bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Cytokines, Cytokine Inhibitors, and Acute-Phase Proteins Following Anti-TNF-α Therapy in Rheumatoid Arthritis
Authors: Charles Peter , Elliott Michael J. , Davis Diana , Potter Alison , Kalden Joachim R. , Antoni Christian , Breedveld Ferdinand C. , Smolen Josef S. , Eberl Gabriele , deWoody Kim , Feldmann Marc , Maini Ravinder N.
Published: December 2022

Treatment with a chimeric mAb to TNF-α has been shown to suppress inflammation and improve patient well-being in rheumatoid arthritis (RA), but the mechanisms of action of such treatment have not been fully explored. Here we show that in vivo administration of anti-TNF-α Ab, using a longitudinal analysis, results in the rapid down-regulation of a spectrum of cytokines, cytokine inhibitors, and acute-phase proteins. Marked diurnal variation in the serum levels of some of these were detected. These results were consistent with the concept of a cytokine-dependent cytokine cascade, and the degree of clinical benefit noted after anti-TNF-α therapy is probably due to the reduction in many proinflammatory mediators apart from TNF-α, such as IL-6, which reached normal levels within 24 h. Serum levels of cytokine inhibitors such as soluble p75 and p55 TNFR were reduced as was IL-1 receptor antagonist. Reductions in acute-phase proteins occurred after serum IL-6 fell and included serum amyloid A, haptoglobin, and fibrinogen. The latter reduction could be of importance, as it is a risk factor for atherosclerosis, which is augmented in RA patients.
Lipid-DNA Complexes Induce Potent Activation of Innate Immune Responses and Antitumor Activity When Administered Intravenously
Authors: Dow Steven W. , Fradkin Lee G. , Liggitt Denny H. , Willson Andy P. , Heath Timothy D. , Potter Terry A.
Published: December 2022

Cationic lipid-DNA complexes (CLDC) are reported to be safe and effective for systemic gene delivery, particularly to the lungs. However, we observed that i.v. injection of CLDC induced immunologic effects not previously reported. We found that even very low doses of CLDC administered i.v. induced marked systemic immune activation. This response included strong up-regulation of CD69 expression on multiple cell types and systemic release of high levels of Th1 cytokines, from both lung and spleen mononuclear cells. CLDC were much more potent immune activators on a per weight basis than either LPS or poly(I:C). The remarkable potency of CLDC appeared to result from enhancement of the immune stimulatory properties of DNA, since cationic lipids alone were without immune stimulatory activity. Systemic treatment with CLDC controlled tumor growth and significantly prolonged survival times in mice with metastatic pulmonary tumors. NK cells accumulated to high levels in the lungs of CLDC-treated mice, were functionally activated, and released high levels of IFN-γ. The antitumor activity induced by CLDC injection was dependent on both NK cells and IFN-γ. Thus, DNA complexed to cationic liposomes becomes highly immunostimulatory and capable of inducing strong antitumor activity when administered systemically.
Essential Role of T Cell NF-κB Activation in Collagen-Induced Arthritis
Authors: Seetharaman Rajalakshmi , Mora Ana L. , Nabozny Gerald , Boothby Mark , Chen Jin
Published: December 2022

NF-κB/Rel proteins are ubiquitous transcription factors that are activated by proinflammatory signals or engagement of Ag receptors. To study the role of NF-κB/Rel signaling in T lymphocytes during autoimmune disease, we investigated type II collagen-induced arthritis (CIA) in transgenic mice expressing a constitutive inhibitor of NF-κB/Rel (IκBα(ΔN)) in the T lineage. Expression of the IκBα(ΔN) transgene was persistently high in adult peripheral lymphoid organs and undetectable in T cell-depleted splenocytes, suggesting the expression of the transgene is restricted to the T lineage. The incidence and severity of CIA were decreased significantly in these IκBα(ΔN) transgenic mice compared with nontransgenic littermates. Inhibition of CIA was not due solely to a decrease in their CD8+ population because transfer of wild-type CD8+ cells into transgenic mice failed to restore disease susceptibility. Protection against disease was associated with a moderate decrease in clonal expansion and a profound and persistent decrease in Ag-induced IFN-γ production in vivo. Consistent with decreased level of anti-type II collagen-specific Abs and IFN-γ, serum levels of IgG2a anti-CII Abs were significantly reduced. However, anti-CII-specific IgG1 levels were normal, indicating that some aspects of T cell help were unaffected. Taken together, these results suggest that inhibition of NF-κB in T cells impairs CIA development in vivo through decreases in type 1 T cell-dependent responses.
Evidence for the Involvement of CD44 in Endothelial Cell Injury and Induction of Vascular Leak Syndrome by IL-2
Authors: Rafi-Janajreh Asimah Q. , Chen Dawei , Schmits Rudolf , Mak Tak W. , Grayson Randolph L. , Sponenberg D. Phillip , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: December 2022

At sites of chronic inflammation seen during infections, autoimmunity, graft-vs-host response, and cytokine therapy, endothelial cell injury is known to occur, the exact mechanism of which is unknown. In the current study we used IL-2-induced vascular leak syndrome (VLS) as a model to investigate whether cytotoxic lymphocytes use CD44 in mediating endothelial cell injury. Administration of IL-2 to wild-type mice triggered significant VLS in the lungs and liver. In contrast, in CD44 knockout (KO) mice, IL-2-induced VLS was markedly reduced in the lungs and liver. IL-2-treated wild-type and CD44 KO mice had similar levels of perivascular infiltration with lymphocytes in the lungs and liver. This suggested that the decrease in VLS seen in CD44 KO mice was not due to the inability of lymphocytes to migrate to these organs. Ultrastructural studies demonstrated extensive endothelial cell damage in the lungs and liver of IL-2-treated wild-type, but not CD44 KO, mice. Moreover, CD44-KO mice exhibited a marked decrease in IL-2-induced lymphokine-activated killer cell activity. The induction of VLS was dependent on the expression of CD44 on immune cells rather than endothelial cells because adoptive transfer of CD44+, but not CD44− spleen cells along with IL-2 into CD44 KO mice triggered VLS. The IL-2-induced VLS was blocked by administration of F(ab′)2 of Abs against CD44. The current study demonstrates that CD44 plays a key role in endothelial cell injury. Blocking CD44 in vivo may offer a novel therapeutic approach to prevent endothelial cell injury by cytotoxic lymphocytes in a variety of clinical disease models.
Chemokine Receptor Expression and Signaling in Macaque and Human Fetal Neurons and Astrocytes: Implications for the Neuropathogenesis of AIDS
Authors: Klein Robyn S. , Williams Kenneth C. , Alvarez-Hernandez Xavier , Westmoreland Susan , Force Thomas , Lackner Andrew A. , Luster Andrew D.
Published: December 2022

Chemokines are believed to play a role in the neuropathogenesis of AIDS through their recruitment of neurotoxin-secreting, virally infected leukocytes into the CNS. Levels of chemokines are elevated in brains of patients and macaques with HIV/SIV-induced encephalitis. The chemokine receptors CCR3, CCR5, and CXCR4 are found on subpopulations of neurons in the cortex of human and macaque brain. We have developed an in vitro system using both macaque and human fetal neurons and astrocytes to further investigate the roles of these receptors in neuronal response to inflammation. Here we report the presence of functional HIV/SIV coreceptors CCR3, CCR5, and CXCR4 on fetal human and macaque neurons and CCR5 and CXCR4 on astrocytes immediately ex vivo and after several weeks in culture. Confocal imaging of immunostained neurons demonstrated different patterns of distribution for these receptors, which may have functional implications. Chemokine receptors were shown to respond to their appropriate chemokine ligands with increases in intracellular calcium that, in the case of neurons, required predepolarization with KCl. These responses were blocked by neutralizing chemokine receptor in mAbs. Pretreatment of neural cells with pertussis toxin abolished responses to stromal-derived factor-1α, macrophage inflammatory protein-1β, and RANTES, indicating coupling of CCR5 and CXCR4 to a Giα protein, as in leukocytes. Cultured macaque neurons demonstrated calcium flux response to treatment with recombinant SIVmac239 envelope protein, suggesting a mechanism by which viral envelope could affect neuronal function in SIV infection. The presence of functional chemokine receptors on neurons and astrocytes suggests that chemokines could serve to link inflammatory and neuronal responses.
Differential Susceptibilities to Chronic Beryllium Disease Contributed by Different Glu69 HLA-DPB1 and -DPA1 Alleles
Authors: Wang Zaolin , White P. Scott , Petrovic Michelle , Tatum Owatha L. , Newman Lee S. , Maier Lisa A. , Marrone Babetta L.
Published: December 2022

Chronic beryllium disease (CBD) is associated with the allelic substitution of a Glu69 in the HLA-DPB1 gene. Although up to 97% of CBD patients may have the Glu69 marker, about 30–45% of beryllium-exposed, unaffected individuals carry the same marker. Because CBD occurs in only 1–6% of exposed workers, the presence of Glu69 does not appear to be the sole genetic factor underlying the disease development. Using two rounds of direct automated DNA sequencing to precisely assign HLA-DPB1 haplotypes, we have discovered highly significant Glu69-containing allele frequency differences between the CBD patients and a beryllium-exposed, nondiseased control group. Individuals with DPB1 Glu69 in both alleles were almost exclusively found in the CBD group (6/20) vs the control group (1/75). Whereas most Glu69 carriers from the control group had a DPB1 allele *0201 (68%), most Glu69 carriers from the CBD group had a non-*0201 DPB1 Glu69-carrying allele (84%). The DPB1 allele *0201 was almost exclusively (29/30) associated with DPA1 *01 alleles, while the non-*0201 Glu69-containing DPB1 alleles were closely associated with DPA1 *02 alleles (26/29). Relatively rare Glu69-containing alleles *1701, *0901, and *1001 had extremely high frequencies in the CBD group (50%), as compared with the control group (6.7%). Therefore, the most common Glu69-containing DPB1 allele, *0201, does not seem to be a major disease allele. The results suggest that it is not the mere presence of Glu69, per se, but specific Glu69-containing alleles and their copy number (homozygous or heterozygous) that confer the greatest susceptibility to CBD in exposed individuals.
An Immunoadhesin Incorporating the Molecule OX-2 Is a Potent Immunosuppressant That Prolongs Allo- and Xenograft Survival
Authors: Gorczynski Reginald M. , Cattral Mark S. , Chen Zhigi , Hu Jiang , Lei Ji , Min Wei-Ping , Yu Gary , Ni Jin
Published: December 2022

We have established that, in mice receiving donor-specific immunization by the portal vein, the increased graft survival seen is associated with the increased expression of a molecule (OX-2) on a subpopulation of dendritic cells (DC), and polarization of cytokine production to type 2 cytokines on Ag-specific restimulation of cells from these mice. Furthermore, infusion of a mAb to OX-2 blocks both the increased graft survival and the altered cytokine production seen. We have constructed an immunoadhesin in which the extracellular domain of OX-2 is linked to the murine IgG2a Fc region, and we have expressed this molecule (OX-2:Fc) in a eukaryotic (baculovirus) expression system. Incubation of lymphocytes with 50 ng/ml OX-2:Fc inhibits a primary mixed lymphocyte reaction in vitro, as assayed by proliferation and induction of cytotoxic T cells, and also alters cytokine production with decreased IL-2 (IFN-γ) production and increased IL-4 (IL-10) production. Similarly, in vivo infusion of OX-2:Fc promotes increased allo- and xenograft (both skin and renal grafts) survival and decreases the Ab response to sheep erythrocytes. Our data suggest this molecule might have clinical importance in allo- and xenotransplantation.
Cutting Edge: CXCR4-Lo: Molecular Cloning and Functional Expression of a Novel Human CXCR4 Splice Variant
Authors: Gupta Shalley K. , Pillarisetti Kodandaram
Published: December 2022

Human CXCR4 is a specific receptor for the CXC chemokine stromal cell-derived factor-1 (SDF-1) and a coreceptor for T cell line tropic strains of HIV-1. Genetic knockouts of CXCR4 and SDF-1 have delineated their critical role during embryonic cardiogenesis, leukopoiesis, and vasculogenesis. Herein, we used bioinformatics and differential strategies like isoform-specific RT-PCR and Northern blots to identify and clone a novel unspliced isoform of human CXCR4, termed CXCR4-Lo. CXCR4-Lo corresponds to a larger ∼4.0-kb mRNA transcript and differs from the known human CXCR4 by the first 9 aa in the functionally important NH2-terminal extracellular domain of the receptor. CXCR4-Lo-transfected rat basophil leukemia-2H3 cells responded to SDF-1 with a transient rise of intracellular Ca2+ concentration and by undergoing chemotaxis. Expression of CXCR4-Lo is noteworthy, as it may have differential affinity as a coreceptor for HIV strains in comparison with CXCR4. Furthermore, CXCR4-Lo may also provide a functional backup to CXCR4 during embryogenesis.
Germinal Center B Cell Apoptosis Requires Both Caspase and Cathepsin Activity
Authors: van Eijk Marco , de Groot Cornelis
Published: December 2022

Follicular dendritic cells (FDCs) select B cells during germinal center (GC) reactions. The B cells that are able to bind to the FDCs receive a signal that leads to the termination of endonuclease activity in the nuclei of those B cells. This signal must be in addition to the signals transferred through the cross-linkage of the B cell receptors and signals resulting from the interactions of the adhesion molecules lymphocyte function-associated Ag-1 and very late Ag-4 with ICAM-1 and VCAM-1, respectively. In this report, we present evidence that the FDCs silence all apoptotic processes in GC B lymphocytes and additionally switch off pre-present endonuclease activity. We also show that GC B cell apoptosis requires cathepsin activity downstream of caspase-3. This cathepsin activity is directly connected to endonuclease activity and therefore may be an interesting target for the antiapoptotic factors produced by FDCs.
Bromelain, from Pineapple Stems, Proteolytically Blocks Activation of Extracellular Regulated Kinase-2 in T Cells
Authors: Mynott Tracey L. , Ladhams Andrew , Scarmato Pierre , Engwerda Christian R.
Published: December 2022

Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3ε mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-γ, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways.
A Composite C/EBP Binding Site Is Essential for the Activity of the Promoter of the IL-3/IL-5/Granulocyte-Macrophage Colony-Stimulating Factor Receptor βc Gene
Authors: van Dijk Thamar B. , Baltus Belinda , Raaijmakers Jan A. M. , Lammers Jan-Willem J. , Koenderman Leo , de Groot Rolf P.
Published: December 2022

The common β-chain (βc) is the main signaling component of the heterodimeric receptors for IL-3, IL-5, and GM-CSF and is primarily expressed on myeloid cells. The proximal βc promoter is regulated by GGAA binding proteins, including PU.1, a hemopoietic specific member of the Ets family. However, it is not likely that PU.1 alone accounts for the myeloid-restricted expression of the βc subunit. Here we describe the identification of a C/EBP binding enhancer that is located 2 kb upstream of the transcription start site. The enhancer contains two elements that bind C/EBPα and -β in U937 cells, while C/EBPε is also bound in extracts of HL-60 cells. Importantly, deletion of the enhancer or mutation of either of one of the C/EBP sites results in a complete loss of promoter activity in cell lines as well as in primary cells, showing the importance of C/EBP members in βc gene activation. We further show that PU.1 has to cooperate with C/EBP proteins to induce βc transcription. Since the βc is already expressed on CD34+ cells, these results demonstrate that both C/EBP and PU.1 are not only important for the myeloid-specific gene regulation at later stages of myeloid differentiation.
Partial IL-10 Inhibition of the Cell-Mediated Immune Response in Chronic Beryllium Disease
Authors: Tinkle Sally S. , Kittle Lori A. , Newman Lee S.
Published: December 2022

Chronic beryllium disease (CBD) provides a human disorder in which to study the delayed type IV hypersensitivity response to persistent Ag that leads to noncaseating pulmonary granuloma formation. We hypothesized that, in CBD, failure of IL-10 to modulate the beryllium-specific, cell-mediated immune response would result in persistent, maximal cytokine production and T lymphocyte proliferation, thus contributing to the development of granulomatous lung disease. To test this hypothesis, we used bronchoalveolar lavage cells from control and CBD subjects to evaluate the beryllium salt-specific production of endogenous IL-10 and the effects of exogenous human rIL-10 (rhIL-10) on HLA expression, on the production of IL-2, IFN-γ, and TNF-α, and on T lymphocyte proliferation. Our data demonstrate that beryllium-stimulated bronchoalveolar lavage cells produce IL-10, and the neutralization of endogenous IL-10 does not increase significantly cytokine production, HLA expression, or T lymphocyte proliferation. Second, the addition of excess exogenous rhIL-10 partially inhibited the beryllium-stimulated production of IL-2, IFN-γ, and TNF-α; however, we measured no change in T lymphocyte proliferation or in the percentage of alveolar macrophages expressing HLA-DP. Interestingly, beryllium salts interfered with an IL-10-stimulated decrease in the percentage of alveolar macrophages expressing HLA-DR. We conclude that, in the CBD-derived, beryllium-stimulated cell-mediated immune response, low levels of endogenous IL-10 have no appreciable effect; exogenous rhIL-10 has a limited effect on cytokine production and no effect on T lymphocyte proliferation or HLA expression.
Identification of Natural Antigenic Peptides of a Human Gastric Signet Ring Cell Carcinoma Recognized by HLA-A31-Restricted Cytotoxic T Lymphocytes
Authors: Suzuki Kazuhiro , Sahara Hiroeki , Okada Yohjiro , Yasoshima Takahiro , Hirohashi Yoshihiko , Nabeta Yuki , Hirai Itaru , Torigoe Toshihiko , Takahashi Shuji , Matsuura Akihiro , Takahashi Nobuaki , Sasaki Aya , Suzuki Manabu , Hamuro Junji , Ikeda Hideyuki , Wada Yoshimasa , Hirata Koichi , Kikuchi Kokichi , Sato Noriyuki
Published: December 2022

Peptides of human melanomas recognized by CD8+ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.
Regulation of IL-15-Stimulated TNF-α Production by Rolipram
Authors: Kasyapa Chitta S. , Stentz Carrie L. , Davey Michael P. , Carr Daniel W.
Published: December 2022

Agents that increase intracellular cAMP have been shown to reduce joint inflammation in experimental arthritis, presumably by lowering the release of proinflammatory cytokines, such as TNF-α. Recent studies suggest that, in joints of patients with rheumatoid arthritis, TNF-α release from macrophages is triggered by their interaction with IL-15-stimulated T lymphocytes. In this report, we analyze the effect of rolipram, a cAMP-specific phosphodiesterase inhibitor, on TNF-α production in this experimental system. Cocultures of U937 cells with IL-15-stimulated T cells, but not control T cells, resulted in increased release of TNF-α. Pretreatment of T cells with rolipram or cAMP analogues inhibited the IL-15-stimulated increases in proliferation, expression of cell surface molecules CD69, ICAM-1, and LFA-1, and release of TNF-α from macrophages. Addition of PMA to T cells dramatically increased the expression of cell surface molecules, but had little or no effect on TNF-α release from either T cells or from cocultures, suggesting that other surface molecules must also be involved in T cell/macrophage contact-mediated production of TNF-α. Addition of PMA synergistically increased the proliferation of IL-15-stimulated T cells and the secretion of TNF-α from IL-15-stimulated T cell/macrophage cocultures. Rolipram and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) blocked these increases. Measurement of protein kinase A (PKA) activity and the use of inhibitory cAMP analogues (RpCPT-cAMP) confirmed that rolipram worked by stimulating PKA. These data suggest that PKA-activating agents, such as rolipram, can block secretion of TNF-α from macrophages by inhibiting T cell activation and expression of surface molecules.
Superoxide Attenuates Macrophage Apoptosis by NF-κB and AP-1 Activation That Promotes Cyclooxygenase-2 Expression
Authors: von Knethen Andreas , Callsen Dagmar , Brüne Bernhard
Published: December 2022

Macrophages are a major source of cytokines and proinflammatory radicals such as superoxide. These mediators can be both produced and utilized by macrophages in autocrine-regulatory pathways. Therefore, we studied the potential role of oxygen radical-regulatory mechanisms in reprogramming macrophage apoptosis. Preactivation of RAW 264.7 cells with a nontoxic dose of the redox cycler 2,3-dimethoxy-1,4-naphtoquinone (5 μM) for 15 h attenuated S-nitrosoglutathione (1 mM)-initiated apoptotic cell death and averted accumulation of the tumor suppressor p53, which is indicative for macrophage apoptosis. Preactivation with superoxide promoted cyclooxygenase-2 induction that was NF-κB and AP-1 mediated. NF-κB activation was confirmed by p50/p65-heterodimer formation, IκB-α degradation, and stimulation of a NF-κB luciferase reporter construct. Furthermore, a NF-κB decoy approach abrogated cyclooxygenase-2 (Cox-2) expression as well as inducible protection. The importance of AP-1 for superoxide-mediated Cox-2 expression and cell protection was substantiated by using the extracellular signal-regulated kinase-inhibitor PD98059 and the p38-inhibitor SB203580, which blocked Cox-2 expression. In corroboration, Cox-2 expression was hindered by a dominant-negative c-jun mutant (TAM67). Protection from apoptosis was verified in human macrophages with the notion that superoxide promoted Cox-2 expression, which in turn attenuated nitric oxide-evoked caspase activation. We conclude that the sublethal generation of oxygen radicals reprograms macrophages by NF-κB and AP-1 activation. The resulting hyporesponsiveness reveals an attenuated apoptotic program in association with Cox-2 expression.
GDKV-Induced Antiphospholipid Antibodies Enhance Thrombosis and Activate Endothelial Cells In Vivo and In Vitro
Authors: Gharavi Azzudin E. , Pierangeli Silvia S. , Colden-Stanfield Margaret , Liu Xiao Wei , Espinola Ricardo G. , Harris E. Nigel
Published: December 2022

Antiphospholipid (aPL) Abs are associated with thrombosis, pregnancy loss, and thrombocytopenia in patients with systemic lupus erythematosus or primary antiphospholipid syndrome (APS). β2-Glycoprotein I (β2GPI), a phospholipid-binding serum protein, is involved in aPL binding to phospholipids. aPL can be generated in mice by immunization with β2GPI, and these Abs are thrombogenic and cause pregnancy loss in mice. The objective of this study is to determine whether aPL induced by immunization with the phospholipid-binding site of β2GPI are thrombogenic and whether they activate endothelial cells (EC) in vivo and in vitro. Murine monoclonal aPL were generated from spleen cells of a mouse immunized with GDKV, a synthetic 15-aa peptide spanning Gly274–Cys288 in the fifth domain of human β2GPI, which represents the phospholipid-binding site of β2GPI. The Abs generated had aPL and anti-β2GPI activities. The effect of these Abs on thrombus formation and on EC activation in vivo was determined using a mouse model of thrombosis and microcirculation that enables examination of the adhesion of leukocyte to EC as an indication of EC activation as well as adhesion molecule expression using in vitro ELISA analysis. Mice injected with this monoclonal aPL showed a significant increase in leukocyte sticking and also produced larger thrombi that persisted longer. Exposure to GDKV-induced aPL for 4 h significantly increased surface Ag expression of E-selectin, ICAM-1, and VCAM-1. These data indicate that aPL induced by immunization with the phospholipid binding site of β2GPI are thrombogenic and activate endothelial cells.
Pancreas-Infiltrating Th1 Cells and Diabetes Develop in IL-12-Deficient Nonobese Diabetic Mice
Authors: Trembleau Sylvie , Penna Giuseppe , Gregori Silvia , Chapman Harold D. , Serreze David V. , Magram Jeanne , Adorini Luciano
Published: December 2022

IL-12 and IL-12 antagonist administration to nonobese diabetic (NOD) mice accelerates and prevents insulin-dependent diabetes mellitus (IDDM), respectively. To further define the role of endogenous IL-12 in the development of diabetogenic Th1 cells, IL-12-deficient NOD mice were generated and analyzed. Th1 responses to exogenous Ags were reduced by ∼80% in draining lymph nodes of these mice, and addition of IL-12, but not IL-18, restored Th1 development in vitro, indicating a nonredundant role of IL-12. Moreover, spontaneous Th1 responses to a self Ag, the tyrosine phosphatase-like IA-2, were undetectable in lymphoid organs from IL-12-deficient, in contrast to wild-type, NOD mice. Nevertheless, wild-type and IL-12-deficient NOD mice developed similar insulitis and IDDM. Both in wild-type and IL-12-deficient NOD mice, ∼20% of pancreas-infiltrating CD4+ T cells produced IFN-γ, whereas very few produced IL-10 or IL-4, indicating that IDDM was associated with a type 1 T cell infiltrate in the target organ. T cell recruitment in the pancreas seemed favored in IL-12-deficient NOD mice, as revealed by increased P-selectin ligand expression on pancreas-infiltrating T cells, and this could, at least in part, compensate for the defective Th1 cell pool recruitable from peripheral lymphoid organs. Residual Th1 cells could also accumulate in the pancreas of IL-12-deficient NOD mice because Th2 cells were not induced, in contrast to wild-type NOD mice treated with an IL-12 antagonist. Thus, a regulatory pathway seems necessary to counteract the pathogenic Th1 cells that develop in the absence of IL-12 in a spontaneous chronic progressive autoimmune disease under polygenic control, such as IDDM.
Cutting Edge: SIV Nef Protein Utilizes Both Leucine- and Tyrosine-Based Protein Sorting Pathways for Down-Regulation of CD4
Authors: Bresnahan Patricia A. , Yonemoto Wes , Greene Warner C.
Published: December 2022

The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.
Vasoactive Intestinal Peptide Synergizes with TNF-α in Inducing Human Dendritic Cell Maturation
Authors: Delneste Yves , Herbault Nathalie , Galea Brice , Magistrelli Giovanni , Bazin Ingrid , Bonnefoy Jean-Yves , Jeannin Pascale
Published: December 2022

We investigated the effects of different neuropeptides on human dendritic cells (DC) maturation. Immature DC, derived from monocytes cultured for 6 days with IL-4 plus GM-CSF, have been exposed to somatostatin, substance P, or vasoactive intestinal peptide (VIP). Among these neuropeptides, only VIP induces the production of bioactive IL-12 and the neoexpression of CD83 on a fraction of the DC population, with an effect significant at 100 and 10 nM, respectively. These effects of VIP are dose-dependent, unaffected by polymixin B, and partly prevented by a VIP receptor antagonist. Although the effects of VIP alone remain modest, it synergizes with TNF-α to induce DC maturation. In the presence of a suboptimal concentration of TNF-α, which has no detectable effect on DC by itself, VIP induces the production of high levels of bioactive IL-12, the neoexpression of CD83 on almost all the DC population (with an effect significant at 10 and 0.1 nM, respectively), and the up-regulation of various adhesion and costimulatory molecule expression. Moreover, DC exposed to VIP plus a suboptimal concentration of TNF-α are as potent as mature DC obtained by treatment with an optimal concentration of TNF-α in stimulating allogenic T cell proliferation. Our data suggest that, in inflammatory sites, VIP may cooperate with proinflammatory mediators, such as TNF-α, to induce DC maturation.
IL-12 Acts Selectively on CD8α− Dendritic Cells to Enhance Presentation of a Tumor Peptide In Vivo
Authors: Grohmann Ursula , Bianchi Roberta , Belladonna Maria L. , Vacca Carmine , Silla Silvia , Ayroldi Emira , Fioretti Maria C. , Puccetti Paolo
Published: December 2022

Previous work has shown that a significant proportion of murine splenic dendritic cells (DC) express a high affinity receptor for IL-12, thus accounting for the adjuvanticity of the cytokine when DBA/2 mice are transferred with syngeneic DC exposed in vitro to rIL-12 and an otherwise poorly immunogenic tumor peptide. In DBA/2 mice, splenic DC consist of 90–95% CD8− and 5–10% CD8+ cells. To detect any difference in IL-12 responsiveness among phenotypically distinct DC subtypes, enriched CD8− (>99% pure) and CD8+ (∼80% pure) populations of DC from DBA/2 spleens were assayed for APC function in vivo following exposure to rIL-12 and tumor peptide in vitro. Unlike unfractionated DC, the CD8− fraction was capable of effective presentation of the peptide even when the cells had not been pretreated with IL-12 before peptide pulsing. The addition of as few as 3% CD8+ cells during pulsing blocked in vivo priming by the CD8− fraction. However, pretreatment of CD8− DC with IL-12 before cell mixing and peptide pulsing ablated the inhibitory effect of the CD8+ fraction. CD8−, but not CD8+, DC showed significant message expression for the β1 and β2 subunits of the IL-12 receptor. These data suggest that a minority population of CD8+ DC, which appeared to secrete IL-10 in vitro, negatively regulates the induction of T cell reactivity by peptide-loaded CD8− DC in DBA/2 mice. However, the CD8− fraction can be primed by IL-12 to overcome the inhibitory effect of the CD8+ subtype.
In Vivo Immunomodulation Following Intradermal Injection with DNA Encoding IL-18
Authors: Kremer Laurent , Dupré Loïc , Wolowczuk Isabelle , Locht Camille
Published: December 2022

IL-18, a recently identified cytokine synthesized by different cell types, including Kupffer cells, activated macrophages, and keratinocytes, induces IFN-γ production by T cells and NK cells. The cDNA encoding IL-18 with its natural signal peptide was cloned under control of the CMV promoter and injected into the skin of mice. A single intradermal injection of this construction led to efficient in vivo expression of IL-18 in cutaneous dermal cells and induced IFN-γ mRNA production, indicating that it was produced in a biologically active form. In addition, a massive cellular infiltrate was observed in the skin 2 days after injection. When the mice were subsequently infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG), they produced lower levels of anti-BCG Abs than control animals. However, in contrast to their lowered humoral immune response, the mice produced higher amounts of Ag-specific IFN-γ after in vitro restimulation, as compared with the controls. Therefore, injection of DNA encoding IL-18 into the skin modulates both Ag-specific humoral and T cell responses upon mycobacterial infection. It increases the Th1 type response, which may be particularly useful for the development of new immunotherapeutic or immunoprotective approaches against infections by intracellular parasites, such as mycobacteria.
Effect of Vascular Endothelial Growth Factor and FLT3 Ligand on Dendritic Cell Generation In Vivo
Authors: Ohm Joyce E. , Shurin Michael R. , Esche Clemens , Lotze Michael T. , Carbone David P. , Gabrilovich Dmitry I.
Published: December 2022

The cytokine FLT3 ligand (FL) enhances dendritic cell (DC) generation and has therefore been proposed as a means to boost antitumor immunity. Vascular endothelial growth factor (VEGF) is produced by a large percentage of tumors and is required for development of tumor neovasculature. We previously showed that VEGF decreases DC production and function in vivo. In this study, we tested the hypothesis that VEGF regulates FL effects on DC generation. In seven experiments, four groups of mice were treated with PBS, VEGF alone (100 ng/h), FL alone (10 μg/day), or with the combination of FL and VEGF. VEGF and PBS were administered continuously for 14 days via s.c. pumps. FL was given s.c. daily for 9 days, beginning on day 4. Tissues were collected and the number, phenotype, and function of lymph node, splenic, and thymic DCs were analyzed on day 14. As expected, treatment with FL resulted in a marked increase in the number of lymph node and spleen DCs and a smaller increase in thymic DC. Pretreatment of mice with VEGF inhibited these FL effects in lymph nodes and thymus by about 50%, whereas spleen DC numbers were undiminished by VEGF. VEGF treatment in vivo also inhibited the ability of FL to increase the number of hemopoietic precursor cells and the level of maturity exhibited by DC derived from these hemopoietic precursor cells in vitro. VEGF inhibited FL-inducible activation of transcription factor NF-κB. These data suggest that VEGF interferes with the ability of FL to promote dendritic cell differentiation from bone marrow progenitor cells in mice and therefore may decrease the therapeutic efficacy of FL in settings where increased numbers of DCs might provide clinical benefits.
The CD28/B7 Interaction Is Not Required for Resistance toToxoplasma gondiiin the Brain but Contributes to the Development of Immunopathology
Authors: Reichmann Gaby , Villegas Eric N. , Craig Linden , Peach Robert , Hunter Christopher A.
Published: December 2022

Infection of C57BL/6 mice with Toxoplasma gondii leads to chronic encephalitis characterized by infiltration into the brain of T cells that produce IFN-γ and mediate resistance to the parasite. Our studies revealed that expression of B7.1 and B7.2 was up-regulated in brains of mice with toxoplasmic encephalitis (TE). Because CD28/B7-mediated costimulation is important for T cell activation, we assessed the contribution of this interaction to the production of IFN-γ by T cells from brains and spleens of mice with TE. Stimulation of splenocytes with Toxoplasma Ag or anti-CD3 mAb resulted in production of IFN-γ, which was inhibited by 90% in the presence of CTLA4-Ig, an antagonist of B7 stimulation. However, production of IFN-γ by T cells from the brains of these mice was only slightly reduced (20%) by the addition of CTLA4-Ig. To address the role of the CD28/B7 interaction during TE, we compared the development of disease in C57BL/6 wild-type (wt) and CD28−/− mice. Although the parasite burden was similar in wt and CD28−/− mice, CD28−/− mice developed less severe encephalitis and survived longer than wt mice. Ex vivo recall responses revealed that mononuclear cells isolated from the brains of chronically infected CD28−/− mice produced less IFN-γ than wt cells, and this correlated with reduced numbers of intracerebral CD4+ T cells in CD28−/− mice compared with wt mice. Taken together, our data show that resistance to T. gondii in the brain is independent of CD28 and suggest a role for CD28 in development of immune-mediated pathology during TE.
The Nonintegrin Laminin Binding Protein (p67 LBP) Is Expressed on a Subset of Activated Human T Lymphocytes and, Together with the Integrin Very Late Activation Antigen-6, Mediates Avid Cellular Adherence to Laminin
Authors: Canfield Stephen M. , Khakoo Aarif Y.
Published: December 2022

A search for genes expressed in activated T cells revealed that the nonintegrin, 67-kDa laminin binding protein (p67 LBP) is expressed on the surface of a subset (10–15%) of activated peripheral blood T cells. Surface p67 LBP expression is detectable by FACS using the anti-p67 LBP mAb, MLuC5, within 6 h of T cell activation with phorbol dibutyrate and ionomycin, peaks 18–36 h postactivation, and persists for 7–10 days. The subset of T cells expressing p67 LBP is composed of mature, single-positive cells (85% CD4+8−, 15% CD4−8+) of memory cell phenotype (100% CD45 RO+/CD45 RA−). The p67 LBP+ T cells also express the integrin α6 chain (CD49f), which is known to associate with p67 LBP on tumor cells. In addition, the p67 LBP+ T cells express the integrin β1, which associates with α6 in the laminin-specific integrin receptor very late activation Ag (VLA)-6 (α6β1). Expression of an exogenous cDNA encoding the 37-kDa LBP precursor (p37 LBPP) confers p67 LBP surface expression on a p67 LBP-negative Jurkat T cell line (B2.7). Expression of p67 LBP induces B2.7 transfectants to adhere to laminin, but avid laminin binding depends on coexpression of VLA-6. Taken together, these data indicate that p67 LBP is an activation-induced surface structure on memory T cells that, together with VLA-6, mediates cellular adherence to laminin.
Evidence That Langerhans Cells in Adult Pulmonary Langerhans Cell Histiocytosis Are Mature Dendritic Cells: Importance of the Cytokine Microenvironment
Authors: Tazi Abdellatif , Moreau Joelle , Bergeron Anne , Dominique Stéphane , Hance Allan J. , Soler Paul
Published: December 2022

Because Langerhans cells (LC) in peripheral tissues are generally “immature” cells with poor lymphostimulatory activity, the contribution of immune responses initiated by LC to the pathogenesis of pulmonary LC histiocytosis (LCH) has been uncertain. In this study we demonstrate that LC accumulating in LCH granulomas are phenotypically similar to mature lymphostimulatory dendritic cells present in lymphoid organs. LC in LCH granulomas intensely expressed B7-1 and B7-2 molecules, whereas normal pulmonary LC and LC accumulating in other pathologic lung disorders did not express these costimulatory molecules. The presence of B7+ LC in LCH granulomas was associated with the expression in these lesions, but not at other sites in the lung, of a unique profile of cytokines (presence of GM-CSF, TNF-α, and IL-1β and the absence of IL-10) that is known to promote the in vitro differentiation of LC into cells expressing a lymphostimulatory phenotype. Finally, LCH granulomas were the only site where CD154-positive T cells could be identified in close contact with LC intensely expressing CD40 Ags. Taken together, these results strongly support the idea that an abnormal immune response initiated by LC may participate in the pathogenesis of pulmonary LCH, and suggest that therapeutic strategies aimed at modifying the lymphostimulatory phenotype of LC may be useful in the treatment of this disorder.
Induction and Visualization of Mucosal Memory CD8 T Cells Following Systemic Virus Infection
Authors: Kim Sung-Kwon , Schluns Kimberly S. , Lefrançois Leo
Published: December 2022

Whether CD8 T cell memory exists outside secondary lymphoid organs is unclear. Using an adoptive transfer system that enables tracking of OVA-specific CD8 T cells, we explored the antigenic requirements for inducing CD8 T cell memory and identified intestinal mucosa memory cells. Although systemic immunization with soluble OVA induced clonal expansion, memory CD8 cells were not produced. In contrast, infection with virus-encoding OVA induced memory CD8 cells in the periphery and the lamina propria and intraepithelial compartments of the intestinal mucosa. Mucosal memory cells expressed a distinct array of adhesion molecules as compared with secondary lymphoid memory cells, suggesting that there may be separate mucosal and systemic memory pools. Mucosal CD8 memory cells rapidly produced IFN-γ after Ag stimulation. Reactivation of memory cells by Ag feeding resulted in increased cell size and up-regulation of CD28 and CD11c. CD8 mucosal memory cells exhibited ex vivo lytic activity that was up-regulated dramatically following Ag reencounter in vivo. Interestingly, reactivation of memory cells did not require CD28-mediated costimulation. The ability of the intestinal mucosa to maintain CD8 memory cells provides a potential mechanism for effective mucosal vaccination.
Nitric Oxide Suppresses Human T Lymphocyte Proliferation Through IFN-γ-Dependent and IFN-γ-Independent Induction of Apoptosis
Authors: Allione Alessandra , Bernabei Paola , Bosticardo Marita , Ariotti Silvia , Forni Guido , Novelli Francesco
Published: December 2022

Human normal and malignant T cells cease to proliferate, down-modulate Bcl-2 expression, and undergo apoptosis when cultured in the presence of NO-donor compounds (sodium nitroprusside and NOC12) for 48 h. At 72 h, cells that evade apoptosis start to proliferate again, overexpress both chains of the IFN-γR, and thus become susceptible to apoptosis in the presence of IFN-γ. By contrast, in the presence of IFN-γ, no apoptosis, but an increase of proliferation was displayed by control cultures of T cells not exposed to NO and not overexpressing IFN-γR chains. The NO-induced cell surface overexpression of IFN-γR chains did not affect the transduction of IFN-γ-mediated signals, as shown by the expression of the transcription factor IFN regulatory factor 1 (IRF-1). However, transduction of these signals was quantitatively modified, because IFN-γ induces enhanced levels of caspase-1 effector death in NO-treated cells. These findings identify NO as one of the environmental factors that critically govern the response of T cells to IFN-γ. By inducing the overexpression of IFN-γR chains, NO decides whether IFN-γ promotes cell proliferation or the induction of apoptosis.
Systemic Expression of Rat Soluble Retinal Antigen Induces Resistance to Experimental Autoimmune Uveoretinitis
Authors: McPherson Scott W. , Roberts Josh P. , Gregerson Dale S.
Published: December 2022

To assess the role of sequestration in the maintenance of the immune privilege of the retina, retrovirally mediated gene transfer was used to express a defined, specific retinal autoantigen, rat soluble retinal Ag (S-Ag), in a systemic, nonsequestered manner. In this study we report the stable, long term transduction of rat retinal S-Ag into PBMC. Tolerance to S-Ag was assayed by challenging the S-Ag chimeric animals with S-Ag peptides in CFA and monitoring the time course and severity of experimental autoimmune uveoretinitis (EAU). The resulting data showed a correlation between the incidence of S-Ag chimerism and the loss of susceptibility to EAU. The development of resistance to EAU induction supports the hypothesis that Ag sequestration contributes to retinal immune privilege.
Comparing the Relative Role of Perforin/Granzyme Versus Fas/Fas Ligand Cytotoxic Pathways in CD8+ T Cell-Mediated Insulin-Dependent Diabetes Mellitus
Authors: Kreuwel Huub T. C. , Morgan David J. , Krahl Troy , Ko Alice , Sarvetnick Nora , Sherman Linda A.
Published: December 2022

CD8+ cytotoxic T cells play a critical role in initiating insulin-dependent diabetes mellitus. The relative contribution of each of the major cytotoxic pathways, perforin/granzyme and Fas/Fas ligand (FasL), in the induction of autoimmune diabetes remains controversial. To evaluate the role of each lytic pathway in β cell lysis and induction of diabetes, we have used a transgenic mouse model in which β cells expressing the influenza virus hemagglutinin (HA) are destroyed by HA-specific CD8+ T cells from clone-4 TCR-transgenic mice. Upon adoptive transfer of CD8+ T cells from perforin-deficient clone-4 TCR mice, there was a 30-fold increase in the number of T cells required to induce diabetes. In contrast, elimination of the Fas/FasL pathway of cytotoxicity had little consequence. When both pathways of cytolysis were eliminated, mice did not become diabetic. Using a model of spontaneous diabetes, which occurs in double transgenic neonates that express both clone-4 TCR and Ins-HA transgenes, mice deficient in either the perforin or FasL/Fas lytic pathway become diabetic soon after birth. This indicates that, in the neonate, large numbers of autoreactive CD8+ T cells can lead to destruction of islet β cells by either pathway.