Characterization of murine macrophage Fc receptor-dependent phagocytosis and antibody-dependent cellular cytotoxicity during in vitro culture with interferons-gamma, alpha/beta and/or fetal bovine serum

Resident and oil-elicited inflammatory peritoneal macrophages (PM phi) from competent C3HeB/FeJ and genetically deficient C3H/HeJ mice were characterized for their Fc receptor (FcR)-dependent binding, phagocytic and ADCC functions during in vitro differentiation under the influence of mouse recombinant interferon-gamma (rIFN-gamma), interferon-alpha/beta (IFN-alpha/beta) fetal bovine serum (FBS), or in serum-free medium. Freshly cultured resident PM phi from C3HeB/FeJ mice had low levels of FcR-mediated phagocytosis in response to mouse monoclonal IgG gamma 2a, IgG gamma 2b or IgG gamma 1, opsonized sheep erythrocytes as compared to oil-elicited inflammatory PM phi from the same strain. Resident PM phi were uniformly upregulated in their FcR-dependent phagocytosis after 24-48 h in vitro culture with FBS to levels approximating that of freshly cultured inflammatory PM phi which were also further upregulated after 24 h in vitro culture with FBS. Both resident and inflammatory PM phi were upregulated largely by an autostimulatory process in that they increased their FcR-mediated phagocytosis in serum-free RPMI-1640 medium without the addition of rIFN-gamma or IFN-alpha/beta, although FBS further augmented FcR upregulation. A synergistic effect of FBS and rIFN-gamma was required for total reconstitution of FcR-mediated phagocytosis of FcR-incompetent C3H/HeJ inflammatory PM phi in that FBS or rIFN-gamma alone only partially reconstituted FcR function, whereas in combination full reconstitution occurred. Thus, macrophages from competent C3HeB/FeJ mice were upregulated in their FcR-mediated functions largely by an autostimulatory process, presumably dependent on endogenous of IFN-beta, whereas, genetically-deficient C3H/HeJ macrophages required exogenous rIFN-gamma in combination with fetal bovine serum for synergistic reconstitution of FcR functions. The uniform upregulation of FcR-dependent effector functions in vitro appears to provide an efficient system for enhanced immune function during differentiation which may be applicable to in vivo situations.     

Macrophage cytoskeleton association with CR3 and CR4 regulates receptor mobility and phagocytosis of iC3b-opsonized erythrocytes.

Alveolar macrophages (AM phi) were examined for CR1 (C3b receptor, CD35), CR3 (iC3b receptor; CD11b/CD18), and CR4 (iC3b receptor; CD11c/CD18) by assays for binding of C3-opsonized sheep erythrocytes (EC3b or EC3bi) and uptake of specific monoclonal antibodies (mAbs). In AM phi isolates from nine normal volunteers, 49% of cells bound EC3b and 71% bound EC3bi. Quantitation of receptors per cell with [125I]mAbs showed 8.5 x 10(4) CR4, 5.1 x 10(4) CR3, and 2.6 x 10(4) CR1. With most AM phi preparations, CR3 was the major receptor mediating attachment of EC3bi, despite the predominance of CR4 antigens. Anti-CR3 inhibited EC3bi rosettes by > or = 50%, whereas anti-CR4 blocked rosettes by < or = 18%. U937 cells differentiated with phorbol myristate acetate resembled AM phi in receptor expression but exhibited almost no CR4-dependent rosetting. Despite the relative inability of CR4 to mediate EC3bi attachment, AM phi ingestion of [51Cr]EC3bi was blocked by either anti-CR3 or anti-CR4. Two lines of evidence indicated that CR3 were more mobile within the membrane than were CR4. Immunofluorescence staining demonstrated patching and occasional capping of CR3, whereas CR4 remained uniformly distributed. This patching and capping of CR3 required the actin cytoskeleton, as it was inhibited by cytochalasin D. Modulation experiments using surfaces coated with anti-CR3 or anti-CR4 also showed that CR3 was more mobile than was CR4. However, there was some variation among AM phi isolates from different donors. In seven isolates, no CR4 modulation was produced with anti-CR4, whereas in six other isolates, CR4 was modulated by 66%. Incubation of cells in cytochalasin D increased modulation of both CR3 and CR4 on mAb-coated surfaces. Cells exhibiting increased mobility of CR4 showed an increased ability to form CR4-dependent EC3bi rosettes. The data are consistent with the hypothesis that CR3 and CR4 exhibit a variable association with the cytoskeleton that regulates their mobility and function. A relatively mobile subset of CR3 and/or CR4 mediates EC3bi attachment, whereas a relatively immobile subset of CR3 and/or CR4 fails to mediate EC3bi attachment but functions to promote ingestion of EC3bi.     

FcR-independent antibody-mediated cellular cytotoxicity

In this study we used palmitate-derivatized antibodies (pal-Ab) to examine the minimum contribution of Fc receptors (FcR) to macrophage (M phi)-mediated lysis and phagocytosis in antibody-dependent cellular cytotoxicity (ADCC). Pal-Ab specific for chicken erythrocytes (CE) were incorporated into the plasma membranes of M phi by insertion of the palmitate hydrocarbon chains into the outer leaflet of the phospholipid bilayer. In this system, the palmitate anchor bypassed the requirement for FcR in antibody-dependent effector-target conjugation and provided a unique opportunity to uncouple antibody-FcR interactions in ADCC. We show that binding of CE targets by P388D1 M phi effector cells through anti-CE pal-F(ab')2 can lead to efficient extracellular target cell lysis, but not phagocytosis. In contrast, pal-F(ab')2-mediated interactions between CE and peritoneal exudate M phi (PEM) activated both ingestion and extracellular lysis of the targets. In normal ADCC, FcR-dependent interactions between CE and either P388D1 cells or PEM triggered both extracellular lysis and phagocytosis. Our results demonstrate that lysis and phagocytosis in CE-directed ADCC by M phi have different minimum requirements for FcR functions. Moreover, our results suggest that FcR-independent triggers on the PEM surface are capable of triggering target cell lysis and internalization following antibody-mediated interactions.     

Modulation of macrophage mannosyl-specific receptors by cultivation on immobilized zymosan. Effects on superoxide-anion release and phagocytosis.

Unopsonized zymosan effectively induces a respiratory burst (O-2 release, hexose monophosphate (HMP) shunt stimulation) in thioglycollate-elicited and BCG-activated macrophages (M phi). These M phi are known to express lectin-like receptors specific for mannose or fucose-terminated glycoconjugates (MFR). A role for the MFR in phagocytosis of zymosan was demonstrated by cultivating M phi on a glutaraldehyde-fixed layer of zymosan, a procedure which depleted M phi of MFR-mediated pinocytic activity, but not other surface antigens (F4/80, Mac-1) or receptors (FcR, C3R). After modulation of MFR, M phi lost the ability to phagocytose zymosan, but ingested antibody or complement-coated zymosan vigorously via alternative receptors. Challenge with free zymosan failed to enhance respiratory burst activity in M phi which had been cultivated on zymosan. Such M phi were also refractory to zymosan taken up by alternative receptors or other ingested particles (EIgG), but responded to a non-particulate challenge, PMA. These studies show that the MFR, like other receptors, can mediate phagocytosis and elicit a respiratory burst in suitably primed M phi, but indicate that phagocytosis via specific receptors (FcR, C3R) need not trigger a respiratory burst.     

Interferon-gamma treatment impairs Fc receptor type II-mediated phagocytosis of human macrophages by a post-receptor-binding mechanism.

The influence of priming by interferon-gamma (IFN-gamma) on FcR expression and function was investigated with human monocyte-derived macrophages. As a ligand specifically interacting with FcRII, bovine IgG1-coated erythrocytes (Bo1-EA) were used. The uptake of these particles by human monocytes and macrophages could be inhibited with anti-FcRII. Moreover, macrophages representing the phenotype that fails to interact with murine IgG1, as revealed in an anti-CD3 IgG1-driven T-cell proliferation assay, had a low avidity for Bo1-EA, and Bo1-EA-macrophage interaction could not be inhibited by anti-FcRII in these cells. Thus, anti-Leu-4 non-responsiveness in a T-cell stimulation assay is associated with an inability of FcRII to interact with bovine IgG1. An influence of IFN-gamma priming on FcRII expression and function was studied, therefore, in anti-Leu-4 responders (bovine IgG1 high responders in the phagocytosis test, susceptible to anti-FcRII treatment). IFN-gamma-primed macrophages from such donors displayed a markedly reduced phagocytosis of Bo1-EA. This reduction was observed both with adherent and with suspended macrophages This type of modulation was not due to a reduced expression of FcRII, nor due to a reduced avidity of expressed FcR to its ligand, as revealed by flow cytometric and rosetting analysis. Since phagocytosis of latex particles and of tanned erythrocytes is little influenced by IFN-gamma priming, our data suggest that IFN-gamma affects FcRII-mediated phagocytosis by a post-receptor-binding mechanism.     

p150/95, Third member of the LFA-1/CR3 polypeptide family identified by anti-Leu M5 monoclonal antibody

Monoclonal antibody (mAb) anti-Leu M5 reacts with a two-chain molecule composed of a 150-kDa alpha subunit noncovalently associated with a 95-kDa beta subunit and probably is specific for an epitope on the 150-kDa alpha chain. This p150/95 antigen is the third member of a family of polypeptides sharing a common 95-kDa beta chain, which includes the lymphocyte function-associated antigen LFA-1 (p177/95) and complement receptor CR3 (Mo1/MAC-1/OKM1; p165/95) antigens. Sequential immunoprecipitation with anti-p95 beta chain mAb specifically removed the antigens detected by anti-LFA-1, anti-CR3 and anti-Leu M5 mAb. Certain patients with recurrent bacterial infections are genetically deficient in expression of the LFA-1 and Mo1 antigens, and have impaired granulocyte function. Granulocytes from a patient with this disease also failed to react with anti-Leu M5. Stimulation of normal granulocytes with f-Met-Leu-Phe, C5a-desArg, or calcium ionophore resulted in increased expression of Mo1 and Leu M5 antigens on the cell surface, but did not significantly increase expression of LFA-1 antigen. In functional assays, anti-Leu M5 did not inhibit T cell-mediated or natural killer cell-mediated cytotoxicity. In addition, anti-Leu M5 neither inhibited the binding of complement-coated particles to CR1 or CR3 nor did it affect the binding of EC3dg to neutrophils (CR4). These studies clearly indicate that the p150/95 antigen recognized by the anti-Leu M5 antibody is a structurally distinct member of the LFA-1/CR3 family.     

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.     

The coordination of signaling during Fc receptor-mediated phagocytosis

Phagocytosis by macrophages can be initiated by Fcgamma receptors (FcR) in membranes that bind to Fc regions of immunoglobulin G (IgG). Activated FcR transduce signals to cytoplasm, which regulate the internalization of IgG-coated particles into plasma membrane-derived vacuoles, phagosomes. Particles internalized by phagocytosis are much larger than FcR, which prompts questions of if and how the receptors are coordinated with each other. FcR-mediated signal transduction entails recruitment of proteins from cytoplasm to the receptor, largely via protein phosphorylation. These FcR signaling complexes then activate proteins that regulate actin, myosin, membrane fusion, and the production of reactive oxygen intermediates. Recent fluorescence microscopic studies of phagocytosis in macrophages indicate that signaling by FcR occurs as a sequence of distinct stages, evident in the spatial and temporal patterns of phosphoinositides, protein kinase C, and Rho-family GTPase activation on forming phagosomes. The coordination of these stages may be regulated by lipids or lipid-anchored proteins, which diffuse away from FcR complexes. Lateral diffusion of FcR-derived signals could integrate FcR-dependent responses over large areas of membrane in the forming phagosome.     

C1q acts synergistically with phorbol dibutyrate to activate CR1-mediated phagocytosis by human mononuclear phagocytes

The adherence of human monocytes and culture-derived macrophages to surfaces coated with complement subcomponent C1q has been previously shown to enhance Fc receptor (FcR)-mediated phagocytosis by these cells. We examined the effects of C1q on C3b/C4b receptor (CR1)-mediated phagocytosis by mononuclear phagocytes. A small percentage of human monocytes cultured in the presence of serum became competent to ingest sheep erythrocytes bearing IgM and C4b (EAC4b). This phagocytic activity was enhanced when these cultured-derived macrophages were adhered to C1q-coated surfaces. However, when cultured in a defined serum-free medium, these cells did not ingest EAC4b, even in the presence of C1q. To investigate this differential responsiveness, we studied the effects of C1q in conjunction with cell-activating agents on CR1 activation. Treatment of serum-free cultured monocytes with phorbol dibutyrate (PDBu), prior to addition of the targets, induced these cells to ingest EAC4b. In addition, when exposed to C1q, both the percentage of these PDBu mononuclear phagocytes ingesting EAC4b and the number of targets ingested increased threefold over the level achieved by macrophages treated with PDBu alone. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine did not activate CR1-mediated phagocytosis and did not substitute for PDBu in causing synergy with C1q. Freshly isolated monocytes adhered to human serum albumin-coated glass slides in the absence or presence of PDBu did not phagocytose EAC4b. Also C1q did not stimulate monocyte CR1-mediated phagocytosis. However, addition of PDBu to cells adherent to the C1q surface triggered phagocytosis of EAC4b. The concentration of PDBu and the time of addition of PDBu relative to addition of the EAC4b targets were found to be important parameters for the achievement of maximal synergy in both the freshly isolated and cultured cell systems. This enhanced phagocytic activity was also seen with cells adhered to the purified collagen-like, pepsin-resistant, fragment of C1q. Since this region was previously shown to interact with C1q surface receptors, it appears that occupancy of this receptor is triggering events contributing to the enhanced cellular function. These experiments suggest that C1q and PDBu promote ingestion via CR1 by different but synergistic mechanisms. These data also demonstrate that the CR1-mediated enhancement of phagocytosis is not specific for FcR-mediated ingestion, but also applies to phagocytosis via CR1.     

Comparison of receptors required for entry of Leishmania major amastigotes into macrophages.

We investigated the mechanisms of entry of amastigotes of Leishmania major from two different sources into macrophages by comparing their use of the Fc receptor (FcR), complement receptor type 3 (CR3), and mannose-fucose receptor (MFR). Amastigotes were obtained from BALB/c mice and SCID mice. FcR involvement was examined by opsonizing L. major with parasite-specific immunoglobulin G (IgG). Antiparasite IgG did not alter the uptake of amastigotes from BALB/c mice since these amastigotes had antibody bound to their surface: IgG1 was the most predominant antibody, followed by IgG2b, IgM, and IgG2a. However, opsonization with antiparasite IgG enhanced the entry of amastigotes that lacked antibody on their surface, namely, amastigotes obtained from SCID mice or from macrophages infected in vitro. These results indicate that the FcR is important for amastigote entry into macrophages. Down-modulation of FcRs onto immune complexes, however, did not reduce the entry of amastigotes containing surface-bound IgG into macrophages. Monoclonal antibodies against the CR3 inhibited the entry of amastigotes from either BALB/c or SCID mice into J774A.1 macrophage-like cells. Simultaneous blocking of FcR and CR3 further increased the inhibition of phagocytosis. Treatment of macrophages with soluble mannan or down-modulating the MFR onto mannan-coated coverslips had no effect on the entry of amastigotes from BALB/c or SCID mice. Thus, the MFR does not appear to be used by amastigotes of L. major. We show that ingestion of amastigotes appears to occur primarily through the FcR and CR3; however, additional receptors may also participate in the uptake of amastigotes.     

Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of high-affinity and low-affinity receptors for IgG by using different erythroid target cells.

Antibody-dependent cellular cytotoxicity (ADCC) and Fc receptor-mediated phagocytosis were determined with human monocytes, monocyte-derived macrophages and activated macrophages, using rabbit IgG-covered sheep red blood cells (EAs) and anti-D-treated human erythrocytes (EAhu) as target cells. Monocyte and macrophage-mediated ADCC were distinguished by different kinetics, monocytes lysing either target more rapidly than macrophages. Macrophage activation by recombinant IFN-gamma (rIFN-gamma) led to a marked increase in ADCC activity against EAhu. This manifested in increased lysis of optimally sensitized target cells, in a sustained lysis of target cells carrying low antibody densities, and as an enhanced resistance to lysis inhibition by competing fluid-phase inhibition by competing fluid-phase IgG. All these effects were less striking when EAs were the target cells. Phagocytosis of EAs by rIFN-gamma-treated cells was strongly suppressed, regardless of the amount of antibody on the target cells, and susceptibility to inhibition by fluid-phase IgG was slightly increased. By comparison, phagocytosis of EAhu was depressed to a lesser degree, and susceptibility to inhibition by fluid-phase IgG was reduced when macrophages were rIFN-gamma treated. These and other experiments suggested that the functional triggering of monocytes and macrophages by EAs involved, at least in part, low-affinity Fc receptors (FcR), whereas EAhu interacted with macrophages via high-affinity FcR. It is shown elsewhere that rIFN-gamma treatment of macrophages increases the expression of high-affinity FcR, but not low-affinity FcR (Jungi, Lerch & Brcic, 1987). Differences in the rIFN-gamma-induced functional alterations assessed with EAhu or with EAs are interpreted therefore as being a consequence of differential involvement of high-affinity FcR and of low-affinity FcR in mediating an effector function. For monitoring rIFN-gamma-induced alterations in the effector capacity EAs are more appropriate targets since up-regulation of high-affinity FcR has a smaller influence on the response to this type of target. Using metabolic inhibitors, ADCC could be dissociated from ingestion suggesting that ADCC is not a post-phagocytic event.     

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.     

Relationship between murine macrophage Fc receptor-mediated phagocytic function and competency for activation for non-specific tumor cytotoxicity

The relationship between Fc receptor (FcR) function and activation of murine macrophage populations for non-specific tumor cytotoxicity was studied. Oil-elicited inflammatory peritoneal macrophages (PM phi) from C3HeB/FeJ mice had higher FcR function upon harvest than resident PM phi from the same strain or elicited PM phi from genetically deficient C3H/HeJ mice. C3HeB/FeJ inflammatory PM phi were uniformly responsive to activation by MAF and the complement activators: LPS, Poly I:C, cobra venom factor (CVF) and zymosan for tumoricidal activity. Resident cells from the same strain and C3H/HeJ-elicited PM phi were uniformly unresponsive to the same activators. In vitro culture of C3HeB/FeJ resident PM phi with fetal bovine serum for 24-48 h produced unregulation of FcR function which coincided with a conversion from an unresponsive to a responsive state for tumoricidal activity. Reconstitution of the FcR function of C3H/HeJ-elicited PM phi during 24-48 h culture with lymphokine or Poly I:C also coincided with the restoration of responsiveness to activation by LPS, CVF, and zymosan for tumor cytotoxicity. Thus, the consistent temporal relationship between upregulated FcR function and the capacity of macrophages to respond to activation for non-specific tumoricidal activity may be more than coincidental. Preincubation of responsive C3HeB/FeJ-elicited PM phi with insoluble immune complex or heat-aggregated IgG was shown to blockade FcR-mediated phagocytosis and to abrogate LPS-mediated tumoricidal activity. Interestingly, FcR blockade by IgG-opsonized sheep erythrocyte conjugates selectively inhibited activation by MAF, LPS, and Poly I:C, but had no inhibitory effect on activation by CVF or zymosan. Similar blockade of C3b receptors produced an identical pattern of selective inhibition of activation. This selective inhibition of non-specific tumoricidal activity by FcR/C3bR blockade suggests the existence of two pathways for antibody-independent activation of macrophages.     

Mab. 198: a monoclonal antibody recognizing the complement type 3 receptor (CR3) in the rabbit.

A mouse monoclonal (Mab.198, IgG1) was generated that recognizes an epitope expressed on rabbit peripheral phagocytes and tissue macrophages. The membrane antigen recognized by Mab.198 consisted of two polypeptide chains of 165,000 and 95,000 molecular weight (MW). This Mab efficiently inhibited complement receptor-mediated granulocyte functions such as phagocytosis of complement-opsonized particulate antigens and zymosan-induced chemiluminescent responses. Fc receptor-mediated phagocyte functions were, on the contrary, barely affected by Mab.198. The cellular distribution, molecular structure and functional characteristics of the membrane antigen defined by Mab.198 suggested that this antibody recognizes the complement type 3 receptor (CR3). We found that Mab.M1/70, specific for mouse CR3 (i.e. CD 11 or Mac-1 antigen), also binds on rabbit phagocytes. However, this Mab recognizes a different CR3 epitope than Mab.198 as shown by cross-blocking experiments. This anti-rabbit CR3 Mab will be useful in the characterization of rabbit complement receptors and their involvement in immune reactions.     

Interaction of Mycobacterium avium complex with human macrophages: roles of membrane receptors and serum proteins.

Invasive, disease-associated members of the Mycobacterium avium complex are facultative intracellular pathogens of mammalian macrophages. The mechanism(s) by which M. avium is ingested by mononuclear phagocytes is unknown. We examined the role of membrane receptors on macrophages as well as the role of opsonic components of the serum (complement, fibronectin, C-reactive protein and fibrinogen in the attachment and ingestion of M. avium by human monocyte-derived macrophages. Preincubation of serum with antibodies against C3 and fibronectin, in contrast to preincubation of serum with antibodies against complement-reactive protein and fibrinogen, significantly reduced the binding of M. avium to macrophages in concentration-dependent manner (57 to 93% and 35 to 61% inhibition by anti-C3 and anti-fibronectin antibody, respectively, in a concentration range of 25 to 100 micrograms/ml). We also observed that incubation of macrophages with OKM1 anti-complement receptor type 3 (CR3) antibody in the presence of serum decreased the binding of M. avium to macrophages by 86% +/- 6%, while the same antibody inhibited binding by 63% +/- 7% in the absence of serum. In contrast, incubation of macrophages with anti-LFA-1, anti-p 150.95, anti-CR1, or anti-Mac-1 had no effect on the ability of M. avium to bind to the cell. In addition, incubation of macrophages with alpha-methyl-D-mannoside was also associated with decreased attachment of M. avium to macrophages. These results provide evidence for the role of three macrophage receptors (CR3, fibronectin, and mannosyl-fucosyl receptors) in the uptake of M. avium by human macrophages.     

Ability of rosetting or non-rosetting individual control and inflammatory macrophages to kill Escherichia coli X43 intracellularly

An autoradiographic method combined with a rosette technique was used to assess the bactericidal activity of individual control and inflammatory peritoneal macrophages (PM phi) in the presence or absence of expression of Fc receptor for IgG (FcR). There was a lack of FcR reactivity in a certain percentage of both categories of PM phi exposed to E. coli X43, a bacterium which is readily phagocytosed in the presence of specific antibody. Both rosetting and non-rosetting PM phi were capable of phagocytosing E. coli X43, but inflammatory PM phi showed a marked reduction in their capacity to ingest these bacteria compared with control PM phi. Once ingested the E. coli X43 were killed equally well by non-rosetting and rosetting control and inflammatory PM phi.     

Opsonin-independent phagocytosis of group B streptococci: role of complement receptor type three.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.     

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.     

Group B streptococcus-induced nitric oxide production in murine macrophages is CR3 (CD11b/CD18) dependent.

Nitric oxide (NO) is produced by murine macrophages in response to cytokines and/or gram-negative bacterial lipopolysaccharide. NO induction by gram-positive bacteria such as group B streptococci (GBS), the major etiologic agents of neonatal pneumonia and meningitis, has received little study. GBS as well as two other gram-positive bacterial species, Staphylococcus aureus and Staphylococcus epidermidis, were found to stimulate NO production in thioglycolate-elicited murine macrophages and in the mouse macrophage cell line J774A.1 in the presence of gamma interferon. Serotype Ia and III GBS were both stimulatory, as were asialo- and type antigen-deficient mutant strains of type III GBS. NO production was dose dependent, inhibitable by L-arginine analogs, and unaffected by polymyxin B. Since phagocytosis by murine and human phagocytes of GBS is dependent on complement receptor type 3 (CR3), the role of CR3 in the NO response to GBS was tested in the CR3-deficient myelomonocytic cell line WEHI-3. GBS did not induce NO, whereas S. aureus or lipopolysaccharide did induce NO in WEHI-3 cells. S. epidermidis, whose nonopsonic phagocytosis is also CR3 dependent, failed to induce NO in WEHI-3 cells. Monoclonal anti-CR3 (anti-CD11b or anti-CD18) in the presence of interferon also induced NO production in thioglycolate-elicited macrophages and in J774A.1 cells but not in WEHI-3 cells. This evidence suggests that ligated CR3 and gamma interferon act synergistically to induce NO production and that CR3 mediates the GBS-induced signal for NO production in interferon-treated macrophages.     

The complement fragment C3d facilitates phagocytosis by monocytes.

Two receptors for fragments of C3 are described for human monocytes: CR1 and CR3, which bind C3b and iC3b, respectively. Recently a leucocyte receptor that binds C3dg has also been described, designated CR4. We previously reported that IgM-sensitized sheep erythrocytes that are heavily coated with C3d (EAC3d) can bind to human monocytes that have been cultured in fetal calf serum (FCS). Here we determine whether such binding of C3d-coated targets can lead to phagocytosis, and identify the specific monocyte receptor involved in C3d binding. We confirm that EAC3d bearing greater than 10,000 C3d/cell bind to FCS-cultured monocytes. Furthermore, using non-cultured monocytes, we demonstrate that C3d enhances rosette formation of IgG-coated E and, like C3b and iC3b, C3d augments IgG Fc receptor-mediated phagocytosis. Less than 100 C3d/cell are capable of enhancing phagocytosis, whereas 10,000 or more C3d/cell are required for rosette formation with cultured cells. These results indicate that the C3d-binding receptor is present on peripheral blood monocytes but has poor affinity for target particles coated only with C3d. Anti-CR2 monoclonal antibodies, which recognize the C3d receptor of lymphocytes, do not block EAC3d rosette formation with monocytes. In contrast anti-Mol, a monoclonal antibody against CR3, inhibits EAC3d rosettes by approximately 42%. Anti-CR1 increases this effect, but complete inhibition is not achieved. Ethylenediamine tetraacetate also markedly reduces EAC3d rosetting, reducing the numbers to less than 5%. Thus, the C3d-binding receptor on monocytes, unlike CR4, is metal dependent. Together these data indicate that CR3 is predominantly responsible for C3d binding to monocytes.