bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombin induces IL-6 production in fibroblasts and epithelial cells. Evidence for the involvement of the seven-transmembrane domain (STD) receptor for alpha-thrombin.
Authors: Sower L E , Froelich C J , Carney D H , Fenton J W , Klimpel G R
Published: January 2023

alpha-Thrombin is a multifunctional serine protease that has an important role in the coagulation cascade, wound healing, and inflammatory response. In this study, we show that thrombin induces IL-6 production in human epithelial cells and fibroblasts. ELISA and Northern blot analyses showed that physiologic concentrations of thrombin (0.1-1 micrograms/ml) induced IL-6 production in human lung fibroblasts, skin fibroblasts, and epithelial cells. Hirudin, a thrombin inhibitor, completely blocked IL-6 induction by thrombin. Treatment of fibroblasts with inactivated diisopropylphosphofluoridate (DIP)-alpha-thrombin, gamma-thrombin, or trypsin had no effect on IL-6 production. In contrast, treatment with the thrombin-tethered ligand receptor peptide TRP-7 (SFLLRNP) induced IL-6 production, but at lower levels than that induced by native alpha-thrombin. Finally, IL-6 pretreatment of lung or skin fibroblasts resulted in the enhanced production of IL-6 following exposure to thrombin. These results suggest that fibroblasts and epithelial cells may represent a significant source of IL-6 in the inflammatory response to tissue injury, and that cytokine production is an important biologic consequence of thrombin's interaction with its seven-transmembrane domain (STD) receptor.
Antigen presentation by epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of CD45RA+ T cells.
Authors: Marelli-Berg F M , Weetman A , Frasca L , Deacock S J , Imami N , Lombardi G , Lechler R I
Published: January 2023

The immunoregulatory effects of alloantigen presentation by tissue parenchymal cells to resting peripheral blood CD4+ T cells was investigated. Coculture of CD45RO+ (memory) and CD45RA+ (naive) T lymphocytes with primary cultures of MHC class II-expressing epithelial cells rendered both populations of T cells hyporesponsive to a subsequent challenge by the same MHC molecule expressed on EBV-transformed lymphoblastoid B cell lines. However, the mechanisms responsible for the allospecific hyporesponsiveness were distinct. For the CD45RO+ T cells, responsiveness was restored by subsequent culture in the presence of IL-2; the addition of IL-2 had no effect on the reactivity of the CD45RA+ T cells. In contrast, the naive T cells were protected from the induction of nonresponsiveness by the presence of a neutralizing anti-CD95 Ab during the culture with thyroid follicular cells. In addition, the hyporesponsive CD45RO+ T cells effected linked suppression, in that they inhibited proliferation against a third-party DR alloantigen when the third-party alloantigen was coexpressed with the DR Ag against which hyporesponsiveness had been induced. These results suggest that recognition of Ag by T cells on tissue parenchymal cells plays an important role in the maintenance of peripheral T cell tolerance, inducing nonresponsiveness in naive and memory T cells by distinct mechanisms.
Depressed Langerhans cell migration and reduced contact hypersensitivity response in mice lacking TNF receptor p75.
Authors: Wang B , Fujisawa H , Zhuang L , Kondo S , Shivji G M , Kim C S , Mak T W , Sauder D N
Published: January 2023

Epidermal Langerhans cells (LC) belong to the dendritic cell family and represent the major APC within the skin. LC capture epicutaneous Ag, migrate into regional lymph nodes, and present Ag to T cells, thereby initiating primary immune response. The migratory properties of LC are an essential component of their function. The molecular mechanisms responsible for LC migration are far less defined. However, evidence has been accumulating to suggest that TNF-alpha, a major proinflammatory cytokine, plays an important role in promoting DC migration. To confirm the role of TNF-alpha in LC migration and to examine which type of TNF receptor signaling is involved in such an event, we utilized gene-targeted knockout mice lacking TNF receptor p55 or p75. The migration of LC was assessed by examining the frequency of hapten-bearing cells in draining lymph nodes following hapten FITC painting, and the accumulation of dendritic cells in draining lymph nodes after intradermal injection of TNF-alpha. While LC migration was normal in p55-deficient mice, the migration was markedly depressed in p75-deficient mice. Receptor p75-deficient mice also demonstrated a hyporesponsiveness in allergen-induced contact dermatitis, but a normal responsiveness in irritant-induced contact dermatitis. These results suggest that p75-dependent signaling plays a crucial role in the migration of LC and in the initiation of cutaneous immune responses.
Potent induction of a neutrophil and eosinophil-rich infiltrate in vivo by human mast cell tryptase: selective enhancement of eosinophil recruitment by histamine.
Authors: He S , Peng Q , Walls A F
Published: January 2023

Tryptase is the most abundant protein constituent of the secretory granules of human mast cells, but little is known of the contribution of this serine proteinase in acute allergic reactions. We have purified tryptase from human lung tissue by immunoaffinity procedures, and have investigated its potential to provoke an inflammatory infiltrate in vivo. Within 6 h of injection into the skin of guinea pigs, the accumulation of large numbers of neutrophils and eosinophils was observed, and those eosinophils closest to the injection site were partially degranulated. Similarly, injection of tryptase into the peritoneum of mice, even in quantities as low as 5 ng, stimulated the ingress of neutrophils. The response was dose dependent at 3, 6, and 16 h, with increases in median numbers of up to 400-fold. At the later time points eosinophil numbers were increased by up to 10-fold, and there were elevations also in the numbers of lymphocytes and macrophages. In both models, the actions of tryptase appeared to be dependent on an intact catalytic site. Coinjection of heparin with tryptase had relatively little effect on tryptase-induced responses. On the other hand, although histamine did not itself stimulate cell accumulation, over a range of concentrations it altered the cellular composition of the infiltrate induced by tryptase. Addition of histamine to tryptase provoked selective increases in eosinophil numbers of up to fivefold in the mouse peritoneum. Tryptase may provide an important stimulus for granulocyte recruitment in allergic disease.
TNF and lymphotoxin-alpha polymorphisms associated with common variable immunodeficiency: role in the pathogenesis of granulomatous disease.
Authors: Mullighan C G , Fanning G C , Chapel H M , Welsh K I
Published: January 2023

A subgroup of common variable immunodeficiency (CVID) patients have distinct clinical features, particularly granulomata splenomegaly, characteristic blood lymphocyte phenotype, and elevated circulating TNF levels. To investigate the genetic basis for this phenotype, 150 CVID patients and 200 controls were genotyped for six biallelic TNF and lymphotoxin-alpha (LT alpha) polymorphisms and eight class I and II HLA loci using PCR and sequence specific primers (PCR-SSP) sequence-specific primers. Clinical and immunophenotypic data were collected for 90 patients to examine associations with CVID patient subgroups. The presence of granulomata (22% of patients) was strongly associated with splenomegaly, T and B lymphopenia, reduced CD4+ CD45RA+ T cells, and CD8+ CD57+ lymphocytosis, confirming the concept of a subgroup of patients with distinct clinical and laboratory features. The uncommon TNF +488A allele was strongly associated with this subgroup (p = 0.0005). The association between "granulomatous" CVID and TNF +488A was independent of HLA class I and II associations. We postulate that the presence of the TNF +488A allele, or alleles in linkage disequilibrium with it, contributes to the high levels of TNF and granulomatous complications characteristic of this subgroup of patients.
Yersinia invasin, a bacterial beta1-integrin ligand, is a potent inducer of lymphocyte motility and migration to collagen type IV and fibronectin.
Authors: Arencibia I , Suárez N C , Wolf-Watz H , Sundqvist K G
Published: January 2023

The Yersinia pseudotuberculosis invasin protein was found to be a potent inducer of pseudopodia formation and chemotactic and haptotactic migration in human T lymphocytes. Checkerboard analysis confirmed that migration was directional. The Yersinia invasin triggered migration of otherwise poorly migratory normal T cells on fibronectin and in particular on collagen type IV, and augmented the migration of leukemic T cell lines on these components. Invasin-induced lymphocyte migration was inhibited by staurosporin that selectively prevented pseudopodia formation but, noteworthy, augmented adhesion. The motogenic and attractant properties of invasin (Inv) were mediated via beta1-integrins, as shown by lack of effect of Inv on the motility of a beta1-integrin-negative lymphoid cell line and inhibition of invasin-induced lymphocyte motility by anti-beta1 Abs. Inv was markedly more effective than the extracellular matrix components fibronectin, collagen type IV, and laminin, which also interact with lymphocyte beta1-integrins, with respect to induction of pseudopodia, chemotaxis, and haptotaxis. Thus, Yersinia invasin is a model ligand for induction of lymphocyte motility via beta1-integrins. The extraordinary capacity of Inv to trigger and guide T lymphocyte motility and potentiate lymphocyte migration to extracellular matrix components may be of pathogenetic significance for the movement of lymphocytes to extraintestinal sites secondary to Yersinia infection.
Biologic activity of proteoglycan macrophage colony-stimulating factor.
Authors: Suzu S , Kimura F , Ota J , Motoyoshi K , Itoh T , Mishima Y , Yamada M , Shimamura S
Published: January 2023

We compared the biologic activities of 85-kDa macrophage-CSF (85-kDa M-CSF), which is fully active, and a proteoglycan M-CSF (PG-M-CSF). Both originate from the same precursor, but the latter retains the carboxyl-terminal portion, which must be proteolytically removed from the precursor to generate 85-kDa M-CSF and which is uniquely modified by a chondroitin sulfate glycosaminoglycan chain. PG-M-CSF supported the formation of murine macrophage colonies such as 85-kDa M-CSF. Furthermore, PG-M-CSF stimulated the proliferation of murine bone marrow macrophages, an M-CSF-dependent murine cell line, and an M-CSF-responsive human cell line established by transfer of the human M-CSF receptor gene. PG-M-CSF and 85-kDa M-CSF had equivalent specific biologic activities on a molar basis in all bioassays. The activity of PG-M-CSF was not affected by enzymatically removing the glycosaminoglycan chain when assayed by the formation of macrophage colonies and proliferation of the bone marrow macrophages. We analyzed the phosphorylation on tyrosine residue(s) of the M-CSF receptor in response to these M-CSFs that trigger mitogenic responses. PG-M-CSF rapidly (within 10 min) induced receptor phosphorylation in human cells with the same potency as 85-kDa M-CSF. These results indicate that PG-M-CSF is not a latent form or precursor of 85-kDa M-CSF but a fully biologically active cytokine.
Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines.
Authors: Sozzani S , Luini W , Borsatti A , Polentarutti N , Zhou D , Piemonti L , D'Amico G , Power C A , Wells T N , Gobbi M , Allavena P , Mantovani A
Published: January 2023

Dendritic cells (DC) are migratory cells that exhibit complex trafficking properties in vivo. The present study was designed to characterize receptor expression and responsiveness to chemoattractants of human DC obtained from PBMC by culture with granulocyte/macrophage-CSF and IL-13. DC expressed appreciable levels of the CCR1, CCR2, and CCR5 receptors for the CC chemokines and the chemokine receptors CXCR1, CXCR2, and CXCR4. DC increased intracellular free calcium and migrated in response to the CC chemokines MCP-3, MCP-4, RANTES, MIP-1alpha, MIP-1beta, and MIP-5/HCC2 and the CXC chemokine SDF-1. In contrast, the CC chemokines MCP-1 and eotaxin had little or no activity in the concentration range tested (up to 1 microg/ml). IL-8 and Gro-beta (CXC) and lymphotactin (C chemokines) were also inactive. DC did not respond to 5-HETE, whereas platelet-activating factor was an active agonist. Selected chemokines active on DC in terms of migration and calcium fluxes were examined for their capacity to modulate endocytosis and Ag presentation. Under conditions in which TNF-alpha was active, MCP-1, MCP-3, MIP-1alpha, and RANTES did not affect these two responses. Thus, among hemopoietic elements, DC respond to a unique set of CC and CXC chemokines, and their responsiveness is restricted to migration with no effect on Ag capture and presentation. Chemokines may play a role in the trafficking of DC under resting or stimulated conditions. Chemokine receptors expressed in DC are likely to underlie HIV infection of this cell type.
Observations on the Proposed Gold Chloride Titration for Determining the Toxicity of Diphtheria Toxin
Authors: Sanderson Everett S. , Yoe John H.
Published: January 2023

Summary and Conclusions

The results of the foregoing experiments may be summarized as follows: Six different diphtheria toxins when tested by the “gold chloride” and guinea pig methods failed to show any parallelism as to toxicity.The green color zone produced in mixtures of gold chloride and toxic broths is not specific for toxin: Inactivated (heated) and non-inactivated toxins gave almost identical gold chloride titration figures.The amount of gold chloride necessary to bring about the maximum green coloration with toxic broths can be entirely altered by changing the initial hydrogen ion concentration and the buffer capacity of the broths.Several batches of uninoculated 1 per cent dextrose extract broth gave a green color zone when mixed with gold chloride.Maximum green color in mixtures of broths and gold chloride is obtained at an approximate pH of 4.5.The gold chloride test does not appear to be a satisfactory method for standardizing bacterial toxins in its present form.
Microglia Are More Efficient Than Astrocytes in Antigen Processing and in Th1 But Not Th2 Cell Activation
Authors: Aloisi Francesca , Ria Francesco , Penna Giuseppe , Adorini Luciano
Published: January 2023

Microglia and astrocytes, two glial cell populations of the central nervous system, present Ag and stimulate T cell proliferation, but it is unclear whether they preferentially activate Th1 or Th2 responses. We have investigated the efficiency of microglia and astrocytes in the presentation of OVA peptide 323-339 or native OVA to Th1 and Th2 cell lines from DO11.10 TCR transgenic mice. Upon stimulation with IFN-γ, microglia express MHC class II molecules, CD40, and ICAM-1 and efficiently present OVA 323-339, leading to T cell proliferation and production of IL-2 and IFN-γ by Th1 and of IL-4 by Th2 cells. IFN-γ-treated astrocytes, which express MHC class II and ICAM-1, present OVA 323-339 less efficiently to Th1 cells but are as efficient as microglia in inducing IL-4 secretion by Th2 cells. However, astrocytes are much less potent than microglia in presenting naturally processed OVA peptide to either T cell subset, indicating inefficient Ag processing. The capacity of astrocytes and microglia to stimulate Th1 and Th2 cells depends on their MHC class II expression and does not involve ICAM-1, B7-1, or B7-2 molecules. However, CD40-CD40L interactions contribute to Th1 activation by microglia. These data suggest that microglia may play a role in the activation of Th1 and Th2 cells, whereas astrocytes would restimulate mainly Th2 responses in the presence of appropriate peptides. This differential capacity of brain APC to restimulate Th1 and Th2 responses may contribute to the reactivation and regulation of local inflammatory processes during infectious and autoimmune diseases.
Kinetics of Establishing the Memory B Cell Population as Revealed by CD38 Expression
Authors: Ridderstad Anna , Tarlinton David M.
Published: January 2023

In this report, we detail changes in the expression of CD38 on murine B cells during the course of a T cell-dependent immune response. CD38 is expressed on all naive B cells but is down-regulated on isotype-switched B cells from both the germinal centers (GCs) and the foci of Ab-forming cells which arise during the first weeks of the response. The down-regulation on GC B cells, however, is reversible since Ag-specific IgG1 B cells with high levels of CD38 are apparent by 2 wk postimmunization. These cells have characteristics that resemble recirculating memory B cells, in that they are small and bind low levels of peanut agglutinin. Such characteristics indicate that the restoration of CD38 levels is coincidental with the transition from GC to memory B cell. Using this observation, we plotted the development of the memory population and the demise of the GC reaction as a function of time after immunization. Our results indicate that the GC reaction ceases gradually over many weeks rather than suddenly, which corresponds with the formation of the memory B cell population. Furthermore, by segregating memory B cells and GC B cells, it was possible to assess the in vitro survival characteristics of each compared with naive B cells. These experiments demonstrated that memory B cell survival in vitro was comparable with naive B cell survival but less than the survival seen for bcl-2-transgenic B cells, whereas GC B cell survival, as expected, was very poor. Hence, by resolving murine Ag-specific memory B cells and GC B cells, we have been able to quantify the development of the memory B cell population.
Differential Activation of T Cells by Natural Antigen Peptide Analogues: Influence on Autoimmune and Alloimmune In Vivo T Cell Responses
Authors: Soares Luis R. B. , Orr Patricia L. , Garovoy Marvin R. , Benichou Gilles
Published: January 2023

Recent studies using synthetic altered peptide ligands (Analogues) have led to the fine dissection of TCR-mediated T cell functions elicited by Ag recognition. Certain Analogues behave as full agonists of the antigenic peptide while others are partial agonists in that they only trigger selected T cell functions. Additionally, peptide Analogues can behave as antagonists by inhibiting functions of T cell clones when coincubated with the wild-type peptide. In fetal thymic organ cultures, synthetic altered peptide ligands can impact T cell repertoire selection. However, the influence of naturally occurring peptide Analogues on T cell immunity in vivo remains hypothetical. We previously reported that, in B10.A mice, immunogenicity and tolerogenicity of the self-MHC class I peptide, Ld 61-80, were influenced by the presentation of a cross-reactive self-peptide, Kk 61-80. Here, we show that Kk 61-80 self-peptide represents a partial agonist of Ld 61-80 in that it induced the proliferation but not the lymphokine production of Ld 61-80-primed T cells. Next, we showed that presentation of Kk 61-80 Analogue peptide mediated T cell tolerance toward Ld 61-80 self-peptide. Alternatively, when Ld protein represented an alloantigen displayed on transplanted cells, immunization with Kk 61-80 Analogue sensitized recipient mice to Ld 61-80 peptide, thus inducing potent immune responses to donor cells. These results show that the presentation of natural Analogue peptides may represent an essential component of T cell responses involved in autoimmunity and transplant rejection.
The Structure of Avian CD5 Implies a Conserved Function
Authors: Koskinen Riitta , Göbel Thomas W. F. , Tregaskes Clive A. , Young John R. , Vainio Olli
Published: January 2023

The chicken CD5 cDNA was isolated by COS cell expression cloning utilizing a novel mAb 2-191. The cDNA contains a 1422-nucleotide open reading frame encoding a mature protein with 32% and 30% identity to mouse and human CD5 polypeptides, respectively. The molecule consists of a 330-amino acid extracellular region with three repeats of the scavenger receptor cysteine-rich domain, a 29-amino acid hydrophobic transmembrane domain, and a 93-amino acid cytoplasmic tail. The cytoplasmic region contains motifs that are highly conserved between species, including several potential phosphorylation sites. The chicken CD5 is a 64-kDa phosphorylated glycoprotein with a protein core of 57 kDa as determined by immunoprecipitation and SDS-PAGE analysis. αβ T cells express a homogeneously high level of CD5, whereas low or intermediate CD5 expression on γδ T cells depends on their tissue location. In contrast to human and mouse, CD5 is found at low levels on all chicken B cells. The high conservation of structural features, as well as signaling motifs, implies a conserved role for CD5 both in lymphocyte development and function.
Phosphatidylinositol 3′-Kinase and SH2-Containing Inositol Phosphatase (SHIP) Are Recruited by Distinct Positive and Negative Growth-Regulatory Domains in the Granulocyte Colony-Stimulating Factor Receptor
Authors: Hunter Melissa G. , Avalos Belinda R.
Published: January 2023

Activation of both positive and “negative” or anti-proliferative signals has emerged as a common paradigm for regulation of cell growth through cell surface receptors that regulate immune responses. SHP-1 and -2 and the novel 5′-inositol phosphatase SHIP have recently been shown to function as growth inhibitory molecules in immune receptor signaling. In the current study, we have identified distinct regions in the granulocyte colony-stimulating factor receptor (G-CSFR) distal to the conserved box 2 motif necessary for mitogenesis, which exert positive and negative influences on growth signaling in Ba/F3 pro-B lymphoid cells. The region spanning amino acids 682 to 715 mediates activation of phosphatidylinositol 3′(PI3)-kinase. Activation of PI3-kinase leads to inhibition of apoptosis, promotion of cell survival, and enhanced proliferative responses to G-CSF. We show that the region of 98 amino acids in the distal tail of the class I G-CSFR down-modulates proliferative signaling, not only in myeloid cell lines, as previously reported, but also in Ba/F3 cells. This same region recruits SHIP to the signaling cascade through a mechanism involving Shc, with the formation of Shc/SHIP complexes. Our data suggest a model in which PI3-kinase and SHIP coordinately regulate growth signaling through the G-CSFR.
FTY720, a Novel Immunosuppressant, Induces Sequestration of Circulating Mature Lymphocytes by Acceleration of Lymphocyte Homing in Rats. I. FTY720 Selectively Decreases the Number of Circulating Mature Lymphocytes by Acceleration of Lymphocyte Homing
Authors: Chiba Kenji , Yanagawa Yoshiki , Masubuchi Yumi , Kataoka Hirotoshi , Kawaguchi Takafumi , Ohtsuki Makio , Hoshino Yukio
Published: January 2023

FTY720, given i.v. or orally at 0.03 mg/kg or more, significantly prolonged skin allograft survival in a dose-dependent manner and showed more potent immunosuppressive activity than cyclosporin A (CsA) or tacrolimus (FK506) in MHC-incompatible rat strains of WKAH donors and F344 recipients. However, unlike CsA or FK506, FTY720 up to 1000 nM did not affect IL-2 production in allogeneic MLC. Within 3 to 24 h after a single oral administration of FTY720 at 0.1 to 1 mg/kg, the number of lymphocytes in the rats was markedly decreased in the peripheral blood and thoracic duct lymph and partially in spleen. By contrast, the number of lymphocytes in peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer’s patches (PP) was significantly increased at the same time. Intravenous transfusion of calcein-labeled rat lymphocytes into rats revealed that FTY720 significantly accelerated lymphocyte homing to PLN, MLN, and PP, dose dependently. Since FTY720-induced lymphocyte homing was completely blocked by simultaneous treatment of the calcein-labeled lymphocytes with mAbs against CD62L, CD49d, and CD11a before the transfusion, the acceleration of lymphocyte homing by FTY720 appears to be mediated by lymphocyte-homing receptors. These findings indicate that FTY720 sequesters circulating mature lymphocytes into PLN, MLN, and PP by acceleration of lymphocyte homing and thereby decreases the number of lymphocytes in peripheral blood, thoracic duct lymph, and spleen. Based on these observations, sequestration of circulating mature-lymphocytes is presumed to be a main mechanism of the immunosuppressive activity of FTY720.
Regulation of TNF-α Production in Activated Mouse Macrophages by Progesterone
Authors: Miller Lance , Hunt Joan S.
Published: January 2023

The purpose of this study was to investigate the relationships between macrophage production of TNF-α and female hormones. Northern blot hybridization experiments showed that the female sex steroid hormone, progesterone, decreases steady state levels of TNF-α mRNA in LPS-activated mouse macrophages (RAW 264.7 and ANA-1 cells) in vitro. The production of intracellular and secreted TNF-α protein, as determined by ELISA, was decreased in both progesterone- and dexamethasone-treated, LPS-stimulated macrophages. Estrogen had no effect on expression of the TNF-α gene in mouse macrophages and did not alter progesterone-mediated suppression. Additional experiments conducted to investigate the mechanism of action of progesterone showed that this hormone, like dexamethasone, elevates steady state mRNA levels of IκBα and increases the levels of IκBα protein that are translocated from the cytoplasm to the nucleus. Thus, progesterone is a potent inhibitor of steady state levels TNF-α mRNA and TNF-α protein production in activated macrophages and may achieve this result through effects on an inhibitor of NF-κB.
Central Nervous System Microglial Cell Activation and Proliferation Follows Direct Interaction with Tissue-Infiltrating T Cell Blasts
Authors: Sedgwick Jonathon D. , Ford Andrew L. , Foulcher Eléna , Airriess Rhonda
Published: January 2023

Central nervous system (CNS)-resident macrophages (microglia) normally express negligible or low level MHC class II, but this is up-regulated in graft-vs-host disease (GvHD), in which a sparse CNS T cell infiltrate is observed. Relative to microglia from the normal CNS, those from the GvHD-affected CNS exhibited a 5-fold up-regulation of characteristically low CD45, MHC class II expression was increased 10- to 20-fold, and microglial cell recoveries were enhanced substantially. Immunohistologic analysis revealed CD4+αβTCR+CD2+ T cells scattered infrequently throughout the CNS parenchyme, 90% of which were blast cells of donor origin. An unusual clustering of activated microglia expressing strongly enhanced levels of CD11b/c and MHC class II was a feature of the GvHD-affected CNS, and despite the paucity of T lymphocytes present, activated microglial cell clusters were invariably intimately associated with these T cells. Moreover, 70% of T cells in the CNS were associated with single or clustered MHC class II+ microglia, and interacting cells were predominantly deep within the tissue parenchyme. Approximately 3.7% of the microglia that were freshly isolated from the GvHD-affected CNS were cycling, and proliferating cell nuclear Ag-positive microglia were detected in situ. Microglia from GvHD-affected animals sorted to purity by flow cytometry and cultured, extended long complex processes, exhibited spineous processes, and were phagocytic and highly motile. These outcomes are consistent with direct tissue macrophage-T cell interactions in situ that lead to activation, proliferation, and expansion of the responding tissue-resident cell.
Alternative Metabolic States in Murine Macrophages Reflected by the Nitric Oxide Synthase/Arginase Balance: Competitive Regulation by CD4+ T Cells Correlates with Th1/Th2 Phenotype
Authors: Munder Markus , Eichmann Klaus , Modolell Manuel
Published: January 2023

Activated murine macrophages metabolize l-arginine via two main pathways that are catalyzed by the inducible enzymes nitric oxide synthase (iNOS) and arginase. We have previously shown that CD4+ T cell-derived cytokines regulate a competitive balance in the expression of both enzymes in macrophages; Th1-type cytokines induce iNOS while they inhibit arginase, whereas the reverse is the case for Th2-type cytokines. Here we addressed the regulation of both metabolic pathways by CD4+ T cells directly. Macrophages were used as APCs for established Th1 and Th2 T cell clones as well as for in vitro polarized Th1 or Th2 T cells of transgenic mice bearing an MHC class II-restricted TCR. Both systems revealed a similar dichotomy in the macrophages; Th1 T cells led to an exclusive induction of iNOS, whereas Th2 T cells up-regulated arginase without inducing iNOS. Arginase levels induced by Th2 T cells far exceeded those inducible by individual Th2 cytokines. Similarly, high arginase levels could be induced by supernatants of Th2 cells stimulated in various ways. Ab blocking experiments revealed the critical importance of IL-4 and IL-10 for arginase up-regulation. Finally, strong synergistic effects between IL-4/IL-13 and IL-10 were observed, sufficient to account for the extraordinarily high arginase activity induced by Th2 cells. Our results suggest that the iNOS/arginase balance in macrophages is competitively regulated in the context of Th1- vs Th2-driven immune reactions, most likely by cytokines without the requirement for direct cell interaction.
IFN-γ Priming Up-Regulates IFN-Stimulated Gene Factor 3 (ISGF3) Components, Augmenting Responsiveness of IFN-Resistant Melanoma Cells to Type I IFNs
Authors: Wong Lee H. , Hatzinisiriou Irene , Devenish Rodney J. , Ralph Stephen J.
Published: January 2023

IFN-stimulated gene factor 3 (ISGF3) mediates transcriptional activation of IFN-sensitive genes (ISGs). The component subunits of ISGF3, STAT1αβ, STAT2, and p48-ISGF3γ, are tyrosine phosphorylated before their assembly into a complex. Subsequently, the ISGF3 complex is translocated to the nucleus. We have recently established that the responsiveness of human melanoma cell lines to type I IFNs correlates directly with their intracellular levels of ISGF3 components, particularly STAT1. In the present study, we show that pretreating IFN-resistant melanoma cell lines with IFN-γ (IFN-γ priming) before stimulation with type I IFN also results in increased levels of ISGF3 components and enhanced DNA-binding activation of ISGF3. In addition, IFN-γ priming of IFN-resistant melanoma cell lines increased expression of type I IFN-induced ISG products, including ISG54, 2′-5′-oligoadenylate synthase, HLA class I, B7-1, and ICAM-1 Ags. Furthermore, IFN-γ priming enhanced the antiviral effect of IFN-β on the IFN-resistant melanoma cell line, MM96. These results support a role for IFN-γ priming in up-regulating ISGF3, thereby augmenting the responsiveness of IFN-resistant melanoma cell lines to type I IFN and providing a molecular basis and justification for using sequential IFN therapy, as proposed by others, to enhance the use of IFNs in the treatment of melanoma.
Heparin-Binding Protein (CAP37) Is Internalized in Monocytes and Increases LPS-Induced Monocyte Activation
Authors: Heinzelmann Michael , Mercer-Jones Mark A. , Flodgaard Hans , Miller Frederick N.
Published: January 2023

Previous studies have shown that the neutrophil-derived heparin-binding protein (HBP), also known as CAP37 or azurocidin, potentiates the LPS-induced release of proinflammatory cytokines (TNF-α, IL-1, and IL-6) from isolated human monocytes. To date, the mechanisms by which HBP enhances LPS-induced monocyte activation have not been elucidated, and it is not known whether HBP also increases the LPS-induced production of other bioactive substances. We studied human monocytes activated by recombinant human HBP and LPS and their interaction with the LPS receptor CD14. We hypothesized that the stimulatory effect of HBP on the LPS-induced release of proinflammatory mediators from monocytes was mediated by specific binding of HBP to monocytes, which resulted in an up-regulation of CD14. Our results demonstrated that HBP alone (10 μg/ml) stimulated the production of TNF-α from isolated monocytes. In addition, HBP had an additive effect on LPS-induced production of TNF-α and PGE2, suggesting a generalized monocyte activation. We used flow cytometry to demonstrate that HBP had a high affinity to monocytes but not to the LPS receptor CD14, and experiments performed at 4°C indicated an energy-dependent step in this process. Confocal microscopy showed that monocytes internalize HBP within 30 min. These data suggest that mechanisms other than increased CD14 expression are responsible for the enhanced release of TNF-α or PGE2 in response to HBP and LPS.
Soluble IL-15 Receptor α-Chain Administration Prevents Murine Collagen-Induced Arthritis: A Role for IL-15 in Development of Antigen-Induced Immunopathology
Authors: Ruchatz Holger , Leung Bernard P. , Wei Xiao-qing , McInnes Iain B. , Liew Foo Y.
Published: January 2023

IL-15 has recently been detected in the synovium of patients with rheumatoid arthritis. IL-15-activated T cells induce significant TNF-α synthesis by macrophages via a cell contact-dependent mechanism, suggesting a key regulatory role for IL-15. Here, we report that the administration of a soluble fragment of IL-15Rα into DBA/1 mice, profoundly suppressed the development of collagen-induced arthritis. This was accompanied in vitro by marked reductions in Ag-specific proliferation and IFN-γ synthesis by spleen cells from treated mice compared with control mice and in vivo by a significant reduction in serum anti-collagen Ab levels. These data directly demonstrate a pivotal role for IL-15 in the development of inflammatory arthritis and also suggest that antagonists to IL-15 may have therapeutic potential in rheumatic diseases.
Thymus Requirement and Antigen Dependency in the “Infectious” Tolerance Pathway in Transplant Recipients
Authors: Onodera Kazuhiko , Volk Hans-Dieter , Ritter Thomas , Kupiec-Weglinski Jerzy W.
Published: January 2023

We have shown that features of infectious tolerance, as originally described in thymectomized mice, may be applied to euthymic rat recipients of heart transplants. We now report on studies aimed at exposing mechanisms underlying the infectious tolerance pathway, with emphasis on the role of thymus and alloantigen. Pretransplant thymectomy diminished the efficacy of CD4-targeted therapy, with donor-specific tolerance induced in ∼50% of recipients. Thymus was required for generation of regulatory T cells under the cover of CD4 mAb therapy and for the ability of these cells to confer infectious tolerance. However, thymus was not mandatory to maintain an infectious-permissive environment in cohorts of adoptively transferred recipients. Intragraft expression of IL-2, IL-4, and IL-10 genes was diminished in euthymic and thymectomized tolerant hosts. However, grafts in the latter group showed significant IFN-γ gene expression, suggesting a less efficient down-regulation of Th1-like cells in the absence of regulatory cells. Indeed, exogenous challenge with rIL-2 or freshly alloactivated spleen cells recreated rejection in thymectomized, but not euthymic, hosts, suggesting that a state of cytokine-responsive anergy contributes to the “noninfectious” form of tolerance in thymectomized rats. The infection-tolerant state did not result from “graft adaptation,” and regulatory T cells restricted for the original alloantigen were exposed to its continuous stimulation. The effective memory for suppression was dependent upon persistent donor-specific alloantigen stimulation; it disappeared within 3 weeks after its removal. Hence, both central and peripheral immune mechanisms, orchestrated by the tolerizing alloantigen, contribute to the infectious tolerance pathway in CD4 mAb-treated rat transplant recipients.
Cooperation Between CD44 and LFA-1/CD11a Adhesion Receptors in Lymphokine-Activated Killer Cell Cytotoxicity
Authors: Matsumoto Goichi , Nghiem Mai P. , Nozaki Naohito , Schmits Rudolf , Penninger Josef M.
Published: January 2023

IL-2-activated NK cells exhibit cytotoxic activity against a wide variety of tumor cells in a non-MHC-restricted fashion and in the absence of prior sensitization. The molecular mechanisms that regulate the cytotoxicity and attachment of activated killer cells to tumor target cells are not known. We provide genetic evidence in CD44−/− and LFA-1−/− mice that the cell adhesion receptors LFA-1 and CD44 regulate the cytotoxic activity of IL-2-activated NK cells against a variety of different tumor cells. This defect in cytotoxicity was significantly enhanced in mice that carried a double mutation of both CD44 and LFA-1. In vitro differentiation, TNF-α and IFN-γ production, and expression of the cytolytic effector molecules perforin and Fas-L were comparable among IL-2-activated NK cells from LFA-1−/−, CD44−/−, CD44−/−LFA-1−/−, and control mice. However, CD44−/−, LFA-1−/−, and CD44−/−LFA-1−/− IL-2-activated NK cells showed impaired binding and conjugate formation with target cells. We also show that hyaluronic acid is the principal ligand on tumor cells for CD44-mediated cytotoxicity of IL-2-activated NK cells. These results provide the first genetic evidence of the role of adhesion receptors in IL-2-activated NK killing. These data also indicate that distinct adhesion receptors cooperate to mediate binding between effector and target cells required for the initiation of “natural” cytotoxicity.
Dissection of Pathways Leading to Antigen Receptor-Induced and Fas/CD95-Induced Apoptosis in Human B Cells
Authors: Lens Susanne M. A. , den Drijver Bianca F. A. , Pötgens Andy J. G. , Tesselaar Kiki , van Oers Marinus H. J. , van Lier René A. W.
Published: January 2023

To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1β-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.
Arachidonic Acid Enhances the Tissue Factor Expression of Mononuclear Cells by the Cyclo-Oxygenase-1 Pathway: Beneficial Effect of n-3 Fatty Acids
Authors: Cadroy Yves , Dupouy Dominique , Boneu Bernard
Published: January 2023

Monocytes express tissue factor (TF) upon stimulation by inflammatory agents. Dietary administration of fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) results in an impairment of TF expression by monocytes. EPA and DHA are metabolized differently from arachidonic acid (AA), the major fatty acid present in cell membranes. We examined the effects of AA on the TF expression of isolated human PBMC, and we determined whether EPA and DHA modulated this phenomenon differently. Nonstimulated PBMC had a low TF-dependent procoagulant activity. When PBMC were incubated with increasing concentrations of AA, the TF-dependent procoagulant activity increased in a dose-dependent manner to 190% at 7.5 μM. Indomethacin, a cyclo-oxygenase inhibitor, totally abolished the stimulating effect of AA, whereas specific pharmacologic inhibitors of cyclo-oxygenase-2 or of 5-lipoxygenase had no inhibitory effect. A thromboxane (TX)A2/endoperoxides receptor antagonist and a TX synthase inhibitor blocked the potentiating effect of AA. Purified PGG2 and carbocyclic TXA2, a TXA2 agonist, enhanced the procoagulant activity of PBMC in a dose-dependent manner whereas, in contrast, PGE2 inhibited it. Finally, contrary to AA, EPA or DHA did not increase TXB2 production or TF expression by PBMC. The TF-dependent procoagulant activity of isolated PBMC was increased by AA through the production of cyclo-oxygenase-1 metabolites; the combined action of PGG2 and TXA2, which potentiated it, was greater than that of PGE2, which inhibited it. Dietary n-3 fatty acids exert part of their beneficial effect by modulating this procoagulant activity differently from AA.
T Helper 1 Response Is Dominant and Localized to the Synovial Fluid in Patients with Lyme Arthritis
Authors: Gross Dawn M. , Steere Allen C. , Huber Brigitte T.
Published: January 2023

Cytokines produced by subsets of CD4+ T helper cells responding to an infection influences the efficiency with which the host is able to mount a protective immune response. In an attempt to elucidate the population of active cells involved in the propagation of Lyme arthritis we have utilized intracellular cytokine staining to analyze the polyclonal immune response at the single cell level. We have determined the Th phenotype in the synovial fluid of patients with a variety of chronic inflammatory arthritides, including patients representative of the spectrum of Lyme arthritis. Th1 cells dominate the immune response in the synovial fluid of patients with Lyme as well as those with rheumatoid or other types of chronic inflammatory arthritis. In addition, the severity of Lyme arthritis directly correlates with the ratio of Th1 to Th2 cells in the synovial fluid, such that the larger the effusion, the higher the ratio (r = 0.67, p < 0.05). These results suggest that Th1 cells play a direct role in the pathogenesis of the inflammatory process seen in Lyme arthritis, and that Th2 cells modulate the pro-inflammatory response generated by Th1 cells in the joint. Finally, we identify Th1 cells specific for outer surface protein A of Borrelia burgdorferi, the agent of Lyme disease. These cells are restricted to patients with Lyme arthritis and are localized to the joint. Furthermore, they persist in patients with prolonged antibiotic treatment-resistant Lyme arthritis, suggesting the possibility of an autoimmune process.
Activation of STAT5 by Lipopolysaccharide Through Granulocyte-Macrophage Colony-Stimulating Factor Production in Human Monocytes
Authors: Yamaoka Kunihiro , Otsuka Takeshi , Niiro Hiroaki , Arinobu Yojiro , Niho Yoshiyuki , Hamasaki Naotaka , Izuhara Kenji
Published: January 2023

LPS is a potent stimulator of monocytes, inducing many of their functions. Although the details of how LPS exerts such functions remain largely unknown, transcription factors such as nuclear factor-κB, nuclear factor-IL-6, and activator protein-1 have been shown to be involved in this process. However, to date it has been thought that no known STAT molecule plays a role in the activation of monocytes by LPS. In this study we examined whether some known STAT molecule is stimulated by LPS, based on the finding that a GAS motif sequence is conserved in the promoter regions of human, mouse, and rat cyclo-oxygenase-2 (COX-2) genes. Consequently, LPS induced activation of STAT5 in human monocytes, and this STAT5 activation occurred in an indirect way via granulocyte-macrophage CSF (GM-CSF) secreted by LPS-stimulated monocytes. Expression of COX-2 protein was partially reduced by treatment of anti-human GM-CSF Ab. Activation of STAT5 was inhibited by either IL-10 or dexamethasone (Dex), but not by aspirin. IL-10 blocked activation of STAT5 indirectly by suppressing GM-CSF production, while Dex inhibited this activation both directly and indirectly. Taken together, these results suggest that in addition to other transcription factors, STAT5 plays an important role in activation of monocytes by LPS, and that STAT5 is another target for IL-10 and Dex to inhibit COX-2 expression in activated monocytes.
Perforin-Deficient CD8+ T Cells Provide Immunity to Listeria monocytogenes by a Mechanism That Is Independent of CD95 and IFN-γ but Requires TNF-α
Authors: White Douglas W. , Harty John T.
Published: January 2023

CD8+ T cells are effective mediators of immunity against Listeria monocytogenes, but the mechanisms by which they provide antilisterial immunity are poorly understood. CD8+ T cells efficiently lyse target cells in vitro by at least two independent pathways. To test the hypothesis that CD8+ T cell-mediated immunity to L. monocytogenes is dependent on perforin or CD95 (Fas, Apo-1), we used C57Bl/6 (B6) and perforin-deficient (PO) mice to generate CD8+ T cell lines specific for the L. monocytogenes-encoded Ag listeriolysin O (LLO). Both lines specifically produce IFN-γ and TNF-α, and mediate target cell lysis in vitro. Cytolysis mediated by the PO-derived CD8+ T cell line is delayed relative to the B6-derived line and is completely inhibited by anti-CD95 Abs. In vivo, PO-derived CD8+ T cells provide specific antilisterial immunity in B6 hosts, CD95-deficient hosts, and IFN-γ-depleted hosts. However, PO-derived CD8+ T cells fail to provide antilisterial immunity in hosts depleted of TNF-α. These results indicate that single Ag-specific CD8+ T cells derived from PO mice can mediate antilisterial immunity by a mechanism that is independent of CD95 or IFN-γ, but requires TNF-α.
IL-12 Reverses Established Antigen-Specific Tolerance of Contact Sensitivity by Affecting Costimulatory Molecules B7-1 (CD80) and B7-2 (CD86)
Authors: Ushio Hiroko , Tsuji Ryohei F. , Szczepanik Marian , Kawamoto Keiko , Matsuda Hiroshi , Askenase Philip W.
Published: January 2023

Cutaneous painting with reactive haptens induces contact sensitivity (CS) responses that are in vivo examples of T cell immunity. In contrast, high dose i.v. administration of the hapten can induce tolerance. We investigated the effect of IL-12 on reversal of this tolerance and attempted to determine in vitro the mechanism of this reversing effect by measuring proliferation and IFN-γ production by CS effector T cells stimulated with hapten-conjugated APC, and we also measured CS ear swelling in vivo. The in vitro responses of T cells to hapten-APC became absent in tolerized mice, paralleling impaired in vivo CS responses. Addition of IL-12 to cultures manifesting this fully established in vitro tolerance completely restored impaired responses of tolerized T cells. The reversing effects of IL-12 were not blocked by anti-IFN-γ mAb, but were blocked by mAbs against B7-1, more strongly by anti-B7-2, and by both Abs together. Additional in vivo ear-swelling response experiments confirmed the reversing effects of IL-12 on established tolerance. To examine whether the IL-12 effect depended on stimulation of IFN-γ, we directly injected IFN-γ into tolerized mice. This partially mimicked but did not fully reconstitute the effects of IL-12. In summary, IL-12 abrogation of established tolerance of CS may have been partially due to endogenous production of IFN-γ, but appeared mainly due to direct activation of the tolerized T cells by affecting signaling through costimulatory molecules B7-1 and B7-2.