bluuebunny/crossref_metadata_2025 huggingface dataset:
Dendritic Cells Generated Either from CD34+ Progenitor Cells or from Monocytes Differ in Their Ability to Activate Antigen-Specific CD8+ T Cells
Authors: Ferlazzo Guido , Wesa Amy , Wei Wei-Zen , Galy Anne
Published: January 2023

Dendritic cells (DC) can be generated in vitro from monocytes (M-DC) or from CD34+ hemopoietic progenitor cells (CD34-DC) but their precursors are not equivalent cells, prompting a comparison of the functional capacities of these APC. Both types of DCs established from the same individuals using the same cytokines displayed a comparable phenotype of mature DC (CD1a+, CD83+, CD86+, CD4+, HLA-DR++, CD14−, CD15− ) and were equally potent stimulators of allogeneic T cell proliferation, being both more powerful than immature M-DCs. An autologous panel of APCs produced in HLA-A2+ individuals, including CD34-DC, M-DC, monocytes, and EBV-lymphoid cell line was comparatively evaluated for presentation of the Erb-B2 peptide E75 to a CTL line. After short exposures (5 h) to E75-loaded APCs, similar levels of intracellular IFN-γ were induced in Ag-specific CD8+ T cells regardless of APC type. In sustained cultures (4–14 days), more Ag-specific T cells were obtained when peptide was presented on CD34-DC (p < 0.05) rather than on M-DC, EBV-lymphoid cell lines, or monocytes, and these effects were dose-dependent. Activated T cells expressed 4-1BB, and the presence of 4-1BB-Ig fusion protein partially blocked Ag-specific CD8+ cell activation after CD34-DC or M-DC presentation. Our results show that 34-DC have a preferential capacity to activate CD8+ T cells and that this property is not strictly correlated to their ability to induce allogeneic T cell proliferation but due to mechanisms that remain to be defined.
β1 Integrin- and Proteoglycan-Mediated Stimulation of T Lymphoma Cell Adhesion and Mitogen-Activated Protein Kinase Signaling by Thrombospondin-1 and Thrombospondin-1 Peptides
Authors: Wilson Katherine E. , Li Zhuqing , Kara Murat , Gardner Kevin L. , Roberts David D.
Published: January 2023

Cell-cell and cell-matrix interactions play important regulatory roles in lymphocyte homeostasis. Thrombospondin-1 (TSP1) is a matricellular protein that differentially promotes the adhesion of resting and activated T cells. In this work, we show that adhesion of Jurkat T cells on substrates coated with TSP1 or TSP1-derived peptides is mediated by β1 integrins, CD47, and heparan sulfate proteoglycans. Interactions with TSP1 or TSP1 peptides stimulated CD3-induced Ras activation and tyrosine phosphorylation of several T cell proteins. The signals from TSP1 and its derived peptides differentially synergized with activation of the TCR to induce phosphorylation of linker for activation of T cells (LAT) and extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 kinases. The phosphorylation of ERK in the presence of full-length TSP1 was transient and dependent on a β1 integrin receptor. Interestingly, peptides derived from the type 1 repeats of TSP1 and a CD47-binding peptide from the carboxyl-terminal domain of TSP1 also stimulated mitogen-activated protein (MAP) kinase phosphorylation. Moreover, the TSP1 heparin-binding peptide synergized with Ab-ligated TCR to transduce signals to the nucleus, detected by activation of AP-1- and Elk-dependent transcription. This TSP1 peptide-dependent activation of AP-1 was inhibited by both heparin and the MAP/ERK kinase inhibitor PD98059, providing a functional link between adhesion molecule interaction and nuclear transactivation events via the MAP kinase pathways. These findings have implications for the role of extracellular TSP1 and TSP1 fragments in the regulation of T cell function during hemostasis, wound repair, and other inflammatory responses.
Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Stimulate the Induction of Th2 Responses by Up-Regulating B7.2 Expression
Authors: Delgado Mario , Leceta Javier , Gomariz Rosa P. , Ganea Doina
Published: January 2023

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two structurally related neuropeptides produced within the lymphoid microenvironment, modulate several immunologic functions. We have recently demonstrated that VIP and PACAP enhance the macrophage costimulatory activity for naive CD4+ T cells exposed to allogeneic or anti-CD3 stimuli through the differential regulation of the B7 costimulatory molecules. In this study, we report on the role of VIP and PACAP on macrophage B7 expression and costimulatory function for Ag-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo. VIP and PACAP up-regulate the costimulatory activity of macrophages for Ag-primed CD4+ T cells. VIP-/PACAP-treated macrophages gain the ability to induce Th2-type cytokines such as IL-4 and IL-5 and reduce Th1-type cytokines such as IFN-γ and IL-2. In vivo administration of VIP or PACAP in Ag-immunized mice reduce the numbers of IFN-γ-secreting cells and enhance the numbers of IL-4-secreting cells. One of the consequences of the VIP-/PACAP-induced shift in cytokine profile is a change in the Ag-specific Ig isotype, increasing IgG1 and decreasing IgG2a levels. Finally, the preferential differentiation into Th2 effector cells after Ag stimulation induced by VIP-/PACAP-treated macrophages is mediated through the up-regulation of B7.2 expression.
TNF-α Inhibits HIV-1 Replication in Peripheral Blood Monocytes and Alveolar Macrophages by Inducing the Production of RANTES and Decreasing C-C Chemokine Receptor 5 (CCR5) Expression
Authors: Lane Brian R. , Markovitz David M. , Woodford Nina L. , Rochford Rosemary , Strieter Robert M. , Coffey Michael J.
Published: January 2023

The pathogenesis of HIV-1 infection is influenced by the immunoregulatory responses of the host. Macrophages present in the lymphoid tissue are susceptible to infection with HIV-1, but are relatively resistant to its cytopathic effects and serve as a reservoir for the virus during the course of disease. Previous investigators have demonstrated that increased serum levels of TNF-α contribute to the clinical symptoms of AIDS and that TNF-α stimulates the production of HIV-1 in chronically infected lymphocytic and monocytic cell lines by increasing HIV-1 gene expression. Although previous studies have suggested that TNF-α may increase HIV-1 infection of primary human mononuclear cells, some recent studies have indicated that TNF-α suppresses HIV-1 infection of macrophages. We now demonstrate that TNF-α suppresses HIV-1 replication in freshly infected peripheral blood monocytes (PBM) and alveolar macrophages (AM) in a dose-dependent manner. As TNF-α has been shown to increase the production of C-C chemokine receptor (CCR5)-binding chemokines under certain circumstances, we hypothesized that TNF-α inhibits HIV-1 replication by increasing the expression of these HIV-suppressive factors. We now show that TNF-α treatment of PBM and AM increases the production of the C-C chemokine, RANTES. Immunodepletion of RANTES alone or in combination with macrophage inflammatory protein-1α and -1β block the ability of TNF-α to suppress viral replication in PBM and AM. In addition, we found that TNF-α treatment reduces CCR5 expression on PBM and AM. These findings suggest that TNF-α plays a significant role in inhibiting monocytotropic strains of HIV-1 by two distinct, but complementary, mechanisms.
NK and CTL Recognition of a Single Chain H-2Dd Molecule: Distinct Sites of H-2Dd Interact with NK and TCR
Authors: Chung Doo Hyun , Dorfman Jeffrey , Plaksin Daniel , Natarajan Kannan , Belyakov Igor M. , Hunziker Rosemarie , Berzofsky Jay A. , Yokoyama Wayne M. , Mage Michael G. , Margulies David H.
Published: January 2023

We generated transgenic mice expressing a single-chain β2-microglobulin (β2m)-H-2Dd. The cell-surface β2m-H-2Dd molecule was expressed on a β2m-deficient background and reacted with appropriate mAbs. It was of the expected m.w. and directed the normal development of CD8+ T cells in the thymus of a broad TCR repertoire. It also presented both exogenously provided and endogenous peptide Ags to effector CD8+ T cells. In tests of NK cell education and function, it failed to reveal any interaction with NK cells, suggesting that the site of the interaction of NK receptors with H-2Dd was disrupted. Thus, the sites of TCR and NK receptor interaction with H-2Dd are distinct, an observation consistent with independent modes of TCR and NK receptor evolution and function.
Tumor Gangliosides Inhibit the Tumor-Specific Immune Response
Authors: McKallip Robert , Li Ruixiang , Ladisch Stephan
Published: January 2023

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5–20 μM inhibited the tumor-specific secondary proliferative response (80–100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63–74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.
The Induction of Virus-Specific CTL as a Function of Increasing Epitope Expression: Responses Rise Steadily Until Excessively High Levels of Epitope Are Attained
Authors: Wherry E. John , Puorro Kristin A. , Porgador Angel , Eisenlohr Laurence C.
Published: January 2023

The role of epitope expression levels in CD8+ T cell priming has been controversial. Yet this parameter is of great importance in the design of rational approaches to optimize CTL responses to a variety of pathogens. In this paper we examine the influence of epitope production on CD8+ T cell priming by exploiting a system that allows a 200-fold range of cell surface epitope expression in vitro with a fixed dose of vaccinia virus. Our results demonstrate that, with the exception of a notable decline at the highest level of epitope, the magnitude of the responding CTL population generated in vivo following equivalent viral infections is essentially proportional to epitope density.
Estradiol Coupling to Human Monocyte Nitric Oxide Release Is Dependent on Intracellular Calcium Transients: Evidence for an Estrogen Surface Receptor
Authors: Stefano George B. , Prevot Vincent , Beauvillain Jean-Claude , Fimiani Caterina , Welters Ingeborg , Cadet Patrick , Breton Christophe , Pestel Joel , Salzet Michel , Bilfinger Thomas V.
Published: January 2023

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17β-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17α-estradiol had no effect. 17β-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17β-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17β-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17β-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-α amplification signal and a weak β signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.
Thymic Transplantation Across an MHC Class I Barrier in Swine
Authors: Haller Gary W. , Esnaola Nestor , Yamada Kazuhiko , Wu Anette , Shimizu Akira , Hansen Andrew , Ferrara Vincent R. , Allison Kenneth S. , Colvin Robert B. , Sykes Megan , Sachs David H.
Published: January 2023

Thymic tissue transplantation has been performed previously in adult mice to induce donor-specific tolerance across allogeneic and xenogeneic barriers. We have now attempted to extend this technique to a large animal preclinical model and describe here our initial studies of allogeneic thymic transplantation in miniature swine. Two miniature swine were thymectomized before thymic tissue transplantation, and two remained euthymic. Donor thymic tissue was harvested from SLA class I-mismatched juvenile pigs and placed into recipient sternocephalicus muscle, kidney capsule, and omentum. A 12-day course of cyclosporin A was started on the day of transplantation. Allogeneic thymic engraftment could only be achieved in euthymic and not in thymectomized miniature swine using this treatment regimen. Both nonthymectomized animals showed good graft development, with evidence of thymopoiesis, as indicated by positive CD1 and host-type SLA class I immunoperoxidase staining of immature graft-infiltrating cells. Both animals also demonstrated donor-specific T cell hyporesponsiveness, as measured by MLR and cell-mediated lympholysis. The thymic grafts continued to develop despite the appearance of high levels of anti-donor specific cytotoxic IgG Abs. Thus, thymic tissue transplanted across an SLA class I barrier can engraft and support host thymopoiesis in euthymic miniature swine. The presence of the host thymus was required for engraftment. These data support the potential of thymic transplantation as part of a regimen to induce donor-specific tolerance to xenogeneic organ grafts.
Regulation of T Cell Homeostasis by Heparan Sulfate-Bound IL-2
Authors: Wrenshall Lucile E. , Platt Jeffrey L.
Published: January 2023

Although IL-2 is commonly thought to promote proliferation of T lymphocytes, mice deficient in IL-2 exhibit splenomegaly, lymphocytosis, and autoimmunity, suggesting this cytokine may have a prominent role in T cell homeostasis. Since the number of T cells in the bloodstream and lymphoid organs is tightly controlled, it is likely that the availability of IL-2 must also be closely regulated. One mechanism altering the local availability of cytokines is association with heparan sulfate, a glycosaminoglycan found on cell surfaces and within extracellular matrices. Here we show that an association between IL-2 and heparan sulfate localizes IL-2 to lymphoid organs such as the spleen. We also show that IL-2, sequestered in this way, contributes to the activation of T lymphocytes and primes T lymphocytes for activation-induced cell death.
Identification and Functional Characterization of the ζ-Chain Dimerization Motif for TCR Surface Expression
Authors: Bolliger Luca , Johansson Britt
Published: January 2023

We recognized a common dimerization motif between the transmembrane (TM) domain of ζ-chain family members and glycophorin A. We have shown that a glycine within the ζ-dimerization motif is critical for ζ-homodimerization and also for its association with the TCR/CD3 complex. Similarly, two residues within the CD3δγ TM domains have proven to be critical for their interaction with the ζ-homodimer. A three-dimensional homology model of the ζ-chain TM domain highlights potential residues preferentially involved either in the ζ2-CD3 or ζ2-TCRαβ association, confirming our experimental findings. These results indicate that, for symmetrical reasons, the ζ-homodimer participates in the TCR/CD3 complex assembly by interacting with CD3γδ TM domains, thereby masking their degradation signals located in the cytoplasmic tails.
Mycobacterium tuberculosis Inhibits IFN-γ Transcriptional Responses Without Inhibiting Activation of STAT1
Authors: Ting Li-Min , Kim Anne C. , Cattamanchi Ashok , Ernst Joel D.
Published: January 2023

IFN-γ activates macrophages to kill diverse intracellular pathogens, but does not activate human macrophages to kill virulent Mycobacterium tuberculosis. We tested the hypothesis that this is due to inhibition of IFN-γ signaling by M. tuberculosis and found that M. tuberculosis infection of human macrophages blocks several responses to IFN-γ, including killing of Toxoplasma gondii and induction of FcγRI. The inhibitory effect of M. tuberculosis is directed at transcription of IFN-γ-responsive genes, but does not affect proximal steps in the Janus kinase-STAT pathway, as STAT1α tyrosine and serine phosphorylation, dimerization, nuclear translocation, and DNA binding are intact in M. tuberculosis-infected cells. In contrast, there is a marked decrease in IFN-γ-induced association of STAT1 with the transcriptional coactivators CREB binding protein and p300 in M. tuberculosis-infected macrophages, indicating that M. tuberculosis directly or indirectly disrupts this protein-protein interaction that is essential for transcriptional responses to IFN-γ. Gamma-irradiated M. tuberculosis and isolated cell walls reproduce the effects of live bacteria, indicating that the bacterial component(s) that initiates inhibition of IFN-γ responses is constitutively expressed. Although lipoarabinomannan has been found to exert effects on macrophages, it does not account for the inhibitory effects of cell walls. These results indicate that one mechanism for M. tuberculosis to evade the human immune response is to inhibit the IFN-γ signaling pathway, and that the mechanism of inhibition is distinct from that reported for Leishmania donovani or CMV, in that it targets the interaction of STAT1 with the basal transcriptional apparatus.
Ig Heavy Chain Complex-Linked Genes Influence the Immune Response in a Murine Cryptococcal Infection
Authors: Lovchik Julie A. , Wilder Julie A. , Huffnagle Gary B. , Riblet Roy , Lyons C. Richard , Lipscomb Mary F.
Published: January 2023

A murine pulmonary infection with Cryptococcus neoformans (Cne) has been used to determine mechanisms regulating effective T cell-mediated immunity in the lungs. In BALB/c and C.B-17 mice, following intratracheal deposition of Cne, the fungus initially grows rapidly and is then progressively cleared from the lungs. Cne clearance in C.B-17 mice requires CD4 and CD8 T cells, IFN-γ, and NO. Clearance in congenic BALB/c mice proceeds more slowly than in C.B-17 mice, even though the only genetic difference between these strains is at the Ig H chain-containing region of chromosome 12. Examination of the pulmonary immune response in the two strains revealed that both cleared lung Cne by T cell-dependent mechanisms and generated equivalent levels of NO. Furthermore, both strains recruited equal numbers of macrophages, lymphocytes, and neutrophils to the lungs, although BALB/c mice recruited higher numbers of eosinophils. Notably, leukocytes isolated from BALB/c lungs during infection secreted lower levels of IFN-γ and higher levels of the Th2 cytokines IL-4 and IL-5 as compared with lung leukocytes from C.B-17 mice. Furthermore, serum levels of IgM, IgG1, IgG2a, and IgG3 anti-Cne Abs generated during infection were significantly greater in BALB/c mice than C.B-17 mice. These data suggest that although both BALB/c and C.B-17 mice clear pulmonary cryptococcosis through T cell-mediated mechanisms, Ig H chain-linked genes in BALB/c mice are associated with a decreased effectiveness of the host response, which we suggest might influence the balance in Th1/Th2 T cell subset development or increase anti-Cne Abs, or both.
Apoptosis of a Human Melanoma Cell Line Specifically Induced by Membrane-Bound Single-Chain Antibodies
Authors: de Inés Concepción , Cochlovius Björn , Schmidt Stefanie , Kipriyanov Sergey , Rode Hans-Jürgen , Little Melvyn
Published: January 2023

CD28 is a key regulatory molecule in T cell responses. Ag-TCR/CD3 interactions without costimulatory signals provided by the binding of B7 ligands to the CD28R appear to be inadequate for an effective T cell activation. Indeed, the absence of B7 on the tumor cell surface is probably one of the factors contributing to the escape of tumors from immunological control and destruction. Therefore, to increase the immunogenicity of tumor cell vaccines, we have expressed anti-CD3 and anti-CD28 single-chain Abs (scFv) separately on the surface of a human melanoma SkMel63 cell line (HLA-A*0201). A mixture of cells expressing anti-CD3 with cells expressing anti-CD28 resulted in a marked activation of allogeneic human PBL in vitro. The apparent induction of a Th1 differentiation pathway was accompanied by the proliferation of MHC-independent NK cells and MHC-dependent CD8+ T cells. PBL that had been cultured together with transfected SkMel63 tumor cells were able to specifically induce apoptosis in untransfected SkMel63 cells. In contrast, three other tumor cell lines expressing HLA-A*0201, including two melanoma cell lines, showed no significant apoptosis. These results provide valuable information for both adoptive immunotherapy and the generation of autologous tumor vaccines.
Inflammatory Cytokines IL-1α, IL-1β, IL-6, and TNF-α Impart Neuroprotection to an Excitotoxin Through Distinct Pathways
Authors: Carlson Noel G. , Wieggel Whitney A. , Chen Jian , Bacchi Annalisa , Rogers Scott W. , Gahring Lorise C.
Published: January 2023

The proinflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α are produced within the CNS, and, similar to the periphery, they have pleotrophic and overlapping functions. We have shown previously that TNF-α increases neuronal survival to a toxic influx of calcium mediated through neuronal N-methyl-d-aspartic acid (NMDA) glutamate-gated ion channels. This process, termed excitotoxicity, is a major contributor to neuronal death following ischemia or stroke. Neuroprotection by this cytokine requires both activation of the p55/TNF receptor type I and the release of TNF-α from neurons, and it is inhibited by the plant alkaloid nicotine. Here, we report that other inflammatory cytokines (IL-1α, IL-1β, and IL-6) are also neuroprotective to excessive NMDA challenge in our system. Neuroprotection provided by IL-1 is distinct from TNF-α because it is inhibited by IL-1 receptor antagonist; it is not antagonized by nicotine, but it is inhibited by a neutralizing Ab to nerve growth factor (NGF). Similar to IL-1, IL-6-mediated neuroprotection is also antagonized by pretreatment with IL-1 receptor antagonist and it is not affected by nicotine. However, neutralizing anti-NGF only partially blocks IL-6-mediated protection. These studies support an important role for distinct but overlapping neuroprotective cytokine effects in the CNS.
Involvement of Phosphatidylinositol 3-Kinase in Stromal Cell-Derived Factor-1α-Induced Lymphocyte Polarization and Chemotaxis
Authors: Vicente-Manzanares Miguel , Rey Mercedes , Jones David R. , Sancho David , Mellado Mario , Rodriguez-Frade Jose Miguel , del Pozo Miguel Angel , Yáñez-Mó María , de Ana Ana Martín , Martínez-A. Carlos , Mérida Isabel , Sánchez-Madrid Francisco
Published: January 2023

The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1α (SDF-1α). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1α receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1α. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1α-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.
Human Transaldolase and Cross-Reactive Viral Epitopes Identified by Autoantibodies of Multiple Sclerosis Patients
Authors: Esposito Maria , Venkatesh Vijay , Otvos Laszlo , Weng Zhiping , Vajda Sandor , Banki Katalin , Perl Andras
Published: January 2023

Multiple sclerosis is mediated by an autoimmune process causing selective destruction of oligodendrocytes. Transaldolase, which is expressed in the brain selectively in oligodendrocytes, is a target of high affinity autoantibodies in serum and cerebrospinal fluid of multiple sclerosis patients. A three-dimensional model of human transaldolase was developed based on the crystal structure of the enzyme from Escherichia coli. To identify immunodominant epitopes, 33 peptides overlapping human transaldolase by 5 amino acids were synthesized. Ab 12484, raised against enzymatically active human transaldolase, recognized antigenic determinants corresponding to linear epitopes (residues 27–31 and 265–290) and α helices (residues 75–98 and 302–329). Four immunodominant peptides harboring charged amino acid residues with topographically exposed side chains were identified by sera from 13 multiple sclerosis patients with predetermined autoreactivity to transaldolase. Autoantibodies binding to the most prominent human transaldolase epitope, between residues 271 and 285, showed cross-reactivity with Epstein-Barr and herpes simplex virus type 1 capsid-derived peptides. Molecular mimicry between immunodominant autoepitopes and viral Ags may be a decisive factor in directing autoimmunity to transaldolase in multiple sclerosis patients.
Involvement of IL-15 in the Pathogenesis of Human T Lymphotropic Virus Type I-Associated Myelopathy/Tropical Spastic Paraparesis: Implications for Therapy with a Monoclonal Antibody Directed to the IL-2/15Rβ Receptor
Authors: Azimi Nazli , Jacobson Steven , Leist Thomas , Waldmann Thomas A.
Published: January 2023

Human T lymphotropic virus type I (HTLV-I) is the causative agent of an inflammatory neurological disease termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). An ongoing lymphocyte activation exists in patients with HAM/TSP, which was demonstrated by the spontaneous proliferation of their PBMC ex vivo. It was shown that spontaneous proliferation present in HAM/TSP is due, in part, to an IL-2/IL-2R autocrine loop. However, addition of Abs against IL-2 or IL-2Rα only partially inhibited the spontaneous proliferation. Since IL-15 is a cytokine with similar functional characteristics to those of IL-2, we reasoned that IL-15 might be an additional growth factor that contributes to the spontaneous proliferation observed in HAM/TSP. In this study, we demonstrated that IL-15 mRNA expression was elevated in PBMC obtained from HAM/TSP patients when compared with those of the normal donors. Furthermore, we showed that the addition of blocking Abs against IL-15 or its receptor inhibited the spontaneous proliferation of HAM/TSP PBMC. Addition of Abs directed toward both IL-15 and IL-2, or their receptors, inhibited the proliferation almost completely. These data suggest the existence of two autocrine loops involving IL-15/IL-15R and IL-2/IL-2R, both contributing to the spontaneous proliferation of HAM/TSP PBMC.
Depressed IL-12-Mediated Signal Transduction in T Cells from Patients with Sézary Syndrome Is Associated with the Absence of IL-12 Receptor β2 mRNA and Highly Reduced Levels of STAT4
Authors: Showe Louise C. , Fox Floyd E. , Williams Donna , Au Karen , Niu Zhutian , Rook Alain H.
Published: January 2023

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-γ) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the β1 and β2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R β1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R β1 and β2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, β2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-α or IFN- γ, which stimulated β2 expression in control PBL. The absence of β2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-α. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.
Vasoactive intestinal peptide (VIP) up-regulates IL-17 generation by mouse CD4 T cells (100.1)
Authors: Dai Qun , Goetzl Edward J
Published: January 2023

VIP produced by cholinergic and sensory nerves and by T cells affects T cell functions through the constitutively-expressed VPAC1 and inducible VPAC2 G protein-coupled receptors. Transgenic mice overexpressing VPAC2 (TG) selectively on CD4 T cells exhibit increased hypersensitivity and immunoinflammatory responses. Present studies were designed to examine the capacity of the VIP-VPAC2 axis to recruit the Th17 inflammatory subset of CD4 T cells. Splenic CD4 T cells isolated from TG and wild-type (WT) mice were incubated with adherent anti-CD3 and anti-CD28 antibodies and cytokines that initiate Th17 development, without and with IL-23 or VIP. IL-17 generation quantified by ELISA was enhanced significantly by 1μM VIP with the same time-course as for IL-23 over five days. IL-17 generation by CD4 T cells of TG mice was significantly greater than by those of WT mice with and without added VIP. Augmentation of IL-17 generation by both VIP and IL-23 was suppressed similarly by IL-27. Thus VIP may be a neuroalternative endogenous stimulus for Th17 development and recruit Th17 cells independently of IL-23 in inflammatory and autoimmune processes. (NIH grant AI029912)
HMGB1 binds to LPS and transfers it to CD14 (100.12)
Authors: Youn Ju Ho , Oh Young Joo , Kim Ah Reum , Shin Jeon-Soo
Published: January 2023

Lipopolysaccharide (LPS) itself released from Gram-negative bacteria poorly induces biological function because of its poor solubility. With help of LPS-binding protein (LBP), the diffusion of LPS monomers from the aggregates is mediated and transferred to CD14 to induce TLR4-mediated signal pathway. Secreted extracellular high mobility group box-1 protein (HMGB1) from activated monocytes acts as a proinflammatory cytokine and serum concentrations of HMGB1 are increased in patients presenting with sepsis and hemorrhagic shock. However, the exact role in triggering inflammation signal transduction on the immune cell surface membrane by secreted HMGB1 is poorly understood. We first show that HMGB1 could bind to three LPS from E. coli, S. typhi and S. minnesota in a concentration-dependent manner by surface plasmon resonance (SPR) and ELISA assay. LBP could inhibit the binding of HMGB1 to LPS in a dose-dependent manner. HMGB1 bound to diphosphryl lipid A moiety containing 2-keto-3-deoxyoctanate from S. minnesota LPS Re595, but poorly to detoxified LPS without lipid A moiety. In addition, HMGB1 could efficiently transfer LPS to CD14 protein in time-dependent and dose-dependent manners using BODIPY-LPS transfer assay and the native gel mobility shift assay. These results suggest that HMGB1 has a novel role in initiating LPS-mediated signaling by catalyzing movement of LPS monomers from LPS aggregates to cell surface CD14 in addition to the previous report of TLR4 signaling by direct HMGB1-TLR4 binding.
Novel immuno-spin trapping-based assay for the analysis of myeloperoxidase (100.13)
Authors: RAMIREZ DARIO C , GOMEZ-MEJIBA SANDRA E , CORBETT JEAN T , MASON RONALD P
Published: January 2023

Myeloperoxidase (MPO) is an important biomarker of inflammatory diseases. The analyses of MPO in biological samples are based on detection of its peroxidase activity, chloramine formation, and MPO-capture immunoassays. We have developed an immuno-spin trapping (IST)-based approach to determine MPO in biological samples. In this approach,

MPO/H2O2/Cl system produces hypochlorous acid (HOCl);HOCl reacts with proteins forming chloramines;chloramines decay forming protein radicals,protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) forming DMPO-protein nitrone adducts, andnitrone adducts are determined using a chemiluminescent direct ELISA with the anti-DMPO antiserum.

We used this assay to quantify MPO in plasma, bronchoalveolar fluid and lung parenchyma of rats exposed to lipopolysaccharide, and compared its sensitivity with a capture-based commercial ELISA kit. Hemoglobin does not interfere and the assay can be used in animal and human samples. The assay has three levels of amplification:

enzymatic formation of HOCl;many DMPO molecules bound per single protein molecule;a sensitive developing system.

The IST-based assay for quantification of MPO will enhance the power of this biomarker for early detection of environmental- and metabolic-induced inflammatory disorders in both experimental and clinical settings. NIH#1K99 ES015414-01
POPULATION GENETICS AND BIOCHEMICAL CHARACTERIZATION OF HUMAN CASPASE-12 (100.16)
Authors: Hermel Evan , Yavari Mehdy , Parsi Katayoun Edalat , Klapstein Kevin Daniel
Published: January 2023

Casp12 encodes the protein caspase 12 (casp12), which has a downregulatory effect upon IL-1 activation and subsequent inflammatory immune reactions. Casp12 is primarily a pseudogene (Casp12p1) in most humans, but ~20% of people of African ancestry have a functional, intact form of Casp12. This allele is increased in African-American individuals with severe sepsis. We examined Casp12 allele distribution in populations of Indians and Central Asians and in a pilot screen found that a small number of Tamils (2 of 10; 20%) possess the intact Casp12 allele. Gujratis, Punjabis and other Central Asians (Baluchi, Iranian, Pakistani or Afghani) were all homozygous for Casp12p1, as were nearly all other Indians (18/19; 95%). Thus, small but distinct populations outside of Africa still harbor Casp12. To examine the biochemical properties of the protein, we expressed recombinant human casp12. By fractionation techniques, casp12 was found exclusively in the cytosol. In order to better characterize the role of casp12 in the inflammatory process, we generated a rabbit polyclonal antiserum to casp12 and used it to determine if the protein interacted directly with components of the inflammasome. Immunoprecipitation of casp12 revealed that the protein did not interact with ASC, Cardinal, or Caspase 5. Casp12 may thus exert its downregulatory effects by direct interaction with IL1.
Prostaglandin E2 induces MMP-9 production in myeloid dendritic cells (100.3)
Authors: Yen Jui-Hung , Ganea Doina
Published: January 2023

Dendritic Cells (DCs) play an important role in the initiation of the immune response. Immature DCs take up antigen, mature and migrate to the regional lymph nodes where they activate antigen-specific naïve T cells. Prostaglandins such as PGE2 are essential for the migration of DCs to the lymph nodes. Matrix metalloproteinases (MMPs) play an essential role in DC migration, through the degradation of extracellular matrix (ECM) and basement membranes. MMP-2, and particularly MMP-9, are essential for dendritic cell migration in vitro through a reconstituted basement membrane (Matrigel), as well as in vivo to the draining lymph nodes. In this study, we show that PGE2 induces membrane-bound and secreted MMP-9 production in BM-DCs through the EP2/EP4-cAMP-PKA signaling pathway. Furthermore, PGE2-induced MMP-9 expression promotes migration of cytokine (TNF-alpha+IFN-alpha)-matured DCs in vitro through Matrigel and to the draining lymph nodes in vivo in B10.A mice. Our results describe a new mechanism for the effect of PGE2 on dendritic cell migration, which could lead to new therapeutic approaches in using DC as vaccination tools.

Grant support: 2RO1 AI0562306 (DG) and Dissertation Fellowship from Rutgers University (J-H Y).
Anaphylatoxins play a key role in the development of experimental lupus cerebritis (100.4)
Authors: Alexander Jessy John , Quigg Richard John
Published: January 2023

Complement activation generates the C3a and C5a anaphylatoxins, which act through specific G-protein coupled receptors (R). Anaphylatoxins correlate with flares in patients with systemic lupus erythematosus. C3aR and C5aR mRNA and protein expression were significantly increased in brains of the lupus mouse model, MRL/lpr mice compared to those of the non-diseased congenic MRL/+ strain. To examine the role of C3aR and C5aR signaling in brain, specific antagonists, N2-[(2,2-diphenylethoxy)acetyl]-L-arginine and acetyl-Phe-(Orn-Pro-D-cyclohexylalanine)-Trp-Arg, respectively, were administered to MRL/lpr mice. Littermate controls were given vehicle alone. Control MRL/lpr mice had increased infiltration of blood cells into brain and an increase in a number of relevant cytokines and cell-bound accessory molecules (γ-IFN, TNF-α, IL-1β, MIP-2, MCP-1, RANTES, TNFR1, and ICAM-1) which were reduced by inhibition of C3aR and C5aR. In addition, the number of apoptotic cells that were increased in MRL/lpr mice compared to MRL/+ mice, was significantly reduced by inhibition of C3aR and C5aR, with a corresponding increase in Iκβα and phosphorylated Akt, and decrease in Bcl-xL and phosphorylated BAD. Thus, pharmacological blockade of anaphylatoxin receptors reduces pathological features in the brain in experimental lupus and represents a potentially viable treatment for human lupus cerebritis.
Nicotinamide as an anti-inflammatory agent: an in vivo and in vitro study in a mouse model of septic shock. (100.7)
Authors: van gool frederic , Galli Mara , Rongvaux Anthony , Fabien Andris , Leo Oberdan
Published: January 2023

Tumor necrosis factor alpha is an important mediator of inflammation. Inhibition of TNF production is beneficial in several inflammatory diseases, and numerous efforts have been devoted in the design of therapies aimed at blocking the production of this cytokine. In the present work, we have analysed the anti inflammatory properties of nicotinamide (NAm), in a mouse model of acute septic shock. NAm was found to protect mice from a lethal injection of LPS, even when administered 2 h post treatment. NAm was found to inhibit TNF secretion while leading to increased IL-10 production in vivo. These observations have been corroborated by in vitro studies, in which NAm was found to inhibit TNF secretion by all the cell types tested. Our observations indicate that NAm only marginally affect TNF mRNA production while inhibiting TNF synthesis, suggesting an effect at a post-transcriptional stage. NAm represents an inhibitor of several classes of NAD dependent enzymes. To confirm the role of a NAD dependent step in the regulation of TNF, we took advantage of a novel pharmacological agent known to inhibit NAD synthesis (FK866). Use of this compound has led us to demonstrate that optimal TNF synthesis requires adequate intracellular NAD concentration. Collectively these studies establish a functional link between metabolism and inflammation, and suggest an important role for NAD dependent enzymes in the regulation of TNF synthesis.
Mast Cell Deficiency Leads to Decreased Expression of Cyclooxygenase-1 (COX1), COX2, Toll-Like Receptor-4 (TLR4) and Endothelial Nitric Oxide Synthase (eNOS) mRNA in Mouse Aorta (100.8)
Authors: Tan Xiaoyu , Idiculla Arun , Smith Donald D , Stechschulte Daniel J , Dileepan Kottarappat N.
Published: January 2023

Mast cells (MC) are well known for their contribution to vascular inflammation and atherosclerosis. We have shown that MC proteases and histamine amplify lipopolysaccharide (LPS)-induced inflammatory responses in human coronary artery endothelial cells via stimulation of TLR4 expression. This study examined mRNA levels of COX1, COX2, TLR4 and eNOS in aortic tissues of MC-deficient (W/Wv) mice and wild type controls (WT) to gain insight into the role of MC in vascular pathophysiology. We also determined the effect of MC degranulation on gene expression in aortas 24 h after intraperitoneal injection of MC degranulation agent compound 48/80 (1 μg/g body wt). We found that, in comparison to WT, aortic tissues of W/Wv mice express lower levels of COX1, COX2, TLR4 and eNOS mRNA. Injection of compound 48/80 resulted in marked stimulation of expression of these genes in aortas of WT, but not in W/Wv mice, confirming the involvement of MC degranulation products in this process. To determine whether a deficiency of MC and/or decreased COX expression in W/Wv mice is associated with changes in prostanoid homoeostasis, the levels of prostacyclin and thromboxane A2 metabolites in urine samples were analyzed. The levels of 6-keto PGF1α and 11-dehydro TXB2 in 24 h urine of both genotypes were comparable when normalized for the volume and creatinine excretion. These results suggest that, although MC deficiency suppresses the expression of COX1 and COX2 genes in the vasculature, the systemic prostanoid homeostasis remains protected.

(Supported by NIH R01-HL070101 and Carey Arthritis Fund)
PGE2 mediates cAMP-dependent Epac1/Rap1 signaling in primary rheumatoid synovial fibroblasts (100.9)
Authors: Kojima Fumiaki , Kapoor Mohit , Yang Lihua , Kawai Shinichi , Crofford Leslie J
Published: January 2023

Exchange protein directly activated by cAMP (Epac1 and Epac2) are recently identified targets for cAMP signaling. Epac activates Rap1 by catalyzing the conversion of GDP-Rap1 to GTP-Rap1, which is independent of classical cAMP/PKA signaling. In this study, we examined the activation of Epac/Rap1 signaling by prostaglandin E2 (PGE2), a proinflammatory lipid mediator, in synovial fibroblasts (RASF) from rheumatoid arthritis (RA) patients. Expression of Epac1 mRNA and protein were observed in RASF, whereas Epac2 expression was absent. Rap1 was also expressed and its active form, GTP-Rap1, was rapidly increased by PGE2 and it was further enhanced in the presence of IBMX, a phosphodiesterase inhibitor. In addition, the effect of PGE2 on Rap1 was mimicked by forskolin, (a direct activator of adenylate cyclase) and butaprost (an EP2 agonist) but not by sulprostone (an EP1 & EP3 agonist), which were correlated to elevation of cAMP. Furthermore, Rap1 activation was also observed by 8-CPT-2Me-cAMP (an Epac agonist) as well as 6-Bnz-cAMP (a PKA agonist). Epac1/Rap1 signaling via EP receptor associated to the elevation of cAMP may act in concert with classical PKA signaling after exposure to PGE2. Epac1/Rap1 signaling may be an alternate signaling pathway in addition to PKA signaling for PGE2 mediated pathophysiology of chronic inflammatory diseases such as RA.

Grant Support: Arthritis Foundation Biomedical Sciences Grant and NIH/NIAMS AR49010.
Interleukin 6 modulates genes associated with fibroblast migration and function (101.10)
Authors: Gallucci Randle M , Luckett Lerin R , Mickle Bethany M
Published: January 2023

Interleukin 6 deficient mice (IL-6KO) display significantly delayed cutaneous wound healing, characterized by decreased granulation tissue formation. Dermal fibroblasts are one of the principal cell types found in granulation tissue and mediate numerous processes during healing including collagen deposition. Polyvinyl alcohol sponge matrices implanted seven days in IL-6KO mice showed far fewer infiltrating fibroblasts as compared to wild-type C57. It was also found that full thickness wounds from IL-6KO mice showed higher expression of MMP-2 and TIMP-2 as compared to wild-type from 1 to 5 days post wounding. Treatment of IL-6KO fibroblast populated free-floating collagen lattices with various concentrations of rmIL-6 showed a dose response decrease in MMP-2 TIMP-2 mRNA and protein expression. The expression of TGFβ’s 1, and 3 as well as TGFβRII, which can mediate collagen formation, were increased in IL-6KO mouse skin treated intradermally with rmIL-6 in a dose response manner. Expression of TGFβ1, 3, and RII were induced as well in IL-6KO fibroblast collagen lattice cultures in a similar manner. These results indicate that IL-6 may influence on collagen deposition during granulation tissue formation in skin wounds. The mechanism of this may involve both the inhibition of MMP function, and the induction of TGFβ genes.

This research was supported by a grant from NIH/NIGMS (5R01GM067745-03).
Adenosine A2a receptor activation reduces lymphocyte-mediated liver injury induced by alpha-galactosylceramide in vivo (101.11)
Authors: Chen Lanlin , Linden Joel M
Published: January 2023

NKT cells play a key role in initiating an inflammatory cascade during liver ischemia/reperfusion injury (IRI). Cytokine production by NKT cells is inhibited by A2A adenosine receptor (A2AAR) activation. To further investigate the mechanism of A2AAR-mediated liver protection, we selectively activated NKT cells in vivo with α-galactosylceramide (α-GalCer, 4 μg/mouse) a glycolipid that is presented by CD1d to invariant NKT cell receptors. Liver injury and lymphocyte activation were assessed by measuring serum alanine transaminase (ALT) which peaked in 18 h, and proinflammatory cytokines in various cells by FACS. Mice treated with the selective A2AAR agonist ATL313 (1 ng/kg/min) prior to and during α-GalCer treatment had significantly reduced serum ALT and interferon (IFN)- 1 _ levels. In NKT cells, ATL313 reduced intracellular IFN-γ, TNF-α and IL-2 by 30–60%. Peak IFN-γ production occurred earlier (< 2 hr) in NKT cells than in NK cells (> 4 hr) suggesting that NK cell activation is downstream of NKT cell activation. All of the effects of ATL313 were abolished in A2AAR KO mice. In IFN-γ KO mice there was a 58% and 70% reduction in TNF-α and IL-2, respectively in response to α-GalCer, suggesting a primary role by IFN-γ in triggering a cytokine cascades that drives liver injury. In summery, selective NKT cell-activation by α-GalCer causes liver injury which is reduced by the A2AAR activation. It appears that ATL313 exerts its action on NKT and possibly other lymphocytes primarily by limiting IFN-γ production from these cells during early inflammatory processes.

Supported by NIH R01 HL37942