Secretory lysosome targeting and induced secretion of human soluble TNF-alpha receptor in murine hematopoietic cells in vivo as a principle for immunoregulation in inflammation and malignancy.
OBJECTIVE
Systemic administration of immunotherapeutics often gives rise to severe side effects. A local deposition, using secretory lysosomes of hematopoietic cells as vehicles for delivery, can overcome this problem. In the present study, the validity of this concept was investigated using retroviral transduction of the human soluble tumor necrosis factor-alpha receptor 1 (hsTNFR1) into murine bone marrow cells, followed by transfer of the genetically modified cells into irradiated mice.


METHODS
Bone marrow cells from donor mice were transduced with retroviral vector containing cDNA for hsTNFR1, together with a transmembrane domain and a tyrosine-sorting signal in order to facilitate the endoplasmic reticulum export and to achieve secretory lysosome loading. Expression of hsTNFR1 in recipient mice was investigated using flow cytometry and Western blot. Enzyme-linked immunosorbent assay was used to measure levels of tumor necrosis factor-alpha, hsTNFR1, and murine TNFR1.


RESULTS
Stable long-term expression of hsTNFR1 was achieved in transplanted mice. Hematopoietic cells, such as natural killer, T and B cells, and neutrophils contained hsTNFR1. Exposure of lipopolysaccaride (in vivo) or phorbole-myristrate esterase (in vitro) induced significant secretion of hsTNFR1. Release of endogeneous murine sTNFR1 did not differ between cells transduced with hsTNFR1 or an "empty" vector.


CONCLUSIONS
Long-term expression in vivo and inducible secretion of hsTNFR1 in murine hematopoietic cells support the potential use of storage organelles in hematopoietic cells as vehicles for targeting inflamed/malignant sites with therapeutically active agents.
Johansson
Asa C M
AC
Department of Hematology, Lund University Hospital, Lund, Sweden.
Nandakumar
Kutty S
KS
Persson
Ann-Maj
AM
Olsson
Inge
I
Hansson
Markus
M
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D016026:Bone Marrow Transplantation; D018076:DNA, Complementary; D016503:Drug Delivery Systems / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D007249:Inflammation / Q000628:therapy; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D009369:Neoplasms / Q000628:therapy; D021382:Protein Sorting Signals / Q000235:genetics; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D047888:Receptors, Tumor Necrosis Factor, Type I