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Address correspondence to Paul Kubes, Immunology Research Group, University of Calgary, 3330 Hospital Dr. NW, Alberta T2N 4N1, Canada. Tel.: (403) 220-8558. Fax: (403) 283-1267. E-mail: pkubes{at}ucalgary.ca
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Abstract |
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Key Words: p38 MAPK; PI3K gamma; chemotaxis; neutrophils; signal transduction
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Introduction |
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Despite the profound structural differences between the many chemoattractants, the receptors for these molecules are similar in that they belong to the seven-transmembrane helix receptor family (Damaj et al., 1996; Prossnitz et al., 1999). Once activated, these receptors transmit their signals to heterotrimeric G proteins, which activate downstream pathways leading to rapid cytoskeletal rearrangements and chemotaxis (Amatruda et al., 1995; Schraufstatter et al., 2001). Phosphoinositide 3-kinase (PI3K)* appears to be a key molecule in one of the intracellular pathways of neutrophil chemotaxis (Hirsch et al., 2000). Although multiple PI3K isoforms exist, chemotactic receptors utilize the p110 catalytic isoform, which is responsible for downstream events including the conversion of membrane phospholipid phosphatidylinositol 4,5-biphosphate into phosphatidylinositol 3,4,5-triphosphate. This leads to the phosphorylation and activation of protein kinase B (also known as PKB or Akt). This is best evidenced by the fact that activation of chemotactic receptors in mice lacking p110
manifests as no phosphorylation or no activation of PKB/Akt, leading to defects in chemotaxis (Hirsch et al., 2000).
However, P110-deficient neutrophils can still chemotax in significant numbers (Hirsch et al., 2000). Indeed, the p38 MAPK pathway has also been demonstrated to play a very important role in neutrophil chemotaxis in humans (Nick et al., 1997) and mice (Cara et al., 2001), and this pathway is still effectively activated in p110
-deficient cells, perhaps explaining neutrophil recruitment to sites of inflammation in these mice (Sasaki et al., 2000). Whether the p38 MAPK and PI3K/Akt pathways are complementary, overlapping, or perhaps even opposing and whether they function in a hierarchical fashion remains unclear. Furthermore, whether there is any physiologic cross-talk between these pathways has never been addressed.
In this study, our data would support the proposed model that end target chemoattractants (fMLP and C5a) function primarily by stimulating p38 MAPK, whereas intermediary chemoattractants (IL-8 and LTB4) primarily function via the PI3K/Akt pathway. These pathways underlie the hierarchical dominance of end target chemoattractants over intermediary chemoattractants inasmuch as activation of the p38 MAPK pathway in neutrophils inhibits the PI3K/Akt pathway. Tonic inhibition of PI3K/Akt is apparent, since inhibition of p38 MAPK causes a marked increase in PI3K/Akt activity and a causally related exaggerated chemotactic response to intermediary chemokines.
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Results |
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Competing gradients of end target and intermediary chemoattractants
In competing gradients consisting of optimal concentrations of fMLP (1 pmol; in right well) and 1/100th optimal concentration of IL-8 (0.1 pmol; in left well), neutrophils preferentially migrated toward fMLP (Fig. 3 a). When optimal concentrations of both fMLP and IL-8 were added, neutrophils still migrated toward fMLP, unexpectedly in much greater numbers. This pattern was repeated with competing gradients consisting of 1/100th optimal concentration of fMLP (0.01 pmol) and optimal concentrations of IL-8 (10.0 pmol). Under these conditions, neutrophils still preferentially migrated toward fMLP, although the effect was less pronounced (Fig. 3 a). It is noteworthy that this lower concentration of fMLP (without IL-8 present) does not induce any migration (Fig. 1 c), suggesting that not only does fMLP overcome any effects of IL-8, but the cells migrate more effectively in the presence of the opposing IL-8 gradient. Neutrophils also migrated toward fMLP in an opposing LTB4 gradient (Fig. 3 a). The other end target chemoattractant, C5a, induced neutrophil chemotaxis in a dominant fashion over optimal doses of LTB4 or IL-8 (Fig. 3 a). When opposing gradients of two end target chemoattractants (fMLP versus C5a) were used, there was no dominance for either chemoattractant, since neutrophils migrated equally well in both directions (unpublished data), consistent with the same rather than different signaling pathways being involved. An identical response was observed when the opposing gradients of the two intermediary chemoattractants (LTB4 versus IL-8) were used (unpublished data). For the remainder of the experiments, fMLP and IL-8 are used to further explore the mechanisms of action. Nevertheless, in numerous instances similar results are observed for C5a and LTB4.
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Changes in downstream events of the IL-8 receptors mediates the chemotactic hierarchy
To determine why end target molecules dominated over intermediary chemoattractants, we next examined whether fMLP directly affects IL-8 receptor number and/or downstream events. Fig. 4 a illustrates that fMLP had absolutely no effect upon either CXCR1 or CXCR2 (the two known IL-8 receptors) at a wide range of concentrations. As a positive control, we show that CXCR1 and CXCR2 were both internalized in response to IL-8 (Fig. 4 a). Clearly, fMLP does not affect IL-8 receptor numbers.
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Effect of PI3K or p38 MAPK inhibition on the fMLP/IL-8 hierarchy
We looked at the effect of PI3K inhibition on the fMLP/IL-8 hierarchy. Two gradients were tested: the first consisting of optimal fMLP (1.0 pmol) and 1/100th optimal IL-8 (0.1 pmol), and the second consisting of 1/100th optimal fMLP (0.01 pmol) and optimal IL-8 (10.0 pmol). In both gradients, neutrophils treated with the PI3K inhibitor LY294002 still preferentially migrated toward fMLP (Fig. 5 a).
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Fig. 5 c demonstrates that alteration in PI3K/Akt was the mechanism by which fMLP induces p38 MAPKinhibited neutrophils to migrate in greater numbers toward the intermediary chemoattractants. First, inhibition of PI3K with LY294002 eliminated p38-inhibited neutrophils from migrating toward IL-8 (Fig. 5 c). To examine whether inhibition of p38 MAPK increased PI3K activity, we examined Akt in the presence and absence of the p38 MAPK inhibitors. Fig. 6 shows that phosphorylation of Akt in response to fMLP and IL-8 peaks at 1 min and returns to control by 5 min. However, when p38 MAPK inhibited neutrophils are stimulated with both fMLP and IL-8, Akt phosphorylation is high and remains high for at least 60 min (Fig. 6). In additional experiments, p38 MAPK was activated by the nonchemotactic end target molecule, LPS, a potent activator of p38 MAPK (Haddad et al., 2001). LPS (10 ng/ml) pretreatment of neutrophils blocked migration to IL-8 (Fig. 7 a) in an identical fashion to fMLP. Inhibiting p38 MAPK with SB203850 before LPS treatment reversed the LPS-induced inhibition of migration to IL-8. However, the number of migrating neutrophils was not enhanced. Fig. 7 b shows that AKT phosphorylation induced by IL-8 was abolished in LPS-treated cells. p38 MAPK inhibition not only restored phosphorylation of AKT in IL-8 and LPS-stimulated cells, but once again the Akt phosphorylation was stronger and more prolonged then the phosphorylation induced by IL-8 alone (see Fig. 4 b for comparison). Clearly, this is a common feature of numerous end target molecules.
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Discussion |
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To study the decision-making (signaling) processes of neutrophils in complex chemotactic fields, we used the under agarose neutrophil migration assay first used by Nelson et al. (1975) and more recently by Campbell et al. (1997) and Foxman et al. (1997)(1999). This system allows for establishment of multiple simultaneous chemotactic fields. There are advantages of this assay over the more widely used Boyden chamber assay. Whereas the latter is used very effectively for large scale screening of compounds, it is a unidirectional fluid phase assay, which does not allow for the effective study of bidirectional decision making by the neutrophils in response to multiple chemotactic agents. Although the concentrations we used may appear larger than what has been used in Boyden chambers, the concentrations cannot be directly compared due to the more limited movement of the chemokines in the agarose versus fluid in a Boyden chamber. Previously, investigators have used this assay to report directional migration of neutrophils (Nelson et al., 1975; Foxman et al., 1997, 1999). We have modified the assay so that we could quantify the number of cells that migrated toward a particular chemotactic cue. This became extremely useful to report enhanced or decreased number of migrating neutrophils in response to various manipulations but required that we design the system in such a manner as to be able to limit and count the number of migrating cells. In this study, we report that end terminal chemoattractants at various concentrations and in various scenarios dominated over the chemotactic potential of intermediary chemoattractants, consistent with the work of Campbell et al., 1997, Foxman et al. (1997)( 1999), and others (Knall et al., 1997) using the same under agarose assay system. We extend the work to show that several of the end target molecules induce chemotaxis via p38 MAPK, whereas in stark contrast the intermediary chemoattractants use the PI3K/Akt pathway in this system. More importantly, a hierarchy between the two pathways exists in that the former has inhibitory impact upon the latter. This may be the underlying problem in serious clinical situations like sepsis in which increased levels of LPS, fMLP, or C5a in the circulation may inhibit chemotaxis toward intermediary chemoattractants, thereby preventing neutrophil infiltrations to sites of infections. Indeed, the inability of neutrophils to emigrate is a well-described (Wagner and Roth, 1999) but unexplained phenomenon in septic patients. In addition, neutrophils migrated toward end target chemoattractants better when intermediary chemoattractants were present in an opposing gradient, suggesting an amplifying loop, which, in the case of fMLP, was at least in part due to increased fMLP receptor number. Along the same lines of cross-talk, we report that inhibition of the p38 MAPK pathway leads to an amplification of the PI3K/Akt pathway in both magnitude and duration. The p38 MAPK inhibition results in neutrophil migration away from end-terminal chemoattractants and toward intermediary chemoattractants in two- to fivefold greater numbers. Finally, and most importantly, the enhanced PI3K/Akt activation induced by p38 MAPK inhibitors is causally related to the increase in neutrophil chemotaxis, since inhibition of PI3K completely inhibited the enhanced chemotaxis in response to intermediary chemokines.
There is some supportive data for individual end terminal chemoattractants using p38 MAPK, whereas intermediary chemoattractants use PI3K/Akt. Previous work has clearly demonstrated that IL-8 activates PI3K/Akt, ERK, and p38 MAPK in primary human neutrophils (Knall et al., 1995; Capodici et al., 1998). Inhibition of PI3K with wortmannin inhibited ERK but not p38 MAPK activation, suggesting that the activation of PI3K/Akt and ERK was distinct from p38 MAPK activation (Knall et al., 1997). Moreover, p38 MAPK activation had no demonstrable role in the induction of neutrophil chemotaxis in response to IL-8, whereas PI3K/Akt was essential for IL-8induced neutrophil chemotaxis (Knall et al., 1997). The bacterial compound fMLP also appears to activate multiple intracellular signaling proteins including PI3K/Akt, ERK, and p38 MAPK (Zu et al., 1998; Yasui and Komiyama, 2001). However, our data suggest that activation of p38 MAPK but not PI3K/Akt blocked fMLP-induced chemotaxis but had little impact on IL-8induced neutrophil chemotaxis. We observed for the first time that when both pathways are activated simultaneously p38 MAPK dominates by depressing PI3K/Akt activity. Furthermore, p38 MAPK could interrupt and turn off previously activated PI3K/Akt in that when neutrophils were placed in a well with IL-8 and fMLP was placed in a distal well, the neutrophils would subsequently respond to fMLP, despite having IL-8/PI3K/Akt activated first. This latter observation is likely critical to neutrophils terminating all activity in response to intermediary chemoattractants and responding to end target chemoattractants.
Our data reveals that cross-talk between p38 MAPK and PI3K/Akt pathways is the inhibitory mechanism of action of end target chemoattractants on intermediary chemoattractant responses. Cross-talk among different signaling pathways is not without precedent. In fact, a significant amount of work has demonstrated that p38 MAPK and PI3K have opposing effects on apoptosis in neutrophils, endothelium, and other cells and that the PI3K/Akt pathway can inhibit p38 MAPK pathway (Berra et al., 1998; Alvarado-Kristensson et al., 2002). Although to date no one to our knowledge has shown that p38 MAPK negatively regulates PI3K/Akt, one study has shown that p38 MAPK can associate with Akt (Rane et al., 2001). In this study, we show that for chemotactic stimuli in neutrophils, p38 MAPK appears to inhibit PI3K. Moreover, when p38 MAPK is inhibited the effects of PI3K/Akt are greatly unmasked as evidenced by both a very substantial increase in Akt activity and a fivefold increase in PI3K-dependent chemotaxis.
A second site of inhibition by fMLP/p38 MAPK on IL-8/PI3K/Akt may be at the IL-8 receptor. Activation of the fMLP receptor has been shown to cross-desensitize G protein activation stimulated by IL-8 (Richardson et al., 1995; Ali et al., 1999). This process is mediated by receptor phosphorylation in which sites on the cytoplasmic end of the IL-8 receptors are phosphorylated by PKC. However, based on data presented herein, we feel that neither phosphorylation of the IL-8 receptor nor internalization is likely to be the reason for neutrophils not responding to IL-8 in the presence of fMLP. First, fMLP had absolutely no effect on the surface expression of either CXCR1 or CXCR2 in our system. Second, placing neutrophils into a local environment of IL-8 (Fig. 3 b) activated the IL-8 receptors and caused rapid homologous desensitization and internalization (Fig. 4). Nevertheless, even after the IL-8 receptors were internalized and phosphorylated by local IL-8, neutrophils still migrated toward fMLP placed in a distal well (in fact in greater numbers than without IL-8 present). Clearly, inhibition of the IL-8 pathway occurred down stream of the receptors and as our study suggests via p38 MAPK. Moreover, mutations at the phosphorylation sites on the IL-8 receptors do not inhibit responses to IL-8 stimulation (Richardson et al., 1998). As such, these authors predicted that another mechanism downstream of the G proteins was involved in maintaining the fMLP/IL-8 hierarchy. Our studies suggest that this mechanism is an inhibition of the PI3K pathway by p38 MAPK. Mechanisms by which p38 MAPK and PI3K/Akt differentially effect migration could include relocalization of CD11/CD18 toward the leading edge of neutrophils (preferentially toward end target chemoattractants), redistribution of CD11/CD18 into local microenvironments (e.g., lipid rafts) leading to improved chemotaxis and/or increased affinity, and/or avidity in response to end target chemoattractants but not intermediary chemokines. Our preliminary work suggests that the p38 MAPK and PI3K/Akt pathways may recruit different subunits of the CD18 glycoprotein complex and that in opposing gradients Mac-1 seems to dominate over LFA-1. This would suggest that the two
subunits of CD18 oppose rather than cooperate with each other. There is much controversy regarding the relative importance of LFA-1 versus Mac-1 with studies indicating a dominant role for either the former or the latter (Gao and Issekutz, 1996; Ding et al., 1999; Hogg and Leitinger, 2001; Seo et al., 2001). Our observation that different chemoattractants may preferentially activate LFA-1 or Mac-1 separately would explain some of this controversy. Although the prevailing view is that LFA-1 and Mac-1 function in a cooperative additive manner, this theory does not explain the curious finding that for a particular stimulus, LFA-1 deficiency in mice reduces neutrophil recruitment, whereas Mac-1 deficiency actually increased neutrophil recruitment (Ding et al., 1999), raising the possibility of some opposing effects of LFA-1 and Mac-1.
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Materials and methods |
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Neutrophil isolation and preparation
Blood was collected from healthy human donors. Erythrocytes were removed using dextran sedimentation (6% dextran/0.9% NaCl) followed by two rounds of hypotonic lysis using ddH2O. Neutrophils were isolated from the resulting cell suspension using Ficol-Histopaque density centrifugation. The entire isolation was done at 4°C. Purified neutrophils were suspended in HBSS at a concentration of 1.0 x 107 cells/ml and were kept on ice until needed. Neutrophils were >95% viable and >97.5% pure as determined by FACS®.
Under agarose assay procedure
The under agarose assay was used as described previously (Nelson et al., 1975; Foxman et al., 1999) with minor modification. Falcon 35 mm x 10 mm culture dishes were filled with 3 ml of a 1.2% agarose solution containing 50% H2CO3-buffered HBSS and 50% RPMI-1640 culture medium with 20% heat-inactivated FCS. After the agarose solidified, three wells, 3.5 mm in diameter and 2.4 mm apart, were cut into a straight line in the gel. The gels were allowed to equilibrate for 1 h in a 37°C/5% CO2 incubator. Unless otherwise noted, the center well was loaded with purified neutrophils, and the outer wells were loaded with varying concentrations of chemoattractant. Once loaded, the gels were incubated for 2 h in a 37°C/5% CO2 incubator, which allowed sufficient time for neutrophils to migrate the entire distance between the wells. Results were recorded using a video camera attached to a ZEISS Axiovert 135 microscope.
For the dose response experiments, the two outer wells were loaded with neutrophils, and the inner well was loaded with chemoattractant. The maximum IL-8 concentration tested was 1.25 µM, which was the concentration of our stock solution.
p38 MAPK inhibitors were added to isolated neutrophils such that the final inhibitor concentration was 30 µM for SKF86002 and 10 µM for SB203580, which have been shown to be the optimal inhibitory concentration without nonspecific effects (Zu et al., 1998). The concentrations of PI3K inhibitors were 100 nM for wortmannin and 50 µM for LY294002 (Niggli and Keller, 1997). All inhibitors were incubated with the neutrophils for 10 min. All neutrophils were stored on ice until needed.
To determine the effects of competing fMLP and IL-8 gradients, one outer well was loaded with fMLP, the second outer well was loaded with IL-8, and the central well was loaded with neutrophils. We examined whether p38 MAPK was dominant over PI3K by adding p38 MAPKinhibited, PI3K-inhibited, or PI3K- and p38 MAPKinhibited neutrophils to the central well and IL-8 and fMLP into the two outer wells. To eliminate the possibility of diffusion rates accounting for preferential migration, p38 MAPKinhibited or untreated neutrophils were loaded into a well containing IL-8, and fMLP was placed in an adjacent well (or vice versa).
To test the role of the two IL-8 receptors (CXCR1 and CXCR2) in the fMLP/IL-8 hierarchy, we treated neutrophils with either 2.5 µg/ml anti-CXCR1 or 10 µg/ml anti-CXCR2 antibodies and loaded these neutrophils into the central well of a gel containing IL-8 and fMLP in its outer wells.
Video analysis
The under agarose assay allows quantification of both random migration (chemokinesis) and directional migration (chemotaxis). To determine if the migration we observed was directional, two target areas were analyzed. Target A was a segment 500 µm x 2,300 µm between the neutrophil containing well and the chemoattractant containing well. Target B was of the same size as the first zone but extended from the neutrophil well to a point directly opposite the chemoattractant containing well (Fig. 1 a). The number of chemotaxing cells was determined by subtracting the number of migrating cells in target B from the number of migrating cells in area A.
IL-8 neutrophil receptor analysis using FACS®
Whole blood was diluted 1/10 with HBSS. Blood was stimulated using fMLP or IL-8 (1100 nM) for 20 min at 37°C. Control tubes (cells alone) received no chemoattractant but were placed at 37°C for 20 min. Anti-CXCR1 or anti-CXCR2 primary antibody was added to experimental tubes at 2.5 and 10 µg/ml, respectively, and isotype IgG2a antibody was added to control tubes. All antibodies were incubated at 37°C for 15 min. Untreated and treated controls were used to ensure stimulation did not induce autofluorescence and nonspecific binding. After labeling, cells were washed and then lysed with Optilyze B solution for 10 min. Cells were washed again and incubated with FITC-labeled secondary antibody. After a 15 min, incubation cells were washed and resuspended in HBSS to a concentration of 1.0 x 106 cells/ml.
To test the effects of the p38 MAPK inhibitor, SB203580, on receptor internalization, neutrophils were treated with 10 µM SB203580 and then labeled using anti-CXCR1 or anti-CXCR2 antibodies as described previously. These cells were stimulated with 0.00010.1 µM fMLP and incubated for 20 min at 37°C. Fluorescent intensity was analyzed using flow cytometry gating on the neutrophil population.
Calcium flux
Isolated human neutrophils at a concentration of 10 million cells/ml were either left untreated or inhibited with the p38 MAPK inhibitor SB203580 for 1 h at 37°C. These cells were labeled with 4 µM fluo-3 dye at 37°C for 30 min with occasional agitation. Cells were washed twice with Hepes buffer with 1 mM calcium (145 mM NaCl, 5 mM KCl, 1 mM MgCl, 10 mM Hepes, and 10 mM glucose) and centrifuged at 1,300 rpm for 6 min. The neutrophils were diluted to one million cells per ml in Hepes buffer and pipetted into 4-mL cuvettes in PerkinElmer fluorescence spectrometer 65010S with an excitation wavelength of 480 nm and emission recorded at 530 nm. Neutrophils were initially stimulated with 100 nM IL-8 or fMLP. Once the calcium levels stabilized, the cells were stimulated a second time with an additional 100 nM IL-8 or fMLP. Calcium flux was measured as the percentage of total calcium (fmax). fmax was determined by treating fluo-3labeled cells with Triton X-100.
Akt blotting
p38 MAPK inhibited and uninhibited neutrophils at a concentration of 4.0 x 107 cells/ml were treated with fMLP and/or IL-8 such that the final concentration of either chemoattractant was 100 nM. Aliquots of these cell suspensions were removed at various time points and added to an equal volume of boiling 2x Laemmli buffer (125 mM Tris HCl, pH 6.8, 8% SDS, 10% ß mercapto ethanol, 17% glycerol, 6.65% bromophenol blue, 5 mM orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Samples were then electrophoresed on an 8% SDSpolyacrylamide gel and transferred to a highbond p+ membrane. Blots were immunoblotted using antiphospho-Akt antibody in TBS-tween with 2% BSA. The blots were then stripped and reprobed with anti-Akt antibodies to show that all lanes were loaded equally. Both blots were visualized on radiography film using a secondary antibody conjugated to HRP and an EPO chemiluminescence kit. Densitometry was performed using Quantity One software.
Statistics
All data are expressed as the arithmetic mean ± SEM. Data was compared using a single sample t test unless otherwise noted. Values of P < 0.05 were considered to be statistically significant.
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Footnotes |
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Acknowledgments |
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Submitted: 22 February 2002
Revised: 15 August 2002
Accepted: 15 August 2002
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References |
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