bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage-Derived Nitric Oxide Regulates T Cell Activation via Reversible Disruption of the Jak3/STAT5 Signaling Pathway
Authors: Bingisser Roland M. , Tilbrook Peta A. , Holt Patrick G. , Kees Ursula R.
Published: January 2023

Nitric oxide (NO) has been invoked as an important pathogenic factor in a wide range of immunologically mediated diseases. The present study demonstrates that macrophage-derived NO may conversely function to fine tune T cell-mediated inflammation via reversible dephosphorylation of intracellular signaling molecules, which are involved in the control of T cell proliferation. Thus, T cells activated in the presence of alveolar macrophages are unable to proliferate despite expression of IL-2R and secretion of IL-2. This process is reproduced by the NO generator S-nitroso-N-acetylpenicillamine and is inhibitable by the NO synthase inhibitor NG-methyl-l-arginine. Analysis of T cell lysates by immunoprecipitation with specific Abs and subsequent immunoblotting indicated marked reduction of tyrosine phosphorylation of Jak3 and STAT5 mediated by NO. Further studies indicated that NO-mediated T cell suppression was reversible by the guanylate cyclase inhibitors methylene blue and LY-83583 and was reproduced by a cell-permeable analogue of cyclic GMP, implicating guanylate cyclase activation as a key step in the inhibition of T cell activation by NO.
The Trophic Action of IL-7 on Pro-T Cells: Inhibition of Apoptosis of Pro-T1, -T2, and -T3 Cells Correlates with Bcl-2 and Bax Levels and Is Independent of Fas and p53 Pathways
Authors: Kim Kyungjae , Lee Chong-kil , Sayers Thomas J. , Muegge Kathrin , Durum Scott K.
Published: January 2023

Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53−/− mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-XL, Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.
Immunologic Characterization of CD7-Deficient Mice
Authors: Lee David M. , Staats Herman F. , Sundy John S. , Patel Dhavalkumar D. , Sempowski Gregory D. , Scearce Richard M. , Jones Dawn M. , Haynes Barton F.
Published: January 2023

Human CD7 is an Ig superfamily molecule that is expressed on mature T and NK lymphocytes. Although in vitro studies have suggested a role for CD7 in lymphoid development and function, the exact function of CD7 in vivo has remained elusive. One patient has been reported with SCID syndrome attributed to CD7 deficiency. To study in vivo functions of CD7, we have generated CD7-deficient mice and assessed their lymphoid development and function. CD7-deficient mice were viable, had normal peripheral blood and spleen lymphocyte numbers, and had normal specific Ab responses with Ag-driven Ig isotype switching. Thymocyte numbers were normal in 4-wk-old, 6-mo-old, and 1-yr-old CD7-deficient mice, but in 3-mo-old CD7-deficient mice, total thymocyte numbers were significantly increased by 60% (p < 0.02) compared with normal age-matched +/+ littermates. CD7-deficient splenocytes proliferated normally in response to various mitogens, including PHA, anti-CD3, Con A, and LPS. While NK cell numbers and cytolytic activity to YAC targets were normal, CD7-deficient mice had lower Ag-induced MHC class I-restricted CTL activity against OVA-transfected target cells than did +/+ control mice. Thus, CD7-deficient mice did not have a SCID syndrome, but rather had transient increases in thymocyte numbers at age 3 mo and altered splenocyte Ag-specific CTL effecter cell activity. These data suggest a role for CD7 in regulating intrathymic T cell development and in mediating CTL effecter function.
IL-16 Activates the SAPK Signaling Pathway in CD4+ Macrophages
Authors: Krautwald Stefan
Published: January 2023

IL-16 has been reported as a modulator of T cell activation and was shown to function as chemoattractant factor. The chemotactic activity of IL-16 depends on the expression of CD4 on the surface of target cells, but the intracellular signaling pathways are only now being deciphered. This report describes IL-16 as an additional activator of the stress-activated protein kinase (SAPK) pathway in CD4+ macrophages. Treatment of these cells with recombinant expressed IL-16 leads to the phosphorylation of SEK-1, resulting in activation of the SAPKs p46 and p54. IL-16 stimulation also leads to the phosphorylation of c-Jun and p38 MAPK (mitogen-activated protein kinase), without inducing MAPK-family members ERK-1 and ERK-2. Interestingly, the IL-16-mediated activation of SAPKs and p38 MAPK in macrophages alone induces no detectable apoptotic cell death. These observations suggest specific regulatory functions of IL-16 distinct from the proinflammatory cytokines TNF-α and IL-1β.
HLA-G Isoforms Produced by Placental Cytotrophoblasts and Found in Amniotic Fluid Are Due to Unusual Glycosylation
Authors: McMaster Michael , Zhou Yan , Shorter Simon , Kapasi Kubra , Geraghty Dan , Lim Kee-Hak , Fisher Susan
Published: January 2023

The human placenta expresses HLA-G, a nonclassical (class Ib) MHC molecule that could play a central role in maternal tolerance of the semiallogeneic fetus. In this work, we report the production of a new mAb, 4H84, that specifically reacts with HLA-G in two formats: immunocytochemistry and immunoblotting. Immunolocalization experiments with 4H84 confirmed our previous finding that cytotrophoblasts within the uterine wall are the only cells in tissue sections of placenta that express the HLA-G protein. Additional experiments showed that both amniocytes and cytotrophoblasts in the amnion-chorion express this protein. Since multiple HLA-G transcripts have been described, we used immunoblotting to study the HLA-G isoforms produced by cytotrophoblasts in vitro and by the amnion-chorion in vivo. Cytotrophoblasts, their conditioned medium, and amniotic fluid samples contained heterodisperse immunoreactive bands (Mr 35,000–50,000). N-deglycosylation by peptide-N-glycosidase F digestion resolved these isoforms into two distinct bands. Cell samples contained primarily an Mr 37,000–42,000 protein, most likely encoded by the full-length mRNA. Conditioned medium and amniotic fluid contained a slightly smaller protein, most likely the secreted form lacking the transmembrane and cytoplasmic regions. Removal of polylactosamine chains by endo-β d-galactosidase digestion significantly reduced the electrophoretic mobility of the immunoreactive bands, suggesting that HLA-G, unlike class Ib molecules studied to date, carries N-acetyllactosamine units. These data show that Mr heterogeneity of HLA-G is due to its novel glycosylation, rather than to the translation of alternatively spliced mRNAs. We postulate that the unusual carbohydrate structures this molecule carries could interact with maternal immune cells and/or stabilize the molecule.
Failure to Activate Cytosolic Phospholipase A2 Causes TNF Resistance in Human Leukemic Cells
Authors: Wu Yu-Ling , Jiang Xu-Rong , Newland Adrian C. , Kelsey Stephen M.
Published: January 2023

Activation of cytosolic phospholipase A2 (cPLA2) by TNF has been shown to be an important component of the signaling pathway leading to cell death. The role of cPLA2 in the cytotoxic action of TNF was investigated in a panel of human leukemic cell lines. TNF could activate cPLA2 only in U937 and HL60 TNF-sensitive leukemic cells, but not in KG1a, CEM, and CEM/VLB100 cells that are relatively resistant to TNF. Pretreatment with 4-bromophenacyl bromide, a cPLA2 inhibitor, rendered U937 and HL60 cell lines resistant to the cytotoxic effect of TNF. Immunoblot and reverse-transcriptase PCR demonstrated that cPLA2 expression was detectable at both transcriptional and translational levels in all leukemic cell lines studied, although CEM and CEM/VLB100 cells expressed cPLA2 mRNA and protein at lower levels. The protein synthesis inhibitor, cycloheximide, increased TNF-induced cPLA2 activity and cytotoxicity in both CEM and CEM/VLB100 cell lines. Low levels of cPLA2 activity in the KG1a cell line could be activated by the cPLA2 activator mellitin, or the calcium ionophore A23187. The data suggest that cPLA2 activity is involved in TNF-induced cytotoxicity in leukemic cells. Resistance to TNF-induced cytotoxicity may involve either protein inhibitors that act upstream of cPLA2 in the TNF-signaling pathway or constitutive defects of cPLA2 itself, possibly involving calcium utilization.
Mechanisms of Macrophage Stimulation Through CD8: Macrophage CD8α and CD8β Induce Nitric Oxide Production and Associated Killing of the ParasiteLeishmania major
Authors: Hirji Nadir , Lin Tong-Jun , Bissonnette Elyse , Belosevic Miodrag , Befus A. Dean
Published: January 2023

Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8α (OX8, 5 μg/ml) or CD8β (341, 10 μg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8α and CD8β stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 μg/ml), PP1 (src family kinase inhibitor, 5 μg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 μg/ml), and Ro 31-8220 (PKC inhibitor, 1 μM) significantly inhibited anti-CD8α- and anti-CD8β-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) (src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8α stimulated NO-dependent macrophage killing of the parasite Leishmania major. For the first time, this work demonstrates that the β-chain of macrophage CD8, in addition to the α-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.
The Role of T Cells in Allografted Tumor Rejection: IFN-γ Released from T Cells Is Essential for Induction of Effector Macrophages in the Rejection Site
Authors: Yoneda Yukio , Yoshida Ryotaro
Published: January 2023

Allografted Meth A tumor rejection is T cell dependent, but T cells are inactive toward the allograft; rather, the main effector cells are allograft-induced macrophages (AIM) with MHC haplotype specificity. Here, we examined the role of T cells in the induction of AIM in the rejection site. On day 4.5 after i.p. transplantation of Meth A fibrosarcoma cells to C57BL/6 (B6) mice, we obtained a kind of precursor of AIM (pro-AIM) from the transplantation site by an enrichment technique involving adherence to serum-coated dishes. The noncytotoxic pro-AIM-rich population put into a diffusion chamber became cytotoxic against Meth A cells after 2 days in the peritoneal cavity of an untreated B6 mouse. Similar activation of the chambered B6 pro-AIM-rich population occurred in IFN-γ −/− B6 mice, whereas there was no activation when chambers containing an IFN-γ −/− mouse-derived pro-AIM-rich population were placed in normal or IFN-γ −/− mice, suggesting that IFN-γ is involved in the activation. RT-PCR experiments demonstrated that among bulk infiltrates T cells were the major producer of IFN-γ; and most of the cells in a T cell-eliminated pro-AIM population in a diffusion chamber kept for 2 days in a B6 mouse did not become AIM. Furthermore, IFN-γ −/− B6 mice could not reject allografted Meth A tumor cells, whereas the grafts were rejected by i.p. injections of IFN-γ into the mutant mice. These results indicate that IFN-γ released from allograft-induced T cells is essential for both the activation of a kind of pro-AIM to AIM in the transplantation site and the rejection of an allografted tumor.
IL-6 Produced by Kupffer Cells Induces STAT Protein Activation in Hepatocytes Early During the Course of Systemic Listerial Infections
Authors: Gregory Stephen H. , Wing Edward J. , Danowski Kristine L. , van Rooijen Nico , Dyer Kevin F. , Tweardy David J.
Published: January 2023

Kupffer cells were the principal source of IL-6 produced in the livers of mice following i.v. inoculation of Listeria monocytogenes. IL-6 mRNA expression and the production of IL-6 were reduced drastically within the nonparenchymal liver cell population derived from mice rendered Kupffer cell depleted by pretreatment with liposome-encapsulated dichloromethylene diphosphonate. A sharp increase in the appearance of activated STAT3 occurred in extracts of purified hepatocytes derived from normal mice infected i.v. with Listeria. Remarkably, the kinetics of this increase overlapped IL-6 mRNA expression by Kupffer cells; each peaked at approximately 30 min postinfection. No increase in STAT3 activation was observed in IL-6-deficient or Kupffer cell-depleted animals. The results of these experiments indicate that the synthesis of IL-6 and the activation of STAT3 within hepatocytes are critical functions of Kupffer cells occurring very early during the course of systemic listerial infections.
Characterization of the Peptide Binding Motif of a Rhesus MHC Class I Molecule (Mamu-A*01) That Binds an Immunodominant CTL Epitope from Simian Immunodeficiency Virus
Authors: Allen Todd M. , Sidney John , del Guercio Marie-France , Glickman Rhona L. , Lensmeyer Gary L. , Wiebe Donald A. , DeMars R. , Pauza C. David , Johnson R. Paul , Sette Alessandro , Watkins David I.
Published: January 2023

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM181–189. Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.
Altered Intestinal Immune System but Normal Antibacterial Resistance in the Absence of P-Selectin and ICAM-1
Authors: Steinhoff Ulrich , Klemm Uwe , Greiner Manuela , Bordasch Karin , Kaufmann Stefan H. E.
Published: January 2023

ICAM-1 and P-selectin are adhesion molecules that regulate leukocyte migration, extravasation to inflammatory sites, and other immune cell interactions. T cell-mediated resistance against acute infection with Listeria monocytogenes and chronic infection with Mycobacterium bovis Calmette-Guérin bacillus was investigated in mutant mice lacking P-selectin and/or ICAM-1. Mice deficient in P-selectin (Psel−/−), ICAM-1 (ICAM−/−), or the combination of both (Psel−/− × ICAM−/−) showed normal bacterial clearance, comparable delayed-type hypersensitivity reactions, and equivalent memory T cell responses. Additionally, the distribution of αβ vs γδ T lymphocyte populations was examined. Normal lymphocyte distributions were noted in thymus, spleen, and blood, whereas mutant mice showed marked alterations in the intestinal intraepithelial (i-IEL) and lamina propria lymphocytes. Differences in i-IEL populations were reflected functionally by differential lytic activities and cytokine productions of i-IEL populations from mutant mice. Despite these changes within the mucosal immune system of mutant mice, their resistance against oral infection with L. monocytogenes was apparently unimpaired. These findings demonstrate that P-selectin and ICAM-1 are critically involved in the shaping of lymphocyte populations of the gut but have only a minor influence on systemic and regional host defense against intracellular bacteria.
IL-4-Dependent Regulation of TGF-α and TGF-β1 Expression in Human Eosinophils
Authors: Elovic Aram E. , Ohyama Hiroe , Sauty Alan , McBride Jim , Tsuji Takanori , Nagai Masazumi , Weller Peter F. , Wong David T. W.
Published: January 2023

TGFs play important roles in wound healing and carcinogenesis. We have previously demonstrated that eosinophils infiltrating into different pathologic processes elaborate TGF-α and TGF-β1. Eosinophils infiltrating hamster cutaneous wounds were found to express TGFs sequentially. In this study, we examined the biologic mediators that may regulate the expression of TGF-α and -β1 by eosinophils. Eosinophils were isolated from the peripheral blood of healthy donors and cultured in the absence or presence of IL-3, IL-4, and IL-5. Cells were analyzed by in situ hybridization and immunohistochemistry. Supernatants from these cultures were assayed for secreted TGF-α and TGF-β1 using TGF-specific ELISAs. IL-3, IL-4, and IL-5 independently up-regulated TGF-β1 mRNA and product expression by eosinophils in all donors. Interestingly, TGF-α production by eosinophils was up-regulated by IL-3 and IL-5 but was down-regulated by IL-4. Consistent with the ability of IL-4 to regulate eosinophil responses, IL-4 signaling molecules are present in human eosinophils. The observation that IL-4 can differentially regulate the expression of TGF-α and TGF-β1 suggests that IL-4 may serve as a physiologic molecular switch of TGF expression by the infiltrating eosinophils in wound healing and carcinogenesis.
Complement Activation Occurs on Subendothelial Extracellular Matrix In Vitro and Is Initiated by Retraction or Removal of Overlying Endothelial Cells
Authors: Hindmarsh Elizabeth J. , Marks Rory M.
Published: January 2023

Vascular endothelium is continuously exposed to plasma complement, which could generate a potent proinflammatory signal if activated on the vascular wall. Normal endothelium, however, expresses an anti-inflammatory phenotype, which includes resistance to complement fixation. As activated endothelium converts to a proinflammatory phenotype, we investigated the effect of cytokines on endothelial susceptibility to complement fixation. Cytokine-treated HUVEC were exposed to human serum as a source of complement, and C3 deposition was quantified. IL-1β and TNF-α in combination with IFN-γ markedly increased endothelial C3 deposition; however, immunofluorescence microscopy revealed that the endothelial cells had retracted, and that bound C3 was concentrated not on cells but in areas of exposed subendothelial extracellular matrix (ECM). Studies with cell-free ECM indicated that complement activation required only ECM exposure and was independent of cellular activation. C3 deposition on ECM was reproduced by reconstituting the alternative pathway, which generated a stable C3 convertase on ECM, but not on endothelial cells. C3b and iC3b were identified on ECM exposed to purified alternative pathway components and serum, respectively. In conditions associated with endothelial disruption, exposure of subendothelial ECM could induce complement fixation and contribute to inflammation and vascular damage.
Complement Receptor 3 of Macrophages Is Associated with Galectin-1-Like Protein
Authors: Avni Orly , Pur Zivit , Yefenof Eitan , Baniyash Michal
Published: January 2023

We have previously identified a 16-kDa protein with a pI of 5.1 (P16/5.1) that is associated with macrophage CR3. Microsequencing of P16/5.1 indicated exclusive homology to the β-galactoside-binding lectin, galectin-1. Abs specific to a galectin-1 unique peptide reacted with P16/5.1. The association of P16/5.1 with CR3 was specifically inhibited by lactose, which binds with high affinity to galectin-1. These data together with similarities in molecular mass and pI suggest that P16/5.1 is galectin-1. Two-color immunofluorescence staining revealed the expression of galectin-1 on the macrophage surface and its colocalization with CR3. However, a surplus of CR3 was free of galectin-1, and some galectin-1 molecules were associated with cell surface receptors other than CR3. Based on these results we propose two models depicting the functional significance of CR3-galectin-1 association: 1) homodimeric galectin-1 possessing a divalent sugar binding site may act as an extracellular adapter molecule that cross-links CR3 with other receptors; and 2) association of galectin-1 with β-galactosides on the extracellular domain of CR3 may modify the binding affinity of the receptor to its ligand. These possibilities are not mutually exclusive and can clarify the mode by which CR3 transmits signals in macrophages.
Novel Role of Transmembrane SCF for Mast Cell Activation and Eotaxin Production in Mast Cell-Fibroblast Interactions
Authors: Hogaboam Cory , Kunkel Steven L. , Strieter Robert M. , Taub Dennis D. , Lincoln Pam , Standiford Theodore J. , Lukacs Nicholas W.
Published: January 2023

Mast cell activation can be induced by multiple mechanisms, including IgE-, complement-, and stem cell factor (SCF)-mediated pathways. In addition, the interaction of mast cells with particular cell populations, such as fibroblasts, have also demonstrated increased mast cell reactivity. In these studies, we have investigated the role of fibroblast-mast cell interaction for induction of histamine release and chemokine production and the specific role of SCF during this interaction. Primary pulmonary fibroblast cell lines were grown in culture and used throughout these studies. Mast cells were grown in parallel with fibroblasts by incubation of bone marrow cells with SCF and IL-3. During mast cell-fibroblast coculture, increased histamine release could be attenuated either by separation of the cell populations using a Trans-Well setup, which did not allow cellular contact, or by specific anti-SCF Ab. In addition, a significant increase in eotaxin, a potent eosinophil-specific C-C chemokine, was also observed during fibroblast-mast cell interaction. The production of eotaxin was cell contact dependent and could be inhibited using an anti-SCF Ab or specific antisense therapy. SCF was constitutively produced from fibroblasts in its transmembrane form and could be induced by TNF. SCF-coated plates induced significant mast cell-derived eotaxin production, whereas soluble SCF induced little or no eotaxin, suggesting a necessity for receptor cross-linking for activation. These studies indicate that fibroblast-mast cell contact plays a role in exacerbation of histamine release and eotaxin production.
Low Grade Rhinovirus Infection Induces a Prolonged Release of IL-8 in Pulmonary Epithelium
Authors: Johnston Sebastian L. , Papi Alberto , Bates Philip J. , Mastronarde John G. , Monick Martha M. , Hunninghake Gary W.
Published: January 2023

Rhinoviruses are important respiratory pathogens implicated in asthma exacerbations. The mechanisms by which rhinoviruses trigger inflammatory responses in the lower airway are poorly understood, in particular their ability to infect the lower airway. Bronchial inflammatory cell (lymphocyte and eosinophil) recruitment has been demonstrated. IL-8 is a potent proinflammatory chemokine that is chemotactic for neutrophils, lymphocytes, eosinophils, and monocytes and may be important in the pathogenesis of virus-induced asthma. Increased levels of IL-8 have been found in nasal samples in natural and experimental rhinovirus infections. In these studies we therefore examine the ability of rhinovirus to infect a transformed lower airway epithelial cell line (A549) and to induce IL-8 protein release and mRNA induction. We observed that rhinovirus type 9 is able to undergo full viral replication in A549 cells, and peak viral titers were found 24 h after inoculation. Rhinovirus infection induced a dose- and time-dependent IL-8 release up to 5 days after infection and an increase in IL-8 mRNA expression that was maximal between 3 and 24 h after infection. UV inactivation of the virus completely inhibited replication, but only reduced IL-8 protein production and mRNA induction by half, while prevention of virus-receptor binding completely inhibited virus-induced IL-8 release, suggesting that part of the observed effects was due to viral replication and part was due to virus-receptor binding. These studies demonstrate that rhinoviruses are capable of infecting a pulmonary epithelial cell line and inducing IL-8 release. These findings may be important in understanding the pathogenesis of rhinovirus-induced asthma exacerbations.
Role of TNFR1 and TNFR2 in TNF-Induced Platelet Consumption in Mice
Authors: Tacchini-Cottier Fabienne , Vesin Christian , Redard Mireille , Buurman Wim , Piguet Pierre F.
Published: January 2023

An injection of TNF in mice induced profound thrombocytopenia, due to an increase of platelet consumption, that was evident after 1 h and lasted for 3 days. This process was evident in mice that were genetically deficient in TNFR2 (p75) but not in mice lacking TNFR1 (p55), indicating that the process is mediated by TNFR1-bearing cells. To explore the site of action of TNF, labeled platelets from TNFR1 −/− or +/+ donors were transferred to TNFR1 −/− or +/+ recipients. TNF induced the consumption of platelets from TNFR1 −/− donors when injected into +/+ recipients, while platelets from +/+ donors were not consumed when present in TNFR1 −/− recipients; this finding indicates that TNF acts on the TNFR1 of host cells but does not act on platelets. The expression of TNFRs is consistent with this interpretation, since TNFRs were not detected on platelets by flow cytometry. In megakaryocytes, the expression of TNFR1 was detected by immunohistochemistry. These results indicate that TNF induces platelet consumption by acting not on platelets directly but on the TNFR1 of other cells, presumably increasing the release of factors with agonist activity for platelets.
Neutrophils Are Major Contributors to Intraparenchymal Lung IL-1β Expression After Hemorrhage and Endotoxemia
Authors: Parsey Merdad V. , Tuder Rubin M. , Abraham Edward
Published: January 2023

Acute lung injury and the acute respiratory distress syndrome (ARDS) are significant causes of morbidity and mortality following sepsis and hemorrhage. Increased IL-1β production in the lung is important in the development of acute inflammatory lung injury. Although neutrophils are an important component of the inflammatory response that characterizes acute lung injury, there is little information to suggest that they are capable of initiating cytokine-mediated immune responses in the lung. To explore the role of neutrophils in the early stages of acute lung injury, we examined IL-1β production by mouse lung neutrophils after hemorrhage and endotoxemia. There was a significant increase in IL-1β expression among intraparenchymal pulmonary neutrophil/mononuclear cells (IPNMC) 1 h after hemorrhage or endotoxemia. IL-1β was detected only in a neutrophil-rich fraction of the IPNMC, but not in T and B lymphocytes positively selected from the IPNMC. Cyclophosphamide (CTX)-treated neutropenic mice expressed significantly less IL-1β in IPNMC after hemorrhage or endotoxemia compared with CTX-untreated controls. Immunohistochemical analysis of lung sections from mice after hemorrhage or endotoxemia revealed IL-1β expression in infiltrating neutrophils. These data indicate that IL-1β-producing neutrophils traffic to the lungs rapidly in response to hemorrhage or endotoxemia and support the concept that proinflammatory cytokine production by lung neutrophils may contribute to the development of lung injury after blood loss and sepsis.
Requirements for αd in IgG Immune Complex-Induced Rat Lung Injury
Authors: Shanley Thomas P. , Warner Roscoe L. , Crouch Larry D. , Dietsch Gregory N. , Clark Darcey L. , O’Brien Maggie M. , Gallatin W. Michael , Ward Peter A.
Published: January 2023

αd is a newly cloned adhesion molecule that forms a heterodimer with CD18. The requirement for αd in IgG immune complex-induced lung injury in rats has been evaluated by the use of blocking polyclonal and monoclonal antibodies to rat αd. Using whole lung extracts, Northern and Western blot analyses have revealed up-regulation of mRNA and αd protein in inflamed lungs. Immunostaining has revealed the presence of αd in lung tissue and in alveolar macrophages as early as 1 h after initiation of the inflammatory reaction. When polyclonal rabbit Ab to rat αd was coinstilled into lung together with Ab to BSA, lung injury (as determined by leakage of [125I]albumin into lung parenchyma) was significantly diminished. In parallel, there was reduced accumulation of neutrophils recoverable in bronchoalveolar lavage (BAL) fluids. These findings were associated with reduced levels of TNF-α as well as NO2−/NO3− in BAL fluids. A hamster mAb to rat αd was also protective in this lung injury model. Anti-αd inhibited in vitro production of NO2−/NO3− by rat alveolar macrophages (stimulated with LPS and IFN-γ) by approximately 60%. These data suggest that, in the lung inflammatory model employed, αd up-regulation occurs in lung macrophages and is necessary for expression of TNF-α, recruitment of neutrophils, and full development of lung injury.
Development of Murine Allergic Asthma Is Dependent Upon B7-2 Costimulation
Authors: Keane-Myers Andrea M. , Gause William C. , Finkelman Fred D. , Xhou X.-d. , Wills-Karp Marsha
Published: January 2023

Allergic asthma is thought to be mediated by CD4+ T lymphocytes producing the Th2-associated cytokines, IL-4, and IL-5. Recently, the costimulatory molecules B7-1 and B7-2, which are expressed on the surface of APC, have been suggested to influence the development of Th1 vs Th2 immune responses. We examined the in vivo role of these costimulatory molecules in the pathogenesis of Th2-mediated allergen-induced airway hyperresponsiveness in a murine model of asthma. In this model, OVA-sensitized A/J mice develop significant increases in airway responsiveness, pulmonary eosinophilia, and pulmonary Th2 cytokine expression following aspiration challenge with OVA as compared with PBS-control animals. Strikingly, administration of anti-B7-2 mAb to OVA-treated mice abolished allergen-induced airway hyperresponsiveness, pulmonary eosinophilia, and elevations in serum IgG1 and IgE levels. Anti-B7-2 treatment of OVA-treated mice reduced both total lung IL-4 and IL-5 mRNA and bronchoalveolar lavage fluid IL-4 and IL-5 protein levels, with no significant changes in IFN-γ message or protein levels. In contrast, treatment with anti-B7-1 mAbs had no effect on allergen-induced airway hyperresponsiveness, IgE production, or cytokine production, however, it significantly suppressed pulmonary eosinophilia. We conclude that B7-2 provides the necessary costimulatory signal required for the development of in vivo allergic responses to inhaled allergen exposure.
Cutting Edge: Cytokine-Dependent Abortion in CBA × DBA/2 Mice Is Mediated by the Procoagulant fgl2 Prothombinase
Authors: Clark David A. , Chaouat Gerard , Arck Petra C. , Mittruecker Hans Willi , Levy Gary A.
Published: January 2023

Spontaneous resorption in the CBA × DBA/2 model is attributed to NK cells, macrophages, and Th1-type cytokines. In vivo depletion of NK cells by anti-asialoGM1 Ab or macrophage depletion by silicon dioxide treatment reduced abortion rates, which could no longer be boosted by injecting TNF-α (which activates NK cells) or IFN-γ (which activates macrophages). TNF-α + γ-IFN coadministration aborted >80% of the embryos whether or not NK cells or macrophages had been depleted or estradiol + progesterone was injected to correct potential reduction in ovarian function by cytokines. The cytokines also aborted IRF1+/+ C57BL/6 but not IRF1−/− females pregnant by IRF1+/+ DBA/2. Both spontaneous and cytokine-boosted abortions in CBA × DBA/2 were blocked by Ab to fgl2 prothombinase expressed by cytokine-stimulated vascular endothelial cells and monocytes; in vivo Ab depletion of granulocytes also prevented TNF-α + IFN-γ-induced abortions. Cytokine-triggered thrombotic/inflammatory processes in maternal uteroplacental blood vessels causes abortion.
CD40 Ligand Exerts Differential Effects on the Expression of Iγ Transcripts in Subclones of an IgM+ Human B Cell Lymphoma Line
Authors: Ford Gregory S. , Yin Chun Hui , Barnhart Bryan , Sztam Kevin , Covey Lori R.
Published: January 2023

The CD40:CD40 ligand (CD40L) interaction plays a critical role in T cell-dependent isotype switching. To elucidate the role of CD40 signaling in the activation of γ germline transcription and as an extension, in targeting Cγ regions for isotype switching, an IgM+ Burkitt lymphoma cell line (Ramos 2G6) was assayed for the up-regulation of germline γ transcripts after CD40L stimulation. Independent Ramos 2G6 subclones that either expressed (Iγ+) or did not express (Iγ−) basal levels of Iγ transcripts were assessed for their transcriptional response to CD40L signaling by contact with either a Jurkat T cell line (D1.1) or a transfected CD40L-expressing epithelial cell line (293/CD40L) in the presence or absence of IL-4. Both Iγ− and Iγ+ Ramos 2G6 subclones cultured with IL-4 and CD40L markedly up-regulated germline transcription predominantly from the γ1, γ2, and γ3 subclasses over levels obtained with IL-4 alone. In addition, these two signals were required to obtain de novo switch recombination. However, incubation with CD40L alone resulted in a substantial increase in germline transcription only in the Iγ+ and not the Iγ− subclones. Observed basal transcription at the γ1 locus also correlated with the ability of not only the γ1 locus, but also the γ2 and γ3 loci, to up-regulate germline transcripts in response to CD40 signaling. These data are consistent with CD40:CD40L contact up-regulating germline transcription only after the B cell has received a signal that alters the transcriptional state of the heavy chain locus.
Natural Killer Cell Development and Function Precede αβ T Cell Differentiation in Mouse Fetal Thymic Ontogeny
Authors: Carlyle James R. , Michie Alison M. , Cho Sarah K. , Zúñiga-Pflücker Juan Carlos
Published: January 2023

Natural killer (NK) cells mediate MHC-unrestricted cytolysis of virus-infected cells and tumor cells. In the adult mouse, NK cells are bone marrow-derived lymphocytes that mature predominantly in extrathymic locations but have also been suggested to share a common intrathymic progenitor with T lymphocytes. However, mature NK cells are thought to be absent in mouse fetal ontogeny. We report the existence of thymocytes with a mature NK cell phenotype (NK1.1+/CD117−) as early as day 13 of gestation, approximately 3 days before the appearance of CD4+/CD8+ cells in T lymphocyte development. These mature fetal thymic NK cells express genes associated with NK cell effector function and, when freshly isolated, display MHC-unrestricted cytolytic activity in vitro. Moreover, the capacity of fetal thymic NK cells for sustained growth both in vitro and in vivo, in addition to their close phenotypic resemblance to early precursor thymocytes, confounds previous assessments of NK lineage precursor function. Thus, mature NK cells may have been inadvertently included in previous attempts to identify multipotent and bipotent precursor thymocytes. These results provide the first evidence of functional NK lymphocytes in mouse fetal ontogeny and demonstrate that NK cell maturation precedes αβ T cell development in the fetal thymus.
Presence of Activated Antigen-Binding B Cells During Immunization Enhances Relative Levels of IFN-γ in T Cell Responses
Authors: Pasare Chandrashekhar , Morafo Vivian , Entringer Maureen , Bansal Pratima , George Anna , Bal Vineeta , Rath Satyajit , Durdik Jeannine M.
Published: January 2023

To examine the influence of Ag presentation by B cells on immune responses, we have used mice transgenic for an Ig heavy chain from a monoclonal anti-azobenzenearsonate (Ars) Ab to deliver Ag to B cells during immunization. A large proportion of transgene-expressing B cells in these mice binds Ars, while transgenic serum Ig shows poor Ars binding. Transgenic B cells present Ars proteins better than their nonhaptenated counterparts. This is associated with an increase in the proliferative responses of transgenic T cells to Ars protein immunization. Although B cell numbers in the transgenic mice are lower, many B cells in them show an activated phenotype, as identified by altered surface levels of peanut agglutinin reactivity, CD23, CD24, CD44, CD62L, and CD86. Even against nonhaptenated immunogens, transgenic responses show significant enhancement in the relative proportions of the Th1 cytokine IFN-γ over the Th2 cytokines IL-4 and IL-10. Haptenated immunogens further enhance the predilection of transgenic mice to produce relatively more IFN-γ. Consistent with this, there is an increase in IgG2a/IgG1 ratios in serum Abs in response to haptenated immunogens in transgenic mice. Adoptive transfer of primed hapten-specific secondary B cells into nontransgenic mice also induces an increase in relative levels of IFN-γ in response to haptenated immunogens. Thus, presentation of immunogen in vivo by activated Ag-binding B cells contributes to enhanced immunogenicity and a Th1 cytokine bias.
Role of Proteasomes in T Cell Activation and Proliferation
Authors: Wang Xin , Luo Hongyu , Chen Huifang , Duguid William , Wu Jiangping
Published: January 2023

The role of proteasomes in T cell activation, proliferation, and apoptosis was investigated using a proteasome-specific inhibitor lactacystin (LAC). Inhibition of the proteasome activity by LAC repressed the mitogen-induced T cell proliferation. The proteasome activity was definitively required for the T cells to progress from the G0 to S phase. It was necessary to optimize the progress from the G1/S boundary to the G2/M phase, but not for the progress from the G2/M phase to the next G1 phase. Probably as a result of a blockage of cell cycle progress, the cycling, but not the resting, T cells underwent apoptosis when treated with LAC. Mechanistically, we have found that cyclin-dependent kinase-2 (CDK2) and the cyclin E-associated kinase (largely CDK2), but not CDK4, in the G1 phase were strongly inhibited by LAC. This could be an important mechanism for the proteasome to regulate the cell cycle. The degradation of cyclin E in the late G1 and early S phases was dependent on the proteasome, although it was unlikely that this accounted for the observed inhibition of T cell proliferation. There was a reduced decay of p27Kip1 in the late G1 phase when the proteasome activity was suppressed, and this might be a contributing mechanism for the observed inhibition of CDK2 activity. Interestingly, p21Cip1 was up-regulated during the G1 phase, and the up-regulation was inhibited by LAC. Our study shows that the proteasome plays pivotal roles in regulating T cell activation and proliferation, and its effect is probably exerted through multiple mechanisms.
Separation of Function Between the Domains of Toxic Shock Syndrome Toxin-1
Authors: Wahlsten Jennifer L. , Ramakrishnan S.
Published: January 2023

Toxic shock syndrome toxin-1 (TSST1) is a superantigenic exotoxin produced by certain strains of Staphylococcus aureus. Structurally, TSST1 is composed of two domains: residues determined by crystallography to directly interact with MHC II molecules reside within the N-terminal domain, while TSST1 residues critical for superantigenicity are within the C-terminal domain. In this study, we expressed the individual N- and C-terminal domains of TSST1 in Escherichia coli and studied their biologic activities. The TSST1 N-terminal domain (TSST(1–87)) did not induce proliferation of human PBLs or release of TNF-β, but did induce TNF-α release. However, TSST1-elicited proliferation and release of both TNF isoforms were inhibited by a molar excess of TSST(1–87). The TSST1 C-terminal domain (TSST(88–194)) did not bind MHC II molecules, yet it elicited production of TNF-α and TNF-β, and induced TCR Vβ-specific proliferation similarly to intact TSST1. When covalently cross-linked to tumor cells, TSST(88–194) elicited a local in vivo antitumor response indistinguishable from TSST1. Although intact TSST1 causes lethal shock in vivo, the individual domains of this molecule may have therapeutic potential: the N-terminal domain to antagonize lymphocyte activation and TNF release during acute TSST1-precipitated toxic shock syndrome, and the C-terminal domain to stimulate antitumor responses without MHC II binding.
Distinct Phospholipases A2 Regulate the Release of Arachidonic Acid for Eicosanoid Production and Superoxide Anion Generation in Neutrophils
Authors: Tithof Patricia K. , Peters-Golden Marc , Ganey Patricia E.
Published: January 2023

Arachidonic acid (AA) released from membrane phospholipids by phospholipase A2 (PLA2) is important as a substrate for eicosanoid formation and as a second messenger for superoxide anion (O2−) generation in neutrophils. Different isoforms of PLA2 in neutrophils might mobilize AA for different functions. To test this possibility, we sought to characterize the PLA2s that are activated by the neutrophil stimuli, Aroclor 1242, a mixture of polychlorinated biphenyls, and A23187, a calcium ionophore. Both Aroclor 1242 and A23187 caused release of [3H]AA; however, O2− production was seen only in response to Aroclor 1242. Eicosanoids accounted for >85% of the radioactivity recovered in the supernatant of A23187-stimulated cells but <20% of the radioactivity recovered from cells exposed to Aroclor 1242. Omission or chelation of calcium abolished A23187-induced AA release, but did not alter AA release in Aroclor 1242-stimulated neutrophils. AA release and O2− production in response to Aroclor 1242 were inhibited by bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2. BEL, however, did not alter A23187-induced release of AA. Cell-free assays demonstrated both calcium-dependent and calcium-independent PLA2 activity. Calcium-independent activity was inhibited >80% by BEL, whereas calcium-dependent activity was inhibited <5%. Furthermore, calcium-independent, but not calcium-dependent, PLA2 activity was significantly enhanced by Aroclor 1242. These data suggest that Aroclor 1242 and A23187 activate distinct isoforms of PLA2 that are linked to different functions: Aroclor 1242 activates a calcium-independent PLA2 that releases AA for the generation of O2−, and A23187 activates a calcium-dependent PLA2 that mobilizes AA for eicosanoid production.
Fever-Range Hyperthermia Enhances L-Selectin-Dependent Adhesion of Lymphocytes to Vascular Endothelium
Authors: Wang Wan-Chao , Goldman Lorin M. , Schleider David M. , Appenheimer Michelle M. , Subjeck John R. , Repasky Elizabeth A. , Evans Sharon S.
Published: January 2023

The L-selectin leukocyte adhesion molecule plays an important role in controlling leukocyte extravasation in peripheral lymph nodes and at sites of tissue injury or infection. Although febrile responses during infection and inflammation are associated with enhanced immune activity, the contribution of fever-range temperatures to controlling lymphocyte recruitment to tissues has not been previously examined. In this report we provide evidence that direct exposure of lymphocytes to fever-range temperatures (38–41°C) in vitro for 9 to 24 h resulted in a >100% increase in L-selectin-dependent adhesion of these cells to lymph node high endothelial venules (HEV). Moreover, culture of lymphocytes under hyperthermia conditions markedly enhanced the ability of these cells to traffic in an L-selectin-dependent manner to peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches. In contrast, febrile temperatures did not increase LFA-1 function as assessed by measuring lymphocyte adhesion to ICAM-1–3T3 transfectants. Fever-range hyperthermia further did not increase L-selectin surface density on lymphocytes or L-selectin-dependent recognition of soluble carbohydrate substrates; however, a marked increase in ultrastructural immunogold-labeling of L-selectin was observed in response to thermal stimuli. These results suggest that elevated temperatures enhance L-selectin adhesion and/or avidity through the regulation of L-selectin conformation or organization in the plasma membrane. Finally, the observed thermal effects on L-selectin adhesion were attributed to soluble factors in the conditioned medium of heat-treated cells. Taken together, these data provide new insight into the potential physiologic role of the febrile response in enhancing lymphocyte recruitment to tissues through the regulation of L-selectin adhesion.
Cutting Edge: Constitutive Activation of TCR Signaling Molecules in IL-2-IndependentHerpesvirus saimiri-Transformed T Cells
Authors: Noraz Nelly , Saha Kunal , Ottones Florence , Smith Susan , Taylor Naomi
Published: January 2023

Both human T cell leukemia virus type I and simian Herpesvirus saimiri (HVS) transform human T cells in vitro. Although IL-2-independent growth in human T cell leukemia virus type I-transformed T cells is associated with constitutive phosphorylation of JAK/STAT kinases, we now demonstrate that different mechanisms may be responsible for the ability of HVS-transformed T cells to proliferate in the absence of exogenous cytokines. The IL-2 independence of an HVS-transformed cell line correlated with constitutive activation of protein tyrosine kinases known to be induced following TCR engagement. Thus, in these cells we observed increased phosphotransferase activity of Lck as well as constitutive tyrosine phosphorylation of the TCR-associated ZAP-70 kinase and expression of the related Syk protein tyrosine kinase. While Syk is generally not expressed in activated T cells, its introduction has been shown to enhance TCR responsiveness. These results suggest that distinct signal transduction cascades can participate in the transition of T cells to IL-2 independence.