bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and kinetic analysis of an epitope-specific shift in the B cell memory response to a multideterminant antigen.
Authors: Press J L , Giorgetti C A
Published: December 2022

Our previous studies showed that the primary and memory B cell responses to the multideterminant antigen poly-(L-Tyr, L-Glu)-poly-D,L-Ala-poly-L-Lys ((T,G)-A-L), differ. The primary response is dominated by antibodies binding side-chain epitopes; there is little antibody response to epitopes on the poly-D,L-Ala-poly-L-Lys backbone of (T,G)-A-L. In contrast, B cells producing A-L+ antibodies constitute approximately a third of the memory response to (T,G)-A-L. To determine the basis of this epitope-specific repertoire shift, we have examined the kinetics of expression of A-L+ B cells and antibodies after in vivo antigen priming and identified VH and V kappa genes used by A-L+ hybridoma antibodies derived from primary vs memory B cells. Kinetic studies, using the splenic focus assay, showed that the clonal frequency of A-L+ B cells remains low (<3% of (T,G)-A-L-specific B cells) 1 wk after Ag priming, increases (9%) by 2 wk, but does not reach the memory frequency (30%) until at least 3 wk after immunization. Molecular analyses showed that both the primary and memory A-L+ antibody responses are heterogeneous, using different VH and V kappa gene families as well as different germ-line genes within a VH gene family. Both H and L chain gene sequences showed somatic mutations in primary as well as memory antibodies. Analysis of antibody binding patterns and somatic mutations in a set of clonally related B cells that use a new germ-line VH gene in the VGAM3.8 family (VGK7, described here), showed a direct correlation between somatic mutation and change in antibody binding specificity. Our results demonstrate how somatic mutation and Ag selection play a role in the development of the memory response to a multideterminant Ag. The data are discussed in the context of the single vs dual lineage models for memory B cell generation.
Prostaglandin E2 limits arachidonic acid availability and inhibits leukotriene B4 synthesis in rat alveolar macrophages by a nonphospholipase A2 mechanism.
Authors: Christman B W , Christman J W , Dworski R , Blair I A , Prakash C
Published: December 2022

Prostaglandin E2 (PGE2), a potent mediator of inflammation released in large amounts by endotoxin-stimulated alveolar macrophages (AM), has been shown to inhibit leukotriene B4 (LTB4) release by activated neutrophils. We investigated the hypothesis that LTB4 synthesis by AM can be regulated by PGE2 and performed experiments to determine the biochemical site of regulation. AM obtained from Sprague-Dawley rats were preincubated with PGE2 before stimulation with the calcium ionophore A23187. LTB4, platelet-activating factor (PAF), lysoPAF, (AA), and 5-hydroxyeicosatetraenoic acid were isolated from AM cells and supernatants, then quantified by gas chromatography/electron capture negative ion mass spectrometry. Stimulated AM released 30.50 +/- 4.52 ng LTB4/10(6) cells and were inhibited by PGE2 in a dose dependent manner. PGE2 (1 microM) inhibited LTB4 synthesis by 37% and decreased release of both 5-hydroxyeicosatetraenoic acid and arachidonic acid by stimulated AM, but did not alter synthesis of PAF or lysoPAF. These mass measurements suggest that PGE2 does not affect the activity of phospholipase A2, PAF acetyltransferase, leukotriene A4 hydrolase. We conclude that PGE2 attenuates LTB4 production in alveolar macrophages by altering the activity of lipases other than phospholipase A2. PGE2-mediated inhibition of LTB4 synthesis by AM may regulate the initiation of lung inflammation.
Recombinant tetravalent group A streptococcal M protein vaccine.
Authors: Dale J B , Chiang E Y , Lederer J W
Published: December 2022

Previous studies have shown that the amino-terminal regions of group A streptococcal M proteins contain primarily protective (opsonic) epitopes and not tissue-cross-reactive epitopes. Limited primary structures from multiple serotypes of M protein containing only protective epitopes could potentially be linked together to form a broadly protective vaccine. The present studies were undertaken to determine the protective immunogenicity of a recombinant, multivalent hybrid molecule containing amino-terminal subunits of types 24, 5, 6, and 19 M proteins. Polymerase chain reaction primers were designed to amplify emm gene fragments ranging from 35 to 113 codons. The PCR products were ligated in tandem and inserted into pKK223-3. The tetravalent M protein that was purified from extracts of Escherichia coli migrated as a single band on SDS-PAGE with an apparent m.w. of 31 kDa. In immunoblot analyses, the hybrid protein reacted with serotype-specific antisera indicating that it contained all four M protein subunits. Rabbits immunized with the purified tetravalent M protein developed significant antibody levels against all four serotypes of native M proteins represented in the hybrid protein. None of the antisera cross-reacted with human tissues. The immune sera also opsonized all four serotypes of group A streptococci. Our data show that a hybrid protein containing subunits from multiple M proteins can evoke broadly protective immune responses without tissue-cross-reactive antibodies.
Induction of IFN-alpha by HIV-1 in monocyte-enriched PBMC requires gp120-CD4 interaction but not virus replication.
Authors: Francis M L , Meltzer M S
Published: December 2022

IFN-alpha is in plasma of HIV-1 infected patients during early and late-stage disease and may play a role in control of virus replication. The stimulus for IFN-alpha production, the cells that produce this cytokine, and the effectiveness of this IFN-alpha response for control of virus infection are not yet defined. Culture fluids from freshly isolated PBMC of HIV-1 seronegative donors contained high levels of IFN-alpha after exposure to 100 to 1000 infectious HIV-1 particles per culture. Levels of IFN-alpha induced by HIV-1 were directly dependent on the number of monocytes in cell preparations: No IFN-alpha was detected from T cell-enriched PBMC. In monocyte cultures, induction of IFN-alpha by HIV-1 was relatively specific: Levels of IL-1 beta, IL-6, IFN-gamma, and TNF-alpha remained at baseline. Capacity of HIV-1 virions to induce IFN-alpha was not dependent on virus replication. IFN-alpha was induced by (a) heat-inactivated HIV-1, (b) virions from 8E5 cells, a cell line that releases noninfectious HIV-1, (c) HIV-1-infected cells fixed in paraformaldehyde, and (d) T cell-tropic HIV-1 that binds to but does not infect monocytes. Capacity of HIV-1 virions and HIV-1 infected cells to induce IFN-alpha was completely inhibited by soluble rCD4 or mAb against CD4 or gp120. Antibodies against CD4, however, did not induce monocytes to produce IFN-alpha. HIV-1-induced IFN-alpha production was inhibited by antibodies against both V3 loop determinants and the CD4 binding site of gp120. Further, sera and purified immunoglobulin from HIV-1 infected patients also inhibited HIV-1-induced IFN-alpha production. These observations suggest that potentially protective antiviral responses associated with IFN-alpha production in HIV-1 infected patients are inhibited by the development of antibodies against gp120.
Trypanosoma cruzi: immunity-induced in mice and rats by trypomastigote excretory-secretory antigens and identification of a peptide sequence containing a T cell epitope with protective activity.
Authors: Taibi A , Plumas-Marty B , Guevara-Espinoza A , Schöneck R , Pessoa H , Loyens M , Piras R , Aguirre T , Gras-Masse H , Bossus M
Published: December 2022

In the present study, we investigate the immunoprotective properties of trypomastigote excretory-secretory Ag (ESA) in experimental models. In the case of BALB/c mice, the immunization with ESA resulted in the reduction of parasitemia during acute infection and a significant level of protection in terms of mortality with more than 60% survival, whereas none of the mice in the control groups survived after 39 days postinfection. The same experiments performed in Fischer rats showed a high degree of protection against acute lethal infection with 100% survival, whereas 20 to 40% of rats in the control groups survived the acute phase of T. cruzi infection. Mouse and rat immune sera presented trypanolytic activity against Trypanosoma cruzi infective forms, and recognized two major parasite components of 85 and 24 kDa. The analysis of specific isotype profiles showed a predominance of IgG1, IgG2a, and IgG2b antibody responses. Rat antisera to ESA were then used to screen a trypomastigote cDNA library. Several clones were identified, all of which encoded for the 24-kDa protein. Using a mAb (Tcr7) produced against the native protein, the 31-kDa recombinant fusion protein was purified by affinity chromatography. The antisera to the recombinant protein used in IFA and immunoelectron microscopy showed that the localization of the 24-kDa protein differs among T. cruzi developmental stages. Protection experiments were performed in BALB/c mice using two synthetic peptides (20-40 and 109-124) derived from the primary sequence of the 24-kDa polypeptide. The results obtained clearly indicated that the peptide 109 to 124 containing a putative T cell epitope represents the most protective epitope, which induced 30 to 50% of protection against mortality during acute infection, whereas the percent survival in the control groups (OVA and 20-40 OVA peptide-immunized mice) was around 16%. Moreover, analysis of T cell proliferation in response to OVA-coupled peptides clearly indicated that only the 109 to 124 peptide had the capacity to induce the proliferation of T cells from peptide-immunized mice. Interestingly, only the 109 to 124-coupled peptide induced the proliferation of T cells from T. cruzi-infected mice.
The role of CD4+ T cells in immunity to malaria sporozoites.
Authors: Weiss W R , Sedegah M , Berzofsky J A , Hoffman S L
Published: December 2022

BALB/c mice are strongly protected against malaria after immunization with Plasmodium yoelii (PY) sporozoites, and this is an important model for malaria vaccine development in humans. This paper explores the role of CD4+ cells in the induction of the antimalarial immune response. The method used has been to treat animals with anti-CD4 mAb before immunization, resulting in a profound depletion of CD4+ T cells. The primary finding is that mice are not protected by sporozoite immunization after depletion of CD4+ cells. Such mice make little antibody to malaria sporozoite Ag, showing the strong T-cell dependence of these humoral responses. However, infusion of hyperimmune serum does not restore immunity to anti-CD4 treated animals. Neither does injection of exogenous IL-2 compensate for the absence of CD4+ cells. However, regrowth of CD4+ cells does allow successful immunization of animals, showing that long-term suppression against malaria Ag has not been induced by immunization in the absence of CD4+ cells. It is thought that infiltrating CD8+ T cells are critical effector cells against PY parasites in the pre-erythrocytic stages. Mice immunized while depleted of CD4+ cells have normal numbers of CD8+ T cells infiltrating their livers. In addition, they have normal numbers of splenic CD8+ CTL directed at the PY circumsporozoite protein. Thus, it appears that although CD8+ cells have been activated in the absence of CD4+ cells, they cannot protect mice against malaria. We conclude that a successful vaccine against the pre-erythrocytic stages of malaria must activate both CD4+ and CD8+ T cells.
Protein tyrosine kinase inhibitors decrease induction of nitric oxide synthase activity in lipopolysaccharide-responsive and lipopolysaccharide-nonresponsive murine macrophages.
Authors: Dong Z , Qi X , Xie K , Fidler I J
Published: December 2022

We investigated tumoricidal activation and induction of nitric oxide synthase (NOS) activity in macrophages from LPS-responsive (C3H/HeN) and -unresponsive mice (C3H/HeJ). Macrophages were incubated in vitro with a synthetic lipopeptide or with LPS and IFN-gamma. LPS and IFN-gamma activated C3H/HeN but not C3H/HeJ macrophages to lyse B16 melanoma cells. In contrast, lipopeptide and IFN-gamma activated macrophages from both strains of mice. Genistein, a specific inhibitor of protein tyrosine kinase, significantly blocked tumoricidal activation of macrophages from both strains of mice. Genistein did not affect tumor cell binding but significantly inhibited the production of nitric oxide. Genistein, herbimycin A, and tyrphostin inhibited the induction of NOS activity in macrophages from both strains of mice. These data suggest that protein tyrosine kinase activity is involved in the signal transduction pathway of LPS and other synthetic bacterial-related immunomodulators at a point preceding triggering of macrophage tumoricidal activation and expression of inducible NOS activity.
Transcriptional down-regulation of tumor necrosis factor-alpha gene expression by a synthetic peptide homologous to retroviral envelope protein.
Authors: Haraguchi S , Good R A , Cianciolo G J , James-Yarish M , Day N K
Published: December 2022

We have previously shown that a synthetic peptide (CKS-17) homologous to retroviral envelope protein suppresses the accumulation of superantigen staphylococcal enterotoxin-induced TNF-alpha mRNA in human PBMC and in highly purified human monocytes. The present study was designed to examine the underlying mechanism(s) by which CKS-17 down-regulates the TNF-alpha mRNA expression using a human acute monocytic leukemia cell line THP-1 stimulated with the superantigen staphylococcal enterotoxin E. A cyclooxygenase inhibitor indomethacin does not reverse the inhibition of TNF-alpha mRNA expression by CKS-17, suggesting that prostaglandins are not responsible for the suppressive action of CKS-17. The inhibitory effect of CKS-17 is, however, significantly blocked by a protein synthesis inhibitor cycloheximide, indicating that CKS-17 requires de novo protein synthesis to induce the suppressive activity. The mRNA stability assays using actinomycin D show that CKS-17 does not decrease the TNF-alpha mRNA stability. Nuclear run-on transcription assays further reveal that CKS-17 suppresses the TNF-alpha mRNA transcription rate. Taken together, these results suggest that the synthetic retroviral peptide CKS-17 down-regulates TNF-alpha mRNA expression through inhibition of transcriptional activation of the TNF-alpha gene, which requires de novo synthesis of a transcriptional repressor protein(s).
Autosomal codominant inheritance and Japanese incidence of deficiency of OKT4 epitope with lack of reactivity resulting from conformational change.
Authors: Takenaka T , Kuribayashi K , Nakamine H , Yoshikawa F , Maeda J , Kishi S , Nakauchi H , Minatogawa Y , Kido R
Published: December 2022

A large Japanese family in which some members were homozygous or heterozygous for OKT4 epitope deficiency was studied. Homozygotes, heterozygotes, and normal individuals were identified by differences in the number of OKT4 epitopes on the surfaces of lymphocytes. This deficiency was transmitted as an autosomal codominant trait. The internalization of CD4 molecules and the production of IL-2 by lymphocytes of these subjects were examined. The OKT4 epitope was not needed for internalization of CD4 molecules, and IL-2 was produced in the same amounts by these different kinds of subjects. DNA from four clones lacking OKT4 established from four individuals of this family was sequenced. As reported elsewhere for different subjects, a single nucleotide substitution (CGG-->TGG) was found in all four cell lines. The mutation results in arginine being replaced by tryptophan. Analysis showed different hydrophobicity at positions 239 and 240 from the control, probably giving rise to a conformational change in CD4 accounting for lack of reactivity with the OKT4 monoclonal antibody. The incidence of homozygotes in the Japanese population was found to be 0.47% by examination of 1478 random samples, and on the basis of this value, the incidence of heterozygotes was estimated to be 12.8%.
Binding and cytotoxicity characteristics of the bispecific murine monoclonal antibody 2B1.
Authors: Weiner L M , Holmes M , Richeson A , Godwin A , Adams G P , Hsieh-Ma S T , Ring D B , Alpaugh R K
Published: December 2022

Bispecific monoclonal antibodies (BsmAb) with specificity for tumor Ag and effector cell trigger molecules have been shown to redirect the cytotoxicity of several peripheral blood mononuclear cell populations against relevant tumor. The BsmAb, 2B1, binds to the extracellular domain of the c-erbB-2 gene product of the HER2/neu proto-oncogene and to CD16. In this report, the binding and cytotoxic characteristics of 2B1 are presented. Maximal saturation binding of 2B1 to PBL and c-erbB-2 expressing SK-OV-3 cells occurred in the 1 microgram/ml concentration range. However, substantial lysis potentiation was observed at 1000-fold lower BsmAb concentrations. Optimal tumor lysis was obtained when the BsmAb, PBL, and target cells were continuously coincubated. When PBL were franked with 2B1, washed, and added to labeled targets, substantially less lysis was observed. These results suggest that the best way to therapeutically exploit the cytotoxic attributes of 2B1 may be to obtain continuous BsmAb exposure to tumor. Approaches based on franking of this BsmAb to PBL may not be warranted.
Cellular and molecular mechanisms of activation of MHC nonrestricted cytotoxic cells by IL-12.
Authors: Cesano A , Visonneau S , Clark S C , Santoli D
Published: December 2022

Freshly separated PBL and two IL-2-dependent clonal T cell lines (TALL-103/2 [TCR-gamma delta+] and TALL-104 [TCR-alpha beta+]) were used to dissect, at the cellular and molecular levels, the mechanisms governing the enhancing effects of IL-12 on MHC nonrestricted cytotoxic activity. IL-12 induced cytotoxicity both in PBL and TALL cells, although less efficiently than IL-2, and the two cytokines displayed additive effects. Unlike IL-2, IL-12 failed to enhance the CD3 or CD2 redirected killing of the TALL cell lines. Analysis of the percentage of E/T conjugates induced by these cytokines showed that IL-12 could increase PBL, but not TALL cell, binding only when used in combination with IL-2. This and the failure of IL-12 to increase the expression of several adhesion molecules on PBL and TALL cells indicated no significant role for IL-12 in the binding step of the lytic interaction. We also excluded that production of TNF-alpha, IFN-gamma, or IL-2 by the IL-12-activated effectors might account for the cytotoxicity-inducing effects of this cytokine. By contrast, IL-12 was found to induce cytotoxic granule formation in PBL and TALL cells and to up-regulate the expression of their cytolytic components, including perforin, serine esterases, and TIA-1, both at the protein and mRNA levels. The costimulatory action of IL-2 and IL-12 on granulogenesis and induction of cytolytic proteins correlated well with the additive effects on cytotoxicity. Studies with cyclohexamide and actinomycin D demonstrated that TALL cells but not PBL require de novo protein synthesis for expression of serine esterases and perforin mRNA after IL-2 and/or IL-12 stimulation, and that both cytokines act directly or indirectly at the transcriptional level. Overall, these data indicate that IL-12 acts mostly at a postbinding level of the lytic interaction, involving enhanced expression of cytotoxic mediators, and that IL-2 and IL-12 use partially distinct pathways to activate the MHC nonrestricted killer function of mature T cells and resting lymphocytes.
Appearance of TCR-alpha beta+ CD4-CD8- skin intraepithelial lymphocytes in radiation bone marrow chimeras.
Authors: Ogimoto M , Matsuzaki G , Yoshikai Y , Tauchi Y , Nomoto K
Published: December 2022

Thymocytes expressing homogeneous TCR V gamma 5/V delta 1 gene without N nucleotide addition (canonical sequence) have been reported to be positively selected in fetal thymus. Because skin intraepithelial lymphocytes (s-IEL) also express canonical sequence, it is proposed that s-IEL are generated by an epidermis-specific homing of these positive selected fetal V gamma 5/V delta 1-bearing thymocytes. We have reported that positive selection of V gamma 5+ thymocytes is not seen in AKR/J-->C3H/He bone marrow chimeras. In this report, we analyzed the phenotype of donor-derived s-IEL from AKR/J-->C3H/He bone marrow chimeras that appeared in the absence of positive selection of V gamma 5-bearing thymocytes. We found interestingly that almost all donor-derived s-IEL in the bone marrow chimeras expressed TCR-alpha beta lack expression of both CD4 and CD8. Furthermore, TCR-alpha beta+ s-IEL were detected in adult-thymectomized bone marrow chimeras, indicating that the TCR-alpha beta+ s-IEL are extrathymically derived. All these results suggest that the s-IEL expressing TCR-alpha beta appear extrathymically in the absence of positive selection of V gamma 5/V delta 1-bearing thymocytes. Our results may also explain the contradicting result between human s-IEL, which are TCR-alpha beta+ CD4- CD8- and murine s-IEL, and suggest the importance of extrathymic T cell differentiation pathway of the TCR-alpha beta lineage in immune surveillance.
Monoclonal c-myc transformed macrophage cell lines. I. Heterogeneity in ability to process and present antigen.
Authors: Trannoy E , Manser T , Cole M D , Daley M J
Published: December 2022

Processing of proteins into immunogenic forms and their subsequent presentation to T cells are mediated by APC. Monocytes and macrophages have long been recognized as one of the APC types. However, little is known about whether functional heterogeneity in processing and presentation exist within the monocyte/macrophage population. Past difficulties in obtaining clonal representatives of these populations have limited investigations in this regard. The c-myc-containing retrovirus MRV, previously shown to immortalize murine macrophages, was used to generate a large panel of macrophage cell clones. Differences observed in cell surface antigen expression and morphology demonstrated phenotypic heterogeneity among these clones. Functional heterogeneity was also observed both before and after IFN-gamma and IL-4 stimulation. The clones differ in their capacity to present several nominal antigens to T cell hybridomas. When parallel variation in ability to present both a nominal antigen and a peptide representing the epitope for which a T cell hybridoma was specific was observed among the clones, this variation correlated with the levels of surface MHC class II antigen the clones expressed. In contrast, diversity in the ability to process and present certain nominal antigens among clones that all presented the corresponding antigenic peptide with similar efficiency did not appear to be due to differences in levels of surface MHC class II molecules. Our results suggest that the macrophage clones are heterogeneous in their ability to both process and present several antigens. The ability to obtain macrophage tissue culture cell lines displaying phenotypic and functional heterogeneity should allow insight into the impact of normal macrophage heterogeneity on the outcome of immune responses in vivo.
Characterization and expression of recombination activating genes (RAG-1 and RAG-2) in Xenopus laevis.
Authors: Greenhalgh P , Olesen C E , Steiner L A
Published: December 2022

The primary repertoire of B and T cells is established by V(D)J recombination. Two closely linked genes, RAG-1 and RAG-2, are essential for this process, and have been identified in mice, humans, and chickens. To study lymphocyte development in Xenopus laevis, we have characterized RAG-1 and RAG-2 in this species and examined their patterns of expression. Degenerate oligonucleotides, based on the known highly conserved RAG-1 sequences, were used to amplify, by the polymerase chain reaction, a segment of Xenopus RAG-1 from genomic DNA. A product of expected size was obtained and used to identify a genomic clone that contained the complete coding region of RAG-1 (1045 codons), and approximately the 3'-half of the coding region of RAG-2. The coding regions of RAG-1 and RAG-2 each lie on a single exon, are in opposite transcriptional orientation, and are separated by approximately 6 kb. The sequence of the remainder of RAG-2 was determined by PCR amplification of genomic DNA, with primers based on sequence analysis of RAG-2 cDNA clones. The predicted Xenopus RAG-1 protein is 71% identical in amino acid sequence to the sequences of each of the mouse, human, and chicken proteins; from position 392 to 1012 the identity is 88%. The coding region of Xenopus RAG-2 (520 codons) is somewhat less conserved among the different species. Tissue-specific expression of Xenopus RAG-1 and RAG-2 was examined both by Northern blotting and by a reverse transcription-polymerase chain reaction assay. In juvenile frogs, the highest levels of RAG-1 and RAG-2 expression were observed in the thymus, with lower levels in liver and spleen, and even lower levels in the kidneys. In adults, the thymus and bone marrow were found to be the principal sites of expression of both genes. RAG-2, but not RAG-1, was expressed in oocytes.
Resolution of blood-stage malarial infections in CD8+ cell-deficient beta 2-m0/0 mice.
Authors: van der Heyde H C , Manning D D , Roopenian D C , Weidanz W P
Published: December 2022

We utilized a definitive model of CD8+ T cell deficiency, the beta 2-microglobulin-deficient (beta 2-m0/0) mouse, to determine whether CD8+ T cells are required in the resolution of blood-stage malaria. In a parallel experiment, C57Bl/6 mice treated with anti-CD8 mAb showed significantly higher levels of parasitemia than untreated C57Bl/6 control mice at several points during the infection. This finding suggests some role for CD8+ cells in containing malaria. However, the beta 2-m0/0 mice, which are genetically blocked from expressing MHC class I or class Ib glycoproteins and therefore have < 2.5% of the normal number of CD8+ T cells, nevertheless resolved infections with three virulence variants of murine Plasmodium. The resolution of Plasmodium chabaudi adami, Plasmodium yoelii yoelii 17X, and Plasmodium chabaudi chabaudi AS infections by beta 2-m0/0 mice in the virtual absence of CD8+ cells demonstrates that these cells are not required to suppress murine malaria and that the suppression mechanism is not MHC class I restricted. The similarity of the time-course for resolution of infection in beta 2-m0/0 and intact control mice with all three subspecies of Plasmodium further supports the lack of a requirement for CD8+ T cells in the suppression of malarial parasitemia.
Role of IL-10 on antigen-presenting cell function for schistosomal egg-specific monoclonal T helper cell responses in vitro and in vivo.
Authors: Flores Villanueva P O , Chikunguwo S M , Harris T S , Stadecker M J
Published: December 2022

Egg Ag-stimulated lymphoid cell culture supernatants from schistosome-infected mice significantly inhibited Ag-specific, MHC-restricted proliferative responses of cloned schistosomal egg Ag-specific CD4+, Th-1-type lymphocytes. The inhibitory molecule in the supernatants was found to be the cytokine IL-10. Maximal IL-10 was produced by cells from mice carrying 8-wk schistosome infections, and none by cells from normal mice. IL-10 exerted its biologic activity on APC, and not directly on the cloned lymphocytes, resulting in the inhibition of Th-1 lymphocyte proliferation, whereas Th-2 responses were not affected. Most significantly, IL-10 also affected Th-1 clone activity in vivo, as measured by the inhibition of schistosomal egg Ag-specific local delayed-type hypersensitivity reactions. IL-10 produced in schistosome-infected individuals may play a role in the generation of APC, such as macrophages in schistosomal egg granulomas, unable to efficiently stimulate, but capable of inducing a state of anergy/unresponsiveness in Th-1 lymphocytes. We believe that this state of Th-1 cell anergy translates into the down-regulation of granulomatous hypersensitivity (immunomodulation) characteristically observed in the evolving schistosomal disease.
Antibodies chaperone circulating IL-6. Paradoxical effects of anti-IL-6 "neutralizing" antibodies in vivo.
Authors: May L T , Neta R , Moldawer L L , Kenney J S , Patel K , Sehgal P B
Published: December 2022

In the baboon or the mouse, a stimulus such as LPS, TNF, or IL-1 typically led to a rapid induction of circulating IL-6, the levels peaked by 2 to 3 h and then declined to near-baseline values by 6 to 8 h. Administration to baboons or mice of "neutralizing" anti-IL-6 mAb followed by an IL-6 inducer led to a marked and sustained increase in circulating IL-6 levels. IL-6 Ag, IL-6 biologic activity, neutralizing anti-IL-6 mAb, and IL-6/anti-IL-6-mAb complexes could all be observed for an extended period of time (beyond 8 h) in the circulation of such animals. Nevertheless, in mice, if the anti-IL-6 mAb had been administered before the IL-6 inducer, there was a reduction in the in vivo IL-6-induced stimulation of fibrinogen levels, indicating that most of the intravascular IL-6 was not readily available for eliciting hepatocyte effects under these experimental conditions. Intraperitoneal administration into mice of mixtures of murine rIL-6 or human rIL-6 together with their respective anti-IL-6 mAb led to a marked increase in the appearance and longevity in the peripheral circulation of the exogenously administered murine or human rIL-6 species in a biologically active form. Varying the ratio of human rIL-6 to anti-human IL-6 mAb indicated that a molar ratio of 1:1 was sufficient for the ability of mAb to chaperone IL-6 in the murine circulation. Human rIL-6 mixed with "neutralizing" mAb in the approximate ratio 1:1 elicited an enhanced fibrinogen response in vivo in the mouse; an IL-6:mAb ratio of 1:125 led to a reduction in the fibrinogen response even though the levels of circulating B9 bioactivity and of human rIL-6-Ag were maximal under these conditions. Gel-filtration chromatographic and Western blotting analyses of IL-6 present in vivo in the mAb-free baboon revealed that although the IL-6 Ag was largely present in high molecular mass complexes of size 400 kDa in association with soluble IL-6 receptor, the B9 bioactivity was largely of low molecular mass (20 kDa). In contrast, in the anti-IL-6 mAb-treated baboon or mouse, the IL-6 Ag and bioactivity were both largely in complexes of 200 kDa. Thus, the binding of IL-6 in the intravascular compartment to other proteins, anti-IL-6 mAb in the present studies, gives IL-6 unexpected biochemical and pharmacologic properties in vivo.
Immobilization of Fc epsilon receptors by wheat germ agglutinin. Receptor dynamics in IgE-mediated signal transduction.
Authors: McCloskey M A
Published: December 2022

Transmembrane signaling initiated by the receptor with high affinity for Fc stem of IgE(Fc epsilon RI) requires the diffusion-dependent cross-linkage and persistent aggregation of the Fc epsilon RI. Disruption or prevention of receptor cross-links at any time during the secretory response quickly terminates secretion. We found that in the rat basophilic leukemia mast cell line, addition of wheat germ agglutinin, a lectin that binds to the Fc epsilon RI alpha subunit, caused a precipitous decline in the lateral diffusional and electrokinetic mobilities of the Fc epsilon RI. Both the unoccupied Fc epsilon RI and IgE-Fc epsilon RI complexes became immobilized, as determined from in situ electromigration and postelectric field relaxation. Immobilization of the Fc epsilon RI by wheat germ agglutinin was accompanied by a ligand-reversible association of 125I-IgE-Fc epsilon RI complexes with the Triton X-100-insoluble cytoskeletal fraction. Wheat germ agglutinin rapidly inhibited Fc epsilon RI-mediated signal transduction and secretion, whether cross-linkage was initiated by multivalent antigen, covalent IgE oligomers, anti-IgE, or anti-Fc epsilon RI antibody. Inhibition of signaling and secretion occurred on simultaneous addition of wheat germ agglutinin and antigen, and also when wheat germ agglutinin was added at increasing times after induction of Fc epsilon RI cross-linkage. Wheat germ agglutinin neither reduced the affinity of anti-DNP IgE for haptenic DNP-lysine nor reversed the binding of IgE to the Fc epsilon RI. Although wheat germ agglutinin caused internalization of the Fc epsilon RI, the onset of inhibition preceded and its extent exceeded that of internalization. Wheat germ agglutinin did not interfere with the secretory apparatus, as indicated by its lack of inhibition of secretion elicited by calcium ionophores. These findings suggest that inhibition of signal transduction is secondary to an initial event linked to immobilization of the Fc epsilon RI. Implications of these results are discussed with respect to the dynamics of Fc epsilon RI aggregation on rat basophilic leukemia cells.
L- and E-selectin can recognize the same naturally occurring ligands on high endothelial venules.
Authors: Mebius R E , Watson S R
Published: December 2022

The selectin family of cell adhesion molecules consists of three members, E-selectin (ELAM-1), L-selectin (LECAM-1), and P-selectin (CD62, GMP140, or PADGEM), which are all involved in binding of leukocytes to endothelial cells. All members have structural similarities and they can all bind to a common carbohydrate epitope, sialyl Lewis X in in vitro assays. To study cross-reactivity of the selectins in more detail we used Ig chimeras of murine and human L- and human E-selectin. All three chimeras bound to the natural ligands of L-selectin on specialized high endothelial venules in both human and murine lymphoid organs as determined by immunohistochemistry and were able to precipitate 50- and 90-kDa sulfated ligands from organ cultures of murine peripheral and mesenteric lymph nodes. This recognition was calcium dependent and inhibitable by mAb specific for the lectin domain of the respective selectin. L- and E-selectin binding to high endothelial venules and to the sulfated ligands was inhibitable by the carbohydrates, fucoidan and sialyl Lewis X, respectively. Although immunoprecipitations showed that both murine and human L-selectin could recognize the sulfated ligands from high endothelial venules more efficiently than human E-selectin at the concentrations used, both L- and E-selectin chimeras could inhibit in vivo lymphocyte trafficking into peripheral lymph nodes equally well.
Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma.
Authors: Suschek C , Rothe H , Fehsel K , Enczmann J , Kolb-Bachofen V
Published: December 2022

We investigated the effects of murine rTNF-alpha, human rIL-1 beta, and rat rIFN-gamma in various concentrations and/or combinations on inducible nitric oxide (NO) production in primary cultures of rat aortic endothelial cells. Northern blot analysis of total RNA from induced and control cultures using the cloned mouse macrophage gene of inducible NO synthase as probe as well as polymerase chain reaction using a specific primer sequence gave a positive signal for activated cells only. A RNA approximately 4.4 kb of length similar to the inducible form of NO synthase in macrophages was labeled. The concentration of nitrite as a stable reaction product of NO in culture supernatants was determined 24 h after incubation with the various cytokines. IL-1 beta alone (40 to 1000 U/ml) induced formation of increasing amounts of nitrite with increasing concentrations of IL-1 beta present. Neither TNF-alpha alone (10 to 2000 U/ml) nor IFN-gamma alone 25 to 500 U/ml) showed significant effects on nitrite production. Simultaneous incubation with low concentrations of TNF-alpha (< or = 100 U/ml) and IL-1 beta abrogated the induction effect of IL-1 beta. Conversely, addition of high concentrations of TNF-alpha (> or = 500 U/ml) led to near maximal levels of nitrite formation even at lowest IL-1 beta concentrations (40 U/ml). In addition, simultaneous incubation of endothelial cells with IFN-gamma plus IL-1 beta and/or TNF-alpha led to near maximal NO production of endothelial cells, even at lowest IFN-gamma concentrations (25 U/ml). We hypothesize that the regulating effect of TNF-alpha may in vivo help to prevent local inflammatory responses from spreading to intact sites.
Altered prostaglandin E2 regulation of cytokine production in atopic dermatitis.
Authors: Chan S C , Kim J W , Henderson W R , Hanifin J M
Published: December 2022

The monocyte-derived inflammatory mediator, prostaglandin E2 (PGE2), can reduce IFN-gamma production, and this in turn may relate to IL-4 up-regulation of IgE synthesis and impaired delayed hypersensitivity in atopy. These abnormalities may relate to the cyclic nucleotide dysregulation in atopic dermatitis (AD), where monocyte cyclic AMP-phosphodiesterase (PDE) activity is increased and the consequent reduction in cAMP levels allows increased inflammatory responsiveness. In this study, we assessed the relationship between PGE2 and IFN-gamma production along with abnormal PDE activity in AD monocytes. Blood mononuclear leukocytes (MNL) from normal and AD donors were cultured for 24 hours, and supernatants were assayed for PGE2 and IFN-gamma by RIA. Spontaneous PGE2, but not leukotriene C4 release, was significantly increased in AD MNL (p < 0.05), although IFN-gamma levels were reduced (p < 0.05). In contrast, purified AD T cells, after removal of PGE2-producing monocytes, produced levels of IFN-gamma significantly higher than in normal T cell cultures. Inhibition of PGE2 synthesis by indomethacin caused increased IFN-gamma production by MNL cultures. We noted a strong negative correlation (r = 0.77) between PDE activity and IFN-gamma production in MNL cultures. We speculate that abnormal cyclic nucleotide metabolism caused by increased PDE activity may allow elevated levels of PGE2 production by AD monocytes. This study demonstrates a regulatory interaction between monocytes and T cells in AD and suggests that PGE2 may be an extracellular messenger between these cells to modulate IFN-gamma production.
Increased sensitivity to TNF-mediated cytotoxicity of BL6 melanoma cells after H-2Kb gene transfection.
Authors: Kim M , Herberman R , Gorelik E
Published: December 2022

Transfection of the H-2Kb and neor genes into BL6-8 (H-2Kb-, H-2Db+) melanoma clone resulted in various phenotypic changes with appearance of soybean agglutinin (SBA) and Grifonia Simplicifolia 1-B4 (GS1B4) lectin binding carbohydrates and loss of melanoma-associated antigen (MAA). In parallel H-2Kb gene-transfected melanoma cells showed increased sensitivity to TNF lysis. To further delineate the ability of H-2Kb gene to induce the phenotypic changes and TNF sensitivity, BL6-8 melanoma clone was transfected with the H-2Kb gene alone without cotransfection with neor gene and transfected cells were selected for adherence to SBA lectin-conjugated agarose beads. Analysis of isolated clones revealed that 38 of 47 tested clones have been found to be expressing the H-2Kb Ag, SBA, and GS1B4 lectin binding carbohydrates but lost MAA, e.g., H-2Kb+, lectin+, MAA-, and in parallel these cells became sensitive to TNF lysis. Although all clones with high expression of H-2Kb Ag were sensitive to TNF lysis, it seems unlikely that H-2K molecules are directly required for or involved in TNF-induced melanoma cell lysis. This conclusion is based on findings that four H-2Kb-transfected clones selected on SBA-agarose beads did not expressed H-2Kb Ag but manifested increase in SBA and GS1B4 lectin binding and loss of MAA and also became sensitive to TNF lysis. It seems that increase in TNF sensitivity is a part of the broad phenotypic changes induced by the H-2K gene that remained stable even in the clones in which the transfected H-2Kb gene was lost or down-regulated. We believe that the effects of the H-2Kb gene on melanoma cell phenotype and TNF sensitivity are indirect and are probably mediated via its inhibition of the melanoma-associated ecotropic retrovirus production and activation of some repressed cellular genes. Study of the mechanisms responsible for TNF sensitivity of BL6 melanoma cells revealed that the H-2Kb gene transfection resulted in an increase in p55 TNF receptor expression. TNF-induced activation of phospholipase A2 and release of arachidonic acid metabolites was observed only in the H-2Kb transfected, but not in BL6-8 melanoma cells transfected with neor or class II H-21Ak genes. TNF resistance of BL6 melanoma cells appeared to be due to a block in transduction of the lytic signal that was reversed after transfection with H-2Kb gene.
Pathway of signal delivery to murine thymocytes triggered by co-crosslinking CD3 and Thy-1 for cellular DNA fragmentation and growth inhibition.
Authors: Nakashima I , Pu M Y , Hamaguchi M , Iwamoto T , Rahman S M , Zhang Y H , Kato M , Ohkusu K , Katano Y , Yoshida T
Published: December 2022

The signal delivery pathway triggered by crosslinking CD3 and Thy-1 together (CD3/Thy-1 crosslinkage) on murine thymocytes for cellular DNA fragmentation/growth inhibition was analyzed. The treatment of thymocytes with herbimycin A as a specific tyrosine kinase inhibitor under suboptimum conditions before the CD3/Thy-1 crosslinkage partially but preferentially inhibited the otherwise promoted tyrosine phosphorylation of p40 and p56. Evidence was then provided that acceleration of the kinase activity of p56lck was involved in the CD3/Thy-1 crosslinkage-triggered signal. Partial characterization of p40 distinguished it from the p43 and p41 MAP kinases, the tyrosine phosphorylation of which was only marginally accelerated. Promotion of DNA fragmentation by the CD3/Thy-1 crosslinkage-triggered signal was actually ablated by the treatment with herbimycin, suggesting the obligatory involvement of the herbimycin highly sensitive kinase activity in the signal pathway. The signal induced by co-crosslinkage of CD3 and Thy-1 was also shown to be negatively biased against mature T lymphocytes, suppressing their CD3-mediated growth response. The negative signal was then found to partially attack the process of c-fos transcription as an earlier nuclear event. Interestingly, this c-fos suppression was prevented by the treatment of thymocytes with herbimycin before stimulation, for accelerated expression of c-fos. It is suggested from these results that the CD3/Thy-1 crosslinkage delivers protein tyrosine kinase-dependent negative signaling for inhibition of early and late nuclear events of both immature thymocytes and mature T lymphocytes.
An activation-responsive element in the murine IL-4 gene is the site of an inducible DNA-protein interaction.
Authors: Tara D , Weiss D L , Brown M A
Published: December 2022

IL-4 is a pleiotropic cytokine whose expression is limited to a subset of activated T cells and cells of the basophil/mast cell lineage. It plays a key role in regulating many immune responses; however, little is known about the intracellular signaling events that lead to the selective and transient IL-4 expression in either of these cell types. In this study, the molecular basis of stimulation-dependent transcription in T cells was explored. To identify cis elements that regulate IL-4 gene transcription, various amounts of the 5' flanking region of the murine IL-4 gene were linked to a chloramphenicol acetyl transferase (CAT) reporter gene and tested for the ability to modulate CAT gene transcription in PMA-stimulated EL-4 T cells. These experiments indicate that multiple positive and negative-acting elements contribute to the overall level of IL-4 transcription. These elements are located both proximal and distal to the transcription initiation site (TIS). An activation responsive element is located within 87 bp of the IL-4 gene TIS. This sequence is sufficient to confer responsiveness to PMA-mediated signals and results in a 10- to 20-fold induction of CAT reporter gene activity compared to activity detected in unstimulated cells. Proteins that specifically bind sequences within this region (-88 to -60) are detected in both unstimulated and stimulated EL-4 T cell nuclear extracts. An additional DNA-protein interaction is detected only when extracts from stimulated cells are analyzed. Base substitutions within the -88 to -60 sequence affect both transactivation function and protein/DNA interactions and demonstrate that sequences between -78 and -69 bp are critical. Together, these data support a model in which T cell activation signals stimulate binding of a nuclear protein(s) to a preexisting IL-4 DNA-protein complex. Proteins detected in these promoter proximal DNA-protein complexes are likely to be key elements in facilitating stimulation-dependent IL-4 transcription.
Pulmonary T helper lymphocytes are CD44hi, CD45RB- effector/memory cells in mice vaccinated with attenuated cercariae of Schistosoma mansoni.
Authors: Coulson P S , Wilson R A
Published: December 2022

Percutaneous exposure of C57Bl/6 strain mice to radiation-attenuated schistosome cercariae stimulates proliferation of Ag-specific T lymphocytes in the draining lymph nodes (LN), followed by an intense leukocytic infiltration into the pulmonary parenchyma and airways. The airway T cells persist at elevated levels at least up to 10 wk and will secrete IFN-gamma and IL-3 upon antigenic stimulation in vitro. We report here that more CD4+ T cells from the airways responded rapidly to mitogen by up-regulating the p55 subunit of the IL-2R than did splenocytes from the same animal, suggesting that the bulk of the pulmonary Th infiltrate comprised previously activated cells. A four-fold higher response to Ag indicated the greater abundance of schistosome-specific cells in the lungs than the spleen. Virtually all of the pulmonary CD4+ T cells expressed high levels of the memory marker CD44 (Pgp-1), in contrast with the situation in the draining LN and circulation where only a minority were in that category. Very few Th cells from the inflamed lung bound antibody to an epitope coded for by the B exon of CD45, unlike those from the LN and circulation; the antibody normally binds strongly to naive and weakly to memory CD4+ T cells. Overall, the airway CD4+ T lymphocytes in the schistosome-vaccinated mouse display the functional and phenotypic characteristics of short-term effector/memory cells. We believe that their presence endows the lung with the ability to respond rapidly to recall Ag in the form of challenge parasites.