Collectins are proteins of the innate immune system involved in pattern recognition and opsonization of foreign particles. Recent evidence suggests a role for other members of the collectin family, specifically SP-A, in the clearance of apoptotic cells 105
Here, we propose that a member of this family, MBL, and a close relative, C1q, bind to apoptotic cells and initiate their uptake into macrophages. MBL was found to bind primarily to apoptotic cells and showed little evidence of interaction with viable cells. C1q has been reported to bind to the surface of a number of different cell types 55106107108109110111112
and here was shown to attach to both viable and apoptotic Jurkat T cells. Despite this binding, only apoptotic cells are engulfed by macrophages. However, a careful examination of the of C1q binding revealed a clustered distribution on apoptotic cells. This was similar to C1q binding shown previously on apoptotic keratinocytes 16
or vascular endothelial cells 17
. On viable Jurkat T cells, C1q binding was diffuse. Reexamination of MBL attachment suggested a similar distribution on apoptotic cells and implicates significantly altered, but to date uncharacterized, surface structures on the membrane blebs of apoptotic cells.
The multifunctional properties and binding sites on MBL and C1q make the orientation of their attachment to the apoptotic cells difficult to clearly identify. The data presented herein suggest that for productive initiation of apoptotic cell engulfment, C1q and MBL bind to the apoptotic cells by their globular domains and to the phagocyte through their collagen-like tails.
The globular head regions of the collectins are known to be involved in pattern recognition and binding and it is suggested that they interact with the ligands on the surface of the apoptotic cells. A report of increased mannose expression on apoptotic hepatocytes and lymphocytes is noteworthy in this regard 23113114115
. It can be inferred from the data that after binding and/or aggregation of the collectins on the apoptotic cell surface, their collagenous tails interact with receptor on the surface of the phagocyte. It seems likely that this interaction is low affinity and requires significant aggregation to mediate signaling and uptake.
Complement components have been shown to bind to and opsonize apoptotic cells 46116117
. Preincubation of the apoptotic cells with C1q or MBL resulted in some increased uptake, suggesting opsonization of the target cells (viable cells, regardless of pretreatment, were not engulfed by macrophages). However, significant ingestion was seen even in the absence of prior opsonization and this uptake was markedly reduced in the presence of anti-C1q or anti-MBL. The data suggest involvement of the two collectin family members even in the absence of added protein and imply production by the macrophages. Macrophages are known to synthesize and secrete C1q 3
and preliminary experiments suggest they may produce MBL as well (data not shown). In vivo, these two proteins will likely be supplied from plasma, and addition of serum to the macrophage uptake system did increase the ingestion of apoptotic, but not viable, cells (data not shown). On the other hand, not all of the uptake is expected to occur via these C1q or MBL processes and, as with most attempts to identify apoptotic uptake receptors by ligand blockade 19118119
, inhibition was never complete.
Preincubation of the phagocytes with the C1q or MBL before addition of apoptotic targets resulted in a reduced phagocytic index. Plating macrophages onto wells coated with C1q or MBL before adding target apoptotic cells yielded the same result, suggested to reflect modulation of the phagocytic receptors. The ability of isolated C1q tails to similarly modulate the functional receptor further supports the concept that it is this portion of the protein that is primarily recognized by the phagocytes.
The redundancy of recognition and uptake mechanisms driven by multiple ligands and receptors makes detailed analysis of any one of these particularly complex. To simplify the examination of C1q- or MBL-induced uptake, a single ligand-coated particle was used. Human erythrocytes were coated with biotin (Eb), and a sandwich was created with avidin and the biotinylated ligand of choice (Ebab-X). These Ebab cells have a number of advantages, including the ability to distinguish binding to the phagocyte surface from engulfment as well as complete absence of binding and engulfment with control erythrocytes. This system demonstrated that Ebab coated with C1q, MBL, or purified C1q collagenous tails both bound to, and were directly taken up into, macrophages.
Several receptors have been postulated to play a role in C1q- or collectin-mediated uptake of particles by macrophages or other cell types. These include CR1, CR3, C1qRp, and cC1qR 50596061656997
. Of these, only antibodies to the cC1qR, or CRT, were found to be effective in inhibiting phagocytosis of apoptotic cells by HMDMs. CRT has been shown by several groups to bind to the collagenous tail regions of MBL, SP-A, conglutinin, and C1q 174
or SPD (unpublished data). In fact, all anti-CRT antibodies tested were effective in inhibiting the apoptotic cell engulfment, including polyclonal antibodies produced against the N-domain of the protein, which contains the region shown to bind C1q collagenous tails 78120
. The efficacy of antibody against the C-domain may result from blockade of collectin or C1q tail binding or perhaps as a consequence of receptor modulation. Antibodies to a wide variety of alternative receptors or binding sites were inactive. Ingestion of Ebab
coated with the purified tail region of C1q, which shares functional homology with the collagenous tail region of MBL and other collectins, was also inhibited by antibody against CRT.
Although CRT serves a number of critical functions within the endoplasmic reticulum 7893
, it has also been shown to be expressed on the surface of many cell types, including human macrophages 49848696121122
. Although cultured HMDMs display much phenotypic heterogeneity 123
, a large portion of the population tested demonstrated surface expression of CRT. However, the mode of access to the cell surface and the mechanism of retention at this site is not at all clear — it may be transported from the endoplasmic reticulum or bind to the cell surface from the extracellular milieu 98124
. However, either route could readily allow for attachment of C1q or MBL collagenous tails. On the other hand, as CRT is not a transmembrane protein, actual signaling for uptake of apoptotic cells must be presumed to involve some other membrane structure recruited for the process.
A recent report has suggested that CRT may bind to the multifunctional receptor, CD91 98
; we show colocalization of CRT with CD91 on the HMDM cell surface ( A). This molecule, also known as the α2m receptor or LRP, is a type 1, 600-kD transmembrane protein made up of an α and a β chain which are noncovalently, but tightly attached. Importantly, the receptor has a 100 amino acid cytoplasm tail that contains two NPXY endocytosis signal sequences 125
. It shares sequence homology in the cytoplasmic tail with other receptors used for endocytosis, including the newly cloned ced-1
gene from Caenorhabiditis elegans
known to play a role in the phagocytosis of apoptotic cells in these organisms 126127
. CD91 is found on the surface of HMDMs (among many other cell types) and appeared to be a prime candidate for signaling for apoptotic cell uptake into these cells. Accordingly, antibodies to CD91 were found effective in inhibiting uptake of apoptotic cells, or Ebab
coated with MBL, C1q, or C1q collagenous tails in exactly the same fashion as shown for anti-CRT antibodies.
The hypothesis, then, is that CRT acts to bind the collagenous tails of C1q and MBL and then signals for uptake due to its interaction with CD91. To support this likelihood, a number of different experiments were performed. Importantly, CD91 could be modulated from the upper surface of macrophages by plating the cells onto wells coated with a ligand for this molecule, α2m. Not only were the upper macrophage surfaces depleted of CD91 (shown by reduction in attachment of Ebab-anti-CD91) but also in CRT (shown by reduction of attachment of anti-CRT Ebab). Plating macrophages onto wells coated with control proteins did not modulate these receptors. Macrophages plated on either α2M or C1q tails showed diminished binding or uptake of Ebab coated with MBL, C1q, C1q tails, or α2m (Ebab-α2m were also shown to be bound and ingested by the macrophages). These macrophages were still able to bind and ingest Ebab coated with anti-Fc receptor antibody (anti-CD32 Ebab). Finally, modulation of the macrophage receptors by plating the macrophages onto wells coated with either α2m or C1q tails blocked uptake of apoptotic cells. It seems reasonable to suggest that this demonstration of a role for CD91 in collectin family mediated ingestion of apoptotic cells may be extended to systems in which these innate immune system, pattern recognition molecules participate in recognition and removal of foreign organisms and cell debris.
We have recently provided evidence that uptake of apoptotic cells through the phosphatidylserine receptor is mediated by a process of macropinocytosis (unpublished data). Data presented herein support the ability of CRT/CD91 ligation by C1q or MBL to induce macropinocytosis and that this is the mechanism by which these collectin family members initiate uptake of apoptotic cells. Thus anti-CRT and anti-CD91 antibodies were shown to directly stimulate macropinocytosis of the water soluble dye Lucifer Yellow. Inclusion of this dye during uptake of apoptotic cells resulted in simultaneous ingestion of apoptotic cell and dye (data not shown). Similarly in these studies, Ebab coated with anti-CRT were shown to be ingested in a fashion that was accompanied by Lucifer Yellow uptake into the same compartment. This could be contrasted with uptake of Ebab via the Fc receptor. Finally, CRT and CD91 were shown capable of mediating bystander uptake of attached cells into macrophages. Erythrocytes coated with an anti-CD36 antibody were found to attach to the macrophage surface without subsequent ingestion ( C). Antibody ligation of either surface CRT or CD91 in this system was shown to drive engulfment of the attached E ( B). The growth factor M-CSF had the same effect on attached erythrocytes (not shown). Antibodies to other surface molecules, including anti-FcR, had no effect on uptake. In these systems involving stimulation of CRT and CD91 with antibody, crosslinking the primary antibody led to an enhanced effect. The multivalency of C1q and the collectins, as well as their localized binding to the apoptotic cell surface, is in keeping with this concept. Similarly, CD91 mediates endocytosis of α2m/proteinase complexes most effectively when crosslinked ( A).
The ability of CRT and CD91 ligation to initiate macropinocytosis, therefore, suggests a mechanism for uptake of apoptotic cells. It may also implicate macropinocytosis, as well as CD91 involvement as an explanation for the known ability of collectins to enhance phagocytosis of foreign particles and organisms 152110
. We also suspect their similar participation in recognition and uptake of cell debris during removal of damaged and necrotic cells. C1q avidly binds via its globular head groups to free mitochondria 128129
. Pattern recognition by collectin family members of the innate immune system could, by engaging CRT and then CD91, provide significantly enhanced signaling potential, not only quantitatively, but also, because of the broad specificity of their globular head recognition domains, to involve a very wide variety of ligands. Finally, CD91 engagement has been shown to initiate maturation of immature, dendritic cells 124
and enhancement of antigen presentation 130
C1q and MBL bind to and facilitate the ingestion of apoptotic cells by human macrophages. The structurally and functionally similar collagenous tails of C1q and MBL bind to CRT, which, in turn is bound to CD91 on the macrophage cell surface. Engagement of this receptor initiates a process of macropinocytosis and eventual engulfment of the apoptotic cell. This mechanism, which involves pattern recognition molecules of the innate immune system, a multifunctional cellular protein, and an evolutionarily conserved clearance receptor, may be an ancient method the body has evolved for ridding itself of a potentially harmful source of self-antigen.