bluuebunny/crossref_metadata_2025 huggingface dataset:
Themicrophthalmiatranscription factor (MITF) contains two N-terminal domains required for transactivation of osteoclast target promoters and rescue ofmimutant osteoclasts
Authors: Mansky Kim C , Marfatia Kavita , Purdom Georgia H , Luchin Alex , Hume David A , Ostrowski Michael C
Published: September 2022

The microphthalmia transcription factor (MITF) regulates gene expression during differentiation of several distinct cell types, including osteoclasts. A structure/function analysis was performed to determine whether transcription activation domains were important for MITF action in osteoclasts. In addition to a previously characterized acidic activation necessary for melanocyte differentiation, the analysis defined a second potential activation domain located between amino acids 140 and 185. This second domain is required for MITF transactivation of two probable targets, the E-cadherin promoter and the tartrate-resistant acid phosphatase promoter, in transient transfection assays. An intact MITF gene rescued differentiation when introduced into osteoclasts derived from mi/mi mice using a retrovirus vector. In parallel experiments, an MITF gene lacking the acidic-activation domain rescued differentiation twofold less efficiently than wild type, and a gene lacking the region between amino acid residues 140 and 185 rescued differentiation tenfold less efficiently than wild type. The results indicate that the N-terminal region of MITF is necessary for activation of gene expression in osteoclasts and provides one mechanism by which this factor regulates distinct target genes in different cell types.
Cloning and sequencing of rabbit leukocyte NADPH oxidase genes reveals a unique p67phoxhomolog
Authors: Gauss Katherine A , Mascolo Patrice L , Siemsen Daniel W , Nelson Laura K , Bunger Peggy L , Pagano Patrick J , Quinn Mark T
Published: September 2022

The NADPH oxidase plays an important role in immune and nonimmune cell functions. Because rabbits represent an established model for studying a number of important disease processes that involve NADPH oxidase activity, we carried out studies to clone and sequence all five rabbit leukocyte NADPH oxidase genes. Comparison of the rabbit sequences with those of other species showed that, with the exception of p67phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67phox had a very divergent C-terminus and was 17 amino acids longer than any other known p67phox homolog. This was surprising, given the high degree of conservation among all of the phox proteins sequenced previously. To evaluate the functional consequences of this difference, wild-type rabbit p67phox and a mutated rabbit p67phox missing the C-terminal 17 amino acids were expressed and analyzed in a cell-free assay. Our results show that the full-length and truncated rabbit p67phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full-length p67phox. These studies contribute to our understanding of the nature of the leukocyte NADPH oxidase in different species and will be valuable in future research using the rabbit model.
Molecular profiles of allograft rejection following inhibition of CD40 ligand costimulation differentiated by cluster analysis
Authors: Damrauer Scott M , DeFina Rachel , He Hongzhen , Haley Kathleen J , Perkins David L
Published: September 2022

Recent technological advances in biomedical research, such as genomesequences and DNA microarrays, have dramatically increased the size of relevant databases. A major challenge is the extraction of a limitednumber of parameters from these databases that can differentiate anddiagnose complex biological states. In a model of cardiactransplantation investigating immunosuppression by inhibition of CD40ligand costimulation, we have applied a combination of clusteralgorithms and self-organizing maps to analyze a panel of 60 candidategenes. Dendrograms generated by cluster analysis distinguisheddifferent molecular bases of rejection. Using self-organizing maps, weidentified nine genes (CD4, CCR3, CCR5, LTβ, MIP-1α, MIP-2, CD8α,IP-10, and RANTES), each with a unique profile of transcriptionalexpression, that reproduce the differentiation of states of rejectionin dendrograms. Using histology and immunohistochemistry, we correlateddifferential regulation of CD4 and CD8 at the levels of mRNA andprotein. Our strategy of data reduction successfully decreased thenumber of genes to nine, which are sufficient to differentiate distinctstates of rejection in our experimental protocol.
Activation of the RON receptor tyrosine kinase protects murine macrophages from apoptotic death induced by bacterial lipopolysaccharide
Authors: Chen Yi-Qing , Zhou Yong-Qing , Wang Ming-Hai
Published: September 2022

RON is a receptor tyrosine kinase activated by macrophage-stimulating protein. We demonstrate here that RON activation inhibits LPS-induced apoptosis of mouse peritoneal macrophages and Raw264.7 cells expressing RON or a constitutively active RON mutant. The antiapoptotic effect of RON was accompanied with the inhibition of LPS-induced production of nitric oxide (NO), a molecule responsible for LPS-induced cell apoptosis. This conclusion is supported by experiments using a chemical NO donor GSNO, in which RON activation directly blocked GSNO-induced apoptotic death of Raw264.7 cells and inhibited LPS-induced p53 accumulation. Furthermore, we showed that treatment of cells with wortmannin, which inhibits phosphatidylinositol (PI)-3 kinase, prevents the inhibitory effect of RON on LPS-induced macrophage apoptosis. These results were confirmed further by expression of a dominant inhibitory PI-3 kinase p85 subunit. These data suggest that by activating PI-3 kinase and inhibiting p53 accumulation, RON protects macrophage from apoptosis induced by LPS and NO. The antiapoptotic effect of RON might represent a novel mechanism for the survival of activated macrophages during inflammation.
Emerging evidence that molecules expressed by mammalian tissue grafts are recognized by the innate immune system
Authors: Fox-Marsh Annette , Harrison Leonard C
Published: September 2022

The innate immune system existed prior to the emergence of adaptive immunity in sharks and higher vertebrates. Homologues of many mammalian innate immune-system elements such as the toll-like receptors exist in species as distant as Drosophila. Selective pressure has led to the development of highly conserved, soluble, and cell-surface receptors that recognize functionally essential molecules shared by microbial pathogens. It is thought that molecular patterns that exquisitely distinguish pathogenic cells from mammalian cells are recognized. Therefore, it would seem unlikely that innate immune-system elements should recognize mammalian tissues. However, there is increasing evidence to suggest that this is the case and that innate immunity promotes rejection of transplanted mammalian tissues, particularly those from other species (xenografts). Evidence for innate recognition of mammalian grafts, the nature of this recognition, and the bi-directional interactions between innate and adaptive immunity that contribute to graft rejection are discussed in this review, with the emphasis on nonvascular xenografts.
Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro
Authors: Soilleux Elizabeth J , Morris Lesley S , Leslie George , Chehimi Jihed , Luo Qi , Levroney Ernest , Trowsdale John , Montaner Luis J , Doms Robert W , Weissman Drew , Coleman Nicholas , Lee Benhur
Published: September 2022

DC-SIGN is a C-type lectin, highly expressed on the surface ofimmature dendritic cells (DCs), that mediates efficient infection of Tcells in trans by its ability to bind HIV-1, HIV-2, and SIV. Inaddition, the ability of DC-SIGN to bind adhesion molecules on surfacesof naïve T cells and endothelium also suggests its involvementin T-cell activation and DC trafficking. To gain further insights intothe range of expression and potential functions of DC-SIGN, weperformed a detailed analysis of DC-SIGN expression in adult and fetaltissues and also analyzed its regulated expression on cultured DCs andmacrophages. First, we show that DC-SIGN expression is restricted tosubsets of immature DCs in tissues and on specialized macrophages inthe placenta and lung. There were no overt differences between DC-SIGNexpression in adult and fetal tissues except that DC-SIGN expression inalveolar macrophages was only present after birth. Similarly, intissues, DC-SIGN was observed primarily on immature (CD83-negative)DCs. Secondly, in the peripheral blood, we found expression of DC-SIGNon a small subset of BDCA-2+ plasmacytoid DC precursors (pDC2),concordant with our finding of large numbers of DC-SIGN-positive cellsin allergic nasal polyps (previously shown to be infiltrated by DC2). Triple-label confocal microscopy indicated that DC-SIGN was colocalizedwith BDCA-2 and CD123 on DCs in nasal polyp tissue. Consistent withthis finding is our observation that DC-SIGN can be up-regulated onmonocyte-derived macrophages upon exposure to the Th2 cytokine, IL-13. In summary, our data demonstrate the relevant populations of DC andmacrophages that express DC-SIGN in vivo where it may impact theefficiency of virus infection and indicate that DC-SIGN expression maybe involved in the Th2 axis of immunity.
IL-12 suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis
Authors: Lee Sujin , Zheng Mei , Deshpande Shilpa , Eo Seong Kug , Hamilton Thomas A , Rouse Barry T
Published: September 2022

Topical application of plasmid DNA encoding IL-12 to the cornea of mice prior to ocular infection with Herpes simplex virus type 1 (HSV) results in diminished corneal immunoinflammatory lesions. Such herpetic stromal keratitis (HSK) reactions in humans represent an important cause of blindness. The effect of IL-12 pretreatment acted via inhibitory effects on corneal neovascularization rather than by inhibiting viral replication or the function of CD4+ T cells that mediate HSK. The antiangiogenesis induced by IL-12 DNA application was mediated indirectly via the cytokine IFN-γ and one or both of two chemokine molecules, IP-10 and MIG. Thus IL-12 DNA administration lacked modulatory effects on HSK in GKO mice, indicating the necessary involvement of IFN-γ induction for antiangiogenesis. In contrast, exposure of GKO mice to IP-10 DNA did suppress the severity of HSK. Furthermore, treatment with specific antisera to IP-10 and MIG in HSV-infected mice abrogated the IL-12-induced inhibitory effect on lesion severity. Taken together, our data indicate that the HSV-induced ocular immunoinflammatory lesions can be modulated by IL-12 and that this effect results from chemokine inhibition of angiogenesis. The use of antiangiogenesis therapy might represent a useful control measure against HSK.
Beryllium fluoride-induced cell proliferation: a process requiring P21ras-dependent activated signal transduction and NF-κB-dependent gene regulation
Authors: Misra Uma Kant , Gawdi Govind , Pizzo Salvatore Vincent
Published: September 2022

We studied the effect of beryllium fluoride on murine peritoneal macrophages and determined its effects on signal transduction and genetic regulation. At low concentration (1–5 nM), BeF2 caused an approximate twofold increase in [3H]thymidine uptake and cell number, but above 5 nM, it showed cytotoxic effects. BeF2 increased cellular inositol (1,4,5)trisphosphate (IP3) and [Ca2+]i about twofold. The rise in [Ca2+]i occurred consequent to release from IP3-sensitive Ca2+ stores and from influx, mainly via L-type channels. A significant increase in the levels of MEK1, ERK1, p38 MAPK, and JNK phosphorylation was observed in BeF2-exposed macrophages. The levels of NF-κB and CREB transcription factors and the proto-oncogenesc-fos and c-myc were also elevated significantly. Intracellular Ca2+ chelation blocked the effect of BeF2. We conclude that BeF2 at low concentration exerts its mitogenic effects in peritoneal macrophages by elevating [Ca2+]i, which triggers the activation of p21ras-dependent MAPK signaling cascades.
Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation
Authors: Lehtonen Anne , Matikainen Sampsa , Miettinen Minja , Julkunen Ilkka
Published: September 2022

GM-CSF signals through JAK2 and STAT5 and stimulates the expression of STAT5 target genes, such as pim-1 and CIS. Analyzed by EMSA, GM-CSF stimulation led to much stronger STAT5 DNA-binding to pim-1 or CIS GAS elements in primary human monocytes compared with mature macrophages. Similarly, GM-CSF-induced expression of pim-1 and CIS mRNAs was much stronger in monocytes. These differencies were not a result of downregulation of the GM-CSF receptor system or STAT5 expression, because monocytes and macrophages readily expressed GM-CSF receptor, JAK2, STAT5A, and STAT5B mRNAs and proteins. Monocytes expressed significant amounts of truncated STAT5 forms that took part in STAT5-DNA complex formation in GM-CSF-stimulated monocytes. This resulted in faster moving STAT5 complexes compared with macrophages in EMSA. Our results demonstrate that STAT5 isoform expression, GM-CSF-induced STAT5 activation, and STAT5 target-gene expression are altered significantly during monocyte/macrophage differentiation.
LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca2+-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells
Authors: Rodríguez-Fernández José Luis , Sánchez-Martín Lorena , de Frutos Cristina Alvarez , Sancho David , Robinson Martyn , Sánchez-Madrid Francisco , Cabañas Carlos
Published: September 2022

Lymphocyte function-associated antigen (LFA-1) is a member of the β2 family of integrins that is selectively expressed on leukocytes. Herein, we show that Ca2+ mobilizing agents A23187, thapsigargin, and ionomycin induce an increase in adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) and activation and redistribution of the proline-rich tyrosine kinase-2 (PYK2) to the microtubule-organizing center (MTOC) in T-lymphoblasts. These effects are similar to those observed upon direct induction of activation of LFA-1 with the stimulatory mAb KIM-127. Most importantly, Ca2+ mobilization did not induce activation of PYK2 when the LFA-1/ICAM-1 interaction was prevented with function-blocking mAb, implying that the Ca2+-induced activation of PYK2 requires integrin engagement. Furthermore, pretreatment of the cells with the Ca2+ chelator EGTA, which depletes the intracellular Ca2+, inhibited the effects of mAb KIM-127 on cell morphology and PYK2 activation. This inhibition with EGTA was not reversed by cross-linking integrin LFA-1 with specific antibodies, indicating that Ca2+ exerts its effects through a target downstream of this integrin. In this regard, immunofluorescence and Western blot analysis showed that Ca2+ chelators affect the organization of the microtubular cytoskeleton and the localization of PYK2 to the MTOC area, suggesting that these agents could inhibit the activation of PYK2 by interfering with the microtubular network of T cells. Taken together, our results demonstrate for the first time an important role for the integrin LFA-1 and the microtubular cytoskeleton in the Ca2+-mediated activation of PYK2 in T-lymphocytes.
β3-Mediated engulfment of apoptotic tumor cells by dendritic cells is dependent on CAMKII: inhibition by HIV-1 Tat
Authors: Poggi Alessandro , Carosio Roberta , Rubartelli Anna , Zocchi Maria Raffaella
Published: September 2022

In this paper, we show that the engulfment of apoptotic tumor cells by DC requires the activation of the calcium-calmodulin kinase II (CAMKII). Indeed, DC phagocytosis of apoptotic lymphoma cells is consistently inhibited by KN62 and KN93, two blockers of CAMKII, but not by the inactive compound KN92. Wortmannin and LY294002, two inhibitors of the phosphatidyl-inositol-3 kinase, slightly decrease the phagocytosis of apoptotic cells, at variance with PD98059, an inhibitor of the mitogen-activated protein kinase. It is interesting that the addition of synthetic HIV-1 Tat, which we demonstrated to inhibit phagocytosis and calcium influx in DC, blocks the activation of CAMKII elicited via β3 integrin, which is involved in apoptotic body engulfment by DC. Experiments performed with Tat-derived peptides showed that this inhibition is mediated by the C-terminal domain of Tat. Finally, pertussis toxin can prevent HIV-1 Tat-mediated inhibition, suggesting the involvement of a guanosine triphosphate-binding (G) protein in DC-mediated phagocytosis.
Rat monocyte-derived dendritic cells function and migrate in the same way as isolated tissue dendritic cells
Authors: Richters C D , Mayen I , Havenith C E G , Beelen R H J , Kamperdijk E W A
Published: September 2022

Dendritic cells (DC) are the most potent antigen-presenting cells and are therefore useful to induce immune responses against tumor cells in patients. DC can be generated in vitro from monocytes using GM-CSF and IL-4, the so-called monocyte-derived DC (MoDC). To achieve antitumor responses, MoDC must be able to migrate to the draining lymph nodes after injection to induce cytotoxic T cells. Therefore, we studied migration of MoDC in a rat model. Functional rat MoDC were generated from PVG-RT7B rats and injected subcutaneously into PVG rats. These rat strains differ only at one epitope of the leukocyte-common antigen, which can be recognized by the antibody His 41. The advantage is that migrated cells can be detected in the draining lymph nodes by staining sections with His 41+; thus, migration is not influenced by labeling procedures. Rat MoDC migrated to the T-cell areas of the draining lymph nodes, just as isolated Langerhans cells or spleen DC do. In contrast, monocytes also migrated to the B-cell areas and the medulla.
Effect of pentoxifylline on polarization and migration of human leukocytes
Authors: Domínguez-Jiménez Carmen , Sancho David , Nieto Marta , Montoya María C , Barreiro Olga , Sánchez-Madrid Francisco , González-Amaro Roberto
Published: September 2022

Leukocyte polarization has a key role in the induction and effector phases of immune response. We assessed the effect of pentoxifylline (PTX) on the polarization and migration of human lymphocytes and neutrophils. A dose-dependent, inhibitory effect on the polarization of lymphoid cells induced by chemokines or IL-15 was found. In addition, PTX interfered with the chemotaxis of peripheral blood T cells and T lymphoblasts. A similar effect was observed on the transendothelial migration of these cells. In addition, the polarization of neutrophils, its adherence to endothelium, and their transendothelial migration, induced by different stimuli, were inhibited by PTX. By contrast, this drug had only a mild effect on endothelial cells and a partial inhibition on the induction of ICAM-1 expression by TNF-α. The inhibitory effect of PTX on leukocyte polarization and extravasation may contribute significantly to the anti-inflammatory and immunoregulatory activity of this drug.
Ambient pCO2 modulates intracellular pH, intracellular oxidant generation, and interleukin-8 secretion in human neutrophils
Authors: Coakley R J , Taggart C , Greene C , McElvaney N G , O'Neill S J
Published: September 2022

Although neutrophils are a critical component of the inflammatory process, their functional regulation is incompletely understood. Of note, although pCO2 varies physiologically and pathologically in the neutrophilic milieu, its affect on neutrophil biological processes is unresolved. We demonstrate here that neutrophils respond to hypo- and hypercarbia, (0.04% and 10%) by increasing and decreasing, respectively, intracellular oxidant production (basally and in response to opsonized Escherichia coli and phorbol esters). Further, hypo- and hypercarbia increase and decrease, respectively, the release of IL-8 from LPS-stimulated cells; both effects are attenuated by the carbonic anhydrase inhibitor, acetazolamide. Anion exchange did not restore pHi under hypocarbic conditions, however partial restoration of pHi under hypercarbic conditions was achieved by Na+/H+ exchange and vacuolar ATPases. Abrogation of pCO2-induced changes in pHi prevented hypocarbia-induced generation of reactive oxidant species. These observations suggest that CO2 modifies neutrophil activity significantly by altering pHi.
Nitric oxide synthase and nitric oxide production in in vivo-derived mast cells
Authors: Gilchrist Mark , Savoie Michelle , Nohara Osamu , Wills Fiona L , Wallace John L , Befus A D
Published: September 2022

Nitric oxide (NO) is a potent mediator synthesized by a variety of cells involved in inflammatory reactions. We investigated the expression of NO synthase (NOS) in rat peritoneal mast cells (PMC). Small amounts of eNOS mRNA were detected basally, whereas neither mRNA for iNOS nor nNOS was detected in unstimulated PMC. Following stimulation by antigen, interferon-γ (IFN-γ), or anti-CD8 antibody, PMC up-regulated iNOS mRNA expression. In situ RT-PCR confirmed that iNOS mRNA originated from PMC. Production of iNOS protein was confirmed in stimulated PMC by immunohistochemistry. Upon stimulation with antigen, IFN-γ, or anti-CD8, nitrite production was increased significantly (8.4±0.6, 7.6±0.9, and 6.6±0.9 μM/2×105 cells/48 h NO2−, respectively; P<0.01), whereas unstimulated PMC released 2.1 ± 0.3 μM/2 × 105 cells/48 h NO2−. These findings demonstrate that in vivo-derived PMC transcribe and translate mRNA for NOS and produce NO.
Regulation of murine macrophage proinflammatory and anti-inflammatory cytokines by ligands for peroxisome proliferator-activated receptor-γ: counter-regulatory activity by IFN-γ
Authors: Alleva David G , Johnson Eric B , Lio Francisco M , Boehme Stefen A , Conlon Paul J , Crowe Paul D
Published: September 2022

The prostaglandin, 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2)1, and thiazolidinediones are ligands for the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)-γ, which mediates anti-inflammatory activity by suppressing murine macrophage (Mφ) production of the inflammatory mediator, nitric oxide (NO). Here, we elucidated this anti-inflammatory activity further by investigating whether PPAR-γ ligands regulated a panel of proinflammatory and anti-inflammatory cytokines produced by primary inflammatory murine Mφ (thioglycollate-elicited peritoneal exudate Mφ; PEM). Thiazolidinediones and 15d-PGJ2 suppressed lipopolysaccharide (LPS)-induced PEM production of NO and IL-12(p40) to a greater extent than IL-6 and TNF-α production. Whereas 15d-PGJ2 showed the greatest extent of suppression of proinflammatory mediator production, the thiazolidinedione, BRL49653, was the most potent compound studied. Surprisingly, treatment with the Mφ-activation cytokine, IFN-γ, prevented PPAR-γ ligands from suppressing the proinflammatory cytokines completely and reduced their suppression of NO production substantially, demonstrating that activation conditions affect PPAR-γ-mediated, anti-inflammatory activity. Western analysis demonstrated that the antagonistic activity of IFN-γ did not involve modulation of PPAR-γ expression but showed that IFN-γ interfered with PPAR-γ ligand regulation of p42/p44 MAP kinase activation and the cytosolic disappearance of NF-κB upon LPS stimulation. Finally, we showed that PPAR-γ ligands did not substantially modulate production of the anti-inflammatory cytokine, IL-10, and that antibody-mediated neutralization of IL-10 did not prevent the ligands from suppressing proinflammatory mediator production. In contrast to studies with noninflammatory human monocytes and Mφ, our results demonstrate that primary murine inflammatory Mφ are extremely sensitive to the anti-inflammatory activity of PPAR-γ ligands. These results suggest that drugs such as thiazolidinediones may be most effective in suppressing Mφ activity early (i.e., in the absence of lymphocyte-derived IFN-γ) in the inflammatory process.
Inhibition of murine macrophage nitric oxide production by synthetic oligonucleotides
Authors: Zhu Fu-Gang , Reich Charles F , Pisetsky David S
Published: September 2022

Synthetic 30-mer phosphorothioate (Ps) oligonucleotides (ODN) comprised of single bases (SdA30, SdC30, SdG30, and SdT30) were assessed for their effects on nitric oxide (NO) production by murine bone marrow macrophages (BMMC) and macrophage cell lines J774 and RAW264.7. Pretreatment of these cells with any of the four Ps ODN inhibited NO production induced by CpG ODN, E. coli DNA (EC DNA), or LPS. This inhibition was time- and dose-dependent and was observed even if the Ps ODN were added as long as 12 h after stimulation. As in the case of stimulatory ODN, inhibition was dependent on backbone structure and length. Thus, all four 30-mer, single-base Ps ODN were inhibitory, and only dG30 among phosphodiester ODN was inhibitory. Together, these observations indicate that Ps ODN can inhibit macrophage production of inflammatory mediators, suggesting a role of these compounds as immunomodulatory agents.
Contribution of phopholipase D and a brefeldin A-sensitive ARF to chemoattractant-induced superoxide production and secretion of human neutrophils
Authors: Káldi Krisztina , Szeberényi Júlia , Rada Balázs K , Kovács Péter , Geiszt Miklós , Mócsai Attila , Ligeti Erzsébet
Published: September 2022

We show that blockers of phospholipase D (PLD) reduce fMLP-triggered exocytosis of secretory vesicles effectively. In accordance with this, the PLD product phosphatidic acid (PA) was able to induce mobilization of secretory vesicles. Although PLD seems to play a role in the release of all neutrophil granule types, exogenous PA alone was not sufficient to activate the exocytosis of primary and secondary granules, suggesting that in the case of these granules, additional signaling factors are required to initiate the secretory responses. The ADP-ribosylation factor (ARF)-inhibitor brefeldin A (BFA) inhibited the fMLP-stimulated O2·− production strongly, whereas it did not influence any of the exocytic responses, and no significant effect of BFA was detected on the O2·− generation induced by other stimuli. On the basis of these results, we propose that upon chemoattractant stimulation, PLD activity is involved in induction of degranulation and O2·− production, but a BFA-sensitive ARF is only required to the activation of the NADPH oxidase. This ARF action seems to participate exclusively in the signaling pathway between the fMLP receptor and the oxidase.
Signaling to localized degranulation in neutrophils adherent to immune complexes
Authors: Nauclér Claes , Grinstein Sergio , Sundler Roger , Tapper Hans
Published: September 2022

The present study demonstrates that the secretion of azurophilic granules occurring during Fc receptor-mediated attachment and spreading of neutrophils is highly localized to the adhering region of the cell. In contrast, the secretion of specific granules occurs in a nonpolarized way. This implies that unique signals are involved in the regulation of azurophilic degranulation. Assembly of actin filaments, as visualized by staining with rhodamine phalloidin, neither hindered nor facilitated degranulation. Further, the azurophilic secretory response remained localized in the presence of cytochalasin B. Release of azurophilic-granule content was inhibited by genistein and erbstatin, inhibitors of tyrosine kinases, and by GF109203X, a protein kinase C (PKC) inhibitor. We could also demonstrate a relative enrichment of syk tyrosine kinase and the PKC isoforms α and β1 in adherent plasma membranes.
The Cbl family of ubiquitin ligases: critical negative regulators of tyrosine kinase signaling in the immune system
Authors: Rao Navin , Dodge Ingrid , Band Hamid
Published: September 2022

The Cbl family of proteins are evolutionarily conserved negative regulators of activated tyrosine kinase-coupled receptors. Antigen receptors are prominent targets of negative regulation by the Cbl family members, Cbl and Cbl-b, which proteins function as ubiquitin ligases. Cbl and Cbl-b contain substrate recognition domains that interact specifically with activated protein tyrosine kinases of the Src and Syk/ZAP-70 families. Cbl-mediated ubiquitination of these kinases leads to their degradation, resulting in attenuation of receptor signals. Cbl may also control activation-induced monoubiquitination of antigen receptors, thus facilitating their delivery to lysosomes for subsequent degradation. Finally, the interactions of Cbl proteins with downstream targets of tyrosine kinases, such as PI-3-kinase and Vav, could provide an additional mechanism to attenuate receptor signaling. By targeting multiple components of antigen receptor signaling for degradation, the Cbl protein family provides a critical mechanism to ensure an appropriate immune response. The hyperresponsiveness of Cbl−/− and Cbl-b−/− lymphocytes and the autoimmune phenotype of Cbl-b−/− mice lend strong support for this proposal. The ability to control early receptor signals through regulated protein degradation provides a novel paradigm of immunoregulation.
Different Gi-coupled chemoattractant receptors signal qualitatively different functions in human neutrophils
Authors: Berger Miles , Budhu Sadna , Lu Emily , Li Yongmei , Loike Devora , Silverstein Samuel C , Loike John D
Published: September 2022

fMLP- or TNF-α-stimulated neutrophils produced H2O2 when they adhered to fibrinogen-coated surfaces but not when they adhered to collagen I-, collagen IV-, or Matrigel-coated surfaces. In contrast, LTB4- or IL-8-stimulated neutrophils did not produce H2O2 when they adhered to any of these surfaces. fMLP and TNF-α were much more potent than LTB4 and IL-8 in stimulating neutrophils to up-regulate and to activate their αMβ2 integrins, as measured by the binding of specific monoclonal antibodies. Pretreatment of neutrophils with pertussis toxin completely blocked their production of H2O2 on fibrinogen-coated surfaces in response to fMLP and their migration through Matrigel in response to fMLP, LTB4, and IL-8. These data show that although the fMLP, LTB4, and IL-8 receptors are coupled to pertussis toxin-sensitive Gα proteins, they signal neutrophils to initiate qualitatively different effector functions. We propose that the qualitative differences in effector functions signaled by different chemoattractants reflect qualitative differences in using G-protein β and/or γ subunits or other factors by their cognate receptors.
Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated fromTrypanosoma cruzitrypomastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-γ-primed-macrophages
Authors: Coelho Patrícia S , Klein André , Talvani André , Coutinho Sibele F , Takeuchi Osamu , Akira Shizuo , Silva João S , Canizzaro Hélia , Gazzinelli Ricardo T , Teixeira Mauro M
Published: September 2022

Glycosylphosphatidylinositol-anchoredmucin-like glycoproteins from Trypanosoma cruzi trypomastigotes (tGPI-mucins) activate macrophages in vitro to produce proinflammatory cytokines, chemokines, and nitric oxide. These effects of tGPI-mucins may be important in the ensuing immune response to T. cruzi. Here, we have sought evidence for a role of tGPI-mucins in mediating leukocyte recruitment in vivo. tGPI-mucins are highly effective in promoting cell recruitment in the pleural cavity of mice primed with IFN-γ-inducing agents but not in naïve mice. Maximal recruitment was observed at a dose between 250 and 1250 ng tGPI-mucins. There was a significant elevation in the levels of MCP-1 in the pleural cavity of primed animals injected with tGPI-mucins, and in vivo neutralization of MCP-1 abolished leukocyte recruitment. Pretreatment with anti-MIP-1α or anti-RANTES had no effect on the recruitment induced by tGPI-mucins. MCP-1 immunoreactivity was detected in pleural macrophages, and macrophages produced MCP-1 in vitro, especially after priming with IFN-γ. Finally, tGPI-mucins induced significant leukocyte recruitment in primed C3H/HeJ but not in TLR2-deficient mice. Together, our results suggest that T. cruzi-derived GPI-mucins in conjunction with IFN-γ may drive tissue chemokine production and inflammation and bear a significant role in the pathogenesis of Chagas disease.
The expression pattern of the ITIM-bearing lectin CLECSF6 in neutrophils suggests a key role in the control of inflammation
Authors: Richard Manon , Veilleux Patricia , Rouleau Michèle , Paquin Robert , Beaulieu André D
Published: September 2022

In our study of the modulation of the expression of inflammation-related genes in neutrophils, we have found a gene called CLECSF6 (C-type lectin superfamily 6). CLECSF6 expresses two mRNA species at low levels in resting neutrophils. Here, we describe for the first time the sequence of the short mRNA version. It lacks amino acids that are likely to affect the functionality of its protein product. GM-CSF, IL-3, IL-4, and IL-13 caused an accumulation of the short CLECSF6 mRNA in neutrophils. The surface expression of the CLECSF6 protein was reduced by TNF-α, IL-1α, LPS, and Matrigel®. CLECSF6 bears the immunoreceptor tyrosine-based inhibition motif (ITIM) involved in signal transduction resulting in the inhibition of leukocyte activation. We propose that some neutrophil activators modulate the expression of CLECSF6 at the mRNA (GM-CSF, IL-3, IL-4, and IL-13) or protein (TNF-α, IL-1α, LPS, and Matrigel®) levels in ways that block ITIM-based transduction of anti-inflammatory signals and therefore promote inflammation.
Increased responsiveness of murine eosinophils to MIP-1β (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors, CCR5 and CCR8
Authors: Oliveira Sandra H P , Lira Sergio , Martinez-A Carlos , Wiekowski Maria , Sullivan Lee , Lukacs Nicholas W
Published: September 2022

In the present study, we investigated the regulation of chemokine-mediated responses and receptor expression on eosinophils from mice. MIP-1α (CCL3) and eotaxin (CCL11) induced a significant and only partially overlapping intracellular calcium flux in antigen-elicited and peripheral blood eosinophils, and MCP-1 (CCL2), MDC (CCL22), MIP-1β (CCL4), and TCA-3 (CCL1) did not. To demonstrate functional use of the specific receptors, we examined chemotactic responses. Peripheral blood eosinophils migrated toward MIP-1α (CCL3) and eotaxin (CCL11) but not MCP-1 (CCL2), MDC (CCL22), MIP-1β (CCL4), and TCA-3 (CCL1). Antigen-elicited eosinophils migrated toward MIP-1α (CCL3) and eotaxin (CCL11), but also migrated in response to MIP-1β (CCL4) and TCA-3 (CCL1), suggesting the up-regulation of additional chemokine receptors on antigen-elicited eosinophils. The up-regulation of the additional chemokine-receptor responses appeared to be in part because of cytokine activation, because TNF-α and/or IL-4 were able to up-regulate CCR1, -3, -5, and -8 mRNA expression in eosinophils as well as migration responses to the appropriate ligands. Using antibodies specific for CCR5 and CCR8, the chemotactic response to MIP-1β and TCA-3, respectively, was reduced significantly. Finally, the expression of these new receptors appears to have an effect on activation and degranulation because MIP-1β (CCL4) and TCA-3 (CCL1) induce significant levels of LTC4 from elicited eosinophils. These results suggest that eosinophils may up-regulate and use additional chemokine receptors during progression of inflammatory, allergic responses for migration and activation.
Protein kinase A regulates β2 integrin avidity in neutrophils
Authors: Jones Samuel L
Published: September 2022

The adhesive phenotype of neutrophils (PMN) depends largely on activating and deactivating intracellular signals regulating β2 integrin avidity for ligand. Our hypothesis is that PKA is a negative regulator of β2 integrin avidity. In this work, we examined the role of PKA in PMN αMβ2 integrin activation. Elevation of cAMP inhibited αMβ2 integrin-dependent adhesion of PMN to immune complexes (IC), but not PMA-induced adhesion. The PKA inhibitor KT5720 reversed the ability of cAMP to suppress adhesion to IC. Moreover, inhibition of PKA activity was sufficient to activate αMβ2 integrin-dependent adhesion and increase β2 integrin expression and binding of the monoclonal antibody CBRM1/5, which recognizes activated αMβ2 specifically. However, PKA activity was necessary for sustained adhesion. Disruption of A kinase-anchoring, protein-PKA binding with a cell-permeant peptide derived from the AKAP Ht31 also activated adhesion. Unlike pharmacologic inhibition of PKA, AKAP peptide-induced adhesion was PKC dependent and did not affect β2 integrin expression or CBRM1/5 binding. These data demonstrate that PKA appears to have a dual role in the mechanism regulating αMβ2 integrin avidity and adhesion.
DC-SIGN, a C-type lectin on dendritic cells that unveils many aspects of dendritic cell biology
Authors: Geijtenbeek Teunis B H , Engering Anneke , van Kooyk Yvette
Published: September 2022

Dendritic cells (DC) are present in essentially every tissue where they operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T cells. It is becoming clear that not all C-type lectins on DC serve as antigen receptors recognizing pathogens through carbohydrate structures. The C-type lectin DC-SIGN is unique in that it regulates adhesion processes, such as DC trafficking and T-cell synapse formation, as well as antigen capture. Moreover, even though several C-type lectins have been shown to bind HIV-1, DC-SIGN does not only capture HIV-1 but also protects it in early endosomes allowing HIV-1 transport by DC to lymphoid tissues, where it enhances trans infection of T cells. Here we discuss the carbohydrate/protein recognition profile and other features of DC-SIGN that contribute to the potency of DC to control immunity.
Selective up-regulation of phospholipase C-β2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors
Authors: Bertagnolo Valeria , Marchisio Marco , Pierpaoli Sabina , Colamussi Maria Luisa , Brugnoli Federica , Visani Giuseppe , Zauli Giorgio , Capitani Silvano
Published: September 2022

In this study, we have investigated the expression of phospholipase C-β2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-β2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-β2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-β2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-β2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-β2 levels can predict the in vivo responsiveness to ATRA of APL patients.
Compound exocytosis of granules in human neutrophils
Authors: Lollike Karsten , Lindau Manfred , Calafat Jero , Borregaard Niels
Published: September 2022

Human neutrophils are of prime importance for the immune defense. Recent data from eosinophils and pancreatic beta cells have indicated that granules, upon exocytosis, occasionally fuse with each other in the cytosol prior to their subsequent fusion with the plasma membrane. This is termed compound exocytosis. We therefore studied exocytosis of single granules from human neutrophils by the high-resolution cell-attached patch-clamp capacitance technique. We found that 1.5% of the capacitance steps was greater than 5 fF, i.e., significantly larger than steps expected for exocytosis of single granules. The mean step size of these events was 20.5 fF, corresponding to compounds formed by at least five granules. The capacitance input from compound steps contributed more than 20% of the total capacitance increase. Electron microscopy captured morphological manifestations of transient exocytic events, confirming the functional results obtained by capacitance measurements. Compound exocytosis may be a mechanism for efficient targeting of release during exocytosis.
Wegener's granulomatosis: antiproteinase 3 antibodies induce monocyte cytokine and prostanoid release—role of autocrine cell activation
Authors: Hattar Katja , Bickenbach Annette , Csernok Elena , Rosseau Simone , Grandel Ulrich , Seeger Werner , Grimminger Friedrich , Sibelius Ulf
Published: September 2022

Antineutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 [PR3; cytoplasmic ANCA (c-ANCA)], a leukocyte serine protease, are highly specific for Wegener's Granulomatosis (WG). A pathogenetic role for c-ANCA has been proposed as a result of their ability of activating neutrophils, whereas their interaction with monocytes is less well characterized. We investigated the influence of monoclonal anti-PR3 antibodies (anti-PR3) and c-ANCA from WG sera on monocyte cytokine and prostanoid release. We found that PR3 was expressed on the surface of isolated monocytes. Anti-PR3 challenge provoked a pronounced release of cytokines with early appearance of tumor necrosis factor α (TNF-α) and interleukin (IL)-1β and delayed release of IL-6, IL-8, and thromboxane A2 (TxA2). The secretory response was reproduced by c-ANCA but not by human and murine control IgG and anti-CD14 antibodies. Because F(ab)2 fragments of anti-PR3 were ineffective, coligation of Fc gamma receptors (FcγR) was apparently mandatory for monocyte activation. Using soluble receptors for TNF-α and IL-1β and a Tx receptor antagonist, we noted that the “early” cytokines functioned as inducers of TxA2, which then activated IL-8 release. In contrast, IL-6 formation was an independent event. We concluded that anti-PR3 antibodies are potent inducers of monocyte cytokine and prostanoid release, and TNF-α, IL-1β, and TxA2 function as facilitators of the secretory response. These mechanisms may contribute to inflammatory tissue injury in WG.
Systemic immunoregulatory and pathogenic functions of homeostatic chemokine receptors
Authors: Müller Gerd , Höpken Uta E , Stein Harald , Lipp Martin
Published: September 2022

The adoptive immune response relies on a precise temporal and spatial positioning of lymphocytes within lymphoid and nonlymphoid tissues. Chemokines, constitutively expressed or induced during inflammation provide a flexible navigation system directing lymphocytes into specific microcompartments. Precision and specificity in this process are achieved by varying patterns of chemokine receptors expressed on the cell surface of lymphocytes in the course of cell differentiation. The chemokine receptors CXCR5 and CCR7 are principal regulators for targeting T cells, B cells, and dendritic cells into secondary lymphoid organs. The analyses of knockout mice have been instrumental in exploring the crucial role of these receptors for the compartmentalization of secondary lymphoid organs into functionally separated T and B cell zones. Aside from the homeostatic recirculation of lymphocytes and inflammatory processes, chemokine receptors are also involved in malignancies such as lymphoproliferative diseases and cancer metastasis. Recent results from our laboratory present evidence for the involvement of CCR7 in the dissemination of neoplastic cells in classic Hodgkin disease. There is also accumulating evidence for the involvement of CXCR5 in the formation of ectopic follicles as observed in lymphomas or autoimmune diseases. In addition, CCR7 and CXCR5 have been identified as useful markers in the classification of functionally distinct subsets of T-helper cells, which will lead to a better understanding of T cell memory and T cell effector function in lymphoid system homeostasis and disease.
Viral chemokine-binding proteins
Authors: Seet Bruce T , McFadden Grant
Published: September 2022

The chemokines are a large family of small signaling proteins that bind to G-protein-coupled receptors (GPCRs) on target cells and mediate the directional migration of immune cells into sites of infection or inflammation. The large DNA viruses, particularly the poxviruses and herpesviruses, have evolved several mechanisms to corrupt the normal functioning of the chemokine network. Two strategies rely on mimicking chemokines or chemokine receptors. A third strategy involves the production of secreted chemokine-binding proteins (CKBPs) that exhibit no sequence similarity to any known host proteins, yet function to competitively bind and inhibit the interactions of chemokines with cognate receptors. Each strategy has provided unique insights into the elusively complex world of the chemokines. Here, we focus on recent advances made in the understanding of secreted CKBPs encoded by poxviruses and herpesviruses. A better understanding of how viral CKBPs function to manipulate the immune response may provide further clues as to how to develop specific therapeutic agents to abrogate chemokine-mediated disease conditions.
Transient expression of Ym1, a heparin-binding lectin, during developmental hematopoiesis and inflammation
Authors: Hung Shuen-Iu , Chang Alice Chien , Kato Ikunoshin , Chang Nan-Chi A
Published: September 2022

Ym1, a secretory protein transiently produced by activated peritoneal macrophages elicited by parasitic infections, has been identified as a novel heparin-binding lectin. X-ray crystallography study revealed that Ym1 has a β/α barrel structure with a carbohydrate-binding cleft similar to that of triose-phosphate isomerases. To further delineate the physiological significance of Ym1, we examined its expression patterns during mouse embryonic development and inflammation states elicited by agents other than parasitic infections in the peritoneal cavity and brain. This is the first report revealing prominent expression of Ym1 in early myeloid precursor cells of hematopoietic tissues—initially in the yolk sac and subsequently in fetal liver, spleen, and bone marrow. In nonhematopoietic systems, Ym1 was not detected in most of the tissues examined, with the exception of lung. Although no expression was detected up to gestation day 16.5 (E16.5), an increasing level of Ym1 expression in lung was detected from E18.5 on and persisted through adulthood. While most resident macrophages in various tissues examined are Ym1-negative, transient expression of Ym1 may be induced in their activated counterparts during inflammation in response to different stimuli in vivo, ranging from various chemical agents to brain injuries. The temporal and spatial expression in myeloid precursors and its transient induction in activated macrophages support the notion that Ym1 may be involved in hematopoiesis and inflammation. In addition, its putative functional association with heparin/heparan sulfate is discussed.