C1q Receptors: Regulating Specific Functions of Phagocytic Cells

Immunobiol., vol. 199, pp. 250-264 (1998)

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA

C1q Receptors: Regulating Specific Functions of Phagocytic Cells 1 ANDREA

J. TENNER

Abstract A Clq receptor that upregulates the phagocytic capacity of professional phagocytes, ClqRp , has been identified, and its primary structure determined by cDNA cloning and sequencing. Monoclonal antibodies that immunoprecipitate this 126,000 Mr polypeptide inhibit the enhancement of phagocytosis triggered not only by C1q but also by mannose binding lectin (MEL) and pulmonary surfactant protein A (SPA) providing critical evidence that this polypeptide is a functional receptor or component of the receptor that mediates this enhancement of phagocytosis. The amino acid sequence, deduced from the cloned cDNA coding for this receptor, indicates that this surface glycoprotein receptor is a novel type I membrane protein of 631 amino acid containing a region homologous to C-type lectin carbohydrate recognition domains,S EGF-like domains, a single transmembrane domain and a 47 amino acid intracellular domain. Expression of this receptor is limited to cells of myeloid origin, platelets and endothelial cells, consistent with a relatively selective function, and making it an attractive candidate for therapeutic modulation of function. A distinct C1q receptor that triggers superoxide in polymorphonuclear leukocytes has been functionally characterized and designated as C1qR02-. Thus, the accumulated data that will be summarized here demonstrate that there are at least two Clq receptor/receptor complexes (ClqRp and C1qRoz-), each triggering distinct cellular responses, that multiple C1q receptors can be expressed on the same, as well as on different, cell types, and that at least one C1q receptor, C1qRp, is capable of responding to multiple ligands.

Clq, a 460,000 Dalton glycoprotein, is a hexamer of 6 identical subunits, each of which contains three distinct polypeptide chains (1). It is one of a few proteins that has an extended collagen-like sequence (repeating Gly-X-Y triplets, where X is often proline and Y is often hydroxylysine or hydroxyproline) contiguous with a noncollagen-like sequence (2). The noncollagen-like domain of Clq mediates the recognition of many, but not all (3-5), activators of Cl, thereby leading to the initiation of the classical complement cascade. Clr and Cls, the classical pathway serine proteases, assemble around the collagen-like region of Abbreviations: CCP = classical complement pathway; Clq-CLP = collagen-like fragment of C1q; CRl = complement receptor (CD35); FeR = Fc receptor; HSA == human serum albumin; MBL == mannose binding lectin; MoAb = monoclonal antibody; SPA = pulmonary surfactant protein A °1998 by Gustav Fischer Verlag

Clq receptor . 251

C1q (3), presumably near the "kink" region such that some portion of amino acids 14-26 of the A chain are capable of binding nonimmunoglobulin-containing activators (4, 5). C1q has also be reported to bind to a number of different cell types to trigger a variety of cellular responses (Table 1). The isolation and characterization of the cell surface «receptor» molecules responsible for mediating these cellular functions has been an active area of investigation in several laboratories. A cellular receptor is generally defined as a molecule to which a specific ligand binds in a saturable and reversible manner, resulting in the generation of a cellular response (6). This last criteria, induction of function, distinguishes a receptor from a simple binding protein which can be defined as a protein with the single criteria of specifically binding another molecule under a given set of conditions. Another class of molecular interactions, generally thought of as distinct from that of receptor-ligand interaction, is that of a biological response/activity modifier, such as an inhibitor or other regulatory protein that binds another molecule to regulate its activity. The regulatory protein, clusterin, would be an example of a complement system biological activity modifier (7). Thus, to designate a protein as a cell surface receptor for a particular ligand, it must be demonstrated that the protein is on the surface of a cell and that its interaction with a ligand results in a cellular response. It is now quite clear that many receptors are complexes of multiple polypeptide chains (e.g., T cell receptor, integrins, cytokine receptors). Table 1. Cellular functions triggered by Clq. Function

Cell Type

Ref.

Enhancement of phagocytosis

Monocyteslmacrophages Neutrophils Endothelial cells Fibroblasts

(15, 16,53-55) (17) (56)

Enhances binding of immune complexes

Mesangial cells Endothelial cells

(57)

(54)

(58,59)

Cellular cytotoxicity

Monocytes

(60-62)

Stimulates oxidative metabolism

Neutrophils Vascular Smooth Muscle

(47-49) (63)

Enhances microbial killing

Monocytes Neutrophils Eosinophils

(48)

Induces expression of adhesion molecules

Endothelial cells Platelet

(58) (65)

Enhances secretion of immunoglobulin

B cells

(66,67)

Stimulates CaH-activated K+ channels & Initiates chemotaxis

Fibroblasts

(68)

Triggers DNA synthesis

Fibroblasts

(69)

Enhances antibody-mediated apoptosis

Mesangial cells

(70)

(53)

(64)

252 . A. J. TENNER Thus, it is possible that a given polypeptide could be a component of a receptor without containing the interaction site involved in the actual binding of the ligand. An example of this type of receptor is the IL-6 receptor, in which gp130 is the signaling component while the 80 kD subunit binds the ligand (8). Both subunits are necessary elements of the receptor complex. Several laboratories have reported the isolation of C1q binding proteins from cells (Table 2). From the current available data, some of these molecules can be classified as receptors (Ex. C1qRp), while others have the properties of a biological response modifier, such as chrondroitin sulfate proteoglycan (CSPG) (9). Still others, while, originally described as cell surface receptors for C1q (10), either are not readily detected on the cell surface (11, 12) (and this volume) or lack definitive functional data indicating the induction of a cellular response. One of these molecules, however, gC1qR, has been shown to inhibit complement mediated lysis of erythrocytes (10) and C1q-induced platelet aggregation (13) in vitro, and thus is categorized in Table 2 as a potential response modifier. CR1 has recently been shown to bind C1q (14), and therefore is classified as a C1q binding protein. However, future studies should determine if CR1 plays a role in mediating a signal to the cell as a result of its interaction with C1q, and thus if it should be designated as a true cellular 'receptor' for C1q. Table 2. C1q binding proteins. Receptor:

C1qRp "C1qR02_

Response modifier:

gC1qR" CSPG

Binding protein:

CR1':cC1qRlcalreticulin*

"- Molecules have been cloned and primary sequence deduced. CSPG = Human B cell derived chondroitin sulfate proteoglycan (9).

C1qRp: A multiligand receptor that enhances phagocytosis One of the first consequences of C1q binding to phagocytic cell surfaces to be documented was the enhancement of phagocytosis of particles that were suboptimally opsonized with IgG or complement (15, 16) (Fig. 1). While first described in human mononuclear phagocytes, these observations have been expanded to include C1q-mediated enhancement of neutrophil phagocytosis by OHKURO, et al. (17). This enhancement of a critical protective function would be most important in early stages of infection or trauma, prior to the enlistment and/or amplification of other 'adaptive' defense mechanisms such as appropriate

Clq receptor . 253

Figure. 1. Clq enhances phagocytic activity. Human macrophages were adhered to control surfaces (left panel) or to surfaces precoated with Clq (10 jlg/ml) (right panel). Sheep erythrocytes suboptimally opsonized with IgG were added and the cells incubated at 37°C for one hour. Targets that were not ingested were removed by washing and by hypotonic lysis. Cells are fixed and stained with Giemsa Wright, and phagocytosis of targets scored (15). The enhancement of phagocytic capacity in the macrophages adhered to Clq (right panel) compared to control wells (left panel) is clearly evident. Similar results are seen with freshly isolated monocytes, although the absolute number of targets ingested is slightly lower.

levels of specific and high affinity IgG. This induction of cell function, like many other immunological responses, requires multivalent presentation of the ligand, Clq, or a conformational change in the ligand induced by aggregation or immobilization of the Clq monomer (18). Early studies demonstrated that the Clq receptor interaction site was contained in the collagen-like domain of Clq, and is normally masked by Clr and Cls in the macromolecular complex Cl (15). Once Clq binds to an activator, however, Clr and Cls are converted to active serine esterases which trigger the initiation of the classical complement pathway. Subsequent regulation by the CI-Inhibitor leads to the dissociation of Clr and Cls from the Clq molecule, thereby exposing the receptor interaction site(s), permitting the induction of cell function. Pepsin digestion of Clq yields an intact collagen-like fragment of Clq (Clq-CLR). This proteolytic fragment of 180,000 Da was shown to be necessary and sufficient for mediating the enhancement of phagocytosis (15). To identify the surface receptor components involved in mediating these functions, monoclonal antibodies to cellular Clq-binding proteins were generated by immu-

254 . A. J. TENNER nizing with material derived from detergent extracts of myeloid cells that had been bound to and eluted from immobilized C1q-CLR (19). The resulting antibodies were subsequently screened for reactivity with myeloid cell surfaces and, importantly, for the ability to inhibit the enhancement of phagocytosis by C1q (19). Two monoclonal antibodies, R139 (IgG2b) and R3 (IgM), were further characterized. These MoAb inhibited C1q-mediated enhancement of phagocytosis, but not phagocytosis or the enhancement of phagocytosis in general, as baseline and fibronectin-mediated enhancement of phagocytosis was not inhibited by R139 (19) or R3 (20). Interestingly, the IgM antibody, R3, when presented as crosslinked by immobilization on a surface, mimicked the C1q-mediated enhancement of phagocytic activity in monocytes, in a dose-dependent manner (NEPoMucENo, et al., unpublished results), confirming the requirement for a multivalent presentation in order to activate this C1q receptor, at least in monocytes. This stimulation of function by crosslinking receptors with a MoAb conclusively defines this cell surface polypeptide recognized by the monoclonal antibody, which we now designate C1qRp, as a component of the C1q receptor that enhances phagocytosis. Anti-C1qRp monoclonal antibodies immunoprecipitated a 126,000 Mr cell surface glycoprotein from detergent extracts of monocytes, neutrophils and the monocyte-like cell line, U937, but not from Raji or CEM lymphoblastoid cell lines (19). Using antibody affinity chromatography and SDS-PAGE, the protein recognized by the monoclonal antibodies was isolated, and the amino acid sequence of the N-terminus and 10 internal proteolytic fragments obtained (21). A cDNA coding for a 631 amino acid protein, in which all of the sequenced peptide fragments were located, was subsequently cloned. Homology searches of both protein and DNA data bases demonstrated that this sequence represents a novel Type 1 surface protein, containing a domain structurally similar to a Ctype lectin carbohydrate recognition domain (a motif common with several other proteins involved in endocytosis), five EGF-domains, a single transmembrane domain and a 47 amino acid intracellular domain (21) (Fig. 2). While the molecular weight from the deduced amino acid sequence is considerably less than the 126,000 Mr seen in SDS-PAGE gels of the mature protein, transfection of the coding region for the 631 amino acid protein into CHO cells resulted in the expression of a protein immunoprecipitated by the anti-C1qRp monoclonal antibodies with the identical relative migration in SDS-PAGE as the mature pro-

N sp

TM

c

Figure 2. C1qRp domain structure. The primary structure of C1qRp deduced from the cDNA sequence contains domains which include a 21 amino acid signal peptide (SP) and a C-type lectin carbohydrate recognition domain (CRD) at the N-terminus, five Epidermal Growth Factor (EGF)-like domains (numbered), including three calcium binding (3, 4 and 5) EGF domains, and a single transmembrane (TM) domain. The putative N-linked glycosylation site is indicated within the second EGF domain (21).

Clq receptor . 255

tein (NEPOMUCENO, et al., unpublished results). Subsequent analyses have demonstrated that the protein contains a high amount of O-linked carbohydrate and additionally contains an unusual distribution of amino acids, both of which contribute to the abnormal migration behavior when subjected to SDS-PAGE (NEPOMUCENO, et al., unpublished results). Mannose binding lectin (MBL) and pulmonary surfactant protein A (SPA), two other proteins with macromolecular structure similar to that of C1q, have also been shown to be able to directly opsonize particles (22-24) [reviewed in (25)] as well as enhance the ingestion of suboptimally opsonized particles bound to cells through FcR and CR1 (15, 16,26,27). It has been postulated that these macromolecules, and perhaps other proteins containing collagen-like sequences, may have evolved prior to IgG as defense recognition molecules (28) and that these molecules may play an important role at early stage of disease and in other conditions where ample antibodies may not be present [for example, prior to generation of anti-carbohydrate IgG in young children (29)]. Since MBL and SPA have collagen-like N-terminal regions, it was hypothesized that, similar to C1q, this region of these molecules was responsible for the interaction of the ligands with the cell to induce the enhancement of phagocytic function, perhaps through a common cell surface receptor component specific for such collagenlike ligands. Preincubation of monocytes with the anti-C1qRp MoAb was sub-

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Figure 3. Anti-C1qRp monoclonal antibody inhibits C1q- and MBL-enhanced phagocytosis. Human monocytes were preincubated with buffer (open bars), control mouse IgG (stripped bars) or R139 (anti ClqRp (hatched bars) prior to addition to wells precoated with 4 pg/ml

HSA, C1q, or MBL. After one hour adherence, EAC4b targets were added along with 10 ng/ml PDBu and. phagocytosis assessed. The error bars represent the standard deviation of duplicate samples from a single experiment, representative of three distinct experiments (27).

256 . A. J. TENNER

sequently shown to inhibit SPA- (30) and MBL- (27) enhanced phagocytosis (Fig. 3), demonstrating that the activation by C1q, MBL and SPA occurs via a receptor with at least one common component, C1qRp • This hypothesis was supported by reports from GEERTSMA, et aI. and SOELL, et aI. demonstrating that C1q can block the binding of SPA- (22) and MBL- (31) coated particles to monocytes. Interestingly, in our reciprocal experiments using purified rMBL (gift from R. A. B. EZEKOWITZ) to compete with 125I-C1q-CLR for binding to U937 cells, monocytes, and neutrophils, only partial inhibition of binding on all three cell types (27) could be demonstrated, suggesting that there may be additional, unique C1q binding sites on these cells, in addition to the MBLISPA shared site (see below). Furthermore, TINa and WRIGHT (24) demonstrated that SP-A stimulation of phagocytosis of specific pathogens is inhibited in monocytes adhered to surface bound C1q, but not in alveolar macrophages similarly treated. Thus, the differentiation state of the macrophage may dictate the surface distribution of C1qRp, its mobility (32) and/or the induction of additional functional receptors. Consistent with this latter hypothesis, SHEPHERD and colleagues have identified a novel 210 kD SPA receptor (33). It will be interesting to determine if other cell surface interactions for MBL, SPA and other collectins [such as SP-D (34)J exist, and if so, how the consequences of ligation of these receptors influence the overall response to specific pathogens, injury and/or homeostasis. Many monocyte activators stimulate the expression of proinflammatory cytokines which can have detrimental side effects. For example, TNF-a enhances HIV expression (35) and thus induction of this proinflammatory cytokine would be undesirable in HIV+ individuals. Studies of the lack of induction of individual cytokines by MBL and SPA have recently been published (31, 36). While these latter studies report on the effect of these ligands on a specific proinflammatory cytokine, we chose to take a boarder approach and, in collaboration with Dr. R. ROCHFORD (Univ. of Michigan), used a cassette of probes and an RNase protection assay to simultaneously assess 8 distinct cytokines. Our in vitro findings indicate that under conditions in which C1q, SPA or MBL enhance phagocytosis in human monocytes expression of proinflammatory cytokines is not induced GASINSKIENE, et aI., submitted) suggesting that monocyte activation by these ligands is, at least to some degree, selective (for enhanced ingestion), and thus more desirable than other pharmacologic agents that additionally promote an inflammatory response. The only other compound with similar immunoregulatory properties (no proinflammatory cytokine induction, but enhancement of microbicidal activity) that has been characterized is a glucose polymer, PGG-Glucan, which has shown significant efficacy in Phase IIII trials in reducing post surgery infections (37, 38). Very little is known about the receptor(s) involved in this effect, although the signaling mechanisms involved in the PGG-Glucan system are now being investigated (39). It is clear however, that the presentation of the ligand (particulate or soluble) and/or the coligation of other receptors influence the response of the mononuclear phagocyte. Several investigators have demonstrated induction of cytokines when MBL or SPA are complexed with pathogenic substances (40,

Clq receptor . 257

41). There is also evidence of some TNF-a secretion by monocytes and IL-6, IL-8 and MCP-1 by endothelial cells upon incubation of with C1q in vitro. These differences may be related to the cell type and state of differentiation of the cells, and thus further investigation will be critical to provide the basis for rational design of therapeutic agents to enhance resistance to infection. FACS analysis using the monoclonal antibodies, as well as Northern blot analysis (Fig. 4) and RT-PCR studies, have demonstrated that a single species of the receptor is present in cells of myeloid origin, in platelets and on endothelial cells, but is not expressed by lymphoid cells or fibroblasts (42). C1 q binds DNA, chromatin and mitochondrial lipids, consistent with a possible role in clearing cellular debris as well as phagocytosis of pathogenic microbes. The presence of this receptor on endothelial cells [originally postulated by ANDREWS, et al. (43)J, is particularly interesting in light of the recent report of HESS, et al. suggesting that at least some subtypes of these cells are capable of phagocytosing apoptotic lymphocytes (44). While the involvement of C1qRp in this function is merely speculative at this time, such a function would be particularly advantageous in many circumstances if no induction of inflammatory cytokines/processes occur. It will be interesting to determine if the frequent development of lupus-like disorders including vasculitis in individuals that are genetically deficient for C1q (45), or rheumatoid vasculitis in individuals that have antibodies to C1q (46) is influenced by a deficiency in the Clq/ClqRp receptor system.

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258 . A. J. TENNER However, it has also been reported that, in vitro, in the presence of immune complexes C1q stimulates endothelial adhesiveness for leukocytes, suggesting distinct consequences depending on the combination of receptors engaged (similar to that described above for pathogens). Development of a C1qRp knock out mouse model, as well as further immunohistochemical studies of the tissue distribution of this receptor, will aid in defining the physiologic function(s) of this receptor.

C1qR02_: Stimulation of 5uperoxide production by C1q As mentioned earlier, C1q elicits additional cellular responses upon interaction with other cell types (Table 1). The selective expression of C1qRp suggests that many of those C1q-induced responses must be mediated by distinct receptors. We and others have demonstrated that C1q, again when presented in a multivalent fashion, can trigger the generation of toxic oxygen radicals by neutrophils (Fig. 5) (47, 48). Generation of toxic oxygen radicals is protective during infection, as these molecules facilitate microbial killing. However, a lack of regulation or failure of the phagosome to close can lead to local tissue destruction, and thus understanding the mechanisms involved in the C1q-triggered oxidase activity should be useful in both avoiding the initiation of this function and/or down regulating it when desired. While the C1q-mediated stimulation of neutrophils shares some common parameters of other neutrophil activation systems, there are several quite distinct parameters that indicate a unique initial signaling pathway from those previously described for NADPH oxidase. The first novel characteristic was that while C1q triggers superoxide production, it does not induce degranulation of primary or secondary granules (49). Other characteristics that are distinct from most (not all) previously characterized activators include the lack of pertussis toxin inhibition and the lack of a requirement for stable adherence to a surface for activation of the oxidase (50). Three separate lines of analysis provide compelling evidence that the C1q receptor that triggers superoxide (C1qRo2J differs in some way from the C1q receptor that modulates phagocytosis (C1qRp ). First, while FACS analysis demonstrated that the monoclonal antibodies recognizing C1qRp were reactive with neutrophils, none of these antibodies either inhibited or mimicked the C1q-induced production of O 2- by neutrophils (20) (in contrast to their effects on C1q-, MBL- and SPA-induced enhancement of phagocytosis). Second, while mimicking C1q in the ability to enhance phagocytosis, neither MBL nor SPA induce superoxide production by neutrophils (49).These results suggest that a receptor-interaction site for C1qRo2_is unique to C1q, whereas a different interaction site, common to MBL, SPA and Clq, exists for ClqRp • Third, more recent studies in our laboratory have utilized defined fragments of the collagenlike region of C1q generated by either trypsin, endoproteinase LysC (endoLysC) or matrix metalloproteinase-2 (MMP-2) digestion to determine that the region of the C1q-CLR flanked by amino acids 42 to 61 on the C chain, which is specifically lost by trypsin cleavage but not by endoLysC or MMP-2 cleavage, is

Clq receptor . 259

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Figure 5. Clq stimulates superoxide production. Human neutrophils (3.5 x lOS/well) were added to wells of a microtiter plate that had been coated with 30 or 100 ]Jg/ml Clq-CLR or buffer only. Production of superoxide was monitored as the superoxide dismutase-inhibitable reduction of cytochrome c as previously described (51).

absolutely required for activation of the neutrophil oxidative burst (51, 52). This region does not appear to be required for effective enhancement of phagocytosis by C1q. We are currently assaying the ability of enzymatically derived C1q fragments to enhance phagocytosis, in efforts to further define the specific ligandreceptor interactions involved in triggering each of these functions. Future identification of the surface molecules comprising C1qR02 _ will be a critical step toward the ability to modulate this powerful protective response, which, if not regulated properly, can cause significant host tissue damage. The general characteristics of each of C1qRp and C1qR02_ are summarized in Table 3. In summary, the ability to upregulate phagocytic activity may be a useful prophylactic approach for individuals who are immunocompromised by genetics, age, as a result of diseaselinfection or during immunosuppressive treatment. Future advances in the molecular characterization of C1qRp , investigation of the

260 . A. J. TENNER Table 3. C1q receptors. C1Rp

C1qRoz_

Function:

Enhances Phagocytosis

Triggers superoxide production

Me: Cellular E:x;pression:

126,000 Me

?

Monocytes Neutrophils Endothelial cells Platelets

Neutrophils Monocytes Smooth muscle cells (?)

C1q MBL SPA

C1q

Ligands:

signaling pathways involved in mediating and regulating the enhancement of phagocytic activity, and the use of animal models should contribute to an understanding of the physiologic significance of this receptor in preventing or limiting disease, and to providing a basis for the design of agonists for the receptor. In addition, further delineation of the specific ligand-receptor interactions involved in triggering each Clq-mediated function will facilitate selective modulation of these responses without the development of undesired inflammation or susceptibility to other pathology (for example, to enhance phagocytic capacity without generating toxic oxygen radicals). Acknowledgements Work from the author's laboratory presented here was supported by grants from the Arthritis Foundation, American Heart Association and NIH AI#41 090. In addition to contributing to the work reviewed here, the author wishes to thank RON NEPOMUCENO and SOL RUIZ for helping to prepare the Figures presented.

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