CD16, also known as the low affinity receptor for IgG (FcRIIIa), is the receptor responsible for the induction of NK antibody-dependent cellular cytotoxicity (ADCC). CD16, expressed by most NK cells, is able to transduce an activating signal through its association with CD3 and FcRI chains. Previous studies with primary NK cells have shown that upon CD16 cross-linking with specific monoclonal antibodies (mAb), the receptor is rapidly internalized by an ATP-independent mechanism that requires an intact actin cytoskeleton. Others have shown that specific mAb-mediated down-regulation of CD16 is blocked by the Zn2+-dependent metalloproteases inhibitor, 1,10-phenanthroline, suggesting that the down-regulation of CD16 from the cell surface is more a consequence of metalloprotease-induced cleavage of receptor rather than internalization. Shedding of CD16 has also been shown after treatment of NK cells with the PKC activator phorbol 12-myristate 13-acetate (PMA). Our studies are focused on determining the effective contribution of internalization and shedding to the ligand-mediated down-regulation of CD16. Our preliminary data suggest that different mAbs that bind CD16 act differently in regard to regulating CD16 expression. One mAb, B73.1, seems to enhance the down modulation of the CD16 from the cell surface without additional stimuli, whereas two other mAbs do not induce any significant down modulation. We are currently investigating whether this loss of receptor expression in case of B73.1 is due to internalization or to the shedding of the receptor from the cell surface due to cleavge by metallo-proteases (MMPs). To address this, we are performing experiments incubating the cells with this mAb for different time points in presence or not or specific MMPs inhibitors. In parallel, we are investigating the mechanisms that control the cell surface expression and intracellular trafficking of CD16. Our preliminary data indicate that CD16 down modulation requires and intact actin cytoskeleton, that the receptor is not recycling back to the cell surface and that Arf6 seems to play a crucial in this down modulation. We surprisingly found that almost 96% of the CD16 cell surface expression is lost upon treatment of primary NK cells with aluminium fluoride(AlF4-), an inhibitor of Arf-6 function. Curiously, very recent data show that treatment with MMPs inhibitors is able to restore up to 55% of the surface expression of CD16 lost upon AlF4- treatment, suggesting that somehow, the Arf-6 mediated down-modulation of CD16 is controlled by MMPs. we are intensely investigating the mechanism by which Arf-6 is regulating CD16 expression. We are also investigating if the impaired CD16 down modulation that we observe upon actin disrupting drug treatment can be restored in the presence of MMPs inhibitors. FcmuR is a member of immunoglobulin gene superfamily that is highly expressed on NK cells and recently it has been shown to be an IgM-specific Fc receptor. Nothing is known about the function of this receptor on NK cells. Our preliminary results show that crosslinking this receptor on primary NK cells leads to phosphorylation of cytoplasmic tyrosine residues, as well as to the activation of intracellular kinases, indicating that ligation of this receptor is capable of activating NK cells. Moreover, the addition of IgM to primary NK cells enhances their killing of K562 tumor cells. We have also learned that exposure of NK cells to IL-2 markedly down-regulates the expression of FcmuR. Since activated NK cells secrete IL-2, this down-modulation may occur in order to avoid excess inflammation and thus may have functional consequences.
