bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2.
Authors: Diefenbach R J , Isenman D E
Published: December 2022

Most evidence points toward there being a shared binding site in complement receptor type 2 (CR2, CD21) for the complement ligand C3dg and the EBV surface envelope glycoprotein gp350/220. Indeed, synthetic peptide studies have suggested that the CR2-binding sites in human C3dg and EBV gp350/220 share a similar sequence motif. The proposed CR2-binding sequence in C3dg is EDPGKQLYNVEA (residues 1199-1210 of mature C3), whereas that in EBV gp350/220 is EDPGFFNVEI (residues identical to C3dg are underlined). To further examine the role of amino acids 1199-1210 in the binding of the C3 fragments iC3b and C3dg to CR2, the following alanine-substitution variants of human C3 were tested in two independent CR2-binding assays: ED1199,1200AA; KQ1203,1204AA; L1205A; Y1206A; NV1207,1208AA; E1209A; and ED-KQ-NV1199,1200-1203,1204-1207,1208AA-AA-AA. Also engineered and tested was a chimeric C3 molecule in which the 1199-1210 sequence (PVPGGYQLTLEA) from the non-CR2-binding trout C3 molecule was grafted onto a human C3 background. Recombinant C3 proteins were expressed transiently in COS-1 cells, deposited as C3b on C3 convertase-bearing sheep erythrocytes and finally converted to cell-bound iC3b or C3dg using factors H and I. Binding of EAC423bi and EAC423dg to CR2 on Raji cells or EAC423dg to soluble CR2 was assessed. In most cases, the substitutions had little effect on CR2-binding activity and even in the case of the most highly substituted variants, the decrease in CR2-binding activity was less than twofold. Thus, contrary to the results anticipated from synthetic peptide studies, the single and multiple substitutions to the C3 sequence tested failed to corroborate a role for the 1199-1210 sequence in the C3dg-CR2 interaction.
Analysis of the New Zealand Black contribution to lupus-like renal disease. Multiple genes that operate in a threshold manner.
Authors: Drake C G , Rozzo S J , Hirschfeld H F , Smarnworawong N P , Palmer E , Kotzin B L
Published: December 2022

F1 progeny of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop an autoimmune process remarkably similar to human systemic lupus erythematosus. Previous studies have implicated major genetic contributions from the NZW MHC and from a dominant NZB gene on chromosome 4. To identify additional NZB contributions to lupus-like disease, (NZB x SM/J)F1 x NZW backcross mice were followed for the development of severe renal disease and were comprehensively genotyped. Despite a 50% incidence of disease, significant associations between the presence of the NZB genotype and disease were noted on chromosomes 1, 4, 7, 10, 13, and 19. The data indicated that multiple NZB genes, in different combinations, contribute to severe renal disease, and that no single gene is required. To further investigate this NZB contribution, NZB x SM/J (NXSM) recombinant inbred (RI) strains were crossed with NZW mice, and F1 progeny were analyzed for the presence of lupus-like renal disease. Interestingly, nearly all of the (RI x NZW)F1 cohorts studied expressed some level of disease. Five RI strains generated a high incidence of disease, similar to (NZB x NZW)F1 mice, and nearly one-half of the cohorts developed disease at intermediate levels. Only two cohorts demonstrated very little disease, supporting the conclusion that multiple genes are capable of disease induction. Experiments correlating the genotypes of these RI strains with their ability to generate disease revealed that none of the disease-associated loci defined by the backcross analysis were present in all five RI strains that generated disease at high levels. Overall, both the backcross data and RI analysis provide additional support for the genetic complexity of lupus nephritis and uphold the conclusion that heterogeneous combinations of contributing NZB genes seem to operate in a threshold manner to generate the disease phenotype.
Selective expansion followed by profound deletion of mature V beta 8.3+ T cells in vivo after exposure to the superantigenic lectin Urtica dioica agglutinin.
Authors: Galelli A , Delcourt M , Wagner M C , Peumans W , Truffa-Bachi P
Published: December 2022

Urtica dioica agglutinin (UDA) is a superantigen that, in vitro, binds to specific carbohydrate structures on class II and induces a sixfold enrichment of V beta 8.3+ BALB/c mice splenic T cells. Superantigens have pleiotropic effects in vivo, causing the activation, proliferation, and deletion of specific T cells, but are heterogenous in regard to their effects on T cell tolerization. We, therefore, compared the responses of peripheral T cells from adult BALB/c mice with the i.v. injection of 50 micrograms UDA or the bacterial superantigen staphylococcal enterotoxin B (SEB) that also recognizes the V beta 8.3 gene product. The data presented indicate that activation, clonal expansion, anergy, and death of V beta 8.3+ T cells occur sequentially after UDA administration. Two days after UDA injection, the proportion of V beta 8.3+ T cells in the periphery is elevated to approximately twice that of normal mice. This expansion occurs in both CD4+ and CD8+ subsets. V beta 8.3+ T cells from UDA-primed mice are anergic to UDA restimulation and fail to proliferate or to produce IL-2. Futhermore, the proliferation of V beta 8.3+ T cells is followed by their rapid disappearance concomitant with their specific elimination by apoptosis. In 1 wk, all CD4+ V beta 8.3+ peripheral T cells are deleted. The decline of V beta 8.3+ T cells in the CD4+ subset is more than in the CD8+ subset. This occurs in thymectomized and in thymus-intact animals. Two months after UDA priming, the percentage of V beta 8.3+ T cells is still lower than in control mice.
A cascade of cytokines is responsible for IL-9 expression in human T cells. Involvement of IL-2, IL-4, and IL-10.
Authors: Houssiau F A , Schandené L , Stevens M , Cambiaso C , Goldman M , van Snick J , Renauld J C
Published: December 2022

We have previously demonstrated that IL-9 induction in human T cells stimulated with PMA and anti-CD3 Ab is mediated by IL-2, as it was blocked by anti-IL-2R Ab. The experiments reported here indicate that anti-IL-2R Ab also inhibited the expression of IL-4, IL-6, and IL-10, thereby raising the possibility that the blockade of IL-9 production by anti-IL-2R mAb could be secondary to the blockade of these cytokines. We found that the inhibition caused by anti-IL-2R Ab on IL-9 production could be reversed by the addition of a combination of IL-4 and IL-10. Moreover, IL-9 production by T cells stimulated with PMA and anti-CD3 Ab was blocked by the addition of anti-IL-4 and anti-IL-10 Abs. Similar results were obtained with T cells cultured on B7-1/Fc gamma RII-transfected fibroblasts in the presence of anti-CD3 Ab. Analysis of cytokine production by different T cell subsets revealed that IL-4, IL-9, and IL-10 were produced only by CD45 Ro+ T cells. Importantly, CD45 Ra+ T cells were capable of producing IL-9 provided that both IL-4 and IL-10 were added to the cultures. The possible relationship between IL-4 production and IL-10 production was clarified by experiments indicating that IL-10 production was inhibited by anti-IL-4 Ab. However, IL-4 was not sufficient to trigger IL-10 production, which required both IL-2 and IL-4. Taken together, our results demonstrate the existence of a cascade of cytokines responsible for IL-9 expression, with IL-2 being required for IL-4 production, a combination of IL-2 and IL-4 for IL-10 production, and a combination of IL-4 and IL-10 for IL-9 production. Kinetics studies of cytokine gene expression in T cells further validated this model.
Induction of protective polyclonal antibodies by immunization with a Plasmodium yoelii circumsporozoite protein multiple antigen peptide vaccine.
Authors: Wang R , Charoenvit Y , Corradin G , Porrozzi R , Hunter R L , Glenn G , Alving C R , Church P , Hoffman S L
Published: December 2022

Monoclonal Abs against the repeat region of the circumsporozoite protein (CSP) completely protect mice against Plasmodium yoelii (Py), but synthetic peptide and recombinant protein vaccines designed to produce only Abs to the PyCSP repeat region have never been reported to consistently provide protection. This lack of protection in the rodent model system has predicted the poor protection achieved in humans after immunization with synthetic peptide and recombinant protein P. falciparum CSP vaccines and has raised serious questions regarding the capacity for vaccine-induced polyclonal Abs against the CSP to consistently protect humans. We now report immunization studies with a multiple Ag peptide vaccine designed to rely on "universal" T epitopes from tetanus toxin to produce T cell help for induction of protective Abs against the repeat region of the PyCSP. When delivered with a nonionic block co-polymer adjuvant, the vaccine protected 78 to 100% of three inbred strains of mice, and 100% of outbred mice against P. yoelii sporozoite challenge. Protection was associated with Ab titer, and passive transfer of purified IgG from immune mice protected naive recipients. Similar protection was achieved when the peptide was encapsulated in liposomes with lipid A and mixed with aluminum hydroxide. By demonstrating for the first time solid protection against P. yoelii by polyclonal Abs against the CSP, these data provide the rationale for assessment of a similarly constructed and formulated P. falciparum CSP multiple Ag peptide vaccine in humans.
Mechanism of drug-induced lupus. I. Cloned Th2 cells modified with DNA methylation inhibitors in vitro cause autoimmunity in vivo.
Authors: Yung R L , Quddus J , Chrisp C E , Johnson K J , Richardson B C
Published: December 2022

Treating activated CD4+ T cells with DNA methyltransferase inhibitors modifies gene expression and induces autoreactivity. Adoptive transfer of viable polyclonal autoreactive cells causes a lupus-like disease, most likely because of one or more effector functions expressed by the autoreactive cells. However, the number of potential effector mechanisms expressed by polyclonal cells is large. To more readily identify responsible mechanisms, we asked if autoimmunity can be induced by using the conalbumin-reactive, cloned Th2 cell line D10.G4.1, treated with 5-azacytidine (5-azaC) or procainamide (Pca). Treated, but not untreated, cells responded to syngeneic APCs without Ag, overexpressed LFA-1, spontaneously lysed syngeneic macrophages, and secreted relatively large amounts of IL-6, small amounts of IL-4, and no detectable IL-2 nor IFN-gamma. Adoptive transfer of treated, but not untreated, cells induced a severe immune complex glomerulonephritis, pulmonary alveolitis, central nervous system abnormalities including fibrinoid necrosis, karyorrhexis, and meningitis, and bile duct proliferation with periportal inflammatory cell infiltration resembling primary biliary cirrhosis. Anti-ssDNA, anti-dsDNA, and anti-histone Abs were also found. These experiments demonstrate that modification of this cloned T cell line with DNA methyltransferase inhibitors is sufficient to cause an autoimmune disease, with features of lupus as well as autoimmune liver disease. The results also raise the possibility that macrophage lysis, IL-6 secretion, and LFA-1 overexpression could contribute to the disease process. This system may be useful in testing the role of these and other pathologic mechanisms in the development of specific autoimmune lesions.
Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor beta- and gamma-chains.
Authors: Adamczewski M , Numerof R P , Koretzky G A , Kinet J P
Published: December 2022

Previous studies using tyrosine phosphatase inhibitors have implicated tyrosine phosphatases in the signal transduction pathway initiated by aggregation of Fc epsilon RI, the high affinity receptor for IgE. To define more precisely a role for the tyrosine phosphatase CD45 in Fc epsilon RI-mediated signaling, we have transfected the three subunits of Fc epsilon RI into wild-type Jurkat and a CD45-deficient Jurkat derivative. Here we demonstrate that CD45 is necessary for the initiation of calcium flux through the transfected Fc epsilon RI. In contrast to the effect of phosphatase inhibitors, the tyrosine phosphorylation levels of beta and gamma after aggregation of Fc epsilon RI are surprisingly reduced, relative to wild-type Jurkat, in the CD45-deficient cells. After reconstitution of the CD45-deficient cells with a chimeric molecule containing the cytoplasmic phosphatase domains of CD45, both the base line and activation-induced tyrosine phosphorylation levels are increased. By examining Lck autophosphorylation, we find that Fc epsilon RI aggregation induces an increase in Lck enzymatic activity only in wild-type Jurkat and the CD45-deficient Jurkat reconstituted with chimeric CD45. This regulation of src-family tyrosine kinase activity may be the means by which CD45 controls aggregation-induced receptor phosphorylation.
Phenotypic characterization of CD4+ T cells that exhibit a transendothelial migratory capacity.
Authors: Brezinschek R I , Lipsky P E , Galea P , Vita R , Oppenheimer-Marks N
Published: December 2022

The phenotype of CD4+ T cells capable of transendothelial migration was determined using an in vitro model system, in which cells migrate through a monolayer of endothelial cells (EC) on collagen gels. A specific subset of resting CD4+ memory T cells was found to migrate. T cells within this subset can be defined by the bright expression of CD11a, CD26, CD44, and CD49d. Additionally, the migratory CD4+ T cell population is largely CD58bright, CD31-, CD62L-, and is also enriched in cells that brightly express CD49c, CD49e, and CD49f. Only a minority of the cells are activated, as indicated by expression of CD69. The EC were found to play a central role in facilitating migration of this subset because selective enrichment of CD11abright, CD26bright, CD44bright, CD4+ T cells was not observed when cells migrated in the absence of EC. Activation of the T cells induced a modest degree of migration of an additional subset of CD45RA+, CD31+ naive T cells. In contrast, TNF-alpha activation of the EC increased the transendothelial migration of an additional subset of activated memory T cells that expressed CD69 and CD62L. Neither activation of the T cells, stimulation of the EC, nor the presence of macrophage inflammatory protein-1 alpha (MIP-1 alpha) or RANTES, however, altered the phenotype of the majority of the migratory CD4+ T cell population, which is characteristic of a particular stage of memory cell differentiation. These results suggest that CD4+ T cells acquire the capacity for transendothelial migration at a specific phase of maturation that is only minimally altered by the activation of either the T cell or the EC, or by the presence of specific chemokines in the subendothelial matrix.
Receptor-specific induction of individual AP-1 components in B lymphocytes.
Authors: Huo L , Rothstein T L
Published: December 2022

The inducible nuclear transcription factor complex, AP-1, typically consists of heterodimers between Jun and Fos proteins. Although components are drawn from families of related molecules, little is known about the physiologic regulation of jun- and fos-related gene products. In particular, it is not known whether expression of individual family members is stimulus-specific or whether the same signaling pathways are responsible for induction of all subunits. To clarify these issues, AP-1 components were examined following activation of primary B lymphocytes through two separate receptors, the surface Ig Ag receptor, and the CD40 receptor for T cell influences. Both forms of stimulation led to expression of JunB and JunD mRNA and protein; however, induction of JunB mediated by anti-Ig Ab was protein kinase C (PKC)-dependent, whereas induction mediated by CD40 ligand was resistant to PKC depletion. The two forms of stimulation diverged even further with respect to Fos expression. Although both stimuli induced c-Fos, expression of FosB was stimulus specific at both the mRNA and protein levels, in that this component was induced by anti-Ig but not by CD40 ligand. Stimulated expression of c-Fos and FosB was in all cases PKC-independent. These results provide evidence for receptor-specific differences in the expression of AP-1 components, primarily with respect to FosB. They also indicate that separate intracellular pathways may be used for induction of jun and fos gene products and that the same transcription factor (junB) may be triggered by two surface receptors through separate pathways that differ in PKC dependence.
Potential involvement of IL-10 in suppressing tumor-associated macrophages. Colon-26-derived prostaglandin E2 inhibits TNF-alpha release via a mechanism involving IL-10.
Authors: Kambayashi T , Alexander H R , Fong M , Strassmann G
Published: December 2022

The administration of murine TNF-alpha to colon (C)-26 bearing mice, significantly protects the host against the catabolic effects of the tumor. This effect of exogenous TNF-alpha can be primarily attributed to tumor lysis rather than to a direct anticachectic action. Murine peritoneal macrophages cultured with the C-26 line or with C-26 culture supernatant do not release TNF-alpha in response to LPS stimulation. The reduction in TNF-alpha levels is associated with a significant increase in IL-10 levels. Single cell suspension of freshly disaggregated C-26 tumor (which contains host macrophages), do not produce TNF-alpha but contain significant levels of PGE2 and IL-10. In contrast, PGE2 but not TNF-alpha or IL-10 can be detected in the C-26.IVX cell line that is used to generate tumors in vivo. Neutralizing anti IL-10 Ab but not isotype-matched Ab, significantly reverses the inhibitory effect of the tumor cells and their culture supernatant on macrophage TNF-alpha release. Additional evidence is presented to indicate that the C-26-derived inhibitory activity is related to PGE2. Taken together, these results support the hypothesis that tumor-derived PGE2 prevents tumor-infiltrating macrophages from producing TNF-alpha, in part by augmenting macrophage IL-10 synthesis.
Co-localization of inducible-nitric oxide synthase and Plasmodium berghei in hepatocytes from rats immunized with irradiated sporozoites.
Authors: Klotz F W , Scheller L F , Seguin M C , Kumar N , Marletta M A , Green S J , Azad A F
Published: December 2022

Both CD8+ T cells and IFN-gamma (IFN-gamma) are important components in the regulation of inducible-nitric oxide synthase (iNOS) which contribute to liver stage anti-malarial activity in rodents immunized with irradiated sporozoites. IFN-gamma, provided by malaria-specific CD8+ T cells, stimulates liver cells to produce nitric oxide (NO) for the destruction of infected hepatocytes or the parasite within these cells. To identify the cell source of iNOS in livers from Brown Norway rats challenged with Plasmodium berghei sporozoites, we probed tissue sections with antisera that recognize iNOS and the malarial exoerythrocytic stage parasite. Immunofluorescence analysis of parasitized livers demonstrate that 1) iNOS was found in infected hepatocytes, not Kupffer or endothelial cells; and 2) a higher proportion of infected hepatocytes express iNOS in immunized rats compared with naive animals after challenge. There was no immunoreactivity to the iNOS antisera in liver sections of immunized rats 15 h after sporozoite challenge, however, iNOS activity was present in 18% of the infected hepatocytes by 24 h and reached 81% by 31 h. In contrast, < 10% of the infected hepatocytes displayed iNOS activity in naive or immune animals 48 h after challenge. We also found a significant decrease in the ability of the immunized animals to express iNOS in response to sporozoite challenge by accelerating the removal of pre-existing irradiated-attenuated parasites from hepatocytes with the antimalarial drug, primaquine. Therefore, induction and maintenance of iNOS activity were dependent on intrahepatic persistence of the irradiated-attenuated parasite. These results suggest that liver-iNOS expression following sporozoite challenge is restricted to the infected hepatocyte and dependent on the presence of the irradiated-attenuated parasite in immune animals.
Conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor-binding subunit essential for tyrosine phosphorylation and proliferation.
Authors: Ronco L V , Doyle S E , Raines M , Park L S , Gasson J C
Published: December 2022

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues and a tryptophan-serine motif (WSXWS) in the extracellular domain and proline-rich cytoplasmic domain. The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, hGM-CSFR, consists of two subunits, alpha (hGM-CSFR alpha), which is required for ligand binding, and beta (hGM-CSFR beta), which is required for signal transduction. Both the alpha and beta subunits are members of the cytokine receptor superfamily. In this study, we analyzed mutations in the conserved amino acids of the alpha subunit to determine their function in signal transduction, as assayed by tyrosine phosphorylation and proliferation. Disruption of either of the conserved disulfide bonds in the extracellular domain abolishes low-affinity binding but not binding to a preformed heterodimeric complex with the beta-chain. Cells expressing receptors with mutations in cysteines 2 or 3 grew as well as cells expressing wild-type receptors in human GM-CSF (hGM-CSF) and phosphorylated the same proteins on tyrosine residues, although the level of phosphorylation may be attenuated; cysteine 3 appears to be required for generation of the true high-affinity binding site. The WSXWS motif and the cytoplasmic domain are required for function of the human GM-CSF receptor, as stable cell lines expressing receptors with these mutations were unable to proliferate continuously in hGM-CSF. Surprisingly, no function for the conserved proline-rich region of the cytoplasmic domain could be ascertained from these studies; cells expressing these receptors were indistinguishable from wild-type in both binding and functional assays.
Inflammatory cytokines induce AIDS-Kaposi's sarcoma-derived spindle cells to produce and release basic fibroblast growth factor and enhance Kaposi's sarcoma-like lesion formation in nude mice.
Authors: Samaniego F , Markham P D , Gallo R C , Ensoli B
Published: December 2022

Homosexual males often present signs of immune activation and are likely to have increased levels of inflammatory cytokines such as IL-1 beta, TNF-alpha, and IFN-gamma. These individuals develop Kaposi's sarcoma (AIDS-KS) more frequently than other HIV-1-infected groups. Our previous work demonstrated that inflammatory cytokines stimulate the growth of spindle cells derived from AIDS-KS lesions (AIDS-KS cells) and that these cells produce high levels of bFGF that mediate autocrine and paracrine (endothelial) cell growth and angiogenesis. Here we show that AIDS-KS cells constitutively produce and release bioactive bFGF in the absence of cell death, and that extracellular bFGF exist in both a soluble and a bound form; the latter can be released by treatment with trypsin, heparin, or heparinase I. Inflammatory cytokines stimulate both the synthesis and release of biologically active bFGF from KS cells and enhance their ability to induce angiogenic KS-like lesions in nude mice. Because bFGF is highly expressed in primary KS lesions, and is a mediator of KS-like lesion formation, these results suggest that the export of bFGF induced by inflammatory cytokines may play a critical role in the induction and progression of KS in HIV-1-infected homosexual men.
Analysis of murine soluble Fc epsilon RII sites of cleavage and requirements for dual-affinity interaction with IgE.
Authors: Bartlett W C , Kelly A E , Johnson C M , Conrad D H
Published: December 2022

The low-affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal sequence homologous to C-type animal lectins. Between this region and the membrane is a repetitive sequence predicted to form an alpha-helical coiled-coil and is termed the stalk region. The Fc epsilon RII is proteolytically cleaved when at the cell surface in this stalk region. Both the 38 Kd and 28 Kd major released fragments were isolated from culture media and N-terminal sequencing demonstrated that the cleavage sites were in the third and fourth repeat domains, respectively. The identified sites show no apparent similarity with the cleavage sites previously identified in human Fc epsilon RII. Recent studies have demonstrated that the intact Fc epsilon RII interacts with IgE with a dual-affinity, resulting from a multivalent interaction with the IgE Fc region; mutant Fc epsilon RII that have a disruption of the alpha-helical coiled-coil have a single low-affinity interaction consistent with a monomeric interaction with IgE. The soluble Fc epsilon RII were shown to interact with IgE with an affinity similar to these mutant Fc epsilon RII. Preparation of a chimeric Fc epsilon RII in which the transmembrane and cytoplasmic regions were replaced with sequences from Ly-49 revealed that these regions played no role in the multimeric association of the Fc epsilon RII necessary for dual-affinity interaction with IgE. In addition, a full-sized soluble Fc epsilon RII construct was expressed, and this molecule demonstrated increased capacity to interact with IgE.
Recently activated naive CD4 T cells can help resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation to secretion of T helper 2-type cytokines.
Authors: Croft M , Swain S L
Published: December 2022

Development of T cells during primary responses was investigated using pigeon cytochrome C-specific naive Th from TCR transgenic mice. Naive CD4 cells did not activate and help resting B cells. This failure was found to be primarily because the resting B cells were incapable of stimulating the naive Th. Provision of a costimulatory signal such as anti-CD28, or addition of APCs that express costimulatory molecules, such as dendritic cells, activated B cells, and B7+ and B7+ICAM(+)-expressing fibroblasts, induced naive Th activation and promoted T cell-dependent help for IgM secretion. T cell activation for as little as 24 h promoted helper activity, and Ig secretion required production of small amounts of IL-4 by the activated naive Th. On initial stimulation, naive Th secrete only IL-2. By mRNA analysis, activated naive Th were also shown to produce IL-4, however induction of IL-4 message only occurred 24 h after initial activation and required additional stimulation with Ag. A single exposure of naive CD4 to Ag/APC followed by 4 to 12 days in culture led to generation of effector Th which secreted IL-2 and some IFN-gamma, and no detectable IL-4 or IL-5, and which could only help B cells to IgM secretion. In contrast, similar cultures that received Ag/APC one or more times during this period generated effector cells capable of secreting easily detectable titers of IL-4 and IL-5, as well as IL-2 and IFN-gamma, and able to now promote IgG1 and IgE responses. Generation of these Th0-like effectors was accompanied by increasing amounts of IL-4 secreted during the culture period after each restimulation, and addition of anti-IL-4 in culture inhibited development of the capacity to produce Th2 cytokines. These studies reinforce the notion that naive CD4 must interact with a costimulatory professional APC, rather than a resting B cell, for initiation of the primary response, but show that such an interaction can result in rapid development of the ability to interact with and provide cognate help to B cells. They also suggest that if activated naive CD4 cells receive multiple stimulations from Ag/APC, enough endogenous IL-4 can be produced to drive differentiation into effectors secreting type 2 cytokines. The existence of such an autocrine feedback mechanism suggests that the amount and availability of Ag could influence the nature and polarization of the Th response.
Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.
Authors: Ford A L , Goodsall A L , Hickey W F , Sedgwick J D
Published: December 2022

Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4+ T cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45lowCD11b/c+ is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45highCD11b/c+) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45highCD11b/c+ transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4+ myelin basic protein (MBP)-reactive T cells. CD45highCD11b/c+ CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture, suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4+ T cells to proliferate or secrete IL-2.
Expression and function of mouse Fas antigen on immature and mature T cells.
Authors: Nishimura Y , Ishii A , Kobayashi Y , Yamasaki Y , Yonehara S
Published: December 2022

We prepared mAbs specific for the mouse Fas Ag (CD95) and used them to analyze the expression and apoptosis-inducing activity of the Fas Ag on murine immunocytes. Cytofluorometry of mouse bone marrow, thymus, and splenocytes using the mAbs indicated that cells of the T lineage, except for bone marrow cells, expressed Fas Ag on the surface. CD4-CD8- undifferentiated thymocytes expressed low levels of Fas Ag. Immature CD4+CD8+ thymocytes and mature CD4+CD8- and CD4-CD8+ thymocytes were highly positive for Fas Ag. CD4+CD8+ thymocytes were specifically sensitive to the apoptosis-inducing activity of anti-Fas, although CD4-CD8-, CD4+CD8-, and CD4-CD8+ thymocytes were resistant. Spleen T cells were resistant to anti-Fas, whereas they expressed Fas Ag. The superantigen, staphylococcal enterotoxin B (SEB) administered to BALB/c mice, induced clonal expansion and successive clonal deletion of spleen T cells bearing the V beta 8 TCR, which specifically reacts to SEB. Such clonal deletion of V beta 8 T cells was highly suppressed in lpr mice, which have defects in the Fas Ag gene. In SEB-administrated BALB/c mice, expression of Fas Ag was significantly enhanced on V beta 8, but not on V beta 6 T cells, which cannot react to SEB. Moreover, V beta 8 T cells in SEB-primed mice were sensitive to the cell-killing activity of anti-Fas, although V beta 6 T cells were resistant. These findings show that the expression level and apoptosis-inducing activity of Fas Ag on peripheral T cells are directly up-regulated by stimulation through the TCR in vivo.
B cell selection and allelic exclusion of an anti-DNA Ig transgene in MRL-lpr/lpr mice.
Authors: Roark J H , Kuntz C L , Nguyen K A , Mandik L , Cattermole M , Erikson J
Published: December 2022

We have used an Ig transgene (VH3H9) that increases the frequency of anti-DNA autoantibodies to address whether the production of antinuclear Abs in systemic lupus erythematosus is the consequence of a breakdown of B cell tolerance. We have shown that nonautoimmune mice regulate anti-DNA B cells, and that lupus-prone MRL-lpr/lpr mice are defective in this regulation. Here we show that a subset of anti-DNA B cells, namely those that stain nuclei in a homogeneous fashion, not only fail to be deleted in MRL-lpr/lpr mice, but undergo preferential clonal expansion. In addition, we describe a surprising finding: the VH3H9 transgene is less efficient at inhibiting endogenous heavy chain gene rearrangement on the autoimmune-prone MRL-lpr/lpr genetic background than on the nonautoimmune BALB/c background.
Retargeting of CTL by an efficiently refolded bispecific single-chain Fv dimer produced in bacteria.
Authors: Kurucz I , Titus J A , Jost C R , Jacobus C M , Segal D M
Published: December 2022

A single-chain bispecific Fv dimer (bs(sFv)2) having specificity for mouse CD3 epsilon chain and human transferrin receptor was produced in bacterial inclusion bodies. To overcome difficulties associated with in vitro protein folding, we used a novel renaturation approach to obtain active bs(sFv)2. The protein was dissolved in the weak ionic detergent sodium lauroylsarcosine, and disulfides were formed by oxidation in air. After oxidation, the bs(sFv)2 exhibited very little covalent aggregation and migrated as a single species in nonreducing SDS-PAGE, suggesting that disulfides were correctly paired. The detergent was removed using an ion exchange resin and the protein fractionated by size exclusion chromatography. The recovered 65-kDa protein was monomeric in non-denaturing solvent, homogeneous by SDS-PAGE, and comprised 15 to 20% of material applied to the gel filtration column. This protein bound specifically to both mouse CD3 epsilon chain and human transferrin receptor with affinities indistinguishable from those of the parental Fabs or single-chain Fvs. The bs(sFv)2 specifically redirected mouse cytotoxic T cells to lyse target cells expressing human transferrin receptor at picomolar concentrations. Bacterially produced and detergent oxidized bs(sFv)2 molecules may therefore provide the abundant amounts of homogeneous active material required to redirect cytotoxic cells against tumors and other unwanted cells in animal models and in patients.
Molecular cloning and functional characterization of human lymphotactin.
Authors: Kennedy J , Kelner G S , Kleyensteuber S , Schall T J , Weiss M C , Yssel H , Schneider P V , Cocks B G , Bacon K B , Zlotnik A
Published: December 2022

We describe the isolation of a cDNA that encodes human lymphotactin (Ltn), a new class of lymphocyte-specific chemokine. Human Ltn shows similarity to some members of the C-C chemokine family but has lost the first and third cysteine residues that are characteristic of the C-C and C-X-C chemokines. Ltn is chemotactic for lymphocytes but not for monocytes, a characteristic that makes it unique among chemokines. In addition, calcium flux desensitization studies indicate that Ltn uses a unique receptor. The human Ltn gene maps to a different chromosome than do the C-C and C-X-C chemokine families. Taken together, these characteristics indicate that Ltn is the first example of a new class of human chemokines with preferential effects on lymphocytes.
Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes.
Authors: Manickan E , Rouse R J , Yu Z , Wire W S , Rouse B T
Published: December 2022

Plasmid DNA encoding proteins represent a convenient novel approach to vaccination. We have investigated this "genetic immunization" approach as a means to protect against herpes simplex virus (HSV) infection using a mouse zosteriform model that mimics several aspects of reactivated HSV infection of humans. After i.m. immunization with plasmid DNA-encoding glycoprotein B (gB), (pc-gB), 80% of BALB/c mice were completely protected and lesions were delayed in the remaining animals. Upon pc-gB vaccination, the animals developed both gB- and HSV-specific IgG Ab response and the isotype examination revealed a predominance of IgG2a. These mice also have low levels (1/16) of HSV-neutralizing Abs. Immune splenocytes obtained from pc-gB-immunized mice, when restimulated in vitro with HSV resulted in production of type 1 cytokines. Evidence for CD(8+)-mediated cytotoxic T lymphocyte response was equivocal. Protection could be adoptively transferred to nude mice recipients by CD4+ T cells from pc-gB-immunized mice but not by CD8+ T cells. Our results demonstrate that genetic immunization is a potent means of inducing protection against HSV and that the mechanism of immunity responsible for clearing virus from cutaneous sites is principally by CD4+ T cells. It is likely that these cells are Th1 cells because type 1 cytokines were the major cytokines detected upon in vitro Ag stimulation.
Contribution of the TCR alpha-chain to the differential recognition of bacterial and retroviral superantigens.
Authors: Daly K , Nguyen P , Hankley D , Zhang W J , Woodland D L , Blackman M A
Published: December 2022

The TCR alpha-chain has been implicated in superantigen recognition by some T cells. In this study, recognition of a retroviral (minor lymphocyte stimulating Ag-1, Mls-1) and a bacterial (staphylococcal enterotoxin B, SEB) superantigen by T cells expressing an identical (transgenic) V beta 8.1 beta-chain and either a V alpha 2+ or V alpha 11+ alpha-chain have been compared. Individual hybridomas were found to vary in their recognition of the two superantigens. Whereas some T cell hybridomas recognized both superantigens comparably, other hybridomas preferentially recognized either Mls-1 or SEB, indicating that the contribution of the TCR alpha-chain to recognition of these superantigens is not equivalent. The degree of heterogeneity in recognition varied between the V alpha 2+ and V alpha 11+ hybridomas, although there were individual hybridomas within each group with distinct patterns of reactivity that varied only in CDR3 alpha. These data are consistent with recent evidence that recognition of Mls-1 and SEB involves distinct TCR/MHC interactions.
Heterogeneity of human mast cells based on cytokine content.
Authors: Bradding P , Okayama Y , Howarth P H , Church M K , Holgate S T
Published: December 2022

Human mast cells can be divided into two distinct phenotypes based on their content of neutral serine proteases, suggesting that they serve differing biologic and pathologic roles. Recently, it has been demonstrated that human mast cells are a source of several pleiotropic cytokines including IL-4, IL-5, IL-6, IL-8, and TNF-alpha, but not all mast cells contain all of these cytokines, suggesting that there is also functional heterogeneity with respect to cytokine expression. In this study, we have examined the relationship between mast cell neutral protease expression and cytokine content using immunohistochemistry. Bronchial mucosal biopsies from five normal subjects and five patients with allergic asthma, and nasal mucosal biopsies from five normal subjects and three patients with allergic rhinitis were embedded in glycol methacrylate. Sections (2 microns) were stained for IL-4, IL-5, and IL-6, adjacent to serial sections stained for tryptase and chymase. The distribution of cytokines among the tryptase+ chymase- mast cells (MCT) and tryptase+ chymase+ mast cells (MCTC) was examined by co-localization of cytokines to MCTC or MCT in serial sections using the camera-lucida. Although IL-4 was distributed among both mast cell phenotypes, it was expressed preferentially by the MCTC subset (overall 85% MCTC:15% MCT). In contrast, IL-5 and IL-6 were restricted almost exclusively to the MCT subset. Immunostaining of isolated skin mast cells (> 99% MCTC) supported these findings, with strong immunoreactivity present for IL-4 but very little for IL-5 or IL-6. These results indicate that in addition to exhibiting heterogeneity with respect to neutral protease content of the secretory granules, human mast cells are also heterogeneous with respect to cytokine content. This suggests that the biologic functions of MCTC and MCT cells differ as a result of their capacity to generate and release different cytokine profiles.
Lipopolysaccharide binding protein and CD14 modulate the synthesis of platelet-activating factor by human monocytes and mesangial and endothelial cells stimulated with lipopolysaccharide.
Authors: Camussi G , Mariano F , Biancone L , De Martino A , Bussolati B , Montrucchio G , Tobias P S
Published: December 2022

The biosynthesis of platelet-activating factor (PAF) during Gram-negative involves the interaction of LPS with the cells of the host. We have investigated the molecular mechanism that controls cell recognition and PAF biosynthetic response to LPS in human monocytes (MO), glomerular mesangial cells (MC), and HUVEC in culture. The synthesis of PAF by MO and MC involves two proteins, plasma LPS binding protein (LBP) and cell membrane CD14 (mCD14). As MO, MC were shown to express the mCD14 molecule by several mAbs. MO and mCD14-positive MC were stimulated to synthesize PAF either by the 63D3 and IOM-2 mAbs or by the natural ligand LBP-LPS complex. Moreover, LeuM3, 28C5, and 18E12 mAbs that were themselves unable to stimulate the synthesis of PAF blocked PAF synthesis initiated by LBP-LPS complex. LBP was required for synthesis of PAF by MO. In MC, which synthesize PAF also after stimulation by LPS alone, the LBP was shown to speed and significantly enhance the synthesis of PAF. The soluble form of CD14 (sCD14), when added to MO stimulated with LBP-LPS complexes, inhibited the synthesis of PAF possibly by competing with mCD14. In contrast, sCD14 was shown to be required for LPS-induced synthesis of PAF by HUVEC, which did not express mCD14. Therefore, membrane receptors (mCD14) and plasma soluble proteins (LBP and sCD14) may enable different human cell types to synthesize PAF after LPS stimulation.
IFN-beta selectively down-regulates transferrin receptor expression in human peripheral blood macrophages by a post-translational mechanism.
Authors: Testa U , Conti L , Sposi N M , Varano B , Tritarelli E , Malorni W , Samoggia P , Rainaldi G , Peschle C , Belardelli F
Published: December 2022

We evaluated the effect of IFN-beta on the expression of transferrin receptor (TfR) during the in vitro differentiation of peripheral blood monocytes to macrophages. IFN-beta exerted a strong inhibitory effect on the expression of TfR. As little as 0.1 IU/ml was sufficient to induce a 40% reduction of transferrin (Tf) binding sites on 7-day cultured macrophages. Scatchard plot analysis revealed that this impaired Tf binding in IFN-beta-treated macrophages was not due to a decreased affinity of the TfR for its ligand but to a reduction in the number of cell surface TfR. IFN-gamma did not exert any significant effect on the expression of TfR, even though it was capable of partially reverting the inhibitory effect of the IFN-beta on Tf binding. To understand the mechanism by which IFN-beta inhibited TfR expression, we examined the expression of TfR mRNA, 125I-Tf binding to detergent-solubilized cells, and TfR cellular distribution. The results of these experiments showed that IFN-beta caused neither a significant alteration of the expression of TfR mRNA nor a decrease of the total content of TfR molecules. Moreover, immunofluorescence analysis of TfR localization indicated that TfR was clustered in an intracellular compartment in IFN-beta-treated macrophages. These data demonstrate that IFN-beta is capable of dramatically down-modulating TfR in macrophages by post-translational mechanisms (i.e., by sequestering this receptor in intracellular compartments).
Chimeric horse/human recombinant C9 proteins identify the amino acid sequence in horse C9 responsible for restriction of hemolysis.
Authors: Tomlinson S , Wang Y , Ueda E , Esser A F
Published: December 2022

Equine C9, in contrast to human C9, has extremely low hemolytic activity against most mammalian erythrocytes, although the amino acid sequences of both proteins show 77% identity. In an attempt to define the region of human C9 responsible for conferring its lytic activity, or conversely, the region of equine C9 responsible for its restriction, recombinant human and equine C9 and four chimeric human/equine C9 proteins were constructed and expressed in COS-7 cells. Recombinant human and equine C9 displayed hemolytic profiles similar to those of the purified native proteins. Exchange of a fragment extending from residues 145 to 290 in horse C9 with the corresponding one from human C9 created a fully hemolytic protein. This region contains the putative hinge region but not the membrane-interacting domain. Nonlytic chimeric C9 proteins inhibited hemolysis and binding of human C9 to EAC1-8 cells, indicating that they bind to their receptor, but subsequent unfolding or insertion into the membrane is impaired. These results suggest that restriction factors, such as glycophorin, CD59, or homologous restriction factor, on erythrocytes may limit the activity of horse C9 by interacting with its hinge region. In support of this conclusion direct binding of CD59 to immobilized horse C9 was detected by ligand blotting, and it was observed that a polyclonal anti-CD59 Ab enhanced human and horse C9-mediated hemolysis of human EAC1-7, but the increase in hemolytic activity of horse C9 by inhibition of CD59 was less than what could be achieved by insertion of the human C9 hinge region into horse C9.
MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response.
Authors: Leite-de-Moraes M C , Herbelin A , Machavoine F , Vicari A , Gombert J M , Papiernik M , Dy M
Published: December 2022

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.