bluuebunny/crossref_metadata_2025 huggingface dataset:
Adoptively transferred CD4+ lymphocytes from CD8 -/- mice are sufficient to mediate the rejection of MHC class II or class I disparate skin grafts.
Authors: Dalloul A H , Chmouzis E , Ngo K , Fung-Leung W P
Published: December 2022

Recent studies revealed that CD4+ cells initiate allograft rejection through direct recognition of allogeneic MHC class II Ags and indirect recognition of MHC peptides processed by self APCs. Both pathways were shown to help CD8+ cells that eventually lysed allogeneic MHC class I-presenting targets. There was little evidence, however, that CD4+ cells are sufficient for graft rejection. We studied skin graft rejection by CD8-deficient (CD8 -/-) mice. We showed that BALB/cJ(H-2d) CD8 -/- mice could reject allogeneic skin from C57BL/6J(H-2b) mice deficient in MHC class I or in MHC class II Ags. To understand the role of CD4+ cells in this process, we isolated them from CD8 -/- mice and transferred them to BALB/cJ nude mice that had been grafted with allogeneic skin (H-2b) from animals deficient in MHC class I or MHC class II. Nude mice injected with CD4+ cells rejected MHC class II and, albeit more slowly, MHC class I disparate skins. We showed in vitro evidence that CD4+ cells were not cytotoxic toward MHC class I or MHC class II disparate targets and that they recognized MHC class I allogeneic targets through indirect recognition. CD4+ cells produced Th1 cytokines, but not IL-4, following stimulation with allogeneic cells. Furthermore, intragraft TNF-alpha was elevated in skin grafted onto nude mice reconstituted with CD4+ cells compared with nonreconstituted mice. This suggests that MHC class II- or MHC class I-guided CD4+ cells alone are sufficient to induce rejection by the generation of cytokine-induced lesions.
pH dependence of MHC class I-restricted peptide presentation.
Authors: Stryhn A , Pedersen L O , Romme T , Olsen A C , Nissen M H , Thorpe C J , Buus S
Published: December 2022

The function of MHC class I molecules is to bind and present antigenic peptides to cytotoxic T cells. Here, we report that class I-restricted peptide presentation is strongly pH dependent. The presentation of some peptides was enhanced at acidic pH, whereas the presentation of others was inhibited. Biochemical peptide-MHC class I binding assays demonstrated that peptide-MHC class I complexes are more stable at neutral pH than at acidic pH. We suggest that acid-dependent peptide dissociation can generate empty class I molecules and that the resulting binding potential can be exploited by a subset of peptide-MHC class I combinations, in some cases leading to considerable peptide exchange. We further speculate that the relative instability of peptide-class I complexes under acidic conditions may affect the outcome of class I-restricted Ag presentation, as less stably associated peptides may dissociate from class I during passage of the acidic trans-Golgi network, and therefore may not be presented. Finally, our results may in part explain how endocytosed proteins can be presented by MHC class I molecules to cytotoxic T cells.
Rat RT6.2 and mouse Rt6 locus 1 are NAD+: arginine ADP ribosyltransferases with auto-ADP ribosylation activity.
Authors: Rigby M R , Bortell R , Stevens L A , Moss J , Kanaitsuka T , Shigeta H , Mordes J P , Greiner D L , Rossini A A
Published: December 2022

RT6 is a glycosylphosphatidylinositol-linked protein found on the surface of mature rat T lymphocytes. Cells that express RT6 have an immunoregulatory function and modulate the expression of autoimmune diabetes mellitus in the BioBreeding rat. A homologue of the rat RT6 gene, designated Rt6, has been identified in the mouse, but expression of mouse Rt6 protein has not been documented. Rat RT6 is known to be a nicotinamide adenine dinucleotide (NAD+) glycohydrolase. We now report that rat RT6.2 and recombinant mouse Rt6 locus 1 proteins possess auto-ADP ribosylation activity. In addition, mouse Rt6 but not rat RT6, catalyzes the ADP ribosylation of exogenous acceptors such as histones. The ADP-ribosyl-protein bonds in auto-ADP-ribosylated rat RT6.2, auto-ADP-ribosylated mouse Rt6, and ADP-ribosylhistone synthesized by Rt6 were stable to HgCl2 and HCl, but labile to NH2OH, consistent with ADP ribosylarginine linkages. To determine if these enzymatic activities could affect the function of rat T cells, the effect of substrate availability on lymphocyte proliferation was examined. An inverse correlation was observed between NAD+ concentration in the medium and the ability of rat T cells to respond to anti-CD3, ConA, and PMA plus ionomycin. The data suggest that lymphocyte surface ADP ribosyltransferases could be involved in signaling and immunoregulatory processes.
Presentation of the protective parasite antigen LACK by Leishmania-infected macrophages.
Authors: Prina E , Lang T , Glaichenhaus N , Antoine J C
Published: December 2022

Macrophages are apparently the only cells that in vivo allow the growth of the intracellular pathogen Leishmania. They are thus generally considered as likely candidates for the presentation of parasite Ag to CD4+ T lymphocytes known to be involved in protective and counterprotective immune responses. In the present study, we examined whether mouse macrophages infected with Leishmania were capable of stimulating T cell hybrids and a T cell clone reacting with the previously identified protective Ag LACK (Leishmania homologue of receptors for Activated C Kinase). This parasite protein is expressed in both promastigote and amastigote stages of Leishmania. We found that IFN-gamma-treated macrophages recently infected with live Leishmania promastigotes were fully competent to activate LACK-reactive T cells. However, at later times of infection, permissive macrophages infected with promastigotes were no longer able to present LACK, in spite of the presence of numerous intracellular parasites. This punctual presentation of LACK was apparently linked with the destruction, at least partial, of the intracellular parasites. In contrast, macrophages infected with live Leishmania amastigotes were always unable to stimulate the LACK-specific T cells. Amastigote-infected macrophages could, however, reactivate the T cells if LACK-delta(1), a recombinant form of LACK, was added as an exogenous protein in the culture medium. Similar results were obtained with all combinations tested involving macrophages from various origins, different activating cytokines (IFN-gamma, granulocyte-macrophage CSF, IL-4), several Leishmania species (L. amazonensis, L. major, L. donovani), and 15 different LACK-reactive T cell hybrids and clones. From these data, it is tempting to propose that the differentiation of promastigotes into amastigotes, which leads to a better survival of the parasites within macrophages, also allows them to go unnoticed by the immune system.
TNF-alpha inactivation of collagen receptors: implications for fibroblast function and fibrosis.
Authors: Chou D H , Lee W , McCulloch C A
Published: December 2022

TNF-alpha inhibits collagen synthesis and at high concentrations stimulates collagenase synthesis in fibroblasts. As fluid from chronic inflammatory lesions contains significant levels of TNF-alpha, it is puzzling why these lesions exhibit dense accumulations of disorganized collagen. In this study we determined if low concentrations of TNF-alpha may inhibit the collagen phagocytic pathway in fibroblasts and thereby contribute to fibrosis. Collagen phagocytosis was measured by flow cytometric assessment of internalized, fluorescent collagen beads. TNF-alpha induced a dose-dependent reduction (optimal dose: 40% at 10 ng/ml; p<0.001) in the proportion of phagocytic cells and a twofold reduction of the number of internalized beads per cell but did not alter the total number of vital cells. TNF-alpha reduced by twofold the degradation of collagen films. Fluid flow shear-force assays demonstrated that TNF-alpha caused a 72% reduction (p < 0.05) in strong binding of collagen-coated beads to cells indicating that TNF-alpha may inactivate receptors and inhibit collagen binding. Furthermore, TNF-alpha reduced cell contact area with collagen substrates by threefold and inhibited reattachment of trypsinized cells by fourfold. Although levels of collagen receptors were increased by TNF-alpha (53% increase in alpha(2) (beta)1 integrin; p<0.001, 20% increase in alpha(1)beta(1)), the receptors were inactivated by the cytokine. The reduced phagocytic activity of TNF-alpha-treated cells was restored to control levels by treatment with the integrin-activating Abs A16G6 and JBS2. TNF-alpha inhibited focal adhesion formation and phosphotyrosine staining in focal adhesions. These effects were replicated by the tyrosine kinase inhibitor genistein, which also inhibited phagocytosis. Collectively, these data indicate that TNF-alpha inhibits adherence and phagocytosis of collagen. These effects are mediated by a reduction in the strength of alpha(2)beta(1) integrin binding to collagen, possibly through tyrosine kinases in focal adhesions. At low concentrations of TNF-alpha (10 ng/ml) that are found in the periphery of chronic inflammatory lesions, we suggest that inhibition of the collagen phagocytic pathway may contribute to fibrosis.
Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages.
Authors: Sanghera J S , Weinstein S L , Aluwalia M , Girn J , Pelech S L
Published: December 2022

Bacterial LPS stimulation of murine macrophages leads to increased tyrosine phosphorylation and activation of the 42- and 44-kDa mitogen-activated protein kinases (MAPK) and the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) and p38, related to the high osmolarity glycerol protein kinase in Saccharomyces cerevisiae (HOG1). LPS caused a rapid increase (10 min) in phosphotransferase activity toward myelin basic protein (MBP), a polypeptide that encompassed the first 169 residues of c-Jun fused to gluthathione S-transferase (GST-c-Jun (1-169)) and 27-kDa heat shock protein (hsp27). MonoQ fractionation of cell extracts resolved phosphotransferase activity peaks toward MBP, GST-c-Jun (1-169), and hsp27, which contained MAPK, SAPK/JNK, and MAPKAPK2, respectively, as indicated by immunoblotting data. In RAW 264.7 macrophages, LPS stimulation of MAPKAPK2, a substrate of p38 HOG1 and MAPK, appeared to occur predominantly via p38 HOG1 and not the MAPK. PMA, which activated the MAPK as potently as LPS, did not strongly activate MAPKAPK2, as assessed by hsp27 phosphorylation. Consistent with p38 HOG1-mediating LPS activation of MAPKAPK2, treatment with LPS, but not PMA, increased the tyrosine phosphorylation of p38 HOG1, a modification known to elevate the enzymatic capacity of this kinase. In LPS-treated cells, the activity of SAPK/JNK was increased 5- to 10-fold, as measured by precipitating SAPK/JNK with Abs or immobilized GST-c-Jun and performing an in vitro kinase assay. In addition, the kinases thought to be upstream of SAPK/JNK, SAPK/ERK kinase 1 (SEK1), and MAPK/ERK kinase kinase 1 (MEKK1), were activated following LPS, but not PMA, exposure (5-fold and 2.5-fold, respectively.
Thymic-independent T cell regeneration occurs via antigen-driven expansion of peripheral T cells resulting in a repertoire that is limited in diversity and prone to skewing.
Authors: Mackall C L , Bare C V , Granger L A , Sharrow S O , Titus J A , Gress R E
Published: December 2022

Thymic regenerative capacity in humans decreases with age, suggesting that thymic-independent pathways of T cell regeneration may predominate during adulthood. Using a murine bone marrow transplantation model, we present evidence that thymic-independent T cell regeneration occurs primarily via expansion of peripheral T cells and is Ag driven since significant expansion of CD4+ or CD8+ transgenic (Tg+)/TCR-bearing cells occurs only in the presence of Ag specific for the TCR. Such expansion resulted in skewing of the regenerated repertoire with 40 to 65% of the regenerated CD4+ or CD8+ T cells expressing the Tg+/TCR in thymectomized hosts after bone marrow transplantation. In experiments in which nontransgenic population are used as T cell inocula, we noted decreased CD4 expansion when Class II MHC was blocked by mAb treatment in vivo, an CD8 expansion failed to occur in Class I MHC-deficient hosts providing evidence that T cell regeneration in thymic-deficient hosts largely occurs via TCR-MHC-mediated selection of peripheral T cell populations. This process results in a T cell repertoire comprised exclusively of T cells recently activated by the antigenic milieu of the host, with negligible numbers of residual "naive" cells bearing TCRs for Ags absent at the time of expansion. These findings have important implications for approached to enhance T cell regeneration in humans and provide evidence that vaccine strategies could skew the T cell repertoire toward a specific antigenic target if administered to thymic-deficient hosts during immune reconstitution.
CD95 engagement releases calcium from intracellular stores of long term activated, apoptosis-prone gammadelta T cells.
Authors: Rovere P , Clementi E , Ferrarini M , Heltai S , Sciorati C , Sabbadini M G , Rugarli C , Manfredi A A
Published: December 2022

Engagement of the CD95 (Apo-1, Fas) membrane receptor is known to induce apoptosis in a variety of sensitive cells, even in the absence of extracellular Ca2+. The signal transduction events implicated in this pathway are poorly understood. We have recently characterized normal human Vgamma9/Vdelta2+ T cell clones that maintain similar levels of CD95 membrane expression throughout the culture. Here we show that 3 wk of culture after in vitro restimulation are necessary for the cells both to die and to acquire the ability to mobilize intracellular Ca2+ upon CD95 ligation. Buffering of intracellular Ca2+ by accumulation of the chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N1,N-tetraacetic acid protects from CD95-triggered apoptosis, suggesting that the two phenomena are causally related. As intracellular Ca2+ release by inhibition of endoplasmic reticulum ATPases induces apoptosis in both recently and long term activated gamma delta cells, the molecular regulation of activation-dependent apoptosis is likely to involve events upstream of CD95-dependent Ca2+ release. The CD95-triggered increase in the intracellular Ca2+ concentration depends on depletion of the same intracellular Ca2+ stores mobilized by ligation of the TCR, and Ca2+ release does not depend on inositol 1,4,5-trisphosphate generation.
The MHC class I genes of the rhesus monkey. Different evolutionary histories of MHC class I and II genes in primates.
Authors: Boyson J E , Shufflebotham C , Cadavid L F , Urvater J A , Knapp L A , Hughes A L , Watkins D I
Published: December 2022

Homologues of the human HLA-A and -B MHC class I loci have been found in great apes and Old World primates suggesting that these two loci have existed for at least 30 million years. The C locus, however, shows some sequence similarity to the B locus and has been found only in gorillas, chimpanzees, and humans. To determine the age of the MHC class I C locus and to examine the evolution of the A and B loci we have cloned, sequenced, and in vitro translated 16 MHC class I cDNAs from two unrelated rhesus monkeys (Macaca mulatta) using both cDNA library screening and PCR amplification. Analyses of these sequences suggest that the C locus is not present in the rhesus monkey, indicating that this locus may be of recent origin in gorillas, chimpanzees, and humans. The rhesus monkey's complement of MHC class I genes includes the products of at least one expressed A locus and at least two expressed B loci, indicating that a duplication of the B locus has taken place in the lineage leading to these Old World primates. Comparison of rhesus monkey MHC class I cDNAs to their primate counterparts reveals fundamental differences between MHC class I and class II evolution in primates. Although MHC class II allelic lineages are shared between humans and Old World primates, no such trans-species sharing of allelic lineages is seen at the MHC class I loci.
Analysis of the CDR3 region of the rearranged IgH chain genes in patients with severe combined immunodeficiency and severe lymphopenia.
Authors: Minegishi Y , Akagi K , Nishikawa K , Okawa H , Yata J
Published: December 2022

Human B cell-negative severe combined immunodeficiency (B-SCID) is a primary immunodeficiency disease characterized by both T and B lymphocytopenia and agammaglobulinemia. Although lymphocytes of B-SCID patients express defective recombinase activity and rarely succeed in making Ag receptors, the molecular defect of the human B-SCID remains to be identified. To gain more insight into the human B-SCID defect and its effect on the Ab repertoire, we examined the Ig heavy (H) chain genes of the peripheral blood leaky B cells from three B-SCID patients. Although the number of B cells in their peripheral blood was very limited, we could obtain 41 productive VDJ recombinations from the 58 joints. The recombinations showed a grossly altered pattern characterized by short N nucleotides, short deleted nucleotides from 5' JH segments, immature JH and D segment utilization, absence of VDDJ recombination, and all but one JH segment equal to germline JH segments. Therefore, Ab repertoire of IgH chain gene in human B-SCID was limited because of restricted junctional and combinatorial diversity and few somatic mutations. Furthermore, unusually long P nucleotides were inserted in junctional sequences, which might indicate that resolution of hairpin is impaired in human B-SCID as in the murine SCID.
In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying viral CD8+ T cell epitopes.
Authors: Fayolle C , Sebo P , Ladant D , Ullmann A , Leclerc C
Published: December 2022

Exogenous Ags enter the endosomal pathway and are presented to CD4+ T cells in association with class II molecules whereas endogenously synthesized Ags, such as viral proteins, are presented to CD8+ T cells in association with MHC class I molecules. Therefore, most CTL activation strategies use live vectors although an alternative possibility could be to deliver the epitope into the cytosol by targeting it to an invasive nonreplicative vector. The adenylate cyclase toxin of Bordetella pertussis is able to invade a large number of eukaryotic cells and to deliver its catalytic domain to the cytosol of the cells. In the present study, we have tested the in vivo immunogenicity of recombinant adenylate cyclase toxins expressing CTL epitopes either from the nucleoprotein of lymphocytic choriomeningitis virus or from the V3 region of HIV-1 gp120. BALB/c mice immunized with these toxins developed high specific CTL responses that were shown to be mediated by class I-restricted CD8+ CTL. The induction of CTL responses by recombinant toxins did not require CD4+ T cells and the cytotoxic activity persisted 2 mo after immunization. The activation of CTL responses by the recombinant adenylate cyclase toxin required the full-length invasive activity of the toxin but did not depend upon its catalytic adenylate cyclase activity as demonstrated with a genetically inactivated recombinant toxin expressing the lymphocytic choriomeningitis virus epitope. This genetically detoxified invasive toxin represents, therefore, an attractive new vector for CTL activation.
Characterization of early IL-12, IFN-alphabeta, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection.
Authors: Orange J S , Biron C A
Published: December 2022

Murine cytomegalovirus (MCMV) infection of mice induces early cytokines. Although certain of these can directly inhibit viral replication, they also can promote defense by activating NK cells. MCMV induces IFN-alphabeta-dependent NK cell cytotoxicity and IL-12-dependent NK cell IFN-gamma production. Studies were initiated to define cytokine-mediated NK and T cell-independent antiviral defense and specific cytokine-elicited NK cell responses during MCMV infections. IFN-alphabeta, TNF, IL-12, and IFN-gamma were all shown to be induced 2 days after infection of immunocompetent mice. Infections of NK and T cell-deficient mice demonstrated that virus-induced IFN-alphabeta, TNF, and IL-12, but not IFN-gamma, were produced independently of these populations, and that IL-12 production occurred in the absence of detectable IFN-gamma. In vivo neutralization studies of IFN-alphabeta, TNF, and IL-12 showed that each of these factors had NK and T cell-independent antiviral functions, as well as specific effects on NK cell responses. Examination of NK cell cytotoxicity, blastogenesis, and IFN-gamma production demonstrated that: IL-12 was required for NK cell IFN-gamma production but not blastogenesis and cytotoxicity; IFN-alphabeta was necessary for NK cell blastogenesis and cytotoxicity but not IFN-gamma production; and TNF facilitated IFN-gamma production but inhibited NK cell cytotoxicity. This work defines the biologic consequences of early cytokine expression during viral infection.
Both N- and C-terminal regions of the bioactive N-terminal fragment of the neutrophil granule bactericidal/permeability-increasing protein are required for stability and function.
Authors: Capodici C , Weiss J
Published: December 2022

An N-terminal fragment (residues 1-199) of the 456-residue human bactericidal/permeability-increasing protein (BPI), isolated after limited proteolysis, exhibits antibacterial and LPS-neutralizing activities equal to or greater than those of holo-BPI. To assess minimal structural requirements for bioactivity, mutant species of BPI were expressed in vivo by transient transfection and in vitro by cellfree transcription/translation. BPI1-456 and BPI1-193 demonstrated the expected antibacterial and LPS-binding activities. Deletion of the N-terminal 12 residues did not diminish BPI function. However, further truncation either from the C-terminus to residue 169 (BPI1-169) or from the N-terminus (BPIdelta15-56) yielded in vitro products with little or no LPS-binding activity and in vivo products that could not be recovered from the culture medium or cellular acid extracts. The possible role of cysteine-175 (the three cysteines in human BPI are at residues 132, 135, and 175) in BPI stability/function was examined by substitution of Cys(175) with serine. Recovery of C175S BPI from extracellular medium was reduced 10-fold, and C175S BPI produced in vitro had little LPS-binding activity. Compared with wild-type holo-BPI and BPI1-193, BPI1-169, BPIdelta15-56, and C175S BPI showed increased susceptibility to cleavage by elastase in the region 1-193 (but not in the region 200-456), indicating conformational changes that may account for the loss of function. These findings suggest that the proteolytic N-terminal fragment of BPI corresponds closely to the minimum functional (antibacterial/anti-LPS) domain of BPI and that residues near both ends of this fragment are essential for structural stability and functional integrity.
Identification of surface molecules associated with physiologic activation of eosinophils. Application of whole-blood flow cytometry to eosinophils.
Authors: Mawhorter S D , Stephany D A , Ottesen E A , Nutman T B
Published: December 2022

Activation is central to the eosinophil's functional role as an immune responder cell. To evaluate such activation in cells freshly isolated from peripheral blood, a method for whole-blood immunostaining and flow cytometry-based eosinophil selection was developed. Simultaneous comparison of purified eosinophils and whole-blood cells revealed significant differences in the levels of expression of various surface molecules, which suggested that the purification process activated the eosinophils. Subsequent analyses were conducted with the whole-blood assay. When eosinophils from helminth-infected persons (n = 18) were compared with those from normal individuals (n = 10), the early activation marker CD69 was found to be significantly increased (geometric mean (GM) = 4.3 vs. 1.0%, p = 0.04). The granulocyte activation marker CD66 was also up-regulated on eosinphils from helminth patients (GM = 53.3 vs. 31.0%, p = 0.044), as was the tetraspan family molecule CD81 (TAPA-1; GM = 79.4 vs. 48.2%, p = 0.02). Conversely, in vivo CD23 (FcepsilonRII) expression on eosinophils was decreased in the presence of parasitic infection (GM = 0.9 vs. 5.7%, p = 0.02). Expression of the eosinophil surface molecules CD69, CD81, and CD23 was significantly enhanced after cytokine stimulation in vitro with IL-3 or GM-CSF. In vivo, specific anthelmintic therapy resulted in decreased CD66 and CD25 expression (p < 0.05 compared with pretreatment) to levels approaching those seen in uninfected normal individuals. These findings indicate the dynamic nature of eosinophil surface molecules and demonstrate an important role for whole-blood staining in developing an understanding of the nature of eosinophil activation and of their role in inflammatory reactions to helminth parasites.
Constitutive production of IL-4 and IL-10 and stimulated production of IL-8 by normal peripheral blood eosinophils.
Authors: Nakajima H , Gleich G J , Kita H
Published: December 2022

To study the capacity and regulation of cytokine production by normal peripheral blood eosinophils, we isolated eosinophils from healthy individuals and stimulated them with immobilized Ig or TNF-alpha, with or without exogenous IL-5. By reverse transcription-PCR, uncultured, freshly isolated eosinophils constitutively expressed mRNA for IL-4, IL-10, and TGF-beta1. Eosinophils stimulated by immobilized secretory IgA, immobilized IgA, immobilized IgG, or TNF-alpha for 3 h expressed mRNA encoding IL-3, IL-4, IL-8, IL-10, granulocyte-macrophage CSF, TNF-alpha, TGF-beta, and RANTES. The mRNA for IL-2, IL-5, or IFN-gamma was not detected. IL-4 and IL-10 protein, but not IL-8, were measurable in lysates of fresh eosinophils or eosinophils cultured with medium alone for 24 h. Eosinophils incubated with immobilized Ig or TNF-alpha released IL-8 protein into the supernatants. In contrast, IL-4 and IL-10 proteins were not detectable. Soluble secretory IgA immune complexes also induced degranulation, as measured by eosinophil-derived neurotoxin, and IL-8 release, but not IL-4 or IL-10 release, from eosinophils. Release of IL-8 protein and storage of IL-4 and IL-10 proteins were enhanced by exogenous IL-5 and inhibited by a transcription inhibitor, actinomycin D. Degranulation of stored granule proteins was not affected by actinomycin D. Therefore, normal peripheral blood eosinophils can transcribe and synthesize several cytokines, including IL-4, IL-8, and IL-10; some are stored, and some are released. These cytokines may play important roles in modulating immune responses in diseases associated with eosinophils.
Characterization of murine intercellular adhesion molecule-2.
Authors: Xu H , Bickford J K , Luther E , Carpenito C , Takei F , Springer T A
Published: December 2022

Rat mAbs were raised against murine intercellular adhesion molecule-2 (ICAM-2). Immune precipitation and purification reveal that the murine ICAM-2 glycoprotein is 55 kDa and is similar in size to human ICAM-2. ICAM-2 is expressed on a variety of leukocyte cell lines, including T and B lymphoma, mastocytoma, and macrophage lines. ICAM-2 is well expressed on endothelioma cell lines, and in contrast to ICAM-1, expression is not increased by inflammatory cytokines. One of the mAb to ICAM-2 partially or completely inhibits binding of cells expressing LFA-1 to purified ICAM-2, and binding of cells expressing ICAM-2 to purified LFA-1. The findings in the mouse are congruent with those in the human, suggesting functional conservation of ICAM-2 across species.
Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2).
Authors: Morton P A , Fu X T , Stewart J A , Giacoletto K S , White S L , Leysath C E , Evans R J , Shieh J J , Karr R W
Published: December 2022

CTLA-4 expressed on activated T cells binds to CD80 (B7-1) and CD86 (B7-2) molecules present on APC with high avidity and appears to deliver a negative regulatory signal to the T cell. We have investigated the kinetics of CTLA-4 binding to CD80 and CD86, together with the effects of selected CTLA-4 mutations on binding activity. The dissociation constants (Kd) for binding of CTLA-4-Ig to CD80 and CD86 transfectants were 8.1 and 6.7 nM, respectively. Surface plasmon resonance was used to determine kinetic parameters of CTLA-4-Ig binding to CD80-Ig and CD86-Ig fusion proteins and revealed enhanced association (ka) and dissociation (kd) rate constants for CD86-Ig compared with CD80-Ig. Furthermore, CD80-Ig and CD86-Ig fusion molecules demonstrated variable abilities to cross-compete for binding to several modified forms of CTLA-4-Ig. Differential binding of CD80 and CD86 to CTLA-4 was further revealed by analysis of 10 discrete CTLA-4 mutants. Five single amino acid substitutions within the CTLA-4 MYPPPY domain exerted modest effects on CD80 binding, but each of these substitutions completely abrogated CD86 binding. In addition, substitutions just N-terminal of the MYPPPY region, and within the CDR1-like region of CTLA-4, eliminated both CD80 and CD86 binding. Hence, CD80 and CD86 bind with different association/dissociation kinetics to similar, but distinct, sites on CTLA-4.
Macrophages have cell surface IL-10 that regulates macrophage bactericidal activity.
Authors: Fleming S D , Campbell P A
Published: December 2022

IL-10, which is secreted by multiple cell types, has regulatory effects on macrophages, including decreasing their ability to kill some microorganisms. The experiments presented here test the hypothesis that endogenous IL-10 inhibits the ability of macrophages to kill the facultative intracellular bacterium, Listeria monocytogenes. We show that the nonbactericidal macrophage hybrid, H36.12j (12j), can kill Listeria after incubation for 3 days with anti-IL-10 mAb. IL-10 was not detected in 12j macrophage supernatants by ELISA. However, flow cytometric analysis revealed high levels of IL-10 on the 12j cell surface. This indicates that macrophages that fail to secrete IL-10 may express IL-10 on the cell surface, and this IL-10 appears to suppress listericidal activity. Surface IL-10 is not unique to the 12j macrophage hybrid and may correlate with the absence of bactericidal activity in other macrophages. For instance, nonlistericidal resident and thioglycolate-elicited peritoneal exudate cells have 24 to 72% IL-10-positive macrophages. In contrast listericidal proteose peptone-elicited peritoneal exudate cells contained < 5% IL-10-positive macrophages. Whether this IL-10 is an integral membrane protein or is bound to IL-10 receptors is not yet known. However, the IL-10 does not elute with acid as other passively bound molecules do, nor does exogenous IL-10 bind to macrophages. In either case, since anti-IL-10 induces nonbactericidal macrophages to become bactericidal, the surface IL-10 is biologically active, and it appears to regulate macrophage bactericidal activity.
Blood clearance kinetics and liver uptake of mononucleosomes in mice.
Authors: Gauthier V J , Tyler L N , Mannik M
Published: December 2022

Nucleosomes generated by apoptosis have become of considerable interest in relation to pathogenesis of systemic lupus erythematosus in mice and humans. Therefore, the fate of circulating mononucleosomes was examined in normal C57Bl/6J mice. The mononucleosomes were prepared from chicken erythrocytes and radiolabeled on the histone component. The removal of nucleosomes from circulation at doses less than 11 micrograms of injected mononucleosomes was rapid, but with increasing doses of injected nucleosomes, the slopes of the removal curves decreased. Liver was the major organ for removal of circulating nucleosomes, accounting for 71.0 to 84.7% of nucleosomes removed from circulation at 10 min. After i.v. injection of nucleosomes, 0.52 +/- 0.15% localized in kidneys. With prior i.v. injection of histones, the glomerular localization of mononucleosomes increased threefold. The clearance of mononucleosomes was decreased sixfold by concurrent injection of ssDNA. These studies show that in mice, circulating mononucleosomes are handled similar to DNA, and they do not avidly localize in glomeruli unless histones have already bound to renal glomeruli.
Regulation of murine macrophage IL-12 production. Activation of macrophages in vivo, restimulation in vitro, and modulation by other cytokines.
Authors: Skeen M J , Miller M A , Shinnick T M , Ziegler H K
Published: December 2022

IL-12 is important in the host response to a variety of pathogens. It plays an adjuvant-like role in an initial immune response as well as a therapeutic role in established infections. Despite its well documented importance, comparatively little is known about the regulation of IL-12 production. In this study, we examined IL-12 production by cultured murine peritoneal macrophages from two perspectives: 1) macrophage activation in vivo, and 2) stimulation of IL-12 secretion in vitro. Macrophages were maximally activated within 48 h in vivo during infection with Listeria. Interestingly, although avirulent or heat-killed Listeria induced only minimal production of IL-12 by macrophages, the immunogenic combination of heat-killed bacteria and rIL-12 was highly stimulatory for IL-12 production. LPS and peritoneal inflammatory agents were also stimulatory, but latex beads were ineffective, indicating that microbial components were essential and phagocytosis alone was insufficient. Restimulation in vitro revealed similar patterns, in that infection and LPS were stimulatory but latex beads were not. A systematic survey of potential stimulatory agents showed that microbial heat shock proteins, crude bacterial extracts, bacterial superantigens, a yeast extract, and dsRNA induced IL-12 in vitro. Other cytokines also influenced IL-12 induction. IFN-gamma, which is up-regulated during infection, acted in synergy with other stimuli, suggesting an amplification loop for IL-12 production, whereas IL-4, IL-10, IL-13, and TGF-beta were inhibitory. The existence of a broad range of stimuli from a wide variety of pathogenic organisms underscores the fundamental importance of IL-12 in host defense.
Regulation of bioactive IL-12 production in lipopolysaccharide-stimulated human monocytes is determined by the expression of the p35 subunit.
Authors: Snijders A , Hilkens C M , van der Pouw Kraan T C , Engel M , Aarden L A , Kapsenberg M L
Published: December 2022

IL-12 enhances IFN-gamma production by NK cells and T lymphocytes and plays a pivotal role in the development of Th1 cells. Bioactive IL-12 (p70) is composed of two subunits, p35, which is only secreted as part of the p70 dimer, and p40, which can also be secreted by itself. The IL-12 subunits are encoded by two separate genes. Therefore, we studied to what extent the expression of each of the subunits contributes to the regulation of IL-12 production. We found that in LPS-stimulated whole blood and purified monocytes, p70 and p40 production are enhanced by IFN-gamma and inhibited by IL-10 and IL-4. However, IFN-gamma and IL-10 had stronger effects on p70 production than on p40 production, and IL-4 affected p40 production more strongly. Concomitantly, in all experimental conditions tested, p40 production greatly exceeded p70 production, suggesting that p35 expression was limiting. Analysis of p35 and p40 mRNA expression by PCR confirmed this notion. Resting purified monocytes expressed neither p40 nor p35 mRNA. The effects of IFN-gamma, IL-10, and IL-4 on the p35 mRNA expression in LPS-stimulated purified monocytes were similar to the effects of these cytokines on p70 protein production, and the p40 mRNA expression corresponded to p40 protein production. Our results imply that production of the two IL-12 subunits is differently regulated for each subunit, mainly at the level of mRNA expression, and that the level of bioactive IL-12 production in monocytes in response to LPS and cytokines is determined by the level of p35 expression.
Augmentation by calmodulin of ADP-ribosylation factor-stimulated phospholipase D activity in permeabilized rabbit peritoneal neutrophils.
Authors: Takahashi K , Tago K , Okano H , Ohya Y , Katada T , Kanaho Y
Published: December 2022

Activation of the membrane-bound phospholipase D (PLD) requires cytosolic factor(s), and ADP-ribosylation factor (ARF) has been identified as a cytosolic PLD activator. In the present study, we demonstrate that calmodulin (CaM) and ARF are both involved in PLD activation in rabbit peritoneal neutrophils. The PLD activity of streptolysin O-permeabilized, cytosol-depleted rabbit neutrophils was significantly enhanced when the permeabilized cells were reconstituted with bovine brain cytosol in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas there was little activation of the enzyme in the absence of cytosol. The GTP gamma S-stimulated PLD activity in the presence of cytosol was augmented on increasing the concentration of free Ca2+. The PLD activity stimulated by GTP gamma S and Ca2+ in this system was inhibited by the calmodulin inhibitor W-7. These findings suggest that CaM plays a role as a cytosolic PLD activator. Moreover, highly purified CaM alone, as well as partially purified ARF alone, promoted a slight stimulation of the PLD activity in permeabilized neutrophils. Interestingly, ARF-stimulated PLD activity was augmented by CaM in the presence of GTP gamma S and Ca2+. This augmentation was again inhibited by W-7, as well as by the structurally unrelated CaM inhibitor trifluoperazine. These data imply that CaM stimulates the PLD activity of rabbit neutrophils in concert with ARF.
Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca(2+)-dependent translocation from cytoplasm to membranes and cytoskeleton.
Authors: van den Bos C , Roth J , Koch H G , Hartmann M , Sorg C
Published: December 2022

MRP8 and MRP14 are two Ca(2+)-binding proteins expressed in myelomonocytic cells. Complexes of MRP8 and MRP14 colocalize with membranes and intermediate filaments in a Ca(2+)-dependent manner. MRP14, unlike MRP8, exists in two isoforms, the smaller of which (MRP14') has been shown to lack the first four amino acids; both MRP14 and MRP14' are also present as phosphorylated forms. As shown in the present work by metabolic labeling of monocytes with [35S]methionine, MRP14 and MRP14' are translated simultaneously. By PCR analysis we found no evidence for the presence of different mRNA species. Since MRP14 is encoded by a single copy gene, our data indicate that MRP14' formation is due to alternative translation of a single mRNA species. Two-dimensional electrophoresis of [32P]orthophosphate-labeled monocyte proteins followed by Western blotting and autoradiography revealed that the two phosphorylated MRP14 isoforms incorporated the bulk of the radioactivity found in monocytic proteins. Using differential centrifugation we demonstrated the presence of distinct isoform patterns in different subcellular locations. Further, in response to elevated Ca2+ concentrations we observed a preferential translocation of phosphorylated MRP14 isoforms from the cytosol toward membranes and the cytoskeleton. This might be caused by altered calcium binding, and indeed, using isoelectric focusing and 45Ca2+ overlay the MRP14 band containing phosphorylated MRP14 revealed increased Ca2+ binding compared with bands containing other MRP14 isoforms. This represents the first evidence for functional differences in phosphorylated MRP14 isoforms compared with nonphosphorylated MRP14 isoforms. These functional differences suggest that MRP14 represents the regulatory subunit of MRP8/MRP14 complexes.
Adoptively transferred CD8+ T lymphocytes provide protection against TMEV-induced demyelinating disease in BALB/c mice.
Authors: Nicholson S M , Dal Canto M C , Miller S D , Melvold R W
Published: December 2022

On intracerebral infection with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV), certain mouse strains develop a chronic demyelinating disease similar both clinically and pathologically to human multiple sclerosis. Other strains remain resistant. We previously established that differential susceptibility to this demyelinating disease exists among BALB/c substrains, with BALB/cAnNCr mice being susceptible while BALB/cByJ mice are resistant. BALB/cByJ mice are rendered susceptible to TMEV-induced demyelination on exposure to low dose gamma-irradiation before TMEV infection. BALB/cAnNCr and irradiated, infected BALB/cByJ animals are protected against TMEV-induced demyelination by the transfer of a splenic population from TMEV-infected BALB/cByJ donors. Resistance to demyelination appears to be mediated by a CD8+ radiosensitive population, which is induced on infection with TMEV and which must act early to establish resistance to TMEV-induced demyelination.
Medullary thymic epithelial cells induce tolerance to intracellular proteins.
Authors: Oukka M , Cohen-Tannoudji M , Tanaka Y , Babinet C , Kosmatopoulos K
Published: December 2022

The role of the medullary thymic epithelial cells in tolerance induction to MHC class I restricted self peptides has been analyzed by studying the beta-galactosidase (beta-gal)-specific cytotoxic T cell response of a transgenic mouse expressing beta-gal in the thymus, skin, and central nervous system (Tg beta-gal mouse). Our results showed that: 1) beta-gal expression in the thymus was limited in a subpopulation of medullary epithelial cells, and bone marrow-derived thymic cells were beta-gal-1; 2) Tg beta-gal mice did not mount an anti-beta-gal CTL response even in the presence of exogenous IL-2, while Tg beta-gal-->B6 chimeras responded to beta-gal as strongly as NTg beta-gal mice; 3) Tg beta-gal mice did not generate CTL against the immunodominant Kb-restricted beta-gal 497-504 peptide; 4) tolerance was due to the thymic epithelial cells that expressed beta-gal because nude mice grafted with thymus from Tg beta-gal mice were also unable to respond to beta-gal; 5) the Tg beta-gal mouse-derived beta-gal+ medullary epithelial TEC.X10 line presented the Kb-restricted beta-gal 497-504 epitope. In conclusion, these results demonstrate that medullary thymic epithelial cells induce a complete tolerance towards class I-restricted self peptides presented on their own surface.
Interaction of IL-15 with the shared IL-2 receptor beta and gamma c subunits. The IL-15/beta/gamma c receptor-ligand complex is less stable than the IL-2/beta/gamma c receptor-ligand complex.
Authors: de Jong J L , Farner N L , Widmer M B , Giri J G , Sondel P M
Published: December 2022

This study was designed to compare the interactions of IL-2 and IL-15 with the IL-2R beta and IL-2R gamma c subunits, as differences in receptor interactions between IL-2 and IL-15 might contribute to the functional differences between these two cytokines. The results suggest the existence of a human IL-15R alpha subunit, although physical evidence of this molecule was not obtained. Proliferation of anti-CD3 (OKT3)-stimulated human PBL was compared for responsiveness to IL-2, IL-15, and F42K, and IL-2 mutant that does not bind the IL-2R alpha chain. F42K was more potent than IL-15 in activating a dose-dependent response. This fact, along with Scatchard binding analyses of IL-15 on OKT3 blasts and YT cells revealing both high and intermediate affinity receptors, supports the existence of IL-15R alpha on these cells. Additional characterization of the IL-15R utilized covalent cross-linking to affinity label IL-2R and IL-15R on YT cells and OKT3 blasts. Consistent with previously reported functional data, IL-2R alpha was not co-precipitated from the [125I]IL-15 receptor-ligand complex, demonstrating that IL-15 does not interact physically with the IL-2R alpha subunit. While IL-2R alpha did co-precipitate with IL-2R beta and IL-2R gamma c in the presence of IL-2, IL-15R alpha did not co-precipitate with the IL-2R beta/gamma c complex. Finally, YT cells equilibrated with IL-2 and then precipitated through IL-2R beta showed that IL-2R beta and IL-2R gamma c co-precipitate in a 1:1 ratio, while only IL-2R beta was found in the immunoprecipitates of YT cells equilibrated with IL-15. This indicates that IL-15 creates a less stable bridge between the IL-2R beta and IL-2R gamma c chains than does IL-2 on YT cells. This result was identical for both surface-iodinated YT cells and immunoprecipitates that were probed for IL-2R beta and IL-2R gamma c on Western blots.
Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response.
Authors: Dong R P , Kameoka J , Hegen M , Tanaka T , Xu Y , Schlossman S F , Morimoto C
Published: December 2022

CD26, a T cell activation Ag, also known as dipeptidyl peptidase IV, is directly associated with adenosine deaminase (ADA) on the surface of T cells and T cell lines. In the present study, we examined both the binding of ADA and CD26 and the functional consequences of this interaction. We found that ADA was associated with CD26 on T cell lines lacking either ADA or dipeptidyl peptidase IV enzymatic activity, indicating that the association between dipeptidyl peptidase IV and ADA did not require enzymatic activity. Moreover, using immunoelectron microscopy, we demonstrated that CD26 and ADA co-localized on the cell surface, but not inside cells, suggesting that CD26 did not transport ADA to the surface. In keeping with this observation, we showed that human CD26-transfected murine pre-B cell lines lacking human ADA acquired ADA from an extracellular source. More importantly, adenosine in the absence of cell surface ADA inhibited T cell proliferation and IL-2 production induced by various stimuli. On the other hand, cells expressing ADA and CD26 on the surface were much more resistant to the inhibitory effect of adenosine. These data suggest that ADA on the cell surface is involved in an important immunoregulatory mechanism by which released ADA binds to cell surface CD26, and this complex is capable of reducing the local concentration of adenosine.
Early signal transduction by the receptor to the chemokine monocyte chemotactic protein-1 in a murine T cell hybrid.
Authors: Dubois P M , Palmer D , Webb M L , Ledbetter J A , Shapiro R A
Published: December 2022

Members of the C-C family of chemotactic cytokines promote chemotaxis and adhesion of leukocytes. In this study, we have identified a murine T cell hybrid that expresses receptors to the chemotactic cytokine monocyte chemotactic protein-1 (MCP-1). This cell line was used to examine MCP-1 receptor-mediated signal transduction events in a homologous system in the absence of interference with other receptors. Our results show that in the 3B4 M1.9 T cell hybrid, MCP-1 receptors mediate intracellular calcium mobilization and extracellular calcium import without detectable increases in total water-soluble inositol phosphates. In addition, MCP-1 regulates the tyrosine phosphorylation of specific substrates at 42 and 44 kDa and induces mobility shift of p42/44 mitogen-activated protein kinases. MCP-1-mediated calcium responses, tyrosine phosphorylation, and the electrophoretic mobility shift of p42/44 mitogen-activated protein kinases can be inhibited by pretreatment of cells with pertussis toxin, indicating a role for Gi-like G proteins in coupling the MCP-1R to signal transduction.