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.
1- 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.
2- 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
3- 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
4- 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.
5- 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.
1- 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.
, 2- 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.
, 5- 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.
6- 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.
7- 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 A
2 (PLA
2), leading to the release of arachidonate from membrane phospholipids.
7- Ferrante A.
- Hii C.S.
- Costabile M.
Regulation of neutrophil functions by long chain fatty acids.
In turn, arachidonate stimulates several responses in leukocytes,
7- Ferrante A.
- Hii C.S.
- Costabile M.
Regulation of neutrophil functions by long chain fatty acids.
, 8- 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,
1- 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.
, 2- 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.
, 5- 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.
9- 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.
, 10- 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.
11- 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.
1- 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.
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,
7- Ferrante A.
- Hii C.S.
- Costabile M.
Regulation of neutrophil functions by long chain fatty acids.
, 8- 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.
, 13- 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.
8- 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.
7- 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,
12- 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,
18Significance of plasma free fatty acid levels in human malaria with parasitaemia.
and levels of 500 μmol/L have been observed under ischemic conditions.
19- 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 PLA
2 (cPLA
2), which releases arachidonate from membrane phospholipids.
20- 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.
2- 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 cPLA
2,
21- 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.
, 22- 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.
5- 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
23- 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
24- 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 PLA
2. In contrast, dexamethasone, by increasing lipocortin-1 (annexin-1) levels, inhibits cPLA
2.
25Dexamethasone down-regulates the 85 kDa phospholipase A2 in mouse macrophages and suppresses its activation.
, 26- 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 cPLA
2 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.
2- 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
5- 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.
27- 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,
5- 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.
28- 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
28- 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
29- 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,
30- 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.
31- 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,
28- 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.
30- 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,
10- 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.
Article info
Publication history
Published online: July 11, 2011
Accepted:
May 23,
2011
Footnotes
Supported by the National Health and Medical Research Council of Australia.
Current address of N.N.G., Children's Medical Research Institute, Cell Signaling Unit, Westmead, Australia; of U.T., Department of Graduate Program, Faculty of Allied Health Sciences, Thammasart University (Rangsit campus), Phatum Thani, Thailand; of O.P., Medical Molecular Biology Unit, Office of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Copyright
© 2011 American Society for Investigative Pathology. Published by Elsevier Inc.