bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting signal and subcellular compartments involved in the intracellular trafficking of HLA-DMB.
Authors: Copier J , Kleijmeer M J , Ponnambalam S , Oorschot V , Potter P , Trowsdale J , Kelly A
Published: December 2022

Evidence suggests that peptide loading onto MHC class II molecules occurs in a specialized late endocytic compartment (MIIC) where HLA-DM predominantly resides and in which MHC class II transiently accumulates before transport to the cell surface. We examined the targeting signals and compartments involved in the intracellular trafficking of human HLA-DM by expressing hybrid molecules comprising the cytoplasmic domain of DMB and luminal and transmembrane domains of CD8 in HeLa cells. A tyrosine-based tetrapeptide motif present in the cytoplasmic domain of DMB targeted hybrid molecules to intracellular vesicles. Mutation of the tyrosine residue to alanine resulted in redistribution of hybrid molecules to the cell surface. Correct intracellular targeting of HLA-DM was crucial for normal function in B cells. Immunoelectron microscopy on ultrathin cryosections showed that CD8-DMB molecules accumulated in late endocytic compartments sharing characteristics with lysosomes, like MHC class II compartments in APCs. Thus far, the exit of DMB from the Golgi complex has not been elucidated. Interestingly, we found that although the mannose 6-phosphate receptor and CD8-DMB contain similar tyrosine signals, no co-localization was observed in the trans-Golgi network, suggesting that these proteins are differentially sorted at this site. Co-transfection of CD8-DMB, HLA-DR alpha, HLA-DR beta, and an invariant chain revealed that HLA-DR molecules accumulated together with CD8-DMB in these lysosomal compartments. The similarity of these lysosomal-like compartments in wild-type and transfected cells suggests that they are part of the normal endocytic pathway in non-APCs.
T cell recognition and tolerance of antibody diversity.
Authors: Eyerman M C , Zhang X , Wysocki L J
Published: December 2022

The capacity of B cells to self-present their Ab variable regions in the context of class II MHC structures suggests a potential regulatory problem. If T cells were able to recognize self-presented Ab, then T cell help might be delivered to B cells independently of a foreign carrier epitope, resulting in a chronic state of unregulated Ab synthesis. For this reason, we have proposed that T cells normally attain a state of tolerance to Ab V region diversity. Here, we tested this idea by performing direct immunizations with unmutated isologous mAb. We also identified and analyzed epitopes recognized by class II MHC-restricted T cell hybridomas that were originally generated against two physiologically mutated isologous mAb. Our results indicate that the class II MHC-restricted T cell repertoire is tolerant of germ-line-encoded Ab diversity and that the physiologic somatic hypermutation process creates immunogenic epitopes in Ab V regions, in some cases by producing class II MHC-binding peptides. In agreement with these findings, we found that germ-line-encoded Ab V regions are presented by endogenous splenic APC at a level that is physiologically significant.
Thymocyte cell fate alteration by high levels of CD8alpha is independent of CD8-lck association.
Authors: Smith D H , Lee N , Lacy E
Published: December 2022

CD8 expression on the surface of developing thymocytes is essential for the positive selection and maturation of CD8 single positive T cells. CD8 is physically associated with the cytoplasmic tyrosine kinase, p56lck and, in addition to increasing the avidity between the TCR and MHC/peptide complex, may transduce intracellular signals. Here we address the extent to which the requirement for CD8 in thymocyte selection depends on its capacity to signal vs its contribution to avidity. We have previously reported that elevated levels of CD8alpha can alter the fate of developing 2C TCR+ thymocytes from positive to negative selection in H-2b mice. We show here that the fate of 2C TCR thymocytes overexpressing a mutant form of CD8alpha that does not bind to p56lck is the same as that of thymocytes overexpressing the wild-type CD8alpha-chain. This finding demonstrates that, for a TCR-MHC system in which the level of CD8alpha is the deciding factor in cell fate, negative selection does not require increased CD8-dependent signaling through p56lck. We have also found that highly elevated levels of CD8alpha expression (10-fold) block thymocyte maturation in both CD8alpha single and CD8alpha/HY TCR double transgenic mice. Although these may be additional examples of CD8alpha overexpression altering cell fate from positive to negative selection, we present arguments in favor of the interpretation that high levels of CD8alpha actually may block positive selection.
Nonsecretory apoptotic killing by human NK cells.
Authors: Vujanovic N L , Nagashima S , Herberman R B , Whiteside T L
Published: December 2022

We defined a novel constitutive mechanism of cell-mediated cytotoxicity, utilized by nonactivated human peripheral blood NK cells against a variety of tumor cell targets resistant to secretory (i.e., perforin/granzyme-mediated) NK cell killing. Untreated NK cells rapidly induced membrane damage and necrosis in K562 target cells (as determined by 51Cr release assay and confirmed by transmission electron microscopy; TEM), in the absence of DNA fragmentation and apoptosis (as assessed by [3H]thymidine release assay and TEM). Chelation of extracellular Ca2+ or paraformaldehyde fixation completely abrogated NK cell secretory activity and necrotic killing. In contrast, NK cells with either normal or impaired secretion consistently exhibited cytotoxicity against a wide variety of solid tumor cell lines in 1-h [3H]thymidine release, but not in 4-h 51Cr release, assays. Thus, the cytotoxicity was attributable to a nonsecretory, cell membrane-mediated mechanism. It appeared to selectively induce DNA fragmentation and apoptosis without plasma membrane damage and necrosis. This was further demonstrated by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and confirmed by TEM. Among defined populations of immune cells, only NK cells showed an inherent ability to rapidly induce apoptotic death in solid tissue-derived malignant cells. This study demonstrates that NK cells are unique immune cells constitutively endowed with multiple mechanisms of destroying abnormal cells.
Beta-amyloid (25-35) peptide and IFN-gamma synergistically induce the production of the chemotactic cytokine MCP-1/JE in monocytes and microglial cells.
Authors: Meda L , Bernasconi S , Bonaiuto C , Sozzani S , Zhou D , Otvos L , Mantovani A , Rossi F , Cassatella M A
Published: December 2022

The molecular mechanisms involved in the development of senile plaques characteristic of aging and Alzheimer's disease are poorly understood. In this study, we examined whether human monocytes and murine microglial cells stimulated with the active fragment of amyloid beta-protein (Abeta(25-35)) express the monocyte chemotactic protein-1 (MCP-1)/JE. We show that upon incubation with Abeta(25-35), human monocytes accumulate MCP-1 mRNA and produce significant amounts of MCP-1. The effect of Abeta(25-35) on MCP-1 secretion was neither mimicked by a scrambled analogue nor affected by polymyxin B sulfate, even though the latter almost completely abolished the effect of LPS on MCP-1 expression. Murine microglial cells stimulated with Abeta(25-35) also expressed high levels of JE mRNA (the murine counterpart of MCP-1) and released bioactive chemotactic factors. In addition, we report that IFN-gamma significantly synergizes with Abeta(25-35) either in human monocytes or in murine microglial cells, and that Abeta(25-35) plus/minus IFN-gamma-mediated early induction of MCP-1 mRNA does not require new protein synthesis. Finally, we provide evidence that the Abeta(25-35)- and Abeta plus IFN-gamma-induced production of MCP-1 is, in large part, mediated in an autocrine fashion by endogenous TNF-alpha. Taken together, our findings uncover another novel biologic action of Abeta(25-35) and might help in better understanding the mechanisms underlying mononuclear phagocyte recruitment and activation into amyloid deposits.
Murine glomerulotropic monoclonal antibodies are highly oligoclonal and exhibit distinctive molecular features.
Authors: Lefkowith J B , Di Valerio R , Norris J , Glick G D , Alexander A L , Jackson L , Gilkeson G S
Published: December 2022

We recently produced a panel of seven glomerular-binding mAbs from a nephritic MRL-lpr mouse that bind to histones/nucleosomes (group I) or DNA (group II) adherent to glomerular basement membrane. To elucidate the molecular basis of their binding and ontogeny, we sequenced their variable (V) regions, analyzed the apparent somatic mutations, and predicted their three-dimensional structures. There were two clonally related sets (3 of 4 in group I, 3 of 3 in group II) both of the VHJ1558 family, and one mAb of the VH 7183 family. V region somatic mutations within clonally related sets had little effect on glomerular binding and did not appear to be selected for based on glomerular binding. The VH regions were most homologous with those from autoantibodies to histones, DNA, or IgG (i.e., rheumatoid factors), the Vkappa regions, with those from autoantibodies to small nuclear ribonucleoproteins (snRNP). The VH regions also exhibited an unusual VD junction (in the group I clonally related set) and an overall high content of charged amino acids (arginine, aspartic acid) in complementarity-determining regions (CDRs), particularly in CDR3. Molecular modeling studies suggested that the Fv regions of these mAbs converge to form a flat, open surface with a net positive charge. The CDR arginines in group I mAbs; appear to be located in Ag contact regions of the binding cleft. In sum, these data suggest that glomerulotropic mAbs are a highly restricted set of Abs with distinctive molecular features that may mediate their binding to glomeruli.
CD8(+) T cell-mediated spontaneous diabetes in neonatal mice.
Authors: Morgan D J , Liblau R , Scott B , Fleck S , McDevitt H O , Sarvetnick N , Lo D , Sherman L A
Published: December 2022

Transgenic mice that express the influenza virus hemagglutinin (HA) on pancreatic islet beta cells (ins-HA) demonstrate tolerance of HA even after immunization with influenza virus. Surprisingly, when Ins-HA mice were mated with a transgenic mouse expressing a TCR specific for an epitope of HA that is restricted by MHC class I H-2Kd (Clone-4 TCR), the resulting double transgenic (Ins-HA x Clone-4 TCR)F1 neonates developed spontaneous autoimmune diabetes immediately after birth and died within 10 days. This represents a unique situation in which all safeguards within the immune system that normally maintain tolerance of self-antigens in the neonate are insufficient.
Beta-chemokine inhibition of monocytotropic HIV-1 infection. Interference with a postbinding fusion step.
Authors: Oravecz T , Pall M , Norcross M A
Published: December 2022

The beta-chemokines RANTES, MIP-1 alpha, and MIP-1 beta have potent suppressive effects on HIV-1 infection resulting from an early postbinding block in virus fusion and entry. Inhibition was observed only with monocytotropic isolates and mapped to the V3 region of the HIV-1 envelope. RANTES did not inhibit virus expression in chronically infected cells or reduce initial virus attachment to the cell membrane. Inhibitory activity required RANTES binding to the target cell but not G protein-mediated signaling or protein tyrosine kinase activity. The results are consistent with a reversible competitive mechanism of virus inhibition that prevents a V3-associated postbinding step in membrane fusion. The data support a role for a RANTES chemokine receptor as a coreceptor for monocytotropic-HIV-1.
Bcl-2 expression inhibits prostaglandin E2-mediated apoptosis in B cell lymphomas.
Authors: Brown D M , Phipps R P
Published: December 2022

Apoptosis is a critical mechanism in the maturation and maintenance of the immune system. However, the process by which cells die remains poorly understood. The proto-oncogene bcl-2 is considered important in determining whether cells enter an apoptotic pathway or survive. In this report, we first examined the differential sensitivity of immature (CH31) and mature (CH12) B cell lymphomas to growth inhibition by PGE2. The CH31 cell line was growth inhibited and underwent apoptosis in response to PGE2, unlike its mature counterpart, CH12. Furthermore, endogenous levels of the anti-apoptotic protein Bcl-2 in CH31 cells were low compared with CH12. To further investigate the role of Bcl-2 in PGE2- and cAMP-mediated cell death, a retroviral vector bearing the human bcl-2 gene was introduced into CH31. High expression of Bcl-2 in CH31 had no effect on growth inhibition induced by PGE2 or dibutyryl cAMP. In contrast, increased expression of Bcl-2 completely inhibited PGE2- and cAMP-mediated DNA fragmentation and nuclear condensation. Finally, cell cycle analysis of Bcl-2-expressing CH31 cells demonstrated that PGE2 increased the percentage of cells in G1, and analysis of synchronized populations revealed that PGE2 acts at all phases of the cell cycle to delay normal progression. These results support the hypothesis that apoptosis induced through PGE2 and cAMP signaling is sensitive to regulation by Bcl-2 in CH31 B cell lymphomas. Furthermore, unlike apoptosis, regulation of PGE2- and cAMP-mediated growth inhibition in B lineage cells is a distinct and Bcl-2-independent mechanism.
Enhanced generation of NK cells with intact cytotoxic function in CD45 exon 6-deficient mice.
Authors: Yamada H , Kishihara K , Kong Y Y , Nomoto K
Published: December 2022

CD45 is a cell membrane-type protein tyrosine phosphatase that is essential for Ag receptor-mediated signaling in both T and B lymphocytes. To characterize roles of CD45 molecules in murine NK cells, we analyzed the development and the cytotoxic functions of NK cells in mice lacking CD45 exon 6 (CD45 -/-). A markedly increased number of NK cells was observed in spleens of the CD45 -/- mice, despite no CD45 surface expression on the NK cells. From the results of mixed bone marrow chimera experiments, it was demonstrated that the expansion of NK cells in CD45 -/- mice was due to the influence of disappeared expression of CD45 in the NK cells per se, but not to the modulation of environmental factors. The NK cells in the CD45 -/- mice had normal cytotoxic activities, including NK and Ab-dependent cell-mediated cytotoxicity activities comparable with those in normal mice (CD45 +/+). Additionally, the CD45 -/- NK cells could functionally differentiate to lymphokine-activated killer cells by culturing with a high dose of IL-2, despite a lack of induced expression of B220. Therefore, these results suggest that CD45 is involved in NK cell development, but is not essential for cytotoxic activities and Fc gamma R-mediated signaling in NK cells.
The binding epitopes of human CD16 (Fc gamma RIII) monoclonal antibodies. Implications for ligand binding.
Authors: Tamm A , Schmidt R E
Published: December 2022

Numerous mAbs have been generated against Fc gamma RIII (CD16), the low-affinity receptor for the Fc part of IgG. Most of the mAbs recognize both the receptor isoforms, transmembranous Fc gamma RIIIA, and glycosylphosphatidylinositol-linked Fc gamma RIIIB. Binding epitopes of some of the mAbs that differentiate between the two neutrophil Ag (NA) alleles of Fc gamma RIIIB (CLB-Gran11 against NA1; GRM1, BL-LGL/1 against NA2 allele) have been mapped on the first, membrane-distal domain of CD16. We demonstrate that mAbs 3G8, B88-9, CLB-Gran1, MEM-154, and LNK16 almost completely block the receptor's interaction with IgG. Using chimeric Fc gamma RIIIB/Fc epsilon RI receptors and molecular modeling, we localized the epitopes of 3G8 and B88-9 on the putative FG loop of the membrane-proximal Ig-like domain, which we have previously identified as the major binding site for IgG. The epitopes of CLB-Gran1 and MEM-154 are shown to reside in proximity to the FG loop (probably BC or C'E loop). The blocking mAb LNK16 was detected to be directed against the putative C' beta-sheet of the membrane-proximal domain, suggesting that additional residues may be involved in IgG binding of Fc gamma RIII.
Characterization of an Ig VH idiotope that results in specific homophilic binding and increased avidity for antigen.
Authors: Yan X , Evans S V , Kaminki M J , Gillies S D , Reisfeld R A , Houghton A N , Chapman P B
Published: December 2022

mAb against GD3 ganglioside demonstrate homophilic binding in which soluble anti-GD3 mAb bind, through the GD3 binding site, to a VH idiotope (designated IdHOM) on solid phase anti-GD3 mAb. In this way, homophilic binding provides a mechanism for amplifying the binding of mAb to cell surface GD3. We show that serine 52a, within CDR2, is required for IdHOM expression, homophilic binding, and high avidity binding to cell surface GD3. Computer modeling based on the crystal structure of anti-GD3 mAb R24 showed serine 52a situated at the mouth of the GD3 binding pocket, but not directly involved with GD3 binding. Substitutions at position 52a predicted to maintain the GD3 binding pocket (e.g., threonine) resulted in the loss of IdHOM expression and homophilic binding and markedly decreased binding to cell surface GD3, but maintained low avidity GD3 binding as measured by ELISA. All other substitutions at position 52a were predicted to significantly distort the GD3 binding pocket and resulted in the loss of both homophilic binding and any detectable avidity for GD3. We have structurally defined IdHOM and conclude that this idiotope is not required for the GD3 binding pocket, but that the idiotope is necessary for homophilic binding, which is required for high avidity binding to cell surface GD3. We speculate that selection of certain VH genes may result in the expression of idiotopes that allow homophilic binding, and this may represent a general mechanism for increasing the avidity of Abs against T cell-independent Ags.
Th1-associated immune responses to beta-galactosidase expressed by a replication-defective herpes simplex virus.
Authors: Brubaker J O , Thompson C M , Morrison L A , Knipe D M , Siber G R , Finberg R W
Published: December 2022

The immunogenic properties of a replication-defective herpes simplex virus HD-2, containing the Escherichia coli lacZ gene under control of the HSV ICP8 early gene promoter were studied in BALB/c mice. Experiments were designed to determine if the HD-2 virus preferentially stimulated either Th1- or Th2-associated immune responses to beta-galactosidase (beta gal). Sera from mice immunized i.p. or s.c. with virus HD-2, beta gal on aluminum phosphate adjuvant, or a control ICP8 deletion mutant, d301, were assayed for total and Ag-specific IgG1 and IgG2a Abs, beta gal-driven lymphocyte proliferation, and in vitro production of the cytokines IFN-gamma, IL-4, and IL-2. Viruses HD-2 and d301 preferentially stimulated the production of total serum IgG2a following two immunizations i.p. or a single immunization s.c., while only HD-2 virus stimulated in vivo production of beta gal-specific IgG2a serum Abs. In contrast, beta gal adsorbed on AIPO4 preferentially stimulated production of Ag-specific IgG1 serum Abs. The HD-2 virus also induced a potent cellular proliferative response to beta gal, which was still pronounced 5 wk after primary immunization. Cultured lymphocytes from HD-2-immunized mice produced IFN-gamma after 5 days in culture with soluble beta gal in an Ag- and dose-dependent fashion. These results demonstrate that replication-defective mutants of HSV can be used as vectors for eliciting Th1-associated immune responses to a heterologous Ag expressed from the viral genome.
Escherichia coli and Pseudomonas aeruginosa induce expansion of V delta 2 cells in adult peripheral blood, but of V delta 1 cells in cord blood.
Authors: Kersten C M , McCluskey R T , Boyle L A , Kurnick J T
Published: December 2022

Human peripheral blood T cells proliferate in response to Escherichia coli and Pseudomonas aeruginosa. We observed that during the first few days after stimulation a large percentage of the responding PBMC were gamma delta T cells. In our study we characterized the early T cell responses of freshly isolated adult and newborn PBMC to soluble preparations of heat-killed E. coli and P. aeruginosa. Specimens from all healthy adults tested showed intense proliferation in response to both bacterial preparations; at 6 days, the responding cells were mainly T cell blasts, of which high percentages (up to 80%) were gamma delta T cells, most expressing V delta 2/V gamma 9. All newborn blood specimens tested also showed T cell proliferative responses, which included a marked expansion of gamma delta T cells, mainly of the V delta 1 subset. Populations of purified V delta 1 and V delta 2 T cells were obtained from adult PBMC following stimulation with E. coli; both subsets proliferated upon rechallenge with the bacterial preparations. Protease treatment of the bacterial preparations did not appreciably affect their ability to induce expansion of gamma delta T cells in either adult or cord blood, indicating that the stimulatory components were not proteins. The response of gamma delta T cells from newborns indicates that prior exposure to bacterial products is not necessary and suggests that gamma delta T cells are important elements in natural immunity to these extracellular organisms.
Anaphylaxis mediated through a humanized high affinity IgE receptor.
Authors: Dombrowicz D , Brini A T , Flamand V , Hicks E , Snouwaert J N , Kinet J P , Koller B H
Published: December 2022

Mast cells and basophils, which are activated by IgE and allergens through the high affinity IgE receptor (Fc epsilon RI), play a prominent role in anaphylaxis in the mouse. Mice deficient in this receptor become resistant to passive anaphylaxis. As a first step in developing an in vivo model that more closely mimics the IgE-mediated responses in man, we used a combination of transgenic and embryonic stem cell technology to generate a mouse line in which the murine Fc epsilon RI alpha-chain has been replaced with its human homologue. We demonstrate here that these mice express a tetrameric high affinity IgE receptor, in which the human alpha-chain associates with the murine beta- and gamma-chains, and that upon triggering with relevant Ag, this receptor mediates the initiation of the expected intracellular events. In addition, we show that the human alpha-chain restores an anaphylactic response to the nonresponsive alpha-deficient parental mouse line. This "humanized" mouse represents a potentially important model system, not only for studying the role of IgE in human immune responses, but also for testing potential therapeutic reagents that can interfere with responses mediated through the human Fc epsilon RI receptor.
Analysis of the gene that encodes the complement regulatory protein, membrane inhibitor of reactive lysis (CD59). Identification of an alternatively spliced exon and characterization of the transcriptional regulatory regions of the promoter.
Authors: Holguin M H , Martin C B , Eggett T , Parker C J
Published: December 2022

Membrane inhibitor of reactive lysis (MIRL, CD59) is an 18-kDa glycosylphosphatidylinositol-anchored protein that regulates formation of the membrane attack complex of complement. The purpose of these studies was to characterize the gene that encodes CD59. Our experiments redefined the structural organization of the gene by identifying a previously unrecognized alternatively spliced exon. Analysis by PCR of cDNA derived from a variety of cultured human cell lines and from PBMC showed that transcripts containing the alternatively spliced exon sequence were expressed concordantly with transcripts lacking that sequence. Primer extension studies demonstrated that the transcriptional start site of alternatively spliced CD59 mRNA is the same as that of transcripts without the alternatively spliced exon sequence, suggesting that expression of both forms of CD59 mRNA is regulated similarly. Analysis of the promoter region showed that the first 70 nucleotides immediately 5' of the transcriptional start site of the CD59 gene are essential for both constitutive and PMA-responsive transcription; however, responsiveness to PMA is cell line specific. Together, these studies have redefined the organization of the CD59 gene and identified regions of the promoter involved in constitutive and PMA-inducible transcription.
IL-11 enhances survival and decreases TNF production after radiation-induced thoracic injury.
Authors: Redlich C A , Gao X , Rockwell S , Kelley M , Elias J A
Published: December 2022

We hypothesized that IL-11 would protect against radiation-induced thoracic injury. To test this hypothesis, we compared the survival of rIL-11 and vehicle-treated control mice after 25 Gy of thoracic irradiation, and initiated studies to elucidate the mechanism of the observed protection. This dose of radiation killed 50% of the control mice during the first 2 wk after irradiation. In contrast, the s.c. administration of rIL-11 resulted in significant radioprotection with 89% of the rIL-11-treated animals surviving the study interval (p < 0.001). This radioprotection was at least partially specific for normal thoracic structures since rIL-11 did not alter the development or radiosensitivity of EMT6 tumor cells growing as lung metastases. TNF mRNA was not detected in normal lungs but was impressively induced after thoracic irradiation. Treatment with rIL-11 abrogated this increase. Parallel in vitro studies demonstrated that rIL-11 inhibits LPS and radiation-induced macrophage TNF protein production and mRNA accumulation. These studies demonstrate that rIL-11 reduces the mortality following thoracic irradiation, without enhancing the development or diminishing the radiosensitivity of pulmonary metastatic tumors. They also demonstrate that rIL-11 inhibits both radiation-induced TNF mRNA expression in vivo and macrophage TNF protein production and mRNA accumulation in vitro, suggesting that the radioprotective effects of rIL-11 may be mediated, at least in part, via the modulation of TNF production.
Identification of mouse granulocyte chemotactic protein-2 from fibroblasts and epithelial cells. Functional comparison with natural KC and macrophage inflammatory protein-2.
Authors: Wuyts A , Haelens A , Proost P , Lenaerts J P , Conings R , Opdenakker G , Van Damme J
Published: December 2022

Neutrophil and monocyte chemotactic factors were isolated from conditioned media of mouse fibroblasts and epithelial cells. Neutrophil chemotactic activities were purified to homogeneity using a four-step chromatographic procedure, and the corresponding proteins were identified by amino acid sequence analysis. Natural forms of the murine chemokines KC and macrophage inflammatory protein-2 were isolated from virus-infected fibroblasts. However, the major neutrophil chemotactic activity from fibroblasts stimulated with endotoxin plus double-stranded RNA and from PMA-treated epithelial cells resided in other 7- and 8-kDa proteins. Amino acid sequence analysis revealed a novel Cys-Xaa-Cys chemokine structure, characterized by the conservation of four cysteines and the Glu-Leu-Arg motif. Based on the completely identified primary structure of this natural protein, this chemokine must be considered to be the murine homologue of human and bovine granulocyte chemotactic protein-2 (GCP-2; 61 and 64% identical residues, respectively). Due to NH2-terminal cleavage, 11 different forms of mouse GCP-2 were discovered. In contrast to human and bovine GCP-2, functional comparison of long and short NH2-terminal forms of mouse GCP-2 demonstrated that truncated mouse GCP-2 (short form) has a higher specific activity in neutrophil activation (gelatinase B release) and chemotaxis assays. Furthermore, mouse GCP-2 was more potent than human GCP-2 on human neutrophils, and more active than murine KC and macrophage inflammatory protein-2 on mouse neutrophils. In view of the absence of a murine homologue for IL-8, NH2-terminally processed GCP-2 can be considered a major neutrophil chemoattractant in the mouse during the inflammatory response.
Eosinophil recruitment following allergen challenge is associated with the release of the chemokine RANTES into asthmatic airways.
Authors: Teran L M , Noso N , Carroll M , Davies D E , Holgate S , Schröder J M
Published: December 2022

Eosinophil infiltration of the airways in response to allergen exposure is a characteristic of bronchial asthma. However, the mechanisms by which these cells are recruited are poorly understood. We have investigated the presence of eosinophil chemotactic activity (ECA) in bronchoalveolar lavage fluid obtained from allergic asthmatics (n = 6) 4 h after endobronchial allergen challenge. ECA was purified by sequential heparin affinity chromatography and reverse-phase HPLC. A single peak of ECA was detected; SDS-PAGE and Western blot analysis showed that the peak contained a protein of 8 kDa and corresponded to the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted). Consistent with this, the ECA was neutralized by an Ab to RANTES. Measurement of RANTES by ELISA in 10x concentrated bronchoalveolar lavage fluid showed increased levels of this chemokine at the allergen site (median, 187 pg/ml; range, 46-263 pg/ml) in comparison with a saline challenge control site (median, 32.5 pg/ml; range, 11-94 pg/ml), P < 0.005. Furthermore, there was a significant correlation between concentrations of immunoreactive RANTES and the number of eosinophils at the allergen challenge site (r = 0.8; p < 0.001), but not at the saline site (r = 0.2; p = 0.12). These results suggest that RANTES in involved in the recruitment of eosinophils into the asthmatic airways after allergen challenge.
The responses of mature T cells are not necessarily antagonized by their positively selecting peptide.
Authors: Ignatowicz L , Kappler J , Parker D C , Marrack P
Published: December 2022

Transgenic mice have been produced in which all detectable MHC class II proteins, IAb, are bound to a single peptide. Hence, all of the CD4+ class II-restricted T cells in these animals are positively selected in the thymus by reaction with this single class II/peptide combination. It has been suggested that the peptide involved in positive selection of a particular T cell might antagonize the responses of that T cell to other peptides, a phenomenon that might serve to inhibit autoimmune reactions. To test this idea, we took advantage of the fact that T cells from the class II single-peptide mice react strongly in primary mixed lymphocyte cultures with IAb molecules from wild-type H-2b mice, i.e., loaded with a heterogeneous collection of self peptides. The responses of the T cells to other class II-bound peptides were not inhibited by high concentrations of the selecting peptide. Therefore, peptides involved in positive selection of T cells do not necessarily inhibit or antagonize the responses of mature T cells to Ag.
Two distinct stimulus-dependent pathways lead to production of soluble murine interleukin-4 receptor.
Authors: Blum H , Wolf M , Enssle K , Röllinghoff M , Gessner A
Published: December 2022

The IL-4R exists in two forms, either membrane bound or as a soluble (s) molecule. Since the sIL-4R binds to its ligand with high affinity, thereby acting as an immunoregulatory molecule, we were interested in the processes leading to its release. First, the release of sIL-4R in the model of murine leishmaniosis was analyzed. Infection of mice with Leishmania major resulted in up-regulation of sIL-4R production by Ag-stimulated CD4+ T cells, with a maximum around 7 days after infection. To clarify the mechanisms underlying sIL-4R release, in vitro studies were performed. After stimulation of naive lymphoid cells with IL-4, sIL-4R release was dependent on up-regulation of spliced IL-4R mRNA, as shown by inhibition with specific antisense oligonucleotides. In contrast to this, no increase in the spliced IL-4R mRNA and no inhibitory influence of antisense oligonucleotides were observed after stimulation of T cells from IL-4-deficient mice with anti-CD3 mAb. Thus, TCR stimulation can lead to IL-4-independent sIL-4R production. Under these conditions proteolytic shedding of membrane-bound IL-4R appears to be the principal mechanism of release, since in contrast to stimulation with IL-4, iodinated sIL-4R could only be immunoprecipitated after cell surface labeling and subsequent TCR stimulation. The common gamma-chain, a component of the IL-4R complex, did not appear to be involved in the pathways leading to sIL-4R expression. This analysis suggests the existence of two differentially regulated pathways of sIL-4R release, possibly having different consequences for the regulation of IL-4 bioactivity.
Intracellular signaling for inducible antigen receptor-mediated Fas resistance in B cells.
Authors: Foote L C , Schneider T J , Fischer G M , Wang J K , Rasmussen B , Campbell K A , Lynch D H , Ju S T , Marshak-Rothstein A , Rothstein T L
Published: December 2022

CD40 ligand-activated B cells are sensitive targets for CD4+ Th1 effector cells that kill in a Fas-dependent fashion. Susceptibility to apoptosis is counteracted by Ag receptor binding that produces a state of resistance to Fas engagement in otherwise sensitive targets. In the present study, protection from Th1-mediated apoptosis was found to be induced by protein kinase C and calcium signals, which in combination mimicked the level of Fas resistance produced by surface Ig engagement. Signaling for Fas resistance did not alter Fas expression. Furthermore, B cells that were protected against Th1-mediated apoptosis were also resistant to apoptosis mediated by soluble, rFas ligand. Taken together, these results indicate that signaling for protection against Fas-mediated apoptosis does not depend on alteration of the interaction between B cell target and Th1 effector populations. Instead, surface IgM-derived protein kinase C and calcium signals appear to produce an intracellular change in the Fas signaling pathway that develops over a period of hours and interferes with the apoptotic process through a mechanism that depends on protein synthesis.
Molecular interaction between CD58 and CD2 counter-receptors mediates the ability of monocytes to augment T cell activation by IL-12.
Authors: Gollob J A , Li J , Kawasaki H , Daley J F , Groves C , Reinherz E L , Ritz J
Published: December 2022

IL-12 stimulates both T and NK cells and is pivotal in the development of the Th1 immune response. In this work, we show that an interaction between CD2 and CD58 on activated T cells and monocytes, respectively, regulates the T cell response to IL-12. B cells provide little IL-12-specific costimulation, and this correlates with the low level of CD58 on B cells relative to monocytes and the lack of significant up-regulation in response to IFN-gamma or PHA activation. CHO cell transfectants expressing CD58 at a level comparable with that found on monocytes restore IL-12 responsiveness to APC-depleted T cells. This effect is not observed with CHO cells expressing CD48, a second CD2 ligand with a low avidity for CD2 relative to CD58. Thus, in addition to augmenting adhesion between T cells and their cognate APCs and facilitating TCR-triggered activation, the CD2-CD58 interaction uniquely optimizes the T cell response to IL-12.
Generation of naturally processed peptide/MHC class I complexes is independent of the stability of endogenously synthesized precursors.
Authors: Goth S , Nguyen V , Shastri N
Published: December 2022

Proteolysis of endogenously synthesized cellular proteins is essential for constitutive display of processed peptide/MHC class I complexes on the APC surface for stimulating CD8+ T cells. However, the extent to which normal protein turnover serves as the source of processed peptides is not clear. To address this question, we used pairs of novel N-end rule substrates that varied in their intracellular stability and served as precursors for generating peptide/MHC class I (OVA257-264/Kb or influenza nucleoprotein 366-374/Db) complexes. Surprisingly, although each of three precursor pairs tested varied profoundly in their intracellular stability, they were indistinguishable in either T cell stimulation assays, or in the amounts of naturally processed peptides in the APC extracts. Our findings demonstrate that the proteolytic turnover of endogenously synthesized proteins is not directly proportional to the generation of processed antigenic peptide/MHC class I complexes.
IL-12 antagonism induces T helper 2 responses, yet exacerbates cardiac allograft rejection. Evidence against a dominant protective role for T helper 2 cytokines in alloimmunity.
Authors: Piccotti J R , Chan S Y , Goodman R E , Magram J , Eichwald E J , Bishop D K
Published: December 2022

IL-12 promotes Th1 development and inhibits the generation of Th2 by inducing IFN-gamma production. In several experimental models, Th2 are preferentially induced in the absence of IL-12. It was proposed that the preferential induction of Th2 by IL-12 antagonism would inhibit Th1-driven rejection responses, thereby promoting allograft acceptance. To test this possibility, mouse cardiac allograft recipients were treated with either anti-IL-12 Abs, or with the IL-12 receptor antagonist p40 homodimer. Unmodified rejection is characterized by a Th1-dominated response, with Th2 cytokines being absent or only weakly expressed within the allograft. Though both forms of IL-12 antagonism induced Th2 cytokine expression within the allograft, these treatments surprisingly exacerbated graft rejection relative to control animals. Interestingly, IL-12 antagonism did not inhibit IFN-gamma gene expression or in vivo sensitization of IFN-gamma-producing cells. Similar observations were made when IL-12 p40 knockout mice were used as allograft donors and recipients, verifying that IL-12 was not required for Th1 development. Further, IL-12 antagonism was associated with strong expression of p40 and weak expression of p35 within the graft. Neither p35 nor p40 mRNAs were detectable in control allografts. These data indicate that while IL-12 antagonism does induce Th2 cytokine expression within cardiac allografts, Th2 cytokines do not play a dominant protective role in the rejection process. Further, the Th2-inducing activity of IL-12 antagonism is not related to decreased IFN-gamma production, but may reflect altered regulation of IL-12 itself.
Short term treatment with soluble neuroantigen and anti-CD11a (LFA-1) protects rats against autoimmune encephalomyelitis: treatment abrogates autoimmune disease but not autoimmunity.
Authors: Willenborg D O , Staykova M A , Miyasaka M
Published: December 2022

Resistance to autoimmune encephalomyelitis was induced by s.c. infusion of myelin basic protein (MBP) in saline in combination with i.p. injections of anti-CD11a (LFA-1) mAbs. This treatment induces resistance to EAE induction, which appears early and persists for at least one month after treatment. Some MBP-CFA-challenged resistant rats showed minimal inflammation in the central nervous system, which was, however, confined to the meninges of the lower spinal cord. Examination of the immune status of MBP-anti-LFA-1 treated rats before encephalitogenic challenge failed to reveal any priming when assessed by Ag driven proliferation and cytokine production by lymphoid cells, and by circulating Ab production. Following challenge of protected rats, lymph node cell proliferation to MBP was unaltered, indicating that reactive cells had not been deleted or energized. Resistance could not be transferred with lymphoid cells from treated rats nor abrogated by cyclophosphamide treatment. In treated rats following challenge, there was a shift in the isotype of anti-MBP Ab produced, from an IgG2a:IgG1 ratio of 2:1 to 1:1, due to an increase in IgG1 production, indicating a possible bias towards a nonpathogenic Th2 CD4+ T cell response. The IgG1 Ab was detected early after challenge suggesting that pretreatment had indeed primed the animals, and had primed them to go down the Th2 pathway following encephalitogenic challenge. The ability to divert immune reactivity from a destructive to a nondestructive response could have important therapeutic implications for autoimmune disease.
Coexpression of B7-1 and antigen blocks tolerance induction to antigen presented by resting B cells.
Authors: Yuschenkoff V N , Sethna M P , Freeman G J , Parker D C
Published: December 2022

To investigate a role for B cells as tolerogenic APCs in peripheral lymphoid organs, we have developed a system in which B cells from mice transgenic for the membrane-bound form of human mu-chain are transferred into nontransgenic recipients. Mice injected with B cells expressing human mu-chain became profoundly tolerant to human mu-chain, as shown by greatly reduced Ab responses following challenge with human mu-chain in adjuvant. Adoptive transfer experiments showed that the recipient's Th cell response to human mu-chain was impaired. When the human mu transgenic spleen cells were activated with LPS before transfer, they no longer induced tolerance. Spleen cells from double-transgenic mice expressing both human mu-chain and the costimulatory molecule B7-1 (CD80) also failed to induce tolerance to human mu-chain. However, neither human mu transgenic LPS blasts nor double-transgenic B cells induced an Ab response or primed for a secondary Ab response to Ag in adjuvant. Therefore, we find that expression of B7-1 together with Ag can interfere with tolerance induction without inducing Ab formation or priming for a secondary Ab response.
Interaction of anti-cholesterol antibodies with human lipoproteins.
Authors: Dijkstra J , Swartz G M , Raney J J , Aniagolu J , Toro L , Nacy C A , Green S J
Published: December 2022

Inoculation of mice with cholesterol-rich liposomes containing the adjuvant monophosphoryl lipid A results in the production of antiserum containing IgM Ab to cholesterol. The specificity of the Ab was to cholesterol and structurally similar sterols containing a 3 beta-hydroxyl group. Anti-cholesterol binding activity was significantly diminished if the 3 beta-hydroxyl was altered by either epimerization, substitution, oxidation, or esterification. A similar specificity for 3 beta-hydroxy-sterols was observed for an anti-cholesterol IgM mAb. Both hyperimmune serum and the mAb reacted with intact human very-low-/intermediate-density lipoprotein (VLDL/IDL) and low-density lipoproteins (LDL), but not high-density lipoproteins (HDL), in an ELISA, but could react with total lipid extracts containing cholesterol that were prepared from all three lipoprotein classes. Functionally, immune serum or the mAb aggregated and induced a fusion-like reaction with VLDL/IDL and LDL at low temperatures: these aggregates result in spherical structures visible with light microscopy. Similarly, binding of anti-cholesterol A to small cholesterol-rich liposomes resulted in the appearance of vesicular structures with approximately 20- to 200-fold increased diameters. These data demonstrate that the anti-cholesterol Ab recognize unesterified cholesterol in VLDL/IDL and LDL; high-density lipoprotein cholesterol in the intact lipoprotein, however, appears to be protected from reaction with these Ab.
Ig light chain gene in the Siberian sturgeon (Acipenser baeri).
Authors: Lundqvist M , Bengtén E , Strömberg S , Pilström L
Published: December 2022

Elasmobranch and teleost fish have their Ig light (L) chain loci organized in multiple clusters (VL-JL-CL). The VL segments of teleosts are in opposite transcriptional orientation to the CL genes, suggesting that in teleosts and elasmobranchs there may have been separate evolutionary events leading to this organization. To address this problem, the IgL locus from the Siberian sturgeon (Acipenser baeri) (representative of a branch between elasmobranchs and teleosts) was investigated. Sequence analysis of cDNA clones shows that sturgeon VL genes are most similar to those of teleosts, but that sturgeon CL genes are more similar to those of the sharks. Southern blot analyses of sturgeon erythrocyte DNA with VL- and CL-specific probes showed that there are more than 20 VL segments in both the tetraploid Siberian sturgeon and the diploid sterlet (Acipenser ruthenus), but only a few CL segments in the genome of the Siberian sturgeon and up to four CL segments in that of the sterlet. Screening of an unamplified genomic library gave more than 300 VL-positive and four CL-positive clones. None of these contained inserts positive for both probes. PCR analysis of a genomic CL clone using IC and CL-specific primers suggested that upstream of the CL segment there are at least seven JL segments. it is concluded that sturgeons have a kappa-like organization of their IgL locus and that the clustered organization of IgL loci in bony fish and sharks arose from two distinct evolutionary events.
IL-4-induced expression of the IL-1 receptor antagonist gene is mediated by STAT6.
Authors: Ohmori Y , Smith M F , Hamilton T A
Published: December 2022

IL-4 alone or in cooperation with LPS can induce the expression of the gene encoding the secreted-type IL-1 receptor antagonist (sIL-1ra) in mononuclear phagocytes. To determine the nuclear signaling mechanisms involved in this response, the region flanking the transcription start site of the human sIL-1ra gene was placed upstream of the luciferase reporter gene, and the function of specific sequence elements was analyzed following transient transfection in the macrophage-like cell line RAW264.7. A region located between -250 and -200 bases relative to the transcription start site was necessary for response to IL-4 alone and for cooperation between IL-4 and LPS. This 50-bp region contains two inverted repeat elements that represent potential binding sites for members of the signal transducer and activator of transcription (STAT) gene family (STAT-binding elements (SBEs)). Site-directed mutagenesis of the distal SBE abolished IL-4 responsiveness, and multiple copies of this motif were able to confer IL-4 sensitivity to luciferase expression in the context of a heterologous (herpes virus thymidine kinase) promoter. Mutation of the proximal SBE in the intact IL-1ra promoter had little or no effect on response to IL-4, and this sequence motif was inactive when examined alone. Electrophoretic mobility shift assays using an oligonucleotide corresponding to the distal SBE identified a single binding activity that was detected in nuclei within 15 min of IL-4 treatment and that was recognized by Ab to STAT6. These results indicate that IL-4-induced STAT6 is required for IL-4-induced transcriptional activation of the sIL-1ra gene.