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Regulation of CRIg Expression and Phagocytosis in Human Macrophages by Arachidonate, Dexamethasone, and Cytokines

  • Nick N. Gorgani
    Correspondence
    Address reprint requests to Nick N. Gorgani, M.D., Ph.D., Department of Immunopathology, Women's and Children's Hospital, 72 King William Rd, North Adelaide, SA 5006, Australia
    Affiliations
    Department of Immunopathology, South Australia Pathology, Women's and Children's Hospital Campus, North Adelaide, Australia

    Discipline of Paediatrics, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia
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  • Umaporn Thathaisong
    Affiliations
    Department of Immunopathology, South Australia Pathology, Women's and Children's Hospital Campus, North Adelaide, Australia

    Discipline of Paediatrics, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia
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  • Violet R.S. Mukaro
    Affiliations
    Department of Immunopathology, South Australia Pathology, Women's and Children's Hospital Campus, North Adelaide, Australia

    Discipline of Paediatrics, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia
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  • Ornnuthchar Poungpair
    Affiliations
    Department of Immunopathology, South Australia Pathology, Women's and Children's Hospital Campus, North Adelaide, Australia

    Discipline of Paediatrics, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia
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  • Amanda Tirimacco
    Affiliations
    Department of Immunopathology, South Australia Pathology, Women's and Children's Hospital Campus, North Adelaide, Australia

    Sansom Institute, University of South Australia, Adelaide, Australia
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  • Charles S.T. Hii
    Affiliations
    Department of Immunopathology, South Australia Pathology, Women's and Children's Hospital Campus, North Adelaide, Australia

    Discipline of Paediatrics, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia
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  • Antonio Ferrante
    Affiliations
    Department of Immunopathology, South Australia Pathology, Women's and Children's Hospital Campus, North Adelaide, Australia

    Discipline of Paediatrics, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia

    Sansom Institute, University of South Australia, Adelaide, Australia
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Open ArchivePublished:July 11, 2011DOI:https://doi.org/10.1016/j.ajpath.2011.05.021
      Although the importance of the macrophage complement receptor immunoglobulin (CRIg) in the phagocytosis of complement opsonized bacteria and in inflammation has been established, the regulation of CRIg expression remains undefined. Because cellular activation during inflammation leads to the release of arachidonate, a stimulator of leukocyte function, we sought to determine whether arachidonate regulates CRIg expression. Adding arachidonate to maturing human macrophages and to prematured CRIg+ macrophages caused a significant decrease in the expression of cell-surface CRIg and CRIg mRNA. This effect was independent of the metabolism of arachidonate via the cyclooxygenase and lipoxygenase pathways, because it was not inhibited by the nonsteroidal anti-inflammatory drugs indomethacin and nordihydroguaiaretic acid. Studies with specific pharmacological inhibitors of arachidonate-mediated signaling pathways showed that protein kinase C was involved. Administration of dexamethasone to macrophages caused an increase in CRIg expression. Studies with proinflammatory and immunosuppressive cytokines showed that IL-10 increased, but interferon-γ, IL-4, and transforming growth factor-β1 decreased CRIg expression on macrophages. This down- and up-regulation of CRIg expression was reflected in a decrease and increase, respectively, in the phagocytosis of complement opsonized Candida albicans. These data suggest that a unique inflammatory mediator network regulates CRIg expression and point to a mechanism by which arachidonate and dexamethasone have reciprocal effects on inflammation.
      Recently, we have defined a new subset of tissue resident macrophages with distinct properties based on the presence of complement receptor immunoglobulin (CRIg) on their surfaces.
      • Gorgani N.N.
      • He J.Q.
      • Katschke Jr, K.J.
      • Helmy K.Y.
      • Xi H.
      • Steffek M.
      • Hass P.E.
      • van Lookeren Campagne M.
      Complement receptor of the Ig superfamily enhances complement-mediated phagocytosis in a subpopulation of tissue resident macrophages.
      CRIg mediates rapid phagocytosis of complement (C3b/iC3b) opsonized pathogens such as Listeria monocytogenes and Staphylococcus aureus and of IgM-coated erythrocytes, leading to effective sequestration of bacteria within Kupffer cells, thus protecting the host by limiting bacterial dissemination and evolution of a cytokine storm.
      • Helmy K.Y.
      • Katschke Jr, K.J.
      • Gorgani N.N.
      • Kljavin N.M.
      • Elliott J.M.
      • Diehl L.
      • Scales S.J.
      • Ghilardi N.
      • van Lookeren Campagne M.
      CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.
      Recombinant CRIg-Fc ameliorates experimental arthritis
      • Katschke Jr, K.J.
      • Helmy K.Y.
      • Steffek M.
      • Xi H.
      • Yin J.
      • Lee W.P.
      • Gribling P.
      • Barck K.H.
      • Carano R.A.
      • Taylor R.E.
      • Rangell L.
      • Diehl L.
      • Hass P.E.
      • Wiesmann C.
      • van Lookeren Campagne M.
      A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis.
      and ischemia/reperfusion injury
      • Chen J.
      • Crispín J.C.
      • Dalle Lucca J.
      • Tsokos G.C.
      A novel inhibitor of the alternative pathway of complement attenuates intestinal ischemia/reperfusion-induced injury.
      and depresses both humoral and cellular immunity.
      • Vogt L.
      • Schmitz N.
      • Kurrer M.O.
      • Bauer M.
      • Hinton H.I.
      • Behnke S.
      • Gatto D.
      • Sebbel P.
      • Beerli R.R.
      • Sonderegger I.
      • Kopf M.
      • Saudan P.
      • Bachmann M.F.
      VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.
      Thus, we regard CRIg as a profound anti-inflammatory protein. Unlike CR3, which requires divalent cations, cytokine-induced preactivation and multimerization, CRIg binds readily with high affinity to monomeric complement breakdown products and opsonized pathogens in a divalent-cation-independent manner. Thus, CRIg-dependent uptake of monomeric complement products and opsonized particles can occur under noninflammatory physiological conditions.
      • Gorgani N.N.
      • He J.Q.
      • Katschke Jr, K.J.
      • Helmy K.Y.
      • Xi H.
      • Steffek M.
      • Hass P.E.
      • van Lookeren Campagne M.
      Complement receptor of the Ig superfamily enhances complement-mediated phagocytosis in a subpopulation of tissue resident macrophages.
      • Helmy K.Y.
      • Katschke Jr, K.J.
      • Gorgani N.N.
      • Kljavin N.M.
      • Elliott J.M.
      • Diehl L.
      • Scales S.J.
      • Ghilardi N.
      • van Lookeren Campagne M.
      CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.
      • Vogt L.
      • Schmitz N.
      • Kurrer M.O.
      • Bauer M.
      • Hinton H.I.
      • Behnke S.
      • Gatto D.
      • Sebbel P.
      • Beerli R.R.
      • Sonderegger I.
      • Kopf M.
      • Saudan P.
      • Bachmann M.F.
      VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.
      Although studies on CRIg expression have focused on tissue macrophages, there is also evidence that monocytes can develop into CRIg+ macrophages.
      • Kim J.K.
      • Choi E.M.
      • Shin H.I.
      • Kim C.H.
      • Hwang S.H.
      • Kim S.M.
      • Kwon B.S.
      Characterization of monoclonal antibody specific to the Z39Ig protein, a member of immunoglobulin superfamily.
      In view of the importance of CRIg in promoting bacterial phagocytosis/clearance in a noninflammatory manner, its regulation on tissue infiltrating macrophages derived from blood monocytes is likely to be of importance.
      We have been interested in studying the immunomodulatory properties of polyunsaturated fatty acids in phagocyte function.
      • Ferrante A.
      • Hii C.S.
      • Costabile M.
      Regulation of neutrophil functions by long chain fatty acids.
      Enrichment of cellular membrane phospholipids with the ω-6 polyunsaturated fatty acid arachidonate leads to a potentially highly inflammatory environment. Many exogenous and endogenous inflammatory mediators induce the activation of phospholipase A2 (PLA2), leading to the release of arachidonate from membrane phospholipids.
      • Ferrante A.
      • Hii C.S.
      • Costabile M.
      Regulation of neutrophil functions by long chain fatty acids.
      In turn, arachidonate stimulates several responses in leukocytes,
      • Ferrante A.
      • Hii C.S.
      • Costabile M.
      Regulation of neutrophil functions by long chain fatty acids.
      • Huang Z.H.
      • Hii C.S.
      • Rathjen D.A.
      • Poulos A.
      • Murray A.W.
      • Ferrante A.
      N-6 and n-3 polyunsaturated fatty acids stimulate translocation of protein kinase Calpha, -betaI, -betaII and -epsilon and enhance agonist-induced NADPH oxidase in macrophages.
      stimulating the release of cytokines and oxygen-derived reactive species and increasing cell-surface receptors. Given that CRIg plays an important role in immunity to infection and the regulation of inflammation,
      • Gorgani N.N.
      • He J.Q.
      • Katschke Jr, K.J.
      • Helmy K.Y.
      • Xi H.
      • Steffek M.
      • Hass P.E.
      • van Lookeren Campagne M.
      Complement receptor of the Ig superfamily enhances complement-mediated phagocytosis in a subpopulation of tissue resident macrophages.
      • Helmy K.Y.
      • Katschke Jr, K.J.
      • Gorgani N.N.
      • Kljavin N.M.
      • Elliott J.M.
      • Diehl L.
      • Scales S.J.
      • Ghilardi N.
      • van Lookeren Campagne M.
      CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.
      • Vogt L.
      • Schmitz N.
      • Kurrer M.O.
      • Bauer M.
      • Hinton H.I.
      • Behnke S.
      • Gatto D.
      • Sebbel P.
      • Beerli R.R.
      • Sonderegger I.
      • Kopf M.
      • Saudan P.
      • Bachmann M.F.
      VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.
      we examined the effects of arachidonate on CRIg expression in macrophages. Its effects were compared with those caused by steroidal and nonsteroidal anti-inflammatory agents and by cytokines that have proinflammatory and anti-inflammatory activities.
      Our findings demonstrated that arachidonate down-regulates CRIg expression in primary human monocytes during their maturation into macrophages, as well as in matured CRIg+ macrophages, in a protein kinase C (PKC)-dependent manner. Of note, although nonsteroidal anti-inflammatory agents had no effect, dexamethasone increased CRIg expression on macrophages. Furthermore, investigations on the effects of IFN-γ, IL-4, IL-10, and TGF-β1 revealed a unique cytokine network operating to control CRIg expression on macrophages.

      Materials and Methods

      Reagents

      Arachidonate was purchased from Cayman Chemical (Ann Arbor, MI). Primers for CRIg were designed using Oligo Perfect Designer (Invitrogen, Carlsbad, CA). Forward and reverse primers used to amplify human CRIg cDNA were 5′-TCCTGGAAGTGCCAGAGAGT-3′ and 5′-TGTACCAGCCACTTCACCAA-3′, respectively. These primers were synthesized by Invitrogen (Mulgrave, Australia). RNeasy Plus total RNA purification kit was purchased from Qiagen (Doncaster, Victoria, Australia; Valencia, CA). Indomethacin, nordihydroguaiaretic acid, wortmannin, GF109203X, and phorbol myristate acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO) and PD98059 from Cell Signaling Technology (Danvers, MA). A mouse monoclonal antibody that recognizes the IgV domain of human CRIg was kindly provided by Dr. Menno van Lookeren Campagne (Genentech, South San Francisco, CA). Recombinant cytokines, IFNγ, and IL-10 were purchased from ProSpec-Tany Technogene (Rehovot, Israel) and IL-4 and TGF-β1 from R&D Systems (Minneapolis, MN).

      Purification of Cells

      Venous blood was collected from healthy volunteers under guidelines of the human ethics committee of the Children, Youth and Women's Health Service. Peripheral blood mononuclear cells (PBMCs) were prepared by passing the heparinized blood through Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden; Little Chalfont, UK). The interface layer containing PBMCs was washed three times in RPMI-1640 medium supplemented with 10% fetal calf serum, glutamine, antibiotics, and 10 mmol/L HEPES, pH 7.4 (RPMI-FCS). Cells were counted using an automated Cell-Dyn 3500R analyzer (Abbott Laboratories, Abbott Park, IL) and viability was determined by counting the number of Trypan Blue-excluding cells, using a hemocytometer. Monocytes were purified from PBMCs by density gradient centrifugation, as described previously.
      • Seager Danciger J.
      • Lutz M.
      • Hama S.
      • Cruz D.
      • Castrillo A.
      • Lazaro J.
      • Phillips R.
      • Premack B.
      • Berliner J.
      Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation.
      • Marantos C.
      • Mukaro V.
      • Ferrante J.
      • Hii C.
      • Ferrante A.
      Inhibition of the lipopolysaccharide-induced stimulation of the members of the MAPK family in human monocytes/macrophages by 4-hydroxynonenal, a product of oxidized omega-6 fatty acids.
      Briefly, PBMCs were washed with RPMI-FCS containing 1 mmol/L EDTA at room temperature. Cells were layered on a 46% iso-osmotic Percoll gradient (GE Healthcare) and spun at 600 × g for 30 minutes at room temperature. The monocyte-containing layer was washed twice with ice-cold RPMI-FCS containing 1 mmol/L EDTA at 600 × g for 5 minutes at 4°C. Monocytes were >90% pure as judged by staining the cells with human monocyte marker CD14.

      Production of Cytokine-Rich Fluids

      PBMCs were cultured in RPMI-FCS medium containing the T-cell mitogen phytohemagglutinin (PHA) for 3 days and then the cell-free culture fluids were harvested and used as a source of cytokines.
      • Staugas R.
      • Harvey D.
      • Ferrante A.
      • Nandoskar M.
      • Allison A.
      Induction of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by Pseudomonas aeruginosa and exotoxin A-induced suppression of lymphoproliferation and TNF, lymphotoxin, gamma interferon, and IL-1 production in human leukocytes.

      Cell Culture

      PBMCs or purified monocytes were cultured in RPMI-FCS in humidified air containing 5% CO2 at 37°C at a density of 0.4 × 106 cells/mL in the presence or absence of inhibitors, arachidonate, PMA, cytokines, or dexamethasone. Arachidonate was kept in ethanol at −70°C until use. On the day of use, arachidonate was brought to room temperature, desired amounts were transferred to sterile and endotoxin-free glass tubes, and ethanol was evaporated under a stream of nitrogen. Fetal bovine serum was added to the tube and mixed gently to dissolve arachidonate before addition to the RPMI-FCS. PMA (5 ng/mL) and dexamethasone (30 ng/mL) were delivered in 0.1% dimethyl sulfoxide and 0.1% ethanol, respectively. In some experiments, inhibitors of arachidonate-activating pathways were incubated with the cells before addition of arachidonate. In other studies, the monocytes were cultured in the presence of cytokine-rich PBMC conditioned medium, rIFN-γ, IL-4, IL-10, and TGF-β1 at 20 ng/mL.

      Purification and Assessment of Quality of Total RNA

      Cells were harvested by gentle scraping and then washed once. The pellets were lysed in RLT Plus buffer (Qiagen) and kept frozen at −70°C until use. Total RNA was isolated using an RNeasy Plus mini kit (Qiagen) according to the manufacturer's instructions. The quality and quantity of total RNA was assessed using an Experion automated electrophoresis system with an RNA StdSens analysis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions.

      Quantitative RT-PCR

      RNA was converted to cDNA using an iScript cDNA synthesis kit (Bio-Rad). The cDNA was then amplified in triplicate reactions with iQ SYBR Green Supermix (Bio-Rad) and 500 nmol/L of each primer pair for CRIg and the housekeeping gene GAPDH, using an iQ5 real-time PCR detection system with iQ5 optical system software version 2.1 (Bio-Rad).

      Examination of CRIg Expression on the Macrophage Surface

      After in vitro differentiation, the monocyte-derived macrophages were harvested, tested for viability, and processed for determining the level of surface CRIg expression essentially as described previously.
      • Gorgani N.N.
      • He J.Q.
      • Katschke Jr, K.J.
      • Helmy K.Y.
      • Xi H.
      • Steffek M.
      • Hass P.E.
      • van Lookeren Campagne M.
      Complement receptor of the Ig superfamily enhances complement-mediated phagocytosis in a subpopulation of tissue resident macrophages.
      Cells were blocked in 10 μg/mL Intragam human immunoglobulin preparation (CSL Ltd, Parkville, Australia) and 5% human serum. Cells were then incubated with monoclonal mouse anti-human CD14-fluorescein isothiocyanate antibody (BD Biosciences, North Ryde, Australia; San Jose, CA) and either anti-human CRIg antibody (Genentech) or phycoerythrin-labeled IgG1 isotype (BD Biosciences) that had been labeled with a Lightning-Link R-phycoerythrin conjugation kit (Innova Biosciences, Cambridge, UK) according to the manufacturer's instructions for 30 minutes at 4°C. The cells were analyzed by flow cytometry using a FACSCalibur system (BD Biosciences).

      Phagocytosis Assay

      To 2.5 × 105 macrophages were added 1 × 106 Candida albicans organisms in a final volume of 0.5 mL in HBSS. Complement containing human blood group AB was added to a final concentration of 10%. The cells were incubated with end-to-end mixing at 37°. After 45 minutes, the unphagocytosed fungi were remove by differential centrifugation at 175 × g for 10 minutes and then the macrophages in the pellet were resuspended and cytocentrifuged on a microscope slide and stained with Giemsa stain. The number of particles in phagocytic vacuoles was then determined.

      Statistical Analysis

      Unpaired comparisons were analyzed using the two-tailed Student's t-test and multiple comparisons were performed using Dunnett's test, with P < 0.05 considered significant.

      Results

      Arachidonate Depresses Expression of CRIg in Macrophages

      Initial studies demonstrated that human peripheral blood monocytes expressed CRIg protein on their surface during their maturation into macrophages in culture. These initial studies showed that cell-surface CRIg expression was maximal by day 7 of culture. To examine whether arachidonate alters this expression, purified monocytes were cultured in the presence of arachidonate. These were then examined for expression levels of this receptor on day 7 using a PE-labeled anti-CRIg monoclonal antibody. By day 7 of culture, the matured macrophages expressed CRIg protein but in the presence of arachidonate at 20 μmol/L there was a significant reduction in this expression (Figure 1A). The CRIg macrophages remained as a single population under these conditions, as can be seen from a representative experimental run (Figure 1B).
      Figure thumbnail gr1
      Figure 1Regulation of CRIg protein levels on the surface of monocyte-derived human macrophages. A: CRIg protein expression on monocyte-derived macrophages cultured for 7 days in the absence or presence of 10 or 20 μmol/L arachidonate was significantly down-regulated at the higher concentration. Down-regulation data are presented as means ± SEM of three experiments, each conducted with cells from a different individual. **P < 0.01. B: A representative experimental run for the data in A shows that the individual populations were maintained. FIU, fluorescence intensity units.

      Effects of Arachidonate on CRIg mRNA Expression

      To determine whether the effects of arachidonate are pretranscriptional, CRIg mRNA expression was examined using quantitative RT-PCR. Although freshly isolated human PBMCs contained no detectable CRIg mRNA, culturing these cells in RPMI-FCS medium for several days resulted in increased CRIg mRNA levels (Figure 2). In all human donor PBMCs that we have tested, a kinetic analysis of increase in CRIg mRNA (data not shown) indicated that CRIg mRNA reached maximum levels at 3 days of culture. Therefore, compared with the noncultured PBMCs, the relative mRNA levels of CRIg increased by approximately 10-fold in cells cultured for 3 days (Figure 2A). The effect of arachidonate on CRIg mRNA levels was then examined. Incubation of PBMCs in the presence of RPMI-FCS medium containing arachidonate resulted in decreased CRIg mRNA levels in an arachidonate concentration-dependent manner (Figure 2B). Similar results were obtained using purified monocytes. Arachidonate again caused a significant decrease in CRIg mRNA expression on monocytes (Figure 2C). Treatment of purified monocytes with arachidonate did not alter the viability and final number of cells (data not shown), nor the quality of RNA samples (data not shown). Similar results were also obtained in PBMC cultures, and none of the agents used in the present study at specified amounts altered cell viability and the quality of the RNA samples (data not shown).
      Figure thumbnail gr2
      Figure 2Regulation of CRIg mRNA levels on PBMCs and purified monocytes. A: Levels of CRIg mRNA, relative to the housekeeping gene GAPDH, increased approximately 10-fold between day 0 (fresh PBMCs) and day 3 after culturing of PBMCs in RPMI-FCS medium. B: After culture for 3 days in the presence of arachidonate, CRIg mRNA levels in PBMCs were significantly decreased in a concentration-dependent manner. C: A similar effect of arachidonate was seen with purified monocytes. Data are presented as means ± SEM of three experiments, each conducted with cells from a different individual. *P < 0.05, **P < 0.01.

      Cytokines Regulate CRIg Expression

      Previous studies have shown that inflammatory infiltrating monocytes/macrophages do not express CRIg.
      • Vogt L.
      • Schmitz N.
      • Kurrer M.O.
      • Bauer M.
      • Hinton H.I.
      • Behnke S.
      • Gatto D.
      • Sebbel P.
      • Beerli R.R.
      • Sonderegger I.
      • Kopf M.
      • Saudan P.
      • Bachmann M.F.
      VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.
      We hypothesized that this could have been caused by the presence of cytokines in inflammatory foci. To mimic such conditions, we tested the effect of a T-cell mitogen, PHA, on CRIg mRNA levels in PBMC cultures. Activation of PBMCs by PHA results in secretion of Th1 cytokines [such as IFNγ, lymphotoxin (LT), IL-2, and tumor necrosis factor (TNF)] that may inhibit CRIg expression during maturation of monocytes to macrophages. Incubation of PBMCs in the presence of PHA resulted in a substantial decrease in CRIg mRNA levels (Figure 3A). In addition, conditioned medium that was recovered from PHA-stimulated PBMC cultures known to contain cytokines
      • Staugas R.
      • Harvey D.
      • Ferrante A.
      • Nandoskar M.
      • Allison A.
      Induction of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by Pseudomonas aeruginosa and exotoxin A-induced suppression of lymphoproliferation and TNF, lymphotoxin, gamma interferon, and IL-1 production in human leukocytes.
      down-regulated CRIg mRNA levels during maturation of purified monocytes to macrophages (Figure 3B).
      Figure thumbnail gr3
      Figure 3Effects of cytokines on CRIg mRNA expression. A: Incubation in the presence of PHA significantly down-regulated CRIg mRNA levels during PBMC culture. B: Conditioned medium from PHA-stimulated PBMCs (PHA-CM) significantly down-regulated CRIg expression during maturation of purified monocyte to macrophages. C: Treatment with 20 ng/mL of recombinant IL-4, TGF-β1, and IFN-γ significantly down-regulated and IL-10 significantly up-regulated CRIg expression during maturation of purified monocytes to macrophages. PBMCs and monocytes were assayed for CRIg mRNA levels after 3 days of culture. Data are presented as means ± SEM of three or four experiments, each conducted with cells from a different individual. *P < 0.05, **P < 0.01, and ***P < 0.001.
      A more extensive investigation using recombinant cytokines revealed unique regulatory characteristics of the cytokine network. Treatment with IFN-γ caused a reduction in CRIg mRNA expression during the maturation of monocytes to macrophages (Figure 3C). Notably, both IL-4 and TGF-β1 caused a decrease in CRIg expression; in contrast, IL-10 increased CRIg expression in macrophages (Figure 3C).

      Arachidonate Regulates CRIg Expression in a PKC-Dependent Manner

      We have shown that arachidonate uses several different intracellular signaling pathways to activate cellular responses, including PI3 kinase
      • Hii C.S.
      • Moghadammi N.
      • Dunbar A.
      • Ferrante A.
      Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family.
      and ERK 1/2.
      • Hii C.S.
      • Ferrante A.
      • Edwards Y.S.
      • Huang Z.H.
      • Hartfield P.J.
      • Rathjen D.A.
      • Poulos A.
      • Murray A.W.
      Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism.
      In human monocyte-derived macrophages, we have previously demonstrated that arachidonate causes the activation of several PKC isozymes.
      • Huang Z.H.
      • Hii C.S.
      • Rathjen D.A.
      • Poulos A.
      • Murray A.W.
      • Ferrante A.
      N-6 and n-3 polyunsaturated fatty acids stimulate translocation of protein kinase Calpha, -betaI, -betaII and -epsilon and enhance agonist-induced NADPH oxidase in macrophages.
      To investigate whether these signaling molecules regulate the expression of CRIg, chemical inhibitors with an acceptable level of specificity were used. The PBMCs were pretreated with the inhibitors for 60 minutes before the addition of arachidonate. After 3 days of culture, the cells were harvested and CRIg mRNA was measured. The data indicate that neither the PI3 kinase inhibitor wortmannin nor the MEK inhibitor PD98059, which blocks ERK signaling
      • Hii C.
      • Stacey K.
      • Moghaddami N.
      • Murray A.
      • Ferrante A.
      Role of the extracellular signal-regulated protein kinase cascade in human neutrophil killing of Staphylococcus aureus and Candida albicans and in migration.
      had any effect on the action of arachidonate (Figure 4A). The p38 inhibitor SB203580 also did not affect CRIg mRNA level (data not shown). The selective PKC inhibitor GF109203X, however, completely reversed the effect of arachidonate in a concentration-dependent manner (Figure 4B). This finding was supported by the action of the direct PKC activator PMA, which, like arachidonate, markedly down-regulated expression of CRIg during maturation of purified monocytes to macrophages (Figure 4C).
      Figure thumbnail gr4
      Figure 4The role of intracellular signaling molecules on arachidonate-induced inhibition of CRIg expression. A: Preincubation with wortmannin (50 nmol/L) or PD98059 (20 μmol/L) did not affect arachidonate (50 μmol/L)-induced inhibition of CRIg mRNA levels in PBMCs after 3 days of culture. B: Preincubation with GF109203X (1 and 2 μmol/L) reversed this response, in a concentration-dependent manner. C: PMA (5 ng/mL) down-regulated expression of CRIg in PBMCs after 3 days of culture. D: Preincubation with indomethacin (10 μmol/L) or with nordihydroguaiaretic acid (10 μmol/L) had no effect on arachidonate (50 μmol/L)-induced inhibition of CRIg mRNA levels in PBMCs after 3 days of culture. The results are presented as means ± SEM of three experiments, each conducted with cells from a different individual. The significance of inhibition and reversal of the decrease by inhibitors, *P < 0.05, **P < 0.01, and ***P < 0.001.

      The Role of Metabolism via Cyclooxygenase and Lipoxygenase Pathways

      One way by which arachidonate may have acted is through its metabolism via the cyclooxygenase and lipoxygenase pathways, as well as generation of eicosanoids, leukotrienes, and prostaglandins.
      • Calder P.C.
      • Yaqoob P.
      • Thies F.
      • Wallace F.A.
      • Miles E.A.
      Fatty acids and lymphocyte functions.
      • Serhan C.N.
      Resolution phase of inflammation: novel endogenous anti-inflammatory and proresolving lipid mediators and pathways.
      This possibility was excluded, however, because nordihydroguaiaretic acid, a lipoxygenase inhibitor, and indomethacin, an inhibitor of cyclooxygenase 1 and 2, did not affect the ability of arachidonate to inhibit CRIg mRNA expression in PBMCs (Figure 4D).

      Effects of the Steroidal Anti-Inflammatory Agent Dexamethasone on CRIg Expression

      In our studies, it was evident that the nonsteroidal anti-inflammatory agents indomethacin and nordihydroguaiaretic acid had no effect on CRIg expression in macrophages. It was therefore of interest to see whether dexamethasone, which is known to alter macrophage function (including the expression of CR3), can alter CRIg expression in macrophages. Previous studies have demonstrated that dexamethasone down-regulates CR3 expression on myeloid cells.
      • Hartnell A.
      • Kay A.B.
      • Wardlaw A.J.
      Interleukin-3-induced up-regulation of CR3 expression on human eosinophils is inhibited by dexamethasone.
      Here, we examined the effects of dexamethasone on the surface expression of CRIg protein during monocyte maturation to macrophages. Purified monocytes were cultured for 7 days in the presence of dexamethasone and then were examined for cell-surface CRIg protein levels by flow cytometry analysis. The results demonstrated that dexamethasone caused severalfold increase in CRIg expression (Figure 5A), which comprised a single macrophage population (Figure 5B).
      Figure thumbnail gr5
      Figure 5Effect of dexamethasone on CRIg expression. A: CRIg protein levels were increased severalfold on monocyte-derived macrophages cultured for 7 days in the presence of dexamethasone. B: A representative experimental run for the data in A shows that the individual populations were maintained. C: In the presence of dexamethasone, CRIg mRNA levels were increased severalfold during 3 days of PBMC culture. D: The effect of dexamethasone on CRIg mRNA levels in PBMC cultures over the first 24 hours of culture was time-dependent. Data are presented as means ± SEM of three experiments, each conducted with cells from a different individual. ***P < 0.001.
      We also examined the effects of dexamethasone at the level of CRIg mRNA by quantitative RT-PCR. Culturing PBMCs in RPMI-FCS in the presence of dexamethasone for 3 days resulted in a profound increase in CRIg mRNA levels (Figure 5C). When analyzing the kinetics of the effects of dexamethasone, the incubation of PBMCs in the presence of dexamethasone resulted in a time-dependent increase in CRIg mRNA levels, which first became evident at 6 hours of culture (Figure 5D).

      Arachidonate and IFN-γ Depress but Dexamethasone Increases Phagocytosis of Complement Opsonized Fungi

      Studies were undertaken to determine whether the changes in CRIg expression induced by arachidonate, IFN-γ, and dexamethasone correspond to changes in phagocytosis of microbial pathogens by macrophages. Monocytes were cultured for 7 days in the presence of arachidonate (100 μmol/L), IFN-γ (20 ng/mL), or dexamethasone (30 ng/mL) and then examined for phagocytic activity. The macrophages were incubated at 37°C with C. albicans and serum complement. After 45 minutes, smears were prepared by cytocentrifugation and stained. The number of fungi in phagocytic vacuoles was scored microscopically.
      The results demonstrated that arachidonate and IFN-γ caused a significant reduction in the phagocytosis of the fungi, compared with control macrophages (Figure 6, A and B). In contrast, dexamethasone caused a significant increase (Figure 6C). When macrophages were scored as those having engulfed >4 fungi, dexamethasone caused a threefold increase and arachidonate and IFN-γ a >70% decrease. Similar results were found when the scoring was based on the number of fungi engulfed per cell (Figure 6).
      Figure thumbnail gr6
      Figure 6Effects of modulating CRIg expression on macrophage phagocytosis. Macrophages were cultured in the presence of 100 μmol/L arachidonate (A), 20 ng/mL IFN-γ (B), or 30 ng/mL dexamethasone (C). After 7 days, the cells were examined for their ability to phagocytose complement opsonized C. albicans. Phagocytosis was scored as both the number of macrophages that had engulfed >4 fungi and the number of fungi engulfed per cell. Data are presented as means ± SEM of four, six, and four experiments, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001.

      Modulation of CRIg Expression on CRIg+ Macrophages by Arachidonate and Dexamethasone

      Although our findings demonstrated a regulatory role for arachidonate and dexamethasone in CRIg expression, these data did not delineate whether these agents act directly on the expression of CRIg or indirectly through affecting the macrophage maturation that leads to changes in CRIg expression. We therefore examined the effects of arachidonate and dexamethasone on CRIg expression in prematured macrophages. Monocytes were allowed to mature in culture and after 3 days were treated with diluent, arachidonate, or dexamethasone. After 24 hours of incubation, the level of CRIg mRNA expression was determined. Arachidonate down-regulated but dexamethasone up-regulated CRIg mRNA levels in the macrophages (Figure 7A). The up-regulation by dexamethasone was time-dependent, becoming first evident at 3 hours of incubation (Figure 7B) and increasing to fourfold by 24 hours.
      Figure thumbnail gr7
      Figure 7Regulation of CRIg mRNA levels on matured human macrophages. A: In 3 days-matured human monocyte-derived macrophages, CRIg mRNA expression was down-regulated by 24-hour exposure to arachidonate, but was up-regulated by 24-hour exposure to dexamethasone. B: The effect of dexamethasone on CRIg mRNA levels on matured human monocyte-derived macrophages was time-dependent, reaching a fourfold increase over 24 hours; data are from a representative experiment conducted in triplicate. C: At the 20 μmol/L concentration, arachidonate significantly down-regulated surface CRIg protein expression on CRIg+ macrophages that matured in the presence of dexamethasone. Data are presented as means ± SEM of three experiments, each conducted with cells from a different individual (A and C). **P < 0.01.
      We also examined whether arachidonate down-regulates surface CRIg protein expression on macrophages. To facilitate these experiments, we enhanced the expression of CRIg by maturing the monocytes in the presence of dexamethasone for 7 days. These macrophages were then used in the experiments to examine the effects of arachidonate. Arachidonate caused a 75% drop in CRIg expression on macrophages within 2 hours (Figure 7C). This suggests that arachidonate also regulates CRIg expression at a post-translational level.

      Discussion

      Our data, from experiments using the proinflammatory fatty acid arachidonate and the steroidal anti-inflammatory dexamethasone, demonstrate that CRIg expression in human macrophages is subject to both positive and negative regulation. At concentrations of ≥20 μmol/L, arachidonate presented to cells in the presence of serum caused a significant decrease in CRIg expression at both the mRNA and protein levels. The findings demonstrate that, among the various signaling pathways that arachidonate activates,
      • Ferrante A.
      • Hii C.S.
      • Costabile M.
      Regulation of neutrophil functions by long chain fatty acids.
      • Huang Z.H.
      • Hii C.S.
      • Rathjen D.A.
      • Poulos A.
      • Murray A.W.
      • Ferrante A.
      N-6 and n-3 polyunsaturated fatty acids stimulate translocation of protein kinase Calpha, -betaI, -betaII and -epsilon and enhance agonist-induced NADPH oxidase in macrophages.
      • Hii C.S.
      • Ferrante A.
      • Edwards Y.S.
      • Huang Z.H.
      • Hartfield P.J.
      • Rathjen D.A.
      • Poulos A.
      • Murray A.W.
      Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism.
      PKC was primarily involved. In human macrophages, we have previously reported that arachidonate causes the translocation of PKCα, -βI, -βII, and -ε to a particulate fraction, which is a hallmark of PKC activation in macrophages.
      • Huang Z.H.
      • Hii C.S.
      • Rathjen D.A.
      • Poulos A.
      • Murray A.W.
      • Ferrante A.
      N-6 and n-3 polyunsaturated fatty acids stimulate translocation of protein kinase Calpha, -betaI, -betaII and -epsilon and enhance agonist-induced NADPH oxidase in macrophages.
      The ability of arachidonate to decrease CRIg expression in macrophages could be totally prevented by the addition of the PKC inhibitor GF109203X, which inhibits both classical and novel PKC isoforms. This conclusion is further supported by the finding that PMA, which uses PKC as its receptor, was also able to cause a decrease in the expression of CRIg. No evidence for a role of PI3 kinase, p38, or ERK1/ERK2 in the regulation of CRIg expression was obtained. Furthermore, the effects of arachidonate were unlikely to depend on the generation of eicosanoids via the cyclooxygenase and lipoxygenase pathways, because the inhibitors of these pathways did not prevent the arachidonate-induced decrease in CRIg expression. It is likely that the effects seen were achieved through the action of arachidonate as a free fatty acid, which is consistent with other effects of arachidonate on other cell-types that are independent of these pathways.
      • Ferrante A.
      • Hii C.S.
      • Costabile M.
      Regulation of neutrophil functions by long chain fatty acids.
      Previously, we have demonstrated that polyunsaturated fatty acids act through the Erb receptor family,
      • Hii C.S.
      • Moghadammi N.
      • Dunbar A.
      • Ferrante A.
      Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family.
      and this may be one means by which arachidonate caused the decrease in CRIg expression in macrophages.
      We found that, although the PKC-activating agents down-regulated the expression of CRIg, the levels of CRIg were up-regulated by dexamethasone. Macrophages that had undergone maturation in the presence of dexamethasone expressed significantly more CRIg on the cell surface than those matured in the presence of vehicle. The increase was accompanied by an increase in CRIg mRNA, observable after 6 hours of incubation with dexamethasone. This demonstrates that the up-regulation of CRIg protein expression was a consequence of dexamethasone increasing the levels of CRIg mRNA. The positive modulation of CRIg expression appeared to be restricted to steroidal anti-inflammatory agents, because nonsteroidal anti-inflammatory agents such as nordihydroguaiaretic acid and indomethacin, in addition to having no effect on the arachidonate-induced effect, had no effect on the basal levels of CRIg. These data therefore reveal a new target of corticosteroids such as dexamethasone, which may contribute to their anti-inflammatory action.
      The antithetical effects of arachidonate and dexamethasone on CRIg expression were not restricted to maturing macrophages. Incubation of in vitro matured macrophages with dexamethasone resulted in a further up-regulation in the expression of CRIg in these CRIg+ macrophages. Notably, arachidonate also decreased the expression of CRIg in macrophages that had been matured in the presence of dexamethasone. The finding that the down-regulation of surface CRIg expression by arachidonate was evident within 2 hours of its addition suggests that this fatty acid also regulates expression at the post-translational level. Overall, the results imply that CRIg expression is subject to both positive and negative modulation throughout the life span of the macrophages.
      The effects of arachidonate on CRIg expression were observed at concentrations that have been reported to prevail in the plasma during infection and inflammation. For example, circulating arachidonate levels in excess of 100 μmol/L have been reported in patients with malaria,
      • Eissen E.U.
      Significance of plasma free fatty acid levels in human malaria with parasitaemia.
      and levels of 500 μmol/L have been observed under ischemic conditions.
      • Yasuda H.
      • Kishiro K.
      • Izumi N.
      • Nakanishi M.
      Biphasic liberation of arachidonic and stearic acids during cerebral ischemia.
      These elevated levels are most likely the consequence of the activation of cytosolic PLA2 (cPLA2), which releases arachidonate from membrane phospholipids.
      • Loeper J.
      • Goy J.
      • Emerit J.
      • Rozensztajn L.
      • Jeny C.
      • Bedu O.
      Acides gras et la peroxydation des lipides dans l'atherosclerose humaine.
      Thus, it is possible that, under these conditions, high levels of circulating arachidonate will result in suppression of CRIg expression on CRIg+ macrophages and so affect the ability of these cells to function. Consistent with this, our previous studies have demonstrated that bacterial infection in the absence of CRIg (eg, in CRIg knockout mice) results in bacterial dissemination, elevated systemic inflammatory cytokine levels, and decreased survival.
      • Helmy K.Y.
      • Katschke Jr, K.J.
      • Gorgani N.N.
      • Kljavin N.M.
      • Elliott J.M.
      • Diehl L.
      • Scales S.J.
      • Ghilardi N.
      • van Lookeren Campagne M.
      CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.
      Thus, arachidonate-mediated down-regulation of CRIg expression is likely to lead to exacerbation of pathogenesis.
      Apart from producing a massive increase in the levels of circulating arachidonate in conditions such as malaria and ischemia, cPLA2 may play a more subtle role in regulating CRIg expression. Consistent with this, lipopolysaccharide, a known activator of cPLA2,
      • Dieter P.
      • Kolada A.
      • Kamionka S.
      • Schadow A.
      • Kaszkin M.
      Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by group IV cytosolic phospholipase A2, but not by group V and group IIA secretory phospholipase A2.
      • Miller A.M.
      • Masrorpour M.
      • Klaus C.
      • Zhang J.X.
      LPS exacerbates endothelin-1 induced activation of cytosolic phospholipase A2 and thromboxane A2 production from Kupffer cells of the prefibrotic rat liver.
      has been reported to cause a decrease in CRIg expression.
      • Vogt L.
      • Schmitz N.
      • Kurrer M.O.
      • Bauer M.
      • Hinton H.I.
      • Behnke S.
      • Gatto D.
      • Sebbel P.
      • Beerli R.R.
      • Sonderegger I.
      • Kopf M.
      • Saudan P.
      • Bachmann M.F.
      VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.
      Both arachidonate
      • Robinson B.S.
      • Hii C.S.
      • Ferrante A.
      Activation of phospholipase A2 in human neutrophils by polyunsaturated fatty acids and its role in stimulation of superoxide production.
      and PMA
      • Li Q.
      • Subbulakshmi V.
      • Oldfield C.M.
      • Aamir R.
      • Weyman C.M.
      • Wolfman A.
      • Cathcart M.K.
      PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes.
      are also known activators of cPLA2 and secretory PLA2. In contrast, dexamethasone, by increasing lipocortin-1 (annexin-1) levels, inhibits cPLA2.
      • Gewert K.
      • Sundler R.
      Dexamethasone down-regulates the 85 kDa phospholipase A2 in mouse macrophages and suppresses its activation.
      • Stone R.M.
      • Imamura K.
      • Datta R.
      • Sherman M.L.
      • Kufe D.W.
      Inhibition of phorbol ester-induced monocytic differentiation and c-fms gene expression by dexamethasone: potential involvement of arachidonic acid metabolites.
      Thus, the PKC-activating agents and dexamethasone may act on a common intracellular target such as cPLA2 to affect their opposing actions on CRIg expression.
      Of note, medium from cultures of PHA-stimulated PBMCs that was rich in Th1 cytokines caused a marked decrease in CRIg expression on macrophages, an effect that was also observed with recombinant IFN-γ. This could be one way by which cytokines amplify the inflammatory response.
      • Helmy K.Y.
      • Katschke Jr, K.J.
      • Gorgani N.N.
      • Kljavin N.M.
      • Elliott J.M.
      • Diehl L.
      • Scales S.J.
      • Ghilardi N.
      • van Lookeren Campagne M.
      CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.
      Not surprisingly, therefore, in experimental inflammation it has been found that at the center of the inflammatory foci the macrophages lack CRIg but those at the periphery show expression
      • Vogt L.
      • Schmitz N.
      • Kurrer M.O.
      • Bauer M.
      • Hinton H.I.
      • Behnke S.
      • Gatto D.
      • Sebbel P.
      • Beerli R.R.
      • Sonderegger I.
      • Kopf M.
      • Saudan P.
      • Bachmann M.F.
      VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.
      . This is consistent with mediator-induced down-regulation of CRIg. Unexpectedly, CRIg expression on macrophages was decreased by the Th2 cytokine IL-4. This may, at least in part, explain our previous finding that IL-4 decreased the phagocytosis of complement opsonized Plasmodium falciparum-infected erythrocytes.
      • Kumaratilake L.M.
      • Ferrante A.
      IL-4 inhibits macrophage-mediated killing of Plasmodium falciparum in vitro A possible parasite-immune evasion mechanism.
      Furthermore, IL-10 and TGF-β1, although considered to be immunosuppressive cytokines, exerted opposite effects on CRIg expression: IL-10 increased but TGF-β1 decreased CRIg expression. The data thus reveal that a unique cytokine network operates to control CRIg expression, and this is likely to increase our understanding of inflammation control.
      It was evident from our results that the changes in CRIg expression induced by arachidonate, dexamethasone, and cytokines have biological relevance. In association with a decrease in CRIg expression induced by arachidonate, there was a significant reduction in phagocytosis of complement opsonized C. albicans. A similar finding was shown for the Th1 cytokine IFN-γ. In contrast, dexamethasone increased the expression of CRIg, and the macrophages showed a corresponding increase in phagocytosis. These changes in phagocytosis were consistent irrespective of the method used to score phagocytosis.
      Our findings are consistent with results from studies conducted with a well-defined inflammatory reaction in mice showing the absence of CRIg+ macrophages in the intensity of the inflammatory foci,
      • Vogt L.
      • Schmitz N.
      • Kurrer M.O.
      • Bauer M.
      • Hinton H.I.
      • Behnke S.
      • Gatto D.
      • Sebbel P.
      • Beerli R.R.
      • Sonderegger I.
      • Kopf M.
      • Saudan P.
      • Bachmann M.F.
      VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.
      supporting the idea that inflammation down-regulates CRIg expression. Consistent with this, it has recently been reported that CRIg expression by hepatic macrophages was down-regulated in chronic hepatitis B patients who had not been treated for at least 6 months, compared with that of normal donors.
      • Guo S.
      • Yang C.
      • Mei F.
      • Wu S.
      • Luo N.
      • Fei L.
      • Chen Y.
      • Wu Y.
      Down-regulation of Z39Ig on macrophages by IFN-gamma in patients with chronic HBV infection.
      Furthermore, consistent with our data, Guo et al
      • Guo S.
      • Yang C.
      • Mei F.
      • Wu S.
      • Luo N.
      • Fei L.
      • Chen Y.
      • Wu Y.
      Down-regulation of Z39Ig on macrophages by IFN-gamma in patients with chronic HBV infection.
      recently reported that IFNγ down-regulated the expression of CRIg on human macrophages. In contrast, CRIg+ macrophages have been shown to be present in clinical samples such as synovial lining layer of rheumatoid arthritis patients
      • Tanaka M.
      • Nagai T.
      • Tsuneyoshi Y.
      • Sunahara N.
      • Matsuda T.
      • Nakamura T.
      • Tsuyama S.
      • Hasui K.
      • FitzGerald O.
      • Matsuyama T.
      Expansion of a unique macrophage subset in rheumatoid arthritis synovial lining layer.
      and foamy macrophages of atherosclerotic plaques,
      • Lee M.Y.
      • Kim W.J.
      • Kang Y.J.
      • Jung Y.M.
      • Kang Y.M.
      • Suk K.
      • Park J.E.
      • Choi E.M.
      • Choi B.K.
      • Kwon B.S.
      • Lee W.H.
      Z39Ig is expressed on macrophages and may mediate inflammatory reactions in arthritis and atherosclerosis.
      including many samples from patients who had received treatments before collection. This is not surprising, because we would expect the expression of CRIg to fluctuate according to the intensity of the inflammatory reaction and the type of treatment being administered.
      Although native CRIg plays a major role in complement-mediated clearance of systemic pathogens and autologous cells without the evolution of a cytokine storm, it appears that CRIg is anti-inflammatory toward T cells. For example, human dendritic cells transfected with CRIg inhibited the proliferation of CD4+ and CD8+ T cells and decreased the expression of activation markers (CD25 and CD69) and secretion of proinflammatory cytokines by these cells.
      • Xu S.
      • Sun Z.
      • Li L.
      • Liu J.
      • He J.
      • Song D.
      • Shan G.
      • Liu H.
      • Wu X.
      Induction of T cells suppression by dendritic cells transfected with VSIG4 recombinant adenovirus.
      In vivo, the number of intrahepatic CRIg+ macrophages in untreated chronic hepatitis B patients was found to be inversely correlated with serum alanine aminotransferase, a marker of liver inflammation, but was positively correlated with plasma hepatitis B viral load,
      • Guo S.
      • Yang C.
      • Mei F.
      • Wu S.
      • Luo N.
      • Fei L.
      • Chen Y.
      • Wu Y.
      Down-regulation of Z39Ig on macrophages by IFN-gamma in patients with chronic HBV infection.
      consistent with CRIg having an anti-T-cell function. On the other hand, others have shown that cross-linking of CRIg (or Z39Ig) on human monocytic THP-1 cells causes nuclear translocation of nuclear factor-κB and increases expression of matrix metallopeptidase-9 and secretion of IL-8, and that PMA treatment of the human myeloid leukemia cell line TF-1A resulted in increased expression of CRIg.
      • Lee M.Y.
      • Kim W.J.
      • Kang Y.J.
      • Jung Y.M.
      • Kang Y.M.
      • Suk K.
      • Park J.E.
      • Choi E.M.
      • Choi B.K.
      • Kwon B.S.
      • Lee W.H.
      Z39Ig is expressed on macrophages and may mediate inflammatory reactions in arthritis and atherosclerosis.
      Apart from the substantial differences that exist between normal macrophages and macrophage cell lines,
      • Marantos C.
      • Mukaro V.
      • Ferrante J.
      • Hii C.
      • Ferrante A.
      Inhibition of the lipopolysaccharide-induced stimulation of the members of the MAPK family in human monocytes/macrophages by 4-hydroxynonenal, a product of oxidized omega-6 fatty acids.
      these data are consistent with activation of macrophages via CRIg as crucial for the clearance of complement opsonized microbial pathogens in the absence of a cytokine storm.
      In summary, this is the first study demonstrating that a major product of the hydrolysis of membrane phospholipids, arachidonate, can down-regulate the expression of CRIg in macrophages. Arachidonate is generated during CRIg-mediated phagocytosis of complement opsonized microbial pathogens, promoting the down-regulation of CRIg and thereby contributing to initiation of the adaptive immune response/T cell activation. The finding that dexamethasone up-regulates CRIg expression may have relevance to the immunosuppressive properties of the steroid. Our results also suggest involvement of a unique cytokine network in the regulation of CRIg expression on macrophages during inflammation. In this manner, CRIg constitutes a control point in the inflammatory responses.

      Acknowledgments

      We thank Sarah Harvey, Christos Marantos, Haley Prime, and Tristan Frank for technical assistance, Dr. Menno van Lookeren Campagne for kindly providing anti-CRIg antibody, and Prof. David Hume (Roslin Institute, University of Edinburgh) for his invaluable discussion on aspects of this work.

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