Increased infectivity of stationary-phase promastigotes of Leishmania donovani: correlation with enhanced C3 binding capacity and CR3-mediated attachment to host macrophages.

This study demonstrated that the greater infectivity of stationary-phase promastigotes of Leishmania donovani is related to increased complement fixation on the parasite surface, resulting in increased binding to host mononuclear phagocytes (MPs) via complement type 3 receptors (CR3). The in vivo infectivity of log- and stationary-phase promastigotes was compared by measuring parasite loads in the livers of BALB/c mice 14 days after i.v. inoculation. The same populations were tested for their ability to bind to resident murine peritoneal macrophages (RPM) in vitro during a 20-min serum-free incubation period. Stationary-phase parasites displayed both higher in vivo infectivity and increased in vitro binding. However, following uptake by RPM, no significant difference in the 72 hr survival of the two populations could be detected. The in vitro binding of log and stationary parasites was uniformly inhibited in the presence of a mAb (M1/70) specific for CR3, confirming that the interaction of this receptor with its ligand, iC3b, plays a vital role in initial attachment of both promastigote populations. Following incubation with a human serum source, the amount of ligand appeared to be greater on the surface of stationary-phase promastigotes, as indicated by their ability to trigger the alternative complement pathway and by solid-phase ELISA measurements using antiserum specific for human C3. Collectively, these findings suggest that the infectivity of L. donovani promastigotes is influenced by the extent of initial attachment to host MPs, as determined by the levels of complement deposition and subsequent CR3-mediated binding.     

Role of macrophage complement and lectin-like receptors in binding Leishmania parasites to host macrophages

This paper reviews briefly work carried out in our laboratory on the relative roles of the macrophage plasma membrane receptor (CR3) for the cleaved third complement component (iC3b) and the mannosyl/fucosyl receptor (MFR) in binding, ingestion and respiratory burst (RB) response elicited by promastigotes versus amastigotes of Leishmania donovani. In the absence of serum soluble inhibitors (mannan, ribonuclease B) of the MFR cause a dose-dependent reduction in the numbers of promastigotes binding to murine resident peritoneal macrophages and in the proportion of bound parasites eliciting a RB response. For amastigotes no consistent reduction in binding in the presence of mannan is observed but the proportion of parasites eliciting a RB is reduced. Serum-independent binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, is also inhibited by the anti-CR3 monoclonal antibody M1/70, by Fab anti-C3, and by an inhibitor of C3 fixation, sodium salicyl hydroxamate. For amastigotes, which are poor activators of the alternative pathway, a lesser effect is observed with all three inhibitors of CR3-mediated binding. The results obtained with these three independent inhibitors provide strong evidence that cleaved macrophage-derived C3 (iC3b), which can be visualised on the parasite surface in electron microscope sections following addition of anti-C3 antibody and a protein A-gold conjugate, mediates binding to CR3. Modulation experiments in which either CR3 or MFR are rendered inaccessible demonstrate that both receptors must be present on the segment of the macrophage membrane with which the parasite makes contact to mediate binding and ingestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of iC3b binding to human polymorphonuclear leucocytes.

We determined in binding assays using monomeric fluid-phase iC3b and Scatchard analysis that iC3b binds to human polymorphonuclear leucocyte type 3 complement receptor (CR3), a low-density/high-affinity receptor (28,200 binding sites, affinity constant (Ka) = 2.1 +/- 0.47 X 10(6) L/M), and to the C3b receptor (CR1), a high-density/low-affinity receptor (54,700 binding sites, Ka = 1.7 +/- 2.04 X 10(5) L/M. Binding of iC3b to CR1 was confirmed by blocking experiments with polyclonal F(ab')2 antibody against CR1, and competitive binding experiments with C3b. Binding of iC3b to CR3 was demonstrated by blocking experiments with the monoclonal antibody OKM10 against the ligand binding site of CR3. Inhibition of both CR1 and CR3 did not completely reduce iC3b binding, indicating the existence of additional iC3b-binding sites on PMN. Using flow cytometric analysis of receptor expression, no positive or negative co-operativity was observed between CR1 and CR3. Expression of both receptors increased in a dose-dependent manner after incubation with f-met-leu-phe or phorbol myristate acetate; however, only CR3 expression was enhanced at very low concentrations of these stimuli. iC3b/CR3 interactions probably play a central role in host defence against microorganisms.     

Leishmania major-human macrophage interactions: cooperation between Mac-1 (CD11b/CD18) and complement receptor type 1 (CD35) in promastigote adhesion.

It has been suggested that the developmental maturation of Leishmania major promastigotes can affect their interaction with human complement receptors. To study this, we measured the adhesion of metacyclic and logarithmic-phase L. major promastigotes to complement receptors expressed on primary macrophages, to recombinant receptors expressed on transfected cells, or to purified complement receptors in a cell-free system. We demonstrate that complement-opsonized promastigotes can bind to both Mac-1 and complement receptor type 1 (CR1) and that the transition of promastigotes from the noninfectious logarithmic phase of growth to the infectious metacyclic stage does not affect this interaction. Furthermore, we show that Mac-1 and CR1 can cooperate to mediate the efficient adhesion of complement-opsonized metacyclic promastigotes to cells expressing both receptors. On human monocyte-derived macrophages, Mac-1 appears to make a quantitatively greater contribution to this adhesion than does CR1, since blocking macrophage Mac-1 diminishes metacyclic promastigote adhesion to a greater extent than does blocking CR1. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic decrease in complement-dependent promastigote adhesion, relative to normal monocytes. The predominance of Mac-1 in these interactions is due, at least in part, to the factor I cofactor activity of CR1, which facilitates the conversion of C3b to iC3b. The stable adhesion of complement-opsonized metacyclic promastigotes to Mac-1 is a prerequisite for phagocytosis by human monocyte-derived macrophages. Blocking Mac-1 on macrophages abrogates the majority of the complement-dependent phagocytosis of promastigotes, whereas blocking CR1 has no detectable effect on phagocytosis. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic reduction in promastigote phagocytosis relative to normal bovine monocytes. We conclude, therefore, that the two complement receptors, Mac-1 and CR1, can cooperate to mediate the initial complement-dependent adhesion of metacyclic promastigotes to human monocyte-derived macrophages and that Mac-1 is the predominant complement receptor responsible for the phagocytosis of complement-opsonized metacyclic promastigotes.     

Cutaneous host defense in leishmaniasis: interaction of isolated dermal macrophages and epidermal Langerhans cells with the insect-stage promastigote.

Leishmania species are obligate intracellular pathogens of mononuclear phagocytes. Successful infection depends on sequestration of the promastigote (insect form) within host cells, allowing transformation into the relatively hardy amastigote stage. Promastigotes are killed readily by circulating phagocytes and nonimmune serum, suggesting that cutaneous infection is initiated within a permissive cell in the epidermis or dermis. From large sections of primate skin dermal macrophages and epidermal Langerhans cells were isolated, and their interaction with promastigotes of Leishmania major was investigated in vitro. Dermal macrophages were readily infected with promastigotes, and successful transformation to and replication of amastigotes was observed. Ingestion of promastigotes by dermal macrophages was not associated with a significant respiratory burst, in contrast to that by other macrophage populations, and was associated with significantly greater survival of parasites. Stimulation of these cells with phorbol myristate acetate or opsonized zymosan revealed that those cells were generally oxidatively deficient. Langerhans cells could not be successfully infected by promastigotes under similar conditions. Examination of these cells for expression of CR3, which has been identified as a potential Leishmania receptor, revealed that Langerhans cells did not express the alpha M subunit of CR3, whereas dermal macrophages were CR3 positive. These data support the concept that dermal macrophages are the site of initiation of Leishmania infection.     

Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes.

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. Two macrophage receptors, the mannose-fucose receptor (MFR) and the receptor for complement component C3bi, CR3, were examined for their roles in the attachment and ingestion of L. donovani by human monocyte-derived macrophages. Two monoclonal antibodies which bind to the human CR3, anti-Mo1 and anti-Mac-1, inhibited both attachment and ingestion of L. donovani promastigotes after preincubation with human monocyte-derived macrophages; attachment was inhibited by 40 and 62% by anti-Mo1 and anti-Mac-1, respectively, and ingestion was inhibited by 34 and 51% by anti-Mo1 and anti-Mac-1, respectively. The interaction between promastigotes and CR3 may not have involved the C3bi-binding site on CR3, however, because a monoclonal antibody which exhibits specificity for this site, OKM10, inhibited promastigote attachment by only 18%. In contrast, OKM1, which is believed to react with the alternate lectinlike binding site on CR3, inhibited ingestion by 65%. MFR activity was inhibited using the soluble MFR ligands, mannan and mannosylated bovine serum albumin, which also inhibited promastigote attachment by 40 and 37%, respectively. The simultaneous inhibition of both CR3 (by anti-Mac-1) and the MFR (by either mannan or mannosylated bovine serum albumin) resulted in a greater decrease in promastigote attachment than inhibition of either receptor alone. Additionally, the reduction of MFR activity by allowing macrophages to adhere to a mannan-coated surface followed by the addition of anti-CR3 antibodies resulted in an 81% inhibition of promastigote ingestion, a greater decrease than was obtained by manipulation of either receptor alone. The results suggest that the MFR and CR3 independently participate in the attachment and ingestion of L. donovani promastigotes by human macrophages.     

Contribution of CR3, CD11b/CD 18 to cytolysis by human NK cells

The complement receptor CR3 molecule functions in direct intercellular contacts mediated by its beta chain, CD18. Similarly to the Fc receptor (CD16), CR3 is a marker of human natural killer cells. We have shown that opsonization of NK targets with iC3b leads to their increased lytic sensitivity. Opsonization could be achieved by incubating certain B and T cell lines in human serum. The expression of CR2 was a prerequisite for C3 fragment fixation. The CR2 negative cell line, P3HR1 could be opsonized by incubation in human serum when induced to express the EBV envelope glycoprotein gp350. C3b or iC3b could also be deposited artificially on cell surfaces by chemical coupling to surface reactive antibodies. Similarly to the function of macrophages and monocytes, contact with opsonized targets exclusively through the iC3b binding site of CR3 did not seem to trigger NK function. We attempted to clarify the functional role of other CR3 ligands. The beta chain of the molecule, CD18, was essential to the NK effect. The NK targets did not seem to interact with the beta-glucan binding epitope on the alpha chain of CR3, CD11b. On the other hand, the cytolytic function could be enhanced through this epitope with the appropriate ligand.     

An interaction between CD16 and CR3 enhances iC3b binding to CR3 but is lost during differentiation of monocytes into dendritic cells

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC.     

Treatment of murine macrophages with interferon-gamma inhibits their ability to bind leishmania promastigotes.

The binding of leishmania promastigotes to macrophages pretreated with interferon-gamma (IFN-gamma) was compared to binding to untreated (resident) cells. IFN-gamma-treated macrophages bound fewer leishmania promastigotes than did untreated cells. The decreased binding was apparent over a wide dose range of parasite inocula when the assays were performed in the absence of exogenous complement. This decrease was specific to leishmania, since treated and untreated macrophages bound comparable amounts of immunoglobulin G- and complement-coated sheep red blood cells. Decreased parasite binding occurred early in the macrophage activation pathway. Pretreatment of macrophages with IFN-gamma for as little as 6 h, a time insufficient to induce other macrophage activation parameters, significantly reduced their ability to bind leishmania promastigotes. To determine the mechanism of this decreased phagocytosis by activated cells, macrophages were pretreated with specific inhibitors before the addition of leishmania. The binding of promastigotes to untreated (resident) macrophages was inhibited by approximately 50% by reagents that blocked either of two macrophage receptors, complement receptor type 3 (Mac-1) or a leishmania species-specific lectin-like receptor. Binding to IFN-gamma-treated macrophage populations, in contrast, was substantially inhibited only by antibody to Mac-1. Saccharides that were 50% inhibitory in the resident cell population, decreased binding by less than 10% in activated cells. The lack of saccharide inhibition by IFN-gamma-treated cells was also reflected in an inability of activated macrophages to bind to beads coated with purified leishmania lipophosphoglycan (LPG). These LPG-coated beads bound well to resident macrophages but poorly to activated cells. Thus, leishmania bind to macrophages by two distinct mechanisms, one that utilizes Mac-1 and a second mechanism that does not depend on complement and is saccharide inhibitable. These two binding mechanisms are distinct and differentially regulated in resident and activated cells.     

Recognition of binding sites on Candida albicans by monoclonal antibodies to human leukocyte antigens.

Candida albicans exhibits examples of human molecular mimicry, including receptors resembling human steroid receptors and human complement receptor (CR)-like receptors that bind the complement fragments C3d and iC3b. To further characterize the epitopes of the Candida human CR-like molecules, a panel of monoclonal antibodies (MAbs) against epitopes within the human CR3 was used. MAbs Mo1, M1/70HL, and 7C3 bound to the germ tube, as assessed by immunofluorescence. MAbs Leu15, 60.1, and 95G8 did not bind. Miscellaneous MAbs against other antigens on human leukocytes (B2, 3D9, and OKT4) did not bind. However, MY9, which recognizes a monocyte antigen, bound extensively to the germ tube. The binding of certain anti-CR3 MAbs suggested limited identity between the Candida CR3-like receptor and the human CR3. The binding of MY9 identified an antigen recognized by a MAb to a human cell antigen not previously known to exist on C. albicans.     

A study of the differential respiratory burst activity elicited by promastigotes and amastigotes of Leishmania donovani in murine resident peritoneal macrophages.

Acridine orange and ethidium bromide and a combination of fluorescent and transmitted light microscopy used in conjunction with the qualitative nitroblue tetrazolium assay for superoxide anion (O2-) release demonstrated dramatic differences in the binding of and respiratory burst (RB) activity elicited by promastigotes and amastigotes of Leishmania donovani in resident peritoneal macrophages (M phi) from C57BL/10ScSn mice. When amastigotes were incubated with M phi for 30 min the number of parasites per 100 M phi was 2-4-fold higher, a higher proportion of M phi became infected and the mean number of parasites per infected M phi was higher than in promastigote infections. RB activity was higher for promastigotes than amastigotes both in terms of the percentage of infected M phi containing formazan positive parasites and the percentage of individual formazan positive parasites. In an attempt to explain the differential response to promastigotes and amastigotes, RB activity was examined for sodium azide-treated, glutaraldehyde-fixed and heat-killed parasites and for various transformation intermediates between amastigotes and promastigotes. Binding and RB activity were also examined in conjunction with competitive binding assays designed to determine the specific receptors involved in ligand binding of both forms of the parasite to the M phi. The results indicate that, while amastigotes may possess an azide-sensitive mechanism which either competes for O2- produced or causes localized inactivation of RB activity, this cannot account for the full magnitude of the difference between the two forms of the parasite. The transformation and competitive binding studies suggest that the more likely explanation lies in both qualitative and quantitative differences in the distribution of surface ligands involved in binding the parasite to the M phi plasma membrane and that the well characterized mannose/fucose receptor may be important in promastigote, but not amastigote, binding and RB activity.     

Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.

Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR2 interaction may suggest a possible immunoregulatory role of HSV glycoprotein C.     

Enhanced binding of immune complexes processed by erythrocyte CR1 (CD 35) receptors to purified CR2 (CD 21) receptors from tonsillar mononuclear cells

The binding of immune complexes (IC) opsonized by serum complement (C) and IC processed by CR1 (CD 35) receptors on human erythrocytes (E) to purified CR2 (CD 21) receptors was compared. Soluble CR2 was prepared from tonsillar mononuclear cells and purified by antibody affinity chromatography. Solid phase CR2 as well as CR2 subjected to PAGE and blotted onto nitro-cellulose membranes bound 125I-labelled BSA anti-BSA IC which had been opsonized by C and processed by CR1 up to ten times more efficiently than IC reacted with serum only. Radiolabelled monomeric C3d also bound to solid phase CR2. The binding of IC to purified and solid phase bound CR2 could be inhibited by anti-CR2 antibodies or by preincubation of the IC with polyclonal antibodies reacting with C3d or C3b/iC3b. Thus, both C3dg and iC3b appeared to mediate binding of IC to CR2. Preincubation of solid phase CR2 with purified monomeric C3d did not inhibit the subsequent binding of E-CR1 processed IC. The data indicate that E-CR1 have an important role in generating IC which bind effectively to CR2 receptors on B lymphocytes.     

Structural and functional relationships among receptors and regulators of the complement system

The classical and alternative pathway of complement activation are regulated by a series of fluid phase and cell-bound factors, some of which at the same time serve as receptors for fragments of C3 and C4. These molecules are factor H, CR1 (C3b/C4b receptor), CR2 (C3d/EBV receptor), C4BP (C4b binding protein), DAF (decay accelerating factor), MCP (membrane cofactor protein; earlier designated p45/70), CR3 (iC3b receptor or Mac-1) and CR4 (protein 150/95). Due to structural, genetic and functional features these factors are members of one or several newly recognized large families of proteins: (1) molecules with 60 amino acids long repeats (H, CR1, CR2, C4BP, DAF); (2) proteins with 1,2-diacylglycerol membrane anchoring (DAF); (3) proteins with a heterodimer structure and preference for ligands containing the tripeptide arginine-glycine-asparagine (CR3, CR4). Recognizing the above mentioned regulators and receptors of the complement system as belonging to these protein families opens new perspectives for further genetic and functional research of mutual interest to complement and noncomplement scientists.     

Family studies of erythrocyte complement receptor type 1 levels: reduced levels in patients with SLE are acquired, not inherited.

It has been claimed that patients with systemic lupus erythematosus (SLE) have an inherited deficiency of erythrocyte complement receptor type 1 (CR1, with ligand binding specificity for C3b, iC3b and C4b). CR1 functions as the only cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg. The activity of this receptor on red cells may be an important mechanism for handling immune complexes which have bound C3b or iC3b. Radioligand binding studies were performed using a monoclonal antibody to CR1, E11, to enumerate these receptors accurately. The results confirmed that patients with SLE have a reduced number of CR1 molecules per red cell, but showed no reduction in CR1 levels amongst their consanguineous relatives. Study of 13 normal families suggested the presence of heritable factors controlling the numbers of erythrocyte CR1 molecules; in particular there was a correlation between mean parental CR1 numbers and CR1 numbers in their children. However, amongst 17 families of 19 patients with SLE, four families were identified in which genotypically 'high CR1' SLE patients had persistently low phenotypes. This is not compatible with the hypothesis that the reduction in erythrocyte CR1 numbers in these patients is inherited.     

The structural basis for complement receptor type 2 (CR2, CD21)-mediated alternative pathway activation of complement: studies with CR2 deletion mutants and vaccinia virus complement-control protein-CR2 chimeras

The role of complement receptor 2 (CR2) short consensus repeats (SCR) in binding of hydrolyzed C3 (iC3) to form an alternative pathway (AP) convertase, and promoting C3 fragment deposition following AP activation, was examined. We used (1) K562 cells transfected with CR2 constructs, where the C3d-binding site of CR2 (SCR1+2) was replaced with the four-SCR vaccinia virus complement control protein (VCP), or truncation mutants thereof, and (2) COS cells transfected with wild-type (wt) CR2, or deletion mutants thereof. AP activation required iC3 binding in both systems. Thus, the VCP-CR2 chimera had an iC3 binding efficiency of 11.4 %, compared to wtCR2, and a relative AP activity of 5.5 %, the truncation mutants being inactive. Of the CR2 mutants, only EK (DeltaSCR10 - 11) had AP activity similar to wtCR2. NN (DeltaSCR6 - 8) and NOP (DeltaSCR6-mid14) had reduced AP activity, but near normal iC3 binding. XB (DeltaSCR3 - 6) and PP (DeltaSCR3-mid14) were inactive in both assays. We conclude that, whilst iC3 binding to CR2 via SCR1 - 4 is essential for AP activation, the efficiency of C3 deposition also depends on the midportion of CR2.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Human Alveolar Macrophages Cultured on Fibronectin in Vitro

We studied the phagocytosis of agarose beads by human alveolar macrophages in terms of the morphology, the receptors involved, and the cellular substrates (plastic or fibronectin) used. Beads coated with C3b (58%) and iC3b (42%) by treatment with serum, were ingested during 45 min by CR1 and CR3 on the macrophages. This ingestion was inhibited 80-90% by the presence of polyclonal F(ab')2 anti-C3 fragments. Since the phagocytosis of both C3b- and iC3b-coated beads was about threefold stronger than for C3b-coated beads (trypsinized serum-treated beads), the results indicate that the CR3 is more phagocytic than the CR1. The phagocytosis of initially complement uncoated beads, which are slowly opsonized with macrophage-produced C3b and iC3b in vitro, was also strongly inhibited (70-80%) by the presence of anti-human C3 F(ab')2 fragments. There was an increased phagocytosis (10-17%) of complement precoated beads by macrophages cultured on the fibronectin substrate versus the plastic substrate. The morphology and rapid phagocytosis of the complement precoated beads was demonstrated by SEM. The general impression was that membranous protrusions stretched towards the beads, which became increasingly enclosed by plasma membrane.     

Co-operation between human CR1 (CD35) and CR2 (CD21) in internalization of their C3b and iC3b ligands by murine-transfected fibroblasts

CR1 and CR2 are expressed as associated proteins on the B-lymphocyte surface. To investigate their respective contributions to the internalization of C3 fragments, transfected murine fibroblasts expressing human CR1, CR2, or both CR1 and CR2 were produced. CR1- and CR1-CR2-expressing cells bound C3b and C3b-dimer whereas CR2- and CR1-CR2-expressing cells bound iC3b and C3de. In all cases, maximum binding was achieved at low ionic strength. CR1-CR2-positive cells internalized two- to threefold more C3b and 1.5-fold more iC3b than CR1- and CR2-single-positive cells, respectively. Internalization of the anti-CR1 antibody J3D3, or C3de was at the same level, in both double-transfected and single-transfected cells. Furthermore, the internalization of C3b dimer by CR1-CR2 cells was impaired in the presence of OKB7, an anti-CR2-blocking antibody, but it was not altered in CR1 cells. Taken together, these findings suggest that CR1 and CR2 collaborate to internalize C3b and iC3b proteins. We suggest that the induction of conformational changes of the ligands enhances their binding to both receptors.     

Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein.

We describe here that parasites of the genus Leishmania contain a cytolytic activity which acts optimally at pH 5.0 to 5.5 and at 37 degrees C in vitro. or the four species examined, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) major presented considerable hemolytic activity, whereas Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis showed little and no hemolytic activity, respectively. The cytolytic factor of L. amazonensis promastigotes was characterized as a protein with no protease-, phospholipase-, or detergent-like activity, probably localized inside membranous vesicles. The use of osmotic protectants revealed the colloid-osmotic nature of hemolysis, which is indicative of pore formation in the membranes of target cells. This putative pore-forming protein also damaged nucleated cells, including macrophages, causing an increase in their membrane permeability with leakage of cytoplasmic proteins. Both promastigotes and amastigotes express this lytic activity, suggesting that the cytolysin may have a function in both stages of this parasite. The pH and temperature required for optimal activity indicate that it might be more effective within the mammalian host, particularly inside the macrophage parasitophorous vacuole. In promastigotes of L. amazonensis, the expression of lytic activity seems to be regulated during their growth in vitro, being maximal at the early stationary phase.     

Complement receptor 3 binds the Borrelia burgdorferi outer surface proteins OspA and OspB in an iC3b-independent manner

Persistence of borreliae within the vertebrate host depends on the fate of interactions between the spirochetes and target cells. The present work demonstrates the direct binding of the Borrelia burgdorferi outer surface proteins OspA and OspB to CR3 and that this binding is independent of iC3b.