bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C mediates activation of nuclear cAMP response element-binding protein (CREB) in B lymphocytes stimulated through surface Ig.
Authors: Xie H , Rothstein T L
Published: December 2022

The cAMP response element-binding protein (CREB) is generally considered to be responsive to elevation of cAMP through the activity of protein kinase A (PKA). Although it is well known that cAMP-raising agents can strongly influence B cell stimulation, the regulation of CREB has been little studied. Recently, cross-linking of surface Ig (sIg) was shown to result in trans-activation of a cAMP response element (CRE)-dependent promoter to which bound B cell CREB. In this study, we explored the mechanism underlying this unexpected linkage between sIg and CREB. We found that sIg cross-linking results in phosphorylation of CREB at Ser133. Although this phosphorylation step is mediated by PKA in pheochromocytoma cells, it depends on protein kinase C (PKC) in B lymphocytes. This conclusion is based on abrogation of sIg-induced CREB Ser133 phosphorylation by long-term phorbol-ester treatment to deplete PKC, and mimicking of sIg-induced CREB phosphorylation and CRE-dependent gene expression by short-term PKC agonism. Furthermore, CD40 ligand (CD40L) and LPS, two PKC-independent forms of B cell stimulation, failed to induce phosphorylation of CREB Ser133. These results suggest that CREB responds to specific surface-receptor signals in B cells and that this response is mediated by PKC. Interestingly, forskolin failed to induce phosphorylation of CREB Ser133 in B cells, although it did so in PC12 pheochromocytoma cells. Taken together with PKC mediation of CREB Ser133 phosphorylation in B cells, these results suggest that the dominant mode of CREB regulation is cell-type specific.
T cell subset-dependent modulation of immunity to Borrelia burgdorferi in mice.
Authors: Keane-Myers A , Nickell S P
Published: December 2022

The possible involvement of specific T cells in resolution of infections with Borrelia burgdorferi (Bb), the causative agent of human Lyme disease, has not been adequately studied. To investigate the potential role of T cell subsets in resistance, we have depleted mice of CD4+ and CD8+ T cell subsets in vivo by the administration of specific mAbs and have examined outcomes after infection with Bb. Our results indicate that CD4+ T cells are required for immunologic control of spirochete levels, because their depletion in both susceptible C3H/HeN and resistant BALB/c mice increased the severity of arthritis and the numbers of spirochetes found in joints and skin, as compared with Bb-infected mice treated with a control mAb. In contrast, the CD8+ T cell compartment, particularly in susceptible C3H/HeN mice, appears to promote the disease process, possibly by interfering with the generation of protective immunity, as abrogation of this subset in vivo led to a reduction in both arthritis and in spirochete levels found in joints and skin when compared with Bb-infected control mice. Our inability to establish a correlation between resistance and Bb-specific IgG Ab levels in these mice raises the possibility that Ab-independent mechanisms are important in protection. These findings suggest that the final outcome in Bb-infected hosts may be the net effect of antagonistic influences exerted by CD4+ and CD8+ T cell subsets.
Human urokinase-type plasminogen activator primes neutrophils for superoxide anion release. Possible roles of complement receptor type 3 and calcium.
Authors: Cao D , Mizukami I F , Garni-Wagner B A , Kindzelskii A L , Todd R F , Boxer L A , Petty H R
Published: December 2022

Urokinase-type plasminogen activator (uPA), which binds to cells via a specific receptor (uPAR), participates in pericellular proteolysis during leukocyte migration. Previous studies have indicated that uPAR is physically associated with CR3 (CD11b/CD18). To test the functional interactions of CR3 and uPAR, we have examined the ability of uPA to elicit changes in cytosolic calcium levels of normal neutrophils, neutrophils from a leukocyte adhesion deficiency (LAD) patient, and 3T3 transfectants expressing CR3, uPAR, or both. We found that calcium levels of neutrophils increased from 106 +/- 6 nM in untreated cells to 199 +/- 25 nM in the presence of uPA. In contrast, no significant change in calcium was observed when neutrophils from an leukocyte adhesion deficiency patient were examined. The uPA-dependent calcium rise was inhibited by mAb directed against either CR3 or uPAR and required intact uPA. To substantiate further these findings, we prepared transfectants expressing genes encoding uPAR, CR3, and both receptors; only cells expressing both receptors experienced a rise in intracellular calcium. Although uPA's calcium signal is insufficient to trigger superoxide production, FMLP dose-dependent superoxide production was greatly enhanced by incubating neutrophils with intact, but not fragmented, uPA. Flow cytometry experiments utilizing an FMLP analogue exclude the possibilities that urokinase binds to the FMLP receptor or up-regulates its expression. We suggest that calcium is a second messenger of uPA, that this message is mediated in a CR3-dependent fashion, and that this signal primes neutrophils for superoxide production.
The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells.
Authors: Fruman D A , Bierer B E , Benes J E , Burakoff S J , Austen K F , Katz H R
Published: December 2022

FK506 and cyclosporin A (CsA) are immunosuppressive agents that inhibit IL-2 production by activated T cells, but only CsA inhibits IgE activation-induced cytokine transcripts in mouse IL-3-dependent, bone marrow-derived mast cells (BMMC). We previously associated the resistance of BMMC to FK506 with a deficiency in the expression of FK506 binding protein (FKBP) 12, a molecule that forms a complex with FK506 capable of inhibiting calcineurin phosphatase activity in vitro. In this report, we establish that FKBP12 mediates FK506 inhibition of both calcineurin phosphatase activity and IgE activation-induced cytokine transcripts in a Kirsten murine sarcoma virus-immortalized mast cell line that is FKBP12 deficient. Overexpression of FKBP12 by transfection enhanced the ability of FK506 to inhibit calcineurin phosphatase activity (IC50 = 2 nM), compared with cells transfected with the expression vector alone (IC50 > 30 nM). The IC50 value for FK506 inhibition of IgE activation-induced transcripts for TNF-alpha decreased from 40 nM in vector control cells to 10 nM in FKBP12 transfectants. Similarly, the IC50 value for inhibition of IL-6 transcripts decreased from > 1000 nM in vector control cells to 35 nM in FKBP12 transfectants. In contrast, activation-elicited release of the secretory granule mediator beta-hexosaminidase was only partially inhibited by FK506 at 1000 nM, regardless of the levels of FKBP12 expressed by the cells. Thus, FKBP12 is the dominant cytosolic protein that mediates FK506 inhibition of TNF-alpha and IL-6 transcripts.
Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung.
Authors: Haviland D L , McCoy R L , Whitehead W T , Akama H , Molmenti E P , Brown A , Haviland J C , Parks W C , Perlmutter D H , Wetsel R A
Published: December 2022

The small-complement C5 activation fragment, C5a, is a potent phlogistic molecule that, on binding to the C5a Receptor (C5aR), mediates contraction of smooth muscle, enhances vascular permeability, and promotes leukocyte functions such as directed chemotaxis, degranulation, mediator release, and production of superoxide anions. Although C5aR expression has traditionally been thought to be limited primarily to myeloid blood cells, including neutrophils, monocytes, macrophages, and eosinophils, we report here that C5aR is expressed by liver and lung cells as well as by cells in several other tissues. By Northern blot analysis, it was determined that mouse liver, baboon liver, human liver, and the human hepatoma-derived cell line HepG2 express a normal size (2.3 kb) C5aR mRNA; in HepG2 cells, the quantity of C5aR mRNA was comparable to that contained in dbcAMP-differentiated U937 cells. HepG2 cells were demonstrated to express the C5aR on their cell surface by flow cytometric and immunofluorescence analyses as well as by 125I-C5a binding assays. The binding data indicated that HepG2 cells express a single class of C5aR with a Kd of 1.18 nM and approximately 28,000 receptors per cell. In vivo expression of C5aR in human liver cells was demonstrated by in situ hybridization and immunohistochemistry analyses. Northern blot analysis of murine and baboon organs shows that, in addition to the liver, other tissues express C5aR mRNA in significant quantities, including the spleen, lung, heart, kidney, and intestine. Moreover, mice treated with LPS show a large increase in C5aR mRNA in all these tissues except the intestine. Immunostaining of human lung tissue demonstrated that bronchial and alveolar epithelial cells, as well as vascular smooth muscle and endothelial cells, also express the C5aR. Collectively, these data indicate that the C5aR is expressed in several different types of cells in liver and lung, and in yet undetermined cell types in spleen, heart, intestine, and kidney. Furthermore, these data suggest that the C5a anaphylatoxin mediates previously unrecognized functions by binding to tissue cells that express the C5aR.
Immature B lymphocytes are deficient in expression of the src-family kinases p59fyn and p55fgr1.
Authors: Wechsler R J , Monroe J G
Published: December 2022

Unlike mature B cells, immature B cells are highly sensitive to tolerance induction and do not proliferate in response to stimulation through their Ag receptors. Our previous studies have suggested that this differential responsiveness is caused by differences in Ag receptor-proximal signaling pathways. Because many of these pathways are regulated by tyrosine phosphorylation, we have compared the expression and activity of tyrosine kinases in immature and mature B cells. Consistent with previous studies, we demonstrate that mature B cells express syk, lyn, blk, hck, and fyn; in addition, we find that they express significant amounts of the src-family kinase p55fgr. Immature B cells from day-3 mouse spleen show similar expression and activity of syk, lyn, blk, and hck. However, these cells have marked deficiencies in fyn and fgr expression. Differential expression of fyn and fgr is also observed in immature cells from the bone marrow, indicating that this phenotype is characteristic of the immature stage of B cell development. High levels of fyn and fgr are detectable in splenic B cells beginning at 4 wk of age, coincident with the appearance of cells that proliferate in response to receptor cross-linking. These observations indicate that expression of the src-family kinases fyn and fgr is regulated developmentally in murine B lymphocytes. Moreover, they suggest that these kinases may be important for mature B cell responses to Ag receptor stimulation, and that their absence may contribute to the unresponsiveness and tolerance susceptibility of immature B cells.
Bcl-2 blocks degranulation but not fas-based cell-mediated cytotoxicity.
Authors: Chiu V K , Walsh C M , Liu C C , Reed J C , Clark W R
Published: December 2022

The ability of bcl-2 in target cells to block cell-mediated cytotoxicity by allospecific CTL was tested. The blocking effect was variable. Because killing by CTL involves two different pathways, degranulation (perforin plus granzymes) and fas, we examined the effect of bcl-2 on these pathways independently. Bcl-2 in target cells blocked apoptotic cell death induced either by cytotoxic granule extracts or by CTL killing under conditions in which the fas pathway is blocked. On the other hand, bcl-2 had no effect on target cell-killing either by Fas-specific mAb or by CTL capable of killing only via the fas pathway. These data suggest bcl-2 may block apoptotic lysis induced by perforin plus granzymes, but not apoptotic lysis induced via the fas pathway. Thus, any analysis of the effect of bcl-2 on apoptotic cell death in target cells killed by CTL must take into account the relative contributions of the degranulation vs fas pathways.
Antigen-independent, integrin-mediated T cell activation.
Authors: Sturmhöfel K , Brando C , Martinon F , Shevach E M , Coligan J E
Published: December 2022

The "outside-in" signals produced by the interaction of integrin molecules with the extracellular matrix (ECM) trigger a multitude of cellular events. The vitronectin receptor (VNR), an alpha v beta 3 heterodimer, functions as a costimulatory molecule for the activation of a subset of V gamma 1.1/C gamma 4-bearing gamma/delta T cells, which have been postulated to recognize a ubiquitous self-antigen. We addressed the question of whether stimulation of these T cells requires both engagement of the VNR by ECM proteins and engagement of the TCR by its Ag. We introduced into a TCR- but VNR+ mutant T cell hybridoma, TG40 (derived from 2B4), a chimeric molecule that contains the cytoplasmic tail of the TCR zeta-chain fused to the cytoplasmic and transmembrane region of either human CD8 or human CD25. The transfectants expressing the chimeric molecules secreted IL-2 constitutively when the VNR was engaged with a ligand, e.g., provided by ECM proteins present in FCS. This constitutive cytokine secretion could be blocked with mAb directed against the VNR, with or the peptide RGD, or by growth in serum-free medium. VNR-mediated cell activation also induced the phosphorylation of the zeta-chain. Signaling through the zeta-chain was required, as cells transfected with a chimera containing only a 22 amino-acid long, truncated zeta-chain did not secrete IL-2 constitutively. Thus, we demonstrated that the binding of the VNR to ECM protein in the presence of the zeta-chain is sufficient to induce cytokine secretion by T cells and does not require the recognition of an Ag by the TCR. Such integrin-mediated, Ag-independent activation of T cells may play a critical role in the potentiation of inflammatory responses.
Resistance of melanoma cell lines to interferons correlates with reduction of IFN-induced tyrosine phosphorylation. Induction of the anti-viral state by IFN is prevented by tyrosine kinase inhibitors.
Authors: Ralph S J , Wines B D , Payne M J , Grubb D , Hatzinisiriou I , Linnane A W , Devenish R J
Published: December 2022

Clinical and experimental studies examining the action of IFNs on human malignant melanomas and melanoma cell lines have shown that this cancer cell type is frequently IFN resistant. In the present study, the IFN responsiveness of five melanoma cell lines, SK-MEL-28, SK-MEL-3, MM96, HT-144, and Hs 294T, as determined by the levels of IFN-induced expression of the antiviral proteins, 100 kDa 2',5'-oligoadenylate synthetase (OAS) and Mx Ag, was shown to correlate with the IFN responsiveness of the five lines measured in antiproliferative and antiviral assays. Three of the lines, SK-MEL-28 (IFN sensitive), SK-MEL-3 (moderately IFN sensitive), and MM96 (IFN insensitive) were analyzed further to ascertain their relative levels of IFN-activated signal transduction. Pretreatment of the three melanoma cell lines with the tyrosine kinase inhibitors, Herbimycin A or Genistein, produced a dose-dependent inhibition of the antiviral action of IFN-alpha, -beta, and -gamma and the induction of OAS by IFN-beta. Thus, induction of the antiviral state in melanoma cells by IFN requires activation of tyrosine kinase-dependent signaling pathways. Furthermore, the IFN responsiveness of three melanoma cell lines could be correlated with the ability to detect by immunoblotting of SDS-PAGE displays of cell lysates, IFN-induced tyrosine phosphorylated cellular proteins in the range m.w. 80 to 130 kDa. This induction was also sensitive to the tyrosine kinase inhibitors Herbimycin A and Genistein. Based on these results, we propose that the IFN-resistant melanoma cell lines examined contain a deficiency early in the IFN signal transduction pathway resulting in a reduced potential for IFN-induced tyrosine phosphorylation and a lack of responsiveness to IFN.
Pyrrolidine dithiocarbamate inhibits intercellular adhesion molecule-1 biosynthesis induced by cytokines in human fibroblasts.
Authors: Kawai M , Nishikomori R , Jung E Y , Tai G , Yamanaka C , Mayumi M , Heike T
Published: December 2022

Intercellular adhesion molecule-1 (ICAM-1), the ligand of lymphocyte function-associated antigen-1, plays an important role in the interactions of a variety of hemopoietic and nonhemopoietic cells, including leukocytes, fibroblasts, and endothelial cells. ICAM-1 is known to be involved in the onset of several diseases such as inflammation, allograft rejection, and so on. In this report, we investigated the effects of dexamethasone, cyclosporin A, FK506, and pyrrolidine dithiocarbamate (PDTC) on the induction of the ICAM-1 gene by cytokines in fibroblasts. PDTC, a potent inhibitor of NF-kappa B, was shown by ELISA and FACS analysis to prevent dramatically the expression of the ICAM-1 gene stimulated by IL-1 alpha, IFN-gamma, and PMA, although the other reagents inhibited it only slightly. Ribonuclease protection assay revealed that PDTC blocked the expression of the ICAM-1 gene at the mRNA level. To elucidate the mechanism of this inhibition, we constructed a series of ICAM-1 promoter deletion mutants linked to the chloramphenicol acetyl transferase gene and analyzed the effect of PDTC on their activities. Transient transfection analysis indicated that the critical region for inhibition by PDTC is an NF-kappa B binding site-like motif (GGGAGGATTCC, ICAM-1 kappa B) that is located at position-540. Electrophoresis mobility shift assay revealed that PDTC actually inhibits the binding of NF-kappa B (or NF-kappa B-like) protein to the ICAM-1 kappa B site. These findings suggest that PDTC inhibits ICAM-1 gene expression by inhibiting the association of NF-kappa B (or NF-kappa B-like) protein with the ICAM-1 kappa B site.
Isolation of nine gene sequences induced by silica in murine macrophages.
Authors: Segade F , Claudio E , Wrobel K , Ramos S , Lazo P S
Published: December 2022

Macrophage activation by silica is the initial step in the development of silicosis. To identify genes that might be involved in silica-mediated activation, RAW 264.7 mouse macrophages were treated with silica for 48 h, and a subtracted cDNA library enriched for silica-induced genes (SIG) was constructed and differentially screened. Nine cDNA clones (designated SIG-12, -14, -20, -41, -61, -81, -91, -92, and -111) were partially sequenced and compared with sequences in GenBank/EMBL databases. SIG-12, -14, and -20 corresponded to the genes for ribosomal proteins L13a, L32, and L26, respectively. SIG-61 is the mouse homologue of p21 RhoC. SIG-91 is identical to the 67-kDa high-affinity laminin receptor. Four genes were not identified and are novel. All of the mRNAs corresponding to the nine cloned cDNAs were inducible by silica. Steady-state levels of mRNAs in RAW 264.7 cells treated with various macrophage activators and inducers of signal transduction pathways were determined. A complex pattern of induction and repression was found, indicating that upon phagocytosis of silica particles, many regulatory mechanisms of gene expression are simultaneously triggered.
In vivo regulation of rat neutrophil apoptosis occurring spontaneously or induced with TNF-alpha or cycloheximide.
Authors: Tsuchida H , Takeda Y , Takei H , Shinzawa H , Takahashi T , Sendo F
Published: December 2022

We previously demonstrated that human TNF-alpha induces rapid apoptosis of human neutrophils. To understand better the in vivo significance of neutrophil apoptosis, we examined spontaneous, recombinant human and mouse TNF-alpha- or cycloheximide-induced apoptosis of normal peripheral blood neutrophils (PBN), PBN from rats injected i.p. with proteose peptone or a streptococcus preparation, OK-432 (inflammatory PBN), peritoneally exudated neutrophils (PEN) obtained after a proteose peptone injection, and normal bone marrow neutrophils. The following observations were made. 1) Normal PBN responded to TNF-alpha, but PEN, normal bone marrow neutrophils, and inflammatory PBN at 12 h after stimulation did not. 2) The sensitivity to TNF-alpha of the inflammatory PBN started to decrease at 3 h, was lowest at 12 h, and was almost restored at 52 h after stimulation. 3) Spontaneous apoptosis of normal and inflammatory PBN reached 25% at 12 h after in vitro incubation, but that of PEN and normal bone marrow neutrophils was very low over this period. 4) The sensitivity to cycloheximide (6 h incubation) was high for normal PBN and bone marrow neutrophils, but low for PEN and inflammatory PBN after 12 h. 5) 125I-rhTNF-alpha binding of bone marrow neutrophils was significantly lower than that of normal and inflammatory PBN and PEN. 6) TNF-alpha-induced apoptosis of normal or inflammatory PBN and bone marrow neutrophils was enhanced by treatment with low doses of cycloheximide that alone were barely able to induce neutrophil apoptosis; however, apoptosis of PEN was not. The mechanisms and in vivo significance of these phenomena are discussed.
Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase.
Authors: Vlahos C J , Matter W F , Brown R F , Traynor-Kaplan A E , Heyworth P G , Prossnitz E R , Ye R D , Marder P , Schelm J A , Rothfuss K J
Published: December 2022

Neutrophils contain a multicomponent NADPH oxidase system that is involved in the production of microbicidal oxidants. Stimulation of human neutrophils with the peptide FMLP activates this respiratory burst enzyme to produce superoxide and also has been shown to result in activation of phosphatidylinositol (Ptdlns) 3-kinase. Treatment of human neutrophils with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a potent and specific inhibitor of Ptdlns 3-kinase, resulted in complete inhibition of Ptdlns 3-kinase activity as well as in inhibition of superoxide production in FMLP-treated neutrophils in suspension; FMLP-stimulated oxidant production in adherent cells was also abolished. Treatment of human neutrophils with PMA resulted in production of superoxide without activation of Ptdlns 3-kinase; LY294002 did not block superoxide production in neutrophils exposed to PMA. In addition, LY294002 did not inhibit cellfree NADPH oxidase activation, CD11b-dependent adhesion, actin polymerization in response to FMLP, or FMLP-induced calcium flux. These results suggest that the signal transduction pathway of the FMLP-receptor involves activation of Ptdlns 3-kinase, which is required for subsequent superoxide production induced by the chemotactic peptide. Furthermore, Ptdlns 3-kinase may be located directly upstream of protein kinase C or other protein kinases, which in turn activate the NADPH oxidase system.
HIV replication in IL-2-stimulated peripheral blood mononuclear cells is driven in an autocrine/paracrine manner by endogenous cytokines.
Authors: Kinter A L , Poli G , Fox L , Hardy E , Fauci A S
Published: December 2022

Replication of HIV is regulated by virus-encoded regulatory proteins, as well as by a variety of cellular factors including cytokines. In the present study, we have investigated the autocrine/paracrine effects of endogenous cytokines on HIV replication in primary PBMCs of healthy HIV seronegative individuals. Addition of rIL-2 to cultures between 0 and 72 h after isolation of PBMCs allowed the replication of primary HIV isolates and laboratory-adapted HIV strains to levels comparable with or greater than those obtained in parallel cultures of autologous PHA-blasts. In this regard, both major cellular targets of HIV infection, CD4+ T lymphocytes and mononuclear phagocytes, were maintained for several weeks in IL-2-stimulated PBMC cultures and virion production was observed in both cell lineages. The kinetics of secretion of several cytokines (such as TNF-alpha, IL-1 beta, IL-6, and IFN-gamma), as well as expression of cellular activation markers, paralleled HIV replication in IL-2-stimulated PBMCs. Endogenous pro-inflammatory cytokines and IFN-gamma played a major role in the regulation of HIV replication in IL-2-stimulated PBMCs, as determined by the ability of several anti-cytokine Abs or antagonists to suppress HIV production; this was not the case in parallel cultures of autologous PHA-blasts. Thus, IL-2-stimulated PBMCs may represent a more physiologic in vitro system than PHA-blasts for the study of HIV infection and replication, and should prove useful in investigating the role of cytokines and other host factors in the regulation of HIV production.
Immobilized anti-CD3 antibody activates T cell clones to induce the production of interstitial collagenase, but not tissue inhibitor of metalloproteinases, in monocytic THP-1 cells and dermal fibroblasts.
Authors: Miltenburg A M , Lacraz S , Welgus H G , Dayer J M
Published: December 2022

In this study we have investigated whether direct cell to cell contact between activated paraformaldehyde-fixed T cell clones obtained from synovial tissue of patients with osteoarthritis (OA) or rheumatoid arthritis and target monocytic cells or dermal fibroblasts influenced the balance between interstitial collagenase and its specific inhibitor tissue inhibitor of metalloproteinases (TIMP) produced by the latter cell types. PHA/PMA-activated fixed T cell clones or their membranes strongly induced the production of collagenase both in monocytic THP-1 cells and in dermal fibroblasts. In contrast, only low levels of TIMP were induced in THP-1 cells and no change of TIMP expression was observed in fibroblasts as a result of stimulation with PHA/PMA-activated T cells or T cell membranes. Anti-CD3-activated T cell clones stimulated the production of collagenase both in THP-1 cells and fibroblasts, whereas TIMP levels were not influenced. Collagenase production in THP-1 cells induced by anti-CD3-activated T cell clones was 1) dependent on the dose of anti-CD3 used to stimulate the T cells, 2) initiated only when CD3 was cross-linked, and 3) inhibited when cyclosporin A was included during T cell activation. Our data collectively indicate that activated T cells in contact with monocytic cells or fibroblasts may alter the balance between interstitial collagenase and its specific inhibitor TIMP. This selective induction of a mediator profile representative of matrix breakdown as a result of target cell interaction with activated T cells may be an important factor in the local process of tissue destruction that characterizes osteoarthritis and rheumatoid arthritis.
Anti-IL-4 antibody prevents graft-versus-host disease in mice after bone marrow transplantation. The IgE allotype is an important marker of graft-versus-host disease.
Authors: Ushiyama C , Hirano T , Miyajima H , Okumura K , Ovary Z , Hashimoto H
Published: December 2022

Induction of a graft-vs-host reaction in irradiated (BALB/c X C57BL/6)F1 mice (CBF1 mice) with bone marrow cells (BMC) plus spleen cells of BALB/c mice leads to bone marrow transplantation--GVHD (BMT-GVHD). BMT-GVHD is characterized by liver disease, splenomegaly, and hypergammopathy. In addition, we found that increased serum IgE and IgG1 levels were correlated with BMT-GVHD such as liver disease and splenomegaly. The allotype of increased IgE levels in BMT-GVHD was IgEa of donor origin, not IgEb of host origin. We also found that in the thymus of murine BMT-GVHD, the CD4+ CD8+ double-positive T cells were decreased, but the CD4+ CD8- or CD4- CD8+ single-positive T cells were increased. Interestingly, double-positive T cells appeared in the spleen, suggesting that abnormal T cell differentiation existed in murine BMT-GVHD. When the recipients were treated with anti-IL-4 Ab (11B11), the increase of IgE and IgG1 was markedly reduced and liver disease and splenomegaly were also prevented. Moreover, abnormal T cell differentiation and maturation were suppressed. These observations suggest that IL-4 plays an important role in immunoregulation or pathogenesis of allogeneic effects, and 11B11 prevents immunodysfunction including T cell differentiation in the thymus or the spleen and autoimmune symptoms in murine BMT-GVHD.
CTL responses of HLA-A2.1-transgenic mice specific for hepatitis C viral peptides predict epitopes for CTL of humans carrying HLA-A2.1.
Authors: Shirai M , Arichi T , Nishioka M , Nomura T , Ikeda K , Kawanishi K , Engelhard V H , Feinstone S M , Berzofsky J A
Published: December 2022

Vaccine development in animal models depends on ability to recognize epitopes seen by human T cells. In this work, we show that CTL responses in transgenic mice expressing human HLA-A2.1 prospectively predict the same four of 11 hepatitis C virus (HCV) structural protein-derived peptides, expressing a sequence motif for HLA-A2.1 binding, that are actually recognized by human A2.1-restricted CTLs. The CTLs also recognized targets endogenously expressing these proteins. Human CTLs from HCV-infected patients, tested by using the same peptides, revealed a virtually identical response repertoire. A highly conserved HCV core peptide was the most immunogenic, and may be a valuable component of a vaccine against a broad range of HCV isolates in HLA-A2-positive patients. These results suggest that, in spite of species differences, the T cell repertoire is plastic enough to allow a similar response when the same class I MHC molecule is presenting the peptide. Thus, the HLA molecule plays the primary role in determining which peptides are recognized by CTLs. This transgenic mouse model is important for the study of HLA-restricted CTL determinants and for an approach to design a potential HCV vaccine.
Structure of the mouse CD72 (Lyb-2) gene and its alternatively spliced transcripts.
Authors: Ying H , Nakayama E , Robinson W H , Parnes J R
Published: December 2022

The complete sequence of the CD72 gene from the C57L mouse, including the 5' and 3' flanking sequences, is reported. The gene spans 6830 base pairs and includes nine exons surrounding eight introns. It does not have an obvious TATAA box, so it belongs to a group of genes with TATA-less promoters that are regulated during mammalian immunodifferentiation. cDNA sequence comparisons among CD72a, CD72b, and CD72c alleles have demonstrated two distinct seven amino acid insertion/deletions among these allelic variants. Based on our genomic sequence studies as well as PCR analyses, we found that different strains of mice can alternatively or exclusively use either of two AG sites surrounding the 21-bp insertion/deletions as 3' splice sites in an allele-specific manner. Other alternative splicing events, such as exon skipping, also contribute to CD72 polymorphism. In mouse splenic B cells there are allele-specific distributions of CD72 mRNAs that contain sequences from both exon 3 and exon 4, from either exon 3 or exon 4, or from neither exon 3 nor 4. It is unclear what the in vivo function might be of the proteins encoded by the mRNA forms lacking these exon sequences.
IL-7 in the cell-mediated immune response to a human pathogen.
Authors: Sieling P A , Sakimura L , Uyemura K , Yamamura M , Oliveros J , Nickoloff B J , Rea T H , Modlin R L
Published: December 2022

The goal of the present study was to investigate the role of IL-7 in regulating immune responses to infection. Leprosy provides a model for understanding human immune responses to infection; the disease presents as a spectrum in which the clinical manifestations correlate with the levels of cell-mediated immunity to the pathogen, Mycobacterium leprae. To determine whether IL-7 is produced at the site of infection in leprosy, we used the PCR to measure IL-7 and IL-7R mRNA in skin lesions. IL-7 mRNA was more strongly expressed in the tuberculoid form of the disease, in which the infection is limited (mean cpm = 48 +/- 8; n = 11), as compared with the progressive lepromatous form (17 +/- 2; n = 11). IL-7R mRNA, both membrane-bound and soluble forms, were also more strongly expressed in tuberculoid lesions, although these differences were not as striking as those for IL-7. The cellular source of IL-7 included Ag-stimulated monocytes and IFN-gamma-induced keratinocytes. M. leprae-induced PBMC responses in tuberculoid patients involved up-regulation of IL-7 and IL-7R mRNA and was IL-7 dependent. In contrast, M. leprae did not induce IL-7 mRNA in lepromatous patients, and their T cell responses were weakly augmented by rIL-7. These data suggest that IL-7, produced at the site of disease, contributes to the cell-mediated immune response to human pathogens.
Overexpression of vascular permeability factor (VPF/VEGF) and its endothelial cell receptors in delayed hypersensitivity skin reactions.
Authors: Brown L F , Olbricht S M , Berse B , Jackman R W , Matsueda G , Tognazzi K A , Manseau E J , Dvorak H F , Van de Water L
Published: December 2022

Delayed hypersensitivity (DH) is a T cell-mediated form of immune response characterized by a predominantly perivascular, mononuclear cell infiltrate. The venules in DH reactions are hyperpermeable to plasma proteins, leading to extravasation of plasma fibrinogen and its extravascular clotting to form a fibrin gel that promotes induration and angiogenesis. The mechanisms responsible for microvascular hyperpermeability in DH are unknown. Recently, a cytokine named vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF) has been implicated in the chronic vascular hyperpermeability and angiogenesis of solid and ascites tumors, healing wounds, rheumatoid arthritis, and psoriasis. These findings suggested that VPF/VEGF might also have a role in the pathogenesis of DH. Two model systems were studied: allergic contact dermatitis to poison ivy in human volunteers and classical tuberculin hypersensitivity in rats. In both, in situ hybridization revealed that the mRNAs encoding VPF/VEGF were strikingly overexpressed in keratinocytes of the epidermis; scattered mononuclear cells infiltrating the dermis also overexpressed VPF/VEGF mRNA, to a greater extent in rat tuberculin than in human contact reactions. In contact reactions, mRNAs for two VPF/VEGF vascular endothelial cell receptors, flt-1 and KDR, were also strikingly overexpressed. Abundant fibrin deposition in both models confirmed that dermal microvessels were indeed hyperpermeable to plasma fibrinogen. These results implicate VPF/VEGF as a potentially important mediator in the pathogenesis of cell-mediated immunity and provide further evidence that products of epithelial cells may regulate the inflammatory response.
IL-4 induction of VCAM-1 on endothelial cells involves activation of a protein tyrosine kinase.
Authors: Palmer-Crocker R L , Pober J S
Published: December 2022

IL-4 triggers tyrosine phosphorylation of a single major substrate (M(r) 145,000) in cultured human endothelial cells (EC) as detected by Western blot of whole cell lysates or of anti-phosphotyrosine immunoprecipitates. Phosphorylation of this substrate depends on IL-4 concentration (appearance at 10 U/ml, maximal at 300 to 1000 U/ml) and time of treatment (onset by 1 min, peak at 5 to30 min, duration of 60 to 120 min). Immunoprecipitation with specific mAb identified the phosphorylated substrate as the IL-4R. Treatment of EC with IL-4 alone causes only a small increase in the expression of vascular cell adhesion molecule-1 (VCAM-1), but IL-4 significantly augments the level of VCAM-1 expression induced by PMA. Pretreatment of EC with herbimycin A (0.5 to 1.0 microgram/ml) for 12 to 18 h abrogates both IL-4-induced tyrosine phosphorylation and IL-4-augmented VCAM-1 expression. This concentration of herbimycin A does not inhibit and may augment PMA-induced VCAM-1 expression in replicate wells. These observations suggest that IL-4 induction of VCAM-1 in EC involves the activation of an as yet unidentified protein tyrosine kinase that phosphorylates the IL-4R.
A 12-kDa protein of Mycobacterium tuberculosis protects mice against experimental autoimmune encephalomyelitis. Protection in the absence of shared T cell epitopes with encephalitogenic proteins.
Authors: Ben-Nun A , Mendel I , Sappler G , Kerlero de Rosbo N
Published: December 2022

Mycobacterium tuberculosis (Mt), routinely used to promote the induction of autoimmune diseases, can also protect against their development. Recently, we demonstrated that purified protein derivative (PPD) is the major fraction of Mt that protects mice against the induction of experimental autoimmune encephalomyelitis (EAE). We have now ascribed the protective activity to a 12-kDa protein purified from PPD. Sequence identity between the first 17 amino acids of the 12-kDa PPD protein and the 10-kDa BCG-a protein of Mt suggested that these proteins are identical or closely related. However, in contrast to the 12-kDa PPD protein, the 10-kDa BCG-a protein did not protect against EAE, nor did it stimulate PPD-specific T cells, suggesting that the 12-kDa PPD protein and the 10-kDa BCG-a protein share some homology but are not identical. The protective activity of the 12-kDa PPD protein correlated with its ability to stimulate PPD-specific T cells. The significance of this correlation is not clear and the mechanism of protection was not fully elucidated. However, N-terminal sequence identity between the 12-kDa PPD protein and the 10-kDa BCG-a protein, which shares 43% homology with GroES stress protein, suggested that the 12-kDa PPD protein may also belong to the bacterial heat-shock protein (hsp) family. Thus, by analogy with protection against arthritis or diabetes by hsp65, the mechanism of protection could be based on shared T cell epitopes with the target self Ag. However, the 12-kDa PPD protein did not stimulate encephalitogenic T lymphocytes. Effective protection against EAE by the 12-kDa PPD protein, in the absence of a stimulatory effect on encephalitogenic T lymphocytes, suggests a potential use for this protein in the therapy of autoimmune diseases.
Anti-TNF-alpha treatment down-regulates the expression of fibronectin and decreases cellular infiltration of cardiac allografts in rats.
Authors: Coito A J , Binder J , Brown L F , de Sousa M , Van de Water L , Kupiec-Weglinski J W
Published: December 2022

Extracellular matrix (ECM) components provide costimulatory signals for T cell activation in vitro, and may be critical for lymphocyte migration and tissue positioning in vivo. We conducted a series of studies in rat recipients of cardiac allografts to evaluate intragraft expression of a prominent ECM protein, fibronectin (FN), and to analyze the effects of infusing a neutralizing anti-TNF-alpha serum on FN expression and lymphocyte migration into the transplants. LBNF1 cardiac allografts were rejected within 8 days in control LEW rats. A prominent immunohistochemical feature of this immune response was the dense deposition of FN at the graft site as early as 3 h, which then peaked at 4 to 6 days. The early 3-h FN deposition (likely plasma FN) was noted before cellular infiltration. Northern blot analysis established that a marked induction of FN mRNA expression occurred in rejecting cardiac allografts at day 4 after transplantation. To determine the source of FN mRNA, we conducted a series of in situ hybridization studies with probes for FN and lysozyme, a macrophage-specific marker. Indeed, the majority of graft-infiltrating cells expressed lysozyme mRNA and FN mRNA. Administration of anti-TNF-alpha serum into LEW hosts (0.5 ml i.v. at days 1 and 3 only) abrogated acute rejection and prolonged cardiac allograft survival to approximately 13 days. This was accompanied by depressed circulating and intragraft TNF-alpha levels, and markedly down-regulated FN mRNA/protein expression patterns, as compared with those in recipients given nonimmune rabbit serum. Anti-TNF-alpha treatment also markedly decreased graft infiltration by ED1+ monocytes/macrophages, OX-8+, and VLA-4+ cells, normally peaking at 4 days. Moreover, we found that the migration of 111In-labeled specifically sensitized lymph node lymphocytes to cardiac allografts in secondary rat recipients conditioned with anti-TNF-alpha serum was significantly decreased, as compared with that in controls. Thus, FN expression by intragraft macrophages occurs within the same interval as cellular infiltration, and may act as an ECM component "signal" for selective homing of recirculating lymphocytes in graft recipients. The results of this study support the notion that in vivo interactions between mononuclear cells and ECM may be vital for the ingress of alloreactive lymphocytes at the graft site, and offer potential novel sites for therapeutic intervention in the control of transplant rejection.
HIV-1 down-regulates CD4 costimulation of TCR/CD3-directed tyrosine phosphorylation through CD4/p56lck dissociation.
Authors: Kanner S B , Haffar O K
Published: December 2022

One consequence of HIV type 1 (HIV-1) infection is the gradual loss of responsiveness of T lymphocytes to Ags both in vitro and in vivo. It has been suggested that the underlying mechanism that contributes to this T cell dysfunction before CD4+ cell decline involves down-regulation of surface receptors, alterations in intracellular redox status, interference by viral Ags, and later in infection, the absence or alteration of specific cytokine production. In this report, we demonstrate that infection of the T-lymphocytic cell line H9 with the LAI isolate of HIV-1 results in profoundly altered regulation of CD4-induced costimulation of TCR/CD3-directed signaling. TCR/CD3-induced tyrosine phosphorylation of the intracellular enzyme phospholipase-C gamma 1 and the surface receptor/substrates CD5 and CD6 was unaffected by virus infection, whereas augmented responses normally observed after the co-ligation of CD4 with TCR/CD3 on T lymphocytes were absent in HIV-1-infected H9 cells. Costimulation of TCR/CD3-induced signaling via MHC class II molecules was also down-regulated in virally infected cells. TCR/CD3 and HLA-DR receptor expression remained intact in infected cultures for at least 3 wk, whereas CD4 surface expression was gradually lost but maintained for up to 1 wk, suggesting that the absence of costimulation early in infection was not surface receptor density-dependent. In HIV-1-infected cells, CD4 was not physically linked with its associated tyrosine kinase p56lck, whereas normal levels of p56lck were readily recovered from the cellular cytoplasm. Similar observations were noted in cultures of H9 cells infected with a field isolate of HIV-1 obtained from cultured PBMC from an infected donor. HIV-1 infection of T lymphocytes thus down-regulates potentially critical early signal transduction events by a mechanism that appears to involve interference of CD4 receptor association with p56lck. A potential outcome of these biochemical effects may include the limited responsiveness of infected T cells to antigenic stimulation observed during HIV-1 infection.
Existence of a soluble form of CD50 (intercellular adhesion molecule-3) produced upon human lymphocyte activation. Present in normal human serum and levels are increased in the serum of systemic lupus erythematosus patients.
Authors: Pino-Otín M R , Viñas O , de la Fuente M A , Juan M , Font J , Torradeflot M , Pallarés L , Lozano F , Alberola-Ila J , Martorell J
Published: December 2022

CD50 (ICAM-3) is a leukocyte differentiation Ag expressed almost exclusively on hemopoietic cells, with a key role in the first steps of immune response. To develop a specific sandwich ELISA to detect a soluble CD50 form (sCD50), two different mAbs (140-11 and 101-1D2) recognizing non-overlapping epitopes were used. sCD50 was detected in the supernatant of stimulated PBMCs, with the highest levels after CD3 triggering. Simultaneously, the CD50 surface expression diminished during the first 24 h. sCD50 isolated from culture supernatant and analyzed by immunoblotting showed an apparent m.w. of 95 kDa, slightly smaller than the membrane form. These data, together with Northern blot kinetics analysis, suggest that sCD50 is cleaved from cell membrane. Furthermore, we detect sCD50 in normal human sera and higher levels in sera of systemic lupus erythematosus (SLE) patients, especially in those in active phase. The sCD50 levels showed a positive correlation with sCD27 levels (r = 0.4213; p = 0.0026). Detection of sCD50, both after in vitro CD3 triggering of PBMCs and increased in SLE sera, suggests that sCD50 could be used as a marker of lymphocyte stimulation.
Derivation of vaccines from mimotopes. Immunologic properties of human hepatitis B virus surface antigen mimotopes displayed on filamentous phage.
Authors: Meola A , Delmastro P , Monaci P , Luzzago A , Nicosia A , Felici F , Cortese R , Galfrè G
Published: December 2022

We have previously reported the identification, using human immune sera, of mimotopes of human hepatitis B virus surface Ag (HBsAg) displayed on filamentous phage. To test if these mimotopes could be useful in developing a vaccine against the human hepatitis B virus (HBV), we have compared the humoral immune response of animals immunized either with a recombinant HBsAg vaccine, or with mimotopes. Immunogens were prepared by fusing the mimotopes on different carrier molecules (phage coat protein pIII and pVIII, recombinant human H ferritin, HBV core peptide) and by synthesizing multiple antigenic peptides carrying the mimotopes' amino acid sequences. These immunogens were injected into mice and rabbits and sera were collected and tested for the presence of HBsAg-specific Abs. Our data confirm that mimotopes can induce a humoral immune response resembling that induced by the original Ag, and HBsAg mimotopes displayed on phage prove to be the best immunogens, inducing the most reproducible and potent immunization. Mimotopes that react as HBV subtype-specific Ags do not show this specificity as immunogen and induce a nonsubtype-restricted response. Furthermore, mimotopes displayed on phage elicit a strong response to HBsAg in a strain of mouse reported to show a low response to it. These results indicate that mimotopes identified from random peptide libraries through utilizing human immune sera could be important leads for the derivation of new vaccines.
Thiol-mediated redox regulation of apoptosis. Possible roles of cellular thiols other than glutathione in T cell apoptosis.
Authors: Sato N , Iwata S , Nakamura K , Hori T , Mori K , Yodoi J
Published: December 2022

Thiol redox status modulates various aspects of cellular function. We demonstrate that oxidation of cellular sulfhydryl (SH) groups induces apoptosis. In Jurkat T cells and human PBL blasts, the fraction of apoptotic nuclei increased after treatment with an SH-specific oxidant, diamide. Analysis of DNA fragmentation and nuclear morphology also indicated that SH oxidation could induce apoptosis. In the apoptosis induced by SH oxidation, the decrease of cellular glutathione was transient and the increase of glutathione disulfide was observed only after apoptotic changes had occurred. Depletion of cellular glutathione with buthionine sulfoximine failed to induce apoptosis, despite a marked decrease of cellular glutathione, which was greater than that observed in apoptosis induced by diamide. Thus, the changes of cellular glutathione or glutathione disulfide may not be the major cause of apoptosis induced by diamide. Intracellular adult T cell leukemia-derived factor/human thioredoxin, another thiol-related antioxidant protein, was oxidized by incubation with diamide. These results suggest that thiol redox status is one of the key factors of the apoptotic pathway in which thiols other than glutathione may play even more critical roles.