bluuebunny/crossref_metadata_2025 huggingface dataset:
PAF-mediated Ca2+ influx in human neutrophils occurs via store-operated mechanisms
Authors: Hauser Carl J , Fekete Zoltan , Adams John M , Garced Matthew , Livingston David H , Deitch Edwin A
Published: September 2022

Many inflammatory mediators activate neutrophils (PMN) partly by increasing cytosolic calcium concentration ([Ca2+]i). Modulation of PMN [Ca2+]i might therefore be useful in regulating inflammation after shock or sepsis. The hemodynamic effects of traditional Ca2+ channel blockade, however, could endanger unstable patients. Store-operated calcium influx (SOCI) is known now to contribute to Ca2+ flux in “nonexcitable” cells. Therefore, we studied the role of SOCI in human PMN responses to the proinflammatory ligand PAF. PMN [Ca2+]i was studied by spectrofluorometry with and without external calcium. We studied the effects of PAF on Mn2+ entry into and on Ca2+ efflux from thapsigargin (Tg)-treated cells. Influx was assessed in the presence and absence of the blockers SKF-96365 (SKF), TMB-8, and 2-APB. Half of PAF [Ca2+]i mobilization occurs via calcium influx. The kinetics of calcium entry were typical of SOCI rather than receptor-mediated calcium entry (RMCE). SKF had multiple nonspecific effects on [Ca2+]i. Inhibition of store emptying by TMB-8 and 2-APB blocked all calcium entry, demonstrating influx was store depletion-dependent. PAF has no direct effect on calcium efflux. Where SOCI is maximal, PAF has no further effect on calcium-channel traffic. PAF-induced calcium signals are highly dependent on SOCI and independent of RMCE. SOCI-specific blockade might modulate PMN-mediated inflammation and spare cardiovascular function in shock and sepsis.
Severe exercise enhances phagocytosis by murine bronchoalveolar macrophages
Authors: Su Shu-Hui , Chen Hsiun-ing , Jen Chauying J
Published: September 2022

Because physical activity affects the immune competency of individuals by an unknown mechanism, we investigated the effect of acute exercise on phagocytosis of bronchoalveolar macrophages (BAMs). Male BALB/c mice, 7–9 weeks old, ran on a treadmill to exhaustion (severe exercise, SE) or at a final speed of 17 m/min for 30 min (moderate exercise, ME). Although both exercise protocols induced differential leukocytosis, 95% leukocytes from lung lavages of both groups were BAMs. The BAM phagocytic capacity of nonopsonized beads increased immediately after SE but not after ME, gradually returning to the basal level after 4 h. SE upregulates the macrophage scavenger receptors (SR-A type I/II and MARCO), CR3, and ICAM-1, but not FcγR. Although the blocking effect of MARCO antibody was most pronounced, that of ICAM-1 antibody was totally reversed by cross-linking CR3. Our results showed that SE, but not ME, activated BAMs and that the enhanced nonopsonized phagocytosis was mainly mediated by scavenger receptors and ICAM-1/CR3.
Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis
Authors: Hattar Katja , Sibelius Ulf , Bickenbach Annette , Csernok Elena , Seeger Werner , Grimminger Friedrich
Published: September 2022

Anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) possess a high sensitivity and specificity for Wegener's granulomatosis. Due to their capacity of directly activating neutrophils, a pathogenetic role for these autoantibodies has been proposed. We investigated the impact of subthreshold concentrations of monoclonal anti-PR3 antibodies (anti-PR3; 0.1 μg/mL) on neutrophil activation elicited by a secondary agent. Preincubation with anti-PR3 resulted in a massive amplification of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukotriene (LT) generation, with a marked increase in the liberation of LTB4, LTA4, and 5-hydroxyeicosatetraenoic acid (5-HETE). This priming commenced within 2.5 min, with a maximum after 5–7.5 min. Moreover, anti-PR3 pretreatment markedly enhanced PMN movement toward fMLP. The priming effect of anti-PR3 toward fMLP challenge was reproduced by c-ANCA, but not by F(ab)2 fragments of the antibodies and isotype-matched control IgG. Generation of superoxide anion and release of elastase were suppressed in anti-PR3-pretreated neutrophils undergoing fMLP challenge. In contrast, neutrophil activation by platelet-activating factor (PAF) or the calcium ionophore A23187 remained unaffected. We conclude that subthreshold concentrations of anti-PR3 antibodies selectively modify neutrophil responses to fMLP, with enhancement of leukotriene generation and chemotaxis, but suppression of respiratory burst and degranulation. Such priming might contribute to localized neutrophil accumulation together with blunted host defense in Wegener's granulomatosis.
Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury
Authors: Nakatani Keigo , Takeshita Seiichiro , Tsujimoto Hiroshi , Kawamura Youichi , Sekine Isao
Published: September 2022

To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil-mediated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbilical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose-dependently by urinary trypsin inhibitor (ulinastatin, UTI) and ONO-5046, which have the ability to inactivate neutrophil elastase, but not by gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban, which have no ability to inactivate neutrophil elastase. In addition, when UTI and ONO-5046 were added to the tumor necrosis factor α-primed neutrophils alone, they showed a dose-dependent inhibition of the intracellular elastase activity, but the other SPI did not, for either flow cytometry or confocal microscopy. Therefore, UTI and ONO-5046 may protect EC against the neutrophil-mediated injury not only by inactivating the extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils and suppressing the production and secretion of activated elastase from them.
Colostral neutrophils express Fcα receptors (CD89) lacking γ chain association and mediate noninflammatory properties of secretory IgA
Authors: Honorio-França Adenilda C , Launay Pierre , Carneiro-Sampaio Magda M S , Monteiro Renato C
Published: September 2022

Colostrum plays an important role in protecting newborn infants against acute gastrointestinal and respiratory infections. IgA antibodies have been considered the major effector component; however, the role of their receptors on colostral phagocytes, especially neutrophils, has not been studied. Here, we demonstrate that CD15+ colostrum neutrophils express IgA Fc receptors (FcαR, CD89) at levels similar to those of blood neutrophils. Most colostral cells (70%) bear secretory IgA (SIgA) on their surface (and intracellularly), whereas blood cells do not. The FcαR on colostral neutrophils was identified as the a.1 isoform with a similar molecular mass (55–75 kDa) as that identified for blood neutrophils. Removal of N-linked carbohydrates revealed a major protein core of 32 kDa for both cell types. In contrast, co-immunoprecipitation and immunoblot experiments using a mild detergent, digitonin, revealed a lack of γ chain association with FcαR (γ-less) exclusively on colostral neutrophils. The functional role of these γ-less FcαR cells was evaluated by measuring superoxide release and killing of SIgA-coated enteropathogenic E. coli. No increase in superoxide release was observed in colostral cells compared with blood neutrophils, whereas optimal release was obtained with PMA stimulation. Furthermore, despite similar bacterial phagocytosis index between both cell types, IgA-mediated bacterial-killing was not detectable with colostral neutrophils, whereas killing was detectable on blood cells. These results reveal exclusive expression of γ-less FcαR on colostral neutrophils associated with receptor hyperoccupation by IgA and with low, bacterial-killing activity, which suggest that this receptor may mediate noninflammatory effects of SIgA.
The endotoxin-binding bactericidal/permeability-increasing protein (BPI): a target antigen of autoantibodies
Authors: Schultz H , Weiss J , Carroll S F , Gross W L
Published: September 2022

The bactericidal/permeability-increasing protein (BPI) is anendotoxin-binding neutrophil leukocyte-granule protein withantibacterial and anti-endotoxin properties. A recombinant form of BPI(rBPI21) has been developed and is being tested as atherapeutic agent to treat Gram-negative bacterial infections andexposure to Gram-negative bacterial endotoxin. BPI is also a targetantigen of anti-neutrophil cytoplasmic autoantibodies (ANCA). BPI-ANCAare present in cystic fibrosis, inflammatory bowel disease, vasculitis,and primary sclerosing cholangitis; presence of BPI-ANCA appearsassociated with a higher inflammatory disease activity and greaterorgan damage. BPI-ANCA as well as ANCA directed at otherneutrophil-granule proteins may exacerbate inflammation by nonspecificeffects of extracellular and cell-associated immune complexes. BPI-ANCAmay further worsen inflammation by reducing the ability of BPI topromote clearance of Gram-negative bacteria and bacterial-associatedendotoxin.
Changes of HSP72-expression in leukocytes are associated with adaptation to exercise under conditions of high environmental temperature
Authors: Fehrenbach Elvira , Niess Andreas Michael , Veith Roman , Dickhuth Hans-Herrmann , Northoff Hinnak
Published: September 2022

Overexpression of the heat shock protein HSP72 providesthermotolerance. We asked if two consecutive endurance runs 1 weekapart (CR1, CR2) and additional environmental heat stress affectHSP72-expression in leukocytes of nonheat-acclimated enduranceathletes. Twelve subjects were allocated randomly into two groups. Group HH completed both runs at 28°C ambient temperature, and groupNH performed CR1 at 18°C and CR2 at 28°C. HSP72-expression wasdetermined by flow cytometry and RT-PCR before and 0, 24, and 48 hafter exercise. Additionally, post-exercise cells were exposed toin vitro heat shock (HS; 2 h, 42°C). The prolonged, high HSP72 protein level after CR1 in HH compared with NH may reflectthermotolerance induced by endurance exercise at high ambienttemperature. Adaptation of cardiocirculatory/thermoregulatory capacityafter CR2 in HH went along with a more rapid down-regulation of HSP72compared with CR1. HSP72 mRNA demonstrated temperature-related changesafter exercise. The reduced HS response in vitro after CR2may represent exercise-related adaptation mechanisms. HSP72concentrations in leukocytes may indicate previous exercise- andtemperature-related stress conditions and adaptation in immunocompetentcells.
Quantitative analysis of chemokine expression by dendritic cell subsets in vitro and in vivo
Authors: Vissers Joost L M , Hartgers Franca C , Lindhout Ernst , Teunissen Marcel B M , Figdor Carl G , Adema Gosse J
Published: September 2022

Upon maturation, dendritic cells (DCs) have to adjust their chemokineexpression to sequentially attract different leukocyte subsets. We usedreal-time quantitative polymerase chain reaction analysis to study indetail the expression of 12 chemokines involved in the recruitment ofleukocytes into and inside secondary lymphoid organs, by DCs indistinct differentiation stages, both in vitro and in vivo. Monocyte-derived immature DCs expressed high levels of DCchemokine 1 (DC-CK1), EBI1-ligand chemokine (ELC), macrophage-derivedchemokine (MDC), macrophage-inflammatory protein (MIP)-1α, and thymusand activation-regulated chemokine (TARC). Upon maturation, DCsup-regulated the expression of DC-CK1 (60-fold), ELC (7-fold), and TARC(10-fold). Activation of DCs by CD40 ligand further up-regulated theexpression of ELC (25-fold). We found that freshly isolated blood DCsexpressed only low levels of interleukin-8, lymphotactin, and MIP-1α. It is interesting that the chemokine profile expressed by activatedCD11c− lymphoid-like as well as CD11c+ myeloidblood DCs mimics that of monocyte-derived DCs. Additionally, purifiedLangerhans cells that had migrated out of the epidermis expressed asimilar chemokine pattern. These data indicate that different DCsubsets in vitro and in vivo can express the same chemokines to attractleukocytes.
Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes
Authors: Zouki Christine , József Levente , Ouellet Sophie , Paquette Yves , Filep János G
Published: September 2022

Recent studies indicate that nitric oxide (NO) or related compounds mayregulate the production of interleukin (IL)-8, a potent proinflammatorychemokine. Here we report that peroxynitrite (ONOO−)formed by a reaction of NO with superoxide mediates IL-8 geneexpression and IL-8 production in IL-1β- and TNF-α-stimulated humanleukocytes in whole blood. The NO synthase inhibitors aminoguanidineand NG-nitro-l-arginine methylester blocked nuclear accumulation of activator protein-1 (AP-1) andnuclear factor (NF)-κB in both polymorphonuclear (PMN) andmononuclear leukocytes and inhibited IL-8 mRNA expression and IL-8release by ∼90% in response to IL-1β and TNF-α. EnhancedONOO− formation was detected in granulocytes, monocytes, and lymphocytes after challenge with IL-1β or TNF-α. The additionof ONOO− (0.2–80 μM) to whole blood increased nuclearaccumulation of AP-1 and NF-κB in PMN and mononuclear leukocytes andaugmented IL-8 mRNA expression and IL-8 production in aconcentration-dependent fashion. Pyrrolidine dithiocarbamate, aninhibitor of NF-κB activation, attenuated ∼70% of IL-8 releaseevoked by IL-1β, TNF-α, or ONOO−. These resultsindicate that ONOO− formation may underlie the action ofcytokines towards IL-8 gene expression in human leukocytes.
Combinatorial signals by inflammatory cytokines and chemokines mediate leukocyte interactions with extracellular matrix
Authors: Vaday Gayle G , Franitza Susanne , Schor Hagai , Hecht Iris , Brill Alexander , Cahalon Liora , Hershkoviz Rami , Lider Ofer
Published: September 2022

On their extravasation from the vascular system into inflamed tissues, leukocytes must maneuver through a complex insoluble network of molecules termed the extracellular matrix (ECM). Leukocytes navigate toward their target sites by adhering to ECM glycoproteins and secreting degradative enzymes, while constantly orienting themselves in response to specific signals in their surroundings. Cytokines and chemokines are key biological mediators that provide such signals for cell navigation. Although the individual effects of various cytokines have been well characterized, it is becoming increasingly evident that the mixture of cytokines encountered in the ECM provides important combinatorial signals that influence cell behavior. Herein, we present an overview of previous and ongoing studies that have examined how leukocytes integrate signals from different combinations of cytokines that they encounter either simultaneously or sequentially within the ECM, to dynamically alter their navigational activities. For example, we describe our findings that tumor necrosis factor (TNF)-α acts as an adhesion-strengthening and stop signal for T cells migrating toward stromal cell-derived factor-1α, while transforming growth factor-β down-regulates TNF-α-induced matrix metalloproteinase-9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF-α, can transmit diverse signals to different subsets of leukocytes, depending on its combination with other cytokines, its concentration, and its time and sequence of exposure. The combinatorial effects of multiple cytokines thus affect leukocytes in a step-by-step manner, whereby cells react to cytokine signals in their immediate vicinity by altering their adhesiveness, directional movement, and remodeling of the ECM.
TSG-14 transgenic mice have improved survival to endotoxemia and to CLP-induced sepsis
Authors: Dias Adriana A M , Goodman Adam R , Dos Santos Jane Lima , Gomes Rachel Novaes , Altmeyer Anne , Bozza Patrícia T , de Fátima Horta Maria , Vilcek Jan , Reis Luiz F L
Published: September 2022

Tumor necrosis factor-stimulated gene 14 (TSG-14)/PTX3 was identifiedoriginally as a TNF-α and IL-1β-stimulated gene from normal, humanforeskin fibroblasts and vascular endothelial cells, respectively. TSG-14 gene encodes a 42-kDa-secreted glycoprotein with acarboxy-terminal half that shares homology with the entire sequence of C-reactive protein (CRP) and serum amyloid P component (SAP),acute-phase proteins of the pentraxin family. Some experimentalevidence suggests that TSG-14 plays a role in inflammation, yet itsfunction and mechanism of action remain unclear. We have generatedtransgenic mice that overexpress the murine TSG-14 gene under thecontrol of its own promoter. From eight transgenic founders, twolineages were derived and better characterized: Tg2 and Tg4, carryingtwo and four copies of the transgene, respectively. TSG-14 transgenicmice were found to be more resistant to the endotoxic shock induced byLPS and to the polymicrobial sepsis caused by cecal ligation andpuncture (CLP). Moreover, macrophages derived from the transgenic miceproduced higher amounts of nitric oxide in response to IFN-γ,TNF-α, and LPS as compared with macrophages from wild-type animals, and the augmented response appears to be the consequence of a higherresponsiveness of transgenic macrophages to IFN-γ. The data shownhere are the first in vivo evidence of the involvement of TSG-14 in the inflammatory process and suggest a role for TSG-14 in thedefense against bacterial infections.
CD8α− and CD8α+ subclasses of dendritic cells undergo phenotypic and functional maturationin vitroandin vivo
Authors: De Smedt Thibaut , Butz Eric , Smith Jeffrey , Maldonado-López Roberto , Pajak Bernard , Moser Muriel , Maliszewski Charles
Published: September 2022

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of naïve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8α expression. We have studied the phenotype and maturation state of mouse splenic CD8α− and CD8α+ DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8α− and CD8α+ DCs into potent and equally efficient activators of naïve CD4+ and CD8+ T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.
Serotonin protects NK cells against oxidatively induced functional inhibition and apoptosis
Authors: Betten Åsa , Dahlgren Claes , Hermodsson Svante , Hellstrand Kristoffer
Published: September 2022

High concentrations of the neurotransmitter serotonin can be found ininflamed and ischemic peripheral tissues, but the role of serotonin inimmunoregulation is largely unknown. Here we report that serotoninprotected human natural-killer (NK) cells from oxidatively inducedinhibition inflicted by autologous monocytes in vitro. Serotoninprotected NK cells from monocyte-mediated apoptosis and suppression ofcytotoxicity and maintained the activation of NK cells induced byinterleukin-2 despite the presence of inhibitory monocytes. A detailedanalysis of these protective effects revealed that serotonin scavengedreactive oxygen species (ROS) derived from theH2O2-myeloperoxidase (-MPO) system. Serotoninshared this scavenger activity with its precursor, 5-hydroxytryptophan(5-HTP); however, serotonin was >10-fold more potent than 5-HTP inprotecting NK cells against functional inhibition and apoptosis. Wepropose that serotonin, by scavenging peroxidase-derived ROS, may serveto protect NK cells from oxidative damage at inflammatory sites.
Stimulation of human neutrophils and monocytes by staphylococcal phenol-soluble modulin
Authors: Liles W Conrad , Thomsen Anni R , O'Mahony D Shane , Klebanoff Seymour J
Published: September 2022

Modulins represent microbial products that stimulate cytokine production in host cells. The modulins responsible for gram-positive sepsis remain poorly understood. Staphylococci release a factor (or factors) that activates nuclear factor-κB and stimulates cytokine production in cells of macrophage lineage. This factor, termed phenol-soluble modulin (PSM), has been recently isolated from culture supernatant of Staphylococcus epidermidis. We examined the effects of PSM on proinflammatory properties of human neutrophils and monocytes in vitro. PSM activated the respiratory (oxidative) burst in neutrophils and primed neutrophils for enhanced respiratory burst activity in response to formyl-methionyl-leucyl-phenylalanine. PSM also stimulated neutrophil degranulation as reflected by increased surface expression of CD11b and CD18, which was accompanied by rapid shedding of l-selectin. Spontaneous apoptosis of both neutrophils and monocytes was inhibited by PSM. Furthermore, PSM also functioned as a chemoattractant factor for both neutrophils and monocytes. Thus, the proinflammatory properties of PSM resemble those of both lipopolysaccharide and bacterial chemotactic peptides. These findings suggest that PSM may play a role in the pathogenesis and systemic manifestations of sepsis caused by staphylococci.
Insights into molecular mechanisms of contact hypersensitivity gained from gene knockout studies
Authors: Wang Binghe , Feliciani Claudio , Freed Irwin , Cai Qinchao , Sauder Daniel N
Published: September 2022

Contact hypersensitivity (CHS), a dendritic-cell (DC)-dependent, T-cell-mediated skin immune response to reactive haptens, has been a subject of intense research for many years. The molecular mechanisms underlying CHS are complicated and are not fully understood. During the past few years, varieties of gene-targeted knockout mice have been used in the study of CHS. Such studies have contributed significantly to our understanding of the mechanisms responsible for the initiation of CHS. This review focuses on insights into molecular requirements for CHS gained from knockout studies.
Monocyte chemoattractant protein-1 enhances HSV-induced encephalomyelitis by stimulating Th2 responses
Authors: Nakajima Hideto , Kobayashi Makiko , Pollard Richard B , Suzuki Fujio
Published: September 2022

Monocyte chemoattractant protein (MCP)-1 has a pathogenic role in herpesvirus-induced encephalomyelitis (HSM). Anti-MCP-1 antibody greatly decreased HSM severity in mice infected with herpes simplex virus type 2 (HSM mice), compared with its effect in control HSM mice treated with rabbit immunoglobulin. HSM severity was markedly enhanced in mice previously treated with a mixture of interleukin (IL) 4 and -10. In response to stimulation with antigen, HSM mouse cells isolated from cerebrospinal fluids (CSF cells) produced IL-4 in culture fluids; however, IL-4 production decreased in CSF cells derived from HSM mice previously treated with anti-MCP-1 antibody. A macrophage population isolated in CSF cells from HSM mice (CSF-Mφ) produced MCP-1 in culture fluids. In response to stimulation with herpesvirus antigen, a population of T cells isolated from CSF cells from HSM mice (CSF-T cells) produced IL-4 into their culture fluids, although MCP-1 was not produced by CSF-T cells stimulated by this antigen. IL-4 production by CSF-T cells was markedly enhanced when they were stimulated with viral antigen in the presence of murine recombinant MCP-1 (rMCP-1). Furthermore, IL-4 was produced in naive splenic T cells cocultured with CSF-Mφ. These results indicate that the severity of HSM is influenced by MCP-1, which stimulates Th2 responses.
Monophosphoryl lipid A stimulated up-regulation of reactive oxygen intermediates in human monocytes in vitro
Authors: Saha D C , Barua R S , Astiz M E , Rackow E C , Eales-Reynolds L-J
Published: September 2022

The production of reactive oxygen and nitrogen intermediates is a common response to infectious challenge in vivo. These agents have been implicated in the modulation of cytokine responses and are produced in large amounts in response to endotoxins produced by a number of infectious agents. The antigen-presenting cell activation caused by these lipopolysacchardies (LPS) has been exploited in the use of these agents as adjuvants. In recent years, less-toxic derivatives have been sought. One such agent, monophosphoryl lipid A (MPL), has been used increasingly in vivo as an adjuvant and as a modulator of the inflammatory process. It is known that this agent modulates the inflammatory response and cytokine production. In addition, we have shown its effect on the production of reactive nitrogen intermediates. In this paper, we show that MPL stimulates the release of high levels of superoxide (O2−) and hydrogen peroxide (H2O2), the latter being greater than that seen with LPS and appearing to be related to the inability of MPL to stimulate catalase activity. When cells were pretreated with LPS or MPL and subsequently challenged with LPS, the production of O2− and H2O2 was inhibited significantly by LPS and MPL. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 μg/ml MPL, the same concentration that induced the maximum release of H2O2 in primary stimulation. In addition, we have shown that following MPL pretreatment, LPS stimulation does not cause the loss of cytoplasmic IκBα, which occurs when human monocytes are cultured with LPS. From our results, we propose a model for the reduced toxicity of MPL.
Different orders for acquisition of apoptotic characteristics by leukocytes
Authors: Hess Krista L , Johnson Jacob D , Cook-Mills Joan M
Published: September 2022

Apoptotic leukocytes undergo cellular changes that are used as markers for “early” versus “late” stages of apoptosis. To ascertain if the order for acquisition of these changes is unique to specific hematopoietic cell types, we compared four leukocyte cell types and the following five apoptotic characteristics: MC540 incorporation, annexin V-FITC binding, propidium iodide (PI) labeling of hypodiploid nuclei, DNA fragmentation by a colorimetric assay, and cell membrane permeability to PI. The order for acquisition of these apoptotic characteristics was significantly different for each of the leukocyte cell types and for the mode of induction of apoptosis. It is interesting that the nuclear changes but not the membrane changes studied in mouse spleen cells required caspase activity. In summary, the acquisition of these apoptotic characteristics occurs through caspase-dependent and caspase-independent mechanisms, and importantly, the order for acquisition of the characteristics is specific for the cell type and for the mode of induction of apoptosis.
Circular minidefensins and posttranslational generation of molecular diversity
Authors: Leonova Larisa , Kokryakov Vladimir N , Aleshina Galina , Hong Teresa , Nguyen Tung , Zhao Chengquan , Waring Alan J , Lehrer Robert I
Published: September 2022

We purified two new minidefensins (RTD-2 and RTD-3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD-1, a circular (θ) defensin previously described by Tang et al. (Science, 286, 498–502, 1999). However, whereas the 18 residues of RTD-1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD-2 and -3 comprise tandem nonapeptide repeats derived from only one of the RTD-1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.
Amoeboid leukocyte crawling through extracellular matrix: lessons from theDictyosteliumparadigm of cell movement
Authors: Friedl Peter , Borgmann Stefan , Bröcker Eva-B
Published: September 2022

Cell movement within three-dimensional tissues is a cycling multistep process that requires the integration of complex biochemical and biophysical cell functions. Different cells solve this challenge differently, which leads to differences in migration strategies. Migration principles established for leukocytes share many characteristics with those described for ameba of the lower eukaryoteDictyostelium discoideum. The hallmarks of amoeboid movement include a simple polarized shape, dynamic pseudopod protrusion and retraction, flexible oscillatory shape changes, and rapid low-affinity crawling. Amoeboid crawling includes haptokinetic adhesion-dependent as well as biophysical migration mechanisms on or within many structurally and functionally different substrates. We describe central aspects of amoeboid movement in leukocytes and the implications for leukocyte crawling and positioning strategies within interstitial tissues.
Vδ1 and Vδ2 γδ T cells express distinct surface markers and might be developmentally distinct lineages
Authors: De Rosa Stephen C , Mitra Dipendra K , Watanabe Nobukazu , Herzenberg Leonore A , Herzenberg Leonard A , Roederer Mario
Published: September 2022

We report here that the two major types of γδ T cells found in human blood, Vδ1 and Vδ2, were found to have markedly different phenotypes. Vδ2 cells had a phenotype typical of most αβ T cells in blood; i.e., they were CD5+, CD28+, and CD57−. In contrast, Vδ1 cells tended to be CD5−/dull, CD28−, and CD57+. Furthermore, although Vδ1 T cells appeared to be “naive” in that they were CD45RA+, they were CD62L− and on stimulation uniformly produced interferon-γ, indicating that they are in fact memory/effector cells. This phenotype for Vδ1 cells was similar to that of intestinal intraepithelial lymphocytes, a subset that can develop in the absence of the thymus. We suggest that the Vδ1 and Vδ2 T cell subsets represent distinct lineages with different developmental pathways. The disruption of the supply of normal, thymus-derived T cells in HIV-infected individuals might be responsible for the shift in the Vδ2/Vδ1 ratio that occurs in the blood of individuals with HIV disease.
Leukocyte elastase in murine and human non-Hodgkin lymphomas
Authors: De Noncourt Pascal , Robledo Olivier , Alain Tommy , Kossakowska Anna E , Urbanski Stefan J , Potworowski Edouard F , St-Pierre Yves
Published: September 2022

Extracellular proteases play a crucial role in the invasive behavior of normal and transformed leukocytes. Thus far, however, most of the attention has been focused on members of the family of matrix metalloproteinases. In this work, we show that lymphoma cells can express leukocyte elastase (LE) and recruit the enzyme at their surface via ICAM-1. The expression of LE by lymphoma cells was augmented significantly by stimulation with IL-6 and IL-13, both of which also induced the expression of MMP-9. Although LE and IL-13 transcripts were detected in several non-Hodgkin's lymphomas, immunohistochemical analysis of lymphoma tissues also showed that LE was strongly expressed in infiltrating leukocytes. Given the spectrum of key molecules that can be cleaved by LE and that LE and MMP-9 are involved in the invasive behavior of normal or transformed leukocytes, our results raise the hypothesis that LE plays a crucial role in the multistep processes of inflammation and lymphoma metastasis.
Differential regulation of transendothelial migration of THP-1 cells by ICAM-1/LFA-1 and VCAM-1/VLA-4
Authors: Ronald John A , Ionescu Carmen V , Rogers Kem A , Sandig Martin
Published: September 2022

The adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressed in atherogenic lesions are thought to regulate monocyte diapedesis. To better understand their specific roles we used function-blocking antibodies and examined in a culture model the morphology, motility, and diapedesis of THP-1 cells interacting with human coronary artery endothelial cells. The number of motile THP-1 cells was reduced only when VCAM-1 or both ICAM-1 and VCAM-1 were blocked. Blockade of ICAM-1 and VCAM-1, either separately or together, reduced to the same degree the distance that THP-1 cells traveled. Diapedesis was reduced only during the simultaneous blockade of both adhesion molecules. Blockade of either ICAM-1 or VCAM-1 inhibited pseudopodia formation, but ICAM-1 blockade induced the formation of filopodia. We suggest that the interactions of endothelial ICAM-1 and VCAM-1 with their ligands differentially regulate distinct steps of diapedesis by modulating the ratio of active and inactive forms of small GTPases such as Rho, Rac, and Cdc42.
Selective attraction of naive and memory B cells by dendritic cells
Authors: Dubois Bertrand , Massacrier Catherine , Caux Christophe
Published: September 2022

In this study, we investigate whether dendritic cells (DC), known to interact directly with T and B cells, might also contribute to the recruitment of B cells through the production of chemotactic factors. We found that B cells responded to several chemokines (CXCL12, CCL19, CCL20, and CCL21), which can be produced by DC upon activation. In addition, supernatant from DC (SNDC) potently and selectively attracted naive and memory B cells but not germinal center (GC) B cells or other lymphocytes (CD4+, CD8+ T cells or NK cells). Production of this activity was restricted to DC and was not increased following DC activation by LPS or CD40 ligand. Surprisingly, the B-cell chemotactic response to SNDC was insensitive to pertussis toxin treatment. In addition, the chemotactic factor(s) appeared resistant to protease digestion and highly sensitive to heat. This suggested that the DC chemotactic factor(s) is different from classical chemoattractants and does not involve Gαi proteins on the responding B lymphocytes. It is interesting that SNDC was able to synergize with several chemokines to induce massive migration of B lymphocytes. These observations show that DC spontaneously produce factors that, alone or in cooperation with chemokines, specifically regulate B-cell migration, suggesting a key role of DC in the recruitment or localization of B lymphocytes within secondary lymphoid organs.
Association of ARF and Rabs with complement receptor Type-1 storage vesicles in human neutrophils
Authors: Chaudhuri Subhendu , Kumar Anoopa , Berger Melvin
Published: September 2022

During neutrophil activation, the properties of the cell are rapidly altered by increases in the surface expression of functionally important receptors and adherence molecules. At the same time, endocytic and phagocytic activities increase. These alterations require precise regulation of membrane and protein movement, which is achieved, at least in part, by bidirectional movement of small transport vesicles. GTP-binding proteins, including Rabs and ADP-ribosylation factors (ARFs), play critical roles in regulating vesicle trafficking in other types of cells. The ability to immunoisolate the “secretory” vesicle subpopulation in which complement receptor type 1 (CR1) is stored allowed us to determine which types of low-molecular-weight GTP-binding proteins interact with these vesicles and under what conditions. CR1-containing vesicles from resting human neutrophils constitutively copurify with Rabs 3a, 4, and 5a, and reversibly bind an ARF, likely ARF1. ARF binding is dependent on free Mg2+ and is enhanced by GTPγS. Mg2+ at 0.4 μM is necessary for half-maximal binding of ARFs to CR1 storage vesicles. Artificial phospholipid vesicles and primary and secondary granules from human neutrophils do not bind ARFs themselves and do not compete for recruitment of ARFs to CR1 vesicles, suggesting that specific membrane environments and/or proteins on these vesicles stabilize the ARF-GTP-Mg2+ complex. Free Ca2+ at 300 nM does not inhibit ARF binding to CR1 storage vesicles, but 10 mM Ca2+ does reduce such binding. These findings suggest that ARF-GTP specifically and reversibly interacts with CR1 storage vesicles in human polymorphonuclear leukocytes and may play a role in regulating their transport.
Rafts and synapses in the spatial organization of immune cell signaling receptors
Authors: Dykstra Michelle , Cherukuri Anu , Pierce Susan K
Published: September 2022

The multichain immune recognition receptors (MIRRs), including the T cell and B cell antigen receptors and the high affinity receptor for IgE, play an important role in immune cell signaling. The MIRRs have no inherent kinase activity, but rather associate with members of the Src-family kinases to initiate signaling. Although a great deal is understood about the biochemical cascades triggered by MIRRs, the mechanism by which signaling is initiated was not known. The evidence now indicates that the Src-family kinases are concentrated in cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, that exclude the MIRRs. Upon ligand-induced crosslinking the MIRRs translocate into rafts where they are phosphorylated. The MIRRs subsequently form highly ordered, polarized structures termed immunological synapses that provide for prolonged signaling. An understanding of the biochemical composition of rafts and synapses and the mechanisms by which these form should lend insight into the regulation of immune cell activation.
Muramyl dipeptide-Lys stimulates the function of human dendritic cells
Authors: Todate Akihito , Suda Takafumi , Kuwata Hiroshi , Chida Kingo , Nakamura Hirotoshi
Published: September 2022

Muramyl dipeptide (MDP)-Lys (L18), a synthetic MDP analogue derived from bacterial cell walls, has been reported to be a potent immunoadjuvant that enhances protective immunity against pathogens and tumors by stimulating immune-competent cells, such as monocytes and macrophages. However, it is not known whether MDP-Lys modulates the function of dendritic cells (DCs), which are the most potent antigen-presenting cells and play a crucial role in initiating T cell-mediated immunity. Therefore, we examined the effects of MDP-Lys on the expression of surface molecules, cytokine production, and antigen-presenting function of human DCs generated from peripheral blood cells in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. We found that MDP-Lys markedly up-regulated the expression of CD80, CD83, CD86, and CD40, but not human leukocyte antigen-DR, and stimulated the production of tumor necrosis factor-α, IL-6, IL-8, IL-10, and IL-12 (p40) by human DCs in a dose-dependent manner. Furthermore, MDP-Lys-treated DCs showed enhanced antigen-presenting function compared with untreated DCs, as assessed by an allogeneic mixed lymphocyte reaction. These results suggested that the immunoadjuvant activity of MDP-Lys in vivo is mediated, in part, by its stimulation of DC function.
A highly conserved c-fmsgene intronic element controls macrophage-specific and regulated expression
Authors: Himes S Roy , Tagoh Hiromi , Goonetilleke Nilukshi , Sasmono Tedjo , Oceandy Delvac , Clark Richard , Bonifer Constanze , Hume David A
Published: September 2022

The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in thefms gene.
p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line
Authors: Werz Oliver , Klemm Jenny , Rådmark Olof , Samuelsson Bengt
Published: September 2022

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.
Interferon-γ receptor 2 expression as the deciding factor in human T, B, and myeloid cell proliferation or death
Authors: Bernabei Paola , Coccia Eliana M , Rigamonti Laura , Bosticardo Marita , Forni Guido , Pestka Sidney , Krause Christopher D , Battistini Angela , Novelli Francesco
Published: September 2022

Theheterodimeric interferon (IFN)-γ receptor (IFN-γR) is formed of two chains. Here we show that the binding chain (IFN-γR1) was highly expressed on the membranes of T, B, and myeloid cells. Conversely, the transducing chain (IFN-γR2) was highly expressed on the surfaces of myeloid cells, moderately expressed on B cells, and poorly expressed on the surfaces of T cells. Differential cell membrane expression of IFN-γR2 determined the number of receptor complexes that transduced the IFN-γ signal and resulted in a different response to IFN-γ. After IFN-γ stimulation, high IFN-γR2 membrane expression induced rapid activation of signal transducer and activator of transcription-1 (STAT-1) and high levels of interferon regulatory factor-1 (IRF-1), which then triggered the apoptotic program. By contrast, low cell membrane expression resulted in slow activation of STAT-1, lower levels of IRF-1, and induction of proliferation. Because the forced expression of IFN-γR2 on T cells switched their response to IFN-γ from proliferative to apoptotic, we concluded that the surface expression of IFN-γR2 determines whether a cell stimulated by IFN-γ undergoes proliferation or apoptosis.
Identification of human CD93 as the phagocytic C1q receptor (C1qRp) by expression cloning
Authors: Steinberger Peter , Szekeres Andreas , Wille Stefan , Stöckl Johannes , Selenko Nicole , Prager Elisabeth , Staffler Günther , Madic Otto , Stockinger Hannes , Knapp Walter
Published: September 2022

CD93 is a ∼120 kDa O-sialoglycoprotein that within the hematopoietic system is selectively expressed on cells of the myeloid lineage. So far, its primary structure and function were unknown. We used retroviral-expression cloning to isolate the CD93 cDNA. Sequence analysis revealed that CD93 is identical to a protein on human phagocytes termed C1q receptor (C1qRp). C1qRp was shown previously to mediate enhancement of phagocytosis in monocytes and was suggested to be a receptor of C1q and two other structurally related molecules. When studying CD93 transductants and control cells, we found that cells expressing CD93 have enhanced capacity to bind C1q. Furthermore, we show that immature dendritic cells (DC) express CD93/C1qRp, and mature DC, known to have reduced capacity for antigen uptake and to have lost the ability to phagocytose, show weak-to-negative CD93/C1qRp expression.
Potential role for Duffy antigen chemokine-binding protein in angiogenesis and maintenance of homeostasis in response to stress
Authors: Du Jianguo , Luan Jing , Liu Hua , Daniel Thomas O , Peiper Stephen , Chen Theresa S , Yu Yingchun , Horton Linda W , Nanney Lillian B , Strieter Robert M , Richmond Ann
Published: September 2022

CXC chemokines, which induce angiogenesis, haveglutamine-leucine-arginine amino acid residues (ELR motif) in the aminoterminus and bind CXCR2 and the Duffy antigen chemokine-bindingprotein. Duffy, a seven transmembrane protein that binds CXC and CCchemokines, has not been shown to couple to trimeric G proteins or totransduce intracellular signals, although it is highly expressed on redblood cells, endothelial cells undergoing neovascularization, andneuronal cells. The binding of chemokines by Duffy could modulatechemokine responses positively or negatively. Positive regulation couldcome through the presentation of chemokine to functional receptors, andnegative regulation could come through Duffy competition withfunctional chemokine receptors for chemokine binding, thus serving as adecoy receptor. To determine whether Duffy has a role in angiogenesisand/or maintenance of homeostasis, we developed transgenic miceexpressing mDuffy under the control of the preproendothelinpromoter/enhancer (PPEP), which directs expression of the transgeneto the endothelium. Two PPEP-mDuffy-transgenic founders wereidentified, and expression of the transgene in the endothelium wasverified by Northern blot, RT-PCR, and immunostaining of tissues. Thephenotype of the mice carrying the transgene appeared normal by allvisual parameters. However, careful comparison of transgenic andnontransgenic mice revealed two phenotypic differences:mDuffy-transgenic mice exhibited a diminished angiogenic response to MIP-2 in the corneal micropocket assay, and mDuffy-transgenic miceexhibited enhanced hepatocellular toxicity and necrosis as comparedwith nontransgenic littermates in response to overdose of acetaminophen(APAP; 400 mg/kg body weight). Morover, APAP treatment was lethal in50% of the mDuffy-transgenic mice 24 h post challenge, and 100%of the nontransgenic littermates survived this treatment at the 24 h time point. Our data suggest that enhanced expression of mDuffy onendothelial cells can lead to impaired angiogenic response tochemokines and impaired maintenance of homeostasis in response to toxicstresses.