bluuebunny/crossref_metadata_2025 huggingface dataset:
Normal sequence of phenotypic transitions in one cohort of 5-bromo-2'-deoxyuridine-pulse-labeled thymocytes. Correlation with T cell receptor expression.
Authors: Lucas B , Vasseur F , Penit C
Published: January 2023

"In vivo" kinetics of T cell differentiation and TCR expression in the normal murine thymus were re-evaluated using a new technique for simultaneous detection of bromodeoxyuridine and two surface markers. The transition from CD4-8- precursors to CD4+8+ immature cells was directly observed during cell proliferation, and shown to proceed through transitory intermediates expressing no or low amounts of CD4. CD3-TCR expression also started during this transition and resulted in the production of a majority of TCRlo cells but also of a significant number (1 to 2 x 10(6) of TCRhi immature (heat-stable Ag+) thymocytes. After cessation of proliferation, the maturational transition from CD4+8+ to CD4+8- and CD4-8+ (in this order) was restricted to TCRhi cells produced during CD4+8+ cell generation. The acquisition of the single positive phenotype preceded HSA down-regulation, suggesting that maturation of TCRhi thymocytes proceeds in two separate steps. The major TCRloCD4+8+ subset appeared a dead end subset and showed no up-regulation of TCR expression at any time.
Transforming growth factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vitro.
Authors: Snapper C M , Waegell W , Beernink H , Dasch J R
Published: January 2023

Addition of transforming growth factor-beta 1 (TGF-beta 1) to in vitro cultures of murine B cells activated with bacterial LPS selectively stimulates IgG2b and IgA class switching and decreases cellular proliferation. To assess a possible role for endogenous TGF-beta in modulating the Ig isotypes produced by LPS-activated cells, we utilized a neutralizing anti-TGF-beta mAb to abrogate endogenous TGF-beta activity. Anti-TGF-beta antibody, over a range of relatively low cell densities, strikingly inhibited both IgG3 and IgG2b production in response to LPS, with little or no change in the concentrations of secreted IgM. This effect of anti-TGF-beta antibody was specific, since it did not occur with an isotype-matched control mAb and was completely reversed with exogenous TGF-beta 1. Optimal IgG3 secretion occurred at concentrations of TGF-beta that were approximately eightfold lower than that necessary for maximal synthesis of IgG2b. Neutralization of endogenous TGF-beta in LPS-activated cultures was associated with an approximately twofold increase in proliferation and viable cell yields, a modest decrease in the percentage of membrane (m)IgG2b+ cells, and a modest increase in the percentage of mIgG3+ cells. This latter finding indicated that TGF-beta was not required for IgG3 class switching, but for maturation of mIgG3+ cells into Ig secretors. Highly purified B cells, obtained by electronic cell sorting, released active TGF-beta in response to LPS and showed a similar marked reduction in LPS-mediated IgG3 and IgG2b secretion in the presence of anti-TGF-beta antibody. Abrogation of endogenous TGF-beta activity in LPS-activated cultures also resulted in a striking reduction in IFN-gamma-mediated IgG2a production, and a more modest decrease in the synthesis of IgG1 and IgE in the presence of IL-4. These data indicate that relatively low concentrations of TGF-beta are essential for stimulating optimal IgG secretion by LPS-activated B cells, in an Ig isotype-nonspecific manner, and may regulate these responses in an autocrine fashion.
Protective immunity in baboons vaccinated with a recombinant antigen or radiation-attenuated cercariae of Schistosoma mansoni is antibody-dependent.
Authors: Soisson L A , Reid G D , Farah I O , Nyindo M , Strand M
Published: January 2023

Mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni exhibit high levels of resistance to challenge infection. We have previously shown that sera from these mice recognize polypeptides that are expressed on the surface of newly transformed schistosomula. We have cloned and sequenced a cDNA that encodes a 62-kDa portion of one of these polypeptides. Vaccination of mice with this 62-kDa polypeptide (designated rlrV-5) elicits high antibody titers and significant resistance to challenge infection. We report here the results of a vaccination trial in baboons with the rlrV-5 or radiation-attenuated cercariae. rlrV-5 was presented either in the form of protein micelles or complexed with the outer membrane protein of meningococcus to form proteosomes. The level of protection achieved in these groups ranged from 0 to 54%, with a mean of 27.7%. In baboons exposed to radiation-attenuated cercariae the level of protection was very high, with a mean of 84%. The resistance observed after vaccination with rlrV-5 or radiation-attenuated cercariae was reflected in the overall histopathology. Vaccination of baboons with rlrV-5 or radiation-attenuated cercariae elicited an antibody response against epitopes exposed on the surface of newly transformed schistosomula. In the case of baboons vaccinated with radiation-attenuated cercariae, this response was not limited to epitopes encompassed by rlrV-5. Analysis of individual baboon sera by ELISA demonstrated that there was a direct correlation between the anti-rlrV-5 titer and resistance to challenge worm burden, suggesting that the immunoprotective mechanism is antibody-dependent.
Inhibition of murine B cell proliferation and down-regulation of protein kinase C levels by a phosphorylcholine-containing filarial excretory-secretory product.
Authors: Harnett W , Harnett M M
Published: January 2023

E-S 62, a major excretory-secretory product of the rodent filarial parasite, Acanthocheilonema viteae, inhibits the polyclonal activation (DNA synthesis) of mouse B cells by mitogenic anti-Ig antibodies. This effect appears to be due at least in part to the phosphorylcholine (PC) moiety of the molecule, because it can be mimicked by PC-BSA or PC-chloride. Activation of the B cells by LPS is not influenced by the presence of E-S 62/PC, suggesting that they may target some aspect of signaling via the Ag receptor. E-S 62/PC failed to inhibit surface Ig-mediated generation of the second messenger, inositol triphosphate, indicating that their target may not be the early biochemical events associated with activation. Exposure to E-S 62/PC was found to lead to a reduction in the level of protein kinase C, an important downstream regulatory enzyme, in anti-Ig-treated cells. This protein-kinase C down-regulation may be the biochemical mechanism underlying E-S 62/PC-mediated inhibition of surface Ig-activated B cells.
Inhibition of human CTL-mediated lysis by fibroblasts infected with herpes simplex virus.
Authors: Posavad C M , Newton J J , Rosenthal K L
Published: January 2023

Previously, we demonstrated that human anti-HSV CTL and allo-antigen-specific CTL were inhibited in lysing their normally sensitive target cells when they were exposed to human fibroblasts (FB) infected with herpes simplex virus (HSV). In this study, the mechanism of inhibition of CTL lytic function by FB infected with HSV-1 (HSV-FB) was studied. CTL exposed to HSV-FB early (2 h) in the infection cycle were inhibited by a mechanism that appears to be distinct from the inhibition of lytic function mediated by HSV-FB at late times (20 h) during the infection cycle. The inhibition of CTL-mediated lysis by FB infected with HSV-1 for 2 h required the expression of ICP4, an immediate-early protein of HSV-1, but not the production of infectious virus or virus-induced shut-off of host protein synthesis. In contrast, the expression of HSV-specific glycoproteins essential for viral infectivity (glycoproteins B, D, H, K, and L), and thus, infectious virus, was required for inhibition of CTL lytic function by FB infected with HSV-1 for 20 h. Further, CTL exposed to FB infected with HSV-1 for 20 h expressed HSV-specific proteins indicating that they were infected with HSV-1. Cell-to-cell spread of HSV-1 appeared to be the major mode of transmission because 1) an insufficient level of HSV-1 was present in the supernatant of HSV-FB to inhibit CTL lytic function; and 2) paraformaldehyde-fixed HSV-FB did not inhibit CTL-mediated lysis. The inhibition of CTL lytic function by HSV-FB may be an important mechanism of HSV-induced immunosuppression, permitting the virus to spread and persist in immunocompetent hosts after primary infection or reactivation of latent HSV.
Phospholipase A2 activity in human neutrophils. Stimulation by lipopolysaccharide and possible involvement in priming for an enhanced respiratory burst.
Authors: Forehand J R , Johnston R B , Bomalaski J S
Published: January 2023

Exposure to LPS, platelet-activating factor, certain cytokines, and other agents can prime human neutrophils for an increased release of superoxide anion (O2-) in response to stimuli. Previous work with LPS has suggested that priming may involve alterations in signal transduction pathways related to the release of O2-. Products derived from the enzymatic activity of phospholipase A2 (PLA2) on membrane phospholipids reportedly activate certain of these signaling events. Thus, PLA2 could play a regulatory role in the release of O2- by neutrophils. We examined this possibility by studying the effect of LPS on neutrophil PLA2 activity. Exposure to LPS triggered a fivefold increase in activity of an endogenous PLA2; control cells incubated under identical conditions without LPS showed no increase. Neutrophil-associated PLA2 activity increased 5 to 10 min after the addition of LPS to the cells and preceded the emergence of the primed state. Quinacrine and p-bromophenacylbromide, inhibitors of PLA2, blocked LPS priming but not the baseline O2- release from unprimed cells. The LPS-provoked increase in PLA2 activity resulted in release of oleic acid (38 +/- 4% above baseline) but not arachidonic, linoleic, or palmitic acid. In contrast, ionomycin resulted in significant release of all four fatty acids. The addition of exogenous PLA2 to neutrophils primed them. Priming was rapid and was 80 +/- 5% complete within 3 min. Thus, LPS and perhaps other agents may mediate their effects on O2- release at least in part by triggering PLA2 activity. PLA2 activation may play a role in regulating production and release of O2- by the human neutrophil.
Regulation of MCP-1/JE gene expression during monocytic differentiation.
Authors: Gruss H J , Brach M A , Schumann R R , Herrmann F
Published: January 2023

We examined the expression of the MCP-1/JE gene in freshly prepared human monocytes and again after either in vitro or in vivo maturation of these cells. We show that previously unstimulated blood monocytes of healthy individuals prepared by adherence procedures display high levels of MCP-1/JE mRNA and protein. Monocytes that were not previously exposed to activational plastic surfaces but were separated from other blood cells by counter-flow centrifugal elutriation expressed severalfold lower MCP-1/JE transcript and protein levels. Treatment of these cells with endotoxin was associated with the down-regulation of MCP-1/JE mRNA and protein levels. In contrast, exposure to IFN-gamma resulted in increased MCP-1/JE gene expression. During the process of in vitro maturation of monocytes into macrophages, a gradual decrease of MCP-1/JE mRNA and protein expression was noted. Both mature (day 8) blood monocyte-derived culture macrophages and peritoneal macrophages completely failed to express the MCP-1/JE gene, which could be restored after exposure to IFN-gamma and cross-linking of macrophage Fc gamma R with murine solid phase IgG2a mAbs; however, endotoxin failed to induce MCP-1/JE expression in these cells.
Phagocytic processing of exogenous particulate antigens by macrophages for presentation by class I MHC molecules.
Authors: Harding C V , Song R
Published: January 2023

Exogenous Ags that are processed in vacuolar endocytic compartments are generally presented by class II MHC molecules and not class I MHC (MHC-I) molecules, which conventionally present cytoplasmic or endogenous Ags. Accordingly, i.v. immunization of C57BL/6 mice with soluble OVA did not elicit a CD8 T cell response. However, i.v. immunization with OVA coupled to Latex particles (Latex-OVA) elicited an OVA-specific CD8 T cell response in vivo (particles from 59 to 2000 nm diameter were effective). In vitro, Latex-OVA was processed by H-2b macrophages and presented by Kb at least 100- to 1000-fold more efficiently than was soluble OVA. Inhibition of phagocytosis by cytochalasin D blocked the processing of Latex-OVA, whereas processing was not blocked by Brefeldin A. Latex-OVA was presented directly by H-2b macrophages or after "regurgitation" of processed OVA peptide from viable MHC-disparate macrophages for binding to surface Kb molecules on fixed H-2b macrophages. Peptide regurgitation was observed during processing of both Latex-OVA and Salmonella typhimurium 14028s that express an OVA fusion protein (Crl-OVA). However, the regurgitation pathway was less efficient than direct processing by viable H-2b macrophages. Thus, macrophages express an alternate pathway that allows MHC-I presentation of vacuolar exogenous particulate Ags, including inert synthetic particles without lipid membranes and intravacuolar bacteria. Peptides from these Ags are released from intracellular compartments to bind to surface MHC-I molecules, but peptide-MHC-I complexes also may be generated within intracellular compartments.
Regulation of human IgG subclass production by cytokines. IFN-gamma and IL-6 act antagonistically in the induction of human IgG1 but additively in the induction of IgG2.
Authors: Kawano Y , Noma T , Yata J
Published: January 2023

In this study, we investigated the roles of IFN-gamma and IL-6 in the induction of IgG subclasses from PBMC stimulated with PWM. The presence of IFN-gamma in the first half of the culture period dramatically suppressed the production of IgG1, whereas spontaneous IgG2 secretion was sharply enhanced by IFN-gamma when the latter was present throughout the culture period. Endogenous IFN-gamma was shown to be effective in IgG1 and IgG2 production because IgG1 production was enhanced and IgG2 production was inhibited by the addition of anti-IFN-gamma Ab. Because IFN-gamma did not act on the PBMC depleted of surface IgG2-bearing cells to enhance IgG2, IFN-gamma is unlikely to act as a switching factor. Differing from IFN-gamma, IL-6 enhanced every IgG subclass production as a nonswitching factor, although each of IgG subclasses required IL-6 differentially; maximal IgG1 responses and IgG4 responses occurred when IL-6 was present for the entire culture period or the last half of the culture period, whereas maximal IgG3 responses required the presence of IL-6 for the entire culture period and maximal IgG2 responses were seen when IL-6 was present solely during the last half of the culture period. IFN-gamma antagonized the synthesis of IgG1 by IL-6 and cooperated with IL-6 to produce IgG2. This observation was further supported by the fact that the IL-6-induced IgG1 and IgG2 enhancement were respectively up-regulated and down-regulated by anti-IFN-gamma. Altogether, the critical roles of IFN-gamma and IL-6 were implicated.
Association of Gm allotypes with the antibody response to the outer membrane proteins of a common upper respiratory tract organism, Moraxella catarrhalis.
Authors: Goldblatt D , Scadding G K , Lund V J , Wade A M , Turner M W , Pandey J P
Published: January 2023

Previously, Gm allotypes have been shown to influence human serum Ig subclass levels as well as the Ab levels achieved after Ag stimulation. The majority of the latter studies have focused on Ab responses to polysaccharide Ags. In this study, we have investigated the relationship between Gm allotypes and naturally occurring serum Ab levels to a bacterial protein Ag, the outer membrane proteins of a common microorganism, Moraxella catarrhalis. In the sera of 160 patients having chronic/recurrent sinusitis, there was a highly significant correlation between the level of specific anti-M. catarrhalis IgG3 level and certain Gm phenotypes. After additional investigation, we found that the presence of G3m(21) homozygosity correlated significantly with lower levels of Ag-specific IgG3. Specific anti-M. catarrhalis IgG3 levels were found to be independent of total serum IgG3 concentrations, and there was no correlation between the serum level of IgG3 and any Gm phenotype. Total IgG and IgG2 that were specific for pneumococcal cell wall polysaccharide also were measured in this group of patients, and no correlation was found between the naturally occurring IgG2 subclass levels to pneumococcal cell wall polysaccharide and the interactive effect of G2m(23) (syn: G2m(n)) and Km(1). Gm allotypes may influence Ab responses other than the anti-carbohydrate responses and, therefore, should be taken into account when investigating IgG subclass responses to protein Ags.
Formation of large molecular weight fragments of DNA is a key committed step of apoptosis in thymocytes.
Authors: Cohen G M , Sun X M , Fearnhead H , MacFarlane M , Brown D G , Snowden R T , Dinsdale D
Published: January 2023

Apoptosis is a process in which cells die in a controlled manner and apparently participate in their own demise. It is best characterized morphologically by condensation of chromatin and biochemically by cleavage of chromatin at internucleosomal regions to yield a classical DNA ladder pattern. Apoptosis was induced in thymocytes by exposure to either the glucocorticoid, dexamethasone, or DNA topoisomerase II inhibitor, etoposide. We describe the formation of large m.w. fragments of DNA, 30 to 50 kilobase pairs in length, in a population of these thymocytes at an early stage of apoptosis before internucleosomal cleavage of DNA. These fragments are absent in normal thymocytes and their formation is dependent on protein synthesis. Their appearance is coincident with the commitment of these cells to apoptosis. The formation of these large fragments is associated with the condensation of chromatin, abutting the nuclear membrane, recognized as one of the earliest ultrastructural signs of apoptosis. Subsequent cleavage of these large fragments gives rise to oligonucleosomal fragments and is independent of protein synthesis. We propose that the formation of large fragments of DNA represents a key committed step in apoptosis, and that it is from these fragments that the archetypal DNA ladders associated with apoptosis are derived.
T lymphocyte T cell-B cell-activating molecule/CD40-L molecules induce normal B cells or chronic lymphocytic leukemia B cells to express CD80 (B7/BB-1) and enhance their costimulatory activity.
Authors: Yellin M J , Sinning J , Covey L R , Sherman W , Lee J J , Glickman-Nir E , Sippel K C , Rogers J , Cleary A M , Parker M
Published: January 2023

Activation-induced cell surface molecules are involved in mediating bidirectional T-B lymphocyte signaling that is important in the induction of T or B lymphocyte effector functions. In this regard, T-BAM/CD40-L is an activation-induced CD4+ T cell surface molecule known to be important in inducing B cell effector functions. This report demonstrates that T-BAM/CD40-L molecules on a Jurkat T cell leukemia subclone (D1.1) or nonlymphoid 293 kidney cell transfectants induce B cells or B-CLL cells to express CD80 (B7/BB-1) in a manner that is specifically inhibited by anti-T-BAM/CD40-L mAb 5C8. Because activation-induced B cell surface molecules, such as CD80, deliver costimulatory signals to T cells that augment T cell proliferation, the functional costimulatory capacity of T-BAM/CD40-L-primed B cells and B-CLL cells was studied. T-BAM/CD40-L-primed B cells or B-CLL cells augment the proliferative responses of allogenic T cells. Furthermore, T-BAM/CD40-L priming is specifically inhibited by mAb 5C8. Together, these studies demonstrate that T-BAM/CD40-L induces CD80 expression on resting B cells or B-CLL cells. Moreover, T-BAM/CD40-L signaling enhances B cell costimulatory capacity. These studies suggest that T-BAM/CD40-L molecules not only induce B cell differentiative processes that result in Ab secretion, but also enable B cells to prime Ag-specific T cells for subsequent clonal expansion.
Anti-IL-4 treatment of Schistosoma mansoni-infected mice inhibits development of T cells and non-B, non-T cells expressing Th2 cytokines while decreasing egg-induced hepatic fibrosis.
Authors: Cheever A W , Williams M E , Wynn T A , Finkelman F D , Seder R A , Cox T M , Hieny S , Caspar P , Sher A
Published: January 2023

Increasing evidence suggests that schistosome egg granulomas are primarily Th2 cellular reactions. Mice infected with Schistosoma mansoni were treated with a neutralizing mAb against IL-4 to evaluate the role of this cytokine in the generation of parasite egg-induced cell-mediated responses and hepatic pathology. Animals treated with anti-IL-4 before egg deposition showed decreased IL-4, IL-5, and IL-10 production in response to in vitro antigenic stimulations and decreased IL-5 and IL-13 mRNA levels in the liver. As observed previously, non-B, non-T cells were a major source of IL-4 in infected mice treated with control mAb, and the diminished IL-4 response in anti-IL-4-treated animals was shown to be caused at least in part by a reduction in the number of these cells, as well as by decreased secretion of IL-4 per cell. In contrast, production of the Th1 cytokines IL-2 and IFN-gamma was elevated in anti-IL-4-treated infected mice in vitro, and the corresponding mRNAs in the liver were increased. Anti-IL-4 treatment did not consistently reduce the size of hepatic granulomas around S. mansoni eggs, but markedly inhibited granuloma formation in the lungs of the same animals after i.v. egg injection. Nevertheless, anti-IL-4-treated infected mice showed consistent and marked reductions in hepatic collagen deposition. These findings indicate that IL-4 plays a major role in the development of the Th2 response in S. mansoni-infected mice and contributes to the pathogenesis of hepatic fibrosis.
Methimazole prevents induction of experimental systemic lupus erythematosus in mice.
Authors: Singer D S , Kohn L D , Zinger H , Mozes E
Published: January 2023

Experimental SLE can be induced in mice by immunization with a human mAb to DNA (16/6Id). Immunized mice develop Abs to the 16/6Id immunogen, DNA, and nuclear Ags. Subsequently, clinical manifestations of disease develop, including leukopenia, proteinuria, and immune complex deposits in the kidney. MHC class I Ags play a critical role in the induction of experimental SLE, as demonstrated by the finding that class I-deficient mice are resistant to disease induction. This finding suggested that agents that reduce MHC class I expression might mitigate experimental SLE in normal mice. These studies report that methimazole, which has been shown to repress class I transcription in some cell lines, reduces class I expression on PBLs in vivo and prevents the development of clinical manifestations of SLE in 16/6Id-immunized mice. These data suggest that methimazole, which has been used in the treatment of Graves' disease, may be useful in the clinical treatment of SLE and other autoimmune diseases.
Lipopolysaccharide-dependent induction of IL-10 receptor expression on murine fibroblasts.
Authors: Weber-Nordt R M , Meraz M A , Schreiber R D
Published: January 2023

Although various biologic activities of IL-10 have been identified, little is known about IL-10's molecular mechanism of action. Herein we report the characterization of IL-10R on different murine cell lines and demonstrate the LPS inducibility of this protein. With the use of purified recombinant murine IL-10 and labeled IL-10-specific mAb, IL-10Rs were detected by flow cytometry. Radioligand binding analyses showed that RAW264.7 cells expressed 238 +/- 87 receptors per cell and bound ligand with a single affinity of 8.3 +/- 2.4 x 10(9) M-1. Similar studies documented IL-10R expression on murine B cells (CH27) and CD4+ Th1 cells but not murine Th2 cells, L929 or WA-17 fibroblasts, or fibrosarcoma cells. Exposure of fibroblasts to LPS-induced cellular IL-10 binding activity in a dose- and time-dependent manner. Radioligand binding analyses performed on LPS-treated fibroblasts showed cellular expression of 325 +/- 59 IL-10 binding sites and a binding affinity of Ka = 7.5 +/- 2.5 x 10(8) M-1. RT-PCR analysis confirmed the induction of the IL-10R. Functional analyses of IL-10R-expressing cells revealed that IL-10 activated a cellular transcription factor in RAW264.7 cells that bound a DNA probe containing the IFN-gamma response region from the Fc gamma RI gene. In contrast, IL-10 did not induce gamma response region binding activity in L929 fibroblasts either treated with LPS or transfected with the murine IL-10R cDNA. These results thus indicate that cellular responses to IL-10 may be influenced by both the external environment and the presence of additional signaling components within the cell.
Recombinant human IL-6 suppresses demyelination in a viral model of multiple sclerosis.
Authors: Rodriguez M , Pavelko K D , McKinney C W , Leibowitz J L
Published: January 2023

We used a murine model of multiple sclerosis (MS) induced by Theiler's murine encephalomyelitis virus (TMEV) to test the effect of IL-6 on central nervous system (CNS) demyelination. Administration of human rIL-6 (2.5 micrograms/dose), beginning one day before infection and then twice daily for 28 days, dramatically reduced demyelination and inflammation in the spinal cord of susceptible SJL/J mice. Benefit also was observed when rIL-6 was used as a therapeutic agent and begun on day 15 after infection, a time in which there is the first evidence of inflammation and demyelination in the spinal cord. Suppression of myelin damage by treatment with rIL-6 was associated with fewer virus Ag-positive cells in the spinal cord. Infectious CNS virus titers, as measured by plaque assay, were reduced in rIL-6-treated animals on day 15 after infection, but not on day 7, 22, or 29 after infection. Total serum Igs and virus-specific Igs, as detected by indirect ELISA, were increased markedly in rIL-6-treated mice, whereas no effect was observed on TMEV-neutralizing Ab titers. In vivo administration of rIL-6 inhibited a murine CNS-demyelinating disease induced by a virus, suggesting that this IL may have application for the treatment of human MS.
Morphologic and functional characterization of perforin-deficient lymphokine-activated killer cells.
Authors: Liu C C , Walsh C M , Eto N , Clark W R , Young J D
Published: January 2023

Mice deficient in perforin, a key mediator of lymphocyte-mediated cytolysis, have recently been generated using the gene knockout technique. CTL and NK cells derived from these mice have been shown to be defective in the granule-dependent cytolytic pathway. To investigate whether the granule-formation process has been altered in these perforin-deficient cytotoxic cells, rendering them defective in using the other granule mediators, we have examined in the present study the morphologic and functional characteristics of perforin-deficient LAK cells. Perforin-deficient LAK cells, similar to wild-type LAK cells, were shown to contain a large number of granules in their cytoplasm. By electron microscopy, the morphology of the granules present in these two cell populations appeared indistinguishable. The complete depletion of perforin in LAK cells derived from perforin gene-knockout mice was further confirmed by immunoelectron microscopy using anti-perforin antiserum. The expression of other cytolytic mediators, present either within the granules (granzymes A and B) or elsewhere (Fas ligand), appeared to be unperturbed, as investigated using the reverse transcription-PCR technique. Like the CTL and NK cells isolated from perforin-deficient mice, perforin-deficient LAK cells could lyse only target cells that express high levels of Fas molecule. Furthermore, these perforin-deficient LAK cells, similar to wild-type LAK cells and a CTL clone, were resistant to perforin-mediated cytolysis.
Transchromosomally derived Ig heavy chains.
Authors: Knight K L , Kingzette M , Crane M A , Zhai S K
Published: January 2023

During an immune response, activated B cells undergo isotype switching and begin to express isotypes other than IgM and IgD. Isotype switching occurs when downstream C gamma, C alpha, or C epsilon genes are rearranged into the S mu chromosomal region, resulting in the deletion of the region in between. These rearrangements usually occur in cis, i.e., intrachromosomally. In previous studies, we analyzed allotypic specificities of rabbit secretory IgA and identified a substantial number of IgA heavy chains with VH and C alpha allotypes that were encoded by VH and C alpha genes in trans. In those studies, however, we could not determine whether the trans association of VH and C alpha occurred during VDJ gene rearrangement or during isotype switching. Here, we cloned rabbit cDNA which encodes these trans IgA heavy chains and determined the chromosomal origin of the VH, JH, and C alpha regions. To determine whether the trans association occurred during VDJ gene rearrangement, we analyzed the nucleotide polymorphism of the JH region and the VH allotype encoded by the cDNA. We found that the VH and JH genes used in the VDJ gene rearrangements were from the same chromosome, indicating that the VH, D, and JH gene rearrangements occurred in cis. Furthermore, we analyzed the DNA polymorphisms of JH and C alpha and showed that the VDJ and C alpha genes encoding the trans IgA molecules were derived from different parental chromosomes. We suggest that the trans association occurred during isotype switching. This study shows that VH and CH can associate transchromosomally as part of a normal immune response.
Immunization with a single T helper cell epitope abrogates Friend virus-induced early erythroid proliferation and prevents late leukemia development.
Authors: Miyazawa M , Fujisawa R , Ishihara C , Takei Y A , Shimizu T , Uenishi H , Yamagishi H , Kuribayashi K
Published: January 2023

Synthetic peptide vaccines containing a single Th cell epitope identified in the gp70 envelope glycoprotein of Friend murine leukemia helper virus induced potent protective immunity against Friend virus infection. H-2a/b mice immunized by a single s.c. injection of the CFA emulsion containing a peptide that represented the N-terminal gp70 epitope recovered slowly from initial development of splenomegaly, and most did not develop late leukemia, whereas most of the control mice given an injection of CFA alone showed sustained leukemic splenomegaly after the challenge with Friend virus. The mice of the same genetic background immunized with the C-terminal Th cell epitope by a single injection of a separate synthetic peptide eliminated virus-producing cells from the spleen within 12 days after inoculation of Friend virus complex, and did not develop early splenomegaly or polycythemia. H-2a/a mice were not protected by immunization with either one of the two synthetic peptides. Earlier production and more rapid class switching of virus-neutralizing Abs were observed in H-2a/b mice immunized with the peptide vaccines after the challenge with Friend virus, compared with the responses of the control mice. Detailed kinetic and immunohistopathologic analyses suggested that Th cells might be directly involved in the growth inhibition and elimination of virus-infected erythroid precursor cells.
Prostaglandin synthase 1 and prostaglandin synthase 2 both participate in activation-induced prostaglandin D2 production in mast cells.
Authors: Kawata R , Reddy S T , Wolner B , Herschman H R
Published: January 2023

Activation of MMC-34 cells, a murine mast cell line, or bone marrow-derived mast cells by aggregation of IgE cell surface receptors or addition of calcium ionophore stimulates prostaglandin (PG) D2 synthesis and secretion. An initial rapid burst of PGD2 synthesis, complete within 30 min, is followed by a slower subsequent production of PGD2 that reaches a maximum 4 to 8 h after activation in MMC-34 cells. PG synthase 1 (PGS-1) message and protein are expressed constitutively in MMC-34 cells and are not modulated by exposure to calcium ionophore or aggregation of IgE receptors. In contrast, activation of MMC-34 or bone marrow-derived mast cells induces expression of the PG synthase 2 (PGS-2) gene. PGS-2 induction following mast cell activation is blocked by dexamethasone. The initial PGD2 burst in activated MMC-34 cells is prevented by aspirin pretreatment, suggesting that constitutive PGS-1 present in mast cells before activation is responsible for the early PGD2 production in response to activation. In contrast, the later phase of PGD2 production is blocked by dexamethasone, cycloheximide, or NS-398, a PGS-2-specific nonsteroidal anti-inflammatory drug that inhibits PGS-2 enzyme activity but not PGS-1 activity. These data demonstrate that mast cell activation results 1) in the induction of PGS-2 gene expression, and 2) in both PGS-1-dependent PGD2 synthesis and PGD2 synthesis that is dependent on the activation-induced synthesis and activity of PGS-2.
A cyclic adenosine 3',5'-monophosphate signal is required for the induction of IL-1 beta by TNF-alpha in human monocytes.
Authors: Lorenz J J , Furdon P J , Taylor J D , Verghese M W , Chandra G , Kost T A , Haneline S A , Roner L A , Gray J G
Published: January 2023

IL-1 beta is a cytokine generally considered to be a major component involved in the pathogenesis of rheumatoid arthritis and other inflammatory diseases. Of the agents found in high concentrations in inflamed rheumatoid arthritis joints, TNF-alpha is among the most strongly implicated as an in vivo inducer of IL-1 beta. Here we report that in human PBMC and in a stable transfectant of the promonocytic cell line, THP-1, TNF-alpha indeed appears to be an inducer of IL-1 beta production, but only in the presence of dibutyryl cAMP or agents such as the PG that elevate intracellular cAMP levels. This TNF-alpha/cAMP pathway regulates IL-1 beta production at the level of transcription and requires a cAMP response element located between -2762 and -2755 bp in the upstream regulatory sequence of IL-1 beta. Because PG, which are known to elevate cAMP levels in vivo, and TNF-alpha are both found in significant quantities in the synovial fluid of rheumatoid arthritis joints, the observed synergistic up-regulation in IL-1 beta synthesis by TNF-alpha/cAMP (PG) may provide valuable insight into the potential pathways involved in the continuous production of IL-1 beta in the chronically inflamed joint.
Vascular cell adhesion molecule-Ig fusion protein selectively targets activated alpha 4-integrin receptors in vivo. Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.
Authors: Jakubowski A , Ehrenfels B N , Pepinsky R B , Burkly L C
Published: January 2023

The alpha 4 beta 1-integrin (CD49d, CD29) constitutively expressed on leukocytes regulates cell migration to inflammatory sites, cell activation, and development through its interactions with two alternate ligands, vascular cell adhesion molecule-1 (VCAM-1; CD106) expressed on cytokine-activated endothelium, dendritic and stromal cells, and the extracellular matrix protein fibronectin. Another alpha 4-integrin receptor, alpha 4 beta 7, expressed on leukocytes also binds VCAM-1 and fibronectin (FN), and controls homing to mucosal tissues through its interactions with mucosal vascular addressin MAdCAM-1. In vitro studies have shown that alpha 4-dependent cell adhesion is regulated by the activation state of the cell and by divalent cations. However, the existence and role of cells with different alpha 4 activation states in vivo have not been defined. Herein we show that a soluble ligand with the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region, VCAM-Ig, binds selectively to activated alpha 4-receptors on murine cells, such as those induced by Mn2+ in vitro. To determine whether the cells identified by VCAM-Ig were required under physiologic conditions, we assessed its anti-inflammatory effect. We show that VCAM-Ig is not bound to the majority of murine alpha 4+ cells after in vivo administration, yet it significantly delays the onset of adoptively transferred autoimmune diabetes. Thus, soluble VCAM-Ig can modify alpha 4-dependent disease progression, apparently by its selective action on cells with activated alpha 4-integrin receptors, thereby providing evidence for distinct alpha 4 activation states in vivo.
Cross-reactive antibodies have both protective and pathogenic potential.
Authors: Limpanasithikul W , Ray S , Diamond B
Published: January 2023

We report the characteristics of five IgM anti-phosphorylcholine (PC) Abs derived from nonautoimmune BALB/c mice. These Abs were chosen on the basis of their unique ability to cross-react with dsDNA. Three Abs protect mice from a lethal pneumococcal infection, and all five Abs deposit in glomeruli and cause renal dysfunction. Analysis of V region gene usage shows that none of the Abs is encoded by the V1 gene segment used to encode the dominant T15+ anti-PC response typically expressed in serum of PC-immunized BALB/c mice. The demonstration of the existence of noncanonical protective anti-PC Abs that also display pathogenic autoreactivity suggests that idiotypically restricted responses may serve to prevent the expression of Abs that react with both foreign and self Ags and so help maintain self-tolerance.
Evidence that the same gamma delta T cells respond during infection-induced and autoimmune inflammation.
Authors: Mukasa A , Lahn M , Pflum E K , Born W , O'Brien R L
Published: January 2023

Inflammatory responses are induced in both testes of a mouse following injection of Listeria monocytogenes into one testis. Although the uninjected testis contains no detectable bacteria, it undergoes an autoimmune attack. Normally, the testis lacks lymphocytes, but in the infected and autoimmune state, both gamma delta and alpha beta T cells are found as infiltrates. Here, we have examined the repertoire of the infiltrating gamma delta T cells, using two different methods, and found a high frequency of V gamma 6/V delta 1 gamma delta T cells in both infected and autoimmune testes. All of these expressed the invariant V gamma 6/V delta 1 TCR previously reported. However, secondary gamma and delta transcripts present within V gamma 6/V delta 1 hybridomas indicated nonclonality. Interestingly, some of these secondary transcripts were derived from gamma gene rearrangements not previously found in this gamma delta T cell subset, implying a difference in its origin. The increase in V gamma 6/V delta 1 cells observed here in both infected and autoimmune testes, together with our previous finding of a preferential response by the same subset in Listeria-infected liver, indicates that their response is triggered by the inflammation rather than by the infectious agent or because they are already resident in the tissue. We and others have previously reported that the presence of gamma delta T cells during certain inflammatory conditions correlates with less host tissue damage. This result, together with the evidence presented here, further implies that a response by the V gamma 6/V delta 1 subset in some way exerts a controlling influence on the host inflammatory response.
VH gene replacement in hyperselected B cells of the quasimonoclonal mouse.
Authors: Cascalho M , Wong J , Wabl M
Published: January 2023

The primary repertoire of the quasimonoclonal mouse is monospecific. However, among peripheral B cells, there is a high frequency of variant cells with V(H) replacements, which are also hypermutated. We show in this work that these hyperselected cells expand in numbers as the animals increase in age, switch their isotypes, and with increasing age become the almost exclusive contributors to the pool of serum Ig. The fraction of such cells is higher in the peritoneum than in peripheral blood, supporting the view that the peritoneum is a site of production of nonspecific serum Ig. We have also isolated and partially sequenced the replacing V(H) gene segments from B220-positive, Id-negative cells, and mu-negative, lambda-positive (i.e., switched) cells, and matched them with their germline counterparts. V(H) families are represented proportional to the number of members in the germline, a finding that is consistent with the idea that environmental Ag pressure maintains the germline repertoire of V gene segment.
CD94/NKG2 inhibitory receptor complex modulates both anti-viral and anti-tumoral responses of polyclonal phosphoantigen-reactive V gamma 9V delta 2 T lymphocytes.
Authors: Poccia F , Cipriani B , Vendetti S , Colizzi V , Poquet Y , Battistini L , López-Botet M , Fournié J J , Gougeon M L
Published: January 2023

Viral, bacterial, protozoal, and cancer-associated Ags elicit strong responses in human gammadelta T lymphocytes. The majority of these cells in the peripheral blood express the Vgamma9Vdelta2-encoded TCR and recognize nonpeptidic phosphoantigens without an apparent MHC restriction. We have shown that Vgamma9Vdelta2 T cells express the inhibitory CD94/NKG2 receptor for HLA class I molecules. The anti-CD94 mAb inhibits 1) the Vgamma9Vdelta2 T cell proliferation in response mycobacterial phosphoantigens and 2) the HIV-induced Vgamma9Vdelta2 T cell expansion. Vgamma9Vdelta2 T cells stimulated with nonpeptidic mycobacterial antigens produce IFN-gamma and TNF-alpha. Signaling through the CD94/NKG2 receptor interferes with the synthesis of these cytokines. The CD94/HLA class I interaction is also involved in the cytotoxic activity of Vgamma9Vdelta2 T cells. The Vgamma9Vdelta2 T cell regulation through the CD94 receptor may be important for the potentially dual function in innate immunity, i.e., 1) NK-like and 2) TCR ligand-induced cytolytic activities.
Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependent degradation are highly efficient inducers of cytolytic T lymphocytes.
Authors: Wu Y , Kipps T J
Published: January 2023

We generated plasmid expression vectors encoding ubiquitin and beta-galactosidase (beta-gal) with different intervening amino acids, allowing for the production of processed protein products that have either stabilizing or destabilizing residues at their N-termini. P815 cells transfected with plasmids encoding beta-gal with a destabilizing N-terminus did not have detectable expression beta-gal unless they were treated with inhibitors specific for the proteasome. Inhibitors of other proteolysis pathways had no such effect. Nevertheless, transfectants expressing beta-gal with different amino acid residues were equally sensitive to cytolysis by a CTL clone specific for a beta-gal peptide presented in the context of H-2Ld. In contrast to vectors encoding native beta-gal, plasmid vectors encoding beta-gal with a destabilizing residue did not induce detectable anti-beta-gal Abs when injected into skeletal muscle of BALB/c mice. However, such vectors were significantly more effective than vectors encoding native beta-gal or beta-gal with a stabilizing residue in stimulating CTL specific for P13.2, a lacZ transfectant of P815. We conclude that incorporation of strategies that enhance proteasome-dependent degradation may generate DNA vaccines that are more effective in inducing cellular immunity against targeted Ags.
Determination of adenosine deaminase binding domain on CD26 and its immunoregulatory effect on T cell activation.
Authors: Dong R P , Tachibana K , Hegen M , Munakata Y , Cho D , Schlossman S F , Morimoto C
Published: January 2023

CD26, a 110-kDa cell surface glycoprotein, exhibits dipeptidyl peptidase IV enzyme activity and plays an important role in T cell costimulation. In the present study, we examined both the exact adenosine deaminase (ADA) binding domain on CD26 and the functional consequences of mutated CD26 transfectants that were deficient for cell surface ADA. Using CD26 deletion, human-rat swap, and point mutations, we found that the residues of L340, V341, A342, and R343 on the CD26 molecule were essential amino acids for ADA binding. When these amino acids were mutated and transfected into Jurkat cells, the resultant CD26 transfectants expressed only CD26, not ADA, on the cell surface. The amount of IL-2 produced by wild-type and mutated CD26 transfectants was almost the same following stimulation with anti-CD3 plus PMA. However, the mutated CD26 transfectants were much more sensitive to the inhibitory effect of adenosine on IL-2 production than were the wild CD26 transfectants. These data suggest that ADA on the cell surface does not directly involve T cell activation. Conversely, CD26 alone does not result in modulating the inhibitory effect of adenosine. Only the ADA bound to CD26 on the cell surface was functional and could counteract the inhibitory effect of elevated extracellular adenosine.
Influence of VH CDR3 arginine and light chain pairing on DNA reactivity of a bacterial DNA-induced anti-DNA antibody from a BALB/c mouse.
Authors: Wloch M K , Clarke S H , Gilkeson G S
Published: January 2023

Anti-DNA induced in BALB/c mice by immunization with bacterial (Escherichia coli) DNA resemble spontaneous anti-DNA from lupus mice in V gene use and cross-reactivity with other nuclear Ags, but lack the high V(H) CDR3 arginine content seen in anti-DNA from lupus mice. Moreover, the induced anti-DNA bind bacterial and mammalian single-stranded (ss) DNA and bacterial double-stranded (ds) DNA, but do not bind mammalian dsDNA. This reactivity profile is in contrast to that of the spontaneously arising anti-DNA of lupus mice, among which mammalian dsDNA reactive Abs are prominent. In this study we demonstrate that the addition of arginine to V(H) CDR3 of an induced anti-DNA confers the mammalian dsDNA binding characteristic of anti-DNA from lupus mice. The ability to confer mammalian dsDNA binding is dependent on both the position of the arginine in V(H) CDR3 and the light chain with which the heavy chain is paired, suggesting the light chain plays a more substantial role in DNA binding by this Ab than has previously been reported for other anti-DNA. Our data support the argument that V(H) CDR3 arginines tend to confer antimammalian dsDNA reactivity, leading to censure of B cells expressing these Abs and provides an explanation for the absence of arginine-rich V(H) CDR3 in the bacterial DNA-induced response.
Suppression of TNF-alpha expression, inhibition of Th1 activity, and amelioration of collagen-induced arthritis by rolipram.
Authors: Ross S E , Williams R O , Mason L J , Mauri C , Marinova-Mutafchieva L , Malfait A M , Maini R N , Feldmann M
Published: January 2023

Rolipram is a type IV phosphodiesterase inhibitor that suppresses inflammation and TNF-alpha production. As anti-TNF-alpha therapy is effective in rheumatoid arthritis, we investigated the effect of rolipram on collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis. Rolipram was administered after the onset of clinical arthritis at doses of 0.5, 3, 5, or 10 mg/kg twice daily, with a dose-dependent therapeutic effect on clinical severity and joint erosion. Immunohistochemical analysis of joints of rolipram-treated mice revealed 67% reduction in TNF-alpha-expressing cells compared with control arthritic mice. In vitro studies using bone marrow-derived macrophages confirmed that rolipram directly suppressed TNF-alpha and IL-12 production following stimulation with IFN-gamma and LPS. The effect of rolipram on T cell activity was studied by measuring Th1/Th2 cytokine production by collagen-stimulated draining lymph node cells from arthritic mice treated in vivo with rolipram. Rolipram reduced IFN-gamma production and increased IL-10, indicating that rolipram down-regulated the ongoing Th1 response to type II collagen. Finally, the effect on CIA of combination therapy was studied using rolipram plus either anti-TNF-alpha or anti-CD4 mAbs. Rolipram plus anti-TNF-alpha was not therapeutically additive, whereas rolipram plus anti-CD4 mAb was clearly additive. This result indicates that the therapeutic effects of rolipram overlap with TNF-alpha blockade, but are complementary to anti-CD4 treatment. It is therefore proposed that a major mechanism of action of rolipram in CIA is suppression of TNF-alpha activity. These findings suggest that type IV phosphodiesterase inhibitors may be effective in pathologic conditions, such as RA, with overexpression of TNF-alpha.