Immunity, Vol. 3, 485-493, October, 1995, Copyright 0 1995 by Cell Press Mannose Binding (MBP) Enhances Mononuclear Function via a Receptor that Contains the 126,000 M, Component of the Clq Receptor

Andrea J. Tenner,’ Susan L. Robinson,7 amino acid sequence. The carboxy half of the MBP mole- and R. Alan B. EzekowitzS cule constitutes a carbohydrate recognition domain (CRD) ‘Department of Molecular Biology and Biochemistry and has features of an ever-growing family of animal C-type University of California lectins (Drickamer, 1987). Irvine, California 92717 The presence of a collagen-like domain in a nonmatrix tAmerican Red Cross polypeptide, while previously somewhat unusual, is now J. H. Holland Laboratory being discovered in an, as of yet, small family of Rockville, Maryland 20855 called collectins (Malhotra et al., 1992; Sastry and Ezeko- *Division of Hematology/Oncology witz, 1993), which contain a region of collagen-like sequence Children’s Hospital (Gly-X-Y) contiguous with recognition domains composed Dana-Farber Cancer Institute of noncollagen-like amino acid sequences (Drickamer et Department of Pediatrics al., 1966; Thiel and Reid, 1989), usually with lectin-like Harvard Medical School properties. In addition to MBP, the other members of this Boston, Massachusetts 02115 family are the pulmonary surfactant proteins, SP-A and SP-D (Shimizu et al., 1992), the classical complement component, Clq, and conglutinin, a protein that binds to Summary the iC3b fragment of complement C3 (Lee et al., 1991; Lachmann, 1967). MBP, SP-A, and Clq all have an inter- Mannose-binding protein (MBP), Clq, the recognition ruption in the Gly-X-Y repeat pattern of the collagen-like component of the classical complement pathway, and sequence (Drickamer et al., 1988), hydroxylated amino pulmonary surfactant protein A (SP-A) are members acids, and short NH,-terminal domains containing in- of a faniily of molecules containing a collagen-like se- terchain disulfide bonds. Electron microscopy has demon- quence contiguous with a noncollagen-like sequence, strated remarkable similarity in macromolecular structure and usually having the properties of a lectin. Clq and (Vosset al., 1988) between Clqand SP-A. With theexcep SP-A have been shown to enhance monocyte FcR- and tion of the glycine residues in every third residue in the CRl-mediated , suggesting that thecom- collagen-like domain, the proteins bear no common se- mon structural features of the collagen-like domains quence identity to one another. Interestingly, all of these may provide a basisforthis immunologically important molecules have been shown to enhance uptake of defined function. Results presented here demonstrate that material (Friis-Christiansen et al., 1990; Krieger, 1992). MBP also enhanced FcR-mediated phagocytosis by SP-A enhances the uptake of lipids by isolated alveolar both monocytes and macrophages, and stimulated type II cells (Wright et al., 1987), while MBP augments the CRl-mediated phagocytosis in human culture-derived uptake by of certain bacteria, viruses, and macrophages and in phorbol ester-activated mono- model polymers in vitro (Kuhlman et al., 1989; Hartshorn cytes. Furthermore, a monoclonal that recog- et al., 1993; Soell et al., 1995). It has been well docu- nizes a 126,000 M, cell surface protein and inhibits mented that Cl q has properties of both an opsonic ligand Clq-enhanced phagocytosis, inhibited the MBP-medi- (mediating binding/recognition of microbes or model parti- ated enhancement of phagocytosis. Thus, the recep- cles by the cells) (Sorvillo et al., 1986) and an activation tors that mediate the enhancement of phagocytosis ligand (enhancing the response of the cell to the binding by MBP and Clq share at least one critical functional of a second ligand to a different receptor) (Bobak et al., component, the 126,000 M, ClqRp. 1987, 1988; Brown, 1986). These effects were mediated via the collagen-like fragment of Clq, as the isolated pep- Introduction sin-resistant fragment, but not the pepsin-sensitive colla- genase-resistant fragments, was effective in enhancing The human mannose-binding protein (MBP) represents a phagocytic activity (Bobak et al., 1987). Tenner et al. nonclonal form of humoral immunity in which discrimina- (1989) previously showed that, like Clq, SP-A can en- tion of self versus nonself relies on the recognition of car- hance FcR- and CR1 -mediated phagocytosis by mononu- bohydrate that are common to pathogens but are clear phagocytes, suggesting that common structural fea- rarely if ever expressed on host cell surfaces or circulating tures of the collagen-like domains may provide a basis for glycoproteins (reviewed by Ezekowitz, 1991). Mannose this biological function. A cellular protein that binds to the binding proteins have been isolated from serum and liver head regions of Clq has recently been described (Ghe- of man and rodents. The predominant circulating form of brehiwet et al., 1994). This indicates that Cl q can interact MBP appears as a multimer of approximately 600,000 Da with cellsvia the collagen-like region (tail) and the globular of a 32,000 Da subunit. Examination of the sequence pre- heads. dicted from rodent and human cDNAs reveals an NH2 Mannose-binding protein is also a member of a family terminus containing cysteine residues, which may, via in- of proteins including the macrophage mannose receptor terchain disulfide bonds, stabilize a collagen helix com- (MR) (Ezekowitz et al., 1990; Taylor et al., 1990), a lectin prised of up to 19 repeats of Gly-X-Y in the contiguous found in the abdominal cavity of the flesh fly Sarcophaga Immunity 466

and a recently described lipopolysaccharide-binding pro- A tein from the hemolymph of the American cockroach, Peri- planeta americana, that have a role in host defense and are up-regulated in response to infection or trauma (Jomori and Natori, 1991; Ezekowitz et al., 1988; Taylor et al., 1989). For example, stable accumulation of Sarcophaga Clq lectin mRNA increases significantly in response to the in- troduction of foreign substances into the abdominal cavity of flesh flies (Takahashi et al., 1985). Similarly, the hepatic synthesis of the human mannose-binding protein as well as the mouse MBPA are up-regulated as part of the acute phase response (Sastry et al., 1991). These similarities HSA between the regulation of the mammalian proteins and those found in insects suggest that the role of the man- nose-binding protein in host defense has primitive origins. The results of experiments described here demonstrate that MBP, like Clq and SP-A, is an activation ligand as well as an opsonic ligand. Furthermore, a 6 (MAb) that recognizes a component of the Clq receptor on myeloid cells that enhances phagocytic activity (Cl qRp) 4ooI-- inhibits the MBP-mediated increase in phagocytosis, sug- gesting that the receptor for these two ligands share at least one polypeptide component. This mechanism of acti- vation may be relevant at sites of inflammation where these molecules can accumulate, and could provide a beneficial enhancement of the phagocytic capacity of acute inflammatory cells. Finally, competitive binding studies demonstrate that Clq and MBP share an interac- tion site on human myeloid cells, lending support to the hypothesis that the similar collagen-like macromolecular structure may mediate a similar function. These ligands may be examples of elements of innate immunity that can function both as a first line of defense prior to production of specific antibody and synergistically with the adaptive Protein Costing Concentntion (&II) immune system in protecting the host from infection. Figure 1. MBP Enhances Monocyte F&Mediated Phagocytosis Human monocytes purified by countercurrent elutriation were added Results to LabTek chambers that had been precoaled with 4, 6, or 16 uglml of HSA (inverted closed triangle), Clq (closed circle), or rMEP (open MBP Enhances FcR-Mediated Phagocytosis inverted triangle). EAlpG targets were added and phagocytosis was assessed. The collagen-like region of Clq, SP-A, and collagen type (A) The percentage of monocytes ingesting at least one & target. IV, but not collagen type I, have been shown to increase (B) The number of targets ingested per 100 monocytes. Values pre- the phagocytosis of opsonized particles by mononuclear sented are the mean + SD of duplicate samples from one of six similar phagocytes(Bobaket al., 1987; Tenner et al., 1989; Brown experiments. and Goodwin, 1988). MBP, an acute phase response pro- tein, has been shown to act directly as an of man- nose-rich pathogens. Given the similarity of macromolecu- adhered. After 1 hr, (IgG)-coated tar- lar structure of MBP with Clq and SP-A, we investigated gets were added and incubated for 30 min at 37%. Figure whether MBP could, like Cl q and SP-A, activate the mono- 1 presents the data from 1 of 6 separate experiments dem- nuclear cell in such a way as to increase the subsequent onstrating that mannose-binding protein enhances FcR- phagocytosis of particles opsonized with IgG. Multivalent mediated phagocytosis in a dose-dependent manner as presentation of Clq or a conformational change induced compared with cells adherent to uncoated surfaces. This by aggregation or immobilization of the Clq monomer increase was seen both as an increase in the percentage seems to be required for the generation of a functional of cells ingesting the target particles (Figure 1A) and as response upon binding to cells (Tenner and Cooper, 1982). an increase in the number of targets ingested per cell This requirement is similar to that seen with CRl, CR2, (Figure 1 B, phagocytic index). Phagocytosis of unopso- FcRII, FcRIII, and many other surface receptors (Wener nized erythrocytes is never seen under any condition et al., 1989; Brunswick et al., 1989). Therefore, human tested. monocytes were added to glass slides previously coated This ability to function as an activation ligand was not with either Clq, MBP, or serum albumin to which the cells specific for freshly isolated monocytes. In vitro culture con- MSP and Clq Enhance Phagocytosis via 126,000 M, Receptor 467

ditions are known to influence macrophage differentiation MAb against ClqRp Inhibits MBP-Mediated (Bobak et al., 1988; Wright and Silverstein, 1982). Isolated Enhancement of Phagocytosis human monocytes cultured in a defined serum-free me- To determine whether the monocyte receptors for Clq dium, HL-1, have less of the characteristics of macro- and MBP that increase the phagocytic response contain a phages than do those cultured for the same period of time common receptor component, the MAb, R139, that inhibits in media containing 10% fetal calf serum (FCS) (Bobak the Clq-mediated enhancement of phagocytosis was et al., 1988). However, MBP was found to enhance the tested for its effect on the MBP-induced increase in phago- ingestion of IgG-coated E, by phagocytes previously cul- cytosis. EA,,&4b targets, rather than EAIO~, were used in tured for 3-7 days with either HL-1 or in media containing these experiments to avoid potential competition with FcR. 10% FCS. In three separate experiments, MBP increased Purified monocytes were preincubated with either buffer, EAIgG phagocytosis 3- to 4-fold more than cells adhered R139 (anti-ClqRp) or a control MAb for 15 min prior to to control surfaces. This degree of enhancement was simi- addition to HSA-, Clq-, MBP-, or fibronectin-coated wells. lar to that seen with Clq, and comparable also to that R139 inhibited both the Clq- and the MBP-mediated en- previously published for SP-A (Tenner et al., 1989). Fur- hancement of phagocytosis but did not effect the fibronec- thermore, results were similar whether native MBP or re- tin-induced increase in phagocytosis (Figure 2). As has combinant MBP was presented to the cells (data not been seen previously (Guan et al., 1991, 1994) the inhibi- shown). tion of number of targets ingested per cell was more pro- nounced (59%-820/o inhibition) (n = 3, p < .OOl) than CR-l-Mediated Phagocytosis Is Modulated by MBP the percentage of cells actively engaged in phagocytosis To determine whether this stimulation of the phagocytic (370/o-81% inhibition) (n = 3, p < ,003). Knowledge of the capacity of monocyteslmacrophages was specific only for signal transduction mechanisms should provide insights targetsengaging the FcR, we tested whethertargets opso- into the basis for these differential inhibitory effects. nized with IgM and C4b that bind to the C3b receptor, CR1 (Bohnsack et al., 1985) were also ingested to a greater Effect of MBP on the Binding of Clq to Monocytes, degree upon interaction of the phagocytes with MBP. U937 Cells, and Neutrophils While monocytes readily bind and ingest IgG-opsonized Since both MBP and Clq triggered a similar enhancement particles and avidly bind CBb-/C4b-coated particles, these of phagocytosis and this was inhibited by the MAb to a “resting” phagocytes do not ingest particles bound via 126,000 M, cell surface protein, it was possible that these CR1 , unless activated to become macrophages or treated ligands were interacting with the same cell surface recep- with tumor-promoting phorbol esters, such as PDBu (Bo- tor. If so, MBP should be able to compete effectively with bak et al., 1988). Thus freshly isolated monocytes were Clq for binding to these cells. Therefore, the ability of added to wells coated with MBP, Clq, or control proteins, MBP to inhibit the binding of 1251-C1q-(collagen-like frag- or treated with buffer only. After 1 hr at 37% EAI,&4b ment)CLF was assessed. Figure 3 presents binding data targets were added to the wells in the presence of 10 nglml from several experiments that demonstrate that MBP re- PDBu. While the absolute amount of phagocytosis varied producibly inhibited the binding of Clq-CLF to monocytes among the experiments (n = 6) MBP (recombinant or by 25%. Similarly, binding to neutrophils (Figure 4) and native) consistently augmented CRl-mediated phagocy- U937 (data not shown) was also inhibited by 25%-420/o. In tosis to roughly the same degree as Cl q. Table 1 presents control experiments, mannan, which would competitively results from several experiments in which a coating solu- block the MBP lectin domain, had no effect on the ability tion of 8-10 pglml of the test protein was used. MBP, of MBP to inhibit ‘251-C1q-CLF on monocytes. That is, in like Clq, increased both the percentage of cells ingesting two additional experiments MBP inhibited Clq-CLF bind- targets (1.8-to 8.1 -fold) and the number of targets ingested ing to monocytes by 61% and 77% alone, and 55% and per effector cell scored (1.9- to 13.9-fold increase) relative 820/o, respectively, in the presence of l-10 mglml man- to human serum albumin (HSA) or bovine serum albumin nan. These data suggest that the inhibition is not due to (BSA) control or uncoated control wells (Table 1). lectin,interaction of MBP with the cell surface or the recep-

Table 1. MBP Enhances CR1 -Mediated Phagocytosis

Uncoated Albumin” c1q Experiment number PercenF I Percent I Percent I Percent I

1 7.5 f 2 12 2 6 451 5*3 26.5 f 9 45.5 + 20 50 * 1 116 k 0 2 9.4 + 4 19 f 11 74.5 f 6 262 + 31 62.5 f 7 214 f 30 3 25 + 6.6 45 * 16 46 f 0 65 + 0.7 46 k 12 100 * 36 4 6k4 6.5 + 9 49 t 4 103 + 35 60 + 9 130 f 40 a HSA in experiments 1 and 4; BSA in experiment 3. ’ Coating concentration for all proteins was 8 uglml, except for MBP in experiment 3, in which the coating concentration was 10 @ml. c Percentage of cells ingesting targets; I, phagocytic index; number of targets ingested per 100 cells counted. Immunity 499

PC.04

HSA Clq MBP Fn 4 6 8 PROTEIN (ug)

Figure 3. Effect of MBP on Clq Binding to Monocytes 1251-Clq-CLF was added to human monocytes in the presence of in- creasing concentrations of rMBP, unlabeled Clq, or unlabeled Clq CLF. After incubation on ice for 30 min, the cells were spun through phthalate cushion and cell-associated radiolabel was determined. The percent of control binding in the presence of MBP (closed circle) (n = 4, three separate experiments), unlabeled Clq (closed triangle) (n = I), or unlabeled Clq-CLF (closed square) (n = 2). Error bars depict standard deviation of the mean. In each individual experiment, samples were run in triplicate or duplicate. Total counts bound in the control, uncompeted samples were 1622-l 1943 cpm depending on the specific activity of the labeled protein.

to inhibit specifically the Cl q-mediated enhancement of phagocytosis, also inhibits the MBP-induced increase in monocyte phagocytic capacity. Thus, the receptor(s) for HSA Clq MBP Fn these two macromolecules that mediate the enhancement of phagocytosis share at least one critical surface polypep- Figure 2. Anti-C1qRp Inhibits Map-Enhanced Phagocytosis tide component. As is becoming increasing evident in Human monocytes were preincubated with buffer (closed bars), R139 many ligandlreceptor systems, receptors are often multi- (anti-Clq&) (hatched bars), or control mouse IgG ((open bars) prior subunit complexes, containing ligand binding compo- to addition to wells precoated with 4 uglml HSA, Clq, rMBP, or fibro- nectin. After 1 hr adherence, EAC4b targets were added along with nents as well as polypeptides that are involved in the sig- 10 nglml PDBu and phagocytosis assessed. The error bars represent naling function of the receptor. Whether the component of the standard deviation of duplicate samples from a single experiment. the phagocytosis modulating receptor (Cl qRp) recognized Results are from one experiment representative of a total of three such by the MAb, R139, is the ligand binding component of experiments, with different donors, R139 antibody preparations, and ligand preparations. the receptor(s) or has another role in receptor signaling remains to be seen. Soell and colleagues (1995) recently showed that MBP- containing complexes are ingested by human monocytes. tor, but rather that a common interaction domain exists The data presented here differ from that report in that within the collagen-like region of these molecules. In addi- here MBP is not associated or bound to the target being tion, the fact that only partial inhibition of Cl q binding was ingested. Rather, cell interaction with MBP alone induces demonstrated supports the hypothesis that there is more as yet unknown intracellular responses, such that upon than one binding site for Clq-CLF on these cell types, and binding of a target to a separate recognition receptor, such that MBP may provide a mechanism for distinguishing the as FcR or CR 1, phagocytosis occurs in a larger percentage various Clq binding sites/receptor types. of the myeloid cell population, with each cell ingesting more targets within a given time period. Levitz et al. (1993) Discussion recently reported that monocytes and PMN cultured on immobilized MBP demonstrated increased binding to The data in this paper demonstrate that MBP can interact Cryptococcus neoformans. It will be interesting to deter- with the mononuclear phagocyte cell surface to enhance mine whether this “activation” receptor is also involved in phagocytosis. In addition, a MAb that recognizes a 126,000 the cell interaction that mediates the enhanced binding, M, cell surface protein and that previously has been shown ingestion, or both of MBP-coated particles. MBP and Clq Enhance Phagocytosis via 126,000 M, Receptor 469

ity of MBP, verified by definition of the structure of the IT PC.06 cocrystals of MBP and its ligand (Weis et al., 1992), is such that it recognizes microbial carbohydrate structures and not mammalian oligosaccharides, this result was not unexpected. While these data suggest a common cell in- P

Figure 4. Effect of MBP on Clq Binding to Neutrophils diates the Clq enhancement of phagocytosis (ClqRp) (Guan et al., 1991) a Clq binding protein with different The percent of control binding is the mean percent control binding calculated from three experiments for rMBP (closed circle) (performed apparent molecular weight (in the 56,000 M, range) has in triplicate or duplicate). In one of the experiments, unlabeled Clq- been purified by several laboratories (Ghebrehiwet et al., CLF was added in increasing concentrations (closed square), whereas 1992; Peerschke and Ghebrehiwet, 1988; Malhotra et al., intwootherexperimentsClqwasaddedasthecontro1 inhibitor(closed 1988). This latter protein has been reported to have se- triangle). Error bars depict standard deviation of the mean. The aver- age maximal binding in each experiment varied from 1952-9623 cpm, quence similarity to calreticulin (Malhotra et al., 1993), and correlating with the specific activity of the Clq-CLF. in solid phase in vitro assays this protein binds multiple members of the collectin family (Malhotra et al., 1990). Polyclonal to the calreticulin-like collectin bind- Previously reported experiments have demonstrated ing protein inhibit adhesion of fibroblasts (Bordin et al., that the Clq cell interaction site that mediates enhanced 1990) and platelets (Peerschke and Ghebrehiwet, 1990) phagocytic capacity is located on the collagen-like portion to Clq-coated surfaces, suggesting a role for this protein of the Clq molecule (Tenner and Cooper, 1980; Bobak in ligand-cell interactions. In contrast, two MAbs to the et al., 1987) and that SP-A enhances FcR- and CRl- 126,000 M, ClqR, have been shown to inhibit Clq- mediated phagocytosis in human monocytes and macro- mediated monocyte function (Guan et al., 1991, 1994) phages (Tenner et al., 1989) and serum-opsonized bacte- but not Clq-mediated 02- production by PMN (Guan et ria by rat alveolar macrophages (van lwaarden et al., al., 1994). Furthermore, unlike Clq, neither SP-A nor MBP 1990). These data are consistent with the hypothesis that immobilized to plastic surfaces trigger 02- production it is the common collagen-like structure of these molecules (Goodman and Tenner, 1992) again suggesting that the that mediates this cell activation. (The globular domains Clq receptors that mediate these different effects are not of SP-A and MBP share some homology with each other identical. Alternatively, this lack of an effect on neutrophil [Ca’+ and carbohydrate binding domains] but not with function by MBP and SP-A may be due to an inappropriate Clq.) Collagen type IV (Brown and Goodwin, 1988) has presentation of the molecules, as Kuhlman et al. (1989) also been shown to increase phagocytic capacity of mono- have shown the enhancement of neutrophil 02- production cytes, while collagen type I does not (Bobak et al., 1987) when Salmonella are preopsonized with MBP prior to addi- indicating that there is some specificity within the collagen- tion to PMN. This type of “receptor diversity” is similar to containing molecules that is critical for recognition by the the well-established demonstration of multiple classes of cellular receptor(s) that mediate this enhancement of Fc and C3 receptors on these cells, which mediate differ- phagocytic function. The ability of MBP to inhibit a portion ent functions. This receptor diversity ultimately should be of the binding of Clq-CLF to these cells suggests that useful in modulating these activities therapeutically. MBP and Clq do interact with a common surface binding Clq is normally present in blood in complex with site. These observations are consistent with the findings ClrZClsn, such that the receptor interaction site of Clq of Soell and colleagues that soluble Clq inhibited the bind- is masked. Upon binding of Clq in Cl to an activating ing of MBP-containing complexes to human monocytes substance such as an immune complex, bacteria, or other (Soell et al., 1995). The degree of inhibition was not in- pathogen, the proenzymes Cl r and Cls are activated and fluenced by interactions of the carbohydrate recognition become susceptible to inactivation by the serum regula- domain of MBP with either the monocyte cell surface or tory protein, Cl inhibitor. This inhibitor forms a covalent Clq, as mannan (which effectively saturates the lectin complex with activated Cl r and activated Cl s, abrogating binding site of MBP) did not effect the percent inhibition enzymatic activity and resulting in the dissociation of acti- of 1251-C1q-CLF by MBP. Since the lectin binding specific- vated Cl r2C1s2 from Clq (reviewed by Cooper, 1985). It Immunity 490

is that region of the collagen-like domain that becomes unit. In addition to advancing the general understanding of exposed as a result of this dissociation of activated Cl r&l sZ, the regulation of host phagocytic function, such receptor- which interacts with specific cell surface receptors (Tenner receptor systems may be amenable to common beneficial and Cooper, 1980; Bobak et al., 1987) leading to the modu- manipulation. lation of cell function. Thus, in addition to its role as the recognition subunit of the first component of the classical Experimental Procedures complement cascade, Clq may provide a direct means Media, Reagents, and Antibodies of triggering or enhancing host defense responses during RPM1 1640 medium was either purchased from GIBCO (Long Island, early stages of disease or infection when antibody or cellu- New York) or from the production laboratory of the American Red lar defense mechanisms have not yet developed (Cooper, Cross (Rockviile, Maryland). HLl medium was purchased from Ven- trex Laboratories(Portland, Maine). FCS andsupplementedcalf serum 1985). As mentioned above, SP-A and MBP have also (SCS) were purchased from Hyclone (Logan, Utah). L-glutamine and been shown to enhance the uptake of specific classes of gentamicin were obtained from M. A. Bioproducts (Waikersville, Mary- molecules (Tenner et al., 1989; Drickamer et al., 1986; land). Sheep erythrocytes are purchased from Colorado Serum (Den- Ezekowitz et al., 1988; Voss et al., 1988; White et al., ver, Colorado). Anti-sheep red blood cells, both IgG and IgM, were 1985). Since the globular regions of SP-A and MBP have purchased from Diamedix (Miami, Florida). The human serum albumin used for the elutriation buffer obtained from the American Red Cross lectin-like properties, which confer a specificity of binding was prepared by Travenol Laboratories, Incorporated (Glendale, Caii- to that region of the molecule, it has recently been sug- fornia). All other reagents used, except where noted otherwise, were gested that these lectins and other proteins containing obtained in the highest quality available from Sigma Chemical Com- collagen-like sequences (including Clq) may have evolved pany (St. Louis, Missouri). The MAb, R139, that recognizes a cell prior to IgG as defense recognition molecules and that surface protein (100,000 M, nonreduced, 126,000 M, reduced), was derived after immunization with partially purified Clq-binding proteins, these molecules also may play an important role at early obtained from affinity purification on Clq-CLF-Sepharose as pre- stages of disease and in other conditions where ample viously reported (Guan et al., 1991). antibodies may not present (for example, prior to genera- tion of anti-carbohydrate IgG in young children [Ezekowitz, Protein Isolation and lodination MBP was isolated from serum or recombinant MBP (rMBP) prepared 1991; Farries et al., 1990; Thiel and Reid, 1989)). as previously described (Ezekowitz et al., 1969 and Super et al., 1992, Phagocytosis is a major host defense mechanism by respectively). Clq was isolated from plasma-derived human serum by which potentially deleterious material is removed and the method of Tenner et al. (1961), modified as described (Young et made accessible to inactivation. The phagocytic capacity/ al., 1991). The preparations used were fully active as determined by potential of a cell can be modulated by cytokines that trig- hemolytic titration and homogeneous as assessed by sodium dodecyl sulfate-poiyacrimide gel electrophoresis. Cl q collagen-like fragments ger cellular differentiation (Gresham et al., 1986; Sampson (Clq-CLF) were obtained by the digestion of Clq with pepsin using et al., 1991) or by other activation ligands, which enhance the procedure of Reid (1976) as modified by Siegel and Schumaker the phagocytic response of the cell upon interaction of a (1963). Protein concentration was determined using an extinction coef- separate (opsonic) ligand with its cell surface receptors ficient (El”) at 260 nm of 6.62 for Clq (Reid et al., 1972) and 2.1 for Clq-CLF (Siegel and Schumaker, 1963). The iodogen method (Pierce, (reviewed by Brown, 1986). This activation is probably rele- Rockford, Illinois) has been determined to be optimal for radiolabeling vant at sites of inflammation where cytokines, MBP, Clq, the Clq-CLF (Guan et al., 1991). The specific activity of the ‘=I-Clq- or other regulatory molecules may accumulate and en- CLF was approximately 0.7-1.5 pC/Ng. All proteins were stored hance the phagocytic capacity of acute inflammatory cells. at -70%. Purified human fibronectin was provided by Drs. S. Huff The molecular events involved in the phagocytic process and K. lngham (American Red Cross Holland Laboratory, Rockville, Maryland). and in its regulation have not been completely elucidated, although it is clear that more than one pathway can be Cells evoked (Gresham et al., 1988,1989; Brown, 1986; Brown For experiments investigating the enhancement of phagocytosis and and Goodwin, 1988; Pommier et al., 1984, 1983). For ex- binding of ligands to monocytes, human peripheral blood monocytes ample, a MAb (B6H12) directed against an integrin- were isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described (Bobak et al., 1986). associated protein (Brown et al., 1990) has been shown to Blood units were collected into CPDAl by the Washington D. C. Re- block the enhancement of phagocytosis induced by many, gional Blood Service of the American Red Cross or by the University but not all, RGD containing extracellular matrix proteins of California at Irvine Medical Center Blood Bank (Orange, California) (Gresham et al., 1989). That MAb (B6H12) does not block or at the University of California at Irvine Medical Plaza, Irvine, Califor- nia. Greater than 92% of the cells in each preparation were monocytes, the Cl q mediated enhancement of phagocytosis, whereas according to size analysis on a Coulter Channelyzer. Macrophages the antibody to ClqRp inhibits Clq- and MBP-mediated, in these experimentswere elutriated monocytes that had been cultured but not fibronectin-mediated, enhancement of phagocyto- in Teflon jars (Savillex Corporation, Minnetonka, Minnesota) at 1 x sis (Guan et al., 1991). Future studies should elucidate lo6 cells/ml in HL-1 culture medium (a serum-free defined medium) further common molecular features of the receptor com- as previously reported (Tenner et al., 1969) or in FCS. On days 2-6 of culture, macrophages were collected and washed three times in plexes or the signal transduction pathways for the mole- phosphate-buffered saline (PBS) before use. Only cultures containing cules that share a similar collagen-like domain, thereby >90% viable cells as assessed by trypan blue exclusion were used. determining whether or not the previously described “Clq” As previously published, the number of cells adhered to the LabTek receptor that mediates the enhancement of phagocytosis chamber slides coated with various proteins did not differ significantly (Bobak et al., 1987). (Guan et al., 1991) is a receptor that binds multiple ligands to trigger the enhancement of cell function or whether the Phagocytosis Assay similarity of receptors involves only the 126,000 M, sub- Sheep erythrocytes bearing IgG anti-sheep red blood cells (EA& or MBP and Clq Enhance Phagocytosis via 126,000 M, Receptor 491

IgM anti-Forssman antibody and C4b (EAC4b) were prepared as pre- by human mononuclear phagocytes. Eur. J. Immunol. 78,2001-2007. viously described (Bohnsack et al., 1985). Phagocytosiswas assessed Bohnsack, J. F., Kleinman, H. K., Takahashi, T., O’Shea, J. J., and as previously described (Bobak et al., 1988; Bohnsack et al., 1985). Brown, E. J. (1985). Connective tissue proteins and phagocytic cell In brief, a-well LabTek chambers were coated with varying concentra- function laminin enhances complement and Fc-mediated phagocyto- tions of Clq, MBP. fibronectin, or BSA or HSA (Bobak et al., 1988). sis by cultured human macrophages. J. Exp. Med. 761, 912-923. Monocytes or macrophages (6.25 x lO’cells/well) are added to each Bordin, S., Ghebrehiwet, B., and Page, R. C. (1990). Participation chamber, and the cells allowed to adhere for 1 hr at 37% in 5% COI. of Clq and its receptor in adherence of human diploid fibroblast. J. Targets were then added (lO’/lOO ul) and incubated for 30 min at Immunol. 745, 2520-2526. 37°C. After removing E that were not cell-associated by washing, non- ingested E were removed by hypotonic lysis. After fixation in 1% glutar- Brown, E., Hooper, L., Ho, T., and Gresham, H. (1990). Integrin- aldehyde and staining with Giemsa, phagocytosis was quantitated associated protein: a 50-kD plasma membrane physically and using light microscopy. In experiments in which monocytes, or mono- functionally associated wtih integrins. J. Cell Biol. 711, 2785-2794. cytescultured in HL-1, wereassessedforCRl-mediatedphagocytosis, Brown, E. J. (1986). The role of extracellular matrix proteins in the 10 nglml PDBu was added with the opsonized targets, as it is well control of phagocytosis. J. Leuk. Biol. 39, 579-591. known that while monocytes can bind to targets via CRI, this receptor Brown, E. J., and Goodwin, J. L. (1988). Fibronectin receptors of must be “activated” to mediate phagocytosis in these less differenti- phagocytes characterization of the Arg-Gly-Asp binding proteins of ated cells. The pretreatment of monocytes or HL-l-derived macro- human monocytes and polymorphonuclear leukocytes. J. Exp. Med. phages with phorbol esters such as PDBu (which is not necessary for 167, 777-793. the more activated serum-cultured macrophages) enables phagocyto- Brunswick, M., June, C. H., Finkelman, F. D., and Mond, J. J. (1989). sis of complement-opsonized targets as previously elaborated (Bobak Different patterns of inositol polyphosphate production are seen in et al., 1988). The number of E targets ingested per 100 effector cells B lymphocytes after cross-linking of slg by anti-lg antibody or by a was defined as the phagocytic index, whereas the percentage of ef- multivalent anti-lg antibodydextran conjugate. J. Immunol. 743,1414- fector cells ingesting at least one E target was defined as the percent 1421. phagocytosis. Each experiment, performed on separate days with dif- Cooper, N. R. (1985). The classical complement pathway: activation ferent donors, used duplicate sample wells per condition. Controls of and regulation of the first complement component. Adv. Immunol. 37, unopsonized E, E opsonized with IgM anti-Forssman alone (E-IgM), 151-216. or both were not ingested by monocytes or macrophages under any conditions. In some experiments, monocytes (5 x 105/ml) were pre- Drickamer, K. (1987). Membrane receptors that mediate glycoprotein treated for 15 min at room temperature with 6-20 ug/ml RI 39 (a mouse endocytosis: Structure and biosynthesis. Kidney Int. 32, [Suppl.] IgGPb monoclonal anti-ClqRp [Guan et al., 19911) or control mouse S-l 67-S-l 80. monoclonal IgGPb prior to a 2-fold dilution and addition to chambers Drickamer, K., Dordal, M. S., and Reynolds, L. (1986). Mannose- as above. binding proteins isolated from rat liver contain carbohydrate- recognition domains linked to collagenous tails complete primary Clq Binding Assays structuresand homologywith pulmonarysurfactantapoprotein. J. Biol. It has been previously established that Clq-CLF contains the func- Chem. 261, 6878-6887. tional cell binding domain. Therefore, all assays of Clq binding to Ezekowitz, R. A. 6. (1991). Ante-antibody immunity. Curr. Biol. 1, 60- cells (5 x 105-1 x 106cells/sample) were performed with the iodinated 62. Clq collagen-like fragment (< or = 0.2 ag ‘251-C1q-CLF/sample). The Ezekowitz, R. A. B., Day, L. E., and Herman, G. A. (1988). A human incubations were carried out in 60% RPMI, 0.02 M HEPES, 1% oval- mannose-binding protein is an acute-phase reactant that shares se- bumin, 40% sucrose, 0.02M HEPES, 1% ovalbumin (p = 0.09) as quence homology with other vertebrate lectins. J. Exp. Med. 767, previously described (Tenner and Cooper, 1980). All other aspects of 1034-l 046. the assay were also as previously described (Tenner and Cooper, 1980; Bobak et al., 1986). Assays are usually done in triplicate, except Ezekowitz, R. A. B., Kuhlman, M., Groopman, J. E., and Byrn, R. A. where noted in the figure legends. In all experiments, the total number (1989). A human serum mannose-binding protein inhibits in vitro infec- of counts bound represented less than 6.5% of the total input radiola- tion by the human immunodeficiency virus. J. Exp. Med. 769, 185- bel. Statistical analysis was performed using the student’s t test. 196. Ezekowitz, R. A. B., Sastry, K., Bailly, P., and Warner, A. (1990). Molec- Acknowledgments ular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and Correspondence should be addressed to A. J. T. The authors thank phagocytosis of yeasts in Cos-1 cells. J. Exp. Med. 772, 1785-1794. Dr. J. Ocariz of the University of California at Irvine Medical Center Farries. T. C., Steuer, K. L. K., and Atkinson, J. P. (1990). Evolutionary Blood Bank and Dr. S. Gupta and Ms. C. Head of the University of implications of a new bypass activation pathway of the complement California at Irvine Medical Plaza Infusion Center for collecting blood system. Immunol. Today 11, 78-80. units for these studies. This work was supported by grants from the Friis-Christiansen, P., Thiel, S., Svehag, S., Dessau, R., Svendsen, American Heart Association, the Arthritis Foundation, National Insti- P., Andersen. O., Laursen, S. B., and Jensenius, J. C. (1990). In vivo tutesof Health grant Al-27168 (A. J. T.), and aGrant-in-Aid from Bristol- and in vitro antibacterial activity of conglutinin, a mammalian plasma Myers Squibb (R. A. B. E.). lectin. Stand. J. Immunol. 31, 453-460.

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