In Vitro and In Vivo Characterization of Ultraviolet Light C-Irradiated Human Platelets in a 2 Event Mouse Model of Transfusion

<div><p>UV-based pathogen reduction technologies have been developed in recent years to inactivate pathogens and contaminating leukocytes in platelet transfusion products in order to prevent transfusion-transmitted infections and alloimmunization. UVC-based technology differs from UVA or UVB-based technologies in that it uses a specific wavelength at 254 nm without the addition of any photosensitizers. Previously, it was reported that UVC irradiation induces platelet aggregation and activation. To understand if UVC-induced changes of platelet quality correlate with potential adverse events when these platelets are transfused into animals, we used a 2-event SCID mouse model in which the predisposing event was LPS treatment and the second event was infusion of UVC-irradiated platelets. We analyzed lung platelet accumulation, protein content in bronchoalveolar lavage fluid as an indication of lung injury, and macrophage inflammatory protein-2 (MIP-2) release in mice received UVC-irradiated or untreated control platelets. Our results showed UVC-irradiated platelets accumulated in lungs of the mice in a dose-dependent manner. High-doses of UVC-irradiated platelets were sequestered in the lungs to a similar level as we previously reported for UVB-irradiated platelets. Unlike UVB-platelets, UVC-platelets did not lead to lung injury or induce MIP-2 release. This could potentially be explained by our observation that although UVC treatment activated platelet surface αIIbβ3, it failed to activate platelet cells. It also suggests lung platelet accumulation and subsequent lung damage are due to different and separate mechanisms which require further investigation.</p> </div

In vivo lung accumulation of UVC-irradiated HPs in the 2-event SCID mouse model.

<p><i>A</i>) Mice were pretreated with an intraperitoneal injection of 3 mg/kg LPS 2 hours before intravenous infusion of untreated control or UVC-irradiated HPs at low (0.2 J/cm²) and high (1.2 J/cm²) doses, respectively. Shown are anti-human CD41 immunohistochemistry staining of lung frozen sections; <i>B</i>) Quantification of pixel intensity of anti-hCD41 staining of images shown in A. Mean ± SE, n=3; <i>C</i>) Lung histology from H&E staining of lung paraffin sections. Shown is a representative of three independent experiments.</p

UVC-irradiated HPs showed reduced in vivo recovery in circulation of SCID mice.

<p>Approximately 1 x 10⁹ untreated (open circle) or UVC-irradiated HPs at a dose of 0.2 J/cm² (filled square) were infused into SCID mice. Blood sampling was subsequently performed at indicated time points and the presence of human platelets in circulation positive for anti-human CD41a staining was detected by flow cytometry. Mean ± SE, n=5.</p

UVC-irradiated HPs did not induce lung injury in the 2-event SCID mouse model.

<p>Mice were treated as described above for the 2-event SCID mouse model. Bronchoalveolar fluid (BALF) was collected 1 hour after platelet infusion and the total protein concentration in BALF was measured using BCA protein assay. Mean ± SE, n=5-10.</p

UVC-irradiated HPs failed to induce MIP-2 release in the 2-event SCID mouse model.

<p>3 hours after HPs infusion mice were euthanized. Blood and BALF were collected and plasma was subsequently isolated as described in Materials and Methods. The concentrations of MIP-2 in plasma (<i>A</i>) and BALF (<i>B</i>) were measured using ELISA. Mean ± SE, n=4-5. </p

UVC irradiation induced platelet aggregation and potentiated ADP induced platelet aggregation.

<p><i>A</i>) Human platelets (HPs) at 1 x 10⁶/µL were exposed to 0.2 (30 seconds), 0.4 (1 minutes), 0.8 (2 minutes), and 1.2 J/cm² (3 minutes) UVC illumination. Single platelet count was measured immediately after light exposure on a Cell-Dyn 3700 blood cell counter. Mean ± SE, n=8. <i>B</i>) in vitro aggregation assay was performed with untreated (Ctrl, open circle) or UVC-exposed platelets (0.2 J/cm², filled square). Y axis represents percent of maximal aggregation (%MA). Mean ± SE, n=3.</p

Host Platelets and, in Part, Neutrophils Mediate Lung Accumulation of Transfused UVB-Irradiated Human Platelets in a Mouse Model of Acute Lung Injury

<div><p>We previously reported that ultraviolet light B (UVB)-treated human platelets (hPLTs) can cause acute lung injury (ALI) in a two-event SCID mouse model in which the predisposing event was Lipopolysaccharide (LPS) injection and the second event was infusion of UVB-treated hPLTs. To delineate contributions of host mouse platelets (mPLTs) and neutrophils in the pathogenesis of ALI in this mouse model, we depleted mPLTs or neutrophils and measured hPLT accumulation in the lung. We also assessed lung injury by protein content in bronchoalveolar lavage fluid (BALF). LPS injection followed by infusion of UVB-treated hPLTs resulted in sequestration of both mPLTs and hPLTs in the lungs of SCID mice, although the numbers of neutrophils in the lung were not significantly different from the control group. Depletion of mouse neutrophils caused only a mild reduction in UVB-hPLTs accumulation in the lungs and a mild reduction in protein content in BALF. In comparison, depletion of mPLTs almost completely abolished hPLTs accumulation in the lung and significantly reduced protein content in BALF. UVB-treated hPLTs bound to host mPLTs, but did not bind to neutrophils in the lung. Aspirin treatment of hPLTs <em>in vitro</em> abolished hPLT accumulation in the lung and protected mice from lung injury. Our data indicate that host mPLTs accumulated in the lungs in response to an inflammatory challenge and subsequently mediated the attachment of transfused UVB-hPLTs. Neutrophils also recruited a small percentage of platelets to the lung. These findings may help develop therapeutic strategies for ALI which could potentially result from transfusion of UV illuminated platelets.</p> </div

Neutrophil depletion reduced platelet sequestration in the lungs.

<p>SCID mouse were pretreated with either Gr-1 mAb (250 ug, i.p.) or isotype control (Iso Ctrl, i.p.) 24 hours before LPS administration (3 mg/kg, i.v.), followed by transfusion of control hPLTs or UVB-treated hPLTs. A: Confocal images of lung sections from SCID mice stained with anti-CD41antibodies (red). Blue fluorescence represents Hoechst33342 stained nuclei. The composite images shown were representative of 6 independent experiments. All images were taken using a Zeiss 710 laser scanning confocal microscope, with a 63×/NA1.4 Plan-Apochromat oil objective. B: Quantification of fluorescence intensity of anti-CD41 antibody staining on mouse lung sections. Data are expressed as means ± SD; n = 6 .* p<0.05; ** p<0.01.</p

UVB treated hPLT infusion does not result in more neutrophil sequestration in the lungs of SCID mice as compared with control hPLTs.

<p><b>A</b>: Confocal images of unfixed, vibratome sections of the lungs from SCID/LYS-eGFP mice. In these mice, neutrophils are labeled in green fluorescence. LPS primed mice were injected with LPS (i.v , 3 mg/kg). The composite images shown were representative of 5 independent experiments. All fluorescent images were taken with a Zeiss 710 laser scanning confocal microscope, with a Plan-Apo 40×/NA1.0 water objective. <b>B</b>: Quantification of LYS-eGFP fluorescence intensity on lung sections from 5 independent experiments. Data are expressed as means ± SD; n = 5. * p<0.05. <b>C</b>: Images of immunohistochemical staining on paraffin sections with anti-Gr-1 antibodies. The composite images shown were representative of 4 independent experiments. All transmitted light images were taken with a Nikon Eclipse E800 microscope (Nikon Co., Ltd., Tokyo, Japan), using a 20×/NA 0.45 Plan Fluor objective.</p

UVB-treated hPLT bind to host mouse PLTs, but not to neutrophils in the lung.

<p><b>A</b>: Live images of whole-mount lungs from SCID/CD41-YFP mice, after LPS injection and injection of either Ctrl PLTs (a–d) or UVB PLTs (e–h). MPLTs were visualized by endogenous YFP expression (in green, a and e). Human PLTs were labeled with CMTMR (in red, b and f). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044829#pone-0044829-g006" target="_blank">Figure 6A-c, g</a> show the merged images of mouse and human platelets in the lung. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044829#pone-0044829-g006" target="_blank">Figure 6A-d, h</a> show 3D reconstructed images of a stack of 10 images (10 uM). When UVB-hPLTs were injected into SCID/CD41-YFP mice, hPLTs in the lung colocalize or in close proximity to mouse PLTs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044829#pone-0044829-g006" target="_blank">Figure 6A-g, h</a>). <b>B</b>: Confocal images of whole-mount lungs from SCID/LYS-eGFP mice, after LPS injection and injection of either Ctrl PLTs (a–c) or UVB PLTs (d–f). Mouse neutrophils were visualized by endogenous eGFP expression (in green, a and d). hPLTs were labeled with CMTMR (in red, b and e). c and f show the merged images of hPLTs and mouse neutrophils in the lung. When UVB-hPLTs were injected into SCID/LYS-eGFP mice, the majority of hPLTs in the lung don't bind to neutrophils (f).</p