Human L-ficolin,a Recognition Molecule of the Lectin Activation Pathway of Complement,Activates Complement by Binding to Pneumolysin,the Major Toxin of Streptococcus pneumoniae

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q^−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.     

The heterocytotoxicity of human serum. I. Activation of the alternative complement pathway by heterologous target cells.

Human serum induces cytolysis of mouse thymus and thymoma cells, and cytostasis of mouse bone marrow and spleen cells, and various methylcholanthrene-induced tumour cells. The latter was manifested by deficient metabolic activity when cultured in the presence of fresh human sera. Decomplementation procedures demonstrated that these heterocytotoxic effects are mediated in part via activation of the alternative complement pathway in human serum samples. The presence of properdin and C3 on the target cell surface was confirmed by immune adherence and indirect immunofluorescent tests. Activation of the alternative complement pathway was elicited by incubation of the human serum with the relevant target cells, resulting in the appearance of the cathodal migrating fragment of the factor B, denoting complement activation. The following publication will present evidence that activation of the alternative complement pathway takes place via an antibody-independent mechanism acting at the cell surface. These and other observations in the literature raise the possibility that activation of the alternative complement pathway by surface cell receptors on tumour cells represents a mechanism of natural immunity versus tumours.     

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg(2+) buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.     

Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle

P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.     

Ala-1: A murine alloantigen of activated lymphocytes

Ala-1 (activated lymphocyte antigen-1) is a murine alloantigen expressed on mitogen-stimulated peripheral T and B cells. C3H/An mice were immunized with PHA-stimulated C58 lymphocytes; reciprocal immunizations were also performed. After multiple absorptions to remove unwanted antibody specificities, the antiserum did not lyse thymocytes, lymph node, or spleen cells, but killed more than 90% of Con A-stimulated T cells and more than 90% of LPS-stimulated B cells in cytotoxicity tests. Quantitative absorption studies confirmed that thymocytes are Ala-1^–, but revealed the presence of some Ala-1 antigen in normal lymph node and spleen populations. The strain distribution of Ala-1 was determined for 23 inbred strains. The reactions of the two reciprocal antisera (C3H anti-C58, and C58 anti-C3H) were mutually exclusive on all strains tested, indicating that the antisera probably recognize antithetical forms of Ala-1. Since thymocytes cultured with Con A do not express Ala-1, whereas peripheral mitogen-stimulated cells do, we propose that Ala-1 is a differentiation antigen, the expression of which is restricted to the late stages of development of T and B cells.Abbreviations used in this paper are Con A concanavalin A - PHA phytohemagglutinin - LPS lipopolysaccharide - C complement - BSA bovine serum albumin - B6 C57BL/6 mice - FBS fetal bovine serum     

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.     

Guinea pig erythrocytes, after their contact with influenza virus, acquire the ability to activate the human alternative complement pathway through virus-induced desialation of the cells

Guinea pig erythrocytes that had been exposed to influenza A virus activated the alternative complement pathway in whole human serum in the absence of natural antibodies. Because all virus particles were eluted from the treated cells, activation was not dependent on antiviral antibodies or on virus particles themselves. The relative capacity of treated erythrocytes to activate the alternative pathway was dependent on the amount of virus to which the cells had been exposed and was directly related to the amount of sialic acid removed from the erythrocyte membrane during incubation with either whole virus particles or purified viral sialidase. C3b bound to cells that had been treated with virus, and P-stabilized amplification convertase sites P,C3b,Bb formed on these cells, exhibited increased resistance to the action of the regulatory proteins beta-1H and C3b Ina compared with C3b and P,C3b,Bb on untreated, nonactivating cells. The acquired resistance of the cell-bound, P-stabilized amplification convertase to decay-dissociation by beta-1H was directly related to the activating capacity of the treated cells in whole serum (r = 0.95) and to the amount of sialic acid removed from the cells by the virus (r = 0.98). Desialation represents a specific alteration of the cell surface by which a nonimmune host, through activation of the alternative pathway, may deposit C3b on a target cell that had been exposed to influenza virus and may lyse virus virus-modified cells during orthomyxovirus infections.     

Natural IgM antibodies are involved in the activation of complement by hypoxic human tubular cells

Ischemia-reperfusion injury (IRI) has a major impact on graft survival after transplantation. Renal proximal tubular epithelial cells (PTEC) located at the corticomedullary zone are relatively susceptible to IRI and have been identified as one of the main targets of complement activation. Studies in mice have shown an important role for the alternative pathway of complement activation in renal IRI. However, it is unclear whether experimental data obtained in mice can be extrapolated to humans. Therefore, we developed an in vitro model to induce hypoxia-reoxygenation in human and mouse PTEC and studied the role of the different pathways of complement activation. Exposure of human PTEC to hypoxia followed by reoxygenation in human serum resulted in extensive complement activation. Inhibition studies using different complement inhibitors revealed no involvement of the alternative or lectin pathway of complement activation by hypoxic human PTEC. In contrast, complement activation by hypoxic murine PTEC was shown to be exclusively dependent on the alternative pathway. Hypoxic human PTEC induced classic pathway activation, supported by studies in C1q-depleted serum and the use of blocking antibodies to C1q. The activation of the classic pathway was mediated by IgM through interaction with modified phosphomonoesters exposed on hypoxic PTEC. Studies with different human sera showed a strong correlation between IgM binding to hypoxic human PTEC and the degree of complement activation. These results demonstrate important species-specific differences in complement activation by hypoxic PTEC and provide clues for directed complement inhibition strategies in the treatment and prevention of IRI in the human kidney.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Activation of the alternative complement pathway by isolated human glomerular basement membrane

The capacity of isolated human glomerular basement membrane (GBM) to initiate surface activation of the human alternative complement pathway was defined by the deposition of C3b under circumstances in which the classical complement pathway was inoperative. The deposition of C3b from normal or C2-deficient serum was time- and magnesium-dependent, implying a role for the alternative pathway. Normal human serum rendered deficient in D did not sustain C3b deposition until its reconstitution with D, indicating an absolute requirement for a protein unique to the alternative pathway and essential to the cleavage activation of the C3 amplification convertase of that pathway. The capacity of the excess control proteins H and I to prevent C3b deposition onto GBM incubated in C2-deficient serum provided further evidence for the direct activation of the alternative pathway in this system. The use of radiolabeled monoclonal antibody to localize the deposited C3b afforded specificity and quantitation of about 100 ng of C3b/mg of GBM. Immunohistochemical analysis with a monoclonal antibody to detect C3b demonstrated its deposition to be confined to the epithelial surface of the GBM.     

Plasmodium berghei: sporozoites are sensitive to human serum but not susceptible host serum.

Human complement was activated by rodent malaria, Plasmodium berghei, sporozoites through the alternative pathway, as revealed by C3 deposition on sporozoites using the fluorescent antibody technique. Sporozoites exposed to fresh human serum decreased in infectivity to HepG2 cells, but those exposed to heated or C3-deficient human serum showed normal infectivity to HepG2 cells. In contrast, C3 deposition was not observed on the sporozoites treated with mouse or rat serum even in the presence of specific polyclonal anti-sporozoite antibody. However, following treatment with trypsin (250 micrograms/ml), 81% of salivary gland sporozoites and 49% of oocyst sporozoites became reactive with mouse serum, and reactive sporozoites deposited mouse C3 on their surface in the presence of 30 mM EGTA and 1 mM Mg2+ without antibody. Concomitantly some sporozoites lost reactivity to anti-circumsporozoite protein monoclonal antibody. These results suggest that P. berghei sporozoites possibly express surface molecules that regulate the complement activation pathway of susceptible hosts but not of nonhosts, and that the putative structures consist of protease-sensitive molecule(s) which are closely associated with the circumsporozoite protein.     

Activation of complement by tetrathyridia of Mesocestoides corti: enhancement by antibodies from infected mice and lack of effect on parasite viability

Tetrathyridia of the cestode Mesocestoides corti were isolated from the peritoneal cavity of infected mice. The parasites activated guinea pig and mouse complement (C) in vitro by both the classical and alternative pathways as shown by quantitative C fixation and crossed immunoelectrophoresis. The ability of tetrathyridia to activate mouse C was enhanced by preincubating the parasites in serum obtained from mice infected with M. corti. Antibodies of the IgG1 class, an immunoglobulin found in profoundly increased amounts in mice infected with M. corti, as well as IgM and IgG2 antibodies, bound to cultured tetrathyridia and facilitated deposition of the third component of C (C3) from dilute mouse serum, presumably via classical pathway activation. The results demonstrate that mouse IgG1 antibodies do not prevent the activation of C by the tetrathyridia or by C-fixing antibodies of other classes which become attached to the tetrathyridia. The activation of C in vitro by tetrathyridia did not affect their ability to grow in mice, even though C3-derived polypeptides could be detected by immunofluorescence on the surface of the parasites.     

Rapid removal of mycoplasma from cell lines mediated by a direct effect of complement

Mycoplasma can be removed from the surface of contaminated human and murine cell lines by incubation for 4 h with human, rabbit, guinea pig, or mouse sera. Several lines of evidence suggest the involvement of complement in this process: (1) The activity can be abrogated by heat treatment (56 degrees C for 45 min). (2) Using monoclonal antibodies directed against C3a and C3b, the deposition of C3b fragments on the surface of mycoplasma-positive cells can be demonstrated after 1 h incubation with human serum. (3) Ca2+ depletion ablates the ability of serum to remove the activity. (4) C2def' sera are inactive while addition of purified C2 reconstitutes the activity. The latter two findings implicate that activation of the classical pathway of complement is responsible for the effect. Antibody, however, is not required as demonstrated by the uncompromised activity of Ig-deficient sera from bursectomized chicken. Treatment with human serum or rabbit serum was used successfully to permanently cleanse 10/10 tumor cell lines of human and of murine origin. The complete removal of mycoplasma was monitored over at least 8 weeks by direct DNA staining and confirmed by agar culture and transfer of supernatants to mycoplasma-free Vero cells followed by DNA staining. Thus the direct interaction of mycoplasma and complement appears to be an effective and rapid means of curing cell lines from mycoplasma.     

Complement is an essential component of the immune response to adeno-associated virus vectors

Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-κB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1β (IL-1β), IL-8, and MIP-1β. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.     

Studies of the properties of a streptococcal preparation, OK-432 (NSC-B116209), as an immunopotentiator

Summary The present study was designed to examine the mechanism by which OK-432 triggers the cytotoxic activity of peritoneal exudate cells (PEC). When OK-432 was incubated with freshly harvested mouse serum, the formation of complexes of OK-432 with the third component of complement (C3) was demonstrated by using ¹³¹I-labeled mouse C3. The formation of C3-OK-432 complexes was totally abolished by a chelating compound, EDTA, which had been shown to inhibit the OK-432 induced activation of the alternative complement pathway. The C3-OK-432 complexes thus obtained bound to the resident PEC, which were subsequently shown to be activated. These activated PEC had augmented cytostatic activity against MM2 cells, a mouse mammary carcinoma.Further, the PEC from mice which had received an IP injection of OK-432 4–5 days previously were cytostatic against MM2 cells and also inhibited the growth of MM2 cells in culture. In contrast, resident PEC stimulated rather than inhibited the ³H-thymidine uptake by MM2 cells and the growth of MM2 cells. The mechanism of PEC (presumably macrophages) activation by OK-432 is discussed.     

Regulation of Alternative Pathway Activation and C3a Production by Adipose Cells

Adipose tissue is a major source of adpisin/factor D of the alternative pathway of complement. Adipose tissue also expresses the two other complement components which are involved in the first activation step of the alternative pathway, factor B and C3, and this step is activated in adipose tissue, producing C3a/Acylation Stimulating Protein (C3a/ASP), a stimulator of triglyceride synthesis. Complement activation is a highly regulated process, however, nothing is known about regulation of complement activation in adipose tissue. To gain insight into the nature of adipose complement activation and its regulation, we have now examined the expression of several complement activation regulatory genes, and analyzed the production of C3a/ASP in lean vs. obese, adipsin-deficient mice. We found that undifferentiated preadipocytes expressed the mRNAs encoding the negative regulatory proteins Crry and factor H, but expression of both genes was decreased upon differentiation. The positive regulator properdin, as well as Crry and factor H, were found in adipose tissue. None of these genes was regulated in murine genetic obesity. To investigate the relative levels of complement activation in lean vs. adipsin-deficient obese mice, we developed a radioimmunoassay for measurement of murine C3a/ASP in plasma. We report that there was no significant difference in the level of C3a in lean vs. obese plasma; however, we found a positive correlation between C3a and plasma triglyceride levels in normal lean mice.     

Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation.     

IL 1 requirement for B cell activation revealed by use of adult serum

Fetal calf serum is an essential component of the culture medium developed by Mishell and Dutton for the immunization of murine spleen cells in vitro. Serum from adult donors (mouse, human, rabbit) does not support antibody synthesis in this system. This "deficiency" of adult serum can be overcome with IL 1. Adult serum in the presence of IL 1 is as effective in stimulating a B cell response against xenogeneic red cells as fetal calf serum. We attribute the capacity of fetal calf serum to support an immune response in the absence of exogenous IL 1 to serum factors that cause macrophages to release IL 1 endogenously. Our findings strengthen the notion that IL 1 plays an essential role in the process of B cell activation and suggests that the use of fetal calf serum should be avoided in studies concerned with the function of interleukin 1.     

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg-EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf-dependent, antibody-independent and complement-mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.     

Trypanosoma brucei: recognition in vitro of two developmental forms by murine macrophages

In vitro, murine macrophages attach to the midgut form of Trypanosoma brucei in the absence of exogenous proteins. Recognition appears to be specific and saturable. Attachment is mediated by a non-Fc, non-C3 receptor on the macrophage plasma membrane. The attachment mechanism is protease sensitive, temperature sensitive, requires the presence of divalent cations, and is functional in fetal calf serum-free medium. The midgut form is also lysed in normal rabbit serum by activating the alternative complement pathway. The form isolated from the blood is neither lysed in normal serum nor is it spontaneously recognized by the macrophage, in vitro. Partial trypsinization of the bloodstream form, however, results in both the triggering of alternative complement pathway lysis and spontaneous uptake into macrophages. Murine macrophages attach to the midgut form of T. brucei by a receptor on the macrophage plasma membrane which is capable of recognizing particulate activators of the alternative complement pathway.