bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement for STAT1 in LPS-induced gene expression in macrophages
Authors: Ohmori Yoshihiro , Hamilton Thomas A
Published: September 2022

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared fromStat1−/− mice, whereas levels of tumor necrosis factor α and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1−/− mice. The reduced LPS-stimulated gene expression seen in Stat1−/− macrophages was not the result of reduced activation of nuclear factor κB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-γ-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages fromIFN-α/βR−/− mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.
Adenoviral gene delivery can inactivate Kupffer cells: role of oxidants in NF-κB activation and cytokine production
Authors: Wheeler Michael D , Yamashina Shunhei , Froh Matthias , Rusyn Ivan , Thurman Ronald G
Published: September 2022

Kupffer cells play a significant role in the pathogenesis of several liver diseases; therefore, a potential therapeutic strategy would be to inactivate the Kupffer cell with a gene-delivery system. Although recombinant adenovirus provides robust, transgene expression in parenchymal cells, whether adenovirus transduces Kupffer cells is unclear. Thus, the purpose of this study was to evaluate this possibility. In animals infected with adenovirus, Kupffer cells were identified positively to express adenoviral transgenes by immunohistochemical techniques and Western blot analysis, indicating that Kupffer cells are transduced in vivo. Indeed, isolated Kupffer cells were transduced in vitro with recombinant adenovirus in a dose-dependent manner. Moreover, adenoviral transduction of Kupffer cells was blocked by inhibitors of αVβ5 integrin, the co-receptor for adenovirus binding, supporting the hypothesis that adenovirus transduces Kupffer cells via an αVβ5 integrin-dependent mechanism. Indeed, it is shown here that Kupffer cells express αVβ5 integrins. In a functional assay, infection of isolated Kupffer cells with adenovirus containing superoxide dismutase or IκBα super-repressor blunted LPS-induced nuclear transcription factor kappa B (NF-κB) activation and tumor necrosis factor α (TNF-α) production but not IL-10 production. Moreover, superoxide production was blocked by expression of superoxide dismutase. These data support the hypothesis that LPS-induced NF-κB activation and TNF-α production in Kupffer cells are oxidant-dependent. These findings suggest that Kupffer cell-targeted approaches may be a potential therapeutic strategy against many inflammatory diseases including early alcohol-induced liver injury.
Polymorphonuclear granulocytes induce myocardial dysfunction during ischemia and in later reperfusion of hearts exposed to low-flow ischemia
Authors: Seligmann Christian , Bock Andreas , Leitsch Tobias , Schimmer Mike , Simsek Yusuf , Schultheiss Heinz-Peter
Published: September 2022

Polymorphonuclear granulocytes (PMNs) are known to contribute to reperfusion injury of the heart. However, whether PMNs compromise myocardial function of hearts exposed to a low-flow ischemia has not been determined. Moreover, not much is known about deleterious effects of PMNs at different times during ischemia and reperfusion. Isolated, working guinea pig hearts were subjected to 30 min of low-flow ischemia and reperfusion. Homologous PMNs were applied as 1-min boluses in the presence of thrombin during either ischemia or the first or fifth minute of reperfusion, and postischemic recovery of external heart work (REHW) and intracoronary PMN retention (PMNR) were quantified. In further experiments, the radical scavenger superoxide dismutase (SOD) was added. Compared with controls without PMNs (REHW, 92.4%), application of PMNs led to a significant loss of myocardial function, which was detected at all three examination times. Moreover, intracoronary PMNR increased significantly in comparison with that of controls with hearts not exposed to ischemia or reperfusion. On the other hand, addition of SOD significantly increased REHW. Intracoronary PMNR was not significantly changed by coapplication of SOD. We conclude that thrombin-stimulated PMNs applied at different times during ischemia and reperfusion significantly impaired cardiac function in hearts exposed to a low-flow ischemia.
Nerve growth factor regulates TNF-α production in mouse macrophages via MAP kinase activation
Authors: Barouch Rina , Kazimirsky Gila , Appel Elena , Brodie Chaya
Published: September 2022

In this study, we examined the expression of nerve growth factor (NGF) and its receptors in mouse macrophages and the mechanisms involved in the effect of NGF on tumor necrosis factor (TNF)-α production. Macrophages expressed NGF and the NGF receptors TrkA and p75. Treatment of J744 cells or peritoneal macrophages with NGF induced a large increase in the production of TNF-α. In addition, NGF induced the secretion of nitric oxide in interferon-γ-treated J774 cells or lipopolysaccharide-treated peritoneal macrophages. The induction of TNF-α production by NGF was blocked by K252a, an inhibitor of the TrkA receptor. NGF induced phosphorylation and activation of extracellular signal-regulated kinase, Erk1/Erk2 and c-Jun amino-terminal kinase, whereas it did not induce phosphorylation of p38 mitogen-activated protein kinase. Inhibition of the MAP kinase-Erk kinase pathway with PD 098059 decreased the secretion of TNF-α by NGF. Our results suggest that NGF has an important role in the activation of macrophages during inflammatory responses via activation of mitogen-activated protein kinases.
Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes
Authors: Yousefi Shida , Cooper Paul R , Potter Suzanne L , Mueck Beatrice , Jarai Gabor
Published: September 2022

The migration of neutrophils into sites of acute and chronicinflammation is mediated by chemokines. We used degenerate-primerreverse transcriptase-polymerase chain reaction (RT-PCR) to analyzechemokine receptor expression in neutrophils and identify novelreceptors. RNA was isolated from human peripheral blood neutrophils andfrom neutrophils that had been stimulated for 5 h withgranulocyte-macrophage colony-stimulating factor or by coculturing withprimary human bronchial epithelial cells. Amplification products werecloned, and clone redundancy was determined. Seven knownG-protein-coupled receptors were identified among 38 clones—CCR1,CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1—as well as a novel gene,EX33. The full-length EX33 clone was obtained, and an insilico approach was used to identify the putative murine homologue. TheEX33 gene encodes a 396-amino-acid protein with limitedsequence identity to known receptors. Expression studies of severalknown chemokine receptors and EX33 revealed that resting neutrophilsexpressed higher levels of CXCRs and EX33 compared with activatedneutrophils. Northern blot experiments revealed that EX33 is expressedmainly in bone marrow, lung, and peripheral blood leukocytes. UsingRT-PCR analysis, we showed more abundant expression of EX33 inneutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression amongleukocytes.
Phosphatidylcholine-specific phospholipase C regulates activation of RAW264.7 macrophage-like cells by lipopeptide JBT3002
Authors: Zhang Fahao , Zhao Guiling , Dong Zhongyun
Published: September 2022

Phospholipase activities are thought to be involved in the activation of macrophages by lipopolysaccharide (LPS). Because our previous studies showed that the synthetic lipopeptide JBT3002 might activate macrophages via signaling pathways similar to those used by LPS, we investigated whether phospholipase activities are required for activation of macrophages by JBT3002. Treatment of RAW264.7 murine macrophage-like cells with JBT3002 stimulated expression of both inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) in a dose-dependent manner. The JBT3002-induced production of nitric oxide and TNF-α was significantly inhibited by tricyclodecan-9-yl xanthogenate (D609), a selective inhibitor of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). JBT3002-induced expression of steady-state mRNA for both iNOS and TNF-α was inhibited by D609. Cells treated with JBT3002 had greater production of diacylglycerol (DAG) in 2 min, which lasted for at least 30 min and could be blocked by D609. Activation of RAW264.7 cells was not affected by butanol, a PC-specific phospholipase D inhibitor, and treatment with JBT3002 did not affect phosphatidic acid formation. RAW264.7 cells treated with DAG analogue 1-oleoyl-2-acetyl-sn-glycerol, in the presence of interferon-γ, produced TNF-α. These results suggested that activation of RAW264.7 cells by JBT3002 requires PC-PLC activity.
Role of activin A in negative regulation of normal and tumor B lymphocytes
Authors: Zipori Dov , Barda-Saad Mira
Published: September 2022

Activin A, a member of the transforming growth factor β superfamily, has a wide spread expression pattern and pleiotropic functions. In thisoverview we summarize data that points to a role of activin A innegative regulation of B lineage lymphocytes. Experiments performed byus and by other groups revealed the capacity of activin A to causeapoptotic death of tumor myeloma cells, through mechanisms of cellcycle inhibition and antagonism with the survival signal ofinterleukin-6. In vitro studies on B lymphocyte generation from bonemarrow stem cells and use of human nasal polyps as a model of inflamedtissue further demonstrate an inhibitory role of activin A in B cellspread and accumulation. These data are analyzed with respect to ourmodel of tissue organization that we term the “restrictin model ofcell growth regulation.” This model assumes a morphogen-like role ofactivin A in the hematopoietic system. Thus, the relative concentrationof biologically functional activin A, in different parts of the tissue, may determine the local B cell content and functional state of thesecells within a specific microenvironment.
Tissue distribution of CD6 and CD6 ligand in cattle: expression of the CD6 ligand (CD166) in the autonomic nervous system of cattle and the human
Authors: Konno A , Ahn J-S , Kitamura H , Hamilton M J , Gebe J A , Aruffo A , Davis W C
Published: September 2022

We studied the tissue distribution of CD6+ lymphocytes and cells expressing the CD6 ligand (also known as activated leukocyte cell adhesion molecule CD166) in calves by immunohistochemistry using an anti-bovine CD6 monoclonal antibody (mAb), a human CD6 (huCD6)-immunoglobulin G1 fusion protein (huCD6-Ig), and an anti-human CD166 (anti-huCD166) mAb. The huCD6-Ig and anti-huCD166 mAb bound to the sympathetic and parasympathetic nerve fibers but not to myelinated nerve fibers in the spinal nerve. Studies with human tissue using the anti-huCD166 mAb yielded identical patterns of labeling. Dense accumulations of CD6+ lymphocytes were present in areas of the thymuses and spleens of calves, in areas innervated by huCD6-Ig+ nerves. The cDNAs encoding the bovine CD166 and CD6 were isolated from the sympathetic ganglion and spleen, respectively. Predicted amino acid residues that are important for human and mouse CD6-CD166 binding were also conserved in bovine CD6 and CD166. Bovine CD166 transcripts were detected by reverse transcriptase-PCR in all the tissues that bound huCD6-Ig. These results show that the bovine orthologue of CD166 was constitutively expressed in the autonomic nervous systems of cattle and suggest that CD6+ lymphocytes adhere to CD166+ autonomic nerve terminals via CD6.
Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation
Authors: Wong Birming , Lumma William C , Smith Anthony M , Sisko John T , Wright Samuel D , Cai Tian-Quan
Published: September 2022

Macrophages secrete matrix metalloproteinase 9 (MMP-9), an enzyme that weakens the fibrous cap of atherosclerotic plaques, predisposing them to plaque rupture and subsequent ischemic events. Recent work indicates that statins strongly reduce the possibility of heart attack. Furthermore, these compounds appear to exert beneficial effects not only by lowering plasma low-density-lipoprotein cholesterol but also by directly affecting the artery wall. To evaluate whether statins influence the proinflammatory responses of monocytic cells, we studied their effects on the chemotactic migration and MMP-9 secretion of human monocytic cell line THP-1. Simvastatin dose dependently inhibited THP-1 cell migration mediated by monocyte chemoattractant protein 1, with a 50% inhibitory concentration of about 50 nM. It also inhibited bacterial lipopolysaccharide-stimulated secretion of MMP-9. The effects of simvastatin were completely reversed by mevalonate and its derivatives, farnesylpyrophosphate and geranylgeranyl pyrophosphate, but not by ubiquinone. Additional studies revealed similar but more profound inhibitory effects with L-839,867, a specific inhibitor of geranylgeranyl transferase. However, α-hydroxyfarnesyl phosphonic acid, an inhibitor of farnesyl transferase, had no effect. C3 exoenzyme, a specific inhibitor of the prenylated small signaling Rho proteins, mimicked the inhibitory effects of simvastatin and L-839,867. These data supported the role of geranylgeranylation in the migration and MMP-9 secretion of monocytes.
Differences in splenic B-lymphocyte ganglioside expression and accessibility in normal and endotoxin-hyporesponsive mice
Authors: Berenson Charles S , Rasp Robin H , Gau Jen-Tzer , Ryan John L , Yohe Herbert C
Published: September 2022

Endotoxin-responsive (C3H/HeN) and -hyporesponsive (C3H/HeJ) murine Blymphocytes purified by adherence to anti-immunoglobulin (“antibodypanning”) possess identical gangliosides but different gangliosidesurface accessibilities. We investigated the distribution and surfaceaccessibility of gangliosides of B lymphocytes purified by adherence toplastic (“plastic panning”) or by subtraction of non-B-lymphocytecomponents. As with antibody panning, there were no entirely new orabsent gangliosides in plastic-panned or subtraction-purified Blymphocytes of each strain. However, striking changes in relativeexpression of five gangliosides were detected with each purificationprotocol. Moreover, five gangliosides of antibody-panned andplastic-panned B lymphocytes but only two gangliosides ofsubtraction-purified B lymphocytes were inaccessible to surfacelabeling. Unlike the situation for antibody-panned B lymphocytes, nointerstrain (HeN vs. HeJ) surface accessibility differences existed ingangliosides of plastic-panned or subtraction-purified cells. Exposureof subtraction-purified B lymphocytes to anti-immunoglobulin failed toelicit changes in ganglioside expression. Murine B lymphocytes havedistinct protocol-dependent differences in glycolipid phenotypewhich likely denote individual subpopulations.
Proinflammatory properties of the human S100 protein S100A12
Authors: Yang Zheng , Tao Tony , Raftery Mark J , Youssef Peter , Di Girolamo Nick , Geczy Carolyn L
Published: September 2022

S100 proteins represent a new class of chemoattractants. Here we extend earlier evidence for the proinflammatory properties of human S100A12. A12 induced migration of monocytoid cells, with optimal activity at 10−10 M and potency of >10−9 M C5a. Neutrophils were poorly responsive, and lymphocyte migration was not affected. Actin polymerization in monocytoid cells was accompanied by a sustained [Ca2+]i flux of a magnitude comparable with C5a. A12 elicited a transient infiltration of neutrophils (4–8 h) and more delayed recruitment of monocytes (8–24 h) in vivo. A12 (∼70 nM) was present in synovial fluid (SF) from rheumatoid arthritis patients, and synovium contained A12-positive neutrophils in the sublining and interstitial region, often surrounding the perivasculature but rarely in the synovial lining layer, although some macrophages were positive. The A12 gene was transiently up-regulated in monocytes by tumor necrosis factor α (6 h); induction by lipopolysaccharide (LPS) was sustained (12–48 h). A12 may contribute to leukocyte migration in chronic inflammatory responses.
Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-α, and NO production by bovine macrophages
Authors: Shoda Lisl K M , Kegerreis Kimberly A , Suarez Carlos E , Mwangi Waithaka , Knowles Donald P , Brown Wendy C
Published: September 2022

The immunogenicity of DNA vaccines is partially attributable to the adjuvant properties of bacterial plasmid DNA (pDNA) for B lymphocytes and professional antigen-presenting cells. In mice, modification of immunostimulatory sequences (ISSs), including CpG motifs, in pDNA vectors or oligodeoxynucleotides can increase or decrease their adjuvant properties. ISSs that stimulate optimal responses reportedly differ for murine and human leukocytes. We have previously characterized the mitogenic properties of oligodeoxynucleotides containing one AACGTT motif for bovine B lymphocytes. We now define cytokine responses by macrophages stimulated with pDNA engineered to contain an ISS comprising two AACGTT motifs. Macrophages activated with CpG-modified pDNA secreted significantly more interleukin-12, tumor necrosis factor-α, and nitric oxide than macrophages stimulated with unmodified pDNA or modified pDNA that contained nucleotides scrambled to remove CpG motifs. Engineered CpG-pDNA or CpG-oligodeoxynucleotides should be useful as vaccines or adjuvants to promote the enhanced type 1 responses important for protection against intracellular pathogens.
Transmembrane proteases in focus: diversity and redundancy?
Authors: Bauvois Brigitte
Published: September 2022

Recent advances have led to the identification and characterization of an array of transmembrane proteases that mediate the proteolysis of various substrates (including bioactive peptides, components of the extracellular matrix, and integral proteins) and cell-cell or cell-matrix adhesion. The membrane proteases known to participate in these processes currently include the ectopeptidases, the membrane-type matrix metalloproteases (MT-MMPs), the ADAM (a disintegrin and metalloprotease) family, the meprins, and the secretases, and this list may be expected to grow. The roles that these molecules play within neoplastic and inflammatory sites are being investigated actively. The capacity of these ectoenzymes to transmit intracellular-transduction signals through the plasma membrane has to be considered. An appreciation of their functional redundancy is emerging.
Signaling pathways initiated in macrophages after engagement of type A scavenger receptors
Authors: Coller Susan P , Paulnock Donna M
Published: September 2022

Scavenger receptors are macrophage cell surface molecules associated with endocytic uptake of lipoproteins and binding of microbial ligands. Macrophage class A scavenger receptors (SR-As) interact with ligands to induce cellular signaling leading to gene transcription and cytokine release. We used inhibitors of early and late signaling to block SR-A-mediated polyinosinic-polycytidilic acid (poly I:C) and lipoteichoic acid (LTA) activation of RAW 264.7 macrophages. Effects of multiple inhibitors on tumor necrosis factor (TNF)-α release were monitored to determine requirements for inflammatory cytokine production. Cycloheximide, monodansylcadaverine, and cytochalasin B all blocked TNF-α release from macrophages stimulated with LTA or poly I:C, whereas monensin only nominally reduced TNF-α production. Selected inhibitors of downstream signaling events reduced SR-A-dependent TNF-α release by >95% after stimulation with either ligand, whereas others were ineffective. The PKC inhibitor H7 reduced LTA-dependent secretion of TNF-α by 94% but inhibited poly I:C-dependent TNF-α production only by 50%. Priming of RAW 264.7 cells with interferon-γ potentiated the response to poly I:C but did not alter inhibitor effects. These results demonstrated that for both ligands tested here, early events of receptor internalization are requisite for cellular activation. The response pattern suggests that tyrosine phosphorylation and activation of the MAP kinase pathway are key components of SR-A-mediated signal transduction cascades.
Identification and comparison of residues critical for cell-adhesion activities of two neutrophil CD66 antigens, CEACAM6 and CEACAM8
Authors: Kuroki Motomu , Abe Hironori , Imakiirei Takayuki , Liao Shaoxi , Uchida Hiroko , Yamauchi Yasushi , Oikawa Shinzo , Kuroki Masahide
Published: September 2022

CEACAM6 (CD66c) and CEACAM8 (CD66b) are cell-adhesion proteins onneutrophils that belong to the human carcinoembryonic antigen (CEA)family. CEACAM6 reveals homophilic adhesion and heterophilic adhesionto other CEACAM family antigens including CEACAM8, CEACAM1, and CEA, whereas CEACAM8 exhibits only heterophilic adhesion to CEACAM6. Here, we investigated and compared structural requirements for the homophilicadhesion of CEACAM6 and heterophilic adhesion between CEACAM6 andCEACAM8 at the amino acid level by using CHO transfectants expressingtheir mutant and chimeric proteins. The NH2-terminal domain(N-domain) of CEACAM6 expressed on a CHO cell was suggested to bind the N-domain of CEACAM6 or CEACAM8 on the opposing cell. Byhomologue-scanning mutagenesis, we found that the locations of thesequences critical for the adhesion of CEACAM6 to itself and to CEACAM8are overlapped and that they are highly similar but not identical tothe locations of the residues previously shown to be essential for thebinding of CEACAM antigens to Opa proteins of pathogenicNeisseriae. Our findings imply that subtle differences inthe N-domain sequences determine the specificity of the CEACAM antigenson neutrophils for interaction with the same or different CEACAMantigens and the bacterial proteins.
Increased CCR4 expression in active systemic lupus erythematosus
Authors: Hase Kayoko , Tani Kenji , Shimizu Teruki , Ohmoto Yasukazu , Matsushima Kouji , Sone Saburo
Published: September 2022

CC chemokine receptor (CCR)4 is selectively expressed on Th2-type Tcells and has been shown to be responsible for Th2-dominant immuneresponses. In this study, we analyzed the expression of CCR4 in activesystemic lupus erythematosus (SLE) patients by FACS analysis usinganti-human CCR4 monoclonal antibody and determined the clinicalrelevance in this disease. Higher expression of CCR4 was found onperipheral blood CD4+ T lymphocytes of active SLE patients than wasfound with healthy controls and inactive SLE patients. The CCR4expression significantly correlated with the SLE disease activity index(SLEDAI) scores. The expression was dramatically decreased after thecorticosteroid therapy in parallel with a serum level ofdouble-stranded DNA antibody and SLEDAI scores. Moreover, we found thatserum levels of IL-10 were increased in active SLE patients andsignificantly correlated with the CCR4 expression. This study suggeststhat Th2 immune response is predominant in the active state of SLE, andCCR4 may have relevance in regard to the disease course in SLEpatients.
BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis
Authors: Moulding Dale A , Akgul Cahit , Derouet Mathieu , White Michael R H , Edwards Steven W
Published: September 2022

The human neutrophil spontaneously undergoes apoptosis, but this type of cell death can be delayed or accelerated by a wide variety of agents. There are wide discrepancies in the literature regarding the expression of the Bcl-2 family of proteins in human neutrophils. Here, we show that A1, Mcl-1, Bcl-XL, and Bad are major transcripts in human neutrophils and that levels of these transcripts are cytokine regulated. However, no Bcl-XL protein was detected in Western blots. Protein levels for the proapoptotic proteins Bad, Bax, Bak, and Bik remained constant during culture, despite changes in the levels of mRNA for these gene products. These proapoptotic proteins were extremely stable, having very long half-lives. In contrast, A1 and Mcl-1 transcripts were extremely unstable (with ∼3-h half-lives), and Mcl-1 protein was also subject to rapid turnover. These results indicate that neutrophil survival is regulated by the inducible expression of the short-lived Mcl-1 and possibly the A1 gene products. In the absence of their continued expression, these prosurvival gene products are rapidly turned over, and then the activity of the stable death proteins predominates and promotes apoptosis.
Toll-like receptor 4-mediated activation of murine mast cells
Authors: McCurdy J D , Lin T-J , Marshall Jean S
Published: September 2022

Toll-like receptors (TLRs) are a family of pattern recognition receptors that are critical for cellular responses to a variety of bacterial, viral, and fungal products. Mast cells are important to host survival in a number of models of bacterial infection and might act as sentinel cells in host defense. We therefore examined the expression of TLRs and associated molecules by murine bone marrow-derived mast cells (BMMCs). BMMCs and the murine mast cell line MC/9 expressed mRNA for TLR2, TLR4, and TLR6 but not TLR5 and for both adapter molecule MD-2 and signaling molecule MyD88 but lacked surface CD14. After activation with the TLR2- and TLR4-dependent stimuli Staphylococcus aureus-derived peptidoglycan and Escherichia coli-derived lipopolysaccharide (LPS), respectively, mast cells produced significant levels of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). To determine whether mast cells require TLR4 for cellular responses to LPS, mast cells were derived from the bone marrow cells of C3H/HeJ and C57Bl/10ScNCr mice containing a point mutation and a null mutation, respectively, in TLR4. Using these models, we demonstrated that the BMMC IL-6 and TNF-α responses to LPS were completely dependent on functional TLR4 with no significant LPS response observed in its absence. These findings have important implications for the mechanism of mast cell responses to pathogens and their products and suggest that different TLR4-expressing cells might have different thresholds for activation with LPS.
Themicrophthalmiatranscription factor and the related helix-loop-helix zipper factors TFE-3 and TFE-C collaborate to activate the tartrate-resistant acid phosphatase promoter
Authors: Mansky Kim C , Sulzbacher Sabine , Purdom Georgia , Nelsen Lori , Hume David A , Rehli Michael , Ostrowski Michael C
Published: September 2022

The microphthalmia transcription factor (MITF) regulates different target genes in several distinct cell types, including osteoclasts. The role of the closely related factors TFE3 and TFEC in MITF action was studied. The TFE3 and TFEC proteins were expressed in osteoclast-like cells, and both could be immunoprecipitated in a complex with MITF. In transient transfection assays, TFE3 and TFEC could collaborate with MITF to superactivate the tartrate resistant acid phosphatase (TRAP) promoter, a target for MITF in osteoclasts. Although TFEC had been thought to act as a repressor, we could demonstrate that TFEC acted as a transactivator when fused to the gal4 DNA-binding domain in a yeast one-hybrid-type assay. Additionally, two mRNA isoforms of MITF, MITF-M and MITF-A, were detected in primary osteoclast-like cells by RT-PCR. In transient transfection assays, the MITF-A and MITF-M isoforms activated the promoter of the TRAP gene to the same extent, and both forms could collaborate equally well with TFE3 to activate the TRAP promoter. These results indicate that although different isoforms of MITF appear to be functionally similar, the TFE3 and TFEC proteins may collaborate with MITF to efficiently regulate expression of target genes in osteoclasts.
Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages
Authors: Raes Geert , De Baetselier Patrick , Noël Wim , Beschin Alain , Brombacher Frank , Hassanzadeh Gh. Gholamreza
Published: September 2022

Alternatively activated macrophages (aaMφ) display molecular and biological characteristics that differ from those of classically activated macrophages (caMφ). Recently, we described an experimental model of murine trypanosomosis in which the early stage of infection of mice with a Trypanosoma brucei brucei variant is characterized by the development of caMφ, whereas in the late and chronic stages of infection, aaMφ develop. In the present study, we used suppression subtractive hybridization (SSH) to identify genes that are expressed differentially in aaMφ versus caMφ elicited during infection with this T. b. brucei variant. We show that FIZZ1 and Ym1 are induced strongly in in vivo- and in vitro-elicited aaMφ as compared with caMφ. Furthermore, we demonstrate that the in vivo induction of FIZZ1 and Ym1 in macrophages depends on IL-4 and that in vitro, IFN-γ antagonizes the effect of IL-4 on the expression of FIZZ1 and Ym1. Collectively, these results open perspectives for new insights into the functional properties of aaMφ and establish FIZZ1 and Ym1 as markers for aaMφ.
Physiological levels of 1α, 25 dihydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors
Authors: Grande Alexis , Montanari Monica , Tagliafico Enrico , Manfredini Rossella , Marani Tommaso Zanocco , Siena Michela , Tenedini Elena , Gallinelli Andrea , Ferrari Sergio
Published: September 2022

Although supraphysiological levels of 1α, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38− hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.
Nitric oxide activates ATP-dependent K+ channels in human eosinophils
Authors: Schwingshackl Andreas , Moqbel Redwan , Duszyk Marek
Published: September 2022

Nitric oxide (NO) affects the function of ion channels in many cell types, but its role in the regulation of eosinophil ion channels is unknown. In this study, we used the perforated patch-clamp method to investigate the effect of endogenous and exogenous NO on eosinophil ion channels. Using the NO synthase inhibitor, N-nitro-l-arginine methyl ester, we showed that endogenous NO did not affect the whole-cell current in eosinophil. However, two NO donors, S-nitroso-glutathione and S-nitroso-N-acetyl penicillamine, activated whole-cell currents via a NO/cGMP-dependent pathway. Ion substitution and pharmacological studies showed that NO-activated currents were carried by K+ ions, likely through ATP-dependent K+ channels (KATP). Although RT-PCR studies showed the expression of several classes of K+ channels in human eosinophils, NO donors affected only KATP channel function. We conclude that NO, at concentrations likely to be encountered in vivo, could prevent eosinophil activation by opening KATP channels.
Post-transcriptional regulation of TNF-α during in vitro differentiation of human monocytes/macrophages in primary culture
Authors: MacKenzie Simon , Fernàndez-Troy Neus , Espel Enric
Published: September 2022

Tumor necrosis factor α (TNF-α), a proinflammatory cytokine, is produced abundantly by monocytes and macrophages. We have compared LPS-stimulated TNF-α production and regulation in freshly isolated human monocytes and macrophages differentiated in vitro. A significant increase in LPS-induced TNF-α protein secretion was observed in macrophages over freshly isolated monocytes without comparable differences in TNF-α mRNA induction. Polysome gradient analysis showed polysome-mRNA distribution did not change, whereas TNF-α mRNA stability increased in macrophages. Tristetraprolin mRNA expression was constitutive and decreased with differentiation-linked kinetics. Blockable LPS-inducible MAP kinase activity (p38, ERK) affected TNF-α biosynthesis differentially at the transcriptional and post-transcriptional level throughout the culture period. We suggest that the increase in TNF-α secretion in macrophages relates to changes in post-transcriptional processing, which is regulated indirectly by the expression of RNA-binding proteins. Changes in gene expression throughout monocytic differentiation equip the cell to act as a more potent producer of this proinflammatory cytokine.
Eicosapentaenoic acid inhibits CSF-induced human monocyte survival and maturation into macrophage through the stimulation of H2O2 production
Authors: Terada Sachiyo , Takizawa Mari , Yamamoto Shigeru , Ezaki Osamu , Itakura Hiroshige , Akagawa Kiyoko S
Published: September 2022

Human studies suggest a beneficial effect of eicosapentaenoic acid (EPA)-supplemented diets on atherosclerotic and atherothrombotic disorders as well as autoimmune and inflammatory diseases and tumors. The effects of EPA on human monocyte survival and maturation into macrophage are not yet known. We studied the effects of EPA on the survival and development into macrophage of human monocyte treated with colony-stimulating factor (CSF). We have found that EPA induces cell death of the monocyte via apoptosis, even in the presence of M-CSF or GM-CSF, and inhibits differentiation from the monocyte to macrophage by inducing H2O2 production. In contrast to the effect of EPA on monocytes, EPA did not induce cell death of monocyte-derived macrophages. Such an apoptosis inducing effect on monocytes by EPA may contribute to the efficacy of EPA in atherosclerosis and autoimmune diseases.
Sorting of neutrophil-specific granule protein human cathelicidin, hCAP-18, when constitutively expressed in myeloid cells
Authors: Bülow Elinor , Bengtsson Niklas , Calafat Jero , Gullberg Urban , Olsson Inge
Published: September 2022

Neutrophil granulocytes carry storage organelles, e.g., azurophil and specific granules. Poorly understood are the mechanisms for retrieval from constitutive secretion followed by sorting for storage. Therefore, we asked whether the specific granule protein human cathelicidin (hCAP-18) could be sorted for storage in other granules when the biosynthetic window is widened to allow this. We observed that hCAP-18 was targeted for storage in lysosome-related organelles when expressed constitutively in the rat basophilic leukemia and the mouse promyelocytic (MPRO) cell lines. In addition, premature release of the antibiotic C-terminal peptide LL-37 was observed. Retention of hCAP-18 was diminished by induction of differentiation of MPRO cells. In conclusion, a specific granule protein with native conformation may be sorted for storage in lysosome-related organelles of myeloid cells and converted prematurely to a supposedly biologically active form.
Inhibition of IL-6 signaling by a p38-dependent pathway occurs in the absence of new protein synthesis
Authors: Ahmed Simi T , Mayer Alexander , Ji Jong-Dae , Ivashkiv Lionel B
Published: September 2022

Negative regulation of cytokine signaling is important for limiting the intensity and duration of cytokine action and for maintaining homeostasis. Several constitutive mechanisms for suppressing cytokine Jak-STAT signaling have been described. Inducible or regulated inhibition of cytokine signaling is equally important, and much attention has been focused on inhibition mediated through the induction of expression of suppressors of cytokine signaling (SOCS proteins). We have previously reported IL-1-induced inhibition of IL-6 signaling in monocytes, and herein we use inhibitors of protein synthesis to demonstrate that inhibition of IL-6 signaling can occur in the absence of new protein synthesis. Surprisingly, some protein synthesis inhibitors themselves inhibited IL-6 signaling rapidly, strengthening the conclusion that IL-6 signaling can be inhibited in the absence of protein synthesis. Inhibition of IL-6 signaling by IL-1 and protein synthesis inhibitors was dependent on the p38 stress kinase, and activation of p38 secondary to inducible expression of MKK6 was sufficient to inhibit IL-6 signaling. Inhibition was specific for IL-6, as induction of STAT activation by IFN-γ, IFN-α, and vanadate was not inhibited. IL-1-induced inhibition of IL-6 signaling was not mediated by the activation of tyrosine phosphatases or by p38-dependent activation of phospholipase A2 or cyclooxygenases, which could lead to indirect inhibition via production of prostaglandins. These results identify an inducible mechanism of inhibition of IL-6 signaling that is direct and independent of induction of negative regulators such as SOCS proteins. A role for p38 in mediating inhibition suggests that multiple cytokines and stress agents that activate p38 pathways in monocytes, such as IL-1, TNF, Toll-like receptors, and Fc receptors, can modulate Jak-STAT signaling by pleiotropic cytokines such as IL-6.
Mechanisms of TNF receptor-associated factor (TRAF) regulation in B lymphocytes
Authors: Bishop Gail A , Hostager Bruce S , Brown Kevin D
Published: September 2022

A key component of signaling by members of the tumor necrosis factor receptor (TNF-R) family is interaction with the cytoplasmic adapter proteins known as TRAFs. Several proteins encoded by microbes also interact with TRAFs. A notable example is the CD40 receptor, expressed on antigen presenting cells and providing key activation signals in T cell-dependent B cell activation. CD40 signals to B cells are mimicked by a constitutively active viral protein produced by the Epstein-Barr virus. For each of these receptors, multiple mechanisms of TRAF regulation contribute to signal transduction and the ultimate effect on the B cell. Recent findings concerning these regulatory mechanisms are summarized in this overview.
IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components
Authors: Ariel Amiram , Novick Daniela , Rubinstein Menachem , Dinarello Charles A , Lider Ofer , Hershkoviz Rami
Published: September 2022

Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, β1 integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-γ (INF-γ) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-γ production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites.
Increased susceptibility to apoptosis and attenuated Bcl-2 expression in T lymphocytes and monocytes from patients with advanced chronic hepatitis C
Authors: Nakamoto Yasunari , Kaneko Shuichi , Kobayashi Kenichi
Published: September 2022

Some viral infections are reported to influence the susceptibility of peripheral blood mononuclear cells (PBMC) to apoptosis, which is related to disease progression. The current study was designed to monitor apoptosis in separated PBMC subsets, CD4+ and CD8+ T lymphocytes, and CD14+ monocytes under apoptotic stimuli in patients with chronic hepatitis C. Apoptosis was induced by serum starvation and by incubation with anti-CD3 antibody and with phorbol 12-myristate 13-acetate. With the escalating severity of liver disease, susceptibility of all PBMC subsets to apoptosis increased under the apoptotic stimulus of serum starvation (P<0.05). Consequently, increased susceptibility to apoptosis was associated with diminished intracellular expression of the antiapoptotic protein Bcl-2 (P<0.05). The current observations demonstrate that the abnormality of PBMC subsets in undergoing apoptosis as a result of the down-regulation of Bcl-2 expression may contribute to viral persistence and progression of liver disease in chronic hepatitis C.
The role of P-selectin in experimental colitis as determined by antibody immunoblockade and genetically deficient mice
Authors: Gironella Meritxell , Mollà Meritxell , Salas Azucena , Soriano Antonio , Sans Miquel , Closa Daniel , Engel Pablo , Salas Antonio , Piqué Josep M , Panés Julián
Published: September 2022

We assessed the effects of genetic ablation of the P-selectin gene in comparison with functional immunoblockade of P-selectin on leukocyte recruitment and the course of disease in dextran sulfate sodium-induced colitis in mice. Compared with control antibody-treated wild-type (WT) mice, WT mice treated with anti-P-selectin antibody and P-selectin−/− mice had significantly decreased leukocyte rolling and adhesion in colonic venules and reduced clinical and pathological colitis scores. These reductions were more pronounced in anti-P-selectin-treated than in P-selectin−/− mice. In colonic endothelium, up-regulation of ICAM-1 was similar in WT and P-selectin−/− mice, but VCAM-1 up-regulation was significantly higher in the latter group. Lung leukocyte infiltration and VCAM-1 expression were increased only in P-selectin−/− colitic mice. Mortality was observed only in P-selectin−/− mice. Therefore, ablation of P-selectin function ameliorates colitis, but this protection is attenuated in P-selectin−/− mice, probably due to compensatory mechanisms that involve up-regulation of other adhesion molecules such as VCAM-1.
Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections
Authors: Gregory Stephen H , Wing Edward J
Published: September 2022

Most bacteria that enter the bloodstream are taken up and eliminated within the liver. The specific mechanisms that underlie the role of the liver in the resolution of systemic bacterial infections remain to be determined. The vast majority of studies undertaken to date have focused on the function of resident tissue macrophages (Kupffer cells) that line the liver sinusoids. Indeed, it is often reported that Kupffer cells ingest and kill the bulk of organisms taken up by the liver. Recent studies indicate, however, that phagocytosis by Kupffer cells is not the principal mechanism by which organisms are eliminated. Rather, elimination depends on the complex interaction of Kupffer cells and bactericidal neutrophils that immigrate rapidly to the liver in response to infection. We discuss the critical role of neutrophil-Kupffer cell interaction in innate host defenses and, conceivably, the development and expression of adaptive immunity in the liver.
Binding of function-blocking mAbs to mouse and human P-selectin glycoprotein ligand-1 peptides with and without tyrosine sulfation
Authors: Thatte Aravinda , Ficarro Scott , Snapp Karen R , Wild Martin K , Vestweber Dietmar , Hunt Donald F , Ley Klaus F
Published: September 2022

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin-expressing endothelial cells under shear flow. Function-blocking monoclonal antibodies (mAbs) against mouse and human PSGL-1 recognize an anionic segment at the N-terminus of PSGL-1. High affinity interaction of PSGL-1 with P-selectin requires sulfation of tyrosines 46, 48, and 51 (human) or 54 and 56 (mouse). We tested binding of two anti-human (KPL1 and PL1) and two anti-mouse (4RA10 and 2PH1) PSGL-1 mAbs to synthetic peptides of N-terminus of human and mouse PSGL-1 and found binding to be independent of tyrosine sulfation. In peptide-blocking experiments, sulfated and nonsulfated human and mouse peptides competed with antibody binding to PSGL-1 expressed on myeloid cells. Arylsulfatase treatment significantly reduced P-selectin binding but had no effect on antibody binding. Our data show, in three independent assay systems, that function-blocking antibodies to mouse or human PSGL-1 do not require sulfation of N-terminal tyrosines for binding.
Molecular characterization and expression analysis of leucine-rich α2-glycoprotein, a novel marker of granulocytic differentiation
Authors: O'Donnell Lynn C , Druhan Lawrence J , Avalos Belinda R
Published: September 2022

Using data obtained from cDNA representational difference analysis to identify genes induced during neutrophilic differentiation of the 32D clone 3G (32Dcl3G) cells, we isolated cDNA clones for murine and human leucine-rich α2-glycoprotein (hLRG), a protein with unknown function purified 25 years ago. Expression of LRG during differentiation of 32Dcl3G cells preceded the expression of lactoferrin and gelatinase but followed myeloperoxidase. LRG transcripts were also detected in human neutrophils and progenitor cells but not in peripheral blood mononuclear cells. Notably, LRG expression was up-regulated during neutrophilic differentiation of human MPD and HL-60 cells but down-regulated during monocytic differentiation of HL-60 cells. The hLRG gene was localized to chromosome 19p13.3, a region to which the genes for several neutrophil granule enzymes also map. The putative promoter region of LRG was found to contain consensus-binding sites for PU.1, C/EBP, STAT, and MZF1. These results suggest that LRG is a novel marker for early neutrophilic granulocyte differentiation.
IL-10 expression profiling in human monocytes
Authors: Williams Lynn , Jarai Gabor , Smith Alexandra , Finan Peter
Published: September 2022

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine with numerous immunomodulatory effects, including the inhibition of proinflammatory cytokine production. The mechanisms by which IL-10 exerts these effects still remain largely unknown. As there is evidence that suggests IL-10-mediated cytokine suppression requires the induction of an intermediate gene, we have used gene-chip technology to identify IL-10-inducible genes in human monocytes. We have been able to identify a total of 19 genes that are up-regulated in response to IL-10. Three of these genes had been identified previously: IL-1ra, suppressors of cytokine signaling-3, and CD163; however, the other 16 represent newly identified IL-10-responsive genes. Further analysis of the regulation of eight of these genes showed a remarkable specificity to regulation by lipopolysaccharides (LPS) and IL-10, but not by other anti-inflammatory mediators such as IL-4 and transforming growth factor-β, suggesting that two diverse stimuli such as IL-10 and LPS may engage common signaling mechanisms.