Bactericidal/permeability-increasing protein promotes complement activation for neutrophil-mediated phagocytosis on bacterial surface

The neutrophil bactericidal/permeability-increasing protein (BPI) has both bactericidal and lipopolysaccharide-neutralizing activities. The present study suggests that BPI also plays an important role in phagocytosis of Escherichia coli by neutrophils through promotion of complement activation on the bacterial surface. Flow cytometric analysis indicated that fluorescein-labelled E. coli treated with BPI were phagocytosed in the presence of serum at two- to five-fold higher levels than phagocytosis of the bacteria without the treatment. In contrast, phagocytosis of the fluoresceined bacteria with or without treatment by BPI did not occur at all in the absence of serum. The phagocytosis stimulated by BPI and serum was dose-dependent. The effect of BPI on phagocytosis in the presence of serum was not observed on Gram-positive bacteria (Staphylococcus aureus). Interestingly, the complement C3b/iC3b fragments were deposited onto the bacterial surface also as a function of the BPI concentration under conditions similar to those for phagocytosis. Furthermore, the BPI-promoted phagocytosis was blocked completely by anti-C3 F(ab')(2) and partially by anti-complement receptor (CR) type 1 and/or anti-CR type 3. These findings suggest that BPI accelerates complement activation to opsonize bacteria with complement-derived fragments, leading to stimulation of phagocytosis by neutrophils via CR(s).     

Role of complement component C1q in phagocytosis of Listeria monocytogenes by murine macrophage-like cell lines.

Listeria monocytogenes is a facultative intracellular pathogen of a great variety of cells. Among them, macrophages constitute the major effector cells of listerial immunity during the course of an infection. Although the molecular bases of L. monocytogenes attachment and entry to phagocytes are not completely understood, it has been demonstrated that C3b significantly increases L. monocytogenes uptake by macrophages via complement receptor type 3. The first component of complement, C1q, is present in organic fluids at a relatively high concentration, and C1q receptor sites in macrophages are also abundant. In the present report, results of studies on the role of C1q in the internalization and infectivity of L. monocytogenes by macrophages are presented. L. monocytogenes uptake is enhanced by prior treatment of bacteria with normal sera. Heated serum or C1q-deficient serum abrogates this enhancement. Purified C1q specifically restored uptake. This effect was blocked by the addition of F(ab')2 anti-C1q antibody but not by an irrelevant matched antibody. Direct binding of C1q to L. monocytogenes was specific, saturable, and dose dependent with both fluorescent and radiolabeled C1q. N-Acetyl-D-alanyl-L-isoglutamine, diaminopimelic acid, and L-rhamnose caused a significant dose-dependent inhibition of C1q binding to bacteria, suggesting that these molecules, at least, are involved in the attachment of C1q to L. monocytogenes cell wall. When C1q binding structures on macrophage-like cells were blocked with saturating concentrations of C1q, the uptake of C1q-opsonized bacteria was less than in untreated cells. These experiments demonstrate that, in addition to other reported mechanisms, L. monocytogenes binds C1q, which mediates enhanced uptake by macrophages through C1q binding structures.     

Contribution of immunoglobulins M and G, complement, and properdin to the intracellular killing of Escherichia coli by polymorphonuclear leukocytes.

The effect of immunoglobulins and complement (C) on phagocytosis and intracellular killing of Escherichia coli was studied in vitro. The incubation system consisted of monolayers of human polymorphonuclear leukocytes and C-resistant, [3H]thymidine-labeled E. coli C source was human serum deprived of immunoglobulins and properdin by immunoabsorption. In the absence of C, only immunoglobulin G-coated bacteria were phagocytosed, whereas immunoglobulin M lacked opsonic activity. In the presence of C, phagocytosis was enhanced; however, immunoglobulin M was now more efficient than immunoglobulin G. Intracellular killing was notably augmented when C was activated by immunoglobulin G- or immunoglobulin M-coated bacteria; in contrast, the alternative activation of C by properdin had no effect on phagocytosis or intracellular killing. These results demonstrate the importance of immunoglobulins together with C not only for phagocytosis but also for efficient intracellular killing.     

Gentamicin kills intracellular Listeria monocytogenes.

The purpose of the experiments described here was to test whether membrane-impermeant antibiotics present in the extracellular milieu could kill bacteria within macrophages. For this, mouse macrophage hybrids and elicited mouse peritoneal macrophages first were allowed to phagocytose the facultative intracellular bacterium Listeria monocytogenes. The cells were incubated with or without gentamicin, and their bactericidal activity was measured. The results show that gentamicin caused normally nonbactericidal macrophages to kill L. monocytogenes. In addition, gentamicin caused listericidal cells to kill significantly more bacteria. To determine whether gentamicin accumulated within macrophages during culture, we tested whether lysates of macrophage hybrids cultured for 72 h in gentamicin-containing medium and then washed could kill Listeria cells. When cultured with 50 to 100 micrograms of gentamicin per ml, but not when cultured with 0 to 5 micrograms of gentamicin per ml, cell lysates were extremely listericidal, demonstrating the presence of intracellular gentamicin. Because gentamicin does not penetrate cell membranes, we hypothesized that it can be internalized by the cell through pinocytosis and can enter the same intracellular compartment as does phagocytosed L. monocytogenes. To test this, macrophages which had phagocytosed L. monocytogenes were incubated with the fluorochrome lucifer yellow to trace pinocytosed medium. About half of the Listeria cells within the macrophages were surrounded by lucifer yellow, indicating delivery of pinocytosed fluid, which could contain antibiotics, to phagosomes containing bacteria. The experiments described here indicate that membrane-impermeant antibiotics can enter macrophages and kill intracellular bacteria. Thus, the use of gentamicin in macrophage bactericidal assays can interfere with the results and interpretation of experiments designed to study macrophage bactericidal activity.     

Listericidal and nonlistericidal mouse macrophages differ in complement receptor type 3-mediated phagocytosis of L. monocytogenes and in preventing escape of the bacteria into the cytoplasm.

Listeria monocytogenes is a facultative intracellular bacterium that escapes phagocytic vesicles and replicates in the cytoplasm, where it becomes coated with F-actin. Macrophages, important anti-Listeria effector cells, are heterogeneous in their ability to kill Listeria. Complement receptor type 3 (CR3) mediates most phagocytosis of Listeria by listericidal macrophages. Experiments described here tested whether nonlistericidal macrophages also phagocytosed Listeria through CR3 and whether the ability of Listeria to escape into the cytoplasm correlated with lack of listericidal activity. We show here that CR3 mediated an average of 66% of the phagocytosis of serum-opsonized Listeria by listericidal peptone-elicited macrophages but only 35% by nonlistericidal thioglycolate-elicited macrophages. In thioglycolate-elicited macrophages, most Listeria were cytoplasmic and actin coated, whereas in peptone-elicited macrophages most were retained in the phagosome. These results indicate that listericidal and nonlistericidal macrophages phagocytose Listeria through different receptors and that nonlistericidal macrophages allow Listeria to escape into the cytoplasm.     

Adherence of Legionella pneumophila to guinea pig peritoneal macrophages, J774 mouse macrophages, and undifferentiated U937 human monocytes: role of Fc and complement receptors.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen of alveolar macrophages. Although previous studies have demonstrated that specific antibody facilitates uptake of L. pneumophila by phagocytic cells, the role of complement has been unclear. Thus, we have examined the relative contributions of Fc gamma- and complement receptor-mediated adherence to guinea pig peritoneal macrophages, U937 human monocytic cells, and J774 mouse macrophage cells. Opsonization of L. pneumophila (Philadelphia 2) with polyclonal immunoglobulin G promoted maximum adherence to guinea pig macrophages. In contrast, incubation in the presence of 20% fresh nonimmune human serum from a single donor did not promote adherence. The results obtained with U937 and J774 cells paralleled those obtained with guinea pig macrophages. In the absence of specific antibody, opsonization with guinea pig complement did not enhance adherence of the Philadelphia 1, Philadelphia 2, or Knoxville strain. However, when complement was added to heat-inactivated, specific antiserum, a fourfold increase in the number of adherent organisms was observed. Blocking studies utilizing membrane receptor-specific monoclonal antibodies demonstrated that both Fc and complement receptors mediated adherence of organisms treated with complement in the presence of specific antibody. These results suggest that complement augments adherence of L. pneumophila only when acting in concert with specific antibody.     

Opsonization of Legionella pneumophila in human serum: key roles for specific antibodies and the classical complement pathway

Legionella pneumophila has previously been shown to require serum factors for efficient uptake by phagocytic cells. In this investigation, the roles of specific antibody and complement in phagocytosis of L. pneumophila by human polymorphonuclear leucocytes (PMN) and tissue macrophages were determined. Opsonization was assessed by quantitating the uptake of [3H]-labelled Legionellae. Compared to other Gram-negative and to Gram-positive bacterial species, L. pneumophila was highly resistant to the opsonic activity of normal pooled human serum (PHS). Of 12 donor sera tested, only four promoted significant L. pneumophila uptake when used at full strength. Experiments with immune antibody, and with human sera deficient in immunoglobulins, or the complement components C2, C3, or C5, revealed that L. pneumophila opsonization was dependent on antibody-mediated activation of the classical complement pathway; activation of the alternative pathway could not be detected. At high concentrations, immune antibody alone could adequately opsonize L. pneumophila. Human alveolar and peritoneal macrophages required very similar amounts and types of opsonins for L. pneumophila phagocytosis as did human PMN. Heating L. pneumophila to temperatures greater than or equal to 80 degrees abolished its resistance to opsonization by diluted PHS; however, activation of complement via the alternative pathway or via other antibody-independent routes remained undetectable. These studies show that, in addition to immune antibody, the classical pathway of complement plays an important role in the opsonization of L. pneumophila. The limited ability of these bacteria to interact with human complement provides a likely explanation for their resistance to opsonization and may be partly based on heat-sensitive structures on the surface of L. pneumophila.     

The role of complement and antibody in opsonization and intracellular killing of Candida albicans

The purpose of this investigation was two-fold. The first, to explore the relationship between ingestion (measured by the phagocytic index method), iodination (measured by the neutrophil iodination micromethod) and intracellular killing (measured by the methylene blue test) of Candida albicans by human polymorphonuclear leucocytes. The second, to determine the effects of complement and antibody on ingestion and intracellular killing of C. albicans. Optimal phagocytosis of C. albicans was observed in fresh untreated human serum. Phagocytosis was present but reduced, in serum depleted of either antibody (by absorption) or complement (by heating at 56 degrees C for 30 min). Whilst in the complete absence of serum, or in FCS the levels were reduced still further. The percentage killed of ingested C. albicans remained constant irrespective of the number of organisms ingested, thus the greater the number ingested, the greater the number killed. Maximal intracellular killing expressed as a percentage of ingested Candida occurred in fresh untreated serum. Intracellular killing did occur in heat-inactivated serum and absorbed serum, although the levels were significantly reduced. The results suggest that C. albicans opsonized in fresh normal PHS are phagocytosed as well as killed more efficiently than those opsonized with only complement or antibody.     

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.     

Participation of immunoglobulins and complement components in the intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes.

Immunoglobulins and complement components are required for optimal ingestion and optimal killing of microorganisms by granulocytes. The degree of opsonization of microorganisms necessary for their ingestion was lower than that required for the killing of these bacteria during the ingestion phase. Killing during this phase was found to depend mainly on the presence of heat-labile opsonins, probably C3b, present on the microorganisms. Extracellular immunoglobulin G (IgG) and C3b were indispensable for optimal intracellular killing after ingestion was complete. This was established with an assay permitting assessment of the course of the number of viable intracellular bacteria independent of the ingestion of new live bacteria. Maximal intracellular killing by human granulocytes of ingested catalase-positive (Staphylococcus aureus and Escherichia coli) or catalase-negative (Streptococcus pyogenes and S. pneumoniae) microorganisms was found only when fresh serum was present extracellularly. Killing was suboptimal in the absence of serum. With heat-inactivated serum, the killing index lay between the indices obtained in the presence and absence of fresh serum. The stimulatory activity of heat-inactivated serum was most probably due to the interaction of IgG with the Fc receptor on the granulocyte membrane, since IgG subclasses IgG1 and IgG3 as well as pFc fragments of IgG stimulated the intracellular killing to the same degree as heat-inactivated serum did. In addition, (Fab1)2 fragments of IgG did not stimulate killing, and reduced killing was observed in the presence of heat-inactivated serum after reduction of the number of Fc receptors. The extra stimulation of the killing process in the presence of fresh serum compared with heat-inactivated serum was due to the interaction between membrane receptors and complement--most probably C3b generated by both the classical and the alternative pathways of complement activation. This conclusion is based on results obtained with sera in which one or both complement pathways were blocked, on the restoration of the killing-stimulatory activity of C3-deficient serum after addition of fresh C3, and on the reduced killing observed in the presence of fresh serum after reduction of the number of C3 receptors by the use of pronase or antigranulocyte serum.     

Opsonin-dependent and independent surface phagocytosis of S. aureus proceeds independently of complement and complement receptors.

We examined the mechanism of surface phagocytosis of Staphylococcus aureus by human polymorphonuclear leucocytes (PMN). Surface phagocytosis of unopsonized bacteria occurred, but was significantly enhanced by the presence of serum. The serum requirement was low, and a maximal effect occurred with serum concentrations of 0.25-0.5%. The opsonic effect of serum was not removed by heat inactivation of complement but was adsorbed, at low serum concentrations, by protein A, indicating that opsonin-dependent surface phagocytosis requires IgG but not C3. The requirement of opsonin-dependent surface phagocytosis for IgG was demonstrated further with purified IgG preparations as the sole opsonin. Activation of PMN by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA) increased opsonin-independent surface phagocytosis by 47% and 66%, respectively, but had no effect on opsonin-dependent surface phagocytosis. Blockade of the PMN iC3b receptor (CR3), which has lectin-like properties, by a panel of monoclonal antibodies against the alpha- and beta-chains of CR3 did not inhibit the surface phagocytosis of opsonized or unopsonized S. aureus, and one antibody (NIMP-R10) enhanced opsonin-independent surface phagocytosis. These results indicate that the mechanism of surface phagocytosis is quite different to that observed in suspension assays. Opsonin-independent surface phagocytosis occurs and is enhanced by PMN activation, opsonin-dependent surface phagocytosis is dependent on IgG and not complement, and neither opsonin-independent nor -dependent surface phagocytosis proceeds through CR3.     

Evaluation of the opsonic requirements for phagocytosis of Streptococcus pneumoniae serotypes VII, XIV, and XIX by chemiluminescence assay.

A luminol-enhanced chemiluminescence assay was used to investigate opsonic requirements for phagocytosis of STreptococcus pneumoniae serotypes VII, XIV, and XIX. After opsonization with whole immune sera (with antibody and total complement pathway), heat-inactivated immune sera (with antibody alone), or magnesium dichloride-ethylene glycol tetraacetic acid-chelated immune sera (with antibody and alternative complement pathway), live S. pneumoniae cells were incubated at 37 degrees C with normal polymorphonuclear leukocytes while serial chemiluminescence measurements were recorded. The amount of chemiluminescence observed correlated closely with evidence of phagocytosis as observed by microscopy. Complement was required for efficient opsonization, since all three serotypes showed a slower rise and less integral chemiluminescence after opsonization with heat-inactivated serum as compared with whole serum. The alternative pathway provided opsonic activity equal to that of the total complement pathway for type XIX, but only intermediate activity for types VII and XIV. Type-specific antibody was also required for effective opsonization of all three serotypes since chemiluminescence was markedly reduced when bacteria were opsonized with antibody-depleted serum (serum absorbed with type-specific S. pneumoniae cells at 4 degrees C). Thus, chemiluminescence proved to be an effective means of defining the requirement for both antibody and complement in the opsonization and phagocytosis of S. pneumoniae.     

Availability of complement bound to Staphylococcus aureus to interact with membrane complement receptors influences efficiency of phagocytosis

Complement-mediated opsonization of encapsulated Staphylococcus aureus (CP+) of the predominant capsule types, 5 and 8, remains poorly understood. Our previous work showed that complement is important for mouse survival of CP+ type 5 bacteremia and that the type 5 capsule inhibits the binding of opsonic C3 fragments to the organism. The importance of complement-mediated opsonization of CP+ was tested by neutrophil phagocytosis assays. Complement-mediated opsonization of CP+ increased phagocytosis by 57% compared to opsonization in complement-inhibited serum. Agar-grown CP+, enhancing capsule expression, was phagocytosed only one-tenth as well as the capsule-negative organisms (CP-), supporting the belief that staphylococcal polysaccharide capsules impair phagocytosis. Despite relatively poor phagocytosis of CP+ compared to CP-, complement activation increased the phagocytosis of CP+ by 103%. Thus, complement in normal human serum may have an important role in opsonizing CP+, even when capsule expression is strong. The ability of bound C3 fragments to interact with complement receptor 1 (CD35) on the membrane of human erythrocytes was tested in an immune adherence assay. S. aureus capsule was able to mask C3 fragments on the organism from binding to complement receptor 1. The inhibition of C3 binding to CP+ and the masking of deposited C3 fragments caused by the presence of capsule was associated with markedly decreased phagocytosis. The addition of anti-capsule antibodies to normal human serum was found to markedly improve the recognition of deposited C3 fragments by complement receptor 1 even when the absolute number of C3 molecules bound to S. aureus was not increased.     

A rapid fluorescent assay to distinguish attached from phagocytized yeast particles

In studies of phagocytosis and its consequences for cell activation, it is important to distinguish those particulate stimuli which are completely ingested and internalized from those which are only attached to phagocytic surfaces. Ingestion can be profoundly influenced by both the type of opsonins on the surface of the stimulus and the expression and activation of receptors on the phagocytes for these opsonins. We report a new fluorescent assay which facilitates rapid and reproducible quantitation of attached versus fully internalized live or dead yeast particles by phagocytes. The assay employs the fluorescent dye diaethanol (Uvitex 2B) which stains chitin on the cell wall of fungi and is excluded from live phagocytes. The diaethanol assay and a standard, previously published methylene blue dye exclusion assay yielded comparable results using human neutrophils or monocytes incubated with heat-killed, serum-opsonized Candida albicans. The diaethanol assay proved useful in distinguishing differences in effects of various opsonins, as human neutrophils selectively opsonized with either pooled human serum (PHS), IgG (heat-inactivated PHS) or complement (IgG-depleted PHS) completely internalized 69.5%, 91.3% and 52.5% of cell-associated zymosan particles respectively. Finally, the new assay permitted comparisons of differing macrophage populations, as resident murine peritoneal macrophages internalized only 10.6% of complement-opsonized, cell-associated zymosan particles compared with 41.7% when the macrophages were elicited with thioglycolate. The assay should prove useful to investigators studying both fungal phagocytosis and killing, as well as to those performing general studies of receptor expression, regulation and function.     

Characterization of the receptor-ligand pathways important for entry and survival of Francisella tularensis in human macrophages

Inhalational pneumonic tularemia, caused by Francisella tularensis, is lethal in humans. F. tularensis is phagocytosed by macrophages followed by escape from phagosomes into the cytoplasm. Little is known of the phagocytic mechanisms for Francisella, particularly as they relate to the lung and alveolar macrophages. Here we examined receptors on primary human monocytes and macrophages which mediate the phagocytosis and intracellular survival of F. novicida. F. novicida association with monocyte-derived macrophages (MDM) was greater than with monocytes. Bacteria were readily ingested, as shown by electron microscopy. Bacterial association was significantly increased in fresh serum and only partially decreased in heat-inactivated serum. A role for both complement receptor 3 (CR3) and Fcγ receptors in uptake was supported by studies using a CR3-expressing cell line and by down-modulation of Fcγ receptors on MDM, respectively. Consistent with Fcγ receptor involvement, antibody in nonimmune human serum was detected on the surface of Francisella. In the absence of serum opsonins, competitive inhibition of mannose receptor (MR) activity on MDM with mannan decreased the association of F. novicida and opsonization of F. novicida with lung collectin surfactant protein A (SP-A) increased bacterial association and intracellular survival. This study demonstrates that human macrophages phagocytose more Francisella than monocytes with contributions from CR3, Fcγ receptors, the MR, and SP-A present in lung alveoli.     

Human polymorphonuclear leucocyte receptors for staphylococcal opsonins

The presence of receptors on the plasma membrane of human polymorphonuclear (PMN) leucocytes for factors related to complement and for the Fc region of immunoglobulin has not been clearly defined for opsonized bacteria. To separate the activity of these two receptors, the uptake of [³H]thymidine labelled staphylococci opsonized with normal serum or heat-inactivated serum was measured. Phagocytosis was depressed when bacteria opsonized with normal serum were incubated with trypsin-treated leucocytes, suggesting that complement receptors of human PMN leucocytes are trypsin-sensitive. Phagocytosis of bacteria opsonized with heat-inactivated serum was not depressed by trypsin, but was blocked by incubating PMN leucocytes with heat-aggregated IgG and by incubating opsonized bacteria with protein A. In experiments performed to quantify the number of bacteria attached to but not ingested by PMN leucocytes, it was shown that both complement and Fc receptors participate in the ingestion phase of phagocytosis. Cell membranes of human PMN leucocytes possess two receptors for opsonized staphylococci; a complement receptor which is utilized when bacteria are opsonized in normal serum and an Fc receptor when bacteria are opsonized in heat-inactivated serum. Both receptors participate in the ingestion as well as the attachment phase of phagocytosis.     

Activation of the human complement alternative pathway by Listeria monocytogenes: evidence for direct binding and proteolysis of the C3 component on bacteria.

The capacity of the intracellular pathogen Listeria monocytogenes to activate the alternative pathway of human complement was examined. Incubation of L. monocytogenes with human serum in optimal conditions (20% Mg2+EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]-chelated serum) consumed (31.3 +/- 3.9)% of C3 hemolytic activity and led to similar amounts of C3 deposition among the 27 strains tested, except for a rough mutant and the penicillin-induced L forms of strain EGD, which bound reduced amounts of C3. The same results were obtained with strains belonging to related species (L. innocua, L. seeligeri, L. welshimeri, and L. ivanovii). Direct evidence is provided that L. monocytogenes induces the deposition of C3b and its cleavage products iC3b and C3d through ester and amide linkages, as demonstrated by the analysis of the released products of radiolabelled purified C3 after treatment with hydroxylamine. Our results clearly demonstrate that L. monocytogenes activates the alternative pathway of human complement, suggesting that bacteria in the blood or in tissues of infected patients are opsonized and targeted to C3 receptor-bearing cells such as macrophages.     

Mechanism of the bactericidal action of myeloperoxidase: increased permeability of the Escherichia coli cell envelope.

A sensitive and highly reproducible assay was utilized to study in vitro interactions of Listeria monocytogenes with resident and activated macrophages. The technique is not compromised by extracellular events and can readily differentiate between the efficiency of ingestion and the postphagocytic fate of bacteria. Heat-labile factors in human or homologous serum markedly enhanced the phagocytosis of Listeria without noticeably affecting the intracellular fate of the microorganisms. The behavior of Listeria within macrophages cultivated from C57BL/6 and BALB/c mouse strains corresponded to previous reports of in vivo growth patterns in inbred mice. Thioglycolate- or caseinate-elicited macrophages, although highly phagocytic, were unable to prevent the proliferation of Listeria. A bactericidal macrophage population was derived from from C57BL/6 mice which had been immunized intraperitoneally with a sublethal dose of L. monocytogenes and subsequently boosted with heat-killed homologous organisms. Elicitation of immune animals produced an increase in the percentage of peroxidase-positive macrophages, but this activity could not be correlated with restriction of intracellular bacterial growth.     

Phagocytic receptors dictate phagosomal escape and intracellular proliferation of Francisella tularensis

Francisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Various in vitro models of Francisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent of Francisella phagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strain Francisella tularensis subsp. tularensis Schu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fcγ receptor (FcγR), respectively. CR3-mediated uptake delayed maturation of the early Francisella-containing phagosome (FCP) and restricted phagosomal escape, while FcγR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate of Francisella by delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.     

Opsonizing activities of IgG,IgM antibodies and the C3b inactivator-cleaved third component of complement in macrophage phagocytosis

Phagocytosis of SRBC by guinea-pig peritoneal macrophages is enhanced by opsonizing IgG antibody alone. IgM antibody requires the presence of bound C3. Treatment of C3b coated SRBC with purified C3b inactivator (yielding EA_IgM C1423d) does not reduce attachment to, and phagocytosis by, peritoneal macrophages. This finding suggests the existence of a C3d receptor on peritoneal macrophages. EC43b intermediates which have been produced by removing IgM antibody by mercaptoethanol treatment and by subsequent removal of C1 and C2, are phagocytosed despite the absence of IgM antibody. Furthermore, treatment of EC43b with C3b inactivator does not change phagocytosis. Thus, IgM antibody does not appear to be a necessary prerequisite for the stimulation of phagocytosis, C3b or C3d alone being sufficient.