bluuebunny/crossref_metadata_2025 huggingface dataset:
A murine cDNA encodes a pan-epithelial glycoprotein that is also expressed on plasma cells.
Authors: Bergsagel P L , Victor-Kobrin C , Timblin C R , Trepel J , Kuehl W M
Published: January 2023

Using a subtractive cDNA approach, we have identified a number of genes expressed in murine plasmacytomas, but not B or pre-B lymphomas. One of these genes, 289A, expresses a 1.8-kb microsomally localized mRNA that encodes a 314-amino-acid protein containing a signal sequence and a hydrophobic transmembrane domain. Sequence comparison suggests that the predicted protein is the murine homologue of a human cell surface pan-epithelial glycoprotein known variously as EGP, GA733-2, KSA, and KS1/4, recognized by mAb HEA125, GA733, KS1/4, CO17-1A, M74, and 323/A3. The 289A mRNA is highly expressed in normal murine tissues containing epithelial cells, and at a low level in plasma cells induced by LPS stimulation of spleen B lymphocytes. It is expressed in 15 of 16 plasmacytomas, but at a much lower level, if at all, in pre-B or B lymphomas. In human B cell lines, 289A detects a 1.5-kb mRNA in the myeloma cell line 8226, but not in Burkitt's lymphoma or lymphoblastoid cell lines. Subsequent FACS analysis of human cell lines with the mAb GA733 and KS1/4 demonstrated concordant expression of the mRNA and the protein. We conclude that 289A is the murine homologue of EGP, GA733-2, KSA, and KS1/4 Ag. Although its expression was previously thought to be restricted to epithelial cells, it is also expressed in plasma cells and is a B lymphocyte differentiation Ag. Because of the multiplicity of names, we propose calling the human gene hEGP314, and the murine gene mEGP314.
Clonal heterogeneity of superantigen reactivity in human V beta 6+ T cell clones. Limited contributions of V beta sequence polymorphisms.
Authors: Goronzy J J , Oppitz U , Weyand C M
Published: January 2023

Superantigens encoded in the genome or released by bacteria have been identified as potent modulators of the murine immune system. High frequencies of mature or immature T cells are activated or intrathymically deleted when superantigens cross-link MHC class II molecules and the V beta element of the TCR. The V beta specificity discriminates superantigens from polyclonal T cell stimulators as well as specific Ag and determines the immunomodulatory role in shaping the T cell repertoire. A similar regulatory function of superantigens in the human immune system is less well established. Here, we have studied a series of human T cell clones sharing the TCR V beta 6 element and describe a surprising heterogeneity in their responsiveness to staphylococcal exotoxins. The V beta 6 gene segment had the ability to respond to all staphylococcal enterotoxins (SE); however, for individual T cell clones, there was a clear predominance of SE C3 reactivity compared to SE B and SE C2. The clonal heterogeneity of SE responsiveness did not correlate to sequence polymorphisms in the fourth hypervariable region of the V beta 6 segment, the presumptive binding site for superantigens. Superantigen reactivity was crucially influenced by the presenting HLA-DR molecule, especially when the superantigen served as a coligand, enhancing or suppressing the Ag-specific activation of the TCR. These data suggest that the correlation between human TCR V beta gene segments and superantigen responses is not stringent. Potential intrathymic deletion mechanisms controlled by superantigens may be less selective in humans and may result in a leakiness influenced by the host HLA-DR molecules.
Differential increase of an alternatively polyadenylated mRNA species of murine CD40 upon B lymphocyte activation.
Authors: Torres R M , Clark E A
Published: January 2023

CD40 is an integral membrane glycoprotein found on the surface of human B lymphocytes. Antibodies specific for CD40 have been shown to augment proliferation of activated B lymphocytes, prevent B lymphocyte apoptosis, and prolong the maintenance of normal B lymphocytes in culture. As a step toward developing an in vivo system to examine CD40 function, a molecular clone encoding the murine homologue of the human CD40 B lymphocyte surface Ag was isolated and characterized. Throughout their open reading frames, the murine and human proteins shared 62% predicted amino acid identity. Within the cytoplasmic domain, which includes a completely conserved region known to be important for signaling by human CD40, the CD40 homologues are 78% identical. The human and murine proteins are members of a new cytokine receptor family, which includes the receptors for nerve growth factor and TNF-alpha, that are homologous in their cysteine-rich extracellular domains. The murine CD40 gene is expressed in B lymphocytes as two mRNA species generated by alternative usage of polyadenylation signals in the 3' untranslated region. The activation of B lymphocytes differentially increases the relative levels of these two mRNA transcripts suggesting a posttranscriptional mechanism for the regulation of CD40 surface expression.
Tumor-bearing mice exhibit a progressive increase in tumor antigen-presenting cell function and a reciprocal decrease in tumor antigen-responsive CD4+ T cell activity.
Authors: Zou J P , Shimizu J , Ikegame K , Yamamoto N , Ono S , Fujiwara H , Hamaoka T
Published: January 2023

Splenic CD4+ T cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 2 to 3 wk after the inoculation with CSA1M cells produced IL-2 and macrophage-activating factor upon in vitro cultures. This lymphokine production was achieved without stimulation of these T cells with exogenous stimulating tumor Ag. However, elimination of APC from spleen cells resulted in almost complete abrogation of the capacity of CD4+ T cells to produce IL-2/macrophage-activating factor. The lymphokine production was regained when APC from CSA1M-bearing mice were added back to cultures. APC from normal or another syngeneic tumor (Meth A)-bearing mice failed to regain the lymphokine production. These observations demonstrated that the lymphokines were produced by CD4+ T cells from CSA1M-bearing hosts through their collaboration with APC binding CSA1M tumor Ag in the tumor-bearing state. The lymphokine-producing capacity of whole spleen cells from tumor-bearing mice reached the maximal level around 2 to 3 wk after tumor implantation but gradually decreased with the progress of tumor-bearing stages. Importantly, tumor-bearing stage-related changes were observed in a different fashion in the capacities of anti-CSA1M CD4+ T cells vs CSA1M tumor Ag-binding APC. The capacity of APC increased with the progress of tumor-bearing stages as demonstrated by the stimulation of CSA1M-immunized T cells with APC from different CSA1M-bearing stages. In contrast, the reactivity of anti-CSA1M T cells to APC from a given CSA1M-bearing stage decreased with the tumor-bearing stage. These results demonstrate a stage-related increase tumor Ag-binding APC function, as well as a reciprocal reduction in tumor Ag-responsive CD4+ T cell activity.
Induction of TCR-gamma delta+ cells from thymocytes stimulated by a fetal liver-derived hepatocyte clone.
Authors: Nanno M , Hata M , Shimada S , Doi H , Satomi S , Yagi H , Nakamura M , Sakata T , Suzuki R , Itoh T
Published: January 2023

We have previously reported that a fetal liver-derived hepatocyte clone, FHC-4D2, can support hematopoiesis in vitro. Here, we show that fetal thymocytes (FT) or adult thymocytes (AT) proliferate on the monolayer of FHC-4D2 cells in the presence of rIL-2. Fresh thymocytes contained few TCR-gamma delta+ cells (< 4% for FT and < 1% for AT); significant numbers of TCR-gamma delta+ cells were detected (2-11% for FT and 15-33% for AT) after the coculture with FHC-4D2 and rIL-2. Although FT-derived TCR-gamma delta+ cells predominantly used the V gamma 5 chain, the major population in AT-derived TCR-gamma delta+ cells used V gamma 1, V gamma 4, or V gamma 7 chains. Both FT- and AT-derived TCR-gamma delta+ cells killed FcR-bearing target cells when incubated with anti-TCR-gamma delta Ab. Half of FT-derived TCR-gamma delta+ cells were CD4-CD8 alpha+8 beta-; the rest were CD4-CD8 alpha-8 beta-. AT-derived TCR-gamma delta+ cells expressed neither CD4 nor CD8 molecules. Separation of thymocytes from FHC-4D2 cells with a membrane filter reduced the proliferative response by two- to threefold. Taken together, these results demonstrate that a fetal hepatocyte clone supports thymocytes to develop preferentially into TCR-gamma delta+ cells in cooperation with rIL-2 through cell-cell contact, that the repertoire and the phenotype of induced TCR-gamma delta+ cells are determined by the age of the mice, and that hepatocytes might thus play an active role in T lymphopoiesis in the fetal liver.
Role of the VLA-4 molecule in T cell costimulation. Identification of the tyrosine phosphorylation pattern induced by the ligation of VLA-4.
Authors: Sato T , Tachibana K , Nojima Y , D'Avirro N , Morimoto C
Published: January 2023

The very late activated Ag (VLA) molecules not only mediate T cell adhesions, but also provide costimulation in a TCR/CD3-dependent manner. However, little is known about the signals mediated by the ligation of VLA molecules. Previous work from our laboratory identified a 105-kDa protein that is predominantly phosphorylated on tyrosine residue upon engagement of VLA-4 in a human T lymphoblastic cell line, H9, and in peripheral T cells. In the present study, we have shown that the A and B epitope of VLA-4 plays a key role in VLA-4-mediated T cell costimulation. Moreover, we have demonstrated that the solid phase cross-linking of VLA-4 using Ab (against A and B) or the CS-1 region of fibronectin, stimulated tyrosine phosphorylation of 140-, 120-, 80- to 70-, 60- to 55-, 50-, and 45-kDa proteins in addition to the 105-kDa protein. In contrast, Ab ligation of the C epitope of VLA-4 mainly induced tyrosine phosphorylation of pp105, weakly induced other protein tyrosine phosphorylation, and additionally induced only minimal T cell costimulation. Using immunoblotting, we have identified some of the tyrosine-phosphorylated proteins to be phospholipase C gamma (pp140), pp125 focal adhesion kinase (pp120), paxillin (pp70 and pp50), p59fyn/p56lck (pp60-55), and mitogen-activated protein kinase (pp45). Since solid phase cross-linking of VLA-4 by B2 epitope-specific Ab induced T cell costimulation most strongly via the CD3 pathway, our results suggested that the above tyrosine-phosphorylated proteins may play an important role in VLA-4-mediated T cell costimulatory signaling events.
Intracellular transport of invariant chain-MHC class II complexes to the peptide-loading compartment.
Authors: Xu X , Song W , Cho H , Qiu Y , Pierce S K
Published: January 2023

Th cells recognize peptide fragments of foreign Ags bound to MHC class II molecules. Upon synthesis in the endoplasmic reticulum, the alpha- and beta-chains of the class II molecules rapidly associate with invariant chains (li). The dissociation of li from class II molecules precedes binding of processed Ag and the formation of SDS-stable alpha beta dimers. We previously showed that functional, processed Ag-class II complexes are assembled in a dense lysosome-like compartment that contains stable class II molecules, but no li, referred to in this work as the peptide-loading compartment. We also identified a separate compartment that contains predominantly SDS-unstable li-class II complexes. Because we were unable to identify known organelle markers associated with this compartment, we refer to it as the X compartment. In this work, we provide results that indicate that the X compartment is composed of transport vesicles that move li-class II complexes to the peptide-loading compartment, where all events in the assembly of processed Ag-class II complexes occur.
Local release of IL-10 by transfected mouse mammary adenocarcinoma cells does not suppress but enhances antitumor reaction and elicits a strong cytotoxic lymphocyte and antibody-dependent immune memory.
Authors: Giovarelli M , Musiani P , Modesti A , Dellabona P , Casorati G , Allione A , Consalvo M , Cavallo F , di Pierro F , De Giovanni C
Published: January 2023

The cDNA coding for mouse IL-10 (mIL-10) was transduced into the parental cells of a spontaneous adenocarcinoma of BALB/c mice (TSA-pc), and clones secreting small, medium, and large quantities of IL-10 were selected. In vivo, both low and high producer clones do not display an enhanced ability to grow in H-2 and non-H-2 incompatible mice. Instead, the intensity of their rejection increases in function of the amount of mIL-10 released. After an initial growth period in syngeneic mice, high producer clones undergo complete rejection due to the combined action of CD8+ lymphocytes, NK cells, and neutrophils. After this rejection, mice are immune to a subsequent challenge with TSA-pc. This memory rests on a strong lytic activity of CD8+ CTL and granulocytes. Following the rejection, mice also develop anti-TSA Ab that guide the granulocytes in TSA-pc memory reaction. A direct comparison shows that although TSA clones engineered to release IL-2 activate CTL and no anti-TSA Ab, those engineered to release IL-4 activate a strong Ab response but not CTL. The kind of cytokine released by the tumors appears to determine the type of response. However, IL-10 high producer cells do not deviate the immune memory, neither toward a Th1 nor a Th2. Both the CTL activity and the Ab responses induced by IL-10 high producer cells are the strongest so far observed in the TSA system.
IL-13 down-regulates CD14 expression and TNF-alpha secretion in normal human monocytes.
Authors: Cosentino G , Soprana E , Thienes C P , Siccardi A G , Viale G , Vercelli D
Published: January 2023

CD14, a glycosylphosphatidylinositol (GPI)-linked protein expressed on monocytes and neutrophils, regulates monocyte-lymphocyte interactions and serves as the LPS receptor. We showed previously that IL-4 down-regulates the expression of human CD14 by acting at the transcriptional level. We now investigate whether CD14 expression could also be regulated by IL-13, another member of the chromosome 5 cytokine gene family. IL-13 dose-dependently inhibited CD14 expression on human monocytes. By contrast, expression of CD23 and CD11b was enhanced strongly. Down-regulation of CD14 involved neither shedding nor activation of endogenous GPI anchor-cleaving enzymes. Indeed, soluble CD14 was not increased in the supernatants of IL-13-stimulated monocytes, and expression of CD55/DAF, another GPI-linked protein, was unaffected by IL-13. CD14 transcript levels were reduced sixfold in IL-13-treated monocytes. These results suggest that IL-13 down-regulates membrane CD14 by suppressing CD14 RNA expression. IL-13-dependent down-regulation of CD14 resulted in the inhibition of CD14-mediated events. Indeed, CD14-mediated release of TNF-alpha was inhibited markedly (approximately 75%) in monocytes stimulated with LPS (100 ng/ml) after a 72-h preincubation with IL-13. However, IL-13 also directly inhibited monokine secretion, because it blocked PMA-induced, CD14-independent TNF-alpha release. Down-regulation of CD14 and TNF-alpha secretion may play a major role in the anti-inflammatory effects of IL-13 on LPS-stimulated monocytes.
Expression of CD25 defines peripheral blood mononuclear cells with productive versus latent HIV infection.
Authors: Borvak J , Chou C S , Bell K , Van Dyke G , Zola H , Ramilo O , Vitetta E S
Published: January 2023

The present studies were designed to further determine whether the CD25 marker could distinguish between cells productively and latently infected with HIV. This was accomplished by combining immunotoxin (IT)-mediated killing of CD25+ cells, highly sensitive indirect immunofluorescence to detect remaining CD25+ cells, and PCR-mediated amplification of proviral DNA in immunotoxin-treated vs untreated HIV-infected cells. Our results demonstrate that: 1) By direct immunofluorescence 3 to 8% of PBMCs are CD25+, whereas by indirect immunofluorescence 30% are CD25+. The increased number of CD25+ cells is due to their detection by the highly sensitive indirect immunofluorescence assay. Up to 60% of the CD25+ cells are CD4+ and 12% are CD8+. 2) Treatment of HIV-infected PBMCs with an anti-CD25 IT for 6 days eliminated both CD25high and CD25low cells and decreased the production of p24 by 99%. 3) Differences in the HIV proviral genome were detected in the unfractionated PBMCs vs PBMCs from which CD25+ cells had been eliminated by IT treatment. Hence, PBMCs containing both CD25+ and CD25- cells express all intermediate proviral species and full-length double-stranded proviral DNA. In contrast, CD25- quiescent cells contain predominantly intermediate species. These results confirm and extend our previous observations that expression of CD25 can distinguish latently infected cells from cells producing virus.
SLE-like autoantibodies and Sjögren's syndrome-like lymphoproliferation in TGF-beta knockout mice.
Authors: Dang H , Geiser A G , Letterio J J , Nakabayashi T , Kong L , Fernandes G , Talal N
Published: January 2023

Mice bearing the TGF-beta 1 null mutation (-/-) develop lymphoid infiltrates in the heart, lungs, salivary glands, and other organs similar to those seen in the pseudolymphoma of Sjögren's Syndrome. We studied sera from -/- mice and found elevated Ab levels to dsDNA, ssDNA, and Sm ribonucleoprotein. No Abs to SSA/Ro or SSB/La and no IgM rheumatoid factor were found. Serum autoantibodies were predominately IgG and were specific as shown by ELISA inhibition studies. Antinuclear Ab patterns on Western blots varied from one mouse to the next, indicating a random process responsible for the diversity. Wild-type and heterozygote mice had no autoantibodies. Ig glomerular deposits were found in -/- mice, indicating that these autoantibodies may be pathogenic. Treatment of -/- mice with dexamethasone or TGF-beta 1 failed to suppress autoantibody production. These mice represent an overlap combining the autoimmune serology of SLE with the tissue infiltrates of SS. Our results support the concept that TGF-beta 1 is an important naturally occurring immunosuppressive cytokine whose absence can lead to a systemic autoimmune disease.
Light chain contribution to specificity in anti-DNA antibodies.
Authors: Ibrahim S M , Weigert M , Basu C , Erikson J , Radic M Z
Published: January 2023

We studied mice expressing one of two H chain transgenes. Both transgenes expressed the same 3H9 anti-DNA VDJ, but differed in their constant domains. The IgM transgene efficiently induced tolerance and selected for a subset of endogenous L chains that prevented dsDNA binding. In contrast, the IgG2b secreted-only H chain allowed expression of a broad range of L chains, most of which yielded anti-dsDNA Ab. To deduce the features of L chains that affect DNA binding, we derived hybridomas from LPS-stimulated splenic B cells from the two transgene lines and compared the V kappa sequences of Ab they secreted. Identification of L chains with related sequences but different binding to ssDNA, dsDNA, and cardiolipin allowed us to pinpoint L chain residues that correlate with enhanced or reduced binding. Arginines at the junction of V kappa 1 or V kappa 8 regions and J kappa 1, and arginines or asparagines in CDR1 or CDR2 enhanced DNA binding. Negatively charged residues at the same positions were found to interfere with binding. Thus, we predict that appropriate amino acids at these positions may form contacts with DNA. The likely locations of contact residues in the combining site were evaluated by inspection of previously determined Ab structures. Our results indicate that L chains in anti-DNA Ab are able to modulate DNA binding and contribute contact sites for additional determinants on a complex autoantigen.
flk2/flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors.
Authors: Hunte B E , Hudak S , Campbell D , Xu Y , Rennick D
Published: January 2023

We have investigated the role of flk2/flt3 ligand (FL) in B cell lymphopoiesis. The ability of FL to stimulate the growth of immature B cells was assessed using distinct populations: CD43lowB220+ pre-B cells, CD43+B220+ pro-B cells, and CD43+B220low progenitors. FL failed to affect the growth of the pro-B or pre-B cells whether used alone or in combination with stem cell factor (SCF) or IL-7. In striking contrast, FL was a potent cofactor for the CD43+B220low progenitor cells, interacting with either IL-7 and/or SCF to stimulate their growth. The combination of FL with IL-7 plus SCF stimulated maximum expansion of these cells, albeit, less than that stimulated in stromal cell cultures. When the CD43+B220low progenitors were divided based on expression of heat stable Ag (CD24) into a CD24- and a CD24+ subset, the FL-responsive cells were contained only within the CD24- subset. FL interacted with SCF or with IL-7 to stimulate their growth resulting in a 20- and 50-fold increase in cellularity, respectively. Since the CD24- subset was the most immature of the B cell populations studied, our data suggest that FL costimulates the expansion of very primitive B cell progenitors.
Regulation of the catalytic subunit (p34PSK-J3/cdk4) for the major D-type cyclin in mature B lymphocytes.
Authors: Tanguay D A , Chiles T C
Published: January 2023

We examined the expression of the cyclin-dependent kinase 4, p34PSK-J3/cdk4 protein, in small dense, activated, and proliferating primary B lymphocytes. A small steady state level of cdk4 synthesis was detected in resting B cells. Stimulation of resting B cells with mitogenic amounts of F(ab')2 fragments of goat anti-mouse IgM (anti-Ig) resulted in increased synthesis of cdk4 protein during the mid to late G1 phase of the cell cycle; LPS or the combination of phorbol ester and calcium ionophore also elevated cdk4 levels. Resting B cells that we rendered competent by treatment with IL 4 or low doses of anti-Ig or, alternatively, were activated by phorbol ester or ionomycin alone also exhibited heightened cdk4 protein levels. Subsequent analysis of potential cdk4 regulatory subunit D-type cyclins revealed that cyclin D2, not cyclin D1 or D3, is expressed in primary mature B lymphocytes. The induction of cyclin D2 synthesis in response to mitogenic anti-Ig paralleled cdk4 expression; however, IL-4 or low dose anti-Ig alone did not increase the rate of de novo cyclin D2 synthesis above that of resting B cells. The significance of the lack of cyclin D2 regulation by competence-inducing growth factors was demonstrated, in that only mitogenic factors that stimulated DNA synthesis 1) led to the formation of stable cyclin D2/cdk4 holoenzyme complexes during G1 phase progression, and 2) afforded the isolation of anti-cyclin D2 or anti-cdk4 immunoprecipitates that phosphorylated retinoblastoma. These findings suggest a role for these proteins during the mid to late G1 phase progression and possibly the G1/S phase transition in primary mature B lymphocytes.
Rhinovirus enters but does not replicate inside monocytes and airway macrophages.
Authors: Gern J E , Dick E C , Lee W M , Murray S , Meyer K , Handzel Z T , Busse W W
Published: January 2023

Potential interactions between rhinovirus (RV) and both the airway macrophage and its precursor cell, the blood monocyte, were investigated in terms of direct binding, intracellular replication, cell survival, and cytokine production. When HeLa cell suspensions are inoculated with RV as a positive control, virus titer increases by 100-fold in the first 24 h, confirming intracellular replication. In contrast, RV titer in monocyte and macrophage suspensions steadily decreased. Despite a lack of productive RV replication, cell-associated RV RNA was detectable using a biotin-labeled cDNA probe as early as 6 h after inoculation. Direct binding of RV16 to macrophages was confirmed using radiolabeled virus, although preincubation with anti-ICAM-1 mAb did not block this interaction. Synthesis of RV RNA, as indicated by [3H]uridine incorporation in actinomycin D-treated cells, was detected in HeLa cells but not macrophages, suggesting that the viral RNA detected inside macrophages was from input virus and was not newly synthesized. RV inoculation did not adversely affect monocyte or macrophage viability. Finally, RV caused macrophage activation, as indicated by the induction of TNF-alpha secretion. These in vitro findings suggest that macrophages interact with major group RV in vivo, and raise the possibility that there is a second cellular receptor for these viruses. Furthermore, macrophages do not serve as permissive host cells during in vivo RV infection, but instead may be active participants in anti-RV immunity and RV-induced airway inflammation.
Effects of Mycoplasma fermentans on the myelomonocytic lineage. Different molecular entities with cytokine-inducing and cytocidal potential.
Authors: Rawadi G , Roman-Roman S , Castedo M , Dutilleul V , Susin S , Marchetti P , Geuskens M , Kroemer G
Published: January 2023

Mycoplasma fermentans is a mycoplasma species that has been accused of serving as a cofactor of AIDS development. Here, we show that M. fermentans affects the function of human monocytes and myelomonocytic cell lines on at least two different levels. Heat-inactivated mycoplasma particles induce inflammatory cytokines such as IL-1, IL-6, and TNF in monocytes, as well as in THP-1 cells. Moreover, M. fermentans induces IL-10 (but not IL-12) in freshly isolated human monocytes. The cytokine-inducing effect is mediated by lipid-associated molecules. In addition, we have detected a novel biologic activity that resides in the nonlipid-associated protein fraction of M. fermentans (approximate molecular mass: 15 to 30 kDa) and that has a cytocidal effect on nondifferentiated myelomonocytic cell lines (U937 cells, HL-60 cells), as well as on actinomycin-D-sensitized monocytes. Death is accompanied by oligonucleosomal DNA fragmentation and loss of chromosomal DNA. U937 and HL-60 cells fail to produce cytokines and rather undergo cell death in response to heat-inactivated M. fermentans, provided that they are kept in a relatively undifferentiated stage. Whereas the cytokine-inducing activity is a general feature of many mycoplasma species, it appears that only a restricted panel of mycoplasma species exert a cell death-inducing activity. In addition to M. fermentans strains, Mycoplasma penetrans, another hypothetical cofactor of AIDS, possess a cytocidal activity. This does not apply to other mycoplasma species, including pathogenic ones such as Mycoplasma pneumoniae and Ureaplasma urealyticum. The cell death-inducing effect of M. fermentans is not mediated by cytokines and obeys different principles than TNF-alpha-mediated apoptosis. Thus, in contrast to TNF-alpha-induced death, it is not accompanied by a decrease in the mitochondrial transmembrane potential and is not inhibited by preincubation with the antioxidant drug N-acetylcysteine. In synthesis, it appears that certain AIDS-associated mycoplasma species perturb the function and/or generation of cells from the myelomonocytic lineage via several distinct pathways.
Structural requirements for mucosal vascular addressin binding to its lymphocyte receptor alpha 4 beta 7. Common themes among integrin-Ig family interactions.
Authors: Briskin M J , Rott L , Butcher E C
Published: January 2023

The mucosal vascular addressin, MAdCAM-1, is an Ig family adhesion receptor preferentially expressed by venular endothelial cells at sites of lymphocyte extravasation in mucosal lymphoid tissues and lamina propria. MAdCAM-1 binds the lymphocyte homing receptor for Peyer's patches, the integrin alpha 4 beta 7. We describe a point mutation within the first Ig domain of MAdCAM-1 that abolishes activation-independent alpha 4 beta 7 binding. This point mutation resides within an eight-amino acid motif with homology to sequences important for the integrin binding ability of the related vascular Ig family members, ICAM-1 and VCAM-1. To understand the contributions of the individual MAdCAM-1 domains, chimeric exchanges with the beta 1 integrin ligand, ICAM-1, were made. The two N-terminal domains of MAdCAM-1 are sufficient to confer alpha 4 beta 7 binding comparable to that of native MAdCAM-1. A chimera containing only the N-terminal Ig domain (domain 1) of MAdCAM-1 can also bind (to a lesser extent) alpha 4 beta 7, but only after integrin (to a lesser extent) alpha 4 beta 7, but only after integrin activation. Conversely, the first domain of ICAM-1 appears sufficient to bind activated LFA-1. These data suggest that the first domain of MAdCAM-1 is sufficient for interaction with alpha 4 beta 7, but that sequences within the second domain support this interaction, either by providing additional contact points for integrin binding or by contributing to the conformation or presentation of the N-terminal domain. The second domain of MAdCAM-1 can also support activation-dependent LFA-1 binding to domain 1 of ICAM-1. The findings parallel studies of VCAM-1 binding to alpha 4 beta 1 and suggest that structural differences exist between vascular Ig-like ligands for alpha 4 vs beta 2 integrins.
Induction and regulation of IL-15 expression in murine macrophages.
Authors: Doherty T M , Seder R A , Sher A
Published: January 2023

IL-15 is a recently described cytokine which resembles IL-2 in its biologic activities, stimulating T cell and NK cell proliferation and activation as well as enhancing B cell expansion and Ab production. Unlike IL-2, IL-15 is not produced by lymphocytes, but instead (at least among cells of the immune system) appears to be synthesized primarily by monocyte/macrophages. We have examined the induction of IL-15 in murine macrophages (by semiquantitative reverse transcriptase-PCR and bioassay) in response to a variety of different macrophage-activating stimuli and compared the regulation of IL-15 production to that of IL-12 and TNF-alpha. Optimal induction of IL-15, in each of the macrophages populations tested, was found to require both priming (IFN-gamma) and triggering (LPS, mycobacteria, or Toxoplasma gondii) stimuli. When compared with IL-12 mRNA synthesis by the same macrophages, IL-15.mRNA production was more resistant to inhibition by the down-regulatory cytokines IL-14, IL-13, and TGF-beta. Moreover, IL-10, which is inhibitory for most other monokines, increased levels of IL-15 mRNA found after stimulation. These data establish IL-15 as a product of the macrophage/monocyte lineage, which is up-regulated on activation. IL-15 could thus play an important role in the initiation of immune responses by microbial agents.
Regulation of complement activity by immunoglobulin. I. Effect of immunoglobulin isotype on C4 uptake on antibody-sensitized sheep erythrocytes and solid phase immune complexes.
Authors: Miletic V D , Hester C G , Frank M M
Published: January 2023

Intravenous Ig, composed principally of IgG, prevents complement attack by inhibiting C3 and C4 uptake onto target cells and tissues. Using two different models, Ab-sensitized SRBC and BSA-anti BSA solid phase immune complexes, we have examined the complement inhibitory capacity of three Ig classes (IgG, IgM, IgA) focussing on inhibition of C4 uptake. It was found that on both a weight and molar basis, monomeric serum IgA and IgM were far more active than IgG (weight efficiency ratios were 1.0, 20.8, and 236.3, and molar efficiency ratios 1.0, 24.0, and 1382.9 for polyclonal IgG, IgA1, and IgM, respectively). Monoclonal IgM were less active than polyclonal IgM (50% inhibition was achieved in SRBC model by 0.022, 0.30, 1.6, and 1.6 mg/ml of polyclonal IgM and monoclonal IgM from patients Lew, Will, and Pri). Secretory IgA was less active than serum IgA1 and similar in inhibitory activity to IgG (weight and molar efficiency ratios 1.5 and 0.6 compared with IgG). All tested preparations were less active in the solid phase immune complex model than in the sensitized cell model. A mixture of Igs of different isotypes was somewhat more active than any isotype alone. These results suggest that polyclonal serum IgA and IgM can also be considered for active therapy in diseases accompanied by the activation of classical complement pathway.
Thioredoxin as a potent costimulus of cytokine expression.
Authors: Schenk H , Vogt M , Dröge W , Schulze-Osthoff K
Published: January 2023

Reduction/oxidation (redox) processes have been implicated in various biologic processes, including signal transduction, gene expression, and cell proliferation. Thioredoxin is one of the major redox-regulatory molecules which because of its dithiol/disulfide exchange activity determines the oxidation state of protein thiols. In this study, we report that treatment of several cell types with thioredoxin strongly enhances the expression of various cytokines. In monocytic Mono Mac6 cells stimulated with the phorbolester tetradecanoyl phorbolacetate (TPA), thioredoxin was found to augment the expression of TNF at the protein and mRNA levels. In this and other cell lines, such as fibrosarcoma and endothelial cells, thioredoxin also dose-dependently increased the synthesis of IL-6. Treatment of TPA-stimulated Mono Mac6 cells resulted in a strong potentiation of secreted IL-1 bioactivity and expression of IL-1 alpha and IL-8 mRNA. In addition, in TPA-activated Molt-4 T cells, an increased expression of IL-2 and IL-2-specific transcripts was detected. These data demonstrate that cytokine synthesis may be tightly controlled by redox-dependent processes. As thioredoxin is readily secreted and taken up by cells, it may play an important role as a costimulatory molecule involved in immune processes.
Modulation of endotoxin-induced histamine synthesis by cytokines in mouse bone marrow-derived macrophages.
Authors: Takamatsu S , Nakashima I , Nakano K
Published: January 2023

The aim of this study was to examine the roles of various cytokines in histamine synthesis by macrophages in bone marrow. Pure (> 99% nonspecific esterase-, CD14-, and Mac-1-positive) macrophage populations were obtained after long term culture of bone marrow cells (bone marrow-derived macrophages). Culture of the cells in the presence of Escherichia coli LPS caused a slight, but dose-dependent, increase in histidine decarboxylase-associated histamine synthesis with a concomitant increase in the expression of CD14, a LPS receptor, as well as the Mac-1 Ag on their surface. Granulocyte/macrophage CSF (GM-CSF) or IL-3 strongly enhanced LPS-induced histamine formation and expression of CD14 on bone marrow-derived macrophages, without affecting the expression of Mac-1 Ag. GM-CSF and IL-3 also caused a marked accumulation of both histidine decarboxylase and IL-6 mRNAs in the cells. Macrophage CSF and IL-1-alpha also potentiated LPS-dependent histamine formation when it was stimulated with GM-CSF or IL-3. In contrast to these cytokines, IFN-gamma suppressed LPS-induced histamine production regardless of whether it was stimulated by GM-CSF or IL-3, and inhibited CD14 expression. Neither IL-6 nor granulocyte CSF had any appreciable effect on LPS-induced histamine production even in combination with GM-CSF or IL-3. These results suggest that GM-CSF and IL-3 enhance LPS-induced histamine production in bone marrow-derived macrophages and that macrophage CSF and IL-1 alpha augment the actions of GM-CSF and IL-3. Possible implication of CD14 molecule in the reactions is discussed.
T cells, but not thymic exposure to HLA-B27, are required for the inflammatory disease of HLA-B27 transgenic rats.
Authors: Breban M , Fernández-Sueiro J L , Richardson J A , Hadavand R R , Maika S D , Hammer R E , Taurog J D
Published: January 2023

Rats transgenic for the human MHC class I gene HLA-B27 are susceptible to a spontaneous multisystem inflammatory disease that resembles human B27-associated disease. This disease requires a high level of expression of the B27 transgene product in cells of hemopoietic origin and can be adoptively transferred to B27 transgenic or nontransgenic rats by transplantation of bone marrow (BM) or fetal liver (FL) cells. To investigate the role played by T cells and the thymus in the disease process, we produced congenitally athymic rnu/rnu F344 rats carrying the disease-prone B27 transgenic locus of the 33-3 line. Transgenic nude rats were protected from disease manifestations. This protection was abated by reconstitution with T cells from euthymic donors of the 33-3 line, with CD4 T cells being more efficient than CD8 T cells in transferring disease. Lethally irradiated, adult-thymectomized (ATX), nontransgenic recipients reconstituted with intact BM, T cell-depleted BM, FL, or nude BM from syngeneic disease-prone lines all developed disease. Pretreatment of the ATX nontransgenic recipients to deplete T cells enhanced the subsequent transferred disease. The inflammatory disease of B27 transgenic rats is thus T cell-dependent. The relevant T cells do not need to encounter B27 in the thymus, and residual radioresistant and/or extrathymically derived host T cells are sufficient to mediate the adoptively transferred disease. The data are most consistent with a model of B27-mediated disease arising from a failure of tolerance and requiring a population of CD4 T cells.
Self-reactive cytotoxic gamma delta T lymphocytes in Graves' disease specifically recognize thyroid epithelial cells.
Authors: Catálfamo M , Roura-Mir C , Sospedra M , Aparicio P , Costagliola S , Ludgate M , Pujol-Borrell R , Jaraquemada D
Published: January 2023

In this paper we report the isolation of a self-reactive cytotoxic gamma delta T cell line, 158RE.2, that originates from the T lymphocyte population infiltrating the thyroid gland of a patient with Graves' disease. Functional data using this cell line demonstrate that gamma delta T cells expanded in the thyroid tissue specifically recognize a ligand expressed by thyroid epithelial cells and cell lines of endocrine epithelial origin. The TCR expressed by these gamma delta T cells--V gamma I/V delta 5--is unusual in peripheral blood lymphocytes, and its specificity is clearly different from that observed in a high percentage of gamma delta T cells from PBL, which express the common TCR V gamma 9/V delta 2. The V gamma I/V delta 5 receptor is involved in the recognition of the ligand expressed by the thyroid cells, but not in the NK-like activity also displayed by 158RE.2. These cells express CD8 alpha alpha dimers, which participate in the thyroid ligand recognition but not in the NK-like activity. The epithelial cell recognition is not restricted by classical MHC class I or class II molecules, although the CD8 alpha alpha participation in the recognition suggests the involvement of nonclassical MHC molecules. These are the first data to be presented on self-reacting gamma delta T cells in human epithelium.
Glutamic acid decarboxylase autoantibodies in stiff-man syndrome and insulin-dependent diabetes mellitus exhibit similarities and differences in epitope recognition.
Authors: Daw K , Ujihara N , Atkinson M , Powers A C
Published: January 2023

Glutamic acid decarboxylase (GAD) is an autoantigen in two autoimmune diseases, insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome (SMS). However, most individuals with one of these diseases do not have the other disease. Prior studies have suggested that the natures of the GAD Abs associated with each of these diseases are different, which may have implications for the autoimmune pathogenesis. We have compared the GAD autoantibody profile and have mapped GAD protein epitope regions in the two diseases using an immunoprecipitation assay with recombinant GAD 65 and GAD 67 proteins, GAD protein fragments, and synthetic GAD peptides, as well as chimeric GAD proteins. Our results indicate that individuals with SMS have GAD Abs in 100- to 500-fold higher titer than individuals with IDDM. The population of GAD Abs in SMS sera is quite complex and includes those that recognize at least three GAD 65 epitope regions located between amino acids 1-16, 188-442, and 442-563. These types of GAD Abs are not found in IDDM sera. All SMS sera also had Ab specificity that binds GAD 67 in a region highly homologous to amino acids 188-442 of GAD 65. In contrast to prior studies that used immunoblotting to measure GAD Abs, we find GAD Abs in SMS sera also target two conformation-dependent regions of GAD 65, one located in the middle and one near the C-terminus of the protein. These two regions of the GAD 65 protein are similar to regions targeted by GAD 65-specific Abs found in individuals with IDDM. These results indicate that although disease-specific epitopes may exist, there is also overlap in the humoral response between the two diseases.
Class I-restricted presentation of an HIV-1 gp41 epitope containing an N-linked glycosylation site. Implications for the mechanism of processing of viral envelope proteins.
Authors: Ferris R L , Buck C , Hammond S A , Woods A S , Cotter R J , Takiguchi M , Igarashi Y , Ichikawa Y , Siliciano R F
Published: January 2023

Uncertainty exists over the site of processing of viral envelope (env) proteins for recognition by CTL. The extracellular domains of env proteins are not present in the cytosol, the site where the class I Ag processing pathway begins. Rather, the ecto-domains of env proteins are cotranslationally translocated into the endoplasmic reticulum during biosynthesis. To clarify the site of processing of viral env proteins, we examined the processing of an HLA B*3501-restricted epitope in the extracellular domain of the HIV-1 env protein. Although this epitope contains an N-linked glycosylation signal sequence, CTL specific for this epitope recognize a nonameric peptide that has not been previously modified by attachment of oligosaccharide. This was demonstrated in two ways. First, an env-specific B*3501-restricted CTL clone recognized a nonglycosylated, synthetic nonamer representing the minimal B*3501-restricted epitope, but not the glycosylated or deglycosylated forms. Second, the naturally processed, B*3501-restricted, env peptide is identical with a nonglycosylated, synthetic nonamer. Thus, the naturally processed form of an env epitope containing an N-linked glycosylation site is derived from env protein that is not glycosylated at the relevant asparagine during biosynthesis. Since the addition of N-linked oligosaccharides occurs only after the glycosylation signal sequence (N-X-S/T) is translocated into the endoplasmic reticulum, the initial processing reaction for this epitope may take place in the cytosol. Low-frequency failure of signal sequence containing polypeptides to engage the translocation apparatus, resulting in synthesis and degradation in the cytosol, may represent an important mechanism for the generation of class I-restricted CTL responses.
IL-10 cooperates with TNF-alpha to activate HIV-1 from latently and acutely infected cells of monocyte/macrophage lineage.
Authors: Finnegan A , Roebuck K A , Nakai B E , Gu D S , Rabbi M F , Song S , Landay A L
Published: January 2023

IL-10 is elevated in HIV-1-infected individuals and has been implicated in disease progression. In this study, we investigated the effects of IL-10 on the activation of HIV-1 from infected monocytes and macrophages. Although IL-10 alone did not induce HIV-1 replication, in the presence of TNF-alpha, IL-10 markedly enhanced virion production from a chronically infected promonocytic cell line (U1) and in acutely infected monocyte-derived macrophages. Neutralizing mAbs to IL-10 and TNF-alpha indicated that both cytokines were essential for the induction and were required to generate a synergistic increase in virus expression. The effects of the two cytokines were distinguishable functionally since pretreatment with TNF-alpha attenuated the cytokine cooperativity, while pretreatment with IL-10 potentiated their cooperativity, suggesting that IL-10 and TNF-alpha play different roles in the activation of virus. Northern blot analysis as well as Ab blocking and cytokine secretion studies indicated that the induction of either endogenous TNF-alpha or IL-10 was not involved in the cooperativity, nor was an up-regulation of TNF-alpha receptors. In combination with TNF-alpha, IL-10 stimulated activating protein-1 (AP-1) and nuclear factor (NF)-kappa B binding activities and cooperated to increase HIV-1 steady-state mRNA levels and enhance long terminal repeat-directed transcription through activation of the NF-kappa B binding sites, suggesting the IL-10 effect occurs at least in part at the transcriptional level. These results indicate that IL-10, in addition to down-regulating the cellular immune response to HIV-1, may also play a role in TNF-alpha-mediated activation of HIV-1 replication in the monocyte/macrophage lineage.
Antigen receptors on immature, but not mature, B and T cells are coupled to cytosolic phospholipase A2 activation: expression and activation of cytosolic phospholipase A2 correlate with lymphocyte maturation.
Authors: Gilbert J J , Stewart A , Courtney C A , Fleming M C , Reid P , Jackson C G , Wise A , Wakelam M J , Harnett M M
Published: January 2023

The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.
IL-10 inhibits nuclear factor-kappa B/Rel nuclear activity in CD3-stimulated human peripheral T lymphocytes.
Authors: Romano M F , Lamberti A , Petrella A , Bisogni R , Tassone P F , Formisano S , Venuta S , Turco M C
Published: January 2023

IL-10 markedly reduces nuclear factor (NF)-kappa B/Rel nuclear activity induced in PBMC by stimulation with the anti-CD3 mAb OKT3. The inhibition is exerted specifically on the NF-kappa B/Rel activation induced by mAb OKT3, and not that produced by PMA. As judged by supershifting the DNA-protein complexes with Abs recognizing specific components of the NF-kappa B/Rel protein family, the p50/p65 (Rel A) heterodimeric form of NF-kappa B is primarily affected. The maximal effect is observed at the IL-10 concentration of 20 U/ml. IL-10 inhibitory activity is exerted on T lymphocytes and is mediated by monocytes. Indeed, monocytes pretreated with IL-10 are able so inhibit NF-kappa B nuclear activity in purified T lymphocytes stimulated with OKT3. Soluble factors do not appear to be involved in the mechanism of inhibition. On the other hand, the up-regulation of CD80 Ag, found on monocytes obtained from PBMC incubated with OKT3, is not detected after addition of IL-10, and the anti-CD28 mAb CLB-CD28/1 restores the NF-kappa B/Rel nuclear activity in IL-10-inhibited lymphocytes. Therefore, the NF-kappa B/Rel inhibition might be ascribed to a lack of cooperation between accessory cells and T lymphocytes, resulting from down-regulation of a costimulatory molecule, such as CD80, produced by IL-10 on activated monocytes. Our results demonstrate that IL-10 can inhibit the induction of NF-kappa B/Rel nuclear activity in CD3-stimulated T lymphocytes. Since inappropriate activation of kappa B-driven genes has a physiopathologic role in a number of diseases, such as HIV infection, our findings support the possibility of using this cytokine to suppress an undesirable activation of these transcription factors.