bluuebunny/crossref_metadata_2025 huggingface dataset:
Shape and shift changes related to the function of leukocyte integrins LFA-1 and Mac-1
Authors: Hogg Nancy , Leitinger Birgit
Published: September 2022

Integrin activity on leukocytes is controlled tightly, ensuring that ligand binding occurs only when leukocytes are in contact with their targets. For an integrinlike LFA-1, this ligand-binding activity comes about as a result of increased integrin clustering. Affinity regulation of integrins also plays a role, but the conformational changes giving rise to increased affinity appear to be secondary to clustering. Conformationally altered LFA-1 can be created artificially by deletion of the I domain, which is the key domain involved in ligand binding for many but not all integrins. Although I domain-deleted LFA-1 (ΔI-LFA-1) cannot bind ligand, it is able to signal constitutively into the cell. One measure of this signaling activity is the ability of ΔI-LFA-1 to activate β1 integrins on the same T lymphocyte. Leukocytes use LFA-1 to migrate across the endothelium. Active β1 integrins may be required subsequently to bind the matrix proteins encountered by leukocytes as they continue their voyage into the tissue interior.
Expression, tissue distribution, and cellular localization of the antiapoptotic TIP-B1 protein
Authors: Berleth Erica S , Masso-Welch Patricia A , Kazim Latif A , Ip Margot M , Mihich Enrico , Ehrke M Jane
Published: September 2022

TIP-B1 is a novel 27-kDa protein isolated from the cytosol of tumornecrosis factor (TNF)-stimulated cells. Cells preincubated with TIP-B1are protected from TNF-induced apoptosis. This study showed that, aswith normal fibroblasts and U937 histiocytic lymphoma, human MCF7mammary adenocarcinoma cells were protected from TNF in aconcentration-dependent manner by pretreatment with either TNF orpurified TIP-B1. Immunoblot and immunohistochemical analyses indicatedexpression of both TIP-B1 mRNA and protein in MCF7 cells and heart, kidney, brain, liver, ovary, uterus, thymus, spleen, lymph node, andmammary gland cells throughout their development. Expression of TIP-B1was heterogeneous, with staining of specific cell types within tissues. Based on the ability of TIP-B1 to protect both normal and tumor cellsfrom TNF-induced apoptosis and its broad tissue distribution, withexpression only in select cells within those tissues, a role for TIP-B1in the regulation of TNF-induced effects is strongly indicated.
Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils
Authors: Asaga Hiroaki , Nakashima Katsuhiko , Senshu Tatsuo , Ishigami Akihito , Yamada Michiyuki
Published: September 2022

Peptidylarginine deiminase, registered as PAD V in the DDBJ/GenBank/EMBL data banks, is expressed in HL-60 cells differentiated into granulocytes or monocytes. We analyzed PAD activities in density-fractionated human peripheral blood cell fractions. PAD activity with similar substrate specificity to that of PAD V was found in the eosinophil and neutrophil fractions, which showed single bands comigrating with authentic PAD V on immunoblotting with an anti-PAD V antibody. Both the biochemical and immunoblotting analyses showed marked enrichment of PAD V in the eosinophil fraction. Its immunoreactivity appeared to localize in eosinophilic granules at high density and in myeloperoxidase-negative cytoplasmic granules of neutrophils at low density, as determined by confocal laser-scanning microscopy. Possible roles of PAD V in myeloid differentiation and granulocyte function are discussed. In addition, we present evidence for the presence of PAD(s) that are antigenically different from PAD V in monocytes and lymphocytes.
Biliary glycoprotein (BGPa, CD66a, CEACAM1) mediates inhibitory signals
Authors: Chen Tie , Zimmermann Wolfgang , Parker James , Chen Ines , Maeda Akito , Bolland Silvia
Published: September 2022

Biliary glycoprotein (BGP, CD66a, CEACAM1) is a member of the carcinoembryonic antigen family (CEA, CD66), a group of transmembrane proteins belonging to the immunoglobulin superfamily. The structural features surrounding the tyrosine residues in the cytoplasmic domain of BGP share similarity with the consensus sequence of the immunoreceptor tyrosine-based inhibition motif (ITIM), the docking site for SHIP, SHP-1, and SHP-2 molecules. Using the well-characterized inhibitory receptor, FcγRIIB, we constructed a FcγRIIB-BGPa chimeric molecule that contained the extracellular and transmembrane domain of FcγRIIB and the cytoplasmic tail of BGPa and expressed it in DT40 B cells. Our results showed that FcγRIIB-BGPa, just like the unmodified FcγRIIB molecule, inhibited calcium influx in activated DT40 B cells. Substitution of tyrosine with phenylalanine (Y459F) in FcγRIIB-BGPa completely abrogated its ability to inhibit calcium influx, indicating that the motif surrounding Y459 is ITIM. The presence of ITIM was also supported by showing that the FcγRIIB-BGPa-mediated inhibitory effect was reduced in SHP-1and SHP-2 mutant DT40 B cells and further diminished in a SHP-1/-2 double-deficient mutant line. The results suggest that SHP-1 and SHP-2 are required for the FcγRIIB-BGPa-mediated inhibitory signals.
Regulation of hematopoiesis by gap junction-mediated intercellular communication
Authors: Montecino-Rodriguez Encarnacion , Dorshkind Kenneth
Published: September 2022

Gap junctions are intercellular channels formed by individual structural units known as connexins (Cx) that allow the intercellular exchange of small molecules between cells. The presence of Cx protein in bone marrow and thymic stromal cells and the demonstration that these cells are functionally coupled have led to the hypothesis that groups of stromal cells in the bone marrow and thymus form a functional syncytium through which their hematopoietic support capacity is coordinated. The validity of this hypothesis was recently tested in a newly developed strain of mice in which the gene encoding Cx43, the principal Cx expressed in hematopoietic tissues, was disrupted. Studies of myelopoiesis and lymphopoiesis in these Cx43-deficient mice revealed that expression of Cx43 in the bone marrow and thymus is critically important during periods of active hematopoiesis, such as during embryogenesis and after recovery from cytoablative treatments. The clinical implications of these observations, as well as issues that remain to be addressed to understand the mechanism(s) by which gap junctions regulate hematopoiesis, are addressed.
Activation of human neutrophils in vitro and dieldrin-induced neutrophilic inflammation in vivo
Authors: Pelletier Martin , Roberge Charles J , Gauthier Marc , Vandal Karen , Tessier Philippe A , Girard Denis
Published: September 2022

Many chemicals of environmental concern are known to alter the immune system and are considered toxic molecules because they affect immune cell functions. Inflammation related to environmental chemical exposure, however, is poorly documented, except that from air pollutants. In this study, we found that the organochlorine insecticide dieldrin could not alter the ability of human neutrophils to phagocytose opsonized sheep red blood cells at nonnecrotic concentrations (0.1, 1, 10, and 50 μM). However, dieldrin was found to increase human neutrophil superoxide production, RNA synthesis, and proinflammatory cytokine interleukin-8 production. The normal apoptotic rate of neutrophils evaluated by both cytology and flow cytometry (CD-16 staining) was not altered by dieldrin treatments, and this was correlated with its inability to inhibit spreading of neutrophils onto glass. Using the murine air pouch model, we found that dieldrin induces a neutrophilic inflammation. Taken together, these results demonstrated that dieldrin is a proinflammatory contaminant. To our knowledge, this is the first report establishing that dieldrin is a contaminant exhibiting proinflammatory properties. In addition, it is the first time that the murine air pouch model has been successfully used to confirm that a chemical of environmental concern can induce an inflammatory response in vivo.
Proinflammatory activity of glycosylphosphatidylinositol anchors derived fromTrypanosoma cruzi: structural and functional analyses
Authors: Almeida Igor C , Gazzinelli Ricardo T
Published: September 2022

A strong activation of macrophages is observed during acute infection with Trypanosoma cruzi. Little is known, however, about the parasite molecules that are responsible for this early activation of innate immunity. Recent studies have shown the stimulatory activity of protozoan-derived glycosylphosphatidylinositol (GPI) anchors on cultured macrophages. In this review, we provide a detailed analysis of the correlation between structure and proinflammatory activity byT. cruzi-derived GPI anchors. We also cover the studies that have identified the Toll-like receptor 2 as a functional GPI receptor and have partially characterized signaling pathways triggered by T. cruzi-derived GPI anchors, which lead to the synthesis of proinflammatory cytokines in macrophages. Finally, we discuss the implications of these findings in resistance and pathogenesis during the infection with T. cruzi.
Leukocytes infiltrating the pancreatic islets of nonobese diabetic mice are transformed into inactive exiles by combinational anti-cell adhesion therapy
Authors: Kommajosyula Sharada , Reddy Shiva , Nitschke Kristina , Kanwar Jagat R , Karanam Muralidhar , Krissansen Geoffrey W
Published: September 2022

Leukocytes infiltrate the pancreatic islets of nonobese diabetic mice, causing β-cell destruction and autoimmune Type I diabetes. Here, wecompletely blocked adoptive transfer of diabetes and reducedspontaneous disease incidence from 71% to 17% by simultaneouslyadministering a combination of antibodies directed against α4, β2,and β7 integrins and their ligands VCAM-1, MAdCAM-1, and ICAM-1 for52 and 28 days, respectively. CD4 and CD8 T cells and macrophages wereexcluded from islets and remained entrapped in a peri-islet location asinactive exiles, no longer expressing normal levels of interferon-γ,interleukin-4, and iNOS. Only IL-10 expression was retained, whichcould aid immunosuppression. Infiltrating leukocytes retained aperi-islet location, even 215 days following suspension of antibodytreatment, potentially forming a barrier to the entry of active, autoantigen-reactive T cells. Combination treatment was effectiveagainst spontaneous disease when administered from 7 days of age butineffective when initiated late in the prediabetic period (day 40 or70). Nevertheless, anti-α4 subunit mAb monotherapy alone was veryeffective, reducing insulitis to levels similar to those obtained withcombinational antibody treatment, suggesting that α4 integrins aremajor receptors contributing to leukocyte infiltration. Treatment withanti-α4 integrin antibody retained some therapeutic benefit whenadministered from days 7, 40, or 70 of age. The results haveimplications for the treatment of diabetes and provide a unique insightinto the fate of disease-forming leukocytes following anti-CAMtherapy.
Membrane trafficking of CD1c on activated T cells
Authors: Salamone María del C , Mendiguren Ana Karina , Salamone Gabriela V , Fainboim Leonardo
Published: September 2022

We investigated the regulation of and the intracellular traffickinginvolved in the membrane expression of CD1c antigen on activated matureT cells. Membrane expression of this glycoprotein was highly regulatedand dependent on the activation state of the cells. The presence of the CD1c antigen on activated peripheral blood mononuclear cells (PBMCs)was confirmed by flow cytometry, reverse transcriptase-PCR (RT-PCR),and immunoperoxidase staining. The RT-PCR analysis of the α3- and3′-untranslated regions of CD1C showed thatphytohemagglutinin (PHA) activation induced expression of transcriptsthat encode the three isoforms (soluble, membrane, andcytoplasmic/soluble). Immunocytochemical studies showed a specificassociation of CD1c with the cell membrane and a cytoplasmic, perinuclear distribution. Although flow-cytometric staining confirmedthe intracellular presence of CD1c, membrane expression on PHA blastcells was not detected. We found that membrane detection of CD1cantigen was temperature dependent. Cell surface binding of theanti-CD1c monoclonal antibody (mAb) was consistently negative at 4 and37°C but was detected at room temperature (18–22°C). Atphysiologic temperatures, activated PBMCs showed intracellularaccumulation of the anti-CD1c mAbs, indicating that CD1c cycled betweencell surface and intracellular compartments. The CD1c exocytosispathway was sensitive to Brefeldin A, cytochalasin B, andchloroquine.
Phosphatidylinositol 3-kinase and ERK are required for NF-κB activation but not for phagocytosis
Authors: García-García Erick , Sánchez-Mejorada Gabriela , Rosales Carlos
Published: September 2022

The molecular events that transduce signals from Fc receptors to the various cellular responses are still poorly defined. We have investigated the role of phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase (ERK) in gene activation and phagocytosis in monocytes. In the THP-1 monocytic cell line, cross-linking of Fc receptors by immune complexes results in activation of the transcription factor NF-κB, via activation of ERK. Activation of both ERK and NF-κB was blocked by wortmannin and LY294002, specific inhibitors of PI 3-K. Wortmannin also inhibited the Fc receptor-mediated increase in the cytosolic calcium concentration, but it did not block immunoglobulin G (IgG)-mediated phagocytosis. In addition, the ERK inhibitor PD98059 did not block phagocytosis of IgG-coated erythrocytes. Both the increase in the cytosolic calcium concentration and phagocytosis depend on an active actin cytoskeleton, as indicated by the total lack of both responses after treatment with cytochalasin B. In contrast, cytochalasin B did not affect Fc receptor-mediated activation of NF-κB. These results identify PI 3-K and ERK as important signaling molecules in the Fc receptor signal transduction pathway of monocytes, which leads to the nucleus for gene activation. These results also suggest that, in contrast to other cell types, unstimulated monocytes do not require PI 3-K and ERK for phagocytosis.
Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals
Authors: Desaulniers Philippe , Fernandes Maria , Gilbert Caroline , Bourgoin Sylvain G , Naccache Paul H
Published: September 2022

The inflammatory response in acute gouty arthritis is in large part a result of the interaction between neutrophils and monosodium urate (MSU) crystals. The tyrosine kinase Syk, which has been largely associated with the phagocytic response by Fc receptors and with spreading mediated by integrins, has been identified as one of the major proteins tyrosine-phosphorylated in human neutrophils upon stimulation by MSU crystals and is known to be mediated in part by the Fc receptor, CD16. This has led to the present examination of the implication of Syk in the activation pathways used by MSU crystals. The tyrosine-phosphorylation patterns induced by MSU crystals and by the ligation of CD16 were inhibited by piceatannol, which, conversely, only slightly delayed but did not diminish the peak of tyrosine phosphorylation induced by cross-linking CD32 or by the addition of fMet-Leu-Phe. Moreover, piceatannol inhibited the activity of Syk as monitored by in vitro kinase assays, by its in situ tyrosine phosphorylation, and by its activity toward exogenous substrates after stimulation by MSU crystals. We also measured the impact of piceatannol on the mobilization of calcium, the production of superoxide anions, and the activity of PLD stimulated by MSU crystals. We noted a distinct inhibition of all these responses by piceatannol. Finally, the morphological changes observed in neutrophils as characteristic of MSU crystal internalization were diminished significantly by piceatannol. The results obtained show that Syk plays a critical and central role in the signal-transduction pathways called upon by MSU crystals subsequent to their interaction with human neutrophils.
The role of CD44 during CD40 ligand-induced dendritic cell clustering and maturation
Authors: Termeer Christian , Johannsen Henning , Braun Thorsten , Renkl Andreas , Ahrens Thomas , Denfeld Ralph W , Lappin Mike B , Weiss Johannes M , Simon Jan C
Published: September 2022

The interaction between CD40 on dendritic cells (DC) and its ligand CD154 has been recognized to be an important feature in the maturation of DC. Here, we were interested in the role of CD44 a surface receptor shown to mediate cell-cell adhesion and binding to Hyaluronic acid (HA). Western blot analysis of human DC stimulated for 3–12 h with CD154 revealed the rapid induction of the 85 kDa standard form of CD44 and an increased HA-binding affinity. Time-lapse video-imaging microscopy of human DC co-cultured on CD154-transfected murine fibroblasts showed that the CD44 up-regulation coincided with the rapid induction of homotypic DC clustering, which did not occur on empty vector-transfected fibroblasts. In this system, addition of anti-CD44s mAbs abrogated DC-cluster formation, thereby inhibiting further maturation, as shown by a reduced TNF-α production and inhibition of CD154-induced MHC class II up-regulation. However, co-incubation with HA-degrading enzymes induced no changes in the CD154-mediated DC clustering and maturation.
Treatment of multiple sclerosis patients with interferon-β primes monocyte-derived macrophages for apoptotic cell death
Authors: Van Weyenbergh Johan , Wietzerbin Juana , Rouillard Dany , Barral-Netto Manoel , Liblau Roland
Published: September 2022

Although interferon (IFN)-β has shown a significant clinical benefit in multiple sclerosis (MS), its mechanism of action remains unclear. We found that IFN-β treatment of patients with MS resulted in a significant increase in apoptotic cell death (measured by annexin V staining and nuclear fragmentation) of monocyte-derived macrophages, as compared with cells derived from patients before treatment. Stimulation of the cells with IFN-β in vitro resulted in an even further increase of annexin V binding, as well as increased Fas (CD 95, APO-1) expression. However, no increased Fas expression, apoptotic monocytes, or monocytopenia were observed upon in vivo treatment. This indicates that IFN-β does not deliver a death signal to monocytes but rather primes for subsequent macrophage apoptosis upon activation or differentiation.
Regulation of the very late antigen-4-mediated adhesive activity of normal and nonreleaser basophils: roles for Src, Syk, and phosphatidylinositol 3-kinase
Authors: Andrews Ronald P , Kepley Christopher L , Youssef Lama , Wilson Bridget S , Oliver Janet M
Published: September 2022

Normal human basophils express the integrin, VLA-4, and cross-linking their high-affinity IgE receptor, FcɛRI, increases their VLA-4-dependent adhesion to VCAM-1-transfected Chinese hamster ovary (CHO) cells. Here we show that the FcɛRI-mediated up-regulation of normal basophil VLA-4 adhesion is abolished by the Src inhibitor, PP1, the Syk inhibitor, ER-27319, and the phosphatidylinositol 3-kinase inhibitor, wortmannin. PP1, but not ER-27319 or wortmannin, also reduces basal adhesion and adhesion stimulated by chemotactic peptide, by Ca++ ionophores, and by phorbol myristate acetate (PMA). Nonreleaser basophils (the consistently Syk-deficient, variably Lyn-deficient, severely degranulation-impaired cells found in about 10% of donors) share the PP1 phenotype of lowered basal adhesion, no FcɛRI-mediated adhesion up-regulation, and reduced adhesive responses to chemoattractant ionophores and PMA. These results implicate Src kinases in the control of basal VLA-4 activity and place Syk and phosphatidylinositol 3-kinase in the pathway linking FcɛRI cross-linking to VLA-4 up-regulation. Both Src and Syk-regulated components of adhesion may be impaired in nonreleaser basophils.
Modes of action of Freund's adjuvants in experimental models of autoimmune diseases
Authors: Billiau Alfons , Matthys Patrick
Published: September 2022

Freund's adjuvants are irreplaceable components of induction protocols of many experimental animal models of autoimmune disease. Apart from the early studies done in the 1950s and 1960s, no further direct investigation on the mode of action of these adjuvants has been undertaken. It is generally assumed that incomplete (IFA) and complete Freund's adjuvant (CFA) act by prolonging the lifetime of injected autoantigen, by stimulating its effective delivery to the immune system and by providing a complex set of signals to the innate compartment of the immune system, resulting in altered leukocyte proliferation and differentiation. Here, we review evidence collected from various types of studies that provide more insight in the specific alterations of the immune response caused by IFA and CFA. Early events include rapid uptake of adjuvant components by dendritic cells, enhanced phagocytosis, secretion of cytokines by mononuclear phagocytes, and transient activation and proliferation of CD4+ lymphocytes. The mycobacterial components within CFA signal T lymphocytes to assume a Th1 profile so that strong delayed-type hypersensitivity against autoantigens develops. In the absence of mycobacteria, T-lymphocyte differentiation tends to assume a Th2 profile with strong antibody production only. The mycobacterial component also accounts for a morphologic and functional remodeling of the haemopoietic system that develops over a period of several weeks and that is characterized by a drastic expansion of Mac-1+ immature myeloid cells. These cells have been found to be associated with enhanced disease in some models but with reduced disease in others. Thus, in experimental autoimmune diseases, CFA-mediated activation of the innate immune compartment is important not only by regulating the early induction phase but also by providing a surplus of effector and regulator cells in the late phase.
Induction of syngeneic graft-versus-host disease in LPS hyporesponsive C3H/HeJ mice
Authors: Flanagan Diana Lowery , Gross Rachel , Jennings C Darrell , Caywood Betty E , Goes Sarah , Kaplan Alan M , Bryson J Scott
Published: September 2022

Syngeneic GVHD (SGVHD) develops following syngeneic bone marrow transplantation and treatment with cyclosporine A. Previous studies have demonstrated a role for IL-12, IFN-γ, and TNF-α in the development of murine SGVHD. Macrophages can be activated to secrete IL-12 and TNF-α via a T-cell-dependent or T-cell-independent pathway (LPS or bacterial products). Studies were designed to determine if LPS participated in the development of SGVHD in C3H/HeN (LPS-responsive) and C3H/HeJ (LPS-hyporesponsive) mice. C3H/HeJ and C3H/HeN mice had similar levels of disease induction and pathology. Following induction of SGVHD, treatment of C3H/HeN, but not C3H/HeJ, mice with a sublethal dose of LPS resulted in mortality. However, neutralization of IL-12 abrogated the development of disease in C3H/HeJ mice, demonstrating that activated macrophages and their products participated in the development of SGVHD in these animals. These data suggested that LPS responsiveness was not a predisposing factor for SGVHD induction.
Senescence in innate immune responses: reduced neutrophil phagocytic capacity and CD16 expression in elderly humans
Authors: Butcher S K , Chahal H , Nayak L , Sinclair A , Henriquez N V , Sapey E , O'Mahony D , Lord J M
Published: September 2022

Elderly humans are more susceptible to bacterial infections because of declining immune status. We have investigated the effect of aging on neutrophil bactericidal responses, comparing neutrophil function in healthy, young (23–35 years) and elderly (>65 years) volunteers. Superoxide generation in response to fMLP was slightly increased in neutrophils from elderly donors, and serum from the elderly was able to opsonize E. coli efficiently. In contrast, phagocytic index was significantly lower in neutrophils from the elderly, compared with young donors (P<0.005). CD11a and CD11b expression was not affected by age, but CD16 was significantly reduced in neutrophils from elderly donors (P<0.0001). CD16 expression and phagocytic index were measured in the same neutrophils using FITC-labeled E. coli, PE-conjugated anti-CD16 antibody, and CD16 expression correlated with phagocytic index (r=0.83;P<0.05). In elderly patients with bacterial infection, CD16 expression remained low. We propose that reduced neutrophil CD16 expression and phagocytosis contribute to human immunesenescence.
Enhancing effects of anti-CD40 treatment on the immune response of SCID-bovine mice toTrypanosoma congolenseinfection
Authors: Haas Karen M , Taylor Katherine A , MacHugh Niall D , Kreeger John M , Estes D Mark
Published: September 2022

African trypansosomes are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. Previous studies have demonstrated that certain breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Because of its extracellular location, the humoral branch of the immune system dominates the response againstTrypanosoma congolense. In the following study, we describe the humoral immune response generated against T. congolense in SCID mice reconstituted with a bovine immune system (SCID-bo). SCID-bo mice infected with T. congolense were treated with an agonistic anti-CD40 antibody and monitored for the development of parasitemia and survival. Anti-CD40 antibody administration resulted in enhanced survival compared with mice receiving the isotype control. In addition, we demonstrate that the majority of bovine IgM+ B cells in SCID-bo mice expresses CD5, consistent with a neonatal phenotype. It is interesting that the percentage of bovine CD5+ B cells in the peripheral blood of infected SCID-bo mice was increased following anti-CD40 treatment. Immunohistochemical staining also indicated increased numbers of Ig+ cells in the spleens of anti-CD40-treated mice. Consistent with previous studies demonstrating high IL-10 production during high parasitemia levels in mice and cattle, abundant IL-10 mRNA message was detected in the spleens and peripheral blood of T. congolense-infected SCID-bo mice during periods of high parasitemia. In addition, although detected in plasma when parasites were absent or low in number, bovine antibody was undetectable during high parasitemia. However, Berenil treatment allowed for the detection of VSG-specific IgG 14 days postinfection in T. congolense-infected SCID-bo mice. Overall, the data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection.
Allogeneic reconstitution after nonmyeloablative conditioning: mitigation of graft-versus-host and host-versus-graft reactivity by anti-CD44v6
Authors: Christ Oliver , Günthert Ursula , Schmidt Dirk-Steffen , Zöller Margot
Published: September 2022

T-cell maturation is accelerated in transgenic mice expressingrat CD44v4-v7 on T cells, the effect being blocked by anti-CD44v6. Thisfinding suggested functional activity of CD44v6 in thymocytedevelopment. We tested the hypothesis by antibody blocking and usingmice with targeted deletion of CD44v6/v7 exon products(CD44v6/v7−/−). When lethally irradiatedCD44v6/v7-competent (CD44v6/v7+/+) mice were reconstitutedsyngeneically, higher numbers of CD44v6/v7−/− thanCD44v6/v7+/+ BMC were required for survival, the period of reconstitution was prolonged, and regain of immunocompetence wasdelayed. Similar findings were observed in lethally irradiated, anti-CD44v6-treated syngeneic CD44v6/v7+/+ hosts. Thus, CD44v6/v7 supports maturation and expansion of hematopoietic progenitorcells. Surprisingly, reconstitution with CD44v6/v7−/− BMCor anti-CD44v6 treatment of the nonlethally irradiated allogeneicCD44v6/v7+/+ host had only a minor impact on survivalrates. When nonlethally irradiated CD44v6/v7−/− hostsreceived an allogeneic graft, survival rates were improved. Thesephenomena have been a result of reduced GvH reactivities when the donorwas CD44v6/v7−/− and reduced HvG reactivities in the CD44v6/v7−/− host. Thus, although a deficit or blockadeof CD44v6/v7 has a negative impact on hematopoietic reconstitution, atransient blockade will be of benefit for the allogeneicallyreconstituted host because of a strong reduction in GvH and HvGreactivities.
(−)Epigallocatechin-3-gallate inhibits leukocyte elastase: potential of the phyto-factor in hindering inflammation, emphysema, and invasion
Authors: Sartor Luigi , Pezzato Elga , Garbisa Spiridione
Published: September 2022

Flavanol (−)epigallocatechin-3-gallate is shown to be a potent naturalinhibitor of leukocyte elastase that may be used to reduceelastase-mediated progression to emphysema and tumor invasion. Thisphyto-factor, abundant in green tea, exerts a dose-dependent, noncompetitive inhibition of leukocyte elastase at a noncytotoxicconcentration and is effective in neutrophil culture. This inhibitionshows an IC50 of 0.4 μM, 30 times higher than theα1-protease inhibitor but lower than other known natural andsynthetic elastase inhibitors. The flavanol inhibits leukocyte elastaseat concentrations of 50, 150, and 2500 times lower than that effectiveon gelatinases (MMP-2 and MMP-9), thrombin, and cathepsin G, respectively, and also blocks elastase-mediated activation of MMP-9.
Myosin-V colocalizes with MHC class II in blood mononuclear cells and is up-regulated by T-lymphocyte activation
Authors: Bizario João C S , Castro Fabíola A , Sousa Josane F , Fernandes Rafael N , Damião Alexandre D , Oliveira Márika K , Palma Patrícia V B , Larson Roy E , Voltarelli Júlio C , Espreafico Enilza M
Published: September 2022

Myosin-V is involved in organelle and vesicle trafficking inSaccharomyces cerevisiae and in other eukaryotic cells from yeast to human. In the present study, we determined by FACS that the major subpopulations of the peripheral blood mononuclear cells express myosin-V with similar fluorescence intensity. Confocal microscopy showed intense labeling for myosin-V at the centrosomal region and a punctate staining throughout the cytoplasm, frequently associated with the central microtubule arrays and the actin-rich cortex. Some degree of overlap with an endolysosomal marker and dynein light-chain 8 k was found at the cell center. Striking colocalization was observed with the major histocompatibility complex (MHC) class II molecules near the cell surface. Treatment with phytohemagglutinin, which induces T-lymphocyte activation, associated with MHC class II expression, increased the levels of myosin-V protein and mRNA for the three members of class V myosins. These data suggest that class V myosins might be involved in relevant functions in the immune response.
Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor
Authors: Bülow E , Nauseef W M , Goedken M , McCormick S , Calafat J , Gullberg U , Olsson I
Published: September 2022

During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein α1-microglobulin (α1-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to α1-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of α1-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.
Intercellular cell adhesion molecule-1 and selectin ligands in acute cardiac allograft rejection: a study on gene-deficient mouse models
Authors: Lácha Jiří , Bushell Andrew , Smetana Karel , Rossmann Pavel , Přibylová Petra , Wood Kathryn , Malý Petr
Published: September 2022

Cell adhesion molecules and their ligands are essential for regulating lymphocyte recirculation and leucocyte emigration into an inflamed or injured tissue. Vascular endothelial selectins as mediators of leucocyte rolling and intercellular cell adhesion molecule-1 (ICAM-1) have been found to be up-regulated on activated endothelium during acute allograft rejection. This study was designed to investigate whether ICAM-1 or selectin-ligand deficiency, or a combination of both, affected graft survival during acute cardiac allograft rejection. To this goal, we performed cardiac transplantation using mice deficient in genes for ICAM-1 or α(1,3)fucosyltransferase Fuc-TVII, representing a model for general absence of selectin-ligand expression, and a newly developed strain with a double mutation in Fuc-TVII and ICAM-1 alleles. Transplantation of a heart from ICAM-1 −/− or Fuc-TVII/ICAM-1 double-mutated mice into allogeneic recipients resulted in limited (2–2.5 days) but nevertheless significant prolongation of the graft survival (P<0.01 and P<0.01 in log-rank test) compared with the survival of unmodified hearts. When ICAM-1 −/− hearts were transplanted into Fuc-TVII −/− recipients, the median survival time was prolonged by 8 days (P<0.01). These data indicate that endothelial ICAM-1 is involved in adhesion events during acute cardiac allograft rejection but reveal that the loss of one type, selectin/leucocyte ligand or selectin/endothelial ligand interaction, does not markedly affect graft survival, thereby suggesting a role for other compensatory adhesion molecule/ligand interactions.
Thioglycollate peritonitis in mice lacking C5, 5-lipoxygenase, or p47phox: complement, leukotrienes, and reactive oxidants in acute inflammation
Authors: Segal Brahm H , Kuhns Douglas B , Ding Li , Gallin John I , Holland Steven M
Published: September 2022

Leukotriene B4 (LTB4) is an easily diffusible proinflammatory chemotactic factor that has been posited to prime the initial inflammatory response for the action of other mediators, including C5a. 5-Lipoxygenase-deficient (5LX−/−) and C5-deficient mice only generated about 50% as much peritoneal leukocytosis as wild-type mice following intraperitoneal (IP) challenge with the sterile irritant, thioglycollate (P<0.005). Pretreatment of C5− mice with the specific 5-lipoxygenase inhibitor, zileuton, reduced peritoneal leukocytosis to almost unstimulated levels, suggesting that LTB4 can act independently of C5a. Previously, LTB4 and C5a have been shown in vitro to be inactivated by metabolites of superoxide. In the current study, we examined the fate of LTB4 in the p47phox−/− mouse model of chronic granulomatous disease (CGD) in which the phagocyte NADPH oxidase is unable to produce superoxide. p47phox−/− mice generated more thioglycollate-elicited peritoneal leukocytosis than wild-type mice. Pretreatment with zileuton caused a 76% reduction in peritoneal leukocytosis in p47phox−/− mice (P<0.005) and a 54% reduction in wild-type mice (P<0.05), whereas pretreatment with dexamethasone or toradol (a cyclooxygenase inhibitor) had no effect. Following IP LTB4 (1 μg/mouse), total recovered peritoneal LTB4 was similar between p47phox−/− and wild-type mice at 10 and 30 min, but was approximately fivefold greater in p47phox−/− mice at 180 min. These data suggest that LTB4 and C5a have separate but overlapping roles in thioglycollate-elicited peritonitis, and at least the leukotriene component is, in turn, regulated by reactive oxidants.
Functional characterization of podia formation in normal and malignant hematopoietic cells
Authors: Fruehauf S , Srbic K , Seggewiss R , Topaly J , Ho A D
Published: September 2022

Hematopoietic cells extend multiple podia of yet unknown function. Our morphological studies using scanning electron microscopy and functional studies using time-lapse video microscopy suggest that podia formed by CD34+ hematopoietic stem cells (HSC) on the bone marrow stroma component fibronectin are characteristic of lamellipodia at the leading edge and uropodia at the trailing edge, cytoskeletal structures that have previously been shown to be responsible for cell locomotion of lymphocytes. In the leukemic cells studied here, stroma-derived factor-1α (SDF-1α) led to a significant eightfold increase in transmigration (BCR-ABL-positive BV173 leukemia cell line;P<0.05) and podia formation in all BCR-ABL-positive leukemic cell lines studied (BV173, K562, 32Dp210) and in two of three BCR-ABL-negative lines (HL60, 32D, not KG1a). We could show that SDF-1α exposure led to a down-regulation of the gene expression of the chemokine receptors CCR4, CXCR4, and CXCR5, which are associated with cell motility and podia formation, indicating a negative feedback control. In BCR-ABL-positive leukemic cells, the effects of SDF-1α on podia formation and cell migration were independent of BCR-ABL-tyrosine kinase activity. Our data are compatible with the hypothesis that formation of specific podia by hematopoietic cells is associated with egression of these cells from the bone marrow.
Hypertonicity suppresses ionophore-induced product formation and translocation of 5-lipoxygenase in human leukocytes
Authors: Bürkert Eva , Rådmark Olof , Samuelsson Bengt , Steinhilber Dieter , Werz Oliver
Published: September 2022

5-Lipoxygenase (5-LO) initiates the biosynthesis of proinflammatory leukotrienes from arachidonic acid (AA). Here, we demonstrate that hypertonicity suppresses ionophore-induced 5-LO product formation reversibly in isolated human polymorphonuclear leukocytes (PMNL) and in Mono Mac 6 cells. Hypertonicity blocked the liberation of AA and abrogated translocation of 5-LO to the nuclear membrane. Accordingly, in the presence of exogenous AA, 5-LO product formation was less affected. The effects of hypertonicity were a result of cell shrinkage and not cytosolic hyperosmolarity. Hypertonicity did not inhibit the rapid increase in intracellular Ca2+ induced by ionophores but prevented the ionophore-induced activation of p38 MAPK-regulated MAPKAP kinases, which can phosphorylate and activate 5-LO (and cPLA2). In summary, we show that hypertonicity blocks agonist-induced release of AA, 5-LO product formation, and translocation and in parallel, prevents activation of p38 MAPK and downstream 5-LO kinases in leukocytes.
Inhibitory actions of glucosamine, a therapeutic agent for osteoarthritis, on the functions of neutrophils
Authors: Hua Jian , Sakamoto Koji , Nagaoka Isao
Published: September 2022

Glucosamine, an amino monosaccharide naturally occurring in the connective and cartilage tissues, contributes to maintaining the strength, flexibility, and elasticity of these tissues. In recent years, glucosamine has been used widely to treat osteoarthritis in humans and animal models. Neutrophils, which usually function as the primary defenders in bacterial infections, are also implicated in the destructive, inflammatory responses in arthritis. In this study, we have evaluated the effects of glucosamine on neutrophil functions using human peripheral blood neutrophils. Glucosamine (0.01–1 mM) dose-dependently suppressed the superoxide anion generation induced by formyl-Met-Leu-Phe (fMLP) or complement-opsonized zymosan and inhibited the phagocytosis of complement-opsonized zymosan or IgG-opsonized latex particles. Furthermore, glucosamine inhibited the release of granule enzyme lysozyme from phagocytosing neutrophils and suppressed neutrophil chemotaxis toward zymosan-activated serum. In addition, glucosamine inhibited fMLP-induced up-regulation of CD11b significantly, polymerization of actin, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). In contrast, N-acetyl-glucosamine, an analogue of glucosamine, did not affect these neutrophil functions (superoxide generation, phagocytosis, granule enzyme release, chemotaxis, CD11b expression, actin polymerization, and p38 MAPK phosphorylation) at the concentrations examined (1–10 mM). Together these observations likely suggest that glucosamine suppresses the neutrophil functions, thereby possibly exhibiting anti-inflammatory actions in arthritis.
ARNO but not cytohesin-1 translocation is phosphatidylinositol 3-kinase-dependent in HL-60 cells
Authors: Bourgoin Sylvain G , Houle Martin G , Singh Indrapal N , Harbour Danielle , Gagnon Steve , Morris Andrew J , Brindley David N
Published: September 2022

Cytohesin-1 and ARNO are guanine nucleotide-exchange factors (GEFs) for ADP-ribosylation factor (Arf). Here, we show that ARNO is expressed in HL-60 cells and established that granulocytic differentiation induced with Me2SO stimulated cytohesin-1 but not ARNO expression. Cytohesin-1 levels in HL-60 granulocytes were similar to those in human neutrophils. Me2SO-differentiated HL-60 cells expressed ARNO and cytohesin-1 isoforms with a diglycine and a triglycine motif in their PH domains, respectively. In vitro, ARNO diglycine and cytohesin-1 triglycine enhanced phospholipase D1 (PLD1) activation by Arf1 with near-maximal effects at 250 nM. These effects were marked particularly at low Mg2+ concentrations. PLD activation was well-correlated with GTP binding to Arf1, and cytohesin-1 was always more potent than ARNO in the PLD- and GTP-binding assays. Increasing Mg2+ concentrations reduced PLD and Arf1 activation by Arf-GEFs. fMetLeuPhe and phorbol 12-myristate 13-acetate stimulated ARNO and cytohesin-1 as well as Arf1 translocation to HL-60 cell membranes. fMetLeuPhe-mediated ARNO recruitment, but not cytohesin-1 and Arf1 translocation, was blocked by phosphatidylinositol 3-kinase inhibitors. The combined results demonstrate that cytohesin-1 triglycine participates in a major phosphatidylinositol 3-kinase-independent pathway linking cell-surface receptors to Arf1 activation and translocation in human granulocytes.
Oxidant-mediated phosphatidylserine exposure and macrophage uptake of activated neutrophils: possible impairment in chronic granulomatous disease
Authors: Hampton Mark B , Vissers Margret C M , Keenan Jacqueline I , Winterbourn Christine C
Published: September 2022

The removal of neutrophils from inflammatory sites is essential for the resolution of inflammation. Surface changes, including phosphatidylserine exposure, label neutrophils for phagocytosis by macrophages. Here, we demonstrate that externalization of phosphatidylserine and uptake by monocyte-derived macrophages occurred in human neutrophils ingesting Staphylococcus aureus. Both processes were dependent on oxidant production from the neutrophil NADPH oxidase. There was no requirement for myeloperoxidase, and H2O2 was identified as the most likely trigger for PS exposure. We hypothesize that clearance of stimulated neutrophils would be delayed in chronic granulomatous disease (CGD) neutrophils, which lack a functional NADPH oxidase. To explore this possibility, heat-killed S. aureus were injected into the peritoneum of CGD and normal mice. Elevated neutrophil numbers were observed in the inflammatory exudate of the CGD animals, consistent with impaired recognition and clearance.
Activation-induced expression of MICA on T lymphocytes involves engagement of CD3 and CD28
Authors: Molinero Luciana L , Fuertes Mercedes B , Rabinovich Gabriel A , Fainboim Leonardo , Zwirner Norberto W
Published: September 2022

MICA is an HLA-related cell stress-regulated antigen recognized by cytotoxic cells expressing the NKG2D molecule. Although resting lymphocytes do not express MICA, it can be induced on PHA-activated T cells. Here, we demonstrate by Western blot that MICA is induced on allogeneic-activated CD4+ and CD8+ T lymphocytes. Blocking activation with anti-HLA class I, anti-HLA-DR, or anti-CD86 mAb affected the expression of MICA slightly. When T cells were stimulated with anti-CD3 or anti-CD28 mAb plus PMA, a sustained up-regulation of MICA was observed by Western blot, RT-PCR, and flow cytometry. The expression of MICA reached a plateau at day 4 after CD3 engagement and at day 3 after anti-CD28/PMA stimulation. Conversely, the proliferative response reached a peak at day 4. Hence, CD3 or CD28 engagement induces MICA expression on T lymphocytes. This activation-induced expression might participate in NKG2D-mediated cytotoxicity toward activated T cells to maintain homeostasis during an ongoing immune response.
Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro
Authors: Allport Jennifer R , Lim Yaw-Chyn , Shipley J Michael , Senior Robert M , Shapiro Steven D , Matsuyoshi Norihisa , Vestweber Dietmar , Luscinskas Francis W
Published: September 2022

Recent evidence has suggested a role for neutrophil proteases during certain inflammatory responses. We demonstrated previously that neutrophil proteases can degrade components of the adherens junctions during neutrophil-endothelial adhesion. We tested the hypothesis that degradation of VE-cadherin at lateral junctions by elastase or MMP-9 facilitates neutrophil transendothelial migration. Neutrophils from MMP-9 or elastase null mice and strain-matched control mice expressed high levels of LFA-1, Mac-1, and L-selectin on their cell surface. Under flow conditions, wild-type and deficient neutrophils rolled, arrested, and transmigrated activated murine endothelium. There was no difference in the total numbers of interacting neutrophils or in the percentage of transmigrated cells. In addition, deficient neutrophils remained capable of degrading murine endothelial VE-cadherin. These results indicate that although neutrophil proteases may play a role in the acute inflammatory response, neutrophil elastase or MMP-9 is not essential for neutrophil transendothelial migration in this murine system.
The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells
Authors: Kwakkenbos Mark J , Chang Gin-Wen , Lin Hsi-Hsien , Pouwels Walter , de Jong Esther C , van Lier René A W , Gordon Siamon , Hamann Jörg
Published: September 2022

The EGF-TM7 family is a group of class B seven-span transmembrane (TM7) receptors expressed predominantly by cells of the immune system. Family members CD97, EMR1, EMR2, EMR3, and ETL are characterized by an extended extracellular region with a variable number of N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain by a stalk. The EGF domain region of the recently identified EMR2 differs from that of CD97 in only 6 out of 236 amino acids. Although small, this difference has been shown to alter ligand specificity. To analyze the structure and cellular distribution of EMR2, a specific monoclonal antibody (2A1) was generated. Use of 2A1 has demonstrated EMR2, like CD97, to be expressed as a heterodimeric receptor consisting of an extracellular α part and a TM7/cytoplasmic β part. Analysis of EMR2 expression on primary blood leukocytes, on hematopoietic cells lines, and in situ revealed a myeloid-restricted profile. Highest expression levels were detected on the more mature CD16+ blood monocytes, on macrophages, and on BDCA-3+ myeloid DC, whereas little if any expression was found on granulocytes. Unlike CD97, no expression was observed on resting or activated lymphocytes. Different expression patterns and the inability of EMR2 to interact with the CD97 ligand CD55 indicate that the molecular twins EMR2 and CD97 likely have nonredundant functions.