Surfactant protein A binds to IgG and enhances phagocytosis of IgG-opsonized erythrocytes

Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein C1q interact. Since complement protein C1q binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to C1q, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to C1q. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding.     

Modular organization of the carboxyl-terminal,globular head region of human C1q A,B, and C chains

The first step in the activation of the classical complement pathway, by immune complexes, involves the binding of the globular heads of C1q to the Fc regions of aggregated IgG or IgM. Located C-terminal to the collagen region, each globular head is composed of the C-terminal halves of one A (ghA), one B (ghB), and one C chain (ghC). To dissect their structural and functional autonomy, we have expressed ghA, ghB, and ghC in Escherichia coli as soluble proteins linked to maltose-binding protein (MBP). The affinity-purified fusion proteins (MBP-ghA, -ghB, and -ghC) bound differentially to heat-aggregated IgG and IgM, and also to three known C1q-binding peptides, derived from HIV-1, HTLV-I, and beta-amyloid. In the ELISAs, the MBP-ghA bound to heat-aggregated IgG and IgM as well as to the HIV-1 gp41 peptide; the MBP-ghB bound preferentially to IgG rather than IgM, in addition to binding beta-amyloid peptide, whereas the MBP-ghC showed a preference for IgM and the HTLV-I gp21 peptide. Both MBP-ghA and MBP-ghB also inhibited C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. However, for IgM-coated erythrocytes, MBP-ghC was a better inhibitor of C1q than MBP-ghB. The recombinant forms of ghA, ghB, and ghC also bound specifically to apoptotic PBMCs. We conclude that the C1q globular head region is likely to have a modular organization, being composed of three structurally and functionally independent modules, which retains multivalency in the form of a heterotrimer. The heterotrimeric organization thus offers functional flexibility and versatility to the whole C1q molecule.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.     

FMMU白化豚鼠免疫学特性研究

目的　通过测定补体含量、免疫球蛋白含量、淋巴细胞的转化率及红细胞免疫功能 ,比较FMMU白化豚鼠和花色豚鼠的免疫学特性。方法　利用自动生化分析仪测定FMMU白化豚鼠和普通花色豚鼠的免疫球蛋白含量 (IgG、IgA、IgM)、C3、C4含量及总补体水平 ;利用淋巴细胞转化实验测定淋巴细胞的转化率 ;利用红细胞免疫复合物 (immunocomplex ,IC)花环形成试验测定两种豚鼠红细胞免疫粘附功能。结果　FMMU白化豚鼠血清中免疫球蛋白含量 (IgG、IgA、IgM)、C3、C4含量及补体总活性均显著低于花色豚鼠 ;FMMU白化豚鼠淋巴细胞转化率比花色豚鼠略低 ;FMMU白化豚鼠红细胞C3bR花环形成率与花色豚鼠差异无显著性 (P >0 0 5 )。而RBC IC花环率显著低于花色豚鼠 (P <0 0 5 )。结论　封闭群FMMU白化豚鼠具有独特的免疫学特性 ,总体免疫学功能低于花色豚鼠。     

Evidence that complement protein C1q interacts with C-reactive protein through its globular head region

C1q acts as the recognition unit of the first complement component, C1, and binds to immunoglobulins IgG and IgM, as well as to non-Ig ligands, such as C-reactive protein (CRP). IgG and IgM are recognized via the globular head regions of C1q (C1qGR), whereas CRP has been postulated to interact with the collagen-like region (C1qCLR). In the present study, we used a series of nine mAbs to C1q, five directed against C1qGR and four against C1qCLR, to inhibit the interaction of C1q with CRP. The F(ab')(2) of each of the five mAbs directed against C1qGR inhibited binding of C1q to polymerized IgG. These five mAbs also successfully inhibited the interaction of C1q with CRP. Moreover, these five mAbs inhibited C1 activation by CRP as well as by polymerized IgG in vitro. In contrast, none of the four mAbs against C1qCLR inhibited C1q interaction with CRP or IgG, or could reduce activation of complement by CRP or polymerized IgG. These results provide the first evidence that the interaction of C1q with CRP or IgG involves sites located in the C1qGR, whereas sites in the CLR do not seem to be involved in the physiological interaction of C1q with CRP.     

A recombinant homotrimer, composed of the alpha helical neck region of human surfactant protein D and C1q B chain globular domain, is an inhibitor of the classical complement pathway

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain. The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen. In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure. Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein. Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB(3), formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. The functional properties of ghB(3) indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. The finding that ghB(3) is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.     

An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors

CD11b/CD18 is a heterodimeric leukocyte surface receptor which functions in both C3bi-ligand binding and homotypic and heterotypic cell adherence. We have examined the effect of several anti-CD11b/18 mAb on phagocytosis of IgG (EIgG) or complement (EC4b) opsonized erythrocytes by polymorphonuclear leukocytes (PMN) and monocytes. F(ab')2 of two mAb (IB4, an anti-beta-chain mAb and Mo-1 an anti-alpha-chain mAb), inhibited both phagocytosis of EIgG and phorbol ester-stimulated phagocytosis of EC4b by PMN and monocytes. These F(ab')2 inhibited the binding of EIgG to monocytes, but they had no effect on binding of EIgG to PMN, or EC4b to either phagocyte. In addition, IB4 inhibited phorbol-ester stimulated phagocytosis of sheep E opsonized with C component 3bi (EC3bi) without inhibiting rosetting of these same targets. These data separate the anti-phagocytic effect of these mAb from effects on phagocyte-target adherence. When PMN were adherent to an anti-CD11b/CD18 F(ab')2-coated surface, EC3bi binding was abolished, but phagocytosis of EIgG or EC4b was unaffected. Subsequent addition of fluid- phase IB4 or Mo-1 F(ab')2 inhibited phagocytosis of EIgG or EC4b by the adherent cells. This suggested that the CD11b/CD18 involved in C3bi rosetting were mobile in the membrane, whereas those involved in phagocytosis of EIgG or EC4b were not. Cytochalasin treatment of PMN during adherence to F(ab')2-coated plates decreased both apical expression of CD11b/18 and subsequent ingestion of EIgG by 70%, suggesting that microfilaments are important in maintaining immobile CD11b/18 on the apical PMN surface. We conclude that there are functionally distinct populations of CD11b/CD18 on monocytes and PMN: one involved in C3bi rosetting and another involved in the process of phagocytosis mediated via several different receptors. CD11b/18 is not required for optimal target binding in all cases, but is always required for ingestion. As with several other integrins, the CD11b/18 molecules involved in phagocytosis have a functional association with the cell cytoskeleton.     

Specific inhibition of the classical complement pathway by C1q-binding peptides.

Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC(50) 2-6 microM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.     

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.     

Interaction of Human Waldenström's IgM Proteins with Guinea Pig and Human Complement1

The activity of purified human Waldenström's IgM proteins to fix complement of human and guinea pig origins was compared at different temperatures using the polystyrene latex particle-adsorption method. It was shown that the interaction of the IgM proteins with complement differed depending on the source of complement and that a pronounced heterogeneity in complement-fixing activity was observed among the IgM proteins when tested with guinea pig complement. Thus, by the use of guinea pig complement, six human IgM proteins examined were classified roughly into two groups, one having a high and the other a low activity at 3 C as well as at 37 C. With human complement, five proteins showed a rather uniform activity at 37 C. However, there was one protein with no detectable activity, suggesting the presence of non-complement-fixing protein in the IgM class. All the six proteins showed no significant activity with human complement at 3 C. No antigenic difference has been found as yet in the Fc or Cμ2 region among these IgM proteins examined.     

Interaction of human Waldenstr?m's IgM proteins with guinea pig and human complement.

The activity of purified human Waldenstr?m's IgM protein to fix complement of human and guinea pig origins was compared at different temperatures using the polystyrene latex particle-adsorption method. It was shown that the interaction of the IgM proteins with complement differed depending on the source of complement and that a pronounced heterogeneity in complement-fixing activity was observed among the IgM proteins when tested with guinea pig complement. Thus, by the use of guinea pig complement, six human IgM proteins examined were classified roughly into two groups, one having a high and the other a low activity at 3 C as well as at 37 degrees C. With human complement, five proteins showed a rather uniform activity at 37 degrees C. However, there was one protein with no detectable activity, suggesting the presence of non-complement-fixing protein in the IgM class. All the six proteins showed no significant activity with human complement at 3 C. No antigenic difference has been found as yet in the Fc or Cmu2 region among these IgM proteins examined.     

The interaction of protein A and Fc fragment of rabbit immunoglobulin G as probed by complement-fixation and nuclear-magnetic-resonance studies.

Protein-A-Fc-fragment complexes were observed in sedimentation-velocity experiments by ultracentrifugation. The interaction was studied by protein-fluorescence-quenching titrations of the Fc fragment with protein A, allowing the dissociation constant to be determined under a variety of conditions. The first component of the complement pathway, C1, is activated by complexes of protein A with rabbit IgG (immunoglobulin G), and the structural basis for this interaction was studied by using n.m.r. (nuclear magnetic resonance). The four Fc-fragment binding sites on protein A were shown to contain aromatic amino acids, and to be connected by mobile hydrophilic regions. Neither n.m.r. nor proton-relaxation-enhancement studies show evidence of a large conformational change of the Fc fragment on binding protein A, and this suggests that the cross-linking of the Fc fragments may be primarily responsible for the activation of component C1. This is supported by the inability of a univalent tryptic fragment of protein A to activate complement fixation by rabbit IgG.     

EAC4 and EAC14 production without purified Ci.

EAC4 and EAC14 of high activity were prepared by treating sensitized red cells with human or guinea pig serum in the presence of TTHA, a chelating agent whid at low pH to maximize C1 and C4 activity and to minimize C3 contamination. Cells prepared with guinea pig complement were contaminated with C2, which could be decayed away at 37 degrees C. Cells sensitized with IgG antibody were more reactive than those sensitized with whole serum or with IgM, and preparations made with TTHA-complement were more reactive than those prepared with purified C1 plus EDTA-complement. EAC14 stored at 0 degrees C for 3 weeks lost very little activity.     

Anticomplementary, Anticoagulatory, and Serum-Protein Precipitating Activity of Sodium Polyanetholsulfonate

Sodium polyanetholsulfonate (SPS) at 7.8 mug/ml completely abolished complement-mediated hemolysis of 1:10 diluted fresh guinea pig and human serum; at least twice as much SPS was required to reduce complement activity in 1:2 diluted human serum. The coagulation of 90 and 20% human blood was inhibited by 250 and 125 mug of SPS per ml, respectively. When added to fresh human serum, SPS precipitated beta 1C-globulin (C3), C4, beta lipoproteins, immunoglobulin IgG, IgM, and IgA, though incompletely.     

Alternative complement pathway activation by C4b deposited during classical pathway activation

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.     

Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies.

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.     

Antigenic specificities of human monoclonal and polyclonal IgM rheumatoid factors. The C gamma 2-C gamma 3 interface region contains the major determinants

The binding site specificity of 12 monoclonal and 11 polyclonal IgM rheumatoid factors (RF) isolated from human plasma or serum has been studied. All IgM RF bound best to sites on IgG and intact Fc. The monoclonal IgM RF did not bind at all to fragments lacking the C gamma 2 or C gamma 3 domains. In contrast, low level binding to the pFc' fragment, composed of the C gamma 3 domain, was seen with seven IgM RF, mainly from patients with rheumatoid arthritis (RA). IgG1 binding appeared to be a requisite specificity of all human IgM RF. IgM RF binding to IgG3 subclass was common among the monoclonal IgM RF. Most RA polyclonal IgM RF but only 2 of the monoclonal IgM RF possessed the IgG1, 2 and 4 binding pattern. Monoclonal IgM RF which bound best to histidine-modified IgG also bound well to IgG3. The 7-kDa fragment D of staphylococcal protein A inhibited the IgG binding of most monoclonal and to a lesser degree polyclonal IgM RF. Thus, the results indicate that the C gamma 2-C gamma 3 interface region of IgG contains the predominant determinants for monoclonal and polyclonal IgM RF. For some monoclonal IgM RF the binding site, even though at the interface of the C gamma 2 and C gamma 3 domains, is not the staphylococcal protein A site. Furthermore, polyclonal IgM RF possess specificities not encountered among the monoclonal IgM RF. These specificities may have special     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

The binding of complement by complexes formed between a rabbit antibody and oligosaccharides of increasing size.

Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.     

Stimulation of human neutrophil Fc receptor-mediated phagocytosis by a low molecular weight cytokine

Human neutrophil Fc receptor-mediated phagocytosis can be markedly enhanced by a low m.w. (less than 10,000) heat-labile cytokine(s) derived from specifically stimulated human mononuclear cells and from a human T cell line, MO(t). PMN incubated with supernatant from control mononuclear leukocyte (MNL) culture bound EIgG (percentage of rosettes = 73.7% +/- 7.1) but did not ingest the attached targets (phagocytic index, PI = 40.7 +/- 9.5) as efficiently as PMN incubated with supernatant from adherent MNL, which had ingested EIgG and were then cocultured with nonadherent MNL (PI = 264.3 +/- 46.3). Cytokine-containing supernatants were fractionated on YM-10 Centricon microconcentrators, and the effluent (YM-10E) was found to contain the phagocytosis-enhancing activity. Optimal Fc receptor-mediated ingestion by YM-10E-stimulated PMN required a critical level of target-bound IgG; stimulation was dose dependent and detectable after 5 min at 37 degrees C with a maximal response by 15 min. Monoclonal antibody 3G8 (anti-PMN Fc receptor) inhibited in a dose-dependent fashion both Fc receptor-mediated rosette formation and ingestion by nonstimulated and YM-10E-stimulated PMN. Solid-phase 3G8 Fab had the same effect. A previously undescribed monoclonal antibody, 1C2, exhibited a different pattern of inhibition. It had no effect on rosetting or ingestion of EIgG by nonstimulated PMN; however, it inhibited EIgG phagocytosis by YM-10E-stimulated PMN down to the level of nonstimulated ingestion without affecting rosette formation. Solid-phase 1C2 had the same effect. These data indicate that phagocytosis mediated by 3G8-positive Fc receptors may be enhanced by cytokine(s) stimulation in a manner requiring the molecule recognized by 1C2. Monoclonal antibodies to the alpha-chain of CR3 had only minimal effects on YM-10E-stimulated ingestion. Fluorescence flow cytometry of YM-10E-stimulated PMN, indirectly stained with 3G8 or 1C2, indicated that cytokine enhancement of EIgG ingestion occurred without an increase in either 3G8 or 1C2 binding sites. These data show that the low avidity Fc receptor, which binds immune complexes, may be functionally modulated at sites of inflammation where PMN and macrophages mediate clearance and destruction of immune complexes and opsonized particles.