bluuebunny/crossref_metadata_2025_split huggingface dataset:
Importance of MEK in Neutrophil Microbicidal Responsiveness
Authors: Downey Gregory P. , Butler Jeffrey R. , Tapper Hans , Fialkow Lea , Saltiel Alan R. , Rubin Barry B. , Grinstein Sergio 
Published: January 2023

Abstract
Exposure of neutrophils to inflammatory stimuli such as the chemoattractant FMLP leads to activation of responses including cell motility, the oxidative burst, and secretion of proteolytic enzymes. A signaling cascade involving sequential activation of Raf-1, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) is also rapidly activated after agonist exposure. The temporal relationship between these events suggests that the kinases may be involved in triggering the effector functions, but direct evidence of a causal relationship is lacking. To assess the role of the MEK/ERK pathway in the activation of neutrophil responses, we studied the effects of PD098059, a potent and selective inhibitor of MEK. Preincubation of human neutrophils with 50 μM PD098059 almost completely (&gt;90%) inhibited the FMLP-induced activation of MEK-1 and MEK-2, the isoforms expressed by neutrophils. This dose of PD098059 virtually abrogated chemoattractant-induced tyrosine phosphorylation and activation of ERK-1 and ERK-2, implying that MEKs are the predominant upstream activators of these mitogen-activated protein kinases. Pretreatment of neutrophils with the MEK antagonist inhibited the oxidative burst substantially and phagocytosis only moderately. In addition, PD098059 antagonized the delay of apoptosis induced by exposure to granulocyte-macrophage CSF. However, the effects of PD098059 were selective, as it failed to inhibit other responses, including chemoattractant-induced exocytosis of primary and secondary granules, polymerization of F-actin, chemotaxis, or activation of phospholipase A2. We conclude that MEK and ERK contribute to the activation of the oxidative burst and phagocytosis, and participate in cytokine regulation of apoptosis.
CD8+ Myelin Peptide-Specific T Cells Can Chemoattract CD4+ Myelin Peptide-Specific T Cells: Importance of IFN-Inducible Protein 10
Authors: Biddison William E. , Cruikshank William W. , Center David M. , Pelfrey Clara M. , Taub Dennis D. , Turner Richard V. 
Published: January 2023

Abstract
The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is due, in part, to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. While it is thought that the inflammatory and demyelination process in MS is the product of Th1-associated cytokines secreted by CD4+ myelin protein-specific T cells present in the CNS, the mechanisms that are responsible for the recruitment and maintenance of these myelin-reactive CD4+ T cells in the CNS have not been elucidated. We have shown previously that CD8+CTL that recognize peptides derived from sequences of the myelin proteolipid protein (PLP) presented by HLA class I molecules can be generated in vitro, and that these PLP-specific CD8+CTL secrete the proinflammatory chemokines macrophage-inflammatory protein-1α and -1β, IL-16, and IP-10. In this study, we demonstrate that soluble products of these PLP-specific CD8+CTL can chemoattract CD4+ T cells that are specific for a myelin basic protein peptide and a PLP peptide, and that the majority of this chemotactic activity is mediated by IFN-inducible protein 10. These results demonstrate that PLP-specific CD8+ T cells can play a role in the recruitment and retention of myelin-derived peptide-specific CD4+ T cells, and indicate that they may play a proinflammatory role in the pathogenesis of MS.
Adhesion Molecule Mechanisms Mediating Monocyte Migration Through Synovial Fibroblast and Endothelium Barriers: Role for CD11/CD18, Very Late Antigen-4 (CD49d/CD29), Very Late Antigen-5 (CD49e/CD29), and Vascular Cell Adhesion Molecule-1 (CD106)
Authors: Shang Xiao-zhou , Lang Bianca J. , Issekutz Andrew C. 
Published: January 2023

AbstractMonocytes migrate through vascular endothelium, and then in connective tissue. As a model of this process, we investigated adhesion molecules involved in monocyte migration through HUVEC and a barrier of human synovial fibroblasts (HSF). Minimal spontaneous monocyte migration (6–7%) occurred through either cell barrier, but this increased markedly (27–35% of added monocytes) when a C5a chemotactic gradient was present. Migration across unstimulated HUVEC was partially inhibited (40%) by mAb to CD18 (β2 integrin) and completely blocked by anti-CD18 plus anti-α4 (CD49d; very late Ag-4 (VLA-4)) mAbs. In contrast, migration across HSF induced by C5a or monocyte chemoattractant protein-1 was not inhibited by mAb to CD18 and was only partially inhibited (33%) in combination with anti-α4 mAb. The CD18- and VLA-4-independent migration across HSF was completely inhibited by mAb to α5 of VLA-5. The inhibitory effect of mAbs to VLA-4 and VLA-5 was on the monocyte and required blockade of CD11/CD18 to be observed. In contrast to HSF, no role for VLA-5 in monocyte transendothelial migration was detected. Both HSF and IL-1-stimulated HUVEC expressed vascular cell adhesion molecule-1 (VCAM-1). However, VLA-4-mediated monocyte migration across HSF was only partially dependent on VCAM-1, in contrast to transendothelial migration, which was completely blocked by anti-VCAM-1 mAbs. In conclusion, unlike transendothelial migration, for which VLA-4 is the alternative mechanism to CD11/CD18 on monocytes, both VLA-4 and VLA-5 can mediate monocyte migration through fibroblast barriers. In addition to VCAM-1, other ligand(s) on HSF are also involved in the VLA-4-mediated migration.
In SCID-hu Mice, Passive Transfer of a Humanized Antibody Prevents Infection and Atrophic Change of Medulla in Human Thymic Implant due to Intravenous Inoculation of Primary HIV-1 Isolate
Authors: Okamoto Yukari , Eda Yasuyuki , Ogura Atsuo , Shibata Shinwa , Amagai Takashi , Katsura Yoshimoto , Asano Toshihiko , Kimachi Kazuhiko , Makizumi Keiichi , Honda Mitsuo 
Published: January 2023

Abstract
Using SCID-hu mice, it was tested whether humanized mAb Rμ5.5 could prevent infection by HIV-1 i.v. inoculation. The Ab that recognizes the IHIGPGRAFYT motif in the principal neutralizing determinant (PND) of HIVMN, as well as the original mouse mAb μ5.5, neutralized HIVMN with high activity. Seven primary field isolates from Japanese hemophiliacs seropositive for HIV-1 clade B were compared for their reactivities to Rμ5.5. Rμ5.5 was effective, particularly against the viruses that matched amino acid sequences of the PND region of HIV-1, and it completely neutralized primary isolates. Moreover, the passive transfer of the Ab elicited protection against challenge by the primary isolates in SCID-hu or hu-PBL-SCID mice after i.v. inoculation with the virus by both quantitative PCR and PBMC-based virus isolation in vitro. Further, inoculation with the Ab also prevented the atrophic change in the medulla of the thymic transplant that was induced by i.v. inoculation of the virus. Thus, the humanized neutralizing Ab Rμ5.5 appears to protect SCID-hu mice from infection by primary field isolates.
Differential Effects of the Rejection of Bone Marrow Allografts by the Depletion of Activating Versus Inhibiting Ly-49 Natural Killer Cell Subsets
Authors: Raziuddin Arati , Longo Dan L. , Mason Llewellyn , Ortaldo John R. , Bennett Michael , Murphy William J. 
Published: January 2023

AbstractNatural killer cells mediate the specific rejection of bone marrow cell (BMC) allografts in lethally irradiated mice. The Ly-49 family of molecules present on subsets of murine NK cells appears capable of binding class I MHC molecules, resulting in transmission of an inhibitory signal to the NK cell. These Ly-49 family members have been shown to have an immunoreceptor tyrosine-based inhibitory motif that is responsible for the inhibitory signal. However, a new Ly-49 family member was found that lacks this motif, Ly-49D, and evidence suggests that this may be an activating receptor. We therefore compared the role of the activating Ly-49 member with NK cells bearing inhibitory Ly-49 receptors in BMC rejection. Depletion of Ly-49D+ NK cells in H-2b mice abrogated their ability to reject H-2d BMC allografts. Similarly, Ly-49C+ NK cells also were shown to mediate the specific rejection of H-2d BMC. When both subsets were depleted, an additive enhancement of BMC engraftment was observed, indicating that both subsets play a role in the rejection of allogeneic H-2-homozygous H-2d BMC. However, rejection of H-2b × d or D8 (H-2b, Dd transgene) BMC allografts was unaffected by Ly-49C+ NK cell depletion in H-2b mice. In marked contrast, depletion of Ly-49D+ NK cells in H-2b mice totally abrogated the rejection of H-2b × d heterozygous BMC in support of in vitro data suggesting that Ly-49D+ NK cells receive activating signals. Therefore, NK subsets demonstrate a differential ability to reject H-2 homozygous and heterozygous BMC.
Cutting Edge: Homologous Recombination of the MHC Class I K Region Defines New MHC-Linked Diabetogenic Susceptibility Gene(s) in Nonobese Diabetic Mice
Authors: Hattori Masakazu , Yamato Eiji , Itoh Naoto , Senpuku Hidenobu , Fujisawa Tomomi , Yoshino Masayasu , Fukuda Masahiro , Matsumoto Eisaku , Toyonaga Tetsushi , Nakagawa Ichiro , Petruzzelli Maria , McMurray Armand , Weiner Howard , Sagai Tomoko , Moriwaki Kazuo , Shiroishi Toshihiko , Maron Ruth , Lund Torben 
Published: January 2023

Abstract
To localize the MHC-linked diabetogenic genes in the nonobese diabetic (NOD) mouse, a recombinational hotspot from the B10.A(R209) mouse was introduced to the region between the MHC class I K and class II A of the NOD mouse with the recombinational site centromeric to the Lmp2/Tap1 complex by breeding the two strains. Replacement of the NOD region centromeric to the recombinational site with the same region in R209 mice prevented the development of diabetes (from 71 to 3%) and insulitis (from 61 to 15%) in the N7 intra-MHC recombinant NOD mice. Similarly, the replacement of the NOD class II A, E and class I D region with the same region in R209 mice prevented the diseases (diabetes, from 71 to 0%; insulitis, from 61 to 3%). In addition to the MHC class II genes, there are at least two MHC-linked diabetogenic genes in the region centromeric to Lmp2.
Spontaneous Human Monocyte Apoptosis Utilizes a Caspase-3-Dependent Pathway That Is Blocked by Endotoxin and Is Independent of Caspase-1
Authors: Fahy Ruairi J. , Doseff Andrea I. , Wewers Mark D. 
Published: January 2023

Abstract
Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 μg/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.
Switch Recombination in a Transfected Plasmid Occurs Preferentially in a B Cell Line That Undergoes Switch Recombination of Its Chromosomal Ig Heavy Chain Genes
Authors: Stavnezer Janet , Bradley Sean P. , Rousseau Norman , Pearson Todd , Shanmugam Ananth , Waite Debra J. , Rogers Paul R. , Kenter Amy L. 
Published: January 2023

Abstract
Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e.g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29μ), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of Sμ-Sα recombination in the plasmid. Recombination of the plasmid in I.29μ cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29μ cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.
Tumor Therapy with Bispecific Antibody: The Targeting and Triggering Steps Can Be Separated Employing a CD2-Based Strategy
Authors: Wild Martin K. , Strittmatter Wolfgang , Matzku Siegfried , Schraven Burkhart , Meuer Stefan C. 
Published: January 2023

Abstract
For tumor therapy with unprimed effector cells, we developed a novel combination of a CD2 × tumor Ag bispecific targeting Ab and an anti-CD2 triggering Ab. These Ab constructs were derived from two novel CD2 mAbs, termed M1 and M2 that, together, but not individually activate T cells. Unlike many other CD2 Abs, M1 and M2 do not interfere with TCR/CD3 triggering nor do they inhibit binding of CD2 to its ligand CD58, thus preserving the physiological functions of these important effector cell molecules. M2 was chemically conjugated with an Ab recognizing the epidermal growth factor-receptor (EGF-R). Incubation of unprimed peripheral blood mononuclear cells with the bispecific F(ab′)2 construct (M2xEGF-R) in the presence of trigger Ab M1 led to efficient selective lysis of EGF-R-positive targets by CTL and NK cells. Importantly, the need for trigger Ab M1 for effector cell stimulation allowed to separate targeting from triggering steps in vitro and should thus enable to focus immune responses to sites of target Ag expression in vivo.
BCL-6-Deficient Mice Reveal an IL-4-Independent, STAT6-Dependent Pathway That Controls Susceptibility to Infection by Leishmania major
Authors: Dent Alexander L. , Doherty T. Mark , Paul William E. , Sher Alan , Staudt Louis M. 
Published: January 2023

Abstract
The BCL-6 gene negatively regulates Th2 responses as shown by the finding that BCL-6-deficient (BCL-6−/−) mice develop a lethal Th2-type inflammatory disease. The response of inbred mouse strains to infection with Leishmania major is under genetic control; BALB/c mice are susceptible and develop a progressive parasite burden, whereas most other common laboratory strains of mice are resistant to infection. We found that BCL-6−/− mice on a resistant genetic background (C57BL/6 × 129 intercrossed mice) were highly susceptible to L. major infection; they resembled BALB/c mice in terms of lesion size, parasite load, and the production of Th2 cytokines. BCL-6−/−IL-4−/− double-mutant mice were also susceptible to L. major infection and produced 10-fold higher levels of the Th2 cytokine IL-13 than IL-4−/− littermate controls. By contrast, BCL-6−/−STAT6−/− double-mutant mice were resistant to L. major infection despite also producing elevated levels of IL-13. These results show that STAT6 is required for susceptibility to L. major infection and suggest that IL-13 signaling through STAT6 may contribute to a nonhealing, exacerbated L. major infection.
Structure and Function of the Glycosaminoglycan Binding Site of Chemokine Macrophage-Inflammatory Protein-1β
Authors: Koopmann Witte , Ediriwickrema Chandrika , Krangel Michael S. 
Published: January 2023

Abstract
The ability of chemokines to bind to glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix is thought to play a crucial role in chemokine function. We investigated the structural basis for chemokine binding to GAGs by using in vitro mutagenesis to identify amino acids of chemokine macrophage-inflammatory protein-1β (MIP-1β) that contribute to its interaction with the model GAG heparin. Among six basic residues that are organized into a single basic domain in the folded MIP-1β monomer, three (R18, K45, and R46) were found to contribute significantly to heparin binding. Of these, R46 was found to play a dominant role, and proved essential for the interaction of MIP-1β with both heparin and heparan sulfate in physiological salt. The results of this mutational analysis have implications for the structure of the MIP-1β-heparin complex, and a comparison of these results with those obtained by mutational analysis of the MIP-1α-heparin interaction suggests a possible structural difference between the MIP-1β-heparin and MIP-1α-heparin complexes. To determine whether GAG binding plays an important role in receptor binding and cellular activation by MIP-1β, the activities of wild-type MIP-1β and R46-substituted MIP-1β were compared in assays of T lymphocyte chemotaxis. The two proteins proved equipotent in this assay, arguing that interaction of MIP-1β with GAGs is not intrinsically required for functional interaction of MIP-1β with its receptor.
Up-Regulation of ICAM-1 by Cytokines in Human Tracheal Smooth Muscle Cells Involves an NF-κB-Dependent Signaling Pathway That Is Only Partially Sensitive to Dexamethasone
Authors: Amrani Yassine , Lazaar Aili L. , Panettieri Reynold A. 
Published: January 2023

Abstract
Although the precise mechanisms by which steroids mediate their therapeutic effects remain unknown, steroids have been reported to abrogate cytokine-induced activation of the transcription factor NF-κB. In some cell types, NF-κB activation is necessary to regulate cytokine-mediated cellular functions. However, compelling evidence suggests that the steroid inhibition of NF-κB is complex and cell specific. Using EMSA, we show that stimulation with TNF-α or IL-1β induces NF-κB DNA-binding activity in human airway smooth muscle cells. TNF-α and IL-1β also increased luciferase activity in airway smooth muscle cells transfected with a reporter plasmid containing κB enhancer elements. Cytokines activated NF-κB by rapidly degrading its cytosolic inhibitor IκBα, which was then regenerated after 60 min. Cytokine-mediated IκBα reappearance was completely blocked by the protein synthesis inhibitor cycloheximide. Inhibition of cytokine-mediated IκBα proteolysis using the protease inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and N-acetyl-l-leucinyl-l-leucinyl-norleucinal also inhibited cytokine-mediated early expression of ICAM-1. Although dexamethasone partially inhibited IL-1β- and TNF-α-induced up-regulation of ICAM-1 at 4 h, dexamethasone had no effect on cytokine-induced ICAM-1 expression at 18–24 h. In addition, neither cytokine-induced degradation or resynthesis of IκBα nor NF-κB DNA-binding activity were affected by dexamethasone. In cells transfected with the luciferase reporter, dexamethasone did not affect TNF-α-induced NF-κB-dependent transcription. Interestingly, cytokine-mediated expression of cyclooxygenase-2 was completely abrogated by dexamethasone at 6 h. Together, these data demonstrate that cytokine-mediated NF-κB activation and ICAM-1 expression involve activation of a steroid-insensitive pathway.
Persistent Expression of Serum Amyloid A During Experimentally Induced Chronic Inflammatory Condition in Rabbit Involves Differential Activation of SAF, NF-κB, and C/EBP Transcription Factors
Authors: Ray Alpana , Ray Bimal K. 
Published: January 2023

AbstractThe serum amyloid A (SAA) protein has been implicated in the pathogenesis of several chronic inflammatory diseases. Its induction mechanism in response to a chronic inflammatory condition was investigated in rabbits following multiple s.c. injections of AgNO3 over a period of 35 days. During unremitting exposure to inflammatory stimulus, a persistently higher than normal level of SAA2 expression was seen in multiple tissues. Induction of SAA was correlated with higher levels of several transcription factor activities. Increased SAA-activating factor (SAF) activity was detected in the liver, lung, and brain tissues under both acute and chronic inflammatory conditions. In the heart, kidney, and skeletal muscle tissues, this activity remained virtually constant. In contrast, CCAAT enhancer binding protein (C/EBP) DNA-binding activity was transiently induced in selective tissues. Higher than normal NF-κB DNA-binding activity was detected in the lung and to a lesser extent in the liver and kidney tissues under both acute and chronic conditions. This result suggested that C/EBP, SAF, and NF-κB are required for transient acute phase induction of SAA whereas SAF and NF-κB activities are necessary for persistent SAA expression during chronic inflammatory conditions.
CD40 Ligand-Activated Human Monocytes Amplify Glomerular Inflammatory Responses Through Soluble and Cell-to-Cell Contact-Dependent Mechanisims
Authors: Kuroiwa Takashi , Lee Eric G. , Danning Carol L. , Illei Gabor G. , McInnes Iain B. , Boumpas Dimitrios T. 
Published: January 2023

Abstract
Monocytes/macrophages play a critical role in the initiation and progression of a variety of glomerulonephritides. We sought to define the interactions between physiologically activated human monocytes and glomerular mesangial cells (MC) by employing a cell culture system that permits the accurate assessment of the contribution of soluble factors and cell-to-cell contact. Human peripheral blood monocytes, primed with IFN-γ and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-α and cocultured with MC. CD40L-activated monocytes induced higher levels of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 synthesis by MC. Separation of CD40L-activated monocytes from MC by a porous membrane decreased the mesangial synthesis of IL-6 by 80% and ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against the β2 integrins, LFA-1 and Mac-1, decreased IL-6 production by 40 and 50%, respectively. Ligation of mesangial surface ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous neutralization of soluble TNF-α and IL-1β decreased MCP-1 production by 55% in membrane-separated cocultures of MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated monocytes (to preserve membrane integrity but prevent secretory activity), cocultured with MC at various ratios, induced IL-6, MCP-1, and ICAM-1 synthesis by MC. Plasma membrane preparations from activated monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition of plasma membrane enhanced TNF-α-induced mesangial IL-6 production by ∼4-fold. Together, these data suggest that the CD40/CD40L is essential for optimal effector function of monocytes, that CD40L-activated monocytes stimulate MC through both soluble factors and cell-to-cell contact mediated pathways, and that both pathways are essential for maximum stimulation of MC.