Lactosylceramide Stimulates Human Neutrophils to Upregulate Mac-1, Adhere to Endothelium, and Generate Reactive Oxygen Metabolites In Vitro
Abstract—Glycosphingolipids (GSLs) and their metabolites play important roles in a variety of biological processes. We have previously reported that lactosylceramide (LacCer), a ubiquitous GSL, stimulates NADPH oxidase–dependent superoxide generation by aortic smooth muscle cells and their consequent proliferation. We postulated that LacCer may upregulate adhesion molecules on human polymorphonuclear leukocytes (hPMNs), perhaps also via NADPH oxidase–dependent reactive oxygen metabolite (ROM) generation. Incubation of hPMNs with LacCer upregulated CD11b/CD18 (Mac-1) and CD11c/CD18, as determined by fluorescence-automated cell sorting. LacCer also stimulated these hPMNs to generate superoxide via NADPH oxidase, as determined by lucigenin-enhanced chemiluminescence. However, the upregulation of Mac-1 by LacCer did not itself appear to be mediated by ROMs, since neither an antioxidant nor an NADPH oxidase inhibitor substantially inhibited the Mac-1 upregulation. However, this Mac-1 upregulation was significantly inhibited by two disparate phospholipase A2 (PLA2) inhibitors. Moreover, LacCer induced arachidonic acid metabolism, which was inhibited by the PLA2 inhibitors, but not by an NADPH oxidase inhibitor. To evaluate the effect of LacCer on hPMN adhesion to endothelium, hPMNs stimulated with LacCer were allowed to adhere to unstimulated human endothelial cell monolayers. LacCer stimulated hPMN adhesion to endothelial cells, which was blocked by anti-CD18 and by the PLA2 inhibitors. We conclude that LacCer stimulates both Mac-1 upregulation and superoxide generation in hPMNs but that ROMs are not the upstream signal for Mac-1 upregulation. This mechanism may well be relevant to acute endothelial injury in inflammation and other pathological conditions.
Glycosphingolipids and their catabolic products are now known to serve as second messengers in a variety of cellular signaling pathways, including those controlling cell proliferation, cell differentiation, cell migration, and programmed cell death.1 2 3 4 5 GlcCer and LacCer are among the ubiquitous GSLs found in eukaryotic cells.5 Elevated levels (2- to 18-fold compared with normal levels) of GSLs, which include LacCer, are found in the plasma of patients with familial hypercholesterolemia.6 7 8 Moreover, calcified or uncalcified plaque intima from human aorta contains high levels of LacCer.8 Oxidized LDLs have also been found to stimulate the synthesis of LacCer.9 Our earlier studies had revealed that LacCer induced the proliferation of aortic smooth muscle cells by activating a p44 mitogen-activated protein kinase,10 which was initiated by the generation of superoxide by a membrane-bound NADPH oxidase.11 These studies had suggested a role for LacCer in the pathogenesis of atherosclerosis via the proliferation of smooth muscle cells, a major cellular component of the atherosclerotic process.
Although neutrophils accumulate during chronic inflammation and myocardial injury due to ischemia and reperfusion,12 the underlying mechanism(s) by which circulating neutrophils initially adhere to the endothelium is not fully understood. We hypothesized that circulating LacCer, or LacCer that had accumulated within the arterial intima, might increase neutrophil-endothelial adhesion by activating neutrophil CD11/CD18. Previously, CD11/CD18 has been shown to be activated rapidly by other inflammatory mediators, such as PAF.13 14 15 Because LacCer may promote vascular smooth muscle cell proliferation by stimulating their generation of superoxide, we evaluated the possibility that LacCer might also induce adhesion molecule expression via superoxide-mediated signaling by hPMNs, which are particularly abundant in “neutrophil” NADPH oxidase.16 17 We also evaluated the effect of LacCer on the adhesion of PMNs to unstimulated endothelium in vitro.
Materials and Methods
Human blood was collected (in 10 U/mL heparin) from laboratory worker volunteers (by a protocol preapproved by the Johns Hopkins University Joint Committee for Clinical Investigation) and centrifuged at 1300g for 10 minutes. The white blood cell layer was removed and layered over cold Accu-prep gradient (Accurate Chemical and Scientific Corp) and centrifuged at 600g for 30 minutes at 4°C to effect leukocyte separation. The RBC+PMN layer was resuspended in RBC lysing buffer (Sigma Chemical Co). After 20 minutes at room temperature, the preparation was centrifuged at 1300g for 2 minutes, and this step was repeated until the PMN pellet was visibly free of RBCs. This suspension was consistently found to be composed of >95% PMNs by microscopic morphology after modified Wright-Giemsa staining (Diff-Quik Stain Set, Baxter), with a viability of >95% by a trypan blue exclusion.
Human umbilical vein ECs (Clonetics Corp) were cultured in plastic flasks in endothelial growth medium with 2% serum (Clonetics), at 37°C, with 5% CO2, passaged at confluence using 0.025% trypsin and 0.53 mmol/L EDTA (GIBCO BRL), and then grown to confluence in 24-well plates. Cells from passages 5 and 6 from these primary cultures were used.
Flow Cytometric Analysis of the Expression of Adhesion Molecules on PMNs
Treated PMNs were rapidly cooled on ice, washed twice with cold washing buffer (PBS containing 0.1% BSA and 0.1% sodium azide), and then incubated with either m-anti-h LFA-1 (CD11a), m-anti-h Mac-1 (CD11b), m-anti-h p150,95 (CD11c), or m-anti-h L-selectin (all from PharMingen), followed by FITC-conjugated monoclonal anti-mouse IgG (American Qualex). The PMNs were gated by forward and side scattering for analysis by FACScan (Becton Dickinson).
Measurement of ROM Generation by PMNs
Lucigenin-enhanced chemiluminescence was used to measure ROM generation by PMNs, and the data were presented as nanomoles superoxide per milligram protein (based on a standard curve, where superoxide was generated by xanthine/xanthine oxidase) as described previously.11 Briefly, 200 mL of HBSS (GIBCO) containing 106 PMNs, 100 mmol/L lucigenin (Sigma), and increasing concentrations of LacCer were added to 96-well plates, and incremental photon emission was monitored every 20 seconds for 20 minutes. The count without PMNs was subtracted from all other values as background. For the inhibition assays, PMNs were preincubated with each inhibitor for 15 minutes at room temperature and then stimulated with LacCer. Bovine erythrocyte SOD (Calbiochem) was used to scavenge extracellularly released superoxide, and DPI (Sigma) and apocynin (Aldrich) were used to inhibit NADPH oxidase.18 19
Assessment of PLA2 Activity
In order to determine whether LacCer activates PLA2 in intact PMNs, PLA2 activity was evaluated by measuring the release of [3H]AA and metabolites from labeled PMNs, as described by Jacobson and Schrier,20 with modification. PMNs (5×107/mL) were incubated with [3H]AA (2 mCi=1×10−11 mol/mL) (NEN) for 90 minutes at 37°C, and the cells were washed three times with calcium-free PBS. PMN (0.5 mL) suspension (2×106/mL HBSS) was first incubated with/without inhibitors for 15 minutes at room temperature, and then 0.5 mL of LacCer (final concentration, 100 nmol/L) in HBSS containing 2% fatty acid–free BSA (Sigma) was added to the cells for 30 minutes at 37°C, after which radioactivity in the supernatant was determined.
PMN-Endothelial Adhesion Assay
PMNs were labeled fluorescently by incubation with 5 mmol/L calcein-AM (Molecular Probes) in calcium-free PBS for 20 minutes at 37°C, washed, and resuspended in HBSS containing 0.2% BSA. These PMNs were then treated with LacCer (or vehicle, 0.05% dimethyl sulfoxide), washed three times with PBS, and then plated on the unstimulated EC monolayers, which had been grown to confluence in 24-well plates. The PMNs (600 mL of 2×106/mL per well) were then incubated with the ECs for 30 minutes at 37°C. Nonadherent PMNs were then removed by gentle washing three times with PBS. The residual adherent PMNs (and ECs) were then lysed using 4 mmol/L Zwittergent (Calbiochem), and the plates were read on a fluorescence plate reader (Millipore) at 480 nm (excitation)/530 nm (emission). The number of adherent PMNs was expressed as the number of PMNs/mm2 EC monolayer, based on the mean fluorescent intensity of each PMN, as determined from a standard curve. To rule out the possibility of adhesion stimulated by EC reactivity in some PMN adhesion assays, the EC monolayers in the half of each 24-well plate were first fixed with 2% buffered formalin for 5 minutes and then washed three times with PBS. The ECs on the other half of the plate were left unfixed but were similarly washed before use for the adhesion assay.
LacCer, ceramide, and GlcCer (all from Sigma) were prepared as described previously11 and used as putative agonists for the stimulation of PMN adhesion molecule expression, assayed by FACScan. To explore the mechanism involved in LacCer upregulation of Mac-1 on PMNs, PMNs were incubated with the following specific inhibitors for 20 minutes at 37°C and then stimulated with 100 nmol/L LacCer for 20 minutes: NAC (a cell-permeable relatively nonselective antioxidant), DPI (a “neutrophil” NADPH oxidase inhibitor), or BAPTA-AM (an intracellular calcium buffer) (Calbiochem) was used to determine whether ROMs, NADPH oxidase activity, or intracellular calcium fluxes mediate this Mac-1 upregulation, respectively. Genistein (Calbiochem), staurosporine (Calbiochem), or BPB (Sigma) and quinacrine dihydrochloride (ICN)20 21 were used for evaluating the possible involvement of tyrosine kinase, PKC, or PLA2, respectively, in LacCer-induced Mac-1 upregulation in hPMNs. Finally, WEB 2086 (a specific PAF-receptor antagonist) (Boehringer Ingelheim) was used to determine the possible involvement of PAF, which is one of the metabolites downstream from PLA2 that can upregulate PMN Mac-1.15 In the inhibition assay of PMN adhesion by BPB or quinacrine, PMNs were first incubated with increasing doses of BPB or quinacrine for 20 minutes, then stimulated with 100 nmol/L of LacCer for 20 minutes, followed by washing three times, and then plated onto formalin-prefixed EC monolayers in the presence of each concentration of BPB or quinacrine to eliminate a possible direct effect of BPB or quinacrine on the ECs. To evaluate the possible role of PMN CD11/CD18 in this adhesion response, PMNs were first incubated with LacCer for 20 minutes at 37°C, washed three times, incubated with monoclonal anti-CD18 F(ab′)2 (mHm23, a generous gift from Dr J. Hildreth, The Johns Hopkins University, Baltimore, Md) for 15 minutes at 37°C, and then plated onto the EC monolayers without washing.
Values were expressed as mean±1 SD. Apparent differences between normally distributed means were evaluated for significance by Student’s t test. Apparent differences in dose-response relations were evaluated by two-way ANOVA. Values of P<.05 were considered to indicate statistical significance.
LacCer Upregulates Mac-1 on PMNs
Treatment of PMNs with LacCer for 20 minutes upregulated CD11b/CD18 (Mac-1) above the level expressed on PMNs incubated for 20 minutes without LacCer in a dose-dependent fashion (Figs 1A⇓ and 3⇓). Maximum stimulation of Mac-1 expression in hPMNs was observed with 100 nmol/L LacCer (Fig 1A⇓). Similarly, LacCer (50 to 100 nmol/L) increased the level of CD11c/CD18 (p150,95) but decreased the level at a higher concentration (200 nmol/L) at this time point (Fig 1B⇓). In contrast, LacCer did not change the levels of CD11a/CD18 (LFA-1) and L-selectin at lower concentrations but downregulated both at higher concentrations (Fig 1C⇓ and 1D⇓). Mac-1 was upregulated time-dependently by 100 nmol/L LacCer (Fig 1E⇓), and maximum stimulation of the Mac-1 level (2.5-fold compared with control) was observed 20 minutes after LacCer stimulation. On the other hand, other GSLs, GlcCer, or ceramide (100 nmol/L) did not alter Mac-1 expression on PMNs, indicating that this Mac-1 upregulation was a response specifically to LacCer (Fig 1F⇓).
LacCer Stimulates PMNs to Generate Superoxide via NADPH Oxidase
Since we had previously found that LacCer activated superoxide generation via NADPH oxidase in human aortic smooth muscle cells, resulting in their proliferation,11 we evaluated whether LacCer also stimulated ROM generation by PMNs, using the lucigenin chemiluminescence assay. A strikingly similar, positive, dose- and time-dependent response was seen in these PMNs (Fig 2A⇓). This neutrophil ROM generation induced by 100 nmol/L LacCer (which had been optimal to upregulate Mac-1 on PMNs) was completely blocked by SOD, indicating its dependence on superoxide generation and inhibited by the NADPH oxidase inhibitors, DPI and apocynin (Fig 2B⇓).
PLA2 Inhibition Inhibits LacCer Upregulation of Mac-1 on PMNs
PLA2 has been found to be involved in the upregulation of Mac-1 by inflammatory mediators,20 and the involvement of calcium, oxidants, tyrosine kinases, or PKC has been suggested in the activation of PLA2.22 23 Therefore, we evaluated the effect of several inhibitors of these mechanisms for their possible role in the LacCer upregulation of Mac-1 in PMNs. The prevention of ROM generation (or calcium flux) by NAC or DPI (or BAPTA-AM), respectively, did not inhibit the LacCer-stimulated Mac-1 upregulation. Moreover, the inhibition of PKC or tyrosine kinase by staurosporine or genistein, respectively, did not inhibit the LacCer upregulation of Mac-1. However, PLA2 inhibition with BPB or quinacrine did block this response in a dose-dependent manner. We also examined the effect of PAF-receptor inhibition with WEB 2086, because PAF is one of the metabolites downstream of PLA2 that is known to upregulate Mac-1. However, no significant inhibition was seen (Fig 3⇓).
LacCer Stimulates Release of AA From PMNs
Because the result shown in Fig 3⇑ suggests that LacCer upregulates Mac-1 through the activation of PLA2, we determined whether LacCer stimulates release of AA from PMNs. [3H]AA, previously incorporated into PMNs, was released into the media by LacCer stimulation, and this release was inhibited by BPB or quinacrine, but not DPI (Fig 4⇓), indicating that superoxide is not needed for LacCer to stimulate PLA2.
LacCer Increases PMN Adhesion to Unstimulated ECs via a CD11/CD18-Dependent Mechanism
Incubation of PMNs with LacCer, followed by their repeated washing, increased PMN adhesion to unstimulated EC monolayers in a dose- and time-dependent manner (Fig 5A⇓ and 5B⇓). Even when these ECs had been prefixed with formaldehyde, the PMN adhesion was comparable to that of the unfixed ECs (Fig 5A⇓), indicating that this increment of PMN adhesion was solely PMN dependent and not due to trace amounts of LacCer carried over in the medium to the ECs. This PMN adhesion was completely blocked by monoclonal anti-CD18 (Fig 5A⇓), indicating that the LacCer-induced PMN adhesion was dependent on CD18. This increase in PMN adhesion was also inhibited by BPB or quinacrine, in a dose-dependent manner (Fig 5C⇓), corresponding to the inhibition of Mac-1 expression by BPB or quinacrine (Fig 3⇑).
The adhesion of monocytes, lymphocytes, and neutrophils to the vascular endothelium is an important physiological event. This phenomenon is initiated by the recognition of ligands such as CD11b/CD18 (Mac-1) protein on neutrophils and monocytes by its receptor, ICAM-1, expressed on the surface of ECs.24 As a response to injury, the leukocytes and the endothelium secrete inflammatory mediators, such as TNF-α, interleukin-8, monocyte chemotactic protein, and PAF. Some of these molecules have been shown to induce ICAM-1 expression in endothelium and CD11b/CD18 (Mac-1) expression in monocytes and neutrophils, thus facilitating the adhesion of large number of neutrophils and monocytes to the endothelium.13 19 24 In the present study, we focused on the effect of LacCer on adhesion molecule expression by PMNs and their consequent adhesion to unstimulated endothelium. We found that the LacCer-mediated increase in the adhesion of PMNs to ECs was associated with, and dependent on, a quantitative change in the expression of CD11/CD18 by these PMNs.
On stimulation, Mac-1 can be mobilized from intracellular secretory vesicles to the cell surface in minutes in PMNs and monocytes by a number of mediators, including leukotriene B4, TNF-α, phorbol esters, calcium ionophore, and PAF.13 15 24 Since substantial levels of ICAM-1, a counterreceptor of PMN CD11/CD18, are constitutively expressed on the surface of the vascular endothelium,24 25 26 27 LacCer activation of only PMN integrins was found to be sufficient to produce a significant increase in PMN-EC adhesion. This tenet was supported by our observation that the adhesion of LacCer-treated PMNs to ECs was similar irrespective of whether ECs were fixed or not fixed with formalin. We have found that incubation of PMNs without LacCer at 37°C for 20 minutes somewhat upregulated Mac-1 (Fig 3⇑), in accordance with previous reports.28 29 Although we cannot exclude the possibility that the upregulation of Mac-1 by LacCer was dependent on this basal state of activation by temperature change or by some other component of the isolation and incubation technique, physiologically relevant levels of adhesion of PMNs to unstimulated endothelium were induced only when stimulation with LacCer was superimposed on this state. Moreover, we did not address the change of affinity of CD11/CD18 directly in the present study. In fact, 200 nmol/L LacCer decreased the expression of adhesion molecules, whereas this concentration induced a higher level of anti-CD18–inhibitable PMN adhesion. Because 200 nmol/L LacCer did not affect cell viability, the discrepancy of the dose-response relation and time course between adhesion molecule expression and PMN adhesion seen might be explained by such a change of affinity or folding and invagination of the plasma membrane as a consequence of continuous strong stimulation.14 29
Alterations in the expression of PMN adhesion molecules and their affinity during their incubation with ECs may also contribute to the observation above. Our findings clearly indicate that LacCer, like other well-known mediators such as PAF,27 may be a major mediator of CD11/CD18 activation and of consequent firm adhesion of leukocytes to the surface of vessels.
We found that the LacCer upregulation of PMN Mac-1 and the consequent adhesion to ECs were both inhibited by two disparate PLA2 inhibitors, consistent with a previous report that the PLA2 inactivation prevents PMN Mac-1 upregulation by other inflammatory mediators, including calcium ionophore, interleukin-8, TNF-α, phorbol esters, and PAF.20 Although it is obvious from previous reports that oxidants may activate PLA2,22 30 LacCer-induced Mac-1 upregulation in PMNs did not appear to be mediated by ROM generation in this system, because neither NAC nor DPI blocked the LacCer-stimulated upregulation of Mac-1. This is also supported by our finding that AA release was not inhibited by DPI. Indeed, it has been suggested that the signal transduction pathway for chemotaxis in PMNs is distinct from the pathway for superoxide production, which involves PLD.31 32 Jacobson and Schrier20 reported that (1) the inhibition of PLA2 prevented PMN Mac-1 upregulation by inflammatory mediators, including calcium ionophore, interleukin-8, TNF-α, phorbol esters, and PAF, (2) AA, the product of PLA2, alone was able to upregulate Mac-1 in PMNs, and (3) inhibitors of cyclooxygenase or 5-lipoxygenase had no effect on this Mac-1 upregulation.20 It is possible that LacCer also uses this common pathway of AA metabolism to upregulate Mac-1 in PMNs. At the present time, it is not known how LacCer initiates this signal event, whether LacCer binds to a receptor on the cell surface, or whether clusters of LacCer interact with the putative receptor. It is suggested that GSLs, eg, LacCer, added exogenously to the culture medium may be incorporated into the plasma membrane, since the lipophilic ceramide moiety is inserted into the lipid bilayer and thus increases the proportion of this lipid by clustering within the cell membrane.33 Further studies are required to elucidate these mechanisms.
LacCer-induced superoxide generation by PMNs is another important finding of the present study, because ROMs are implicated in a wide variety of biological processes, including the activation of nuclear factor-κB, a transcription factor for a number of inflammatory mediators, including adhesion molecules,34 and in injury to the endothelium (which has been implicated in inflammation and other pathological conditions). Our finding that SOD, a cell-impermeant superoxide scavenger, blocked LacCer-induced lucigenin chemiluminescence suggests that superoxide is released from the PMNs into the media. This superoxide appears to be generated by NADPH oxidase, because two disparate inhibitors of NADPH oxidase (DPI and apocynin) inhibited superoxide generation in response to LacCer. On the other hand, smooth muscle cells released superoxide intracellularly in response to LacCer also generated by NADPH oxidase.11
In summary, neutrophils clearly contribute to acute inflammation and injury, which are documented to lead to chronic inflammation.12 Consequently, LacCer may well play an important role in these disease states by stimulating both superoxide generation and adhesion molecule activation in circulating neutrophils.
Selected Abbreviations and Acronyms
|ICAM-1||=||intercellular adhesion molecule-1|
|PKC||=||protein kinase C|
|PLA2, PLD||=||phospholipase A2 and D|
|RBC||=||red blood cell|
|ROM||=||reactive oxygen metabolite|
|TNF-α||=||tumor necrosis factor-α|
This study was supported by grants from the National Institutes of Health (DK-31764 and DK-31722). The authors thank Dr James Hildreth for providing us with mHm23 and Elise H. Smith for assistance with EC culture.
Reprint requests to Gregory B. Bulkley, MD, Department of Surgery, The Johns Hopkins University School of Medicine, Blalock 685, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287-4685.
- Received September 23, 1997.
- Accepted December 17, 1997.
- © 1998 American Heart Association, Inc.
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