Volume 13, Issue 1, Pages 108-117 (January 2006) Rapid Kupffer cell death after intravenous injection of adenovirus vectors  Elanchezhiyan Manickan, Jeffrey S. Smith, Jie Tian, Thomas L. Eggerman, Jay N. Lozier, Jacqueline Muller, Andrew P. Byrnes  Molecular Therapy  Volume 13, Issue 1, Pages 108-117 (January 2006) DOI: 10.1016/j.ymthe.2005.08.007 Copyright © 2005 Terms and Conditions

FIG. 1 Decrease in Kupffer cells in the liver after intravenous delivery of adenovirus vector. (A) Immunostaining for the macrophage marker F4/80 (green) at 10 min after 1012 vp/kg AdV. Sections were counterstained with the DNA stain Hoechst 33342 (blue). pv, portal vein. (B) Immunostaining for F4/80 at 6 h after AdV, showing depletion of KCs. Scale bar, 100 μm. (C) Time course of KC depletion after 1012 vp/kg AdV (n = 3 per group). (D) Dose response of KC depletion at 6 h (n = 3 per group). *P < 0.05 vs control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2006 13, 108-117DOI: (10.1016/j.ymthe.2005.08.007) Copyright © 2005 Terms and Conditions

FIG. 2 Predosing enhances liver expression after AdV, but does not substantially affect the vector biodistribution. (A) Mice were predosed with buffer or various doses of a GFP-expressing AdV, followed 1 h later with 1012 vp/kg of a β-galactosidase-expressing AdV. Mice were sacrificed 3 days later, and β-galactosidase protein was assayed (n = 5 per group). *P < 0.01 vs buffer predose, log-transformed data. (B) Mice were predosed with buffer or 1012 vp/kg of a GFP-expressing AdV, followed 1 h later with 1012 vp/kg of a β-galactosidase-expressing AdV. Mice were sacrificed 1 h later, and the copy number of the β-galactosidase AdV was measured by quantitative PCR for lacZ (n = 5 per group; no significant differences found in any organ between buffer and AdGFP predoses). Molecular Therapy 2006 13, 108-117DOI: (10.1016/j.ymthe.2005.08.007) Copyright © 2005 Terms and Conditions

FIG. 3 Toxic effects of intravenous adenovirus vector on Kupffer cells. All mice were dosed with 1012 vp/kg AdV unless otherwise indicated. F4/80 staining is shown in green. (A) When mice were given a predose of buffer, KC uptake of a subsequent dose of Cy3–AdV (red) was strong. (B) However, a predose of AdV blocked KC uptake of Cy3–AdV almost completely. Intravenous predosing with 1011 vp/kg of unlabeled AdV was followed 1 h later by 1012 vp/kg of Cy3–AdV, and mice were sacrificed after another hour. (C) KCs in the liver lost their membrane integrity after intravenous AdV, as evidenced by uptake of intravenously-administered PI (red), shown here at 10 min after AdV. (D) No uptake of PI was seen in the liver after buffer injection. (E) AdV caused trypan blue to be taken up by large numbers of sinusoidal cells in the liver, shown here 30 min after injection of AdV. Sections were counterstained with eosin. (F) There was a lack of trypan blue uptake in mice injected with buffer. (G) After AdV, numerous TUNEL-positive (red) Kupffer cells were seen in the liver, shown here at 30 min. (H) TUNEL staining was not seen in mice injected with buffer. (I) After injection of Cy3–AdV (red), occasional Cy3–AdV-containing F4/80+ macrophages were seen exiting the liver through the central veins, shown here at 1 h. cv, central vein. (J) In lungs, substantial numbers of Cy3–AdV-containing macrophages were found at later times, shown here at 5 h. (K) A normal lung from a mouse 5 h after injection of buffer, showing little F4/80 positivity. (L) In mice predosed with unlabeled AdV followed by Cy3–AdV, many bright F4/80+ cells were seen in the lungs, but they had low or negative uptake of Cy3–AdV. Mice were predosed with 1011 vp/kg, injected with 1012 vp/kg Cy3–AdV 1 h later, and sacrificed after a further 5 h. Scale bar, 50 μm. Molecular Therapy 2006 13, 108-117DOI: (10.1016/j.ymthe.2005.08.007) Copyright © 2005 Terms and Conditions

FIG. 4 Rapid and severe ultrastructural changes in Kupffer cells after intravenous injection of AdV. (A) A normal KC in a mouse injected 60 min earlier with buffer. (B) Only 10 min after AdV injection, KCs showed necrotic changes, with loss of the plasma membrane, marked vacuolization and disorganization of the cytoplasm, condensation of chromatin, and separation of the nuclear membrane from the chromatin. Note extrusion of chromatin from the nucleus (arrow). The outlined area is shown in D at higher magnification. (C) By 60 min after AdV, chromatin in KCs was completely degraded, leaving only the nuclear matrix (arrowhead), which was enclosed by a disrupted nuclear membrane. KC mitochondria were disrupted and swollen. The nucleus of an adjacent endothelial cell was normal, although swelling of the endothelium was often noted immediately adjacent to KCs (arrow). (D) Enlargement of B showing numerous virions associated with a KC at 10 min. (E) In contrast to the very large numbers of virions that were found within KCs, much smaller numbers of virions could be seen associated with hepatocyte microvilli in the space of Disse, with rare examples of virions being endocytosed into hepatocytes (arrow), shown here at 60 min. The asterisk (*) indicates the sinusoidal lumen. KC, Kupffer cell; EC, endothelial cell; H, hepatocyte; P, platelet; mv, microvilli. Scale bar, 2.0 μm (A–C); 630 nm (D); or 390 nm (E). Molecular Therapy 2006 13, 108-117DOI: (10.1016/j.ymthe.2005.08.007) Copyright © 2005 Terms and Conditions

FIG. 5 Rapid elevation of serum LDH after intravenous adenovirus vector in mice and rhesus macaques. (A) Time course of LDH elevation after intravenous injection of buffer or 1012 vp/kg AdV in mice (n = 4 per data point). Mice at time 0 are uninjected controls. (B) Dose response of LDH elevation, 30 min after injection of AdV in mice. ALT was unaffected (n = 5 per data point). (C) Mice that were predosed with AdV failed to show any additional LDH response to a second dose of AdV (P > 0.05). Mice were predosed, given a second dose 1 h later, and sacrificed after an additional 1 h (n = 4 or 5 per group). (D) In rhesus macaques, elevation of LDH was also seen after intravenous injection of AdV, in a dose- and time-dependent manner (n = 3 per dose group). *P < 0.05 vs buffer-injected animals at the same time point. Molecular Therapy 2006 13, 108-117DOI: (10.1016/j.ymthe.2005.08.007) Copyright © 2005 Terms and Conditions

FIG. 6 Effects of KC killing on the innate cytokine response. Mice were predosed, dosed 1 h later, and then sacrificed after a further 5 h. Serum IL-6 and IL-12 p70 were measured by ELISA (n = 3 or 4 per group). Molecular Therapy 2006 13, 108-117DOI: (10.1016/j.ymthe.2005.08.007) Copyright © 2005 Terms and Conditions