Intrakines--evidence for a trans-cellular mechanism of action

Human CXCR4 is the receptor for the CXC chemokine SDF-1alpha and also acts as a coreceptor for T lymphotropic HIV-1 strains. Blocking the surface expression of this receptor via an intrakine approach has recently been shown to efficiently prevent HIV-1 infection of T cells. The CXC-chemokine gene is fused to an endoplasmic reticulum retention signal (KDEL) that retains the newly synthesized chemokine and its receptor within the cell, where both are subsequently degraded. We constructed MoMuLV-based vectors containing the SDF-KDEL construct driven by the "MND" long terminal repeat, using eGFP as a marker gene (MND-SDF-KDEL-IRES-eGFP) and a control vector (MND-X-IRES-eGFP). CEM human T lymphoblastic leukemia cells were transduced with the intrakine vector or the control vector. We detected a marked downregulation of CXCR4 expression in the cells transduced with the intrakine vectors as opposed to the cells transduced with the control vector. However, the eGFP-negative fraction of the cells transduced with the intrakine vector displayed the same CXCR4 downregulation as the eGFP-positive fraction, suggesting an effect in trans. The possibility of this being due to eGFP being silenced while SDF-KDEL was still expressed was excluded by Southern and Northern blot analyses. Upon cultivating the control cells with supernatant of the cells transduced with the intrakine vector, we observed a downregulation of CXCR4 expression on the control cells. Experiments using rhSDF-1alpha showed downregulation by the supernatant to be comparable to that achieved by the exogenous addition of 30 ng/ml SDF-1alpha. To assess the bioactivity of the secreted substance in the supernatant, a chemotaxis assay was performed. The transmigration observed was, once again, within the range of that achieved by the addition of 30 ng/ml SDF-1alpha. We conclude that the intrakine SDF-KDEL, apart from acting within the cell, is also in part secreted and causes the downregulation of the receptor by acting like a secreted chemokine.