Host platelets and, in part, neutrophils mediate lung accumulation of transfused UVB-irradiated human platelets in a mouse model of acute lung injury

Abstract

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 in vitro 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.

Figure 1. UVB treated hPLTs sequestered in the lungs of LPS primed SCID mice whereas TRAP (4 µM) activated hPLTs did not.

A: Confocal images of lung sections of healthy (a to d) and septic (e to h) SCID mice infused with control human platelets (Ctrl PLT), UVB-treated human platelets (UVB PLT) or TRAP (4 uM) activated human platelets (Representative images from five independent experiments). In healthy mice, there were few hPLTs in the lung in any treatment group (a to d). Sequestration of significant number of hPLTs in the lung of LPS primed mice infused with UVB treated hPLTs was observed (g), whereas a much lower numbers of Ctrl PLT (f) or TRAP-activated hPLTs (h) were sequestered in the lung. Sections were stained with anti-hCD41 antibodies (red). Blue fluorescence represents Hoechst33342 stained nuclei. All images were taken using a Zeiss 710 laser scanning confocal microscope, with a 63×/NA1.4 Plan-Apochromat oil objective. B: Quantification of the fluorescence intensity of anti-hCD41 antibody staining on mouse lung sections. Data are expressed as means ± SD; n = 5. ** p<0.01.

Figure 2. UVB treated hPLT infusion does not result in more neutrophil sequestration in the lungs of SCID mice as compared with control hPLTs.

A: 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: Quantification of LYS-eGFP fluorescence intensity on lung sections from 5 independent experiments. Data are expressed as means ± SD; n = 5. * p<0.05. C: 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.

Figure 3. Neutrophil depletion reduced platelet sequestration in the lungs.

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.

Figure 4. Infusion of UVB-treated hPLTs resulted in retention of both mouse and human PLTs in the lungs of LPS primed SCID mice.

A: Confocal images of mouse lung sections. More mouse PLTs were sequestered in the lungs of SCID; CD41-YFP mice after UVB-treated human platelet injection (A-b,d), compared with the control (A-a, c), visualized by endogenous CD41-YFP fluorescence on fresh frozen sections (A-a, b) or anti-YFP antibody staining on fixed frozen sections (A-c, d). More hPLTs were sequestered in the lungs of the mice after UVB-treated hPLT injection (A-f), compared with the control (A-e), visualized by anti-hCD41 antibody staining on fixed frozen sections. The composite images shown were representative of 5 independent experiments. B: Quantification of pixel intensity of endogenous YFP fluorescence on SCID; CD41-YFP mouse lung sections. All images were taken using a Zeiss 710 laser scanning confocal microscope, with a 63×/NA1.4 Plan-Apochromat oil objective. Data are expressed as means ± SD; n = 5. **p<0.01.

Figure 5. Mouse PLT depletion abolished hPLT sequestration in the lungs of LPS primed SCID mice.

A: Confocal images of mouse lung or liver sections. GPIb antibody treatment abolished hPLT sequestration in the lungs of septic SCID mice (A-c,d vs. a, b), visualized by anti-hCD41 antibody staining. Yet GPIb antibody treatment did not abolish platelet accumulation in the liver (A-g, h vs e, f). Neutrophil count, visualized by endogenous LYS-eGFP fluorescence, in the lung was similar before and after PLT depletion (A-k vs i, l vs j). The composite images shown were representative of 5 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 pixel intensity of anti-hCD41 antibody staining on SCID; CD41-YFP mouse lung sections shown in A-a to d. C: Quantification of fluorescence pixel intensity of anti-hCD41 antibody staining on SCID; CD41-YFP mouse liver sections shown in A-e to h. Data are expressed as means ± SD; n = 5. *p<0.05;**p<0.01.

Figure 6. UVB-treated hPLT bind to host mouse PLTs, but not to neutrophils in the lung.

A: 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). Figure 6A-c, g show the merged images of mouse and human platelets in the lung. Figure 6A-d, h 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 (Figure 6A-g, h). 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).

Figure 7. Treatment with aspirin in vitro prior to UV exposure abolished UVB-hPLT sequestration in the lung.

A: Confocal images of SCID mouse lung sections. Pre-treatment with aspirin in vitro abolished UVB-induced hPLT sequestration in the lung (A-c), compared with vehicle control (A-d). All sections were stained with anti-hCD41 antibodies (red). Blue fluorescence represents TO-PRO-3 stained nuclei. The composite images shown were representative of 3 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-hCD41 antibody staining on mouse lung sections shown in A, n = 3. C: Aspirin treatment in vitro inhibited hPLTs aggregation induced by TRAP (20 µM). Maximum Aggregation (MA) was shown in solid black bar and Dis-Aggregation (DA) was shown in solid grey bar, n = 4. All data are expressed as means ± SD, ** p<0.01, * p<0.05.

Figure 8. Depletion of mouse neutrophils or platelets and aspirin treatment prior to UV exposure all protected mice from pulmonary vascular permeability change.

BALF protein concentration (ug/ml) was measured in different experimental conditions as indicated. N = 3 for LPS+Aspirin/UVB PLT and LPS+DMSO/UVB PLT, n = 5 for all other treatment groups. All data are expressed as means ± SD, ** p<0.01, * p<0.05.