 from purified and unpurified peripheral blood basophils. Protein Fv absorbed with protein A-Sepharose coated with polyclonal IgG did not induce histamine secretion, whereas removal of putative contaminating Ig did not modify the releasing activity. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE). There was an excellent correlation (Spearman rank coefficient (rs) = 0.83; p < 0.001) between the maximal percent histamine release induced by protein Fv and that induced by anti-IgE from basophils. Preincubation of basophils with either protein Fv or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with heterologous stimulus. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released histamine in response to anti-IgE and protein Fv. A monoclonal IgE purified from a myeloma patient (patient ADZ) blocked both anti-IgE- and protein Fv-induced releases, whereas human polyclonal IgG and a monoclonal IgG purified from another myeloma patient (patient ZEG) selectively blocked protein Fv-induced secretion. Protein Fv also induced the release of preformed (histamine and tryptase) and de novo synthesized mediators (sulfidopeptide leukotriene C4 and/or PGD2) from mast cells purified from human lung parenchyma and skin tissues. There was a significant correlation between the maximal percent histamine release induced by protein Fv and anti-IgE from skin mast cells (rs = 0.63; p < 0.01). There was also an excellent correlation between histamine and tryptase release caused by protein Fv from both lung (rs = 0.80; p < 0.001) and skin mast cells (rs = 0.70; p < 0.01). Thus, we established that protein Fv acts as a novel activator of human basophils and mast cells presumably by interacting with the VH domain of the IgE.
Age-associated changes in binding of human B lymphocytes to a VH3-restricted unconventional bacterial antigen.
Authors: Silverman G J , Sasano M , Wormsley S B
Published: December 2022

We have recently demonstrated that there is a site on Staphylococcal protein A (SpA) that interacts with B cell Ig receptors in a manner comparable with known T cell superantigens, because this binding specificity is restricted to Fab with VH3 H chains and most VH3 Ig bind SpA. In the present studies, SpA was used as a phenotypic marker for VH3 expression by human lymphoid cells. As expected, this Fab-mediated binding specificity was completely inhibited by certain VH3 antibodies but not by antibodies from other VH families. In multiparameter flow cytometric analyses, this binding activity was demonstrated to be highly prevalent among B cells (14 to 54%), and was more common among IgM-bearing B cells compared with IgG-bearing B cells. In all studies, Fab-mediated binding of SpA was uniformly expressed by a greater proportion of CD5-positive B cells than CD5-negative B cells. The proportion of B lymphocytes with this VH3-restricted binding capacity was found to undergo age-associated changes, because a large proportion of the peripheral B cells of neonates (mean +/- SD, 46.0 +/- 2.9%) bind this site, but two 10-mo-old subjects and older children had significantly lower binding levels (29.0 +/- 3.5%) that were the same as binding levels by adult peripheral B lymphocytes (30.2 +/- 3.3%). In immunohistochemical studies, tonsilar B cells that bind this site on SpA were shown to be common in mantle zones and germinal centers of secondary follicles. We speculate that Fab-mediated SpA binding represents a fundamental and primitive binding capacity that is part of the human preimmune repertoire, and we discuss the implications for the observed age-dependent shift in Fab-mediated binding of SpA by peripheral blood B cells.
Androgen-induced expression of the peripheral blood gamma delta T cell population in the chicken.
Authors: Arstila T P , Lassila O
Published: December 2022

Unlike alpha beta T cells, the physiologic significance of gamma delta T cells has remained elusive. In avian species they comprise a large circulating T cell subset. Here we report that in chicken around the time of sexual maturation (4 to 6 mo of age) a significant increase of the gamma delta T cells takes place in male but not in female chickens. The frequency of gamma delta T cells increases both in peripheral blood and spleen, but not in intestinal epithelium. This expansion is independent of MHC haplotype, being observed in various inbred and MHC-recombinant strains. Furthermore, administration of testosterone to young female chickens induces an equivalent increase in the frequency of gamma delta T cells in peripheral blood. These results indicate that sex, through androgens, has an effect on the gamma delta T cell numbers in a species, in which these cells form a major subset of peripheral lymphocytes.
Predominant role of tumor necrosis factor-alpha in human monocyte IL-10 synthesis.
Authors: Wanidworanun C , Strober W
Published: December 2022

In previous studies it has been shown that the bacterial endotoxin LPS induces an initial burst of inflammatory cytokine synthesis in human monocytes, which is followed by substantial IL-10 production; the IL-10 then down-regulates the inflammatory cytokine production as well as IL-10 production itself. Herein we tested the hypothesis that IL-10 production in human monocytes is under control of one of the cytokines induced by LPS. Accordingly, we cocultured purified human peripheral blood monocytes with a panel of cytokines including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, granulocyte macrophage-CSF, transforming growth factor-beta, and IFN-alpha and then measured IL-10 mRNA production using a semiquantitative reverse transcription-polymerase chain reaction technique. We found that TNF-alpha had a major effect on IL-10 mRNA production, inducing a 20- to 120-fold increase over baseline production. In contrast, IL-1 alpha, IL-1 beta, IL-6, granulocyte macrophage-CSF, transforming growth factor-beta, and IFN-alpha had little effect (< 3-fold). The induction of IL-10 mRNA by TNF-alpha in monocytes was dose dependent and began between 8 and 24 h after the addition of TNF-alpha; this suggests that the increased IL-10 mRNA level was due to de novo mRNA synthesis rather than mRNA stabilization; this latter finding was corroborated by actinomycin-D time course studies, which showed that the half-life of IL-10 was less than 1 h and was not significantly altered by TNF-alpha. These studies concerning IL-10 mRNA induction by TNF-alpha were corroborated by studies of IL-10 protein secretion: TNF-alpha alone, but not IL-1 alpha, IL-1 beta, or IL-6 induces substantial IL-10 secretion. Furthermore, LPS induces a large amount of IL-10 secretion that is largely inhibited (50 to 75%) by anti-TNF-alpha but not by antibodies to other inflammatory cytokines. Finally, TNF-alpha augments LPS-induced IL-10 secretion. Taken together, these findings suggest that TNF-alpha is unique among the inflammatory cytokines in its role as an inducer of IL-10 in human monocytes, as such, it induces a molecule that provides negative feedback to its own production.
Class II MHC transcriptional mutants are defective in higher order complex formation.
Authors: Riley J L , Boss J M
Published: December 2022

Human class II MHC genes are regulated in part by a series of conserved upstream sequence elements termed the X1, X2, and Y boxes. Class II MHC transcriptional mutant B cell lines have been defined that differ with regard to the presence of RFX, an X1 box DNA-binding activity. To further characterize these mutant cell lines, we tested the ability of these conserved upstream elements to respond to the presence of known transcriptional activation domains. A series of HLA-DRA promoter reporter constructions carrying Gal4 binding sites and GAL4 fusion protein expression vectors were cotransfected into both wild type B cells and mutant B cells representing the two RFX phenotypes. Regardless of RFX or class II phenotype, the activation domains of GAL4-VP16 and GAL4-E1a could synergistically stimulate expression of constructions containing both the X2 and Y boxes. GAL4-VP16- and GAL4-E1a-mediated expression was inhibited by the presence of the X1 box in cells that contained RFX. In mutant cells that lacked RFX, GAL4-VP16 activation was not inhibited. In the RFX-positive class II mutant cell line RJ2.2.5, the X1 box inhibitory activity could be overcome by separating the Gal4 site from the X1 box by two additional helical turns, suggesting that the RFX factor is bound at the X1 site and sterically interferes with activation. This was not the case in wild type B cells, suggesting that a stable higher order complex forms in wild type cells and not in the mutant cells.
Granuloma eosinophils enhance IL-5 production by lymphocytes from mice infected with Schistosoma mansoni.
Authors: Metwali A , Elliott D , Blum A M , Weinstock J V
Published: December 2022

IL-5 is essential for granuloma eosinophilia in mice infection with schistosomiasis. The granulomas constitutively make IL-5 that originates from granuloma CD4+ T lymphocytes. This observation prompted us to learn whether non-T cell elements in the granuloma can promote constitutive IL-5 production. Neither granuloma eosinophils nor spleen cells from infected mice constitutively produced detectable levels of IL-5. Yet, mixing spleen cells with granuloma eosinophils resulted in spontaneous IL-5 secretion without promoting IL-4 production. Moreover, the admixed cultures were more responsive to anti-CD3 or soluble egg Ag than were cultures containing spleen cells alone. IL-5 ELISPOT assays in Transwell chambers showed that the cellular origin of IL-5 in the admixed cultures was the spleen cells, and that the phenomenon did not require cell-to-cell contact with granuloma eosinophils. Also, admixed cultures of granuloma eosinophils and spleen cells released less IL-5 in the presence of mAb that specifically blocked the biologic activity of either IL-2, IL-2R, or IAk. However, adding rIL-2 to spleen cell cultures at concentrations up to 100 U/ml could not stimulate secretion of IL-5. Because eosinophil supernatants contained no IL-2 as assessed by CTLL-2 bioassay, the release of IL-5 in response to eosinophils was not likely secondary to eosinophil secretion of IL-2. Thus, it appears that eosinophils produce a soluble substance that, with IL-2 and ongoing class II MHC/TCR interaction, enhances lymphocyte IL-5 output.
Mycobacteria-macrophage interactions. Macrophage phenotype determines the nonopsonic binding of Mycobacterium tuberculosis to murine macrophages.
Authors: Stokes R W , Haidl I D , Jefferies W A , Speert D P
Published: December 2022

During tuberculosis, host defenses may be determined, in part, by the capacity of resident, elicited, and activated macrophages to bind and ingest Mycobacterium tuberculosis. We have investigated the mechanism by which macrophages bind M. tuberculosis and other mycobacteria in a serum-free system. The extent of binding of M. tuberculosis to macrophages was dependent on the phenotype of the macrophage; thioglycollate-elicited and immune-activated macrophages bound mycobacteria poorly, whereas resident macrophages bound mycobacteria efficiently. Within 'freshly' explanted macrophage populations (from 2 to 24 h in vitro) poor binding of mycobacteria correlated with poor binding of C3bi-coated particles, but not with variations in the level of complement receptor 3 (CR3) expression. Induction of C3bi-coated particle binding in thioglycollate-elicited macrophages by PMA was not accompanied by enhanced M. tuberculosis binding. Inhibition of M. tuberculosis binding by resident macrophages could only be achieved using a mAb recognizing an epitope within CR3 distinct from that which recognizes C3bi. Our results suggest that nonopsonic binding of M. tuberculosis is mediated by a site within CR3, which is distinct from the C3bi binding site. In addition, we show a variation in the capacity of different macrophage phenotypes to bind mycobacteria nonopsonically. These data suggest that heterogeneity in macrophage-mediated clearance of M. tuberculosis may be a significant factor in the progression of tuberculosis.
Regulation of integrin-mediated myeloid cell adhesion to fibronectin: influence of disulfide reducing agents, divalent cations and phorbol ester.
Authors: Davis G E , Camarillo C W
Published: December 2022

Three different agents, dithiothreitol (DTT), Mn2+, and phorbol ester (TPA), were found to induce HL-60 cell adhesion to fibronectin through distinct mechanisms. The binding of HL-60 cells to fibronectin and a 120-kDa fibronectin fragment is completely dependent on the alpha 5 beta 1 integrin, the adhesion activators, and appropriate divalent cations such as Mg2+. Mn2+ alone was able to induce maximal adhesion in the absence of these other activators. With any of the three activators, Ca2+ inhibited adhesion to fibronectin substrates by inhibiting alpha 5 beta 1-fibronectin binding. DTT and Mn2+ were both found to enhance the binding of fibronectin to purified alpha 5 beta 1, which suggests that both agents can directly stimulate the integrin-ligand binding reaction. TPA acts by inducing intracellular phosphorylation whereas neither DTT nor Mn2+ induced protein phosphorylation. TPA-treated HL-60 cells adhere and spread on fibronectin substrates, whereas DTT- and Mn(2+)-treated cells adhere but do not spread. The actin cytoskeletal inhibitor, cytochalasin B, markedly blocks TPA-induced adhesion, has an intermediate effect on DTT-induced adhesion, and has a minimal effect on Mn(2+)-induced adhesion. Collectively, the data suggest that TPA seems to act by inducing phosphorylation events that lead to cytoskeletal changes and alpha 5 beta 1 integrin activation. In contrast, DTT and Mn2+ seem to act primarily by directly influencing the alpha 5 beta 1-fibronectin binding reaction. These studies characterize in detail a regulatory system for studying leukocyte alpha 5 beta 1-fibronectin adhesion and identify DTT as a novel activator of alpha 5 beta 1-fibronectin binding.
Modulation of murine macrophage function by IL-13.
Authors: Doherty T M , Kastelein R , Menon S , Andrade S , Coffman R L
Published: December 2022

Activated macrophages are important effector cells for immune response to many parasites and immune responses are strongly modulated in part by the effect of Th cell-derived cytokines on macrophages. Th1-derived cytokines such as IFN-gamma are strong stimulators of macrophage activation, while cytokines produced by Th2 cells, including IL-4 and IL-10, have been shown under some conditions to inhibit macrophage activities associated with inflammatory responses. IL-13, a recently described cytokine produced by Th2 cells, is also capable of down-modulating macrophage activity in a manner similar to that previously described for IL-4. Treatment of activated macrophages with IL-13 reduces the production of inflammatory monokines in response to IFN-gamma or LPS, both potent stimulators of these factors. In addition, IL-13 decreases the production of nitric oxide by activated macrophages. Nitric oxide has been implicated in both macrophage cytotoxicity and macrophage-associated immunosuppression. The suppression of nitric oxide by IL-13 leads to a decrease in parasiticidal activity by activated macrophages. However, our data indicate that IL-13 has pleiotropic effects, while the inflammatory potential of activated macrophages is significantly reduced, the potential of other macrophage subsets is unimpaired. These data indicate that IL-13 could be a potent modulator of immune responses in vivo, with effects that may embrace both macrophage suppressive and macrophage potentiating functions.
Nitric oxide signaling: a possible role for G proteins.
Authors: Lander H M , Sehajpal P K , Novogrodsky A
Published: December 2022

We have previously reported various inductive effects of nitric oxide on human PBMC. We describe a novel and potentially important mechanism of nitric oxide signaling-through direct activation of guanine nucleotide-binding proteins (G proteins). We have found that nitric oxide treatment of membranes isolated from fresh human PBMC enhances the ability of these membranes to hydrolyze [gamma-32P]GTP and bind [gamma-35S]GTP. In addition, treatment of whole cells with nitric oxide yielded membranes with enhanced GTPase activity. Furthermore, the GTPase activity of pure, recombinant Gs alpha, Gi alpha 1, and p21ras was greatly enhanced by nitric oxide. In support of the existence of this pathway in whole cells, we found that the G protein inhibitor, GDP-beta-S, blocked NF-kappa B translocation induced by nitric oxide or LPS in permeabilized cells. In addition, nitric oxide greatly reduced the pertussis toxin-mediated ADP-ribosylation of 45- and 41-kDa proteins in membranes of these cells. Because G proteins play a central role in many diverse signaling systems, activation by an endogenous and inducible oxidant may represent a novel signaling pathway.
Engraftment of human synovium into severe combined immune deficient mice. Migration of human peripheral blood T cells to engrafted human synovium and to mouse lymph nodes.
Authors: Rendt K E , Barry T S , Jones D M , Richter C B , McCachren S S , Haynes B F
Published: December 2022

To determine the feasibility of using the C.B-17 scid/scid (severe combined immune deficient, SCID) mouse as a recipient of human synovial xenografts, we have engrafted human synovium under the renal capsule of SCID mice, and determined synovial graft survival and histologic characteristics 4 to 7 wk after tissue implantation. Both normal and inflammatory synovial tissue grew well in SCID mice and maintained histologic and phenotypic components of the fresh synovial tissue before implantation. However, the number of T cells in synovial grafts decreased after implantation. To determine whether leukocytes could migrate to human synovial xenografts, either allogenic or autologous PBMC were injected in the peritoneum of SCID mice bearing synovial xenografts. We found that 7 days after i.p. injection of autologous or allogeneic PBMC, injected T cells had selectively migrated to human synovial grafts and to SCID mouse lymph nodes. Our data demonstrate that normal and inflammatory human synovial tissues will grow in SCID mice and serve as recipients for autologous and allogenic peripheral blood human T cells injected i.p. into engrafted mice.
CD4+8- and CD4-8+ mature thymocytes require different post-selection processing for final development.
Authors: Petrie H T , Strasser A , Harris A W , Hugo P , Shortman K
Published: December 2022

Two primary types of TCR-alpha/beta+ T cells are found in the peripheral lymphoid system; CD4+8- T cells, with MHC-class II restricted TCR, and CD4-8+ T cells, which are MHC-class I restricted. Both lineages develop in the thymus from a series of common precursors. However, the precise stage at which they diverge, and the combination of factors that regulates such divergence, are not well defined. The up-regulation of CD3/TCR to high mature levels is thought to be an early event associated with positive selection for self-MHC recognition. Using purified cells from bcl-2 transgenic mice in order to overcome the limitations imposed by cell death on normal thymocytes, we find that a minor subset of CD4+8+ thymocytes expressing high levels of CD3/TCR gives rise to both CD4+8- and CD4-8+ mature cells upon intrathymic transplantation, but only to CD4-8+ in culture. Thus, in addition to demonstrating the dual lineage potential of this subset, these findings show that additional post-selection processing events are required for the production of mature thymocytes, and that CD4+8- and CD4-8+ subsets differ in the types of processing required.
Priming does not change promoter sequence requirements for IFN induction or correlate with the expression of IFN regulatory factor-1.
Authors: Rosztoczy I , Pitha P M
Published: December 2022

Pretreatment of L929 cells with IFN enhances Sendai virus-mediated induction of IL-6, TNF-alpha, and IFNA and IFNB genes. The priming effect could be demonstrated at both the RNA and protein levels and the former did not require cellular protein synthesis. Priming increased the Sendai virus-mediated induction of a murine IFNA4 promoter-chloramphenicol acetyltransferase gene hybrid plasmid (A4CAT) in transiently transfected cells, and deletion analysis showed that the identical DNA sequence was required for the inducibility in primed and unprimed cells. Cotransfection of A4CAT plasmid with interferon regulatory factor-1 (IRF-1) expression plasmid increased CAT expression, however, the IRF-1-mediated expression was further enhanced by priming. These results show that the identical inducible element present in the promoter region of IFNA4 gene is required for both the inducibility and the priming effect; however, no direct correlation between the enhancement of expression of the IRF-1 gene and enhancement of expression of IFN, IL-6, and TNF-alpha genes in the primed cells was observed. We suggest that priming facilitates inducer-mediated posttranscriptional modulation of various transcriptional factors that play a role in stimulation of transcription of these genes.