bluuebunny/crossref_metadata_2025 huggingface dataset:
NF-κB Activation in CD27 Signaling: Involvement of TNF Receptor-Associated Factors in Its Signaling and Identification of Functional Region of CD27
Authors: Yamamoto Hiroyasu , Kishimoto Tadamitsu , Minamoto Seijiro
Published: December 2022

CD27 belongs to TNF receptor family, and it is unique in this family for its disulfide-linked homodimerization of 55-kDa monomers. In the present study we demonstrate that overexpression of CD27 in 293 cells induces a low level of NF-κB activation, and the ligation of the receptor by its corresponding ligand (CD70) augments this signal dramatically. Either TNF receptor-associated factor-2 (TRAF2) or TRAF3 binds to the CD27 molecule from the coimmunoprecipitation experiment. This NF-κB activation signal is inhibited by dominant negative TRAF2 or intact TRAF3, indicating that TRAF2 and TRAF3 works as a mediator and an inhibitor, respectively. The activated NF-κB complex contains at least two components, p50 and p65, but not p52. All these phenomena have also been observed in the TNF receptor type II, CD30 and CD40 signaling system, indicating that this receptor family uses the common or similar molecules for this signal. Finally, we identified the 13-amino acid alignment in the cytoplasmic region of the CD27 molecule (residues 238–250 amino acids), which is critical for the NF-κB activation signal and also for its association with TRAFs. This amino acid alignment contains the EEEG sequence, which is essential for interaction of CD30 or CD40 with TRAFs (TRAF1 and TRAF2, but not TRAF3), and also contains the PIQED sequence, which is similar to PXQXT that is known to be necessary for interaction of TNF receptor II and CD30 with TRAFs (TRAF1, 2, and 3).
Autocrine/Paracrine IFN-αβ Mediates the Lipopolysaccharide-Induced Activation of Transcription Factor Stat1α in Mouse Macrophages: Pivotal Role of Stat1α in Induction of the Inducible Nitric Oxide Synthase Gene
Authors: Gao Jian Jun , Filla Michael B. , Fultz Marion J. , Vogel Stefanie N. , Russell Stephen W. , Murphy William J.
Published: December 2022

We have examined the role of Stat1α in the induction by LPS of the mouse inducible nitric oxide synthase (EC 1.14.13.39) gene. LPS induced both the tyrosine phosphorylation of Stat1α and the production of nitric oxide in a time- and dose-dependent manner. The phosphorylation of Stat1α elicited by LPS differed from that observed using IFN-γ or IFN-β, in that LPS induced less phosphorylated protein and the time course of induction was much delayed (2–4 h compared with 30 min). Cycloheximide inhibited LPS-mediated Stat1α phosphorylation. In addition, cell culture supernatants derived from macrophages treated with LPS for 4 h could be transferred to naive macrophage cultures resulting in rapid (30 min), rather than delayed (4 h), phosphorylation of Stat1α. Together, these results implicated an autocrine/paracrine effector protein(s) in the phosphorylation process. LPS stimulated phosphorylation of Stat1α in peritoneal macrophages derived from IFN-γ-knockout mice, negating any possibility that IFN-γ was the mediator. By contrast, neutralizing Ig raised against mouse IFN-αβ inhibited both the delayed LPS-mediated phosphorylation of Stat1α and the rapid induction of phosphorylation induced by supernatants from LPS-stimulated cultures. Collectively, these results show that LPS-induced IFN-αβ production, Stat1α activation, and nitrite accumulation closely parallel one another, suggesting that indirect activation of transcription factor Stat1α by IFN-αβ is a critical determinant of LPS-mediated inducible nitric oxide synthase gene expression.
Recognition of Two Overlapping CTL Epitopes in HIV-1 p17 by CTL from a Long-Term Nonprogressing HIV-1-Infected Individual
Authors: Harrer Thomas , Harrer Ellen , Barbosa Peter , Kaufmann Friedemann , Wagner Ralf , Brüggemann Susanne , Kalden Joachim R. , Feinberg Mark , Johnson R. Paul , Buchbinder Susan , Walker Bruce D.
Published: December 2022

HIV-1 infection has been shown to elicit strong CTL responses in some infected persons, but few data are available regarding the relationship between targeted epitopes and in vivo viral quasispecies. In this study, we examined the CTL response in a person infected for 15 yr with a CD4 count persistently >500 cells/μl. The dominant in vivo activated CTL response was directed against two overlapping Gag CTL epitopes in an area of p17 known to be essential for viral replication. The 9-mer SLYNTVATL (amino acids 77–85) was recognized in conjunction with HLA-A2, whereas the overlapping 8-mer TLYCVHQR (amino acids 83–91) was recognized by HLA-A11-restricted CTL. Analysis of in vivo virus sequences both in PBMC and plasma revealed the existence of sequence variation in this region, which did not affect viral replication in vitro, but decreased recognition by the A11-restricted CTL response, with maintenance of the A2-restricted response. These results indicate that an essential region of the p17 protein can be simultaneously targeted by CTL through two different HLA molecules, and that immune escape from CTL recognition can occur without impairing viral replication. In addition, they demonstrate that Ag processing can allow for presentation of overlapping epitopes in the same infected cell, which can be affected quite differently by sequence variation.
Attenuation of HLA-DR Expression by Mononuclear Phagocytes Infected withMycobacterium tuberculosisIs Related to Intracellular Sequestration of Immature Class II Heterodimers
Authors: Hmama Zakaria , Gabathuler Reinhard , Jefferies Wilfred A. , de Jong Gary , Reiner Neil E.
Published: December 2022

MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-γ increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRα, HLA-DRβ, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II αβ dimers—dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment—was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.
Role of the Hypervariable Region in Streptococcal M Proteins: Binding of a Human Complement Inhibitor
Authors: Johnsson Eskil , Berggård Karin , Kotarsky Heike , Hellwage Jens , Zipfel Peter F. , Sjöbring Ulf , Lindahl Gunnar
Published: December 2022

Antigenic variation allows pathogenic microorganisms to evade the immune system of the infected host. The variable structure must play an important role in pathogenesis, but its function is in most cases unknown. Here, we identify a function for the surface-exposed hypervariable region of streptococcal M5 protein, a virulence factor that inhibits phagocytosis. The hypervariable region of M5 was found to bind the human complement inhibitor FHL-1 (factor H-like protein 1), a 42-kDa plasma protein. Plasma absorption experiments with M5-expressing bacteria showed that the interaction with FHL-1 occurs also under physiologic conditions. Studies of another extensively characterized M protein, M6, indicated that this protein also has a binding site for FHL-1 in the hypervariable region. The complement-inhibitory function of FHL-1 was retained after binding to streptococci, suggesting that bound FHL-1 protects bacteria against complement attack. All available data now indicate that FHL-1, or another human complement inhibitor, binds to the hypervariable region of M proteins. These findings provide insights into the forces that drive antigenic variation and may explain why the hypervariable region of M protein is essential for phagocytosis resistance. Moreover, these data add to a growing body of evidence that human complement inhibitors are major targets for pathogenic microorganisms.
Two Lineages of Mannose-Binding Lectin-Associated Serine Protease (MASP) in Vertebrates
Authors: Endo Yuichi , Takahashi Minoru , Nakao Miki , Saiga Hidetoshi , Sekine Hideharu , Matsushita Misao , Nonaka Masaru , Fujita Teizo
Published: December 2022

Mannose-binding lectin-associated serine protease (MASP) is a newly identified member of the serine protease superfamily. MASP is involved in host defense against pathogens through a novel system of complement activation, designated the lectin pathway. To elucidate the origin of the lectin pathway and the molecular evolution of MASP, we cloned six MASP cDNAs from five vertebrate species going from mammal to cyclostome. An alignment of the amino acid sequences deduced from the cDNAs revealed the presence of two different lineages of the MASP gene. This classification was supported by the precise correlation with two types of exon organization for the protease domain. One of the two lineages is unique in that a single exon encodes the protease domain, unlike most other serine proteases. All members of this group, termed the AGY type, have an AGY codon at the active site serine. A phylogenetic tree suggests that the AGY type diverged from another lineage, termed the TCN type, before the emergence of primitive vertebrates. Furthermore, the presence of MASP or MASP-like sequences in most vertebrate species suggests that the lectin pathway functions extensively in vertebrates and that its origin is traced back to the invertebrate stage.
Inhibition by Dexamethasone of Antigen-Induced c-Jun N-Terminal Kinase Activation in Rat Basophilic Leukemia Cells
Authors: Hirasawa Noriyasu , Sato Yukako , Fujita Yuhko , Mue Suetsugu , Ohuchi Kazuo
Published: December 2022

Antigen stimulation of IgE-sensitized rat basophilic leukemia RBL-2H3 cells induced activation of c-Jun N-terminal kinase (JNK) within a few minutes with maximum activity attained 40 min later. The increase in JNK activity was accompanied with an increase in phosphorylation of c-Jun in the cells. The Ag-induced JNK activation was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin (10–100 nM) and LY 294002 (100 μM) but not by the protein kinase C inhibitors calphostin C (1 and 3 μM) and Ro 31-8425 (1 and 3 μM). Pretreatment with dexamethasone (10 and 100 nM) for 18 h inhibited the Ag-induced increase in JNK activity in a concentration-dependent manner. At least 6 h of preincubation with dexamethasone was necessary to inhibit the Ag-induced JNK activation. The phosphorylation of c-Jun induced by the Ag stimulation was reduced by pretreatment with dexamethasone without reduction of the content of c-Jun protein. The Ag-induced activation of the JNK kinase kinase mitogen-activated protein kinase-extracellular signal-regulated kinase kinase-1 was also inhibited by pretreatment with dexamethasone at 10 and 100 nM. These findings indicate that dexamethasone reduces JNK protein level and inhibits the Ag-induced activation of JNK resulting in the inhibition of c-Jun phosphorylation.
Induction of Cyclooxygenase-2 by Secretory Phospholipases A2 in Nerve Growth Factor-Stimulated Rat Serosal Mast Cells Is Facilitated by Interaction with Fibroblasts and Mediated by a Mechanism Independent of Their Enzymatic Functions
Authors: Tada Kinji , Murakami Makoto , Kambe Terumi , Kudo Ichiro
Published: December 2022

Mast cells exhibit a biphasic (immediate and delayed) eicosanoid-biosynthetic response after stimulation with particular cytokines or FcεRI (high affinity receptor for IgE) cross-linking. Treatment of rat serosal connective tissue mast cells (CTMC) with nerve growth factor (NGF) induced only the delayed phase of PGD2 generation that depended on inducible cyclooxygenase-2 (COX-2), but not constitutive COX-1, even though the subcellular distributions of these isoforms were similar. Experiments using several phospholipase A2 (PLA2) isozyme-specific probes and inhibitors suggested that both constitutive cytosolic PLA2 and inducible type IIA secretory PLA2 (sPLA2) are involved in NGF-initiated, COX-2-dependent, delayed PGD2 generation in rat CTMC. A type IIA sPLA2 inhibitor, but neither cytosolic PLA2 nor COX inhibitors, reduced, while adding exogenous type IIA sPLA2 augmented, NGF-induced COX-2 expression and its attendant PGD2 generation, indicating that the sPLA2-mediated increase in delayed PGD2 generation was attributable mainly to enhanced COX-2 expression. Type IIA sPLA2 and its close relative type V sPLA2 associated with fibroblastic cell surfaces increased NGF-induced COX-2 expression more efficiently than the soluble enzymes, revealing a particular juxtacrine sPLA2 presentation route. Surprisingly, catalytically inactive type IIA sPLA2 mutants, which were incapable of promoting arachidonic acid release from cytokine-primed cells, retained the ability to enhance COX-2 expression in CTMC, indicating that the COX-2-inducing activities of sPLA2 are independent of their catalytic functions.
STCP-1 (MDC) CC Chemokine Acts Specifically on Chronically Activated Th2 Lymphocytes and Is Produced by Monocytes on Stimulation with Th2 Cytokines IL-4 and IL-13
Authors: Andrew David P. , Chang Ming-shi , McNinch Jennifer , Wathen Scott T. , Rihanek Marynette , Tseng Julia , Spellberg Jason P. , Elias Chester G.
Published: December 2022

STCP-1 stimulated T cell chemoattractant protein-1 (STCP-1) (macrophage-derived chemokine; MDC), a recently described CC chemokine for chronically activated T lymphocytes, was found to act specifically on a subset of memory CD4 lymphocytes that displayed a Th2 cytokine profile. Also, STCP-1, thymus and activation regulated chemokine (TARC), eotaxin, and eotaxin-2 acted specifically on in vitro derived Th2 lymphocytes, while IP-10 (IFN-γ-inducible 10-kDa protein) showed some preference for Th1 lymphocytes. The corresponding receptors for eotaxin, TARC, and IP-10 are also differentially expressed on Th1 and Th2 lymphocytes. In desensitization Ca flux experiments, TARC and STCP-1 bound to a common receptor and therefore at least one chemokine receptor for STCP-1 is CCR4. STCP-1 expression is restricted to immune cells. Dendritic cells, B cells, and macrophages produce STCP-1 constitutively, while NK cells, monocytes, and CD4 lymphocytes produce STCP-1 upon appropriate stimulation. Production of STCP-1 is positively modulated by Th2 cytokines IL-4 and IL-13 but inhibited by IL-10.
Thrombin Receptor Activation Inhibits Monocyte Spreading by Induction of ETB Receptor-Coupled Nitric Oxide Release
Authors: Srivastava Kamal D. , Magazine Harold I.
Published: December 2022

The effect of thrombin receptor activation on monocyte conformation was evaluated using the human monocyte cell line, THP-1, and the thrombin mimetic peptide, Trap-14. Treatment of THP-1 cells with Trap-14 induced rapid rounding of ameboid cells adherent to fibronectin-coated slides, whereas cell rounding was abrogated in the presence of the nitric oxide synthase inhibitor, NG-nitro-l-arginine or the endothelin B receptor antagonist, BQ-788. Endothelin-1 (ET-1) levels in the culture supernatant increased markedly within minutes of Trap-14 exposure with a concomitant loss in cellular ET-1 immunoreactivity. Importantly, loss of ET-1 immunoreactivity was blocked by pretreatment with the vesicle translocation inhibitor, nocodazole. Trap-14 potently induced the release of NO from THP-1 cells, whereas NO release was ablated by preincubation with BQ-788. These data demonstrate that thrombin receptor activation may inhibit cellular spreading as a result of autocrine ET-1 release and subsequent endothelin B receptor-dependent NO production, and suggest that initial exposure of inflammatory cells to thrombin may limit cellular activation and recruitment.
Prevention of Th2-Like Cell Responses by Coadministration of IL-12 and IL-18 Is Associated with Inhibition of Antigen-Induced Airway Hyperresponsiveness, Eosinophilia, and Serum IgE Levels
Authors: Hofstra Claudia L. , Van Ark Ingrid , Hofman Gerard , Kool Mirjam , Nijkamp Frans P. , Van Oosterhout Antoon J. M.
Published: December 2022

Allergic asthma is thought to be regulated by Th2 cells, and inhibiting this reponse is a promising mode of intervention. Many studies have focused on differentiation of Th cells to the Th1 or Th2 subset in vitro. IL-4 is essential for Th2 development, while IL-12 induces Th1 development, which can be enhanced by IL-18. In the present study, we investigated whether IL-12 and IL-18 were able to interfere in Th2 development and the associated airway symptoms in a mouse model of allergic asthma. Mice were sensitized with OVA using a protocol that induces IgE production. Repeated challenges by OVA inhalation induced elevated serum levels of IgE, airway hyperresponsiveness, and a predominantly eosinophilic infiltrate in the bronchoalveolar lavage concomitant with the appearance of Ag-specific Th2-like cells in lung tissue and lung-draining lymph nodes. Whereas treatments with neither IL-12 nor IL-18 during the challenge period were effective, combined treatment of IL-12 and IL-18 inhibited Ag-specific Th2-like cell development. This inhibition was associated with an absence of IgE up-regulation, airway hyperresponsiveness, and cellular infiltration in the lavage. These data show that, in vivo, the synergistic action of IL-12 and IL-18 is necessary to prevent Th2-like cell differentiation, and consequently inhibits the development of airway symptoms in a mouse model of allergic asthma.
Retrovirally Transduced Bone Marrow-Derived Dendritic Cells Require CD4+ T Cell Help to Elicit Protective and Therapeutic Antitumor Immunity
Authors: De Veerman Marijke , Heirman Carlo , Van Meirvenne Sonja , Devos Sophie , Corthals Jurgen , Moser Muriel , Thielemans Kris
Published: December 2022

It has been extensively documented that murine dendritic cells loaded with tumor-associated Ag (TAA)-derived peptides or protein can prime Ag-specific CD8+ cytotoxic T cells in vivo and can elicit Ag-specific immunity. Optimal presentation of TAA might be achieved by retroviral transduction of DCs allowing long term and stable expression of the TAA-peptides as well as the presentation of multiple epitopes in the context of MHC class I and/or class II molecules. Here we show that retroviral transduction of bone marrow-derived dendritic cells (DCs) with chicken OVA cDNA or the reporter gene green fluorescent protein retained their potent stimulatory capacity and that the transduced DCs could process and present the endogenously expressed OVA protein. The DCs transduced with cDNA encoding native OVA protein presented OVA-derived peptides in the context of MHC class I as well as MHC class II and induced a strong Ag-specific CTL response. DCs expressing a cytosolic form of OVA presented OVA peptides only in the context of MHC class I and failed to induce an OVA-specific CTL response in vivo when they had been cultured in the absence of exogenous protein. Immunization with retrovirally transduced DCs resulted in an Ag-specific immunity and rejection of a tumor cell challenge and a significant survival advantage in tumor-bearing mice. These results obtained in this rapidly lethal tumor model suggest that DCs transduced with TAA may be useful for tumor immunotherapy and underscore the importance of the simultaneous delivery of T cell help in the development of Ag-specific cytotoxic T-cells.
CD28-Deficient Mice Are Highly Resistant to Collagen-Induced Arthritis
Authors: Tada Yoshifumi , Nagasawa Kohei , Ho Alexandra , Morito Fumiaki , Ushiyama Osamu , Suzuki Noriaki , Ohta Hideaki , Mak Tak Wah
Published: December 2022

CD28 provides a critical costimulatory signal in Ag-specific T cell activation. Recent studies have revealed an important role for CD28 in the development of autoimmune diseases. We have examined the role of CD28 in collagen-induced arthritis (CIA) by inducing CIA in CD28-deficient DBA/1 mice. CD28-deficient mice never developed arthritis and showed markedly decreased levels of IgG and IgM anti-type II collagen (CII) Abs. In addition, the CD28+/− mice had similar levels of IgG1 and IgG2a anti-CII Abs, whereas in the CD28-deficient mice the level of IgG1 anti-CII Abs was decreased compared with that of IgG2a. IFN-γ production by lymph node cells in response to CII was also reduced. CD28-deficient mice were either immunized four times with CII in CFA to augment Ag loading or given low doses of IL-12 to enhance Th1 type responses. Both treatments resulted in a very low incidence of CIA development and minimal disease. CD28-deficient mice developed arthritis from injection of lymph node cells from CII-immunized wild-type mice, followed by immunization with CII in CFA. Taken together, these results indicate that costimulation of CD28 cannot be replaced by repeated activation through TCR or other costimulatory molecules. Thus, CD28 plays a critical role in both cellular and humoral immunity against CII and is indispensable for the development of CIA.
A Novel Mechanism of CD4 Lymphocyte Depletion Involves Effects of HIV on Resting Lymphocytes: Induction of Lymph Node Homing and Apoptosis Upon Secondary Signaling Through Homing Receptors
Authors: Wang Liqiang , Chen Jenny J. Y. , Gelman Benjamin B. , Konig Rolf , Cloyd Miles W.
Published: December 2022

Recently, we reported that abortive HIV infection of resting human T lymphocytes up-regulated expression of CD62L, the receptor for homing to lymph nodes (LNs), and enhanced homing of these cells from the blood into the LNs (Wang et al., 1997, Virology 228:141). This suggested that HIV-induced homing of resting lymphocytes (which comprise >98% of all lymphocytes) may be a major mechanism for the reduction of CD4+ lymphocytes in the blood of infected individuals. This mechanism also could be partially responsible for the lymphadenopathy that often develops at the same time that CD4+ lymphocytes are disappearing from the blood. In this study, we show that secondary signaling through the homing receptors (CD62L, CD44, CD11a) of abortively infected resting CD4+ T lymphocytes induced apoptosis. These signals would occur as the cells home into the LNs. Apoptosis did not occur after secondary signaling through some other receptors (CD26, CD4, CD45, and HLA class I) or in HIV-exposed resting CD8+ lymphocytes signaled through the homing receptors. These findings indicate that HIV-induced homing of resting CD4+ lymphocytes to LNs results in death of many of these cells. This was confirmed in the LNs of SCID mice that were i.v. injected with HIV-exposed resting human lymphocytes. Thus, these effects of HIV upon binding to resting CD4+ T lymphocytes, which are not permissive for HIV replication, may significantly contribute to their depletion in vivo. These findings also offer an explanation for the bystander effect observed in the LNs of AIDS patients, whereby cells not making virus are dying.
Enhanced Epidermal Langerhans Cell Migration in IL-10 Knockout Mice
Authors: Wang Binghe , Zhuang Lihua , Fujisawa Hiroshi , Shinder Gayle A. , Feliciani Claudio , Shivji Gulnar M. , Suzuki Hirotake , Amerio Paolo , Toto Paola , Sauder Daniel N.
Published: December 2022

The migration of epidermal Langerhans cells (LC) to lymph nodes (LN) is critical in the initiation of contact hypersensitivity (CHS) responses. Studies suggest that contact allergen-induced epidermal proinflammatory cytokines, including IL-1 and TNF-α, play important roles in promoting LC migration. Contact allergens also induce epidermal anti-inflammatory cytokines such as IL-10. Since IL-10 down-regulates proinflammatory cytokine production and inhibits CHS, we hypothesized that IL-10 might inhibit LC migration. To test this hypothesis, IL-10 knockout (KO) mice were epicutaneously sensitized with the hapten, FITC, and 24 h later hapten-bearing cells in the draining LN were examined. The number of hapten-bearing cells in the LN was significantly greater in IL-10 KO mice than in wild-type mice. The mutant mice also had an exaggerated CHS to FITC. Pretreatment with anti-TNF-α Ab or IL-1R antagonist significantly reduced the number of hapten-bearing cells in the LN, suggesting that IL-10 modulation of LC migration involves IL-1 and TNF-α. Moreover, IL-10 KO mice demonstrated a greater increase in TNF-α, IL-1α, and IL-1β mRNAs in the allergen-exposed epidermis, and keratinocytes derived from the mutant mice were able to produce higher amounts of TNF-α and IL-1α protein. These data suggest that IL-10 plays an inhibitory role in LC migration and that this effect may occur via the down-regulation of TNF-α and IL-1 production.
Redirecting Effector T Cells Through Their IL-2 Receptors
Authors: Lustgarten Joseph , Marks James , Sherman Linda A.
Published: December 2022

Fusion proteins constructed of a tumor-specific Ab joined to IL-2 (Ab-IL-2) have been used in the past to deliver cytokine directly to the site of tumor cells in vivo. These molecules mimic the activity of IL-2 and assist in activating and expanding antitumor effector cells. To enhance the cytolytic activity of CTL specific for peptide epitopes of the Her-2/neu tumor Ag presented by HLA-A*0201 molecules, a fusion protein was constructed consisting of a single chain Ab specific for Her-2/neu, linked to IL-2 (neu-Ab-IL-2). When added to a mixture of tumor cells and Her-2/neu-specific CTL, the protein was found to augment lysis of tumor cells. In addition, the hybrid molecule also promoted lysis of Her-2/neu expressing tumors by non-tumor-specific cloned T cell lines, including Th1 CD4 cells. Analysis of the mechanism of cytotoxicity revealed that the fusion protein mediates the formation of stable conjugates between T cells expressing IL-2R and tumor cells expressing Her-2/neu, resulting in lysis through the Fas-Fas ligand pathway. Lysis induction was independent of specific engagement by the TCR. When tested for its ability to enhance tumor cell eradication by Her-2/neu-specific CD8+ T cells in an adoptive transfer model in SCID mice, neu-Ab-IL-2 facilitated the elimination of tumor cells in vivo. Surprisingly, the combination of non-tumor-specific CD8+ T cells and fusion protein also induced a significant delay of tumor growth. This represents a novel approach for redirecting non-tumor-specific T cells to eliminate tumors.
Opsonic Complement Component C3 in the Solitary Ascidian,Halocynthia roretzi
Authors: Nonaka Masaru , Azumi Kaoru , Ji Xin , Namikawa-Yamada Chisato , Sasaki Makoto , Saiga Hidetosi , Dodds Alister W. , Sekine Hideharu , Homma Miwako K. , Matsushita Misao , Endo Yuichi , Fujita Teizo
Published: December 2022

The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates. To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid. The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester. AsC3 has a two-subunit chain structure, and the α-chain is cleaved at a specific site near to the N terminus upon activation. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity. These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.
Characterization of Human Endotoxin Lipopolysaccharide Receptor CD14 Expression in Transgenic Mice
Authors: Hetherington Christopher J. , Kingsley Paul D. , Crocicchio Francesco , Zhang Pu , Rabin Michael S. , Palis James , Zhang Dong-Er
Published: December 2022

CD14 is a major receptor for the bacterial endotoxin LPS. Since CD14 is specifically and highly expressed on the surface of monocytic cells, it has been used as a monocyte/macrophage differentiation marker. To identify elements that are critical for the direction of the tissue-specific expression of CD14, an 80-kb genomic DNA fragment containing the coding region of the CD14 gene, as well as a considerable amount of both upstream and downstream sequence, was used to generate transgenic mice. The analysis of mice from six different founder lines demonstrated that this genomic DNA fragment was sufficient to direct human CD14 gene expression in a monocyte-specific manner among hematopoietic cells. Furthermore, the data lead us to a new finding that CD14 is highly expressed in the human liver, a primary organ involved in the acute phase response. These transgenic mice provide a useful model to analyze the biological function of human CD14.
Enhanced Interaction of HLA-DM with HLA-DR in Enlarged Vacuoles of Hereditary and Infectious Lysosomal Diseases
Authors: Lem Lawrence , Riethof David A. , Scidmore-Carlson Marci , Griffiths Gillian M. , Hackstadt Ted , Brodsky Frances M.
Published: December 2022

Following biosynthesis, class II MHC molecules are transported through a lysosome-like compartment, where they acquire antigenic peptides for presentation to T cells at the cell surface. This compartment is characterized by the presence of HLA-DM, which catalyzes the peptide loading process. Here we report that the morphology and function of the class II loading compartment is affected in diseases with a phenotypic change in lysosome morphology. Swollen lysosomes are observed in cells from patients with the hereditary immunodeficiency Chediak-Higashi syndrome and in cells infected with Coxiella burnetii, the rickettsial organism that causes Q fever. In both disease states, we observed that HLA-DR and HLA-DM accumulate in enlarged intracellular compartments, which label with the lysosomal marker LAMP-1. The distribution of class I MHC molecules was not affected, localizing disease effects to the endocytic pathway. Thus, cellular mechanisms controlling lysosome biogenesis also affect formation of the class II loading compartment. Analysis of cell surface class II molecules revealed that their steady-state levels were not reduced on diseased cells. However, in both disease states, enhanced interaction between HLA-DR and HLA-DM was detected. In the Chediak-Higashi syndrome cells, this correlated with more efficient removal of the CLIP peptide. These findings suggest a mechanism for perturbation of Ag presentation by class II molecules and consequent immune deficiencies in both diseases.
Evidence for a Role of IL-17 in Organ Allograft Rejection: IL-17 Promotes the Functional Differentiation of Dendritic Cell Progenitors
Authors: Antonysamy Mary A. , Fanslow William C. , Fu Fumin , Li Wei , Qian Shiguang , Troutt Anthony B. , Thomson Angus W.
Published: December 2022

IL-17 is a T cell-derived cytokine that stimulates stromal cells and macrophages to secrete proinflammatory cytokines. We hypothesized that IL-17 might play a role in alloimmune responses, and that interference with its activity might suppress allograft rejection. IL-17R:Fc or control IgG was added at the start of mouse MLR or was administered i.p. (100–500 μg/day) for different durations post-transplant to murine recipients of MHC-mismatched cardiac allografts. IL-17R:Fc (50–200 μg/ml) markedly inhibited T cell proliferation in vitro and significantly prolonged nonvascularized cardiac allograft median survival time from 13 to 20 days (100 μg/day; days 0 and 1) or to 19 days (100–300 μg/day; days 0–4). Survival of vascularized grafts was also extended significantly from 10.5 to 19 days by IL-17R:Fc (500 μg/day; days 0–6). To address a possible mechanism by which IL-17 may promote alloreactivity, we examined the influence of IL-17 on the differentiation and function of bone marrow-derived cells propagated in granulocyte-macrophage CSF with or without IL-4 to promote dendritic cell (DC) growth. A minor proportion of CD11c+ DC expressed the IL-17R. IL-17 promoted the maturation of DC progenitors, as evidenced by increased cell surface expression of CD11c, costimulatory molecules (CD40, CD80, CD86), and MHC class II Ag, and allostimulatory capacity. IL-17 had a lesser effect on the phenotype and function of more fully differentiated myeloid DC. These findings suggest a role for IL-17 in allogeneic T cell proliferation that may be mediated in part via a maturation-inducing effect on DC. IL-17 appears to be a novel target for therapeutic intervention in allograft rejection.
Chemotactic Migration Triggers IL-8 Generation in Neutrophilic Leukocytes
Authors: Siddiqui Rafat A. , Akard Luke P. , Garcia J. G. N. , Cui Yi , English Denis
Published: December 2022

Neutrophils recovered from inflammatory exudates possess increased levels of IL-8, but exposure of neutrophils to chemoattractants results in only a modest stimulation of IL-8 generation. This study was undertaken to explore the hypothesis that IL-8 generation in these cells is dependent upon the process of migration. Neutrophils synthesized up to 30 times as much IL-8 during migration in response to a gradient of diverse chemoattractants than they did when stimulated directly by the attractants in the absence of a gradient. This IL-8 response was dependent on migration since it was not observed in cells exposed to concentration gradients of chemoattractants under conditions that prevented cell movement. While actinomycin-D (1 μg/ml) had little influence on the generation of IL-8 during chemotaxis, the protein synthesis inhibitor cycloheximide (10 μg/ml) markedly blunted the accumulation of cell-associated IL-8, suggesting that new protein synthesis from preexisting mRNA was responsible for the effect. Consistent with this interpretation, migrating cells incorporated over 10 times as much [3H]leucine into IL-8 as did nonmotile neutrophils exposed to chemoattractants. A substantial portion of the IL-8 generated during chemotaxis was released upon subsequent metabolic stimulation. Thus, the IL-8 synthesized during chemotaxis is uniquely positioned to exert a regulatory influence on inflammatory processes governed by neutrophilic leukocytes responding to inflammatory and infectious stimuli.
Adhesion-Dependent Degranulation of Neutrophils Requires the Src Family Kinases Fgr and Hck
Authors: Mócsai Attila , Ligeti Erzsébet , Lowell Clifford A. , Berton Giorgio
Published: December 2022

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-α and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck−/−fgr−/− PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-α but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck−/−fgr−/− PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr−/− or hck−/− PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck−/−fgr−/− or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.
Involvement of MIIC-Like Late Endosomes in B Cell Receptor-Mediated Antigen Processing in Murine B Cells
Authors: Drake James R. , Lewis Timothy A. , Condon Krista B. , Mitchell Richard N. , Webster Paul
Published: December 2022

Currently, the involvement of classical vs novel endocytic compartments in the phenomenon of B cell receptor (BCR)-mediated Ag processing is a matter of considerable debate. In murine B cells, class II vesicles (CIIV) represent a novel endocytic compartment involved in BCR-mediated Ag processing and class II peptide loading. Alternatively, in human B cells, the MHC class II-enriched compartment (MIIC) represents a lysosome (L)-like endocytic compartment that appears to be involved in this process. Presently, the relationship between CIIV, MIIC, and classical endosomes and L remains to be determined. Using density gradient centrifugation, a subcellular compartment morphologically and immunologically similar to human MIIC has been identified, isolated, and characterized in murine B cells. These MIIC-like vesicles represent a population of class II-positive late endosomes (LE) and are distinct from CIIV. MIIC-like LE are uniquely marked by the thiol protease cathepsin B, and along with mature L, appear to be the major repository of DM molecules in these cells. Importantly, both MIIC-like LE and CIIV isolated from Ag-pulsed B cells contain BCR-internalized Ag as well as antigenic peptide-class II complexes.
T Cell Activation Signals Up-Regulate p38 Mitogen-Activated Protein Kinase Activity and Induce TNF-α Production in a Manner Distinct from LPS Activation of Monocytes
Authors: Schafer Peter H. , Wang Liwen , Wadsworth Scott A. , Davis Janet E. , Siekierka John J.
Published: December 2022

p38 mitogen-activated protein kinase (MAPK) (p38) is involved in various cellular responses, including LPS stimulation of monocytes, resulting in production of proinflammatory cytokines such as TNF-α. However, the function of p38 during antigenic stimulation of T cells is largely unknown. Stimulation of the human Th cell clone HA-1.70 with either the superantigen staphylococcal enterotoxin B (SEB) or with a specific antigenic peptide resulted in p38 activation and the release of TNF-α. MAPK-activated protein kinase-2 (MAPKAPK-2), an in vivo substrate for p38, was also activated by T cell signaling. SB 203580, a selective inhibitor of p38, blocked p38 and MAPKAPK-2 activation in the T cell clone but did not completely inhibit TNF-α release. PD 098059, a selective inhibitor of MAPK kinase 1 (MEK1), blocked activation of extracellular signal-regulated kinase (ERK) and partially blocked TNF-α production by the clone. In human peripheral T cells, p38 was not activated by SEB, but rather by CD28 cross-linking, whereas in the human leukemic T cell line Jurkat, p38 was activated by CD3 and CD28 cross-linking in an additive fashion. TNF-α production by peripheral T cells in response to SEB and anti-CD28 mAb correlated more closely with ERK activity than with p38 activity. Therefore, various forms of T cell stimulation can activate the p38 pathway depending on the cells examined. Furthermore, unlike LPS-stimulated monocytes, TNF-α production by T cells is only partially p38-dependent.
Inhibition of Th1 Polarization by Soluble TNF Receptor Is Dependent on Antigen-Presenting Cell-Derived IL-12
Authors: Becher Burkhard , Blain Manon , Giacomini Paul S. , Antel Jack P.
Published: December 2022

Th1-polarized CD4+ T cells are considered central to the development of a number of target-directed autoimmune disorders including multiple sclerosis. The APC-derived cytokine IL-12 is a potent inducer of Th1 polarization in T cells. Inhibition of IL-12 in vivo blocks the development of experimental allergic encephalomyelitis, the animal model for multiple sclerosis. Based on previous work that suggests that the production of IL-12 by activated human central nervous system-derived microglia is regulated by autocrine TNF-α, we wanted to determine whether inhibition of TNF could induce a reduction of Th1 responses by its impact on systemic APCs. We found that soluble TNFR p75-IgG fusion protein (TNFR:Fc) inhibited production of IFN-γ by allo-Ag-activated blood-derived human CD4 T cells. We documented reduced IL-12 p70 production by APCs in the MLR. By adding back recombinant IL-12, we could rescue IFN-γ production, indicating that TNFR:Fc acts on APC-derived IL-12. Consistent with an inhibition of the Th1 polarization, we found a decreased expression of IL-12R-β2 subunit on the T cells. Furthermore, the capacity of T cells to secrete IFN-γ upon restimulation when previously treated with TNFR:Fc is impaired, whereas IL-2 secretion was not altered. Our results define a TNF-dependent cytokine network that favors development of Th1 immune responses.
Presence of Pentoxifylline During T Cell Priming Increases Clonal Frequencies in Secondary Proliferative Responses and Inhibits Apoptosis
Authors: Gupta Manisha , George Anna , Sen Ranjan , Rath Satyajit , Durdik Jeannine M. , Bal Vineeta
Published: December 2022

Naive T cells appear to be primed by specific Ag to differentiate into either effectors or memory cells. We have been analyzing the factors involved in this differential commitment in the priming of alloresponsive human T cells in vitro and have shown that the presence of a phosphodiesterase inhibitor, pentoxifylline (POX), during priming results in a decrease in the primary response and enhancement in the secondary proliferative response. We now show that the POX-mediated effect can be mimicked by dibutyryl cAMP. The secondary response enhancement is due to the effects of POX on the T cells rather than the APCs, because even fixed APCs can prime T cells in the presence of POX. POX affects T cells directly by increasing clonal frequency rather than the burst size of the secondary responders. The known inhibition of IL-2 production by POX is not responsible for this effect, because exogenous IL-2 supplementation does not block it. The presence of POX during priming alters the outcome of T cell activation, resulting in a lower frequency of cells expressing IL-2Rα (CD25) and a decrease in their subsequent apoptosis, and this anti-apoptotic effect is consistent with the enhanced commitment of T cells to secondary responsiveness by POX.
Antigen Recognition Influences Transendothelial Migration of CD4+ T Cells
Authors: Marelli-Berg Federica M. , Frasca Loredana , Weng Ling , Lombardi Giovanna , Lechler Robert I.
Published: December 2022

The functional significance of MHC class II expression by vascular endothelial cells remains obscure. In this study the possibility that Ag presentation by endothelial cells (EC) influences T cell transmigration, facilitating the recruitment of Ag-specific T cells into tissues, was investigated. The frequencies of T cells with specificity for an HLA-DR alloantigen, or for the recall Ag tetanus toxoid (TT), were measured in peripheral blood CD45RO+ (memory) CD4+ T cells before and after transmigration through γ-IFN-treated EC monolayers. Frequencies of anti-DR17, IL-2-secreting T cells were fourfold higher in the T cells that transmigrated through a monolayer of DR17-expressing EC. Similar increases were seen in TT-specific, DR7-restricted T cells that transmigrated through TT-pulsed, DR7-expressing EC. To examine more directly the effects of cognate recognition of Ag presented by EC, T cell clones were used. For clones that proliferated in a costimulation-independent manner to Ag presented by EC, cognate recognition arrested transmigration. In contrast, Ag presentation by EC to B7-dependent T cell clones, which do not proliferate following cognate recognition of EC, enhanced the rate of transendothelial migration. These data suggest that Ag presentation by EC may serve to augment the recruitment of Ag-specific T cells into tissues and that proliferation and transmigration are mutually exclusive T cell responses.
IFN-γ Down-Regulates MHC Expression and Antigen Processing in a Human B Cell Line
Authors: O’Neil Deborah , Swanton Charles , Jones Andrew , Medd Patrick G. , Rayment Neil , Chain Benjamin
Published: December 2022

IFN-γ is a crucial mediator in the induction of cell-mediated Th1-type responses but is predominantly a negative regulator of B cell differentiation and proliferation. This cytokine is therefore a key factor in determining Th1 vs Th2 differentiation. This study investigates the action of IFN-γ in modulation of HLA-DR expression and Ag presentation by EBV-transformed human B cell lines. In contrast to its action on the monocyte/macrophage, IFN-γ down-regulates surface MHC expression on these B cells, and this regulation is posttranscriptional. In parallel with MHC down-regulation, there is a reduced capability to process and present exogenous protein and peptide Ag to T cell hybridomas. IFN-γ does not change the rates of fluid phase endocytosis or exocytosis in this model system but correlates with an up-regulation of the lysosomal enzymes cathepsins B and D.
Galectin-1 Specifically Modulates TCR Signals to Enhance TCR Apoptosis but Inhibit IL-2 Production and Proliferation
Authors: Vespa Glaucia N. R. , Lewis Linda A. , Kozak Katherine R. , Moran Miriana , Nguyen Julie T. , Baum Linda G. , Miceli M. Carrie
Published: December 2022

Galectin-1 is an endogenous lectin expressed by thymic and lymph node stromal cells at sites of Ag presentation and T cell death during normal development. It is known to have immunomodulatory activity in vivo and can induce apoptosis in thymocytes and activated T cells (1–3). Here we demonstrate that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis, but antagonizes TCR-induced IL-2 production and proliferation in a murine T cell hybridoma and freshly isolated mouse thymocytes, respectively. Although CD4+CD8+ double positive cells are the primary thymic subpopulation susceptible to galectin-1 treatment alone, concomitant CD3 engagement and galectin-1 stimulation broaden susceptible thymocyte subpopulations to include a subset of each CD4−CD8−, CD4+CD8+, CD4−CD8+, and CD4+CD8− subpopulations. Furthermore, CD3 engagement cooperates with suboptimal galectin-1 stimulation to enhance cell death in the CD4+CD8+ subpopulation. Galectin-1 stimulation is shown to synergize with TCR engagement to dramatically and specifically enhance extracellular signal-regulated kinase-2 (ERK-2) activation, though it does not uniformly enhance TCR-induced tyrosine phosphorylation. Unlike TCR-induced IL-2 production, TCR/galectin-1-induced apoptosis is not modulated by the expression of kinase inactive or constitutively activated Lck. These data support a role for galectin-1 as a potent modulator of TCR signals and functions and indicate that individual TCR-induced signals can be independently modulated to specifically affect distinct TCR functions.