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* Center for Experimental and Molecular Medicine and
Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
Department of Internal Medicine 1, Medical University of Vienna, Vienna, Austria; and
¶ Cardiovascular Research Center and Department of Pharmacology, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The prompt initiation of host defense mechanisms is essential for the host to survive. The innate immune system, that is responsible for mounting an immediate response to invading pathogens, is considered the central element of host defense in peritonitis. LPS is a major constituent of the outer cell wall of Gram-negative bacteria, such as E. coli, and the principal inducer of inflammatory responses to these pathogens. CD14, TLR4, and MD-2 make up the LPS receptor complex involved in the cellular recognition of and signaling by LPS (3, 4, 5, 6, 7, 8). LPS-binding protein (LBP)3 greatly augments the transfer of LPS to the CD14-TLR4 complex. Recently, we demonstrated the pivotal role of LBP during murine E. coli peritonitis: the rapid recruitment of polymorphonuclear cells (PMNs) to the site of infection critically depended on the presence of LBP and mice lacking LBP displayed a greatly increased bacterial outgrowth and dissemination that led to early death (9).
PMNs are key phagocytes required for the immediate elimination of bacteria. One of the most powerful weapons generated by activated PMNs is reactive oxygen species that are used for antibacterial defense. However, in parallel with the killing of bacteria, free radicals may damage host molecules, and in particular induce lipid peroxidation. Oxidized phospholipids (OxPL) are generated in vivo at sites of acute and chronic inflammation (10, 11, 12). Accumulating evidence suggests that OxPL are not merely by-products of the inflammatory response, but can actively regulate inflammation (13). Most previous studies (13, 14, 15) focused on the proinflammatory effects of OxPL, which are thought to play a role in initiating and maintaining chronic inflammation such as atherosclerosis. However, it is increasingly recognized that OxPL at the same time possess potent anti-inflammatory properties, which include the direct antagonism of LPS recognition by cells of the innate immune system. Indeed, OxPL effectively inhibit the interaction of LPS with LBP, CD14, and TLR4 (16). The biological significance of this finding was further underlined by the observation that the exogenous administration of OxPL could prevent mortality of mice exposed to high doses of LPS; in this respect, OxPL reproduced the LPS-resistant phenotype of mice lacking LBP, CD14, or TLR4 (16, 17, 18, 19, 20).
The biological role of OxPL during Gram-negative infection with viable bacteria has not been studied so far. We therefore decided to investigate the effects of OxPL during E. coli-induced abdominal sepsis in vivo and the role of CD14 herein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Pathogen-free 9- to 11-wk-old male C57BL/6 wild-type (WT) mice were purchased from Harlan Sprague Dawley (Horst). CD14 gene-deficient (CD14–/–) mice were obtained from The Jackson Laboratory (21) and backcrossed to C57BL/6 background six times. Age- and sex-matched mice were used in all experiments. The Animal Care and Use Committee of the University of Amsterdam (Amsterdam, The Netherlands) approved all experiments.
Phospholipids
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) and dimyristoylphosphatodylcholine (DMPC) were purchased from Sigma- Aldrich. DMPC was used as negative control due to the fact that DMPC lacks unsaturated fatty acids and thus cannot be oxidized. Oxidized PAPC (OxPAPC) was generated by air oxidation as described recently and stored in chloroform at –70°C (16). The extent of PAPC oxidation was routinely confirmed by electrospray ionization-mass spectrometry (22), and only preparations showing a reproducible pattern of lipid oxidation products were used in the study. These preparations were routinely tested in in vitro assays for biological activity as well as LPS content using the Limulus assay. Directly before use, OxPAPC were dried in glass tubes under the stream of N2 and resuspended in NaCl by vortexing.
Induction of peritonitis
Peritonitis was induced as described previously (9, 23, 24). In brief, E. coli O18:K1 was cultured in Luria-Bertani medium (Difco) at 37°C, harvested at mid-log phase, and washed twice before inoculation. Mice were injected i.p. with 1–2 x 104 CFU E. coli in 200 µl of sterile saline. The inoculum was plated on blood agar plates to determine viable counts. OxPAPC (12.5 mg/kg) or control lipids (DMPC; 12.5 mg/kg in 200 µl of NaCl) or the same volume of carrier were injected i.p. immediately before bacterial inoculation.
Monitoring of mortality and enumeration of bacteria
In survival studies, 8–12 mice per treatment group were inoculated with E. coli. Because mortality occurs primarily between 24 and 48 h after infection in this model, mortality was assessed every 2 h in this period; thereafter, mortality was monitored every 6 h. In separate studies, mice were sacrificed 4 or 20 h after infection; at these time points, mice were anesthetized by inhalation of isoflurane and peritoneal lavage was performed with 5 ml of sterile isotonic saline using an 18-gauge needle. Lavage fluid was collected in sterile tubes and put on ice. After collection of peritoneal fluid, deeper anesthesia was induced by i.p. injection of 0.07 ml/g mixture of 0.315 mg/ml fentanyl, 10 mg/ml fluanisone (both Janssen), and 5 mg/ml midazolam (Roche). After opening the abdomen blood was drawn from the lower caval vein and collected in sterile tubes containing heparin and immediately placed on ice. Liver lobes were harvested and homogenized at 4°C in four volumes of sterile saline using a tissue homogenizer (BioSpec Products). CFU were determined from serial dilutions of peritoneal lavage, liver homogenates, and blood, plated on blood agar plates and incubated at 37°C for 16 h before colonies were counted.
Cell counts and differentials
Cell counts, determined on each peritoneal lavage sample stained with Türck’s solution (Merck), were counted in a hemocytometer (Türck counting chamber). The cells were then diluted to a final concentration of 1 x 105 cells/ml and differential cell counts were performed on cytospin preparations stained with Giemsa.
Cytokine/chemokine assays
Cytokines and chemokines (TNF-
, IL-6, IL-10, and MCP-1) were measured using the cytometric bead array multiplex assay (BD Biosciences) according to the manufacturer’s instructions. The detection limits were 5 pg/ml. Keratinocyte-derived chemokine (KC), MIP-2, and IL-1
concentrations were determined using commercially available ELISA (R&D Systems).
Bacterial killing assay
Bacterial killing was determined according to a protocol published recently (25). In brief, RAW 264.7 cells (American Type Culture Collection) were mobilized using 5 mg/ml lidocaine in PBS, washed, and plated in 24-well plates at a density of 2 x 105 cells/well. Cells were allowed to adhere for 2 h at 37°C and washed thoroughly with serum-free RPMI 1640. E. coli O18:K1 were added at a multiplicity of infection of 100 and spun onto cells at 2000 rpm for 5 min, after which plates were placed at 37°C for 10 min. Each well was then washed five times with ice-cold PBS to remove extracellular bacteria. To determine bacterial uptake after 10 min, triplicate of wells were lysed with sterile H2O and designated as t = 0. Prewarmed serum-free RPMI 1640 with or without 50 µg/ml OxPAPC was added to remaining wells and plates were placed at 37°C for 5, 10, 30, 60, or 90 min, after which cells were again washed five times with ice-cold PBS and lysed as described above. Cell lysates were plated in serial-fold dilutions on blood agar plates and bacterial counts were enumerated after 16 h. Bacterial killing was expressed as the percentage of killed bacteria in relation to t = 0 (percent killing = 100 – ((no. of CFU at time x/no. of CFU at time 0) x 100)).
Phagocytosis assays
Phagocytosis was evaluated in essence as described before (26, 27). Peritoneal lavage was performed in WT and CD14–/– mice (n = 8/strain) using 5 ml of sterile saline. Lavage fluid was collected in sterile tubes and put on ice. Peritoneal macrophages (PM) were washed, counted, and resuspended in RPMI 1640 at a final concentration of 1 x 106 cells/ml. Cells were then allowed to adhere in 12-well microtiter plates (Greiner) overnight. Adherent monolayer cells were washed thoroughly with HBSS and incubated with FITC-labeled heat-killed E. coli (O18:K1, 1 x 108 CFU/ml) in the presence or absence of 50 µg/ml OxPAPC at 37°C or 4°C for 30 min. Immediately thereafter, cells were put on ice, washed in PBS, suspended in Quenching solution (ORPEGEN Pharma), and analyzed using a FACSCalibur (BD Biosciences). To obtain primary PMNs, WT mice (n = 8) were injected i.p. with 4% proteose peptone (Difco). The next day mice were anesthetized by inhalation of isoflurane (Abbott Laboratories) and peritoneal lavage was performed with 5 ml of sterile saline using an 18-gauge needle. Lavage fluid was collected in sterile tubes, washed twice, and cells were seeded in tissue culture plates to get rid of adherent macrophages. Nonadherent cells were counted after 2 h and consisted of >95% PMNs. Phagocytosis assay was performed as described above. The phagocytosis index of each sample was calculated: (mean fluorescence x percent positive cells at 37°C) minus (mean fluorescence x percent positive cells at 4°C). The same procedure was followed when using RAW 264.7 cells with the exception of different incubation times (up to 240 min) and an additional proteinase K (Promega) step (250 µg/ml at room temperature for 30 min) to remove extracellular bacteria before FACS analysis. In some experiments, LPS (100 ng/ml, from E. coli O55:B5; Sigma-Aldrich) was added during overnight adherence. When indicated, anti-SR-A Ab (clone 2F8; Serotec) or isotype control (Serotec) was added at 10 µg/ml. Phagocytosis of fluorescent polystyrene microspheres (FluoroSpheres, 1 µm; Molecular Probes) was performed with RAW 264.7 cells that were coincubated with increasing doses of OxPAPC (10–50–100 µg/ml), DMPC (50 µg/ml) or carrier at 37°C. Uptake was analyzed by FACS after 120 min and related to 4°C controls as described above.
Endocytosis/pinocytosis assays
Cellular uptake of HRP (Sigma-Aldrich), FITC-dextran (Molecular Probes), Lucifer yellow (LY; Molecular Probes), or mannosylated BSA-FITC (Sigma-Aldrich) was determined as reported earlier (28). RAW 264.7 cells were cultured in 12-well plates at a concentration of 0.5 x 106 cells/well and allowed to adhere overnight in RPMI 1640 supplemented with 10% FCS. The next day, cells were washed thoroughly in serum-free RPMI 1640 and FITC-dextran (1 mg/ml), LY (1 mg/ml), or mannosylated BSA-FITC (10 µg/ml) was added and cells were incubated at 37°C for indicated times. In some experiments, excess mannan (from Saccharomyces cerevisiae; Sigma-Aldrich) was added at a concentration of 3 mg/ml. At indicated time points, cells were put on ice, washed thoroughly with cold PBS, and analyzed by FACS analysis. Cells pulsed at 4°C were used to determine background uptake. The uptake index of each sample was calculated: (mean fluorescence x percent positive cells at 37°C) minus (mean fluorescence x percent positive cells at 4°C). To determine uptake of HRP, cells were cultured as described above, HRP was added at indicated concentrations and incubated for 3 h at 37°C. Next, cells were washed four times and lysed with 0.05% Triton X-100 in 10 mM Tris buffer (pH 7.4) for 30 min. The enzyme activity of the lysate was measured using o-phenylenediamine and H2O2 as substrate.
Statistical analysis
Differences between groups were calculated using the Mann-Whitney U test or one-way ANOVA. For survival analysis, Kaplan-Meier analysis followed by log rank test was performed. Values are expressed as mean ± SEM. A p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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OxPL have been shown to improve survival during murine endotoxemia due to their capacity to reduce the bioavailability of LPS, thereby attenuating overwhelming systemic inflammation (16). We consequently were interested in studying the effect of OxPL, such as OxPAPC, on the course of septic peritonitis induced by viable Gram-negative bacteria, i.e., microorganisms that express LPS. Mice were inoculated i.p. with E. coli along with OxPAPC or vehicle and observed for 5 days. As depicted in Fig. 1A, OxPAPC-treated mice started to succumb as early as 20 h after infection while all control mice remained alive until t = 38 h. In total, 92% (11 of 12) of OxPAPC-treated mice died vs only 42% (5 of 12) of the control animals (p = 0.004). Thus, the administration of OxPAPC rendered mice more susceptible to E. coli peritonitis.
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To obtain insight in the mechanism underlying the accelerated and higher mortality of mice that received OxPAPC, we repeated this experiment and sacrificed mice 4 or 20 h after infection to enumerate bacterial counts in peritoneal lavage fluid (PLF; the primary site of infection), blood, and liver (to evaluate to which extent the infection became systemic). Already 4 h after induction of peritonitis, bacterial counts in OxPAPC-treated mice were up to 1 log higher than in controls, but the differences between groups did not reach statistical significance (control vs 1 x 103 CFU/ml OxPAPC): PLF, 2.0 ± 0.6 vs 10.2 ± 3.6; blood, 1.8 ± 0.8 vs 20.9 ± 9.6; liver, 8.8 ± 6.0 vs 27.8 ± 12.5). Thereafter, E. coli grew exponentially in all body compartments but much faster in OxPAPC-treated animals and significantly increased CFU counts were recovered from PLF, blood, and liver from these mice 20 h after infection (Fig. 1, B–D). Therefore, the i.p. administration of OxPAPC is associated with an increased bacterial outgrowth and dissemination during E. coli peritonitis.
OxPL transform WT mice into a CD14-deficient phenotype
Because earlier findings pointed toward the fact that OxPL inhibit the interaction of LPS with LBP and CD14 (16) and because LPS is a major immunogenic component of E. coli (29), we next aimed to clarify whether the effects of OxPAPC described above could be explained by a functional CD14 blockade in the presence of these phospholipids. For this purpose, CD14–/– and WT mice were treated with OxPAPC or control lipids (DMPC) and infected i.p. with E. coli. Survival studies disclosed that CD14–/– mice were highly susceptible to E. coli peritonitis and succumbed quickly (Fig. 2A). The administration of OxPAPC transformed WT mice into a CD14–/– phenotype, whereas no additional effect on lethality was observed in CD14–/– animals treated with OxPAPC (Fig. 2B). Of note, to exclude potential nonspecific effects of lipids, we used control lipids (DMPC) instead of carrier (NaCl) in this set of experiments. As expected, DMPC-treated mice behaved exactly like mice that received carrier in earlier experiments, i.e., they showed an improved survival when compared with OxPAPC-treated animals. Mortality rates were slightly higher in this experiment (compared with Fig. 1A), due to the, in retrospect, higher number of bacteria mice were infected with. We then repeated this experiment and enumerated bacterial counts 20 h after infection. CD14–/– mice treated with control lipids (DMPC) displayed significantly increased bacterial counts in PLF, liver, and blood when compared with DMPC-treated WT mice (Fig. 2, C–E). OxPAPC treatment enhanced bacterial outgrowth in WT mice, confirming the experiments shown in Fig. 1 (again illustrating the fact that DMPC treatment results in identical results as carrier treatment). Importantly, OxPAPC did not further increase bacterial loads in CD14–/– mice (Fig. 2, C–E). In addition, although the numbers of E. coli CFU tended to be higher in blood and livers of OxPAPC-treated CD14–/– mice than in OxPAPC-treated WT mice (Fig. 2, D and E), the differences between groups did not reach statistical significance. Together, these findings underline the importance of CD14 during Gram-negative peritonitis and lay emphasis on CD14 as a potential target molecule that might explain the detrimental effects induced by OxPL in vivo.
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Having shown that OxPL impaired outcome, we then asked which factors might be involved in the outgrowth and spread of E. coli. Responses accountable for an appropriate innate immune response during peritonitis include the local production of proinflammatory cytokines and chemokines at the site of the infection (27, 30, 31). Because OxPL can inhibit LPS bioavailability (16), we considered it possible that OxPAPC would impair the early cytokine/chemokine response to E. coli, thereby enhancing susceptibility to abdominal sepsis. To address this issue, we measured IL-6, TNF-, IL-1, MCP-1, KC, MIP-2, and IL-10 in PLF and plasma. As shown in Table I, OxPL did not impair the early induction of proinflammatory cytokines after i.p. infection with E. coli. Peritoneal IL-6, TNF-, MCP-1, and KC concentrations were even higher in OxPL-treated mice at t = 4 h, which indicates that a suppressed early immune response cannot explain differences in outcome and bacterial elimination (Table I). Because PMN influx to the peritoneal cavity is a critical component of the innate immune response during peritonitis, we next determined leukocyte counts in PLF. However, 4 h after induction of peritonitis, we did not find any indication for an impaired leukocyte influx (Table II). On the contrary, OxPAPC-treated mice had the tendency to attract more leukocytes than control mice, likely as a result of the higher bacterial load in these mice. Thus, the number of PMN at the site of infection does not explain differences in bacterial clearance and outcome. When examining the local peritoneal and systemic cytokine/chemokine response in WT and CD14–/– mice at t = 20 h, we identified an enormously increased release of IL-6, TNF-, and MCP-1 in PLF and plasma of CD14–/– animals after 20 h (Fig. 3), whereas MIP-2 and KC levels were similar in all groups (data not shown). These values by far exceeded the levels measured in WT mice treated with OxPAPC. Of interest, CD14–/– control mice (i.e., DMPC treated) had the highest cytokine/chemokine concentrations, whereas CD14–/– animals that received OxPAPC showed less pronounced elevations (Fig. 3). Therefore, OxPAPC modestly reduced the cytokine response in CD14–/– but not in WT mice in vivo. In contrast, CD14–/– animals displayed a severely impaired ability to attract PMNs and macrophages to the peritoneal cavity, irrespective of OxPAPC treatment (Table III). Even 20 h after the induction of peritonitis when vast amounts of bacteria were encountered in all mice, the number of PMNs and macrophages was significantly reduced in the absence of CD14. Thus, although OxPAPC did not impair the attraction of inflammatory cells to the site of infection, CD14 crucially contributes to this important host defense mechanism.
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Having shown that OxPAPC affects CD14-independent pathways of the innate immune response, we next aimed to investigate the functional and cellular alterations induced by OxPAPC. Microscopic evaluation of infiltrating leukocytes in PLF of OxPAPC-treated mice revealed a high proportion of cells covered by enormous amounts of bacteria (Fig. 4A). Although bacteria were also visible in samples from control mice, the number and close association with leukocytes was much less impressive. This led us to examine whether OxPAPC influences functional properties of leukocytes, such as killing or phagocytosis of bacteria. Using primary PM and PMNs from C57BL/6 mice or RAW 264.7 PM, we studied both phagocytosis and bacterial killing in the presence or absence of OxPAPC. As depicted in Fig. 4B, the addition of OxPAPC following uptake of bacteria did not affect the killing properties of macrophages. However, OxPAPC impaired the ability of phagocytes to internalize E. coli in a time-dependent manner (Fig. 4, C and D). In addition, both primary PM and PMNs showed an impaired phagocytosis of FITC-labeled E. coli in the presence of OxPAPC (Fig. 4, E and F). Of importance, CD14 did not interfere with the phagocytic properties of macrophages or PMNs, as indicated by unaltered phagocytosis rates of primary cells from CD14–/– mice (Fig. 4E). Together, these data showed that OxPAPC inhibits phagocytosis of E. coli but does not influence bacterial killing.
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To further understand the impact of OxPL on phagocytosis, we then asked whether our finding of impaired phagocytosis in the presence of OxPAPC is specific for pathogens such as E. coli or reflects a more general phenomenon. Because we performed all phagocytosis assays (Fig. 4) under serum-free conditions using nonopsonized bacteria, the possibility that OxPAPC interferes with Fc receptor or complement-mediated phagocytosis is unlikely. However, scavenger receptors (SR)-A have been identified as important receptors that specifically contribute to Fc receptor-independent phagocytosis of E. coli (32). We therefore investigated the potential contribution of SR-A and found, in accordance with earlier reports (32), that blocking SR-A with anti-SR-A Abs significantly reduced uptake of E. coli by macrophages (Fig. 5A). The addition of OxPAPC alone showed exactly the same inhibitory effect as anti-SR-A alone and no additive effect was observed when the combination of both reagents was tested (Fig. 5A). We then tried to more closely imitate the in vivo situation seen during peritonitis, when PM encounter LPS or bacteria that activate them before they phagocytose whole bacteria, and preincubated macrophages with LPS before the addition of E. coli. As expected, phagocytosis of E. coli was tremendously increased when macrophages were preactivated by LPS (Fig. 5B; compare y-axes of A and B). In addition, when anti-SR-A or OxPAPC were added to these activated macrophages, the inhibitory action of OxPAPC clearly exceeded that of anti-SR-A (Fig. 5B). Together these findings indicate that OxPAPC either interferes with phagocytosis receptors other than SR-A or that OxPAPC uses additional (possibly activation-dependent) pathways. To assess SR-A-independent phagocytosis and the role of OxPAPC herein, we next investigated the uptake of polystyrene microspheres and identified that OxPAPC dose-dependently inhibited the uptake of these microspheres (Fig. 5C). Importantly, control lipids (DMPC) did not show this effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Host defense in peritonitis is a delicate balance between proinflammatory pathways aimed at the rapid elimination of bacteria and anti-inflammatory pathways intended to prevent systemic inflammation (36). Any imbalance in pro- or anti-inflammatory mediators might prove harmful. We initially considered it conceivable that OxPAPC, due to their anti-inflammatory properties described in models of acute inflammation (16), might represent an endogenous mediator that assists preventing overwhelming inflammation and, therefore, would reduce mortality in mice suffering from severe peritonitis. Our findings, however, could not confirm this hypothesis but rather disclosed an important, although detrimental, role for OxPAPC in host defense against E. coli in vivo. In the presence of OxPAPC, a higher proportion of mice succumbed and this was accompanied by an increased bacterial load in all organs tested. The important attraction of phagocytes to the peritoneal cavity was not compromised by OxPAPC. It has been reported earlier (11, 14, 37) that OxPL themselves induce the production of chemokines such as IL-8, KC, and MCP-1 in vitro as well as in vivo. In our hands, the addition of OxPAPC during E. coli peritonitis in vivo was accompanied by increased peritoneal MCP-1 and KC concentrations, which may have contributed to the elevated number of PMNs and monocytes/macrophages in peritoneal fluid early (t = 4 h) after induction of peritonitis.
OxPAPC can inhibit the interaction of LPS with CD14 (16). Because CD14 is a major component of the LPS-signaling complex on innate immune cells and an important player in host defense against Gram-negative bacterial infections (3, 38, 39, 40, 41, 42), we were interested in studying whether our observations of increased bacterial outgrowth and mortality in OxPAPC-treated mice were linked to an inhibitory action of OxPL on the interaction of LPS with CD14. Using CD14 gene-deficient mice, we first were able to demonstrate an important role of CD14 in the innate immune response during E. coli peritonitis in vivo. Analogous to our earlier studies, where we investigated the role of LBP in this infection model and in line with earlier reports in Salmonella peritonitis, an inadequate onset of inflammation in CD14–/– mice led to early systemic dissemination and increased bacterial outgrowth (9, 43). Although bacterial dissemination and survival rates were identical in CD14–/– and OxPAPC-treated mice, the underlying mechanisms differed. OxPAPC administration had no influence on the number of PMNs attracted to the peritoneal cavity but recruited PMNs imposed covered by bacteria. This inspired us to investigate the functional properties of PMNs and macrophages and led us to discover an impaired phagocytosis by professional phagocytes in the presence of OxPAPC. Indeed, RAW cells as well as primary PMNs and PM were less capable of effectively phagocytosing E. coli in the presence of OxPAPC. Although CD14 was attributed to phagocytosis of Gram-negative bacteria by an earlier report (44), we could not disclose a role for this receptor. In line, our laboratory previously found no effect of a blocking anti-CD14 Ab i.v. administered to human subjects on phagocytosis by blood monocytes and PMNs (42). However, CD14 is known to participate in the elimination of apoptotic cells and OxPAPC, due to its capacity to interfere with CD14, is certainly a candidate molecule that might interact with this process (45). In fact, one report (46) showed an impaired phagocytosis of apoptotic cells in the presence of minimally modified low-density lipoprotein that bind to CD14 and concurrently enhance the uptake of oxidized low-density lipoprotein. Nevertheless, the potential impact of OxPL on phagocytosis of bacteria has not been investigated thus far and we hereby report for the first time that OxPAPC interfere with the elimination of bacteria in vivo and that this mechanism does not depend on CD14.
Another receptor that crucially contributes to opsonin-independent phagocytosis of E. coli is SR-A (32). SR-A recognizes a wide range of polyanions including LPS from Gram-negative bacteria. Although not known so far, the possibility exists that OxPAPC interferes with the binding of polyanions to SR-A, thereby preventing the phagocytosis of Gram-negative bacteria. However, our data from activated macrophages clearly demonstrate that OxPAPC inhibits phagocytosis to a greater degree than blocking Ab against SR-A. Addition of OxPAPC to anti-SR-A further reduced the uptake of E. coli, thus indicating different inhibitory pathways. Of note, although the class B SR CD36 has been shown to bind OxPAPC (47), CD36 is not involved in the phagocytosis of E. coli (48), which precluded us from investigating CD36 as a target receptor that might explain the effects of OxPAPC observed in this study.
Our data indicate that the inhibitory action of OxPAPC is not restricted to phagocytosis of E. coli. Considering the multitude of endocytotic pathways elicited by macrophages, we found OxPAPC to not only impair phagocytosis of bacteria but also polystyrene particles as well as receptor-mediated and fluid-phase endocytosis. These findings definitely underline the broad, and potentially harmful, impact of oxidation products generated during bacterial infections or chronic inflammation in vivo. Although the pathophysiological impact of these findings remains to be established, we propose a detrimental role for OxPL during bacterial infections. Beside infections, OxPL are found predominantly during chronic inflammation such as atherosclerosis. The observations described in this study of impaired endocytosis in the presence of OxPL could explain the very recent finding of diverse bacterial products within a single plaque specimen from patients with coronary heart disease (49). The possibility exists that OxPL, present in atherosclerotic plaques, prevent the effective elimination of bacteria that are encountered during asymptomatic phases of bacteremia, such as after dental procedures or translocation from the intestines, thus leading to the accumulation of intracellular bacterial products that in turn might contribute to ongoing inflammation.
It should be noted that although OxPAPC and CD14 deficiency negatively influenced the outcome of E. coli peritonitis by different mechanisms, OxPAPC did not further impair host defense in CD14–/– mice. Importantly, however, OxPAPC and CD14 deficiency both impacted on PMNs and macrophages, and although OxPAPC profoundly diminished the capacity of CD14–/– PMNs and macrophages to phagocytose E. coli in vitro, apparently this immune suppressing effect did not further impact on the outcome of CD14–/– mice in vivo due to the fact that these animals had very few PMNs and macrophages in their peritoneal cavity and as a consequence already had a severely hampered cellular immune response.
In conclusion, we demonstrate that OxPAPC reduce host defense against abdominal sepsis caused by E. coli most likely by inhibiting the phagocytosing capacity of cells involved in innate immunity by a CD14-independent mechanism. Although OxPL might be able to prevent overwhelming inflammation in settings of sterile inflammatory disorders, our results suggest that OxPL generated at sites of inflammation impair the innate immune response to bacterial infections.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was in part supported by the Austrian Fonds zur Förderung der Wissenschafllichen Forschung Grant P18232-B11 (to S.K.). 
2 Address correspondence and reprint requests to Dr. Sylvia Knapp, Research Center for Molecular Medicine of the Austrian Academy of Sciences, Department of Internal Medicine 1, Medical University Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. E-mail address: sknapp{at}cemm.oeaw.ac.at or sylvia.knapp{at}meduniwien.ac.at
3 Abbreviations used in this paper: LBP, LPS-binding protein; WT, wild type; OxPAPC, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; KC, keratinocyte-derived chemokine; LY, Lucifer yellow; SR, scavenger receptor; DMPC, dimyristoylphosphatodylcholine; OxPL, oxidized phospholipid; PLF, peritoneal lavage fluid; PM, peritoneal macrophage; PMN, polymorphonuclear cell.
Received for publication August 17, 2006. Accepted for publication October 26, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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) and CD14–/– (
) mice were treated with 12.5 mg/kg control lipids (DMPC) or OxPAPC and infected with 1.2 x 104 CFU E. coli i.p. PLF and plasma TNF-
) as compared with DMPC (
). D, Representative histogram after incubation with mannosylated BSA-FITC at t = 30 min (thin line, 4C control; thick line, 37°C carrier; filled gray, 37°C OxPAPC). E, Fluid-phase pinocytosis of LY is inhibited in the presence of OxPAPC (10, 50, or 100 µg/ml, 
) as compared with carrier (•). A representative histogram at t = 120 min is depicted in F (thin line, 4°C control; thick line, 37°C carrier; filled gray, 37°C 10 µg/ml OxPAPC). Mean ± SEM. *, p < 0.05 vs carrier.