bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that secretory phospholipase A2 plays a role in arachidonic acid release and eicosanoid biosynthesis by mast cells.
Authors: Fonteh A N , Bass D A , Marshall L A , Seeds M , Samet J M , Chilton F H
Published: December 2022

This study investigated the role of secretory PLA2 (sPLA2) in arachidonic acid (AA) release and lipid mediator generation in mouse bone marrow-derived mast cells. Initial studies indicated that mast cells contain multiple PLA2 activities and secreted PLA2 activity upon Ag stimulation. The secreted PLA2 activity is blocked by DTT and by a group II PLA2-neutralizing Ab. Mast cells incubated with groups I or II PLA2 selectively release polyunsaturated fatty acids, such as AA, into supernatant fluids. The bulk of AA released by sPLA2 is derived from phosphatidylethanolamine. The fatty acids released by extracellular PLA2 mimic those found in supernatant fluids after Ag stimulation of mast cells. Incubation of mast cells with PLA2 generates cyclooxygenase (CO) products but no 5-lipoxygenase (5-LO) products. Ag, but not PLA2, induces the translocation of 5-LO to cellular membranes and the formation of 5-LO products. The addition of sPLA2 to Ag-stimulated mast cells increases the synthesis of 5-LO products. Octadeuterated AA (2H8AA) added to the outside of cells to trace extracellular AA metabolism is rapidly converted to CO products. Addition of sPLA2, or Ag in combination with 2H8AA reduces the quantity of 2H8AA converted to deuterated CO products, when compared with adding 2H8AA alone. The reduction of deuterated CO products can be accounted for with increases in nondeuterated CO products. 2H8AA is only converted to 5-LO produces when mast cells are activated with Ag. The amount of 2H8AA converted to deuterated 5-LO products is reduced by the addition of PLA2 to Ag-stimulated cells. The competitive formation of deuterated and nondeuterated products observed when mast cells are incubated with 2H8AA, indicates that extracellular AA released by sPLA2 is used for both CO and 5-LO product formation. Taken together, these data reveal a role for sPLA2 in the release of AA and demonstrate that AA released by this mechanism is used for eicosanoid generation.
Induction of monocyte chemotactic proteins MCP-1 and MCP-2 in human fibroblasts and leukocytes by cytokines and cytokine inducers. Chemical synthesis of MCP-2 and development of a specific RIA.
Authors: Van Damme J , Proost P , Put W , Arens S , Lenaerts J P , Conings R , Opdenakker G , Heremans H , Billiau A
Published: December 2022

Monocyte chemotactic proteins (MCP) belong to a group of structurally and functionally related factors, called chemokines. To facilitate additional characterization of the recently identified MCP-2, the 76-residue protein was chemically synthesized. The synthetic 7-kDa monomeric protein was chemotactic for monocytes at 1 nM and was biochemically similar to natural MCP-2. Sensitive radioimmunoassays for both MCP-1 and MCP-2 were developed. These RIAs were specific in that no cross-reactivity could be observed, and other chemokines or cytokines were not detected. Induction of MCP-1 and MCP-2 in human diploid fibroblasts and peripheral blood leukocytes as well as osteosarcoma, epidermal carcinoma, and melanoma cells by the cytokines IL-1 beta, IFN-beta, and IFN-gamma and cytokine inducers such as dsRNA, virus, endotoxin, mitogen, and phorbol ester was studied. In connective tissue cells, IL-1 beta was the best inducer of MCP-1, but IFN-gamma was a superior inducer of MCP-2. Mononuclear cells also proved to be a source of MCP-1 and MCP-2 when stimulated by most of the inducers tested. Granulocytes, however, were inefficient producers. Measles virus induced MCP-1 and MCP-2 in most cell types. In general, the yields of MCP-2 were at least 10-fold lower than those of MCP-1. It is concluded that, although MCP-2 is often coproduced with MCP-1, regulation of expression of the two chemokines is not identical. It remains to be studied under which pathological conditions MCP-2 is released in vivo and whether MCP-1 and MCP-2 can activate different target cells.
Evidence for defects in V(D)J rearrangements in patients with severe combined immunodeficiency.
Authors: Abe T , Tsuge I , Kamachi Y , Torii S , Utsumi K , Akahori Y , Ichihara Y , Kurosawa Y , Matsuoka H
Published: December 2022

We investigated the patterns of DNA rearrangements at loci for Ig JH genes in patients with severe combined immunodeficiency (SCID). Four SCID patients without B cells (B- SCID) and four SCID patients with B cells (B+ SCID) were examined. Bone marrow cells of these patients were transformed with EBV. The majority of the transformed cells from three B- SCID patients had the germline configuration at their JH gene loci. The rearranged fragments from one patient were analyzed extensively. The rearranged regions in all of the fragments had a common structure wherein two fragments derived from the JH-S mu region were connected inversely. The possible presence of rearranged forms of VHDJH and DHQ52JH sequences in bone marrow cells of two B- SCID patients were examined directly by the polymerase chain reaction (PCR) method. In one patient, we found neither a VHDJH sequence nor a DHQ52JH sequence within the range of sensitivity of the PCR method. In another patient, we found a VHDJH sequence at an extremely low level and DHQ52JH sequences at a relatively low level. Either RAG-1 or RAG-2 gene was not expressed in the B- SCID-derived cell lines. B+ SCID patients did not show any abnormalities in terms of VHDJH rearrangements. These results indicate that B- SCID may be caused by defects in factors involved in V(D)J rearrangements.
Macrophage-derived nitric oxide is involved in the depressed concanavalin A responsiveness of splenic lymphocytes from rats administered morphine in vivo.
Authors: Fecho K , Maslonek K A , Coussons-Read M E , Dykstra L A , Lysle D T
Published: December 2022

The present study examined the role of macrophage-derived nitric oxide in the suppressive effect of in vivo morphine administration on Con A-stimulated proliferation of splenic lymphocytes in rats. The results showed that concentrations of nitrite are significantly greater in Con A-stimulated splenocyte cultures from morphine-treated rats than in cultures from saline-treated rats, and that the depletion of macrophages from splenocyte cultures abolishes the suppressive effect of morphine on Con A-stimulated proliferation. Moreover, the addition of NG-monomethyl-L-arginine (NMMA) to Con A-stimulated splenocyte cultures attenuates the suppressive effect of morphine on mitogenic responsiveness. The addition of excess L-arginine to splenocyte cultures containing NMMA reverses the effect of NMMA and restores morphine's suppressive effect on Con A-stimulated proliferation, but the addition of D-arginine to splenocyte cultures containing NMMA does not restore the suppressive effect of morphine. Taken together, these findings demonstrate that the suppressive effect of in vivo morphine administration on Con A-stimulated proliferation of splenic lymphocytes involves macrophage-derived nitric oxide.
Expression of homing and adhesion molecules in infiltrated islets of Langerhans and salivary glands of nonobese diabetic mice.
Authors: Faveeuw C , Gagnerault M C , Lepault F
Published: December 2022

The nonobese diabetic mouse is a relevant model for insulin-dependent diabetes mellitus which results from the destruction of pancreatic beta cells by mononuclear cells infiltrating the islets of Langerhans. Other organs such as salivary glands display inflammatory infiltration. Using immunohistochemical and flow cytometry analyses, we have studied the expression of diverse homing and adhesion molecules in salivary glands and the pancreas in nonobese diabetic mice. In salivary glands, ICAM-1 was expressed by endothelial and dendritic cells within the lymphocytic infiltration. HEV-like structures expressing PNAd were observed in the areas of lymphocytic infiltration whereas MAdCAM-1 was absent. Lymphocytes infiltrating salivary glands expressed LFA-1 and Pgp-1 although Mel-14 Ag was absent. In infiltrated islets, ICAM-1 was expressed by endothelial cells, dendritic cells, and mononuclear cells. We confirm the presence of HEV-like structures expressing MAdCAM-1 and PNAd in inflamed islets. With regard to peripheral lymphocytes, the proportion of CD4 and CD8 cells expressing Mel-14 was decreased in the infiltrated islets, whereas the expression of LFA-1, Pgp-1, and LPAM-1/2 was increased. B lymphocytes exhibited up-regulation of LPAM-1/2. Moreover, the proportion of CD4, CD8, and B lymphocytes expressing CD69 was increased in the pancreas. These results indicate that first, infiltration of islets of Langerhans results at least partly from modifications of adhesion molecule expression in the pancreas, which allow extravasation of mononuclear cells into the islets via at least three different pathways; and second, that activated cells are concentrated in the infiltrates as compared with peripheral lymphoid organs.
CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. Potential role in regulating helper effector function.
Authors: Yellin M J , Sippel K , Inghirami G , Covey L R , Lee J J , Sinning J , Clark E A , Chess L , Lederman S
Published: December 2022

The T-BAM/CD40-L molecule on CD4+ T cells interacts with B cell CD40 molecules to deliver contact-dependent signals that drive B cell activation and Ig secretion. Cell surface T-BAM/CD40-L expression is transient and may be closely regulated in order to limit the activation and clonal selection of noncognate B cells. We demonstrate that B cells, but not non-B cells, rapidly and specifically down-modulate surface T-BAM/CD40-L expression in a contact-dependent and temperature-sensitive manner that renders T cells unable to activate resting bystander B cells. Because the ability to down-modulate T-BAM/CD40-L correlated with CD40 expression, the role of CD40 molecules in down-modulating its ligand was directly assessed. Anti-CD40 mAb, but not control mAb, block B cell-induced T-BAM/CD40-L down-modulation. Furthermore, CD40+ nonlymphoid transfectants specifically down-modulate surface T-BAM/CD40-L expression. B cells induce T-BAM/CD40-L internalization into cytoplasmic compartments in a process that is inhibited by cytochalasin B. Pretreatment of activated T cells with lysosomotropic agents does not affect CD40-induced down-modulation of surface T-BAM/CD40-L but results in a marked accumulation of T-BAM/CD40-L in cytoplasmic vesicles. Together, these studies strongly suggest that CD40 induced T-BAM/CD40-L down-modulation occurs, in part, by receptor-mediated endocytosis followed by lysosomal degradation and may represent a mechanism to regulate CD4+ T cell helper effector functions.
Kinetics and specificity of peptide-MHC class II complex displacement reactions.
Authors: de Kroon A I , McConnell H M
Published: December 2022

The peptide-induced acceleration of the dissociation of pre-formed complexes of the detergent-solubilized mouse class II molecules IEd and IEk with fluorescein-labeled peptides was investigated using high-performance size exclusion chromatography. While it is generally believed that functional complexes of MHC class II alpha beta heterodimers and peptides have a 1:1 stoichiometry, the data provide qualitative as well as quantitative kinetic evidence that the enhancement of the release of one peptide by a second peptide is due to a two-peptide intermediate. Different combinations of peptides were tested for their ability to accelerate each other's release from IEd. The importance of positive charge for the interaction with IEd was confirmed by the finding that not only dynorphin 1-13 but also poly-L-lysine (14-19 mer) and a peptide corresponding to a mitochondrial presequence (net charge +6) efficiently enhance the release of pre-bound peptides. SDS-PAGE analysis revealed that the efficiently displacing peptides do not stabilize the IEd alpha beta heterodimer at acidic pH, in contrast to the IEd-restricted antigenic peptide HEL 107-116. The data support a mechanism in which the second peptide binds specifically to the pre-formed class II-peptide complex, which, depending on the properties of the peptides involved, leads to the destabilization of the complex and the release of the first peptide.
Requirements for L-selectin in neutrophil-mediated lung injury in rats.
Authors: Mulligan M S , Miyasaka M , Tamatani T , Jones M L , Ward P A
Published: December 2022

L-selectin requirements in three models of acute lung injury in rats have been identified: systemic activation of complement after intravenous infusion of cobra venom factor (CVF) and intrapulmonary deposition of IgG or IgA immune complexes. In the CVF model of lung injury, treatment of rats with hamster monoclonal IgG anti-rat-L-selectin (HRL-1) induced significant neutropenia, necessitating the use of F(ab')2 fragments, which did not cause neutropenia. Treatment of rats with F(ab')2 anti-L-selectin (HRL-1) resulted in significant reductions in lung permeability and hemorrhage in the CVF model. Morphologically, this treatment abrogated adhesive interactions of neutrophils with the pulmonary vascular endothelium. In the IgG immune complex model of injury, the parameters of injury were significantly reduced as a result of treatment with HRL-1. In both models protection was associated with reductions in lung myeloperoxidase content. Treatment of rats with a F(ab')2 form of hamster monoclonal IgG non-blocking anti-L-rat selectin, HRL-2, failed to show protective effects in the CVF and IgG immune complex models of lung injury. In the IgA immune complex model of injury, which is neutrophil-independent and related to toxic products from pulmonary macrophages, no protective effects of anti-L-selectin (HRL-1) were found. Therefore, in neutrophil-dependent and oxygen radical mediated lung injury, L-selectin plays a requisite role in tissue recruitment of neutrophils. In the neutrophil-independent model of lung injury, no requirement for L-selectin appears to exist.
Modulation of thrombospondin receptor expression during HL-60 cell differentiation.
Authors: Suchard S J , Mansfield P J , Dixit V M
Published: December 2022

Thrombospondin (TSP), a multifunctional homotrimeric glycoprotein of approximately 450,000 M(r), is a component of the extracellular matrix that mediates the adhesive interactions of several different cell types including hematopoietic progenitor cells. We have used the promyelocytic leukemia HL-60 cell line to examine TSP receptor expression during differentiation of leukocytes along either the monocyte/macrophage or the polymorphonuclear leukocyte (PMN) pathway. 125I-labeled TSP binding to undifferentiated or differentiated HL-60 cells was time-dependent reaching saturation by 45 min. Undifferentiated HL-60 cells expressed a single class of heparin-inhibitable TSP receptors. Treating HL-60 cells with PMA induced their differentiation to macrophage-like cells and resulted in a concomitant 10-fold increase in TSP receptor expression. As with undifferentiated cells, a single class of heparin-inhibitable receptors was observed. Treating HL-60 cells with DMSO induced their differentiation to PMN-like cells and resulted in a fivefold increase in TSP receptor expression. However, in this case two classes of binding sites were apparent on PMN-like cells, only 40% of which were heparin inhibitable. This is reminiscent of TSP binding to normal peripheral blood PMN (S.J. Suchard, L.A. Boxer, and V.M. Dixit. 1991. J. Immunol. 147:651). In parallel studies, we also examined TSP synthesis during HL-60 cell differentiation. Undifferentiated HL-60 cells synthesized and secreted TSP as assessed by immunoprecipitation. TSP synthesis increased about fourfold when cells were differentiated toward PMN-like cells. In contrast, TSP was not detected in macrophage-like cells. RNase protection assays showed that TSP transcript levels paralleled TSP protein expression during differentiation. These findings suggest that expression of both TSP and TSP receptors are differentially regulated during blood cell maturation.
Tissue-specific regulation of IL-4 mRNA expression in human tonsil.
Authors: Secrist H , Egan M , Peters M G
Published: December 2022

The cellular and molecular regulation of IL-4 mRNA expression in human tonsillar T lymphocytes was examined to define the mechanisms responsible for biphasic IL-4 mRNA expression in this lymphoid organ. Tonsillar T cells expressed IL-4 mRNA in a biphasic manner with peaks at 8 and 24 h after PHA stimulation. De novo protein synthesis was not required for IL-4 mRNA expression because cycloheximide treatment of tonsillar MNC did not ablate the response. Nuclear runoff assays demonstrated transcription of the IL-4 gene at 8 and 24 h, which was not affected by addition of actinomycin D. Separation of T cells into naive (CD45RAhi/CD29lo) and primed (CD45RAlo/CD29hi) subpopulations revealed that although naive and primed T cells expressed IL-4 mRNA at 8 h, the 24-h peak of IL-4 mRNA expression was solely due to primed (CD45RAlo/CD29hi) Th cells. This effect was tissue specific and IL specific in that 1) primed peripheral blood T cells had only one peak of IL-4 mRNA expression at 8 h and 2) in primed tonsillar T cells, mRNA expression of IL-2, IL-6 and IL-2 receptor and c-myc was not delayed. Thus, IL-4 mRNA expression in the tonsil differs depending on the surface expression of different isoforms of the leukocyte common Ag. The tissue- and stimulus-specific regulation of IL-4 mRNA in different lymphoid tissues may play an important role in regional immunoregulation.
Functional and ligand binding specificity of the rabbit neutrophil IL-8 receptor.
Authors: Thomas K M , Taylor L , Prado G , Romero J , Moser B , Car B , Walz A , Baggiolini M , Navarro J
Published: December 2022

IL-8 mediates migration and activation of neutrophils. This study describes the functional and ligand binding specificity of the human intercrine peptides IL-8, neutrophil-activating peptide 2 (NAP-2), melanoma growth stimulatory activity (GRO), and platelet factor 4 (PF4) to rabbit neutrophils and mammalian cell lines transfected with rabbit IL-8 receptor cDNA (F3R). Rabbit neutrophil membranes bound 125I-labeled IL-8 and 125I-labeled NAP-2 but did not bind 125I-labeled GRO or 125I-labeled PF4. Rabbit neutrophils mobilized intracellular Ca2+ in response to IL-8 and NAP-2 but not to GRO or PF4. Monkey kidney cells (COS-7) and hamster lung fibroblasts (CCL-39) were transiently and stably transfected with the rabbit neutrophil IL-8 receptor F3R cDNA. COS-7 cells transfected with F3R cDNA bound 125I-labeled IL-8 but did not bind other IL-8-related peptides such as 125I-labeled NAP-2, 125I-labeled GRO, or 125I-labeled PF4. Furthermore, bound 125I-labeled IL-8 was only displaced by unlabeled IL-8 but not by unlabeled NAP-2, GRO alpha, or PF4. Consistent with this observation, stably transfected CCL 39 cells expressing F3R cDNA mobilized Ca2+ only in response to IL-8. We conclude that F3R cDNA encodes a functional IL-8 receptor isotype with strict ligand binding specificity for IL-8, that rabbit neutrophils do not bind human GRO alpha, and it is suggested that rabbit neutrophils contain in addition to the F3R protein another IL-8 receptor isotype with broad ligand specificity or a distinct NAP-2 receptor.
Naturally processed peptides longer than nine amino acid residues bind to the class I MHC molecule HLA-A2.1 with high affinity and in different conformations.
Authors: Chen Y , Sidney J , Southwood S , Cox A L , Sakaguchi K , Henderson R A , Appella E , Hunt D F , Sette A , Engelhard V H
Published: December 2022

An equilibrium binding assay was used to directly measure the relative affinities of naturally processed 9-mer, 10-mer, and 12-mer peptides for the human class I MHC molecule HLA-A2.1. The peptides exhibited a range of affinities with IC50 values of 11 to 214 nM. The mode of interaction between these peptides and HLA-A2.1 was examined using peptides in which Asp had been substituted for suspected anchor residues. Regardless of length, the previously identified Leu at position 2 relative to the amino terminus was critical for peptide binding. While the carboxyl terminal residue was also critical for the binding of a 9-mer peptide, it was much less important in the binding of longer peptides. Additional residues close to the carboxyl terminus that contained aliphatic hydrocarbon side chains were of similar or greater importance in peptide binding. In addition, residue at position 3 also appeared to be important for the binding of longer peptides. The data suggest that different naturally occurring longer peptides can bind in different conformations to class I MHC molecules. While one of these is similar to the kinked conformation described by others, another conformation would involve an extension of the carboxyl terminus out of the class I binding site. The ability of MHC molecules to accommodate the same peptide in different conformations would appear to have distinct advantages to the immune system.
T cell determinant structure of myelin basic protein in B10.PL, SJL/J, and their F1S.
Authors: Bhardwaj V , Kumar V , Grewal I S , Dao T , Lehmann P V , Geysen H M , Sercarz E E
Published: December 2022

Recent experiments have shown that during the course of chronic experimental allergic encephalomyelitis, there is a shift in the determinant hierarchy away from the dominant to other subdominant and cryptic self determinants. It was therefore of interest to define the pattern of dominance for mouse myelin basic protein in the three commonly used experimental allergic encephalomyelitis model strains of mice, i.e., B10.PL, SJL/J, and (SJL x B10.PL)F1. Our studies indicate that many cryptic determinants are demonstrable, which only activate T cells on injection as individual peptides and not with the native protein. The core amino acid residues of the various determinants are defined and range in size between 5 and 10 amino acids. Interestingly, there is a bias toward H-2u-restricted response vis-a-vis the H-2s-restricted response in the (SJL x B10.PL)F1 strain. The TCR V beta 8.2 gene segment was not predominantly used for responses to other determinants, although some B10.PL and (SJL x B10.PL)F1 cell lines expressed V beta 8.2 more than others. This study represents the most comprehensive analysis so far of the pattern of dominant and cryptic proliferative T cell determinants and their core sequences for mouse myelin basic protein.
Induction of allograft tolerance in rats by an HLA class-I-derived peptide and cyclosporine A.
Authors: Nisco S , Vriens P , Hoyt G , Lyu S C , Farfan F , Pouletty P , Krensky A M , Clayberger C
Published: December 2022

T cell recognition of MHC molecules initiates a cascade of events resulting in allograft rejection. CTLs damage the graft by targeting nonself-MHC class I molecules. We and others have previously shown that small synthetic peptides corresponding to regions of certain MHC class I molecules can inhibit the CTL response against MHC class I alloantigens in vitro. Here we report that rat heart allografts survived survived indefinitely when transplanted into recipients treated with a synthetic peptide corresponding to residues 75-84 of (B7.75-84) in combination with a subtherapeutic dose of cyclosporine A. Furthermore, this treatment induced long-term donor-specific tolerance that was mediated by anergic cells, indicating that such peptides may have potential as therapeutics for human organ transplantation.
Superantigen-like properties of an antibody bispecific for MHC class II molecules and the V beta domain of the T cell antigen receptor.
Authors: Yagi J , Nakata M , Uchiyama T , Nishikawa M , Mizushima Y , Nishioka K , Ito K , Yagita H , Okumura K , Janeway C A
Published: December 2022

A working model for the action of superantigens (SAg) is that they are simple proteins having binding sites for both MHC class II molecules and the V beta domain of the TCR. Binding of a SAg to both molecules cross-links the TCR, inducing a biologic response. In this study, we have tested this working model using a SAg mimic consisting of a hybrid Ab bispecific for the murine MHC class II molecule I-E and the V beta 8 domain of the murine TCR. The bispecific Ab activates V beta 8-bearing T cells only in the presence of I-E molecules on APC when tested in vitro. The effect of the bispecific Ab in vivo revealed both clonal deletion and a reduction in the responsiveness of V beta 8-bearing T cells. Thus, the results suggest that molecules distinct from SAg that can bind to both MHC class II molecules and the V beta domain of the TCR can mimic the biologic actions of a SAg.
Monomeric homologue of mammalian CD28 is expressed on chicken T cells.
Authors: Young J R , Davison T F , Tregaskes C A , Rennie M C , Vainio O
Published: December 2022

A mAb recognizing a 40- to 44-kDa monomeric molecule on the surface of chicken T cells was used to screen a cDNA expression library made from Con A-stimulated chicken spleen cells. The sequence of the cDNA obtained encoded a molecule having 50% amino acid sequence identity with mammalian CD28, but the cysteine residue involved in the inter-chain bridge of the mammalian CD28 homodimer was not conserved in the chicken sequence. The molecule produced in transfected COS-7 cells was also recognized by another mAb that had previously been thought to recognize an avian homologue of CD2. The sequence data establish that this molecule is a homologue of mammalian CD28 in the strict evolutionary sense.
Lipopolysaccharide and cytokine augmentation of human monocyte IgA receptor expression and function.
Authors: Shen L , Collins J E , Schoenborn M A , Maliszewski C R
Published: December 2022

Receptors for IgA (Fc alpha R) are found on phagocytic cells in the peripheral blood and tissues associated with mucosal areas where IgA Abs constitute a major line of defense. Because Fc alpha R are capable of triggering protective functions of monocytes and neutrophils, such as phagocytosis and the oxidative burst, they may be important in amplifying the antimicrobial effects of IgA. Various cytokines play a role in regulating function and FcR expression of monocytes, macrophages, and neutrophils. The present studies examine the modulation of monocyte Fc alpha R by LPS and cytokines. LPS strongly up-regulated monocyte Fc alpha R expression. TNF and IL-1, produced in response to LPS, promoted Fc alpha R increase, as did GM-CSF; whereas IFN-gamma down-regulated Fc alpha R. Increased receptor expression was accompanied by augmented IgA-mediated phagocytosis. An increase in Fc alpha R-specific mRNA was detected in monocytes treated with TNF, IL-1, GM-CSF, and LPS; whereas message was reduced in cells treated with IFN-gamma. Monocyte-derived macrophages and cells of the Monomac 6 monocyte-like line expressed greater numbers of Fc alpha R than monocytes but were less responsive to LPS and TNF. Cell lines THP-1 and U937, which expressed similar or lower levels of Fc alpha R than monocytes, displayed an increase in Fc alpha R in response to LPS and, to various degrees, to TNF, IL-1, and GM-CSF. These results indicate that Fc alpha R on monocytes are modulated by endotoxin and an array of cytokines distinct from those that regulate expression of FcR for IgG.
The NF-beta A-binding element, not an overlapping NF-IL-6-binding element, is required for maximal IL-1 beta gene expression.
Authors: Buras J A , Monks B G , Fenton M J
Published: December 2022

NF-beta A is a monocyte, neutrophil, and B cell-specific nuclear protein that is involved in regulation of the IL-1 beta gene. These studies further define the functional role of NF-beta A in RAW264.7 monocytic cells by using transient transfection analysis. We showed that NF-beta A was able to activate transcription from a heterologous promoter in a distance-independent and dose-dependent manner. NF-beta A also appeared to function in a positionally independent manner within the IL-1 beta cap-site proximal (CSP) promoter. NF-beta A was required for maximal IL-1 beta gene expression directed by the upstream LPS-inducible enhancer element. Deletion of the NF-beta A-binding sequence resulted in an 80% reduction in basal reporter gene activity and an 86% reduction in LPS-inducible reporter gene activity in constructs containing only the enhancer and CSP promoter. Other regulatory elements located between the enhancer and the cap site were not able to substitute functionally for the absence of NF-beta A. Recently, other investigators have reported that IL-1 beta CSP promoter function was decreased by introducing multiple mutations within both the NF-beta A-binding sequence, and a putative overlapping NF-IL-6-binding sequence. We have found that these mutations predominantly affect NF-beta A binding. Furthermore NF-beta A, and not NF-IL-6, was required for supporting basal and LPS-inducible transcription from a minimal IL-1 beta CSP promoter (positions -58 to +11). This promoter region did not appear to direct monocyte-specific IL-1 beta gene expression because reporter constructs containing the IL-1 beta CSP promoter were also active in transiently transfected HeLa cells.
Functional and phenotypic change of T cells in murine acquired immune deficiency.
Authors: Kanagawa O , Gayama S , Vaupel B
Published: December 2022

Infection of susceptible C57BL/6 mice with defective LP-BM5 murine leukemia virus causes disease termed murine acquired immune deficiency syndrome (MAIDS). The disease is characterized by lymphoadenopathy, hyperimmunoglobulinemia, and immune deficiency in both T and B cell functions. The development of disease requires the presence of mature T cells, especially CD4 T cells, and B cells. It has previously been shown that a B cell tumor line derived from MAIDS mouse stimulated a large fraction of unprimed T cells based on TCR V beta chain expression. This stimulatory activity was assumed to be mediated by a superantigen encoded by MAIDS virus. The stimulation of T cells by viral superantigen was thought to play a role in the development of the disease. To examine the role of T cell reactivity to MAIDS superantigen, we used TCR transgenic mice. There are two distinct T cell populations which can be distinguished based on their TCR expression and function in the TCR transgenic mice, one bearing the transgene derived alpha- and beta-chain TCR that is nonreactive to MAIDS superantigen and the other bearing an endogenous alpha- but transgene-derived beta-chain TCR that is reactive to superantigen. Unlike T cells found in noninfected TCR transgenic mice, anergic T cells expanding in virally infected TCR transgenic mice are homogeneous for the TCR phenotype, indicating the presence of a selection of T cells based on their TCR expression. T cell hybridomas established by fusing T cells from virus-infected transgenic mice to thymoma cell line are also anergic. We found mRNA of defective LP-BM5 virus in a majority of T cell hybridomas from virus-infected mice but not from noninfected mice. By using in vitro infection of T cell clones with recombinant virus containing LP-BM5 MAIDS virus gag gene, we have demonstrated that virus infection directly abrogated the Ag-specific reactivity of T cells. The establishment of anergic T cell hybridomas and the in vitro infection of T cells with recombinant viruses would be a useful tool in the analysis of biochemical and molecular mechanisms of T cell dysfunction in MAIDS.
Bovine conglutinin gene exon structure reveals its evolutionary relationship to surfactant protein-D.
Authors: Liou L S , Sastry R , Hartshorn K L , Lee Y M , Okarma T B , Tauber A I , Sastry K N
Published: December 2022

Bovine conglutinin (BC), a member of the mammalian C-type collectin subfamily, is a serum protein synthesized in liver that is believed to play a role in natural host defense. Previously, we have characterized a full length BC cDNA and we now describe the partial characterization of a genomic clone that encodes for the BC gene (CGN1). BC is encoded by nine exons spanning > 11 kb and has been localized previously to band 18 of bovine (Bos taurus) chromosome 28. Genomic sequencing demonstrated that the signal peptide/amino-terminal domain, the carbohydrate recognition domain, and the linking peptide, a domain between the collagenous region and the carbohydrate recognition domain, are each encoded by a single exon. The collagenous domain is split into five exons, with the 5' most region being located within the exon that also encodes the signal peptide/amino terminus. The remaining four collagenous domain exons are tandemly arranged with lengths of 117, 108, 108, and 117 bp, respectively. Overall, the BC genomic organization is very similar to that of the human surfactant protein-D gene, SFTP4. On the basis of identical collagen domain structures, we suggest that conglutinin and bovine surfactant protein-D evolved from a gene duplication event occurring in Bovidae after divergence from other mammals.
Antigen receptors on Thy-1+ CD3- CS-initiating cell. In vitro desensitization with hapten-amino acid or hapten-Ficoll conjugates, versus hapten-protein conjugates, suggests different antigen receptors on the immune cells that mediate the early and late components of murine contact sensitivity.
Authors: Garssen J , Van Loveren H , Kato K , Askenase P W
Published: December 2022

In murine contact sensitivity (CS) models, cutaneous immune responses are caused by the activity of two different Ag-specific Thy-1+ CD5+ cells. These two different cell subsets act in an obligate sequence to mediate separate early and late components of CS that are accompanied respectively by skin swelling responses at 2 and 24 h after local challenge with the hapten. The early-acting CS-initiating cells are not conventional T cells inasmuch as they are surface negative for CD4, CD8, and CD3. In contrast to this non-MHC-restricted, CS-initiating cell, the classical late-acting CS effector T cell that is recruited locally is CD3+, CD4+, CD8-, and is MHC-restricted. In our study, we have conducted experiments in which a mixture of immune CS-initiating and CS-effector cells were desensitized by incubation in vitro at 37 degrees C with various hapten-amino acid and hapten-carrier conjugates, before i.v. transfer and subsequent ear challenge of normal recipient mice. Desensitization was achieved with multivalent complexes of the relevant hapten conjugated to a variety of unrelated nonmurine carrier proteins, and, in some instances, with monovalent hapten conjugated to amino acid. Because the CS-mediating cells were desensitized in a hapten-specific manner, but these same cells transferred CS reactivity that was hapten/MHC-class II specific, it was suggested previously that these results argued in favor of a two-receptor model for T cell recognition of Ag, one receptor for hapten and the other for MHC. Our study suggests that these findings need to be reinterpreted because CS reactions are mediated by two different, Thy-1+ Ag-specific cells that act in an obligate sequence. Our data suggest that the late-acting Ag/MHC-specific CS-effector T cells are not hapten-specific. In fact, desensitization with hapten alone has a locus of action solely on the Ag receptor of the first-acting Thy-1+ CS-initiating cells--which are therefore truly hapten-specific and which are required for local recruitment of the Ag/MHC-specific late CS-effector T cells.
Modulation of fibronectin and thymic stromal cell-dependent thymocyte maturation by retinoic acid.
Authors: Meco D , Scarpa S , Napolitano M , Maroder M , Bellavia D , De Maria R , Ragano-Caracciolo M , Frati L , Modesti A , Gulino A
Published: December 2022

Retinoic acid (RA) controls the differentiation of a variety of cell types, although its role in influencing T cell development and the mechanisms potentially involved have not been thoroughly investigated. To study the ability of RA to modulate T cell development, we established a thymic stromal cell line (TC-1S) that supports the phenotypic maturation of CD4-8- double negative (DN) or CD3-4-8- triple negative (TN) thymocyte precursors. Cocultures of either DN or TN thymocytes on a monolayer of TC-1S cells resulted in the appearance of thymocytes with a more mature phenotype (CD4+8+ double positive, CD4+ or CD8+ single positive, and CD3(low) cells). Double negative T cell contact with TC-1S cells also increased the production of fibronectin (FN) by the thymic stroma and the expression of the VLA-4 FN receptor on the DN cells. Ab-mediated inhibition of the interaction between FN and its receptors significantly reduced the level of induced T cell maturation. Addition of RA either to TC-1S cells alone or to the coculture with DN cells decreased stromal cell FN expression, antagonized DN cell-induced increase in stromal cell FN production and significantly inhibited in vitro thymocyte maturation. The effects of RA were likely mediated by RA acid receptors alpha and gamma expressed both in DN thymocytes and TC-1S cells. Together these data suggest that FN/VLA-4 interaction may be an important component of stromal cell-dependent thymocyte phenotypic differentiation and that this interaction can be one of the targets for the influence of RA in T cell development.
Modulation of signaling via the B cell antigen receptor by CD21, the receptor for C3dg and EBV.
Authors: Luxembourg A T , Cooper N R
Published: December 2022

CD21 is the receptor for C3dg and EBV. Several reports have shown that these CD21 ligands, and certain anti-CD21 mAb, trigger B cell activation, particularly when combined with Ag receptor ligation. However, the characteristics, biologic functions, and importance of this CD21-signaling pathway are unknown. We have used a model we recently developed to study B cell activation induced by complex particulate Ag, such as immune complexes and viruses, to begin to examine these questions. In the current studies, we incubated purified small resting B cells with 100-nm latex beads bearing various combinations of CD21 ligands and mAbs to CD19, CD35, and the Ag receptor. CD21, CD19, and CD35 have all been implicated in modulating membrane IgM initiated signaling. Beads coated with mAb to the C3dg/EBV-binding portion of CD21, but not mAb to other portions of the CD21 molecule, triggered B cell homotypic aggregation. Beads coated with the same CD21 ligands, although inactive alone, synergized with anti-IgM mAb in greatly increasing (20- to 180-fold) mRNA levels of the c-fos nuclear proto-oncogene. Signaling via CD21 was tyrosine kinase dependent. Levels of c-myc mRNA were not altered by CD21 ligands. Anti-CD19 and anti-CD35 mAb did not augment signaling via membrane IgM as assessed by changes in c-fos mRNA levels. These findings indicate that CD21 ligands binding to the C3dg/EBV-binding site of CD21 markedly augment B cell activation initiated by Ag receptor ligation via a selective, c-fos-dependent signaling pathway.
HLA restriction and TCR usage of T lymphocytes specific for a novel candidate autoantigen, X2 MBP, in multiple sclerosis.
Authors: Voskuhl R R , Robinson E D , Segal B M , Tranquill L , Camphausen K , Albert P S , Richert J R , McFarland H F
Published: December 2022

Previous investigations of the major 18.5-kDa isoform of myelin basic protein (MBP) as a target autoantigen in multiple sclerosis (MS) have failed to identify an epitope uniformly recognized with higher frequency in MS patients compared with controls. Because remyelination has been observed in MS plaques, we were prompted to investigate T cells specific for myelin protein isoforms with up-regulated expression during remyelination. We have recently described such T cells that recognize the exon 2-encoded region of MBP (X2 MBP), a sequence included in the 21.5- and 20.2-kDa isoforms of MBP. These cells were shown to be CD4+, HLA class II restricted, and cytolytic. In members of one multiplex MS family, X2 MBP-specific T lymphocytes were as prevalent as T cells specific for immunodominant regions within the major 18.5-kDa isoform of MBP. The present study characterizes X2 MBP-specific T cell responses in additional multiplex MS family members as well as in heterogeneous (non-familial) MS patients and in healthy controls. The frequencies of X2 MBP-specific T cells in each of the affected family members from two of three MS families were significantly increased as compared with both the heterogeneous MS group and the healthy control group. Also, X2 MBP-specific T cell lines from affected family members were primarily restricted to molecules encoded by the DR2/DQw1 allele. Although TCR usage was generally heterogeneous, there was evidence of intraindividual sequence identity. These data suggest that: 1) Myelin proteins with up-regulated expression during the course of disease should be considered as candidate autoantigens in MS. 2) The functional basis for the association of DR2/DQw1 inheritance with MS susceptibility may be related to presentation of autoantigens by this allele. 3) TCR therapy will need to be individually tailored to target the most prevalent autoantigen-specific response.
Octamer factors exert a dual effect on the IL-2 and IL-4 promoters.
Authors: Pfeuffer I , Klein-Hessling S , Heinfling A , Chuvpilo S , Escher C , Brabletz T , Hentsch B , Schwarzenbach H , Matthias P , Serfling E
Published: December 2022

The promoters of IL-2 and IL-4 genes contain multiple binding sites for octamer factors. In peripheral T lymphocytes and several T cell lines, both the ubiquitous Oct factor Oct-1 and the lymphocyte-specific factor Oct-2 are expressed and bind to the IL-2 and IL-4 promoters. Prominent octamer binding sites of IL-2 and IL-4 promoters are their upstream promoter sites (UPS) which share 14 identical nucleotides. Multiple copies of the IL-2 and IL-4 UPS act as inducible enhancers in T cells, and their induction is inhibited by the immunosuppressant cyclosporin A (CsA). Closely linked to the octamer site, the IL-2 UPS contains a non-canonical AP-1 binding (TRE) site, and mutation in either site to a non-functional factor binding site impairs the induction of the IL-2 promoter. The binding of AP-1 and octamer factors to the IL-2 UPS DNA overlaps, and the tight association and functional cooperation of octamer with AP-1 factors is of crucial importance for the inducible IL-2 UPS activity. Introduction of five or ten spacer nucleotides between both IL-2 UPS sites results in a drastic reduction of inducible UPS activity, both in the loss of suppression by CsA and stimulation by the Ca(2+)-dependent phosphatase calcineurin. Within the IL-4 UPS the Oct and TRE-like motifs are separated by a binding site of nuclear factor of activated T cells (NF-AT). This site shares nine out of ten bp with an IL-2 NF-AT site. The strong binding of NF-ATp to the IL-4 UPS site suppresses the simultaneous binding of Oct factors to the IL-4 UPS. Because the two other Oct binding sites of IL-4 promoter show a similar sequence configuration, the binding of NF-AT seems to prevent the simultaneous binding of Oct factors to the IL-4 promoter. By contrast, both classes of factors bind simultaneously to the IL-2 promoter, and their tight association with AP-1 enhances the IL-2 promoter activity.
Association of autoantibodies to topoisomerase I and the phosphorylated (IIO) form of RNA polymerase II in Japanese scleroderma patients.
Authors: Satoh M , Kuwana M , Ogasawara T , Ajmani A K , Langdon J J , Kimpel D , Wang J , Reeves W H
Published: December 2022

Autoantibodies to RNA polymerases (RNAP) I and III are highly specific for scleroderma (SSc), whereas autoantibodies to RNAP II are associated with systemic lupus erythematosus (SLE) and overlap syndromes, as well as SSc. The specificities of autoantibodies to RNAP I, II, and III in 129 SSc sera were investigated in the present study. Immunoprecipitation and pulse-chase analysis demonstrated several patterns of autoantibody recognition of RNAPs. Some sera immunoprecipitated RNAP II only after its largest subunit was phosphorylated, suggesting that they contained autoantibodies that recognized an epitope carrying a phosphoamino acid. Autoantibody recognition of all three classes of RNAPs was influenced strongly by race. Although in SLE, autoantibodies to the phosphorylated form of RNAP II (RNAP IIO) were identified in all races, in SSc, these autoantibodies were seen in 21% of Japanese and 5% of Black patients, but never in Caucasians. A striking association of anti-RNAP IIO with anti-topoisomerase I (topo I) autoantibodies was found in Japanese and Black SSc, but not SLE, patients. However, anti-topo I Abs were not associated with anti-RNAP IIO in Caucasians. Japanese SSc patients who were positive for both anti-RNAP IIO and anti-topo I Abs had a significantly higher frequency of diffuse disease, pigmentation changes, flexion contractures, and acro-osteolysis than patients having autoantibodies to topo I alone, and were diagnosed at a younger age (p < 0.05). These data suggest that genetic factors (possibly HLA-linked) influence autoantibody specificity, and that different autoantibody fine specificities may either cause, or be predictive of, different clinical outcomes.
Comparison of two promoters controlling expression of secreted or intracellular IL-1 receptor antagonist.
Authors: Butcher C , Steinkasserer A , Tejura S , Lennard A C
Published: December 2022

IL-1R antagonist can be expressed as a secreted glycoprotein (sIL-1Ra) or as an intracellular nonglycosylated form (icIL-1Ra). The two isoforms only differ in their N-terminal sequences and are thought to originate, by selection of alternative first exons, from a single gene (IL-1RN). We have previously described a clone that encodes the exons and promoter for sIL-1Ra. In this work, we describe and characterize a clone that contains the further upstream, alternative first exon used to generate icIL-1Ra. Sequences upstream to this alternative exon are demonstrated to have promoter activity in the human colon epithelial cell line HT29, whereas the sIL-1Ra promoter has undetectable activity. Both the icIL-1Ra and sIL-1Ra promoters were active when transfected into THP-1 cells. Neither promoter was responsive to PMA or LPS when introduced into HT29 or THP-1 cells. Activity of the endogenous icIL-1Ra and sIL-1Ra promoters was also investigated. In agreement with the transfected icIL-1Ra promoter, the endogenous icIL-1Ra gene is active in HT29 cells, though induced by PMA. In contrast to the transfected icIL-1Ra promoter, the endogenous icIL-1Ra gene is inactive in THP-1 cells and induced by PMA. Our data complete the structure of the human IL-1RN gene, demonstrating that the two forms of IL-1Ra are produced from the same gene by use of alternative first exons. We also show that there are two distinct promoters that control IL-1Ra expression, that lie upstream to each alternative first exon.