bluuebunny/crossref_metadata_2025 huggingface dataset:
Substance P selectively activates TNF-alpha gene expression in murine mast cells.
Authors: Ansel J C , Brown J R , Payan D G , Brown M A
Published: December 2022

There is increasing evidence that the neurologic system is capable of modulating a wide range of immunologic responses, including certain inflammatory processes in the lung, gastrointestinal tract, and skin. It has been proposed that secreted neuropeptides such as substance P (SP) may mediate these neuroinflammatory interactions by binding to and stimulating immune cells such as mast cells and lymphoid cells. SP is secreted in a variety of tissues by an extensive network of neurosensory C and A5 fibers in response to a wide range of noxious stimuli and injury. Previous studies to examine the effect of SP on mast cells have focused on its role in triggering histamine release and mediating immediate hypersensitivity responses. Recently it was demonstrated that mast cells are also capable of secreting multiple cytokines including TNF-alpha, IL-1, IL-3, IL-4, IL-6, and GM-CSF. In this study we tested the possibility that SP may also influence mast cell-mediated late inflammatory events by modulating the production of one or several of these cytokines. Our results indicate that SP induces TNF-alpha mRNA expression and TNF-alpha secretion in a dose-dependent manner in a murine mast cell line, CFTL12. Likewise, SP stimulates TNF-alpha secretion in freshly isolated murine peritoneal mast cells. The induction of mast cell TNF-alpha is selective, since SP does not stimulate the production of IL-1, IL-3, IL-4, IL-6, or GM-CSF in these cells. The CFTL 12 mast cell line constitutively expresses high levels of SP receptor mRNA which is not modulated by PMA/cycloheximide treatment or SP. These results further support the concept that the neurologic system modulates inflammatory events by neuropeptide-mediated mast cell cytokine release.
Transforming growth factor-beta inhibits E-selectin expression on human endothelial cells.
Authors: Gamble J R , Khew-Goodall Y , Vadas M A
Published: December 2022

Transforming growth factor-beta (TGF-beta), a pleiotropic cytokine that is elaborated in the active form upon co-culture of endothelial cells and pericytes or smooth muscle cells, has been shown to decrease the adhesiveness of endothelial cells for neutrophils, lymphocytes, and tumor cells. The mechanism whereby TGF-beta inhibits the adhesiveness of human endothelial cells was investigated. TGF-beta inhibited the basal E-selectin (formerly ELAM-1) expression by 55 +/- 7% and TNF-stimulated expression by 57 +/- 4%. Similar decreases of IL-1-stimulated expression were also seen. Peak inhibition was seen at TGF-beta doses between 0.2 and 2 ng/ml. Both TGF-beta 1 and -beta 2 were functional. The effectiveness of TGF-beta in inhibiting E-selectin expression was dependent on cell density and incubation time. TGF-beta also inhibited E-selectin mRNA levels in endothelial cells. TGF-beta had no effect on the expression of VCAM-1 and ICAM-1, but was additive with IL-4 in inhibiting the expression of E-selectin. The expression of E-selectin has been shown to mediate several aspects of the inflammatory response involving neutrophils and memory T lymphocytes. Perivascular TGF-beta appears to act as an inhibitor of the expression of the endothelium-specific selectin, E-selectin, and therefore of inflammatory responses involving neutrophils and (a subset of) lymphocytes.
Increased Fc alpha R expression and IgA-mediated function on neutrophils induced by chemoattractants.
Authors: Hostoffer R W , Krukovets I , Berger M
Published: December 2022

The recent reports of receptors for IgA on blood and mucosal phagocytes suggest a more active role then previously thought for IgA in host defense. Using a mAb and flow cytometry we determined the expression of the Fc alpha R on resting and chemoattractant-stimulated blood neutrophils and compared them with mucosal neutrophils. Fc alpha R expression on blood neutrophils could be rapidly up-regulated in vitro, increasing three to fourfold within minutes of exposure to the chemoattractants FMLP (optimal at 10(-8) M) and zymosan activated serum (a source of C5a). The level of Fc alpha R expression found on neutrophils obtained from bronchoalveolar lavage of cystic fibrosis patients with chronic lung infection was almost identical to that found on blood neutrophils from the same patients maximally activated in vitro. The rise in Fc alpha R expression induced by 10(-8) M FMLP was rapid, with a plateau in 15 to 20 min, and was not inhibited by 10 mg/ml of cycloheximide or puromycin, suggesting that the mechanism of up-regulation involves translocation from intracellular storage pools. Ionomycin (2 mM) plus 1.2 mM CaCl2 also increased expression of Fc alpha R, and its effects were inhibited by EDTA. Trifluoperazine, an inhibitor of calmodulin and/or protein kinase C-dependent processes, blocked the increase in Fc alpha R expression induced by FMLP, but the effects of the chemoattractant were not blocked by EDTA, suggesting that intracellular stores of calcium are important in the physiologic regulation of Fc alpha R expression. Neutrophil superoxide production could be induced by aggregated IgA, and was increased if the neutrophils were pretreated with FMLP, correlating with the increase in Fc alpha R expression. The superoxide response to a suboptimal dose of aggregated IgG was unaffected by FMLP pretreatment, and antibodies to Fc gamma R failed to block the superoxide production induced by IgA. Thus, the increase in phagocyte surface expression of Fc alpha R induced by chemoattractants in vitro and on mucosal cells in vivo, and the concomitant increase in IgA-mediated function may greatly facilitate the defense of mucosal surfaces.
HLA A2 restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection.
Authors: Nayersina R , Fowler P , Guilhot S , Missale G , Cerny A , Schlicht H J , Vitiello A , Chesnut R , Person J L , Redeker A G , Chisari F V
Published: December 2022

Inasmuch as the hepatitis B virus (HBV) is not directly cytopathic for the infected hepatocyte, it is generally presumed that viral clearance and liver cell injury during viral hepatitis are due to a CTL response to HBV encoded Ag presented by HLA class I molecules. We have previously examined the peripheral blood CTL response to two HBV nucleocapsid epitopes in patients with acute and chronic viral hepatitis, one of which is restricted by HLA-A2, whereas the other is dually restricted by HLA-A31 and Aw68. In this study, we defined the HLA-A2-restricted CTL response to the hepatitis B surface Ag (HBsAg) by using a panel of HBsAg-derived synthetic peptides containing the ideal HLA-A2.1 binding motif (-L------V). Several novel aspects of HBV immunobiology and pathogenesis are evident from this study. First, the peripheral blood CTL response to HBV-encoded Ag is remarkably polyclonal and multispecific in most patients with acute hepatitis. Indeed, HLA-A2-restricted CTL specific for as many as four envelope epitopes and one nucleocapsid epitope were found to be present simultaneously in individual patients with acute viral hepatitis. Second, HBV-specific CTL are not detectable in the peripheral blood in a minority of patients with acute hepatitis, nor have we detected a CTL response in any of the patients with chronic hepatitis that we have studied thus far. Although the cellular and molecular basis for CTL nonresponse remains to be determined, the data suggest that it may contribute to viral persistence. Third, the diversity and the specificity of the CTL response is determined in part by the coding sequence of the viral genome present in each infected patient. Indeed, the apparent nonresponse of some acutely infected patients to at least one HBsAg-specific CTL epitope actually reflects infection by a viral variant that contains a critical substitution in one of the anchor residues within the epitope. Finally, at a fundamental level, the data suggest that the presence of the HLA-A2.1-binding motif in a peptide may not be sufficient for binding; and the capacity of a peptide to bind the class I molecule does not guarantee that it will be immunogenic.
The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes.
Authors: Engel P , Nojima Y , Rothstein D , Zhou L J , Wilson G L , Kehrl J H , Tedder T F
Published: December 2022

CD22 is a B lineage-restricted member of the Ig superfamily that serves as an adhesion receptor expressed by mature B lymphocytes. In this study, the ability of different cell types to attach to COS cells transiently transfected with a full-length CD22 cDNA (COS-CD22) was examined to determine the cellular distribution of the ligand for CD22. T and B lymphocytes, monocytes, erythrocytes, and neutrophils formed specific rosettes with COS-CD22 cells at 4 degrees C. A panel of 33 new mAb directed against CD22 were developed to examine the regions of CD22 that mediate adhesion. Four of these mAb, HB22-7, -22, -23, and -33 (at 1 to 5 micrograms/ml) specifically blocked adhesion (75 to 95%) of all cell types to COS-CD22 cells. Each of these mAb cross-blocked each other's binding, suggesting that ligand binding occurs through a single region of CD22. These mAb also identify a region of CD22 distinct from those defined by previously described CD22 mAb. CD22-mediated adhesion of cell lines to COS-CD22 cells was independent of CD45RO and CDw75 expression, and it was not inhibited by mAb against known integrins. Although alpha-2,6-linked sialic acid expressed on the surface of COS cells did not serve as a ligand for CD22, the CD22 ligand may contain a critical sialic acid determinant, as neuraminidase treatment of all target cells eliminated CD22-mediated adhesion. CD22-mediated adhesion was Ca2+/Mg2+ independent, again suggesting that integrins were not involved. An inhibitory substance for CD22-mediated adhesion was found to be present in FCS and some ascites fluid. Analysis of CD22 mRNA and protein revealed that although multiple mRNA splice variants of CD22 mRNA can be detected, only a single protein isoform was detected on the cell surface. Therefore, although the identity of the CD22 ligands remains incompletely characterized, it is possible that a single major ligand is expressed by RBC and leukocytes, which binds to a single region of CD22.
Direct effects of IL-10 on subsets of human CD4+ T cell clones and resting T cells. Specific inhibition of IL-2 production and proliferation.
Authors: de Waal Malefyt R , Yssel H , de Vries J E
Published: December 2022

The direct effects of IL-10 on the proliferation and lymphokine production of human peripheral blood T cells and CD4+ T cell clones representing the Th0, Th1-like, and Th2-like Th cell subsets were investigated in the absence of professional APC. IL-10 partially inhibited the proliferative responses of CD4+ human T cell clones induced by anti-CD2 or anti-CD3 mAb cross-linked on CD32 (Fc gamma RII)-transfected mouse L cells. Transfection of ICAM-1 or LFA-3 in CD32+ L cells resulted in enhanced proliferative responses of CD4+ T cell clones after activation by anti-CD3 mAb, whereas transfection of B7 in CD32+ L cells enhanced proliferative responses of CD4+ T cell clones after activation by anti-CD2 mAb. In addition, B7 expression on CD32+ L cells was required for activation of small resting T cells by anti-CD3 or anti-CD2 mAb. IL-10 inhibited the proliferation of T cell clones induced by anti-CD2 or anti-CD3 mAb on CD32+ L cells expressing these accessory molecules, indicating that interactions of LFA-3, ICAM-1, and B7 with their ligands on T cells did not overcome the inhibitory effects of IL-10. Inhibition of proliferation of T cell clones by IL-10 was in all instances completely neutralized by relatively low concentrations of IL-2, whereas IL-4 was ineffective. IL-10 did not affect the expression of the TCR/CD3 complex, CD2, LFA-1, CD28, or IL-2R alpha- or beta-chains, nor did it inhibit the induction of the latter two molecules on T cells after activation. Inhibition of proliferation was found to be the result of specific inhibition of IL-2 production by the responding T cell subsets, which occurred at the mRNA level. The production and mRNA levels of IL-4, IL-5, IFN-gamma, and granulocyte/macrophage-CSF were not affected by IL-10. Taken together, these results indicate that IL-10/IL-10R interaction on CD4+ T cell clones and peripheral blood T cells results in signaling pathways that specifically interfere with activation processes leading to IL-2 production. These direct inhibitory effects on IL-2 production by activated T cells may contribute to the general immunosuppressive activities of IL-10.
Activation of CD8-dependent cytotoxic T lymphocyte adhesion and degranulation by peptide class I antigen complexes.
Authors: Kane K P , Mescher M F
Published: December 2022

Activation of CTL requires engagement of both the TCR and the CD8 coreceptor. Immobilized class I proteins and in vitro-formed peptide class I Ag complexes have been used to examine the relative contributions of TCR and CD8 to the adhesion and response of cloned, class I-restricted CTL. The extent of degranulation was found to be directly proportional to the concentration of peptide used to pulse class I, suggesting that activation is a direct function of TCR occupancy level. In contrast, activation of degranulation as a function of the amount of class I on the surface displayed a marked threshold density dependence. Essentially the same density dependence was found for the response of CTL to fluid phase anti-TCR mAb and non-Ag class I, indicating that CD8-class I interaction must exceed a threshold before effective cosignaling can occur. Adhesion and degranulation of CTL was minimal in response to in vitro peptide-class I complexes prepared at a class I density below the threshold. However, the same density of peptide class I initiated both adhesion and response if additional non-Ag class I was coimmobilized on the same surface at levels above threshold. Thus, when surface levels of peptide class I complex are low, as is likely to be the case under physiologic conditions, the level of TCR occupancy achieved is, by itself, insufficient to mediate cell adhesion or activate degranulation. The results demonstrate, however, that low TCR occupancy is sufficient to provide the signal to prime CD8. Provided that the surface density of class I is sufficiently high, CD8 then mediates strong adhesion and provides the costimulatory signal(s) to activate response.
Detection of a glycosylation-dependent ligand for the T lymphocyte cell adhesion molecule CD2 using a novel multimeric recombinant CD2-binding assay.
Authors: Parish C R , Recny M A , Knoppers M H , Waldron J C , Warren H S
Published: December 2022

The CD2 molecule plays an important role in T cell adhesion by interacting with the ligands CD58 (LFA-3) and CD59. In order to detect additional ligands for CD2, potentially of low binding affinity, we have prepared a highly fluorescent, multimeric form of rCD2 whose binding to cells can be quantified by flow cytometry. Initial studies demonstrated that binding of multimeric rCD2 to cells was CD2-specific, concentration and time dependent, and saturable. The negative charge on cells was also found to play a critical role in the efficiency of multimeric rCD2 binding. Analysis of binding of multimeric rCD2 to 17 CD58+ cell types revealed that only 8 of the cells exhibited binding. Failure of multimeric rCD2 to interact with the other cells could not be explained by differences in CD58 expression, suggesting that, in terms of CD2 binding, there are qualitative differences in CD58 on different cell types. Binding of multimeric rCD2 to six of the seven reactive cells was virtually totally inhibited by CD58 mAb pretreatment, whereas binding to the erythroleukemic line K562 was only partially blocked, suggesting the existence of another CD2 ligand. Subsequent studies demonstrated that the putative new ligand is not CD59, and that it interacts with a different region of the CD2 molecule than CD58, probably a site located between the T11(1) and T11(2) epitopes. The binding affinity of CD2 for the new ligand is 10-fold lower than for CD58 and, based on studies with truncated rCD2, the binding site for the new ligand is located within the amino-terminal 105 amino acids of the CD2 polypeptide. Unlike CD58, the new ligand is tunicamycin sensitive suggesting that it contains a N-linked carbohydrate structure that is essential for functional activity.
Regulation of the tyrosine kinase-dependent adhesion pathway in human lymphocytes through CD45.
Authors: Wagner N , Engel P , Tedder T F
Published: December 2022

Cell-cell adhesive interactions involve numerous receptor/ligand interactions that play a crucial role in the development of immune function. Engagement of multiple cell-surface molecules on B lymphocytes generates intracellular signals through a tyrosine kinase-dependent pathway that activates cell-surface adhesion receptors and thereby induces homotypic cell-cell adhesion. Homotypic adhesion is mediated in part through LFA-1/ICAM-1 and other heretofore unknown adhesion receptors. In this study, evidence of a regulatory role for CD45 in the induction of homotypic adhesion is suggested. A new mAb (HAB-1) was developed that inhibits homotypic adhesion in B cell lines induced through MHC class I and class II, CD19, CD20, CD21, CD40, and Leu-13 cell-surface molecules. Although binding of this mAb strongly inhibited cell-surface Ag-induced homotypic adhesion at mAb concentrations as low as 0.1 microgram/ml, it exhibited no effect on homotypic adhesion induced by phorbol esters. Binding of the HAB-1 mAb to lymphocytes altered the pattern of cellular protein tyrosine phosphorylation, but did not have a global inhibitory effect on cell activation because it did not have major effects on the growth of mitogen-activated lymphocytes. Immunoprecipitation studies revealed that the HAB-1 mAb identified an epitope present on all isoforms of CD45. The HAB-1 mAb may identify a unique epitope of CD45 because this mAb had a unique staining pattern when assessed by indirect immunofluorescence staining. The HAB-1 mAb was similar to some other CD45 mAb that have the capacity to amplify CD2-induced proliferation of blood lymphocytes. However, only 1 of 12 other anti-CD45 mAb tested had a similar inhibitory effect on adhesion. Homotypic adhesion of lymphocytes may therefore be governed by a regulatory system of cell-surface molecules that generate positive and negative signals that either trigger adhesion or, like CD45, directly down-regulate adhesion. This highlights the significance of adhesive events that result from surface molecules being engaged by their natural ligands during lymphocyte activation.
Developmental and cytokine-mediated regulation of MHC class II gene promoter occupancy in vivo.
Authors: Kara C J , Glimcher L H
Published: December 2022

The class II genes of the major histocompatibility complex are a family of genes whose expression is regulated developmentally in cells of the B lineage and by IFN-gamma in many other cell types. Using the approach of in vivo footprinting, which allows for the examination of protein-promoter interactions within intact cells, we demonstrated a transition from unoccupied to occupied to once again unoccupied class II promoters in cell lines representing the developmental pathway of B cells. IFN-gamma treatment of HeLa cells led to increased promoter occupancy of the DR alpha and DR beta promoters at the same sites that are constitutively bound in mature B cells. No IFN-gamma-specific binding site was induced. Additionally, an octamer element in the DR alpha gene displayed preferential binding in B cells. These results demonstrate that changes in the transcription of the class II genes are associated with changes in factor binding at the promoter in vivo. Moreover, given the ubiquity of class II promoter binding proteins, these results suggest that throughout B cell development and upon IFN-gamma stimulation, the accessibility of class II promoter DNA is subject to regulation.
Genetic basis of human complement C8 beta deficiency.
Authors: Kaufmann T , Hänsch G , Rittner C , Späth P , Tedesco F , Schneider P M
Published: December 2022

The eighth component of human complement (C8) is a serum protein consisting of three chains (alpha, beta, and gamma) and encoded by three different genes, C8A, C8B, and C8G. C8A and C8B are closely linked on chromosome 1p, whereas C8G is located on chromosome 9q. In the serum the beta subunit is non-covalently bound to the disulfide-linked alpha-gamma subunit. Patients with C8 beta deficiency suffer from recurrent neisserial infections such as meningitis. Exon-specific polymerase chain reaction (PCR) amplification with primer pairs from the flanking intron sequences was used to amplify all 12 C8B exons separately. No difference regarding the exon sizes was observed in a C8 beta-deficient patient compared with a normal person. Therefore, direct sequence analysis of all exon-specific PCR products from normal and C8 beta-deficient individuals was carried out. As a cause for C8 beta deficiency, we found a single C-T exchange in exon 9 leading to a stop codon. An allele-specific PCR system was designed to detect the normal and the deficiency allele simultaneously. Using this approach as well as PCR typing of the Taql polymorphism located in intron 11, five families with 7 C8 beta-deficient members were investigated. The mutation was not found to be restricted to one of the two Taql RFLP alleles. The mutant allele was observed in all families investigated and can therefore be regarded as a major cause of C8 beta deficiency in the Caucasian population. In addition, two C8 beta-deficient patients were found to be heterozygous for the C-T exchange. The molecular basis of the alleles without this point mutation also causing deficiency has not yet been defined.
Structural similarity of antibody variable regions from immune and autoimmune anti-DNA antibodies.
Authors: Krishnan M R , Marion T N
Published: December 2022

Anti-DNA antibodies have been successfully induced in normal, nonautoimmune mice by immunization with complexes formed with a DNA-binding peptide, Fus1, and native, B form, mammalian DNA. Fus1 is a 27-amino acid peptide from the internal domain of a ubiquitin fusion protein from Trypanosoma cruzi. The structure of this peptide is homologous to the consensus amino acid sequence for a DNA-binding, "zinc finger" motif, and the peptide binds to DNA. A panel of six anti-DNA antibody-producing hybridomas, two IgM and four IgG2a, have been generated from a single BALB/c mouse immunized with Fus1-DNA. The V region structures for both H and L chains of the induced anti-DNA antibodies are highly homologous if not identical to the V region structures of spontaneous anti-DNA antibodies from autoimmune (NZB x NZW)F1 mice. The DNA specificities of the anti-DNA antibodies were also similar to those of autoimmune anti-DNA antibodies. Three of the four induced IgG antibodies bound equally well to native and denatured DNA. These results demonstrate that antibody specific for nDNA can be induced with immunogenic complexes of native DNA. They also demonstrate that monoclonal representatives of the induced anti-DNA antibodies have serologic and structural characteristics similar if not identical to those of spontaneous anti-DNA antibodies from autoimmune mice. The experimental system described here should provide insight not only about the structural basis for autoimmunity to DNA but also the function of anti-DNA antibody in the immunopathology of SLE.
Monoclonal, anti-domain and anti-peptide antibodies assign the molecular weight 160,000 granulocyte membrane antigen of the CD66 cluster to a mRNA species encoded by the biliary glycoprotein gene, a member of the carcinoembryonic antigen gene family.
Authors: Stoffel A , Neumaier M , Gaida F J , Fenger U , Drzeniek Z , Haubeck H D , Wagener C
Published: December 2022

mAb directed against the CD66 cluster of granulocyte differentiation Ag recognize Ag of the carcinoembryonic Ag family. A major Ag in extracts from granulocyte membranes bound by CD66 antibodies exhibits a relative molecular mass of 160,000. According to recent data, this Ag may be a product of the biliary glycoprotein (BGP) gene that belongs to the CEA gene family. As a result of alternative splicing, the BGP gene is transcribed into at least seven distinct mRNA species. To identify splice variants of BGP, antisera were raised against the A2 domain expressed in bacteria and to a peptide comprising the C-terminal 23 amino acids encoded by the 3' exon of the BGP gene. The antisera and an mAb specific for members of the BGP family were used to identify potential BGP splice variants in granulocyte membranes. For comparison, the binding of antibodies to Ag purified from human bile was investigated. In the membrane preparation from granulocytes, the only Ag identified by the mAb, the domain antiserum and the peptide antiserum, was the Ag of M(r) 160,000 recognized by a CD66 antibody. These results indicate that the M(r) 160,000 granulocyte membrane Ag of the CD66 cluster is the product of the BGP-specific mRNA containing all coding sequences of the BGP gene. Among two major biliary glycoproteins present in human bile, the M(r) 115,000 Ag contains the A2 domain, whereas the domain is lacking in the "classical" biliary glycoprotein of M(r) 85,000. None of the bile Ag bound the peptide antiserum.
Use of the most JH-proximal human Ig H chain V region gene, VH6, in the expressed immune repertoire.
Authors: Varade W S , Marin E , Kittelberger A M , Insel R A
Published: December 2022

VH6, the most 3' human H chain V region gene, is preferentially expressed in the preimmune repertoire of the developing human fetus. To determine whether VH6 contributes to the immune repertoire, we amplified and sequenced from human spleen RNA VH6 rearrangements that were isotype-switched to IgG. Eighteen distinct in-frame VH6 clones were sequenced. Definitive assignment to C gamma 1 could be made in the majority of clones, and all had undergone extensive somatic hypermutation in the V region with an average of 14 (range, 2 to 25) mutations/281 bp in the VH6 segment. The overall frequency of somatic mutation was 5.2% of the total nucleotides sequenced. Replacement somatic mutations were targeted to the complementarity determining region, and the complementarity determining region had higher replacement to silent mutation ratios, consistent with antigenic selection. Silent somatic mutations were also significantly more frequent in the complementarity determining region compared to the framework region. Five clones were derived from the same VH6Dxp'1JH6 rearrangement, each containing unique and shared mutations. All DH segments used by independently arising clones were unique, and all DH reading frames were seen. N segments were lacking at either the 5'DH-JH junction or the 3'DH-JH junction in 7 of 13 independent rearrangements. DH-DH recombinations were frequently observed, and there was biased usage of JH3b and JH4b in the clones. The data imply that a high degree of diversity arises from use of this 3'-VH gene in the immune repertoire.
Differences in cytokine response and induction of nitric oxide synthase in endotoxin-resistant and endotoxin-sensitive mice after intravenous gram-negative infection.
Authors: Evans T J , Strivens E , Carpenter A , Cohen J
Published: December 2022

Previous reports have suggested that the endotoxin-resistant C3H/HeJ strain of mouse is more susceptible to infection than is the endotoxin-sensitive parent strain, C3H/HeN, although they have never been compared in an i.v. model of sepsis. We therefore have used these mouse strains in an i.v. model of Gram-negative sepsis to compare their sensitivities to infection, their cytokine responses, and the levels of induction of the enzyme nitric oxide synthase assayed in their livers. By using i.v. infection with Escherichia coli we have found that both strains are approximately equally sensitive to this organism, despite the C3H/HeJ mice having a markedly attenuated TNF-alpha response. IFN-gamma levels after infection were identical in the two strains; the levels of nitric oxide synthase induced in their livers were about fourfold greater in the C3H/HeJ mice. This difference could not be explained by differences in bacterial load. These experiments suggest that factors other than TNF-alpha are important in determining outcome from Gram-negative sepsis and that TNF-alpha is not a major factor in the induction of hepatic nitric oxide synthase after infection in vivo.
Virus-specific cytotoxic T cell-mediated lysis of lymphocytes in vitro and in vivo.
Authors: Kyburz D , Speiser D E , Aebischer T , Hengartner H , Zinkernagel R M
Published: December 2022

Virus-specific CTL play a major role in early antiviral protection against lymphocytic choriomeningitis virus (LCMV). When mice are infected with high doses of certain LCMV isolates, the initiated CTL response may vanish before the virus is eliminated completely. To evaluate the possibility that this may be because of CTL lysing CTL, we studied the susceptibility to lysis of LCMV-specific CTL clones and of primary immune spleen cells in vitro and in vivo. Confirming earlier reports, CTL were lysed in vitro when incubated with their specific Ag peptide; lysis was MHC restricted because T cell clones derived from allogeneic bone marrow chimeras that do not express the correct restriction element were not susceptible to mutual CTL lysis. The density of CTL cultures correlated with the degree of inactivation of CTL, indicating that CTL lysed each other mutually rather than committed suicide. To assess CTL-mediated lysis of CTL in vivo, mice were infected s.c. into the footpad with LCMV, and shortly before the CTL-dependent footpad swelling developed by day 6 to 7, specific peptide was injected locally. The regional lymph node (LN) was reduced in size, contained about 10 times fewer cells, and only 2% to 3% of the lytic activity when compared with several control LN. This drastic local effect was observed within a few hours and was accompanied by DNA fragmentation. Thus, in this model system, peptide loading of lymph node cells may lead to CTL-mediated cytotoxicity and death of T cells (including CD8+ effector cells and other LN cells) in vivo. These results may suggest that, during overwhelming virus infections, lysis of CTL passively loaded with relevant released peptides possibly may contribute to the impairment of immune responses.
Induction of diabetes is influenced by the infectious virus and local expression of MHC class I and tumor necrosis factor-alpha.
Authors: Ohashi P S , Oehen S , Aichele P , Pircher H , Odermatt B , Herrera P , Higuchi Y , Buerki K , Hengartner H , Zinkernagel R M
Published: December 2022

To study self reactivity, a transgenic mouse model has been established in which the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) is expressed in the beta-islet cells of the pancreas (rat insulin promoter (RIP)-gp). These mice (H-2b) do not spontaneously develop diabetes; however, infection with the LCMV strain WE rapidly induces hyperglycemia. In this study, comparative analysis of H-2k RIP-gp-transgenic animals demonstrated that the haplotype influences the incidence and kinetics of diabetes and alters the requirement for the CD4+ T cell subset. This study also showed that the properties of the virus expressing the self target Ag determined whether hyperglycemia occurred in RIP-gp-transgenic mice. Various LCMV strains were able to induce diabetes in RIP-gp-transgenic animals, whereas infection with a recombinant vaccinia virus expressing LCMV-gp (vacc-gp) did not induce diabetes. However, vacc-gp could induce diabetes in double (RIP-gp/TCR)-transgenic mice, where the majority of CD8+ T cells expressed a receptor specific for LCMV-gp, suggesting that a critical number of self-reactive T cells must be activated to induce disease. Notably, histologic analysis of pancreata taken various days after LCMV or vacc-gp infections indicated that induction of diabetes coincided with an increase in MHC class I expression on the islets of Langerhans. Additional studies with vacc-gp were done to determine other factors that possibly enhance autoimmune attack. Transgenic mice expressing both LCMV-gp and TNF-alpha under the control of the RIP were infected with vacc-gp, and 50% of RIP-gp/TNF-alpha-transgenic animals became hyperglycemic. These data suggest that the increased local lymphocyte traffic as a result of TNF-alpha expression attracts activated gp-specific T cells, enhancing the possibility of hyperglycemia. Collectively, these results demonstrate that the induction of diabetes in this model is influenced by the MHC haplotype, the infectious agent, TNF-alpha expression, the level of MHC class I expression, and the induction of a threshold number of self-reactive CTL.
Role of CD25+ and CD25-T cells in acute HIV infection in vitro.
Authors: Ramilo O , Bell K D , Uhr J W , Vitetta E S
Published: December 2022

T cell activation plays a major role in the ability of the HIV to remain latent or establish a productive infection. The alpha-chain of the IL-2R (CD25, Tac, p55) is expressed on activated but not resting T cells and therefore represents an ideal marker to distinguish activated from resting T cells. The present studies were designed to define the role of CD25+ (activated) and CD25- (resting) T cells in an acute HIV infection in vitro. This objective was accomplished by selectively killing CD25+ cells with an anti-CD25-ricin A chain immunotoxin (RFT5-deglycosylated ricin A (dgA)) either before or after HIV infection, and then determining the effect of eliminating these cells on the secretion of viral p24 Ag. Three major findings have emerged from this study: 1) Elimination of the small population (3 to 5%) of activated, CD25+ cells present in normal PBMC before HIV infection results in a 99% reduction in p24 secretion. 2) RFT5-dgA, an immunotoxin directed against CD25, kills HIV-infected CD25+ cells. Elimination of the CD25+ cells after infection with HIV virtually stops viral production and the spread of the infection in these cultures. This was confirmed by coculturing RFT5-dgA-treated PBMC with H9 cells. 3) When RFT5-dgA-treated PBMC (CD25-cells) were infected with HIV and then activated with a solid phase anti-CD3 mAb, the levels of p24 produced were comparable with those of PBMC from which the CD25+ cells had not been eliminated. Taken together these findings suggest that both activated (CD25+) and resting/quiescent (CD25-) cells can be infected with HIV but that only the CD25+ cells produce viral proteins in the absence of additional activation.
Phenotypic and functional characterization of T cell lines specific for individual respiratory syncytial virus proteins.
Authors: Alwan W H , Record F M , Openshaw P J
Published: December 2022

Mice sensitized to individual respiratory syncytial (RS) virus proteins show distinct patterns of immunity and pulmonary pathology when challenged with live virus. To explore the immune mechanisms responsible for the differences in postvaccination disease, BALB/c (H-2d) mice were primed by scarification with recombinant vaccinia viruses (rVV) expressing the major glycoprotein (G), fusion protein (F), phosphoprotein (P), nucleoprotein (N), or second matrix (22K) protein of RS virus. Ag-stimulated spleen cell cultures gave rise to CD3+TCR-alpha beta + T cell lines. Those from rVV-F-primed mice contained a mixture of CD8+ and CD4+ T cells, whereas those specific to G, N, and P were mostly CD4+. In contrast, 22K-specific lines were mostly CD8+. F- and 22K-specific lines showed virus-specific CTL activity against H-2d targets, but the lines from rVV-G-, -N-, and -P-primed mice did not. The F-specific lines contained Th cells that released an excess of IL-2 and some IL-3 but little IL-4 or IL-5 (i.e., a "Th1" pattern). In contrast, the G-specific line released IL-3, IL-4, and IL-5 but little IL-2 (i.e., a "Th2" pattern). The 22K line contained IL-3-releasing T cells. Staining of T cell subsets for CD45RB varied in intensity in different lines, consistent with the cytokine secretion profiles of the Th cells that they contained. Because different RS virus proteins (given in the same form by the same route) prime for functionally different T cells, the ways in which individual proteins are processed and presented might be important in determining these patterns of immunity.
Role of thiols in the presentation of a snake toxin to murine T cells.
Authors: Maillère B , Cotton J , Mourier G , Léonetti M , Leroy S , Ménez A
Published: December 2022

We isolated and characterized two T hybridomas specific for a highly stable snake toxic protein. One hybridoma, called T1C9, is I-E(d)-restricted and stimulated by both the native and reduced and carboxymethylated (RCM) toxins and by synthetic fragments containing the region 24-36. The other hybridoma, called T1B2, is I-A(d)-restricted and stimulated by the native toxin, only. Neither the RCM toxin nor any of the initial synthetic peptides used in our study could stimulate it. We show that this lack of effect is associated with the presence, in the epitope-containing fragment, of irreversible blocking groups on cysteine residues. Indeed, when the fragment 32-49 has its cysteines involved in either intra-(32-49SS) or mixed disulfides, a stimulation of T1B2 was observed. Fixed APC do not present native toxin to either hybridomas but present RCM toxin to T1C9. Strikingly, fixed APC present the peptide 32-49SS to T1B2; however, we show that this is possible only because the peptide disulfide is reduced. The thiol dependence of this epitope suggests that the native toxin can stimulate T1B2 only after disulfide reduction. This reaction may constitute a major step during the processing of the toxin and more generally of any disulfide-containing Ag.
MHC class II molecules deliver costimulatory signals in human T cells through a functional linkage with IL-2-receptors.
Authors: Odum N , Kanner S B , Ledbetter J A , Svejgaard A
Published: December 2022

MHC class II-positive T cells are found in tissues involved in autoimmune and infectious disorders. Because stimulation of class II molecules by mAb or bacterial superantigens induces protein tyrosine phosphorylation through activation of PTK3 in T cells, we hypothesized that class II signals play a regulatory function in T cell activation. Here, we show that cross-linking HLA-DR and -DP but not -DQ molecules by immobilized mAb enhanced proliferative T cell responses to IL-2. In contrast, class II stimulation had no effect on IL-4-induced proliferation. The costimulatory effect was most pronounced at low concentrations of IL-2, was blocked by IL-2R mAb, and was at least partly mediated through an up-regulation of IL-2 high affinity receptors. As expected, activation of IL-2R by IL-2 induced tyrosine phosphorylation of several proteins including p56lck, and class II cross-linking by mAb induced tyrosine phosphorylation of specific substrates including PLC-gamma 1. Combined stimulation of IL-2R and class II molecules had an additive effect on tyrosine phosphorylation. Pretreatment of T cells with a protein tyrosine kinase inhibitor, herbimycin A, inhibited IL-2 and class II-induced proliferation suggesting that class II costimulation of IL-2 responses may involve activation of tyrosine kinases. Taken together, the present data suggest that extensive cross-linking of class II molecules delivers costimulatory signals that enhance IL-2 sensitivity in human T cells.
Isotypic residues in the membrane proximal domain of MHC class II beta-chains control activation of CD4+ T cells.
Authors: Sant A J
Published: December 2022

To investigate the role that isotypic residues play during interactions between CD4+ T cells and MHC class II molecules, interisotypic MHC class II beta-chains have been generated in which the beta 1 domain is derived from I-A and the beta 2, transmembrane, and intracytoplasmic regions are derived from I-E. Interisotypic or wild-type A beta genes have been transfected into L cells with the genes encoding the wild-type A alpha-chain. Transfectants bearing the recombinant beta-chain thus express an MHC class II dimer in which the amino-terminal domains that control TCR and peptide-binding interactions are wild-type A alpha and A beta, whereas the membrane proximal domains are derived from A alpha and E beta. L cells expressing this recombinant class II molecule or wild-type A alpha A beta have been compared functionally for their ability to stimulate Ag-specific T cell hybridomas and normal T cell clones and to activate primary alloreactive and superantigen-specific T cells. Ag-specific T cell hybridomas vary in their ability to be activated by the recombinant class II molecule, and sensitivity to the isotypic form of the membrane proximal domain correlates with expression of the CD4 molecule. CD4+ T cells distinguish between the wild-type and recombinant dimers, whereas CD4- T cells react equivalently with both. The recombinant class II molecules are defective in activation of normal T cell clones and are totally deficient in activation of primary alloreactive and superantigen-reactive T cells, but stimulate CD4- alloreactive T cell hybridomas equivalently. Together, the results from these experiments suggest that the interisotypic dimers possess normal TCR and peptide interactions, but altered CD4-dependent accessory interactions necessary for activation of normal T cells. These findings indicate that isotypic residues in the membrane proximal domains of MHC class II control CD4-linked accessory function and that this accessory function is most critical for freshly isolated CD4 lymphocytes.
T cell-macrophage cognate interaction in the activation of macrophage effector function by Th2 cells.
Authors: Stout R D , Suttles J
Published: December 2022

The in vitro induction of cytostatic/cytotoxic activity in macrophages generated in spleen cell cultures requires a signal cascade initiated by costimulation with both LPS and IFN-gamma. Th2 lymphocytes, although they do not produce IFN-gamma, can provide the signals necessary for induction of cytostatic activity in IFN-gamma-primed macrophages. These signals appear to be delivered by cognate interaction between the Th2 cells and macrophages in that: 1) they are not delivered by culture supernatants of Th2 cells activated 6 or 20 h by Con A or by immobilized anti-CD3 mAb; 2) they are not delivered if cell contact between Th2 cells and macrophages is prevented; and 3) they can be delivered by paraformaldehyde-fixed activated Th2 cells. Paraformaldehyde-fixed resting Th2 cells cannot stimulate activation of INF-gamma-primed macrophages. The Th2 cells must be activated at least 3 h before fixation to acquire macrophage stimulatory activity. Optimal macrophage-stimulating activity is attained after 6-h activation of the Th2 and declines thereafter. Although the activation of IFN-gamma-primed macrophages by viable resting Th2 displays Ag specificity and MHC restriction, the activation of IFN-gamma-primed macrophages by paraformaldehyde-fixed activated Th2 is neither Ag specific nor MHC restricted. These observations suggest that T cell-mediated activation of macrophages can involve a signaling cascade of Ag-specific and Ag-nonspecific adhesion events comparable to those hypothesized to occur in T cell-mediated B cell activation.
Regulation of the expression of human C epsilon germline transcript. Identification of a novel IL-4 responsive element.
Authors: Ichiki T , Takahashi W , Watanabe T
Published: December 2022

Transcriptional regulation for Ig H chain germline transcripts induced by cytokines is a topic of recent interest for the understanding of the mechanism of class switch recombination. Among human B cell lines examined, we have found that a human IgM-producing B cell line, DND39 (EBV negative) expressed germ-line transcripts of epsilon constant gene (C epsilon) when stimulated with IL-4. In our study, the regulatory element responsible for the expression of IL-4-induced human C epsilon germ-line transcript was determined using DND39 cells. To identify the IL-4 responsive promoter/enhancer element, deletion analysis of the upstream region of the germ-line exon (I epsilon) of the C epsilon germ-line transcript which is located 5' to the switch region, was performed by using a luciferase gene as a reporter. Deletion analysis showed that a DNA fragment which lies between -215 and -154 bp upstream from the most 3' transcriptional initiation site of human I epsilon gene is fully responsible for the induction of germ-line transcripts by IL-4. According to a mutational analysis, the DNA fragment between -163 and -152 bp, was identified to be a novel IL-4 responsive element in a human C epsilon gene. Electrophoretic gel mobility shift assay showed the presence of IL-4-induced nuclear factor that specifically bound to this IL-4 responsive element. This novel IL-4 responsive element and an IL-4-induced DNA binding protein may play an important role for the induction of C epsilon germ-line transcript as well as class switching to IgE.
Differential expression of 13 IgA-heavy chain genes in rabbit lymphoid tissues.
Authors: Spieker-Polet H , Yam P C , Knight K L
Published: December 2022

Molecular cloning techniques have recently demonstrated that rabbit has 13 different IgA C alpha H chain genes. This is in contrast to human and mouse that have only two and one C alpha heavy chain genes, respectively. In previous studies, nucleotide sequence analysis indicated that the 13 rabbit C alpha genes were potentially functional, and in vitro expression experiments showed that at least 12 of these genes were expressible. To understand the role of these multiple IgA isotypes we analyzed RNA of various lymphoid tissues for the presence of mRNA representing each of the multiple C alpha genes. We used the RNase protection assay with probes that are specific for the 13 different C alpha genes and we consistently found that at least 10 of the C alpha genes are expressed, albeit at different levels, in gut (small intestines), appendix, mesenteric lymph node, and mammary tissue. However, in salivary gland (submandibular), only seven of these genes are expressed at significant levels and in lung and tonsil only one C alpha gene, C alpha 4, is expressed at a level comparable to its expression in other tissues. Analysis of RNA of Peyer's patch showed differences in the level of C alpha gene expression between different animals and between different Peyer's patches of the same rabbit; in some cases, most of the C alpha genes were expressed, but in some cases only C alpha 4 was expressed at a significant level. Inasmuch as C alpha 4 is the 5' most C alpha gene we propose that IgA-producing cells are derived from B cells that have initially undergone isotype switching to C alpha 4 and we discuss various mechanisms that could explain switching to the more 3' C alpha genes.