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From the Laboratory of Immunopathology,* the Centre d’Immunologie de Marseille Luminy, INSERM/Centre National de la Recherche Scientifique/Université de la Méditerranée, and the Haematology Laboratories, Faculty of Medicine, IFR48, Marseille, France; and the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, The Netherlands
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Abstract |
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MPs are submicron membrane elements, mainly expressing PS at their surface, as well as surface antigens from their cell of origin.4 Originating from physiological processes, MPs are found in the circulation of healthy subjects,5-7 but their numbers can be increased in various pathological conditions such as thrombotic4,5,8 or infectious diseases.7,9-13 These MPs derive from activated blood cells and may carry at their surface procoagulant7,13 and proadhesive properties,14 suggesting that they could be implicated in the pathogenesis of these diseases. Such a role has indeed been recently demonstrated in mice in which the involvement of procoagulant MPs in the development and growth of thrombi was assessed in vivo.15 In addition in the experimental model of mouse malarial infection, a correlation between thrombocytopenia, increased numbers of platelet-derived MPs, and expression of cerebral complications has been reported.16
The syndrome of cerebral malaria (CM), the major fatal complication of plasmodium infection, invariably occurs, in susceptible mice strains, at day 7 after infection with Plasmodium berghei ANKA (PbA) and is unrelated to parasite counts in the blood.17 The fatal outcome is generally attributed to the sequestration of activated blood cells (notably monocytes/macrophages, parasitized erythrocytes, and platelets) in cerebral vessels consequent to immune responses in the host.18 However, the pathogenic mechanisms underlying the occurrence of cerebral lesions are still incompletely understood. Along this line of thought and in particular considering the suggestion of a causal relationship between MPs and the clinical severity of malaria, we set out to investigate whether a reduction of cellular ability to vesiculate, as that evidenced in the absence of the ABCA1 transporter, had any effect on the course of experimental malarial infection in mice.
In this study we provide evidence that the sole absence of ABCA1 gene converts the susceptibility of the DBA/1 strain of mice to complete resistance to CM. This is accompanied by a lower reactivity of cells within the brain vessels and a reduced cellular vesiculation, as assessed both in vivo, by lower levels of plasma MPs during malarial infection, and in vitro on restimulation of platelets and macrophages. These findings are thus consistent with a pathogenic role of MPs in the development of cerebral complications.
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Materials and Methods |
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ABCA1 WT (ABCA1+/+) and ABCA1 KO (ABCA1–/–) DBA/1 mice (12 to 14 weeks old) were generated in our laboratory as previously described.1 Apo-AI–/– mice (Apoa1tm1Unc) in C57BL/6J background19 and C57BL/6J control strain mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were maintained under pathogen-free conditions in our facilities. Mice were infected by intraperitoneal injection of 106 pRBC. Blood was collected from the tail for determination of parasitemia, or from retro-orbital puncture under anesthesia for study of hematological parameters. On the onset of the neurological phase, ie, days 7 to 11, after anesthesia, brains were sampled, included in Tissue-Tek (Leica Microsystèmes, Rueil-Malmaison, France), snap-frozen in liquid nitrogen, and then stored at –80°C.
Immunohistochemistry
For immunostaining, 7-µm frozen sections were incubated overnight at 4°C with primary monoclonal antibodies directed against murine ICAM-1 (3E2), VCAM-1(429), CD40 (3/23), LFA-1(M17/4) (Pharmingen, San Diego, CA), and CD41 (MWReg30)20 after saturation with normal rabbit serum. After washing, sections were incubated for 1 hour at room temperature with biotinylated goat polyclonal antibodies anti-rat or anti-hamster IgG, followed by the addition of HRPO-avidin (anti-rat or anti-hamster ABC kits; Vector, Peterborough, England). Color reaction was obtained by the addition of diaminobenzidine substrate-chromogen (Sigma, Saint-Quentin Fallavier, France). Slides were counterstained with Mayer’s hematoxylin before permanent mounting with Entellan (Merck, Brussels, Belgium). Slides were pictured at x400 magnification using an Eclipse 800 microscope (Nikon, Champigny-sur-Marne, France) and a digital camera; vessel labeling was then analyzed by quantitative digitalized image analysis using Lucia software (Nikon). At least 10 brains were sampled per group and computerized image analysis was performed on an average of 10 microphotographs per mouse.
Tumor Necrosis Factor (TNF) Assay
On the same samples, diluted 1:2 in the recommended buffer, TNF levels were measured using the Quantikine ELISA kit (R&D Systems, Lille, France). Macrophage cell culture supernatants were diluted similarly.
HDL Analysis
Lipid analysis was performed as described.21 Briefly, serum concentrations of cholesterol were determined using enzymatic colorimetric assays (Roche Diagnostics, Mannheim, Germany). The distribution of cholesterol over the different lipoproteins in serum was analyzed by fractionation of 30 µl of serum of each mouse using a Superose 6 column (3.2 x 30 mm, Smartsystem; Pharmacia, Uppsala, Sweden). Total cholesterol content of the effluent was determined using enzymatic colorimetric assays (Roche Diagnostics), taking the efficiency of recovery from the column into account.
MP Analysis
Blood samples were collected on 200 µl of 0.129 mol/L sodium citrate. Platelet-free plasma was prepared as previously described.5 Briefly, samples were spun down at 1500 x g for 15 minutes, followed by a 2-minute centrifugation at 13,000 x g, were then labeled with fluorescein isothiocyanate (FITC)-annexin V (Beckman Coulter Immunotech, Marseille, France) for 15 minutes at room temperature, and then diluted in 500 µl of binding buffer. Samples were then analyzed using an Epics XL flow cytometer (Beckman Coulter, Villepinte, France).
Platelet-free plasma from either uninfected or PbA-infected ABCA1–/– and ABCA1+/+ was incubated with specific monoclonal antibodies directed against endothelium (
vß3, CD51; Becton Dickinson Pharmingen), monocytes (CD14, Becton Dickinson Pharmingen), and platelets (GPIIbIIIa, CD41), followed by a FITC-F(ab)'2-goat anti-rat secondary antibody. After addition of phosphate-buffered saline (PBS), samples were analyzed by flow cytometry and the percentages of each cell-derived MPs were determined.
Procoagulant Activity of MPs
Plasma from PbA-infected mice on day 8 after infection were prepared as described above (ABCA1+/+, n = 14; ABCA1–/–, n = 10) and centrifuged twice at 20,000 x g for 1 hour. After each step, MP pellet was resuspended in PBS. Pellets from centrifugation 1 and 2 were pooled. The total MP number present in each pellet was quantified by FITC-annexinV labeling. Numbers of MPs of each sample were adjusted, by dilution with Owren-Koller buffer (Diagnostica Stago, France), to the lowest number obtained. Procoagulant activity of MPs was quantified as the reduction of Howell clotting time of a normal mouse plasma pool (N plasma) using a modified method published by Combes and colleagues.5 Briefly, 75 µl of N plasma were mixed, in a glass tube containing a magnet, with 37.5 µl of Owren-Koller buffer (control) or 37.5 µl of MP suspension (assay). Clotting reaction was initiated by the addition of 37.5 µl of 100 mmol/L CaCl2. The reaction was performed in a water bath under stirring at 37°C and the time for the clot to form was recorded. Procoagulant activity was expressed as the percentage of reduction of the control clotting time: (1-ratio assay/control) x 100.
Proinflammatory Potential of MPs
MP pellet were prepared as above, resuspended in Dulbecco’s modified Eagle’s medium-F12, and counted by flow cytometry using FITC-annexin V labeling. Noninfected mouse peritoneal exudate cells (100,000 macrophages/well) were seeded overnight in RPMI 10%-fetal calf serum medium at 37°C. Adherent cells were washed and co-cultured in 96-well flat-bottomed culture plates with either RPMI medium alone or supplemented with lipopolysaccharide (LPS) (2 µg/ml), MPs (from infected or noninfected ABCA1+/+ mice) in a 1:7 ratio (macrophage:MP) for 3 hours. Plates were then centrifuged at 1000 x g for 5 minutes and supernatants were harvested and kept frozen at –20°C until TNF assay.
Platelet Activation Assay
After blood collection from noninfected mice, platelet-rich plasma was prepared by centrifugation at 200 x g during 15 minutes. Platelet-rich plasma was then activated 45 minutes at 37°C with either calcium ionophore (A23187, 5 µmol/L; Sigma) or ADP (10 µmol/L, Sigma). After activation, plasma was prepared by two successive centrifugations at 1500 x g during 15 minutes and 13,000 x g during 2 minutes. Plasma was then labeled with FITC-annexin V and MPs were counted by flow cytometry.
Macrophage Activation Assay
Macrophages from noninfected mice were collected 4 days after intraperitoneal injection with 3% thioglycollate (Invitrogen, Cergy-Pontoise, France). Thioglycollate-induced macrophages were purified by peritoneal cavity washes using ice-cold Hanks’ balanced salt solution (Invitrogen). Contaminating red blood cells were removed by incubation for 10 minutes at 37°C in the dark in lysis buffer (NH4Cl, 8.26 g/L–1; NaHCO3, 1.00 g/L–1, ethylenediaminetetraacetic acid (4 Na) 0.037 g/L–1, pH 7.3). After washes, cells were allowed to adhere in 24-well flat-bottom culture plates at a density of 1 x 105 cells per well, overnight, in medium (Dulbecco’s modified Eagle’s medium/F12 and 10% fetal calf serum, Invitrogen). Cells were then washed and placed in serum-free medium (Dulbecco’s modified Eagle’s medium/F12) for 2 hours. Medium was then discarded and cells were activated either overnight with LPS (10 µg/ml–1) or for 6 hours with A23187 (5 µmol/L, Sigma). Activation supernatant was then removed, centrifuged 2 minutes at 13,000 x g to pellet cells in suspension, and labeled with FITC-annexin V before flow cytometric analysis. All these experiments were performed three times on cells from five mice per group.
Statistical Analysis
Survival curves (Kaplan-Meier), nonparametric Mann-Whitney U-tests, and 
2 tests were performed with GraphPad Prism 4.0 software and P < 0.05 was considered significant. Results are expressed as mean ± SEM or SD as mentioned. Linear regression was performed to compare regression lines from MP procoagulant activities.
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Results |
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Series of ABCA1 KO (ABCA1–/–) and ABCA1 WT (ABCA1+/+) DBA/1 mice were infected by PbA and subsequently monitored for the development of cerebral signs. The ABCA1+/+ mice died within the same time frame as CM susceptible mice of C57BL/6 strain, ie, during the second week of infection, referred to as the "neurological" phase (Figure 1a)
. As expected, ABCA1 WT mice presented evident clinical signs of neurological syndrome; these were not seen in ABCA1 KO mice, despite identical levels of parasitemia on day 8 of infection (18% and 17%, respectively).17
In ABCA1–/– mice, death occurred during the third week of PbA infection in the context of severe anemia (Figure 1a)
. The evaluation of the cumulative incidence of the neurological syndrome revealed a complete protection in ABCA1–/– mice, whereas ABCA1+/+ and heterozygous ABCA1+/– mice showed an incidence of 88% and 75%, respectively.
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). These observations suggest that contrary to their wild-type counterpart, on PbA infection, ABCA1–/– mice behave like other CM-R mice such as BALB/c or DBA-2. Immunopathology of Brain Lesions of ABCA1–/– Mice
To better characterize the protection of ABCA1–/– mice, we analyzed by immunohistochemistry brain specimens sampled at day 7 after infection, to address the activation status of three cells types involved in CM pathogenesis: endothelium, leukocytes, and platelets. In infected mice endothelium, the up-regulation of ICAM-1 and VCAM-1 induced by infection was significantly lower in ABCA1–/– compared to ABCA1+/+ mice (P < 0.001; Figure 2a
, top and bottom left). The presence of intravascular TNF was also significantly reduced in ABCA1–/– mice (P < 0.0001; Figure 2a
, bottom right). In contrast, the expression of CD40 (Figure 2a
, top right), and of iNOS (data not shown) was comparable in ABCA1+/+ and ABCA1–/– mice.
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, top left; P = 0.0001). In addition, LFA-1 expression was significantly lower in microvessels from ABCA1–/– mice than in those from wild-type controls (Figure 2b
, top right; P < 0.0001). This suggests lower numbers of sequestered leukocytes, or of platelets, because LFA-1 is also expressed on their surface.22,23
Indeed, platelets were found in reduced numbers in the vascular lumen of infected ABCA1–/– mice, as quantitated by GPIIbIIIa staining (Figure 2b
, bottom right; P < 0.0001). Overall these data indicate an impaired cellular activation in ABCA1–/– mice during the malarial infection. The reduction in the sequestration of leukocytes in the brain vessels of ABCA1 KO mice on infection prompted us to investigate the role of bone marrow (BM)-derived cells into the generation of resistance in ABCA1 KO mice. We thus assessed the response to PbA infection into BM chimeric mice between the two strains. However, total body irradiation modified the survival curves in the control grafts, ie, ABCA1 WT into WT and ABCA1 KO into KO (not shown and N.H. Hunt, personal communication), thus hampering a conclusive interpretation of the results. Nonetheless, the chimeras harboring ABCA1 KO BM into ABCA1 WT recipients died between day 8 and day 22 after infection (five of seven with clinical signs of CM). Conversely, grafting BM from ABCA1 WT mice into ABCA1 KO resulted in the death of all mice (n = 7) by severe anemia without signs of CM.
Because the ABCA1 transporter plays a key role in lipid metabolism and its functional impairment results in drastically reduced plasma HDL levels, it was mandatory to investigate whether protection against CM occurred as a mere consequence of dyslipidemia. We thus studied the response to PbA infection of Apo-AI-deficient mice, which also show low levels of plasma HDL.19
Despite similar incidence of the neurological syndrome in the wild-type corresponding strains, Apo-AI–/– mice developed CM similarly to these susceptible strains (Table 1)
, showing that the low levels of HDL cannot explain by themselves the protection of ABCA1–/– mice.
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Another feature of ABCA1–/– mice is the impaired capacity to produce erythrocytic MPs in response to agonists.1
We first turned our attention to plasma MP levels, quantitated by flow cytometry on day 7 or 8 after infection, as a reflection of the capability of blood cells to vesiculate in response to PbA infection. We observed a dramatic rise in MP plasma levels in ABCA1+/+ mice, whereas it was significantly less marked in their ABCA1–/– counterparts (P = 0.03 versus 0.009; Figure 3a
). Using flow cytometry, we then estimated the cell origin of MPs present in the plasma. This phenotyping indicated that MPs were mainly of platelet origin, whereas monocyte and endothelial MPs accounted for less than 30% (Figure 3b)
. Interestingly, the cellular origin of MPs was similar in both ABCA1–/– mice and in ABCA1+/+ mice. In addition, although the number of total plasma MPs was different between ABCA1+/+ and ABCA1–/– mice on infection, the cell origin of these MPs was not significantly modified by this infection (Figure 3b)
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The procoagulant activity of MPs isolated from PbA-infected mice was determined using a modified protocol of the Howell clotting time. The procoagulant activity was expressed as the capacity of a suspension of MPs to reduce the clotting time of normal plasma. MPs from PbA-infected ABCA1+/+ mice reduced more strongly the clotting time than those from PbA-infected ABCA1–/– (Figure 3c
, left; P = 0.02). Moreover when increasing numbers of MPs were added to the control plasma, we found, in both mouse strains, a linear relationship between the reduction of the clotting time and the number of MPs (Figure 3c
, right). A linear regression between the two curves showed that although the slopes are not significantly different, the elevations are highly significantly different (P = 0.0065), indicating that the two curves are distinct and that the ABCA1+/+ curve is above the ABCA1–/– one. Altogether, these data and the observed higher numbers of circulating MPs in ABCA1+/+ mice, converge on a higher circulating MP-derived procoagulant activity in the plasma of ABCA1+/+ mice.
MPs from PbA-Infected ABCA1+/+ Mice Display a Higher Proinflammatory Activity than Those MPs from Noninfected Mice
When added to macrophages at a ratio of 1:7 (macrophage:MP), MPs isolated from the plasma of noninfected mice induced a significantly lower release of TNF (Figure 3d
; P = 0.028) than MPs isolated from PbA-infected mice. As a control, macrophages were incubated with medium alone or LPS and, as expected, LPS induced a significantly higher TNF release than either medium alone or MPs from PbA-infected mice (Figure 3d
; P = 0.004 and P = 0.04, respectively). These data show that MPs produced in conditions of infection display a proinflammatory potential that is not present on the MPs produced in physiological conditions.
Cellular Responses to Vesiculation Agonists
Platelets and monocyte/macrophages are two cell types known to play a crucial role in the pathogenesis of the cerebral syndrome. As we observed a reduced number of MPs in the plasma, we assessed whether these two cell types present an altered response to in vitro stimulation by agonists of vesiculation as it has already been shown for erythrocytes.1
Platelets were purified from whole blood and activated with ADP and A23187. In parallel, thioglycollate-induced peritoneal macrophages from noninfected mice were restimulated in vitro with calcium ionophore (A23187) and LPS. Although both platelets (Figure 4a)
and macrophages (Figure 4b)
purified from ABCA1+/+ mice showed the significant increase in vesiculation similar to the one known to occur in human cells,11
in ABCA1–/– mice, platelets showed a complete lack of vesiculation and macrophages presented a reduced capacity to release MPs when submitted to the agonists. Moreover, stimulation-induced MP release was statistically higher in ABCA1+/+ than in ABCA1–/– mice for both platelets (ADP, P = 0.04; A23187, P = 0.01) and macrophages (LPS, P = 0.013; A23187, P = 0.024). These data thus confirm and extend the previous findings of reduced membrane vesiculation from ABCA1–/– cells in vitro and evidence a similar lack of response after in vivo stimulation of the animals by plasmodial antigens.
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Discussion |
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On malaria infection, in ABCA1–/– mice, we evidenced a lower activation status of the cells present in the brain lesion, as showed by the weaker up-regulation of endothelial adhesion molecules in brain microvessels such as ICAM-1 and VCAM-1, the reduced leukocyte sequestration, as well as the ablated platelet accumulation. The endothelial alteration, subsequent to inflammatory cytokine release, is a crucial step in the pathogenesis of CM.18,24
The absence of elevated TNF levels in the plasma of PbA-infected ABCA1 KO mice could therefore partly explain this impaired cell reactivity. A similar lack of ICAM-1 up-regulation correlated with protection has been found in TNFR2-deficient mice25
and, more recently, in a series of cytokine knockout (TNF, interleukin-12, or interferon-
) and T-cell-deficient Rag1–/– mice, on infection by PbA.26
Besides, ICAM-1–/– mice infected by PbA do not develop CM.27
A modulation of lymphotoxin (LT) could also be envisaged since recent data have shown that LT–/– mice are protected against the neurological syndrome.28
The lack of platelet accumulation in brain vessels from PbA-infected ABCA1–/– mice could also be explained by the fact that platelets from ABCA1–/– mice display an impaired platelet aggregation in whole blood on in vitro ADP stimulation29
and thus present a compromised platelet function.
Because of its flippase activity, ABCA1 is one of the determinants of the lipid transbilayer remodeling, and thereby is involved in MP production. Indeed, ABCA1 deficiency led to a reduced externalization ability and consecutive membrane vesiculation of red blood cells.1
In addition to this in vitro role on vesiculation, we demonstrated here that ABCA1 is necessary for an enhancement of the in vivo vesiculation during the course of PbA infection. This is consistent with the previous demonstration that reducing platelet-derived MP levels via caspase inhibitors lowers CM incidence.16
Moreover, we found that platelets and macrophages of ABCA1-deficient mice are significantly less reactive in terms of MP production when restimulated in vitro. Indeed, the ABCA1 transporter controls the outward translocation of PS at the plasma membrane3
and is thus likely to affect the vesiculation process. On the other hand, we provide evidence that, on infection, MPs can be envisaged as pathogenic elements in view of their procoagulant and proinflammatory properties. Indeed, we demonstrated here that, added at identical numbers, MPs derived from the plasma of PbA-infected ABCA1+/+ mice displayed a higher procoagulant activity than those derived from the plasma of ABCA1–/– mice. This suggests that because the absolute number of MPs in the plasma is higher in WT than in KO mice, the global circulating procoagulant activity is therefore higher in WT. This is compatible with a higher susceptibility of these mice to develop thrombotic events within the vessels where the endothelial activation is the highest. However, ABCA1 knockout mice produce less TNF during infection (Figure 1b)
and this may explain their relative resistance to CM. This finding is consistent with the importance of proinflammatory cytokines in CM.
Similarly, we demonstrated that MP isolated from PbA-infected mice display a higher proinflammatory activity than those isolated from noninfected mice. This suggests that MPs produced during malarial infection could be pathogenic also via the induction of the release of proinflammatory mediators, such as TNF, by circulating mononuclear cells. This would maintain an inflammatory atmosphere within the vessels, in a way that favors a worsening of the endothelial lesion occurring during CM. To further assess this pathogenic role of MPs, it will be worth assessing whether resistance to CM invariably accompanies the down-modulation of genes that, like ABCA1, control vesicle formation from membranes.
The finding that ABCA1 deletion confers a complete protection against CM, associated to an impaired MP production, orientates toward new therapeutic approaches to the search for candidate genes controlling vesiculation. Vesiculation also is implicated in diseases such as atherosclerosis in which one of the main goals of research is to prevent the thrombogenic effect of PS in the atherosclerotic plaque by preventing both MP release and PS exposure at the cell surfaces. Thus, lower MP levels are likely to reduce the incidence of CM and thrombotic diseases.4,30 Although these data may appear contradictory with the known anti-atherogenic properties of the ABCA1,31 one has to keep in mind that CM is an acute thrombotic event limited to microvessels whereas atherosclerosis is a chronic alteration of cell lipid metabolism essentially expressed in macrovessels. Taken together, our data provide new insights in CM pathogenesis and suggest that interventions on the ABCA1 gene product could be of interest, not only in severe malaria, but more widely in thrombotic disorders.
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Footnotes |
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Supported by the French Ministry of Research (PAL+ Program, to G.E.G. and G.C.), the United Nations Development Program/World Bank/World Health Organization Special Programe for Research and Training in Tropical Diseases (Tropical Disease Research, to G.E.G. and G.C.), and the Association de Lutte Contre le Cancer (grant no. 4245 to G.C.). The Centre d’Immunologie de Marseille Luminy is supported by institutional grants from INSERM and Centre National de la Recherche Scientifique.
V.C., N.C., and M.A. contributed equally to the work; and G.E.G. and G.C. directed the work equally.
Accepted for publication September 21, 2004.
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