bluuebunny/crossref_metadata_2025 huggingface dataset:
Islet-specific T cell clones transfer diabetes to nonobese diabetic (NOD) F1 mice.
Authors: Peterson J D , Pike B , McDuffie M , Haskins K
Published: December 2022

To investigate diabetes resistance to T cell-mediated disease transfer, we administered islet-specific T cell clones to the F1 progeny of nonobese diabetic (NOD) mice that were crossed with various nondiabetes-prone inbred mouse strains. We investigated four diabetogenic CD4+ T cell clones and all induced insulitis and full development of diabetes in (SWR x NOD)F1, (SJL x NOD)F1, and (C57BL/6 x NOD)F1 mice. In contrast, (BALB/c x NOD)F1 and (CBA x NOD)F1 mice were susceptible to disease transfer by some T cell clones but not others, and (C57/L x NOD)F1 mice seemed to be resistant to both insulitis and disease transfer by all of the clones tested. Disease induced by the T cell clones in susceptible F1 strains was age dependent and could only be observed in recipients younger than 13 days old. Full or partial disease resistance did not correlate with the presence or absence of I-E, different levels of Ag expression in islet cells, or differences in APC function. The results from this study suggest that there may be multiple factors contributing to susceptibility of F1 mice to T cell clone-mediated induction of diabetes, including non-MHC-related genetic background, the immunologic maturity of the recipient, and individual characteristics of the T cell clones.
CD4+ and CD8+ alpha beta, and gamma delta T cells are cytotoxic effector cells of beta 2-microglobulin-deficient mice against cells having normal MHC class I expression.
Authors: Udaka K , Marusić-Galesić S , Walden P
Published: December 2022

Despite original reports that describe the absence of CD8+ CTLs in mice having homozygous deficiency in the beta 2-microglobulin gene, strong cytotoxic activities against cells with normal beta 2-microglobulin expression were observed in these animals. This cytotoxicity was reproducibly induced by immunizations with allogeneic or syngeneic cells. MHC class II expression by the stimulator cells was not required. The effector population contained alpha beta T cells with CD4+ or CD8+ phenotypes, as well as gamma delta T cells with CD8- and CD8+ phenotypes. Both alpha beta T cell types recognized MHC class I molecules. The gamma delta T cells seemed to respond to molecules that are affected by the absence of beta 2-microglobulin, but that are not encoded by classical MHC loci.
Human TCR-gamma/delta alloreactive response to HLA-DR molecules. Comparison with response of TCR-alpha/beta.
Authors: Flament C , Benmerah A , Bonneville M , Triebel F , Mami-Chouaib F
Published: December 2022

We have analyzed the human gamma/delta T cell alloreactive response to class II HLA-DR molecules and attempted to compare this response with that mediated by the TCR-alpha/beta counterparts. Several gamma/delta CTL clones from a healthy individual were generated in mixed lymphocyte reactions against an EBV-transformed B cell line termed E418. Fine specificity and primary TCR structure of 10 representative clones (all CD4- CD8 +/-) were then determined. Functional studies, with the use of B cell lines homozygous for HLA-DR (DR1-10), indicated that all gamma/delta T cell clones specifically reacted with HLA-DR2 molecules. In addition, five clones were able to cross-react with subtypes of HLA-DR8. Extended panel target experiments, including lymphoblastoid cells expressing various HLA-DR2 subtypes, showed that the T cell clones displayed distinct fine specificities. Clones with broad (Dw2, Dw12, Dw21, Dw8.1, and Dw8.2) or in contrast, more restricted (DRB1*1501 or DRB1*1503) specificity were identified. Furthermore, amino acid substitutions at predicted peptide binding site position 30 and TCR-interacting position 67 of the DRB*1 beta-chain seemed to affect alloresponse of some T cell clones. With respect to TCR-gamma/delta structure, diversity in gene segment usage was observed, with the predominance of T cells using a V3-J gamma 2/V1-J delta 1+ receptor. A smaller fraction of the cells expressed TCR comprising V gamma 9 and V delta 1 regions. In contrast, the V delta 3 gene segment was used by a minority of the cells, and the V delta 2 was not expressed by any T cell clone. Together, the present data indicate that similarly to TCR-alpha/beta, human TCR-gamma/delta lymphocytes may recognize in a highly specific fashion a particular HLA-DR heterodimer. T cell clones cross-reacting with other HLA-DR molecules were also identified. Despite some degree of heterogeneity, V gene segment use by alloreactive clones seemed to be nonrandom. No obvious correlation between TCR gene use and HLA-DR alloreactivity could be identified. Moreover, our results suggest that similarly to TCR-alpha/beta cells, foreign MHC-bound peptides may contribute to TCR-gamma/delta alloreactive response.
A molecule expressed on accessory cells, activated T cells, and thymic epithelium is a marker and promoter of T cell activation.
Authors: Izon D J , Jones L A , Eynon E E , Kruisbeek A M
Published: December 2022

T cell maturation results in part from direct cell-cell interactions between developing thymocytes and thymic stromal cells. Identification of the cell surface molecules involved in these interactions has been approached by production of mAbs reactive to thymic stromal cell surface Ags. A mAb against one such Ag, mouse thymic stroma (MTS) mAb MTS 23, stains a subset of thymic medullary epithelium by immunohistology. In addition, it was found to detect, by flow cytometry, an Ag constitutively expressed on peripheral B cells and macrophages as well as thymic and splenic dendritic cells. This Ag was also up-regulated on T cells and thymocytes within 24 to 48 h after activation. We then investigated whether the Ag identified by MTS 23 may represent a functional accessory molecule. MTS 23 was able to block up to 75% of T cell proliferation in soluble anti-CD3 and Ag-induced responses in a dose-dependent manner, but not under conditions in which no APCs were required. The molecule detected by this mAb has an apparent molecular mass of 120 kDa under reducing and nonreducing conditions. On the basis of these molecular properties and expression pattern, it is therefore postulated that MTS 23 may detect a novel accessory molecule important for T cell activation. Its expression on thymic epithelium is consistent with the notion that T cell development is not solely a consequence of unique molecular interactions, but also of signals arising from combinations of interactions involving molecules also expressed extrathymically.
Activation of the Ig germ-line gamma 1 promoter. Involvement of C/enhancer-binding protein transcription factors and their possible interaction with an NF-IL-4 site.
Authors: Lundgren M , Larsson C , Femino A , Xu M , Stavnezer J , Severinson E
Published: December 2022

Ig isotype switching in B lymphocytes is preceded by transcription of the corresponding unrearranged, or germ-line (GL), CH gene. The promoter of mouse GL C gamma 1 transcripts has been shown to be located within a 349-bp KpnI/Bg/II fragment, extending from -147 to +202 bp relative to the first transcription initiation site. By the electrophoretic mobility shift assay, we have analyzed nuclear extracts from three B cell lines and splenic B cells for the presence of proteins binding to this fragment. We show that they give different patterns of DNA binding, implying significant complexity in the regulation of this locus. We focused on the sIgM+ mouse B lymphoma line L10A6.2 that has been shown able to confer responsiveness of the GL gamma 1 promoter to phorbol ester plus IL-4. Activation of this cell line results in altered expression of several nuclear DNA-binding complexes involving two members of the C/enhancer-binding protein (EBP) family of transcription factors, namely C/EBP beta (nuclear factor (NF)-IL-6/LAP) and Ig/EBP-1 (C/EBP gamma). The complexes bind to two C/EBP elements, one at about -115 bp and one near the first RNA start site. In normal B cells stimulated by LPS or IL-4, new complexes appear that bind to C/EBP and NF-IL-4 elements, respectively, located within the -125/-101 region. The -125/-101 segment previously has been shown to be required for transcriptional activation. We discuss these findings in relation to the presence of consensus C/EBP binding sites in other IL-4-regulated promoters.
TNF receptor signal transduction. Ligand-dependent stimulation of a serine protein kinase activity associated with (CD120a) TNFR60.
Authors: VanArsdale T L , Ware C F
Published: December 2022

TNF is a pluripotent cytokine that mediates activities through two distinct receptors of 55 to 60 kDa (CD120a, known as TNFR60) and 75 to 80 kDa (CD120b, known as TNFR80). These receptors share homology in the extracellular ligand binding region; however, the cytoplasmic domains are distinct and lack any inherent enzymatic activity, which suggests that ligand binding and subsequent receptor clustering leads to the association of active signaling molecules with TNFRs. To test this hypothesis, we isolated TNFRs by immunoprecipitation and examined the immune complexes for the presence of associated phosphoproteins and protein kinase activity. In the U-937 monocytic cell line, prelabeled with 32PO4, TNF induces the association of several phosphoproteins with TNFR60, but not TNFR80. The TNFR60 immune complexes also contain a TNF-dependent serine protein kinase activity, which was detected by an in vitro kinase assay, that phosphorylates proteins of 125, 97, 85, and 60 kDa, which are of apparent molecular masses that are similar to those of TNF-induced phosphoproteins that coprecipitate with TNFR60. Association of serine protein kinase activity with TNFR60 is rapid and dependent on the concentration of TNF. Proteins of molecular mass similar to the 125- and 97-kDa protein kinase substrates seem to be associated with TNFR60 immune complexes only after exposure of U-937 cells to TNF. The TNFR60-associated protein kinase activity is inhibited by staurosporine, but not by the protein kinase A and C inhibitors, HA-1004 and H7. Staurosporine greatly enhanced the sensitivity of U-937 cells to the cytotoxic effect of TNF. These results suggest a serine protein kinase(s), and, possibly, other TNF-dependent TNFR60-associated proteins may be involved in mediating signals through TNFR60 in response to ligand binding.
Erythrocyte complement receptor type 1 and interactions between immune complexes, neutrophils, and endothelium.
Authors: Beynon H L , Davies K A , Haskard D O , Walport M J
Published: December 2022

The deposition of immune complexes (IC) may play an important part in the pathogenesis of vasculitis. An increase in permeability of the vascular endothelial lining is a prerequisite for IC deposition. We used an in vitro model to examine the effects of interactions between IC, neutrophils, and endothelium on the integrity of endothelial cell monolayers. Human umbilical vein endothelial cells were grown to confluence on an FITC-labeled matrix, and monolayer integrity was assessed by the exclusion of an 125I-anti-FITC Ab. Alteration in endothelial monolayer permeability was associated with increased uptake of 125I-anti-FITC Ab, expressed as a percentage of the maximal uptake of Ab onto the FITC-matrix from which endothelial cells had been stripped. Neither resting nor cytokine-stimulated endothelial cells bound hepatitis B surface Ag (HBsAg/anti-HBsAg) IC. Immune complexes were shown to activate neutrophils to induce a 9.5% increase in the permeability of IL-1 beta-stimulated endothelium. This increase in endothelial permeability was abrogated by the addition of RBC bearing normal complement receptor type 1 (CR1) numbers (3.2%). This protective effect was shown to be related to the binding of IC to erythrocyte CR1 and was reduced by CR1 blockade using polyclonal rabbit anti-CR1 Abs. These observations demonstrate that IC are capable of directly activating neutrophils to induce increases in endothelial permeability, which may facilitate the deposition of circulating IC. The results support the hypothesis that the binding of IC to erythrocyte CR1 may inhibit damaging interactions between IC, neutrophils, and endothelium.
Quantitation of ICAM-1 expression in mouse lung during pneumonia.
Authors: Burns A R , Takei F , Doerschuk C M
Published: December 2022

In the systemic circulation, neutrophil emigration into sites of acute inflammation is mediated through the leukocyte adhesion complex, CD11/CD18. ICAM-1 is an inducible endothelial ligand for CD11a/CD18 and CD11b/CD18. Streptococcus pneumoniae elicits neutrophil emigration through a CD18-independent mechanism whereas Escherichia coli endotoxin elicits emigration through a CD18-dependent mechanism in rabbit lungs. To determine whether ICAM-1 is up-modulated in the lung during CD18-independent and CD18-dependent emigration, ultrastructural immunogold-labeling studies were performed on BALB/c mice given airway instillates of S. pneumoniae or E. coli endotoxin. Ultrathin cryosections of frozen lung tissue were immunogold labeled with the mAb YN1/1.7.4 against the murine homologue of human ICAM-1. Gold particles on the plasma membranes of alveolar endothelial and epithelial cells were quantitated by transmission electron microscopy. Capillary endothelial ICAM-1 expression did not change during neutrophil emigration toward S. pneumoniae, a CD18-independent pathway in rabbits. In contrast, ICAM-1 expression increased 4.2-fold in response to E. coli endotoxin (known to elicit CD18-dependent emigration in mice), suggesting that the mechanism of adhesion may be regulated by the expression of endothelial rather than neutrophil adhesion molecules. Constitutive expression of ICAM-1 on alveolar epithelial cells was 22-fold greater than on capillary endothelium. Epithelial expression was mainly restricted to type I pneumocytes, whereas type II pneumocytes, the precursors of type I cells, expressed little or no ICAM-1. However, during pneumonia, type II but not type I pneumocytes showed increased ICAM-1 expression, suggesting that ICAM-1 expression represents an early differentiation even in response to epithelial injury.
Association of IgA-Fc receptors (Fc alpha R) with Fc epsilon RI gamma 2 subunits in U937 cells. Aggregation induces the tyrosine phosphorylation of gamma 2.
Authors: Pfefferkorn L C , Yeaman G R
Published: December 2022

We investigated the possibility that IgA-binding chains of Fc alpha R on monocytic cells are physically associated with gamma 2 subunits of Fc epsilon RI (Fc epsilon RI gamma 2 or gamma 2). Fc alpha R was precipitated from lysates of IFN gamma-treated U937 cells, subclone 10.6, and probed by immunoblotting with Ab against human gamma 2. Fc alpha R was precipitated through anti-Fc alpha R mAbs A59 or A62, through A62 from lysates that had been exhaustively precleared of high affinity IgG-Fc receptors (Fc gamma RI) and of low affinity Fc gamma RII, and through anti-Fc alpha R mAb A77 from Fc gamma RI-precleared lysates of untreated 10.6 cells. precipitation was also performed through F(ab')2 A77 and through the native ligand of the receptor, hlgA. In all cases, Fc alpha R precipitates contained co-isolated 22-kDa gamma 2 (unreduced). The Fc alpha R alpha-chain/gamma 2 complex did not readily dissociate in 1% Nonidet P-40 as did Fc gamma RI alpha-chain/gamma 2, suggesting a novel aspect to the Fc alpha R subunit interaction. Specific Fc alpha R aggregation on cells triggered a robust respiratory burst and the tyrosine phosphorylation of several proteins. Among them was phospho-gamma 2, which migrated as a 24- to 28-kDa gamma 2 phosphoprotein on gels and was detected as a 28-kDa phosphoprotein by anti-phosphotyrosine immunoblot. Aggregated Fc alpha Rs that were precipitated from Fc alpha R-triggered cells also contained a phosphoprotein of the same mobility and immunoreactivity, as did aggregated Fc gamma RI from which the 28-kDa phosphoprotein could be more readily eluted and identified (as phospho-gamma 2). We concluded that myelocytic Fc alpha Rs are multichain complexes containing gamma 2 subunits that are tyrosine phosphorylated upon Fc alpha R aggregation. As IgA is the predominant Ig on mucosal surfaces, gamma-subunits may play an important role in mucosal immunity involving leukocytic Fc alpha R.
Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide.
Authors: Satta N , Toti F , Feugeas O , Bohbot A , Dachary-Prigent J , Eschwège V , Hedman H , Freyssinet J M
Published: December 2022

Endotoxin-stimulated monocytes can elicit a dual procoagulant response. They express tissue factor and expose phosphatidylserine in the outer leaflet of the plasma membrane. Tissue factor, a membrane glycoprotein, is the cellular trigger of blood coagulation reactions. Phosphatidylserine is an essential anionic phospholipid for surface amplification of thrombin generation. In this study the distribution of these two procoagulant entities between activated monocytes and derived microparticles was assessed after stimulation by LPS. The presence of CD14, CD11a, and CD18, and possible associated adhesion potential were examined, particularly on microparticles. Tissue factor was evidenced by using a specific functional assay and flow cytometry. Phosphatidylserine exposure was monitored through its catalytic activity in a thrombin generation assay and by flow cytometry with the use of FITC-conjugated annexin V, a protein probe of anionic phospholipids. CD14, CD11a, and CD18 were detected by flow cytometry. The interaction of microparticle CD11a/CD18 with intracellular adhesion molecule-1 was demonstrated by using immobilized recombinant intracellular adhesion molecule-1 fusion protein. The major part of tissue factor and phosphatidylserine-dependent procoagulant activity was associated with microparticles after LPS stimulation. This was confirmed by flow cytometry. The presence of functional CD11a/CD18, and CD14 on microparticles testifies to an associated adhesion potential. These results show that membrane vesiculation could be responsible for dissemination of inducible monocyte procoagulant activities and suggest that derived microparticles could also participate in endothelium stimulation. This emphasizes the role of monocyte as a central element in the coupling between inflammation/infection and thrombosis.
T cell-independent and T cell-dependent B cell activation increases IFN-gamma R expression and renders B cells sensitive to IFN-gamma-mediated inhibition.
Authors: Abed N S , Chace J H , Cowdery J S
Published: December 2022

We have studied the relationship between B cell activation and the ability of IFN-gamma to inhibit B cell differentiation. The LPS activation of conventional and CD5+ B cells resulted in increased IFN-gamma R expression and increased the ability of IFN-gamma to inhibit LPS-induced B cell differentiation correlated with increased IFN-gamma R expression. We detected increased B cell IFN-gamma R expression 12 h after activation, and maximal IFN-gamma R expression was observed at 24 h. Activation of B cells by F(ab2)' anti-IgM induced a similar increase in IFN-gamma R expression. In autoimmune New Zealand Black mice, both conventional and CD5+ B cells showed a pattern of IFN-gamma R expression similar to that seen in DBA/2 mice, and both populations of B cells were sensitive to inhibition by IFN-gamma. To examine the role of IFN-gamma in the regulation of T cell-dependent B cell responses, we activated B cells with the CDC35 T cell line (which is specific for rabbit IgG). When rabbit anti-mouse Ig-treated B cells were activated by CDC35 T cells, we found that B cells exhibited increased IFN-gamma R expression by 48 h; we also found that IFN-gamma inhibited CDC35-mediated IgM secretion to a degree similar to IFN-gamma inhibition of T cell-independent B cell differentiation. Additionally, IFN-gamma inhibited CDC35-stimulated B cells even in the presence of exogenous IL-4 and IL-5. This study establishes the importance of IFN-gamma as a regulator of both T cell-independent and T cell-dependent B cell differentiation.
Analysis of IL-2 gene regulation in c-fos transgenic mice. Evidence for an enhancement of IL-2 expression in splenic T cells stimulated via TCR/CD3 complex.
Authors: Ochi Y , Koizumi T , Kobayashi S , Phuchareon J , Hatano M , Takada M , Tomita Y , Tokuhisa T
Published: December 2022

Effects of the deregulated c-fos gene product on IL-2 expression were studied in splenic T cells from c-fos transgenic (H2-c-fos) mice. IL-2 gene expression and IL-2 production by H2-c-fos T cells stimulated with immobilized anti-CD3 Abs were enhanced and prolonged as compared with those by control T cells. Activator protein-1 activity in nuclear extract from the H2-c-fos T cells was also higher than that from the control cells. There was no significant difference in the activity of other transcription factors including IL-2 kappa B, NFAT, and Oct-1, between the H2-c-fos and the control T cells. However, activity of a negative regulatory element binding factor (NRE-A) in the H2-c-fos T cells was much lower than that in the control cells. These results suggest that c-Fos/activator protein-1 is a major regulatory factor for IL-2 gene expression in splenic T cells activated through the TCR/CD3 complex.
Demonstration of organic anion transport in T lymphocytes. L-lactate and fluo-3 are target molecules.
Authors: Sommer F , Bischof S , Röllinghoff M , Lohoff M
Published: December 2022

In this paper, we describe for the first time the existence of organic anion transport in T lymphocytes, exemplified by the transmembrane transport of the anions L-lactate and the Ca2+ indicator fluo-3. The transport of either anion was found to be inhibitable by probenecid, a common blocker of organic anion transport. Transport of L-lactate was observed in long-term cultured T cell lines, as well as in freshly ex vivo isolated T cells, and occurred via a saturable, pH-dependent, and stereospecific process. L-Lactate uptake was dependent on the activation state of the T cells, because activation of T cells by Con A strongly enhanced accumulation of L-lactate from the medium. Because L-lactate may be transported bidirectionally through the T cell membrane in vivo, different physiologic roles of L-lactate transport are discussed. L-Lactate uptake may serve as an alternative source of energy in an inflamed, glucose-deficient tissue or may represent a prerequisite for the earlier-published immunoregulatory function of this molecule on T cells. On the other hand, release of L-lactate emerging from glycolysis could be necessary to avoid acidification of the cell. The fact that the Ca2+ indicator fluo-3 is also transported through the cellular membranes of long-term cultured T cells via organic anion transport has important implications for the determination of Ca2+ influx into T cells. Even though the transport of both molecules, L-lactate and fluo-3, represents organic anion transport, evidence is presented that confirms that the respective transport systems are different.
Lack of allelic exclusion permits autoreactive B cells to escape deletion.
Authors: Iliev A , Spatz L , Ray S , Diamond B
Published: December 2022

The R4A-gamma 2b transgenic mouse harbors the gene for the gamma 2b heavy chain of an anti-dsDNA Ab. Approximately 80% of B cells expressing the transgene display allelic exclusion. Although the transgenic mice have little to no detectable serum anti-DNA activity, splenic B cells can be stimulated in vitro with LPS to secrete anti-DNA Ab. Hybridomas derived from LPS-stimulated splenic B cells were analyzed for expression of the transgene and for DNA binding. All nine transgene-encoded anti-DNA-producing lines were found to express an endogenous IgM heavy chain. Of 19 randomly selected lines producing a transgene-encoded non-DNA binding Ab, none expressed a second heavy chain. The tight correlation between lack of allelic exclusion and anti-dsDNA specificity provides strong support for the hypothesis that a major function of allelic exclusion is to prevent the maintenance of a pool of potentially activatable autoreactive cells.
Identification of the IgA-binding region in streptococcal protein Arp.
Authors: Johnsson E , Andersson G , Lindahl G , Hedén L O
Published: December 2022

Cell surface proteins that bind to the Fc part of human IgA are expressed by different species of pathogenic streptococci. The most extensively characterized streptococcal IgA-binding protein is the Streptococcus pyogenes protein Arp4, a member of the M protein family. Here we describe work that identifies the IgA-binding region in this streptococcal protein. A comparison of the amino acid sequences of protein Arp4 and four other IgA-binding proteins of S. pyogenes first made possible the identification of a putative IgA-binding region. Site-specific mutagenesis and generation of deletions were then used to show that Arp4 derivatives lacking different parts of the putative IgA-binding region had lost the ability to bind IgA. Conclusive evidence for the localization of the IgA-binding region was obtained through the characterization of a chimeric protein, in which the putative IgA-binding region of Arp4 had been introduced into another S. pyogenes cell surface protein that does not bind IgA. Our data show that a region comprising 29-amino acid residues in the N-terminal part of Arp4 is necessary and sufficient for IgA-binding capacity. Competitive inhibition experiments with synthetic peptides indicated that the C-terminal half of this 29 residue region may be most important for the IgA-binding property of Arp4. These results identify, for the first time, the ligand-binding region in an Fc alpha binding protein.
Five putative subclasses of swine IgG identified from the cDNA sequences of a single animal.
Authors: Kacskovics I , Sun J , Butler J E
Published: December 2022

We report the sequences of more than 40 partial and complete swine C gamma cDNAs obtained by PCR cloning of first strand cDNA, and from a cDNA expression library, all from a single animal. These seem to represent five IgG subclasses, that can be grouped into two clusters; one contains IgG1 and IgG3 and the other, IgG2a, IgG2b and IgG4. IgG2a and IgG2b differ by only three amino acids, but single strand conformational polymorphism analyses of PCR-amplified IgG2-specific segments in animals of different breeds, argues for their putative subclass status. Major subclass differences are found in the hinge and C gamma 3, but differences in upper hinge length, associated with segmental flexibility in the IgGs of other species, are absent. All subclasses have identical middle hinge segments that can accommodate three interheavy chain disulfide bridges. The putative swine IgG subclasses have their greatest similarity with those of the human except for the near absence of hinge region variation. Swine subclasses such as ruminant IgG2a, have a lower hinge deletion which in human IgG1, contains one of the motifs believed necessary for interaction with Fc gamma Rs. IgG1 was the most frequently encountered subclass cDNA (25 of 43) and the single swine-mouse hybridoma tested had a sequence identical to IgG1. Partial sequence analyses of genomic clones identified one clone identical with the IgG1/IgG3 subclass cluster, two identical to the IgG2b/IgG4 subclass group, and two identical to each other but different from any of the expressed sequences reported here. Genomic blots suggest that up to eight C gamma genes are present in the genome.
Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages.
Authors: Jun C D , Choi B M , Hoon-Ryu  , Um J Y , Kwak H J , Lee B S , Paik S G , Kim H M , Chung H T
Published: December 2022

The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone. On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.
Contribution of tyrosine kinases to the selective orientation of human CD4+ bifunctional cloned T cells toward proliferation or cytolytic function.
Authors: Eljaafari A , Soula M , Dorval I , Pirenne H , Quelvennec E , Bernard A , Fagard R , Sterkers G
Published: December 2022

We show that T cell activation of human CD4+ cloned T cells through the CD2 molecule can induce either autocrine proliferation or cytolysis, depending on the pair of anti-CD2 mAbs used for stimulation, that is, D66/T11(1) or GT2/T11(1), respectively. As the earliest biochemical event after CD2 stimulation is likely the induction of tyrosine phosphorylation of various proteins, we investigated whether differential activation of protein tyrosine kinases (PTKs) could contribute to the selective induction of each function. Results show that herbimycin A, a potent PTK inhibitor, markedly decreased the induction of both proliferation and cytolysis. This implies a regulatory role for tyrosine phosphorylation in the induction of each function by CD2. However, that PTKs are differentially activated upon induction of proliferation by D66/T11(1) or cytotoxic function by GT2/T11(1) emanated from two different approaches. First, immunoblotting total cellular extracts with an anti-phosphotyrosine mAb showed different patterns of tyrosine phosphorylation depending on the pair of CD2 mAbs used for stimulation. Second, a differential activation of p56lck, a src-related PTK, was observed after stimulation with D66/T11, and GT2/T11(1). Although induction of proliferation by D66/T11(1) was correlated with increased Lck activity, this was not observed when cells were triggered to lyse by GT2/T11(1). Thus, by providing striking correlative evidences linking differences in PTK activation with induction of different functions in bifunctional cloned T cells, our results strongly suggest that PTKs may contribute to the selective orientation of T cell functions at a single-cell level.
Immunological tolerance to a defined myelin basic protein antigen administered intrathymically.
Authors: Goss J A , Nakafusa Y , Roland C R , Hickey W F , Flye M W
Published: December 2022

To explore the mechanisms responsible for the development of tolerance to allografts after intrathymic (IT) injection of alloantigen, the well-defined model of experimental autoimmune encephalomyelitis (EAE), which mimics the human autoimmune disease multiple sclerosis, was used. This inflammatory neurologic syndrome is initiated by myelin basic protein (MBP)-reactive CD4+ T lymphocytes restricted to self-MHC class II molecules. Naive adult, EAE-susceptible Lewis (RT1(1) rats were treated IT, i.v., or i.p. with a single dose (100 micrograms) of guinea pig-myelin basic protein (GP-MBP 1-176) in PBS plus 1 ml rabbit anti-rat lymphocyte serum i.p. Twenty-one days later, all rats were challenged by intradermal hind footpad injections of 50 micrograms GP-MBP in PBS emulsified in CFA. Only IT, but not i.p. or i.v., administration of GP-MBP plus anti-lymphocyte serum conferred marked resistance to a subsequent systemic challenge of GP-MBP, as demonstrated by the prevention of weight loss and paralysis characteristic of EAE. The IT administration dramatically decreased the size and number of histologic perivascular infiltrates observed per visual field in spinal cord of the tolerant animals and decreased GP-MBP-specific T lymphocyte in vitro proliferation (p < 0.01), whereas proliferation to a nonspecific mitogen (Con A) was not altered. With the addition of rIL-2, the decreased Ag-specific proliferative responses of IT-treated animals increased to control levels. Adoptive transfer of 100 x 10(6) splenocytes from tolerant hosts i.v. to naive syngeneic Lewis rats challenge with 100 micrograms GP-MBP in CFA had no effect on clinical or histologic EAE. Exposure of MBP to maturing thymocytes results in functionally immunounresponsive lymphocytes and prevention of autoimmune EAE.
Cell surface P- and E-selectin support shear-dependent rolling of bovine gamma/delta T cells.
Authors: Jutila M A , Bargatze R F , Kurk S , Warnock R A , Ehsani N , Watson S R , Walcheck B
Published: December 2022

The vascular selectins P- and E-selectin are inducible adhesion proteins expressed by endothelial cells that have been shown to support shear-dependent rolling of myeloid cells. This interaction is thought to be a prerequisite event for subsequent steps, such as tight adhesion/aggregation and transendothelial cell migration, involved in the accumulation of leukocytes into tissues. Certain lymphocyte subsets have also been shown to bind the vascular selectins, but the importance of this interaction in mediating shear-dependent rolling, as described for myeloid cells, has not been demonstrated. We expand on our earlier observation that bovine gamma/delta T cells bind E-selectin by showing that this interaction leads to a reproducible rolling event in assays done under shear forces that approximate those that occur in vivo. E-selectin, expressed by L cell transfectants or cytokine-stimulated human and bovine endothelial cells, equally supports the shear-dependent rolling interaction. The lymphocyte adhesion proteins L-selectin, CD44, and CD2 do not contribute to this event. Neuraminidase treatment of the gamma/delta T cells or addition of EDTA to the assay completely blocks the rolling interaction. We further show for the first time that P-selectin expressed by thrombin-activated platelets or a soluble P-selectin/human Ig chimera specifically binds gamma/delta T cells. The P-selectin interaction is similar to the rolling event mediated by E-selectin--it requires divalent cations and sialic acid on the lymphocyte, it lacks involvement of L-selectin and CD44, and rolling occurs under physiologic shear conditions. These results provide the documentation that the vascular selectins can support shear-dependent rolling of a lymphocyte subset and that P-selectin mediates the adhesion of gamma/delta T cells.
T lymphocyte development and function in dogs with X-linked severe combined immunodeficiency.
Authors: Somberg R L , Robinson J P , Felsburg P J
Published: December 2022

Canine X-linked severe combined immunodeficiency disease (XSCID) is characterized by a failure to thrive, thymic dysplasia, and a lack of a T lymphocyte mitogenic response. As in human XSCID, affected dogs in our colony have a mutation in the IL-2R-gamma gene. This mutation dramatically altered T lymphocyte development, because XSCID thymi were severely reduced in size and cellularity, contained an increased proportion of immature CD4-CD8- thymocytes, a decreased proportion of intermediate CD4+CD8+ thymocytes, and a normal proportion of CD4+CD8- and CD4-CD8+ thymocytes. XSCID thymi were also deficient in the percentage of CD3-L+ thymocytes. Interestingly, several XSCID dogs had normal percentages of CD3-L+ PBL. Although the mutation did not interfere with IL-2 production, PHA-activated XSCID PBL demonstrated severely diminished IL-2 binding and were nonresponsive to IL-2. These results indicate that the lack of a functional IL-2R-gamma chain in dogs with XSCID primarily affects developing CD4-CD8- thymocytes as they acquire the cell surface Ag CD3-L and interferes with the ability of peripheral T lymphocytes to bind and respond to IL-2.
Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase.
Authors: Wang J , Nemoto E , Kots A Y , Kaslow H R , Dennert G
Published: December 2022

This report demonstrates that incubation of cytotoxic T cells with NAD causes suppression of their ability to proliferate in response to stimulator cells or to lyse targets. Effects are evident after incubation for 3 h with concentrations of NAD as low as 1 microM and are sustained for many hours after removal of NAD from culture media. Suppression is a result of the failure of CTL to form specific conjugates with targets as well as a lower level of activation in response to TCR-mediated stimulation, although TCR-mediated transmembrane signaling is demonstrable. Metabolites of NAD such as nicotinamide, ADP-ribose, and cyclic-ADP-ribose have no detectable effect, indicating that NAD-glycohydrolase or ADP-ribose cyclase do not mediate suppression. Incubation of intact CTL with [32P]NAD leads to incorporation of 32P into a particulate, subcellular fraction, a reaction that is not inhibitable by ADP-ribose. Hydroxylamine, but not mercuric ion releases [32P]ADP-ribose, whereas phosphodiesterase releases [32P]AMP from the particulate subcellular fraction, suggesting that labeling is a result of enzymatic mono-ADP-ribosylation of arginines. In support of this, treatment of intact CTL with phosphatidylinositol-specific phospholipase C releases an arginine-specific ADP-ribosyltransferase and causes insensitivity to ecto-NAD suppression. These results suggest that a GPI-anchored ADP-ribosyltransferase uses ecto-NAD to ADP-ribosylate proteins that regulate CTL function.
The role of NF-Y and IRF-2 in the regulation of human IL-4 gene expression.
Authors: Li-Weber M , Davydov I V , Krafft H , Krammer P H
Published: December 2022

Activity of the IL-4 promoter was shown to be regulated by multiple cis-acting elements. In this study, two additional regulatory elements, a CCAAT element and a 15-nucleotide element homologous to the IFN- and virus-stimulation response element (ISRE), were identified in the human promoter region at -195 to -172. The ISRE-homologous sequence was shown to interact with two nuclear proteins, IRF-2, a transcriptional repressor of the IFN genes, and an NF-1-like factor. Mutations of the ISRE site increased overall IL-4 promoter activity twofold, suggesting a possible negative role of IRF-2 in the regulation of IL-4 transcription. The CCAAT element was found to interact with NF-Y, a nuclear factor essential for expression of MHC class II genes. Mutations of the CCAAT element at -195 to -172 resulted in a substantial decrease of IL-4 promoter activity. Furthermore, NF-Y was also found to bind to the previously described NF-ATp binding site of the IL-4 promoter (-79 to -62, originally described as "P element"), and the previously described P-binding complex NF-P was shown to contain NF-Y. These findings suggest that NF-Y may play an important role in the regulation of IL-4 gene expression.
Bovine C4b binding protein. Molecular cloning of the alpha- and beta-chains provides structural background for lack of complex formation with protein S.
Authors: Hillarp A , Thern A , Dahlbäck B
Published: December 2022

C4b binding protein (C4BP) regulates the complement system. It also interacts with anticoagulant protein S and with serum amyloid P component. Human C4BP is composed of seven identical 70-kDa alpha-chains and one 45-kDa beta-chain. The binding site for C4b is located on the alpha-chain, whereas the beta-chain binds protein S. Nothing is known about the structure and function of bovine C4BP. No complexed form of protein S was detected by using a gel filtration chromatography system combined with Western blotting. Bovine cDNA clones encoding the C4BP alpha- and beta-chains were isolated from a bovine liver cDNA library. Three overlapping alpha-chain clones predicted a 562-amino acid residues-long mature polypeptide. The overall amino acid sequence similarity with the human alpha-chain was 61%. Like its human counterpart, the bovine alpha-chain is composed of eight contiguous short consensus repeat units, each of approximately 60 amino acid residues, and a carboxyl-terminal nonrepeat region. One bovine beta-chain clone was found and characterized. It predicted a mature bovine beta-chain of 181 amino acid residues. The identity with the human beta-chain was 65% at the amino acid level. A noteworthy difference between bovine and human beta-chains was that the bovine beta-chain only contained two short consensus repeats compared with three in human beta-chain. Sequence alignment indicates that the region corresponding to residues 1-60 (repeat 1) in the human beta-chain is absent in the homologous bovine polypeptide. Because the short consensus repeats of the human beta-chain contain the binding site for protein S, the lack of one repeat unit in the bovine beta-chain may provide a clue to the lack of complex formation between C4BP and protein S in bovine plasma.
L-selectin and very late antigen-4 integrin promote eosinophil rolling at physiological shear rates in vivo.
Authors: Sriramarao P , von Andrian U H , Butcher E C , Bourdon M A , Broide D H
Published: December 2022

Adherence of eosinophils to vascular endothelium and their accumulation at sites of allergen challenge are hallmarks of allergic inflammation. However, the molecular mechanisms mediating eosinophil adhesion under conditions of blood flow are not well understood. The present studies were performed to identify the receptors on human eosinophils involved in initiating adhesion to activated endothelium at physiologic shear rates in vivo. We have compared the relative contribution of L-selectin, VLA-4 (CD49d), and CD18 integrins in mediating eosinophil adhesion to microvascular endothelial cells in the rabbit mesentery by using intravital video microscopy. Eosinophils were found to roll in venules, but not arterioles, and this rolling could be stimulated by activation of endothelium with IL-1. In contrast to neutrophil rolling, which is predominantly L-selectin-dependent, eosinophil rolling was mediated by L-selectin, and also VLA-4. mAbs to L-selectin and VLA-4 alpha, but not CD18, significantly inhibited eosinophil rolling in vivo. The inhibition of VLA-4-mediated eosinophil rolling was not caused by modulation of eosinophil L-selectin or CD18 expression. This inhibition also was not caused by nonspecific inhibitory effect of the Abs studied, because the anti-VLA-4 mAbs inhibited eosinophil (VLA-4+) but not neutrophil (VLA-4-) rolling in the mesenteric venules. These results demonstrate that early events of eosinophil adhesion, i.e., rolling, are mediated by multiple adhesion receptors, including L-selectin and VLA-4, at physiologic shear rates in vivo.
Apoptotic cell death induced by intracellular proteolysis.
Authors: Williams M S , Henkart P A
Published: December 2022

To mimic the injection of granzymes into target cells by cytotoxic lymphocytes or the activation of endogenous proteases in programmed cell death, the proteases chymotrypsin, proteinase K, or trypsin were loaded into the cytoplasm of several different cell types using the osmotic lysis of pinosomes technique. Internalization of these proteases caused cell lysis within several hours, accompanied by extensive nuclear damage in most but not all combinations of target cells and proteases. This nuclear damage, quantitated by DNA release from nuclei, was associated with apoptotic features including DNA fragmentation into nucleosomal ladders, chromatin condensation, nuclear fragmentation, and membrane blebbing. Agents reported to block programmed cell death, including aurintricarboxylic acid, inhibitors of energy metabolism, and protein or RNA synthesis, failed to block this protease-induced death, although some inhibited nuclear damage. In separate experiments, introduction of staphylococcal nuclease into cells led to near complete (at least 75% of total) nucleosomal DNA fragmentation within 6 to 8 h. Condensation of chromatin did not accompany this fragmentation to the same extent, and there was approximately a 10-h lag between half-maximal DNA fragmentation and 50% loss of membrane integrity. The results suggest that activation of intracellular proteases during cell death by any molecular pathway could give rise to apoptotic morphology and DNA fragmentation.
HIV-1 suppression of hematopoiesis in vitro mediated by envelope glycoprotein and TNF-alpha.
Authors: Maciejewski J P , Weichold F F , Young N S
Published: December 2022

Dysmorphic marrow morphology and bone marrow failure are common in AIDS patients, but the mechanism of HIV-1 effects on blood cell production is unclear. Experiments to test the susceptibility of hematopoietic progenitor cells to HIV-1 infection have led to conflicting results. We found that hematopoietic colony formation by burst-forming units-erythroid and CFU-GM was equivalently inhibited by both active and heat-inactivated, noninfectious virus. Inhibition was dependent on the presence of macrophages and was not observed in cultures derived from highly enriched CD34+ cells. We hypothesized that TNF-alpha, produced by mononuclear phagocytes after contact with HIV-1 or gp120 and itself a potent suppressor of hematopoiesis, might mediate this effect. The addition of anti-TNF-alpha neutralizing Abs to marrow cultures abrogated inhibition by gp120 or virus. In contrast, neutralizing Abs to Il-4, IFN-alpha, and TGF-beta failed to improve colony formation. TNF-alpha was released from blood monocytes and marrow mononuclear cells stimulated by gp120. TNF-alpha is increased in the blood of patients with late stage AIDS and may mediate many of the symptoms of the disease. Our data do not support a requirement of direct infection of hematopoietic progenitor cells by HIV-1 for the inhibition of hematopoiesis in vitro. We propose instead an indirect mechanism of viral suppression of hematopoiesis as a result of TNF-alpha induction by virus or viral envelope glycoprotein. The importance of local TNF-alpha production in patients' marrow is amenable to clinical testing.
Characterization and expression of alternatively spliced IgE heavy chain transcripts produced by peripheral blood lymphocytes.
Authors: Batista F D , Efremov D G , Burrone O R
Published: December 2022

We have investigated the IgE heavy chain isoforms produced in vivo by analyzing the epsilon mRNA species present in unstimulated PBL and expressing them individually in a myeloma cell line. Seven epsilon mRNA species were identified by using reverse transcription-PCR, cloning, and sequencing analysis. These species included the classical secreted (epsilon CH4-S) and membrane-bound (epsilon CH4-M1'-M2) IgE and five alternatively spliced epsilon transcripts. At the protein level, the five alternatively spliced epsilon transcripts (epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, epsilon CH4'-1-M2, and epsilon CH3-13-CH4) corresponded to four epsilon heavy chain isoforms, in which various parts of the CH4 domain were replaced by new stretches of amino acids at the carboxyl termini. The same epsilon mRNA species also were present in the IgE producing myeloma cell line U266. However, except for the classical membrane and secreted IgE, the corresponding proteins could not be identified. To further characterize the epsilon CH4-S, epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, and epsilon CH4-M1'-M2 species, we expressed them as chimeric mouse/human anti-4-hydroxy-5-iodo-3-nitrophenacetyl Abs in a mouse myeloma cell line. Only the classical secreted and membrane isoforms were found to be secreted or expressed on the cell surface, respectively, and the other forms were retained within the cells and degraded. These data suggest that some of the epsilon mRNA isoforms produced by PBL are aberrantly spliced mRNAs, the protein products of which are eliminated by post-translational events.