A requirement for PI 3-kinase activity in FcγR-mediated phagocytosis is well established 121820
. Because SHIP has been shown to downmodulate the function of receptors that activate PI 3-kinase, and FcγRIIB contains a SHIP-binding site, it is not surprising that SHIP is capable of downmodulating FcγR-mediated phagocytosis. In contrast, little is known about the role of PI 3-kinase and SHIP in CR3-mediated phagocytosis. The results of this study are consistent with a role for both PI 3-kinase and SHIP in regulating CR3-mediated phagocytosis.
The involvement of PI 3-kinase in β2
integrin function was suggested by studies demonstrating a requirement for this family of enzymes in β2
integrin–dependent adhesion 212238
. Sensitivity to PI 3-kinase inhibitors was dependent on the nature of both the stimulus for adhesion and the adhesive substrate. Addition of immune complexes to human neutrophils resulted in a twofold increase in β2
integrin surface expression and a sixfold increase in the expression of a β2
integrin activation-dependent epitope, both of which were sensitive to WM 22
. These results are consistent with a requirement for PI 3-kinase in immune complex–induced inside-out signaling of β2
integrins in neutrophils. Because β2
integrin function in most phagocytic leukocytes requires prior or concurrent activation, it would be difficult to discern an independent requirement for PI 3-kinase in β2
integrin outside-in signaling. The use of peritoneal macrophages from C57BL/6 mice and COS cells transfected with CR3 in this study circumvents this problem; in the case of the former, CR3 phagocytic function is apparent in the absence of additional stimuli. This capacity is sustained over several weeks while the cells are maintained in culture. The phagocytic capacity of COS cells transfected with CR3 is even greater, and essentially all of the CR3-expressing cells demonstrate high levels of surface expression of the activation-dependent epitope, CBRM1/5. The WM sensitivity of phagocytosis of EC3bi in these two cell types attests to a role for PI 3-kinase in outside-in signaling via CR3.
The finding that SHIP is capable of modulating FcγR-mediated phagocytosis is consistent with the recruitment of SHIP to the phosphorylated ITIM of FcγRIIB, which is likely to occur during binding of IgG. This model is supported by a recent study 11
. However, it is unclear how SHIP becomes recruited to phagosomes during engagement of CR3. Mutational analysis indicates that both the SH2
domain and the NPXY motifs of SHIP are required for SHIP-mediated downmodulation of CR3-dependent phagocytosis, suggesting that the residues therein contribute to SHIP targeting. Several different proteins have been shown to interact with these motifs. The SH2
domain of SHIP has been shown to be required for the interaction of SHIP with FcγRIIB 8
, Shc 29
, Gab 1/2 39
domain–bearing protein tyrosine phosphatase 40
, and Dok-3 41
. The NPXY motifs of SHIP mediate interactions with Shc 42
and the p85 subunit of PI 3-kinase 43
. For the most part, the significance of these interactions in vivo is unknown. Because SHIP translocates to the cytoskeleton after the clustering of CR3, it is anticipated that the identification of specific and direct SHIP–protein interactions that accompany CR3 ligation will be difficult. Further studies will be needed to address the mechanism of SHIP recruitment to the phagosome during CR3-mediated phagocytosis.
Ligation of either CR3 or FcγR leads to an increase in PtdIns(3,4,5)P3
and PI 3-kinase activity is required for phagocytosis (1, 2, 18, 20, and this study). However, it is unknown which product of PI 3-kinase is essential for phagocytosis. PI 3-kinase catalyzes the addition of phosphate to the D-3 positions of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate to form phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and PtdIns(3,4,5)P3
, respectively. Of these, SHIP is known only to hydrolyze PtdIns(3,4,5)P3 4445
. Therefore, the reciprocal roles of PI 3-kinase and SHIP in phagocytosis suggests that PtdIns(3,4,5)P3
is the relevant target of these phosphoinositide-modulating enzymes. A similar role for PtdIns(3,4,5)P3
has been proposed for signaling via the B cell antigen receptor in B lymphocytes and FcεRI in mast cells 89
. By binding the PH domain on members of the Tec family of tyrosine kinases, PtdIns(3,4,5)P3
contributes to the calcium signaling pathway in these cells 567
. The identity of PH domain proteins that positively regulate phagocytosis is unknown, although several proteins that contain putative PtdIns(3,4,5)P3
-binding PH domains 46
are expressed in macrophages (4748
; Greenberg, S., unpublished results).
It is possible that SHIP modulates phagocytosis by decreasing cellular d
-inositol 1,3,4,5-tetrakisphosphate (Ins[1,3,4,5]P4
) content, although a role for Ins(1,3,4,5)P4
hydrolysis by SHIP in vivo is lacking. It is not known whether Ins(1,3,4,5)P4
is generated during the course of FcγR- or CR3-mediated phagocytosis. One study found a minimal and delayed increase in this phosphoinositide after the addition of serum-opsonized zymosan to human neutrophils 49
has been shown to inhibit exocytosis in neurons, which has been attributed to the inhibition of synaptotagmin function by the binding of Ins(1,3,4,5)P4 50
. Nonneuronal forms of synaptotagmin have also been implicated in exocytosis, and a recent study has shown that these isoforms of synaptotagmin bind Ins(1,3,4,5)P4
in vitro 51
. Overexpression of SHIP might result in a decrease in basal or stimulated Ins(1,3,4,5)P4
levels, thereby enhancing exocytosis. Because phagocytosis requires the addition of new membrane during phagosome formation (i.e., exocytosis [1
]), it is unlikely that the mechanism of inhibition of phagocytosis by SHIP is via this mechanism. However, there are likely to be other cellular targets of Ins(1,3,4,5)P4
, including PH domains that are capable of interacting with this water-soluble phosphoinositide in vitro 5354
. It is possible that PtdIns(3,4,5)P3
interact with a similar spectrum of PH domain–containing proteins in vivo.
Although this study did not address the precise mechanism by which products of PI 3-kinase and SHIP affect CR3-mediated phagocytosis, the lack of inhibition by WM of focal accumulations of F-actin beneath phagocytic cups strongly suggests that pseudopod extension and/or phagosomal closure requires PI 3-kinase activity. In this respect, CR3-mediated phagocytosis resembles FcγR-mediated phagocytosis 1
. There are obvious differences in the morphology of phagosomes formed by clustered FcγRs and CR3: CR3-containing phagosomes appear to “sink” into the cell without demonstrating obvious pseudopod extension 1655
. Because the membrane surface area that is internalized during phagocytosis is a function only of the number and size of the particles engulfed, the requirement for new membrane in CR3- and FcγR-mediated phagocytosis must be similar. It is therefore likely that CR3-mediated phagocytosis requires exocytic insertion of new membrane 152
, and that this process requires PI 3-kinase activity similar to FcγR-mediated phagocytosis 1
Our findings are somewhat divergent from a recent study in which LFA-1–mediated adhesion to intracellular adhesion molecule (ICAM)-1 was enhanced in clones of the IL-3–dependent cell line, DA-ER, stably over-expressing SHIP 56
. There are many possible explanations for this apparent disparity, including differences in the nature of the cell type used and differences in the integrins (CR3 versus LFA-1) and substrates (iC3b versus ICAM-1) that were studied. Although neither study addressed the in vivo consequences of modulation of SHIP activity on phagocytosis or leukocyte adhesion, it is interesting to speculate that the infiltration of leukocytes observed in the lungs of SHIP−/−
may reflect, in part, enhanced β2
integrin–dependent adhesive interactions with the endothelium.
Because SHIP plays a role in hematopoietic cell development and has been implicated in signaling via c-fms, the receptor for CSF-1 45
, it is possible that enhanced phagocytosis in SHIP−/−
cells is due to an alteration in their state of differentiation or activation. While this is difficult to test definitively, the observation that surface FcγR and CR3 expression and particle binding was unaltered in SHIP−/−
cells suggests that this is not the case. In addition, the use of COS cells in some of the assays avoids the possible contribution of SHIP to CSF-1–dependent signaling.
In conclusion, we have presented evidence for a previously unsuspected role of SHIP in phagocytosis mediated by CR3, and for phagocytosis mediated by FcγRs. Together with data suggesting that PI 3-kinase is required for β2 integrin outside-in signaling, the relative expression and activities of PI 3-kinase(s) and SHIP in different macrophage populations are likely to determine the strength of the phagocytic signal. As CR3 has been implicated in other cellular functions besides phagocytosis, the expression of SHIP may influence other CR3-dependent activities, including leukocyte migration into inflammatory foci.