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Blood, 1 July 2005, Vol. 106, No. 1, pp. 150-157. Prepublished online as a Blood First Edition Paper on March 15, 2005; DOI 10.1182/blood-2005-01-0023.
IMMUNOBIOLOGY
The role of endothelial PI3K
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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) in neutrophils plays a critical role in the directed migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3K activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class-Ib PI3K function in neutrophils of p110–/– mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in wild-type (WT) neutrophil attachment to and 17-fold increase in rolling velocities on p110–/– microvessels in vivo in response to tumor necrosis factor alpha (TNF
). This alteration in adhesion was further augmented by a deficiency in p110
, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokine-stimulated endothelium. Interestingly, E-selectin–mediated adhesion in p110–/– mice was impaired by more than 95%, but no defect in nuclear factor kappa B (NF-
B)–induced gene expression was observed. These findings suggest a previously unrecognized partnership between class-I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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, 
, , or ) is in association with a particular regulatory subunit (designated p50, p55, p85, or p101).1,6,7 Functionally, all class-I PI3Ks catalyze the phosphorylation of phosphatidylinositol (4,5)–bisphosphate (PIP2) to form phosphatidylinositol (3,4,5)–trisphosphate (PIP3) in response to activation of either receptor tyrosine kinases (RTKs) or G-protein–coupled receptors (GPCRs), which ultimately regulates a diverse array of biologic functions. In regards to innate immunity, one major role of PI3Ks is to support chemoattractant-directed migration of neutrophils, macrophages, and specific populations of mast cells into sites of inflammation.8-11 Mechanistically, activation of this intracellular signaling pathway is essential for reorganization of cytoskeleton and membrane structure in response to such agonists, events that result in cell polarity and pseudopodia extension.12-14 In particular, it is believed that PI3K,a class-Ib PI3K, predominates in this process, as recruitment of neutrophils into inflamed tissues was reduced by more than 60% in p110-null mice as compared with wild-type (WT) controls.9-11 These studies, however, did not take into account the contribution of other cell types required for neutrophil trafficking, including vascular endothelium.
The recruitment of neutrophils to sites of inflammation relies not only on their ability to undergo transendothelial cell migration, but also upon a dynamic interplay between adhesion molecules expressed on these cells and the inflamed vessel wall.15,16 In this regard, the endothelial lining does not act as a permissive barrier between the blood and tissues but rather regulates neutrophil trafficking in response to various stimuli. For instance, endothelial cell activation by the proinflammatory cytokine tumor necrosis factor alpha (TNF) is a key event for the initiation of a well-orchestrated series of adhesive interactions involved in neutrophil recruitment, a process that relies in part on the de novo expression of E-selectin on the luminal surface of the vessel.17,18 E-selectin is one of 3 structurally related molecules that mediate the reversible capture (ie, tethering) by and subsequent rolling of leukocytes on the inflamed vessel wall in response to hydrodynamic flow.19 It does so by virtue of its ability to interact with constitutively expressed counterreceptors on human neutrophils such as P-selectin glycoprotein ligand-1 (PSGL-1).20 Maximal expression of E-selectin on cytokine-stimulated endothelium occurs within 4 hours and decays by 24 hours; surface expression parallels that of mRNA and is reliant in part on the nuclear factor kappa B (NF-B) and IB regulatory system.21-24 NF-B is a transcription factor that exists in the cytoplasm of cells in an inactive form due to its association with I kappa B alpha (IB). Degradation of IB in response to TNF stimulation enables NF-B to translocate into the nucleus where it can activate gene expression.
Class-I PI3Ks are known to contribute to specific biologic processes in endothelium such as angiogenesis.25 This signaling pathway, however, was not thought to be a major mediator of proinflammatory processes elicited by TNF.26 For instance, controversy exists surrounding the role of class-I PI3Ks in regulating NF-B–mediated gene expression of adhesion molecules such as E-selectin. Previously, it has been reported that the pan–class-I PI3K inhibitor LY294002 did not prevent the transcription of an NF-B–dependent E-selectin promoter-reporter gene.27 By contrast, transfection of HepG2 cells with a dominant-negative mutant of the p85 regulatory subunit, critical for the function of class-Ia PI3Ks, resulted in both the inhibition of the PI3K signaling pathway and TNF-induced expression of the reporter gene, NF-B/chloramphenicol acetyltransferase (CAT).28 Recently, we have suggested that class-I PI3Ks may indeed be involved in regulating the proadhesive state of vascular endothelium in response to cytokine stimulation.29 In particular, genetic deletion of the p110 catalytic subunit in mice significantly reduced neutrophil tethering to and increased rolling velocities of these cells on cytokine-stimulated venular endothelium by a yet-to-be-identified mechanism. The significance of this observation, however, and the potential contribution of its gamma counterpart to this process, remain to be determined.
In the current study, we explore the contribution of PI3K activity in endothelium in supporting neutrophil trafficking into sites of inflammation. This was accomplished using in vitro and in vivo experimental approaches that evaluated the effect that p110 deficiency in endothelium has on promoting E-selectin expression and interactions with neutrophils. The reported observations broaden our understanding of signal transduction pathways that contribute to the multistep adhesion cascade.
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Materials and methods |
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Antibodies used in experiments included CL3 and CL37 (anti–human E-selectin, inhibitory and noninhibitory, respectively; ATCC, Manassas, VA), 9A9 (function-blocking anti–murine E-selectin; Klauss Ley, University of Virginia), platelet-endothelial cell adhesion molecule (PECAM) 1.3 (anti–human PECAM-1; Peter Newman, University of Wisconsin, Milwaukee), and fluorescein isothiocyanate (FITC)–conjugated goat F(ab')2 anti–mouse immunoglobulin (Ig) (CALTAG Laboratories, Burlingame, CA). The following rat monoclonal antibodies (mAbs) to mouse proteins were purchased from BD Pharmingen (Franklin Lakes, NJ): FITC-conjugated RB6-8C5 (Gr-1) and biotinylated 10E9.6 (E-selectin). Qdot525 streptavidin conjugate was obtained from Quantum Dot (Hayward, CA). Recombinant murine and human TNF were obtained from PeproTech (Rocky Hill, NJ) and R&D Systems (Minneapolis, MN), respectively. Murine E-selectin, human P-selectin, or human intercellular adhesion molecule 1 (ICAM-1) expressed as Fc chimeric proteins were obtained from R&D Systems, Genetics Institute (Cambridge, MA), or ICOS (Bothell, WA), respectively. Bay 11-7082 and LY294002 were purchased from EMD Biosciences (San Diego, CA). The p110 inhibitor, IC87114, and recombinant p110 proteins were synthesized and purified as described.30 Rabbit anti-p110 and p110 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).
Animals
p110–/–/GFP–/+ mice and their WT littermate controls have been described29 and were used between 8 and 12 weeks of age. p110–/–/GFP–/+ mice were generated in a similar manner.9 Mice in which P-selectin was genetically deleted were obtained from Jackson Laboratories (Bar Harbor, ME) and mated with p110–/–/GFP–/+ animals to generate the double knockout. All animals were handled in accordance with policies administered by the National Institutes of Health and the Washington University Institutional Animal Care and Use Committee.
Fetal liver reconstitution
Matings were determined by detection of a copulation plug (designated 0.5 days gestation). All mice were 8 to 10 weeks old with a genetic background of C57BL/6 x 129/Sv. Male mice deficient in p110, p110, or both catalytic subunits were lethally irradiated (950 rad) and reconstituted with fetal liver cells from WT littermates expressing green fluorescent protein (GFP–/+).31 Briefly, embryos were harvested 14.5 days after coitus, fetal livers dispersed, and the cell suspension centrifuged, washed, and resuspended in Dulbecco modified Eagle medium (DMEM). Cells from each liver were injected into 2 mice that had been irradiated on the same day. Experiments were performed 8 to 10 weeks after injection.
Neutrophil purification
Mouse bone marrow (BM) polymorphonuclear cells (PMNs) were isolated by discontinuous Percoll gradient centrifugation as previously described.29,32,33 The resulting cell populations in all p110 genotypes were equivalent for expression of the granulocyte marker Gr-1 (81% to 86% positive).
LPS-induced lung inflammation
Intratracheal instillation of lipopolysaccharide (LPS; 10 µg/g body weight) was performed as previously described.29 At 6 hours after the challenge, mice were euthanized, bronchoalveolar lavage (BAL) fluid collected, samples centrifuged, and resuspended in 0.6 mL phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 5 mM EDTA (ethylenediaminetetraacetic acid), pH 7.4. Samples were then placed in 24-well tissue-culture plates (Becton Dickinson, Franklin Lakes, NJ) and the number of fluorescent neutrophils determined per unit area (0.7 mm2)by fluorescent microscopy (Nikon X10 and Eclipse TE 300 microscopes; Nikon, Melville, NY). A minimum of 4 fields of view were recorded on Hi-8 videotape and subsequently analyzed using a PC-based interactive image analysis system (Image Pro Plus). For E-selectin–blocking experiments, 100 µg F(ab')2 9A9 in PBS was administered by intravenous route just prior to instillation of LPS.
TNF measurement
Whole blood was collected from p110–/– mice or WT controls by cardiac puncture. LPS (250 ng/mL final concentration) or PBS was added to equivalent volumes of blood and samples incubated at 37°C for 6 hours. Supernatant was obtained by centrifugation and subsequently analyzed for TNF by enzyme-linked immunosorbent assay (ELISA; Biosource International, Camarillo, CA). Values were normalized to absolute neutrophil counts contained in each blood sample.
Intravital microscopy
The surgical preparation of animals for all in vivo studies was performed using standard techniques.29 An inflammatory response in the cremaster muscle (CM) venules of mice was induced by an intrascrotal injection of recombinant murine TNF (20 ng/mouse). The tissue was surgically exposed 3 hours after cytokine stimulation and viewed on an intravital microscope (IV-500; Mikron Instruments, San Diego, CA). The tissue was kept moist by superfusion with thermocontrolled (37°C), bicarbonate-buffered saline. Green fluorescent protein (GFP)–expressing cells were visualized in the microcirculation through a 60x/0.90
water immersion objective lens on a LumPlan FL microscope (Olympus, Melville, NY) using an intensified camera (VE1000SIT; Dage mti) and epifluorescence illumination. Rolling fraction was defined as the percentage of cells that interact with a given region of venule as compared with the total number of cells that enter that vessel (interacting and noninteracting) during the same time period. Venular shear rates were determined from optical Doppler velocimeter measurements of centerline erythrocyte velocity. Video images were recorded using a Hi8 VCR (Sony) and analysis of performed using a PC-based image analysis system.
Laminar flow assays
Human umbilical vein endothelial cells (HUVECs; passage 2-3), grown on fibronectin-coated glass coverslips, were pretreated with IC87114 (2 µM), LY294002 (10 µM), Bay 11-7082 (10 µM), or vehicle control (dimethyl sulfoxide [DMSO]) for 1 hour prior to stimulation with TNF (5 ng/mL, 4 hours). Peripheral blood neutrophils were isolated from healthy volunteers and infused over the endothelial cell monolayer that was incorporated into a parallel plate flow chamber (GlycoTech, Gaithersburg, MD) for 5 minutes at a shear rate of 200 s–1.29 Approval was obtained from the Washington University institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki. Neutrophil-endothelial cell interactions were recorded and analyzed as previously described.29 For E-selectin blockade, HUVECs were incubated with mAb CL3 (50 µg/mL, 15 minutes) prior to adhesion assays.
For flow studies involving recombinant protein, polystyrene plates were coated overnight with 100 µg/mL protein A (Sigma) at 4°C, then washed, and finally incubated with E- or P-selectin or ICAM-1-Fc chimeric proteins diluted to a concentration of 20 µg/mL (PBS, 0.1% BSA, pH 7.4) for 2 hours at 37°C. Nonspecific interactions were blocked with rabbit Ig (50 µg/mL) for 30 minutes at 37°C. Murine neutrophils (1 x 106/mL; Hanks balanced salt solution [HBSS], 10 mM Hepes [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 1 mM CaCl2, 0.5% BSA, pH 7.4) were infused over the selectin substrates at a shear rate of 200 s–1. The number of cells that attached over 5 minutes was determined and expressed per unit area.
NF-B p50 nuclear translocation assay
Cultured HUVECs (passage 3) were incubated for 16 hours in 0.5% fetal calf serum (FCS) containing medium 199. Cells were pretreated with vehicle (DMSO) or 10 µM of IC87 114, LY294002, or BAY 11-7082 for 2 hours before stimulation with TNF (10 ng/mL) for 30 minutes. Cells were harvested by trypsin digestion and nuclear extracts were prepared by using the TransFactor extraction kit (BD Bioscience/CLONTECH, Mountain View, CA) according to the manufacturer's instructions. After centrifugation at 20 000g for 5 minutes at 4°C, supernatants (nuclear extracts) were assayed for p50 content. An equal amount of nuclear extracts (10 µg) was added to incubation wells precoated with the DNA-binding consensus sequence. The presence of translocated p50 subunit was then assessed by using Mercury TransFactor kit (BD Biosciences/CLONTECH).
Immunoprecipitation and PI3K activity assay
Spleens from WT mice were pulverized in liquid nitrogen–cooled mortar and solubilized in PI3-kinase lysis buffer (50 mM Tris-HCl [pH 7.4]), 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, and a cocktail of inhibitors to serine and cysteine proteases (Complete Mini; Roche Applied Science, Indianapolis, IN). HUVEC lysates were prepared as described.29 Lysates were precleared with protein A–Sepharose and aliquots of the supernatants were incubated with antibodies specific for p110 and p110, or control antibody for 1 hour at 4°C, followed by addition of protein A–Sepharose for 2 hours at 4°C. Precipitates were washed once with lysis buffer, twice with 0.1 M Tris-HCl, pH 7.4; 5 mM LiCl; and 0.1 mM sodium orthovanadate and once with PI3-kinase buffer containing 20 mM Hepes, pH 7.4, 10 µM ATP, 5 mM MgCl2, plus 50 µg/mL horse IgG (Pierce, Rockford, IL). Lipid kinase activity was determined as previously described.30 The radioactive product PIP3 was captured onto a 96-well polyvinylidene difluoride filter plate (Millipore, Billerica, MA) and the bound radioactivity was quantitated with Microbeta Liquid Scintillation Counter (PerkinElmer Life Sciences, Boston, MA).
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Western blot analysis
HUVECs and the murine endothelioma cell line bEND3.1 (ATCC) lysates were prepared as described for the PI3K function assay. Recombinant p110, , , and proteins (20 ng/lane) and cell lysates (100 µg/lane) were electrophoresed in precast 8% polyacrylamide gels (Invitrogen Life Technologies, Carlsbad, CA), transferred electrophoretically to a polyvinylidene difluoride membrane (Invitrogen), and immunoblotted with p110 antibody as described previously.29
Statistical analysis
A Student t test was used for statistical comparisons. Statistical significance was set at P values less than .05.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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–/– chimeric mice
To determine if a "nonleukocyte" component of PI3K activity contributes to neutrophil accumulation at sites of inflammation, we evaluated the recruitment of these cells into LPS-treated lungs of p110–/– mice reconstituted with fetal liver cells (FLCs) from GFP-expressing WT littermates. Circulating white blood cell (WBC) counts and absolute neutrophil counts (mean ± standard deviation [SD]) of reconstituted animals were 10.1 K/µL ± 1.4 K/µL and 3.4 K/µL ± 0.8 K/µL, respectively, values equivalent to that of WT-matched controls (10.0 K/µL ± 2.1 K/µL and 2.8 K/µL ± 0.3 K/µL, respectively). Moreover, more than 95% of circulating GR-1 (+) cells in whole blood of all chimeric animals expressed GFP, which is consistent with complete reconstitution of the granulocyte population with p110+/+ neutrophils (data not shown). Intratracheal instillation of LPS into WT littermates resulted in an 11.5-fold increase in the number of fluorescent cells in BAL fluid 6 hours after challenge as compared with animals treated with normal saline (Figure 1A). Similar results were obtained in WT mice reconstituted with WT FLCs (data not shown). Complete absence of the p110 catalytic subunit, however, significantly reduced neutrophil counts (
84%). Surprisingly, LPS-induced recruitment of these cells was still mitigated (45%) despite reconstituting p110–/– animals with WT FLCs. This finding was not restricted to PI3K, as the activity of PI3K (class-Ia PI3K) in other cell types also contributes to the inflammatory cell infiltrate, albeit not to the extent observed for the former (Figure 1B). The importance of endothelium in neutrophil recruitment is demonstrated by the ability of a function blocking F(ab')2 to E-selectin (9A9), an adhesion molecule expressed on inflamed endothelium, to reduce BAL fluid cell counts by 70% (Figure 1C). Although TNF generated in response to LPS is required for expression of E-selectin, absence of PI3K activity did not alter the ability of leukocytes to secrete this proinflammatory cytokine (Figure 2).34,35
p110 is expressed in vascular endothelium
As class-I PI3K activity has been shown to exist in endothelium, it is conceivable that the PI3K may be present in this cell type and thus participate in neutrophil trafficking. To demonstrate for the first time that the p110 catalytic subunit not only is expressed in endothelium but is functional, we performed immunoprecipitation analysis and measured the activity of PI3K purified from proliferating vascular endothelial cells. Western blot analysis revealed the presence of this class-Ib isoform in both HUVECs and the murine endothelioma cell line bEND3.1 (Figure 3Aii,iii, respectively). Moreover, the immunoprecipitated material was functional as measured by its ability to generate PIP3 (Figure 3B). Importantly, the activity of p110 could be blocked by pan–class-I PI3K inhibitor LY294002 (10 µM), but not by IC87114 (10 µM) which is selective for p110 (Figure 3B-C). This is consistent with previous results demonstrating a 58-fold selectivity of IC87114 for p110 than for p110.30 By contrast, inhibitory concentration (IC50) values for LY294004 vary among the 4 class-I PI3Ks by only about 10-fold.
PI3K in endothelium is required for efficient neutrophil capture and rolling
The existence of p110 as a functional complex in vascular endothelium suggests that it may play an important role in mediating the neutrophil recruitment in response to proinflammatory stimuli. Thus, we sought to elucidate the potential mechanism(s) by which PI3K may regulate such an event by observing the behavior of GFP-expressing granulocytes in microcirculation of TNF-stimulated venules of mice chimeric for p110 activity. An absence of this catalytic subunit in endothelium alone resulted in the identical reduction in the number of fluorescent cells that attached to and rolled on the inflamed vessel wall as compared with animals lacking p110 in both cell types (Figure 4A). This suggests that PI3K in neutrophils does not play a role in this process. Interestingly, there was greater impairment in the attachment of WT neutrophils in p110–/– versus p110–/– chimeric animals (70% versus 55%), suggesting that the endothelial component of class-I PI3K activity may contribute to the differences observed in the LPS-induced acute lung injury model (Figure 4C,F). In addition to a defect in neutrophil attachment, rolling velocities of p110–/– versus p110–/– chimeric animals were increased by 17.5- and 7.5-fold, respectively, values also comparable to that observed in their nonreconstituted counterparts (Figure 4B,D). It appears, however, that the activity of both class-I PI3K isoforms is required for optimal attachment and rolling of neutrophils, as there was a greater perturbation in these adhesive parameters in mice lacking both catalytic subunits. Rolling fractions and velocities (mean ± SD) of neutrophils in p110–/–/–/– mice reconstituted with WT FLCs (GFP–/+) were 12.5% ± 4.3% and 136 µm/s ± 26.6 µm/s, respectively (Figure 4E,G). By contrast, values in reconstituted p110-or -deficient mice were 24% ± 5.2% versus 44.6% ± 7.7% and 95.1 µm/s ± 29 µm/s versus 44 µm/s ± 12.8µm/s, respectively. Thus, a lack of both p110 and p110 resulted in a more than 85% decrease in neutrophil attachment to and an approximately 23-fold increase in rolling velocities as compared with WT controls. We conclude from these observations that PI3K plays a significant role in regulating the proadhesive state of cytokine-stimulated vascular endothelium and that the activity of both class-Ia and Ib PI3Ks are required for optimal interactions between neutrophils and inflamed vessel wall.
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The observed reduction in attachment and increase in rolling velocities of WT neutrophils in animals deficient in class-Ia or Ib PI3Ks suggested that an alteration in selectin-mediated adhesion had occurred. Although P-selectin expressed on endothelium predominates in the initial capture of circulating neutrophils in acute tissue injury, it is E-selectin that accounts for the phenotypically slow rolling movements of these cells in microvessels of TNF-stimulated CM.36 To determine whether class-I PI3Ks contribute to E-selectin–mediated recruitment of neutrophils, we next evaluated the behavior of GFP-expressing granulocytes in P-selectin–deficient mice that lacked p110 activity (P-selectin–/–/p110–/–) or had received the p110-specific inhibitor IC87114. In regards to the latter, we had previously demonstrated the specificity of this compound for this class-Ia PI3K isoform.29 To provide additional evidence that IC87114 directly blocks the function of p110, we measured the catalytic activity of this enzyme isolated from spleen extracts of WT animals in the absence or presence of this inhibitor. By comparison to vehicle control, incubation of immunoprecipitated p110 with 10 µM of IC87114, which inhibits more than 95% of delta activity, reduced PIP3 production by more than 90% (Figure 5A).
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stimulation of the CM resulted in a more than 88% reduction in neutrophil attachment to and rolling on inflamed venular endothelium as compared with vehicle treatment alone (Figure 5B). Mean plasma level of the compound 4 hours after oral administration was 4.9 µM ± 2.7 µM, a concentration known to inhibit more than 85% of p110 but less than 5% of p110 activity.30 The requirement for E-selectin is demonstrated by the ability of the function-blocking mAb 9A9 to abrogate interactions between circulating granulocytes and the vessel wall in animals that received vehicle control. Thus, p110 activity is required for E-selectin–dependent adhesion of neutrophils. Moreover, the critical interplay between this adhesion molecule and class-I PI3K activity is further demonstrated in animals deficient in both P-selectin and p110. Attachment of GFP-expressing neutrophils to TNF-stimulated venules in the CM of these animals was impaired by more than 95% (Figure 5C).
To demonstrate that either genetic deletion of p110 or blockade of p110 activity in these animals does not prevent surface expression of E-selectin, which could account for the observed reduction in neutrophil adhesion, we next evaluated the accumulation of fluorescent semiconductor nanocrystals encapsulated in phospholipid micelles (Qdots) coupled to an antibody that recognizes this selectin molecule. In the absence of cytokine stimulation, no immunofluorescence was detected on the vessel wall (Figure 5Dii). By contrast, TNF-induced stimulation resulted in the deposition of the Qdots/antibody conjugate on microvessels in P-selectin–/– animals treated with IC87114 or deficient in p110 (Figure 5Diii,iv, respectively). The specificity of the interaction was confirmed by the lack of immunofluorescence staining in TNF-stimulated venules of E-selectin–/– mice treated with vehicle control (Figure 5Dv) or the p110 selective inhibitor (Figure 5Dvi).
Class-I PI3K activity is not required for NF-B–mediated expression of E-selectin
To further extend our in vivo observation that class-I PI3K activity may not be required for cytokine-induced expression of E-selectin, we performed flow cytometric analysis on TNF-stimulated (4 hours) HUVECs pretreated with vehicle control, IC87114 (2 µM to 50 µM), or LY294002 (10 µM). No difference in E-selectin expression was noted in the presence or absence of the inhibitors (Figure 6A-B). By contrast, TNF-induced expression of E-selectin was abrogated by Bay 11-7082 (10 µM), a small molecule inhibitor that impairs NF-B nuclear translocation and thus E-selectin gene transcription.37 Further evidence in support of our finding that class-I PI3K activity does not participate significantly in the expression of this adhesion molecule was provided by evaluating NF-B nuclear translocation in TNF-stimulated HUVECs. In contrast to Bay 11-7082, treatment with either the nonspecific or isoform selective class-I PI3K inhibitors LY294002 or IC87114, respectively, did not prevent nuclear localization of NF-B as determined by an ELISA that detects the p50 subunit of this transcription factor (Figure 6C). Both inhibitors nevertheless reduced the ability of TNF-stimulated HUVEC monolayers to capture untreated neutrophils in vitro by over 45% at a wall shear rate of 200 s–1, whereas the E-selectin–blocking antibody CL3 and Bay 11-7082 (10 µM) impaired attachment by 90% and 100%, respectively (Figure 6D). These results suggest that the role of class-I PI3Ks in E-selectin–mediated neutrophil attachment differs from transcriptional regulation by NF-B.
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or activity in leukocytes does not impair selectin-dependent capture in flow as observed in vivo, we next evaluated the attachment of purified p110–/– or p110–/– neutrophils to surface-immobilized selectin-Fc chimeras using a parallel plate flow chamber apparatus. Despite the lack of either class-I isoform activity, neutrophils from mutant animals accumulated equally well on these substrate and at levels comparable to that of WT controls (Figure 7A-B). |
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has been shown to be important in a number of biologic processes essential for neutrophil function in an innate immune response. Foremost is its ability to support directed migration of these cells upon GPCR activation by various chemoattractants. Our results provide conclusive evidence that PI3K activity in vascular endothelium is also essential for neutrophil recruitment, as its activity is required for efficient selectin-mediated adhesion, an important event in the multistep process that enables neutrophils to traffick into inflamed tissues. Confirmation that the activity of this class-Ib PI3K in neutrophils did not contribute to the observed impairment in adhesion was demonstrated by (1) similar alterations in rolling fraction and velocities in TNF-stimulated venules in the CM of p110–/– mice as compared with those reconstituted with WT FLCs, and (2) similar levels of attachment of p110–/– neutrophils to surface-immobilized E- or P-selectin in flow as compared with WT control cells. The biologic significance of our findings is revealed by the persistent reduction in neutrophil accumulation in an LPS-induced acute lung injury model that used animals chimeric for class-Ib PI3K activity. Moreover, the requirement for E-selectin to promote neutrophil infiltration into inflamed lung and the profound impact that p110 deficiency has on this interaction supports a defect in endothelial cell–mediated adhesion as the mechanism for the reduced accumulation of neutrophils in this acute lung injury model. That said, the greater perturbation in selectin-dependent adhesion of WT neutrophils on endothelium deficient in p110 and p110 suggests that optimal adhesive interactions require the activity of both PI3K isoforms.
How might PI3K participate in regulating the proadhesive state of TNF-stimulated endothelium? It is conceivable that its activity may contribute to surface expression of adhesion molecules, as it has been suggested that PI3Ks, in general, play a pivotal role in TNF-mediated activation of NF-B–dependent genes.23,28 In this scenario, stimulation of the type-1 TNF receptor results in activation of class-I PI3Ks and subsequent generation of PIP3. Consequently, this leads to the phosphorylation of Akt, a downstream target of PI3Ks, which in turn activates NF-B and thus transcription of genes necessary for key immune and inflammatory responses. Indeed, our previous results concur with the observation that class-I PI3K activity is essential not only for phosphorylation of Akt but also for the activation of PDK1 in endothelium in response to TNF. Both Akt and PDK1 are cytoplasmic proteins that contain pleckstrin homology domains, enabling them to be recruited to the plasma membrane where they can directly interact with PIP3. Activation of these protein kinases is essential for many of the downstream signaling events associated with PI3K activity. By contrast, our current results do not support a role for class-I PI3K–induced activation of NF-B, as neither p110 nor its counterpart contributes significantly to this process. This is evident by the inability of the pan–class-I PI3K inhibitor LY249004 or -specific inhibitor IC87114 to (1) prevent nuclear translocation of this transcription factor in HUVECs in response to TNF, and (2) alter the level of expression of E-selectin on this cell type. Moreover, this adhesion molecule was still present on cytokine-stimulated venules in the CM of p110–/– and p110–/– mice despite the significant reduction in selectin-dependent recruitment of neutrophils. Thus, an NF-B–independent mechanism must account for the majority of alteration in E-selectin–mediated adhesion in animals lacking class-Ia or Ib PI3Ks.
In addition to gene regulation, it has been shown that cytoskeletal-mediated distribution of adhesion molecules such as E-selectin on the surface of cytokine-stimulated endothelium plays a role in regulating its proadhesive state. Disruption of microtubule assembly in endothelial cells with colchicine before or after TNF activation altered the location but not expression of E-selectin, an event that reduced neutrophil attachment as assayed under static conditions.38 Moreover, linkage of E-selectin to the cytoskeleton may also be of biologic relevance in terms of strengthening interactions between these cells. In fact, multivalent ligation of E-selectin either by antibodies or surface-bound leukocytes results in its association with components of the actin cytoskeleton via its cytoplasmic domain, an event believed to confer greater resistance to mechanical deformation.39 These data suggest that microtubule dynamics contribute to the functioning of E-selectin on endothelium. In terms of signaling pathways, the small guanosine triphosphate–binding protein Rho has been shown to be required for clustering of E-selectin in the vicinity of leukocytes once they are bound to the endothelial surface.40 Although the involvement of p110 or in this process has not been demonstrated to date, class-I PI3Ks are known to contribute to the clustering of cell surface receptors on chemokine-stimulated lymphocytes.41 Thus, a precedent exists for the involvement of these lipid kinases in regulating the distribution and function of adhesion molecules on cells. Current studies are focused on elucidating the role of PI3K and in promoting E-selectin interactions with components of the cytoskeleton and subsequent effects on surface distribution and function.
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activity contributes in part to the innate immune response by regulating the ability of selectin molecules expressed on TNF-stimulated endothelium to efficiently capture circulating neutrophils. Moreover, we have clarified a controversial issue regarding the involvement of class-I PI3Ks in NF-B–mediated gene regulation. |
Acknowledgements |
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Footnotes |
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Prepublished online as Blood First Edition Paper, March 15, 2005; DOI 10.1182/blood-2005-01-0023.
Supported by National Institutes of Health grant HL63 244-05 and the American Lung Association (T.G.D.) and the Medical Research Council and Biotechnology and Biological Sciences Research Council (M.T.).
K.D.P., J.D., and J.S.H are employed by ICOS Corporation, the company whose potential product was studied in the present work.
An Inside Blood analysis of this article appears at the front of the issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Thomas G. Diacovo, Department of Pediatrics and Department of Pathology, Columbia University, College of Physicians and Surgeons, 3959 Broadway, CHC115, New York, NY 10032; e-mail: td2142{at}columbia.edu.
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V. Pinho, R. de Castro Russo, F. A. Amaral, L. P. de Sousa, M. M. Barsante, D. G. de Souza, J. C. Alves-Filho, D. C. Cara, J. S. Hayflick, C. Rommel, et al. Tissue- and Stimulus-Dependent Role of Phosphatidylinositol 3-Kinase Isoforms for Neutrophil Recruitment Induced by Chemoattractants In Vivo J. Immunol., December 1, 2007; 179(11): 7891 - 7898. [Abstract] [Full Text] [PDF]
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C. D. Wrann, N. A. Tabriz, T. Barkhausen, A. Klos, M. van Griensven, H. C. Pape, D. O. Kendoff, R. Guo, P. A. Ward, C. Krettek, et al. The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling C5a-Mediated Activation of Innate Immune Functions J. Immunol., May 1, 2007; 178(9): 5940 - 5948. [Abstract] [Full Text] [PDF]
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W. Swat, V. Montgrain, T. A. Doggett, J. Douangpanya, K. Puri, W. Vermi, and T. G. Diacovo Essential role of PI3K{delta} and PI3K{gamma} in thymocyte survival Blood, March 15, 2006; 107(6): 2415 - 2422. [Abstract] [Full Text] [PDF]
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K. S. Lee, H. K. Lee, J. S. Hayflick, Y. C. Lee, and K. D. Puri Inhibition of phosphoinositide 3-kinase {delta} attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model FASEB J, March 1, 2006; 20(3): 455 - 465. [Abstract] [Full Text] [PDF]
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| Copyright © 2005 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
activity in neutrophil trafficking
indicates WT littermate (GFP–/+ p110
, p110
) 1 hour prior to induction of inflammation with TNF
