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Kupffer Cell Isolation for Nanoparticle Toxicity Testing
Published on: August 18, 2015
Diphtheria toxin-murine granulocyte-macrophage colony-stimulating factor-induced hepatotoxicity is mediated by
Marlena M Westcott1, Ralph J Abi-Habib, Kimberley A Cohen
1Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Abstract:
DT388GMCSF, a fusion toxin composed of the NH2-terminal region of diphtheria toxin (DT) fused to human granulocyte-macrophage colony-stimulating factor (GMCSF) has shown efficacy in the treatment of acute myeloid leukemia. However, the primary dose-limiting side effect is liver toxicity. We have reproduced liver toxicity in rats using the rodent cell-tropic DT-murine GMCSF (DT390mGMCSF). Serum aspartate aminotransferase and alanine aminotransferase were elevated 15- and 4-fold, respectively, in DT390mGMCSF-treated rats relative to controls. Histologic analysis revealed hepatocyte swelling; however, this did not lead to hepatic necrosis or overt histopathologic changes in the liver. Immunohistochemical staining showed apoptotic cells in the sinusoids, and depletion of cells expressing the monocyte/macrophage markers, ED1 and ED2, indicating that Kupffer cells (KC) are targets of DT390mGMCSF. In contrast, sinusoidal endothelial cells seemed intact. In vitro, DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes. Two related fusion toxins, DT388GMCSF, which targets the human GMCSF receptor, and DT390mIL-3, which targets the rodent IL-3 receptor, induced a less than 2-fold elevation in serum transaminases and did not deplete KC in vivo. In addition, DTU2mGMCSF, a modified form of DT390mGMCSF with enhanced tumor cell specificity, was not hepatotoxic and was significantly less toxic to KC in vivo and in vitro. These results show that DT390mGMCSF causes liver toxicity by targeting KC, and establish a model for studying how this leads to hepatocyte injury. Furthermore, alternative fusion toxins with potentially reduced hepatotoxicity are presented.
Insights
DT390mGMCSF causes liver toxicity by targeting Kupffer cells (KC), leading to hepatocyte injury. Alternative fusion toxins show reduced hepatotoxicity, offering safer treatment options for acute myeloid leukemia.
Area of Science:
- Biochemistry
- Toxicology
- Immunology
Background:
- DT388GMCSF, a diphtheria toxin (DT) fusion protein with human granulocyte-macrophage colony-stimulating factor (GMCSF), shows promise for acute myeloid leukemia (AML).
- A significant dose-limiting side effect of DT-based fusion toxins is liver toxicity.
Purpose of the Study:
- To investigate the mechanism of liver toxicity induced by DT390mGMCSF, a rodent-specific fusion toxin.
- To identify the cellular targets responsible for DT390mGMCSF-induced hepatotoxicity.
- To evaluate alternative fusion toxins for reduced liver toxicity.
Main Methods:
- DT390mGMCSF was administered to rats to assess liver toxicity.
- Serum transaminases (AST, ALT) were measured.
- Liver histology and immunohistochemistry were performed to identify cellular changes and target cells.
- In vitro cytotoxicity assays were conducted on primary rat Kupffer cells (KC) and hepatocytes.
- Related fusion toxins (DT388GMCSF, DT390mIL-3, DTU2mGMCSF) were evaluated for hepatotoxicity and KC toxicity.
Main Results:
- DT390mGMCSF significantly elevated serum transaminases and caused hepatocyte swelling in rats.
- Immunohistochemistry revealed apoptotic cells in sinusoids and depletion of KC (ED1+, ED2+), indicating KC as the primary target.
- DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes in vitro.
- DT388GMCSF and DT390mIL-3 showed minimal liver toxicity and did not deplete KC.
- DTU2mGMCSF, a modified toxin with enhanced tumor specificity, was not hepatotoxic and had reduced KC toxicity.
Conclusions:
- DT390mGMCSF induces liver toxicity by selectively targeting and depleting Kupffer cells.
- This study establishes a model for understanding DT390mGMCSF-mediated hepatocyte injury.
- Development of alternative fusion toxins, such as DTU2mGMCSF, offers potential for safer AML therapies with reduced hepatotoxicity.
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