Mechanism and intervention of murine transfusion-related acute lung injury caused by anti-CD36 antibodies

Anti-CD36 Abs have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36 Ab–mediated TRALI, and potential therapies have not yet been identified. Here, we developed a murine model of anti-CD36 Ab–mediated TRALI to address these questions. Administration of mouse mAb against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab′)2 fragments, induced severe TRALI in Cd36+/+ male mice. Predepletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 Abs increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36–mediated TRALI. Administration of GZ1 F(ab′)2, antioxidant (N-acetyl cysteine, NAC), or C5 blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36–mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab′)2 after TRALI induction, significant improvement was achieved when mice were treated postinduction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.


Introduction
Transfusion-related acute lung injury (TRALI) is a life-threatening syndrome that occurs within 6 hours of a blood transfusion and is characterized by acute respiratory distress and the development of noncardiogenic pulmonary edema (1). The 2-hit model (2,3) is the most widely accepted hypothesis: it suggests that TRALI is the result of 2 independent events that trigger a cascade of immune reactions leading to vascular endothelium damage, pulmonary capillary fluid leakage, and, consequently, lung edema (1,4). Risk factors such as inflammation present in a patient can act as the first hit, whereas the second hit can be conveyed by transfusion-specific factors that can be divided into Ab-and non-Ab-mediated TRALI (1,(4)(5)(6). Abs against HLA class II and HNA-3 are the major clinical drivers of severe TRALI (7,8).
In the classical Ab-mediated TRALI model, neutrophils have been implicated as important factors (9)(10)(11), whereas in some studies, authors have reported that monocytes and macrophages, rather than neutrophils, play crucial roles in the development of TRALI (12,13). Significant controversy, however, still exists concerning the role of platelets in TRALI (14). Recent studies in murine models have demonstrated that osteopontin, secreted by macrophages, was critically required for TRALI induction via stimulation of neutrophil recruitment into the lungs (15) and that Fc-mediated complement activation was an important feature of TRALI (16).
Anti-CD36 Abs have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36 Ab-mediated TRALI, and potential therapies have not yet been identified. Here, we developed a murine model of anti-CD36 Ab-mediated TRALI to address these questions. Administration of mouse mAb against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab′) 2 fragments, induced severe TRALI in Cd36 +/+ male mice. Predepletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 Abs increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36-mediated TRALI. Administration of GZ1 F(ab′) 2 , antioxidant (N-acetyl cysteine, NAC), or C5 blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36-mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab′) 2 after TRALI induction, significant improvement was achieved when mice were treated postinduction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36. Individuals lacking CD36 on platelets and on monocytes (type I deficiency) generate anti-CD36 Abs (hereafter referred to as anti-CD36) due to immunization after blood transfusion or during pregnancy. Type I CD36 deficiency is extremely rare in White people but is found at relatively high frequency among Asians (>0.5%) (17,18). In Asians, anti-CD36 plays a critical role in the pathological mechanism of platelet transfusion refractoriness and fetal/neonatal alloimmune thrombocytopenia (FNAIT) (19)(20)(21)(22).
In Japan, TRALI cases caused by anti-CD36 have been reported (23,24); incubation of monocytes with anti-CD36 plasma derived from a female blood donor that induced TRALI in patients resulted in the production of the pro-inflammatory mediators LTB 4 and TNF-α (23). This reaction was dependent on the presence of human lung microvascular endothelial cells (HLMVECs), indicating the role of cellular crosstalk between monocytes and lung endothelial cells. Nevertheless, the exact mechanism of anti-CD36mediated TRALI remains unknown.
Although TRALI is a leading cause of transfusion-related death, specific treatments are currently unavailable (1,4). Hence, supportive measures remain the mainstay for patients with TRALI (25). In recent years, animal models have been established to study the mechanism of TRALI and to evaluate specific treatments. Some drugs, including aspirin, intravenous immunoglobulin (IVIG), anti-osteopontin Ab, and IL-10, have been shown to prevent TRALI in a mouse model of anti-MHC class I-mediated TRALI (10,15,26,27). However, no promising preclinical approaches have been reported for other Ab specificities.
In this study, we developed a murine model to study the pathomechanism of anti-CD36-mediated TRALI and to subsequently test different possible agents for efficacy in the treatment of severe TRALI caused by anti-CD36 Abs.

Results
Mouse and human anti-CD36 Abs induce severe TRALI in a 2-hit mouse model. To establish a TRALI model in mice, mAb GZ1 (IgG2a; 0.4 mg/kg BW) was first administered i.v. to naive Cd36 +/+ male mice. No significant changes in rectal temperature or lung wet/dry (W/D) weight ratios were observed ( Figure 1, A and B), suggesting that anti-CD36 alone could not induce TRALI. However, a significant decrease in rectal temperature (GZ1-vs. isotype-treated: 32.12°C ± 0.89°C vs. 38.16°C ± 0.47°C; P < 0.0001) ( Figure 1A) and increase in lung W/D weight ratios (GZ1-vs. isotype-treated: 8.10 ± 1.00 vs. 4.50 ± 0.08; P < 0.0001) ( Figure 1B) were detected when Cd36 +/+ male mice were pretreated with a low dose of LPS (0.1 mg/kg administered i.p.) and subsequently injected with mAb GZ1 (i.e., the 2-hit model), when compared with isotype controls. This phenomenon was not detected in Cd36 -/male mice. Administration of LPS alone in male and female Cd36 +/+ mice did not increase lung W/D weight ratios.
Survival rate (SR) analysis using Kaplan-Meier curves showed that more than half of the mice did not survive beyond 2 hours after administration of mAb GZ1 IgG, compared with 100% survival in isotype or mAb GZ1 F(ab′) 2 -treated cohorts (SR: 45% vs. 100%; P < 0.01) ( Figure 1I). Pathological analysis of the lungs showed the presence of pulmonary edema and alveolar septal thickening in male mice primed with LPS and treated with mAb GZ1 IgG but not with F(ab′) 2 fragments. In Cd36 -/male mice, no pathological change was observed ( Figure 1J).
To test the effects of human anti-CD36 Abs in this animal model, 3 sera samples from transfusion-immunized patients (anti-CD36 patients 1, 2, and 3 [hereafter, anti-CD36 1, 2, and 3, respectively]) were tested for binding to mouse platelets by flow cytometry. Ab binding was observed with Cd36 +/+ but not Cd36 -/-JCI Insight 2023;8(6):e165142 https://doi.org/10.1172/jci.insight.165142 mouse platelets, similar to the result with mAb GZ1 (Figure 2A). When purified IgG from anti-CD36 1 and 2 sera samples was injected into LPS-primed Cd36 +/+ mice, increases in lung W/D weight ratios and decreases in rectal temperatures were observed (Figure 2, B and C). These reactions were not observed in Cd36 -/mice. Unfortunately, anti-CD36 3 serum could not be tested in this model, because of limited materials. These results demonstrated that both murine and human anti-CD36 Abs can induce severe TRALI in male, but not female, mice in a 2-hit model.
Monocytes and complement are important pathogenic factors for murine TRALI induced by anti-CD36. To assess the contributions of different recipient blood cell types and complement on the mechanism of anti-CD36mediated TRALI, monocytes, neutrophils, platelets, and complement were depleted in vivo before TRALI induction with mAb GZ1 (Supplemental Figure 2).
All monocyte-and complement-depleted mice survived 2 hours after TRALI induction (SR, 100%) ( Figure 3C). In contrast, the SR of neutrophil-depleted mice at 2 hours was not significantly different from that of nondepleted mice (65% vs. 45%; P > 0.05) ( Figure 3C). Interestingly, even lower SRs were observed in platelet-depleted mice compared with nondepleted mice (10% vs. 45%; P < 0.01) ( Figure 3C), likely due to abundant hemorrhages found in the lung alveoli, indicating the protective role of platelets in anti-CD36mediated TRALI, most probably by maintaining vascular integrity.
Lung histological analysis of neutrophil-and platelet-depleted mice revealed fibrin deposition (28) and interalveolar capillary hyperemia resembling that observed in nondepleted TRALI mice. As noted above, hemorrhages in the alveoli were found in platelet-depleted mice. In contrast, no histological signs of acute lung injury were observed in monocyte-or complement-depleted mice; lung histology resembled that observed in naive mice ( Figure 3D).
Anti-CD36-mediated murine TRALI is associated with increased plasma C5a levels. In this study, strong TRALI was observed in male, but not in female, mice. However, female mice injected with GZ1 could also develop TRALI after transfusion with plasma from male mice, with significantly decreased rectal temperature (GZ1vs. isotype-treated: 32.34°C ± 0.33°C vs. 38.00°C ± 0.42°C; P < 0.0001) ( Figure 4A) and increased lung W/D weight ratio (GZ1-vs. isotype-treated: 6.40 ± 1.07 vs. 4.71 ± 0.11; P < 0.01) ( Figure 4B), compared with isotype control, and increased protein in BAL, documented by the lung histology (Supplemental Figure 3, A and B). In contrast, TRALI did not occur when female mice were transfused with heat-inactivated male plasma ( Figure 4, A and B), indicating that female mice possess lower complement level than do male mice (29).
Human monocytes primed with human anti-CD36 sera impair human pulmonary endothelial cell permeability. Next, we asked whether anti-CD36 could induce monocyte activation and thereby alter endothelial permeability. First, we found that binding of mAb GZ1 onto monocytes caused ROS generation that could be inhibited by antioxidant N-acetyl cysteine (NAC) ( Figure 5A). Furthermore, analysis of supernatant from monocytes after treatment with anti-CD36 3 serum from a patient with TRALI showed significantly higher TNF-α concentrations compared with monocytes treated with control serum (260. 13 Figure 5B). Treatment of HLMVECs with the supernatant increased transendothelial permeability, as shown by higher influx of fluorescently labeled BSA through the HLMVEC monolayer in the Transwell assay in comparison with HLMVECs treated with control supernatant (MFI: 227.67 ± 8.69 vs. 177.67 ± 13.22, respectively; P < 0.0001) ( Figure 5C). This phenomenon was not found when HLMVECs were treated with anti-CD36 3 serum directly. These findings were confirmed by transendothelial resistance (TEER) analysis: HLMVECs treated with supernatant from monocytes primed with anti-CD36 3 had lower TEER than did HLMVECs treated with control serum ( Figure 5D).
Upregulation of CD36 expression on monocytes after adhesion on TNF-α-activated endothelial cells. Subsequently, we measured the effect of TNF-α on CD36 expression by quantitative reverse transcription PCR. Significant downregulation of CD36 expression was found on monocytes, but not on HLMVECs, after TNF-α treatment ( Figure 5E). Real-time quantitative PCR showed that monocytes expressed significantly more CD36 transcript than did HLMVECs (26.30 vs. 22.22, respectively; P < 0.0001) ( Figure 5F). When monocytes were co-cultured with TNF-α-treated HLMVECs, significant upregulation of CD36 expression, compared with monocytes that had adhered to untreated HLMVECs, was observed, as shown by immunoblotting (0.97 vs. 0.63, respectively; P < 0.01) ( Figure 5G). In a control experiment, no change in CD14 expression on monocytes was found.
Administration of each inhibitor before TRALI prevented the development of TRALI; neither significant decrease of rectal temperature nor significant increase in lung W/D weight ratios was observed when compared with mice that did not receive these inhibitors ( Figure 6, A and B). In contrast, administration of GZ1 F(ab′) 2 after TRALI did not prevent TRALI. No significant difference in the lung W/D weight ratios between TRALI mice receiving GZ1 F(ab′) 2 and untreated TRALI mice was found (7.88 ± 0.65 vs. 8.10 ± 1.00; P > 0.05) ( Figure 6B). Significant improvement was detected when injected NAC was administered together with inhalation of atomized NAC. Although the lung W/D weight ratios were not significantly lower than in the untreated TRALI cohort (NAC vs. untreated: 7.31 ± 1.04 vs. 8.10 ± 1.00; P >0.05) (Figure 6B), the survival of mice treated with NAC after TRALI significantly improved (85% vs. 45%, respectively; P < 0.01). No significant improvement was achieved with NAC solution alone (data not shown). This improvement in lung W/D weight ratios became more significant when mAb BB5.1 was administered after TRALI compared with the untreated TRALI group (5.33 ± 1.04 vs. 8.10 ± 1.00, respectively; P < 0.0001) ( Figure 6B). Accordingly, all mice survived after induction treatment with mAb BB5.1 (SR, 100%), but only 85% and 70% survived after treatment with NAC and GZ1 F(ab′) 2 , respectively ( Figure 6C).

Discussion
In this study, we developed a murine model of anti-CD36-induced TRALI and found that murine mAbs and human Abs against CD36 induced severe TRALI in Cd36 +/+ male mice, but not in Cd36 +/+ female and Cd36 -/male mice. This TRALI response was only observed in the presence of a low dose of LPS (i.e., the 2-hit model) (31) and was characterized by increased lung W/D weight ratios, decreased rectal temperatures, decreased Pao 2 and Sao 2 , and increased protein, chemokine (KC, MIP-2), and cytokine (TNF-α) concentrations in BAL. This reaction is not only dependent on the sex of the mice but also on the Fc part of the Abs, the presence of monocytes, ROS generation by monocytes, TNF-α production, and C5 complement activation. That anti-CD36 from human sera could also induce TRALI in our murine model is likely due to the high degree of homology (approximately 85%) between the human and mouse CD36 protein (32) and because most CD36 Abs react with immunodominant epitopes (20,33).
Meanwhile, the contribution of different cell types to the pathogenesis of TRALI has become increasingly clear (1). It is commonly believed that Ab-mediated TRALI is the result of pulmonary damage caused by neutrophils. However, the pathogenic role of other cells, including monocytes, macrophages, endothelial cells, and platelets, has been well studied (1,14,15,(34)(35)(36)(37). Nevertheless, these cellular pathways remain complex and somehow depend on Ab specificities (1).
Here, we found that anti-CD36 could still induce TRALI in the absence of neutrophils. In other TRALI models, neutrophil depletion completely protected mice from anti-MHC class I-mediated TRA-LI (10). This discrepancy is most probably due to the fact that neutrophils do not express CD36 antigens (38). The SR of neutrophil-depleted mice, however, was higher when compared with that of nondepleted mice (SR, 65% vs. 45%, respectively), indicating that neutrophils do contribute to the severity of TRALI. In a previous study, researchers demonstrated HLA-DR is inducible in human neutrophils both in vitro and in vivo by IFN-γ or GM-CSF (39), suggesting that neutrophils may play a more important role in anti-HLA class II-mediated TRALI (40). Whether this is also true for CD36 is currently unknown.
Several studies have shown that monocytes are indispensable for the mechanism of Ab-mediated TRALI (12,15,41). Sachs et al. (42) showed that Abs against MHC class II antigens, which are expressed on monocytes but not on neutrophils, triggered TRALI in an ex vivo rat model. In anti-MHC class I murine models, depletion/inactivation of monocytes/macrophages completely suppressed TRALI (12, 13, 15, 41). Here, we  test (B, E, and G), or with 1-way ANOVA with Bonferroni's correction for multiple comparisons (C and F). **P < 0.01, ****P < 0.0001. showed that depletion of monocytes completely abolished anti-CD36-induced TRALI. Normal lung W/D weight ratios and rectal temperatures were detected in monocyte-depleted mice. All mice survived (SR, 100%), indicating a pivotal role for monocytes in anti-CD36-mediated murine TRALI.
It has been demonstrated that gp91phox KO mice are completely protected from TRALI induced by anti-MHC class I Abs, suggesting that the formation of ROS is important for the development of TRALI (10). An additional study in mice showed the important role of CD36 in H 2 O 2 -induced lung injury (43). We found that anti-CD36 could induce ROS production, which could be inhibited by the antioxidant NAC, both in vitro and in vivo.
The role of platelets in TRALI has been less well defined (14,36). Some studies have found recipient platelets to be pathogenic (26,44), whereas other studies have found them to be dispensable for the onset of TRALI (12,45). This discrepancy may have been caused by different factors, including different experimental methodologies. In our murine model, platelet depletion was induced by anti-GPIb-α administration, and this depletion aggravated the severity of anti-CD36-induced TRALI: SR of the mice in this cohort (10%) was much lower than that of the controls. Besides signs of TRALI, bleeding tendency was observed in our mice, indicating that platelets may maintain vascular integrity in anti-CD36-mediated TRALI (46). Furthermore, we found that F(ab′) 2 fragment of anti-CD36 could not induce TRALI, indicating the role of Fcγ receptors (FcγRs). In a previous study, researchers demonstrated that Fc block (mAb 2.4G2) against CD16/CD32 could prevent anti-MHC Ab-induced TRALI (12). Similarly, pretreatment with mAb 2.4G2 prevented the development of TRALI in our mice, a finding that strengthens the idea of a role of FcγR in anti-CD36-mediated TRALI. Along this line, prophylactic injection of IVIG also prevented mice's TRALI development (47). The question of whether activating or inhibitory FcγR is involved is intriguing and needs further investigation (48).
Our previous study demonstrated that anti-HNA-3a could bind to CTL-2 expressed on endothelial cells and could induce endothelial barrier disturbance (49). In contrast, the results of our in vitro experiments suggested a minor role of endothelial CD36 in anti-CD36-mediated TRALI. First, we found low CD36 expression on the surface of HLMVECs. Second, direct priming with anti-CD36 sera did not alter the permeability and resistance of HLMVECs. However, we found that priming of monocytes with anti-CD36 Abs caused TNF-α secretion in the supernatant and upregulation of CD36 expression on monocytes when monocytes interacted with TNF-α-activated endothelial cells. Incubation of HLMVECs with TNF-α-rich supernatants from anti-CD36-primed monocytes increased endothelial permeability and decreased endothelial resistance, a finding that supports the idea of an important pathogenic role for monocytes.
Increasing evidence indicates that complement may be an important mediator of lung injury in TRALI; however, this has not yet been systematically investigated (50). One recent study demonstrated that TRALI depends on the ability of the Ab Fc domain to induce complement activation (16) on the surface of endothelial cells, as shown in the murine model of anti-MHC class I-mediated TRALI (51). Accordingly, we found that the F(ab′) 2 fragments of mAb GZ1 did not induce TRALI, and depletion of complement or inhibition of C5 completely prevented the occurrence of anti-CD36-mediated TRALI. The role of C5 may explain the observation that anti-CD36 did not cause TRALI in female mice, which is in agreement with reports in the literature (29); female mice had much lower plasma C5 levels than did male mice. However, studies showed that estrogen could suppress lung inflammatory responses through an effect on vascular cell adhesion molecules and pro-inflammatory mediators (52), which might affect the development of TRALI. To reduce this possibility, only female mice without any signs of estrus were used in this study. Furthermore, we found that anti-CD36 could trigger generation of C5a, which is involved in the recruitment of several inflammatory leukocyte types (53). C5a enhanced TNF-α release by LPS-stimulated monocytes and macrophages (54,55), and recent data showed that binding of C5a to C5aR on monocytes could lead to ROS generation and secretion of inflammatory cytokines such as IL-1β (56). In this respect, our in vitro experiment showed that anti-CD36 generated ROS production in monocytes. Strait et al. (12) found that anti-MHC I Abs bound to endothelial cells activated complement, leading to C5a production and recruitment of monocytes to the lungs of mice, resulting in endothelial cell damage. Based on our current in vitro results, this pathway might not apply for anti-CD36-mediated TRALI, because anti-CD36 did not directly alter endothelial permeability of HLMVECs, most probably due to very low CD36 expression on these cells. However, the exact contribution of anti-CD36 bound to endothelial cells needs to be further studied in vivo.
To date, specific treatment for TRALI is unavailable (4). In this study, we examined 3 different inhibitors. Administration of the competitive inhibitor GZ1 F(ab′) 2 was only effective to prevent TRALI, but not as a post-TRALI remedy. This condition seemed different from FNAIT, in which deglycosylated GZ1 has been shown to prevent fetal death caused by anti-CD36 (20). Significant inhibition, however, was observed before TRALI onset, as well as after TRALI, in our study when NAC was administered i.v. together with inhalation of atomized NAC.
Among complement proteins, C5 plays a major role in complement-mediated inflammation (30). Therefore, anti-C5 treatments represent a favored target for the development of anti-complement drugs for different diseases (57)(58)(59)(60). Recently, Zelek et al. (30) demonstrated that mAb BB5.1, which is specific for mouse C5, could effectively inhibit C5 activation and prevent C5a accumulation in mice. In comparison with untreated mice, we found that administration of mAb BB5.1 not only was able to prevent but also could therapeutically treat anti-CD36 murine TRALI.
Overall, our observations suggested the following mechanism and intervention of anti-CD36-mediated TRALI (Figure 7): interaction between monocytes and TNF-α-activated lung endothelial cells leads to the upregulation of CD36 expression on monocytes, resulting in increased Ab binding, triggering complement activation, ROS generation, and cytokine production, which are all responsible for the severe endothelial dysfunction in TRALI.
In conclusion, we shed light on the pathomechanism of anti-CD36-mediated TRALI using a 2-hit murine model. In this model, we prevented the occurrence of TRALI by prophylactic injection of GZ1 F(ab′) 2 , NAC, or mAb BB5.1. More interestingly, we also achieved a good therapeutic effect of mAb BB5.1 on established anti-CD36-mediated TRALI. The potential for effective therapy reinforces the need for increased awareness of anti-CD36-induced TRALI, particularly in Asian populations. Although some therapeutic approaches are suggested by our work, more research on the detailed mechanism with sera from different patients with TRALI should be performed to expand the relevance of these findings.

Methods
Mice. WT C57BL/6J mice were purchased from the Laboratory Animal Centre of the Sun Yat-Sen University. Cd36 -/mice were purchased from Jackson Laboratory (B6.129S1-Cd36 tm1Mfe /J). All mice, aged 8-10 weeks, were housed under specific pathogen-free conditions.
Analysis of BALs. BALs were performed as previously described (9), with minor modifications. The BAL was performed at 30 minutes after administration of anti-CD36 or the isotype control. The protein contents of BALs were quantified using a bicinchoninic acid assay using BSA as a standard (Thermo Fisher Scientific). CXCL2/ MIP-2, TNF-α, and KC concentrations were measured by ELISA (Quantikine ELISA Kit, R&D Systems).
Lung W/D weight ratios. The W/D weight ratio of the lungs of mice was defined as a parameter of pulmonary edema. At 2 hours after anti-CD36 infusion, mice were anesthetized using 2, 2, 2-tribromoethanol (Avertin) i.p. (2% final in PBS), and the chest cavity was exposed. The whole lung was removed, weighed, dried in an oven at 60°C for 72 hours, and reweighed. The lung W/D weight ratio was calculated by the following formula: net wet weight/net dry weight.
Histology. At 2 hours after TRALI induction with anti-CD36, mice lungs were removed and fixed overnight with 4% paraformaldehyde (Sigma-Aldrich). Lung sections (8 μm) were stained with H&E and examined with a Leica DMI3000 B inverted microscope.
Complement ELISAs. Whole blood samples were collected from mice 2 hours after the infusion with mAb GZ1 or LPS, for controls. Naive mice were treated as the control. Serum was isolated after blood coagulation on ice for 2 hours and stored at -80°C until use. ELISAs for mouse C3 (catalog D721061, Sangon Biotech), C5 (catalog ab264609, Abcam), and C5a (catalog D721063, Sangon Biotech) were used according to manufacturers' instructions.
Plasma transfer. Plasma transfusion was performed as previously described (12) with minor modification. Blood samples were collected from the tail veins of male mice into 3.2% sodium citrate-containing tubes (1:10 dilution) at 4°C. Plasma was separated, pooled, and stored at -80°C. Heat-inactivated pooled plasma (56°C for 30 minutes) was used as the control. The mAb GZ1 or isotype control was diluted in 300 μL of the pooled plasma for TRALI induction, as described above.
Measurement of cytokine secretion in vitro. PBMCs were isolated from healthy CD36-positive blood donors using Ficoll-Paque premium (GE Healthcare). PBMCs were primed with anti-CD36, and the culture supernatant was analyzed for cytokine secretion as described in initial studies (61). Briefly, human anti-CD36 3 (20%, vol/vol) was incubated with Cd36 +/+ PBMCs for 20 hours at 37°C, and then the culture supernatant was collected, centrifuged at 13,000g for 5 minutes in 4°C, and stored at -80°C until use. AB serum samples from healthy individuals were treated as the control. The concentration of TNF-α in the supernatant was measured by solid phase ELISA (R&D Systems).
Endothelial permeability assay. A permeability assay was performed as described previously with minor modifications (62). Primary HLMVECs (catalog 3000, Sciencell) were maintained in endothelial cell medium (Sciencell). Endothelial cells at passage 5 were then plated onto Transwells (6.5 mm diameter, 0.4 mm