bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoreactivity and function of oligosaccharides in cobra venom factor.
Authors: Gowda D C , Petrella E C , Raj T T , Bredehorst R , Vogel C W
Published: December 2022

Cobra venom factor (CVF) is the nontoxic C-activating glycoprotein in cobra venom. It is a structural and functional analogue of complement component C3. Previous work has established that the major oligosaccharide chain of CVF is a symmetric fucosylated biantennary complex-type N-linked chain containing an alpha-galactosylated Le(x) antigenic epitope, Gal alpha 1-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1, a novel carbohydrate structure. We show that naturally occurring anti-alpha-Gal Ab present in normal human serum binds to CVF. In view of a possible human application of CVF, we studied the effect of this anti-alpha-Gal Ab on CVF function as well as the effect of oligosaccharide modification or removal on CVF activity and immunoreactivity with the anti-alpha-Gal Ab. The immunoreactivity of CVF with the anti-alpha-Gal Ab is abolished upon de-alpha-galactosylation or complete deglycosylation. De-alpha-galactosylated CVF and deglycosylated CVF were found to be equally active in forming a stable C3/C5 convertase with human factor B and in decomplementing human serum indicating that the oligosaccharide chains of CVF are not required for its C-activating function. Consistent with the unaltered functional activity, deglycosylation of the molecule caused only minor changes in secondary structure as judged by far UV circular dichroism analysis. However, the oligosaccharide chains appear to contribute to the thermal stability of CVF, because deglycosylation caused the molecule to be more sensitive to temperature. Inasmuch as rCVF produced in mammalian cells can be expected to contain sialylated oligosaccharide chains, we also prepared sialylated CVF by enzymatic sialylation of de-alpha-galactosylated and de-alpha-fucosylated CVF with 2,6-sialyltransferase. It was found that the secondary structure and the activity of sialylated CVF were unchanged compared to native CVF.
Sublytic complement attack exposes C-reactive protein binding sites on cell membranes.
Authors: Li Y P , Mold C , Du Clos T W
Published: December 2022

C-reactive protein (CRP) is an acute phase serum protein synthesized by the liver. CRP has been localized to acute inflammatory sites and has been postulated to facilitate the removal of damaged cells. CRP binds to a number of ligands that may be present in inflammatory sites, and the extent to which individual ligands are involved in its binding to tissue sites is unknown. Complement activation is important in the tissue damage in many inflammatory conditions causing cell membrane damage and recruitment of inflammatory cells. This paper describes the binding of CRP to complement-damaged cell membranes. Raji cells activate the alternative complement pathway resulting in the deposition of C3b and membrane attack complexes (MAC) on the cell membrane. However, Raji cells are relatively resistant to killing by human complement. Treatment of Raji cells with human serum led to calcium-dependent phosphocholine-inhibitable CRP binding. CRP binding was eliminated by depletion of C3, C5, or C8 and reduced by depletion of C9 from serum. CRP binding preceded cell death and co-localized with MAC on cell membranes. CRP binding to complement-treated liposomes required phosphatidylcholine in addition to the MAC indicating that membrane phospholipids rather than the MAC proteins provide the binding sites for CRP. However, for both liposomes and Raji cells disruption of the lipid bilayer by complement attack was required for CRP binding to occur. These results support the hypothesis that CRP binding at sites of inflammation may be mediated by exposed phospholipids on damaged cell membranes.
Mast cell and neutrophil expression of dog mast cell protease-3. A novel tryptase-related serine protease.
Authors: Yezzi M J , Hsieh I E , Caughey G H
Published: December 2022

In previous work the primary structure of a previously unknown protease was deduced from the sequence of a dog mastocytoma cDNA. The predicted preproprotein shares some features with mast cell tryptases but is no more than 49% identical in sequence to known trypsin-like enzymes, including dog tryptase. This study explores the expression of this protein, termed dog mast cell protease-3 (dMCP-3). A polyclonal Ab was raised to a peptide corresponding to residues 166-181 of the deduced sequence. Anti-dMCP-3(166-181) Ig recognizes dMCP-3 expressed as a CheY fusion protein in Escherichia coli and binds to a approximately 36-kDa protein in extracts of dog mastocytomas. The Ab does not recognize dog tryptase, dMCP-3s closest known relative in mastocytoma cells. When used with fluorescein-conjugated and alkaline phosphatase-conjugated secondary Abs, anti-dMCP-3(166-181) Ig yields punctate cytoplasmic staining in mastocytoma cells, suggesting localization to intracellular granules. Staining is greatly reduced by preincubation with synthetic dMCP-3 peptide, supporting the specificity of the Ab. Immunohistochemical staining of normal dog tissues reveals scattered dMCP-3 reactive cells in skin, intestine, trachea, and lung parenchyma. Double staining with Ab and methylene blue shows that anti-dMCP-3(166-188) Ig recognizes extravascular mononuclear tissue cells with metachromatic granules. In addition, cytoplasmic staining is seen in polymorphonuclear leukocytes within vessels in tissue sections and in leukocytes harvested from blood. Hybridization of dMCP-3 cDNA to dog skin RNA provides further evidence of dMCP-3 gene transcription in normal tissue. Thus, this study provides immunochemical evidence of dMCP-3 expression in dog mast cell tumors, normal tissue mast cells, and neutrophils.
Elevated in vivo and in vitro secretion of CD8-alpha molecules in patients with systemic lupus erythematosus.
Authors: Linker-Israeli M , Hyun S , Ozeri-Chen T , Wallace D J , Banks K , Klinenberg J R
Published: December 2022

Endogenously activated CD8+ cells contribute to the aberrant immune regulation that characterizes SLE. Because stimulation of CD8+ cells with lectin/Ag triggers release of CD8-alpha molecules (sCD8), we measured, in patients with SLE, serum sCD8 content, its correlation with disease activity, and the in vitro release of sCD8 by SLE PBMCs. sCD8 levels, measured by ELISA in sera of 50 SLE patients, were higher than normal in 16 out of 21 mildly active and 15 out of 15 active SLE patients. sCD8 correlated positively with the clinical index for disease activity (r = 0.57, p = 0.001) and with sIL-2R levels (r = 0.52, p < 0.0001), and negatively with serum C3 levels (r = -0.5, p < 0.04). Freshly isolated SLE PBMCs had higher than normal CD8-alpha mRNA levels and secreted high levels of sCD8 in vitro (p < 0.05 vs control PBMC). sCD8 in vitro release by SLE PBMCs may be modulated by non-CD8+ cells. Thus, anti-CD2 mAb inhibited sCD8 release, whereas anti-HLA mAb increased it in unseparated PBMCs, but not in CD8+ enriched cultures. Moreover, sCD8 release increased significantly in PBMC cultures enriched for CD8+ DR+ cells by negative selection. Added-back monocytes decreased sCD8 to original levels, but not after glutaraldehyde fixation, nor in the presence of anti-HLA mAb. Further, lectin-induced IgG production and proliferation were reduced in the presence of sCD8, suggesting that the soluble CD8 molecules may be immunoregulatory. Because the high sCD8 levels in sera of active SLE likely reflect pathogenic cell activation, serum CD8 content may be an additional serologic activity marker, and its study could provide insights into mechanisms of disease.
Idiotypic intravaginal vaccination to protect against candidal vaginitis by secretory, yeast killer toxin-like anti-idiotypic antibodies.
Authors: Polonelli L , De Bernardis F , Conti S , Boccanera M , Gerloni M , Morace G , Magliani W , Chezzi C , Cassone A
Published: December 2022

The principles of idiotypic (Id) vaccination were used to immunize against vaginitis caused by Candida albicans, a widespread and sometimes intractable disease in women. To this aim, a murine mAb (KT4, IgG1) neutralizing in vitro the anti-Candida activity of a yeast killer toxin (YKT) was used as an Id vaccine to elicit Abs with toxin-like activity in a rat vaginitis model. Nonimmunized and isotype-matched, irrelevant mAb-immunized rats served as controls. An effective protection was obtained in Id-vaccinated animals, as demonstrated by a highly significant decrease in vaginal Candida CFU compared with controls. The protection was associated with rising vaginal titers of anti-idiotypic Abs (IdAb), prevalently of the IgA isotype, that were able to passively transfer the protective state to nonimmunized animals. The vaginal IdAb possessed YKT-like activity because they were able to kill in vitro the challenging fungal cells, and this killing was neutralized by the mAb KT4. Overall, these data demonstrate that secretory IdAb elicited by intravaginal Id vaccination with mAb KT4 protected the rats from the infectious challenge with Candida albicans by molecular mimicking YKT activity as its internal image.
B7 expression on thymic medullary epithelium correlates with epithelium-mediated deletion of V beta 5+ thymocytes.
Authors: Degermann S , Surh C D , Glimcher L H , Sprent J , Lo D
Published: December 2022

Recent evidence suggests that I-E+ thymic epithelium, especially medullary epithelium, can induce partial deletion of superantigen-reactive T cells expressing TcR V beta 5, V beta 11, and V beta 17. To seek further information on this issue, we constructed bone marrow chimeras in which MHC class II I-E is expressed on thymic epithelial cells at various levels and locations; the chimeras were reconstituted with stem cells from TcR V beta 5 transgenic mice. Intrathymic deletion of V beta 5 T cells was restricted to relatively mature T cells (expressing high TcR levels), and the degree of deletion correlated with the density of I-E expression in the thymic medulla rather than in the thymic cortex; selective I-E expression in medullary epithelium caused prominent deletion. Interestingly, immunostaining of normal and chimeric mice revealed that expression of B7 (the ligand for CD28) is largely restricted to a subset of medullary epithelial cells; these cells are I-E+ and co-express a specific carbohydrate bound by the lectin UEA-1. B7 expression was lower in thymuses of class II-deficient mice (A beta b-/-) and T-cell-deficient mice (SCID), suggesting that B7 expression is up-regulated during CD4+ thymocyte selection. In support of this idea, B7 expression in the thymus was restored to a normal level in bone marrow reconstituted SCID mice. Because B7 expression correlates with a costimulatory signal for T cells, selective expression of B7 and related antigens on I-E+ medullary epithelium may explain why these cells play a more prominent role in V beta deletion than cortical epithelium.
Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo.
Authors: Hamann A , Andrew D P , Jablonski-Westrich D , Holzmann B , Butcher E C
Published: December 2022

Lymphocyte recirculation through different organs is thought to be regulated by adhesion molecules ("homing receptors") recognizing tissue-specific vascular addressins on endothelium. Here we show that the alpha 4/beta 7-integrin has a key role in the migration of mouse lymphocytes to mucosal sites. Homing to Peyer's patches but not to peripheral lymph nodes is inhibited by Fab fragments of mAb PS/2 against the alpha 4-integrin chain, by mAb DATK32 recognizing a combinatorial epitope on the alpha 4/beta 7-integrin, and by mAb FIB30 against the beta 7-chain. The Abs significantly reduce homing of lymphocytes to the intestine, as well. The migration of immunoblasts to gut and gut-associated lymphoid tissue also involves the alpha 4/beta 7-integrin heterodimer. Another anti-alpha 4 Ab, R1-2, which blocks lymphocyte binding to Peyer's patches in the Stamper-Woodruff frozen section assay and lymphocyte adhesion to VCAM-1 and fibronectin, has only minor effects on lymphocyte traffic in vivo. Anti-VCAM-1 Ab as well as the fibronectin peptide CS-1 are without influence on the migration to Peyer's patches or intestine, in contrast to Ab against the mucosal addressin MAdCAM-1. Thus, homing to gut-associated sites is regulated by the alpha 4/beta 7-integrin heterodimer interacting with the vascular addressin, MAdCAM-1, and not with fibronectin or VCAM-1 as counterstructures. Inhibition of homing to Peyer's patches and intestine by the anti-integrin Abs studied was only partial. L-selectin also participates in the homing of small lymphocytes to mucosal sites, especially Peyer's patches, but does not contribute substantially to the localization of blasts into the intestinal wall. The results support a major, but not exclusive role of the alpha 4/beta 7-integrin in lymphocyte traffic to mucosal sites.
Role of dermal cells from normal and ultraviolet B-damaged skin in induction of contact hypersensitivity and tolerance.
Authors: Kurimoto I , Arana M , Streilein J W
Published: December 2022

With conventional regimens for induction of contact hypersensitivity with highly reactive haptens, circumstantial evidence implicates both epidermal and dermal APC. However, similar applications of hapten to skin that has been treated with an acute, low dose UVB radiation protocol induce contact hypersensitivity only in certain genetically defined strains of mice, termed UVB-resistant. Moreover, mice that fail to acquire contact hypersensitivity when hapten is painted on UVB-exposed skin display hapten-specific tolerance. In the present study, dermal cell suspensions have been prepared from normal and UVB-exposed mouse skin as a means of identifying the cell(s) that: 1) provide APC function after UVB radiation in UVB-resistant mice; and 2) confer tolerance after UVB radiation in UVB-susceptible mice. The results confirm that the normal murine dermis contains Ia+ cells that, when hapten-derivatized in vitro and injected s.c. into naive, syngeneic mice, possess typical Ag-presenting function. Cells with similar function are retained in the dermis of UVB-resistant mice after exposure to UVB radiation, whereas dermal cells of UVB-exposed skin of UVB-susceptible mice display no ability to induce contact hypersensitivity. Instead, the latter dermal cells, when hapten-conjugated and injected s.c., induce tolerance. Thus, doses of UVB radiation that deplete the epidermis of Langerhans cells do not deplete the dermis of UVB-resistant mice of contact hypersensitivity-inducing APC, but do confer upon cells within the dermis of UVB-susceptible mice the capacity to induce unresponsiveness.
Gene transfer studies of T cell receptor-gamma delta recognition. Specificity for staphylococcal enterotoxin A is conveyed by V gamma 9 alone.
Authors: Loh E Y , Wang M , Bartkowiak J , Wiaderkiewicz R , Hyjek E , Wang Z , Kozbor D
Published: December 2022

gamma delta T cells bearing the V gamma 9 gene segment have been shown to recognize staphylococcal enterotoxin A (SEA) and a range of other Ags including mycobacterial Ags. We have established an experimental system to analyze the recognition properties of human TCR-gamma delta on a molecular level by transferring the receptor from its original T cell into a Jurkat T cell host that does not express an endogenous TCR. Three groups of transfectants that express the same delta-chain, V delta 1, but different gamma-chains (V gamma 9-J2-C gamma 2, V gamma 3-J2-C gamma 2, and V gamma 9-JP-C gamma 1) together with the endogenous CD3 were obtained. The transfectant T cells each expressing different gamma delta receptors all produced IL-2 after stimulation with plastic bound anti-CD3 Ab, but only those expressing V gamma 9 responded to stimulation with SEA in the presence of an autologous lymphoblastoid B cell line. In addition, transfectants that expressed V delta 2 combined with V gamma 9 could also respond to SEA. These results indicate that the V gamma 9 portion of the receptor, independent of the J region and C region or the delta-chain, is responsible for recognizing SEA.
Soluble and membrane-bound TNF-alpha are involved in the cytotoxic activity of B cells from tumor-bearing mice against tumor targets.
Authors: Lopez-Cepero M , Garcia-Sanz J A , Herbert L , Riley R , Handel M E , Podack E R , Lopez D M
Published: December 2022

Splenic B cells from BALB/c mice bearing mammary adenocarcinomas are capable of performing Ab-dependent cellular cytotoxicity. Effector-target conjugation after 18 h results in minimal cytoplasmic damage, whereas extensive nuclear disintegration is observed. To determine whether splenic B cells from tumor-bearing mice can effect direct cytotoxicity against tumor cells, L929 and WEHI 164 cells were used as targets. B lymphocytes from tumor-bearing mice, but not from normal animals, were capable of lysing these two types of tumor cells. However, only a low level of cytotoxicity could be detected when the nontumorigenic 3T3 cells were used as targets. To elucidate the mechanism of cytotoxicity of these killer B cells, RNase protection assays were performed using perforin, granzyme A, TNF-alpha, and lymphotoxin probes. No perforin, granzyme A, or lymphotoxin RNA could be detected in purified preparations of B cells from normal and tumor-bearing mice. B cells from normal mice did not have TNF-alpha RNA. In contrast, B cells from tumor bearers expressed TNF-alpha RNA. TNF-alpha could be detected in supernatants from both unstimulated and stimulated tumor bearers' splenic B cells, as measured by ELISA, and its lytic activity was neutralized by anti-TNF-alpha Ab. Western blots revealed the presence of TNF-alpha on the surface of the killer B cells. Paraformaldehyde-fixed B cells from tumor-bearing mice but not from normal animals were able to lyse TNF-alpha-sensitive tumor targets. This cytotoxicity was neutralized by anti-TNF-alpha Ab. These results suggest that TNF-alpha in soluble and membrane-bound forms may be involved in the mechanism of cytotoxicity exerted by B cells from tumor-bearing mice.
T cell-induced Ig allotypic suppression in mice. Basis for emergence or tolerization, during the perinatal period, of natural T cells specific to the IgG2ab allotype.
Authors: Majlessi L , Marcos M A , Benaroch P , Denoyelle C , Bordenave G
Published: December 2022

We have previously described an anti-IgG2ab T cell activity in normal Igha/a mice. Their congenic partner at the Igh locus (Ighb/b) and Igha/b hybrids bred from them, do not display this T cell activity but express IgG2ab. As these mice are supposed to possess the same genetic elements related to this potential T cell repertoire, only somatic selection mechanisms could be responsible for their different behavior. In this study, we investigated the basis for the emergence (in Igha/a mice) or tolerization (in Ighb/b-congenic mice and in Igha/b hybrids) of these natural anti-IgG2ab T cells. Stringent perinatal B cell deprivation in Ighb/b and Igha/b mice resulted in the emergence of anti-IgG2ab T cells, as these individuals could be subjected to autoimmune, T cell-mediated IgG2ab suppression. Furthermore, the acquisition of anti-IgG2ab T cell activity was drastically reduced in Igha/a mice, perinatally exposed to IgG2ab; thus, the presence of this allotype leads to tolerization of these specific T cells.
Function of adhesion molecules lymphocyte function-associated antigen-3 and intercellular adhesion molecule-1 on human epidermal Langerhans cells in antigen-specific T cell activation.
Authors: Teunissen M B , Rongen H A , Bos J D
Published: December 2022

In addition to the interaction between the TCR and the MHC/Ag complex on the APC, optimal T cell activation also requires interaction between adhesion molecules on the APC and their ligands on T cells. We determined the presence of adhesion molecules on human epidermal Langerhans cells (LC) and their role in Ag-specific T cell activation. Freshly isolated LC did not display ICAM-1 (CD54), ICAM-2, LFA-1 (CD11a), and LFA-3 (CD58), as detected by double-color FACS analysis, using HLA-DR expression for LC identification. Upon culture, LC clearly expressed ICAM-1 and LFA-3, both already detectable after 1 day, reaching a plateau at day 2. ICAM-2 and LFA-1 were undetectable on cultured LC and attempts to induce this expression by different culture conditions remained unsuccessful. mAb against ICAM-1, LFA-1, LFA-3, and CD2, continuously present during culture, inhibited the T cell proliferative response to Candida albicans presented by cultured LC. Pretreatment of LC and/or T cells with mAb indicated that anti-ICAM-1 and anti-LFA-3 inhibited at the LC level, whereas anti-LFA-1 and anti-CD2 inhibited at the T cell level. The mAb-induced inhibition was dose-dependent, but a total blockade of the response was never achieved. Time-course observations revealed that ICAM-1 and LFA-3 on LC only functioned during the initiation phase of T cell activation. Our study demonstrates that both ICAM-1 and LFA-3 on LC considerably contribute to the generation of a T cell response. The high expression of these accessory molecules enable LC, at least in part, to perform their powerful Ag-presenting function.
Regulation of antibody specificity to Plasmodium falciparum merozoite surface protein-1 by adjuvant and MHC haplotype.
Authors: Chang S P , Nikaido C M , Hashimoto A C , Hashiro C Q , Yokota B T , Hui G S
Published: December 2022

An effective malaria vaccine must be capable of eliciting a protective immune response in individuals of diverse genetic makeup. In this report, we describe the co-regulation of immune responsiveness to growth-inhibitory Plasmodium falciparum merozoite surface protein-1 (MSP-1) epitopes by MHC-linked immune response genes and by the adjuvant used in MSP-1 vaccine formulations. When congenic mice differing in MHC haplotype were immunized with MSP-1 either in CFA or incorporated into a synthetic monophosphoryl lipid A (LA-15-PH)-liposome formulation, mice of different haplotypes produced anti-MSP-1 Abs capable of inhibiting P. falciparum growth. Mice of H-2b and H-2ja haplotypes produced Abs possessing high levels of inhibitory activity upon immunization with MSP-1 in LA-15-PH/liposomes whereas these haplotypes produced noninhibitory Abs when immunized with MSP-1 in CFA. Conversely, H-2d haplotype mice produced inhibitory Abs when immunized with MSP-1 in CFA but not when immunized with MSP-1 in LA-15-PH/liposomes. The LA-15-PH/liposome adjuvant was more effective than CFA in inducing growth-inhibitory Abs. The level of parasite growth inhibition observed for a particular mouse strain correlated with Ab titers against conserved, C-terminal MSP-1 epitopes, which appear to be important targets for Ab-mediated inhibition in mice immunized with both adjuvant formulations. Our results suggest that adjuvant formulation and MHC genes act in a reciprocal manner to control immune responsiveness to specific epitopes, and raise the possibility of manipulating genetically-controlled responsiveness to vaccine Ags by utilizing alternative adjuvants in vaccine formulations.
Characterization of the E-selectin ligand on NK cells.
Authors: Pinola M , Renkonen R , Majuri M L , Tiisala S , Saksela E
Published: December 2022

In this study we demonstrate that human CD56+CD16+/CD3- NK cells adhere to the E-selectin expressed by stimulated HUVEC in a sialidase- and Ca(2+)-dependent manner, and express a silylated Lex adhesion structure. We have characterized this sLe(x) epitope on NK cell in detail and show here that the sLe(x) on NK cells was not recognized by the CSLEX1 Ab, but was readily identified by two anti-di-sLe(x) Abs, KM-93 and FH-6. Furthermore, cleaving sialic acid with a sialidase treatment revealed a pool of Le(x) epitopes on the NK cells surface, providing further proof that NK cells express sLe(x) epitopes. Extensive protease treatments did not cleave the sLe(x) epitope from NK cells, which suggests that it could be linked to a lipid backbone. This di-sLe(x) was able to mediate adhesion to E-selectin, suggesting that it represents an essential part or is closely related to a selectin ligand on NK cells. We were also able to show that NK cells possess several alpha 2,3 sialyltransferases and alpha 1,3 or alpha 1,3/4 fucosyltransferases. These enzymes are crucial in the synthesis of sLe(x) epitopes on cell surfaces. Taken together, we provide evidence that NK cells have a di-sLe(x) oligosaccharide capable of adhesion to E-selectin, and NK cells have the machinery (i.e., relevant transferases) to generate these sialylated Lewis oligosaccharides.
Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus.
Authors: Emlen W , Niebur J , Kadera R
Published: December 2022

SLE is a disease characterized by the generation of an immune response to intact nuclear Ags, especially components of the nucleosome, histones and DNA. The process of programmed cell death, or apoptosis, is characterized by cleavage of chromatin into oligonucleosomes and release of these nucleosomes into the extracellular space. To address the question of whether altered apoptosis might provide a source of extracellular nuclear Ags in SLE, we have examined apoptosis of lymphocytes isolated from patients with SLE, patients with rheumatoid arthritis (RA), and normal controls. Apoptosis was measured by three independent methods and confirmed by gel electrophoresis. Freshly isolated lymphocytes (t0) showed low levels of apoptosis. However, lymphocytes from SLE patients demonstrated a significant increase in the number of apoptotic cells at t0 compared with normal controls and RA patients. In tissue culture, lymphocytes from all patient groups underwent apoptosis, but the rate of apoptosis of lymphocytes derived from SLE patients was 2.35-fold faster than apoptosis of lymphocytes from normal controls or RA patients. The increased rate of apoptosis could not be accounted for by corticosteroid or cytotoxic medication. There was a significant correlation between SLE disease activity as measured by the systemic lupus activity measure and rate of apoptosis in vitro. The release of intact nucleosomes during apoptosis was measured by ELISA; lymphocytes from SLE patients released increased amounts of nucleosomal material into the extracellular space in direct proportion to the rate of apoptosis. Abnormal apoptosis of lymphocytes in SLE may provide a source of extracellular nuclear Ag to drive the immune response and to allow the formation of immune complexes. The demonstration of altered in vitro apoptosis of lymphocytes derived from SLE patients raises the possibility that abnormalities of apoptosis may contribute to the pathogenesis of SLE.
A positively selecting thymic epithelial cell line lacks costimulatory activity.
Authors: Vukmanović S , Stella G , King P D , Dyall R , Hogquist K A , Harty J T , Nikolić-Zugić J , Bevan M J
Published: December 2022

The participation of costimulatory molecule interactions in positive selection of T lymphocytes was addressed by assessing the ability of a positively selecting thymic epithelial cell (TEC) line, 427.1, to stimulate allospecific CTL responses. Stimulation of H-2s spleen cells with the H-2b expressing 427.1 line does not result in the generation of cells capable of lysing H-2b target cell lines. The level of expression of MHC class I molecules by 427.1 is lower than that found in other stimulatory TEC lines. However, this finding does not account for the nonstimulatory phenotype. Up-regulation of MHC class I did not result in stimulation, and fusion of 427.1 cells with stimulatory TEC resulted in a line with low MHC class I molecule expression and stimulatory phenotype. The TEC line 427.1 does not express the costimulatory molecule B7/BB1, and transfection of the B7/BB1-encoding DNA results in expression of the molecule and conversion into a stimulatory phenotype, demonstrating directly that the non-stimulatory phenotype is a result of lack of costimulation. However, B7/BB1 expression does not improve the ability of 427.1 TECs to induce positive selection. Intrathymic injection of the B7/BB1 transfected, compared with mock transfected 427.1 cells, rescued fewer CD8+ mature thymocytes in beta 2-microglobulin negative mice. Therefore, unlike the peripheral T cell responses to Ag, positive selection of T cells in the thymus may not depend on the costimulation provided by the presenting cell.
Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins.
Authors: Kast W M , Brandt R M , Sidney J , Drijfhout J W , Kubo R T , Grey H M , Melief C J , Sette A
Published: December 2022

We have measured the binding affinity for five HLA-A alleles: HLA-A1 (A*0101), A2.1 (A*0201), A3 (A*0301), A11 (A*1101), and A24 (A*2401); of a set of all possible nonamer peptides (n = 240) of human papillomavirus type 16 E6 and E7 proteins. High affinity binding peptides were identified for each of the alleles, thus allowing us to select several candidates for CTL-based vaccines. Moreover, this unbiased set of peptides allowed an evaluation of the predictive value of HLA motifs derived either from the analysis of sequencing of pools of naturally processed peptides or from the binding analysis of polyalanine nonameric peptides that differed in the amino acids (aa) present at the anchor positions. Whereas pool sequencing-derived motifs were present in only 27% of high affinity binders, the more expanded motif, based on analysis of different aa substitutions at the anchor positions, was present in 73% of high affinity binders. Furthermore, it was found that the presence of anchor residues in a peptide was in itself not sufficient to determine binding to MHC class I molecules, because the majority of motif-containing peptides failed to bind to the relevant MHC. Finally, specific HLA motifs were used to predict peptide binders of 8, 10, and 11 aa in length. Several high affinity binding peptides were identified for each of the various peptide lengths, indicating a significant size heterogeneity in peptides capable of high affinity binding to HLA-A molecules.
Co-ligation of CD31 and Fc gamma RII induces cytokine production in human monocytes.
Authors: Chen W , Knapp W , Majdic O , Stockinger H , Böhmig G A , Zlabinger G J
Published: December 2022

The CD31 (PECAM-1) cell surface glycoprotein is considered to be involved in intercellular recognition and adhesion. Cytokines play a major role in cellular interactions, and therefore it was of interest to study whether engagement of CD31 affects synthesis and release of proadhesive cytokines. Here we demonstrate that immobilized CD31 mAb 1B5 induces the release of TNF-alpha, IL-1 beta, and IL-8 from human PBMCs. CD11b mAb VIM12 and HLA-D mAb VID1, both of which are of the same Ig subclass as mAb 1B5 (IgG1), as well as nonbinding isotype control mAb VIAP, were ineffective. That the effect was caused by the mAb, but not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and coculture with polymyxin B, which did not abolish TNF-alpha release. Cytokine production through intact mAb 1B5 was completely blocked by soluble F(ab) fragments of anti-IgG Fc gamma RII mAb IV.3, suggesting a significant contribution of that FcR. Cross-linking of neither CD31 nor Fc gamma RII molecules with the respective F(ab) fragments induced TNF-alpha release, but nonbinding control IgG1 Ab was able to restore the response of PBMC to 1B5 F(ab) fragments, when both Ab preparations were coated concomitantly. Therefore, only coligation of CD31 and Fc gamma RII appears to transduce activation signals leading to cytokine production. Our findings thus indicate a novel functional aspect of CD31 molecules that might play an important role in the propagation of an ongoing immune response as well as in the regulation of cell-cell interactions during inflammatory reactions.
Induction, characterization, and functional coupling of the high affinity chemokine receptor for RANTES and macrophage inflammatory protein-1 alpha upon differentiation of an eosinophilic HL-60 cell line.
Authors: Van Riper G , Nicholson D W , Scheid M P , Fischer P A , Springer M S , Rosen H
Published: December 2022

Eosinophilic differentiation of a pro-eosinophilic HL-60 cell line resulted in the induction of a high affinity RANTES/macrophage inflammatory protein-1 alpha receptor. The induced receptor is biochemically indistinguishable in RANTES equilibrium-binding studies from the monocytic receptor expressed on THP-1 cell membranes. Continued expression of the receptor requires the continuous presence of the inducing stimulus, and receptor site number declines without a loss of binding affinity with a t1/2 of 11.5 h on withdrawal of the inducing stimulus. The induced receptor is capable of three physiologic measures of receptor coupling, namely, ligand-induced Ca2+ fluxes, priming of the respiratory burst, and chemotaxis. Dose-dependent Ca2+ fluxes were elicited upon increasing concentrations of RANTES and MIP-1 alpha whereas no response was measured upon addition of MIP-1 beta or MCP-1. In addition, desensitization studies demonstrated that previous exposure to either RANTES or MIP-1 alpha almost completely inhibits a Ca2+ flux upon subsequent exposure to either ligand. Priming of the respiratory burst to PMA in differentiated cells by human rRANTES was more effective than priming by IL-5 or granulocyte-macrophage-CSF, whereas undifferentiated cells failed to secrete superoxide anion. In addition, differentiated cells underwent chemotaxis in response to RANTES. This provides the first evidence for the induction of a C-C chemokine receptor upon eosinophilic differentiation of a leukocyte cell line, and is in keeping with the demonstrated ability of human RANTES to induce the rapid formation of eosinophilic inflammatory sites.
Determination of the primary structures of human skin chymase and cathepsin G from cutaneous mast cells of urticaria pigmentosa lesions.
Authors: Schechter N M , Wang Z M , Blacher R W , Lessin S R , Lazarus G S , Rubin H
Published: December 2022

This study establishes the primary structure of human skin chymase and provides further evidence for the presence of a cathepsin G-like proteinase within human mast cells. The amino acid sequence of human skin chymase was established by protein methods and by analysis of PCR amplification products obtained with cDNA-derived from urticaria pigmentosa (UP) lesions. UP is a disease characterized by skin lesions containing high numbers of mast cells. Proteolytic digests of human chymase purified from normal skin yielded 10 resolvable peptides that were sequenced by automated Edman degradation. The amino acid sequences for these peptides combined with the sequence obtained for the protein's NH2-terminal region (35 residues) accounted for 137 residues of the human skin chymase sequence. This partial amino acid sequence corresponded to the sequence of human heart chymase, a proteinase isolated from heart tissue with immunologic and hydrolytic properties similar to skin chymase. PCR amplification of UP-derived cDNA with primers based on the cDNA structure of heart chymase demonstrated a single amplification product of expected size which was subcloned and sequenced. The amino acid sequence (135 residues) deduced from this product was identical to that of heart chymase in the region between the primers. This sequence, along with that established for the purified protein, constituted 99% of the heart chymase primary structure, strongly indicating that human skin and heart chymases have identical primary structures. Amplification of the same UP-cDNA with primers coding for the NH2- and COOH-terminal sequences of human neutrophil cathepsin G also produced a specific amplification product which was sequenced. The deduced amino acid sequence between the primers was identical to that reported for neutrophil cathepsin G, indicating that the protein of cutaneous mast cells previously shown to be immunologically cross-reactive with neutrophil cathepsin G has a comparable amino acid sequence. UP-cDNA demonstrating amplification products for cathepsin G did not demonstrate amplification products for human neutrophil elastase, suggesting that the cathepsin G PCR amplification product was not derived from neutrophils or monocytes possibly contaminating the lesion. These studies provide further evidence that human skin mast cells contain two different chymotrypsin-like proteinases.
Vascular endothelial growth factor. A cytokine modulating endothelial function in rheumatoid arthritis.
Authors: Koch A E , Harlow L A , Haines G K , Amento E P , Unemori E N , Wong W L , Pope R M , Ferrara N
Published: December 2022

Angiogenesis is important in the proliferation of inflammatory synovial tissue. Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that is also angiogenic in vivo. We examined the potential role of VEGF in mediating chemotaxis and proliferation of endothelial cells in rheumatoid arthritis (RA) using samples of synovial tissue and synovial fluid from 55 arthritic patients. Synovial fluid VEGF by ELISA was higher in RA synovial fluids (386 +/- 122 ng/ml) (SE) compared with osteoarthritis (OA) synovial fluids (< 0.8 ng/ml) (p < 0.05) or synovial fluids from patients with other arthritides (6.6 +/- 2 ng/ml). In addition to its known mitogenic properties, we found that human rVEGF was chemotactic for HUVECs at concentrations above 0.02 nM. Incubation of RA synovial fluids with neutralizing anti-VEGF resulted in 23 to 66% (mean 53 +/- 4%) inhibition of HUVEC chemotaxis. Conditioned medium from four of five RA synovial tissue explants was mitogenic for bovine adrenal capillary endothelial cells. Anti-VEGF neutralized from 19 to 42% (mean 28 +/- 4%) of this mitogenic activity. To determine the cellular source of VEGF in synovial tissue, we employed immunohistochemistry. VEGF+ cells were rarely (< 1%+) found in normal synovial tissues. In contrast, RA and OA synovial tissues exhibited VEGF+ lining cells (8% and 13%, respectively). A few synovial tissue macrophages were VEGF+ in both RA and OA (5% and 2%, respectively). These results elucidate a newly described function for VEGF as a potent chemotaxin for endothelial cells as well as a role for VEGF in RA-associated endothelial migration and proliferation.
60-kDa Ro protein autoepitopes identified using recombinant polypeptides.
Authors: Saitta M R , Arnett F C , Keene J D
Published: December 2022

The human Ro ribonucleoprotein is a clinically important yet poorly understood autoantigen. The contribution of Ro autoantibodies to pathogenesis of autoimmune disease remains unclear, as do the stimuli that initiate and maintain the response. Recent evidence suggests that patient anti-Ro responses target individual proteins, including a 60- and a 52-kDa species, within the complex in a disease-specific manner. However, Ro antisera retain considerable heterogeneity in their recognition of both continuous and discontinuous epitopes on the protein components. Previous characterization of Ro autoepitopes has primarily involved solid phase assays, which are of limited value in identifying discontinuous epitopes. To address the heterogeneity of Ro and the issue of discontinuous autoepitopes, we have generated 10 overlapping recombinant polypeptides of the human 60-kDa Ro protein and compared their reactivities using a soluble immunoprecipitation assay. Seven different epitopes, both continuous and discontinuous, were distinguished and seven distinct patterns of reactivity were discerned among the sera from 12 patients. These patterns of reactivity showed no relationship to clinical diagnosis but did correlate with the titer of Abs against recombinant 60-kDa Ro and with the concomitant presence of Abs directed against recombinant 52-kDa Ro protein. Sera that immunoprecipitated only the full-length 60-kDa protein had low or undetectable anti-60 kDa titers by ELISA and immunoblot using recombinant Ag, demonstrating a predominant recognition of discontinuous epitopes. These data indicate that autoantibody responses to the 60-kDa Ro Ag can preferentially target discontinuous epitopes and that the ability to recognize continuous epitopes is accompanied by the appearance of 52-kDa Ro autoantibodies.
Inhibition of experimental autoimmune encephalomyelitis by MHC class II binding competitor peptides depends on the relative MHC binding affinity of the disease-inducing peptide.
Authors: Wauben M H , Joosten I , Schlief A , van der Zee R , Boog C J , van Eden W
Published: December 2022

Blocking of the Ag presentation function of MHC molecules by competitor peptides has been proposed as a potential immunotherapy for MHC-associated autoimmune diseases. Despite the fact that successful inhibition of experimental autoimmune encephalomyelitis (EAE) by coimmunization with competitor peptides had been achieved, it remained questionable whether the in vivo activity of such peptides was solely the result of MHC blockade. In the peptide MBP72-85-induced EAE model in Lewis rats, we designed a single amino acid-substituted analogue of MBP72-85 with a superior MHC binding capacity, and with the capacity to activate encephalitogenic MBP72-85-specific T cells. Subsequently, two well-defined competitor peptides, one EAE related and one non-EAE related, were studied for their respective efficacies to inhibit the in vitro proliferation of an encephalitogenic T cell clone induced by the original MBP72-85 or the superior MHC binding analogue peptide. It appeared that the response to MBP72-85 was inhibited very efficiently by both competitor peptides, whereas the response to the superior MHC binding analogue peptide was not. Co-immunization of either the related or non-related competitor peptide together with MBP72-85 inhibited EAE induction in a concentration-dependent manner. In such protected rats, polyclonal T cell responses against MBP72-85 were dramatically decreased. However, EAE induced by the stronger MHC binding MBP72-85 analogue could not be inhibited by either of the competitor peptides. Moreover, in these rats, T cell priming for both MBP72-85 and the MBP72-85 analogue was not inhibited. These results show that competition for MHC binding in vivo could lead to inhibition of EAE induction.
Cellular and molecular mechanisms of the IL-12-induced increase in allospecific murine cytolytic T cell activity. Implications for the age-related decline in CTL.
Authors: Bloom E T , Horvath J A
Published: December 2022

The mechanism(s) by which IL-12 augments CTL generation and the potential for IL-12 to alter the age-related decline in CTL activity were assessed using primary CTL stimulated by allogeneic cells. IL-12 at 2 U/ml enhanced the generation of allospecific CTL response in young adult mice by 1.5- to 5-fold, and optimal augmentation was achieved and maintained with as low as 0.1 U/ml IL-12. In contrast to the enhancement of MHC-unrestricted lytic activity, which was most efficient when IL-12 was added near the end of the 5 day culture period, nearly equivalent augmentation of Ag-specific CTL was induced when IL-12 was added from 1 to 5 days before harvest. The augmentation of CTL by IL-12 was not associated with increased incorporation of [3H]TdR. Increased IFN-gamma production was not required for IL-12 to augment CTL activity because when three different Abs against mouse IFN-gamma were each included in the MLC at concentrations abolishing recoverable IFN-gamma protein, they did not inhibit the IL-12 effect but, under some conditions, further increased CTL activity. Ab to IL-2 also did not alter the IL-12 effect. Depletion of ASGM1+ cells from the responding population before stimulation did not significantly alter the response to IL-12. However, depletion of CD8+ T cells from effector cells obtained after stimulation substantially reduced the IL-12-induced augmentation of CTL. Thus, the results suggested that IL-12 augments CTL activity primarily through an effect on T cells, and the augmentation was associated with an approximate doubling of perforin levels, which was not caused by a significantly increased proportion of CD8+ cells. Finally, IL-12 was effective for moderating the age-related decrease in CTL activity, suggesting a potential role for immunomodulation by IL-12.
Recombinant CD40 ligand exerts potent biologic effects on T cells.
Authors: Fanslow W C , Clifford K N , Seaman M , Alderson M R , Spriggs M K , Armitage R J , Ramsdell F
Published: December 2022

Murine and human CD40 ligand (CD40L) were recently cloned, expressed, and shown to possess potent activity on human and murine B cells, including stimulation of proliferation and Ig secretion in the presence of cytokines. In addition to its action on B lymphocytes, this report demonstrates that CD40L induced both CD4+ and CD8+ T cells isolated from murine lymphoid tissues to proliferate in the presence of submitogenic dosages of Con A, PHA, CD3 mAb, and TCR-alpha beta mAb. The presence of CD40L during suboptimal TCR stimulation resulted in increased expression of the activation Ags IL-2R alpha and CD69 and increased IL-2 production. Taken together, these results show that CD40L is a potent activator of murine T cells and suggest that CD40L is involved in the regulation of T cell function mediated through T:T cell interaction.
The induction of peripheral tolerance by the chronic activation and deletion of CD4+V beta 5+ cells.
Authors: Fink P J , Fang C A , Turk G L
Published: December 2022

In C57BL/6 mice transgenic for a rearranged gene encoding a V beta 5+ beta-chain of the TCR, transgene expression among CD4+ cells decreases with age, such that approximately 40% of CD4+ cells express an endogenous beta-chain gene in 8-mo-old mice. A similar deletion of V beta 5+ cells is observed among CD4+ cells from nontransgenic littermates. V beta 5+ T cells are deleted intrathymically in I-E+Mtv-9+ strains of mice, but this chronic deletion occurs in the lymphoid periphery, in the absence of I-E. We now demonstrate the increased expression of the activation markers CD44 and VLA-4 among CD4+V beta 5+ cells, in the absence of either an increase in size or IL-2 receptor expression. Functional as well as phenotypic differences distinguish CD4+ from CD8+ cells in older V beta 5+ transgenic mice. Relative to their CD8+ counterparts, CD4+V beta 5+ cells are hyporesponsive to plate-bound anti-V beta 5 Abs, and this anergy is partially reversible by the addition of exogenous IL-2. These data suggest the deletion of CD4+V beta 5+ cells is the result of a process that includes their activation, loss of function, and their eventual removal. To investigate the involvement of the principal V beta 5 superantigen Mtv-9 in this chronic deletion, we have derived several lines of V beta 5+I-E-Mtv-9- mice. Transgene expression also declines with age in CD4+ T cells in these mice, clearly demonstrating that the chronic deletion of CD4+V beta 5+ cells does not require Mtv-9. There is considerable variation in the kinetics and efficiency of CD4+V beta 5+ deletion between lines of Mtv-9- transgenic mice that is not from differences in the profiles of endogenous mammary tumor proviruses nor readily explained by environmental differences that influence proviral expression. These results suggest the existence of genetic factors other than mammary tumor proviruses that influence the deletion of CD4+V beta 5+ cells in the absence of I-E.
In vivo administration of insulin-like growth factor-I stimulates primary B lymphopoiesis and enhances lymphocyte recovery after bone marrow transplantation.
Authors: Jardieu P , Clark R , Mortensen D , Dorshkind K
Published: December 2022

Recombinant human insulin-like growth factor-I (IGF-I) (100 micrograms/day) was infused by osmotic minipumps into mice to evaluate its effects on bone marrow B lymphopoiesis. IGF-I treatment resulted in a significant increase in the total number of bone marrow B lineage cells by 2 wk post-treatment in both normal, adult mice and animals that had been lethally irradiated and reconstituted with syngeneic bone marrow. Effects were still apparent at wk 3, even though pumps delivered IGF-I for only 14 days. In addition, the results confirm previous observations that IGF-I treatment results in an increase in numbers of splenic B cells and suggest that this is in part the result of stimulation of B cell proliferation in that organ.