Our work suggests that at least for DCs, surface contact is sufficient to introduce an activation signal across the plasma membrane, leading to an intracellular signaling cascade that involves Syk. It is well established that lipid raft aggregation significantly facilitates many signaling events. In IgE-mediated mast cell activation, one theory states that the function of surface receptors is to bring together sufficient amounts of lipid rafts of which cholesterol is an important component (Brown, 2006
; Brown and London, 1998
). The function of different membrane lipids in segregating surface proteins is well-known (Zacharias et al., 2002
). ITAM-containing receptors are attracted to cholesterol rich regions on the cell surface. However, because lipid rafts prior to receptor ligation are small (less than 60 nM in diameter), random accumulation of receptors with activating motifs such as ITAM at any given point is low, balanced by efficient dispersion (Kusumi et al., 2004
). If cholesterol is stabilized, the aggregation of lipid rafts may by themselves allow certain membrane events to take place, leading to cellular activation. Evidence supporting this mechanism comes from the assay where GM1, another component of lipid rafts, was crosslinked. The crosslinking induced significant membrane reorganization (Dietrich et al., 2001
). This process is clearly not activated by protein receptor interaction. Syk membrane recruitment is triggered by protein receptor ligation that leads to phosphorylation of ITAMs, such as those present in BCR and FcR (Abram and Lowell, 2007
). However, basal level ITAM phosphorylation is commonly observed. Since MSU engages a large continuous membrane surface, it may trigger sufficient recruitment of Syk to the inner leaflet (). This hypothesis also explains why control cell lines do not interact strongly with MSU crystal as Syk is a hematopoietic kinase. Approaching the issue from a different angle, in several separate studies, it has been reported that membrane sorting occurs under the influence of membrane curvature and force (van Meer and Vaz, 2005
), leading to highly concentrated lipid species confined in a space, such as an elongated tube, as a consequence. Reasoning from this prospective, the binding of any particular lipid moiety may not be crucial, as long as an external object has affinity to the plasma membrane. Because such an affinity based contact inevitably leads to membrane curvature. We noted with interest that in a most recent paper, it was shown that Shiga toxin may bind to surface structure, which leads to invagination and endocytosis as a pure lipid membrane event (Romer et al., 2007
). Under our experimental settings, we have collected some evidence to suggest that MSU may interact with cholesterol. Whether it occurs under any biological conditions, and if so whether it has any biological meanings remains to be ascertained. However, even if another membrane moiety plays some or a large part of crystal binding, our proposal of force-induced lipid sorting may still stand. As one of our many possibilities, we would like to suggest on theoretical grounds that MSU crystal surface with a collective zeta potential of −37.4 mV (data not shown) might allow hydrogen bonding with properly packed cholesterol following lipid sorting. This suggestion is certainly speculative and solid surface chemistry work is needed to delineate the interaction.
One possible/hypothetical scheme of the receptor independent Syk kinase activation
Regarding the possibility of cholesterol sorting mediated by the crystal, some theoretical considerations may suggest that such an interaction could take place, unlike other H-bond forming partners. One feature of membrane lipids is their propensity to produce dense molecular “packing” into a structure that could even be aligned with the lattice of the crystal. This would allow for a large number of H-bonds to occur per given surface area explaining the strong adhesive interaction. H-bonds formed with protein molecules are by comparison dispersed, because individual polypeptide chains cannot be sorted to such a density. Another detail that may facilitate this steady interaction is the ability of cholesterol to increase the bending rigidity of bilayer membranes and thus strongly suppresses their thermal undulations (manuscript in preparation). This in turn significantly reduces the entropic repulsion between the approaching surfaces. How these factors are incorporated into our specific case will be a topic of study for years to come.
We propose a two step, receptor-independent APC activation mechanism as follows: 1. MSU forms a spatially tight inter-molecular bonding (possibly hydrogen bond) with cholesterol within the first 30 seconds or less and the binding leads to lipid sorting due to the fluidity of the membrane. The force at this stage by our measurement is no more than 200 pN. 2. Lipid sorting aggregates ITAM-containing receptors which are preferentially segregated into cholesterol/sphingolipid rich regions of lipid rafts. It should be noted that ITAM-containing receptors are common in immune cells. 3. On DCs, Syk kinase is recruited by this accumulation and subsequently turns on PI3K. Syk is a hematopoietic kinase, and its functional association with downstream PI3Ks is unique in DCs or other phagocytes (Greenberg and Grinstein, 2002
), and 4. This activation may trigger activities mediated by PI3Ks that is tightly linked to actin/microfilament movement, and the structural alterations brought about by the cytochalasin sensitive entities lead to auto amplification, permitting further accumulation of signaling molecules at the plasma membrane with higher degrees of phosphorylation, and larger contacts to be formed with the crystal. The force at this stage is in the nN range. Since these two steps are experimentally continuous, they manifest as a two phased force curve change. It is important to note that while Syk and PI3K are required for the binding, it was interesting the ITAM DKO and Src TKO DCs retained the affinity, although in some cases they were lower than WT DCs. We would like to suggest that additional ITAM-containing membrane structures, such as GVPI and WSL-1 (Gardiner et al., 2008
; Lohi and Lehto, 1998
) and Src kinases, such as Src itself and Abi and Csk provided the residual functionality (Hughes, 1996
It is worth further emphasizing that the central hypothesis of our work is the ability of sorted lipid domains to attract ample signaling molecules to a highly localized area on the cell surface. The ITAM containing adaptor molecules, often associated with lipid rafts, are an important intermediate although their identities may not be crucial. They serve to nonspecifically attract Syk kinases to the surface as they do in the receptor-based Syk recruitment. How the specific activation events mediated by this generic kinase recruitment differ from other receptor mediated counterparts need to be analyzed in greater details in the future. From our work, it appears that an event akin to phagocytosis does take place.
An important question yet to be addressed is how "complete" the signaling cascade is in such a lipid based interaction, in terms of common markers of DC activation (CD86, cytokine and phagocytic capacities etc). The pronase treatment is harsh and cells die within 2 hours and before any subsequent activation events can be measured. Any evidence deduced from assays without complete removal of surface proteins should be viewed with suspicion of residual receptor based activities. This report therefore, is incomplete in this regard due to the limits of our technologies as far as blocking all protein based interactions without affecting overall state of cellular viability. Syk mediated PI3K activities are well documented (Aderem and Underhill, 1999
; Greenberg and Grinstein, 2002
; Underhill and Ozinsky, 2002
). However, MSU mediated "complete" DC activation requires Nalp3 inflammasomes (Martinon et al., 2006
). Information directly linking these two categories of signaling events is yet scarce and suggestive. For instance MAPK activation is triggered by Syk (Wan et al., 1996
) which in turn may be linked to inflammasomes and other cellular activations (Shaw et al., 2008
). In our view, our study illustrates the link between surface lipid sorting and Syk kinase activation. Since the Syk/PI3K pathway is suggested to be inflammatory such as in the case of FcR gamma mediated phagocytosis, MSU mediated lipid sorting by inference may be linked to "inflammatory phagocytosis". Associations with other downstream events are a matter of speculation, and they only serve as "readouts" for this study. It is entirely possible that Nalp3 mediated DC activation depends on some protein-based recognition of the crystal surface, and the lipid binding merely serves as a pre-requisite for other events to take place.
This report provides an explanation of how particulate materials, even without any known mechanism, may trigger strong DC activation, in cases independent of opsonization and antibody binding (). Broadly speaking, there is no reason to rule out this class of cellular interaction in other signaling settings, including those with receptors, as ligation forces/membrane contact are integral parts of signal exchange. It was a surprise finding that DCs showed ever-increasing affinity to a solid structure without its surface proteins (). Being adoptive to the enormous variances of binding targets and being independent of the surface proteins, our results seem to imply that outer membrane undergoes some form of lipid sorting/reconfiguration to maximize the binding affinity following each contact, although this suggestion needs intense scrutiny. How membrane lipids engage diverse solid surfaces with increasing binding strength as a consequence of intracellular signaling is puzzling and will be a topic of future studies.
Our work has several implications. First, crystal-induced diseases, such as gout and calcium salt deposition, are historic topics of medicine that so far have not been explained at molecular levels. Our work provides a testable hypothesis for that line of research. Second, phagocytosis has long been regarded as a receptor-based event (Stuart and Ezekowitz, 2008
). That reasoning at times falls short in explaining why nearly all particulate substances including man-made synthetic materials, once entering tissues, trigger strong innate recognition. Our work puts this important topic under new light. Lastly, it should be pointed out that some of the best known adjuvants are crystals. Alum, the only FDA approved human vaccine enhancer, is a mixture of aluminum salt crystals. Despite its extensive usage in human and significant research on its cellular modulation (Jordan et al., 2004
), we still do not have any understanding on its facilitating effect on antigen uptake. Membrane lipid interaction is a clear candidate for its mechanism. Overall, our work suggests that innate recognition has an extension unrelated to TLR/PAMPs.
Beyond proposing a new mechanism for Syk mediated DC activation, several lines of techniques employed in this work will likely have value for research in immunology. AFM based force measurements (cell hesion) are a valid alternative to conventional imaging analysis and other readings of cellular activation, as they obtain quantitative binding affinities in real time and the contacts are reversible on the fly, unlike stationary readings of imaging methods. They also have the advantage of efficiency as optimized experiments can be carried out in hours or minutes rather than days, and in theory, with one cell. The nickel mesh based trapping enables readings for non-adherent cells, which to our knowledge, has never been attempted before. We therefore believe these biophysical approaches will add functional tools for immunologic research and beyond.