bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunodominance in the graft-vs-host disease T cell response to minor histocompatibility antigens.
Authors: Korngold R , Wettstein P J
Published: December 2022

Immunodominance controls the generation of CTL in the C57BL/6By (B6) anti-BALB.B H-2b-matched strain combination. Despite the potential of responding to numerous individual minor histocompatibility (H) Ag on BALB.B APC, the focus of the CTL response is largely specific for only a limited number of target Ag. These minor H Ag could be distinguished by their differential expression on a panel of target cells from the CXB recombinant inbred strains, the E, G, I, J, and K (all H-2b), which express different composites of the original BALB minor H Ag. A hierarchy was observed in which first-order immunodominant Ag were present on both CXBK and CXBG cells, whereas second-order dominant Ag were found on CXBE, CXBJ, and CXBI cells. To test whether immunodominance also plays a role in the development of lethal graft-vs-host disease (GVHD) directed to multiple minor H Ag, B6 T cells were transplanted along with T cell depleted bone marrow, to irradiated (825 rad) recipients of either the BALB.B or CXB recombinant inbred strains. The results indicate that a hierarchy of immunodominance does exist in GVHD, but it differs from that predicted from the in vitro CTL studies. GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients. Presensitization of B6 donor mice to CXBG or CXBK splenocytes 3 wk before transplant did not significantly increase the overall GVHD potential in the corresponding CXBG or CXBK recipients. Evidence for second-order immunodominance was provided by the transfer of CXBE T cells and ATBM to irradiated CXBG and BALB.B recipients with resultant, potent GVHD.
Cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation.
Authors: Percopo C M , Hooks J J , Shinohara T , Caspi R , Detrick B
Published: December 2022

The retinal pigment epithelial (RPE) cell has long been considered an important regulatory cell, maintaining physiological and structural balance within the retina. We have previously shown that the RPE cell may also be important in autoimmunity and transplantation. These cells can be induced by cytokines to express MHC class II Ag in ocular inflammatory and autoimmune conditions. In this report we show that isolated rat RPE cells can be induced to express class II Ag following incubation with rat rIFN-gamma. The ability of RPE cells to present Ag was determined by both T cell proliferation assays and IL-2 production. Only the Ia-positive RPE cells can present retinal Ag (S-Ag and interphotoreceptor-binding protein) to specifically sensitized rat Th cells. Moreover, the ability of chloroquine to inhibit this activity suggests that the RPE cell is also capable of processing Ag prior to Ag presentation. These studies indicate that cytokine-mediated activation of RPE cells may be a basic component of ocular immunity and an important aspect of RPE cell transplantation.
Analysis of IL-1 and TNF-alpha gene expression in human rheumatoid synoviocytes and normal monocytes by in situ hybridization.
Authors: MacNaul K L , Hutchinson N I , Parsons J N , Bayne E K , Tocci M J
Published: December 2022

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.
cAMP mediates IL-1-induced lymphocyte penetration through endothelial monolayers.
Authors: Turunen J P , Mattila P , Renkonen R
Published: December 2022

Endothelial cell incubated with IL-1 have been shown adhere more lymphocytes than nontreated endothelial cells. Here we demonstrate that IL-1 can also increase lymphocyte penetration through endothelial monolayers in vitro. IL-1 induced a transient increase in the number of lymphocytes penetrated through the endothelial monolayer into a filter in a time- and dose-dependent manner. This effect could be mimicked by increasing the cytosolic cAMP levels in the endothelial cells either by forskolin or dibutyryl-cAMP. Concomitantly we were able to show that IL-1 increased the cytosolic cAMP levels in endothelial cells. An inhibitor of adenylate cyclase, ddAdo, decreased both the IL-1-induced cAMP elevation and lymphocyte penetration. A protein kinase A inhibitor HA 1004 could inhibit the IL-1-induced lymphocyte penetration, where as protein kinase C (N-(2-guamidino-ethyl)-5-isoquinolinesyl foamide hydrocloride) and calcium-calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalensulfanamide) inhibitors had no effect. Adding dibutyryl-cGMP or calcium ionophore to the endothelial cells could not mimic IL-1-induced penetration and finally IL-1 did not induce PKC translocation in endothelial cells. These data support the view that IL-1 acts via cAMP as a second messenger in regard to lymphocyte penetration through endothelial cells. The above data demonstrate that IL-1-induced lymphocyte penetration through endothelial cells and that this IL-1-induced signal is transduced via cAMP in endothelial cells.
Sphingosine potentiates IL-1-mediated prostaglandin E2 production in human fibroblasts.
Authors: Ballou L R , Barker S C , Postlethwaite A E , Kang A H
Published: December 2022

IL-1 stimulates PGE2 production in human fibroblasts by stimulating arachidonic acid (AA) mobilization and cyclooxygenase synthesis. Cyclooxygenase is the first enzyme in the pathway that converts AA to PGE2. To examine the role of protein kinase C (PKC) in IL-1-mediated PGE2 production, we treated cells with PMA, which stimulated PGE2 production suggesting a positive role for PKC activation in the regulation of PGE2 synthesis. Therefore, we tested the effect of sphingosine, a PKC inhibitor, on IL-1-induced PGE2 production. Alone, sphingosine had little effect on PGE2 production. However, when sphingosine was added with IL-1, or IL-1 was added to sphingosine-pretreated cells, PGE2 production increased severalfold, suggesting that the inhibition of PKC results in enhanced IL-1-mediated PGE2 production; structural analogs of sphingosine did not potentiate the IL-1 effect. In cells made deficient in PKC by prolonged exposure to PMA, IL-1-mediated PGE2 production was enhanced compared with normal cells, further suggesting that functional PKC is not required for, and may down-modulate, IL-1-mediated PGE2 production. These findings also suggest that PMA and IL-1 stimulate PGE2 synthesis via fundamentally different pathways. In separate studies on the effect of IL-1 on AA mobilization, we found that IL-1 induced an increase in phospholipase A2 (PLA2) activity and that cycloheximide blocked the increase, suggesting the requirement for new protein synthesis. We also found that the PLA2 activity increased as a result of IL-1 exposure was further stimulated by sphingosine. Thus, in addition to its primary effects on the cell, which are likely mediated via PKC, we present evidence suggesting that sphingosine may also play a role in potentiating an IL-1-induced PLA2 activity, resulting in increased availability of AA for conversion to PGE2.
Rapid increase of the human IFN-gamma receptor phosphorylation in response to human IFN-gamma and phorbol myristate acetate. Involvement of different serine/threonine kinases.
Authors: Mao C , Merlin G , Ballotti R , Metzler M , Aguet M
Published: December 2022

Various cell surface receptors are phosphorylated upon binding of their ligand, and this phosphorylation seems to be involved in the signal transduction or in the feedback regulation of this signal. The possibility of a phosphorylation of the human IFN-gamma receptor (hu-IFN-gamma-R) has been investigated with 32P-labeled whole Raji cells and receptor purification either by immunoprecipitation with an anti-hu-IFN-gamma-R polyclonal antiserum or by affinity chromatography. The hu-IFN-gamma-R was found to be phosphorylated at a basal level. Upon incubation of the cells with recombinant hu-IFN-gamma, a dose-dependent two-fold increase of this phosphorylation was observed. Phosphoamino acid analysis by TLC showed that the same amino acids, serine and threonine, are phosphorylated at a basal level and after incubation with hu-IFN-gamma. Protein kinase C and Ca2+/calmodulin-dependent kinase pathways have been reported in some cases to be involved in the signal transduction pathway of hu-IFN-gamma. Both pathways involved the activation of a serine/threonine kinase and therefore we have investigated the possibility of hu-IFN-gamma-R phosphorylation by these kinases. PMA, an activator of protein kinase C, induced a rapid increase of the receptor phosphorylation in Raji cells, whereas the Ca2+ ionophore A23187 did not. PMA-induced hu-IFN-gamma-R phosphorylation was not associated with any effect on expression or inactivation of the receptor. PMA alone did not mimic the hu-IFN-gamma effect in Raji cells as measured by induction of IP-10 gene expression, a high specific marker of hu-IFN-gamma response. But the protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine, reduced this IFN-gamma-induced expression. However, H7 and staurosporine treatment as well as protein kinase C depletion suppressed PMA-induced receptor phosphorylation, whereas constitutive and hu-IFN-gamma-induced phosphorylation remained unchanged. Our results suggest that the serine/threonine kinase involved in the hu-IFN-gamma-R phosphorylation induced by IFN-gamma is different from protein kinase C.
Modulation of IgM secretion and H chain mRNA expression in CH12.LX.C4.5F5 B cells by adrenocorticotropic hormone.
Authors: Bost K L , Clarke B L , Xu J C , Kiyono H , McGhee J R , Pascual D
Published: December 2022

The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP.
Differences in transcriptional enhancers of HIV-1 and HIV-2. Response to T cell activation signals.
Authors: Tong-Starksen S E , Welsh T M , Peterlin B M
Published: December 2022

T cell activation results in high levels of HIV replication and is thought to be one mechanism leading to the conversion from latent to active viral infection. In HIV-1, the sequences that respond to these signaling events are found in the long terminal repeat (LTR) and comprise the transcriptional enhancer, which contains two conserved binding sites for the nuclear factor kappa B (NF kappa B). The corresponding region in the second AIDS retrovirus, HIV-2, contains a conserved and a divergent NF kappa B binding site. We demonstrate that the HIV-1 LTR responds better than the HIV-2 LTR to T cell activation signals. These qualitative differences in the response to T cell activation are reproduced not only when HIV-1 or HIV-2 enhancers are placed upstream of a heterologous promoter but also when these enhancers are switched between their respective LTR. In electrophoretic mobility shift assays, NF kappa B binds to both conserved sites in the HIV-1 transcriptional enhancer and only to the single conserved site in the HIV-2 transcriptional enhancer. Instead of NF kappa B, the activator protein 3 binds to the divergent site in HIV-2. In conclusion, HIV-1 and HIV-2 are differentially regulated by T cell activation signals, and this difference may account for the longer period of viral latency observed with HIV-2 than with HIV-1 infection.
Heterogeneity in direct cytotoxic function of L3T4 T cells. TH1 clones express higher cytotoxic activity to antigen-presenting cells than TH2 clones.
Authors: Chang J C , Zhang L , Edgerton T L , Kaplan A M
Published: December 2022

In the process of generating culture supernatant from T cell clones, with anti-CD3 antibodies and the B lymphoma A20 as APC, a striking difference in the stimulation of TH1 and TH2 clones was observed, i.e., TH2 clones produced higher levels of lymphokines than TH1 clones. This prompted us to test the hypothesis that differential killing of APC (thus the removal of stimuli) by T cells led to differential T cell activation. By studying a panel of five TH1 and seven TH2 clones, it was demonstrated that TH1 clones mediated significantly higher levels of cytotoxicity toward A20 cells in the presence of soluble anti-CD3 antibody (as opposed to immobilized anti-CD3). Although T cell clones could, when activated with immobilized anti-CD3, produce lymphokines cytotoxic to A20 cells, experiments in which lymphokine production was blocked indicated that T cell clones, in the presence of soluble anti-CD3, mediated killing of A20 through direct cytotoxicity. A higher level of cytotoxicity, by TH1 compared with TH2 clones, was not restricted to anti-CD3 or a particular target cell type, because it also occurred with Con A- or Ag-dependent killing (a monocyte-macrophage cell line), and LPS blasts. Furthermore, the higher cytotoxic activity of TH1 clones compared with TH2 clones was independent of the stage of T cell activation and was unlikely a result of the length of in vitro culture. High levels of killing of APC led to low levels of T cell activation, the significance of which may be as a negative feedback mechanism in the immune response. Other biologic relevancies of higher cytotoxic activity in TH1 vs TH2 cells were also discussed.
Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor.
Authors: Tan P , Anasetti C , Martin P J , Hansen J A
Published: December 2022

To determine IL-2 requirement for activation of suppressor cells, PBMC were primed in one-way MLR in the presence of 10 micrograms/ml anti-IL-2R beta-chain antibody 2A3 (CD25) or control antibody, then irradiated and added as regulators in a fresh MLR. Cells primed in the presence of antibody 2A3 suppressed the proliferative response to fresh autologous lymphocytes to specific alloantigen but had no effect on the response to cells from third party donors. Priming in the presence of an antibody of irrelevant specificity induced only limited suppressor activity. Activated suppressor cells did not show cytolytic activity specific for the stimulators when tested at the time of the suppressor cell assay. To identify the subset(s) responsible for suppression, cells primed in the presence of antibody 2A3 were separated into CD4+/CD45RA+, CD4+/CD45RA-, and CD8+ subsets, which were irradiated and then tested. The suppressive activity was found predominantly in the CD4+/CD45RA+ subset, whereas CD8+ cells had some activity and CD4+/CD45RA- cells had none. No subset suppressed the response of autologous cells to third-party cells. When primed CD4+/CD45RA+ cells were cocultured with fresh autologous lymphocytes depleted of CD8+ cells, no suppression was observed, indicating that, although the CD4+/CD45RA+ cells can function as inducers of suppressors, they cannot function as suppressor-effectors. Conversely, CD8+ cells activated in MLR in the presence of 2A3 caused suppression, regardless of whether the fresh autologous responder population contained CD8+ cells. CD4+/CD45RA+ and CD8+ subsets isolated after priming in the presence of 2A3 also demonstrated Ag-specific suppression in the generation of cytotoxic T lymphocytes whereas CD4+/CD45RA- cells had no activity. Our data are consistent with the model that suppression of alloreactivity requires the cooperation of two types of cells, a CD4+/CD45RA+ suppressor-inducer and a CD8+ suppressor-effector population. Activated Tsi and fresh Tse or activated Tse alone can suppress lymphocyte proliferation and generation of CTL in response to specific Ag. Activation of Ag-specific T suppressor-inducer and T suppressor-effector cells appears to be relatively IL-2 independent and presumably require one or more other growth factors.
Bone marrow-derived cells are essential for intrathymic deletion of self-reactive T cells in both the host- and donor-derived thymocytes of fully allogeneic bone marrow chimeras.
Authors: Yoshikai Y , Ogimoto M , Matsuzaki G , Nomoto K
Published: December 2022

The fate of self-reactive T cells was examined in both the host- and donor-derived thymocytes of fully allogeneic bone marrow (BM) chimeras of two strain combinations of AKR/J (H-2k, IE+, Thy-1.1, Mls-1a2b) and C57BL/6 (H-2b, IE-, Thy-1.2, Mls-1b2b). Sequential appearance of host- and donor-derived T cells occurred in the thymus of both AKR----B6 and B6----AKR chimeras in which 5 x 10(6) of T cell-depleted BM cells were used to reconstitute recipients lethally irradiated with 950 rad. Thymocytes bearing V beta 6 high, which recognize MHC class II IE-binding Ag encoded by Mls-1a allele, were detected in neither host- nor donor-derived thymocytes of AKR-B6 chimeras in which Mls-1a and IE were expressed only by the BM-derived cells. Thymocytes bearing V beta 11high capable of recognizing IE were also deleted in the host- and donor-derived thymocytes of the AKR----B6 chimeras. One million of BM cells were inadequate to deletion of the B beta 6high and V beta 11high T cells in the host-derived thymocytes of these chimeras. On the other hand, significant number of V beta 6high and V beta 11high thymocytes were detected in both the host- and donor-derived thymocytes in B6----AKR chimeras where sufficient dose of IE- stem cells were used to reconstitute irradiated Mls-1aIE+ recipients. These results suggest that clonal deletion of the host- and donor-reactive T cells in both the host- and donor-derived thymocytes is an important mechanism for the induction of transplantation tolerance in allogeneic BM chimeras and that BM-derived APC may be essential for the intrathymic elimination of both the host- and donor-reactive T cells in the BM chimeras.
Differential regulation of IL-1 production in human monocytes by IFN-gamma and IL-4.
Authors: Donnelly R P , Fenton M J , Finbloom D S , Gerrard T L
Published: December 2022

We have demonstrated that IL-4 markedly inhibits IL-1 production by highly purified normal human monocytes. When added to monocyte cultures, IL-4 suppressed LPS-induced IL-1 production in a time- and dose-dependent manner. Concentrations of IL-4 as low as 100 pg/ml reduced IL-1 production by approximately 50%, and doses of 1 ng/ml or higher suppressed IL-1 production by more than 90%. Maximal inhibition required that IL-4 be added before or simultaneous with LPS. Northern dot blot analyses revealed that IL-4 not only dramatically reduced the steady-state IL-1 beta mRNA levels in LPS-stimulated monocytes, but also those of TNF-alpha and IL-6. The inhibitory effect was not stimulus-specific because IL-4 suppressed IL-1 production induced by a variety of monocyte activation stimuli, including LPS, PMA, and Staphylococcus aureus Cowan strain. Monocytes expressed a relatively small number of high affinity IL-4R (approximately 150/cell; Ka = 3.15 +/- 1.13 x 10(10) M-1) indicating that relatively few receptors are necessary to generate the inhibitory effect. IL-4 enhanced monocyte MHC class II Ag (HLA-DR) expression in a manner similar to that of IFN-gamma. However, although both IFN-gamma and IL-4 up-regulated HLA-DR expression, they exhibited opposite effects on IL-1 production: IFN-gamma significantly enhanced monocyte IL-1 production induced by submaximal concentrations of LPS; whereas, IL-4 suppressed IL-1 production. Moreover, IL-4 largely neutralized the potentiating effect of IFN-gamma suggesting that IL-4 may be an effective antagonist of certain IFN-gamma-induced effects. Together these findings demonstrate that the relative levels of IFN-gamma and IL-4 may profoundly influence the state of monocyte activation by differentially regulating the expression of IL-1.
Cytokine regulation of C3 and C5 production by the human type II pneumocyte cell line, A549.
Authors: Rothman B L , Despins A W , Kreutzer D L
Published: December 2022

A growing body of literature suggests that a variety of cell products (e.g., cytokines, C components, etc.) likely play an important role during inflammation and host defense by locally regulating the diverse functions of recruited (i.e., immunologic cells) as well as tissue cells. Previously, a number of investigations have demonstrated the ability of immunologic cells to produce C components in vitro, and further studies have identified a variety of cytokines that can regulate C component production by these cells. Recently, we have demonstrated the ability of lung tissue cells, including epithelial cells and fibroblasts, to synthesize and secrete numerous C components and complement regulatory proteins in vitro. Additionally, we have demonstrated that C component production can be modulated by a variety of factors including endotoxin and serum. In our studies we investigated the effects of specific cytokines, i.e., IL and IFN, on the production of the third (C3) and fifth (C5) C components by the continuous cell line of human type II pneumocytes (A549). Specifically, using sensitive ELISA we demonstrated that A549 pneumocytes exposed to IL-1 alpha, IL-1 beta, or IL-2 induced a dose-dependent, more than twofold, increase in C3 production and a 50% decrease in C5 production when compared to control (untreated) A549 cells. Interestingly, IFN-alpha significantly decreased both C3 and C5 production, i.e., 38 and 71%, respectively, in a dose-dependent manner. IFN-gamma had no effect on C3 production, but significantly decreased C5 production by A549 pneumocytes by 84%. These data not only demonstrate that cytokines have the capability to modulate C3 and C5 production by human type II pneumocytes in vitro, but that C3 and C5 production by these cells can be regulated independently by different cytokines. In vivo, cytokine modulation of C component production by local tissue cells likely plays an important role in the regulation of inflammation and host defense within the lung.
Natural killer (NK) cell subsets in the mouse. NK-1.1+/LGL-1+ cells restricted to lysing NK targets, whereas NK-1.1+/LGL-1- cells generate lymphokine-activated killer cells.
Authors: Mason L H , Mathieson B J , Ortaldo J R
Published: December 2022

Our laboratory has recently identified a novel Ag, LGL-1, that is expressed on a major population of mouse NK cells. Two color immunofluorescence analysis has demonstrated that spleen cells consist of two major subsets of NK cells. We have identified an NK-1.1+/LGL-1+ subset that consists of 50% of the total NK cells and an NK-1.1+/LGL-1- subset comprising the remaining 50%. Because numerous reports have identified NK cells as the major cell type mediating lymphokine-activated killing (LAK), the NK-1.1+/LGL-1+ and NK-1.1+/LGL-1- subsets were examined for their contribution toward LAK generation, as defined by their ability to lyse P815 tumor targets. Antibody plus C depletion experiments with the use of anti-LGL-1 indicated that LGL-1+ cells were not found on LAK precursor or effector cells. Two-color cell sorting experiments were also performed to separate freshly isolated NK-1.1+/LGL-1+ spleen cells from the NK-1.1+/LGL-1- subset. It was found that the vast majority of LAK activity (greater than 95%) is derived from the NK-1.1+/LGL-1- cells. Cell sorting of LAK effectors also demonstrated that the NK-1.1+/LGL-1- cells mediated the vast majority of lysis against P815 targets. Similar results were obtained when NK cell subsets were analyzed for their contribution toward ADCC. These findings may prove important in understanding and further elucidating the contribution of NK cells to the LAK phenomenon. Our data also indicates that subsets of NK cells exist that may function differently in response to stimulation by various lymphokines and cytokines.
Bovine natural killer-like cell responses against cell lines expressing recombinant bovine herpesvirus type 1 glycoproteins.
Authors: Palmer L D , Leary T P , Wilson D M , Splitter G A
Published: December 2022

The murine retrovirus shuttle vector pZipNeo SV(X)1 was used to construct plasmids encoding the three major surface glycoproteins of bovine herpesvirus-1 (BHV-1). Each plasmid was transfected into D17, a canine osteosarcoma cell line sensitive to lysis by bovine NK-like cells when infected with BHV-1. After selection in G418 sulfate, cell lines expressing the recombinant gene products were sorted by flow microfluorimetry, radioimmunoprecipitated, and analyzed by SDS-PAGE for fidelity as compared to the native viral glycoproteins. Two of the three genetically engineered cell lines (gI and gIV) could successfully serve as targets to detect bovine NK-like cytolysis. These findings support and extend a previous study from our laboratory indicating a role for BHV-1 glycoproteins in the cytolytic response by bovine NK-like cells. Additionally, this study demonstrated that individual proteins are recognized by these effector cells, and that recombinant glycoproteins can direct cytolytic activity in the absence of host cell infection-associated proteins. This is the first known report of Ag directed cytotoxicity by bovine null (non-B, non-T) cells.
IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma.
Authors: Thornhill M H , Haskard D O
Published: December 2022

Alteration in the surface membrane of endothelial cells (EC) is a feature of endothelial activation both at sites of inflammation in vivo and after stimulation with cytokines in vitro. The effects of stimulating EC with IL-1 or TNF include enhanced adhesiveness for polymorphonuclear leukocytes (PMN) and T cells, the induction of EC leukocyte adhesion molecule-1 (ELAM-1) expression, and the increased expression of intercellular adhesion molecule-1 (ICAM-1) and the 1.4C3 Ag. In contrast, IFN-gamma stimulation increases EC binding of T cells but not PMN and enhances ICAM-1 expression but not ELAM-1 or 1.4C3 Ag expression. Recently we have reported that the T cell-derived cytokine IL-4 also increases EC adhesiveness for T cells but not PMN. In this study we have examined the effect of IL-4 on the expression of several cytokine-inducible EC activation Ag, by using a previously described ELISA technique. IL-4 modulation of activation Ag expression was concentration dependent, optimal at around 100 U/ml, and exhibited a unique pattern compared to that seen with the other cytokines. Although, IL-4 stimulation increased 1.4C3 Ag expression (p less than 0.001), it significantly inhibited constitutive ICAM-1 expression (p less than 0.01) and did not induce ELAM-1. Furthermore, IL-4 exhibited significant synergy with IL-1 or TNF in inducing 1.4C3 Ag expression (p less than 0.001) but inhibited the increased expression of ICAM-1 produced by IL-1, TNF, or IFN-gamma (p less than 0.01) and inhibited the induction of ELAM-1 by IL-1 and TNF (p less than 0.001). In contrast, IL-4 had no effect on the expression of EC HLA-class I, -DR, -DP, or -DQ and neither enhanced nor inhibited the effect of IFN-gamma on the expression of these molecules. Finally, although IL-4 alone caused little if any shape change in EC monolayers, it strongly synergized with TNF or IFN-gamma in causing a change in shape to a more fibroblastic morphology. These observations indicate that IL-4 increases EC adhesiveness for T cells by the induction of a different adhesion molecule to ICAM-1. Furthermore, the ability of IL-4 to both enhance and inhibit the expression of activation Ag on EC already activated by IL-1, TNF, or IFN-gamma suggests that it may be important in altering the quality of inflammatory responses such as may occur during the development and maintenance of chronic or immune-mediated inflammation.
CD38 molecule: structural and biochemical analysis on human T lymphocytes, thymocytes, and plasma cells.
Authors: Alessio M , Roggero S , Funaro A , De Monte L B , Peruzzi L , Geuna M , Malavasi F
Published: December 2022

The structure of the CD38 molecule has been evaluated by one- and two-dimensional gel analysis and by enzymatic digestions. The source of the Ag was mainly membrane preparations obtained from MLC cells, from normal thymocytes, and from the plasmocytoma line LP-1. Membranes were solubilized in NP-40 and the extracts fractionated by immunoaffinity chromatography [using a specific anti-CD38 antibody (A10 mAb) covalently linked to Sepharose protein A]. The purified Ag migrated as a single chain of Mr = 45,000 not associated with beta 2-microglobulin. Two-dimensional IEF gel electrophoresis revealed five spots (isoelectric point (pI) range: 6.5 to 6.9). After neuraminidase treatment, the mobility of the five polypeptides shifted to a more basic pI. Endoglycosidase-H treatment reduced the Mr of CD38 by 20%, revealing a broader band centered at Mr = 36,000. Treatment of CD38 molecule with V8 Staphylococcus aureus protease yielded a single dominant band at Mr = 38,000 which was still reactive with A10 mAb. The CD38 molecular was trypsin-resistant in both denatured or native conditions. These results clearly show the glycoprotein nature of CD38 molecule, which includes 2 to 4 N-linked oligosaccharide chains containing sialic acid residues. Furthermore, the present data indicate that the CD38 molecule does not display an apparent biochemical polymorphism among the different CD38+ cells or lines.
Role of recurrent hydrophobic residues in catalysis of helix formation by T cell-presented peptides in the presence of lipid vesicles.
Authors: Lu S , Reyes V E , Lew R A , Anderson J , Mole J , Humphreys R E , Ciardelli T
Published: December 2022

We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.
DF3 antigen, a human epithelial cell mucin, inhibits adhesion of eosinophils to antibody-coated targets.
Authors: Hayes D F , Silberstein D S , Rodrique S W , Kufe D W
Published: December 2022

Although mucins provide lubrication and physical protection for epithelial cell surfaces, other functional roles for these large glycoproteins are unknown. One human mucin, designated DF3 Ag, is detectable on apical surfaces of normal epithelial secretory cells and in normal milk, urine, and plasma. The present studies have examined the effects of DF3 Ag purified from both normal and malignant sources on the antibody-dependent cytotoxicity of Schistosoma mansoni by eosinophils (ADCC-E) and on the adherence of eosinophils to inert antibody-coated targets. DF3 Ag purified from tissue culture supernatant of a human breast carcinoma cell line or from human milk inhibited ADCC-E in a concentration-dependent manner, with half-maximal inhibitory activity at 3 to 10 micrograms/ml. Inhibition of ADCC-E was specific for the DF3 mucin, because no inhibition was observed with two other unrelated, circulating glycoproteins: carcinoembryonic Ag and alpha 1-acid glycoprotein. Inhibition was not a result of direct cytotoxicity of the DF3 Ag for eosinophils, as demonstrated by the lack of detectable effect of the mucin on cellular trypan blue exclusion or PMA-induced H2O2 release. The inhibitory effect was time dependent, requiring the presence of DF3 Ag in the ADCC-E culture for at least 4 h, beginning within the first 2 h of eosinophil-schistosomula interaction. Furthermore, inhibition was not a result of interaction between DF3 Ag and the activating lymphokine. These data suggest that inhibition of ADCC-E by DF3 Ag is a result of interference of adhesion of eosinophils to Ig-coated targets. In this regard, purified DF3 tumor Ag prevents eosinophil adherence to human Ig-conjugated Sepharose 4B beads. Preincubation of the inert Ig-coated targets with DF3 Ag did not inhibit subsequent eosinophil adherence, suggesting that DF3 Ag interacts with a moiety present on the eosinophil. Inhibition of adhesion occurred at 37 degrees C, but was also observed at 4 degrees C. These results suggest that DF3 Ag acts as an immunomodulating agent. Because activated eosinophils can damage surrounding normal tissues as well as infectious organisms, DF3 Ag may serve to protect secretory epithelium from the cytotoxic effects of activated inflammatory cells.
Expression and function of a 90-kilodalton homodimeric molecule (10D1 antigen) on human thymocytes.
Authors: Kobata T , Yagita H , Matsuda H , Seki M , Ota Y , Katagiri M , Okumura K
Published: December 2022

The 10D1 Ag is a 90-kDa homodimeric molecule specifically expressed on a subpopulation of human T cells, and is involved in an alternative pathway of T cell activation. In the present study, we have examined the expression and function of the 10D1 Ag on human thymocytes. Three-color FMF analysis showed that the 10D1 Ag was highly expressed on minor but distinct subpopulations of double-negative and CD4 single-positive thymocytes, and weakly on a part of double-positive thymocytes, but not on CD8 single-positive thymocytes. In double-negative thymocytes, the vast majority of 10D1+ cells were immature thymocytes of CD7+2+3- phenotype. Interestingly, 10D1 mAb could induce the proliferation of CD4 single-positive thymocytes in the presence of goat anti-mouse Ig to cross-link the 10D1 Ag. The treatment of thymocytes with OKT4 mAb plus C but not with OKT8 mAb plus C totally abrogated the proliferative response induced by 10D1 mAb, indicating that the 10D1-responsible thymocytes were of CD4+8- phenotype. This 10D1 mAb-induced thymocyte proliferation was perfectly dependent on the endogenous IL-2/IL-2R system since a complete inhibition was observed with anti-IL-2 and anti-IL-2R mAb. The proliferating CD4 single positive thymocytes predominantly expressed the IL-2R alpha (p55) but not a detectable level of the IL-2R beta (p75). These results indicate that, although the 10D1 Ag can be detected on the CD7+2+3-4-8- thymocytes, its functional expression is restricted to a minor more mature CD4+ thymocyte population as well as in peripheral blood T cells, and the implications of these findings are discussed.
Capsaicin-sensitive primary sensory neurons are potent modulators of murine delayed-type hypersensitivity reactions.
Authors: Girolomoni G , Tigelaar R E
Published: December 2022

Capsaicin, a neurotoxin that depletes primary sensory neurons (polymodal nociceptors) of neuropeptides, was used to explore the role of such neurons on the expression of delayed-type hypersensitivity reactions in mice. BALB/c mice received s.c. injections with capsaicin (100 mg/kg) and tested 1 to 2 wk later exhibited insensitivity to chemically induced irritation (greater than 80% reduction in the eye-wiping response for more than 15 wk) as well as loss (greater than 95% reduction) of the ear swelling response to topical capsaicin. Early (less than or equal to 4 h) ear swelling to topical DNFB and oxazolone was also markedly reduced by capsaicin pretreatment, suggesting neurogenic inflammation as a major component of the early irritant reaction to haptens. In contrast, capsaicin-pretreated mice exhibited enhanced contact sensitivity (CS) reactions to oxazolone (greater than 90%) and DNFB (greater than 50%) and enhanced delayed-type hypersensitivity reactions to SRBC (greater than 20%). Adoptive transfer experiments revealed that CS augmentation was not due to generation of increased numbers and/or activity of effector T cells. Histologic studies as well as experiments measuring migration of 51Cr-labeled, Ag-nonspecific cells showed increased edema and enhanced cell localization in CS elicitation sites in capsaicin-pretreated mice. These results indicate that peptidergic neurons, via neuropeptide release, regulate the expression of T cell-mediated, delayed-in-time, cutaneous inflammatory reactions. The net effect of these neurons on the late (cellular) phase of such responses seems to be suppressive, because their impairment results in augmented reactions.
Neurogenic inflammation, vascular permeability, and mast cells. II. Additional evidence indicating that mast cells are not involved in neurogenic inflammation.
Authors: Kowalski M L , Sliwinska-Kowalska M , Kaliner M A
Published: December 2022

Activation of cutaneous sensory nerves induces vasodilatation and vascular permeability, i.e., neurogenic inflammation. We examined the histology and possible mast cell involvement in cutaneous neurogenic inflammation induced by electrical nerve stimulation (ENS). Three lines of evidence indicated that mast cells were not involved in rodent cutaneous neurogenic inflammation induced by electrical stimulation of the saphenous nerve. 1) Most mast cells (86.5% of all mast cells in the dorsal skin of the paw) were found in the deep dermis, whereas vessels developing increased vascular permeability after nerve stimulation (visualized with the supravital dye Monastral blue B, a macro-molecular tracer) were localized predominantly in the superficial dermis. By contrast, i.v. substance P, which also causes increased cutaneous vascular permeability, predominantly caused deeper vessels to leak. As analyzed by electron microscopy, the vessels that developed permeability in response to nerve stimulation, and were thereby stained with Monastral blue B, were found to be exclusively postcapillary venules. 2) Disodium cromoglycate (DSCG), a mast cell stabilizing compound, inhibited the cutaneous vascular permeability induced by intradermal injections of anti-IgE in a dose-dependent manner. By contrast, vascular permeability induced by ENS was not influenced by disodium cromoglycate treatment. 3) ENS and i.v. substance P both induced cutaneous vascular permeability in mast cell-deficient W/Wv mice, despite the fact that their skin contained only 4.7% of the mast cells present in their normal +/+ litter mates. The magnitude of ENS-induced vascular permeability responses in W/Wv mice were similar to control +/+ and BALB/c mice. This study supports our earlier observations suggesting that mast cell activation is not essential for the initial, vascular permeability phase of neurogenic inflammation in rodent skin.
Mechanisms underlying the anti-inflammatory effects of essential fatty acid deficiency in experimental glomerulonephritis. Inhibited release of a monocyte chemoattractant by glomeruli.
Authors: Rovin B H , Lefkowith J B , Schreiner G F
Published: December 2022

Injection of nephrotoxic serum into rats results in glomerular inflammation and proteinuria. Rats placed on an essential fatty acid (EFA)-deficient diet are protected from the glomerular macrophage infiltration and the ensuing proteinuria. To account for this protection, we studied EFA-deficient rats to determine if there were defects in macrophage chemotaxis. We also investigated the possibility that EFA deficiency diminishes the production of a glomerular chemoattractant for monocytes. In microchemotaxis assays EFA-deficient macrophages migrated normally. EFA-deficient serum did not appear to contain a chemotactic inhibitor. Cultured glomeruli from control and control nephritic rats were found to elaborate a chemoattractant for monocytes. This chemoattractant activity was markedly enhanced after induction of nephritis, was heat stable, was not altered by inhibition of cyclooxygenase, lipoxygenase, or platelet-activating factor, and did not depend on C or the glomerular inflammatory cell infiltrate. EFA-deficient glomeruli harvested from animals receiving injections with nephrotoxic serum produced markedly less chemoattractant activity than glomeruli from control nephritic animals. Lipid extraction of nephritic glomeruli from control animals yielded chemoattractant activity in the organic phase. Extracts of EFA-deficient nephritic glomeruli had considerably less activity. We propose that EFA deficiency attenuates glomerular inflammation by inhibiting the ability of glomeruli to produce a specific lipid monocyte chemoattractant after exposure to a nephritic stimulus.
Genetic variability at the human tumor necrosis factor loci.
Authors: Webb G C , Chaplin D D
Published: December 2022

Variability in the structure of the human tumor necrosis factor (TNF-alpha) or lymphotoxin (TNF-beta) genes may contribute to the functional polymorphism of the HLA gene complex. We have characterized an allelic restriction fragment length polymorphism (RFLP) of the TNF-beta gene by using the restriction endonuclease NcoI. Digestion of genomic DNA with NcoI and Southern blotting by using TNF-alpha gene probes show 5.4-kb and 10.5-kb hybridizing fragments. In Caucasian populations, the 10.5-kb fragment is present in 64 to 72% of haplotypes. The polymorphic NcoI site is located within the first intron of the TNF-beta gene. Additional restriction fragment variability was demonstrated by digestion with AccI; however, this restriction fragment variability was not allelic in nature. Rather, it was a consequence of variable DNA methylation at AccI sites within and upstream of the TNF-beta gene. In peripheral blood leukocytes, methylation of the TNF-beta AccI sites was greatest in neutrophils (TNF-beta nonproducers), and lowest in T lymphocytes (the major producers of TNF-beta). These results suggest strongly that variation in DNA methylation may play an important role in regulation of the expression of the TNF-beta gene.
Accessory cell-independent stimulation of human T cells by streptococcal M protein superantigen.
Authors: Kotb M , Majumdar G , Tomai M , Beachey E H
Published: December 2022

Stimulation of T cells by superantigens has been reported to be dependent on the presence of APC where binding to class II molecules is a prerequisite to recognition by the TCR. We examined the response of human T cells and a leukemic T cell line, Jurkat to the superantigen, streptococcal M protein. We show that immobilized or cross-linked streptococcal M protein stimulates Jurkat cells (V beta 8), but not normal purified human T cells, to produce IL-2. Activation of purified T cells by this superantigen required costimulatory signals provided by PMA, IL-1, and IL-6. These cytokines and growth factors alone can induce IL-2 production by T cells; however, proliferation occurred only in the presence of superantigen, which together with PMA, IL-1, and IL-6 induced the expression of IL-2R alpha on T cells. Similar results were obtained when the response of purified T cells to another known superantigen, staphylococcal enterotoxin B were examined, indicating that this phenomenon is not unique to M protein. Superantigens interact with a large number of T cells with particular V beta, and thus provide excellent models for studies of the role of biochemical events and signal transduction in T cell activation. Understanding these events may also explain the pathogenesis of autoimmune diseases associated with certain superantigens, such as streptococcal M protein that is thought to be involved in rheumatic fever and rheumatic heart disease.
IL-2 protects against anti-CD3-induced cell death in human medullary thymocytes.
Authors: Nieto M A , González A , López-Rivas A , Diaz-Espada F , Gambón F
Published: December 2022

In recent years, several studies have confirmed the clonal elimination of thymocytes with receptors that recognize Ag and MHC molecules present on the membrane of thymic stromal cells, a process that may be relevant to the establishment of self-tolerance. In our work, we show that anti-CD3 treatment of single positive CD4+ or CD8+ human medullary thymocytes (obtained by anti-CD1a plus C) induces their apoptotic death. Some events commonly associated with the early steps of normal activation (IL-2R expression, increase in cytoplasmic Ca2+) are also induced after anti-CD3 treatment. Nevertheless, IL-2 is not secreted by these activated cells. The addition of exogenous IL-2 inhibits the apoptosis induced by anti-CD3. We suggest that the lack of secretion of IL-2 by medullary thymocytes may be a physiologic mechanism implicated in the process of negative selection that leads to tolerance.