Published online before print May 22, 2003
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* Institute of Immunology, Philipps University, Marburg, Germany; and
Institute of Laboratory Medicine, Leopoldina-Hospital, Schweinfurt, Germany
Correspondence: Andreas Kaufmann, Ph.D., Philipps-University Marburg, Institute of Immunology, Robert-Koch-Str. 17, D-35037 Marburg, Germany. E-mail: kaufmana{at}mailer.uni-marburg.de
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(MIP-1), CCL4/MIP-1ß, and CCL5/regulated on activation, normal T expressed and secreted (RANTES), all chemokines binding to these two receptors. The CCL3/MIP-1-induced shifts of intracellular calcium concentrations declined gradually to almost undetectable levels, and most conspiciuously, the chemotactic response to CCL3/MIP-1, CCL4/MIP-1ß, and CCL5/RANTES was lost after infection with active influenza virus. Inactivated virus particles did not significantly alter the responsiveness induced by CCR1 and CCR5 agonists. Despite the inability of chemokine receptors to elicit migration, phosphorylation of protein kinase B was not altered in virus-infected monocytes. Thus, influenza A virus infection rapidly abolishes the functional responsiveness of monocytes and prevents an adequate response of the infected cells to chemokine stimulation.
Key Words: chemokines • migration • monocytes/macrophages
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(TNF-), interleukin (IL)-1, and IL-6 [6
7
8
9
] and led to a strong and selective induction of mononuclear cell-attracting chemokines [10
].
Chemokines are potent, low molecular weight chemoattractant cytokines, which are considered to be the main factors recruiting effector cells during inflammatory diseases [11
12
13
14
]. The subfamily of CC chemokines, such as CCL2/monocyte chemotactic protein 1 (MCP-1), CCL3/macrophage inflammatory protein 1 (MIP-1), CCL4/MIP-1ß, and CCL5/regulated on activation, normal T cell expressed and secreted (RANTES), preferentially acts on mononuclear cells [15
]. The major role of CCL2/MCP-1 and CCL3/MIP-1 as monocyte and lymphocyte chemoattractants in host defense has previously been shown in knockout models [16
, 17
]. In particular, CCL3/MIP-1-deficient animals were found to display an impaired recruitment of monocytes in the respiratory tract after influenza A infection.
Chemokine-mediated migration of leukocytes depends on the expression of specific cell-surface receptors. All chemokine receptors are members of the seven-transmembrane domain rhodopsin-like superfamily of receptors and are coupled to guanine 5'-triphosphate (GTP)-binding proteins [18
]. Monocytes/macrophages have recently been shown to express the CC chemokine receptors CCR1, CCR2, and CCR5 [19
]. Most chemokine receptors are not specific for only one ligand but promiscuously bind more than one chemokine. Although CCL2/MCP-1 is a specific ligand for CCR2, CCL3/MIP-1, CCL4/MIP-1ß, and CCL5/RANTES signaling is mediated via CCR1 and CCR5.
In our present study, we analyzed the expression of the CC chemokine receptors CCR1, CCR2, and CCR5 and the functional responsiveness of monocytes to chemokines in the course of an influenza A virus infection. We found that the chemokine response of the cells was gradually lost during an infection with active replicating virus, independent of CCR up- or down-regulation.
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The monocytes were resuspended at a density of 2 x 106 cells/ml in RPMI 1640 supplemented with 2 mM glutamine, 25 mM HEPES, 1% (vol/vol) nonessential amino acids, 2 mM pyruvate, 50 U/ml penicillin, and 50 mg/ml streptomycin (all from Biochrom KG, Berlin, Germany). The cell suspension (2 ml) was plated into Teflon tubes (Savillex, Minnetonka, MN) and was cultured at 37°C in the presence of 5% CO2 in humidified air [20 ].
All reagents used during cell preparation and culture were essentially free of contaminating endotoxin, as determined by the Limulus test (BioWhittaker, Walkersville, MD).
Virus preparation and exposure to human monocytes
Influenza A virus strain A/PR/8 (H1N1) was kindly donated by Dr. Hans-Dieter Klenk (Institute of Virology, Philipps University, Marburg, Germany) and was propagated and purified as previously outlined in detail [6
]. Aliquots of the virus were inactivated for 30 min at 56°C in a water bath or were UV-inactivated on ice by radiation at 254 nm for 30 min. Infectivity was assessed by a standard plaque assay as a cytopathic effect on confluent cultures of mycoplasma-free Madin Darby canine kidney II cells with a 0.5% agarose overlay [21
]. Human monocytes were infected by exposure to two multiplicity of infection (MOI) A/PR/8 virus (two plaque-forming units per cell) or were stimulated with an equivalent dose of inactivated virus particles for 1 h under serum-free conditions [6
, 7
, 9
]. Thereafter, 2% AB serum (Sigma, Munich, Germany) was added. After the incubation times, the cultures were kept on ice for 30 min, extensively resuspended, washed twice in phosphate-buffered saline, and immediately used for further experiments.
Chemokine receptor surface expression
Cells were incubated with biotinylated anti-human CCR1, CCR2, CCR5 (R&D Systems, Wiebaden, Germany), or isotype-specific goat anti-mouse (PharMingen, Hamburg, Germany) monoclonal antibody, washed twice, and subsequently incubated with streptavidin-phycoerythrin (PE; PharMingen). To amplify the received fluorescence signal, cells were further incubated with biotinylated antiavidin D antibody (Vector Laboratories, Burlingame, CA), followed by a second incubation with streptavidin-PE conjugate. The samples were analyzed on a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany).
Intracellular Ca2+ concentrations
[Ca]i changes in Fura-2-loaded cells were monitored after chemokine stimulation by excitation wavelengths at 340 and 380 nm and an emission wavelength at 510 nm on a fluorescence spectrometer (BMG Laboratory Technologies GmbH, Offenburg, Germany), as described elsewhere [22
], according to the technique reported by Grynkiewicz et al. [23
].
Chemotaxis assay
Cell migration was assayed in quadruplicate, using a 48-well microchemotaxis chamber technique (Neuro Probe, Gaithersburg, MD). The chemotactic response of infected monocytes was assayed as follows: The chemoattractant stimulus (27 µl), diluted in RPMI 1640, supplemented with 0.1% bovine serum albumin, was placed into the lower chamber. Supplemented RPMI 1640 lacking chemokines was used to exclude unspecific chemokinesis (negative control). After separation by polyvinylpyrrolidone-free polycarbonate filters with 5 µm pores (Neuro Probe), the upper chamber was filled with 50 µl cell suspension (2x106 cells/ml). The chamber was incubated at 37°C in air with 5% CO2 for 60 min. Thereafter, filters were removed, fixed in methanol, and stained with hematoxylin (Sigma). The total number of migrated cells per well was densitometrically calculated by a computer-assisted imaging system (Vilber Lourmat, distributed by Fröbel, Wasserburg, Germany). Results are expressed as the difference of total number of chemokine-induced cell migration and migration to control medium (specific chemotaxis).
Protein kinase B (PKB) phosphorylation
Human monocytes (5x106/well) were cultivated for 20 h in six-well tissue-culture plates. After two washes, the monocytes were left untreated, infected with two MOI of influenza A virus, or stimulated with an equivalent dose of 56°C-inactivated virus for 6 h. Thereafter, cells were stimulated for 5 min with CCL3/MIP-1, formyl-Met-Leu-Phe (fMLP), or left untreated and subsequently lysed in a protein extraction reagent (M-PERTM, Pierce, Rockford, IL) supplemented with a protease-inhibitor mixture (CompleteTM, Roche Diagnostics, Mannheim, Germany). For phosphatidylinositol-3 kinase (PI-3K) inhibition experiments, cells were incubated with the indicated concentrations of LY294002 before stimulation with CCL3/MIP-1. Samples (10 µg total protein/lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following electrophoresis, resolved proteins were transferred electrophoretically to polyvinylidene difluoride membranes and were then blocked for 2 h at room temperature with Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBST) and 5% (w/v) nonfat powdered milk. Thereafter, blots were incubated overnight at 4°C with a 1:1000 dilution of phospho-PKB-specific antibody (recognizing only the activated form of PKB after phosphorylation at position Ser473) or an antibody specifically recognizing endogenous PKB, independent of phosphorylation (New England Biolabs, Frankfurt/M., Germany). The blots were washed with TBST (3x5 min) and incubated with alkaline phospatase-linked secondary antibody immunoglobulin G anti-rabbit [1:1000 in 5% (w/v) nonfat milk in TBST] for 1 h at room temperature. Bound antibody was visualized using the 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium mixture (Sigma fastTM, Sigma Chemical Co., St. Louis, MO), according to the manufacturer’s instructions.
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Figure 1. Cell-surface expression of CCR1, CCR5, and CCR2 after influenza A virus infection. Human monocytes were purified from peripheral blood mononuclear cells by counter-flow centrifugation and were cultivated in Teflon vessels (5x106/2 ml) in the absence or presense of influenza A/PR/8 (two MOI) or an equivalent dose of inactivated virus particles. (A) Six hours after infection, monocytes were stained with specific antibodies and analyzed for CCR1, CCR2, and CCR5 expression using FACScan analysis. (B) The number of surface-bound receptors at 1, 3, 6, and 10 h after exposure to virus is shown as the mean fluorescence intensity (MFI). Untreated cells were used as control. A representative analysis out of four different donors is shown.
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. At different times after virus treatment, monocytes were loaded with the calcium-sensitive dye Fura-2 AM, and shifts in intracellular calcium concentrations were determined spectrofluorometrically. As expected, the responsiveness of virus-infected cells to the chemokine CCL2/MCP-1, a specific ligand for CCR2, declined dramatically (Fig. 2A
, middle panel), as detectable shifts in intracellular calcium concentrations disappeared already as soon as 3 h after virus infection. This finding implies that the onset of unresponsiveness to CCL2/MCP-1 preceded the disappearence of CCR2 (Fig. 1B
, right panel). In parallel to the diminished surface expression of CCR2 observed after treatment with inactivated virus particles, intracellular calcium shifts induced by CCL2/MCP-1 declined but were still detectable for up to 10 h (Fig. 2A
, right panel). The responsiveness of virus-infected cells to CCL3/MIP-1, a chemokine that mediates its signal via high-affinity binding to CCR1 and CCR5, lasted longer: The shifts of CCL3/MIP-1-induced intracellular calcium concentrations in virus-infected cells decreased gradually and were moderately reduced but not abolished after 10 h (Fig. 2B
, middle panel). On first sight, this finding was not in line with the apparent unchanged or even enhanced CCR1 and CCR5 expression on the protein level. In contrast, stimulation of monocytes with inactivated virus particles did not affect the CCL3/MIP-1-induced calcium shifts (Fig. 2B
, right panel), corresponding with the unchanged or enhanced CCR1 and CCR5 expression pattern observed after treatment with inactive virus.
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Figure 2. Chemokine-induced shifts of intracellular calcium in influenza A virus-infected monocytes. Purified monocytes were left untreated (Control), infected with two MOI of influenza A/PR/8, or treated with inactivated virus. At the indicated time, cells were washed, loaded with Fura-2 AM, and subsequently stimulated with CCL2/MCP-1 (A) or CCL3/MIP-1 (B). Intracellular Ca2+ mobilization in response to the chemokines was measured by a fluorescence spectrometer. Arrows indicate the addition of chemokines (all used at 50 ng/ml). The [Ca2+]i-dependent changes of fluorescence were expressed as the ratio at excitation wavelengths 340 and 380 nm. Figures show representative data out of four independent experiments.
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after infection with influenza A virus as well as to the nonchemokine chemotaxin fMLP. After 1 h, the chemotactic response of virus-infected cells to CCL2/MCP-1, CCL3/MIP-1, and fMLP remained unaltered when compared with the untreated control (Fig. 3
, top). However, at 3 h after infection, the migration toward both chemokines was almost entirely lost, whereas fMLP still induced a strong migration. Again, unresponsiveness of CCL2/MCP-1 clearly preceded the down-regulation of CCR2 receptor expression on the cell surface. This early onset of migratory unresponsiveness was even more surprising for CCL3/MIP-1: Despite an enhanced expression of specific receptors and detectable shifts of intracellular calcium, the infected cells stopped migration in response to CCL3/MIP-1 as soon as 3 h after virus infection. In contrast, the fMLP-induced migration was unaffected for at least 6 h and remained detectable for up to 10 h after infection.
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Figure 3. Chemotaxis of virus-infected monocytes to CCL2/MCP-1 and CCL3/MIP-1. The chemotactic response of virus-infected monocytes was assayed by a 48-well microchemotaxis chamber technique. At the indicated times after infection (two MOI) or stimulation with equivalent doses of influenza virus inactivated by UV light or treatment at 56°C, cell migration in response to CCL2/MCP-1, CCL3/MIP-1 (50 ng/ml), or fMLP (10-8M) was determined. Mock-infected monocytes were used as control. The results are expressed as the difference of chemokine-induced cell migration and migration to control medium (specific chemotaxis±SD). A representative analysis out of four is shown.
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, which correlates with the unchanged or up-regulated CCR1 and CCR5 expression and the undisturbed calcium shifts. Thus, in contrast to infected cells where migration was rapidly stopped in response to chemokines, monocytes treated with inactive virus particles were still able to respond to chemokines, and CCL2/MCP-1 as well as CCL3/MIP-1 elicited an adequate migration.
Strong reduction of CCL4/MIP-1ß and CCL5/RANTES induced migration after influenza A virus infection
In addition to CCL3/MIP-1, the chemokines CCL4/MIP-1ß and CCL5/RANTES also induce cell activation via the chemokine receptors CCR1 and CCR5. To evaluate if CCL4/MIP-1ß and CCL5/RANTES responses are also affected by viral infection, we further examined the migration of virus-infected cells toward these chemokines.
As shown in Figure 4
, the chemotactic responsiveness of virus-infected monocytes induced by the CCR1 and CCR5 agonists CCL3/MIP-1, CCL4/MIP-1ß, and CCL5/RANTES declined dramatically when compared with untreated control cells. As soon as 3 h after virus treatment, the infected cells nearly stopped migration in response to CCL3/MIP-1, CCL4/MIP-1ß, and CCL5/RANTES. In contrast, monocytes stimulated with UV-inactivated virus particles exhibited strong chemotactic responsiveness similar to untreated control cells. Thus, viral infection completely diminished the responsiveness to CCR1 and CCR5 agonists despite unchanged cell-surface expression of both receptors.
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Figure 4. Chemotactic response induced by CCL4/MIP-1ß and CCL5/RANTES. Three hours after infection, migration of virus-treated monocytes was assayed as described. Cells were left untreated, infected with two MOI influenza A virus, or stimulated with equivalent doses of UV-inactivated virus particles. The CCR1 and CCR5 agonists CCL3/MIP-1, CCL4/MIP-1ß, and CCL5/RANTES (all at 50 ng/ml) were used as chemotactic stimuli. The chemotaxin fMLP (10-8M) was used as positive control. The results are expressed as the difference of chemokine-induced cell migration and migration to control medium (specific chemotaxis±SD).
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mediated phosphorylation of PKB after influenza A virus infection
subunits from trimeric G proteins after chemokine-induced receptor activation [24
]. Therefore, monocytes were treated with infectious or inactivated virus and restimulated with CCL3/MIP-1. Cells without retreatment or stimulation with fMLP were used as negative and positive controls, respectively. The induced phosphorylation of PKB at serine 473 was assayed by Western blot technique. As shown in Figure 5
(left panel), activation of PKB by CCL3/MIP-1 was undisturbed, even after infection with replicating influenza A virus. Similar amounts of phosphorylated PKB after CCL3/MIP-1 restimulation were detected in control cells, virus-infected cells, as well as monocytes treated with inactivated virus particles. In addition, pretreatment of monocytes with the chemotactic tripeptide fMLP did not influence the CCL3/MIP-1-mediated PKB activation. Furthermore, restimulation with fMLP also induced an enhanced PKB phosphorylation in control cell as well as in monocytes treated with active and inactivated virus. In contrast, as expected as a result of receptor desensitization, only a weak PKB activation was observed in fMLP-pretreated monocytes after homologous restimulation. Equal protein loading was controlled by determining total levels of endogenous PKB (Fig. 5
, right panel). The unaffected ability of CCL3/MIP-1 to mediate PKB phosphorylation even in virus-infected monocytes points to an undisturbed receptor state.
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Figure 5. Phosphorylation of PKB in monocytes infected with influenza A virus. Human monocytes (5x106/3 ml) were stimulated with fMLP (10-8M), infected with two MOI of influenza A/PR/8, or treated with an equivalent dose of heat-inactivated virus particles. Untreated cells were used as control. After 6 h, cells were washed and restimulated with medium (background control), CCL3/MIP-1 (200 ng/ml), or fMLP (10-8M) for 5 min and subsequently lysed in protein extraction reagent. Phosphorylation of PKB (left panel) was assayed by Western blot. Equal loading was controlled by detecting total amounts of PKB (right panel). One representative analysis out of three experiments is shown.
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-induced phosphorylation of PKB after pretreatment with LY294002, a selective inhibitor of PI-3K [25
]. For this purpose, virus-infected monocytes were preincubated with different doses of LY294002 for 1 h and subsequently restimulated with CCL3/MIP-1. Uninfected cells were used as controls. The chemokine-induced phosphorylation of PKB was assayed by Western blot. As shown in Figure 6
, preincubation with the PI-3K inhibitor LY294002 dose-dependently diminished the strong CCL3/MIP-1-induced PKB phosphorylation to nearly undetectable values in virus-infected as well as uninfected control cells, thus indicating PKB as a downstream effector of PI-3K activity, excluding PI-3K-independent activation of PKB, and furthermore, clearly supporting an undisturbed ligand binding and initiation of receptor signaling down to PI-3K activation even in virus-infected cells.
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Figure 6. Inhibition of PI-3K activity by LY294002. Human monocytes (5x106/3 ml) were left untreated or infected with two MOI of influenza virus for 6 h. One hour before restimulation with CCL3/MIP-1 (200 ng/ml), cells were incubated with different amounts of LY294002 as indicated. Five minutes after chemokine restimulation, cells were lysed in protein extraction reagent, and 10 µg/lane total proteins was assayed by Western blot for PKB phosphorylation at serine 473 and total PKB to confirm equal loading. The blots shown are representative of three independent experiments with cells from different donors.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In studying the infection of human monocytes by influenza A virus, we could previously show a strong expression of TNF-, IL-1, IL-6, and IL-10 within 10 h [6
7
8
9
]. Thereafter, 70–80% of infected monocytes died by apoptosis within 24–36 h [26
27
28
29
]. In addition, an influenza A virus infection led to a highly selective induction of mononuclear leukocyte-attracting CC chemokines and most remarkably, to a suppression of neutrophil-attracting CXC chemokines [10
, 30
]. After having established the release of these effector molecules, we next examined the responder cells in terms of chemokine receptor expression and function.
The present study shows that at least two groups of chemokine receptors can be identified that respond differently to an influenza A virus infection of monocytes. Conversely, CCR1 and CCR5, which were consistently or even enhanced, expressed a down-regulation and rapid disappearance of CCR2 (Fig. 1)
, preceded by a strong reduction of CCL2/MCP-1-induced Ca2+ flux (Fig. 2)
and chemotactic activity (Fig. 3)
. A dissociation between receptor expression and functional response became particularly apparent by studying CCR1 and CCR5: Unimpaired cell-surface expression (Fig. 1)
together with intact CCL3/MIP-1-dependent receptor activation (Fig. 5)
counteracted the decreased Ca2+ flux (Fig. 2)
and the loss of monocyte migration (Figs. 3
and 4)
induced by the CCR1 and CCR5 agonists CCL3/MIP-1, CCL4/MIP-1ß, and CCL5/RANTES.
We had previously reported that an influenza A virus infection of monocytes stimulated the production of chemokines, e.g., CCL2/MCP-1 and CCL3/MIP-1 [10
]. On the basis of these results, one could speculate that a ligand-mediated down-regulation of CCR expression had occurred, which as has been shown previously [31
], could have led to a progressive loss of functional receptors on the cell surface by homologous desensitization. However, this effect seems to be unlikely, as virus-induced chemokine release did not start earlier than 4 h after infection [10
]. At this time, we already detected a total chemotactic unresponsiveness of influenza-infected monocytes to the chemokines CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1ß, and CCL5/RANTES. (Figs. 3
and 4) . Furthermore, when influenza A virus was inactivated at 56°C or UV light, and monocytes were exposed to these replication-defective virus particles, chemokine production still persisted [30
] without impairing Ca2+ flux or chemokine-induced migration. Thus, an unrestricted infectivity with subsequent virus replication was required to render monocytes unresponsive to secondary chemokine stimulations. This was not simply the result of a decreased viability of cells, as virus-infected monocytes strongly migrated toward fMLP (Figs. 3
and 4)
. Furthermore, a simple exhaustion of GTP-mediated signal pathways was not the underlying cause of unresponsiveness. This assumption is further underlined by the finding that chemotaxis toward CCR1 and CCR5 agonists ceased within 3 h after infection, but the Ca2+ flux persisted up to 10 h. In this regard, previously published studies [32
, 33
] showed that cell migration and calcium flux were independently regulated by different signal-transduction pathways: PI-3K-deficient neutrophils exhibit reduced migratory capacities but showed an intact calcium release after chemokine stimulation. Conversely, results obtained with neutrophils lacking phospholipase C (PLC) indicated that PLC pathways may play an important role in chemoattractant-mediated regulation of intracellular calcium concentrations but not in chemotaxis [32
]. However, as replicating influenza virus did not impair phosphorylation of PKB, one of the known major downstream events of PI-3K signaling, a defective PI-3K activation is unlikely. This is further supported by our experiments with the PI-3K-specific inhibitor LY294002 (Fig. 6)
, which revealed that even in virus-infected monocytes, PI-3K activity is necessary for chemoattractant-mediated activation of PKB. In summary, this accounts for an intact receptor/ligand interaction and an undisturbed chemokine-mediated induction of signaling pathways upstream of PI-3K activation. Thus, as-yet unknown signaling events downstream of PI-3K activation appear to be impaired by virus-specific and replication-dependent factors.
Our results that virus-infected monocytes do not respond to chemokines are supported by a recent publication showing that herpes simplex virus-1 (HSV-1)-infected, immature dendritic cells (DCs) failed to react to the CCR7-specific ligand CCL19/Epstein-Barr-induced 1 ligand chemokine [34 ]. The authors concluded that this mechanism prevents the migration of infected DCs to secondary lymphatic organs. However, an analysis of CCR7 mRNA or protein expression after HSV-1 infection has not been performed in these studies.
In conclusion, infection of human monocytes with influenza A virus was accompanied by a substantial reduction of their chemokine-mediated responses. An unchanged or even enhanced expression of chemokine receptors did not counteract this process. When considering the essential role of monocytes/macrophages and DCs in antiviral immunity, it is tempting to speculate that impairing migration and activation by chemokines is a general strategy of viruses to evade the immune response. However, the infected host may also profit from migratorily passive monocytes, as it would arrest the virus-bearing cells in a confined area and would block spreading of infectious viruses to lymphoid and other organs.
Received November 18, 2002; revised March 26, 2003; accepted April 1, 2003.
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in chemoattractant-mediated signal transduction Science 287,1046-1049 in inflammation Science 287,1049-1053This article has been cited by other articles:
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