bluuebunny/crossref_metadata_2025 huggingface dataset:
Three intracellular signals for cytotoxic T lymphocyte-mediated killing. Independent roles for protein kinase C, Ca2+ influx, and Ca2+ release from internal stores.
Authors: Haverstick D M , Engelhard V H , Gray L S
Published: December 2022

Ligation of the TCR after interaction of a CTL with a relevant target cell results in a change in CTL shape with reorientation and secretion of cytoplasmic granules. Secretion can be induced by treating CTL with the combination of a phorbol ester to activate protein kinase C and ionomycin to increase the intracellular Ca2+ concentration by ion-selective permeabilization of both the plasma membrane and internal Ca2+ storage depots. Treatment of cloned murine CTL with PMA induced a change in cell shape without movement of cytoplasmic granules, whereas increased intracellular [Ca2+] after treatment with ionomycin resulted in granule movement without shape change. Granule movement induced by ionomycin did not lead to secretion in the absence of PMA. Direct activation of the TCR resulted in increased intracellular Ca2+ predominately from influx of extracellular Ca2+ with a substantially smaller contribution from release of Ca2+ from internal stores. By independently inhibiting either component of the TCR-initiated increase in intracellular Ca2+, it was determined that both sources of Ca2+ were required for granule movement and secretion. Thus, increases in intracellular Ca2+ concentration mediated by these two sources appear not to be functionally equivalent. Taken together, these results indicate that the CTL functional response that normally occurs after interaction with an antigenically relevant target cell is regulated by three independent signals.
Cytokine production by mature and immature thymocytes.
Authors: Fischer M , MacNeil I , Suda T , Cupp J E , Shortman K , Zlotnik A
Published: December 2022

We have studied the ability of subpopulations of activated thymocytes to produce four cytokines (IL-2, IL-4, IFN-gamma and TNF-alpha) which are believed to play roles in T cell development. Supernatants from various thymocyte subsets activated with calcium ionophore and PMA were tested for these cytokines. All CD3hi thymocyte subsets (CD4+8-, CD4-8- and CD4-8+) produced high titers of these four cytokines except CD3+4-8+ thymocytes, which did not produce IL-4. In contrast, CD4+8+ thymocytes did not produce any detectable cytokines. CD3-4-8- thymocytes produced IL-2, IFN-gamma, and TNF-alpha (but not IL-4) when activated by calcium ionophore + PMA and IL-1. We then separated CD3-4-8- thymocytes into IL-2R+ and IL-2R-. CD3-4-8-IL-2R+ thymocytes only produced small amounts of IL-2 when activated with calcium ionophore + PMA + IL-1, whereas CD3-4-8-IL-2R- thymocytes did not require IL-1 to produce IL-2, IFN-gamma, and TNF-alpha. Finally, CD4-8+3- thymocytes (an immature population believed to be an intermediate between CD3-4-8- and CD4+8+ thymocytes) only produced marginally detectable levels of IL-2 upon stimulation with calcium ionophore, PMA, and the addition of IL-1 did not result in increased levels of cytokine production. These observations indicate discrete patterns of cytokine production by the subsets studied and suggest specific controls of cytokine gene expression during T cell development.
GTP-binding proteins transduce signals generated via human FC gamma receptor IIIA (CD16).
Authors: Procopio A D , Paolini R , Vecchio I , Frati L , Santoni A
Published: December 2022

This study demonstrates that GTP-binding proteins regulate Fc gamma RIII-mediated signal transduction and inositol phosphate (IPn) generation in human NK cells. In addition the cross-linking of CD16 by mAb, guanosine 5'-o-3-thiophosphate induced 1,4,5 inositol trisphosphate (IP3) release in permeabilized NK cells and their membranes. By contrast, guanosine 5'-o-2-thiophosphate, almost completely inhibited IP3 generation induced by cross-linking with anti-CD16 mAb. Pretreatment of NK cells with 10 to 100 ng/ml Vibrio cholerae toxin (Ctx) almost completely inhibited the generation of IP3 and of other Ipn as well as Fc gamma RIII-operated cell functions such as antibody-dependent cell-mediated cytotoxicity against antibody-coated P815 mastocytoma cells. Isolated B subunit of Ctx was inactive. Bordetella pertussis toxin (0.1 to 1 microgram/ml) only marginally affected IP3 release and antibody-dependent cell-mediated cytotoxicity. Ctx increased cAMP levels in NK cells. However, inhibition of IP3 release preceded the rise of cAMP. Moreover, cAMP analogues (8-chlor-cAMP, 8-bromo-cAMP, dibutiryl-cAMP), as well as intracellular cAMP-enhancing agents (PGE1, PGE2, and forskolin) did not mimicked the effects of Ctx on IP3 generation, suggesting that the adenylate cyclase pathway is not responsible for the early effects of Ctx on Fc gamma RIII-mediated signalling. Overall these results demonstrate that signal transduction via Fc gamma RIII is mediated by Ctx-sensitive cellular membrane GTP-binding protein.
Divergent regulation of phospholipase C-alpha and phospholipase C-gamma transcripts during activation of a human T cell line.
Authors: Goldfien R D , Seaman W E , Hempel W M , Imboden J B
Published: December 2022

Ligand-induced activation of T cells results in stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC). A structurally diverse family of PI-PLC isoforms has recently been defined, and more than one isoform is frequently coexpressed in a single cell or tissue, suggesting that different forms may play distinct roles in cellular activation, proliferation, or differentiation. We show here that both PLC-alpha and PLC-gamma are expressed in rat splenic T cells and in Jurkat cells (a human T cell line). Activation of Jurkat cells with the combination of PMA and PHA leads to increased expression of PLC-alpha message and decreased expression of PLC-gamma message after 4 h of stimulation. The increase in PLC-alpha transcripts was detectable at 4 h, maximal at 6 h, and remained elevated for at least 24 h. The decrease in PLC-gamma message was transient, with a maximal effect at 4 h, and a return to basal levels by 6 h. Changes in PI-PLC transcripts were also induced by the combination of PMA and the calcium ionophore, ionomycin. These data demonstrate that the expression of transcripts for PLC-alpha and PLC-gamma can be differentially regulated during a cellular response, and raise the possibility that these two isoforms of PI-PLC subserve distinct functions in T cell activation.
Expression of phospholipase C isozymes by murine B lymphocytes.
Authors: Hempel W M , DeFranco A L
Published: December 2022

Cross-linking of membrane (m) Ig, the B cell receptor for Ag, activates protein tyrosine phosphorylation and hydrolysis of phosphotidylinositol 4,5-bisphosphate. The latter signal transduction pathway is an important mediator of antigen receptor engagement. The initial event in this pathway is the activation of phospholipase C (PLC). The identity of the isozyme of PLC used in B cells and the mechanism by which it becomes activated are currently unknown. The cDNA encoding five different isozymes have been cloned. As a first step in identifying the isozyme of PLC that is coupled to mIgM, murine cDNA fragments for the five cloned PLC isozymes were generated by the polymerase chain reaction (PCR), cloned, and used to screen a panel of B cell lines representing different stages of development for PLC mRNA expression. All the B cell lines tested expressed high levels of PLC alpha and PLC gamma 2 mRNA, whereas PLC beta and PLC delta mRNA expression were undetectable by both Northern blot and PCR analysis. PLC gamma 1 had a more complicated pattern of mRNA expression. PLC gamma 1 mRNA expression was lower than that observed for PLC alpha or PLC gamma 2 mRNA and varied widely among different cell lines. The pattern of PLC gamma 1 mRNA expression did not correlate with the developmental stage of the cell lines. The pattern of PLC gamma 1 protein expression in the panel of B cell lines correlated with the pattern of PLC gamma 1 mRNA expression. PLC gamma 1 expression was very low in several B cell lines, despite the fact that these cell lines show mIgM-stimulatable PLC activity. The variable and in some cases very low expression of PLC gamma 1 suggests that it may not be the form of PLC that is activated by mIgM. In contrast, PLC alpha and PLC gamma 2 were abundantly expressed in all B cell lines tested. This observation is consistent with the possibility that PLC alpha or PLC gamma 2 is activated by mIgM.
Cytochalasins enhance the proliferation of CD4 cells through the CD3-Ti antigen receptor complex or the CD2 molecule through an effect on early events of activation.
Authors: Matsuyama T , Yamada A , Deusch K , Sleasman J , Daley J F , Torimoto Y , Abe T
Published: December 2022

Cytochalasins are known to inhibit or enhance the proliferation of T cells induced by mitogens in a concentration-dependent fashion. To clarify the mechanism by which cytochalasins enhance T cell proliferation, we examined which activation pathways and events in signal transduction were affected by cytochalasins. We also examined subsets of CD4 cells for a preferential response to cytochalasins. Cytochalasins enhanced the proliferation of CD4 cells induced by optimal doses of anti-CD3 antibody or suboptimal doses of anti-CD2 antibodies. Cytochalasins, at low concentrations, enhanced the rise in intracellular Ca2+ and production of IP3 in CD4 cells activated by anti-CD2 or CD3 antibodies. Cytochalasins also enhanced the modulation of CD3 induced by anti-CD3 antibody. These results suggest that cytochalasins enhance the proliferation of CD4 cells by affecting early events in signal transduction after activation through the CD3-Ti Ag-receptor complex or CD2 molecule. At the doses used, cytochalasins appear to interact with cytochalasin-binding sites in the cell membrane. Cytochalasins predominantly enhanced CD3-mediated proliferation in the CD29-subset of CD4 cells.
Recognition of peptides that are immunopathogenic but cryptic. Mechanisms that allow lymphocytes sensitized against cryptic peptides to initiate pathogenic autoimmune processes.
Authors: Lipham W J , Redmond T M , Takahashi H , Berzofsky J A , Wiggert B , Chader G J , Gery I
Published: December 2022

Interphotoreceptor retinoid binding protein (IRBP) is a glycoprotein that localizes in the retina and induces inflammatory changes in this tissue in immunized animals. Certain IRBP-derived peptide determinants are also immunopathogenic, and we have previously shown that these determinants could be either immunodominant or cryptic. Lymphocytes sensitized against the cryptic peptides do not recognize whole IRBP in vitro, and yet these lymphocytes must recognize the protein in vivo to initiate the autoimmune pathogenic process. We have examined here two hypothetical explanations for this dissociation: 1) It is possible that when IRBP is processed in vitro, immunodominant peptide determinants compete with the cryptic ones and inhibit their interaction with the MHC molecules on the APC. This explanation was ruled out here by the finding that the immunodominant peptide 1179-1191 ("W10") did not inhibit the response to a cryptic one, 1158-1180 ("R4"), when added at equivalent and even moderately higher concentrations. 2) The second hypothesis proposes that the cryptic antigenic sites are not generated from IRBP by the APC in vitro, whereas enzymes in the retina digest the protein to yield fragments that generate these antigenic sites upon processing by the APC. In line with this hypothesis, we have found that cleavage of IRBP by certain endoproteinases (Asp-N, Glu-C, or V-8) produced molecules that were recognized in culture by lymphocytes sensitized to the immunopathogenic but cryptic peptide R4. This study, therefore, describes a putative Ag processing mechanism that results in IRBP recognition and, consequently, the initiation of an autoimmune process by lymphocytes sensitized against a cryptic peptide. Furthermore, experiments with R4 and other cryptic peptides have shown that cleavage fragments of up to 38 residues in length can be presented by APC, to stimulate lymphocytes sensitized against these peptides. No responses were stimulated, however, by fragments of 75 or more residues. The data thus provide new insights into the processing and presentation of cryptic peptide determinants by APC.
Release of the cytokines colony-stimulating factor-1, granulocyte-macrophage colony-stimulating factor, and IL-6 by cloned murine vascular smooth muscle cells.
Authors: Schrader J W , Moyer C , Ziltener H J , Reinisch C L
Published: December 2022

Vascular smooth muscle cells were cultured from the mesenteric arteries of MRL lpr/lpr, MRL +/+, CBA/J, or C3H/HeJ mice and evaluated for their ability to synthesize a range of cytokines. Vascular smooth muscle cells of MRL +/+, MRL lpr/lpr, and CBA/J origin released biologically significant amounts of CSF-1 and IL-6 and relatively low but detectable amounts of granulocyte macrophage-CSF (GM-CSF) but not IL-2, IL-3, or IL-4. Vascular smooth muscle cells of C3H/HeJ origin produced lower amounts of CSF-1 and IL-6, and GM-CSF was barely detectable. Production of these cytokines did not require the exogenous growth factors present in FCS and occurred, although at lower levels, in serum-free medium supplemented with insulin, transferrin, and albumin. Cloned lines of MRL +/+ vascular smooth muscle cells, with electron microscopic and immunochemical properties of vascular smooth muscle cells, produced CSF-1, IL-6, and GM-CSF, establishing that vascular smooth muscle cells were a direct source of CSF-1, IL-6, and GM-CSF. These observations highlight the need for experiments to directly address the question of whether vascular smooth muscle cells constitutively produce these cytokines under physiologic conditions in vivo and suggest that vascular smooth muscle cells may participate actively in inflammation by releasing cytokines that are active on lympho-hemopoietic and other cells.
Retinoic acid-induced growth inhibition of a human myeloma cell line via down-regulation of IL-6 receptors.
Authors: Sidell N , Taga T , Hirano T , Kishimoto T , Saxon A
Published: December 2022

In this report we demonstrate that retinoic acid (RA) down-regulated the number of IL-6R on human leukocyte cell lines, including the myeloma cell line AF10, and two B cell hybridomas that correspond to cells at earlier stages of B cell development. Using AF10 cells, whose growth was determined to be mediated by the autocrine action of IL-6, we found that RA reduction of IL-6R was concentration-dependent over a range of 10(-11) to 10(-5) M and corresponded to the ability of the retinoid to inhibit cell proliferation. The down-regulation of IL-6R number by RA was accompanied by reduced IL-6R mRNA expression. RA did not affect endogeneous IL-6 synthesis or secretion from AF10 cells. However, addition of exogenous rIL-6 could overcome RA-induced growth inhibition. Menthol, a structurally unrelated compound to RA, also suppressed IL-6R expression and, correspondingly, inhibited cell growth. Taken together, our results suggest that the antiproliferative action of RA on AF10 cells is caused by reduction of IL-6R expression and subsequent inhibition of IL-6-mediated autocrine growth. These findings suggest the possibility that down-regulation of IL-6R is a means by which RA can modulate immune function.
IL-8 gene expression and production in human peripheral blood lymphocyte subsets.
Authors: Smyth M J , Zachariae C O , Norihisa Y , Ortaldo J R , Hishinuma A , Matsushima K
Published: December 2022

We have investigated IL-8 mRNA expression and IL-8 production in highly purified subsets of peripheral blood lymphocytes. T cells stimulated with PHA, ionomycin, or PMA alone failed to express IL-8 mRNA. However T cells stimulated with a combination of PMA and ionomycin or PMA and PHA expressed IL-8 mRNA in a PMA dose-dependent manner and maximally after 3 to 6 h of culture. Induction of IL-8 mRNA appeared to be specifically in the CD4+ T cell subset. Surprisingly, however, T cells were not induced to secrete significant levels of IL-8 polypeptide, even in the presence of accessory monocytes. In addition, immunoprecipitation analysis of PMA/ionomycin-treated T cell lysates detected only minor levels of cellular IL-8 Ag thereby suggesting that in T cells, the production of IL-8 was inhibited at the posttranscriptional level. By contrast, CD3- large granular lymphocytes (LGL) were both induced to express IL-8 mRNA and secrete biologically active IL-8 upon specific stimulation with IL-2 and ligand (anti-CD16 mAb) for the NK cell receptor for IgG-Fc (CD16), or upon nonspecific stimulation with PMA. IL-2 and anti-CD16 mAb synergistically induced IL-8 expression in LGL. Other nonactivating LGL-specific mAb did not induce LGL IL-8 secretion. The amount of IL-8 produced by activated LGL was donor variable, but generally 5 to 10 times less than that secreted by monocytes. The ability of LGL to release IL-8 and a large number of other cytokines further supports the hypothesis that LGL may contribute to both inflammatory and immunologic responses.
Superinduction of IL-2 gene transcription in the presence of cycloheximide.
Authors: Zubiaga A M , Muñoz E , Huber B T
Published: December 2022

Lymphokine production is regulated both at the transcriptional and the posttranscriptional level. To date, it has been shown that the protein synthesis inhibitor cycloheximide (CHX) up-regulates IL-2 expression in T cells by stabilizing its mRNA. In this report we have examined the effect of CHX on IL-2 at the transcriptional level. We have found that CHX has a positive regulatory function in IL-2 transcription, which is dependent on prior activation of this gene. This is not due to posttranslational conversion of inactive NFkB into its active form by CHX, because a clustered mutation in the kB-like sequence in the IL-2 enhancer that abrogates NFkB binding does not affect the up-regulation of IL-2 transcription. These results favor the hypothesis that, in addition to positive factors, negative elements regulate IL-2 transcription. Furthermore, we have tested the effect of CHX on IL-4 and granulocyte-macrophage-CSF transcription of both lymphokines. These results suggest that transcriptional up-regulation by CHX may be specific for IL-2 with respect to lymphokine expression.
Upstream elements of the MHC class II E beta gene active in B cells.
Authors: Finn P W , Kara C J , Grusby M J , Folsom V , Glimcher L H
Published: December 2022

The MHC class II (Ia) molecules are heterodimeric cell surface glycoproteins that have a limited tissue distribution and are essential for an Ir. These proteins are known to both display a limited tissue distribution and be responsive to up-regulation by cytokines such as IL-4. To begin to identify the molecular basis for both of these phenomena in B cells, we performed a deletional analysis of the upstream region of the murine E beta gene. We show that there are several cooperating positive as well as negative control elements in the E beta upstream region. The sequence between -2679 and -66 is a strong transcriptional element containing several sequences that act synergistically as positive elements. The negative elements are located more proximal to the transcription start site. Furthermore, two consensus motifs, termed X and Y, which are located in the proximal upstream region of all class II genes, are necessary for the transcriptional activity of more distal elements. Additionally, we identify an IL-4-responsive element located within 666 bp upstream of the transcription initiation site.
Sequence of C region of L chains from Xenopus laevis Ig.
Authors: Zezza D J , Mikoryak C A , Schwager J , Steiner L A
Published: December 2022

A cDNA expression library, prepared from Xenopus laevis splenocytes, was screened with antibodies to Xenopus Ig. One clone, lambda XIg23, reacted with antibodies to IgY and to IgM; the insert hybridized to approximately 1.3-kb RNA from spleen, the approximate size expected for L chain mRNA. An additional clone, lambda XIg31, was identified by cross-hybridization. The inserts of lambda XIg23 and lambda XIg31 begin in the third framework region of the V region and extend through the C region to the poly(A) tail. Except for a single nucleotide difference, the two C region sequences are identical. The amino acid sequence of the C region was compared with the sequences of a variety of C kappa and C lambda, as well as to C region sequences of L chains from Rana catesbeiana and from two species of shark. The Xenopus C region resembles mouse and human C kappa slightly more than C lambda. The similarity of the Xenopus and Rana C regions to each other is approximately the same as that of either amphibian sequence to mammalian CL. The data are discussed in terms of the evolution of kappa and lambda C regions.
Germinal center B cells and antibody production in the bone marrow.
Authors: Dilosa R M , Maeda K , Masuda A , Szakal A K , Tew J G
Published: December 2022

In secondary antibody (Ab) responses, Ag processing and presentation occur in secondary lymphoid organs but most serum Ab is produced by cells in the bone marrow. Plasma cells in the bone marrow are derived from B cells activated by Ag in secondary lymphoid organs. We hypothesized that germinal center (GC) B cells, which acquire Ag from follicular dendritic cells in draining lymph nodes during the first few days of the secondary response, migrate to the bone marrow to terminally differentiate and produce specific Ab. To test this we looked for GC B cells in the thoracic duct lymph and in peripheral blood after secondary challenge using the peanut agglutininhi phenotype and blast cell morphology as markers for GC B cells. In addition, GC B cells were injected i.v. into naive recipients to determine if they would home to the bone marrow. Finally, to determine if the bone marrow environment supports maturation and Ab production by GC B cells, we cocultured GC B cells with bone marrow cells or bone marrow supernatants. The results indicate that blast cells bearing the GC B cell phenotype were present in both the thoracic duct and the peripheral blood 3 days after antigenic challenge. Day 3 peripheral blood cells secreted specific Ab, whereas cells isolated on day 0, 8, or 11 did not. Furthermore, in adoptive transfer experiments, only the day 3 GC B cells produced specific Ab and migrated to the bone marrow of naive mice. Finally, either bone marrow cells or factor(s) produced by bone marrow cells markedly enhanced Ab production by day 3 GC B cells. These data support the hypothesis that during the first few days after secondary challenge GC B cells seed the bone marrow and differentiate into plasma cells which produce the large quantities of Ab typical of secondary responses.
Identification of a novel human thymocyte subset with a phenotype of CD3- CD4+ CD8 alpha + beta-1. Possible progeny of the CD3- CD4- CD8- subset.
Authors: Hori T , Cupp J , Wrighton N , Lee F , Spits H
Published: December 2022

We investigated responsiveness to cytokines and differentiating potential of early human T cell precursors in vitro. Human CD3- CD4- CD8- (triple negative) thymocytes were highly purified by using magnetic bead columns and cell sorting. These cells proliferated for the first 3 to 4 days and then remained viable for up to 14 days in the presence of IL-7, IL-2 or IL-4 had only limited growth-promoting activity on these cells and could not maintain the cell viability. We followed the phenotypic change of triple negative thymocytes during culture with IL-7. After 7 to 14 days of culture with IL-7, a considerable proportion became CD4+ CD8+ (double positive). These cells were found to be CD3- CD4+ CD8 alpha+ beta- in contrast to common double positive thymocytes, which express low levels of CD3 and both alpha- and beta-chains of CD8. By using four-color immunofluorescence and multi-parameter cytofluorometric analysis, we could identify this novel subset in fresh thymocytes. These results suggest that the CD3- CD4+ CD8 alpha+ beta- subset exists physiologically in the human thymus and may represent an intermediate stage between triple negative and common double positive thymocytes.
Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice.
Authors: Cremer M A , Terato K , Seyer J M , Watson W C , O'Hagan G O , Townes A S , Kang A H
Published: December 2022

To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.
Clonal analysis of the peripheral T cell compartment of the SCID-hu mouse.
Authors: Vandekerckhove B A , Krowka J F , McCune J M , de Vries J E , Spits H , Roncarolo M G
Published: December 2022

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.
Modulation of IL-2- and IL-4-dependent human B cell proliferation by cyclic AMP.
Authors: Vazquez A , Auffredou M T , Galanaud P , Leca G
Published: December 2022

The second messenger cAMP is a modulator of cellular growth possessing both inhibitory and stimulatory properties. In this report, we show that IL-2- and IL-4-dependent DNA synthesis of anti-mu-activated human B cells is modulated in opposite ways by agents increasing intracellular levels of cAMP. Forskolin and 2'-O-dibutyriladenosine-3',5'-cyclic monophosphate had no proliferative effect by themselves. Nevertheless they decreased IL-2-driven proliferation and increased IL-4-mediated DNA synthesis. IL-4 and cAMP each inhibited the IL-2-dependent proliferation with similar patterns of reactivity. Both IL-4 and forskolin needed to be present during the first 48 h of culture to display inhibitory activity, and preactivation of B cells for 16 h with forskolin and IL-4 did not prevent further B cell response to IL-2. This suggests that cAMP and IL-4 directly interact with IL-2 signaling. In addition, we show that the cAMP-dependent protein kinase inhibitor N-(2-methylamino-ethyl)-5-iso-quinoline-sulfamide reversed the IL-4-inhibitory effect on IL-2-driven proliferation. Our data suggest that the IL-4-inhibitory signal to IL-2-driven human B cell proliferation involves cAMP-dependent protein kinase activation.
The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes.
Authors: Chonn A , Cullis P R , Devine D V
Published: December 2022

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.
Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides.
Authors: Zanetti M , Litteri L , Griffiths G , Gennaro R , Romeo D
Published: December 2022

The antimicrobial polypeptides Bac7 and Bac5 (bactenecins) are stored in the large granules of bovine neutrophils as precursor forms, or probactenecins. Maturation of probactenecins has been investigated by studying the effects of stimulus-induced degranulation on this process. Stimulation of neutrophils with PMA, which is a secretagogue for specific and large granules but not for azurophils, induces a substantial discharge of uncleaved probactenecins in the extracellular medium, as revealed by Western blot analysis. When neutrophils are exposed to opsonized bacteria in the presence of cytochalasin B, resulting in exocytosis of the content of azurophils in addition to that of specific and large granules, probactenecins are secreted and rapidly converted into the corresponding mature antimicrobial peptides. Such a conversion is prevented if serine proteases, stored in the azurophils, are inhibited by pretreatment of neutrophils with PMSF. Phagocytosis, while causing a rapid discharge of the contents of azurophil and of the large granules into phagocytic vacuoles, as indicated by immunogold electron microscopy, also induces cleavage of probactenecins into mature peptides, as revealed by Western blot analysis. We conclude that the final processing of the storage forms of bactenecins arises from their interaction with the serine protease(s) of azurophils during bacteria-induced degranulation of neutrophils.
Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL.
Authors: Binder D , Kündig T M
Published: December 2022

T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell-dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc-IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN-gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro.
Macrophage cytostatic effect on Trypanosoma musculi involves an L-arginine-dependent mechanism.
Authors: Vincendeau P , Daulouède S
Published: December 2022

Peritoneal macrophages from mice infected with an extracellular parasite, Trypanosoma musculi were effective in inhibiting parasite proliferation in vitro. This trypanostatic activity could be suppressed by NG monomethyl-L-arginine (NGMMA), a specific inhibitor of a biochemical pathway synthesizing L-citrulline and inorganic nitrogen oxides from L-arginine. Macrophages exerted this in vitro antiproliferative effect from the 10th day of infection on and this activity was maximum around 14th day of infection. Nitrite production paralleled development of macrophage trypanostatic activity. Macrophages collected from BCG-infected mice or treated with IFN-gamma in vitro also exerted a trypanostatic activity which was suppressed by NGMMA. A trypanostatic activity suppressed by NGMMA was also exerted by splenic macrophages from T. musculi-infected mice. Trypanostatic activity of IFN-gamma-treated macrophages was reduced by addition of anti-TNF-alpha showing the participation of TNF-alpha in IFN-gamma-mediated macrophage trypanostatic activity. Nitric oxide (NO) gas inhibited T. musculi proliferation. Addition of excess iron reversed the trypanostatic effect of both macrophages and NO gas. All these data showed that, as reported for a broad spectrum of microorganisms, activated macrophages displayed an antimicrobial effect on trypanosomes through the L-arginine: NO pathway that could participate in controlling infection in T. musculi-infected mice before appearance of antibody-dependent mechanisms. NO production by activated macrophages could trigger iron loss from critical target enzymes in trypanosomes.
Identification of enhancer sequences 3' of the rabbit Ig kappa L chain loci.
Authors: Hole N J , Harindranath N , Young-Cooper G O , Garcia R , Mage R G
Published: December 2022

The rabbit is useful for studies of Ig L chain gene expression because of a great disparity in expression of two isotypic forms of the kappa L chain. Normally, K1 is expressed at high levels and K2 is almost silent; expression of K2 increases in mutant or experimentally allotype-suppressed animals. The reasons for the preferential utilization of the K1 isotype have not been fully elucidated. We were interested in looking for second enhancers 3' of the C kappa genes because the absence of a 3' enhancer in the K2 locus could explain the preferential utilization of the K1 isotype. However, we found a strong region of enhancer activity about 7 kb downstream of the C kappa 2 gene. Sequences in this region are highly conserved between rabbit, man, and mouse. There also appears to be a homologous 3' enhancer region in the rabbit K1 locus. We also confirmed earlier reports that the rabbit K1 intron enhancer is inactive in transient transfections into mouse B cells but find that the same construct has low but significant activity in a human B cell line. In a comparable construct the K2 intron enhancer is without activity suggesting possible differential activity of the intronic enhancers.
Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56).
Authors: Lanier L L , Chang C , Azuma M , Ruitenberg J J , Hemperly J J , Phillips J H
Published: December 2022

The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N-CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell-mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets.
Weak base amines can inhibit class I MHC-restricted antigen presentation.
Authors: Michalek M T , Benacerraf B , Rock K L
Published: December 2022

This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.
The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.
Authors: Tohma S , Hirohata S , Lipsky P E
Published: December 2022

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.
T cell lines specific for an immunodominant epitope of human basic protein define an encephalitogenic determinant for experimental autoimmune encephalomyelitis-resistant LOU/M rats.
Authors: Hashim G , Vandenbark A A , Gold D P , Diamanduros T , Offner H
Published: December 2022

The LOU/M rat (RT-1w) haplotype, although resistant to an encephalitogenic challenge of guinea pig myelin basic protein (Gp-BP)/CFA and unresponsive to Gp-BP, responded strongly to human (Hu)-BP. Both T cell and antibody responses focused on the 110-129 determinant of Hu-BP, and T cells specific for this epitope transferred clinical and histologic experimental autoimmune encephalomyelitis (EAE) to naive LOU/M rats. Moreover, EAE could be induced actively with Hu-BP and a synthetic Hu-S110-129 peptide in CFA, but only with co-immunomodulation by pertussis toxin or cyclophosphamide. Analysis of TCR V region genes revealed the predominant use of the V beta 8.5-J beta 2.3 gene combination, with extensive N region additions to both D beta 1 and D beta 2. These results define the Hu-BP 110-129 peptide sequence as the major encephalitogenic epitope for the LOU/M strain of rat previously considered resistant to EAE, and support the idea that the encephalitogenic property of BP and other CNS Ag for a given MHC is encompassed within immunodominant T cell epitopes. Furthermore, the TCR sequence data indicate the predominant use of a different V beta 8 subfamily member (V beta 8.5) than the V beta 8.2 gene used preferentially by several other rat strains and the PL/J mouse in the T cell response to BP.
Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-1-dependent and -independent binding mechanisms.
Authors: Thornhill M H , Wellicome S M , Mahiouz D L , Lanchbury J S , Kyan-Aung U , Haskard D O
Published: December 2022

The adhesion of lymphocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for lymphocytes, cytokines may regulate lymphocyte accumulation and hence the nature and progression of inflammatory responses. IL-1, TNF, IFN-gamma, and IL-4 each increase EC adhesiveness for T cells when used alone in adhesion assays in vitro. As cytokines are more likely to act in combination at sites of inflammation in vivo, we have studied the stimulating effect of different combinations of cytokines on EC adhesiveness for T cells and polymorphonuclear leukocytes (PMN). Acting alone IL-1, TNF, IFN-gamma, and IL-4 each significantly enhanced EC adhesiveness for T cells (p less than 0.005), whereas only IL-1 (p less than 0.005) and TNF (p less than 0.005) but not IFN-gamma or IL-4 significantly enhanced adhesiveness for PMN. When EC were stimulated with optimal concentrations of TNF in combination with IL-4 or IFN-gamma, there was a significant further increase in adhesiveness for T cells (p less than 0.003), but not PMN, over that seen with TNF alone. The additive effect of TNF and IL-4 was more marked than that of TNF and IFN-gamma. Although approximately equal proportions of T cells and PMN bound to TNF-stimulated EC, nearly double the proportion of T cells compared with PMN bound EC preincubated with TNF and IL-4 together. A similar interaction with IL-4 or IFN-gamma was exhibited by lymphotoxin. mAb-inhibition studies indicated that the extra increase in binding caused by stimulating EC with TNF and IL-4 in combination was mediated by VCAM-1 whereas that caused by stimulating with TNF and IFN-gamma in combination was substantially mediated through leukocyte function-associated Ag-1- and VCAM-1-independent mechanisms. These observations suggest that whereas IL-1 and TNF alone are unselective in terms of leukocyte adhesion to EC, the combination of TNF (or LT) with IL-4 or IFN-gamma may be of key importance in determining the recruitment of a lymphocyte-predominant infiltrate in immune mediated inflammation, and in initiating the transition from acute to chronic inflammation.