* Department of Immunology, Lerner Research Institute, and
The Cole Eye Institute, The Cleveland Clinic Foundation, Ohio;
Division of Biology, University of California, San Diego; and
Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Bethesda, Maryland
Correspondence: Andrew C. Larner, The Cleveland Clinic Foundation, Department of Immunology, NB3-30, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: larnera{at}ccf.org
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/ß and type II IFN-
induce the expression of early response genes through activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Stat) pathway. Although IFNs regulate a variety of other signaling cascades, little is known about how they contribute to the biological activities of these cytokines. In this study, we demonstrate that IFN-ß or IFN- induces the phosphorylation of the serine/threonine kinase Akt in primary human peripheral blood monocytes. Abrogation of the IFN-stimulated Akt activation by phosphatidylinositol-3 kinase (PI-3K) inhibitors prevents IFN-induced adhesion in these cells, and IFN activation of the Stat1-dependent guanylate-binding protein (GBP) gene is not affected. Importantly, Stat1-deficient bone marrow macrophages displayed a similar level of IFN--stimulated adhesion compared with macrophages derived from wild-type littermates. These findings demonstrate for the first time that IFN stimulation of a PI-3K signaling cascade modulates the ability of these cytokines to regulate monocyte adhesion, and this process does not require the expression of Stat1, a primary mediator of IFN- signaling.
Key Words: Jak • Akt • cytokine • MAPK • ERK
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß and IFN- [1
]. As such, a kinase-inactive mutant of Jak1 can sustain IFN--inducible Stat activation and Stat-regulated gene expression but does not support the antiviral activity of this cytokine [2
]. Similarly, certain deletions of the IFN- receptor 2, which do not affect IFN- activation of the Jak/Stat pathway, result in the loss of the antiviral activity of IFN- [3
]. Recent reports of Stat1-independent, IFN-induced gene expression or inhibition of B cell lymphopoiesis by IFN-/ß in the absence of Stat1 highlight the potential importance of alternative second messengers in mediating some of the biological actions of IFNs.
One well-characterized, "alternative" signaling cascade regulated by IFNs is the mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) pathway. ERKs are responsible for serine phosphorylation of Stat1, Stat3, and Stat4, which maximizes their transcriptional activation potential [4
,5
]. IFN-ß and IFN- activate Raf-1 as well as B-Raf, two serine kinases ultimately responsible for the activation of p42MAPK [6
]. Activation of Raf-1 by IFNs requires Jak1 and Stat1 [6
,7
].
Additional signaling mediators stimulated by IFNs include phospholipase A2 [8
], certain isoforms of protein kinase C [9
], and phosphoinositide-3 kinase (PI-3K) [10
11
12
13
]. The guanosine 5'-triphosphate exchange protein Vav and the adaptor proteins IRS-1, c-Cbl, and CrkL are tyrosine-phosphorylated in response to IFN- and IFN- [14
15
16
]. IFN-/ß and IFN- also increase the activation of Rap1 and Rac1 [17
,18
]. The biological effects of stimulation of these cascades in the IFN response are unclear.
In an attempt to elucidate the role of alternative signaling cascades regulated by IFNs, we focused on PI-3K. Activation of PI-3K and subsequently Akt is well-known to play a critical role in cell growth and survival [19
]. Akt is also involved in chemotaxis as well as angiogenesis [20
,21
]. Although IFNs are in most instances inhibitors of cell growth, several reports indicate that IFN protects B cells and macrophages from apoptosis [22
23
24
25
], raising the possibility that IFN activation of PI-3K might be involved in this process. IFNs are also well-known activators of intercellular adhesion molecule-1 (ICAM-1) expression [26
], as well as chemoattractants such as monocyte chemoattractant protein (MCP)-1 and MCP-3. Adhesion and extravasation of monocytes from blood vessels at the site of injury are important functions that must occur for these cells to mediate their inflammatory actions. It has been known that IFN- promotes macrophage and dendritic cell adherence to basement membrane proteins such as laminin or collagen [27
,28
]. As IFNs are among the most potent stimuli of monocyte activation, these cells provide a useful model to examine the role of PI-3K in IFN action.
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Extract preparation and Western blotting
Primary monocytes were incubated in RPMI 1640 with 10% fetal calf serum (FCS) and were not starved before incubation with IFNs. Cells (107) were washed with ice-cold phosphate-buffered saline (PBS) and resuspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM NaF, 0.5 mM dithiothreitol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mM ß-glycerol phosphate, and 1 mM vanadate). BMDM were plated on 60 mm dishes in complete media [Dulbecco’s modified Eagle’s medium (DMEM), 20% FCS, supplemented with 30% L-cell media containing macrophage-colony stimulating factor (M-CSF)]. Cells were not starved before treatment with IFN-. Lysates were cleared by centrifugation at 12,000 g for 20 min at 4°C, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before transfer to Immobilon membranes. The membranes were incubated with the appropriate antibodies. Western blots were developed using enhanced chemiluminescence.
Northern blots and RNase protection assays
RNA was prepared from cells using RNAzol. cDNAs for guanylate-binding protein (GBP) and actin were used as probes for Northern hybridization or RNase protection assays using standard procedures [30
31
32
].
Adhesion assays
Elutriated monocytes (106/well) were plated in 24-well polystyrene tissue-culture dishes in RPMI 1640 and 10% FCS. Cells were incubated in the presence or absence of IFN-ß or IFN- for 4 h. Each plate was washed with 1 ml PBS, two times, and the number of cells remaining on the well was counted after removing them from the plate with trypsin–EDTA. In those samples incubated with LY294002 or wortmannin, the compounds were added 30 min before the addition of IFNs. Samples were assayed in triplicate. Adhesion of BMDM was assayed using polycarbonate membranes. BMDM adhesion was performed using a Boyden chamber containing 8.0 µm pore polyvinylpyrrolidone-free polycarbonate membranes. IFN- (5 ng/ml) in DMEM/1% FCS was placed in the lower wells, and cells (50 µl 1.25x105 cells/ml suspension in DMEM/1% FBS) were placed in the upper wells. Chambers were incubated for 2 h at 37°C in a 5% CO2 humidified incubator. The filter was fixed (10% buffered formalin), stained (Gills hematoxylin), and mounted between two glass slides. The number of cells adhering per well was counted, and SEM was calculated from quadruplicate samples.
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triggers a PI-3K-dependent phosphorylation of Akt, we used primary human peripheral blood monocytes isolated from normal volunteers. Cells were incubated with IFN- or IFN-ß for 1 h, and whole cell lysates were prepared, resolved by SDS-PAGE, and transferred to Immobilon membranes. The membranes were incubated with a phospho-specific antibody that detects Akt only when phosphorylated at Serine 473, which is required for full activation of Akt (Fig. 1A
, upper panel). Very little or no basal phosphorylation of Akt was observed in untreated monocytes, and incubation with IFN- or IFN-ß induced a robust phosphorylation of the enzyme. Reprobing the blot with an antibody, which recognizes phosphorylated and unphosphorylated Akt, demonstrated that equal amounts of protein were present in all samples (Fig. 1A
, lower panel). Similar results were seen when THP-1 cells were incubated with IFN- or IFN-ß (Fig. 1B)
. Phosphorylation of Akt by IFN-ß or IFN- occurred rapidly within 30 min after exposure of monocytes to these cytokines (Fig. 1C)
. The enzyme remained phosphorylated for at least 2 h in the presence of IFN- or IFN-ß. IFN-stimulated phosphorylation of Akt was inhibited when monocytes were preincubated 30 min with the PI-3K inhibitors wortmannin or LY294002, demonstrating that activation of PI-3K is required for IFN-stimulated phosphorylation of Akt.
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Figure 1. IFN-ß and IFN- stimulate phosphorylation of Akt in primary monocytes and THP-1 cells. Primary monocytes (A) or THP-1 cells (B) were incubated with IFN-ß (1000 units/ml) or IFN- (10 ng/ml) for 30 min before preparation of cell lysates. The immunoblots (WB) were probed with phosphoserine 473-specific Akt antisera (P-AKT). To ensure equal loading of protein, the blots were reprobed with antisera that recognize all forms of Akt (lower panels). (C) Kinetics of IFN-ß- or IFN--stimulated phosphorylation of Akt in primary monocytes. Cells were incubated with IFNs for 30, 60, or 120 min before preparation of cell extracts. Separate aliquots of cells were also incubated for 60 min with 10 µM LY294002 (LY) or 100 nM wortmannin (W) before the addition of IFN for 60 min.
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or IFN-ß for 3 h in the presence or absence of LY294002. As expression of the GBP gene is stimulated by IFN-ß and IFN- in a Stat1-dependent manner [35
], a Northern blot of these samples was probed for the presence of GBP mRNA. IFN-ß- and IFN--stimulated expression of this mRNA was unaffected by inclusion of LY294002 during the incubation of primary monocytes (cf., Fig. 2
, lanes 2 and 3 or 5 and 6), suggesting that the PI-3K pathway is not modulating the transcriptional activity of Stat-dependent genes in primary monocytes (Fig. 2A)
. It is interesting that in the monocytic leukemia cell line, THP-1, LY294002 inhibited IFN-ß- and IFN--stimulated expression of GBP, suggesting differences in PI-3K-dependent signaling events between primary and transformed monocytes (Fig. 2B)
. The amount of GBP mRNA in the presence of IFN and LY294002 relative to IFN alone is displayed below each blot. The relative amounts of IFN-induced GBP expression were arbitrarily set at 100. All values were normalized to the amount of actin mRNA in each sample. To exclude that nonspecific toxic effects were accounting for the inhibitory effects of LY294002 in THP-1 cells, we analyzed IFN-ß-stimulated expression of ISG15, a gene whose activation requires tyrosine phosphorylation of Stat1 and Stat2. Although IFN-ß-stimulated expression of GBP was inhibited more than 80% by LY294002, inhibition of ISG15 transcription was only 30%, suggesting that the effects of this compound in THP-1 cells are Stat1-specific. Similar results were seen in monocytes and THP-1 cells that were incubated with IFN-ß or IFN- for 6 h (data not shown). Incubation of THP-1 cells with LY294002 also had no effect on the ability of IFN-ß or IFN- to stimulate tyrosine phosphorylation of Stat1 (Fig. 2C)
. We are presently exploring the underlying mechanism that accounts for these cell-specific effects.
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Figure 2. IFN-induced expression of GBP RNA is inhibited by LY294002 in THP-1 cells but not in primary monocytes. Primary monocytes (A) or THP-1 cells (B) were incubated with IFN-ß or IFN- for 3 h before preparation of RNA. LY294002 (LY) was added 60 min before IFN as in Figure 1
. Northern blots were probed for GBP or actin. IFN-ß-stimulated expression of ISG15 was analyzed by RNase protection using actin as an internal control. The relative amount of GBP or ISG15 RNA in the sample derived from IFN-treated cells was arbitrarily set at 100, shown at the bottom of the blot. The amount of these RNAs in cells exposed to IFN and LY294002 was measured relative to those samples derived from cells incubated with only IFN. All calculations were normalized to levels of actin RNA. The relative values of each RNA were determined using a phosphoimager. (C) IFN-ß- and IFN--stimulated tyrosine phosphorylation (PY) of Stat1 was analyzed in THP-1 cells incubated with or without 10 µM LY294002 for 30 min before the addition of IFN-ß or IFN- for 30 min. Cell extracts were prepared and immunoblotted with antisera that recognizes only the tyrosine-phosphorylated form of Stat1.
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activation of PI-3K in their biological actions in monocytes, we focused on their adherence properties. The ability of monocytes to adhere and migrate to sites of inflammation is essential for their proinflammatory activities. Their adherence to blood vessels is also intimately involved in endothelial cell proliferation and atherogenesis [28
]. The tyrosine kinase Pyk2, which has been reported to be important for monocyte spreading and motility [36
], is activated by treatment of cells with IFN- [37
]. Although IFN- induces the adherence of murine peritoneal macrophages and rat dentritic cells to fibronectin and collagen [27
,28
], the effects of IFN- and IFN-/ß on the adherence properties of primary human monocytes have not been examined. Elutriated monocytes attach to plastic in greater numbers after exposure to IFNs. To determine whether IFN-ß- or IFN--stimulated attachment is PI-3K-regulated, elutriated monocytes were placed in polystyrene dishes and incubated with or without these cytokines for 4 h. Unattached cells were removed from the wells, and attached cells were then counted (see Fig. 3
). Incubation of monocytes with IFN- or IFN-ß increased the number of attached cells by 
2.5-fold. It is important that although incubation of cells with the PI-3K inhibitor LY294002 in the absence of IFN-ß or IFN- had no effect on the number of attached cells, this inhibitor completely prevented the enhanced attachment of cells observed in the presence of IFN-ß or IFN-.
 View larger version (29K): [in a new window] |
Figure 3. IFN-stimulated attachment of monocytes is reversed by the PI-3K inhibitor LY294002. Primary monocytes (106) were plated on 24-well tissue-culture plates. Prior to the addition of IFN- (10 ng/ml) or IFN-ß (1000 units/ml), some samples were exposed to LY294002 (Ly; 10 µm) or dimethyl sulfoxide. Cells were incubated with or without IFNs for 3 h, and numbers of cells that remained attached to the dishes after washing with PBS were determined. The difference between untreated and IFN-- or IFN-ß-incubated cells with respect to differences in adherence had a P < 0.0005.
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- and IFN-ß-stimulated Stat1-dependent gene expression did not appear to be modulated by Akt in primary monocytes, we wanted to examine a possible role of Stat1 activation in cell adhesion. To address this issue, we used primary murine BMDM. A previous study demonstrated that IFN- stimulated adhesion of these cells to laminin and type IV collagen-coated surfaces [28
]. BMDM were harvested from femurs and placed in culture for 6–8 days in the presence of M-CSF before being plated on polycarbonate filters in the absence or presence of IFN-. After 2 h, the filters were washed, and attached cells were counted ( Fig. 4
). Although incubation of macrophages with IFN- stimulated a twofold increase in attached cells compared with cells not exposed to the cytokine, we were unable to demonstrate a consistent IFN-ß-stimulated adhesion of BMDM to the filters (data not shown). IFN--stimulated adhesion of BMDM was dose-dependent (Fig. 4A)
. Incubation of macrophages from Stat1-deficient mice with IFN- induced their adhesion to a similar extent as cells isolated from wild-type littermates (Fig. 4B)
. IFN--stimulated adhesion of wild-type and Stat1-deficient BMDM was also reversed in the presence of LY294002. To further examine the role of PI-3K in IFN--stimulated adhesion, we assayed the IFN--stimulated phosphorylation of Akt. Western blots were probed for phosphorylated Akt using cell lysates that were prepared from wild-type and Stat1-/- cells incubated with or without IFN- for 30 min (Fig. 4C)
. IFN--stimulated phosphorylation of Akt was similar in BMDM from wild-type and Stat1 null mice, suggesting that activation of the PI-3K pathway does not require the expression of Stat1.
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Figure 4. IFN--stimulated adhesion of murine BMDM does not require the expression of Stat1. BMDM isolated from wild-type or Stat1 null littermates were cultured in M-CSF for 6–8 days before being plated on polycarbonate membranes. (A) Macrophages were incubated with various concentrations of IFN- for 2 h before counting the numbers of cells attached to the membrane. (B) BMDM from wild-type (WT) and Stat1 null mice (KO) were incubated with or without IFN- (10 ng/ml) for 2 h. Samples incubated with LY294002 (Ly) were incubated with drug for 30 min before the addition of IFN-. The difference between untreated and IFN--incubated cells with respect to differences in adherence had a P < 0.001 in macrophages from wild-type mice and P < 0.005 in macrophages from Stat1 null mice. (C) BMDM isolated from wild-type or Stat1-deficient mice (KO) were incubated with IFN- (10 ng/ml) for 30 min. Cell extracts were prepared and analyzed by immunoblot (WB) for phosphorylated Akt (P-AKT; upper panel) or total Akt (lower panel).
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to induce GBP RNA expression in THP-1 cells. However, there was no such effect of this PI-3K inhibitor on GBP expression in primary monocytes. The results highlight the highly cell-specific effects of PI-3K with regard to its role in the regulation of the activity of Stat proteins (i.e., Stat1). It also might reflect the reported role of nuclear factor-
B in activation of ISGs by IFN-ß through a PI-3K-regulated event [38
].
The ability of monocytes to adhere to surfaces such as endothelial cells is an intrinsic property necessary for their activation and migration to sites of inflammation. Incubation of monocytes with IFN- or IFN-ß for 4 h induced a substantial increase in the numbers of attached cells. Furthermore, this enhanced adherence was reversed in the presence of the PI-3K inhibitor LY294002. These results define a clear, biological response that is regulated by IFNs, which requires the activity of PI-3K. IFN- also stimulates cell adhesion and phosphorylation of Akt in murine BMDM. In BMDM isolated from mice that do not express Stat1, we observed no alterations in the ability of IFN- to stimulate cell adhesion or activation of Akt. Furthermore, the PI-3K inhibitory LY294002 prevented IFN--stimulated adhesion of Stat1 null macrophages. Previous reports indicate that a number of genes are regulated by IFN- in the absence of Stat1 [29
,39
]. The results presented here extend these observations to define a clear, biological action of this cytokine that does not require the activation or expression of Stat1.
At the moment, the downstream targets of IFN activation of PI-3K, which enhance the ability of monocytes and macrophages to adhere, are unknown. IFN activation of Akt may or may not be involved in mediating the increased adhesion. Although incubation of cells with the protein synthesis inhibitor cycloheximide prevents IFN-stimulated cell adhesion (data not shown), the role of IFN-stimulated genes in cell adhesion of macrophages is unclear. If the synthesis of new RNAs is required, one possible target is fibronectin, whose expression is induced by treatment of Stat1 null BMDM with IFN-. Fibronectin has been shown to enhance spreading of macrophages on plastic [28
]. IFN- induction of ICAM-1 appears to be Stat1-dependent [40
]. We are presently attempting to identify target genes and/or proteins regulated by IFN- or IFN-ß, which require activation of PI-3K for enhanced monocyte adherence.
Received October 25, 2002; revised December 20, 2002; accepted January 2, 2003.
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