Binding of the 68-kilodalton protein of Mycobacterium avium to alpha(v)beta3 on human monocyte-derived macrophages enhances complement receptor type 3 expression.

Attachment to and uptake by host cells are important early events in the pathogenesis of intracellular organisms such as Mycobacterium avium. Monocyte-derived macrophages (MDM) are known to express multiple surface receptors that play a role in binding to and uptake of M. avium. These include complement receptor type 3 (CR3), fibronectin receptor, mannose receptor, and transferrin receptor. In addition to these, we have previously reported that the integrin receptor alpha(v)beta3 also plays a role in binding to M. avium in a nonopsonic environment. Further, we have shown that a 68-kDa surface protein of M. avium binds to human monocytes and plays a role in attachment of M. avium to MDM. The present study provides direct evidence that this protein mediates attachment of M. avium to MDM by binding to alpha(v)beta3. Using the technique of cell surface enzyme-linked immunosorbent assay, we have shown that the M. avium 68-kDa protein inhibits the binding of monoclonal antibodies (MAb) against alpha(v)beta3 to MDM compared to control proteins such as ovalbumin and laminin (P < 0.05). Dual-labeling studies were performed to demonstrate that after phagocytosis, alpha(v)beta3 is present along with M. avium in phagosomes of M. avium-infected MDM. In addition, we have demonstrated that this interaction between alpha(v)beta3 and the M. avium 68-kDa protein resulted in enhancement of CR3 expression, which is known to play a role in complement-mediated uptake of M. avium. Attachment of MDM to wells coated with the M. avium 68-kDa protein resulted in a twofold increase in CR3 expression compared to attachment of MDM to wells coated with ovalbumin. This enhancement was completely inhibited by pretreatment of MDM with MAb against alpha(v)beta3. In summary, M. avium binds to MDM via alpha(v)beta3 with the help of the M. avium 68-kDa protein, and this ligation enhanced the expression of CR3 on MDM. Since CR3 has been known to play a role in M. avium uptake, enhanced expression of this receptor mediated by M. avium-alpha(v)beta3 interaction indicates a complex mechanism of communication among different receptors that participate in M. avium attachment and uptake. These findings add to current understanding of the roles played by multiple receptor-ligand systems in uptake and pathogenesis of intracellular pathogens such as M. avium.     

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.     

Roles of complement and complement receptor type 3 in phagocytosis of Listeria monocytogenes by inflammatory mouse peritoneal macrophages.

Listeria monocytogenes is a facultative intracellular bacterium that is phagocytosed by and can proliferate within cells of the mononuclear phagocyte system. However, the receptors used by macrophages to internalize this organism have not been identified. In the experiments described here, the contributions of serum complement component C3 and macrophage complement receptor type 3 (CR3) to opsonization and phagocytosis of L. monocytogenes by mouse inflammatory peritoneal macrophages were studied. An assay which allowed the distinction of adherent versus internalized bacteria was used to show that following mixing of L. monocytogenes with inflammatory macrophages, greater than 95% of the bacteria bound were internalized by these phagocytes. When immunofluorescent antibodies to C3 and immunoglobulin were used, C3 but not immunoglobulin was detected on L. monocytogenes following incubation in normal serum or ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetracetic acid-Mg(2+)-chelated serum. When macrophages were incubated with 5% serum and L. monocytogenes in a standard assay, approximately 80% of the phagocytosis was inhibited by heat-inactivated serum or by the addition of F(ab')2 anti-C3 antibody. The role of macrophage CR3 was demonstrated by the ability of anti-CR3 monoclonal antibody M1/70 to decrease phagocytosis to the same levels as those seen with heat-inactivated serum. These experiments indicated that in the presence of normal serum, L. monocytogenes is phagocytosed by inflammatory macrophages primarily because CR3 on these cells binds to C3 deposited on the bacterial surface.     

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.     

Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins

Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the alpha5, alphaV, beta1, and beta3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells.     

Platelet adhesion to radiofrequency glow-discharge-deposited fluorocarbon polymers preadsorbed with selectively depleted plasmas show the primary role of fibrinogen

Fluorocarbon radio-frequency glow-discharge (RFGD) treatment has previously been shown to cause decreased platelet adhesion despite the presence of adsorbed fibrinogen on the surfaces. In this study platelet adhesion to fluorocarbon RFGD-treated surfaces preadsorbed with human plasma was further examined. A series of plasma deposited fluorocarbon thin films were made by varying the C3F6/CH4 ratio in the monomer feed. The surfaces were preadsorbed with plasma, serum, or plasma selectively depleted of fibronectin, vitronectin, or Von Willebrand factor, and platelet adhesion was measured. We also measured fibrinogen adsorption to the surfaces from plasma, monoclonal antibody binding to adsorbed fibrinogen and SDS elutability of the adsorbed fibrinogen. The antibodies used bind to the three putative platelet binding sites on fibrinogen, namely, M1 antibody binds to the dodecapeptide at the C-terminus of the gamma chain, gamma (402-411), R1 antibody binds to a sequence in the Aalpha chain (87-100) which includes RGDF at Aalpha (95-98) and R2 antibody binds a sequence in the Aalpha chain (566-580) which includes RGDS at Aalpha (572-575). Fibrinogen was found to play a decisive role in mediating platelet adhesion to the fluorocarbon surfaces contacting plasma. Few platelets adhered to the fluorocarbon surfaces preadsorbed with serum, while preadsorption with plasma selectively-depleted of either fibronectin, vitronectin, or von Willebrand factor did not decrease platelet adhesion significantly. Replenishment of exogenous fibrinogen to serum restored platelet adhesion, while replenishment of the other proteins had no effect. Platelet adhesion to the fluorocarbon surfaces was lower than to PET or the methane glow-discharge-treated PET. However, there was no apparent correlation between platelet adhesion and the amount of fibrinogen adsorption or monoclonal antibody binding to surface-bound fibrinogen.     

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.     

C3bi/CR3 is a Main Ligand-Receptor Interaction in Attachment and Phagocytosis of C3-Coated Particles by Mouse Peritoneal Macrophages

We investigated the relative role of C3bi-CR3 interaction in the binding and phagocytosis of EAC43 by mouse peritoneal macrophages. Anti-Mac-1 F(ab')2 markedly inhibited the binding and lymphokine-induced phagocytosis of both EAC43b and EAC43bi. Fifty per cent inhibition of attachment and phagocytosis occurred at 1 microgram/ml of anti-Mac-1 F(ab')2 in the incubation media. On the other hand, EIgG binding and phagocytosis were not inhibited at all even at a concentration of 10 micrograms/ml. Depletion of divalent cations from the incubation media abolished EAC43b and EAC43bi rosettes but not EIgG rosettes or phagocytosis. These data suggested that both EAC43b and EAC43bi binding to macrophages were mediated via CR3. Because a drastic decrease of EAC43bi rosettes was observed in the case of EAC43bi cells prepared with smaller amounts of C3, a small contamination of C3bi molecules on EAC43b, itself, cannot explain the efficient attachment of EAC43b. We propose that EAC43b on the macrophage surface can be quickly converted to EAC43bi, forming EAC43bi rosettes, and that those erythrocytes are vigorously ingested by lymphokine-activated macrophages. In accordance with this hypothesis, we demonstrated that EAC43b was converted to EAC43bi in the medium in which macrophages had been incubated.     

Monoclonal antibodies that recognize distinct epitopes of the macrophage type three complement receptor differ in their ability to inhibit binding of Leishmania promastigotes harvested at different phases of their growth cycle.

The macrophage receptor CR3 has been shown by several investigators to be involved in the binding of Leishmania promastigotes to host macrophages. This receptor is known to recognize iC3b and to mediate direct lectin-like attachment of particles such as yeast zymosan. In the present study, two anti-CR3 monoclonal antibodies, M1/70 and 5C6, which ligate different epitopes of murine CR3, have been used in conjunction with sodium salicyl hydroxamate (Saha; inhibits covalent ester linkages of C3 to an activator surface) to block binding of L. donovani and L. major promastigotes harvested at different phases of their growth cycle. M1/70 inhibited all promastigote binding. 5C6 and Saha blocked in parallel only the binding of peanut agglutinin (PNA)-positive late log and early stationary phase parasites. These results suggest that the binding PNA-positive parasites to CR3 is iC3b-mediated, while entry of the more infective PNA-negative late stationary phase promastigotes into host macrophages may involve direct lectin-like binding to CR3.     

Growth within macrophages increases the efficiency of Mycobacterium avium in invading other macrophages by a complement receptor-independent pathway.

Infections caused by organisms of the Mycobacterium avium complex occur in approximately 50 to 60% of patients with AIDS. M. avium is an intracellular pathogen that survives and multiplies within mononuclear phagocytes. In this study, we investigated the uptake of M. avium grown within macrophages (intracellular growth M. avium [IG]) by a second macrophage compared with M. avium cultured in broth (extracellular growth M. avium [EG]). The results showed that IG was six- to eightfold more efficient than EG in entering macrophages. In addition, while an anti-CR3 antibody was able to inhibit approximately 60% of EG uptake by macrophages, it failed to inhibit the entry of IG. In contrast to EG, IG uptake into macrophages was significantly inhibited in the presence of anti-beta1-integrin and anti-transferrin receptor antibodies. Entry into macrophages by alternate receptors was associated with resistance to tumor necrosis factor alpha (TNF-alpha) stimulation. While stimulation with TNF-alpha resulted in inhibition of the growth of EG, it was not associated with inhibition of intracellular growth of IG. Investigation of the reason why M. avium is able to sense the changes in the intracellular environment triggering a change to the invasive phenotype suggests a direct relationship with macrophage apoptosis. These results suggest that intracellular growth is associated with novel mechanisms of M. avium uptake of macrophages and that those mechanisms appear to offer advantages to the bacteria in escaping the host defense.     

Fibronectin increases Trypanosoma cruzi amastigote binding to and uptake by murine macrophages and human monocytes.

Trypanosoma cruzi amastigotes present receptors for human fibronectin as indicated by the saturable binding of [125I]fibronectin to this form of the parasite. Scatchard analysis indicates that the number of fibronectin receptors per amastigote was 1.3 x 10(3) with a Kd of approximately 2.3 nM. Addition of physiological concentrations of fibronectin to amastigote-macrophage cocultures significantly increased the binding of amastigotes to murine macrophages. This increase was evidenced in both the number of amastigotes bound to macrophages and the percentage of macrophages containing bound amastigotes. The uptake of amastigotes by either murine macrophages or human blood monocytes was also increased in the presence of exogenous fibronectin. The increase induced by fibronectin was blocked when amastigotes were pretreated with the RGDS tetrapeptide of the fibronectin cell attachment site. Furthermore, the ability of fibronectin to enhance amastigote binding to and uptake by macrophages was inhibited by the F(ab')2 fragment of anti-fibronectin immunoglobulin G (IgG) but not by an irrelevant anti-human IgG F(ab')2 fragment. Pretreatment of either amastigotes or macrophages with fibronectin also resulted in a significant increase in amastigote binding to and uptake by macrophages. These results suggest that fibronectin may play a role in facilitating the attachment and ingestion of T. cruzi amastigotes by macrophages and monocytes in chagasic tissue lesions.     

Serum and Plasma Fibronectin Binds to Complement Reacted Immune Complexes Primarily via C1q

The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phase with serum at 37 degrees C caused attachment of Clq and C3b. Addition of EDTA to the serum inhibited the binding of C3b, but not Clq, whereas substitution of the anti-BSA IgG on the solid phase with the F(ab')2 fragments abrogated the Clq, but not the C3b binding. Fibronectin binding was observed after incubation of the solid-phase IC with serum or plasma at conditions where Clq was also bound, whereas only minor amounts of fibronectin bound to the solid phase IC via C3b. Purified fibronectin showed a dose-dependent binding to solid-phase IC pretreated with Clq or fibronectin-depleted serum, confirming that the binding of fibronectin to IC largely occurred via Clq. Significantly smaller amounts of fibronectin were bound to solid-phase IC incubated with plasma instead of serum, despite the higher fibronectin concentration in plasma. This difference was found not to be due to a fibrinogen-fibronectin interaction in plasma. Binding of fibronectin to preformed fluid phase IC incubated with serum was demonstrated by SDS-PAGE analysis of PEG-precipitated IC.     

Fibronectin attachment protein is necessary for efficient attachment and invasion of epithelial cells by Mycobacterium avium subsp. paratuberculosis

Attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by two epithelial cell lines were enhanced by soluble fibronectin (FN). Peptide blocking of the FN attachment protein (FAP-P) inhibited the internalization of M. avium subsp. paratuberculosis. Disruption of FAP-P expression significantly reduced attachment and ingestion of M. avium subsp. paratuberculosis by T-24 and Caco-2 cells. The results indicate that the interaction between FN and FAP-P facilitates attachment and internalization of M. avium subsp. paratuberculosis by epithelial cells.     

Characterization of inhibition of Mycobacterium avium replication in macrophages by normal human serum.

Serum from some AIDS patients permits the rapid multiplication of Mycobacterium avium in cultured human macrophages. Serum from human immunodeficiency virus-negative individuals inhibits replication. The characteristics of the serum-induced inhibition were examined here. M. avium 7497 serovar 4 grew exponentially in macrophages when they were cultured in serumless medium. Growth was measured by determining the CFU after infected macrophages were lysed at 0 to 7 days after infection. Normal AB serum (5 to 10%) added to infected macrophages resulted in an initial 4-day lag of bacterial growth followed by rapid replication from 4 to 7 days. Serum also inhibited bacterial replication in medium without macrophages. This inhibition was not biphasic but was sustained over 7 days. Macrophage-associated M. avium became less responsive to serum inhibitor within 24 h after infection of macrophages. Within 2 days of culture, M. avium no longer responded to inhibitor. Replication of macrophage-derived M. avium (Vi) was in some instances serum inhibitable and at other times was enhanced by serum, when it was used to infect fresh macrophages. The Vi phenotype remained serum inhibitable without macrophages. Preinfection of macrophages with heat-killed M. avium did not alter serum-induced bacterial inhibition or escape from inhibition.     

Apoptosis of Mycobacterium avium-infected macrophages is mediated by both tumour necrosis factor (TNF) and Fas, and involves the activation of caspases

Mycobacterium avium causes disseminated infection in AIDS patients and several forms of infection in immunocompetent hosts. Recent studies have shown that M. avium infection of macrophages in vitro leads to apoptosis of significant numbers of infected cells. Several strains of M. avium used to infect human macrophages for 5 days (multiplicity of infection of 10) triggered 28-46% higher levels of apoptosis than observed with uninfected macrophages at the same time points. Mycobacterium avium strains unable to replicate intracellularly (rep-) resulted in a 15% rate of apoptosis, while M. smegmatis-infected monolayers showed the same percentage of apoptotic cells as the uninfected macrophage control. The presence of anti-TNF-alpha antibody reduced apoptosis to 17% and the presence of anti-Fas antibody reduced apoptosis to 10%. When both antibodies were used together, the apoptosis level was 5% above the control. Treatment with TGF-beta also reduced the number of apoptotic cells in infected monolayers. If intracellular growth was inhibited, apoptosis of macrophages decreased significantly. It was also shown that apoptosis was associated with IL-1 beta-converting enzyme (ICE) activation and was significantly reduced by a caspase inhibitor. Gaining understanding of the mechanisms of M. avium-associated apoptosis of macrophages will provide important insight into M. avium pathogenesis.     

Role of Complement Receptors in Uptake of Mycobacterium avium by Macrophages In Vivo: Evidence from Studies Using CD18-Deficient Mice

Mycobacterium avium is an intracellular pathogen that has been shown to invade macrophages by using complement receptors in vitro, but mycobacteria released from one cell can enter a second macrophage by using receptors different from complement receptors. Infection of CD18 (beta(2) integrin) knockout mice and the C57 BL/6 control mice led to comparable levels of tissue infection at 1 day, 2 days, 1 week, and 3 weeks following administration of bacteria. A histopathological study revealed similar granulomatous lesions in the two mouse strains, with comparable numbers of organisms. In addition, transmission electron microscopy of spleen tissues from both strains of mice showed bacteria inside macrophages. Our in vivo findings support the hypothesis that M. avium in the host is likely to use receptors other than CR3 and CR4 receptors to enter macrophages with increased efficiency.     

The roles of complement receptors type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) in the regulation of the immune complex-elicited respiratory burst of polymorphonuclear leukocytes in whole blood

The binding of immune complexes (1C) to polymorphonuclear leukocytes (PMN) and the consequent respiratory burst (RB) were investigated in whole blood cell preparations suspended in 75% human serum, using flow cytometry. Blockade of the complement receptor (CR)1 receptor sites for C3b on whole blood cells using the monoclonal antibody (mAb) 3D9 resulted in a 1.9-fold increase in the IC-elicited PMN RB after 5 min of incubation, rising to 3.1-fold after 40 min. This enhancement was not due to increased IC deposition on PMN. Blockade of CR3 abrogated the mAb 3D9-induced rise in RB activity and inhibited the IC binding to PMN in a whole blood cell preparation, with or without mAb 3D9, by approximately 40% from 15–40 min while reducing their RB over 40 min to approximately one third. Blockade of CR1 on either erythrocytes (E) or leukocytes, before mixing the populations, revealed that the potentiation of the RB by mAb 3D9 was associated with abrogation of E-CR1 function, whereas blockade of leukocyte-CR1 had a diminishing effect. Exposure to IC at high concentrations induced release of both specific and azurophilic granule contents from PMN. The latter was CR3 dependent in that blockade of the receptor inhibited the lactoferrin release by one third during 40 min of incubation. In conclusion, CR3 plays a significant role in the IC-mediated generation of an RB and release of specific granules by PMN, while CR1 on whole blood cells, primarily E CR1, restricts the IC-elicited RB in PMN. We propose that CR1 in whole blood promotes the degradation of IC-bound iC3b to C3dg, thereby rendering the IC inaccessible for binding to CR3.     

Inhibition by monoclonal anticomplement receptor type 1 on interactions between senescent human red blood cells and monocytic-macrophagic cells

The effect of different murine monoclonal antibodies (Mab) specific for the glycoprotein complement receptor type 1 (CR1), type 2 (CR2), and type 3 (CR3) on the adhesion to and on the phagocytosis of human senescent red blood cells (S-RBC) by monocytes or by monocyte-derived macrophages (M phi) was investigated. Murine Mab anti-CR3 (anti-Leu 15 and OKM1) were found to inhibit, in the same order of magnitude, on one hand, the Fc receptors (FcR)-dependent rosetting and phagocytosis, and, on the other hand, the S-RBC rosetting and phagocytosis by adherent monocytes. Thus, the specific involvement of the CR3 epitopes recognized by Mab anti-Leu 15 or by OKM1 in the interactions between S-RBC and monocyte/macrophage could not be demonstrated. Murine Mab anti-CR1 was found to be a significant inhibitor of binding to and of phagocytosis of S-RBC (but not of young [Y] RBC) by monocytes or M phi, whereas Mab OKM5 carrying the same isotype as Mab anti-CR1, but a different specificity, was devoid of any significant inhibitory effect. Furthermore, Y-RBC or S-RBC opsonized with Mab anti-CR1 did not form FcR-dependent rosettes and were not internalized by monocytes; in addition, preincubation of phagocytes with Mab anti-CR1 did not inhibit FcR-dependent rosetting and phagocytosis. These results suggest that the effect of anti-CR1 is mediated through a specific binding to CR1 and not through an FcR blockade. As the role of specifically bound IgG on phagocytosis of human S-RBC by macrophages has previously been demonstrated by several authors, the present study suggests that monocyte-macrophage complement receptor type 1 may act in synergy with Fc receptors in the recognition of S-RBC by macrophages. It is shown in addition that the tripeptide Arg-Gly-Asp, identical to the region of iC3b recognized by CR3 and by several adhesion-promoting receptors that are structurally similar to CR3, such as fibronectin or vitronectin, is a significant inhibitor of the binding to and the phagocytosis of S-RBC by monocytic-macrophagic cells.     

Fibronectin attachment protein homologue mediates fibronectin binding by Mycobacterium avium subsp. paratuberculosis

Attachment of Mycobacterium avium subsp. paratuberculosis to host tissue and penetration of mucosal surfaces are pivotal events in the pathogenesis of Johne's disease. Fibronectin (FN) binding is required for attachment and internalization of several mycobacteria by epithelial cells in vitro. The objective of this study was to further characterize the FN binding activity of M. avium subsp. paratuberculosis. Although the bacteria bound FN poorly at pH above 7, brief acid pretreatment greatly enhanced FN binding within the pH range (3 to 10) studied. A 4.6-kbp fragment from an M. avium subsp. paratuberculosis genomic library was found to contain a 1,107-bp open reading frame that shows very high nucleotide sequence identity with that of the FN attachment protein (FAP) gene of M. avium subsp. avium. Pretreatment of FN with an FN-binding peptide from M. avium subsp. avium FAP abolished FN binding, indicating that M. avium subsp. paratuberculosis binds FN in a FAP-dependent manner. Pretreatment of M. avium subsp. paratuberculosis with anti-FAP immunoglobulin G did not abrogate FN binding; blocking occurred only when anti-FAP was added together with FN. FAP was detected by immunofluorescence only in lipid-extracted M. avium subsp. paratuberculosis. Western blotting and immunoelectron microscopy revealed that FAP is located near the interior of the cell envelope of M. avium subsp. paratuberculosis. The results indicate that a FAP homologue mediates the attachment of FN to M. avium subsp. paratuberculosis. Further, given the subcellular location of FAP, it is considered that this protein operates at the terminus of a coordinated FN binding system in the cell envelope of M. avium subsp. paratuberculosis.     

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.