IC-21 Macrophages Research Articles

S73 Macrophages as vehicles for delivering cell therapy to injured lung

IntroductionInjury to the alveolar epithelium underlies a number of important lung diseases, exemplified by the syndromes of acute lung injury and acute respiratory distress syndrome, which currently have a poor...

Effects of cell culture density on phagocytosis parameters in IC-21 macrophages

Cell culture density is shown to alter the parameters characterizing phagocytic activity of cells in vitro. Phagocytosis index (PI, mean number of beads per cell in the bead-containing population) and phagocytosis percent (PP, percentage of bead-containing cells in cell population under study) for IC-21 macrophages incubated in the presence of non-opsonized 2-μm fluorescent latex beads were determined using fluorescent microscopy and ImageJ software specially adapted for the purpose. Under control conditions (DMEM without serum), increase in cell culture density was accompanied with a decrease of both parameters of the phagocytic activity. At a mean density of 4 cells/105 μm2 (9 cells per a viewfield) PI was 7.1 ± 0.2 beads/cell and at 20 cells/105 μm2 (40 cells per a viewfield) PI dropped to 4.6 ± 0.1 beads/cell. PP was less sensitive, varied in the range of 95–100% but also decreased as the cell density grew. At any density, PI was 1.5–2 times higher than the expected value (number of beads per µm2 × cell contour area); apparently this divergence can be accounted for by cell locomotion and capture of a larger number of beads than could drop onto a motionless cell with a constant contour area. Increase in cell density was also accompanied by a decrease of the cell contour area (Sc), which amounted to 750 ± 16 μm2 at a density of 4 cells/105 μm2 and 346 ± 4 μm2 at a density of 20 cells/105 μm2. As the bead concentration was the same in all experiments, density-dependent decrease in PI and PP may be related with the observed decrease in cell contour area. Yet, the bead number per cell area unit (PI/Sc) was bigger at higher density and PI/Sc was higher in cells with smaller Sc. Thus, individual (specific) activity of the cells did not lessen with an increase of the cell culture density in the range studied (4–20 cells/105 μm2). Reduction of the cell contour area may reflect alteration in cell adhesion to the substrate as well as competitive relations between adhesion and phagocytic processes. The data obtained imply that cell culture density has to be controlled as a factor notably altering the phagocytic activity parameters. The effects of serum, methyl-β-cyclodextrin, and carbenoxolon reported earlier [Golovkina et al. 2009. Biol membrany. 26 (5), 379–386] are re-evaluated and confirmed here.

A Cell Profiling Framework for Modeling Drug Responses from HCS Imaging

The authors present an unsupervised, scalable, and interpretable cell profiling framework that is compatible with data gathered from high-content screening. They demonstrate the effectiveness of their framework by modeling drug differential effects of IC-21 macrophages treated with microtubule and actin disrupting drugs. They identify significant features of cell phenotypes for unsupervised learning based on maximum relevancy and minimum redundancy criteria. A 2-stage clustering approach annotates, clusters cells, and then merges them together to form super-clusters. An interpretable cell profile consisting of super-cluster proportions profiled at each drug treatment, concentration, or duration is obtained. Differential changes in super-cluster profiles are the basis for understanding the drug's differential effect and biology. The authors' method is validated by significant chi-squared statistics obtained from similar drug-treated super-cluster profiles from a 5-fold cross-validation. In addition, drug profiles of 2 microtubule drugs with equivalent mechanisms of action are statistically similar. Several distinct trends are identified for the 5 cytoskeletal drugs profiled under different conditions.

The role of integrin α Dβ 2 (CD11d/CD18) in monocyte/macrophage migration

Integrin α Dβ 2 (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin α Mβ 2 (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of α Dβ 2 in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of α Dβ 2 and examined their migration to various extracellular matrix proteins. When expressed at a low density, α Dβ 2 on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with β 1/β 3 integrins to support cell migration. However, its increased expression on the α Dβ 2-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of α Dβ 2 with anti-α D blocking antibodies restores β 1/β 3-driven cell migration by removing the excess α Dβ 2-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-α D antibody. These results demonstrate that the density of α Dβ 2 is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of α Dβ 2 contribute to monocyte migration while α Dβ 2 upregulation on differentiated macrophages may facilitate their retention at sites of inflammation.

Adsorbed serum albumin is permissive to macrophage attachment to perfluorocarbon polymer surfaces in culture

Monocyte/macrophage adhesion to biomaterials, correlated with foreign body response, occurs through protein-mediated surface interactions. Albumin-selective perfluorocarbon (FC) biomaterials are generally poorly cell-conducive because of insufficient receptor-mediated surface interactions, but macrophages bind to albumin-coated substrates and also preferentially to highly hydrophobic fluorinated surfaces. Bone marrow macrophages (BMMO) and IC-21, RAW 264.7, and J774A.1 monocyte/macrophage cells were cultured on FC surfaces. Protein deposition onto two distinct FC surfaces from complex and single-component solutions was tracked using fluorescence and time-of-flight secondary ion mass spectrometry (ToF-SIMS) methods. Cell adhesion and growth on protein pretreated substrates were compared by light microscopy. Flow cytometry and integrin-directed antibody receptor blocking were used to assess integrins critical for monocyte/macrophage adhesion in vitro. Albumin predominantly adsorbs onto both FC surfaces from 10% serum. In cultures preadsorbed with albumin or serum-dilutions, BMMO responded similar to IC-21 at early time points. Compared with Teflon AF, plasma-polymerized FC was less permissive to extended cell proliferation. The beta(2) integrins play major roles in macrophage adhesion to FC surfaces: antibody blocking significantly disrupted cell adhesion. Albumin-mediated cell adhesion mechanisms to FC surfaces could not be clarified. Primary BMMO and secondary IC-21 macrophages behave similarly on FC surfaces, regardless of preadsorbed protein biasing, with respect to adhesion, cell morphology, motility, and proliferation.

Dying cells program their expedient disposal: serum amyloid P component upregulation in vivo and in vitro induced by photodynamic therapy of cancer

Serum amyloid P component (SAP) is known as a prototypic acute phase reactant in the mouse and the protein that binds to dying cells securing their swift disposal by phagocytes. Treatment of solid tumors by photodynamic therapy (PDT) triggers SAP production in the liver of host mice, its release in the circulation and accumulation in PDT-targeted lesions. In the present study, mouse Lewis lung carcinoma (LLC) cells treated in vitro by PDT are shown to upregulate their gene encoding SAP. This effect was manifested following PDT treatment mediated by various types of photosensitizers (Photofrin, BPD, mTHPC, ALA). Generated SAP protein was not detected in tissue supernatants but remained localized to producing PDT-treated cells. The upregulation of SAP gene was observed also in untreated IC-21 macrophages after they were co-incubated for 4 h with PDT-treated LLC cells. Based on these findings, SAP that accumulates in PDT-treated tumors may originate from both systemic sources (released from the liver as acute phase reactant) and local sources; the latter could include tumor cells directly sustaining PDT injury and macrophages invading the tumor that become stimulated by signals from these affected tumor cells. Since SAP gene upregulation in LLC cells increased with the lethality of PDT dose used for their treatment, we propose that cells sensing they are inflicted with mortal injury can turn on molecular programs insuring not only that they die an innocuous form of death (apoptosis) but also that once they are dead their elimination is (facilitated by SAP) swift and efficient.

Macrophage Serum‐Based Adhesion to Plasma‐Processed Surface Chemistry is Distinct from That Exhibited by Fibroblasts

Plasma-polymerized films deposited from AlAm, HxAm, NVP, NVFA, AA and FC were compared to TCPS and PS surfaces in supporting cellular attachment, viability, and proliferation in serum-based culture in vitro for extended periods of time (>7 d). Surface patterns were created using multi-step depositions with physical masks. Cell adhesion in the presence of serum was compared for (monocyte-) macrophage and fibroblast cell lines. Cellular response was tracked over time, reporting adhesive behavior, proliferative rates, and morphological changes as a function of surface chemistry. Micropatterned surfaces containing different surface chemistries and functional groups (e.g. -NH(2), -COOH, -CF(3)) produced differential cell adhesive patterns for NIH 3T3 fibroblasts compared to J774A.1, RAW 264.7 or IC-21 (monocyte-) macrophage cell types. Significantly, macrophage adhesion is substantial on surfaces where fibroblasts do not adhere under identical culture conditions.

Engineered Polymeric Nanoparticles for Receptor-Targeted Blockage of Oxidized Low Density Lipoprotein Uptake and Atherogenesis in Macrophages

Strategies to prevent the uptake of modified low density lipoproteins (LDLs) by immune cells, a major trigger of inflammation and atherogenesis, are challenged by complex interfacial factors governing LDL receptor-mediated uptake. We examine a new approach based on a family of "nanoblockers", which are designed to examine the role of size, charge presentation, and architecture on inhibition of highly oxidized LDL (hoxLDL) uptake in macrophages. The nanoblockers are macromolecules containing mucic acid, lauryl chloride, and poly(ethylene glycol) that self-assemble into 15-20 nm nanoparticles. We report that the micellar configuration of the macromolecules and the combined display of anionic (carboxylate) groups in the hydrophobic region of the nanoblockers caused the most effective inhibition in the uptake of hoxLDL by IC21 macrophages. The nanoblockers primarily targeted SR-A and CD36, the major scavenger receptors and modulated the "atherogenic" phenotype of cells in terms of the degree of cytokine secretion, accumulation of cholesterol, and "foam cell" formation. These studies highlight the promise of synthetically engineered nanoblockers against oxidized LDL uptake.

Titanium particles and surface‐bound LPS activate different pathways in IC‐21 macrophages

It is still unknown if wear-debris particles themselves induce osteolysis or if they serve a functional role as receptors for ligands that incite an inflammatory response that ultimately leads to bone resorption. In this study, commercially pure titanium particles (cpTi) were subjected to a serial combination of different cleaning methods to remove Lipopolysaccharide (LPS) or were incubated in LPS solutions of known concentrations. Then, the response of the macrophage cell line IC-21 to the cleaned particles, LPS-bound Ti particles, and soluble LPS was examined. It was found that cleaned particles up to 1000 particles per cell did not stimulate macrophages to release Tumor necrosis factor-alpha (TNF-alpha) or Interleukin 6 (IL-6), but they significantly increased the release of Prostaglandin E(2) (PGE(2)) when the particle concentration was higher than 500 particles per cell. At one particle per cell, Ti particles bound with LPS stimulated the release of IL-6 and TNF-alpha by macrophages. The level of released cytokines was dependent on, and correlated with, the amount of LPS present on the particles. The macrophages were more sensitive to soluble LPS than to particle-bound LPS, and the simultaneous addition of cleaned Ti particles did not have additional effects on the effects of soluble LPS. This study shows evidence that, cpTi particles and LPS have distinct mechanisms of action on the IC-21 macrophages, but that both contribute to the development of an inflammatory response.

Nanoscale Anionic Macromolecules Can Inhibit Cellular Uptake of Differentially Oxidized LDL

Nanoscale particles could be synthetically designed to potentially intervene in lipoprotein matrix retention and lipoprotein uptake in cells, processes central to atherosclerosis. We recently reported on lipoprotein interactions of nanoscale micelles self-assembled from amphiphilic scorpion-like macromolecules based on a lauryl chloride-mucic acid hydrophobic backbone and poly(ethylene glycol) shell. These micelles can be engineered to present varying levels of anionic chemistry, a key mechanism to induce differential retentivity of low-density lipoproteins (LDL) (Chnari, E.; Lari, H. B.; Tian, L.; Uhrich, K. E.; Moghe, P. V. Biomaterials 2005, 26, 3749). In this study, we examined the cellular interactions and the ability of carboxylate-terminated nanoparticles to modulate cellular uptake of differentially oxidized LDL. The nanoparticles were found to be highly biocompatible with cultured IC21 macrophages at all concentrations examined. When the nanoparticles as well as LDL were incubated with the cells over 24 h, a marked reduction in cellular uptake of LDL was observed in a nanoparticle concentration-dependent manner. Intermediate concentrations of nanoparticles (10(-6) M) elicited the most charge-specific reduction in uptake, as indicated by the difference in uptake due to anionic and uncharged nanoparticles. At these concentrations, anionic nanoparticles reduced LDL uptake for all degrees of oxidation (no oxidation, mild, high) of LDL, albeit with qualitative differences in the effects. The anionic nanoparticles were particularly effective at reducing the very high levels of uptake of the most oxidized level of LDL. Since complexation of LDL with anionic nanoparticles is reduced at higher degrees of LDL oxidation, our results suggest that anionic nanoparticles interfere in highly oxidized (hox) LDL uptake, likely by targeting cellular/receptor uptake mechanism, but control unoxidized LDL uptake by mechanisms related to direct LDL-nanoparticle complexation. Thus, anionically functionalized nanoparticles can modulate the otherwise unregulated internalization of differentially oxidized LDL.

The Role of Integrin αDβ2 in Macrophage Migration.

Integrin αDβ2 (CD11d/CD18), the most recently discovered member of the β2 sub-family of adhesion receptors, is strongly upregulated on macrophage foam cells which underscores its potential role in atherosclerosis. However, the contribution of αDβ2 to monocyte/macrophage adhesive reactions and the significance of its overexpression on these cells remain unknown. Recently we characterized αDβ2 as a multiligand receptor capable of binding many extracellular matrix proteins with the recognition specificity overlapping that of the major myeloid-specific integrin αMβ2 (Mac-1). We hypothesized that the αDβ2 ability to bind numerous ligands in the extracellular matrix and its capacity to be upregulated to high density on the surface of macrophages may modulate cell adhesiveness and, thus, affect migration. To evaluate the role of αDβ2 in migration, we generated model and natural cells expressing different densities of αDβ2 and tested their migration to different extracellular matrix proteins. In vitro studies demonstrated that αDβ2 expressed at low densities, either on the surface of HEK293 cells or the mouse macrophage cell line IC-21, supported migration which was partially inhibited by anti-αD function-blocking antibodies. Furthermore, β1 and β3 integrins expressed on HEK293 cells and IC-21 macrophages, respectively, contributed to migration because anti-β1 and anti-β3 antibodies inhibited migration. Increased expression of αDβ2 on the surface of HEK293 cells and its upregulation by PMA on IC-21 macrophages resulted in the inhibition of cell migration. Ligation of αDβ2 with anti-αD antibodies restored β1- and β3-driven cell migration by means of removing restraints imposed by the excess of the αDβ2-ligand adhesive bonds. To test the possibility that progressive upregulation of αDβ2 can block macrophage migration in vivo, we assessed the effect of anti-αD function blocking antibodies using the thioglycollate-induced peritonitis model. More than 4-fold upregulation of αDβ2 was detected on macrophages in 72 h after thioglycollate stimulation and, similar to in vitro studies, the numbers of migration macrophages increased in the presence of anti-αD antibodies. These results demonstrate that the density of αDβ2 can modulate cell migration and suggest that low levels of αDβ2 can contribute to monocyte migration while αDβ2 upregulation on differentiated macrophages might facilitate their retention at the site of inflammation.

Survival ofSalmonella entericaSerovar Typhimurium within Late Endosomal-Lysosomal Compartments of B Lymphocytes Is Associated with the Inability To Use the Vacuolar Alternative Major Histocompatibility Complex Class I Antigen-Processing Pathway

Gamma interferon (IFN-gamma)-activated macrophages use an alternative processing mechanism to present Salmonella antigens to CD8(+) T lymphocytes. This pathway involves processing of antigen in a vacuolar compartment followed by secretion and loading of antigenic peptides to major histocompatibility complex class I (MHC-I) molecules on macrophage cell surface and bystander cells. In this study, we have shown that B lymphocytes are not able to process Salmonella antigens using this alternative pathway. This is due to differences in Salmonella enterica serovar Typhimurium-containing vacuoles (SCV) when comparing late endosomal-lysosomal processing compartments in B lymphocytes to those in macrophages. The IFN-gamma-activated IC21 macrophage cell line and A-20 B-cell line were infected with live or dead Salmonella enterica serovar Typhimurium. The SCV in B cells were in a late endosomal-lysosomal compartment, whereas SCV in macrophages were remodeled to a non-characteristic late endosomal-lysosomal compartment over time. Despite the difference in SCV within macrophages and B lymphocytes, S. enterica serovar Typhimurium survives more efficiently within the IFN-gamma-activated B cells than in activated macrophage cell lines. Similar results were found during in vivo acute infection. We determined that a lack of remodeling of late endosomal-lysosomal compartments by live Salmonella infection in B lymphocytes is associated with the inability to use the alternative MHC-I antigen-processing pathway, providing a survival advantage to the bacterium. Our data also suggest that the B lymphocyte late endosome-lysosome environment allows the expression of Salmonella virulence mechanisms favoring B lymphocytes in addition to macrophages and dendritic cells as a reservoir during in vivo infection.

Phosphorylation of Tumor Necrosis Factor Receptor 1 (p55) Protects Macrophages from Silica-induced Apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.

Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages.

The lethal factor (LF) component of Bacillus anthracis lethal toxin (LeTx) cleaves mitogen activated protein kinase kinases (MAPKKs) in a variety of different cell types, yet only macrophages are rapidly killed by this toxin. The reason for this selective killing is unclear, but suggests other factors may also be involved in LeTx intoxication. In the current study, DNA membrane arrays were used to identify broad changes in macrophage physiology after treatment with LeTx. Expression of genes regulated by MAPKK activity did not change significantly, yet a series of genes under glycogen synthase kinase-3-beta (GSK-3beta) regulation changed expression following LeTx treatment. Correlating with these transcriptional changes GSK-3beta was found to be below detectable levels in toxin-treated cells and an inhibitor of GSK-3beta, LiCl, sensitized resistant IC-21 macrophages to LeTx. In addition, zebrafish embryos treated with LeTx showed signs of delayed pigmentation and cardiac hypertrophy; both processes are subject to regulation by GSK-3beta. A putative compensatory response to loss of GSK-3beta was indicated by differential expression of three motor proteins following toxin treatment and Kif1C, a motor protein involved in sensitivity to LeTx, increased expression in toxin-sensitive cells yet decreased in resistant cells following toxin treatment. Differential expression of microtubule-associating proteins and a decrease in the level of cellular tubulin were detected in LeTx-treated cells, both of which can result from loss of GSK-3beta activity. These data provide new information on LeTx's overall influence on macrophage physiology and suggest loss of GSK-3beta contributes to cytotoxicity.

Cellular signaling during the macrophage invasion by Trypanosoma cruzi.

We have reported that protein tyrosine kinases play an important role in the invasion of Trypanosoma cruzi into primary resident macrophages. In the present study we carry out immunofluorescence assays, using monoclonal anti-phosphotyrosine antibodies, to reveal an accumulation of tyrosine-phosphorylated residues at the site of parasite association with the macrophage surface, colocalizing with host cell F-actin-rich domains. SDS-PAGE analysis of macrophage cell line IC-21 tyrosine phosphoproteins, labeled with [(35)S] L-methionine, revealed several peptides with increased levels of phosphorylation upon interaction with the parasite. Among them, were detected bands of 140, 120, 112, 94, 73, 67, and 56 kDa that match the molecular weights of proteins described as being tyrosine phosphorylated during events that lead to actin assembly in mononuclear phagocytes. The pretreatment of IC-21 macrophages with the tyrosine kinase inhibitor tyrphostin 23 inhibited trypomastigote uptake showing that tyrosine phosphorylation is important for the parasite penetration in this particular cell line. Immunofluorescence microscopy, using antibodies against p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), placed this enzyme also in the same sites, in accordance to what is reported for phagocytosis. We suggest that once the components of T. cruzi trypomastigotes surface are recognized by macrophage receptors, they trigger the activation of a tyrosine phosphorylation cascade, PI 3-kinase recruitment, and assembly of actin filaments at the site of initial cell-to-cell contact, resembling the events described during phagocytosis. These achievements support the model for a phagocytic-like actin-dependent invasion mechanism for T. cruzi trypomastigotes into macrophages.

Silica-induced apoptosis in murine macrophage: involvement of tumor necrosis factor-alpha and nuclear factor-kappaB activation.

Alveolar macrophages play a critical role in silica-induced lung fibrosis. Silica exposure induces tumor necrosis factor (TNF)-alpha release and nuclear factor (NF)-kappaB activation, and apoptotic mechanisms have been implicated in silica-induced pathogenesis. To characterize potential relationships between these signaling events, we studied their induction in two murine macrophage cell lines. The RAW 264.7 macrophage cell line was more sensitive, and the IC-21 macrophage cell line more tolerant to silica exposure (0.2 or 1 mg/ml for 6 h) as evidenced by significantly higher apoptotic responses in RAW 264.7 (P < 0.05). RAW 264.7 macrophages exhibited enhanced TNF-alpha production and NF-kappaB activation in response to silica, whereas IC-21 macrophages did not produce TNF-alpha in response to silica and did not induce NF-kappaB nuclear binding. Inhibition of NF-kappaB in RAW 264.7 cells with BAY11-7082 significantly increased apoptosis while inhibiting TNF-alpha release. In addition, TNF-alpha and NF-kappaB activation, but not apoptosis, were induced by lipopolysaccharide (LPS) in both cell lines, and NF-kappaB inhibition reduced LPS-induced TNF-alpha release. These data suggest that TNF-alpha induction is dependent on NF-kappaB activation in both cell lines. However, silica can induce apoptosis in murine macrophages, independently of TNF-alpha stimulation, as in IC-21 macrophages. Furthermore, NF-kappaB activation in macrophages may play dual roles, both pro- and antiapoptotic during silica injury.

Estradiol signaling via sequestrable surface receptors.

Estradiol (E(2))-signaling is widely considered to be exclusively mediated through the transcription-regulating intracellular estrogen receptor (ER) alpha and ERbeta. The aim of this study was to investigate transcription-independent E(2)-signaling in mouse IC-21 macrophages. E(2) and E(2)-BSA induce a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) of Fura-2 loaded IC-21 cells as examined by spectrofluorometry. These changes in [Ca(2+)](i) can be inhibited by pertussis toxin, but not by the ER-blockers tamoxifen and raloxifene. The E(2)-signaling initiated at the plasma membrane is mediated through neither ERalpha nor ERbeta, but rather through a novel G protein-coupled membrane E(2)-receptor as revealed by RT-PCR, flow cytometry, and confocal laser scanning microscopy. A special feature of this E(2)-receptor is its sequestration upon agonist stimulation. Sequestration depends on energy and temperature, and it proceeds through a clathrin- and caveolin-independent pathway.

Murine cytomegalovirus M78 protein, a G protein-coupled receptor homologue, is a constituent of the virion and facilitates accumulation of immediate-early viral mRNA

The M78 protein of murine cytomegalovirus exhibits sequence features of a G protein-coupled receptor. It is synthesized with early kinetics, it becomes partially colocalized with Golgi markers, and it is incorporated into viral particles. We have constructed a viral substitution mutant, SMsubM78, which lacks most of the M78 ORF. The mutant produces a reduced yield in cultured 10.1 fibroblast and IC21 macrophage cell lines. The defect is multiplicity dependent and greater in the macrophage cell line. Consistent with its growth defect in cultured cells, the mutant exhibits reduced pathogenicity in mice, generating less infectious progeny than wild-type virus in all organs assayed. SMsubM78 fails to efficiently activate accumulation of the viral m123 immediate-early mRNA in infected macrophages. M78 facilitates the accumulation of the immediate-early mRNA in cycloheximide-treated cells, arguing that it acts in the absence of de novo protein synthesis. We conclude that the M78 G protein-coupled receptor homologue is delivered to cells as a constituent of the virion, and it acts to facilitate the accumulation of immediate-early mRNA.

PYK2 is an adhesion kinase in macrophages, localized in podosomes and activated by beta(2)-integrin ligation.

Pyk2 is a member of the focal adhesion kinase (FAK) family, highly expressed in the central nervous system and haemopoietic cells. Although Pyk2 is homologous to FAK, its role in signaling pathways was shown to be distinct from that of FAK. We show here that Pyk2 is highly expressed in peritoneal IC-21 macrophage and is tyrosine phosphorylated in response to cell attachment to fibronectin and fibrinogen. Upon IC-21 cell adhesion, Pyk2 tyrosine phosphorylation is inhibited by blocking antibodies to the integrin subunits alpha(M) and beta(2). Furthermore, Pyk2 is rapidly tyrosine phosphorylated in response to ligation of beta(2) integrins by antibodies. In migrating macrophages, Pyk2 localizes to perinuclear regions and to podosomes, where it is clustered with tyrosine phosphorylated proteins. Furthermore, in the podosomal ring structure, which surrounds the central actin core, Pyk2 co-localizes with vinculin, talin, and paxillin. In the podosomes, Pyk2 also co-localizes with the integrin alpha(M)beta(2). Lastly, reduction of Pyk2 expression in macrophages leads to inhibition of cell migration. We propose that Pyk2 is functionally linked to the formation of podosomes where it mediates the integrin-cytoskeleton interface and regulates cell spreading and migration.

Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages.

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca(2+)], which is due to release of Ca(2+) from intracellular Ca(2+) stores. This Ca(2+) mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.