Our findings reveal that TRPV2 participates in the very earliest steps of macrophage phagocytosis. In the specific case of FcγR-mediated phagocytosis, TRPV2 channels recruited to the nascent phagosome in response to initial FcγR engagement by IgG mediate membrane depolarization, which in turn triggers PIP2 synthesis and the consequent actin depolymerization required for efficient higher-order clustering of occupied FcγR. In the absence of such clustering binding, and thus phagocytosis of IgG-coated particles are impaired.
The molecular mediator(s) of macrophage depolarization evoked by phagocytic substrates have remained unclear. Fcγ receptor-evoked depolarization in J774 cells was shown to result predominantly from Na+
influx through a pathway selective for small monovalent cations. Similar depolarization responses were observed in phospholipid vesicles reconstituted with purified Fcγ receptor, prompting the suggestion that the receptor itself formed the channel 26
. Other studies have reported that in response to immune complexes or prolonged artificial depolarization macrophages exhibit a relatively nonselective cationic current 6, 24
. Similar currents were seen in response to reactive oxygen species or ADP-ribose, leading to speculation that the depolarization-evoked current might be mediated by TRPM2. However, the significant NMDG permeability of the current was not characteristic of TRPM224
and, in related microglia, depolarization evoked currents pharmacologically resemble TRPV1 29
. Also potentially involved in these responses is the potassium channel ether-a-go-go-related gene (ERG), whose inhibition potentiates macrophage depolarization 25
. We do not yet understand the relationship between depolarization responses mediated by TRPV2 and those reported in these other studies. However, the fact that KCl treatment rescues the depolarization, binding and phagocytosis defects in TRPV2 deficient macrophages suggests that other ion channels can circumvent the requirement for TRPV2, if properly engaged.
Although artificial membrane depolarization facilitates phagocytic receptor binding25
, the underlying mechanism is not clear. We found that depolarization achieved via TRPV2 or KCl increases PIP2 synthesis, apparently by directly or indirectly activating PI(4)K. The polyamine spermine also activates PI(4)K 30
, thus potentiation of this enzyme by membrane depolarization might be attributable to local surface charge effects at the inner plasma membrane leaflet 20
. Depolarization-evoked PIP2 synthesis can be inhibited by adenosine, which is more potent for type II PI(4)K than type III subtypes, and has previously been shown to block latex bead-evoked phagosomal PIP2 synthesis in J774 macrophage cells 28
. However, we cannot exclude the possible involvement of other PI(4)K isoforms in this process. PIP2 promotes controlled actin depolymerization during phagocytosis 27
. Our data indicate that this effect on actin is the basis by which depolarization promotes FcγR clustering. Similar effects of TRPV2-mediated depolarization on the cytoskeleton might underlie the defects in chemoattractant-evoked motility observed in the absence of TRPV2 13, 14
Normally, higher-order FcγR clustering depends not only on controlled actin dynamics 27
, but also on oligomeric occupancy of neighboring FcγRs 21
. Consistent with this notion, neither monomeric IgG, KCl, nor low-dose actin depolymerizing agents alone were sufficient to trigger FcγR clustering or TRPV2 co-localization with FcγR in our experiments. However, when monomeric IgG was combined with either of the other two stimuli, both clustering and co-localization proceeded normally. This suggests that initial FcγR oligomerization is not essential for subsequent clustering, provided that partial actin depolymerization is achieved in another way. Yet, TRPV2 recruitment, which is required for normal IgG-evoked actin depolymerization in the absence of an exogenous depolarizing stimulus, appears to require oligomeric FcγR occupancy. We also show that TRPV2 recruitment to the nascent phagosome requires Src family kinase signaling, as well as signaling through PI(3)K, Akt, and PKC ζ. Although the exact sequence must be established, Src family kinase activation is one of the earliest events in FcγR signaling, and triggers PI(3)K activation 21
, while Akt is generally recruited by PI(3)K-synthesized PIP3. PKC ζ, which is also required for PIP3-dependent GLUT4 translocation in adipocytes 31
, likely acts downstream of Akt.
In our experiments, we measured clustering of the low-affinity FcγR (FcγRII/ FcγRIII). These are the isotypes thought to be primarily responsible for FcγR-mediated phagocytosis of IgG opsonized substrates (Indik, 1995). Another isotype, FcγRI, can bind monomeric IgG2a with high affinity, but is typically saturated under physiological conditions. Since our experiments use mixed isotype IgG, further work is necessary to discern any contributions of FcγRI to the clustering we observed.
We cannot exclude additional roles for TRPV2 in phagocytosis beyond substrate binding. A recent proteomics study detected TRPV2 in early endosomal fractions purified from bone marrow-derived macrophages and J774 cells 32
. Another group identified a Ca2+
current in artificially enlarged early endosomes in HEK293 embryonic kidney-derived cells that shared biophysical features with TRPV2, leading them to speculate that TRPV2 or a similar channel facilitates early endosome fusion and maturation 33
. Studies to evaluate later functions for TRPV2 in phagocytosis are therefore warranted.
The other major finding of our study is that mice lacking TRPV2 are profoundly defective in defense against Listeria
infection, exhibiting both impaired bacterial clearance and accelerated mortality. These results, which might be attributable to both the phagocytosis and motility defects observed in TRPV2KO macrophages, support a critical role for this channel in early innate immunity against Listeria
and, perhaps, other intracellular pathogens. The increased organ bacterial load in Listeria
infected TRPV2KO mice also suggests that one or more of the multiple pathways by which Listeria
enters nonphagocytic parenchymal cells 34
are TRPV2 independent. Together, these findings make TRPV2 an attractive potential target for the treatment of microbial infection and other inflammatory and immune conditions that involve macrophages.