Calreticulin has been shown to bind the collagenous tails of members of the collectin family of pattern recognition molecules. Its presence on cell surfaces then, implies a potential role for recognition of the collectins, and anything they may have bound via their globular head groups. In addition to foreign organisms, collectins bind to apoptotic cells and can mediate their uptake into phagocytic cells via this calreticulin-mediated recognition of the collagenous domains. As a non-transmembrane protein, calreticulin cannot transduce intracellular signals by itself but appears to use for this uptake and removal, a partner, CD91, which does exhibit appropriate intracellular signaling domains and function.

Introduction

The process of apoptosis, or programmed cell death, leads to numerous alterations in and on the cell that ultimately result in its clearance from the tissue. In simple terms, apoptosis involves steps that lead to destruction of the nuclear replicative apparatus as well as surface changes that allow the cell to be recognized and removed. While the signaling pathways that initiate these changes are complex, the end result under normal circumstances, is removal of the cell (Fig. 15.1). Studies over the last twenty years have shown this removal to represent a phagocytic event and have begun to define the receptors and mechanisms involved, as well as the usual consequences of the event. Some recent reviews on these subjects include.¹⁷

Figure 1

A macrophage ingesting apoprotic neutrophils. The electron micrograph was taken from a resolving pulmonary inflammatory response and depicts an apoptotic neutrophil in contact with the macrophage surface and apoptotic debris within phagosomes.

Tissues in metazoan animals do not remain static after development but are increasingly recognized to undergo turnover and remodeling, even in the adult. While this capacity may be more limited in mammals than, for example, amphibians, nevertheless, cell death, removal and replacement appear to be generally critical elements in normal tissue homeostasis. Key to these processes is the quiet, non-inflammatory and potentially regenerative nature of apoptosis and removal, which usually occurs in situ on a cell by cell basis. Thus, while professional phagocytes such as macrophages and immature dendritic cells are certainly most efficient at recognizing and internalizing apoptotic cells, the majority of cell types in the body seem capable of performing this function—including those from endodermal, epidermal or mesodermal origin. Removal is efficient and, usually, sporadic. This means that it has generally escaped notice and even now is very hard to identify when examination of tissues is limited to only snapshots in time. A marked example of the efficacy of this process is the normal removal of circulating neutrophils which has been estimated to encompass more than twice the total numbers within the circulation every 24 hours.⁸ Massive remodeling, apoptosis and cell removal occurs during embryonic development, metamorphosis in amphibians, involution of the mammary gland or uterus after lactation or parturition in mammals, during the establishment and maintenance of the immune system and after every inflammatory response.

Clear, local examples of abnormal apoptotic cell removal are only beginning to be appreciated in mammals, likely because of significant redundancies in the removal processes. On the other hand, genetic studies in the nematode Caenorhabditis elegans have led to the identification of at least seven genes that contribute to apoptotic cell clearance. Most of these represent molecules now known to participate in signaling pathways involved in the ingestion of apoptotic cells. Only one of the identified genes has been implicated as a potential recognition molecule for apoptotic cells. This has led to speculation that even in this group of animals, there may be substantial redundancy in the receptors involved in specifically recognizing apoptotic cells with less redundancy in the signaling. It is this apoptotic cell recognition where calreticulin comes in, but, in an indirect fashion i.e., via recognition and binding of the collectin family of molecules to apoptotic cells, followed then by their ability to interact with cell surface calreticulin.

Recognition that calreticulin might play a role in apoptotic cell recognition and clearance comes from a concatenation of initially disparate observations. In 1997 Korb et al reported the binding of C1q to apoptotic keratinocytes and showed that this was localized to membrane blebs.⁹ This led us to question the possible role of C1q and, indeed other members of the broadly defined collectin family of molecules, in apoptotic cell recognition. From a totally different direction, Botto, Walport and colleagues were investigating the potential links between C1q levels and autoimmune disease, particularly Systemic lupus erythematosus, SLE. They generated a C1qa^-/- mouse and indeed showed it to have autoimmune abnormalities. In addition, they presented evidence for increased numbers of apoptotic cells in the kidneys of these animals¹⁰—a phenomenon that was entirely in keeping with defective apoptotic cell clearance. In support of this concept, deliberate instillation of apoptotic cells into the peritoneum of C1q^-/- animals revealed a clearance defect that could be restored by re-addition of C1q.¹¹ Paradoxically, later studies have suggested that although C1q may participate in apoptotic cell clearance at a number of sites in the body, keratinocyte removal itself may not use this mechanism¹² since this latter was unaffected in the knockout mice.

From the other direction, a series of studies over many years had led to the recognition that one of the key receptors for C1q on cells was in fact calreticulin. Identification of cellular C1q receptors has had a long, complicated history and has generated numerous potential candidates. Ghebrehiwet and colleagues first characterized a binding molecule they called cC1qR,^13,14 because it appeared to recognize the collagenous tails of the C1q (see also ref. 15), in contrast to globular head binding “receptors” they called gC1qR. This cC1qR was later shown to be cell surface calreticulin.^16,17 The cC1qR (and thus, calreticulin) binds not only C1q, but also surfactant protein A (SP-A), mannose binding lectin (MBL) and conglutinin, i.e., members of the collectin family. It also interacts with another member of this family, surfactant protein D (SPD) (Eggleton personal communication and ref. 20). In fact a binding domain on calreticulin was identified that could be blocked with specific peptides.¹⁸

Putting this together led to experiments showing that C1q participated in the uptake of apoptotic cells by human monocyte-derived macrophages in vitro by a mechanism that could be blocked by anti-calreticulin antibodies.¹⁹ This in turn directed attention to the possibility that other members of the collectin family could also mediate apoptotic cell uptake using the same mechanisms. Calreticulin-dependent uptake of apoptotic cells was shown in vitro for MBL¹⁹ well as for SP-A (ref. 20 and see also ref. 21) and SP-D.²⁰ In addition, SP-D^-/- mice exhibited an apoptotic cell clearance defect in the lungs.²⁰

So much for the history, now let us examine the mechanisms involved.

The Collectin Family of Pattern Recognition, Innate Immune System, Molecules

Collectins are a family of multimeric, multifunctional, pattern-recognition molecules with a common three dimensional structure (see for example refs. 22–30). Globular head groups containing the recognition units are linked to long, remarkably similar, collagen-like tails by a hinge region. True collectins (SP-A, SP-D, MBL, conglutinin etc) exhibit C lectin activity in their heads—hence the name. C1q is similar in structure but has no lectin function. Unlike the others, the collagen tails here comprise three slightly different chains in contrast to the homo-trimers of the true collectins. C1q used to be thought different too because of its ability to interact with C1r and C1s to initiate the classical complement pathway. More recently a similar activity has been shown for MBL interacting with its own activation components.^22,28,31,32 For simplicity, therefore, we will consider these together as members of the collectin family.

C1q, MBL, SP-A and SP-D all have been shown to bind to the surface of apoptotic cells. However, the nature of this binding is not simple. Surprisingly, very little is known about the surface changes on cells that occur during apoptosis. Loss of the normal membrane phospholipid asymmetry is the most well characterized change and results in exposure of normally inner leaflet phosphatidylserine (PS).^33,34 The exact mechanisms are not understood in detail but the process seems to be driven by an increase in bidirectional phospholipid movement (scrambling) accompanied by a block of the normally rectifying aminophospholipid translocase so that any PS that reaches the outer leaflet is not returned to the inside.^35,36

In addition to carbohydrates and proteins, each of these four collectin family proteins has been shown to bind phospholipids, including in some cases, phosphatidylserine itself (see reviews noted above). Whether PS is indeed the ligand for one or more of the collectin family remains to be determined. On the other hand, altered glycosylation of the apoptotic cell surface may also contribute. Uptake of apoptotic cells by the liver was shown to involve altered carbohydrates^37,38 and the MBL binding to apoptotic Jurkats was shown susceptible to mannosidase (unpublished observations).

In our hands, only MBL exhibited a marked increase in total binding to the surface of apoptotic cells compared with their viable counterparts^19,20—the others bound to both viable and apoptotic cells. This should be contrasted with data reported by others for SP-A.²¹ Whether the contrasting results reflect different cells or assays, the point is that the globular head groups do appear capable of interacting with ligands on normal cells and, likely have significant roles to play as a consequence. One example would be the increasing recognition that SP-A exhibits a potent anti-inflammatory effect in the lungs presumably as a function of stimulation by its globular head group of specific surface molecules (“receptors”). When initially encountered in our studies of C1q binding this lack of quantitative difference between binding to viable versus apoptotic cells posed a conceptual problem in trying to understand a potential role for C1q in apoptotic cell uptake. However, careful examination of the distribution of binding showed that the C1q attached to apoptotic cells in a highly localized, aggregated fashion. This fits the original description of C1q binding to keratinocyte blebs⁹ and in later studies, by its globular heads, to apoptotic blebs on endothelial cells.³⁹ MBL, SP-A and SP-D all attach to apoptotic cells in a similar, highly localized fashion.^19,20

Two important points emerge from these observations. For one, the data support primary interaction of the collectin family with apoptotic cells via their globular heads, thereby leaving their collagenous tails for engagement of the phagocyte. Secondly, the localized, aggregated, distribution may have important implications for modes of signaling to those phagocytes.

Collectin Interaction with Cell Surface Calreticulin

This subject is treated at length in refs. 16–17 and 40 in which the specific sites on the calreticulin that interact with the collagen-like tails of the collectins are defined and discussed. In Chapters 5 and 7 as well as in ref. 40 are considered the complex issues of calreticulin expression on the surface of cells. At this point in time, a wide variety of cells can be shown to exhibit this molecule on the surface. How the molecule gets there is a subject of some discussion. An increasing consensus seems to be emerging that it is actively transported to the membrane surface (see for example ref. 41) although the mechanisms involved are not yet clear. Calreticulin's KDEL, endoplasmic reticulum retrieval sequence, was thought to imply an alternative mechanism. For example, any cells dying in a culture or cell isolate might be expected to release some calreticulin from the ER which could then bind to putative surface ligands for this molecule on the surrounding cells. A number of arguments have been mounted against this possibility, including the observed uniformity of calreticulin amount from cell to cell. It is worth noting that gp96, another ER, KDEL-containing chaperone, is also becoming recognized as a widely distributed cell surface protein,⁴² raising the same types of questions regarding how it gets there. The comparison is made even more relevant by the observation that both calreticulin and gp96 bind to CD91 (see below). An intriguing new possibility for mechanism of surface expression comes from the suggestion that the endoplasmic reticulum can exhibit transient connections with the plasma membrane, particularly during phagocytic events (DesJardins, M, personal communication). On the other hand, while the origin of cell-surface calreticulin is important to understand, how it got there may make no difference to its potential role in collectin binding and apoptotic cell removal.

Binding of collectin-coated particles or apoptotic cells was blocked by pretreatment with anti-calreticulin antibodies. In a further series of studies, the role of cell membrane calreticulin was demonstrated by using the receptor modulation approach pioneered by Silverstein.⁴³ Plating macrophages on ligands or antibody for a “receptor” (in this case calreticulin) leads to accumulation or sweeping of that receptor to the underneath of the cell leaving the upper surface selectively depleted of that receptor and any molecules it might be closely associated with.^19,20 In our case, we confirmed the efficacy of the modulation by probing the upper surface for remaining calreticulin with erythrocytes coated with anti-calreticulin antibodies. Plating macrophages onto any of the collectin family members led to selective depletion of calreticulin from the upper surface. Furthermore, plating on isolated collagenous tails from either C1q or SP-A had the same effect, supporting the orientation proposed, namely that the collectins bind the apoptotic cells by their globular heads and interact with calreticulin on the phagocytic cell via their tails. In all of these modulation experiments, removal of upper surface calreticulin was also accompanied by decreased uptake of apoptotic cells.

Interaction of Calreticulin with CD91/LRP As a Mechanism for Initiating Apoptotic Cell Internalization

As a non-transmembrane surface molecule, calreticulin presumably needs a partner for mediating intracellular signaling and internalization. A potential candidate for this has arisen in studies by Srivastava's group.^42,44,45 They have been investigating the role of heat shock proteins (chaperones) in the induction of immune responses and were also searching for a transmembrane signaling molecule (receptor) that would bind and internalize heat shock proteins and any peptides that may be attached to them. The prime candidate turned out to be CD91 (Fig. 15.2). Since calreticulin was one of the chaperones examined by them for peptide presentation, their demonstration that calreticulin bound to CD91 opened the potential that this molecule was responsible for collectin/calreticulin signaling and ultimately, for uptake of apoptotic cells.

Figure 2

Scheme showing the potential orientation of collectins, calreticulin and CD91/LRP in the recognition and uptake of apoptotic cells.

CD91, also known as LDL receptor-related protein (LRP) and as one of the α-2 macroglobulin receptors (α2MR), is a large receptor comprising two non-covalently associated chains. The transmembrane chain exhibits signaling domains in its cytoplasmic region that seem to be involved in one key function of the molecule, namely internalization of bound ligands. The extracellular α-chain contains numerous potential binding sites for a multitude of proteins. These are summarized in the recent review by Hertz⁴⁶ and include LDL, α2 macroglobulin, heat shock proteins, calreticulin, glycosaminoglycans and many more. Since many of these proteins themselves can bind other molecules (peptides, proteins, lipids etc) the whole system represents a potentially diverse, highly amplified mechanism for uptake and clearance—see Figure 15.3.

Figure 3

CD91/LRP as a clearance receptor. A large number of molecules interact with CD91/LRP and many of these in turn are promiscuous in their own ligands.

To address the involvement of CD91 in apoptotic cell removal, similar approaches were used^19,20 as previously outlined for calreticulin. Thus, for C1q, MBL, SP-A and SP-D attached to erythrocytes, anti-CD91 effectively blocked uptake into macrophages. Coating the erythrocytes with anti-calreticulin, calreticulin itself or α2-macroglobulin as an alternative CD91 ligand, also initiated uptake in an anti-CD91 inhibitable fashion. Plating macrophages onto surfaces coated with calreticulin, α2-macroglobulin, collectins or collectin collagenous tails all modulated CD91 from the upper surface of the cells as determined by diminished attachment and internalization of erythrocytes with attached anti-CD91 antibody. Finally calreticulin and CD91 were seen to co-localize on macrophage surfaces by immunofluorescence.

From these observations it is suggested that CD91 may act as a general candidate for internalization not only for lipoproteins, a2-macroglobulin, heat shock proteins and the like, but also for calreticulin and molecules that bind to this, including the collectins. This pathway would presumably apply not only to opsonization of apoptotic cells by the collectins but also to clearance of cell debris and foreign organisms. In this regard it is noteworthy that C1q (via its globular head groups) is an excellent ligand for mitochondria—interacting with not only mitochondrial proteins but also lipids such as cardiolipin.⁴⁷⁴⁹ When one remembers the likely evolutionary origin of mitochondria, this recognition becomes even more logical. We suspect in addition that the other collectins, through their extensive lipid and carbohydrate recognition properties will also be important in cell debris uptake. This potentially makes CD91 the ultimate in removal of dead cells and their components.

As mentioned above, much information on apoptotic cell clearance has come from genetic studies in C. elegans. This animal does contain a molecule homologous with mammalian calreticulin⁵⁰ and it has been suggested to play a role in certain forms of cell death, but more from its calcium sequestering activity than any implication of possible cell surface properties.⁵¹ The original genetic screens did not show association of calreticulin with apoptotic cell removal and reports of calreticulin RNAi experiments in the worm were not accompanied by suggestions of abnormalities in cell clearance.⁵¹

CED-1 on the other hand may represent a more interesting story. Its recent cloning suggested that, unlike the other nematode molecules mentioned above as part of signal cascades, it might serve as a cell surface receptor.⁵² Initial attempts to find a mammalian equivalent (using the whole gene as template) led to a suggested relationship with an endothelial cell scavenger receptor, SREC). Unfortunately the putative signaling domains did not match well. On the other hand, a search of the mammalian databases based on the intracellular domain of CED-1 led to demonstration of significant homology with the intracellular domain of CD91.⁵³ In fact the worm and mammalian domains serve equally to bind to CED6 (or its mammalian equivalent, GULP), an adapter molecule shown to be involved in apoptotic cell uptake in both C. elegans and mammalian systems.⁵⁴

The dissimilarity between CD91 and CED-1 in their extracellular domains might suggest that, although the signaling domains are related, the organisms have developed quite different approaches to actually recognizing the apoptotic cells surface. In the case of CED-1 this may be direct binding but in mammalian systems, via a complex set of bridging molecules including calreticulin and the collectins. On the other hand one might ask whether bridging molecules linking apoptotic cells to CED-1 might exist in C. elegans and/or whether CD91 has an additional ability to directly recognize surfaces structures on apoptotic cells.

Mechanisms of Uptake and Signaling

This subject has been discussed at some length in recent papers and reviews (e.g., refs. 1,2,55,56). Current data are in keeping with a mechanism that is akin to stimulated macropinocytosis, not unlike processes involved in uptake of pathogenic Salmonella into epithelial cells⁵⁷ (Fig. 15.4). Stimulated macropinocytosis can be distinguished from phagocytosis induced through the Fc receptor which involves a “zipper” mechanism and results in close apposition between phagosomal membranes and the ingested particle. Macropinocytosis on the other hand leads to more spacious phagosomes and concurrent ingestion of extracellular fluid. It can also result in bystander uptake, i.e., stimulation of macropinocytosis by receptor ligation can mediate ingestion of any molecule or particle previously attached to the membrane in the local vicinity. Direct ligation of either calreticulin or CD91 on the cell can induce macropinocytosis and the ingestion of water soluble dye from the medium.¹⁹

Figure 4

Proposed two-step recognition and uptake process for apoptotic cells. Tethering ligands cooperate with internalization receptors to optimize ingestion of these large particles. The uptake mechanism is proposed to be a form of stimulated macropinocytosis. (more...)

Macropinocytosis also appears to be a key mechanism by which materials are taken up into immature dendritic cells, although here, the process seems to be constituitively turned on. Supply of calreticulin to such dendritic cells leads to uptake, processing and presentation of any associated peptides. Thus, addition of calreticulin isolated from different tumors to dendritic cells allowed them to induce tumor specific responses to appropriate T cells.⁵⁸ This type of experiment further supports the potential involvement of calreticulin-mediated uptake, not only in clearance of intracellular materials but also in participation in the interface between the innate and adaptive immune responses.

Although we do not know in detail the signaling pathways involved in uptake induced by collectin/calreticulin/CD91, there does seem to be a common step in the requirement for rac-1 (equivalent to C. elegans CED-10) as seen in both apoptotic cell ingestion and macropinocytosis in general. As suggested above, GULP or CED-6 may be a necessary adaptor protein, but how specific this is to CD91 is not yet known. Since the collectins bind to apoptotic cells in a localized, aggregated fashion, and are ineffective when attached more diffusely to non-apoptotic cells, we have hypothesized that the collagenous tails also need to be aggregated in order to deliver an appropriate signal to the cell (see Fig. 5). This could mean the calreticulin and/or the CD91 must be crosslinked to initiate signaling or merely reflection of an avidity issue. Developing our understanding of the signaling pathways will be important and we suspect that they will be different from those involved in more classical phagocytic mechanisms. The very broad evolutionary distribution of these apoptotic cell and macropinocytotic uptake processes as well as their presence in most cell types suggest a strikingly “primitive” process.

Figure 5

Collectins are innate immune system molecules that recognize and bind foreign materials and also apoptotic cells nd cell debris through their golbular heads. This presents their collagenous tails to calreticulin/CD91 complexes on phagocytes to initiate (more...)

Conclusions

The data support a crucial role for cell-surface calreticulin in recognition and removal of apoptotic cells, cell debris and intracellular constituents. Via the action of the collectin family of “defense collagens” the spectrum of recognized structures is broadened enormously. The collectin family of molecules has long been implicated in enhancing phagocytosis, in part by acting as opsonins (e.g., refs. 59,60). Since the time of Metchnikov, science approached phagocytosis from an infectious and protective bias. While in no way diminishing the importance in host defense, newer information on so-called “innate immune system molecules”, including the collectins, suggests that a clear distinction between recognition of “foreign” and “self ” in these systems may be significantly blurred. Removal of autologous damaged cells and debris is a critically important clean-up mechanism leading to restoration of normal structure and function. Beyond even this function, however, we are now coming to recognize that cells are produced in the body in excess, both during development and in the adult. These excess cells, if not “used” or after they have performed their normal functions, are efficiently removed. As discussed above, we hypothesize an important role for cell surface calreticulin in this process. Thus, it seems reasonable to suggest that some of the profound developmental abnormalities seen in calreticulin^-/- mice (e.g., ref. 61) may be due not only to its absence from the endoplasmic reticulum, but also because of abnormal apoptotic cell clearance. From an evolutionary perspective, parsimonious utilization of similar genes and processes for recognition and uptake of unwanted cells, dead cells and cell debris as well as for foreign organisms makes sense. The multiple roles in metazoa for calreticulin makes this a fascinating molecule, not only from the functional perspective but also in this evolutionary context. As already suggested, it would seem well worth while to question directly its role in direct or indirect recognition of dying cells and their products, even in organisms as diverse as invertebrates and plants.

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