bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the chemokine RANTES by a human bronchial epithelial cell line. Modulation by cytokines and glucocorticoids.
Authors: Stellato C , Beck L A , Gorgone G A , Proud D , Schall T J , Ono S J , Lichtenstein L M , Schleimer R P
Published: December 2022

The chemokine RANTES is a potent chemoattractant for eosinophils, lymphocytes, and monocytes, and has been detected recently in the epithelium of human airways mucosa. We have studied, therefore, the expression of RANTES mRNA and protein in the human bronchial epithelial cell line BEAS-2B. Using Northern blot analysis, RANTES mRNA was not detectable in unstimulated BEAS-2B cells. Incubation of cells with TNF-alpha or IFN-gamma induced the expression of RANTES mRNA and protein within 16 h. The combination of TNF-alpha and IFN-gamma displayed a marked synergism in inducing RANTES expression. Pretreatment of cells with the glucocorticoid budesonide (10(-10)-10(-7) M) for 24 h inhibited expression of RANTES mRNA and protein stimulated by either TNF-alpha or TNF-alpha plus IFN-gamma in a concentration- and time-dependent manner. Nonglucocorticoid steroids did not inhibit RANTES mRNA expression. Production of RANTES by epithelium could contribute to the mechanism of selective cellular recruitment occurring in the airways during inflammation, thus playing a relevant role in the pathogenesis of diseases such as asthma, rhinitis, and polyposis. The down-regulation of RANTES production by glucocorticoids in epithelial cells may contribute to the efficacy of these compounds in reducing cellular infiltration and, ultimately, to their anti-inflammatory properties.
Differential requirement of CD28 costimulation for activation of murine CD8+ intestinal intraepithelial lymphocyte subsets and lymph node cells.
Authors: Gelfanov V , Lai Y G , Gelfanova V , Dong J Y , Su J P , Liao N S
Published: December 2022

The CD8+CD4- (CD8+) murine small intestinal intraepithelial lymphocytes (IELs) contain two subpopulations, one expressing alpha alpha-CD8 homodimers and another alpha beta-CD8 heterodimers. In this study, plate-bound anti-TCR beta-chain (TCR-beta) mAb alone or combined with anti-CD28 mAb is used as a model system to study activation requirement of these two CD8+ IEL subsets. In contrast to CD8+ lymph node (LN) cells that require both TCR and CD28 triggering for activation, alpha beta-CD8+ IELs proliferate and produce IL-2 and IFN-gamma when stimulated with anti-TCR-beta mAb alone, and soluble CTLA-4 Ig has no effect on their responses. On the other hand, alpha alpha-CD8+ IELs neither make IL-2 or IFN-gamma nor proliferate even when both stimuli are provided. However, alpha alpha-CD8+ IELs are capable of proliferation in both CD8+ IEL subsets is lower than in CD8+ LN cells, which contributes to the weaker and delayed response of CD8+ IELs.
Cyclic AMP signaling pathways are important in IL-1 beta transcriptional regulation.
Authors: Chandra G , Cogswell J P , Miller L R , Godlevski M M , Stinnett S W , Noel S L , Kadwell S H , Kost T A , Gray J G
Published: December 2022

An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.
Phosphorylated immunoreceptor signaling motifs (ITAMs) exhibit unique abilities to bind and activate Lyn and Syk tyrosine kinases.
Authors: Johnson S A , Pleiman C M , Pao L , Schneringer J , Hippen K , Cambier J C
Published: December 2022

Signal transduction by T and B cell Ag receptors and certain receptors for Ig Fc regions (Fc gamma RI, hFc gamma RIIA, Fc gamma RIII, Fc alpha R, and Fc epsilon RI) involves a conserved sequence motif, termed an immunoreceptor tyrosine-based activation motif (ITAM) and found in multiple receptor chains. Phosphorylation of the two ITAM tyrosines is a critical event in signal transduction. To address the function of this phosphorylation, we assessed the ability of nonphosphorylated and biphosphorylated ((p)2ITAM) ITAM peptides to bind and modify the activity of src and syk family kinases in vivo and in vitro. All (p)2ITAMs, but not their nonphosphorylated counterparts, induced extensive protein tyrosine phosphorylation in permeabilized cells. However, the patterns of proteins phosphorylated differed among (p)2ITAMs. This phosphorylation was found to reflect activation of the src family kinase Lyn, but not Syk. In vitro studies using purified Lyn showed that src family kinase activation resulted from a direct interaction with (p)2ITAM. Binding studies demonstrated clear differences in binding specificity of (p)2ITAMs. Most strikingly, Ig alpha (p)2ITAM and TCR-zeta c and CD3 epsilon (p)2ITAMs exhibit inverse binding preferences for src and syk family kinases. Taken together, these findings demonstrate a novel mechanism by which src family tyrosine kinases are activated, and are consistent with the possibility that different ITAMs may preferentially activate distinct signaling pathways as a consequence of distinct effector Src homology 2 domain (SH2) binding preference.
Non-sense and missense mutations in the structural genes of complement component C1q A and C chains are linked with two different types of complete selective C1q deficiencies.
Authors: Petry F , Le D T , Kirschfink M , Loos M
Published: December 2022

To shed light on the molecular basis of two different types of complete C1q deficiency, we performed extensive Southern blot analysis and sequenced all exons of the genes coding for the A, B, and C chains of C1q on two groups of deficient patients. In one family with three cases of complete C1q deficiency we found a point mutation in the codon for glutamine (CAG) at position 186 of the A chain that led to a termination codon (TAG). No gene products of any of the three genes were found in the patients' sera, indicating that full length polypeptides of the A, B, and C chains are required to form and secrete functional C1q. A second point mutation was found in a patient with a complete functional C1q defect. The abnormal C1q molecule had been shown to have a low m.w. of approximately 150,000 and a sedimentation coefficient of below 7S. The mutation occurred in the codon for glycine in position 6 of the C chain where a single base exchange (G-->A transition) has led to an arginine residue. In the parents and son of patient G we could demonstrate the heterozygous state of the mutation by the occurrence of both bases in question, G and A. The interruption of the collagen-like triplet motif Gly-X-Y and, even more likely, the introduction of a large positively charged side chain at the N-terminus of the polypeptide may inhibit the assembly of three structural subunits consisting of two A-B dimers and one C-C dimer to form the 11S C1q macromolecule.
Identification of a mouse protein homologous to the human CD6 T cell surface protein and sequence of the corresponding cDNA.
Authors: Robinson W H , Prohaska S S , Santoro J C , Robinson H L , Parnes J R
Published: December 2022

CD6 is a 105/130 kDa monomeric T cell surface glycoprotein that has been shown to play a role in human T cell activation. Recently a partial mouse CD6 cDNA sequence was described. We have isolated full-length cDNA clones including the initiation codon and sequence encoding the full signal peptide, as well as an additional 39 amino acids within the cytoplasmic domain as compared to the previously reported clone. The predicted full-length mouse CD6 protein contains 665 amino acids and has the features of a type I integral membrane protein. The extracellular domain of mouse CD6 is composed of three repeated cysteine-rich domains similar to those in human CD6, mouse and human CD5, and other members of a family of proteins whose prototype is the type I macrophage scavenger receptor. In marked contrast to the previously published human CD6 sequence, the mouse sequence predicts a long cytoplasmic tail that is not closely related to other proteins and possesses two proline-rich motifs containing the SH3-domain binding consensus sequence, three protein kinase C phosphorylation site motifs, nine casein kinase-2 phosphorylation site motifs, and a serine-threonine-rich motif repeated three times. Northern blot analysis revealed that mouse CD6 mRNA is expressed predominantly in thymus, lymph node, and spleen. A polyclonal antiserum was raised against mouse CD6 by gene gun plasmid DNA immunization of rabbits with the mouse CD6 cDNA in an expression vector. In immunofluorescence analysis this polyclonal antiserum positively stained the surface of cells transfected with the mouse CD6 cDNA in an expression vector, as well as most normal mouse thymocytes and peripheral T cells. CD6 protein is expressed on most CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes, and is expressed at highest levels on mature CD3high thymocytes. The expression of mouse CD6 in thymocytes and peripheral T cells correlates closely with the expression of the related CD5 molecule. The polyclonal rabbit anti-mouse CD6 Abs immunoprecipitated a major polypeptide of 128 kDa from resting and 130 kDa from PMA- and FCS-activated mouse thymocytes and lymph node cells; it is likely that this increase in size upon activation is due to phosphorylation of mouse CD6 as has been described for human CD6. These data demonstrate that mouse thymocytes and T cells express a 130-kDa cell surface protein homologous to human CD6.
IL-7 stimulates protective immunity in mice against the intracellular pathogen, Toxoplasma gondii.
Authors: Kasper L H , Matsuura T , Khan I A
Published: December 2022

Cytokines, in particular IFN-gamma and IL-12, are important in host protection against infection with Toxoplasma gondii. This parasite is a major cause of congenital infection and morbidity in immunosuppressed persons, especially those with AIDS. IL-7, a monomeric protein produced by bone marrow stromal cells and fetal thymus, is able to induce the proliferation of pro-B cells and CD4+ and CD8+ T cells, and to enhance cytotoxicity of CTL and NK cells. Inbred mice were infected with a lethal dose of T. gondii and given IL-7 twice daily. Mice treated with IL-7 beginning at the time of infection survived, whereas mice either treated after infection or not treated died. Phenotypic analysis of splenocytes identified an expansion of NK (asialo GM1+) cells and CD8+ T cell populations. In vivo depletion of NK (asialo GM1+) and CD8+ T cells showed that cells expressing these phenotypes were important for maintaining protection against the parasite. IFN-gamma depletion resulted in complete reversal of the protective effect of IL-7 administration. In vivo depletion of endogenous IL-7 enhanced susceptibility to infection. Cytokine analysis by semiquantitative reverse-transcriptase PCR showed that IL-7 enhances the IFN-gamma response and furthermore reverses the parasite-mediated down-regulatory response on IL-2. These observations indicate that exogenous administration of human rIL-7 is able to protect mice against acute parasite challenge by stimulating IFN-gamma production and augmenting the CD8+ T cell-mediated CTL response.
CD8highCD57+ T lymphocytes in normal, healthy individuals are oligoclonal and respond to human cytomegalovirus.
Authors: Wang E C , Moss P A , Frodsham P , Lehner P J , Bell J I , Borysiewicz L K
Published: December 2022

CD8+CD57+ lymphocytes from normal peripheral blood are divided into T cells expressing high levels of CD8 and NK cells expressing low levels of surface CD8. Increased numbers of CD8highCD57+ T cells correlate with previous exposure to human cytomegalovirus (HCMV) infection, but no virus-specific function or function for CD8highCD57+ cells has been recorded. We have studied the TCR repertoire and responses of the CD8highCD57+ population induced by virus-infected fibroblasts in healthy individuals. Using three-color flow cytometry of PBL, we detected restricted TCRBV usage in the CD8highCD57+ subset of 11/15 subjects, compared with 1/15 in the CD8+, CD57- subset. The results of anchored PCR and sequencing also showed oligoclonality of TCR; 40-70% of CD8highCD57+ lymphocytes (10-20% of total CD8+ T cells) were derived from single clones. Such expansions were stable with time and detected in one subject over a 2-yr period. Functionally, CD8highCD57+ lymphocytes proliferated strongly to HCMV-, but not HSV-, VZV-, or influenza-infected fibroblasts in an MHC-unrestricted manner in vitro, including preferential augmentation of particular in vivo oligoclonally expanded subpopulations. HCMV-specific MHC-restricted CTL were detected, but limiting dilution analysis showed that these were a minority (< 10%) and not the oligoclonal subsets. In contrast, depletion of oligoclonally expanded CD8highCD57+ subpopulations, resulted in the increase of HCMV-specific CTL, suggesting functional heterogeneity in these cells.
Molecular analyses of the association of CD4 with two members of the transmembrane 4 superfamily, CD81 and CD82.
Authors: Imai T , Kakizaki M , Nishimura M , Yoshie O
Published: December 2022

Previously, we have shown that CD81 and CD82, two members of the transmembrane 4 superfamily, form multimolecular membrane complexes by associating with each other and with CD4 or CD8 in T cells. In the present study, we further analyzed the molecular basis of the CD4 association with CD81 and CD82 by co-precipitation experiments. First, we examined the regions of CD4 involved in the association with CD81 and CD82 by employing chimeric proteins generated from CD4 and CD2. It was confirmed that CD4, but not CD2, was capable of binding with CD81 and CD82 in transfected cells. We found that the cytoplasmic region of CD4 was sufficient for the chimeric proteins to co-precipitate CD81, while both the cytoplasmic and extracellular regions of CD4 were required for them to efficiently co-precipitate CD82. We next found, by using truncated CD4 lacking the C-terminal 31 amino acids or mutated CD4 with the cysteine residues at 394 and 397 replaced by serine, that the p56lck binding site or the covalent modification with palmitic acid was not necessary for CD4 to associate with CD81 and CD82. Finally, we found that the binding of p56lck to CD4 strongly inhibited its association with CD81 and CD82. It is, therefore, suggested that CD4 exists at least in two physical states, one associated with p56lck and another associated with CD81 and CD82 in the absence or uncoupling of p56lck.
IL-8 induces neutrophil chemotaxis predominantly via type I IL-8 receptors.
Authors: Hammond M E , Lapointe G R , Feucht P H , Hilt S , Gallegos C A , Gordon C A , Giedlin M A , Mullenbach G , Tekamp-Olson P
Published: December 2022

IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation. IL-8 reacts with neutrophils via two distinct types of IL-8-R. Receptor-specific Abs were raised against peptides derived from the first extracellular domain of each IL-8-R. Anti-IL-8-R1 and anti-IL-8-R2 selectively block 125I-IL-8 binding to rIL-8-R type 1 or 2, respectively. The anti-peptide Abs were used to assess the role of each receptor in the chemotactic response of neutrophils to GRO alpha and to IL-8. Anti-IL-8-R2 blocks GRO alpha-induced chemotaxis of neutrophils. Chemotaxis to GRO alpha is not inhibited by anti-IL-8-R1. Thus GRO alpha stimulates chemotaxis exclusively through IL-8-R2 and independently of IL-8-R1. Surprisingly, anti-IL-8-R1 inhibits the majority (78 +/- 3%) of IL-8-induced neutrophil chemotaxis. Only a minor proportion of IL-8-induced chemotaxis (29 +/- 5%) is inhibited by anti-IL-8-R2. These findings indicate that chemotaxis to IL-8 is mediated predominantly by type 1 IL-8-Rs and suggest that IL-8-R1 is an appropriate target for therapeutic strategies to limit neutrophil influx in diseases where neutrophils contribute to pathophysiology.
Identification of a novel class of mammalian Fc gamma receptor.
Authors: Zhang G , Young J R , Tregaskes C A , Sopp P , Howard C J
Published: December 2022

A cDNA encoding an Ig receptor that conferred the ability to bind erythrocytes sensitized with IgG2, but not IgG1, was cloned by screening a cattle alveolar macrophage library, made in the vector pCDM8, expressed in COS-7 cells. A search of the PIR database indicated a greater level of similarity with human Fc alpha R than with any other FcR. The percentage of identical amino acids was 41% and nucleotides 56%. This high similarity is between the extracellular and transmembrane domains; the cytoplasmic tails are unrelated. Similarities with human Fc gamma RI, Fc gamma RII, Fc gamma RIII, Fc epsilon RI, or bovine Fc gamma RII were less than 28%. COS-7 cells transfected with the cloned plasmid bound erythrocytes specifically sensitized with IgG2 but not with IgG1. In tests with heat-aggregated bovine Igs, IgG2 purified from serum bound to transfected COS-7 cells but IgG1 from serum and IgA from tracheobronchial secretions did not. Human serum IgA also failed to bind to transfected COS-7 cells although it did bind to bovine neutrophils and monocytes. Only aggregated bovine IgG2 inhibited the binding of IgG2-sensitized erythrocytes to COS-7 cells transfected with the plasmid. These observations indicate that the FcR bound IgG2 specifically and was not a receptor for IgA. Thus, this receptor, which we have named bovine Fc gamma 2R, represents a novel class of mammalian Fc gamma R, the evolution of which is likely to have been influenced by the truncated hinge of the bovine IgG2 molecule. Analysis of genetic divergence of the genes encoding bovine and human Fc gamma R and Fc alpha R indicated that the novel bovine gene and the human Fc alpha R gene probably evolved from a common ancestor, which is not shared by other Fc gamma R.
The cytokines IL-4, IFN-gamma, and IL-12 regulate the development of subsets of memory effector helper T cells in vitro.
Authors: Bradley L M , Yoshimoto K , Swain S L
Published: December 2022

We analyzed the development of cytokine-producing effector T cells from resting CD4 memory cells. Previously we showed that such memory effectors are induced in vivo upon re-exposure to Ag. Here we demonstrate that effectors arise in vitro when memory CD4 cells are restimulated with Ag in the presence cytokines. Resting splenic CD4 cells from KLH-primed mice that were depleted of naive cells by adult thymectomy and were exclusively of memory phenotype initially secreted high titers of IL-2 and low levels of IL-4 and IFN-gamma in response to Ag. When memory CD4 cells were restimulated for 3 to 4 days in cultures containing rIL-2 and Ab to block endogenous IFN-gamma and IL-4 secretion, Th0-like effectors that produced greatly increased levels of IL-2, IL-4, and IFN-gamma developed. rIL-4 together with rIL-2 and anti-IFN-gamma induced Th2-like cells that secreted primarily IL-4. In contrast, Th1-like effectors that produced IL-2 and IFN-gamma developed in the presence of rIL-2 and anti-IL-4. Addition of rIFN-gamma further enhanced priming for IFN-gamma secretion. rIL-12 also induced effectors that produced high levels of IFN-gamma, but little IL-2. Thus, cytokines direct the development of effector subsets from memory CD4 cells. Our results suggest that memory and naive CD4 cells undergo parallel development following Ag stimulation, initially secreting predominantly IL-2 and differentiating in response to IL-4, IFN-gamma, and IL-12 into polarized effector subsets.
Glucocorticoid-mediated regulation of protein phosphorylation in primary human T cells. Evidence for induction of phosphatase activity.
Authors: Paliogianni F , Hama N , Balow J E , Valentine M A , Boumpas D T
Published: December 2022

Glucocorticoid hormones (GC) have profound effects on the development and homeostasis of the immune system. In this communication we present evidence that GC regulate Ca(2+)-mediated pathways of T cell activation by a mechanism that involves abrogation of the autophosphorylation of the multifunctional Ca2+/calmodulin kinase (CaM kinase II) and induction of protein phosphatase activity. Primary human T cells were stimulated with the combination of ionomycin and phorbol ester in the presence or absence of dexamethasone (Dex) (10(-6)-10(-12) M). Stimulation of T cells resulted in a rapid activation of CaM kinase II and protein kinase C (PKC) activity as determined by the phosphorylation of synthetic peptide substrates recognized by these enzymes. Dex inhibited the activity of CaM kinase II but not PKC activity in a dose-dependent fashion (minimum effective dose 10(-10) M). Stimulation of 32P-labeled T cells induced a rapid increase in the phosphorylation level of CaM kinase II which was inhibited by Dex. The inhibitory effect of Dex on this enzyme was fully reversed in the presence of the phosphatase inhibitor okadaic acid (250 nM) or RU 486, a glucocorticoid antagonist. These results suggest that GC inhibit the activation of CaM kinase during T cell activation through a mechanism that involves both the GC receptor and protein phosphatases 2A and/or 1. Inhibition of protein phosphorylation through the induction of protein phosphatase activity may represent a novel mechanism for the diverse effects of GC on eukaryotic cells.
TCR/CD3 complex-mediated signal transduction pathway in T cells and T cell lines from patients with systemic lupus erythematosus.
Authors: Vassilopoulos D , Kovacs B , Tsokos G C
Published: December 2022

We studied the TCR/CD3 complex-mediated signal transduction pathway in freshly isolated T cells and T cell lines from patients with systemic lupus erythematosus (SLE). The peak and 5-min anti-CD3 mAb-mediated free intracytoplasmic Ca2+ concentration ([Ca2+]i) increase was statistically significant higher in fresh T cells from SLE patients than in control T cells. Increased CD3-mediated [Ca2+]i responses were observed in T cells from patients with SLE but not in T cells from other rheumatic diseases. Furthermore, significantly increased CD3-mediated [Ca2+]i responses were observed in T cell lines from SLE patients but not from controls. Although the [Ca2+]i response did not correlate with the global SLE disease activity or individual clinical manifestations, it was significantly higher in the group of patients who were not on treatment. Both CD4+ and CD8+ T cell subsets from peripheral blood cells and T cell lines displayed higher CD3-mediated [Ca2+]i responses than their normal counterparts. The peak of the response occurred earlier in the patient than in the normal group. The amount of Ca2+ that was released from the intracellular stores was higher in lupus than control T cells. The TCR/CD3-induced production of inositol phosphate metabolites in SLE cells was comparable with controls. The sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin-induced [Ca2+]i response was similar in both SLE and normal T cells. Our experiments demonstrate for the first time a definite abnormality in the early steps of the TCR/CD3-mediated signal transduction pathway in T cells from SLE patients that involves increased release of Ca2+ from intracellular stores.
A novel chemokine, macrophage inflammatory protein-related protein-2, inhibits colony formation of bone marrow myeloid progenitors.
Authors: Youn B S , Jang I K , Broxmeyer H E , Cooper S , Jenkins N A , Gilbert D J , Copeland N G , Elick T A , Fraser M J , Kwon B S
Published: December 2022

A new member of the beta-chemokine family, macrophage inflammatory protein (MIP)-related protein-2 (MRP-2) was isolated from a murine macrophage cell line, RAW 264.7. MRP-2 is composed of 122 amino acids of which the first 21 residues constitute a putative signal sequence. The putative mature protein is composed of 101 amino acids with a molecular weight of 11,600. MRP-2 is structurally similar to MIP-related protein-1 (MRP-1) (C10) and MIP-1 alpha. MRP-2 shows a 50.8% sequence identity at the protein level to MRP-1 and 46.3% identity to MIP-1 alpha. MRP-2 detects approximately 1.3 kilobase mRNA from monocyte and macrophage cell lines but does not detect the mRNA from T and B cells. The MRP-2 gene termed Scya9 was mapped to the central region of mouse chromosome 11 near the Scya1 and Scya2 genes, which are also members of the beta-chemokine superfamily. The Scya gene cluster was located between neurofibromatosis type 1 (Nf1) and myeloperoxidase (Mpo). rMRP-2 significantly suppressed colony formation by murine and human bone marrow granulocyte-macrophage (CFU-granulocyte-macrophage), erythroid (burst-forming unit-E), and multipotential (CFU-granulocyte-erythroid-macrophage-megakaryocyte) progenitor cells stimulated by combinations of growth factors.
Incomplete T cell receptor V beta allelic exclusion and dual V beta-expressing cells.
Authors: Balomenos D , Balderas R S , Mulvany K P , Kaye J , Kono D H , Theofilopoulos A N
Published: December 2022

Recent studies have documented incomplete TCR V alpha-chain allelic exclusion and dual V alpha-bearing T cells. Herein, we show that V beta allelic exclusion is also incomplete, since a significant proportion of peripheral T cells express dual V beta in both TCR transgenic and normal mice. Studies in TCR transgenic mice indicated that although a small proportion of T cells escaped allelic exclusion in the thymus, dual V beta-expressing cells expanded dramatically in the periphery with age, and such expanded cells had an activated phenotype. Although not as pronounced, age-related increases in dual V beta-bearing cells were also observed in normal mice. These findings may have important implications for TCR selection and specificity, age-related repertoire changes, and autoimmune disease pathogenesis.
Human follicular dendritic cells enhance cytokine-dependent growth and differentiation of CD40-activated B cells.
Authors: Grouard G , de Bouteiller O , Banchereau J , Liu Y J
Published: December 2022

Germinal centers constitute microanatomic subunits within secondary follicles where B cells undergo somatic mutations, isotype switch and affinity selection. This allows the generation of memory B cells and plasma cells, whose Igs bind to the eliciting Ag with a high affinity. T cells and follicular dendritic cells (FDCs) are thought to play key roles in the germinal center reaction. To study effects of FDCs on B cell growth and differentiation, we have isolated FDC-lymphocyte clusters from human tonsils by enzymatic digestion and centrifugation of the resulting cell suspension through BSA gradient. Irradiated FDC-lymphocyte clusters induced moderate proliferation of autologous B cells. IL-2 was the only cytokine able to enhance B cell proliferation cocultured with FDCs. When B cells were activated by soluble anti-CD40 Ab with or without IL-2, IL-3, IL-4, IL-10 or IL-13, addition of FDCs increased B cell proliferation. In the presence of FDCs, maximal B cell proliferation was observed in anti-CD40 stimulated cultures supplemented with either IL-4 + IL-10 or IL-2 + IL-10. Cultures performed in the presence of IL-2 and IL-10 resulted in high levels of Ig production in the presence of FDCs. In conclusion, the present study demonstrates that freshly isolated human FDCs can enhance the growth and differentiation of CD40-activated B cells.
Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28.
Authors: Hurtado J C , Kim S H , Pollok K E , Lee Z H , Kwon B S
Published: December 2022

The expression of the murine T cell Ag 4-1BB, a member of the TNF-R family, is induced by T cell activation. Previously, we and others had shown that signaling through 4-1BB enhanced proliferative T cell responses. To investigate a potential role for the interaction of 4-1BB with its ligand (4-1BBL) in T cell activation, we studied the ability of a soluble chimera of 4-1BB (4-1BBFc) to interfere with proliferative responses and cytokine production in models of activation dependent in intercellular interactions. The potential blocking effect of 4-1BBFc was compared with that of the chimeric molecule CTLA-4Ig, a reagent known to interfere with the interaction of CD28 (and/or CTLA-4) with B7 costimulatory receptors. In this study, we report that 4-1BBFc partially blocked both the activation of unfractionated splenocytes triggered by soluble anti-CD3 (anti-CD3s), and the more physiologically relevant responses to alloantigen. In addition, we show that both chimeric molecules partially blocked proliferative responses and IL-2 secretion by highly purified resting T cells activated with anti-CD3s in the presence of fixed accessory cells that express B7 receptors and 4-1BBL. Furthermore, in this model system, the blocking capacity of 4-1BBFc and CTLA-4Ig appears to correlate with the relative expression of their respective cognate receptors (4-1BBL and B7) on the accessory cell. Simultaneous addition of both blocking reagents produced an additive effect in the model systems studied.
Rapamycin inhibits ribosomal protein synthesis and induces G1 prolongation in mitogen-activated T lymphocytes.
Authors: Terada N , Takase K , Papst P , Nairn A C , Gelfand E W
Published: December 2022

We investigated the effects of rapamycin (RAP) on cell cycle progression and protein synthesis in mitogen-activated primary T lymphocytes. Stimulation of resting human T lymphocytes with phorbol ester and calcium ionophore rendered cells capable of initiating DNA synthesis within 30 h; roughly 60% of the cells entered the first G2/M phase of the cell cycle within 96 h. Addition of RAP delayed the entry into S phase by 9 h, although a similar percentage (approximately 50%) of cells entered the first G2/M phase and proliferated. On this basis, we concluded that RAP primarily induced a G1 prolongation without blocking cell cycle progression. Addition of the co-mitogens to resting T lymphocytes up-regulated the translation of ribosomal protein mRNA concurrent with activation of p70s6k. RAP inhibited this translational up-regulation of ribosomal protein mRNA as well as the activation of p70s6k without affecting translation of nonribosomal protein mRNA. RAP also prevented the synthesis and accumulation of ribosomal proteins. Further, this failure to increase ribosomal proteins, which probably reflects the failure to increase numbers of ribosomes, resulted in suppression of the synthesis of total cellular protein and a delay in the escalation of cell size. RAP-treated cells eventually initiated DNA synthesis when cell size became equivalent to that of the control cells entering S phase of the cell cycle. Thus, inhibition of protein synthesis caused by the primary inhibition of ribosomal protein mRNA translation probably explains the effect of RAP on cell cycle progression of mitogen-activated resting T lymphocytes.
Down-regulation of the afferent phase of T cell-mediated pulmonary inflammation and immunity by a high melanin-producing strain of Cryptococcus neoformans.
Authors: Huffnagle G B , Chen G H , Curtis J L , McDonald R A , Strieter R M , Toews G B
Published: December 2022

The interaction(s) between cryptococcal virulence factors and leukocytes involved in generating protective cell-mediated immunity is not well defined. Intratracheal inoculation of Cryptococcus neoformans strain 52 induced a vigorous T cell-mediated pulmonary inflammatory response that controlled the growth of the organism. In contrast, strain 145 induced a pulmonary inflammatory response that was delayed in onset, slower to develop, and ineffective in controlling the infection. In addition, the expansion of cryptococcus-specific lymphocytes in the pulmonary lymph nodes and titer of specific Abs in the serum of strain 145-infected mice were both diminished markedly. Of the known cryptococcal virulence factors, these two strains differed only in melanin production (52-low and 145-high). Heat-killed strain 145 cryptococci (HKC-145) that had been rendered melanin-negative induced TNF-alpha production by alveolar macrophages in vitro and stimulated vigorous cryptococcus-specific lymphoproliferation. In contrast, high melanin-containing HKC-145 inhibited TNF-alpha production and lymphoproliferation. In vivo, mice infected with melanin low strain 52, but not melanin high strain 145, had elevated levels of TNF-alpha in the bronchoalveolar lavage fluid. Mice co-infected with strains 145 and 52 generated a pulmonary inflammatory response resulting in increased long-term survival. Taken together, these studies demonstrate that melanin does not protect cryptococci from being eliminated in vivo by recruited, activated effector cells; but melanin can inhibit the recognition of the organism by host defenses, thereby down-regulating the afferent phase of T cell-mediated immunity, e.g., TNF-alpha production and lymphoproliferation.
Diversity of Ig light chain clusters in the sandbar shark (Carcharhinus plumbeus).
Authors: Hohman V S , Schluter S F , Marchalonis J J
Published: December 2022

The genes encoding Ig chains in elasmobranchs are arranged in clusters or cassettes, an organizational pattern dramatically different from the mammalian translocon gene arrangement. Cluster gene arrangements, which have now been found in non-elasmobranchs as well, pose interesting dilemmas for understanding the mechanisms of Ig gene expression and regulation in terms of allelic exclusion and clonal selection. We have sequenced five lambda genomic clones encoding complete sandbar shark (Carcharhinus plumbeus) lambda L chain gene loci. While the coding regions among all five clones are highly homologous, the noncoding regions have significant differences that allowed us to identify two types of lambda L chain clusters. The noncoding regions are < 60% identical between groups, while the three clones belonging to the first group share > 95% identity in their noncoding regions. The second group is more diverse and may be comprised of several related subgroups. The two clones in this group share approximately 85% identity in the noncoding regions. Variations in the promoter region, including octamer and TATA box orientation and position, are identified between the two groups and may have implications for the molecular regulation of Ab production. Our results show the sandbar shark lambda L chain family to be a complex and diverse system.
Differential transcription efficiency of two Ig VH promoters in vitro.
Authors: Buchanan K L , Hodgetts S I , Byrnes J , Webb C F
Published: December 2022

Each Ig variable region gene segment is transcribed from its own unique promoter. While all of these promoters share common consensus elements that contribute to the B cell-specific expression of these genes, the DNA sequence of each promoter is distinct. In this study, we have directly compared the transcription efficiencies of two murine heavy chain (VH) promoters in a murine B cell in vitro transcription system. We found that the promoters differed in both transcription efficiency and the ability to bind specific protein complexes. While some of the transcription differences may be attributed to differences in basal promoter elements, the spacing between the octamer and the heptamer consensus elements was found to be important. Others have reported a direct correlation between transcription efficiency and the probability that individual variable region gene segments will rearrange. Our studies may be of direct importance to those interested in identifying B cell-specific transcription factors and may ultimately help to explain differences in the expression of some VH gene segments.
Macrophage activation by culture in an anoxic environment.
Authors: Albina J E , Henry W L , Mastrofrancesco B , Martin B A , Reichner J S
Published: December 2022

The extracellular amino acid composition of experimental wounds in rats during peak macrophage infiltration bears the imprint of the elevated arginase activity present in wound fluid: L-arginine is found in this space in concentrations markedly lower, and L-ornithine in concentrations markedly higher, than those that are detectable in plasma. No evidence, in the form of L-citrulline or NO2- accumulation, can be found at this time for nitric oxide synthase (NOS) activity. Wound-derived macrophages, however, metabolize L-arginine through both arginase and NOS in culture. Given the requirements of NOS for O2 and the reduced O2 tension in wounds, experiments were performed to determine the role of O2 availability on the metabolism of L-arginine by wound-derived macrophages. Results demonstrated that, beyond inhibiting NOS, culture of wound-derived macrophages in an anoxic environment provided an activation signal, markedly increasing total L-arginine metabolism, arginase activity, NOS protein content, and the release of TNF-alpha and IL-6. Neither resident nor Corynebacterium parvum-elicited peritoneal macrophages responded to anoxic culture with increases in L-arginine utilization, arginase activity or, in the case of resident macrophages, in NOS protein content. The enhanced TNF-alpha and IL-6 release induced by anoxia in wound-derived macrophages was also found in resident peritoneal macrophages. Anoxia appears to act, then, as an inducer of activation-associated traits in macrophages obtained from different sites.
Differential regulation of transforming growth factor beta-1 gene expression by glucocorticoids in human T and glial cells.
Authors: Batuman O A , Ferrero A , Cupp C , Jimenez S A , Khalili K
Published: December 2022

The inflammatory process in the brain requires bidirectional interaction of the immune and nervous systems. Evidently, astrocytic glial cells play an important role in facilitating this communication by releasing immunomodulators and cytokines. Expression of transforming growth factor beta-1 (TGF beta-1), a potent inhibitor of T cell function and glial cell proliferation, is highly regulated in T cells and is believed to be an important component in the molecular interaction between the immune and nervous systems. Comparative analysis of TGF beta-1 gene expression in human T and glial cells by Northern hybridization and S1 nuclease protection assay showed that dexamethasone (DM) caused a significant decrease in the basal and PMA-induced levels of TGF beta-1 mRNA in glial cells but not in T cells. This reduction correlated with a lower level of TGF beta-1 protein production. Transient transfection assay using deletion constructs of the 5' TGF beta-1 gene promoter-containing sequences between -453 and +11 bp identified a region spanning -160 to -60 bp as a potential sequence responsive to regulation by DM in T cells, whereas in glial cells, the overall transcriptional activity of the 5' TGF beta-1 promoter was reduced after DM treatment, but promoter activity within each construct remained constant in response to DM. Thus, a DM-responsive region could not be identified within the TGF beta-1 promoter in glial cells. These findings suggest that TGF beta-1 gene expression is differentially regulated by distinct regulatory elements in T and glial cells, and that extracellular stimulators, including glucocorticoids, can utilize the TGF beta-1-regulatory pathway to affect the functions of neural and immune cells.
Ultraviolet B radiation up-regulates the expression of IL-15 in human skin.
Authors: Mohamadzadeh M , Takashima A , Dougherty I , Knop J , Bergstresser P R , Cruz P D
Published: December 2022

Ultraviolet B (UVB) radiation is a potent modulator of skin-related immune responses, particularly those involving the synthesis and the secretion of cytokines. The discovery of a new T cell mitogen, IL-15, prompted use to investigate its expression in skin and to examine the effects of UVB radiation on such expression. RNA from unirradiated and UVB-irradiated epidermal and dermal sheets derived from human foreskin as well as from unirradiated and UVB-irradiated skin cell populations were assayed for IL-15 expression by semiquantitative RT-PCR. Constitutive levels of IL-15 mRNA were detected in dermal sheets, but not in epidermal sheets. Following UVB treatment, IL-15 mRNA was induced in epidermal sheets and enhanced in dermal sheets. UVB-inducible epidermal expression of IL-15 mRNA was traced to HLA-DR- cells (presumably keratinocytes) and not to HLA-DR+ cells (Langerhans cells). Cultured keratinocytes and dermal fibroblasts displayed basal levels of IL-15 mRNA that were also up-regulated following UVB exposure. Immunoblot analysis revealed secretion of IL-15 protein by keratinocytes that was enhanced following UVB treatment. These results constitute the first report of IL-15 mRNA expression and protein production in human skin. In addition to expanding the known influence of UVB radiation on the capacity of keratinocytes and dermal fibroblasts to express immunomodulatory cytokines, these findings suggest a new mechanism by which UVB can promote Ag-independent T cell responses via elaboration of IL-15.
T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex. Effects of conservative amino acid substitutions at anchor position 1 of influenza matrix protein19-31.
Authors: Wu S , Gorski J , Eckels D D , Newton-Nash D K
Published: December 2022

T cells recognize peptide fragments of Ags bound to MHC-encoded molecules. Pockets in the MHC peptide-binding groove accommodate a limited set of amino acid side chains present at anchor positions in peptide; however, the functional significance of accommodation of different side chains at an anchor position in peptide is not clear. A panel of T cell clones was evaluated to test the effect of conservative amino acid substitution at a primary peptide anchor position. Results of T cell stimulation studies were correlated with two well studied characteristics of the peptide/MHC complex, which are the affinity of peptide binding to MHC and the stability of the resulting complex upon PAGE in the presence of SDS. We found that formation of a functional complex required neither high affinity peptide binding nor SDS stability. Furthermore, T cell clones differed in their ability to recognize individual peptide variants, suggesting that some structural aspect of the peptide/MHC complex is influenced by interactions between peptide anchor residues and MHC pockets.
Chemotactic activity of mycobacterial lipoarabinomannans for human blood T lymphocytes in vitro.
Authors: Berman J S , Blumenthal R L , Kornfeld H , Cook J A , Cruikshank W W , Vermeulen M W , Chatterjee D , Belisle J T , Fenton M J
Published: December 2022

A crucial early event in tuberculosis is the ingestion of Mycobacterium tuberculosis (Mtb) by alveolar macrophages. Chemotactic factors released by infected macrophages are likely to initiate a granulomatous response, a key feature of host resistance to tuberculosis. To date, the role of mycobacterial products in regulating the granulomatous response has not been clearly defined. Here we report that the mycobacterial cell wall glycophospholipid lipoarabinomannan (LAM) could specifically induce human peripheral blood T cell chemotaxis in vitro. Both terminally mannosylated LAM isolated from Mtb and LAM lacking the terminal mannosyl units isolated from an avirulent mycobacterium could induce T cell migration in the absence of serum. In contrast, terminally mannosylated LAM isolated from Mycobacterium bovis BCG failed to induce T cell chemotaxis. These observations represent the first report that LAM is capable of directly inducing biologic responses in human T cells. Flow cytometry analysis revealed that CD4+, CD8+, and CD45RO+ lymphocytes were present in the migrating cell populations at ratios similar to those found in nonmigrating cells. The chemotactic response was found to require new protein synthesis, and could be blocked by inhibitors of protein tyrosine kinases at concentrations that did not affect random migration. Acyl groups at the reducing terminus of LAM appear to be required for the chemotactic activity of this mycobacterial glycolipid. Lastly, culture supernatants from human alveolar macrophages infected in vitro with a virulent strain of Mtb could induce T cell migration. Much of the migratory activity present in these supernatants could be blocked using a mAb against LAM, suggesting that LAM is one of the chemotactic factors released by Mtb-infected alveolar macrophages.
Down-regulation by IL-4 and up-regulation by IFN-gamma of mast cell induction from mouse spleen cells.
Authors: Hu Z Q , Zenda N , Shimamura T
Published: December 2022

While investigating an involvement of other factors aside from endogenous IL-3 and prostaglandin E (PGE) in mast cell induction from mouse splenocytes, we found that the mast cell induction was inversely proportional to IL-4 levels and tended to directly proportionate IFN-gamma levels in the supernatants recovered on days 2 and 4. Thereafter, we examined the effects of rIFN-gamma, rIL-4, and rIL-10 on mast cell induction. IFN-gamma and IL-10 dose-dependently induced mast cells. Time course study showed an importance of adding rIFN-gamma into the cultures at the early phase (on days 0 and 2 of a 12-day culture). When endogenous IFN-gamma at the early phase was neutralized by anti-IFN-gamma Ab, all stimulants, including rIFN-gamma, rIL-10, and PGE1, failed to induce mast cells. On the contrary, rIL-4 dose-dependently suppressed the mast cell induction by rIFN-gamma, rIL-10, LPS, PGE, and dibutyryl cAMP. The inhibitory effect of IL-4 was observed when IL-4 was added into the cultures at the early phase, but not after day 4. The suppressive action of IL-4 was diminished completely by the addition of neutralizing anti-IL-4 Ab. IL-12, a key regulator of IFN-gamma and IL-4 production, also induced mast cells. These results revealed, for the first time, that IFN-gamma is crucial for the survival and/or differentiation of splenic mast cell precursors and that IL-4 is a key inhibitor for the precursors, although IFN-gamma is not a mast cell growth factor and IL-4 is a growth factor for immature and mature mast cells.